Comprehensive Investigation of 225 Patients with Myeloid Malignancies and Erythroid Hyperplasia (≥50%) Demonstrates That Acute Erythroid Leukemia (AEL, according WHO Classification 2008) Differs Significantly From MDS but Overlaps with Other AML Subtypes and Pure AEL Regarding Clinical and Genetic Features.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1672-1672
Author(s):  
Ulrike Bacher ◽  
Claudia Haferlach ◽  
Tamara Alpermann ◽  
Susanne Schnittger ◽  
Wolfgang Kern ◽  
...  
Keyword(s):  
Who Classification ◽  
Molecular Genetic ◽  
Erythroid Cells ◽  
Employment Equity ◽  
Not Otherwise Specified ◽  
Erythroid Hyperplasia ◽  
Genetic Abnormalities ◽  
Genetic Features ◽  
Equity Ownership ◽  
Who 2008

Abstract Abstract 1672 Introduction: According to the WHO 2008, acute erythroid leukemia (AEL) is defined by erythroid hyperplasia ≥50% and ≥20% of myeloblasts of non-erythroid cells but <20% of all nucleated cells. “Pure AEL” is defined by ≥80% of erythropoiesis without relevant myeloblasts. Cases with erythroid hyperplasia ≥50% and ≥20% of myeloblasts of all nucleated cells are classified as acute myeloid leukemia (AML-MRC/NOS), while presence of <20% of myeloblasts of non-erythroid cells assigns cases to myelodysplastic syndrome (MDS). As the separation of MDS/AML with erythroid hyperplasia in different categories is still under debate, we studied 225 patients with MDS/AML and ≥50% erythroid cells in bone marrow (BM) for cytomorphology, cyto-/molecular genetics, and prognosis. Patients/Methods: The cohort consisted of 225 pts (f/78; m/147; median age, 68.8 yrs; 18.5–88.4 yrs) with BM erythroid hyperplasia (≥50%) and different myeloid subtypes strictly defined according to WHO 2008: MDS: n=107; AML-MRC/NOS: n=32; AEL: n=79, pure AEL: n=7 (the WHO cohort “AML with recurrent genetic abnormalities” was excluded). All pts were investigated by MGG staining of BM and chromosome banding/FISH. In addition, we performed analysis for the NPM1 mutations (n=126 investigated), FLT3-ITD (n=135), MLL-PTD (n=136), and NRAS mutations (n=90). Results: MDS subtypes were as follows: RA: n=18; RARS: n=18; RCMD: n=21; RCMD-RS (WHO 2001): n=26; RAEB-1: n=22; RAEB-2: n=2. Most AML pts were categorized as “AML with myelodysplasia related changes; AML-MRC” (27/30 cases; 90%); 3 pts were classified as “AML, not otherwise specified; AML-NOS”, 2 pts were not evaluable for this aspect. We first compared the MDS cohort (n=107) with the AML cohort (all 118 pts with AML-MRC/NOS, AEL, and pure AEL): Overall survival (OS) was better in MDS than in the AML cohort (median: not reached vs. 13.9 months; p<0.001). In contrast, OS showed no significant differences across the AML-NOS/MRC, AEL, and pure AEL subgroups (9.3 vs. 13.9 vs. 6.1 months; n.s.). In the total cohort, aberrant karyotypes (KTs) were detected in 105/225 pts (46.7%) and were associated with inferior median OS when compared to normal KTs (aberrant KTs: 12.5 months vs. normal KTs: not reached; p<0.001). Aberrant KTs were more frequent in the AML categories when compared to MDS (69/118; 58.5%; vs. 36/107; 33.6%; p<0.001), but showed no significant differences across the different AML subgroups: AML-MRC/NOS: 20/32; 62.5%; AEL: 44/79; 55.7%; pure AEL: 5/7; 71.4%; n.s.). Performing cytogenetic risk categorization according to revised MRC criteria (Grimwade, 2010) for the whole cohort, unfavorable KTs showed an inferior prognosis compared to intermediate KTs (unfav. KTs: 65/225; 28.9%; median OS: 7.6 months; vs. intermed. KTs: 160/225; 71.1%; not reached; p<0.001). The pts from the AML cohort more frequently had unfav. KTs than those with MDS (AML cohort: 50/118; 42.4% vs. MDS: 15/107; 14.0%; p<0.001). Unfav. KTs were similarly distributed in the AML cohort (AML-MRC/NOS: 14/32; 43.8%; AEL: 32/79; 40.5%; pure AEL: 4/7; 57.1). Regarding the molecular markers, we detected the NPM1mut in 25/126 investigated (19.8%; MDS: 0/43; AML cohort: 22/91; 24.2%), FLT3-ITD in 5/135 (3.7%; MDS: 0/43; AML cohort: 5/92; 5.4%), MLL-PTD in 12/136 (8.8%; MDS: 2/44; 4.5%; AML: 10/92; 10.9%), and NRASmut in 4/90 (4.4%; MDS: 1/42; 2.4%; AML: 3/48; 6.2%). Mutation frequencies did not differ significantly in the MDS vs. AML categories or across the AML-MRC/NOS, AEL, and pure AEL subgroups. Conclusions: MDS with erythroid hyperplasia (≥50%) was clearly separated from the AML cohort (consisting of AML-MRC/NOS, AEL, and pure AEL, all with ≥50% of erythropoiesis) by less adverse cytogenetics and by improved survival. In contrast, no significant differences were observed across the different acute leukemia subentities regarding prognosis and cyto-/molecular genetic features. These data support the separation of MDS and AML with ≥50% of erythroid precursors according to the WHO classification. However, with respect to different AML subgroups, the separation to AEL, pure AEL, and AML-MRC/-NOS having ≥50% erythropoiesis seems arbitrary: these AML subtypes show no significant differences regarding prognosis or genetic risk profiles. This argues in favor of a combined group of AML with erythroid hyperplasia aiming to facilitate the definition for clinical studies and the development of therapeutic strategies. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership, Research Funding. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

The Recently Suggested MDS/MPN Subgroup “Refractory Anemia with Ring Sideroblasts Associated with Marked Thrombocytosis (RARS-T)” Demonstrates Unique Clinical, Cytogenetic, and Molecular Aspects: A Comprehensive Analysis of 57 Cases Strictly Defined According to WHO Criteria

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3081-3081
Author(s):  
Ulrike Bacher ◽  
Johanna Flach ◽  
Claudia Haferlach ◽  
Tamara Alpermann ◽  
Wolfgang Kern ◽  
...  
Keyword(s):  
Who Classification ◽  
Normal Karyotype ◽  
Favorable Outcome ◽  
Refractory Anemia ◽  
Employment Equity ◽  
Who Criteria ◽  
Ring Sideroblasts ◽  
Equity Ownership ◽  
Wbc Count ◽  
Who 2008

