Patrick Wuchter

Adoptive cell therapies using chimeric antigen receptor or T cell receptor-transgenic T cells (TCR-T) are effective in treating hematological malignancies but have limited efficacy in solid tumors, particularly brain tumors. A major challenge of adoptive T cell therapy in brain tumors is lack of access to the target antigen, which is necessary for restimulation and persistence. Here, we addressed this challenge by using an mRNA vaccine encoding the cognate antigen to induce in vivo restimulation of transferred TCR-T cells in glioma models. Delivery of mRNA encoding the immunodominant MHC class I ovalbumin epitope SIINFEKL to splenic dendritic cells initiated strong expansion of adoptively transferred ovalbumin-specific OT1 CD8+ T cells to up to 80% of all circulating CD8+ T cells. RNA vaccination markedly improved T cell infiltration and retained interferon-stimulated gene signature in the brain, suppressing glioblastoma growth. The therapeutic efficacy of this approach was further demonstrated in an experimental model for a first-in-class TCR-T cell therapy targeting an HLA-A2-restricted immunodominant epitope of the human glioblastoma stemness antigen PTPRZ1. Co-administration of human dendritic cells transfected with PTPRZ1-mRNA led to rejection of orthotopically implanted primary human gliomas endogenously expressing PTPRZ1 and HLA-A2 after adoptive transfer of PTPRZ1-TCR-T cells. In summary, these findings indicate that mRNA vaccines can boost TCR-T cell therapy against glioblastoma.

Acute myeloid leukaemia (AML) in older or unfit patients is commonly treated with hypomethylating agents (HMA) plus venetoclax (VEN), but prolonged VEN exposure often causes substantial haematological toxicity. We retrospectively analysed 61 elderly AML patients treated with HMA + VEN for 7 (7d), 14 (14d) or > 14 days (> 14d) per cycle. Best overall response rate was 76% and did not differ significantly between schedules ( p = 0.38). Median overall survival (OS) was numerically longer with 7d (28.4 months) than with 14d (11.4 months) or > 14d (6.7 months), although unadjusted differences were not significant ( p = 0.23). At cycle level, the 7d schedule was associated with fewer infections and more complication‐free cycles than both longer schedules, while bleeding events were similar. Grade 4 cytopenias and platelet transfusion requirements were also lower with 7d. In an adjusted Cox model, prior HMA exposure, non‐favourable ELN 2024 risk and age ≥ 75 years were associated with inferior OS, whereas the 7d schedule remained associated with improved OS versus > 14d (HR 0.33, p = 0.03). Higher recent grade 4 neutropenia burden was independently associated with inferior OS. These findings suggest that abbreviated VEN exposure may improve tolerability without clear loss of efficacy in AML patients ineligible for intensive chemotherapy.

Introduction: Cryopreservation of peripheral‐blood‐derived hematopoietic stem and progenitor cells (PBSCs) has been used for decades, primarily for autologous transplantation. Now, in the era of new cellular therapies, the cryopreservation of allogeneic PBSCs may also become relevant. While the defining quality‐control parameters are assessed before cryopreservation, in thawed cryopreserved samples the viability of total nucleated cells is often the only parameter analyzed. We therefore evaluated the recovery rates of total and viable CD45 ⁺ and CD34 ⁺ cells after cryopreservation. Methods: Thirty‐nine samples of allogeneic PBSC products were analyzed before and after cryopreservation. Post‐thaw viability was assessed using trypan blue exclusion. To investigate the influence of different preanalytical preparation methods, PBSCs were resuspended in phosphate‐buffered saline (PBS) or human serum albumin (HSA), and the cells suspended in HSA were tested without incubation or following incubation at 37°C for 1 h or 2 h. The number and viability of CD45 ⁺ and CD34 ⁺ cells were evaluated by flow cytometry and compared with pre‐thaw values. Results: After cryopreservation, we observed significant decreases in both total and viable CD45 ⁺ cell numbers. In contrast, the difference in total CD34 ⁺ cell numbers was rather small, whereas the median recovery rate of viable CD34 ⁺ cells after resuspension in PBS was 70.1%. Relevant differences between the preanalytical treatment groups could not be observed. The viability values as determined by trypan blue viability staining and flow cytometry were not correlated. Conclusion: Determination of the CD34 ⁺ cell recovery rate after thawing is feasible and may be used as an additional parameter for quality control for cryopreserved allogeneic PBSCs. However, the results of currently used viability assays vary considerably, making comparisons between different cell processing units and cryopreservation protocols difficult. Therefore, available protocols should be carefully evaluated based on “good‐manufacturing‐practice” (GMP) regulations before implementation.

