Complete Bone Marrow Remission Research Articles

Comprehensive analysis of m6A methylome alterations after azacytidine plus venetoclax treatment for acute myeloid leukemia by nanopore sequencing

N6 adenosine methylation (m6A), one of the most prevalent internal modifications on mammalian RNAs, regulates RNA transcription, stabilization, and splicing. Growing evidence has focused on the functional role of m6A regulators on acute myeloid leukemia (AML). However, the global m6A levels after azacytidine (AZA) plus venetoclax (VEN) treatment in AML patients remain unclear. In our present study, bone marrow (BM) sample pairs (including pre-treatment [AML] and post-treatment [complete remission (CR)] samples) were harvested from three AML patients who had achieved CR after AZA plus VEN treatment for Nanopore direct RNA sequencing. Notably, the amount of m6A sites and the m6A levels in CR BMs was significantly lower than those in the AML BMs. Such a significant reduction in the m6A levels was also detected in AZA-treated HL-60 cells. Thirteen genes with decreased m6A and expression levels were identified, among which three genes (HPRT1, SNRPC, and ANP32B) were closely related to the prognosis of AML. Finally, we speculated the mechanism via which m6A modifications affected the mRNA stability of these three genes. In conclusion, we illustrated for the first time the global landscape of m6A levels in AZA plus VEN treated AML (CR) patients and revealed that AZA had a significant demethylation effect at the RNA level in AML patients. In addition, we identified new biomarkers for AZA plus VEN-treated AML via Nanopore sequencing technology in RNA epigenetics.

CD7 CAR-T Therapy Followed By a Second Transplant for T-Cell Acute Lymphoblastic Leukemia or Lymphoblastic Lymphoma Patients Who Relapsed after a Prior Transplant

Background The therapeutic options for patients with T-cell acute lymphoblastic leukemia or lymphoblastic lymphoma (T-ALL/LBL) who relapse after hematopoietic stem cell transplantation (HSCT) are currently restricted. CD7-targeted chimeric antigen receptor (CAR) T-cell therapy provides a promising avenue towards remission, yet the long-term prognosis for these patients still needs to be improved. The advantages of progressing to a second transplant post-CAR-T treatment remain uncertain due to limited data. In this study, we analyzed the clinical characteristics and transplantation outcomes of 16 T-ALL/LBL patients who, having relapsed after their initial transplantation, achieved complete remission (CR) in bone marrow (BM) and CR/partial remission (PR) in extramedullary lesions via CD7 CAR-T therapy and then opted for a second allogeneic HSCT (allo-HSCT) at our center. Our aim is to explore strategies to enhance the survival rate of these patients. Methods From January 2021 to January 2023, we analyzed T-ALL/LBL patients who underwent second transplantation at Hebei Yanda Lu Daopei Hospital. The patients included in this study met the following criteria: they had relapsed following their first HSCT and had subsequently undergone CD7 CAR-T therapy prior to the second transplantation. The follow-up cut-off date was till June 1, 2023. The median follow-up duration after the second transplantation was 178 days, ranging from 9 to 500 days. Results A total of 16 patients including 13 with T-ALL and 3 with T-LBL were enrolled. There were 14 males and 2 females. The donor of first transplantation was autologous(n=3), sibling identical(n=2), unrelated donor(n=2), or haplo-identical (n=9). After the first transplantation, 8 patients relapsed in bone marrow (BM) and 8 relapsed both in BM and extramedullary sites. Of these, 13 patients received autologous CD7 CAR-T, one patient received donor-derived CD7 CAR-T, and one patient was treated with universal CAR-T cells. After the CD7 CAR-T therapy, all 16 patients (100%) achieved minimal residual disease (MRD)-negative CR in BM, and 6 out of 8 patients (75%) achieved CR in extramedullary lesions. Only two patients exhibited PR in the extramedullary lesions. The median age at the time of the second transplantation was 28 years (range 14-42 years). The median time from diagnosis to the second transplantation was 683 days (range 385-2672 days). The median interval between the first and second transplantation was 438 days (range 229-2540 days), and the median interval from CD7 CAR-T infusion to the second transplantation was 72 days (range 31-92 days). For the second transplantation, 13 cases had haplo-identical donors and 3 cases had unrelated donors. The conditioning regimens utilized were either total body irradiation (TBI)-based (n=9), busulfan-based (n=6), or melphalan-based (n=1). Following the 2 nd transplant, the 1-year overall survival(OS) and leukemia-free survival(LFS) were 58.5% (95% CI, 28.9-85.0%) and 51.4% (95% CI, 22.4-79.9%), respectively(Figure 1). The 1-year relapse incidence (RI) and non-relapse mortality (NRM) were 15.9% (95% CI, 4.4-57.8%) and 31.5% (95% CI, 13.2-74.9%), respectively. The cumulative incidence of CMV and EBV viremia at day 100 were 75.0% (95% CI, 56.5-99.5%) and 19.8% (95% CI, 7.2-54.6%), respectively. The cumulative incidence of all grades acute graft-versus-host disease (aGVHD) and 3-4 grade aGVHD at day 100 were 56.3% (95%CI, 36.5-86.7%) and 25% (95%CI, 10.7-58.4%), respectively. One-year chronic GVHD(cGVHD) was observed in 25.1% of the cases (95%CI, 9.3-67.4%), while moderate to severe cGVHD was not found in any patient. Following the 2 nd transplant, a total of 6 patients died, including 3 from infection, 2 from relapse and 1 from intracranial hemorrhage. Conclusions Our initial findings suggest that even in refractory/relapsed T-ALL/LBL patients who have experienced relapse following previous HSCT, MRD-negative complete remission can potentially be achieved utilizing CD7 CAR-T cell therapy. The subsequent implementation of a second allo-HSCT appears feasible. Combining CAR-T therapy with a follow-up second HSCT presents a strategy for these heavily pre-treated patients, offering promising prospects for survival. However, further evaluation of this treatment approach is required, necessitating long-term monitoring and a larger patient cohort.

Decitabine in Combination with Aclacinomycin, Cytarabine and Granulocyte Colony-Simulating Factor (CAG) Had a High Response Rate in Children with Advanced Myelodysplastic Syndrome and Acute Myeloid Leukemia with Myelodysplasia-Related Changes

Advanced myelodysplastic syndrome (MDS) is a rare disease in children with poor prognosis. Allogeneic transplantation is the only curative option. However, there has been no established treatment prior to transplant. We conducted a retrospective study exploring the response rates and safety of decitabine combined with aclacinomycin, cytarabine and granulocyte colony-stimulating factor (CAG) for induction treatment of advanced MDS and acute myeloid leukemia with myelodysplasia-related changes (MDR-AML) in children. Patients from 1 to 14 years administrated in Pediatric Blood Disease Center of CAMS Blood Disease Hospital between January 1the 2019 and December 31th 2022 were screening. All diagnosed with MDS-EB or MDR-AML according to WHO 2016 criteria and treated with decitabine combined with CAG were identified. The efficacy of one-cycle treatment of MDS-ED was evaluated based on The International Working Group Response Criteria 2006. The response for MDR-AML was evaluated according to the criteria for AML published by ELN. The response of MDR-AML was defined as complete remission (CR) or CR with incomplete count recovery (CRi). The response of MDS-EB was defined as CR or bone marrow CR with hematological improvement (HI). A total of 13 patients, including MDS-EB (n=7) and MDR-AML (n=6), were enrolled for analysis. 5-day decitabine was administrated, combined with modified CAG regimen for 7 days. Eventually 12 patients of those were available for evaluation. For patients with MDS-EB, four achieved CR and three reached marrow CR with HI. Four patients with MDR-AML, three achieved CR and two reached CRi. The overall response rate was 100%(12/12). The most frequent adverse events were myelosuppression and suppression-induced infections, but no infection-related mortality was recorded. There had been no cardiotoxic event happened, either. Only one patient experienced fatal bleeding in early stage after treatment. The median follow-up time was 16 months (range 1-48). At last follow-up, 10 patients who had underwent allo-HCT were alive without disease. The remaining 2 patients died of disease progression. The 2-year estimated overall survival was 62.9%. Decitabine combined with modified CAG had a high response rate for children with advanced MDS and MDR-AMl, and it can bridge patients to curative allogeneic transplantation.

