Clinical relevance of double heterozygosity revealed by next-generation sequencing of homologous recombination repair pathway genes in South African breast cancer patients.

PURPOSE: Genetically predisposed breast cancer (BC) patients represent a minor but clinically meaningful subgroup of the disease, with 25% of all cases associated with actionable variants in BRCA1/2. Diagnostic implementation of next-generation sequencing (NGS) resulted in the rare identification of BC patients with double heterozygosity for deleterious variants in genes partaking in homologous recombination repair of DNA. As clinical heterogeneity poses challenges for genetic counseling, this study focused on the occurrence and clinical relevance of double heterozygous BC in South Africa. METHODS: DNA samples were diagnostically screened using the NGS-based Oncomine™ BRCA Expanded Research Assay. Data was generated on the Ion GeneStudio S5 system and analyzed using the Torrent Suite™ and reporter software. The clinical significance of the variants detected was determined using international variant classification guidelines and treatment implications. RESULTS: Six of 1600 BC patients (0.375%) tested were identified as being bi-allelic for two germline likely pathogenic or pathogenic variants. Most of the variants were present in BRCA1/2, including two founder-related small deletions in three cases, with family-specific variants detected in ATM, BARD1, FANCD2, NBN, and TP53. The scientific interpretation and clinical relevance were based on the clinical and tumor characteristics of each case. CONCLUSION: This study increased current knowledge of the risk implications associated with the co-occurrence of more than one pathogenic variant in the BC susceptibility genes, confirmed to be a rare condition in South Africa. Further molecular pathology-based studies are warranted to determine whether clinical decision-making is affected by the detection of a second pathogenic variant in BRCA1/2 and TP53 carriers.

Navigating the genetic landscape of breast cancer in South Africa amidst a developing healthcare system.

Breast cancer is a significant global health issue as it represents the leading cause of death in women worldwide. In 2021, the World Health Organization established the Global Breast Cancer Initiative framework with the aim to reduce the breast cancer mortality rate by the year 2040. In countries with developing healthcare systems, such as South Africa, the implementation of first-world technologies has been slow. We provide an overview of the strides taken to improve the cost-effectiveness of genetic service delivery for breast cancer patients in South Africa - from advances in the technology utilized for BRCA founder genotyping to variant screening in moderate-to high-penetrance genes. We furthermore reflect on research undertaken to improve accessibility by means of population-directed point-of-care genetic testing that is ideal for use in a primary healthcare setting. We also report on a pilot study utilizing exome sequencing at the intersection between research and service delivery. Finally, we discuss and conclude on the controversies, research gaps, and future prospects based on the most recent developments in first-world countries that are implementable in developing countries to improve early detection of breast cancer and overall disease management.

Implementation of multigene panel testing for breast and ovarian cancer in South Africa: A step towards excellence in oncology for the public sector.

Translation of genomic knowledge into public health benefits requires the implementation of evidence-based recommendations in clinical practice. In this study, we moved beyond BRCA1/2 susceptibility testing in breast and ovarian cancer patients to explore the application of pharmacogenetics across multiple genes participating in homologous recombination DNA damage repair. This involved the utilisation of next-generation sequencing (NGS) at the intersection of research and service delivery for development of a comprehensive genetic testing platform in South Africa. Lack of international consensus regarding risk categorization of established cancer susceptibility genes and the level of evidence required for prediction of drug response supported the development of a central database to facilitate clinical interpretation. Here we demonstrate the value of this approach using NGS to 1) determine the variant spectrum applicable to targeted therapy and implementation of prevention strategies using the 15-gene Oncomine™ BRCA Expanded Panel, and 2) searched for novel and known pathogenic variants in uninformative cases using whole exome sequencing (WES). Targeted NGS performed as a routine clinical service in 414 South African breast and/or ovarian cancer patients resulted in the detection of 48 actionable variants among 319 (15%) cases. BRCA1/2-associated cancers were identified in 70.8% of patients (34/48, including two double-heterozygotes), with the majority (35.3%, 12/34) representing known South African founder variants. Detection of actionable variants in established non-BRCA1/2 risk genes contributed 29% to the total percentage (14/48), distributed amongst ATM, CHEK2, BARD1, BRIP1, PALB2 and TP53. Experimental WES using a virtually constructed multi-cancer NGS panel in 16 genetically unresolved cases (and four controls) revealed novel protein truncating variants in the basal cell carcinoma gene PTCH1 (c.4187delG) and the signal transmission and transduction gene KIT (c.930delA) involved in crucial cellular processes. Based on these findings, the most cost-effective approach would be to perform BRCA1/2 founder variant testing at referral, followed by targeted multigene panel testing if clinically indicated and addition of WES in unresolved cases. This inventive step provides a constant flow of new knowledge into the diagnostic platform via a uniquely South African pathology-supported genetic approach implemented for the first time in this context to integrate research with service delivery.

