Genetic Mutation Analysis of Parkinson’s Disease Patients Using Multigene Next-Generation Sequencing Panels

Abstract Abstract 724 Background: Chromosome content identifies two pathogenic pathways, each occurring in about half of patients with MGUS and multiple myeloma (MM). Hyperdiploid MM (HRD) has 48–75 chromosomes with multiple trisomies selectively involving chromosomes 3, 5, 7, 9, 11, 15, 19 and 21; only 10% of these HRD tumors have primary IgH translocations and no frequent focal genetic mutations have been identified. In contrast primary IgH translocations are identified in over 70% of non-hyperdiploid MM (NHRD). Rearrangements of MYC have been detected by FISH in only 16% of untreated MM, but over 90% of MM cell lines, identifying a late role for MYC in the progression of MM. The introduction of a MYC transgene into a mouse strain predisposed to MGUS results in mice that uniformly develop MM, suggesting a distinct early role of MYC in the progression of MGUS to MM. We report here that rearrangements in a 4Mb region surrounding MYC are present in 70% of HRD, representing the most frequent focal genetic mutation in this genetic subtype of MM. Results: We analyzed the MMRC reference collection of gene expression (Affymetrix Hu133Plus2) and copy number (Agilent 244k CGH) data and performed FISH to identify MYC rearrangements with IgH or IgL loci in 218 patients with untreated and relapsed MM. We found MYC rearrangements in 48% of MM (identified only by FISH in 5%, only by aCGH in 33%, and by both FISH and aCGH in 10%), including 43% of untreated, and 51% of relapsed MM. Using a hyperdiploid index calculated from the median copy number of the chromosomes involved in trisomies we determined that rearrangements of MYC were present in 70% of the top third, 35% of the middle third, and 25% of the bottom third. Using the paired gene expression data we found that the expression of MYC was approximately two-fold higher in the samples with rearrangements compared to those without rearrangements (p<0.001) and about three-fold higher in MM tumors without rearrangements compared to MGUS (p<0001). Using paired RNA and DNA from the MMRC reference collection we determined in 22 informative patients that MYC rearrangements are associated with monoallelic expression of MYC (p<0.01), consistent with cis-dysregulation of MYC. Analysis of the various changes on aCGH, and fine mapping of the genetic architecture of the rearrangements using next generation sequencing identifies a promiscuous array of rearrangements that often result in the introduction of an enhancer within the MYC locus, resulting in its cis-dysregulation. Since they cannot be comprehensively identified by either CGH or FISH alone, more sensitive techniques, such as next generation sequencing approaches, will be required to comprehensively identify all MYC rearrangements in MM. Conclusions: Rearrangements of MYC are the most frequent focal genetic mutation in untreated MM and are particularly prevalent in hyperdiploid MM. While only one third involve an immunoglobulin locus, they all result in cis-dysregulated expression of MYC, and may be one mechanism responsible for the progression of MGUS to MM. Tumors lacking MYC rearrangements bi-allelically over-express MYC by a trans mechanism including potentially inactivating mutations of BLIMP1/PRDM1, or activating mutations of IRF4. We propose two largely non-overlapping pathogenic pathways in MM: HRD associated with frequent MYC rearrangements, and NHRD associated with frequent primary IgH translocations. The prevalence of MYC rearrangements increases with tumor progression, identifying a role for MYC both early and late in tumorigenesis. As therapies that have been reported to target MYC (e.g., IMiDs®, bortezomib, bromodomain inhibitors) are used in the clinic, it will be important to associate their effect with the presence or absence of MYC rearrangements. Disclosures: Bergsagel: Constellation Pharmaceuticals: Consultancy. Keats:Tgen: Employment.

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Mutation Analysis in 200 Cases of Newly Diagnosed Myelodysplastic Syndrome By Next Generation Sequencing: Association with Established Prognostic Variables and IPSS-R

Blood ◽

10.1182/blood.v126.23.1703.1703 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1703-1703

Author(s):

Kankana Ghosh ◽

Parsa Hodjat ◽

Priyanka Priyanka ◽

Beenu Thakral ◽

Keyur P. Patel ◽

...

