CP0201

Anatomía Patológica: receptores

hormonales

Vicente Peg
A. Patológica, H.U. Vall d’Hebron (Barcelona)
Objetivos
• Conocer los principales pasos de la técnica de
inmunohistoquímica.
• Conocer los criterios para la valoración de los receptores
hormonales.
• Conocer los cambios de las últimas guías de valoración de
ASCO/CAP.
Revisión histórica del papel de los r.
hormonales

REVIEWS

K.E. Lukong, BBA Clinical, 2017

Timeline | Milestones in the development of tamoxifenFig.and 4. Cancer and breast cancer pioneers: 400 CE – 1900. a) Hippocrates (engraving by Peter Paul Rubens, 1638https://en.wikipedia.org/wiki/Hippocrates); b) Galen (Source: http://
raloxifene
famousbiologists.org/galen/); c) René Descartes (Source: Hammond, N. (2006). Descartes - The life of Rene Descartes and its place in his times. Tls-Times Lit Suppl, 27–27); d)
Bernardino Ramazzini (source: http://www.britannica.com/biography/Bernardino-Ramazzini); e) Henri François Le Dran (source: https://pictures.royalsociety.org/image-rs-10246); f)
Clomiphene and nafoxidine identified SelectivePeter
1972: Tamoxifen crisis point. Johannes oestrogen-receptor
Müller (source: modulation recognized. 1991: Raloxifene crisis point.
http://www.britannica.com/biography/Johannes-Peter-Muller); g) William Stewart Halsted (source: Rutkow, I.M. (2000). William Stewart Halsted -
as potential antifertility agents. Moments in surgical history. Arch Surg-Chicago 135, 1478–1478.); h) George Thomas Beatson (image from Wellcome Library, London; Photograph by T. & R. Annan & Son; http://
Tamoxifen wellcomeimages.org/indexplus/page/Home.html).
Tamoxifen demonstrated to block the Raloxifene New selective
identified as a human oestrogen receptor, prevent identified as oestrogen-receptor
Discovery of MER25 and potential antifertility mammary cancer and recommended an anti- modulators
clomiphene, the first anti-oestrogens. agent. as a long-termcancers
adjuvant therapy. oestrogen. investigated.
[26]. It is now known from recent epidemiological studies that who argued that surgery was the only method to treat breast cancer.
women who nurse their children are less likely to develop premeno- This practice lasted well into the 20th century and eventually led to
pausal breast cancer and therefore the high incidence in nuns reported the application of radical mastectomy or extensive removal of the
by Ramazzini was likely because they did not have children and not be- breast.
1950 1960 1970 cause of celibacy.
1980 Another absurd postulation 1990 came from Friedrich
Hoffmann of Prussia (1660–1742) and Giovanni Morgagni of Italy 5. 1800–1899
(1682–1771). Hoffmann hypothesized that women who had regular
sex but still developed cancer were practicing “vigorous” sex that caused 5.1. Cancer originates from normal tissue
lymphatic blockage. Morgagni, who was one of the first to perform an
Clomiphene approved for the induction of Tamoxifen approved for the Tamoxifen demonstrated to Development of Tamoxifen approved by the FDA
autopsy and to lay the foundation for scientific oncology, theorized that Major advances in human pathology and safety during surgery as
ovulation, but development as treatment for treatment of advanced breast confer survival advantage in raloxifene as a for the reduction of the risk of
advanced breast cancer discontinued because cancer and the induction of breast cancerpatients.
breast-cancer was caused bytreatment
curdledfor milk. The cause
breast breastofcancer
breast cancer women.
in high-risk well as progress in oncology marked the 19th century. For instance, in
of concerns about potential side effects. ovulation. was also blamed on pus-filledcancer inflammations
discontinued.in the breast (Johanes de 1838, German pathologist Johannes Muller (1801–1858) (Fig. 4) pro-
Gorter, 1689–1762), depressive mental disordersRaloxifene (Claude-Nicolas
approved byLethe FDA posed that cancer cells developed from the blastema between the nor-
Cat, 1700–1768) and childlessness (Lorenz Heister, 1683–1758).for prevention of osteoporosis. mal tissues and not from the lymphatic system, and later Rudolph
Virchow (1821–1902) demonstrated that tumors were composed of
Tamoxifen and raloxifene are the pioneering selective oestrogen-receptor modulators, but both
4.3. Removal went
of the through
tumor a crisis“metastasis”
to prevent point at which decisions were made as tocells
terminate development; however, success occurred when the medicines were essentially re-invented for the focused application. Tamoxifen was transformedwas
whether to
(Reviewed
frompromoted
an
Jordan VC, Nat Rev Drug Discov. 2003
in [19]). It was during this time that hand-washing
and the pasteurization technique was invented as a pre-
antifertility drug to an anti-breast-cancer drug and raloxifene was converted from an anti-breast-cancer drug to a treatment for osteoporosis.
In the mid-18th century, Henri Le Dran (1685–1770) (Fig. 4), a lead- Laboratory milestones are
cautionary measure during surgery. Joseph Lister (1827–1912) intro-
highlighted in blue and clinical milestones are highlighted in green. ing French physician, realized that cancer was not a systemic disease but duced the concept of surgical antisepsis using carbolic acid spray;
a local affliction that progressed in stages. In 1757, he proposed the sur- aseptic techniques were adopted for the first time by the Baltic German
gical removal of the breast tumor before it spread to the lymph nodes of surgeon Ernst von Bergmann (1836–1907); and surgical masks and
the armpits. This resonated with the views of Claude-Nicolas Le Cat, sterile rubber surgical gloves were also introduced [19]. However, a
The link between ovarian function and the develop- by the mid-1960s Clomid (clomiphene citrate mixed
ment of breast cancer has been known for more than a isomers) had become established as a standard therapy
century8,9. The initial clinical observations were sub- for the induction of ovulation19.
sequently complemented by the laboratory finding that Although the scientists at Merrell envisaged many
early ovariectomy can reduce the incidence of mammary clinical applications for anti-oestrogens, such as regula-
10
Biomarcadores en cáncer de mama
VOLUME 34 • NUMBER 10 • APRIL 1, 2016

