Impact of post-transplant cyclophosphamide and splenomegaly on primary graft failure and multi-lineage cytopenia after allogeneic hematopoietic cell transplantation.

Primary graft failure (PGF) and multi-lineage cytopenia (MLC) increase the risk of nonrelapse mortality in allogeneic hematopoietic cell transplants (HCT). We evaluated the impact of post-transplant cyclophosphamide (PTCy) and splenomegaly on PGF and MLC for hematological malignancies. This study included patients with PTCy (N=84) and conventional graft-vs.-host disease prophylaxis (N=199). The occurrence of splenomegaly varied widely, ranging from 17.1 % (acute myeloid leukemia) to 66.7 % (myeloproliferative neoplasms). Ten patients (N=8 in the PTCy and N=2 in the non- PTCy) developed PGF, and 44 patients developed MLC (both N=22). PTCy and severe splenomegaly (≥20 cm) were risk factors for PGF (odds ratio (OR): 10.40, p<0.01 and 6.74, p=0.01 respectively). Moreover, severe splenomegaly was a risk factor for PGF in PTCy patients (OR: 10.20, p=0.01). PTCy (hazard ratio (HR) 2.09, p=0.02), moderate (≥15, <20 cm, HR 4.36, p<0.01), and severe splenomegaly (HR 3.04, p=0.01) were independent risk factors for MLC. However, in subgroup analysis in PTCy patients, only mild splenomegaly (≥12, <15 cm, HR 4.62, p=0.01) was a risk factor for MLC. We recommend all patients be screened for splenomegaly before HCT, and PTCy is cautioned in those with splenomegaly.

IMPROVE-BMT: A Pilot Randomized Controlled Trial of Prehabilitation Exercise for Adult Hematopoietic Stem Cell Transplant Recipients.

Background: There is limited evidence on the effects of aerobic and resistance training exercise interventions to improve physical function and patient-reported outcomes prior to autologous and allogeneic hematopoietic stem cell transplant (HSCT). IMPROVE-BMT was a single-site, pilot randomized controlled trial investigating the feasibility, acceptability, and safety of a pragmatic resistance training exercise program prior to HSCT compared to usual HSCT care. Secondary aims included differences in physical function between the exercise group (EX) and usual care control group (UC). Methods: Outcome measurements were assessed: prior to HSCT, on/around day of HSCT admission, +30 days post-HSCT, and +100 days post-HSCT. The exercise intervention was a home-based exercise program that incorporated resistance-band and bodyweight exercises. Results: Acceptability among participants was 83%; exercise adherence averaged at 92%; and there were zero exercise-related adverse or serious adverse events. The average pre-transplant exercise phase was 6.28 weeks (2.71-18.29 weeks). EX (n = 36) demonstrated larger increases in the six-minute walk test distance, short physical performance battery scores, and 30-s chair stands compared to UC (n = 38) and demonstrated significant within-group improvements for the six-minute walk test, the short physical performance battery, the 30-s chair stands, and the timed up-and-go test. Conclusions: IMPROVE-BMT demonstrates that pragmatic exercise is highly feasible for HSCT recipients and can potentially lead to enhanced recovery that may not be achievable in non-exercisers.

Assessment of chimerism by next generation sequencing: A comparison to STR/qPCR methods.

Chimerism analysis is used to evaluate patients after allogeneic hematopoietic stem cell transplant (allo-HSCT) for engraftment and minimal measurable residual disease (MRD) monitoring. A combination of short-tandem repeat (STR) and quantitative polymerase chain reaction (qPCR) was required to achieve both sensitivity and accuracy in the patients with various chimerism statuses. In this study, an insertion/deletion-based multiplex chimerism assay by next generation sequencing (NGS) was evaluated using 5 simulated unrelated donor-recipient combinations from 10 volunteers. Median number of informative markers detected was 8 (range = 5 - 11). The limit of quantitation (LoQ) was determined to be 0.1 % recipient. Assay sample number/batch was 10-20 and total assay time was 19-31 h (manual labor = 2.1 h). Additionally, 50 peripheral blood samples from 5 allo-HSCT recipients (related: N = 4; unrelated: N = 1) were tested by NGS and STR/qPCR. Median number of informative markers detected was 7 (range = 4 - 12). Results from both assays demonstrated a strong correlation (Y = 0.9875X + 0.333; R2 = 0.9852), no significant assay bias (difference mean - 0.08), and 100 % concordant detection of percent recipient increase ≥ 0.1 % (indicator of increased relapse risk). NGS-based chimerism assay can support all allo-HSCT for engraftment and MRD monitoring and simplify clinical laboratory workflow compared to STR/qPCR.

