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.

Incorporating functional genomics into the pathology-supported genetic testing framework implemented in South Africa: A future view of precision medicine for breast carcinomas.

A pathology-supported genetic testing (PSGT) framework was established in South Africa to improve access to precision medicine for patients with breast carcinomas. Nevertheless, the frequent identification of variants of uncertain significance (VUSs) with the use of genome-scale next-generation sequencing has created a bottleneck in the return of results to patients. This review highlights the importance of incorporating functional genomics into the PSGT framework as a proposed initiative. Here, we explore various model systems and experimental methods available for conducting functional studies in South Africa to enhance both variant classification and clinical interpretation. We emphasize the distinct advantages of using in vitro, in vivo, and translational ex vivo models to improve the effectiveness of precision oncology. Moreover, we highlight the relevance of methodologies such as protein modelling and structural bioinformatics, multi-omics, metabolic activity assays, flow cytometry, cell migration and invasion assays, tube-formation assays, multiplex assays of variant effect, and database mining and machine learning models. The selection of the appropriate experimental approach largely depends on the molecular mechanism of the gene under investigation and the predicted functional effect of the VUS. However, before making final decisions regarding the pathogenicity of VUSs, it is essential to assess the functional evidence and clinical outcomes under current variant interpretation guidelines. The inclusion of a functional genomics infrastructure within the PSGT framework will significantly advance the reclassification of VUSs and enhance the precision medicine pipeline for patients with breast carcinomas in South Africa.

Pathology-supported genetic testing for the application of breast cancer pharmacodiagnostics: family counselling, lifestyle adjustments and change of medication.

BACKGROUND: Pathology-supported genetic testing (PSGT) enables transitioning of risk stratification from the study population to the individual. RESEARCH DESIGN AND METHODS: We provide an overview of the translational research performed in postmenopausal breast cancer patients at increased risk of osteoporosis due to aromatase inhibitor therapy, as the indication for referral. Both tumor histopathology and blood biochemistry levels were assessed to identify actionable disease pathways using whole exome sequencing (WES). RESULTS: The causes and consequences of inadequate vitamin D levels as a modifiable risk factor for bone loss were highlighted in 116 patients with hormone receptor-positive breast cancer. Comparison of lifestyle factors and WES data between cases with vitamin D levels at extreme upper and lower ranges identified obesity as a major discriminating factor, with the lowest levels recorded during winter. Functional polymorphisms in the vitamin D receptor gene contributed independently to therapy-related osteoporosis risk. In a patient with invasive lobular carcinoma, genetic counseling facilitated investigation of the potential modifying effect of a rare CDH1 variant co-occurring with BRCA1 c.66dup (p.Glu23ArgfsTer18). CONCLUSION: Validation of PSGT as a three-pronged pharmacodiagnostics tool for generation of adaptive reports and data reinterpretation during follow-up represents a new paradigm in personalized medicine, exposing significant limitations to overcome.

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.

Immuno-Thrombotic Complications of COVID-19: Implications for Timing of Surgery and Anticoagulation.

Early in the coronavirus disease 2019 (COVID-19) pandemic, global governing bodies prioritized transmissibility-based precautions and hospital capacity as the foundation for delay of elective procedures. As elective surgical volumes increased, convalescent COVID-19 patients faced increased postoperative morbidity and mortality and clinicians had limited evidence for stratifying individual risk in this population. Clear evidence now demonstrates that those recovering from COVID-19 have increased postoperative morbidity and mortality. These data-in conjunction with the recent American Society of Anesthesiologists guidelines-offer the evidence necessary to expand the early pandemic guidelines and guide the surgeon's preoperative risk assessment. Here, we argue elective surgeries should still be delayed on a personalized basis to maximize postoperative outcomes. We outline a framework for stratifying the individual COVID-19 patient's fitness for surgery based on the symptoms and severity of acute or convalescent COVID-19 illness, coagulopathy assessment, and acuity of the surgical procedure. Although the most common manifestation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is COVID-19 pneumonitis, every system in the body is potentially afflicted by an endotheliitis. This endothelial derangement most often manifests as a hypercoagulable state on admission with associated occult and symptomatic venous and arterial thromboembolisms. The delicate balance between hyper and hypocoagulable states is defined by the local immune-thrombotic crosstalk that results commonly in a hemostatic derangement known as fibrinolytic shutdown. In tandem, the hemostatic derangements that occur during acute COVID-19 infection affect not only the timing of surgical procedures, but also the incidence of postoperative hemostatic complications related to COVID-19-associated coagulopathy (CAC). Traditional methods of thromboprophylaxis and treatment of thromboses after surgery require a tailored approach guided by an understanding of the pathophysiologic underpinnings of the COVID-19 patient. Likewise, a prolonged period of risk for developing hemostatic complications following hospitalization due to COVID-19 has resulted in guidelines from differing societies that recommend varying periods of delay following SARS-CoV-2 infection. In conclusion, we propose the perioperative, personalized assessment of COVID-19 patients' CAC using viscoelastic hemostatic assays and fluorescent microclot analysis.

