Feasibility Of Next-generation Sequencing Research Articles

Next-generation sequencing using tissue specimen collected with a 1.1 mm-diameter cryoprobe in patients with lung cancer.

Next-generation sequencing (NGS) analysis is considered standard for lung cancer diagnosis in clinical practice. Little is known about the feasibility of NGS using tumour tissue sampled with a 1.1 mm-diameter cryoprobe. We aimed to investigate the suitability of specimens obtained by transbronchial cryobiopsy (TBC) using a 1.1 mm-diameter cryoprobe for NGS analysis. Patients with lung cancer who underwent TBC using a 1.1 mm-diameter cryoprobe for NGS testing between October 2020 and April 2023 were enrolled. A 4.0- or 3.0 mm-diameter bronchoscope with radial probe endobronchial ultrasound and virtual bronchoscopic navigation was used to detect peripheral lung lesions. All procedures were performed under fluoroscopic guidance. Data were analysed retrospectively. A total of 56 patients underwent TBC using a 1.1 mm cryoprobe for NGS testing, during the study period. Most patients (98%) were in the advanced stage of lung cancer (recurrent or inoperable disease of stages III or IV). The diagnostic yield of NGS for DNA and RNA sequencing was 95% each (53 of 56). Moderate bleeding was noted in three patients (5%) and none of the study patients developed life-threatening complications, such as pneumothorax or lung infection. TBC using a 1.1 mm-diameter cryoprobe is a useful and safe tool for NGS analysis, for both DNA and RNA sequencing.

A glimpse into the feasibility of next generation sequencing for newborn screening of lysosomal and other diseases with second-tier biochemical assays as part of the screening process

A glimpse into the feasibility of next generation sequencing for newborn screening of lysosomal and other diseases with second-tier biochemical assays as part of the screening process

Abstract 5790: Genomic landscape of circulating tumor DNA alterations in patients with paraganglioma and pheochromocytoma

Abstract Background: Paragangliomas (PGLs) and Pheochromocytomas (PCCs) are rare neuroendocrine tumors (NETs). PGLs arise from chromaffin cells in the ganglia of the autonomic nervous system, while PCCs arise from chromaffin cells in the adrenal medulla. The genomic landscapes of PGLs and PCCs have not been well studied. Thus, the aim of this study is to report differences of mutational occurrences and confirm the feasibility of next generation sequencing (NGS) testing circulating tumor DNA (ctDNA) in patients with PGL and PCC. Patients and Methods: Molecular alterations in 46 plasma samples were evaluated using a commercially available ctDNA assay (Guardant360® or Guardant360® CDx) across multiple institutions from 2016-2021. The tests detect single nucleotide variants and indels in 54-83 genes with copy number amplifications and fusions in selected genes. Results: Of the 24 PGL patients, there was a median age of 55 (range:28-78) years and 14 (58%) patients were male. Of the 22 PCC patients, there was a median age of 56 (range:28-86) years and 12 (54.5%) patients were male. Genetic alterations were identified in 16 (67%) PGL and 17 (77%) PCC patients. Of the 16 PGL samples with alterations, TP53 associated genes were most commonly altered (44%), followed by ATM (25%), FGFR2 (19%), APC (13%), BRAF (13%), BRCA1 (13%), CCND2 (13%), FGFR3 (13%), IDH2 (13%), KRAS (13%), PDGFRA (13%), RB1 (13%), TERT(13%), ALK (6%), ARID1A (6%), BRCA2 (6%), CCND1 (6%), CDK6 (6%), CDK12 (6%), EGFR (6%), FGFR1 (6%), KIT (6%), MET (6%), NF1 (6%), NRAS (6%), PIK3CA (6%), PTEN (6%), and ROS1 (6%). Of the 17 PCC samples with alterations, TP53 was most commonly altered (41%), followed by ATM (35%), NF1 (24%), FGFR1 (18%), APC (13%), EGFR (12%), MET (12%), MYC (12%), NOTCH1 (12%), PDGFRA (12%), TSC1 (12%), AR (6%), ARID1A(13%), BRAF (6%), BRCA1 (6%), BRCA2 (6%), CCND1 (6%), CDK6 (6%), CHEK2 (6%), ERBB2 (6%), EZH2 (6%), FGFR2 (6%), IDH2 (6%), KIT (6%), KRAS (6%), NRAS (6%), NTRK1 (6%), NTRK2 (6%), and VHL (6%). 21% of PGL and 41% of PCC patients reported alterations associated with therapies approved in other indications including genes in the MAPK pathway and the homologous recombination repair pathway. Conclusions: Liquid biopsy is a non-invasive method that can provide personalized treatment options for patients. In this study, we found that evaluation of ctDNA was feasible among individuals with advanced PGL and PCC. We report a high rate of homologous recombinant deficiencies that are in need of evaluation in future Percentage refers to frequency of patients with an alteration detected. Citation Format: Lana Khalil, Jill Tsai, Leylah Drusbosky, Olatunji Alese, Maria Diab, Mehmet Akce, Christina Wu, Olumide Babjide Gbolahan, Bassel El-Rayes, Walid Shaib. Genomic landscape of circulating tumor DNA alterations in patients with paraganglioma and pheochromocytoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5790.

