Comprehensive Molecular Profiling of T-Cell Acute Lymphoblastic Leukemia Identifies Mutations in 76 (97.4%) of 78 Patients

Abstract Abstract 2548 Introduction: At present, the diagnosis of T-ALL is based on immunophenotyping and specific chromosomal rearrangements. However, the knowledge about recurrent somatic mutations is limited. Patients and Methods: We studied a cohort of 78 adult T-ALL cases (n=33 early, n=33 cortical, n=2 mature T-ALL, n=10 subtype not available), including 57 males and 21 females. Age ranged from 18.8–87.7 yrs (median: 42 yrs). A deep-sequencing assay was used to investigate for specific molecular alterations in genes involved in transcriptional regulation: NOTCH1, FBXW7, CDKN2A, DNMT3A, FLT3-ITD, FLT3-TKD, NPM1, PTEN, and RUNX1. Further, chromosome banding analysis and FISH with probes for DNMT3A (2p23), SEC63 (6q21), MYB (6q23), CDKN2A (9p21), PTEN (10q23), CDKN1B (12p13) and TP53 (17p13), as well as CDKN2B promoter methylation analyses were performed. Results: Cytogenetic data was available in 68 patients: normal karyotype: n=22 (2 of these harbored a PICALM-MLLT10-rearrangement), SIL-TAL1-rearrangement: n=3, t(5;14)(q35;q32): n=2, t(10;14)(q24;q11)/t(7;10)(q34;q24): n=9, t(10;11)(p13;q21): n=3, other abnormalities n=29. Importantly, molecular mutations were detected in 67/78 patients (85.6%). In detail, NOTCH1 was the most frequently mutated gene (55/77 cases, 71.4%). Other alterations were detected in DNMT3A (16/78; 20.5%); RUNX1 (13/78; 16.6%); FBXW7 (11/75; 14.6%); PTEN (7/78; 10.0%); CDKN2A (3/58; 5.2%); FLT3-ITD (2/78; 2.5%); and FLT3-TKD (1/70; 1.4%). By FISH analyses, heterozygous deletions of the following loci were observed: DNMT3A (1/43; 2.3%), SEC63 (7/43; 16.3%), PTEN (1/32, 3.1%), CDKN1B (8/43; 18.6%) and TP53 (3/43; 7.0%). CDKN2A deletions were detected in 30/72 (41.6%) cases: n=14 heterozygous, n=15 homozygous, n=1 showed a clone with a heterozygous and a subclone with a homozygous deletion. Further, the CDKN2B promoter methylation status was analyzed. Here, 36/74 (48.6%) cases demonstrated hypermethylation. As such, when combining molecular mutations, CDKN2A deletions, and CDKN2B hypermethylation, in median 2 alterations per case were observed (range 1–5). Moreover, almost every patient (76/78) harbored at least one aberration resulting in a mutation rate of 97.4%. Interestingly, considering alterations in the group of cyclin-dependent kinase inhibitors (CDKN2A/1B deletions, CDKN2A mutations, and CDKN2B hypermethylation), 61/78 (78.2%) cases carried at least one such alteration. With respect to associations amongst molecular mutations, no specific pattern was observed except for a strong correlation between RUNX1 and DNMT3A mutations, i.e. 6/13 RUNX1 mutated cases concomitantly harbored DNMT3A mutations (p=0.021). Furthermore, we observed that DNMT3A and RUNX1 alterations were associated with higher age (DNMT3A: mean±SD 60.9±16 vs. 39.6±16 years; p<0.001; RUNX1: mean±SD 55.4±18 vs. 41.7±18 yrs; p=0.013) whereas PTENmut were associated with younger age (mean±SD 32.9±10 vs. 45.0±19 yrs; p=0.019). With regard to cytogenetics, DNMT3A was significantly correlated with normal karyotype (9/23, 39.1% vs. 6/45, 13.3%; p=0.028). Moreover, RUNX1mut were associated with lower WBC count (mean±SD 26.4±41 vs. 63.4±90 cell count G/L; p=0.025). With respect to immunophenotypes, cases with RUNX1mut showed a trend to be associated with early T-ALLs (9/23, 39.1% vs. 6/45, 13.3%; p=0.082). CDKN2B hypermethylation was significantly correlated with early T-ALLs (21/32, 65.6% vs. 10/31, 32.2%; p=0.012). In contrast, FBXW7mut were associated with the cortical subgroup (1/32, 3.1% vs. 9/32, 28.1%; p=0.013). With regard to clinical outcome, patients with RUNX1mut had a shorter overall survival (OS) compared to RUNX1wt patients (alive at 2 yrs: 44.4% mutated vs. 64.0% wild-type, p=0.011). Further, for NOTCH1mut cases (alive at 2 yrs: 67.4% mutated vs. 33.6% wild-type, p=0.060) a trend towards a better OS was detectable. Conclusions: 1. T-ALL is characterized by a high number of genetic alterations since 46/68 (67.6%) showed cytogenetic aberrations. In addition, at least one molecular alteration was observed in 76/78 (97.4%) patients. 2. The most frequent alterations observed were mutations in NOTCH1, DNMT3A, RUNX1 and FBXW7. 3. The cyclin-dependent kinase inhibitors were altered by deletion, mutation or hypermethylation in 78.2% of cases. 4. RUNX1 mutations are associated with shorter and NOTCH1 mutations with longer OS. Disclosures: Grossmann: MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Weissmann:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Artusi:MLL Munich Leukemia Laboratory: Employment. Schindela:MLL Munich Leukemia Laboratory: Employment. Stadler:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Download Full-text

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 ◽

10.1182/blood.v116.21.953.953 ◽

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.

