Molecular Classification of AML-MRC Reveals a Distinct Profile and Identifies MRC-like Patients with Poor Overall Survival

Background: AML with myelodysplasia related changes (AML-MRC) is as specific WHO category with poor prognosis. It requires ≥ 20% of blasts, and (1) the history of MDS or MDS/MPN, or (2) "MDS related cytogenetic abnormalities", or (3) multilineage dysplasia. Drugs such as Vyxeos® have been approved by FDA and EMA only for treatment of t-AML or AML-MRC. However, counting blasts or grading dysplasia in clinical routine is hampered by limited reproducibility due to different levels of expertise and small phenotypic alterations, challenging upfront treatment decisions. Cytogenetics is not available in all cases and has 5-10 days of turnaround time (TAT). In contrast, next-generation sequencing (NGS) panels for AML are now broadly available at faster TAT. Aim: (1) Use machine learning to define a molecular AML-MRC signature; (2) compare the impact of conventional WHO definitions and molecular factors on classification and outcome. Patients and Methods: Gold standard routine AML diagnosis was performed on 739 cases. Overall survival (OS) data was available for 619 patients. Amplification-free whole genome sequencing was performed on HiSeqX and NovaSeq with median coverage of 106x. Gender-matched reference DNA was used for unmatched normal variant calling with Strelka2. Pindel was used for FLT3-ITD. For variant classification, we applied a GnomAD cutoff of 0.0005 and filtered on protein-truncating and (likely) pathogenic variants from databases. Results: According to WHO standards 165/739 (22%) cases fulfilled MRC criteria (96 male; 69 female). The non-MRC cohort (n=574) represents a heterogeneous AML population incl. the WHO defined recurrent cytogenetic abnormalities or t-AML (301 male, 273 female). Median age was higher in the MRC cohort (73 [22-90] vs. 64 [18- 93] years, p<.001) and OS was significantly shorter (median 6 vs. 23 months, p<.001). Mutation analysis was limited to 73 frequently mutated genes, in order to allow application of our model on prospective diagnostic cases analyzed by common routine panels. In the MRC group, up to seven mutations were found per patient and an average of 2.7 genes per patient were mutated. The most frequently mutated gene in AML-MRC was TP53 (62/165, 38%) as expected by the inclusion of complex karyotypes. TP53 mutations were associated with shorter OS in the MRC cohort (median: 3 vs. 11 months, p =.001). We used machine learning (ML) approaches to identify with LASSO regression and 10-fold cross-validation the most informative features to distinguish between MRC and patients without MRC. The dataset was randomly divided into a training (90%) and test set (10%) and the procedure was repeated 500 times to cover all the variance in the dataset and to extract the most reliable factors. Factors with the highest weight on AML-MRC prediction were mutations in TP53, RUNX1, SETBP1, splicing factors and epigenetic regulators, and absence of mutations in NPM1, CEBPA and others (s. figure). In order to allow our model to be used in a routine diagnostic workflow, we also used the genes identified by ML but classified mutations by a simpler point system (≥2 points as cutoff for MRC, s. figure). This allowed us to identify 83% (137/165 by ML) and 70% (116/165 by points) of cases currently defined as MRC solely by molecular genetics. Including cytogenetic data and patient's history in an informed genetic model results in 99% (164/165 by ML) and 96% (159/165 by points) of true positive MRC definition. However, the molecular models classified 112 (ML) and 80 (points) of the 574 non-MRC cases, as being AML-MRC. Even after excluding AML with recurrent cytogenetic abnormalities and t-AML, 14% (82/574 DL) or 11% (63/574 points) show a MRC-like molecular profile. In both models MRC-like patients had dismal outcome analogous to AML-MRC (median OS: 6 months for both) and significantly inferior to remaining non-MRC patients (6 vs. 35 months, s. figure). Conclusions: (1) Using patients' history and genetic information instead of morphology allow to identify 96-99% of AML-MRC as defined in WHO today. In the future, extended NGS panels (e.g. incl. fusion gene detection) will allow fast and standardized AML-MRC classification even without chromosome banding analysis. (2) The molecular MRC-like pattern can be found in >10% of patients currently not classified as AML-MRC but with comparably poor OS. This suggests considering MRC treatment strategies for patients with MRC-like molecular profile. Disclosures Baer: MLL Munich Leukemia Laboratory: Employment. Walter:MLL Munich Leukemia Laboratory: Employment. Stengel:MLL Munich Leukemia Laboratory: Employment. Hutter:MLL Munich Leukemia Laboratory: Employment. Meggendorfer: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.

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Influence of Karyotype On Overall Survival in Patients with Higher-Risk Myelodysplastic Syndrome Treated with Azacitidine or a Conventional Care Regimen.

Blood ◽

10.1182/blood.v114.22.1755.1755 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1755-1755 ◽

Cited By ~ 5

Author(s):

Ghulam J Mufti ◽

Steven D. Gore ◽

Valeria Santini ◽

Pierre Fenaux ◽

Lewis R. Silverman ◽

...

Keyword(s):

