Uses and Limitations of Immunohistochemistry in Breast Pathology
Moderator: Dr. Anna Sapino and Dr. Nour Sneige
Section 5 -
Pathology of Hereditary Breast Cancer: Immunohistochemical Markers
Jocelyne Jacquemier, Emmanuelle Charaffe Jauffret, Laetitia
Huiart, Daniel Birnbaum, François Eisinger, Luc Xerri and Hagay
Hereditary breast cancer is known to account for 5 to 10% of all breast cancer, and approximately, two
thirds of these hereditary forms are thought to be due to BRCA1/BRCA2 genes. In fact BRCA1 (17q21) is
involved in 15 to 45% of all hereditary breast cancer and in over 80% of hereditary breast and ovarian
cancer; whereas BRCA2 (13q 12-13) is thought to be three times less frequently involved. For a BRCA1/2
mutation carrier, the lifetime risk of developing a breast or ovarian cancer ranges from 40 to 85% and 10
to 63%, respectively
Before deciding whether there is an indication for genetic testing, risk
assessments are carried out by a geneticist based on the specific familial and clinical characteristics
of the patients. Risk modelling tools such as Cancer gene can also be used to evaluate the probability
of identifying a mutation in a family
Genetic tests are usually carried out first on the family
member with the highest probability of being a mutation carrier. Certain pathological features can help
to distinguish breast tumors with BRCA1 mutations from those with BRCA2 mutations. Tumors with BRCA1 are
high grade cancers, with high mitotic index, pushing margins and lymphocytic infiltrate, and the
medullary profile, whereas BRCA2 mutations cases are more heterogenous, relatively high grade less
Before microarrays and tissue-arrays technologies immunohistochemistry had suggested a specific
relation with the absence of estrogen receptor and the p53 expression in BRCA1. Recent microarray
studies had identified in these estrogen receptor negative profile a new sub-phenotype close to the BRCA1
profile with absence of ERBB2 expression and a basal cytokeration expression .
Could the new results issued from gene expression profiling help to better predict breast cancer
associated with a germline heritance mutation, or to better understand the role of these BRCA genes in
breast cancer in general?
Immunohistochemical and Molecular Characteristics:
Both BRCA1 and BRCA2 have been suggested to have a role in transcriptional regulation and several
potential BRCA1 target genes have been identified. The nature of these genes suggests that loss of BRCA1
could lead to inappropriate proliferation, consistent with high mitotic grade. BRCA1 and BRCA2 have been
also implicated in DNA repair and regulation of centrosome number. Loss of either of these functions
would be expected to lead chromosomal instability.
Recent progress in immunohistochemistry and molecular biology techniques has enabled in depth
investigation for molecular pathology of these tumors.
Familial Invasive Breast Cancer Related to BRCA1: Immunohistochemistry
ER negativity is characteristic of BRCA1 tumors . From 63 to 90% has been reported in the literature.
It has been suggested that this relationship could be due to the high frequency of Grade III, but the
ER negativity, is four to eight times higher in BRCA1grade III than in Sporadic Grade III. In the
consortium study published by Lakhani et al 90% of BRCA1 related breast cancer were ER negative
 compared to 35% of control cases. These extreme difference was observed among patients less 45
years old. This tendency was confirmed in this study by the high frequency of PR negativity 79% in BRCA1
carriers to 41% in sporadic cases.
P53 and BRCA1 are tumor suppressor genes, and both are involved in many cellular processes ranging
from DNA double–strand repair, to cell arrest, apoptosis, and transcriptional regulation. A direct
functional link between p53 and BRCA1 was initially suggested by the observation that loss of p53 can be
partially rescue embryonic lethality in BRCA1 knockout mice .
Several studies have demonstrated the higher incidence of p53 immunostaining in BRCA1 carcinomas. P53
has been detected in 37-77% of BRCA1 carriers, for 20% in sporadic cases. Comparison with the panel of
p53 mutation BRCA1 reveals significant difference in distribution and base changes. This suggests that
BRCA1/2 mutations influence the type and distribution of p53 mutations in breast cancers. The presence
of these altered amino acids on the non –DNA –binding side of the p53 molecule could represent an
interaction surface for another, unknown protein.
HER-2 expression is very rare in BRCA1 carcinomas, reported frequency is from 0 to 3.7% in considering
the 3+ expression. Few studies has been done in FISH for HER2. One study reported that 19% of the BRCA1
had a low level of HER2 amplification with a ratio between 2 to 3.1, but high level of amplification was
not observed. That was confirmed by the fact that two studies has not observed amplification of BRCA1
carcinomas . However a frequent monosomy of chromosome 17 has been observed from 35-61% in BRCA1
carcinomas. This can represent the second hit of inactivation in a proportion of BRCA1.