Feasibility of next generation sequencing (NGS) from Uzbekistan: A preliminary data analysis.

e14579 Background: Molecular testing has become an important part of management of cancer patents. Due to local nonavailability and financial constrains NGS is picking up slowly in Uzbekistan. An attempt was being made to see feasibility and analyze next generation sequencing data from local population. Here we report data from first such big analysis. Methods: 76 advanced cancer patients were enrolled and tissue samples were sent for NGS. The analysis were sent to the lab for performing FOUNDATIONONER CDx (F1CDx) - the NGS based in vitro diagnostic device for detection of substitutions, insertion and deletion alterations(indels), and copy numbers alterations(CNAs) in 324 genes and select gene rearrangements, as well as genomic signatures including microsatellite instability(MSI), tumor mutational burden(TMB), and for selected forms of ovarian cancer, loss of heterozygosity(LOH) score, using DNA isolated from formalin-fixed, paraffin-embedded(FFPE) tumor tissue specimens. Results: Out of 76 patients (35 male 41female) there were 15/76 lung cancer patients, 13/76- breast cancer, 12/76 - colorectal, 5/76 - ovarian and other malignancies. In cohort of lung cancer, the multiple gene abnormalities detected were - EGFR-5/15; ALK-3/15; ROS-0; KRAS-1/15; NRAS-0; BRAF-1/15; MET-1/15; NTRK-3/15. In breast cancer population - BRCA1 mutation was found in - 3/13; BRCA2 in 1/13; ERBB-3/13; and ESR-1/13. In group with colorectal cancer the gene abnormalities located were - BRAF-1/12; KRAS-7/12: G12D-1; G12V-1; G12C-1; NRAS-0; MSI-3/7; MMR: MLH-1; MSH2-2; MSH3-2; MSH6-2. In ovarian cancer findings included - BRCA1 in 0/5; BRCA2-1/5; PALB-1/5; CHEK-1/5; PARP1-1/5. The most frequent genetic alteration observed in the whole group was TP53 (50/76). Commonest hereditary gene alterations besides TP53 were seen in ATM gene -12/76. A large number of Variant of Unknown Significance were also recorded. Conclusions: It is feasible to perform NGS analysis using reference laboratories. There is population of patients, willing to undergo specialized tests. Actionable molecular data information can be collected and utilized for patient care. No unique markers could be found due to small sample size.

Improving the diagnostic yield of biliary stricture biopsies using a directional atherectomy device.

e16167 Background: Diagnosing the pathologic etiology of biliary strictures presents challenges that have been well-documented. Accuracy of traditional brushing and fine needle aspirate techniques range from 44% to 73%.2 Over the last 2 decades the use of atherectomy catheter biliary biopsy has demonstrated high sensitivity and positive predictive values with a low complication rate. This endoluminal approach allows for larger volume histologic samples, allowing for the potential to obtain adequate tissue for next generation sequencing (NGS). The aim of this retrospective review of a single-center’s experience was to define the accuracy, ease of evaluation, and the ability to obtain NGS for personalized treatment. Methods: A retrospective review was performed under a waiver from the Institutional Review Board. A search of medical records from 1/2016 to 2/2021 yielded 20 biopsies in 19 patients. The 20 biopsies were completed by 4 interventional radiologists with between 2-11 years of experience. All available samples were analysed by a pathologist with greater than 10 years of experience and graded for adequacy and feasibility of NGS. Results: The patient population was 53% female with average age of 65.1 years. 12/19 (63%) patients had prior non-atherectomy biopsy attempts. The average ease of evaluation score for the 19 biopsies was 7.8/9, compared to 6.2/9 for the brushing specimens obtained from a subset of the same patients. Seven biopsies were positive for malignancy, ten were benign and three were non-diagnostic. The 7 biopsies that rendered a definitive diagnosis of malignancy had an average ease of evaluation score 8.7/9. The overall accuracy rate was 78.9%, and 71% of malignant samples were deemed adequate for NGS. There were two biopsy related complications; one pseudoaneurysm treated with embolization and one biliary drain dislodgement. Conclusions: Biliary biopsy using an atherectomy device has similar accuracy to traditional techniques; however, it allows for improved diagnostic yield and increased tissue specimen with 71% of samples adequate for NGS testing.