Organ Involvement in BPDCN Is Associated with Sun Exposure History, TET2 and RAS Mutations, and Survival

Introduction Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare, aggressive hematologic neoplasm that can involve skin, bone marrow (BM), central nervous system (CNS), and non-CNS extramedullary disease (EMD) sites. Recent findings show that ultraviolet light (UV) exposure may contribute to the development of BPDCN (Griffin, Nature 2023). Conflicting data exist as to whether anatomic involvement is clinically relevant, and genetics have not been incorporated into disease site prognostic models. In addition, the role of non-overt (<5%, microscopic) BM involvement remains unclear as trials have only regarded ≥5% as evidence of BPDCN. Methods We assessed associations between organ involvement, UV exposure, molecular characteristics, and outcome in patients with biopsy-confirmed BPDCN seen at Dana-Farber Cancer Institute between 2006-2022. UV exposure was classified as high vs. low by an independent reviewer blinded to outcomes, based on medical, social, and occupational history. We classified 3 groups at diagnosis: systemic without skin (either BM with ≥5% BPDCN cells or EMD and no skin involvement), skin only (with no overt BM [<5% BPDCN cells] and no EMD), or skin and systemic; groups were classified irrespective of CNS involvement at diagnosis. Next generation sequencing was performed on blood/BM at diagnosis. Comparisons were performed by Fisher's exact test. Survival was calculated from treatment initiation and comparisons made by the log-rank test. Uni- and multivariable Cox regression models were fit for association with overall survival (OS), with SCT as a time varying covariate. Results Overall, we included 66 consecutive patients with a median age at diagnosis of 68 years (interquartile range [IQR] 61-76). The majority (n= 57, 86%) of patients were male. Disease distribution at diagnosis involved skin (n=52, 79%), BM (n=50, 76% [39 (59%) with ≥5% BPDCN cells]) and EMD (n=30, 45%), ( Table, Figure). The median time from first symptom to diagnosis was 61 days (IQR 35-134) and from diagnosis to treatment was 30 days (IQR 20-46). The first therapeutic regimen was tagraxofusp (n=30, 45%), AML (n=12, 18%) or ALL (n=12, 18%) chemotherapy, or other treatment (n=12, 18%). Complete remission (CR) after first treatment was documented in 35 (57%) with 20/35 consolidated in CR1 with SCT (57%); however, after a median of 6.1 months from response assessment (IQR 2.4-8.1 months), 22/35 who achieved CR relapsed, with skin (n=13, 54%), BM (n=12, 50%), EMD (n=5, 21%), or CNS (n=3, 13%) as relapse sites. The median number of therapies was 2 (0-6), and 35 (53%) had SCT at any time. The pattern of post-SCT relapse included disease involvement in skin (8/14, 57%), BM (5/14, 36%), EMD (3/14, 21%) and CNS (3/14, 21%). Patients with skin only at diagnosis (n=19) vs. any systemic involvement (n=47) were more frequently ≥75 years (9/19 [47%] vs. 9/47 [9%], p=0.032), had lower rates of complex karyotype (0 vs. 12 [32%], p=0.022) and activated signaling mutations ( N/KRAS, FLT3, CBL, CKIT, S H2B3; 1 [8%] vs. 14 [40%], p=0.04), mainly driven by lower NRAS (0 in skin only vs. 10 [29%] with any systemic, p=0.044, Figure). In contrast, those with any skin involvement had higher rates of UV exposure (27 [59%] vs. 3 [23%], p=0.03) and TET2 mutations (28 [72%] vs. 3 [33% ], p=0.051) compared to those with no skin involvement, consistent with the recently aforementioned link between UV exposure and selection for TET2 mutated plasmacytoid dendritic cells. The median OS of the entire group was 18.2 months (95% CI 13-24) and did not statistically differ between presentation groups (skin only: 23.5 [95% CI 13-NR]; systemic + skin 20.4 [95% CI 11-26]; systemic only 17.5 [95% CI 9-22], p=0.14). In a multivariable analysis, age <60 (HR 0.35 [95% CI 0.13-0.94], p=0.038) was associated with improved OS, whereas BM involvement ≥5% (but not microscopic involvement) was independently associated with worse OS (HR 2.65 [95% CI 1.1-6.36]). Conclusions Specific organ involvement in BPDCN is associated with UV exposure, age, cytogenetic and molecular characteristics. Skin is the most common relapse site post CR or SCT. Survival is better in younger patients (age <60 years), and worse in those with ≥5% BM involvement after adjustment for age and SCT. These data establish additional factors that, in combination, have prognostic value in BPDCN, and provide independent evidence that sun exposure influences BPDCN genetics and clinical presentation.

P342: BASELINE GENE EXPRESSION ANALYSIS OF RELAPSED ACUTE B-LYMPHOBLASTIC LEUKEMIA PATIENTS TREATED WITH INOTUZUMAB OZOGAMICIN

Background: Despite introduction of novel agents, relapsed/refractory B-cell acute lymphoblastic leukemia (R/R B-ALL) carries dismal outcome. Inotuzumab ozogamicin (InO) is a humanized anti-CD22 monoclonal antibody conjugated to calicheamicin approved for R/R B-ALL able to obtain high CR rates but of short duration. Mechanisms underlaying InO resistance are largely unknown. Aims: To characterize the baseline differentially expressed genes in a series of R/R B-ALL patients in relation with patient response to InO in order to individuate potential pathways involved in resistance. Methods: Gene expression profile of 18 R/R B-ALL patient samples was analyzed with RNA-seq, before InO exposure. All patients received at least 1 InO course. Patient population was divided in poor/non-responders (NR) and responders (R), based on InO response. R were defined as patients with duration of response (DoR) ≥3 months after bone marrow (BM) complete remission (CR) achievement. NR were defined either refractory or with a DoR <3 months after CR achievement. The list of significant (p<0.05) differentially expressed genes (DEGs) in NR, as compared to R whose absolute fold change (FC) was ≥2, was analyzed. P values were corrected with the Benjamini-Hochberg algorithm (false discovery rate; FDR). Gene expression results were analyzed with QIAGEN Ingenuity Pathway Analysis (IPA). Results: Eighteen R/R BM samples of CD22-positive B-ALL were analyzed with RNA-seq. Eight patients were defined as NR and 10 patients as R. Patient disease characteristics in NR and R groups were homogeneous for age, sex, number of Philadelphia (Ph)-positive ALL patients, duration of 1st remission, number of previous therapy lines including hematopoietic stem cell transplantation (HSCT) and Blinatumomab. At the time of InO therapy all patients had morphological BM relapse and all were CD22-positive. Median CD22 expression percentage on leukemic blasts was 100% (range 100-100) in NR patients and 100% (range 70-100) in R patients (p=0.177). Two patients in R group had a CD22 expression blast percentage of 70% and 76%. Median CD22-fluorescent intensity (CD22-FI) in NR as compared to R was 75.38 (IQR 59.58, 89.51) and 136.51 (IQR 114.38, 151.57) respectively, with significantly lower values in NR group (p = 0.04). Overall, 370 genes were differentially expressed (p<0.05) in NR, as compared to R patients, of which 32 were significantly differentially expressed (corrected p < 0.05, FC ≥2). Thirty-one were down- and 1 was up-regulated in NR vs R. DEGs were involved in basophil differentiation, carbon dioxide transport, erythrocyte and myeloid cell development, heme metabolic and porphyrin-containing compound biosynthetic processes, cation and cellular ion homeostasis. Both IPA upstream regulator and regulator effect analysis identified the serine/threonine homeodomain-interacting protein kinase-2 (HIPK2) as predicted downregulated. The inhibition of HIPK2 was predicted to be the causal upstream condition of the under-expression of six DEGs from the set (FECH, ANK1, SCL4A1, EPOR, GATA1, HBZ) with activation Z score of -2.449 and p value of overlap = 1.02E-09. No difference in terms of HIPK2 expression was appreciated in the two groups, suggesting downregulation at post-transcriptional level. Image:Summary/Conclusion: A unique pattern of gene expression signature based on HIPK2 downregulation was identified in poor responders to InO, providing potentially important insights in mechanisms of resistance. HIPK2 downregulation needs to be further validated. CT and CP contributed equally

