Marrow Blasts Research Articles

Overall Survival in Patients with Ccus Depends on Presence of Anemia

Introduction Clonal cytopenia of undetermined significance (CCUS) is a recently recognized hematological disorder characterized by the presence of one or more cytopenias, evidence of clonal hematopoiesis and not fulfilling the criteria for a myeloid neoplasm. In contrast to myelodysplastic syndromes (MDS) where 95% of the patients are anemic at time of diagnosis, that is only the case for around 60% of patients with CCUS (Weeks et al. Prediction of Risk for Myeloid Malignancy in Clonal Hematopoiesis. NEJM Evid. 2023). Furthermore, as recent studies on survival in CCUS primarily included younger patients (Weeks et al. NEJM Evid. 2023) or population-based cohorts not specifically referred for diagnostic work up of unexplained cytopenia (UC) (Rossi et al. Clinical relevance of clonal hematopoiesis in persons aged ≥80 years, Blood 2021) additional survival data on patients with CCUS are warranted. In this study, we investigated survival outcomes in patients with CCUS referred for primary work up of UC. Methods We included 241 patients with CCUS and compared them to 144 patients with low-risk MDS, defined as having less than 5% blasts in the bone marrow. Bone marrow biopsies and next-generation sequencing (NGS) were performed in all patients and cytogenetic analyses using G-band karyotyping was done in 362 (94%) of cases. For NGS, we used a gene panel with the most commonly mutated genes in MDS. The patients were included at 6 different institutions in Denmark from 2013 and onwards. A cut-point of 5 years follow-up was chosen for more robust survival data, and patients were censored at this time point if followed for more than 5 years. Patients with CCUS were stratified into 3 groups dependent on the type of cytopenia present at diagnosis. CCUS patients with pancytopenia (n = 35), and CCUS patients without pancytopenia were split based on whether they were anemic (n = 143) or not (n =63). We compared overall survival between the CCUS groups and low-risk MDS in a Cox proportional hazards model adjusted for age, sex, number of variants and presence of high-risk variants. Results Of the 241 CCUS patients, 178 (74%) were anemic (hemoglobin < 12 and 13 g/dL for women and men, respectively), while this was the case for 130 (90%) of the patients with MDS. A total of 772 variants were identified in the entire cohort with a median of 2 (IQR: 1-3) per patient for both CCUS and MDS patients. The overall survival at 5 years was 45% for low-risk MDS patients and 62% for CCUS patients. For CCUS patients without anemia, CCUS patients with anemia and CCUS patients with pancytopenia overall survival at 5 years was 85%, 52% and 55%, respectively (p < 0.0001). In the adjusted Cox proportional hazards model, we found a significantly superior overall survival for CCUS patients without anemia compared to those with anemia (HR: 0.28, 95%-CI: 0.13-0.58, p = 0.001) and pancytopenia (HR: 0.22, 95%-CI: 0.09-0.53, p = 0.001) and patients with low-risk MDS (HR: 0.18, 95%-CI: 0.09-0.38, p < 0.001). There was no significant survival difference between CCUS patients with pancytopenia and low-risk MDS patients (HR: 0.67, 95%-CI: 0.46-1.50, p = 0.5). Of the remaining variables, age was the strongest prognostic factor with hazard ratio increasing with a factor 1.05 (95%-CI: 1.03-1.07, p < 0.001) per year aged. Predicted survival probabilities for a 65-year-old and 80-year-old person demonstrate a substantial difference in overall survival across all 3 categories of CCUS (figure 1). Discussion Here we show that the presence of anemia impacts overall survival among CCUS patients. We observe that CCUS patients without anemia, representing 26% of CCUS patients in our cohort, have a superior overall survival compared to those with anemia, even with a comparable mutational profile. Our findings indicate that other factors than mutational signatures impact overall survival. Moreover, the prevalence of CCUS increases with age, and our findings demonstrate a considerable difference in survival between a 65-year-old and 80-year-old person diagnosed with CCUS. This raises the concern that prediction models can overestimate the survival rates if the elderly group of CCUS patients is not well represented in these models. In conclusion, we show that age and hemoglobin levels are important factors in overall survival for CCUS patients, and that patients with CCUS without anemia have a superior overall survival and should be considered as a separate group within the CCUS category.

Clinical Characteristics of Long-Term Survivors Treated with BCMA CAR T Cell

Introduction Anti-BCMA(B-cell maturation antigen) CAR-T therapy, a novel and efficacious method for treating relapsed/refractory multiple myeloma (RRMM), has been approved and applied in clinics. Here, we report the survival analysis and clinical characteristics associated with long-term survivors (complete response at 18 months) after CAR-T cell therapy. Methods Between 1 January 2018 and 1 February 2023, 22 consecutive patients with relapsed/refractory MM were enrolled in a clinical trial of BCMA CAR T cells (registered at www.clinicaltrials.gov as #NCT04500431). This study was approved by the Tongji Hospital of Tongji University Ethics Committee, and written informed consent was provided by each patient. They all were treated with a single infusion of CAR-T cells. FC chemotherapy regimen was administered before transfusion. The median number of CAR-T cells transfused was 5.0 (range 4.0-6.6) ×10 6/kg. Long-term efficacy and related clinical characteristics were evaluated. Results In the 22 patients treated, the overall response rate was 77% (17 of 22). With a median follow-up of 24 months (range, 4-52), the median PFS were 17 months. The adverse events within 30 days after infusion were well tolerated. Toxic hematologic effects were the most common grade ≥3 events, including neutropenia (68%), anemia (54%), and thrombocytopenia (36%).Only 1 patient experienced grade 1 neurologic toxicity.No patients experienced severe infection within 30 days after infusion. Detailed patient baseline characteristics are described in Tables 1. During follow-up after CAR T-cell therapy, the complete response rate was 36.3%(8/22), 40.9(9/22) and 27.2(6/22) at 12 months, 18 months and 24 months, respectively. At 18 months, the patients achieved the highest complete response rate. Baseline characteristics, such as age, sex, isotype, ECOG, high-risk cytogenetics, prior lines of therapy, percentage of plasma blasts in bone marrow, serum monoclonal protein and extramedullary disease in the 9 complete response patients at 18 months were similar to other progressive disease 13 patients. However, the significant differences of International Staging system stage(p=0.034) and Durie-Salm staging system(p=0.002) at baseline were observed between the 9 complete response patients and 13 progressive disease patients at 18 months(Table 1). The International Staging system stage(area under curve 0.80) and Durie-Salm staging system(area under curve 0.86) predicted the outcome of patients at 18 months well. Durie-Salmon Staging system was published in 1975, which used commonly available clinical parameters including calcium, hemoglobin, bone lesions, creatinine level and serum or urine monoclonal protein. The ISS staging system was proposed in 2005 based on baseline serum levels of beta2-microglobulin (β2M) and albumin. And there was no statistically significant difference in the baseline serum levels of beta2-microglobulin, albumin, calcium, bone lesions and creatinine level between the 9 complete response patients and 13 progressive disease patients at 18 months. As figure 1 illustrates, the hemoglobin of 9 complete response patients was higher than that of 13 progressive disease patients with statistical significance at 9 time-points: PreFC(117.6±20.7 vs 94.6±23.6 , p = 0.028), D -4(113.6±15.9 vs 86.0±25.7 , p = 0.016), D -3(107.8±11.8 vs 84.6±23.0 , p = 0.027), D -2(117.6±20.7 vs 94.6±23.6 , p = 0.0009), D -1(111.0±19.9 vs 81.8±19.0 , p = 0.013), D0(107.1±20.4 vs 83.0±22.8 , p = 0.029), D1(107.7±19.1 vs 84.2±24.3 , p = 0.032), D4(104.1±14.9 vs 82.7±19.9 , p = 0.015), D5(103.4±19.1 vs 82.2±18.3 , p = 0.018). In the univariate Cox regression analysis, the hemoglobin at PreFC(hazard ration (HR) = 0.97; 95% confidence interval (CI): 0.95-1; p = 0.029), D -4(HR=0.96 ;95% CI:0.92-1, p = 0.012), D -3 (HR=0.97 ;95% CI:0.94-1, p = 0.029), D -2(HR=0.93 ;95% CI:0.89-1, p = 0.003), D -1(HR=0.97 ;95% CI:0.95-1, p = 0.047), D0(HR=0.97 ;95% CI:0.94-1, p = 0.038), D1(HR=0.97 ;95% CI:0.94-1, p = 0.044), D4(HR=0.95 ;95% CI:0.92-1, p = 0.01) and D5(HR=0.97 ;95% CI:0.94-1, p = 0.027) were relative factors of prognosis. Conclusions These long-term follow-up data in patients after BCMA CAR -T therapy suggest that baseline ISS ,DS staging and hemoglobin at 9 time-points display opposing association with long-term progression-free survival.

Use of CLL1 and CD45RA As Biomarkers for Leukemia Stem Cells in Acute Myeloid Leukemia Re-Induction Therapy Decision Making

Background: The decision to re-induce patients with newly diagnosed acute myeloid leukemia (AML) currently relies on morphological evaluation of leukemia blasts in post treatment bone marrow, with more than 5% bone marrow blast counts indicating necessity of re-induction therapy. Our previous study showed that changes in Leukemia Stem Cell (LSC) subsets, identified by CLL1 (CD371) or CD45RA as specific markers, are highly correlated with therapeutic outcome. In this study, we focus on newly diagnosed AML patients undergoing induction therapy to determine whether information gained from LSC subset analysis can aid in decision making for re-induction therapy. Methods: We analyzed flow cytometry data from bone marrow aspirate in patients with newly diagnosed AML of all types except acute promyelocytic leukemia (M3) before and after induction therapy from a single institution. “Leukemic blasts” were identified by morphology and leukemia-associated immunophenotype by Multicolor Flow cytometry (MFC). Hematopoietic stem cells (HSCs) gating was based on CD45 dim/SSC and CD34 +CD38 low/- expression. Leukemic stem cells (LSC) subset was analyzed using % of CD371 (CLL-1) or CD45RA positive cells over total HSCs (%CLL1 +/CD34 +CD38 - or %CD45RA +/CD34 +CD38 -). Newly diagnosed AML with >50% (CLL1 +/CD34 +CD38 - or CD45RA +/CD34 +CD38 -) on diagnosis was selected for further study. Fold change of LSC subset was calculated using LSC percentage post-induction divided by LSC percentage on diagnosis. Chart review was conducted to determine correlation between LSC subset change, bone marrow blasts count and clinical course. Mann-Whitney test was used for statistical analysis. Results: Forty-eight newly diagnosed AML (non-M3) cases, who received at least one cycle of induction therapy with both pre- and post-treatment bone marrow biopsy were reviewed. Twenty-seven cases were identified with CLL1 +/CD34 +CD38 - or CD45RA +/CD34 +CD38 - above the 50% threshold on diagnosis. Four cases were later excluded due to inadequate CD34 +CD38 - cellular events on flow cytometry (<20) in post-treatment bone marrow samples. As shown in figure 1, in all the 9 patients with post induction blasts counts of less than 5% (complete remission, CR), post induction LSC subset is reduced to less than 0.5× of LSC subset on diagnosis. Thus, post induction LSC subset less than 0.5× of diagnosis is used as cut off for “adequate reduction”. In 14 patients with residual blasts of more than 5%, there is a wide range of LSC subset change. Interestingly, 6 of them showed adequate reduction of LSC subsets, similar to patients who achieved CR. Among them, one was observed and showed CR on repeat bone marrow biopsy at a later time point without further therapy. The other 5 patients were given re-induction and achieved CR after a second cycle. Eight patients with residual blasts did not achieve adequate reduction of LSC subset (residual LSC subset > 0.5× of diagnosis). Five achieved CR on subsequent re-induction. Three had persistent disease and failed to achieve CR despite 3 cycles of induction therapy. Conclusion: In newly diagnosed AML with high percent of CLL1 +/CD34 +CD38 - or CD45RA +/CD34 +CD38 - LSC subset on diagnosis, monitoring LSC subset offers additional information regarding efficacy of induction therapy. All the patients with CR showed residual LSC subset of 0.5-fold or less compared to LSC subset on diagnosis (defined as “adequate reduction” of LSC subset). For patients who did not achieve remission but adequately reduced LSC subset, which indicates efficacy of induction therapy, observation with repeat bone marrow biopsy, or re-induction with similar regimen can be considered. For patients with no adequate reduction of LSC subset, intensified chemotherapy or alternative therapy with different mechanism of action needs to be considered to maximize the possibility of achieving CR on re-induction. A multi-institutional study with more patients is needed to confirm these findings and validate these LSC biomarkers for this important treatment decision making in AML.

