TruSeq Custom Amplicon Research Articles

MM-356 Deep Coverage Targeted Next Generation Sequencing Approach to Determine Somatic Mutational Spectrum of Recurrently Mutated Genes in Newly Diagnosed Multiple Myeloma in a Large Indian Cohort

To determine the mutational spectrum of recurrently mutated genes in a large cohort of Indian patients presenting with multiple myeloma (MM) in our center by performing deep coverage-targeted NGS. Samples of 151 multiple myeloma patients (149 NDMM and 6 follow-up) were sequenced. Patients were diagnosed with MM per IMWG criteria 2014. The study was initiated following approval from the institute's ethical committee, and written informed consent was obtained from all patients. BMA was subjected to plasma cell isolation using CD138+ MACS. DNA underwent automated extraction using the Maxwell System. Mutations were tested on a commercially available 47-gene custom panel (Illumina) specifically designed for MM. Libraries were prepared using a TruSeq Custom Amplicon Low Input Kit (Illumina). DNA (25 ng) was hybridized with oligos specific for targeted regions of interest. Libraries were pooled. Paired-end sequencing was carried out on Illumina's MiSeq sequencer using micro flow cell with V3 chemistry and MS600 V3 series reagent cartridge. Sequenced reads were mapped to GRCh37/Hg19 reference and data obtained as FASTQ files and processed with MiSeq Reporter. As output, .vcf files were generated. Genomic variants were annotated by ANNOVAR. Sequencing coverage of 4771 X (Range 2063X-10980X) revealed different types of variants (exonic, intronic, splice region, 3' and 5' UTR, and upstream gene). Pathogenicity was checked by COSMIC. At least one pathogenic mutation was found in 77% (116/151) of patients (median 1 mutation per patient, range 0-7) and 51% (24/47) of genes. Forty-five percent (68/151) of patients had mutations in more than one gene. The most frequently mutated genes were KRAS, NRAS, BRAF, TP53, DIS3, FAT4, FAT3, and DNAH5. Genomic aberrations found were compared with the frequency of mutations as per the MMRF and TCGA datasets and related literature; good concordance was found (especially in MAPK pathway genes). BRAF and RAS mutations were mutually exclusive. Few mutations were validated by Sanger sequencing. The high frequency of mutations in MAPK pathway genes (KRAS, NRAS, BRAF) suggested the importance of this pathway in myelomagenesis and therapeutics. Follow-up sample sequencing may help deduce clonal evolution during disease course. Identification of clinically relevant target driver mutations may aid in prognosis and therapy personification.

Evidence of NF-ΚB Pathway Activation in Patients with Advanced, High Molecular Risk Myelofibrosis

Evidence of NF-ΚB Pathway Activation in Patients with Advanced, High Molecular Risk Myelofibrosis

Genomic Profiling In Fit, Elderly Patients Receiving Oral Fludarabine, Oral Cyclophosphamide and Intravenous Rituximab (ALLG CLL5 OFOCIR trial) As Initial Treatment Of Chronic Lymphocytic Leukemia (CLL)

BackgroundAnalysis for genomic alterations by fluorescence in-situ hybridization (FISH) allows prognostic stratification in chronic lymphocytic leukemia (CLL). Recurrent mutations in genes including NOTCH1, SF3B1, BIRC3 and others have recently been identified in patients with CLL by next-generation sequencing and may provide independent prognostic information (Rossi D et al. Blood 2013). The prognostic impact of these mutations following first-line therapy chemotherapy has not been consistent in the literature (Oscier D et al. Blood 2012; Ysebaert L et al. ASH Abstract 2012; Stilgenbauer S et al. ASH Abstracts 2012) and the prognostic significance of these mutations in elderly patients requiring therapy is not clearly defined (Chiaretti S et al. ASH Abstracts 2012). AimWe aimed to describe the incidence of genomic alterations by FISH and multiplexed targeted resequencing of genes with potential prognostic importance in an elderly population of patients requiring frontline treatment for CLL. MethodsPre-treatment peripheral blood and bone marrow aspirate samples were obtained from patients enrolled on a phase II randomized clinical trial investigating oral fludarabine, oral cyclophosphamide and i.v. rituximab (poFCivR) tolerance in previously untreated fit elderly patients with CLL (Australasian Leukemia and Lymphoma Group [ALLG] CLL5, ACTRN12608000404325). Peripheral blood lymphocytes were purified by Ficoll gradient separation. Bone marrow aspirate samples were analyzed for CLL-associated genomic changes with a Vysis CLL FISH probe kit (Abbott, Des Moines, IL). DNA was extracted from peripheral blood samples and we performed targeted genome sequencing of genes including TP53, ATM, NOTCH1, SF3B1, BIRC3, MYD88, XPO1 and FBXW7 using a TruSeq Custom Amplicon Design Panel on a MiSeq Sequencer as per manufacturer's protocol (Illumina, San Diego, CA). DNA mutation data was analyzed and compared to reported mutations in the literature and COSMIC database. Non-synonymous mutations with a mutation frequency >10% are reported. Results120 patients were enrolled into the CLL5 study and 78 samples were available for analysis. In these patients, FISH analysis identified 17p deletion in 10 pts (13%), 11q deletion in 6 pts (8%), trisomy 12 in 15 pts (19%), deletion 13q in 28 pts (36%), no abnormality (n=14, 18%), and not performed (n=5, 6%). By targeted sequencing, mutations were identified in TP53 (n=7, 9%), ATM (n=7, 9%), NOTCH1 (n=12, 15%), SF3B1 (n=10, 13%), MYD88 (n=6, 8%), BIRC3 (n=2, 3%), XPO1 (n=6, 8%), FBXW7 (n=4, 5%). Thirty patients (38%) did not have a mutation in one of these genes. Mutation distribution is demonstrated in Figure 1. Five of 7 mutations in TP53 occurred in the DNA-binding core domains (exons 5-8) and one mutation in the homo-oligomerization domain and one in exon 11. The majority of NOTCH1 mutations were identified in the PEST domains (11 of 12) and all but one mutation in SF3B1 were identified in the PP2A repeats 5-9. Correlation with patient characteristics, IGHV mutational sequencing and clinical outcome data is proceeding. [Display omitted] ConclusionWe demonstrate a high proportion of genomic mutations in fit patients age 65 years and older undergoing first-line therapy for CLL. By comparison with data from Italian CLL trials, UK LRF CLL4 and the CLL8 study, our study demonstrates similar mutational frequencies in previously described recurrently mutated genes. These mutations are assessable by massive parallel sequencing and may potentially offer useful predictive information in patients requiring therapy for CLL. AcknowledgmentsSupported by grants from CLL Global Research Foundation and Cancer Institute of NSW / Northern Translational Cancer Research Unit. Disclosures:Mulligan:Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Comparison of Variant Interpretation between Manual Expert Curator and Automated in-Silico Classification Using ACMG/AMP Guidelines in Myeloid Malignancies in a Routine NGS Setting

