Types Of Mutations Research Articles

Novel CD20 Mutations As a Mechanism of Resistance to CD20-CD3 Targeted Therapies in Non-Hodgkin's Lymphoma

Non-Hodgkin's Lymphoma (NHL) is one of the most common blood cancer types in the world. CD20 is a B cell restricted lineage marker that is a well-established target for the treatment of NHL and anti-CD20 agents are included as in standard of care regimens. Given the success of these targeted therapies, strategies to further enhance therapeutic activity are being explored, including the CD20-CD3 bispecific molecule, mosunetuzumab, which is approved for treatment of relapsed and refractory Follicular Lymphoma in adults who have received >2 lines of treatment. Mosunetuzumab acts by redirecting CD3+ T cells to engage and eliminate CD20-expressing B cells. As with other targeted therapies, mutations and loss of target expression have been described following mosunetuzumab therapy [1]. Here we describe the impact of novel mutations identified in patient biopsy samples from the mosunetuzumab monotherapy trial, GO29781 that were located within the extracellular and transmembrane domains of CD20 on protein expression, localization and T-cell mediated killing. NHL cell lines, SU-DHL-16 and MAVER-1, were engineered using CRISPR-Cas9 system to create CD20 knock out (KO) cell lines. Wild-type (WT) or mutant CD20 were exogenously re-expressed in the CD20 KO cell lines and in the CD20 negative acute lymphoblastic leukemia cell line, REH, to create isogenic cell lines for further analysis. Complete CD20 loss was observed in cells expressing CD20 P160fs (frameshift) and Q187* (truncating) mutations. CD20 missense mutations located in the extracellular domain (C167G, K175E) expressed CD20 protein levels comparable to CD20 WT cells and membrane localization was confirmed by flow cytometry and immunofluorescence microscopy. To test the functional effect of CD20 mutations, in vitro co-culture cell killing assays were carried out using healthy donor CD8+ T cells, isogenic cell lines (3:1 effector to tumor cell ratio) and a proof-of-concept CD20-CD3 bispecific antibody. Differences in T-cell mediated killing were observed across mutation types. CD20 WT cell lines demonstrated 95-99% cell killing following 48 hour treatment while the CD20 KO, frameshift and truncating mutations all showed negligible T-cell mediated killing (~10-17% cell death). Cells expressing the missense mutations in the extracellular domain showed resistance to T-cell mediated killing (~30-45% cell death) compared to CD20 WT cells. T-cell activation was assessed by CD69 (early) and CD25 (late) surface markers. In all co-cultures, the level of CD69+ T cells was equivalent, while the CD69+CD25+ CD8 T cells were only significantly increased in the CD20 WT co-cultures, consistent with the high level of T-cell mediating killing. We have confirmed that these mutations can play a role in the development of resistance through both loss of target protein expression and interference within the anti-CD20 binding site. These studies help to characterize mechanisms of resistance to mosunetuzumab that could be extended to other anti-CD20 targeted agents. [1] Schuster et al. Journal of Clinical Oncology 2022, 40 (16), 7526.

Ras Activity Is Significantly Elevated in Patients with Primary Myelofibrosis and Is Agnostic to the Presence of RAS and JAK pathwaymutations

Myelofibrosis (MF) is a myeloproliferative stem cell disorder arising from various clonal mutations in early myeloid-committed hematopoietic progenitor cells. The subsequent acquisition of additional mutations and cytokine signaling then heralds the recruitment of fibroblasts to the marrow space, leading to collagen deposition. MF disease behavior varies widely and hematopoietic stem cell transplantation remains the only curative therapeutic option. In non-transplant candidates, managing cytopenic disease is uniquely challenging as patients develop resistance to commonly-utilized Jak-inhibitors. Expanding the molecular landscape of the disease beyond Jak/Stat signaling with the goal of identifying new therapeutic targets has, accordingly, been an area of growing interest in MF research. Recently-published studies, including results from our own institution, have identified mutations that potentially activate Ras signaling as being associated with features of advanced MF (higher somatic mutation burden) as well as with shorter overall survival and progression to AML. The goals of our study is to expand on this prior data, characterize the activity of Ras signaling and determine its role in patients with MF. Peripheral blood mononuclear cells (PBMCs) from 27 patients with myelofibrosis (MF) at a single institution underwent integrated genomic-transcriptomic sequencing analyses using the University of Michigan MiOncoSeq platform. PBMCs were separated by Ficoll® followed by cell lysis and protein quantification. Ras activity was measured by the binding of effector Ras binding domain (RBD) via ELISA. Patients with MF were compared to healthy donors and to patients with either polycythemia vera (PV) or essential thrombocythemia (ET). Ras activity in MF was examined across different mutations and with respect to various clinical parameters. Multivariate analyses were performed to assess the impact of each individual mutation on Ras activity. Accompanying statistical analyses for all assessments were conducted using two-way unpaired t-tests. Overall, Ras activity in 21 assessable patients with MF was 1.64 fold higher than in PV/ET (12 patients, p=0.0462) and 1.74 fold higher than healthy donors (13 volunteers, p=0.0304). While the majority of patients in the MF cohort had driver mutations, including in JAK2 (73%) and CALR (15.4%), the individual types of driver mutations did not impact Ras activity. Within the Ras/MAP Kinase signaling pathway, mutations were present at a range of variant allele frequencies (VAF), including 2 patients with NRAS (one with VAF 42%, one <5%), 2 KRAS (both <5%), 2 NF1, 1 CBL and 1 RAF1 (VAFs not assessed). The individual impact of various driver mutations, epigenetic regulators and splicing factors as assessed by multivariate analysis identified a trend toward enhanced Ras activity with mutations in RAS, CALR, ASXL1, TET2 and TP53, suggesting that in addition to RAS, other mutations also activate Ras signaling in MF. With regards to clinical parameters, Ras activity was weakly correlated with spleen size and high molecular risk mutation (HMR) presence, although this did not reach statistical significance in this cohort due to a relatively small sample size. Next, we performed a differential enrichment of myeloid and lymphoid cells from PBMCs using EasySep™ and measured Ras activity in lymphoid and myeloid enriched cells. We observed that the ratio of myeloid:lymphoid activity in patients with MF with RAS/MAP Kinase pathway mutations is 3.8 times higher than in patients without such mutations (p=0.0012), suggesting that RAS pathway mutations have a predominant role in myeloid cells. Our results show that the majority of patients with MF have hyperactive Ras signaling compared to PV/ET or healthy donors, regardless of the presence of alterations in the Ras signaling pathway. This underscores the importance of functional assessment of Ras activity in patients with MF and provides evidence that targeting Ras signaling in MF may lead to therapeutic benefit. We are currently expanding our analysis to a larger cohort of MF patients and performing preclinical testing of Ras/MAP Kinase pathway inhibitors as a means of overcoming Jak-inhibitor resistance.

Different Mutations in SF3B1 Gene Have Specific Transcriptomic Characteristics in Myelodysplastic Neoplasms

