|Year : 2022 | Volume
| Issue : 1 | Page : 117-121
Histopathological characterization of carcinoma breast with BRCA1/2 sequence variation in a Tertiary Care Center in Kerala, South India
Reeba Mary Issac1, Prema Saldanha2, Jessy Mangalathu Mathai1, Jency Mathews3, Rebecca Mathews1, Bindu Kumari4, Tiju Chacko5
1 Department of Pathology, Pushpagiri Institute of Medical Sciences and Research Center, Tiruvalla, Kerala, India
2 Department of Pathology, Yenepoya Medical College, Yenepoya University, Mangaluru, Karnataka, India
3 Department of Surgical Oncology, Pushpagiri Institute of Medical Sciences, Tiruvalla, Kerala, India
4 Department of Pathology, Sree Uthradom Thirunal Academy of Medical Sciences, Trivandrum, Kerala, India
5 Department of Molecular Biology, Dianova Laboratories, Kottayam, Kerala, India
|Date of Submission||30-Aug-2021|
|Date of Acceptance||18-Nov-2021|
|Date of Web Publication||11-Mar-2022|
HOD & Professor, Department of Pathology, Yenepoya Medical College, Yenepoya University, Derlakatte, Mangaluru, Karnataka
Source of Support: None, Conflict of Interest: None
Background: Hereditary breast cancers constitute around 5%–10% of all breast cancers. The most commonly mutated genes in hereditary breast and ovarian cancer syndrome are the BRCA1 and BRCA2 genes. There has not been any research into the histopathological features of BRCA1/2 mutations in breast cancer in Kerala. The goal of this work was to use next-generation sequencing to look into the range of BRCA1/2 genetic variants and to discover characteristic histopathologic features associated with BRCA1/2 sequence variations. Methods: Fifty female patients diagnosed with carcinoma breast were screened for BRCA1/2 variants by next-generation sequencing. Patients were selected irrespective of age or family history of breast or ovarian cancer. The histopathological features of BRCA1/2 associated breast cancer cases were also studied. Results: We identified sequence variants in BRCA1 and BRCA2 genes in 6 women (6/50, 12%). The sequence variants included two deleterious mutations and five variants of uncertain significance. It was observed that most of the patients with BRCA1/2 sequence variants presented with aggressive tumor characteristics. Statistical significance was noted in BRCA1/2 carriers with tumors of higher histologic grade (P = 0.048). Conclusion: Morphological and immunohistochemical features of BRCA1/2-associated breast cancer may be valuable to predict mutation carrier status for appropriate therapeutic decision-making.
Keywords: BRCA1, BRCA2, histopathology, immunohistochemistry, pathogenic mutation, variant of uncertain significance
|How to cite this article:|
Issac RM, Saldanha P, Mathai JM, Mathews J, Mathews R, Kumari B, Chacko T. Histopathological characterization of carcinoma breast with BRCA1/2 sequence variation in a Tertiary Care Center in Kerala, South India. Biomed Biotechnol Res J 2022;6:117-21
|How to cite this URL:|
Issac RM, Saldanha P, Mathai JM, Mathews J, Mathews R, Kumari B, Chacko T. Histopathological characterization of carcinoma breast with BRCA1/2 sequence variation in a Tertiary Care Center in Kerala, South India. Biomed Biotechnol Res J [serial online] 2022 [cited 2023 Jan 28];6:117-21. Available from: https://www.bmbtrj.org/text.asp?2022/6/1/117/339367
| Introduction|| |
Germline mutation in BRCA1 and BRCA2 genes is the most common cause for hereditary breast cancer which forms a part of a genetic tumor syndrome called as hereditary breast and ovarian cancer syndrome (HBOC). The average risk for breast cancer in BRCA1 mutation carriers by the age of 70 years is 65% and BRCA2 carriers is 45%. BRCA1 and BRCA2 genes are tumor suppressor genes located on chromosome 17q21 and 13q12.3. The BRCA1 gene contains 24 coding exons spread over 80 kb. The BRCA2 gene contains 27 coding exons spread over 84 kb of genomic DNA. They play a primary role in DNA double-strand break repair by homologous recombination pathway. When the BRCA1/2 genes are nonfunctional, DNA repair occurs through alternate mechanisms which are error prone leading to the development of genetic aberrations and increased risk for the development of malignant neoplasms in multiple organs. The presence of these mutations substantially increases the likelihood of developing cancers of the breast, ovary, fallopian tube, endometrium, prostate, pancreas, stomach, and colon. Ashkenazi Jews have an increased risk of developing HBOC compared to the general population. This is due to a high frequency of BRCA1/2 mutations seen in this ancestry. The founder mutations described in them are 185delAG and 5382insC in BRCA1 and 6174delT in BRCA2 gene. These founder mutations have been identified in several other population all over the world.