Download Full-text

Characterization of MDS Harboring TET2 Mutations and/or TET2 Deletions

Blood ◽

10.1182/blood.v128.22.4288.4288 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4288-4288 ◽

Cited By ~ 1

Author(s):

Claudia Haferlach ◽

Anna Stengel ◽

Manja Meggendorfer ◽

Wolfgang Kern ◽

Torsten Haferlach

Keyword(s):

Overall Survival ◽

Relative Risk ◽

Molecular Genetic ◽

Normal Karyotype ◽

Prognostic Impact ◽

Complex Karyotype ◽

Wild Type ◽

Employment Equity ◽

Total Cohort ◽

Equity Ownership

Background: TET2 mutations and deletions have been reported in MDS. A detailed analysis of the prognostic impact of TET2 deletions and their association to TET2 mutations is lacking. Aim: To characterize MDS with TET2 mutations (mut) and/or TET2 deletions (del) with respect to accompanying cytogenetic and molecular genetic abnormalities and their impact on prognosis. Patients and Methods: First 788 unselected MDS cases (cohort A) were evaluated. As in this cohort only 8 cases with TET2 deletion were detected, further MDS were screened for TET2 deletions. In total 77 MDS harboring a TET2 deletion were identified and included in cohort B. Both cohorts were analyzed by chromosome banding analysis, FISH, genomic arrays and mutation analysis of TET2. Cases from cohort A were also analyzed for mutations in ASXL1, ATM, BCOR, BRCC3, CBL, CTCF, DNMT3A, ETV6, EZH2, FBXW7, IDH1, IDH2, JAK2, KRAS, LAMB4, MPL, NCOR1, NCR2, NF1, NRAS, PHF6, PRPF8, PTPN11, RAD21, RUNX1, SETBP1, SF3B1, SMC3, SRSF2, STAG2, TET2, TP53, U2AF1 and ZRSR2. Results: In cohort A 248 cases (31%) with TET2mut were identified. TET2del and a normal karyotype were more frequent in MDS with TET2mut as compared to those with TET2 wild-type (wt) (3% vs 1%, p=0.006; 89% vs 78%, p<0.001). SF3B1 and ASXL1 were frequently mutated in both TET2mut and TET2wt MDS (32% and 34%, 22% and 18%, respectively). In MDS with TET2mut compared to MDS with TET2wt the following genes were less frequently mutated: ATM (0.5% vs 3%, p=0.05), DNMT3A (9% vs 15%, p=0.02), ETV6 (0.5% vs 3%, p=0.03), IDH1 (0.5% vs 3%, p=0.02), IDH2 (1% vs 5%, p=0.002), TP53 (2% vs 7%, p=0.004), U2AF1 (4% vs 9%, p=0.04), while the following genes were more frequently mutated: CBL (6% vs 2%, p=0.01), EZH2 (8% vs 2%, p<0.001), SRSF2 (27% vs 12%, p<0.001), and ZRSR2 (15% vs 3%, p<0.001). Overall spliceosome genes were more frequently mutated in TET2mut than in TET2wt MDS (77% vs 56%, p<0.001). In the total cohort A neither TET2mut nor TET2del had an impact on overall survival (OS). In TET2mut MDS and TET2wt MDS SF3B1mut were associated with favorable outcome, while TP53mut were associated with shorter OS in both subsets (table 1). However in TET2mut MDS mutations in RUNX1 (p<0.0001), CBL (p=0.001), and U2AF1 (p=0.03) were independently associated with shorter OS, while in TET2wt MDS mutations in KRAS (p=0.03), EZH2 (p=0.02), NRAS (p=0.02), SRSF2 (p=0.007), IDH2 (p=0.05), and ASXL1 (p=0.01) were independently associated with shorter OS. In cohort B 40/77 (52%) MDS with TET2del also harbored a TET2mut. The 4q deletion encompassing the TET2 gene was < 10 MB in size and thus cytogenetically cryptic in 77% of cases with TET2mut, while the TET2 deletion was cryptic in only 24% of cases without TET2mut. A normal karyotype was present in 37 cases (48%), a complex karyotype in 29 (38%) and other abnormalities in 11 cases (14%). TET2mut were frequent in cases with a normal karyotype (68% vs aberrant karyotype: 32%, p<0.001) and were rare in cases with a complex karyotype (13%). Relating the mutation load of TET2mut to the proportion of cells with TET2del as determined by FISH revealed in 60% of cases that both TET2 alterations were present in the main clone, while in 23% of cases the TET2mut was present in a subclone only and in 17% the TET2del was observed in a subclone only. In the subset of patients with TET2del in a subclone only, 83% showed a normal karyotype and none a complex karyotype, while in the subset of cases with TET2mut in a subclone only, 43% showed a normal and 29% a complex karyotype. In the total cohort B the presence of a TET2mut in addition to the TET2del had no prognostic impact, while the presence of a complex karyotype was associated with shorter OS (RR: 8.0, p=0.004). Conclusions: 1) TET2 deletions are rare in TET2 mutated MDS (3%). 2) TET2 mutations are frequent in MDS with TET2 deletion (52%). 3) TET2 mutations are highly correlated to a normal karyotype and are rare in complex karyotype. 3) Neither TET2 mutations nor TET2 deletions have a prognostic impact in MDS. 4) In TET2 mutated MDS mutations in RUNX1, TP53, CBL, and U2AF1 have the strongest negative independent impact on OS, which in TET2 wild-type MDS is the case for mutations in TP53, KRAS, EZH2, NRAS, SRSF2, IDH2 and ASXL1. Table The relative risk of parameters significantly (p<0.05) associated with overall survival are depicted in TET2 mutated and TET2 wild-type MDS Table. The relative risk of parameters significantly (p<0.05) associated with overall survival are depicted in TET2 mutated and TET2 wild-type MDS Disclosures Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Stengel: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.

Download Full-text

In CML Patients with Good Response to TKIs Other Gene Mutations Are Frequently (37%) Present in Addition to Philadelphia Negative, Cytogenetically Aberrant Clones but Are Rare (4%) in Cases with MMR and Normal Karyotype

Blood ◽

10.1182/blood.v124.21.3126.3126 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3126-3126 ◽

Cited By ~ 1

Author(s):

Susanne Schnittger ◽

Manja Meggendorfer ◽

Niroshan Nadarajah ◽

Tamara Alpermann ◽

Wolfgang Kern ◽

...

