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Recent Advances in Histopathology 23 (2015) (PDF) (UnitedVRG)
Recent Advances in Histopathology 23 (2015) (PDF) (UnitedVRG)
Histopathology 23
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Preface
We are delighted to present Recent Advances in Histopathology 23 after careful selection of the most
important developments within the field.
The practice of pathology is a constantly evolving speciality. To reflect this, one of the main goals
of this edition is to review the rapid development and application of molecular techniques to
complement the morphological assessment of tissue diagnosis, prognosis and response to treatment.
We have commissioned a wide range of chapters, in order to provide a comprehensive update
of key topics in histopathology for those preparing for their pathology postgraduate exams. We
believe that both generalist as well as specialist consultant and academic histopathologists will find
information of relevance to their daily practice and continued professional development.
This volume of Recent Advances in Histopathology continues the series role as a key resource for
pathologists, both in the UK and internationally, who wish to remain up to date with current trends
in modern histopathology. We are especially grateful to the contributors, who responded to our
invitation with enthusiasm and produced well written and interesting chapters in a timely fashion. The
next edition is already under development, and we welcome chapter suggestions from our readers.
Massimo Pignatelli
Patrick Gallagher
March 2014
iii
Contents
Chapter 1
Chapter 2
17
Chapter 3
31
Chapter 4
Pathology of obesity
47
Chapter 5
61
Chapter 6
73
Chapter 7
87
Chapter 8
103
Chapter 9
117
Chapter 10
135
Chapter 11
149
Chapter 12
159
Chapter 13
169
Index
181
Chapter 1
Antibody-mediated rejection of
solid organ allografts
Margaret M Burke, Desley AH Neil, Annalisa Angelini
IntroductIon
Transplantation is the treatment of choice for end-stage organ failure. Overall survival rates
in the UK [1] (Table1.1) are comparable to those from the international registries. However,
despite improved management of acute cellular rejection (ACR) and of the complications
of immunosuppression, antibody-mediated rejection (AMR) remains a significant cause of
morbidity and mortality early and late after transplantation.
Margaret M Burke, MB, FRCPath, Department of Pathology, Harefield Hospital, Royal Brompton & Harefield NHS
Foundation Trust, London, UK
Email: M.Burke@rbht.nhs.uk (for correspondence)
Desley AH Neil, BMedSc, MBBS, PhD, FRCPath, Department of Cellular Pathology, Queen Elizabeth Hospital
Birmingham, Birmingham, UK
Annalisa Angelini, MD, Heart Transplant Pathology Unit, Department of Cardiac, Thoracic and Vascular Sciences,
University of Padua, Padua, Italy
Table 1.1 Mean patient survival 1, 5 and 10 years after solid organ transplantation
(UK data 19982010*)
Organ
Kidney
Adult (DBD)
Adult (living donor)
Paediatric (DBD)
Paediatric (living donor)
95.75
98.50
99.25
98.50
88.3
95.5
98.3
96.6
75
90
94
93
Heart
Adult
Paediatric
81.25
92.50
71
81.6
55
59
Liver
Adult (DBD)
Paediatric (DBD)
88.25
92.75
74.6
86.3
59
81
Lung
Adult
Paediatric
78
NA
53
NA
31
NA
Pathology
are the most prevalent, with least risk to the graft [6]. De novo DSAs develop in 2030% of
transplant recipients, usually to Class II or combined Class I and II HLA antigens [4].
De novo non-HLA antibodies may develop, such as anti-vimentin against endothelium,
and mediate graft damage. Anti-MICA (MHC Class I related chain A) may be associated
with AMR and late allograft failure. Other non-HLA antibodies may be markers of previous
graft injury with exposure of self antigens [2]. Transplants have been done successfully
across the ABO blood barrier raising questions about differences in the immune response
to blood group (carbohydrate) antigens versus HLA (glycoprotein) antigens and endothelial
susceptibility to both. Antibodies to glutathione S-transferase T1 (GSTT1) develop in liver
transplant recipients with an allograft genetically mismatched for this protein [7].
CDL is a specific but not sensitive test for detection of anti-HLA antibodies. A recent
important advance in technology is the solid phase assay (Luminex assay) which uses flow
cytometry to detect antibodies to HLAs by the use of single antigens bound to polystyrene
beads. With its greater sensitivity compared with CDL assays, it has led to an increase in the
number of detectable anti-HLA antibodies. Many of these are DSAs, but not all are harmful
to the graft [6]. Recently the addition of labelled C4d or C1q to the Luminex assay has
enabled detection of C4d-fixing or C1q-fixing DSAs which appear to be strongly associated
with poor allograft survival compared with noncomplement-fixing DSAs [6,8]. In a further
refinement of the test single antigen kits to individual HLAs can enable identification of
individual DSAs which can then be quantified by their mean fluorescence intensity (MFI)
level. Increasingly the MFI is used to monitor the impact of DSA-depleting therapies. Whilst
there is considerable interest in these functional assays, there is no agreement as yet on
their potential value in individual recipients or, in the case of MFIs, what level should be set
as the threshold for significance.
Nonetheless these advances raise the possibility of developing systems of risk stratification
providing there is international standardization of methodology and interpretation of results.
Thus, development of personalised immunosuppression and desensitization in high-risk
DSA-positive potential recipients may become feasible in the future [9].
pAthology
The pathologist is a key member of the clinical team managing allograft recipients. Regular
updates and access to results of biopsy reproducibility studies using digital technology
[10,11] are available in international forums and publications such as those led by the
Banff Foundation for Allograft Pathology (http://www.banfffoundation.org/) and the
International Society for Heart and Lung Transplantation (ISHLT) Annual Scientific
Meetings (http://www.ishlt.org/). This ensures continual refinements of grading systems
for biopsy diagnosis of AMR in the different organs in the light of outcome data from
international multicentre studies.
In 2004, a National Institute of Health conference on diagnosis of AMR in solid
organ allografts proposed a generic classification system based on a multidisciplinary
approach using serology for DSAs, C4d deposition in tissues, histopathological changes
and graft dysfunction [12] (Table 1.2). Recent molecular studies, mainly in kidney, have
highlighted the importance of the histopathological features of AMR in the absence
of C4d positivity, and the combination of C4d and histology without a proven DSA in
diagnosing AMR [3].
Table 1.2 Modified NIH proposal for working classification of antibody-mediated rejection*
Antibody-mediated rejection:
Allograft dysfunction, histopathological findings, capillary C4d deposition, circulating DSAs (HLA or non-HLA)
Asymptomatic antibody-mediated rejection:
Histopathological findings, capillary C4d deposition, circulating DSAs (HLA or non-HLA)
Silent/latent antibody-mediated response:
Capillary C4d deposition and/or circulating DSAs (HLA or non-HLA)
*Adapted from Takemoto et al. [12].
Molecular studies in kidney suggest that C4d or DSAs alone with positive histopathology may equate with antibodymediated rejection [3,17]
DSAs, donor-specific antibodies; HLA, human leucocyte antigens.
Kidney
The presentation of renal AMR may be acute (aAMR) with rapid reduction in renal
function, often associated with proteinuria, or may be more insidious [13]. Ongoing
Pathology
untreated, partially treated or multiple episodes of aAMR can result in structural changes
in the allograft. Once these develop the process is called chronic AMR (cAMR) [9]. The
histological features depend on the timing of the biopsy.
By Banff criteria the diagnosis of renal AMR is multidisciplinary and requires DSA
positivity, C4d deposition in peritubular capillaries (PTCs) and histological features of
AMR [3,19]. If only two of the three features are present, or the pathologist is unaware of the
DSA data then it is classified as suspicious of AMR. However these criteria are increasingly
recognised as missing AMR with clinical impact [13,20].
Histopathology
In aAMR microcirculatory changes occur in PTCs and glomerular capillaries (GCs),
termed peritubular capillaritis and transplant glomerulitis, respectively (Figure 1.1a and
b), with accumulation of macrophages and T lymphocytes [13,21]. In severe cases, there is
thrombotic microangiopathy (TMA) with thrombi in arterioles and/or glomeruli [13]. The
other cause of TMA in this setting is acute calcineurin inhibitor (CNI) toxicity. Fibrinoid
necrosis in larger vessels is recognised as a feature of AMR [3] as is transmural and intimal
Figure 1.1 Acute renal antibody-mediated rejection. (a) Glomerulitis: Periodic acid Schiff-stained section of
glomerulus showing segmental capillary loops plugged with inflammatory cells (arrow). Most other capillary loops
are empty. (b) Peritubular capillaritis: Haematoxylin and eosin-stained section of vacuolated tubular epithelium
indicating acute tubular injury, interstitial oedema separating the tubules and peritubular capillaritis (arrows) with
intravascular inflammatory cells. (c) C4d-immunostained section showing deposition on endothelium of glomerular
capillaries and (d) on peritubular capillaries.
arteritis [13]. Peritubular capillaritis should be assessed only in cortex as it is less specific in
medulla, where it may be seen in ascending infection.
In cAMR there is reduplication of glomerular basement membrane (GBM) and
peritubular capillary basement membranes (PTCBMs) [13,22]. On light microscopy, this
is seen as double contours of GBMs, termed transplant glomerulopathy. Other causes
of double contours must be excluded such as recurrent or de novo immune complex
glomerulonephritis and chronic TMA due to other causes. Chronic vascular rejection,
consisting of fibromuscular intimal thickening, with minimal elastosis, is seen as a
consequence of cAMR, although other factors such as increased cold ischaemia time and
CMV infection may be involved.
Immunohistochemistry
C4d deposition is seen in PTCs and/or GCs (Figure 1.1 c and d) [23], but only peritubular
capillary deposition is assessed as part of Banff criteria for diagnosis of AMR. By IF, Banff
criteria state that 50% of PTCs need to show positive staining to be considered positive
[3]. However, recent evidence suggests that any staining has an impact on long-term graft
survival [23]. On IF GC C4d deposition may be difficult to assess because of glomerular
autofluorescence. The presence of glomerular C4d positivity increases the risk of long-term
graft loss. The medulla is the most sensitive area in which to detect C4d deposition in PTCs.
C4d deposition in atrophic areas has been shown to affect long-term graft survival [3]. With
the recent understanding of the role of natural killer (NK) cells in AMR, a marker for NK
cells may help in diagnosis [20].
Electron microscopy
The earliest feature of AMR is swelling of ECs, with increase in organelles of GC and PTC.
Within glomeruli this is associated with or followed by electron-lucent widening and
accumulation of debris on the subendothelial side of the GBM [24,25]. With more severe,
AMR there is necrosis of ECs and accumulation of platelets, both features of TMA.
Untreated glomerular changes progress with deposition of a new layer of GBM just
under the EC layer (Figure 1.2a). There may be interposition of mesangial cells in the
GBM, but there are no electron dense deposits, a feature which allows differentiation from
immune complex-mediated reduplication of the GBM. Parallel changes occur in PTCs with
reduplication of the PTC basement membrane (Figure 1.2b) [13,22]. Three to four layers
are evidence of early cAMR and five or more layers are diagnostic of cAMR [3,24]. The
ultrastructural features of cAMR precede the light microscopic features of double contours.
Heart
The incidence of cardiac AMR is estimated as 1020%. It may occur early or late
after transplantation. By recently established ISHLT criteria, diagnosis is based on
morphological and immunopathological criteria alone [14,15].
Pathology
GBM
Epi
Background
Several studies over the last two decades have correlated microvascular inflammation and
capillary C4d positivity with DSA positivity, graft dysfunction, CAV and a poor outcome
[26,27]. Diagnostic criteria for AMR were included in the 2005 revision of ISHLTs 1990 working
formulation for biopsy diagnosis of rejection [28]. In 2010, the requirements outlined in 2005
for DSA positivity and graft dysfunction were dropped after acceptance of asymptomatic AMR
as an entity, with the diagnosis of AMR to be made henceforth by pathological criteria alone
[2]. In 2011, a preliminary grading system for AMR was drawn up and published by members
of ISHLTs Pathology Council [14] with publication of the definitive version in 2013 [15]. It
remains subject to review as experience of its utility increases.
Histopathology
Diffuse myocardial microvascular inflammation is the hallmark of cardiac AMR (Figure
1.3a) and is associated with capillary C4d deposition and, often, circulating DSAs with/
without cardiac allograft dysfunction [14]. In pAMR1 (h+) and pAMR2 capillaries are
distended, with lumens narrowed by plump activated ECs and plugs of IV macrophages
[26,29] (Figure 1.3b and c). In pAMR3 there is oedema, vasculitis, haemorrhage,
microvascular thrombosis, capillary damage and myocyte necrosis with neutrophil
infiltration and karyorrhectic debris, usually in the clinical setting of profound irretrievable
allograft dysfunction. Occasional IV macrophages may be also seen in the quiescent state,
ACR, ischaemic injury, infection and healing biopsy sites.
Figure 1.3 Cardiac antibody-mediated rejection. (a) Haematoxylin and eosin-stained section of myocardium which
shows diffuse microvascular inflammation. (b) Capillary endothelial cells are prominent (short arrows) and lumens
contain mononuclear cells (long arrows). (c) The intravascular cells are CD68-positive, thus confirming them as
macrophages. (d) There is diffuse capillary C4d deposition.
Immunopathology
Antibodies for biopsy evaluation of AMR are C4d and CD68 using IHC and C3d, C4d and
HLA-DR using IF [14,15]. Some pathologists use either HLA-DR by IF or CD31/CD34 by
IHC to assess capillary integrity which may be lost in pAMR3 or after repeated episodes
of lesser grades of pAMR. In pAMR1 (i+) and pAMR2 a positive C4d result by IHC and IF
is multifocal (>50% of intact myocardium) or diffuse capillary deposition of any intensity
[14,15] (Figure 1.3d). Diffuse weak capillary C4d deposition may reflect previous or
resolving AMR. Focal strong C4d deposition by IHC (>1050% of intact myocardium) may
represent evolving AMR and the pathologist should recommend close monitoring of graft
function and peripheral blood DSA studies. In pAMR3 C4d deposition may be absent, weak
or multifocal because of endothelial damage. All C4d-positive cases should be followed
up by IHC/IF until negative. Perimyocytic C4d deposition is occasionally observed, but its
significance is currently unknown. Capillaries in Quilty lesions, scars and ischaemic injury
may also stain but are not relevant to diagnosis of AMR at this time. Results of IF staining
with the monoclonal antibody to C3d may predict AMR [30]. Results of IHC staining with
the polyclonal antibody to C3d are neither specific nor sensitive for AMR and are not
recommended at this time.
Pathology
The potential screening value of the histological features of AMR is poor [31,32], but
when confirmed by IHC, CD68-positive IV macrophages predict myocardial capillary C4d
deposition, circulating DSA and clinical symptoms early, but not late after transplantation
[29]. Although IV macrophages are typical for cardiac AMR, IV T lymphocytes have also
been identified, raising the question of evolving ACR, perhaps potentiating AMR [33] C4d
positivity by IF and/or IHC, without morphological changes, (i.e. pAMR1 (i+)], may predict
AMR [30], correlate with DSA production [27], graft dysfunction and loss and lead to later
accelerated CAV. Prospective detection of C4d and C3d in allograft biopsies using IF also
predicts AMR, allograft vasculopathy and increased mortality [30]. One study using paraffin
section IHC showed, surprisingly, that C3d, but not C4d deposition predicts CAV [34].
Liver
The true incidence and impact of AMR in the liver are unknown due to the lack of large
prospective studies assessing DSAs. This has also held back the interpretation of the
pathology, in particular the interpretation of C4d staining [35]. Its diagnosis and prevalence
are the major topics for discussion at the forthcoming Banff allograft pathology meeting in
August 2013 where further guidelines may be established. Currently, the diagnosis of AMR
in the liver should be based on graft dysfunction, a proven DSA and histological features
suggestive of AMR with a positive C4d stain in portal or sinusoidal microvasculature.
Background
As hyperacute rejection rarely occurred with livers transplanted against a positive crossmatch, the belief that the liver was protected from AMR has long been held despite
early studies showing an increased risk of graft loss in the first year from rejection [16].
Preformed DSAs are associated with worse allograft survival. Their incidence ranges from
10.5% to 22.2% and a positive pretransplant CDL cross-match occurs in about 10% of
recipients [36]. Preformed DSAs are cleared relatively quickly post-transplant (17 days)
in 6585% of patients. Persistent DSA is associated with steroid-resistant rejection, AMR
and the development of chronic rejection. De novo DSAs occur in up to 10% of recipients
in the first year post-transplant and around 50% longer term. They are associated with
increased risk of acute and chronic rejection, particularly in the presence of a high MFI,
with progressive graft fibrosis and the development of cirrhosis [37].
Autoantibodies of the type found in autoimmune hepatitis, such as smooth muscle
actin, but with an atypical staining pattern, are found in up to 75% of paediatric and adult
recipients [37]. Antibodies to GSTT1, a protein expressed in hepatocytes and bile ducts
but lacking in 20% of Caucasians and up to 58% of non-Caucasians, develop when there
is a genetic mismatch between the donor and recipient, the donor having the wild type
10
gene and the recipient the null genotype. They develop prior to the development of graft
inflammation, and only with a mismatched graft, suggesting that they are allogeneic
although their histological pattern is different to HLA DSAs [7].
Histopathology
The histological features suspicious of AMR are portal tract changes indicative of duct
obstruction portal oedema and a ductular reaction (Figure 1.4a), often accompanied
by a neutrophilic infiltrate. Sinusoidal inflammation is less clearly defined; however,
microvascular inflammation, with an increase in inflammatory cells, possibly macrophages
or neutrophils, has been found [36,38]. The presence of perivenulitis in ACR should raise
suspicion of coexistent AMR [38]. Features of evolving chronic rejection (dysplastic/sick
bile ducts and progressive paucity of bile ducts often in the setting of unresolving acute
rejection) should also prompt IHC for C4d deposition and serology for DSAs [16,35].
In the GSTT1-related DSA setting plasma cells are prominent, and in recipients with
chronic hepatitis autoantibodies are often detected. HLA DSAs have also been detected
in patients with this pattern, particularly when fibrosis is severe with bridging and
cirrhosis. Areas with unexplained coagulative necrosis may also be an indication of AMR.
In recipients with preformed antibodies and a positive cross-match nonspecific changes
resembling preservation-reperfusion injury may be seen in biopsies taken early posttransplant, including hepatocyte swelling, cholestasis and platelet microthrombi.
Immunopathology
The interpretation of C4d staining is still incompletely understood due to a lack of
correlation with DSAs in most studies [35]. C4d may be deposited in portal and/or
sinusoidal microvasculature (Figure 1.4b and c) [3840]. It has been shown that IF is more
sensitive than IHC but the staining patterns differ, with portal vascular staining dominating
in IHC and sinusoidal staining in IF [36,39]. In the setting of chronic rejection, C4d
deposition often appears to be sinusoidal in areas of central perivenulitis [38].
Lung
The true incidence of pulmonary AMR is unknown as definitions and diagnostic criteria are
currently lacking. Recently recommendations for biopsy interpretation have been published
in the first attempt to reach international consensus on pathology [17]. Two multicentre
studies are in progress with updates expected from the forthcoming Banff Foundation Allograft
Pathology Conference in August 2013 and at ISHLTs Annual Scientific Meeting in 2014.
The lung allograft is subject to challenge from both the recipients immune system and
the environment. This is reflected in overlapping patterns of pathology, with many potential
causes. Consequently, the diagnosis of pulmonary AMR is one of exclusion, made in a
multidisciplinary context and based on graft dysfunction, proven circulating DSAs and
histopathological features suggestive of AMR whether or not capillary C4d is detected (the
triple test).
Background
Known patterns of immunologic injury in the lung associated with de novo DSAs
include persistent/recurrent ACR of all grades, lymphocytic bronchiolitis and
Pathology
BD
b
Figure 1.4 Possible mixed hepatic rejection. (a)
Haematoxylin and eosin-stained section of a portal
tract which is inflamed with bile duct (BD) and venous
(v) inflammation features of acute cellular rejection.
Portal oedema and marginal ductular reaction (arrows)
raise the possibility of concurrent antibody-mediated
rejection. (b) C4d-immunostained section of a portal
tract showing staining of portal microvasculature and
(c) (from another patient) of liver parenchyma showing
C4d deposition on sinusoidal endothelium.
Histopathology
Interpretation of lung allograft pathology is on routinely fixed and processed transbronchial
lung biopsies and may be difficult because the biopsies are often small and fragmented
and may not fully inflate despite gentle agitation immediately after immersion in formalin.
Histopathological patterns which should prompt staining for C4d deposition are given
in Table 1.3 [17]. Chief amongst them is neutrophilic capillaritis, defined as patchy or
11
12
diffuse dense neutrophilic septal infiltrates with karyorrhectic debris. There may also be
fibrin platelet thrombi in the microvasculature, alveolar haemorrhage and flooding of
neutrophils into adjacent alveolar spaces. Neutrophilic capillaritis should be distinguished
from neutrophilic margination, i.e. neutrophilic infiltrates in the septae in the absence
of karyorrhexis and fibrin. Both may produce a histopathological picture of diffusely
thickened and cellular alveolar septae which should prompt higher power examination for
the above features (Figure 1.5a).
Immunopathology
Although the evidence for the role of capillary C4d deposition in pulmonary AMR is not
robust, it is still advised as part of allograft biopsy evaluation to enable pathologists to
gain more experience in its interpretation (Figure 1.5b). A scoring system analogous
to that for renal and cardiac biopsies (distribution of staining >50% = positive) is
suggested. Nonspecific C4d deposition in elastin in the alveolar walls is a significant
problem interfering with assessment of capillary deposition. Its deposition in hyaline
membranes may reflect other mechanisms of activation such as the nonimmune
mannose-lectin pathway of complement activation, perhaps explaining its occurrence
in infection, sepsis and reperfusion injury in both transplanted and nontransplanted
lungs [43].
Recommendations
Because of overlapping patterns of pathology with many different causes, a definition
and grading system for pulmonary AMR cannot be recommended at this time [17].
However, neutrophilic capillaritis +/ capillary C4d deposition should be reported as
Conclusion
Figure 1.5 Diagnosing pulmonary antibody-mediated rejection (AMR). (a) Haematoxylin and eosin-stained section
of a transbronchial lung biopsy. The alveolar septae are slightly thickened with increased cellularity due to a sparse
mixed inflammatory infiltrate. The picture is nonspecific; possible causes include early bacterial or viral infection.
AMR cannot be excluded. (b) C4d staining showed diffuse capillary deposition. The recipient had presented with
decreasing lung function despite treatment of acute cellular rejection and was found to be donor-specific antibodypositive. In the absence of other causes, especially infection, the clinicopathological diagnosis was findings
consistent with pulmonary AMR.
findings suggestive of AMR provided the diagnosis is put in its clinical context and
correlated with the results of contemporaneous DSA results (the triple test). Other
causes such as infection, acute lung injury of any cause and drug-related changes
should be excluded. If serological and clinical data are not available at the time of
reporting, a recommendation for serological testing should be made. Future critical
appraisal of this approach may enable progress towards a definition and an agreed
grading system for pulmonary AMR.
conclusIon
The pathological hallmarks of AMR have been described in several, but not all solid
organ allografts and are still evolving. Currently, microscopic features common to all
are capillary endothelial activation, microvascular inflammation, IV inflammatory cells
(nearly always macrophages) and capillary deposition of C4d. Molecular assessment
of biopsies in renal allograft recipients has revealed new information on aspects of
histopathlogical features of importance but does not, as yet, have a well-defined role in
diagnosis.
A standard approach to diagnosis and management of AMR should include correlation
of the biopsy findings with serological evidence of DSAs and assessment of allograft
function. This requires a multidisciplinary team approach by clinicians, immunologists
and pathologists. The advent of technology to quantitate circulating DSAs and to
determine their ability to fix complement and their IgG subtypes suggests that risk
stratification in an individual recipient should be possible, perhaps leading to personalised
immunosuppression in high-risk cases.
13
14
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26. Lones MA, Czer LS, Trento A, et al. Clinical-pathologic features of humoral rejection in cardiac allografts: a
study in 81 consecutive patients. J Heart Lung Transplant 1995; 14:151162.
27. Smith RN, Brousaides N, Grazette L, et al. C4d deposition in cardiac allografts correlates with alloantibody. J
Heart Lung Transplant 2005; 24:12021210.
28. Stewart S, Winters GL, Fishbein MC, et al. Revision of the 1990 working formulation for the standardization
of nomenclature in the diagnosis of heart rejection. J Heart Lung Transplant 2005; 24:17101720.
29. Fedrigo M, Feltrin G, Poli F, et al. Intravascular macrophages in cardiac allograft biopsies for diagnosis of
early and late antibody-mediated rejection. J Heart Lung Transplant 2013; 32:404409.
30. Tan CD, Sokos GG, Pidwell DJ, et al. Correlation of donor-specific antibodies, complement and its regulators
with graft dysfunction in cardiac antibody-mediated rejection. Am J Transplant 2009; 9:20752084.
31. Hammond ME, Stehlik J, Snow G, et al. Utility of histologic parameters in screening for antibody-mediated
rejection of the cardiac allograft: a study of 3,170 biopsies. J Heart Lung Transplant 2005; 24:20152021.
32. Fedrigo M, Gambino A, Benazzi E, et al. Role of morphologic parameters on endomyocardial biopsy to
detect sub-clinical antibody-mediated rejection in heart transplantation. J Heart Lung Transplant 2011;
30:13811388.
33. Fedrigo M, Leone O, Burke M, et al. Inflammatory cell burden and phenotype in endomyocardial biopsies
from patients with antibody-mediated rejection (AMR) - An AECVP multicenter study. J.Heart Lung
Transplant 2013;32(4S):S19.
34. Moseley EL, Atkinson C, Sharples LD, Wallwork J, Goddard MJ. Deposition of C4d and C3d in cardiac
transplants: a factor in the development of coronary artery vasculopathy. J Heart Lung Transplant 2010;
29:417423.
35. Bellamy CO. Complement C4d immunohistochemistry in the assessment of liver allograft biopsy samples:
applications and pitfalls. Liver Transpl 2011; 17:747750.
15
16
36. Kozlowski T, Andreoni K, Schmitz J, Hayashi PH, Nickeleit V. Sinusoisdal C4d deposits in liver allografts
indicate an antibody-mediated response: diagnostic considerations in the evaluation of liver allografts.
Liver Transpl 2012; 18:641658.
37. Evans HM, Kelly DA, McKiernan PJ, Hubscher S. Progressive histological damage in liver allografts following
pediatric liver transplantation. Hepatology 2006; 43:11091117.
38. Bellamy CO, Herriot MM, Harrison DJ, Bathgate AJ. C4d immunopositivity is uncommon in ABO-compatible
liver allografts, but correlates partially with lymphocytotoxic antibody status. Histopathology 2007;
50:739749.
39. Lunz J, Ruppert KM, Cajaiba MM, Isse K, et al. Re-examination of the lymphocytotoxic crossmatch in liver
transplantation: can C4d stains help in monitoring? Am J Transplant 2012; 12:171182.
40. Musat AI, Agni RM, Wai PY, et al. The significance of donor-specific HLA antibodies in rejection and
ductopenia development in ABO compatible liver transplantation. Am J Transplant 2011; 11:500510.
41. Badesch DB, Zamora M, Fullerton D, et al. Pulmonary capillaritis: a possible histologic form of acute
pulmonary allograft rejection. J Heart Lung Transplant 1998; 17:415422.
42. Astor TL, Galantowicz M, Phillips A, Palafox J, Baker P. Pulmonary capillaritis as a manifestation of acute
humoral allograft rejection following infant lung transplantation. Am J Transplant 2009; 9:409412.
43. DeNicola MM, Weigt SS, Belperio JA, et al. Pathologic findings in lung allografts with anti-HLA antibodies. J
Heart Lung Transplant 2013; 32:326332.
Chapter 2
The maternal death autopsy
Sebastian Lucas
Introduction
In the UK, the current maternal mortality rate is 11.4/100,000 maternities, resulting in
about 120 deaths a year [1]. About 80% are autopsied by instruction from a coroner or fiscal
(consented autopsies are most unusual). Thus, such autopsies are relatively rare, and the
appropriate trend is for them to be examined in regional centres by specialist pathologists.
This is the ideal since the range of possible causes of death is very wide (Table 2.1); the
evaluation of causation can be complex, requiring much histopathology and thinking time;
and the medical, social and legal consequences of such deaths are profound, prolonged
and expensive.
18
a. Uterine atony
b. Abruption of the placenta
c. Placenta praevia
d. Abnormally adherent placenta
i. Placenta accreta, increta, percreta
e. Genital tract trauma tear, laceration
i. Spontaneous or iatrogenic forceps, episiotomy
f. Retained placental material
g. Rupture of the uterus
i. Spontaneous or iatrogenic
h. Life support for PPH
i. Transfusion-associated lung injury ( TRALI), fluid overload
Peripartum dilated
cardiomyopathy (PPCM)
Amniotic fluid embolism
syndrome (AFES)
Early pregnancy deaths
Sepsis
Obstetric anaesthesia
a. General anaesthesia
i. Cardiac or ventilatory problems
b. Epidural (spinal) anaesthesia
ii. Infection
iii.Dural puncture, cerebrospinal fluid (CSF) leakage and subdural
haemorrhage
Air embolism
Choriocarcinoma and
hydatidiform mole
Ovarian hyperstimulation
syndrome (OHSS)
Acute fatty liver of pregnancy
(AFLP)
Indirect
Venous thromboembolism
Cardiac
a.Dissection of aorta
b.Dissection of coronary , splenic and other abdominal arteries
Other diseases
Indirect
Coincidental
Definition
Examples
Pre-eclampsia
Amniotic fluid embolism
Genital tract trauma
Postpartum haemorrhage
Genital tract sepsis
Dissection of aorta
Sudden cardiac death with a
morphologically normal heart
Congenital heart disease
Venous thromboembolism
HIV/AIDS
Homicide
Road collision
Illicit drug toxicity
Most cancers
Some suicides
always helpful. Although virtually all maternal autopsies are medicolegal, coroners do not
place obstacles in the way of the investigation, being increasingly only too pleased to have
them sorted out unambiguously.
