Plasma versus tissue tumor mutational burden as biomarkers of durvalumab plus tremelimumab response in patients with metastatic colorectal cancer in the CO.26 trial.

PURPOSE: Tissue derived tumor mutation burden (TMB) of ≥10 mutations/Mb is a histology agnostic biomarker for the immune checkpoint inhibitor (ICI) pembrolizumab. However, the dataset on which this was validated lacked colorectal cancers (CRCs), and there is limited evidence for immunotherapy benefit in CRC using this threshold. PATIENTS AND METHODS: CO.26 was a randomized phase II study of 180 patients comparing durvalumab and tremelimumab (D+T, n=119 patients) versus best supportive care (BSC, n=61 patients). ctDNA sequencing was available for 168 patients (n=118 D+T, n=50), of which 165 had evaluable plasma TMB (pTMB). Tissue sequencing was available for 108 patients. Optimal thresholds for stratifying patients based on overall survival were determined using a minimal p-value approach. This report includes the final overall survival analysis. RESULTS: Tissue TMB ≥10 mutations/Mb was not predictive of benefit from D+T compared to BSC in microsatellite stable (MSS) metastatic CRC (HR 0.71 [95% CI:0.28-1.80], p=0.47). No tissue TMB threshold could identify a high TMB group that benefited from ICI. In contrast, plasma TMB (pTMB) ≥28 mutations/Mb was predictive of benefit from D+T (HR=0.34 [95%CI:0.13-0.85], p=0.022), as was clonal pTMB ≥10.6 mutations/Mb (HR=0.10 [95%CI:0.014-0.79], p=0.029) and subclonal pTMB ≥25.9/Mb (HR=0.20 [95% CI:0.061-0.69], p=0.010). Higher pTMB was associated with length of time on cytotoxic agents (p=0.021) and prior anti-EGFR exposure (p=2.44x10-06). CONCLUSION: pTMB derived from either clonal or subclonal mutations may identify a group more likely to benefit from immunotherapy, though validation is required. Tissue TMB provided no predictive utility for immunotherapy in this trial.

Variant Classification Discordance: Contributing Factors and Predictive Models.

An ever-growing catalog of human variants is hosted in the ClinVar database. In this database, submissions on a variant are combined into a multisubmitter record; and in the case of discordance in variant classification between submitters, the record is labeled as conflicting. The current study used ClinVar data to identify characteristics that would make variants more likely to be associated with the conflict class of variants. Furthermore, the Extreme Gradient Boosting algorithm was used to train classifier models to provide prediction of classification discordance for single submission variants in ClinVar database. Population allele frequency, the gene harboring the variant, variant type, consequence on protein, variant deleteriousness score, first submitter identity, and submission count were associated with conflict in variant classification. Using such features, the optimized classifier showed accuracy on the test set of 88% with the weighted average of precision, recall, and f1-score of 0.84, 0.88, and 0.85, respectively. There were pronounced associations between variant classification discordance and allele frequency, gene type, and the identity of the first submitter. The study provides the predicted discordance status for single-submitter variants deposited in ClinVar. This approach can be used to assess whether single-submitter variants are likely to be supported, or in conflict with, future entries; this knowledge may help laboratories with clinical variant assessment.

Implementing Next-Generation Sequencing Process Changes to Increase Capacity and Improve Timeliness of Molecular Biomarker Profiling for Lung Cancer Patients.

