Abstract: Monitoring of measurable residual disease (MRD) provides prognostic information in patients with Nucleophosmin1-mutated (NPM1mut) acute myeloid leukemia (AML) and represents a powerful tool to evaluate treatment effects within clinical trials. We determined NPM1mut transcript levels (TLs) by quantitative reverse-transcription polymerase chain reaction and evaluated the prognostic impact of NPM1mut MRD and the effect of gemtuzumab ozogamicin (GO) on NPM1mut TLs and the cumulative incidence of relapse (CIR) in patients with NPM1mut AML enrolled in the randomized phase 3 AMLSG 09-09 trial. A total of 3733 bone marrow (BM) samples and 3793 peripheral blood (PB) samples from 469 patients were analyzed. NPM1mut TL log10 reduction ≥ 3 and achievement of MRD negativity in BM and PB were significantly associated with a lower CIR rate, after 2 treatment cycles and at end of treatment (EOT). In multivariate analyses, MRD positivity was consistently revealed to be a poor prognostic factor in BM and PB. With regard to treatment effect, the median NPM1mut TLs were significantly lower in the GO-Arm across all treatment cycles, resulting in a significantly greater proportion of patients achieving MRD negativity at EOT (56% vs 41%; P = .01). The better reduction in NPM1mut TLs after 2 treatment cycles in MRD positive patients by the addition of GO led to a significantly lower CIR rate (4-year CIR, 29.3% vs 45.7%, P = .009). In conclusion, the addition of GO to intensive chemotherapy in NPM1mut AML resulted in a significantly better reduction in NPM1mut TLs across all treatment cycles, leading to a significantly lower relapse rate.

