Liquid Biopsy Versus Tissue Biopsy to Determine Front Line Therapy in Metastatic Non-Small Cell Lung Cancer (NSCLC).

In the last decade, non-small-cell lung cancer (NSCLC) treatment has improved with the approval of multiple therapies to target specific genetic alterations. Though, next generation sequencing (NGS) has traditionally been conducted from tissue biopsy samples, developing data supports the use of plasma-based circulating tumor DNA (ctDNA), also known as "liquid biopsy," to complement tissue biopsy approaches in guiding front-line therapy. This study is a retrospective analysis of 170 new NSCLC patients treated at 2 cancer centers within a 5-year period who received both tissue and liquid biopsy NGS as standard of care. Based on a treatment schema defined by testing sufficiency, biomarker detection, and turnaround time (TAT), physicians based the majority of their treatments on liquid biopsy results (73.5%) versus tissue biopsy (25.9%). Liquid biopsy NGS returned results on average 26.8 days faster than tissue and reported higher testing success. For guideline-recommended biomarkers, liquid biopsy was 94.8% to 100% concordant with tissue. In comparing testing modalities, a liquid-first approach identified guideline-recommended biomarkers in 76.5% of patients versus 54.9% in a tissue-first approach. There was no significant difference in time-to-treatment, or survival outcomes (overall survival and progression free survival) based on liquid versus tissue biopsy findings. This research demonstrates that liquid biopsy NGS is an effective tool to capture actionable genetic alterations in NSCLC. Due to its high concordance to tissue, faster TAT, and similarity in outcomes and time-to-treatment, liquid biopsy can be used either as a first-line test or concordantly with tissue biopsy to guide treatment decisions in NSCLC.

Clinical exome-based panel testing for medically actionable secondary findings in a cohort of 383 Italian participants.

Background: Next-generation sequencing-based genetic testing represents a great opportunity to identify hereditary predispositions to specific pathological conditions and to promptly implement health surveillance or therapeutic protocols in case of disease. The term secondary finding refers to the active search for causative variants in genes associated with medically actionable conditions. Methods: We evaluated 59 medically actionable ACMG genes using a targeted in silico analysis of clinical exome sequencing performed in 383 consecutive individuals referred to our Medical Genetics Unit. A three-tier classification system of SFs for assessing their clinical impact and supporting a decision-making process for reporting was established. Results: We identified SFs with high/moderate evidence of pathogenicity in 7.0% (27/383) of analyzed subjects. Among these, 12/27 (44.4%) were carriers of a high-risk recessive disease allele. The most represented disease domains were cancer predisposition (33.3%), cardiac disorders (16.7%), and familial hypercholesterolemia (12.5%). Conclusion: Although still debated, ensuring during NGS-based genetic testing an opportunistic screening might be valuable for personal and familial early management and surveillance of medically actionable disorders, the individual's reproductive choices, and the prevalence assessment of underestimated hereditary genetic diseases.

Correlation of changes in subclonal architecture with progression in the MMRF CoMMpass study.

Multiple myeloma (MM) is a heterogeneous plasma cell proliferative disorder that arises from its premalignant precursor stages through a complex cascade of interactions between clonal mutations and co-evolving microenvironment. The temporo-spatial evolutionary trajectories of MM are established early during myelomatogenesis in precursor stages and retained in MM. Such molecular events impact subsequent disease progression and clinical outcomes. Identification of clonal sweeps of actionable gene targets in MM could reveal potential vulnerabilities that may exist in early stages and thus potentiate prognostication and customization of early therapeutic interventions. We have evaluated clonal evolution at multiple time points in 76 MM patients enrolled in the MMRF CoMMpass study. The major findings of this study are (a) MM progresses predominantly through branching evolution, (b) there is a heterogeneous spectrum of mutational landscapes that include unique actionable gene targets at diagnosis compared to progression, (c) unique clonal gains/ losses of mutant driver genes can be identified in patients with different cytogenetic aberrations, (d) there is a significant correlation between co-occurring oncogenic mutations/ co-occurring subclones e.g., with mutated TP53+SYNE1, NRAS+MAGI3, and anticorrelative dependencies between FAT3+FCGBP gene pairs. Such co-trajectories may synchronize molecular events of drug response, myelomatogenesis and warrant future studies to explore their potential for early prognostication and development of risk stratified personalized therapies in MM.

