Transcript capture and ultradeep long-read RNA sequencing (CAPLRseq) to diagnose HNPCC/Lynch syndrome.

PURPOSE: Whereas most human genes encode multiple mRNA isoforms with distinct function, clinical workflows for assessing this heterogeneity are not readily available. This is a substantial shortcoming, considering that up to 25% of disease-causing gene variants are suspected of disrupting mRNA splicing or mRNA abundance. Long-read sequencing can readily portray mRNA isoform diversity, but its sensitivity is relatively low due to insufficient transcriptome penetration. METHODS: We developed and applied capture-based target enrichment from patient RNA samples combined with Oxford Nanopore long-read sequencing for the analysis of 123 hereditary cancer transcripts (capture and ultradeep long-read RNA sequencing (CAPLRseq)). RESULTS: Validating CAPLRseq, we confirmed 17 cases of hereditary non-polyposis colorectal cancer/Lynch syndrome based on the demonstration of splicing defects and loss of allele expression of mismatch repair genes MLH1, PMS2, MSH2 and MSH6. Using CAPLRseq, we reclassified two variants of uncertain significance in MSH6 and PMS2 as either likely pathogenic or benign. CONCLUSION: Our data show that CAPLRseq is an automatable and adaptable workflow for effective transcriptome-based identification of disease variants in a clinical diagnostic setting.

Somatic copy number alteration and fragmentation analysis in circulating tumor DNA for cancer screening and treatment monitoring in colorectal cancer patients.

BACKGROUND: Analysis of circulating free DNA (cfDNA) is a promising tool for personalized management of colorectal cancer (CRC) patients. Untargeted cfDNA analysis using whole-genome sequencing (WGS) does not need a priori knowledge of the patient´s mutation profile. METHODS: Here we established LIquid biopsy Fragmentation, Epigenetic signature and Copy Number Alteration analysis (LIFE-CNA) using WGS with ~ 6× coverage for detection of circulating tumor DNA (ctDNA) in CRC patients as a marker for CRC detection and monitoring. RESULTS: We describe the analytical validity and a clinical proof-of-concept of LIFE-CNA using a total of 259 plasma samples collected from 50 patients with stage I-IV CRC and 61 healthy controls. To reliably distinguish CRC patients from healthy controls, we determined cutoffs for the detection of ctDNA based on global and regional cfDNA fragmentation patterns, transcriptionally active chromatin sites, and somatic copy number alterations. We further combined global and regional fragmentation pattern into a machine learning (ML) classifier to accurately predict ctDNA for cancer detection. By following individual patients throughout their course of disease, we show that LIFE-CNA enables the reliable prediction of response or resistance to treatment up to 3.5 months before commonly used CEA. CONCLUSION: In summary, we developed and validated a sensitive and cost-effective method for untargeted ctDNA detection at diagnosis as well as for treatment monitoring of all CRC patients based on genetic as well as non-genetic tumor-specific cfDNA features. Thus, once sensitivity and specificity have been externally validated, LIFE-CNA has the potential to be implemented into clinical practice. To the best of our knowledge, this is the first study to consider multiple genetic and non-genetic cfDNA features in combination with ML classifiers and to evaluate their potential in both cancer detection and treatment monitoring. Trial registration DRKS00012890.

Constitutional chromothripsis of the APC locus as a cause of genetic predisposition to colon cancer.

PURPOSE: Approximately 20% of patients with clinical familial adenomatous polyposis (FAP) remain unsolved after molecular genetic analysis of the APC and other polyposis genes, suggesting additional pathomechanisms. METHODS: We applied multidimensional genomic analysis employing chromosomal microarray profiling, optical mapping, long-read genome and RNA sequencing combined with FISH and standard PCR of genomic and complementary DNA to decode a patient with an attenuated FAP that had remained unsolved by Sanger sequencing and multigene panel next-generation sequencing for years. RESULTS: We identified a complex 3.9 Mb rearrangement involving 14 fragments from chromosome 5q22.1q22.3 of which three were lost, 1 reinserted into chromosome 5 and 10 inserted into chromosome 10q21.3 in a seemingly random order and orientation thus fulfilling the major criteria of chromothripsis. The rearrangement separates APC promoter 1B from the coding ORF (open reading frame) thus leading to allele-specific downregulation of APC mRNA. The rearrangement also involves three additional genes implicated in the APC-Axin-GSK3B-ß-catenin signalling pathway. CONCLUSIONS: Based on comprehensive genomic analysis, we propose that constitutional chromothripsis dampening APC expression, possibly modified by additional APC-Axin-GSK3B-ß-catenin pathway disruptions, underlies the patient's clinical phenotype. The combinatorial approach we deployed provides a powerful tool set for deciphering unsolved familial polyposis and potentially other tumour syndromes and monogenic diseases.

Highly sensitive liquid biopsy Duplex sequencing complements tissue biopsy to enhance detection of clinically relevant genetic variants.

Background: Liquid biopsy (LB) is a promising complement to tissue biopsy for detection of clinically relevant genetic variants in cancer and mosaic diseases. A combined workflow to enable parallel tissue and LB analysis is required to maximize diagnostic yield for patients. Methods: We developed and validated a cost-efficient combined next-generation sequencing (NGS) workflow for both tissue and LB samples, and applied Duplex sequencing technology for highly accurate detection of low frequency variants in plasma. Clinically relevant cutoffs for variant reporting and quantification were established. Results: We investigated assay performance characteristics for very low amounts of clinically relevant variants. In plasma, the assay achieved 100% sensitivity and 92.3% positive predictive value (PPV) for single nucleotide variants (SNVs) and 91.7% sensitivity and 100% PPV for insertions and deletions (InDel) in clinically relevant hotspots with 0.5-5% variant allele frequencies (VAFs). We further established a cutoff for reporting variants (i.e. Limit of Blank, LOB) at 0.25% VAF and a cutoff for quantification (i.e. Limit of Quantification, LOQ) at 5% VAF in plasma for accurate clinical interpretation of analysis results. With our LB approach, we were able to identify the molecular cause of a clinically confirmed asymmetric overgrowth syndrome in a 10-year old child that would have remained undetected with tissue analysis as well as other molecular diagnostic approaches. Conclusion: Our flexible and cost-efficient workflow allows analysis of both tissue and LB samples and provides clinically relevant cutoffs for variant reporting and precise quantification. Complementing tissue analysis by LB is likely to increase diagnostic yield for patients with molecular diseases.