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

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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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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Integrated Diagnostic Approach for Suspected Myelodysplastic Syndrome As a Basis for Advancement of Diagnostic Criteria

Blood ◽

10.1182/blood.v128.22.299.299 ◽

2016 ◽

Vol 128 (22) ◽

pp. 299-299 ◽

Cited By ~ 2

Author(s):

Wolfgang Kern ◽

Manja Meggendorfer ◽

Claudia Haferlach ◽

Torsten Haferlach

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Diagnostic Criteria ◽

Mutation Analysis ◽

Diagnostic Approach ◽

Employment Equity ◽

Flow Cytometric ◽

Equity Ownership ◽

Genetic Results

Abstract Background: Myelodysplastic syndromes (MDS) comprise a heterogeneous group of diseases diagnosed and classified based on cytomorphology and cytogenetics according to the WHO classification. Flow cytometry and mutation analysis may provide additional diagnostic potential. Aim: To correlate the diagnostic results derived from flow cytometry and mutation analysis with those of cytomorphology and cytogenetics in patients with suspected MDS. To estimate the impact of these findings on the cytomorphologic reevaluation during follow up. Methods: Between February 2008 and July 2016 bone marrow samples from a total of 1681 patients with cytopenias and suspected MDS were prospectively analyzed by a combined diagnostic approach. This included in all cases cytomophology and cytochemistry, cytogenetics based on chromosome banding analysis supplemented by FISH analysis, flow cytometric assessment according to ELN criteria (Westers et al., Leukemia 2012) and mutation analysis for ASXL1, EZH2, RUNX1 and TP53which represent the prognostically most important molecular markers both in the pivotal study on molecular genetics in MDS (Bejar et al. NEJM 2011) and in a large multicenter study (Bejar et al., ASH 2015). Patients diagnosed with non-MDS hematologic malignancies were excluded. Patients´ age ranged from 17 to 95 years (median 72) and male:female ratio was 1.27. Results: 816/1681 (49%) patients were diagnosed with MDS based on cytomorphology. An aberrant karyotype was found in 319/1681 (19%) patients. Flow cytometry was in agreement with MDS in 889/1681 (54%) patients. The number of patients with mutations in the respective genes were 193/1681 (12%) for ASXL1, 37 (2%) for EZH2, 84 (5%) for RUNX1 and 69 (4%) for TP53. At least one of these mutations was present in 318/1681 (19%) patients and one, two and three genes were mutated in 261 (16%), 49 (3%) and 8 (1%) patients, respectively. Comparison between cytomorphology and flow cytometry revealed concordant results in 1300 (77%) patients (both positive for MDS in 667 (40%) and both negative for MDS in 633 (38%) patients). Cytomorphology diagnosed MDS while flow cytometry was negative (C+F-) in 149 (9%) cases and flow cytometry was in agreement with MDS while cytomorphology was negative (F+C-) in 232 (14%) cases. Analyzing genetic results in these discordant cases revealed an aberrant karyotype in 34/149 (23%) of C+F- cases and in 30/232 (13%) of F+C- cases, respectively. At least one of the four analyzed genes was found mutated in 19/149 (13%) of C+F- cases and in 37/232 (15%) of F+C- cases, respectively. Combining these findings, an aberrant karyotype or at least one mutated gene were found in 45/149 (30%) of C+F- cases and in 55/232 (24%) of F+C- cases, respectively. In contrast, in cases rated MDS by both cytomorphology and flow cytometry (C+F+) an aberrant karyotype or at least one mutated gene were found in 354/667 (53%) cases while this was true for 61/633 (10%) C-F- cases only (p<0.001). Follow-up analyses of bone marrow samples by cytomorphology were available for 116 cases initially not diagnosed with MDS by cytomorphology. 40 of them were initially rated in agreement with MDS by flow cytometry. Median follow-up time was 1.0 year. In 29 patients MDS was diagnosed by cytomorphology at follow-up. In the total of 116 patients with follow-up analyses the Kaplan-Meier estimate of probability of MDS was 40% at 2 years. Probability of MDS at 2 years was non-significantly higher in cases initially rated in agreement with MDS by flow cytometry as compared to others (48% vs. 35%). The respective impact of the presence of an aberrant karyotype or at least one mutated gene was even higher (2 year probability of MDS 71% vs. 23%, p<0.001). Combining flow cytometric and genetic results revealed the highest probability of MDS in case of positivity for both (F+G+, 81% at 2 years), followed by G+F- (65%), F+G- (29%) and F-G- (20%, p=0.002). Conclusion: In patients with cytopenia not diagnosed with MDS by cytomorphology the presence of cytogenetic aberrations and molecular mutations typically associated with MDS reveals a high probability of development of MDS, particularly if in parallel flow cytometric evaluation is in agreement with MDS. Further study is warranted aiming at a respective extension of diagnostic criteria. Disclosures Kern: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer: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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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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High-Throughput Sequencing to Identify Cytogenetic and Molecular Genetic Aberrations in 24 AML Exomes