Role of Actionable Genes in Pursuing a True Approach of Precision Medicine in Monogenic Diabetes.

Monogenic diabetes is a genetic disorder caused by one or more variations in a single gene. It encompasses a broad spectrum of heterogeneous conditions, including neonatal diabetes, maturity onset diabetes of the young (MODY) and syndromic diabetes, affecting 1-5% of patients with diabetes. Some of these variants are harbored by genes whose altered function can be tackled by specific actions ("actionable genes"). In suspected patients, molecular diagnosis allows the implementation of effective approaches of precision medicine so as to allow individual interventions aimed to prevent, mitigate or delay clinical outcomes. This review will almost exclusively concentrate on the clinical strategy that can be specifically pursued in carriers of mutations in "actionable genes", including ABCC8, KCNJ11, GCK, HNF1A, HNF4A, HNF1B, PPARG, GATA4 and GATA6. For each of them we will provide a short background on what is known about gene function and dysfunction. Then, we will discuss how the identification of their mutations in individuals with this form of diabetes, can be used in daily clinical practice to implement specific monitoring and treatments. We hope this article will help clinical diabetologists carefully consider who of their patients deserves timely genetic testing for monogenic diabetes.

Regorafenib, an investigational agent for the treatment of cholangiocarcinoma.

INTRODUCTION: Cholangiocarcinoma is a prevalent gastrointestinal cancer with a high mortality rate. A limited number of cholangiocarcinoma patients are diagnosed with early-stage disease but unfortunately, most patients present with an advanced-stage disease which is not amenable to curative surgical resection. AREAS COVERED: We discuss regorafenib, a multi-kinase inhibitor, which has been investigated as a therapeutic agent in advanced stage biliary tract cancer patients in phase II trials. We examined the efficacy and toxicity of this agent and its potential in this patient population in the future. We also provide further insights on novel approaches to optimize the efficacy of regorafenib in cholangiocarcinoma patients. EXPERT OPINION: The recent phase II trials of single-agent regorafenib in advanced stage biliary tumors revealed a modest activity in non enriched patient population and is currently part of the national comprehensive cancer network (NCCN) guidelines (Level 2B) in the refractory setting. However, more opportunities for this agent exist in combination approaches with other therapeutics such as immune checkpoint inhibitors. It is also important to recognize that the paradigm has significantly shifted for targeted therapy to more specific and more potent tyrosine kinase inhibitors targeting specific actionable genes.

Genetic alterations and their association with clinicopathologic characteristics in advanced breast carcinomas: focusing on clinically actionable genetic alterations.

Breast carcinomas (BCs) are genetically heterogeneous and associated with numerous mutations which can be used to predict outcomes and initiate targeted therapies. We investigated clinicopathologic characteristics associated with gene mutations detected using the FoundationOne CDx assay in a cohort of 223 clinically advanced BCs (66 locally recurrent and 157 metastatic) from our institution. One hundred fifty unique mutations were identified (total 1008) in the cohort, with the most prevalent (>10%) including TP53 (53.8%), PIK3CA (35%), MYC (22%), CCND1 (19.7%), FGF19 (19.7%), FGF4 (16.6%), FGF3 (16.1%), ZNF703 (14.8%), ESR1 (13.9%), FGFR1 (13.5%), PTEN (12.1%), and CDH1 (10.8%). ERBB2 genetic alteration was most common in human epidermal growth factor receptor 2 (HER2)-positive BCs, and GATA3 and ESR1 mutations were only identified in hormone receptor-positive BC. Mutations enriched in triple-negative BCs (TNBCs) included TP53, PTEN, RB1, and CDKN2A/B. CDH1 mutation was predominantly found in lobular carcinomas, and PIK3CA mutation was also enriched. Mutations enriched in metaplastic carcinomas with heterologous mesenchymal differentiation included TP53, PTEN, MCL1, CDKN2A/B, and NOTCH2. An increase in mutations of CCND1, FGF19, FGF4, FGF3, ESR1, and EMSY was identified in metastatic BCs compared with locally recurrent BCs. Overall, PIK3CA was the most frequent clinically actionable genetic alteration (35%), followed by MYC (22%), CCND1 (19.7%), and FGF3/FGF4/FGFR1 (16%). In conclusion, our study provides genetic insight into the biology of advanced BCs and summarizes their most frequent clinically actionable genetic alterations, generating useful genomic information for potential improvement of patient management.