Abstract: Background: Recently, the oral multitargeted small molecule FLT3 inhibitor midostaurin (M) was approved for treatment of FLT3-mutated AML in combination with standard chemotherapy. In the international RATIFY (NCT00651261) trial, addition of M led to superior overall and event-free survival compared to placebo, thus defining a new standard of care in this AML subset (Stone RM et al. NEJM 2017). Although not powered for subgroup analyses, M showed consistent effects across all FLT3 mutation strata [tyrosine kinase domain (TKD); internal tandem duplication (ITD) with low (0.05-0.7; ITDlow) or high ( 〉 0.7; ITDhigh) allelic ratio] suggesting significant off-target activity beyond FLT3 inhibition. Aim: We aimed to comprehensively profile the mutational landscape of FLT3 mutated (FLT3mut) AML in a large, well characterized cohort of patients (pts) treated within the RATIFY trial using a high-throughput targeted sequencing (HTS) approach. Methods: HTS was performed on the entire coding region of 262 genes involved in hematologic malignancies including 20 genes that encode kinases targeted by M (M kinome, MK). Pretreatment peripheral blood (PB; 14%) or bone marrow (BM; 86%) specimens were available from 475 (66%) of 717 FLT3mut AML RATIFY pts. Libraries were prepared using SureSelectXT custom solutions (Agilent). Paired-end sequencing was carried out on a HiSeq platform (Illumina). FLT3 mutation (mut) status was available for all pts [TKD: 24%; ITD: 76% (ITDlow: 45%; ITDhigh:31%)], and cytogenetic data for 454 pts (96%). Results: An average sequencing depth of 978x was obtained for all pts. In sum, 1815 mut (missense: 49%; indels: 40%; nonsense: 7%; other: 3%) were identified with a mean of 3.8 mut per pt (FLT3 strata; TKD: 4; ITDlow: 4; ITDhigh: 3.6).Overall, recurrent mut ( 〉 5% of all pts) were found in NPM1 (61%), DNMT3A (39%), WT1 (21%), TET2 (12%), RUNX1 (11%), NRAS (11%), PTPN11 (9%), ASXL1 (8%), IDH1 (8%), IDH2 (7%; R140 only), and SMC1A (6%). In contrast, TP53 (1%) and biallelic CEPBA (1%) mut were rare events. This was also true for aberrations of the MK (7% in total) with KIT (2%), MAP3K11 (1%), and NTRK3 (1%) being most frequently mutated. When stratified according to FLT3mut type, mut in NRAS (24% vs 7%, p 〈 .0001), SMC1A (10% vs 4%, p=.02), and KIT (4% vs 1%, p=.02) occurred significantly more often in TKD than ITD groups, respectively, whereas WT1 (13% vs 24%, p=.018) was more frequently co-mutated in the ITD group. In general, pts in the TKD group had significantly more mut in genes encoding for cohesin (TKD: 29% vs ITD: 16%, p=.004) and signaling (TKD: 40% vs ITD: 24%, p=.001) proteins compared to ITD pts, who had significantly more mut in transcription genes (TKD: 37% vs ITD: 48%, p=.03). Based on the mut and cytogenetic data, we next sought to assign all FLT3mut pts to the 11 recently defined molecular AML classes (Papaemmanuil E et al. NEJM 2016). The majority fell into two classes, namely the NPM1 (N; 62%) and the chromatin-spliceosome (CS; 15%) classes, underscoring the significance of FLT3mut as the driver in these particular genomic classes. Other class-defining lesions were rare or absent in this cohort [inv(16): 2%; t(8;21): 2%; t(11q23;x): 2%; t(6;9): 1%, TP53-aneuploidy: 1%; CEBPAbiallelic: 1%; IDH2R172: 0%]. In 14% of all pts categorization was not possible (no or 〉 1 class-defining lesion), emphasizing the need for further refinement of this classification. When focusing on these two groups, N and CS had comparable FLT3mut patterns (TKD: 24% vs 21%; ITDlow: 44% vs 45%; ITDhigh: 32% vs 33%), whereas N more frequently correlated with a normal karyotype (N: 91% vs CS: 63%). With respect to clinical characteristics, no differences between N and CS in terms of age, white blood cells, platelets, PB and BM blasts, as well as history of MDS were observed. Conclusion: In this comprehensive sequencing approach, we could further delineate the molecular pattern of FLT3mut AML. Here, FLT3-ITD and -TKD groups exhibited remarkable differences in cooperating pathways, highlighting distinct biologic features in the leukemogenesis of FLT3mut AML. Overall, mut of MK genes were rare events, not fully explaining the complexity of M off-target effects. Understanding the underlying disease mechanism will potentially provide useful information on prognosis and prediction of response to M. Further analyses including correlation with clinical outcome are ongoing. Support: U10CA180821, U10CA180861, U10CA180882, U24CA196171 Disclosures Bullinger: Janssen: Speakers Bureau; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Pfizer: Speakers Bureau; Sanofi: Research Funding, Speakers Bureau; Amgen: Honoraria, Speakers Bureau; Bayer Oncology: Research Funding. Gathmann:Novartis: Employment. Larson:Ariad/Takeda: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; BristolMyers Squibb: Consultancy, Research Funding. Medeiros:Genentech: Employment; Celgene: Consultancy, Research Funding. Tallman:ADC Therapeutics: Research Funding; AROG: Research Funding; BioSight: Other: Advisory board; Orsenix: Other: Advisory board; AbbVie: Research Funding; Daiichi-Sankyo: Other: Advisory board; Cellerant: Research Funding. Tiecke:Novartis: Employment. Pallaud:Novartis: Employment. Ehninger:Cellex Gesellschaft fuer Zellgewinnung mbH: Employment, Equity Ownership; GEMoaB Monoclonals GmbH: Employment, Equity Ownership; Bayer: Research Funding. Ganser:Novartis: Membership on an entity's Board of Directors or advisory committees. Stone:Otsuka: Consultancy; Jazz: Consultancy; Cornerstone: Consultancy; Fujifilm: Consultancy; Arog: Consultancy, Research Funding; Pfizer: Consultancy; Sumitomo: Consultancy; Novartis: Consultancy, Research Funding; Ono: Consultancy; Orsenix: Consultancy; Merck: Consultancy; Argenx: Other: Data and Safety Monitoring Board; AbbVie: Consultancy; Agios: Consultancy, Research Funding; Amgen: Consultancy; Astellas: Consultancy; Celgene: Consultancy, Other: Data and Safety Monitoring Board, Steering Committee. Thiede:AgenDix: Other: Ownership; Novartis: Honoraria, Research Funding. Döhner:AROG Pharmaceuticals: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; AROG Pharmaceuticals: Research Funding; Pfizer: Research Funding; Bristol Myers Squibb: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Celator: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Bristol Myers Squibb: Research Funding; Sunesis: Consultancy, Honoraria, Research Funding; Astellas: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Astex Pharmaceuticals: Consultancy, Honoraria; Astex Pharmaceuticals: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Pfizer: Research Funding; Seattle Genetics: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Celator: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Agios: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Jazz: Consultancy, Honoraria.