Background:In recent years, with the adoption of next generation sequencing, clinical laboratories are performing an increasing catalogue of genetic testing. This shift in paradigm was accompanied by new challenges in variant interpretation. Evaluating the pathogenicity of a variant is challenging given the plethora of types of genetic evidence that laboratories have to consider. Deciding how to weigh each type of evidence is difficult, and standards are needed. In 2015, ACMG/AMP published guidelines with 28 criteria that include evidence derived from population data, case-control analyses, functional data, computational predictions, allelic data, segregation studies, and de novo observations to assess variants with conflicting data.in different sources.Aims:We set out to evaluate in myeloid malignancies how the ACMG/AMP guidelines compare to an accredited laboratory approach to variant classification and explore the variance in the use and interpretation of the pathogenicity criteria. This may reduce variability in reporting across different labs and improve upon turn-around time of NGS results.Patient cohort and method:We have selected 50 cases with myeloid malignancies and with an interesting and complex mutational pattern, harboring variants which were difficult to classify during routine workup, analyzed either with a 26 genes myeloid panel (ThunderStorm Target Enrichment library; Raindance, Billerica, MA) or a 63 genes panel (TruSeq Custom Amplicon; Illumina, San Diego, CA). Alignment and Variant calling was performed with JSI SeqPilot (JSI Medisys, Ettenheim, Germany). Expert curators in the lab annotated each variant manually in a 3-tier system (pathogenic, uncertain significance, benign) given the lab's SOP for variant classification. Each variant was checked against the following databases: COSMIC (v76), ClinVar, dbSNP (v147) and IARC TP53 (r17). Population frequency information was extracted from ExAC (gnomAD) and 1,000 genomes. Disease-associated frequency of mutations was extracted from the lab's own database based on 235,669 variants classified between 2006 and 2017. Mutation impact prediction was performed using PolyPhen-2, SIFT and VEP.For this study, VCF files from Seqpilot were analyzed in QCI interpret software (Qiagen, Hilden, Germany), which computes variant classification based on ACMG/AMP professional guidelines without human intervention. In contrast to the lab's 3-tier system, a 5-tier system is proposed (Pathogenic, Likely pathogenic, Uncertain significance, Likely benign, Benign).Results:Among the 50 cases 747 variants were classified in our labs routine workflow according SOPs and accreditation according EN ISO15189, subsequent to the elimination of sequencing artefacts. 443 were classified as benign, 55 with variant of uncertain significance (VUS) and 248 as pathogenic by the expert curators. Using the computed classification yielded 395 benign variants, 16 likely benign, 143 with uncertain significance, 86 likely pathogenic and 107 pathogenic variants. To be able to compare, pathogenic/likely pathogenic and benign/likely benign were binned together. In 87% (651/747) of instances both approaches are concordant. Strikingly, there were no strong discordance calls, with a mismatch between pathogenic and benign (see Table 1). Manual interrogation of a subset of the discrepant calls revealed that in those instances, public data was scarce and/or in-silico mutation impact predication was unavailable. In some instances the pathogenic call in the manual approach was due to disease-specific frequency data of that particular variant in the lab's database.Conclusion:This study systematically evaluated the performance of manual curation against fully automated ACMG/AMP guideline variant classification and found a high concordance rate of 87% between both approaches. With more curated data and more comprehensive knowledge bases, the automated classification will even improve further. The automated approach seems to be more cautious, thus the bias towards more uncertain significance calls, which is preferable to miscalls. The guidelines seem to yield results sufficiently good for clinical use, especially for labs with little experience in variant classification and a big step forward regarding standardization. [Display omitted] DisclosuresNadarajah:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Dissection of Clonal Evolution By Temporal Mutation Profiling in Chronic Lymphocytic Leukemia Patients Treated with Ibrutinib

Introduction: The irreversible Bruton tyrosine kinase (BTK) inhibitor ibrutinib is changing the paradigm of the treatment of chronic lymphocytic leukemia (CLL) with remarkable outcomes in first line as well as in relapsed CLL, including high risk patients with TP53 aberration. While the majority of patients demonstrate durable responses, with a median follow up of 3 years, approximately 18% of them develop resistance and relapse. Mutations in the BTK and PLCG2 genes emerged as predominant mechanisms conferring secondary ibrutinib resistance with BTK and/or PLCG2 mutations detected in the majority of resistant patients, often even 10 months before the clinical relapse. Given the very poor outcome of patients discontinuing ibrutinib, comprehensive understanding of the mechanisms of ibrutinib resistance and changes in the clonal architecture induced by the selective pressure of the drug are of major clinical importance. To dissect the clonal evolution in the context of all relevant mutation targets in CLL, we performed a temporal mutation profiling of 31 genes in paired pre- and post-treatment samples of patients receiving ibrutinib therapy.Methods: Sequential samples from 18 patients treated with single-agent ibrutinib were included in this study. Pre-treatment peripheral blood samples were available in all patients, with post-treatment samples at 18 months for 6 patients, at 12 months for 7 patients, at 8 months for 3 patients and at 3 months for 2 patients, with a median follow up of 12 months. Targeted deep NGS analysis of 31 recurrently mutated target genes, including ATM, BCOR, BIRC3, BRAF, BTK, CHD2, DDX3X, EGR2, EIF2A, EP300, FBXW7, HIST1H1E, IGLL5, KLHL6, KMT2D, LRP1B, MAPK1, MED12, MGA, MYD88, NFKBIE, NOTCH1, PLCG2, POT1, RIPK1, RPS15, SAMHD1, SF3B1, TP53, XPO1 and ZMYM3 was performed using the TruSeq Custom Amplicon approach (Illumina) on genomic DNA specimens extracted from peripheral blood mononuclear cells. Variant calling was performed using the VariantStudio3.0 application (Illumina) and only variants supported with more than 100 mutant reads were considered. Our cohort represented a heavily pretreated patient group with a median of 2.5 (range: 1-5) lines of prior therapies.Results: The ultra-deep NGS analysis revealed a total of 473 somatic mutations in the 18 paired samples with a median allelic depth of 25.550x across the 31 genes analyzed. The post-treatment samples carried a considerably higher number of mutations compared to the pre-treatment samples (114 vs 359 mutations) with more than half of the variants representing subclonal alterations with variant allele frequencies (VAF) of <10%. The most frequently mutated genes at baseline included TP53 (50%) , NOTCH1 (50%) and POT1 (28%) with similarly high mutation frequencies in the post-treatment samples. MYD88, BIRC3 and SF3B1 mutations were enriched in the post-ibrutinib samples with mutation frequencies of 11% vs 33%, 11% vs 39% and 11% vs 50%, respectively. Interestingly, XPO1 mutations emerged only in the post-treatment samples of five (28%) patients. As for the BTK and PLCG2 mutations, none of the pre-treatment samples carried a mutation in either of these genes, however, we detected emerging subclonal BTK and PLCG2 mutations (with VAFs between 1.5% and 11%) in 28% (5/18) and 33% (6/18) of the post-treatment samples, respectively. A subset of cases harbored multiple mutations in both genes with some of these representing novel BTK and PLCG2 mutations affecting previously unreported amino acid residues. Scrutiny of the clonal evolution revealed that all individual cases were characterized by almost unique combinations of mutations as well as patterns of emerging and disappearing mutant subclones upon the selective pressure of ibrutinib.Conclusions: The ultra-deep NGS analysis performed on paired samples of CLL patients treated with ibrutinib provided a significant insight into the mutational repertoire as well as subclonal dynamics of the leukemic compartment. Furthermore, it has revealed a profound subclonal heterogeneity that seems to be further enhanced by ibrutinib therapy, as demonstrated by a higher frequency of subclonal variants in the post-treatment samples. The clinical significance and the potential role of the novel BTK and PLCG2 variants identified in our study will require a longer follow up time, as currently all patients harboring these subclonal variants are still on ibrutinib therapy. DisclosuresNo relevant conflicts of interest to declare.

