Next-generation Sequencing Gene Panel Research Articles

Is gene panel sequencing more efficient than clinical-based gene sequencing to diagnose autoinflammatory diseases? A randomized study.

The aim of this study was to compare the effectiveness of the gene-panel next-generation sequencing (NGS) strategy versus the clinical-based gene Sanger sequencing for the genetic diagnosis of autoinflammatory diseases (AIDs). Secondary goals were to describe the gene and mutation distribution in AID patients and to evaluate the impact of the genetic report on the patient's medical care and treatment. Patients with AID symptoms were enrolled prospectively and randomized to two arms, NGS (n=99) (32-55 genes) and Sanger sequencing (n=197) (one to four genes). Genotypes were classified as 'consistent/confirmatory', 'uncertain significance' or 'non-contributory'. The proportion of patients with pathogenic genotypes concordant with the AID phenotype (consistent/confirmatory) was significantly higher with NGS than Sanger sequencing [10 of 99 (10·1%) versus eight of 197 (4·1%)]. MEFV, ADA2 and MVK were the most represented genes with a consistent/confirmed genotype, whereas MEFV, NLRP3, NOD2 and TNFRSF1A were found in the 'uncertain significance' genotypes. Six months after the genetic report was sent, 54 of 128 (42·2%) patients had received effective treatment for their symptoms; 13 of 128 (10·2%) had started treatment after the genetic study. For 59 of 128 (46%) patients, the results had an impact on their overall care, independent of sequencing group and diagnostic conclusion. Targeted NGS improved the diagnosis and global care of patients with AIDs.

Novel inactivating follicle-stimulating hormone receptor mutations in a patient with premature ovarian insufficiency identified by next-generation sequencing gene panel analysis

ObjectiveTo find the genetic etiology of premature ovarian insufficiency (POI) in a patient with primary amenorrhea and hypergonadotropic hypogonadism.DesignCase report.SettingUniversity hospital.Patient(s)A Belgian woman aged 32 years with POI at the age of 17, her parents, and her sister whose POI was diagnosed at age 29.Intervention(s)Analysis of a panel of 31 genes implicated in POI (POIGP) using next-generation sequencing (NGS), Sanger sequencing, and in vitro functional study.Main Outcome Measure(s)Gene variants, family mutational segregation, and in vitro functional impact of the mutant proteins.Result(s)The analysis of the gene panel using NGS identified the presence of two novel follicle-stimulating hormone receptor (FSHR) missense mutations at a compound heterozygous state in the affected patient: c.646 G>A, p.Gly216Arg, and c.1313C>T, p.Thr438Ile. Sanger sequencing showed the presence of each mutation at heterozygous state in the patient’s parents and at heterozygous compound state in the affected sister. Both substituted amino acids (Gly216 and Thr438) were conserved in FSHR of several vertebrate species as well as in other glycoproteins receptors (TSHR and LHCGHR), suggesting a potentially important role in glycoprotein receptor function. An in vitro functional study showed similar results for both variants with more than 90% reduction of their cell surface expression and a 55% reduction of their FSH-induced cyclic adenosine 3′:5′ monophosphate (cAMP) production compared with the wild-type FSHR.Conclusion(s)The analysis of a gene panel of 31 genes implicated in POI allowed us to identify two novel partially inactivating mutations of FSHR that are likely responsible for the POI phenotype of the proband and of her affected sister.

Targeted next-generation sequencing of adult gliomas for retrospective prognostic evaluation and up-front diagnostics.

We aimed to reclassify a population-based cohort of 529 adult glioma patients to evaluate the prognostic impact of the 2016 World Health Organization (WHO) central nervous system tumour classification. Moreover, we evaluated the feasibility of gene panel next-generation sequencing (NGS) in daily diagnostics of 225 prospective glioma patients. The retrospective cohort was reclassified according to WHO 2016 criteria by immunohistochemistry for IDH-R132H, fluorescence in situ hybridization for 1p/19q-codeletion and gene panel NGS. All tumours of the prospective cohort were subjected to NGS analysis up-front. The entire population-based cohort was successfully reclassified according to WHO 2016 criteria. NGS results were obtained for 98% of the prospective patients. Survival analyses in the population-based cohort confirmed three major prognostic subgroups, that is, isocitrate dehydrogenase (IDH)-mutant and 1p/19q-codeleted oligodendrogliomas, IDH-mutant astrocytomas and IDH-wildtype glioblastomas. The distinction between WHO grade II and III was prognostic in patients with IDH-mutant astrocytoma. The survival of patients with IDH-wildtype diffuse astrocytomas carrying TERT promoter mutation and/or EGFR amplification overlapped with the poor survival of IDH-wildtype glioblastoma patients. Gene panel NGS proved feasible in daily diagnostics. In addition, our study confirms the prognostic role of glioma classification according to WHO 2016 in a large population-based cohort. Molecular features of glioblastoma in IDH-wildtype diffuse glioma were linked to poor survival corresponding to IDH-wildtype glioblastoma patients. The distinction between WHO grade II and III retained prognostic significance in patients with IDH-mutant diffuse astrocytic gliomas.