Overall Survival ◽

Board Of Directors ◽

Research Funding ◽

Conventional Care ◽

Complex Karyotype ◽

Trisomy 8 ◽

Employment Equity ◽

Advisory Committees ◽

Cytogenetic Abnormalities ◽

Equity Ownership

Abstract Abstract 1755 Poster Board I-781 Background Karyotypic abnormalities are common in myelodysplastic syndromes (MDS), and specific chromosomal abnormalities are associated with poor prognosis. The phase III AZA-001 study (Lancet Oncol, 2009) showed azacitidine (AZA) prolonged overall survival (OS) regardless of IPSS cytogenetic risk category. This analysis assessed the effects of specific cytogenetic abnormalities on OS in patient (pt) subgroups treated with AZA or a conventional care regimen (CCR). Methods Pts with higher-risk MDS (FAB RAEB, RAEB-t, or CMML and IPSS Int-2 or High) were enrolled and randomized to receive AZA or CCR. CCR comprised 3 treatments: best supportive care only, low-dose ara-C, or induction chemotherapy. Erythropoietins were prohibited. OS was determined in subgroups of pts with del 5/5q-, del 7/7q-, or trisomy 8, each as part of a non-complex karyotype (<3 cytogenetic abnormalities) or as part of a complex karyotype (≥3 cytogenetic abnormalities). OS was also analyzed in pts with combinations of del 5/5q- and/or del 7/7q- as part of non-complex or complex karyotypes (Table). Pt karyotype was determined at baseline. OS was assessed using Kaplan-Meier methods. A stratified Cox proportional hazards regression model was used to estimate hazard ratios (HRs) and associated 95% confidence intervals (CI). Results A total of 358 pts were enrolled (AZA 179, CCR 179). Of them, 153 had normal karyotypes (AZA 77, CCR 76). Median OS in pts with normal karyotypes was not reached at 21.1 months with AZA vs 17.2 months (95%CI: 15.2 – 24.1 months) with CCR; HR = 0.63 (95%CI: 0.39 – 1.03). Of remaining pts, 136 had del 5/5q-, del 7/7q-, and/or trisomy 8 as part of a non-complex or complex karyotype. AZA was associated with longer OS vs CCR in all subgroups of pts with non-complex cytogenetics, with HRs ranging from 0.20 (95%CI: 0.06 – 0.65) to 0.51 (95%CI: 0.05 – 4.74) (Table). In both the AZA and CCR treatment groups, pts in all subgroups with non-complex karyotypes had substantially longer OS than pts with complex karyotypes. Pts with complex karyotypes in some subgroups had longer OS with AZA vs CCR: median OS in pts with del 5/5q-, del 5/5q- WITHOUT del 7/7q-, or trisomy 8 as part of a complex karyotype treated with AZA survived 5.1, 8.0, and 12.4 months longer, respectively, than their counterparts who received CCR. HRs with AZA vs CCR in pts with complex cytogenetics ranged from 0.42 (95%CI: 0.10 – 1.69) to 0.55 (95%CI: 0.29 – 1.05). Conclusions These findings support earlier data showing effectiveness of AZA in higher-risk MDS pts with complex or non-complex karyotypes. Major gains in OS were obtained with AZA vs CCR (12-18 months longer OS with AZA) for the following categories: del 7/7q- (non-complex), del 7/7q- WITHOUT del 5/5q- (non-complex), and trisomy 8 (non-complex and complex). Pts with trisomy 8 treated with AZA experienced a 3-fold increase in median OS compared with similar pts who received CCR. Longer OS (AZA 15.3 vs CCR 7.3 months) was also obtained for pts with del5/5q- WITHOUT del7/7q- as part of a complex karyotype. The worse cytogenetic categories, del 7/7q- and del 5/5q- AND del 7/7q-, both with complex karyotype, were associated with the poorest OS regardless of treatment. Pt subgroups in this post hoc analysis were small and heterogeneous; confirmation of these findings in larger pt samples is warranted. Disclosures Mufti: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Gore:Celgene: Consultancy, Equity Ownership, Research Funding; Johnson & Johnson: Research Funding. Santini:Celgene: Honoraria. Fenaux:Celgene: Honoraria, Research Funding; Ortho Biotech: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Cephalon: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; MSD: Honoraria, Research Funding; Epicept: Honoraria, Research Funding. Skikne:Celgene: Employment, Equity Ownership. Hellstrom-Lindberg:Celgene: Research Funding. Seymour:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Beach:Celgene: Employment, Equity Ownership. Backstrom:Celgene: Employment, Equity Ownership. Fernando:Celgene: Employment, Equity Ownership.

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Prognostic Value of “Monosomal Karyotype” in Comparison to “Complex Aberrant Karyotype” in AML: A Study on 824 Cases with Aberrant Karyotype

Blood ◽

10.1182/blood.v118.21.418.418 ◽

2011 ◽

Vol 118 (21) ◽

pp. 418-418

Author(s):

Claudia Haferlach ◽

Tamara Alpermann ◽

Susanne Schnittger ◽

Wolfgang Kern ◽

Torsten Haferlach

Keyword(s):

Cox Regression ◽

Complex Karyotype ◽

Employment Equity ◽

Cytogenetic Abnormalities ◽

Cox Regression Analysis ◽

Poor Risk ◽

Risk Patients ◽

Equity Ownership ◽

The Impact ◽

Monosomal Karyotype

Abstract Abstract 418 Background: Several classifications based on cytogenetics have been proposed in AML. Typically 3 major categories for prognostication are defined: favorable, intermediate and unfavorable. The assignment to the unfavorable group shows minor differences between the different cytogenetic classifications currently used, however certain cytogenetic subgroups are assigned to the unfavorable subgroup concordantly: −5/5q−, 7q−/−7, −17/abn17p, inv(3)(q21q26)/t(3;3)(q21;q26) and complex karyotype (CK). With respect to CK 3 definitions are used: ≥3, ≥4 or ≥5 unrelated abnormalities. Recently, a so-called “monosomal karyotype” (MSK) defined as a karyotype showing “two or more distinct autosomal chromosome monosomies or one single autosomal monosomy in the presence of structural abnormalities” was introduced (Breems et al. JCO 2008). It was suggested that patients with MSK have a poor outcome being even inferior to CK. Aim: We here evaluated the prognostic power of differently defined cytogenetic subsets in order to identify the best definition for the prognostically most unfavorable subgroup. Patients: From our initial cohort of newly diagnosed AML (n=1,959) patients with t(15;17), t(8;21) or inv(16) (n=170) and AML with normal karyotype (n=965) were excluded. Thus, 824 patients with cytogenetic abnormalities remained for further investigation. Results: 428/824 (51.9%) patients showed an intermediate risk karyotype according to revised MRC criteria (MRC-I) (Grimwade et al. Blood 2010), while the remaining 396/824 (48.1%) cases belonged to the unfavorable MRC group (MRC-U). 162/824 cases (19.7%) fulfilled the criteria of MSK. According to MRC, 4 of these 162 cases with MSK were classified MRC-I while 158 were classified MRC-U. The overlap in classification between CK and MRC-U differed depending on the number of aberrations used to define CK. As such, the number of cases with CK was 272 (33.0%; MRC-I: 17, MRC-U: 255) using ≥3 clonal aberrations, and decreased to 222 (26.9%; all MRC-U) patients using ≥4 clonal aberrations or 196 (23.8%; all MRC-U) cases when applying the criterion of ≥5 clonal aberrations, respectively. Univariate Cox regression analysis revealed that unfavorable cytogenetics as defined by MRC-U, MSK, CK defined as ≥3, ≥4 or ≥5 unrelated abnormalities were all significantly associated with inferior OS as compared to the respective remaining intermediate group (for all p<0.001). Hazard ratios were 1.61, 1.93, 1.68, 1.94, and 1.92, respectively. Median OS in the respective categories was 8.5, 5.7, 6.3, 5.7, and 5.7 months, respectively. We then performed further analyses within the unfavorable risk group defined according to MRC and tested the impact of the 4 definitions for unfavorable subsets. In each comparison the median OS was significantly shorter for the subset with MSK, or CK defined as ≥3, '4 or ≥5 unrelated abnormalities as compared to the remaining MRC-U cases (5.7 vs 11.7 mo p=0.005; 6.3 vs 10.6 mo, p=0.031; 5.7 vs 11.0 mo, p=0.003; 5.7 vs 10.9 mo, p=0.006). Furthermore OS of patients within MRC-U excluding cases with MSK, or CK with ≥3, ≥4 or ≥5 unrelated abnormalities did not differ from patients with cytogenetic abnormalities assigned to MRC-I (median OS 11.7, 10.6, 11.0 and 10.9 mo, respectively vs 21.1 mo, p=0.072, p=0.16, p=0.28, and p=0.11, respectively). Within the MRC-U cohort only 124 cases fulfilled both criteria: MSK and CK≥4 (median OS 5.3 mo), 97 were CK≥4 only (median OS 6.3 mo) and 35 MSK only (median OS 6.7 mo). OS did not differ between these 3 subgroups but was significantly shorter for all comparisons to patients included in none of these subgroups (p<0.001, p=0.009, p=0.012, respectively). On the other hand OS of the 33 cases with 3 unrelated abnormalities did not differ from MRC-U cases with 1 or 2 abnormalities (18.9 vs 10.6, p=0.48). Conclusions: All definitions of very poor risk AML patients allow to identify a subset within MRC-U that shows significantly shorter OS than the remaining MRC-U cases. However, “complex karyotype defined as ≥4 unrelated abnormalities” is the best parameter as it identifies the largest proportion of very poor risk patients. Even more important, the application of the monosomal karyotype for prognostication and clinical guidance in AML misses 24.5% of the very poor risk patients identified based on CK ≥4. This may lead to suboptimal treatment decisions in this clinically proven very high risk patients. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Alpermann: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.