Abstract Abstract 3081 Introduction: In the new WHO 2008 classification, “refractory anemia with ring sideroblasts associated with marked thrombocytosis” (RARS-T) represents a provisional entity defined by platelets ≥450 ×109/l (being lowered from 600 ×109/l; WHO 2001), proliferation of large megakaryocytes, bone marrow (BM) blasts <5%, and ring sideroblasts ≥15% of nucleated erythropoiesis. The separation of RARS-T from other myeloproliferative/myelodysplastic neoplasms is still under debate. Patients: To further characterize this subtype and to evaluate whether its separate position in the WHO classification is justified from biologic/genetic aspects, we analyzed 57 patients with a diagnosis of RARS-T (strictly defined according to WHO 2008 criteria) for peripheral blood parameters, BM morphology, cyto-/molecular genetics, and clinical profiles. The study cohort consisted of 34 females and 23 males (median age, 76 years, range, 51–92 yrs; 52 de novo; 5 therapy-associated). At the time of analysis, all pts were at diagnosis or therapy naïve. Patients with a sole del(5q) or >5% of blasts were excluded according to WHO criteria. Methods: All BM samples underwent May Giemsa Gruenwald and iron stainings. Chromosomal banding analysis (and FISH if needed) were performed in 56/57 cases. PCR was done for the following markers: JAK2V617F (investigated: n=47), MPLW515 (n=46), NRAS (n=24), TET2 mutations (TET2mut, n=14), MLL-PTD (n=13), FLT3-ITD (n=12), and CBL (n=16). Result: Median WBC count was 7.9 ×109/l (range, 3.1–60.0 ×109/l), median hemoglobin (Hb) level was 10 g/dl (range, 6–13 g/dl), and median platelet count was 572 ×109/l (range, 454-1, 737 ×109/l). The median ring sideroblast count was 60% (range, 18–92%). Karyotypes (KT) were as follows: normal KT: n=52 (52/56; 93%); +8: n=2; -Y: n=1. The most frequent mutation was the JAK2V617F (18/47; 38%); an MPLW515 mutation was detected in 3/46 (7%). From the 46 pts being analyzed both for the JAK2 as for the MPLmut, 21 (45.6%) were observed with one of both markers; there was no coincidence of the JAK2 and the MPL mutations. Furtheron, 5/14 (36%) had a TET2 mutation. Coincidences of molecular markers were observed in 3 pts who had a JAK2V617F and a TET2mut in parallel (TET2mut: 3/10; 30% in JAK2mut pts; vs. 2/4 in JAK2 wildtype pts; n.s.). No patient had a JAK2V617F and MPLW515 in parallel. There was no mutation of the NRAS, MLL-PTD, FLT3, or CBL genes in pts investigated for these markers. A positive JAK2V617F mutated status correlated significantly with higher platelets (p=0.038; T-test), whereas no significant correlations were observed for the respective medians taken as thresholds for leukocytes (≥7.9 ×109/l vs. <7.9 ×109/l vs.), Hb (≥10.0 g/dl vs. <10.0 g/dl), or ring sideroblast percentages (≥60% vs. <60%). All 3 pts with MPLW515mut had platelets ≥600×109/l. Cytogenetic aberrations were independent from the JAK2mut status (normal karyotype: 17/45 JAK2mut; 38%; vs. aberrant KT: 1/2 JAK2mut; n.s.) and the MPLmut status (normal KT: 3/44 MPLmut; 7%; vs. aberrant KT: 0/2; n.s.). Higher WBC count (≥7.9 ×109/l) was correlated to a higher Hb level (≥10 g/dl) (p=0.47) and to higher platelets (≥600 ×109/l) (p=0.011). The patients with RARS-T had a favorable outcome with 84.6% being alive at 2.5 years. Conclusion: Investigation of 57 patients strictly fulfilling the criteria of the WHO 2008 classification was able to confirm the unique profile of RARS-T in all aspects: patients with the RARS-T had a normal karyotype (>90% of all cases), had no prognostically adverse cytogenetic alterations, and frequently showed mutations of the JAK2 (V617F) or MPL (W515K/L) genes (45.6% in total). The molecular profile was even more homogeneous in RARS-T cases with ≥600 ×109/l platelets (the WHO threshold from 2001) due to significantly higher proportions of JAK2V617F positive cases when compared to cases with platelets between 450 and <600 ×109/l. However, from clinical aspects, patients with RARS-T had a favorable outcome in our study independent of the molecular state or the number of platelets. These data support to include RARS-T as definite subtype in the next edition of the WHO classification. The frequent occurrence of TET2 mutations in our cohort has to be noted for future diagnostic and classification approaches. Therefore, in cases suspicious for RARS-T but without evidence of a JAK2V617F, molecular screening should be performed including analysis for alterations of the TET2 and MPL genes. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership, Research Funding. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

scholarly journals A Comprehensive Cytogenetic and Molecular Genetic Characterization of Patients with T-PLL Revealed Two Distinct Genetic Subgroups and JAK3 Mutations As an Important Prognostic Marker

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1639-1639
Author(s):  
Anna Stengel ◽  
Wolfgang Kern ◽  
Melanie Zenger ◽  
Karolina Perglerová ◽  
Susanne Schnittger ◽  
...  
Keyword(s):  
T Cell ◽  
Prognostic Marker ◽  
Array Cgh ◽  
Molecular Genetic ◽  
Employment Equity ◽  
Copy Number State ◽  
Tp53 Mutations ◽  
Genetic Abnormalities ◽  
Equity Ownership

