Peripheral Blood Counts Research Articles

Venetoclax in Combination with Intermediate Doses of Cytarabine in Consolidation Phase for Acute Myeloid Leukemia Patients in First Complete Remission; Results of the Part 1 of the Phase 1/2 Multicentric Covenidac Study

For authors: Please: L Gastaud and P Peterlin: co-primary, H dombret et C Recher: co-last Background: The French ALFA-FILO AML intergroup launched the BIG-1 trial (EudraCT No.: 2014-000699-24) in 2015 to evaluate different intensive chemotherapy (ICT) strategies aiming at improving the survival of younger adults with acute myeloid leukemia (AML). All patients (pts) with previously untreated non-APL and non-CBF AML aged 18-60 years were eligible for this trial. The trial design included several randomization types. Results of the R1 and R2 randomizations, namely idarubicin vs daunorubicin for induction and IDAC vs HDAC for consolidation, will be reported at this meeting. In patients who achieved first complete remission (CR), randomizations R4 consisted of nested randomized Phase 2 or 1/2 studies evaluating the addition of new drugs to the IDAC/HDAC consolidation cycles. The protocol was designed to allow several sequential multicenter R4 evaluations of various new agents over the trial period. Venetoclax (VEN) has proved its efficacy when combined to azacitidine or low dose cytarabine in unfit AML pts (DiNardo et al. 2020; Wei et al. Blood 2020). Its role in fit patients treated with ICT is still under investigation. The COVENIDAC Phase 1/2 R4 sub-study is evaluating the safety and efficacy of VEN added to IDAC during the consolidation cycles of CR pts. We report herein the result of the Phase 1 part. Methods: The primary objective of this Phase 1 study was to determine the maximum tolerated dose (MTD) of VEN in combination with IDAC during the consolidation cycles, which will be the recommended dose for the Phase 2 (RP2D). Dose limiting toxicities (DLTs) were assessed during the first cycle of consolidation (C1) according to a 3+3 design. A maximum of 3 consolidation cycles was planned depending on the AML risk group. Hematological DLT was defined as no peripheral blood count recovery within 56 days from day 1 of the cycle. Non-hematological DLTs were defined as Grade ≥4 toxicities or Grade ≤3 toxicities which have not recovery by day 56 and deemed related to VEN per investigator opinion. VEN was started at dose level (DL) 1: 100 mg/d from day 1 to 8. In the absence of DLTs at DL1, 3 others DL were planned: 200 mg/d from day 1 to 8 (DL2); 400 mg/d from day 1 to 8 (DL3), and 400 mg/d from day 1 to 14 (DL4). IDAC was administrated at 1.5 gr/m² twice daily on day 1/3/5 for DL1, DL2 and DL3 pts, then on day 1/2/3 for DL4 pts after a protocol amendment. Main inclusion criteria were: pts in first CR following 1 or 2 courses of induction chemotherapy fit for further ICT; favorable and intermediate risk AML as defined by the protocol; ECOG PS ≤2; written informed consent. Measurable residual disease (MRD) was assessed by multiparametric flow cytometry (MFC) in all pts as well as NPM1-qPCR in NPM1-mutated pts. Results: Between 06/2021 and 04/2022, 15 pts from 9 centers were screened and enrolled. Pt characteristics were as follows: median age, 42 years (IQR, 30-49); 2 males/7 females; ECOG-PS, 0 in 10 pts and 1 in 5 pts; favorable/intermediate/adverse ELN-2017 risk, 9/2/4. Neutropenia and thrombocytopenia durations are shown in Table 1. Only 1 hematological DLT was observed at DL3 (platelet count of 96 giga/L at day 56 in a patient who was infected by Covid-19). Overall, there were very few non hematological AEs ( Table 2, no grade 4) and only 4 SAEs (1 related to IDAC/VEN during C1, 3 non-related). No non-hematological DLT was observed. No patient died during the DLT observation period, 1 experienced a septic shock (C2, DL2). One pt received only C1 (due to DLT), 7 received 2 cycles, and 7 received 3 cycles. Median time between initiation of subsequent consolidation cycles was 37.5 days (IQR, 35-41) for C1/C2 and 40 days (IQR, 35-43) for C2/C3. One pt had to stop VEN during C3. Six pts received an allogeneic HSCT (5 in CR1, 1 after salvage). With a median follow-up of 13.5 months, 3 pts relapsed and 2 died. MRD results will be presented. Conclusion: VEN/IDAC combination appears to be feasible and safe as consolidation therapy, with expected thrombocytopenia and neutropenia durations. These durations appeared even shorter at DL4, when IDAC was given on 3 consecutive days. VEN at 400 mg/d from day 1 to 14 combined with IDAC on day 1/2/3 was thus retained as RP2D for the randomized Phase 2 with RFS as primary endpoint. This phase 2 has already recruited 29 of the 200 pts planned.

Variability of bone marrow biopsy reporting affects accuracy of diagnosis of myeloproliferative neoplasms: data from the ALLG MPN01 registry

The Philadelphia-negative myeloproliferative neoplasms (MPN) are a heterogeneous group of overlapping bone marrow disorders defined by characteristic peripheral blood counts and bone marrow morphological findings in conjunction with recurrent somatic mutations. The accurate diagnosis and subclassification of MPN relies upon careful reporting of bone marrow morphology combined with ancillary information in an integrated pathology report. This co-operative trial group study ALLG MPN01 (ANZCTR:12613000138785), led by the Australasian Leukaemia & Lymphoma Group (ALLG), aimed to describe the current approach to diagnosis of MPN in routine practice. Specifically, we assessed the frequency with which bone marrow biopsies were performed, and the adherence of reporting pathologists to recommendations contained in the revised 2016 WHO classification pertaining to MPN. We reviewed the diagnosis of 152 patients from eight institutions who were enrolled in a national MPN registry of the ALLG between 2010 and 2016. The ALLG MPN01 registry is now closed to recruitment. Key features were extracted from pathology reports provided to the registry. Bone marrow biopsies were performed in 112/152 cases (74%). The pathological information entered was concordant with the stated clinical diagnosis in 75/112 cases (67%). The main reasons for discordant results were incomplete descriptions of megakaryocyte topography and morphology, inconsistent grading of reticulin fibrosis, and failure to integrate the available morphological and ancillary clinicopathological information. In this retrospective audit, 26% of MPN patients did not undergo a diagnostic bone marrow biopsy. In those who did, the specific MPN subtype may not have been reported correctly in 33% of cases, as evidenced by inconsistent features reported or insufficient information to assess. A more standardised approach to bone marrow reporting is required to ensure accuracy of MPN diagnoses and consistent reporting to cancer registries and clinical trials.

