Lisa Pleyer

TCL1 mice are the most commonly used preclinical model for chronic lymphocytic leukemia (CLL), a B-cell malignancy characterized by clonal CD5 ⁺ B-lymphocyte accumulation. B-cell receptor (BCR) sequencing identifies two important risk markers, immunoglobulin heavy chain variable region (IGHV) mutational status and receptor stereotypy. Despite the clinical importance in patients, no broad examinations of BCR repertoires have been conducted in TCL1 mice. We analysed BCR repertoires of 85 TCL1 mice, primarily comprising CLL clones using IGHV1 and IGHV11 (27.3% and 49.1% of CLL clones, respectively). Interestingly, TCL1 mice with dominant IGHV1 CLL clones showed significantly higher levels of CD4 ⁺ T-cells, and increased exhaustion levels (PD-1) on splenic CD8 ⁺ T-cells compared to IGHV11 CLL clones. Furthermore, cancer related pathways (p53, MTORC and KRAS) were distinctly regulated in IGHV1 CLL clones which also exhibited significantly decreased survival (hazard ratio 2.6) in female mice. Additionally, mice with dominant IGHV1 CLL clones displayed an almost twofold inguinal lymph node enlargement. In conclusion, we identified molecular, phenotypical and immunological differences between IGHV1 and IGHV11 CLL clones, which are key to consider for preclinical studies using the TCL1 mouse model. Furthermore, our data suggests that IGHV1 CLL clones model the nodal form of human CLL.

Chronic myelomonocytic leukemia (CMML) is a heterogeneous disease presenting with either myeloproliferative or myelodysplastic features. Allogeneic hematopoietic cell transplantation (allo-HCT) remains the only potentially curative option, but the inherent toxicity of this procedure makes the decision to proceed to allo-HCT challenging, particularly because patients with CMML are mostly older and comorbid. Therefore, the decision between a nonintensive treatment approach and allo-HCT represents a delicate balance, especially because prospective randomized studies are lacking and retrospective data in the literature are conflicting. International consensus on the selection of patients and the ideal timing of allo-HCT, specifically in CMML, could not be reached in international recommendations published 6 years ago. Since then, new, CMML-specific data have been published. The European Society for Blood and Marrow Transplantation (EBMT) Practice Harmonization and Guidelines (PH&G) Committee assembled a panel of experts in the field to provide the first best practice recommendations on the role of allo-HCT specifically in CMML. Recommendations were based on the results of an international survey, a comprehensive review of the literature, and expert opinions on the subject, after structured discussion and circulation of recommendations. Algorithms for patient selection, timing of allo-HCT during the course of the disease, pretransplant strategies, allo-HCT modality, as well as posttransplant management for patients with CMML were outlined. The keynote message is, that once a patient has been identified as a transplant candidate, upfront transplantation without prior disease-modifying treatment is preferred to maximize chances of reaching allo-HCT whenever possible, irrespective of bone marrow blast counts.

The current gold standard of response assessment in patients with myelodysplastic syndromes (MDS), chronic myelomonocytic leukemia (CMML), and acute myeloid leukemia (AML) is morphologic complete remission (CR) and CR with incomplete count recovery (CRi), both of which require an invasive BM evaluation. Outside of clinical trials, BM evaluations are only performed in similar to 50% of patients during follow-up, pinpointing a clinical need for response endpoints that do not necessitate BM assessments. We define and validate a new response type termed "peripheral blood complete remission" (PB-CR) that can be determined from the differential blood count and clinical parameters without necessitating a BM assessment. We compared the predictive value of PB-CR with morphologic CR/CRi in 1441 non-selected, consecutive patients diagnosed with MDS (n = 522; 36.2%), CMML (n = 132; 9.2%), or AML (n = 787; 54.6%), included within the Austrian Myeloid Registry (aMYELOIDr; NCT04438889). Time-to-event analyses were adjusted for 17 covariates remaining in the final Cox proportional hazards (CPH) model. DeepSurv, a CPH neural network model, and permutation-based feature importance were used to validate results. 1441 patients were included. Adjusted median overall survival for patients achieving PB-CR was 22.8 months (95%CI 18.9-26.2) versus 10.4 months (95%CI 9.7-11.2) for those who did not; HR = 0.366 (95%CI 0.303-0.441; p <.0001). Among patients achieving CR, those additionally achieving PB-CR had a median adjusted OS of 32.6 months (95%CI 26.2-49.2) versus 21.7 months (95%CI 16.9-27.7; HR = 0.400 [95%CI 0.190-0.844; p =.0161]) for those who did not. Our deep neural network analysis-based findings from a large, prospective cohort study indicate that BM evaluations solely for the purpose of identifying CR/CRi can be omitted.

Background: Next generation sequencing (NGS) has become indispensable for diagnosis, risk stratification, prognostication, and monitoring of response in patients with myeloid neoplasias. Guidelines require bone marrow evaluations for the above, which are often not performed outside of clinical trials, indicating a need for surrogate samples. Methods: Myeloid NGS analyses (40 genes and 29 fusion drivers) of 240 consecutive, non-selected, prospectively collected, paired bone marrow/peripheral blood samples were compared. Findings: Very strong correlation (r = 0.91, p < 0.0001), high concordance (99.6%), sensitivity (98.8%), specificity (99.9%), positive predictive value (99.8%), and negative predictive value (99.6%) between NGS analyses of paired samples was observed. A total of 9/1321 (0.68%) detected mutations were discordant, 8 of which had a variant allele frequency (VAF) ≤ 3.7%. VAFs between peripheral blood and bone marrow samples were very strongly correlated in the total cohort (r = 0.93, p = 0.0001) and in subgroups without circulating blasts (r = 0.92, p < 0.0001) or with neutropenia (r = 0.88, p < 0.0001). There was a weak correlation between the VAF of a detected mutation and the blast count in either the peripheral blood (r = 0.19) or the bone marrow (r = 0.11). Interpretation: Peripheral blood samples can be used to molecularly classify and monitor myeloid neoplasms via NGS without loss of sensitivity/specificity, even in the absence of circulating blasts or in neutropenic patients.

