Introduction: Axicel is approved for treatment of r/r PMBCL beyond the 2nd treatment line (>2L). However, real-world data on axicel safety and efficacy in this orphan entity is limited. Here, we present a comparison of standard-of-care axicel treatment in r/r PMBCL >2L versus diffuse large B-cell lymphoma, not otherwise specified (DLBCL). Methods: The previously published German Lymphoma Alliance (GLA)/ German Registry for Stem Cell Transplantation (DRST) dataset (Bethge et al., Blood 2022) was screened for PBMCL patients treated with axicel. Investigators were consulted for follow-up data and confirmation of diagnosis. Axicel-treated patients with DLBCL from the same dataset served as comparators. Results: Altogether, 13 patients with PMBCL were identified in the total sample of 173 patients treated with axicel, along with 131 DLBCL patients. PMBCL patients were significantly younger (median age 39 (20–48) years versus 60 (20–83) years for DLBCL), but were comparable to DLBCL for all other baseline parameters, including performance status, prior treatment lines, prior transplantations, IPI, LDH, and disease status at lymphodepletion. Specifically, in the PMBCL population, IPI was high/high-intermediate in 54% of patients and 92% had active disease at lymphodepletion due to omission of bridging attempts in 38% and unsuccessful bridging in 54% of patients. Only 18% each had received prior radiotherapy and checkpoint inhibitor treatment, respectively. With rates of grade ≥3 CRS and neurotoxicity of 15% each, and 8% of patients without neutrophil recovery at day 30, safety outcomes of the PMBCL patients did not significantly differ from the DLBCL cohort. Non-relapse deaths did not occur in the PMBCL group. Regarding efficacy, the best overall response rate (ORR) in PMBCL was 85%, with 54% complete responses (CR), and thus not significantly different from the DLBCL cohort (ORR 71%, CR 42%). With a median follow-up of 35 months, 2-year progression-free survival (PFS) and overall survival (OS) of the PMBCL group was 54% and 75%, respectively, and compared favorably with DLBCL outcomes (PFS 26%, p = 0.069; OS 36%, p = 0.011). Of note, in the PMBCL group, progression events beyond 8 months did not occur (Figure), and patients not responding to bridging had 2-year PFS similar to patients without bridging or with bridging response (57% vs. 50%). Keywords: Aggressive B-cell non-Hodgkin lymphoma, Cellular therapies Conflicts of interests pertinent to the abstract. M. Schubert Educational grants: Kite/Gilead F. A. Ayuk Honoraria: Gilead, BMS, Janssen, Takeda, Therakos/Mallinckrodt Research funding: Therakos/Mallinckrodt W. Bethge Honoraria: Gilead M. von Bonin Honoraria: Gilead, Novartis, Janssen, Takeda, Daiichi Sankyo V. Vucinic Honoraria: Novartis, Kite/Gilead, Janssen, BMS/Celgene. Educational grants: Kite/Gilead, Janssen, BMS/Celgene E. M. Wagner-Drouet Consultant or advisory role: Kite/Gilead, Novartis M. Subklewe Consultant or advisory role: Amgen, BMS, Gilead, Janssen, Miltenyi, Novartis, Pfitzer, Seagen Honoraria: Amgen, BMS, Gilead, Miltenyi, Novartis, Pfitzer, Takeda Research funding: Amgen, BMS, Gilead, Miltenyi, Novartis, Roche, Seagen C. D. Baldus Honoraria: Gilead, Novartis, BMS/Celgene Educational grants: Gilead D. Mougiakakos Consultant or advisory role: AbbVie, Roche, BMS, Hexal, Novartis, MSD, Celgene, Janssen, Pfizer, AstraZeneca, Jazz Pharma M. S. Topp Consultant or advisory role: Astra Zeneca, BMS, Kite/Gilead, Janssen, Roche, Novartis Research funding: Kite/Gilead, Regeneron, Roche G. Wulf Honoraria: Gilead, Novartis. Takeda, Clingen, Amgen N. Kröger Honoraria: Kite/Gilead, Jazz Pharma, MSD, Neovii, Novartis, Riemser, Pfizer, BMS Research funding: Neovii, BMS, Novartis, Riemser P. Dreger Honoraria: Novartis, Kite/Gilead, BMS, Miltenyi