Keyword(s):

Kinase Inhibitors ◽

Gene Mutations ◽

Clinical Importance ◽

Normal Karyotype ◽

Imatinib Treatment ◽

Mutation Load ◽

Employment Equity ◽

Cytogenetic Aberration ◽

Equity Ownership ◽

Over Time

Abstract Background: In chronic myeloid leukemia (CML) clonal chromosome aberrations in metaphases not carrying a t(9;22)(q34;q11) have been described during treatment with tyrosine kinase inhibitors (TKI), so-called Philadelphia-negative (Ph-) clones. Very rarely transformation to MDS was observed in patients carrying such Ph- clones but mainly restricted to patients harboring -7. Overall, the clinical significance of this phenomenon remains obscure. Aim: 1) Analyze in a large cohort of TKI-treated CML patients who developed Ph- clones the presence and occurrence of molecular mutations over time. 2) Evaluate whether molecular mutations are also present in CML patients who were at least in major molecular remission (MMR) and presented with a normal karyotype. Patients and Methods: First Cohort: 51 CML patients (pts, 23 males, 28 females; median age: 60 yrs, range: 37-84 yrs) with response to TKI (imatinib only: n=32, nilotinib only: n=2, imatinib and dasatinib or nilotinib: n=11, all three TKIs: n=6) who developed Ph- clones. Cytogenetics in these pts were as follows: +8 sole (n=24), -Y (n=8), -7 sole (n=4), +9 (n=2), other trisomies (n=4), 9 had other aberrations including some with combinations of two different clones (n=4). In median these abnormalities were present in 30% (range 8-100%) of analyzed metaphases. BCR-ABL1 levels at the time point of analysis were between 0 and 3.8 (median: 0.023) according to international scale. Second Cohort: 50 CML pts (24 males, 26 females; median age: 56 yrs, range: 21-83 yrs), who were at least in MMR and without development of any cytogenetic aberration after 3 years of imatinib treatment. Median time from start of therapy to analysis was 2.6 years (range 3 months to 14 yrs). All cases were analyzed with a pan-myeloid gene panel of 29 genes: ASXL1, BCOR, BRAF, CBL, DNMT3A, ETV6, EZH2, FLT3 (TKD), IDH1, IDH2, JAK2, KIT, KRAS, MLL-PTD, NOTCH1, NPM1, NRAS, PRPF40B, PTPN11, SF1, SF3A1, SF3B1, SRSF2, TET2, TP53, U2AF1, U2AF2 and ZRSR2. Either complete coding genes or hotspots were first amplified by a microdroplet-based assay (RainDance, Billerica, MA) and subsequently sequenced with a MiSeq instrument (Illumina, San Diego, CA). In addition, RUNX1 was sequenced on the 454 NGS platform (454 Life Sciences, Branford, CT). Results: In the first cohort 28 mutations were found in 19 patients, as 5 patients had 2 and 2 patients even 3 mutations.Thus,in 19/51 pts (37.3%) ≥1 mutation was detected. Median mutation load was 11.5% (range: 2-56%). In detail, mutations in the following genes were detected: ASXL1 (n=9), DNMT3A (n=7), RUNX1 (n=3), NRAS (n=2), TET2 (n=2) and one each in CBL, EZH2, IHD1, PRPF40B, and TP53. Subsequently, these mutations were evaluated in samples from earlier or later time points (18 pts with a total of 235 samples, range: 3-20 samples/pt). In 12 cases a sample from diagnosis of CML was available. In 2 cases a CBL and an ASXL1 mutation were already detectable at low levels, 1.4% and 2%, respectively, at the time of diagnosis and later increased with decreasing BCR-ABL1 levels. In all other 10 cases the mutations were not detectable at diagnosis and were for the first time detectable during TKI treatment (in median after 24 months after diagnosis, range 2-73 months). In the remaining 6 cases date of occurrence could not be determined by backtracking as all earlier samples available were positive for the respective mutation. However, the over time mutation levels were inversely related to BCR-ABL1 expression indicating the presence in independent clones. Within the second cohort with cases in MMR that remained cytogenetically normal only in 2 of the 50 pts (4%) mutations were detected. In one patient a DNMT3A mutation was detected that could be monitored for 8 years with constant low mutation load (3-6%). This was not detectable at diagnosis and occurred after 6 months on imatinib. Very similarly, in the second case a TET2 mutation was first detected after 6 months on imatinib with a mutation load of 2% that very slowly increased to 7% within 8 years. Conclusions: 1) In CML patients that develop Ph- clones other mutations occur in 37.3%. 2) In contrast, in randomly selected CML pts with MMR that are cytogenetically normal, molecular mutations can be detected in only 4%. 3) The clinical importance of molecular mutations in CML in MMR remains unclear. 4) However, these results implicate that chromosomal aberrations are an indicator for genomic instability, also at the molecular level. Disclosures Schnittger: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Alpermann: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.

Download Full-text

The MDM2 Inhibitor NVP-CGM097 Is Highly Active in a Randomized Preclinical Trial of B-Cell Acute Lymphoblastic Leukemia Patient Derived Xenografts

Blood ◽

10.1182/blood.v126.23.797.797 ◽

2015 ◽

Vol 126 (23) ◽

pp. 797-797 ◽

Cited By ~ 5

Author(s):

Elizabeth C Townsend ◽

Tiffany DeSouza ◽

Mark A. Murakami ◽

Joan Montero ◽

Kristen Stevenson ◽

...

Keyword(s):