BACKGROUND: Faced with expansion of molecular tumor biomarker profiling, the molecular genetics laboratory at Kingston Health Science Centre experienced significant pressures to maintain the provincially mandated 2-week turnaround time (TAT) for lung cancer (LC) patients. We used quality improvement methodology to identify opportunities for improved efficiencies and report the impact of the initiative. METHODS: We set a target of reducing average TAT from accessioning to clinical molecular lab report for LC patients. Process measures included percentage of cases reaching TAT within target and number of cases. We developed a value stream map and used lean methodology to identify baseline inefficiencies. Plan-Do-Study-Act cycles were implemented to streamline, standardize, and automate laboratory workflows. Statistical process control (SPC) charts assessed for significance by special cause variation. RESULTS: A total of 257 LC cases were included (39 baseline January-May 2021; 218 post-expansion of testing June 2021). The average time for baseline TAT was 12.8 days, peaking at 23.4 days after expansion of testing, and improved to 13.9 days following improvement interventions, demonstrating statistical significance by special cause variation (nonrandom variation) on SPC charts. CONCLUSIONS: The implementation of standardized manual and automated laboratory processes improved timeliness of biomarker reporting despite the increasing volume of testing at our center.

Future Role of Health Technology Assessment for Genomic Medicine in Oncology: A Canadian Laboratory Perspective.

Genome-based testing in oncology is a rapidly expanding area of health care that is the basis of the emerging area of precision medicine. The efficient and considered adoption of novel genomic medicine testing is hampered in Canada by the fragmented nature of health care oversight as well as by lack of clear and transparent processes to support rapid evaluation, assessment, and implementation of genomic tests. This article provides an overview of some key barriers and proposes approaches to addressing these challenges as a potential pathway to developing a national approach to genomic medicine in oncology.

Programmed Cell Death Protein 1 Inhibitors and MET Targeted Therapies in NSCLC With MET Exon 14 Skipping Mutations: Efficacy and Toxicity as Sequential Therapies.

Introduction: NSCLC with MET exon 14 skipping mutation (METex14) is associated with poor outcomes. Integration of novel targeted therapies is challenging because of barriers in testing and drug access. We, therefore, sought to characterize the treatment patterns, outcomes, and emerging issues of treatment sequencing in patients with METex14-mutant NSCLC. Methods: We reviewed all NSCLC cases with METex14 alterations between 2014 and 2020 across four Canadian cancer centers. Demographics, disease characteristics, systemic therapy, overall response rates (ORRs), survival, and toxicity were summarized. Results: Among 64 patients with METex14-mutant NSCLC, the median overall survival was 23.1 months: 127.0 months in stage 1, 27.3 months in resected stage 2 and 3, and 16.6 months in unresectable stage 3 or 4 disease, respectively. In patients with advanced disease, 22% were too unwell for systemic treatment. MET tyrosine kinase inhibitors (TKIs) were administered to 28 patients with an ORR of 33%, median progression-free survival of 2.7 months, and 3.8 months for selective TKIs. Programmed cell death protein-1 (PD-1) inhibitors were given to 25 patients-the ORR was 44% and progression-free survival was 10.6 months. No responses were seen with subsequent MET TKIs after initial TKI treatment. Grade 3 or higher toxicities occurred in 64% of patients who received MET TKI after PD-1 inhibitors versus 8% in those who did not receive PD-1 inhibitors. Conclusions: Many patients with advanced METex14 NSCLC were too unwell to receive treatment. PD-1 inhibitors seem effective as an initial treatment, although greater toxicity was seen with subsequent MET TKIs. Thus, timely testing for METex14 skipping and initial therapy are imperative to improving patient survival.

Optimizing the number of variants tracked to follow disease burden with circulating tumor DNA assays in metastatic colorectal cancer.