Blood ◽

10.1182/blood.v124.21.2389.2389 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2389-2389

Author(s):

Sonja Althammer ◽

Andreia de Albuquerque ◽

Niroshan Nadarajah ◽

Manja Meggendorfer ◽

Susanne Schnittger ◽

...

Keyword(s):

Copy Number ◽

Research Funding ◽

Framework Programme ◽

Employment Equity ◽

Seventh Framework Programme ◽

Equity Ownership ◽

Diagnostic Work ◽

Gains And Losses ◽

Work Up ◽

Diagnostic Work Up

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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Prognostic Molecular Stratification in Relapsed/Refractory Multiple Myeloma - Results of the Pomalidomide Mukseven (NCT02406222) Biomarker Trial

Blood ◽

10.1182/blood-2019-123640 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4327-4327

Author(s):

James Croft ◽

Andrew Hall ◽

Amy L Sherborne ◽

Katrina Walker ◽

Sidra Ellis ◽

...

Keyword(s):

Multiple Myeloma ◽

Flow Cytometry ◽

T Cell ◽

High Risk ◽

Real World ◽

Research Funding ◽

Unmet Need ◽

Employment Equity ◽

Equity Ownership

Background Treatment of relapsed/refractory multiple myeloma (RRMM) remains challenging as durable remissions are achieved in patient sub-groups only. Identifying patients that are likely to benefit prior to or early after starting relapse treatments remains an unmet need. MUKseven is a trial specifically designed to investigate and validate biomarkers for treatment optimization in a 'real-world' RRMM population. Design In the randomized multi-center phase 2 MUKseven trial, RRMM patients (≥2 prior lines of therapy, exposed to proteasome inhibitor and lenalidomide) were randomized 1:1 to cyclophosphamide (500 mg po d1, 8, 15), pomalidomide (4 mg days 1-21) and dexamethasone (40 mg; if ≥75 years 20 mg; d1, 8, 15, 21) (CPomD) or PomD and treated until progression. All patients were asked to undergo bone marrow (BM) and peripheral blood (PB) bio-sampling at baseline, cycle 1 day 14 (C1D14, on-treatment) and relapse. For biomarker discovery and validation, IGH translocations were profiled by qRT-PCR, copy number aberrations by digital MLPA (probemix D006; MRC Holland), GEP by U133plus2.0 array (Affymetrix), PD protein markers by IHC and PB T-cell subsets by flow cytometry for all patients with sufficient material. Primary endpoint was PFS, secondary endpoints included response, OS, safety/toxicity and biomarker validation. Original planned sample size was 250 patients but due to a change in UK standard of care during recruitment with pomalidomide becoming available, a decision was made to stop recruitment early. Results In total, 102 RRMM patients were randomized 1:1 between March 2016 and February 2018. Trial entry criteria were designed to include a real-world RRMM population, permitting transfusions and growth factor support. Median age at randomization was 69 years (range 42-88), 28% of patients had received ≥5 prior lines of therapy (median: 3). Median follow-up for this analysis was 13.4 months (95% CI: 12.0-17.5). 