Post Allogeneic Stem Cell Transplantation Research Articles

Donor Lymphocyte Infusion (DLI) post allogeneic stem cell transplant (allo-SCT) in Acute Myeloid Leukemia (AML) and High-Grade Myelodysplastic Syndrome (MDS). A longitudinal retrospective study using peripheral blood (PB) CD34+ and CD3+ donor chimerism (DC) monitoring

IntroductionThis longitudinal study was based on the outcomes of Donor Lymphocyte Infusion (DLI) for falling peripheral blood (PB) CD34+ and CD3+ donor chimerism (DC). MethodsFrom 2012 to 2018, data was collected from the BMT database and electronic medical records (EMR). The primary objective was to compare the indication for DLI based on falling PB CD34+ or CD3+ DC in patients post allo-SCT for AML and MDS and their overall survival (OS). Results18/70 patients met the inclusion criteria. Indications for DLI were i) falling PB CD34+ DC ≤ 80 % with morphological relapse, ii) falling PB CD34+ DC ≤ 80 % without morphological relapse and iii) falling PB CD3+ DC ≤ 80 % without falling PB CD34+ DC. Log rank analysis showed falling PB CD34+ DC and morphological relapse had significantly lower OS. Linear regression demonstrated better OS post DLI if there was PB CD34+ and CD3+ chimerism response at 30 days (p = 0.029), GVHD (p = 0.032) and tapering immunosuppression at the time of falling DC (p = 0.042). ConclusionDLI for PB CD34+ DC values ≤ 80 % and morphological relapse had the lowest OS. In this study, full DC was achieved after DLI even with a PB CD3+DC value as low as 13 %, provided the PB CD34+ DC remained > 80 %. Further research is vital in CD34+ DC as a biomarker for disease relapse and loss of engraftment.

Antiviral cellular therapy for enhancing T-cell reconstitution before or after hematopoietic stem cell transplantation (ACES): a two-arm, open label phase II interventional trial of pediatric patients with risk factor assessment

Viral infections remain a major risk in immunocompromised pediatric patients, and virus-specific T cell (VST) therapy has been successful for treatment of refractory viral infections in prior studies. We performed a phase II multicenter study (NCT03475212) for the treatment of pediatric patients with inborn errors of immunity and/or post allogeneic hematopoietic stem cell transplant with refractory viral infections using partially-HLA matched VSTs targeting cytomegalovirus, Epstein-Barr virus, or adenovirus. Primary endpoints were feasibility, safety, and clinical responses (>1 log reduction in viremia at 28 days). Secondary endpoints were reconstitution of antiviral immunity and persistence of the infused VSTs. Suitable VST products were identified for 75 of 77 clinical queries. Clinical responses were achieved in 29 of 47 (62%) of patients post-HSCT including 73% of patients evaluable at 1-month post-infusion, meeting the primary efficacy endpoint (>52%). Secondary graft rejection occurred in one child following VST infusion as described in a companion article. Corticosteroids, graft-versus-host disease, transplant-associated thrombotic microangiopathy, and eculizumab treatment correlated with poor response, while uptrending absolute lymphocyte and CD8 T cell counts correlated with good response. This study highlights key clinical factors that impact response to VSTs and demonstrates the feasibility and efficacy of this therapy in pediatric HSCT.

Post-transplant cyclophosphamide, calcineurin inhibitor, and mycophenolate mofetil compared to anti-thymocyte globulin, calcineurin inhibitor, and methotrexate combinations as graft-versus-host disease prophylaxis post allogeneic stem cell transplantation from sibling and unrelated donors in patients with acute myeloid leukemia: a study on behalf of the Acute Leukemia Working Party of the European Society for Blood and Marrow Transplantation.

Post-transplant cyclophosphamide plus calcineurin inhibitor (CNI)(tacrolimus or cyclosporine A) plus mycophenolate mofetil (PTCy/TAC or CSA/MMF) and anti-thymocyte globulin plus CNI (tacrolimus or cyclosporine A) plus methotrexate (ATG/TAC or CSA/MTX) are common graft-versus-host disease (GVHD) prophylaxis regimens. We compared the two regimens in patients with acute myeloid leukemia (AML) undergoing allogeneic transplantation from matched siblings or unrelated donors. 402 received PTCy/TAC or CSA/MMF and 5648 received ATG/TAC or CSA/MTX. Patients in the PTCy-based group were younger (48.7 vs. 51.5 years, p = 0.024) and there was a higher frequency of patient cytomegalovirus seropositivity and female donor to male patient combination in this group (77.8% vs. 71.8%, p = 0.009 and 18.4% vs. 14.4%, p = 0.029, respectively). More patients in the PTCy-based group received reduced-intensity conditioning (51.5% vs. 41%, p < 0.0001). No differences were observed in the incidence of acute GVHD grade II-IV and III-IV (21.2% vs. 20.4%, p = 0.92 and 8.1% vs. 6%, p = 0.1) or 2-year total and extensive chronic GVHD (33.7% vs. 30%, p = 0.09 and 10.7% vs. 11.2%, p = 0.81) between the groups. In the multivariate analysis, all transplant outcomes did not differ between the groups. PTCy/CNI/MMF and ATG/CNI/MTX are alternative regimens for GVHD prophylaxis in AML patients.