The efficiency of an autologous, as well as an allogeneic, HSC graft is mainly determined by the number of CD34⁺ cells present. The dose of transplanted CD34⁺ cells per kg body weight (bw) determines the kinetics of the neutrophil and platelet engraftment after auto-HCT (Weaver et al. Blood 86:3961–9, 1995). The measurement of CD34⁺ cells by flow cytometry is, therefore, an important method to assess the graft quantity and quality.

The intravenous infusion of patient’s own HSC (autologous SCT) to restore BM damage is the basic principle of high-dose chemotherapy, since otherwise the patient would expect long-lasting aplasia with life-threatening infections. Therefore, a sufficient collection of HSC before application of high-dose therapy is mandatory. Since HSC expresses CD34 on their surface, the number of CD34+ cells in the transplant material is considered as an indicator of the HSC content.The aim of infusion of HSC from a donor (allogeneic SCT) is to restore BM damage and to treat the patient’s disease. It represents a permanent cellular immunotherapy by adding a graft versus tumor effect in malignant diseases.

Introduction: Autologous stem cell transplantation is a successful routine procedure with only a small number of non-engraftment cases, although the time to hematopoietic recovery may vary considerably across patients. While CD34 has been the decisive marker for enumerating hematopoietic stem and progenitor cells (HSPCs) for more than 30 years, the impact of CD34-positive cellular subpopulations in autologous HSPC grafts on hematopoietic reconstitution remains unclear. Methods: The two-color ISHAGE protocol represents the current gold standard for CD34+ cell enumeration but includes only the number of viable CD45+/CD34+ cells relative to the body weight of the recipient. We adapted a multicolor flow cytometry marker panel for advanced characterization of CD34 subpopulations in retained samples of autologous peripheral blood stem cell products (n = 49), which had been cryostored for a wide range from 4 to 15 years. The flow cytometric analysis included CD10, CD34, CD38, CD45, CD45RA, CD133, and viability staining with 7AAD. The findings were correlated with clinical engraftment data, including reconstitution of leukocytes, neutrophils, and platelets after transplantation (TPL). Results: We demonstrated that the identification of autologous HSPC subpopulations by flow cytometry after cryopreservation is feasible. Regarding the distribution of HSPC subpopulations, a markedly different pattern was observed in comparison to previously published data obtained using fresh autologous material. Our data revealed the largest ratio of lympho-myeloid progenitors (LMPPs) after freezing and thawing, followed by multipotent progenitors and erythroid-myeloid progenitors. A high ratio of LMPPs, representing an immature stage of differentiation, correlated significantly with early neutrophilic granulocyte and leukocyte engraftment (p = 0.025 and p = 0.003). Conversely, a large ratio of differentiated cells correlated with late engraftment of neutrophilic granulocytes (p = 0.024). Overall, successful engraftment was documented for all patients. Conclusion: We established an advanced flow cytometry panel to assess the differentiation ability of cryostored autologous peripheral blood stem cell grafts and correlated it with timely hematopoietic reconstitution. This approach represents a novel and comprehensive way to identify hematopoietic stem and progenitor subpopulations. It is a feasible way to indicate the engraftment capacity of stem cell products.