In this prospective study (NCT01595295), 272 patients treated with azacitidine completed 1456 EuroQol 5-Dimension (EQ-5D) questionnaires. Linear mixed-effect modelling was used to incorporate longitudinal data. When compared with a matched reference population, myeloid patients reported more pronounced restrictions in usual activities (+28%, p < 0.0001), anxiety/depression (+21%, p < 0.0001), selfcare (+18%, p < 0.0001) and mobility (+15%, p < 0.0001), as well as lower mean EQ-5D-5L indices (0.81 vs. 0.88, p < 0.0001), and lower self-rated health on the EuroQol Visual Analogue Scale (EQ-VAS) (64 vs. 72%, p < 0.0001). After multivariate-adjustment, (i) the EQ-5D-5L index assessed at azacitidine start the predicted time with clinical benefit (TCB) (9.6 vs. 6.6 months; p = 0.0258; HR = 1.43), time to next treatment (TTNT) (12.8 vs. 9.8 months; p = 0.0332; HR = 1.42) and overall survival (OS) (17.9 vs. 12.9 months; p = 0.0143; HR = 1.52); (ii) Level Sum Score (LSS) predicted azacitidine response (p = 0.0160; OR = 0.451) and the EQ-5D-5L index showed a trend (p = 0.0627; OR = 0.522); (iii) up to 1432 longitudinally assessed EQ-5D-5L response/clinical parameter pairs revealed significant associations of EQ-5D-5L response parameters with haemoglobin level, transfusion dependence and hematologic improvement. Significant increases of the likelihood ratios were observed after addition of LSS, EQ-VAS or EQ-5D-5L-index to the International Prognostic Scoring System (IPSS) or the revised IPSS (R-IPSS), indicating that they provide added value to these scores.

Acute myeloid leukemia (AML) is a hematopoietic malignancy characterized by altered myeloid progenitor cell proliferation and differentiation. As in many other cancers, epigenetic transcriptional repressors such as histone deacetylases (HDACs) are dysregulated in AML. Here, we investigated (1) HDAC gene expression in AML patients and in different AML cell lines and (2) the effect of treating AML cells with the specific class IIA HDAC inhibitor TMP269, by applying proteomic and comparative bioinformatic analyses. We also analyzed cell proliferation, apoptosis, and the cell-killing capacities of TMP269 in combination with venetoclax compared to azacitidine plus venetoclax, by flow cytometry. Our results demonstrate significantly overexpressed class I and class II HDAC genes in AML patients, a phenotype which is conserved in AML cell lines. In AML MOLM-13 cells, TMP269 treatment downregulated a set of ribosomal proteins which are overexpressed in AML patients at the transcriptional level. TMP269 showed anti-proliferative effects and induced additive apoptotic effects in combination with venetoclax. We conclude that TMP269 exerts anti-leukemic activity when combined with venetoclax and has potential as a therapeutic drug in AML.

Background: Azacitidine is the treatment backbone for patients with acute myeloid leukemia, myelodysplastic syndromes and chronic myelomonocytic leukemia who are considered unfit for intensive chemotherapy. Detailed reports on adverse events in a real-world setting are lacking. Aims: To analyze the frequency of adverse events in the Austrian Registry of Hypomethylating agents. To compare real-world data with that of published randomized clinical trials. Results: A total of 1406 patients uniformly treated with a total of 13,780 cycles of azacitidine were analyzed. Hematologic adverse events were the most common adverse events (grade 3–4 anemia 43.4%, grade 3–4 thrombopenia 36.8%, grade 3–4 neutropenia 36.1%). Grade 3–4 anemia was significantly more common in the Registry compared to published trials. Febrile neutropenia occurred in 33.4% of patients and was also more common in the Registry than in published reports. Other commonly reported adverse events included fatigue (33.4%), pain (29.2%), pyrexia (23.5%), and injection site reactions (23.2%). Treatment termination due to an adverse event was rare (5.1%). Conclusion: The safety profile of azacitidine in clinical trials is reproducible in a real-world setting. With the use of prophylactic and concomitant medications, adverse events can be mitigated and azacitidine can be safely administered to almost all patients with few treatment discontinuations.

Acute myeloid leukemia (AML) cells can secrete trophic factors, including extracellular vesicles (EVs), instructing the stromal leukemic niche. Here, we introduce a scalable workflow for purification of immunomodulatory AML-EVs to compare their phenotype and function to the parental AML cells and their secreted soluble factors. AML cell lines HL-60, KG-1, OCI-AML3, and MOLM-14 released EVs with a peak diameter of approximately 80 nm in serum-free particle-reduced medium. We enriched EVs >100x using tangential flow filtration (TFF) and separated AML-derived soluble factors and cells in parallel. EVs were characterized by electron microscopy, immunoblotting, and flow cytometry, confirming the double-membrane morphology, purity and identity. AML-EVs showed significant enrichment of immune response and leukemia-related pathways in tandem mass-tag proteomics and a significant dose-dependent inhibition of T cell proliferation, which was not observed with AML cells or their soluble factors. Furthermore, AML-EVs dose-dependently reduced NK cell lysis of third-party K-562 leukemia targets. This emphasizes the peculiar role of AML-EVs in leukemia immune escape and indicates novel EV-based targets for therapeutic interventions.