Pioneering BRCA1/2 Point-Of-Care Testing for Integration of Germline and Tumor Genetics in Breast Cancer Risk Management: A Vision for the Future of Translational Pharmacogenomics.

Research performed in South African (SA) breast, ovarian and prostate cancer patients resulted in the development of a rapid BRCA point-of-care (POC) assay designed as a time- and cost-effective alternative to laboratory-based technologies currently used for first-tier germline DNA testing. In this study the performance of the new assay was evaluated for use on a portable screening device (ParaDNA), with the long-term goal to enable rollout at POC as an inventive step to meet the World Health Organization's sustainable development goals for Africa. DNA samples for germline testing were obtained retrospectively from 50 patients with early-stage hormone receptor-positive breast cancer referred for genomic tumor profiling (MammaPrint). Currently, SA patients with the luminal-type breast cancer are not routinely selected for BRCA1/2 testing as is the case for triple-negative disease. An initial evaluation involved the use of multiple control samples representing each of the pathogenic founder/recurrent variants included in the BRCA 1.0 POC Research Assay. Comparison with a validated laboratory-based first-tier real-time polymerase chain reaction (PCR) assay demonstrated 100% concordance. Clinical utility was evident in five patients with the founder BRCA2 c.7934delG variant, identified at the 10% (5/50) threshold considered cost-effective for BRCA1/2 testing. BRCA2 c.7934delG carrier status was associated with a significantly younger age (p=0.03) at diagnosis of breast cancer compared to non-carriers. In three of the BRCA2 c.7934delG carriers a high-risk MammaPrint 70-gene profile was noted, indicating a significantly increased risk for both secondary cancers and breast cancer recurrence. Initiating germline DNA testing at the POC for clinical interpretation early in the treatment planning process, will increase access to the most common pathogenic BRCA1/2 variants identified in SA and reduce loss to follow-up for timely gene-targeted risk reduction intervention. The ease of using cheek swabs/saliva in future for result generation within approximately one hour assay time, coupled with low cost and a high BRCA1/2 founder variant detection rate, will improve access to genomic medicine in Africa. Application of translational pharmacogenomics across ethnic groups, irrespective of age, family history, tumor subtype or recurrence risk profile, is imperative to sustainably implement preventative healthcare and improve clinical outcome in resource-constrained clinical settings.

Exome Sequencing in a Family with Luminal-Type Breast Cancer Underpinned by Variation in the Methylation Pathway.

Panel-based next generation sequencing (NGS) is currently preferred over whole exome sequencing (WES) for diagnosis of familial breast cancer, due to interpretation challenges caused by variants of uncertain clinical significance (VUS). There is also no consensus on the selection criteria for WES. In this study, a pathology-supported genetic testing (PSGT) approach was used to select two BRCA1/2 mutation-negative breast cancer patients from the same family for WES. Homozygosity for the MTHFR 677 C>T mutation detected during this PSGT pre-screen step was considered insufficient to cause bilateral breast cancer in the index case and her daughter diagnosed with early-onset breast cancer (<30 years). Extended genetic testing using WES identified the RAD50 R385C missense mutation in both cases. This rare variant with a minor allele frequency (MAF) of <0.001 was classified as a VUS after exclusion in an affected cousin and extended genotyping in 164 unrelated breast cancer patients and 160 controls. Detection of functional polymorphisms (MAF > 5%) in the folate pathway in all three affected family members is consistent with inheritance of the luminal-type breast cancer in the family. PSGT assisted with the decision to pursue extended genetic testing and facilitated clinical interpretation of WES aimed at reduction of recurrence risk.

Impact of MammaPrint on Clinical Decision-Making in South African Patients with Early-Stage Breast Cancer.

The aim of the study was to evaluate the impact of MammaPrint on treatment decision-making in patients with breast cancer. Clinicopathologic information of all breast cancer patients referred for MammaPrint testing in South Africa was collected from 2007 until 2014. A total of 107 patients (109 tumors) with estrogen receptor/progesterone receptor positive and human epidermal growth factor receptor-2 negative tumors were selected with tumors ≥10 mm, or when 1-3 nodes were involved without extra-nodal extension. None of the clinical indicators correlated significantly with the MammaPrint risk classification, which changed the decision for adjuvant chemotherapy in 52% of patients. Of 60 patients who were clinically high risk, 62% had a low-risk MammaPrint result and of the 47 clinically low -risk patients 40% had a high-risk MammaPrint result. This study indicates that MammaPrint could reduce the need for adjuvant chemotherapy by 17% using the selection criteria stipulated. The significant impact on treatment decisions confirmed the clinical utility of MammaPrint independent of standard clinicopathologic risk factors as supported by long-term clinical outcome studies.

