Early Cancer Detection in Li-Fraumeni Syndrome with Cell-Free DNA.

People with Li-Fraumeni syndrome (LFS) harbor a germline pathogenic variant in the TP53 tumor suppressor gene, face a near 100% lifetime risk of cancer, and routinely undergo intensive surveillance protocols. Liquid biopsy has become an attractive tool for a range of clinical applications, including early cancer detection. Here, we provide a proof-of-principle for a multimodal liquid biopsy assay that integrates a targeted gene panel, shallow whole-genome, and cell-free methylated DNA immunoprecipitation sequencing for the early detection of cancer in a longitudinal cohort of 89 LFS patients. Multimodal analysis increased our detection rate in patients with an active cancer diagnosis over uni-modal analysis and was able to detect cancer-associated signal(s) in carriers prior to diagnosis with conventional screening (positive predictive value = 67.6%, negative predictive value = 96.5%). Although adoption of liquid biopsy into current surveillance will require further clinical validation, this study provides a framework for individuals with LFS. SIGNIFICANCE: By utilizing an integrated cell-free DNA approach, liquid biopsy shows earlier detection of cancer in patients with LFS compared with current clinical surveillance methods such as imaging. Liquid biopsy provides improved accessibility and sensitivity, complementing current clinical surveillance methods to provide better care for these patients. See related commentary by Latham et al., p. 23. This article is featured in Selected Articles from This Issue, p. 5.

Retrospective Analysis of Canadian Adults with Tuberous Sclerosis Complex.

BACKGROUND: Our study goal was to characterize the relative frequencies of molecular and phenotypic traits of tuberous sclerosis complex (TSC) in a Canadian adult population. Previous studies have sought to identify TSC-related genotypic and phenotypic trends in pediatric cohorts, but little is known about clinical manifestations and severity when it presents in adults. METHODS: We conducted a retrospective chart review of adult patients seen at the TSC clinic at the University Health Network genetics clinics (Toronto, Ontario) to compare trends in the relative frequency of TSC manifestations with genotype. RESULTS: Fifty-one patients were eligible for this study. Eight patients had a pathogenic/likely pathogenic variant in the tuberous sclerosis complex 1 (TSC1) gene, 18 had a tuberous sclerosis complex 2 (TSC2) pathogenic/likely pathogenic variant, 6 patients had multiple variants identified in TSC1/TSC2 or TSC2/PKD1, 11 had no mutation identified (NMI) and 8 had no genetic testing done. Patients with a pathogenic/likely pathogenic variant in TSC2 presented with an increased involvement of multiple systems and a higher frequency of TSC-related manifestations relative to the other mutation groups. CONCLUSION: Previous studies comparing the wide phenotypic variability with TSC genotype have mainly comprised pediatric cohorts. With a focus on adults, we found trends to be similar across previous literature. An informed multidisciplinary approach should be taken to ensure proper surveillance and management of adults with TSC until a correlation between genotype and phenotype, especially past infancy, is better understood.

Current and new frontiers in hereditary cancer surveillance: Opportunities for liquid biopsy.

At least 5% of cancer diagnoses are attributed to a causal pathogenic or likely pathogenic germline genetic variant (hereditary cancer syndrome-HCS). These individuals are burdened with lifelong surveillance monitoring organs for a wide spectrum of cancers. This is associated with substantial uncertainty and anxiety in the time between screening tests and while the individuals are awaiting results. Cell-free DNA (cfDNA) sequencing has recently shown potential as a non-invasive strategy for monitoring cancer. There is an opportunity for high-yield cancer early detection in HCS. To assess clinical validity of cfDNA in individuals with HCS, representatives from eight genetics centers from across Canada founded the CHARM (cfDNA in Hereditary and High-Risk Malignancies) Consortium in 2017. In this perspective, we discuss operationalization of this consortium and early data emerging from the most common and well-characterized HCSs: hereditary breast and ovarian cancer, Lynch syndrome, Li-Fraumeni syndrome, and Neurofibromatosis type 1. We identify opportunities for the incorporation of cfDNA sequencing into surveillance protocols; these opportunities are backed by examples of earlier cancer detection efficacy in HCSs from the CHARM Consortium. We seek to establish a paradigm shift in early cancer surveillance in individuals with HCSs, away from highly centralized, regimented medical screening visits and toward more accessible, frequent, and proactive care for these high-risk individuals.

