Functional interrogation of Lynch syndrome-associated MSH2 missense variants via CRISPR-Cas9 gene editing in human embryonic stem cells.

Lynch syndrome (LS) predisposes patients to cancer and is caused by germline mutations in the DNA mismatch repair (MMR) genes. Identifying the deleterious mutation, such as a frameshift or nonsense mutation, is important for confirming an LS diagnosis. However, discovery of a missense variant is often inconclusive. The effects of these variants of uncertain significance (VUS) on disease pathogenesis are unclear, though understanding their impact on protein function can help determine their significance. Laboratory functional studies performed to date have been limited by their artificial nature. We report here an in-cellulo functional assay in which we engineered site-specific MSH2 VUS using clustered regularly interspaced short palindromic repeats-Cas9 gene editing in human embryonic stem cells. This approach introduces the variant into the endogenous MSH2 loci, while simultaneously eliminating the wild-type gene. We characterized the impact of the variants on cellular MMR functions including DNA damage response signaling and the repair of DNA microsatellites. We classified the MMR functional capability of eight of 10 VUS providing valuable information for determining their likelihood of being bona fide pathogenic LS variants. This human cell-based assay system for functional testing of MMR gene VUS will facilitate the identification of high-risk LS patients.

RNA-Seq for the detection of gene fusions in solid tumors: development and validation of the JAX FusionSeq™ 2.0 assay.

Whole transcriptome sequencing (RNA-Seq) has gained prominence for the detection of fusions in solid tumors. Here, we describe the development and validation of an in-house RNA-Seq-based test system (FusionSeq™ 2.0) for the detection of clinically actionable gene fusions, in formalin-fixed paraffin-embedded (FFPE) specimens, using seventy tumor samples with varying fusion status. Conditions were optimized for RNA input of 50 ng, shown to be adequate to call known fusions at as low as 20% neoplastic content. Evaluation of assay performance between FFPE and fresh-frozen (FF) tissues exhibited little to no difference in fusion calling capability. Performance analysis of the assay validation data determined 100% accuracy, sensitivity, specificity, and reproducibility. This clinically developed and validated RNA-Seq-based approach for fusion detection in FPPE samples was shown to be on par if not superior to off-the-shelf commercially offered assays. With gene fusions implicated in a variety of cancer types, offering high-quality, low-cost molecular testing services for FFPE specimens will serve to best benefit the patient and the advancement of precision medicine in molecular oncology. KEY MESSAGES: A custom RNA-Seq-based test system (FusionSeq™ 2.0) for the detection of clinically actionable gene fusions, Evaluation of assay performance between FFPE and fresh-frozen (FF) tissues exhibited little to no difference in fusion calling capability. The assay can be performed with low RNA input and neoplastic content. Performance characteristics of the assay validation data determined 100% accuracy, sensitivity, specificity, and reproducibility.

Molecular profiling of CNS tumors for the treatment and management of disease.

The World Health Organization (WHO) has defined more than 130 distinct central nervous system (CNS) tumor entities, of which glioblastoma is the most fatal primary brain tumor. However, the correlation of the molecular signatures of glioblastoma with clinical significance for precision medicine is not well-known. How, and to what extent these variants may affect clinical decision making remains uncertain. Here, we evaluate 48 glioblastomas submitted for testing using the JAX ActionSeq™ Next-generation sequencing (NGS) panel. We identified 131 clinically significant variants (Tier I and II) across 30 of the 212 genes (14%). TP53, EGFR, PTEN, IDH1 were the most commonly mutated genes; EGFR, CDK4 amplifications, and CDKN2A deletion were the most frequently detected copy-number alterations. CDK4/6 and PI3K inhibitors were among the most commonly reported drug class with FDA approved therapies and investigational therapies, which is consistent with the frequencies of these genes in our cohort. Overall, our study established the molecular profiles of glioblastoma based on the 2017 joint consensus guidelines by AMP/ASCO/CAP and provides the potential implications for targeted therapeutic options currently available.

Mutation Yield of a Custom 212-Gene Next-Generation Sequencing Panel for Solid Tumors: Clinical Experience of the First 260 Cases Tested Using the JAX ActionSeq™ Assay.

