Molecular monitoring of T-cell kinetics and migration in severe neurotoxicity after real-world CD19-specific chimeric antigen receptor T cell therapy

CD19-specific chimeric antigen receptor (CD19-CAR) T-cell therapies mediate durable responses in late-stage B-cell malignancies, but can be complicated by a potentially severe immune effector cell-associated neurotoxicity syndrome (ICANS). Despite broad efforts, the precise mechanisms of ICANS are not entirely known, and resistance to current ICANSdirected therapies (especially corticosteroids) has been observed. Recent data suggest that inflammatory cytokines and/or targeting of cerebral CD19-expressing pericytes can disrupt the blood-brain barrier and facilitate influx of immune cells, including CAR T cells. However, specific tools for CD19-CAR T-cell analysis within often minute samples of cerebrospinal fluid (CSF) are not broadly available. Here, we applied our recently developed digital polymerase chain reaction assays to monitor CD19-CAR T-cell kinetics in CSF and blood in real-world patients with neurotoxicity. Consistently, we observed a CAR T-cell enrichment within CSF in ICANS patients with further progressive accumulation despite intense corticosteroid- containing immuno-chemotherapies in a subset of patients with prolonged and therapy-resistant grade 3-4 neurotoxicity. We used next-generation T-cell receptor-b sequencing to assess the repertoire of treatment-refractory cells. Longitudinal analysis revealed a profound skewing of the T-cell receptor repertoire, which at least partly reflected selective expansion of infused T-cell clones. Interestingly, a major fraction of eventually dominating hyperexpanded T-cell clones were of non-CAR T-cell derivation. These findings hint to a role of therapy-refractory T-cell clones in severe ICANS development and prompt future systematic research to determine if CAR T cells may serve as ‘door openers’ and to further characterize both CAR-positive and non-CAR T cells to interrogate the transcriptional signature of these possibly pathologic T cells

Autologous haematopoietic stem cell transplantation for multiple sclerosis: a position paper and registry outline

Background: While substantial progress has been made in the development of disease-modifying medications for multiple sclerosis (MS), a high percentage of treated patients still show progression and persistent inflammatory activity. Autologous haematopoietic stem cell transplantation (AHSCT) aims at eliminating a pathogenic immune repertoire through intense short-term immunosuppression that enables subsequent regeneration of a new and healthy immune system to re-establish immune tolerance for a long period of time. A number of mostly open-label, uncontrolled studies conducted over the past 20 years collected about 4000 cases. They uniformly reported high efficacy of AHSCT in controlling MS inflammatory disease activity, more markedly beneficial in relapsing-remitting MS. Immunological studies provided evidence for qualitative immune resetting following AHSCT. These data and improved safety profiles of transplantation procedures spurred interest in using AHSCT as a treatment option for MS. Objective: To develop expert consensus recommendations on AHSCT in Germany and outline a registry study project. Methods: An open call among MS neurologists as well as among experts in stem cell transplantation in Germany started in December 2021 to join a series of virtual meetings. Results: We provide a consensus-based opinion paper authored by 25 experts on the up-to-date optimal use of AHSCT in managing MS based on the Swiss criteria. Current data indicate that patients who are most likely to benefit from AHSCT have relapsing-remitting MS and are young, ambulatory and have high disease activity. Treatment data with AHSCT will be collected within the German REgistry Cohort of autologous haematopoietic stem CeLl trAnsplantation In MS (RECLAIM). Conclusion: Further clinical trials, including registry-based analyses, are urgently needed to better define the patient characteristics, efficacy and safety profile of AHSCT compared with other high-efficacy therapies and to optimally position it as a treatment option in different MS disease stages. Keywords: Autologous haematopoietic stem cell transplantation (AHSCT), multiple sclerosis, registry study, treatment recommendation

Standardized monitoring of cytomegalovirus-specific immunity can improve risk stratification of recurrent cytomegalovirus reactivation after hematopoietic stem cell transplantation

