High-Throughput Sequencing to Identify Cytogenetic and Molecular Genetic Aberrations in 24 AML Exomes

Abstract Introduction: In acute myeloid leukemia (AML), the karyotype and the molecular mutation profile are the strongest parameters for classification and prognostication. Yet, diagnostic analyses rely on chromosome analysis and sequencing of a constantly growing number of genes. Aim: To evaluate whether whole exome sequencing (WES) can reliably identify copy number states and molecular mutations in a single-step procedure. Patients and Methods: The cohort included 24 AML with an aberrant karyotype at initial diagnosis (ID) who achieved cytogenetic remission (CR) after chemotherapy. Patients showed complex karyotype (n=6), 11q23/MLL-rearrangement (n=4), t(15;17)(q24;q21) (n=4), inv(16)(p13q22) (n=4), t(8;21)(q22;q22) (n=3), and 3q26/EVI1-rearrangement (n=3). For WES DNA was extracted from bone marrow and treated with the TruSeq Exome enrichment kit targeting 201,071 exons. 2x100 bp paired-end sequencing was performed on an Illumina HiSeq machine (Illumina, San Diego, CA) at Fasteris (Geneva, Switzerland). After mapping the sequenced reads with Burrows-Wheeler Aligner [Li&Durbin, Bioinformatics, 2009], variants where called with GATK [McKenna et al., Genome Res., 2010] and copy number variations (CNV) were detected by Excavator [Magi et al., 2013, Genome Biol.]. For validation of the detected variants, 21 leukemia related genes were screened by amplicon sequencing (Illumina MiSeq, or Roche 454, Branford, CT). Array-based comparative genomic hybridization (aCGH) using 12x270K microarrays (Roche NimbleGen, Madison, WI) or 4 x 180K microarray slides (Agilent Technologies, Santa Clara, CA) was performed on all samples. We called CNV using default settings as well as fixed thresholds on the probe medians (0.3 for gains and -0.5 for losses on probe medians and at least 10 probes per segment). Results: The targeted regions were covered by 86 reads on average, while 90% of the bases were covered by at least 15 reads. By comparing ID and CR we detected an average of 15 somatic single nucleotide variants and short indels per patient (range 4-25), affecting 303 genes in total, including genes involved in leukemogenesis. After excluding polymorphisms we screened the mutated genes for recurrence among all cases. Four genes were mutated in at least 3 samples: WT1 (n=5), TP53 (n=4), NRAS (n=3) and TNS1 (n=3). Fourteen genes were mutated in 2 samples: ASXL2, DSCAM, GATA2, IDH2, KIT, OR4C5, POU4F1, LOC93432, RPTOR, SMC1A, SYNE2, TET2, TTN and USP9X. Mutations in OR4C5, LOC93432, SYNE2, TTN and USP9X have not been associated with AML yet. They were rated as damaging according to the SIFT algorithm [Ng and Henikoff, Genome Res., 2003]. In a prior diagnostic work-up 21 different genes had been screened and revealed 16 mutations affecting 7 genes. WES identified 14 mutations correctly (the 2 remaining mutations were covered by reads only insufficiently) and did not call any mutation in genes classified as negative in the routine diagnostic work-up. We further compared CNV derived from WES and aCGH in all 24 patients. Gains and losses detected by aCGH involved 2.65 and 1.40 billion bp, respectively. 96% of bp involved in these CNV were also detected by WES. Of the regions in which WES could not reproduce CNV calls, 15% did not contain exons. WES called gains and losses covering in total 2.56 and 1.47 billion bp, respectively. With aCGH we detected 98% of the gains and 86% of the losses. Regions missed by aCGH did show concordant signal that did not pass the fixed thresholds. However, while relaxing the thresholds to default settings, aCGH reproduces 99% of the WES results. Thus, an excellent concordance was observed (R = 0.99, p < 2.2e-16). We further analysed 19 cytogenetically balanced rearrangements that caused 42 breakpoints in affected chromosomes in 17 patients. As most breakpoints occur in non-coding regions, WES in general is limited in detecting these balanced rearrangements. However, short CNV were detected by WES in 10 cases and confirmed by aCGH. Conclusion: WES was capable of delineating molecular mutation profiles and of robustly detecting copy number states in AML at diagnosis. We suggest that WES in combination with multiplex RT-PCR-based techniques for the detection of recurrent fusion transcripts is a promising approach for a future diagnostic work-up for AML classification and prognostication. This project has been funded by the Seventh Framework Programme (FP7/2007-2013) under grant agreement n. 306242. Disclosures Althammer: MLL Munich Leukemia Laboratory: Employment; Seventh Framework Programme (FP7/2007-2013): Research Funding. de Albuquerque:MLL Munich Leukemia Laboratory: Employment; Seventh Framework Programme (FP7/2007-2013): Research Funding. Nadarajah:MLL Munich Leukemia Laboratory: Employment; Seventh Framework Programme (FP7/2007-2013): Research Funding. Meggendorfer:MLL Munich Leukemia Laboratory: Employment; Seventh Framework Programme (FP7/2007-2013): Research Funding. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership; Seventh Framework Programme (FP7/2007-2013): Research Funding. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership; Seventh Framework Programme (FP7/2007-2013): Research Funding. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership; Seventh Framework Programme (FP7/2007-2013): Research Funding. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership; Seventh Framework Programme (FP7/2007-2013): Research Funding.

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In Myelodysplastic/Myeloproliferative Neoplasms Aberrant Antigen Expression As Assessed by Multiparameter Flow Cytometry Is Related to Molecular Genetic Mutations

Blood ◽

10.1182/blood.v118.21.5152.5152 ◽

2011 ◽

Vol 118 (21) ◽

pp. 5152-5152

Author(s):

Wolfgang Kern ◽

Susanne Schnittger ◽

Tamara Alpermann ◽

Claudia Haferlach ◽

Torsten Haferlach

Keyword(s):

