Comparative Analyses of Targeted Myeloid Cancer Next-Generation Sequencing Panel in Fresh Blood, Bone Marrow and FFPE Material.

Next-generation sequencing is a vital tool for personalized diagnostics and therapies in cancer. Despite numerous advantages, the method depends on multiple parameters regarding the sample material, e.g., sample fixation. A panel's ability to ensure balanced pre-amplification of the regions of interest is challenging, especially in targeted sequencing approaches, but of significant importance to its applicability across hematological malignancies and solid tumors. This study comparatively evaluated the technical performance of the commercially available OncomineTM Myeloid Panel in fresh and Formalin-fixed paraffin-embedded (FFPE) material by using an Ion Torrent™ Personal Genome Machine™ System and Ion GeneStudio S5 System platform. In total, 114 samples were analyzed, including 55 fresh materials and 59 FFPE samples. Samples were sequenced with a minimum of one million reads. Amplicons with coverage below 400 reads were classified as underperforming. In fresh material, 49/526 amplicons were identified as performing insufficiently, corresponding with 18 genes. Using FFPE material, 103/526 amplicons underperformed. Independent of input material, regions in 27 genes, including ASXL1, BCOR and BRAF, did not match quality parameters. Subsequently, exemplary mutations were extracted from the Catalogue of Somatic Mutations in Cancer database. This technical evaluation of the OncomineTM Myeloid Panel identified amplicons that do not achieve adequate coverage levels and which need to be considered when interpreting sequencing.

Different treatment strategies versus a common standard arm (CSA) in patients with newly diagnosed AML over the age of 60 years: a randomized German inter-group study.

A randomized inter-group trial comparing more intensive treatment strategies to a common standard arm 3 + 7 (CSA) was conducted in patients with non-M3 AML. Untreated patients ≥ 60 years were allocated to the CSA (n = 132) or to the study group arms (n = 1154) of the AMLCG (TAD/HAM versus HAM/HAM ± G-CSF followed by TAD and maintenance) and the OSHO (intermediate-dose ara-C/mitoxantrone followed by ara-C/mitoxantrone). Median age of the 1147 eligible patients was 69 (range 60-87) years. CR/CRi status at 90 days was not significantly different between the CSA (54% (95%CI: 45-64)) and the study group arms (53% (95%CI: 47-60) and 59% (95%CI: 58-63)). The five-year event-free survival (EFS) probability (primary endpoint) was 6.2% (95%CI: 2.7-14.0) in the CSA, 7.6% (95%CI: 4.5-12.8) in study group A and 11.1% (95%CI: 9.0-13.7) in B. The 5-year OS was 17.2% (95%CI: 11.0-26.9), 17.0% (95%CI: 2.0-23.9), and 19.5% (95%CI: 16.7-22.8) in CSA, study group A and B, respectively. Neither study group differed significantly from the CSA regarding EFS, OS, or relapse-free survival. In multivariate analyses, allocation to the treatment strategy was not significantly associated with the time-to-event endpoints. The evaluation of more intensive treatment strategies did not show clinically relevant outcome differences when compared to CSA.

Addendum to the German Consensus Recommendations on Ponatinib in the Treatment of Chronic Myeloid Leukemia.

BACKGROUND: Based on the new data from the primary analysis of the OPTIC (Optimizing Ponatinib Treatment in CP-CML) trial on dose optimization of ponatinib in patients with chronic phase (CP)-CML, the German consensus paper on ponatinib published in 2020 (Saussele S et al., Acta Haematol. 2020) has been updated in this addendum. SUMMARY: Focus is on the update of efficacy and safety of ponatinib, reflecting the new data set, as well as the update of the benefit-risk assessment and recommendations for ponatinib starting dose in CP-CML - provided that the decision to use ponatinib has already been made. Furthermore, based on OPTIC and additional empirical data, the expert panel collaborated to develop a decision tree for ponatinib dosing, specifically for intolerant and resistant patients. The recommendations on cardiovascular management have also been updated based on the most recent 2021 guidelines of the European Society of Cardiology (ESC) on cardiovascular disease prevention in clinical practice. KEY MESSAGES: The OPTIC data confirm the high efficacy of ponatinib in patients with CP-CML and provide the basis for individualized dose adjustment during the course of treatment.