Abstract: Patients with acute myeloid leukemia (AML) and a FLT3 internal tandem duplication (ITD) have poor outcomes to current treatment. A phase 2 hypothesis-generating trial was conducted to determine whether the addition of the multitargeted kinase inhibitor midostaurin to intensive chemotherapy followed by allogeneic hematopoietic cell transplantation (alloHCT) and single-agent maintenance therapy of 12 months is feasible and favorably influences event-free survival (EFS) compared with historical controls. Patients 18 to 70 years of age with newly diagnosed AML and centrally confirmed FLT3-ITD were eligible: 284 patients were treated, including 198 younger (18-60 years) and 86 older (61-70 years) patients. Complete remission (CR) rate, including CR with incomplete hematological recovery (CRi) after induction therapy, was 76.4% (younger, 75.8%; older, 77.9%). The majority of patients in CR/CRi proceeded to alloHCT (72.4%). Maintenance therapy was started in 97 patients (34%): 75 after alloHCT and 22 after consolidation with high-dose cytarabine (HiDAC). Median time receiving maintenance therapy was 9 months after alloHCT and 10.5 months after HiDAC; premature termination was mainly a result of nonrelapse causes (gastrointestinal toxicity and infections). EFS and overall survival at 2 years were 39% (95% confidence interval [CI] , 33%-47%) and 34% (95% CI, 24%-47%) and 53% (95% CI, 46%-61%) and 46% (95% CI, 35%-59%) in younger and older patients, respectively. EFS was evaluated in comparison with 415 historical controls treated within 5 prospective trials. Propensity score-weighted analysis revealed a significant improvement of EFS by midostaurin (hazard ratio [HR], 0.58; 95% CI, 0.48-0.70; P & lt; .001) overall and in older patients (HR, 0.42; 95% CI, 0.29-0.61). The study was registered at www.clinicaltrials.gov as #NCT01477606.

Abstract: Vitamin D (VitD) deficiency is common in patients with hematologic malignancies undergoing allogeneic transplantation (alloSCT), but its prognostic relevance is unclear. Patients and Methods The impact of pretransplant VitD status on overall survival, relapse mortality, and nonrelapse mortality was investigated retrospectively in a cohort of 492 patients undergoing alloSCT at our center from 2002 to 2013. VitD deficiency was defined as a serum level of 25-hydroxyvitamin D3 〈 20 ng/mL (equivalent to 〈 50 nM) before alloSCT and was assessed using accredited laboratory methods and a standard chemiluminescent immunoassay. Results were validated in an independent cohort of 398 patients diagnosed with myeloid malignancies. Results A total of 396 (80%) and 348 (87%) patients had VitD deficiency before alloSCT in the training and validation cohort, respectively. In the training cohort, VitD deficiency was significantly associated with inferior overall survival (hazard ratio [HR], 1.78; P = .007) in multivariable analysis. This was due to a higher risk of relapse (HR, 1.96; P = .006) rather than nonrelapse mortality. A significant association of pretransplant VitD deficiency with higher relapse rates was observed only in patients diagnosed with myeloid (HR, 2.55; P = .014) but not with lymphatic diseases (HR, 1.60; P = .147). A similar impact of pretransplant VitD deficiency on relapse risk in myeloid diseases was also observed in an independent patient cohort (HR, 2.60; P = .017). Validation of the effect of VitD deficiency on relapse in patients with myeloid malignancies was successful. Conclusion Pretransplant VitD deficiency was associated with a higher risk of relapse in patients allografted for myeloid malignancies. Prospective studies on VitD status and correction of VitD deficiency in the setting of alloSCT are highly warranted.

Abstract: The immune system can recognize and attack cancer cells, especially those with a high load of mutation-induced neoantigens. Such neoantigens are abundant in DNA mismatch repair (MMR)-deficient, microsatellite-unstable (MSI) cancers. MMR deficiency leads to insertion/deletion (indel) mutations at coding microsatellites (cMS) and to neoantigen-inducing translational frameshifts. Here, we develop a tool to quantify frameshift mutations in MSI colorectal and endometrial cancer. Our results show that frameshift mutation frequency is negatively correlated to the predicted immunogenicity of the resulting peptides, suggesting counterselection of cell clones with highly immunogenic frameshift peptides. This correlation is absent in tumors with Beta-2-microglobulin mutations, and HLA-A*02:01 status is related to cMS mutation patterns. Importantly, certain outlier mutations are common in MSI cancers despite being related to frameshift peptides with functionally confirmed immunogenicity, suggesting a possible driver role during MSI tumor evolution. Neoantigens resulting from shared mutations represent promising vaccine candidates for prevention of MSI cancers.

