Use Of Next-generation Sequencing Panel Research Articles

Abstract 3398: Clinical use of next-generation sequencing panel in pediatric oncology patients

Abstract Background: Next-generation sequencing (NGS) technology is gradually expanding in cancer diagnosis and therapeutic decision-making. However, reports on the clinical performance and utility of pan-cancer NGS panel in pediatric patients are still lacking. Methods: We analyzed the patients who underwent brain tumor or pan-cancer NGS panel sequencing at Seoul National University Children’s Hospital. DNA-based NGS panel testing was started in April 2018, and RNA panel was added from January 2022. Single nucleotide variants/small insertions and deletions (SNV/INDEL), fusion, copy number alteration (CNA), microsatellite instability (MSI) and tumor mutational burden (TMB) were evaluated. Results: A total of 140 patients were analyzed between August 2018 and March 2022. The median age at diagnosis was 8.5 (range 0.0-31.8) years. The diagnoses were CNS tumors in 56 (40.0%), sarcoma in 45 (32.1%), other solid tumors in 36 (25.7%) and histiocytosis in 3 (2.1%) patients. At the time of NGS testing, 115 patients (75.2%) were newly diagnosed and 38 patients (24.8%) were in relapsed or refractory status. In all patients, 85.7% had at least one pathogenic or likely pathogenic variant. Diagnosis was refined or changed in five patients (3.6%) after the NGS panel results were reported. Oncogenic gene fusions were discovered in 45 (32.1%) patients. EWSR1, BRAF, NTRK and RET-related fusions were the most common. Frequent SNV/INDEL included BRAF, CTNNB1 and TP53 mutations. Common CNA were CDKN2A and CDKN2B loss, MYCN amplification, CCND3 amplification and PTEN loss. Nine (6.4%) patients were identified with germline alteration, additionally. No patient had MSI, and two patients had high TMB. Fifteen patients (10.7%) were enrolled in clinical trials or compassionate use program according to molecular profiling (DAY101 [BRAF inhibitor] in 3, selpercatinib in 3, palbociclib in 2, olaparib in 2, alectinib in 1, repotrectinib in 1, larotrectinib in 1, atezolizumab in 1 and erdafitinib in 1). Two (1.4%), one (0.7%) and one (0.7%) patients received vemurafenib, larotrectinib, and pembrolizumab with non-reimbursement. Best responses included two complete response (1 high grade glioma [HGG] and 1 renal cell carcinoma), five partial responses (2 low grade glioma, 1 HGG, 1 Langerhans cell histiocytosis, and 1 papillary thyroid carcinoma), two stable disease and six progressive diseases. Four patients were not evaluable due to short duration of drug administration. One-year progression free survival and overall survival was 54.5% and 52.7%, respectively. Conclusions: Application of NGS panel in pediatric cancer aid in diagnosis, treatment decision, clinical trial enrollment and germline risk determination. Although there are not many cases linked with molecular target-based therapy, it is leading to clinical benefits in pediatric patients and more understanding genomic profiling of pediatric cancer. Citation Format: Jung Yoon Choi, Hyun Jin Park, Bo Kyung Kim, Kyung Taek Hong, Jaemoon Koh, Sung-Hye Park, Jeong Mo Bae, Hongseok Yun, Hyoung Jin Kang. Clinical use of next-generation sequencing panel in pediatric oncology patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3398.

Chapter 25 - Hereditary neuropathy

The hereditary neuropathies, collectively referred as Charcot-Marie-Tooth disease (CMT) and related disorders, are heterogeneous genetic peripheral nerve disorders that collectively comprise the commonest inherited neurological disease with an estimated prevalence of 1:2500 individuals. The field of hereditary neuropathies has made significant progress in recent years with respect to both gene discovery and treatment as a result of next-generation sequencing (NGS) approach. These investigations which have identified over 100 causative genes and new mutations have made the classification of CMT even more challenging. Despite so many different mutated genes, the majority of CMT forms share a similar clinical phenotype, and due to this phenotypic homogeneity, genetic testing in CMT is increasingly being performed through the use of NGS panels. The majority of patients still have a mutation in one the four most common genes (PMP22 duplication-CMT1A, MPZ-CMT1B, GJB1-CMTX1, and MFN2-CMT2A). This chapter focuses primarily on these four forms and their potential therapeutic approaches.