P779: OPTIMIZING TREATMENT OUTCOMES FOR CHILDREN WITH HIGHER-RISK MYELODYSPLASTIC SYNDROME UNDERGOING ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION

Background: Myelodysplastic syndrome (MDS) are rare clonal hematopoietic disorders in children. Risk stratification system for adults is unfit for children. Refractory anemia with excess blasts(RAEB), acute myeloid leukemia with myelodysplasia-related changes(AML-MRC), monosomy 7 and complex karyotype always indicate poor prognosis. Hypomethylating agent (HMA) improved survivals of high-risk MDS in adults, and decitabine as part of conditioning regimen for allogenetic hematopoietic stem cell transplantation(allo-HSCT) was associated with favorable outcomes. However, these findings have not been tested in pediatric cohorts broadly. Aims: To evalute the efficacy and safety of hypomethylating agents as a part of pre-transplant treatments and contidioning regimens in children with higher-risk MDS. Methods: Patients from 1 to 14 years of age who were diagnosed with higher-risk MDS and underwent allo-HSCT consecutively between February 2019 and August 2020 in Pediatric Blood Diseases Center were enrolled. All identified patients were retrospectively reviewed with a collection of clinical data, laboratory test results, and outcome. Results: We reviewed 18 pediatric patients with higher-risk MDS, including 7 with RAEB, 8 with AML-MRC, and 3 with refractory cytopenia of childhood with monosomy 7. Regardless of initial diagnosis, monosomy 7 is the most common cytogenetic aberration (8/18, 44.4%). Next-generation sequencing (NGS) was performed on 15 specimens and the most frequent detection of mutation gene was SETBP1, followed by ETV6. Before transplantation, ten patients received monotherapies or combined chemotherapies with HMA, two patients received AML-like chemotherapies, and nine of those achieved bone marrow complete remission. The stem cell sources came from matched sibling donor (n=1), haploidentical donor (n=11), and unrelated cord blood (n=6), respectively. Low-dose decitabine-containing myeloablative conditioning regimens were administered in all cases. About 50% of patients (n=9) experienced Grade II- IV acute graft versus host diseases post-transplant. The cumulative incidence of transplantation-related mortality (TRM) was 11.1%. During a median follow-up of 10.5 (range, 2–36) months after transplantation, 15 of 18 patients were alive. At time of last follow-up, all survivors had resolution of hematologic disorder without relapse. Summary/Conclusion: In summary, administration of hypomethylating agents to allogeneic SCT resulted in excellent outcomes in children with higher-risk MDS.

First-in-Human Clinical Study of a Novel CD7-Targeted Chimeric Antigen Receptor (CAR)-T Cell Therapy for Refractory/Relapsed Mixed Phenotype Acute Leukemia (MPAL)

IntroductionMixed phenotype acute leukemia (MPAL) is a rare subtype of acute leukemia with features of both acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). The blast cells of MPAL express multilineage immunophenotypic markers and may have a shared B/T/myeloid phenotype. CD7 is expressed by the leukemic blasts and malignant progenitor cells of approximately 30% of AML patients. MPAL is usually associated with a relatively poor prognosis which is even worse compared to either AML or ALL, and even among patients that undergo allogeneic hematopoietic stem cell transplantation (allo-HSCT), underscoring the need for new therapies for these patients. CAR-T cell therapy has shown to be efficacious with a tolerable safety profile in a subset of hematological malignancies. Here, we explored the safety and efficacy of CAR-T therapy in treating refractory/relapsed (r/r) MPAL patients with CD7-positive in a phase I dose-escalation clinical study (https://clinicaltrials.gov NCT04938115).MethodsPeripheral blood (PB) mononuclear cells were obtained from patient themselves or patient transplant donors for those who relapsed post-transplant by leukapheresis. T-cells were purified using CD3+ magnetic beads or CD4+ and CD8+ magnetic beads. The second-generation CD7 CAR with a 4-1BB costimulatory domain was manufactured per the manufacturer's protocol. This phase I dose escalation study was initiated to explore the safety and efficacy of CD7 CAR-T in treating patients with MPAL. Prior to the CAR-T cells infusion, patients received systemic bridging chemotherapy due to rapid disease progression and then all patients received intravenous fludarabine (30mg/m 2/d) and cyclophosphamide (300mg/m 2/d) (FC) lymphodepleting chemotherapy for 3 consecutive days (Day -5 to Day -3). The median time from leukapheresis to CAR-T cell infusion was 14 days.ResultsFrom Nov. 2020 to June 2021, 5 adult acute leukemia patients with CD7-positive (4 MPAL, 1 FLT3-mutated AML) were enrolled and infused with CD7 CAR T-cells, one with a low-dose (1.5×10 5/kg), 2 with a medium dose (5.0×10 5/kg), and 3 with a high dose infusion (1.0×10 6/kg). The median transduction efficiency of products was 70.4% (range: 32.0%-96.9%). As of July 30, 2021 cut-off date, the median observation time was 37 days (range: 35-232 days). Characteristics of enrolled patients are shown in Table 1. The median age was 33 years (range: 21-37 years), and the median bone marrow (BM) blasts percentage by morphology was 28.0% (range: 2.5-51.5%) at enrollment. Four patients had prior allo-HSCT history including one who had extramedullary disease (EMD) who relapsed following a 2 nd transplant and had also received CD19 CAR-T cell therapy prior to enrollment in the current trial. The median interval period from prior transplant to CD7 CAR-T cell infusion was 16 months (range: 8-23 months). Following infusion, the median peak of circulating CD7 CAR-T cell was 1.17×10 5copies/μg (0.651~7.04×10 5copies/μg) genomic DNA which occurred around Day 21 (Day14 - Day21) and 59.2% (4.28%~74.58%) occurring on Day18 (Day11-Day21) by q-PCR and flow cytometry respectively (Fig.1).Four weeks post CD7 CAR-T cell infusion, 4/5 patients achieved complete remission (CR) or CR with incomplete blood recovery (CRi) in BM and all achieved minimal residual disease (MRD)-negative CR. One patient who relapsed post 2 nd transplant, also with EMD, achieved CR in BM, however, remission was not achieved in EMD through PET-CT evaluation on Day 35.One MRD-negative CR patient subsequently underwent consolidative allo-HSCT 120 days post CD7 CAR T-cell infusion and has remained in MRD-negative CR. Other 3 patients without consolidative allo-HSCT remained in MRD-negative CR till the last follow-up. All 5 patients developed cytokine release syndrome (CRS)-three of 5 patients had grade I CRS, one patient had grade II and one patient had grade III CRS. None of the patients had neurotoxicity. Among the 4 patients who had prior allo-HSCT, 1 developed grade I graft-versus-host disease post CAR-T therapy.ConclusionThis study demonstrated that CD7-targeted CAR-T therapy offers an opportunity to achieve CR for CD7-positive MPAL patients even for those who relapsed post-transplant. Safety was manageable, however, more data on additional patients and longer observation times are needed to further evaluate the efficacy of CD7 CAR-T products. [Display omitted] DisclosuresBa: SenlangBio: Current Employment.