16 patients remained on trial at time of analysis (median number of cycles: 19.5; range 8-28). More patients achieved ≥PR with CPomD compared to PomD: 70.6% (95% CI: 56.2-82.5%) vs. 47.1% (CI: 32.9-61.5%) (P=0.006). Median PFS was 6.9 months (CI: 5.7-10.4) for CPomD vs. 4.6 months (CI: 3.5-7.4) for PomD, which was not significantly different as per pre-defined criteria. Follow-up for OS is ongoing and will be presented at the conference. High-risk genetic aberrations were found at following frequencies: t(4;14): 6%, t(14;16)/t(14;20): 2%, gain(1q): 45%, del(17p): 13%. Non-high risk lesions were present as follows: t(11;14): 22%, hyperdiploidy: 44%. Complete information on all high-risk genetic markers was available for 71/102 patients, of whom 12.7% had double-hit high-risk (≥2 adverse lesions), 46.5% single-hit high-risk (1 adverse lesion) and 40.8% no risk markers, as per our recent meta-analysis in NDMM (Shah V, et al., Leukemia 2018). Median PFS was significantly shorter for double-hit: 3.4 months (CI: 1.0-4.9) vs. single-hit: 5.8 months (CI: 3.7-9.0) or no hit: 14.1 months (CI: 6.9-17.3) (P=0.005) (Figure 1A). GEP was available for 48 patients and the EMC92 high-risk signature, present in 19% of tumors, was associated with significantly shorter PFS: 3.4 months (CI: 2.0-5.7) vs. 7.4 (CI: 3.9-15.1) for EMC92 standard risk (P=0.037). Pharmacodynamic (PD) profiling of cereblon and CRL4CRBN ubiquitination targets (including Aiolos, ZFP91) in BM clots collected at baseline and C1D14 is currently ongoing. Preliminary results for the first 10 patients demonstrate differential change of nuclear Aiolos (Figure 1C), with a major decrease in Aiolos H-scores in 7/10 patients from baseline to C1D14 and reconstitution at relapse. T-cell PB sub-sets were profiled at baseline and C1D14 by flow cytometry. Specific sub-sets increased with therapy from baseline to C1D14, e.g. activated (HLA-DR+) CD4+ T-cells, as reported at last ASH. CD4+ T-cell % at baseline was associated with shorter PFS in these analyses in a multi-variable Cox regression model (P=0.005). PD and T-cell biomarker results will be updated and integrated with molecular tumor characteristics and outcome. Discussion Our results demonstrate that molecular markers validated for NDMM predict treatment outcomes in RRMM, opening the potential for stratified delivery of novel treatment approaches for patients with a particularly high unmet need. Additional immunologic and PD biomarkers are currently being explored. Disclosures Croft: Celgene: Other: Travel expenses. Hall:Celgene, Amgen, Janssen, Karyopharm: Other: Research funding to Institution. Walker:Janssen, Celgene: Other: Research funding to Institution. Pawlyn:Amgen, Janssen, Celgene, Takeda: Other: Travel expenses; Amgen, Celgene, Janssen, Oncopeptides: Honoraria; Amgen, Celgene, Takeda: Consultancy. Flanagan:Amgen, Celgene, Janssen, Karyopharm: Other: Research funding to Institution. Garg:Janssen, Takeda, Novartis: Other: Travel expenses; Novartis, Janssen: Research Funding; Janssen: Honoraria. Couto:Celgene Corporation: Employment, Equity Ownership, Patents & Royalties. Wang:Celgene Corporation: Employment, Equity Ownership. Boyd:Novartis: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Pierceall:Celgene: Employment. Thakurta:Celgene: Employment, Equity Ownership. Cook:Celgene, Janssen-Cilag, Takeda: Honoraria, Research Funding; Janssen, Takeda, Sanofi, Karyopharm, Celgene: Consultancy, Honoraria, Speakers Bureau; Amgen, Bristol-Myers Squib, GlycoMimetics, Seattle Genetics, Sanofi: Honoraria. Brown:Amgen, Celgene, Janssen, Karyopharm: Other: Research funding to Institution. Kaiser:Takeda, Janssen, Celgene, Amgen: Honoraria, Other: Travel Expenses; Celgene, Janssen: Research Funding; Abbvie, Celgene, Takeda, Janssen, Amgen, Abbvie, Karyopharm: Consultancy.