Multiomics Characterization of Normal CD34+ Hematopoietic Progenitor Cells (HPC) and Blasts in the Bone Marrow (BM) and Peripheral Blood (PB) of Patients with Acute Myeloid Leukemia (AML)

BACKGROUNG: Multiparameter flow cytometry (MFC) is traditionally used to asses MRD in AML. However, its sensitivity and specificity lag behind other methods and this precludes a broader application in routine practice. It could be hypothesized the emerging multiomics such as single-cell RNA and antibody sequencing could uncover new differences from normal and leukemia associated immunophenotypes (DfN/LAIP). In addition, the identification of DfN/LAIP may be easier in PB when compared to the BM due to the scarcity of CD34+ HPC in the former sample. AIM: Uncover new DfN/LAIP for improved MFC discrimination between normal CD34+ HPC and blasts in the BM and PB of AML patients. METHODS: A total of 47 samples were analyzed in this study. Namely, paired BM and PB from 5 young healthy adults (20-23yo) and 5 PB samples from older healthy adults (47-88yo); paired BM and PB of 11 newly diagnosed AML (44-79yo) patients, and paired BM and PB collected in 5 of the 11 AML patients at the time of MRD assessment. We used fluorescence activated cell sorting (FACS) to isolate CD34+ HPC from the BM and PB of healthy adults as well as blasts from AML patients. scCITE-Seq with a panel of 600 genes and 50 oligo-conjugated antibodies, selected according to their role in stem cell identification, lineage differentiation, clonal hematopoiesis and aberrant expression in myeloid neoplasms was performed. After quality control filtering, a total of 111,334 cells were analyzed and differential expression of genes and antigens was considered if an adjusted P-value <.05 and a log 2FoldChange (FC) >|1| were observed. RESULTS: We first analyzed the phenotype of paired BM and PB 32,637 CD34+ HPC from younger healthy adults. There were 82 deregulated genes, 50 overexpressed in BM and 32 in PB. CLEC12A, CEBPA and HBB were amongst the top overexpressed genes in BM, whereas CRHBP, ADGRG6 and MEIS1 showed the highest upregulation in PB. These results suggest partial transcriptional overlap between BM and PB CD34+ HPC, with the former showing reduced differentiation. There were no differences in antigen expression. Next, we analyzed the potential effect of aging in the phenotype of 34,102 CD34+ HPC from the PB of young and older healthy adults. There were 34 genes differentially expressed including BCORL1, CBLB, HRAS and KDM6A. These findings might reflect an upregulation of genes associated with clonal hematopoiesis. Furthermore, there were 13 antigens differentially expressed including CD9, CD10, CD33, CD38, CD62L and CD371. The comparison of 56,387 blasts in paired BM and PB samples from newly diagnosed AML patients showed near overlap with only HBB being differentially expressed. There were no differences in antigen expression. We then investigated the extent of phenotypic differences between normal CD34+ HPC vs blasts in the BM and PB of healthy adult's vs AML patients. There were 224 and 15 differentially expressed genes between BM cell types, whereas in PB the numbers were 182 and 11, respectively. The markers that contributed the most in the discrimination between CD34+ HPC vs blasts in PB were WT1, ADM, HMOX1, DDIT3, HOPX, CD371, CD45RA, CD133 and CD47. The overlap between DfN/LAIP identified in PB and BM was 31%. Four of the five patients monitored after treatment were MRD positive in BM using MFC. Of note, BM and PB CD34+ HPC isolated from the five AML patients (including one MRD negative in BM) clustered separately of normal BM and PB CD34+ HPC from healthy adults. There was near overlap between BM and PB cells from AML patients. The markers that contributed the most in the discrimination between PB CD34+ HPCfrom healthy adult's vs resistant cells from AML patients were CXCL16, LILRA5, MME, CD274, CTSL, MINPP1 and AHSP. CONCLUSIONS: Normal CD34+ HPC in PB showed partial overlap with the BM counterpart, with a few phenotypic differences probably related to less differentiation and the effects of aging and clonal hematopoiesis. The phenotype of BM and PB blasts from newly diagnosed AML patients nearly overlapped, which indicates that DfN/LAIP may be equally applicable in BM and PB. Accordingly, the markers included in the scRNA/Ab-seq panel effectively discriminated CD34+ HPC from blasts regardless of the sample of origin. Furthermore, we showed phenotypic divergence between PB CD34+ HPC of healthy adults' vs AML patients after treatment. Taken together, this study identified new DfN/LAIP markers for improved assessment of MRD using MFC in the PB of AML patients.

Real-World Outcomes Using Front-Line Midostaurine in Combination with Intensive Chemotherapy for Patients Aged ≥ 60 Years Old with FLT3 Mutated Acute Myeloid Leukemia

BACKGROUND: The prognosis of patients with acute myeloid leukemia (AML) varies depending on the presence of mutations in the FLT3 tyrosine kinase. Internal tandem duplications (ITD) and mutations in the tyrosine kinase domain (TKD) result in continuous activation of FLT3, leading to uncontrolled blast proliferation. Since the publication of the clinical trial CALGB 10603 (RATIFY), it has been established that adding midostaurine to intensive chemotherapy improves the overall survival (OS) of patients with mutated FLT3. However, it is worth noting that this study only included patients 18-59 years old. Subsequently, a phase II clinical trial (AMLSG 16-10 trial) conducted a subgroup analysis suggesting that midostaurine may also benefit to patients between 60 and 70 years of age. Nevertheless, these findings have not been studied in real-world clinical practice. METHODS : This is multicentric non-interventional retrospective real-world study including patients ≥60 years of age with newly diagnosed FLT3-mutated AML treated with intensive chemotherapy and midostaurine (50 mg orally twice daily, d8-22) between 1st 2017 and July, 30 th 2021 from the French Innovate Leukemia Organization (FILO) and the Programa Español de Tratamientos en Hematología (PETHEMA). The primary objective was to evaluate the efficacy of midostaurine in combination with intensive chemotherapy in patients over 60 years old. We assessed the rate of complete remission (CR) or complete remission with incomplete hematologic recovery (CRi) after induction therapy, OS and event-free Survival (EFS). RESULTS: A total of 191 patients (FILO Group 122 (63.9%); PETHEMA Group 69 (36.1%)), aged between 60 and 77 years, were enrolled in the study. The patients had a median age of 67.8 years (range 60.1; 77.6). Among them, 134 (70.2%) were aged less than 70 years, while 57 (29.8%) were aged 70 years or older. 159 (83.3%) had a performance status (ECOG) of 1 or lower, while 24 (12.6%) had an ECOG of 2 or higher. 151 (79.1%) had de novo acute myeloid leukemia (AML), and 40 (20.9%) had secondary AML at diagnosis. The median white blood cell count at diagnosis was 26.5 x 10^9/L [5.99; 107]. The median percentage of blasts in the bone marrow at diagnosis was 76% [55; 88.5]. The frequency of FLT3-ITD mutation was 154 (80.6%), FLT3-TKD mutation was 44 (23%), and double mutations were observed in 7 (3.7%) patients. The median FLT3 ITD/wt ratio was 57% [22.5 - 78]. 119 (62.3%) were NPM1 muted. Regarding the intensive chemotherapy schedule in induction, 162 (84.8%) patients were treated with idarubicin-based regimens, 18 (9.4%) with daunorubicin-based regimens, and 8 (4.2%) with CPX-351. After induction, 150 (78.5%) of the patients achieved complete remission/complete remission with incomplete hematologic recovery (CR/CRi). Of those patients, 96 (64%) proceeded to intensive chemotherapy schedule in consolidation, with 83 (86.5%) of them receiving midostaurine. In contrast, 32 (21.3%) received non-intensive chemotherapy consolidation, with 27 (84.4%) receiving midostaurine. Additionally, 37 (24.7%) of the patients who achieved CR/CRi after induction underwent a hematopoietic-cell transplantation (HCT), with 33 (89.2%) undergoing allogeneic HCT and 4 (12.1%) undergoing autologous HCT. The median follow-up of the patients was 32 months. OS for the entire population was 23 months with a 2-years OS of 49.1% (95%CI: 41.5-56.7). Median EFS for the entire population was 11.1 months with a 2-years EFS of 37.1%. (95%CI: 29.6-44.5). For patients undergoing allogeneic HCT after achieving CR/CRi following induction, the median OS was not reached, with a 2-year OS of 69% (95% CI: 54.6-88.3). In contrast, for patients who achieved CR/CRi after induction but did not undergo allogeneic HCT, the OS was 32.6 months with a 2-year OS of 50.2% (95% CI: 48.4-68.2). CONCLUSIONS: This real-life study suggests that the addition of midostaurine to intensive chemotherapy regimens in elderly patients could result in acceptable CRc, OS and EFS. In order to establish the benefit of midostaurin, we will compare these results with a historical control cohort.