Abstract: Background and Aims: DNA mismatch repair-deficient cancers show microsatellite instability (MSI) and accumulate high numbers of somatic insertion/deletion mutations at repetitive sequence stretches. The high mutational load of MSI cancers leads to the generation of multiple frameshift peptide (FSP) neoantigens, which are highly immunogenic and recognized by the immune system as foreign antigens. MSI cancers respond particularly well to treatment with immune checkpoint inhibitors such as anti-PD-1 antibodies. We systematically analyzed immune evasion phenomena in MSI cancers and aimed to identify factors that are related to and potentially determine PD-1 expression on tumor-infiltrating lymphocytes, focusing on alterations of HLA class I and II antigen presentation pathways in cancer cells. Methods: Microsatellites located in Beta2-microglobulin (B2M) and the HLA class II-regulatory genes RFX5 and CIITA were analyzed for mutations in MSI colorectal cancer specimens (n = 53). HLA class I and II antigen expression was examined by immunohistochemistry. In addition, tumor-infiltrating lymphocytes (CD3-positive T cells, PD-1-positive T cells) were quantified using a semi-automated system. Results: We related B2M mutation and HLA class II antigen expression status of MSI colorectal cancer specimens (n = 56) to CD3- and PD-1 positive T cell infiltration in the tumor. PD-1-positive T cell infiltration was significantly higher in B2M-mutant (mt) compared to B2M-wild type (wt) tumors (median: 22.2 cells per 0.25 mm2 in B2M-mt vs. 2.0 cells per 0.25 mm2 in B2M-wt, Wilcoxon's rank sum test p = 0.002). Increasing PD-1-positive T cell infiltration was significantly related to an increased likelihood of B2M mutation and loss of HLA class I antigen expression (OR = 1.81). In contrast, HLA class II antigen expression status was not related to the proportion of PD-1-positive lymphocytes, but significantly associated with enhanced overall T cell infiltration. Conclusions: These results suggest that immune evasion mediated by B2M mutation-induced loss of HLA class I antigen expression predominantly occurs in an environment of activated PD-1-positive T cell infiltration, supporting the validity of the immunoediting concept in MSI colorectal cancers. Moreover, B2M mutation status may predict therapy resistance against anti-PD-1 therapy. Note: This abstract was not presented at the conference. Citation Format: Matthias Kloor, Jonas Janikovits, Julia Krzykalla, Axel Benner, Niels Grabe, Fabian Echterdiek, Meike Mueller, Sandrina Koerner, Aysel Ahadova, Magnus von Knebel Doeberitz. Immune evasion and PD-1-positive T cell infiltration in DNA mismatch repair-deficient colorectal cancer [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A085.

Abstract: Analysis of the efficiency and toxicity of cyclophosphamide‐based stem cell mobilization in patients with relapsed multiple myeloma ( RMM ). Methods Peripheral blood stem cells ( PBSC s) were mobilized with high dose cyclophosphamide (2 g/m 2 daily on days 1 and 2) and G‐ CSF plus pre‐emptive/rescue plerixafor in RMM patients (first to third relapse) treated within the Re LA psE trial of the German‐Speaking Myeloma Multicenter Group ( GMMG ). Results Mobilization was initiated with high‐dose cyclophosphamide (HD‐CY) and G‐ CSF in 30 patients. Fifteen patients received additional pre‐emptive/rescue administration of plerixafor. Stem cell collection was successful (≥2×10 6 CD 34+ cells per kg bw) in 77% (23/30 patients). Patients with prior high‐dose melphalan collected a significantly lower median total number of PBSC s than patients without prior high‐dose melphalan (3.3×10 6 vs 17×10 6 CD 34+ cells/kg bw). Toxicity of HD‐CY was frequent with 12 serious adverse events ( SAE ) in 37% of patients (11/30 patients). Infections accounted for the majority of SAE reports. In two patients, SAE s were lethal (septic shock). Conclusions These data proof feasibility of PBSC collection at relapse but emphasize the importance of collection and storage of additional PBSC transplants during first‐line treatment when mobilization is more efficient and less toxic.

Abstract: To evaluate the prognostic impact of gene expression levels ( EL s) of two tumor suppressor genes, sprouty 4 ( SPRY 4, located on 5q) and lysine methyltransferase 2C ( KMT 2C, located on 7q) in correlation with clinical characteristics and genetic abnormalities assessed at initial diagnosis in acute myeloid leukemia ( AML ). Method Gene expression levels were measured on cDNA by RT ‐ qPCR from diagnostic bone marrow samples of 275 intensively treated adult AML patients (median age, 48 years). Results KMT 2C EL s were significantly lower in abn7q/‐7 ( P  = .001), whereas SPRY 4 EL s were not associated with abn5q/‐5. Higher KMT 2C and SPRY 4 EL s were significantly associated with lower genetic risk groups as defined by the European LeukemiaNet classification. Additionally, KMT 2C EL s were lower in cytogenetically normal patients with DNMT 3A ( P  = .01) or FLT 3 ‐ ITD mutations ( P  = .05). KMT 2C EL s were not associated with prognosis, whereas higher SPRY 4 EL s showed a favorable impact on event‐free ( EFS , P  = .01), relapse‐free ( RFS , P  = .01) and in‐trend on overall survival ( P  = .06) for cytogenetically abnormal patients, which was confirmed in multivariable analysis for EFS ( HR , 0.84; 95%‐ CI , 0.73‐0.97; P  = .02) and RFS ( HR , 0.85; 95%‐ CI , 0.73‐0.98; P  = .02). Conclusion Our data indicate that KMT 2C EL s are associated with specific genetic features and that SPRY 4 EL s may add prognostic information.