Angela Jacobson

Prevalence and Spectrum of Germline Cancer Susceptibility Gene Mutations Among Patients With Early-Onset Colorectal Cancer.

Importance Hereditary cancer syndromes infer high cancer risks and require intensive cancer surveillance, yet the prevalence and spectrum of these conditions among unselected patients with early-onset colorectal cancer (CRC) is largely undetermined. Objective To determine the frequency and spectrum of cancer susceptibility gene mutations among patients with early-onset CRC. Design, Setting, and Participants Overall, 450 patients diagnosed with colorectal cancer younger than 50 years were prospectively accrued from 51 hospitals into the Ohio Colorectal Cancer Prevention Initiative from January 1, 2013, to June 20, 2016. Mismatch repair (MMR) deficiency was determined by microsatellite instability and/or immunohistochemistry. Germline DNA was tested for mutations in 25 cancer susceptibility genes using next-generation sequencing. Main Outcomes and Measures Mutation prevalence and spectrum in patients with early-onset CRC was determined. Clinical characteristics were assessed by mutation status. Results In total 450 patients younger than 50 years were included in the study, and 75 gene mutations were found in 72 patients (16%). Forty-eight patients (10.7%) had MMR-deficient tumors, and 40 patients (83.3%) had at least 1 gene mutation: 37 had Lynch syndrome (13, MLH1 [including one with constitutional MLH1 methylation]; 16, MSH2; 1, MSH2/monoallelic MUTYH; 2, MSH6; 5, PMS2); 1 patient had the APC c.3920T>A, p.I1307K mutation and a PMS2 variant; 9 patients (18.8%) had double somatic MMR mutations (including 2 with germline biallelic MUTYH mutations); and 1 patient had somatic MLH1 methylation. Four hundred two patients (89.3%) had MMR-proficient tumors, and 32 patients (8%) had at least 1 gene mutation: 9 had mutations in high-penetrance CRC genes (5, APC; 1, APC/PMS2; 2, biallelic MUTYH; 1, SMAD4); 13 patients had mutations in high- or moderate-penetrance genes not traditionally associated with CRC (3, ATM; 1, ATM/CHEK2; 2, BRCA1; 4, BRCA2; 1, CDKN2A; 2, PALB2); 10 patients had mutations in low-penetrance CRC genes (3, APC c.3920T>A, p.I1307K; 7, monoallelic MUTYH). Importantly, 24 of 72 patients (33.3%) who were mutation positive did not meet established genetic testing criteria for the gene(s) in which they had a mutation. Conclusions and Relevance Of 450 patients with early-onset CRC, 72 (16%) had gene mutations. Given the high frequency and wide spectrum of mutations, genetic counseling and testing with a multigene panel could be considered for all patients with early-onset CRC.

Improving performance of multigene panels for genomic analysis of cancer predisposition

Purpose:Screening multiple genes for inherited cancer predisposition expands opportunities for cancer prevention; however, reports of variants of uncertain significance (VUS) may limit clinical usefulness. We used an expert-driven approach, exploiting all available information, to evaluate multigene panels for inherited cancer predisposition in a clinical series that included multiple cancer types and complex family histories.Methods:For 1,462 sequential patients referred for testing by BROCA or ColoSeq multigene panels, genomic DNA was sequenced and variants were interpreted by multiple experts using International Agency for Research on Cancer guidelines and incorporating evolutionary conservation, known and predicted variant consequences, and personal and family cancer history. Diagnostic yield was evaluated for various presenting conditions and family-history profiles.Results:Of 1,462 patients, 12% carried damaging mutations in established cancer genes. Diagnostic yield varied by clinical presentation. Actionable results were identified for 13% of breast and colorectal cancer patients and for 4% of cancer-free subjects, based on their family histories of cancer. Incidental findings explaining cancer in neither the patient nor the family were present in 1.7% of subjects. Less than 1% of patients carried VUS in BRCA1 or BRCA2. For all genes combined, initial reports contained VUS for 10.5% of patients, which declined to 7.5% of patients after reclassification based on additional information.Conclusions:Individualized interpretation of gene panels is a complex medical activity. Interpretation by multiple experts in the context of personal and family histories maximizes actionable results and minimizes reports of VUS.Genet Med 18 10, 974–981.