Recurrence of cytomegalovirus reactivation remains a major cause of morbidity and mortality following allogeneic hematopoietic stem cell transplantation. Monitoring cytomegalovirus-specific cellular immunity using a standardized assay might improve the risk stratification of patients. A prospective multicenter study was conducted in 175 intermediate- and high-risk allogeneic hematopoietic stem cell transplant recipients under preemptive antiviral therapy. Cytomegalovirus-specific cellular immunity was measured using a standardized IFN-γ ELISpot assay (T-Track® CMV). Primary aim was to evaluate the suitability of measuring cytomegalovirus-specific immunity after end of treatment for a first cytomegalovirus reactivation to predict recurrent reactivation. 40/101 (39.6%) patients with a first cytomegalovirus reactivation experienced recurrent reactivations, mainly in the high-risk group (cytomegalovirus-seronegative donor/cytomegalovirus-seropositive recipient). The positive predictive value of T-Track® CMV (patients with a negative test after the first reactivation experienced at least one recurrent reactivation) was 84.2% in high-risk patients. Kaplan-Meier analysis revealed a higher probability of recurrent cytomegalovirus reactivation in high-risk patients with a negative test after the first reactivation (hazard ratio 2.73; p=0.007). Interestingly, a post-hoc analysis considering T-Track® CMV measurements at day 100 post-transplantation, a time point highly relevant for outpatient care, showed a positive predictive value of 90.0% in high-risk patients. Our results indicate that standardized cytomegalovirus-specific cellular immunity monitoring may allow improved risk stratification and management of recurrent cytomegalovirus reactivation after hematopoietic stem cell transplantation. This study was registered at www.clinicaltrials.gov as #NCT02156479

Ruxolitinib plus extracorporeal photopheresis (ECP) for steroid refractory acute graft-versus-host disease of lower GI-tract after allogeneic stem cell transplantation leads to increased regulatory T cell level

Acute graft-versus-host disease (aGVHD) is a serious complication after stem cell transplantation and is associated with high non-relapse mortality. If steroid treatment as first-line therapeutic approach fails, treatment options are limited. In retrospective studies, ruxolitinib, a selective Janus kinase 1/2 inhibitor as well as extracorporeal photopheresis (ECP) could show high efficacy in treatment of steroid refractory acute and chronic GVHD. Here, we report single-center experience of combining JAK-inhibitor treatment with ECP in 18 patients with severe steroid refractory aGVHD of lower GI-tract. The treatment was well tolerated and no severe cytopenia (grade IV) occurred, in three patients grade III cytopenia could be observed. Response was complete or partial in 44% and 11%, respectively, resulting in an estimated 2 year overall survival of 56%. Steroids were tapered rapidly with a median time of 2 days for halving of dosage avoiding additional steroid-associated side effects. Under treatment with ruxolitinib and ECP, an increased level of regulatory T cells could be observed elucidating direct effects of this treatment on immune response

Treosulfan-Based Conditioning Regimen for Second Allograft in Patients with Myelofibrosis

Relapse after allogeneic hematopoietic stem cell transplantation (AHSCT) in myelofibrosis (MF) patients remains as a significant issue despite advances in transplantation procedures and significant prolongation in survival. Second AHSCT is a potential treatment option but associated with high treatment-related mortality and novel less toxic conditioning regimens are needed. In 33 MF patients with relapse after AHSCT and failure to donor lymphocyte infusion (DLI) we investigated treosulfan (36–42 g/m2) in combination with fludarabine and anti-thymocyte globulin (ATG) as conditioning regimen for a second AHSCT with matched related (n = 2), unrelated (n = 23), or mismatched unrelated (n = 8) donors. All patients achieved leukocyte engraftment after a median of 11 days, and 56 ± 13% experienced acute GVHD grade II–IV at day 100. The therapy-related mortality at day 100 and at 3 years was 16% and 31%, respectively. The cumulative incidence of relapse at 5 years was 16%, resulting in a 5-year disease-free and overall survival of 45% and 47%, respectively. Treosulfan-based conditioning for second allograft in relapsed MF patients resulted in about 50% of the patients in long-term freedom from disease