Myeloproliferative Neoplasms ◽

Molecular Genetic ◽

Antigen Expression ◽

Multiparameter Flow Cytometry ◽

Employment Equity ◽

Aberrant Expression ◽

Equity Ownership ◽

Diagnostic Work ◽

Work Up ◽

Diagnostic Work Up

Abstract Abstract 5152 Background: Immunophenotyping by multiparameter flow cytometry (MFC) is increasingly used in the diagnostic work-up of patients with cytopenias and suspected myelodysplastic syndromes (MDS). Myelodysplastic/myeloproliferative neoplasms (MDS/MPN) comprise a group of diseases with some features of MDS and is separately classified in the current WHO system. While the immunophenotype of chronic myelomonocytic leukemia has been described in detail, data is scarce on the use of MFC in myelodysplastic/myeloproliferative neoplasms, unclassifiable (MDS/MPNu) as well as on refractory anemia with ring sideroblasts and thrombocytosis (RARS-T), which is a provisional entity in the current WHO classification. Aim: To assess patients with MDS/MPNu and RARS-T for MDS-related aberrant immunophenotypes in the context of a comprehensive diagnostic work-up including cytomorphology, cytogenetics, and molecular genetics. Patients and Methods: A total of 91 patients were analyzed in parallel by cytomorphology, cytogenetics, and MFC applying an antibody panel designed to diagnose MDS. MFC was used to detect expression of mature antigens in myeloid progenitors; abnormal CD13-CD16- and CD11b-CD16-expression patterns, aberrant expression of myeloid markers and reduced side scatter signal in granulocytes; reduced expression of myelomonocytic markers in monocytes; aberrant expression of CD71 in erythroid cells; as well as expression of lymphoid markers in all myeloid cell lines. In 77/91 patients molecular genetic markers were investigated. The median age of the patients was 75.1 years (range, 35.3–87.4). The male/female ratio was 60/31. Six patients had RARS-T and 85 had MDS/MPNu. Results: In 54/91 (59.3%) patients MFC identified an MDS-immunophenotype. This was true in 4/6 (66.7%) RARS-T and in 50/85 (58.8%) MDS/MPNu (n.s.). Cases with MDS-immunophenotype displayed aberrancies significantly more frequently than those without as follows: in myeloid progenitor cells (number of aberrantly expressed antigens, mean±SD: 0.5±0.6 vs. 0.2±0.4, p=0.002), granulocytes (2.7±1.3 vs. 1.2±1.1, p<0.001), and monocytes (1.7±1.2 vs. 0.5±0.7, p<0.001). Accordingly, there was a significant difference in the total number of aberrantly expressed antigens (4.9±2.4 vs. 2.0±1.4, p<0.001). The presence of an aberrant karyotype was not related to an MDS-immunophenotype which was observed in 11/18 (61.1%) cases with aberrant karyotype and in 43/73 (58.9%) with normal karyotype (n.s.). Mutations in RUNX1 and TET2 as well as FLT3-ITD were predominantly present in cases with an MDS-immunophenotype (10/33, 30.3%) and occurred less frequently in cases without (1/7, 9.1%, n.s.). In detail, RUNX1 mutations were present in 4/26 (10.3%) vs. 0/2, TET2 mutations were present in 4/6 (66.7%) vs. 1/2 (50%), and FLT3-ITD was present in 3/29 (10.3%) vs. 0/5. Accordingly, in cases with RUNX1 or TET2 mutations or with FLT3-ITD a significantly higher number of aberrantly expressed antigens was observed as compared to cases with none of these mutations (mean±SD, 6.4±2.0 vs. 4.4±2.5, p=0.024). In contrast, JAK2V617F mutations occurred at identical frequencies in patients with and without MDS-immunophenotype (11/38, 28.9% vs. 9/31, 29.0%). Regarding prognosis, the presence of an MDS-immunophenotype had no impact on overall survival. Conclusions: These data demonstrates that MDS-related aberrant antigen expression is present in the majority of patients with RARS-T and MDS/MPNu. While there is no association between the presence of an MDS-immunophenotype and the detection of JAK2 mutations cases with an MDS-immunophenotype tended to more frequently carry mutations in RUNX1 and TET2 as well as FLT3-ITDs. These data therefore suggests that MDS/MPNu may be subdivided based on molecular genetics and on the immunophenotype into cases with MDS-related features and those without. Further analyses are needed to validate these findings and their potential significance in RARS-T. Disclosures: Kern: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Alpermann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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Use Of Flow Cytometry To Identify Myelodysplasia In Peripheral Blood

Blood ◽

10.1182/blood.v122.21.2774.2774 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2774-2774

Author(s):

Wolfgang Kern ◽

Richard Schabath ◽

Tamara Alpermann ◽

Claudia Haferlach ◽

Susanne Schnittger ◽

...

Keyword(s):

Flow Cytometry ◽

Peripheral Blood ◽

Antigen Expression ◽

Employment Equity ◽

Aberrant Expression ◽

Equity Ownership ◽

Diagnostic Work ◽

Work Up ◽

Diagnostic Work Up

Abstract Background Flow cytometry (FC) is increasingly used in diagnostic work-up of bone marrow (BM) from patients with suspected or proven myelodysplastic syndrome (MDS). Data on FC in peripheral blood (PB) is scarce. Aims Evaluate the use of FC for PB in suspected or proven MDS by comparison to BM analyzed during follow-up. Methods PB of 157 patients (pts) with suspected MDS was analyzed by FC applying ELN criteria defined recently for diagnosis of MDS in BM (Westers et al., Leukemia 2012). For all pts during follow-up at least one BM sample was evaluable by morphology, cytogenetics, and FC in parallel to confirm or exclude MDS (according to WHO 2008 criteria). Pts were then grouped according to results obtained from BM analysis during follow-up time points into 1) proven MDS (n=96), 2) no MDS (n=32), and 3) MPN, MDS/MPN, or “MDS possible” (presence of dysplastic features by morphology but not sufficient to diagnose MDS) (n=29) (median time to MDS confirmation, 0.9 months, range, 0.1-53.0; median time to last BM assessment without confirmation of MDS; 0.8 months, range, 0.2-23.0). Results First, results of FC on PB were compared between pts with finally proven MDS (n=96) by BM vs. those with no MDS by BM as diagnosed during follow-up. All 34 pts with myeloid progenitor cells (MPC) by FC in PB had finally proven MDS. However, in addition 62/94 (66.0%) of those without MPC (p<0.0001) also had proven MDS. Thus, the presence of MPC in PB was at least strongly indicative of MDS while there were also cases with MDS without MPC in PB. Moreover, besides the presence of MPC in PB, 17 of these 34 cases in addition displayed an aberrant antigen expression on MPC. Focusing on granulocytes we first analyzed side-scatter (SSC) signals in granulocytes as ratio of mean SSC signals granulocytes/lymphocytes (G/L). While for BM samples a reduced SSC ratio G/L had been described which reflects hypogranulation, we indeed found similar data for PB with a significantly lower SSC ratio G/L in pts with proven MDS as compared to those without (mean±SD 5.7±1.1 vs. 6.3±1.0, p=0.015). More strict, a mean SSC ratio G/L of 3.9 was found to most specifically identify pts with MDS: all 6 cases with a ratio <3.9 had MDS. Regarding aberrant antigen expression in granulocytes, MDS was more frequently diagnosed among cases with vs. without the following features: aberrant CD11b/CD16 expression pattern (43/46 investigated, 93.5% vs. 53/82, 64.6%; p=0.0002), lack of CD10 expression (37/43, 86.0% vs. 59/85, 69.4%; p=0.052), CD56 expression (19/21, 90.5% vs. 77/107, 72.0%; p=0.098). Cumulating this data, ≥2 aberrantly expressed antigens on granulocytes were found indicative of MDS: 42/45 (93.3%) of pts with aberrant expression of ≥2 antigens had MDS while only 54/83 (65.1%) of those with 0 or 1 aberrantly expressed antigen had finally proven MDS (p=0.0003). Regarding aberrant antigen expression in monocytes, pts with the following features more frequently had MDS as compared to those without: reduced expression of HLA-DR, CD13, CD11b, or CD15, aberrant expression of CD2 or CD34 (as single makers all n.s.). However, cumulating this data also resulted in a significant relation to a diagnosis of MDS during follow-up: 31/36 (86.1%) of pts with aberrant expression of ≥2 antigens on monocytes were diagnosed MDS vs. 65/92 (70.7%) of those without (p=0.052). Integrating the data for the different cell compartments, pts were separated according to the presence of the following 4 criteria: 1) presence of MPC in PB by FC, 2) aberrant expression of ≥1 antigen in MPC in PB, 3) aberrant expression of ≥2 antigens in granulocytes in PB, and 4) aberrant expression of ≥2 antigens in monocytes in PB: 68/76 (89.5%) of pts with ≥1 of these criteria had MDS, which was the case in 28/52 (53.8%) of cases fulfilling none of these criteria (p<0.0001). Strengthening the selection to presence of ≥2 of the criteria, all such 36 cases had MDS which was true for 60/92 (65.2%) of those with ≤1 criterion (p<0.0001). Applying these criteria to the set of remaining 29 pts with MPN, MDS/MPN, or possible MDS, 17 (58.6%) of them fulfilled ≥1 criterion which was true for 8/32 (25.0%) of pts not diagnosed MDS (p=0.010). Conclusions FC reveals MDS-related findings in PB samples using a specific panel targeting 10 antigens and may be used to identify pts with a high probability of MDS. Further studies with direct comparison of PB and BM should clarify the role of PB analysis by FC in the diagnostic work-up of pts with suspected MDS. Disclosures: Kern: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schabath:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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ETV6 Rearrangements Are Recurrent Genetic Events In Myeloid Malignancies and In AML Are Associated with NPM1- and RUNX1-Mutations and An Intermediate Prognosis

