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Kaplan-Meier estimation of (A) overall survival of BRAFV600E mCRC NGS cohorts and (B) progression free survival of BRAFV600E mCRC NGS cohorts from BC Cancer and MD Anderson shows no difference in overall or progression free survival.

Prevalence of BRAFV600E mutated colorectal cancer by IHC testing within VCH population-based cohort.

Kaplan-Meier estimation of overall survival of mCRC patients with BRAFV600E mutations detected by (A) IHC (BRAFIHC) and (B) NGS (BRAFNGS) demonstrates no survival difference between those with dMMR versus pMMR for BRAFIHC and BRAFNGS within individual groups.

Clinical characteristics of patients with BRAFV600E mCRC receiving first-line palliative chemotherapy.

Multivariate analysis of interaction between BRAF and MMR status in patients with metastatic colorectal cancer.

Kaplan-Meier estimation of overall survival of mCRC patients with BRAFV600E mutations detected by NGS (BRAFNGS) and two IHC (BRAFIHC) subsets: patients that received any NGS testing and patients that were never NGS tested.

Multivariate analysis of Progression Free Survival of patients with BRAFV600E mCRC with therapy forced into model.

Purpose: BRAF V600E mutations portend poor prognosis in metastatic colorectal cancer (mCRC); however, the true prevalence and prognosis are unknown, as unwell patients may not undergo BRAF sequencing. Experimental Design: We reviewed a population-based cohort of 1,898 patients with colorectal cancer that underwent reflexive IHC mismatch repair (MMR) and BRAFV600E testing. Outcomes among IHC-detected BRAFV600E mCRC (BRAFIHC) were compared with patients with next-generation sequencing (NGS)–identified BRAFV600E-mutated mCRC from two institutions (BRAFNGS) with patients spanning from 2004 to 2018. Results: All-stage population prevalence of BRAFV600E was 12.5% (238/1,898) and did not differ between early and metastatic stages (P = 0.094). Prevalence among mCRC was 10.6% (61/575), of whom 51 (83.6%) were referred to oncology and 26 (42.6%) had NGS testing. BRAFIHC had worse median overall survival (mOS) than BRAFNGS [5.5 vs. 20.4 months; HR, 2.90; 95% confidence interval (CI), 1.89–4.45; P < 0.0001], which persisted in multivariate analysis (P < 0.0001). Across a combined NGS and IHC cohort, BRAFV600E tumors with deficient MMR showed worse mOS compared with MMR proficient tumors (8.9 vs. 17.2 months; HR, 1.46; 95% CI, 0.96–2.27; P = 0.043). In this combined cohort, first-line progression-free survival was 5.9 months, with minimal differences between regimens. Within the population-based cohort, attrition between treatment lines was high with only 60.7% receiving first-line chemotherapy and 26.2% receiving second line. Conclusions: Patients with BRAFV600E-mutated mCRC have a worse prognosis than previously suggested, potentially arising from referral bias for testing. High attrition between lines of therapy suggests efficacious therapies need to be prioritized early for patients to benefit.

In metastatic colorectal cancer (mCRC),This retrospective review evaluated patients in British Columbia, Canada with mCRC andAge at diagnosis, sex, anatomic location and stage at diagnosis did not differ by

Objectives Non-invasive prenatal testing requires the presence of fetal DNA in maternal plasma. Understanding how preexamination conditions affect the integrity of cell-free DNA (cfDNA) and fetal fraction (FF) are a prerequisite for test implementation. Therefore, we examined the adjusted effect that EDTA and Streck tubes have on the cfDNA quantity and FF. Methods A total of 3,568 maternal blood samples across Canada were collected in either EDTA, or Streck tubes, and processing metrics, maternal body mass index (BMI), gestational age and fetal karyotype and sex were recorded. Plasma samples were sequenced using two different sequencing platforms in separate laboratories. Sequencing data were processed with SeqFF to estimate FF. Linear regression and multivariate imputation by chained equations were used to estimate the adjusted effect of tube type on cfDNA and FF. Results We found a positive association between cfDNA quantity and blood shipment time in EDTA tubes, which is significantly reduced with the use of Streck tubes. Furthermore, we show the storage of plasma at −80 °C is associated with a 4.4% annual relative decrease in cfDNA levels. FF was not associated with collection tube type when controlling for confounding variables. However, FF was positively associated with gestational age and trisomy 21, while negatively associated with BMI, male fetus, trisomy 18, Turners syndrome and triploidy. Conclusions Preexamination, maternal and fetal variables are associated with cfDNA quantity and FF. The consideration of these variables in future studies may help to reduce the number of pregnant women with inconclusive tests as a result of low FF.

