Lymphocyte Recovery Research Articles

Characteristics and influencing factors of immunological non-responders in HIV-1-infected patients receiving antiretroviral therapy: a cross-sectional study in Guangxi.

The prevalence of AIDS and mortality rates among HIV-infected individuals in Guangxi remain relatively high, potentially due to impaired CD4 + cell recovery. This study aims to identify factors hindering CD4 + cell recovery in people living with HIV. We conducted a retrospective study on people living with HIV in Guangxi, China, with data collection extending to the end of 2023. CD4 + T cells were categorized into "immunological responders" and "non-responders" based on CD4 + cell recovery after treatment. Multivariate logistic regression was used to analyze factors associated with the development of immunological non-responders. Recovery of CD4 + T lymphocytes was assessed using the Generalized Additive Mixed Model (GAMM) and Generalized Estimating Equations (GEE). Additionally, multivariate Cox regression identified factors influencing survival rates. Our findings indicated a 52.44% incidence of immunological non-responders after two years of treatment. Factors such as age, sex, education, occupation, infection route, pre-treatment CD4 + T cell count, HIV subtype, and treatment regimen were linked to immunological non-response. Specifically, male gender, education up to high school, farming occupation, heterosexual transmission, CRF01_AE subtype, pre-treatment CD4 + T cell count < 200/µL, and the 3TC + EFV + TDF regimen were identified as significant risk factors. Statistically significant differences in CD4 + T lymphocyte recovery rates were observed among different HIV subtypes (P < 0.05). Beyond age, sex, ethnicity, occupation, subtype, and treatment regimen, being an immunological non-responder was found to be a risk factor for both mortality and accelerated disease progression. The study highlights the complexity of factors affecting CD4 + cell recovery post-HIV treatment in Guangxi and underscores the need for vigilant clinical monitoring of people living with HIV, particularly those with low pre-treatment CD4 + T cell levels.

Unraveling the Impact of Sarcopenia-Induced Lymphopenia on Treatment Response and Prognosis in Patients with Stage III Non-Small Cell Lung Cancer: Insights for Optimizing Chemoradiation and Immune Checkpoint Inhibitor.

Sarcopenia is a poor prognostic factor in non-small cell lung cancer (NSCLC). However, its prognostic significance in patients with NSCLC receiving immune checkpoint inhibitors (ICIs) and its relationship with lymphopenia remain unclear. We aimed to investigate the prognostic role of sarcopenia and its effect on lymphocyte recovery in patients with stage III NSCLC treated with concurrent chemoradiotherapy (CCRT) followed by ICI. We retrospectively evaluated 151 patients with stage III NSCLC who received definitive CCRT followed by maintenance ICI between January 2016 and June 2022. Sarcopenia was evaluated by measuring the skeletal muscle area at the L3 vertebra level using computed tomography scans. Lymphocyte level changes were assessed based on measurements taken before and during CCRT and at 1, 2, 3, 6, and 12 months post-CCRT completion. Even after adjusting for baseline absolute lymphocyte count through propensity score-matching, patients with pre-radiotherapy (RT) sarcopenia (n=86) exhibited poor lymphocyte recovery and a significantly high incidence of grade ≥3 lymphopenia during CCRT. Pre-RT sarcopenia and grade ≥3 lymphopenia during CCRT emerged as prognostic factors for overall survival and progression-free survival, respectively. Concurrent chemotherapy dose adjustments, objective response after CCRT, and discontinuation of maintenance ICI were also analyzed as independent prognostic factors. Our results demonstrated an association between pre-RT sarcopenia and poor survival, concurrent chemotherapy dose adjustments, and impaired lymphocyte recovery after definitive CCRT. Moreover, CCRT-induced lymphopenia not only contributed to poor prognosis but may have also impaired the therapeutic efficacy of subsequent maintenance ICI, ultimately worsening treatment outcomes.

Lymphocyte recovery after bendamustine therapy in patients with mantle cell lymphoma. Results of a retrospective analysis and prognostic impact in the CAR-T era.

Bendamustine in combination with rituximab (BR) or with rituximab and cytarabine (R-BAC) is the standard first-line immunochemotherapy in mantle cell lymphoma (MCL) for elderly patients and patients ineligible for intensive regimens or autologous transplantation. As bendamustine causes prolonged lymphopenia and the literature lacks evidence of its persistence in patients with MCL, this retrospective analysis aims to estimate the lymphocyte recovery time, also in view of potential immunotherapy with CAR-T cells. Data were collected from 44 consecutive MCL patients who received bendamustine (BR or R-BAC) as first-line therapy at the Hematology Unit of Sapienza University Hospital between May 2011 and April 2022. Twenty patients (45%) were treated with R-BAC and 24 (55%) with BR. At baseline, the median lymphocyte count was 1795/µl (range: 370-11730/µL). One month after the end of therapy, it was 450/µl (range: 50-3300/µl) and 3 months after 768/µl (range: 260-1650/µl). After 6 and 9 months, we observed a gradual increase in median lymphocyte count of 900/µl (range: 370-2560/µl and 130-2770/µl, respectively). After 12 months median lymphocyte count was 1256/µl (range: 240-4140/µl). Median lymphocyte count at 1, 3, 6, and 9 months post-treatment was significantly lower than baseline but showed recovery by the 12 months. This finding is crucial for MCL patients considering CAR-T cell therapy, suggesting a minimum 9-month interval between bendamustine administration and leukapheresis.

Abstract 27: Physiologic Oxygen Expansion Enhances Lymphocyte and Neutrophil Recovery Following Transplantation

Abstract 27: Physiologic Oxygen Expansion Enhances Lymphocyte and Neutrophil Recovery Following Transplantation

Prospective Study of the Recovery of CD4+ T cell Subsets and Their Relationship to the Overall Survival Following Allogeneic Hematopoietic Stem Cell Transplantation.

