Comprehensive Gene Panel Research Articles

Validation of Ion TorrentTM Inherited Disease Panel with the PGMTM Sequencing Platform for Rapid and Comprehensive Mutation Detection.

Quick and accurate molecular testing is necessary for the better management of many inherited diseases. Recent technological advances in various next generation sequencing (NGS) platforms, such as target panel-based sequencing, has enabled comprehensive, quick, and precise interrogation of many genetic variations. As a result, these technologies have become a valuable tool for gene discovery and for clinical diagnostics. The AmpliSeq Inherited Disease Panel (IDP) consists of 328 genes underlying more than 700 inherited diseases. Here, we aimed to assess the performance of the IDP as a sensitive and rapid comprehensive gene panel testing. A total of 88 patients with inherited diseases and causal mutations that were previously identified by Sanger sequencing were randomly selected for assessing the performance of the IDP. The IDP successfully detected 93.1% of the mutations in our validation cohort, achieving high overall gene coverage (98%). The sensitivity for detecting single nucleotide variants (SNVs) and short Indels was 97.3% and 69.2%, respectively. IDP, when coupled with Ion Torrent Personal Genome Machine (PGM), delivers comprehensive and rapid sequencing for genes that are responsible for various inherited diseases. Our validation results suggest the suitability of this panel for use as a first-line screening test after applying the necessary clinical validation.

Identification of very rare clinically actionable KRAS variants in colorectal cancer patients using a comprehensive large gene panel.

e15632 Background: While commercially available hotspot testing detects over 99% of the activating mutations in KRAS located at codons 12, 13, 61, and 146, rare oncogenic variants beyond these regions can significantly impact treatment decisions in colorectal cancer (CRC). Herein we showcase the utility of comprehensive gene panel testing in identifying low frequency yet clinically relevant KRAS alterations to enable informed clinical treatment decisions. Methods: 575 CRC specimens were analyzed using CANCERPLEX over a three-year period (KEW, Inc; Cambridge, MA). CANCERPLEX is a comprehensive large gene panel comprising 435 cancer associated genes. A rare KRAS alteration was defined as a variant with a frequency > 0.02% in COSMIC. Variants with clinical actionability in CRC were defined as activating mutations with inferred therapeutic resistance to anti-EGFR antibody therapy. The analytical sensitivity and specificity for single nucleotide polymorphisms (SNPs) in CANCERPLEXwas 99.2% and > 99.9%, respectively. Results: KRAS Q22K, L19F, and G60D were identified as rare oncogenic variants in our analysis of 575 CRC cases. Q22K, L19F, and G60D has been previously reported in 0.009% (7/73000), 0.017% (13/73000), 0.003% (2/73000), respectively, in CRC cases (COSMIC). All three variants have been reported to have transforming potential in vitro and in vivo and trigger activation of downstream oncogenic pathways [PMID:11095964;26284123; 17150185; 20949621]. As activating mutations in KRAS predict lack of response to EGFR antibody therapies, cetuximab and panitumumab were reported as contraindications in all three cases. While not a rare variant, we detected activating mutations in codon 146 in 3% (17/575) of our cohort; strikingly, 5 of the KRAS A146 variants were not identified by prior less comprehensive modes of RAS testing. Conclusions: Low frequency, clinically relevant KRAS variants in CRC may be overlooked using commercially available RAS hotspot assays. In this study we demonstrate that a comprehensive gene panel may detect clinically consequential rare KRAS mutations that may define patients’ treatment and clinical course.

Comprehensive genomic transcriptomic tumor-normal gene panel analysis for enhanced precision in patients with lung cancer.

A CMS approved test for lung cancer is based on tumor-only analysis of a targeted 35 gene panel, specifically excluding the use of the patient’s normal germline tissue. However, this tumor-only approach increases the risk of mistakenly identifying germline single nucleotide polymorphisms (SNPs) as somatically-derived cancer driver mutations (false positives). 621 patients with 30 different cancer types, including lung cancer, were studied to compare the precision of tumor somatic variant calling in 35 genes using tumor-only DNA sequencing versus tumor-normal DNA plus RNA sequencing. When sequencing of lung cancer was performed using tumor genomes alone without normal germline controls, 94% of variants identified were SNPs and thus false positives. Filtering for common SNPs still resulted in as high as 48% false positive variant calling. With tumor-only sequencing, 29% of lung cancer patients had a false positive variant call in at least one of twelve genes with directly targetable drugs. RNA analysis showed 18% of true somatic variants were not expressed. Thus, sequencing and analysis of both normal germline and tumor genomes is necessary for accurate identification of molecular targets. Treatment decisions based on tumor-only analysis may result in the administration of ineffective therapies while also increasing the risk of negative drug-related side effects.

