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Higher γδ T cell counts in patients with malignancies are associated with better survival. However, γδ T cells are rare in the blood and functionally impaired in patients with malignancies. Promising results are reported on the treatment of various malignancies with in vivo expansion of autologous γδ T cells using zoledronic acid (zol) and interleukin-2 (IL-2). Here we demonstrated that zol and IL-2, in combination with a novel genetically engineered K-562 CD3scFv/CD137L/CD28scFv/IL15RA quadruplet artificial antigen-presenting cell (aAPC), efficiently expand allogeneic donor-derived γδ T cells using a Good Manufacturing Practice (GMP) compliant protocol sufficient to achieve cell doses for future clinical use. We achieved a 633-fold expansion of γδ T cells after day 10 of coculture with aAPC, which exhibited central (47%) and effector (43%) memory phenotypes. In addition,90% of the expanded γδ T cells expressed NKG2D, although they have low cell surface expression of PD1 and LAG3 inhibitory checkpoint receptors. In vitro real-time cytotoxicity analysis showed that expanded γδ T cells were effective in killing target cells. Our results demonstrate that large-scale ex vivo expansion of donor-derived γδ T cells in a GMP-like setting can be achieved with the use of quadruplet aAPC and zol/IL-2 for clinical application.

Little is known about whether risk classification at diagnosis predicts post-hematopoietic cell transplantation (HCT) outcomes in patients with acute myeloid leukemia (AML). We evaluated 8709 patients with AML from the CIBMTR database, and after selection and manual curation of the cytogenetics data, 3779 patients in first complete remission were included in the final analysis: 2384 with intermediate-risk, 969 with adverse-risk, and 426 with KMT2A-rearranged disease. An adjusted multivariable analysis detected an increased risk of relapse for patients with KMT2A-rearranged or adverse-risk AML as compared to those with intermediate-risk disease (hazards ratio [HR], 1.27; P = .01; HR, 1.71; P < .001, respectively). Leukemia-free survival was similar for patients with KMT2A rearrangement or adverse risk (HR, 1.26; P = .002, and HR, 1.47; P < .001), as was overall survival (HR, 1.32; P < .001, and HR, 1.45; P < .001). No differences in outcome were detected when patients were stratified by KMT2A fusion partner. This study is the largest conducted to date on post-HCT outcomes in AML, with manually curated cytogenetics used for risk stratification. Our work demonstrates that risk classification at diagnosis remains predictive of post-HCT outcomes in AML. It also highlights the critical need to develop novel treatment strategies for patients with KMT2A-rearranged and adverse-risk disease.

Key Points Ofatumumab with glucocorticoid therapy for cGVHD resulted in 62.5% ORR at 6 months and 53% FFS at 12 months.Safety was observed with ofatumumab plus glucocorticoid for initial therapy., Visual Abstract, Standard initial therapy of chronic graft vs. host disease (cGVHD) with glucocorticoids results in suboptimal response. Safety and feasibility of therapy with ofatumumab (1000 mg IV on days 0 and 14) and prednisone (1 mg/kg/day) was previously established in our phase I trial (n = 12). We now report the mature results of the phase II expansion of the trial (n = 38). The overall NIH severity of cGVHD was moderate (63%) or severe (37%) with 74% of all patients affected by the overlap subtype of cGVHD and 82% by prior acute cGVHD. The observed 6 month clinician-reported and 2014 NIH-defined overall response rates (ORR = complete + partial response [CR/PR]) of 62.5% (1-sided lower 90% confidence interval=51.5%) were not superior to pre-specified historic benchmark of 60%. Post-hoc comparison of 6 month NIH response suggested benefit compared to more contemporaneous NIH-based benchmark of 48.6% with frontline sirolimus/prednisone (CTN 0801 trial). Baseline cGVHD features (organ involvement, severity, initial immune suppression agents) were not significantly associated with 6-month ORR. The median time to initiation of second-line therapy was 5.4 months (range 0.9-15.1 months). Failure-free survival (FFS) was 64.2% (95% CI 46.5-77.4%) at 6 months and 53.1% (95% CI 35.8-67.7%) at 12 months, whereas FFS with CR/PR at 12 months of 33.5% exceeded a benchmark of 15% in post-hoc analysis, and was associated with greater success in steroid discontinuation by 24 months (odds ratio 8 (95% CI 1.21-52.7). This single-arm phase II trial demonstrated acceptable safety and potential efficacy of the upfront use of ofatumumab in combination with prednisone in cGVHD. This trial was registered at www.clinicaltrials.gov as #NCT01680965.