Data on the association between chimeric antigen receptor (CAR)-T-cell kinetics and patient outcome in the nontrial setting are missing, mainly due to the lack of broadly available CAR-T-cell diagnostic quantification tools. We performed prospective quantification of axicabtagene ciloleucel (axi-cel) in 21 patients treated for aggressive B-cell lymphoma at our clinic. Median peak CAR-T-cell count was 16.14 CAR-T cells/µL. Patients with 16.14/μL or higher peak CAR-T cells (strong expanders) had more day-30 objective responses (91% vs 40%, P = .02). In univariate analysis, peak CAR-T cell ≥ 16.14 (P < .001), normal platelet counts at start of lymphodepletion (P < .001), no prior stem cell transplant (P = .04), and peak CAR-T cells as continuous variable (P = .03) were associated with better progression-free survival (PFS). After adjusting for platelet counts and prior stem cell transplantation, peak CAR-T cells below median was still associated with shorter PFS (relative risk, 0.15, 95% confidence interval, 0.04-0.59, P = .007). Low platelet counts also maintained significant impact on PFS. Our data demonstrate association of axi-cel levels and outcome in a nontrial setting and for the first time use a cutoff to segregate weak and strong expanders with respective outcomes.

PURPOSEClonal hematopoiesis of indeterminate potential (CHIP) occurs in the blood of approximately 20% of older persons. CHIP is linked to an increased risk of hematologic malignancies and of all-cause mortality; thus, the eligibility of stem-cell donors with CHIP is questionable. We comprehensively investigated how donor CHIP affects outcome of allogeneic hematopoietic stem-cell transplantation (HSCT).METHODSWe collected blood samples from 500 healthy, related HSCT donors (age ≥ 55 years) at the time of stem-cell donation for targeted sequencing with a 66-gene panel. The effect of donor CHIP was assessed on recipient outcomes, including graft-versus-host disease (GVHD), cumulative incidence of relapse/progression (CIR/P), and overall survival (OS).RESULTSA total of 92 clonal mutations with a median variant allele frequency of 5.9% were identified in 80 (16.0%) of 500 donors. CHIP prevalence was higher in donors related to patients with myeloid compared with lymphoid malignancies (19.2% v 6.3%; P ≤ .001). In recipients allografted with donor CHIP, we found a high cumulative incidence of chronic GVHD (cGVHD; hazard ratio [HR], 1.73; 95% CI, 1.21 to 2.49; P = .003) and lower CIR/P (univariate: HR, 0.62; 95% CI, 0.40 to 0.97; P = .027; multivariate: HR, 0.63; 95% CI, 0.41 to 0.98; P = .042) but no effect on nonrelapse mortality. Serial quantification of 25 mutations showed engraftment of 24 of 25 clones and disproportionate expansion in half of them. Donor-cell leukemia was observed in two recipients. OS was not affected by donor CHIP status (HR, 0.88; 95% CI, 0.65 to 1.321; P = .434).CONCLUSIONAllogeneic HSCT from donors with CHIP seems safe and results in similar survival in the setting of older, related donors. Future studies in younger and unrelated donors are warranted to extend these results. Confirmatory studies and mechanistic experiments are warranted to challenge the hypothesis that donor CHIP might foster cGVHD development and reduce relapse/progression risk.