Abstract 5671: Alignment of TMB measured on clinical samples: Phase IIB of the Friends of Cancer Research TMB Harmonization Project

Abstract Introduction: Tumor mutational burden (TMB) is the number of somatic mutations per megabase in a tumor's genome and has shown promise as a predictive biomarker of response to immune checkpoint inhibitors across several cancers. TMB is typically measured by whole exome sequencing (WES TMB) or by targeted next-generation sequencing gene panels (panel TMB). As more assays are developed to estimate TMB, harmonization is emerging as an unmet need and is a key goal of the Friends of Cancer Research (Friends) TMB Harmonization Project. Phase I of the Harmonization Project demonstrated correlation between panel TMB and WES TMB using TCGA data and defined theoretical sources of variability across panels. In phase IIA, sustainable TMB reference standard materials generated from human derived cell lines were used to characterize variability in TMB measurements across panels and assessed for utility in TMB alignment. Phase IIB aims to characterize variability in TMB measurements in clinical samples and to establish best practices for estimating and aligning TMB in order to improve consistency across panels. Methods: Fifteen laboratories (16 targeted gene panels) at different stages of development participated in phase IIB. Thirty formalin-fixed paraffin-embedded (FFPE) samples with >30% tumor content were acquired; tumor DNA was isolated by a single reference lab. TMB values were calculated for DNA extracted from lung (N=10), bladder (N=10), and gastric tumors (N=10) using WES and a uniform bioinformatics pipeline agreed upon by all Consortium members. DNA samples were also sent to all laboratories, and each used their own sequencing and bioinformatics pipelines to estimate TMB from the genes represented in their respective panels. For each tumor sample, a median across panel TMB estimates was calculated; individual panel TMB estimates were translated to fold-changes relative to the sample median to quantify variability. Association between WES TMB (reference) and panel TMB will be assessed by regression analysis; dependence of association on cancer type was investigated. Results: A subset of tumor samples (9 bladder, 7 lung, and 5 gastric) was analyzed using 11 panels at the time of abstract submission. Median panel TMB values ranged 0.60 - 40.26 across samples, with median of median values of 5.35. Fold-change from sample-level medians ranged 0x - 6.67x. Assessment of these clinical samples by WES and all 16 gene panels, as well as regression analysis results, are forthcoming. Conclusions: The Friends TMB Harmonization Project has made substantial progress in characterization of TMB measurement variability and association between WES TMB and panel TMB. These are important steps toward alignment of TMB estimates generated by different gene panels which may improve the interpretation of findings within clinical development programs and ultimately enhance the usefulness of this predictive biomarker in clinical decision making. Citation Format: Diana M. MERINO, Laura M. Yee, Lisa M. McShane, P. Mickey Williams, Tomas Vilimas, Rajesh Patidar, J. Carl Barrett, Shu-Jen Chen, Jen-Hao Cheng, Jeffrey M. Conroy, Dinesh Cyanam, Kenneth R. Eyring, David A. Fabrizio, Vincent Funari, Elizabeth P. Garcia, Sean T. Glenn, Christopher D. Gocke, Vikas Gupta, Lisa M. Haley, Matthew D. Hellmann, Laurel Keefer, Lauryn R. Keeler, Brett Kennedy, Alexander J. Lazar, Laura E. MacConaill, Kristen L. Meier, Arnaud Papin, Naiyer A. Rizvi, Ethan Sokol, Phillip Stafford, John F. Thompson, Warren Tom, Victor J. Weigman, Mingchao Xie, Chen Zhao, Mark D. Stewart, Jeff Allen. Alignment of TMB measured on clinical samples: Phase IIB of the Friends of Cancer Research TMB Harmonization Project [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5671.