Clonal Progression during First Course of Chemotherapy in Acute Myeloid Leukemia Suboptimal Responders Heralds Reinduction Failure and Early Death

INTRODUCTIONOptimal response to induction chemotherapy for acute myeloid leukemia (AML) is achievement of complete remission (CR) and minimal residual disease (MRD) negativity. Suboptimal responses include achievement of CR but MRD positivity (CRMRD+ patients) as well as failure to achieve CR. The prospect is bleak, especially in the latter group, but intensified treatment can cure some of these. Careful deliberation of patient fitness, drug toxicities and level of residual disease is required prior to choice of reinduction therapy. HYPOTHESISWe hypothesized that by studying clonal architecture changes during AML treatment, subclonal development or progression would add another dimension to the chemotherapy response assessment beyond that of merely a quantitative reduction in blasts or a given MRD marker. PATIENTS AND METHODS25 uniformly treated (“3+7” like) AML patients were included, 7 CRMRD+ (flow cytometry-determined MRD levels between 0.02% and 1%), and 18 who did not achieve CR after the first course of chemotherapy, of whom 17 received reinduction chemotherapy. Median time from diagnosis to CR evaluation after first course of chemotherapy: 33 days (range: 20-74 days)For the CRMRD+ patients; MRD cells from bone marrow samples taken after 1st treatment course were isolated using a FACSAria III based on the appropriate leukemia associated phenotype determined at diagnosis.All samples were analyzed using a 26-gene panel (ASXL1, BCOR, CALR, CBL, CSNK1A1, DNMT3A, ETV6, EZH2, FLT3-TKD, GATA2, IDH1, IDH2, JAK2, KIT, KRAS, MPL, NPM1, NRAS, RUNX1, SETBP1, SF3B1, SRSF2, TET2, TP53, U2AF1 and ZRSR2) . Furthermore, CSFR3, GATA1, PTPN11 and WT1 were analyzed in the group who did not achieve CR. The TruSeq Custom Amplicon kit was used for library prep and samples were sequenced using the MiSeq instrument. RESULTSIn the CRMRD+ group clonal architecture in unsorted diagnosis samples were compared to clonal architecture in the FACS products. In 3 informative cases, a development in the clonal architecture was seen. In two cases, a subclone positive for TET2 only persisted even if the fully mutated clones of the patients (one TET2 +/ NRAS +, one TET2 +/ NPM1 +/ IDH2 +) disappeared. Both patients relapsed. In the third patient; a IDH2 + SRSF2 + clone persisted, a IDH2 + SRSF2 + BCOR + clone disappeared. In the remaining 4 patients no mutations were detected in the FACS product.For the group of patients not in CR clonal architecture development between diagnosis and regeneration after first course of chemotherapy could be studied in 16/18 cases. Two cases were negative for all aberrations tested. 9/16 cases were clonally stable, displaying a reduction in mutation load comparable to leukemic burden reduction. In the remaining 7 cases, clonal development was seen: In 4 cases, one or more subclones were selectively eradicated leaving the more resistant clones. NPM1 was the only marker that was eradicated in more than one clone (n=2) and TET2 was the only marker that persisted in more than one clone (n=2). In 3/7 cases subclones emerged during chemotherapy; a WT1 + NRAS + clone in a NRAS + background, a SETBP1 + TET2 + ASXL1 + RUNX1 + clone in an RUNX1 + background and a DNMT3A + NPM1 + clone in a NPM1 + background. The 2-year survival of patients who received further chemotherapy was significantly different in the three groups (Clonally stable (n=9): 0.58, clonal architecture development but no emerging subclones (n=4): 0.25, AMLs with emerging subclones (n=3): 0 (last patient died after 224 days), P = 0.01 (Log-rank test)). CONCLUSIONThis study provides proof of the concept of subclonal architecture changes even in patients with minute amounts of residual leukemia (CRMRD+ group) and we were able to study the subclonal architecture changes in three of these patients.In patients not reaching CR, clonal architecture development was a rather common occurrence (7/16 cases). In three patients we observed that not only did a subclone resist chemotherapy, it grew during treatment. Even in this small study, this finding proved to be a significant marker of ultimate treatment failure.If corroborated in larger studies, we envisage that an approach like the one applied here can assist physicians' decisions in the choice of further treatment in patients experiencing suboptimal response to induction chemotherapy. DisclosuresMeggendorfer:MLL Munich Leukemia Laboratory: Employment. Koch:Munich Leukemia Laboratory: Employment. Fasan:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Mutations in DNMT3A, U2AF1, and EZH2 Identify Intermediate-Risk Acute Myeloid Leukemia Patients with Poor Outcome after CR1