More attention in this chapter is directed to the direct deaths; indirect and coincidental
deaths encompass the range of pathologies encountered in any women, although there are
aspects where pregnancy accentuates the pathology.
19
20
Pathology
The important autopsy pathology is in the lungs. Amniotic fluid, amniotic and fetal
squamous cells and fetal hair embolise to the small vessels of the lungs. They may be
seen easily on haematoxylin and eosin stains, but it is critical to perform supportive
special stains: Alcian blue to show amniotic acid mucin, and high molecular weight
keratin immunohistochemistry (e.g. LP34) to demonstrate the squames. An endothelial
CD31 immunostain completes the panel, to distinguish embolic squames from sloughed
endothelial cells (Figure 2.1).
In the renal glomeruli, fibrin thrombi are usually found in the capillary lumens,
reflecting the common disseminated intravascular coagulation (DIC) that is part of the AFE
syndrome (Figure 2.2). The uterus will usually show much mucosal bleeding, and perhaps
AF material in the mural veins. In principle, it may be possible to demonstrate the location
of entry of AF into the uterine veins, e.g. via a caesarean incision or mucosal split, but in
practice this rarely pertains.
The pathogenesis is debated [4]. Traditionally, the amniotic material embolising into the
lung is thought to trigger an acute anaphylactic response with cardiopulmonary shut down,
whilst also triggering the clotting cascade and consumptive coagulopathy. But some argue
that AFE syndrome is merely an example of systemic inflammatory response syndrome
(SIRS) from inappropriate release of endogenous inflammatory mediators, an abnormal
maternal immune response to fetal antigens.
Table 2.3 Differential diagnosis of sudden collapse in pregnancy, during or after delivery
Venous thromboembolism
Amniotic fluid embolism
Hypovolaemic shock from haemorrhage
Cardiac arrhythmia, e.g. SADS, ischaemic heart disease
Fulminant sepsis
Eclampsia
Arterial/aortic rupture
Air embolism
SUDEP sudden unexpected/unexplained death in epilepsy
Negligence aspects
Medicolegally, AFE is important since it used as a defence against claims of clinical
negligence where there has been fatal peri- or postpartum haemorrhage (PPH); for
whatever the cause of the haemorrhage, AFE would make it inevitably fatal. So it is
important to look for AFE in all cases where it might be a relevant factor, to prove or
exclude it.
21
22
Figure 2.2 Thrombotic microangiopathy in the kidney glomerulus. (a) Disseminated intravascular coagulation
fibrin thrombi in amniotic fluid embolism syndrome (haematoxylin and eosin). (b) Platelet thrombi in thrombotic
thrombocytopaenic purpura immunohistochemical stain against CD61.
Pathology
Brain
In about 60% of fatalities, there is deep intracerebral haemorrhage, without pre-existing
berry aneurysm, or other predisposing lesion. Diffuse cortical petechial haemorrhages
are another version, particularly prominent in the occipital lobes. Otherwise there is brain
swelling and diffuse cerebral oedema.
Kidney
The lesion of glomerular endotheliosis is characteristic and unique to PET [7]. The endothelial
cells are swollen, making the glomerular capillaries appear bloodless (Figure 2.3). The
glomerulus may also herniate into the proximal tubule. Endothelial cells may be vacuolated
with lipid (best seen with electron microscopy). With silver staining, basement membrane
thickening and remodelling produces a string-of-beads appearance.
Liver
Grossly the liver shows blotchy focal or confluent haemorrhagic necrosis [8]. Histologically,
there is periportal fibrin deposition, haemorrhage and hepatocyte necrosis, a lesion unique
to PET.
Placental abruption
Abruption of the uterus will have been heralded clinically. It leaves a clot between the
maternal placental surface and uterus which usually indent both. It is often accompanied
by a severe coagulopathy.
Creta syndromes
The placenta creta syndromes commonly follow from previous caesarean section, with
the fibrotic scar rendering the decidua suboptimal. The placental villi then attach direct to
the uterine muscle (accreta), or invade further into the myometrium (increta) and, rarely,
through it (percreta). Torrential bleeding follows when the placenta is detached in the
23
24
third stage of labour. Histological examination of the uterus bed confirms the diagnosis
(Figure 2.4).
Uterine rupture
Rupture of the uterus is classically a consequence of big baby, small pelvis and prolonged
labour. Modern obstetrics mandates caesarean section, so this is rarely seen in the UK.
Previous Caesarean section is a risk factor. Drugs that enhance uterine contraction, used
in termination, labour and postpartum, can sometimes result in rupture; these include
misoprostol and oxytocics. The rupture is typically lateral.
Abortion
Spontaneous abortion or miscarriage (delivery before 24 weeks gestation without
iatrogenic induction) may be septic or aseptic. Causes of death include ascending genital
tract sepsis, uterine haemorrhage and molar pregnancy [1].
Legal, i.e. medically safe, termination of pregnancy has a minimal mortality. But
occasional fatality results from rupture of the uterus due to misoprostol, or comorbidity
such as taking cocaine. Unsafe (criminal) abortion is fortunately rare in the UK, but can
cause death from infection and haemorrhage.
Sepsis
Sepsis in pregnancy is complicated because it is not one syndrome but several, with different
pathogeneses [1]. In severe and fatal cases, the end result is bacteraemic septic shock and
multiorgan failure, often with DIC. The syndromes and pathologies are depicted in Table 2.4 [9].
Examining the placenta is critical for sepsis autopsies, ideally with microbiology culture
as well as histopathology. Pre-evisceration maternal blood cultures taken as aseptically as
possible from the neck vessels or heart are necessary, and should be part of the standard
protocol for all maternal autopsies. And check on any predeath cultures done since they
will be even more useful if positive.
In category 4 sepsis, note that there is no inflammation of the genital tract despite the
high bacterial load (Figure 2.5). This form of toxic shock sepsis accounted for one-third of
fatal sepsis in the last UK triennial report [1], and the pregnancy per se may not actually be
relevant to its occurrence in predisposed individuals.
In postdelivery genital tract sepsis, it is often not clear how the infection (most
commonly group A Streptococcus) entered the body. The Semelweis syndrome of
infection from the hands of health care workers directly into the genital tract is rare in
modern obstetrics in high-income countries. Inadvertent contamination by the mothers
hand, from her nasal carriage of community-acquired organisms, is probably frequent.
Case definition
Pathology
1. Unsafe abortion
Unsafe/illegal termination
of pregnancy
Clostridium spp
2. Ruptured membranes
(genital tract sepsis)
3. Postdelivery (genital
tract sepsis)
Vaginal or caesarean
delivery or termination of
pregnancy; a well interval
of one or more days; with
genital tract infection
evidenced by clinical,
microbiological and
histopathological features
Group A Streptococcus
pyogenes (GAS)
4. Community-acquired
sepsis
GAS, pneumococcus
5. Postpartum sepsis
related to birth process
but genital tract not
involved
25
26
Cardiovascular disease
In the UK, cardiac and vascular diseases are the commonest category of maternal death
[1]. Weakening of the walls of the aorta and some medium or large arteries (most often
the splenic or coronary artery) result in aneurysm, dissection and rupture usually in the
third trimester. The aetiology appears to be multihit: an inherent predisposition combined
with progesterone-associated weakening of the media. Histologically, there is elastic
degeneration, deposits of mucin and attenuated muscle. The outcome is usually a sudden
unexpected collapse from shock (Table 2.3).
Cardiac disease
Cardiac disease is increasing in prevalence in pregnancy [1]. The causes are listed in
Table 2.1. Whilst ischaemic heart disease is explicable on the grounds of lifestyle, obesity
and the increasing age of pregnant women, sudden unexpected arrhythmic cardiac death
syndrome is a puzzle. Yet significant numbers of women die suddenly in the third trimester
or after delivery, the autopsy is negative and the heart is morphologically normal. We
postulate that these may represent potentially inheritable cardiac conditions, such as long
QT syndrome. Thus, it is essential in this scenario to exclude all other possible causes of
death, including cocaine and other stimulatory drugs, and to retain a piece of frozen spleen
tissue for later DNA analysis. The blood relatives will be examined in a cardiac genetic clinic
to determine whether there is a recognised genetic disease.
Congenital heart disease (CHD) is increasingly better managed, and thus pregnancy
has become safer. But the large shift of blood volume and changing intravascular pressures
that take place physiologically, just after delivery, still mean that those with CHD and
pulmonary hypertension are at significant risk of cardiac arrest.
Peripartum cardiomyopathy
This is defined as heart failure during the last month of pregnancy and up to 5 months
postdelivery, with all other causes excluded. It is a dilated cardiomyopathy with the usual
nonspecific histology. Aetiologically, the current view is of an oxidative proapoptotic stress
on myocytes, related to prolactin [10]. Technically, PPCM is a direct maternal death.
Pregnancy-associated infections
As well as genital tract and other acute bacterial sepsis syndromes (see above), there are
other important associations, but the immunopathology is not well understood. Because
pregnancy is a relative immunodepressed state with regard to cell-mediated immunity,
viral infections (herpes simplex, viral hepatitis, influenza), listeriosis and tuberculosis
may be more aggressive than in the nonpregnant woman. However, there is no proof of a
general immunodepression in pregnancy that predisposes to bacterial infections that are
countered by neutralising antibody responses. Two viral syndromes are discussed below.
Epidemic influenza
The 2009-2010 pandemic of type A/H1N1 influenza demonstrated the impact of pregnancy
upon the clinical manifestations of H1N1 infection. It affected mainly third trimester
pregnant woman, who became severely ill from influenza pneumonitis and acute
lung injury, requiring treatment in intensive care. Many acquired secondary bacterial
pneumonia. Proportionately, pregnancy was the pre-eminent risk factor for death with
H1N1 infection with, roughly, a x100 relative risk of death compared with nonpregnant
women [12].
27
28
HIV/AIDS
This is not a significant issue in the UK, where nearly all HIV-positive pregnant women
(about 1500 pa) are identified before delivery, and treated for their benefit as well as
reducing the risk of maternofetal transmission of HIV [1]. But in low-income countries with
high HIV prevalence (e.g. sub-Saharan Africa), it is a major (and preventable) contribution
to mortality, increasing the maternal mortality rates by about 10-fold [13]. The typical
scenario is of late presentation with advanced HIV disease at around the time of delivery,
and death shortly after from tuberculosis or other opportunistic infections; or from sepsis
or complications of abortion.
Conclusion
This account is necessarily selective. It is likely that the rank order of types of death will
change as obstetrics and populations change; for example, increasing maternal age and
obesity [1,14] should impact on indirect maternal death rates. The maternal autopsy will
continue to provide powerful information on such future trends.
References
1. Cantwell R, Clutton-Brock T, Cooper G et al. Saving Mothers Lives: Reviewing maternal deaths to make
motherhood safer: 2006-2008. The Eighth Report of the Confidential Enquiries into Maternal Deaths in the
United Kingdom. BJOG 2011;118 (Suppl 1):1-203.
2. Lucas S. Guidelines on autopsy practice. Scenario 5: maternal death, G100. London: The Royal College of
Pathologists, 2010.
References
3. Knight M, Tuffnell D, Brocklehurst P, Spark P, Kurinczuk JJ. UK obstetric surveillance system. Incidence and
risk factors for amniotic-fluid embolism. Obstet Gynecol 2010; 115:910917.
4. Clark SL. Amniotic fluid embolism. Clin Obstet Gynecol 2010; 53:322328.
5. Sibai B, Dekker G, Kupferminc M. Pre-eclampsia. Lancet 2005; 365:785799.
6. Zeeman GG. Neurologic complications of pre-eclampsia. Sem Perinatol 2009; 33:166172.
7. Mirza FG, Cleary KL. Pre-eclampsia and the kidney. Sem Perinatol 2009; 33:173178.
8. Joshi D, James A, Quaglia A, Westbrook RH, Heneghan MA. Liver disease in pregnancy. Lancet 2010;
375:594605.
9. Lucas S. The autopsy pathology of sepsis-related death. In: Fernandez R (ed.), Severe Sepsis and Septic
Shock - Understanding a Serious Killer. Rijeka: InTech, 2012.
10. Hilfiker-Kleiner D, et al. 16-kDa prolactin and bromocriptine in post-partum cardiomyopathy. Curr Heart
Fail Rep 2012; 9:174182.
11. Hunt BJ, Thomas-Dewing RR, Bramham K, Lucas SB. Preventing maternal deaths due to acquired
thrombotic thrombocytopenic purpura. J Obstet Gynaecol Res 2013; 39:347350.
12. Moran NF, Moodley J. The effect of HIV infection on maternal health and mortality. Int J Gynaecol Obstet
2012; 119:S2629.
13. Lucas SB. Predictive clinicopathological features derived from systematic autopsy examination of patients
who died with A/H1N1 influenza infection in the UK 200910 pandemic. Health Technol Assess 2010;
14:83114.
14. Knight M, Kurinczuk JJ, Spark P, Brocklehurst P. UK Obstetric Surveillance System. Extreme obesity in
pregnancy in the United Kingdom. Obstet Gynecol 2010; 115:989997.
29
Chapter 3
Classification and treatment of
non-small-cell lung carcinoma
Golda Shelley-Fraser, Nidhi Bhatt, Adam Dangoor and Matthew Sephton
Introduction
Lung cancer mortality remains a major health issue causing over a million deaths worldwide
in 2000 according to WHO data [1]. Cancer trends have been steadily changing in recent
times. Whilst lung cancer in wosmen remains low in developing countries, incidence and
mortality in women have almost doubled in developed countries over a 30-year period.
Latest data from WHO GLOBOCAN 2008 [2] shows that lung cancer has surpassed breast
cancer as the leading cause of cancer death in women. Alternatively, incidence in men
has plateaued in developed countries. Changing lung cancer trends have been related to
increasing incidence of smoking in women, particularly of cigarettes with lower tar and
nicotine that are also reportedly associated with increase in adenocarcinoma incidence.
Investigative studies into vast survival differences in adenocarcinoma subtypes (as in
2004 WHO classification) in last few years have resulted in major advancements in the
management of non-small-cell lung carcinoma (NSCLC).
Adenocarcinoma
In 2011, the International Association for the Study of Lung Cancer, American Thoracic
Society and European Respiratory Society (IASLC/ATS/ERS) jointly developed a
multidisciplinary approach to classification and management of adenocarcinoma [3]. In
essence, a diagnostic approach for former bronchioloalveolar carcinoma (BAC) has been
developed (Figure 3.1). There is emphasis on approach to small specimens for diagnosis as
well as to optimise tissue for molecular tests.
Preinvasive neoplasia
Atypical adenomatous hyperplasia (AAH)
This is defined as a 5mm area of atypical pneumocyte proliferation lining centriacinar
alveoli with minimal septal widening. The lesional cells are believed to be type 2
32
Adenocarcinoma
in situ
Minimally
invasive
adenocarcinoma
Lepidic
predominant
adenocarcinoma
BAC
Invasive
adenocarcinoma
with a lepidic
component
Invasive
mucinous
adenocarcinoma
pneumocytes and/or Clara cells. They have a hobnail appearance and a high nuclear:
cytoplasmic ratio.
AAH can be multifocal and is often an incidental finding in the background of NSCLCs.
It may be difficult to differentiate from type 2 pneumocyte hyperplasia, but AAH is discrete
unlike the ill-defined and patchy nature of the latter.
Invasive neoplasia
Minimally invasive adenocarcinoma (MIA)
MIA is a small (3cm) adenocarcinoma with lepidic growth pattern and 1 foci of invasion
measuring 5mm. Invasion is defined by the presence of angulated tumour nests or
acini within a desmoplastic or myofibroblastic stroma. By definition, it lacks pleural and
lymphovascular invasion and shows no evidence of tumour necrosis. As for AIS, diagnosis
Adenocarcinoma
should be made only after the entire lesion has been examined pathologically. If strict
diagnostic criteria are used, MIA has a 100% 5-year DFS.
Figure 3.2 Subtypes of invasive nonmucinous adenocarcinoma. (a) Lepidic component of a lepidic predominant
adenocarcinoma seen confined to the alveolar lining with no evidence of stromal invasion. (b) Lepidic
predominant with possible acinar component. (c) Acinar predominant adenocarcinoma showing glandular
profiles invading desmoplastic stroma. (d) Papillary predominant adenocarcinoma showing intra-alveolar tumour
papillae with fibrovascular cores. (e) Micropapillary predominant adenocarcinoma showing intra-alveolar tufts with
no stromal cores. (f ) Solid adenocarcinoma showing no morphological evidence of glandular differentiation.
33
34
Colloid adenocarcinoma
This is characterised by extracellular mucin lakes that expand and/or destroy existing
alveolar spaces and contain groups of malignant mucinous epithelium, which are often
difficult to visualise. The mucinous epithelium is of goblet cell or columnar type.
Fetal adenocarcinoma
This is characterised by cribriform and tubular structures composed of columnar cells with
clear cytoplasm that recapitulate fetal lung tubules and resemble early secretory phase
endometrium. Morular metaplasia, as in endometrioid adenocarcinoma, is also present.
Pure variants are usually seen in younger age groups. They are associated with
-catenin mutations and show nuclear (as opposed to membranous) -catenin [5]. In the
authors experience, they are often seen as a minor component in otherwise conventional
adenocarcinomas; these have normal membranous -catenin expression.
Enteric adenocarcinoma
This variant is defined as the presence of enteric differentiation (acinar/cribriform
architecture and intra-acinar necrosis) in at least 50% of the tumour. Presence of other
Figure 3.3 Mucinous lung neoplasia. (a) Mucinous atypical adenomatous hyperplasia. (b) Mucinous
adenocarcinoma in situ. (c) Mucinous minimally invasive adenocarcinoma. (d) Invasive mucinous adenocarcinoma.
(e) Enteric adenocarcinoma. (f ) Fetal adenocarcinoma.
35
36
This is a diagnosis of exclusion that can only be accurately made on a surgical specimen
rather than biopsy due to the heterogeneity of lung carcinomas. WHO has separated LCC
into subtypes: large cell neuroendocrine carcinoma, large cell undifferentiated carcinoma,
basaloid carcinoma, lymphoepithelioma-like carcinoma, clear cell carcinoma and LCC
with rhabdoid differentiation.
Large cell neuroendocrine carcinomas express neuroendocrine markers including
synaptophysin, chromogranin and CD56, although only expression of one is required
for diagnosis. Large cell undifferentiated carcinoma usually expresses cytokeratins
and approximately half express TTF-1; neuroendocrine markers are negative. Basaloid
carcinoma is rare, resembling its cutaneous counterpart. Lymphoepithelial-like carcinoma
of the lung shows a syncytial growth pattern and a prominent lymphocytic infiltrate;
there is an association with EpsteinBarr virus. Clear cell carcinoma shows prominent
glycogen containing clear cytoplasm and should be distinguished from metastatic renal cell
carcinoma. In LCC with rhabdoid differentiation, at least 10% of tumour cells should show
rhabdoid differentiation.
Adenosquamous carcinoma
Adenosquamous carcinoma is defined as carcinoma showing both squamous and
glandular differentiation, with each component comprising at least 10%, according to WHO
criteria. They represent between 0.4% and 4% of NSCLCs and are associated with cigarette
smoking. They often present as a peripheral mass and can show central scar formation.
Histologically, they must show both squamous and glandular differentiation. The
two components may be separate or may mingle. Diagnosis on biopsy is dependent
on sampling; definitive diagnosis is best made on the surgical specimen. On
immunohistochemistry, the adenocarcinoma component should express TTF-1 and CK7,
whilst the squamous component is usually decorated by p63 and CK5/6.
Sarcomatoid carcinoma
Sarcomatoid carcinomas are a group of poorly differentiated non-small cell carcinomas.
They represent approximately 1% of non-small cell carcinomas and are strongly associated
with tobacco smoking. WHO divides this subtype into 5: pleomorphic carcinoma, spindle
cell carcinoma, giant cell carcinoma, carcinosarcoma and pulmonary blastoma.
They commonly arise centrally, although pleomorphic carcinomas tend to be peripheral
showing a predilection for chest wall involvement. Pleomorphic carcinoma is composed of
non-small cell carcinoma either SCC, adenocarcinoma or LCC admixed with a population
of malignant spindle or giant cells, the latter occupying at least 10% of the total tumour
volume.
Spindle cell carcinoma is composed wholly of malignant spindle cells, and likewise
giant cell carcinoma consists solely of malignant giant cells. Carcinosarcoma is a biphasic
tumour composed of both epithelial and sarcomatoid elements; the carcinoma component
is generally SCC. The sarcomatoid component may display chondroid, rhabdoid or
osteoid differentiation. Pulmonary blastoma is a primitive tumour composed of epithelial
and mesenchymal foci. The epithelial component may resemble fetal adenocarcinoma;
squamous morulae may be included. The stromal cell component is undifferentiated and
resembles blastema. The epithelial elements usually express cytokeratins and the blastema
cells express vimentin and smooth muscle actin.
Multiple tumours
Multiple tumours can present challenges. All tumours must be identified macroscopically,
measured accurately and their relationship to one another established. The tumours
need to be compared microscopically to determine whether they have similar histological
appearances. If similar they are likely to represent intrapulmonary metastases, if not
synchronous primaries are favoured (Table 3.1) [8,9].
Pleural invasion
TNM 7th edition made recommendations for evaluation of visceral pleural invasion. If
there is no suggestion of pleura involvement, the tumour is classified as PL0. If the tumour
invades beyond the outer elastic layer, it is classified as PL1. If tumour extends to the
pleural surface, it is PL2. The tumour is classified as PL3 when there is parietal pleural
involvement. The use of elastic stains is advocated. PL1 or PL2 upstages an otherwise T1
tumour to T2 and PL3 upstages a tumour to T3 (Figure 3.4) [10].
Table 3.1 Comparison of TNM 6th edition classification with the changes in TNM 7th edition
Description
2cm
T1
T1a
>23cm
T1
T1b
5cm
T2
T2a
>57cm
T2
T2b
>7cm
T2
T3
T3
T3
T4
T3
T4
T4
M1
T4
T4
M1a
M1
M1a
Distant metastases
M1
M1b
37
38
Figure 3.4Demonstration
of visceral pleural invasion. (a)
The pleura appears to be intact
without invasion on haematoxylin
and eosin staining; the elastic
stain (EVG) demonstrates invasion
of tumour beyond the outer
elastic layer (PL1). (b) Tumour
extending to the visceral pleural
surface (PL2).
Problems in diagnosis
Adenocarcinoma
Interobserver reproducibility in subtypes
Several recent studies have investigated interobserver variability amongst experts in
diagnosing the newly described INA subtypes. Whilst these have reported high level of
agreement (approaching 90%) in identifying a single pattern as the predominant pattern
[11,12], similar agreement is not achievable in identifying all patterns and even less so
in determining the percentage proportion of each subtype (authors experience). The
prognostic significance of the latter is debatable but may be useful in distinguishing
between a metachronous/synchronous primary and a metastasis.
Assessment of invasion
Distinguishing between AIS and LPA is usually straightforward but separating AIS and MIA
& MIA and LPA may be quite challenging even after the entire lesion has been examined
histologically [1]. An elastin stain has been recommended to differentiate alveolar collapse
from alveolar destruction by invasion, but this is often difficult to interpret.
Problems in diagnosis
Differentiating between a metastatic SCC in the lung from a primary tumour can be
difficult. Immunohistochemistry is not particularly helpful in this matter, although a
metastasis from a head and neck tumour may express p16 and/or HPV if HPV related.
Otherwise, architectural features such as vascular invasion, a well-circumscribed lesion
and the lack of an in situ component favour a metastasis over a primary lesion.
Adenosquamous carcinoma
Common pitfalls in the diagnosis of adenosquamous carcinoma are entrapped benign
bronchial glands within a SCC and squamous dysplasia in an adenocarcinoma. Diagnosis
is straight forward if squamous differentiation is associated with acinar, papillary or
lepidic growth patterns of adenocarcinoma. The diagnosis is more made difficult when the
adenocarcinoma component shows a solid growth pattern. Mucin droplets can be seen
in SCC and so in this situation more than five mucin droplets per high-power field are
required for a diagnosis [1].
The main differential diagnosis is mucoepidermoid carcinoma, in particular a highgrade mucoepidermoid carcinoma. Low-grade mucoepidermoid carcinomas are generally
easy to separate from adenosquamous carcinoma as they lack significant cytological atypia.
Also, mucoepidermoid carcinomas tend to be centrally located, whereas adenosquamous
carcinomas are generally peripheral.
39
40
Treatment of NSCLC
First-line chemotherapy
The management of metastatic NSCLC has grown in complexity in the past 5 years, guided
by the increasing influence from histological and molecular analyses. In the past, it was
considered sufficient to have obtained histological confirmation of the diagnosis of lung
cancer and having simply differentiated between NSCLC and small cell lung cancer. This
was commonly possible on bronchial washings and brushings. Small cell lung cancer is
traditionally treated with a platinum agent, cisplatin or carboplatin, in combination with
a second agent, such as etoposide. For the first-line treatment of patients with metastatic
NSCLC, the platinum agents have commonly been combined with one other agent such
as gemcitabine, paclitaxel or vinorelbine in a platinum doublet, regardless of histological
subtype.
These regimens have been shown in phase III studies to have comparable efficacy
with varying side effect profiles [1315]. Until 2008-2009, patients in the United Kingdom
were commonly treated with a platinum and gemcitabine combination for all histological
subtypes of NSCLC (Figure 3.6), with median overall survival and progression-free
survival in the ranges of 9.59.9 months and 4.65.5 months respectively [14]. With respect
to whether cisplatin or carboplatin should be the platinum-containing agent of choice,
the British Thoracic Oncology Group (BTOG) trial BTOG2 showed that carboplatin
and gemcitabine were not inferior to the higher dose of cisplatin (80mg/m2) used in
combination with gemcitabine in one of the trial arms in terms of median overall survival
[16] and therefore carboplatin/gemcitabine remained a popular treatment option for
metastatic NSCLC until 2009.
In 2008, the importance of identifying the histological subtype of NSCLC in guiding the
management was shown. Scagliotti et al. [17] identified that overall survival in patients
with NSCLC receiving cisplatin/pemextrexed was noninferior to cisplatin/gemcitabine
(median survival 10.3 vs. 10.3 months respectively). However, the study identified that
patients with adenocarcinoma subtype had a 1.7 month improved overall survival if
they received cisplatin /pemetrexed, rather than cisplatin/gemcitabine (12.6 vs. 10.9
months respectively). In the case of large-cell carcinoma histology, the improvement in
NSCLC
For palliative chemotherapy
NSCLC confirmed
First line:
carboplatin/gemcitabine
Second line:
erlotinib or docetaxel
Histopathological/
molecular analysis
Progressive disease
Treatment of NSCLC
overall survival with cisplatin/pemetrexed was even more marked compared to cisplatin/
gemcitabine (10.4 vs. 6.7 months respectively). In contrast, the reverse situation was seen
in patients with squamous cell histology, where there was significantly worse survival with
cisplatin/pemetrexed versus cisplatin/gemcitabine (9.4 vs. 10.8 months respectively).
This was the first prospective phase III study in NSCLC to show survival differences
based on histological subtype. It is thought that the reason for this difference in efficacy of
chemotherapy regimens is based on significantly increased levels of thymidylate synthetase
(TS) in squamous cell histology. Increased levels of TS have been shown in preclinical data
to correlate with reduced sensitivity to pemetrexed. The work from Scagliotti et al. shifted
the need from simply diagnosing NSCLC, to the need to identify the histological subtype,
in order for the optimal chemotherapy regimen to be recommended for the patient.
The National Institute for Health and Clinical Excellence (NICE) in the United Kingdom
approved the use of pemetrexed in combination with cisplatin for the first-line treatment of
patients with locally advanced or metastatic NSCLC with non-squamous histology [18].
41
42
study was performed in a population known to have high rates of EGFR gene mutations
(Asian patients, non-smokers or former light smokers), but the subsequent EURTAC study
demonstrated similar results in the European population using a different EGFR TKI,
erlotinib (Tarceva), where median progression-free survival was 9.7 months with erlotinib
versus 5.2 months with chemotherapy in EGFR mutation positive patients [23].
In addition to IPASS and EURTAC, other studies have confirmed the additional benefit of
using first-line EGFR TKIs in patients with EGFR mutations [24]. It has also been shown that
this treatment is commonly better tolerated than cytotoxic chemotherapy and therefore
knowing the EGFR mutation status of a patient with metastatic NSCLC is of key importance
in guiding the appropriate treatment, in addition to knowing the histological subtype of
NSCLC. Rates of EGFR mutations are higher in nonsquamous subtypes (approximately
10% in adenocarcinomas [25] versus 3.4% in squamous cell subtypes [26]), and therefore
it is common tumours of the nonsquamous subtype that are sent for EGFR mutation
analysis. The importance of testing appropriate samples for EGFR mutation analysis has
implications for the need of larger sample sizes during diagnostic procedures.
Treatment of NSCLC
Description
Gefitinib
EGFR inhibitor
Used as first-line treatment in EGFR mutation positive patients
Erlotinib
EGFR inhibitor
Used as first-line treatment in EGFR mutation positive patients
Used as second-line treatment after progression with chemotherapy
Crizotinib
Afatinib
NSCLC, non-small cell lung cancer; EGFR, epidermal growth factor receptor; ALK, anaplastic lymphoma kinase; c-Met,
mesenchymal-epithelial transition factor; HGFR, hepatocyte growth factor receptor; EMLA4, echinoderm microtubuleassociated protein-like 4; Her2, human epidermal growth receptor 2; Her4, human epidermal growth factor receptor 4.