Phase I Trial Combining Venetoclax with G-CSF, Cladribine, Cytarabine, and Mitoxantrone (CLAG-M) for Patients with High-Grade Myeloid Neoplasms

Background: Venetoclax in combination with hypomethylating agents or low dose cytarabine is a safe and effective treatment for newly diagnosed older, unfit patients with acute myeloid anemia (AML). Combining venetoclax with high intensity combination chemotherapy may increase response rates and eventual chance for cure. Previous studies have combined venetoclax with fludarabine, cytarabine, idarubicin, and G-CSF (FLAG-ida) with 89% of patients achieving a composite complete remission (CR), of which 93% were MRD negative (DiNardo et. al, AJH, 2022). We report the preliminary results of a phase 1 study examining the addition of venetoclax to a high intensity induction regimen, CLAG-M (cladribine, cytarabine, mitoxantrone, G-CSF), for fit patients with newly diagnosed adverse risk or relapsed/refractory high-grade myeloid neoplasms. Methods: Subjects with AML or other high-grade myeloid neoplasm (≥10% myeloid blasts in peripheral blood or marrow) were eligible if they had (1) new diagnosis with adverse risk disease by European LeukemiaNet 2017 criteria or (2) relapsed/refractory (R/R) disease. Venetoclax was administered on day 1-14 with a 4-day ramp-up. All patients received standard neutropenic prophylaxis and venetoclax was dose-adjusted for concomitant use of CYP3A4 inhibitors. Dose level (DL) is defined in Table 1. Dose limiting toxicity (DLT) was defined as grade >4 non-heme toxicity, prolonged aplasia > 49 days, or grade >3 non-heme toxicity lasting >48h and resulting in a treatment delay beyond day 56. Two subjects were started at DL 2 and a Bayesian optimal interval (BOIN) design was used for subsequent doses, with a true targeted DLT rate of 30%. Re-induction was allowed for persistent disease after the first cycle; subjects with CR were eligible to receive up to three cycles of venetoclax plus cladribine and cytarabine (CLAG) at the same dose level as induction. Target accrual is 20 subjects. Events were defined as death, relapse, or failure to achieve CR/CRi on post induction evaluation. The study was approved by the local IRB, and subjects were consented in accordance with the Declaration of Helsinki. Results: At time of data cutoff (7/6/23), 15 subjects (5 with newly diagnosed and 10 with R/R AML) were enrolled. Median age was 65 (range: 30-74) years. Five were newly diagnosed and 10 were R/R. Two subjects were enrolled at DL 1, 6 at DL 2, 5 at DL 3, and 2 at DL 4 as per the BOIN design. Two DLTs occurred: grade 4 respiratory failure at DL 2 and grade 5 sepsis at DL 3. Treatment related grade 3 adverse events (AE) included febrile neutropenia (5 subjects), dyspnea (2), mucositis (1), hypoxia (1), delirium (1), syncope (1), tumor lysis syndrome (1), and sepsis (1). Non-treatment related grade 3 AEs included hypokalemia (1 patient) and pericarditis (1). 28-day mortality was 7%. Twelve subjects received 1 cycle and 3 subjects received 2. Overall response rate (CR + CRi) was 73%. Two subjects achieved CR without MRD, 1 CR with MRD, 3 CRi without MRD, 5 CRi with MRD, 1 MLFS without MRD, 2 had persistent disease, and 1 died before evaluation. Median overall survival from treatment start was 7.4 months (95% CI 4.2 months - not reached [NR]). Median follow up of survivors was 6 months. Median event free survival was 3.3 months (95% CI 1.2 months - NR). Four subjects (1 newly diagnosed, 3 R/R) went on to receive allogeneic hematopoietic cell transplant (HCT). The administration of venetoclax presented challenges for inpatient and outpatient dosing, as medication diaries showed subjects underdosing venetoclax when taking concomitant posaconazole due to the complexity of the dosing regimen. Discussion: Preliminary results show that venetoclax can be safely added to a high intensity regimen for fit, high-risk patients without adversely affecting standard therapies including subsequent allogeneic HCT. Grade 3 and higher toxicity was common. In this preliminary analysis, the overall response rate was 73%, which is comparable to that observed with other chemotherapy regimens with the addition of venetoclax. A protocol amendment is in process to simplify venetoclax dosing with concomitant CYP3A4 inhibitors. This study will ultimately determine the MTD for subsequent phase II study to evaluate the therapeutic efficacy of venetoclax and CLAG-M.

Five-Year Follow up Results of Phase II Clinical Trial Evaluating Ruxolitinib (RUX) and Azacitidine (AZA) Combination Therapy in Patients (pts) with Myelodysplastic Syndrome/Myeloproliferative Neoplasms (MDS/MPNs)

Introduction: MDS/MPN represent a distinct category of Philadelphia chromosome negative myeloid neoplasms that share pathological and morphological characteristics with both MDS and MPN. Very few prospective studies have been conducted in pts with MDS/MPN, with currently available data showing a median overall survival (OS) of less than two years in this challenging subset. We conducted a phase 2 clinical trial evaluating the combination of JAK-inhibitor RUX with demethylating agent AZA in pts with MDS/MPN. The interim analyses (Assi et al, AJH 2018) showed objective responses in 57% pts and a median duration of response (DOR) of 8 months (mos). Herein we report the long-term follow up of a larger cohort of pts treated on this prospective ongoing clinical trial. Methods: We conducted an open label single-arm phase 2 clinical trial (NCT01787487) at the MD Anderson Cancer Center, Houston, evaluating the use of RUX-AZA in newly diagnosed or previously treated MDS/ MPN. Adults aged ≥ 18 years with MDS/MPN categorized as Intermediate 1-2 or High-risk by Dynamic International Prognosis Scoring System (DIPSS) (Passamonti et al, Blood 2010) were included. Pts previously exposed to RUX and/or AZA were excluded. RUX 5-20mg twice daily (per RUX label depending upon platelet counts at initiation) was given in 28-day cycles, and AZA 25mg/m2 (SQ or IV) Days 1- 5 was added starting cycle 4. Cycles were repeated every 4-6 weeks depending on count recovery. Response assessment was carried out using the 2015 International Consortium Proposal of response MDS/MPN criteria (Savona M et al, Blood 2015) and best objective response was tabulated. Progression free survival (PFS) included treatment change because of disease progression or inadequate response, transformation to acute myeloid leukemia (AML) or death and calculated from the time of therapy initiation. Results: From May 2013 to August 2022, 52 pts with a median age of 68 years (range, 39-82 years) were treated on trial. The data cutoff was July 15, 2023 ( Table 1). 32 pts (61%) were males, and 9 (75%) pts were white. 50 pts (96%) had ECOG PS<2. Baseline median bone marrow (BM) blasts were 4.5% (range 0-19%), with 19 (37%) carrying JAK2 mutations, and 16 (31%) with an abnormal karyotype. Intermideate-2 DIPSS was the most prevalent risk category, seen in 23 (44%) pts. At least one cytopenia was present in 32 (61%) pts, and three (6%) pts had bicytopenia. 24 of 49 (49%) had palpable splenomegaly, and 3 pts had undergone prior splenectomy. After a median follow up of 60 months (mos) (95% CI: 50.0-71.3) from therapy initiation, 18 (35%) pts are alive, and 1 pt is on active trial therapy. Objective responses per the MDS/MPN international consensus response criteria were observed in 28 (54%) pts and included clinical improvement in 14 (50%) pts, partial marrow response in 12 (43%) pts, optimal marrow response and cytogenetic complete remission in 1 (4%) pt each. The DOR in the 28 pts with objective response to therapy (censored for death in pts who died without evidence of progression) was 35 mos (95% CI: NE-73.5). The median OS in this group of responders was 36.4 mos (95% CI: NE-77.2). Five of these pts died without confirmed disease progression at the time of death. For the full cohort the median PFS was 13.4 mos (95% CI: 7.9-18.8) and median OS was 31.8 mos (95% CI: 18.5-44.9 ) (Figure 1). Transformation to AML occurred in 12 (23%) pts, with the median time to transformation being 13.2 mos (range 2.9-71.3) and all of them have died at the time of last follow-up. 8 (15%) pts underwent allogenic stem cell transplant (ASCT) as subsequent therapy following treatment with AZA-RUX amongst whom 6 (75%) had shown objective responses prior to transplant. For the pts who underwent ASCT, median PFS from the initiation of AZA-RUX was 40.8 months and median OS was not reached. Conclusion: Long term follow up from this phase II prospective clinical trial demonstrates durable disease responses with the RUX-AZA combination in MDS/MPN, with the responses translating into improved survival outcomes.

Bone Marrow Cellularity at Diagnosis Predicts Outcomes of Patients with Myelodysplastic Syndrome with Allogeneic Hematopoietic Stem Cell Transplantation

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only known curative therapy for myelodysplastic syndromes (MDS). The decision of allo-HSCT depends on prognostic scoring models such as revised international prognostic scoring system (IPSS-R) which include cytogenetic abnormalities, cytopenia, and % of bone marrow (BM) blasts. Recently, the prognostic role of genomic profile in MDS patients is arising. However, it is hard to conduct the next generation sequencing to all MDS patients in the real-world. Therefore, we aimed to evaluate the prognostic impact of BM cellularity at diagnosis as a new clinical prognostic factor of MDS with allo-HSCT. A total of 122 patients with MDS patients who underwent allo-HSCT between July 2009 to August 2022 in single center were analyzed retrospectively. Overall survival (OS) was analyzed using the Kaplan-Meier survival curves according to the BM cellularity at diagnosis. The potential prognostic parameters were evaluated for OS by uni-and multivariate cox-regression analysis. All patients were eligible for allo-HSCT and median age of 61.5 years (range: 19-71). Median follow-up duration was 36.9 (IQR 17.6-72.4) months. The 3-year and 5-year OS were 60.1% (95% CI 51.6-69.0) and 52.5% (95% CI 42.8-61.3), respectively. IPSS-R risk group was classified as very low (6, 4.9%), low (15, 12.2%), intermediate (40, 32.8%), high (37, 30.3%) and very high (24, 19.7%). Seventeen patients (13.9%) had complex karyotype at diagnosis. Seventy-seven patients (63.1%) were treated with hypomethylating agents before allo-HSCT. Most patients (70, 57.4%) had less than 5% BM blasts at allo-HSCT. The majority (74, 60.7%) received reduced intensity conditioning. OS significantly differed between patients with hypercellular BM and normo- to hypocellular BM at diagnosis by the log-rank test ( P <0.001) (Figure 1). In multivariate analysis for OS, complex karyotype at diagnosis ( P<0.001; HR 6.00; 95% CI 3.08-11.66), age ( P=0.04; HR 1.03; 95% CI 1.00-1.06) and hypercellular BM at diagnosis ( P=0.005; HR 2.16; 95% CI 1.26-3.72) were associated with worse outcome (Table 1). In this study, we demonstrated the BM hypercellularity at diagnosis independently predicts the inferior outcome of MDS patients with allo-HSCT. Although further validation with a larger cohort would be needed, it could be helpful to decide the treatment strategy of transplantation eligible MDS patients.