Initiation of universal tumor screening for Lynch syndrome in colorectal cancer patients as a model for the implementation of genetic information into clinical oncology practice

Lynch syndrome confers a hereditary predisposition to colorectal and other cancers. Universal tumor screening (UTS) for Lynch syndrome is recommended by several professional societies, but the implementation can be complex. This article describes the evaluation, process development, and initiation of Lynch syndrome UTS at a tertiary referral cancer center.

Deep sequencing with intronic capture enables identification of an APC exon 10 inversion in a patient with polyposis

Purpose:Single-exon inversions have rarely been described in clinical syndromes and are challenging to detect using Sanger sequencing. We report the case of a 40-year-old woman with adenomatous colon polyps too numerous to count and who had a complex inversion spanning the entire exon 10 in APC (the gene encoding for adenomatous polyposis coli), causing exon skipping and resulting in a frameshift and premature protein truncation.Methods:In this study, we employed complete APC gene sequencing using high-coverage next-generation sequencing by ColoSeq, analysis with BreakDancer and SLOPE software, and confirmatory transcript analysis.Results:ColoSeq identified a complex small genomic rearrangement consisting of an inversion that results in translational skipping of exon 10 in the APC gene. This mutation would not have been detected by traditional sequencing or gene-dosage methods.Conclusion:We report a case of adenomatous polyposis resulting from a complex single-exon inversion. Our report highlights the benefits of large-scale sequencing methods that capture intronic sequences with high enough depth of coverage—as well as the use of informatics tools—to enable detection of small pathogenic structural rearrangements.Genet Med 16 10, 783–786.

Family Studies for Classification of Variants of Uncertain Classification: Current Laboratory Clinical Practice and a New Web-Based Educational Tool

Multi-gene cancer panels often identify variants of uncertain clinical significance (VUS) that pose a challenge to health care providers in managing a patient’s cancer risk. Family segregation analysis can yield powerful data to re-classify a VUS (as either benign or pathogenic). However, financial and personnel resources to coordinate these studies are limited. In an informal assessment we found that family studies for variant classification are done by most clinical genetics laboratories that offer hereditary cancer panel testing. The process for family studies differs substantially across laboratories. One near universal limitation is that families usually have too few individuals for an informative co-segregation analysis. A unique and potential resource-saving approach is to engage patients and their families in expanding their own pedigrees for segregation analysis of their VUS. We describe a novel public educational tool (FindMyVariant.org) designed to inform patients and genetic counselors about strategies to improve the probability of variant classification using familial segregation. While the web tool is designed to be useful for any gene, the project was primarily focused on VUS’s returned in cancer risk genes. FindMyVariant.org is a resource for genetic providers to offer motivated families who are willing to gather information about their family relationships and history. Working alongside clinical or research genetic laboratories, the information they collect may help reclassify their VUS using segregation analysis.

Assessment of Tumor Sequencing as a Replacement for Lynch Syndrome Screening and Current Molecular Tests for Patients With Colorectal Cancer

