Amplicon-based Next-generation Sequencing Panel Research Articles

Identification of causative gene variants for patients with known monogenic diabetes using a targeted next-generation sequencing panel in a single-center study.

We aimed to verify the usefulness of targeted next-generation sequencing (NGS) technology for diagnosing monogenic diabetes in a single center. We designed an amplicon-based NGS panel targeting 34 genes associated with known monogenic diabetes and performed resequencing in 56 patients with autoantibody-negative diabetes mellitus diagnosed at < 50years who had not been highly obese. By bioinformatic analysis, we filtered significant variants based on allele frequency (< 0.005 in East Asians) and functional prediction. We estimated the pathogenicity of each variant upon considering the family history. Overall, 16 candidate causative variants were identified in 16 patients. Among them, two previously known heterozygous nonsynonymous single-nucleotide variants associated with monogenic diabetes were confirmed as causative variants: one each in the GCK and WFS1 genes. The former was found in two independent diabetes-affected families. Two novel putatively deleterious heterozygous variants were also assumed to be causative from the family history: one frameshift and one nonsynonymous single-nucleotide variant in the HNF4A gene. Twelve variants remained as candidates associated with the development of diabetes. Targeted NGS panel testing was useful to diagnose various forms of monogenic diabetes in combination with familial analysis, but additional ingenuity would be needed for practice. The online version contains supplementary material available at 10.1007/s13340-023-00669-3.

Systematic KIT Sequencing Identifies Unsuspected Systemic Mastocytosis with Associated Hematological Neoplasm