Engraftment and Measurable Residual Disease Monitoring after Hematopoietic Stem Cell Transplantation: Comparison of Two Chimerism Test Strategies, Next-Generation Sequencing versus a Combination of Short-Tandem Repeats and Quantitative PCR.

Chimerism testing supports the study of engraftment and measurable residual disease (MRD) in patients after allogeneic hematopoietic stem cell transplant. In chimerism MRD, relapse can be predicted by increasing mixed chimerism (IMC), recipient increase ≥0.1% in peripheral blood, and proliferating recipient cells as a surrogate of tumor activity. Conventionally, the combination of short-tandem repeat (STR) and quantitative PCR (qPCR) was needed to ensure assay sensitivity and accuracy in all chimerism status. We evaluated the use of next-generation sequencing (NGS) as an alternate technique. The median numbers of informative markers in unrelated/related cases were 124/82 (NGS; from 202 single-nucleotide polymorphism), 5/3 (qPCR), and 17/10 (STR). Assay sensitivity was 0.22% (NGS), 0.1% (qPCR), and 1% (STR). NGS batch (4 to 48 samples) required 19.60 to 24.80 hours and 1.52 to 2.42 hours of hands-on time (comparable to STR/qPCR). NGS assay cost/sample was $91 to $151, similar to qPCR ($99) but higher than STR ($27). Using 56 serial DNAs from six post-transplant patients monitored by the qPCR/STR, the correlation with NGS was strong for percentage recipient (y = 1.102x + 0.010; R2 = 0.968) and percentage recipient change (y = 0.892x + 0.041; R2 = 0.945). NGS identified all 17 IMC events detected by qPCR (100% sensitivity). The NGS chimerism provides sufficient sensitivity, accuracy, and economical/logistical feasibility in supporting engraftment and MRD monitoring.

The addition of doxycycline to fluoroquinolones for bacterial prophylaxis in autologous stem cell transplantation for multiple myeloma.

BACKGROUND: Bacterial prophylaxis with a fluoroquinolone (FQ) during autologous stem cell transplant (ASCT) is common, although not standardized among transplant centers. The addition of doxycycline (doxy) to FQ prophylaxis was previously linked to reduced neutropenic fever and bacteremia in multiple myeloma (MM) patients undergoing ASCT although several confounders were present. We compared the incidence of neutropenic fever and bacteremia between MM patients variably receiving prophylaxis with FQ alone and FQ-doxy during ASCT. METHODS: Systematic retrospective chart review of MM patients who underwent ASCT between January 2016 and December 2021. The primary objective was to determine the effect of bacterial prophylaxis on neutropenic fever and bacteremia within 30 days of ASCT. Multivariable logistic regression for neutropenic fever and univariate logistic regression for bacteremia accounted for differences in subject characteristics between groups. RESULTS: Among 341 subjects, 121 received FQ and 220 received FQ-doxy for prophylaxis. Neutropenic fever developed in 67 (55.4%) and 87 (39.5%) subjects in the FQ and FQ-doxy groups, respectively (p = .005). Bacteremia was infrequent, with 5 (4.1%) and 5 (2.3%) cases developing in the FQ and FQ-doxy groups, respectively (p = .337). Among Gram-negative bacteremia events, 7/7 Escherichia coli strains were FQ-resistant, and 5/7 were ceftriaxone-resistant. CONCLUSION: The FQ-doxy prophylaxis group had fewer cases of neutropenic fever than the FQ group, however, there was no significant difference in bacteremia. High rates of antibiotic resistance were observed. An updated randomized controlled trial investigating appropriate prophylaxis for ASCT in the context of current oncology standards and changing antimicrobial resistance rates is warranted.

Clofarabine Preconditioning followed by Allogeneic Transplant Using TBI and Post-Transplant Cyclophosphamide for Relapsed Refractory Leukemia.