Abstract Background: T-cell prolymphocytic leukemia (T-PLL) is a rare, mature T-cell neoplasm with poor prognosis. Only few T-PLL cases have been analyzed with regard to cytogenetic and molecular genetic aberrations so far. Therefore, we performed a comprehensive characterization of patients with T-PLL, including the identification of potential correlations between the respective markers and their impact on prognosis. Methods: The cohort comprised 47 T-PLL cases (32 male, 15 female). Median age was 69.8 years (range: 32.7-86.6 years). Diagnosis of T-PLL was assigned by immunophenotyping and cytomorphology. All 47 patients were further investigated using (i) chromosome banding analysis (CBA), (ii) interphase FISH to determine the copy number state for TP53 and ATM and chromosomal rearrangements of TCRA/D and TCL1 and (iii) array CGH. Next-generation amplicon deep-sequencing was performed to analyze mutations in ATM,BCOR, TP53 (n=47, respectively); JAK1 (n=44) and JAK3 (n=45) were analyzed by Sanger sequencing. Clinical follow-up data was available for 43 patients. Results: In all 47 cases, chromosomal abnormalities and/or molecular mutations were detected. Combining CBA and FISH data, an inv(14)(q11q32)/t(14;14)(q11;q32) was observed in 37/47 (78.7%) cases, a t(X;14)(q27;q11) in 3 cases (6.4%) and an i(8)(q10) in 17/47 (36.2%) cases. ATM deletions were detected in 27/47 (57.5%), TP53 deletions in 11/47 (23.4%) patients. Array CGH analyses revealed additional gains and losses of specific chromosomal regions, mainly affecting 7q (deletions in region 7q34-7q36; n=16), 12p (deletions in 12p12-12p13; n=11) and 22q (deletions in 22q11-q12 with a concomitant gain of 22q12-q13; n=8). Regarding molecular analyses, the most frequently mutated gene was ATM (34/47; 72.3%). Mutations in TP53 were found in 7/47 (14.9%) and in BCOR in 4/47 (8.5%) patients. Mutations of JAK1 were found in 3/44 (6.8%), and of JAK3 in 8/45 (17.8%) cases. ATM and TP53 frequently carried a mutation of one allele and a deletion of the other: 23/34 (67.6%) cases with ATM mutation also showed an ATM deletion and in 5/7 (71.4%) cases with TP53 mutation also a TP53 deletion was detected. Regarding chromosomal aberrations, all cases with i(8)(q10) harbored a TCRA/D rearrangement and an ATM mutation, whereas TP53 mutations were not present in any case with i(8)(q10). ATM mutations were found to be correlated to TCRA/D rearrangements (33/40 TCRA/D+ cases, 82.5%; 1/7 TCRA/D- cases, 14.3%; p<0.001). In contrast, TP53 mutations were predominantly observed in patients without TCRA/D rearrangement (4/7 TCRA/D- cases, 57.1%; 3/40 TCRA/D+ cases, 7.5%; p=0.008). Additionally, all three JAK1 mutations were detected in cases with a TCRA/D rearrangement. When splitting the cohort into patients ≤60 years (n=13) and >60 years (n=34), JAK1 mutations (0/12 vs. 3/32) and mutations/deletions in the TP53 gene were detected exclusively in patients >60 years (TP53mut: 0/13 vs. 7/34; TP53del: 0/13 vs. 11/34). JAK3 mutations were also found predominantly in older patients (1/12; 8.3% vs. 7/33; 21.2%). Median overall survival (OS) was 27.4 months. No influence on OS was found for mutations and/or deletions of ATM, TP53, BCOR orJAK1 or aberrations of chromosomes 8 or 14. The age of patients was found to impact OS (median OS, ≤60 years: 29.0 months vs. >60 years: 15.9 months), although this was not significant (p=0.077). However, OS was found to be significantly shorter in patients with JAK3 mutation compared to patients without JAK3 mutation (median OS, 5.1 months vs. 29.1 months; p=0.009). Conclusions: Genetic abnormalities were revealed in all 47 cases with T-PLL. Two distinct genetic subgroups of T-PLL were identified: A large subset, comprising 81% of patients, showed abnormalities involving the TCRA/D locus activating the proto-oncogenes TCL1 (14q32) or MTCP1 (Xq28). This subgroup had higher frequencies of i(8)(q10) and of ATM mutations, while the second group was characterized by a higher frequency of TP53 mutations (figure). Further, JAK3 mutations were identified as an important prognostic marker, showing a significant negative impact on OS. Figure 1: Genetic abnormalities in T-PLL Figure 1:. Genetic abnormalities in T-PLL mut=mutation, del=deletion, TCRA/D=rearrangements involving TCRA/D, TCL1=rearrangements involving TCL1, MTCP1= rearrangements involving MTCP1 Disclosures Stengel: MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Zenger:MLL Munich Leukemia Laboratory: Employment. Perglerová:MLL2 s.r.o.: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Impact of Expression of BAALC, CDKN1B, ERG, EVI1, and MN1 on Prognosis and Their Association with Karyotype, FLT3-ITD, NPM1 and MLL-PTD Status In Adult AML: A Comprehensive Study on 286 Cases

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 953-953
Author(s):  
Claudia Haferlach ◽  
Alexander Kohlmann ◽  
Sonja Schindela ◽  
Tamara Alpermann ◽  
Wolfgang Kern ◽  
...  
Keyword(s):  
Molecular Genetic ◽  
Normal Karyotype ◽  
Genetic Alterations ◽  
Complex Karyotype ◽  
Employment Equity ◽  
Aberrant Expression ◽  
Cox Regression Analysis ◽  
Total Cohort ◽  
Equity Ownership ◽  
Low Expression

Abstract Abstract 953 Introduction: The WHO classification in 2008 listed for the first time aberrant expression of genes as molecular genetic alterations affecting outcome in AML. High expression of BAALC, ERG and MN1 were shown thus far to be associated with unfavorable outcome in normal karyotype AML (AML-NK). In addition high EVI1 expression was suggested to predict poor outcome. Recently, our group identified low expression of CDKN1B as a favorable prognostic marker. The aim of this study was to evaluate the expression of BAALC, CDKN1B, ERG, EVI1 and MN1 in AML comprising all cytogenetic risk groups with respect to their association with distinct cytogenetic and known molecular genetic subgroups and their impact on prognosis. Patients/Methods:: Expression levels of BAALC, CDKN1B, ERG, EVI1 and MN1 were determined by oligonucleotide microarrays (HG-U133 Plus 2.0, Affymetrix) in 286 AML (t(15;17) n=15; t(8;21) n=16; inv(16) n=7; normal karyotype n=99; 11q23/MLL-rearrangements n=10; complex karyotype n=51; other abnormalities n=88). Patients were further analyzed for mutations in NPM1, FLT3-ITD, CEPBA and MLL-PTD. Results: Expression of BAALC, CDKN1B, ERG, EVI1 and MN1 varied significantly between genetic subgroups: While t(15;17), t(8;21) and 11q23/MLL-rearrangements were associated with low CDKN1B expression, AML-NK and NPM+ cases showed a higher CDKN1B expression. Lower BAALC expression was observed in AML with t(15;17), 11q23/MLL-rearrangement and AML-NK as well as in FLT3-ITD+ AML and in NPM1+ AML, while in AML with other abnormalities a higher BAALC expression was observed. ERG expression was lower in AML with 11q23/MLL-rearrangement and normal karyotype, while it was higher in AML with complex karyotype. Low EVI1 expression was observed in AML with t(15;17), t(8;21), inv(16) and AML-NK, while it was higher in AML with 11q23/MLL-rearrangements. Low MN1 expression was associated with t(15;17), t(8;21) and AML-NK, while it was increased in cases with inv(16) or other abnormalities. Next, Cox regression analysis was performed with respect to overall survival (OS) and event free survival (EFS). In the total cohort high BAALC and ERG expression as continuous variables were associated with shorter OS and EFS while CDKN1B, EVI1 and MN1 had no impact. Furthermore the cohort was subdivided into quartiles of expression for each gene. After inspection of the survival curves the cut-off for high vs low expression was set as follows: BAALC: 75th percentile, CDKN1B: 25th percentile, ERG and MN1: 50th percentile. For EVI1 expression pts were separated into expressers (n=44) and non-expressers (n=242). Low CDKN1B expression was associated with longer OS and EFS in the total cohort (p=0.005, not reached (n.r.) vs 14.9 months (mo); p=0.013, 31 vs 9.7 mo). High BAALC expression had no impact on OS, but was associated with shorter EFS in the total cohort as well as in AML with intermediate cytogenetics and AML with other abnormalities (p=0.032, 6.2 vs 13.0 mo; p=0.027, 5.1 vs 11.3 mo; p=0.006, 2.3 vs 14.8 mo). High ERG expression was significantly associated with shorter OS and EFS in the total cohort (p=0.002, 12.5 mo vs n.r.; p=0.001, 8.1 vs 15.7 mo) as well as in AML-NK (p=0.001, 11.3 mo vs n.r.; p=0.010, 7.2 vs 22.1 mo). OS was also shorter in AML with unfavorable karyotype (p=0.048, median OS 9.3 mo vs n. r.). With respect to MN1 high expressers had a significantly shorter OS and EFS in the total cohort (p=0.004, 12.3 mo vs. n.r.; p=0.001, 8.1 vs 16.7 mo) as well as in AML-NK (p=0.001, 9.7 mo vs n.r.; p=0.001, 5.1 vs 22.1 mo). In a multivariate analysis including CDKN1B, ERG and MN1 all parameters retained their impact on OS as well as on EFS, while BAALC lost its impact on EFS. Adding MLL-PTD, NPM1+/FLT3-ITD-, favorable and unfavorable karyotype into the model demonstrated an independent significant adverse impact on OS for MLL-PTD (p=0.027, relative risk (RR): 2.38) and ERG expression (p=0.044, RR: 1.59) only. In the respective analysis for EFS only favorable karyotype showed an independent association (p=0.002, RR: 0.261). Conclusion: 1) Expression of BAALC, CDKN1B, ERG, EVI1 and MN1 varies significantly between cytogenetic subgroups. 2) BAALC as a continuous variable and CDKN1B, ERG and MN1 as dichotomized variables are independently predictive for OS and EFS in AML. 3) ERG expression even retains its independent prediction of shorter OS if cytogenetic and other molecular genetic markers are taken into account. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Schindela:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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