Blood ◽

10.1182/blood.v116.21.2758.2758 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2758-2758

Author(s):

Claudia Haferlach ◽

Susanne Schnittger ◽

Wolfgang Kern ◽

Torsten Haferlach

Keyword(s):

De Novo ◽

Normal Karyotype ◽

Employment Equity ◽

Childhood All ◽

Myeloid Malignancies ◽

Equity Ownership ◽

De Novo Aml ◽

Diagnostic Work ◽

Work Up

Abstract Abstract 2758 Introduction: The ETV6 gene (formerly TEL) is located in the chromosomal band 12p13 and is a frequent target of deletions and chromosomal translocations in both myeloid and lymphoid leukemias. In ALL the most frequent partner gene of ETV6 is RUNX1. ALL with ETV6-RUNX1 fusions are observed in 20% of childhood ALL and are associated with favorable outcome. In contrast ETV6 rearrangements are less frequent and not well described in myeloid malignancies. Therefore, the aim of this study was to analyze ETV6 rearrangements in myeloid malignancies with respect to frequency, partner genes and impact on prognosis. Patients/Methods: 55 cases with ETV6 rearrangements were identified in a total cohort of 9,550 cases (0.5%) with myeloid malignancies (de novo AML: n=3,090, s-AML: 486, t-AML: 222, MDS: n=3,375, MDS/MPN overlap: n=210, CMML: n=447, MPN: n=1,720) which had been sent to our laboratory between 08/2005 and 07/2010 for diagnostic work-up. In all cases chromosome banding analysis was performed and in cases with abnormalities involving 12p13 FISH was carried out in addition to verify the ETV6 rearrangement. Results: ETV6 rearrangements were observed in 31 patients with de novo AML (1.0% of investigated cases), 8 with s-AML (1.7%), 5 with t-AML (2.3%), 6 with MDS (0.2%) and 5 with MPN (0.3%). No ETV6 rearrangements were detected in the cohorts of MDS/MPN or CMML. ETV6 rearrangements were significantly more frequent in s-AML and t-AML as compared to de novo AML (p<0.001). Median age in AML was 59.9 years. In 15 cases with de novo AML FAB-subtypes were available: M0: n=8, M1: n=4, M2: n=1, M4: n=1, and M7: n=1. Thus, ETV6 rearrangements are closely related to immature AML subtypes. In 25/55 cases (45.5%) the ETV6 rearrangement was the sole abnormality. Recurrent additional abnormalities were 7q-/-7 in 10 cases and del(5q) in 8 cases. 36 different partners of ETV6 were observed, recurrent partners were located on 3q26 (EVI1, n=11), 5q33 (PDGFRB, n=4), 22q12 (n=3), 2q31 (n=2), 5q31 (ACSL6, n=2), 12p12 (n=2), 17q11 (n=2). Molecular analysis was performed in addition in AML with ETV6 rearrangements for mutations in NPM1 (n=26 investigated), FLT3-ITD (n=33), FLT3-TKD (n=11), MLL-PTD (n=25) and RUNX1 (n=7). NPM1-mutations were observed in 5 cases (19.2%), FLT3-ITD in 3 cases (9.1%), FLT3-TKD in 2 cases (18.2%), MLL-PTD in 1 case (4%) and RUNX1 mutations in 4 cases (57.1%), respectively. Clinical follow-up data was available of 47 cases. No differences in overall survival (OS) and event-free survival (EFS) were observed in cases with ETV6 rearrangement whether or not additional cytogenetic abnormalities or 7q-/-7 or del(5q) were present. Next 30 de novo AML with ETV6 rearrangement were compared to 819 AML without ETV6 rearrangement. Based on cytogenetics cases were assigned into 9 subgroups: 1) t(15;17)(q22;q21), n=48; 2) t(8;21)(q22;q22), n=29; 3) inv(16)(p13q22)/t(16;16)(p13;q22), n=19; 4) 11q23/MLL abnormalities, n=28; 5) inv(3)(q21q26)/t(3;3)(q21;q26), n=6; 6) normal karyotype, n=424; 7) complex karyotype, n=71; 8) other abnormalities, n=194 and 9) ETV6 rearrangements, n=30. Median OS was not reached for groups 1, 2, 3, 4, and 6 and was 10.6 mo, 11.8 mo, 32.2 and 26.3 mo for groups 5, 7, 8, and 9 respectively. OS at 2 yrs was 95.6%, 96.3%, 76.6%, 64.9%, 26.7%, 63.3%, 23.9%, 58.5% and 60.1% for groups 1–9, respectively. The respective data for median EFS were: not reached for groups 1 and 2 and 15.9 mo, 13.5 mo, 5.1 mo, 16.6 mo, 7.5 mo, 12.5 mo and 14.0 mo for groups 3–9, respectively. Conclusions: ETV6 rearrangements are rare in myeloid malignancies. ETV6 is rearranged with a large variety of partner genes. The highest frequency of ETV6 rearrangements was observed in s-AML and t-AML. OS and EFS of AML with ETV6 rearrangements are comparable to AML with normal karyotype. Thus, the detection of ETV6 rearrangements is associated with in intermediate prognosis. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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Complete or Partial Deletions of IKZF1 Occur In 28% of Blast Crisis CML and Are the Only Recurrent Submicroscopic Alteration Associated with Disease Progression: An Analysis of 43 Cases Using High-Resolution Genome-Wide Copy Number DNA Arrays and Molecular Assays

Blood ◽

10.1182/blood.v116.21.4172.4172 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4172-4172

Author(s):

Vera Grossmann ◽

Melanie Zenger ◽

Alexander Kohlmann ◽

Susanne Schnittger ◽

Wolfgang Kern ◽

...