Genetic and biochemical features of the monogenic hereditary urolithiasis

Urolithiasis is a common urological problem. In most cases, this multifactorial pathology develops due to the combination of inherited low-penetrance gene variants and environment factors such as urinary tract infections and unbalanced diet. However, some cases are monogenic. These hereditary forms of urolithiasis manifest in childhood, and are characterized by multiple, bilateral and recurrent kidney stones and progress to chronic renal failure relatively early. Due to widening acceptance of exome and gene panel sequencing, substantially larger percentages of urolithiasis cases are now attributed to hereditary causes, up to 20% among patients of 18 years old or younger. Here we review genetic and biochemical mechanisms of urolithiasis, with an emphasis on its hereditary forms, including fermentopathies (primary hyperoxaluria, adenine phosphorobosyltransferase deficiency, phosphoribosyl-pyrophosphate-synthetase deficiency, xanthinuria, Lesch-Nihan syndrome) and these caused by membrane transport alterations (Dent's disease, familial hypomagnesia with hypercalciuria and nephrocalcinosis, hypophosphatemic urolithiasis, distal tubular acidosis, cystinuria, Bartter's syndrome). We suggest a comprehensive gene panel for NGS diagnostics of the hereditary urolithiasis. It is expected that accurate and timely diagnosis of hereditary forms of urolithiasis would enable the counselling of the carriers in affected families, and ensure personalized management of the patients with these conditions.

Comprehensive next generation sequencing assay and bioinformatic pipeline for identifying pathogenic variants associated with hereditary cancers.

e13105 Background: Diagnosis of hereditary cancer syndromes involves time-consuming comprehensive clinical and laboratory work-up, however, timely and accurate diagnosis is pivotal to the clinical management of cancer patients. Germline genetic testing has shown to facilitate the diagnostic process, allowing for identification and management of individuals at risk for inherited cancers. However, the laboratory diagnostics process requires not only development and validation of comprehensive gene panels to improve diagnostic yields, but a quality driven workflow including an end-to-end bioinformatics pipeline, and a robust process for variant classification. We will present a gene panel for the evaluation of hereditary cancer syndromes, conducted utilizing our novel end-to-end workflow, and validated in the CLIA-approved environment. Methods: A targeted Next-Generation Sequencing (NGS) panel consisting of 130 genes, including exons, promoters, 5’-UTRs, 3’-UTRs and selected introns, was designed to include genes associated with hereditary cancers. The assay was validated using samples from the 1000 genomes project and samples with known pathogenic variants. Elements software was utilized for end-to-end bioinformatic process ensuring adherence with the CLIA quality standards, and supporting manual curation of sequence variants. Results: Preliminary data from our current panel of genes associated with hereditary cancer syndromes revealed high sensitivity, specificity, and positive predictive value. Accuracy was confirmed by analysis of known SNVs, indels, and CNVs using 1000 Genomes and samples carrying pathogenic variants. The bioinformatics software allowed for an end-to-end quality controlled process of handling and analyzing of the NGS data, showing applicability for a clinical laboratory workflow. Conclusions: We have developed a comprehensive and accurate genetic testing process based on an automated and quality driven bioinformatics workflow that can be used to identify clinically important variants in genes associated with hereditary cancers. It's performance allows for implementation in the clinical laboratory setting.

Next-generation sequencing of non-small cell lung cancer using a customized, targeted sequencing panel: Emphasis on small biopsy and cytology.

Background:Next-generation sequencing (NGS) with a multi-gene panel is now available for patients with lung adenocarcinoma, but the performance characteristics and clinical utility of this testing are not well-described. We present the results of an extended 467 gene panel in a series of advanced, highly selected nonsmall cell lung cancer (NSCLC) patients using a range of specimens, including predominantly small biopsy and cytology specimens.Materials and Methods:A retrospective review of 22 NSCLC biopsies sent for NGS using an extended gene panel from January 2014 to July 2015. The customized NGS panel sequences 467 cancer-associated genes with exonic and intronic sequences obtained from purified tumor DNA. Genomic alterations, patient characteristics, and success of testing were determined.Results:The majority of samples tested were metastatic lung adenocarcinoma on final pathology. Of the 22 specimens tested, 5 (22.7%) were surgical resections and 17 (77.3%) were small biopsy and cytology specimens. Twenty-one (95%) of the specimens were adequate for full sequencing and yielded a total of 204 genomic alterations (average 8.9 per tumor), of which 17 (average 0.81 per tumor) were actionable and/or clinically relevant. Genomic alterations were found most commonly in the TP53, EGFR, EPHB1, MLL3, APC, SETD2, KRAS, DNMT3A, RB1, CDKN2A, ARID1A, EP300, KDM6B, RAD50, STK11, and BRCA2 genes.Conclusions:NGS using a comprehensive gene panel was performed successfully in 95% of all NSCLC cases in this series, including 94% small biopsy and cytology specimens and 100% surgical resections. This custom assay was performed on a range of tumor specimens and demonstrates that small specimens are able to provide a similar depth of information as larger ones. As many patients present at an advanced stage and only small specimens are obtained, the information these provide has the potential for guiding treatment in highly selected patients with advanced lung adenocarcinoma.