Purpose:In this first-in-human, phase I, GVHD prevention trial (NCT02891603), we combine pacritinib (PAC), a JAK2 inhibitor, with sirolimus to concurrently reduce T-cell costimulation via mTOR and IL6 activity. We evaluate the safety of pacritinib when administered with sirolimus plus low-dose tacrolimus (PAC/SIR/TAC) after allogeneic hematopoietic cell transplantation.Patients and Methods:The preclinical efficacy and immune modulation of PAC/SIR were investigated in xenogeneic GVHD. Our phase I trial followed a 3+3 dose-escalation design, including dose level 1 (pacritinib 100 mg daily), level 2 (pacritinib 100 mg twice daily), and level 3 (pacritinib 200 mg twice daily). The primary endpoint was to identify the lowest biologically active and safe dose of pacritinib with SIR/TAC (n = 12). Acute GVHD was scored through day +100. Allografts included 8/8 HLA-matched related or unrelated donor peripheral blood stem cells.Results:In mice, we show that dual JAK2/mTOR inhibition significantly reduces xenogeneic GVHD and increases peripheral regulatory T cell (Treg) potency as well as Treg induction from conventional CD4+ T cells. Pacritinib 100 mg twice a day was identified as the minimum biologically active and safe dose for further study. JAK2/mTOR inhibition suppresses pathogenic Th1 and Th17 cells, spares Tregs and antileukemia effector cells, and exhibits preliminary activity in preventing GVHD. PAC/SIR/TAC preserves donor cytomegalovirus (CMV) immunity and permits timely engraftment without cytopenias.Conclusions:We demonstrate that PAC/SIR/TAC is safe and preliminarily limits acute GVHD, preserves donor CMV immunity, and permits timely engraftment. The efficacy of PAC/SIR/TAC will be tested in our ongoing phase II GVHD prevention trial.

The table shows maximum acute GVHD organ stage and overall grade observed through day +100 after transplant on the PAC/SIR/TAC phase I trial.

The table shows the characteristics of patients treated on the PAC/SIR/TAC phase I trial.

Impact of JAK2/mTOR blockade on human NK cell function and proliferation. A) Graph shows the cytolytic activity ({plus minus}SEM) of NK cells after 4 hours of culture with K562 target cells, while exposed to DMSO, ruxolitinib (lμM), pacritinib (1.25μM), sirolimus 10ng/ml, or a combination of pacritinib plus sirolimus. B) Human NK cells (105 ) were stimulated with a cytokine cocktail of recombinant human (rh) IL-2 (200 IU/ml) and rhll-15 (10 ng/ml), in the presence of DMSO, ruxolitinib, pacritinib, sirolimus, or pacritinib plus sirolimus. Drugs were added once on day 0. Cytokines were replenished on day +3. Bar graph shows NK cell proliferation ({plus minus}SEM) on day +5 of the culture using a colorimetric assay.

The table shows the pharmacokinetic analysis of pacritinib concentration over time.

The table shows pacritinib adherence during treatment period according to dose level on the PAC/SIR/TAC phase I trial.

Safety measures monitored on trial including (A) QTcF, (B) cardiac ejection fraction, (C) prothrombin time, (D) partial thromboplastin time, (E) and thrombin time

The table shows adverse events per CTCAE version 5 encountered during the PAC/SIR/TAC phase I trial.