Impact of Genemutations and Dynamics Methylation of CDKN2B and DLC-1 Promoters on Treatment Response with Azacitidine

Introduction: Azacitidine (AZA) is a DNA hypomethylating agent used to treat high-risk myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) in patients who are not eligible for intensive strategies. In view of the variability of drug response, last years it has been studied the genes mutations as possible response biomarkers. In addition, although there seems to be no association between promoter genes methylation and response, dynamics methylation before and after AZA treatment could be a predictable tool of clinical response. CDKN2B gene is a regulator of cell cycle and its aberrant hypermethylation is recurrent in myeloid diseases. DLC-1 gene plays a critical role in cell migration and proliferation and although its abnormal methylation is common in different kinds of cancers, its role in hematological malignancies is slightly studied.Aim: To assess the predictive value of gene mutations and dynamics methylation of CDKN2B and DLC-1 promoters on AZA response in a series of patients with myeloid neoplasms.Methods: We studied 61 patients with MDS and 16 patients with AML who received AZA treatment. According to the International Working Group (IWG) 2006 criteria for MDS, responders were defined as patients achieving complete remission (CR), bone marrow CR (mCR), partial remission (PR) or stable disease with hematologic improvement (SDHI). Ten genes with prior implication in the pathogenesis of myeloid diseases were analyzed: DNMT3A , IDH1 , IDH2 , ASXL1 , SETBP1 , TP53 , SF3B1 , SRSF2 , U2AF1 and ETNK1. For methylation assay, EZ DNA Methylation-Gold kit (Zymo Research) was employed for the bisulfite treatment of DNA. The methylation levels of CDKN2B and DLC-1 were studied with unmethylated and methylated primers, positive and negative controls and analysis of the PCR products in a QIAxcel System (Qiagen). In all patients CDKN2B and DLC-1 methylation were analyzed pre-AZA treatment and in 57 patients with post-AZA DNA sample the methylation assay was also possible at that point. P values <0.05 were considered as statistically significant .Results: Of the 77 patients analyzed, 30 patients (39%) showed AZA response (median cycles=6): CR=13, mCR=2, PR=8, and SDHI=7. Stable disease and treatment failure were observed in 19 (25%) and 28 patients (36%), respectively. Pre-treatment, genetic mutations were presented in 63 patients (82%) and methylationof CDKN2B and DLC-1 promoters were detected in 32 (42%) and 37 (48%) patients respectively (Figure 1). Patients with mutations in DNA methylators (DNMT3A , IDH1/2 ) showed a significant worse AZA response than the remaining patients (9% vs. 44%, P =0.026) and furthermore DNMT3A patients presented a lower overall survival (OS) compared with DNMT3A patients (8 vs . 19 months, P =0.042). All SETBP1 patients also did not show AZA response and moreover transformed to AML in a few months. On the other hand, TP53 mutations did not influence the drug response although they were associated with complex karyotypes (92% vs. 8%, P <0.001) and with a lower OS (10 vs. 21 months, P =0.043). Conversely, splicing genes mutations (SF3B1 , SRSF2 and U2AF1 ) were related to favourable karyotypes (78% vs. 54%, P =0.007), higher OS (28 vs. 13 months, P =0.038) and lower DLC-1 methylation (33% vs . 64%, P =0.010). Interestingly, patients with DLC-1 methylation showed higher levels (>5%) of bone marrow blasts (74% vs . 42%, P =0.005), higher frequency of complex karyotypes (29% vs. 9%, P =0.002) and lower OS (15 vs . 30 months, P =0.044). In addition,dynamics methylation of DLC-1 was significantly associated with the AZA response: 59% patients who reduced DLC-1 methylation levels post-treatment, responded favourably to the drug and 95% patients who maintained or increased them, did not respond (P =0.002; Figure 2). Finally, CDKN2B methylated patients had a significant co-occurrence with favourable karyotypes (80% vs. 45%, P =0.014), presented a higher OS (28 vs. 13 months, P =0.018) but its dynamics methylation was not associated with the AZA response (P =0.07).Conclusion: In our seriesof patients treated with AZA, DNMT3A and IDH1/2 mutations have a negative impact on drug response. DLC-1 dynamics methylation is associated with the AZA response and clinical outcome.The combination of point mutations and monitoring methylation could contribute to give light to the heterogeneity of AZA response in myeloid neoplasms. [Display omitted] DisclosuresSolano:Neovii: Honoraria.

Gynecological Myeloid Sarcoma: Literature Review and a Case Report

Myeloid sarcoma, also known as chloroma or granulocytic sarcoma, is a rare disease characterized by the proliferation of immature myeloid cells in extramedullary lesions. Chloroma is more commonly observed in patients with acute myeloid leukemias, other myeloproliferative neoplasms, or myelodysplastic syndrome. However, it can also manifest itself as solitary tumor. Sarcoma can develop in different organs and tissues, but most frequently it appears in lymph nodes, soft tissues, and bones. Myeloid sarcoma with primary gynecological lesion is very rarely mentioned. In literature cases of cervical lesions are described. The present article summarizes the literature data concerning different aspects of myeloid sarcoma diagnosis and treatment. The issue under discussion is the role of chemotherapy, radiotherapy, surgery, and bone marrow transplantation in the treatment of this malignant tumor. It appears that whatever the primary tumor localization, the best treatment options are chemotherapy and allogeneic bone marrow transplantation (allo-BMT). A promising trend is the use of novel targeted drugs improving outcomes of treatment. The article provides a case report of a female patient with cervical myeloid sarcoma and concomitant bone marrow involvement, as well as the description of clinical course, diagnosis, and treatment. The patient received chemotherapy with subsequent allo-BMT. The pre-transplant therapy enabled allo-BMT with the deepest response possible. The patient achieved PET-and MRD-negative complete remission of cervical myeloid sarcoma and bone marrow.

An Immune Senescence and Exhaustion-Related RNA Profile Predicts Clinical Outcomes in Acute Myeloid Leukemia