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.

The cost impact of unselective vs selective MammaPrint testing in early-stage breast cancer in Southern Africa.

BACKGROUND: MammaPrint (MP) has been applied in South Africa (SA) for decision-making in early-stage hormone receptor-positive breast cancer since 2006. The cost-impact of MP in SA has not been assessed. AIM: To assess different MP testing strategies for cost-minimization in early-stage breast carcinoma using a funder perspective. METHODS: Clinico-pathologic information was extracted from a prospectively collected database. Clinical risk stratification was done using Adjuvant Online! (AOL) and the Predict V2.1 algorithm (www.predict.nhs.uk). An unselected MP testing strategy was compared to a selective strategy, testing only clinically high risk (cHigh) patients. Excluding human epidermal growth factor receptor-2 positive tumours, the costs for chemotherapy treatment and MP using funding data were used to evaluate the financial impact of these strategies. RESULTS: In 583 patients with 601 tumours, 52% were clinically low risk (cLow) (AOL) while the average Predict 10-year survival with chemotherapy was 2.9%. MP correlated strongly with Predict and 318 (60%) patients were MP low risk. Unselective testing allowed omission of chemotherapy in 44 (8.4%) patients but escalated cost by 57.7%. Using a selective testing strategy, only 251 would be tested, de-escalating treatment in 138 (55%) and reducing cost by 19.5%. Considering a Predict value up to 3.2% as cHigh, cost would be up to 7.3% (p = 0.0467) lower with a selective testing strategy. CONCLUSION: MP allowed reduction in the use of adjuvant chemotherapy. Unselective use of MP increases overall costs. A selective testing strategy through clinical risk stratification using AOL/Predict results in substantial cost saving.

CYP19A1 rs10046 Pharmacogenetics in Postmenopausal Breast Cancer Patients Treated with Aromatase Inhibitors: One-year Follow-up.

BACKGROUND: Significant individual variation in bone loss associated with aromatase inhibitors (AIs) emphasizes the importance of identifying postmenopausal breast cancer patients at high risk for this adverse effect. The study explores the clinical relevance of genetic variation in the Cytochrome P450 19A1 (CYP19A1) gene in a subset of South African patients during the first year of taking AIs for estrogen receptor (ER)-positive breast cancer. METHODS: The study population consisted of ER-positive breast cancer patients on AIs, followed in real-life clinical practice. Body mass index was measured and bone mineral density (BMD) was determined at baseline and at month 12. CYP19A1 genotyping was performed using real-time polymerase chain reaction analysis of rs10046, extended to Sanger sequencing and whole exome sequencing in 10 patients with more than 5% bone loss at month 12 at the lumbar spine. RESULTS: After 12 months of AI treatment, 72 patients had completed BMD and were successfully genotyped. Ten patients (14%) experienced more than 5% bone loss at the lumbar spine over the study period. Genotyping for CYP19A1 rs10046 revealed that patients with two copies of the A-allele were 10.79 times more likely to have an ordinal category change of having an increased percentage of bone loss or no increase at the lumbar spine, compared to patients with the GA or GG genotypes (CI of 1.771- 65.830, p=0.01). None of the 34 patients without lumbar spine bone loss at month 12 were homozygous for the functional CYP19A1 polymorphism. At the total hip region, patients with the AA genotype were 7. 37 times more likely to have an ordinal category change of having an increased percentage of bone loss or no increase (CI of 1.101- 49.336, p=0.04). CONCLUSION: Homozygosity for the CYP19A1 rs10046 A-allele may provide information, in addition to clinical and biochemical factors that may be considered in risk stratification to optimize bone health in postmenopausal breast cancer women on AIs. Further investigation is required to place the clinical effect observed for a single CYP19A1 gene variant in a genomic context.

Pioneering Informed Consent for Return of Research Results to Breast Cancer Patients Facing Barriers to Implementation of Genomic Medicine: The Kenyan BRCA1/2 Testing Experience Using Whole Exome Sequencing.