As more clinically-relevant genomic features of myeloid malignancies are revealed, it has become clear that targeted clinical genetic testing is inadequate for risk stratification. Here, we develop and validate a clinical transcriptome-based assay for stratification of acute myeloid leukemia (AML). Comparison of ribonucleic acid sequencing (RNA-Seq) to whole genome and exome sequencing reveals that a standalone RNA-Seq assay offers the greatest diagnostic return, enabling identification of expressed gene fusions, single nucleotide and short insertion/deletion variants, and whole-transcriptome expression information. Expression data from 154 AML patients are used to develop a novel AML prognostic score, which is strongly associated with patient outcomes across 620 patients from three independent cohorts, and 42 patients from a prospective cohort. When combined with molecular risk guidelines, the risk score allows for the re-stratification of 22.1 to 25.3% of AML patients from three independent cohorts into correct risk groups. Within the adverse-risk subgroup, we identify a subset of patients characterized by dysregulated integrin signaling and RUNX1 or TP53 mutation. We show that these patients may benefit from therapy with inhibitors of focal adhesion kinase, encoded by PTK2, demonstrating additional utility of transcriptome-based testing for therapy selection in myeloid malignancy., Several genomic features have been found for acute myeloid leukaemia (AML) but targeted clinical genetic testing fails to predict prognosis. Here, the authors generate an AML prognostic score from RNA-seq data of patients, which successfully stratifies AML patients and which may provide guidance for therapeutic strategies.

Next-generation sequencing assays are capable of identifying cancer patients eligible for targeted therapies and can also detect germline variants associated with increased cancer susceptibility. However, these capabilities have yet to be routinely harmonized in a single assay because of challenges with accurately identifying germline variants from tumor-only data. We have developed the Oncology and Hereditary Cancer Program targeted capture panel, which uses tumor tissue to simultaneously screen for both clinically actionable solid tumor variants and germline variants across 45 genes. Validation using 14 tumor specimens, composed of patient samples and cell lines analyzed in triplicate, demonstrated high coverage with sensitive and specific identification of single-nucleotide variants and small insertions and deletions. Average coverage across all targets remained >2000× in 198 additional patient tumor samples. Analysis of 55 formalin-fixed, paraffin-embedded tumor samples for the detection of known germline variants within a subset of cancer-predisposition genes, including one multiexon deletion, yielded a 100% detection rate, demonstrating that germline variants can be reliably detected in tumor samples using a single panel. Combining targetable somatic and actionable germline variants into a single tumor tissue assay represents a streamlined approach that can inform treatment for patients with advanced cancers as well as identify those with potential germline variants who are eligible for confirmatory testing, but would not otherwise have been identified.

PURPOSE: BRAF(V600E) mutations portend poor prognosis in metastatic colorectal cancer (mCRC); however, the true prevalence and prognosis are unknown, as unwell patients may not undergo BRAF sequencing. PATIENTS AND METHODS: We reviewed a population-based cohort of 1898 patients with CRC that underwent reflexive immunohistochemistry (IHC) mismatch repair (MMR) & BRAF(V600E) testing. Outcomes among IHC detected BRAF(V600E) mCRC (BRAF(IHC)) were compared to patients with next generation sequencing identified BRAF(V600E) mutated mCRC from two institutions (BRAF(NGS)) with patients spanning from 2004-2018. RESULTS: All-stage population prevalence of BRAF(V600E) was 12.5% (238/1898) and did not differ between early and metastatic stages (p=0.094). Prevalence among mCRC was 10.6% (61/575), of whom 51 (83.6%) were referred to oncology and 26 (42.6%) had NGS testing. BRAF(IHC) had worse median overall survival (mOS) than BRAF(NGS) (5.5 vs 20.4 months, hazard ratio (HR) 2.90, 95% confidence interval (CI) 1.89-4.45, p

