Utilization Of Next-generation Sequencing Research Articles

Racial variation in molecular profile of advanced gastrointestinal cancers.

236 Background: Heterogeneity in the tumor molecular profile based on race is poorly understood. We sought to review the utilization of next generation sequencing (NGS) in patients with advanced gastrointestinal (GI) malignancies treated at a rural academic center and analyze inter racial variations in the molecular tumor profile. Methods: We conducted a retrospective review of patients with advanced GI malignancies that underwent NGS between 2015 to 2018 at East Carolina University.104 patients met eligibility criteria but 8 patients were excluded due to insufficient tissue sampling. Patients with colorectal, gastric, pancreatic, biliary, small intestinal and esophageal cancers were included. Targeted NGS using Caris Life Sciences¨ platform was performed to obtain molecular analysis. We conducted descriptive univariate analysis, cox regression and Kaplan-Meier survival curve analysis. Results: Median age at diagnosis was 64yrs and 64% of patients were black. The study cohort had 41% (n=39) with colon cancer, 18%(17) gastric cancer, 30% (29) pancreatic cancer, 6%(6) biliary cancer, 4%(4) small intestinal cancer and 1%(1) esophageal cancer. 60% (55) had de novo Stage IV disease. Median overall survival (OS) was 25 months (mo), 30 mo in blacks and 32 mo in whites (p value =0.46). Microsatellite stability was seen in 94% (87) and instability in 3% (3). Overall cohort had mutations (mut) in KRAS (50%), TP53 (64%), BRAF (4%), and ERBB amplification (3%). On the cox regression model APC mutation was associated with worse outcome. Black patients had more alterations in KRAS, TP53 (both not significant), and APC (p=0.02). Conclusions: In our analysis we observed inter racial variations in molecular profile of advanced GI malignancies. Black patients had increased rates of APC, KRAS and TP 53 mut. Further studies are required to analyze the impact of these molecular variations on outcomes. Results. [Table: see text]

Limited bias effect of intratumoral heterogeneity on genetic profiling of hepatocellular carcinoma.

Utilization of next-generation sequencing (NGS) to identify potential therapeutic targets and then prescribe matched agents provides new hope for patients with advanced cancer, such as hepatocellular carcinoma (HCC). However, intratumoral heterogeneity (ITH) challenges precise genomic profiling and may lead to target treatment failure. This study aims to evaluate whether and to what extent would genetic profiling be biased by ITH in HCC. We datamined publications focusing on the ITH of HCC and extracted the sequencing and clinicopathological information to make data reanalysis. Potential therapeutic targets and driver genes in HCC were specially pooled as reference to analyze the bias effect of ITH on genetic profiling. Five studies which analyzed ITH using NGS of multi-site samples were enrolled, with a total of 207 tumor samples from 36 HCC patients. The ITH ranged from 5.21% to 88.27% and no correlations between ITH extent and sample numbers, sequencing depth, or clinicopathological parameters were observed. In total, 72 therapeutic and 15 candidate driver genes were pooled as reference. Totally, 38.8% HCCs were found to be drugable in single-site sample, of which only 19.4% might be biased by ITH. Of the driver genes, 86% could be detected in single-site sample. HCC is a highly heterogeneous disease. While ITH indeed hinders comprehensive and precise HCC genome profiling, it has limited influences on identification of actionable and driver mutations. Single-site sampling/biopsy assayed with targeted deep sequencing might be efficient in the clinical management of HCC.

Analysis of the utilization of next generation sequencing in gynecologic malignancies

Objective: We investigated the utilization of next-generation sequencing (NGS) for gynecologic malignancies at our institution.

Loss-of-function mutations in FGF8 can be independent risk factors for holoprosencephaly.