Introduction: Intermediate-risk acute myeloid leukemia (IR-AML) is a clinically heterogeneous disease, for which optimal post-remission therapy has been debated. Several studies using next-generation sequencing have identified important prognostic information and led to modifications in risk stratification. However, the utility of this genetic information in decision making for IR-AML has yet to be elucidated. We investigated the clinical relevance of recurrent driver gene mutations in a well-characterized cohort of IR-AML patients in first complete remission (CR1) after induction chemotherapy.Methods: A total of 1,589 AML patients treated at the Cleveland Clinic (CC) between 2002 and 2016 were screened for eligibility, of whom 825 were in European Leukemia Net (ELN) IR category. Of them, 100 IR-AML patients, who had mutational information at diagnosis and achieved CR after ≥ 1 round of intensive induction were included. CR was defined as <5% bone marrow blasts, absolute neutrophil counts ≥ 1x109/L, and platelet counts ≥ 100x109/L. CR with inadequate count recovery (CRi) was same as CR, except for residual neutropenia or thrombocytopenia. The induction regimen was 7+3, and patients with persistent disease at day 14 marrow were re-induced with 7+3 or 5+2. Patients who did not receive intensive therapy or achieve CR/CRi were excluded. A TruSeq Custom Amplicon panel targeting exons of 62 genes was used for deep sequencing at diagnosis. We used The Cancer Genome Atlas (TCGA) data for validation of our findings.Results: In the CC cohort, median age was 58.5 (24-75) years, 64% had normal cytogenetics, and 31% required >1 induction cycles to achieve CR1. DNMT3A and FLT3-ITD mutations were the most common (19%), followed by RUNX1 (15%), TET2 (12%), IDH2 (12%), NPM1 (12%), ASXL1 (11%), and U2AF1 (11%). The frequency of persistent disease after initial induction was higher in patients with mutations in DNMT3A (47% vs 27%, p= .03) and U2AF1 (55% vs 28%, p= .01), while CRi was more common in patients with BCOR mutation (50% vs 8.5%, p= .02). In univariable analysis, patients with mutations in DNMT3A, U2AF1, and EZH2, and patients who achieved CRi after induction had worse overall (OS) and relapse-free survival (RFS). Patients who had hematopoietic cell transplant (HCT) in CR1 achieved better OS (HR, 0.4; 95% CI, 0.2-0.7; p=0.003) and RFS (HR, 0.3; 95% CI, 0.1-0.6; p< 0.001) than those who did not. Other clinical factors analyzed, such as age, sex, cytogenetics, and the number of induction courses to achieve CR1 were not significant predictors for survival after CR1. To evaluate the impact of these three mutations on risk stratification, we compared patients whose AML harbored any of the mutations with those who did not. The median OS and RFS for patients with mutations were 13 and 9 months, respectively, compared to 41 and 16 months in patients without (p< 0.0001 and p= .003). Similarly, TCGA patients with mutations had significantly poorer OS (median, 9.8 vs 46.8 months, p= .0002) and RFS (median, 9.3 vs 17 months, p= .02). In multivariable analysis, mutations maintained an independent effect on survival (Table 1).Since mutations and HCT were the major predictors of outcome after CR1, we analyzed the outcomes of patients harboring mutations in relation to HCT (Figure 1). In both cohorts, patients who did not have the mutations and underwent HCT had the best outcomes. In the CC cohort, median OS and RFS for patients who had mutations and HCT were 14 and 11 months, respectively, compared to 7.5 and 5 months in patients who did not undergo HCT (p= .04 for OS and p= .002 for RFS). In the TCGA cohort, patients who harbored mutations and did not undergo HCT had significantly worse OS (median, 6.3 vs 24.6 months, p= .001) and RFS (median, 7.8 vs 12.5 months, p= .003). In view of the recently proposed ELN 2017 criteria that place IR patients with RUNX1 or ASXL1 mutations into the adverse risk, we explored outcomes of these patients at the time of CR1. Presence of these mutations did not predict OS and RFS in our cohort, and performance of HCT did not confer a significant survival benefit in patients with these mutations.Conclusions: In two independent cohorts, DNMT3A, U2AF1, and EZH2 mutations were predictors of relapse and OS in IR-AML at CR1, and performance of HCT translated into survival benefit. Our results provide evidence of clinical utility in considering mutation screening to stratify IR-AML patients to guide therapeutic decisions. [Display omitted] DisclosuresSekeres:Celgene: Membership on an entity's Board of Directors or advisory committees. Gerds:CTI BioPharma: Consultancy; Incyte: Consultancy. Maciejewski:Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Speaker Fees; Ra Pharma: Consultancy; Apellis Pharmaceuticals: Consultancy. Advani:Pfizer: Consultancy; Takeda/ Millenium: Research Funding.

Genetic Characterization and Clinical Impact of Loss of MHC Class I and II Expression in De Novo Diffuse Large B-Cell Lymphoma

Introduction: Immune recognition by T cells requires the presentation of processed peptides in association with major histocompatibility complex class I (MHC-I) and class II (MHC-II) molecules. Among the tumor immune escape mechanisms described to date, alterations in the expression of MHC molecules play a crucial role in the development of diffuse large B-cell lymphoma (DLBCL). Loss of MHC-I or -II expression in DLBCL can occur through multiple mechanisms, including deletion of the MHC-I and -II locus, loss of function mutations of B2M, and genetic alterations of CIITA ; however, the role of additional genetic and epigenetic mechanisms remains largely unknown. With regard to clinical significance, a number of studies have reported that low MHC-II expression on tumor cells is associated with shorter survival in DLBCL, while the prognostic effect of MHC-I expression has not been systematically evaluated in a large, homogeneously treated cohort. In addition, although the frequency of loss of MHC-II expression differs between ABC- and GCB-DLBCL cell of origin (COO) subtypes, the prognostic impact remains ill-defined. Here, we conducted a comprehensive phenotypic and genetic analysis in a population-based DLBCL cohort to investigate the genetic background and clinical relevance of loss of MHC-I and -II expression.Method: We analyzed biopsies from 347 patients newly diagnosed with de novo DLBCL and uniformly treated with R-CHOP at the BC Cancer Agency. High-resolution copy number analysis was performed using Affymetrix SNP 6.0 arrays. Mutation status was determined using deep targeted re-sequencing of the coding exons of 57 genes with a Truseq Custom Amplicon assay (Illumina) and/or Fluidigm Access Array chips, and RNAseq. Immunohistochemical staining of MHC-I and -II was performed on tissue microarrays (n=332). COO was assigned by gene expression (Lymph2Cx assay) in 323 cases (183 GCB, 104 ABC and 36 unclassifiable).Results: We found the following MHC-I and -II staining patterns:membrane-positive (n=188 (57%) and n=233 (72%), respectively), cytoplasmic only-positive / membrane-negative (n=86 (26%) and 31 (10%), respectively) and negative staining for both membrane and cytoplasmic (n=55 (17%) and n=59 (18%), respectively). Overall, membranous MHC-I and -II expression was not detected in 141/329 (43%) and 90/323 (28%) cases, respectively. While the frequency of loss of MHC-I expression was not different between COO subtypes, loss of MHC-II expression occurred more often in ABC-DLBCL (36% vs 22%, p=0.026). This is consistent with previous observations showing that MHC-II expression is reduced during plasmacytic differentiation and in ABC-DLBCL. Loss of MHC-I expression was associated with B2M mutation and deletion as well as with HLA-C deletions (all p <0.001, Fisher's exact test). Interestingly, mutations of EZH2 and GNA13 were detected more frequently in cases that had loss of MHC-I expression (p <0.001 and p=0.004, respectively). Moreover, mutations of CIITA and RFXAP (components of the MHC-II enhanceosome), and mutations of PRDM1 and TNFAIP3, ABC-DLBCL-specific mutations, were significantly enriched in MHC-II negative cases. Of note, mutations of B2M and EZH2 were significantly associated with loss of MHC-II expression, as well (p=0.001 and p=0.016, respectively). The associations between EZH2 mutation and MHC-I and -II expression were also observed when the analysis was restricted to GCB-DLBCL (both p <0.0001).With a median follow up of 6.5 years for living patients, the loss of MHC-I expression was not associated with survival in the entire DLBCL cohort, as well as within each COO subtype. Although there was also no significant survival difference according to the MHC-II expression status in the entire DLBCL cohort, loss of MHC-II expression was associated with an inferior 5y-time to progression (TTP, 59% vs 79%, p=0.007) in the GCB subtype. Interestingly, in the ABC subtype, loss of MHC-II expression was associated with a superior 5y-TTP (64% vs 40%, p=0.03).Conclusion: Our approach using next generation sequencing and high resolution SNP arrays uncovered the genetic background, as well as the clinical impact of loss of MHC-I and-II expression in a uniformly R-CHOP-treated large population-based cohort of DLBCL patients. Our results warrant further studies investigating a potential mechanism of EZH2 mutations regulating MHC-I and -II expression in DLBCL. DisclosuresSavage:Bristol-Myers Squibb: Honoraria; Seattle Genetics: Consultancy, Honoraria; Roche: Research Funding; Celgene: Consultancy; Merck: Honoraria. Sehn:Celgene: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Roche/Genentech: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria. Morin:Epizyme, Inc: Consultancy. Connors:Genentech: Research Funding; Bayer Healthcare: Research Funding; Lilly: Research Funding; Merck: Research Funding; Seattle Genetics: Research Funding; Amgen: Research Funding; Janssen: Research Funding; F Hoffmann-La Roche: Research Funding; NanoString Technologies: Research Funding; Bristol-Myers Squibb: Research Funding; Cephalon: Research Funding; Takeda: Research Funding; NanoString Technologies, Amgen, Bayer, BMS, Cephalon, Roche, Genentech, Janssen, Lilly, Merck, Seattle Genetics, Takeda,: Research Funding. Scott:Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; BCCA: Patents & Royalties: Patent describing molecular subtyping of DLBCL licensed to NanoString Technologies. Patent describing measurement of the proliferation signature in MCL.. Steidl:Affimed Therapeutics: Consultancy; Juno Therapeutics: Consultancy.