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Azacitidine (AZA) Prolongs Overall Survival in Older Patients with Acute Myeloid Leukemia (AML) with Poor Prognostic Karyotypes Compared with Conventional Care Regimens (CCR)

Blood ◽

10.1182/blood.v128.22.1638.1638 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1638-1638 ◽

Cited By ~ 2

Author(s):

Hartmut Döhner ◽

Paresh Vyas ◽

John F. Seymour ◽

Valeria Santini ◽

Richard M. Stone ◽

...

Keyword(s):

Overall Survival ◽

Board Of Directors ◽

Older Patients ◽

Research Funding ◽

Complex Karyotype ◽

Employment Equity ◽

Advisory Committees ◽

Cytogenetic Abnormalities ◽

Poor Risk ◽

Equity Ownership

Abstract Background: Karyotype is the strongest independent prognostic factor for survival in AML. The randomized phase 3 AZA-AML-001 study of older patients with AML showed AZA prolonged overall survival (OS) compared with CCR (10.4 vs 6.5 months, respectively; P=0.101) (Dombret et al, Blood, 2015). In a prospective subanalysis of the study, AZA was shown to meaningfully prolong OS by 3.2 months compared with CCR (P=0.0185) in the subgroup of patients with NCCN-defined poor-risk cytogenetics (Döhner et al, Blood, 2014: Abstract 621). Aim: This analysis evaluates treatment effects of AZA vs CCR on OS in subgroups of patients with specific cytogenetic abnormalities as well as in patient subgroups defined by cytogenetic risk per modified European LeukemiaNet (ELN) recommendations (not considering molecular markers) (Döhner et al, Blood, 2010). Methods: Patients aged ≥65 years with newly diagnosed AML (>30% bone marrow [BM] blasts), ECOG performance status score ≤2, intermediate- or poor-risk cytogenetics per NCCN 2009 criteria, and WBC count ≤15x109/L were randomized to receive AZA (75 mg/m2/day [d] x7d/28d) or CCR: intensive chemotherapy (cytarabine 100-200mg/m2IV x7d + anthracycline IV x3d induction), low-dose ara-C (20mg SC BID x10d/28d), or best supportive care only. Karyotypes obtained from BM were reviewed centrally by an independent cytogeneticist. OS was evaluated in subgroups of patients with frequent specific abnormalities, including -5/del(5q), -7, -7/del(7q), abnormal (17p) or complex karyotype (based on specific abnormalities, patients may have been evaluated in more than one category). OS was also assessed for patients in ELN-defined karyotype risk subgroups: Intermediate (Int)-I (normal karyotype), Int-II (all abnormalities not classified as Favorable or Adverse), and Adverse karyotype. OS was assessed using Kaplan-Meier methods and compared using a weighted log-rank test. Results: Centrally reviewed cytogenetic data were available for 485/488 patients (99.4%). In all, 220 patients (45.4%; AZA n=114, CCR n=106) had Int-I karyotype, 111 patients (22.9%; AZA n=53, CCR n=58) had Int-II karyotype, and 154 patients (31.8%, AZA n=73, CCR n=81) had Adverse karyotype (Figure 1). OS was comparable between AZA and CCR in patients with Int-I karyotype (14.1 vs 10.1 months, respectively; hazard ratio [HR] 0.83, 95%CI 0.60, 1.1; P=0.44) and patients with Int-II karyotype (8.9 vs 9.6 months; HR 1.19, 95%CI 0.79, 1.8; P=0.78). There was a significant 2.4-month median OS difference in favor of AZA in patients with Adverse karyotype (5.3 vs 2.9 months with CCR; HR 0.71, 95%CI 0.51, 0.99; P=0.046; Figure 2), with 1-year survival rates of 29.1% vs 14.7% for AZA and CCR, respectively. AZA was associated with longer median OS and higher 1-year survival compared with CCR for all subgroups of patients with the specific cytogenetic abnormalities under study: -5/del(5q), -7, -7/del(7q), abnormal (17p), and complex karyotype, with HRs ranging from 0.54 to 0.69(Table). Median OS in the CCR arm was less than 3 months for each of these subgroups. Similar to what has been reported in MDS (Ravandi et al, Cancer, 2009), AML patients with chromosome 7 abnormalities responded particularly well to AZA, with an improvement in median OS of 4.1 months over CCR. Patients with complex karyotypes also had meaningful improvements in OS, with ~15% more AZA-treated patients alive at 1 year than CCR patients. Conclusions: Prognosis is dismal for older AML patients with adverse karyotypes, and is especially poor for patients with complex karyotypes. Median OS and 1-year survival in patients with ELN-defined Adverse karyotype treated with AZA were almost double those of patients treated with CCR. AZA-treated patients with the specific cytogenetic abnormalities and/or complex karyotype in this analysis had a 31-46% reduction in risk of death vs CCR, and proportions of patients alive at 1 year were 11-22% greater with AZA. These data suggest AZA should be the preferred treatment for older patients with AML and adverse karyotypes. Disclosures Seymour: Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Speakers Bureau; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding, Speakers Bureau; Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Santini:Astex: Consultancy; Amgen: Consultancy; Onconova: Consultancy; Novartis: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding. Stone:Celator: Consultancy; Novartis: Consultancy; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Xenetic Biosciences: Consultancy; Agios: Consultancy; Amgen: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Consultancy; Jansen: Consultancy; Pfizer: Consultancy; ONO: Consultancy; Juno Therapeutics: Consultancy; Merck: Consultancy; Roche: Consultancy; Seattle Genetics: Consultancy; Sunesis Pharmaceuticals: Consultancy. Al-Ali:Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Morrill:Celgene: Employment, Equity Ownership. Songer:Celgene: Employment, Equity Ownership. Weaver:Celgene Corporation: Employment, Equity Ownership. Skikne:Celgene: Employment, Equity Ownership. Beach:Celgene Corporation: Employment, Equity Ownership. Dombret:Agios: Honoraria; Ambit (Daiichi Sankyo): Honoraria; Menarini: Honoraria; Menarini: Honoraria; Servier: Honoraria; Sunesis: Honoraria; Karyopharm: Honoraria; Kite Pharma.: Honoraria, Research Funding; Astellas: Honoraria; Janssen: Honoraria; Seattle Genetics: Honoraria; Roche/Genentech: Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Pfizer: Honoraria; Ariad: Honoraria, Research Funding; Novartis: Honoraria; Celgene: Consultancy, Honoraria; Jazz Pharma: Honoraria, Research Funding.