NSCLC
For systemic therapy
EGFR mutation
positive
First line:
gefitinib
or
erlotinib
Histopathological/
molecular analysis
Progressive disease
ALK
positive
First line:
crizotinib
Non-squamous
histology
First line:
platinum/
pemetrexed
Squamous
histology
First line:
platinum/gemcitabine
or
vinorelbine
Second line:
docetaxel
or
erlotinib
Figure 3.7 Typical management of metastatic non-small cell lung cancer (NSCLC) in 2013. There is increasing
influence of pathological and molecular analyses on the management. This is a typical situation and is by no means
extensive. NSCLC, non-small cell lung cancer; EGFR, epidermal growth factor receptor; ALK, anaplastic lymphoma
kinase.
43
44
References
1. Travis WD, Brambilla E, Muller-Hermelink HK, et al., eds. Pathology and genetics. Tumours of the lung,
pleura, thymus and heart. World Health Organisation Classification of Tumour. Lyon: IARC Press, 2004.
2. Ferlay J, Shin HR, Bray F, et al. GLOBOCAN 2008 v1.2, Cancer Incidence and Mortality Worldwide: IARC
CancerBase No. 10 [Internet]. Lyon, France: International Agency for Research on Cancer, 2010. http://
globocan.iarc.fr. (Last accessed April 2013.)
3. Travis WD, Brambilla E, Noguchi M, et al. International Association for the Study of Lung Cancer/American
Thoracic Society/European Respiratory Society International Multidisciplinary Classification of Lung
Adenocarcinoma. J Thorac Oncol 2011; 6:244285.
4. Warth A, Muley T, Meister M, et al. The Novel Histologic International Association for the Study of
Lung Cancer/American Thoracic Society/European Respiratory Society Classification System of Lung
Adenocarcinoma is a stage-independent predictor of survival. J Clin Oncol 2012; 30:14381446.
5. Nakatani Y, Masudo K, Miyagi Y, et al. Aberrant nuclear localization and gene mutation of beta-catenin
in low-grade adenocarcinoma of fetal lung type: up-regulation of the Wnt signaling pathway may be a
common denominator for the development of tumors that form morules. Mod Pathol. 2002;15:617624.
6. Goldstraw P, Crowley J, Chansky K, et al. The IASLC Lung Cancer Staging Project: proposals for the revision
of the TNM stage groupings in the forthcoming (seventh) edition of the TNM classification of malignant
tumours. J Thorac Oncol 2007 2:706714.
7. Groome PA, Bolejack V, Crowley JJ, et al. The IASLS Lung Cancer Staging Project: validation of the proposals
for revision of the T. N. and M descriptors and consequent stage groupings in the forthcoming (seventh)
edition of the TNM classification of malignant tumours. J Thorac Oncol 2007; 2:694705.
References
8. Postmus PE, Brambilla E, Chansky K, et al. The IASLC Lung Cancer Staging Project: proposals for revision of
the M descriptors in the forthcoming (seventh) edition of the TNM classification of lung cancer. J Thorac
Oncol 2007;2:686693.
9. Travis WD. Reporting lung cancer pathology specimens. Impact of the anticipated 7th edition of TNM
classification based on recommendations of the IASLC Staging Committee. Histopathology 2009; 54:311.
10. Travis WD, Brambilla E, Rami-Porta R, et al. Visceral pleural invasion: pathologic criteria and use of elastic
stains: proposal for the 7th edition of the TNM classification for lung cancer. J Thorac Oncol 2008; 3:1384
1390.
11. Thunnissen E, Beasley MB, Borczuk AC, et al. Reproducibility of histopathological subtypes and invasion in
pulmonary adenocarcinoma. An international interobserver study. Mod Pathol 2012; 25:15741583.
12. Warth A, Stenzinger A, von Brunneck A-C, et al. Interobserver variability in the application of the novel
iaslc/ats/ers classification. Eur Respir J 2012; 40:12211227.
13. Kelly K, Crowley J, Bunn PA, Jr., et al. Randomized phase III trial of paclitaxel plus carboplatin versus
vinorelbine plus cisplatin in the treatment of patients with advanced non-small-cell lung cancer: a
Southwest Oncology Group trial. J Clin Oncol 2001; 19:32103218.
14. Scagliotti GV, De Marinis F, Rinaldi M, et al. Phase III randomized trial comparing three platinum-based
doublets in advanced non-small-cell lung cancer. J Clin Oncol 2002; 42854291.
15. Schiller JH, Harrington D, Belani CP, et al. Comparison of four chemotherapy regimens for advanced nonsmall-cell lung cancer. N Engl J Med 2002; 346:9298.
16. Ferry D, Billingham LJ, Jarrett HW, et al. S85British Thoracic Oncology Group Trial, BTOG2: randomised
phase III clinical trial of gemcitabine combined with cisplatin 50 mg/m2 (GC50) vs cisplatin 80 mg/m2
(GC80) vs carboplatin AUC 6 (GCb6) in advanced NSCLC. Thorax 2011; 66:A41.
17. Scagliotti GV, Parikh P, von Pawel J, et al. Phase III study comparing cisplatin plus gemcitabine with cisplatin
plus pemetrexed in chemotherapy-naive patients with advanced-stage non-small-cell lung cancer. J Clin
Oncol 2008; 26:35433551.
18. National Institute for Health and Care Excellence (NICE). Lung cancer (non-small-cell, first line treatment)
pemetrexed (TA181). London: NICE, 2009.
19. Lynch TJ, Bell DW, Sordella R, et al. Activating mutations in the epidermal growth factor receptor
underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 2004; 350:21292139.
20. Shepherd FA, Rodrigues Pereira J, Ciuleanu T, et al. Erlotinib in previously treated non-small-cell lung
cancer. N Engl J Med 2005; 353:123132.
21. National Institute for Health and Care Excellence (NICE). Lung cancer (non-small-cell) - erlotinib (TA162).
London: NICE, 2008.
22. Mok TS, Wu YL, Thongprasert S, et al. Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma. N
Engl J Med 2009; 361:947957.
23. Rosell R, Carcereny E, Gervais R, et al. Erlotinib versus standard chemotherapy as first-line treatment
for European patients with advanced EGFR mutation-positive non-small-cell lung cancer (EURTAC): a
multicentre, open-label, randomised phase 3 trial. Lancet Oncol 2012; 13:239246.
24. Zhou C, Wu YL, Chen G, et al. Erlotinib versus chemotherapy as first-line treatment for patients with
advanced EGFR mutation-positive non-small-cell lung cancer (OPTIMAL, CTONG-0802): a multicentre,
open-label, randomised, phase 3 study. Lancet Oncol 2011; 12:735742.
25. Marchetti A, Martella C, Felicioni L, et al. EGFR mutations in non-small-cell lung cancer: analysis of a large
series of cases and development of a rapid and sensitive method for diagnostic screening with potential
implications on pharmacologic treatment. J Clin Oncol 2005; 23:857865.
26. Miyamae Y, Shimizu K, Hirato J, et al. Significance of epidermal growth factor receptor gene mutations in
squamous cell lung carcinoma. Oncol Rep. 2011; 25:921928.
27. Soda M, Choi YL, Enomoto M, et al. Identification of the transforming EML4-ALK fusion gene in non-smallcell lung cancer. Nature 2007; 448:561566.
28. Kwak EL, Bang YJ, Camidge DR, et al. Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer.
N Engl J Med 2010; 363:169316703.
29. Solomon B, Varella-Garcia M, Camidge DR. ALK gene rearrangements: a new therapeutic target in a
molecularly defined subset of non-small cell lung cancer. J Thorac Oncol. 2009; 4:14501454.
30. Shaw AT, Yeap BY, Solomon BJ, et al. Effect of crizotinib on overall survival in patients with advanced nonsmall-cell lung cancer harbouring ALK gene rearrangement: a retrospective analysis. Lancet Oncol 2011;
12:10041012.
45
Chapter 4
Pathology of obesity
Eve P Fryer, Ian SD Roberts, Clare Verrill
Introduction
The prevalence of obesity worldwide is increasing at an alarming rate. In the USA, 35.7%
of adults are obese [1] and obesity accounts for approximately 280,000325,000 deaths
annually [2]. Degrees of obesity are also rising; for example in the USA, numbers of
people in body mass index (BMI) categories >40 and >50 have increased by 50% and 75%
respectively between 2000 and 2005 [3]. Pathologists will therefore inevitably encounter
the effects of obesity in their surgical diagnostic work or in their post-mortem practice and
increasingly, pathologists will be asked to perform autopsies in those who have undergone
bariatric surgery.
Post-mortems in the obese are challenging and it is not just the logistical and
physical challenges of dealing with larger body sizes which are important, but also
there are a number of fatal conditions which are effectively exclusive to obesity such as
obstructive sleep apnoea (OSA), obesity hypoventilation syndrome (OHS) and obesity
cardiomyopathy. These conditions are likely being under-recognised by pathologists, who
attribute death to other conditions such as nonsignificant coronary artery atherosclerosis.
In addition, there are a number of conditions for which obesity is a significant, but lesser
known independent risk factor such as venous thromboembolic disease. It is important
that the role obesity plays in these deaths is acknowledged by inclusion in the medical
certificate of cause of death. This, however, is difficult to implement when, e.g., body
height and weight are not being recorded in approximately half of coronial autopsy reports
in the United Kingdom [4].
The concept that adipose tissue is not an inert substance, but an organ which has
multisystemic effects when present in increased amounts in obesity, is gaining acceptance.
As a consequence, obese individuals are therefore not just larger than normal, but
have altered physiology. Adipose tissue has an endocrine role, secreting adipokines
and promoting inflammation by secretion of cytokines, which are implicated in the
development of cancer, atherosclerosis and thrombosis.
Eve P Fryer, FRCPath, Consultant Pathologist, Oxford University Hospitals NHS Trust, Oxford, UK
Ian SD Roberts, FRCPath, Professor of Cellular Pathology, University of Oxford, Consultant Pathologist, Oxford
University Hospitals NHS Trust, Oxford, UK
Clare Verrill, FRCPath, Consultant Pathologist, Oxford University Hospitals NHS Trust, Oxford, UK
Email: Clare.Verrill@OUH.nhs.uk (for correspondence)
48
Pathology of obesity
Respiratory system
Obstructive sleep apnoea
OSA is a condition of recurrent episodes of apnoea (interrupted air flow) due to obstruction
of the upper airway during sleep, followed by transient awakening to restore airway patency
[7]. The condition occurs in the obese because of accumulation of fat in the neck region.
The diagnosis is suggested by a history of snoring and observed apnoeas. The diagnosis
is confirmed by polysomnography and it is usually treated by continuous positive airway
pressure (CPAP). The condition is exacerbated by smoking (which increases inflammation
of the upper airways), sedative drugs and alcohol consumption (as they worsen the
apnoeas and arterial de-saturations).
Recurrent apnoeas cause chronic alveolar hypoxia, which can result in pulmonary
artery constriction with subsequent pulmonary hypertension and cor pulmonale. Right
ventricular failure is usually accompanied by left ventricular failure due to the direct effects
of obesity or hypertension. Far from just causing poor quality sleep, OSA has numerous
effects, being an independent risk factor for congestive cardiac failure, hypertension,
myocardial infarction, cardiac arrhythmias and sudden death [7]. It is also associated with
increased rates of car and work-related accidents [8].
Table 4.1 The WHO 2000 classification of obesity and other commonly used terminology
BMI (kg/m2)
<18.5
Underweight
Underweight
18.5024.99
Normal
Normal range
25.0029.99
Overweight
Overweight
30.0034.99
Obese
Obese class I
35.0039.99
Obese class II
40.00
Morbid obesity
50.00
Superobesity
60.00
Super-superobesity
Respiratory system
These deaths are often challenging as autopsy findings may be limited to obesity only, but
death can be attributed to OSA provided a thorough approach is taken. The circumstances
of the death are crucial to the diagnosis and therefore details such as exact body position
must be actively sought from the Coroner. In a textbook case, the pathologist would be
provided with a history of sleep apnoea during life, the body would be found in bed with
the deceased on their back (supine), and they may have ingested alcohol or other sedatives
at a level which alone is insufficient to account for death. There may be right ventricular or
biventricular failure. If there is no history of sleep apnoea, it is still possible to attribute death
due to the condition, but as the definitive test (polysomnography) is performed during life,
this becomes more difficult. Features of the case such as being found deceased in bed in the
supine position and a history from relatives suggestive of sleep apnoea, such as snoring, may
support the diagnosis. A post-mortem where OSA is being considered as the cause of death
requires full histological and toxicological assessment.
The suggested criteria for issuing a cause of death at post-mortem due to OSA are [9,10]
as follows:
1. A diagnosis during life of OSA, even in the absence of respiratory failure. Without an
established diagnosis, an appropriate history, e.g. snoring
2. The circumstances of the death, in particular death always in bed, during sleep in the
supine position, often whilst not using CPAP
3. Absence of specific autopsy findings such as an acute cardiac or cerebral event
4. Evidence of intoxication with alcohol or other sedatives
49
50
Pathology of obesity
2. Sudden death in the absence of a clear alternative cause of death such as an acute
cardiac or cerebral event
3. Features of pulmonary hypertension and cor pulmonale such as right ventricular
hypertrophy and dilatation. In everyday practice, right ventricular hypertrophy can be
defined by the Fulton technique as an isolated right ventricular weight of >65g or a left:
right ventricular weight ratio of <2.33.3:1.
Heart
Obesity cardiomyopathy
Obesity directly causes alterations in the structure and function of the heart even in the
absence of other conditions associated with obesity such as hypertension. The mechanism
responsible is an increase in total blood volume and cardiac output because of the high
metabolic activity of excess adipose tissue [12]. The high cardiac output state results in
ventricular dilatation and left and right ventricular hypertrophy with systolic dysfunction.
The term obesity cardiomyopathy is used when these changes lead to congestive cardiac
failure
If systemic hypertension is also present, this further promotes left ventricular
dilatation and hypertrophy, the effect being additive to that of obesity, not synergistic [12].
Hypertensive heart disease can be differentiated from obesity cardiomyopathy by the left
ventricular hypertrophy being concentric in hypertension versus eccentric in obesity.
There may be a history during life of hypertension and there may be changes suggestive of
hypertension in other organs such as the kidney [9].
Obesity cardiomyopathy typically occurs in those who have severe and long-standing
obesity. The modes of death are either progressive congestive heart failure or sudden
arrhythmic cardiac death. Previously, fat infiltration in the myocardium was proposed as
the cause of obesity cardiomyopathy. This is now largely discredited as fat can be seen in the
myocardium of the right ventricle in normal hearts. The currently accepted model of obesity
cardiomyopathy is multifactorial, involving metabolic disturbances (insulin resistance,
Heart
increased free fatty acid levels and increased levels of adipokines), activation of the renin
angiotensinaldosterone and sympathetic nervous systems, myocardial remodelling and
small vessel disease [13].
Obesity cardiomyopathy is most likely to be confused with dilated cardiomyopathy
at post-mortem, and obviously the distinction is important as dilated cardiomyopathy
has genetic implications. Dilated cardiomyopathy shows ventricular dilatation with
an inadequate degree of left ventricular hypertrophy (left ventricle wall thickness
<10mm), whereas obesity cardiomyopathy tends to show left ventricular hypertrophy
and dilatation (wall thickness >10mm) [9]. Microscopically, in dilated cardiomyopathy
there is myocardial fibrosis that is not usually seen in obesity cardiomyopathy. Fat may be
present in the right ventricle in obesity cardiomyopathy, but not usually in the left ventricle
unless it surrounds blood vessels in the outer wall (which is considered normal) [9]. It is
recommended that a sample of heart and spleen is frozen for genetic studies in case of an
inherited cardiomyopathy.
Defining cardiomegaly in obesity is difficult. Typically in obesity cardiomyopathy the
heart weight is markedly above that predicted for body weight and typically >800900g.
However, it may be close to that predicted for body weight, but always greater than if
one accepts a fixed value of an upper limit of normal such as 400g for females and 500g
for males [14]. It is known that heart weight increases with body weight due to left and
right ventricular hypertrophy and tables have been proposed which allow calculation of
predicted heart weight from the body weight [15]. At some point, however, even though the
heart weight appears proportional to the body weight, this must be considered potentially
pathological as the increased ventricular mass predisposes to arrhythmia. In the authors
experience, there is a relatively high proportion of deaths in the obese in which there are few
findings, namely obesity, an apparent arrhythmic death and left ventricular hypertrophy.
Criteria for issuing a cause of death due to obesity cardiomyopathy [9,16] are as follows:
1. Heart weight increased over value predicted for normal body weight
2. Left ventricular or biventricular hypertrophy and dilatation of atria and ventricles. Small
foci of interstitial fibrosis may be present, but not extensive ischaemic fibrosis
3. There may be marked fat in the right ventricle usually in the epicardial surface
and extending in with blood vessels (in the absence of fibrosis which suggests
arrhythmogenic right ventricular cardiomyopathy), often up to the trabeculae. These
changes usually affect the anterior and lateral wall and less so the posterior wall
(Figure 4.2)
4. Exclusion of significant coronary artery disease, myocarditis, myocardial infarction or
other clear alternative cause of death
51
52
Pathology of obesity
Liver
Obesity causes a spectrum of changes within the liver from simple steatosis through to
nonalcoholic steatohepatitis (NASH), fibrosis, cirrhosis and hepatocellular carcinoma.
The majority of patients with morbid obesity will have steatosis (91% in a recent systematic
review). In the same review, the prevalence of NASH was 37%, but this was extremely
variable between individual studies [22]. NASH is associated with increased mortality
compared with the normal population, partly due to an increase in liver-related deaths, but
it has also recently emerged as a predictor of cardiovascular disease [23].
The histological features of NASH are steatosis, ballooning of hepatocytes, lobular
inflammation, Mallory bodies and a pericellular pattern of fibrosis. The NAFLD activity
score can be useful in determining if a given case meets the criteria for a diagnosis of
steatohepatitis. [24] Histological assessment cannot reliably distinguish between alcoholic
steatohepatitis and NASH. However, features said to favour alcohol over nonalcoholic
causes are neutrophils, prominent Mallory bodies and extensive zone 3 fibrosis. Marked
steatosis with less severe steatohepatitis with nuclear vacuolation (possibly suggesting
insulin resistance) favours NASH over alcohol [23].
The mechanism of NASH development remains uncertain, but has been proposed to
involve insulin resistance, iron accumulation, oxidative stress and hepatocyte death with an
Kidney
imbalance in anti- and proinflammatory factors [25]. The current model suggests that two
hits are required, with the first hit being peripheral insulin resistance, leading to hepatic
steatosis. These lipid-laden hepatocytes are then vulnerable to a combination of second
hits caused by cytokines, oxidative stress or genetic factors. Adipose tissue is a source of
inflammation and therefore a plausible cause of the second hit.
Kidney
A high BMI is associated with an increased risk of chronic kidney disease (CKD).
This association is partly explained by the increased frequency of hypertension and
diabetes mellitus in obesity. In addition, high BMI results in an increased renal mass,
glomerulomegaly and hyperfiltration-associated injury [26]. Histologically, this manifests
as an increase in mesangial matrix and secondary focal segmental glomerulosclerosis
(FSGS) with sclerosis and hyalinosis adjacent to the vascular pole of the glomerulus
(Figure 4.3). This pattern of glomerular injury is associated with proteinuria and
progressive interstitial fibrosis and tubular atrophy. The proteinuria may be in the
Figure 4.3 Effects of obesity on the kidney. An obese 42-year-old man (weight 140kg) presented with nephrotic
proteinuria (6g/day). Renal biopsy shows marked glomerulomegaly (a) with focal segmental sclerosis and hyalinosis
adjacent to the vascular pole (b). An obese 71-year-old man with type II diabetes mellitus developed acute renal
failure (serum creatinine rising from 200 to 500mol/L) shortly after starting on orlistat. Renal biopsy shows a
diabetic nephropathy with nodular glomerulosclerosis (c), together with intratubular calcium oxalate crystals,
indicative of an oxalate nephropathy (d).
53
54
Pathology of obesity
nephrotic range (>3.5g/24 hours) but without features of the nephrotic syndrome. At
electron microscopy, there is effacement of podocyte foot processes that is in proportion
to the severity of proteinuria and is generally less extensive than that seen in primary
FSGS [27].
In addition to the above structural changes, obesity exacerbates injury associated
with many primary renal diseases. For example, in a recent series of patients with IgA
nephropathy, a high BMI was found to be an independent predictor of progressive loss
of renal function [28]. Obesity is also associated with an increased risk of renal stone
disease [29].
Weight loss following bariatric surgery is associated with reduction in proteinuria
and stabilisation of CKD [30]. However, gastric bypass is associated with a risk of enteric
hyperoxaluria and oxalate nephropathy [31]. This complication typically presents as
acute renal failure, often on a background of CKD. Histologically, there are abundant
calcium oxalate crystals within tubules, with associated acute and chronic tubular injury
(Figure 4.3). Prognosis is generally poor, with most patients requiring renal replacement
therapy. Similarly, oxalate nephropathy may result from orlistat therapy for obesity [32].
This drug reduces digestion of dietary fats by inhibiting the production of gastric and
pancreatic lipase. Intestinal absorption of oxalate is normally limited by the formation
of insoluble calcium oxalate in the bowel lumen. Enteric hyperoxaluria develops as a
result of reduced absorption of fats and bile acids. In the presence of fat malabsorption,
free fatty acids within the colonic lumen bind calcium ions, thus increasing oxalate
absorption.
Cancer
Obesity is associated with increased risk of several cancer types: breast, endometrial,
colorectal, pancreatic, gastric, oesophageal adenocarcinoma (related to Barretts
oesophagus), cholangiocarcinoma, hepatic, melanoma and renal cancer. Insulin resistance,
hyperinsulinaemia, increased insulin-like growth factor (IGF), elevated steroid and peptide
hormones and systemic inflammation as a result of adipocytokine production, e.g. leptin
and tumour necrosis factor alpha (TNF-a), all appear to play a role in the development of
malignancy in the obese.
Infection
Production of adipocytokines by excess fat disturbs the balance between adipose tissue and
the immune system by causing dysregulated immune response, impaired chemotaxis and
altered macrophage differentiation [33].
Bariatric surgery
Obesity is an established risk factor for surgical site infection, hospital acquired
infections and skin infection. It also predisposes to urinary tract infections [33]. It is not
certain whether obesity affects the risk and outcomes of community acquired infections
such as pneumonia, bacteraemia and sepsis [33].
Bariatric surgery
The current National Institute for Clinical Excellence (NICE) guidelines on surgery in
obesity state that bariatric surgery should be offered to those with a BMI of 40kg/m2 or
over, and also to those patients with a BMI of 3540kg/m2 who also have obesity-related
complications, such as diabetes mellitus [36]. Bariatric surgery reduces overall mortality
by approximately 40% with reductions in deaths from heart disease, diabetes mellitus
and cancer, together with improvement in cardiac function and reversal of obesity
cardiomyopathy [37]. Mortality from bariatric surgery is low; a meta-analysis of 361 studies
comprising 85,048 patients showed a mortality at <30 days (early) of 0.28% and 30 days to 2
years (late) of 0.35% [38].
Increasingly, pathologists will be asked to perform autopsies following bariatric
surgery. The cause of death may be directly or indirectly related to the surgery, related to
complications of obesity or unrelated. Bariatric surgical options are all usually performed
laparoscopically and are divided into restrictive procedures (laparoscopic gastric banding
or vertical sleeve gastrectomy) or combined restrictive and malabsorption procedures
(most commonly Roux-en-Y gastric bypass) [39]. Laparoscopic gastric banding creates
a small gastric pouch, which restricts calorie intake proximal to a band (Figure 4.4a).
This is connected to a subcutaneous access port and can be adjusted by changing the
volume of saline within the band. Complications of gastric banding include nausea and
vomiting, dehydration with resultant electrolyte disturbances and/or prerenal failure. Rare
complications are gastrointestinal haemorrhage, aspiration, erosions caused by ischaemia
due to pressure on the gastric wall by the band which may result in gastric necrosis (which
requires resection), oesophageal or gastric perforation, ulcers, band slippage or abdominal
sepsis.
Insertion of a gastric balloon (to result in a feeling of fullness) can be performed
endoscopically. The balloons can deflate and rupture with migration and rarely cause
small bowel obstruction. Vertical sleeve gastrectomy involves creating a sleeve of stomach
with a lesser capacity along the greater curvature (Figure 4.4b), and tube stricture is the
main late complication specific to this procedure. Other complications are similar to the
55
Pathology of obesity
56
Aspiration
Oesophagus
Gastric pouch
Perforation
Erosion due to
ischaemia
Gastric necrosis
Band slippage
Ulceration
Band
Subcutaneous
access port
Nausea +
vomiting
Electrolyte
disturbances
Stomach
Duodenum
Oesophagus
Sleeve of stomach
Tube stricture
Stomach
Duodenum
Oesophagus
Bowel ischaemia
Perforation
Marginal ulcers
Anastomotic leak
Anastomotic stricture (late)
Haemorrhage
Cholelithiasis
Stomach
Liver
Duodenum
Nausea + vomiting
Malabsorption
Jejunum
Jejunal ulceration
Intestinal obstruction
secondary to
adhesions or internal
herniation
Small intestinal
bacterial overgrowth
Conclusion
technical complications of Roux-en-Y gastric bypass and would include anastomotic leak,
haemorrhage and perforation.
Roux-en-Y gastric bypass creates a small gastric pouch. The distal stomach and proximal
small bowel are bypassed by anastomosing the gastric pouch to the jejunum. The other limb
is then anastomosed to the small bowel (see Figure 4.4c). Immediate complications include
anastomotic leak, perforation, intestinal ischaemia, bleeding or pulmonary embolism. Late
complications include intestinal obstruction secondary to adhesions or internal herniation,
as patients with a Roux-en-Y bypass have several mesenteric defects created by the surgery
through which a small bowel loop can pass and become obstructed or strangulated,
anastomotic stricture and marginal ulceration (ulcers which occur at the gastrojejunal
anastomosis of uncertain, but probably multifactorial origin). Autopsy studies have also found
a high incidence of nonischaemic arrhythmic cardiac deaths after bariatric surgery [40].
The National Confidential Enquiry into Patient Outcome and Death (NCEPOD)
published an audit of 29 UK post bariatric surgery deaths in 2012. Two of the deaths
followed gastric balloon insertion; one death due to gastric rupture on removal of the
device, the other a cardiac arrest associated with a large heart. Of the other 27 deaths, the
commonest causes of death were: anastomotic leak and sepsis (10 deaths), pulmonary
thrombo-embolism (6 deaths), intra-abdominal haemorrhage (3 deaths) and malnutrition
from short bowel (2 deaths). Deaths which were either directly or indirectly related to the
surgery occurred at all time points, including many years after surgery [41].
Cholelithiasis (cholesterol gall stone formation) is a frequent consequence of the rapid
weight loss following bariatric surgery. Roux-en-Y surgery can be complicated by dumping
syndrome and Roux-en-Y stasis syndrome (chronic abdominal pain, nausea and vomiting
due to stasis of solids in the stomach), and nutritional deficiencies are common. Small
intestinal bacterial overgrowth (SIBO) syndrome is characterised by an increased number
(usually 105 colony forming units of bacteria per mL of proximal jejunal aspirate) and/or
abnormal type of bacteria in the small bowel. It shows increased incidence in the morbidly
obese when compared with nonobese subjects and can complicate gastric bypass surgery.
SIBO may be clinically asymptomatic or cause diarrhoea or anaemia. In more severe cases,
there are features of malabsorption and malnutrition. The gold standard for diagnosis
during life is microbiological assessment of jejunal aspirate, although the hydrogen breath
test is also commonly used.
Conclusion
Pathologists will increasingly encounter obesity-related pathology in their practice, with
many conditions specific to obesity. The pathology of obesity-related conditions is complex
and often multifactorial, requiring specific knowledge of these conditions and a careful
thorough approach to diagnosis.
57
58
Pathology of obesity
Post-mortems in the obese require specific attention, as findings in conditions such as OSA
may be limited and it is the circumstances of the death which are key to the diagnosis.
Obesity directly affects the heart, resulting in left ventricular hypertrophy that may progress
to obesity cardiomyopathy when congestive cardiac failure is present.
Pathologists will increasingly encounter autopsy cases where there has been bariatric
surgery and although mortality for these laparoscopic procedures is low, there are a number
of complications which can occur and could result in death, although death may be a
consequence of obesity itself or be entirely unrelated to the surgery.
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United States. JAMA 2012; 282:15301538
3. Sturm R. Increases in morbid obesity in the USA: 2000-2005. Public Health 2007; 121:492496.
4. National Confidential Enquiry into Patient Outcome and Death. The Coroners Autopsy: Do we deserve
better? NCEPOD: London, 2006.
5. World Health Organisation. The problem of overweight and obesity. WHO 2000 whglibdoc.who.int/trs/
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6. Byard RW. The complex spectrum of forensic issues arising from obesity. Forensic Sci Med Pathol 2012; 8:
402-13.
7. Azagra-Calero E, Espinar-Escalona E, Barrera-Mora JM, Llamas-Carreras JM, Solano-Reina E. Obstructive
sleep apnoea syndrome (OSAS). Review of the literature. Med Oral Patol Cir Bucal 2012; 17:e925-9.
8. Young T, Blustein J, Finn L, Palta M. Sleep-disordered breathing and motor vehicle accidents in a
population-based sample of employed adults. Sleep 1997; 20:608613.
9. Fryer EP, Roberts ISD, Sheppard MN, Verrill C. Postmortem examination in the morbidly obese.
Histopathology 2014; 64: 200-210.
10. Robinson GV, Stradling JR, Davies RJO, Roberts ISD. Post mortem diagnosis of fatal obstructive sleep
apnoea. Histopathology 2004; 45:12.
11. Chau EHL, Lam D, Wong J, Mokhlesi B, Chung F. Obesity hypoventilation syndrome. A review of
epidemiology, pathophysiology and perioperative considerations. Anesthesiology 2012; 117:118.