Myelodysplastic neoplasms (MDS) are hematopoietic stem cell (HSC) disorders characterized by ineffective hematopoiesis, peripheral cytopenia, and increased tendency to leukemic transformation. Somatic mutations in spliceosome machinery occur in approx. 50% of MDS patients. Among these, up to 30% of MDS patients carry a mutation in SF3B1. According to the 2022 classification system, MDS-SF3B1 is now considered as separate MDS subtype. Mutated SF3B1 perturbs mitochondrial function, resulting in apoptosis and ineffective erythropoiesis. The most common mutation of SF3B1, K700E, accounts for 50% of the variants, with additional codons (such as 666 and 662) acting as hotspot sites. Different mutations seem to be associated with distinct clinicopathological features. However, the prognostic implication of different SF3B1 mutations in MDS remains controversial and needs further clarification. This study investigated differences in clinical and molecular features of MDS-SF3B1 with different types of SF3B1 mutations. To investigate a direct impact of different point mutations in SF3B1, the study was based on four isogenic NALM6 cell lines, the wildtype (wt) and three cell lines, each with different SF3B1 mutation (CRISPR/Cas9-edited K700E, K666N, and H662Q). To understand phenotypic manifestations of the mutations in the disease, we also analyzed clinical and transcriptional characteristics of 146 lower-risk MDS patients. Using RNA-seq in the cell lines and CD34+ BM patient cells (17 patients with no mutation, 11 patients with K700E SF3B1 mutation, 5 patients with K666N/E SF3B1 mutation), we investigated effects of different SF3B1 mutations on RNA splicing events and gene expression levels. Initially, outcome analysis confirmed the previously described observation that patients with a SF3B1 mutation have favorable outcomes compared to those with other splicing factor mutations. Further, the analysis showed that specific mutations in SF3B1 tend to lead to different prognoses; patients with K700E had longer survival compared to those with other SF3B1 mutations (mean progression-free survival was 64.8 vs. 29.1 months). Analysis of RNA splicing in NALM6 showed that K666N and H662Q mutations share similar characteristics whereas K700E mutation is more similar to wt-cells. Most significant changes were found in retained introns (RI); increased RI inclusion levels were detected in K666N/H662Q cells compared to K700E/wt cells. In patients, RI events were also strongly increased in K666N/E compared to K700E samples (Fig. 1). A significant overlap was identified between cell line and patient data; out of 173 genes with higher RI inclusion level in K666N-NALM6 cells, 77 genes had higher RI inclusion also in patients. Interestingly, 12 out of these 77 genes were related to mitochondrial functions (e.g., CREBZF, DDX1, MRPL23, MUTYH, and RAF1). RNA-seq data further showed significant alternations in gene expression levels in the four isogenic cell lines and also in patient samples with different mutations. Surprisingly, a more pronounced difference was detected in gene expression between patients with different SF3B1 mutations than between patients with no mutation and with any SF3B1 mutation (irrespective of its type), suggesting that specific SF3B1 mutations result in different phenotypes. Pathway analyses showed that deregulated genes in samples with K700E vs. other hotspot mutations were significantly enriched in electron transport, cell cycle, proliferation, quiescence, and HSC differentiation. Importantly, expression of multiple mitochondria-related genes was altered between K700E and K666N/H662Q NALM6 cells (334 genes, FDR < 0.01; Fig. 2), indicating different metabolic characteristics of the cells with non-K700E mutations. For example, several subunits of mitochondrial respiratory complexes I and IV were upregulated in K700E cells, pointing to differences in energetic metabolism. Altogether, our data support the hypothesis that only K700E mutation of SF3B1 may have a positive prognostic value and that other SF3B1 mutations may influence cells differently, specifically affecting their mitochondrial functions. Based on our results, we conclude that prognostic implication of different SF3B1 mutations in MDS will need additional refinements in future. Supported by AZV CR (NU20-03-00412), GA CR (20-19162S), and MH CZ-DRO (00023736).

Efficacy of Demethylated Drug Combine with Low Dose Chemotherapy in Juvenile Myelomonocytic Leukemia

Objection：Juvenile myelomonocytic leukemia (JMML) is a rare pediatric leukemia with a median survival time of less than 2 years. Currently, hematopoietic stem cell transplantation (HSCT) is the only possible cure of JMML. Most of the newly diagnosed pediatric patients with JMML have high tumor burden, and rapid disease progression, and may not tolerate HSCT. In the present study, we aimed to explore the effection of Demethylated drug in combination with low-dose chemotherapy on JMML patients before HSCT. Methods：A retrospective analysis of 16 JMML patients was performed from Jul 2017 to Nov 2021. All patients were given Demethylated drug in combination with low-dose chemotherapy, 10 patients decitabine at a dose of 20 mg/m2 for 5 days, supplemented with cytarabine (50-100 mg/m2×3~5 days), and /or etoposide (50 mg/m2×3~5 days), 6 patients Azacytidine at a dose of 75mg/m2 for 7 days combined with homoharringtonine 2mg/m2 for 5-7 days. Each treatment interval was 4 weeks, bridging to HSCT after three~four courses of treatment. Results：The median age of onset of the 16 JMML patients was 36 months (range 6-51 months), the male to female ratio was 14:2, and the median volume of the spleen at diagnosis was 357cm3 (range: 175-588) by Three-dimensional ultrasound of spleen. The median white blood cell (WBC) count was 39 (range 2-128) × 109/L, the median hemoglobin 84 (range 47-110) g/l, and the median platelet 36 (range 16-75) × 109/L，the median monocyte count was 8 (range 2-26) × 109/L at diagnosis, the median RDW-CV was 19 (15-30)%, the median RDW-SD 56 (45-77) fl, the median HbF 37(1-76)%. Chromosome was performed, of which 13 cases had normal karyotype. 2 cases had tirsomy 8, 1 case had structural abnormality in chrosome 9. Next-generation sequencing results showed that 11(68.8%) cases carried PTPN11 mutations, 6 (37.5%) cases NF1 mutation, 5 (31.3%) KRAS mutation, 4 (25%) NRAS mutation, 1 case without one of the Ras pathway genes. 8 cases (50%) had more than two types of canonical JMML mutations, among them 6 cases (75%) had PTPN11 and NF1 co-mutation. 10 cases had secondary gene mutations, 3 (3/10) cases JAK3 mutation, 2 (2/10) SETBP1 mutation, 2 (2/10) SH2B3 mutation, 2(2/10) ASXL1 mutation, 2 (2/10) ARID1A mutation, 1 (1/10) SRSF2 mutation. The median treatment course was three cycles (range: 1-6), and the response rate was 68.8% (11/16) after one cycle and 81.8% (9/11) after three cycles. One case achieved complete remission (CR) after four courses of therapy. The WBC and monocyte counts were significantly decreased after treatment (P<0.001), and the spleen volume showed a decreasing tendency. Eleven patients received HSCT, 9 cases were implanted successfully and 2 cases failed, of which 1 case was relieved by secondary transplantation and 2 patients died of grade IV graft-versus-host disease (GVHD) after transplantation. The median follow-up time was 31 months. The 2-year progression-free survival rate was 75.0±10.8%. Conclusions：Demethylated drug in combination with low-dose chemotherapy could reduce JMML patients' tumor burden, improve the general condition, and obtain a clinical response rate of 81.8% after 3 cycles therapy. Therefore, such a combination regimen could be used as a therapeutic option for JMML before HSCT.

Myelodysplasia-Related Gene Mutations for NGS-Based MRD Assessment in Acute Myeloid Leukemia

Introduction: Measurable residual disease (MRD) plays an increasing role for treatment management of AML patients. While next-generation-sequencing (NGS) based MRD assessment can be well standardized, there is uncertainty about the type of gene mutations that associate with prognosis and are useful for MRD assessment. The 2022 ELN recommendations assign patients with at least one mutation in the genes ASXL1, BCOR, EZH2, RUNX1, SF3B1, SRSF2, STAG2, U2AF1 or ZRSR2 to the subgroup of myelodysplasia-related gene mutations (MRGM), which is associated with poor prognosis. We aimed to define the prognostic effect of MRD after induction chemotherapy in AML patients with MRGM using MRG mutations for MRD assessment by NGS. Methods: We retrospectively collected bone marrow and peripheral blood samples from AML patients in complete remission (CR) or CR with incomplete hematologic recovery (CRi) after one to two cycles of 7+3-based standard induction chemotherapy (IC) with at least one MRGM detected at diagnosis by myeloid panel sequencing covering 48 AML associated genes. Patients treated with one course of IC were included if they proceeded to allogeneic hematopoietic cell transplantation (alloHCT) already after one cycle. Molecular MRD status was assessed using targeted NGS and bioinformatics error-correction with 0.01% sensitivity as described before using only MRGM as MRD markers. Results: MRD was assessed on 104 adult newly-diagnosed AML patients (median age 58.6) with a total of 166 MRGM, resulting in a median of one MRGM per patient (range 1-4). 43 (41.3%) patients had secondary or therapy-related AML and 20 (19.2%) had myelodysplasia-related cytogenetic abnormalities. 68 (65.4%) patients were MRD positive for at least one MRGM after one (18 patients (17.3%)) or two (86 patients (82.7%)) cycles of intensive chemotherapy, while 36 (34.6%) became MRD negative. Measured variant allele frequencies (VAF) of positive samples ranged from 0.02-43.1% (median VAF 1.34%).MRD positive patients were older and more likely to be male compared to MRD negative patients, while cytogenetic risk, WBC count, Hgb, platelet count and ECOG status at diagnosis were similar to MRD negative patients. 53 (77.9%) MRD positive and 23 (63.9%) MRD negative patients underwent alloHCT in first CR/CRi.Median follow-up was 3.35 years. OS (P = 0.92), RFS (P = 0.99) and CIR (P = 0.98) were similar in MRD positive (at least one MRGM detectable) and negative (no MRGM detectable) patients (Figure 1). OS, RFS and CIR were also similar between MRD positive and negative patients, when MRD negativity was defined by conversion of at least one MRGM to negativity (55 (52.9%) MRD positive, 49 (47.1%) MRD negative), and when ASXL1 was excluded from the analysis. As variants with VAF >5% in CR/CRi samples may indicate clonal hematopoiesis, we excluded all variants with VAF >5% and repeated the analysis with the 88 (84.6%) remaining patients. Again, OS, RFS and CIR were similar for MRD positive and negative patients. We next reasoned that reduction of VAF correlates with the sensitivity of leukemic cells and possibly with depth of remission and long-term outcome. The fold-reduction of VAFs from diagnosis to the time of MRD analysis was calculated for all variants. For patients with several MRGMs, the variant with the largest fold-reduction was considered including undetectable variants. Patients with more or less than a 2-log, 3-log or 4-log reduction (log 10) had similar OS, RFS and CIR, respectively. Evaluating each MRGM individually, only RUNX1-MRD associated with RFS and by trend CIR (comparing MRD positive to negative patients for OS, HR 3.41, 95% CI 0.43-26.82, P = 0.24; RFS, HR 4.37, 95% CI 1.07-17.79, P = 0.04; CIR, HR 3.30, 95% CI 0.79-13.82, P = 0.1). Conclusion: 65.4% of AML-MRGM patients in CR/CRi remained MRD positive when MRGMs were used for MRD assessment by NGS. MRD positivity was associated with age above 60 years and male sex.Using all MRG mutations for MRD assessment at a threshold of 0.01% or by various degrees of log-reduction was not predictive of outcome after intensive induction chemotherapy. This limits the number of potential MRD markers for NGS analysis before alloHCT, although individual markers like RUNX1 may be of prognostic value.