Abstract LB101: New assay technology and utility of peripheral blood vs bone marrow for molecular MRD in MDS

Abstract Background Rare tumor-specific mutations in patient samples serve as excellent markers to monitor the course of malignant disease and responses to therapy. However, improved assay techniques are needed for broad adoption in routine clinic. We describe herein - superRCA assays - providing rapid and highly specific detection of DNA sequence variants present at very low frequencies in DNA samples using a standard flow cytometer for readout. We demonstrate and compare the assay against ddPCR for precise, ultra-sensitive detection of single-nucleotide mutant sequences from malignant cells to follow the course of MDS patients following allogeneic stem cell transplantation, both in bone marrow and peripheral blood. Methods This study is based on patient material from the Nordic MDS study NMDSG14B (NCT02872662). The patients in this retrospective study have previously undergone sequencing (TruSight, Illumina) upon diagnosis, after which patient specific driver mutations (1-4 per patient) has been chosen and analyzed longitudinally to monitor molecular MRD using ddPCR (Bio-Rad). These longitudinal samples were re-analyzed using superRCA mutation assays (Rarity Bioscience). Both quarterly bone marrow aspirates and monthly peripheral blood samples post allogeneic stem cell transplantation were included to investigate to what extent frequent blood derived molecular MRD gives equivalent relapse information as bone marrow. For each mutation, sensitivity was assessed through serial dilution and wild type control, with the limit of detection calculated as Mean(wt)+3*SD(wt). Samples were analyzed in duplicates or triplicates and sample to sample variation was calculated as SD/Mean. Results A total of 22 relapse patients and 21 different mutations were analyzed, covering single nucleotide variants, insertions, deletions, as well as mutations with very high GC in ASXL1 and SRSF2. The analysis demonstrating lower background and limit of detection for the superRCA assays compared to the equivalent ddPCR data. With spike-in dilution series, we demonstrated the superRCA assay could detect single point mutations as low as 1 in 100,000 with the flow cytometer readout. Mean background for superRCA was 0,0016% in wild type controls, and mean limit of detection was 0,0023%. From available serial dilutions, the mean sample to sample variation (CV) at 0,016% was 22,87% and 29,32% at 0,001% dilution.In longitudinal sample analysis of relapse patients, superRCA assay detected remaining mutations after initial treatment and clearly revealed the remaining malignant clone, subsequently leading to a relapse. The superRCA assay also demonstrated the feasibility of detecting corresponding leukemia mutations in the PBMCs when such mutations were present in bone marrow. Conclusions The data shows comparable mutant allele frequency results between superRCA and ddPCR upon diagnosis and relapse. The data further demonstrates superRCA mutation assays for the selected driver mutations can reach to very low limit of detection and increasing mutations could be detected in both blood and bone marrow predicting the relapse of MDS post transplantation in clinical samples. To further study the utility of using peripheral blood for molecular MRD in MDS patients, more data is needed and studies are ongoing. Citation Format: Linus Bosaeus, Lei Chen, Magnus Tobiasson, Caroline Lindholm, Lucia Cavelier, Eva Hellström Lindberg. New assay technology and utility of peripheral blood vs bone marrow for molecular MRD in MDS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB101.

Treatment of post-allogeneic hematopoietic stem cell transplant cytopenias with sequential doses of multipotent mesenchymal stromal/stem cells

Background aimsCytopenias after allogeneic stem cell transplantation (allo-SCT) are a common complication, the underlying pathogenic mechanisms of which remain incompletely understood. Multipotent mesenchymal stromal/stem cell (MSC) therapy has been successfully employed in the treatment of immune-related disorders and can aid in the restoration of the hematopoietic niche. MethodsA phase II clinical trial to assess the efficacy and safety of administering four sequential doses of ex-vivo expanded bone marrow MSCs from a third-party donor to patients with persistent severe cytopenias after allo-SCT was performed. ResultsThe overall response rate on day 90 was 75% among the 27 evaluable patients (comprising 12 complete responses, 8 partial responses, and 7 with no response). The median time to respond was 14.5 days. Responses were observed across different profiles, including single or multiple affected lineages, primary or secondary timing, and potential immune-mediated or post-infectious pathophysiology versus idiopathic origin. With a median follow-up for surviving patients of 85 months after MSC infusion, 53% of patients are alive. Notably, no adverse events related to MSC therapy were reported. ConclusionsIn summary, the sequential infusion of third-party MSCs emerges as a viable and safe therapeutic option, exhibiting potential benefits for patients experiencing cytopenias following allo-SCT.

A phase II study of belumosudil for chronic graft-versus-host disease in patients who failed at least one line of systemic therapy in China

BackgroundChronic graft-versus-host disease (cGVHD) is an immune-related disorder that is the most common complication post-allogenic hematopoietic stem cell transplant. Corticosteroids with or without calcineurin inhibitors (CNIs) remain the mainstay of cGVHD treatment for first-line therapy. However, for many patients, cGVHD symptoms cannot be effectively managed and thus require second-line therapy. Currently, there is no approved treatment for second-line cGVHD treatment in China. In this study, belumosudil, a highly selective and potent rho-associated coiled-coil-containing protein kinase-2 inhibitor demonstrated to be effective for cGVHD in the United States and other Western countries, is investigated in patients with cGVHD in China for its overall benefit–risk balance.MethodsThis multicenter, open-label phase II study evaluated the safety, efficacy, and pharmacokinetics of oral belumosudil 200 mg once daily in cGVHD patients who had been treated with at least one line of systemic therapy in China. The primary endpoint was overall response rate (ORR); each individual patient’s response was assessed by the investigator using the 2014 National Institutes of Health consensus criteria. Secondary endpoints were duration of response (DOR), time to response (TTR), changes in Lee Symptom Scale (LSS) score, organ response rate, corticosteroid dose change, CNI dose change, failure-free survival, time-to-next-treatment, overall survival, and safety.ResultsThirty patients were enrolled in the study with a median follow-up time of 12.9 months. ORR was 73.3% (95% confidence interval: 54.1–87.7%) and all responders achieved partial response. Median DOR among responders was not reached and median TTR was 4.3 weeks (range: 3.9–48.1). Fifteen patients (50.0%) achieved clinically meaningful response in terms of reduction in LSS score by ≥ 7 points from baseline. Corticosteroid and CNI dose reductions were reported in 56.7% (17/30) and 35.0% (7/20) of patients, respectively. Most treatment-emergent adverse events (TEAEs) were mild to moderate in severity, with 11 patients (36.7%) experiencing grade ≥ 3 TEAEs. The most common grade ≥ 3 TEAE was pneumonia (n = 5, 16.7%).ConclusionsBelumosudil treatment demonstrated a favorable benefit–risk balance in treating cGVHD patients who previously have had standard corticosteroid therapy in China where approved second-line setting is absent.Trial registrationClinicalTrials.gov identifier NCT04930562.