The immunological response of Egyptians to coronavirus disease-19 infection: a cohort study of lymphocyte populations and peripheral blood counts

Background The worldwide pandemic of COVID-19 infection that started in 2019 still lays its shadows over all populations of the world. COVID-19 infection presented with a spectrum of symptoms that varied from wave to wave, and also led to a wide number of long-term sequelae. Many immune system cells and cytokines were implicated in COVID-19 pathophysiology. Thus, many immuno-modulator and immuno-suppressive drugs were used in the management of severe cases. Lymphocytes are the key players of immune system, the change in their count and different subsets is expected to vary with COVID-19 infection. Objective The current study aimed to evaluate the role of peripheral blood lymphocyte subsets in predicting the outcome of COVID-19 patients and to investigate their correlation with different clinical and laboratory variables. Materials and methods The study included 64 patients hospitalized with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). They were stratified according to in-hospital mortality into survivors and nonsurvivors. Demographic, clinical and laboratory data were collected. Flowcytometric evaluation of lymphocyte subsets was done on admission. Results and conclusion Nonsurvivors showed lower relative lymphocyte count, higher absolute neutrophil count, and higher neutrophil to lymphocyte ratio (NLR) compared with survivors (P = 0.034, 0.006, 0.011; respectively). NLR at a cut off 15.3 had a sensitivity of 70.59% and specificity of 61.29% for predicting mortality in COVID-19 patients. The relative and absolute counts of lymphocyte subsets did not show a statistically significant difference between the two groups. Platelet count showed statistically significant positive correlation with absolute counts of total T lymphocytes, T helper, T cytotoxic, and B lymphocytes. The platelet to lymphocyte ratio (PLR), NLR and D-dimer results were negatively correlated with the total T lymphocytes, T helper, T cytotoxic, naïve T cytotoxic and B lymphocyte absolute counts. The NLR, absolute neutrophil count and platelet count may serve as adjuvant predictors of survival in COVID-19 disease. Although lymphocyte subsets did not differ statistically across survival groups, their correlation with other possible prognostic markers may justify further investigation on their role in COVID-19 pathophysiology.

Outsmart™ IL-2/15, a Novel Cytokine Designed to Enhance ROR1-Specific CAR T Anti-Tumor Activity While Minimizing Treg Activation and Systemic Cytokine Exposure

Introduction: Effective treatment with chimeric antigen receptor T (CAR-T) cells against solid and some liquid tumors will require the safe production of immune-modulating cytokines to promote CAR-T cell expansion and to stimulate endogenous anti-tumor immune responses within the tumor microenvironment. OutSmart™ IL-2/15 is a computationally designed, CD8α-targeted, IL-2-based cytokine produced by engineered T cells via a T cell activation-dependent promoter. Here, we demonstrate that inducible production of CD8-targeted IL-2/15 drives proliferation and anti-tumor efficacy of ROR1-specific CAR-T cells, stimulates bystander CD8 + T and NK cells, and minimizes activation of immune suppressive T regulatory cells (Tregs). Methods and Results: Wild-type (wt) IL-2 was modified using Rosetta protein design software package to eliminate IL-2Rα binding while retaining native IL-2Rβγ binding interfaces. Out of 10,355 designs, 38 were prioritized based on in silico analysis and ML-guided structure prediction, and evaluated for IL-2R binding as recombinant proteins. Consistent with the design criteria, none of the designs bound IL-2Rα, and 33 (87%) retained IL-2Rβγ binding and signaling activity. pSTAT5 analysis showed wt IL-2 highly activated CD4 +CD25 +FoxP3 + Treg cells, whereas the novel IL-2 designs showed a >3-log reduction Treg stimulation. These designs were iteratively optimized to generate a version exhibiting improved thermostability and IL-2Rβγ binding affinity equivalent to wt IL-2. To increase the potency against immune effector cells, but not Tregs, an anti-CD8α VHH was tethered to the modified IL-2 molecules (CD8-IL-2/15) to mimic high-affinity IL-2Rα binding and selectively activate CD8 + T and NK cells. This prosthetic binding interaction dramatically increased CD8 + T cell stimulation and CD8α + NK cell activation (>2-logs and 1-log by pSTAT5, respectively), while retaining its reduced capacity to stimulate Tregs. CD8-IL-2/15 gene expression was subsequently placed under the control of a T cell activation-dependent promoter and integrated into a lentiviral construct with a constitutively expressed ROR1-specific CAR. T cells transduced with a lentivirus expressing CD8-IL-2/15 and a ROR1-targeting CAR showed inducible cytokine production following exposure to ROR1 + tumor cells (H1975) leading to enhanced CAR-T cell proliferation and anti-tumor killing during repeat tumor challenge assays in vitro. Using immune-deficient mice (NSG) engrafted with H1975 tumors, inducible CD8-IL-2/15 enhanced ROR1-specific CAR-T cells showed durable tumor control at low doses (1e6 - 4e6 CAR T cells/animal), which was comparable to wt IL-2, whereas mock T cells and ROR1 CAR only T cells failed to control tumor growth ( Figure 1). In addition, CAR-T expansion with CD8-IL-2/15 increased >4-fold over wt IL-2 (p<0.0001), measured by cell counts in peripheral blood. Conclusions: In summary, OutSmart™ IL-2/15 is a genetic module that produces CD8-IL-2/15 in response to T cell activation, promoting robust CAR-T expansion and enhanced anti-tumor efficacy. Furthermore, local production of a CD8-IL-2/15 enhances the effector function of bystander CD8 + T cells and NK cells, but only minimally activates Treg cells due to removing the IL-2Rα binding interface. Inducible production of CD8-IL-2/15 dramatically improves the potency of a ROR1 CAR for treating ROR1 + solid and liquid cancers. More generally, OutSmart™ protein design methods and control technologies can be applied to create other designed cytokines for oncology and beyond.

Single-Cell Multi-Omics Reveals Type-2 Functionality in Maintaining CAR T Cell Longevity Associated with 8-Year Leukemia Remission

Despite a high response rate in chimeric antigen receptor (CAR) T therapy for acute lymphocytic leukemia (ALL), ~50% of patients relapse within the first year, representing an urgent question to address in the next stage of cellular immunotherapy. The pioneering clinical trials conducted at Upenn/CHOP provide a unique opportunity to examine the molecular determinants of ultra-long CAR T persistence. We performed single-cell multi-omics profiling of ~700k pre-infusion CD19-targeted 4-BB CAR T cells from 82 pediatric ALL patients and 6 healthy donors. Patient demographics were collected between Sep-2012 to July-2022. To uncover the hallmarks of CAR T longevity, we correlated the single-cell atlas with the duration of B-cell aplasia (BCA), a widely used pharmacodynamic measurement of CAR T persistence, and classified all the patients into 5 groups (Table 1). Five patients in our cohort have exhibited a median BCA duration of 8.4 years (BCA-L group) and another 11 patients continue to have BCA with a median time of 5.1 years (BCA-O group). To remove potential confounding variables between trial design, 42 patients including the 5 BCA-L were analyzed as the Discovery Cohort, while the other 40 patients including the 11 BCA-O were used as Validation Cohort. Our analysis of CD19-specific stimulated CAR T cells from the Discovery Cohort revealed a prominent role of type-1 function, which was highly represented but had no discernible correlation with long-term persistence. Unexpectedly, we identified that elevated type-2 functionality was significantly associated with BCA-L patients maintaining CAR T persistence of 8.4 years, and this signature was robustly presented in the BCA-O patients in our Validation Cohort. Higher type-2 cytokine level of long-term persistent CAR T cells was independently validated using both flow cytometry and multiplexed secretomic assay. Through ligand-receptor interaction analysis, type-2 cytokines were found to regulate a cluster of Tim-3+terminal effector cells showing over activation of cytotoxicity, impaired immune function, elevated exhaustion signature and diminished proliferative potentiality, and CAR T cells from BCA-L patients showed reduced dysfunctional hallmarks. We next conducted in vitro functional studies to assess the impact of adding type-2 cytokine (IL-4) during CAR-specific activation on CAR T fitness. The addition of 10ng/mL IL-4 significantly boosted the proliferation and mitigated dysfunction of CAR T cells from 6 patients in the BCA2 group that only mediated short-term response (~3 months) and had negligible impact on their type-1 functionality. Remarkably, the functional profile of short-term BCA2 CAR T cells after IL-4 supplementation exhibited a comparable pattern to that of long-term BCA-L CAR T cells, particularly in terms of type-2 pathway, oxidative phosphorylation metabolism, PI3K/AKT signaling, mTOR signaling, and cell cycle regulation. To investigate the response of the host following CAR T infusion, we performed a comprehensive proteomic profiling of post-infusion serum proteins in a total of 41 patients. Baseline measurements (-2 to 0 days before CAR T infusion) showed a significantly higher level of type-2 cytokines in long-term patients. From the rapid expansion period (day 6-8) until 11 days post-infusion, we consistently observed significantly higher levels of circulating type-2 cytokines in 5-year or 8-year relapse-free responders. Conversely, no difference was observed in type-1 cytokines. In a leukemic NSG mouse model, both type-2 high and low CAR T cells were highly effective in reducing tumor burden in the first two weeks. However, type-2 high CAR T had significantly superior expansion, and the absolute CAR+ cell counts in peripheral blood were 10-fold higher than type-2 low CAR T at day 8 and day 12. To mimic tumor cell relapse, a second dose of tumor cells were injected into mice on day 17. Notably, type-2 high CAR T cells demonstrated a potent capability to elicit recall responses upon leukemia rechallenge, significantly prolonging the survival of tumor-bearing mice compared to type-2 low CAR T. The superior performance could be associated with their decreased exhaustion and increased memory phenotype. Our findings provide key insights into the mediators of CAR T longevity and suggest a potential therapeutic strategy to maintain long-term remission by enhancing type-2 functionality in CAR T cells.