Impact of Mutational Burden and IPSS-M on Response to ESAs in Lower Risk Myelodysplastic Neoplasms

INTRODUCTION Lower risk myelodysplastic neoplasms (LR-MDS), as defined by IPSS-R, are frequently presenting with anemia which is primarily treated with erythropoiesis stimulating agents (ESAs). However, response rates are suboptimal due to primary resistance or subsequent loss of response, leading to transfusion dependency and poor quality of life. In 2022 a new prognostic system has been developed considering number and type of somatic mutations in MDS (IPSS-M), showing a stronger prognostic power in MDS. AIMS We aimed to assess whether IPSS-M risk score could predict ESA response in IPSS-R LR-MDS patients. METHODS In this multicentric European study we evaluated baseline IPSS-M score in 397 LR-MDS cases from 14 different centers (Italy, Germany, France and Spain) and correlated with ESA response assessed with IWG criteria. Mutational data were obtained by different next generation sequencing (NGS) panels targeting genes recurrently mutated in myeloid neoplasia and necessary to calculate IPSS-M. RESULTS Median age of patients was 74 years with male preponderance (54.5%). Serum EPO levels (sEPO) were available in 277/397 cases (mean 157U/L, <200 U/L in 80% of the cases). When transfusion history (TH) was known (252/397), 44.6% of cases were transfusion dependent (TD). The majority of the cases received erythropoietin-α and darbepoetin-α with response rate of 52.6% (HI-E) according to IWG criteria and a median duration of response of 30 months (data from 165 responders with follow up information). We observed that IPSS-M score, but not the number of mutations, was significantly lower in ESA responders compared to non-responders (median -1,08 vs -0,63 respectively , p<0.0001). The very low (VL) IPSS-M risk group was enriched in ESA responders (74 vs 26% p=0.036), while groups from moderate low (ML) to very high (VH) were more enriched with non-responders (37.5 vs 62% p=0.001; 45.4 vs 54.6% p=0.045; 29.6 vs 70,4% p=0.0017; 25 vs 75% p=0.021 respectively, Fig.1). In cases with known TH (n=252), non-TD (NTD) ESA responders had lower IPSS-M scores (mean -1,17) compared to i) NTD ESA non-responders (mean -0,61 , p<0.0001), ii) TD ESA responders (mean -0,93, p=0,025) and iii) TD ESA non-responders (mean -0,54, p<0.0001). As observed in other settings, 17% of the cases (n=67) were re-stratified to higher risk IPSS-M categories (8%, 13,4% and 44,3% of IPSS-R very low, low and intermediate risk respectively). Among upstaged cases, most were ESA non-responders especially in the moderate high (MH, 67%), high (H, 61%) and very high-risk (VH, 100%) groups. Only 4% of cases (n=17) were down-staged, all from IPSS-R intermediate risk group, and showed response rates similar to IPSS-R intermediate cases who remained ML or MH. Finally, IPSS-M score, sEPO and TD correlated with response in multivariate logistic regression analysis ( p=0.005, p=0.004 and p=0.014 respectively), but only IPSS-M score and TD were predictive (OR 0.51 and 0.34 respectively). Based on this model, we designed a ROC curve (AUC 0.79) where a cut-off of 0.46 showed a sensitivity of 0.9 and specificity of 0.67 in discriminating responders from non-responders (Fig.2). CONCLUSIONS To our knowledge, this is the first report correlating IPSS-M with ESA response in a large number of LR-MDS cases. IPSS-M scores exhibited predictive power, with lower values in ESA responders compared to non-responders and in NTD compared to TD cases. We also observed that higher IPSS-M categories were inversely correlated with ESA response. A fraction of cases with scarce ESA response was upstaged to IPSS-M higher risk groups. The number of mutations did not correlate with ESA response, while IPSS-M score, which considers the prognostic weight of specific mutations, showed predictive significance. In the multivariate analysis IPSS-M score and TD were strong predictors of ESA response, irrespective of baseline sEPO, potentially defining at diagnosis LR-MDS for whom novel agents like luspatercept could be considered as first line therapeutic option.

Deciphering Thrombosis Risk in Vexas Syndrome Patients through Thrombin Generation Assay Andthromboelastography

Introduction: VEXAS syndrome (for Vacuoles in myeloid progenitors, E1 ubiquitin ligase, X-linked, Autoinflammatory manifestations and Somatic) is the consequence of the clonal expansion of hematopoietic stem or progenitor cells with somatically acquired mutation of UBA1. VEXAS is a rare disease characterized by a variety of autoinflammatory manifestations and frequently associated with other hematological malignancies. Venous (VTE) and arterial thrombosis (AT) are recurrently observed in disease course and may affect up to 50% of patients. The exact pathophysiological mechanism underlying thrombosis genesis in UBA1 mut patients is not yet elucidated. Thrombin generation assay (TGA) and thromboelastography (ROTEM) are global hemostasis assays that might be used to identify hemostatic profiles at risk of thrombosis (Ay C et al. J Clin Oncol 2011). In this study, we aimed to evaluate TGA and ROTEM as potential surrogate markers of clot formation in UBA1 mut patients to predict their risk for thrombosis. Methods: TGA and ROTEM assays were performed in UBA1 mut patients between March 2022 and July 2023 , in Hospices Civils de Lyon, France. Patients were tested whether or not they had a personal history of VTE/AT. For patients already treated with oral anticoagulant interfering with exploratory assays, a temporary heparin switch was realized. TGA allows to evaluate concentration of generated thrombin through time, resulting in a dynamic thrombin generation curve, from which various parameters can be determined that describe thrombin activity, such as endogenous thrombin potential (ETP; area under the TG curve). Regarding ROTEM, analyses have been performed on citrated tubes using the ROTEM delta. For each test, the parameters CT (clotting time in seconds), A5 and A10 (measured clot amplitude at 5 and 10 minutes in mm), angle alpha (velocity in °), maximum clot firmness (in mm) and LI30 (% clot lysis at 30 minutes) have been measured. Results: We screened 15 UBA1 mut patients, while only 14 were evaluable for TGA/ROTEM (1 patient excluded due to concomitant use of apixaban the day of TGA). Five out of 15 (33%) had an history of VTE (N=3) or AT (N=2) at the time of TGA/ROTEM blood tests. With a median follow-up of 11.8 months after hmostastic explorations, none of screened patients experienced VTE/AT. On the 14 evaluable patients, TGA/ROTEM exploration was performed in 4 of them at the time of diagnosis prior any treatment onset (including steroids). Active disease (defined by increased CRP and/or clinical manifestations) at the time of TGA did not influence significantly time to peak (mins), peak thrombin (nM) and endogeneous thrombin potential (ETP, nM. min) either in platelet rich- and platelet poor- plasma. Interestingly, patients with prior history of VTE/AT (N=4) had significantly higher ETP compared to those without thrombosis history (N=10) either in platelet rich- and platelet poor-plasmas. Moreover, UBA1 mut patients without thrombosis history exhibited similar ETP values than controls ( figure 1). There was no significant correlation between type of UBA1 mutation, UBA1 allelic frequency, concomitant MDS, type of treatment at the time of TGA assay. Regarding ROTEM analysis, there was no significant differences in clot formation between patients with or without thrombosis history in EXTEM, NATEM, and FIBTEM channels. However, in TEMACT channel (activation as in EXTEM but with a plasminogen activator), we observed higher MCF (Maximum Clot Firmness) in UBA1 mutpatients with prior history of VTE/AT ( Figure 2). Acquired and inherited thrombophilia (protein C and S activities, lupus anticoagulant, anticardiolipine antibodies, anti-beta2 GP1 antibodies, factor V Leiden and prothrombin G20210A mutations, antithrombin evels) were explored in 8 out 15 of our patients. Two of them harbored lupus anticoagulant but without antiphospholipid antibodies. They do not have eitherthrombosis history or higher ETP levels. Conclusions: Based on these preliminary results, TGA and more specifically ETP might identify UBA1 mutpatients at risk of VTE or AT. ROTEM identified an hypofibrinolytic phenotype UBA1 mutpatients with thrombosis history paving the way for future explorations. The small sample size and the retrospective (post-VTE/AT) nature of these explorations are main limitations of this study, which need to be confirmed with a larger follow-up.