Tumors arising in BRCA1 and BRCA2 carriers do have certain histologic and molecular features. BRCA1-associated breast cancer usually has aggressive tumor characteristics with higher histologic grade, prominent lymphocytic infiltrate, and triple-negative phenotype. They usually express epithelial keratins CK5/6 which is referred to as the basal-like molecular subtype. The histologic patterns observed in BRCA1 carriers are usually invasive carcinoma of no special type (NST), invasive breast carcinoma with medullary pattern, and metaplastic carcinomas. BRCA2-associated breast cancer is usually hormone receptor positive with luminal A molecular subtype. They lack HER2 expression. These characteristic pathological features in BRCA1/2 carriers are useful for mutation prediction which may help triage patients toward testing and also to assess the pathogenicity of BRCA variants of uncertain significance.
Only very few studies have been done worldwide exploring the tumor characteristics in BRCA1/2 carriers. As of now, there is only one study from Kerala regarding BRCA1/2 mutation in breast cancer which focused only on the prevalence of the mutation. This study was undertaken to assess the morphological and immunohistochemical characteristics in carcinoma breast patients with sequence variants in BRCA1/2 genes.
| Methods|| |
The present study consecutively recruited 50 women with a histopathological diagnosis of invasive breast carcinoma after obtaining informed consent. The study was undertaken between March 2019 and March 2020 in the department of pathology of a tertiary care center in Kerala. Approval was obtained from the institutional ethics committee (PIMSRC/E1/388A/33/2014). Epidemiological data were collected from these patients using a prestructured questionnaire. Four milliliters blood sample was collected for molecular testing.
Clinical parameters such as age, gender, family history of breast cancer, tumor stage, number of lesions, and regional lymph node involvement were noted. Histopathologic parameters such as tumor subtype, histologic grade, and lymphocytic infiltrate were evaluated. Hormone receptor expression, Her2 overexpression, and Ki-67 proliferation were studied by immunohistochemical staining on formalin-fixed paraffin-embedded tissues.
Genetic testing was done in an NABL accredited laboratory (DDRC SRL Diagnostic Services, Ernakulam). DNA was isolated from the given whole blood for mutation analysis. Concentration of isolated DNA was analyzed using Qubit fluorometer. The DNA extracted from the blood sample was tested for mutations by next-generation sequencing technology. Variants discovered were reported in accordance with ACMG guidelines (American College of Medical Genetics and Genomics).
The association between BRCA1/2 gene mutation and patient characteristics including morphologic and immunohistochemical findings was tested for statistical significance using Fisher's/Chi-square tests except for age which was done using Mann–Whitney U-test. P value of less than 0.05 was considered statistically significant.
| Results|| |
Descriptive analysis of patient data was done [Table 1]. It was noted that maximum number of patients were above 50 years of age (48%). Family history of breast cancer was observed in six cases where three patients had a history of breast cancer in the mother, three in the sibling (sister), and one in a second degree relative (cousin). Multifocality was noted in six cases. Only 48% of cases showed positive evidence of regional lymph node involvement.
|Table 1: Clinicopathological characteristics of BRCA1/2-positive and BRCA1/2-negative study subjects|
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Mutation analysis done revealed clinically significant BRCA1 and BRCA2 sequence variants in 12% (6/50) of women. Pathogenic mutations were identified in 2 women (4%) and variant of uncertain significance in 5 women (10%). One study participant had both pathogenic mutation and variant of uncertain significance. It was observed that median age at diagnosis of invasive breast cancer was 34 years among BRCA1 and 54.5 years among BRCA2 sequence variant carriers [Table 2] and [Table 3].
|Table 2: Descriptive statistics of age of patients with BRCA1 sequence variants|
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|Table 3: Descriptive statistics of age of patients with BRCA2 sequence variants|
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Pathogenic mutations identified were c. 68_69delAG in BRCA1 gene and c.5645C >A in BRCA2 gene. Variant of uncertain significance identified in BRCA1 gene was c.3995G >T and c.1593G > A. Variant of uncertain significance identified in BRCA2 gene was c.6937 + 27G >C, c.5986G >A, and c.4165T >G.