It has been suggested that the low incidence of HER2 amplification in BRCA1 carcinomas may be due to a
physical codeletion of one HER2 allele and nearby sequences during the loss of heterozygosity at the
Apoptosis and cell cycle.
Deregulation of apoptosis plays an important role in the pathogenesis and progression of breast cancer,
as well as in the response of tumors therapeutic intervention. If BCL2 is commonly expressed in ER
positive breast sporadic cancer, BCL2 and BAX have a low rate of expression in BRCA1 associated tumor,
by contrast to the high level of Caspase 3. The level of Caspase 3 to appreciate the apoptotic index
is associated with high grade tumors .
Cell-cycle progression is governed by cyclin dependent kinases, that are activated by cyclin binding
and inhibitors (CDKI). CyclinD1 which is known to be up regulated by estrogen, is mostly detected in low
grade ER positive tumors. That explain by contrary the low level of positivity of Cyclin D1 in BRCA1
In an other hand Overexpression of Cyclin E and A has been related to BRCA1 phenotype
In sporadic carcinoma the expression of P-Cadherin and high molecular cytokeratin CK5/6, CK14, and
Ck17, correspond to the basal myoepithelial phenotype.
A basal epithelial phenotype is found in about 15 % of all invasive breast cancer. Microarray have
shown that this phenotype is associated with breast cancer that express neither estrogen receptor, nor
ERBB2  forming the new targeted group of triple negative tumors. Foulkes et
al.  has tested the hypothesis that this phenotype is related to BRCA1 germline mutation.
Out of a series of 292 breast cancer 76 have had this profile. 88% of the 17 BRCA 1 mutated cases the
Cytokeratin 5/6 was positive against 45% for the non BRCA1 cases. In this series seven cases were
Medullary and four of them BRCA1 mutated.
Predictive factors of a tumor associated to BRCA1 mutation
Rapid diagnosis of hereditary breast cancer with germ line mutation is necessary to allow the best
management of these patients for whom molecular based methods are time consumer and costly.
With an original multifactor approach we have investigated by immunohistochemistry 21 markers to
develop a simple procedure for a rapid identification of high risk patients. The 21 markers are divided
into 5 groups: Three commercial anti-Brca1 protein, Three markers associated with the loss of X
chromosome inactivation, 6 markers related to basal, myoepithelial or mesenchymal status, 6 markers
commonly examined in breast cancer and finally 3 markers out of studies on breast common gene expression.
Tissue-microarrays is used to compare 27 confirmed BRCA1 mutation tumours with 81 sporadic breast
cancer ones matched on age.
The results showed a strong correlation between the BRCA1 mutation–associated patients and the loss
of X inactivation (LYS27), confirmed the value of MS110 as a good BRCA1 mutation IHC marker as well as
ER,PR,P53,Bcl2,and Ki167, propose a specific myoepithelio-mesenchymal hypotheses for BRCA1 tumours
oncogenesis, and validate the CDC47 protein by IHC. The confirmation of these results will lead to a
new rapid and economic pre selection method far better than family history for the BRCA1 mutation
associated patients with an optimal management of these patients and their families.
After adjustment on the variable of pairing "age " chosen before beginning the study, the variables
significantly linked to BRCA1 mutation state were ;
Grade 3, MS110, Lys27 and Vimentin with a very important Odds Ratio, even more than 30 in cumulated
Immunohistochemical Profile of Familial Invasive Breast Cancer Related to BRCA2
The frequency of ER and PR expression in BRCA2 tumors has been reported as similar to that in sporadic
breast tumors. This percentage decreases with the increasing age. Foulkes et al  analyzing tumors
less than 45 years found a significant difference with the sporadic cases. The BRCA2 carriers having a
higher frequency of ER positive. But some contradictory results has been obtained and these results need
to be confirm.
The results concerning BRCA2 are less conclusive than for BRCA1. It has been reported than mutation
of p53 was observed in 29 to 63% for only 20% in sporadic controls. However there is no difference in
the location of p53 mutations.
Concerning HER2 for BRCA2 carriers, a lot of variation has been obtained. Expression of HER2 is less
frequent for Palacios (0-3%) confirmed by the absence of amplification, but for Armes et al this
expression is equal to sporadic cases. In contrast to BRCA1, BRCA2 infrequently showed chromosome 17
monosomy, while around 20% were polysomic .