The utilization of next generation sequencing has been shown to accelerate gene discovery in human disease. However, our confidence in the correct disease-associations of rare variants continues to depend on functional analysis. Here, we employ a sensitive assay of human FGF8 variants in zebrafish to demonstrate that the spectrum of isoforms of FGF8 produced by alternative splicing can provide key insights into the genetic susceptibility to human malformations. In addition, we describe novel mutations in the FGF core structure that have both subtle and profound effects on ligand posttranslational processing and biological activity. Finally, we solve a case of apparent digenic inheritance of novel variants in SHH and FGF8, two genes known to functionally coregulate each other in the developing forebrain, as a simpler case of FGF8 diminished function.

Evaluation of changes to the Rickettsia rickettsii transcriptome during mammalian infection.

The lifecycle of Rickettsia rickettsii includes infection of both mammalian and arthropod hosts, with each environment presenting distinct challenges to survival. As such, these pathogens likely have distinctive transcriptional strategies for infection of each host. Herein, we report the utilization of next generation sequencing (RNAseq) and bioinformatic analysis techniques to examine the global transcriptional profile of R. rickettsii within an infected animal, and to compare that data to transcription in tissue culture. The results demonstrate substantial R. rickettsii transcriptional alteration in vivo, such that the bacteria are considerably altered from cell culture. Identification of significant transcriptional changes and validation of RNAseq by quantitative PCR are described with particular emphasis on known antigens and suspected virulence factors. Together, these results suggest that transcriptional regulation of a distinct cohort of genes may contribute to successful mammalian infection.

Candidate gene studies of diabetic retinopathy in human.

Diabetic retinopathy (DR) is a multifactorial disease with complex pathophysiology. It is the main cause of blindness among the people in productive age. The purpose of this literature review is to highlight recent achievements in the genetics of diabetic retinopathy with particular focus on candidate gene studies. We summarized most of the available published data about candidate genes for diabetic retinopathy with the goal to identify main genetic aspects. We conclude that genetic studies reported contradictory findings and no genetic variants meet criteria of a diagnostic marker, or significantly elucidate the root of DR development. Based on these findings it is important to continue with the research in the field of DR genetics, mainly due to the fact that currently new possibilities and approaches associated with utilization of next-generation sequencing are available.

Abstract 15: Target next sequencing profiling of pediatric solid tumors: Potential use for the identification of actionable mutations

Abstract The utilization of next generation sequencing for the identification of actionable gene mutations to guide the choice of therapy towards compounds that selectively inhibit specific genomic alterations is increasingly successful in adult oncology. Yet, limited progress has been achieved in pediatric oncology. Certain pediatric tumor types have shown limited, if any, improvement in overall survival, despite the efforts of institutional and cooperative group clinical trials. For most patients with recurrent pediatric malignancy, the outcome remains dismal. It is clear that a better understanding of the basic biology of these tumors is needed, and that through this understanding, novel and targeted therapeutic approaches may prove beneficial. The utility of systematic genetic profiling for individual tumors has not yet been demonstrated. Genetic screening may not only predict actionable targeted therapy, but also may define alterations that predict resistance to certain therapeutic agents, thereby avoiding the toxicity of salvage therapy with limited hope for benefit. To fill this gap, our group has conducted a pilot study to assess the potential utility of a custom targeted next generation sequencing panel for the identification of directly actionable gene mutations in recurrent pediatric malignancies. Using an “in house” custom panel, we sequenced the exomes of 156 genes (for a total of 5,610 amplicons) frequently mutated in solid tumors, including 29 genes with direct therapeutic target agents. As a proof of principle, we retrospectively analyzed five cases. Tissue sections from paraffin embedded blocks of recurrent pediatric tumors and matching tumor free tissue were microdissected and analyzed using the custom Ion Proton gene panel for the identification of potential targetable sequence variants. All cases were associated with the presence of mutations in actionable genes: ABL1, AKT2, ALK, CDKN2A, EGFR, ERBB2, ERB4, FGFR3, JAK3, NOTHC1, NRAS, PI3CA, RET. Interestingly, four of the five analyzed samples contained mutations in more than one actionable target. These observations suggest that clonal evolution analysis may be of particular importance for these specific tumor types. Our pilot study suggests that in recurrent pediatric malignancies, for which the standard of care offers limited hope for cure, targeted gene sequencing for the identification of actionable targets may provide important information to support therapeutic decision making. Note: This abstract was not presented at the conference. Citation Format: Adam S. Levy, Michael Roth, Nicole Patterson, Erwin Scott, Wilber Quispe-Tintaya, Michelle R. Ewart, Alex Maslov, Aaron Golden, Cristina Montagna. Target next sequencing profiling of pediatric solid tumors: Potential use for the identification of actionable mutations. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr 15.