Background: Systemic mastocytosis (SM) can occur on its own or in association with non-mast cell disorders (SM with associated hematological neoplasm, SM-AHN). Because extensive bone marrow involvement by non-mast cell disease may obscure mast cells in routinely-stained biopsies, it is possible that SM-AHN is under-recognized. Given the high association between KIT D816V mutations and SM, we asked if systematic evaluation for somatic KIT mutations in patients with known or suspected hematologic malignancies could identify unsuspected cases of SM-AHN.Methods: We queried 5754 clinical next generation sequencing (NGS) samples from 4173 unique patients with known/suspected hematologic neoplasms evaluated at our institution between 1/1/15 and 6/30/17 using a custom, 95-gene, amplicon-based NGS panel (PMID 27339098). Exons 8-9, 11 and 17 of KIT were analyzed. In patients with a somatic KIT mutation but without known SM, marrows were assessed for SM with mast cell tryptase and CD25 immunohistochemistry (IHC).Results: We identified 37 somatic KIT mutations in 35 patients (Figure), including non-D816V (n=9) and D816V (n=26; 1 case with concurrent D816Y).Non-D816V KIT mutations occurred in 9 patients: 5 core-binding factor-rearranged acute myeloid leukemia (CBF-AML) [418_419insFF (n=1); 417_419TYDdelinsI (n=1); Y823C (n=1); D816Y (n=2; 1 with also V559D)], 1 AML with myelodysplasia-related changes (AML-MRC; 419delD), 1 acute promyelocytic leukemia (N822K), 1 chronic myelomonocytic leukemia (R634W), and 1 mast cell leukemia (D816Y). In addition to the patient with mast cell leukemia, 2 patients with CBF-AML had known SM [418_419insFF; D816Y and concurrent V559D]. IHC performed for this study on the remaining 6 cases did not reveal multifocal, dense aggregates of abnormal mast cells.Of the 26 cases with the D816V variant, 18 had an established diagnosis of SM [SM without AHN (n=6); SM with suspected AHN, based on marrow morphology and/or abnormal peripheral counts (n=5); SM-AHN where AHN = chronic myelomonocytic leukemia (n=6) or myelodysplastic syndrome (n=1)]. In the 3 remaining cases of CMML or MDS with KIT D816V (marked on Figure), SM was not clinically or pathologically suspected. IHC performed following identification of the KIT D816V mutation by NGS revealed clusters of spindled, CD25+ mast cells, consistent with SM-AHN. All 3 patients also had a serum tryptase >20 ng/mL and splenomegaly, although these parameters may be influenced by the AHN. In 1 patient, retrospective review of prior liver/lymph node biopsies revealed previously unrecognized aggregates of spindled, CD25+ mast cells accounting for unexplained fibrosis. The patient was started on Midostaurin following SM diagnosis with significant clinical response. The final 5 cases with the D816V variant were AML. This included 4 de novo or therapy-related AMLs in which the KIT mutation was not present at initial diagnosis. In 2 cases, the mutation appeared in peripheral blood at time of relapse after stem cell transplant (concurrent bone marrow biopsy not performed; pre-transplant marrows showed no evidence of SM by IHC). In the other 2 cases, there was no IHC evidence of SM in leukemic marrows bearing the KIT D816V mutation. The final AML was AML-MRC with known KIT mutation; increased, CD25+ mast cells were present at time of AML but were not spindled or clustered.None of the cases of isolated SM had additional driver mutations, whereas 15 of 16 cases of known or suspected SM-AHN exhibited driver mutations in genes that are recurrently mutated in myeloid neoplasms. In the SH-AHN cases, the KIT D816V variant allele fraction varied from subclonal to equivalent to levels of other driver mutations. A group of 30 cases of CMML (n=15) and MDS (n=15) without KIT mutations were used as controls. None of these cases showed IHC evidence of SM.Conclusion: We find that detection of the KIT D816V mutation in CMML or MDS is invariably associated with SM, even in cases in which SM is not clinically suspected. Our data strongly suggest that identification of the KIT D816V mutation in patients with a diagnosis of CMML or MDS should prompt additional testing for underlying SM given the potential prognostic and therapeutic implications. [Display omitted] DisclosuresAster:Eli Lilly and Company: Membership on an entity's Board of Directors or advisory committees. Steensma:Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Research Funding; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Incyte: Equity Ownership; Onconova: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy; Takeda: Consultancy; H3 Biosciences: Consultancy. Lindsley:Takeda Pharmaceuticals: Consultancy; Jazz Pharmaceuticals: Consultancy; MedImmune: Research Funding. DeAngelo:Celgene: Research Funding; ARIAD: Consultancy, Research Funding; Glycomimetics: Research Funding; Blueprint Medicines: Honoraria, Research Funding; Immunogen: Honoraria, Research Funding; BMS: Consultancy; Takeda Pharmaceuticals U.S.A., Inc.: Honoraria; Shire: Honoraria; Novartis Pharmaceuticals Corporation: Consultancy, Honoraria, Research Funding; Incyte: Consultancy, Honoraria; Amgen: Consultancy, Research Funding; Pfizer Inc.: Consultancy, Honoraria, Research Funding.

Activity of Venetoclax-Based Therapy in CMML and CMML with Blast Transformation

Activity of Venetoclax-Based Therapy in CMML and CMML with Blast Transformation

Next Generation Sequencing-Based Mutational Profile of Therapy-Related AML and De Novo AML: Results from a Single Pediatric Institution in Argentina

