Characterization of Del(14)(q24q32) in Mature B-Cell Neoplasms By Array-CGH, Cytogenetics and Molecular Mutations

Abstract Background: Deletions of 14q occur recurrently in mature B-cell neoplasms at a low frequency of 1.5% (Reindl et al., BJH 2010). In about one-third of these cases breakpoints show a clustering at 14q24.1 (centromeric) and at 14q32.3 (telomeric). Limited genetic data is available on this rare subgroup. Aim: To characterize del(14)(q24q32) using array based comparative genomic hybridization (aCGH) and to analyze cytogenetical and molecular characteristics. Patients and Methods: 34 patients with mature B-cell neoplasms and del(14)(q24q32) by chromosome banding analysis were analyzed. Median age was 72 years (range: 45-94 years). Male:female ratio was 1.4:1. All cases were analyzed by aCGH (Agilent, Waldbronn, Germany) and for mutations in MYD88, NOTCH1, TP53, and SF3B1 as well as for IGHV mutational status by direct Sanger sequencing. IGHV mutated (mut) cases without stereotypic VH3-21 were classified as IGHV favourable (IGHV fav). For statistical analysis of CLL patients data were compared with a cohort of 1,136 untreated CLL patients without del(14q). Results: Patients with del(14)(q24q32) were immunophenotypically classified as follows: 26 (59%) had CLL (n=20) or CLL/PL (n=6), 6 (18%) had splenic marginal zone lymphoma (SMZL), one patient had two B-cell neoplasms (SMZL and CLL/PL: 66% and 10% infiltration, respectively) and one patient had monoclonal B-cell lymphocytosis that progressed to CLL within two years. Analysis with aCGH showed that centromeric breakpoints were detected within a region of 970 kb (69,135,775 - 70,106,558) and telomeric breakpoints localized within a region of 712 kb (105,618,017 - 106,329,974). The median length of the deletions was 36.9 Mb (range: 35.5-37.1). Interestingly, the same breakpoints were present in 4 patients, each: 69,248,772 - 106,329,974 (cluster 1) and 69,271,436 - 106,329,974 (cluster 2). Genes located at the centromeric breakpoint that may be activated by juxtaposition to the IGH locus include FUT8. Its expression contributes to cancer malignancy. Interesting candidate genes located within the deleted region are PPP1R13B (p53 interaction) and NUMB (NOTCH1 interaction). In addition to del(14)(q24q32) 39 cytogenetic aberrations were detected in 23 patients. Two changes were recurrent: trisomy 12 (+12; n = 14) and del(13q) (n = 3). All patients of cluster 1 had +12 (cluster 1 vs. non-cluster 1: 100% vs. 33%, p=0.022). Mutations were detected in TP53 (n = 5, 15%) and NOTCH1 (n = 12, 35%). In SF3B1 a variant and in MYD88 no mutation were found. All mutated cases were CLL or CLL/PL cases only (n.s.). No molecular or cytogenetic differences were detected between CLL or CLL/PL and SMZL. Additionally, the IGHV mutational status was determined in CLL and CLL/PL cases (unmutated in 18 (69%), mutated in 8 (31%)). Further statistical analyses were performed in cases with CLL or CLL/PL with vs. without del(14)(q24q32). NOTCH1 mut (42% vs. 12%, p<0,001) and +12 (46% vs. 14%, p<0.001) were more frequent in patients with del(14q) vs. without. Of note, NOTCH1 mut were not associated with +12 in patients with del(14q) (with vs. without +12: 21% vs. 45%, n.s.), whereas patients without del(14q) showed a significant association (31% vs. 9%, p<0.001). Furthermore, del(13q) was rare in patients with del(14q) (4% vs. 61%, p<0.001) and IGHV fav was detected in less cases (35% vs. 60%, p=0.014). In 978 cases (events = 373; del(14q): n=13, events = 11) data on time to treatment (TTT) was available. TTT was shorter in patients with vs. without del(14q) (4 vs. 95 months, p<0.001). This was also the case when TP53 disrupted (del(17p) and TP53 mut) and +12 cases were excluded from the del(14q) cohort (17 vs. 95, p<0.001). For Cox regression analyses cytogenetic aberrations were hierarchically classified as follows: del(14q), del(17p), del(11q), +12, del(13q) sole. Univariate analysis of cytogenetic subgroups and molecular mutations identified that del(14q), TP53 disrupted, del(11q), +12, NOTCH1 mut, and SF3B1 mut have significant negative impact on TTT and del(13q) sole and IGHV fav are positive prognosticators. Multivariate analysis showed independent impact of del(14q) (HR: 5.2, p<0.001), del(11q) (HR:1.5, p=0.037), SF3B1 mut (HR: 1.5, p=0.008), and IGHV fav (HR: 0.3, p<0.001). Conclusions: del(14)(q24q32) are deletions with a very low variability in breakpoints in mature B-cell neoplasms. In CLL patients they are associated with +12, NOTCH1 mut and independently with a very short TTT. Disclosures Jeromin: MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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Impact of FLT3 Mutation Status and Other Genetic Parameters In Acute Promyelocytic Leukemia (APL) with t(15;17)(q22;q12)/PML-RARA.