Blood ◽  
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.

RAP-536 Promotes Terminal Erythroid Differentiation and Reduces Anemia in a Murine Model of Myelodysplastic Syndromes

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3796-3796 ◽  
Author(s):  
Rajasekhar NVS Suragani ◽  
Robert Li ◽  
Dianne Sako ◽  
Asya Grinberg ◽  
R. Scott Pearsall ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Erythroid Differentiation ◽  
Severe Anemia ◽  
Ineffective Erythropoiesis ◽  
Employment Equity ◽  
Erythroid Hyperplasia ◽  
Erythroid Precursors ◽  
Equity Ownership ◽  
Terminal Erythroid Differentiation

Abstract Abstract 3796 Myelodysplastic syndromes (MDS) are a group of hematopoietic stem cell disorders characterized by peripheral blood cytopenias such as anemia, neutropenia or thrombocytopenia. Ineffective erythropoiesis due to increased proliferation and abortive maturation of precursors leads to severe anemia, the most common cytopenia observed in MDS syndromes. Despite elevated erythropoietin (EPO) and erythroid hyperplasia, MDS patients are often given recombinant EPO therapy to stimulate erythropoiesis. However, only a small proportion of patients respond to EPO therapy. Frequent blood transfusions as supportive care result in iron overloading and recently iron overloading is also linked to enhanced progression to AML. Therefore, alternative therapies are necessary to treat anemia in MDS patients. Signaling by members of the TGFβ superfamily are known regulators of erythropoiesis. We developed ACE-536, a ligand trap consisting of a modified activin receptor Type IIB extracellular domain linked to a human Fc domain. In vitro assays revealed that ACE-536 inhibits smad 2/3 ligands of the signaling pathway but not smad 1/5/8 ligands. Dose dependent studies using ACE-536 in mice, rats and monkeys revealed that ACE-536 treatment resulted in increased red blood parameters but did not affect other cell types. These data suggests that ACE-536 inhibits smad 2/3 phosphorylation modulating the expression of downstream genes involved in erythroid development pathway. BFU-E and CFU-E colony formation assays from bone marrow and spleen in mice following ACE-536 treatment revealed that ACE-536 did not affect the proliferation stages of erythropoiesis. In mice, terminal erythroid differentiation analysis by flow cytometry at 72hrs following RAP-536 (10mg/kg) treatment demonstrated decreased basophilic and increased ortho- and poly-chromatophilic erythroblasts and reticulocytes compared to VEH treatment. Cell cycle analysis of bone marrow and splenic erythroblasts counterstained with BrdU and 7-AAD after RAP-536 (10mg/kg, for 24 hours) or VEH treatment to EPO pre-treated (1500 units/kg, for 40 hours) mice (N=5/group) revealed that EPO+RAP-536 treatment resulted in significant decrease in S-phase and increase in G1/G2-phases of cell cycle compared to EPO+VEH treatment. In addition, EPO+RAP-536 treatment resulted in a greater increase in RBC parameters than either of the treatments alone. Together, these results demonstrate that ACE-536 increases red blood cell formation by promoting maturation of late stage erythroblasts. We then investigated the effect of ACE-536 on anemia in NUP98-HOXD13 (NHD13) transgenic murine model of MDS. NHD13 mice develop anemia, neutropenia and lymphopenia, with normal or hyper cellular bone marrow. A Majority of the mice die by 14 months due to severe pancytopenia or progression to acute myeloid leukemia. In this study, mice were divided into three groups based on age. Early (∼4 months old), mid (∼8 months old) and late stage (∼10 months) groups were randomized and dosed with either RAP-536 at 10 mg/kg or VEH twice per week for 6–8 weeks. NHD13 mice in each group had severe anemia characterized by reduced RBC, Hemoglobin and HCT and compared to wild-type littermates prior to treatment. Treatment of RAP-536 for 6–8 weeks significantly increased RBC parameters and reversed anemia at all stages. Peripheral blood smear analysis revealed no indication of increased leukemic progression due to RAP-536 treatment. Cell differential and flow cytometric evaluation of erythroid precursors from bone marrow demonstrated decreased erythroid precursors and hyperplasia after RAP-536 treatment compared to vehicle treated control. Our data demonstrate that RAP-536 can increase hematology parameters by enhancing maturation of terminally differentiated red blood cells. We have shown RAP-536 corrects ineffective erythropoiesis, decreases erythroid hyperplasia and normalizes myeloid: erythroid ratios without enhanced progression to AML in a murine MDS model. Therefore ACE-536 may represent a novel treatment for anemia associated with MDS, particularly in patients that are refractory to EPO therapy. ACE-536 has completed Phase I clinical trials in healthy human volunteers and Phase II study in MDS patients is planned. Disclosures: Suragani: Acceleron Pharma Inc: Employment, Equity Ownership. Li:Acceleron Pharma Inc: Employment, Equity Ownership. Sako:Acceleron Pharma Inc: Employment, Equity Ownership. Grinberg:Acceleron Pharma Inc: Employment, Equity Ownership. Pearsall:Acceleron Pharma Inc: Employment, Equity Ownership. Kumar:Acceleron Pharma Inc: Employment, Equity Ownership.