12. Alpert MA. Obesity cardiomyopathy: pathophysiology and evolution of the clinical syndrome. Am J Med
Sci 2001; 321:225236.
13. Wong C, Marwick TH. Obesity cardiomyopathy: pathogenesis and pathophysiology. Nat Clin Pract
Cardiovasc Med 2007; 4:436443.
14. Fabre A, Sheppard MN. Sudden adult death syndrome and other non-ischaemic causes of sudden cardiac
death. Heart 2006; 92:316320.
15. Gaitskell K, Perera R, Soilleux EJ. Derivation of new reference tables for human heart weights in light of
increasing body mass index. J Clin Path 2011; 64:358362.
16. Hookana E, Junttila MJ, Puurunen VP, et al. Causes of nonischaemic sudden cardiac death in the current era.
Heart Rhythm 2011; 8:15701575.
17. Poirier P, Giles TD, Bray GA, et al. Obesity and cardiovascular disease: pathophysiology, evaluation and
effect of weight loss. Circulation 2006; 113:898918.
18. Templeton AH, Carter KLT, Sheron N, Gallagher PJ, Verrill C. Sudden unexpected death in alcohol misuse
an unrecognised public health issue? Int J Environ Res Public Health 2009; 6:30703081.
19. De Koning L, Merchant AT, Pogue J, Anand SS. Waist circumference and waist to hip ratio as predictors of
cardiovascular events: meta-regression analysis of prospective studies. Eur Heart J 2007; 28:850856.
20. Behn A, Ur E. The obesity epidemic and its cardiovascular consequences. Curr Opin Cardiol 2006;
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Future Cardiol 2010; 6:16.
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23. Levene AP, Goldin RD. The epidemiology, pathogenesis and histopathology of fatty liver disease.
Histopathology 2012; 61:141152.
24. Kleiner DE, Brunt EM, Van Natta M, et al. Design and validation of a histological scoring system for
nonalcoholic fatty liver disease. Hepatology 2005; 41:13131321.
25. Tran A, Gual P. Non-alcoholic steatohepatitis in morbidly obese patients. Clin Res Hepatol Gastroenterol
2013; 37:1729.
26. Amann K, Benz K. Structural renal changes in obesity and diabetes. Semin Nephrol 2013; 33:2333.
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epidemic. Kidney Int 2001; 59:14981509.
28. Kataoka H, Ohara M, Shibui K, et al. Overweight and obesity accelerate the progression of IgA
nephropathy: prognostic utility of a combination of BMI and histopathological parameters. Clin Exp
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29. Ahmed MH, Ahmed HT, Khalil AA. Renal stone disease and obesity: what is important for urologists and
nephrologists? Renal Failure 2012; 34:13481354.
30. Alexander JW, Goodman HR, Hawver LR, Cardi MA. Improvement and stabilization of chronic kidney
disease after gastric bypass. Surg Obes Relat Dis 2009; 5:237241.
31. Nasr SH, DAgati VD, Said SM, et al. Oxalate nephropathy complicating roux-en-y gastric bypass: an
underrecognized cause of irreversible renal failure. Clin J Am Soc Nephrol 2008; 3:16761683.
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39. Neff KJH, le Roux CW. Bariatric surgery: a best practice article. J Clin Pathol 2013; 66:9098.
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(accessed 04/02/14)
59
Chapter 5
Stratified medicine for cancer:
the role of the histopathologist
Emily Shaw, Margaret Ashton-Key, Peter WM Johnson
Introduction
Cellular pathologists have been supporting and refining the process of accurate disease
classification for decades, with methods such as immunohistochemistry and in situ
hybridisation adopted in order to supplement morphological assessment of tumours as
part of the diagnostic process. Stratified cancer medicine involves predictive analysis: the
characterisation of tumours according to the presence or absence of specific molecular
abnormalities that are associated with differential treatment responses, in order to offer
appropriately targeted therapy and avoid unnecessary treatment. This is being applied to
an increasing number of solid tumour types to complement the traditional morphological
organ or tissue of origin-based assessment of tumours. Thanks to recent advances in
genomic technology that have opened up new possibilities for molecular taxonomy in
cancer, this is a rapidly evolving area which is having a direct impact on histopathology
practice. This represents an important part of the wider concept of delivering more
personalised or precision medicine across many different disease areas in the current
postgenomic era, since the elucidation and publication of the first human genome
over a decade ago. This review aims to summarise advances in this area with a focus on
solid tumours in adult patients. A detailed review of the clinical trials in this area and
applications in neuropathology, haematolymphoid or paediatric malignancies are beyond
the scope of this article.
Many cancer genomes have now been sequenced and published, including as part
of the International Cancer Genome Consortium (http://icgc.org). Cancer genome
sequencing projects have generated findings of clinical and therapeutic relevance,
e.g. the identification of the mutated BRAF oncogene as a key driver in just over half of
Emily Shaw, BM(Hons), MRCP(UK), Specialty Registrar in Histopathology, Salisbury Hospital NHS Foundation Trust
and Clinical Lead for the Stratified Medicine Programme, Cancer Research UK, London, UK
Margaret Ashton-Key, DM, FRCPath, Consultant Histopathologist and Honorary Senior Lecturer, University Hospital
Southampton NHS Foundation Trust, Southampton, UK
Peter WM Johnson, MD, FRCP, Chief Clinician for Cancer Research UK, London and Professor of Medical Oncology
at Southampton University Faculty of Medicine and University Hospital Southampton NHS Foundation Trust ,
Southampton, UK
Email: ecshaw@doctors.org.uk (for correspondence)
62
Table 5.1 Terms used to classify and describe the effects of gene mutations in cancer
Timing
Functional effect
Prediction of treatment
response
Importance in
carcinogenesis
Mutation nomenclature
Mutations are conventionally described using nomenclature agreed by the HGVS (Human Genome Variation
Societys) with reference to both the coding and protein changes. The following example is for the most common
mutation in the BRAF gene, a point mutation due to a single nucleotide substitution
Coding (c.): This is the description of the abnormality at DNA nucleotide level, according to the numbered
nucleotide position on the sense DNA strand (5 to 3 direction) of the reference genome:
For example c.1799T>A refers to substitution of adenine for thymine at position 1799
Protein (p.): This describes the abnormality at protein coding, amino acid level, according to the number of the
affected amino acid. The reference amino acid is denoted by its one or three letter code at the start of the sequence
and the mutant amino acid follows the position number at the end of the sequence:
For example p.V600E or Val600Glu refers to coding for glutamate rather than valine at codon 600
Introduction
malignant melanomas. The timescale from this discovery to the licensing of the BRAF
inhibitor vemurafenib was encouragingly short in drug development terms. Apart from
the immunomodulator therapy ipilimumab, this represents the first effective treatment
option for patients with advanced melanoma. One of the challenges in making sense
of the deluge of data from genome sequencing studies is in distinguishing key driver
mutations from nonpathogenic bystander or passenger mutations, particularly since
cancer is characterised by genomic instability, with each cancer containing anything
from 1000 to 100,000 different point mutations [1], some of which are private to that
tumour, making assessment of their role in pathogenesis difficult. There is an increasing
requirement for the cellular pathologist to be conversant in the language describing
the effects of genetic abnormalities identified through cancer genome screening, and
some of the terminology is summarised in Table5.1. Recent research into cancer
genomic evolution gives insights into the degree of spatial and temporal heterogeneity
and complexity [2]; challenges to the delivery of personalised cancer medicine through
genomics.
Histopathologists are accustomed to the use of molecular markers for diagnostic
purposes, e.g. characteristic chromosomal translocations in soft tissue tumours and
haematolymphoid malignancies. An exemplar for the application of newer predictive
molecular markers was the introduction of HER2 testing in breast cancer to identify
patients who may benefit from treatment with trastuzumab (Herceptin, Roche). Table 5.2
summarises the currently licensed therapeutic agents for use in patients with tumours
bearing specific genetic aberrations.
Table 5.2 Cancer medicines active against specific genetic abnormalities and currently
approved for use worldwide.
Drugs
Disease indication
t(15;17) translocation
Imatinib
KIT/PDGFRA mutation
Trastuzumab
Trastuzumab emtansine
(antibody-drug conjugate)
Pertuzumab
Breast cancer
Metastatic gastric cancer
(trastuzumab only)
Cetuximab
Panitumumab
Gefitinib
Erlotinib
Crizotinib
EGFR mutation
Vemurafenib
63
64
Historical background
In the past, pharmaceutical companies have usually provided initial funding following the
introduction of a new predictive marker, often through a limited number of laboratories.
When this funding has ended, new service and funding agreements have had to be found.
The United Kingdom National External Quality Assurance Scheme (UK NEQAS) for
Molecular Genetics and Immunocytochemistry and in situ hybridisation have been quick
to establish quality assurance schemes for new markers and currently there are established
disease-based schemes encompassing analysis of HER2, EGFR, KRAS, BRAF and KIT.
Testing has either been delivered by histopathology or molecular genetics laboratories, and
increasingly by unified departments of molecular pathology.
Brief description
of method
Comparative
DNA input
required
Comparative
sensitivity
Limit of
detection*
Main
advantages
Main dis
advantages
Amplification
and sequencing
of polymerase
chain reaction
(PCR) products
by selective
incorporation of
chain-terminating
dideoxynucleotides
during in vitro
DNA replication
Low
(~100ng)
Lowest
1020%
Identification
of known and
novel variants
Labourintensive;
may miss lowlevel variants
Pyrosequencing
Sequencing
by synthesis:
detection of the
luminescence
released when a
pyrophosphatelabelled nucleotide
molecule is
incorporated
during DNA
synthesis
Low
(~100ng)
High
5%
Can also
be used for
targeted
mutation
detection
Fast method
with real-time
readout
Comparatively
high
sequencing
error rate
Nextgeneration
sequencing
Massively parallel
sequencing of
thousands-millions
of amplified
DNA molecules
using capture- or
amplicon-based
approaches
High
(~500ng)
High
(dependent
on read
depth)
10%
Highest
throughput
technology,
enabling
greater scope
of analysis
up to whole
exome/
genome
Larger input
quantities
of DNA
required and
complex data
interpretation
requirements
Screening methods using comparison of mutated with normal DNA (detection of all variants, known and
unknown)
High-resolution
DNA melting
analysis (HRM)
Screening of
samples using
comparison of the
melting curves
of PCR products
against known
normal samples
Low
(~100ng)
Low
5%
Quick,
melting
products
can be
sequenced to
identify exact
abnormality
Nonspecific
amplification
of products
can lead to
missed calls
Single-strand
conformational
polymorphism
analysis (SSCP
or SSCA)
Heat-denatured
PCR products are
compared with
known samples
using capillary
electrophoresis
and analysed
according to
electrophoretic
mobility
Low
(~100ng)
High
110%,
varies by
mutation
Established
technique,
low-cost
Technical
parameters
(e.g.
temperature,
gel
composition)
must be
strictly
controlled
65
66
Selective
amplification
of sequences
containing a
defined mutation
over those that
do not
High
(~500ng)
Highest
<18%,
varies by
mutation
Fast and
sensitive
technique
Only detects
predefined
hotspot
mutations
Fragment
length analysis
DNA fragment
length analysed
against size
standards to
detect deletions
and insertions
Low
(~100ng)
High
12%
Fast and
sensitive
technique
Cannot be
used to
detect point
mutations
those samples that are not normal, for further work to characterise the precise abnormality
present if required. Determination of the sequence can be achieved by conventional
Sanger (dideoxy-) sequencing, which is considered to be more labour intensive and have
a lower sensitivity than more modern next-generation sequencing technologies. The limit
of detection for direct sequencing is generally considered to be 20%, i.e. a mutation must
be present in 20% of the DNA within a sample to be confident of picking it up by direct
sequencing analysis. It is currently uncertain what effect if any mutations present at a
low frequency within a tumour have on overall biological behaviour, and therefore whether
there is a threshold of significance. Pyrosequencing is a similar but slightly more sensitive
technique for sequence determination and mutation detection, with an estimated limit of
detection of 5% [6].
Methods for targeted analysis include amplification refractory mutation system (ARMS),
which is a technique in combination with real-time quantitative PCR to selectively amplify
those sequences containing a defined mutation over those that do not, i.e. are wild-type as
well as fragment length analysis. Fragment length analysis can be used to detect insertions
or deletions but not point mutations.
The analytical sensitivity of the mutation detection methods in use currently is between
75% and 90% for sequencing and HRM analysis and >90% for pyrosequencing, SSCP,
fragment length analysis, next-generation sequencing and allele-specific PCR [7]. The
choice of technique involves a trade-off balancing analytical sensitivity (ability to correctly
detect mutation-positive cases) and limit of detection (minimum detectable percentage of
mutant vs. wild-type alleles in a sample) with the specificity of the method.
Following analytical and clinical interpretation, the output of the molecular analysis
is formulated into a report for the requesting clinician or pathologist. The International
Organization for Standardization (ISO), the College of American Pathologists and UK
NEQAS have all issued guidance on the contents of this report (summarised in Table5.4)
[8]. In some centres, the report is sent to the referring clinician only and filed in the
patient record, and in others the report is received by the histopathologist and issued as
a supplementary report or integrated molecular pathology report. Since the mutational
Patient information
Three-point identifiers, e.g. patient name, date of birth and reference number
Nature of sample, tissue and tumour type, percentage content tumour nuclei
as assessed by referring pathologist, clinical indication for analysis, name and
address of referrer
Results
Contact information
profile is an attribute of the tumour rather than the patient, the latter approach seems more
logical and may allow the molecular results to be further interpreted in the context of the
morphological and immunohistochemical features of the tumour.
67
68
Breast cancer
Ascertaining the HER2 status of invasive breast cancer has now been the standard of care
for over a decade, and this is achieved using immunohistochemical assessment of protein
expression in the majority of cases. In situ hybridisation can be used to confirm gene
amplification and is generally reserved for cases with equivocal immunohistochemistry
results. In the past few years, there has been interest in using gene expression profiling
in breast cancer to provide risk stratification in addition to traditional histopathological
parameters such as grade, vascular invasion and lymph node involvement. Data from
gene expression profiling tools such as Oncotype DX (21 genes, Genomic Health) and
MammaPrint (70 genes, Agendia) can be used to identify a subset of patients with such
a good prognosis that they can be spared adjuvant chemotherapy since adverse effects
would be likely to outweigh the potential benefits. A recent NICE appraisal of gene
expression arrays in breast cancer approved Oncotype DX as cost-effective for use in the
in certain situations, but recommended further research to establish the utility of the IHC4
panel (immunohistochemistry for oestrogen and progesterone receptors, HER2 and the
proliferation marker Ki67) [21]. In terms of molecular taxonomy, a landmark study using
expression arrays led to classification of breast cancer into five molecular subtypes [22].
These were further expanded into ten subtypes in the METABRIC study published last year
[23], and although this work has led to mechanistic insights and the discovery of new driver
mutations in breast cancer, translation to the clinic is still some way off.
Melanoma
The BRAF gene encodes a serine/threonine kinase, an enzyme activated by
phosphorylation and responsible for transferring phosphate groups to other proteins to
modulate their function that is part of the Raf kinase family. Over 90% of BRAF mutations
are in codon 600, the commonest being V600E. The BRIM3 trial recently provided evidence
that patients with previously untreated, unresectable stage IIIC/IV melanoma with
V600E mutation had improved overall and progression-free survival with vemurafenib
when compared with standard dacarbazine therapy [24]. An unexpected finding was the
increased risk of cutaneous squamous cell carcinoma in patients receiving vemurafenib
therapy, and a possible mechanism for this is paradoxical stimulation of events in a related
cellular pathway in epidermal cells with wild-type BRAF. Ongoing studies are focusing on
improving the durability of response to BRAF inhibitors by trialling them in combination
with other targeted agents acting on related pathways. The MEK pathway also shows
overactivity in melanomas harbouring BRAF mutations and phase III clinical trials of MEK
inhibitors are currently underway [25].
69
70
Conclusion
The characterisation of tumour genomes to identify oncogenic drivers and therapeutic
targets is beginning to have a significant impact on the management of people with cancer,
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lung cancer who harbour EML4-ALK. J Clin Oncol 2009; 27:4247.
17. Popat S, Vieira de Arajo A, Min T, et al. Lung adenocarcinoma with concurrent exon 19 EGFR mutation
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18. Kwak EL, Bang Y-J, Camidge DR, et al. Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer.
N Engl J Med 2010; 363:16931703.
19. Katayama R, Shaw AT, Khan TM, et al. Mechanisms of acquired crizotinib resistance in ALK rearranged lung
cancers. Sci Transl Med 2012; 4:120ra17. doi:10.1126/scitranslmed.3003316
20. Mosse YP, Balis FM, Lim MS, et al. Efficacy of crizotinib in children with relapsed/refractory ALK driven
tumours including anaplastic large cell lymphoma and neuroblastoma. A Childrens Oncology Group phase
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cancer. N Eng J Med 2013; 368:11991209.
Chapter 6
Mucosal pathology of the gastric
cardia and Barretts oesophagus
James J Going
IntroductIon
Barretts columnar-lined oesophagus has a long history [13], but only since Norman
Barrett, Philip Allison and others drew attention to it in the mid-twentieth century [46] did
it attract increasing notice, further stimulated by the emergence of a strong association with
oesophageal adenocarcinoma [7].
Adenocarcinomas arising from mucosa of Barretts oesophagus and the gastric cardia
(junctional cancers) present late, and are often inoperable. As surgery, chemotherapy and
radiotherapy are only moderately effective, prognosis is poor and lethality high. This state
of affairs is a challenge to do better.
As the dietary austerities of the Second World War and after receded into history, the
incidence of oesophageal and junctional adenocarcinoma, previously rare, began to
increase in Western societies [8]. This increase has been absolute, with increasing obesity
(identified by epidemiological studies as an important risk factor; [9]) leading to reflux
of acid and bile into the distal oesophagus, injuring the native oesophageal mucosa, and
in some people stimulating glandular (columnar) metaplasia. Barretts oesophagus
is however only identified in a minority of people with reflux, and conversely may be
present in people who have never experienced troublesome reflux symptoms. Continuing
reflux with ongoing mucosal injury and inflammation are likely to further promote
adenocarcinogenesis.
The increase in oesophageal adenocarcinoma and junctional gastric cancer has also
been inversely related to distal gastric cancer associated with Helicobacter pylori infection,
as improved social conditions reduced the number of people infected with H. pylori during
childhood.
The response of the medical profession to the challenge of oesophageal and junctional
adenocarcinoma has focused on endoscopic and biopsy surveillance in the small minority
of people known to have Barretts columnar-lined oesophagus, the only definitely known
precursor of oesophageal adenocarcinoma apart from obesity and reflux.
James J Going, BSc, PhD, MRCP, FRCPath Senior Lecturer in Pathology and Honorary Consultant Pathologist,
University of Glasgow and Southern General Hospital, and Department of Pathology, Southern General Hospital,
Glasgow, UK
Email: going@udcf.gla.ac.uk (for correspondence).
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This seems to present a tantalising opportunity for early detection and prevention of
these unpleasant cancers, but unfortunately, as most of them occur in people not known
to have had Barretts oesophagus, this can never have much impact on mortality from
these diseases. Conversely, not only will most people with Barretts oesophagus not die of
oesophageal adenocarcinoma, most will never even know they had it. At least as many will
die of colon cancer.
There are many other challenges. Even the definition of Barretts oesophagus is
contested, and the level of cancer risk associated with it, the diagnosis of dysplasia can be
problematical, and the role of biomarkers in risk stratification remains unclear. Much of the
available evidence is of poor quality [10].
Some junctional cancers arise in a biological context similar to Barretts oesophagus,
in patients with normal gastric mucosa, whilst others develop on a background of chronic
gastritis, intestinal metaplasia and glandular atrophy [11] resembling in these respects
distal gastric cancer of intestinal type. This review is chiefly concerned with Barretts
oesophagus and junctional cancers arising on a comparable background. The Siewert
classification is not easily related to biological context and will not be considered further.
This review will examine: (1) Mucosal phenotypes of Barretts columnar-lined
oesophagus and the gastric cardia. (2) Diagnosis of Barretts oesophagus. (3) Is there a
case for population screening for Barretts oesophagus? (4) How dangerous is Barretts
oesophagus? (5) The case for postascertainment endoscopic and biopsy surveillance of
Barretts oesophagus. (6) The challenge of dysplasia diagnosis in Barretts oesophagus. (7)
Advanced imaging and detection of Barretts dysplasia. (8) The potential of biomarkers to
improve risk stratification in Barretts oesophagus. (9) Endoscopic treatment of the highrisk oesophagus and cardia.
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a well-developed brush border are almost never seen, and Paneth cells are seldom
conspicuous.
Postascertainment surveillance
next biopsy. Also, there is almost no spatiotemporal data relating intestinal metaplasia
in Barretts oesophagus with dysplasia and its progression. The relationship between
intestinal metaplasia and dysplasia has been questioned [15], and the occurrence of
genetic and epigenetic abnormalities similar to those in cancers in nonintestinal glandular
mucosa also calls in question the idea that intestinal type mucosa only is at increased risk
of malignancy [16].
Accurate assessment of patient-specific cancer risk in Barretts oesophagus will require
better knowledge of controls of mucosal phenotype (cell fate specification) and the
evolution of dysplastic clones than is presently available. This is unlikely to be reducible to
whether intestinal metaplasia is present or absent, so requiring intestinal metaplasia as a
necessary precondition for Barretts oesophagus is too rigid to justify on present evidence.
It may also hinder understanding of carcinogenesis in metaplastic oesophageal mucosa
and at the oesophagogastric junction, and its use to define Barretts columnar-lined
oesophagus is illogical.
Published studies of adenocarcinoma risk in Barretts oesophagus yield a wide variety of
estimates. Provenzale et al. [17] demonstrated publication bias and proposed an incidence
of symptomatic adenocarcinoma in Barretts oesophagus about 0.5% per annum, a figure
widely accepted since then, but even this lower figure has been challenged more recently
by several allegedly population-based studies.
Reported incidence of adenocarcinoma in Barretts oesophagus is between 0% and 3.5%
per annum [18,19]. Risk estimates in some studies were probably overestimated (small
size, short follow-up, variable definitions, inclusion of prevalent cancers and high-grade
dysplasia, HGD) [20]. Current surveillance guidelines are based on an adenocarcinoma
risk 0.5% per annum estimated from a meta-analysis of available data to the year 2000 [21].
A more recent meta-analysis by Desai et al. [22] excluding prevalent cancers and HGD
reported an incidence of adenocarcinoma of 0.33% per annum. These figures are important
because estimates of early adenocarcinomas (EAC) incidence influence the degree to which
endoscopic and biopsy surveillance are likely to be beneficial in Barretts oesophagus.
Bhat et al. [23] reported cancer risk estimates for an unselected cohort of Barretts
oesophagus (not all with intestinal metaplasia, ascertained from pathology reports) in
Northern Ireland, with mean follow-up of 7 years. After excluding baseline HGD and EAC,
combined EAC, HGD and cancer of the gastric cardia risk estimate was 0.13% per annum
(CI 0.100.16). When the analysis was restricted to cases with intestinal metaplasia and
visible Barretts mucosa, the incidence of EAC was 0.18% per annum and the combined
incidence of HGD, EAC and gastric cardia cancer was 0.33% per annum, identical to the
estimate of Desai et al. [22].
The risk of adenocarcinoma amongst Barretts oesophagus patients in a populationbased study from Denmark [24] was the same at 0.12% per annum. The relative risk of
adenocarcinoma amongst Barretts oesophagus patients compared with the general
population was 11.3%. These findings have been said to call into question current screening
and surveillance regimens. However, de Jongh et al. [25] found a progression rate more
typical of previous estimates at about 0.4% per annum in a Dutch population study.
postascertaInMent surveIllance
For patients known to have Barretts oesophagus, current guidelines continue to recommend
surveillance endoscopy and biopsy every 35 years for patients without dysplasia; every
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612 months for low-grade dysplasia (LGD) and quarterly for HGD [26,27] in the plausible,
but unproven expectation that HGD and low-stage adenocarcinoma (pT1a/b) will be more
amenable to treatment. There are however no controlled trials of surveillance efficacy, so the
extent to which current strategies are optimal (or even justified) is unknown.
If the risk of progression to adenocarcinoma is really much lower than previously
reported, the value of surveillance is called into question, particularly in Barrett patients
without dysplasia, in whom even quinquennial surveillance may not be cost-effective [28].
The wide variation in reported EAC progression rate in Barretts oesophagus [20] increases
doubt about effectiveness and cost-effectiveness of current surveillance strategies [17,29].
Most studies of cost-effectiveness of Barretts surveillance have assumed an EAC incidence
of 0.5% per annum, which as mentioned above may be an overestimate [3033]. Continuing
reflux, age >50 years, maleness, Caucasian ethnicity, elevated body mass index (BMI) and
especially dysplasia are additional adenocarcinoma risk factors. Diagnostic accuracy of
dysplasia also needs to be considered.
Figure 6.3 Low-grade dysplasia, missed in routine practice. Left panel: No dysplasia. Right panel: fragmented
dysplastic surface epithelium and keratin debris. Reported as no dysplasia. Disruption of mucosal architecture
probably made this dysplastic focus harder to recognise. Subsequent biopsies confirmed persistent low-grade
dysplasia.
dysplasia diagnosis, which in reality was probably pseudoincident, in the sense that it had
probably been present all along, but not been detected by an inefficient biopsy protocol.
Careful implementation of Seattle biopsy protocol is demanding for patient,
endoscopist, histology laboratory and interpreting pathologist. It remains to be shown that
alternative approaches are better. Unfortunately, in white light endoscopy, flat dysplasia is
hard to see but if reliable endoscopic visualisation of dysplastic Barretts mucosa could be
achieved, considerable efficiency might be achieved, with improved predictive value and
cost-effectiveness.
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Ploidy
Abnormal cellular DNA content (aneuploidy) is associated with increased risk of
adenocarcinoma. Reid et al. [43] showed that patients without aneuploid cells have a low
risk of progression, whilst patients in whom baseline biopsies demonstrated aneuploidy,
tetraploidy, or HGD progressed to cancer over 5 years in 43% and 59% of cases. In some
specialist centres, flow cytometry to assess ploidy is undertaken in the assessment
of Barretts mucosa, but it is not widely used probably for reasons including cost,
reimbursement issues and technical challenges. Flow cytometry divorces DNA content
measurements from morphology. Image cytometry of nuclei from thick sections, however,
allows some histological control, and image cytometry on histological sections gives best
correlation of DNA content with morphology, but introduces its own problems with nuclear
truncation and overlapping.
Molecular markers. TP53 is usually wild type in nondysplastic Barretts mucosa [44].
One TP53 allele may be inactivated by mutation and loss of heterozygosity at TP53 is
common in adenocarcinomas [44,45]. Overexpression of p53 (and sometimes circulating
p53 antibodies) is associated with progression to adenocarcinoma [46], and patients with
TP53 loss of heterozygosity (LOH) are more likely to progress to aneuploidy, HGD and
adenocarcinoma [47]: Thirty-seven per cent of patients with baseline p53 LOH progressed
to adenocarcinoma, against 3% without. Prevalence of LOH increases from 6% in
nondysplastic Barretts mucosa to 57% in HGD.
Aberrant tumour suppressor gene (TSG) promoter methylation is also an
adenocarcinoma risk biomarker [48]. In a multicenter study of 200 patients by Jin et al.
[49], an eight-biomarker panel predicted about half of HGDs and adenocarcinomas, with
reasonable specificity. Although these results appear encouraging, nonstandardised
methodology and an almost complete lack of prospective trials are problematical. Without
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29. Somerville M, Garside R, Pitt M, Stein K. Surveillance of Barretts oesophagus: is it worthwhile? Eur J Cancer
2008; 44:588599.
30. Sonnenberg A, Soni A, Sampliner RE. Medical decision analysis of endoscopic surveillance of Barretts
oesophagus to prevent oesophageal adenocarcinoma. Aliment Pharmacol Ther 2002; 16:4150.
31. Inadomi JM, Sampliner R, Lagergren J, et al. Screening and surveillance for Barrett esophagus in high-risk
groups: a cost-utility analysis. Ann Intern Med 2003; 138:176186.
32. Inadomi JM, Somsouk M, Madanick RD, et al. A cost-utility analysis of ablative, therapy for Barretts
esophagus. Gastroenterology 2009; 136:21012114.
33. Das A, Wells C, Kim HJ, et al. An economic analysis of endoscopic ablative therapy for management of
nondysplastic Barretts esophagus. Endoscopy 2009; 41:400408.
34. Curvers WL, fen Kate FJ, Krishnadath KK, et al. Low grade dysplasia in Barretts oesophagus: overdiagnosed
and underestimated. Am J Gastroenterol 2010; 105:15231530.
35. Wani S, Falk GW, Post J, et al. Risk factors for progression of low grade dysplasia in patients with Barretts
oesophagus. Gastroenterology 2011; 141:11791186
36. Abela JE, Going JJ, Mackenzie JF, et al. Systematic four-quadrant biopsy detects Barretts dysplasia in more
patients than nonsystematic biopsy. Am J Gastroenterol 2008; 103:850855.
37. Wolfsen HC, Crook JE, Krishna M, et al. Prospective, controlled tandem endoscopy study of narrow band
imaging for dysplasia detection in Barretts Esophagus. Gastroenterology 2008; 135:2431.
38. Curvers WL, van den Broek FJ, Reitsma JB, et al. Systematic review of narrowband imaging for the detection
and differentiation of abnormalities in the esophagus and stomach (with video). Gastrointest Endosc 2009;
69:307317.
39. Curvers WL, Herrero LA, Wallace MB, et al. Endoscopic tri-modal imaging is more effective than standard
endoscopy in identifying early-stage neoplasia in Barretts esophagus. Gastroenterology 2010; 139:1106
1114.
