Molecular characterization of breast cancer in young Brazilian women.

To evaluate the distribution of molecular subtypes of breast tumors diagnosed in young Brazilian women and to analyze the frequency of loss of heterozygocity (LOH) in BRCA1 among different molecular subtypes of early-onset breast cancer.



Samples from 72 cases of invasive breast carcinoma diagnosed in women aged between 19 and 40 years were evaluated using an immunohistochemical panel of biomarkers. Three intragenic BRCA1 locus microsatellites, D17S1322, D17S1323, and D17S855, were PCR amplified from matched normal (lymphocyte) and tumor DNAs for (LOH) analysis.



We found 13 cases (18%) that had an immunohistochemical profile consistent with being basal-like. Forty cases (55%) were luminal A type; 11% (8 cases) were luminal B type, 13% (9 cases) were HER2-overexpressing tumors and two cases were ER-/HER2- carcinomas lacking basal marker expression. Four of the 16 informative cases at D17S1322, one of the four informative cases at D17S855, and none of the five informative cases at D17S1323 displayed LOH (four basal-like and one Luminal A). Microsatellite instability (MSI) at D17S855 and D17S1322 was found in two cases (one a basal-like and one Luminal A).



In our study, basal-like tumor was the second most frequent molecular type among young Brazilian women and was only observed in women diagnosed under the age of 35 years. There was no significant difference of LOH at BRCA1 locus rates between basal-like breast tumors and not-basal-like breast tumors (p=0.62). LOH in BRCA1 and MSI in these breast cancers were not frequent but may indicate a small group of breast cancers with a specific molecular makeup.

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title: Loss of Heterozygosity in Bilateral Breast Cancer
abstract: Women who develop bilateral breast cancer at an early age are likely to harbour germline mutations in breast cancer susceptibility genes. The aim of this study was to test for concordant genetic changes in left and right breast cancer of young women (age <50) with bilateral breast cancer that may suggest an inherited breast cancer predisposition. Microsatellite markers were used to test for loss of heterozygosity (LOH) in left and right tumours for 31 women with premenopausal bilateral breast cancer. Markers adjacent to or within candidate genes on 17p (p53), 17q (BRCA1), 13q (BRCA2), 11q (Ataxia Telangiectasia-ATM) and 3p (FHIT) were chosen. Mutational testing for BRCA1 and BRCA2 was performed for cases where blood was available. Concordant LOH in both left and right tumours was demonstrated for at least one of the markers tested in 16/31(54%) cases. Where allelic loss was demonstrated for both left and right breast cancer, the same allele was lost on each occasion. This may suggest a common mutational event. Four cases showed concordant loss of alleles in both left and right breast cancer at D17S791 (BRCA1). BRCA1 mutations were identified in two of these cases where blood was available. Four cases showed concordant LOH at D13S155 (BRCA2). Concordant LOH was further demonstrated in seven cases for D11S1778 (ATM) and four cases for D3S1300 (which maps to the FHIT gene), suggesting a possible role for these tumour suppressor genes in this subgroup of breast cancer patients. No concordant allelic loss was demonstrated for D17S786 suggesting that germline mutations in p53 are unlikely in such cases of bilateral breast cancer.