Predictive Modeling of Hematopoietic Stem Cell Mobilization and Collection Based on Peripheral Blood Marker

Background: Autologous stem cell transplantation (ASCT) is the major transplantation therapy for patients with lymphoma and MM, and the success of auto-HSCT depends on the mobilization efficiency of peripheral blood HSC. In clinical practice, CD34 + cell count in peripheral blood is often monitored to predict the collection effect and determine the collection time. However, prediction of poor mobilizers is still difficult . Methods: We retrospectively analyzed 108 patients with lymphoma and multiple myeloma(MM) at Chongqing University Cancer Hospital who underwent autologous hematopoietic stem cell (HSC) collection between January 2021 to August 2022. There are two groups: collection success group and collection failure group. Compared the differences of clinical characteristics and impact of peripheral blood related detection indexes before autologous HSC collection. Logistic regression analysis was used to establish a prediction model for the successful collection by multi-marker and further evaluated multi-marker prediction models. Results : There was no significant difference in sex, disease type, disease stage, age, disease status before mobilization, mobilization drugs and chemotherapy courses between the successful collection group and the failed collection group ( P> 0.05). The proportion of patients with mobilization days less than 5 days was higher in the successful collection group ( P< 0.05). The platelet count (PLT), mononuclear cell count (MNC) and absolute CD34 + cell count in the successful collection group were significantly higher than those in the failure group. The sensitivity, specificity and area under the curve (AUC) of combining three indexes were 80.9%, 95.9% and 0.944, respectively. Applied the Logistic regression models to the absolute count of PLT, MNC and CD34 + cells to construct the multi-marker prediction algorithms. The 3-marker model of PLT+MNC+CD34 + cells had the lowest AIC, BIC and high AUC. A prediction model was established based on the absolute count of PLT, MNC and CD34 + cells. Conclusion :Patients with less than 5 days of mobilization have a higher success rate of collection. PLT, MNC and CD34 + cell counts before collection can reflect HSC mobilization in patients with lymphoma and MM, and the combination of the three can improve the accuracy of prediction of collection effect.The predictive model based on the absolute number of PLT, MNC and CD34 + cells have good predictive value. Our study suggests that multi-markers are important diagnostic tools for predicting successful collection.

Oral Decitabine/Cedazuridine with Venetoclax in High-Risk Myelodysplastic Syndrome or Chronic Myelomonocytic Leukemia: Analysis By Different Response Criteria

Introduction: Patients with higher-risk myelodysplastic syndromes (HR-MDS) have poor survival. The standard of care for HR-MDS are hypomethylating agents (HMA). The addition of venetoclax (Ven) to HMA has been shown to improve outcomes in acute myeloid leukemia (AML) and potentially in HR-MDS. The combination of oral decitabine/cedazurridine (DEC-C) with Ven has been shown to induce high response rates in patients with HR-MDS. Herein we analyze the responses of patients treated with DEC-C with Ven according to different response criteria. Additionally, we analyze the cytogenetic responses and mutation dynamics. Methods: We analyzed patients included in a phase I/II single center clinical trial (NCT04655755). diagnosed with treatment-naïve MDS (IPSS intermediate-2 or high) or CMML with excess blasts. Responses were analyzed by the International Working Group (IWG) criteria for MDS of 2006 and 2023, as well as the European LeukemiaNet (ELN) criteria for AML (2022). Cytogenetic responses and mutations were analyzed by conventional cytogenetics/FISH and NGS, respectively. Results: Between January 2021 and January 2023, 39 patients were enrolled. The median age of the entire cohort was 71 years old (range, 27-94), with 26 (70%) male patients. The WHO 2016 diagnosis was MDS with excess-blasts 1 (n=6, 16%), MDS with excess-blasts 2 (n=24, 65%), CMML-2 (n=6, 16%) and atypical chronic myeloid leukemia (n=1, 3%). The cytogenetic risk according to IPSS was good (n=11, 30%), intermediate (n=12, 32%) and adverse (n=14, 38%). The most common mutations found were ASXL1 (n=18, 49%), RUNX1 (n=14, 38%), SRSF2 (n=11, 30%), TET2 (n=8, 22) and TP53 (n=7, 19%). According to the IWG-2006 classification, the overall response rate was 95%, with 17 (43%) patients achieving complete remission (CR) and 20 (52%) patients achieving marrow CR (mCR). When using the IWG-2023 criteria, the overall response rate was 82%, with 23 (59%) patients achieving CR and 9 (23%) achieving CR with limited count recovery/CR with partial hematologic recovery. Of note, 5 patients achieved mCR according to the IWG-2006 but did not achieved CR by the IWG-2023 due to the peripheral blood count. Moreover, we did not find differences in overall survival (OS) between CR and mCR (OS of 60% for both groups, p=0.5). When using the ELN 2022 criteria for acute myeloid leukemia, the ORR was 80% with 2 patients not responding and 2 patients in a morphologic leukemia-free status (MLFS) (Figure 1). Both IWG-2006 and 2023 define cytogenetic response as complete disappearance of chromosomal abnormalities present at diagnosis. 14 patients out of 29 (48%) with cytogenetic abnormalities achieved cytogenetic response after a median of 2 courses of treatment (1-9). The IPSS cytogenetic risk of patients responding was good in 1 (7%) patient, intermediate in 8 (57%) patients and poor in 5 (36%) patients, including 3 patients with complex karyotype. Among patients not achieving cytogenetic response, 5 (33%) and 10 (67%) patients had an intermediate and adverse IPSS cytogenetic risk, respectively. Although molecular responses are not defined in the IWG-2023, we studied the molecular dynamics between diagnosis and best response. 23 patients of the study had molecular data in paired samples at diagnosis and at CR while on treatment. When analyzing the dynamics of each individual mutation, STAG2 (11/11, 100%), NRAS (3/3, 100%), BCOR (4/5, 80%) and IDH1 (2/3, 67%) showed the higher rate of clearance from diagnosis to CR. On the contrary, ZRSR2 (3/4, 75%), U2AF1 (3/4, 75%), TP53 (3/4, 75%), SRSF2 (5/7, 71%) and ASXL1 (10/14, 71%) showed the higher rate of persistence from diagnosis to CR (Figure 2). Among all patients in the study, 19 (49%) proceeded to hematopoietic stem cell transplantation (HSCT). Eleven patients had molecular data after HSCT, all achieving complete mutation clearance after HSCT. Conclusion: DEC-C with Ven achieves a high rate of responses, although with the IWG-2023 criteria responses are lower due to the disappearance of mCR and more strict criteria in terms of peripheral blood count recovery. Almost half of patients with cytogenetic abnormalities can acquire cytogenetic responses, including poor risk cytogenetics. Subclonal mutations (like STAG2 or NRAS) can be cleared with this combination while founding mutations (like ASXL1 or TP53) tend to persist, although HSCT has a potential to eliminate them.