The dynamics of B-cell reconstitution post allogeneic hematopoietic stem cell transplantation: A real-world study.

The immune reconstitution after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is crucial for preventing infections and relapse and enhancing graft-versus-tumor effects. B cells play an important role in humoral immunity and immune regulation, but their reconstitution after allo-HSCT has not been well studied. In this study, we analyzed the dynamics of B cells in 252 patients who underwent allo-HSCT for 2 years and assessed the impact of factors on B-cell reconstitution and their correlations with survival outcomes, as well as the development stages of B cells in the bone marrow and the subsets in the peripheral blood. We found that the B-cell reconstitution in the bone marrow was consistent with the peripheral blood (p=0.232). B-cell reconstitution was delayed by the male gender, age >50, older donor age, the occurrence of chronic and acute graft-versus-host disease, and the infections of fungi and cytomegalovirus. The survival analysis revealed that patients with lower B cells had higher risks of death and relapse. More importantly, we used propensity score matching to obtain the conclusion that post-1-year B-cell reconstitution is better in females. Meanwhile, using mediation analysis, we proposed the age-B cells-survival axis and found that B-cell reconstitution at month 12 posttransplant mediated the effect of age on patient survival (p=0.013). We also found that younger patients showed more immature B cells in the bone marrow after transplantation (p=0.037). Our findings provide valuable insights for optimizing the management of B-cell reconstitution and improving the efficacy and safety of allo-HSCT.

Detection of mixed chimerism on blood film morphology in a patient with Chediak-Higashi syndrome post allogeneic stem cell transplant

Detection of mixed chimerism on blood film morphology in a patient with Chediak-Higashi syndrome post allogeneic stem cell transplant

Severe Late Agranulocytosis Post-Allogeneic Stem Cell Transplant during Tacrolimus Taper

Severe Late Agranulocytosis Post-Allogeneic Stem Cell Transplant during Tacrolimus Taper

Retroperitoneal lymphadenopathy: An underappreciated contributor to acute kidney failure in patients with cancer

Introduction: Retroperitoneal lymphadenopathy represents a unique and infrequent complication within various malignancies. This case report exemplifies the intricate diagnostic challenges that arise when identifying nondilated obstructive nephropathy in the context of retroperitoneal lymphadenopathy due to diffuse large B-cell lymphoma (DLBCL), particularly when conventional imaging fails to reveal the hallmark feature of hydronephrosis. Case description: We present a 66-year-old male with relapsed chronic lymphocytic leukemia post-allogeneic stem cell transplantation. His direct admission to the hematology/oncology service was due to failure to thrive. Despite the absence of hydronephrosis on imaging, the patient developed volume overload and increased serum creatinine levels. An ultrasound unveiled mild bilateral collecting system dilation, while laboratory findings did not provide valuable insight into the underlying etiology. A Lasix renal scan confirmed split renal function with minimal bilateral excretion. High clinical suspicion resulted in an MRI scan showing interval enlargement of retroperitoneal lymph nodes and pelvicaliectasis, which prompted urological and interventional radiology involvement. Conclusions: This case underscores the complexity of diagnosing retroperitoneal lymphadenopathy and pelvicaliectasis, in DLBCL, particularly when hydronephrosis is absent on imaging. It emphasizes the significance of clinical acumen, collaboration among specialties, and the use of advanced imaging techniques. Timely recognition of nondilated obstructive nephropathy in the setting of retroperitoneal lymphadenopathy is pivotal for guiding appropriate management strategies and optimizing patient outcomes in the intricate clinical setting.

Evaluating the Feasibility and Acceptability of a Psychosexual Intervention for Couples Post-allogeneic Hematopoietic Stem Cell Transplantation.

Allogeneic hematopoietic stem cell transplantation (HSCT) can have a long-term impact on patients' sexual functioning and consequently, their relationship satisfaction. This study aimed to examine the feasibility and acceptability of a 5-session psychosexual intervention for HSCT survivors and their partners. To be eligible patients were required to be more than 3 months post-allogeneic HSCT. The intervention comprised 2 components: (1) A psychosexual education session exploring medical/behavioral treatment options for sexual dysfunction, delivered by a specialist nurse; (2) A 4-session Emotionally-Focused Therapy-based relationship education program for couples delivered by a clinical psychologist. Measures assessing relationship quality, sexual functioning, anxiety, and depression were administered pre- and post-intervention. Feasibility was measured via participation rate, adherence and compliance with completing main measures. Acceptability of the intervention was assessed via patient/partner satisfaction surveys. Eight of 85 (9.4%) eligible patients and their partners participated in the study. Reasons for declining participation included: feeling uncomfortable due to sensitive nature of intervention; did not have any relationship/sexual function issues; and experiencing ongoing medical issues. Six of 8 couples (75%) attended at least 4 intervention sessions and 5 of 8 (62.5%) completed the main study measures post-intervention. Four couples reported that sexual satisfaction and relationship satisfaction was somewhat or much better post-intervention; 1 reported no change. Feasibility criteria were not met, with low enrolment rate, however the intervention was deemed acceptable. Couples who participated adhered to the intervention and considered it beneficial. Further strategies to identify and manage sexual concerns are required.