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Combinatorial Effect of HDAC6i and Ibrutinib Therapy in CLL Murine Model

Blood ◽

10.1182/blood.v128.22.2035.2035 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2035-2035

Author(s):

Kamira Karen Maharaj ◽

John J. Powers ◽

Susan Deng ◽

Alex Achille ◽

Mibel Pabon-Saldana ◽

...

Keyword(s):

Overall Survival ◽

B Cells ◽

Cell Lines ◽

Adoptive Transfer ◽

Single Agent ◽

Tumor Burden ◽

Employment Equity ◽

Bcr Signaling ◽

Equity Ownership ◽

The Impact

Abstract Ibrutinib, a Bruton tyrosine kinase (BTK) inhibitor is now FDA approved for front line and relapsed and refractory CLL. Although this drug has been shown to be successful in controlling the disease, most patients only reach partial responses (PR) likely secondary to activation of alternative and redundant BCR signaling pathways. In the past several years, epigenetic changes in CLL have gained special attention (Tong et al 2010) because of their intricate interplay with previously described genetic events and its active role in the regulation of pathogenesis and immune-related pathways (Stilgenbauer et al 2002). Recent publications have described the differential expression of specific HDACs in CLL, as well as the impact of global histone deacetylase activity and its relation with progression and overall survival (Van Damme et al 2014, Yang et al 2015). Previously, we had reported that expression of HDAC6 is increased in CLL patient samples and cell lines. We have also demonstrated that modulation of this HDAC, effects cell proliferation and viability in CLL cell lines. Additionally, treatment with HDAC6i (ACY-738 - a potent and selective HDAC6i) demonstrated increased overall survival in euTCL1 mice, a murine CLL model. Treatment of euTCL1 mice (both adoptive transfer and aging model) with ACY-738 as a single agent resulted in 1) overall survival advantage, 2) reduction of tumor burden, 3) reduction in PD-L1 expression in the malignant B cell population and 4) decreased circulating Treg numbers. Furthermore, we demonstrated that in an in vitrostudy, treatment of HDAC6i with ibrutinib in CLL cell lines render strong synergistic cell killing. In our current study of this combinatorial approach, using the adoptive transfer euTCL1 model receiving Ibrutinib in drinking water and ACY-738 in feed, we demonstrate a further decrease in tumor burden when compared to single agent treatment with either compound alone. This observed effect on tumor burden occurred in conjunction with decreases in co-inhibitory molecules and circulating Treg frequency. The combination was well tolerated and no significant toxicity was observed. Since aberrant over-expression of HDAC6 in CLL cell lines and patient's samples have already been demonstrated (Van Damme et al 2012, Sahakian et al 2012) we sought to understand its mechanistic role in BCR survival pathways of CLL. Our studiesin normal B cells isolated from C57BL/6 and HDAC6KO mice, demonstrated a reduction in phosphorylation of BTK, ERK, and AKT. Similar results were observed when we compared euTCL1 to euTCL1/HDAC6KO B cells. Additionally,we observed decreased phosphorylation of ERK and SYK in MEC2-HDAC6KD cells when compared to parental control CLL cells. Moreover, RNA-Seq studies of the eu-TCL1/HDAC6KO versus euTCL1 B cells showed several key BCR signaling proteins altered by the deletion of HDAC6. In conclusion, these results from our preclinical CLL models suggest that combinatorial therapy of Ibrutinib plus HDAC6i show synergistic inhibition of BCR signaling and therefore a better overall treatment outcome. Disclosures Quayle: Acetylon Pharmaceuticals: Employment, Equity Ownership. Jones:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Pinilla-Ibarz:Gilead: Consultancy, Speakers Bureau; Pharmacyclics: Consultancy, Speakers Bureau; Novartis: Consultancy; Abbvie: Consultancy, Speakers Bureau; Novartis: Consultancy; Janssen: Consultancy, Honoraria; Gilead: Consultancy, Speakers Bureau.

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Is Renal Impairment Still a Poor Prognostic Marker in Myeloma Care?: A Population Based Study Including 1542 Patients

Blood ◽

10.1182/blood.v120.21.5033.5033 ◽

2012 ◽

Vol 120 (21) ◽

pp. 5033-5033

Author(s):

Katarina Uttervall ◽

Johan Andreasson ◽

Johan Liwing ◽

Per Näsman ◽

Johan Aschan ◽

...

