Testing For Cancer Predisposition Genes Research Articles

An analysis of the impact of annual cancer genetic testing guideline updates on a past patient population.

Germline genetic testing for cancer predisposition genes has become an essential component of cancer treatment and risk reduction. The National Comprehensive Cancer Network (NCCN) releases annual genetic testing guidelines that identify characteristics of patients that could be affected by a hereditary cancer syndrome. These guidelines have broadened over time and the implications for past patients of cancer genetics clinics are not well understood. This study is a retrospective chart review aimed at determining the percentage and characteristics of past patients that meet updated NCCN guidelines (Breast, Ovarian, and Pancreas [BOP] v1.2022 and Colorectal [CRC] v1.2021), patients that attended a follow-up appointment, and patients who went on to receive genetic testing. Clinical data and characteristics were compared between the study population as a whole and the cohort of patients that met updated NCCN guidelines BOP v1.2022 and CRC v1.2021. The study population consisted of 280 patients with 76 (27.1%) patients meeting updated NCCN guidelines BOP v1.2022 and CRC v1.2021. The year of initial cancer genetic counseling appointment was statistically significant (p = 0.023) with patients more likely to meet NCCN guidelines BOP v1.2022 and CRC v1.2021 with earlier initial cancer genetic counseling appointments. In the cohort that met updated NCCN guidelines BOP v1.2022 and CRC v1.2021, the most common reason was a change in the NCCN guidelines (BOP or CRC) (54/76, 71.1%) with triple-negative breast cancer diagnosed at any age being the most impactful guideline change (19/54, 35.2%). Twenty-one patients attended a follow-up appointment (7.5%) and of those that received genetic testing (17/21, 81%) most received negative results (13/17, 61.9%), with one pathogenic, low penetrance result (1/17, 5.9%, CHEK2 p.I157T). Provider-initiated follow-up was attributed to most follow-up appointments (16/21, 76.2%) implying patients do not tend to follow-up on their own. Education to non-genetics providers as well as targeted implementation of follow-up protocols possibly managed by genetic counseling assistants and utilizing electronic medical record (EMR) patient messaging could lead to improved patient follow-up.

Therapeutic applications of germline testing for cancer predisposition genes in Asia in the real world.

10614 Background: Germline genetic testing is traditionally done in patients suspected with hereditary cancer syndrome for enhanced cancer surveillance and/or prevention. In recent years, germline genetic testing is increasingly performed for therapeutic indication, given the approval of PARP inhibitor in advanced cancer patients with germline BRCA1/2 or other homologous recombination repair (HRR) gene mutations, and immunotherapy for patients with mismatch repair gene mutations. We evaluated the clinical utility of actionable (treatable) pathogenic germline mutations (PGM) in a tertiary cancer center in Asia. Methods: We conducted a retrospective review of cancer patients who underwent germline genetic testing at the National University Cancer Institute, Singapore, Cancer Genetics Clinic. The primary objectives were to determine the prevalence of potentially actionable PGM in cancer predisposition genes and their therapeutic applications, with focus on BRCA, HRR and mismatch repair genes. Results: From 2000 till 2022, 1154 cancer patients underwent germline genetic testing, with the majority (81.9%) tested with multi-gene panels comprising 9-134 genes. 93% (n = 1069) fulfilled conventional criteria for genetic testing; 7% (n = 81) were tested specifically for treatment decisions. 411 (35.6%) patients tested positive for a PGM, of which 334 (81%) were potentially actionable. BRCA1/2 mutations accounted for 62.3% of actionable mutations, followed by mismatch repair (18%; MLH1 8.7%, MSH2 6%, MSH6 2.1%, PMS2 1.2%), and other HRR genes (19.7%: RAD51 6.3%, ATM 4.8%, PALB2 3%, BRIP1 2.4%, others 3.3%). Among patients with BRCA/HRR gene mutations, breast (60%) and ovarian cancer (26.7%) were the commonest primary cancer. 152 germline positive patients have advanced cancers, and 79 (52%) patients received germline directed therapies (PARP inhibitor: n = 75; immunotherapy: n = 4). Median duration of immunotherapy in the 4 mismatch repair gene carriers was 20.5 months (range 5-40). Among germline BRCA/HRR mutation carriers with advanced cancer who were treated with PARP inhibitor, ovarian cancer was the commonest (n = 49), followed by breast (n = 14) then prostate (n = 6). Median PARP inhibitor treatment duration was 8 months (range 1-76). Among germline BRCA/HRR mutation carriers who received platinum-based chemotherapy, pathological complete response rate in the neoadjuvant setting was 53% (n = 17 breast cancers; TNBC [n = 11], other subtypes [n = 6]) and objective response rate was > 80% in the advanced setting (n = 71; ovarian = 51, breast = 9, others = 11). Conclusions: About one-third of cancer patients tested in a high risk Cancer Genetics Clinic in a tertiary cancer centre in Asia carry PGM, of which ~80% are potentially actionable. > 50% advanced cancer patients with actionable PGM received germline directed therapies, confirming the clinical utility of germline testing in the real world.