The patient with pathogenic mutation in BRCA1 gene (c.68_69delAG) was 31 years old with no family history of breast cancer and presence of metastasis in three axillary lymph nodes. She also had variant of uncertain significance in BRCA2 gene (c.6937 + 27G >C). Pathologic characteristics were large tumor size(>2 cm), invasive carcinoma of NST-histologic type, Grade 3 histologic grade, presence of lymphovascular emboli, large, prominent lymphocytic infiltrate, presence of necrosis, triple-negative phenotype, and high proliferation index [Figure 1].
|Figure 1: Micrographs of carcinoma breast with pathogenic mutation in BRCA1 gene. (a) Invasive carcinoma of no special type (×10). (b) Higher magnification showing high histologic grade and lymphocytic infiltrate (×40)|
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The patient with pathogenic mutation in BRCA2 gene (c.5645C >A) was 50 years old with family history of breast cancer in the mother of the proband and with presence of metastasis in two axillary lymph nodes. Pathologic characteristics were large tumor size (>2 cm), invasive carcinoma of NST-histologic type, Grade 3 histologic grade, presence of lymphovascular emboli, prominent lymphocytic infiltrate, presence of necrosis, triple-negative phenotype, and high proliferation index.
The patient with variant of uncertain significance in BRCA1 gene (c.3995G > T) was 34 years old with no family history of breast cancer and presence of metastasis in three axillary lymph node. Pathologic characteristics were large tumor size (>2 cm), invasive carcinoma of NST-histologic type, Grade 3 histologic grade, presence of lymphovascular emboli, prominent lymphocytic infiltrate, presence of necrosis, triple-negative phenotype, and high proliferation index.
The patient with variant of uncertain significance in BRCA1 gene (c.1593G >A) was 53 years old with no family history of breast cancer and absence of regional lymph node involvement. Pathologic characteristics were tumor size <2 cm, invasive carcinoma of NST-histologic type, Grade 1 histologic grade, no lymphovascular emboli, presence of necrosis, presence of ductal carcinoma in situ, triple negative phenotype, and high proliferation index.
The patient with variant of uncertain significance in BRCA2 gene (c.5986G >A) was 59 years old with no family history of breast cancer and absence of regional lymph node involvement. Pathologic characteristics were large tumor size (>2 cm), invasive carcinoma of NST-histologic type, Grade 2 histologic grade, no lymphovascular emboli, hormone receptor positivity, absence of Her2 overexpression, and low proliferation index.
The patient with variant of uncertain significance in BRCA2 gene (c.4165T >G) was 70 years old with no family history of breast cancer and presence of metastasis in four axillary lymph nodes. Pathologic characteristics were large tumor size (>2 cm), invasive carcinoma of NST-histologic type, Grade 2 histologic grade, presence of lymphovascular emboli, hormone receptor positivity, absence of HER2 overexpression, and high proliferation index.
Significant association was observed only for histological grade of the tumor (P = 0.048) with BRCA1/2. High-grade tumors had significantly higher proportion of BRCA (14.3%) when compared to low-grade tumors (11.1%).
| Discussion|| |
Most prior research on BRCA1/2-associated cancers focused on a highly selected group.
However, in this study, participants were chosen without regard to their age or family history of cancer. We analyzed data on the pathology of breast tumors arising in women with BRCA1 and BRCA2 sequence variants. Only very few studies have assessed tumor pathology in mutation carriers worldwide. There is also lack of data regarding histopathological characteristics of BRCA1/2 gene mutated breast cancer in Indian women.
All genetic variant level interpretations in this study were mapped to five categorization groups while undergoing BRCA genetic testing: benign, likely benign, variant of unknown significance (VUS), likely pathogenic, and pathogenic. The present study has described tumor pathology in both pathogenic mutation and variant of uncertain significance carriers.
Studies on pathologic characteristics of variant of uncertain significance in BRCA1 and BRCA2 genes are limited. More than 1500 VUS have been identified in BRCA1 and BRCA2 genes. With the advent of next-generation sequencing for multigene testing, the rate of identification of VUS has increased overtime. Many of these VUS will be reclassified into benign or pathogenic mutation with more studies in future.