Donor Lymphocyte Infusion and Molecular Monitoring for Relapsed Myelofibrosis After Hematopoietic Cell Transplantation

Hematopoietic cell transplantation (HCT) is a curative approach for myelofibrosis patients, but relapse is a major cause of treatment failure. We investigated the effect of donor lymphocyte infusion (DLI) in 37 patients with molecular (n = 17) or hematological relapse (n = 20) after HCT. Patients received median of 2 (range, 1–5) cumulative DLI (total of 91 infusions). Median starting dose was 1 × 106 cells/kg, escalated by half-log ≥6 weeks if no response nor graft-versus-host disease (GvHD) occurred. Median time to first DLI was 40 weeks for molecular relapse versus 145 weeks for hematological relapse. Overall molecular complete response (mCR) at any time was 73% (n = 27) and was significantly higher for initial molecular relapse (88%) versus hematological relapse (60%; P = 0.05). The 6-year overall survival was 77% versus 32% (P = 0.03). Acute GvHD 2–4 occurred in 22% and half of the patients achieved mCR without any GvHD. All patients who relapsed from mCR achieved after first DLI could be salvaged with subsequent DLI, showing long-term survival. No second HCT was needed for molecular relapse versus 6 for hematological relapse. This comprehensive and largest study to date suggests molecular monitoring together with DLI as standard of care and a crucial approach to achieve excellent outcomes in relapsed myelofibrosis

Enhanced immune reconstitution of γδ T cells after allograft overcomes negative impact of pre-transplant MRD positive status in AML patients.: γδ T cells and MRD+ AML.

BACKGROUND Minimal residual disease (MRD) prior to allogeneic stem cell transplantation (allo-SCT) in AML is a poor risk factor for outcome. The γδ T cells represents a unique minority lymphocyte population which is preferentially located in peripheral tissues, can recognize antigens in non-MHC restricted manner and plays a "bridging" role between innate and adaptive immune system. OBJECTIVES In this study, we investigated a potential graft-vs-leukaemia effect of γδ T cells reconstitution post-transplant in AML patients with pre-transplant positive minimal/measurable disease status (MRD+). STUDY DESIGN We investigated a potential graft-vs-leukaemia effect of γδ T cells reconstitution post-transplant in AML patients with pre-transplant positive MRD+. MRD assessment was performed in 202 patients (MRD+, n=100) with multicolored flow cytometry ("different from normal" strategy). Analysis for absolute concentrations of CD3+, CD4+, CD8+, NK, and γδ T cells were performed by flow cytometry according to an internal protocol at day +30 and +100 post-transplant. Differences between categorical and continuous variables were determined by Chi-square and Student's T-test, respectively. The Mann-Whitney test was used to compare medians of continuous variables. Spearman correlation was used for nonparametric assessment of correlation between different cell subsets during immune reconstitution. Kaplan Meier survival analysis and Cox regression analysis were used to investigate the associations between immune reconstitution and survival outcomes. Grays' analysis was used to compute incidences of relapses, non-relapse mortality (NRM) and graft-vs-host disease (GvHD). RESULTS Follow-up for survivors was 28 months (3-59). Younger age (≤58) of recipient and donor (<30), sex mismatch, matched donors, CMV reactivation and ATG were associated with a faster γδ T cell reconstitution. In multivariable analysis for MRD+ patients, higher than median level of γδ T cells on days +30 and +100 resulted in a significant improved leukaemia-free (HR 0.42, p=0.007 and HR 0.42, p=0.011, respectively) and overall survival (HR 0.44, p=0.038 and HR 0.33, p=0.009, respectively). Further, higher γδ T cell level on day +30 led to significant reduced risk of relapse (HR 0.36, p=0.019). No impact of γδ T cell level on day +30 and +100 could be seen in MRD- patients and no correlation with occurrence of graft-versus-host disease could be observed. CONCLUSION An enhanced immune reconstitution of γδ T cells post-transplant may overcome the higher relapse risk of pre-transplant MRD+ patients with AML