Assessment Of Human Equilibrative Nucleoside Transporter 1 (hENT1) – Expression By Flow Cytometry In Acute Myeloid Leukemia and Myelodysplastic Syndromes and Correlation To Clinical Outcome In Acute Myeloid Leukemia Patients Treated With Intensive Chemotherapy

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2582-2582 ◽  
Author(s):  
Frauke Bellos ◽  
Bruce H. Davis ◽  
Naomi B. Culp ◽  
Birgitte Booij ◽  
Susanne Schnittger ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Myeloid Leukemia ◽  
De Novo ◽  
Molecular Genetic ◽  
Newly Diagnosed ◽  
Employment Equity ◽  
Equity Ownership ◽  
De Novo Aml ◽  
Very High ◽  
Acute Myeloid

Abstract Background Nucleoside analogs depend on cellular hENT1 expression for entry into cells and cytotoxic activity. Studies suggest low cellular hENT1 levels correlate with poor response to such chemotherapies in solid tumors, data on AML and MDS is scarce. Aim To examine hENT1 expression by multiparameter flow cytometry (MFC) in newly diagnosed AML and MDS and correlate results to morphologic, cytogenetic (CG) and molecular genetic (MG) findings. To examine hENT1 expression with respect to clinical outcome in AML patients (pts) treated with intensive cytarabine-based chemotherapy (CHT). Methods We studied pts with newly diagnosed AML (n=145) and MDS (n=96), 133/108 male/female, median age 67.3 (AML) and 73.3 years (MDS). CG was done in 130 AML and 86 MDS. Pts included 107 de novo AML, 9 t-AML, 29 s-AML; FAB: 9 M0, 27 M1, 50 M2, 9 M3, 21 M4, 8 M4eo, 7 M5, 14 not classified; by CG (MRC): 21 favorable, 75 intermediate, 34 adverse. 91 were de novo MDS, 5 t-MDS; 1 RARS, 17 RCMD-RS, 37 RCMD, 3 5q- syndrome, 3 RAEB-1, 5 RAEB-2, 1 CMML, 24 not classified; 2 IPSS-R very low, 55 IPSS-R low, 8 IPSS-R intermediate, 8 IPSS-R high, 13 IPSS-R very high. hENT1 expression was quantified by a novel four color intracellular staining assay using monoclonal antibodies against hENT1, CD45, CD64 and myeloperoxidase. Median fluorescence intensities (MFI) of hENT1 were determined in myeloid progenitors (MP), granulocytes (G) and monocytic cells (Mo) and correlated to hENT1 MFI in lymphocytes to derive hENT1 index (index). Results No correlation of index to age, gender, hemoglobin level or counts for blasts, WBC or platelets was detected. In AML, we generally saw higher index by trend in the more favorable prognostic subgroups. M3/t(15;17)/PML-RARA+ displayed higher index in MP than non-M3 AML (4.24 vs 2.56, p<0.001). G index was lower in M0 (3.01) vs M1, M2, M4 and M4eo (5.66, 4.34, 5.35, 4.77; p=0.01, 0.028, 0.004, 0.043, respectively) and in M2 compared to M1 and M4 (4.34. vs 5.66 and 5.35, p=0.01 and 0.033, respectively). M2 showed lower MP index than M5 (2.42 vs 2.99, p=0.016). Considering CG, index in MP was higher in favorable vs intermediate and adverse pts (3.05 vs 2.58 and 2.53, p=0.034 and 0.023, respectively), Mo index was higher ín favorable vs adverse pts (3.17 vs 2.71, p=0.044). By MG, higher index in Mo and G was observed in RUNX1-RUNX1T1+ AML (4/83, 4.32 vs 3.04, p=0.01; 8.16 vs 4.60, p=0.002, respectively). Higher index for MP was found in FLT3-ITD mutated (mut) (18/111; 3.19 vs 2.62, p=0.012), CEPBA mut (4/26, 3.15 vs 2.35, p=0.004) and for Mo in NPM1 mut AML (23/104; 3.72 vs 2.84, p=0.02), whereas lower index for MP was found in RUNX1mut pts (13/65; 2.17 vs 2.59, p=0.031). De novo AML displayed higher MP index than s-AML (2.7 vs 2.28, p=0.008). Using lowest quartile of index for MP (2.1185) as cut-off, AML pts in the MRC intermediate group treated with CHT (n=38) had inferior OS if MP index was below vs above this cut-off (OS at 6 months 63% vs. 95%, p=0.017, median follow up 4.6 months). MDS showed lower Mo and MP index than AML (2.68 vs 2.96, p=0.021, 1.84 vs 2.65, p<0.001, respectively). By IPSS-R, significance was reached for higher index in Mo and MP in very low risk compared to low risk pts (3.39 vs 2.54, p=0.013 and 4.07 vs 1.78, p<0.001, respectively), MP in very low compared to intermediate and high risk pts (4.07 vs 1.95, p=0.004; 4.07 vs 1.76, p=0.002), and MP and G in very low vs very high risk pts (4.07 vs 1.71, p=0.005; 5.86 vs 3.85, p=0.001, respectively). IPSS-R intermediate vs poor and very poor showed lower G index (5.47 vs 3.59, p=0.018 and vs 3.85, p=0.034 respectively). Conclusion AML with genetic and molecular genetic good risk profile had higher hENT1 expression in MP, G and Mo, suggesting a causal mechanism for better response to CHT and better outcome. Consequently, AML with poor risk molecular genetics (RUNX1 mut) showed lower levels of hENT1 in MP. The detection of higher levels in FLT3-ITD mut pts is in line with reportedly good response to CHT, overall worse outcome being mostly due to early relapses. Strikingly, we saw differences in outcome in pts treated with CHT according to hENT1 expression with shorter OS in pts with low index for MP. Higher index in de novo AML than s-AML and MDS may be causal for better response to nucleoside-based CHT in de novo AML. Data for MDS may be interpreted accordingly, lower risk cases showing higher index in MP, G and Mo. Further analyses are needed to explore hENT1 expression in AML and MDS more comprehensively. Disclosures: Bellos: MLL Munich Leukemia Laboratory: Employment. Davis:Trillium Diagnostics, LLC: Equity Ownership. Culp:Trillium Diagnostics, LLC: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

scholarly journals The RUNX1 Gene Is Altered in 26% of AML Patients Either By Translocation, Mutation, Gain or Deletion

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 123-123
Author(s):  
Claudia Haferlach ◽  
Niroshan Nadarajah ◽  
Wolfgang Kern ◽  
Susanne Schnittger ◽  
Torsten Haferlach
Keyword(s):  
De Novo ◽  
Intermediate Risk ◽  
Missense Mutations ◽  
Employment Equity ◽  
Runx1 Gene ◽  
Genetic Abnormalities ◽  
Partner Gene ◽  
Different Types ◽  
Equity Ownership ◽  
De Novo Aml