Multi-Hit TP53 Mutations in Myeloid Neoplasms: Prognostic Impact of Morphologic Subtype Designation and Variant Allele Frequency

Background: Biallelic TP53 alterations result from either sequence variations or deletions involving TP53 and are not necessarily synonymous with “multi-hit TP53”. According to the 2022 international consensus classification (ICC) for myeloid neoplasms (MN), multi-hit TP53 signifies i) the presence of two or more distinct TP53 mutations, each with variant allele frequency (VAF) ≥10%, ii) a single TP53 mutation with VAF ≥50%, or iii) a single TP53 mutation with VAF ≥10% accompanied by a cytogenetically-apparent del(17p13.1), copy-neutral loss of heterozygosity (LOH) at the 17p TP53 locus, or, in the absence of LOH information, complex karyotype ( Arber et al. Blood 2022;140: 1200). The current study is focused on MN with multi-hit TP53 and examines the additional prognostic impact of morphologic subtype designation, bone marrow (BM) or peripheral blood (PB) blast percentage, TP53 VAF, and MN with diagnostic qualifiers (i.e., therapy-related, or secondary progressing from myelodysplastic/myeloproliferative/overlap syndromes/neoplasms). Methods : The current study was conducted under an institutional review board approved minimum risk protocol that allowed retrospective collection and analysis of data from Mayo Clinic patient records. Multi-hit TP53 was defined as per ICC criteria as outlined above ( Arber et al. Blood 2022;140: 1200). Morphologic subtype designations of MN were according to ICC criteria and assigned at the time of TP53 detection. NGS and cytogenetic information was available in all study patients. Survival analyses were calculated from time of TP53 mutation detection. Conventional statistical methods were applied using JMP Pro 16.0.0 software (SAS Institute, Cary, NC, USA). Results : Initial screening flagged 143 patients with biallelic TP53 abnormalities derived from formal laboratory reports of NGS data and cytogenetic studies; of these, 130 met ICC criteria for multi-hit TP53: pure erythroid leukemia (PEL; N=24), acute myeloid leukemia (AML)-not PEL (N=54), myelodysplastic syndromes (MDS; N=36), MDS/AML (N=11), and other MN (N=5). Of these 130 informative cases, 128 (98%) harbored complex/monosomal karyotype (CK/MK). Further analysis excluded patients with “other” MN (N=5) and those without CK/MK (N=2), the latter to mitigate the confounding effect of CK/MK on survival and the former because of small sample size and disease heterogeneity. Clinical and laboratory characteristics of the 124 study patients are outlined in table 1. Survival analysis stratified by ICC-defined MN subtypes revealed the prognostic relevance of morphological distinction between PEL vs “AML-not PEL” (p<0.01; HR 2.4) and PEL vs “ TP53-muated MDS/AML” (p=0.02; HR 2.3) while survival was similar between “AML-not PEL” and “ TP53-mutated MDS/AML” (p=0.9; Figure 1a). Survival in “ TP53-mutated MDS” was significantly longer, compared to PEL (p<0.01; HR 0.2), “ TP53-mutated AML-not PEL” (p=0.01; HR 0.5), and “ TP53-mutated MDS/AML” (p=0.07; HR 0.5), the latter with borderline significance (Figure 1a). Multivariable analysis confirmed the independent prognostic relevance of ICC subtype designation (p<0.01) and also revealed additional negative prognostic contribution from advanced age (p=0.02), male gender (p=0.02), MN, secondary (p<0.01), MN, therapy-related (p=0.03), and DNMT3A mutation (p=0.02). Significance was retained in all instances, with the exception of MN, therapy-related (p=0.15), when analysis was repeated after excluding PEL cases (Figure 1b). Conclusions : The current study confirms the prognostic validity of the ICC morphologic subtype designation and TP53 VAF classification threshold, in the context of multi-hit TP53. The study also highlights the prognostic distinction between PEL and “ TP53-mutated AML-not PEL” and the prognostic alignment between the latter and “ TP53-muated MDS/AML”, both of which displayed inferior survival, compared to “ TP53-mutated MDS”. The study also suggests additional prognostic contribution from secondary and therapy-related qualification while the observation regarding DNMT3A mutation requires validation with higher number of informative cases.

Safety, Pharmacodynamic, and Anti-Tumor Activity of SL-172154 As Monotherapy and in Combination with Azacitidine (AZA) in Relapsed/Refractory (R/R) Acute Myeloid Leukemia (AML) and Higher-Risk Myelodysplastic Syndromes/Neoplasms (HR-MDS) Patients (pts)

Background: CD47 blocking antibodies and SIRPα-Fc fusion proteins have demonstrated activity in combination with azacitidine (AZA) in previously untreated (UnTx) HR-MDS and AML pts. SL-172154 (SIRPα-Fc-CD40L), a hexameric, bi-functional fusion protein consisting of SIRPα domains linked to CD40L domains through an inert Fc linker demonstrated improved anti-tumor activity in comparison to naked CD47 blocking antibodies in pre-clinical studies [de Silva et al. Cancer Immunol Res 2020]. SL-172154, as a CD47 inhibitor, requires combination with AZA to enhance pro-phagocytic signals on leukemic stem cells/blasts thereby potentiating macrophage phagocytosis in AML/HR-MDS. Here we report the results from the Phase 1 Dose Escalation. cohorts for SL-172154 monotherapy [SL-mono] and for SL-172154 + AZA combination [SL-AZA] in pts with HR-MDS or AML. Methods: The objectives of the dose escalation cohorts include evaluation of safety, dose-limiting toxicity (DLT), recommended phase 2 dose, pharmacokinetic and pharmacodynamic effects and efficacy per IWG 2006 for MDS and ELN 2017 for AML. SL-172154 was administered IV weekly of a 28-day cycle as monotherapy or with AZA 75 mg/m 2 for 7 days per cycle. mTPI-2 was used for dose escalation with a DLT threshold of 20%. Pts ≥ 18 years old with R/R HR-MDS or R/R AML were eligible for dose escalation cohorts. In addition, UnTx pts with TP53 mutant (TP53m)-MDS were eligible for SL-AZA cohorts. Results: As of May 25, 2023, 37 pts were enrolled (19 in SL-mono; 18 in SL-AZA cohorts): 14 women; median age 70 years [range 44-81]. 27 pts had R/R AML (16 de novo; 11 secondary), 5 had R/R MDS, and 5 were UnTx TP53m-MDS. 23 (85%) of 27 R/R AML pts had adverse genetic abnormalities per 2017 ELN classification, including 9 (33%) with TP53m or del 17. The median number of prior lines of AML therapy was 2 [range 1-4]; 25 (93%) had received venetoclax (VEN) and 23 (85%) had received a hypomethylating agent (HMA). All 5 pts with R/R MDS had previously received HMA and 2 pts were previously treated with VEN. SL-172154 was dose escalated as monotherapy or with AZA as shown in Table 1. Infusion-related reactions (IRRs) were the most common SL-172154-related treatment-emergent AEs (TEAEs) reported in 13 (68%) and 8 (44%) pts in SL-mono and SL-AZA cohorts, respectively. IRRs (51 events) were Grade (G) 1 or 2 except for 3 G3 events (1 each at 6 mg/kg SL-mono and SL-AZA; 1 at 3 mg/kg SL-mono). Other SL-172154-related TEAEs observed in ≥ 3 pts were AST increased (4; 21%), ALT increased (3; 16%) and nausea (3; 17%) in SL-mono cohorts, and nausea (3; 17%) in SL-AZA cohorts. All events of AST/ALT increased were transient. Only one DLT (G3 IRR in the 6 mg/kg SL-AZA cohort) was reported, which required a dose reduction to 3 mg/kg. As of July 10, 2023, in SL-mono cohorts, 1 pt with R/R AML post 7+3, FLAG and VEN/AZA achieved Morphologic Leukemia-Free State (blast reduction from 19% to <5%) after 1 cycle of 6 mg/kg SL-172154 and proceeded to allogeneic hematopoietic cell transplant (allo-HCT) after cycle 2. In 12 evaluable pts with R/R AML receiving SL-AZA, although no objective responses (OR) were observed, relative reduction in bone marrow (BM) blasts from baseline was reported in 2/5 pts at 1 mg SL-AZA (-50%, -75%) and 5/7 pts at 3 mg SL-AZA (ranging from -35% to -90%). One pt underwent an allo-HCT. In 4 evaluable pts with UnTx TP53m-MDS, the ORs were 1 complete remission (CR), 1 marrow CR, and 2 stable disease. 2 of them proceeded to allo-HCT. SL-172154 induced elevations in serum IL-12p40, IP-10, IL-8, IL-10, MIP3α and MCP1: greater response at 3 mg/kg compared to 1 mg/kg while similar between 3 and 6 mg/kg. In the BM, SL-172154 induced a dose-dependent increase in phagocytic cells (CD11b, HLA-DR, CD16, CD36 and CD64) within mature myeloid immune cell compartments (CD45highCD34-). Reduction in leukemic blasts (CD45lowCD34+) was associated with an increase in mature myeloid and phagocytic cell phenotypes (3 mg/kg SL-AZA > 1 mg/kg SL-AZA). Conclusions: SL-172154 was well tolerated up to 3 mg/kg as monotherapy and in combination with AZA. Preliminary efficacy signals were detected in UnTx TP53m-MDS and R/R AML. A response to SL-172154 monotherapy, dose-dependent increases in serum cytokines and accumulation of mature myeloid cells in BM suggest a potential role for CD40 stimulation. Based on the safety, preliminary efficacy and pharmacodynamic activity, 3 mg/kg SL-172154+ AZA is being evaluated in treatment naïve pts with TP53m AML and HR-MDS.