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Sensitive Monitoring of Minimal Residual Disease Status in CEBPA-Mutated Acute Myeloid Leukemia Using Amplicon Deep-Sequencing

Blood ◽

10.1182/blood.v118.21.2517.2517 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2517-2517

Author(s):

Alexander Kohlmann ◽

Vera Grossmann ◽

Annette Fasan ◽

Elisa Stopp ◽

Tamara Alpermann ◽

...

Keyword(s):

Deep Sequencing ◽

Residual Disease ◽

Normal Karyotype ◽

Initial Diagnosis ◽

Employment Equity ◽

Clone Size ◽

Equity Ownership ◽

Conventional Methods ◽

Biallelic Mutations

Abstract Abstract 2517 Introduction: CEBPA (CCAAT/enhancer binding protein alpha) encodes a member of the basic region leucine zipper (bZIP) transcription factor family essential for myeloid differentiation. CEBPA mutations occur predominantly in AML with a normal karyotype and CEBPA mutated AML has been included as provisional entity in the WHO classification. Cases with biallelic mutations were reported as being associated with a favorable clinical outcome, thus patients are spared from allogeneic transplantation in first CR. Screening for CEBPA mutations in patients with AML is often performed applying a combination of fragment length analysis, DHPLC and subsequent direct sequencing using Sanger technique (conventional methods). Study Design: Next-generation amplicon deep-sequencing (454 Life Sciences, Branford, CT) is a more sensitive quantitative detection method than Sanger sequencing and thus was used to analyze 144 samples from 29 CEBPA mutated AML patients with a normal karyotype. For a longitudinal analysis starting at diagnosis and following the course of treatment bone marrow (n=134) or peripheral blood (n=10) samples were obtained between 5/2006 and 6/2011. The sequencing assay targeted the complete coding region of CEBPA, covered with 4 amplicons, and was performed using genomic DNA extracted from mononuclear cells. In median, 711 reads per amplicon were generated using the NGS assay, thereby allowing a sensitive quantitative assessment of the CEBPA mutational burden in order to monitoring minimal residual disease (MRD). In median, 4 time points per patient (range: 2–9) were included with a median time span of 9.5 months (range: 1–45 months). The median sampling interval was 2 months (range: 0.3–45 months). Results: First, we evaluated the concordance of mutation detection by comparing data from NGS and conventional methods using the samples at initial diagnosis. In all 29 AML patients NGS concordantly detected the mutations known from conventional methods, i.e. in total 26 frame-shifts, 15 in-frame alterations, 8 missense, and 2 nonsense mutations. Further, at initial diagnosis, deep-sequencing detected the mutations with a median burden of 44% sequencing reads (range 3%–88%) and thus already allowed a quantitative assessment of the mutational load. There was no difference observed for 6 patients with monoallelic vs. 21 cases with biallelic mutations (excluding 2 cases with homozygous alterations). We next investigated the distribution of clones and their underlying kinetics of clone size reduction during subsequent high-dose chemotherapy cycles. Overall, 26/29 cases were evaluable and the clone size was assessed by NGS at the second analysis point during course of disease–in median 63 days from time of diagnosis (range 10–215 days): (i) In 16/26 cases, deep-sequencing was not able anymore to detect the mutations as observed at diagnosis. 14 of these 16 negative cases stayed in complete molecular remission till the end of follow-up (median follow-up 6.5 months, range 1–34.2; 2/14 cases with allogeneic stem cell transplantation). (ii) Interestingly, in 4/26 cases residual disease with clones ranging from 8%–50% was indicative of non-response to treatment. In this subgroup 3/4 patients were characterized by resistant disease or early relapse (1 case excluded due to short follow-up). (iii) In the remainder group of 6/26 patients with mutations still detectable in a range of 0.12%–3.7%, complete molecular remission status was achieved at subsequent time points. However, in this group also 3 relapses were observed including 2 cases with allogeneic stem cell transplantation. Of note, in 3/6 cases from the latter group, NGS had outperformed conventional methods and was able to still detect residual clones enabling a superior monitoring of therapy response. In all cases with biallelic mutations both clones responded in parallel with similar kinetics. Moreover, 5 patients were investigated following relapse of AML or non-response to therapy. In all 5/5 analyses including 2 monoallelic and 3 biallelic alterations the same mutations as harbored at initial diagnosis remained detectable. Conclusion:CEBPA mutations provide increasing clinical utility for the detection of MRD. We here demonstrated that deep-sequencing is a suitable unbiased and robust method to accurately detect and quantify CEBPA aberrations enabling an individualized monitoring of disease status and treatment efficacy. Disclosures: Kohlmann: MLL Munich Leukemia Laboratory: Employment; Roche Diagnostics: Honoraria. Grossmann:MLL Munich Leukemia Laboratory: Employment. Fasan:MLL Munich Leukemia Laboratory: Employment. Stopp:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Schindela:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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Cytogenetic and Molecular Genetic Clonal Evolution in CLL Is Associated with an Unmutated IGHV Status and Frequently Leads to a Combination of Loss of 17p and TP53 mutation