Background: The number of somatic mutations detectable in circulating tumor DNA (ctDNA) is highly heterogeneous in metastatic colorectal cancer (mCRC). The optimal number of mutations required to assess disease kinetics is relevant and remains poorly understood. Objectives: To determine whether increasing panel breadth (the number of tracked variants in a ctDNA assay) would alter the sensitivity in detecting ctDNA in patients with mCRC. Design: We used archival tissue sequencing to perform an in silico assessment of the optimal number of tracked mutations to detect and monitor disease kinetics in mCRC using sequencing data from the Canadian Cancer Trials Group CO.26 trial. Methods: For each patient, 1, 2, 4, 8, 12, or 16 of the most clonal (highest variant allele frequency) somatic variants were selected from archival tissue-based whole-exome sequencing and assessed for the proportion of variants detected in matched ctDNA at baseline, week 8, and progression timepoints. Results: Data from 110 patients were analyzed. Genes most frequently encountered among the top four highest VAF variants in archival tissue were TP53 (51.9% of patients), APC (43.3%), KRAS (42.3%), and SMAD4 (9.6%). While the frequency of detecting at least one tracked variant increased when expanding beyond variant pool sizes of 1 and 2 in baseline (p = 0.0030) and progression (p = 0.0030) ctDNA samples, we observed no significant benefit to increases in variant pool size past four variants in any of the ctDNA timepoints (p < 0.05). Conclusion: While increasing panel breadth beyond two tracked variants improved variant re-detection in ctDNA samples from patients with treatment refractory mCRC, increases beyond four tracked variants yielded no significant improvement in variant re-detection.

Consensus Recommendations to Optimize the Detection and Reporting of NTRK Gene Fusions by RNA-Based Next-Generation Sequencing.

The detection of gene fusions by RNA-based next-generation sequencing (NGS) is an emerging method in clinical genetic laboratories for oncology biomarker testing to direct targeted therapy selections. A recent Canadian study (CANTRK study) comparing the detection of NTRK gene fusions on different NGS assays to determine subjects' eligibility for tyrosine kinase TRK inhibitor therapy identified the need for recommendations for best practices for laboratory testing to optimize RNA-based NGS gene fusion detection. To develop consensus recommendations, representatives from 17 Canadian genetic laboratories participated in working group discussions and the completion of survey questions about RNA-based NGS. Consensus recommendations are presented for pre-analytic, analytic and reporting aspects of gene fusion detection by RNA-based NGS.

A Novel Approach to Detect IDH Point Mutations in Gliomas Using Nanopore Sequencing: Test Validation for the Clinical Laboratory.

The use of standard next-generation sequencing technologies to detect key mutations in IDH genes for glioma diagnosis imposes several challenges, including high capital cost and turnaround delays associated with the need for batch testing. For both glioma testing and testing in other tumor types where highly specific mutation identification is required, the high-throughput nature of next-generation sequencing limits the feasibility of using it as a primary approach in clinical laboratories. We hypothesized that third-generation nanopore sequencing by Oxford Nanopore Technologies has the capability to overcome these limitations. This study aimed to develop and validate a nanopore-based IDH mutation detection assay for clinical practice using glioma formalin-fixed, paraffin-embedded (FFPE) tissue. Glioma FFPE (n = 66) samples with confirmed IDH gene mutational status were sequenced on the MinION device using an amplicon-based approach. All cases were concordant when compared with the reference results. Limit of blank and limit of detection for the variant allele fraction were 1.5% and 3.3%, respectively, at 500× read depth per gene. Total sequencing cost per sample was CAD$50 to CAD$134 with results being available in 9 to 15 hours. These findings demonstrate that nanopore-sequencing technology can be leveraged to develop low-cost, high-performance clinical sequencing-based assays with quick turnaround times to support the detection of targeted mutations in FFPE tumor tissue.

CANTRK: A Canadian Ring Study to Optimize Detection of NTRK Gene Fusions by Next-Generation RNA Sequencing.