Rapid discrimination between tuberculosis and sarcoidosis using next-generation sequencing

ObjectivesTuberculosis (TB), an infectious disease caused by Mycobacterium tuberculosis (MTB), has similar clinical, radiological, and histopathological characteristics to sarcoidosis (SA). Accurately distinguishing SA from TB remains a clinical challenge. MethodsA total of 44 TB patients and 47 SA patients who were clinically diagnosed using chest radiography, pathological examination, routine smear microscopy, and microbial culture were enrolled in this study. The MTB genome was captured and sequenced directly from tissue specimens obtained upon operation or biopsy, and the feasibility of next-generation sequencing (NGS) for the MTB genome in the differential diagnosis of TB from SA was evaluated. ResultsUsing a depth >10× and coverage >15% of the sequencing data, TB patients were identified via the NGS approach directly using operation or biopsy specimens without clinical pretreatment. The sensitivity, specificity, and concordance of the NGS method were 81.8% (36/44), 95.7% (45/47), and 89.0% (81/91), respectively (kappa = 0.78, 95% confidence interval 0.65–0.91; P<0.001). ConclusionsThis study established an improved NGS strategy for rapidly distinguishing patients with TB from those with SA and has potential clinical benefits.

Fanconi Anemia Gene Variants in Patients with Gonadal Dysfunction.

Fanconi anemia (FA) is a multisystem disease, characterized by the triad of physical abnormalities, bone marrow failure, and increased risk for malignancy. In the past few years, data has accumulated regarding fertility issues in FA patients, mostly due to gonadal dysfunction, which is prevalent in FA patients reaching puberty. It seems that attenuated FA phenotype lacking the classical manifestations often is presented with POI or azoospermia. Searching the literature, we summarized data regarding FA patients presenting as suffering from sub/infertility due to gonadal dysfunction, with or without other FA symptoms. We present a summary of the patients having biallelic pathogenic variants in FA genes FANCA, FANCM, BRCA2, and XRCC2 that presented with gonadal dysfunction with or without other phenotypic features of FA. Some were in mosaic, while some are considered hypomorphic, enabling residual protein function. There are also a few descriptions of POI associated with monoallelic pathogenic variants in FANCA, BRCA2, and FANCL. We conclude that the diagnosis of FA in gonadal dysfunction patients is of utmost importance due to its actionability. Follow-up strategies in FA patients are designed to discover early stages of leukemias and solid tumors and thus save lives. The feasibility of next-generation sequencing (NGS) can now ease this diagnostic procedure. An open question is the justification of performing NGS for all isolated azoospermia/POI patients.

Feasibility of next-generation sequencing (Oncomine™ DX Target Test) for the screening of oncogenic mutations in advanced non-small-cell lung cancer patients.

The Oncomine™ Dx Target Test based on next-generation sequencing has been approved for the screening of oncogenic mutations in advanced non-small-cell lung cancer patients. We assessed the tissue sample factors that affect the success rate of Oncomine™ Dx Target Test companion diagnostics and the feasibility of using biopsy specimens for Oncomine™ Dx Target Test companion diagnostics in advanced non-small-cell lung cancer patients. Ninety-nine biopsy samples were subjected to genetic testing using the Oncomine™ Dx Target Test companion diagnostics to detect v-raf murine sarcoma viral oncogene homologue B1 mutations (Cohort 1), and 136 biopsy samples were examined using Oncomine™ Dx Target Test companion diagnostics for the detection of multiple oncogenic mutations (Cohort 2) between July 2018 and April 2020. We retrospectively collected clinical and pathological data, including tissue size and tumour cell content. The success rate was 77% (76/99) in Cohort 1 and 93% (127/136) in Cohort 2. In Cohort 1, the success rate was significantly associated with the tumour cell content: the success rate was 63% for samples with a tumour cell content of <20%, whereas it was 83% for samples with a tumour cell content of 20% or higher (P=0.0446). The tissue size also affected the success rate: a success rate of 57% was obtained for tissue sizes <4mm2, whereas a success rate of 95% was obtained for tissue sizes of 4mm2 or larger (P<0.0001). In Cohort 2, the success rate was 100% when tumour specimens with a tissue size of 4mm2 or larger were used. Tissue size and tumour cell content were significantly associated with the success rate of Oncomine™ Dx Target Test companion diagnostics.

Application of DNA barcoding and metabarcoding for species identification in salmon products

ABSTRACT Mislabelling is a significant manifestation of food fraud. Traditional Sanger sequencing technology is the gold standard for seafood species identification. However, this method is not suitable for analysing processed samples that may contain more than one species. This study tested the feasibility of next-generation sequencing in identifying mixed salmon products. Salmon samples containing up to eight species were amplified using 16S rRNA mini-barcode primers, and sequenced on an Illumina HiSeq2500 platform. All species were accurately identified, and mixtures as low as 1% (w/w) could be detected. Furthermore, this study conducted a market survey of 32 products labelled as salmon. For pure and mixed fish products, Sanger and next-generation sequencing techniques were respectively used for species identification, and for NGS results, we also used real-time PCR method to cross-validate the mixed products to further verify the accuracy of the DNA metabarcoding technology established in this study. DNA barcoding and metabarcoding of commercial salmon food products revealed the presence of mislabelling in 16 of 32 (50%) samples. The developed DNA barcoding and metabarcoding methods are useful for the identification of salmon species in food and can be used for quality control of various types of salmon products.