Apoptosis and cell cycle.
With regard to apoptotic markers BCL2 and BAX were both present in BRCA2, confirming the good
association with ER.
Cyclin D1 was expressed from 27% for Osin to 55% for Armes
. To conclude CyclinD1 has been
observed in a higher incidence in BRCA2 than in BRCA1, notion confirmed by the cDNA microarray analysis
. This notion was not contradictory since CyclinD1 is a protein induced by estrogen, and this
association with estrogen receptors has been clearly demonstrated.
Non BRCA1 /BRCA2 familial breast cancer
A Substantial proportion of families with multiple cases of breast cancer is not attributable to these
two genes (non BRCA1/2familes).We have characterized the pathology of 82 breast cancers from non
–BRCA1/2 families. Breast cancers were characterized by a lower grade, fewer mitoses, less nuclear
polymorphism, more tubule formation, less lymphocytic infiltrate, more infiltrating lesion.
Non BRCA1/2 breast cancer differ from BRCA1/2 tumors, from non familial cancer, but this last
differences may be attributable to various type of bias
Major improvement in the efficacity of BRCA1/BRCA2 mutation screening using morphological features of
breast cancer,a family history of breast cancer and or ovarian cancer is the main criterion used in
screening gene carriers. However ascertaining a patient's family history is a difficult task, which
significantly restricts the use of this clinical parameter in clinical practice. In this context it was
established that BRCA1-BCs show a specific morphoclinical pattern. The differences are more evident for
BRCA1and has been more extensively studied. One of the most important applications of this information
would be its use as a guide for genetic testing.
In multivariate analysis the two more discriminant morphoclinical parameters
available for establishing the BRCA1 status, in addition to an early age, are Estrogen receptor
negativity (ER-) poor tumor differenciation (TD3)
. The medullaty phenotype had also a strong factor
of presumption. In order to evaluate the economic impact, we tested the efficacity of these two
parameters as BRCA1 germline mutation indicators in a population based of women who developed a breast
cancer by the age of 35 years, regardless of their family history. A high rate of 28.6% of BRCA1
mutations was found to occur in the group of tumors with both ER-and TD3 versus only 3.6% in tumors with
other profiles (p=0.007)
Adding to these parameters the basal phenotype increased the likelihood to be BRCA1. In this sense
Lakhani suggested that bases on ER, CK5/6, CK14, conduct to better sensibility and positive predictive
value than familial history.
In our hand the association of Grade3, MS110 negativity, Lys 27 negativity and vimentin positivity
conduct to a probability of BRCA1 mutation close to 80%.
A major limitation of the use of the morphological criteria in clinical setting is the lack of
specific features of BRCA2, and non BRCA1/2 carcinomas. Most familial cancers are not associated with
BRCA1or BRCA2 germline mutations, so it is particularly important to best define these familial
In an other hand the identification of BRCA1 can conduct to a best prescription of chemotherapy. The
loss of BRCA1 function is associated with sensitivity to DNA damaging chemotherapy (MytomycinC and
Cisplatin), and may be associated with resistance to spindle poisons (Taxanes and Vinca Alkaloids). The
High percentage of BRCA1 that overexpressed EGFR (67%)
raises the possibility of specific EGFR
- Armes JE, Trute L, White D, Southey MC, Hammet F, Tesoriero A, Hutchins AM, Dite GS, McCredie MR, Giles GG, Hopper JL, Venter DJ. Distinct molecular pathogeneses of early-onset breast cancers in BRCA1 and BRCA2 mutation carriers: a population-based study. Cancer Res. 1999; 59: 2011-2017.
- Osin PP, Lakhani SR. The pathology of familial breast cancer: Immunohistochemistry and molecular analysis. Breast Cancer Res. 1999; 1: 36-40.
- Eisinger F, Bressac B, Castaigne D, Cottu PH, Lansac J, Lefranc JP, Lesur A, Nogues C, Pierret J, Puy-Pernias S, Sobol H, Tardivon A, Tristant H, Villet R. Identification and management of hereditary breast/ovarian cancers (2004 update). Bull Cancer 2004; 91: 219-237.