Critical assessment of secondary findings in genes linked to primary arrhythmia syndromes.

As comprehensive sequencing technologies gain widespread use, questions about so-called secondary findings (SF) require urgent consideration. The American College of Medical Genetics and Genomics has recommended to report SF in 59 genes (ACMG SF v2.0) including four actionable genes associated with inherited primary arrhythmia syndromes (IPAS) such as catecholaminergic polymorphic ventricular tachycardia, long QT syndrome, and Brugada syndrome. Databases provide conflicting results for the purpose of identifying pathogenic variants in SF associated with IPAS at a level of sufficient evidence for clinical return. As IPAS account for a significant proportion of sudden cardiac deaths (SCD) in young and apparently healthy individuals, variant interpretation has a great impact on diagnosis and prevention of disease. Of 6381 individuals, 0.4% carry pathogenic variants in one of the four actionable genes related to IPAS: RYR2, KCNQ1, KCNH2, and SCN5A. Comparison of the databases ClinVar, Leiden Open-source Variant Database, and Human Gene Mutation Database showed impactful differences (0.2% to 1.3%) in variant interpretation improvable by expert-curation depending on database and classification system used. These data further highlight the need for international consensus regarding the variant interpretation, and subsequently management of SF in particular with regard to treatable arrhythmic disorders with increased risk of SCD.

Pathogenic and Uncertain Genetic Variants Have Clinical Cardiac Correlates in Diverse Biobank Participants.

Background Genome sequencing coupled with electronic heath record data can uncover medically important genetic variation. Interpretation of rare genetic variation and its role in mediating cardiovascular phenotypes is confounded by variants of uncertain significance. Methods and Results We analyzed the whole genome sequence of 900 racially and ethnically diverse biobank participants selected from a single US center. Participants were equally divided among European, African, Hispanic, and mixed races/ethnicities. We evaluated the American College of Medical Genetics and Genomics medically actionable list of 59 genes, focusing on the cardiac genes. Variation was interpreted using the most recent reports in ClinVar, a database of medically relevant human variation. We identified 19 individuals with pathogenic or likely pathogenic variants in cardiac actionable genes (2%) and found evidence of related clinical correlates in the electronic health record. Participants of African ancestry, compared with those of European ancestry, had more variants of uncertain significance in the medically actionable genes including the 30 cardiac actionable genes, even when normalized to total variant count per person. Longitudinal measures of left ventricle size from ≈400 biobank participants (1723 patient-years) were correlated with genetic findings. The presence of ≥1 uncertain variant in the actionable cardiac genes and a cardiomyopathy diagnosis correlated with increased left ventricular internal diameter in diastole and in systole. In particular, MYBPC3 was identified as a gene with excess variants of uncertain significance. Conclusions These data indicate that a subset of uncertain genetic variants may confer risk and should not be considered benign.

Establishment and characterization of novel patient-derived extraskeletal osteosarcoma cell line NCC-ESOS1-C1.