Blood ◽

10.1182/blood.v116.21.1685.1685 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1685-1685

Author(s):

Susanne Schnittger ◽

Claudia Haferlach ◽

Tamara Alpermann ◽

Wolfgang Kern ◽

Torsten Haferlach

Keyword(s):

Platelet Count ◽

Acute Promyelocytic Leukemia ◽

Univariate Analysis ◽

Promyelocytic Leukemia ◽

Prognostic Impact ◽

Employment Equity ◽

Transcript Levels ◽

Cytogenetic Aberrations ◽

Mutational Status ◽

Equity Ownership

Abstract Abstract 1685 Acute promyelocytic leukemia (APL) with t(15;17)(q22;q12)/PML-RARA displays the most favourable entity among the different subtypes of acute myeloid leukemia with a five year overall survival (OS) of more than 80%. However, around 10–20% still experience relapse. In order to find pretreatment parameters that may be predictive for outcome in APL, we have comprehensively analyzed 147 PML-RARA-positive patients (pts) at diagnosis. All pts were treated with ATRA in addition to standard chemotherapy. Patients were selected according to availability of cytogenetics, FLT3-ITD mutational status and FLT3-TKD mutational status. The cohort was comprized of 85 males and 62 females. Median white blood cell count (WBC) was 1,8×109/L (range: 0.2–183.4 × 109/L), median platelet count was 30.0 (range: 1.0–228.0 × 109/L) and median hemoglobin level (Hb) was 9.7 (range: 3.6–16.4 g/dL). In 115 pts bone marrow smears were available: 68 were classified as M3 and 47 as M3v (FAB criteria). According to PML-RARA fusion type 89 pts had a bcr1 (long variant), 52 a bcr3 (short variant) and 6 a bcr2 (exon6) breakpoint. Diagnostic %PML-RARA/ABL1 transcript levels were heterogeneously distributed ranging from 0.6 to 96.7 (median: 18.5). In 57/147 pts (38.8%) additional cytogenetic aberrations (ACA) were detected (+8/+8q: n=26, i(17q): n=11, 9q-: n=3, complex: n=2, all others: n=15). A FLT3-ITD was detected in 47 pts (32.0%) and a FLT3-TKD mutation in 19 pts (12.9%). Thus, a total of 65 pts (44.2%) had a mutated FLT3 status (one case revealed both ITD and TKD mutations). FLT3-ITD was highly associated with bcr3 breakpoints (32 FLT3-ITD vs. 20 FLT3wt compared to 14 FLT3-ITD vs 75 FLT3wt in bcr1 and 1 FLT3-ITD vs. 5 FLT3wt in bcr2; p<0.001). Furthermore, FLT3-ITD was associated with higher WBC (mean: 33,153 compared to 5,170 × 109/L in FLT3wt pts, p<0.001) and a lower platelet count (mean: 30,351 vs. 63,324 × 109/L in FLT3wt pts, p=0.001). All parameters mentioned above were analyzed for a possible impact on OS and EFS. Median follow up time of this cohort was 16 months. OS was significantly better in males (2 year OS: 94.2% vs. 78.5% in females; p=0.038). Age as a continuous variable was found significantly related to both OS and EFS (p=0.002, each). Overall, the presence of ACAs had no impact on OS or EFS. In a next step the different ACAs as defined above were evaluated separately. The only group with significantly shorter OS and EFS was the one including the non recurrent “other” ACAs (2 years OS/EFS: 63.6% each, compared to 87.5%/81.4% in the remaining 5 cytogenetic groups, p=0.014/p=0.040). Importantly, these differences exclusively are due to four early deaths in this “other non recurrent” group. No significant effect on OS or EFS was found for WBC, Hb, platelet count, M3/M3v, PML-RARA breakpoint, diagnostic %PML-RARA/ABL1 transcript levels as well as FLT3-ITD, FLT3-TKD or combined FLT3-ITD/TKD status. However, when the FLT3-ITD/wildtype ratio was taken into account a significantly worse EFS was found for those with a FLT3-ITD/wildtype ratio >0.5 (n=21; 2 years EFS: 61.2% vs. 83.5% in the combined group with FLT3wt or FLT3-ITD/wildtype ratio < 0.5, p=0.009). Parameters with significant impact in univariate analysis were included into the multivariate analyses. For OS this was performed for gender, age and “other non recurrent ACA”. All three parameters were proven independent prognostic factors (p=0.026, RR: 0.24; p=0.004, RR: 1.44/decade; and p=0.013, RR: 4.32, respectively). For EFS age, FLT3-ITD/wildtype ratio >0.5, and “other non recurrent ACA” were analyzed (p=0.003, RR: 1.41/decade; p=0.077, RR: 2.73, and p=0.049, RR: 2.46, respectively). In conclusion, specific treatment in APL is extremely efficient what results in minor prognostic impact of otherwise established pretreatment parameters like WBC count, additional cytogenetic aberrations and mutated FLT3 status. Age is the strongest prognostic factor for OS and EFS. Non-recurrent ACAs are associated with an inferior OS. Most importantly, FLT3-ITD mutations with high allelic burden of more than 0.5 are associated with a shorter EFS. This data should be confirmed in controlled prospective studies to draw final conclusions for clinical decision making. Disclosures: Schnittger: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership, Research Funding. Alpermann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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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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Higher Expression of Nuclear Cereblon in Bone Marrow Biopsies of Patients with Multiple Myeloma Treated with Imids in the HOVON-87/Nmsg-18 Trial Is Associated with Longer PFS and OS

Blood ◽

10.1182/blood-2019-128584 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3071-3071

Author(s):