Keyword(s):

Disease Progression ◽

Copy Number ◽

Chromosome Banding ◽

Blast Crisis ◽

Chronic Phase ◽

Partial Deletion ◽

Employment Equity ◽

Monosomy 7 ◽

Equity Ownership ◽

Gains And Losses

Abstract Abstract 4172 In advanced CML, approximately 80% of patients develop additional non-random cytogenetic abnormalities in Ph+ cells. However, little is known about molecular mutations leading to disease progression. Only limited changes that occur in the clonal evolution of the chronic phase to blast crisis, both in gene expression patterns and DNA copy number alterations, have been described. Novel technologies allow comprehensive detection of additional molecular genomic aberrations. To identify recurrent submicroscopic gains and losses, as well as copy neutral loss of heterozygocity (CN-LOH), we employed whole-genome 2.7M arrays (Affymetrix, Santa Clara, CA) to study 22 cases with blast crisis CML (n=14 myeloid; n=6 lymphoblastic; n=2 not specified) and 18 cases of untreated patients in chronic phase, matched for age and gender. In four cases, the analyses were performed on paired samples. We first investigated the occurrence of CN-LOH and observed in two patients with blast crisis a recurrent CN-LOH for 1p (both ranging to the telomere as being typical for acquired CN-LOH). Secondly, all aberrations identified by chromosome banding analysis were also detected by microarray analysis. The microarray used provided copy number estimates for 2.7 million markers, thus, even very small gains and losses were identified. As such, 39 submicroscopic variations were found, which were not detectable by chromosome banding analysis. Aberrations occurred predominantly in blast crisis CML: number of alterations in chronic phase vs. blast crisis, n=4 vs. n=9 gains, n=7 vs. n=16 losses, and n=0 vs. n=3 CN-LOHs; e.g. gains for AKT2, MLLT4, ELN, and losses of CBFB, MLLT10 or MYC, respectively. Besides microdeletions in the breakpoint region of BCR (n=5) and ABL1 (n=5), the only recurrent submicroscopic alteration was a deletion confined to a subset of exons from the IKZF1 gene, located on the short arm of chromosome 7 (7p12.2), and was observed in three patients in the cohort of blast crisis. In addition, in three patients of blast crisis cohort a complete or partial monosomy 7p had been identified. IKZF1 encodes for a transcription factor which is an important regulator of lymphoid cell differentiation. To further investigate the role of IKZF1 deletions, additional 21 patients with blast crisis CML (n=14 myeloid; n=4 lymphoblastic; n=3 not specified) were screened. In this independent cohort, two patients showed monosomy 7 in chromosome banding analyses and in four patients deletions of internal IKZF1 exons were identified by PCR using specific primer pairs for the common deletions in the IKZF1 gene as described by Iacobucci et al. (Blood, 114:2159-67, 2009). In total, for both cohorts of blast crisis CML, in 28% (12/43) of cases either complete loss of IKZF1 due to cytogenetic aberrations leading to loss of 7p or intragenic IKZF1 deletions were observed. Of note, these aberrations never occurred concomitantly. In detail, a complete deletion of IKZF1 was detected in 5 patients due to a derivative chromosome 7 or a monosomy 7: 2/5 cases with lymphoblastic blast crisis and 2/5 cases with myeloid blast crisis (1/5 case not specified). A partial deletion of IKZF1 was found in 7 cases: 5/7 cases with partial IKZF1 deletions presented with lymphoblastic blast crisis and 1/7 case with myeloid blast crisis (1/7 case data not specified). In detail, in five cases the common partial deletion of exons 4–7 was detected, resulting in a dominant negative isoform, one case was harboring the exons 2–7 deletion, and another case was carrying both of the two mutations. In three of the seven cases with an intragenic IKZF1 deletion, matched DNA from the first diagnosis and chronic state was available. The respective IKZF1 deletions were not detectable in these specimens indicating that IKZF1 deletions developed during disease progression. Moreover, in one patient we performed a serial analysis of 8 time points across different disease states (interval from diagnosis to most recent investigation: 2.2 years). Here, exons 2–7 deletion was only detected at the stage of blast crisis. In addition, in 4 of 7 cases with a partial deletion of IKZF1 a point mutation in BCR-ABL1 (E279K, T315I, F317L, Q252H) was detected. In conclusion, these results underline a pathogenetic contribution of IKZF1 deletions to disease progression in CML and therefore constitute an important progression marker to blast crisis. Disclosures: Grossmann: MLL Munich Leukemia Laboratory: Employment. Zenger:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership, Research Funding.

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Flow Cytometric Immunophenotyping in Myelodysplasia: Discovery and Diagnosis

Blood ◽

10.1182/blood.v124.21.sci-24.sci-24 ◽

2014 ◽

Vol 124 (21) ◽

pp. SCI-24-SCI-24

Author(s):

Arjan A Van de Loosdrecht ◽

Theresia M. Westers

Keyword(s):