Abstract P2-02-05: Comparison of non-breast and ovarian cancer phenotypes of BRCA1/2 mutation carriers across multi-gene panels

Abstract Background: BRCA1/2 germline mutations account for the majority of hereditary breast and ovarian cancers. Until 2013, the only way to identify individuals with BRCA mutations was through single gene testing. With multi-gene panel testing (MGPT) including BRCA1/2, BRCA mutations are being identified at an increased rate. To date, the phenotype of BRCA1/2 mutation carriers includes an increased prevalence of breast, ovarian, prostate, and pancreatic cancer, as well as melanoma. The phenotype, however, of mutation carriers identified by panel testing is not well understood. Methods: All sequential cases submitted to our laboratory for MGPT including BRCA1/2 between June 2013 and June 2015 and BRCA1/2 single gene testing between June 2013 and February 2015 were retrospectively reviewed. Data from 77,345 test request forms were reviewed; probands with a BRCA1 or BRCA2 mutation were selected and analyzed. Probands with no personal history of cancer/not provided (n=528) were excluded. Results: Of 2,967 BRCA1/2 positive probands, 2,439 (82.2%) had a personal history of cancer, with 2,794 cancers reported. On all tests completed, with the exception of a pancreatic cancer focused panel, breast and/or ovarian cancer were the most commonly observed cancer types (n=2,364, 84.6%). On single gene testing (n=739), prostate (n=16, 2.2%), colorectal (n=8, 1.1%), and pancreatic (n=7, 0.9%) cancers were the other most frequent cancers reported. Across all MGPT cases (n=1,700), additional observed cancers included uterine (n=48, 2.8%), colorectal (n=46, 2.7%), pancreatic (n=37, 2.2%), melanoma (n=29, 1.7%), and thyroid (n=27, 1.6%). For breast cancer-specific gene panel cases (n=934), additional cancers included thyroid (n=12, 1.3%) and uterine cancer (n=10, 1.1%). For gynecologic cancer gene panel cases (n= 482), additional cancers included uterine (n=21, 4.4%), colorectal (n=11, 2.3%), and melanoma (n=11, 2.3%). Cases tested via a pancreatic cancer gene panel (n=25,) had pancreatic cancer (n=17, 68%) reported most frequently, followed by breast (n=6, 24%), colorectal (n=3, 12%), and melanoma (n=3, 12%). For comprehensive cancer gene panel cases (n=254), colorectal (n=28, 11.0%), uterine (n=17, 6.7%), pancreatic (n=14, 5.5%), prostate (n=10, 3.9%), thyroid (n=10, 3.9%), and a variety of rare tumor types (n=15, 5.9%) were among the additional cancers reported. Discussion: As expected, the majority of cancers reported on almost all tests were breast and/or ovarian cancer. More specifically, the observed cancers for tumor specific cancer panels, matched the relevant cancer type (i.e. pancreatic cancer most commonly reported in those tested via a pancreatic cancer panel). However, on the comprehensive cancer panels, other than breast and ovarian cancer, there appears to be a more even distribution of various cancer types, including rare tumors, in probands who have a BRCA1 or BRCA2 mutation. Further studies should be conducted to examine the phenotypes of BRCA mutation carriers identified via comprehensive cancer panels to determine the association and prevalence of unexpected cancer types with BRCA1/2 mutations. This could have important implications for determining the most appropriate genetic test and management of BRCA mutation carriers. Citation Format: Jackson M, Yusuf A, Espenschied C, Paudyal T, Profato-Partlow J. Comparison of non-breast and ovarian cancer phenotypes of BRCA1/2 mutation carriers across multi-gene panels [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-02-05.

CML Patients with Primary Resistance or Suboptimal Response to TKI Therapy Have Variants in Genes Affecting Drug Absorption and Metabolism