Cell of Origin and Genomic Profile of Primary Central Nervous System Lymphoma Determined Using the Nanostring Lst Assay and Ultra Deep Targeted Next Generation Sequencing

Introduction: Primary central nervous system lymphoma (PCNSL) is characterized by a consistently inferior outcome compared to systemic diffuse large B-cell lymphoma (DLBCL) with a median overall survival of 2-4 years. Therefore, PCNSL represents a significant clinical challenge with an urgent need for better biomarkers and novel therapeutic targets. The distinction of DLBCL into cell of origin (COO) categories based on patterns of gene expression reminiscent of germinal center B-cell (GC-) type, activated B-cell (ABC-) type, with a small number of “unclassified” cases, has profound prognostic and potential therapeutic implications with many novel therapies displaying differential efficacy between the GC and ABC groups. Therefore, the precise determination of COO is becoming increasingly important for the appropriate patient stratification for novel targeted therapies. The NanoString LST assay is a digital gene-expression based test for COO assignment, representing a more accurate means of COO determination compared to standard immunohistochemistry (IHC) based algorithms, and was recently successfully applied in nodal DLBCLs. In the present study, we tested the LST assay on a cohort of PCNSL patients and performed a complementary targeted genomic profiling on a subset of these patients.Methods: The COO of 77 PCNSL patients was determined with the NanoString LST assay using RNAs extracted from FFPE samples and compared with the results obtained by the Hans IHC algorithm. The genomic profiles of 62 PCNSL cases were determined using ultra-deep targeted next generation sequencing (NGS) of 13 recurrently mutated target genes, including CARD11, CCND3, CD79B, CSMD2, CSMD3, IRF4, C-MYC, MYD88, PAX5, PIM1, PRDM1, PTPRD and TP53 using the TruSeq Custom Amplicon approach (dual strand workflow) on a HiSeq4000 Instrument (Illumina) to an average depth of 30.000x. Only variants supported by more than 100 mutant reads with an allele frequency (VAF) of >5% were considered.Results: The LST assay resulted in the following subtype calls: 80.5% ABC, 12.9% GC and 6.5% unclassified (UC). Using the Hans IHC algorithm, 94.8% (73/77) of the PCNSL cases were classified as ABC subtype with 5.2% (4/77) representing the GC subtype. Interestingly, the COO classification obtained using the LST assay showed a different outcome in 16.9% (13/77) of the cases: Out of the 12 cases classified as ABC subtype with the IHC algorithm, 7 cases presented as GC subtype and 5 showed an UC profile using the LST assay. On the other hand, one case characterized as GC subtype with the IHC assay was classified as ABC when analyzed using the LST assay. As for the mutation analysis, we detected a total of 402 somatic mutations in the 62 PCNSL cases across the 13 genes analyzed with individual cases harboring mutations in 4 genes on average (range: 1-11). The most frequently mutated genes were MYD88 (76%), PIM1 (55%) and PRMD1 (50%). The mutation frequencies in the remaining genes were as follows: CD79B (40%), PTPRD (32%), MYC (31%), PAX-5 (31%), CARD11 (26%), TP53 (26%), IRF4 (23%), CCND3 (19%), CSMD2 (19%) and CSMD3 (18%). Analyzing the mutation profiles of the ABC and GC subtypes, we observed an enrichment of CD79B (47% vs 20%) and PIM1 (59% vs 20%) in cases with ABC subtype, with IRF4 mutations being present in ABC cases only (29% vs 0%). None of the other genes demonstrated a considerable difference between the GC and ABC subtypes with regards to the mutation frequency.Conclusions: We successfully applied, for the first time, the NanoString LST assay for COO determination using FFPE derived RNA samples from a cohort of 77 PCNSL patients. Interestingly, the LST assay revealed a lower proportion of patients with ABC subtype compared our IHC findings (80.5% vs 94.8%). This may reflect the genuine biology of these cases, as the NanoString LST assay represents the most reliable COO determination approach compared to the original gold standard GEP method. The frequent mutations revealed in components of different actionable pathways including the BCR pathway, cell cycle regulation and CNS development highlights the role of these variants in the pathogenesis of PCNSL. Considering the emergence of novel therapies with selective activity in the GC and ABC patient groups the precise assignment of COO and the complementary mutation analysis will most likely have an important role in guiding the appropriate patient management in PCNSL. DisclosuresStorhoff:NanoString Technologies: Employment, Equity Ownership. Ghali:NanoString Technoliges: Employment.