P1012: A NUMBER OF CONGENITAL ERYTHROCYTOSIS ARE MULTIGENIC

Background: Imbalance in erythropoiesis and oxygen homeostasis could lead to the onset of Erythrocytosis, a clinical condition characterized by persistently raised hemoglobin (Hb) and hematocrit (Ht) levels. Nowadays, a high number of patients are classified as “Idiopathic Erythrocytosis” (IE) meaning that it is not possible to identify the cause of hematocrit increase. Recently, HFE mutations has been found to be often present in idiopathic erythrocytic patients. The HFE mutations could facilitate the increase in red cell mass, and this indicates the possible involvement of Iron Metabolism in causes of IE. Aims: To identify the possible mutations of IE patients using our gene panel for Next Generation Sequencing (NGS). Methods: In 118 patients with IE, regularly followed in our surgery, we used a gene panel for NGS composed by fifteen genes: JAK2, EGLN1 (PHD2), EPO-R, FTL, FTH, ASXL1, HFE, HFE2, TFR2, HAMP, SLC40A1, SLC11A2, VHL, BPMG, EPAS1 (HIF-2α). We analyzed all the exons parts of these genes. The approach used for the library preparation was a multiplexing PCR and data were analyzed by bioinformatics tools. In all patients, the mutations found were confirmed with Sanger Sequencing. Results: In 78 out of the 118 patients (66%) evaluated, we found one 1 mutated gene in 55 patients (7 EGLN1, 1 VHL, 34 HFE, 6 TFR2, 3 JAK2 and 4 EPO-R), 2 mutated genes in 18 patients (1 EGLN1/JAK2, 4 EGLN1/HFE, 2 EPAS1/HFE, 1 EPAS1/JAK2, 1 EPAS1/EGLN1, 2 EPO-R/HFE, 2 HFE/JAK2, 1 JAK2/TFR2, 2 with TFR2/EGLN1, 1 VHL/HFE, 1 TFR2/HFE) and 3 mutated genes in 5 patients (1 EGLN1/EPO-R/HFE, 1 VHL/HFE/EGLN1, 1 EPAS1/EPO-R/HFE, 1 EPAS1/HFE/EGLN1 and HFE/TFR2/JAK2). All the mutations found were germline. Interestingly, 21 out of the 27 (78%) patients carrying HFE mutation displayed 2 or more mutations. Summary/Conclusion: The use of NGS panels in various clinical conditions has frequently demonstrated that multiple mutations coexist in various patients and that the monogenic diseases are quite rare. This study is the first at our best knowledge that gives evidence of more than one mutated gene in erythrocytotic patients, and this is the results of the use of an appropriate NGS panel. The data here reported suggest that we can confirm that HFE mutations are common in IE patients, in some cases associated with other mutations, but the present data ask some questions: HFE mutations alone can induce erythrocytosis? or other genetic imbalances are needed? The presence of multiple mutations could lead to a greater severity of the disease? A significant number of IE patients displayed TFR2 mutations, giving more relevance to the hypothesis of the iron metabolism implication in IE genesis. Finally, the presence of 11 patients carrying germlines JAK2 mutations is surprising, and we suppose it will be necessary to strictly watch these patients in the suspicion of a future appearance of a polycythemia vera. The present study demonstrates the need to extend the biomolecular knowledge in patients with idiopathic erythrocytosis.