Racial Disparities in Family Variant Testing for Cancer Predisposition Genes

Purpose: Despite the substantial clinical impact of genetic testing, racial disparities exist in the delivery of this service. Here, we partnered with a commercial laboratory (Invitae) to establish whether there are racial disparities in the uptake of family variant testing (FVT). We also investigated if providing FVT at no cost impacts rates of cascade testing in Black and White families. Methods: This is a retrospective analysis comparing rates of FVT in self-reporting Black probands to self-reporting White probands who underwent germline genetic testing for genes associated with hereditary cancer through Invitae. All Black and White patients found to have a pathogenic/likely pathogenic variant P/LPV) in a hereditary cancer syndrome gene were identified up to one year before and up to one year after FVT became no-charge in 1/2017. The proportion of probands with at least one at- risk family member who underwent FVT was compared between Black and White probands using logistic regression, including the interaction between covariates of cost and race. Results: Between 1/2016 and 1/2018, 8,530 Black and 87,846 White probands underwent genetic testing. Of these, 9.3% (n =791) Black probands and 11.4% (n=9,998) White probands had a P/LPV identified. The uptake of FVT, defined by percentage of positive probands with at least one family member undergoing testing, was significantly lower in Black participants compared to White participants (11.9% versus 21.7%, odds ratio 0.5, 95% CI 0.4-0.6, p<0.001). Period of testing before or after FVT was no-charge did not impact this difference (p=0.23 for the interaction). FVT rates were significantly lower in Black patients compared to White patients both before (8.1% versus 18.7%, OR 0.4, 95% CI 0.2-0.6, p<0.001) and after (13.6% versus 23.1%, OR 0.5, 95% CI 0.4-0.7, p<0.001) testing became no-charge. Conclusion: While FVT rates were low overall, they were significantly lower in Black families compared to White families. Cost of FVT did not have a significant impact on the racial disparity seen, suggesting additional barriers exist. Recognizing these disparities and determining the contributing factors are crucial to developing tailored interventions that would ultimately advance racial equity in cancer care.

Unique ER PR expression pattern in breast cancers with CHEK2 mutation: a hormone receptor and HER2 analysis based on germline cancer predisposition genes

PurposeEstrogen-receptor (ER) and progesterone-receptor (PR) expression levels in breast cancer, which have been principally compared via binomial descriptors, can vary widely across tumors. We sought to characterize ER and PR expression levels using semi-quantitative analyses of receptor staining in germline pathogenic variant (PV) carriers of cancer predisposition genes.MethodsWe conducted a retrospective chart review of patients who underwent germline genetic testing for cancer predisposition genes at a tertiary cancer center genetics clinic. We performed comparisons of semi-quantitative ER and PR percentage staining levels across carriers and non-carriers of cancer predisposition genes.ResultsBreast cancers from BRCA1 PV carriers expressed significantly lower ER (15.2% vs 78.2%, p < 0.001) and lower PR (6.8% vs 41.1%, p < 0.001) staining compared to non-PV carriers. Similarly, breast cancers of BRCA2 (66.7% vs 78.2%, p = 0.005) and TP53 (50.6% vs 78.2%, p = 0.015) PV tumors also displayed moderate decreases in ER staining. Conversely, CHEK2 tumors displayed higher ER (93.1% vs 78.2%, p = 0.005) and PR (72% vs 48.8%, p = 0.001) staining when compared to non-PV carriers. We observed a wide range of dispersion across the ER and PR staining levels of the carriers and noncarriers. ER and PR ranges of dispersion of CHEK2 tumors were uniquely narrower than all other groups.ConclusionThe findings of our study suggest that precise expression levels of ER and PR in breast cancers can vary widely. These differences are further augmented when comparing expression staining across PV and non-PV carriers, suggesting potentially unique tumorigenesis and progression pathways influenced by germline cancer predisposition genes.

Breast cancer ER, PR, and HER2 expression variance by germline cancer predisposition genes.