Ruth Wester ◽

M Duin ◽

King Hong Lam ◽

Suzana Couto ◽

Yan Ren ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Cox Regression ◽

Employment Equity ◽

Advisory Committees ◽

Cytoplasmic Staining ◽

Cox Regression Analysis ◽

Cytogenetic Aberrations ◽

Equity Ownership ◽

Response Groups

Introduction Response to treatment in patients with multiple myeloma (MM) is variable. With increasing possibilities of treatment regimens, predictive factors for response are important. Immune modulating agents (IMiDs) require Cereblon (CRBN) for activity. Therefore, the aim of this study was to identify the genes involved in the CRBN pathway which predict the response to therapy with IMiDs. Methods Paraffin embedded bone marrow (BM) biopsies were used from newly diagnosed patients included in HOVON-87/NMSG-18 trial obtained at inclusion. In this trial, elderly patients with MM were randomized between treatment with Melphalan-Prednisone (MP)-Thalidomide (MPT) followed by thalidomide maintenance versus MP-Lenalidomide (MPR) followed by lenalidomide maintenance (Zweegman et al. Blood 2016;127:1109-1116). BM biopsies were stained with a fully automated dual color, bright-field immunohistochemical assay for CRBN, its neosubstrates Ikaros and Aiolos and the downstream targets IRF4 and c-MYC. CD138 was used to identify MM plasma cells in the BM samples. For CRBN, both nuclear and cytoplasmic staining was evaluated. The distribution and intensity of the immunostaining was assessed using the H-score. The H-scores were calculated using the following formula: [1 × (% cells 1+) + 2 × (% cells 2+) + 3 × (% cells 3+)] and range from 0-300 (0-600 for combined cytoplasmic-nuclear CRBN H-score). For the Cox regression analysis H-scores were corrected by dividing these by a factor 100: hazard rates were considered per 100 points increase of the H-score. Protein levels of the CRBN pathway were compared between patients with complete response (CR) or very good partial response (VGPR) vs partial response (PR) and no change/progressive disease (NC/PD). High-risk cytogenetic aberrations (FISH) were defined as having deletion of 17p, and/or translocation t(4;14) and/or t(14;16). Statistical analysis was done using univariate and multivariate Cox regression analysis for progression free survival (PFS) and overall survival (OS), and the Mann-Whitney test for comparing response groups. Kaplan-Meier survival curves were generated to illustrate survival. Results BM samples obtained at diagnosis from 149 patients were evaluated. Seventy-one patients were treated in the thalidomide arm vs 78 patients in the lenalidomide arm. Median age was 73 years [range 60-90]. Revised ISS stages I/II/III were 12%/80%/8% respectively. At the time of analysis, median follow up of the 45 patients still alive was 83 months [range 23 - 114 months]. Best response on protocol treatment was sCR/CR in 22%, VGPR in 30%, PR in 36% and NC/PD in 12%. Protein expression across the response groups showed higher nuclear CRBN in patients who responded better (sCR/CR/VGPR; median H-score: 178 (49-273)) compared to patients with a worse response (PR/NC/PD; median H-score: 157 (67-251)), albeit not statistically significant (Mann-Whitney p-value=0.06). Higher H-score of nuclear staining of CRBN was associated with a longer PFS and OS, with a hazard ratio (HR) of 0.52 for PFS (95% confidence interval (CI)=0.37-0.86, p<0.001) and a HR of 0.56 for OS (95% CI=0.36-0.78; p<0.01). Patients with the top quartile nuclear CRBN levels had a median PFS of 21 months longer compared to patients with the lowest quartile (38 months vs 17 months). In terms of OS, patients with the highest quartile nuclear CRBN expression demonstrated a median survival that was 2 times as high as found in patients with the lowest quartile (75 months vs 35 months; Figure 1). In addition, cytoplasmic staining of CRBN was associated with improved PFS (HR = 0.66 (95% CI=0.47-0.94; p=0.02), but not with OS (HR = 0.73 (CI=0.48-1.11); p=0.14). None of the other markers were associated with survival. In a multivariate analysis (which included study arm (MPT vs MPR), nuclear CRBN, high-risk cytogenetic aberrations and R-ISS), nuclear CRBN remained independently associated with OS as well as R-ISS and study arm. For PFS, only nuclear CRBN remained statistically significant after backward selection. Despite treatment arm being a statistically significant term in the multivariate Cox model for OS, no relation was found for treatment arm and nuclear CRBN, in terms of OS. Conclusions In this study we demonstrate that higher expression of nuclear CRBN in myeloma cells in BM of patients with MM was associated with a superior PFS and OS. Disclosures Couto: Celgene Corporation: Employment, Equity Ownership, Patents & Royalties. Ren:Celgene Corporation: Employment, Equity Ownership. Wang:Celgene Corporation: Employment, Equity Ownership. Thakurta:Celgene: Employment, Equity Ownership. Zweegman:Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding. Broyl:Celgene, amgen, Janssen,Takeda: Honoraria. Sonneveld:Amgen: Honoraria, Research Funding; BMS: Honoraria; Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Karyopharm: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; SkylineDx: Research Funding.

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Cytogenetic aberrations and their prognostic value in a series of 330 splenic marginal zone B-cell lymphomas: a multicenter study of the Splenic B-Cell Lymphoma Group

Blood ◽

10.1182/blood-2010-02-267476 ◽

2010 ◽

Vol 116 (9) ◽

pp. 1479-1488 ◽

Cited By ~ 119

Author(s):

Marta Salido ◽

Cristina Baró ◽

David Oscier ◽

Kostas Stamatopoulos ◽

Judith Dierlamm ◽

...