Abstract 816: Utility and validation of a comprehensive cost-effective targeted DNA panel including FLT3-ITDs, CALR and CEBPA on a next-generation sequencing (NGS) platform for hematological malignancies

Abstract A clinical next generation sequencing (NGS) assay comprises various factors taken into consideration including selection of appropriate markers thereby panel size, nucleic acid input, assessment and interpretation, and workflow. The downstream benefits of the development of such panels therefore involve improvement in prognosis, patients' outcome, and benefit from targeted therapy. Myeloid malignancies such as myeloproliferative neoplasms (MPN), myelodysplastic syndrome (MDS), MDS/MPN, acute myeloid leukemia (AML) are associated with somatic mutations in around 25 to 50 genes. Herein, we describe clinical validation of a DNA based targeted NGS assay utilizing QIAact Myeloid DNA UMI panel in combination with QIAGEN GeneReader NGS system. The assay employs unique molecular index technology (UMI) into a gene specific, primer based target enrichment technology enabling sequencing of specific regions of interest providing an integrated solution by simultaneously assessing many candidate genes for actionable mutations. The QIAact Myeloid DNA UMI panel is a 25 gene panel for markers of known clinical significance, including single nucleotide variants (SNV), and large insertion/deletion mutations. At the lab, 40 individual AML specimens with known mutation profiles were tested. The limit of detection (LOD) was assessed by sequencing Seraseq myeloid mutation DNA mix as positive control. For accuracy, inter-run and intra-run controls were also implemented. All samples were previously characterized by using another 54 gene NGS panel. The samples were prepared using QIAact Myeloid DNA UMI panel kits, sequenced with QIAGEN GeneReader NGS system, and mutations identified using the QIAGEN Clinical Insight (QCI) Analyze software suite, adjusted specifically for variant calling. Variant calling was accurate and reproducible at allele frequencies ≥5%. Limit of detection (LOD) studies also determined that input of 40ng DNA was optimal for high analytical sensitivity. High positive and negative percentage agreement with prior results was observed across all variant categories. The assay was also able to identify 52bp deletion CALR type 1 variant, and CEBPα amplicons mutations which require separate bi-directional Sanger sequencing for identification. Also, the assay was able to identify FLT3 ITDs up to 101bp insertion which are detected by performing PCR separately. Hence, the study presents the data establishing the QIAact Myeloid DNA UMI panel suitable for implementation as a routine clinical NGS test for myeloid malignancies. The optimized chemistry allows high-throughput analytical sensitivity for detection of highly relevant mutations including large Insertion/Deletion (InDel). Citation Format: Meenakshi Ahluwalia, Ashis Mondal, Nikhil Sahajpal, Allan Njau, Kimya Jones, Pankaj Kumar Ahluwalia, Yasmeen Jilani, Ravindra Kolhe. Utility and validation of a comprehensive cost-effective targeted DNA panel including FLT3-ITDs, CALR and CEBPA on a next-generation sequencing (NGS) platform for hematological malignancies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 816.

Second-Tier Next Generation Sequencing Integrated in Nationwide Newborn Screening Provides Rapid Molecular Diagnostics of Severe Combined Immunodeficiency.

Severe combined immunodeficiency (SCID) and other T cell lymphopenias can be detected during newborn screening (NBS) by measuring T cell receptor excision circles (TRECs) in dried blood spot (DBS) DNA. Second tier next generation sequencing (NGS) with an amplicon based targeted gene panel using the same DBS DNA was introduced as part of our prospective pilot research project in 2015. With written parental consent, 21 000 newborns were TREC-tested in the pilot. Three newborns were identified with SCID, and disease-causing variants in IL2RG, RAG2, and RMRP were confirmed by NGS on the initial DBS DNA. The molecular findings directed follow-up and therapy: the IL2RG-SCID underwent early hematopoietic stem cell transplantation (HSCT) without any complications; the leaky RAG2-SCID received prophylactic antibiotics, antifungals, and immunoglobulin infusions, and underwent HSCT at 1 year of age. The child with RMRP-SCID had complete Hirschsprung disease and died at 1 month of age. Since January 2018, all newborns in Norway have been offered NBS for SCID using 1st tier TRECs and 2nd tier gene panel NGS on DBS DNA. During the first 20 months of nationwide SCID screening an additional 88 000 newborns were TREC tested, and four new SCID cases were identified. Disease-causing variants in DCLRE1C, JAK3, NBN, and IL2RG were molecularly confirmed on day 8, 15, 8 and 6, respectively after birth, using the initial NBS blood spot. Targeted gene panel NGS integrated into the NBS algorithm rapidly delineated the specific molecular diagnoses and provided information useful for management, targeted therapy and follow-up i.e., X rays and CT scans were avoided in the radiosensitive SCID. Second tier targeted NGS on the same DBS DNA as the TREC test provided instant confirmation or exclusion of SCID, and made it possible to use a less stringent TREC cut-off value. This allowed for the detection of leaky SCIDs, and simultaneously reduced the number of control samples, recalls and false positives. Mothers were instructed to stop breastfeeding until maternal cytomegalovirus (CMV) status was determined. Our limited data suggest that shorter time-interval from birth to intervention, may prevent breast milk transmitted CMV infection in classical SCID.