10526 Background: The association between breast cancer characteristics and survival with estrogen receptor (ER) and progesterone receptor (PR) expression has been primarily studied via binomial categories, ER-positive and ER-negative. In order to better characterize germline genetic influences on these markers, we investigated their IHC expression semi-quantitatively in cancer predisposition germline pathogenic variant (PV) carriers of the following genes: BRCA1, BRCA2, PALB2, TP53, PTEN, CDH1, ATM, CHEK2, and Lynch syndrome genes. The HER2 expression was also analyzed. Methods: We conducted a retrospective chart review of patients with germline panel genetic testing for cancer predisposition genes at Moffitt Cancer Center’s GeneHome clinic. Inclusion criteria included 1) women ≥18 years old, 2) breast cancer diagnosis, 3) cancer predisposition germline panel genetic test results, 4) available ER and PR expression levels, and 5) available HER expression and/or amplification status. ER, PR, and HER2 status were compared between PV carriers and non-PV carriers via Mann-Whitney U at p&gt;0.05. Results: A total of 847 cases were reviewed for the study. Among 658 patients with a breast cancer diagnosis and complete ER PR data, 365 cases (55.5%) were non-PV carriers and 293 cases (44.5%) carried a PV in at least one of the genes listed above. Among 635 cases with available HER2 expression/amplification status, 355 (55.9%) cases were non-PV carriers and 288 (45.4%) cases were PV-carriers. When compared with non-PV carrier controls, BRCA1 PV carriers’ breast tumors had significantly lower ER and/or PR expression. Further, BRCA2 and TP53 PV tumors also displayed moderately lower ER expression. Contrarily, CHEK2 tumors displayed higher ER and PR expression compared to controls. Further, BRCA1 and BRCA2 PV carriers were more likely to have HER2- breast cancers. Conclusions: Differences in ER, PR, HER2 expression levels were observed in germline PV carrier breast cancers, signaling differential impacts by germline PVs on the tumor evolution process. It is likely that tumor differences in PV carriers influence responses to therapies, including hormone therapy, anti-HER2 therapy, and subsequent survival.[Table: see text]

Abstract LB-304: Oncologic therapy for solid tumors alters the risk of clonal hematopoiesis

Abstract Solid tumor patients often suffer from cytopenias and are at risk for therapy-related myeloid neoplasms (tMN). Somatic mutations in leukemia-associated genes can occur in normal healthy individuals, referred to as clonal hematopoiesis (CH). CH is associated with cytopenias, risk of leukemia and cardiovascular disease. We and others have shown that CH is frequent in cancer patients. Characterization of the relationship between exposure to specific oncologic regimens and CH and how these relate to cytopenias and tMN risk would inform treatment decisions and tMN prevention strategies. To determine the relationship between CH and oncologic therapy we interrogated CH in a cohort of 9045 solid tumor patients. Subjects were sequenced using a targeted panel of cancer-associated mutations used to screen tumor samples against a blood control sample. Mutation detection was performed on blood-derived sequencing data using the matched tumor as a comparator and accounted for background sequencing error rates. CH was identified in 23% of patients. In multivariate regression analyses adjusted by age, CH was more often found in current smokers (OR=1.20, 95%CI=1.07-1.35, p&amp;lt;0.001) and less often found in Asians compared to Whites (OR=0.72, 95%CI=0.56-0.89, p&amp;lt;0.001). Smoking was associated with CH mutations in ASXL1 (OR=3.75, 95%CI=2.73-5.17, p&amp;lt;0.001). There was a higher proportion of patients with CH among those who had received chemotherapy (OR=1.14, 95%CI=1.02-1.26, p=0.02) and those who had received external beam radiation therapy (OR=1.45, 95%CI=1.28-1.63, p&amp;lt;0.001) prior to blood collection. Mutations in the DNA repair/cell cycle pathway (including TP53, PPM1D and CHEK2) were more common among patients who received chemotherapy and radiation therapy prior to IMPACT testing compared to those who were treatment naïve (p&amp;lt;0.001). Exposure to prior cytotoxic chemotherapy (OR=1.20, 95%CI=1.02-1.30; p=0.007) and radiation therapy (OR=1.6, 95%CI=1.4-1.9, p&amp;lt;0.001) was associated with having CH while exposure to immunotherapy and targeted therapy was not. Increasing cumulative dose of chemotherapy overall and cytotoxic therapy was associated with a higher likelihood of CH (p=0.015 and p=0.007 respectively). There was evidence of specific gene, treatment and dosage effects. To further examine the relationship between oncologic therapy and clonal evolution of CH, we collected 375 sequential samples at least 18 months apart. T mean change in VAF of CH mutations per year was found to increase by 0.17% in patients who did not receive further therapy during the follow-up time and 0.49% in those who were exposed to cytotoxic chemotherapy. A subset of patients with CH were consented to germline testing for cancer predisposition genes (N=6368). We observe a higher rate of CH among patients with a germline mutation in the cell cycle/DNA repair pathway (i.e. TP53) when compared to patients without germline mutations (OR=3.7, 95% CI: 1.35-9.35, p-value=0.01). CH is frequent in solid tumor patients and can be reliably detected when a matched tumor normal targeted gene sequencing approach is performed. Beyond age, CH is strongly associated with race, smoking and importantly prior exposure to oncologic therapy with evidence of specific treatment effects. Screening of CH in cancer cohorts is critical to the development of future clinical guidelines and risk-adapted prevention strategies for tMN. Note: This abstract was not presented at the meeting. Citation Format: Kelly Bolton, Ryan Ptashkin, Lior Braunstein, Teng Gao, Sean M. Devlin, Daniel Kelly, Catherine Coombs, Minal Patel, Matahi Moarii, Elsa Bernard, Antonin Berthon, Laura Boucai, Dominik Glodzik, Axel Martin, Zsofia Stadler, Michael Walsh, Diana Mandelker, Akshar Patel, Jessica Schulman, Gunes Gundem, Aijazuddin Syed, Maria Arcila, David B. Solit, Mark E. Robson, Marc Ladanyi, Choonsik Lee, John Philip, Dean Bajorin, Montserrat Garcia-Closas, Stuart Gardos, David Hyman, Martin Tallman, Mariko Yabe, Kenneth Offit, Howard Scher, Virginia Klimek, Luis Diaz, Nilanjan Chatterjee, Michael F. Berger, Lindsay Morton, Ross Levine, Ahmet Zehir, Elli Papaemmanuil. Oncologic therapy for solid tumors alters the risk of clonal hematopoiesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-304.