Keyword(s):

B Cell ◽

Prognostic Value ◽

Marginal Zone ◽

Univariate Analysis ◽

Collaborative Study ◽

Prognostic Significance ◽

B Cell Lymphoma ◽

Splenic Marginal Zone Lymphoma ◽

B Cell Lymphomas ◽

Cytogenetic Aberrations

We conducted a retrospective collaborative study to cytogenetically characterize splenic marginal zone lymphoma (SMZL) and ascertain the prognostic value of chromosomal aberrations. Of 330 cases, 72% displayed an aberrant karyotype, 53% were complex, and 29% had a single aberration. The predominant aberrations were gains of 3/3q and 12q, deletions of 7q and 6q and translocations involving 8q/1q/14q. CD5 expression was detected in 39 of 158 cases (25%). The cytogenetic makeup of the CD5+ group differed significantly from that of the CD5− group. Cases with unmutated IGHV were significantly associated with deletions of 7q and TP53. A strong association was noted between usage of the IGVH1-2 and deletion 7q, 14q alterations, and abnormal karyotype. On univariate analysis, patients with more than or equal to 2 aberrations, 14q alterations, and TP53 deletions had the shortest survival; 7q deletion did not affect survival. On multivariate analysis, cytogenetic aberrations did not retain prognostic significance; the parameters negatively affecting survival were hemoglobin and age. In conclusion, the cytogenetic profile of SMZL is distinct from other B-cell lymphomas. Complexity of the karyotype, 14q aberrations, and TP53 deletions are poor prognostic indicators and may be considered together with other clinicobiologic parameters to ascertain the prognosis of SMZL.

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Landscape Of Genetic Lesions In 944 Patients With Myelodysplastic Syndromes

Blood ◽

10.1182/blood.v122.21.521.521 ◽

2013 ◽

Vol 122 (21) ◽

pp. 521-521 ◽

Cited By ~ 3

Author(s):

Yasunobu Nagata ◽

Vera Grossmann ◽

Yusuke Okuno ◽

Ulrike Bacher ◽

Genta Nagae ◽

...

Keyword(s):

Myelodysplastic Syndromes ◽

Cox Regression ◽

Molecular Model ◽

Clonal Evolution ◽

Univariate Analysis ◽

Gene Mutations ◽

Risk Groups ◽

Employment Equity ◽

Linear Predictor ◽

Equity Ownership

Abstract Background Myelodysplastic syndromes (MDS) are a heterogeneous group of myeloid neoplasms characterized by varying degrees of cytopenias and a predisposition to acute myeloid leukemia (AML). With conspicuous clinical and biological heterogeneity in MDS, an optimized choice of treatment based on accurate diagnosis and risk stratification in individual patients is central to the current therapeutic strategy. Diagnosis and prognostication in patients with myelodysplastic syndromes (MDS) may be improved by high-throughput mutation/copy number profiling. Methods A total of 944 patients with various MDS subtypes were screened for gene mutations and deletions in 104 known/putative genes relevant to MDS using targeted deep-sequencing and/or array-based genomic hybridization. Impact of genetic lesions on overall survival (OS) was investigated by univariate analysis and a conventional Cox regression, in which the Least Absolute Shrinkage and Selection Operator (lasso) was used for selecting variables. The linear predictor from the Cox regression was then used to assign the patients into discrete risk groups. Prognostic models were constructed in a training set (n=611) and confirmed using an independent validation cohort (n=175). Results After excluding sequencing/mapping errors and known or possible polymorphisms, a total of 2,764 single nucleotide variants (SNVs) and insertions/deletions (indels) were called in 96 genes as high-probability somatic changes. A total of 47 genes were considered as statistically significantly mutated (p<0.01). Only 6 genes (TET2, SF3B1, ASXL1, SRSF2, DNMT3A, and RUNX1) were mutated in >10% of the cases. Less common mutations (2−10%) involved U2AF1, ZRSR2, STAG2, TP53, EZH2, CBL, JAK2, BCOR, IDH2, NRAS, MPL, NF1, ATM, IDH1, KRAS, PHF6, BRCC3, ETV6, and LAMB4. Intratumoral heterogeneity was evident in as many as 456 cases (48.3%), even though the small number of gene mutations available for evaluation was thought substantially to underestimate the real frequency. The number of observed intratumoral subpopulations tended to correlate with the number of detected mutations and therefore, advanced WHO subtypes and risk groups with poorer prognosis. Mean variant allele frequencies (VAFs) showed significant variations among major gene targets, suggesting the presence of clonogenic hierarchy among these common mutations during clonal evolution in MDS. The impact of these genetic lesions on clinical outcomes was initially investigated in 875 patients. In univariate analysis, 25 out of 48 genes tested significantly affected overall survival negatively (P<0.05), and only SF3B1mutations were associated with a significantly better clinical outcome. Next, to evaluate the combined effect of these multiple gene mutations/deletions, together with common clinical/cytogenetic variables used for IPSS-R, OS was modeled by a conventional Cox regression. A total of 14 genes, together with age, gender, white blood cell counts, hemoglobin, platelet counts, cytogenetic score in IPSS-R, were finally selected for the Cox regression in a proportional hazard model and based on the linear predictor of the regression model, we constructed a prognostic model (novel molecular model), in which patients were classified into 4 risk groups showing significantly different OS (“low”, “intermediate”, “high”, and “very high risk”) with 3-year survival of 95.2%, 69.3%, 32.8%, and 5.3%, respectively (P<0.001). These results demonstrated that the mutation/deletion status of a set of genes could be used as variables independent of clinical parameters to build a clinically relevant prognostic score. When applied to the validation cohort, the novel molecular model was even shown to outperform the IPSS-R. Conclusions Large-scale genetic and molecular profiling by cytogenetics, NGS and array-CGH not only provided novel insights into the pathogenesis and clonal evolution of MDS, but also helped to develop a powerful prognostic model based on gene mutations and other clinical variables that could be used for risk prediction. Molecular profiling of multiple target genes in MDS is feasible and provides an invaluable tool for improved diagnosis, biologic subclassification and especially prognostication for patients with MDS. Disclosures: Grossmann: MLL Munich Leukemia Laboratory: Employment. Bacher:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Alpermann:MLL Munich Leukemia Laboratory: Employment. Roller:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Kohlmann: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.