Background Chemotherapy refractoriness and disease relapse continue to be significant obstacles to therapeutic success in AML. We have recently identified bone marrow (BM) transcriptomic profiles that stratify patients with AML into an immune-infiltrated and an immune-depleted subtype and that refine the accuracy of survival prediction in response to conventional chemotherapy beyond that afforded by cytogenetic and molecular prognosticators (Vadakekolathu J, et al. Sci. Transl. Med. 2020; 12: eaaz0463). Importantly, AML CD8+ T cells exhibit features of immune exhaustion and senescence (IES), including the upregulation of natural killer (NK) cell-associated transcripts, which persist only in chemotherapy non-responders (Knaus HA, et al. JCI Insight 2018; 3: e120974). The aim of the current study was to determine whether IES correlate with immune infiltration and with clinical outcomes in AML. Methods We used prior knowledge and gene set enrichment analysis (GSEA) to derive a 68-gene IES signature score (computed as the cohort-wide average of gene expression) from publicly available RNA-sequencing datasets (TCGA and Beat-AML Master Trial; 162 and 281 AML patients, respectively). The wet-lab AML cohorts used in this study included a total of 382 BM samples from children and adults with AML treated with curative intent. BMs were collected at time of diagnosis, complete remission (CR) and relapse (PMCC, SAL and CHOP series). BM RNAs were profiled on the nCounter platform using the PanCancer Immune Profiling Panel (NanoString Technologies, Seattle, WA). Results In the AML discovery cohort, the IES score significantly correlated with BM lymphoid infiltration (R=0.958) and with CD8+ T cell (R=0.88), cytotoxic T cell (R=0.90) and tumor inflammation signature scores (R=0.82, P&amp;lt;0.0001 for all; Fig. 1A). The novel IES gene signature showed minimal overlap with previously discovered interferon (IFN)-related gene sets with prognostic relevance in AML (Fig. 1B) and was higher in TP53-mutated (P&amp;lt;0.0001) and RUNX1-mutated (P=0.0071) compared with WT AML. Overall survival (OS) was significantly shorter in patients with higher than median IES scores (Fig. 1C). IES signature scores were also associated with worse clinical outcomes in Beat-AML cases (median OS of 10.6 and 21.1 months, respectively, in patients with higher and lower than median IES scores; Fig. 1D) and were higher in primary induction failures (PIF) compared with chemotherapy responders (Fig. 1E). The top IES genes associated with survival prediction both in TCGA and Beat-AML, although not overlapping with published IFN-related gene sets, were still significantly enriched in type I/II IFN signaling pathways and in molecular functions indicative of heightened ion-gated channel activity, as shown by protein-protein interaction network analyses. The above findings were validated in independent wet-lab cohorts comprising adults (PMCC series; n=290; SAL series; n=46) and children (CHOP series; n=46) with AML. In the ELN adverse risk subgroup (PMCC cohort), both relapse-free survival (RFS) and OS were significantly shorter in patients with higher than median compared with lower than median IES scores (median RFS time of 6.93 versus not reached [log-rank P=0.0053], and median OS of 10.5 months versus 18 months [log-rank P=0.0011], respectively). In contrast, the IES signature score failed to stratify survival in patients with ELN favorable and intermediate risk. Finally, a pairwise comparison of matched diagnostic, CR and relapse BM samples (SAL series; n=22 patients and CHOP series; n=46 patients) showed significantly higher IES signature scores at time of CR, congruent with chemotherapy-induced acceleration of IES, and at time of post-chemotherapy relapse compared with disease onset (Fig. 1F). Notably, CD8A, TBX21, a Th1 transcription factor, and markers of NK cells and cytotoxic T lymphocytes, including KLRK1, KLRD1, KLRC2, GNLY, and granzymes, were among the top ranked immune genes associated with AML relapse (Fig. 1G). Conclusions Patients with immune-infiltrated AML exhibit features of IES, which may correlate with adverse-risk molecular features, chemotherapy refractoriness and shorter survival. Molecular circuits reflective of IES might also underpin AML relapse after conventional induction chemotherapy. IES T cells could be functionally rejuvenated by novel immunotherapies being investigated in AML. Figure 1 Disclosures Church: NanoString Technologies, Inc.: Current Employment. Davidson-Moncada:Macrogenics: Current Employment. Tasian:Incyte Corporation: Research Funding; Aleta Biotherapeutics: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Research Funding. Gojo:Amphivena: Research Funding; Merck: Research Funding; Genentech: Research Funding; Amgen: Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees. Luznik:WindMil Therapeutics: Patents &amp; Royalties: Patent holder; AbbVie: Consultancy; Merck: Research Funding, Speakers Bureau; Genentech: Research Funding. Rutella:Kura Oncology: Research Funding; MacroGenics, Inc.: Research Funding; NanoString Technologies, Inc.: Research Funding.

Clinical Profile of IMGN632, a Novel CD123-Targeting Antibody-Drug Conjugate (ADC), in Patients with Relapsed/Refractory (R/R) Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN)

Clinical Profile of IMGN632, a Novel CD123-Targeting Antibody-Drug Conjugate (ADC), in Patients with Relapsed/Refractory (R/R) Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN)

Anti-CD38 Monoclonal Antibody Daratuzumab Improve Responses to Chemotherapy-Refractory Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN): A Case Report

Background Blastic plasmacytoid dendritic cell neoplasm (BPDCN) accounts for 0.44% of hematologic malignancies and is a rare aggressive myeloid neoplasm which shows high rates of cutaneous lesions, bone marrow involvement and lymphadenectasis. Central nervous system (CNS) recurrence can occur when disease progress, preventative intrathecal treatment was essential, whereas optimal treatment of relapsed or refractory BPDCN is still poorly defined. Since a transmembrane glycoprotein, CD38, was defined as a cell receptor which is express not only on lymphoid tissues but also on myeloid cells, Daratumumab (Dara) , the first-in-class human-specific anti-CD38 IgG1 mAb approved for the treatment of MM, had been currently reported to use in treating other hematological malignancies such as Acute Promyelocytic Leukemia (APL), T-acute lymphoblastic leukemia and BPDCN. Case presentation A 66-year-old male patient was admitted to hospital due to fatigue and palpitation, no cutaneous lesion and B symptoms were presented. Physical examination showed painless enlarged lymph nodes on neck, armpits and groin from multiple bilateral. Blood routine: WBC 2.18×109/L, HGB 83g/L, PLT 57×109/L. Bone marrow aspiration showed 79.5% of unclassified cells with irregular nuclei and immature chromatin pattern. Flow cytometry showed 82.26% abnormal cells were positive for CD56,CD123,CD38,CD303,CD304,TCL-1,CD5,CD4. Fluorescence in situ hybridization(FISH) showed P53/CEP17 1.2% positive on amplification, while chromosome showed normal karyotype. Next Generation Sequencing(NGS): TET2/JAK1/KMT2D/PHF6/TP53 mutation. BPDCN of stage Ⅳ/group A was diagnosed. Induction regimen VDCAP was suspended because the patient suffered from infection and grade IV cytopenia on the 3rd day of chemotherapy. Then 7 cycles of CDOP with 5 months of oral BCL-2 inhibitor Venetoclax were applied, autologous stem cell cryopreservation was conducted in the 4th to 5th cycle chemotherapy interval. Bone marrow aspiration after 8 cycles indicated that the patient received bone marrow complete remission, however minimal residual disease test harbored 0.65% abnormal dendritic cells. The patient suffered from headache and sight lost due to disease central nervous system (CNS) recurrence during preparation of autologous stem cell transplantation. The CNS relapse was confirmed by lumbar puncture, the cerebrospinal fluid White blood cell count is 278 x10e6 copies/L and flow cytometry testing for these abnormal white cells was in accord to BPDCN phenotype, meanwhile BM morphology showed 48% of abnormal dendritic cells of typical BPDCN immunophenotype with CD38 expression by flow cytometry either. After treatment with Two cycles of Daratuzumab-VRd regimen and one cycle of HD-MTX(3g/m2) in addition to Temolozomide 140mg/d for 7 days, the disease re-assessment was done, bone marrow aspiration showed 10% residual tumor cells, the amount of WBC in CSF reduced to 14 x10e6 copies/L. Therapeutic response reached Partial Remission (PR) and the patient's state is good. Conclusion BPDCN is rare, therefore no standard treatment was the first-in-class recommendation. Regimens for ALL, AML and NHL had been used for the induction therapy. Although remission rate of ALL regimens are as high as 90%, the duration of response was short, with a median survival of only 12-14 months. Targeted therapy like BCL-2 inhibitor Venetoclax, CD38 monoclonal antibody Daratuzumab and CD123-direct cytotoxin Targraxofusp had been reported to be sensitive to disease. This is the secondly reported case that treated with Daratuzumab. All two cases seemed to be effective. Currently, there is no standardized treatment for BPDCN, novel targeted therapies may improve the prognosis of BPDCN in the near future. This case also indicating that CD38 CART therapy also might be rational for the treatment on myeloid neoplastic. Disclosures No relevant conflicts of interest to declare.