BACKGROUNDChronic myeloid leukemia in chronic phase (CML CP) is successfully treated with tyrosine kinase inhibitors (TKIs) but a proportion of patients show primary or secondary resistance to TKI therapy. BCR-ABL1 mutations are the primary cause for secondary resistance to TKI therapy, but the causes for primary resistance are poorly known. Putative variants in genes affecting drug absorption, distribution, metabolism and excretion (ADME) have not been comprehensively analyzed. We set out to screen CML CP patients who either had primary resistance to TKI therapy or suboptimal response with large ADME gene panel to examine if these genes play a role in poor TKI responses.METHODSWe selected samples from 21 CML CP patients treated in our institute who either had primary resistance (N=6) or responded suboptimallyto TKI treatment (n=15) but did not have BCR-ABL1 mutations. DNA samples from the time of diagnosis were sequenced with massively parallel sequencing using a comprehensive custom ADME gene panel covering exons and introns of 382 genes. Over 200 healthy volunteers who had been sequenced with the same gene panel served as population controls. Loss-of-function and novel variants were validated using capillary sequencing.We tested the differences of variant allele frequencies between CML patients and healthy controls by performing the Cochran-Armitage trend test using additive coding (0,1,2) for genotypes homozygous for the major allele, heterozygous, and homozygous for the minor allele, respectively. CML patients with optimal response (n=24) were used as a control group.RESULTSSequencing of 21 poor responding CML CP patients revealed a total of 658 nonsynonymousgene variants. Majority of the variants (94%) were missense variants whose effects on gene function are uncertain. Eigthvariants were previously unreported. 18 variants were predicted to lead to a complete loss-of-function of the genes affected. Principal component analysis did not reveal clear global differences between the CML patients and healthy controls in the frequencies of ADME gene variants. However, the Cochran-Armitage trend test showed a number of variants whose frequencies in CML samples were significantly different from the healthy controls (table 1). Two of the loss-of-function variants were in the top 10 variants, whose frequencies differed between CML patients and healthy controls. ACAD11 rs41272317 variant was observed in 14.2% of patients whereas its frequency in healthy controls was 0.5% (p=0.003). Similarly DPYD rs3918290 was observed in 14.2% of patients and 3.3% of healthy controls (p=0.006). ACAD11 encodes an acyl-CoA dehydrogenase enzyme with a preference for carbon chain lengths between 20 and 26, and has been shown to be an important metabolic target for p53 pro-survival function. DPYD codesdihydropyrimidine dehydrogenase, which is involved in the metabolism ofpyrimidines and the anticancer agent 5-fluorouracil. Sanger sequencing confirmed both variants in patient samples. From the top10 missense variants, SLC15A1 rs144553892 variant was significantly more often observed in poor responding CML patients than in healthy controls (14.2 and 0.9%, respectively, p=0.003). SLC15A1 is a solute carrier family 15 member 1 which has a known function as a co-transporter foroligopeptides. Recently solute carriers have been identified to have significant impact on cancer therapy. Screening of additional CML patients is ongoing to confirm the frequency of these variants in optimally responding CML patients.CONCLUSIONSCML patients with primary resistance or suboptimal response to TKI therapy have mutations in genes affecting drug intake and metabolism, which can be causally associated with poor therapy responses. Validation in larger patient cohorts is warranted. [Display omitted] DisclosuresPorkka:Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding. Mustjoki:Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Ariad: Research Funding.

Novel Mutations in Patients with Blast Crisis or Accelerated Phase Chronic Myeloid Leukemia

Introduction: Progression of chronic myeloid leukemia (CML) to blast crisis (BC) results from the acquisition of additional driver mutations, which still are poorly understood. In the tyrosine kinase inhibitor (TKI) era, BC-CML remains a challenging clinical entity with very poor prognosis and short survival. In addition, there is an unmet need for identification of progression-related genetic mutations and potentially targetable pathways.Patients and methods: Bone marrow samples from 16 patients with BC-CML (myeloid n=13, lymphoid n=2, unknown phenotype n=1) and 2 patients with accelerated phase (AP) were collected from the Helsinki University Hospital, Finland and National Cancer Institute, Cairo University, Egypt. In addition, skin biopsy samples were collected for germline variant controls. Whole exome sequencing (WES) (n=8) was done with Agilent or NimbleGen exome capture kits and deep-targeted sequencing with the NimbleGen (SeqCap EZ Design) comprehensive cancer gene panel (n=10) using an Illumina HiSeq instrument. The panel was comprised of 578 driver genes with documented association to common and rare cancers (gathered from Sanger and NCBI tests databases). All mutated genes identified by WES were included in the panel, as well as genes previously reported to be mutated in BC-CML.Results: We identified 55 mutations in 33 driver genes (average: 3 per patient, range 0-7). Of the identified 33 mutated genes, 27 were ranked ≥1 in the Gene Ranker Cancer scoring system (http://cbio.mskcc.org/tcga-generanker), where genes with a score of 1 have a documented association with cancer in a cancer gene database and higher scores indicate more frequent incidence of gene mutations in different cancers. Core-binding factor (CBF) aberrations (RUNX1 mutations and inv [16]) were the most recurrent variants (n=6 in 5 patients, 27.7% of the patients) followed by ABL1 mutations (n=4 in 4 patients, 22%) and BCOR mutations (n=4 in 3 patients, 16.7%). Other recurrent mutations included FLT3, IKZF1, and NOTCH1 mutations which all were found in 2 cases. Some of the discovered mutations have not been reported in BC-CML patients before, such as mutations to MTOR, PTPRJ, CD274 (PD-L1), IL21R, SETD2 and ZRSR2 genes. In silico analysis of the targeted genes showed that many of the affected genes interact with each other in different pathways and also with ABL1. The top pathways affected were associated with key biological functions: regulation of hematopoiesis (11 genes affected), leucocyte differentiation (9 genes) and transcriptional regulation (11 genes).Conclusion: The genomic landscape of advanced phases of CML (BC and AP) shows complex heterogeneity with a broad range of genes affected leading to dysregulation of multiple molecular pathways that have an impact on treatment responses and disease biology. Such complexity suggests that a personalized approach maybe the best treatment option for these patients. DisclosuresHeckman:Celgene: Research Funding; Pfizer: Research Funding. Porkka:Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding. Mustjoki:Pfizer: Honoraria, Research Funding; Ariad: Research Funding; Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding.