In our study, sequence variants were noted in both BRCA1 and BRCA2 genes. Pathogenic mutation in BRCA1/2 genes was observed in 4% of cases. Variant of uncertain significance in BRCA1/2 genes was noted in 10% of cases. All the cases with sequence variants presented with invasive carcinoma of NST. Both the cases with pathogenic mutation in BRCA1 and BRCA2 were of triple-negative phenotype. Patients with VUS in BRCA1 gene in two cases also presented with triple-negative breast cancer. Patients with VUS in BRCA2 gene presented with hormone receptor positivity in two cases and triple-negative phenotype in one case. High proliferation index was observed in all patients except for one case with VUS in BRCA2 gene. Tumor size was more than 2 cm in all patients except for one case with VUS in BRCA1 gene. Patients with pathogenic mutation in both genes presented with high-grade tumors. Patients with VUS in BRCA1/2 genes also presented with high-grade tumors except for one case which had Grade 1 tumor. Lymphovascular emboli were noted in both patients with pathogenic mutation, one patient with VUS in BRCA1 gene and two patients with VUS in BRCA2 gene.
According to a study by Fountzilas et al., histological type in BRCA1 and BRCA2 carriers was also predominantly of the ductal type. In comparison to BRCA1 tumors, a higher percentage of BRCA2 tumors were human epidermal growth factor receptor 2 (HER2) positive. These findings are consistent with our study.
In another study by Mavaddat et al., it was observed that proportion of hormone receptor-negative breast cancers reduced with age at diagnosis in BRCA1 carriers but rose with age at diagnosis in BRCA2 carriers. In BRCA1 carriers, the proportion of triple-negative cancers dropped with age at diagnosis, whereas in BRCA2 carriers, the proportion of triple-negative tumors increased with age at diagnosis. Hormone receptor-negative tumors were seen in both BRCA1 and BRCA2 carriers.
In a retrospective review study done by You et al., it was noted that BRCA-positive familial breast cancers were mostly ductal carcinomas with high nuclear grade and lymph node metastases, whereas BRCA-negative cancers had a lower proliferation index. BRCA1 carriers were more likely to have the triple-negative phenotype, whereas BRCA2 carriers were more likely to have the luminal phenotype. This is in contrast to our finding where BRCA2 pathogenic mutation carrier was of triple-negative phenotype.
In a Chinese study, it was observed that breast cancers with BRCA1 gene mutation exhibited high nuclear grade tumors. About 72.7% of BRCA1 mutant breast cancers were triple negative, compared to 25.5% of BRCA2 mutation breast cancers. HER2 expression was lower in BRCA1 and BRCA2 mutant breast tumors. In BRCA1 mutation breast cancer, the positive expression of cytokeratin 5/6 (CK5/6) and epidermal growth factor receptor was around 50%. Breast tumors with BRCA1 mutations were less likely to be androgen receptor (AR) positive. These findings are similar to another study done in China where they too found out that BRCA1 mutant tumors presented with a high nuclear grade, estrogen receptor/progesterone receptor/HER2 negative, low Ki-67, and positive CK5/6. In addition, they also observed that BRCA1-mutated cancers displayed lower CHEK2 immunohistochemical expression and higher cytoplasmic BRIT1 expression than BRCA2-mutated malignancies. Nuclear PARP-1 expression was also substantially higher in BRCA1/2-associated cancers than in non-BRCA1/2 mutant malignancies.
Cecener et al. demonstrated hormone-positive features in most of BRCA1 and BRCA2 mutation carriers in Turkish breast cancer patients. Triple-negative breast cancer is found in up to 80% of patients with positive BRCA1 mutations, according to another study from Poland. These findings are in line with what we found in our research.
BRCA genes were found to be mutated in 65% of cases in a study on breast cancer patients in Northwestern Romania. Invasive ductal carcinoma was found in 97.5% of patients. Their results showed BRCA1 mutation association with a low percentage of estrogen receptor and progesterone receptor, an increased Ki67 index, and a HER2-negative status which is in concordance to our study findings.
| Conclusion|| |
Our data indicate that there are characteristic histopathological features in women with sequence variation in BRCA1/2 genes. In our study, we reported the pathological and immunohistochemical features of breast cancer patients from South India with BRCA1/2 sequence variation. These findings support previous findings in the pathology of BRCA-related cancers and add to our understanding of the link between histologic entities and mutations in these genes, which could have therapeutic implications. Larger databases of mutation carriers should allow for more detailed tumor analysis. Pathological indicators, in addition to family history, may be effective in identifying patients who are likely to possess a BRCA1/2 germline mutation from breast cancer patients in general.