Oncologic Therapy for Solid Tumors Alters the Risk of Clonal Hematopoiesis

Background: Solid tumor patients are at a heightened risk for developing therapy-related myeloid neoplasms (tMN). Recent studies show evidence of somatic mutations in leukemia-associated genes in normal healthy individuals, referred to as clonal hematopoiesis (CH). We and others have shown that clonal hematopoiesis (CH) is also frequent in cancer patients. A detailed characterization of the relationship between exposure to specific oncologic regimens, the molecular features of CH presentation and how these relate to tMN risk is warranted to inform treatment decisions, early detection and prevention strategies.Methods: To determine the relationship between CH and oncologic therapy, we performed a systematic interrogation of CH in a cohort of 17,478 solid tumor patients with clinical, outcome and molecular profiling by MSK-IMPACT. MSK-IMPACT is a targeted panel of cancer-associated mutations used to screen tumor samples against a blood control sample. Mutation detection was performed on blood derived sequencing data (median coverage at 600x) using the matched tumor as a comparator and accounted for background sequencing error rates.Results: Overall, 40% of the 17,478 patients were treatment naïve prior to IMPACT testing, 37% had received chemotherapy alone, 17% had received radiation therapy and 18% had received both. CH was identified in 4013 (23%) of patients, median VAF was 4% (range=1-80%). The vast majority (76%) had a single mutation whereas 9% had two and 5% had three or more. The number of mutations correlated with clone size (p-value=<0.001). The proportion of patients with CH greatly increased with each decade of life (p<0.001). In multivariate regression analyses adjusted by age, CH was more often found in former or current smokers (p=0.03) than in non-smokers and in Whites compared to Asians (p=0.005) and Hispanics (p=0.005). There were no significant differences by gender. Rates of CH varied greatly by solid tumor type but when limited to treatment naïve patients, no significant differences remained, suggesting that this was driven by different treatment exposures. As previously reported in healthy patient cohorts, CH in the DNA hydroxymethylation pathway predominated (52% of total CH mutations). There was a higher proportion of patients with mutations in the DNA repair/cell cycle pathway (including TP53, PPM1D and CHEK2) and among patients who received chemotherapy and radiation therapy prior to IMPACT testing compared to those who were treatment naïve (p<0.001). Exposure to prior cytotoxic chemotherapy (OR=1.2, 95%CI=1.0-1.3; p=0.03) and radiation therapy (OR=1.6, 95%CI=1.4-1.9, p<0.002) was associated with having CH while exposure to immunotherapy and targeted therapy was not. There was evidence of specific gene, treatment and dosage effects. To further examine the relationship between oncologic therapy and clonal evolution of CH mutations, we have collected 375 sequential samples collected at least 18 months apart. A subset of patients with CH were consented to germline testing for cancer predisposition genes (N=1835). We observe a lower rate of CH among patients with a germline mutation in the homologous recombination deficiency pathway (ie BRCA1, BRCA2) (OR=0.66, 95% CI: 0.43-0.99, p-value=0.05) and a higher rate of CH among patients with a germline mutation in the cell cycle/DNA repair pathway (i.e. CHEK2, TP53) when compared to patients without germline mutations (OR=1.6, 95% CI: 1.0-2.6, p-value=0.05). In analyses stratified by prior treatment, patients with germline mutations in the cell cycle/DNA repair pathway (i.e. CHEK2, TP53) with prior radiation therapy exposure were more likely to have CH compared to patients with no germline mutations who were exposed to radiation therapy (OR=4.1,95%CI=1.1-17.0, p-value for interaction=0.04).Conclusions: CH is frequent in solid tumor patients and can be reliably detected when a matched tumor normal targeted gene sequencing approach is performed. Beyond age, CH is strongly associated with race, smoking and importantly prior exposure to oncologic therapy with evidence of specific treatment effects. Taken together, we show that screening of CH in cancer cohorts is critical to the development of future clinical guidelines, the development of risk-adapted treatment decisions, surveillance programs and definition of patient subsets at highest risk for tMN. DisclosuresCoombs:Incyte: Other: Travel fees; DAVA Oncology: Honoraria; Abbvie: Consultancy; H3 Biomedicine: Honoraria; AROG: Other: Travel fees. Tallman:Daiichi-Sankyo: Other: Advisory board; Cellerant: Research Funding; BioSight: Other: Advisory board; AROG: Research Funding; Orsenix: Other: Advisory board; AbbVie: Research Funding; ADC Therapeutics: Research Funding. Yabe:Y-mAbs Therapeutics: Consultancy. Levine:Celgene: Consultancy, Research Funding; Novartis: Consultancy; Gilead: Honoraria; Isoplexis: Equity Ownership; Epizyme: Patents & Royalties; Prelude: Research Funding; C4 Therapeutics: Equity Ownership; Janssen: Consultancy, Honoraria; Imago: Equity Ownership; Qiagen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Research Funding; Loxo: Consultancy, Equity Ownership.