Epigallocatechin-3-Gallate (EGCG) Ameliorates Ineffective Hematopoiesis of Myelodysplasia Syndrome (MDS)

Epigallocatechin-3-gallate (EGCG) is a gallate ester obtained by condensation of gallic acid with the (3R)-hydroxy group of (-)-epigallocatechin, extracted from green tea, exhibiting beneficial effects as an apoptosis and cell differentiation inducer helping counteract uncontrolled proliferation and impaired differentiation characteristic of leukemia and myelodysplastic syndrome. Clinical trials in patients with chronic lymphoid leukemia, low grade B-lymphomas and acute myeloid leukemia with myelodysplasia related changes were carried out showing positive results. This study aimed to evaluate the in vivo effect of EGCG in a mice model of myelodysplastic syndrome (NHD13 mice). B6.SJL-PtprcaPep3b/BoyJ mice (PepBoy) received 9,5Gy irradiation followed by transplantation of bone marrow cells from NUP98-HOXD13 mice by i.v. caudal vein injection; disease establishment was confirmed at day 15 by blood cytopenia. To assess the EGCG effects on MDS, 10-week-old female NUP98-HOXD13 mice (NHD13 +) and their littermate WT mice (NHD13 -) were treated for 4 weeks with EGCG (50mg/Kg/day 5x/week i.p.) or vehicle. The animals were then sacrificed, and bone marrow and spleen were obtained for flow cytometry and histology analyses. Automated hematological counts and clinical biochemistry parameter analysis were done. After signing the written informed consent, a 71-year-old man diagnosed with high-risk MDS, with 9% blasts in the bone marrow, underwent a daily oral 800mg EGCG treatment. This study was approved by the Ethical Committee of University of Campinas and by the National Research Ethics Committee ( CAAE: 40656720.5.0000.5404). During this period, the patient underwent weekly clinical assessments and laboratory studies to monitor any potential toxicity. Peripheral blood samples were obtained before and after treatment to observe any alteration in cell counts and immune cell composition. Before treatment, NDH13 + exhibited a reduction in number of leukocytes, platelets, and hemoglobin levels, in accordance with the literature. EGCG treatment ameliorated hematological parameters, showed by increased leukocyte numbers (monocytes and neutrophils) and platelets with no signals of liver and kidney toxicity detected by histology and serum biochemistry. Untreated mice showed increased osteoprogenitor, osteoblast and endothelial cells in the bone marrow, corroborating the literature. EGCG treatment did not alter the frequency of these cells, HSC or immature cells (CD34 + or CD117 +). However, EGCG induced extramedullary hematopoiesis in spleen and no changes in the percentage/frequency of Foxp3 + regulatory T cells (CD4 +CD25 + and/or CD4 +FOXP3 +). Additionally, the patient treated by ECGC for one month exhibited reduction of regulatory T cells CD4 +CD25 + and CD4 +FOXP3 +, and increased CD8 + T cells (CD3 +CD8 +) and natural killer cells (CD16 +CD56 +) accompanied by increased mature leukocytes (monocytes and neutrophils), with no changes in hemoglobin and platelet number. Our initial findings indicate that EGCG induces improved hematological parameters in MDS and might shift immune cells to a more cytotoxic phenotype. The extramedullary hematopoiesis observed in mice may be due to reduction of CXCR4 in bone marrow cells, as previously described by our group. In summary, EGCG might be a promising adjuvant in the treatment of MDS. Further research and clinical studies are warranted to validate and fully explore EGCG therapeutic benefits. Fig1a EGCG ameliorates NHD13 and transplanted mice hematological parameters. At the end of the EGCG treatment (50mg/kg/day i.p) or vehicle (Ctrl), NHD13 + and transplanted (PepBoy) mice were bled from the retro-orbital plexus. EGCG induced an increased the number of leukocytes ( A), monocytes ( B), neutrophils ( C) and the number of platelets ( D) using the CELL-DYN Emerald Hematology System counter. EGCG represented by the green column, and the vehicle by the gray color. Statistical significance ( Mann-Whitney test) is indicated; n=6 per group. Fig1b. EGCG improved the immune system of HR-SMD patient. A 71-years-old man with HR-SMD received EGCG treatment (800 mg/Kg/day) for 30 days. Peripheral blood counts were evaluated at day 0 and day 30. EGCG ameliorated hematological parameters - increased mature leukocytes (A) - monocytes (B) and neutrophils (C), with no changes in hemoglobin (D) and platelets

Severe Congenital Neutropenia-Type 5: Impaired T Cell Proliferation, Aberrant Th1 Cytokine Production, Abnormal Megakaryocytes, and Impaired Platelet Granule Formation in a Patient with VPS45 Deficiency Caused By Uniparental Isodisomy

Introduction VPS45 is essential in early endosome formation with rabenosyn-5 and syntaxin 16. VPS45 deficiency is one of the inborn errors of immunity characterized by severe congenital neutropenia and myelofibrosis. Impaired production and function of neutrophils, increased cell death, and abnormal hematopoiesis have been reported, whereas there are several undetermined pathophysiologies, such as T and/or NK lymphopenia or myelofibrosis. Here, we experienced a girl with VPS45 deficiency presenting with neutropenia at birth, progressive T and NK lymphopenia, hepatosplenomegaly, and myelofibrosis in infancy. We examined the pathophysiology of VPS45 deficiency focusing on hematologic disorder, T cell function, and myelofibrosis. Methods We sequentially evaluated peripheral blood and bone marrow cell counts and morphologic features. Genetic analysis was performed using DNA from peripheral blood. We evaluated T cell proliferation using the CFDA-SE cell tracer kit with the stimulation of PHA-L or anti-CD3/CD28 beads. Serum cytokine analysis was performed by beads array. Protein expression was examined in platelets from the patient and the healthy control by western blot analysis. Results Case: The patient was born to nonconsanguineous Japanese parents. She received a blood examination for mild respiratory distress at birth. This examination demonstrated neutropenia, the emergence of myeloid cells, and nucleated red blood cells in the peripheral blood, whereas mature neutrophils were confirmed in the bone marrow. Genetic analysis demonstrated homozygous pathogenic variants of p.E238K in VPS45. The same heterozygous variant was confirmed in her father, but not in her mother. SNP array analysis demonstrated that she inherited chromosome 1 only from her father and developed VPS45 deficiency by paternal uniparental isodisomy. At 7 months, she demonstrated T and NK lymphocytopenia, macrothrombocytopenia, and hepatosplenomegaly. Bone marrow examination demonstrated the presence of blast cells, micro or separated megakaryocytes, and pseudo-Pelger-Huet anomaly. Electronic microscopy of peripheral blood demonstrated abnormal granules in neutrophils and macro-thrombocytes with reduced alpha granules and open channel enlargement. Elevated interferon-gamma, IL-12, and IL-23 levels, representing T helper 1 (Th1) activation were found. We initiated ruxolitinib at 7 months to suppress aberrant Th1 activation and extra-medullar hematopoiesis which could be associated with massive hepatosplenomegaly. Ruxolitinib was effective for hepatosplenomegaly and reduced Th1 cytokine levels without significant complications. She received hematopoietic cell transplantation with bone marrow from an HLA-matched unrelated donor 30 days after the initiation of ruxolitinib. T cell function: With anti-CD3/CD28 beads stimulation, the patient's CD8-positive cells were significantly less proliferative. Either CD4-positive or CD8-positive cells did not proliferate with PHA-L stimulation. Platelet analysis: Western blot analysis of platelet demonstrated a reduced amount of alpha granule proteins (von Willebrand factor and PF4), not of p-selectin. Discussion VPS45 deficiency was characterized by severe congenital neutropenia and myelofibrosis. Furthermore, T cell deficiency, especially CD8-positive T lymphopenia was observed in some patients with VPS45 deficiency. Our study suggested that T cell lymphopenia would be associated with impaired TCR signaling and mitogen response, although the detailed mechanism remains to be elucidated. The impaired platelet alpha granule formation confirmed by electron microscopy and western blot analysis in our patients was compatible with the previous VPS45 deficiency patient. A similar manifestation is observed in gray platelet syndrome which is also associated with affected endosomal transport. Affected endosomal transport would be responsible for myelofibrosis. We hypothesized that massive hepatosplenomegaly was attributable to the hyper-activated Th1 cells and extra-medullar hematopoiesis associated with JAK1 and JAK2 signaling. Indeed, JAK1/ JAK2 inhibitor, ruxolitinib improved hepatosplenomegaly in this patient. Conclusion VPS45 deficiency could broadly affect not only neutropenia and myelofibrosis but T cell, NK cell, megakaryocyte, and platelet function.