40. Sharma P, Meining AR, Coron E, et al. Real-time increased detection of neoplastic tissue in Barretts
esophagus with probe-based confocal laser endomicroscopy: final results of an international multicenter,
prospective, randomized, controlled trial. Gastrointest Endosc 2011; 74:465472.
41. Shaheen NJ, Sharma P, Overholt BF, et al. Radiofrequency ablation in Barretts esophagus with dysplasia. N
Engl J Med 2009; 360:22772222
42. Moyes LH, Going JJ. Still waiting for predictive biomarkers in Barretts oesophagus. J Clin Pathol 2011;
64:742750.
43. Reid BJ, Levine DS, Longton G, et al. Predictors of progression to cancer in Barretts oesophagus: baseline
histology and flow cytometry identify low and high risk patient subsets. Am J Gastroenterol 2000;
95:16691676.
44. Blount PL, Ramel S, Raskind WH, et al. 17p allelic deletions and p53 protein overexpression in Barretts
adenocarcinoma. Cancer Res 1991; 51:54825486.
45. Hamelin R, Flejou JF, Muzeau F, et al. TP53 gene mutations and p53 protein immunoreactivity in malignant
and premalignant Barretts esophagus. Gastroenterology 1994; 107:10121018.
46. Cawley HM, Meltzer SJ, De Benedetti VM, et al. Anti-p53 antibodies in patients with Barretts esophagus or
esophageal carcinoma can predate cancer diagnosis. Gastroenterology 1998; 115:1927.
47. Reid BJ, Prevo LJ, Galipeau PC, et al. Predictors of progression in Barretts esophagus II: baseline 17p
(p53) loss of heterozygosity identifies a patient subset at increased risk for neoplastic progression. Am J
Gastroenterol 2001; 96:28392848.
48. Sato F, Jin Z, Schulmann K, et al. Three-tiered risk stratification model to predict progression in Barretts
esophagus using epigenetic and clinical features. PloS One 2008; 3:e1890 88.
49. Jin Z, Cheng Y, Gu W, et al. A multicenter, double-blinded validation study of methylation biomarkers for
progression prediction in Barretts esophagus. Cancer Res 2009; 69:41124115.
50. Phoa KYN, van Vilsteren FG, Pouw RE, et al. Radiofrequency ablation in Barretts oesophagus with
confirmed low-grade dysplasia: Interim results of a European multicenter randomised controlled trial
(SURF). Gastroenterology 2013; 155:s187.
Chapter 7
Pathology of regenerative and
neoplastic hepatocellular nodules
Alberto Quaglia
Primary liver tumours are currently classified into epithelial, mixed/uncertain origin,
mesenchymal, germ cell and lymphomas [1]. Epithelial tumours are further subdivided
into hepatocellular and biliary, based on their resemblance to their normal epithelial
counterparts. This does not imply exclusive derivation from mature hepatocytes
or cholangiocytes. The liver contains a reserve regenerative compartment made of
hepatic progenitor cells believed to reside in the canal of Hering [2]. This progenitor cell
compartment is capable of activating, proliferating and differentiating into both hepatocyte
and cholangiocyte lineages, particularly when mature epithelial cells are damaged or
their replication is inhibited [3]. Hepatic progenitors are activated in many chronic
liver disorders [4] and may be targeted by carcinogenesis. Neoplastic transformation of
progenitor cells leads to neoplastic progenies which maintain the ability to differentiate
into both lineages . This in turn explains the observation of hepatic progenitor cell features
in hepatocellular neoplasms at an early stage (dysplastic nodules) or advanced tumours
with mixed hepatocellular and cholangiocellular phenotypes [3,5,6]. Concepts such
as tumour acquisition of progenitor cell features by de-differentiation, bone marrow
derivation of a proportion of hepatic progenitor/stem cells, cell fusion and cancer
cooperation add a further layer to the intricate pathogenesis and phenotypic complexity of
liver tumours [3,711].
The 4th edition of the WHO classification of tumours of the digestive system [1]
subdivides hepatocellular epithelial tumours into benign tumours [hepatocellular
adenoma (HCA), focal nodular hyperplasia (FNH)], malignancy-associated and
premalignant lesions (large cell change, small cell change, dysplastic nodules) and
malignant tumours [hepatocellular carcinoma (HCC) and variants, hepatoblastoma,
undifferentiated]. These categories partly overlap and partly divert from previous
classifications, in particular the widely adopted Working Party 1995 [12] consensus based
on the classification of hepatocellular nodules into regenerative lesions [monoacinar (e.g.
nodular regenerative hyperplasia), multiacinar, lobar/segmental hyperplasia, cirrhotic
nodule, FNH] and dysplastic or neoplastic lesions (HCA, dysplastic focus, low-grade/highgrade dysplastic nodule) and HCC. Considerations on the general definition of terms such
as tumour, nodule, regenerative, neoplastic, benign, dysplastic and malignant are beyond
Alberto Quaglia, MD, PhD, FRCPath, Institute of Liver Studies, Kings College Hospital, London, UK
Email: alberto.quaglia@nhs.net (for correspondence)
88
the purpose of this review, which for sake of simplicity and authors personal preference
maintains the time honoured distinction of regenerative versus neoplastic lesions.
This review also focuses on the more practical issues of differentiating histologically
between these various entities, in adult patients. The reader is referred to textbooks
[1315] or topical reviews [1618] for more comprehensive systematic descriptions of the
histopathology of hepatocellular lesions, and the most recent advances in their molecular
biology aspects [19,20].
The diagnosis of liver nodules rests on proper clinicopathological correlation. A full
clinical history, close correlation with imaging findings and ideally an adequate sample
of lesional tissue along with a separate sample of nonlesional tissue to assess the status of
background liver should be the basis for their histological interpretation.
Figure 7.1 Reticulin stain (Gordon and Sweet). (ac) Acute liver failure due to acetaminophen overdose. Serial
biopsies from native liver during its recovery phase following auxiliary transplantation (donor right lobe attached to
recipients left lobe). (a) 29 days, (b) 146 days, and (c) 350 days. Acute necrosis with sparing of periportal hepatocytes
results in an initial phase of hepatocellular proliferation, acute inflammation and reticulin collapse with parenchymal
nodular transformation. Resolution of inflammation, matrix reabsorption and hepatic plates remodelling
and growth lead eventually to full reconstitution of the liver mass, involution of the graft and withdrawal of
immunosuppression. (d) Regenerative nodule in nodular regenerative hyperplasia. The nodular area is flanked by
atrophic plates with condensation of reticulin fibres but no bridging fibrosis. (e) Regenerative nodule in advanced
stage chronic hepatitis C. The nodular area is surrounded by bridging fibrous septa. (f ) Primary biliary cirrhosis.
Bridging fibrosis linking up portal tracts generate a jigsaw puzzle pattern rather than round nodules.
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In advanced chronic liver disease, fibrosis progression leads to the formation of fibrous
septa, which encase regions of hepatic parenchyma to form nodules (cirrhotic nodules),
the size and shape of which depend essentially on the underlying disease. For example, in
chronic biliary disorders the regenerative nodules are typically in a jig-saw puzzle pattern,
the fibrous septa connecting portal tracts reciprocally, and sparing the hepatic venules
(Figure 7.1). In chronic hepatitis C, the nodules are of small size due to porto-portal and
porto-central bridging with loss of the vascular anatomical relationships (Figure 7.1). Twocell-thick hepatic plates are often present, and irrespective of the aetiology there may be
deposition of granules of copper-binding protein at the interface with the fibrous septa. This
indirect sign of advanced fibrosis may be helpful in the interpretation of core needle biopsy
specimens, in which nodule formation is not readily apparent due to the small size of the
sample or the large size of the nodules or both.
Some cirrhotic nodules may enlarge, and become dominant compared with the adjacent
regenerative nodules, to the point that they stand out at macroscopic examination of a
surgical specimen or may even be identified on imaging (large regenerative nodules,
macroregenerative nodules in older terminology). Sinusoidal capillarisation and individual
arteries may be present but are usually focal. In conditions such as BuddChiari syndrome
and primary sclerosing cholangitis, large areas or segments with preserved vascular outflow,
biliary drainage or portal vein supply in combination may become hyperplastic, particularly
in comparison with adjacent underperfused or poorly drained areas. This may cause, in
some cases, severe distortion of the lobar anatomy (Figure 7.2).
Nodular regenerative hyperplasia is the term used to describe thickened hepatic plates
arranged in a nodular configuration, often irrespective of specific lobular zonal boundaries
and with intervening atrophic hepatic plates, dilated sinusoids to a variable extent but no
bridging fibrosis. It typically occurs when small portal veins are obliterated, in the context,
for example, of systemic microvascular disease. The nodular hepatic plates are probably
those with preserved portal perfusion, whereas the atrophic ones are those affected by the
obliteration of their small portal vein tributaries (Figure 7.1). Areas of nodular regenerative
hyperplasia can become dominant and form regenerative masses, which can be identified
on imaging, causing difficulties in the differential diagnosis with HCC. Terms such as
adenomatous hyperplasia or partial nodular transformation have been used in the past to
name such lesions. These masses resemble FNH and probably share a similar pathogenesis
[24,25].
FNH is considered to be the typical example of a localised regenerative process, leading
to the formation of a mass identifiable on imaging and rarely even symptomatic. It is
usually solitary, but multiple FNH can occur in the context of vascular malformations
and coexist with intracranial tumour. It usually develops in noncirrhotic livers of women
3040 years of age, but FNH-like lesions have been described in cirrhosis [26,27]. It is
considered a benign hyperplastic regenerative process secondary to a local abnormality
of blood flow due to a vascular malformation or obliteration and arterialisation. This is
supported by its polyclonality, increased ANGPT1/ANGPT2 ratio, and association with
vascular disorders such as BuddChiari syndrome, hereditary haemorrhagic telangiectasia,
portal vein thrombosis, portal vein atresia and congenital portosystemic shunt [28,29].
Macroscopically, FNH can range in size from a few millimetres to several centimetres.
It usually shows a pale, micronodular and firm parenchymal surface, with or without
a central scar. There is usually no capsule, the periphery of the lesion merging with the
adjacent liver. Microscopically, it consists of nodules of benign-looking hepatocytes
separated by fibrous septa containing a ductular reaction, a lymphoid infiltrate and
aberrant vascular structures. Bioulac-Sage et al. [30] have recently shown a fairly
characteristic pattern of glutamine synthetase expression, typically patchy, highlighting the
hepatocytes away from the fibrous septa (Figure 7.3b). There may be a degree of sinusoidal
capillarisation by CD34 immunostaining. Associated features include steatosis, siderosis
and cholate stasis.
The differential diagnosis of regenerative nodules depends on the clinical context. In
cirrhotic patients, large regenerative nodules need to be differentiated from dysplastic
nodules and HCC. Dominant, mass-forming areas of nodular regenerative hyperplasia
and FNH may mimic radiologically HCA, HCC or nonhepatocellular lesions. These specific
differential diagnoses are discussed below.
Figure 7.3 (a) A 32-year-old woman transplanted for glycogen storage disease type 1. A 10-mm hepatocellular
adenoma showed diffuse staining for glutamine synthetase. No nuclear or cytoplasmic staining for -catenin was
identified. Please note background liver on the right hand side showing normal glutamine synthetase expression by
perivenular hepatocytes. (b) 52-year-old woman. Liver resection for focal nodular hyperplasia. In comparison with
picture (a), the pattern of glutamine synthetase is typically map-like.
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significant increase or decrease in some changes in comparison with the adjacent tissue.
These changes include siderosis, copper/copper-binding protein deposits, steatosis, clear
cell change, MalloryDenk bodies, increased trabecular thickness, increased cell density,
pseudoglandular structures, nuclear hyperchromasia or irregularity of the nuclear contour,
cytoplasmic basophilia, increased proliferative rate within areas of a nodule or compared
with background liver and loss of the reticulin stroma. Large cell change, originally called
liver cell dysplasia [40], describes the presence, usually in the context of chronic liver
disease, of more or less demarcated areas of liver parenchyma in which hepatocytes show
large atypical nuclei with preserved nuclearcytoplasmic ratio. It has been shown to be
associated with a risk of development of HCC, particularly in patients with viral hepatitis,
although its association with cholestasis and senescence suggests that this change may
have more than one pathogenesis [41]. Small cell change refers to the presence of areas
of increased cell density due to a reduction in hepatocyte size and increased nuclear
cytoplasmic ratio, often associated with nuclear hyperchromasia and irregularity of the
nuclear contour. A relationship between small cell change and progenitor cells has been
proposed on the basis of a similar immunohistochemical profile [42]. Small cell change is
considered to be a true precursor of HCC. In some cases, nodules fulfilling the criteria of
dysplastic nodules harbour foci of overt HCC in a nodule-in-nodule pattern.
According to a recent consensus paper [43], dysplastic nodules are further subdivided
into low-grade and high-grade lesions. Low-grade dysplastic nodules may be
indistinguishable from large regenerative nodules, differing only by the focal presence of
some of the dysplastic changes described above. High-grade dysplastic nodules may be
indistinguishable from early HCC probably because they represent part of a continuum.
Recent studies have shown that identification of stromal invasion, i.e. the presence
of tumour cells inside portal tracts or fibrous septa, may be of help in differentiating
between dysplastic nodules and HCC, but in the authors opinion, its interpretation is not
straightforward, even with the aid of immunohistochemistry [44].
Recent molecular studies [45] have led to the introduction of an immunohistochemical
panels including heat-shock protein 70, glypican 3 and glutamine synthetase, deemed to
be useful in differentiating dysplastic nodules from overt HCC [46]. Lack of staining with all
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three markers supports a high-grade dysplastic nodule, staining for one marker supports
well-differentiated HCC but does not exclude a high-grade dysplastic nodule, and staining
for two or three markers favours a well-differentiated HCC over a high-grade dysplastic
nodule. Clathrin heavy chain [47] and annexin-A2 [48] have been shown to improve the
performance of this panel.
To summarise, rather than separating categorically dysplastic nodules from HCC,
the currently accepted view is that HCC development and the transition between
dysplasia and HCC can be defined by three phases [43]: (1) high-grade dysplastic nodule
characterised by absence of stromal invasion, iso or hypovascularised with individual
arteries and portal perfusion; (2) well-differentiated (early) HCC, vaguely nodular with
indistinct margins, individual arteries, residual portal perfusion and iso-hypovascularity
on imaging, with or without stromal invasion; (3) moderately differentiated (progressed)
distinctly nodular HCC, with or without stromal invasion, with individual arteries, no portal
tracts and characteristic imaging features of hypervascularity in the arterial phase, and
hypovascularity in the venous phase. Vascular invasion may or may not be present.
Despite widespread interest in the concept of dysplastic nodule, the practical
diagnostic implications are largely dependent on local clinical practice. In view of the
current clinicoradiological criteria, small nodular lesions in cirrhotic patients are rarely
biopsied, or may be biopsied during local ablative procedure. The histological differential
diagnosis between large regenerative nodule, dysplastic nodule and HCC in this
setting may be challenging, severely limited by sampling error and essentially based on
clinicopathological correlation. The practical issues on the diagnosis of dysplastic nodules
in livers removed at transplantation are tempered, as the clinical emphasis in this context is
on the identification of histological predictors of tumour recurrence after transplantation,
which usually applies to overt HCC. Identification of incidental dysplastic nodule in
resection specimens of HCC from patients with compensated cirrhosis may confirm field
change and risk of malignant transformation in the remaining liver.
Small cell change and large cell change are part of a constellation of features which are
occasionally seen incidentally, either in a biopsy specimen not targeted radiologically to
a specific lesion or in sections from surgical specimens outside the boundaries of lesions
identified macroscopically. These changes include the iron-free foci in livers of patients
with genetic hemochromatosis described by Deugnier et al. in 1994 [49], the areas of
severe irregular regeneration of hepatocytes described by Shibata et al. in 1998 [50] or the
dysplastic foci characterised by any of the changes characteristic of dysplastic nodules,
in areas not >1mm. The general view is that these changes should at least be mentioned
in a histology report as they may indicate an increased risk of HCC. How this risk can be
quantified or whether and how it should dictate further clinical management is not clear.
Gene expression profiling has recently produced a prognostic gene expression signature to
predict the development of HCC [51].
Hepatocellular adenoma
HCA is considered to be a benign neoplasm of hepatocytes, although the term benign
contrasts the concept that this lesion carries a risk of malignant transformation. The main
risk factor for developing HCA is exposure to oestrogens or androgens. Outside the context
of hormonal stimulation, HCA is associated with conditions such as glycogen storage
disorder [52,53], and familial adenomatous polyposis. HCA can be single or multifocal, in
Hepatocellular adenoma
which case the term adenomatosis may apply. HCAs vary in size and are usually fairly well
circumscribed, with a soft cut surface often similar to background liver, although in many
cases areas of haemorrhage, necrosis or fibrosis may be present. Microscopically, HCA
are composed of hepatic plates similar to those in the background liver and intervening
individual arteries, but usually no portal tracts. Areas of necrosis, haemorrhage or fibrosis
are variably present. Some adenomas are rich in Dubin-Johnson-like pigment, possibly due
to a defect in excretion of organic anions [54]. More specific changes are associated with
the subtypes of HCA recently described on the basis of a correlation between molecular,
histological and clinical features [55,56] (Table 7.1). The subtype associated with mutations
of HNF1a is characteristically steatotic and lacks expression of fatty-acid binding protein.
Small microadenomas with similar characteristics are often found in the adjacent liver.
The inflammatory subtype, previously considered a variant of FNH [57], may be associated
with mutations of the IL6ST gene and tends to occur in associations with obesity, alcohol
consumption and systemic inflammatory signs and symptoms. It is characterised by
sinusoidal ectasia (telangiectasia), an intralesional inflammatory cell infiltrate often related
to intralesional arteries, a ductular reaction and strong expression of serum amyloid A
(SAA) and C-reactive protein (CRP), particularly in comparison with the background liver.
Steatosis may or may not be present. A small percentage of inflammatory adenomas may
harbour mutations of the b-catenin gene, which characterise a third subtype of HCA. The
histological features of this b-catenin-activated variant include pseudogland formation,
nuclear atypia, variable, often very focal nuclear and cytoplasmic staining for b-catenin
and diffuse expression of glutamine synthetase (Figure 7.3). This subtype of HCA occurs
usually in men, is considered to be at risk of malignant transformation and may be very
difficult to differentiate from well-differentiated HCC. The fourth category of HCA includes
all adenomas which do not fit into any of the three categories above.
As in the case of regenerative nodules and FNH, HCA and in particular its inflammatory
subtype may be overlooked in a biopsy sample, particularly if clinical details are lacking
and there is no sample of background liver for comparison. Particular attention needs to
be paid to the presence of individual arteries, and the spots of connective tissue containing
arteries and ductular reaction typical of the inflammatory subtype, which may be easily
mistaken for portal tracts, or FNH septa. Immunohistochemistry for serum amyloid A or
C-reactive protein is usually helpful, as they are both strongly expressed in inflammatory
adenoma, in contrast to FNH or background liver which are negative. Staining for
glutamine synthetase is also effective as, in background liver, it decorates the perivenular
Histological features
Clinical features
-catenin mutated
HNF1 mutated
Bleeding
Unclassified
Nonspecific features
Variable
95
96
hepatocytes, and in FNH those away from the septa (antiseptal), generating a fairly typical
map-like pattern (Figure 7.3) [30]. The differential diagnosis between HCA and HCC is
described below.
Hepatocellular carcinoma
HCC is considered to be the most common type of primary malignant liver tumour in
adults, although its incidence shows marked geographical variation, depending essentially
on the distribution of its risk factors. HCC is more common in males. The main risk factor
is cirrhosis and the main association is with hepatitis B virus (HBV) and hepatitis C virus
(HCV) infection, aflatoxin and alcohol, but any inherited or chronic liver condition increases
the risk of developing HCC. HCC can affect children with inherited metabolic conditions
such as tyrosinaemia, hypercytrullinaemia, biliary atresia, Bylers disease, bile salt export
pump (BSEP) deficiency and a-1-antitrypsin deficiency. Regression of fibrosis does not
eliminate the risk of HCC. HCC can occur in chronic viral hepatitis at a precirrhotic stage.
This is more frequent in HBV than HCV infection [15]. HCC does occur in noncirrhotic,
sometimes elderly patients. A good proportion of these patients have signs or history of
iron overload, past exposure to HBV or alcohol to excess. Diabetes, obesity and in more
general terms the metabolic syndrome are considered to be significant risk factors for the
development of HCC in livers without significant fibrosis [58]. Some patients, however,
may not have any risk factor or sign of liver injury [59,60]. HCC can develop in patients
taking anabolic steroids, but may show regression after hormonal withdrawal [61]. The
fibrolamellar variant of HCC is a well-recognised entity, which affects young adults, without
underlying history of liver disease. This variant of HCC has peculiar radiological and
histological characteristics, spread and behaviour, and its pathogenesis remains obscure.
HCC tends to be larger in noncirrhotic patients than in cirrhotic ones, partly due to
surveillance programmes in patients with chronic liver disease, leading to its identification
at a relatively early stage. HCC can grow into three main patterns. The nodular pattern
is the one usually observed in cirrhotic liver and consists of an expanding mass welldemarcated from the surrounding tissue often by interposition of a capsule and with
a multilobulated cut surface. The transition between dysplastic nodules and HCC,
along with the concept of early vaguely nodular and progressed distinctly nodular
HCC, has already been discussed. The term satellite is commonly used to describe the
presence of small tumoural nodules in the vicinity of the main mass. Whether multiple
nodular masses represent multifocal disease (either synchronous or metachronous) or
intrahepatic spread may be difficult to establish. A well-differentiated tumour, or presence
at its periphery of well-differentiated areas or even areas suggestive of origin from a
dysplastic nodule (e.g. nodule-in-nodule pattern) supports a multicentric origin in early
stage tumours [15]. The infiltrative pattern is usually observed in noncirrhotic livers and
consists of a large mass which occupies a good proportion of a lobe or more than one
lobe. The term massive also applies to tumours of this size. There is often involvement of
large portal vein branches. HCC in a diffuse pattern is rare, usually observed in cirrhotic
livers and consists of multiple nodules which may mimic the cirrhotic nodules, may not be
visible on imaging and difficult to identify macroscopically. The term cirrhotomimetic is
often used to describe this pattern [62].
Microscopically, HCC is defined by its resemblance to normal hepatocytes, according
broadly to the traditional Edmondson and Steiner criteria [63] . HCC, however, can
Hepatocellular carcinoma
be very heterogeneous and characterised by areas with different growth patterns and
degree of differentiation. Tumour cells are often arranged in a trabecular, pseudoacinar
or solid pattern. Well-differentiated HCC resembles normal hepatocytes with a similar
nuclearcytoplasmic ratio, similar nucleolated nuclei, well-demarcated cell borders
and eosinophilic cytoplasm. The cytoplasm may show steatosis or clarification, and
sometimes accumulation of MalloryDenk bodies, or eosinophilic globular inclusions.
Formation of canalicular bile plugs is a diagnostic feature of HCC, although nonlesional
cholestatic hepatocellular rosettes may become entrapped within the tumour and
mistaken for a tumour component. Staining for CEA using a polyclonal antibody, or
CD10, is the most commonly used method to demonstrate canaliculus formation, but
its expression is not hepatocellular specific; poorly differentiated tumours may not form
canaliculi, and distinction between a canalicular and membranous pattern is based on
subjective interpretation and may not be straightforward. In this respect, the usage of
more hepatocellular-specific canalicular markers appears promising. Two recent studies
have shown excellent specificity of BSEP expression [64,65] in distinguishing between
HCC and its extrahepatic mimics. Outside canalicular expression, arginase-1 [66] has been
noted to have superior sensitivity and specificity than Hep-Par-1 (hepatocyte paraffin
1). Cytokeratins panels (8/18, 19, 7 and 20) are of limited use due to frequent aberrant
HCC expression and considerable overlap with extrahepatic tumours. Alpha-fetoprotein
immunohistochemical detection is often focal and its sensitivity is too low. Glypican-3
may complement Hep-Par-1 in the diagnosis of poorly differentiated HCC [67], but it is
expressed in non-neoplastic liver parenchyma [68] and extrahepatic tumours [67], and
should not be used in isolation. The histological diagnosis of poorly differentiated HCC may
depend ultimately on the identification of better-differentiated areas in resected tumours.
Vascular invasion remains an important prognostic factor. Expression of stem cell markers,
biliary cytokeratins and a gene expression profile similar to hepatoblast identifies a subtype
of HCC with poor prognosis [69]. HCC can grow inside large bile ducts [70].
Fibrolamellar carcinoma is a well-known variant of HCC. It affects usually young adults
and is not associated with underlying chronic liver disease. It is characterised by fibrous
stroma and hepatoid tumour cells of large size, with eosinophilic cytoplasm, cytoplasmic
inclusions and scanty mitotic activity. A recent series has shown that fibrolamellar
carcinoma does not have a better survival than conventional HCC in children [71]. Other
variants include lymphocyte-rich, clear cell, scirrhous and sclerosing HCC. The term
mixed or combined hepatocholangiocellular carcinoma refers to tumours with mixed
hepatocholangiocellular phenotype, or tumours with progenitor cell features.
The differential diagnosis depends on the clinical context. In cirrhotic patients, most
nodular lesions are hepatocellular, but there are exceptions. Caturelli et al [39] showed that
a small proportion of small nodules identified by imaging in cirrhotic patients and suspect
for dysplastic nodules or HCC, were of other nature (e.g. lymphomas, haemangiomas).
Of note cholangiocarcinoma has been described in non-biliary cirrhosis [72]. The issues
regarding the diagnosis of dysplastic nodules and early HCC are described above.
In non-cirrhotic liver the differential diagnosis is usually with other primary epithelial
or non-epithelial tumours (focal nodular hyperplasia, hepatocellular adenoma,
cholangiocarcinoma, angiomyolipoma) or with metastases. FNH is usually the easiest
to differentiate, due to its architecture and bland appearance of lesional hepatocytes. In
contrast, the differential diagnosis with HCA can be challenging for the following reasons:
1) HCAs are considered to be at risk of malignant transformation, particularly those
97
98
Future directions
The classification of HCA based on molecular, clinical and histological correlation [18]
has shown how the approach to the diagnosis of hepatocellular tumour is changing.
Recent studies using high-throughput molecular techniques have classified HCC into
subcategories associated with specific aetiologies and specific molecular pathways. A
recent meta-analysis of gene expression profiles worldwide identified three robust HCC
Future directions
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Chapter 8
Serrated lesions of
colon and rectum
Ashraf EK Ibrahim, Mark J Arends
Introduction
The adenoma is the most frequent precursor of colorectal cancer (CRC) and it is high-risk
adenomas of large size, with villous architecture and high-grade dysplasia that are more
likely to progress to invasive adenocarcinoma. This conventional adenomacarcinoma
sequence is the most common pathway of colorectal neoplastic progression, accounting
for around 7080% of CRC development and it is associated with accumulation of
mutations, DNA deletions/losses, DNA gains and amplifications, with aneuploidy due to
chromosomal instability (CIN), as well as other genetic and epigenetic changes, in a wide
range of cancer-related genes [13]. Molecular genetic analyses have identified many, but
not all, of these molecular changes, including the most frequent genetic and epigenetic
alterations, such as those affecting APC (~80%), KRAS (~40%), TP53 (5060%), SMAD4
(4060%) and others [1,4,5].
In addition to the conventional adenomacarcinoma pathway with underlying
CIN as the main type of genomic instability, there are other pathways of neoplastic
progression (Figure 8.1), such as lesions displaying microsatellite instability (MSI) or
aberrant methylation of CpG islands within gene promoters the CpG island methylator
phenotype (CIMP) [6]. The morphologically serrated pathways represent alternative
routes of colorectal neoplastic progression. The right-sided sessile serrated neoplasia
pathway involves formation of hyperplastic polyps, sessile serrated lesions (SSLs) and
serrated adenomas (of usual type), which may progress to serrated adenocarcinoma,
whereas the left-sided traditional serrated neoplasia pathway includes hyperplastic
polyps and traditional serrated adenomas (TSAs) which may evolve into invasive serrated
adenocarcinomas. The serrated pathway lesions are also characterised by particular genetic
and molecular characteristics, including MSI and CIMP in some, although these are not
as fully characterised at the molecular level as those found in the conventional adenoma
carcinoma pathway (Table 8.1).
Ashraf EK Ibrahim, MBChB (Hons), MSc, PhD, FRCS, FRCPath, University of Cambridge, Department of Pathology,
Addenbrookes Hospital, UK
Email: aeki2@cam.ac.uk (for correspondence)
Mark J Arends, MBChB (Hons), BSc (Hons), PhD, FRCPath, MA, University of Edinburgh, Department of Pathology,
Institute of Genetics & Molecular Medicine, Western General Hospital Campus, Edinburgh, UK
104
Table 8.1 WHO classification [7] and their commonly used synonyms in histopathological
interpretation of serrated polyps and serrated polyposis
WHO classification
Hyperplastic polyp
105
106
Mixed polyps
Mixed polyps contain a mixture of foci of hyperplastic polyp and foci of dysplastic adenoma,
either conventional adenoma or serrated adenoma (Figure 8.3). They were originally thought
to represent collision lesions between a hyperplastic polyp and a nearby adenoma. However,
currently they are regarded as displaying the development of a focus of dysplasia within a preexisting hyperplastic polyp [13]. As such, they represent links between hyperplastic polyps
and either serrated adenomas, of usual type, or TSAs in the serrated neoplasia pathways.