INTRODUCTION: Obtaining informed consent from study participants and disseminating the findings responsibly is a key principle required for ethically conducted clinical and genetic research. Reports from African researchers providing feedback on insights gained during the return of whole exome sequencing (WES) results to breast cancer patients treated in resource-limited settings is lacking. AIM: The empirical process used to fill this gap in relation to BRCA1/2 variant detection using WES provided unique insights incorporated into a pathology-supported genetic testing algorithm for return of research results to Kenyan breast cancer patients. METHODS: The Informed consent form approved by the Moi Teaching and Referral Hospital in Kenya was adopted from a translational research study conducted in South Africa. Initially, the informed consent process was piloted in 16 Kenyan female patients referred for breast surgery, following a community-based awareness campaign. A total of 95 female and two male breast cancer patients were enrolled in the study from 2013 to 2016. Immunohistochemistry (IHC) results of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER2) status were obtained from hospital records. DNA of patients with a family history of cancer was extracted from saliva and screened for pathogenic variants in the BRCA1/2 genes as the first step using WES. RESULTS: Ten patients approached for participation in this study declined to sign the informed consent form. Data on IHC used as a proxy for molecular subtype were available in 8 of 13 breast cancer patients (62%) with a family history of cancer. Five BRCA1/2 variants of uncertain clinical significance were detected, as well as a pathogenic BRCA2 variant (c.5159C > A; S1720∗) in a female patient eligible for return of WES results. CONCLUSION: Experience gained during the qualitative pilot phase was essential to overcome challenges associated with the translation of sophisticated genetic terms into native African languages. Detection of a pathogenic BRCA2 variant in a patient with familial breast cancer, frequently associated with hormone receptor-positive breast carcinoma as reported in this case, led to a high level of confidence on which to base risk management in future. Implementation of new technologies alongside standard pathology provides a practical approach to the application of genomic medicine in Africa.

The tumor genetics of acral melanoma: What should a dermatologist know?

Dermatologists stand at the gateway of individualization of classification, treatment, and outcomes of acral melanoma patients. The acral melanoma genetic landscape differs in vital ways from that of other cutaneous melanomas. These differences have important implications in understanding pathogenesis, treatment, and prognosis. The selection of molecularly targeted therapy must be adapted for acral melanoma. It is also critical to recognize that tumor development is far more complex than an isolated event, reliably treated by a medication acting on a single target. Tumors exhibit intratumor genetic heterogeneity, metastasis may have different genetic or epigenetic features than primary tumors, and tumor resistance may develop because of the activation of alternative genetic pathways. Microenvironmental, immune, and epigenetic events contribute and sustain tumors in complex ways. Treatment strategies with multiple targets are required to effectively disrupt the tumor ecosystem. This review attempts to translate the current molecular understanding of acral melanoma into digestible concepts relevant to the practice of dermatology. The focus is tumor genetics defining potentially treatable cancer pathways, contextualized within the relevant pathologic and molecular features.

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.

The relationship between measurement of in vivo brain glutamate and markers of iron metabolism: A proton magnetic resonance spectroscopy study in healthy adults.

Fundamental human studies which address associations between glutamate and iron metabolism are needed. Basic research reports associations between glutamate and iron metabolism. Human studies report sex differences in iron metabolism and glutamate concentrations, which suggest that these relationships may differ by sex. We hypothesised associations would be apparent between in vivo glutamate and peripheral markers of iron metabolism, and these associations would differ by sex. To test this, we recruited 40 healthy adults (20 men, 20 women) and measured (a) standard clinical biomarker concentrations for iron metabolism and (b) an in vivo proxy for glutamate concentration, glutamate with glutamine in relation to total creatine containing metabolites using proton magnetic resonance spectroscopy studies with a two-dimensional chemical shift imaging slice, with voxels located in bilateral dorsolateral prefrontal cortices, anterior cingulate cortices and frontal white matter. Only the female group reported significant associations between peripheral markers of iron metabolism and Glx:tCr concentration: (a) right dorsolateral prefrontal cortex Glx:tCr associated positively with serum transferrin (r = .60, p = .006) and negatively with transferrin saturation (r = -.62, p = .004) and (b) right frontal white matter Glx:tCr associated negatively with iron concentration (r = -.59, p = .008) and transferrin saturation (r = -.65, p = .002). Our results support associations between iron metabolism and our proxy for in vivo glutamate concentration (Glx:tCr). These associations were limited to women, suggesting a stronger regulatory control between iron and glutamate metabolism. These associations support additional fundamental research into the molecular mechanisms of this regulatory control.

Identification of an iron-responsive subtype in two children diagnosed with relapsing-remitting multiple sclerosis using whole exome sequencing.