The Canadian NTRK (CANTRK) study is an interlaboratory comparison ring study to optimize testing for neurotrophic receptor tyrosine kinase (NTRK) fusions in Canadian laboratories. Sixteen diagnostic laboratories used next-generation sequencing (NGS) for NTRK1, NTRK2, or NTRK3 fusions. Each laboratory received 12 formalin-fixed, paraffin-embedded tumor samples with unique NTRK fusions and two control non-NTRK fusion samples (one ALK and one ROS1). Laboratories used validated protocols for NGS fusion detection. Panels included Oncomine Comprehensive Assay v3, Oncomine Focus Assay, Oncomine Precision Assay, AmpliSeq for Illumina Focus, TruSight RNA Pan-Cancer Panel, FusionPlex Lung, and QIAseq Multimodal Lung. One sample was withdrawn from analysis because of sample quality issues. Of the remaining 13 samples, 6 of 11 NTRK fusions and both control fusions were detected by all laboratories. Two fusions, WNK2::NTRK2 and STRN3::NTRK2, were not detected by 10 laboratories using the Oncomine Comprehensive or Focus panels, due to absence of WNK2 and STRN3 in panel designs. Two fusions, TPM3::NTRK1 and LMNA::NTRK1, were challenging to detect on the AmpliSeq for Illumina Focus panel because of bioinformatics issues. One ETV6::NTRK3 fusion at low levels was not detected by two laboratories using the TruSight Pan-Cancer Panel. Panels detecting all fusions included FusionPlex Lung, Oncomine Precision, and QIAseq Multimodal Lung. The CANTRK study showed competency in detection of NTRK fusions by NGS across different panels in 16 Canadian laboratories and identified key test issues as targets for improvements.

Development of a comprehensive approach to adult hereditary cancer testing in Ontario.

BACKGROUND: Genetic testing for hereditary cancer susceptibility has advanced over time due to the discovery of new risk genes, improved technology and decreased cost. In the province of Ontario, testing eligibility criteria were initially developed to include hereditary breast, ovarian and colorectal cancer syndromes. The rapid evolution of genetic technologies has facilitated the ability to interrogate a large number of genes concurrently. This, coupled with new knowledge about risk genes, necessitated a coordinated approach to expanding the scope of genes and indications tested and synchronisation of access and test utilisation across the province as required in a publicly funded universal healthcare system. METHODS: Ontario Health-Cancer Care Ontario convened expert working groups to develop a standardised and comprehensive cancer gene list for adults and accompanying hereditary cancer testing (HCT) criteria using an evidence-based framework and broad laboratory and clinical genetics engagement. RESULTS: A standardised 76-cancer-gene panel, organised into 13 larger disease site panels and 25 single/small gene panels, was developed and endorsed by the working groups. Provincial genetic testing eligibility criteria were updated to align with the new panels and to guide clinical decision-making. In the first year following the implementation of these changes, 10 564 HCT panels were performed with an overall mutation detection rate of 12.2%. CONCLUSION: Using an evidence framework and broad clinical engagement to develop and endorse an updated guidance document, cancer genetic testing for adults in Ontario is now standardised and coordinated across the province.

Circulating Tumor DNA Identifies Diverse Landscape of Acquired Resistance to Anti-Epidermal Growth Factor Receptor Therapy in Metastatic Colorectal Cancer.

PURPOSE: Anti-epidermal growth factor receptor (EGFR) antibodies are effective treatments for metastatic colorectal cancer. Improved understanding of acquired resistance mechanisms may facilitate circulating tumor DNA (ctDNA) monitoring, anti-EGFR rechallenge, and combinatorial strategies to delay resistance. METHODS: Patients with treatment-refractory metastatic colorectal cancer (n = 169) enrolled on the CO.26 trial had pre-anti-EGFR tissue whole-exome sequencing (WES) compared with baseline and week 8 ctDNA assessments with the GuardantOMNI assay. Acquired alterations were compared between patients with prior anti-EGFR therapy (n = 66) and those without. Anti-EGFR therapy occurred a median of 111 days before ctDNA assessment. RESULTS: ctDNA identified 12 genes with increased mutation frequency after anti-EGFR therapy, including EGFR (P = .0007), KRAS (P = .0017), LRP1B (P = .0046), ZNF217 (P = .0086), MAP2K1 (P = .018), PIK3CG (P = .018), BRAF (P = .048), and NRAS (P = .048). Acquired mutations appeared as multiple concurrent subclonal alterations, with most showing decay over time. Significant increases in copy-gain frequency were noted in 29 genes after anti-EGFR exposure, with notable alterations including EGFR (P < .0001), SMO (P < .0001), BRAF (P < .0001), MET (P = .0002), FLT3 (P = .0002), NOTCH4 (P = .0006), ERBB2 (P = .004), and FGFR1 (P = .006). Copy gains appeared stable without decay 8 weeks later. There were 13 gene fusions noted among 11 patients, all but one of which was associated with prior anti-EGFR therapy. Polyclonal resistance was common with acquisition of ≥ 10 resistance related alterations noted in 21% of patients with previous anti-EGFR therapy compared with 5% in those without (P = .010). Although tumor mutation burden (TMB) did not differ pretreatment (P = .63), anti-EGFR exposure increased TMB (P = .028), whereas lack of anti-EGFR exposure resulted in declining TMB (P = .014). CONCLUSION: Paired tissue and ctDNA sequencing identified multiple novel mutations, copy gains, and fusions associated with anti-EGFR therapy that frequently co-occur as subclonal alterations in the same patient.