Introduction: In metastatic colorectal cancer (mCRC), RAS mutations impart inferior survival and resistance to anti-epidermal growth factor receptor (EGFR) antibodies. KRAS G12C inhibitors have been developed and we evaluated how KRAS G12C differs from other RAS mutations. Patients and Methods: This retrospective review evaluated patients in British Columbia, Canada with mCRC and RAS testing performed between 1 January 2016 and 31 December 2018. Sequencing information from The Cancer Genome Analysis (TCGA) was also obtained and analysed. Results: Age at diagnosis, sex, anatomic location and stage at diagnosis did not differ by RAS mutation type. Progression free survival on first chemotherapy for patients with metastatic KRAS G12C tumours was 11 months. Median overall survival did not differ by RAS mutation type but was worse for both KRAS G12C (27 months) and non-G12C alterations (29 months) than wildtype (43 months) ( p = 0.01). Within the TCGA, there was no differential gene expression between KRAS G12C and other RAS mutations. However, eight genes with copy number differences between the G12C and non-G12C RAS mutant groups were identified after adjusting for multiple comparisons ( FITM2, PDRG1, POFUT1, ERGIC3, EDEM2, PIGU, MANBAL and PXMP4). We also noted that other RAS mutant mCRCs had a higher tumour mutation burden than those with KRAS G12C mutations (median 3.05 vs 2.06 muts/Mb, p = 4.2e–3) and that KRAS G12C/other RAS had differing consensus molecular subtype distribution from wildtype colorectal cancer (CRC) ( p Conclusion: KRAS G12C tumours have similar clinical presentation to other RAS mutant tumours, however, are associated with differential copy number alterations.

Non-invasive prenatal aneuploidy testing (NIPT) by next-generation sequencing of circulating cell-free DNA in maternal plasma relies on chromosomal ratio (chrratio) measurements to detect aneuploid values that depart from euploid ratios. Diagnostic performances are known to depend on the fraction of fetal DNA (FF) present in maternal plasma, although how this translates into specific quantitative changes in specificity/positive predictive values and which other variables might also be important is not well understood.To explore this issue, theoretical relationships between FF and various measures of diagnostic performances were assessed for a range of parameter values. Empirical data from three NIPT assays were then used to validate theoretical calculations.For a given positivity threshold, dramatic changes in specificity and positive predictive values (PPV) as a function of both FF and the coefficient of variation (CV) of the chrratio measurement were observed. Theoretically predicted and observed chrratio z-scores agreed closely, confirming the determinant impact of small changes in both FF and chrratio CV.Evaluation of NIPT assay performances therefore requires knowledge of the FF distribution in the population in which the test is intended to be used and, in particular, of the precise value of the assay chrratio CV for each chromosome or genomic region of interest. Laboratories offering NIPT testing should carefully measure these parameters to ensure test reliability and clinical usefulness in interpreting individual patients' results.

Clinical reporting of solid tumor sequencing requires reliable assessment of the accuracy and reproducibility of each assay. Somatic mutation variant allele fractions may be below 10% in many samples due to sample heterogeneity, tumor clonality, and/or sample degradation in fixatives such as formalin. The toolkits available to the clinical sequencing community for correlating assay design parameters with assay sensitivity remain limited, and large-scale empirical assessments are often relied upon due to the lack of clear theoretical grounding. To address this uncertainty, a theoretical model was developed for predicting the expected variant calling sensitivity for a given library complexity and sequencing depth. Binomial models were found to be appropriate when assay sensitivity was only limited by library complexity or sequencing depth, but functional scaling for library complexity was necessary when both library complexity and sequencing depth were co-limiting. This model was empirically validated with sequencing experiments by using a series of DNA input amounts and sequencing depths. Based on these findings, a workflow is proposed for determining the limiting factors to sensitivity in different assay designs, and the formulas for these scenarios are presented. The approach described here provides designers of clinical assays with the methods to theoretically predict assay design outcomes a priori, potentially reducing burden in clinical tumor assay design and validation efforts.