JOURNAL OF CLINICAL ONCOLOGY A S C O S P E C I A L A R T I C L E

Use of Biomarkers to Guide Decisions on Adjuvant Systemic

Therapy for Women With Early-Stage Invasive Breast Cancer:
American Society of Clinical Oncology Clinical
Practice Guideline
Lyndsay N. Harris, Nofisat Ismaila, Lisa M. McShane, Fabrice Andre, Deborah E. Collyar,
Ana M. Gonzalez-Angulo, Elizabeth H. Hammond, Nicole M. Kuderer, Minetta C. Liu, Robert G. Mennel,
Catherine Van Poznak, Robert C. Bast, and Daniel F. Hayes

Author affiliations appear at the end of this

article. A B S T R A C T
Published online ahead of print at
Purpose
www.jco.org on February 8, 2016.
To provide recommendations on appropriate use of breast tumor biomarker assay results to guide
Clinical Practice Guideline Committee decisions on adjuvant systemic therapy for women with early-stage invasive breast cancer.
“[…] No biomarker except for estrogen receptor, progesterone receptor, and
approval: September 21, 2015.

Editor’s note: This American Society of

Methods
A literature search and prospectively defined study selection sought systematic reviews, meta-

human epidermal growth factor receptor 2 was found to guide choices of

Clinical Oncology clinical practice
guideline provides recommendations analyses, randomized controlled trials, prospective-retrospective studies, and prospective com-
based on the comprehensive review and parative observational studies published from 2006 through 2014. Outcomes of interest included

specific treatment regimens. Treatment decisions should also consider

analyses of the relevant literature for each overall survival and disease-free or recurrence-free survival. Expert panel members used informal
recommendation. Additional information,
which may include a data supplement
consensus to develop evidence-based guideline recommendations.
with additional evidence tables, a Results
disease stage, comorbidities, and patient preferences”.
methodology supplement, slide sets,
clinical tools and resources, and links to
patient information at www.cancer.net, is
The literature search identified 50 relevant studies. One randomized clinical trial and 18 prospective-
retrospective studies were found to have evaluated the clinical utility, as defined by the guideline, of
available at: www.asco.org/guidelines specific biomarkers for guiding decisions on the need for adjuvant systemic therapy. No studies that
and www.asco.org/guidelineswiki. met guideline criteria for clinical utility were found to guide choice of specific treatments or
Authors’ disclosures of potential conflicts regimens.
of interest are found in the article online at
Recommendations
www.jco.org. Author contributions are
found at the end of this article.
In addition to estrogen and progesterone receptors and human epidermal growth factor receptor 2,
the panel found sufficient evidence of clinical utility for the biomarker assays Oncotype DX,
Reprint requests: American Society of
Clinical Oncology, 2318 Mill Road, Suite
EndoPredict, PAM50, Breast Cancer Index, and urokinase plasminogen activator and plasminogen
800, Alexandria, VA 22314; guidelines@ activator inhibitor type 1 in specific subgroups of breast cancer. No biomarker except for estrogen
asco.org receptor, progesterone receptor, and human epidermal growth factor receptor 2 was found to guide
Corresponding author: American Society choices of specific treatment regimens. Treatment decisions should also consider disease stage,
of Clinical Oncology, 2318 Mill Rd, Suite comorbidities, and patient preferences.
800, Alexandria, VA 22314; e-mail:
guidelines@asco.org. J Clin Oncol 34:1134-1150. © 2016 by American Society of Clinical Oncology
 Determinación de los receptores hormonales
Immunocytochemical Analysis of Estrogen
Receptors in Human Breast Carcinomas
Evaluation of 130 Cases and Review of the Literature Regarding
Concordance With Biochemical Assay and Clinical Relevance
D. Craig Allred, MD; Mario A. Bustamante, MD; Craig O. Daniel, MD; Harold V. Gaskill, MD; Anatolio B. Cruz, Jr, MD

\s=b\An estrogen receptor\p=n-\immunocytochemicalassay (ER-ICA)