Acute myeloid leukemia patients with induction failure or relapsed refractory disease have minimal chance of achieving remission with subsequent treatments. Several trials have shown the feasibility of clofarabine-based conditioning in allogeneic stem cell transplants (allo-HSCT) for non-remission AML patients. Pre-transplant conditioning with clofarabine followed by reduced-intensity allo-HSCT has also demonstrated a potential benefit in those patients with human leukocyte antigen (HLA)-identical donors, but it is not commonly used in haploidentical and mismatched transplants. In this case report, we describe our experience of seven cases of non-remission AML who received clofarabine preconditioning followed by an allo-HSCT with PTCy. The 2-year overall survival and disease-free survival was 83.3% (95% confidence interval (CI): 27.3-97.9%) and 85.7% (95% CI: 33.4-97.9%). Median days of neutrophil and platelet recovery were 16 (range of 13-23) and 28 (range of 17-75), respectively. The cumulative incidence of grade II-IV acute graft-versus-host disease (GVHD) at day 100 and chronic GVHD at 1-year showed 28.6% (95% CI: 8-74.2%) and 28.6% (95% CI: 3-63.9%), respectively. The two-year relapse rate was 14.3% (95% CI: 2.14-66.6%). One-year GVHD-free relapse-free survival (GFRS) at 1-year was 71.4% (95% CI: 25.8-92%). Our patients showed successful outcomes with clofarabine preconditioning to reduce the leukemic burden at the pre-transplant period followed by PTCy to reduce GVHD resulting in lower relapsed rate and better GFRS in these patients.

Acute Myeloid Leukemia Causes T Cell Exhaustion and Depletion in a Humanized Graft-versus-Leukemia Model.

Allogeneic hematopoietic stem cell transplantation (alloSCT) is, in many clinical settings, the only curative treatment for acute myeloid leukemia (AML). The clinical benefit of alloSCT greatly relies on the graft-versus-leukemia (GVL) effect. However, AML relapse remains the top cause of posttransplant death; this highlights the urgent need to enhance GVL. Studies of human GVL have been hindered by the lack of optimal clinically relevant models. In this article, we report, the successful establishment of a novel (to our knowledge) humanized GVL model system by transplanting clinically paired donor PBMCs and patient AML into MHC class I/II knockout NSG mice. We observed significantly reduced leukemia growth in humanized mice compared with mice that received AML alone, demonstrating a functional GVL effect. Using this model system, we studied human GVL responses against human AML cells in vivo and discovered that AML induced T cell depletion, likely because of increased T cell apoptosis. In addition, AML caused T cell exhaustion manifested by upregulation of inhibitory receptors, increased expression of exhaustion-related transcription factors, and decreased T cell function. Importantly, combined blockade of human T cell-inhibitory pathways effectively reduced leukemia burden and reinvigorated CD8 T cell function in this model system. These data, generated in a highly clinically relevant humanized GVL model, not only demonstrate AML-induced inhibition of alloreactive T cells but also identify promising therapeutic strategies targeting T cell depletion and exhaustion for overcoming GVL failure and treating AML relapse after alloSCT.

CD26lowPD-1+ CD8 T cells are terminally exhausted and associated with leukemia progression in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a devastating blood cancer with poor prognosis. Novel effective treatment is an urgent unmet need. Immunotherapy targeting T cell exhaustion by blocking inhibitory pathways, such as PD-1, is promising in cancer treatment. However, results from clinical studies applying PD-1 blockade to AML patients are largely disappointing. AML is highly heterogeneous. Identification of additional immune regulatory pathways and defining predictive biomarkers for treatment response are crucial to optimize the strategy. CD26 is a marker of T cell activation and involved in multiple immune processes. Here, we performed comprehensive phenotypic and functional analyses on the blood samples collected from AML patients and discovered that CD26lowPD-1+ CD8 T cells were associated with AML progression. Specifically, the percentage of this cell fraction was significantly higher in patients with newly diagnosed AML compared to that in patients achieved completed remission or healthy controls. Our subsequent studies on CD26lowPD-1+ CD8 T cells from AML patients at initial diagnosis demonstrated that this cell population highly expressed inhibitory receptors and displayed impaired cytokine production, indicating an exhaustion status. Importantly, CD26lowPD-1+ CD8 T cells carried features of terminal exhaustion, manifested by higher frequency of TEMRA differentiation, increased expression of transcription factors that are observed in terminally exhausted T cells, and high level of intracellular expression of granzyme B and perforin. Our findings suggest a prognostic and predictive value of CD26 in AML, providing pivotal information to optimize the immunotherapy for this devastating cancer.