Importance Universal tumor screening for Lynch syndrome (LS) in colorectal cancer (CRC) is recommended and involves up to 6 sequential tests. Somatic gene testing is performed on stage IV CRCs for treatment determination. The diagnostic workup for patients with CRC could be simplified and improved using a single up-front tumor next-generation sequencing test if it has higher sensitivity and specificity than the current screening protocol. Objective To determine whether up-front tumor sequencing (TS) could replace the current multiple sequential test approach for universal tumor screening for LS. Design, Setting, and Participants Tumor DNA from 419 consecutive CRC cases undergoing standard universal tumor screening and germline genetic testing when indicated as part of the multicenter, population-based Ohio Colorectal Cancer Prevention Initiative from October 2015 through February 2016 (the prospective cohort) and 46 patients with CRC known to have LS due to a germline mutation in a mismatch repair gene from January 2013 through September 2015 (the validation cohort) underwent blinded TS. Main Outcomes and Measures Sensitivity of TS compared with microsatellite instability (MSI) testing and immunohistochemical (IHC) staining for the detection of LS. Results In the 465 patients, mean age at diagnosis was 59.9 years (range, 20-96 years), and 241 (51.8%) were female. Tumor sequencing identified all 46 known LS cases from the validation cohort and an additional 12 LS cases from the 419-member prospective cohort. Testing with MSI or IHC, followed by BRAF p.V600E testing missed 5 and 6 cases of LS, respectively. Tumor sequencing alone had better sensitivity (100%; 95% CI, 93.8%-100%) than IHC plus BRAF (89.7%; 95% CI, 78.8%-96.1%; P = .04) and MSI plus BRAF (91.4%; 95% CI, 81.0%-97.1%; P = .07). Tumor sequencing had equal specificity (95.3%; 95% CI, 92.6%-97.2%) to IHC plus BRAF (94.6%; 95% CI, 91.9%-96.6%; P > .99) and MSI plus BRAF (94.8%; 95% CI, 92.2%-96.8%; P = .88). Tumor sequencing identified 284 cases with KRAS, NRAS, or BRAF mutations that could affect therapy for stage IV CRC, avoiding another test. Finally, TS identified 8 patients with germline DPYD mutations that confer toxicity to fluorouracil chemotherapy, which could also be useful for treatment selection. Conclusions and Relevance Up-front TS in CRC is simpler and has superior sensitivity to current multitest approaches to LS screening, while simultaneously providing critical information for treatment selection.

Using Somatic Mutations from Tumors to Classify Variants in Mismatch Repair Genes

Present guidelines for classification of constitutional variants do not incorporate inferences from mutations seen in tumors, even when these are associated with a specific molecular phenotype. When somatic mutations and constitutional mutations lead to the same molecular phenotype, as for the mismatch repair genes, information from somatic mutations may enable interpretation of previously unclassified variants. To test this idea, we first estimated likelihoods that somatic variants in MLH1, MSH2, MSH6, and PMS2 drive microsatellite instability and characteristic IHC staining patterns by calculating likelihoods of high versus low normalized variant read fractions of 153 mutations known to be pathogenic versus those of 760 intronic passenger mutations from 174 paired tumor-normal samples. Mutations that explained the tumor mismatch repair phenotype had likelihood ratio for high variant read fraction of 1.56 (95% CI 1.42-1.71) at sites with no loss of heterozygosity and of 26.5 (95% CI 13.2-53.0) at sites with loss of heterozygosity. Next, we applied these ratios to 165 missense, synonymous, and splice variants observed in tumors, combining in a Bayesian analysis the likelihood ratio corresponding with the adjusted variant read fraction with pretest probabilities derived from published analyses and public databases. We suggest classifications for 86 of 165 variants: 7 benign, 31 likely benign, 22 likely pathogenic, and 26 pathogenic. These results illustrate that for mismatch repair genes, characterization of tumor mutations permits tumor mutation data to inform constitutional variant classification. We suggest modifications to incorporate molecular phenotype in future variant classification guidelines.

Polyposis Caused by Low APC Mosaicism

Purpose: To present a patient with familial adenomatous polyposis (FAP) caused by a low level of somatic mosaicism. Case description: A twenty-one year old female presented with rectal bleeding and abdominal pain. She underwent a colonoscopy and esophagogastroduodenoscopy which revealed extensive polyposis. There was no family history of polyps or early onset colon cancer in her family. Methodology: Next-generation sequencing (NGS) analysis was performed using the ColoSeqTM panel on DNA extracted from both peripheral blood lymphocytes and colonic polyps. RESULTS: Molecular analysis detected the p.E1408X deleterious mutation in the APC gene in in 12 of 276 (4%) reads of the DNA in the peripheral blood leukocytes and in 30% of the DNA from colonic polyps. Conclusion: We report that low level of 4% APC mosaicism led to florid polyposis. Our report highlights the power of deep next-generation sequencing to identify mosaic mutations that are missed by traditional approaches. Though somatic APC mosaicism has previously been reported to cause polyposis syndrome in a few cases, it has been underestimated as a cause of polyposis coli. This case should reinforce the need to search for mosaicism in all patients with a personal history of polyposis and no family history.