Genetic panel screening in patients with clinically unclassified systemic autoinflammatory diseases.

Systemic autoinflammatory diseases (SAIDs) may not always present with typical clinical findings of a monogenic disease. We aimed to genetically screen and diagnose these clinically unclassified patients by next-generation sequencing (NGS) analysis. A total of 64 patients who had clinical findings of a periodic fever syndrome but did not meet the clinical diagnostic criteria for any SAID or had clinical findings for more than one monogenic SAID were identified as "clinically unclassified SAIDs." NGS panel analysis, including 16 genes, was performed in these patients. Patients, who could not be classified as one of the defined SAID after the result of the NGS gene analysis, were identified as "undefined SAID." The most common autoinflammatory symptoms in unclassified SAID patients were abdominal pain (60.9%), arthralgia (48.4%), urticarial rash (43.8%), myalgia (40.6%), oral aphthae (28.1%), and conjunctivitis (20.3%), respectively. In the result of the NGS gene panel screening, pathogenic, likely pathogenic variants, or VUS (variants of uncertain significance) were detected in 36 of 64 patients in at least one gene in the NGS panel. A total of 15 patients were diagnosed with a monogenic SAID according to both phenotypic and genotypic data; 12 patients as FMF, two patients as FCAS, and one patient as TRAPS, respectively. A total of 49 patients who did not meet the classification criteria including genetic results for a monogenic SAID were followed as undefined SAID. The classification criteria described for SAIDs so far unfortunately do not cover all patients with signs of periodic fevers. The NGS gene panel appears to be a useful diagnostic tool for some of the patients with clinically unclassified SAID findings. Key Points • The classification criteria described for SAIDs do not cover all patients with signs of periodic fevers • The use of the undefined SAID nomenclature will benefit clinicians for diagnosis and initiating early treatment • The NGS panel appears to be a useful diagnostic tool in patients with clinically unclassified SAIDs.

Barriers to prescribing targeted therapies for NSCLC patients with highly actionable gene variants in the VA National Precision Oncology Program.

2005 Background: Next-Generation Sequencing (NGS) gene panels are often completed to guide therapeutic decisions for patients with advanced stage non-small cell lung cancer (NSCLC). Patients with highly-actionable gene variants may experience improved therapeutic treatments and reduced toxicities with use of targeted agents. Ensuring appropriate prescription of targeted therapies is therefore of high importance. We sought to identify barriers to targeted agent use within the Veterans Health Affairs’ (VHA) National Precision Oncology Program (NPOP). Methods: A retrospective evaluation examined the cohort of NSCLC patients who underwent NGS multi-gene panels through NPOP between July 2015 and February 2019. A level of evidence for drug actionability was assigned to each observed oncogenic gene variant using an artificial intelligence offering (IBM Watson for Genomics: WfG). WfG level 1 and 2A evidence was reviewed by NPOP staff to exclude gene variants that did not conform to NPOP level 1 and 2A definitions. Anti-neoplastic drug prescriptions and oncology provider notes were obtained for all included patients from the VHA Corporate Data Warehouse. Review of clinical notes of patients who did not receive targeted agents was performed to categorize the reason(s). Results: Of 1764 NSCLC patients who successfully underwent NGS gene panel testing, 156 (8.9%) received therapeutic level 1 (7.3%) or 2A (1.6%) options for targeted agents based on WfG evidence analysis. In total, 117 (6.6%) patients had NPOP level 1 and 2A gene variants, all within ALK, BRAF, EGFR, ERBB2, MET, and RET. Of these, 49 (41.2%) patients were not prescribed available targeted agents. The three most common reasons were: (1) treating provider did not comment on NGS results (30.7%), (2) patient did not carry a diagnosis of advanced stage disease (18.4%), and (3) patient had begun an alternative systemic therapy prior to completion of sequencing (16.3%). No patient was denied access to a level 1 or 2A targeted drug due to utilization-management review. Conclusions: A substantial minority of patients with advanced NSCLC bearing highly-actionable gene variants are not prescribed available targeted agents. Further provider- and pathologist-directed educational effort are needed, as well as implementation of health informatics systems to provide near real-time decision support for test ordering and interpretation.

Targeted Next-Generation Sequencing in a Large Cohort of Genetically Undiagnosed Patients with Neuromuscular Disorders in Spain.