Prevalence of Clinically Relevant Germline BRCA Variants in a Large Unselected South African Breast and Ovarian Cancer Cohort: A Public Sector Experience.

Breast cancer is a multifaceted disease that currently represents a leading cause of death in women worldwide. Over the past two decades (1998-2020), the National Health Laboratory Service's Human Genetics Laboratory in central South Africa screened more than 2,974 breast and/or ovarian cancer patients for abnormalities characteristic of the widely known familial breast cancer genes, Breast Cancer gene 1 (BRCA1) and Breast Cancer gene 2 (BRCA2). Patients were stratified according to the presence of family history, age at onset, stage of the disease, ethnicity and mutation status relative to BRCA1/2. Collectively, 481 actionable (likely-to pathogenic) variants were detected in this cohort among the different ethnic/racial groups. A combination of old (pre-2014) and new (post-2014) laboratory techniques was used to identify these variants. Additionally, targeted genotyping was performed as translational research revealed the first three recurrent South African pathogenic variants, namely BRCA1 c.1374del (legacy name 1493delC), BRCA1 c.2641G>T (legacy name E881X) and BRCA2 c.7934del (legacy name 8162delG). This initial flagship study resulted in a cost-effective diagnostic test that enabled screening of a particular ethnic group for these variants. Since then, various non-Afrikaner frequent variants were identified that were proven to represent recurrent variants. These include BRCA2 c.5771_5774del (legacy name 5999del4) and BRCA2 c.582G>A, both Black African founder mutations. By performing innovative translational research, medical science in South Africa can adopt first-world technologies into its healthcare context as a developing country. Over the past two decades, the progress made in the public sector enabled a pivotal shift away from population-directed genetic testing to the screening of potentially all breast and ovarian cancer patients, irrespective of ethnicity, family history or immunohistochemical status. The modifications over the years complied with international standards and guidelines aimed at universal healthcare for all. This article shares all the cohort stratifications and the likely-to pathogenic variants detected.

Mutations in BRCA-related breast and ovarian cancer in the South African Indian population: A descriptive study.

Knowledge of the genetic landscape of a specific population group is vital for population-specific diagnosis and treatment of familial breast cancer. Although BRCA-related diagnostic testing has long been implemented in South Africa, the genotyping approach previously failed for the SA Indian population as it was based on other SA population groups. Because this population is uniquely admixed, the lack of population-specific data resulted in the implementation of comprehensive mutation screens for BRCA1/2. A total of 223 female patients were screened for clinically actionable variants. High-resolution melting analysis (HRMA) was used to screen 88 patients for DNA alterations in the coding and splice site boundaries of BRCA1 exons 2-9, BRCA1 exons 11-23, BRCA2 exons 2-9 and BRCA2 exons 12-27. The protein truncation test (PTT) was used to screen the three larger exons (BRCA1 exon 10 and BRCA2 exons 10 and 11) for protein termination changes. Multiplex ligation-dependent probe amplification (MLPA) was used to determine the presence of larger indels and possible copy number differences. Next Generation Sequencing (NGS) was performed on the remaining 135 samples. All potential variants were confirmed by performing Sanger DNA sequencing. The search revealed 28 different pathogenic heterozygotic variants, together with nine variants of unknown significance (VUS). The results suggested that the SA Indian population represents a different genetic admixture compared to that of mainland India, as only five pathogenic variants corresponded to those reported for mainland India. Familial breast cancer testing for SA Indian patients should therefore be performed as comprehensively as possible as the pathogenic variants seem to be family- rather than population-specific. Furthermore, predictive testing of family members will contribute to relieve the financial burden on the country's healthcare system, as increased surveillance and appropriate management could prevent disease.