Germline whole genome sequencing in adults with multiple primary tumors.

Multiple primary tumors (MPTs) are a harbinger of hereditary cancer syndromes. Affected individuals often fit genetic testing criteria for a number of hereditary cancer genes and undergo multigene panel testing. Other genomic testing options, such as whole exome (WES) and whole genome sequencing (WGS) are available, but the utility of these genomic approaches as a second-tier test for those with uninformative multigene panel testing has not been explored. Here, we report our germline sequencing results from WGS in 9 patients with MPTs who had non-informative multigene panel testing. Following germline WGS, sequence (agnostic or 735 selected genes) and copy number variant (CNV) analysis was performed according to the American College of Medical Genetics (ACMG) standards and guidelines for interpreting sequence variants and reporting CNVs. In this cohort, WGS, as a second-tier test, did not identify additional pathogenic or likely pathogenic variants in cancer predisposition genes. Although we identified a CHEK2 likely pathogenic variant and a MUTYH pathogenic variant, both were previously identified in the multigene panels and were not explanatory for the presented type of tumors. CNV analysis also failed to identify any pathogenic or likely pathogenic variants in cancer predisposition genes. In summary, after multigene panel testing, WGS did not reveal any additional pathogenic variants in patients with MPTs. Our study, based on a small cohort of patients with MPT, suggests that germline gene panel testing may be sufficient to investigate these cases. Future studies with larger sample sizes may further elucidate the additional utility of WGS in MPTs.

Oncologist-led germline genetic testing for uveal melanoma.

PURPOSE: To report the genotype and phenotype of a cohort of unselected uveal melanoma (UM) patients who had germline multi-gene panel genetic testing, including the BAP1 gene, from a large multi-ethnic cancer centre. We describe the central role of the medical genetics clinic in collaboration with oncologists in a mainstreaming model to facilitate genetic testing, counselling and streamlining of patients with hereditary cancer predisposition. METHODS: A retrospective chart review of clinical and genetic findings of unselected UM patients who had germline genetic testing between December 2019 and October 2021 was conducted. Extracted DNA from peripheral blood samples were analyzed with a multi-gene panel that included at least six genes associated with hereditary melanoma. The correlation between the genotype and the phenotype of the cohort was evaluated. Statistical analysis comprised descriptive and comparative statistics with significance assigned at p < .05. The genetics clinic streamlined patients among the relevant oncology clinics for cancer screening in germline BAP1 positive individuals. RESULTS: In unselected UM patients, 3.5% (4/114) tested positive for a BAP1 pathogenic variant. Germline BAP1 status was associated with a family history of mesothelioma (p = .0015) and metastatic disease (p = .017). There were no other significant associations between the patient- or tumour-related characteristics and germline BAP1 results. CONCLUSION: A germline BAP1 mutation was detected in 3.5% of unselected UM patients. The oncologist-initiated and genetics-led mainstreaming model is a straightforward process and can be utilized for offering genetic testing to all UM patients.

Developing a disease-specific annotation protocol for VHL gene curation using Hypothes.is.