The term neuromuscular disorder (NMD) includes many genetic and acquired diseases and differential diagnosis can be challenging. Next-generation sequencing (NGS) is especially useful in this setting given the large number of possible candidate genes, the clinical, pathological, and genetic heterogeneity, the absence of an established genotype-phenotype correlation, and the exceptionally large size of some causative genes such as TTN, NEB and RYR1. We evaluated the diagnostic value of a custom targeted next-generation sequencing gene panel to study the mutational spectrum of a subset of NMD patients in Spain. In an NMD cohort of 207 patients with congenital myopathies, distal myopathies, congenital and adult-onset muscular dystrophies, and congenital myasthenic syndromes, we detected causative mutations in 102 patients (49.3%), involving 42 NMD-related genes. The most common causative genes, TTN and RYR1, accounted for almost 30% of cases. Thirty-two of the 207 patients (15.4%) carried variants of uncertain significance or had an unidentified second mutation to explain the genetic cause of the disease. In the remaining 73 patients (35.3%), no candidate variant was identified. In combination with patients’ clinical and myopathological data, the custom gene panel designed in our lab proved to be a powerful tool to diagnose patients with myopathies, muscular dystrophies and congenital myasthenic syndromes. Targeted NGS approaches enable a rapid and cost-effective analysis of NMD- related genes, offering reliable results in a short time and relegating invasive techniques to a second tier.

Comparison of Annotation Services for Next-Generation Sequencing in a Large-Scale Precision Oncology Program.

Next-generation sequencing (NGS) multigene panel testing has become widespread, including the Veterans Affairs (VA), through the VA National Precision Oncology Program (NPOP). The interpretation of genomic alterations remains a bottleneck for realizing precision medicine. We sought to examine the concordance for pathogenicity determination and clinical actionability of annotation services in NPOP. Unique gene variants were generated from NGS gene panel results using two sequencing services. For each unique gene variant, annotations were provided through N-of-One (NoO), IBM Watson for Genomics (WfG), and OncoKB. Annotations for pathogenicity (all three sources) and actionability (WfG and OncoKB) were examined for concordance. Cohen's kappa statistic was calculated to measure agreement between annotation services. Among 1,227 NGS results obtained between 2015 and 2017, 1,388 unique variants were identified in 117 genes. The genes with the largest number of variants included TP53 (270), STK11 (92), and CDKN2A (81). The most common cancer type was lung adenocarcinoma (440), followed by colon adenocarcinoma (113). For pathogenic and likely pathogenic variants, there was 30% agreement between WfG and NoO (kappa, -0.26), 76% agreement between WfG and OncoKB (kappa, 0.22), and 42% agreement between NoO and OncoKB (kappa, -0.07). For level 1 drug actionability of gene variant-diagnosis combinations, there was moderate agreement between WfG and OncoKB (96.9%; kappa, 0.44), with 27 combinations identified as level 1 by both services, 58 by WfG alone, and 6 variants by OncoKB alone. There is substantial variability in pathogenicity assessment of NGS variants in solid tumors by annotation services. In addition, there was only moderate agreement in level 1 therapeutic actionability recommendations between WfG and OncoKB. Improvement in the precision of NGS multigene panel annotation is needed.

Abstract P4-09-11: PIK3CA somatic alterations in 412 chinese invasive breast cancers: Higher frequency of mutant H1047R detected by next generation sequencing compared to breast cancer in caucasians

Abstract Purpose: Somatic alterations of PIK3CA gene is an important therapeutic target in breast cancer (BC). The PI3Kα-specific inhibitor alpelisib was remarkably active against PIK3CA-mutated, HR-positive/HER2-negative BC in the SOLAR-1 trial. We hypothesized that PIK3CA alterations in Chinese BC patients across different molecular subtypes might differ from other ethnic groups and this information would be useful for selecting anti-PI3K therapy. Methods: The molecular profile of the PIK3CA gene was analyzed in 412 Chinese untreated invasive BC patients with ER/HER2 molecular subtypes, using a 540 gene next-generation sequencing panel. The results were compared to the molecular profile of Caucasian breast cancer patients in The Cancer Genome Atlas(TCGA-white). Results: Compared to wild type, PIK3CA alterations were more frequent in the ER+ subtype (49.3%, p=0.024), and tumors with low ki67 proliferation (58.3%, p=0.007) and low histological grade (grade I/II/III 80%, 53.4%, 35.9%, p<0.001). Compared to TCGA Caucasian race (TCGA-white), Chinese BC patients had a higher alteration frequency (45.6% vs. 34.7%, p<0.001). The p.H1047R mutation predominantly occurred among three hot spots (p.E545K, p.E542K and p.H1047R) for Chinese BC compared to that of the TCGA-white cohort (66.1% vs 43.7%, p=0.01). Nine novel mutation sites of PIK3CA were observed in the Chinese cohort that was absent among Caucasian BC patients. Among the four ER/HER2 molecular subtypes, ER+/HER2+ tumors harbored the most PIK3CA alterations (51.6%), while ER-/HER2- harbored the least (30.0%). However the latter presented the highest frequency of copy number amplification (19.05%). Conclusion: PIK3CA alterations prevail in nearly half of Chinese BC patients and has some different molecular features compared to that of Caucasian BC patients. The PIK3CA distribution patterns differed among four ER/HER2 subtypes. The results provide more insights for evaluating the results of PIK3CA inhibitors. Citation Format: Minghan Jia, Ning Liao, Bo Chen, Guochun Zhang, Yulei Wang, Xiaoqing Chen, Liping Guo, Li Cao, Hsiaopei Mok, Chongyang Ren, Kai Li, Cheukfai Li, Lingzhu Wen, Jiali Lin, Guangnan Wei, Zhou Zhang, Charles M Balch. PIK3CA somatic alterations in 412 chinese invasive breast cancers: Higher frequency of mutant H1047R detected by next generation sequencing compared to breast cancer in caucasians [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-09-11.