The contribution of large genomic rearrangements in BRCA1 and BRCA2 to South African familial breast cancer.

BACKGROUND: Pathogenic variants that occur in the familial breast cancer genes (BRCA1/2) lead to truncated ineffective proteins in the majority of cases. These variants are mostly represented by small deletions/insertions, nonsense- and splice-site variants, although some larger pathogenic rearrangements occur. Currently, their contribution to familial breast cancer (BC) and ovarian cancer (OVC) in South Africa (SA) is unknown. METHODS: Seven hundred and forty-four patients affected with BC or OVC were screened for larger genomic rearrangements (LGRs) by means of multiplex ligation-dependent probe amplification or Next Generation Sequencing using the Oncomine™ BRCA research assay. RESULTS: The patients represented mostly medium to high-risk families, but also included lower risk patients without a family history of the disease, diagnosed at an early age of onset (< 40 years). Eight LGRs were detected (1.1%); seven in BRCA1 with a single whole gene deletion (WGD) detected for BRCA2. These eight LGRs accounted for 8.7% of the 92 BRCA1/2 pathogenic variants identified in the 744 cases. The pathogenic LGRs ranged from WGDs to the duplication of a single exon. CONCLUSIONS: Larger rearrangements in BRCA1/2 contributed to the overall mutational burden of familial BC and OVC in SA. Almost a quarter of all pathogenic variants in BRCA1 were LGRs (7/30, 23%). The spectrum observed included two WGDs, one each for BRCA1 and BRCA2.

Globally Rare BRCA2 Variants With Founder Haplotypes in the South African Population: Implications for Point-of-Care Testing Based on a Single-Institution BRCA1/2 Next-Generation Sequencing Study.

Breast cancer patients historically benefitted from population-based genetic research performed in South Africa, which led to the development of founder-based BRCA1/2 diagnostic tests. With the advent of next-generation sequencing (NGS) technologies, the clinical utility of limited, targeted genetic assays were questioned. The study focused on mining NGS data obtained from an extensive single-institution NGS series (n=763). The aims were to determine (i) the prevalence of the most common recurrent/founder variants in patients referred for NGS directly; and (ii) to explore the data for inferred haplotypes associated with previous and potential new recurrent/founder variants. The identification of additional founder variants was essential for promoting and potentially advancing to rapid founder-based BRCA1/2 point-of-care (POC) technology as a time- and cost-effective alternative. NGS revealed actionable BRCA1/2 variants in 11.1% of patients tested (BRCA1 - 4.7%; BRCA2 - 6.4%), of which 22.4% represented variants currently screened for using first-tier targeted genetic testing. A retrospective investigation into the overall mutation-positive rate for an extended cohort (n=1906), which included first-tier test results, revealed that targeted genetic testing identified 74% of all pathogenic variants. This percentage justified the use of targeted genetic testing as a first-tier assay. Inferred haplotype analysis confirmed the founder status of BRCA2 c.5771_5774del (rs80359535) and c.7934del (rs80359688) and revealed an additional African founder variant (BRCA2 c.582G>A - rs80358810). A risk-benefit analysis using a questionnaire-based survey was performed in parallel to determine genetic professionals' views regarding POC testing. This was done to bridge the clinical implementation gap between haplotype analysis and POC testing as a first-tier screen during risk stratification of breast and ovarian cancer patients. The results reflected high acceptance (94%) of BRCA1/2 POC testing when accompanied by genetic counselling. Establishing the founder status for several recurrent BRCA2 variants across ethnic groups supports unselected use of the BRCA POC assay in all SA breast/ovarian cancer patients by recent local and international public health recommendations. Incorporating POC genotyping into the planned NGS screening algorithm of the Department of Health will ensure optimal use of the country's recourses to adhere to the set standards for optimal care and management for all breast cancer patients.