However, following the publication of European guidelines in 2011 [14], there is now
agreement in the UK and participating European countries to use the term Sessile
Serrated Lesion as the preferred term for this lesion. SSLs have some similarities to larger
hyperplastic polyps in that they show a hyperplastic or serrated polyp-like appearance
with some unusual architectural features, of which the key feature is the presence of
horizontal orientation of the deep part of the crypts (just above the muscularis mucosae),
often forming L-shapes (or boot shapes), or inverted T-shapes, or anchor shapes (Figures
8.48.6). However, in hyperplastic polyps the pattern of serration extends around half
way to two-thirds of the way down the crypts (Figure 8.2), in SSLs the pattern of serration
extends down to or nearly to the base of the crypts (Figures 8.58.6). Another key feature
is the lack of conventional or genuine nuclear dysplasia, although the lesional cells may
have some mild nuclear enlargement and nuclear atypia that does not amount to true
dysplasia. Some of the crypts may also show dilatation, sometimes with the most dilated
segment near to the crypt base (Figures 8.48.6). SSLs are more frequent in the right colon
107
108
and more common in females than males. Sometimes they may be large, sessile, yellow in
colour endoscopically or poorly defined [15].
SSLs may show evidence of abnormal proliferation on MIB-1/Ki-67 immunostaining,
but have a normal subepithelial collagen plate. In some SSLs, there may be evidence of
loss of MLH1 expression by immunohistochemistry. They may express MUC5AC or MUC6
by immunohistochemistry. SSLs may sometimes be found in HPS [16]. The distribution of
SSLs in the large bowel shows a right-sided predominance with involvement of the caecum
and ascending colon in 51% of SSL cases, transverse colon in 16%, descending colon in
11%, sigmoid colon in 12% and rectum in 10% [17].
SSLs may sometimes have foci of dysplasia, representing links in the sessile serrated
neoplasia pathway from SSL to serrated adenoma of usual type. In the WHO (2010)
classification [7] SSPs with cytological atypia replaced the designation mixed polyps and
may show either low-grade or high-grade dysplasia (Table 8.1). There is some uncertainty
about the frequency of SSLs relative to other colorectal polyps and this reaches 7% in some
series, although observer reproducibility is an issue, due to lack of familiarity with the
diagnostic criteria, as well as the nomenclatural confusion leading to mislabelling of SSLs
as adenomas [15,16].
Pathways of pathogenesis
filiform TSA that shows multiple and prominent thin, elongated projections of TSA-like
dysplastic epithelium with serrated contours [21].
Serrated adenocarcinoma
Serrated adenocarcinomas have been reported to account for approximately 7.5% of all
CRCs, but 17.5% of right-sided colonic adenocarcinomas, although in our experience
and practice the proportions are lower than those reported in the literature. Serrated
adenocarcinomas represent the end-points of the serrated pathways: the majority of
left-sided serrated colorectal carcinomas are microsatellite stable (MSS) or show only
low-frequency MSI (MSI-L) and arise from TSAs [22,23], whereas right-sided serrated
adenocarcinomas appear to arise from SSLs/serrated adenomas (Figure 8.8) and show
a high-frequency MSI (MSI-H) with 30% microsatellite markers displaying changes in
length [22, 2426]. Serrated adenocarcinomas are usually considered to be more prevalent
within the right colon, although in one series over 50% were observed in the distal colon
[22]. Morphologically, serrated carcinomas show serrated architecture often in irregularly
elongated crypt-like structures, but little tubular or papillary growth pattern, with abundant
eosinophilic cytoplasm and are commonly associated with a mucinous component [22]
(Figures 8.9 and 8.10).
Pathways of pathogenesis
Serrated colorectal neoplasia pathways
Overall, it has been estimated that around 2030% CRCs arise via the serrated neoplasia
pathways. The pathways of development of serrated neoplasms can be divided into
two distinct pathways, one that mostly occurs on the right side of the large bowel and
results in sessile serrated neoplastic lesions and the other on the left side or distal bowel
and generates TSAs and associated carcinomas [27,28] (Figure 8.1). In the right-sided
sessile serrated neoplasia pathway, the initial lesion is a hyperplastic polyp (often of
109
110
Figure 8.10Serrated
adenocarcinoma. There is invasion
by malignant epithelium forming
irregularly branching, elongated,
serrated crypt-like structures. At the
upper right of the picture, there
is evidence of extracellular mucin
secretion indicative of a mucinous
component (magnification x50).
Molecular abnormalities
microvesicular type), which may expand and develop into a SSL. Within this, there may
evolve a focus of dysplasia forming a serrated adenoma (of usual type) and a subset of
these may progress to a proximal serrated carcinoma (50% of which are predominantly
mucinous) and these usually show MSI-H, with silencing of MLH1 expression by promoter
hypermethylation as a part of the CIMP-H phenotype (see below), occurring in the
sporadic or hereditary non-polyposis colorectal cancer (HNPCC)/Lynch syndrome (LS)
setting [28,29].
The left-sided traditional serrated neoplasia pathway starts with distal hyperplastic
polyps, which may progress in some cases to formation of TSAs, some of which may evolve
into distal serrated carcinomas that are MSS or MSI-L with a low-frequency CpG island
methylation phenotype low (CIMP-L), without methylation of the MLH1 promoter, found
in the sporadic or non-HNPCC/LS context [28,29].
Molecular abnormalities
Some of the most frequent molecular alterations in colorectal tumourigenesis include
those affecting APC which is mutated and sometimes deleted in around 80% conventional
(non-serrated) adenomas and carcinomas [30]. This occurs as a very early event in
association with normal epithelium changing to monocryptal or oligocryptal adenomas, as
seen in FAP coli, an inherited syndrome of bowel adenoma and carcinoma susceptibility
due to germline APC mutation [31]. KRAS mutations are found in around 40% colorectal
adenomas and carcinomas, and they occur mostly during the early stages of adenoma
growth and can synergise either with APC mutations or defective mismatch repair (MMR)
to accelerate colorectal tumour formation [32,33]. In contrast, TP53 mutations are observed
in approximately 5060% cancers, often occurring as a late event around the time of
transition from conventional adenoma to carcinoma [34,35]. Many other genetic and
epigenetic changes can occur during this adenoma-carcinoma sequence, including MMR
abnormalities, usually due to acquired MLH1 promoter hypermethylation in 15% sporadic
CRC, as well as in 24% CRC due to inherited mutations in MMR genes in HNPCC/LS, in
addition there is loss of a large part of chromosome 18, containing the SMAD4 and SMAD2
genes, occurring in around 60% CRC, as well as low- or very low-frequency mutations to
a wide range of cancer-associated genes [3638]. Other specific changes to SSLs and TSAs
have been described such as loss of expression and promoter methylation of SLIT2, which
contributes to malignant transformation by E-cadherin degradation [39].
111
112
partner MSH6 [41,42]. This leads to defective MMR which appears to be selected due to a
reduction in susceptibility to DNA-damage-induced apoptosis [43,44].
MSH2 and MLH1 are the two most frequently mutated genes in LS, accounting for
4045% LS families each, and minor ones being MSH6 and PMS2 (<10% LS families
each), with rare LS families having other affected genes [45]. The resulting failure to
repair DNA replication-associated errors in these tumours produces a high frequency of
mutations, either as single base mismatches or in regions of short tandem DNA repeats
(the repeat units often being 1-4bp in length), known as microsatellites, such as repetitive
polynucleotide tracts often of the form (A)n or (CA)n. Thus, DNA extracted from such
tumours shows variation in length (both shorter and longer) of a significant proportion of
microsatellites, a phenomenon known as MSI [45].
MSI-H with 30% abnormal microsatellite markers is the hallmark of cancers in LS and
is also observed in 15% of sporadic CRCs, due to somatically acquired MLH1 promoter
hypermethylation, resulting in transcriptional silencing of both copies of MLH1 [11]. The
majority of sporadic CRC exhibiting MSI-H develops in the right side of the large bowel
(caecum, ascending colon and transverse colon) [46]. Deficiency in MMR produces an
increase in the mutation rate (100x 1000x the normal mutation rate), sometimes called
a mutator phenotype, which leads to mutations accumulating in genes that play key
roles in the regulation of growth of normal colonic epithelia and tumour development,
and these are selected during carcinogenesis as part of a progressive deregulation of
growth control [3].
Lesion size
Recommended surveillance
interval (years)
Hyperplastic polyp
<10mm
10
<10mm
SSL
>10mm
Any size
Any size
Any size
113
114
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Chapter 9
An update on the pathology of
chronic inflammatory
bowel disease
Roger M Feakins, Neil A Shepherd
Introduction
Chronic inflammatory bowel disease (CIBD), in the context of this chapter at least,
represents the range of pathology with classical chronic ulcerative colitis (UC) at one end
and classical Crohns disease at the other. Pathologically, there is very much a spectrum
and there may well be equivocal pathology. In biopsy material, the latter is now termed
inflammatory bowel disease unclassified (IBDU) [1], whereas the term indeterminate
colitis is reserved for equivocal cases of CIBD in resection material [2]. The latter remains
a controversial diagnosis. Many do not like the term but it can be a useful category, when
strictly restricted to resection material, because there is good evidence that the natural
history is like that of UC in the majority of cases. Thus, whilst one can expect increased
complications compared with UC, particularly pelvic sepsis, when ileal pouch construction
is undertaken, there are distinctly dissimilar results for this category compared with
Crohns disease.
Here, we concentrate on diagnostic and management issues of CIBD pathology.
Histology is generally regarded as the gold standard for the diagnosis of CIBD and for the
distinction between UC and Crohns disease. However, clinicians have many other data
on which to make a clinical assessment of such cases. This includes clinical, endoscopic,
radiological and microbiological evidence. So, on the one hand, a biopsy report may simply
provide corroborative evidence for a diagnosis made from many sources of information. On
the other hand, pathologists should understand that a strongly worded pathological report
will often mean that a diagnosis of CIBD or of one of the main types of CIBD is irrevocably
appended to a patient and that this may be inappropriate. Perhaps this is most apposite to
mimics of CIBD, as the microscopic appearances in many conditions, perhaps particularly
infective, can appear very similar to those of CIBD.
Roger M Feakins, MD, FRCPI, FRCPath, Pathology and Pharmacy Building, Royal London Hospital, London, E1
2ES, UK
Neil A Shepherd, DM, FRCPath, Gloucestershire Cellular Pathology Laboratory, Cheltenham General Hospital,
Cheltenham, UK
Email: neil.shepherd@glos.nhs.uk (for correspondence)
118
Accordingly, pathologists should understand that colonoscopic biopsies are often just
a small piece of the evidence that the clinician has for the diagnosis and management of
CIBD patients. Also, it is very important that pathologists avoid inappropriate terminology.
Diagnostic summaries of unqualified colitis can be distinctly misleading. Admittedly, many
gastroenterologists equate the term colitis with ulcerative colitis, but there are many other
causes. The pathologists primary role, in biopsy material, is to make a diagnosis of CIBD
and not colitis. They should then determine whether the features favour UC or Crohns
disease. If they are equivocal, a designation of IBDU is appropriate. Once again, it must be
emphasised that pathologists should be diligent to exclude the many mimics of CIBD and
thus, as ever in gastrointestinal pathology, the context of the pathological appearances is
all-important. This underpins the importance of the pathologist receiving accurate clinical,
endoscopic and imaging information to ensure that an inappropriate diagnosis of CIBD is
not made in cases where there is strong mimicry of the pathological changes of CIBD.
Table 9.1 Histological features that help discriminate between IC and CIBD
Histological
feature
Reliability for
discriminating
CIBD from IC
Favours
Frequency
in IC (%)
Frequency
in CIBD
(%)
Reproducibility
Comments
Basal
plasmacytosis
High
CIBD
06
69100
Moderate/good
Best predictor of
CIBD
Normal in caecum.
Equivalent to loss
of the plasma cell
gradient and to
transmucosal CI
Normal
architecture
High
IC
85
Moderate
Crypt distortion
High
CIBD
030
28100
Variable
Crypt branching
High
CIBD
10
79
Variable
Crypt atrophy
High
CIBD
015
1033
Variable
Villiform mucosal
surface
High
CIBD
07
1127
Variable
Granulomas
Moderate
CIBD
02
627
Good
Exclude crypt
rupture
Does not
distinguish
between UC
and IC
Basal lymphoid
aggregates
Fair
CIBD
07
1835
Moderate
Difficult to
distinguish from
normal aggregates
Fair
CIBD
02
116
Moderate/good
Uncommon;
limited data
Paneth cell
metaplasia
Doubtful
NA
415
Variable
Normal in right
colon
Probably a feature
of longstanding
inflammation
Crypt abscesses
None
NA
4754
7590
Moderate
Mucin depletion
None
NA
70
92
Variable
Loss of parallelism
Caution next to
crypt abscess or
lymphoid follicle
Crypt shortening
and/or wide crypt
spacing
IC, infective colitis; CI, chronic inflammation; CIBD, chronic inflammatory bowel disease; UC, ulcerative colitis.
119
120
features of the two diseases. Above all else, UC is characteristically continuous, whereas
Crohns disease is notably patchy or discontinuous. Evidence of small bowel involvement
strongly favours Crohns disease, unless the features suggest backwash ileitis. The
thickness of the bowel wall can also be easily appreciated macroscopically.
Although UC is classically continuous, it is important to understand that there may
be discontinuous disease in UC. The caecal patch lesion is now well recognised [12] and
the appendiceal skip lesion is also a characteristic feature of more extensive UC [13].
Furthermore, treatment can have a profound effect on the distribution of disease in UC.
This is seen both macroscopically and microscopically. More powerful immunosuppressive
agents and monoclonal antibodies can produce a notable discontinuity of active disease in
UC and this may readily be apparent macroscopically.
Crohns disease
Disease in continuity
Vascularity seldom
Serositis common
121
122
Prevalence in ileum
Comment
Refs
Granulomas (noncryptolytic)
B and R
Specific (unless
cryptolytic).
However there are
other causes, e.g.
infection
[1517]
Giant cells
5% inflamed CD bxs
[15]
Indicated CD in
one study
[17]
Suggested CD in
one study
[17]
Submucosal inflammation
Fissure ulcers
0% UC resections
[16]
Regarded as
favouring CD
[17]
0% UC resections
Favours CD
[16]
0% UC resections
Favours CD
[16, 17]
Favours CD
[17]
Favours CD
[17]
Favours CD
[17]
Favours CD
[17]
Neural hyperplasia
Ileal features favouring UC
Mild ileal mucosal injury with
moderate/markedly active caecal
colitis
Characteristic of
ileal UC in one
study
[17]
Characteristic of
ileal UC in one
study
[17]
Both
2% of inflamed CD bxs
1% UC resections
Not specific; a
sign of chronic
ulceration
[15,16]
[15]
Architectural
changes
distinguish Crohns
disease from
infection but not
from neoplasia,
endometriosis
[15]
Associated with
decreased goblet
cells
Increased eosinophils
[15]
Neutrophil polymorphs
[15]
Cryptitis
[15]
10.5% UC resections
[16]
Focal inflammation
[15]
3% UC resections
[16]
Erosions NOS
2% UC resections
Villous atrophy
1.5% UC resections
[15]
[16]
[16]
CD, Crohns disease; bx, biopsy; UC, ulcerative colitis; LP, lamina propria; NOS, not otherwise specified; CIBD, chronic
inflammatory bowel disease.
We would particularly identify the importance of the connective tissue changes in Crohns
disease. For instance, fat wrapping is now regarded as pathognomonic of Crohns disease.
It is very common in small intestinal Crohns disease, but it is less common and more
difficult to identify in colonic Crohns disease [14]. Particularly in the small intestine, other
connective tissue changes are well recognised macroscopically. The bowel wall thickening
is caused by both fibrosis and proliferation of smooth muscle in the submucosa (obliterative
muscularisation of the submucosa, OMUS) and neuronal and vascular changes may also be
evident macroscopically [14].
Ileum
Ileal involvement by CIBD is considerably more common in Crohns disease than in
UC in biopsies and strongly favours the former. However, ileal UC may be more easily
detected in resections than in biopsies: there was a prevalence of 17% in one report. The
term backwash ileitis for ileal UC has been questioned, as the evidence for a backwash
mechanism is weak. For example, some patients do not have contiguous caecal and
proximal colonic UC. As regards specific histological features, the presence of noncryptolytic granulomas favours Crohns disease over UC but ileal microscopic changes are
otherwise not usually very informative. Table 9.3 lists some of the features seen in ileal
mucosa that may have discriminant value between UC and Crohns disease, often based
on limited published evidence or the opinions of experts [1517]. Initial suggestions that
ileal involvement predicts an increased risk of pouchitis and of carcinoma in patients with
UC have not been confirmed by subsequent studies [15,16,18,19].
123
124
125
126
now considerable evidence that previous appendicectomy protects patients against the
subsequent occurrence of UC [29]. Many have argued that the reason for this is that the
removal of immunocompetent tissue of the appendix reduces the likelihood of subsequent
UC. We wonder if this is more likely to be because of the influence of faecal stasis on
the generation of mucin changes that lead to UC. We note the association of UC with
sites of faecal stasis notably the rectum, diverticular disease in the sigmoid colon and
the appendix and have postulated that this could be a factor in the protection of the
appendicectomised patient against UC.
The pathological assessment of the appendix is also important in the differential
diagnosis of CIBD. The appendix is now well recognised to be one of the skip lesions
of UC [13]. Indeed, the pathology of the involved appendix in UC is very characteristic.
Just like that in the affected colon and rectum, there is diffuse mucosal pathology with
no semblance of transmural inflammation (Figure 9.4). The pathological assessment of
such an appendix provides powerful evidence towards a diagnosis of UC in colectomy
specimens.
Florid transmural inflammation in the form of lymphoid aggregates with focal active
inflammation, with or without ulceration, might suggest Crohns disease when seen in an
appendicectomy specimen. However, caution is appropriate in this situation. Chronic or
grumbling acute appendicitis can lead to very similar pathological appearances. We have
seen many cases suggestive of involvement of the appendix by Crohns disease only to find
that the clinicians favour a grumbling appendix and subsequent follow-up has not shown
any evidence of Crohns disease elsewhere. The diligent pathologist requires to be cautious
in this situation and should only suggest a diagnosis of Crohns disease when observing
such changes in an appendicectomy specimen. We believe that this appendiceal scenario
is similar to that seen in resection specimens of sigmoid colon afflicted by diverticular
disease. In this situation, also, there can be profound mimicry of Crohns disease but most
cases appear only to represent mimicry of Crohns disease by complicated diverticular
disease rather than true multifocal Crohns disease [3033].
In a similar vein, caution is appropriate with granulomatous appendicitis. Whilst the
presence of well-formed epithelioid cell granulomata, especially with other features of
Crohns disease such as focal ulceration and transmural inflammation, might well indicate
Crohns disease, it has been shown that granulomatous appendicitis, per se, is more likely
to represent a chronic acute appendicitis rather than true Crohns disease [34]. So, in
this situation, one should give a guarded report and advise clinicians to seek evidence of
Crohns disease elsewhere.
127
128
Over the years, there has been considerable controversy as to whether the presence
of pathological changes of Crohns disease, at resection margins, is of influence in
determining whether or not that patient is likely to suffer recurrence of the disease. In fact,
there is now substantial evidence that most features of Crohns disease, such as granulomas
and active inflammation, do not have any influence over the recurrence or prognosis
when demonstrated at or close to an excision margin [12,37]. We would therefore suggest
that histological sections taken of resection margins are of little value for determining
likely recurrence or prognosis of Crohns disease. Nevertheless, there is a small amount of
evidence to suggest that Crohns disease-associated myenteric plexitis may be predictive of
recurrence [38,39].
It appears that granulomas cause particular diagnostic problems when they are present
in pathological specimens where there has been previous surgery. For instance, they
are a characteristic feature of diversion proctocolitis, whether the initial indication for
surgery was UC or Crohns disease, being especially seen in the defunctioned rectum after
colectomy for UC [40]. Indeed, well-circumscribed granulomas are almost a normal
feature in the pelvic ileal reservoir mucosa, especially within Peyers patches and lymphoid
aggregates. A pathologist should be very cautious before making a diagnosis of Crohns
disease in pathological specimens where there has been previous surgery.
We have already emphasised the striking mimicry of Crohns disease that can occur
in complicated diverticular disease. Indeed, well-formed epithelioid cell granulomata
certainly occur in this situation. Furthermore, rather characteristic transmural
inflammation, not necessarily in the form of rosaries but rather migrating radially away
from inflamed diverticula, is seen in complicated diverticular disease. It can be seen that
the combination of granulomas, focal active inflammation, transmural inflammation
and even fissuring ulceration and fistulae, in complicated diverticular disease, can cause
profound mimicry of Crohns disease. As with granulomatous appendicitis, the diligent
clinician and pathologist should seek evidence of Crohns disease elsewhere.
In contradistinction to well circumscribed basal and submucosal granulomas,
cryptolytic granulomas are certainly not specific to Crohns disease (Figure 9.5). These have
been recognised in infective enterocolitis, diversion proctocolitis, diverticular colitis, the
pouch mucosa and pouchitis and secondary colitis. Furthermore, cryptolytic granulomas
are increasingly recognised in active UC [12,41].
129
130
Table 9.5 Features of CIBD-related colorectal carcinoma (CRC) and other CRC (%) [42,44,45].
Category of CRC
Sporadic
MSS
Sporadic
MSI
Lynch
syndrome
(hereditary
MSI)
CIBDrelated
MSS
CIBDrelated
MSI
UC
related
Crohns
related
Yes
Yes
>10
>10
>10
Rare
<50
Rare
Clinical feature
Mean age <55
No
No
Yes
Yes
Multiplicity of
tumours
Rare
010
>10
>10
Right-sided
<50
>50
>50
<50
Lack of necrosis
<50
>50
>50
>50
>50
>50
Crohns-like
reaction
Rare
>50
>50
>50
2550
2550
>50
2550
Mucinous/
presence of mucin
<50
>50
4156
>50
29
>50
>50
46
Well differentiated
Rare
>40
432
>40
>40
>40
>40
>40
Pathological
feature
Rare
>10
>10
>10
>10
>10
Medullary
phenotype
Rare
>20
>20
Rare
Rare
Rare
Pouch cancer
IPAA may be offered to patients with UC who have had a colectomy. It is associated with
a reduced risk of CRC. Carcinoma in the pouch itself is rare and is more likely if there was
preoperative colorectal neoplasia or if there is PSC. Pouch cancer is most likely to arise
in the columnar cuff at the pouchanal junction and thereby likely represents neoplasia
arising in the small segment of lower rectum that is retained at the lower aspect of the
ileal pouch. So, when compared with other UC-associated CRCs, pouch carcinoma is very
similar but is more likely to have a Crohns-like peritumoral reaction [46,47].
Lymphoma
Suggestions that the risk of intestinal and extraintestinal lymphoma is increased in longstanding CIBD are not supported by population studies. However, there is an increased
131
132
Appendiceal neoplasia
Appendiceal involvement by CIBD is common. As in the large bowel, chronic mucosal
inflammation may be a risk factor for the development of neoplasia. Indeed, case reports
have suggested an association between CIBD and appendiceal epithelial neoplasms. A
larger study showed that patients with CIBD and synchronous colorectal neoplasia had
a 15-fold increase in the prevalence of appendiceal cystadenoma, compared with those
without CIBD, and an 8-fold increase compared with those with uncomplicated CIBD.
However, the risk in CIBD overall was not increased significantly compared with non-CIBD
patients. Appendiceal neuroendocrine tumours have the same prevalence in CIBD patients
as in the general population [50,51].
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Chapter 10
Diagnosis and therapy of
gastrointestinal MALT lymphoma
Andrew C Wotherspoon, Larissa Sena T Mendes
IntroductIon
In 1983, Isaacson and Wright first reported the existence of a group of indolent B-cell
lymphomas that formed a specific clinicopathological entity and occurred specifically at
extranodal sites [1,2]. These lymphomas structurally showed many of the features of native
lymphoid tissue found distributed along the small intestine, appendix and colorectum.
The most prominent concentration of native mucosa-associated lymphoid tissue (MALT)
is seen in the terminal ileum in the form of Peyers patches, and for this reason the term
lymphomas of MALT applied to this group of lymphoma. A mucosa is not a prerequisite
for these lymphomas as the disease defining features are organisation, cell morphology,
immunophenotype and molecular characteristics [35].
Since that first description, many studies have unravelled the aetiopathogenesis, clinical
behaviour, histological features and molecular mechanisms associated with this group of
tumours. The recognition that some of these lymphomas may respond to antibiotic therapy
means that accurate diagnosis is essential if one is to avoid under treatment of other
lymphoma types. Furthermore, strategies are needed to assess response and determine if
and when more conventional therapies should be applied to cases diagnosed as resistant to
eradication therapies [3,6].
Andrew C Wotherspoon, MB, BCh, FRCPath, Department of Histopathology, Royal Marsden Hospital, London, UK
Email: andrew.wotherspoon@rmh.nhs.uk (for correspondence)
Larissa Sena T Mendes, MD, Department of Histopathology, Royal Marsden Hospital, London, UK
136
The lymphoid tissue in the terminal ileum is the prototype of MALT. There are three
intimately related components to this tissue: the B-cell and T-cell areas in the mucosa and
the draining mesenteric lymph nodes. The Peyers patches in the terminal ileum consist
of lymphoid follicles that occupy the full thickness of the mucosa with the base facing
the muscularis mucosae (Figure 10.1a). The germinal centre has a lower dark zone and
luminal light zone, which shares the organisational characteristics of secondary follicles in
lymph nodes (Figure 10.1bc). The mantle surrounds the germinal centre and is broader
in the subepithelial area. Outside the mantle zone is a further layer of B cells (Figure
10.1bc) that is not seen in peripheral lymph nodes and is characteristically only seen in
the spleen, mesenteric lymph nodes and extranodal lymphoid tissue. This is the marginal
zone which is composed of cells that are slightly larger than mantle zone cells with more
abundant pale cytoplasm. The marginal zone is also broader at the luminal aspect, and
marginal zone B cells are seen to enter the epithelium over the dome of the follicle to form
a lymphoepithelium (Figure 10.1bc). The epithelium in this area contains a population
of specially adapted cells, M cells, which facilitate the transport of large molecules across
the epithelial barrier to enhance and maintain the integrity of intestinal immunity. The
area around the follicle contains T cells, plasma cells and accessory cells. Scattered
intraepithelial T cells are present in the gut epithelium throughout the intestine. MALT
lymphomas recapitulate this structural organisation [4,5].
figure 10.1 (a) Peyers patches in the terminal ileum. (b) Germinal centre
surrounded by a thin layer of small lymphocytes with scant cytoplasm
forming the mantle zone. Outlining this layer, there is the marginal zone
with slightly larger cells and more abundant cytoplasm. (c) Marginal zone
cells enter the overlying epithelium forming a lymphoepithelium. (d)
Acquired mucosa-associated lymphoid tissue: presence of a lymphoid
follicle and reactive lymphoid infiltrate surrounding and entering the
gastric epithelium.
As MALT lymphoma typically arises in areas that are devoid of constitutive organised
lymphoid tissue, acquisition of this is the first step along the path of lymphomagenesis.
Organised lymphoid tissue is usually acquired in the context of either infection or
autoimmune disease. In the stomach organised lymphoid tissue of MALT-type is acquired
most frequently in association with infection by Helicobacter pylori (Hp) but can be seen
with infection by Helicobacter heilmannii and in patients with Sjogrens syndrome. The
acquired lymphoid tissue shares all the features of native MALT with a reactive germinal
centre, mantle and thin marginal zone (Figure 10.1d). There may be infiltration of the
epithelium by marginal zone B cells to form a lymphoepithelium, but the specialised M
cells are not seen [911].
137
138
usually either IgM or IgA and rarely IgG. The cells are typically negative for CD5, CD23,
cyclinD1, CD10 and bcl-6 (Figure 10.3bc). Very occasional cases with expression of
CD5 have been described. There is expression of bcl-2 protein. Expression of CD43 is
seen in approximately 50% of cases (Figure 10.3d). Staining of FDC can be achieved with
antibodies to CD21 and/or CD23 (Figure 10.3c) and staining for cytokeratin may help to
highlight lymphoepithelial lesions (Figure 10.3ef). Staining with antibodies to bcl-6 and
CD10 can highlight residual germinal centres [3,7]. There is light chain restriction (kappa
more frequent than lambda) (Figure 10.3gh).
The presence of a significant number of large cells in clusters or sheets (generally 20 or
more large cells) should provoke a diagnosis of diffuse large B cell lymphoma. It is crucial
to distinguish such clusters from residual naked aggregates of follicle centre cells. This can
be confidently achieved by demonstrating the presence of CD10 and bcl-6 with lack of bc-2
staining characteristic of residual follicle centre cells [20,12].
Immunoglobulin heavy chain and light chain genes show clonal rearrangement and
there is a high load of somatic hypermutation [6]. MALT lymphomas are characterised by
several recurrent chromosomal translocations, which involve the nuclear factor-kappa B
(NF-kB) signalling pathway [6,21]. Of these, the most frequent is the translocation t(11;18)
(q21;q21) which creates a novel functioning fusion product by translocating the amino
terminus region of apoptosis inhibitor 1 (API1) gene to the carboxyl terminus of MALT1.
139
140
The resulting fusion product has the ability to activate the canonical and noncanonical
NF-kB pathways. This translocation appears specific to MALT lymphoma and is present
in about 25% of gastric MALT lymphomas, but the frequency in nongastric GI MALT
lymphoma is less certain. When the t(11;18)(q21;q21) is present, it is almost always the sole
genetic abnormality [22,23].
Other chromosomal translocations have been described including t(1;14)(p22;q32),
found in up to 4% of gastric MALT lymphomas, translocating the BCL-10 gene into to
come under the influence of the promoter of the immunoglobulin heavy chain gene. The
resulting protein also leads to activation of NF-kB pathway. When this translocation is
present there are often further genetic changes particularly trisomies of chromosomes
3, 12 and 18 [9,21,24]. The architectural organisation, immunophenotype and presence
of somatic hypermutation (indicating antigen selection) all point to an origin of MALT
lymphoma from postgerminal centre marginal zone B cells [5,7,10].
dIfferentIaL dIagnosIs
With advances in the understanding of NHL, the underlying genetic abnormalities and the
increasing development of more subtype-specific treatment options, the accurate diagnosis
of these entities has become more important.
atypia, Dutcher bodies and formation of genuine lymphoepithelial lesions have been shown to
be highly associated with MALT lymphoma rather than reactive infiltrates [16,27,28].