Flow cytometry of DNMT1 as a biomarker of hypomethylating therapies.

The 5-azacytidine (AZA) and decitabine (DEC) are noncytotoxic, differentiation-inducing therapies approved for treatment of myelodysplastic syndrome, acute myeloid leukemias (AML), and under evaluation as maintenance therapy for AML postallogeneic hematopoietic stem cell transplant and to treat hemoglobinapathies. Malignant cell cytoreduction is thought to occur by S-phase specific depletion of the key epigenetic regulator, DNA methyltransferase 1 (DNMT1) that, in the case of cancers, thereby releases terminal-differentiation programs. DNMT1-targeting can also elevate expression of immune function genes (HLA-DR, MICA, MICB) to stimulate graft versus leukemia effects. In vivo, there is a large inter-individual variability in DEC and 5-AZA activity because of pharmacogenetic factors, and an assay to quantify the molecular pharmacodynamic effect of DNMT1-depletion is a logical step toward individualized or personalized therapy. We developed and analytically validated a flow cytometric assay for DNMT1 epitope levels in blood and bone marrow cell subpopulations defined by immunophenotype and cell cycle state. Wild type (WT) and DNMT1 knock out (DKO) HC116 cells were used to select and optimize a highly specific DNMT1 monoclonal antibody. Methodologic validation of the assay consisted of cytometry and matching immunoblots of HC116-WT and -DKO cells and peripheral blood mononuclear cells; flow cytometry of H116-WT treated with DEC, and patient samples before and after treatment with 5-AZA. Analysis of patient samples demonstrated assay reproducibility, variation in patient DNMT1 levels prior to treatment, and DNMT1 depletion posttherapy. A flow-cytometry assay has been developed that in the research setting of clinical trials can inform studies of DEC or 5-AZA treatment to achieve targeted molecular pharmacodynamic effects and better understand treatment-resistance/failure.

At-Home Foscarnet Administration in Patients with Cytomegalovirus Infection Post-Allogeneic Stem Cell Transplantation: A Unicentric, Safe, and Feasible Program.

Cytomegalovirus (CMV) infection is a relevant cause of morbimortality in patients receiving allogeneic stem cell transplantation (allo-HCT). Foscarnet (FCN) is an effective drug against CMV administered intravenously and usually on an inpatient basis. The Home Care Unit (HCU) for hematologic patients at our hospital designed an at-home FCN administration model to avoid the hospitalization of patients requiring FCN treatment. This study analyzes whether the at-home administration of FCN is as safe and effective as its hospital administration. We collected and compared demographic, clinical, analytical, and economic data of patients with CMV infection post-allo-HCT who received FCN in the hospital (n = 16, 17 episodes) vs. at-home (n = 67, 88 episodes). The proportions of patients with cured CMV infections were comparable between the two groups (65.9% vs. 76.5%, p = 0.395). The median duration of FCN treatment was 15 (interquartile range [IQR] 9-23) and 14 (IQR 11-19) days in the HCU and inpatient cohorts, respectively (p = 0.692). There were no significant differences in the FCN toxicities between groups except for hypocalcemia (26.1% vs. 58.8%, p = 0.007), which was more prevalent in the inpatient cohort. A significant cost-effectiveness was found in the HCU cohort, with a median savings per episode of EUR 5270. It may be concluded that home administration of FCN is a safe, effective, and cost-efficient therapeutic option for patients with CMV infection and disease.

Post Allogeneic Stem Cell Transplant Outcomes Following Response to Hypomethylating Agent Therapy in Myelodysplastic Syndromes Are Predicted By Persistent International Prognostic Scoring System-Molecular Risk