High Risk ◽

Research Funding ◽

Severe Dysplasia ◽

Diagnostic Score ◽

Flow Cytometric ◽

Transfusion Requirements ◽

Diagnostic Work ◽

Work Up ◽

Diagnostic Work Up ◽

Myeloid Progenitors

Abstract Diagnosis of myelodysplastic syndromes (MDS) is based on the integration of results from diagnostic tools. Initial laboratory assessments in patients with suspected MDS comprise analysis of peripheral blood and bone marrow with the gold standard of cytomorphology, conventional cytogenetics and interphase fluorescence-in-situ hybridization (FISH). Single nucleotide polymorphism (SNP) array and next-generation sequencing (NGS) are emerging techniques. The pathological hallmark of MDS is dysplasia. Flow cytometry (FC) can identify aberrancies in antigen expression and differentiation patterns that are indicative of dysplasia. FC is regarded instrumental and now even recommended in the diagnostic work-up of (suspected) MDS, especially when dysplasia by cytomorphology is minimal and cytogenetics shows no (typical) abnormalities. No single FC marker has been identified that is specific for MDS. The WHO-2008 classification recommends the presence of three or more FC aberrancies as highly suggestive for MDS. Minimal requirements to analyze dysplasia by FC have been proposed by the European LeukemiaNet (ELNet) working group (ELNet iMDS-Flow. ELNet recommendations should enable a categorization of FC results in cytopenic patients as "normal", "suggestive of", or “high probability of” MDS. FC as a single technique is not sufficient for the diagnosis of MDS and should be part of an integrated diagnostic work-up. Within the ELNet-iMDS-Flow group, a score based on four cardinal parameters was validated in MDS patients with <5% blasts and non-clonal cytopenic controls. This diagnostic score comprises: a) SSC of granulocytes (reflecting granularity) as ratio to lymphocytes; b) percentage of CD34+ myeloid progenitors of nucleated cells; c) percentage of B cell progenitors within the total CD34+ compartment; and d) expression of CD45 on CD34+ myeloid progenitors as ratio to lymphocytes. An abnormal value for every single parameter is assigned 1 point; MDS is highly suggestive when a score of 2 or more is obtained. Sensitivity of this diagnostic score was 70% and specificity 92%. It was demonstrated that this score also separates distinct subgroups with respect to prognosis within IPSS-R risk groups. Most flow scores mainly incorporate markers that cover the myelomonocytic lineage, e.g. the flow cytometric scoring system (FCSS). The latter separates patients with no and mild-to-moderate dysplasia from those with severe dysplasia. The FCSS has recently been shown to add significantly in separating patients into low or high risk disease in the revised IPSS subgroups. Finally, FCSS originally designed as a prognostic score, but can also be applied as a diagnostic score as combined with the 4-parameter FC diagnostic score to further improve sensitivity and specificity. Flow profiles based on the myelomonocytic lineage may fail to recognize MDS patients that exclusively show erythroid and/or megakaryocytic dysplasia. Analysis of megakaryocytes is hampered by their paucity. Recent advances shows that when adding erythroid markers such as CD71, CD36 and CD117/CD105 may add significantly to the diagnostic score of MDS by FC. Interestingly, aberrancies in the myelomonocytic lineage have been shown in patients with solely erythroid dysplasia with impact on prognosis. In addition, aberrant FC profile of myeloid progenitors has been associated with high transfusion requirements and disease progression as well as with a short duration of response or even lack of response to growth factor and azacitidine treatment. In conclusion, FC is a useful tool in the diagnostic work-up of MDS. Further studies on the value of FC in diagnosis, prognosis and predicting response to treatment are ongoing, as well as correlation of FC results with data obtained by SNP and NGS within prospective multicenter clinical trials in low and high risk MDS. Disclosures Van de Loosdrecht: celgene: Honoraria, Research Funding; alexion: Research Funding.

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Clinical Relevant Alterations Identified By Comprehensive Genomic Profiling Can Potentially Improve Therapeutic Option and Change Prognosis in Hematologic Malignancies

Blood ◽

10.1182/blood.v128.22.5109.5109 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5109-5109

Author(s):

Jie He ◽

Minal Patel ◽

Irene Phillip ◽

Mark Bailey ◽

Ellin Berman ◽

...

Keyword(s):

Targeted Therapies ◽

Copy Number ◽

Research Funding ◽

Standard Of Care ◽

Genomic Profiling ◽

Employment Equity ◽

Comprehensive Genomic Profiling ◽

High Concordance ◽

Equity Ownership ◽

Copy Number Amplification

Abstract Background: Hybrid capture based comprehensive genomic profiling (CGP) in a spectrum of human leukemias has led to a deepened understanding of biological mechanism of leukemogenesis and improved risk stratification and has provided additional options for targeted therapy beyond standard chemotherapy or transplantation. However, most adults with leukemia still relapse after initial therapy, and incorporating comprehensive genomic profiling results into clinical practice still requires clear guidelines and evidence on how this compares with the conventional diagnosis approach. Here we analyzed a consecutive series of 116 acute leukemia patients profiled by FoundationOne Heme to learn how the results compared with current standard of care diagnosis and how any additional insights into the genomic alterations may lead to a changed or improved diagnosis, therapy and prognosis. Methods: A total of 116 consecutive newly diagnosed or relapse/refractory leukemia patients from Memorial Sloan Kettering Cancer Center were profiled by FoundationOne Heme. DNA and RNA integrated next-generation sequencing was performed in a CLIA-certified, CAP-accredited, NYS-approved laboratory for comprehensive genomic profiling. All captured libraries were sequenced to high depth averaging 569X for DNA (405 genes) and >3M unique pairs for RNA (265 genes). Somatic variants identified included short variants, copy number amplification and loss and rearrangements. Ninety-nine patients had Karyotyping and/or FISH performed at the same time to allow comparing results between platforms. Results: The age of this clinical sample cohort range from 19 to 85, with median age of 54. The diagnosis included AML (49), ALL (29), LGL (12), CML (7), MDS (4), and 16 other subtypes. CGP was performed at the time of diagnosis (47 patients, 41%), relapsed/refractory (58 patients, 50%), stable disease (7 patients, 6%) and complete remission (3 patients, 3%). CGP successfully reported 308 alterations in 103 patients with an average of 3.0 alterations per patient, including 158 base substitutions, 75 indels, 21 copy number amplification or loss, and 54 rearrangements. High concordance of known translocations, including BCR-ABL1, RUNX1T1-RUNX1, PML-RARA, MLLT10-MICALM, MLL-rearrangement, ETV-6 rearrangement and EVI1 rearrangement, were observed between FoundationOne Heme and Karyotpye/FISH in 76 out of 78 cases (97%). In addition to genomic abnormalities identified by Karyotype/FISH, CGP identified additional clinically relevant alterations in 61 cases (59%). These alterations included genes associated with new targeted therapies, such as FLT3, IDH1/2, KRAS/NRAS/BRAF and KIT and known/novel prognostic biomarkers, including TP53, NF1, CDKN2A/B, and RB1 (Figure 1). Novel fusions involved in NUP98, ABL1, PDGFRB, JAK2 were found in 12 patients, and 4 known/novel Ph-like ALL signature fusions were identified which can inform the use of clinically available targeted therapies for these patients with high-risk ALL. Conclusion: CGP has high concordance with standard of care testing (Karyotyping/FISH) with respect to the detection of known translocations/fusion genes. More importantly, CGP allowed for the identification of additional clinically relevant genomic alterations in a substantial fraction of leukemia patients seen in routine clinical care. These data demonstrate CGP can inform novel therapeutic interventions, improve accuracy of clinical diagnosis, and provide added value to improve prognosis prediction in adult leukemia. Figure 1 Figure 1. Disclosures He: Foundation Medicine, Inc: Employment, Equity Ownership. Bailey:Foundation Medicine, Inc: Employment, Equity Ownership. Park:Amgen: Consultancy; Genentech/Roche: Research Funding; Juno Therapeutics: Consultancy, Research Funding. Douer:Shire: Consultancy, Speakers Bureau; Pfizer: Consultancy; Jazz Pharmaceuticals: Honoraria; Gilled Sciences, Inc: Consultancy; Spectrum: Consultancy. Nahas:Foundation medicine: Employment. Otto:Foundation Medicine, Inc: Employment. Vergilio:Foundation Medicine: Employment. Mughal:Foundation Medicine: Employment, Equity Ownership. Ross:Foundation Medicine, Inc: Employment. Stephens:Foundation Medicine, Inc: Employment, Equity Ownership. Miller:Foundation Medicine: Employment, Equity Ownership. Lipson:Foundation Medicine, Inc: Employment.

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Distinct, Ethnic, Clinical, and Genetic Characteristics of Myelodysplastic Syndromes with Der(1;7)

Blood ◽

10.1182/blood-2019-129943 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5392-5392 ◽

Cited By ~ 1

Author(s):

Rurika Okuda ◽

Hideki Makishima ◽

Yasuhito Nannya ◽

Yotaro Ochi ◽

Tetsuichi Yoshizato ◽

...