Cohesin Haploinsufficiency Promotes Reduced Latency in Inv(16) Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a cancer of the bone marrow that affects 20,000 adults in the United States alone, with global rates increasing over the last 30 years (Heimbruch et al. 2021, Dong et al. 2020). Approximately 15% of adult AML patients harbor the chromosomal aberrations t(8;21) or inv(16), which affect the core binding complex members RUNX1 and CBFB, respectively. Together, RUNX1 and CBFB function as a transcription factor that regulates key lineage genes and proliferation in hematopoietic cells (Pulikkan and Castilla 2018). t(8;21) and inv(16) result in the expression of the fusion proteins AML1-ETO and CBFB-SMMHC, both of which have dominant negative effects on native RUNX1 as well as novel transcriptional roles (Surapally et al. 2021). Although both t(8;21) and inv(16) result in AMLs with similar characteristics, they differ in the number and type of co-occurring mutations (Opatz et al. 2020). More mutations per clone are observed in t(8;21) vs. inv(16) AMLs. Additionally, several studies have highlighted a difference between the rates of mutation of cohesin genes and t(8;21) vs. inv(16) (Faber et al. 2016, Opatz et al. 2020, Duployez et al. 2016, Jahn et al. 2020, Qin et al. 2022). Cohesin is comprised of the subunits SMC3, SMC1A, RAD21, and STAG2. The complex has a prominent role in hematopoietic cells in chromatin organization and DNA looping events that regulate gene expression and self-renewal (Heimbruch et al. 2021). Cohesin mutations are found in approximately 10% of AML cases (Ley et al 2013). Interestingly, cohesin mutations are found in 18-27% of t(8;21) AMLs, but in 0-4% of inv(16) AMLs (Faber et al. 2016, Opatz et al. 2020, Duployez et al. 2016, Jahn et al. 2020, Qin et al. 2022). The low rate of co-mutation between inv(16) and cohesin genes suggests potential synthetic lethality. However, it is also possible that cohesin mutations occur at a higher rate than reported due to biases in the age, ethnicity, or treatment status of the patients included in genome/exome sequencing studies. To test if cohesin mutations and inv(16) are synthetic lethal, we used an inv(16) knock-in mouse model ( Mx1-Cre;inv(16)) combined with a cohesin-deficient mouse ( Mx1-Cre; Smc3 fl/+) (Kuo et al. 2006, Viny et al. 2015). We followed inv(16);Smc3wt and inv(16);Smc3 -/+ animals post-Cre activation and monitored mice for leukemic development. We found that inv(16);Smc3 -/+ mice developed AML with reduced latency compared to inv(16);Smc3wt mice, suggesting cooperativity rather than synthetic lethality. To determine the mechanism by which Smc3 haploinsufficiency accelerates inv(16) leukemias, we performed ATAC sequencing and single cell RNA sequencing on hematopoietic stem and progenitor cells (HSPCs). ATAC-seq revealed a large increase in open chromatin in promoter regions in inv(16);Smc3 -/+ vs. inv(16);Smc3wt HSPCs. These open regions were enriched for binding motifs of the Ets family member Fli1. Increased expression of Fli1 was observed by western blotting in inv(16);Smc3 -/+ HSPCs. Interestingly, CBFB-SMMHC binding sites commonly contain Ets motifs (Mandoli 2014), suggesting that the increased Fli1 expression and binding site accessibility in inv(16);Smc3 -/+ may drive the observed decrease in latency. scRNA-seq revealed cluster-specific differences between inv(16);Smc3wt and inv(16);Smc3 -/+ cells. In several clusters, inv(16);Smc3 -/+ cells showed a loss of expression of genes involved in apoptosis and differentiation. Of particular interest is Gata2, as loss of Gata2 generates a more aggressive phenotype in AML (Saida et al. 2020). Therefore, Smc3 haploinsufficiency may result in Gata2 deregulation, thereby impacting disease latency. Currently, we have two potential explanations for the observed phenotype: Deregulated binding of Fli1 and its downstream transcriptional pathways and decreased expression of Gata2 in sub-populations of inv(16);Smc3 -/+ cells. Which of these mechanisms mediates the reduced latency in inv(16);Smc3 -/+ animals is an area of active investigation. Collectively, our studies show that a lack of observed co-mutation does not necessarily indicate synthetic lethality. We propose that cohesin mutations may be more common in inv(16) leukemias than has been reported and suggest that a careful consideration of age, ethnicity, and treatment status may be warranted when evaluating rates of co-mutation.

Discovery of JNJ-88549968, a Novel, First-in-Class CALRmutxCD3 T-Cell Redirecting Antibody for the Treatment of Myeloproliferative Neoplasms

Myeloproliferative neoplasms (MPNs) are clonal malignant disorders of hematopoiesis arising in the hematopoietic stem cell (HSC) compartment that are characterized by excessive production of mature blood cells of the myeloid lineage. Transformation to secondary acute myeloid leukemia (sAML) represents a significant cause of death among MPN patients and this transformation occurs mainly from the clone carrying the disease phenotype driver mutation. Current treatment options for MPN patients are not curative and are limited to symptomatic treatment. Therefore, identification of novel therapeutic approaches with a clear disease-modifying effect for the treatment of MPNs and intercepting their progression to sAML is an unmet medical need. Mutations in JAK2, thrombopoietin receptor (MPL), and calreticulin (CALR) are phenotypic drivers in the pathogenesis of MPN. CALR mutations (CALRmut) are the second most frequent in MPN. CALRmut are insertions or deletions resulting in a frameshift in the last exon of the gene, causing a loss of the KDEL ER-retention motif and generation of a 36 amino acid positively charged C-terminal neoantigen. Due to loss of the KDEL motif, CALRmut are not confined to the ER and through interaction with MPL are trafficked to the cell surface where they induce persistent MPL activation and oncogenicity. Immunotherapies engaging T cells, such as bispecific cluster of differentiation 3 (CD3) redirection antibodies, show promising response rates in the clinic. Here, we describe JNJ-88549968, a novel first in class T-cell redirecting bispecific antibody that selectively targets CALRmut with the potential to achieve cures by eliminating MPN clones. The mechanism of action of JNJ-88549968 is to act as a bridge between CALRmut MPN cancer cells and T cells, inducing T-cell activation with subsequent T-cell-mediated cytotoxicity to CALRmut cancer cells in vitro and in vivo. JNJ-88549968 recognizes CALRmut epitopes common to all known CALRmut types. Cell surface localization of CALRmut was confirmed in CD34 + cells from CALRmut MPN patients. In-depth characterization of T cells from CALRmut MPN patients, using CyTOF and functional assays, confirmed their fitness and functionality. These data validate T-cell redirection as a rational therapeutic strategy for MPN patients carrying CALRmut. JNJ-88549968 demonstrated selective binding to CALRmut cell lines and no measurable binding to CALR wild type cells. JNJ-88549968 led to CALRmut-selective T-cell activation and cytotoxicity to CALRmut-engineered cell lines in vitro. The activity of JNJ-88549968 was also explored in an autologous setting using CD34 + cells isolated from CALRmut MPN patients as target cells together with T cells isolated from the same patients. JNJ-88549968 elicited concentration-dependent cytotoxicity of patient-derived CALRmut CD34 + cells. JNJ-88549968-mediated cytotoxicity was observed against all tested CALRmut CD34 + cancer cells and was independent of the type of CALR mutation. Moreover, JNJ-88549968 mediated robust in vivo efficacy in two independent CALRmut-positive xenograft murine leukemia models. In an established disseminated model, treatment with JNJ-88549968 significantly increased lifespan (ILS) compared to vehicle-treated control mice. Secreted CALRmut protein can be found in CALRmut MPN patient plasma. Evaluation of the impact of soluble CALRmut on JNJ-88549968 activity, including CALRmut-patient derived whole blood studies, indicated no effect on the activity of JNJ-88549968 in vitro. Taken together, JNJ-88549968 is a novel first-in-class bispecific T-cell redirection antibody investigated for the treatment of CALRmut MPN. JNJ-88549968 is currently being advanced for clinical investigation in patients with MPN.