Germline ERBB2 Variants Associate with Myeloproliferative Neoplasms

Background: The myeloproliferative neoplasms (MPN) and myelodysplastic syndrome (MDS) are usually sporadic diseases, however, familial cases are well-described. Approximately 10% of MPN cases display familial clustering, and there is a 5-7 fold increased risk of developing an MPN among first degree relatives of MPN patients. While familial MDS appears to be less common, identification of multiple predisposition genes has led to the incorporation of this entity into the most recent WHO classification of myeloid neoplasms. Identification of predisposing germline mutations in the myeloid malignancies has led to a better understanding of the pathophysiology of these diseases and improved clinical care. However, many familial MDS and MPN cases exist in which the inherited genetic lesion is unknown. Our recent investigation of a multigenerational family with inherited MDS/AML marked by erythroid hyperplasia identified a missense variant in ERBB3 that co-segregated with the disease phenotype (1). In this study, we investigated a kindred with an autosomal dominant familial cancer syndrome characterized by both MPN and solid tumor malignancies.Methods: Whole exome sequencing was performed via the Baylor-Hopkins Center for Mendelian Genomics at Center for Inherited Disease Research at Johns Hopkins University. Saliva or peripheral blood samples were used as source DNA. Sequence analysis and variant calling/filtering was performed as previously described (1). Targeted sequencing of the ERBB genes was performed via MiSeq using an Illumina TruSeq Custom Amplicon (v1.5) designed to sequence the entire open reading frame of selected genes. Analysis was performed using Illumina BaseSpace TruSeq applications. Germline variants were identified via variant allele frequency and heterozygous, nonsynonymous variants with a minor allele frequency < 0.01 and a depth > 40x were prioritized.Results: The proband was diagnosed with a JAK2 V617F positive MPN at age 37. Subsequent personal cancer history included melanoma and renal carcinoma. Family history was significant for ovarian cancer and melanoma in her mother, MPN and melanoma in her maternal aunt, and bladder cancer in her maternal uncle (Figure 1A). Exome sequencing of the three family members affected with melanoma was performed in order to identify the predisposing inherited variant in this family. Sequence analysis produced 71 candidate rare, heterozygous, nonsynonymous single nucleotide variants (SNV), including one in the gene ERBB2 . Genotyping of 2 additional family members for the ERBB2 c.3182T>C;p.L1061P SNV established that it co-segregates with disease (Figure 1A). To further investigate the prevalence of rare germline ERBB2 and ERBB3 variants in both MPN and MDS, we performed targeted sequencing of these genes in two discovery cohorts: 1) 96 cases of MPN, of which 63 had a 1st or 2nd degree relative with a family history of MPN and 2) 19 cases of erythroid MDS/AML. Five out of 96 (5.2%) cases of MPN were found to have ERBB2 variants, while 3 of 96 (3.1%) had ERBB3 variants. Of the 19 erythroid MDS/AML cases, no ERBB2 variants were identified, while 1 had an ERBB3 variant. Further, one MPN case with a germline ERBB2 c.G3250T:p.D1084Y variant had a family history of breast cancer in her mother, who also carried this variant (Figure 1B). We analyzed a validation cohort of 630 cases of hematologic malignancies (approximately 20% MPN) sequenced as part of their clinical evaluation at the Sidney Kimmel Cancer Center. Similar to the findings in our discovery cohorts, rare germline ERBB2 variants were enriched in MPN, found in approximately 5% of MPN compared to only 1.8% of all other myeloid disorder diagnoses. In contrast, germline ERBB3 variants were present in 1.6% of MPN compared to 1.8% of the other myeloid disorders (Table).Conclusions: We have identified germline ERBB2 variants that co-segregate with disease in two kindreds with familial cancer syndromes manifesting as a mixed MPN and solid tumor phenotype. In addition, we show that germline ERBB2 and ERBB3 variants are present in patients with hematologic malignancies at a higher incidence than expected in the general population, and ERBB2 variants are associated with an MPN phenotype. Together, these data implicate these genes in the predisposition to myeloid malignancy as well as other familial cancer syndromes. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract PO-084: Genetic profiling of rat gliomas and cardiac schwannomas from cell phone radiofrequency radiation exposure using a targeted next-generation sequencing gene panel

Abstract The cancer hazard associated with exposure to cell phone radiofrequency radiation (RFR) was examined using lifetime exposure in Sprague-Dawley (SD) rats at the Ramazzini Institute (RI), Italy. There were increased incidences of gliomas in the brain and schwannomas in the heart. In order to understand the translational relevance of these rat tumors for human disease, we examined the top 23 orthologous cancer genes mutated in human gliomas using a custom built a next-generation sequencing gene panel for rats based on Illumina’s TruSeq Custom Amplicon Technology. SD rat tissues (gliomas =15; cardiac schwannomas=9, interim (1 year) sacrificed non-tumor brain tissues from RFR exposed rats =30, control brain and heart control tissues from lifetime exposure=10 each, and age-matched control brain tissues from interim sacrificed rats=10) from the RI-RFR cancer bioassay were examined in this study. The deepSNV R-package with various filtering criteria and the read depth of &amp;gt;1000x were used to identify single-nucleotide variants (SNVs) in the rat gliomas and schwannomas. At 5% allelic frequency, there were an average of 43 SNVs per rat glioma and point mutations were detected in 9 genes (Arid1a, Cic, Tert promoter, Tp53, Atrx, Nf1, Pdgfra, Pi3kr1, and Setd2) in at least 3 or more rat gliomas based on population frequency. Five genes (Nf1, Tert promoter, Setd2, Arid1a, and Pdgfra) harbored SNVs in interim sacrificed brain tissues that were also present in RFR-exposed gliomas from lifetime exposure. Interestingly, no mutations were detected in hotspot regions of Idh1, Idh2, Egfr or Braf. In contrast to most human gliomas which harbor mutations in IDH1 and IDH2 genes, the rat gliomas seem to be Idh1 wild type with mutations in the other glioma-related genes. Primary cardiac tumors are extremely rare in humans. With this targeted NGS panel (at allelic frequency of 2.5%), there were an average of 146 SNVs per rat cardiac schwannoma and unique point mutations were detected in Cic, Egfr, Arid1a, Nf1, Setd2, Cdkn2a, Erbb2, Atrx, Pdgfra, and Notch1 in 3 or more cardiac schwannomas. A subset of the SNVs (Arid1a, Tp53, and Nf1) in rat gliomas was confirmed in human gliomas in the COSMIC database supporting the translational relevance of the rat gliomas for human disease. In addition, several SNVs from rat gliomas and cardiac schwannomas were found in various human cancers including carcinomas, hematopoietic neoplasms, melanomas and neuroendocrine tumors. In conclusion, the rat gliomas appear to share genetic alterations with a subtype of IDH1 wildtype human gliomas and rat primary cardiac schwannomas also harbor mutations in some of the queried cancer genes. Citation Format: Ramesh C. Kovi, Andrea Vornoli, Ashley Brooks, Thai Vu T. Ton, Miaofei Xu, Eva Tibaldi, Federica Gnudi, Jian-Liang Li, Robert C. Sills, John R. Bucher, Fiorella Belpoggi, Arun R. Pandiri. Genetic profiling of rat gliomas and cardiac schwannomas from cell phone radiofrequency radiation exposure using a targeted next-generation sequencing gene panel [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-084.

Rare, Protein-Altering Variants in AS3MT and Arsenic Metabolism Efficiency: A Multi-Population Association Study.

Background:Common genetic variation in the arsenic methyltransferase (AS3MT) gene region is known to be associated with arsenic metabolism efficiency (AME), measured as the percentage of dimethylarsinic acid (DMA%) in the urine. Rare, protein-altering variants in AS3MT could have even larger effects on AME, but their contribution to AME has not been investigated.Objectives:We estimated the impact of rare, protein-coding variation in AS3MT on AME using a multi-population approach to facilitate the discovery of population-specific and shared causal rare variants.Methods:We generated targeted DNA sequencing data for the coding regions of AS3MT for three arsenic-exposed cohorts with existing data on arsenic species measured in urine: Health Effects of Arsenic Longitudinal Study (HEALS, ), Strong Heart Study (SHS, ), and New Hampshire Skin Cancer Study (NHSCS, ). We assessed the collective effects of rare (allele frequency ), protein-altering AS3MT variants on DMA%, using multiple approaches, including a test of the association between rare allele carrier status (yes/no) and DMA% using linear regression (adjusted for common variants in 10q24.32 region, age, sex, and population structure).Results:We identified 23 carriers of rare-protein-altering AS3MT variant across all cohorts (13 in HEALS and 5 in both SHS and NHSCS), including 6 carriers of predicted loss-of-function variants. DMA% was 6–10% lower in carriers compared with noncarriers in HEALS [ (95% CI: , )], SHS [ (95% CI: , )], and NHSCS [ (95% CI: , )]. In meta-analyses across cohorts, DMA% was 8.7% lower in carriers [ (95% CI: , )].Discussion:Rare, protein-altering variants in AS3MT were associated with lower mean DMA%, an indicator of reduced AME. Although a small percentage of the population (0.5–0.7%) carry these variants, they are associated with a 6–10% decrease in DMA% that is consistent across multiple ancestral and environmental backgrounds. https://doi.org/10.1289/EHP8152