Research Funding ◽

Lymphoblastic Leukemia ◽

Wild Type ◽

Employment Equity ◽

Highly Active ◽

Cell Acute Lymphoblastic Leukemia ◽

Equity Ownership ◽

Relapsed Disease ◽

Mdm2 Inhibitor

Abstract The majority of drugs tested in clinical trials for hematologic malignancies ultimately fail to confer benefit. In many cases, this results from the inability to accurately identify patients most likely to benefit and mechanisms that mediate resistance. At the same time, clinical trials commonly fail to capture essential specimens at early and late timepoints that could be used to identify predictors of response, pharmacodynamic markers of on-target activity and targetable resistance pathways. To overcome these challenges, we have developed a repository of >200 patient-derived xenografts (PDXs) of leukemias and lymphomas now available through the DFCI Public Repository of Xenografts (PRoXe; http://www.proxe.org). Because PDX models can be developed and propagated from a large number of patients, randomized in vivo studies using adequate numbers of PDXs offer the first opportunity to capture the diversity of disease biology in pre-clinical drug testing. We tested the novel MDM2 inhibitor CGM097, which is currently undergoing clinical testing in solid tumors, in 24 B-cell acute lymphoblastic leukemia (B-ALL) PDXs (including hypodiploid, near haploid, MLL- rearranged, CRLF2-rearranged, and BCR-ABL models) in a randomized, phase II-like trial. Only a small subset of B-ALLs harbor de novo TP53 mutations, suggesting that MDM2 antagonists may have broad activity in this disease. Each PDX was injected into 4 NOD.SCID.IL2Rɣ-/- (NSG) mice. Upon engraftment (>2% hCD45+/hCD19+ cells in the peripheral blood), mice were randomized to vehicle or CGM097 treatment arms. One animal from each treatment arm was sacrificed 26 hours after beginning treatment to examine pharmacodynamic endpoints. The remaining two mice continued on daily therapy until moribund. CGM097 markedly improved overall survival (median 73 vs 28 days for vehicle; p=0.0008). All 19 models with survival benefit from CGM were TP53 wild-type. Among 6 models (all TP53 wild-type) derived from patients with relapsed disease, the median survival improvement compared to vehicle was 53 days (p=0.0059), consistent with robust single-agent activity in relapsed disease. Specimens at the 26 hour timepoint and upon progression to moribund were captured from the majority of mice in the trial, allowing for comprehensive characterization of the trial population. Dynamic BH3 profiling (Montero et al. Cell 2015), in which CGM097 or vehicle is added to leukemia cells harvested from mice and the effect of CGM097 on "priming" for apoptosis was performed on 10 models and demonstrated 100% accuracy in predicting response to CGM097. To characterize the effects of MDM2 inhibition on p53-dependent gene expression, we measured expression of 120 p53-related genes using a custom Nanostring gene expression panel. Differential expression analysis identified 11 genes that were significantly upregulated (p≤0.05) by CGM097 treatment at the 26 hour timepoint, including the canonical p53 targets BBC3, CDKN1A and MDM2. All mice treated with CGM097 ultimately became moribund from progressive leukemia. Targeted deep sequencing identified acquired TP53 mutations in only 2 leukemias after progression on CGM097. This indicates that p53 mutation is not the primary genetic driver of resistance to MDM2 inhibition in B-ALL PDXs. Despite this, CGM097-dependent transcriptional changes were largely abolished in the majority of leukemias collected from mice upon progression on CGM097. In summary, we established a paradigm for "Phase II-like" trials in panels of human leukemia PDX models. With this approach, we defined CGM097 as a highly active agent across the diverse spectrum of TP53-wildtype B-ALL, and established 19 independent models of acquired resistance that are the ideal reagents for defining mechanisms and then testing combinations in vivo that overcome those mechanisms. The same paradigm could be applied as a new standard for pre-clinical testing of drugs to minimize the empiric nature of current drug development strategies. Disclosures Barzaghi-Rinaudo: Novartis Institutes for Biomedical Research: Employment. Letai:AbbVie: Consultancy, Research Funding; Tetralogic: Consultancy, Research Funding; AstraZeneca: Consultancy, Research Funding. Jeay:Novartis: Employment, Equity Ownership. Wuerthner:Novartis: Employment, Equity Ownership. Halilovic:Novartis: Employment, Equity Ownership.

Download Full-text

CHFR promoter methylation indicates poor prognosis in stage II microsatellite stable colorectal cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.15_suppl.e14503 ◽

2013 ◽

Vol 31 (15_suppl) ◽

pp. e14503-e14503

Author(s):

Manon van Engeland ◽

Arjen Cleven ◽

Sarah Derks ◽

Muriel XG Draht ◽

Kim M Smits ◽

...

Keyword(s):

Colorectal Cancer ◽

Promoter Methylation ◽

Poor Prognosis ◽

Cpg Island ◽

Methylation Status ◽

Genetic Alterations ◽

Stage Ii ◽

Prognostic Impact ◽

Wild Type ◽

Cpg Island Methylation

e14503 Background: Data on the prognostic significance of promoter CpG island methylation in colorectal cancer (CRC) are conflicting, possibly due to associations between methylation and other factors affecting survival such as genetic alterations and use of adjuvant therapy. Here we examine the prognostic impact of promoter methylation in CRC patients treated with surgery alone in the context of microsatellite instability (MSI), BRAF- and KRAS mutations. Methods: 173 CRCs were analyzed for promoter methylation of 19 tumor suppressor- and DNA repair genes, the CpG island methylator phenotype (CIMP), MSI, the exon 15 V600E BRAF mutation and KRAS codon 12 and 13 mutations. Results: Unsupervised hierarchical clustering based on methylation status of 19 genes revealed three subgroups: cluster 1 (CL1, 57% (98/173) of CRCs), cluster 2 (CL2, 25% (43/173) of CRCs) and cluster 3 (CL3, 18% (32/173) of CRCs). CL3 had the highest methylation index (0.25, 0.49 and 0.69 respectively, p=<0.01) and was strongly associated with CIMP (p<0.01). After stratification for tumor stage, MSI and BRAF status, no statistically significant differences in survival between CL1, CL2 and CL3 nor between CIMP and non-CIMP CRCs were detected. Analyzing genes separately revealed that CHFR promoter methylation was associated with a poor prognosis in stage II, MSS, BRAF wild-type CRCs: HR=3.89 (95% CI =1.58-9.60, p=0.003) and HR=2.21 (95% CI =1.09-4.48, p=0.03) in a second population-based study (n=151). Conclusions: CHFR promoter CpG island methylation, which is associated with MSI, also occurs frequently in MSS CRCs and is a promising prognostic marker in stage II, MSS, BRAF wild-type CRCs.

Download Full-text

RUNX1 Mutations Reduce the Expression of CEBPA in Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v118.21.2420.2420 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2420-2420 ◽

Cited By ~ 1

Author(s):

Vera Grossmann ◽

Alexander Kohlmann ◽

Frank Dicker ◽

Katrin Butschalowski ◽

Wolfgang Kern ◽

...

Keyword(s):