Molecular profiling of early-stage colorectal cancer (CRC) using targeted next generation sequencing (NGS) to predict signatures of recurrence.

242 Background: While the 5-year recurrence rate in early stage CRC is low (12%) and there is currently limited role of adjuvant chemotherapy in such cases, a unique subset of patients (pts) will have late recurrences. To identify molecular signatures predictive of late recurrence after pts undergo intended curative resection, we employed a 22 targeted gene NGS panel in pts with early CRC. Association between mutation status and recurrence free survival (RFS) was analyzed. Methods: Pts with stage I-II CRC had their tumor prospectively sequenced between 09/2015-12/2018 by an ion torrent targeted 22 gene hotspot NGS panel, including KRAS, EGFR, BRAF, PIK3CA, AKT1, ERBB2/4, PTEN, NRAS, STK11, MAP2K1, ALK, DDR2, CTNNB1, MET, TP53, SMAD4, FBX7, NOTCH1, and FGFR1/2/3. Associations were analyzed with unadjusted p-values (p) and Benjamini & Hochberg adjusted (BHp) shown. Results: Clinical and pathologic data from 180 pts were analyzed: median age 66 (range 24-86), male (47%), stage I (41%), stage II (69%), left (54%) vs right (36%) sided primary tumors, and microsatellite stable (85%). 35 (19%) pts had adjuvant therapy (n = 21 rectal, n = 14 colon). Pathological mutations were found in 160 (89%) of pts, including TP53 (56%), KRAS (44%), PIK3CA (22%), BRAF (12%), SMAD4 (8%), MET (6%) and NRAS (3%). There was only 1 case of ERBB2 mutation. 33 pts (18%) had evidence of recurrence. 36 month RFS was 82%. Common sites of recurrence included liver (13 pts, 39%), lung (10 pts, 30%), and bone (2 pts, 6%). Alterations in MET cDNA and protein were associated with recurrence-free survival (RFS) (HR = 4.1; p =0.0026, BHp= 0.057). Interestingly, while TP53 mutations are typically associated with worse prognosis in metastatic colorectal cancers, it was not associated with RFS (HR = 0.8; p = 0.55, BHp= 0.98). There was also no association between the number of gene alterations and RFS (p = 0.45). Conclusions: These data highlight that targeted NGS tumor profiling of early stage CRC, including sequencing MET among other genes, may be utilized alongside known prognostic pathological factors to predict pts with a higher risk of recurrence and may facilitate tailored adjuvant chemotherapy to mitigate this risk.

Clinical utility of targeted next generation sequencing (NGS) to predict response to neoadjuvant therapy (NT) in rectal cancer (RC).

237 Background: NGS analysis has identified molecular alterations that have predictive and prognostic implications in advanced RC. However, its role in identifying predictive signatures of chemoradiotherapy response in RC remains largely unexplored. Neoadjuvant rectal cancer (NAR) score, a composite score available after resection, is a validated short-term surrogate endpoint for long-term outcomes. Herein, we identified gene alterations in early stage RC with NGS analysis and correlated mutations with tumor response using the NAR scoring system. Methods: Patients (pts) with stage I-III RC completing NT followed by resection (2011-2018) had their tumor prospectively sequenced by an ion torrent targeted 22 gene NGS panel ( AKT1, ALK, BRAF, CTNNB1, DDR2, EGFR, ERBB2/4, FBXW7, FGFR1/2/3, KRAS, MAP2K1, MET, NOTCH1, NRAS, PIK3CA, PTEN, SMAD4, STK11, TP53). NAR scores were calculated based on clinical and pathologic staging data input, yielding 3 cohorts: low (score < 8, pCR or near CR), intermediate (8-16), or high ( > 16, poor or no response) NAR scores. Associations were drawn using Two-sided Fisher’s exact. Both unadjusted (p) and Benjamini & Hochberg adjusted (BHp) p-values are shown. Results: 51 pts with RC were analyzed: median age 59 (24-81), male (59%), clinical stage I (6%), II (25%) and III (69%). Pathological mutations were found in 94% of pts, including TP53 (76%), KRAS (49%), PIK3CA (10%), NRAS (6%), FGFR3 (4%), MET (4%). Post NT 8 (16%) pts had low NAR scores, including 6 (12%) cases with pCR, 26 (51%) intermediate, and 17 (33%) high NAR scores. Gene alterations in FGFR3 (p =0.022, BHp = 0.15) and NRAS (p =0.026, BHp = 0.15) were associated with NAR score, while no differences were observed with mutations in TP53 or KRAS. Additionally, 17 (33%) pts had recurrences [13% of pts with low NAR scores, 31% intermediate, 47% high], with metastases primarily to lung (53%) and/or liver (35%). Conclusions: This study suggests that pts with RC who derived favorable responses to standard NT could have a targeted molecular profile, namely alterations in FGFR3 and NRAS. Conversely, pts that lack this profile may benefit from novel therapeutic approaches to improve NAR scores and long-term outcomes.