O1-5-5 - Implementation of clinical sequencing using a comprehensive gene panel (OncoPrimeTM)

O1-5-5 - Implementation of clinical sequencing using a comprehensive gene panel (OncoPrimeTM)

Abstract 5234: Rare deleterious germline mutations of candidate genes associated with risk of familial esophageal squamous cell carcinoma (ESCC) by targeted sequencing

Abstract Background and aims: Esophageal cancer (EC) is a deadly disease with poor survival rates of only ∼10%. The EC incidence remains highest in the Shanxi and Henan regions near the Tang Hang Mountain area in Northern China. In endemic esophageal squamous cell carcinoma (ESCC) regions, common familial clustering and historical migration patterns suggest an inherited ESCC genetic susceptibility. We hypothesize that cancer predisposition gene(s) (CPGs) carrying rare deleterious variants play a crucial role in ESCC genetic susceptibility and aim to identify the underlying CPGs of familial ESCC from Henan by applying the targeted sequencing approach. Methods: Our discovery set utilized NGS technology to capture a comprehensive gene panel critical for ESCC by targeted re-sequencing of 1271 selected genes (11 Mb) and sequenced for the germline variants in the blood DNA from 222 familial ESCC individuals and 124 Henan non-cancer controls. Familial ESCC was defined as at least two family members developed ESCC. The custom designed panel contains a cancer panel of 578 gene list (4Mb) and a custom list of 693 (7Mb) ESCC-specific genes derived from various approaches including microarray profiling in FH+ ESCC primary tissues, homozygosity-by-descent (HBD), SNP array-based analysis by both GWAS and candidate gene approaches of our own set of MassArray SNP association data. The data from 346 individuals were processed using the analysis pipeline following the GATK guideline. Briefly, raw fastq reads were mapped using the Burrows-Wheeler Aligner (BWA). PCR duplicates are marked using Picard. The raw variants are called by GATK HaplotypeCaller using a joint genotyping of all samples. Results: Preliminary data analysis for the 346 samples resulted in the final call set comprised of 52328 rare variants. There are 26486 rare variants present in the 222 FH+ ESCC cases, but absent in the 124 controls. The average sequencing coverage was 88X. Principal component analysis (PCA) and multidimensional scaling (MDS) indicated a homogenous population structure between the case and the control populations. Targeted re-sequencing results of the 1271 selected genes in 222 familial ESCC individuals identified a recurrent missense variant in AIM1L (Absent in Melanoma 1-like) in 14/222 (6.3%) of FH+ ESCC patients but absence in the 124 non-cancer control. Data obtained by a melting curve-based genotyping platform by LC480 and LightSNiP assay for high throughput validation of this recurrent SNP in larger sample cohort are underway and will be presented. Conclusions: A SNP in AIM1L may be associated with higher risk for ESCC in Henan and validation in larger cohort is required. The data may provide valuable insight for understanding genetic susceptibility to this deadly cancer. Acknowledgements: We acknowledge the Asian Fund for Cancer Research for grant support. Citation Format: Josephine Mun Yee Ko, Lu wen Ni, Sheyne, Sta Ana Choi, Lisa Chan Lei, Li Dong Wang, Maria Li Lung. Rare deleterious germline mutations of candidate genes associated with risk of familial esophageal squamous cell carcinoma (ESCC) by targeted sequencing. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5234.

Abstract 1384: A comprehensive gene panel for precise diagnosis and treatment of childhood cancer