Von Hippel-Lindau (VHL) disease is a rare, autosomal dominant disorder that predisposes individuals to developing tumors in many organs. There is significant phenotypic variability and genetic variants encountered within this syndrome, posing a considerable challenge to patient care. The lack of VHL variant data sharing paired with the absence of aggregated genotype-phenotype information results in an arduous process, when characterizing genetic variants and predicting patient prognosis. To address these gaps in knowledge, the Clinical Genome Resource (ClinGen) VHL Variant Curation Expert Panel (VCEP) has been resolving a list of variants of uncertain significance within the VHL gene. Through community curation, we crowdsourced the laborious task of variant annotation by modifying the ClinGen Community Curation (C3)-developed Baseline Annotation protocol and annotating all published VHL cases with the reported genotype-phenotype information in Hypothes.is, an open-access web annotation tool. This process, incorporated into the ClinGen VCEP's workflow, will aid in their curation efforts. To facilitate the curation at all levels of genetics expertise, our team developed a 4-day biocuration training protocol and resource guide. To date, 91.3% of annotations have been completed by undergraduate and high-school students without formal academic genetics specialization. Here, we present our VHL-specific annotation protocol utilizing Hypothes.is, which offers a standardized method to present case-resolution data, and our biocuration training protocol, which can be adapted for other rare disease platforms. By facilitating training for community curation of VHL disease, we increased student engagement with clinical genetics while enhancing knowledge translation in the field of hereditary cancer. Database URL: https://hypothes.is/groups/dKymJJpZ/vhl-hypothesis-annotation.

CIViCdb 2022: evolution of an open-access cancer variant interpretation knowledgebase.

CIViC (Clinical Interpretation of Variants in Cancer; civicdb.org) is a crowd-sourced, public domain knowledgebase composed of literature-derived evidence characterizing the clinical utility of cancer variants. As clinical sequencing becomes more prevalent in cancer management, the need for cancer variant interpretation has grown beyond the capability of any single institution. CIViC contains peer-reviewed, published literature curated and expertly-moderated into structured data units (Evidence Items) that can be accessed globally and in real time, reducing barriers to clinical variant knowledge sharing. We have extended CIViC's functionality to support emergent variant interpretation guidelines, increase interoperability with other variant resources, and promote widespread dissemination of structured curated data. To support the full breadth of variant interpretation from basic to translational, including integration of somatic and germline variant knowledge and inference of drug response, we have enabled curation of three new Evidence Types (Predisposing, Oncogenic and Functional). The growing CIViC knowledgebase has over 300 contributors and distributes clinically-relevant cancer variant data currently representing >3200 variants in >470 genes from >3100 publications.

Clinical implementation of genetic testing in adults for hereditary hematologic malignancy syndromes.

PURPOSE: As research on hereditary hematologic malignancy syndromes (HHMS) are accumulating, cancer genetics clinics are identifying more adult hematology patients with an inherited component to their disease. However, investigations for HHMS are complex, and there is no formal consensus on genetic testing criteria. METHODS: We developed genetic testing criteria for adult hematology patients through a comprehensive literature review and our experience at the Princess Margaret Cancer Centre. We validated our criteria by applying them retrospectively to patients referred to our clinic for HHMS assessment. RESULTS: Our genetic testing criteria are comprehensive of myeloid malignancies, lymphoid malignancies, and bone marrow failure, including age at diagnosis, family history, and genetic test results in blood and bone marrow. Of the 104 patients who met the criteria, 26% had at least 1 actionable variant in any gene associated with an increased risk of cancer and 13% had an actionable variant resulting in an HHMS diagnosis. A total of 15 patients had incidental findings, including 11 patients with a pathogenic variant associated with carrier status for an autosomal recessive disorder and 4 patients with a mosaic result. CONCLUSION: Our high gene positivity rate shows the utility of a broad approach to germline testing in an adult hematology population.

LZTR1 molecular genetic overlap with clinical implications for Noonan syndrome and schwannomatosis.