Abstract Background: In AML four types of acquired alterations of the RUNX1 gene have been described: 1. translocations involving RUNX1 leading to fusion genes such as RUNX1-RUNX1T1, 2. molecular mutations, 3. amplifications of RUNX1, 4. Partial or complete deletions of the RUNX1 gene. Aim: To determine the frequency of different RUNX1 alterations and to characterize the spectrum of accompanying genetic abnormalities. Patients and Methods: We screened 726 de novo AML patients (pts) for RUNX1 deletions (del) and translocations using a dual color break-apart probe covering the 5' and 3' part of RUNX1 (MetaSystems, Altlussheim, Germany) and in addition evaluated RUNX1 mutations (mut) by Sanger or next-generation amplicon deep-sequencing. Median age was 67 yrs (range: 18 to 100 yrs). For all patients cytogenetics was available and categorized according to MRC criteria (Grimwade et al. Blood 2010). Partial deletions of RUNX1 as detected by FISH were confirmed by array CGH (Agilent Technologies, Santa Clara, CA). Results: In 89/726 pts (12.3%) abnormalities of the RUNX1 gene were detected by FISH: 10 pts (1.4%) showed a deletion encompassing the whole RUNX1 gene while additional 9 pts (1.2%) showed a partial loss of one RUNX1 copy. A gain of one RUNX1 copy was present in 45/726 (6.2%) pts. In 3 pts a gain of the 5' part of RUNX1 was accompanied by a loss of the 3' part while in 2 pts one copy of the 3' part was gained accompanied by a loss of the 5' part. A translocation affecting the RUNX1 gene was detected in 31 pts (4.3%). The partner gene was RUNX1T1 in 29 pts and located on 16q13 and 18p11 in one pt each. One pt with a RUNX1 translocation also showed a 5' RUNX1 deletion. In 110/726 pts (15.2%) a RUNX1mut was detected. Of these, 16 pts showed two and 5 pts three mutations in RUNX1. Thus, in total 136 mutations were detected in 110 pts: 58 (42.6%) were frameshift, 42 (30.9%) missense, 21 (15.4%) nonsense, 9 (6.6%) splice-site and 6 (4.4%) in-frame insertions/deletions. The RUNX1mut was homozygous in 15 pts, these were predominantly missense mutations (9/15; 60%). Within the subset of pts with RUNX1mut 2 harbored an additional RUNX1del and 9 pts a gain of a RUNX1 copy, while no RUNX1 translocation was present. In AML FAB type M0 both RUNX1mut and RUNX1del showed the highest frequencies (33.3% and 14.8%). 48.4% and 45.2% of cases with RUNX1 translocations were FAB type M1 and M2. While RUNX1mut were most frequent in the cytogenetic intermediate risk group (19.1%; favorable: 2.2%, adverse: 9.7%), RUNX1del were most frequent in pts with adverse risk cytogenetics (9.7%; favorable: 1.1%, intermediate: 0.8%). A comparable distribution was observed for a gain of RUNX1 copies (adverse: 19.4%, favorable: 4.5%, intermediate: 2.6%). With respect to additional molecular mutations all types of RUNX1 alterations were mutually exclusive of NPM1mut. Further, the frequency of DNMT3Amut and CEBPAmut was significantly lower in pts with RUNX1 alterations as compared to those without (14.3% vs. 34.3%; p<0.0001 and 6.4% vs. 13.4%; p=0.012). However, some striking differences between the different types of RUNX1 alterations were detected: ASXL1mut were significantly more frequent in pts with RUNX1mut (36.7%) but rather infrequent in pts with RUNX1del, gain and translocation (12.5%, 6.1%, and 6.7%). A comparable association was noticed for SF3B1mut which were frequent in RUNX1mut pts (23.8%) and rather infrequent in pts with RUNX1del, gain and translocations (0%, 10.5%, and 0%). In contrast, pts with either RUNX1del or RUNX1 gains showed a significantly higher TP53mut frequency (66.7% and 35.3%) as compared to RUNX1mut or RUNX1 translocated pts (7.1% and 4.8%). In the total cohort median overall survival (OS) was 18.7 months and differed significantly between the different types of RUNX1 alterations: for RUNX1 translocations, mutations, gains and deletions it was 35.5, 14.1, 12.4 and 4.3 months. Conclusions: 1. The RUNX1 gene is altered in 26% of AML. 2. All types of RUNX1 alterations predominantly occur in AML M0, M1 and M2 and are rare in the remainder AML. 3. They are mutually exclusive of NPM1 mutations and show a negative association with DNMT3A mutations. 4. While RUNX1 mutations were most frequent in patients with intermediate risk cytogenetics, RUNX1 deletions and gains were most frequent in patients with adverse cytogenetics. 5. Outcome differs significantly and is best in patients with RUNX1 translocations and worst in cases with RUNX1 deletions. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

scholarly journals Clonal Analysis of Human Bone Marrow CD34+ Cells Edited By BCL11A-Targeting Zinc Finger Nucleases Reveals Clinically Relevant Levels of Fetal Globin Expression in Edited Erythroid Progeny

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3234-3234 ◽  
Author(s):  
Kai-Hsin Chang ◽  
Timothy Sullivan ◽  
Mei Liu ◽  
Xiao Yang ◽  
Chao Sun ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Zinc Finger ◽  
Zinc Finger Nucleases ◽  
Erythroid Cells ◽  
Coding Region ◽  
Employment Equity ◽  
Cd34 Cells ◽  
F Cells ◽  
Equity Ownership ◽  
Patent Applications