Comprehensive Cancer-Predisposition Gene Testing in an Adult Multiple Primary Tumor Series Shows a Broad Range of Deleterious Variants and Atypical Tumor Phenotypes

Multiple primary tumors (MPTs) affect a substantial proportion of cancer survivors and can result from various causes, including inherited predisposition. Currently, germline genetic testing of MPT-affected individuals for variants in cancer-predisposition genes (CPGs) is mostly targeted by tumor type. We ascertained pre-assessed MPT individuals (with at least two primary tumors by age 60 years or at least three by 70 years) from genetics centers and performed whole-genome sequencing (WGS) on 460 individuals from 440 families. Despite previous negative genetic assessment and molecular investigations, pathogenic variants in moderate- and high-risk CPGs were detected in 67/440 (15.2%) probands. WGS detected variants that would not be (or were not) detected by targeted resequencing strategies, including low-frequency structural variants (6/440 [1.4%] probands). In most individuals with a germline variant assessed as pathogenic or likely pathogenic (P/LP), at least one of their tumor types was characteristic of variants in the relevant CPG. However, in 29 probands (42.2% of those with a P/LP variant), the tumor phenotype appeared discordant. The frequency of individuals with truncating or splice-site CPG variants and at least one discordant tumor type was significantly higher than in a control population (χ2 = 43.642; p ≤ 0.0001). 2/67 (3%) probands with P/LP variants had evidence of multiple inherited neoplasia allele syndrome (MINAS) with deleterious variants in two CPGs. Together with variant detection rates from a previous series of similarly ascertained MPT-affected individuals, the present results suggest that first-line comprehensive CPG analysis in an MPT cohort referred to clinical genetics services would detect a deleterious variant in about a third of individuals.

The ICR639 CPG NGS validation series: A resource to assess analytical sensitivity of cancer predisposition gene testing.

The analytical sensitivity of a next generation sequencing (NGS) test reflects the ability of the test to detect real sequence variation. The evaluation of analytical sensitivity relies on the availability of gold-standard, validated, benchmarking datasets. For NGS analysis the availability of suitable datasets has been limited. Most laboratories undertake small scale evaluations using in-house data, and/or rely on in silico generated datasets to evaluate the performance of NGS variant detection pipelines. Cancer predisposition genes (CPGs), such as BRCA1 and BRCA2, are amongst the most widely tested genes in clinical practice today. Hundreds of providers across the world are now offering CPG testing using NGS methods. Validating and comparing the analytical sensitivity of CPG tests has proved difficult, due to the absence of comprehensive, orthogonally validated, benchmarking datasets of CPG pathogenic variants. To address this we present the ICR639 CPG NGS validation series. This dataset comprises data from 639 individuals. Each individual has sequencing data generated using the TruSight Cancer Panel (TSCP), a targeted NGS assay for the analysis of CPGs, together with orthogonally generated data showing the presence of at least one CPG pathogenic variant per individual. The set consists of 645 pathogenic variants in total. There is strong representation of the most challenging types of variants to detect, with 339 indels, including 16 complex indels and 24 with length greater than five base pairs and 74 exon copy number variations (CNVs) including 23 single exon CNVs. The series includes pathogenic variants in 31 CPGs, including 502 pathogenic variants in BRCA1 or BRCA2, making this an important comprehensive validation dataset for providers of BRCA1 and BRCA2 NGS testing. We have deposited the TSCP FASTQ files of the ICR639 series in the European Genome-phenome Archive (EGA) under accession number EGAD00001004134.

CoverView: a sequence quality evaluation tool for next generation sequencing data

Quality assurance and quality control are essential for robust next generation sequencing (NGS). Here we present CoverView, a fast, flexible, user-friendly quality evaluation tool for NGS data. CoverView processes mapped sequencing reads and user-specified regions to report depth of coverage, base and mapping quality metrics with increasing levels of detail from a chromosome-level summary to per-base profiles. CoverView can flag regions that do not fulfil user-specified quality requirements, allowing suboptimal data to be systematically and automatically presented for review. It also provides an interactive graphical user interface (GUI) that can be opened in a web browser and allows intuitive exploration of results. We have integrated CoverView into our accredited clinical cancer predisposition gene testing laboratory that uses the TruSight Cancer Panel (TSCP). CoverView has been invaluable for optimisation and quality control of our testing pipeline, providing transparent, consistent quality metric information and automatic flagging of regions that fall below quality thresholds. We demonstrate this utility with TSCP data from the Genome in a Bottle reference sample, which CoverView analysed in 13 seconds. CoverView uses data routinely generated by NGS pipelines, reads standard input formats, and rapidly creates easy-to-parse output text (.txt) files that are customised by a simple configuration file. CoverView can therefore be easily integrated into any NGS pipeline. CoverView and detailed documentation for its use are freely available at github.com/RahmanTeamDevelopment/CoverView/releases and www.icr.ac.uk/CoverView

CoverView: a sequence quality evaluation tool for next generation sequencing data.