Feasibility of next-generation sequencing test for patients with advanced NSCLC in clinical practice.

BackgroundThe usefulness of the Oncomine Dx Target test (Oncomine Dx), a next‐generation sequencing (NGS) test, has already been proven in clinical trials. However, NGS requires high‐quality tumor samples and takes a long time to generate results. The feasibility of NGS for use in advanced non‐small cell lung cancer (NSCLC) patients in clinical practice has not yet been determined.MethodsPatients serially diagnosed with advanced NSCLC were evaluated in our hospital. The Oncomine Dx, Cobas EGFR mutation test (Cobas EGFR), and ALK‐IHC were performed. The patients were divided into four sets: the full analysis set (FAS) that referred to patients diagnosed with NSCLC, the intent to perform companion diagnostics (CDx) set (IPS) that referred to patients in which CDx had been ordered regardless of sample quality, the per‐performed CDx set (PPS) that referred to patients who could undergo CDx regardless of the results, and the per‐completed CDx set (CCS) that referred to patients in which informative results were received from the CDx.ResultsThe total number of patients analyzed in the study was 167. The IPS/FAS of Oncomine Dx (80.2%) was lower than that of the ALK‐IHC (85.0%) and Cobas EGFR (92.8%). The CCS/FAS of Oncomine Dx (65.9%) was lower than that of the ALK‐IHC (82.0%) and Cobas EGFR (92.2%). PPS/IPS and CCS/PPS of the Oncomine Dx with nonsurgical biopsy ranged between 78.6% and 90.9%, which was lower than those patients who underwent surgical resection (95.0% and 100%).ConclusionsThe feasibility of Oncomine Dx in clinical practice was lower than the other CDx. The feasibility of Oncomine Dx will increase by improving the biopsy procedure.Key pointsSignificant study findings The usefulness of a next‐generation sequencing (NGS) test has been proven in clinical trials.The feasibility of NGS is lower than other diagnostics in clinical practice especially with regard to nonsurgical biopsy. What this study adds It is necessary to improve the feasibility of NGS in clinical practice.To improve NGS feasibility, turnaround time must be shortened, and larger samples must be obtained during surgical procedures.

Feasibility and clinical impact of routine molecular testing of gastrointestinal cancers at a tertiary centre with a multi-gene, tumor-agnostic, next generation sequencing panel

Background High-throughput sequencing technologies are increasingly used in research but limited data are available on the feasibility and value of these when routinely adopted in clinical practice. Material and methods We analyzed all consecutive cancer patients for whom genomic testing by a 48-gene next-generation sequencing (NGS) panel (Truseq Amplicon Cancer Panel, Illumina) was requested as part of standard care in one of the largest Belgian cancer networks between 2014 and 2019. Feasibility of NGS was assessed in all study patients, while the impact of NGS on the decision making was analyzed in the group of gastrointestinal cancer patients. Results Tumor samples from 1064 patients with varying tumor types were tested, the number of NGS requests increasing over time (p < .0001). Success rate and median turnaround time were 91.4% and 12.5 days, respectively, both significantly decreasing over time (p ≤ .0002). Non-surgical sampling procedure (OR 7.97, p < .0001), tissue from metastatic site (OR 2.35, p = .0006) and more recent year of testing (OR 1.79, p = .0258) were independently associated with NGS failure. Excluding well-known actionable or clinically relevant mutations which are recommended by international guidelines and commonly tested by targeted sequencing, 57/279 (20.4%) assessable gastrointestinal cancer patients were found to have tumors harboring at least one actionable altered gene according to the OncoKB database. NGS results, however, had a direct impact on management decisions by the treating physician in only 3 cases (1.1%). Conclusions Our findings confirm that NGS is feasible in the clinical setting with acceptably low failure rates and rapid turnaround time. In gastrointestinal cancers, however, NGS-based multiple-gene testing adds very little to standard targeted sequencing, and in routine practice the clinical impact of NGS panels including genes which are not routinely recommended by international guidelines remains limited.

Blood-based next-generation sequencing analysis of neuroendocrine neoplasms.