Safety and Tolerability of AZD0466 As Monotherapy for Patients with Advanced Hematologic Malignancies. Results from a Phase I/II Trial

Background: Overexpressionof B-cell lymphoma 2 (BCL-2)-related pro-survival proteins allows cancer cells to evade apoptosis, and targeting these proteins has become important in anticancer therapy. BCL-2 inhibition by venetoclax (VEN) is beneficial in many patients (pts) with acute myeloid leukemia (AML), but resistance is common due to upregulation of other pro-survival proteins such as BCL-extra-large (BCL-xL) and myeloid cell leukemia-1. Dual inhibition of BCL-2/xL has potential for broader activity than agents such as VEN. However, BCL-xL is critical for platelet survival and inhibition can cause dose-limiting thrombocytopenia (Konopleva and Letai. Blood 2018). To broaden therapeutic activity, we developed AZD0466, a drug-dendrimer conjugate in which the BCL-2/xL dual inhibitor AZD4320 is covalently conjugated to a pegylated poly-L-lysine dendrimer and gradually released by hydrolysis. Preclinically, AZD4320 showed greater inhibition of tumor growth than VEN and navitoclax in VEN-resistant xenograft models (Balachander et al. Clin Cancer Res 2020). A first-in-human study (NCT04214093) in pts with advanced solid tumors indicated that AZD0466 is well tolerated with no reported dose-limiting toxicities (DLTs). We present preliminary data from NIMBLE, a Phase I/II trial of AZD0466 in pts with advanced hematologic malignancies (NCT04865419). Methods: Module 1, Part A of NIMBLE is a dose-escalation study of AZD0466 monotherapy investigating its preliminary safety and tolerability, pharmacokinetics (PK), and preliminary efficacy. Dose escalation and de-escalation of AZD0466 follow a modified toxicity probability interval (mTPI-2) design. Eligible pts are ≥18 years old with relapsed/refractory (R/R) AML, acute lymphoblastic leukemia (ALL), or high-risk myelodysplastic syndrome. AZD0466 is administered IV with a dose ramp-up on Days (D) 1, 4, and 8, reaching the target dose on D8 of Cycle 1, with weekly administrations thereafter. Cycle duration is 35 days for Cycle 1 and 28 days for subsequent cycles. Pts remain on study until disease progression, unacceptable toxicity, or withdrawal of consent. Resul ts: As of May 31, 2023, 31 pts (n=25 AML, n=6 ALL; median age 69 years) had received ≥1 dose of AZD0466 at target doses of 300 mg (n=4), 600 mg (n=4), 1200 mg (n=7), 2400 mg (n=5), 3600 mg (n=9), and 5400 mg (n=2). Median treatment duration was 4.1 weeks (range 0.1−29.6). Twenty-two pts (71%) had prior VEN and 11 (35%) had prior hematopoietic stem cell transplantation. Treatment-emergent adverse events (TEAEs) of any grade reported in >10% of pts are displayed in Table 1. One Grade 5 intracranial hemorrhage unrelated to AZD0466 was reported. Four pts experienced a serious adverse event (SAE) possibly related to AZD0466 (alanine aminotransferase [ALT] increase, pneumonia, troponin T increase, and uncoded SAE). Grade ≥3 TEAEs possibly related to AZD0466, reported in 12 pts (39%), included febrile neutropenia (n=3), platelet count decrease (n=2), diarrhea, thrombocytopenia, ALT increase, blood creatinine phosphokinase increase, pneumonia, gamma-glutamyltransferase increase, lipase increased, and troponin T increase (n=1, each). DLTs of asymptomatic troponin increase were observed in two pts (3600 mg [n=1], 5400 mg [n=1]). Evaluation of maximum concentration and area under the concentration-time curve revealed dose-proportional exposure to AZD4320. Released AZD4320 represented 1-5% of total AZD4320 and had a longer half-life than total AZD4320. Preclinical modeling and PK data predicted that 5400 mg may be required to drive efficacy. As a surrogate of BCL-xL on-target activity, transient aggravation of thrombocytopenia post-dosing was observed across all dose cohorts followed by platelet recovery prior to the next dose. Platelet reduction was in line with preclinical models showing a substantial reduction at lower doses with a ceiling effect at higher doses. As of July 6, 2023, no complete responses have been observed. However, preliminary bone marrow (BM) blast reductions of >50% were observed in four pts (1200 mg [n=1], 2400 mg [n=2], 3600 mg [n=1]), indicating antileukemia activity of AZD0466. Conclusions: Given the observed DLTs of asymptomatic increase in troponin levels, and the lack of significant clinical benefit, the study was terminated.

Impact of Type of Hypomethylating Agent (HMA) Used on Outcomes of Patients (Pts) with Higher-Risk Myelodysplastic Syndromes/Neoplasms (HR-MDS) - a Large, Multicenter, Retrospective Analysis

JPB and TK Co-first; RK and AMZ are Co-senior authors Introduction HMAs remain the mainstay for frontline treatment of HR-MDS. Azacitidine (AZA) and decitabine (DEC; including oral cedazuridine/decitabine) are the only FDA-approved HMAs. However, AZA and DEC have not been compared directly in randomized trials. In this study, we aimed to assess the clinical outcomes of pts with HR-MDS treated with different HMA regimens, focusing on overall survival (OS) and treatment responses. Methods The VALIDATE database includes pts with HR-MDS treated with HMA-based therapies in the frontline setting from 14 specialized MDS centers. HR-MDS pts treated with HMA-based therapies in the frontline setting were included. HR-MDS was defined as having an IPSS ≥1.5 or IPSS-R >3.5 (n=213 pts excluded). Pts were excluded from the survival analysis if age at diagnosis was <18 years (n=1), bone marrow (BM) blasts ≥20% or unknown at HMA initiation (n=61), or if survival status, follow-up time, date of HMA initiation, or HMA type was unknown (n=28). To be included in the analysis of response based on IWG 2023 criteria (Zeidan A et al, Blood 2023), pts had to have a BM response assessment within 180 days after HMA initiation to allow determination of response (n=290 pts excluded). Time to event analyses were estimated using the Kaplan-Meier method and treatment groups (AZA vs DEC monotherapy) were compared by log-rank test and assessed from the time of HMA initiation. Multivariable Cox regression models were performed among pts treated with AZA and DEC monotherapy to identify predictors of response and OS. This study was supported by an independent research grant from AbbVie. Results 1,223 pts were screened of whom 919 were included in the survival analysis. Median age was 68 years (Range [R]: 19-95) with 66% males. Our cohort was enriched for pts with adverse genetic features including complex karyotype (38%) and TP53 mutations (36%). Overall, 76% of pts were treated with HMA monotherapy (56% AZA, 20% DEC) and 24% received HMA-based combination therapy (HMA/VEN: 15%, other HMA combinations: 9%). 38.2% underwent allogeneic hematopoietic cell transplant (allo-HCT). The median HMA duration was 5 cycles (R: 1 - 94). Due to the small number and the heterogeneity of pts receiving HMA combinations, as well the multiple partner drugs used in these pts ( Table), we compared OS and responses only between pts treated with AZA (n = 512 pts) or DEC monotherapy (n = 186 pts). In unadjusted analyses, median OS differed by treatment type (p = 0.002) and was 19.8 months (mo) with AZA (95% CI: 17.0 - 23.1 mo) and 14.3 mo with DEC (95% CI: 11.2 - 18.5 mo). Among 629 pts evaluable for response, rates of complete remission (CR) and overall response (ORR) were 14.8% and 48.6% for AZA monotherapy and 5.6% and 50.4% for DEC monotherapy, respectively. In a Cox multivariable regression model ( Figure) adjusted for age, sex, TP53 mutation status, complex karyotype, IPSS-M category (compared to very high risk), and receipt of allo-HCT, there was no difference in OS when comparing AZA and DEC monotherapy (Hazard ratio [HR]: 0.95, 95% CI: 0.72 - 1.26; p = 0.740). Variables associated with adverse OS were male sex (HR: 1.52; 95% CI: 1.16 - 2.00; p = 0.002) and presence of TP53 mutation (HR: 1.50, 95% CI: 1.05 - 2.14; p = 0.027). Conversely, receipt of allo-HCT (HR: 0.26, 95% CI: 0.19 - 0.37; p<0.001) and IPSS-M moderate-high (HR: 0.57, 95% CI: 0.37 - 0.87; p=0.009) and moderate-low (HR: 0.58, 95% CI: 0.36 - 0.95; p = 0.031; both compared to IPSS-M very high risk) were associated with improved OS. Similarly, there were no statistically significant differences in ORR between AZA and DEC (OR: 1.05, 95% CI: 0.66 - 1.68; p = 0.832) in a Cox multivariable regression model adjusted for age, sex, TP53 mutation, complex karyotype, IPSS-M category, and treatment type. Conclusions: Among pts included in the real-world VALIDATE database, there were no significant difference in OS or ORR (IWG 2023) between AZA- and DEC-treated pts in adjusted analyses. Other factors (e.g., TP53 mutations, complex karyotype) are substantially more relevant to outcomes than the specific HMA used. The small number of pts and heterogeneity of partner drugs in HMA-based combinations precluded robust analyses or conclusions regarding differences in efficacy. Additional analyses evaluating the impact of combinations and molecular subtypes on response and survival will be presented during the meeting as more pts are added to the database.

Low Disease Burden Pre-CAR-T Cell Therapy for Children and AYA with B-ALL Is Associated with an Impaired Outcome When Obtained through Intensification of the Bridging Therapy

Rationale. The role of bridging therapy, i.e., treatment between apheresis and lymphodepletion (LD) preceding tisagenlecleucel (tisa-cel) infusion for relapsed/refractory B cell acute lymphoblastic leukemia (R/R BCP-ALL) is still unclear. We report a retrospective study on our experience on 81 children, adolescents, and young adults with R/R BCP-ALL who underwent apheresis with an intent to receive tisa-cel infusion, between March 2016 and October 2020. Methods. Bridging therapy intensity was classified into two intensity groups. A high intensity bridging (HIB) regimen, used in 23 patients (pts), was defined as the use of more than 4 intravenous anti-leukemic drugs per week and/or the need of secondary intensification of bridging therapy, the latter being the case in 19 pts (83%). A low-intensity bridging (LIB), in general a combination of vincristine, steroids +/-asparaginase, was used in 58 pts. The aim of the study was to evaluate the impact of HIB versus LIB on tisa-cel efficacy. Factors associated with a failure to infuse tisa-cel were also explored. Results. Patients had a median age of 15 years (range: 1-19). Tisa-cel indication was a primary refractory disease for 5 pts (6%), a first post-allo-HSCT relapse for 47 pts (58%) and a second relapse or more for 29 pts (36%). Patients who received a HIB therapy were more likely to have high-risk genetic features (39% vs 12%, p= .01) and/or a primary refractory disease (17% vs 2%, p=.02). Grade 3+ non-infectious and infectious adverse events during bridging therapy were also more likely in this group, albeit in a non-statistically significant way (35% vs 16 %, p= .07 and 52% vs 34 %, p= .21; respectively). Nine patients (11%) were not infused (disease progression: 3 pts; toxicity: 5 pts (sinusoidal obstruction syndrome: 1 pt, encephalopathy: 2 pts, fungal infection; 1 pt, hemoptysis: 1 pt); and CD19 negativation: 1 pt). A high pre-apheresis tumor burden ( p=.01), the occurrence ( p=.01), and the number of grade ≥ 3 adverse events during bridging period ( p<.01) were associated with a higher risk of tisa-cel non-infusion, while HIB was not ( p=.73). The 72 remaining patients (89%) finally received tisa-cel infusion, 52 (64%) after a LIB therapy and 20 (25%) after a HIB therapy. Despite a comparable BM disease burden at apheresis between the LIB and the HIB groups, patients presenting a high disease burden prior to LD (bone marrow (BM) blasts > 50%) were more frequent in the HIB group ( p< .01). When compared to patients who received a LIB, patients treated with a HIB had a worst, albeit non-statistically different cumulative incidence of relapse (CIR) (SHR 1.94, 95% CI [0.95-3.91], p=.07), an inferior 18-month event-free survival (EFS) (26.9% vs 54.1%; p=.02) and a lower 18-month overall survival (OS) (50.9% vs 88.8%; p<.01). Considering a threshold at 50% BM blasts, patients who presented a high disease burden prior to LD had poorer early response rates (D28 minimal residual disease (MRD) negativity: 85% vs 69%, p< .01), a higher, albeit non-statistically different CIR (SHR 2.27, 95% CI [0.98-5.27], p=.06), an inferior 18-month EFS (26.9% vs 51.3%; p=.02) and a lower 18-month OS (22.6% vs 91.5%; p<.01). We then asked if the bridge intensity, aimed at achieving a low disease burden before LD, could influence patient outcomes. Median percentages of BM blast infiltration were similar after LIB (0%) and HIB therapy (1%, p=.87) in patients with ≤50% BM blast at LD. In these patients, a prior HIB regimen was associated with a higher rate of detectable MRD at D28 (40% vs 8%, p=.02), an inferior 18-month EFS (18.0% vs 57.5%; p=.02) and a higher, albeit non-statistically different CIR (SHR 2.05, 95%CI [0.87-4.88], p=.1). The 18-month OS did not differ between groups (74.0% vs 94.6%; p=.15), suggesting that these patients may respond to further lines of treatment after relapse. The outcome of patients presenting a low disease burden at LD after a HIB regimen was thus similar to those with a high disease burden at LD. Conclusion. This study confirms the prognostic value of a high disease burden pre-LD but also reveals that a low disease burden prior tisa-cel therapy is associated with an impaired outcome when obtained through intensification of the bridging therapy. Additional post-tisa-cel therapies should be considered in this group of patients.