title: Biophenotypes and survival of BRCA1 and TP53 deleted breast cancer in young women
abstract: The aim of this study was to examine the loss of heterozygosity (LOH) of BRCA1 (17q21) and TP53 (17p13.1) in early-onset breast cancer patients; to correlate biopathological characteristics with molecular alterations; and to investigate the survival of LOH-related cancers.BRCA1 and TP53 LOH were evaluated in 78 early-onset breast cancers (≤40 years, Group 1) and 80 patients with age <55 years (Group 2). Cases were characterized for multiple biological markers (ER, PR, proliferation index (PI), NEU and p53). LOH was carried out on microdissected paraffin embedded tissues; microsatellites D17S855 (BRCA1) and D17S786 (TP53) were amplified by fluorescent PCR and analyzed by an automated DNA sequencer. Early-onset breast cancers showed a higher frequency of ductal histotype (89,7% vs. 56,3% p<0.001), node-positive (53,8% vs. 38,7%), larger size (p=0.017), higher mitotic rate (p=0.025), higher nuclear and final grade (p=0.01 and p=0.001, respectively). D17S855 LOH was 32,8% in group 1 vs. 21% in group 2; D17S786 LOH was 50,7% vs. 31.3% (p=0.03), respectively. BRCA1 LOH was correlated with higher PI (p=0.032) and higher p53 expression (p<0.001) in group 1 and with higher NEU expression (p=0.028) in group 2. TP53 LOH was correlated with p53 overexpression (p=0.03) in group 1. A worse clinical outcome in early-onset LOH related cancers emerged from follow-up data: TP53 and BRCA1 LOH were associated with a shorter relapse free interval (RFI) (p=0.03) and a poorer overall survival (OS) (p=0.04), respectively. This study underlines different biological profiles in the two age groups investigated, probably reflecting different mechanisms of carcinogenesis. In accordance with adverse histopathological features in early-onset patients, LOH-related cancers have an unfavorable prognosis.

title: [Clinical and economic issues in adjuvant chemotherapy for HER-2 positive breast cancer].
abstract: To examine efficacy figures and drug expenditure for adjuvant chemotherapy in human epidermal growth factor receptor 2 (HER-2) positive breast cancer, in the Brazilian supplemental health insurance market.



We obtained efficacy data (disease free survival at 3-years) and drug cost estimate for current adjuvant strategies in HER-2 positive breast cancer: Finland Herceptin (FinHER), National Surgical Adjuvant Breast and Bowel Project (NSAPB-31), North Central Cancer Treatment Group (N9831), Herceptin Adjuvant (HERA) and Breast Cancer International Research Group (BCIRG-006). We estimated clinical impact measures--number needed to treat (NNT) and absolute risk reduction (ARR)--and total drug cost by protocol to avoid one single cancer recurrence.



The largest ARR was 11.7% (95% CI: 2.2% to 21.2%) in the FinHER study, and the smallest in the nonanthracycline arm of the BCIRG trial, 4.9% (95% CI: 1.8% to 8.1%). The NNT was 8 (95% CI: 3 to 28) in the FinHER, 8 (95% CI: 7 to 11) in the NSABP-31/N9831, 12 (95% CI: 9 to 18) in the HERA, 14 (95% CI: 11 to 24) in the BCIRG/Anthracycline, and 17 (CI 95% CI: 12 to 34) in the BCIRG/Nonanthracycline. Drug cost to avoid one single cancer recurrence would be R$ 418,285.44 with the FinHER regimen, R$ 1,716,789.44 with the NSABP-31/N9831, R$ 2,481,891.58 with the HERA, R$ 2,963,634.62 with the BCIRG/Anthracycline, and R$ 3,930,520.43 with the BCIRG/Nonanthracycline (exchange rate: R$ 1.00=USD 0.56).



From an economic viewpoint, four to seven times more patients could benefit by using a short-course of trastuzumab at the initial adjuvant chemotherapy cycles (FinHER regimen) than by the prolonged trastuzumab administration as used in other adjuvant schedules.

title: Rabbit antibodies for hormone receptors and HER2 evaluation in breast cancer.
abstract: Novel rabbit monoclonal antibodies (RabMab) for estrogen (ER), progesterone (PR) receptors and HER2 evaluation by immunohistochemistry have recently been commercially released. We compared the RabMab anti-ER, anti-PR and anti-HER2 to mouse monoclonal antibodies (Mab) using tissue microarrays (TMA) of breast carcinomas.