Sample tracking and identity are essential when processing multiple samples in parallel. Sequencing applications often involve high sample numbers, and the data are frequently used in a clinical setting. As such, a simple and accurate intrinsic sample tracking process through a sequencing pipeline is essential. Various solutions have been implemented to verify sample identity, including variant detection at the start and end of the pipeline using arrays or genotyping, bioinformatic comparisons, and optical barcoding of samples. None of these approaches are optimal. To establish a more effective approach using genetic barcoding, we developed a panel of unique DNA sequences cloned into a common vector. A unique DNA sequence is added to the sample when it is first received and can be detected by PCR and/or sequencing at any stage of the process. The control sequences are approximately 200 bases long with low identity to any sequence in the National Center for Biotechnology Information nonredundant database (30 bases) and contain no long homopolymer (7) stretches. When a spiked next-generation sequencing library is sequenced, sequence reads derived from this control sequence are generated along with the standard sequencing run and are used to confirm sample identity and determine cross-contamination levels. This approach is used in our targeted clinical diagnostic whole-genome and RNA-sequencing pipelines and is an inexpensive, flexible, and platform-agnostic solution.

Formalin fixation is the standard method for the preservation of tissue for diagnostic purposes, including pathologic review and molecular assays. However, this method is known to cause artifacts that can affect the accuracy of molecular genetic test results. We assessed the applicability of alternative fixatives to determine whether these perform significantly better on next-generation sequencing assays, and whether adequate morphology is retained for primary diagnosis, in a prospective study using a clinical-grade, laboratory-developed targeted resequencing assay. Several parameters relating to sequencing quality and variant calling were examined and quantified in tumor and normal colon epithelial tissues. We identified an alternative fixative that suppresses many formalin-related artifacts while retaining adequate morphology for pathologic review.

3579 Background: BRAFV600E ( BRAF) mutations (mts) portend poor prognosis in mCRC and patients (pts) may die before ascertainment. Since 2014, Vancouver Coastal Health (VCH) has performed reflex hereditary screening of CRCs with BRAF and mismatch repair (MMR) immunohistochemistry (IHC). We evaluated this BRAF mt population-based cohort ( BRAFPOP) to establish the true prognosis of BRAF mts in mCRC. Methods: We reviewed all mCRCs from VCH between 4/2014 and 5/2018 for BRAF by IHC (VE1 antibody). Overall survival (OS) from stage IV diagnosis was compared to mCRCs with next generation sequencing (NGS) determined BRAF mts ( BRAFNGS) from BC Cancer & MD Anderson. BRAFNGS OS did not differ by center (p = 0.77). Results: See table for BRAF cohort baseline characteristic comparison. BRAFPOP pts had worse OS than BRAFNGS pts (HR 2.5, 95% CI 1.6 – 3.9, P < 0.0001). Median OS for all BRAF mt pts was 17.9 mos. Both groups had worse OS than wild type pts (P < 0.0001). 52 (81%) of BRAFPOP pts were referred to oncology, 40 (63%) received chemotherapy, and 12 (19%) had NGS BRAF testing. BRAFPOP pts who had NGS testing with BRAF mts had OS comparable to other BRAFNGS pts (P = 0.89) and better OS than BRAFPOP pts that never had NGS testing (HR 0.37, 95% CI 0.18-0.76, P = 0.030). Pts with BRAF mts and MMR deficiency (dMMR) (n = 40) had worse OS than MMR proficiency (pMMR, n = 202) (1.6, 95% CI 1.0-2.5, P = 0.011). This was driven by BRAFPOP dMMR pts (HR 1.9, 95% CI 0.9-4.0, P = 0.036) as no difference was seen by MMR in BRAFNGS pts (HR 1.3, 95% CI 0.8-2.2, P = 0.30). Conclusions: Current estimates of prognosis for mCRC with BRAF mts likely underestimate its impact due to referral bias for NGS testing. BRAF mts with dMMR are associated with worse prognosis than pMMR. This appears driven by BRAFPOP pts. [Table: see text]