Introduction: The International Prognostic Scoring System-Molecular (IPSS-M) ( Bernard NEJM Evidence 2022) and the 2023 International Working Group (IWG) response criteria for myelodysplastic syndrome (MDS) ( Zeidan Blood 2023) are used to more accurately assess prognosis and therapeutic response in MDS. However, it is unknown if these tools can be used to predict outcomes post-allogeneic hematopoietic stem cell transplant (HCT). We sought to understand the impact of pre-HCT IPSS-M on post-HCT outcomes in patients (pts) with MDS who responded to hypomethylating agent (HMA) therapy. Methods: Pts with MDS treated with HMA (azacitidine or decitabine) who received an HCT post-HMA at Dana-Farber Cancer Institute from January 2014 to December 2020 were included. Response to HMA was assessed by 2023 IWG response criteria and was defined as complete remission (CR) + CR with bi-lineage blood count recovery (CRbi) + CR with uni-lineage blood count recovery (Cruni) + CR with partial hematological recovery (CRh). Combined clinical and molecular risk was assessed by IPSS-M at times of diagnosis and of HCT, the latter using the bone marrow biopsy and sequencing data collected closest to HCT. High risk was defined as IPSS-M moderately high, high, and very high, whereas low risk was defined as IPSS-M very low, low, and moderately low. Results: A total of 148 pts with MDS who received HMA and underwent subsequent HCT were included. Median age was 64 years (range 26-79) and 61.5% were men. Pts were diagnosed with MDS-EB1 (26.4%), MDS-EB2 (45.3%), and other MDS subtypes (28.3%). IPSS-M at time of diagnosis was very low (2%), low (10.1%), moderate low (6.1%), moderate high (8.8%), high (31.8%), and very high (22.3%). IPSS-M pre-HCT was very low (12.8%), low (8.8%), moderate low (10.8%), moderate high (12.8%), high (20.3%), and very high (10.8%). IPSS-M at diagnosis and pre-HCT could not be calculated because of missing molecular data in 18.9% and 23.6% of pts, respectively. Pts received a median of 4 HMA cycles (range 1-20) and were treated with azacitidine for 7 days (54.1%), decitabine for 5 days (39.2%), and other schedules (6.7%). Prior to HCT, IWG 2023 responses were: CR (15.5%), CRbi (14.9%), CRuni (19.6%), CRh (0.7%), partial remission (PR: 1.4%), hematological improvement (HI: 9.5%), and no response (38.5%). Most pts received HCT from either a matched unrelated (60.8%) or matched related donor (18.2%) with reduced intensity conditioning (74.3%). Pts received graft versus host disease prophylaxis with tacrolimus (tac)/methotrexate (MTX) (54.7%), tac/MTX/sirolimus (19.6%), post-transplant cyclophosphamide/mycophenolate mofetil/tac (15.5%), tac/sirolimus (8.1%), other (2%). Median HCT-comorbidity index (CI) was 2 (range 0-13) and 48.6% pts had HCT-CI ≥ 3. For the entire cohort, the median follow-up time among ongoing survivors was 48.3 months (range 5.5-101.3). Among pts who responded to HMA per IWG 2023 criteria (CR/CRbi/CRuni/CRh), those who had high risk by IPSS-M prior to HCT had significantly shorter median overall survival (OS) (27 months; 95% CI 7.5-51) compared to pts with a low risk by IPSS-M (not reached; p=0.016) ( Figure A). Cumulative incidence of relapse (CIR) at 4 years was 66% for pts with high risk and 31% for low risk (p=0.034) ( Figure B). Pts with response to HMA with high risk by IPSS-M had lower OS (4-year OS: 27% versus 53%; p=0.016) and progression-free survival (4-year PFS: 19% versus 50%; p=0.018) but similar non-relapse mortality (NRM) (4-year NRM: 16% vs. 19%; p=0.66) post-HCT compared to pts with low risk at time of HCT. Conclusion: For pts with MDS who achieve a response to HMA prior to HCT, combined clinical/molecular risk, as assessed by IPSS-M, has an important prognostic impact on post-HCT outcomes. Pre-HCT risk should be evaluated for prognostication and to guide patient care, including future prospective studies evaluating novel agents for post-HCT therapy.

New Assay Technology and Utility of Peripheral Blood Vs Bone Marrow for Molecular MRD in AML and MDS

Background Rare tumor-specific mutations in patient samples serve as excellent markers to monitor the course of malignant disease and responses to therapy in clinical routine, and improved assay techniques are needed for broad adoption. We describe herein - superRCA assays - which provides for rapid and highly specific detection of DNA sequence variants present at very low frequencies in DNA samples using a standard flow cytometer for readout. We demonstrate and compare the assay against ddPCR for precise, ultra-sensitive detection of single-nucleotide mutant sequences from malignant cells to follow the course of AML and MDS patients following allogeneic stem cell transplantation, both in bone marrow and peripheral blood. Methods This study is based on patient material from the Nordic MDS study NMDSG14B (NCT02872662)and AML samples from the U-CAN biobank. The patients in this retrospective study have all previously undergone sequencing (TruSight panel, Illumina) upon diagnosis, after which patient specific driver mutations (1-4 per patient) has been chosen and analyzed longitudinally to monitor molecular MRD using ddPCR (Bio-Rad). These longitudinal samples were re-analyzed using superRCA mutation assays (Rarity Bioscience), a novel and ultra-sensitive technique for mutation detection using flow cytometer for readout. Both quarterly bone marrow aspirates and monthly peripheral blood samples post allogeneic stem cell transplantation were included to investigate to what extent frequent blood derived molecular MRD gives equivalent relapse information as bone marrow. For each mutation, sensitivity was assessed through serial dilution and wild type control, with the limit of detection calculated as Mean(wt)+3*SD(wt). Samples were analyzed in duplicates or triplicates and sample to sample variation was calculated as SD/Mean. Resul ts A total of 22 relapse patients and 21 different mutations were analyzed, covering single nucleotide variants, insertions, deletions, as well as mutations with very high GC in ASXL1 and SRSF2. The analysis demonstrating lower background and limit of detection for the superRCA assays compared to the equivalent ddPCR data. With spike-in dilution series, we demonstrated the superRCA assay could detect single point mutations as low as 1 in 100,000 with the flow cytometer readout. Mean background for superRCA was 0,0016% in wild type controls, and mean limit of detection was 0,0023%. From available serial dilutions, the mean sample to sample variation (CV) at 0,016% was 22,87% and 29,32% at 0,001% dilution. In longitudinal sample analysis of relapse patients, superRCA assay detected remaining mutations after initial treatment and clearly revealed the remaining malignant clone, subsequently leading to a relapse. The superRCA assay also demonstrated the feasibility of detecting corresponding leukemia mutations in the PBMCs when such mutations were present in bone marrow. Conclusions The data shows comparable mutant allele frequency results between superRCA and ddPCR upon diagnosis and relapse. The data further demonstrates superRCA mutation assays for the selected driver mutations can reach to very low limit of detection and increasing mutations could be detected in both blood and bone marrow predicting the relapse of MDS post transplantation in clinical samples. The low detection limit and high precision of superRCA are consequences of the highly selective genotyping of the repeated target sequences in combination with high input capacity and the large numbers of products that may be conveniently analyzed by flow cytometry. To further study the utility of using peripheral blood for molecular MRD in MDS and AML patients, more data is needed and studies are ongoing.