Diagnosis of 4 novel cases of adult acid sphyngomielynase deficiency by screening with next-generation sequencing gene panels

Diagnosis of 4 novel cases of adult acid sphyngomielynase deficiency by screening with next-generation sequencing gene panels

Assessment of the clinical utility of four NGS panels in myeloid malignancies. Suggestions for NGS panel choice or design.

The diagnosis of myeloid neoplasms (MN) has significantly evolved through the last few decades. Next Generation Sequencing (NGS) is gradually becoming an essential tool to help clinicians with disease management. To this end, most specialized genetic laboratories have implemented NGS panels targeting a number of different genes relevant to MN. The aim of the present study is to evaluate the performance of four different targeted NGS gene panels based on their technical features and clinical utility. A total of 32 patient bone marrow samples were accrued and sequenced with 3 commercially available panels and 1 custom panel. Variants were classified by two geneticists based on their clinical relevance in MN. There was a difference in panel's depth of coverage. We found 11 discordant clinically relevant variants between panels, with a trend to miss long insertions. Our data show that there is a high risk of finding different mutations depending on the panel of choice, due both to the panel design and the data analysis method. Of note, CEBPA, CALR and FLT3 genes, remains challenging the use of NGS for diagnosis of MN in compliance with current guidelines. Therefore, conventional molecular testing might need to be kept in place for the correct diagnosis of MN for now.

Applications of Next Generation Sequencing to the Analysis of Familial Breast/Ovarian Cancer.

Next generation sequencing (NGS) provides a powerful tool in the field of medical genetics, allowing one to perform multi-gene analysis and to sequence entire exomes (WES), transcriptomes or genomes (WGS). The generated high-throughput data are particularly suitable for enhancing the understanding of the genetic bases of complex, multi-gene diseases, such as cancer. Among the various types of tumors, those with a familial predisposition are of great interest for the isolation of novel genes or gene variants, detectable at the germline level and involved in cancer pathogenesis. The identification of novel genetic factors would have great translational value, helping clinicians in defining risk and prevention strategies. In this regard, it is known that the majority of breast/ovarian cases with familial predisposition, lacking variants in the highly penetrant BRCA1 and BRCA2 genes (non-BRCA), remains unexplained, although several less penetrant genes (e.g., ATM, PALB2) have been identified. In this scenario, NGS technologies offer a powerful tool for the discovery of novel factors involved in familial breast/ovarian cancer. In this review, we summarize and discuss the state of the art applications of NGS gene panels, WES and WGS in the context of familial breast/ovarian cancer.

Investigating diagnostic sequencing techniques for CADASIL diagnosis

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a cerebral small vessel disease caused by mutations in the NOTCH3 gene. Our laboratory has been undertaking genetic diagnostic testing for CADASIL since 1997. Work originally utilised Sanger sequencing methods targeting specific NOTCH3 exons. More recently, next-generation sequencing (NGS)-based technologies such as a targeted gene panel and whole exome sequencing (WES) have been used for improved genetic diagnostic testing. In this study, data from 680 patient samples was analysed for 764 tests utilising 3 different sequencing technologies. Sanger sequencing was performed for 407 tests, a targeted NGS gene panel which includes NOTCH3 exonic regions accounted for 354 tests, and WES with targeted analysis was performed for 3 tests. In total, 14.7% of patient samples (n = 100/680) were determined to have a mutation. Testing efficacy varied by method, with 10.8% (n = 44/407) of tests using Sanger sequencing able to identify mutations, with 15.8% (n = 56/354) of tests performed using the NGS custom panel successfully identifying mutations and a likely non-NOTCH3 pathogenic variant (n = 1/3) identified through WES. Further analysis was then performed through stratification of the number of mutations detected at our facility based on the number of exons, level of pathogenicity and the classification of mutations as known or novel. A systematic review of NOTCH3 mutation testing data from 1997 to 2017 determined the diagnostic rate of pathogenic findings and found the NGS-customised panel increases our ability to identify disease-causing mutations in NOTCH3.