Special techniques may be helpful in such cases. Immunophenotypic demonstration
of light chain restriction is characteristic of a neoplastic infiltrate. When expression of
CD43 is demonstrated on the B-cell population, it can be used as a marker of a neoplastic
population. Molecular studies may be useful. Although some studies have suggested that
clonal populations can be demonstrated in reactive infiltrates, adoption of the European
BIOMED-2 primer/protocol has made detection of clonal population more specific and
accurate. Optimised protocol/heteroduplex analysis has made false positive results from
biopsies with reactive infiltrates very rare if the analysis is conducted appropriately.
Detection of a clonal population in a suspicious lymphoid infiltrate should be considered
highly indicative for the diagnosis of MALT lymphoma [15,29].
141
142
eradication therapy and the strategy for managing cases where there is antibiotic resistance
in the organism should be determined by current international guidelines [3436]. There
has been a suggestion that, in a similar way to gastric MALT lymphoma, IPSID may be
associated with infection by Campylobacter jejuni. Whilst definitive evidence is still lacking,
it is certainly true that a proportion of cases of IPSID in early stages will respond to broad
spectrum antibiotic therapy [7,26].
No specific relationship of extragastric classical GI lymphoma to infective organisms
has been shown. Reports of response of MALT lymphoma in the colorectum to antibiotic
therapy exist. In some cases, there is associated gastric Hp infection, but it is unlikely that the
Hp and lymphoma are causally related and the response is likely to be serendipitous [37].
Prediction of gastric MALT lymphoma cases that will respond to Hp eradication alone
would clearly be important to the management of these patients. In reality no clinical,
pathological or molecular feature will absolutely predict those cases that will not respond to
antibiotic based regimes alone. The time to response is also very variable with some patients
showing complete response at the time of first control endoscopy to assess Hp status, whilst
others only regress after months or even years following successful eradication. Success of Hp
eradication should be confirmed by current clinical guidelines but could include a urea breath
test as the most accurate determinant of presence of small residual Hp colonies [35,36].
Absence of Hp infection is likely to be associated with a lack of response, although
there are exceptions and these may be due to the presence of other, as yet undetermined,
infective organisms [15]. The determination of Hp status at diagnosis is therefore crucial to
patient management. Whilst histological evaluation, culture and stool polymerase chain
reaction (PCR)-based studies are all effective detection methods for Hp infection, there
may be cases where previous partial or complete eradication therapy may give a negative
result in patients previously Hp positive. It has been shown that more accurate assessment
of Hp status in the context of MALT lymphomagenesis is achieved by serological based
studies as circulating antibodies may be present up to 2 years following eradication [15,35].
In general, cases with deeper infiltration of the gastric wall by lymphoma with or without
local lymph node involvement are less likely to respond to eradication therapy. This feature
is best determined using endoscopic ultrasound [15,38,39].
Whilst no specific immunophenotypic feature has been shown to predict response to
Hp eradication therapy alone, molecular studies have a role. Whilst some cases of response
have been recorded, the presence of the t(11;18)(q21;q21) is generally a specific predictor
that response to Hp eradication alone is unlikely [40]. This translocation is present in only
approximately 3% of cases responding to Hp eradication therapy alone, whilst it is detected
in about 50% of cases in stage IE and in about 70% of cases in stage IIE or above that do not
respond to this approach [15,39].
The timing when the translocation should be investigated remains controversial,
either at diagnosis or when more conventional treatment is proposed. Assessment is best
performed using interphase fluorescence in situ hybridisation with MALT1 dual-colour
break-apart and API2-MALT1 dual-colour fusion probes or by reverse transcriptionpolymerase chain reaction (RTPCR) of the API2-MALT1 fusion transcript [15,31].
When eradication therapy is deemed to have failed or delayed response is considered
unlikely, more conventional standard antilymphoma therapeutic approaches are
indicated [15]. Surgery is no longer considered an option due to the multifocal nature
of the disease [41]. Radiotherapy has been shown to be a highly effective therapy in
gastric MALT lymphomas. Standard chemotherapy using several regimes with or without
immunotherapy has been shown to be effective in the treatment of MALT lymphoma. Of
note, is the fact that the presence of t(11;18)(q21;q21) is associated with high frequency
of treatment failure in patients given single oral alkylating agents (chlorambucil or
cyclophosphamide) or thalidomide [15,18].
High-grade transformation may occur in MALT lymphoma, although this is very rare in
cases with t(11;18)(q21;q21) [3,7]. Whilst progression to diffuse large B-cell lymphoma has
generally been considered an indication for combination chemotherapy, there is increasing
evidence that these lymphomas may respond to Hp eradication alone. This remains to be
confirmed and such an approach is only recommended in patients unfit for other therapies
or in the context of an appropriate clinical trial [20,42].
Morphological features
Recommendation
No need of additional therapy
NC no change
143
144
latter group would represent patients in whom, if the absence of change is prolonged,
radiotherapy/chemotherapy may be used at an earlier stage [33,43].
Some biopsies show persistent lymphoid infiltrate, but with reduced density compared
with diagnostic biopsy and with the development of fine fibrosis in the lamina propria with
loss of glands associated with destruction following lymphoepithelial lesion formation. This
is the group considered to be showing responding residual disease (Figure 10.4c). Whilst
this appearance persists, a watchful waiting approach can be continued [18,43].
In many cases, there will be near complete absence of lymphoid infiltrate with only
the presence of occasional lymphoid aggregates within the lamina propria, mainly at the
base of the mucosa (Figure 10.4b). It has been shown that in the majority of cases these
aggregates contain a small population of neoplastic B cells that are derived from the
lymphoma clone. This group has been labelled probable minimal residual disease (pMRD).
Follow-up studies of cases with this residual infiltrate have indicated that this has no
clinical significance in terms of disease progression, and further therapy in these cases is
not indicated [6,15,44].
Sequential follow-up biopsies are indicated in all cases of gastric MALT lymphoma.
Occasional relapses can be seen and these may be associated with reinfection/regrowth of
Hp. In these cases, repeat eradication therapy has proven effective. In some cases, there is
Conclusion
concLusIon
MALT lymphoma represents a specific lymphoma entity with characteristic clinical,
pathologic and molecular features. The GI tract is the area most frequently involved by
MALT lymphoma with the majority of cases occurring in the stomach. In the stomach, the
greater numbers of cases are associated with Hp infection and eradication of the organism
results in lymphoma regression for most of them, although time to regression can be highly
variable. Certain characteristics, including depth of gastric wall involvement, local lymph
node involvement and presence of the t(11;18)(q21;q21), predict for lack of response to
eradication therapy, in which case radiotherapy or chemotherapy but not surgery is highly
effective in inducing lymphoma regression. Histological assessment of post eradication
gastric biopsies should be performed within the context of the GELA assessment scheme
to provide information about the tempo and direction of travel of potential lymphoma
regression. For extragastric GI MALT lymphoma, antibiotic-based therapy may be effective
in a proportion of cases but generally chemotherapeutic strategies are frequently required.
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146
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marginal zone B-cell lymphoma of MALT. Gut 2011; 60:747758.
Papadaki L, Wotherspoon AC, Isaacson PG. The lymphoepithelial lesion of gastric low-grade B-cell
lymphoma of mucosa-associated lymphoid tissue (MALT): an ultra-structural study. Histopathology 1992;
21:415421.
Isaacson PG, Wotherspoon AC, Diss T, Pan LX. Follicular colonization in B-cell lymphoma of mucosaassociated lymphoid tissue. Am J Surg Pathol 1991; 15:819828.
Copie-Bergman C, Wotherspoon AC. MALT lymphoma pathology, initial diagnosis and post treatment
evaluation. In: Cavalli F, Stein H, Zucca E (eds), Extranodal lymphomas pathology and management.
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Liu H, Ye H, Dogan A, et al. T(11;18)(q21;q21) is associated with advanced MALT lymphoma that expresses
nuclear BCL10. Blood 2001; 98:11821187.
Ye H, Liu H, Raderer M, et al. High incidence of t(11;18)(q21;q21) in Helicobacter pylori-negative gastric
MALT lymphoma. Blood 2003; 101:25472550.
Lucas PC, Kuffa P, Gu S, et al. A dual role for the API2 moiety in API2-MALT1-dependant NK-kappa B
activation: heterotypic oligomerization and TRAF2 recruitment. Oncogene 2007; 26:56435644.
Tian MT, Gonzalez G, Scheer B, et al. BCL10 can promote survival of antigen-stimulated B-lymphocytes.
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28. Zucca E, Bertoni F, Roggero E, et al. Molecular analysis of the progression from Helicobacter pyloriassociated chronic gastritis to mucosa-associated lymphoid tissue lymphoma of the stomach. NEngl J Med
1998; 338:804810.
29. Hummel M, Oeschger S, Barth TF, et al. Wotherspoon criteria combined with B cell clonality analysis by
advanced polymerase chain reaction technology discriminates overt gastric marginal zone lymphoma
from chronic gastritis. Gut 2006; 55:782787.
30. El-Zimaity HM, El-Zaatari FA, Dore MP, et al. The differential diagnosis of early gastric mucosa-associated
lymphoma: polymerase chain reaction and paraffin section immunophenotyping. Mod Pathol 1999;
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31. Dreyling M, Thieblemont C, Gallamini A, et al. ESMO guidelines consensus conference on malignant
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32. Wotherspoon A, Doglioni C, Diss T, et al. Regression of primary low-grade B-cell gastric lymphoma of
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33. Hussell T, Isaacson PG, Crabtree JE, et al. The response of cells from low grade B cell gastric lymphomas of
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34. Chen LT, Lin JT, Tai JJ, et al. Long-term results of anti-Helicobacter pylori therapy in early stage gastric highgrade transformed MALT lymphoma. J Natl Cancer Inst 2005; 97:13451353.
35. Malfertheiner P, Megraud F, OMorain CA, et al. Management of Helicobacter pylori infection the
Maastricht IV/Florence Consensus Report. GUT 2012; 61:646664.
36. Nakamura S, Sugiyama T, Matsumoto T, et al. Long-term clinical outcome of gastric MALT lymphoma after
eradication of Helicobacter pylori: a multicentre cohort follow-up study of 420 patients in Japan. Gut 2012;
61:507513.
37. Niino D, Yamamoto K, Tsuruta O, et al. Regression of rectal mucosa-associated lymphoid tissue (MALT)
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38. Fishbach W, Goebeler-Kolve ME, Greiner A. Diagnostic accuracy of EUS in the local staging of primary
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45. Capelle LG, de Vries AC, Looman CW, et al. Gastric MALT lymphoma: epidemiology and high
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Chapter 11
Medical revalidation for
histopathologists
Peter Furness
Peter Furness, BM, BCh, PhD, FRCPath, Consultant Histopathologist, University Hospitals of Leicester; Revalidation
Lead, University Hospitals of Leicester, Leicester, Leicestershire, UK. Revalidation Lead, East Midlands; Former vice
Chair and Revalidation Lead, the Academy of Medical Royal Colleges, London, UK
Email: pnf1@le.ac.uk
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since the early 1990s. Proposals based mainly on a requirement to undertake CPD were
well advanced, but had not been implemented, at the time when Harold Shipman was
convicted. It became obvious that the system would be expected to catch the next Harold
Shipman so the GMCs plans were put on hold pending an investigation of the background
of Shipmans crimes by Dame Janet Smith.
Dame Janets report identified, as expected, the need for a system to check up on
the performance of doctors every few years [1]. But she was highly critical of the GMCs
proposals. As a consequence, those plans were abandoned.
The next major development was the publication, by the then Chief Medical Officer Sir
Liam Donaldson, of a White Paper entitled Trust, Assurance and Safety: The Regulation of
Health Professionals in the 21st Century [2]. This was a very broad-ranging document, but
it included proposals for regular checks on the performance of doctors. The whole process
was called revalidation, but it was initially split into two components. Relicensing was to
be a check that a doctor remained competent at the level of basic registration with the GMC
as a medical practitioner. Recertification was to be a check that specialists were operating
at an appropriate level for their specialty. The Medical Royal Colleges, which provide
postgraduate curricula and examinations in all the medical specialties in the UK, were
asked to deliver recertification. This seemed reasonable, as the Medical Royal Colleges are
the bodies that set the standard for postgraduate specialist medical practice in the UK.
However, the Medical Royal Colleges regarded this as a poisoned chalice. The
opportunity for the profession to set and test against its own standards was appealing; but
the risks and logistic requirements were substantial. The White Paper did not specify how
doctors would be evaluated. There appeared to be an assumption that doctors would have
to sit examinations comparable to those which the Colleges already delivered; but this
ignored the high level of medical specialisation in the UK. In histopathology alone, it would
have been necessary to deliver a separate examination for every organ system, every year.
Other medical specialties had the same problem. The Colleges had already suffered an
increasing level of regulatory bureaucracy in relation to their postgraduate curricula and
examinations. Legal challenges by unsuccessful candidates were becoming increasingly
common and expensive. For the Colleges, examinations had become increasingly complex
and expensive to run. And the Colleges are professional membership organisations,
maintained largely by subscriptions from their Fellows. It is one thing for an organisation
funded by its Fellows to tell an aspiring young doctor that he or she has not quite reached
the required standard; it is quite another to tell established Fellows who for many years
have paid their College subscription fees that they are no longer fit to work. The Colleges
declined to do as they were asked.
In the White Paper and in subsequent discussions a consensus emerged that, largely
as a result of the problem of extreme medical specialisation, doctors should be expected
to prove their worth only in relation to their own individual scope of practice. It was soon
recognised that the huge diversity of individual medical practice excluded the option of
sitting examinations at intervals. Furthermore, examinations could not cover the crucial
aspect of what doctors actually do, rather than what they are capable of doing under
examination conditions. The idea that revalidation should be based on an annual review
of the whole of a doctors individual practice gained ground. This would be based on the
annual appraisal which was already demanded by most NHS contracts of employment; but
the process of medical appraisal would have to be enhanced and made more formal if it
was to satisfy the requirements of the GMC.
As a result, the GMC took the lead in defining how annual appraisals might satisfy this
role. The Medical Royal Colleges became sources of advice on how the GMCs general
requirements should be interpreted in the context of a specific specialty [3]. It was
recognised that if revalidation was to be based on appraisals of a doctors actual practice, the
division into relicensing and recertification was unhelpful, so those terms were dropped.
To deliver this, appraisals would have to fulfil two tasks that, in an educational context,
are usually kept separate; the summative question (Is this doctor fit to practise?) and the
formative question (Can this competent doctor be helped to improve his/her performance
even more?). A decision was made that medical appraisal should be made to answer both
questions, but this has led to some confusion. Some individuals and organisations have
pursued their laudable aims of improving overall standards by demanding more and more
from doctors, using the implied but inappropriate threat of removal of the doctors licence
to practise medicine.
So it is important to start a discussion of the UKs new revalidation processes by making
this summative/formative distinction absolutely clear. Everyone including the GMC,
Royal Colleges and patients wants every doctor to work to the highest level they can. That
is the formative element of appraisal, and for most doctors it should be the major part of the
process. The question of whether or not a doctor keeps a licence to practise is ultimately set
by the GMCs Fitness to Practise Panels [4,5] not by the appraiser or by the Royal Colleges,
although the Colleges may give advice to the GMC. That is the summative question. Those
who wish to use medical appraisal to raise the bar of medical practice should recognise
that if appraisals are calibrated to deliver an opinion that is significantly higher than the
level set by the GMC, we will have a system where appraisal suggests that a doctor is not
fit to practice, but then the GMC overturns that opinion. Then what happens? The doctor
returns to work with a grievance. No one benefits.
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care, including monitoring and responding to adverse clinical incidents and complaints.
So it should be recognised that the outcome of appraisals is only one of the factors that ROs
are expected to consider when making a recommendation.
ROs only have three options when making a recommendation to the GMC [4].
1. Recommend revalidation. This is clearly the option preferred by all concerned, but the
doctor must have contributed to the appraisal process all the information that the GMC
demands. Note that the RO merely recommends; the GMC makes the final decision
2. Recommend deferral. This is appropriate when, for some legitimate reason, there is not
yet enough information on which to base a revalidation recommendation. There must
be a plan in place to obtain that information, as the deferral recommendation must
include an indication of when the information will be available and the delay must be
no >1 year.
3. Failure to engage. There is a degree of vagueness here, because there is no option called
Engaging, but not really trying hard enough. It is yes or no. However, if an RO feels that
a doctor is not engaging with the process, the RO should discuss this with the GMCs
local Employment Liaison Advisor (ELA) even if the problem is identified early in the
revalidation cycle. If the ELA is convinced that there is a problem, the GMC will contact
the doctor concerned directly to warn of the consequences. One possible consequence
is that the doctors revalidation date will be brought forward, potentially to only a few
months away. If the doctor still does not comply with the requirements of revalidation,
the next step would be GMC action for failure to engage, an administrative process
which can result in the removal of the doctors licence to practise medicine without
the involvement of any formal assessment of competence by the GMC. So doctors who
think they need not worry about revalidation in the early years of the cycle may find
their revalidation date brought forward dramatically.
There is no option of making a formal do not revalidate recommendation. That is because
any RO who is unable to make any of the above three recommendations should already
have brought their concerns in relation to the doctors standard of practice to the attention
of the GMC. If the GMC is investigating a doctors performance, the revalidation process is
halted.
could cover all the GMCs requirements. The resultant list of required information is described
in the GMCs publication on Supporting Information for Medical Appraisal and Revalidation
[11]. Guidance on how these general requirements should be interpreted by pathologists has
been published by the Royal College of Pathologists [16] and is only summarised here.
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essential. It is arguable that the detailed reports from EQA schemes should be discussed in
confidence with the appraiser, as low scores on specific cases may be important guides to
focus on CPD activity. But such schemes test what pathologists can do, in a rather artificial
setting; they do not test what pathologists actually do, which is what matters to patients.
So audit remains a requirement. A variety of other sources of information may be relevant,
including turnaround times, rates of seeking second opinions, numbers of supplementary
reports issued, reflective review of the investigation of individual difficult cases and
more. All must be interpreted with caution, and if possible compared against relevant
benchmarks.
Significant events
Any serious mishap in the previous year must be discussed. For a histopathologist, this
could be a wrong diagnosis, a misplaced specimen, a seriously delayed report, an accident
in the laboratory and many more. But the number and magnitude of such incidents will
very rarely be a point of major concern. As with complaints, the GMC is rather more
interested in knowing that when things do go wrong, doctors react appropriately and take
action to minimise damage to patients and to prevent a recurrence. As a result, in medical
appraisal the negative implications of something going wrong can be turned into a very
positive affirmation that the doctor handled the problem well.
Statement of health
All doctors are expected to ensure that their own health does not compromise the care of other
patients. The GMC expects a standard statement to be signed to confirm that this is happening.
Statement of probity
This is another standard GMC statement. Superficially, it seems a little absurd; if someone
formally declares they are telling the truth, how can we know they are not lying? But the
power of this statement lies in the context. If any of the other information provided at
an appraisal is found to be deliberately incorrect, or in some respects even if it is merely
incomplete, this probity statement is demonstrably false. The consequence could be a very
rapid referral to the GMCs disciplinary procedures.
Table 11.1 Statements the appraiser is asked to confirm to the Responsible Officer. If any of
these statements cannot be made, the appraiser is asked to explain why
An appraisal has taken place that reflects the whole of a doctors scope of work and addresses the principles and
values set out in Good Medical Practice
Appropriate supporting information has been presented in accordance with the Good Medical Practice Framework
for Appraisal and Revalidation and this reflects the nature and scope of the doctors work
A review that demonstrates appropriate progress against last years personal development plan has taken place
An agreement has been reached with the doctor about a new personal development plan and any associated
actions for the coming year
No information has been presented or discussed in the appraisal that raises a concern about the doctors fitness to
practise
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been delivered, but is planned for next year. But any serious concerns should be escalated
by the appraiser to the RO.
References
References
1. Smith J. The Shipman inquiry, third report. Death certification and the investigation of deaths by coroners.
London, Home Department and Secretary of State for Health, 2003.
2. Department of Health. The White Paper, Trust Assurance and Safety: the regulation of health professionals.
London: Department of Health, 2007.
3. Academy of Medical Royal Colleges. Revalidation. In: Review 2012-2013. London: Academy of Medical
Royal Colleges, 2013.
4. General Medical Council. Ready for revalidation. Making revalidation recommendations: the GMC
responsible officer protocol. Guide for responsible officers. Manchester: General Medical Council, 2012.
5. General Medical Council. The investigation process. http://www.gmc-uk.org/concerns/the_investigation_
process.asp. (Last accessed 22 March 2013.)
6. General Medical Council. Ready for revalidation. Meeting the GMCs requirements for revalidation.
Manchester: General Medical Council, 2013.
7. Health Care and Associated Professions, Doctors. The medical profession (responsible officers)
amendment) regulations 2013. Statutory Instruments 2013: 391.
8. General Medical Council. My designated body. http://www.gmc-uk.org/doctors/revalidation/12387.asp.
(Last accessed 22 March 2013.)
9. NHS Revalidation Support Team (RST). http://www.revalidationsupport.nhs.uk/index.php. (Last accessed
22 March 2013.)
10. NHS Revalidation Support Team. Medical appraisal guide. A guide to medical appraisal for revalidation in
England. London: NHS Revalidation Support Team, 2013.
11. General Medical Council. Ready for revalidation. Supporting information for appraisal and revalidation.
Manchester: General Medical Council, 2012.
12. General Medical Council. Revalidation. http://www.gmc-uk.org/doctors/revalidation.asp. (Last accessed 22
March 2013.)
13. General Medical Council. Ready for revalidation. The Good medical practice framework for appraisal and
revalidation. Manchester: General Medical Council, 2012.
14. General Medical Council. Good medical practice. http://www.gmc-uk.org/guidance/good_medical_
practice.asp. (Last accessed 22 March 2013.)
15. NHS Revalidation Support Team. Testing and piloting. http://www.revalidationsupport.nhs.uk/events/
about_the_rst/our_performance/Testingandpiloting.php. (Last accessed 22 March 2013.)
16. Royal College of Pathologists. Supporting information for appraisal and revalidation: guidance for
pathologists. London: Royal College of Pathologists, 2012.
17. General Medical Council. Colleague and patient feedback for revalidation. http://www.gmc-uk.org/
doctors/revalidation/colleague_patient_feedback_intro.asp. (Last accessed 22 March 2013.)
18. Follett B, Paulson-Ellis M. A review of appraisal, disciplinary and reporting arrangements for senior NHS and
university staff with academic and clinical duties. London: Department of Education, 2001.
19. Coroners and Justice Act 2009. Notification, certification and registration of deaths. Chapter 2. In: Coroners
and Justice Act 2009, Chapter 25, Part 1. London: Coroners and Justice Act, 2009.
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Chapter 12
Molecular testing for human
papilloma virus
Karin Denton
Introduction
Human papilloma virus (HPV) is an ancient DNA virus infection with which has been
known for many years to be an essential step in the development of cervical cancer [1,2].
The molecular biology of HPV infection and disease progression is understood in detail
and this has allowed the development of numerous testing strategies. There are now over
140 recognised types of HPV and more than 80 of these have been fully sequenced. Typing
is based on variance demonstrated in DNA sequencing, >10% being required to define
a new type. Types are numbered in relation to order of discovery; there is no relation to
phylogeny.
In terms of oncogenesis, HPV 16 is the type involved in most cases of cervical cancer
(5362%), with HPV 18 present in 1315%. The frequency of different HPV types present
in cases of cervical cancer varies in different countries and also probably varies on a much
more local level [3,4]. Other HPV types are causative in fewer cases. In total 14 types are
recognised as being associated with cervical carcinogenesis, these are 16, 18, 31, 33, 35, 39,
45, 51, 52, 56, 58, 59, 68 and 66. These are collectively referred to as high-risk HPV.
Dr Karin Denton, MBChB FRCPath, Director, Cancer Screening Quality Assurance (SW), Public Health England, UK
Email: karin.denton@phe.gov.uk (for correspondence)
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Immunisation
All four UK countries, in common with elsewhere in the world, have implemented a
programme of HPV immunisation. From 2008 HPV immunisation was offered to girls
aged 1213 through to 18. The only country to implement a national schools-based
immunisation programme earlier was Australia in 2007. Initially the UK procured a
bivalent vaccine against HPV 16 and 18 only, but in 2012 a quadrivalent vaccine including
also HPV 6 and 11, which are responsible for most cases of genital warts, was nationally
commissioned. Coverage in 1213 years olds, delivered to girls through their schools,
has been very high (around 80% in the areas with highest coverage) but the catch up
programme to older girls has never enjoyed such high coverage (around 3035%).
All data suggest that these vaccines are highly effective in preventing infection with HPV
type 16 and 18 in a naive population, and immunisation will definitely have an impact on
the prevalence of HPV-related disease in the future. However, it is important to note that
whilst immunised women have started to be screened in Scotland and Wales; in England
this has not yet occurred as they have not reached the age of 25. Furthermore, the effect will
be limited initially due to low coverage in this cohort, and high prevalence of active HPV
infections at the time of immunisation in 1618 years olds. Women immunised at age 1213
will not reach screening age in England until 2020.
Triage
HPV testing in triage and test of cure have been extensively evaluated [7]. The previous
model for the treatment of low-grade abnormalities was to repeat cytology in 6 months.
The rationale for this was to detect HPV persistence, but it had the disadvantage of poor
compliance with follow-up, delay and inconvenience for women, and also low detection rate
of high-grade disease in women eventually referred to colposcopy. Triage allows women
who are HPV negative to revert to routine recall as their risk of having significant disease is
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extremely low, and allows selection of the higher risk women for immediate colposcopy,
reducing problems with attendance. However, the detection rate of CIN2+ at colposcopy is
still low at around 15% [7]. Triage is cost-effective and is popular with women.
Test of cure
Previously, women having treatment of high-grade CIN would have annual cytology tests
for 10 years, a significant imposition. Although the detection rate of residual disease was
low, it was always felt that these women were at higher risk than average and needed
enhanced surveillance. Around 85% of women have negative results for both cytology and
HPV at their first follow-up visit, and the facility to return these women to routine recall is
hugely beneficial.
Primary screening
HPV testing has been evaluated in primary screening in many countries for many years.
Several large randomised controlled trials have been undertaken to assess whether this
type of screening gives better outcomes than cytology screening [12,14,15]. Most trials have
used the oldest and best-established HPV test, Hybrid Capture II (Quiagen). It has always
been apparent that the sensitivity of HPV testing for CIN with this technology is very high,
but the specificity is low. HPV infection in the absence of CIN2+ is extremely common with
50% of 1820 years olds testing positive at cytology [16] Even by age 25 this figure is unlikely
to drop below 30%.
viruses which do have an E3). E4 disrupts cytokeratins; E5 allows interaction with growth
factor receptors. The two genes of interest in oncogenesis are E6 which is involved in p53
degradation and E7 which binds the retinoblastoma gene, Rb. The L1 and L2 genes code for
capsid proteins. The L1 capsid protein is used in vaccines to generate an immune response.
Hybrid Capture II
In this test, RNA probes react to DNA targets for 13 high-risk HPV types. The RNADNA
hybrids are detected with a monoclonal antibody to the hybrids and this is then subject
to chemiluminescent detection. There are manual and automated versions available,
but there is still a significant operator requirement. Results are very reproducible and the
system is robust and reliable.
Original evaluations were performed using a cut-off of 1 RLU, but following evaluation in
the ARTISTIC trial [16] a cut-off of 2 RLU is used in England. This is only a semi-quantitative
method, and again the key is to correlate RLU score not with presence of high-risk HPV,
but with presence of CIN2+. It should be noted that in fact quantitation of HPV testing on
cervical cytology samples is illusory, because the samples themselves vary enormously in
cellularity.
Genprobe Aptima
This is the only approved method which detects RNA rather than DNA. The significance
of this is that it should be more specific. HPV infection in its early stages does not lead to
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integration with host DNA. Whilst other HPV tests detecting viral DNA will be positive,
until E6/E7 has been integrated, this test will remain negative. Integration is the first key
step in oncogenesis. Genprobe uses a transcription-mediated amplification methodology.
The system is highly automated and capable of high-volume testing. Genprobe is also used
for other virological investigations including hepatitis, HIV and Chlamydia, and again
these platforms have been widely implemented in laboratories.
Hologic Cervista
This utilises a novel detection method, invader probe chemistry. This achieves a 110
million fold amplification of HPV DNA. HPV 16 and 18 typing can also be performed as a
reflex using genotype-specific probes.
Worldwide applications
The five tests described above are only applicable to organised testing in rich countries. Not
only are the tests relatively expensive (though price is reducing with market forces), they
require reliable power sources, mains water and a relatively clean environment. The testing
equipment is large, fragile and not easy to move.
In resource poor settings, which have by far the greatest incidence of cervical cancer,
other tests may be more appropriate. There is extensive experience that introducing
cytology-based screening in these settings is difficult, but limited HPV testing is feasible.
There are at least two HPV tests under development which may well be better suited to
these settings. These are the Care HPV test which uses Hybrid Capture II chemistry and
the lateral flow test. Development of both is being supported by the Bill and Melinda Gates
Foundation. Both require no electricity, running water or refrigeration and give a fast
result.
Biomarkers
Because of the poor specificity of a positive HPV test result for CIN2+, attention has turned
to indirect markers to see if these could be used to refine the result. Of course, cytology
could be included as an indirect marker and in fact retains the highest specificity of any test
of CIN2+. This is the reason that in the UK HPV primary screening pilots which commenced
in 2013, cytology is being used to triage those samples which test positive for HPV.