MEDB-72. MOLECULAR CHARACTERIZATION OF MEDULLOBLASTOMAS IN A SINGLE INSTITUTION

INTRODUCTION: The four molecular groups (WNT, SHH, Group 3 and Group 4) in medulloblastoma have been well established for the past decade. New subgroups within the four principal molecular groups have recently been discovered and recognized by WHO classification of Central Nervous System Tumours (5th edition). Subgroups were reported to have distinct somatic copy-number aberrations and clinical outcomes. This further classification could be helpful to refine prognostication and potentially provide risk stratification for treatment planning. AIM: To interrogate archival medulloblastoma samples using Oncoscan Microarray Assay, correlate with clinical features and consider the assay for clinical use. METHODS: Thirty-one archival samples with histological diagnosis of medulloblastoma and molecular grouping results from NanoString were retrieved and evaluated with Oncoscan Microarray Assay. Twenty-six were subjected to DNA methylation profiling to compare the results. Eight cases also had molecular data from next-generation sequencing (NGS) done with the in-house Ampliseq Childhood Cancer Panel. Correlation was made with clinical characteristics and outcomes of these 31 patients. RESULTS: OncoScan microarray showed distinct differences in the copy number profiles of the 31 medulloblastoma samples. Seventeen samples could be further classified into one of 12 subgroups. However, further subgrouping was challenging without first determining the main molecular group especially amongst non-WNT/SHH tumours. DNA methylation results provided corroboration with the Oncoscan subgrouping results in 25 of 26 samples. NGS panel detected additional genetic alterations in 5 of 8 samples. CONCLUSIONS: Oncoscan Microarray Assay showed potential in providing additional molecular information for further subgrouping of medulloblastoma, but was insufficient for determining the main molecular groups. Moving forward, molecular characterization could instead be done through use of NGS panel and DNA methylation, which provides tumour epigenetic profiling on top of copy number variants. These could be used alongside the NanoString platform, which is performed routinely for all medulloblastomas at our centre.

Recent Advances in the Molecular Biology of Systemic Mastocytosis: Implications for Diagnosis, Prognosis, and Therapy.

In recent years, molecular characterization and management of patients with systemic mastocytosis (SM) have greatly benefited from the application of advanced technologies. Highly sensitive and accurate assays for KIT D816V mutation detection and quantification have allowed the switch to non-invasive peripheral blood testing for patient screening; allele burden has prognostic implications and may be used to monitor therapeutic efficacy. Progress in genetic profiling of KIT, together with the use of next-generation sequencing panels for the characterization of associated gene mutations, have allowed the stratification of patients into three subgroups differing in terms of pathogenesis and prognosis: (i) patients with mast cell-restricted KIT D816V; (ii) patients with multilineage KIT D816V-involvement; (iii) patients with “multi-mutated disease”. Thanks to these findings, new prognostic scoring systems combining clinical and molecular data have been developed. Finally, non-genetic SETD2 histone methyltransferase loss of function has recently been identified in advanced SM. Assessment of SETD2 protein levels and activity might provide prognostic information and has opened new research avenues exploring alternative targeted therapeutic strategies. This review discusses how progress in recent years has rapidly complemented previous knowledge improving the molecular characterization of SM, and how this has the potential to impact on patient diagnosis and management.

Next-Generation Sequencing Improves Diagnosis, Prognosis and Clinical Management of Myeloid Neoplasms.

Molecular diagnosis of myeloid neoplasms (MN) is based on the detection of multiple genetic alterations using various techniques. Next-generation sequencing (NGS) has been proved as a useful method for analyzing many genes simultaneously. In this context, we analyzed diagnostic samples from 121 patients affected by MN and ten relapse samples from a subset of acute myeloid leukemia patients using two enrichment-capture NGS gene panels. Pathogenicity classification of variants was enhanced by the development and application of a custom onco-hematology score. A total of 278 pathogenic variants were detected in 84% of patients. For structural alterations, 82% of those identified by cytogenetics were detected by NGS, 25 of 31 copy number variants and three out of three translocations. The detection of variants using NGS changed the diagnosis of seven patients and the prognosis of 15 patients and enabled us to identify 44 suitable candidates for clinical trials. Regarding AML, six of the ten relapsed patients lost or gained variants, comparing with diagnostic samples. In conclusion, the use of NGS panels in MN improves genetic characterization of the disease compared with conventional methods, thus demonstrating its potential clinical utility in routine clinical testing. This approach leads to better-adjusted treatments for each patient.