A Single-Arm, Open-Label, Multicenter Study to Assess Molecular Response of P1101 Therapy in Patients with Polycythemia Vera and Elevated Hematocrit: Results from 12-Month Core Study

Background: The treatment goals of Polycythemia Vera (PV) have been focused on reducing the thrombotic and hemorrhagic risks and preventing disease transformation. A short-term way to confirm the effect of treatment is to check peripheral blood count remission. Recently, published researches have demonstrated that JAK2 driver mutation is associated with not only disease progression but hematologic response. Although it is still controversial whether a decrease in JAK2 mutation should be one of the treatment goals or not, the molecular response (MR) is frequently assessed in clinical trials. Moreover, the interest in treatment is moving toward disease modification. Ropeginterferon alfa-2b(P1101) showed it was more effective in achieving durable hematological and molecular remissions than hydroxyurea and well tolerated during long-term application. However, there are limited data on the clinical relevance of MR during P1101 and the efficacy and safety of P1101 in Eastern Asians. Aims: The aim of this study is to evaluate clinical and molecular response, and the association between efficacy and MR. In addition, the safety and tolerability of P1101 were also collected. Methods: This single-arm, open-label study has been performed in 16 hospitals in Korea. Patients were eligible if 19 years or older with PV diagnosed by WHO's 2016 criteria, requiring cytoreductive therapy and elevated hematocrit (Hct) (>45%). Patients were treated with P1101, S.C. q2w, at a starting dose of 250 µg, followed by 350 µg(week 2), 500 µg(week 4), and thereafter until week 48 (core study period). The JAK2 Val617Phe allele burden was assessed every 3 months. The chi-square test was used to determine any association between complete hematologic response (CHR) and MR using the Phi coefficient as a measure of strength association. Results: With a data cut-off date of 10 Jul 2023, a total of 99 patients were enrolled and 77 patients are under treatment including extension period in the study. Twenty patients dropped out including 2 patients due to adverse events and 2 patients completed core study. Total 95 patients were included in this analysis as FA (full analysis) set: 52 (54.7%) patients was HU-naïve and 43 (45.3%) patients was HU-resistant/intolerant(R/I). The median age was 58 years (range, 26-81) and 53.7% were male. The percentagesof patients with low and high risk were 56.8% and 43.2%, repectively. Until now, 94, 87, 79, and 60 patients were evaluable at 3, 6, 9, and 12 months, respectively. CHR was achieved in 25 (27%) of 94, 40 (46%) of 87 patients, 45 (57%) of 79 patients, and 38 (63%) of 60 patients at 3, 6, 9, and 12 months, respectively. Twenty-eight (32%) of 88, 29 (36%) of 80 patients, 32 (52%) of 62 patients, and 23 (61%) of 38 patients achieved the MR at 3, 6, 9, and 12 months, respectively. The Phi Coefficient for determining of association between CHR and MR was 0.56 (P<0.0001), 0.48 (P<0.0001), 0.4735 (P=0.0002), and 0.4938 (P=0.0041) at each time point (Table 1). Regarding the calculated absolute change from baseline of JAK2 allele burden at each time point, a faster and deeper decreasing pattern was shown in CHR responders compared to non-responders (Table 2). Regarding the Hct, platelets, and WBC, the mean value of all parameters significantly decreased to the normal range after 6 months and were consistent in normal until 12 months. Moreover, there is also no significance difference between HU naïve vs R/I and low vs High risk. In terms of safety, a total of 160 treatment-emergent adverse events (TEAEs) and 84 treatment-related adverse events (TRAE) were reported. The most common TEAEs (%, n/95) were alopecia (18%, 17/95) and adverse events related to liver function (16%, 15/95), respectively. Onset time of TRAEs distributed 8, 22, 27, 18, and 8 during week 0-4, 4-12, 12-24, 24-36, and 36-48, respectively. There were no unexpected TEAEs. Two serious adverse drug reactions (1 hepatotoxicity and 1 bipolar disorder) were reported. Conclusion: The updated data showed consistent results with the previous 2022 ASH meeting abstract that ropeginterferon alfa-2b therapy, with rapid dose optimization, induced hematological response, reduced JAK2 allele burden, and was well tolerated in Korean patients. Moreover, we clarified the association between efficacy and MR as assessed by reduction in JAK2 allele burden. More data will be updated during the meeting.

Clinical Manifestations Are Evolving and Progressive in Patients with Vexas Syndrome