ER-ICA-Negative-DCCA-Positive
on frozen sections of 130 samples (45% ofTotal) Recently, promising alternate methods for measuring the
performed
was human ER content have become available based on the development
DCCA
Fine-
n 130
=
breast carcinoma. A standard dextran-coated charcoal
(DCCA) wasER
results between
( + ) Benign
performed
Unexplained
Tissue in Specimen]—
on the same samples. Concordance of
the tests was 91%. The sensitivity and specificity
assay
False of monoclonal antibodies that are specific for ER and their use
( + ) DCCA
in immunocytochemical assays (ICAs).7,8 By using JER-ICA-Positivean ER¬
With <50% Negative Cells)
-in = 46)
of the ER-ICA, compared with the DCCA, were 92% and 89%, ICA, pathologists can directly visualize the receptor status of
(All ER-ICA-Positive)
respectively. We describe the ER-ICA technique(55% and review the individual tumor cells by light microscopy on a variety of (n 69)
ER-ICA-Positive-DCCA-Negative
literature regarding the use of the ER-ICA in evaluating breast
Total) histological preparations, including frozen sections, fine-nee¬
=

nce, % respect Cells

cancer with Tumor to theRare in Specimen
agreement of results with the DCCA, dle aspirates, and permanent sections. Studies that have used
the nature ofPremenopausal the ability"Masks" an ER-ICA have shown that almost all ER-positive tumors (n 23)
=

discordant results,(Estrogen
response to hormone
Improper therapy,
Handling Specimen
to predictER)
|_False
the clinical
andofthe ability to predict disease\x=req-\ ( ) DCCA
contain varying proportions of ER-positive and ER-negative
free survival. The combined experience of many studies has
-

cells and varying concentrations of receptor within ER-posi¬

shown that the Unexplained
ER-ICA is a highly specific and sensitive method tive cells. An ER-ICA is a sensitive reproducible method of
for measuring the level of ERs in breast tumors with a high level
Early experience has suggested evaluating this receptor heterogeneity that, in early studies,
Fig 4.—Types DCCA.
of agreement with theand
that the ER-ICA can predict the proportions of discordant
response to hormone and therapy
results is
and
between
emergingthe as a useful new discriminant in recognizing ER- (n 42)
=

dextran-coated charcoal
disease-free survival, as well as assay
II"
or better (DCCA) the
than the DCCA. The estrogen receptor-
positive tumors with a relatively high risk of recurrence. In
immunocytochemical
evaluation assay (ER-ICA)
of receptor heterogeneity, made possiblecalculated from contrast,
by the ER\x=req-\ a the
literature re¬ DCCA provides quantitative information re¬
ICA,view that involved
may enhance our ability
the toDCCA and ER-ICA
discriminate studies
ER-positive tumors of nearlygarding3000the ER concentration in the entire specimen, but it is
total
a relatively high risk of recurrence.
withand 400 discordant frozen sections of breast carcinomas. Possible unable to address the ER heterogeneity of tumor cells within
Negative Low (Arch Surg. 1990;125:107-113)
Intermediate High for discordant results are alsothelisted. specimen. An ER-ICA is also able to evaluate the ER
mechanisms responsible content inPlus small tumors and retrospectively on paraffin blocks
signPositive
cancer ispositive;
indicates the mostminus sign,new negative.
Breast
plasm
ICA Score
ER women
and the second most
common
lethal
malignant
malignant neoplasm
in the United States, with 142 900 new cases and
neo¬
in
of fixed tumors, both ofwhich are not possible by the DCCA.
We report our experience with an ER-ICA on 12 frozen sec¬
tions of 130 samples of breast carcinoma and a comparison of
18 24
43 300 deaths projected forwere
1989.'
discordant byER-ICA-negative/DCCA-positive,
It has been known for these results with the DCCA. In addition, review is Time,
provid¬
Follow-up mo
Fig 3.—Comparison of the estrogen receptor cases
-

almost a (ER)
a
valuesthatobtained
some breast cancers will favorably
andand century ed that summarizes the published experience regarding the
the dextran-coated charcoal assay (DCCA)
respond slightly
to the ER-immunocyto-
hormonal more than half were
manipulation when, ER-ICA-positive/DCCA-
in 1896, Sir George concordance of resultsFig 5. Predictive
between the ER-ICA andofDCCA,
value the estrogen
the receptor-immunocytochemi¬
chemical assay (ICA) performed onBeaston negative.
frozen sections
reported Pari
the and
ofregression Posey19
130 breast recentlybreast
of metastatic looked cancermore closely
ability of intothe ER-ICA
—

predict(ER-ICA)
caltoassay regarding
the response disease-free survival in 111 patients
to hormone