Keyword(s):

Bone Marrow ◽

Next Generation Sequencing ◽

Myelodysplastic Syndrome ◽

Mutation Analysis ◽

Conflicts Of Interest ◽

International Prognostic Scoring System ◽

P Value ◽

Newly Diagnosed ◽

Next Generation ◽

Generation Sequencing

Abstract INTRODUCTION Myelodysplastic syndrome (MDS) is known to have numerous genomic aberrations that predict response to treatment and overall survival. We aimed to assess various mutations in newly diagnosed MDS cases by next generation sequencing (NGS) and their association with various well-established clinicopathologic parameters and the Revised International Prognostic Scoring System (IPSS-R). MATERIALS AND METHODS We performed molecular studies on DNA extracted from bone marrow aspirate specimens in 200 newly diagnosed treatment naïve MDS patients presenting at a single institution from 08/2013 to 03/2015 as part of routine clinical work up in a CLIA certified molecular diagnostics laboratory. Cases met criteria for MDS per WHO 2008 criteria. The entire coding sequences of 28 genes (ABL1, ASXL1, BRAF, DNMT3A, EGFR, EZH2, FLT3, GATA1, GATA2, HRAS, IDH1, IDH2, IKZF2, JAK2, KIT, KRAS, MDM2, MLL, MPL, MYD88, NOTCH1, NPM1, NRAS, PTPN11, RUNX1, TET2, TP53, WT1) were sequenced using a NGS-based custom-designed assay using TruSeq chemistry on Illumina MiSeq platform. FLT3 internal tandem duplications (ITD) and codon 835/836 point mutation were detected by PCR followed by capillary electrophoresis. CEBPA mutation analysis was performed by PCR followed by Sanger sequencing on 186 patients. RESULTS Median age was 67 years. Patients included 139 males (69.5%) and 61 females (30.5%). Hematologic parameters are as follows [median (range)]: Hb 9.6 g/dL (5-16.7), platelets 75 K/μ L (5-652), WBC: 2.8 K/μ L (0.4-20.8), ANC 1.3 K/μ L (0.0 -12.0), AMC 0.2 K/μ L (0.0-3). Bone marrow (BM) blasts [median (range)] were 4% (0-19). Of 192 patients with cytogenetic analysis performed, 65 (33.85%) had diploid karyotype, 53 (27.6%) had one, 21 (10.93%) had two, 13 (6.77%) had three, 40 (20.83%) had > three abnormalities. IPSS-R risk categorization of the 200 cases is as follows: very low (17 cases, 8.5%), low (46, 23%) intermediate (42, 21%), high (47, 23.5%), very high (48, 24%). Mutations identified by NGS are as detailed in Table 1. Of the 4 patients with FLT mutations detected, the breakdown is as follows: FLT3 ITD (3, 75%), FLT3 D835 (1, 25%), FLT3, ITD + D835 (0, 0%). CEBPA mutation was detected in 12 of 186 (6.45%) cases assessed. CEBPA was detected in 12 (6.45%). Sixty three (31.5%) cases had no mutations detected in the genes analyzed by NGS or PCR, 80 (40%) had mutations in one, 42 (21%) had mutations in two, 8 (4%) in three and 7 (3.5%) in > three genes. We found positive associations between mutated genes and various parameters as detailed in Table 2. No association was found between frequency of any particular mutation and the IPSS-R score. CONCLUSIONS: MDS is a heterogeneous group of myeloid neoplasms at the genetic level. Multiple genetic mutations in a large subset of cases likely indicate clonal evolution. A subset of mutations has significant association with well-established clinico-pathologic parameters like WBC and BM blast percentage. With longer follow-up, we could use this data to refine IPSS-R. Table 1. Number of cases % cases TP53 46 23 TET2 33 16.5 RUNX1 27 13.5 ASXL1 25 12.5 DNMT3A 17 8.5 EZH2 12 6 IDH2 8 4 IDH1 7 3.5 NRAS 7 3.5 JAK2 5 2.5 FLT3 4 2 PTPN11 3 1.5 EGFR 2 1 MPL 2 1 WT1 2 1 GATA2 1 0.5 KIT 1 0.5 KRAS 1 0.5 MYD88 1 0.5 NPM1 1 0.5 BRAF 1 0.5 Table 2. Mutated genes p value WBC ASXL1 <0.042 AEC TET2 <0.016 BM blast % RUNX1, CEBPA <0.008, p<0.02 BM myelocyte % TP53, TET2, RUNX1, DNMT3A <0.014, <0.014, <0.015, <0.038 AEC: absolute eosinophil count, BM: bone marrow Disclosures No relevant conflicts of interest to declare.

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