OBJECTIVE: The study aimed to retrospectively evaluate the positive yield rate of a custom 212-gene next-generation sequencing (NGS) panel, the JAX ActionSeq™ assay, used in molecular profiling of solid tumors for precision medicine. METHODS: We evaluated 261 cases tested over a 24-month period including cancers across 24 primary tissue types and report on the mutation yield in these cases. RESULTS: Thirty-three of the 261 cases (13%) had no detectable clinically significant variants. In the remaining 228 cases (87%), we identified 550 clinically significant variants in 88 of the 212 genes, with four of fewer clinically significant variants being detected in 62 of 88 genes (70%). TP53 had the highest number of variants (125), followed by APC (47), KRAS (47), ARID1A (20), PIK3CA (20) and EGFR (18). There were 38 tier I and 512 tier II variants, with two genes having only a tier I variant, seven genes having both a tier I and tier II variant, and 79 genes having at least one tier II variant. Overall, the ActionSeq™ assay detected clinically significant variants in 42% of the genes included in the panel (88/212), 68% of which (60/88) were detected in more than one tumor type. CONCLUSIONS: This study demonstrates that of the genes with documented involvement in cancer, only a limited number are currently clinically significant from a therapeutic, diagnostic and/or prognostic perspective.

Molecular profiling of gynecologic cancers for treatment and management of disease - demonstrating clinical significance using the AMP/ASCO/CAP guidelines for interpretation and reporting of somatic variants.

Molecular features of gynecologic cancers have been investigated in comprehensive studies, but correlation of these molecular signatures with clinical significance for precision medicine is yet to be established. Towards this end, we evaluated 95 gynecologic cancer cases submitted for testing using The JAX ActionSeq™ NGS panel. Molecular profiles were studied and compared to TCGA datasets to identify similarities and distinguishing features among subtypes. We identified 146 unique clinically significant variants (Tier I and II) across 45 of the 212 genes (21%), in 87% (83/95) of cases. TP53, PTEN, ARID1A, PIK3CA and ATM were the most commonly mutated genes; CCNE1 and ERBB2 amplifications were the most frequently detected copy-number alterations. PARP inhibitors were among the most commonly reported drug class with clinical trials, consistent with the frequency of DNA damage-response pathway mutations in our cohort. Overall, our study provides additional insight into the molecular profiles of gynecologic cancers, highlighting regulatory pathways involved, raising the potential implications for targeted therapeutic options currently available.

Evaluating gene fusions in solid tumors - Clinical experience using an RNA based 53 gene next-generation sequencing panel.

Given the known association of gene fusions with solid tumor morbidity and the need to clarify the role of fusions in therapeutic, prognostic and diagnostic outcomes, we reviewed the positive yield rate for fusions in solid tumors using cases that were referred to our laboratory for clinical testing. We retrospectively evaluated results from 183 solid tumor samples that were received during a 24 month period for testing using the FusionSeq™ assay, an RNA-based Next Generation Sequencing (NGS) panel of 53 genes known to form fusions in solid tumors. Positive yield rate (actionable fusions) was evaluated for all samples tested, as a correlate for clinical utility. Twenty five fusions (actionable, variants of uncertain significance - VUS, and benign) were identified, of which 7 were classified as actionable gene fusions, resulting in an overall positive yield rate of ∼3.8% (7/183). Sixteen mostly novel fusions were classified and reported as VUSs. Five fusion events were classified as false positives, occurring due to mispriming or wild-type read through while 2 were classified as likely benign. Additionally 68% of fusions (17 of 25) detected in our study were present in prostate, colorectal, and gynecological cancers, suggesting that the frequency of fusions identified is dependent on specific tumor type. The high number of novel fusions identified highlights the potential for fusions in precision medicine.

Technical and Regulatory Considerations for Taking Liquid Biopsy to the Clinic: Validation of the JAX PlasmaMonitorTM Assay.

The standard of care in oncology has been genomic profiling of tumor tissue biopsies for the treatment and management of disease, which can prove to be quite challenging in terms of cost, invasiveness of procedure, and potential risk for the patient. As the number of available drugs in oncology continues to increase, so too does the demand for technologies and testing applications that can identify genomic alterations targetable by these new therapies. Liquid biopsies that use a blood draw from the diseased patient may offset the many disadvantages of the invasive procedure. However, as with any new technology or finding in the clinical field, the clinical utility of an analytical test such as that of the liquid biopsy has to be established. Here, we review the clinical testing space for liquid biopsy offerings and elucidate the technical and regulatory considerations to develop such an assay, using our recently validated PlasmaMonitorTM test.