Blood ◽

10.1182/blood.v128.22.3213.3213 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3213-3213

Author(s):

Claudia Haferlach ◽

Sabine Jeromin ◽

Niroshan Nadarajah ◽

Melanie Zenger ◽

Wolfgang Kern ◽

...

Keyword(s):

Median Time ◽

Tp53 Mutation ◽

Clonal Evolution ◽

Molecular Genetic ◽

Normal Karyotype ◽

Employment Equity ◽

Mutation Status ◽

Equity Ownership ◽

Mutation Analyses

Abstract Background: The clinical course in CLL is very heterogeneous ranging from stable disease to a rather rapid progression requiring treatment. The acquisition of genetic abnormalities termed clonal evolution (CE) is likely to correlate with clinical progression and might be used to guide treatment strategies. Aim: The aim of this study was to evaluate the frequency of CE on the cytogenetic (CCE) and molecular genetic (MCE) levels and its association with the IGHV mutation status and clinical outcome. Methods: 179 CLL cases were selected on the basis that chromosome banding analysis (CBA) and mutation analyses in TP53 and SF3B1 all having been performed at least at two time points. The median age at first evaluation was 72 years (range: 46-95). The first time point of analysis was at primary diagnosis (n=131) or during course of disease but prior to any treatment (n=48). In all patients interphase FISH was performed with probes for 17p13 (TP53), 13q14 (D13S25, D13S319, DLEU), 11q22 (ATM), and the centromeric region of chromosome 12 and the IGHV mutation status was evaluated. A total of 465 CBA, 417 TP53 and 424 SF3B1 mutation analyses were evaluated. The median number of samples per patient was 2 (range: 2-9). The time between samples ranged from 1 month to 9.8 years (median 21 months). For all patients clinical follow-up data was available with a median follow-up of 7.4 years and 5-year overall survival (OS) of 88%. Results: At first investigation CBA revealed a normal karyotype in 31 (17%) patients. In cases with an aberrant karyotype the pattern of abnormalities was typical for CLL: del(13q); 51% (homozygous: 15%), +12: 18%, del(11q): 16%, and del(17p): 5%. A complex karyotype (≥3 abnormalities) was present in 18%. The IGHV status was unmutated (IGHV-U) in 56% of cases and TP53 and SF3B1 mutations were detected in 10% and 15%, respectively. CCE was observed in 63/179 patients (35%). The median time to CCE was 46 months (range 3-111). The most frequent abnormalities gained during CCE were loss of 17p (14/63; 22%), 13q (11/63; 18%), and 11q (10/63; 16%). Acquired loss of 17p was more frequent in SF3B1mutated CLL (19% vs 6%, p=0.04). MCE was observed in 29/179 cases (16%). TP53 and SF3B1 mutations were acquired during the course of the disease in 23 (14%) and 7 (5%) cases, respectively. The median time to MCE was 61 months (range 1.5-109). Of note, in 2 cases with TP53 deletion a TP53 mutation was acquired and in 2 cases with TP53 mutation a TP53 deletion was acquired. In 12 CLL both a TP53 deletion and a TP53 mutation were acquired (table). CCE and MCE were significantly associated with IGHV-U (p=0.003; p<0.001) and with each other (p<0.001). In more detail, in 71% of cases with CCE and 90% of cases with MCE an IGHV-U was present. Thus, CCE and MCE were less frequent in IGHVmut CLL (23% and 4%). In 30% of CLL with CCE also MCE occurred. In addition CCE was associated with an aberrant karyotype at first investigation (p<0.001). CCE occurred in only 3% of CLL with a normal karyotype but in 42% of CLL with an aberrant karyotype. Time to treatment was significantly shorter in patients with CCE, MCE and both compared to the respective patients without (2.1 vs 5.5 yrs, p=0.004; 1.8 vs 4.8 yrs, p=0.07; 2.2 vs 5.3 yrs; p=0.04). While no impact of CCE on OS was observed in patients with a mutated IGHV status, in patients with an unmutated IGHV status a tendency to shorter OS was observed in cases with CCE compared to those without (7 year OS: 67% vs 83%; p=0.2). No impact on OS was observed for MCE. This may be due to rather short follow up after CE. However, if CCE and MCE resulted in CLL harboring both TP53 deletion and TP53 mutation 5 year OS was significantly shorter than in CLL with neither TP53 deletion nor TP53 mutation (75% vs 91%, p=0.03). Conclusions: 1) We observed CCE in 35% and MCE in 16% of CLL. 2) The pattern of cytogenetic abnormalities acquired during the course of the disease is similar to the pattern observed in CLL at diagnosis, however the frequency varies with del(17p) being the most frequently gained in CE. 3) CCE and MCE were highly correlated to IGHV-U. 4) In 25% of CLL with CCE and MCE CE resulted in the co-occurrence of TP53 deletion and TP53 mutation, which was associated with a significantly shorter OS emphasizing the necessity to reevaluate the TP53 status during the course of the disease to guide treatment. 5) The frequency and impact of CE needs to be further studied in unselected patient cohorts in which CBA and mutational analysis is performed on a regular basis. Table Table. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Jeromin:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Zenger:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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DIAGNOSTIC UTILITY OF FLOW CYTOMETRY IN MYELODYSPLASTIC SYNDROMES.

