Spectrum and prevalence of BRCA1/2 germline mutations in Pakistani breast cancer patients: results from a large comprehensive study.

BACKGROUND: Pathogenic germline mutations in BRCA1 and BRCA2 (BRCA1/2) account for the majority of hereditary breast and/or ovarian cancers worldwide. To refine the spectrum of BRCA1/2 mutations and to accurately estimate the prevalence of mutation in the Pakistani population, we studied 539 breast cancer patients selected for family history and age of diagnosis. METHODS: Comprehensive screening for BRCA1/2 germline mutations was performed using state-of-the-art technologies. RESULTS: A total of 133 deleterious mutations were identified in 539 families (24.7%), comprising 110 in BRCA1 and 23 in BRCA2. The prevalence of BRCA1/2 small-range mutations and large genomic rearrangements was 55.4% (36/65) for families with breast and ovarian cancer, 27.4% (67/244) for families with two or more cases of breast cancer, 18.5% (5/27) for families with male breast cancer, and 12.3% (25/203) for families with a single case of early-onset breast cancer. Nine mutations were specific to the Pakistani population. Eighteen mutations in BRCA1 and three in BRCA2 were recurrent and accounted for 68.2% (75/110) and 34.8% (8/23) of all identified mutations in BRCA1 and BRCA2, respectively. Most of these mutations were exclusive to a specific ethnic group and may result from founder effects. CONCLUSIONS: Our findings show that BRCA1/2 mutations account for one in four cases of hereditary breast/ovarian cancer, one in five cases of male breast cancer, and one in eight cases of early-onset breast cancer in Pakistan. Our study suggests genetic testing of an extended panel of 21 recurrent BRCA1/2 mutations for appropriately selected patients and their families in Pakistan.

High prevalence and predominance of BRCA1 germline mutations in Pakistani triple-negative breast cancer patients.

BACKGROUND: Women harboring BRCA1/2 germline mutations have high lifetime risk of developing breast/ovarian cancer. The recommendation to pursue BRCA1/2 testing is based on patient's family history of breast/ovarian cancer, age of disease-onset and/or pathologic parameters of breast tumors. Here, we investigated if diagnosis of triple-negative breast cancer (TNBC) independently increases risk of carrying a BRCA1/2 mutation in Pakistan. METHODS: Five hundred and twenty-three breast cancer patients including 237 diagnosed ≤ 30 years of age and 286 with a family history of breast/ovarian cancer were screened for BRCA1/2 small-range mutations and large genomic rearrangements. Immunohistochemical analyses were performed at one center. Univariate and multiple logistic regression models were used to investigate possible differences in prevalence of BRCA1/2 mutations according to patient and tumor characteristics. RESULTS: Thirty-seven percent of patients presented with TNBC. The prevalence of BRCA1 mutations was higher in patients with TNBC than non-TNBC (37 % vs. 10 %, P < 0.0001). 1 % of TNBC patients were observed to have BRCA2 mutations. Subgroup analyses revealed a larger proportion of BRCA1 mutations in TNBC than non-TNBC among patients 1) diagnosed at early-age with no family history of breast/ovarian cancer (14 % vs. 5 %, P = 0.03), 2) diagnosed at early-age irrespective of family history (28 % vs. 11 %, P = 0.0003), 3) had a family history of breast cancer (49 % vs. 12 %, P < 0.0001), and 4) those with family history of breast and ovarian cancer (81 % vs. 28 %, P = 0.0005). TNBC patients harboring BRCA1 mutations were diagnosed at a later age than non-carriers (median age at diagnosis: 30 years (range 22-53) vs. 28 years (range 18-67), P = 0.002). The association between TNBC status and presence of BRCA1 mutations was independent of the simultaneous consideration of family phenotype, tumor histology and grade in a multiple logistic regression model (Ratio of the probability of carrying BRCA1/2 mutations for TNBC vs. non-TNBC 4.23; 95 % CI 2.50-7.14; P < 0.0001). CONCLUSION: Genetic BRCA1 testing should be considered for Pakistani women diagnosed with TNBC.

Deleterious RAD51C germline mutations rarely predispose to breast and ovarian cancer in Pakistan.

RAD51C plays a key role in homologous recombination-mediated DNA repair and maintenance of genomic stability. Biallelic RAD51C mutations cause Fanconi anemia, and monoallelic mutations predispose women to breast and ovarian cancer. Genetic variability of RAD51C and its impact in Asian populations have been poorly studied. Here, we report the results of comprehensive mutational screening of the RAD51C gene in 348 BRCA1/2-negative breast and/or ovarian cancer patients from Pakistan. Mutation analysis of the complete RAD51C-coding region was performed using denaturing high-performance liquid chromatography analysis, followed by DNA sequencing of variant fragments. Three novel protein-truncating mutations, c.204T>A, c.225T>G, and c.701C>G, were identified. c.204T>A was found in one out of 22 (4.5 %) early-onset (≤45 years of age) ovarian cancer patients and c.225T>G in one out of 119 (0.8 %) patients from breast cancer only families. c.701C>G was found in a 60-year-old control with no family history of breast/ovarian cancer. Furthermore, three novel in silico-predicted potentially functional mutations, a missense mutation, c.873T>G, a variant in 5'UTR, c.1-34T>G, and a recurrent intronic variant, c.965+21A>G, were identified. The missense mutation was observed in a patient with bilateral breast cancer from a breast and ovarian cancer family (HBOC), the 5'UTR variant was noted in an early-onset breast cancer patient, and the intronic variant in one early-onset breast cancer patient and one ovarian cancer patient from a HBOC family. Five of the six mutations described were not detected in 400 healthy controls. These findings suggest that RAD51C plays a marginal role in breast and ovarian cancer predisposition in Pakistan. Reliable estimation of the clinical implications of carrying a deleterious RAD51C mutation will require identification of additional mutation-positive patients/families.