The immune recovery (IR) following the allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an advancement procedure that fundamentally correlated to the curative success. It is critical to understand the interfering factors in IR to prevent the HSCT-related mortality. The factors influencing CD4+T cell recovery post (HSCT) are not well totally characterized. This work was conducted to analyze the kinetics of CD4+T cell subsets recovery post HSCT, and to correlate their reconstitution with different factors that may influence the overall survival following HSCT. We prospectively evaluated the clinical outcomes and CD4+ T lymphocyte subtypes regeneration kinetics at different time-points of 22 patients with allogeneic HSCT for malignant and non-malignant diseases from 2007 to 2008. Statistics (means, minimal, and maximal values) were used to describe patient baseline characteristics. Results were presented as absolute count of CD4+ T cells, % of naive and memory subsets, and p-values. Thymus-independent pathways were responsible for the rapid recovery of memory CD4+T cells less than 6 months after HSCT. Thymus-dependent pathways were activated between 6 and 12 months in the majority of patients with increasing counts of naive CD4+ T cell. Furthermore, increasing patient age and chronic GVHD predicted the slow naive T cell recovery and also predicted high memory T cell numbers. The proper CD4 + reconstitution was associated with younger age, a non-malignant disease and a lower incidence of acute graft-versus-host disease ≥ grade 2. Additionally, the CD4+ T lymphocytes recovery ≥ 200/µl was associated with a higher overall survival. Different factors affected the IR post the allo-HSCT. The CD4+ count ≥ 200/µl was a simple IR predictor of overall survival and better clinical outcome following allogeneic HSCT.

Predictive Factors for Early Immune Recovery in NHL Patients after Autologous Transplantation: A Multicenter Prospective Study.

Early lymphocyte recovery as manifested by an absolute lymphocyte count at d+15 (ALC-15) ≥ 0.5 × 109/L after autologous hematopoietic stem cell transplantation (AHCT) has been associated with a better outcome. This prospective multicenter study aimed to clarify factors associated with ALC-15 ≥ 0.5 × 109/L after AHCT among 178 patients with non-Hodgkin lymphoma. The mobilization capacity, as manifested by peak blood CD34+ cell numbers > 45 × 106/L correlated with higher ALC-15 levels (p = 0.020). In addition, the amount of CD3+CD4+ T cells > 31.8 × 106/kg in the infused graft predicted ALC-15 ≥ 0.5 × 109/L (p < 0.001). Also, the number of infused graft CD3+CD8+ T cells > 28.8 × 106/kg (p = 0.017) and NK cells > 4.4 × 106/kg was linked with higher ALC-15 (p < 0.001). The two-year progression-free survival after AHCT was significantly better in patients with ALC-15 ≥ 0.5 × 109/L (74 vs. 57%, p = 0.027). The five-year OS in patients with higher ALC-15 was 78% vs. 60% in those with lower ALC-15 (p = 0.136). To conclude, the mobilization capacity of CD34+ cells and detailed measures of graft cellular content mark prognostic tools that predict ALC-15 ≥ 0.5 × 109/L, which is associated with a better outcome in NHL patients after AHCT.

Allogeneic Hematopoietic Cell Transplant For Bone Marrow Failure or Myelodysplastic Syndrome in Dyskeratosis Congenita/Telomere Biology Disorders: Single-Center, Single-Arm, Open-Label Trial of Reduced-Intensity Conditioning Without Radiation

BackgroundDyskeratosis congenita/Telomere biology disorders (DC/TBD) often manifest as bone marrow failure (BMF) or myelodysplastic syndrome (MDS). Allogeneic hematopoietic cell transplant (alloHCT) rescues hematologic complications, but radiation and alkylator-based conditioning regimens cause diffuse whole-body toxicity and may expedite DC/TBD-specific non-hematopoietic complications. Optimization of conditioning intensity in DC/TBD to allow for donor hematopoietic cell engraftment with the least amount of toxicity remains a critical goal of the alloHCT field. Objectives/Study DesignWe report prospectively collected standard alloHCT outcomes from a single-center single-arm open-label clinical trial of bone marrow or peripheral blood stem cell alloHCT for DC/TBD-associated BMF or MDS. Conditioning was reduced intensity (RIC) including alemtuzumab 1mg/kg, fludarabine 200 mg/m2, and cyclophosphamide 50 mg/kg. A previous single-arm open-label phase II clinical trial for the same patient population conducted at the same center, differing only by inclusion of 200 centigray of total body irradiation (TBI), served as a control cohort. ResultsThe Non-TBI cohort included 10 patients (ages 1.7-65.9 years, median follow-up of 3.9 years) compared to the control TBI cohort which included 12 patients (ages 2.2-52.2 years, median follow-up of 10.5 years). Baseline characteristics differed only in total CD34+ cells received, with a median of 5.6 (Non-TBI) compared to 2.6 (TBI) x 106/kg (p=0.02; no difference in total nucleated cells). The cumulative incidence of day +100 grade II-IV acute and 4-year chronic graft-versus-host disease (GvHD) were low at 0 and 10% (Non-TBI) and 8 and 17% (TBI), respectively (acute, p=0.36; chronic, p=0.72). Primary graft failure was absent. Secondary non-neutropenic graft failure occurred in one (Non-TBI cohort). The Non-TBI cohort demonstrated delayed achievement of full donor chimerism but superior lymphocyte recovery. There was no difference in 4-year overall survival at 80% (Non-TBI) and 75% (TBI; p=0.78).MDS as an indication for alloHCT was uncommon, but overall associated with poor outcomes. There were 3 MDS patients in the Non-TBI cohort: 1 relapsed and died at day+387; 1 relapsed at day+500 and is alive 5.5 years later following salvage with a 2nd alloHCT; 1 relapsed at day+1093 and is alive at day +100 after a 2nd alloHCT. There was 1 MDS patient in the TBI cohort who achieved 100% donor myeloid engraftment without relapse but died at day+827 from a bacterial infection in the setting of immune mediated cytopenia. ConclusionElimination of TBI from the RIC regimen for DC/TBD was not associated with significant changes in rates of graft failure, GvHD, and overall survival, but was associated with delayed achievement of full donor chimerism and improved lymphocyte reconstitution. For DC/TBD-associated BMF, TBI appears to be dispensable. Optimal approaches to DC/TBD-associated MDS remain unclear. Larger cohorts are needed to better assess the unique contribution of TBI and donor CD34+ cell dose. Longer follow-up is required to assess differences in DC/TBD complications and late effects.

