Vimentin and laminin expression is associated with basal-like phenotype in both sporadic and BRCA1-associated breast carcinomas

AIMS: To determine whether basal-like phenotype and vimentin and/or laminin are related in both sporadic/familial (BRCA1 or BRCA2 mutated) tumours. METHODS: 230 non-familial and 28 hereditary node-negative invasive breast carcinomas were immunohistochemically analysed for oestrogen receptors (ER), progesterone receptors (PR), cytokeratin 5/6 (CK5/6), epidermal growth factor receptors (EGFR), Ki67, p53, vimentin and laminin, using tissue microarrays. Tumours were considered to have basal-like phenotype if they were ER negative and HER2 negative, but positive for CK5/6 and/or EGFR. RESULTS: In sporadic tumours, vimentin expression was found in 77.8% cases with basal-like phenotype and 15.5% of non-basal cases (p<0.001). In familial cases, vimentin was expressed in 83.3% basal-like cancers and 16.7% of non-basal tumours (p<0.001). Vimentin expression was more frequent in BRCA1 than BRCA2 mutation carriers. Vimentin expressing tumours were associated with poor prognosis (p = 0.012) among patients not receiving adjuvant chemotherapy and showed a trend for local recurrence or visceral but not bone metastasis (p = 0.021). Laminin expression was also related to basal-like phenotype in both sporadic/familial cases (p<0.001 and p = 0.007, respectively), but neither with prognosis nor recurrence pattern in sporadic cancers. CONCLUSIONS: Vimentin and laminin expression is associated with basal-like phenotype in breast cancer. Expression of vimentin and laminin is characteristic of BRCA1 associated tumours. Since vimentin and laminin staining is widely used by pathologists for diagnostic purposes, thus demonstrating the robustness of their specific antibodies, the immunohistochemical evaluation of these two molecules could be used in identification of basal-like breast tumours in both sporadic/familial cases.     

High follicular phase luteinizing hormone levels in young healthy BRCA1 mutation carriers: implications for breast and ovarian cancer risk

BRCA1 mutation carriers have up to 80% life-time risk of developing breast cancer and 20-40% risk of developing ovarian cancer. High LH levels have been linked to increased risks of both breast and ovarian cancers in some studies and it is unknown whether gonadotropin levels are associated with BRCA1 mutation status. The aim of the study was to explore whether gonadotropin levels were associated with BRCA1 mutation status among healthy 40-year-old-women from hereditary breast cancer families. All women completed a questionnaire including information on reproductive factors and OC use. We measured height, weight, breast volumes, and plasma levels of LH, FSH, and estradiol (E2) once during menstrual cycle days 5-10 and once again during cycle days 18-23 in 43 non-carriers from BRCA1 families, 20 BRCA1 mutation carriers, and 101 women from non-BRCA1/2 families. The strongest predictors of high LH levels among BRCA1 mutation carriers and non-carriers during cycle days 5-10 were being a BRCA1 mutation carrier (p=0.002), lack of current OC use (p=0.003), and being nulliparous (p=0.01), adjusted for age and menstrual cycle day when the samples were obtained. This association was seen both in non-OC users and current OC users but was only significant in the former group (p=0.005). Because of multiple analyses it is possible that our finding is a result of a Type 1 statistical error. After a permutation test the new adjusted p value in non-OC users was 0.05. FSH and E2 were similar in non-carriers, BRCA1 mutation carriers and women from non-BRCA1/2 families. We found significantly elevated LH levels in the follicular phase among young healthy BRCA1 mutation carriers compared with non-carriers from BRCA1 families. This is a small study and confirmatory studies are warranted to establish whether elevated LH levels are part of the BRCA1 phenotype and may be manipulated in order to reduce cancer risks in BRCA1 mutation carriers.     