Patterns of cancer cell sphere formation in primary cultures of human oral tongue squamous cell carcinoma and neck nodes.

Recently a sub-population of cells with stem cell characteristics, reported to be associated with initiation, growth, spread and recurrence, has been identified in several solid tumors including oral tongue squamous cell carcinoma (OTSCC). The aim of our pilot study was to isolate CD44+ cancer stem cells from primary cultures of OTSCC and neck node Level I (node-I) biopsies, grow cell spheres and observe their characteristics in primary cultures. Parallel cultures of hyperplastic lesions of tongue (non-cancer) were set up as a control. Immunohistochemistry was used to detect CD44/CD24 expression and magnetic activated cell sorting to isolate CD44+ cell populations followed by primary cell culturing. Both OTSCC and node-I biopsies produced floating spheres in suspension, however those grown in hyperplastic and node-I primary cultures did not exhibit self-renewal properties. Lymph node metastatic OTSCC, express higher CD44/CD24 levels, produce cancer cell spheres in larger number and rapidly (24 hours) compared to node negative OTSCC (1 week) and non-cancer specimens (3 weeks). In addition, metastatic OTSCC have the capacity for proliferation for up to three generations in primary culture. This in vitro system will be used to study cancer stem cell behavior, therapeutic drug screening and optimization of radiation dose for elimination of resistant cancer cells.

Constitutional CHEK2 mutations are infrequent in early-onset and familial breast/ovarian cancer patients from Pakistan.

BACKGROUND: Less than 20% of Pakistani women with early-onset or familial breast/ovarian cancer harbor germ line mutations in the high-penetrance genes BRCA1, BRCA2 and TP53. Thus, mutations in other genes confer genetic susceptibility to breast cancer, of which CHEK2 is a plausible candidate. CHEK2 encodes a checkpoint kinase, involved in response to DNA damage. METHODS: In the present study we assessed the prevalence of CHEK2 germ line mutations in 145 BRCA1/2-negative early-onset and familial breast/ovarian cancer patients from Pakistan (Group 1). Mutation analysis of the complete CHEK2 coding region was performed using denaturing high-performance liquid chromatography analysis, followed by DNA sequencing of variant fragments. RESULTS: Two potentially deleterious missense mutations, c.275C>G (p.P92R) and c.1216C>T, (p.R406C), were identified (1.4%). The c.275C>G mutation is novel and has not been described in other populations. It was detected in a 30-year-old breast cancer patient with a family history of breast and multiple other cancers. The c.1216C>T mutation was found in a 34-year-old ovarian cancer patient from a family with two breast cancer cases. Both mutations were not detected in 229 recently recruited BRCA1/2-negative high risk patients (Group 2). CONCLUSION: Our findings suggest that CHEK2 mutations may not contribute significantly to breast/ovarian cancer risk in Pakistani women.

Identification of the deleterious 2080insA BRCA1 mutation in a male renal cell carcinoma patient from a family with multiple cancer diagnoses from Pakistan.

Pathogenic germ line mutations in the BRCA1 and BRCA2 genes confer elevated risk of breast and ovarian cancer in females and have a link with variety of cancers other than breast cancer in males. Here we report the first case of a 45-year-old Pakistani male with renal cell carcinoma who was identified to harbor a disease-associated BRCA1 germ line mutation, 2080insA. Detailed description of the family history, clinical presentation and histopathological features of the renal cell carcinoma are presented.

The impact of culture and sociological and psychological issues on Muslim patients with breast cancer in Pakistan.

Breast cancer is the most common form of cancer in Muslim women in Pakistan. The impact of the initial diagnosis, culture, religion, and psychosocial and psychological aspects of the disease is not well established. This qualitative study examined the experience and coping strategies used by patients with breast cancer in relation to its impact on their physical, mental health, religious, and family issues. Thirty patients with breast cancer were interviewed. Data were analyzed using thematic analysis. The patient's experience of breast cancer focused on the range of emotions felt throughout the illness trajectory, the importance of religion and family support on coping strategies used to manage the adverse effects of chemotherapy, and also the financial concerns. This is the first study to examine Pakistani Muslim women's views on the lived experience of breast cancer. This article provides clarification of the voiced experiences of women with breast cancer. The data not only highlight the role of religion and family support as essential coping strategies but also emphasize the issues of isolation, aggression, and anger as common responses to chemotherapy. Unique features of this study are women's need to seek spiritual support for their illness and the overriding innate characteristic of maternal responsibility. These cultural features require further analysis and research.