● Resultados similares.
cancer specimens. A value of 5 fmol/mg the was
in patients nature
who of
considered
received a small
to be series
a of
an oophorectomy.2 discordant
Since then, it has results and
test therapy, and the ability with breast were followed for 3 years. The ER-ICA-
of the ER-ICA to predict
cancer who disease-free
positive DCCA test result in this study.
become
foundThe ER-ICA
established
wellthat thatscored
the progression of some breast
all ER-ICA-negative/DCCA-positive
was results(DFS).
survival in¬ positive tumors are stratified into those with greater than 50% negative
according to the method of Reiner et al.'0
cancers is
volvedand partially dependent on the interaction of various
growth factorsDCCA
low-positive that could be cells and
receptors on the explained
hormones values
with specific on MATERIALS ANDthose
METHODSwith less than 50% negative cells (modified from
thecells
basis of the residual
themselves. ER-positive benign
breast Walker et al22).
epithelium
● Permite ver la heterogeneidad.
tumor Specimens
in the
specimens.
Many studies within theDisclosure
apparently "false-posi¬ past 15 yearsofhave
suchshown that
about half of all breast carcinomas One hundred thirty consecutive samples of breast carcinoma ob¬
In addition to
presenting tive"
a comparison of theresults
microscopic results be¬
is due estrogen receptors
to the visualiza¬
andDCCA
that 50% to 60% of these tumors direct
from biopsies or showed stabilization of their disease in response
possess
(ERs) will stabilize or
tained regression mastectomies were acquired or
between Octo¬
tween the ER-ICA and DCCA in 130
tion
regress
ofER-positive speci¬ possible by
frozen-section
when treated with cells
a
made
variety therapies designed
of the to
ber
ER-ICA. 1987 and
In February
antiestrogen therapy. Although
our 1989,
to from women who underwent operations these results involved
● Similar capacidad de predicción deOnly
respuesta arelatively
HT y DFS. patients,
at the University of Texas Medical Center Hospital and the Audie
mens of breast carcinoma, we have reviewed
series, English
the DCCA
the ofaverage estrogenvalueER-ICA-negati¬
of thewithsix its small numbers the 74%
reduce the level circulating or interfere positive predic¬
Murphy Veterans Administration Hospital in San of The
Antonio, Tex.
regarding
literature the level offunction.3"5
ve/DCCA-positive
agreement less thanthe
between
In contrast,
samples 10% of ER-negative
fmol/mg.
was 9 tumors one of these
specimens were received tive value
significantly higher
in the of the ER-ICA
frozen-section room within is
10 min¬ than the 50%
DCCA and ER-ICA measured inthat respond antiestrogen
frozentosections,
samples could therapy. Other studies have shown
permanent
tentatively explained by
be the
utes of excision and immediately divided into three separate portions
of ER- that is
patients with ER-positive tumors enjoy a longer disease- commonly
presence
of tissue to 60% associated
for the diagnostic permanent sections, the DCCA, and the with the DCCA. The ER¬
sections, and fine-needle aspirates
freeofsurvival
breast(DFS)
positive benign cells,
tumors. Table
and longer 1 the
while
overall other
survival than five were discordant
patients ER-ICA. for
compared favorably ICA alsopositive to the 65% and 86%
shows the individual and combinedwith concordance
unresolved
ER-negative of results
tumors.6
reasons. ForThe
analysis by Posey19 Pari and of ER-
negative predictive
these reasons, the evaluation DCCA DCCA values that were derived from the
of the ER status has become a standard in the man¬
 by Breslow [30], was used to evaluate the prognos- tients with ER-IHC negat
tic role of ERDCC and ER-IHC. Significance tests that of ER-IHC positive
Concordancias métodos bioquímicos - IHQ 2.40). On the other hand, D
Table 2. Patient distribution according to ER status associated with ERDCC
panel: X2 = 1.66, p = 0.198)
ERDCC + ERDCC − ard ratio was 1.32, but the
(279 pts) (126 pts)
included the value of one (
ER-IHC + (250 pts) 227 pts 23 pts
possible to conclude that
ER-IHC − (155 pts) 52 pts 103 pts higher risk of relapse th
males.
Molino A et al, Breast Cancer Res Treat, 1997

● Puede determinarse en una gran variedad de muestras y con menos material

(PAAF, bloques celulares, citologías, bloques de parafina…).
● Todo el receptor se analiza, no soloindicate
Please la parteauthor’s
extracelular. Independiente
corrections in blue,desetting errors i
esteroides endo/exógenos y de 125883
tamoxifeno.
BREA ART.NO 805-96 ORD.NO 230263.Z
● Sencillo, no requiere equipamiento especial.
Inmunohistoquímica (1990)

Inmunología + histología + química

Evidenciar la existencia de una proteína
en un tejido, mediante la visualización de
la unión entre antígeno y anticuerpo.
Anticuerpo 1º Enmascaramiento Desenmascaramiento Bloqueo de la
por fijación en formol antigénico peroxidasa endógena.
Antígeno

Tejido
Incubación anticuerpo Revelado
Peroxidasa Incubación con secundario
anticuerpo primario
Anticuerpo2º

DAB
Pasos de la técnica inmunohistoquímica

Preparación del
tejido

Desenmascaramiento

Incubación Ac

Detección
Pasos de la técnica inmunohistoquímica

Preparación del
· Epítopos y morfología deben preservarse. tejido
· Formaldehído: uniones químicas entre las
proteínas del tejido que detiene los procesos
celulares y congela los componentes celulares, Desenmascaramiento
evitando su degradación.
· 6-72h.
· Bloques de parafina, cortes 4-10 µm (máx. 6 sem)
Incubación Ac

· Fijadores alternativos.
· Criocortes: procesamiento más corto, mejor
Detección
preservación epítopos pero peor morfología.
Métodos de fijación alternativos
Moelans et al / Formaldehyde Substitute Fixatives

A B C D

E F G H

I J K L

❚Image 2❚ H&E staining after neutral buffered formalin (NBF) fixation and fixation with 3 alternative fixatives (F-Solv, FineFIX,
Moelans CB et al, Am J Clin Pathol, 2011
and RCL2). A-D, Kidney tissue sample. Note that erythrocytes are absent in C and D (A, ×5; B, ×5; C, ×5; D, ×5). E-H, Paneth
Pasos de la técnica inmunohistoquímica

Preparación del tejido

· Fijación en formol enmascara los epítopos.