Introduction Vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic (VEXAS) syndrome is caused by somatic mutations in UBA1 and characterized by systemic autoinflammation and hematologic manifestations. Clinically, disease progression appears heterogenous, with differing degrees of organ involvement seen in individual patients over time. Disease mortality is high, and to date only UBA1 variant, presence of MDS or significant cytopenia, and clonal hematopoiesis have been associated with worse overall survival. Prospective data are currently lacking on evolution of symptoms and organ involvement. We characterized disease evolution over time and looked at the benefit of prescribed therapies in our prospective cohort of VEXAS patients. Methods Forty-four VEXAS patients with a confirmed UBA1 mutation have been enrolled in our study at the National Institutes of Health (NIH) since 2019. We collected demographic data and clinical history, and performed standardized evaluations including peripheral blood counts, inflammatory markers, serum protein electrophoresis, and bone marrow biopsies. All patients were screened for eligibility and suitability for hematopoietic stem cell transplant (HSCT) on a phase II clinical trial (NCT05027945). Patients who did not undergo HSCT received recommendations for medical therapy. Results Subjects were male with median age of 67 years (50 - 79) and 95% were of White ethnicity. The most common UBA1 variant was M41T (n=19, 43%) followed by M41L (n=15, 34%), M41V (n=7, 16%), and splice motif mutations (n=4, 7%). Median variant allele fraction (VAF) of the cohort was 73%; M41T (76%), M41L (70%), M41V (58%), splice (61%). Thirty-six (82%) patients initially presented with skin lesions and/or arthralgias. Other initial manifestations included fever (27%), fatigue (25%), chondritis (14%), pulmonary (11%) and ocular/orbital (9%). Interestingly, only one patient presented with symptomatic anemia. At the time of first NIH clinic visit patients had manifestations of VEXAS disease for median of 6 years (range 1 - 11). Only 20% (9/44) had no active symptoms. Fatigue (39%) was most common followed by skin rash, arthralgias, pulmonary, and neurologic manifestations (Figure). Twenty-three percent had developed transfusion dependent anemia. Eleven patients (25%) had bone marrow findings meeting criteria for myelodysplastic syndrome (MDS); one patient had MDS-EB1. Venous thromboembolism was documented in 18 patients (40%). Thirteen patients (30%) had plasma cell disorder, four of whom had multiple myeloma (MM). All except one patient were receiving prednisone at NIH evaluation, with median dose of 20mg (range 5mg-50mg). Sixteen patients (36%) had elevated CRP despite glucocorticoids and steroid-sparing agents. In 66% of patients, concomitant steroid-sparing agents were used, most frequently IL-6 inhibitors (n=9), or JAK inhibitors (n=6). Other agents included erythropoietin stimulating agents (n=2), canakinumab (n=2), adalimumab (n=2), methotrexate (n=2), and azacytidine, azathioprine, colchicine, dapsone, ustekinumab in one patient each. Two patients were on MM directed therapies. At last follow-up, 8 patients (18%) had died, 6 patients (14%) were post HSCT, and 9 patients (20%) were pursuing HSCT. The indication for HSCT was refractory inflammation in two patients and progressive marrow failure in four. Twenty-one patients continued medical management: 15 (35%) with controlled inflammation on their respective regimens and 6 (14%) with progressive hemato-inflammatory manifestations. Of these six patients, one was non-compliant with therapy other than prednisone and other five were deemed unfit for HSCT due to poor performance status (n=2), age (n=2), or kidney disease (n=1). Conclusion Clinical manifestations of VEXAS syndrome evolve and progress with time. Severe cytopenia is rare at disease onset. Our observations indicate that biologics directed at JAK-STAT pathway, IL-6, IL-1 and hypomethylating agents may provide qualitative benefit in one-third of cases evidenced by controlled disease, but are not steroid sparing. HSCT is a curative approach, but, the timing is not well established. Most challenging are patients who have exhausted all effective steroid-sparing agents, require high dose of glucocorticoids, and are not a HSCT candidate. Development of effective therapies for VEXAS is a research priority to improve outcomes.

ROS-Driven Structural and Functional Fibrinogen Modifications Are Reverted By JAK Inhibition in Myeloproliferative Neoplasms

Myeloproliferative neoplasms (MPNs) are bone marrow diseases characterized by excess clonal hematopoiesis resulting in elevated peripheral blood counts. Subtypes include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). They share clinical features (e.g., splenomegaly, thrombotic complications, risk of leukemic transformation), and a common molecular basis: the dysregulation of the JAK-STAT pathway, which confers a hypersensitivity of hematopoietic stem cells (HSC) to the action of growth factors and cytokines, and, as a result, an overproduction of blood cells. Genetic analysis of patients with MPNs revealed that >95% of patients with PV and about 50% of patients with ET or PMF carry an activating point mutation in the JAK2 gene (JAK2-V617F). Despite clinical evidence indicating a higher susceptibility of MPN patients to develop thrombotic disease, the underlying pathogenetic mechanisms are not fully understood. It appears that thrombosis is the result of a severe inflammatory response associated to remarkable oxidative stress in the presence of endothelial dysfunction and a procoagulant environment. Hypercoagulability is a consequence of disrupted hemostasis and fibrinolysis due to damage of the endothelium and defective fibrinolysis. Evidence suggests that overproduction of ROS and a deprived antioxidant system play a major role in the pathogenesis of MPN, as well as in the resistance to tyrosine-kinase inhibitor therapy. Moreover, systemic inflammation is thought to be the main trigger of thrombosis and seems to be mainly mediated by pro-inflammatory cytokines along with endothelial cell dysfunction. Recent studies have provided evidence that fibrinogen plays a multifaceted role in inflammatory responses and autoimmunity. The ability of fibrinogen to participate in the inflammatory response depends on its specific interaction with integrins, the leukocyte cell surface adhesion receptors expressed on neutrophils, monocytes, macrophages, and several subsets of lymphocytes. In MPN patients, endothelial cell dysfunction, increased reactive oxygen species (ROS) production, and proinflammatory cytokines have been reported, together with impaired fibrinolysis. We also investigated the potential role of oxidative stress and inflammation in the promotion of thrombosis. In order to study the mechanisms of inflammation-induced thrombosis in MPN patients, the aim of the present study was to investigate the role of oxidation-induced structural and functional fibrinogen modifications in MPN patients. The eventual counteracting effects of ruxolitinib, a first-in-class JAK inhibitor, were also assessed. 40 MPN patients and 40 age- and sex-matched healthy control subjects were included in the study. We measured specific oxidative stress markers andtheir modifications in plasma, and functional and structural parameters in purified fibrinogen fractions. Marked signs of oxidative stress in plasma (P<0.01 versus controls) and, correspondingly, an increased extent of fibrinogen oxidation (P<0.01 versus controls) were observed in patients. Furthermore, fibrinogen fractions purified from MPN patients exhibited significantly reduced clotting ability and decreased susceptibility to plasmin-induced lysis (P<0.01 versus controls). Alterations in fibrinogen secondary structure, as suggested by circular dichroism spectroscopy, and in fibrin clot architecture were also identified. Importantly, clot structure obtained by stimulated emission depletion (STED) super-resolution microscopy indicated modifications in fiber diameter and clot porosity in MPN patients (P<0.01 versus controls). In patient samples, fibrinogen clot analysis and oxidative-dependent changes reveal novel structural and functional fibrinogen modifications which may favor thrombotic complications in MPNs. In patients receiving ruxolitinib, a significant improvement in redox status was observed, accompanied by a significant improvement in fibrinogen structural and functional features, suggesting a potential impactof ruxolitinib on cardiovascular prevention in MPNs.