B-NHL Cases in a Tertiary Pediatric Hematology-Oncology Department: A 20-Year Retrospective Cohort Study.

Non-Hodgkin lymphoma (NHL) is among the five most common pediatric cancer diagnoses in children and adolescents and consists of a heterogeneous group of lymphoid tissue malignancies -with B-cell-derived NHL accounting for nearly 80% of cases. Novel and high-throughput diagnostic tools have significantly increased our understanding of B-NHL biology and molecular pathogenesis, leading to new NHL classifications and treatment options. This retrospective cohort study investigated 17 cases of both mature B-cell NHL (Burkitt lymphoma or BL; Diffuse large B-cell lymphoma or DLBCL; Primary mediastinal large B-cell lymphoma or PMBCL; Follicular lymphoma or FL) and immature B-cell progenitor NHL (B-lymphoblastic lymphoma or BLL) that were treated in a tertiary Pediatric Hematology-Oncology Department during the last 20 years. Modern NHL protocols for children, adolescents, and young adults, along with the addition of rituximab, are safe and efficient (100% overall survival; one relapse). Elevated ESR was more prevalent than elevated LDH. Analyses have focused on immune reconstitution (grade ≥3 infections, lymphocyte and immunoglobulin levels recovery) and body-mass-index changes post-treatment, late effects (in 53% of patients), and the presence of histology markers BCL2, BCL6, CD30, cMYC, and Ki-67%. One patient was diagnosed with a second malignant neoplasm (papillary thyroid cancer).

Enrichment of T-lymphocytes from leukemic blood using inertial microfluidics toward improved chimeric antigen receptor-T cell manufacturing

Chimeric antigen receptor cell therapy is a successful immunotherapy for the treatment of blood cancers. However, hurdles in their manufacturing remain including efficient isolation and purification of the T-cell starting material. Herein, we describe a one-step separation based on inertial spiral microfluidics for efficient enrichment of T-cells in B-cell acute lymphoblastic leukemia (ALL) and B-cell chronic lymphocytic leukemia patient's samples. In healthy donors used to optimize the process, the lymphocyte purity was enriched from 65% (SD ± 0.2) to 91% (SD ± 0.06) and T-cell purity was enriched from 45% (SD ± 0.1) to 73% (SD ± 0.02). Leukemic samples had higher starting B-cells compared to the healthy donor samples. Efficient enrichment and recovery of lymphocytes and T-cells were achieved in ALL samples with B-cells, monocytes and leukemic blasts depleted by 80% (SD ± 0.09), 89% (SD ± 0.1) and 74% (SD ± 0.09), respectively, and a 70% (SD ± 0.1) T-cell recovery. Chronic lymphocytic leukemia samples had lower T-cell numbers, and the separation process was less efficient compared to the ALL. This study demonstrates the use of inertial microfluidics for T-cell enrichment and depletion of B-cell blasts in ALL, suggesting its potential to address a key bottleneck of the chimeric antigen receptor-T manufacturing workflow.

Prognostic value of hemogram parameters in osteosarcoma: The French OS2006 experience.

Previous studies have shown that neutrophil-to-lymphocyte (NLR) ratio at diagnosis and early lymphocytes recovery on doxorubicin-based chemotherapy, may impact the outcome in patients with osteosarcoma (OST). This study aimed to evaluate the prognostic value of hemogram parameters in patients with OST treated with high-dose methotrexate and etoposide/ifosfamide (M-EI) chemotherapy. We retrospectively analyzed the prognostic value of various hemogram parameters at diagnosis and during therapy in a large consecutive cohort of patients with OST included in the French OS2006 trial and treated with M-EI chemotherapy. A total of 164 patients were analyzed. The median age was 14.7years (interquartile range [IQR]: 11.7-17). Median follow-up was 5.6years (IQR: 3.3-7.7years). Three-year event-free survival (EFS) and overall survival (OS) were 71.5% (95% confidence interval [CI]: 64%-78%) and 86.4% (95% CI: 80%-91%), respectively. In univariate analysis, blood count parameters at diagnosis and early lymphocyte recovery at Day 14 were not found prognostic of survival outcomes. By contrast, an increase of NLR ratio at Day 1 of the first EI chemotherapy (NLR-W4) was associated with reduced OS in univariate (p=.0044) and multivariate analysis (hazards ratio [HR]=1.3, 95% CI: 1.1-1.5; p=.002), although not with EFS. After adjustment on histological response and metastatic status, an increase of the ratio NLR-W4 of 1 was associated with an increased risk of death of 30%. We identified NLR-W4 as a potential early biomarker for survival in patients with OST treated with M-EI chemotherapy. Further studies are required to confirm the prognostic value of NLR and better identify immune mechanisms involved in disease surveillance.