BRCA1 Mutations in Familial Ovarian Cancer

BRCA1 mutation research in ovarian and breast cancer 17q21-linked families has yielded a large number of germline sequence variations. Somatic mutations have been uncommonly reported. We screened 81 probands with primary ovarian, peritoneal, or fallopian tube carcinoma for BRCA1 mutations. The study group was intentionally biased by the inclusion of 29 probands with a family history of ovarian and/or breast carcinoma, 13 probands diagnosed on or before age 45, seven individuals with a metachronous breast cancer and 51 tumors with BRCA1 LOH. Tumor and/or germline DNA was screened by modified techniques of single-strand confirmation polymorphism analysis, and abnormal banding patterns were sequenced to confirm mutations. Twenty-one (25.9%) BRCA1 sequence variations were identified. Eight mutations were somatic including seven null mutations. Apart from classical hereditary ovarian/breast cancer, a family history of ovarian/breast cancer defines a subset of ovarian cancer individuals with a significant likelihood of either a germline or a somatic BRCA1 gene sequence variation.     

BRCA1-methylated sporadic breast cancers are BRCA-like in showing a basal phenotype and absence of ER expression

BRCA1 mutations have been associated with hereditary breast cancer only. Recent studies indicate that a subgroup of sporadic breast cancer might also be associated with reduction in BRCA1 mRNA levels and protein expression. However, the mechanism of reduced mRNA and protein expression is yet not fully elucidated. This study aims to assess BRCA1 protein expression and the role of BRCA1 promoter methylation in sporadic breast cancer in North Indian population and to correlate these with known prognostic factors and molecular profiles of breast cancer. BRCA1 protein expression was normal (>50?% tumour cells) in 41 (43?%) cases, reduced (20-50?% tumour cells) in 33 (35?%) cases and absent/markedly reduced (<20?% tumour cells) in 21 (22.1?%) cases. Cases which were negative for BRCA1 protein were more frequently positive for basal markers (29 versus 5?%) and were more often ER-negative (62 versus 39?%) than BRCA1-positive tumours. Methylation of BRCA1 promoter region was seen in 11/45 cases (24?%). All 11 cases showing BRCA1 methylation had absent (eight cases) or reduced (three cases) BRCA1 protein expression. BRCA1 protein-negative tumours were more frequently basal marker-positive and ER-negative, highlighting the 'BRCAness' of sporadic breast cancer with loss of BRCA1 protein expression through promoter hypermethylation similar to hereditary breast cancer with BRCA1 mutations. Loss of BRCA1 in sporadic breast cancer suggests that therapeutics targeting BRCA1 pathway in hereditary breast cancer like PARP inhibitors might be used as therapeutic targets for sporadic breast tumours.     

BRCA1 and BRCA2 sequence variants in Chinese breast cancer families

A series of 45 high-risk breast cancer patients, consisting of 25 affected individuals from 16 families in China with at least two cases of breast cancer and 20 cases of breast cancer diagnosed under age 35 without reported family history, were studied for germline mutations of the BRCA1 and BRCA2 genes. Thirteen of the 16 families contained at least one case diagnosed under age 50. Three distinct protein truncating sequence variants, likely to be disease-associated, were identified: two novel mutations in BRCA1 (1584G>T and 5028delC), and a previously reported mutation in BRCA2 (7883delTTAA). Additional sequence variants identified included common polymorphisms, and several variants of unknown clinical significance, including a novel BRCA1 alteration. Based on models for predictive testing using allele frequencies and risks estimated in Western populations, our results suggest that BRCA1/2 mutations account for a somewhat smaller fraction of breast cancer cases in Tianjin than in the Caucasian populations studied. This difference could be the result of a lower penetrance of BRCA1/2 mutations due to the surrounding environmental and hormonal milieu, or a lower frequency of mutations in this population. Larger, more detailed, studies will be necessary to determine which factors underlie this difference.     

BRCA1 mutations in Algerian breast cancer patients: high frequency in young, sporadic cases

Breast cancer rates and median age of onset differ between Western Europe and North Africa. In Western populations, 5 to 10 % of breast cancer cases can be attributed to major genetic factors such as BRCA1 and BRCA2, while this attribution is not yet well defined among Africans. To help determine the contribution of BRCA1 mutations to breast cancer in a North African population, we analysed genomic DNA from breast cancer cases ascertained in Algiers. Both familial cases (at least three breast cancers in the same familial branch, or two with one bilateral or diagnosed before age 40) and sporadic cases less than 38 years of age were studied. Complete sequencing plus quantitative analysis of the BRCA1 gene was performed. 9.8 % (5/51) of early-onset sporadic and 36.4 % (4/11) of familial cases were found to be associated with BRCA1 mutations. This is in contrast 10.3 % of French HBOC families exhibiting a BRCA1 mutation. One mutation, c.798_799delTT, was observed in two Algerian families and in two families from Tunisia, suggesting a North African founder allele. Algerian non-BRCA1 tumors were of significantly higher grade than French non-BRCA tumors, and the age at diagnosis for Algerian familial cases was much younger than that for French non-BRCA familial cases. In conclusion, we observed a much higher frequency of BRCA1 mutations among young breast cancer patients than observed in Europe, suggesting biological differences and that the inclusion criterea for analysis in Western Europe may not be applicable for the Northern African population.     