Abstract We have created and are validating a targeted gene panel that utilizes 20ng of FFPE DNA and 10 ng of FFPE RNA in a simple, 3-tube Ion AmpliSeq assay suitable for sequencing on the Ion Torrent platform with rapid turn around time (<3 days in lab) and automated report generation that is designed specifically to improve pediatric cancer diagnosis, identify prognostic features, and detect genomic alterations (including mutations, insertions, deletions, gene amplification, and RNA expression levels) that match current targeted therapeutic targets. The core content is identical to the Oncomine Comprehensive Assay as employed by the NCI MATCH program. In addition, a total of 128 genes known to be genomically altered in pediatric cancer are interrogated (either hotspots or coding exons), 21 genes frequently amplified in pediatric cancer, and 157 gene fusions (including over 1,500 variants) unique to these tumors are included. Unique to this panel are 22 pharmacogenomic gene polymorphisms that influence cytotoxic drug metabolism, to guide patient-specific dosing. Content was chosen to cover all common pediatric malignancies, including pediatric leukemia (ALL, AML, and multiple variants within each), lymphoma, brain tumors, neuroblastoma, and sarcomas, by a working group composed of pediatric oncologists and pathologists. Specific genomic alterations were identified from a comprehensive search of the pediatric cancer literature by the panel and categorized by level of evidence (FDA approved drug, open clinical trial, or peer-reviewed literature identifying a feature in a pediatric cancer). This panel includes 45 of the 51 gene targets identified by the COG TAP committee for pediatric cancer specific gene target-therapeutic agent matching. In contrast to the NCI MATCH program, which currently employs the Ion Torrent Personal Genome Machine and Ion 318 Chip, this panel is intended for use on the newly available Ion S5 platform, due to its significantly enlarged content. Verification of individual feature identification by orthogonal assays (Sanger sequencing, PCR, FISH, SNP arrays) is in process. The assay will be released initially as an LDP, with intended updated content as new features and therapeutics are identified. Unlike current panels for specific features like single gene mutations or known gene translocations, this panel is intended to detect all currently known genomic alterations with level I, II, or III evidence in pediatric cancer, as well as the ability to detect new gene mutations and fusion variants (subject to secondary assay validation). Also unique is the ability to measure gene amplification and RNA expression levels in one comprehensive panel starting with 30 ng or less of nucleic acid extracted from routine clinical FFPE specimens. This is the only comprehensive gene panel for pediatric cancer that encompasses diagnostic, prognostic, pharmacogenomic, and therapeutic targets in one assay. Citation Format: Timothy J. Triche, Jackie Biegel, Alex Judkins, Tracy Busse, Alan Wayne, Deepa Bhojwani, Shahab Asgharzadeh, Matthew Oberley, David Parham. A comprehensive gene panel for precise diagnosis and treatment of childhood cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1384.

Unbiased targeted next-generation sequencing molecular approach for primary immunodeficiency diseases

Molecular genetics techniques are an essential diagnostic tool for primary immunodeficiency diseases (PIDs). The use of next-generation sequencing (NGS) provides a comprehensive way of concurrently screening a large number of PID genes. However, its validity and cost-effectiveness require verification. We sought to identify and overcome complications associated with the use of NGS in a comprehensive gene panel incorporating 162 PID genes. We aimed to ascertain the specificity, sensitivity, and clinical sensitivity of the gene panel and its utility as a diagnostic tool for PIDs. A total of 162 PID genes were screened in 261 patients by using the Ion Torrent Proton NGS sequencing platform. Of the 261 patients, 122 had at least 1 known causal mutation at the onset of the study and were used to assess the specificity and sensitivity of the assay. The remaining samples were from unsolved cases that were biased toward more phenotypically and genotypically complicated cases. The assay was able to detect the mutation in 117 (96%) of 122 positive control subjects with known causal mutations. For the unsolved cases, our assay resulted in a molecular genetic diagnosis for 35 of 139 patients. Interestingly, most of these cases represented atypical clinical presentations of known PIDs. The targeted NGS PID gene panel is a sensitive and cost-effective diagnostic tool that can be used as a first-line molecular assay in patients with PIDs. The assay is an alternative choice to the complex and costly candidate gene approach, particularly for patients with atypical presentation of known PID genes.

Inherited mutations in cancer susceptibility genes are common among survivors of breast cancer who develop therapy‐related leukemia

Risk factors for the development of therapy-related leukemia (TRL), an often lethal late complication of cytotoxic therapy, remain poorly understood and may differ for survivors of different malignancies. Survivors of breast cancer (BC) now account for the majority of TRL cases, making the study of TRL risk factors in this population a priority. Subjects with TRL after cytotoxic therapy for a primary BC were identified from the TRL registry at The University of Chicago. Those with an available germline DNA sample were screened with a comprehensive gene panel covering known inherited BC susceptibility genes. Clinical and TRL characteristics of all subjects and those with identified germline mutations were described. Nineteen of 88 survivors of BC with TRL (22%) had an additional primary cancer and 40 of the 70 survivors with an available family history (57%) had a close relative with breast, ovarian, or pancreatic cancer. Of the 47 subjects with available DNA, 10 (21%) were found to carry a deleterious inherited mutation in BRCA1 (3 subjects; 6%), BRCA2 (2 subjects; 4%), TP53 (tumor protein p53) (3 subjects; 6%), CHEK2 (checkpoint kinase 2) (1 subject; 2%), and PALB2 (partner and localizer of BRCA2) (1 subject; 2%). Survivors of BC with TRL have personal and family histories suggestive of inherited cancer susceptibility and frequently carry germline mutations in BC susceptibility genes. The data from the current study support the role of these genes in TRL risk and suggest that long-term follow-up studies of women with germline mutations who are treated for BC and functional studies of the effects of heterozygous mutations in these genes on bone marrow function after cytotoxic exposures are warranted. Cancer 2016;122:304-311. © 2015 American Cancer Society.