Early recovery of natural killer cells post T-cell depleted allogeneic stem cell transplantation using alemtuzumab “in the bag”

BackgroundAllogeneic stem cell transplantation (SCT) is a critical therapy for haematological malignancy but may lead to acute and chronic graft versus host disease (GvHD). T-cell depletion with alemtuzumab, either in vivo or ex vivo, reduces the incidence of GvHD but is a risk factor for disease relapse and poor immune reconstitution. Natural killer (NK) cells are the first lymphocytes to recover. Classical NK cells make up >90% of the normal circulating population and can directly kill neoplastic or virally infected cells while the regulatory subset makes up <10%, secretes cytokines and is not cytotoxic. The recovery and balance of these subsets post SCT remains controversial, with most studies analysing patients who received unmanipulated grafts and in vivo immunosuppression. ObjectiveThe aim was to assess the early recovery of NK cells in 18 consecutive patients receiving ex vivo T-cell depleted SCT and to compare the results to 25 individuals receiving haploidentical non-T cell depleted grafts. MethodsAll patients received myeloablative conditioning. After stem cell collection, the stem cells of the T cell depleted group were treated “in the bag” with alemtuzumab (CAMPATH 1H) at a concentration of 1mg/108 mononuclear cells and thereafter immediately infused. For those receiving non-T cell depleted grafts, GvHD prophylaxis was with post infusion therapeutic doses of cyclophosphamide. Blood samples were collected at days 21, 28 and 90. Complete blood counts were performed on an automated analyser while lymphocyte and NK subsets were examined using multiparameter flowcytometry. NK cells were defined as lymphocytes which were CD3-/CD56+. The classical subset was recognised as CD56dim/CD16+ while the regulatory population as CD56bright/CD16-. The results for both transplant types were compared at all time points using SPSS v8 statistical software. ResultsThe recovery of lymphocytes was slow in both groups. Those receiving non-T cell depleted grafts had significantly higher T cell counts at day 21 and 28 when compared to the T cell depleted group (P < 0.05). In contrast, NK cells in the ex vivo T-cell depleted patients recovered rapidly and by day 21 was no different to normal (p > 0.05), while the non-T cell depleted group had significantly decreased numbers (p < 0.001), only recovering at day 90. Both groups had abnormal NK cell subset ratios with significantly elevated percentages of regulatory cells (p < 0.05). However, significant differences were observed between the two groups with those receiving T cell depleted grafts having lower percentages of regulatory cells as well as higher numbers of classical NK cells at day 21 and 28 (p < 0.01). ConclusionThis study of ex vivo T-cell depleted SCT's demonstrates that NK cells recover quicker when compared to those receiving unfractionated grafts. These results may have implications for GvHD and the GvL effect which warrants further study.

Infection risk and antimicrobial prophylaxis in bendamustine-treated patients with indolent non-Hodgkin lymphoma: An Australasian Lymphoma Alliance study.

Infection and lymphopenia are established bendamustine-related complications. The relationship between lymphopenia severity and infection risk, and the role of antimicrobial prophylaxis, is not well described. This multicentre retrospective study analysed infection characteristics and antimicrobial prophylaxis in 302 bendamustine-treated indolent non-Hodgkin lymphoma patients. Lymphopenia (<1 × 109/L) was near universal and time to lymphocyte recovery correlated with cumulative bendamustine dose. No association between lymphopenia severity and duration with infection was observed. Infections occurred in 44% of patients (50% bacterial) with 27% hospitalised; 32% of infections occurred ≥3 months post bendamustine completion. Infection was associated with obinutuzumab and/or maintenance anti-CD20 therapy, prior therapy and advanced stage. Twenty-four opportunistic infections occurred in 21 patients: ten varicella zoster virus (VZV), seven herpes simplex virus (HSV), one cytomegalovirus, one progressive multifocal leucoencephalopathy, one nocardiosis, one Pneumocystis jiroveci pneumonia (PJP) and three other fungal infections. VZV/HSV and PJP prophylaxis were prescribed to 42% and 54% respectively. Fewer VZV/HSV infections occurred in patients receiving prophylaxis (HR 0.14, p = 0.061) while PJP prophylaxis was associated with reduced risk of bacterial infection (HR 0.48, p = 0.004). Our study demonstrates a significant infection risk regardless of lymphopenia severity and supports prophylaxis to mitigate the risk of early and delayed infections.

Impact of HIV-1 genetic diversity on disease progression: a prospective cohort study in Guangxi.

The high proportion of AIDS cases and mortality rates in Guangxi underscores the urgency to investigate the influence of HIV-1 genetic diversity on disease progression in this region. Newly diagnosed HIV-1 patients were enrolled from January 2016 to December 2021, and the follow-up work and detection of CD4+T lymphocytes were carried out every six months until December 2022. Multivariate logistic regression was used to analyze the factors affecting pre-treatment CD4+T lymphocyte counts, while local weighted regression models (LOESS) and generalized estimating equation models (GEE) were conducted to assess factors influencing CD4+T Lymphocyte Recovery. Cox regression analysis was utilized to examine the impact of subtypes on survival risk. Additionally, HIV-1 env sequences were utilized for predicting CXCR4 and CCR5 receptors. The study encompassed 1867 individuals with pol sequences and 281 with env sequences. Our findings indicate that age over 30, divorced/widowed, peasant, heterosexual infection, CRF01_AE, long-term infection, and Pre-treatment Viral load >10000 copies/ml were factors associated with higher risk for pre-treatment CD4+T lymphocyte decline. Specifically, male gender, age over 30, heterosexual infection (HETs), long-term infection, CRF01_AE, and Pre-treatment CD4 T cell counts below 350/µL were identified as risk factors impeding CD4+T lymphocyte recovery. Pre-treatment CD4+T lymphocyte counts and recovery in individuals infected with CRF01_AE were lower compared to CRF07_BC and CRF55_01B. Additionally, CRF01_AE and CRF08_BC subtypes exhibited higher mortality rates than CRF07_BC, CRF55_01B, and other subtypes. Notably, CRF01_AE demonstrated the highest percentage of CXCR4 affinity ratios. This research unveils the intricate influence of HIV-1 gene diversity on CD4+T lymphocyte dynamics and clinical outcomes. It highlights the multifaceted nature of HIV infection in Guangxi, providing novel insights into subtype-specific disease progression among HIV-infected individuals in this region.

