Akute myeloische Leukämie II
V544 C-type like lectin molecule 1 (CLL-1)-spezifische chimäre Antigen-Rezeptor (CAR) exprimierende T-Zellen für die Behandlung der akuten myeloischen Leukämie (AML)
Nicola Schanda, Heidelberg, D
Introduction: The application of genetically modified T cells expressing chimeric antigen receptors (CARTs) has emerged as promising treatment approach in particular for patients with B-cell malignancies. However, similar progress has not yet been achieved in the treatment of acute myeloid leukemia (AML). C-type lectin-like molecule 1 (CLL-1) is expressed on leukemic bulk and stem cells of most AML patients, but absent on normal hematopoietic stem cells, indicating the therapeutic potential of CLL-1-targeting CAR T-cells in AML treatment. The aim of this project was to evaluate the anti- leukemic efficacy of different CLL-1-specific CAR constructs against AML in vitro.
Methods: Using a published CLL1-specific single chain variable fragment (scFv), we generated a panel of CLL-1-CARs, that consisted of CD28 as a co-stimulatory domain, the TCR derived CD3z chain and one of four different spacers (short, intermediate, long and long-flexible), that differed in size and structural composition. After retroviral transduction of activated T cells, derived from peripheral blood mononuclear cells of healthy donors, transduction efficacy, phenotype and expression of markers associated with T cell exhaustion of all CLL-1-CART products were evaluated by flow cytometry. Short- term cytotoxicity was measured by a chromium-51 release assay whereas long-term killing and expansion capacity upon repeated antigen stimulation by CLL-1-positive tumor cells was evaluated in a serial coculture assay.
Results: CLL-1-CARTs were efficiently generated for all different CAR constructs. While transduction efficiency was over 90% in all constructs, the short-spacer CAR was expressed at a higher mean fluorescence intensity and also showed the lowest expansion rate prior to antigen stimulation. While all CLL-1-CARTs expanded and efficiently eliminated tumor cells in an antigen-dependent manner, even when repeatedly challenged with tumor cells, outgrowth of AML cells occurred at first in the long-spacer CARTs after the second coculture. Analysis of exhaustion and phenotype markers (TIM3, LAG3, PD-1) revealed a comparable expression profile in all constructs.
Conclusion: CLL-1-CARTs exhibited potent anti-leukemic activity against AML in vitro. While functional differences between spacer compositions were detectable in the serial co-culture assay, in vivo studies are ongoing to identify the most efficient construct for early-phase clinical testing in patients with CLL-1 positive AML.
V333 Überwinden von Therapieresistenz in FLT3 Wildtyp akuter myeloischer Leukämie durch eine neue Venetoclax-Kombinationsstrategie mit Gilteritinib
Christina Schmidt, Heidelberg, D
Introduction: Venetoclax-based treatment strategies are effective in elderly AML patients, but upfront resistance as well as relapse following initial response remain major challenges.
Methods: We performed high throughput drug screening to identify the most effective venetoclax combination partners by analyzing venetoclax in combination with 64 different drugs on primary blasts from 30 high-risk AML patients. We validated our findings in sensitive and resistant cell lines and carried out mechanistic investigations by proteomics and immunoblotting.
Results: Drugs directly or indirectly targeting the anti-apoptotic protein Mcl-1 showed strong effects on the viability of AML cells when combined with venetoclax. Among these drugs, gilteritinib appeared as an attractive combination partner for venetoclax with high potential of fast clinical translation. The combination drug screen identified gilteritinib and venetoclax as a highly efficacious and synergistic combination in FLT3 wildtype AML. In cell lines, the combination of gilteritinib and venetoclax increased apoptosis, reduced viability, and diminished colony-formation capacity. The combination treatment led to decreased phosphorylation of the signaling protein ERK as well as to downregulation of the anti- apoptotic protein Mcl-1. The latter was due to increased phosphorylation at serine 159 which was associated with proteasomal degradation. The combination of venetoclax and gilteritinib overcame resistance in cell lines insensitive to venetoclax and azacytidine as well as in AML samples of patients with refractory disease.
Conclusion: In summary, our results identified gilteritinib as a potential therapy combination partner for venetoclax in FLT3 wildtype AML patients.
V656 Venetoclax in Kombination mit HMA oder LDAC in der rezidiverten/refraktären AML - Erfahrung aus zwei universitären Zentren in Deutschland
Krischan Braitsch, München, D
Introduction: Patients with relapsed/refractory acute myeloid leukemia (r/r AML) or patients unfit for intensive therapy are challenging to treat. Venetoclax (VEN) combined with hypomethylating agents (HMA) has shown encouraging results in unfit AML patients. We conducted a retrospective study of 71 AML patients treated with VEN between 2018 and 2021 at two university hospital sites with the aim to assess clinical characteristics and outcome.
Methods: Baseline patient characteristics including genetics, previous treatment lines, courses of VEN treatment and outcome data were collected by medical chart review. The median time of follow-up was 7.2 months.