Quality assurance and quality control are essential for robust next generation sequencing (NGS). Here we present CoverView, a fast, flexible, user-friendly quality evaluation tool for NGS data. CoverView processes mapped sequencing reads and user-specified regions to report depth of coverage, base and mapping quality metrics with increasing levels of detail from a chromosome-level summary to per-base profiles. CoverView can flag regions that do not fulfil user-specified quality requirements, allowing suboptimal data to be systematically and automatically presented for review. It also provides an interactive graphical user interface (GUI) that can be opened in a web browser and allows intuitive exploration of results. We have integrated CoverView into our accredited clinical cancer predisposition gene testing laboratory that uses the TruSight Cancer Panel (TSCP). CoverView has been invaluable for optimisation and quality control of our testing pipeline, providing transparent, consistent quality metric information and automatic flagging of regions that fall below quality thresholds. We demonstrate this utility with TSCP data from the Genome in a Bottle reference sample, which CoverView analysed in 13 seconds. CoverView uses data routinely generated by NGS pipelines, reads standard input formats, and rapidly creates easy-to-parse output text (.txt) files that are customised by a simple configuration file. CoverView can therefore be easily integrated into any NGS pipeline. CoverView and detailed documentation for its use are freely available at github.com/RahmanTeamDevelopment/CoverView/releases and www.icr.ac.uk/CoverView.

The Quality Sequencing Minimum (QSM): providing comprehensive, consistent, transparent next generation sequencing data quality assurance.

Next generation sequencing (NGS) is routinely used in clinical genetic testing. Quality management of NGS testing is essential to ensure performance is consistently and rigorously evaluated. Three primary metrics are used in NGS quality evaluation: depth of coverage, base quality and mapping quality. To provide consistency and transparency in the utilisation of these metrics we present the Quality Sequencing Minimum (QSM). The QSM defines the minimum quality requirement a laboratory has selected for depth of coverage (C), base quality (B) and mapping quality (M) and can be applied per base, exon, gene or other genomic region, as appropriate. The QSM format is CX_BY(P Y)_MZ(P Z). X is the parameter threshold for C, Y the parameter threshold for B, P Y the percentage of reads that must reach Y, Z the parameter threshold for M, P Z the percentage of reads that must reach Z. The data underlying the QSM is in the BAM file, so a QSM can be easily and automatically calculated in any NGS pipeline. We used the QSM to optimise cancer predisposition gene testing using the TruSight Cancer Panel (TSCP). We set the QSM as C50_B10(85)_M20(95). Test regions falling below the QSM were automatically flagged for review, with 100/1471 test regions QSM-flagged in multiple individuals. Supplementing these regions with 132 additional probes improved performance in 85/100. We also used the QSM to optimise testing of genes with pseudogenes such as PTEN and PMS2. In TSCP data from 960 individuals the median number of regions that passed QSM per sample was 1429 (97%). Importantly, the QSM can be used at an individual report level to provide succinct, comprehensive quality assurance information about individual test performance. We believe many laboratories would find the QSM useful. Furthermore, widespread adoption of the QSM would facilitate consistent, transparent reporting of genetic test performance by different laboratories.

The spectrum of BRCA1 and BRCA2 alleles in Latin America and the Caribbean: a clinical perspective.

Hereditary cancer predisposition gene testing allows the identification of individuals at high risk of cancer that may benefit from increased surveillance, chemoprevention, and prophylactic surgery. In order to implement clinical genetic strategies adapted to each population’s needs and intrinsic genetic characteristic, this review aims to present the current status of knowledge about the spectrum of BRCA pathogenic variants in Latin American populations. We have conducted a comprehensive review of 33 studies published between 1994 and 2015 reporting the prevalence and/or spectrum of BRCA1 (OMIM 113705) and BRCA2 (OMIM 600185) variants. The combined sample size for these studies consisted of 4835 individuals from 13 countries in Latin America and the Caribbean, as well as in Hispanics in the United States. A total of 167 unique pathogenic variants have been reported in the existing literature. In unselected breast cancer cases, the prevalence ranged from 1.2 to 27.1 %. Some countries presented a few recurrent pathogenic variants, while others were characterized by diverse, non-recurrent variants. The proportion of BRCA pathogenic variants shared between Hispanics in the United States and Latin American populations was estimated at 10.4 %. Within Latin America and the Caribbean, 8.2 % of the BRCA variants reported were present in more than one country. Countries with high prevalence of BRCA pathogenic variants may benefit from more aggressive testing strategies, while testing of recurrent variant panels might present a cost-effective solution for improving genetic testing in some, but not all, countries.Electronic supplementary materialThe online version of this article (doi:10.1007/s10549-015-3629-3) contains supplementary material, which is available to authorized users.