Clinical analysis of decitabine combined with ruxolitinib regimen in treatment of newly diagnosed chronic myelomonocytic leukemia

Objective To analyze the clinical efficacy of decitabine combined with ruxolitinib regimen in the treatment of patients with newly diagnosed chronic myelomonocytic leukemia (CMML), and explore the effects of gene mutations on the prediction of efficacy and prognosis. Methods From March 2016 to August 2018, five cases of newly diagnosed patients with CMML admitted to the Department of Hematology, Ruijin Hospital North Affiliated to Shanghai JiaoTong University School of Medicine were selected as study subjects. Among them, there were 3 male patients and 2 female patients, with a median age of 60 years. In this study, the treatment regimens were deccitabine combined with rucotinib: deccitabine 20 mg/(m2·d), d1~3; rucotinib 5~10 mg/d, d1~28, 4 weeks as a course of treatment. At the end of each treatment course, the efficacy was evaluated based on the bone marrow cell morphology of the patients. Adverse reactions related to treatment were observed. The variant allele frequency (VAF) of 22 types of acute myeloid leukemia (AML)/myelodysplastic syndrome (MDS) related genes was detected before and after treatment. The follow-up period was up to May 1, 2019, with an interval of 1 month. The clinical features and efficacy of 5 patients with CMML were retrospectively analyzed, as well as the changes of related VAF of genes before and after treatment. The procedure of this study is accordance with the requirement of the revised World Medical Association Declaration of Helsinki in 2013. Consent form was obtained from all subjects. Results ① Among the 5 patients received the combination of descitabine and rucotinib regimen, 3 patients obtained overall response, among which patient 2 received complete remission (CR), patient 3 received hematologic improvement (HI), and patient 5 received bone marrow complete remission (mCR). Patient 4 had no effect. Patient 1 had disease progression (PD). All patients′ spleen size decreased (extent of reduction>50%). ② All the 5 patients showed myelosuppression after chemotherapy, among which patient 3 had the most severe myelosuppression and the longest period of myelosuppression, up to 2 months. Chest CT results of patient 1, 3 and 4 showed pulmonary infections, which was improved after active anti-infection treatment. Patient 1 and 4 had diarrhea. Patient 3 presented mild liver function impairment. Patient 5 presented constipation. And all symptoms improved after symptomatic and supportive treatment. ③ The results of VAF of mutations of patients before and 6 months after treatment showed, JAK2 V617F VAF (17% to 0), SRSF2 P95H VAF (44% to 39%) were decreased, and CBL H398P VAF (15% to 60%), TET2 Q273fs VAF (87% to 95%) were increased in the bone marrow specimens of patient 1. In patient 2, SRSF2 P95L VAF (69% to 49%) and CBL R420Q VAF (45% to 1%) were decreased, ASXL1 G710fs VAF (47% to 49%) was almost unchanged, and TET2 L1721W VAF (32% to 50%) was increased. In patient 4, SRSF2 P95L VAF (29% to 7%), TET2 Q705X VAF (95% to 62%) were decreased, and ASXL1 G642fs VAF (18% to 39%) was increased. The results of VAF of mutations of patients before and 2 months after treatment showed, CBL W408S VAF (29% to 31%) in the bone marrow specimens of patient 4 showed almost no change. In patients 5, TET2 R550X VAF (21% to 13%) and CBL C396R VAF (44% to 26%) were decreased, while TET2 C1298Y VAF (22% to 13%) was almost unchanged. Conclusions Decitabine combined with ruxolitinib in the treatment of newly diagnosed CMML can effectively improve the clinical symptoms and the curative effect. There is still a large degree of variability in the evaluation of clinical efficacy based on the VAF of single gene mutations, and this needs to be verified by prospective studies with large samples. Key words: Leukemia, myelomonocytic, chronic; Mutation; Treatment outcome; Decitabine; Ruxolitinib

Adult Case Series of Extramedullary Relapsing Acute Myeloid Leukemia (AML) after Allogeneic Stem Cell Transplantation in Whom a Rapid Response and a Reasonably Long Remission Could be Achieved with Gemtuzumab Ozogamycin (GO)

Gemtuzumab Ozogamycin (GO) is a drug conjugated monoclonal antibody which targets CD33 an antigen highly expressed on the surface of AML blasts. GO is approved for the treatment of both newly-diagnosed and relapsed AML. Despite growing evidence regarding its efficacy and safety among AML patients there is limited data about the use of GO in isolated extra-medullary relapsed AML. Extramedullary AML remains as an unmet clinical need regarding the poor prognosis and lack of a standard therapeutic approach. Our cases demonstrated a rapid and long lasting response with a favorable toxicity profile, in patients who were treated with GO as a single agent after an isolated extramedullary relapsing disease. Our first case was a 40-years-old woman admitted to our outpatient clinic with pain in her joints and redness in her back and stomach. She was diagnosed with CD33-positive Acute Myeloid Leukemia according to the immunophenotyping of bone marrow blasts. Idarubicin and ARA-C (7+3) combination initiated as a frontline induction therapy and morphological remission was achieved after the first cycle of induction. After the first cycle of consolidation chemotherapy with high dose ARA-C she has relapsed with skin myeloid sarcomas and diffuse bone marrow infiltration. Soon after the relapsing disease she was put on to ida-flag salvage chemotherapy and she responded well with the disappearance of skin lesions and morphological complete remission of bone marrow. After achieving response, an allogeneic stem cell transplantation (HSCT) was applied from her sibling donor with a myeloablative conditioning. Unfortunately patient had a relapsing extramedullary disease with reappearance of skin lesions even with an ongoing acute skin gvhd (Figure) soon after allogeneic HSCT. Bone marrow biopsy revealed no increase in blast count. We have decided to initiate a novel targetted therapy to control the extramedullary disease. As her blasts infiltrating the skin were universally CD 33 positive we considered to put her on GO therapy with the approval obtained from health authority. GO was applied according to the dosage approved by FDA for relapsing disease. After the first cycle of GO she had a rapid disappearance of all skin lesions just complicated with a febrile neutropenic episode and re-activation of CMV infection which was controlled with Gancyclovir. She has retained a complete remission regarding extramedullary disease throughout the repeated courses of GO for 3 times, and she is still in remission for 4 months after the first appearance of skin lesions (Figure). Our second case was a 24-years-old boy who admitted to our outpatient clinic with a worsening fatigue and shortness of breath. He was also diagnosed with CD33-positive Acute Myeloid Leukemia according to the immunophenotyping of bone marrow blasts and was able to achieve a morphological CR after frontline induction therapy with 7+3 protocol. After two cycles of high dose ARA-C consolidation he was transplanted from a matched unrelated donor because of an intermediate cytogenetic risk profile. At the 14th month of allogeneic transplantation he had relapsed with a bone marrow blast count of 90% and harboring a monosomy on the 10th chromosome. After the first salvage chemotherapy with IDA-FLAG protocol he has achieved a morphological CR and we have decided to proceed with an alternative donor transplantation. But unfortunately soon after discharge, he has admitted to the emergency clinic with a new onset headache and nausea and vomiting. A cranial CT revealed multiple foci of solid masses with peripheral edema. We have performed a lumbar puncture and CSF fluid revealed a high number of CD33 positive blastic cells (over 100 cells per HPF). At the time of central nervous system (CNS) relapse his bone marrow was free of blastic infiltration. We decided to initiate GO treatment with the diagnosis of isolated central nervous system relapse of AML, accompanying intra-thecal chemotherapy via an Omaya Reservoir. He has received two cycles of GO which was complicated with neutropenic fever and grade 4 leukopenia and thrombocytopenia, and his CNS lesions also responded well and clinically he had no CNS related signs or symptoms. The patient received additional GO with continued response but unfortunately after a severe pneumonia he passed away at intensive care unit. Our case series documented a clinically relevant therapeutic field for GO in isolated extramedullary relapse of AML. Disclosures No relevant conflicts of interest to declare.