Abstract Sickle cell disease (SCD) is one of the most common inherited blood disorders and is caused by a mutation at the adult beta globin gene resulting in substitution of valine for glutamic acid at position 6 in the encoded protein. While SCD can be cured by hematopoietic stem cell transplant (HSCT), complete donor chimerism is not required to achieve clinical benefits. Stable mixed chimerism of 10-15% in bone marrow or peripheral blood nucleated cells with >70% donor-derived RBCs has been reported to achieve transfusion independence and a symptom-free state in a SCD patient. It has also been proposed that SCD can be treated by reactivating developmentally silenced fetal gamma globin to form fetal hemoglobin (alpha2gamma2, HbF), which inhibits polymerization of HbS. The effect of HbF is predicted to be maximal when HbF content per cell exceeds 10 pg (~30% of total Hb). Furthermore, pathology is prevented when protective F cells (>30% HbF per cell) constitute >70% of total RBCs. We hypothesize that in a gene therapy setting, if >15% of SCD patients' autologous HSCs are programmed to produce protective F cells during erythropoiesis, it will translate into >70% protective F cells in circulation and provide significant alleviation of clinical symptoms. Genome wide association studies have identified BCL11A as a major modifier of HbF levels. Subsequent studies have shown that BCL11A plays a critical role in the fetal to adult globin developmental switch and in repressing fetal globin expression in adult erythroid cells. Conditional inactivation of BCL11A in adult erythroid cells leads to high levels of pan-cellular fetal globin expression and correction of hematologic and pathologic defects in a humanized SCD mouse model. Previously, we have reported that zinc finger nucleases (ZFNs) targeting BCL11A either in the coding region or the GATAA motif in the erythroid-specific enhancer efficiently disrupt the BCL11A locus in human primary CD34+ cells following electroporation of ZFN-encoding mRNA. Elevated fetal globin expression in bulk erythroid cultures was observed following disruption. To determine what percentage of HSPCs have been modified and whether the HbF/F cell content has reached the hypothesized therapeutic level, we analyzed erythroid cells clonally derived from ZFN-transfected CD34+ cells. Genotype of each clonal culture was determined by deep sequencing and globin production was analyzed by a highly sensitive UPLC method. We found that up to 80% of the BFU-Es had both BCL11A alleles edited, half of which had KO/KO alleles (either out of frame mutations for coding region or elimination of the GATAA motif in the enhancer). BCL11A coding KO/KO cells expressed on average 79.1% ± 12.2% fetal globin (Mean ± SD) whereas GATAA motif enhancer region KO/KO cells expressed approximately 48.4% ± 14.1% fetal globin, in comparison with 14.5% ± 9.6% in WT/WT cells . These levels of fetal globin should be sufficiently high to confer protection against HbS polymerization in sickle cells. WT/KO cells in both coding and enhancer editing experiments showed an intermediate phenotype with fetal globin averaging 26.9%± 9.9% and 25.79% ± 12.6%, respectively. Interestingly, when background (WT/WT) fetal globin level was subtracted, the fetal globin levels in WT/KO cells are comparable to those observed in patients with BCL11A haploinsufficiency, which average 14.6%± 10.3%. Together, our data demonstrate that genome editing of BCL11A using highly efficient ZFNs can lead to clinically relevant levels of fetal globin expression in KO/KO erythroid cells. If the frequency of KO/KO BFU-Es we observed in vitro reflects the frequency of KO/KO HSCs in bone marrow after autologous transplantation, genome editing of BCL11A has the potential to provide significant clinical benefit for patients with SCD. Disclosures Chang: Biogen: Employment, Equity Ownership. Sullivan:Biogen: Employment, Equity Ownership. Liu:Biogen: Employment, Equity Ownership. Yang:Biogen: Employment, Equity Ownership. Sun:Biogen: Employment, Equity Ownership. Vieira:Biogen: Employment, Equity Ownership. Zhang:Biogen: Employment. Hong:Biogen: Employment, Equity Ownership. Chen:Biogen: Employment, Equity Ownership. Smith:Biogen: Employment, Equity Ownership. Tan:Biogen: Employment, Equity Ownership. Reik:Sangamo BioSciences: Employment, Equity Ownership, Patents & Royalties: Patent applications have been filed based on this work. Urnov:Sangamo BioSciences: Employment, Equity Ownership, Patents & Royalties: Patent applications have been filed based on this work. Rebar:Sangamo BioSciences: Employment. Danos:Biogen: Employment, Equity Ownership. Jiang:Biogen: Employment, Equity Ownership.

Cytogenetic and Molecular Genetic Shifts in 27 Genes Investigated By NGS Depict Specific Routes from MDS to s-AML in 38 Patients with Paired Samples

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2378-2378
Author(s):  
Andreia De Albuquerque ◽  
Manja Meggendorfer ◽  
Wolfgang Kern ◽  
Karolina Perglerová ◽  
Tamara Alpermann ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Molecular Genetic ◽  
Employment Equity ◽  
Data Set ◽  
Kit Mutation ◽  
Hematological Disorders ◽  
Secondary Acute Myeloid Leukemia ◽  
Paired Samples ◽  
Mutation Profile ◽  
Equity Ownership

Abstract Background: Myelodysplastic syndromes (MDS) are a heterogeneous group of hematological disorders that can progress to secondary acute myeloid leukemia (s-AML). We performed a comprehensive approach based on cytomorphology, cytogenetics and amplicon deep sequencing using a newly developed pan-myeloid gene panel in order to better clarify this process. Methods: Paired samples from 38 patients at initial diagnosis of MDS and at progression to s-AML were analyzed for the following genes: ASXL1, BCOR, BRAF, CBL, DNMT3A, ETV6, EZH2, FLT3TKD,FLT3-ITD, GATA1, GATA2, IDH1, IDH2, JAK2, KIT, KRAS, MLL-PTD, MPL, NPM1, NRAS, PHF6, RUNX1, SF3B1, SRSF2, TET2, TP53, U2AF1,WT1 and ZRSR2. With exception of RUNX1 and ZRSR2 (sequenced on the 454 NGS platform (454 Life Sciences, Branford, CT)) and MLL-PTD and FLT3-ITD (analysed by PCR), all remainder genes were sequenced using a microdroplet-based assay (RainDance, Lexington, MA) and the MiSeq sequencing instrument (Illumina, San Diego, CA). A median coverage of 7,626 reads (range 174-12,256) per gene was achieved. The lower limit of detection was set at 3%. Results: At MDS diagnosis, patients were classified according to WHO: RARS (n=2), RCMD (n=3), RCMD-RS (n=8), RAEB-1 (n=12), RAEB-2 (n=12), and MDS with isolated 5q deletion (n=1). Progression to s-AML was defined by blasts &gt;20% in bone marrow or peripheral blood. Transformation to AML occurred at a median time of 18 months (range 2 – 73) after MDS diagnosis. According to chromosome banding analysis and FISH, 14 (36.8%) MDS and 22 (57.9%) s-AML patients had an aberrant karyotype. Thirteen patients (34.2%; 8 with normal and 5 with already aberrant karyotype) gained chromosome abnormalities during progression to s-AML. All samples presented at least one mutation. The most frequent mutated genes were identical for MDS and s-AML: ASXL1 (36.8% / 42.1%); RUNX1 (36.8% / 39.5%); SF3B1 (23.7% / 23.7%); SRSF2 (47.4% / 47.4%) and TET2 (34.2% / 34.2%). Mutations in GATA1 and MPL were not found. Only one KIT-mutation was detected in a patient at MDS stage, while FLT3-TKD mutations were identified in s-AML samples only (7.9%; 3/38 patients). Results for MLL-PTD and FLT3-ITD were available for 58 samples (76.3%). The incidence of MLL-PTD was low (4.5%, 1/22 for MDS and 8.3%; 3/36 in s-AML). FLT3-ITD mutations were exclusively detected in 5 patients (14.3%) at s-AML stage. A total of 21 (55.3%) patients won at least 1 mutation during progression to s-AML. Surprisingly none of the gene mutation frequencies differed significantly between the MDS and s-AML stages. Therefore, we further compared this data set to an independent cohort of 944 newly diagnosed MDS patients (Haferlach T et al, Leukemia 2014). WHO diagnosis categories were comparable between both cohorts. However, mutation frequencies differed: in the present cohort the following mutations were more frequent than in the published cohort: GATA2 (5.3% vs 0.7%; P=0.044), IDH2 (18.4% vs 3.9%; P=0.001), RUNX1 (36.8% vs 10.6%; P=&lt;0.001) and SRSF2 (47.4% vs 17.5%; P=&lt;0.001), for ASXL1 (P=0.078), ETV6 (P=0.068), NRAS (P=0.064), suggesting a role of these genes as s-AML drivers. MDS patients with DNMT3Amut transformed faster to AML than DNMT3A wild-type (wt) cases (median of 8 vs 19 months; P= 0.022). Intriguingly, also SF3B1mut were associated with a faster s-AML progression compared to SF3B1wt (median 8 vs 19 months; P= 0.008). All mutations that were lost at the time of s-AML transformation were small clones (mutation load ≤10%), which hypothetically could have succumbed to more aggressive clones leading to transformation to AML. In detail, in 4 patients with mutation loss there was an increase in the loads of the remaining mutations, whereas their karyotype remained stable during s-AML transformation. On the other hand, the remaining 3 patients with mutation loss at the time of s-AML diagnosis had stable mutation loads in the remaining mutated genes but had a cytogenetic progression. Conclusions: 1) The molecular mutation profile remains stable between MDS and s-AML state in most of the cases and mutations were gained or lost in only a few cases. 2) FLT3-ITD was only detected at AML stage. 3) Mutations in GATA2, IDH2, RUNX1 and SRSF2 seem to predispose to s-AML transformation and mutations in DNMT3A and SF3B1 lead to a significantly faster s-AML progression. 4) Shifts in the karyotype were detected in more than one third of patients underlining its impact on s-AML transformation. Disclosures De Albuquerque: MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Perglerová:MLL2 s.r.o.: 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.