Final Report: Somatic Mutations and HMGCLL1 Haplotype Are Not Associated with Molecular Relapse-Free Survival in Patients with Chronic Myeloid Leukemia Who Attempt Treatment-Free Remission

Background Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell disorder resulting from the BCR::ABL1 rearrangement. Patients with CML have a comparable life expectancy to that of age-matched individuals in the general population when they achieve an optimal response with tyrosine kinase inhibitor (TKI) therapy. With the aim of improving the quality of life of patients who are significantly affected by drug-related adverse events and financial burden, the practical goal of CML treatment has shifted to treatment-free remission (TFR), defined as the successful discontinuation (DISC) of TKI therapy without losing leukemia control. TKI treatment DISC is a safe procedure but requires appropriate candidate selection and frequent molecular monitoring. Several biomarkers were investigated, but no definite biomarker has yet been successfully identified and validated. We have investigated somatic mutations before TKI discontinuation and HMGCLL1 haplotype as a potential biomarker for TFR, given that they were previously described as predictors of CML outcomes. Patients and methods A total of 155 patients who attempted TKI discontinuation for TFR from 5 countries were included: Canada (n=71), Brazil (n=30), Argentina (n=28), Italy (n=23), and the Czech Republic (n=3). We applied our in-house single molecule-molecular inversion probe (smMIP) panel which is a barcoded error-corrected DNA-based sequencing with a limit of detection up to 0.2%. The panel encompasses 40 genes with 332 amplicon probes: epigenetic modifiers (n=7), activation signaling (n=12), myeloid transcription factor (TF; n=5), spliceosome (n=3), tumor suppressor (n=3), cohesion (n=4), miscellaneous (n=3) and 8 HMGCLL1 single nucleotide polymorphisms (SNPs). Two groups were constructed based on these eight SNPs: group 1: CTCAGGCA (C- haplotype) or group 2: ACGTAATG (A-haplotype). Samples were collected prior to TKI DISC when patients met the criteria for TFR attempt. Molecular relapse-free survival (mRFS) was calculated from the date of TKI DISC to the date of confirmed loss of deep molecular response (DMR). Loss of DMR includes single-time loss of major molecular response (MMR) or two consecutive episodes of loss of molecular response 4 log or deeper response (MR4). Kaplan-Meier survival estimate was used to evaluate mRFS and Cox's proportional hazard regression model was applied. Results The median MR4 duration prior to TKI DISC was 7.2 years (range 4.7-9.3), while overall TKI treatment duration was 8.5 years (range 6.0-11.6). With a median follow-up of 831 days (range: 460-1,415), out of the 155 patients, 68 (43.9%) lost response, among whom 6 lost MMR only, 12 lost MR4 only, and 50 lost MMR and MR4, both. Median time to MMR loss was 95 days (range 83.5-137). A total of 71 mutations were detected in 51 patients (32.9%). TET2 mutation was the most frequent mutation, detected in 17 cases (24%) followed by ASXL1 (n=14; 20%) and DNMT3A (n=12; 17%). When grouped by relevant biological pathways involved, mutations in epigenetic genes (n=42, 61%) were the most frequently involved. mRFS rate was 64% (95% CI [55.8-71.1%]) and 56% [48.2-64.0%] at 6 and 12 months, respectively. During the univariate analysis, no significant difference was observed in mRFS according to the presence, number, or type of somatic mutations, or the HMGCLL1 haplotype. Additionally, there were no significant differences in mRFS based on age, sex, Sokal risk group, line of TKI therapy, or TKI drug type prior to TKI DISC. However, MR4 duration prior to TKI DISC was associated with mRFS rate: patients with≥7.3 years (n=71) showed higher mRFS: 74.09% [62.05-82.82%] compared to those <7.3 years (n=74) with mRFS rate of 44.36% [32.54 -55.51] (p <0.01, HR 0.36 [0.21-0.63]). In addition, a longer duration of TKI treatment before TKI DISC was associated with higher mRFS: patients with ≥ 8.3 years of treatment (n=85) showed 71.8% mRFS [60.4-80.4], compared to 42.5% mRFS [30.5-54.0%] in those with <8.3 years (n=71) (p<0.01, HR 0.48 [0.31-0.74]). Multivariate analyses for mRFS confirmed that MR4 duration and the total duration of TKI treatment before TKI DISC were associated with TFR outcomes, but not the presence of epigenetic mutations, any mutation, or HMGCLL1 haplotype (Table1). Conclusion In CML, somatic mutations before attempting TFR are frequently observed. However, our current study does not support their prognostic significance as TFR-predictive biomarkers.

Cancer Genomic Profiling and Minimal Residual Disease Monitoring By Cell-Free DNA Sequencing in Pediatric Leukemia

Introduction: Cell-free DNA (cfDNA) sequencing has been reported as a promising non-invasive tool for detecting cancer and monitoring treatment response. We sought to evaluate the potential of a cfDNA-based strategy for detecting diverse classes of somatic mutations relevant to pediatric leukemia and cancers. Methods: Plasma samples collected at diagnosis from 135 acute lymphoblastic leukemia (ALL) and 34 acute myeloid leukemia (AML) patients were evaluated, including 114 B-ALL and 21 T-ALL. We also studied diagnostic plasma samples from 38 solid tumor and 19 brain tumor patients, and plasma collected from 12 patients with B-ALL at days 15, 22 and end of induction chemotherapy (EOI). Plasma cfDNA extraction and library preparation were performed with the NucleoSnap cfDNA kit and ThruPLEX Tag-Seq HV kit, respectively. Next-generation sequencing was performed using a capture panel covering 355 pediatric cancer genes (including noncoding regions covering hotspots of structural variation) and 8,103 heterozygous population polymorphisms. We developed analytic approaches for de novo detection of all types of somatic mutations, including focal copy number alterations, rearrangements, and large chromosomal abnormalities. Whole-genome sequencing (WGS) was also performed for 10 cfDNA samples from leukemia. Data were compared with corresponding whole-genome, exome and transcriptome tumor cell sequencing data at diagnosis. Results: At diagnosis, plasma cfDNA yield was higher for leukemias than for solid and brain tumors (median, 142 ng/ml, 7.16 ng/ml, and 7.07 ng/ml, respectively. The average coverages of leukemia cfDNA sequencing were 790X by capture panel post error suppression and 69X by WGS. We found that 100% cases of leukemia at diagnosis had detectable circulating tumor DNA (ctDNA) in plasma cfDNA, and the median ctDNA purity was 77%, comparable to primary tumor purity from diagnostic sequencing. Overall, our panel covered 86% somatic variants present in the tumor samples. Of those covered events, capture-based cfDNA sequencing detected 98% SNVs/indels, 95% focal copy gains/losses, 100% internal tandem duplications, 95% chromosomal translocations, and 99% aneuploidy. In addition, capture-based sequencing identified cfDNA-specific variants such as NRAS/KRAS SNVs and a IKZF1 deletion that were subclonal in plasma cfDNA. Of those uncovered events, 85% were structural variants with their breakpoints in intronic/noncoding regions, and cfDNA WGS identified all somatic events present in the 10 sequenced tumor samples. To detect minimal residual disease (MRD) in B-ALL patients during induction, we increased cfDNA sequencing depth for low VAF events, and the detection limit was 0.4% for SNV/indel and structural variations. Compared to the MRD results from flow cytometry, somatic mutations were detected in 7/8 MRD-positive and 1/4 MRD-negative patients on day 15, 3/4 MRD-positive patients on day 22, and 0/2 MRD-positive patients at EOI. We also sequenced cfDNA from peripheral blood and bone marrow plasma for 2 B-ALL patients on day 0, day 15, and EOI, with comparable ctDNA purity and dynamics observed between the two compartments. In a patient with concurrent ALL and neuroblastoma (NBL) who experienced NBL relapse four months after the primary diagnosis, cfDNA genomic profiling identified ctDNA derived from ALL (52% of total plasma cfDNA), primary NBL (20%), and relapsed NBL (10%), suggesting that genetic variations of multiple tumors could be detected in a single cfDNA sample. Conclusion: Our study has demonstrated the utility of cfDNA sequencing to identify both sequence and structural somatic mutations in childhood acute leukemia. This approach will be useful to monitor disease, as well as providing a non-invasive diagnostic approach.