Impact of Salvage and Bridging Therapy in Adult Patients with Refractory B-Cell Acute Lymphoblastic Leukemia (B-ALL) Referred for Anti-CD19 CAR T-Cell Therapy: An Intention to Treat Analysis

Background: Prognosis of relapsed/refractory (R/R) B-cell acute lymphoblastic leukemia (ALL) is poor, particularly after allogeneic hematopoietic cell transplantation (alloHCT). In this setting, several CAR T-cell products targeting CD19 (CART19) have been able to achieve complete response (CR) rates of 70 to 90%. However, a significant proportion of these patients still relapse. Several studies focus on the effect of the CART19 product on efficacy, however, information on how salvage therapy (ST) and bridging therapy (BT) can modulate the outcomes of CART19 therapy is scarce. Methods: We report the outcomes of all adult patients with R/R B-ALL intended for treatment (aphaeresis performed) with available CART19 products in a single institution from Jul 2017 to Jul 2023. This includes patients intended to receive the academic varnimcabtagene autoleucel (var-cel) in the CART19-BE-01 trial and the consecutive compassionate use and hospital exemption programs, as well as with commercially available tisagenleuceucel (tisa-cel). Lymphodepletion was performed with fludarabine (90 or 120 mg/m 2) and cyclophosphamide (900 or 1000 mg/m 2), followed by the infusion var-cel or tisa-cel, respectively. We analyzed all ST (treatment administered after last relapse before CART19-apheresis) and BT (treatment administered after apheresis and before CART19 infusion) performed, and analyzed the impact of BT on PFS and OS after apheresis. Results: A total of 74 adult B-ALL patients underwent leukapheresis and CART19 (var-cel/tisa-cel) production. At screening patients had a median age of 34 years (19-78), 45% were females, with a median of 3 prior lines of therapy (2-7) including inotuzumab (69%), blinatumomab (27%) and allo-HCT (84%). After last relapse, 92% of patients received ST with a median of 1 (0-3) treatment line, including inotuzumab (48%), high-dose chemotherapy (28%), low-dose chemotherapy (21%), TKIs (12%), blinatumomab (9%), radiotherapy (7%) and DLIs (4%). The overall response to ST prior apheresis was MRD-negative CR (28%), MRD-positive CR (22%), partial response (4%) and progressive disease (PD) in 46%. After apheresis was secured, BT was performed in 76% of patients with a median of 1 (0-2) bridging line, including low-dose chemotherapy (44%), intrathecal therapy (23%), high-dose chemotherapy (15%), TKIs (15%), inotuzumab (14%), radiotherapy (3.5%), blinatumomab (1.8%) and DLIs (1.8%). BT was not administered in 24% (8/74) of patients mainly due to morphological remission at screening (83%) and frailty (11%). The overall response to BT prior CART19 infusion was MRD-negative CR (31%), MRD-positive CR (12%), partial response (7%) and progressive disease (47%), with a 3% treatment related mortality (TRM) events attributed to BT. Inotuzumab administration occurred either as ST or BT to CART19 in 86% of patients (the remaining 14% was previouly given as bridge to allo-HCT), and was able to achieve MRD-negative CR in 42% of patients. The overall control of the disease after BT either worsened (51%), remained similar (14%), or improved (35%). After this, 12% (n=9) of CART19 treatments were not performed due to progressive disease (33%), TRM (22%), therapeutic alternative (22%), and production failure (11%). Infused patients achieved an MRD-negative CRR of 90% with a median PFS of 11,2 months. There were no differences in OS between patients with ≥5% or <5% bone marrow (BM) blast counts either before apheresis (ITT, figure 1) or after BT prior to CART19 infusion. However, despite the lack of apparent impact of this disease burden categorization on the OS at both timepoints, a statistically significant difference was observed after categorizing patients regarding the response to BT (figure 2), with a median PFS of 6.6 vs 18.6 months if the overall disease status of the patients either worsened or remained stable/improved after BT (p=0.0002). Conclusions: To the best of our knowledge this could be one of the first studies focusing on the impact of ST and BT in adult patients with R/R B-ALL referred for CART19 therapy. These results suggest that the potentially synergistic impact of bridging therapy may be mediated more by the modulation exerted by BT on the proliferative kinetics of the disease rather than on the absolute tumour burden of the patient. More detailed results on the independent impact of each type of BT will be provided in the final presentation.

Results of a Phase 1/2 Study of Lower Dose CPX-351 for Patients with Int-2 or High Risk IPSS Myelodysplastic Syndromes and Chronic Myelomonocytic Leukemia after Failure to Hypomethylating Agents

INTRODUCTION: Hypomethylating agents (HMAs) are the standard of care for patients (pts) with higher-risk myelodysplastic syndromes (HR-MDS) and chronic myelomonocytic leukemia (CMML). HMA failure (HMA-F) is associated with poor outcomes and median survival of 4 to 6 months. CPX-351 is a drug liposomal encapsulation of cytarabine (araC) and daunorubicin (dauno) at a synergistic 5:1 molar ratio approved for the treatment of pts with therapy-related acute myeloid leukemia (AML) or AML with MDS-related changes. Here we present the results of a phase I-II study of lower dose CPX-351 for HR-MDS and CMML after HMA-F. METHODS: We conducted a phase I/II clinical trial of CPX-351 for pts with MDS or CMML after HMA-F with Int-2 or High risk by IPSS, or Int-1 with >10% bone marrow (BM) blasts. The study included an initial phase I dose-escalation portion, following a 3+3 design, followed by a phase II dose expansion portion. Dose escalation included 4 dose levels of CPX-351: 10 units/m 2 (dauno 4.4mg/m 2 and araC 10mg/m 2), 25 units/m 2 (dauno 11mg/m 2 and araC 25mg/m 2), 50 units/m 2 (dauno 22mg/m 2 and araC 50mg/m 2) and 75 units/m 2 (dauni 33mg/m 2 and araC 75mg/m 2). Therapy was administered intravenously on days 1, 3 and 5 of 28-day cycles during induction and on days 1 and 3 of re-induction or consolidation. Re-induction was allowed in pts not achieving response after induction. The primary end point was to evaluate safety and determine the maximum tolerated dose of CPX-351. Responses were evaluated following 2006 IWG criteria. The Kaplan-Meir product-limit method was used to estimate median survival. RESULTS: Between June 2019 and September 2022, 23 pts have been treated: 15 in the phase I portion, and 8 in the phase II. A total of 17 pts had MDS, and 6 had CMML. Four (17%) pts had intermediate-1 risk IPSS and >10% blasts, 15 (65%) had intermediate-2 and 4 (17%) had high-risk IPSS. Median number of prior therapies was 1 (range 1-4) including 4 (17%) pts with failure to prior venetoclax therapy and 2 (9%) with prior HSCT. In the phase I portion, 3 pts received CPX-351 at dose level 1, 3 at dose level 2, 3 at dose level 3 and six at dose level 4. One pt treated at dose level 4 developed grade 2 congestive heart failure with grade 2 reduction in left-ventricular ejection fraction (LVEF). No additional DLTs were detected during the 28-day DLT evaluation window. The initial two pts treated at the P2RD of 75units/m 2 during phase II experienced cardiac complications: grade 3 congestive heart failure without reduction in LVEF and grade 3 right-sided heart failure, respectively. Study was amended to continue further treatment in phase II at dose level 3 (50units/m 2). Most common adverse events (AEs) were lower extremity edema (n=12, 52%), febrile neutropenia (n=11, 48%), dyspnea (n=10, 44%), mucositis (n=10, 44%), constipation (n=9, 39%), diarrhea (n=9, 39%), and generalized muscle weakness (n=9, 39%). Most common grade 3-4 AEs were febrile neutropenia (n=11, 48%), neutropenia (n=5, 22%), lung infection (n=4, 17%), diarrhea (n=4, 17%) and thrombocytopenia (n=3, 13%). The 4-week and 8-week cumulative incidences of mortality were 0% and 4%, respectively. Median number of days to cycle 2 was 47 days (range 30-83). Dose reductions of CPX-351 occurred in 4 (17%) pts. Median number of cycles was 3 (range 1-9). Median cycles to best response was 1 (range 1-3). Among pts with HR-MDS, the ORR was 71% (n=12): CR in 1 (6%), mCR with HI in 1 (6%) and mCR in 11 (59%) pts (Fig 1A). Among pts with BM blast reduction to <5% by the end of cycle 1, 11 (65%) and 9 (53%) had ANC recovery to >0.5x10 9/L and >1x10 9/L, respectively, and 8 (47%) and 6 (35%) had platelet recovery to >50x10 9/L and >100x10 9/L, respectively. Based on 2023 IWG response criteria, the ORR was 59% (n=10) including CR in 1 (6%), CR bilineage (CRbi) in 4 (24%) and CR unilineage in 5 (29%). Of 11 (65%) pts with baseline cytogenetic abnormalities, 2 (18%) and 2 (18%) achieved complete and partial cytogenetic responses, respectively. Three (13%) pts underwent HSCT at time of best response. Among pts with CMML (n=6), only one (17%) achieved response. Median response duration was 4.5 months (95% CI 0.7-8.2 months) and the median OS was 12.6 months (95% CI 3.6-21.7 months) (Fig 1B). CONCLUSIONS: Lower doses of CPX-351 in HR-MDS after HMA-F can induce clinically meaningful responses and be used as a bridge to allogeneic stem-cell transplant. Therapy is associated with median OS of 12.6 months in this population with expected survival of 4-6 months.