Results: The median age at beginning of VEN treatment was 74 (21-85) years. Most patients [n=32 (45%)] were assigned to the adverse ELN2017 risk group. The majority of patients [n=56 (79%)] received prior treatment before VEN including intensive chemotherapy [n=29 (41%)] and allogeneic stem cell transplantation [n=26 (37%)]. Treatment with >4 cycles HMA/low-dose cytarabine (LDAC) was applied in n=25 (35%) patients before VEN combination therapy. In n=14 patients (20%) VEN was started during the first or second treatment cycle. The median initial VEN dosage was 400mg (50-800mg); VEN was combined with azacytidine [n=32 (45%)], decitabine [n=18(25%)] or LDAC [n=20 (28%)]. VEN was started after progression to HMA/LDAC treatment in n=35 (49%) patients. In 60% of patients, VEN was discontinued or dose-adjusted during treatment. Among patients with an evaluable response [n=56 (79%)], 16 (29%) achieved complete remission (CR) or CR with incomplete hematologic recovery (CRi), 24 (43%) had a partial remission (PR) or stable disease (SD), and 16 (29%) were refractory. The median overall survival (OS) was 5.4 months. OS was highly influenced by remission status (Figure 1A) and significantly longer in patients with less than 2 prior treatment lines (Figure 1 B). HMA pretreatment for up to 4 cycles before VEN initiation had no effect on OS in our cohort. Conclusions: Our real-world data show that VEN combinations are feasible treatment regimens also for heavily pretreated AML patients. Further studies with larger cohorts are warranted.
V661 Primäre natürliche Killer-Zellen, die einen C-type like lectin molecule 1 (CLL-1)-spezifischen chimären Antigen-Rezeptor exprimieren, zeigen eine potente antileukämische Aktivität gegen akute myeloische Leukämie (AML)
David Sedloev, Heidelberg, D
Introduction: Acute myeloid leukemia (AML) is an aggressive hematological malignancy associated with a poor prognosis, particularly in patients with relapsed and refractory disease. Natural killer (NK) cell-based immunotherapy is emerging as promising component of cancer treatment. However, in AML patients, responses to unmodified allogeneic NK cell products are only moderate. C-type like lectin molecule 1 (CLL-1) is a promising surface antigen for targeted therapy of AML as it is expressed on the majority of AML patients. In contrast, expression is low or even absent on healthy hematopoietic stem and progenitor cells. The goal of our study was to investigate whether CLL-1-specific chimeric antigen receptor (CAR) expression can enhance the functionality of activated allogeneic NK cells (NKs).
Methods: Activated NKs from eleven healthy donors were transduced with a panel of second- generation CLL-1-specific CAR constructs that all contained a CD28 co-stimulatory and the the CD3Zeta chain but differed in size and structural composition of the extracellular spacer domain. After successful generation of CLL1-CAR NK cells by retroviral transduction, we assessed the stability of CAR expression during expansion. Further, short- and long-term in-vitro cytotoxicity, proliferation and cytokine secretion were analyzed.
Results: Using a gamma-retroviral vector system, activated NKs from all donors could be efficiently transduced with all CAR constructs (>90% CAR-positive cells for all conditions) and CAR expression remained stable at high levels during NK cell expansion. CLL1-CAR-NKs and non-transduced NKs (NT- NKs) both eliminated AML cells lines in short-term killing assays, but only CLL1-CAR NKs showed long- term control of tumor cell growth upon serial stimulation with tumor cells. Increased anti-tumor efficacy of CLL1-CAR NKs compared to NT-NKs was associated with significantly increased cell expansion and higher levels of cytokine secretion . NKs expressing a CAR containing a long spacer with additional flexibility mediated superior cytotoxic functionality and expansion compared to other constructs. Conclusion: CLL1-specific CAR-NKs exhibit potent anti-tumor efficacy against primary AML cell lines in- vitro and in-vivo studies to confirm these promising results are ongoing. Further, viability and functional capacity of CLL1-CAR constructs are strongly influenced by their structural composition.
V301 Anwendung und Nutzen der RNA-Sequenzierung im diagnostischen Umfeld
Claudia Haferlach, München, D
Introduction: RNA sequencing (RNAseq) has become a powerful tool for genome wide fusion transcript detection and gene expression analyses outperforming targeted assays. The detected transcriptional events are often crucial for risk stratification, MRD monitoring and targeted therapy in hematological malignancies.
Methods: RNAseq was performed for 572 AML, 279 ALL and 46 patients with eosinophilia for the detection of fusion transcripts. 101 bp paired-end reads were produced on a NovaSeq 6000 system (Illumina, San Diego, CA) with a yield between 35-125 million paired reads per sample.
Results: In AML, RNAseq was applied for detection of fusion transcripts with potential therapeutic implications in cases so far lacking entity-defining abnormalities (as assessed by standard cytogenetics and/or molecular genetics). In 54/264 (20%) of these cases a fusion was detected, comprising 94 novel fusions, some involving genes that are potential targets for personalized therapy (e.g. RBPMS-FGFR1). In ALL, RNAseq was used for classification of cases into fusion-defined subgroups, as 97% of recurrent risk- stratifying fusions could be identified, assigning 76/279 samples to their respective entities. Similar to AML, 57 novel fusions were detected which might have diagnostic and/or therapeutic potential. Moreover, RNAseq detected a number of cryptic fusions in ALL (e.g. SET-NUP214, ABL1-NUP214) or fusions which were missed by conventional cytogenetics due to e.g. insufficient cell proliferation (e.g. PAX5-JAK2, KMT2A-USP2). In patients with eosinophilia, RNASeq aimed at detecting the heterogenous landscape of all different fusion transcripts, known for myeloid/lymphoid neoplasm with eosinophilia. In 33/46 patients the diagnosis was based on an entity-defining fusion transcript. Furthermore, in two samples with eosinophilia novel fusion genes could be detected (PTPRC-MPL, TNIP1-PFGRB), leading to the diagnosis of a clonal disease, but also offering potential treatment options.
Conclusions: We conclude that RNAseq is a valuable diagnostic tool for either correct subclassification of cases and for identification of gene fusions with prognostic and/or therapeutic impact. It furthermore has the advantage that these analyses can be performed “transcriptome wide”. In particular in cases so far lacking entity-defining or therapeutically targetable genetic abnormalities using standard methods or in cases in which conventional methods failed, RNAseq is superior to these techniques.