Real-World Effectiveness and Safety of Hetrombopag for Delayed Platelet Engraftment Post-Allogeneic Hematopoietic Stem Cell Transplantation

Background: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) serves as a critical therapeutic approach for various malignant and non-malignant hematopoietic disorders. Notwithstanding successful hematopoietic recovery in most patients following allo-HSCT, a subset may encounter delayed platelet engraftment (DPE), a prevalent and potentially serious complication that stems from myeloablative conditioning. Occurring in approximately 5%-37% of allo-HSCT recipients, DPE is a known harbinger of increased transplant-related mortality and diminished quality of survival. Despite its clinical significance, a well-established standard treatment protocol for post-allo-HSCT DPE remains elusive. Multiple platelet transfusions, often administered in such cases, not only exacerbate the economic burden but also induce toxicities, thereby undermining the patient's quality of life. An alternative to platelet transfusion lies in the utilization of thrombopoietin-receptor agonists (TPO-RAs), advocated for enhancing platelet engraftment. These agents interact with the thrombopoietin receptor, stimulating intracellular signaling pathways inclusive of JAK/STAT, PI3K/AKT, and ERK1/2, which in turn, promote megakaryocyte proliferation and differentiation, thereby augmenting platelet production. Hetrombopag, a novel, orally administered, non-peptide, small-molecule TPO-RA, has been validated to effectively boost platelet engraftment and reduce the necessity for platelet transfusions post-allo-HSCT in a pilot study. Our study sought to investigate the effectiveness and safety of hetrombopag in treating DPE following allo-HSCT within a real-world setting. Methods: In this retrospective study, we evaluated adult patients with hematopoietic disorders who underwent allo-HSCT and subsequently experienced DPE in our center, treated with hetrombopag between March 2022 and April 2023. Given the lack of a consensus definition, we deemed DPE as a platelet count (PLT) &amp;lt; 20 × 10 9/L or a significant and rapid decreasing trend past day 28 post-allo-HSCT. Hetrombopag dosages of 2.5 mg, 5 mg, or 7.5 mg/day were prescribed based on individual patient conditions. Response criteria post-treatment were as follows: complete response: PLT &amp;gt; 50 × 10 9/L without platelet transfusion; partial response: PLT between 20 and 50 × 10 9/L without platelet transfusion; no response: PLT &amp;lt; 20 × 10 9/L. Results: A total of 20 patients were included in the analysis, with a median age of 52, and 60% of them were male. Hetrombopag administration commenced at a median of 58 days following allo-HSCT at a median dose of 5 mg/day, with a median treatment duration of 44 days. The initial platelet count was 24.5 × 10 9/L and serum ferritin levels stood at 3351.1 ng/mL. Post-hetrombopag treatment, platelet counts escalated to 67.5 × 10 9/L while serum ferritin levels decreased to 2726.0 ng/mL. Of the 20 patients, 13 patients (65%) exhibited a complete response, 6 patients (30%) achieved a partial response, and 1 patient (5%) displayed no response. Hetrombopag was well tolerated with only 5 patients reporting increased alanine aminotransferase/aspartate aminotransferase or total bilirubin levels (all grade 1 or 2), and the association with hetrombopag was undetermined. Conclusion: Our findings suggest that hetrombopag yields a sustained response with acceptable tolerability in patients who experienced DPE following allo-HSCT, and could potentially offer a novel approach for managing such patients. Future large-scale, prospective studies are warranted to further assess the potential benefits of hetrombopag for improving patient outcomes following allo-HSCT.

Inotuzumab Ozogamicin in Lymphoid B Cell Malignancy：a Single Center Analysis in the Real World

Background : Recent developments in immunoconjugate usage has changed the landscape of lymphoid B-cell malignancy therapy. Historically, those who relapse from this condition have a dismal prognosis and limited treatment options. One recent development is the inotuzumab ozogamicin (INO), a humanized anti-CD22 monoclonal antibody conjugated to the cytotoxic antibiotic calicheamicin, which has the potential to reduce the overall toxicity of intensive regimens for ALL, as well as to show therapeutic effects in lymphoid B-cell malignancy. Aims and Methods: The study included B cell-acute lymphoblastic leukemia (B-ALL) (n=23), B/ myeloid mixed phenotype acute leukemia (MPAL) (n=1) and B-cell lymphoblastic lymphoma (B-LBL) (n=3) adult patients who received INO in our center from June 2022 to June 2023. Intravenous INO was given 1 or more doses at the dose of 0.6mg/m2, its clinical efficacy and adverse reactions were explored. Results: In our center, INO was strategically employed in various therapeutic scenarios: induction (n=2), maintenance/consolidation (n=6), minimal residual disease (MRD) clearance (n=5), and the management of relapsed/refractory lymphoid B-cell malignancies (n=14). Of the 27 patients, 14 were subjected to a combination regimen of INO and chemotherapy for recurrent and refractory lymphoid B-cell malignancy. The composition of these patients comprised of 4 individuals who experienced a relapse post-standard chemotherapy, 1 patient with a relapse post-allogeneic hematopoietic stem cell transplantation (allo-HSCT), 4 patients with a relapse post-autologous hematopoietic stem cell transplantation (auto-HSCT), and the remaining 5 patients were categorized as refractory cases. The median number of doses administered was 2 (1-3). Complete remission (CR) or CR with incomplete hematologic recovery (CRi) was observed in 9 out of 14 patients (64.3%) within a median duration of 30 (16-78)days. Partial response (PR) was noted in 2 of the 14 patients (14.3%), rendering the overall response rate (ORR) to be 78.6%. Negative MRD was observed in 7 out of 14 patients (50.0%). Of the 9 patients who achieved CR/CRi, 4 (44.4%) relapsed again with 2 fatalities. The remaining 2 patients attained negative MRD following a second allo-HSCT or INO retreatment. The two patients who achieved PR proceeded to receive chimeric antigen receptor T cell (CART) treatment, with one of them subsequently undergoing allo-HSCT after achieving CR. For the three non-responders (NR), one patient unfortunately passed away while the other two patients remained alive and were either participating in clinical trials or undergoing allo-HSCT after CART. For MRD clearance, it was successfully achieved in 4 out of 5 patients (80.0%)treated with INO within a median period of 45 (28-52)days. However, 2 patients reverted to MRD positivity and 1 died. The other four patients underwent allo-HSCT with neither mortality nor recurrence observed. Moreover, two patients (100%) were treated with a VICLP induction regimen combined with INO, with MRD negativity observed post-treatment. Regarding the remaining six patients, three patients were treated with INO as consolidation therapy due to high-risk factors or the intention to undergo auto-HSCT. Another 3 B-ALL patients treated with INO as maintenance therapy after auto-HSCT or after the remission of recurrence after transplantation. One patient unfortunately experienced a second recurrence four months following INO maintenance. The remaining five patients achieved and have since sustained negative MRD status, preceding INO treatment. Fortunately, no treatment-related fatalities or treatment discontinuation due to adverse events were observed. Notably, 19 out of 27 patients (70.4%) experienced Grade 3-4 hematological toxicity, while 1 out of 24 patients (4.2%) exhibited Grade 3 alanine aminotransferase (ALT) elevation. Veno-occlusive disease (VOD) was not observed. Furthermore, two patients developed septicemia, one each from Escherichia coli and Klebsiella pneumoniae, respectively. Conclusions: Given the well tolerance and improvement in response rates, InO may be an important treatment option not only for patients with relapsed/refractory lymphoid B-cell malignancy, but also for induction, maintenance and MRD clearance treatment. Key words: inotuzumab ozogamicin, acute lymphoblastic leukemia, hematopoietic stem cell transplantation, CD22