IMPROVE-BMT: a protocol for a pilot randomised controlled trial of prehabilitation exercise for adult haematopoietic stem cell transplant recipients.

INTRODUCTION: Haematopoietic stem cell transplant (HSCT) in adults is an intensive medical procedure for a variety of haematological malignancies. Although there is a large body of evidence demonstrating the negative effects of HSCT on physical function and psychosocial parameters, there is limited evidence on the impact of HSCT on body composition and bone health. Further, aerobic and resistance-training exercise interventions aimed at improving physical function and patient-reported outcomes largely take place during the peritransplant and post-transplant period. Prehabilitative exercise, or exercise prior to medical treatment, has been successfully deployed in presurgical candidates and other tumour sites, yet there is a paucity of evidence on the effect of prehabilitation in HSCT patients. The aim of this study is to investigate the feasibility, acceptability and safety of a resistance training exercise programme in patients with haematological malignancies prior to HSCT. METHODS AND ANALYSIS: IMpact of PRehabilitation in Oncology Via Exercise-Bone Marrow Transplant is a single-site, pilot randomised controlled trial of an exercise intervention compared with usual care. The primary aim is to assess the feasibility, acceptability and safety of the resistance-training exercise intervention prior to HSCT. Secondary aims include evaluating the differences in physical function, body composition, bone mineral density and patient-reported outcomes between the exercise group and usual care control group. Outcome measurements will be assessed: prior to HSCT, on/around day of HSCT admission, +30 days post-HSCT and +100 days post-HSCT. The exercise intervention is a home-based resistance training exercise programme that incorporates resistance band and body weight exercises. The primary outcomes will be reported as percentages and/or mean values. The secondary outcomes will be analysed using appropriate statistical methods to portray within-group and between-group differences. ETHICS AND DISSEMINATION: The study has Penn State College of Medicine approval. Results will be disseminated through scientific publication and presentation at exercise-related and oncology-related scientific meetings. TRIAL REGISTRATION NUMBER: NCT03886909.

Donor HLA mismatch promotes full donor T-cell chimerism in the allogeneic stem cell transplant with reduced-intensity conditioning and post-transplant cyclophosphamide GVHD prophylaxis.

Full donor T-cell chimerism (FDTCC) after allogeneic stem cell transplant (allo-SCT) has been associated with improved outcomes in hematologic malignancy. We studied if donor human leukocyte antigen (HLA) mismatch improves achievement of FDTCC because mismatched HLA promotes donor T-cell proliferation where recipient T-cells had been impaired by previous treatment. Patients (N = 138) received allo-SCT with reduced-intensity conditioning (RIC) from 39 HLA mismatched donors (16 unrelated; 23 haploidentical) with post-transplant cyclophosphamide (PTCy) or 99 matched donors (21 siblings; 78 unrelated) with PTCy (N = 18) or non-PTCy (N = 81). Achievement of FDTCC by day 100 was higher with HLA mismatched donors than matched donors (82.1% vs. 27.3%, p < 00,001), which was further improved with 200 cGy total body irradiation (87.9%) or lymphoid (versus myeloid) malignancy (93.8%). Since all mismatched transplants used PTCy, FDTCC was higher with PTCy than non-PTCy (68.4% vs. 25.7%, p < 0.00001), but not in the matched transplant with PTCy (38.9%), negating PTCy as the primary driver. Lymphocyte recovery was delayed with PTCy than without (median on day + 30: 100 vs. 630/µL, p < 0.0001). The benefit of FDTCC was not translated into survival outcomes, especially in myeloid malignancies, possibly due to the insufficient graft-versus-tumor effects from the delayed lymphocyte recovery. Further studies are necessary to improve lymphocyte count recovery in PTCy transplants.

Post-transplant cyclophosphamide alters immune signatures and leads to impaired T cell reconstitution in allogeneic hematopoietic stem cell transplant.