Two TMA containing breast carcinomas were built. Sections were immunostained using anti-ER and anti-PR, Mab and RabMab. The sections stained for ER and PR were evaluated considering positive those tumors in which more than 1% of the tumor cell nuclei stained moderate or strong. For HER2, the immunostained sections were evaluated using the ASCO/CAP guidelines for HER2. Chromogenic in situ hybridization (CISH) was used as the gold standard for HER2 evaluation. CISH was evaluated using the Zymed HER2 CISH interpretation guidelines.



RabMab against ER have similar staining patterns compared to the 6F11 (Mab), but stronger than 1D5 (Mab) from three different suppliers. The RabMab against PR provide stronger and sharper immunohistochemical signals compared to Mab. The detection of HER2 protein overexpression was more prevalent with the polyclonal antibodies and RabMab than with the Mab. These were more specific than the RabMab, which were more sensitive when compared to CISH.



The novel RabMab against ER and PR showed higher intensity of staining than the Mab. The RabMab against HER2 is more sensitive than Mab, however, Mab presented more specificity than RabMab when compared to CISH for HER2 evaluation of breast carcinomas.

title: Distinct molecular pathogeneses of early-onset breast cancers in BRCA1 and BRCA2 mutation carriers: a population-based study.
abstract: Breast cancers arising in women with and without a germline mutation in the BRCA1 or BRCA2 gene display different histological features, which suggests unique mechanisms of molecular pathogenesis: We used a molecular pathological analysis to define the genetic abnormalities relevant to these specific pathogeneses. Tumor material was studied from 40 women with breast cancer diagnosed before 40 years of age, sampled from a population-based study and stratified by BRCA1 and BRCA2 germline mutation status. Cases were not selected for family history or ethnic origin, and none were known to be genetically related. Thus, germline mutation itself is likely to impact on the molecular pathogenesis of these tumors, with no substantial influence due to modifying genetic or environmental factors. Breast cancers occurring in BRCA1 mutation carriers had significantly higher levels of p53 expression, including the preinvasive (carcinoma in situ) stage of disease, compared with cancers occurring in BRCA2 mutation carriers or women with no detectable germline mutation. These cancers also had a higher proliferation rate as measured by Ki-67 antibody. Expression of the prognostic factors c-erbB-2, cyclin D1, and estrogen receptor was significantly less common in BRCA1 mutation carriers. Lower levels of cyclin D1 were also found in cancers from BRCA2 mutation carriers compared with non-mutation carriers. Direct p53 mutation analysis revealed mutations in 18% of all of the early-onset breast cancers within the study and included rare insertion and deletional mutations in cancers from BRCA1 mutation carriers. Our data indicate that a BRCA1 breast cancer phenotype may be recognized by an exceptionally high proliferation rate and early and frequent p53 overexpression but infrequent selection for overexpression of several other prognostic factor proteins known to be involved in breast oncogenesis. In contrast, breast cancers arising in BRCA2 mutation carriers have a more heterogeneous phenotypic profile.

title: Relationship of patients' age to histopathological features of breast tumours in BRCA1 and BRCA2 and mutation-negative breast cancer families
abstract: IntroductionOur aim was to evaluate the relationship of patients' age to histopathological features of hereditary breast tumours in a series of breast cancer families not selected for age at diagnosis. In sporadic breast cancer, tumours from premenopausal patients have been shown to differ from those of postmenopausal patients, but this phenomenon has been little studied among familial patients.MethodsRepresentative areas of all available breast cancer tissue specimens (n = 262) from 25 BRCA1, 20 BRCA2, and 74 non-BRCA1/2 breast cancer families were punched into a tissue microarray. Immunohistochemical staining of oestrogen receptor, progesterone receptor, ERBB2, and p53 as well as the histology and grade of tumours in these three groups of families were studied in different age groups and compared with each other.ResultsWe found that only breast cancers from young (<50 years) BRCA1+ patients represent features documented as being typical of BRCA1-associated cancers, such as high tumour grade, negativity for oestrogen and progesterone receptors, and overexpression of p53. Among the BRCA2 families, the opposite was found, with a significantly higher frequency of tumours negative for oestrogen and progesterone receptors among the older patients than among the other groups, but no distinctive tumour characteristics among the younger BRCA2 patients.ConclusionTumours of BRCA1 and BRCA2 carriers aged 50 years or more differed significantly from those of younger carriers. This difference may reflect different biological behaviour and pathways of tumour development among the older and the younger BRCA1 and BRCA2 patients, with impact also on prognosis and survival.