Mediterranean Journal of Hematology and Infectious Diseases ◽

10.4084/mjhid.2017.017 ◽

2017 ◽

Vol 9 (1) ◽

pp. e2017017 ◽

Cited By ~ 3

Author(s):

Matteo Della Porta ◽

Cristina Picone

Keyword(s):

Flow Cytometry ◽

Myelodysplastic Syndromes ◽

Qualitative Evaluation ◽

Normal Karyotype ◽

Accurate Method ◽

Specificity And Sensitivity ◽

Sensitivity Data ◽

Fcm Analysis ◽

Diagnostic Work ◽

Work Up

The pathological hallmark of myelodysplastic syndromes (MDS) is marrow dysplasia, which representsthe basis of the WHO classiﬁcation of these disorders. This classiﬁcation provides clinicians with a useful tool for deﬁning the different subtypes of MDS and individual prognosis. The WHO proposal has raised some concern regarding minimal diagnostic criteria particularly in patients with normal karyotype without robust morphological markers of dysplasia (such as ring sideroblasts or excess of blasts). Therefore, there is clearly a need to reﬁne the accuracy to detect marrow dysplasia. Flow cytometry (FCM) immunophenotyping has been proposed as a tool to improve the evaluation of marrow dysplasia. Rationale for the application of FCM in the diagnostic work up of MDS is that immunophenotyping is an accurate method for quantitative and qualitative evaluation of hematopoietic cells and that MDS have been found to have abnormal expression of several cellular antigens. To become clinically applicable, FCM analysis should be based on parameters with sufﬁcient speciﬁcity and sensitivity, data should be reproducible between different operators and the results should be easily understood by clinicians. In this review, we discuss the most relevant progresses in detection of marrow dysplasia by FCM in MDS

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Highly Sensitive Detection of Minimal Residual Disease in Acute Lymphoblastic Leukemia Using Next-Generation Sequencing of Immunoglobulin Heavy Chain Variable Region

Blood ◽

10.1182/blood.v118.21.2540.2540 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2540-2540

Author(s):

Malek Faham ◽

Tom Willis ◽

Martin Moorhead ◽

Victoria Carlton ◽

Jianbiao Zheng ◽

...