IntroductionTherapy-related acute myeloid leukemia (t-AML) and de novo AML (dnAML) are a group of aggressive diseases with poor outcome, in spite of recent advance in therapy. However, the alterations in different genes seems to be associated with this dismal prognosis. Recently, different groups have shown that t-AML in adult patients has a mutational profile distinct from dnAML. TP53 mutations were mostly described in t-AML. Next-generation sequencing (NGS) is a fast and accurate method used to demonstrate the presence of multiple alterations in several genes. The comparison of t-AML and dn-AML mutational profiles could be useful for improving our understanding of the molecular genetics events and in the outcome of each group as well.Objectives: 1-To describe the incidence of pathogenic variants in t-AML and dn-AML using a customized NGS panel, 2-to study correlations of these mutations with classical genetic features in each group of AMLs, 3-to compare the mutational profile of both diseases, and 4-to evaluate the prognostic impact of these mutations.Patients and MethodsA total of 136 available DNA samples from non-APL dn-AML and 11 t-AML pediatric patients were retrospectively analyzed. The samples were sequenced on a Miseq (Illumina) platform, using a customized amplicon-based NGS panel (Illumina) including 65 genes. A total of 172,360 bp of DNA target regions were covered with 1,207 amplicons with a mean coverage of 1,308. Sequences obtained were analyzed by in-house bioinformatics pipeline, with particular analysis for NPM1, CEBPA and FLT3 genes, using Pindel and ITDseek algorithms.ResultsAt least one pathogenic variant was detected in 100 out of 136 (74%) dn-AML analyzed cases, and 11 out of 11 (100%) t-AML patients. Overall, 317 mutations were found in 49 genes with a mean of 3.2 variants per patient for dn-AML, and 53 mutations in 26 genes with a mean of 4.8 variants per patient for t-AML. The most frequently mutated genes were: BCORL1 (5.4%, p=ns), FLT3 (5.0%, p=ns), KMT2A (5.0%, p=ns), BCOR (4.7%, p=0.0126), RB1 (4.4%, p=ns) for dn-AML, and KDM6A (15.1%, p<0.00001), BCOR (9.4%), CUX1 (9.4%, p=0.0020), BCORL1 (5.7%), FLT3 (5.7%) for t-AML. TP53 mutations were only detected in 5 dnAML cases. In the group of patients with dnAML, the distribution of classical genetic abnormalities was: KMT2A/11q23 (24%), RUNX1-RUNX1T1 (22%), Normal karyotype (19%), CBFB-MYH11 (9%). The most frequent mutations in normal karyotype were: FLT3 (38.1%, p=0.0019), CEBPA (27.3%, p=0.0001), NPM1 (28.6%, p<0.00001) and WT1 (23.8%, p=0.0001). In the group of t-AML, the genetic features were: KMT2A/11q23 (45%, p=ns) and Normal karyotype (9%, p=ns). CBFA2T2 mutation was statistically associated with the presence of RUNX1-RUNX1T1 in dn-AML (p=0.0002). No other significant association between variants and genetic groups was observed. Complete remission was achieved in 92% of cases in dn-AML and 72% in t-AML (median follow-up: 21 and 5 months respectively). Leukemia-free survival probability (LFSp) was 60(5)% [High-Risk: 55(6)%; Low-Risk: 71(10)%] for dn-AML and 13(11)% for t-AML (p=0.0008). In dn-AML group, LFSp(SE) of patients with at least one deleterious variant was 60(6)%, whereas those without any variant showed LFSp(SE): 58(10)(p=ns). In the normal karyotype group, the genotype NPM1/CEBPA-mutated plus FLT3/WT1-wild-type disclosed a LFSp(SE) of 86(13)% while remaining patients showed a LFSp(SE) of 42(14)% (p=ns).ConclusionsWe describe the incidence of mutations detected by NGS in a series of pediatric t-AML and dn-AML in our country. In spite of the limited number of patients, the mutation profile of t-AML seems to be different from the de novo counterpart. BCOR mutations were significantly associated with dn-AML cases, while KDM6A and CUX1 variants with t-AML patients. In contrast with adult reports, non TP53 mutations were observed in our group of t-AML patients. There was a significantly higher incidence of mutations in FLT3, CEBPA, NPM1 and WT1 within the normal karyotype group of dn-AML. The combined genotype NPM1/CEBPA-mutated plus FLT3/WT1-wild-type showed a trend to a better LFSp in dn-AML. The molecular genetic profile characterization of these heterogeneous diseases allows expansion of our understanding about the leukemogenic mechanisms involved. DisclosuresNo relevant conflicts of interest to declare.