Long-Term Efficacy and Immune Reconstitution with Bendamustine As a Lymphodepleting Agent for Axicabtagene Ciloleucel (Axi-Cel) in Patients with Refractory or Relapsed Large B-Cell Lymphoma (LBCL)

Introduction: Lymphodepletion (LD) is crucial for the expansion and persistence of CAR T cells. Fludarabine in combination with cyclophosphamide (FC) is the LD standard regimen. A national fludarabine shortage in 2022 necessitated the exploration of alternative LD regimens. Bendamustine (benda) is a purine analog and an alkylating chemotherapy that has been used as an LD agent for tisagenlecleucel (Ghilardi et al.,2022). However, there is no data for benda LD in other commonly used CART products for relapsed or refractory non-hodgkin lymphoma (rel/ref NHL). Early safety, efficacy, and expansion kinetics data were presented previously (Bharadwaj, ASH 2022). We are now reporting long-term efficacy and immune reconstitution data. Methods: 84 consecutive patients with relapsed/refractory LBCL treated with axi-cel were included in this single-institution study. 27 patients who received Bendamustine lymphodepletion (90mg/m2 on days -4 and -3) were compared to a preceding 57-patient FC cohort. Response, survival, and cytopenia data were collected closest to the 12-month time point for all patients who had not progressed. Response was assessed using the Lugano 2014 criteria, while toxicities were evaluated using the CTCAE version 5.0 and the ASTCT grading. To reduce the influence of confounding variables in this study, a covariate balance propensity score weighting approach (IPTW) was implemented. Covariates included age, sex, presence of bridging therapy, IPI score at apheresis, number of prior therapy lines, absolute lymphocyte count at leukapheresis, prior auto SCT, and pre-lymphodepletion LDH, ALC, ANC, and platelet count. Continuous outcomes were compared using the Mann-Whitney U test, categorical outcomes using chi-square tests, and time-to-event outcomes using a log-rank test weighted in the IPTW sample. Statistical significance was considered when p &amp;lt; 0.05. All analyses were conducted in R 4.3.0. Results: There was no significant difference in the best complete response (CR) rate and best overall response rate (ORR) between the two groups, with a CR rate of 71.9% for FC and 74% for bendamustine (p = 0.182), and an ORR of 89.4% for FC and 85.1% for bendamustine (p = 0.502). For the FC group, 9-month progression-free survival (PFS) and overall survival (OS) were 63.16% (95% CI: 49.29% to 74.19%) and 78.95% (95% CI: 65.92% to 87.45%), respectively with a median follow-up of 15.5 months (IQR: 13.6, 16.8). 9-month PFS and OS point-estimates for the bendamustine group were 70.37% (95% CI: 49.4% to 83.94%) and 77.41% (95% CI: 56.41% to 89.18%), respectively, with a median follow-up of 9.5 months (IQR: 8.6, 9.8). No significant difference was found between groups in either 9 months PFS ( P=0.055) or 9 months OS ( P=0.6290). Median PFS and OS have not been achieved for either group [Figure 1]. Bendamustine was effective in achieving severe lymphodepletion, with a median absolute lymphocyte count (ALC) nadir of 0.11 K/ul (IQR: 0.04, 0.17) occurring within one day after axi-cel infusion. In comparison, the FC group had a lower median ALC nadir of 0.03 K/ul (IQR: 0.01, 0.06) occurring on the day of axi-cel infusion ( P=0.01). Both cohorts showed a similar pattern of lymphocyte recovery over 1, 3, and 6 months. Median ALC counts at 9-12 months post-infusion indicated better lymphocyte recovery in the bendamustine group with a median ALC of 1.55 K/ul (IQR: 1.04, 1.94) compared to 0.96 K/ul (IQR: 0.58, 1.23) in the FC group ( P=0.043) [Figure 2]. There was no significant difference in the recovery of absolute neutrophil count, hemoglobin, and platelets. Updated efficacy analysis and immune reconstitution data including T-cell, B-cell, and NK cell recovery will be reported at the time meeting, as all patients will have completed at least one year of follow-up by September 2023. Discussion: Bendamustine lymphodepletion prior to axi-cel therapy showed comparable efficacy and safety outcomes to the standard FC regimen in relapsed/refractory LBCL patients. Bendamustine demonstrated effective lymphodepletion and better lymphocyte recovery at 9-12 months post-infusion compared to FC cohort. These findings suggest that bendamustine may be an acceptable alternative lymphodepleting regimen. Prospective studies, ideally randomized control trial, are warranted.

Hyperbaric Oxygen (HBO) Effects on Blood Count Recovery and Post-Transplant Outcomes Following High-Dose Therapy and Autologous HSPC Transplantation for Multiple Myeloma: A Multicenter Phase II Randomized Clinical Trial