Megakaryocytes Have Immune Characteristics in Murine Immune Bone Marrow Failure

Introduction Megakaryocytes (MKs) have familiar roles in the bone marrow (BM) microenvironment, including platelet production, maintenance of hematopoietic stem and progenitor cells (HSPCs) function, and more recent evidence suggests MK engagement in immune responses. Immune aplastic anemia (AA) is characterized by oligoclonal expansion of T-cells and death of HSPCs leading to bone marrow failure (BMF). As MKs demonstrate niche maintenance and immune effector function, we explored the potential roles of MKs in the development of AA in a murine model of immune BMF. Methods BMF was induced in CByB6F1 mice by intravenous infusion of lymph node (LN) cells from major histocompatibility complex mismatched C57BL/B6 (B6) donor mice at a dose of 30-40x10 6 cells/recipient. No irradiation was given to recipients to avoid extra damage to MKs and CByB6F1 recipient mice were sacrificed at D7, D10, D14, and D17 following LN cell infusion (Chen J, et al. Blood. 2004). Peripheral blood (PB) samples, tibias, femurs, and sterna were harvested at each timepoint. Immunophenotyping of MKs and T cells was performed by flow cytometry on flushed BM cells and two-photon excitation microscopy (2P) imaging was performed on fixed sternal samples, using fluorescence-labelled CD41 antibody to visualize the physical location of MKs in BM. To enrich for MKs, BM cells were filtered through a 10 µm pluriStrainer™ through which most lymphocytes, red blood cells, and other small cells were eluted while MKs and other large size cells remained on the strainer. Large cells and residual small cells were subsequently assessed by single cell RNA sequencing (sc-RNAseq) on the Parse Bio Evercode™ platform based on combinatorial barcoding technology, which was able to accommodate the large size of MKs, as no partitioning step is needed in the analysis workflow. Results BMF was successfully induced as measured by decreasing PB counts from point of lymphocyte infusion accompanied by increasing expansion of CD4 and CD8 T-cells and marked reduction of total BM cells. MKs persisted throughout the course of BMF as measured by both flow cytometry and 2P (Figure 1A) despite PB thrombocytopenia and progressive marrow hypocellularity. Concomitantly, MKs identified by large size on forward scatter versus side scatter and CD41+ expression had progressive upregulation of immune effector markers IA-IE and CD53 over the course of BMF. Following sc-RNAseq and bioinformatics analysis of enriched BM cells at D14, cell clustering identified a distinct cell cluster representing MKs (Figure 1B). MK identity was supported by co-expression of transcripts encoding platelet factor 4, TUBB1, Glycoprotein 5, and Itga2b (CD41). This MK cell population was distinct from macrophages which highly expressed Fcgr4, Cd5l, Ccr5, Cd74 and Gbp5 genes. Gene expression profiling demonstrated that MKs in BMF mice showed upregulation of immune pathways such as interferon gamma and interferon alpha response and downregulation of pathways related to platelet activation and platelet homeostasis, as compared with normal mice. Conclusions In a murine model of BMF, using multiple modalities, we demonstrate that MKs cease platelet production, manifested by reduction in peripheral blood platelet count and downregulation of gene expression pathways related to platelet production, while concurrently acquiring characteristics of immune effector cells. This suggests that MKs are active participants in immune BMF.

Assessment and predictive ability of the absolute neutrophil count in peripheral blood for in vivo CAR T cells expansion and CRS

BackgroundChimeric antigen receptor (CAR) T cell therapy is an advanced and effective immunotherapy for relapsed or refractory B-cell malignancies. High expansion of CAR T cells in vivo and durable antitumor...

IMPACT OF BLOOD PARAMETERS ON BCR/ABL1 P210 TESTING IN PATIENTS WITH CHRONIC MYELOID LEUKEMIA IN TURKIYE

Objective: Chronic myeloid leukemia (CML) is one of the most common hematological malignancies derived from the BCR/ABL1 fusion gene. Patients with CML generally manifest leukocytosis with basophilia and neutrophilia. The verification of CML is often based on the detection of BCR/ABL1 fusion. We aimed to investigate the impact of peripheral blood (PB) differential and complete blood count (CBC) on BCR/ABL1 p210 test ordering in patients with suspected CML. 
 Methods: We performed a retrospective assessment of patients tested for the first time for BCR/ABL1 p210 fusion. We obtained clinical and laboratory findings of 235 patients from the database of our clinic. BCR/ABL1 p210 fusion was detected by quantitative real-time polymerase chain reaction (RT-qPCR). We implemented t-tests or Mann–Whitney U tests for the comparison of continuous data. We plotted the receiver operating characteristic (ROC curves) and calculated the area under the ROC curve (AUC) for each parameter. 
 Results: Among 235 patients, 25 (%10.6) received a new diagnosis of CML. CML patients had significantly increased white blood cell count (WBC) with differential. Absolute basophil count showed the highest area under the ROC curve (AUC) value of 0.829, which had a cut-off value of 0.3 × 103/ µL. 76.00% of CML cases had an absolute basophil count of ≥0.3 × 103/µL, while 95.24% of the non-CML cases had an absolute basophil count of

A German-Wide Systematic Study on Mobilization and Collection of Hematopoietic Stem Cells in Poor Mobilizer Patients with Multiple Myeloma prior to Autologous Stem Cell Transplantation

Introduction: In patients with a clinical indication for autologous hematopoietic stem cell transplantation (ASCT), sufficient mobilization of CD34+ precursor cells into peripheral blood is essential to ensure adequate hematopoietic stem cell (HSC) collection prior to intensive therapy. However, with standard granulocyte-colony stimulating factor (G-CSF)-based mobilization schemes, an important minority of patients fail to mobilize sufficient (e.g., >10/µL) CD34+ cell counts into the peripheral blood and are considered as poor mobilizers (PM). Because failure to achieve sufficient CD34+ cell mobilization can negatively affect important clinical treatment endpoints, the use of plerixafor (PLX) was approved to increase CD34+ mobilization in PM patients. Methods: The German non-interventional, multicenter, open-label, prospective OPTIMOB study evaluated HSC mobilization strategies prior to planned ASCT in adult patients with hematologic malignancies (lymphomas or multiple myeloma [MM]) focusing on PM patients. PM patients were defined as follows: (1) never achieving ≥20 CD34+ cells/µL before 1st apheresis, (2) receiving PLX at any timepoint of mobilization, (3) their initially planned stem cell yield had to be reduced, or (4) they had not received apheresis due to low CD34+ count in peripheral blood. Results: 168 of 475 MM patients (35%) participating in the OPTIMOB study were classified as PM, and 155 of them (92%) received PLX (PM+PLX) during the study. PM patients were 40–78 years old, slightly more often male (n = 97, 58%), mostly newly diagnosed (n = 146, 87%) and received highly individualized previous treatments. Ninety-four of the PMs underwent chemotherapy mobilization (65%), and 51 patients (35%) received steady-state mobilization with G-CSF only during 1st mobilization attempt. 92% of the total PM population (n = 155) underwent apheresis, 78% of them (n = 117) achieved >2.0 × 106 CD34+ cells/kg body weight on the 1st day of apheresis. PM+PLX had a higher median total collection result than those PM patients without PLX support (7.2 vs. 5.7 × 106 CD34+ cells/kg body weight). In total, ASCT was performed in 136 PM+PLX (88%) versus 8 PM−PLX patients (62%). Conclusion: The OPTIMOB study showed that a considerable proportion of adult MM patients in Germany are PMs. Even though most of PMs were supported with PLX in the OPTIMOB study, PM-PLX also successfully mobilized HSCs, allowing ASCT in majority of all PMs. However, further analyses are required for treatment optimization in PMs.

The Role of the IL-33/ST2 Axis in CpG-Induced Macrophage Activation Syndrome.

Macrophage activation syndrome (MAS) is a fatal inflammatory condition, which is often associated with the elevation of multiple proinflammatory cytokines and multiple organ dysfunction. Previous studies have shown that ST2 contributes to T cell overactivation and plays a detrimental role in mouse models of primary hemophagocytic lymphohistiocytosis. The purpose of this study was to investigate the role of the IL-33/ST2 axis in a mouse model of MAS induced by repeated injections of cytosine-phosphate-guanine (CpG). Serum cytokines were determined using the cytometric bead array by flow cytometry. IL-33 and ST2 were detected by immunohistochemistry and real-time quantitative PCR in the liver and spleen of mice. CD3 and F4/80 in the liver were detected by immunohistochemistry. Inflammatory macrophages and effector memory T lymphocytes were detected by flow cytometry. The CpG-induced MAS model was successfully induced after repeated CpG injections, presenting with hypercytokinemia and hepatosplenomegaly. The numbers of IL-33 positive cells in the liver and spleen decreased significantly, while the expression of ST2 in the liver tended to increase in the mice with MAS. IL-33 and St2 knockout mice showed similar levels of hepatosplenomegaly, peripheral blood count, and cytokine storm when compared with wild-type (WT) mice after induction of MAS. There were also no significant differences in liver pathology (including inflammatory cell infiltration of CD3 and F4/80) and levels of splenic inflammatory macrophages and effector memory T cells between the WT and knockout mice. These results suggested that IL-33 decreased in the liver and spleen tissues of MAS mice. Further results suggest that IL-33 and St2 knockout mice have no treatment potential in CpG-induced MAS. Thus, the IL-33/ST2 axis has little effect on the prognosis of CpG-induced MAS.