Dear Editor,Mobilizing hematopoietic stem and progenitor cells from the bone marrow into the peripheral blood, collecting them sufficiently by leukapheresis, and transplanting them in an allogeneic or autologous setting as “stem cell graft” is a routine procedure in collection facilities such as in many blood transfusion establishments and transplant centers worldwide. Patients with multiple myeloma and malignant lymphoma are the main candidates for an autologous stem cell transplantation. Today’s induction regimen in both entities have been improved significantly over the last two decades, but the application of high-dose chemotherapy followed by autologous stem cell rescue still represents an essential part of the therapeutic regimen. In this context, the collection of an optimal stem cell product is an important prerequisite.This special issue of Transfusion Medicine and Hemotherapy provides a detailed insight into state-of-the art stem cell mobilization, collection, and processing techniques. Regarding patients with multiple myeloma, Jantunen et al. comprehensively analyzed current mobilization strategies [1]. The impact of clinical parameters and induction regimens on peripheral blood stem cell (PBSC) mobilization in a large cohort of multiple myeloma patients has been evaluated by Sauer and colleagues [2]. In a more specific approach, Sauer et al. [3] assessed in a second study the effectiveness of autologous stem cell collection after daratumumab-VTD versus VCD. For the entity of mantle cell lymphoma, Turunen et al. examined the cellular composition and the clinical outcome after autologous transplantation [4].In general, more than 80% of patients succeed in collecting an autologous PBSC graft. However, 10–20% mobilize insufficiently and are so-called “poor mobilizer” [5], but a harmonized definition has not been found yet. Strategies to overcome this problem include the application of plerixafor, either used preemptively or as a rescue strategy [6].However, comprehensive information on poor mobilizing patients regarding incidence, current treatments, and mobilization strategies is lacking. The German prospective, multicenter, open-label, non-interventional OPTIMOB study addressed this lack of knowledge by analyzing mobilization and collection parameters in a large cohort of adult, transplant-eligible, good and poor mobilizing patients with lymphoma or multiple myeloma. In this special issue, the results of this huge national study with 28 recruiting study centers are presented in two articles: Bittrich et al. [8] described the results in patients with multiple myeloma, whereas Kriegsmann et al. [9] showed the results of the cohort of lymphoma patients.A state-of-the-art collection of PBSCs includes a system of quality control and benchmarking. Approaches to quantitatively assess the effectiveness of autologous leukapheresis sessions have been developed, and some allow not only to calculate the collection efficiency (CE2) but also to manage the duration of the apheresis session [10‒12]. While 2.0 × 106 CD34+ cells/kg body weight (bw) is uniformly accepted as minimum for one autologous transplant, the question of whether more cells for a second or third transplantation or a mere “backup” transplant should be provided is handled divergent. In the allogeneic setting, clinical aspects of both the donor and the recipient have to be considered, e.g., bw differences. Kayser et al. [13] analyzed a formula, alongside other parameters, to predict the outcome of hematopoietic stem cell collection in healthy allogeneic donors. As the number of requested CD34+ cells may vary between 4.0 and over 8.0 × 106 CD34+ cells/kg bw (i.e., if the transplant will be further processed), this approach may help streamline the apheresis session and reduce the burden for the donor.For decades, CD34 is the decisive marker for enumeration of PBSC grafts, but efforts have been made to develop further marker combinations for a more comprehensive assessment of the graft composition and hence quality (e.g., tri-lineage engraftment). As a step in this direction, Heuer et al. analyzed PBSC subpopulations by flow cytometry and correlated them with clinical outcome after autologous transplantation [14]. Taken together, this special issue of Transfusion Medicine and Hemotherapy gives an overview on some of the currently most relevant issues related to procuring PBSC grafts.The authors have no conflicts of interest to declare.There is no funding related to this article.K.K. and P.W. wrote the manuscript.

