Variant classification changes over time in the clinical molecular diagnostic laboratory setting.

BACKGROUND: Variant classification in the setting of germline genetic testing is necessary for patients and their families to receive proper care. Variants are classified as pathogenic (P), likely pathogenic (LP), uncertain significance (VUS), likely benign (LB) and benign (B) using the standards and guidelines recommended by the American College of Medical Genetics and the Association for Molecular Pathology, with modifications for specific genes. As the literature continues to rapidly expand, and evidence continues to accumulate, prior classifications can be updated accordingly. In this study, we aim to characterise variant reclassifications in Ontario. METHODS: DNA samples from patients seen at hereditary cancer clinics in Ontario from January 2012 to April 2022 were submitted for testing. Patients met provincial eligibility criteria for testing for hereditary cancer syndromes or polycystic kidney disease. Reclassification events were determined to be within their broader category of significance (B to LB or vice versa, or P to LP or vice versa) or outside of their broader category as significance (ie, significant reclassifications from B/LB or VUS or P/LP, from P/LP to VUS or B/LB, or from VUS to any other category). RESULTS: Of the 8075 unique variants included in this study, 23.7% (1912) of variants were reassessed, and 7.2% (578) of variants were reclassified. Of these, 351 (60.7%) variants were reclassified outside of their broader category of significance. Overall, the final classification was significantly different for 336 (58.1%) variants. Importantly, most reclassified variants were downgraded to a more benign classification (n=245; 72.9%). Of note, most reclassified VUS was downgraded to B/LB (n=233; 84.7%). CONCLUSIONS: The likelihood for reclassification of variants on reassessment is high. Most reclassified variants were downgraded to a more benign classification. Our findings highlight the importance of periodic variant reassessment to ensure timely and appropriate care for patients and their families.

Validation of low-pass genome sequencing for prenatal diagnosis.

OBJECTIVE: Chromosomal microarray (CMA), while considered the gold standard for detecting copy number variants (CNVs) in prenatal diagnostics, has its limitations, including the necessity to replace aging microarray equipment, low throughput, a static design, and an inefficient multi-day workflow. This study evaluates the feasibility of low-pass genome sequencing (LP-GS) as a potential replacement for CMA in prenatal diagnostics. METHODS: We comprehensively compared LP-GS at 10x and 5x average depths with CMA in a prenatal laboratory. We examined parameters, including concordance, sensitivity, specificity, workflow efficiency, and cost-effectiveness. RESULTS: We found a high degree of agreement between LP-GS and CMA for detecting CNVs and absence of heterozygosity. Furthermore, compared to CMA, LP-GS increased workflow efficiency and proved to be cost-neutral at 10x and cost-effective at 5x. CONCLUSION: Our study suggests that LP-GS is a promising alternative to CMA in prenatal diagnostics, offering advantages, including a more efficient workflow and scalability for larger testing volumes. Importantly, for clinical laboratories that have adopted next-generation sequencing in a separate capacity, LP-GS facilitates a unified NGS-centric approach, enabling workflow consolidation. By offering a single, streamlined platform for detecting a broad range of genetic variants, LP-GS may represent a critical step toward enhancing the diagnostic capabilities of prenatal laboratories.

Recontact to return new or updated PALB2 genetic results in the clinical laboratory setting.

OBJECTIVE: The purpose of this study was to recontact individuals with clinically actionable test results identified through a retrospective research study and to provide a framework for laboratories to recontact patients. METHODS: Genetic testing was conducted on 2977 individuals originally referred for BRCA1 and BRCA2 hereditary breast and ovarian cancer testing that had a negative genetic test result. A gene panel was used to identify pathogenic variants in known or newly discovered genes that could explain the underlying cause of disease; however, analysis was restricted to PALB2 for the purposes of this study. A patient recontact decision tree was developed to assist in the returning of updated genetic test results to clinics and patients. RESULTS: Novel clinically actionable pathogenic variants were identified in the PALB2 gene in 18 participants (0.6%), the majority of whom were recontacted with their new or updated genetic test results. Eight individuals were unable to be recontacted; five individuals had already learnt about their new or updated findings from genetic testing outside the context of this study; three individuals prompted cascade testing in family members; two individuals were deceased. CONCLUSION: Novel pathogenic variants in PALB2 were identified in 18 individuals through retrospective gene panel testing. Recontacting these individuals regarding these new or updated findings had a range of outcomes. The process of conveying genomic results within this framework can be effectively accomplished while upholding patient autonomy, potentially leading to advantageous outcomes for patients and their families.

A model for the return and referral of all clinically significant secondary findings of genomic sequencing.

Secondary findings (SFs) identified through genomic sequencing (GS) can offer a wide range of health benefits to patients. Resource and capacity constraints pose a challenge to their clinical management; therefore, clinical workflows are needed to optimise the health benefits of SFs. In this paper, we describe a model we created for the return and referral of all clinically significant SFs, beyond medically actionable results, from GS. As part of a randomised controlled trial evaluating the outcomes and costs of disclosing all clinically significant SFs from GS, we consulted genetics and primary care experts to determine a feasible workflow to manage SFs. Consensus was sought to determine appropriate clinical recommendations for each category of SF and which clinician specialist would provide follow-up care. We developed a communication and referral plan for each category of SFs. This involved referrals to specialised clinics, such as an Adult Genetics clinic, for highly penetrant medically actionable findings. Common and non-urgent SFs, such as pharmacogenomics and carrier status results for non-family planning participants, were directed back to the family physician (FP). SF results and recommendations were communicated directly to participants to respect autonomy and to their FPs to support follow-up of SFs. We describe a model for the return and referral of all clinically significant SFs to facilitate the utility of GS and promote the health benefits of SFs. This may serve as a model for others returning GS results transitioning participants from research to clinical settings.