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Comprehensive Analyses of Mantle Cell Lymphoma with a 17 Gene Panel Reveal an Accumulation of TP53 Mutations in SOX11 Negative Patients

Blood ◽

10.1182/blood.v124.21.3012.3012 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3012-3012

Author(s):

Manja Meggendorfer ◽

Wolfgang Kern ◽

Claudia Haferlach ◽

Torsten Haferlach ◽

Susanne Schnittger

Keyword(s):

B Cell ◽

Cell Lymphoma ◽

Gene Mutations ◽

Gene Panel ◽

Prognostic Impact ◽

Employment Equity ◽

Mutational Status ◽

Mantle Cell ◽

Sox11 Expression ◽

Equity Ownership

Abstract Introduction: Mantle cell lymphoma (MCL) belongs to the mature B-cell neoplasms and is characterized by t(11;14)(q13;q32)/IGH-CCND1 rearrangement resulting in overexpression of CyclinD1 that is encoded by CCND1. CCND1 is a weak oncogene, requiring additional cooperating oncogenic events. MCL show mainly an aggressive course of disease, although a subset of patients have been identified with an indolent clinical course. This has been associated with a mutated IGHV status and the lack of SOX11 expression. However, some additional gene mutations have been identified in recent years, but the underlying biological heterogeneity is still under debate. Also the prognostic impact of SOX11 remains controversially discussed. Aim: To analyze 1) recently identified molecular mutations in CCND1, UBR5, WHSC1 for their specificity for MCL, 2) in more detail SOX11 negative MCL to get more insights in this group of MCL by sequencing a 17 gene panel. Patients and Methods: In total 184 patients with mature B-cell neoplasms were investigated. All cases were diagnosed according to WHO classification by cytomorphology, immunophenotyping and cytogenetics. 81 cases were diagnosed as MCL, 78 as chronic lymphocytic leukemia/prolymphocytic (CLL/PL), and 25 as CLL. The cohort comprised 67 females and 117 males. CyclinD1 and SOX11 expression levels were quantified by real time PCR. The 17 gene panel included ATM, BIRC3, BRAF, CCND1, FBWX7, IGHV, KLHL6, KRAS, MYD88, NOTCH1, NRAS, POT1, SF3B1, TP53, UBR5, WHSC1, and XPO1. Next generation sequencing was performed on MiSeq instruments (Illumina, San Diego, CA), except for CCND1, UBR5, WHSC1, and IGHV mutational status, which were analyzed by Sanger sequencing. The latter 4 genes and gene expression levels were analyzed in the total cohort, while the 17 gene panel was applied to 26 MCL patients only (13 SOX11 negative and 13 SOX11 positive cases matched for CCND1 mutations and IGHV mutation status). Results: 23/184 (13%) patients had CCND1 mutations, while only 3/184 patients (2%) carried a UBR5 mutation, and 4/184 patients (2%) a WHSC1 mutation. Of note, CCND1 and UBR5 mutations occurred exclusively in MCL patients, WHSC1 mutations were found in MCL and CLL/PL. Therefore CCND1 mutation was, as expected, specific for MCL in comparison to the other 2 mature B-cell neoplasms (p<0.001). Of 81 MCL patients 68 (84%) showed SOX11 overexpression, 23 (28%) had CCND1 mutations and 3 (4%) each had UBR5 and WHSC1 mutations, respectively. The IGHV mutational status was evaluable in 73/81 cases, revealing 32/73 (44%) patients with a mutated IGHV status. A negative correlation of CCND1 mutations and SOX11 overexpression was found: 8/13 (62%) SOX11 negative patients were CCND1 mutated as compared to 15/68 (22%) SOX11 positive patients (p=0.007). Furthermore, CCND1 mutations were more frequent in patients with a mutated IGHV status than in those with unmutated status (15/32 (47%) vs. 8/41 (20%), p=0.021). Accordingly, SOX11 overexpression occurred more often in patients with an unmutated than with a mutated IGHV status (88% vs. 75%; n.s.). Regarding clinical data, more males were SOX11 positive (51/54, 94% vs. 17/27, 63%; p=0.001), and correspondingly more females were CCND1 mutated (12/27, 63% vs. 11/54, 20%; p=0.036). To get more insight in the SOX11 negative patients (n=13) we addressed all these cases and a SOX11 positive control group, matched for IGHV mutational status and CCND1 mutations (n=13), by comprehensive mutational analyses. Overall, beside CCND1 the most frequently mutated gene was TP53 (8/26, 31%), followed by ATM (6/26, 23%), BIRC3 (2/21, 10%), and KRAS (2/26, 8%). No mutations were detected in any of the other genes analyzed. Addressing differences in gene mutations between SOX11 negative and SOX11 positive cases revealed that TP53 mutations were found more frequently in SOX11 negative cases (6/13, 46% vs. 2/13, 15%; n.s.), while ATM mutations were more frequent in SOX11 positive cases (5/13, 39% vs. 1/13, 8%; n.s.). Conclusions: 1) CCND1 mutations are specifically found in MCL, correlate with SOX11 negativity and IGHV mutated status, and are more frequent in females. 2) TP53 is frequently mutated in SOX11 negative patients and its prognostic impact has to be further evaluated. 3) Thus, the differences in clinical course between SOX11 positive and negative MCL patients might correlate with a different spectrum of additional molecular alterations. Disclosures 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. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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FLT3 ITD Signaling Profiles in AML Samples Harboring Mutations.