Molecular Characterisation of Participants in the Phazar Trial Reveals Prognostic Impact of Mutations in Advanced-Phase-MPN

Molecular Characterisation of Participants in the Phazar Trial Reveals Prognostic Impact of Mutations in Advanced-Phase-MPN

Performance evaluation of an amplicon-based next-generation sequencing panel for BRCA1 and BRCA2 variant detection.

BackgroundAs next‐generation sequencing (NGS) technology matures, various amplicon‐based NGS tests for BRCA1/2 genotyping have been introduced. This study was designed to evaluate an NGS test using a newly released amplicon‐based panel, AmpliSeq for Illumina BRCA Panel (AmpliSeq panel), for detection of clinically significant BRCA variants, and to compare it to another amplicon‐based NGS test confirmed by Sanger sequencing.MethodsWe reviewed BRCA test results done by NGS using the TruSeq Custom Amplicon kit from patients suspected of hereditary breast/ovarian cancer syndrome (HBOC) in 2018. Of those, 96 residual samples with 100 clinically significant variants were included in this study using predefined criteria: 100 variants were distributed throughout the BRCA1 and BRCA2 genes. All target variants were confirmed by Sanger sequencing. Duplicate NGS testing of these samples was performed using the AmpliSeq panel, and the concordance of results from the two amplicon‐based NGS tests was assessed.ResultsNinety‐nine of 100 variants were detected in duplicate BRCA1/2 genotyping using the AmpliSeq panel (sensitivity, 99%; specificity, 100%). In the discordant case, one variant (BRCA1 c.3627dupA) was found only in repeat 1, but not in repeat 2. Automated nomenclature of all variants, except for two indel variants, was in consensus with Human Genome Variation Society nomenclature.ConclusionOur findings confirm that the analytic performance of the AmpliSeq panel is satisfactory, with high sensitivity and specificity.

Abstract 5742: Comparison of variants identified by next-generation sequencing panels: A 48-gene panel for myeloid neoplasms and a 35-gene panel for hematologic neoplasms

Abstract Background: Identification of genomic variants by molecular testing plays an important role in classification, risk stratification, and management of myeloid neoplasms. Next-generation sequencing (NGS) assays that target frequently mutated genes are commonly used in clinical settings. Owing to the complexity of NGS workflow, the performance requirements of each NGS assay must be validated for the intended use. However, the scarcity of optimal validation samples challenges efficient validation of NGS assays. We previously reported development and validation of a 48-gene NGS panel for molecular profiling of myeloid neoplasms including acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and myeloproliferative neoplasms (MPN). In this study, we compared the variant calls of a clinically validated 48-gene NGS panel for myeloid neoplasms to those of an independently developed and clinically validated 35-gene NGS panel for hematologic neoplasms. Method: The 48-gene NGS assay is a hybrid capture-based NGS assay using an Illumina NextSeq 500; it was developed to detect single nucleotide variants (SNVs), insertions/deletions (indels), and FLT3 internal tandem duplications (FLT3-ITD). The 35-gene NGS assay HemeSEQ (performed by med fusion, Lewisville, TX) uses Illumina TruSeq custom-amplicon library preparation chemistry, which is sequenced on an Illumina MiSeq. A total of 33,812 bp in 26 genes overlapped between the 2 assays; genes included ASXL1, CALR, FLT3, IDH1, IDH2, JAK2, NPM1, RUNX1, SF3B1, TET2, and TP53. For this study, 141 specimens harboring at least 1 pathogenic mutation within the overlapping genes were selected and sequenced by the 2 assays in CLIA-certified and CAP-accredited laboratories. Detected variants included those for which a targeted therapy or an experimental drug is available. Results: In total, 4.8 million individual base calls (33,812 bp X 141 specimens) were compared. A total of 1,094 variants (278 unique) were concordantly detected by both assays: 1,007 (219 unique) SNVs, and 87 (59 unique) indels; no calls were discordant. Thus, sensitivity was 100% (95% CI: 99.7-100) and specificity was 100% (95% CI: 99.9-100) for overlapping genes. In addition, agreement in variant allele frequency between the 2 assays was good (R2=0.986). The 48-gene assay detected an additional 36 pathogenic mutations from 26 specimens in 14 non-overlapping genes including PTPN11, ATM, BCOR, NF1, and STAG2. Conclusions: Of 4.8 million base calls compared, variant calls were 100% concordant between the 2 NGS assays, and variant allele frequency was in good agreement. The 48-gene assay detected an additional 36 pathogenic mutations in 14 myeloid neoplasm related genes. This study highlights the reliability of robustly validated NGS assays in detection of potentially actionable mutations in clinical settings. Citation Format: Sun Hee Rosenthal, Hansook Chong, Charles Ma, Daniel Sugganth, Renius Owen, Joseph Catanese, Christopher Elzinga, Andrew Grupe, Tracey Freitas, Jennifer Graham, Kristen Champion, Amanda Larsen, Frederick Racke, Felicitas Lacbawan. Comparison of variants identified by next-generation sequencing panels: A 48-gene panel for myeloid neoplasms and a 35-gene panel for hematologic neoplasms [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 5742.

Targeted Informatics for Optimal Detection, Characterization, and Quantification of FLT3 Internal Tandem Duplications Across Multiple Next-Generation Sequencing Platforms

Assessment of internal tandem duplications in FLT3 (FLT3-ITDs) and their allelic ratio (AR) is recommended by clinical guidelines for diagnostic workup of acute myeloid leukemia and traditionally performed through capillary electrophoresis (CE). Although significant progress has been made integrating FLT3-ITD detection within contemporary next-generation sequencing (NGS) panels, AR estimation is not routinely part of clinical NGS practice because of inherent biases and challenges. In this study, data from multiple NGS platforms—anchored multiplex PCR (AMP), amplicon [TruSeq Custom Amplicon (TSCA)], and hybrid-capture—were analyzed through a custom algorithm, including platform-specific measures of AR. Sensitivity and specificity of NGS for FLT3-ITD status relative to CE were 100% (42/42) and 99.4% (1076/1083), respectively, by AMP on an unselected cohort and 98.1% (53/54) and 100% (48/48), respectively, by TSCA on a selected cohort. Primer analysis identified criteria for ITDs to escape detection by TSCA, estimated to occur in approximately 9% of unselected ITDs. Allelic fractions under AMP or TSCA were highly correlated to CE, with linear regression slopes near 1 for ITDs not duplicating primers, and systematically underestimated for ITDs duplicating a primer. Bias was alleviated in AMP through simple adjustments. This article provides an approach for targeted computational FLT3-ITD analysis for NGS data from multiple platforms; AMP was found capable of near perfect sensitivity and specificity with relatively accurate estimates of ARs.