RUNX1 Mutated AML Are Associated with a Distinct Pattern of Cytogenetic and Additional Molecular Genetic Abnormalities

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 124-124
Author(s):  
Niroshan Nadarajah ◽  
Susanne Schnittger ◽  
Wolfgang Kern ◽  
Torsten Haferlach ◽  
Claudia Haferlach
Keyword(s):  
High Frequency ◽  
De Novo ◽  
Male Gender ◽  
The Other ◽  
Typical Pattern ◽  
Employment Equity ◽  
Cytogenetic Abnormalities ◽  
Genetic Abnormalities ◽  
Equity Ownership ◽  
De Novo Aml

Abstract Background: Mutations in RUNX1 have been reported in 5 to 20% of AML. The aim of this study was to analyse a large cohort of RUNX1 mutated AML in detail with respect to accompanying cytogenetic and moleculargenetic abnormalities, mutation type and mutation load. Patients and Methods: We investigated 468 AML with RUNX1 mutations (mut) all identified during diagnostic work-up in our laboratory. Sequencing was performed by either Sanger or next-generation sequencing. Median age was 71.9 yrs (range 18-91 yrs), male : female ratio 297 : 171. 369 patients had de novo AML, 75 s-AML following MDS, 24 t-AML. For all patients (pts) cytogenetics was available and categorized according to MRC (Grimwade et al. Blood 2010). Mutation data was available for NPM1 (n=455), MLL-PTD (n=454), CEBPA (n=449), FLT3-TKD (n=422), WT1 (n=394), FLT3-ITD (n=362), ASXL1 (n=293), TP53 (n=204), DNMT3A (n=143), TET2 (n=143), and SF3B1 (n=99). Data on FAB subtype was available in 342 cases (73.1%). Results: The most frequent FAB subtype in RUNX1mut AML was AML M2 (145/342; 42.4%), followed by M1 (23.1%), M4 (15.8%), M0 (15.5%), other subtypes were rare (<2%). Cytogenetics were favorable in 2 (0.4%), intermediate in 397 (84.8%) and adverse in 69 (14.7%) pts. Most frequent cytogenetic abnormalities were +8 (76; 34.7%), +13 (46; 21.0%), +21 (15; 6.8%), +11 (12; 5.5%), -7 (22; 10%), del(7q) (18; 8.2%), del(5q) (12; 5.5%). Only 12 pts (5.5%) showed a complex karyotype (> 3 aberrations). The frequency of all other abnormalities was <5%. Two pts showed a t(15;17)(q24;q21) and 3 MLL rearrangements (partner genes on 7q32, 17q21, 19p13). None of the other recurrent cytogenetic abnormalities according to WHO classification was present. 249 (53.2%) pts had a normal karyotype. ASXL1mut were the most frequent accompanying mutations (36.5%). Mutation frequencies for the other genes were: SF3B1 (27.3%), TET2 (26.6%), FLT3-ITD (17.7%), DNMT3A (16.1%), MLL-PTD (10.8%), CEBPA (6.9%; single mutated (sm): 5.3%, double mutated (dm): 1.6%), WT1 (5.8%), FLT3-TKD (5.5%), TP53 (4.4%), and NPM1 (1.1%). While mutations in ASXL1 and TET2 frequently occurred concomitantly (37.5% of ASXL1mut cases also harboured a TET2mut, p=0.032), ASXL1mut and SF3B1mut rarely co-occurred (only 2 ASXL1mut were SF3B1mut, p=0.001). There were no differences in mutation frequencies and cytogenetic abnormalities observed between de novo AML, s-AML and t-AML. In 368 cases (78.6%) one RUNX1 mutation was detected, 81 pts (17.3%) showed two, 16 cases (3.4%) three, and 3 cases (0.6%) four. The difference in mutation loads between the 2 mutations with the highest load was ≤10% in 55/100 pts, suggesting that both mutations were present in the same clone. In total 592 RUNX1 mutations were detected, 242 (40.9%) were frameshift, 206 (34.8%) missense, 83 (14.0%) nonsense, 37 (6.3%) in frame insertion/deletions, 23 (3.9%) splice-site and 1 no stop change. No association between the types of 1st and 2nd mutations was observed. The mutations were homozygous in 65 pts (13.9%), these were predominantly missense mutations (38; 58.5%). A significantly higher frequency of homozygous mutations was observed in pts with +13 (28.3%, p=0.006), +21 (46.7%; p=0.002), AML M0 (35.8%; p<0.001) and M1 (22.8%; p=0.014) while they were less frequent in M2 (4.8%; p<0.001) and M4 (3.7%, p=0.017). Survival analyses were restricted to 203 de novo AML pts who were treated with intensive chemotherapy (median overall survival (OS) 20.4 months (mo)). Median OS was significantly longer in female than in male pts (45.6 vs 16.3 mo; p=0.003) and in pts ≤ 60 vs >60 yrs (44.4 vs 16.1 mo; p<0.001). Shorter OS was observed for pts with del(5q) (1.8 vs 21.1 mo; p=0.002) and adverse cytogenetics (12.4 vs 23.3 mo, p=0.016). Including these 4 parameters into a multivariate Cox regression analysis revealed that age, male gender and del(5q) were independently associated with shorter OS (relative risk: 1.8, 1.6, 3.4; p: 0.01, 0.038, 0.028) Conclusions: RUNX1 mutated AML: 1. is associated with a myeloid rather than monocytic differentiation, 2. shows a typical pattern of cytogenetic abnormalities with a high frequency of +8 and +13, 3. has a typical pattern of additional molecular mutations with a high frequency of accompanying ASXL1 und SF3B1 mutations, 4. is nearly mutually exclusive of NPM1 and CEBPAdm mutations and other entity defining genetic abnormalities, 5. Male gender, age >60 yrs and del(5q) were negative prognostic factors. Disclosures Nadarajah: 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.

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