Keyword(s):

Myelodysplastic Syndromes ◽

High Frequency ◽

Copy Number ◽

Research Funding ◽

Trisomy 8 ◽

Employment Equity ◽

Monosomy 7 ◽

Asian Populations ◽

Myeloid Neoplasms ◽

Equity Ownership

der(1;7)(q10;p10) is a recurrent chromosomal abnormality found in a wide variety of myeloid neoplasms observed in as high as 6% of myelodysplastic syndromes (MDS) in Asian populations, while rarely observed in Caucasian populations. It is thought to be generated by a recombination between two highly homologous centromere alphoid sequences which lead to an unbalanced abnormality of monosomy of 7q and trisomy of 1q. However, despite the presence of -7q, der(1;7) has been associated with a better prognosis compared to monosomy 7 or other del(7q) (-7/del(7q)). In addition to its association with +8 and del(20q), frequent RUNX1 mutations and a paucity of mutated TP53 have been reported in der(1;7) tumors, but otherwise, the molecular features of this abnormality have been poorly characterized in the literature. This is most likely because it is very rare in Caucasians, even though it represents one of the most prevalent lesions among Asian populations. The purpose of our study is to clarify the frequency and mutational landscape of der(1;7) in myeloid neoplasms on the basis of targeted-capture sequencing. A total of 1,707 MDS cases, including 944 German and 763 Japanese cases, were enrolled, from which we identified 73 (4.0%) cases with der(1;7). The prevalence was >20 times higher in Japanese (9.0%) than German (0.43%) cohorts (p<0.0001). We also identified a strong male predominance in der(1;7)-positive cases (90.4%) compared to negative cases. Also including an additional 22 cases, somatic mutations and copy number abnormalities in der(1;7) were interrogated in a total of 95 cases, which included 84 (88.4%) with MDS, 9 (9.5%) with AML, and 2 (2.1%) with MPN. Among MDS patients, 29 were low-risk, 47 were high-risk, and the rest were not specified. In mutation analysis, at least one mutation was detected in 98% of der(1;7) cases, most frequently affecting RUNX1 (42%), followed by EZH2 (26%), and ETNK1 (25%). Copy number analysis showed a high frequency of del(20q) and trisomy 8 in der(1;7) cases: 27.4% and 18.9% respectively. On the basis of mutant cell fractions, most of these mutations were present in subclones acquired within the major population harboring der(1;7). In particular, most of the EZH2 (7q35-q36) mutations were thought to be secondary events in der(1;7)-positive cases, while representing initial events acquired before UPD(7q) or -7/del(7q) in der(1;7)-negative cases. Of interest, der(1;7) was associated with a low frequency of TP53 mutations, which were seen only in 3% of cases with der(1;7), whereas highly prevalent in non-der(1;7) cases with -7/del(7q) (52%), which is concordant with a better clinical outcome was observed in der(1;7) cases compared with non-der(1;7) cases with monosomy 7 or other del(7q). Another unique feature of der(1;7) positive MDS was an extremely high frequency of RUNX1 mutations. However, the most prominent finding with secondary mutations in der(1;7) cases is the frequent hot spot mutation in ETNK1, which were originally reported in 8.8% of myeloid neoplasms with MPN features, like SETBP1 mutations. ENTK1 mutations were found in as many as 25% (23/95) of der(1;7) cases, while rarely seen in -7/del(7q) (1/89) (p<0.0001) or amp(1q) (2/68) (p=0.0001). Despite the high frequency of trisomy 8 observed in der(1;7) cases, none were associated with ETNK1 mutations. In addition, all of the RAS pathway mutations (positive in 16 cases) were observed in der(1;7) cases with wild-type ETNK1, while none were in ETNK1-mutant cases. Morphologically, these ETNK1-mutated der(1;7) cases presented with an increased eosinophil count in peripheral blood (760.9/ul vs. 78.1/ul) (p<0.001), compared to those without EKNK1 mutations, suggesting that ENTK1-mutated der(1;7) cases represent a novel disease entity within der(1;7), characterized by unique genetic features and increased eosinophils. In conclusion, der(1;7) is a genetically and clinically distinct subset of myeloid neoplasms, which showed unique features that are distinct from MDS cases in -7 and other del(7q). Especially, ETNK1 mutations subdivided cases with der(1;7) into two groups of genetically distinct subsets as shown in Figure 1. In the future, inhibition of the kinase activity in ETNK1 could be a novel therapeutic strategy in such a previously unrecognized subset as characterized by der(1;7) and eosinophilia. Figure 1 Disclosures Kern: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Baer:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Atsuta:Janssen Paharmaceutical K.K.: Honoraria; Mochida Pharmaceutical Co. Ltd: Honoraria; Kyowa Kirin Co., Ltd: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria. Handa:Ono: Research Funding. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Ogawa:Qiagen Corporation: Patents & Royalties; Kan Research Laboratory, Inc.: Consultancy; ChordiaTherapeutics, Inc.: Consultancy, Equity Ownership; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding; Asahi Genomics: Equity Ownership; RegCell Corporation: Equity Ownership.

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Long-Term Follow-up Identifies Double Hit and Key Mutations As Impacting Progression Free and Overall Survival in Multiple Myeloma

Blood ◽

10.1182/blood-2018-99-113045 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 110-110 ◽

Cited By ~ 1

Author(s):

Cody Ashby ◽

Eileen Boyle ◽

Ruslana G. Tytarenko ◽

Hongwei Wang ◽

Adam Rosenthal ◽

...

Keyword(s):