Abstract 2962: Clinical relevance of circulating cell-free DNA using amplicon-based next-generation sequencing panel in colorectal cancer patients with liver metastasis

Abstract Background: Recent advances in the next-generation sequencing technique (NGS) for the detection of cell-free DNA (cfDNA) will be possible to provide the utility as diagnostic and prognostic biomarkers of cancer. We conducted the study of feasibility to detect plasma cfDNA from patients with metastatic colorectal cancer (mCRC) using NGS panel and investigated their relationship with clinicopathological factors. Methods: A total of 101 mCRC patients with liver metastasis, who had been treated with chemotherapy, were enrolled in this study from February to June in 2017. We investigated i) frequency of detectable mutations in plasma cfDNA, ii) concordance rate of RAS mutation between the DNA extracted from tissues and the plasma cfDNA, iii) relationship between the mutation allele frequencies (MAF) and clinicopathological factors including tumor location, metastatic site, number of metastatic organs, tumor marker (CEA and CA19-9), LDH level, D-dimer level and sum of the tumor diameter measured based on RECIST ver1.1 criteria. Sequencing of plasma cfDNA were performed using Ion Torrent™ Oncomine™ Colon cfDNA Assay. Results: Mutations in plasma cfDNA were detected in 87.1% (88/101) of patients. The frequencies of plasma cfDNA mutation at TP53, KRAS, APC, and PIK3CA were 68.3%, 38.6%, 23.7%, and 14.8%, respectively. RAS mutational concordance rate between DNA extracted from tumor and cfDNA was 76.2% (77/101). MAF were significantly associated with CEA (P&amp;lt;0.0001), CA19-9 (P=0.006), LDH (P&amp;lt;0.0001) and number of metastatic organs (P&amp;lt;0.0001). Patients with liver or lymph node metastasis had significantly higher MAF compared with those without metastases (Liver: P&amp;lt;0.0001, Lymph node: P=0.008). Positive correlations between CEA level, CA19-9 level, LDH level, tumor diameter and MAF were observed. (CEA: r=0.52, CA19-9: r=0.34, LDH: r=0.55, tumor diameter: r=0.57) Conclusions: Our results suggested that this cfDNA Assays could detect mutations at a high rate of mCRC patients undergoing chemotherapy and cfDNA analysis using NGS panel could be useful method of diagnostic biomarkers to monitor the change of RAS status and tumor burden. &amp;lt;!–EndFragment–&amp;gt; Citation Format: Hiroki Osumi, Eiji Shinozaki, Hitoshi Zembutsu, Yoshinori Takeda, Takeru Wakatsuki, Takashi Ichimura, Yumiko Ota, Izuma Nakayama, Mariko Ogura, Mitsukuni Suenaga, Daisuke Takahari, Keisho Chin, Akio Saiura, Kensei Yamaguchi. Clinical relevance of circulating cell-free DNA using amplicon-based next-generation sequencing panel in colorectal cancer patients with liver metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2962.

Clinical characteristics of non-small cell lung cancer harboring mutations in exon 20 of EGFR or HER2

Unlike common epidermal growth factor receptor gene (EGFR) mutations that confer sensitivity to tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC), mutations in exon 20 of either EGFR or the human EGFR2 gene (HER2) are associated with insensitivity to EGFR-TKIs, with treatment options for patients with such mutations being limited. Clinical characteristics, outcome of EGFR-TKI or nivolumab treatment, and the presence of coexisting mutations were reviewed for NSCLC patients with exon-20 mutations of EGFR or HER2 as detected by routine application of an amplicon-based next-generation sequencing panel. Between July 2013 and June 2017, 206 patients with pathologically confirmed lung cancer were screened for genetic alterations including HER2 and EGFR mutations. Ten patients harbored HER2 exon-20 insertions (one of whom also carried an exon-19 deletion of EGFR), and 12 patients harbored EGFR exon-20 mutations. Five of the 13 patients with EGFR mutations were treated with EGFR-TKIs, two of whom manifested a partial response, two stable disease, and one progressive disease. Among the seven patients treated with nivolumab, one patient manifested a partial response, three stable disease, and three progressive disease, with most (86%) of these patients discontinuing treatment as a result of disease progression within 4 months. The H1047R mutation of PIK3CA detected in one patient was the only actionable mutation coexisting with the exon-20 mutations of EGFR or HER2. Potentially actionable mutations thus rarely coexist with exon-20 mutations of EGFR or HER2, and EGFR-TKIs and nivolumab show limited efficacy in patients with such exon-20 mutations.

Diagnosis of monogenic liver diseases in childhood by next-generation sequencing.

