HOW TO CLASSIFY EPITHELIAL

NEUROENDOCRINE NEOPLASIA

PRESENTED BY: MODERATOR:

Dr. Neelesh Nagayach Dr. Upasana Uniya
Dr. Keshav Rajak
 Recent Advances in Histopathology 25th

Guido Rindi
Chapter 8: How to classify epithelial
neuroendocrine neoplasia. MD PhD, Anatomic Pathology Unit, Univ. Of
Cattolica del Sacro, Roma, Italy.

Frediano Inzani

MD PhD, Anatomic Pathology Unit, Fondazione

Policlinico University, Roma, Italy

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 ?Difficulties
?Evolution of the classification
?Nuroendocrine tumour…grade
?Nuroendocrine Carcinoma
?Spectrum of neuroendocrine tumour
?Carcinoid Tumour
?Carcinoid Syndrome

?If you don’t suspect it, you cant detect it.

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 NEUROENDOCRINE
About this
NEOPLASIA:
template DEFINITION

Neuroendocrine neoplasia (NEN) is a tumour category defined by-

 Characteristic histological and ultrastructural features.
 Diffuse and consistent expression in the neoplastic cells of markers of neuroendocrine (NE)
differentiation.
 IHC expression of neuroendocrine markers reflects the presence of components of cellular
organelles.
 These organelles are responsible for biosynthesis and storage of neuroendocrine components.
 Associated with the two regulated pathways of secretion that are normally observed in neurons
and neuroendocrine epithelial cells.
 Ultrastructurally recognisable organelles representing these secretory pathways are-
1.Large dense-core vesicles (LDCV) and
2.Small synaptic-like vesicles (SSV).

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 LDCV: Store, process and release peptide hormones and monoamines
(e-g. secretin, serotonin)
 SSV: small mediators such as synaptic vesicles (gamma-aminobutyric
acid (GABA), taurine, choline)
 The presence of these vesicles suggest the epithelial as well as neural
lineage derivation.
 Markers associated with LDCV and SSV are proteins associated with
these structures and include the chromogranin family peptides (A, B)
within LDCV, and synaptophysin in SSV.
 These popular NE markers are widely used in pathology practice with
well-defined efficacy and limitations.

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 Chromogranin A (CgA) is probably the most specific marker being reported only
in NE cells and neurons.
 ***However, as for other proteins, largely depends on the amount of antigenic protein
present within a given neoplastic cell.
 This implies that while a well-differentiated NE tumour cell has well developed
LDCVs and consequently large amounts of CgA antigen.
 Poorly differentiated NE tumour cells have few and poorly developed LDCVs
and consequently little or no CgA antigen, which can be difficult to visualize in
tissue sections.
 On the contrary, synaptophysin while extremely sensitive and less specific,
being present in non-NE neoplasia of the adrenal cortex, pancreas and lung

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 You can
Transcription
also split your
Factor
content

Transcription factors: upstream proteins that control gene expression.

◉ Recently entered the world of diagnostic pathology and proved extremely
useful in determining either cancer tissue origin or even cancer type.
◉ A typical example is thyroid transcription factor 1 (TTF-1), which has a high
degree of tissue specificity for thyroid but also for lung, where it is
expressed in adenocarcinoma and neuroendocrine neoplasm.
◉ Along this line, the transcription factor insulinoma-associated protein 1
(INSM1) was recently found to be expressed in NEN with a high degree of
specificity and sensitivity.
◉ Becoming a successful second-generation NE marker adopted in clinical
practice.
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 Limitations
In twooforTranscription
three columnsFactors

◉ As TTF-1 is expressed
in poorly differentiated
NEN of multiple sites.
◉ INSM-1 in sarcomas
and in rare non
endocrine thoracic
carcinomas.
Other IHC markers has been used in the past
for definition of NE differentiation.
1.CD56 (neural cell adhesion molecule,
NCAM) which is still often used as a general
NE marker but lacking in specificity.
2.Protein gene product 9.5 (PGP9.5) and
neuron specific enolase (NSE), these are
now obsolete.