Data sharing to improve concordance in variant interpretation across laboratories: results from the Canadian Open Genetics Repository.

BACKGROUND: This study aimed to identify and resolve discordant variant interpretations across clinical molecular genetic laboratories through the Canadian Open Genetics Repository (COGR), an online collaborative effort for variant sharing and interpretation. METHODS: Laboratories uploaded variant data to the Franklin Genoox platform. Reports were issued to each laboratory, summarising variants where conflicting classifications with another laboratory were noted. Laboratories could then reassess variants to resolve discordances. Discordance was calculated using a five-tier model (pathogenic (P), likely pathogenic (LP), variant of uncertain significance (VUS), likely benign (LB), benign (B)), a three-tier model (LP/P are positive, VUS are inconclusive, LB/B are negative) and a two-tier model (LP/P are clinically actionable, VUS/LB/B are not). We compared the COGR classifications to automated classifications generated by Franklin. RESULTS: Twelve laboratories submitted classifications for 44 510 unique variants. 2419 variants (5.4%) were classified by two or more laboratories. From baseline to after reassessment, the number of discordant variants decreased from 833 (34.4% of variants reported by two or more laboratories) to 723 (29.9%) based on the five-tier model, 403 (16.7%) to 279 (11.5%) based on the three-tier model and 77 (3.2%) to 37 (1.5%) based on the two-tier model. Compared with the COGR classification, the automated Franklin classifications had 94.5% sensitivity and 96.6% specificity for identifying actionable (P or LP) variants. CONCLUSIONS: The COGR provides a standardised mechanism for laboratories to identify discordant variant interpretations and reduce discordance in genetic test result delivery. Such quality assurance programmes are important as genetic testing is implemented more widely in clinical care.

Multigene panel testing for hereditary breast and ovarian cancer in the province of Ontario.

PURPOSE: The aim of this study was to determine the diagnostic yield of multigene panel testing among patients referred with hereditary breast and ovarian cancer (HBOC). METHODS: Patients who met provincial eligibility criteria were tested at the Advanced Molecular Diagnostic Laboratory at Mount Sinai Hospital, Toronto. Gene sequencing and exon-level copy number variant (CNV) analysis was performed. The referring physician had the opportunity to choose between several different gene panels based on patient phenotype. Cases were included in the analysis based on personal and family history of cancer and the type of panel ordered. RESULTS: 3251 cases that received panel testing were included in this analysis. Overall, 9.1% (295) had a positive (pathogenic or likely pathogenic) result and 27.1% (882) had an inconclusive result (variant of uncertain significance). The genes with the highest prevalence of positive results were in BRCA2 (2.2%, 71/3235), BRCA1 (1.9%, 62/3235), and CHEK2 (1.4%, 40/2916). Of the positive cases, 9.8% (29) had a pathogenic or likely pathogenic variant in a gene associated with Lynch syndrome (MSH6, MSH2, MLH1, or PMS2). CONCLUSIONS: Our overall positive yield is similar to that reported in the literature. The yield of inconclusive results was three times that of positive results. By testing more individuals in families with HBOC and through data-sharing efforts, the clinical significance of most variants may eventually be determined and panel testing for monogenic cancer predisposition syndromes will have greater utility.

Canadian genetic healthcare professionals' attitudes towards discussing private pay options with patients.

BACKGROUND: Just as there is inconsistency with respect to coverage of genomic testing with insurance carriers, there is interprovincial discrepancy in Canada. Consequently, the option of private pay (e.g., self pay) arises, which can lead to inequities in access, particularly when patients may not be aware of this option. There are currently no published data regarding how the Canadian genetics community handles discussions of private pay options with patients. The purpose of this study was to assess the attitudes of genetic healthcare professionals (GHPs: medical geneticists, genetic counselors, and genetic nurses) practicing in Canada toward these discussions. METHODS: An online survey was distributed to members of the Canadian College of Medical Geneticists and the Canadian Association of Genetic Counsellors to assess frequencies, rationale, and ethical considerations regarding these conversations. Quantitative data were analyzed using descriptive statistics. RESULTS: Of 144 respondents, 95% reported discussing private pay and 65% reported working in a clinic without a policy on this issue. There were geographic and practice-specific differences. The most common circumstance for these discussions was when a test was clinically indicated (e.g., but funding was denied) followed by when the patient initiated the conversation. The most frequently discussed tests included: multi-gene panels (73% of respondents), noninvasive prenatal testing (62%), and pre-implantation genetic diagnosis (58%). Although 65% felt it was ethical to discuss private pay, 35% indicated it was "sometimes" ethical. CONCLUSION: With the increasing availability of genomic technologies, these findings inform how we practice and demonstrate the need for policy in this area.