Keyword(s):

Flow Cytometry ◽

Next Generation Sequencing ◽

Lymphoblastic Leukemia ◽

Patient Specific ◽

Next Generation ◽

Conventional Pcr ◽

Employment Equity ◽

Equity Ownership ◽

Generation Sequencing

Abstract Abstract 2540 Background: Minimal Residual Disease (MRD) assessment is increasingly used for treatment stratification since it is a strong predictor of outcome in Acute lymphoblastic leukemia (ALL). The most widely used MRD assays include flow cytometric detection of aberrant immunophenotypes and PCR amplification of patient-specific antigen-receptor sequences. The latter approach has proven to provide reliable clinical information but requires the development of patient-specific reagents which is laborious, time-consuming, and generates assays with variable sensitivities. In addition, this methodology may miss clonal changes that can occur during the course of the disease, such as the emergence of subclones as well as genetic evolution. To overcome these limitations, we developed a universal amplification assay with a sequencing readout that eliminates the need for patient-specific reagents, allows the assay to detect leukemic cells that have genetically evolved, and has a higher sensitivity than conventional tests. Methods and Results: To amplify all the IgH sequences, we developed a PCR assay to amplify all alleles of all the V and J segments with very low amplfication bias. Amplified molecules were then subjected to clonal sequencing to obtain >1 million reads to measure the frequency of the different IgH clonotypes in the sample. It should be noted that current next generation sequencing costs of this deep sequencing are similar to those of an MRD test conducted by flow cytometry. We tested the sensitivity of the method by in serial dilutions of genomic DNA from a leukemia sample known to carry two IgH clonotypes with into genomic DNA obtained from peripheral blood sample from a healthy donor. The material from the dilution series was then sequenced and analyzed to measure the level of these clones. The leukemic clonotypes could be readily detected even when diluted 1 million fold. To directly compare the our method to established MRD assays in ALL, we studied diagnostic and follow-up samples from 10 ALL patients whose MRD levels have been previously assessed by both real-time PCR amplification of IgH genes and flow cytometry. The results of these tests were not disclosed until completion of the deep sequencing analysis. The follow up samples were collected during (n = 3) or at the end of remission induction therapy (n = 4), or during continuation therapy (n = 3). Samples were processed similarly to identify the leukemia-specific sequence in the diagnostic samples and determine the level of these sequences in the follow up samples. The sequencing-based method identified all 5 samples that were MRD-positive according to flow cytometry and PCR (Figure 2), with highly concordant estimates of MRD levels. Notably, among the remaining 5 samples, scored as MRD-negative by both flow cytometry and conventional PCR, the sequencing method detected residual leukemic sequences at a very low level (∼10−6) in one of the samples. The other 4 samples were MRD-negative by all three methods. Studies with a larger cohort of ALL samples are ongoing. Contrary to conventional PCR-based MRD testing, the sequencing technology allows for the detection of leukemic clones that evolve by V replacement or other mechanisms. In this study, we identified clonotypes in several of the diagnostic samples that appeared to be the result of V replacement. These and other newly appearing related clones can be monitored in subsequent samples using the generic amplification and sequencing assay. Conclusions: We developed a highly sensitive and specific MRD detection method based on next-generation sequencing of IgH genes. This method has substantial advantages over conventional PCR MRD in that it eliminates the need for patient-specific reagents, can follow genetic evolution, and has potential for higher sensitivity. Disclosures: Faham: Sequenta Inc: Employment, Equity Ownership. Willis:Sequenta Inc: Employment, Equity Ownership. Moorhead:Sequenta Inc: Employment, Equity Ownership. Carlton:Sequenta Inc: Employment, Equity Ownership. Zheng:Sequenta Inc: Employment, Equity Ownership.

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