The PRIME Trial: PARP Inhibition in IDH Mutant Effectiveness Trial. a Phase II Study of Olaparib in Isocitrate Dehydrogenase (IDH) Mutant Relapsed/Refractory Acute Myeloid Leukemia and Myelodysplastic Syndrome

Background: Recurring mutations have been identified in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) which translate to therapeutic targets. Isocitrate dehydrogenase-1 and -2 (IDH1/2) mutations occur in ~20% of AML, and up to 12% of patients with MDS. Three conserved mutational hotspots in the IDH enzymes alter their function and lead to the production of (R)-2-hydroxyglutarate (2HG), an oncometabolite with numerous downstream effects, including impaired DNA damage repair. Specifically, homologous recombination (HR) is impaired by inhibiting the function of histone demethylases that are critical for HR and recruitment of the HR machinery to sites of DNA damage. In HR deficient tumors poly-ADP ribose polymerase (PARP) enzymes mediate a key salvage pathway. PARP inhibition in HR deficient tumors leads to synthetic lethality via simultaneous inhibition of HR and SSB mediated DNA repair. Our group previously demonstrated synthetic lethality with PARP inhibition in IDH mutant cells lines, and other IDH mutant models including primary patient-derived cell lines and genetically-matched tumor xenografts. Study Design and Methods: The PRIME trial (NCI10264) is a proof of concept, biomarker-driven, multi-institution, phase II open label clinical trial to assess the overall response of IDH1/2 mutant relapsed/refractory AML and MDS to PARP inhibitor monotherapy with olaparib. The clinical trial is executed by the Experimental Therapeutics Clinical Trials Network of the NCI. The Cancer Therapy Evaluation Program will provide olaparib. Eligibility criteria include documented IDH1 or IDH2 mutation in blood or bone marrow within 30 days of enrollment based on mutational testing by PCR or sequencing in a CLIA certified laboratory and willingness to undergo a bone marrow biopsy. Patients will be treated with olaparib 300 mg q12hrs each day of a 28-day cycle, using a tablet formulation, until disease progression, unacceptable toxicity, withdrawal of consent or death. Blood and bone marrow samples for 2-HG analysis will be collected prior to starting therapy and after 1 cycle (28 days), cycle 2, 3, 6, 9, 12 or when there is concern for disease progression (Figure 1). A Simon two-stage optimal design will be used to test the null (ORR=10%) versus the alternative hypothesis (ORR=40%) in each arm. In the first stage, 9 patients will be accrued in each arm. If one or fewer responses are observed in these 9 patients, that arm will be stopped early for futility. Otherwise, 11 additional patients will be accrued for a total of 18 in each arm. We reject the null hypothesis if at least 5 responses are observed in these 20 patients. In each arm, we have approximately 90% power to detect a 30% increase in ORR at a one-sided type I error rate of 0.05. Primary endpoint: Overall response rate (ORR) of 40%, i.e., a 30% ORR improvement (40% vs. historical control ORR = 10%) based on MDS International Working Group 2006 criteria and AML MDS International Working Group 2003 criteria after 6 cycles of treatment. Cumulative ORR will include complete remission, complete remission with incomplete blood count recovery, partial response, and bone marrow complete remission. Secondary endpoints: Progression-free survival (the interval between the time of initiation of olaparib to the time of documentation of olaparib failure or last follow-up) and overall survival (the interval between the time of initiation of olaparib to the time of death or last follow-up) for the trial. Exploratory studies: The PRIME trial will also test the utility of 2-HG and DNA damage markers such as γ-H2AX as potential biomarkers of response to olaparib. Using multiple viability assays on leukemia cell lines and bone marrow cultures we will assess synergistic therapeutic combinations to further improve outcomes in this patient population. To confirm efficacy in vivo without undue toxicity, promising combination therapies will be confirmed in cytokine-humanized immunodeficient "MISTRG" mice. We will also examine the impact of PARP inhibitors on the genomic, proteomic, metabolomic and immunologic landscape of IDH 1/2-mutant hematologic malignancies using DNA whole exome sequencing (WES), RNA-Seq, and liquid chromatography-mass spectrometry assessment of oncometabolites. Disclosures Bindra: Cybrexa: Consultancy, Equity Ownership. Prebet:pfizer: Honoraria; pfizer: Honoraria; pfizer: Honoraria; Boehringer Ingelheim: Research Funding; pfizer: Honoraria; Tetraphase: Consultancy; novartis: Honoraria; novartis: Honoraria; Genentech: Consultancy; Boehringer Ingelheim: Research Funding; novartis: Honoraria; Boehringer Ingelheim: Research Funding; Agios: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding; pfizer: Honoraria; novartis: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; novartis: Honoraria. OffLabel Disclosure: We will be using PARP inhibitors as a novel therapy for patients with relapsed or refractory AML and high risk MDS based on preclinical data.

Impact of Clinical Features, Cytogenetics, Genetic Mutations and Methylation of CDKN2B and DLC-1 Promoters on Treatment Response to Azacitidine

Introduction : Azacitidine (AZA) is a DNA hypomethylating agent used in myeloid neoplasms, however approximately half of patients show treatment failure or relapse. Last years, several studies have showed that genetic mutations may influence on response and survival of the treated patients. Other biomarkers that have traditionally been associated with the response to AZA are the recovery of the platelet count and the presence of abnormalities in the chromosome 7. Finally, the methylation dynamics of genes promoters could be a useful tool to predict the clinical response. Aim: To assess the predictive value on response to AZA of clinical features, cytogenetics, genetic mutations and the methylation dynamics of CDKN2B and DLC-1 promoters in a series of patients with myeloid neoplasms. Methods: We studied 85 patients with myelodysplastic syndromes (MDS) and 28 patients with acute myeloid leukemia (AML) who received AZA. Treatment schedules included 5-azacytidine 75 mg/m2/day for 7 days (n=62) and 5-azacytidine 75 mg/m2/day for 5 days (n=51). According to the International Working Group (IWG) 2006 criteria for MDS, responders were defined as patients achieving complete remission (CR), bone marrow CR (mCR), partial remission (PR) or stable disease with hematologic improvement (SDHI). Sixteen genes with prior implication in the pathogenesis of myeloid diseases were analyzed: NPM1, FLT3, CEBPA, TET2, DNMT3A, IDH1, IDH2, ASXL1, EZH2, RUNX1, SETBP1, TP53, SF3B1, SRSF2, U2AF1 and ETNK1. For methylation assay, EZ DNA Methylation-Gold kit (Zymo Research) was employed for the bisulfite treatment of DNA. Methylation of CDKN2B and DLC-1 were qualitatively studied pre- and post-AZA with unmethylated and methylated primers and analysis of the PCR products in a QIAxcel System (Qiagen). Overall survival (OS) was measured from start of AZA treatment until the last follow-up or death from any cause. P values &lt;0.05 were considered as statistically significant. Results: Of the 113 patients analyzed, 46 patients (41%) showed response to AZA (median cycles=6): CR=19, mCR=6, PR=12, and SDHI=9. Stable disease and treatment failure were observed in 20 (17%) and 47 patients (42%), respectively. Analysis of clinical features showed that platelet doubling after the first AZA cycle was significantly associated with a better response (58% vs. 31% responders, P=0.041). Forty-seven patients (42%) had chromosomal abnormalities. A positive co-occurrence between del(7q)/-7 and del(17p) was found (n=6, P&lt;0.001) and was significantly associated with a worse response to AZA (P=0.039). Pre-treatment, genetic mutations were presented in 98 patients (87%) and methylation of CDKN2B and DLC-1 promoters were detected in 50 (44%) and 37 (33%) patients respectively (Figure 1). Patients with SF3B1 mutations showed a better response to AZA (68% vs. 35% responders, P=0.008) and a higher OS (30 vs. 15 months, P=0.019) than the remaining patients. In contrast, patients with mutations in transcription factors (RUNX1, SETBP1, NPM1) showed a worse response to AZA (20% vs. 47% responders, P=0.014) and a lower OS (10 vs. 19 months, P=0.010). Splicing genes mutations (SF3B1, SRSF2 and U2AF1) were related to favourable karyotypes (90% vs. 59%, P&lt;0.001) and a lower DLC-1 methylation (31% vs. 57%, P=0.006). Interestingly, patients with DLC-1 methylation showed higher levels (&gt;5%) of bone marrow blasts (63% vs. 40%, P=0.016), higher frequency of complex karyotypes (40% vs. 15%, P=0.002), a positive co-occurrence with TP53 alterations (38% vs. 16%, P=0.009) and its methylation dynamics showed a strong trend towards predict the response to AZA: 57% of patients without DLC-1 methylation or who reduced it post-treatment responded favourably to the drug and 65% of patients who maintained or increased it did not respond (P=0.053). In addition, leukemia transformation was higher in MDS patients with DLC-1 methylation post-AZA than in the remaining MDS patients (58% vs. 21%, P=0.002). Conclusion: The combination of chromosomal alterations and genetic mutations together with the monitoring of platelet count and DLC-1 methylation could contribute to give light in myeloid neoplasms to the heterogeneity observed both in the response to AZA and in patient survival. Samples and data from patients included in this study were provided by the INCLIVA Biobank (PT13/0010/0004). Disclosures Hernandez Boluda: Incyte: Other: Travel expenses paid.