BACKGROUND: Multiple sclerosis is a disorder related to demyelination of axons. Iron is an essential cofactor in myelin synthesis. Previously, we described two children (males of mixed ancestry) with relapsing-remitting multiple sclerosis (RRMS) where long-term remission was achieved by regular iron supplementation. A genetic defect in iron metabolism was postulated, suggesting that more advanced genetic studies could shed new light on disease pathophysiology related to iron. METHODS: Whole exome sequencing (WES) was performed to identify causal pathways. Blood tests were performed over a 10 year period to monitor the long-term effect of a supplementation regimen. Clinical wellbeing was assessed quarterly by a pediatric neurologist and regular feedback was obtained from the schoolteachers. RESULTS: WES revealed gene variants involved in iron absorption and transport, in the transmembrane protease, serine 6 (TMPRSS6) and transferrin (TF) genes; multiple genetic variants in CUBN, which encodes cubilin (a receptor involved in the absorption of vitamin B12 as well as the reabsorption of transferrin-bound iron and vitamin D in the kidneys); SLC25A37 (involved in iron transport into mitochondria) and CD163 (a scavenger receptor involved in hemorrhage resolution). Variants were also found in COQ3, involved with synthesis of Coenzyme Q10 in mitochondria. Neither of the children had the HLA-DRB1*1501 allele associated with increased genetic risk for MS, suggesting that the genetic contribution of iron-related genetic variants may be instrumental in childhood MS. In both children the RRMS has remained stable without activity over the last 10 years since initiation of nutritional supplementation and maintenance of normal iron levels, confirming the role of iron deficiency in disease pathogenesis in these patients. CONCLUSION: Our findings highlight the potential value of WES to identify heritable risk factors that could affect the reabsorption of transferrin-bound iron in the kidneys causing sustained iron loss, together with inhibition of vitamin B12 absorption and vitamin D reabsorption (CUBN) and iron transport into mitochondria (SLC25A37) as the sole site of heme synthesis. This supports a model for RRMS in children with an apparent iron-deficient biochemical subtype of MS, with oligodendrocyte cell death and impaired myelination possibly caused by deficits of energy- and antioxidant capacity in mitochondria.

Baseline bone health status in multi-ethnic South African postmenopausal breast cancer patients at initiation of aromatase inhibitor therapy: A descriptive study.

INTRODUCTION: Osteoporosis (OP) risk factor assessment and bone mineral density (BMD) testing are frequently omitted at baseline in aromatase inhibitor (AI) studies, which may lead to misinterpretation of AI associated bone loss. The present study describes bone health of South African postmenopausal women of predominantly Mixed Ancestry, prior to AI treatment. METHODS: This descriptive baseline study, nested in a prospective AI cohort study, included postmenopausal women with endocrine sensitive breast cancer, aged 50 to 80 years. A baseline questionnaire documented demographic-, medical-, lifestyle- and fracture history. Body weight was assessed clinically, and body composition and BMD measured via dual energy absorptiometry (DXA). Data was analysed in STATA 14 using descriptive and inferential statistics. RESULTS: 101 participants were recruited, with a mean age of 61±7 years. Nearly a third (n = 32) of women at baseline fulfilled global criteria for bone protection (BMD T-score ≥-2SD (n = 18); BMD T-score -1.5SD to < -2SD with risk factors (n = 14). Lower body weight, body mass index (BMI), fat mass index and lean mass index were significantly associated with the participants with a BMD measurement in keeping with a diagnosis of OP (p <0.001). Low vitamin D was present in 93% of the cohort tested (n = 95), whilst deficient vitamin D status (<20ng/ml) was documented in 52 women (55%). CONCLUSIONS: In this study, a third of postmenopausal women considered for AI therapy fulfilled international criteria for bone protective pharmacological intervention. This emphasizes the need for clinical risk and BMD assessment in postmenopausal breast cancer patients at baseline. Body composition and bone health associations highlight bone fragility associated with lower body weight.

Fractional anisotropy of white matter, disability and blood iron parameters in multiple sclerosis.