Retrospective genotype-phenotype analysis in a 305 patient cohort referred for testing of a targeted epilepsy panel.

PURPOSE: Epilepsy is a diverse neurological condition with extreme genetic and phenotypic heterogeneity. The introduction of next-generation sequencing into the clinical laboratory has made it possible to investigate hundreds of associated genes simultaneously for a patient, even in the absence of a clearly defined syndrome. This has resulted in the detection of rare and novel mutations at a rate well beyond our ability to characterize their effects. This retrospective study reviews genotype data in the context of available phenotypic information on 305 patients spanning the epileptic spectrum to identify established and novel patterns of correlation. METHODS: Our epilepsy panel comprising 377 genes was used to sequence 305 patients referred for genetic testing. Qualifying variants were annotated with phenotypic data obtained from either the test requisition form or supporting clinical documentation. Observed phenotypes were compared with established phenotypes in OMIM, published literature and the ILAEs 2010 report on genetic testing to assess congruity with known gene aberrations. RESULTS: We identified a number of novel and recognized genetic variants consistent with established epileptic phenotypes. Forty-one pathogenic or predicted deleterious variants were detected in 39 patients with accompanying clinical documentation. Twenty-five of these variants across 15 genes were novel. Furthermore, evaluation of phenotype data for 194 patients with variants of unknown significance in genes with autosomal dominant and X-linked disease inheritance elucidated potentially disease-causing variants that were not currently characterized in the literature. CONCLUSIONS: Assessment of key genotype-phenotype correlations from our cohort provide insight into variant classification, as well as the importance of including ILAE recommended genes as part of minimum panel content for comprehensive epilepsy tests. Many of the reported VUSs are likely genuine pathogenic variants driving the observed phenotypes, but not enough evidence is available for assertive classifications. Similar studies will provide more utility via mounting independent genotype-phenotype data from unrelated patients. The possible outcome would be a better molecular diagnostic product, with fewer indeterminate reports containing only VUSs.

Genomic Profiling of Two Histologically Distinct Rare Urothelial Cancers in a Clinical Setting to Identify Potential Therapeutic Options for Treatment and Management of Disease.

Molecular profiling of urothelial cancers for therapeutic and prognostic potential has been very limited due to the absence of cancer-specific targeted therapies. We describe here 2 clinical cases with a histological diagnosis of an invasive sarcomatoid and a poorly differentiated carcinoma favoring urothelial with some neuroendocrine differentiation, two of the rarer types of urothelial cancers, which were evaluated for mutations in 212 genes for single-nucleotide variants and copy-number variants and 53 genes for fusions associated with solid tumors. In both cases, we identified variants in 2 genes, ARID1A and CDKN2A, indicative of the role of dysregulation of chromatin remodeling and cell cycle control as being common features of bladder cancer, consistent with the proposed model of tumorigenesis in these rare, highly aggressive pathological subtypes. The presence of a KRAS mutation in the poorly differentiated cancer and a TP53 mutation in the sarcomatoid tumor is indicative of a distinctive profile and adds a potential layer of molecular stratification to these rarer histological subtypes. We present a comparative analysis of the histological, clinical, and molecular profile of both cases and discuss the potential to delineate these tumors at the molecular level keeping in mind the possible therapeutic implications.

Clinical utility of a 377 gene custom next-generation sequencing epilepsy panel.

Epilepsy is one of the most common neurological disorders with about 500 genes thought to be involved across the phenotypic spectrum (Busch et al. 2014; Ran et al. 2014), which includes monogenic, multigenic, epistatic and pleiotropic phenotype manifestations (Busch et al. 2014; Thomas et al. 2014), driving the need for a comprehensive diagnostic test. Next-generation sequencing (NGS) allows for the simultaneous investigation of a large number of genes, making it a very attractive option for a condition as diverse as epilepsy at a low cost compared to traditional Sanger sequencing (Lemke et al. 2012; Németh et al. 2013). Our 377 gene epilepsy NGS test was developed to include genes known to cause or have published association with epilepsy and seizure-related disorders. Given the scale of information that is generated, the efficacy of an NGS panel depends on a number of factors, including the genes present on the panel, prebioinformatic and postbioinformatic analysis protocols, as well as reporting criteria, prompting the current study, a retrospective analysis of 305 cases tested for the epilepsy panel.