Post-Transplantation Multicolored Flow Cytometry–Minimal Residual Disease Status on Day 100 Predicts Outcomes for Patients With Refractory Acute Myeloid Leukemia

BACKGROUND Patients with relapsed/refractory AML have a dismal prognosis. Allogeneic stem cell transplantation (allo-SCT) provides a curative approach, however the overall survival (OS) remains low (20-40%). In this setting, though some effective approaches have been evaluated in the recent years, the management of such patients still remains challenging. OBJECTIVES In this study we evaluated the predictive role of post-transplant day +100 MRD detection for post-transplant outcomes for patients with refractory AML. STUDY DESIGN Fifty-six adult patients with refractory AML (median age 58, 20-76; male, 61%) who underwent allo-SCT were included in this retrospective monocentric study. Twenty-nine patients (52%) received FLAMSA-based conditioning. MRD was assessed using multicolored flow cytometry (MFC) according to ELN guidelines ("different from normal" and LAIP). The sensitivity of the method was 10-4 - 10-5. The median marrow blast count at allo-SCT was 25% (range 6-91%). At day +100 post-transplant, 40 patients (71%) experienced MFC-MRD negativity, 16 patients (29%) were MRD positive. All included patients survived at least 100 days post-transplant without relapse. Uni- and multivariate analysis based on Kaplan-Meier and Cox proportional hazards method were performed. RESULTS The median follow-up was 16 months (range 3-66). The post-transplant day 100 MRD negative patients received rather two allografts (27% vs 6%, p=0.08). In multivariate analysis, day +100 MRD status (negative vs positive) (OS: 0.23 (0.1-0.54), p=0.001; relapses: 0.20 (0.1-0.49), p=0.0005) and FLAMSA vs other regimens (0.34 (0.1-0.83), p=0.018; relapses: 0.43 (0.17-1.1), p=0.07) independently impacted post-transplant survival. CONCLUSIONS We suggest that post-transplant day +100 MFC-MRD detection plays predictive role in refractory AML patients and may help to define possible candidates for early post-transplant interventions aiming to decrease the relapse risk and improve survival

Individualized busulfan dosing improves outcomes compared to fixed dose administration in pre-transplant MRD positive AML patients with intermediate risk undergoing allogeneic stem cell transplantation in CR.

Pre-transplant minimal residual disease (MRD) impacts negatively on post-transplant relapse risk in AML. Therapeutic drug monitoring by calculating area-under-the-curve (AUC) was developed to optimize busulfan exposure. Here, we compared post-transplant outcomes after individualized versus fixed busulfan dosage in intermediate-risk AML who achieved CR prior to allograft focusing on pre-transplant flow-MRD. 87 patients (median, 56 years) with intermediate-risk AML and pre-transplant flow-MRD ("different from normal") were included. 32 patients received individualized busulfan; 54 fixed dosage. Individualized dosage was adjusted in 25/32 patients: increased, n = 18/25 (72%); decreased: n = 7/25 (28%). After median follow-up of 27 months, we observed lower 3-year relapses (6%, 2-19% vs 35%, 23-49% p = 0.02), improved 3-year LFS (78%, 54-91% vs 55%, 40-70% p = 0.009) and -OS (82%, 60-93% vs 69%, 54-81% p = 0.05) after individualized compared to fixed Bu. NRM and acute GvHD were not different. In multivariate analysis, fixed Bu showed unfavorable impact on OS (HR 4.6, p = 0.044), LFS (HR 3.6, p = 0.018) and relapses (HR 3.6, p = 0.033). Fixed Bu also had unfavorable impact on LFS (3.6, 1.1-12.6, p = 0.041) in pre-transplant MRD-positive patients. Individualized, AUC-based, busulfan is associated with lower relapses in intermediate-risk AML patients allografted in CR and may overcome pre-transplant MRD-positivity