Prevalence of NTRK Fusions in Canadian Solid Tumour Cancer Patients.

INTRODUCTION: Neurotrophic tyrosine receptor kinase (NTRK) gene fusions occur in ~ 0.3% of all solid tumours but are enriched in some rare tumour types. Tropomyosin receptor kinase (TRK) inhibitors larotrectinib and entrectinib are approved as tumour-agnostic therapies for solid tumours harbouring NTRK fusions. METHODS: This study investigated the prevalence of NTRK fusions in Canadian patients and also aimed to help guide NTRK testing paradigms through analysis of data reported from a national clinical diagnostic testing program between September 2019 and July 2021. RESULTS: Of 1,687 patients included in the final analysis, NTRK fusions were detected in 0.71% (n = 12) of patients representing salivary gland carcinoma (n = 3), soft tissue sarcoma (n = 3), CNS (n = 3), and one in each of melanoma, lung, and colorectal cancer. All three salivary gland carcinomas contained ETV6-NTRK3 fusions. Thirteen (0.77%) clinically actionable incidental findings were also detected. Two of the 13 samples containing incidental findings were NTRK fusion-positive (GFOD1-NTRK2, FGFR3-TACC3 in a glioblastoma and AFAP1-NTRK2, BRAF c.1799T>A in a glioma). The testing algorithm screened most patient samples via pan-TRK immunohistochemistry (IHC), whereas samples from the central nervous system (CNS), pathognomonic cancers, and confirmed/ putative NTRK fusion-positive samples identified under research protocols were reflexed straight to next-generation sequencing (NGS). CONCLUSION: These findings highlight the benefit and practicality of a diagnostic testing program to identify patients suitable for tumour-agnostic TRK inhibitor therapies, as well as other targeted therapies, due to clinically actionable incidental findings identified. Collectively, these findings may inform future guidance on selecting the appropriate testing approach per tumour type and on optimal NTRK testing algorithms.

Precision Oncology in Canada: Converting Vision to Reality with Lessons from International Programs.

Canada's healthcare system, like others worldwide, is immersed in a process of evolution, attempting to adapt conventional frameworks of health technology assessment (HTA) and funding models to a new landscape of precision medicine in oncology. In particular, the need for real-world evidence in Canada is not matched by the necessary infrastructure and technologies required to integrate genomic and clinical data. Since healthcare systems in many developed nations face similar challenges, we adopted a solutions-based approach and conducted a search of worldwide programs in personalized medicine, with an emphasis on precision oncology. This search strategy included review articles published between 1 January 2016 and 1 March 2021 and hand-searches of their reference lists for relevant publications back to 1 December 2005. Thirty-nine initiatives across 37 countries in Europe, Australasia, Africa, and the Americas had the potential to lead to real-world data (RWD) on the clinical utility of oncology biomarkers. We highlight four initiatives with helpful lessons for Canada: Genomic Medicine France 2025, UNICANCER, the German Medical Informatics Initiative, and CANCER-ID. Among the 35 other programs evaluated, the main themes included the need for collaboration and systems to support data harmonization across multiple jurisdictions. In order to generate RWD in precision oncology that will prove acceptable to HTA bodies, Canada must take a national approach to biomarker strategy and unite all stakeholders at the highest level to overcome jurisdictional and technological barriers.