BACKGROUND: Noonan syndrome (NS) is a genetic disorder characterized by developmental delays, typical facial gestalt and cardiovascular defects. LZTR1 variants have been recently described in patients with NS and schwannomatosis, but the association, inheritance pattern and management strategy has not been fully elucidated. Here, we review the contribution of LZTR1 in NS and describe a patient with a novel, likely pathogenic variant in LZTR1. CASE PRESENTATION: A female patient was diagnosed with clinical NS at 8 months of age. She presented in adulthood when a brain and spine MRI identified plexiform neurofibromas; however, she did not meet the clinical criteria for Neurofibromatosis type 1. No pathogenic variants were identified through molecular genetic analysis of NF1, SPRED1 and a multigene NS panel. Whole exome sequencing at age 23 identified a novel de novo likely pathogenic heterozygous variant in the LZTR1 gene denoted as c.743G>A (p.Gly248Glu). Serial MRIs have shown stable imaging findings and the patient is being followed clinically by cardiology, neurology and medical genetics. CONCLUSIONS: We identified a novel mutation in the LZTR1 gene, not previously reported in association with NS. This report provides additional evidence to support for the assessment of schwannomatosis in patients with LZTR1-NS and may have overlap with Neurofibromatosis type 1.

Large scale genotype- and phenotype-driven machine learning in Von Hippel-Lindau disease.

Von Hippel-Lindau (VHL) disease is a hereditary cancer syndrome where individuals are predisposed to tumor development in the brain, adrenal gland, kidney, and other organs. It is caused by pathogenic variants in the VHL tumor suppressor gene. Standardized disease information has been difficult to collect due to the rarity and diversity of VHL patients. Over 4100 unique articles published until October 2019 were screened for germline genotype-phenotype data. Patient data were translated into standardized descriptions using Human Genome Variation Society gene variant nomenclature and Human Phenotype Ontology terms and has been manually curated into an open-access knowledgebase called Clinical Interpretation of Variants in Cancer. In total, 634 unique VHL variants, 2882 patients, and 1991 families from 427 papers were captured. We identified relationship trends between phenotype and genotype data using classic statistical methods and spectral clustering unsupervised learning. Our analyses reveal earlier onset of pheochromocytoma/paraganglioma and retinal angiomas, phenotype co-occurrences and genotype-phenotype correlations including hotspots. It confirms existing VHL associations and can be used to identify new patterns and associations in VHL disease. Our database serves as an aggregate knowledge translation tool to facilitate sharing information about the pathogenicity of VHL variants.

VHL mosaicism: the added value of multi-tissue analysis.

Von Hippel-Lindau disease (VHL) is an autosomal dominant, inherited syndrome with variants in the VHL gene causing predisposition to multi-organ benign and malignant neoplasms. A germline VHL variant is identified in 95-100% of individuals with a clinical diagnosis of VHL. Here, we present the case of an individual with a clinical diagnosis of VHL disease where peripheral blood DNA analysis did not detect a VHL variant. Sequencing of four tumor tissues (ccRCC, pheochromocytoma, lung via sputum, liver) revealed a VHL c.593 T > C (p.Leu198Pro) variant at varying allele fractions (range: 10-55%) in all tissues. Re-examination of the peripheral blood sequencing data identified this variant at 6% allele fraction. Tumor analysis revealed characteristic cytomorphological, immunohistochemical reactivity for alpha-inhibin, and CAIX, and reduced pVHL reactivity supported VHL-related pseudohypoxia. This report of a rare case of VHL mosaicism highlights the value of tissue testing in VHL variant negative cases.

GeneTerpret: a customizable multilayer approach to genomic variant prioritization and interpretation.

BACKGROUND: Variant interpretation is the main bottleneck in medical genomic sequencing efforts. This usually involves genome analysts manually searching through a multitude of independent databases, often with the aid of several, mostly independent, computational tools. To streamline variant interpretation, we developed the GeneTerpret platform which collates data from current interpretation tools and databases, and applies a phenotype-driven query to categorize the variants identified in the genome(s). The platform assigns quantitative validity scores to genes by query and assembly of the genotype-phenotype data, sequence homology, molecular interactions, expression data, and animal models. It also uses the American College of Medical Genetics and Genomics (ACMG) criteria to categorize variants into five tiers of pathogenicity. The final output is a prioritized list of potentially causal variants/genes. RESULTS: We tested GeneTerpret by comparing its performance to expert-curated genes (ClinGen's gene-validity database) and variant pathogenicity reports (DECIPHER database). Output from GeneTerpret was 97.2% and 83.5% concordant with the expert-curated sources, respectively. Additionally, similar concordance was observed when GeneTerpret's performance was compared with our internal expert-interpreted clinical datasets. CONCLUSIONS: GeneTerpret is a flexible platform designed to streamline the genome interpretation process, through a unique interface, with improved ease, speed and accuracy. This modular and customizable system allows the user to tailor the component-programs in the analysis process to their preference. GeneTerpret is available online at https://geneterpret.com .