Limitation of study
Our study has limitations as the results were derived from a small sample size. This could explain the reason for lack of statistical significance with the histopathological features except for tumor grade. More research in future using larger sample sizes is required for better understanding of morphological and immunohistochemical characteristics of BRCA gene mutated breast cancer.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Petrucelli N, Daly MB, Feldman GL. Hereditary breast and ovarian cancer due to mutations in BRCA1 and BRCA2. Genet Med 2010;12:245-59.
Antoniou A, Pharoah PD, Narod S, Risch HA, Eyfjord JE, Hopper JL, et al.
Average risks of breast and ovarian cancer associated with BRCA1 or BRCA2 mutations detected in case series unselected for family history: A combined analysis of 22 studies. Am J Hum Genet 2003;72:1117-30.
Miki Y, Swensen J, Shattuck-Eidens D, Futreal PA, Harshman K, Tavtigian S, et al
. A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1. Science 1994;266:66-71.
Hodgson A, Turashvili G. Pathology of hereditary breast and ovarian cancer. Frontiers in Oncology 2020;10:1-11.
Lakhani SR, Ellis IO, Schnitt SJ, Tan PH, van de Vijver MJ, eds. WHO classification of tumours of the breast, 4th ed. Geneva: World Health Organization, 2012.
Hartge P, Struewing JP, Wacholder S, Brody LC, Tucker MA. The prevalence of common BRCA1 and BRCA2 mutations among Ashkenazi Jews. Am J Hum Genet 1999;64:963-70.
Dilani L, Valerie AW, Reiko W and Ian AC. WHO Classification of Tumours, Breast tumours. 5th ed. Lyon: International Agency for Research on Cancer, 2019.
Lebo MS, Zakoor KR, Chun K, Speevak MD, Waye JS, McCready E, et al.
Data sharing as a national quality improvement program: Reporting on BRCA1 and BRCA2 variant-interpretation comparisons through the Canadian Open Genetics Repository (COGR). Genet Med 2018;20:294-302.
Welsh JL, Hoskin TL, Day CN, Thomas AS, Cogswell JA, Couch FJ, et al.
Clinical decision-making in patients with variant of uncertain significance in BRCA1 or BRCA2 genes. Ann Surg Oncol 2017;24:3067-72.
Eggington JM, Bowles KR, Moyes K, Manley S, Esterling L, Sizemore S, et al
. A comprehensive laboratory-based program for classification of variants of uncertain significance in hereditary cancer genes. Clin Genet 2014;86:229-37.
Fountzilas E, Konstantopoulou I, Vagena A, Apostolou P, Papadimitriou C, Christodoulou C, et al.
Pathology of BRCA1 and BRCA2-associated breast cancers: Known and less known connections. Clin Breast Cancer 2020;20:152-9.
Mavaddat N, Barrowdale D, Andrulis IL, Domchek SM, Eccles D, Nevanlinna H, et al.
Pathology of breast and ovarian cancers among BRCA1 and BRCA2 mutation carriers: Results from the consortium of investigators of modifiers of BRCA1/2 (CIMBA). Cancer Epidemiol Biomarkers Prev 2012;21:134-47.
You C, Xiao Q, Zhu X, Sun Y, Di G, Liu G, et al.
The clinicopathological and MRI features of patients with BRCA1/2 mutations in familial breast cancer. Gland Surg 2021;10:262-72.
Xinyi Z, Tian T, Xu C, Guangqi Q, Menghong S, Jiong W, et al
. Comparative study on the pathological features of BRCA1/2 mutation and non-BRCA1/2 mutation breast cancers in China. China Oncol 2019;29:142-7.
Zhu X, Tian T, Ruan M, Rao J, Yang W, Cai X, et al.
Expression of DNA damage response proteins and associations with clinicopathologic characteristics in Chinese familial breast cancer patients with BRCA1/2
mutations. J Breast Cancer 2018;21:297-305.
Cecener G, Takanlou LS, Takanlou MS, Egeli U, Eskiler GG, Aksoy S, et al
. Clinicopathologic features and genetic characteristics of the BRCA1/2 mutation in Turkish breast cancer patients. Cancer Genet 2020;240:23-32.
Domagala P, Huzarski T, Lubinski J, Gugala K, Domagala W. Immunophenotypic predictive profiling of BRCA1-associated breast cancer. Virchows Arch 2011;458:55-64.
Goidescu IG, Eniu DT, Caracostea GV, Cruciat G, Stamatian F. Associations of pathogenic mutations responsible for breast cancer risk with histology and immunohistochemistry in Romanian population. Rev Română Med Lab 2018;26:165-75.
[Table 1], [Table 2], [Table 3]