Multiple Myeloma ◽

Copy Number ◽

Research Funding ◽

Risk Groups ◽

Employment Equity ◽

Risk Status ◽

Equity Ownership ◽

Double Hit ◽

Mutation Braf

Abstract Introduction: The study of multiple myeloma (MM) genomics has identified many abnormalities that are associated with poor progression free survival (PFS) and overall survival (OS). Copy number abnormalities have been extensively studied in many datasets with long follow-up, however, the prognostic impact of mutations have not been extensively studied and available datasets have generally had a relatively short follow-up of 22-25 months, with one dataset being up to 5.4 years. These analyses have identified a range of mutations that are associated with prognosis, making it important to extend these observations in larger studies with robust diagnostic technologies. Methods: Samples from newly diagnosed MM patients enrolled in Total Therapy trials (n=199) were sequenced on a targeted panel consisting of 140 genes and additional regions of interest for copy number, as well as tiling of the Ig and MYC loci for detection of translocations. Samples were sequenced to a median depth of 452x using 2x75 bp paired end reads. Reads were aligned to hg19 and mutations called using Strelka and filtered with fpfilter. Translocations were called by Manta, and copy number determined by read depth ratio and loss of heterozygosity comparison with a patient matched non-tumor sample. Additional copy number data were generated by ultra-low pass whole genome sequencing (median 0.5x). Events in <2% of patients were not considered for further analysis. Risk groups including international staging system (ISS), revised-ISS, IMWG risk groups, and Double Hit MM (biallelic TP53 or amp1q with ISS III) were defined. Results: The median follow-up for this dataset was 8 years, with a median PFS of 6 years and OS of 11 years. The median age was 60.6 years and risk groups were comparable to other studies with 29.1% of patients with ISS III and 20% with high IMWG risk status. In a univariate analysis the markers with highest hazard ratios (HR) for PFS were Double Hit (9%, HR 5.2; 95% CI 2.79-9.76), abnormal BIRC3 (5%, 2.89; 1.32-6.32), ISS III (29%, 2.88; 1.65-5.02), mutation BRAF (11%, 2.26; 1.3-3.93), mutation LRP1B (6%, 2.23; 1.39-3.58), mutation DIS3 (9%, 2.2; 1.22-3.97), bi-allelic inactivation CYLD (10%, 2.04; 1.01-4.10), and high IMWG risk (20%, 2.01; 1.29-3.13). For OS the markers with highest HR were ISS III (5.21; 2.46-11.07), mutation KMT2C (3%, 4.4; 1.37-14.14), t(14;16) (4%, 3.83; 1.38-10.62), mutation EGR1 (4%, 3.58; 1.28-10.00), Double Hit (3.24; 1.65-6.40), mutation BRAF (2.89; 1.57-5.33), mutation LRP1B (2.49; 1.19-5.24), rearrangements surrounding MYC (46%, 2.49; 1.50-4.11), and high IMWG risk (2.11; 1.26-3.53). In a multivariate analysis for PFS Double Hit (HR 4.37, 95% CI 2.31-8.26), loss of BIRC2/3 (5%, 3.95; 1.69-9.21); mutation LRP1B (3.21; 1.53-6.72), mutation DIS3 (2.44; 1.31-4.53), ISS III (2.29; 1.22-4.32), mutation BRAF (2.28; 1.24-4.18) contributed to the model. For OS, ISS III (3.15;1.40-7.06); 1q21 amp (6%, 2.988; 1.01-8.86); mutation LRP1B (2.90; 1.33-6.35), Double Hit (2.51; 1.05-6.01), deletion CDKN1B (10%, 2.44; 1.15-5.16), and mutation BRAF (2.25; 1.13-4.48) contributed to the model. Conclusion: We confirm the clinical relevance of Double Hit risk status that constitutes 9% of patients; median PFS of 2 vs. 7 years (P<0.0001), and OS 3 vs. 13 years (P=0.0003). With long follow-up and deep sequencing additional mutated genes associated with adverse outcome were identified including BRAF (11%), DIS3 (9%), LRP1B (6%) and KMT2C (3%). Further, inactivation of NF-κB regulators (CYLD, BIRC2/3) were associated with poor PFS or OS. Patients with a BRAF mutation had a median PFS of 2 vs. 7 years (P=0.003), and OS of 6 vs 13 years (P=0.0004), indicating a potential useful intervention for BRAF inhibitors. Disclosures Ortiz: Celgene Corporation: Employment, Equity Ownership. Flynt:Celgene Corporation: Employment, Equity Ownership. Barlogie:Celgene: Consultancy, Research Funding; European School of Haematology- International Conference on Multiple Myeloma: Other: travel stipend; International Workshop on Waldenström's Macroglobulinemia: Other: travel stipend; Myeloma Health, LLC: Patents & Royalties: : Co-inventor of patents and patent applications related to use of GEP in cancer medicine licensed to Myeloma Health, LLC; Dana Farber Cancer Institute: Other: travel stipend; ComtecMed- World Congress on Controversies in Hematology: Other: travel stipend; Millenium: Consultancy, Research Funding; Multiple Myeloma Research Foundation: Other: travel stipend. Thakurta:Celgene Corporation: Employment, Equity Ownership. Morgan:Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Janssen: Research Funding.

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Significance of Flow Cytometric and Mutational Findings in Patients with Cytopenias and Limited or No Signs of Myelodysplasia By Cytomorphology

Blood ◽

10.1182/blood.v126.23.1671.1671 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1671-1671

Author(s):

Wolfgang Kern ◽

Manja Meggendorfer ◽

Claudia Haferlach ◽

Susanne Schnittger ◽

Torsten Haferlach

Keyword(s):

Flow Cytometry ◽

Normal Karyotype ◽

Initial Assessment ◽

Employment Equity ◽

Initial Flow ◽

Flow Cytometric ◽

Equity Ownership ◽

Diagnostic Work ◽

Work Up

Abstract Introduction: The diagnosis of myelodysplastic syndromes (MDS) has been clearly defined by the WHO classification but remains a challenge in a significant number of cases with cytomorphologically borderline findings and normal karyotype. Furthermore, flow cytometry is capable of identifying MDS-specific aberrant antigen expression yet its value in these borderline cases as well as in those even without cytomorphologic findings of myelodysplasia remains to be clarified. Follow-up analyses as well as extension of diagnostic work-up to screening for molecular mutations may give further insight. Aims: Assess the significance of cytomorphologically borderline dysplastic changes and of flow cytometric MDS-related findings in the absence of a clear-cut diagnosis of MDS by screening for molecular mutations and by diagnostic reassessment during follow-up. Patients and methods: Bone marrow samples of 322 patients were assessed for suspected MDS by cytomorphology, flow cytometry and cytogenetics in parallel from 08/2005 to 11/2014 which 1) did not reveal a definite diagnosis of MDS by cytomorphology, 2) had a normal karyotype and 3) had at least one follow-up bone marrow assessment. By cytomorphology, 159 (49%) cases had borderline dysplastic findings while 163 (51%) had no sign of MDS. By flow cytometry, 138 (43%) cases had findings in agreement with MDS according to ELN criteria (Westers et al., Leukemia 2012; at least three aberrantly expressed antigens), 141 (44%) had borderline findings (one or two aberrantly expressed antigens) and 43 (13%) had no signs of MDS. A total of 699 follow-up samples were analyzed (median 2/patient). The median follow-up amounted to 3.0 years. In 147/322 patients (46%) screening for molecular mutations was performed on the initial samples, respectively, targeting a total of 20 genes (median 4 genes/patient, range 1-20). Analyzed genes were ASXL1, TET2, RUNX1, SRSF2, BCOR, DNMT3A, IDH2, NPM1, SF3B1, TP53, ZRSR2, CBL, CSF3R, ETV6, KDM6A, KRAS, MLL, SETBP1, SMC3 and U2AF1. Results: A total of 145 patients (45%) were diagnosed with MDS by cytomorphology during follow-up. The median duration until diagnosis amounted to 3.4 years. Regarding initial cytomorphology, more cases with borderline dysplastic findings were diagnosed MDS at follow-up than those without any dysplastic findings (82/159 (52%) vs 63/163 (39%), p=0.025). However, the duration until diagnosis of MDS did not differ significantly between the two groups (median 2.6 vs 3.4 years). Regarding initial flow cytometry, more cases with findings in agreement with MDS were diagnosed MDS by cytomorphology at follow-up than those without (80/138 (58%) vs 65/184 (35%), p<0.001) while there was no difference between cases with one or two aberrantly expressed antigens at initial assessment vs those with none (51/141 (36%) vs 14/43 (33%), n.s.). The duration until diagnosis of MDS significantly differed between the groups as defined by flow cytometry and was shortest in cases in agreement with MDS at initial assessment and longest in those without any aberrantly expressed antigen (median 1.9 vs 4.1 vs 5.6 years, p<0.001). Overall survival (OS) for all cases was 80% at 5 years. While initial cytomorphologic results revealed no impact on OS, patients with an initial flow cytometric result in agreement with MDS tended to have a shorter OS (5 year OS 70% vs 88%, p=0.12). Molecular screening revealed mutations in 21/147 patients (14%) at initial assessment. Mutated genes included ASXL1 (mutated in 6 patients), TET2 (6), RUNX1 (3), SRSF2 (3), as well as 2 cases each for BCOR, DNMT3A, IDH2, NPM1, SF3B1, TP53 and ZRSR2 and 1 case each for CBL, CSF3R, ETV6, KDM6A, KRAS, MLL, SETBP1, SMC3 and U2AF1. The percentage of patients with at least one mutation did not differ between cases with borderline dysplastic findings by cytomorphology as compared to those without any dysplastic findings. In contrast, significantly more cases with findings in agreement with MDS by flow cytometry had at least one mutation as compared to those with one or two aberrantly expressed antigens as well as to those with none (15/71 (21%) vs 6/58 (10%) vs 0/18, p=0.012). Conclusions: This data strongly supports the need to define the role of flow cytometry in the diagnostic work-up in suspected MDS and argues for an integrated approach with cytomorphology and cytogenetics. Implementation also of molecular data on mutations may further improve the validity of MDS diagnostics. Disclosures Kern: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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The Multiple Myeloma Genome Project: Development of a Molecular Segmentation Strategy for the Clinical Classification of Multiple Myeloma