No association to sudden infant death syndrome detected by targeted amplicon sequencing of 24 genes.

The aim was to identify genetic variants associated with sudden infant death syndrome (SIDS) that can cause disease or introduce vulnerability. Genes reported in a previous SIDS study to have altered messenger ribonucleic acid (mRNA) expression in SIDS were investigated. Samples from 81 SIDS (56 male/28 female) with a median age of 4months (range 0.75-9months) were analysed using Illumina TruSeq custom amplicon for 24 selected genes. Samples were collected from autopsy at Oslo university hospital from children whom died suddenly and unexpectedly from 1988 to 2006. The controls were the germline variation database, Norgene (no description of cases available). After filtering for rare variants, there were a total of 38 variants in the 81 SIDS cases and 462 variants in the 789 controls. After the filtration and curation steps, we found 36 rare variants. The overall occurrence of rare variants for all the SIDS samples was lower than for the Norgene population. There was no association between rare variants in the included genes and SIDS. Although not statistically significant, two of the SIDS cases had a rare variant in the MyD88 gene: rs746651350, rs200424253.

SAT-069 Advantages of Next Generation Sequencing (NGS) in Hypophosphatemic Disorders Diagnosis. First Case of SLC9A3R1 Gene Pathogenic Variant Detected in a Pediatric Patient

Background: Hereditary hypophosphatemic rickets (HHR) is a group of inherited disorders characterized by hypophosphatemia due to renal-phosphate wasting and impairment of vitamin D metabolism, rickets and disproportioned short stature. Different genetic defects are known to cause HHR, but similar clinical and biochemical features were reported. Dominant-X-linked HR (XLHR) is the most frequent form, with an incidence of 1 in 20.000, although dominant and recessive autosomal forms are also described. XLHR is caused by inactivating mutations in the PHEX gene (located at Xp22.1), encoding a endopeptidase which regulates the phosphaturic secretion. Affected individuals present with a broad phenotypic spectrum, ranging from isolated hypophosphatemia up to severe symptoms of rickets. Therefore NGS studies represent an useful tool for molecular diagnosis characterization Aim: to develop a reliable NGS diagnostic tool for HHR and related disorders. Patients and Methods: we develop a NGS panel including 13 genes related with HHR or other hypophosphatemic disorders, using Illumina TruSeq Custom Amplicon technology.We analyzed 12 patients which have been sent to our laboratory for molecular genetic testing under suspicion of HHR based on clinical phenotype and laboratory studies but with no proven mutation in PHEX gene by Sanger sequencing or MLPA analysis or other hypophosphatemic disorder. Results: A previously reported pathogenic variant (p.Arg153Gln) was found in SLC9A3R1 gene encoding NHERF1cotransporter, which interact with phosphate and sodium renal transporter NaPi2a in a 13 months old girl. There are only 5 reported cases with alterations in this gene and all of them were adult patients with nephrolithiasis. The patient was referred to our hospital due to hypercalcemia. She had poor weight gain and laboratory findings showed high serum calcium (16,6 mg/dl), mild serum phosphate (3.9 mg/dl), very low parathyroid hormone (PTH) (< 3 ng/ml), normal 25OHvit D (40 ng/ml) levels, and elevated urinary calcium/ creatinine rate (2), and low phosphate tubular reabsorption (85%). Ultrasound showed nephrolithiasis. Since she had hypophosphatemia and renal phosphate wasting with symptomatic severe PTH independent hypercalcemia probably secondary to excessive calcitriol production with hypercalciuria, a molecular alteration of CYP24A1 or SLC34A1genes was suspected. Conclusion: NGS allowed to report for the first time the identification of a mutation in the SLC9A3R1 gene in a pediatric patient. An early diagnosis might improve long term outcome starting the right therapy to avoid progression of nephrolithiasis and nephrocalcinosis and chronic renal failure.

Mutations in Myosin 5B in Children With Early-onset Cholestasis.

Mutations in Myosin 5B (MYO5B) are known to be associated with microvillous inclusion disease (MVID) a genetic cause of neonatal intractable diarrhoea. More recently, they have been reported in children with cholestasis but without typical gastrointestinal symptoms of MVID. We describe our series of children with cholestasis and mutations in MYO5B. Clinical, laboratory, and histological data were collected from patients with cholestasis and pathogenic mutations in MYO5B, found by next generation sequencing (NGS) but with minimal gastrointestinal disease. Six patients (3 boys) were identified. Median age at presentation was 19 months (range, 3-92). Presenting features were jaundice, pale stools, pruritus, and failure to thrive. Patients 5 and 6 had intractable diarrhoea until the age of 3 and 7 years, respectively, but currently are on full enteral diet with no intestinal symptoms. Median values for serum total bilirubin were 55 μmol/L (2-500), alanine aminotransferase 73I IU/L (32-114), γ-glutamyltransferase 7 IU/L (7-10), and serum bile acids 134 μmol/L (18-274). Three patients underwent 1 or more types of biliary diversion for symptom control. Median follow-up was 5 years (2-22). At most recent follow-up, they all reported pruritus while on antipruritics. Patient 1 had a liver transplant. We identified 6 patients, with mutations in MYO5B, early-onset cholestasis and pruritus, with variable response to biliary diversion without typical MVID.

Detection of BRCA1/2 large genomic rearrangement including BRCA1 promoter-region deletions using next-generation sequencing

BackgroundGermline mutations in BRCA1 and BRCA2 (BRCA1/2) have been conventionally analyzed by Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA). Nowadays, next-generation sequencing (NGS) is increasingly being used in clinical genetics. The aim of this study was to evaluate the performance of NGS BRCA1/2 assays by comparing them with the conventional method. Materials and methodsWe did BRCA1/2 NGS assays of 108 breast and/or ovarian cancer patients whose BRCA1/2 mutation had been previously analyzed by Sanger sequencing and MLPA using TruSeq Custom Amplicon Design AFP2. Single-nucleotide variations (SNVs) and small insertions or deletions (InDels) were evaluated. In addition, we analyzed large genomic rearrangements (LGRs) using a coverage-based algorithm as well as a revised BRCA1/2 NGS assay (BRCAaccuTest PLUS), which additionally covered a BRCA1 promoter region. ResultsThe NGS BRCA1/2 assay detected all 20 SNVs and 21 small InDels in 56 patients. Among seven LGRs detected by MLPA, six exonic LGRs were well identified by both NGS BRCA1/2 assays. One pathogenic LGR, located on a BRCA1 promoter region, was successfully identified using revised BRCAaccuTestPLUS. ConclusionsThese results indicated that an NGS BRCA1/2 assay could detect most LGRs including BRCA1 promoter-region deletion as well as SNVs and small InDels. Therefore, it was applicable to clinical BRCA1/2 mutation tests.