title: Population-based estimate of the average age-specific cumulative risk of breast cancer for a defined set of protein-truncating mutations in BRCA1 and BRCA2. Australian Breast Cancer Family Study.
abstract: The average breast cancer risk for carriers of a germ-line mutation in BRCA1 or BRCA2 (penetrance) has been estimated from the multiple-case families collected by the Breast Cancer Linkage Consortium (BCLC) to be approximately 80% to age 70. However, women now being tested for these mutations do not necessarily have the intense family history of the BCLC families. Testing for protein-truncating mutations in exons 2, 11, and 20 of BRCA1 and exons 10 and 11 of BRCA2 was conducted in a population-based sample of 388 Australian women with breast cancer diagnosed before age 40. Onset of breast cancer was analyzed in the known and potential mutation-carrying first- and second-degree female relatives of cases found to carry a mutation. Of the 18 mutation-carrying cases (9 BRCA1 and 9 BRCA2), only 5 (1 BRCA1 and 4 BRCA2) had at least one affected relative, so family history of breast cancer was not a strong predictor of mutation status in this setting. The risk in mutation carriers was, on average, 9 times the population risk [95% confidence interval (CI), 4-23; P < 0.001]. Penetrance to age 70 was 40% (95% CI, 15-65%), about half that estimated from BCLC families. By extrapolation, approximately 6% (95% CI, 2-20%) of breast cancer before age 40 may be caused by protein-truncating mutations in BRCA1 or BRCA2. Breast cancer risk in BRCA1 or BRCA2 mutation carriers may be modified by other genetic or environmental factors. Genetic counselors may need to take into account the family history of the consultand.

title: Placental Cadherin and the Basal Epithelial Phenotype of BRCA1-Related Breast Cancer
abstract: BRCA1-related breast cancer frequently has a basal epithelial phenotype, and P-cadherin is a basal marker. We undertook a detailed evaluation of the relationship among P-cadherin, prognostic markers in breast cancer, and outcome.  This study was restricted to 292 cases of first primary invasive breast cancer diagnosed in Ashkenazi Jewish women between 1980 and 1995. All available blocks were stained for P-cadherin, and 261 were included in the final statistical analyses, including 27 germ line BRCA1 mutation carriers and 8 BRCA2 mutation carriers. Descriptive analyses were done followed by survival analyses and a Poisson regression analysis.  P-cadherin was present in 80 of the 261 breast cancers (31%) and was more frequently present in tumors that have a basal epithelial phenotype [i.e., high-grade, estrogen receptor– and KIP1 (p27Kip1)–negative tumors, with expression of cytokeratin 5/6, cyclin E, TP53, and presence of BRCA1 mutations and vascular nests (all P < 0.001)]. In a univariate survival model, expression of P-cadherin was associated with a relative risk (RR) of death from breast cancer at a 10-year follow-up of 2.9 (95% confidence interval, 1.8-4.7; P < 0.0001) and was a predictor of poor univariate survival in both lymph node–negative and –positive breast cancers. In a multivariate analysis, the effect of P-cadherin levels was not independent of other basal-related markers. Multivariable interaction modeling showed that P-cadherin positivity was highly predictive of a poor prognosis in small, node-negative breast cancers (RR, 7.1; P = 0.006).  P-cadherin is a marker for basal-like breast cancers and is strongly associated with the presence of a BRCA1 mutation. It is an adverse prognostic factor, particularly in small, node-negative breast cancers.