Extraskeletal osteosarcoma (ESOS) is a rare mesenchymal malignancy producing osteoid and bone in soft tissue without skeletal attachment. ESOS exhibits chemoresistance and poor prognosis, and is distinct from osseous osteosarcoma. The biological characteristics of ESOS are not fully understood, and patient-derived cell lines of ESOS are not available from public cell banks. Here, we established a novel cell line of ESOS and characterized its genetic and biological characteristics as well as examined its response to anti-cancer reagents. The cell line was established using tumor tissue from a 58-year-old female patient with ESOS, and named as NCC-ESOS1-C1. Phenotypes relevant to malignancy such as proliferation and invasion were examined in vitro, and genetic features were evaluated using the NCC Oncopanel assay. The response to inhibitors was monitored by screening of an anti-cancer reagent library. The cells constantly proliferated, showing spheroid formation and invasion capabilities. The NCC Oncopanel revealed the presence of actionable mutations in PIK3CA. Library screening revealed the presence of anti-cancer reagents with significant anti-proliferative effects on NCC-ESOS1-C1 at a low concentration. In conclusion, we established and characterized a novel ESOS cell line, NCC-ESOS1-C1. This cell line will be a useful resource for basic research and preclinical studies.

Identification Of Actionable Genetic Targets In Primary Cardiac Sarcomas.

BACKGROUND: Primary cardiac tumors are extremely rare; most are myxomas with a benign prognosis. However, primary sarcomas are highly aggressive and treatment options are limited. Radical surgery is often not feasible and conventional therapies provide only modest results. Due to the rare nature of primary cardiac tumors, there are no proper randomized studies to guide treatment. Their complexity requires alternative approaches in order to improve treatment efficacy. METHODS: We isolated DNA from 5 primary cardiac sarcomas; the quality of DNA from 3 of them was sufficient to perform high-resolution single nucleotide polymorphism (SNP) array analysis. RESULTS: In the present study, molecular karyotyping revealed numerous segmental chromosomal alterations and amplifications affecting actionable genes that may be involved in disease initiation and/or progression. These include chromosomal break flanking AKT2 in undifferentiated pleomorphic rhabdomyosarcoma, chromosomal break in promoter of TERT, and gain of CDK4 and amplification of MDM2 in inflammatory myofibroblastic tumor. We detected segmental break flanking MOS in high-grade myxofibrosarcoma. In addition, the high number of chromosomal aberrations in high-grade myxofibrosarcoma may cause multiple tumor-specific epitopes, supporting the study of immunotherapy treatment in this type of aggressive tumor. CONCLUSION: Our results provide a genetic rationale that supports an alternative, personalized therapeutic management of primary cardiac sarcomas.

New Challenges in Tumor Mutation Heterogeneity in Advanced Ovarian Cancer by a Targeted Next-Generation Sequencing (NGS) Approach.

Next-generation sequencing (NGS) technology has advanced knowledge of the genomic landscape of ovarian cancer, leading to an innovative molecular classification of the disease. However, patient survival and response to platinum-based treatments are still not predictable based on the tumor genetic profile. This retrospective study characterized the repertoire of somatic mutations in advanced ovarian cancer to identify tumor genetic markers predictive of platinum chemo-resistance and prognosis. Using targeted NGS, 79 primary advanced (III-IV stage, tumor grade G2-3) ovarian cancer tumors, including 64 high-grade serous ovarian cancers (HGSOCs), were screened with a 26 cancer-genes panel. Patients, enrolled between 1995 and 2011, underwent primary debulking surgery (PDS) with optimal residual disease (RD < 1 cm) and platinum-based chemotherapy as first-line treatment. We found a heterogeneous mutational landscape in some uncommon ovarian histotypes and in HGSOC tumor samples with relevance in predicting platinum sensitivity. In particular, we identified a poor prognostic signature in patients with HGSOC harboring concurrent mutations in two driver actionable genes of the panel. The tumor heterogeneity described, sheds light on the translational potential of targeted NGS approach for the identification of subgroups of patients with distinct therapeutic vulnerabilities, that are modulated by the specific mutational profile expressed by the ovarian tumor.

The Genomic Landscape and Pharmacogenomic Interactions of Clock Genes in Cancer Chronotherapy.