Myeloid Leukemia ◽

Normal Karyotype ◽

Transactivation Domain ◽

Wild Type ◽

Runt Domain ◽

Employment Equity ◽

Expression Intensity ◽

Frame Shift ◽

Equity Ownership ◽

Acute Myeloid

Abstract Abstract 2420 Acute myeloid leukemia (AML) is characterized by a clonal expansion of myeloid progenitor cells in peripheral blood and bone marrow. CEBPA (CCAAT/enhancer binding protein alpha) is an essential transcription factor for granulocytic differentiation and encodes a protein exclusively expressed in the myeloid lineage. Mutations in CEBPA, which have a loss-of-function character, occur in 5–14% of patients with AML and result in a cellular differentiation block. CEBPA function can also be affected by mutations in other oncogenes or directly by control of its expression. Various fusion genes have an impact on CEBPA function, e.g. RUNX1-RUNX1T1, which suppresses CEBPA mRNA expression. RUNX1 contains two different domains: the RUNT domain (DNA binding site) and the TAD domain (transactivation domain). RUNX1-RUNX1T1 negatively regulates CEBPA via the RUNT domain by inhibition of autoregulation. Here, we studied whether CEBPA mRNA expression is affected by the mutational status of RUNX1. First, we investigated 209 AML cases using gene expression microarray profiling (Affymetrix HG-U133 Plus 2.0). These included cases with normal karyotype (n=93), with t(8;21) (n=16), with t(15;17) (n=15), with trisomy 8 sole (n=12), with trisomy 13 sole (n=10), with complex aberrant karyotype (n=10), and with various other genetic abnormalities (n=53). The RUNX1 mutation status was analyzed in 178 cases, excluding those cases with t(8;21) and t(15;17), using conventional Sanger sequencing or an amplicon-based next-generation deep-sequencing assay (454 Life Sciences, Branford, CT). In this selected cohort, 41/178 (23%) cases harbored RUNX1 molecular mutations. The median CEBPA expression intensity in all patients was 670 (range 48 – 5,244). In RUNX1 mutated cases the CEBPA expression was significantly lower compared to RUNX1 wild-type cases (mean±SD 429±395 vs. 998±717; P<0.001). Of note, we also confirmed that cases harboring a RUNX1-RUNX1T1 translocation presented a lower CEBPA expression than patients without (n=16 vs. 194, mean±SD 292±216 vs. 950±808; P<0.001), whereas PML-RARA mutated cases showed enhanced expression (n=15 vs. 195, mean±SD 1940±1290 vs. 819±690; P=0.005). In order to validate our results, we subsequently investigated an independent cohort of 151 AML cases exclusively presenting with a normal karyotype. A quantitative real-time RT-PCR assay (Taqman®, Life Technologies, Carlsbad, CA) was established and the expression of CEBPA was normalized against the expression of the control gene ABL1. The median CEBPA expression intensity was 148 (range 21 – 960). Repeatedly, the CEBPA expression was significantly lower in RUNX1 mutated cases (n=81) compared to RUNX1 wild-type cases (n=70) (mean±SD 155±98 vs. 220±182; P=0.008). Additionally, we were interested whether the localization of the mutations in RUNX1 showed any impact on the CEBPA expression. However, no difference was detected between CEBPA expression levels, when RUNX1 mutations were located either outside or within the DNA binding domain (RUNT) (n=46 cases), or behind the RUNT and within the TAD (transactivation domain) domain (n=35 cases) (mean±SD 161±107 vs. 148±87; P=0.559), respectively. In addition, also when comparing missense mutations (n=19) against frame-shift and nonsense alterations (n=62), no difference could be detected (mean±SD 177±114 vs. 148±93; P=0.268). Of note, of the 46 mutations located in the RUNT domain 16 were missense, 5 were nonsense, and 24 were frame-shift mutations, whereas in the TAD domain exclusively nonsense and frame-shift mutations (n=17) were found, which most probably reflects the distinct domain functions. In contrast, separating cases in heterozygous (n=60) or homozygous (n=21) mutations (including those cases with 2 mutations, n=7), we observed a trend towards a lower expression in cases with homozygous mutations (mean±SD 123±88 vs. 166±100; P=0.084). In conclusion, we demonstrated for the first time a significant negative effect of RUNX1 mutations on the expression levels of CEBPA, similarly to the RUNX1-RUNX1T1 fusion which had been reported previously. Moreover, we did not detect any correlations with respect to the localization of the mutations in RUNX1 and CEBPA expression. Disclosures: Grossmann: MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Dicker:MLL Munich Leukemia Laboratory: Employment. Butschalowski: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. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Download Full-text

Comparison Of Mutation Patterns Between Diagnosis and Relapse In 556 Adult Patients With AML Shows High Variability Of Stability

Blood ◽

10.1182/blood.v122.21.4978.4978 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4978-4978

Author(s):

Susanne Schnittger ◽

Niroshan Nadarajah ◽

Tamara Alpermann ◽

Christiane Eder ◽

Alexander Kohlmann ◽

...

Keyword(s):