Establishment of an NPM1 Mutation Copy Number Estimator for Xpert ® NPM1 Mutation Test

Objectives: The nucleophosmin ( NPM1)is the most mutated gene (~30%) in Acute Myeloid Leukemia (AML) 1. Three NPM1 mutations (type A, B, and D) represent ~84% in NPM1-mutated AML cases while other uncommon subtypes occupy ~16% 2. Xpert® NPM1 mutation, an automated cartridge-based test for measuring NPM1 mutation transcript levels (type A, B and D), is standardized to quantify the amount of mutated NPM1 relative to ABL1 control gene based on delta Ct in peripheral blood 3. Since mutated NPM1 level is crucial for risk assessment, medication selection, and ongoing therapeutic monitoring in AML 4,5, it is important to obtain the NPM1 mutation copy number (CN). The aim of this work is to develop NPM1 mutation CN estimator and to compare %NPM1 mutation/ABL1 reporting between delta Ct-based and CN-based methods. Methods: Five levels of NPM1 mutations (A, B, D) and ABL1 IVT-RNA panels as well as two lots of Xpert® NPM1 mutation tests were used to generate standard curves for CN and %CN reporting. The cell lysates from cell lines carrying either NPM1 mutation A, B, or D and AML clinical samples containing NPM1 mutations were examined to evaluate the CN and %CN between two lots of the Xpert® NPM1 mutation tests and to compare the delta Ct-based and CN-based methods for reporting %NPM1 mutation/ABL. Results: Linearity was demonstrated in Ct vs CN input for NPM1 mutation and ABL1 with R 2 above 0.96 for Lot1 and Lot2. Less than 3-fold difference was exhibited for CN and %CN across two lots of Xpert® NPM1 mutation test. Less than 3-fold difference was observed in %NPM1 mutation/ABL1 reporting between delta Ct-based and CN-based approaches. Conclusion: An NPM1 mutation copy number estimator for Xpert® NPM1 mutation test was established, which will provide diagnostic and prognostic values for NPM1-mutated AML patients.

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.

ITGB3 Promotes the Progression of JAK2V617F-Positive Myeloproliferative Neoplams By Regulating Calreticulin to Inhibit Cellular Autophagy

O bjectives: Integrins are integral cell-surface proteins composed of an alpha chain and a beta chain, of which integrin beta 3 (ITGB3) was known to participate in cell adhesion as well as cell-surface mediated signaling and plays vital roles in different tumors. However, the role of ITGB3 in JAK2V617F-positive classical myeloproliferative neoplasms (cMPNs) remians unclear, and the aim of this study was to explore the effect of ITGB3 on cellular autophagy in JAK2V617F-positive cMPN cell line and its underlying mechanisms. Methods: ITGB3 expression in bone marrows from JAK2V617F-positive cMPN patients compared to normal controls were detected by quantitative reverse-transcription PCR (qPCR) and further validated by western blotting. Cell models of cMPNs were constructed using human HEL cell lines carrying JAK2V617F mutation and cytokine-dependent wild type BaF3 cells (Jak2 V617F BaF3 cells) with ectopic expression of Jak2 V617F seperately to perform phenotype and mechanism investigation. The putative targets of STAT5a on the promoters of ITGB3 were disclosed by public databases and a dual-luciferase reporter assay and chromatin immunoprecipitation assay (ChIP) was used to analyze the binding of STAT5a to ITGB3 promoter region. To observe the role of ITGB3 played in cMPN progression, stable-transfected HEL cell line with ITGB3 knockdown or overexpression were established by lentivirus packaging and cell transfection. Cell phenotype including cell proliferation, apoptosis and cell cycle distribution were detected by soft agar colony formation assay, BrdU staining, flow cytometry and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays. The variations of autophagic flux after ITGB3 dysregulation in HEL cells was analyzed using mRFP-GFP-LC3 adenovirus transfection and fluorescence microscopy, and LC3-II/I ratio as well as autophagy-related protein were detected by western blotting. Transmission electron microscopy was used to observe the formation of intracellular autophagosomes and autophagic lysosomes. RNA sequencing and GSEA analysis were applied to predict the downstream pathway and putative targets of ITGB3. Western blotting, immunofluorescence and qPCR were performed to detect the transcription and expression levels of genes. Results: 1.Western blotting and qRT-PCR detection showed increased expression of ITGB3 in bone marrow of JAK2V617F-positive cMPN patients compared to normal controls (A-B). Also, the evaluation of ITGB3 in a variety of myeloid tumor cell lines (HEL, HL60, K562, KG-1a) and wild-type BaF3 cells as well as Jak2 V617F BaF3 cells revealed that ITGB3 protein level is the highest in HEL (which harboring the JAK2V617F) and Jak2 V617F BaF3 cells (C-F). 2. Dual-luciferase reporter assay showed that STAT5a significantly induced the transcription activities of promoters of ITGB3 and inhibition of STAT5a reduced the transcription activities of promoters of ITGB3 (G-H). ITGB3 knockdown (I) in HEL cells reduced the proliferation capacity (J-L), promoted early apoptosis (M-O) and caused cell cycle arrest (P-Q). While ITGB3 overexpression in HEL cells (R) reduced the cell cycle arrest (S) and apoptosis (T-U). 3. Autophagic flux analysis showed that ITGB3 knockdown promoted the autophagic flux in HEL cells (V-X). Inhibition of ITGB3 increases the synthesis and degradation of autophagosomes and accelerates the process of cellular autophagy (Y-Z). Further identification of DEGs in ITGB3-dysregulated HEL cells (AA-AB) reveals that ITGB3 inhibits the expression of calreticulin (AC-AE), and ITGB3 might be involved in the regulation of autophagy in HEL cells through the regulation of calreticulin. Conclusions: 1. ITGB3 is significantly activated in HEL cell lines carrying the JAK2V617F mutation. 2. ITGB3 protein acts as a pro-oncogene in JAK2V617F-positive myeloproliferative neoplams progression by regulating calreticulin to inhibit autophagy, which deserves more in-depth study in JAK2V617F mutation-positive cMPNs.

The Relapse Mechanisms and Genomic Landscape Differ in KMT2A-r Pediatric Leukemia in Relation to Relapse Time

The genomic landscape and mechanisms driving relapse in KMT2A-rearranged ( KMT2A-r) infant and childhood acute lymphoblastic (ALL) and acute myeloid leukemia (AML) are not completely understood. We therefore studied 36 KMT2A-r ALL (n=19) and AML (n=17) patients of which 25 relapsed and 11 remained in remission. Twenty diagnose-relapse-germline trios and 5 multiple relapse samples were analyzed by whole genome (WGS) and whole exome sequencing (WES) and 30 patients longitudinally by using patient-specific mutations identified by WGS/WES, including the KMT2A-r (average coverage 3300X). The mutational burden increased from diagnosis to relapse and relapse evolved through branching evolution. Relapse was seeded by multiple diagnostic clones in 56%, by a single sweeping clone detected at diagnosis in 22%, and by a single sweeping clone not detected at diagnosis in 22%. Notably, the evolutionary patterns correlated to relapse time, where multiple diagnostic clones seeding relapse were connected to an earlier relapse with all very early relapse ALL (3/3, relapse <9 months from diagnosis) and half of the early AML relapse showing this pattern (2/4, relapse <1 year in complete remission, CR1). By contrast, later relapse was connected to a sweeping clone at relapse with 67% of early relapse ALL (>9 months from diagnosis) and 40% of late relapse AML (>1 year in CR1) showing this pattern. Pathway analysis showed that cell cycle genes, glucocorticoid signalling, purine metabolism, mismatch repair, and B-cell differentiation, were enriched in early relapse ALL (83%, 5/6) and included TP53, CREBBP, NT5C2 PMS2, PRPS2, NR3C1, IKZF1, with none of the very early relapse infant ALL harboring such alterations (n=4). Further, TP53 and IKZF1alterationsco-occurred (n=4/4). These results were validated in public data sets of 98 KMT2A-r ALL infants (n=84) and children (n=14) at diagnosis and relapse (n=24) and showed that 50% of early relapse ALL, and none of the 8 very early relapse ALL, had such alterations. Ultra-deep sequencing did not detect the CREBBP, NT5C2, PRPS2or TP53 mutations at diagnosis and manual inspection of the WGS reads failed to detect the PMS2 and NR3C1 deletions. In AML, TP53 and CCND3 alterations were maintained, and gain of WT1 was seen in late relapse AML. Signalling mutations were the most common type of mutations at diagnosis (64%) and relapse (56%) and the frequency was similar in patients that remained in remission and in those that relapsed (55% versus 60%). One infant ALL and four AML patients had multiple relapses, allowing us to study how the genetic landscape evolved across consecutive relapses. This showed a stepwise replacement of clones during treatment in agreement with a fitter clone that evolves under chemotherapeutic selective pressure. Longitudinal analysis allowed sensitive detection of residual leukemia cells and showed that the relapse clone could be detected at diagnosis in 64% of patients. Further, infants with >10% of molecularly detectable leukemia cells after induction therapy, had a high risk of a very early relapse. Ultra-deep sequencing allowed detection of the relapse clone up to 4 months before relapse. In 11 of the 30 patients (3 remission and 8 relapse), low-frequency KMT2A-fusion positive leukemic cells were found at remission outside of the MRD time points. Our longitudinal data also provided unique insights into clonal response to treatment by showing that 1) a change in therapy can favour the eradication of one clone and expansion of another, 2) a clone that initially was the most sensitive clone to therapy, was the one that eventually caused relapse, and 3), a diagnostic clone can be undetectable for a long time before expanding to cause relapse, suggesting that molecular monitoring with personal mutations is a powerful tool to follow response to therapy. These results provide new biological insights into the relapse mechanisms in KMT2A-r leukemia. The data shows different clonal evolution patters depending on when in time the patient relapsed, with very early relapse ALL being seeded by multiple diagnostic subclones and a paucity of acquired genetic alterations at relapse. By contrast, early relapse ALL was characterized by a single diagnostic clone seeding relapse by a clonal sweep along with acquired mutations in chemoresistance-associated genes. To validate and extend these findings, we are currently analyzing 11 additional infant relapse samples with WGS.