Validation and comparison of two NGS assays for the detection of EGFR T790M resistance mutation in liquid biopsies of NSCLC patients.

Analysis of circulating cell-free DNA (cfDNA) derived from peripheral blood (“liquid biopsy”) is an attractive alternative to identify non-small cell lung cancer (NSCLC) patients with the EGFR T790M mutation eligible for 3rd generation tyrosine kinase inhibitor therapy.We evaluated two PCR-based next generation sequencing (NGS) approaches, one including unique molecular identifiers (UMI), with focus on highly sensitive EGFR T790M mutation detection. Therefore, we extracted and sequenced cfDNA from synthetic plasma samples spiked with mutated DNA at decreasing allele frequencies and from 21 diagnostic NSCLC patients. Data evaluation was performed to determine the limit of detection (LoD), accuracy, specificity and sensitivity of both assays.Considering all tested reference dilutions and mutations the UMI assay performed best in terms of LoD (1% vs. 5%), sensitivity (95.8% vs. 81.3%), specificity (100% vs. 93.8%) and accuracy (96.9% vs. 84.4%). Comparing mutation status of diagnostic samples with both assays showed 81.3% concordance with primary mutation verifiable in 52% of cases. EGFR T790M was detected concordantly in 6/7 patients with allele frequencies from 0.1% to 27%. In one patient, the T790M mutation was exclusively detectable with the UMI assay.Our data demonstrate that both assays are applicable as multi-biomarker NGS tools enabling the simultaneous detection of primary EGFR driver and resistance mutations. However, for mutations with low allelic frequencies the use of NGS panels with UMI facilitates a more sensitive and reliable detection.

Points to Consider in the Clinical Use of NGS Panels for Mitochondrial Disease: An Analysis of Gene Inclusion and Consent Forms

Mitochondrial next generation sequencing (NGS) panels offer single-step analysis of the numerous nuclear genes involved in the structure, function, and maintenance of mitochondria. However, the complexities of mitochondrial biology and genetics raise points for consideration in clinical use of these tests. To understand the current status of mitochondrial genetic testing, we assessed the gene offerings and consent forms of mitochondrial NGS panels available from seven US-based clinical laboratories. The NGS panels varied markedly in number of genes (101-1204 genes), and the proportion of genes causing "classic" mitochondrial diseases and their phenocopies ranged widely between labs (18 %-94 % of panel contents). All panels included genes not associated with classic mitochondrial diseases (6 %-28 % of panel contents), including genes causing adult-onset neurodegenerative disorders, cancer predisposition, and other genetic syndromes or inborn errors of metabolism. Five of the panels included genes that are not listed in OMIM to be associated with a disease phenotype (5 %-49 % of panel contents). None of the consent documents reviewed had options for patient preference regarding receipt of incidental findings. These findings raise points of discussion applicable to mitochondrial diagnostics, but also to the larger arenas of exome and genome sequencing, including the need to consider the boundaries between clinical and research testing, the necessity of appropriate informed consent, and the responsibilities of clinical laboratories and clinicians. Based on these findings, we recommend careful evaluation by laboratories of the genes offered on NGS panels, clear communication of the predicted phenotypes, and revised consent forms to allow patients to make choices about receiving incidental findings. We hope that our analysis and recommendations will help to maximize the considerable clinical utility of NGS panels for the diagnosis of mitochondrial disease.