- Berry DA, Iversen ES, Jr., Gudbjartsson DF, Hiller EH, Garber JE, Peshkin BN, Lerman C, Watson P, Lynch HT, Hilsenbeck SG, Rubinstein WS, Hughes KS, Parmigiani G. BRCAPRO validation, sensitivity of genetic testing of BRCA1/BRCA2, and prevalence of other breast cancer susceptibility genes. J.Clin.Oncol. 2002; 20: 2701-2712.
- Antoniou AC, Pharoah PD, McMullan G, Day NE, Stratton MR, Peto J, Ponder BJ, Easton DF. A comprehensive model for familial breast cancer incorporating BRCA1, BRCA2 and other genes. Br.J.Cancer 2002; 86: 76-83.
- Breast Cancer Linkage Consortium. Pathology of familial breast cancer: differences between breast cancers in carriers of BRCA1 or BRCA2 mutations and sporadic cases. Lancet 1997; 349: 1505-1510.
- Lakhani SR, Jacquemier J, Sloane JP, Gusterson BA, Anderson TJ, van de Vijver MJ, Farid LM, Venter D, Antoniou A, Storfer-Isser A, Smyth E, Steel CM, Haites N, Scott RJ, Goldgar D, Neuhausen S, Daly PA, Ormiston W, McManus R, Scherneck S, Ponder BA, Ford D, Peto J, Stoppa-Lyonnet D, Easton DF, . Multifactorial analysis of differences between sporadic breast cancers and cancers involving BRCA1 and BRCA2 mutations. J.Natl.Cancer Inst. 1998; 90: 1138-1145.
- Lakhani SR. The pathology of familial breast cancer: Morphological aspects. Breast Cancer Res. 1999; 1: 31-35.
- van de Vijver MJ. The pathology of familial breast cancer: The pre-BRCA1/BRCA2 era: historical perspectives. Breast Cancer Res. 1999; 1: 27-30.
- Hedenfalk I, Duggan D, Chen Y, Radmacher M, Bittner M, Simon R, Meltzer P, Gusterson B, Esteller M, Kallioniemi OP, Wilfond B, Borg A, Trent J. Gene-expression profiles in hereditary breast cancer. N.Engl.J.Med. 2001; 344: 539-548.
- Honrado E, Benitez J, Palacios J. Histopathology of B. Crit Rev.Oncol.Hematol. 2006; 59: 27-39.
- Foulkes WD, Stefansson IM, Chappuis PO, Begin LR, Goffin JR, Wong N, Trudel M, Akslen LA. Germline BRCA1 mutations and a basal epithelial phenotype in breast cancer. J.Natl.Cancer Inst. 2003; 95: 1482-1485.
- Lakhani SR, Gusterson BA, Jacquemier J, Sloane JP, Anderson TJ, van de Vijver MJ, Venter D, Freeman A, Antoniou A, McGuffog L, Smyth E, Steel CM, Haites N, Scott RJ, Goldgar D, Neuhausen S, Daly PA, Ormiston W, McManus R, Scherneck S, Ponder BA, Futreal PA, Peto J, Stoppa-Lyonnet D, Bignon YJ, Stratton MR. The pathology of familial breast cancer: histological features of cancers in families not attributable to mutations in BRCA1 or BRCA2. Clin.Cancer Res. 2000; 6: 782-789.
- Lidereau R, Eisinger F, Champeme MH, Nogues C, Bieche I, Birnbaum D, Pallud C, Jacquemier J, Sobol H. Major improvement in the efficacy of BRCA1 mutation screening using morphoclinical features of breast cancer. Cancer Res. 2000; 60: 1206-1210.
- Lakhani SR, Reis-Filho JS, Fulford L, Penault-Llorca F, van d, V, Parry S, Bishop T, Benitez J, Rivas C, Bignon YJ, Chang-Claude J, Hamann U, Cornelisse CJ, Devilee P, Beckmann MW, Nestle-Kramling C, Daly PA, Haites N, Varley J, Lalloo F, Evans G, Maugard C, Meijers-Heijboer H, Klijn JG, Olah E, Gusterson BA, Pilotti S, Radice P, Scherneck S, Sobol H, Jacquemier J, Wagner T, Peto J, Stratton MR, McGuffog L, Easton DF. Prediction of BRCA1 status in patients with breast cancer using estrogen receptor and basal phenotype. Clin.Cancer Res. 2005; 11: 5175-5180.
- van der GP, Bouter A, van der ZR, Menko FH, Buerger H, Verheijen RH, van der WE, van Diest PJ. Re: Germline BRCA1 mutations and a basal epithelial phenotype in breast cancer. J.Natl.Cancer Inst. 2004; 96: 712-713.