Desenmascaramiento

· Tratamiento del tejido con calor, enzimas,

detergentes o combinación de los anteriores.
Incubación Ac

Detección
Pasos de la técnica inmunohistoquímica

· 2 tipos de anticuerpos: Preparación del tejido

- policlonales: diferentes epítopos. Muy potentes, fondo.
- monoclonales: un único epítopo. Menos potentes, difíciles
de interpretar si poco epítopo presente o si son poco
específicos. Desenmascaramiento
· Optimización de la concentración:
- exceso: aumento del riesgo de uniones de baja
especificidad una vez que se ha saturado el epítope.
Incubación Ac
- déficit: según la especificidad puede que las señales sean
imperceptibles y aumente el riesgo de falsos negativos.
· Otros moléculas empleadas: péptidos, fragmentos de Ac,
moléculas pequeñas… Detección
Pasos de la técnica inmunohistoquímica

· Objetivo: representación visual de las proteínas. Preparación del tejido

· Sistema más frecuente de detección: Ac secundario (de
una especie diferente) unido a una molécula.
· Muestras parafinadas: reacciones enzimáticas que
Desenmascaramiento
generan un precipitado cromogénico en el tejido en el sitio
de unión del Ac (HRP, AP, DAB, BCIP/NBT unidos al AC
secundario). La reacción permanece durante largo tiempo.
· Muestras congeladas: Ac secundarios unidos a un Incubación Ac
fluoróforo. Fluorescencia temporal pero permiten usar
varios Ac a la vez.
· Posibilidad de amplificar la señal mediante Ac con varias
moléculas “linker” (ej. Polímeros de biotina). Detección
Patrones de tinción
Discordancias
ECOG 2197: IHC/RT-PCR Concordance

Table 2. Concordance of Central RT-PCR by Oncotype DX and Central and Local IHC for ER Status
Central IHC" Central IHC# Local IHC" Local IHC#
Total Oncotype Total Oncotype
Measure No. % No. % DX No. % No. % DX
Central RT-PCR" 404 99 50 14 454 414 95 45 13 459
Central RT-PCR– 5 1 310 86 315 21 5 296 87 317
Total central IHC 409 360 769 435 341 776
Concordance, % 93 91
95% CI 91% to 95% 89% to 93%
Kappa, % 86 83
95% CI 82% to 89% 79% to 87%
Central IHC" 382 89 27 8 409
Central IHC# 48 11 312 92 360
Total local IHC 430 339 769
Concordance, % 90
95% CI 88% to 92%
Kappa, % 80
95% CI 76% to 85%

Abbreviations: RT-PCR, reverse-transcriptase polymerase chain reaction; IHC, immunohistochemistry; ER, estrogen receptor.

Badve SS et al, JCO, 2008

relapse was censored at the time of death without relapse, new primary cancer
in the opposite breast, or at the time the patient was last evaluated for relapse.
Five-year recurrence rates were estimated from the Cox proportional hazards
regression models, on the basis of the empirical cumulative hazard estimate of
the survival function. Associated 95% CIs were obtained by employing the
normal theory approximation to the logarithm of the survival estimates. Anal-
yses of time to relapse were weighted because inferences were for the full E2197
study population. All statistical tests were two sided, and P ! .05 was consid-
ered significant.
RESULTS
Between 14% (central IHC) and 13% (local IHC) of samples
classified as ER negative by IHC are ER positive by central RT-PCR,
whereas 1% and 5% of RT-PCR–negative cases were ER positive by
local and central IHC, respectively.
The distribution of AS by central IHC compared with central
RT-PCR is shown in Figure 1. Although correlation exists between the
measures, for the 14% of discordant cases where RT-PCR was positive
and the central IHC assay by the AS was negative, the RT-PCR values
were often high and not close to the 6.5 cutoff. For these discordant
cases, RT-PCR measurements range from 6.5 to 10.4 units.
PR concordance. Concordance was high between central IHC and
 support for pathology material collection and imposed no restrictions on the
investigators with respect to trial data. The manuscript was prepared by the median follow-up.
authors, who had full access to the data and who made final decisions on Local Hormone Receptor Assessment
content, while the Steering Committee (including a minority membership of

Discordancias
Novartis employees) reviewed the manuscript and offered changes.
Of entered patients, 98% had ER-positive tumors as determined
locally, and 93% had steroid hormone receptors assessed locally using

Table 2. Numbers of Patients As Classified by Local and Central Table 3. Numbers of Patients As Classified by Local and Central
Assessment of Estrogen Receptor Status Assessment of Progesterone Receptor Status
Central Estrogen Receptor Status Central Progesterone Receptor Status
Local Estrogen Local Progesterone
Receptor Status 0 1%-9% ! 10% Total Receptor Status 0 1%-9% ! 10% Total
Negative 24 8 73 105 Negative 371 308 544 1,223
Positive 66 54 5,980 6,100 Positive 183 247 3,584 4,014
Total 90 62 6,053 6,205 Total 554 555 4,128 5,237

NOTE. Of 6,291 patients, the status of 86 is unknown either by local (n " 3) NOTE. One thousand fifty-four of 6,291 patients’ status unknown by local
or central (n " 83) assessment (not tabulated). (n " 952), central (n " 94), or both (n " 8) assessments not tabulated.