Keyword(s):

Overall Survival ◽

Renal Impairment ◽

Prognostic Marker ◽

Control Group ◽

Line Treatment ◽

Employment Equity ◽

Significant Difference ◽

Study Population ◽

Equity Ownership ◽

The Impact

Abstract Abstract 5033 Background: Renal impairment (RI) is a relatively common feature of multiple myeloma (MM) and it has been shown in several studies that RI at diagnosis correlates to inferior survival, significant morbidity and increased early death rate. Aims: To understand the impact of RI on survival in the era of novel agents and to evaluate the efficacy of bortezomib-based treatment in patients presenting with RI. The primary endpoint of the study was overall survival (OS) in the whole population and renal response (RR) in 1st, 2nd and 3rd line treatment in a selected center (Karolinska). Time to progression (TTP), time to next treatment (TTNT), overall survival (OS) and MM response were the secondary endpoints. Methods: The study population included all patients diagnosed with MM since earliest January 2000 until latest June 2011 at 6 university clinics, 3 regional centers and 4 local hospitals in Sweden. A S-Creatinine limit of 177 μmol/L was chosen as cutoff for OS calculations. For a detailed analysis on RR all patients with MM with a S-Creatinine >=130 μmol/L that were diagnosed in clinical practice between January 2000 and July 2010 at Karolinska Huddinge and between January 2005 and July 2010 at Karolinska Solna were selected. This S-Creatinine limit was selected in an attempt to cover all MM patients with GFR <50 mL/min. The study population was divided into those receiving bortezomib and those receiving other drug combinations (control group). Results: The population consisted of 1642 patients, but S-Creatinine values were missing for 100 patients resulting in a study population of 1542 patients. Patients with S-Creatinine >=177 μmol/L (n=267) had a significantly worse median OS of 1. 9 years 95% CI[1. 5;2. 6] compared to S-Creatinine <177 μmol/L (n=1275), with a median OS of 4. 1 years 95% CI[3. 8;4. 4]; (p=<0. 001). Patients with >=177 in S-Creatinine receiving bortezomib (n=60) had a median OS of 2. 6 years compared to 1. 7 years in the control group (n=206) (p=0. 05). Of the 1542 patients, 556 were diagnosed and treated at our center. Ninety-five of these patients had a S-Creatinine >=130 μmol/L at diagnosis. Of the 95 patients 52 also had RI in 2nd line and 25 in 3rd line treatment resulting in a total of 172 treatment occasions where anti-myeloma treatment was given to patients with RI. There was no significant difference regarding age, sex, hemoglobin, β2-microglobulin, calcium, or albumin between the bortezomib-group and the control group. In the bortezomib-group, in 1st line treatment, 11 of 12 patients (92%) improved their GFR compared to 57 of 83 (69%) in the control group (p=0. 049). In the 2nd and 3rd treatment line the overall RR was 19% and 43% in the bortezomib treated compared to 23% and 28% in the control group (p=0. 749, and p=0. 257). When analyzing all treatment lines together the MM response was better in the bortezomib-group with significantly higher overall response rate of 80%, compared to 55% (p=0. 026). Median TTP in 1st, 2nd and 3rd line in the bortezomib-group was 18, 6 and 10 months and in the control group 11, 10 and 8 months. Median survival time was 3. 4 years for the control group, whereas 62% of the bortezomib treated patients still were alive at median time of follow up. Conclusion: RI is still an important prognostic marker in MM despite modern treatment with a proteasome inhibitor. Bortezomib-based regimens can partly overcome the negative impact of RI by a higher frequency of RR and MM responses, as well as an improved median OS in comparison to other treatment regimens for MM patients with RI. Disclosures: Liwing: Janssen-Cilag: Employment, Equity Ownership. Näsman:Janssen-Cilag: Consultancy. Aschan:Janssen-Cilag: Employment, Equity Ownership.

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Impact of Amotosalen/UVA Pathogen Inactivated Platelet Components on Outcomes after Allogeneic Hematopoetic Stem Cell Transplantation: A Single Center Analysis

Blood ◽

10.1182/blood-2019-122330 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1167-1167

Author(s):

Andreas S. Buser ◽

Laura Infanti ◽

Andreas Holbro ◽

Joerg Halter ◽

Sabine Gerull ◽

...

Keyword(s):

Stem Cell ◽

Cox Regression ◽

Univariate Analysis ◽

Conditioning Regimen ◽

Disease Status ◽

Risk Scores ◽

Employment Equity ◽

Stem Cell Source ◽

Equity Ownership ◽

The Impact

Background: Platelet component (PC) transfusion is required for allogeneic hematopoietic stem cell transplantation (HCT) recipients. Contamination with infectious pathogens (bacteria, viruses, or protozoa) and T-cells is a risk factor for transfusion-transmitted infection (TTI) and transfusion associated graft-versus-host disease (TA-GVHD). Pathogen inactivation (PI) treatment of PC with amotosalen-UVA (PI-PC, INTERCEPT Blood System, Cerus Corp) in platelet additive solution (PAS) without bacterial screening, gamma irradiation, CMV serology, and with 7-day storage has been the standard of care in Switzerland since 2011 to manage risk of TTI and TA-GVHD. PI-PC have replaced conventional PC (C-PC) prepared in PAS with gamma irradiation and 5 day storage. We previously reported platelet usage in two consecutive five year periods at the University Hospital of Basel. Mean PI-PC dose was higher (3.0 vs. 2.8 x 1011, p=0.001) and mean storage duration longer (4.2 vs. 3.4 days: p=0.001) than with C-PC. PC expiration wastage was reduced with 7-day PI-PC storage vs. 5-day storage (1.5% vs. 8.7%). For HCT recipients, days of PC support; PC use per patient; and RBC use per patient were similar, despite 24.3% lower corrected count increments (CCI) with PI-PC. Now, we report the impact of these observations on treatment related mortality (TRM) and overall survival (OS) 100 days after HCT. Patients and Methods: A two-period retrospective cohort study was conducted to evaluate PI-PC impact on outcomes of consecutive first allogeneic HCT recipients from January 2006 to December 2010 (Period 1, P1), when gamma-irradiated apheresis C-PC were used, and Period 2 (P2) from January 2011 to December 2017, when apheresis and whole blood-derived PI-PC were used. The review utilized 100-day OS and 100-day TRM to determine the impact of PI-PC on HCT outcomes. Descriptive statistics were used for continuous variables and log-rank analysis for survival outcomes. Univariate analysis was performed using Pearson χ2 statistics. Multivariate Cox regression modelling analyses included: PC period (P1, P2), donor match (HLA identical/twin, matched related, matched unrelated), disease state (early, intermediate, late), and conditioning regimen (reduced intensity, myeloablative) with TRM as the outcome. This was an IRB approved single-center analysis. Results: In P1 and P2, 256 and 557 consecutive first-time allogeneic HCT recipients were included, respectively. By univariate analysis, the distribution of European Group for Bone Marrow Transplantation (EBMT) risk scores (grouped 0-2, 3-4, 5-7) and mean patient age were higher during P2 (p = 0.001 and p <0.001, respectively). Primary disease status (p = 0.039); stem cell source (p <0.001); GVHD prophylaxis with ATG (p <0.001); total body irradiation (p <0.001); and conditioning regimen (p <0.001) were different between P1 and P2. Donor match (p=0.084) and disease status (p = 0.628) were similar in P1 and P2. TRM at day 100 post HCT was significantly less (31/557, 5.5%) for PI-PC recipients in P2 vs. C-PC recipients in P1 (37/256, 14.5%, p<0.001). Overall proportion of survivors at day 100 post HCT was significantly greater for PI-PC recipients (507/557, 91.0 %) compared to C-PC recipients (209/256, 81.6%, p <0.001). By multivariate Cox regression analysis, P2 with PI-PC component support was associated with improved TRM (p = 0.001; adjusted hazard ratio 0.433; 95% confidence interval: 0.262, 0.716). Donor match (p = 0.019), disease state (p = 0.022), and myeloablative conditioning (p = 0.034) were associated with significantly poorer TRM (Table). Stem cell source was not significant (p=0.157) in the model. Hemorrhage was reported as cause of death in 1/50 (2.0%) patients during P2 with PI-PC and 4/47 (8.5%) patients during P1 with C-PCs. Conclusions: Universal implementation of PI-PC in routine with extended storage to 7 days in P2 was associated with reduced TRM and better overall survival 100 days post HCT, despite transplantation of older patients with higher EBMT risk scores. Multivariate analysis revealed an adjusted hazard ratio of 0.433 (95% C.I. 0.262, 0.716) for TRM by 100 days, suggesting better outcomes in P2. This retrospective analysis at a single site indicated that PI-PC treated with amotosalen /UVA stored up to 7 days did not have a negative impact on TRM and OS in HCT recipients, and was an integral part of improving clinical outcomes at our institution. . Table. Disclosures Heim: Novartis: Research Funding. Irsch:Cerus Corporation: Employment, Equity Ownership. Lin:Cerus Corporation: Employment, Equity Ownership. Benjamin:Cerus Corporation: Employment, Equity Ownership. Corash:Cerus Corporation: Employment, Equity Ownership.