One-Pot Era-CRISPR/Cas12a System for Rapid and Sensitive Detection of Multiple NPM1 Mutations

Nucleophosmin 1 (NPM1) gene c.863_864 4-bp insertion mutations occur in 30% of adult acute myeloid leukemia (AML) patients, which is associated with a unique pathological phenotype and good prognostic significance. The detection of NPM1 mutations is important for risk stratification, treatment guidance, and minimal residual disease monitoring in AML. However, the current detection methods have their limitations in terms of equipment requirements, sensitivity, turnaround time, price, etc. Moreover, simultaneous detection of multiple NPM1 mutation types is challenging. Here, we developed a one-pot ERA-CRISPR/Cas12a-based assay in which only one guide crRNA is needed to detect hundreds of NPM1 gene variants, covering over 97% of NPM1-mutated AML cases. By optimizing multiple parameters, this assay finally achieved a sensitivity of as low as 0.01% within 30 min detection under constant 39℃, without wild-type related cross signal. In clinical detection, 21 NPM1 mutation positive cases from 70 AML patient were successfully screened out using our method, including two cases not detected by Sanger sequencing. This method is one-step, rapid, sensitive, specific, inexpensive, and without the need of Sequencer or temperature cycler. It holds the potential to assist clinicians in point of care testing.

Calr Variant Allele Frequency in Essential Thrombocythemia: Molecular Associations and Impact on Disease Phenotype and Outcome

Background. A mutation in calreticulin ( CALRm) is found in 30-35% of patients (pts) with Essential Thrombocythemia (ET). There are 2 main CALRm mutation types, Type 1/Type 1 like (T1) a 52bp deletion, and Type 2/Type 2 like (T2) a 5bp insertion. Other atypical mutations (T3) occur in a minority of pts. Recent study ( Guglielmelli P et al, BCJ 2023) in pts with myelofibrosis (MF) showed that higher CALR variant allele frequency (VAF) was associated with anemia at diagnosis and during FU, and to features of more advanced disease (CD34 + cell counts, ASXL1 mutation (mut), >2 mutated myeloid genes, and shorter leukocytosis-free survival. Aim. To evaluate whether the CALRm VAF in pts with ET was associated with predefined major clinical outcomes: evolution to MF, transformation to acute leukemia (AML), thrombosis, major bleeding, and survival (OS). Patients and Methods. Diagnosis of ET was strictly according to 2023 WHO and ICC to avoid mis-inclusion of prefibrotic MF. CALRm VAF was determined by capillary gel electrophoresis as the ratio (%) of areas under the curve of CALRm/ CALRm+ CALRwt. A panel of 45 myeloid neoplasm-associated genes was sequenced by NGS. Results. A total of 281 CALRm ET pts were identified from CRIMM (Florence, I), Quebec MPN Research Group centers' (Canada) and Mayo Clinic (Rochester, US) databases; 152 (54%), 101 (36%) and 28 (10%) were T1, T2 and T3 CALRm, respectively. Overall FU was 8.6y (0.3-39.4), median survival was not reached. Rate of death at 10y, 20y and 30y was 8%, 15% and 25%. MF transformation occurred in 50 pts (18%), AML in 2 (0.7%), 25 pts (8.9%) died. A major thrombotic event before or at ET diagnosis occurred in 17 (6.0 %) pts (14 arterial and 3 venous), whereas 28 pts (10.0%) had >1 major thrombosis during follow-up (53.6% arterial, 46.4% venous). Major bleeding occurred in 19 pts (6.8%; 16 at or before diagnosis). Ascertainment of correlation of CALRm VAF with predefined outcomes by continuous variable analysis (ROC curve) highlighted a significant correlation of VAF >60% with MF-free survival (MFS), with HR of 2.85 (95%CI, 1.4-5.6; P= .002. Fig. 1A). On the contrary, there was no impact of CALRm VAF as continuous variable on AML, thrombosis, bleeding and OS. We therefore used a threshold of >60% to compare main clinical and hematologic characteristics at diagnosis and outcomes during FU in pts categorized as CALR-high and CALR-low. Of all pts, 21 (7.5%) harbored a VAF >60%; of these, 52%, 19% and 29% were T1, T2, T3, respectively, compared to 54%, 37%, 8% of CALR-low (P= .008). We found no difference in age, gender, constitutional symptoms, splenomegaly, arterial and venous thrombosis at diagnosis and FU, IPSET revised risk stratification, and bleeding events. Hemoglobin was lower (12.8 g/dL (8.4-14.0) versus 13.6 (10.1-16.0); P=.02), while leukocytes and platelets did not differ. The proportion of pts with additional mutations in myeloid genes was significantly greater in CALR-high pts compared to CALR-low (66.7% vs 30.2%; P=0.01), as it was the proportion of pts with >2 mut myeloid genes (25% vs 11.6%, P=0.04). During FU, 52% of CALR-high pts (n=11) transformed to MF compared to 15% (n=39) of CALR-low (P<.0001); AML and death not differ. Anemia (<10g/dL) during the FU developed in 69% of CALR-high pts vs 18%, leukocytosis (>15x10 9/L) in 46% vs 8% and splenomegaly (greater than 5 cm from LCM) in 50% vs 12% (P<.0001 for all). The HR for anemia-free survival was 2.87 (95%CI, 1.3-6.3), for leukocytosis HR 3.71 (95%CI, 1.3-10.5) and splenomegaly HR 3.40 (95%CI, 1.2-9.2), all P<.01. MFS was significantly shorter in T1 (HR 2.0; P=0.04) or T3 (HR 2.7; P=0.03) using T2 as reference. Finally, we compared C ALR-mut, JAK2V617F mut and triple-negative (TN) ET pts. ROC analysis indicated a JAK2VAF >35% ( JAK2-high) as the best cutoff for shorter MFS. As in Fig. 1B, the MFS survival of CALR-high and JAK2-high was superimposable, and significantly shorter that in CALR/JAK2-low and TN pts. Conclusions. A CALR VAF >60% in pts with ET is associated with greater risk and shorter time to MF progression, development of anemia and splenomegaly. Similar findings in JAK2-high pts reinforce that that accumulation of mutated CALR and JAK2 alleles, evident since diagnosis, is detrimental for evolution to post-ET myelofibrosis. Longitudinal studies after diagnosis might provide further information about the kinetics of allele accumulation.