Genetic landscape of adult Langerhans cell histiocytosis with lung involvement

The clinical significance of the BRAF V600E mutation in adult Langerhans cell histiocytosis (LCH), including pulmonary Langerhans cell histiocytosis (PLCH), is not well understood. Similarly, the spectrum of molecular alterations involved in adult LCH has not been fully delineated. To address these issues, we genotyped a large number of adult LCH biopsies and searched for an association of identified molecular alterations with clinical presentation and disease outcome.Biopsies from 117 adult LCH patients, 83 with PLCH (median age 36.4 years, 56 females, 38 multisystem disease, 79 single system disease, 65 current smokers) were genotyped for the BRAF V600E mutation. In 69 cases, LCH lesions were also genotyped by whole-exome sequencing (WES) or targeted gene panel next-generation sequencing (NGS). Cox models were used to estimate the association of baseline characteristics with the hazard of LCH progression.MAPK pathway alterations were detected in 59 out of 69 cases (86%) (BRAF V600E mutation: 36%, BRAF N486_P490 deletion: 28%, MAP2K1 mutations: 15%, isolated NRAS Q61 mutations: 4%), while KRAS mutations were virtually absent in PLCH lesions. The BRAF V600E mutation was not associated with LCH presentation at diagnosis, including smoking status and lung function, in PLCH patients. BRAF V600E status did not influence the risk of LCH progression over time.Thus, MAPK alterations are present in most lesions from adult LCH patients, particularly in PLCH. Unlike reports in paediatric LCH, BRAF V600E genotyping did not provide additional information on disease outcome. The search for alterations involved in the MAPK pathway, including BRAF deletions, is useful for guiding targeted treatment in selected patients with refractory progressive LCH.

Orthogonal Comparison of Four Plasma NGS Tests With Tumor Suggests Technical Factors are a Major Source of Assay Discordance

Discordance between plasma and tumor variant calling has been attributed primarily to tumor heterogeneity, whereas technical variables remain largely unexplored. To measure these variables, we tested four next-generation sequencing (NGS) gene panel assays for mutations in circulating tumor DNA (ctDNA) using replicate sets of 24 plasma samples and compared the results with matched tumor-normal tissue pairs. Our orthogonal approach identified false-negative (FN) and false-positive (FP) variants with high confidence and revealed substantial variability among the ctDNA assays, with a range of sensitivity (38% to 89%) and positive predictive value (36% to 80%). Most discordance in our cross-vendor study was observed below 1% variant allele frequency. FP variants displayed mutational biases and tended to be novel variants not found in somatic databases. Of the 56 unique variants called by all four ctDNA assays, 41 (68%) resulted from technical discordance. These findings suggest that most NGS assay discordance is a result of technical variations and, to a lesser extent, biologic factors such as clonal hematopoiesis of indeterminate potential and tumor heterogeneity.

The relevance of tumor mutation profiling in interpretation of NGS data from cell-free DNA in non-small cell lung cancer patients

Studies have indicated that detection of circulating tumor DNA (ctDNA) prior to treatment is a negative prognostic marker in non-small cell lung cancer (NSCLC). ctDNA is currently identified by detection of tumor mutations. Commercial next-generation sequencing (NGS) assays for mutation analysis of ctDNA for routine practice usually include small gene panels and are not suitable for general mutation analysis. In this study, we investigated whether mutation analysis of cfDNA could be performed using a commercially available comprehensive NGS gene panel and bioinformatics workflow. Tumor DNA, plasma DNA and peripheral blood leukocyte DNA from 30 NSCLC patients were sequenced. In two patients (7%), tumor mutations in cfDNA were immediately called by the bioinformatic workflow. In 13 patients (43%), tumor mutations were not called, but were present in ctDNA and were identified based on the known tumor mutation profile. In the remaining 15 patients (50%), no concordant mutations were detected. In conclusion, we were able to identify tumor mutations in ctDNA from 57% of NSCLC patients using a comprehensive gene panel. We demonstrated that sequencing paired tumor DNA was helpful to interpret data and confirm ctDNA, and thus increased the ratio of patients with detectable ctDNA. This approach might be feasible for mutation analysis of ctDNA in routine diagnostic practice, especially in case of suboptimal plasma quality and quantity.