Background: Neuroendocrine neoplasms (NENs) are a heterogeneous group of neoplasms that span from well-differentiated neuroendocrine tumors (NETs) to highly aggressive neoplasms classified as neuroendocrine carcinomas (NECs). The genomic landscape of NENs has not been well studied. The aim of this study is to confirm the feasibility of next generation sequencing (NGS) testing circulating tumor DNA (ctDNA) in patients with NENs and characterize common alterations in the genomic landscape. Results: Of the 320 NEN patients, 182 (57%) were male with a median age of 63 years (range: 8-93) years. Tumor type included pancreatic NET (N = 165, 52%), gastrointestinal NEC (N = 52, 16%), large cell lung NEC (N = 21, 7%), nasopharyngeal NEC (N = 16, 5%) and NEC/NET not otherwise specified (N = 64, 20%). ctDNA NGS testing was performed on 338 plasma samples; 14 patients had testing performed twice and 2 patients had testing performed three times. Genomic alterations were defined in 280 (87.5%) samples with a total of 1,012 alterations identified after excluding variants of uncertain significance (VUSs) and synonymous mutations. Of the 280 samples with alterations, TP53 associated genes were most commonly altered (N = 145, 52%), followed by KRAS (N = 61, 22%), EGFR (N = 33, 12%), PIK3CA (N = 30, 11%), BRAF (N = 28, 10%), MYC (N = 28, 10%), CCNE1 (N = 28, 10%), CDK6 (N = 22, 8%), RB1 (N = 19, 7%), NF1 (N = 19, 7%), MET (N = 19, 7%), FGFR1 (N = 19, 7%), APC (N = 19, 7%), ERBB2 (N = 16, 6%) and PTEN (N = 14, 5%). Conclusions: Evaluation of ctDNA was feasible among individuals with NEN. Liquid biopsies are non-invasive methods that can provide personalized options for targeted therapies in NEN patients. Patients and Methods: Molecular alterations in 338 plasma samples from 320 patients with NEN were evaluated using clinical-grade NGS of ctDNA (Guardant360®) across multiple institutions. The test detects single nucleotide variants in 54-73 genes, copy number amplifications, fusions, and indels in selected genes.

Feasibility of next-generation sequencing in clinical practice: results of a pilot study in the Department of Precision Medicine at the University of Campania ‘Luigi Vanvitelli’

BackgroundThe emerging role of next-generation sequencing (NGS) targeted panels is revolutionising our approach to cancer patients, providing information on gene alterations helpful for diagnosis and clinical decision, in a short time and with acceptable costs.Materials and methodsIn this work, we evaluated the clinical application of FoundationOne CDx test, a hybrid capture-based NGS. This test identifies alterations in 324 genes, tumour mutational burden and genomic signatures as microsatellite instability. The decision to obtain the NGS assay for a particular patient was done according to investigator’s choice.ResultsOverall, 122 tumour specimens were analysed, of which 84 (68.85%) succeeded. The success rate was influenced by type of specimen formalin-fixed paraffin embedded (FFPE block vs FFPE slides), by origin of the sample (surgery vs biopsy) and by time of fixation (<5 years vs ≥5 years). The most frequent subgroups of effective reports derived from colorectal cancer (25 samples), non-small-cell lung cancer (16 samples), ovarian cancer (10 samples), biliary tract cancer (9 samples), breast cancer (7 samples), gastric cancer (7 samples). The most frequent alterations found in whole population referred to TP53 (45.9%), KRAS (19.6%) and APC (13.9%). Furthermore, we performed an analysis of patients in whom this comprehensive genomic profiling (CGP) had a relevance for the patient’s disease.ConclusionsOn our opinion, CGP could be proposed in clinical practice in order to select patients that could most benefit from the analysis proposed, like patients with good performance status without any available treatments or with unexpected resistance to a therapy.

Blood-based next-generation sequencing analysis of neuroendocrine tumors.