Radiotherapy of head-and-neck squamous cell carcinoma (HNSCC) can cause considerable normal tissue injuries, and mesenchymal stromal cells (MSCs) have been shown to aid regeneration of irradiation-damaged normal tissues. However, utilization of MSC-based treatments for HNSCC patients undergoing radiotherapy is hampered by concerns regarding potential radioprotective effects. We therefore investigated the influence of MSCs on the radiosensitivity of HNSCCs. Several human papillomavirus (HPV)-negative and HPV-positive HNSCCs were co-cultured with human bone marrow-derived MSCs using two-dimensional and three-dimensional assays. Clonogenic survival, proliferation, and viability of HNSCCs after radiotherapy were assessed depending on MSC co-culture. Flow cytometry analyses were conducted to examine the influence of MSCs on irradiation-induced cell cycle distribution and apoptosis induction in HNSCCs. Immunofluorescence stainings of γH2AX were conducted to determine the levels of residual irradiation-induced DNA double-strand breaks. Levels of connective tissue growth factor (CTGF), a multifunctional pro-tumorigenic cytokine, were analyzed using enzyme-linked immunosorbent assays. Neither direct MSC co-culture nor MSC-conditioned medium exerted radioprotective effects on HNSCCs as determined by clonogenic survival, proliferation, and viability assays. Consistently, three-dimensional microwell arrays revealed no radioprotective effects of MSCs. Irradiation resulted in a G2/M arrest of HNSCCs at 96 h independently of MSC co-culture. HNSCCs’ apoptosis rates were increased by irradiation irrespective of MSCs. Numbers of residual γH2AX foci after irradiation with 2 or 8 Gy were comparable between mono- and co-cultures. MSC mono-cultures and HNSCC-MSC co-cultures exhibited comparable CTGF levels. We did not detect radioprotective effects of human MSCs on HNSCCs. Our results suggest that the usage of MSC-based therapies for radiotherapy-related toxicities in HNSCC patients may be safe in the context of absent radioprotection.

The hypomethylating agent 5-azacytidine (5-Aza) is a standard-of-care for patients with higher-risk myelodysplastic syndromes (MDS). Although an initial response is induced in approximately 50% of 5-Aza treated patients, subsequent relapse is almost certain. Recently, inhibitors of anti-apoptotic BCL-2 protein family members have shown therapeutic potential in acute myeloid leukemia (AML) and higher-risk MDS. Alvocidib (Alv), a CDK9 inhibitor and indirect transcriptional repressor of the anti-apoptotic factor MCL-1, has shown anti-leukemic effects in a phase 1 study of patients with AML (Lee DJ et al, Expert Opin Investig Drugs 2019; Zeidner JF et al, Leuk Res 2015). A phase 1b/2 study with Alv and 5-Aza or decitabine in higher-risk patients with MDS was recently completed (NCT03593915); however, biomarkers for response to the Alv and 5-Aza combination are not well characterized. To identify potential biomarkers of response, we performed a comprehensive in vitro assessment of Alv and 5-Aza combination using a clinically well-characterized cohort of n=45 higher-risk patients with MDS and n=11 healthy controls (HC). CD34+ cells were purified from bone marrow (BM) aspirates using positive selection with MACS beads. CD34+ cells of HC were obtained from femur head replacement surgery bone specimens. After 4 days of expansion in SFEM II medium containing StemSpan Myeloid Expansion Supplement, cells were treated with 5-Aza for 48h, Alv for 24h or their combination (5-Aza for 48h followed by Alv for 24h). Cell viability was determined using CellTiter-Glo (CTG) and Annexin-V apoptosis assays. MCL-1 dependency of MDS samples was assessed using MS1 peptide-based assay. Recurrent myeloid neoplasia mutations in 67 genes were assessed in BM mononuclear cells using NGS panel deep sequencing. The combination of 5-Aza+Alv had an additive cytotoxic effect on CD34+ MDS cells in CTG assay (median cell viability = 74%, 73.8% and 55% for 5-Aza, Alv and combination respectively, pCitation Format: Vladimir Ryabov, Nanni Schmitt, Qingyu Xu, Alexander Streuer, Johann-Christoph Jann, Alina Wein, Eva Altrock, Verena Nowak, Nadine Weimer, Julia Obländer, Iris Palme, Ahmed Jawhar, Ali Darwich, Patrick Wuchter, Christel Weiss, Georgia Metzgeroth, Jason M. Foulks, Laurenz Steiner, Mohamad Jawhar, Wolf-Karsten Hofmann, Daniel Nowak. Mutations in the ASXL1 and ZRSR2 genes are associated with the response to the combination of alvocidib and 5-azacytidine in higher-risk myelodysplastic syndromes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6257.