Immunophenotyping characteristics and outcome of COVID-19 patients: peripheral blood CD8+T cell as a prognostic biomarker for patients with Nirmatrelvir.

Nirmatrelvir has been authorized for the treatment of both hospitalized and non-hospitalized COVID-19 patients. However, the association between T lymphocyte subsets and the outcome of hospitalized COVID-19 patients treated with oral Nirmatrelvir has not been investigated. The objective of this study was to examine whether lymphocyte subsets could serve as biomarkers to assess the risk of mortality in COVID-19 patients undergoing Nirmatrelvir treatment, with the aim of enhancing medication management for COVID-19 patients. We conducted a retrospective cohort study at the Xiangya Hospital of Central South University in China between December 5, 2022 and January 31, 2023. The study reported demographic, clinical, T lymphocyte subsets, and inflammatory cytokine data of COVID-19 patients. We evaluated the associations of T lymphocyte subsets on admission with the composite outcome or death of patients using univariate and multivariable Cox regression analyses with hazards ratios (HRs) and 95% confidence intervals (CIs). We identified 2118 hospitalized COVID-19 patients during the study period, and conducted a follow-up of up to 38 days. Of these, 131 patients received Nirmatrelvir, with 56 (42.7%) in the composite outcome group, and 75 (57.3%) in the non-composite outcome group. Additionally, 101 (77.1%) patients were discharged, while 30 (22.9%) died. Our results showed a significant decrease in the CD3+, CD4+, and CD8+ T cell counts of patients in the composite outcome group and mortality group compared to the non-composite outcome group and discharged group, respectively. Multivariate Cox regression analysis showed that the significant decrease in CD8+ T cell count in peripheral blood was independently associated with the composite outcome in COVID-19 patients treated with Nirmatrelvir, with an HR of 1.96 (95%CI: 1.01-3.80). The significant decrease in CD4+ and CD8+ T cell counts in peripheral blood increased the hazard of developing mortality, with HRs of 6.48 (95%CI: 1.47-28.63) and 3.75 (95%CI: 1.27-11.11), respectively. Our study revealed a significant positive correlation between a decrease in CD8+ T cell counts and progression and mortality of hospitalized COVID-19 patients treated with Nirmatrelvir. Lower counts (/μL) of CD8+ T cell (<201) were associated with a higher risk of in-hospital severity and death. Our findings may provide valuable references for physicians in optimizing the use of Nirmatrelvir.

Importance of TREC and KREC as molecular markers for immunological evaluation of down syndrome children

Recurrent and severe infections occurred in children with Down Syndrome (DS) due to immunological parameter defects have been reported. The aim of the study is to evaluate the importance of using T-cell receptor excision circle (TREC) and kappa-deleting recombination excision circle (KREC) as molecular markers for immunological investigation of children with DS. The study included 40 non-disjunction trisomy 21 confirmed DS children, and 25 healthy controls. Peripheral blood (PB) was analyzed for lymphocyte subpopulations by flow cytometry, serum immunoglobulin levels, and TREC and KREC copy numbers using quantitative real-time PCR. DS patients showed significantly lower absolute counts of PB T lymphocytes, T helper lymphocytes, T cytotoxic lymphocytes, B lymphocytes, and Natural killer cells, and lower serum IgA, IgG, and IgM levels compared to healthy controls. Copy number of TREC and KREC showed no significant differences between DS patients and healthy controls. There is a significant positive correlation between TREC copy number with a percentage and absolute count of helper T lymphocytes in patients. Also, the KREC copy number was significantly negatively correlated with the age of patients. These findings suggest that copy numbers of TREC and KREC could be useful as molecular markers for immunological evaluation of patients with DS.

Q-HAM: a multicenter upfront randomized phase II trial of quizartinib and high-dose Ara-C plus mitoxantrone in relapsed/refractory AML with FLT3-ITD

BackgroundAbout 50% of older patients with acute myeloid leukemia (AML) fail to attain complete remission (CR) following cytarabine plus anthracycline-based induction therapy. Salvage chemotherapy regimens are based on high-dose cytarabine (HiDAC), which is frequently combined with mitoxantrone (HAM regimen). However, CR rates remain low, with less than one-third of the patients achieving a CR. FLT3-ITD has consistently been identified as an unfavorable molecular marker in both relapsed and refractory (r/r)-AML. One-quarter of patients who received midostaurin are refractory to induction therapy and relapse rate at 2 years exceeds 40%. The oral second-generation bis-aryl urea tyrosine kinase inhibitor quizartinib is a very selective FLT3 inhibitor, has a high capacity for sustained FLT3 inhibition, and has an acceptable toxicity profile.MethodsIn this multicenter, upfront randomized phase II trial, all patients receive quizartinib combined with HAM (cytarabine 3g/m2 bidaily day one to day three, mitoxantrone 10mg/m2 days two and three) during salvage therapy. Efficacy is assessed by comparison to historical controls based on the matched threshold crossing approach with achievement of CR, complete remission with incomplete hematologic recovery (CRi), or complete remission with partial recovery of peripheral blood counts (CRh) as primary endpoint. During consolidation therapy (chemotherapy and allogeneic hematopoietic cell transplantation), patients receive either prophylactic quizartinib therapy or measurable residual disease (MRD)-triggered preemptive continuation therapy with quizartinib according to up-front randomization.The matched threshold crossing approach is a novel study-design to enhance the classic single-arm trial design by including matched historical controls from previous clinical studies. It overcomes common disadvantages of single-armed and small randomized studies, since the expected outcome of the observed study population can be adjusted based on the matched controls with a comparable distribution of known prognostic and predictive factors. Furthermore, balanced treatment groups lead to stable statistical models. However, one of the limitations of our study is the inability to adjust for unobserved or unknown confounders.Addressing the primary endpoint, CR/CRi/CRh after salvage therapy, the maximal sample size of 80 patients is assessed generating a desirable power of the used adaptive design, assuming a logistic regression is performed at a one-sided significance level α=0.05, the aspired power is 0.8, and the number of matching partners per intervention patient is at least 1. After enrolling 20 patients, the trial sample size will be recalculated in an interim analysis based on a conditional power argument.ConclusionCurrently, there is no commonly accepted standard for salvage chemotherapy treatment. The objective of the salvage therapy is to reduce leukemic burden, achieve the best possible remission, and perform a hemopoietic stem-cell transplantation. Thus, in patients with FLT3-ITD mutation, the comparison of quizartinib with intensive salvage therapy versus chemotherapy alone appears as a logical consequence in terms of efficacy and safety.Ethics and disseminationEthical approval and approvals from the local and federal competent authorities were granted. Trial results will be reported via peer-reviewed journals and presented at conferences and scientific meetings.Trial registrationClinicalTrials.gov NCT03989713; EudraCT Number: 2018-002675-17.