Validation of the BRCA1 antibody MS110 and the utility of BRCA1 as a patient selection biomarker in immunohistochemical analysis of breast and ovarian tumours

BRCA1 protein measurement has previously been evaluated as a potential diagnostic marker without reaching a conclusive recommendation. In this study, we applied current best practice in antibody validation to further characterize MS110, a widely used antibody targeting BRCA1. Antibody specificity was investigated using different biochemical validation techniques. We found that BRCA1 could not be reliably detected using immunoprecipitation and Western blot in endogenously expressing cells. We used immunohistochemistry on formalin-fixed paraffin-embedded cell pellets to establish compatibility with formalin-fixed paraffin-embedded samples. We demonstrated that in transfected cells and cell lines with known genetic BRCA1 status, MS110 successfully detected BRCA1 giving the expected level of staining in immunohistochemistry. Following this, we investigated the use of BRCA1 protein measurement by immunohistochemistry in a cohort of triple negative breast and serous ovarian tumour samples to explore the use of BRCA1 protein measurement by immunohistochemistry for patient stratification. Using MS110 in repeated standardized experiments, on serial sections from a panel of patient samples, results demonstrated considerable run-to-run variability. We concluded that in formalin-fixed tissue samples, MS110 does detect BRCA1; however, using standard methodologies, BRCA1 expression levels in tissue samples is incompatible with the use of this protein as a statistically robust patient selection marker in immunohistochemistry. These results demonstrate the need for further development to deliver BRCA1 protein quantification by immunohistochemistry as a patient stratification marker.     

Prevalence of BRCA1 and BRCA2 mutations in Korean breast cancer patients

The incidence of breast cancer in Korea has been increasing in recent years, such that it is now the most common female cancer. Breast cancer in Korea is characterized by an earlier age of onset than in Western countries, suggesting that it would be related with genetic background. We assayed germline mutations in the BRCA genes to evaluate their genetic pathology in Korean breast cancer patients. The study subjects consisted of 173 patients at clinically higher risk and 109 unselected patients. Germline mutations in the entire coding sequences of the BRCA1 and BRCA2 genes were analyzed by Conformation-Sensitive Gel Electrophoresis (CSGE), and any aberrantly-sized band was sequenced. BRCA mutations were present in 12.7% of the high risk patients, compared with 2.8% of the unselected patients. Among high risk patients, mutations were most prevalent in patients with a family history of breast or first-degree ovarian cancer (22.1%), followed by those with male breast cancer (20%), bilateral breast cancer (20%), multiple organ cancer including breast (13%) and younger breast cancer patients (aged<35 yr) (8.1%). Moreover, BRCA mutations were detected in 34.8% of patients having two high risk factors. These findings suggest that BRCA gene mutation analysis should be performed on Korean patients with high-risk factors for breast cancer.     

Germline mutations of BRCA1 and BRCA2 in Korean breast and/or ovarian cancer families

Germline mutations in the BRCA1 and BRCA2 genes are responsible for the predisposition and development of familial breast and/or ovarian cancer. Most mutations of BRCA1 and BRCA2 associated with breast and/or ovarian cancer result in truncated proteins. To investigate the presence of BRCA1 and BRCA2 germline mutations in Korean breast and/or ovarian cancer families, we screened a total of 27 cases from 21 families including two or more affected first- or second-degree relatives with breast and/or ovarian cancer. PTT, PCR-SSCP, and DHPLC analysis, followed by sequencing were used in the screening process. In nine families, we found BRCA1 and BRCA2 germline mutations that comprised four frameshift mutations and five nonsense mutations. All nine mutations led to premature termination producing shortened proteins. Among the nine mutations, three novel BRCA1 mutations (E1114X, Q1299X, 4159delGA) and two novel BRCA2 mutations (K467X, 8945delAA) were identified in this work.     