Cancer chronotherapy, treatment at specific times during circadian rhythms, endeavors to optimize anti-tumor effects and to lower toxicity. However, comprehensive characterization of clock genes and their clinical relevance in cancer is lacking. We systematically characterized the alterations of clock genes across 32 cancer types by analyzing data from The Cancer Genome Atlas, Cancer Therapeutics Response Portal, and The Genomics of Drug Sensitivity in Cancer databases. Expression alterations of clock genes are associated with key oncogenic pathways, patient survival, tumor stage, and subtype in multiple cancer types. Correlations between expression of clock genes and of other genes in the genome were altered in cancerous versus normal tissues. We identified interactions between clock genes and clinically actionable genes by analyzing co-expression, protein-protein interaction, and chromatin immunoprecipitation sequencing data and also found that clock gene expression is correlated to anti-cancer drug sensitivity in cancer cell lines. Our study provides a comprehensive analysis of the circadian clock across different cancer types and highlights potential clinical utility of cancer chronotherapy.

Which Results to Return: Subjective Judgments in Selecting Medically Actionable Genes.

BACKGROUND: Advances in genomics have led to calls for returning information about medically actionable genes (MAGs) to patients, research subjects, biobank participants, and through screening programs, the general adult population. Which MAGs are returned affects the harms and benefits of every genetic testing endeavor. Despite published recommendations of selection criteria for MAGs to return, scant data exist regarding how decision makers actually apply such criteria. METHODS: The process and criteria used by researchers when selecting MAGs for a preventive genomic sequencing program targeting the general adult population were examined. The authors observed and audio-recorded the gene selection meetings, and analyzed meeting transcripts, gene scoring sheets, and meeting handouts. RESULTS: To select MAGs, the committee imported, from a preexisting project, "a semiquantitative metric" that scores genes on five criteria. Numerous subjective judgments and conceptual challenges in defining and applying the five criteria complicated the selection process. Criteria-related challenges also included the limited evidence available about facts fundamental to the scoring decisions and the emergence and application of criteria that were not part of the original metric. CONCLUSIONS: When identifying MAGs appropriate for screening and return, decision makers must expect and prepare to address such issues as the inevitability of subjective judgments, limited evidence about fundamental decision-making elements, the conceptual complexity of defining criteria, and the emergence of unplanned criteria during the gene selection process.

Pancreatic cancer actionable genes in precision medicine and personalized surgery.

Pancreatic ductal adenocarcinoma (PDAC) is a deadly cancer with an overall 5-year survival rate less than 5% due to the poor early diagnosis and lack of effective therapeutic options. The most effective therapy remains surgery, however post-operative survival could be enhanced with effective adjuvant therapy. The massive information gained from Omics techniques on PDAC at the beginning of the 21st century is a remarkable accomplishment. However, the information gained from the omics data, including next generation sequencing data, has yet to successfully affect care of patients suffering with PDAC. Therefore, we propose the development of an actionable genomic platform that matches a patient's PDAC clinically actionable genes with potential targeted adjuvant therapies. Using this platform, PDX1 has been identified as a potential actionable gene for PDAC, therefore, RNAi therapy, gene therapy and small inhibitory drugs, all targeting PDX1, serve as potential targeted adjuvant therapies. Preclinical studies support the hypothesis that identification of PDAC actionable genes could permit translation of a patient's genomic information into precision targeted adjuvant therapy for PDAC.

Actionable Genes, Core Databases, and Locus-Specific Databases.