Blood ◽

10.1182/blood.v114.22.1588.1588 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1588-1588 ◽

Cited By ~ 1

Author(s):

M. D. Minden ◽

Steven M. Kornblau ◽

David B. Rosen ◽

Aileen Cleary Cohen ◽

Urte Gayko ◽

...

Keyword(s):

Tyrosine Kinase ◽

Signaling Pathways ◽

Intracellular Signaling ◽

Univariate Analysis ◽

Receptor Expression ◽

Molecular Testing ◽

Employment Equity ◽

Mutational Status ◽

Equity Ownership ◽

The Impact

Abstract Abstract 1588 Poster Board I-614 Background Mutations in the receptor tyrosine kinase (RTK) Fms-like tyrosine kinase 3 (FLT3) gene are among the most common somatic mutations in AML with FLT3 internal tandem duplications (ITDs) occurring in 20-35% of adult and 5-15% of pediatric AML. While the presence of FLT3 ITD mutation does not appear to influence outcome to induction chemotherapy, this mutation has been shown to confer a poor prognosis with significantly shorter disease free and relapse free survival. For patients with intermediate risk cytogenetically normal AML, molecular testing for FLT3 ITD has recently been incorporated into the National Comprehensive Cancer Network (NCCN) guidelines for clinical practice. However, while molecular testing can identify a subset of patients at high risk for relapse, there remains clinical heterogeneity likely due to differences in activation of signal transduction networks. Objectives This study tested the ability to use single cell network profiling (SCNP), in which cells are perturbed with extracellular modulators and their response ascertained by multiparametric flow cytometry, to identify a more clinically predictive functional readout of activation state, intracellular signaling capabilities and pathway dysregulation in the context of FLT3 mutational status. Methods Modulated SCNP was performed sequentially on two independent sets of patient samples (n=32 peripheral blood and n=85 bone marrow samples respectively). 304 and 201 “node-metric” i.e. modulated read outs of dynamic elements on individual proteins in signaling pathways were measured in the two sets respectively. These were derived from pathways known to be relevant to Flt3 WT and Flt3-ITD signaling (e.g. Ras-Raf-Erk-S6, PI3K-Akt-S6, STATs), as well as in-vitro chemotherapeutic induction of apoptosis (cleaved PARP, cleaved caspases), phosphatases, drug transporters (e.g. MDR-1, ABCG2) and expression of growth factor RTKs (e.g. Flt3R, c-Kit). Results In the first study, univariate analysis revealed 76 nodes out of 304 tested that distinguished FLT3 ITD from FLT3 WT patient samples (i.e. AUC of ROC >0.7; p<0.05). Analysis of false discovery rate showed this frequency to be significantly greater than the number of nodes that can be expected by chance (p=0.0009). Although several nodes were found to be correlated, many were independent of each other and represented multiple signaling pathways. Importantly, multivariate analysis showed that combinations of independently predictive nodes improved stratification over the single nodes (AUC of ROC up to 0.99) with respect to distinguishing WT and ITD FLT3 samples. Independent analysis of a second set of samples, revealed several nodes in common between the 2 studies which distinguish FLT3 ITD from WT, including etoposide/c-PARP (apoptosis), IL-27/p-STAT3, 5 (JAK/STAT pathways) and Flt3L/p-S6 (Ras/Erk/mTOR/S6 or PI3K/mTor/S6 pathways). In both sample sets, Flt3 receptor expression did not differ significantly between FLT3 ITD and FLT3 WT samples. Conclusions Pathway analysis by SCNP revealed significant differences in signaling in FLT3 ITD relative to WT AML samples across multiple pathways. We propose that a functional signature of FLT3 signaling is distinct from the existing molecular typing and may improve the ability to predict prognostic outcomes in individual AML patients. The impact of other important prognostic, molecular markers within the FLT3 context (e.g. NPM1) are currently under investigation. Disclosures Kornblau: Nodality, Inc.: Consultancy. Rosen:Nodality, Inc.: Employment, Equity Ownership. Cleary Cohen:Nodality Inc.: Employment, Equity Ownership. Gayko:Nodality, Inc.: Employment, Equity Ownership. Putta:Nodality, Inc.: Employment, Equity Ownership. Woronicz:Nodality, Inc.: Employment, Equity Ownership. Fantl:Nodality, Inc.: Employment, Equity Ownership. Cesano:Nodality, Inc.: Employment, Equity Ownership.

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Duohexabody-CD37, a Novel Bispecific Antibody with a Hexamerization-Enhancing Mutation Targeting CD37, Demonstrates Superior Complement-Dependent Cytotoxicity in Preclinical B-Cell Malignancy Models

Blood ◽

10.1182/blood-2018-99-114723 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4170-4170

Author(s):

Simone C. Oostindie ◽

Hilma J. Van Der Horst ◽

Marije B. Overdijk ◽

Kristin Strumane ◽

Sandra Verploegen ◽

...