Abstract 5242: Utilization of next-generation sequencing to identify clinically-relevant mutations in cell-free circulating tumor DNA from patients with advanced pancreatic cancer

Abstract Pancreatic cancer is a uniquely lethal malignancy characterized by frequent mutations in KRAS, CDKN2A, SMAD4, TP53 and many other genes. Molecular characterization is complicated by relatively few patients presenting with surgically resectable disease, frequent absence of metastases in easily accessible sites, and short patient survival. We have shown that KRAS mutation can be detected in cell-free, circulating tumor DNA (ctDNA) isolated from the plasma in a subset of patients and is associated with poor prognosis. The ability to simultaneously detect multiple pancreatic cancer-specific mutations in ctDNA would open a new avenue for detection of clinically-relevant mutations. We collected blood from 28 patients with advanced pancreatic cancer prior to treatment on a phase II clinical trial of pharmacodynamically separated gemcitabine and erlotinib. DNA was extracted from both plasma and available matched FFPE tumor specimens and evaluated with the GeneRead DNA QuantiMIZE System (Qiagen). The mutation status of 160 commonly-mutated genes was determined using an amplicon sequencing assay. Sequencing libraries were prepared from DNA samples (40 ng input) by targeted enrichment of exonic regions using the Qiagen GeneRead Human Comprehensive Cancer Panel, which encompasses >744 kb genomic content with ∼8,000 amplicons. Next-generation sequencing (NGS) was performed on the Illumina MiSeq for validation analyses of a subset of matched tumor-ctDNA samples, and subsequently on the Illumina HiSeq 2500 for ultra-deep sequencing (1,000X) of the entire sample set. Read mapping, variant discovery, and functional annotation were performed with the Qiagen GeneRead Variant Calling Pipeline. The technical success of this sequencing assay for ctDNA, as well as FFPE DNA, was demonstrated by the resulting data meeting the expected NGS quality standards, including a high percentage of mappable reads (∼90%). In addition, the integrity of the sequencing data obtained from ctDNA samples was supported by detection of 82-94% of the variants found in each matched tumor. Somatic, non-synonymous variants were identified in 29 different genes and at allele frequencies typically <0.5. While 52-68% of the mutations identified in the tumor specimen could also be detected in the cell-free ctDNA, not all mutations of potential clinical relevance in tumor specimens were detected in the ctDNA specimens. Updated results from ultra-deep NGS analysis will be presented. The results demonstrate the feasibility of using a new targeted NGS assay for the simultaneous identification of mutations in 160 cancer-related genes in ctDNA. The sensitivity and specificity of variant identification achieved with this technique are potentially clinically relevant, and its optimization could provide a true liquid biopsy for molecular characterization of pancreatic cancer. Citation Format: Clifford G. Tepper, Ryan R. Davis, Stephenie Y. Liu, Rebekah A. Tsai, Irene M. Hutchins, Philip C. Mack, Thomas J. Semrad. Utilization of next-generation sequencing to identify clinically-relevant mutations in cell-free circulating tumor DNA from patients with advanced pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5242. doi:10.1158/1538-7445.AM2015-5242

Clinical application of whole-genome sequencing in patients with primary immunodeficiency

This report illustrates the value of whole genome sequencing (WGS) in elucidating the genetic cause of disease in patients with primary immunodeficiency (PID). As sequencing costs decline, we predict that utilization of next generation sequencing (NGS) in the clinical setting will increase.

