ALBERT

Description: Background: T effector cells (Teff) within the stem cell graft in allogeneic hematopoietic stem cell transplantation (HSCT) can elicit disabling acute graft-versus-host disease (aGvHD) contributing to transplant-related mortality. Teff as donor lymphocyte infusion (DLI) are a therapeutic option to re-induce complete remission (CR) after leukemia relapse. Usually DLIs are given in dose escalating regimens until CR is achieved or first signs of aGvHD develop. To monitor the DLI induced allo-immune response and the efficacy of aGvHD treatment is a clinical challenge, since no established biomarkers are available. T regulatory cells (Tregs) are thought to play an important role in balancing immune responses and studies have shown effects of adoptive Treg transfer as a therapeutic option in GvHD. T cell receptor (TCR) sequencing of T cell subsets, such as Tregs, after DLI might allow the identification of clones inducing control of GvHD. Here we report data of 29 DLI patients with a median follow-up of 〉1 year allowing us to thoroughly analyze differences of the TCR repertoire in patients with or without aGvHD. Aims: We aimed to analyze Treg TCR diversity and clonality changes over time as a potential biomarker for development and treatment response of aGvHD following DLI. Patients and Methods: The study cohort consisted of 29 leukemia patients after HSCT who received DLI treatment for recurrence of disease, molecular relapse or high risk phenotype. Blood samples were taken before and after DLI. A median of 4 (range 2-6) blood samples per patient were available for TCR-sequencing. The last sample was taken at a median of 123 (27-530) days post DLI. After generation of PBMCs CD4+CD127-CD25+ Tregs were FACS-sorted for cDNA-based CDR3-region amplification of the TCR-β chain. CDR3 amplicons were sequenced on the Illumina MiSeq platform and annotated using the IMGT.org database. Further bioinformatic analyses were based on VDJtools and the tcR R-package. Results: In 18/29 patients we observed clinical symptoms of aGvHD, with blood samples available of the acute onset in 12/18 cases. Treg frequencies and absolute numbers did not differ between aGvHD and noGvHD samples. Treg TCR diversity, assessed via inverse Simpson's diversity index (1/D) increased on average by 322% at the first occurrence of aGvHD compared to the previous sample (Figure 1A, B). Stratifying for aGvHD severity (total grade 1-2 vs. 3-4) did not reveal any group differences. However, stratifying by organ involvement (skin vs. GI/liver) showed a more pronounced increase of 1/D in patients with aGvHD of the GI tract and/or the liver. Moreover, in 11 subjects blood samples were available a median of 7 (3-14) days prior to aGvHD diagnosis. Already at this preclinical time point we detected an increased 1/D of +361% (Figure 1A, B). Again, aGvHD organ involvement significantly affected the result, with GI/liver involvement mainly driving this effect (+567% vs. +1% 1/D). In contrast, patients who did not develop aGvHD at any time after DLI showed on average a slight decrease of -8% 1/D compared to the previous time point. Next, we analyzed all aGvHD patients with at least one sample available after initiation of treatment (local and/or systemic steroids). Control or remission of aGvHD symptoms was accompanied by decreased 1/D of on average -58% compared to the previous samples (Figure 2). In 9/11 patients we saw a focusing of the Treg TCR repertoire; in the other two (Figure 2, red lines) no or only partial clinical response to systemic steroids was reported. Conclusion: Our data describe detailed changes in the Treg compartment on the TCR level following development and treatment of aGvHD. Currently, aGvHD diagnosis following DLI treatment relies solely on clinical symptoms, deciding whether further dose increments of DLI can be administered. As our data suggest, Treg TCR sequencing may support transplant specialists with (1) detection of patients at risk for aGvHD, even prior to clinical symptoms, (2) identification of patients eligible for further dose increments (compare Figure 1B), and (3) assessment of aGvHD treatment with guidance whether higher dosage of steroids and/or alternative immunosuppressive treatment might be required (compare Figure 2, red lines). Future prospective studies are needed to replicate these findings in a large cohort, potentially enabling identification of specific Treg TCR clones controlling the allo-immune response in aGvHD. Disclosures Ganser: Novartis: Membership on an entity's Board of Directors or advisory committees. Koenecke:BMS: Consultancy; abbvie: Consultancy; Amgen: Consultancy; Roche: Consultancy.