Optimizing the delivery of genetic and advanced diagnostic testing in the province of Ontario: challenges and implications for laboratory technology assessment and management in decentralized healthcare systems.

AIMS: The Canadian province of Ontario provides full coverage for its residents (pop.14.8 M) for hospital-based diagnostic testing. Historical governance of the healthcare system and a legacy scheme of health technology assessment (HTA) and financing has led to a suboptimal approach of adopting advanced diagnostic technology (i.e. protein expression, cytogenetic, and molecular/genetic) for guiding therapeutic decisions. The aim of this research is to explore systemic barriers and provide guidance to improve patient and care provider experiences by reducing delays and inequity of access to testing, while benefitting laboratory innovators and maximizing system efficiency. MATERIALS AND METHODS: A mixed-methods approach including literature review, semi-structured interviews, and a multi-stakeholder forum involving patient representatives (n = 1), laboratory leaders (n = 6), physicians (n = 5), Ministry personnel (n = 4), administrators (n = 3), extra-provincial experts, and researchers (n = 7), as well as pharmaceutical (n = 5) and diagnostic companies (n = 2). The forum considered evidence of good practices in adoption, implementation, and financing laboratory services and identified barriers as well as feasible options for improving advanced diagnostic testing in Ontario. RESULTS: Overarching challenges identified included: barriers to define what is needed; need for a clear approach to adoption; and the need for more oversight and coordination. Recommendations to address these included a shift to an anticipatory system of test adoption, creating a fit-for-purpose system of health technology management that consolidates existing evaluation processes, and modernizing the governance and financing of testing so that it is managed at a care-delivery level. CONCLUSIONS: The proposals for change in Ontario highlight the role that HTA, governance, and financing of health technology play along the continuum of a health technology life cycle within a healthcare system where decision-making is highly decentralized. Resource availability and capacity were not a concern - instead, solutions require higher levels of coordination and system integration along with innovative approaches to HTA.

Alignment of Fellowship Training with Practice Patterns for Molecular Pathologists: A Report of the Association for Molecular Pathology Training and Education Committee.

In the two decades since Accreditation Council for Graduate Medical Education-accredited Molecular Genetic Pathology fellowships began, the field of clinical molecular pathology has evolved considerably. The American Board of Pathology gathered data from board-certified molecular genetic pathologists assessing the alignment of skills and knowledge gained during fellowship with current needs on the job. The Association of Molecular Pathology conducted a parallel survey of program directors, and included questions on how various topics were taught during fellowship, as well as ranking their importance. Both surveys showed that most training aligned well with the practice needs of former trainees. Genomic profiling of tumors by next-generation sequencing, bioinformatics, laboratory management, and regulatory issues were topics thought to require increased emphasis in training. Topics related to clinical genetics and microbiology were deemed less important by those in practice, perhaps reflecting the increasing subspecialization of molecular pathologists. Program directors still viewed these topics as important to provide foundational knowledge. Parentage, identity, and human leukocyte antigen testing were less important to both survey audiences. These data may be helpful in guiding future adjustments to the Molecular Genetic Pathology curriculum and Accreditation Council for Graduate Medical Education program requirements.

Results of the phase I CCTG IND.231 trial of CX-5461 in patients with advanced solid tumors enriched for DNA-repair deficiencies.