BACKGROUND—Many mutations that contribute to the pathogenesis of acute myeloid leukemia (AML) are undefined. The relationships between patterns of mutations and epigenetic phenotypes are not yet clear. METHODS—We analyzed the genomes of 200 clinically annotated adult cases of de novo AML, using either whole-genome sequencing (50 cases) or whole-exome sequencing (150 cases), along with RNA and microRNA sequencing and DNA-methylation analysis. RESULTS—AML genomes have fewer mutations than most other adult cancers, with an average of only 13 mutations found in genes. Of these, an average of 5 are in genes that are recurrently mutated in AML. A total of 23 genes were significantly mutated, and another 237 were mutated in two or more samples. Nearly all samples had at least 1 nonsynonymous mutation in one of nine categories of genes that are almost certainly relevant for pathogenesis, including transcriptionfactor fusions (18% of cases), the gene encoding nucleophosmin (NPM1) (27%), tumorsuppressor genes (16%), DNA-methylation–related genes (44%), signaling genes (59%), chromatin-modifying genes (30%), myeloid transcription-factor genes (22%), cohesin-complex genes (13%), and spliceosome-complex genes (14%). Patterns of cooperation and mutual exclusivity suggested strong biologic relationships among several of the genes and categories. CONCLUSIONS—We identified at least one potential driver mutation in nearly all AML samples and found that a complex interplay of genetic events contributes to AML pathogenesis in individual patients. The databases from this study are widely available to serve as a foundation for further investigations of AML pathogenesis, classification, and risk stratification. (Funded by the National Institutes of Health.) The molecular pathogenesis of acute myeloid leukemia (AML) has been studied with the use of cytogenetic analysis for more than three decades. Recurrent chromosomal structural variations are well established as diagnostic and prognostic markers, suggesting that acquired genetic abnormalities (i.e., somatic mutations) have an essential role in pathogenesis. 1,2 However, nearly 50% of AML samples have a normal karyotype, and many of these genomes lack structural abnormalities, even when assessed with high-density comparative genomic hybridization or single-nucleotide polymorphism (SNP) arrays 3-5 (see Glossary). Targeted sequencing has identified recurrent mutations in FLT3, NPM1, KIT, CEBPA, and TET2. 6-8 Massively parallel sequencing enabled the discovery of recurrent mutations in DNMT3A 9,10 and IDH1. 11 Recent studies have shown that many patients with

Background: Chimeric transcripts, including partial and internal tandem duplications (PTDs, ITDs) and gene fusions, are important in the detection, prognosis, and treatment of human cancers. Results: We describe Barnacle, a production-grade analysis tool that detects such chimeras in de novo assemblies of RNA-seq data, and supports prioritizing them for review and validation by reporting the relative coverage of co-occurring chimeric and wild-type transcripts. We demonstrate applications in large-scale disease studies, by identifying PTDs in MLL, ITDs in FLT3, and reciprocal fusions between PML and RARA, in two deeply sequenced acute myeloid leukemia (AML) RNA-seq datasets. Conclusions: Our analyses of real and simulated data sets show that, with appropriate filter settings, Barnacle makes highly specific predictions for three types of chimeric transcripts that are important in a range of cancers: PTDs, ITDs, and fusions. High specificity makes manual review and validation efficient, which is necessary in large-scale disease studies. Characterizing an extended range of chimera types will help generate insights into progression, treatment, and outcomes for complex diseases., Simon Fraser University. Bioinformatics for Combating Infectious Disease Project, Simon Fraser University (Graduate Fellowship), Pacific Century Institute (Graduate Scholarship), Genome Canada (Firm), Canadian Institutes of Health Research, Genome British Columbia (Firm) (Grant #121AML), Provincial Health Services Authority (British Columbia, Canada), BC Cancer Foundation

1582Background: ctDNA is a potentially superior means of classifying tumor genotype but predictors of Con with atDNA are not well established. We conducted a prospective multicenter study to determ...

Motivation: Computational identification of genomic structural variants via high-throughput sequencing is an important problem for which a number of highly sophisticated solutions have been recently developed. With the advent of high-throughput transcriptome sequencing (RNA-Seq), the problem of identifying structural alterations in the transcriptome is now attracting significant attention. In this article, we introduce two novel algorithmic formulations for identifying transcriptomic structural variants through aligning transcripts to the reference genome under the consideration of such variation. The first formulation is based on a nucleotide-level alignment model; a second, potentially faster formulation is based on chaining fragments shared between each transcript and the reference genome. Based on these formulations, we introduce a novel transcriptome-to-genome alignment tool, Dissect (DIScovery of Structural Alteration Event Containing Transcripts), which can identify and characterize transcriptomic events such as duplications, inversions, rearrangements and fusions. Dissect is suitable for whole transcriptome structural variation discovery problems involving sufficiently long reads or accurately assembled contigs. Results: We tested Dissect on simulated transcripts altered via structural events, as well as assembled RNA-Seq contigs from human prostate cancer cell line C4-2. Our results indicate that Dissect has high sensitivity and specificity in identifying structural alteration events in simulated transcripts as well as uncovering novel structural alterations in cancer transcriptomes., Pacific Institute for the Mathematical Sciences (Fellowship)