Despite the increased usage of post-transplant cyclophosphamide (PTCy) in allogeneic hematopoietic stem cell transplantation (allo-HSCT), our knowledge of immune reconstitution post-allo-HSCT in the setting of PTCy is limited. Adequate immune reconstitution is the key to a successful transplant. In this study, we aim to investigate the effect of PTCy on the reconstitution of each immune component; more focus was placed on the immunophenotype and functions of T cells. Using blood samples from patients who underwent allo-HSCT under regimens containing PTCy (n = 23) versus those who received no PTCy (n = 14), we examined the impact of PTCy on the post-transplant immune response. We demonstrated a distinct T cell immune signature between PTCy versus non-PTCy group. PTCy significantly delayed T cell reconstitution and affected the T cell subsets by increasing regulatory T cells (Treg) while reducing naïve T cells. In addition, we observed remarkable enhancement of multiple inhibitory receptors (TIGIT, PD-1, TIM-3, CD38, CD39) on both CD4+ and CD8+ T cells on day 30 post-transplantation in patients who received PTCy. Importantly, upregulation of PD-1 on CD8 T cells was persistent through day 180 and these T cells were less functional, manifested by reduced cytokine production upon anti-CD3/CD28 stimulation. Furthermore, we found a significant correlation of T cell immune phenotypes to clinical outcome (disease relapse and GVHD) in patients who received PTCy. Our novel findings provide critical information to understand the mechanism of how PTCy impacts immune reconstitution in allo-HSCT and may subsequently lead to optimization of our clinical practice using this treatment.

A novel clinically relevant graft-versus-leukemia model in humanized mice.

The prognosis for acute myeloid leukemia (AML) relapse post allogeneic hematopoietic stem cell transplantation (alloSCT) is dismal. Novel effective treatment is urgently needed. Clinical benefit of alloSCT greatly relies on the graft-versus-leukemia (GVL) effect. The mechanisms that mediate immune escape of leukemia (thus causing GVL failure) remain poorly understood. Studies of human GVL have been hindered by the lack of optimal clinically relevant models. Here, using our large, longitudinal clinical tissue bank that include AML cells and G-CSF mobilized donor hematopoietic stem cells (HSCs), we successfully established a novel GVL model in humanized mice. Donor HSCs were injected into immune-deficient NOD-Cg-Prkdcscid IL2rgtm1Wjl /SzJ (NSG) mice to build humanized mice. Immune reconstitution in these mice recapitulated some clinical scenario in the patient who received the corresponding HSCs. Allogeneic but HLA partially matched patient-derived AML cells were successfully engrafted in these humanized mice. Importantly, we observed a significantly reduced (yet incomplete elimination of) leukemia growth in humanized mice compared with that in control NSG mice, demonstrating a functional (but defective) GVL effect. Thus, for the first time, we established a novel humanized mouse model that can be used for studying human GVL responses against human AML cells in vivo. This novel clinically relevant model provides a valuable platform for investigating the mechanisms of human GVL and development of effective leukemia treatments.

Alloantigen-activated (AAA) CD4+ T cells reinvigorate host endogenous T cell immunity to eliminate pre-established tumors in mice.

BACKGROUND: Cancer vaccines that induce endogenous antitumor immunity represent an ideal strategy to overcome intractable cancers. However, doing this against a pre-established cancer using autologous immune cells has proven to be challenging. "Allogeneic effects" refers to the induction of an endogenous immune response upon adoptive transfer of allogeneic lymphocytes without utilizing hematopoietic stem cell transplantation. While allogeneic lymphocytes have a potent ability to activate host immunity as a cell adjuvant, novel strategies that can activate endogenous antitumor activity in cancer patients remain an unmet need. In this study, we established a new method to destroy pre-developed tumors and confer potent antitumor immunity in mice using alloantigen-activated CD4+ (named AAA-CD4+) T cells. METHODS: AAA-CD4+ T cells were generated from CD4+ T cells isolated from BALB/c mice in cultures with dendritic cells (DCs) induced from C57BL/6 (B6) mice. In this culture, allogeneic CD4+ T cells that recognize and react to B6 mouse-derived alloantigens are preferentially activated. These AAA-CD4+ T cells were directly injected into the pre-established melanoma in B6 mice to assess their ability to elicit antitumor immunity in vivo. RESULTS: Upon intratumoral injection, these AAA-CD4+ T cells underwent a dramatic expansion in the tumor and secreted high levels of IFN-γ and IL-2. This was accompanied by markedly increased infiltration of host-derived CD8+ T cells, CD4+ T cells, natural killer (NK) cells, DCs, and type-1 like macrophages. Selective depletion of host CD8+ T cells, rather than NK cells, abrogated this therapeutic effect. Thus, intratumoral administration of AAA-CD4+ T cells results in a robust endogenous CD8+ T cell response that destroys pre-established melanoma. This locally induced antitumor immunity elicited systemic protection to eliminate tumors at distal sites, persisted over 6 months in vivo, and protected the animals from tumor re-challenge. Notably, the injected AAA-CD4+ T cells disappeared within 7 days and caused no adverse reactions. CONCLUSIONS: Our findings indicate that AAA-CD4+ T cells reinvigorate endogenous cytotoxic T cells to eradicate pre-established melanoma and induce long-term protective antitumor immunity. This approach can be immediately applied to patients with advanced melanoma and may have broad implications in the treatment of other types of solid tumors.