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A Complete Remission (CR) Is Not a Prerequisite for Prolonged Survival after Autotransplants for Multiple Myeloma.

Blood ◽

10.1182/blood.v104.11.926.926 ◽

2004 ◽

Vol 104 (11) ◽

pp. 926-926 ◽

Cited By ~ 2

Author(s):

Guido Tricot ◽

Maureen Reiner ◽

Jeffrey Sawyer ◽

John Crowley ◽

Bart Barlogie

Keyword(s):

Overall Survival ◽

Acute Leukemia ◽

Tumor Burden ◽

Time Dependent ◽

Prolonged Survival ◽

P Value ◽

High Dose ◽

Cytogenetic Abnormalities ◽

Variate Analysis ◽

The Impact

Abstract In acute leukemia prolonged survival is impossible without obtaining a CR. Based on the acute leukemia model, myeloma therapy has gradually been intensified with the aim to increase the CR rate as a first important step to improve overall survival (OS). Although patients with abnormal metaphase cytogenetics have a significantly inferior outcome in terms of event-free and overall survival, the CR rate is similar for patients with and without cytogenetic abnormalities, indicating that CR may not be a good prognostic indicator of ultimate outcome. To address the importance of obtaining a CR for OS, we analyzed our Total Therapy I (VADx3-high dose cyclophosphamide 6g/m2 with stem cell collection-EDAP-melphalan-based tandem transplants-α interferon maintenance) data in those patients who had not received any treatment prior to enrollment (N=155), received at least one transplant (N=135) and were alive one year after the first transplant (N=132). Kaplan-Meier curves were generated using a 1 year landmark to compensate for the guaranteed time of CR patients, but thereby excluding patients who died within the first year after the first autotransplant (N=3). The 1-year landmark was chosen because the large majority of CR patients (75%) had achieved their CR at 1 year after the first transplant. In addition, a time-dependent co-variate analysis for CR was performed, including the 135 patients. The median follow-up of these patients was 10.5 years. The 9 year OS after the landmark, i.e., 10 years after the first transplant, was 41% (95% confidence interval: 26, 55) for CR patients versus 37% (26, 47) for no CR patients (i.e., PR and <PR) with a logrank p value of 0.71 (Figure 1). Using a time-dependent co-variate analysis for CR, achieving a CR was not significantly related to OS (Hazard Ratio: 0.83; p value 0.39). Only the presence of metaphase cytogenetic abnormalities (HR: 2.0; p=0.005), LDH > 190 U/L (upper limit of normal) (HR: 2.0; p=0.01) and CRP >4.0mg/L (HR: 1.6; p=0.03) were significant for OS. When the importance of CR was assessed separately for patients with (N=43) and without (N=84) abnormal cytogenetic (cytogenetic information was missing on 5 patients), no survival benefit for CR patients was seen in either subgroup (p values 0.52 and 0.32, respectively) and similarly, using the time-dependent co-variate analysis for CR, there was no significant benefit for OS of attaining a CR in either group (p value: 0.7 and 0.5, respectively). We conclude that prolonged survival (>10 years) is observed in a substantial proportion of myeloma patients receiving a tandem autotransplant-based regimen, irrespective of the completeness of response to tandem transplants. The inherent genetic features of the myeloma and the impact on the micro-environment of the myeloma cells appear to be more important than the absolute tumor burden reduction accomplished by tandem transplants. Our findings may also be a reflection of the insensitivity of CR as an assessment of remaining tumor burden in myeloma and a new definition of CR may be required. Figure Figure

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Variation in Health-Related Quality of Life by ECOG Performance Status and Fatigue Among Patients with Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v118.21.4591.4591 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4591-4591 ◽

Cited By ~ 1

Author(s):

Chris L. Pashos ◽

Christopher R Flowers ◽

Mark Weiss ◽

Nicole Lamanna ◽

Charles M Farber ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Performance Status ◽