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 CYTOLOGICAL AND HISTOLOGICAL FEATURES

◉ The cyto-histology of NEN depends on the degree of

tumour cell differentiation.
1}Well differentiated neuroendocrine tumour
cells are-
-Medium in size.
-Round to polygonal (sometimes spindle shaped)
-Abundant eosinophilic or amphophilic cytoplasm
-Monomorphic round to oval nuclei with dispersed ‘salt &
pepper chromatin’
-Absence of evident or prominent nucleoli
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◉ Cell monomorphism: most of the cells look same, this is the most
relevant cyto-/histological feature.
◉ Although low to moderate atypia may be observed.
◉ In these cases, the cells tend to be more irregular with abnormal shape
and increased nuclear to cytoplasm ratio.
◉ Mitoses are usually low.
◉ Tumour necrosis is absent.
◉ If detectable/evident mitotic activity and/or focal, spotty necrosis: They
are associate with 1.relative loss of monomorphism 2.higher histological
grade, and 3.clinical malignancy.
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 THE HISTOLOGICAL STRUCTURES OBSERVED IN-

1.Well-differentiated NEN: are variable and includes-

solid islet, trabecular, ribbon and glandular patterns-most often with an admixture of all
types.
 These histological patterns are overall defined as 'organoid,

 The stroma is usually delicate with abundant vessels. The growth is expansive, or
irregularly expansive, with some degree of infiltration in neighboring normal tissue.

 Tumours with increased stromal density and evident aggression of adjacent non-
neoplastic tissue tend to have higher histological grade and associate with clinical
malignancy.

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Specific sites-

 In the digestive tract the glandular structure is usually reported in the duodenum and
the solid islet structure in the ileum.

 The structure itself cannot be adopted as a reliable and unique criterion tor site assignment.

So These histological patterns are observed in NETs in all systems and, as such,
represent a morphological landmark for well-differentiated NEN.

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2.Poorly differentiated carcinoma

Cells display severely atypical features, with high nucleus/cytoplasm ratio evident ,often atypical
mitoses and frequent tumour-necrosis

They are characterised by large nuclei with a variable amount Of

cytoplasm.
in most instances,
the cytoplasm is a subtle rim and then cells are defined as small.
when the cytoplasm is abundant, this feature makes it a large cell.
nuclei are characterised by dispersed, salt and pepper chromatin.

in small cells nucleoli are usually faint, if discernible at all, while evident and prominent in large
cells.
Small cells are usually frail, nuclear crushing and moulding being the rule in cytological and small
biopsy .

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large cells relatively have abundant cytoplasm.
a mixture of small and large cells can also be observed.

The predominent poorly differentiated cell type (small or large) determines whether to
diagnose a case as either small- or large-cell neuroendocrine carcinoma .

Poorly differentiated carcinoma usually shows a solid structure with abundant tumour
nacrosis with a highly irregular perimeter for which the term "geographical chart" necrosis
is used.

the stroma is usually thick and poor in vessels.

these features are observed in neuroendocrine carcinoma at all different anatomical

sites
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The universal classification principles

 The world health organization- international agency for the research on cancer (WHO-IARC)
proposed a common classification frame for the NEN at all anatomical sites, that is now officially
endorsed.
 The system defined the cancer category, the family/class, the types, the eventual subtypes, and the
grade.

 The cancer category was defined as NEN, which signifies a neoplastic growth in which tumour cells
express markers of NE differentiation.

 The NE families/classes comprise the well-differentiated NET and the poorly differentiated NEC.

 The NEN type is based on grade for NET and as such is site-specific.

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 Subtypes are recognized only for NET.

 The separation of NEN in two groups relies on morphology.

 Irrespective of the site of origin, well-differentiated NETs are characterised by a very low
mutational burden and notably by the absence of abnormality of common cancer driver genes.

 Along the same line, poorly differentiated NECs display mutations of common cancer driver
genes invariably including P-53 and RB.

 Notably, gene expression profiling correlates with histological grade, clinical malignancy, &
progression from a high-grade well-differentiated to a poorly differentiated neoplasm.