Keyword(s):

B Cell ◽

Research Funding ◽

Bispecific Antibody ◽

Lymphocytic Leukemia ◽

Single Point Mutation ◽

B Cell Malignancies ◽

Employment Equity ◽

Healthy Human ◽

Complement Dependent Cytotoxicity ◽

Equity Ownership

Abstract CD37 is a tetraspanin plasma membrane protein abundantly expressed on B-cells and represents a promising therapeutic target for the treatment of B-cell malignancies. Although complement-dependent cytotoxicity (CDC) has proven to be a powerful Fc-mediated effector function for killing hematological cancer cells, CD37 antibody-based therapeutics currently in clinical development are poor inducers of CDC. Here we present DuoHexaBody-CD37, a novel humanized IgG1 bispecific antibody targeting two different CD37 epitopes, with an E430G hexamerization-enhancing mutation, for the potential treatment of B-cell malignancies. The natural process of antibody hexamer formation through intermolecular Fc-Fc interactions between IgG molecules after cell surface antigen binding can be improved by introducing a single point mutation such as E430G in the IgG Fc domain, thereby facilitating more efficient C1q binding and complement activation (Diebolder et al., Science 2014; de Jong et al., PLoS Biol 2016). The hexamerization-enhancing mutation E430G was introduced into two humanized CD37 monoclonal antibodies (mAbs) that bind non-overlapping CD37 epitopes. Different antibody formats and combinations, including the single antibodies, combinations of the mAbs and bispecific mAbs were tested for their capacity to induce CDC and antibody-dependent cellular cytotoxicity (ADCC). The bispecific hexamerization-enhanced antibody variant DuoHexaBody-CD37, showed superior CDC activity compared to the single hexamerization-enhanced mAbs and the combination thereof, both in vitro over a range of different B-cell lines, and ex vivo in tumor cell samples obtained from patients with chronic lymphocytic leukemia (CLL). In a CDC assay using tumor cells obtained from a relapsed/refractory CLL patient who received prior treatment with rituximab, ibrutinib and idelalisib, DuoHexaBody-CD37 induced almost complete lysis (84% lysis at a concentration 100 µg/mL), thereby outperforming the single HexaBody molecules (15% and 23% lysis) and the combination (57%) (Figure 1). In addition to its potent CDC activity, DuoHexaBody-CD37 was also capable of inducing potent ADCC of Daudi cells (EC50 = 12.3 ± 9.5 ng/mL), as assessed using peripheral blood mononuclear cells from 8 healthy human donors in a standard chromium release assay. In assays using whole blood from 6 healthy human donors, DuoHexaBody-CD37 showed efficient B-cell binding and potent and specific depletion of the B-cell population (98% ± 1.3% depletion at 10 µg/mL, EC50 = 0.85 ± 0.284 µg/mL). Furthermore, DuoHexaBody-CD37 induced significant inhibition of tumor growth in vivo in Daudi-luc Burkitt's lymphoma and JVM-3 CLL mouse xenograft models, at doses as low as 0.1 and 1 mg/kg (p<0.05), respectively. In summary, we present a novel therapeutic antibody that, for the first time, combines proprietary DuoBody® and HexaBody® platforms. DuoHexaBody-CD37 induced highly potent CDC and efficient ADCC in preclinical models, suggesting that DuoHexaBody-CD37 may serve as a potential therapeutic mAb for the treatment of human B-cell malignancies. Disclosures Oostindie: Genmab: Employment, Equity Ownership. Van Der Horst:Genmab: Research Funding. Overdijk:Genmab: Employment, Equity Ownership. Strumane:Genmab: Employment, Equity Ownership. Verploegen:Genmab: Employment, Equity Ownership. Lindorfer:Genmab: Research Funding. Cook:Genmab: Research Funding. Chamuleau:Gilead: Research Funding; BMS: Research Funding; celgene: Research Funding; Genmab: Research Funding. Mutis:Gilead: Research Funding; Celgene: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genmab: Research Funding; Novartis: Research Funding; OnkImmune: Research Funding. Schuurman:Genmab: Employment, Other: Warrants. Sasser:Genmab: Employment, Equity Ownership. Taylor:Genmab: Research Funding. Parren:Genmab: Equity Ownership; Lava Therapeutics: Employment. Beurskens:Genmab: Employment, Equity Ownership. Breij:Genmab: Employment, Equity Ownership.

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EZH2 Mutations and Impact on Clinical Outcome Analyzed in 1604 Patients with Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2018-99-114421 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1528-1528

Author(s):

Sebastian Stasik ◽

Jan Moritz Middeke ◽

Michael Kramer ◽

Christoph Rollig ◽

Alwin Krämer ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Board Of Directors ◽