Negative Impact ◽

Melting Curve ◽

Normal Karyotype ◽

Genetic Alterations ◽

Prognostic Impact ◽

Fusion Genes ◽

Employment Equity ◽

Disease Evolution ◽

Equity Ownership ◽

The Impact

Abstract Background In acute myeloid leukemia (AML) molecular mutations are becoming increasingly important as markers for classification, risk stratification and disease monitoring. Frequencies and prognostic impact of most of the currently known mutations have been widely studied. In contrast, the stability during disease evolution and the role of single markers at relapse are less clear. Aim 1) To compare the patterns of molecular mutations between diagnosis and relapse. 2) To analyze the impact of single mutations on time to relapse (TTR). Patients and Methods We investigated paired diagnostic and relapse samples in a cohort of 556 adult AML cases that were selected based on confirmed relapse with available samples from both time points (483 de novo, 33 t-AML, 40 s-AML). The cohort comprized 255 females and 301 males; median age: 63.0 years (range: 18.6-85.2 years). In total, 5,726 paired analyses were performed (mean: 10.3/pt, range: 2-21). Besides analyses for fusion genes (n=114) the following genes were analyzed in paired samples for mutations in respective numbers: ASXL1: n=464, CBL: n=99, CEBPA: n=374, DNMT3A: n=244, FLT3-ITD: n=534, FLT3-TKD: n=461, IDH1: n=469, IDH2: n=418, KIT: n=34, KRAS: n=49, MLL-PTD: n=165, NPM1: n=343, NRAS: n=105, RUNX1: n=356, TET2: n=243, WT1: n=153, others: n=1,101. Mutations were analyzed by amplicon deep-sequencing, direct Sanger sequencing, gene scan, conventional PCR, quantitative real time PCR or melting curve analyses. In addition, chromosome banding analysis data was available in 552 cases. Results 629 relapses were detected in the 556 patients (pts). 67 pts had a second and 6 even a third relapse. At diagnosis the subtypes according to cytogenetics were as follows: PML-RARA (n=10), RUNX1-RUNX1T1 (n=24), CBFB-MYH11 (n=23), MLL-translocations (n=30), DEK-CAN (n=3), other recurrent translocations (n=14), complex karyotype (n=30), other aberrations (n=107), normal karyotype (NK, n=311). In 515/556 (92.6%) pts at least one mutation (mut) was detected at diagnosis (mean: 2.3; range:1-5). In detail, the most frequent markers were mut in: NPM1: n=233, FLT3-ITD: n=217, fusion genes: n=114, DNMT3A: n=101, RUNX1: n=98, TET2: n=81, FLT3-TKD: n=58, ASXL1: n=54, IDH2R140: n=46, MLL-PTD: n=44, WT1: n=42, TP53: n=26, biallelic CEBPA(bi): n=28, monoallelic CEBPA(mono): n=23. At relapse a similar incidence of 88.6% (558/630) mutated pts (mean: 2.0: range 1-6) was detected. However, the mutational pattern changed at relapse in 381/629 (60.6%) of the pts. On the marker level a change was seen for 309 (24.5%) of the mut, with gain of 142/1,263 new mut (11.3%) and a loss of 167 (13.2%) mut that were detectable at diagnosis. Different degrees of stability were observed. Compared to all other markers a stable pattern was found for CEBPAbi: p=0.025; NPM1: p<0.001; MLL-PTD: p=0.006; and fusion genes: p<0.001. This is supporting the respective definition of entity defining mut in the WHO classification. Instead, a low stability (for gain and/or loss) compared to all others was seen for CEBPAmono: p=0.002, FLT3-ITD: p<0.001, FLT3-TKD: <0.001, NRAS: p<0.001, TP53: p<0.001 (only for gain), WT1: p<0.001. This is in accordance with the concept of typical “secondary” mutations, leading to acceleration of the disease. An intermediate stability was detected for TET2, DNMT3A, RUNX1, ASXL1,IDH2R140 (see figure). Of note, of 200 FLT3-ITD positive pts that retained an FLT3-ITD at relapse, only 6 (3%) were stable with respect to mutational load whereas 134 (67%) showed an increase of the FLT3-ITD/wildtype load, and 60 (30%) a decrease showing a further quality of genetic instability. Between the various disease entity defining groups there were no relevant differences in TTR. In contrast, an impact on shorter TTR was seen for mutations in DNMT3A (median TTR: 8.7 vs 12.1 months (m), p=0.058), FLT3-ITD (7.6 vs 12.2 m, p<0.001), RUNX1mut (10.1 vs 12.8 m, p=0.038) and TET2mut (9.9 vs 10.9 m, p=0.049). In the normal karyotype group an effect was only seen for FLT3-ITD (7.3 vs 13.6, p<0.001) and TET2mut (9.9 vs 12.3 m, p=0.025). Conclusions 1) Genetic alterations in AML can be subdivided into stable, intermediate and unstable markers. Entity defining markers according to WHO (fusion genes, NPM1mut, CEBPAbi) are stable between diagnosis and relapse. 2) A significant negative impact on TTR was shown only for secondary mutations (FLT3-ITD, TET2mut, DNMT3Amut and RUNX1 mut). Disclosures: Schnittger: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Eder:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Kuznia:MLL Munich Leukemia Laboratory: Employment. Weissmann:MLL Munich Leukemia Laboratory: Employment. Fasan:MLL Munich Leukemia Laboratory: Employment. Weber:MLL Munich Leukemia Laboratory: Employment. Albuquerque:MLL Munich Leukemia Laboratory: Employment. Jeromin:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Dicker: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.

Download Full-text

Comprehensive Genetic Analyses Of 213 PMF Patients Revealed 13% Of SRSF2 Mutations Being Strongly Correlated To ASXL1 Mutations

Blood ◽

10.1182/blood.v122.21.2828.2828 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2828-2828

Author(s):

Manja Meggendorfer ◽

Tamara Alpermann ◽

Christiane Eder ◽

Claudia Haferlach ◽

Wolfgang Kern ◽

...

Keyword(s):

Molecular Marker ◽

Scoring System ◽

Sanger Sequencing ◽

Gene Mutations ◽

Normal Karyotype ◽

Prognostic Impact ◽

Wild Type ◽

Employment Equity ◽

Cytogenetic Aberrations ◽

Equity Ownership

Abstract Introduction Primary myelofibrosis (PMF) is a myeloproliferative neoplasm, characterized amongst others by stem-cell derived clonal myeloproliferation, bone marrow fibrosis, anemia, splenomegaly, constitutional symptoms and leukemic progression. Diagnosis is based in most cases on cytomorphology/histology demonstrating fibrosis as well as on mutations in JAK2 or MPL. The Dynamic Prognostic Scoring System (DIPSS)-plus is the current base for prognostication using different clinical parameters including karyotype. Furthermore, molecular genetic alterations are currently addressed to provide additional prognostic information. Recently, besides JAK2 and MPL further gene mutations have been described in a limited number of patients, including ASXL1 and SRSF2. Aim To analyze in a large cohort the frequency of SRSF2 and ASXL1 mutations in PMF patients, and to identify their prognostic impact in the context of other previously described gene mutations. Patients and Methods Diagnosis was done according to WHO classification. The cohort comprised 82 female and 131 male patients. In all cases a BCR-ABL rearrangement was excluded by RT-PCR or fluorescence in situ hybridization. JAK2V617F mutation was analyzed in all cases by melting curve analysis, MPLW515 mutation was subsequently analyzed in JAK2V617 wild type (wt) patients. In addition, we analyzed all patients for SRSF2 mutations by Sanger sequencing of the mutational hot spot region coding for amino acid Pro95. Cytogenetics was available in 139 patients. Patients were grouped in favorable (n=121) and unfavorable (n=18) karyotypes based on the DIPSS-plus-scoring system. Based on the previously described correlation of SRSF2mut with ASXL1mut and SETBP1mut in other myeloid entities, SRSF2 mutated cases were also analyzed for mutations in ASXL1 and SETBP1by Sanger sequencing. Follow-up data was available for 136 patients. Results 56% (120/213) of the patients showed JAK2V617F mutations and 18.0% (16/89) of JAK2wt patients carried a mutation in MPLW515 summing up to 65.1% of patients with an already established molecular marker. Of note, SRSF2 was mutated in 12.7% (27/213) of all PMF patients. Patients with SRSF2 mutation had higher white blood cell counts in comparison to SRSF2wt patients (20.00x109/L vs. 7.35x109/L; p=0.005), but there was no correlation to gender, age, hemoglobin level, platelet count or % of myeloblasts in the peripheral blood. In 17 SRSF2mut cases the karyotype was available, 12 were normal karyotype, while two cases showed an unfavorable karyotype according to DIPSS-plus with +8 and i(17)(q10), respectively. The remaining three aberrations belong to the favorable aberration group (del(20q), del(13q), and der(14)). There was no correlation of SRSF2 mutations to the cytogenetic subgroups normal karyotype (n=91) or DIPSS categories favorable and unfavorable aberrations. SRSF2 mutations were also equally distributed between both JAK2V617 or MPLW515 mutated and wild type cases. 18/27 SRSF2mut cases carried also either a JAK2 or MPL mutation, while 9 cases showed no additional JAK2 or MPL mutation. Therefore 30.6% patients remain that carry no mutation in at least one of the three genes investigated first. Interestingly, ASXL1 was frequently mutated in SRSF2 mutated patients (16/23 analyzed SRSF2mut patients) while none of the 24 analyzed SRSF2 mutated cases showed a mutation in SETBP1. To evaluate a potential influence of gene mutations on clinical outcome the overall survival (OS) was calculated. We could confirm that JAK2V617F had no prognostic impact. The same was true for MPLW515 mutations. In contrast to other studies we could not find any impact of SRSF2mutations on OS. Only cytogenetics, i.e. the normal karyotype showed a trend to a prognostic relevance: the median 3 year OS was 70.8% in patients with normal karyotype (n=56) but 58.8% in patients with cytogenetic aberrations (n=29; p=0.153). Conclusion 1) SRSF2 is mutated in 13% of PMF patients. 2) SRSF2 mutated patients show frequently an additional ASXL1 mutation but no coincidence with SETBP1. 3) The prognostic relevance of cytogenetic aberrations was confirmed, while the molecular marker SRSF2 shows no impact on prognosis. Disclosures: Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Eder:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Download Full-text