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 GRADING
About this&template
STAGING

 The classification tools on the basis of the current site-specific classifications are grading and
staging.
 The grading system of NEN is based on proliferation assessment by-
1. Mitotic count per 2mm²
2. Ki67 (MIB-1 clone) percentage
3. Presence of necrosis

-Assessment of proliferation is done on areas of the highest frequency for mitoses

and in areas of the highest nuclear labeling ( hot spots) for ki67.

-Though the presence of necrosis is not quantitatively defined.

In some classifications it is described as
- spotty, when only focally present
- diffuse when extensive.
While proliferation assessment is consistently used to define grade in all classifications, necrosis is
not used for grading of digestive system NEN.
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The staging systems are organ specific.
Well differentiated NEN:
 In Non-endocrine organs-NET-specific staging systems have been devised
for digestive system only.
At all other sites, staging system devised for non-neuroendocrine cancers are
applied.

For Endocrine organs- There is no staging for NEN in the pitutiary, while it
is defined in the thyroid for medullary carcinoma and in the adrenals for
adrenal medulla.
Poorly differentiated NEN: At all anatomical sites follows that devised for
non-neuroendocrine cancer.

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 CURRENT SITE
About
SPECIFIC
this template
CLASSIFICATIONS

THREE-TIERED CLASSIFICATIONS:
1.Head and neck organs-
In the upper airways and the salivary glands, the current classifications define
NET MITOSES PER 2mm² Ki67 Index %
 Well differentiated NEN as NET G1-G3.
-NET G1-G2 display well-differentiated morphology G1 <2 <20%
G2 2-10 <20%
-The presence of necrosis upgrades to NET G2,
G3 >10 >20%
independently from proliferative activity.

NEC MITOSES PER 2mm² Ki67 Index %

 Poorly differentiated NENs are defined as NEC. Small >10 >70%

Cell
Large >10 >50%
Cell

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2.Thorax organs:
 The classification system for the thoracic organs (lung and thymus) defines NET as
"carcinoid’’.
 Typical and atypical based on- morphology, proliferation features and presence of necrosis.
 Mitotic count and necrosis are the main diagnostic criteria.

Carcinoid Mitoses per 2mm² Ki67 Index % Necrosis Equivalent to

Typical <2 <5% No evidence NET G1

Atypical 2-10 <30% Evident NET G2
(Spotty)

 The term "carcinoid tumour NOS" is adopted for small biopsies, in metastatic setting or in
consultation when only representative slides of the surgical specimen are available.

 The "carcinoid tumour NOS" equalises a generic definition of NET.

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 NECs are defined as small cell lung cancer (SCLC) when of small cell type and LCNEC, when
of large cell type.

NEC Cell type Cytomorphology Mitoses per Ki67 index % Necrosis Equivalent to
2mm²
SCLC Small Poorly >10 30-100 Abundant NEC small
Differentiated cell type
LCNEC Large Poorly >10 30-100 Abundant NEC large
Differentiated cell type

 Cases with well-differentiated morphology but with proliferative activity above the AC threshold
and often with necrosis are provisionally classified as "carcinoid tumours/NET with elevated
mitotic counts and/or Ki67 proliferation index.

 As such, these cases equalize NET G3.

 Currently, they are classified as LCNEC with an explanatory note, stating the presence of
carcinoid tumour morphology along with a detailed mitotic count and Ki67 index.

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3.digestive organs:
 In the digestive system (Gastroenteric and Pancreatobiliary tract) the classification defines
 Well-differentiated NEN as NET and
 Poorly differentiated NEN as NEC.
 All NETs display well-differentiated morphology.

NET MITOSES PER 2mm² Ki67 Index % NEC MITOSES PER 2mm² Ki67 Index %

G1 <2 <03% Small >20 >20%

Cell
G2 2-20 <3-20%
Large >20 >20%
G3 >20 >20% Cell

 Whenever feasible, mitotic count is done on 10 mm² areas and the number of mitoses per 2mm² is
defined.
 Ki67% is established on a minimum of 500 counted cells.

 Necrosis is not a classification item.