Clinical Practices ◽

Employment Equity ◽

Ecog Performance Status ◽

Advisory Committees ◽

Equity Ownership ◽

Ecog Ps ◽

The Impact

Abstract Abstract 4591 Introduction: Clinicians and investigators appreciate the value of measuring HRQOL for monitoring CLL and the impact of treatments, and commonly use ECOG performance status (PS) and clinician-reported patient fatigue as surrogates for HRQOL in clinical practice. However, limited data exist on the relationships between PS, fatigue, and HRQOL in CLL patients (pts) undergoing treatment in clinical practices. We examined the associations between these measures and 3 psychometrically validated, patient-reported, HRQOL instruments: the Brief Fatigue Inventory (BFI), EQ-5D, and Functional Assessment of Cancer Therapy-Leukemia (FACT-Leu). Methods: Data were collected as part of Connect CLL®, a prospective observational registry initiated in March 2010 involving US practices. Data on pt demographics and clinical characteristics were provided by clinicians. HRQOL was self-reported by pts at enrollment using the BFI, EQ-5D, and FACT-Leu. Mean BFI, EQ-5D and FACT-Leu scores were analyzed by ECOG PS and clinician-reported fatigue. Differences in HRQOL scores between sub-cohorts were assessed by ANOVA. Results: HRQOL data were reported by 604 pts enrolled from 10 academic, 148 community, and 3 government centers. Pts were predominantly male (62%) and white (90%); mean age was 70 (standard deviation 11) years. BFI data (scale: 0 [no fatigue] - 10 [worst fatigue]) indicated that on average pts report that global fatigue, fatigue severity and fatigue-related interference worsen by ECOG severity (Table 1) and are statistically associated with clinician-reported fatigue (Table 2). Mean EQ-5D overall HRQOL as measured by a Visual Analogue Scale (VAS) from 0 (worst) to 100 (best) worsens by ECOG severity and is significantly worse in pts with fatigue. Mean EQ-5D domain scores (scale: 1 [no problem], 2 [some problems], 3 [incapacity]) indicated that pain/discomfort, mobility and usual activities increase in severity as ECOG worsens and in pts with fatigue. FACT-Leu domains except social/family were statistically worse with worse ECOG PS and in pts with fatigue. Conclusions: Initial results from Connect CLL® indicate that HRQOL worsens with worsening ECOG PS, especially in physical / functioning domains, pain/discomfort, and mobility, and worsens across multiple domains among pts whose physicians reported fatigue. Future analyses should be conducted on how HRQOL, PS and fatigue may change over time with changes in CLL, and how they are influenced by therapies. These results may serve as baseline reference. Disclosures: Pashos: Celgene: Membership on an entity's Board of Directors or advisory committees. Flowers:Genentech/Roche (unpaid): Consultancy; Celgene: Consultancy; Millennium/Takeda: Research Funding; Wyeth: Research Funding; Novartis: Research Funding. Weiss:Celgene: Membership on an entity's Board of Directors or advisory committees. Lamanna:Celgene: Membership on an entity's Board of Directors or advisory committees. Farber:Celgene: Membership on an entity's Board of Directors or advisory committees. Kipps:Igenica: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Abbot Industries: Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Genentech: Research Funding; GSK: Research Funding; Gilead Sciences: Consultancy, Research Funding; Amgen: Research Funding. Lerner:Celgene: Membership on an entity's Board of Directors or advisory committees. Kay:Celgene: Membership on an entity's Board of Directors or advisory committees. Sharman:Celgene: Membership on an entity's Board of Directors or advisory committees. Grinblatt:Celgene: Membership on an entity's Board of Directors or advisory committees. Flinn:Celgene: Membership on an entity's Board of Directors or advisory committees. Kozloff:Celgene: Membership on an entity's Board of Directors or advisory committees. Swern:Celgene Corporation: Employment, Equity Ownership. Kahn:Celgene Corporation: Employment, Equity Ownership. Street:Celgene: Employment, Equity Ownership. Sullivan:Celgene: Employment, Equity Ownership. Keating:Celgene: Membership on an entity's Board of Directors or advisory committees.

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Cytogenetic Clonal Evolution in MDS Is Associated with Shifts towards Unfavorable Karyotypes According to IPSS and Shorter Overall Survival: A Study on 988 MDS Patients Studied Sequentially by Chromosome Banding Analysis

Blood ◽

10.1182/blood.v118.21.968.968 ◽

2011 ◽

Vol 118 (21) ◽

pp. 968-968 ◽

Cited By ~ 1

Author(s):

Claudia Haferlach ◽

Melanie Zenger ◽

Tamara Alpermann ◽

Susanne Schnittger ◽

Wolfgang Kern ◽

...

Keyword(s):

Overall Survival ◽

Chromosome Banding ◽

Cox Regression ◽

Clonal Evolution ◽

Employment Equity ◽

Cox Regression Analysis ◽

Banding Analysis ◽

Chromosome Banding Analysis ◽

Equity Ownership ◽

Acquired Abnormalities

Abstract Abstract 968 Background and Aim: The karyotype is one of the most important prognostic factors in MDS with respect to survival and evolution to AML and may change during the course of the disease. The aim of this study was to evaluate 1. the frequency of acquisition of additional chromosome abnormalities during the course of the disease (clonal evolution), 2. the pattern of acquired genetic abnormalities, 3. the association of karyotype at diagnosis and clonal evolution and 4. the impact of clonal evolution on transformation to AML and overall survival (OS). Patients and Methods: 988 MDS patients were evaluated by chromosome banding analysis (CBA) during the course of their disease. According to IPSS 729 (73.8%) cases showed a favorable karyotype, 146 (14.8%) patients an intermediate karyotype and 113 (11.4%) cases an unfavorable karyotype at first investigation. Progression to AML occurred in 180 of 988 patients during follow-up. Results: 2,454 chromosome banding analyses were performed in 988 cases (mean: 2.48 per case, range: 2–9). The median time between the first and the last evaluation was 12.5 months (range 1–60.6 months). Overall, in 171 of 988 patients (17.3%) clonal evolution was observed. Clonal evolution was detected between 1 and 56 months (median 14.3 months) after first evaluation and occurred later in patients with favorable than in patients with intermediate or unfavorable karyotype (mean 19.8 mo vs 15.5 mo vs 10.5 mo, favorable vs intermediate p=0.07, intermediate vs unfavorable p=0.05 and favorable vs unfavorable p<0.001). The abnormalities most frequently acquired during the course of the disease were +8, 7q−/−7, and gain of 21q detected in 29 cases each, followed by loss of 12p (n=22), 5q (n=14), 17p (n=19), and 20q (n=12). Other recurrently acquired abnormalities were +13 (n=12), +1q (n=12), +3q (n=12), −3q (n=10). Clonal evolution was strongly associated with cytogenetic IPSS category: Clonal evolution occurred in 100/729 cases with upfront favorable cytogenetics (13.7%), in 32/146 patients (21.9%) with upfront intermediate cytogenetics, but in 39/113 cases (34.5%) with upfront unfavorable cytogenetics (p<0.001). In 100 patients with favorable cytogenetics and clonal evolution karyotype was intermediate at second evaluation in 43 cases (43%), unfavorable in 25 cases (25%) and stayed favorable in the remaining 32 patients (32%). In 32 patients with intermediate cytogenetics and clonal evolution karyotype shifted to unfavorable at second evaluation in 11 cases (34.4%) and stayed intermediate in 21 patients (65.6%). Progression to AML was more frequent in patients with clonal evolution as compared to patients without (52/171 (30.4%) vs 128/817 (15.7%); p<0.001). In Cox regression analysis the IPSS karyotype at first evaluation, the IPSS karyotype at second evaluation, clonal evolution and progression to AML were associated with OS (relative risk: 2.12, 2.15, 1.87, and 6.6; p<0.001, p<0.001, p=0.011, p<0.001, respectively). In multivariate Cox regression analysis the IPSS karyotype at second evaluation and progression to AML were independently associated with shorter OS (relative risk: 2.0, and 6.1; p=0.013, p<0.001, respectively). Clonal evolution was associated with shorter OS (median 130.4 months vs not reached, OS at 5 years 72.3%vs 82.9%, p=0.01). Also in the subset of patients without transformation to AML outcome was inferior in patients with clonal evolution as compared to those without clonal evolution (OS at 5 years 78.2% vs 83.0%, p=0.05). Conclusions: 1. Clonal evolution was observed in 17.3% of patients with MDS. 2. The pattern of acquired abnormalities resembles the pattern observed in MDS at primary evaluation. 3. A higher frequency of clonal evolution and a shorter time to clonal evolution is observed in higher cytogenetic IPSS scores determined at first evaluation. 4. Clonal evolution is significantly associated with transformation to AML and shorter OS. 5. Sequential cytogenetic analyses allow the identification of subsets of MDS patients with a higher risk for transformation to AML and thus might guide treatment decisions in future. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Zenger:MLL Munich Leukemia Laboratory: Employment. Alpermann: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.