Background: By reducing systemic erythropoietin (EPO) levels, in vivo studies showed that hyperbaric oxygen (HBO) promoted homing of transplanted umbilical cord hematopoietic stem/progenitor cells (HSPC) to the bone marrow. In a pilot study, we demonstrated that HBO in autologous HSPC transplantation (auto-HSPC) was well tolerated. Time to absolute neutrophil (ANC) recovery was correlated with HBO-mediated reduction in EPO levels. Methods: In this phase II multicenter clinical trial, patients with multiple myeloma (MM) receiving high-dose melphalan and auto-HSPC were randomized 1:1 between HBO and no HBO. HBO was given as 100% oxygen, 2.5 ATA for a total of 90 min, in a single treatment on Day 0, 6 hours before cell infusion. The study's primary objective was to evaluate the effect of HBO on blood count recovery, filgrastim use, blood transfusions, and length of hospitalization (LOH). Exploratory objectives include HBO effects on EPO and IL-15 measured at several time points (pre-conditioning, Day 0 pre-HBO, 6- and 8-hours following HBO, and Days 1-3, 7 and 15 post-transplant). In addition, we explore HBO effects on Day 15 NK cell recovery. Results: A total of 99 patients were enrolled, with 52 randomized to HBO. Baseline characteristics were well balanced except for cell dose, with higher odds that patients with lower cell dose were treated with HBO. There was insufficient evidence of a difference between the two groups regarding time to neutrophil recovery, filgrastim use, blood transfusions, or LOH. Patients randomized to receive HBO were 1.7 (95% CI: 1.1-2.7) times more likely to have lymphocyte recovery (ALC) to &amp;gt; 0.5 k/µL by any given day compared to control (Figure-1). EPO levels (only University of Rochester Medical Center patients) were lower for HBO versus placebo on Day 1 (unadjusted p = 0.02), but there was insufficient evidence of a difference at other time points (Figure-2). Regarding immune reconstitution, IL-15 levels were not significantly different between the two groups. Significant correlation was found between Day 2 IL-15 levels and ALC recovery (p=0.01). We observed higher median NK cell count at Day 15 in the HBO arm (246 vs. 183), but this did not reach statistical significance (P=0.59). Conclusions: HBO resulted in lower EPO levels at Day +1 of auto-HSPC, translating to faster ALC recovery with no effect on neutrophil recovery. Longer follow-up is needed to determine if this will impact progression free survival. Higher NK cell recovery in the HBO arm compared to the non-HBO was not significant to account for the improvement in ALC recovery in the HBO arm. A pilot study (NCT04862676) is ongoing to determine if multiple HBO treatments can keep EPO levels low beyond Day +1 and impact outcomes.

PB CD8 T N and CD4 T EM on day30 after Allo-HSCT Is a Predictive Marker for the Development of aGVHD

Introduction ： Acute GVHD (aGVHD) is a common complication 20-60 days after allo-HSCT and can be life-threatening in some cases. It has been reported to be associated with T-cell immune reconstitution, but the detailed mechanisms are not clear. Early post-transplant lymphocyte recovery is mainly due to homeostatic peripheral expansion (HPE), which is variable depending on various factors such as donor source, recipient's age. T cell-related cytokines, such as sIL-2R , IL-7 and TNFr1 are known biomarkers for the aGVHD, but there are few reports that predict the aGVHD incidence rate using the peripheral blood (PB) lymphocyte subset. We evaluated the effect of PB lymphocyte subset in early post-transplant period on aGVHD. Methods ： We retrospectively analyzed 83 patients (40 in AML, 12 in ALL, 1 in MPAL, 13 in MDS, 2 in MPN, 9 in malignant lymphoma/LPD and 3 in AA) who underwent the allo-HSCT in our hospital from 2013 to 2023. The median age of patients was 44 years (range:17-66). Sixteen received BM, 47 received PB (including 28 with haploidentical (9 with ATG, 19 with PTCY)), and 20 received CB. GVHD prophylaxis was FK506+MTX in 61 patients, CyA+MTX in 3 and MMF+FK506+CY in 19. The median observation period was 495 [38-2983] days. PB lymphocyte subset (Naïve Tcell (T N):CD45RA+CD62L+, Central memory Tcell (T CM) :CD45RA-CD62L+, Effector memory Tcell (T EM) :CD45RA-CD62L-, Effector memory Tcell re-expressed CD45RA (T EMRA) :CD45RA+CD62L-, Th1:CD3+4+CXCR3+CCR6-, Th2:CD3+4+CXCR3-CCR6-, Th17:CD3+4+CXCR3-CCR6+) at day30 (range:28-35) after transplantation were analyzed by FCM. Results ： Twenty-five patients had grade II-IV aGVHD, and 11 had grade Ⅲ-IV aGVHD. Patients with Grade II-IV aGVHD exhibited significantly lower proportion of CD8+T N as compared to non aGVHD groups (GVHD+: 17.7 ± 13.3%; GVHD-: 30.9 ± 16.3%; p &amp;lt; 0.001) . In contrast, the proportion of CD4+T EM and CD8+T EM were higher in the Grade II-IV acute GVHD group (GVHD+: 30.9 ± 23.1%; GVHD-: 21.9 ± 15.6%; p=0.04, GVHD+: 36.1 ± 16.2%; GVHD-: 22.7 ± 14.7%; p &amp;lt; 0.001, respectively). We found a strong statistical correlation between the proportion of CD8+T N and CD8+T EM. The cumulative incidences of Grade II-IV and III -IV acute GVHD at day + 100 for CD8+T N &amp;lt;20% group was 56.9% and 29.6% compared with 14.2% and 6.3% for the CD8+T N &amp;gt;20% group (p &amp;lt; 0.001 and p &amp;lt; 0.01). Similarly, the cumulative incidences of Grade II-IV and III -IV acute GVHD at day + 100 for CD4+T EM &amp;gt;30% group was 48.1% and 32.6% compared with 22.2% and 5.9% for the CD4+T EM &amp;lt;30% group (p= 0.02 and p &amp;lt; 0.01). Two-year NRM was higher in the CD8+T N &amp;lt;20% group than in the CD8+T N &amp;gt;20% group (26.3% vs 4.2%, p=0.008). The two-year cumulative incidence of relapse (CIR) was comparable between the two groups, but tended to be lower in the mismatched CR group (CD8+T N &amp;lt;20%: 33.1% versus CD8+T N &amp;gt;20%: 43.4%, p=0.31). Conclusion ： PB CD8 T N and CD4 T EM fraction on day30 is a predictive biomarker for the development of acute GVHD.