Abstract 4705: Integrating germline cancer predisposition gene testing into routine clinical practice - The Mainstreaming Cancer Genetics programme

Abstract Over 100 cancer predisposition genes with clinical utility are recognised. Testing of these genes is increasingly in demand and is one of the major activities of Clinical Genetics. However, in most countries, testing is very restricted with respect to the number of genes and the number of people tested. Furthermore, the value of genetic testing in individuals with cancer is underappreciated; it provides important information with respect to the cause and optimal treatment of the current cancer, and the risk and optimal management of future cancer. Moreover, testing cancer patients followed by cascade testing of mutation carriers is an effective and efficient way of identifying unaffected mutation carriers in whom screening and risk-reducing strategies can be deployed. The Mainstreaming Cancer Genetics (MCG) Programme is a UK national cross-disciplinary initiative to develop the NGS assays, analytical and interpretive pipelines, clinical infrastructure, training, ethical and evaluation processes required for routine genetic testing to be integrated into cancer patient care. In collaboration with Illumina we have developed a NGS panel targeting 97 cancer predisposition genes with &amp;gt;1500 probes (called the TruSight cancer panel) and have completed detailed evaluation of performance, sensitivity and specificity under different levels of multiplexing, coverage and throughput. We have developed bespoke analytical pipelines developed for high-throughput clinical diagnostic data analysis (called GAMA) and clinical interpretation (called CIGMA). We are implementing a new mixed-model of cancer gene testing whereby consent for medical testing (i.e. in cancer patients) is undertaken by trained oncologists, with only the mutation carriers seen by geneticists. Predictive testing (i.e. in unaffected individuals) continues to be undertaken in genetics. We have developed protocols and e-learning modules to deliver the required training for oncologists to consent for testing. The system is faster and cheaper, whilst retaining flexibility and robustness to provide increased input where required. We are completing a 6 month pilot that has included &amp;gt;100 patients and we are acquiring patient and clinician feedback, the results of which will be presented. www.mcgprogramme.com Citation Format: Nazneen Rahman, The Mainstreaming Cancer Genetics Consortium. Integrating germline cancer predisposition gene testing into routine clinical practice - The Mainstreaming Cancer Genetics programme. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4705. doi:10.1158/1538-7445.AM2014-4705

Abstract B08: Predictors of delay to single-site confirmatory testing for familial BRCA1/2 mutations

Abstract Background: Genetic testing for cancer predisposition genes like BRCA1/2 allows the quantification of future cancer risks and the adoption of preventive measures such as chemoprevention, screening, and prophylactic surgeries to mitigate risks. First-in-family mutations in BRCA1/2 are generally identified in a proband via full-sequence analysis. Once a familial mutation is identified, probands may share test information with family members to alert them of risks. Family members may then pursue single-site testing (SST) to determine if the familial mutation was inherited. Despite the high cancer risks associated with BRCA1/2 mutations, research suggests many family members remain untested or experience delays to confirmatory SST. Few studies have characterized the diverse factors influencing transmission of risk information and uptake of SST within families. Methods: Probands undergoing full-sequence BRACAnalysis by Myriad Genetic Laboratories (MGL) who were found to have unique mutations in BRCA1/2 were randomly selected across 6 years (~50 per year, 2007-2012) from the MGL BRCA1/2 database. In the same time frame, putative “relatives” were identified by linking the proband unique mutation to all downstream requests for SST at the same site. Clinical characteristics for probands and putative relatives were extracted from the MGL Test Request Form (TRF). Pedigrees were independently constructed by two members of the research team for each family based on personal/family history data. Disputed family structures were resolved through group consensus after input from a third member of the research team. Results: Linked data for 298 probands and 701 linked relatives undergoing SST were included in analyses. Overall ≥1 relative was tested in 265/298 (89%) families. Probands were primarily women (97%) of Western/Northern European ancestry (56%). Proband mean age of testing was 49.6 years [range 22-87], and over 90% had a personal history of cancer: [breast (73%) or ovarian (12%), multiple cancers (22%)]. A median 2 relatives were tested per proband [range 0-13]. Median number of days between proband and first relative tested was 75 (2.5 mos). Median time until first test decreased by an average of 11 days per year from 2007-2012 (p=0.01) and there was a trend toward increased SST testing in year one (39% families had ≥1 SST in the first year of follow-up in 2007 vs 78% in 2012, p=0.001). Siblings (n=275/701, 39%) were overall the most frequently tested relative followed by children (22%), parents (11%) and cousins (11%), while delay was shortest for parents (mean 136 days) and longest for cousins (mean 460 days). Univariate analyses found that relationship to proband (p&amp;lt;0.0001), sex (p=0.05), and age (p=0.01) were associated with SST delay. A linear multivariable model found, compared to siblings, parents had significantly shorter times to testing (p&amp;lt;0.001) and cousins (p=0.02) and nieces/nephews (p&amp;lt;0.001) significantly longer. Male sex was associated with longer delay (p=0.03), and a personal history of ovarian cancer (compared to breast) with a shorter delay (p=0.005). Conclusion: Uptake of SST in BRCA1/2 families peaks in year 1 but occurs over many years and primarily among first-degree relatives. Testing delays have decreased over time (2007-2012) and may indicate more frequent sharing of high risk information in families, greater access (e.g. insurance, geographic) to BRCA1/2 SST for relatives, and/or greater awareness of the options for risk reduction once BRCA1/2 status is determined. Over an observation period ≥1 and ≤5 years, a striking 11% of positive BRCA1/2 tests were not followed by a SST. Citation Format: Michael J. Hall, Kim Rainey, Allison Davis, Elizabeth Handorf, Rajesh Kaldate, Kelsey Moyes, Mary B. Daly. Predictors of delay to single-site confirmatory testing for familial BRCA1/2 mutations. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr B08.