Integrated Analysis of Global DNA Methylation and Transcription Reveals a Leukemia Subtype with Extreme Hypermethylation Associated with Silencing of Regulators of Differentiation

Blood ◽

10.1182/blood-2018-99-118521 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2608-2608

Author(s):

Claudia Gebhard ◽

Roger Mulet-Lazaro ◽

Lucia Schwarzfischer ◽

Dagmar Glatz ◽

Margit Nuetzel ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Promoter Methylation ◽

Research Funding ◽

Promoter Hypermethylation ◽

Ctcf Binding ◽

P Value ◽

Global Dna Methylation ◽

Employment Equity ◽

Equity Ownership

Abstract Acute myeloid leukemia (AML) represents a highly heterogeneous myeloid stem cell disorder classified based on various genetic defects. Besides genetic alterations, epigenetic changes are recognized as an additional mechanism contributing to leukemogenesis, but insight into the latter process remains minor. Using a combination of Methyl-CpG-Immunoprecipitation (MCIp-chip) and MALDI-TOF analysis of bisulfite-treated DNA in a cohort of 196 AML patients we previously demonstrated that (cyto)genetically defined AML subtypes, including CBFB-MYH11, AML-ETO, NPM1-mut, CEBPA-mut or IDH1/2-mut subtypes, express specific DNA-methylation profiles (Gebhard et al, Leukemia, 2018). A fraction of AML patients (5/196) displayed a unique abnormal hypermethylation profile that was completely distinct from any other AML subtype. These patients present immature leukemia (FAB M0, M1) with various chromosomal aberrations but very few mutations (e.g. no IDH1/2, KRAS, DNMT3A) that might explain the CpG island methylator phenotype (CIMP) phenotype. The CIMP patients showed high resemblance with a recently reported CEBPA methylated subgroup (Wouters et al, 2007 and Figueroa et al, 2009), which we confirmed by MCIp-chip and MALDI-TOF analysis. To explore the whole range of epigenetic alterations in the CIMP-AML patients we performed in-depth global DNA methylation and gene expression analyses (MCIp-seq and RNA-seq) in 45 AML and 12 CIMP patients from both studies. Principle component analysis and t-distributed stochastic neighbor embedding (t-SNE) revealed that CIMP patients express a unique DNA-methylation and gene-expression signature that separated them from all other AMLs. We could discriminate promoter methylation from non-promoter methylation by selecting MCIp-seq peaks within 3kb around TSS. Promoter hypermethylation was highly associated with repression of genes (PCC = -0.053, p-value = 0.00075). Hypermethylation of non-promoter regions was more strongly associated with upregulation of genes (PCC = 0.046, p-value = 4.613e-06). Interestingly, differentially methylated regions also showed a positive association with myeloid lineage CTCF binding sites (27% vs 18% expected, p-value < 2.2e-16 in a chi-square test of independence). Methylation of CTCF sites causes loss of CTCF binding, which has been reported to disrupt boundaries between so-called topologically associated domains (TADs), allowing enhancers located in a particular TAD to become accessible to genes in adjacent TADs and affect their transcription. Whether this is the case is under investigation. In this study we particularly focused on the role of hypermethylation of promoters in CIMP-AMLs. Promoters of many transcriptional regulators that are involved in the differentiation of myeloid lineages of which several are frequently mutated in AML were hypermethylated and repressed, including CEBPA, CEBPD, IRF8, GATA2, KLF4, MITF or MAFB. Notably, HMGA2, a critical regulator of myeloid progenitor expansion, exhibited the largest degree of CIMP promoter hypermethylation compared to the other AMLs, accompanied by a reduction in gene expression. Moreover, multiple members of the HOXB family and KLF1 (erythroid differentiation) were methylated and repressed as well. In addition, these patients frequently showed hypermethylation of many chromatin factors (e.g. LMNA, CHD7 or TET2). Hypermethylation of the TET2 promoter could result in a loss of maintenance DNA demethylation and therefore successive hypermethylation at CpG islands. We carried out regulome-capture-bisulfite sequencing on CIMP-AMLs compared to other AML samples and normal blood cell controls and confirmed methylation of the same transcription and chromatin factor promoters. We conclude that these leukemias represent very primitive HSCPs which are blocked in differentiation into multiple hematopoietic lineages, due to the absence of regulators of these lineages. Although the underlying cause for the extreme hypermethylation signature is still subject to ongoing studies, the consequence of promoter hypermethylation is silencing of key lineage regulators causing the differentiation arrest in these cells. We argue that these patients may particularly benefit from therapies that revert DNA methylation. Disclosures Ehninger: Cellex Gesellschaft fuer Zellgewinnung mbH: Employment, Equity Ownership; GEMoaB Monoclonals GmbH: Employment, Equity Ownership; Bayer: Research Funding. Thiede:AgenDix: Other: Ownership; Novartis: Honoraria, Research Funding.