Viale G et al, JCO, 2007

3848 JOURNAL OF CLINICAL ONCOLOGY

● 3650 pcts del estudio BIG 1-98 (letrozol vs tamoxifeno vs combinación).

Downloaded from ascopubs.org by 83.46.235.29 on May 1, 2020 from 083.046.235.029
Copyright © 2020 American Society of Clinical Oncology. All rights reserved.
● 97% tumores RE + (definido como ≥ 10%).
● 69% de los negativos (n=105) fueron positivos.
Guías ASCO/CAP
Special Article

American Society of Clinical Oncology/College of

American Pathologists Guideline Recommendations for
Immunohistochemical Testing of Estrogen and
Progesterone Receptors in Breast Cancer
M. Elizabeth H. Hammond; Daniel F. Hayes; Mitch Dowsett; D. Craig Allred; Karen L. Hagerty; Sunil Badve; Patrick L. Fitzgibbons;
Glenn Francis; Neil S. Goldstein; Malcolm Hayes; David G. Hicks; Susan Lester; Richard Love; Pamela B. Mangu; Lisa McShane;
Keith Miller; C. Kent Osborne; Soonmyung Paik; Jane Perlmutter; Anthony Rhodes; Hironobu Sasano; Jared N. Schwartz;
Fred C. G. Sweep; Sheila Taube; Emina Emilia Torlakovic; Paul Valenstein; Giuseppe Viale; Daniel Visscher; Thomas Wheeler;
R. Bruce Williams; James L. Wittliff; Antonio C. Wolff

N Purpose.—To develop a guideline to improve the

accuracy of immunohistochemical (IHC) estrogen receptor
(ER) and progesterone receptor (PgR) testing in breast
cancer and the utility of these receptors as predictive
markers.
Methods.—The American Society of Clinical Oncology
and the College of American Pathologists convened an
international Expert Panel that conducted a systematic
review and evaluation of the literature in partnership with
Cancer Care Ontario and developed recommendations for
optimal IHC ER/PgR testing performance.
Results.—Up to 20% of current IHC determinations of
ER and PgR testing worldwide may be inaccurate (false
negative or false positive). Most of the issues with testing
variables, thresholds for positivity, and interpretation
criteria.
Recommendations.—The Panel recommends that ER and
PgR status be determined on all invasive breast cancers and
breast cancer recurrences. A testing algorithm that relies
on accurate, reproducible assay performance is proposed.
Elements to reliably reduce assay variation are specified. It
is recommended that ER and PgR assays be considered
positive if there are at least 1% positive tumor nuclei in the
sample on testing in the presence of expected reactivity of
internal (normal epithelial elements) and external controls.
The absence of benefit from endocrine therapy for women
with ER-negative invasive breast cancers has been con-
firmed in large overviews of randomized clinical trials.
 Receptores hormonales ASCO/CAP
Table 6. Reporting Elements for ER and PgR IHC Assays
Patient identification information*
Physician identification*
Date of service*
Specimen site and type*
Specimen identification (case and block number)*
Fixative
Cold ischemia time (time between removal and fixation)
Duration of fixation
Staining method used
Primary antibody and vendor
Assay details and other reagents/vendors
References supporting validation of assay (note: most commonly, these will be published studies performed by others that the testing
laboratory is emulating)
Status of FDA approval
Controls (high protein expression, low-level protein expression, negative protein expression, internal elements or from normal breast tissue
included with sample)
Adequacy of sample for evaluation
Results*
Percentage of invasive tumor cells exhibiting nuclear staining3
Intensity of staining: strong, medium, or weak
Interpretation:
Positive (for ER or PgR receptor protein expression), negative (for ER or PgR protein expression), or uninterpretable
Internal and external controls (positive, negative, or not present)
Standard assay conditions met/not met (including cold ischemic time and fixation parameters)
Optional score and scoring system
Comment: Should explain reason for uninterpretable result and or any other unusual conditions, if applicable; may report on status of
any DCIS staining in the sample; should also provide correlation with histologic type of the tumor; may provide information about
laboratory accreditation status
Abbreviations: ER, estrogen receptor; PgR, progesterone receptor; IHC, immunohistochemistry; FDA, US Food and Drug Administration.
Hammond
* Report should contain these elements as a minimum. Other information must be available MEH et for
in the laboratory al, Arch Pathol
review Lab
and/or Med,on
appear 2010
the patient
accession slip.
3 There is no recommendation in this guideline concerning whether specimens containing only ductal carcinoma in situ should be tested for ER/PgR.