Genetic analysis of the BRCA1 region in a large breast/ovarian family: refinement of the minimal region containing BRCA1

We have analyzed a single multi-affected breast/ovarian cancerpedigree (BOV3) and have shown consistent inheritance of markerson chromosome 17q with the disease confirming that this familyis due to the BRCA1 gene. Analysis of 17q hapiotypes shows arecombination event in a bilateral breast cancer case whichsuggests that the BRCA1 gene lies distal to D17S857; D17S857is thus the new proximal boundary for the region containingBRCA1. Combining this information with previously publishedmapping information suggests that BRCA1 is contained in a regionestimated at 1 – 1 .5 Mb in length. All seven breast tumour/bloodpairs examined from this family show loss of heterozygosityin the tumours. The allele retained in each tumour was fromthe disease-bearing chromosome implicating BRCA1 as a tumoursuppressor gene. We have sequenced the 17ß-oestradioldehydrogenase genes (EDH17B1 and EDH17B2) which have been suggestedas candidate genes for BRCA1 in four members of this family.No germline mutations were detected.     

CGH analysis of familial non-BRCA1/BRCA2 breast tumors and mutation screening of a candidate locus on chromosome 17q11.2-12

Mutations in the known predisposing breast cancer genes BRCA1 and BRCA2 account for only a small proportion (<10%) of breast cancer families in the Stockholm region of Sweden. This study aims to identify novel predisposing genes in non-BRCA1/BRCA2 breast cancer families. We have employed comparative genomic hybridization (CGH) and loss of heterozygosity (LOH) data in combination with data from a recently carried out genome wide linkage scan, in an effort to identify chromosomal regions harboring potential breast cancer genes. CGH revealed loss of chromosome 17 and chromosome 6q to be a frequent event in high-risk breast cancer families, while gain of 8q was most prevalent in low-risk families. The loss of chromosome 17 was consistent with previous LOH studies and so this region was investigated further. Disease was shown to be linked to chromosome 17 in those families exhibiting loss of the chromosome in both CGH and LOH analyses. An overlapping region of linkage was determined to lie between markers D17S1294 and D17S1293, fine mapping of the region delineated a region between markers D17S1880 and D17S1293. Ten genes were determined to lie within this 1.5 Mb region and families were screened for germline mutations in these genes. In conclusion, we have investigated one possible small region on chromosome 17 for its involvement in high-risk non-BRCA1/BRCA2 breast cancer families. No predisposing mutations were identified in the 10 genes investigated in this study, however further analysis of chromosome 17 is warranted.     

Loss of nuclear BRCA1 localization in breast carcinoma is age dependent

Abstract.Patients carrying a germ line mutation in the BRCA1 gene are predisposed to breast cancer. Somatic BRCA1 mutations were almost never reported in sporadic breast tumors, but several authors have described a decrease in BRCA1 mRNA and protein. In order to further investigate the possible role of BRCA1 in sporadic breast cancer, an improved immunohistochemical protocol was developed and applied on tissue sections obtained from 102 cancer patients belonging to two nonoverlapping age groups (less than 40 and more than 60 years). Based on the obtained BRCA1 specific nuclear staining we could distinguish two categories of breast cancer. The staining was present in almost all the nuclei of the normal and the cancer cells in about 50% of young (less than 40 years old) as well as older patients (more than 60 years old). Thus, BRCA1 does not seem to be involved in the genesis of these cancers. In the second category of patients, either a fraction of, or all tumor cells showed no nuclear staining. In this category, no nuclear BRCA1 staining could be observed in the tumor cells of 14 (27%) young and 3 (6%) older patients. Among six young patients bearing a breast tumor showing no BRCA1 nuclear staining at all, one was found to carry a BRCA1 germline mutation. Taken together, our results suggest that the molecular pathway in which the BRCA1 protein participates is disturbed in about 50% of sporadic breast cancer, this effect being more pronounced in tumors of young patients.     