BCOR mutation and TLS-ERG expression in acute myeloid leukemia with monoclonal immunoglobulinemia.

Acute myeloid leukemia (AML) originates from the abnormal clonal proliferation of myeloblasts. Immunoglobulin is secreted by B cells. AML with monoclonal antibody often indicates a poor prognosis. Here we report a case of BCOR mutation and TLS-ERG expression in AML with monoclonal immunoglobulinemia. After chemotherapy, the patient achieved bone marrow complete remission. BCOR mutation and TLS-ERG fusion gene in patient's bone marrow were not detected, at the same time, peripheral blood monoclonal immunoglobulin also disappeared. BCOR mutation or TLS-ERG fusion gene expression is associated with poor prognosis, AML with monoclonal immunoglobulin may have the same prognostic significance.

Efficacy analysis of decitabine monotherapy or combined with arsenic trioxide in treatment of myelodysplastic syndromes

Objective To analyze the therapeutic effect and tolerability of decitabine monotherapy or combined with arsenic trioxide for the treatment of patients with myelodysplastic syndromes (MDS). Methods Clinical characteristics of 32 patients with primary MDS in North Hospital of Ruijin Hospital Affiliated to Medical College of Shanghai Jiaotong University from January 2014 to April 2017 were retrospectively analyzed. The clinical data of these patients were collected, and the patients were followed up. Decitabine combined with arsenic trioxide was used in 23 cases, decitabine (20 mg·m-2·d-1) and arsenic trioxide (0.16 mg/kg) were administrated from day 1 to day 5 and was repeated every 4-6 weeks. For the remaining 9 cases, only decitabine was applied, decitabine(20 mg·m-2·d-1) was administrated from day 1 to day 5 and was repeated every 4-6 weeks. The clinicopathological characteristics and the effect of genetic mutations on the efficacy of treatment were investigated. Results Of the 32 patients with primary MDS, 18 were male and 14 were female. The patients were 17-72 years old with a median age of 56 years old. Genetic analysis revealed 10 cases with TP53 mutations, 8 cases with TET2 mutations, 4 cases with U2AF1 mutations, 3 cases with RUNX1 mutations, 3 cases with ASXL1 mutations, 2 cases with NRAS mutations, 2 cases with DNMT3A mutations and 1 case with JAK2 V617 mutation. The follow-up time was 2-23 months with a median follow-up time of 8 months. A total of 21 cases (65.6%) attained treatment response. Among them, there were 10 cases (31.3%) with complete remission (CR), 5 cases (15.6%) with bone marrow complete remission (MCR), and 6 cases (18.7%) with hematological improvement. There was no significant difference in the efficiency and CR rate between the combination group and the monotherapy group (P = 0.441, P = 0.681). Ten cases were found to have TP53 mutations, of which 7 cases had CR. Multivariate analysis demonstrated that TP53 mutation was an independent risk factor for CR (P = 0.037). All patients developed myelosuppression after treatment, of which 16 cases developed pulmonary infection. Conclusions Decitabine combined with arsenic trioxide in the treatment of MDS is effective and well tolerated. The therapeutic effects of decitabine monotherapy or decitabine combined with arsenic trioxide for treatment of patients with TP53 mutations are better than the average levels. Key words: Myelodysplastic syndromes; Decitabine; Arsenic trioxide; Curative effect

Clinical effect of CCLG-ALL2008 regimen in treatment of children and adolescents aged >10 years with acute lymphoblastic leukemia

To study the long-term clinical effect of CCLG-ALL2008 regimen in the treatment of children and adolescents, aged >10 years, with newly diagnosed acute lymphoblastic leukemia (ALL). A retrospective analysis was performed for the clinical data of 150 ALL children and adolescents aged >10 years who were treated with CCLG-ALL2008 regimen from April 2008 to April 2015. The Kaplan-Meier method was used to analyze overall survival (OS) rate and event-free survival (EFS) rate. Among the 150 children and adolescents, there were 87 (58.0%) boys and 63 (42.0%) girls, with a median age of 11 years (range 10-15 years). Of the 150 children and adolescents, 84 (56.0%) had intermediate risk and 66 (44.0%) had high risk; 122 (81.3%) had B-lineage acute lymphoblastic leukemia (B-ALL) and 28 (18.7%) had T-lineage acute lymphoblastic leukemia (T-ALL). The fusion gene test yielded positive results in 51 children and adolescents (34.0%), among whom 16 (31%) had positive BCR-ABL, 11 (22%) had positive TEL-AML1, 8 (16%) had positive E2A-PBX1, and 16 (31%) were positive for other fusion genes. The complete remission rate was 96.0% (144/150) after one course of treatment with CCLG-ALL2008 regimen. The median follow-up time was 52 months (range 3-122 months). The 5-year OS rate was 79.0%±3.5%, and the 5-year EFS rate was 67.3%±4.1%. There were no significant differences in 5-year OS and EFS rates between the children with intermediate or high risk, as well as between the children with B-ALL or T-ALL (P>0.05). The children and adolescents who achieved complete remission of bone marrow at the end of induction therapy had significantly higher 5-year OS and EFS rates than those who did not achieve complete remission (P<0.05). In ALL children and adolescents aged >10 years, CCLG-ALL2008 regimen can help to achieve high complete remission rate, 5-year OS rate and 5-year EFS rate. The children and adolescents failing to achieve complete remission at the end of induction therapy tend to have a poor prognosis.

Blinatumomab in the Treatment of Acute Lymphoblastic Leukemia: Russian Multicenter Clinical Trial

Background. Recent advances in the treatment of relapsed/refractory acute lymphoblastic leukemia (R/R ALL) are attributed to the implementation of immunotherapy methods which include blinatumomab, the bispecific engager of a patient's endogenous T-cells (Blincyto™, Amgen®) (BC). Aim. To assess BC efficacy and toxicity in the treatment of R/R ALL patients with persistence of minimal tumor clone before and after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Materials &amp; Methods. The trial included 66 B-ALL patients with CD19+ aged 18 to 72 years, 23 (35 %) of them with measurable minimal residual disease (MRD+) and 43 (65 %) with R/R ALL. In 18 (27 %) patients BC was administered after prior allo-HSCT. Results. In the overall group 2-year overall survival (OS) and disease-free survival (DFS) in patients with response to BC treatment were 53 % and 38 % respectively. In the R/R ALL group complete remission (CR) was achieved in 29 (67 %) patients including 24 (83 %) patients with negative MRD. CR rate was higher in standard cytogenetic risk group (73 %) in comparison with high-risk group (59 %). In patients with more or less than 50 % blast cells in bone marrow CR rate was 85 % and 61 %, respectively. When BC was administered after prior allo-HSCT and without it CR rate was 80 % and 60 %, respectively. In R/R ALL patients with response to BC 2-year OS and DFS were 40 % and 26 %, respectively, in the MRD+ group of ALL patients they were 66 % and 51 %, respectively. Relapse rate was lower in the group with allo-HSCT than in the group without it, i.e. 21 % vs. 55 %. Adverse events of grade 3-4 were observed in 25 (38 %) patients. In 11 (16 %) patients BC therapy had to be discontinued, in 5 (7 %) patients it was terminated prior to the scheduled date. Conclusion. BC efficacy is higher in the MRD+ group and in R/R ALL patients with smaller tumor mass. BC treatment after allo-HSCT yields remissions in most patients and can be combined with immune-adoptive therapy.