Somatic and Germline HLA Determinants of Immune Surveillance and Escape in Myelodysplastic Syndromes

While the contribution of canonical myeloid genetic drivers to myelodysplastic syndromes/neoplasms (MDS) pathobiology is well established, other genetic events operative under special circumstances, and by itself not constituting intrinsic drivers, may affect the disease course. These include genetic defects conveying resistance to specific therapies, clonally restricted DNA repair alterations favoring the accumulation of additional mutations, and/or lesions facilitating the resistance to inflammation or immune escape. Here, we focus on this latter aspect as it may shed light on the pressure exerted by immune tumor surveillance or acquired resistance to cancer immune therapies, a field that has lagged in myeloid neoplasia as compared to the dramatic changes obtained in solid tumors. We hypothesize that during MDS ontogeny and leukemia evolution a spectrum of immunological mechanisms may shape the oncogenic landscape and facilitate the emergence of clones with diverse immunogenic potential as targets, leading to selective immunotherapeutic liabilities. We accrued a clinically and molecularly well annotated cohort of 101 MDS patients at various stages of the disease spectrum [N=51 low risk (LR)-MDS, N=32 high risk (HR)-MDS, N=18 secondary AML-sAML)]. This cohort included specific clinical sub-entities wherein the contribution of immune forces appears to be a prominent feature i.e., hypocellular MDS (hMDS, N=9) or MDS with co-occurring T large granular lymphocytosis (LGL) (N=5). By combining human leukocyte antigen (HLA) and T cell receptor (TCR) immunosequencing at disease onset, we leveraged our bioinformatics expertise to impute HLA mutations and losses, HLA evolutionary divergence (HED), and characteristics of T cell repertoires. The median age of the whole cohort was 71.2 years (IQR: 63.4-77.7), and median bone marrow (BM) blast count was 3% (1-11%). Cytogenetics revealed normal karyotype in 41 patients (40%), while complex karyotype was observed in 21 (20%) and partial or complete loss of chromosome 7 in 10 cases (10%). Comparisons with a cohort of 784 ethnicity-matched healthy individuals (HC), showed no differences in terms of locus-specific HED configurations. However, when HED scores were analyzed across the disease spectrum, we observed a heterogeneous distribution in MDS patients for the HLA-C locus, with higher values in hypoplastic and LGL-associated MDS as compared to HR diseases (p= 0.0068) and sAML (p= 0.0409). The screening for immune evasion highlighted the presence of both somatic mutations and losses in the HLA region in 14 patients, with both type of alterations found in 1 case. Of those, 6 patients (20% LGL-MDS, 5% LR-MDS and 17% sAML) harbored HLA mutations (in HLA-A, B, C and DPB1 loci), whereas HLA losses were observed in 9 patients (22% hMDS, 5% LR-MDS, 10% HR MDS, 11% sAML) almost exclusively in class II alleles (chiefly DPB1, DRB1 and DPA1). When exploring the degree of immunocompetence by TCR immunosequencing as a proxy, we found a higher degree of diversity (imputed via inverse Simpson index) as compared to healthy controls (p=1.5e-07), possibly in relationship to diverse T cell responses across the disease spectrum. However, no difference between MDS sub-entities was found. Of interest is that the maximal productive frequency (an indicator of the degree of T cell expansion) was higher in patients with hMDS and LGL-associated MDS as compared to HR-MDS (p=0.0079). Finally, inter-sample comparisons of shared clonotypes showed a high degree of overlap with regards to TCR repertoires among MDS as compared to healthy subjects. Our data emphasize the adaptive immune-molecular network underpinning MDS development. HLA constellations and their interactions with TCR repertoires may create selective immune pressure. Somatic HLA aberrations may serve as a marker of ongoing immune pressure and correspond to oligoclonal T-cell expansions and variations in TCR diversity. While virtually no HLA mutations have been previously found in AML/MDS, we show a relatively higher frequency of HLA aberrations in hMDS and MDS-LGL, immunologically stressed scenarios reminiscent of aplastic anemia and post-allogenic transplant AML/MDS cases, whereby respectively 36% 1 and 38% 2 of patients harbored HLA lesions as a mean of escape from cytotoxic lymphocytes or allogeneic Graft-vs-Leukemia reactions.

Results of a Phase II Study of Cladribine, Low Dose Cytarabine and Venetoclax, Alternating with Azacitidine and Venetoclax in Patients with Higher Risk Chronic Myelomonocytic Leukemia or Myelodysplastic Syndromes

INTRODUCTION: Clinical responses to hypomethylating agents (HMAs) in patients (pts) with higher-risk myelodysplastic syndromes (HR-MDS) and myeloproliferative chronic myelomonocytic leukemia (CMML) remain short-lived, with high risk of transformation to acute myeloid leukemia (AML). Prior data suggests use of the purine analogue cladribine can induce monocyte apoptosis and is active in AML, particularly in combination with low dose cytarabine (LDAC) and venetoclax. We aimed to evaluate the safety and activity of cladribine, LDAC and venetoclax in HR-MDS and CMML. METHODS: We designed a phase II basket clinical trial of cladribine combined with LDAC and venetoclax alternating with azacitidine and venetoclax for pts with HR-MDS or CMML. Pts were divided into 4 cohorts: cohort A: MDS with int-1 to high risk by IPSS and >5% blasts with prior HMA-failure (HMA-F); cohort B: CMML-1/CMML-2 with prior HMA-F; cohort C: previously untreated MDS with Int-2 or High risk by IPSS and >10% bone marrow (BM) blasts; cohort D: previously untreated CMML-2 or CMML-1 with at least one high-risk feature (extramedullary disease, splenomegaly of >5cm below costal margin, platelets <100x10 9/L, Hgb level <10g/dL, WBC >13x10 9/L, clonal cytogenetic abnormality [other than monosomy Y]). HMA-F was defined as no response after 6 cycles of therapy with HMA or relapse/progression after any number of cycles. Induction consisted of cladribine 5mg/m 2 daily IV on days 1-3, cytarabine 20mg s.c bid days 1-5 and venetoclax 400mg daily days 1-5. Re-induction was allowed in pts not achieving response after induction. Consolidations consisted of azacitidine 75mg/m 2 daily days 1-7 with venetoclax 400mg daily days 1-7 alternating with cladribine 5mg/m 2 days 1-3, cytarabine 20mg s.c bid days 1-5 and venetoclax 400mg days 1-5 every two 28-day cycles. The primary end point was to evaluate safety, efficacy, and tolerability of the combination. Responses were evaluated following 2006 IWG criteria. The Kaplan-Meir product-limit method was used to estimate median survival. RESULTS: Between October 2022 and June 2023, 20 pts have been treated: 10, 5, 3 and 2 in cohorts A, B, C and D, respectively. Thirteen pts had MDS, and 7 had CMML. Median age was 74 years (range 52-83). Among MDS pts, 8 (62%) had int-2 and 5 (38%) had high risk by IPSS. By the Molecular IPSS, 10 (79%) had very high, 1 (8%) had high, 1 (8%) had moderate high and 1 (8%) had low risk. Among CMML pts, 4 (57%) had high, 2 (29%) had intermediate-2 and 1 (14%) had intermediate-1 risk by the CPSS Molecular score. Among HMA-F cohorts, median number of prior therapies was 1 (range 1-4), median cycles of prior HMA was 6 (range 2-63) and 4 (27%) pts had failure to prior venetoclax therapy. Most common adverse events (AEs) were hypoalbuminemia (40%), nausea (40%), sinus bradycardia (40%), alanine aminotransferase increase (35%), constipation (35%), lower extremity edema (35%), fatigue (35%) and hypocalcemia (35%) Most common grade 3-4 AEs were leukopenia (20%) and febrile neutropenia (15%). The 4-week and 8-week cumulative incidences of mortality were 0%. Median number of cycles was 2 (range 1-6). Median cycles to best response was 1 (range 1-3). Among HMA naïve pts (cohorts C and D), the ORR was 80% (n=4): CR in 2 (40%), mCR in 2 (40%). Based on 2023 IWG response criteria, responses in cohort C (n=3) included: CR in 2 (67%) and CR bilineage (CRbi) in 1 (33%). Based on 2015 IWG MDS/MPN criteria, responses in cohort D (n=2) included optimal marrow response and clinical benefit in 1 (50%) and 1 (50%) pts, respectively. Among HMA-F pts (cohorts A and B) the ORR was 20%: 1 CR (10%) and 1 (10%) mCR with HI. Median follow up is 5.3 months (95% CI 2.4-81). Median EFS is 2.1 months in cohorts A and B and not reached in C and D (Fig 1A). Median OS has not been reached for all cohorts (Fig 1B). At the time of data cut-off, all pts in cohorts C and D discontinued study to proceed with hematopoietic stem-cell transplantation (HSCT). Among cohorts A and B, 2 pts (13%) discontinued study to proceed with HSCT, 5 (33%) due to transformation to AML, 2 (13%) due to no response, 2 (13%) due to physician choice, 1 (7%) due to patient choice and 2 (13%) pts remain on study. CONCLUSIONS: Preliminary data suggests the use of cladribine, LDAC and venetoclax can be safely administered in pts with very high risk MDS and CMML with early promising results in pts with de novo disease.