Yield of Repeat Blood Cultures in Acute Myeloid Leukemia Patients Following Allogeneic Hematopoietic Stem Cell Transplantation

Introduction: Unnecessary repeat blood culture collection has been demonstrated to increase health care costs, length of stay, duration of antimicrobial exposure, and risk of anemia and result in unnecessary removal of vascular catheters. The aim of this study was to characterize the yield of new pathogen identification in acute myeloid leukemia (AML) patients with known bacteremia in the post-allogeneic hematopoietic stem cell transplant (HSCT) period. Methods: This was a retrospective study of AML patients who underwent allogeneic HSCT at Taussig Cancer Center from January 1 st 2019 to December 31 st 2022. Patients who experienced an occurrence of febrile neutropenia (FN) within 35 days of date of HSCT and had positive initial blood cultures within 24 hours of diagnosis of FN were included in the study. Patients who had at least 1 additional set of repeat cultures drawn following initial blood cultures were included in the study. Blood cultures were collected through a peripheral site via venipuncture or through a central line. The study was approved by the Institutional Review Board. Data were collected on baseline patient characteristics, AML disease characteristics, HSCT characteristics, and infection characteristics. Febrile neutropenia was defined as recorded temperature ≥ 100.4°F with concurrent ANC &amp;lt;1500 cells/uL. Each patient day with a recorded temperature ≥100.4°F and ANC &amp;lt;1500 cells/μL was considered as a day of FN. An episode of FN was considered to be resolved following 72 hours of defervescence. Cultures drawn within 24 hours of onset of FN were considered the initial blood cultures. All subsequent sets of blood cultures were considered repeat blood cultures. A positive set of blood culture was defined as &amp;gt;50% of all blood cultures in that set growing an organism. Cultures drawn from all peripheral and central sites on the same day of FN were considered as one set of blood cultures. All patients received post-HSCT anti-bacterial prophylaxis with bactrim or ciprofloxacin. Results: A total of 154 AML patients who underwent HSCT at Taussig Cancer Center were screened for the study. 50/154 (32.5%) of those patients met inclusion criteria and were included in the study. Among the 50 patients included in the study, there were 50 episodes of FN with positive initial blood cultures that were diagnosed within 35 days of HSCT. There were 120 total days of FN among the 50 episodes of FN with an average of 2.4 days per episode. 50 initial sets of blood cultures and 96 sets of repeat blood cultures were drawn between the 50 episodes of FN with an average of 2.9 total sets of blood cultures per episode. 12/96 (12.5%) of repeat blood cultures sets were positive for a pathogen. 3/96 (3.1%) of repeat blood cultures sets grew a pathogen that differed from the pathogen which grew in the preceding positive blood culture. The median duration of febrile neutropenia at time of repeat positive culture was 3.5 days. 30-day mortality rate from infection was 6.0% (3/50). Conclusion: Even amongst bacteremic AML patients in the post-HSCT period, the yield of repeat blood cultures was low for detecting new pathogen identification. Repeat blood cultures should be guided by thorough evaluation of emerging clinical signs and symptoms.

Zinc Status Affects T Cell Reconstitution in Patients Receiving Naïve T Cell Depleted Allogeneic HSCT