A Computational Method to Classify Variants of Uncertain Significance Using Functional Assay Data with Application to BRCA1

Besides revealing cancer predisposition variants or the absence of any changes, genetic testing for cancer predisposition genes can also identify variants of uncertain clinical significance (VUS). Classifying VUSs is a pressing problem, as ever more patients seek genetic testing for disease syndromes and receive noninformative results from those tests. In cases such as the breast and ovarian cancer syndrome in which prophylactic options can be severe and life changing, having information on the disease relevance of the VUS that a patient harbors can be critical. We describe a computational approach for inferring the disease relevance of VUSs in disease genes from data derived from an in vitro functional assay. It is based on a Bayesian hierarchical model that accounts for sources of experimental heterogeneity. The functional data correlate well with the pathogenicity of BRCA1 BRCT VUSs, thus providing evidence regarding pathogenicity when family and genetic data are absent or uninformative. We show the utility of the model by using it to classify 76 VUSs located in the BRCT region of BRCA1. The approach is both sensitive and specific when evaluated on variants previously classified using independent sources of data. Although the functional data are very informative, they will need to be combined with other forms of data to meet the more stringent requirements of clinical application. Our work will lead to improved classification of VUSs and will aid in the clinical decision making of their carriers.

Acceptance of genetic testing for hereditary breast ovarian cancer among study enrollees from an African American kindred

Clinical availability of genetic testing for cancer predisposition genes is generating a major challenge for U.S. health care systems to provide relevant genetic services to underserved populations. Here we present rates of study enrollment and utilization of genetic testing in a research study on BRCA1 testing acceptance in one large kindred. We also present data on baseline access to genetic information as well as enabling and obstructing factors to study enrollment. The study population included female and male members of an African American kindred based in the rural southern United States with an identified BRCA1 mutation. A combination of quantitative and qualitative data were collected and analyzed. Of the 161 living, eligible, and locatable kindred members, 105 (65%) enrolled in the study. Family, personal, and educational motivations were the most commonly endorsed reasons for study participation. The most commonly cited reasons for refusal to participate in the study were: lack of interest, time constraints, and negative experiences with prior participation in genetic research. Eighty three percent of the participants underwent BRCA1 testing. In multiple logistic regression analysis, age 40-49 (odds ratio (OR) = 6.9; 95% confidence interval (CI) = 1.2-39.5), increased perceived risk of being a BRCA1 mutation carrier (OR = 4.1; 95% CI = 1.1-14.6), and high cancer genetics knowledge levels (OR = 1.5; 95% CI = 1.1-2.3) were associated with BRCA1 testing acceptance. The results of this study indicate that cognitive and demographic factors may influence genetic research participation and genetic testing decisions among African Americans who are at increased risk of carrying a deleterious BRCA1 mutation.

Does Genetic Counseling Have Any Impact on Management of Breast Cancer Risk?

Despite there being an increasing literature on the impact of cancer genetic counseling on risk perception and mental health, there is a lack of data describing impact on risk management. Genetic counseling and testing for cancer predisposition genes aims to improve the future health of those at high risk through appropriate surveillance and screening. However, management of breast cancer risk in women with a family history of this disease is an area of controversy. Counseling services may recommend specific risk management options to women, who then rely on their local screening service to make provision. This study investigated the impact of genetic counseling on management of breast cancer risk in women attending Cancer Family Clinics. A total of 293 women attending four genetic clinics were enrolled. Rates of breast self-examination, clinical breast examination, mammography, biopsy, detected cancers, and other screenings were documented. Participants' perceived benefits and barriers to mammography were assessed along with cancer worry. Results show that rates of mammography, clinical breast examination, and breast self-examination were increased following clinic attendance (p < 0.001). Women in the under 35 age-group had limited access to screening. Rates for biopsy and detected cancers were low. Women reported positive attitudes to mammography, with few reported barriers. Contrary to previous studies, there was no evidence that anxiety about breast cancer impedes uptake of health surveillance methods. Genetic counseling had a positive impact on management of breast cancer risk. Whether this translates into future health gains remains to be established.

Genetic predisposition to cancer

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