4110 Background: Neuroendocrine tumors (NET) comprise around 2% of all malignant tumors of the gastrointestinal system. The genomic landscape of NET has not been well studied. The aim of this study was to confirm the feasibility of next generation sequencing (NGS) using ctDNA in NET and characterize common alterations in the genomic profile. Methods: Molecular alterations in 114 plasma samples from 114 patients with NET using clinical-grade NGS of ctDNA (Guardant360Ò) across multiple institutions were evaluated. The test detects single nucleotide variants in 54-73 genes, copy number amplifications, fusions, and indels in selected genes. Results: A total of 114 NET patients were evaluated, of which 64 (56.1%) were female. Mean age was 59.7 years with a range between 23-89 years. ctDNA NGS testing was performed on 114 plasma samples; 1 patient had testing performed twice. Genomic alterations were defined in 94 (n = 94/114, 82.5%) samples with a total of 289 alterations identified after excluding variants of uncertain significance (VUSs) and synonymous mutations. Alterations were identified in at least one sample from 83 patients; TP53 associated genes were most commonly altered (n = 83/289, 28.7%), followed by KRAS (n = 22, 7.6%), PI3CA (n = 15, 5.2%), CCNE1 (n = 15, 5.2%), BRAF (n = 13, 4.5%), MYC (n = 12, 4.1%), ERBB2 (n = 11, 3.8%), APC (n = 10, 3.5%), EGFR (n = 10, 3.5%), MET (n = 10, 3.5%), PTEN (n = 9, 3.1%), RB1 (n = 9, 3.1%), CDK6 (n = 7, 2.4%), AR (n = 5, 1.7%), ARID1A (n = 5, 1.7%), FGFR1 (n = 5, 1.7%), and PDGFRA (n = 5, 1.7%). Other genomic alterations of low frequency, but clinical relevance included: CDK4 (n = 4, 1.3%), NF1 (n = 4, 1.3%), RAF1 (n = 4, 1.3%), GNAS (n = 3, 1.0%), KIT (n = 3, 1.3%), BRCA2 (n = 2, 0.7%), CCND2 (n = 2, 0.7%), CTNNB1 (n = 2, 0.7%), JAK2 (n = 2, 0.7%), NRAS (n = 2, 0.7%), SMAD4 (n = 2, 0.7%), and TERT (n = 2, 0.7%). Alterations in AKT1, ALK, ATM, BRCA1, CCND1, CDKN2A, FGFR2, MTOR, RHOA, SMO and STK11 were all reported once (n = 1, 0.3%). Conclusions: Evaluation of ctDNA is feasible among individuals with NET. Liquid biopsies are not invasive and can provide personalized options for targeted therapies in NET patients.

Next Generation Sequencing (NGS) in metastatic castration resistant prostate cancer (mCRPC) to identify targets for individualized treatment: Is it feasible in daily routine?

194 Background: Systemic of mCRPC consists of various approaches such as docetaxel, abiraterone, enzalutamide, Radium-223 and cabazitaxel. If all treatment options are exhausted, molecular profiling of biopsies from progressive metastases might enable an individualized approach with targeted therapies. It was the purpose of our study to examine (1) the feasibility of NGS in daily routine, (2) the frequency of treatable mutations and (3) the therapeutic outcome. Methods: From 1/2016 to 12/2017, 214 patients with mCRPC were treated; 52 pts progressed after exposure to all approved systemic therapies. All men underwent biopsies of the progressing lesions to perform mutational analysis via NGS using 9 Multiplex PCR amplicons (AR, ATM, BRCA1/2, CTNNB1, DLL3, PIK3CA, PTEN, RAD51C, TP53) which generation was done with a GeneRead DNAseq Custom Panel V2 (Qiagen). Creation of the library was done with the use of GeneRead DNA Library I Core, GeneRead DNA I Amp Kits and NEXTflex-96 DNA Barcode Adapter. Results: Following lesions were biopsied n = 8 (15.4%) liver, N = 4 (7.7%) lung, n = 4 (7.7%) bone, n = 36 (69.2%) lymph node. Sufficient material was received in all cases. 31 (59.7%) demonstrated wild type in all amplicons vorhanden, n = 21 (40.3%) demonstrated at least 1 potentially treatable mutation: BRCA1/2 or ATM n = 11, AR n = 7, DLL3 n = 3, p53 n = 9, Rad51C n = 4, CTNNB1 n = 4, PTEN/PIK3CA n = 4. Results were discussed in our molecular and clincical phase-I tumor board. Median overall survival oft he pateint cohort is 18.5 (6-34) months. Conclusions: NGS analysis of biopsy samples from progressing metastatic sites is feasible in mCRPC presuming an interdisciplinary setting in the diagnostic and therapy decision making process. NGS results in the identification of treatable mutations in more than one third of patients.

Prenatally diagnosed TTN mutation with repeated bilateral club foot by whole exome sequencing

With significant advances in genomic technologies over recent years, Next-generation sequencing (NGS) has become an important tool not only for gene discovery and research but also for clinical genetic diagnosis of rare disorders and single-gene-disorder analysis. Many hundreds of single-gene disorders have been described that can manifest prenatally and will comprise a large portion of undiagnosed, chromosomally normal cases but underlying cause of the abnormality still remains unknown for the large majority of cases with an abnormal ultrasound. However, there has been little experience and study in using trio whole exome sequencing (WES) for prenatal diagnosis. In this report the NGS method was applied for the prenatal genetic diagnosis of unidentified life-threatening phenotype in one family and confirmed the pattern of inheritance of titinopathy as well as two novel TTNgene variations. The report presented in these pages offers a unique and valuable perspective on relevance and feasibility of NGS in prenatal diagnosis field.

Comparison of somatic mutation profiles from cell free DNA (cfDNA) versus tissue in metastatic urothelial carcinoma (mUC).