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 TWO-TIERED CLASSIFICATIONS

◉ The only current two-tiered classification system devised for Medullary Thyroid
Carcinoma (MTC).
◉ A well-differentiated NET of the thyroid composed of calcitonin producing parafollicular ©
cells.
◉ This classification divides MTC in low-grade and high-grade based on proliferation
features and presence of necrosis.
◉ Proliferation is assessed by mitotic count per 2 mm² and ki67 proliferative index.

MTC Mitoses per Ki67 index % Necrosis

2mm²
Low-grade <5 <5% Absent

High-grade >5 >5% Present

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 You Diagnostic
can also split
Challenges
your content

 Challenges includes the correct definition of grading in cytology and biopsy material.
 Cytology and biopsy samples are per se difficult to interpret due to the limits imposed by the
paucity of material and its quality.
 The distinction between high-grade NET and NEC.
 Morphology of NET G3 is sometimes difficult to interpret and to distinguish from NEC.
 The absence of features of high proliferative activity does allow the distinction between NET and
NEC, while specific grading is usually not possible.
 Correctly diagnosing metastases and clearing confusion with mimickers.
 In case of metastatic NEN, the origin may be difficult/impossible to define.
 Morphological features may provide clues, however, only the expression of a site-specific
transcription factor and/or the cytokeratin expression profile may provide evidence for specific
site definition.

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 In two orCont...
three columns

 Several other lesions, non-neoplastic as well as neoplastic, epithelial as well as non-epithelial

cancer, may mimic NEN morphology.
 Mimickers do vary according to site.

Well differentiated forms Poorly differentiated forms

Pancrea Solid pseudo-papillary neoplasm Poorly differentiated SCC

s Acinar cell carcinoma
adenocarcinoma
GIT Granular cell tumour
Epithelioid GIST lymphoma
Thymus Thymoma
melanoma

Lung, Plasma cell neoplasia germ cell tumours

Prostate Adenocarcinoma
Ewing’s sarcoma

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 KEY POINTS

 Suspect NEN, suspicion is based on either cytological and/or histological features.

 Cytological preparation:
 1.Well-differentiated forms: clean smears, low atypia, cell monomorphism, abundant cytoplasm, and
round nuclei.
 Attention should be paid to the periphery where nuclei may be crushed as a needle or smearing
effect, potentially suggesting nuclear fragility and poor cell differentiation.
 2.Poorly differentiated forms: dirty smears, cell debris, nuclear crushing, and moulding at all smear
sites, scarce cytoplasm, evident atypia and mitoses.
 Histological preparation:
 1.Well-differentiated forms: organoid growth, absence of necrosis, cell monomorphism, low or no
atypia.
 2.Poorly differentiated forms: solid structure, abundant necrosis, severe cytological atypia, frequent
mitoses, organoid structures often at periphery with large ribbons/sheets.

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 And tablesCont…
to compare data

 Consider mimickers. Many different cancer types are found at different sites as potential
mimickers. Mimickers display a non-NE immunophenotype.

 Establish the diagnosis. Use IHC for the NEN epithelial versus neural lineage definition.
 For epithelial (CK and/or Vimentin) and
 For neural, choose at least two NE markers, usually Chromogranin A and Synaptophysin.

 Other useful markers are:

 INSM1 & Somatostatin receptor type 2 or 5 (all sites, all differentiation and grades)

 CD56 (thorax)
 SATB2 and TTF1 (both well and poorly differentiated forms of thoracic tumours)

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 Cont...

 Specific site transcription factors like CDX2 in the digestive tract and pancreas (both well and poorly
differentiated forms)

 lineage-specific, e.g. pituitary-specific PIT1, GATA3 for neuroepithelial NEN paraganglioma and
parathyroid.

 Define differentiation. Define the differentiation state, and the NEN type whether NET or NEC. For NEC
define whether large or cell type.

 Define grade. For well-differentiated forms, define the grade using the organ-specific classifications and
determine the mitotic index per 2 mm², the ki67% and, when required the presence of necrosis.

 Define NEN. Adopt organ-specific terminology.

 Define stage. When available, adopt organ-specifc, NET staging (digestive system); otherwise staging for
non-neuroendocrine cancer. consider that NECs are always staged as non-neuroendocrine cancer.
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Thanks!

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