Adoption of next-generation sequencing (NGS) in a diagnostic context raises numerous questions with regard to identification and reports of secondary variants (SVs) in actionable genes. To better understand the whys and wherefores of these questioning, it is necessary to understand how they are selected during the filtering process and how their proportion can be estimated. It is likely that SVs are underestimated and that our capacity to label all true SVs can be improved. In this context, Locus-specific databases (LSDBs) can be key by providing a wealth of information and enabling classifying variants. We illustrate this issue by analyzing 318 SVs in 23 actionable genes involved in cancer susceptibility syndromes identified through sequencing of 572 participants selected for a range of atherosclerosis phenotypes. Among these 318 SVs, only 43.4% are reported in Human Gene Mutation Database (HGMD) Professional versus 71.4% in LSDB. In addition, 23.9% of HGMD Professional variants are reported as pathogenic versus 4.8% for LSDB. These data underline the benefits of LSDBs to annotate SVs and minimize overinterpretation of mutations thanks to their efficient curation process and collection of unpublished data.

Incidental diagnosis of HLRCC following investigation for Asperger Syndrome: actionable and actioned.

Incidental findings are inevitable as clinical research and practice transitions from a single gene approach to a genomic approach. A novel deletion of the Fumarate Hydratase (FH) gene was identified in a 22 year old male who underwent a molecular karyotype as part of an autism spectrum disorder research project. This unexpected result implies a predisposition to Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC), a rare, autosomal dominant condition and has unforeseen implications for him and his family. We review the typical features and management of HLRCC and discuss the challenges that face health professionals, as genetic testing advances and becomes more accessible.

Somatic copy number alterations detected by ultra-deep targeted sequencing predict prognosis in oral cavity squamous cell carcinoma.

BACKGROUND: Ultra-deep targeted sequencing (UDT-Seq) has advanced our knowledge on the incidence and functional significance of somatic mutations. However, the utility of UDT-Seq in detecting copy number alterations (CNAs) remains unclear. With the goal of improving molecular prognostication and identifying new therapeutic targets, we designed this study to assess whether UDT-Seq may be useful for detecting CNA in oral cavity squamous cell carcinoma (OSCC). METHODS: We sequenced a panel of clinically actionable cancer mutations in 310 formalin-fixed paraffin-embedded OSCC specimens. A linear model was developed to overcome uneven coverage across target regions and multiple samples. The 5-year rates of secondary primary tumors, local recurrence, neck recurrence, distant metastases, and survival served as the outcome measures. We confirmed the prognostic significance of the CNA signatures in an independent sample of 105 primary OSCC specimens. RESULTS: The CNA burden across 10 targeted genes was found to predict prognosis in two independent cohorts. FGFR1 and PIK3CAamplifications were associated with prognosis independent of clinical risk factors. Genes exhibiting CNA were clustered in the proteoglycan metabolism, the FOXO signaling, and the PI3K-AKT signaling pathways, for which targeted drugs are already available or currently under development. CONCLUSIONS: UDT-Seq is clinically useful to identify CNA, which significantly improve the prognostic information provided by traditional clinicopathological risk factors in OSCC patients.

Clinical next generation sequencing to identify actionable aberrations in a phase I program.

PURPOSE: We determined the frequency of recurrent hotspot mutations in 46 cancer-related genes across tumor histologies in patients with advanced cancer. METHODS: We reviewed data from 500 consecutive patients who underwent genomic profiling on an IRB-approved prospective clinical protocol in the Phase I program at the MD Anderson Cancer Center. Archival tumor DNA was tested for 740 hotspot mutations in 46 genes (Ampli-Seq Cancer Panel; Life Technologies, CA). RESULTS: Of the 500 patients, 362 had at least one reported mutation/variant. The most common likely somatic mutations were within TP53 (36%), KRAS (11%), and PIK3CA (9%) genes. Sarcoma (20%) and kidney (30%) had the lowest proportion of likely somatic mutations detected, while pancreas (100%), colorectal (89%), melanoma (86%), and endometrial (75%) had the highest. There was high concordance in 62 patients with paired primary tumors and metastases analyzed. 151 (30%) patients had alterations in potentially actionable genes. 37 tumor types were enrolled; both rare actionable mutations in common tumor types and actionable mutations in rare tumor types were identified. CONCLUSION: Multiplex testing in the CLIA environment facilitates genomic characterization across multiple tumor lineages and identification of novel opportunities for genotype-driven trials.