4533 Background: Next-generation sequencing (NGS) of cfDNA is an emerging non-invasive strategy to define tumor mutation profiles that counters spatial and temporal limitations of sequencing single tissue specimens. We examined the feasibility of NGS of cfDNA in mUC and compared mutation profiles from cfDNA to results of tissue NGS previously performed in the clinical setting. Methods: Plasma cfDNA was collected in mUC pts and analyzed using a capture-based NGS assay (MSK-IMPACT) targeting 341-468 genes. NGS profiles from cfDNA and archival tumor tissue (using the same assay) were analyzed in parallel with an established bioinformatics pipeline to identify somatic variants. Results: In 26 pts, NGS analysis of cfDNA detected ≥1 somatic mutations (range 1-21) in 69% (18/26). For 15 pts, NGS data was available from archival tissue (11 primary tumors, 3 metastases, and matched primary/metastatic tissue in 1 case). The interval between cfDNA and tissue collection ranged from 35 days to &gt; 4 yrs. 73% (11/15) of pts received intervening treatment, including 47% (7/15) with chemotherapy, 67% (10/15) with immunotherapy, and 40% (6/15) with both. In 40% (6/15), cfDNA harbored alterations not found in archival tumor tissue. In 73% (11/15), some mutations within archival tissue were not detected in cfDNA, including hotspot HER2 S310F and FGFR3 S249C mutations. Tumor and cfDNA mutation profiles were identical in 20% (3/15), with the tumor/cfDNA interval in this group ranging from 35 days to &lt; 1.5 yrs. Somatic alterations including hotspot ERCC2 P463A and PIK3CA E545K mutations were detected in cfDNA from 3 pts where archival tumor tissue NGS failed. Thus, cfDNA identified new mutations in 50% (9/18) of pts for whom cfDNA identified somatic mutations and tissue NGS was previously attempted. Conclusions: NGS of cfDNA from mUC pts is feasible and successfully detected actionable alterations when archival tumor sequencing failed. The differences between tumor and cfDNA mutation profiles in many pts may reflect tumor evolution or intratumor heterogeneity. Mutation profiles in mUC may be incompletely assessed with NGS of archival tissue, and further investigation of plasma cfDNA for genomic profiling is warranted.

Next-generation sequencing of a large gene panel in patients initially diagnosed with idiopathic ventricular fibrillation

Idiopathic ventricular fibrillation (IVF) is a rare primary cardiac arrhythmia syndrome that is diagnosed in a resuscitated cardiac arrest victim, with documented ventricular fibrillation, in whom no underlying cause is identified after comprehensive clinical evaluation. In some patients, causative genetic mutations are detected that facilitate patient treatment and follow-up. The feasibility of next-generation sequencing (NGS) has increased with its greater availability and decreasing costs. The aim of this study was to assess the diagnostic yield of NGS in patients with IVF. A total of 33 patients initially diagnosed with IVF were included (mean age 53 ± 15 years; 14(42%) men). In all included patients, NGS of 33 genes and the DPP6 haplotype revealed no pathogenic mutations. Genetic screening comprised NGS of a panel of 179 additional genes. Variants with a minor allele frequency of <0.05% were assessed for pathogenicity by using existing mutation databases and in silico predictive algorithms. In 1 of 33 patients, a likely pathogenic mutation was detected. The added yield of genetic testing with NGS of 179 additional genes is 3% in patients with IVF. In 15% of patients, 1 or multiple variants of uncertain clinical significance were detected. The added yield of genetic screening of extended NGS panels in patients initially diagnosed with IVF is minimal. Routine analysis of large diagnostic NGS panels is therefore not recommended.

Abstract 4561: Cancer preventive effect of antioxidants for spontaneous liver cancer in XPA(-/-) mice