Description: Fourteen patients with myeloid leukemia (12 with acute and 2 with chronic myelogenous leukemia) were transplanted from their HLA-identical (n = 9) or haploidentical (n = 5) family donors with CD34-enriched stem cells (HSCT) without further immunosuppression. In order to induce a graft versus leukemia (GvL) effect and to investigate the possibility of controlling graft-versus-host disease (GvHD), the standard transplantation protocol was adapted to include transfusion of gene-modified donor T-cells after HSCT in 11 patients, 3 did not receive any T-cells. Donor-T-cells were transduced with the replication-deficient retrovirus SFCMM-3 which expresses the herpes simplex thymidine kinase (HSV-Tk) as a suicide gene and the truncated low affinity nerve growth factor receptor (ΔLNGFR) for selection purposes. After transfusion, SFCMM-3 transduced T-cells were detectable in all 11 patients by PCR and FACS analyses immediately after transfusion and during the follow up period (range: 1.1-11 years). Two of 9 patients developed acute GvHD: one of the skin, grade 1, 56 days after transfusion of the transduced cells, the other grade II which was successfully treated with ganciclovir. Loss of bcr-abl gene expression was achieved in one patient after expansion of transduced cells. Donor chimerism was stabilized after transfusion of transduced cells in all patients treated. In one patient a cytomegalovirus-reactivation was treated by the transfusion of gene-modified donor T-cells also indicating effectiveness of treatment. To date, 5 patients have relapsed, and died, one after a second HSCT. Two of the patients without transduced T-cells died due to relapse and 7 patients are alive and well and in complete clinical remission. Thus we have shown that transfusion of transduced T-cells is effective and safe in a long follow-up of 11 years post transfusion. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction: The combination treatment of venetoclax (VEN) with both low-dose cytarabine (LDAC) and hypomethylating agents (HMA) in untreated primarily elderly AML patients yielded promising response rates leading to its approval for newly diagnosed AML patients who are 75 years or older, or who have comorbidities that preclude use of intensive induction chemotherapy. Prolonged cytopenias are of potential concern in venetoclax treated patients, especially in patients who underwent allogeneic hematopoietic cell transplantation (alloHCT) prior venetoclax treatment. Objective: To compare hematologic recovery in patients treated with VEN in combination with intensive and non-intensive chemotherapy regimens for the treatment of relapsed or refractory (R/R) acute myeloid leukemia (AML) depending on the pretreatment status for alloHCT. Methods: In this retrospective controlled study (www.clinicaltrials.gov NCT03662724), we included patients aged 18 years or older with R/R acute leukemia previously treated with VEN (days 1-7) combined with intensive salvage chemotherapy (fludarabine, cytarabine, idarubicin - FLAVIDA) or VEN combined with non-intensive regimens, namely HMA or LDAC. Eighty-one patients who were treated with FLA-IDA for R/R AML served as control for the intensively treated patients included in this analysis. Responses were evaluated per revised International Working Group criteria for AML. Main outcome measure was the rate of objective response (complete remission [CR] + CR with incomplete blood count recovery [CRi] + partial remission [PR] + morphologic leukemia-free state (MLFS; defined as less than 5% blasts in an aspirate sample). Safety and efficacy analyses included all patients who received at least one cycle of VEN combination treatment. This study was approved by the local Ethics Review Committee in accordance with the Declaration of Helsinki. Results: Between January 2017 and May 2019 49 patients with a median age of 59 years (range 18-80) received VEN with either FLA-IDA (n=14), HMA (n=31) or LDAC (n=4) and had safety and efficacy outcomes reported. The patient cohort was a high-risk cohort of relapsed (n=24) and refractory (n=25) patients. The analysis included 24 patients (49%) with secondary AML and two patients with biphenotypic acute leukemia (BAL). Twenty-two patients (45%) had received prior alloHCT and 7 (14%) had relapsed