complex tests, which include all predictive cancer factor
assays. This legislation also requires application of
external controls to assure compliance with CLIA stan-
dards. These external controls include required successful
performance on external proficiency surveys (or alterna-
tive external assessment of assay accuracy) and on-site
biennial inspection of laboratories performing highly
complex tests with defined criteria and actions required
when performance is deemed deficient. On-site inspec-
testing reagents and kits, which have potentially high
impact on patient mortality and morbidity, have been the
subject of several guidance documents and reports
referencing FDA opinion on the subject.34
After review of the legislation and applicable regula-
tions, the Panel agreed that the current regulatory
framework provided sufficient justification for the guide-
line recommendations without modification, just as it had
for the previously published ASCO/CAP HER2 guide-
 ing in terms (scored on a scale of 0-5) and staining intensity (scored on a
ing become scale of 0-3). The proportion and intensity were then summed

Valoración semi-cuantitativa
classified as
ing system
to produce total scores of 0 or 2 through 8. A score of 0 -2 was
regarded as negative while 3 - 8 as positive (Figure-1).3,4 Idea
slides were conceived from original paper.4
ositive and
ggests that
positive is at
that if pre
equivocally

r cases over
orted in the
Figure-1: Diagramatic representation of Interpreation of AllredAllred
Score.DC et al, Arch Surg, 1990
ospital were

Results
These 860 cases studied for ER immuno-stains
xed paraffin
included core needle biopsies, lumpectomies, mastectomies
ine clinical and wide local excision biopsy specimens. Of these, 767
(clone D07, (89%) cases were infiltrating ductal carcinomas, 60Kinsel
(7%)LBwere
. A positive et al, Cancer Res, 1989
infiltrating lobular carcinomas and 33 (4%) were minor
er known to variants of breast cancer.
atch. Inbuilt
! The frequency distribution of ER
ER staining
immunohistochemical results based on estimated percentage
trol primary
of tumour cells by conventional methods showed 457 (53%)
es where no
to be completely negative, 251 (29%) were intermediate to
Guías ASCO/CAP 2020
 Guías ASCO/CAP 2020 ER/PgR Testing in Breast Cancer Update

TABLE 1. Summary of All Recommendations (continued)

2010 Recommendation Updated Recommendation
Use of SOPs, including routine use of external control materials with SOPSs should be used that include routine use of external control materials
each batch of testing and routine evaluation of internal normal with each batch of testing and routine evaluation of internal normal
epithelial elements or the inclusion of normal breast sections on each epithelial elements or the inclusion of normal breast sections (or other
tested slide, wherever possible. appropriate control) on each tested slide, wherever possible. External
controls should include negative and positive samples as well as samples
with lower percentages of ER expression (such as tonsil). On-slide
controls are recommended.
Regular, ongoing assay reassessment should be done at least Regular, ongoing assay reassessment should be done at least semiannually
semiannually (as described by Fitzgibbons et al12 and more recently (as described in Fitzgibbons et al12). Revalidation is needed whenever
Torlakovic ); revalidation is needed whenever there is a significant
13
there is a significant change to the test system.13.
change to the test system.
Ongoing competency assessment and education of pathologists. Ongoing competency assessment and education of pathologists is required.
Optimal external proficiency assessment Optimal external proficiency assessment
Mandatory participation in external proficiency testing program with at The laboratory performing ER and PgR testing must participate in external
least two testing events (mailings) per year. proficiency testing or alternative performance assessment as required by
its accrediting organization.
Satisfactory performance requires at least 90% correct responses on
graded challenges for either test.
Optimal laboratory accreditation Optimal laboratory accreditation
On-site inspection every other year with annual requirement for self- On-site inspection every other year should be undertaken with annual
inspection. requirement for self-inspection.
Clinical Question 2. What additional strategies can promote optimal performance, interpretation, and reporting of IHC assays, particularly in cases with low
ER expression?
No specific recommendations were specified in 2010 for low ER Laboratories should include ongoing quality control using SOPs for test
expression cases. evaluation prior to scoring (readout) and interpretation of any case, as
defined in the checklist in Figure 1. Allison KH et al, JCO, 2020
Interpretation of any ER result should include evaluation of the concordance
with the histologic findings of each case. Clinicians should also be aware
 its accrediting organization.
Satisfactory performance requires at least 90% correct responses on
graded challenges for either test.
Optimal laboratory accreditation Optimal laboratory accreditation

Guías ASCO/CAP 2020

On-site inspection every other year with annual requirement for self-
inspection.
On-site inspection every other year should be undertaken with annual
requirement for self-inspection.
Clinical Question 2. What additional strategies can promote optimal performance, interpretation, and reporting of IHC assays, particularly in cases with low
ER expression?
No specific recommendations were specified in 2010 for low ER Laboratories should include ongoing quality control using SOPs for test
expression cases. evaluation prior to scoring (readout) and interpretation of any case, as
defined in the checklist in Figure 1.
Interpretation of any ER result should include evaluation of the concordance
with the histologic findings of each case. Clinicians should also be aware
of when results are highly unusual/discordant and work with pathologists
to attempt to resolve or explain atypical reported findings (Table 3 is an
aid in this process).
Laboratories should establish and follow an SOP stating the steps the
laboratory takes to confirm or adjudicate ER results for cases with weak
stain intensity or # 10% of cells staining (Data Supplement 2, Figure 1
provides an example SOP).
The status of internal controls should be reported for cases with 0%-10%
staining. For cases with these results without internal controls present
and with positive external controls, an additional report comment is
recommended (Table 2).
Clinical Question 3. Are other ER expression assays acceptable for identifying patients likely to benefit from endocrine therapy?
No assays other than IHC are recommended as testing platforms. Validated IHC is the recommended standard test for predicting benefit from
endocrine therapy. No other assay types are recommended as the
primary screening test for this purpose.
Clinical Question 4. Should DCIS be routinely tested for hormone receptors?
ER and PgR testing of DCIS is optional (no formal recommendation made ER testing in cases of newly diagnosed DCIS (without associated invasion) is
to test or not test). recommended to determine potential benefit of endocrine therapies to
reduce risk of future breast cancer; PgR testing is considered optional.