Bendamustine Is Less Toxic Than Fludarabine-Cyclophosphamide-Based Lymphodepletion before CD28-Costimulated CART19 through Reduced Inflammatory Cytokines

Introduction: Lymphodepletion (LD) is an integral component of anti-CD19 chimeric antigen receptor T cell (CART19) immunotherapy, creating the proper environment and cytokine milieu for CART engraftment and function. We previously demonstrated that bendamustine (Benda) LD is as effective as standard fludarabine and cyclophosphamide (Flu/Cy) LD prior to 4-1BB-costimulated tisagenlecleucel but is characterized by significantly reduced cytokine-release syndrome (CRS), neurotoxicity (ICANS), and hematological toxicities. However, whether Benda LD is also effective and safe in CD28 costimulated CART19, and the mechanism for the reduced toxicities associated with Benda LD remain unknown. Methods: We retrospectively evaluated the outcomes of 59 consecutive non-Hodgkin lymphoma patients (pts), including large B cell lymphomas (LBCL, n=48) and follicular lymphoma (FL, n=11), treated with commercial CD28 costimulated axicabtagene ciloleucel (axi-cel) at the University of Pennsylvania between January 2018 and March 2023. Data cut-off was June 30 th, 2023. LD regimens were Flu 30 mg/m 2/day and Cy 500 mg/m 2/day x 3 days or Benda 90 mg/m 2/day x 2 days. LD choice was at the treating physician's discretion, partially determined by Flu shortage. Pts were evaluated for 3-month response (Lugano criteria), survival, and toxicities (ASTCT, CTCAE) in the 30 days after axi-cel infusion. For hematological toxicities, we analyzed lymphocyte, neutrophil, platelet, and hemoglobin levels from the first day of LD to 30 days after axi-cel infusion. For long-term hematological toxicities, we retrieved blood values at 6 months in pts with ongoing remission. Moreover, we analyzed serum samples collected pre- and post-LD (CART19 infusion day) for changes in circulating cytokines (Luminex) and metabolites (Liquid Chromatography/Mass Spectrometry) in a second cohort of 32 pts undergoing CART19. Results: Of 59 pts, 37 received Flu/Cy while 22 Benda LD. The two groups were balanced for most clinical features. Responses with Flu/Cy and Benda LD were similar, with complete remission rates of 51.4% and 50.0%, respectively. Progression-free and overall survivals were similar in the two LD groups. However, any-grade CRS occurred in 91.9% of pts who received Flu/Cy versus 72.7% of pts who received Benda LD; any-grade ICANS after Flu/Cy occurred in 43.2% and after Benda LD in 18.2% of pts ( Fig.1A). These results were confirmed in both LBCL and FL cohorts. Both LD regimens induced profound lymphopenia by the time of axi-cel infusion. However, Flu/Cy LD induced a deeper reduction in median lymphocyte counts (Flu/Cy: 0.00x10 9/L; Benda, 0.20x10 9/L). Additionally, Flu/Cy was associated with a higher incidence of neutropenia of grade ≥3 (100.0% vs 54.5%). Consequently, Flu/Cy pts were more likely to develop infections (78.4% vs. 27.3%), and neutropenic fever (78.4% vs. 13.6%) than Benda receiving pts. By evaluating long-term hematological toxicities, 75.0% of Flu/Cy pts did not have lymphocyte recovery to normal values at 6 months compared to 37.5% with Benda LD. Mechanistically, both Flu/Cy (n=7) and Benda (n=25) LD regimens induced an increase in the levels of cytokines facilitating CART proliferation (IL2, IL7, IL15). Overall, Flu/Cy LD pts had a higher increase in inflammatory cytokine levels compared to Benda LD pts, including a higher increase in the levels of cytokines previously associated with CRS and ICANS (IL15, IL8, and MCP1, Fig.1B). Moreover, after LD, several indispensable anabolic metabolites, including carnitine esters, were decreased in pts receiving Flu/Cy. We also found that NADH and nicotinamide riboside, critical elements for cellular redox balance, were reduced in pts exposed to Flu/Cy LD. We speculate that the higher cytotoxicity/cytopenias, induced by Flu/Cy LD, causes a faster utilization of the above-mentioned metabolites in order to reconstitute the hematopoietic cell compartment to physiological levels, resulting in a higher patient susceptibility to additional metabolic stress. Conclusions: In this retrospective comparison,Benda LD is a safer alternative to Flu/Cy for CART19 immunotherapy with similar efficacy and significantly reduced toxicities. Our data can serve as guidance to clinicians for the selection of the best regimen for lymphodepletion. Our conclusions are supported by our mechanistic studies showing reduced inflammatory cytokines after Benda LD.

Neutrophil to lymphocyte ratio (NTLR) predicts local control and overall survival after stereotactic body radiotherapy (SBRT) in metastatic sarcoma

The neutrophil to lymphocyte ratio (NTLR) and absolute lymphocyte count (ALC) recovery are prognostic across many cancers. We investigated whether NLTR predicts SBRT success or survival in a metastatic sarcoma cohort treated with SBRT from 2014 and 2020 (N = 42). Wilcox Signed Rank Test and Friedman Test compare NTLR changes with local failure vs. local control (N = 138 lesions). Cox analyses identified factors associated with overall survival. If local control was successful, NLTR change was not significant (p = 0.30). However, NLTR significantly changed in patients with local failure (p = 0.027). The multivariable Cox model demonstrated higher NLTR before SBRT was associated with worse overall survival (p = 0.002). The optimal NTLR cut point was 5 (Youden index: 0.418). One-year overall survival in SBRT metastatic sarcoma cohort was 47.6% (CI 34.3%–66.1%). Patients with an NTLR above 5 had a one-year overall survival of 37.7% (21.4%–66.3%); patients with an NTLR below 5 had a significantly improved overall survival of 63% (43.3%–91.6%, p = 0.014). Since NTLR at the time of SBRT was significantly associated with local control success and overall survival in metastatic sarcoma treated with SBRT, future efforts to reduce tumor inhibitory microenvironment factors and improve lymphocyte recovery should be investigated.