Myeloid Leukemia ◽

Research Funding ◽

Employment Equity ◽

Advisory Committees ◽

Mutational Status ◽

Equity Ownership ◽

Acute Myeloid

Abstract Purpose: The enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase and key epigenetic regulator involved in transcriptional repression and embryonic development. Loss of EZH2 activity by inactivating mutations is associated with poor prognosis in myeloid malignancies such as MDS. More recently, EZH2 inactivation was shown to induce chemoresistance in acute myeloid leukemia (AML) (Göllner et al., 2017). Data on the frequency and prognostic role of EZH2-mutations in AML are rare and mostly confined to smaller cohorts. To investigate the prevalence and prognostic impact of this alteration in more detail, we analyzed a large cohort of AML patients (n = 1604) for EZH2 mutations. Patients and Methods: All patients analyzed had newly diagnosed AML, were registered in clinical protocols of the Study Alliance Leukemia (SAL) (AML96, AML2003 or AML60+, SORAML) and had available material at diagnosis. Screening for EZH2 mutations and associated alterations was done using Next-Generation Sequencing (NGS) (TruSight Myeloid Sequencing Panel, Illumina) on an Illumina MiSeq-system using bone marrow or peripheral blood. Detection was conducted with a defined cut-off of 5% variant allele frequency (VAF). All samples below the predefined threshold were classified as EZH2 wild type (wt). Patient clinical characteristics and co-mutations were analyzed according to the mutational status. Furthermore, multivariate analysis was used to identify the impact of EZH2 mutations on outcome. Results: EZH2-mutations were found in 63 of 1604 (4%) patients, with a median VAF of 44% (range 6-97%; median coverage 3077x). Mutations were detected within several exons (2-6; 8-12; 14-20) with highest frequencies in exons 17 and 18 (29%). The majority of detected mutations (71% missense and 29% nonsense/frameshift) were single nucleotide variants (SNVs) (87%), followed by small indel mutations. Descriptive statistics of clinical parameters and associated co-mutations revealed significant differences between EZH2-mut and -wt patients. At diagnosis, patients with EZH2 mutations were significantly older (median age 59 yrs) than EZH2-wt patients (median 56 yrs; p=0.044). In addition, significantly fewer EZH2-mut patients (71%) were diagnosed with de novo AML compared to EZH2-wt patients (84%; p=0.036). Accordingly, EZH2-mut patients had a higher rate of secondary acute myeloid leukemia (sAML) (21%), evolving from prior MDS or after prior chemotherapy (tAML) (8%; p=0.036). Also, bone marrow (and blood) blast counts differed between the two groups (EZH2-mut patients had significantly lower BM and PB blast counts; p=0.013). In contrast, no differences were observed for WBC counts, karyotype, ECOG performance status and ELN-2017 risk category compared to EZH2-wt patients. Based on cytogenetics according to the 2017 ELN criteria, 35% of EZH2-mut patients were categorized with favorable risk, 28% had intermediate and 37% adverse risk. No association was seen with -7/7q-. In the group of EZH2-mut AML patients, significantly higher rates of co-mutations were detected in RUNX1 (25%), ASXL1 (22%) and NRAS (25%) compared to EZH2-wt patients (with 10%; 8% and 15%, respectively). Vice versa, concomitant mutations in NPM1 were (non-significantly) more common in EZH2-wt patients (33%) vs EZH2-mut patients (21%). For other frequently mutated genes in AML there was no major difference between EZH2-mut and -wt patients, e.g. FLT3ITD (13%), FLT3TKD (10%) and CEBPA (24%), as well as genes encoding epigenetic modifiers, namely, DNMT3A (21%), IDH1/2 (11/14%), and TET2 (21%). The correlation of EZH2 mutational status with clinical outcomes showed no effect of EZH2 mutations on the rate of complete remission (CR), relapse free survival (RFS) and overall survival (OS) (with a median OS of 18.4 and 17.1 months for EZH2-mut and -wt patients, respectively) in the univariate analyses. Likewise, the multivariate analysis with clinical variable such as age, cytogenetics and WBC using Cox proportional hazard regression, revealed that EZH2 mutations were not an independent risk factor for OS or RFS. Conclusion EZH mutations are recurrent alterations in patients with AML. The association with certain clinical factors and typical mutations such as RUNX1 and ASXL1 points to the fact that these mutations are associated with secondary AML. Our data do not indicate that EZH2 mutations represent an independent prognostic factor. Disclosures Middeke: Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees. Rollig:Bayer: Research Funding; Janssen: Research Funding. Scholl:Jazz Pharma: Membership on an entity's Board of Directors or advisory committees; Abbivie: Other: Travel support; Alexion: Other: Travel support; MDS: Other: Travel support; Novartis: Other: Travel support; Deutsche Krebshilfe: Research Funding; Carreras Foundation: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees. Hochhaus:Pfizer: Research Funding; Incyte: Research Funding; Novartis: Research Funding; Bristol-Myers Squibb: Research Funding; Takeda: Research Funding. Brümmendorf:Janssen: Consultancy; Takeda: Consultancy; Novartis: Consultancy, Research Funding; Merck: Consultancy; Pfizer: Consultancy, Research Funding. Burchert:AOP Orphan: Honoraria, Research Funding; Bayer: Research Funding; Pfizer: Honoraria; Bristol Myers Squibb: Honoraria, Research Funding; Novartis: Research Funding. Krause:Novartis: Research Funding. Hänel:Amgen: Honoraria; Roche: Honoraria; Takeda: Honoraria; Novartis: Honoraria. Platzbecker:Celgene: Research Funding. Mayer:Eisai: Research Funding; Novartis: Research Funding; Roche: Research Funding; Johnson & Johnson: Research Funding; Affimed: Research Funding. Serve:Bayer: Research Funding. Ehninger:Cellex Gesellschaft fuer Zellgewinnung mbH: Employment, Equity Ownership; Bayer: Research Funding; GEMoaB Monoclonals GmbH: Employment, Equity Ownership. Thiede:AgenDix: Other: Ownership; Novartis: Honoraria, Research Funding.

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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.

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