Incidental findings from cancer next generation sequencing panels.

Next-generation sequencing (NGS) technologies have facilitated multi-gene panel (MGP) testing to detect germline DNA variants in hereditary cancer patients. This sensitive technique can uncover unexpected, non-germline incidental findings indicative of mosaicism, clonal hematopoiesis (CH), or hematologic malignancies. A retrospective chart review was conducted to identify cases of incidental findings from NGS-MGP testing. Inclusion criteria included: 1) multiple pathogenic variants in the same patient; 2) pathogenic variants at a low allele fraction; and/or 3) the presence of pathogenic variants not consistent with family history. Secondary tissue analysis, complete blood count (CBC) and medical record review were conducted to further delineate the etiology of the pathogenic variants. Of 6060 NGS-MGP tests, 24 cases fulfilling our inclusion criteria were identified. Pathogenic variants were detected in TP53, ATM, CHEK2, BRCA1 and APC. 18/24 (75.0%) patients were classified as CH, 3/24 (12.5%) as mosaic, 2/24 (8.3%) related to a hematologic malignancy, and 1/24 (4.2%) as true germline. We describe a case-specific workflow to identify and interpret the nature of incidental findings on NGS-MGP. This workflow will provide oncology and genetic clinics a practical guide for the management and counselling of patients with unexpected NGS-MGP findings.

The evolving role of germline genetic testing and management in prostate cancer: Report from the Princess Margaret Cancer Centre international retreat.

Prostate cancer is a significant cause of cancer mortality. It has been well-established that certain germline pathogenic variants confer both an increased risk of being diagnosed with prostate cancer and dying of prostate cancer.1 There are exciting developments in both the availability of genetic testing and opportunities for improved treatment of patients.On August 19, 2020, the Princess Margaret Cancer Centre in Toronto, Ontario, hosted a virtual retreat, bringing together international experts in urology, medical oncology, radiation oncology, medical genetics, and translational research, as well as a patient representative. We are pleased to provide this manuscript as a review of those proceedings for Canadian clinicians.We highlighted several needs for future research and policy action based on this meeting:Increased access to funding for germline testing for the common genetic disorders associated with increased risk of prostate cancer.More research into identifying genetic factors influencing risk stratification, treatment response, and outcomes of prostate cancer within Canadian populations at higher genetic risk for prostate cancer.Added awareness about genetic risk factors among the Canadian public.Development of patient-specific and reported outcomes research in tailored care for patients at increased genetic risk of prostate cancer.Creation of multidisciplinary clinics that specialize in tailored care for patients at increased genetic risk of prostate cancer.

Early-onset renal cell carcinoma in PTEN harmatoma tumour syndrome.

Individuals with PTEN hamartoma tumour syndrome (PHTS), including Cowden syndrome (CS), are susceptible to multiple benign hamartomas and an increased risk of cancer, particularly breast, endometrial, and thyroid. As a result, individuals undergo enhanced surveillance for early detection of these cancers. However, less commonly occurring cancers, such as colorectal and kidney, have insufficient guidelines for early detection. Currently, screening for kidney cancer via renal ultrasound begins at 40 years of age, because there were only rare cases of elevated risk in prospective series under 40. There have, however, been accumulating reports of kidney cancer in individuals with CS in their 30s, illustrating a need to lower the age of surveillance. We present additional evidence of renal cell carcinoma in two individuals with CS in their early twenties, and propose a reassessment of the abdominal surveillance in patients with PHTS. We propose biannual screening for kidney cancer beginning at 20 years of age.