KPT-8602 Induced Hematopoietic Differentiation in U2AF1 S34F Mutant Cells and Myelodysplastic Syndromes Bone Marrow Stem and Progenitor Cells

Myelodysplastic syndromes (MDS) are a group of clonal bone marrow (BM) neoplasms characterized by ineffective hematopoiesis, cytopenia's, and a high risk of progression to acute myeloid leukemia. Current standard therapies include erythropoiesis stimulating agents for lower risk disease and DNMTi for higher risk MDS; however, less than half of patients respond and even the best responses are transient and non-curative. The majority of MDS patients harbor at least one of ~40 commonly found somatic gene mutations, most of which occur in splicing factor genes such as U2AF1, which also have non-canonical roles in nuclear export. KPT-8602 (Eltanexor) is a novel selective inhibitor of nuclear export (SINE) that acts by disrupting nuclear export of cargo relevant in MDS. We hypothesized that U2AF1 S34 F mutant cells would be selectively sensitive to treatment with KPT-8602. We created immortalized murine hematopoietic progenitor cells by retroviral transduction of stimulated bone marrow (BM) cells isolated from U2AF1 WT or U2AF1 S34F mice with a HOXB8_ER vector. Estrogen drives the hematopoietic transcription factor immortalizing the cells in a progenitor state, however, upon estrogen withdrawal the cells will differentiate and eventually die. This differentiation can be driven by treatment with specific growth factors such as erythropoietin (EPO) for erythroid differentiation or granulocyte macrophage colony stimulating factor (GM-CSF) for myeloid differentiation. KPT-8602 exposure induced differentiation and increased hematopoiesis in murine cell models and primary MDS samples. U2AF1 WT and U2AF1 S34F cells were plated in medium lacking estrogen and treated with GM-CSF (10nM) or EPO (1nM) with or without KPT-8602 (10nM) for 72h, then stained with Ly6C, CD11b, Ter119, and CD71 for flow cytometry analysis. In addition, we performed colony forming capacity assays in both the murine cells and primary MDS BM stem and progenitor cells and age-comparable healthy donors, with 14 day KPT-8602 exposure. Treatment with GM-CSF significantly increased the expression of Ly6C in both U2AF1 WT and U2AF1 S34F cells (WT fold change untreated vs GMCSF =1.9, p=0.03; S34F fold change untreated vs GMCSF =2.3, p<0.01). However, addition of KPT-8602 caused significantly more Ly6C expression than GM-CSF alone in the mutant cells (WT fold change GMCSF vs combination =3.1, p=0.06; S34F fold change GMCSF vs combination =4.0, p<0.01). There were no significant changes in CD11b expression with KPT-8602 treatment. We did not find a statistically significant increase in erythroid markers assessed by Ter-119 and CD71 after EPO treatment, however, KPT-8602 treatment alone showed a trend of increased expression of Ter-119 and CD71 in the mutant cells that fell just below statistical significance (Ter119 p=0.08 for WT, p=0.09 for S34F; CD71 p=0.21 for WT, p=0.06 for S34F). Additionally, we looked at selective toxicity of U2AF1 S34Fby WST-1 reagent and found no differences in toxicity between the WT or mutant cells with KPT-8602. However, U2AF1 WT and U2AF1 S34F progenitor cells showed an increase in colony forming capacity (n=1 with technical duplicates) when plated in the presence of KPT-8602 showing an average fold change in colony growth of 1.4 for U2AF1 WT and 1.3 for U2AF1 S34F underscoring the ability of KPT-8602 to induce differentiation. To confirm these results were not cell line artifacts, we next treated primary BM stem and progenitor cells from MDS patients (n=2) and healthy donors (n=4). MDS patient 1 (age 73y) was diagnosed with MDS-MLD-EB1 and harbored mutations in TP53, ASXL1, BCOR, TET2 and PPM1D. MDS patient 2 (age 79y) was diagnosed with MDS/MPN and harbored an SF3B1 mutation. Healthy donors (n=4) were all male and had a median age of 65y. Treatment with KPT-8602 (10nM) significantly increased burst and colony forming unit-erythroid (BFU-E/CFU-E) in MDS primary cells (p=0.02) with no significant increase in the healthy donors (p=0.57) (BFU-E/CFU-E fold increase was 1.1 for healthy donors and 3.5 for MDS). Collectively, these findings suggest that KPT-8602 promotes myeloid and erythroid differentiation and further validates the use of KPT-8602 as a therapeutic strategy for MDS. Future studies will fully elucidate the specific mechanisms by which KPT-8602 acts in the context of splicing factor mutations, and other commonly found MDS mutation types.

Antibiotic resistance of Helicobacter pylori in Nanjing, China: a cross-section study from 2018 to 2023.

The increasing prevalence of antibiotic resistance in cases of Helicobacter pylori (H. pylori) infection has emerged as a significant global issue. This study offers a comprehensive examination of the alterations in drug resistance exhibited by H. pylori in the Nanjing region of China during the preceding five years. Another important objective is to investigate the influence of levofloxacin medication history on genotypic and phenotypic resistance. This research screened 4277 individuals diagnosed with H. pylori infection between April 2018 and May 2023. The phenotype and genotypic resistance were evaluated using the Kirby-Bauer disk diffusion and PCR method. The most recent primary resistance rates for metronidazole, clarithromycin, levofloxacin, amoxicillin, furazolidone, and tetracycline were recorded at 77.23% (2385/3088), 37.24% (1150/3088), 27.72% (856/3088), 0.52% (16/3088), 0.19% (6/3088), and 0.06% (2/3088), respectively. For the recent five years, we observed a notable upsurge in the rate of metronidazole resistance and a slight elevation of clarithromycin and levofloxacin resistance. The documented resistance rates to single-drug, dual-drug, triple-drug, and quadruple-drug regimens were 35.39%, 28.32%, 25.72%, and 0.21%, respectively. The prevalence of multidrug-resistant strains escalated, rising from 37.96% in 2018 to 66.22% in 2023. The rate of phenotypic and genotypic resistance rate (57.10% and 65.57%) observed in strains obtained from patients without a levofloxacin treatment history was significantly lower than the rate in strains obtained from those with a history of levofloxacin treatment (88.73% and 94.74%). The prevailing gyrA mutations were primarily N87K (52.35%, 345/659), accompanied by D91N (13.96%, 92/659), and closely followed by D87G (10.77%, 71/659). For gyrA mutations, the 91-amino acid mutants exhibit a higher likelihood of discrepancies between phenotypic and genotypic resistance than the 87-amino acid mutants. The extent of antibiotic resistance within H. pylori remains substantial within the Nanjing region. If levofloxacin proves ineffective in eradicating H. pylori during the initial treatment, its use in subsequent treatments is discouraged. The employment of levofloxacin resistance genotype testing can partially substitute conventional antibiotic sensitivity testing. Notably, predicting phenotypic resistance of levofloxacin through PCR requires more attention to the mutation type of gyrA to improve prediction accuracy.

Myopathy with Lactic Acidosis and Sideroblastic Anemia Caused By a Novel PUS1mutation

Mutations in the mitochondrial aminoacyl tRNA synthetases (ARSs) are associated with various clinical phenotypes, including myopathy, lactic acidosis, and sideroblastic anemia (MLASA), which is a rare autosomal recessive disorder. Due to oxidative phosphorylation disorder and abnormal iron metabolism in the skeletal muscle and erythroid bone marrow lines, patients show progressive muscle weakness, exercise intolerance, lactic acidosis, siderocyte anemia, and growth retardation, etc. Herein, we report a case of MLASA caused by a novel homozygous missense mutation (c.597G>T p.R199S) in the pseudouridine synthase 1 ( PUS1) gene, causing the loss of protein function. The patient's parents had a consanguineous marriage, with a grandmother in common. All mutations were heterozygous without disease. While the patient was homozygous for the mutation and developed symptoms of anemia, no specific treatment exists for this genetic disease. Deferrization therapy can reduce iron overload, which leads to reduced heme synthesis in such patients but cannot treat the disease. Consanguineous marriage often causes autosomal recessive genetic diseases and prohibiting it can reduce the incidence of these diseases. The present patient's condition was caused by a mutation in nuclear PUS1, which manifested as SA, hyperlactatemia, iron overload, and muscle damage. PUS1 mutations can be missense, nonsense, or frameshift. We identified a novel missense mutation type in PUS1 in this patient, located on chromosome 12, c.597G>T (p.R199S), which is a homozygous mutation. This was confirmed by the PUS1 analysis revealing homozygosity for the novel mutation. Both the parents and grandmothers were heterozygous for the mutation, but none of them had a clinical manifestation. Combining the patient's family history, medical history, laboratory examination, and genetic characteristics of the disease, the mutation was identified as a pathogenic variant, and the clinical manifestations and genetic findings of the patient expanded the genotype-phenotype spectrum of MLASA. The possibility of MLASA should be suspected in cases of progressive muscle weakness, growth retardation, exercise intolerance in childhood, SA, and lactic acidosis before or after puberty. With the recognition of MLASA and the advent of NGS, identifying patients with PUS1 mutations characterizing incomplete MLASA syndrome became easier, thus further expanding the phenotypic and genotypic heterogeneities of these mitochondrial disorders. Targeting NGS may also significantly improve the prenatal and preimplantation genetic diagnosis of MLASA, providing better genetic counseling for parents. Genetic diseases lack effective treatment measures, and the curative effect is unsatisfactory. Therefore, prevention is more important. Prevention strategies include avoiding consanguineous marriage, genetic counseling, genetic testing for carriers, and a prenatal diagnosis.