[The coronavirus disease 2019 (COVID-19) and hematologic oncology or BMT practice at our center in US].

The coronavirus disease 2019 (COVID-19) pandemic has exerted a considerable impact in our region; thus, we have been performing only emergency transplants in March 2020. At present, all inpatients and surgical patients are being tested and screened for COVID-19. If they are found to be positive, they are transferred to the COVID-19 ward, where a specialized team manages them. Team-based care allows the hematology/oncology teams to perform their regular duties. In particular, for post-transplant patients, treatment decisions are made through discussion with infectious disease specialists, and in principle, the patients are treated using the same protocol as that used for the general COVID-19 infected patients. Currently, vaccination is being promoted at a rapid pace based on the Centers for Disease Control and Prevention Guidelines (CDC) guidelines. At our institution, when a situation of suspected nosocomial COVID-19 infection occurred, all healthcare workers were tested. Thereafter, all hospitalized patients were tested every week for COVID-19, and we were able to overcome the situation. Although definitive measures for COVID-19 are yet to be established, signs of an end to the infection are beginning to appear with a wider availability of vaccines.

Improved outcome in AML relapse after allogeneic transplant with high-intensity chemotherapy followed by 2nd allogeneic stem cell transplant or donor lymphocyte infusion.

Acute myeloid leukemia (AML) relapse after allogeneic stem cell transplant (alloSCT) remains a major therapeutic challenge. While patients with longer remission after initial alloSCT are recommended to receive cell therapy (CT) such as 2ndalloSCT or donor lymphocyte infusion (DLI), survival for patients who relapse within 6 months of alloSCT has been dismal. We evaluated the outcomes of AML relapse after alloSCT to assess the impact of different treatments on long-term survival. One hundred and seventy-two patients with AML underwent alloSCT at the Penn State Cancer Institute from January 2014 to August 2019. Sixty-nine patients relapsed (median age, 60 years; range, 10-75). Of these, 4 patients underwent 2ndalloSCT, and 26 received DLI. One-year overall survival (OS) in all cases was 20.3% (95% CI: 11.8-30.4%). Patients with ECOG performance status (PS) 0-2 at relapse showed a better 1-year OS than those with PS 3-4. Median OS for patients who received chemotherapy only or chemotherapy with CT was 74 or 173.5 days, respectively (p < 0.001). Relapsed patients receiving conventional re-induction chemotherapy were categorized as the high-intensity chemotherapy (H) group, while those receiving treatments such as hypomethylating agents or targeted agents were categorized as the low-intensity chemotherapy (L) group. The H group showed a better 1-year OS compared with the L group. Patients who received H + CT showed a better 1-year OS of 52.9% than the other 3 groups (p < 0.001). Even for patients with post-alloSCT remission duration of less than 6 months, the statistical significance was preserved. Factors including age, donor source at 1stalloSCT, time to relapse, blast counts, PS at relapse, and treatment type after post-alloSCT relapse were used for a multivariate analysis, and matched or mismatched related donor and H + CT after alloSCT were identified as independent factors associated with OS. These findings support the use of H + CT as the treatment option of choice for AML patients who relapse after alloSCT when feasible.