Identification of a novel Afipia species isolated from an Indian flying fox.

An old world fruit bat Pteropus giganteus, held in captivity and suffering from necrosis of its wing digits, failed to respond to antibiotic therapy and succumbed to the infection. Samples submitted to the National Centre for Foreign Animal Disease were tested for viral infection. Vero E6 cells exhibited minor but unique cytopathic effects on second blind passage, and full CPE by passage four. Utilizing an unbiased random amplification technique from cell culture supernatant, we identified a bacterium belonging to the Bradyrhizobiaceae. Purification of cell culture supernatant on TY media revealed a slow growing bacterial isolate. In this study using electron microscopy, 16S rRNA gene analysis and whole genome sequencing, we identify a novel bacterial species associated with the site of infection belonging to the genus Afipia. This genus of bacteria is very diverse, with only a limited number of species characterized. Afipia felis, previously described as the etiological agent to cause cat scratch disease, and Afipia septicemium, most recently shown to cause disease in humans, highlight the potential for members of this genus to form a branch of opportunistic pathogens within the Bradyrhizobiaceae. Increased utilization of next generation sequencing and genomics will aid in classifying additional members of this intriguing bacterial genera.

Next-Generation Sequencing-Based Genetic Testing For Familial Partial Lipodystrophy

ABSTRACT Objective: Familial partial lipodystrophy (FPL) of the Dunnigan type (FPLD) is an autosomal dominant condition characterized by fat loss in the limbs and trunk, fat accumulation in the head and neck, and early onset diabetes mellitus. Here we describe the establishment and utilization of next-generation sequencing (NGS)-based genetic testing for FPLD. Methods: We describe NGS-based mutational analysis of the lamin A/C (LMNA) gene, followed by confirmation through Sanger sequencing. Results: We report a patient and her mother with accumulation of fat in the neck and face and loss of fat in the limbs and trunk typical of FPLD2, with young onset diabetes mellitus without ketoacidosis. Both subjects had elevated homeostasis model assessment estimated insulin resistance (HOMA-IR) values and serum triglyceride levels, indicating insulin resistance. Dual energy X-ray absorptiometry confirmed typical fat redistribution. NGS-based mutational analysis of the LMNA gene in these patients revealed a hot spot missense mutation (c.1444C>T, p.Arg482Trp) that was further confirmed by Sanger sequencing. Conclusion: A high index of clinical suspicion is essential to make an accurate clinical diagnosis in subjects with diabetes at an early age. FPLD should be considered in the differential diagnosis of monogenic diabetes with lipodystrophy. Ion torrent NGS offers flexibility and a multiplexing option that provides a robust and inexpensive platform for screening single-gene disorders. Abbreviations: FPL familial partial lipodystrophy FPLD familial partial lipodystrophy of the Dunnigan type NGS next-generation sequencing

Abstract 3428: Validation and utilization of next generation sequencing in the clinical assessment of gliomas

Abstract Background. Identifying prognostic and potentially therapeutic genetic alterations in gliomas is crucial to improve clinical outcome and guide future targeted therapies in this devastating disease. Here, we establish the feasibility of next generation sequencing (NGS) profiling of glioma samples in a clinical setting. Methods. We optimized and validated a 47 gene panel for use on both fresh and Formalin Fixed Paraffin embedded (FFPE) tissue specimens in a CLIA-certified laboratory. Validation was performed in 27 samples by comparing mutation detection on two different NGS platforms, and we have since incorporated routine molecular tumor profiling into the standard workup of glioma patients seen at our institution. Results. From analysis of 120 clinical samples, we found disease-associated changes in 90 patients (75%). Overall 184 changes including disease-associated point mutations and variants of unclear significance were identified across 98 samples, resulting in an average of 1.85 mutations per sample and a range of 1- 6 mutations. This included 49 amplifications across several genes (EGFR, PDGFRA and KIT). Conclusions. The final validated assay allows for a cost effective and efficient analysis of a spectrum of clinically relevant and actionable biomarkers including point mutations, insertions, deletions and gene amplifications. Given the high fraction of tumors presenting with known disease associated changes, routine genomic profiling has the promise of improving patient outcomes and allow for access to targeted therapies. Citation Format: MacLean P. Nasrallah, Maria Martinez-Lage, Alan Fox, Shrey Sukhadia, Arati Desai, Donald M. O'Rourke, Steven Brem, David Roth, Jennifer J.D. Morrissette, Robert D. Daber. Validation and utilization of next generation sequencing in the clinical assessment of gliomas. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3428. doi:10.1158/1538-7445.AM2014-3428