title: BRCA1 mutations and other sequence variants in a population-based sample of Australian women with breast cancer
abstract: SummaryThe frequency, in women with breast cancer, of mutations and other variants in the susceptibility gene, BRCA1, was investigated using a population-based case–control-family study. Cases were women living in Melbourne or Sydney, Australia, with histologically confirmed, first primary, invasive breast cancer, diagnosed before the age of 40 years, recorded on the state Cancer Registries. Controls were women without breast cancer, frequency-matched for age, randomly selected from electoral rolls. Full manual sequencing of the coding region of BRCA1 was conducted in a randomly stratified sample of 91 cases; 47 with, and 44 without, a family history of breast cancer in a first- or second-degree relative. All detected variants were tested in a random sample of 67 controls. Three cases with a (protein-truncating) mutation were detected. Only one case had a family history; her mother had breast cancer, but did not carry the mutation. The proportion of Australian women with breast cancer before age 40 who carry a germline mutation in BRCA1 was estimated to be 3.8% (95% Cl 0.3–12.6%). Seven rare variants were also detected, but for none was there evidence of a strong effect on breast cancer susceptibility. Therefore, on a population basis, rare variants are likely to contribute little to breast cancer incidence.

title: Extracolonic cancer in hereditary nonpolyposis colorectal cancer.
abstract: It has been hypothesized that in some but not all families with hereditary nonpolyposis colorectal cancer (HNPCC) there is a high risk of certain cancers other than colon cancer.



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In addition to early onset cancers of the colorectum, HNPCC family members are at increased risk for cancers of other gastrointestinal tract organs, and, especially in some families, cancers of the upper urologic and female genital tract.

title: Molecular Pathologic Analysis Enhances the Diagnosis and Management of Muir-Torre Syndrome and Gives Insight Into Its Underlying Molecular Pathogenesis
abstract: The Muir-Torre syndrome (MTS) is an autosomal dominantly inherited disorder, characterized by visceral malignancies and sebaceous skin lesions. In a subset of MTS families the disease is due to an underlying DNA mismatch-repair defect. We have identified a MTS family whose spectrum of reported neoplasia included adenocarcinomas of numerous gastrointestinal sites, carcinomas of the endometrium, ovary and breast, papillary transitional cell carcinoma of the ureter, a range of cutaneous tumors, as well as keratoacanthomas. All tumors were tested for microsatellite instability and immunohistochemically stained for expression of MLH1 and MSH2 proteins. All tumors were found to be microsatellite unstable and lacking in MSH2 protein expression. The subsequent mutation detection focused on hMSH2, and a germline mutation was identified (CAA→TAA, Gln→STOP, codon 337). This mutation was subsequently found in a family member with a single skin lesion only. We propose that the combination of immunohistologic and microsatellite instability analysis can be exploited to screen individuals with characteristic skin lesions even before development of visceral tumors and to direct the subsequent germline mutation search. The profile of microsatellite instability and the genes rendered dysfunctional differed between tumor samples, suggesting that the molecular pathogenesis varied between lesions, despite a common germline mutation.

title: Molecular genetic evidence of the occurrence of breast cancer as an integral tumor in patients with the hereditary nonpolyposis colorectal carcinoma syndrome
abstract: The hereditary nonpolyposis colorectal carcinoma (HNPCC) syndrome is an autosomal dominant genetic disorder caused by the inheritance of a mutation in one of a family of genes encoding DNA mismatch repair (MMR) proteins. HNPCC manifests as genetic instability in linked tumors. Clinically, the syndrome is characterized by early onset malignancies, primarily of the colon and endometrium, with an increased incidence of tumors at other gastrointestinal sites, upper urologic tract, ovary, and pancreas as well. However, the inclusion of breast cancer as an integral tumor of this syndrome is controversial.