Blood ◽

10.1182/blood.v128.22.196.196 ◽

2016 ◽

Vol 128 (22) ◽

pp. 196-196

Author(s):

Brian A Walker ◽

Mehmet K. Samur ◽

Konstantinos Mavrommatis ◽

Cody Ashby ◽

Christopher P Wardell ◽

...

Keyword(s):

Multiple Myeloma ◽

Copy Number ◽

Research Funding ◽

Genome Project ◽

Rna Seq ◽

Medical Sciences ◽

Employment Equity ◽

Segmentation Strategy ◽

Equity Ownership ◽

Support Research

Abstract Introduction Segmenting multiple myeloma (MM) into subgroups with distinct pathogenesis and clinical behavior is important in order to move forward with advancements in therapy and implement a targeted therapy approach. Current technologies have elucidated five major translocation groups, which have a varying effect on prognosis: t(4;14), t(6;14), t(11;14), t(14;16) and t(14;20) along with recurrent copy number changes including deletion of CDKN2C (1p32.3) and TP53 (17p13.1) as well as gain or amplification of 1q21. However, minor translocation and mutational groups are poorly described because sample numbers are limited in small datasets. The availability of multiple sets of high quality mutation data associated with clinical outcomes has provided a unique opportunity in MM whereby clustering mutational data with chromosomal aberrations in the context of gene expression we can develop a molecular classification system to segment the disease into therapeutically meaningful subgroups. The Multiple Myeloma Genome Project (MGP) is a global collaborative initiative that aims to develop a molecular segmentation strategy for MM to develop clinically relevant tests that could improve diagnosis, prognosis, and treatment of patients with MM. Materials and methods We have established a set of 2161 patients for which whole exome sequencing (WES; n=1436), Whole Genome Sequencing (WGS; n=708), targeted panel sequencing (n=993) and expression data from RNA-Seq and Gene Expression arrays (n=1497) were available. These data were derived from the Myeloma XI trial (UK), Intergroupe Francophone du Myeloma/Dana-Faber Cancer Institute (MA), The Myeloma Institute (AR) and the Multiple Myeloma Research Foundation (IA1 - IA8). We assembled all data on a secure site and analyzed it using a streamlined and consistent pipeline using state of the art tools. First, BAM were converted to FASTQ using Picard tools v2.1.1 to extract read sequences and base quality scores. Next, all reads were realigned to the human genome assembly hg19 using BWA-mem. Duplicate marking and sorting was performed using Picard tools v2.1.1. For QAQC we use FASTQC and Picard tools. We identified somatic single nucleotide variants and indels with Mutect2 using default parameters. Translocations and large chromosomal aberrations were identified using MANTA and breakdancer and inferred copy number abnormalities and homozygous deletions using Sequenza v2.1.2 and ControlFreeC. Results We have begun to integrate these diverse large genomic datasets with various correlates. Samples were stratified by RNA-seq expression values and WES/WGS to identify the main cytogenetic groups with high concordance. In addition to the main translocation groups, translocations into MAFA, t(8;14), were detected in 1.2% of samples by both RNA-seq and WES/WGS. RNA-seq also detected fusion transcripts, including the known Ig-WHSC1 transcript in t(4;14). However, a proportion of identified in-frame fusion genes involved kinase domains consistent with activation of the Ras/MAPK pathway, which may be clinical targets for therapy. The main recurrent mutations included KRAS and NRAS, and negative regulators of the NF-κB pathway. In addition we identified recurrent copy number abnormalities and examined the interaction of these with mutations. This highlighted the interaction of the recurrent changes at 1p, 13q, and 17p with mutation of genes located within these regions, specifically indicating bi-allelic inactivation of CDKN2C, RB1 and TP53. Using WGS and RNA-Seq data we identified recurrent translocations and fusion genes that can be used to instruct therapy. Based on these data and the presence of homogeneous inactivation of key tumor expressed genes we will present clinically relevant clusters of MM that can form the basis of future risk and molecular targeted trials. Interaction of mutation with expression patterns has identified distinct expression signatures associated with mutational groups. Conclusions We have established the largest repository of molecular profiling data in MM along with associated clinical outcome data. Integrated analyses of these are enabling generation of clinically meaningful disease segments associated with differing risk. The MGP intends to build a global network by expanding collaboration with leading MM centers around the world and incorporating additional datasets through current and new collaborations. Disclosures Mavrommatis: Discitis DX: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Employment, Equity Ownership. Ashby:University of Arkansas for Medical Sciences: Employment. Ortiz:Celgene: Employment. Towfic:Celgene: Employment, Equity Ownership; Immuneering Corp: Equity Ownership. Amatangelo:Celgene: Employment, Equity Ownership. Yu:Celgene: Employment, Equity Ownership. Avet-Loiseau:celgene: Consultancy; janssen: Consultancy; sanofi: Consultancy; amgen: Consultancy. Jackson:Janssen: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Other: Travel support, Research Funding, Speakers Bureau; MSD: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Speakers Bureau; Takeda: Consultancy, Honoraria, Other: Travel support, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Speakers Bureau. Thakurta:Celgene: Employment, Equity Ownership. Munshi:Takeda: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Merck: Consultancy; Pfizer: Consultancy; Oncopep: Patents & Royalties. Morgan:Univ of AR for Medical Sciences: Employment; Janssen: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Bristol Meyers: Consultancy, Honoraria.

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