Multiple sclerosis (MS) is a disorder related to myelin damage, which can be investigated by neuroimaging techniques such as fractional anisotropy (FA), a measure of microstructural white matter properties. The objectives of this study were to investigate (1) the relationship between FA and disability using an extremes of outcome approach, and (2) whether blood iron parameters were associated with FA and/or disability. Patients diagnosed with MS (n = 107; 14 males and 93 females) had iron parameter tests and disability determinations using the Expanded Disability Status Scale (EDSS). FA was recorded in 48 white matter tracts in 11 of the female patients with MS and 12 female controls. RESULTS: In patients with high disability scores the mean FA was significantly lower (0.34 ± 0.067) than in the control group (0.45 ± 0.036; p = 0.04), while patients with low disability had mean FA values (0.44 ± 0.014) similar to controls (p = 0.5). Positive associations were found between FA and the iron parameters serum iron, ferritin and percentage transferrin saturation (%Tfsat) in all the white matter tracts. For % Tfsat, the associations were highly significant in 14 tracts (p < 0.01; r-values 0.74-0.84) and p < 0.001 (r = 0.83) in the superior fronto occipital fasciculus (LH). In the whole patient group a trend was found towards an inverse association between the EDSS and the %Tfsat (r = -0.26, p = 0.05) after excluding male gender and smoking as confounders, suggesting reduced disability in the presence of higher blood iron parameters. Additionally, significant inverse associations between disease duration and haemoglobin (p = 0.04) as well as %Tfsat (p = 0.02) suggested that patients with MS may experience a decrease in blood iron concentrations over time.

Clinical Overestimation of HER2 Positivity in Early Estrogen and Progesterone Receptor-Positive Breast Cancer and the Value of Molecular Subtyping Using BluePrint.

PURPOSE: Human epidermal growth factor receptor 2 (HER2) positivity is an important prognostic and predictive indicator in breast cancer. HER2 status is determined by immunohistochemistry and fluorescent in situ hybridization (FISH), which are potentially inaccurate techniques as a result of several technical factors, polysomy of chromosome 17, and amplification or overexpression of CEP17 (centromeric probe for chromosome 17) and/or HER2. In South Africa, HER2-positive tumors are excluded from a MammaPrint (MP; Agendia BV, Amsterdam, Netherlands) pretest algorithm. Clinical HER2 status has been reported to correlate poorly with molecular subtype. The aim of this study was to investigate the correlation of clinical HER2 status with BluePrint (BP) molecular subtyping. METHODS: Clinico-pathologic and genomic information was extracted from a prospectively collected central MP database containing records of 256 estrogen receptor-positive and/or progesterone receptor-positive tumors. Twenty-one tumors considered HER2 positive on immunohistochemistry or FISH were identified for this study. RESULTS: The median age of patients was 56 years (range, 34 to 77 years), with a median tumor size of 16 mm (3 to 27 mm). Four (19%) tumors were confirmed HER2-enriched subtype, six (29%) were luminal A, and 11 (52%) were luminal B. The positive predictive values of HER2/CEP17 ratio ≥ 2 and HER2 copy number ≥ 6 were only 29% and 40%, respectively. The differences in means for HER2/CEP17 ratio were significant between BP HER2-enriched versus luminal (P = .0249; 95% CI, 0.12 to 1.21) and MP high-risk versus low-risk tumors (P = .0002; 95% CI, 0.40 to 1.06). CONCLUSION: Of the 21 tumors considered clinically HER2 positive, only four were HER2-enriched subtype with BP, indicating an overestimation of HER2 positivity. FISH testing has a poor positive predictive value.

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.

Multiple sclerosis-like diagnosis as a complication of previously treated malaria in an iron and vitamin D deficient Nigerian patient.

In contrast to malaria, multiple sclerosis (MS) is infrequently found in Black Africans. We describe a 29 year old Nigerian female who developed an MS-like condition with symptoms similar to relapsing-remitting MS following malaria infection, leading to a diagnosis of MS. However, absence of hyperintense lesions in the brain and spinal cord presented a conundrum since not all the diagnostic criteria for MS were met. Pathology supported genetic testing (PSGT) was applied to combine family and personal medical history, lifestyle factors, and biochemical test results for interpretation of genetic findings. This approach provides a means of identifying risk factors for different subtypes of demyelinating disease. The patient was subsequently treated according to an individualised intervention program including nutritional supplementation as well as a change in diet and lifestyle. Deficiencies of vitamin B12, iron and vitamin D were addressed. Genetic analysis revealed absence of the HLA DRB1*1501 allele, considered to be the most prominent genetic risk factor for MS. Extended mutation analysis identified variations in three genes in the folate-vitamin B12 metabolic pathway, which could have increased the patient's sensitivity to the antifolate drugs used to treat the malaria. A glutathione-S-transferase GSTM1 null allele, previously associated with neurological complications of malaria, was also detected. Furthermore, a heterozygous variation in the iron-related transmembrane protease serine 6 (TMPRSS6) gene, rs855791 was found, which could have impacted the patient's iron status following two successive blood donations and exposure to malaria preceding the MS diagnosis. PSGT identifies relevant risk factors for demyelinating disorders resembling MS and uses the data for individualised treatment programs, and to systematically build a database that can provide evidence in large patient cohorts. Follow-up investigations may be suggested, such as whole exome sequencing in selected cases, to ensure that remyelination and restoration of function are achieved.