Abstract PL07-02: Rational cancer drug development for targeted drugs.

Abstract The discovery and the development of novel targeted agents for cancer therapeutics is rapidly evolving as a result of our improved understanding of cancer biology. Identification of the diverse genetic changes in different tumors including specific mutations that drive tumor growth is occurring at a rapid pace. This analysis coupled with a better understanding of the diverse mechanisms that lead to drug resistance may result in improved therapeutic interventions. In addition, recent data has provided new insights into identifying the diverse cell types that comprise the tumor microenvironment, characterizing the role of the immune system in both stimulating and inhibiting cancer growth and deciphering new mechanisms such as epigenetics and RNA-based silencing that are involved in tumorgenesis. These insights have provided us with new therapeutic options which have in some cases resulted in impressive clinical benefit. Even with these advances, a number of challenges remain in order to develop more effective cancer therapeutics. These include better defining key cellular targets that drive the growth of specific tumors, identifying appropriate patients with these genetic changes so as to better tailor therapeutic interventions, discovering more efficacious agents that are able to inhibit targets while providing a reasonable margin of safety so as to allow combination with other agents, and developing novel trial designs and endpoints incorporating biomarkers in order to facilitate clinical testing of targeted therapeutics. The use of biomarkers is critical in order to identify the key growth drivers of specific tumors and select the appropriate patients for therapy. In phase I trials, it is critical to use both pharmacokinetic and pharmacodynamic measures to establish the biologically effective dose of the drug being tested in terms of its ability to inhibit the target and alter tumor biology. Tumor biopsies and/or the use of surrogate tissues and appropriate in vitro assays are critical for this analysis. It is important to define whether a targeted agent alone will be sufficient to alter tumor growth or whether it will need to be combined with other targeted agents or chemotherapy. Preclinical data with appropriate cell lines that have been genetically characterized is essential to inform the clinical studies. Another major issue for both small and large molecule targeted therapeutics is the emergence of drug resistance. Repeat biopsies coupled with genetic analysis of tumors are important to better understand the mechanisms leading to drug resistance. This will inform the use of different therapeutic combinations or new agents to either reverse or prevent resistance. Finally, novel clinical trials designs are being developed given the changing paradigm in cancer drug development from a cytotoxic focus to one utilizing targeted agents. Biopsy and analysis of tumor tissues is critical to characterize genetic changes in tumors and identify specific patient subgroups that may benefit from treatment with targeted agents. The I-SPY 2 and the BATTLE trials are examples of adaptively designed trials where clinical response is correlated with a patient's tumor genetic profile. The utilization of next generation sequencing and the quantitative analysis of both RNA and protein expression in tumor tissues both pre and post treatment are critical to better understand how genetic changes correlate with clinical outcomes. As the development of novel targeted agents evolves, the regulatory landscape will also need to change. In addition to utilizing overall survival as the primary endpoint, endpoints such as progression free survival and molecular responses will become increasingly important. The use of in vitro diagnostics will be necessary to help guide therapeutic development in order to identify appropriate patients for specific therapies. As targeted agents move into earlier stages of disease treatment, appropriate statistical and correlative endpoints that predict clinical benefit will need to be utilized. Thus, as new insights allow a better understanding of the complexity of cancer biology, the discovery and clinical development of new therapeutics will also need to evolve in order to improve clinical outcomes for patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr PL07-02.