CX-5461 is a G-quadruplex stabilizer that exhibits synthetic lethality in homologous recombination-deficient models. In this multicentre phase I trial in patients with solid tumors, 40 patients are treated across 10 dose levels (50-650 mg/m2) to determine the recommended phase II dose (primary outcome), and evaluate safety, tolerability, pharmacokinetics (secondary outcomes). Defective homologous recombination is explored as a predictive biomarker of response. CX-5461 is generally well tolerated, with a recommended phase II dose of 475 mg/m2 days 1, 8 and 15 every 4 weeks, and dose limiting phototoxicity. Responses are observed in 14% of patients, primarily in patients with defective homologous recombination. Reversion mutations in PALB2 and BRCA2 are detected on progression following initial response in germline carriers, confirming the underlying synthetic lethal mechanism. In vitro characterization of UV sensitization shows this toxicity is related to the CX-5461 chemotype, independent of G-quadruplex synthetic lethality. These results establish clinical proof-of-concept for this G-quadruplex stabilizer. Clinicaltrials.gov NCT02719977.

Clinical Utility of Multigene Profiling Assays in Early-Stage Invasive Breast Cancer: An Ontario Health (Cancer Care Ontario) Clinical Practice Guideline.

OBJECTIVE: The purpose of this guideline is to determine the clinical utility of multigene profiling assays in individuals with early-stage invasive breast cancer. METHODS: This guideline was developed by Ontario Health (Cancer Care Ontario)'s Program in Evidence-Based Care (PEBC) through a systematic review of relevant literature, patient- and caregiver-specific consultation and internal and external reviews. Recommendation 1: In patients with early-stage estrogen receptor (ER)-positive/human epidermal growth factor 2 (HER2)-negative breast cancer, clinicians should consider using multigene profiling assays (i.e., Oncotype DX, MammaPrint, Prosigna, EndoPredict, and the Breast Cancer Index) to help guide the use of systemic therapy. Recommendation 2: In patients with early-stage node-negative ER-positive/HER2-negative disease, clinicians may use a low-risk result from Oncotype DX, MammaPrint, Prosigna, EndoPredict/EPclin, or Breast Cancer Index assays to support a decision not to use adjuvant chemotherapy. Recommendation 3: In patients with node-negative ER-positive/HER2-negative disease, clinicians may use a high-risk result from Oncotype DX to support a decision to offer chemotherapy. A high Oncotype DX recurrence score is capable of predicting adjuvant chemotherapy benefit. Recommendation 4: In postmenopausal patients with ER-positive/HER2-negative tumours and one to three nodes involved (N1a disease), clinicians may withhold chemotherapy based on a low-risk Oncotype DX or MammaPrint score if the decision is supported by other clinical, pathological, or patient-related factors. Recommendation 5: The evidence to support the use of molecular profiling to select the duration of endocrine therapy is evolving. In patients with ER-positive disease, clinicians may consider using a Breast Cancer Index (H/I) high assay result to support a decision to extend adjuvant endocrine therapy if the decision is supported by other clinical, pathological, or patient-related factors.

BRCA1 Variant Assessment Using a Simple Analytic Assay.

BACKGROUND: We previously developed a biological assay to accurately predict BRCA1 (BRCA1 DNA repair associated) mutation status, based on gene expression profiles of Epstein-Barr virus-transformed lymphoblastoid cell lines. The original work was done using whole genome expression microarrays, and nearest shrunken centroids analysis. While these approaches are appropriate for model building, they are difficult to implement clinically, where more targeted testing and analysis are required for time and cost savings. METHODS: Here, we describe adaptation of the original predictor to use the NanoString nCounter platform for testing, with analysis based on the k-top scoring pairs (k-TSP) method. RESULTS: Assessing gene expression using the nCounter platform on a set of lymphoblastoid cell lines yielded 93.8% agreement with the microarray-derived data, and 87.5% overall correct classification of BRCA1 carriers and controls. Using the original gene expression microarray data used to develop our predictor with nearest shrunken centroids, we rebuilt a classifier based on the k-TSP method. This classifier relies on the relative expression of 10 pairs of genes, compared to the original 43 identified by nearest shrunken centroids (NSC), and was 96.2% concordant with the original training set prediction, with a 94.3% overall correct classification of BRCA1 carriers and controls. CONCLUSIONS: The k-TSP classifier was shown to accurately predict BRCA1 status using data generated on the nCounter platform and is feasible for initiating a clinical validation.

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.