The Efficacy and Prognosis of Advanced Myelodysplastic Syndrome in Children

Pediatric myelodysplastic syndrome (MDS) is a clonal hematopoietic stem cell neoplasm with an annual incidence of 1-4 cases/million. The low incidence of advanced MDS in children and poor prognosis attract our attention. However, there are limited reports on the scheme and efficacy of pediatric advanced MDS. In this study, we retrospectively analyzed the efficacy of chemotherapy in pediatric advanced MDS and assessed the prognosis of patients. According to 2016 World Health Organization (WHO) classification of haematolymphoid tumours, 30 newly diagnosed pediatric advanced MDS patients during Dec 2007 to Apr 2022 from our single center were selected as the research object. The clinical data and prognosis were analyzed. The median age of patients is 8 years (range: 1-15years). The median bone marrow blast is 15.75% (range: 5-29.5%), while the median peripheral blood blast is 4% (range: 0-29%). Cytogenetic analysis was available for 29 patients, 69% (20/29) cases had abnormal karyotype. Monosomy 7 (37.9%) was the most common cytogenetic abnormality. Out of the total, 7 patients were treated with CAG/HAG regimen (C: aclarubicin 6mg d1-14, H: homoharringtonine 1mg/m 2d1-14, A: cytarabine 10mg/m 2 q12h d1-14, and G: Granulocyte Colony-stimulating Factor 200mg/m 2 d1-14 ), 8 patients with decitabine (20mg/m 2 d1-5), 11 patients with decitabine and CAG/HAG, 4 patients with AML-like chemotherapy (Daunorubicin+cytarabine+etoposide ) or other. The evaluation of response of therapy was available in 25 patients, 5 cases (20%) in partial remission (PR), 11 cases (44%) in complete remission (CR), 9 cases in(36%)non-remission (NR). The total response rate of chemotherapy is 60%, with CAG/HAG therapy 33%, decitabine therapy 43%, combined CAG/HAG and decitabine therapy 78%, AML-like chemotherapy 100%. The total complete remission rate of chemotherapy is 44%, with CAG/HAG therapy 17%, decitabine therapy 43%, combined CAG/HAG and decitabine therapy 56%, AML-like chemotherapy 67%. Patients receiving CAG/HAG in combination with decitabine had higher response rates compared to CAG/HAG or decitabine alone (OR=17.5, 95% CI 1.2-250.3, P=0.035). With a median follow-up of 20 months (range: 1-158months), 23 patients received HSCT after chemotherapy, and they had a higher 2-year overall survival rate compared to those who did not undergo transplantation (86% vs 0%, p<0.001). Decitabine combined with CAG/HAG improve the response rate of advanced pediatric MDS. Chemotherapy bridged hematopoietic stem cell transplantation significantly improves the prognosis of advanced pediatric MDS.

Lymphopenia Confers Poorer Prognosis in Myelodysplastic Neoplasms (MDS) with Very Low and Low IPSS-M

Introduction: In addition to MDS cell-intrinsic genetic lesions, we and others found increasing evidence of dynamic alterations in both adaptive and innate immunity in the disease genesis and/or progression (Boy et al., Nat Commun 2023;14:588). Although a few reports have suggested a prognostic impact of absolute lymphocyte count (ALC) in both del(5q) and non-del(5q) MDS, lymphopenia is not captured by current prognostic scores. Moreover, the association between lymphopenia and somatic mutations has not been reported. In this work, we describe the prevalence of lymphopenia in MDS patients, the interaction with molecular features, and the impact of lymphopenia on patient outcomes. Methods: We retrospectively included all MDS patients followed in our center (Saint-Louis Hospital, Paris, France) between January 2015 and January 2022 with a molecular evaluation. According to the local laboratory threshold, lymphopenia was defined as ALC <1.5 G/L. Low (LR), intermediate (IR), and high-risk (HR) MDS were defined according to IPSS-M (LR: IPSS-M Low and Very Low, scores < -0.5; IR: IPSS-M Moderate Low and Moderate High, scores -0.5 to 0.5; and HR: IPSS-M High and Very High, scores >0.5 respectively).Survival analyses were estimated between MDS diagnosis until death from any cause (OS) or last follow-up, or until AML transformation (LFS) or last follow-up. Allogeneic stem cell transplantation was considered a censoring event. Results: Our cohort included 372 MDS patients (45% females, median age 72 IQR[65-79]) with 230 (62%), 70 (19%), and 72 (19%) patients with LR, IR and HR-MDS respectively. Compared to IPSS-R, molecular IPSS reclassified 175 patients (47%, with 97 (26%) upstaged and 78 (21%) downstaged). The median ALC in the whole cohort was 1.20 IQR[0.87-1.60] G/L. Lymphopenia was highly prevalent in all MDS subgroups (68%, 79% and 75% in LR, IR and HR-MDS respectively), and ALC inversely correlated with higher IPSS-M (Spearman rho=-0.13, p=0.01). Lymphopenia was associated with lower ANC (median 1.90 IQR[1.1-3.4] versus 2.70 IQR[1.6-4.2], p=0.01) and monocyte count (median 0.31 IQR[0.17-0.46] versus 0.42 IQR[0.25-0.78], p=0.04). Lymphopenia was not significantly associated with an excess of marrow blasts (78/267, 29% versus 21/105, 21% in patients with or without lymphopenia respectively, p=0.09). However, its prevalence was significantly lower in MDS with ringed sideroblasts (RS) (27/49 (55%) versus 240/267 (90%), p<0.01). Consistently, SF3B1 mutations were more prevalent in MDS patients without lymphopenia (29/107 (27%) versus 27/265 (10%), p<0.01), while the rest of the somatic mutational landscape did not differ between the 2 groups. The median follow-up of the entire cohort was 4.1 IQR[3.7-4.6] years. Among IPSS-M categories, lymphopenia adversely impacted OS and LFS in LR-MDS patients (median 8.8 years vs not reached for OS, p=0.02; median 12.3 years versus not reached, p=0.02 for LFS) but not in IR (p=0.34 and p=0.49 for OS and LFS respectively) or in HR-MDS (p=0.82 and p=0.83) patients ( Figure 1). In SF3B1 mutated LR-MDS patients (n=46), the adverse prognostic impact of lymphopenia at diagnosis was still significant for both OS (p=0.03) and LFS (p=0.03). Conclusion: In this single-center retrospective analysis, lymphopenia was highly prevalent at MDS diagnosis in all IPSS-M subgroups and ALC negatively correlated with disease severity. The presence of lymphopenia adversely impacted OS and LFS in the LR-MDS subgroup, potentially suggesting a significant role of host immunity in early disease phase and possibly adding a prognostic parameter to IPSS-M.

Chronic Myelomonocytic Leukemia (CMML) with AML Typical Mutations ( NPM1, FLT3 or CEBPA) Identify a High-Risk CMML Group Independent of Molecular-Cpss

CMML is rarely associated with AML typical mutations such as NPM1, FLT3, CEBPA and IDH1/2 (mutAML-CMML). It is unknown what its clinical meaning is and if its presence should change our management into an AML-type treatment. The aim was to describe the characteristics, prognosis and treatment of mutAML-CMML patients. We studied 83 CMML patients of Hospital Clinic diagnosed between 1998-2022, with Next Generation Sequencing (NGS). We included 53 patients with mutAML-CMML from other institutions. We identified 31 patients with mutated IDH2 (all c.419G>A p.(Arg140Gln)), 13 with IDH1, 14 with NPM1,7 with CEBPA (4 bZIP) and 11 with FLT3 (3 FLT3-ITD and 7 FLT3-TKD: 6 TKD1 and 2 TKD2 variants, all different from p.(Asp835Tyr) except one). Patient characteristics and outcome of the group with mut IDH1/2 was not significantly different from wt IDH. Those patients with mut NPM1, FLT3 and/or CEBPApresented adverse characteristics and worse prognosis. Therefore, we grouped the patients with these mutations into a category named mutCFN (n=26) (Table 1). The mutCFN incidence in our series was 9%. The mutCFN patients (n=26) vs wtCFN (n=96) were younger (63 vs 71 years, p=0.004), had more leucocytes (16 vs 7.7x10 9/L, p=0.015), monocytes (2.9 vs 1.7x10 9/L, p=0.006), anemia (9.1 vs 11.2 g/L, p<0.001) and bone marrow blasts (11.5 vs 4%, p<0.001). They belonged more frequently to myeloproliferative variant (57.7 vs 30.2%, p=0.012), CMML-2 (69.2 vs 14.7%, p<0.001), high risk CPSS and CPSS-Mol (72 vs 31%, p<0.001; 88.2 vs 44.9%, p=0.001). They showed more transfusion dependence (69.2 vs 22.9%, p<0.001). Co-mutational representation is shown in Figure 1. mutCFN patients have a different co-mutational pattern from wtCFN with more frequently mutated DNMT3A (42.9 vs 6.3%, p<0.001), SF3B1 (15.8 vs 2.3%, p=0.04), WT1 (10 vs 0%, p=0.03), and less TET2 (24 vs 58.3%, p=0.003) . mutCFN patients received treatment more frequently than wtCFN (84.6 vs 36.5%, p<0.001) and earlier (0.97 vs 4.6 months, p=0.005). They were more frequently treated with chemotherapy (53.8 vs 7.3%, p<0.001) and alloSCT (61.5 vs 11.5%, p<0.001). The median follow-up was 9.3 years (95% CI, 8-11). The 2-year cumulative incidence of AML (CIR-AML) transformation was higher in mutCFN patients (44% vs 13%, p<0.001) and overall survival (OS) was shorter (25 vs 38 months, p=0.057). Besides, when those who received and alloSCT were censored at time of alloSCT, mutCFN showed an inferior survival (9.6 vs 35.7 months, p<0.01). Multivariate analysis confirmed mutCFN as an adverse prognostic factor independent of age and CPSS-Mol (HR 2.42, IC 95% 1.23-4.76, p=0.011). Because CFN mutation showed independent prognostic value not captured by CPSS-Mol, we added to CPSS-Mol 1 point if patient had mutCFN. We derived a modified CPSS-Mol-CFN score and identified five groups with different median OS of 85 (95% CI, 54-117) vs 74 (95% CI, 53-94) vs 40 (95% CI, 17-63) vs 22 (95% CI, 11-33) vs 15 (95% CI, 5-25) months, p<0.001with a better predictive capacity than CPSS-Mol (C-index 0.66 vs 0.64). Nowadays, there is no consensus between the two recent 2022 ICC and WHO classifications, but the identification of NPM1 or CEBPA mutations in cases of CMML could define AML. We analysed the group of patients who harboured bZIP- CEBPA or NPM1 mutations (“mutCN”) (n=17) and we found that these patients retained its chemosensitivity (90%CR) and those treated with chemotherapy had better OS (27 vs 9 months, p<0.001). 10 mutCFN patients received alloSCT with a median OS post-alloSCT of 21 months (95% CI, 16-26) and a 2year-CIR-AML of 37.5% (95% CI, 15-61). 5 patients received targeted therapy for a post-transplant relapse: 3 received sorafenib after a molecular relapse with prolonged responses and 2 received an IDH inhibitor, one of them persisting with a complete response after 4 years. We analyzed 5 paired samples (diagnostic CMML-AML transformation). All of the AML related mutations were retained at AML transformation and emerged new mutations in genes of signaling ( FLT3, NRAS), transcription ( RUNX1, CEBPA) and splicing ( STAG2) pathways. Independently of CPSS-Mol, mutCFN CMML patients have a different profile, closer to AML and with worse prognosis. They could benefit from more aggressive AML-treatment based on chemotherapy and alloSCT when possible. Interestingly, some patients may benefit from approved targeted therapy for AML FinancingISCIII PI19/01476 PI22/01660, co-financed EU, Emili-Letang HCPB