Next-generation sequencing (NGS) has opened up novel diagnostic opportunities for children with unidentified, but suspected inherited diseases. We describe our single-center experience with NGS diagnostics in standard clinical scenarios in pediatric hepatology. We investigated 135 children with suspected inherited hepatopathies, where initially no causative pathogenic variant had been identified, with an amplicon-based NGS panel of 21 genes associated with acute and chronic hepatopathies. In 23 of these patients, we detected pathogenic or likely pathogenic variants in 10 different genes. We present 6 novel variants. A total of 14 of these patients presented with the characteristic phenotype of the related hepatopathy. Nine patients showed only few or atypical clinical symptoms or presented with additional signs. In another 13 out of 135 cases, we detected variants of unknown significance (VUS) in 9 different genes. Only 2 of these patients showed characteristic phenotypes conclusive with the detected variants, whereas 11 patients showed unspecific or atypical phenotypes. Our multi-gene panel is a fast and comprehensive tool to diagnose inherited pediatric hepatopathies. We also illustrate the challenge of dealing with genetic variants and highlight arising clinical questions, especially in patients with atypical phenotypes.

Concurrent somatic mutations in driver genes were significantly correlated with lymph node metastasis and pathological types in solid tumors.

To demonstrate the mutational profiles in solid tumors, we profiled 165 solid tumor samples, including 9 cancer types and 4 sample types, by using amplicon-based next-generation sequencing panel covering 48 highly mutated tumorigenesis-related genes that were deep sequenced at an average coverage of 2000×. Both tumor and sample types had significant effect on tumor genetic mutational profiles. Concurrent driver mutations were frequently detected in solid tumor, concentrating on both modes of action driver genes (activating or loss of function). Furthermore, in non-small cell lung cancer (NSCLC), concurrent driver mutations were also significantly correlated with the lymph node metastasis status and pathological types. Higher frequency of lymph node metastasis was observed in patients with NSCLC with concurrent mutations on at least two driver genes. In addition, patients with lung adenocarcinoma were more likely to harbor concurrent driver mutations than patients with lung squamous and large cell carcinoma. Multiple mutations in the epidermal growth factor receptor gene were more frequently detected in patients with refractory NSCLC compared to untreated naive ones. Therefore, concurrent multiple driver mutations, rather than a single genetic mutation, should be investigated extensively to probe novel genetic biomarkers with clinical benefits.

High-throughput detection of clinically targetable alterations using next-generation sequencing

Next-generation sequencing (NGS) has revolutionized the therapeutic care of patients by allowing high-throughput and parallel sequencing of large numbers of genes in a single run. However, most of available commercialized cancer panels target a large number of mutations that do not have direct therapeutic implications and that are not fully adapted to low quality formalin-fixed, paraffin-embedded (FFPE) samples. Here, we designed an amplicon-based NGS panel assay of 16 currently actionable genes according to the most recent recommendations of the French National Cancer Institute (NCI). We developed a panel of short amplicons (<150 bp) using dual-strand library preparation. The clinical validation of this panel was performed on well-characterized controls and 140 routine diagnostic samples, including highly degraded and cross-linked genomic DNA extracted from FFPE tumor samples. All mutations were detected with elevated inter-laboratory and inter-run reproducibility. Importantly, we could detect clinically actionable alterations in FFPE samples with variant allele frequencies as low as 1%. In addition, the overall molecular diagnosis rate was increased from 40.7% with conventional techniques to 59.2% with our NGS panel, including 41 novel actionable alterations normally not explored by conventional techniques. Taken together, we believe that this new actionable target panel represents a relevant, highly scalable and robust tool that is easy to implement and is fully adapted to daily clinical practice in hospital and academic laboratories.