Description: Background and Aim: Deletion of 5q is the most frequent cytogenetic aberration in MDS and is associated with distinct clinical characteristics, disease course and sensitivity to lenalidomide. The serine-threonine kinase CSNK1A1 is located in the commonly deleted region at 5q32 and has been described as a tumor-suppressor gene in colon cancer and acute myeloid leukemia through regulation of ß-catenin and p53. Recently, missense mutations in CSNK1A1 have been described in individual patients with del(5q) MDS. The aim of our study was to characterize the frequency and potential prognostic impact of CSNK1A1mutations in MDS and AML following MDS. Methods: 192 patients with MDS or AML following MDS (sAML) and deletion of chromosome 5q and 406 patients with MDS/sAML without deletion of chromosome 5q were included in the current analysis (n=598 in total). Patients with MDS (n=442) or sAML (n=156) were cytogenetically characterized by chromosome banding analysis and molecularly analyzed for mutations in exon 3 and 4 of CSNK1A1, the region critical for the kinase function, by Sanger sequencing. Patients with mutated CSNK1A1 were also analyzed for mutations in TP53 by next-generation or Sanger sequencing. Results: CSNK1A1 mutations were found in 17 (8.9%) of 192 MDS patients with del(5q). The mutation frequency was similar between patients with isolated del(5q) (n=153) and patients with concurrent cytogenetic aberrations or missing additional cytogenetic information (n=39)(9.2% vs 7.7%, P=.7). No mutation of CSNK1A1 was found in any of 406 MDS/sAML patients without del(5q). Thirteen patients (76%) had missense mutations affecting amino acid E98 in exon 3 of CSNK1A1. Of these, the glutamic acid to lysin substitution was the most frequent amino acid substitution (n=7). All mutations of glutamic acid 98 had a high probability to be damaging to the protein based on PolyPhen2 predictions (scores 0.922 to1). One patient had an Asn86Tyr mutation concurrently with the Glu98Ala mutation. Four patients (24%) had missense mutations affecting aspartic acid 140 in exon 4 of CSNK1A1. These mutations had moderate PolyPhen2 prediction scores (0.558-0.798). Three of the 17 CSNK1A1 mutated patients had additional cytogenetic aberrations besides del(5q), i.e. one trisomy 8, one trisomy 11, and one monosomy 7. None of the CSNK1A1 patients had a concurrent TP53 mutation. Del(5q) patients with wildtype or mutated CSNK1A1 had a similar median age (73.3 vs 77.5 years, P=.15). 70% and 59% of wildtype and mutated CSNK1A1 patients had female sex, respectively (P=.33). The WBC count was similar between wildtype and mutated CSNK1A1patients (3.9 vs 4.6, P=.47). Survival information was available for 155 patients with del(5q) (81%) including 16 patients (94%) with mutated CSNK1A1. Median follow-up from the time of sample harvest was 2.02 years. The probability of survival at 2 years was 41% for CSNK1A1 mutated and 72% for CSNK1A1wildtype patients (P=.059, log-rank test), suggesting a potential negative prognostic impact of CSNK1A1 mutations in del(5q) MDS patients. Conclusion: CSNK1A1 mutations are highly specific for MDS patients with del(5q) and are one of the most frequent recurrent genetic aberrations in these patients. Our survival analysis suggests that CSNK1A1 mutations have an unfavorable prognostic effect in patients with MDS and del(5q); however, the prognostic impact has to be confirmed in additional patients. Mutation analysis of exon 3 and 4 of CSNK1A1 should be included in the routine workup of MDS patients with deletion of 5q. Disclosures Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Kobbe:Celgene: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Medac: Other; Astellas: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Neovii: Other. Haferlach:MLL: Equity Ownership.