Prednisolone Intake Differentially Regulates IL12p40 and IL18 - Impact on Severe Chronic Gvhd, Relapse and Mortality

Background: Chronic graft-versus-host disease (cGVHD) is an immune-mediated complication after allogeneic stem cell transplantation (alloSCT). It is associated with graft-versus-leukemia effects and lower relapse rates, but in its severe form also with increased morbidity and mortality. The early interferon-gamma (IFNγ)-response marker CXCL9 is increased in mild and severe cGVHD and strongly correlates with IDO activity (1). Interleukin (IL)-18 is an innate, IFNγ inducing cytokine that associates with poor outcome of allografted patients (2), and with severe fibrosing cGVHD (1). The adaptive immunological arm of IFNγ induction relies on IL12 that is produced during cognate interactions between dendritic cells and T cells. IL12 kinetics is poorly characterized after alloSCT. We hypothesized that adaptive (IL12p40) and innate (IL18) immune mediators and their modulation by immunosuppressive drugs (ATG, prednisolone) impact cGVHD severity and outcome. Methods: Patients before alloSCT were included in an observational study allowing collection of clinical data and blood before and for one year after alloSCT. The training cohort included 350 patients transplanted before 2018 at our institution. The validation cohort consisted of 205 patients transplanted after 2018. Patients were eligible if they were not relapsed and did not receive donor leukocyte infusions (DLI) until d180, and did not develop refractory acute GVHD. Cytokines that belonged to the IFNγ pathway (IFNγ, CXCL9, CXCL10, IL12p40, IL12EBI3, IL17, IL18, IL23) or to innate inflammatory pathways (TNFa, IFNa, IL1b, activin, follistatin) were measured by R&amp;D ELISAs. Cumulative (equivalent) prednisolone dosages between d0 and d100 after alloSCT were calculated from electronic patient records. Flow cytometry of PBMC of 100 patients on day+180/onset cGVHD were performed using 24-gate multicolor staining. Mild, severe fibrosing and severe gastro-intestinal (GI) cGVHD were diagnosed according to simplified NIH criteria (training cohort only). Overall survival, non-relapse mortality and time to relapse from time of blood sampling were calculated by multivariable Cox regression analyses. Results: Median time of blood sampling at onset of cGVHD (n=180) was day +168 (range 41-723). Control patients (n=169) were collected on day +180 (171-183). Mild cGVHD was recorded in 133 patients, severe fibrosing cGVHD in 39 patients, and isolated GI-cGVHD in 8 patients. Receiver-operator characteristic (ROC) curves showed that only CXCL9, IL18 and IL12p40 were significantly linked to severe cGVHD. IL18 was associated with severe fibrosing cGVHD, whereas CXCL9 and IL12p40 were correlated to any grade cGVHD. In multivariable Cox analyses including IL12p40, IL18, age, mismatch, recipient and donor gender, disease stage, ATG, MTX, and diagnosis, IL18 associated with poor OS (HR per log2 increase 1.81 (1.19-2.74, p=0.005), whereas IL12p40 associated with superior OS (HR 0.53 (0.35-0.81), p=0.003). This could be validated in the independent cohort. Time course analyses of serum levels of CXCL9, IL12p40 and IL18 revealed a strong increase of CXCL9 and IL12p40 toward onset of cGVHD, but no stimulation of IL18 levels. Cumulative prednisolone intake&amp;gt;100 mg until day+100 significantly reduced IL12p40 serum levels on d180/at onset of cGVHD, without influencing IL18 production (Figure 1). In contrast, ATG reduced both, IL18 and IL12p40 at day180/onset of cGVHD. This might explain the reduced incidence of severe fibrosing cGVHD in patients receiving ATG prophylaxis (days-3 to -1 before alloSCT, 4% vs. 23%, p&amp;lt;0.001, n=343), whereas no effect of early prednisolone intake was noticed (p=0.916, n=137). Multicolor flow cytometry showed that IL12p40, but not IL18 correlated with blood counts of T-lymphocyte populations, including CD4+, CD8+ and γδ-Tcells. IL12p40 and γδ-Tcells were associated with reduced relapse rates. In summary, CXCL9, IL12p40 and IL18 are associated with severe cGVHD. IL18 correlated with severe fibrosing cGVHD and increased mortality, whereas IL12p40 was associated with reduced NRM, reduced relapse risk, improved lymphocyte recovery and γδ-T-cells, and was affected by prednisolone intake but not ATG. Conclusion: These results may help to understand and potentially separate the GVL effects from mortality/morbidity that associate with cGVHD. 1)PMID34755129 2)PMID31680001

Results of phase 2 randomized multi-center study to evaluate the safety and efficacy of infusion of memory T cells as adoptive therapy in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pneumonia and/or lymphopenia (RELEASE NCT04578210)

Background aimsThere are currently no effective anti-viral treatments for coronavirus disease 2019 (COVID-19)–hospitalized patients with hypoxemia. Lymphopenia is a biomarker of disease severity usually present in patients who are hospitalized. Approaches to increasing lymphocytes exerting an anti-viral effect must be considered to treat these patients. Following our phase 1 study, we performed a phase 2 randomized multicenter clinical trial in which we evaluated the efficacy of the infusion of allogeneic off-the-shelf CD45RA− memory T cells containing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cells from convalescent donors plus the standard of care (SoC) versus just the SoC treatment. MethodsEighty-four patients were enrolled in three Spanish centers. The patients were randomized into the infusion of 1 × 106/kg CD45RA− memory T cells or the SoC. We selected four unvaccinated donors based on the expression of interferon gamma SARS-CoV-2–specific response within the CD45RA− memory T cells and the most frequent human leukocyte antigen typing in the Spanish population. ResultsWe analyzed data from 81 patients. The primary outcome for recovery, defined as the proportion of participants in each group with normalization of fever, oxygen saturation sustained for at least 24 hours and lymphopenia recovery through day 14 or at discharge, was met for the experimental arm. We also observed faster lymphocyte recovery in the experimental group. We did not observe any treatment-related adverse events. ConclusionsAdoptive cell therapy with off-the-shelf CD45RA− memory T cells containing SAR-CoV-2–specific T cells is safe, effective and accelerates lymphocyte recovery of patients with COVID-19 pneumonia and/or lymphopenia. Trial registrationNCT04578210