CD4+ T cell Aurora kinase A activation as a GVHD resistance mechanism. A) Graph shows frequency of CD4+, pHistone 3 serine 10+ (pH3ser10) (downstream of CD28, and parallel to mTOR) T cells (mean{plus minus} SEM) at day +21 among patients treated on dose level 1 or dose level 2 of the phase I trial compared to baseline control values. B) Graph depicts the frequency (mean {plus minus} SEM) of CD4+, pH3ser10+ T cells recovered from NSG spleens at day +14, following transplantation with human PBMCs and then treated with vehicle, pacritinib, 531-201, sirolimus, pacritinib plus sirolimus, or 531-201 plus sirolimus for 2 weeks.

The table shows the maximum chronic GVHD organ involvement and overall score per NIH Diagnosis and Staging Criteria through full extent of study follow up on the PAC/SIR/TAC phase I trial.

Impact of JAK2 or STAT3 inhibition plus mTOR blockade on DC-allostimulated human T cell production of IFNγ, IL-13, and GM-CSF. 5-day alloMLRs were treated with DMSO vehicle control, pacritinib 1.25μM, S3I-201 5μM, sirolimus 10ng/ml, pacritinib plus sirolimus, or S3I-201 plus sirolimus. The graphs show the concentrations of A) IFNγ, B) IL-13, and C) GM-CSF (mean{plus minus}SEM) as measured in the supernatants of the culture medium by a cytokine multiplex assay.

Extended Methods including information and details regarding flow cytometry, cell lines, allogeneic mixed leukocyte reactions, Treg Suppression Assays, Xenogeneic GVHD modeling, and key reagents.

The table shows donor chimerism in unsorted bone marrow, and peripheral blood CD3 and CD33 populations from patients treated on the PAC/SIR/TAC phase I trial.

Serial measures of absolute neutrophil count (ANC), hemoglobin (Hgb) and platelet counts (PLT) through complete pacritinib treatment period

The table shows donor/recipient CMV serostatus.

Impact of JAK2/mTOR blockade on human T cell responses toward CMV, EBV, Influenza, and tetanus stimulation. A, B) Graphs show the recall proliferation of CD4+ and CD8+ T cell proliferation in response to CEFT peptides. C, D) Graph shows the frequency of IFNy+, CD107a+ CD4+ or cos+ T cells after stimulation with CEFT peptides.

Post-transplantation cyclophosphamide (PTCy) is being increasingly used for graft-versus-host disease (GVHD) prophylaxis after allogeneic hematopoietic cell transplantation (allo-HCT) across various donor types. However, immune reconstitution and infection incidence after PTCy-based versus conventional GVHD prophylaxis has not been well studied. We evaluated the infection density and immune reconstitution (ie, absolute CD4+ T cell, CD8+ T cell, natural killer cell, and B cell counts) at 3 months, 6 months, and 1 year post-HCT in 583 consecutive adult patients undergoing allo-HCT with myeloablative (n = 223) or reduced-intensity (n = 360) conditioning between 2012 and 2018. Haploidentical (haplo; n = 75) and 8/8 HLA-matched unrelated (MUD; n = 08) donor types were included. GVHD prophylaxis was PTCy-based in all haplo (n = 75) and in 38 MUD allo-HCT recipients, whereas tacrolimus/methotrexate (Tac/MTX) was used in 89 and Tac/Sirolimus (Tac/Sir) was used in 381 MUD allo-HCT recipients. Clinical outcomes, including infections, nonrelapse mortality (NRM), relapse, and overall survival (OS), were compared across the 4 treatment groups. The recovery of absolute total CD4+ T-cell count was significantly lower in the haplo-PTCy and MUD-PTCy groups compared with the Tac/MTX and Tac/Sir groups throughout 1 year post-allo-HCT (P = .025). In contrast, CD19+ B-cell counts at 6 months and thereafter were higher in the haplo-PTCy and MUD-PTCy groups compared with the Tac/MTX and Tac/Sir groups (P

