Section 5 -

Pathology of Hereditary Breast Cancer: Immunohistochemical Markers Jocelyne Jacquemier, Emmanuelle Charaffe Jauffret, Laetitia Huiart, Daniel Birnbaum, François Eisinger, Luc Xerri and Hagay Sobol Marseille, France

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 [1, 2, 3]. 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 [4, 5]. 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 tubule formation [6, 7, 8, 9]. 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 [10]. 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 Hormone receptors: 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 [7] 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 : 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 [11]. 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. HER2. 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 [11]. 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 BRCA1 locus. 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 [11]. 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 carcinomas. In an other hand Overexpression of Cyclin E and A has been related to BRCA1 phenotype Basal 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 [10] forming the new targeted group of triple negative tumors. Foulkes et al. [12] 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 rate. Immunohistochemical Profile of Familial Invasive Breast Cancer Related to BRCA2 Hormone receptors. 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 [12] 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. P53. 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. HER2: 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 [11]. 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 [1, 2] . To conclude CyclinD1 has been observed in a higher incidence in BRCA2 than in BRCA1, notion confirmed by the cDNA microarray analysis [10]. 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 [5, 13] . Conclusions 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) [14]. 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) [14]. 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 categories. 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). 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