Abstract Background: The recent discovery of the somatically acquired calreticulin mutation in about 30% of the myelofibrosis patients provided a new diagnostic marker, which can also be used as marker of minimal residual disease (MRD) following curative treatment with allogeneic stem cell transplantation (ASCT). More than 80% of CALR mutations are of two types: type-1 variants result from a 52-bp deletion and produce the protein change p.L367fs*46, and type-2 variants are caused by a 5-bp insertion and produce the protein change p.K385fs*47. Whereas Sanger as well as next-generation sequencing (NGS) readily allow detection of CALR mutations in newly diagnosed patients, their applicability for MRD detection is limited. Real-time quantitative PCR (qPCR) has therefore been suggested as a potential alternative. In order to combine the increased sensitivity of qPCR with the excellent accuracy of digital PCR (dPCR) we developed a duplex dPCR assay detecting the CALR type-2 mutation in combination with its wild-type allele. Methods and Patients: To address sensitivity and reliability of the novel dPCR assay we first tested it on a new, UKE-1 derived cell line harboring one copy of the CALR type-2 mutation (UKE-1CALR2), which was established by lentiviral gene transfer and single-cell sorting. We generated serial dilutions of UKE-1CALR2 in buffy-coat (BC) cells, isolated DNA and submitted it to dPCR. Using 120 ng EcoRI-restricted genomic DNA we detected up to one UKE-1CALR2 in 10,000 BC cells (0.01%) indicating excellent sensitivity. As expected the detection limit could be further increased by applying the p.K385fs*47-specific dPCR as a singleplex assay. Results: Using our new technique we next performed MRD analysis in CALR+ patients who underwent allogeneic stem cell transplantation and compared results with respective qPCR data. Out of 143 patients with myelofibrosis who underwent allogeneic SCT 92 were JAK2V617 positive, 4 MPL positive and 35 CALR positive. Out of these 35 patients 21 harbored the CALR type-1 and eight the CALR type-2 mutation. In seven out of those eight patients both qPCR and digital PCR could be applied for MRD monitoring after SCT. In 3/7 patient qPCR as well as dPCR were negative on day +20, +100 and +180 after transplantation, respectively. In 2 patients dPCR remained positive at days +100 and +180 days before turning negative, whereas qPCR was already negative. In one patient dPCR remained positive 6 months after SCT, whereas qPCR was negative since day +80. None of the above patients had experienced clinical relapse. In contrast, there was one patient who relapsed 28 months after transplantation. That patient was MRD-negative by qPCR until one months before relapse, whereas dPCR, which initially also became negative after SCT (day +180), converted to positivity already 1 year after transplantation and was steadily increasing until clinical relapse. Conclusion: Our data indicates that the new CALR type-2-mutation specific dPCR assay combines excellent accuracy with high sensitivity thus allowing the monitoring of deep molecular remission and the early detection of MRD in relapsing patients with myelofibrosis after stem cell transplantation * BF and AB contributed equally Disclosures No relevant conflicts of interest to declare.

In this retrospective study, we evaluated donor lymphocyte infusions given for relapsed ( n =48) or persistent ( n =15) myeloma following non-myeloablative allogeneic stem cell transplantation (Allo-SCT). Twenty-four of 63 patients (38.1%) responded: 12 patients (19.0%) with a partial response (PR) and 12 patients (19.0%) with a complete response (CR). Overall survival after donor lymphocyte infusions (DLI) was 23.6 months (1.0–50.7+). Median overall survival for non-responding patients was 23.6 months and has not been reached for the patients responding to DLI. In responders, progression-free survival after DLI was 27.8 months (1.2–46.2+). Patients with a PR had a median progression-free survival of 7.0 months, whereas patients with a CR to DLI had a median progression-free survival of 27.8 months. Major toxicities were acute graft-versus-host disease (GVHD) (38.1%) and chronic GVHD (42.9%). Seven patients (11.1%) died from treatment-related mortality. The only significant prognostic factors for response to DLI were the occurrence of acute and chronic GVHD. There was a trend towards significance for time between transplantation and DLI, and response. Donor lymphocyte infusion following non-myeloablative Allo-SCT is a valuable strategy for relapsed or persistent disease.

Peripheral T lymphocytes are a target of choice for many gene therapeutic strategies. Retrovirus-mediated transduction allows genomic integration and long-term expression of transgenes in target cells. Over many years, low transduction efficiency into primary T lymphocytes has limited clinical application of existing protocols. Recently, gene transfer rates > 50% have been achieved facilitating clinical studies. More attention is thus being focused on the ability of gene-modified cells to carry out innate as well as conferred functions in vivo and the influence of culture conditions, retroviral vector and host response thereon.

Current gene therapeutic protocols directed towards the treatment of inherited disorders (eg ADA-SCID) and viral infections (eg AIDS), as well as adoptive immunotherapy approaches are based on the use of genetically modified lymphocytes. Since only insufficient transduction of T cells is obtained using existing techniques, the development of more efficient gene transfer protocols into these cells is of great importance. We present here a protocol for the highly efficient transduction of human primary T cells at high densities (1 x 106/ml) by retroviral infection. Using retroviral vectors encoding a truncated human low-affinity nerve growth factor receptor (DeltaLNGFR) as a gene transfer marker, we obtained transduction frequencies of more than 70% of CD3+ cells after two cycles of infection. Our protocol is based on the use of FBS-free media for both the production of retrovirus-containing supernatant and the cultivation of the primary T cells. Since the protocol presented here works just as efficiently under large-scale conditions, it may be easily adapted to clinical needs and 'good manufacturing practice' (GMP) standards.