title: Microsatellite instability in hereditary and sporadic breast cancers
abstract: Sporadic cancers and familial breast cancers are characterized by an increase in genetic instability. Little is known about whether mismatch repair defects accompany this genetic instability. We investigated invasive and/or in situ breast cancers from 30 women with deleterious BRCA1/2 mutations and unclassified variant BRCA1/2 alterations. Forty cases of sporadic breast cancers were also investigated, including 7 medullary carcinomas. Malignant and benign lesions were examined from all cases to better understand tumor progression. Automated immunohistochemistry, with antibodies directed against hMLH1 and hMSH2, was used to screen cases for possible mismatch repair defects. When loss of expression was noted, DNA ploidy was performed by cytomorphometry. DNA, after laser microdissection, was extracted from a majority of familial cases and their corresponding controls, and microsatellite instability analysis was performed. None of the familial or sporadic cases had loss of hMSH2 expression. All but one lesion, a DCIS arising in a deleterious BRCA2 mutation carrier, had loss of hMLH1 expression and a tetraploid profile by image cytomorphometry. There was no MSI in any explored lesions (n = 34), as determined by molecular analysis, including the DCIS with loss of hMLH1 expression. We conclude that DNA mismatch repair defects involving hMLH1 and hMSH2 underexpression are extremely rare events in sporadic and familial breast cancer. Mismatch repair gene mutations may be secondary random events in breast cancer progression.

title: [Loss of heterozygosity and microsatellite instability in the region including BRCA1 of breast cancer in Chinese].
abstract: To shed light on the relationship between BRCA1 gene and breast cancer in Chinese Han women.



Four microsatellites DNA (D17S855, D17S579, D17S1327 and THRA1) within the BRCA1 gene were used as polymorphic markers. A study of loss of heterozygosity(LOH) and microsatellite instability(MSI) at the above- mentioned 4 microsatellites of 50 breast cancer patients was conducted by using PCR -PAUGE-DNA silver staining (polymerase chain reaction-polyacrylamide urea gel electrophoresis) method.



Twenty-nine or 58% of the informative cases showed LOH; 35.71%, 15. 38%, 18.18%, and 26.19% of the informative cases showed positive LOH at the D17S855, D17S579, D17S1327 and THRA1 loci respectively. The rate of MSI was 46%, and the rates of MSI at the four loci were 16%, 18%, 18% and 12% respectively. Further study on the associations between the phenomena of LOH and MSI and different clinical stages revealed that MSI was an early event in mammary tumorigenesis while LOH occurred at a later stage.



All of these suggest that breast cancer in Chinese be somehow linked to BRCA1.

title: Genetic instability of BRCA1 gene at locus D17S855 is related to clinicopathological behaviors of gastric cancer from Chinese population.
abstract: To investigate genetic instability of gene BRCA1 at locus D17S855, and their relationship with clinicopathological characteristics of gastric cancer in Chinese population.



Microsatellite instability (MSI) and loss of heterozygosity (LOH) of gene BRCA1 at locus D17S855 were compared between 37 samples of gastric cancer and corresponding non-cancerous gastric tissue.



MSI at locus D17S855 was positive in 7 of 37 samples of gastric cancer (18.95%). MSI had a close relationship with TNM staging but no relation with lymph node metastasis, histological type or tumor differentiation. MSI positive frequency in TNM I + II (31.58%, 6/19) was much higher than that in TNM III + IV (5.56%, 1/18), (P < 0.05). LOH positive rate was 18.92% (7/37). LOH had no relationship to histological type, tumor differentiation or lymph node metastasis, but LOH positive rate in TNM III + IV was 33.33% (6/18), much higher than that in TNM I + II ( 5.26%, 1/19), (P < 0.05). BRCA1 protein was expressed in 14 of 37 samples of gastric cancer. The positive rates of BRCA1 protein in TNM I + II and TNM III + IV were 57.89% and 16.67%, respectively, (P < 0.05). The positive rate of BRCA1 protein was 77.78% in high differentiation samples, 30.77% in middle differentiation and 12.50% in lower differentiation samples, (P < 0.05).



MSI of BRCA1 gene could be used as a molecular marker in early phases of sporadic gastric cancer in Chinese population. LOH occurs at later period of gastric cancer, therefore, it could be used as prognostic factor.