Molecular Analysis of Diamond Blackfan Anemia and Genotype-Phenotype Correlation: Experience from the Canadian Inherited Marrow Failure Registry

Background/Objectives: Diamond Blackfan anemia (DBA) is an inherited disorder characterized by chronic hypoproductive anemia, physical malformations, and an increased risk of malignancies. At least 12 DBA genes have been identified, which include various ribosomal protein genes and the transcription factor GATA1. The aims of our study were (1) to identify the mutation spectrum of DBA patients, utilizing a cohort of patients enrolled on the Canadian Inherited Marrow Failure Registry (CIMFR) and (2) to determine whether specific hematological abnormalities, malformations, and outcomes are associated with specific mutations.Methods: Patients were enrolled on the CIMFR, which is a multicenter cohort study of inherited bone marrow failure syndromes (IBMFS). Genetic testing was performed using one or more of the following tests: Sanger sequencing, next generation sequencing (NGS) DBA gene panel, a comprehensive NGS IBMFS gene panel developed in our laboratory, or comparative genetic hybridization (CGH). Severity of the hematological disease was dichotomized according to a patient's requirement for chronic treatment: those who were maintained on corticosteroids, blood transfusions, or received a hematopoietic stem cell transplantation were considered to have a more severe phenotype than those who did not require hematological treatment. Chi-square tests with a Fisher's exact test correction were used to compare genetic groups with at least 5 patients on observed phenotypes.Results: 71 patients with DBA have been enrolled in our registry. A causal mutation has been identified in 36 of these patients, with the following rates: RPS19 (n=11), RPL11 (n=7), RPL5 (n=6), RPS26 (n=5), RPL35a (n=2), RPS24 (n=2), and one of each RPS7, RPS29, RPS17. Remarkably, a substantial number of patients in our population-based cohort (19.4%) had mild hematological phenotype requiring no therapy. Patients with RPL11 mutations tended to have a less severe DBA phenotype, while patients with RPS19 mutations tended to have a more severe phenotype (P =0.04). In terms of non-hematological malformations, we found no differences in cardiac, stature and craniofacial malformations across the groups compared (all p>0.1). However, patients with RPL5 mutations had significantly more hand malformations (P =0.02), and patients with RPS26 mutations had more genitourinary malformations (P =0.04). To control for the impact of mutation severity on the observed phenotype, we compared the prevalence of mutations that are predicted to result in truncated or lack of protein from the respective allele (large copy-number variation, nonsense, or indel frameshift) to mutations that are predicted to be hypomorphic or affect function (splicing, indel/inframe and, missense) between mutation categories. There were no differences among genetic groups in the severity of their mutations (P =0.58).Conclusions: Mutations in a wide spectrum of ribosomal protein genes underlie DBA cases in Canada, which approximate those observed by other registries in Western countries. Patients with DBA caused by RPL11 mutations tended to have a milder hematological phenotype, while patients with RPS19 mutation tended to have a more severe phenotype. Mutations in RPS26 and RPL5 are associated with genitourinary and hand malformations, respectively. Our findings may help improve counseling of DBA patients and their family. Future studies are needed to replicate our results and determine whether these findings can help personalize care. DisclosuresLipton:Ariad: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Novartis Pharmaceuticals: Consultancy, Research Funding.

O1-13-6 - Introduction of clinical sequence using a comprehensive cancer gene panel (OncoPrime) to daily clinical practice

O1-13-6 - Introduction of clinical sequence using a comprehensive cancer gene panel (OncoPrime) to daily clinical practice

MG-129 Our experience ofin silicogene panel testing for clinically heterogeneous disorders using exome sequencing

Background The implementation of next generation sequencing technology (NGS) in a molecular diagnostics laboratory has resulted in a major transformation in service delivery. The ability to generate accurate sequence data from thousands of genes in a single experiment has facilitated the development of comprehensive gene panel tests for genetically heterogeneous disorders. Previous methodology of gene panel testing included Sanger sequencing, which was time-consuming, labour-intensive and costly and therefore restricted the number of genes that were included in a panel. Methods/results At The Hospital for Sick Children (Toronto, Canada), we have developed and clinically validated in silico gene panels using whole exome sequencing (WES) for several heterogeneous disorders including hereditary spastic paraplegia (HSP), connective tissue and bone disorders (CT), hereditary hearing loss (HL), Noonan syndrome (NS) and autoimmune disorders (AI). Our laboratory’s current strategy of using WES has several advantages including: (1) standardised workflows that facilitate the development of additional gene panels; (2) expansion of current gene panel as new disease associations are discovered; and (3) building of an internal database of allele frequencies to aid in interpretation of variants. Through our validation studies we have shown high reproducibility and accuracy with >99% sensitivity for detection of single nucleotide variants (SNVs) and >95% for detection of small insertion/deletions (indels). Conclusions NGS technologies have enabled our laboratory to expand our testing menu to include genes that comprise part of the differential diagnoses for several disorders which will lead to improved detection rates, increased genetic diagnoses and ultimately better clinical care.