Two-Year Experience of Performing a Next-Generation-Sequencing Based Panel Test in an Academic Medical Center and Its Clinical Impact

Abstract Our ability to interrogate a broad array of genetic alterations in myeloid neoplasm has increased significantly with the advance in next-generation sequencing (NGS). In addition to morphologic examination, flow cytometry and cytogenetics, NGS-based testing can add additional useful information to the diagnostic workup. With improved turnaround time, decreasing costs and an expanding knowledge of the therapeutic and prognostic significance of the detected variants, NGS-based panel testing has increasingly played a major role in the management of patients with myeloid neoplasm. Rapid Heme Panel (RHP) is a custom, 95-gene, amplicon-based NGS panel (PMID: 27339098) that was launched at the Center for Advanced Molecular Diagnostics (CAMD), Brigham and Women's Hospital and Dana-Farber Cancer Institute in August of 2014. RHP covers a total territory of ~200 KB, including hotspots of oncogenes and whole coding exons of tumor suppressor genes that are frequently mutated in myeloid and lymphoid malignancies. Single nucleotide variants, insertions/deletions up to 52-bp, and copy number variations are detected. In the two years following its launch, over 5,000 RHP was performed and reported with an average turnaround time of 7.2 days from time of receipt into the lab. The specimen failed rate is &lt;0.5% and sample repeat rate is &lt;0.2%. Over half of the specimens came from patients with a known myeloid disease: 25% with acute leukemia, 15% with a myelodysplastic syndrome (MDS), 10% with a myeloproliferative neoplasm (MPN) and a minor fraction each with a variety of other myeloid neoplasms such as paroxysmal nocturnal hemoglobinuria (PNH), aplastic anemia, systemic mastocytosis or chronic myeloid leukemia (CML). Twenty percent of the specimens came from patients with known lymphoid malignancies such as hairy cell leukemia, chronic lymphocytic leukemia, lymphoplasmacytic lymphoma, or splenic marginal zone lymphoma. The remainder 25-30% of the specimens came from patients with abnormal blood count (CBC) such as anemia, neutropenia, thrombocytopenia, leukocytosis, thrombocytosis and/or abnormal serum protein electrophoresis (SPEP) where a myeloid or a lymphoid neoplasm was suspected. Greater than 98% of the time, the test was ordered by a hematologist/oncologist. Among patients with a prior diagnosis, &gt;80% of them had at least one pathogenic alterations identified by RHP while about 30% of the patients with abnormal CBC or abnormal SPEP had positive findings. RHP results have been used to (1) provide eligibility for enrollment into clinical trials of targeted therapies; (2) monitor effect of therapy by quantifying variant allele fraction; (3) identify disease progression with detection of emergence of new variants; (4) evaluate post-transplant status by following allele fractions of pre-transplant pathogenic variants; (5) shorten the time and cost to diagnosis by establishing clonality and identification of disease-defining alterations. Disclosures No relevant conflicts of interest to declare.

Elucidation of Additional Mutations By Next-Generation Sequencing Is of Clinical Significance in Patients with Rare MPNs and MDS/MPN Overlap Syndromes