Abbreviations: ASCO, American Society of Clinical Oncology; CAP, College of American Pathologists; DCIS, ductal carcinoma in situ; ER, estrogen receptor;
HER2, human epidermal growth factor receptor 2; IHC, immunohistochemistry; NBF, neutral buffered formalin; PgR, progesterone receptor; QA, quality
assurance SOP, standard operating procedure. Allison KH et al, JCO, 2020

determine hormone receptor status, including (but not Articles were excluded from the systematic review if they
necessarily limited to): specific assay performance, were (1) meeting abstracts not subsequently published in
 cases, the selected course of action should be considered cell nuclei are immunoreactive. A sample may be deemed
by the treating provider in the context of treating the in- uninterpretable for ER or PgR if the sample is inadequate
dividual patient. Use of the information is voluntary. ASCO (insufficient cancer or severe artifacts present, as de-

RE débilmente positivos (1-10%)

provides this information on an “as is” basis, and makes no termined at the discretion of the pathologist), if external and

TABLE 2. Additional Recommended Reporting Comments for Specific Scenarios

Result
1%-10% cells staining
No internal controls and ER is 0%-10%
Abbreviations: ER, estrogen receptor; IHC, immunohistochemistry.
Additional Recommended Comment
The cancer in this sample has a low level (1%-10%) of ER expression by IHC. There are limited
data on the overall benefit of endocrine therapies for patients with low level (1%-10%) ER
expression, but they currently suggest possible benefit, so patients are considered eligible for
endocrine treatment. There are data that suggest invasive cancers with these results are
heterogeneous in both behavior and biology and often have gene expression profiles more
similar to ER-negative cancers.
No internal controls are present, but external controls are appropriately positive. If needed,
testing another specimen that contains internal controls may be warranted for confirmation of
ER status.
Allison KH et al, JCO, 2020

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 Guías ASCO/CAP 2020 ER/PgR Testing in Breast Cancer Update

TABLE 3. Invasive Breast Cancer Histopathologic Concordance With ER Staining

Highly Unusual ER-Negative Results Highly Unusual ER-Positive Results
Low-grade invasive carcinomas of no special type (also known as invasive Metaplastic carcinomas of all subtypes
ductal carcinoma)
Lobular carcinomas (classic type) Adenoid cystic carcinomas and other salivary gland–like carcinomas of
the breast
Pure tubular, cribriform, or mucinous carcinomas Secretory carcinoma
Encapsulated papillary and solid papillary carcinomas Carcinomas with apocrine differentiation

NOTE. If a result is considered highly unusual/discordant, additional steps should be taken to check the accuracy of the histologic type or grade as well as
the preanalytic and analytic testing factors. This workup may include second reviews and repeat testing. If all results appear valid, the result can be reported
with a comment noting that the findings are highly unusual and testing of additional samples may be of value to confirm the findings.
Abbreviation: ER, estrogen receptor.

consensus; Evidence quality: High; Strength of rec-
ommendation: Strong).
Recommendation 2.3
Laboratories should establish and follow an SOP stating the
steps the laboratory takes to confirm or adjudicate ER
results for cases with weak stain intensity or # 10% of cells
staining; Data Supplement 2, Figure 1 provides an example
SOP. (Type: Informal consensus; Evidence quality: High;
Strength of recommendation: Strong).
Recommendation 2.4
The status of internal controls should be reported for cases
with 0% to 10% staining. For cases with these results
negative results such as negative or absent internal con-
trols, evaluation of controls is considered an essential part
of this process.118 If internal controls are negative, or there
Allison KH et al, JCO, 2020
are no internal controls and the external positive controls do
not have appropriate staining, the assay has failed and
needs to be troubleshot. In addition, correlation with any
prior patient-specific ER results on a breast cancer would
be considered relevant. There are data to support that
second reviews and digital quantitative image analysis
reads can be used to improve reproducibility and accuracy
in a pathologist’s scoring (readout) and interpretation, so
these can be useful components of an SOP for these cases;
however, the Expert Panel acknowledges that current data
on these topics are not specific enough to distinguish ER
Valor diagnóstico (HDU vs CDIS bajo grado)
Conclusiones
● RE estudiados desde los años 70´s (IHQ desde los
90´s).
● Biomarcador recomendado (ca infiltrante y CDIS).
● Positividad si ≥ 1%.
● Nueva categoría “ER low positive” si 1-10%.