Another indirect test which has attracted much attention is P16, a cell cycle protein
which is over expressed when E7 binds to pRB, and which can be detected with
immunohistochemistry. Detection of P16 in histology samples is a useful means to confirm
CIN in difficult cases, and to assist with grading. Dectection in cytology samples, either
on its own or in combination with a proliferation marker, Ki67, looks very promising as a
way of improving the specificity of a HPV positive, cytology low-grade sample [20,21]. An
evaluation in the NHS is due to start in 2013. A similar concept underlies ProExC (Beckton
Dixon), a composite marker comprising identifiction of mini chromosome maintenance
(MCM) protein and topoisomerase II (TOP2A) a marker of cell proliferation.
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References
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2. Walboomers JM, Jacobs MV, Manos MM, et al. Human papillomavirus is a necessary cause of invasive
cervical cancer worldwide. J Pathol 1999; 189:129.
3. Kirschner B, Junge J, Holl K, et al. HPV- genotypes in invasive cervical cancer in Danish women. Acta Obstet
Gynecol Scand 2013 Jun 13. doi: 10.1111/aogs.12197
4. Wheeler CM, Hunt WC, Joste NE, et al. Human papillomavirus genotype distributions: implications for
vaccination and cancer screening in the United States. J Natl Cancer Inst 2009; 101:475487.
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47:864871.
13. Castanon A, Ferryman S, Patnick J, Sasieni P. Review of cytology and histopathology as part of the NHS
Cervical Screening Programme audit of invasive cervical cancers. Cytopathology 2012; 23:1322.
14. Leinonen MK, Nieminen P, Lnnberg S, et al. Detection rates of precancerous and cancerous cervical
lesions within one screening round of primary human papillomavirus DNA testing: prospective
randomised trial in Finland. Br Med J 2012; 345:e7789.
15. Ogilvie GS, Krajden M, van Niekerk DJ, et al. Primary cervical cancer screening with HPV testing compared
with liquid-based cytology: results of round 1 of a randomised controlled trial -- the HPV FOCAL Study. Br J
Cancer 2012; 107:19171924
References
16. Kitchener HC, Almonte M, Thomson C, et al. HPV testing in combination with liquid-based cytology in
primary cervical screening (ARTISTIC): a randomised controlled trial. Lancet Oncol 2009; 10:672682
17. Stoler MH, Wright TC, Sharma A, et al. High-risk human papillomavirus testing in women with ASC-US
cytology: results from the ATHENA HPV study. Am J Clin Pathol 2011; 135:468475.
18. Cuzick J, Cadman L, Mesher D, Austin J, Ashdown-Barr L, Comparing the performance of six human
papillomavirus tests in a screening population. Br J Cancer 2013; 108:908913.
19. Preisler S, Rebolj M, Untermann A, et al. HPV detection in Sure Path samples using Roche Cobas real-time
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20. Schmidt D, Bergeron C, Denton KJ, Ridder R; European CINtec Cytology Study Group. p16/ki-67 dual-stain
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mildly abnormal Papanicolaou cytology study. Cancer Cytopathol 2011; 119:158166.
21. Denton KJ, Bergeron C, Klement P; European CINtec Cytology Study Group. The sensitivity and specificity
of p16(INK4a) cytology vs HPV testing for detecting high-grade cervical disease in the triage of ASC-US and
LSIL pap cytology results. Am J Clin Pathol 2010; 134:1221.
167
Chapter 13
Tensins in health and disease
Maham Akhlaq, Hannah Thorpe, Mohammad Ilyas
Introduction
The organs of multicellular organisms execute specialist functions and usually consist of a
mixture of cells embedded in extracellular matrix (ECM) and arranged in a complex threedimensional structure. During organogenesis, cells need to migrate to the appropriate
topographical location and stay there in order to maintain tissue integrity (although
some further movement may be required during organ turn-over). Following injury, cells
may need to migrate into damaged areas and then undergo remodelling into the normal
structure as part of tissue repair. The processes of organogenesis, organ homeostasis and
repair require modulation of cell adhesion to enable cell motility and maintain cell stasis.
Cell adhesion can be viewed as intercellular adhesion (i.e. adhesions between cells) or
cell-to-matrix adhesion. The former is maintained through intercellular junctions, whilst
the latter is maintained predominantly through focal adhesions. In both cases, these
represent points of contact between cells or between the cell and the ECM. They consist of
multimolecular complexes located at the cell membrane which are linked internally to the
cytoskeleton. In response to environmental cues, these complexes can be assembled or
disassembled thereby allowing the cells to become attached/detached from each other and
the ECM which in turn regulates cell motility. An in-depth description of the intercellular
junctions is beyond the scope of this review, but they are briefly described and shown
figuratively in Table 13.1 and Figure 13.1.
Maham Akhlaq, MBBS, MPhil, Division of Pathology, School of Molecular Medical Sciences, University of
Nottingham, Queens Medical Centre, Nottingham, UK
Hannah Thorpe, BSc, MSc, Division of Pathology, School of Molecular Medical Sciences, University of Nottingham,
Queens Medical Centre, Nottingham, UK
Mohammad Ilyas, BSc, MBChB, PhD, FRCPath Division of Pathology, School of Molecular Medical Sciences,
University of Nottingham, Queens Medical Centre, Nottingham, UK
Email: mohammad.ilyas@nottingham.ac.uk (for correspondence)
170
Table 13.1 Intercellular junctions and adhesions acting to regulate cellular adhesion and motility
Cell junction
Tight junctions
Adherens junctions
Gap junctions
Composed of connexin proteins which form a channel structure between neighbouring cells.
Clusters of channels in specialised cellular regions permits passage of small molecules and
ions between adjacent cells, regulating numerous physiological events
Desmosomes
Tight junctions :
Claudin
Occludin
ZO proteins
Desmosomes :
Desmocollin
Desmoglein
Intermediate filaments
Intermediate
filaments
Actin cytoskeleton
Gap junctions :
Connexins
Adherens junctions :
Cadherins
-catenin
-actinin
Actin filaments
-subunits. There are at least 24 different combinations seen and, depending on the
combination, the integrins act as receptors for a variety of different ECM proteins.
Integrins are transmembrane molecules which are attached to the actin cytoskeleton
[1]. This attachment is mediated by a network of proteins clustered on the cytoplasmic
side of the focal adhesions and includes proteins which have structural and signalling
roles (Figure13.2). These include kinases, phosphatases and adaptor molecules and
demonstrate that, in addition to mediating a physical connection from the cell interior to
Extracellular matrix
FAK
p130 cas
Talin
Paxillin
Tensin
Vinculin
-actin
in
Zyxin
Cell
Actin fibres
exterior, focal adhesions also serve roles as signalling platforms, both of which properties
play a pivotal role in regulating cell adhesion.
The life-cycle of focal adhesions can be viewed as consisting of assembly, maturation
and disassembly. Initial binding of integrin heterodimers with the appropriate extracellular
ligand will activate the integrins. Some integrins, such as 1, occur in a high affinity form,
whilst others, such as 3, may require conformational change before activation (known as
outside-in activation). Either way, activation will result in recruitment of adaptor proteins
and cytoplasmic changes resulting in activation of other integrin receptors (known as
inside-out activation) and clustering of integrin receptors around the site of cellECM
contact [2]. The clustering and inside-out activation seem to be mediated by molecules
such as talin and may be metalion dependent [3]. It results in a positive feedback loop
and the formation of early focal adhesions, also known as nascent adhesions, where the
ECM and the cellular cytoskeleton are first linked [2]. The nascent adhesions may undergo
dissolution or, following Rac-dependent actomyosin contraction, may expand to form focal
complexes. Rapid formation and dissolution of nascent adhesions and focal complexes is
a feature of migratory cells but, in the appropriate cellular context, they may progress to
mature focal adhesions which are characterised by contraction of myosin and activation
and recruitment of molecules such as vinculin [4]. This exerts tension on the focal
adhesions and stabilizes the connection with the actin cytoskeleton. In stable nonmotile
cells, the maturation of the focal adhesions is completed by the formation of large actin
stress fibres and the recruitment of Tensins and, in this state, they are sometimes referred
to as fibrillar adhesions [5]. Disassembly of focal adhesions can be mediated through
ubiquitination and proteasomal degradation of talin or degradation of the ECM thereby
removing the signals for integrin activation.
Cell adhesion is critical for cell motility with constant cell attachment at the leading
edge of the cell and detachment at the posterior edge assisting the cyclic process of
cellular protrusion, attachment and traction. The first stage of cell migration involves the
formation of lamellipodia at the leading edge of the cell which protrudes and attaches to
the substratum via focal adhesions. Contact regions at the rear of the cell detach due to
disassembly of focal adhesions and the bulk of the cell follows to relieve tension giving
171
172
a shift in cell position [6]. Focal adhesion-mediated cell migration is enabled in part by
activation of Ras homology family member A (RhoA) and subsequently Rho-associated
protein kinase (ROCK), leading to phosphorylation of downstream substrates including
Myosin2. RhoA promotes stress fibre formation contributing to actomyosin contractility
mechanisms which together with activation of mDia1 (also via RhoA) promotes the
formation of thick, parallel actin stress fibres extending from focal adhesion regions.
Together in concert with other signalling axes, continuous cycles of stress fibre attachment
and detachment drive cell migratory processes.
Tensin proteins are localised at focal adhesions and are amongst the molecules acting
as molecular bridges between integrins and the actin cytoskeleton. The Tensin family
members play pivotal roles in key cellular processes including adhesion, migration,
proliferation, differentiation and apoptosis [7]. Recent years have seen an accumulating
interest in Tensin biology and this is the subject of the remainder of this review.
263
463
Binds PP1
888
989
1735 aa
SH2
ABD II
Tensin 1
Weakly caps barbed ends Bind DLC1
1410 aa
of F-actin filaments
SH2
ABD I
(N-Terminus)
Protein Kinase C1
PTB
PTB
Tensin 2
(C1ten) Bind DLC1
1445 aa
SH2
ABD I
Tensin 3
Cten
PTB
715 aa
SH2
Bind DLC1
(C-Terminus)
PTB
Bind DLC1
Figure 13.3 A detailed diagrammatic representation of the Tensin family protein structure and its binding partners.
Cten lacks the N-terminal common region, which contains the actin-binding activity, one of the two focal adhesiontargeting regions and the phosphatase-like domain.
Tensin 1 has 60% homology with chick Tensin and contains SH2 and ABD regions
similar to the tumour suppressor phosphatase and Tensin homolog (PTEN) [10]. Tensin 1
has been shown to have actin-binding capabilities through two distinct binding sites in the
ABD at residues 1263 and 263463, although an extra actin-binding site is also present in
the central region at residues 889989. The latter region shares high sequence homology
to insertin (an actin capping protein which retards globular actin polymerisation) and has
therefore been proposed to function similarly. However, later data have contradicted this
suggesting that a barbed end capping mechanism is responsible for actin polymerisation
and depolymerisation through this region [11]. Although PTB domains are known for
binding phosphorylated tyrosines, it is through this domain that Tensin 1 (and the other
Tensins) binds to the NPXY motif on the cytoplasmic tail of -integrins independently
of phosphorylation. The SH2 domain, also located at the C terminus, enables Tensin 1 to
bind phosphorylated tyrosine residues on proteins such as focal adhesion kinase (FAK),
deleted in liver cancer (DLC) 1 and phosphoinositide 3 (PI3) kinase. Tensin 1 also contains
phosphorylated tyrosine residues thereby linking an SH2 containing cytoskeletal protein
with signal transduction to the actin cytoskeleton. At both the N- and C-termini, there
are focal adhesion-binding (FAB) domains which are required for localisation to focal
adhesions.
The structure of Tensins 2 and 3 are similar to that of Tensin 1. Neither contains a central
insertisn region and therefore may not fulfil an actin-capping role as seen in Tensin 1. The
central region of Tensin 2 is found to be proline rich and could potentially act as a binding
site for proteins containing Src homology 3 (SH3) or WW domains [12]. Tensin 2 is the
173
174
only member of the Tensin family that has a protein kinase C domain at the N-terminus.
Tensin 3 contains 32 tyrosine residues, 13 of which are predicted to be potential sites of
phosphorylation and possible candidates for signal transduction [9].
Cten shows a high degree of homology at the C-terminus and is thereby included in the
Tensin family of proteins albeit as a more distant and more recently evolved relative. In
contrast to Tensins 13, it does not possess the N-terminus ABD domains which suggest
that Cten still contains the signalling component of other Tensins but lacks the actinbinding capability and thus may play a novel role in cellular processes [7]. Although focal
adhesion localisation is common amongst all the identified Tensin family members, Cten
has also been detected in the nucleus [13].
As the general structure of the Tensin family of proteins comprises both domains for
binding to cytoskeletal proteins and signal transduction components, they fascinatingly
provide the first link between these two cellular modules. The Tensins have each been
linked to multiple upstream and downstream signalling factors and have also been shown
to differentially interact with such components to aid in mediating their response. Lack of
embryonic lethality in murine knockout models suggests functional redundancy of Tensins,
but the divergent regions also suggest distinct roles.
Focal adhesions
Tensin 1 and Tensin 2
Paxillin and Tallin
Tyrosine phosphorylated
proteins increased
Tensin 2
Tallin
Paxillin
Leading edge
Direction of movement
Focal adhesions:
Actomyosin mediated
contractility
Matrix remodelling
Actin polymerisation
Tensin 2
DLC1
Rno A
ROCK
MLCP
Retraction
Fibrillar adhesions:
Tensin1 and Tensin 3
Tyrosine phosphorylated
proteins decreased
Stress fibers
Actin filaments
Focal adhesions
Fibrillar adhesions
Figure 13.4 In a moving fibroblast Tensin 2 is located towards the leading cell edge causing matrix remodelling,
whilst Tensin 1 and Tensin 3 are present in the cell body in the fibrillar adhesions.
form a complex with DLC1. This results in an inhibition of DLC1 which no longer promotes
Rho-GAP activity, leading to increased RhoA-GTP-mediated signal transduction via ROCK
and subsequent motility [16] (Figure 13.5).
In addition to DLC1, the Tensin proteins can activate and are targets of a variety of other
signalling molecules. FAK has been found in complex with Tensin 1 as well as vinculin,
suggesting that Tensin 1 might mediate signal transduction through this interaction [17].
Conversely, growth hormone stimulation has been shown to cause FAK-mediated tyrosine
phosphorylation of Tensin 1 [18]. Forced expression of Cten in epithelial cell lines has been
shown to cause upregulation and stabilisation of both FAK and integrin-linked kinase (ILK)
together with an associated stimulation of cell motility [19]. Knockdown of FAK and ILK
when Cten has been overexpressed and has resulted in loss of cell motility suggesting that
part of the functional effect of Cten is mediated through these proteins [19,20].
The induction of cell motility by forced expression of Cten is associated with epithelial
mesenchymal transition. This is accompanied by downregulation of E-cadherin and
demonstrates that there is crosstalk between adherens junctions and focal adhesions,
although the signalling pathways involved in this interaction have not been clarified [21].
Obvious candidates would be the ILK and FAK signalling pathways, but, either way, it
demonstrates how activity at the different adhesion junctions can be coordinated to allow
cell adhesion and migration in accordance with cellular requirements.
175
-catenin
ABD
Paxillin
Tensin 3
Tallin
E
G
F
E
G
F
R
-catenin
Tallin
-catenin
SH2 PTB
-catenin
DLC1
EGF stimulation
E-cadherin
E
G
F
Steady state
E-cadherin
176
DLC1
ILK
FAK
Actin filaments
Rho A
GTP
AKT
pathway
Rho A
GTP
ROCK
Cell rest
ROCK
Cell migration
Figure 13.5 Epidermal growth factor stimulation causes a switch from Tensin 3 to Cten and promotes cell
migration by the Rho A-activated Rho-associated protein kinase pathway. Cten also increases motility by
detachment of cellcell E-cadherin junctions.
177
178
Conclusion
Tensins are emerging at the forefront as regulators of cell migration. Their ability to both
bind the actin cytoskeleton and mediate signal transduction events at focal adhesion regions
conveys their importance in regulating cell adhesion and migratory processes. The Tensin
familys role in regulation of cell migration in turn extends to their involvement in cancer
metastasis; however, differential activity in different tissues exemplifies the complexity of
such signalling cascades. Further investigations are warranted to help define the place of
this gene family in the realms of cell migration and tumorigenicity. The targeting of the cell
migratory machinery by using the Tensin family of genes presents an interesting therapeutic
target for anticancer therapies and with further characterisation may provide alternative
targets in areas of need including EGFR inhibitor resistant tumours and possibly overcome
problems associated with toxic anti-Src therapies under development.
References
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8. Chen H, Duncan IC, Bozorgchami H, Lo SH. Tensin1 and a previously undocumented family member,
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9. Cui Y, Liao YC, Lo SH. Epidermal growth factor modulates tyrosine phosphorylation of a novel tensin family
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10. Chen H, Lo SH. Regulation of tensin-promoted cell migration by its focal adhesion binding and Src
homology domain 2. Biochem J. 2003; 370:10391045.
11. Lo SH, Janmey PA, Hartwig JH, Chen LB. Interactions of tensin with actin and identification of its 3 distinct
actin-binding domains. J Cell Biol 1994; 125:10671075.
12. Kay BK, Williamson MP, Sudol M. The importance of being proline: the interaction of proline-rich motifs in
signaling proteins with their cognate domains. FASEB J: Official Publication of the Federation of American
Societies for Experimental Biology 2000; 14:231241.
13. Liao Y-C, Chen N-T, Shih Y-P, Dong Y, Lo SH. Up-regulation of C-terminal tensin-like molecule promotes the
tumorigenicity of colon cancer through beta-Catenin. Cancer Res 2009; 69:45634566.
14. Yam JW, Ko FC, Chan CY, Jin DY, Ng IO. Interaction of deleted in liver cancer 1 with tensin2 in caveolae and
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15. Clark K, Howe JD, Pullar CE, et al. Tensin 2 modulates cell contractility in 3D collagen gels through the
RhoGAP DLC1. J Cell Biochem 2010; 109:808817. s
16. Cao X, Voss C, Zhao B, Kaneko T, Li SS. Differential regulation of the activity of deleted in liver cancer 1
(DLC1) by tensins controls cell migration and transformation. Proc Natl Acad Sci USA 2012; 109:14551460.
17. Yamashita M, Horikoshi S, Asanuma K, et al. Tensin is potentially involved in extracellular matrix
production in mesangial cells. Histochem Cell Biol 2004; 121:245254.
18. Zhu T, Goh EL, Lobie PE. Growth hormone stimulates the tyrosine phosphorylation and association of p125
focal adhesion kinase (FAK) with JAK2. Fak is not required for stat-mediated transcription. J Biol Chem
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19. Albasri A, Al-Ghamdi S, Fadhil W, et al. Cten signals through integrin-linked kinase (ILK) and may promote
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20. Al-Ghamdi S, Cachat J, Albasri A, et al. C-terminal tensin-like gene functions as an oncogene and
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21. Albasri A, Seth R, Jackson D, et al. C-terminal Tensin-like (CTEN) is an oncogene which alters cell motility
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Index
A
Abortion, 24
Acetaminophen overdose, and liver failure, 88, 89
Acinar predominant adenocarcinoma (APA), 33, 33
Acute cellular rejection (ACR), 1 see also Antibodymediated rejection (AMR); Transplantation
Adenocarcinoma, 31, 32
adenocarcinoma in situ, 32
atypical adenomatous hyperplasia, 3132
fetal, 34, 35
in Barretts oesophagus, 73, 77 (see also Barretts
oesophagus)
invasive mucinous, 3435, 35
invasive nonmucinous, 33, 3334
minimally invasive, 32
Adenosquamous carcinoma, 36, 39
Adiponectin, 52
Afatinib, 43
AFES see Amniotic fluid embolism syndrome (AFES)
Amniotic fluid embolism syndrome (AFES), 20, 2021,
21, 22
Amoebic colitis, 120, 120
Amplification refractory mutation system (ARMS),
66, 66
AMR see Antibody-mediated rejection (AMR)
Anaplastic lymphoma kinase (ALK), 4243
Antibody-mediated rejection (AMR), 1
and acute cellular rejection, 1
antibodies in pathogenesis of, role of, 23
capillary endothelial cells in, 2
cardiac, 69, 8
classification of, 3, 4
in liver, 910, 11
pathological diagnosis of, 34
pulmonary, 1013, 12, 13
renal, 46, 5, 7
severity of, 4
ATRA (all-trans retinoic acid), 63
Atypical adenomatous hyperplasia (AAH), 3132
Autofluorescence imaging (AFI), 79
B
Bariatric surgery, 5557, 56
and cholelithiasis, 57
deaths after, 55, 57
laparoscopic gastric banding, 55, 56
Roux-en-Y gastric bypass, 56, 57
vertical sleeve gastrectomy, 55, 56, 57
C
Campylobacter jejuni, 142
Cancer
in Barretts oesophagus, risk of, 7677
gene mutations in, 6163 (see also Gene
mutations, in cancer)
genome screening, 6163, 62
lung (see Lung cancer)
obesity and, 54
Tensin proteins in, role of, 177178
Cancer Research UK Stratified Medicine Programme,
42
Carcinosarcoma, 36
Cardiac allograft vasculopathy (CAV), 4
Cardiac antibody-mediated rejection, 69
capillary endothelial cells and lumen, 7, 8
diffuse capillary deposition, 8, 8
diffuse microvascular inflammation in, 7, 8
grading, 7
ISHLT criteria, 6, 7
Cell adhesion, 169, 170, 170 see also Focal adhesions;
Tensin proteins
Cervical cancer, 159 see also Human papilloma virus
(HPV)
Cetuximab, 63
Chemotherapy, for treatment of non-small-cell lung
carcinoma, 4041
Chronic inflammatory bowel disease (CIBD), 117118
amoebic colitis and, 120, 120
appendiceal neoplasia in, 132
appendix and, 125127, 126
basal plasmacytosis in, 118
colorectal carcinoma in, 129, 130, 130
Index
182
F
Fetal adenocarcinoma, 34, 35
Fibrillar adhesions, 172, 174
Fibrolamellar carcinoma, 97
Flow cytometry, 82
Focal adhesions, 169172, 171 see also Tensin
proteins
fibrillar adhesions, 172
function of, 171172
life-cycle of, 171
nascent adhesions, 172
structure of, 169170, 171
Focally enhanced gastritis (FEG), 125
Focal nodular hyperplasia (FNH), 91, 91
Formalin fixed, paraffin embedded (FFPE) tumour
tissue, 64, 69
Fragment length analysis, 66
D
Dilated cardiomyopathy, 51
Disseminated intravascular coagulation (DIC), 20, 22
Donor-specific antibodies (DSAs), 1
Dumping syndrome, 57
Dysplasia
in Barretts oesophagus, 7879, 7981
in chronic inflammatory bowel disease, 131
Dysplastic nodules, 9294, 93
as HCC precursors, 92
high-grade, 9394
large cell change, 93
low-grade, 93
small cell change, 93
Index
I
Imatinib, 63
Immunofluorescence (IF), 4
Immunohistochemistry (IHC), 4
Immunoproliferative small intestinal disease (IPSID),
140
Infection, obesity and, 5455
Influenza, 27
Integrins, 169170
Intrahepatic cholangiocarcinoma, 98
Invasive mucinous adenocarcinoma (IMA), 3435, 35
Invasive nonmucinous adenocarcinoma (INA), 33,
3334
K
KRAS mutations, in colorectal adenomas, 111
L
Large cell carcinoma (LCC), 3536, 39
Lepidic predominant adenocarcinoma (LPA), 33, 33
Leptin, 52
Luminex assay, 3
Lung cancer, 31 see also Non-small-cell lung
carcinoma (NSCLC)
molecular analysis in, 6768
Lymphocytic oesophagitis (LE), 124, 124, 125
Lynch syndrome (LS), 111112
M
MALT lymphoma, 135137, 136
gastrointestinal, 137140
and reactive lymphoid infiltrates, 140141
and small B cell lymphomas, 141
Maternal deaths, 17
abortion and, 24
amniotic fluid embolism syndrome and, 20,
2021, 21, 22
autopsy in, role of, 17, 19
cardiovascular disease and, 2627
causes of, 1819
classification of, 19
coincidental, 17, 19
definition of, 17
direct, 17, 19, 2026
epidemic influenza and, 27
HIV/AIDS and, 28
hypertensive diseases of pregnancy and, 2123,
23
indirect, 17, 19, 2628
peri- and postpartum haemorrhage and, 2324,
24
sepsis and, 25, 25, 26
thrombotic thrombocytopaenic purpura and, 27
venous thromboembolism and, 26
Mean fluorescence intensity (MFI), 3
Melanoma, 69
Metabolic syndrome, obesity and, 54
Micropapillary predominant adenocarcinoma (MPA),
33, 34
Microsatellite instability (MSI), 103 see also Serrated
pathway lesions
Minimally invasive adenocarcinoma (MIA), 32
Miscarriage, 24
Mucoepidermoid carcinoma, 39
183
Index
184
N
Narrow band imaging (NBI), 79
Neutrophilic capillaritis, 1112
and neutrophilic margination, 12
Next-generation sequencing, 65
Nodular regenerative hyperplasia, 89, 9091
Nonalcoholic steatohepatitis (NASH), 5253
Non-small-cell lung carcinoma (NSCLC), 31
adenocarcinoma, 3135
adenosquamous carcinoma, 36
diagnosis of, problems in, 3839
large cell carcinoma, 3536
sarcomatoid carcinomas, 3637
squamous cell carcinoma, 35
staging of, 37, 37, 38
treatment of, 40, 4043, 43, 43
NSCLC see Non-small-cell lung carcinoma (NSCLC)
O
Obesity, 47
adipose tissue in, 47
bariatric surgery in, 5557, 56
body mass index and, 47, 48
cancer and, 54
degrees of, 47
fatal conditions with, 47
heart, effect on, 5052, 52
infection and, 5455
kidney, effect on, 53, 5354
liver, effect on, 5253
metabolic syndrome and, 54
and mortality rate, 47
respiratory system, effect on, 4850
venous thromboembolic disease and, 5455
WHO classification of, 48, 48
Obesity cardiomyopathy, 5051, 52
Obesity hypoventilation syndrome (OHS), 4950, 50
Obliterative bronchiolitis (OB), 4
Obstructive sleep apnoea (OSA), 4849
Oncotype DX, 69
P
Panel-reactive antibodies (PRAs), 2
Panitumumab, 63
Papillary predominant adenocarcinoma (PPA), 33, 33
P16, detection of, 165
Peri- and postpartum haemorrhage, 18, 2324, 24
creta syndromes and, 2324, 24
genital tract trauma and, 24
placental abruption and, 23
placenta praevia and, 23
retained placenta and, 23
uterine atony and, 23
uterine rupture and, 24
R
Radio frequency ablation (RFA), 82, 83
Ras homology family member A (RhoA), 172
Regenerative nodules, 8892, 8991
acetaminophen overdose and, 88, 89
in chronic biliary disorders, 89, 90
in core needle biopsy specimen, 92
differential diagnosis of, 9192
liver injury and, 88, 89
in nodular regenerative hyperplasia, 89, 9091
Renal antibody-mediated rejection, 46
acute, 4, 56
Banff criteria, 5, 6
chronic, 5, 6, 7
glomerulitis, 5, 5
peritubular capillaritis, 5, 56
transplant glomerulopathy, 6
Revalidation, medical, 149151
appraisal and, 151152
colleague and patient feedback, 154
continuing professional development, 153
documentation from previous appraisals, 153
documentation in, 155, 155156
General Medical Council (GMC) on, 149151
historical perspective on, 149150
impact of, 156
process for medical appraisal for, 152153
quality improvement activities, 153154
recommendation, 156
relicensing and revalidation, 150
Responsible Officer (RO), 151152
scope of work, 153
Index
S
Sanger (dideoxy-) sequencing method, 65, 66
Sarcomatoid carcinomas, 3637
Seattle biopsy protocol, 78
Sentinel sites project, 160
Sepsis, 25
classification and pathology, 25
postpartum, 26
in pregnancy, 25
Serrated pathway lesions, 103, 104, 105
hyperplastic polyps, 104106, 105
immunohistochemistry in, diagnosis of, 113
mixed polyps, 106, 106
molecular abnormalities, 111113
serrated adenocarcinomas, 109, 110
sessile serrated polyps, 106108, 107108
surveillance intervals after, 113, 113
traditional serrated adenomas, 108109, 109
WHO classification, 104
Serrated polyposis syndrome see Hyperplastic
polyposis syndrome (HPS)
Sessile serrated lesions (SSLs), 106108, 107108
Single-strand conformational polymorphism analysis
(SSCP/SSCA), 6466, 65
Sjgrens syndrome, 137
Small cell lung cancer, 40
Small intestinal bacterial overgrowth (SIBO)
syndrome, 57
Solid predominant adenocarcinoma (SPA), 33, 34
Spindle cell carcinoma, 36
Spontaneous abortion, 24
T
Tensin proteins, 172 see also Focal adhesions
in carcinogenesis, role of, 177178
cell adhesion and motility, role in, 174175, 175,
176
cell survival and apoptosis and, 176
members of Tensin gene family, 172, 173
regulation of, 176, 177
structure of, 172174, 173
Thrombotic thrombocytopaenic purpura (TTP), 27
Thymidylate synthetase (TS), 41
TP53, 82
Traditional serrated adenomas (TSAs), 103, 108109,
109
Transplantation, 1, 2 see also Antibody-mediated
rejection (AMR)
Trastuzumab, 63
Trastuzumab emtansine, 63
Tyrosine kinase inhibitors, in clinical use in NSCLC,
4142, 43
U
Ulcerative colitis (UC), 117 see also Chronic
inflammatory bowel disease (CIBD)
Uterine atony, 23
Uterine rupture, 24
V
Vemurafenib, 63, 63
Venous thromboembolism (VTE), 18, 26
obesity and, 55
185