BRCA1 and BRCA2 germline mutations in Korean patients with sporadic breast cancer

In order to evaluate the role of BRCA1 and BRCA2 germline mutations in Korean patients with sporadic breast cancer, 97 patients with sporadic breast cancer were analyzed for mutations in the BRCA1 and BRCA2 coding regions, by using a combination of fluorescent-conformation sensitive gel electrophoresis (F-CSGE) and direct sequencing. Fifty-five distinct sequence variants were detected, which included three pathogenic truncating mutations, 15 missense mutations, 16 polymorphisms, and 21 intronic variants. Twenty-six of these variants have never been previously reported and may be of Korean-specific origin. Two pathogenic BRCA1 mutations (c.922_924delinsT, c.5445G>A) and one pathogenic BRCA2 mutation (c.2259delT) were observed, and two of these (BRCA1 c.5445G>A and BRCA2 c.2259delT) are novel. The total prevalence of germline pathogenic mutations in BRCA1 and/or BRCA2 in Korean sporadic breast cancer is estimated to be about 3.1%. Considering that the majority of breast cancer cases are sporadic, the present study will be helpful in the evaluation of the need for the genetic screening of germline BRCA mutations in sporadic breast cancer patients. Further study using a larger sample size is required to determine the merits of genetic diagnosis and counseling in breast cancer patients.     

Novel germline mutations in the BRCA1 and BRCA2 genes in Indian breast and breast-ovarian cancer families

The two major hereditary breast/ovarian cancer predisposition tumor suppressor genes, BRCA1 and BRCA2 that perform apparently generic cellular functions nonetheless cause tissue-specific syndromes in the human population when they are altered, or mutated in the germline. However, little is known about the contribution of BRCA1 and BRCA2 mutations to breast and/or ovarian cancers in the Indian population. We have screened for mutations the entire BRCA1 and BRCA2 coding sequences, and intron-exon boundaries, as well as their flanking intronic regions in sixteen breast or breast and ovarian cancer families of Indian origin. We have also analyzed 20 female patients with sporadic breast cancer regardless of age and family history, and 69 unrelated normal individuals as control. Thus a total of 154 samples were screened for BRCA1 and BRCA2 mutations using a combination of polymerase chain reaction-mediated site directed mutagenesis (PSM), polymerase chain reaction-single stranded conformation polymorphism assay (PCR-SSCP) and direct DNA sequencing of PCR products (DS). Twenty-one sequence variants including fifteen point mutations were identified. Five deleterious pathogenic, protein truncating frameshift and non-sense mutations were detected in exon 2 (c.187_188delAG); and exon 11 (c.3672G>T) [p.Glu1185X] of BRCA1 and in exon 11 (c.5227dupT, c.5242dupT, c.6180dupA) of BRCA2 (putative mutations - four novel) as well as fourteen amino acid substitutions were identified. Twelve BRCA1 and BRCA2 missense variants were identified as unique and novel. In the cohort of 20 sporadic female patients no mutations were found.     

BRCA1 and BRCA2 mutations correlate with TP53 abnormalities and presence of immune cell infiltrates in ovarian high-grade serous carcinoma

We characterized BRCA1 and BRCA2 status (mutation/methylation) in a consecutive series of cases of ovarian carcinoma in order to identify differences in clinicopathological features, molecular characteristics, and outcome between the pelvic high-grade serous cancers with (i) germline or somatic mutations in BRCA1 or BRCA2, (ii) methylation of BRCA1, and (iii) normal BRCA1 or BRCA2. In all, 131 women were identified prospectively, who were undergoing surgical staging and agreed to germline testing for BRCA1 and BRCA2 mutations. Histopathology, germline and somatic BRCA1 or BRCA2 mutations, BRCA1 methylation, and BRCA1 and BRCA2 mRNA expression levels distinguished four subgroups. In all, 103 cases were high-grade serous carcinoma and of these 31 (30%) had germline or somatic BRCA1 or BRCA2 mutations (20% BRCA1 and 10% BRCA2) (group 1), 21 (20%) had methylation of BRCA1 (group 2), and in 51 (50%) there was no BRCA loss (group 3). Group 4 consisted of 28 cases of non-high-grade serous, none of which had BRCA loss. BRCA1 and BRCA2 mRNA expression levels correlated with designated group (P=0.0008). Among high-grade serous carcinomas, there were no differences between groups 1-3 with respect to stage, ascites, CA125 level, platinum sensitivity, cytoreduction rate, neoadjuvant chemotherapy, or survival. Tumors with BRCA1 or BRCA2 mutations had increased immune infiltrates (CD20 and TIA-1) compared with high-grade serous without mutations (P=0.034, 0.027). TP53 expression differed between groups (P<0.0001), with abnormal TP53 expression in 49/50 tumors from groups 1 and 2. Wild-type TP53 expression was associated with worse outcome in high-grade serous (P<0.001). BRCA loss (mutation/methylation) is a common event in the pelvic high-grade serous (50%). TP53 abnormalities and increased immune cell infiltrates are significantly more common in high-grade serous with germline and somatic mutations in BRCA1 or BRCA2, compared with tumors lacking BRCA abnormalities.     