Abstract 76: Molecular profiling of AML patient derived xenograft models with deep sequencing using a 109 AML associated gene panel and a 409 gene comprehensive cancer panel

Abstract We have developed and validated a 109 AML associated gene panel NGS assay based on sequence capture technologies. The overall assay sensitivity was 99.%, and overall assay specificity was 100% when sequenced with reference “gold” standard NA12878. Assay analytical accuracy was evaluated with diluted cell line samples harboring mutations in this gene panel, all diluted mutations with minor allele frequencies were detected, analytical limit of detection (LOD) is < 5%, and assay reproducibility is 96.8%. The assay also demonstrated robustness in sequencing 10 commercial AML patient samples (whole blood and bone marrow) and detecting mutations. Therefore, the AML gene panel deep sequencing assay is sensitive and accurate and is ready to be used for preclinical and clinical studies. We subsequently sequenced 15 AML patient derived xenograft (PDX) models with this assay. These models were also deep sequenced with a 409 comprehensive cancer gene panel based on multiplex amplicon technologies. The mutation profiles of each model will be summarized in this presentation using two sequencing platforms. By analysis of the mutation profile of each model, sub-clones of mutations were revealed in the early passages of these models. Two of these models with FLT-3 mutations were later treated with Quizartinib and Crenolanib separately, the treated mouse models were profiled with these two targeted gene panels; clonal evolution of mutations in these models treated with FLT-3 inhibitors was documented. Deep sequencing with target gene panel is a powerful tool to decipher the mutation sub-clones in the early passages of PDX models, and understand the acquired resistance of drug treatment. Citation Format: Stephen Huang, Paul Lira, Kai Wang, Cathy Zhang, Amy Jackson-Fisher, Keith Ching, Paul Rejto. Molecular profiling of AML patient derived xenograft models with deep sequencing using a 109 AML associated gene panel and a 409 gene comprehensive cancer panel. [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 76. doi:10.1158/1538-7445.AM2015-76

The detection of clinically relevant cancer mutations using a high depth, augmented, comprehensive cancer gene panel.

e12547 Background: Genetic testing is an increasingly accepted form of molecular testing in cancer. Specific genetic lesions can determine prognosis and have diagnostic value, assisting in therapeutic decisions. Next-generation sequencing facilitates the detection of a wide array of somatic mutations, copy number alterations (CNAs), and gene fusions all in a single assay. With increased sequencing depth, mutations present in only a fraction of the neoplastic tissue can be identified. Detection of these mutations after sequencing DNA and RNA involves a complex set of variant detection algorithms tailored specifically for cancer analysis. Methods: We designed a comprehensive cancer panel using an augmented target enrichment strategy optimized for even coverage across the entire span of gene content. This approach includes multiple nucleic acid enrichments to deal with high-GC content and includes custom oligonucleotides designed to compensate for challenging regions. The panel includes over 1,200 cancer-rel...

A comprehensive genomic approach for neuromuscular diseases gives a high diagnostic yield.

Neuromuscular diseases (NMDs) are a group of >200 highly genetically as well as clinically heterogeneous inherited genetic disorders that affect the peripheral nervous and muscular systems, resulting in gross motor disability. The clinical and genetic heterogeneities of NMDs make disease diagnosis complicated and expensive, often involving multiple tests. To expedite the molecular diagnosis of NMDs, we designed and validated several next generation sequencing (NGS)-based comprehensive gene panel tests that include complementary deletion and duplication testing through comparative genomic hybridization arrays. Our validation established the targeted gene panel test to have 100% sensitivity and specificity for single nucleotide variant detection. To compare the clinical diagnostic yields of single gene (NMD-associated) tests with the various NMD NGS panel tests, we analyzed data from all clinical tests performed at the Emory Genetics Laboratory from October 2009 through May 2014. We further compared the clinical utility of the targeted NGS panel test with that of exome sequencing (ES). We found that NMD comprehensive panel testing has a 3-fold greater diagnostic yield (46%) than single gene testing (15-19%). Sanger fill-in of low-coverage exons, copy number variation analysis, and thorough in-house validation of the assay all complement panel testing and allow the detection of all types of causative pathogenic variants, some of which (about 18%) may be missed by ES. Our results strongly indicate that for molecular diagnosis of heterogeneous disorders such as NMDs, targeted panel testing has the highest clinical yield and should therefore be the preferred first-tier approach.