Download Full-text

Focal Adhesion Kinase Inhibitors Reverse the Stromal Adhesion Phenotype of Ikaros-Mutant B-ALL, Induce Apopotosis, and Synergize with ABL1 Tyrosine Kinase Inhibitors: A New Paradigm for Pathogenesis and Therapy of High-Risk B-ALL

Blood ◽

10.1182/blood.v124.21.285.285 ◽

2014 ◽

Vol 124 (21) ◽

pp. 285-285 ◽

Cited By ~ 1

Author(s):

Ila Joshi ◽

Nilamani Jena ◽

Toshimi Yoshida ◽

Leto Paraskevopoulou ◽

Zhihong Zhang ◽

...

Keyword(s):

High Risk ◽

B Cell ◽

Focal Adhesion ◽

Research Funding ◽

Kinase Inhibitors ◽

Dominant Negative ◽

Employment Equity ◽

Equity Ownership ◽

B Lymphoid ◽

Inhibitor Treatment

Abstract B-cell acute lymphoblastic leukemia (B-ALL) is a malignancy of precursor B-lymphocytes affecting both children and adults. Deletions and dominant-negative mutations in IKZF1, the gene encoding the Ikaros transcription factor, are found in ~85% of Ph+ B-ALL and in some cases of Ph– B-ALL, and are associated with poor prognosis. Genomic studies of high-risk Ph– or “Ph-like” B-ALLs have revealed frequent mutation and activation of TK genes and signaling pathways. While ABL1 tyrosine kinase inhibitors (TKIs) such as dasatinib and imatinib have been added to chemotherapy regimens for Ph+ B-ALL, over half of these patients will still relapse, which correlates with residual disease burden in the bone marrow (BM) following induction therapy. Hence, new therapeutic strategies are needed for patients with Ikaros-mutant, high-risk Ph+ and Ph– B-ALL. Using mice with a conditional Ikzf1 mutation (Ike5fl) where the recombined allele is similar to the dominant-negative Ik6 mutant found in human B-ALL, we demonstrated recently that Ikaros DNA-binding function is required in the B-lymphoid lineage for transition from the large to small pre-B cell stage of differentiation, and that arrest at this stage of development can give rise to B-ALL (Joshi et al., Nat. Immunol. 2014;15:294). The survival and proliferation of Ikaros mutant pre-B cells is dependent on increased integrin-mediated stromal adhesion and activation of focal adhesion kinase (FAK). FAK is a non-receptor TK, downstream of integrins and growth factor receptors, which plays important roles in cancer stem cell biology, the tumor microenvironment and tumorigenesis. VS-4718 and VS-6063 (defactinib) are potent, orally bioavailable FAK inhibitors that inhibit tumor growth and metastasis in preclinical models, and are currently under evaluation in clinical trials in patients with various solid tumors. VS-6063 has demonstrated tolerability and preliminary signs of clinical activity as a single agent and in combination with paclitaxel in phase I trials (ASCO, 2014). Here, we show that BCR-ABL1 cooperates with Ikzf1 mutation to accelerate B-leukemogenesis in mice. BCR-ABL1+ Ikaros-mutant B-ALLs exhibit stroma-mediated resistance to ABL1 TKIs, while the FAK inhibitors VS-4718 and VS-6063 are effective in blocking stromal adhesion and inducing apoptosis in both mouse and human Ikaros-mutant B-ALL samples. To test whether dysregulation of TK signaling cooperates with Ikzf1 mutation in the pathogenesis of high-risk B-ALL, we isolated BM B-lymphoid progenitor cells from wild-type (WT), IkE5fl/+ CD2-Cre, and IkE5fl/fl CD2-Cre donors, transduced them with BCR-ABL1 retrovirus and transplanted the cells into recipient mice. We observed a dramatic acceleration of precursor B-lymphoid leukemia induced by BCR-ABL1 in IkE5Δ/+ and particularly in IkE5Δ/Δ donor cells that correlated with a striking (~30-fold) increase in the frequency of engrafting leukemia-initiating or leukemic stem cells (LSCs). Relative to Ikzf1 WT BCR-ABL1+ leukemic cells, Ikzf1-mutant BCR-ABL1+ blasts showed significant resistance to imatinib and dasatinib that was dependent on the presence of OP9 stroma. The effect of FAK inhibition, using the FAK inhibitors VS-4718, VS-6062, and VS-6063 (Verastem), was first tested on murine B-ALL cells (genotypes Ikzf1 mutant, Ikzf1 mutant BCR-ABL1+, and Ikzf1 WT BCR-ABL1+) grown on OP9 stroma. FAK inhibitor treatment abolished stromal adhesion of Ikzf1-mutant B-ALL and induced apoptosis in non-adherent cells, but had little effect on Ikzf1 WT B-ALL cells. VS-4718 and VS-6063 were each synergistic with dasatinib in reducing the viability of Ikzf1-mutant BCR-ABL1+ B-ALL cells cultured on OP9 stroma. For primary human B-ALL samples grown on OP9 stroma, IKZF1-mutant cells were also more sensitive to FAK inhibitor treatment than WT IKZF1 WT B-ALL, with or without BCR-ABL1 expression. Collectively, these observations suggest a new model to explain the pathogenesis of high-risk B-ALL and its resistance to therapy. B-ALLs with IKZF1 mutations may be resistant to TKIs and to chemotherapy by virtue of their stromal adhesion phenotype, resulting in failure to eliminate BM LSCs. Inhibition of FAK signaling in Ph+ or Ph–IKZF1-mutant B-ALL may reverse the stromal-mediated resistance to ABL1 TKIs and/or chemotherapy. Therefore, FAK inhibitors warrant further investigation for the treatment of high-risk IKZF1-mutant B-ALL patients. Disclosures Joshi: Verastem: Research Funding. Yoshida:Verastem, Inc.: Research Funding. Paraskevopoulou:Verastem, Inc.: Research Funding. Zhang:Verastem, Inc.: Research Funding. Krause:Glycomimetics. Inc.: Research Funding. Shapiro:Verastem: Employment, Equity Ownership. Weaver:Verastem: Employment, Equity Ownership. Pachter:Verastem Inc.: Employment, Equity Ownership. Georgopoulos:Verastem, Inc.: Research Funding.

Download Full-text