Low frequency of BRCA1 germline mutations in 45 German breast/ovarian cancer families.

In this study we investigated 45 German breast/ovarian cancer families for germline mutations in the BRCA1 gene. We identified four germline mutations in three breast cancer families and in one breast-ovarian cancer family. among these were one frameshift mutation, one nonsense mutation, one novel splice site mutation, and one missense mutation. The missense mutation was also found in 2.8% of the general population, suggesting that it is not disease associated. The average age of disease onset in those families harbouring causative mutations was between 32.3 and 37.4 years, whereas the family harbouring the missense mutation had an average age of onset of 51.2 years. These findings show that BRCA1 is implicated in a small fraction of breast/ovarian cancer families suggesting the involvement of another susceptibility gene(s).     

CGH-targeted linkage analysis reveals a possible BRCA1 modifier locus on chromosome 5q

Women with germline mutations in BRCA1 have a greatly elevated risk of breast and ovarian cancer. However, considerable variation in the degree of breast cancer risk associated with a BRCA1 mutation has been observed, suggesting that modifiers of BRCA1 penetrance may exist. We hypothesized that the modifier genes might be located in regions of allelic imbalance in the tumors of BRCA1 mutation carriers, as have been reported on chromosomes 4p, 4q and 5q. In order to determine whether novel genetic modifiers of BRCA1-associated breast cancer penetrance in these regions exist, we used non-parametric linkage analysis methods to determine whether allele sharing of chromosomes 4p, 4q and 5q was observed preferentially within BRCA1 mutation families in women with BRCA1 mutations and breast cancer. No significant linkage on chromosome 4p or 4q was observed associated with breast cancer risk in BRCA1 mutation carriers. However, we observed a significant linkage signal at D5S1471 on chromosome 5q (P = 0.009) in all the families analyzed together. The significance of this observation increased in the subset of families with an average of breast cancer diagnosis less than 45 years (P = 0.003). These results suggest the presence of one or more genes on chromosome 5q33-34 that modify breast cancer risk in BRCA1 mutation carriers. The approach described here may be utilized to identify penetrance modifiers in other autosomal dominant syndromes.     

Screening for BRCA1 and BRCA2 mutations in Eastern Finnish breast/ovarian cancer families

Familial aggregation is thought to account for 5-10% of all breast cancer cases, and high penetrance breast and ovarian cancer susceptibility genes BRCA1 and BRCA2 explain < or =20% of these. Hundreds of mutations among breast/ovarian cancer families have been found in these two genes. The mutation spectrum and prevalence, however, varies widely among populations. Thirty-six breast/ovarian cancer families were identified from a population sample of breast and ovarian cancer cases among a relatively isolated population in Eastern Finland, and the frequency of BRCA1/BRCA2 germline mutations were screened using heteroduplex analysis, protein truncation test and sequencing. Five different mutations were detected in seven families (19.4%). Two mutations were found in BRCA1 and three in BRCA2. One of the mutations (BRCA2 4088insA) has not been detected elsewhere in Finland while the other four, 4216-2nt A-->G and 5370 C-->T in BRCA1 and 999del5 and 6503delTT in BRCA2, are recurrent Finnish founder mutations. These results add to the evidence of the geographical differences in distribution of Finnish BRCA1/BRCA2 mutations. This screen also provides further evidence for the presumption that the majority of Finnish BRCA1/BRCA2 founder mutations have been found and that the proportion of BRCA1/BRCA2 mutations in Finnish breast/ovarian cancer families is around 20%.