Delayed immune reconstitution (IR) is a major clinical problem contributing to post-allogeneic hematopoietic stem cell transplant (allo-HSCT) morbidity and mortality. Specifically, CD4 T cell reconstitution is linked with improved clinical outcomes. As the primary organ of T cell production, the thymus is a crucial contributor to T cell reconstitution after HSCT generating a broad T cell receptor (TCR) repertoire selected through central tolerance However, T cell reconstitution can be hindered by poor thymus function as a consequence of age, transplant conditioning, or graft versus host disease (GVHD). At present, no drugs are available to boost T cell recovery, while many of the factors limiting IR are still unknown. Zinc (Zn), the second most abundant trace element in our body, is important for normal T cell development. Zn deficiency (ZD) in humans and mice is characterized by poor circulating T cells and depressed thymic function, which are both restorable after reintroduction of Zn with the diet. Despite having multiple risk factors for ZD, there are no published data focusing on ZD prevalence among allo-HSCT recipients. Based on our previous observations, we hypothesized that peri-HSCT Zn levels correlate with IR. In this study, we analyzed serum Zn levels of allo-HSCT patients who received naïve T cell (T N) depleted grafts as a strategy to prevent aGVHD (Bleakley et al., 2022, NCT00914940). This population is particularly interesting for studying IR because in these patients, circulating T N at d+365 after HSCT will primarily be of donor hematopoietic stem cell origin generated in the recipient thymus. Samples and clinical data were prospectively collected. Fifty-two patients had peri-HSCT serum samples available for Zn measures, as pre-HSCT and/or day +80 samples. Zn measures were performed through mass-spectrometry at the University of Washington Hospital-Biochemistry Lab. ZD was defined for Zn&amp;lt;70ug/dL as defined by the WHO. T cell populations (CD4 +, CD8 +, T N CD4 + and CD8 +, T regulatory cells, NK cells, B cells) were analyzed by flow cytometry at day+365. DNA was extracted from PBMC pellets collected at d+365 and were analyzed in qPCR for the expression of signal-joint T cell receptor excision circles (sjTRECs), being the gold standard for measuring thymic-derived T cells. We studied the association between Zn levels at baseline and day+80 and parameters of IR through linear regression. Comparisons between two groups were analyzed by t-test. Median age at transplant was 47 (range 6-61). Twenty-eight patients received myeloablative conditioning with total body irradiation (TBI) of 13.2 Gy (53.8%) and 24 (46.2%) received an intermediate intensity conditioning regimen with 4Gy TBI. Forty-one patients (78.8%) had pre-HSCT and 46 (88.5%) had day+80 Zn measures; 38 patients (73.1%) had both pre-HSCT and day+80 Zn measures. Median pre-HSCT and day+80 Zn levels were, respectively, 71 (range 60-105) and 80 (range 36-112); at baseline, 13 patients (25%) had ZD (&amp;lt;70ug/dL, according to WHO), whereas seven (13.3%) were ZD at day+80. Median sjTRECs at day +365 were 3.33/mg of DNA (range 0.00-16,704.54). As expected, sjTRECs at day+365 significantly correlated with age at transplant (r 2=0,3824; p&amp;lt;0.0001), whereas Zn levels preHCT or day80 did not. The number of circulating CD4 + and CD8 + T N at day+365 showed significant correlation with age. Zinc levels did not correlate with IR of absolute CD4 +, CD8 +, NK, T regs, and B cells at day+365, although we observed a trend for lower CD4 + counts at day 365 with lower day+80 Zn levels. Day+80 serum Zn levels was associated with sjTRECs at day+365 (Figure 1A. r 2=0.2643; p=0.0019) and lower pre-HSCT Zn significantly correlated with worse CD8 +T N reconstitution at day+365 (Figure 1B. r 2=0.1433; p= 0.0327), with clear trends in the same direction also for pre-HSCT Zn and CD4 + T N and day+80 Zn and CD4 + T N. In conclusion, we found that a non-negligible population of patients undergo allo-HSCT with unrecognized ZD. Peri-transplant Zinc status negatively affects IR, especially on the thymic-derived T cell compartment. Early recognition of ZD and zinc supplementation might help prevent morbidity related to poor IR in a proportion of patients. A multivariable analysis looking at other clinical data and outcomes is ongoing. Validation of these findings is needed on a larger number of patients, including a cohort of patients receiving T N-replete HSCT.

Clinically Significant CMV Infection in Allogeneic Stem-Cell Transplant Recipients: A Single Center Experience

Introduction: Cytomegalovirus (CMV) infection is one of the major causes of concern in allogeneic stem cell transplantation (allo-HSCT). Here we report the clinically significant CMV infections among allo-HSCT recipients at the National Center for Cancer Care and Research (NCCCR) in Qatar. Methods: A retrospective review of the electronic health records of all malignant hematology patients who underwent allo-HSCT at NCCCR was performed from September 2017 to December 2022. Results: Fifty-one malignant hematology patients have been transplanted during the data collection period. Patient characteristics are summarized in Table 1. The median age was 34 years (range 14-53 years). CMV-serostatus was positive in fifty recipients (98%) and all donors. Thirty-two patients (62.7%) have received matched-related grafts. The most frequent indication for HSCT was acute myeloid leukemia, followed by acute lymphoblastic leukemia. Forty-six patients (92%) underwent myeloablative conditioning therapy. Graft-versus-host disease (GvHD) prophylaxis used in the non-haploidentical group was cyclosporine and short-course methotrexate while haploidentical recipients received cyclosporine, mycophenolate mofetil, and post-transplant cyclophosphamide. Letermovir prophylaxis was accessible for use for high-risk patients from February 2022. CMV-PCR was monitored twice per week for all patients until day +100. The clinically significant CMV infection rate was 51%. The cumulative incidence of CMV infection in haploidentical and non-haploidentical groups was 70% and 30%, respectively (figure 1). The median CMV-PCR viral load was 1783.5 IU/mL (range 133- 9600 IU/mL), with the earliest CMV reactivation on day -1 of transplantation. The clinically significant CMV infection incidence was significantly higher in patients who did not receive letermovir prophylaxis. Preemptive antiviral therapy was initiated to prevent CMV disease in all indicated recipients. Foscarnet was the preferred first-line pre-emptive treatment option for patients during cytopenia. No CMV disease or CMV-related mortality occurred in our population. Conclusion: This is the first report from Qatar to report CMV infection post-allogeneic stem cell transplantation. Future in-depth analysis of our population and treatment responses is needed.