Multiple myeloma (MM) is the second most common blood cancer in adults leading to 117,000 deaths every year. Major breakthroughs in clinical research of the past decades transformed the diagnosis and treatment of MM improving the survival rates and overall quality of life of patients. Unfortunately, scientific advancements are not distributed equally around the globe leading to disparities in the treatment outcomes between different regions of the world. Management of MM in low- and middle-income countries represents a big challenge for healthcare providers considering the economic, technological, and infrastructural restraints in comparison to developed countries. Many standards of practice, including diagnostic tools and therapeutic regimens, are not available in developing regions of the world. As an example of an upper-middle-income country, Armenia has been witnessing considerable progress in the diagnosis and treatment of MM, including but not limited to the establishment of autologous stem cell transplant (ASCT), accessibility to modern anti-myeloma medications, and improved diagnostic and monitoring workup. Despite significant improvements, there is still a need for refinement in the management of MM. The aim of this review article is to discuss the latest developments and the current diagnosis and treatment of MM in Armenia as an example of a resource-limited region.

Higher infused total nucleated cell dose (TNC) in allogeneic bone marrow transplant (BMT) with post-transplant cyclophosphamide (PTCy) is associated with improved overall survival. As many centers prefer peripheral blood stem cell grafts (PBSCT) with PTCy, the effect of cell dose on outcomes with this platform also requires elucidation. We retrospectively evaluated 144 consecutive adult patients who received allogeneic T-cell replete PBSCT with PTCy-based graft-versus-host disease (GVHD) prophylaxis for a hematologic malignancy from 2012-2018. The infused CD34+ cell dose was stratified into low ( 10 × 106/kg) dose level groups. In multivariate analysis, the low CD34+ cell dose group had worse non-relapse mortality (HR = 4.51, 95% CI: 1.92-10.58, p 5 × 106 cells/kg may result in improved survival. Thus, this study supports targeting a CD34+ cell dose of >5 × 106 cells/kg for allogeneic PBSCT with PTCy.

Prognostic factors associated with clinical outcomes of acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) patients with central nervous system (CNS) involvement are unknown. We retrospectively studied the characteristics and outcomes of 66 (18 pediatric and 48 adult) patients with CNS leukemia with ALL (n = 41) or AML (n = 25). The median age of patients at diagnosis of CNS leukemia was 30 (range, 1–69) years. Nearly two-third patients had CNS involvement at the initial diagnosis of leukemia. Complete remission of CNS leukemia was attained in 58 (88%) patients, and probability of overall survival at 36 months after the diagnosis of CNS leukemia was 43% for the entire cohort. We identified that achieving remission of systemic leukemia and having CNS leukemia diagnosed and treated before allogeneic transplantation were the factors associated with CNS leukemia remission. Prognostic factors associated with better overall survival in patients with CNS leukemia included pediatric age, diagnosis of CNS leukemia before receiving allogenic transplantation, achieving clearance of systemic or CNS leukemia, receiving no cranial radiation in conjunction with intrathecal chemotherapy (IT), and receiving IT consolidation after achieving remission of CNS leukemia. Our findings show that patients with CNS leukemia are at considerable risk of mortality. Awareness of modifiable prognostic factors such as avoidance of cranial radiation whenever possible and use of IT consolidation can result in improved outcomes in subset of patients with CNS leukemia.

Reduced-intensity conditioning (RIC) allogeneic haematopoietic cell transplantation (allo-HCT) is a curative option for select relapsed/refractory Hodgkin lymphoma (HL) patients; however, there are sparse data to support superiority of any particular conditioning regimen. We analyzed 492 adult patients undergoing human leucocyte antigen (HLA)-matched sibling or unrelated donor allo-HCT for HL between 2008 and 2016, utilizing RIC with either fludarabine/busulfan (Flu/Bu), fludarabine/melphalan (Flu/Mel140) or fludarabine/cyclophosphamide (Flu/Cy). Multivariable regression analysis was performed using a significance level of