Abstract γ-Hydroxy-1, N2-propanodeoxyguanosine (γ-OHPdG), a widely studied acrolein-derived cyclic adduct arisen from oxidized ω-3 and ω-6 polyunsaturated fatty acids (PUFAs), is a mutagenic lesion that is repaired by nucleotide excision repair (NER) pathway. C3H/HeN xeroderma pigmentosum group A (XPA) knockout mice which are deficient on NER pathway showed a significant elevation in liver tumors. We hypothesized that accumulation of γ-OHPdG is playing a role in liver tumorigenesis of XPA(-/-) mice; suppressing the formation of γ-OHPdG by dietary antioxidants should result in the prevention of liver cancer in this model. In this work, we investigated the chemoprotective efficacy of three antioxidants: α-lipoic acid, polyphenon E and vitamin E against liver tumorigenesis in XPA(-/-) mice. Autopsy at 19 months of age revealed that all the antioxidants exhibited significant inhibition effects on the development of liver tumors. Polyphenon E showed the most potent effects with 80% of the mice showing complete protection. The formation γ-OHPdG was measured in the livers of XPA(-/-) mice. An age-dependent increase of γ-OHPdG was found in the liver DNA of XPA(-/-) mice, but not in lungs (a non-target tissue). All the antioxidants decreased the formation of γ-OHPdG. Immunohistochemistry (IHC) for Ki-67 (proliferation index marker) was performed on all the liver samples, and the results showed that dietary antioxidants significantly decrease the proliferation in the liver tumors. α-Lipoic acid and vitamin E showed significant inhibitory effect, although to a lesser extent than Polyphenon E, against the spontaneous liver carcinogenesis in XPA gene-deficient mice model. A correlation between the cancer preventive efficacy and suppression of γ-OHPdG adduct level is observed, indicating a possible role of γ-OHPdG in the liver cancer development in this model. We also explored the feasibility of next-generation sequencing (NGS) to validate the mutation frequency changes as a result of feeding the antioxidant diets. This work was supported by the NCI grant: RO1-CA-134892. Y.F. thanks the Prevent Cancer Foundation for his fellowship (Marcia and Frank Carlucci Charitable Foundation Award in Cancer Prevention and Early Detection) Note: This abstract was not presented at the meeting. Citation Format: Ying Fu, Shana Silverstein, Marcin Dyba, Heidi Coia, Elizabeth Sinclair, Jishen Pan, Bhaskar Kallakury, Michael D. Johnson, Fung-Lung Chung. Cancer preventive effect of antioxidants for spontaneous liver cancer in XPA(-/-) mice. [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 4561. doi:10.1158/1538-7445.AM2015-4561

Abstract 3882: Clinical exome and transcriptome sequencing for identification of actionable cancer targets: A pilot study for children and young adults with relapsed or refractory solid tumors

Abstract Background. With technological advances such as next-generation sequencing, recent gains in understanding pediatric cancer can aid in treatment decisions, especially in the setting of relapse. To discover expressed, clinically significant mutations for pediatric patients with relapsed tumors, we performed a pilot trial using a combination of whole exome sequencing (WES) of tumor/normal DNA and whole transcriptome sequencing (WTS) of tumors, complemented by single nucleotide polymorphism (SNP) arrays. Objectives. We identified 48 pediatric and young adult patients with relapsed or refractory solid tumors with matched tumor/normal samples. Our goals were to determine the feasibility of performing comprehensive genomic analyses in this population, to compare the genomic profile of relapsed tumors to prior reports of primary tumors, and to delineate the percentage of patients with actionable mutations. Actionable changes were defined as reportable germ line mutations determined by the American College of Medical Genetics (ACMG), a change in diagnosis, and somatic changes targetable by FDA approved medications or drugs undergoing clinical trials. Methods. WES was performed on matched tumor and normal samples to identify germ line and somatic mutations. WTS was performed on tumor samples to identify fusion genes, gene expression profiling, and expressed variants. SNP arrays were performed to identify copy number changes. Sanger validation confirmed reportable mutations. Results. In the exome, we noted a median of 33 somatic mutations per sample (range 1-375), a higher mutational burden compared to previously reported primary pediatric malignancies. Transcriptome data further refined results to a median of 7 expressed somatic mutations per sample (range 0-95). The majority of patients had one oncogenic driver. Sequencing relapsed tumors at multiple time points showed the continued presence of driver mutations but a shift in passenger mutations. Eleven of the 48 patients (23%) had a targetable mutation, such as ALK, BRAF, GNAQ, GNA11, IDH1, MTOR, PIK3CA, and SKP2. Two patients (4%) had a change in diagnosis due to the presence or absence of diagnostic fusion genes. In the germ line of 5 patients (10%) we discovered mutations in ACMG-reportable genes MUTYH, SCN5A, TP53, and MLH1. A total of 16 patients (33%) had actionable mutations. Conclusions. Our study showed the feasibility of next-generation sequencing in relapsed pediatric solid tumors, with actionable mutations detected in a third of our patients. We demonstrated the utility in using exome and transcriptome sequencing with SNP arrays. Implementation of these techniques has the potential to change the practice of precision medicine. In summary, we have developed a prototype that will be utilized to design a national Pediatric Match trial in collaboration with the Children's Oncology Group. Citation Format: Wen-I Chang, Andrew S. Brohl, Rajesh Patidar, Jack F. Shern, Jun S. Wei, Young K. Song, Hongling Liao, Jimmy Lin, Sivasish Sindiri, Li Chen, Berkley Gryder, Marielle E. Yohe, Shile Zhang, Melinda S. Merchant, Brigitte C. Widemann, Javed Khan. Clinical exome and transcriptome sequencing for identification of actionable cancer targets: A pilot study for children and young adults with relapsed or refractory solid tumors. [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 3882. doi:10.1158/1538-7445.AM2015-3882