Incorporating microarray assessment of HER2 status in clinical practice supports individualised therapy in early-stage breast cancer.

Accurate determination of human epidermal growth factor receptor-2 (HER2) status is essential for optimal selection of breast cancer patients for gene targeted therapy. The analytical performance of microarray analysis using TargetPrint for assessment of HER2 status was evaluated in 138 breast tumours, including 41 fresh and 97 formalin-fixed paraffin embedded (FFPE) specimens. Reflex testing using immunohistochemistry/in situ hybridization (IHC/ISH) in four discordant cases confirmed the TargetPrint results, achieving 100% agreement regardless of whether fresh tissue or FFPE specimens were used. One equivocal IHC/ISH case was classified as HER2-positive based on the microarray result. The proven clinical utility in resolving equivocal and borderline cases justifies modification of the testing algorithm under these circumstances, to obtain a definitive positive or negative test result with the use of microarrays. Determination of HER2 status across three assay platforms facilitated improved quality assurance and led to a higher level of confidence on which to base treatment decisions.

Genomic medicine and risk prediction across the disease spectrum.

Genomic medicine is based on the knowledge that virtually every medical condition, disease susceptibility or response to treatment is caused, regulated or influenced by genes. Genetic testing may therefore add value across the disease spectrum, ranging from single-gene disorders with a Mendelian inheritance pattern to complex multi-factorial diseases. The critical factors for genomic risk prediction are to determine: (1) where the genomic footprint of a particular susceptibility or dysfunction resides within this continuum, and (2) to what extent the genetic determinants are modified by environmental exposures. Regarding the small subset of highly penetrant monogenic disorders, a positive family history and early disease onset are mostly sufficient to determine the appropriateness of genetic testing in the index case and to inform pre-symptomatic diagnosis in at-risk family members. In more prevalent polygenic non-communicable diseases (NCDs), the use of appropriate eligibility criteria is required to ensure a balance between benefit and risk. An additional screening step may therefore be necessary to identify individuals most likely to benefit from genetic testing. This need provided the stimulus for the development of a pathology-supported genetic testing (PSGT) service as a new model for the translational implementation of genomic medicine in clinical practice. PSGT is linked to the establishment of a research database proven to be an invaluable resource for the validation of novel and previously described gene-disease associations replicated in the South African population for a broad range of NCDs associated with increased cardio-metabolic risk. The clinical importance of inquiry concerning family history in determining eligibility for personalized genotyping was supported beyond its current limited role in diagnosing or screening for monogenic subtypes of NCDs. With the recent introduction of advanced microarray-based breast cancer subtyping, genetic testing has extended beyond the genome of the host to also include tumor gene expression profiling for chemotherapy selection. The decreasing cost of next generation sequencing over recent years, together with improvement of both laboratory and computational protocols, enables the mapping of rare genetic disorders and discovery of shared genetic risk factors as novel therapeutic targets across diagnostic boundaries. This article reviews the challenges, successes, increasing inter-disciplinary integration and evolving strategies for extending PSGT towards exome and whole genome sequencing (WGS) within a dynamic framework. Specific points of overlap are highlighted between the application of PSGT and exome or WGS, as the next logical step in genetically uncharacterized patients for whom a particular disease pattern and/or therapeutic failure are not adequately accounted for during the PSGT pre-screen. Discrepancies between different next generation sequencing platforms and low concordance among variant-calling pipelines caution against offering exome or WGS as a stand-alone diagnostic approach. The public reference human genome sequence (hg19) contains minor alleles at more than 1 million loci and variant calling using an advanced major allele reference genome sequence is crucial to ensure data integrity. Understanding that genomic risk prediction is not deterministic but rather probabilistic provides the opportunity for disease prevention and targeted treatment in a way that is unique to each individual patient.