Introduction:Recently, next-generation sequencing (NGS) has revolutionized the molecular characterization and understanding of several hematologic entities, including myeloproliferative neoplasms (MPN) and myelodysplastic syndrome (MDS)/MPN overlap syndromes. Nevertheless, the frequency and clinical impact of the mutations detected by NGS, remain largely unclear, especially in rare MPN which were analyzed in this study.Methods:Thus, we established a novel amplicon-based NGS panel, comprising genes that are known to be recurrently mutated in MPN and/or MDS/MPN. Hot spot regions or all exons of the following 32 genes were chosen: ABL, ASXL1, BARD, CALR, CBL, CEBPA, CHEK2, CSF3R, DNMT3A, ETNK1, ETV6, EZH2, IDH1, IDH2, JAK2, KIT, KRAS, MPL, NFE2, NRAS, PDGFRA, PTPN11, RUNX1, SETBP1, SF3A1, SF3B1, SH3B2 (LNK), SRSF2, TCF12, TET2, TP53, U2AF1. After establishing this panel, peripheral blood samples of 19 patients, which were diagnosed with CMML(10), aCML(2), MPNu(1), MDS/MPNu or other MPN(6), were analyzed on a MiSeq® illumina sequencer. Variants were only analyzed if the absolute coverage at each SNV site was >50 reads, and the absolute coverage of the mutant allele was 10 or more reads and its relative coverage exceeded 10%.Results:Mean coverage of the run was 1516 reads with good Phred-score quality parameters (>84% of called bases with Q-score >= 30). In 300 bidirectional cycles, a yield of nearly seven gigabases of sequencing data was reached. One out of 19 analyzed patients was excluded from analysis due to insufficient DNA quality. In 89% of the samples(16/18), mutations were detected which had not previously been known to be present in these patients. TET2 (50%, 9/18) and SETBP1 mutations were the most common (44%, 8/18). As expected, TET2 mutations were spread over the entire gene and SETBP1 mutations were restricted to the known hot spot region (exon 4, c.2602-c.2620). Additionally, CSF3R mutations were detected in 22% (4/18) of patients. Epigenetic regulator genes were also affected as EZH2 mutations were detected in 17% (3/18), ASXL1 mutations in 39% (7/18) and IDH1/2 mutations were found in 6% (1/18) of all samples, whereas DNMT3A mutations were not present. Further mutations were found in the following genes: CBL (11%), ETV6 (6%), JAK2V617F (6%), KRAS (11%), NRAS (11%), PTPN11 (6%), SH2B3 (6%) and SRSF2 (11%). Besides previously known mutations, several novel variants could be detected. All but one patient harbored more than one of these mutations.Furthermore, clinical correlates and morphologic and cytogenetic subtypes of each patient were available to associate with the NGS data of individual patients. For example, the one patient with a solitary NRAS c.35G>A (amino acid: p.G12D) mutation showed the most aggressive clinical course in our cohort with transformation to AML only 7 months after first diagnosis of CMML. Moreover, CSF3R mutations have been shown to confer sensitivity to ruxolitinib and may thus open up new avenues of treatment for our patients.Conclusion:In a cohort of unclassified MPN and rare MDS/MPN subtypes, NGS is a powerful tool to characterize samples more extensively. Our data suggests that a more comprehensive understanding of the mutational spectrum may have important clinical impact in individual patients, including diagnosis, prognosis, and more specific treatment. DisclosuresIsfort:Pfizer: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Other: Travel; Ariad: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Other: Travel; BMS: Honoraria; Mundipharma: Other: Travel; Amgen: Other: Travel; Hexal: Other: Travel. Brümmendorf:Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Ariad: Consultancy, Honoraria; Patent on the use of imatinib and hypusination inhibitors: Patents & Royalties. Koschmieder:Novartis: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding.

Evaluation of an amplicon-based next-generation sequencing panel for detection of BRCA1 and BRCA2 genetic variants.

The recent advances in the next-generation sequencing (NGS) technology have enabled fast, accurate, and cost-effective genetic testing. Here, we evaluated the performance of a targeted NGS panel for BRCA1/2 sequencing and confirmed its applicability in routine clinical diagnostics. We tested samples from 88 patients using the TruSeq custom panel (Illumina Inc, USA) and a MiSeq sequencer (Illumina) and compared the results to the outcomes of conventional Sanger sequencing. All 1015 sequence variations identified by Sanger sequencing were detected by NGS, except for one missense variant that might have been missed due to a rare mutation on a primer-binding site. One deletion variation, c.1909+12delT of BRCA2, was falsely called in all samples due to a homopolymer error. In addition, seven different single-nucleotide substitutions with low variant frequencies (range: 16.2-33.3%) were falsely called by NGS. In a separate batch, 10 different false-positive variations were found in five samples. The overall sensitivity and positive predictive value of NGS were estimated to be 99.9 and 87.5%, respectively. The false-positive results could be excluded by setting quality and alternative allele ratio filters and/or by visual inspection using the IGV software. Targeted NGS panel for BRCA1 and BRCA2 showed an excellent agreement with Sanger sequencing results. We therefore conclude that this NGS panel can be used for routine diagnostic method in a clinical genetic laboratory.

Coverage analysis in a targeted amplicon-based next-generation sequencing panel for myeloid neoplasms

AimsPCR amplicon-based next-generation sequencing (NGS) panels are increasingly used for clinical diagnostic assays. Amplification bias is a well-known limitation of PCR amplicon-based approaches. We sought to characterise lower-performance amplicons in...