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Landmark Analyses of DNMT3A Mutations in Hematological Malignancies

Blood ◽

10.1182/blood.v118.21.407.407 ◽

2011 ◽

Vol 118 (21) ◽

pp. 407-407

Author(s):

Vera Grossmann ◽

Alexander Kohlmann ◽

Claudia Haferlach ◽

Tamara Alpermann ◽

Melanie Wild ◽

...

Keyword(s):

Overall Survival ◽

Mutation Rate ◽

Mutation Frequency ◽

Cpg Methylation ◽

Homozygous Mutation ◽

Wild Type ◽

Employment Equity ◽

Acute Leukemias ◽

Equity Ownership

Abstract Abstract 407 CpG methylation is an epigenetic modification that is important for cellular development. The DNMT3A gene, located on chromosome 2p23.3, encodes for a DNA methyltransferase and plays a central role in de novo CpG methylation. Recently, DNMT3A has been reported to be mutated in 22% of AML and 8% of MDS (Ley et al., N Engl J Med, 2010; Walter et al., Leukemia, 2011). Further, DNMT3A mutations were observed to be associated with a short overall survival in both diseases, respectively. In order to determine the role of DNMT3A mutations in leukemia we investigated two different entities by next-generation sequencing: 145 AML patients and 83 cases harboring a T-cell acute lymphoblastic leukemia (T-ALL). We applied an amplicon based deep-sequencing assay (454 Life Sciences, Branford, CT) in combination with the 48.48 Access Array technology (Fluidigm, South San Francisco, CA). The peripheral blood or bone marrow samples were obtained from untreated patients. The AML cohort was restricted to cases with normal karyotype (CN-AML). 87/145 (60%) cases were specifically selected to be wild-type for NPM1, FLT3-ITD, CEBPA, and MLL-PTD, whereas 58/145 (40%) samples were mutated in NPM1 (n=33) or double-mutated in NPM1 and FLT3-ITD (n=25). In our cohort of AML cases without mutations in NPM1, FLT3-ITD, CEBPA, and MLL-PTD, we observed a DNMT3A mutation frequency of 17.2% (15/87 cases). The DNMT3A mutation rate in the NPM1 mutated/FLT3 wild-type cases (16/33, 48.5%, P=0.001) and NPM1/FLT3-ITD mutated cases (19/25, 76%, P<0.001) was significantly higher, confirming the association of DNMT3A mutations with NPM1 and FLT3-ITD mutations that had been reported previously (Ley et al.). Interestingly, also in the cohort of T-ALL we detected patients that carried a DNMT3A mutation (16/83, 19.3%), which is very similar to the mutation frequency in AML, and has not been described yet. To further address the biology of DNMT3A mutations in acute leukemias we combined the AML and T-ALL cohorts and identified in total 31 distinct missense mutations in 65 patients (49 AML, 16 T-ALL). Most frequently, amino acid R882 located in exon 23 was mutated (n=29 cases). In addition, we identified 7 frame-shift alterations, 5 nonsense and 2 splice-site mutations. Moreover, 9 of the 65 mutated cases had two independent mutations. Focusing on AML, only three (6.1%) of the 49 DNMT3A-mutated cases were observed to harbor two different mutations concomitantly. In contrast, in the cohort of T-ALL we detected two different mutations in 6/16 (37.5%, P=0.003) cases. Further, in the cohort of AML, no homozygous mutation was detected, however, in the T-ALL group, two cases harbored a homozygous mutation. Therefore, only 3/49 AML (6.1%) cases, but 8/16 T-ALL (50%) cases showed biallelic mutation status (P<0.001). With respect to overall survival, no association was seen in the complete cohort of CN-AML cases (n=145). After limiting this cohort to the cases without mutations in NPM1, FLT3-ITD, CEBPA and MLL-PTD (n=87), an inferior survival was observed for DNMT3A-mutated patients as compared to DNMT3A wild-type patients (n=15 vs. n=72; alive at 2 years: 27.9% vs. 56.6%; P=0.048). Remarkably, also in the cohort of T-ALL a worse survival for patients with DNMT3A mutations was seen which has not been reported thus far (n=13 vs. n=64; alive at 1 years: 28.6% vs. 80.9%; P=0.001). Subsequently, we were interested whether gain-of-function mutations of the DNMT3A gene were associated with trisomy 2 and acquired uniparental disomy (aUPDs) of the short arm of chromosome 2 where DNMT3A is located. As such, we investigated 9 cases harboring a trisomy 2 (AML n=4, MDS n=4, and CMML n=1) and one MDS patient harboring an aUPD 2p, as confirmed by SNP microarray analyses (SNP Array 6.0, Affymetrix, Santa Clara, CA). Not all, but 3/9 cases with trisomy 2 harbored a DNMT3A mutation (one AML, MDS, and CMML case each), suggesting that duplication of DNMT3A mutations can enhance the effect of the mutation. Moreover, the single case with aUPD 2p also showed a mutation, further suggesting that LOH leading to loss of the wild-type DNMT3A may be another mechanism of disease leading to progression of leukemia. In conclusion, we here report on a high mutation rate of DNMT3A in both AML and T-ALL and independently confirmed an inferior overall survival in these two entities, respectively. This indicates a significant role of DNMT3A alterations in myeloid as well as in lymphoid neoplasms. Disclosures: Grossmann: MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Alpermann:MLL Munich Leukemia Laboratory: Employment. Wild:MLL Munich Leukemia Laboratory: Employment. Weissmann:MLL Munich Leukemia Laboratory: Employment. Eder:MLL Munich Leukemia Laboratory: Employment. Dicker: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.

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