Uncoupling protein 2 deficiency of non-cancerous tissues inhibits the progression of pancreatic cancer in mice.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a disease of the elderly mostly because its development from preneoplastic lesions depends on the accumulation of gene mutations and epigenetic alterations over time. How aging of non-cancerous tissues of the host affects tumor progression, however, remains largely unknown. METHODS: We took advantage of a model of accelerated aging, uncoupling protein 2-deficient (Ucp2 knockout, Ucp2 KO) mice, to investigate the growth of orthotopically transplanted Ucp2 wild-type (WT) PDAC cells (cell lines Panc02 and 6606PDA) in vivo and to study strain-dependent differences of the PDAC microenvironment. RESULTS: Measurements of tumor weights and quantification of proliferating cells indicated a significant growth advantage of Panc02 and 6606PDA cells in WT mice compared to Ucp2 KO mice. In tumors in the knockout strain, higher levels of interferon-γ mRNA despite similar numbers of tumor-infiltrating T cells were observed. 6606PDA cells triggered a stronger stromal reaction in Ucp2 KO mice than in WT animals. Accordingly, pancreatic stellate cells from Ucp2 KO mice proliferated at a higher rate than cells of the WT strain when they were incubated with conditioned media from PDAC cells. CONCLUSIONS: Ucp2 modulates PDAC microenvironment in a way that favors tumor progression and implicates an altered stromal response as one of the underlying mechanisms.

Combined BCL-2 and PI3K/AKT Pathway Inhibition in KMT2A-Rearranged Acute B-Lymphoblastic Leukemia Cells.

Numerous hematologic neoplasms, including acute B-lymphoblastic leukemia (B-ALL), are characterized by overexpression of anti-apoptotic BCL-2 family proteins. Despite the high clinical efficacy of the specific BCL-2 inhibitor venetoclax in acute myeloid leukemia (AML) and chronic lymphocytic leukemia (CLL), dose limitation and resistance argue for the early exploration of rational combination strategies. Recent data indicated that BCL-2 inhibition in B-ALL with KMT2A rearrangements is a promising intervention option; however, combinatorial approaches have not been in focus so far. The PI3K/AKT pathway has emerged as a possible target structure due to multiple interactions with the apoptosis cascade as well as relevant dysregulation in B-ALL. Herein, we demonstrate for the first time that combined BCL-2 and PI3K/AKT inhibition has synergistic anti-proliferative effects on B-ALL cell lines. Of note, all tested combinations (venetoclax + PI3K inhibitors idelalisib or BKM-120, as well as AKT inhibitors MK-2206 or perifosine) achieved comparable anti-leukemic effects. In a detailed analysis of apoptotic processes, among the PI3K/AKT inhibitors only perifosine resulted in an increased rate of apoptotic cells. Furthermore, the combination of venetoclax and perifosine synergistically enhanced the activity of the intrinsic apoptosis pathway. Subsequent gene expression studies identified the pro-apoptotic gene BBC3 as a possible player in synergistic action. All combinatorial approaches additionally modulated extrinsic apoptosis pathway genes. The present study provides rational combination strategies involving selective BCL-2 and PI3K/AKT inhibition in B-ALL cell lines. Furthermore, we identified a potential mechanistic background of the synergistic activity of combined venetoclax and perifosine application.

Exploring Thiazolopyridine AV25R: Unraveling of Biological Activities, Selective Anti-Cancer Properties and In Silico Target and Binding Prediction in Hematological Neoplasms.

Thiazolopyridines are a highly relevant class of small molecules, which have previously shown a wide range of biological activities. Besides their anti-tubercular, anti-microbial and anti-viral activities, they also show anti-cancerogenic properties, and play a role as inhibitors of cancer-related proteins. Herein, the biological effects of the thiazolopyridine AV25R, a novel small molecule with unknown biological effects, were characterized. Screening of a set of lymphoma (SUP-T1, SU-DHL-4) and B- acute leukemia cell lines (RS4;11, SEM) revealed highly selective effects of AV25R. The selective anti-proliferative and metabolism-modulating effects were observed in vitro for the B-ALL cell line RS4;11. Further, we were able to detect severe morphological changes and the induction of apoptosis. Gene expression analysis identified a large number of differentially expressed genes after AV25R exposure and significant differentially regulated cancer-related signaling pathways, such as VEGFA-VEGFR2 signaling and the EGF/EGFR pathway. Structure-based pharmacophore screening approaches using in silico modeling identified potential biological AV25R targets. Our results indicate that AV25R binds with several proteins known to regulate cell proliferation and tumor progression, such as FECH, MAP11, EGFR, TGFBR1 and MDM2. The molecular docking analyses indicates that AV25R has a higher binding affinity compared to many of the experimentally validated small molecule inhibitors of these targets. Thus, here we present in vitro and in silico analyses which characterize, for the first time, the molecular acting mechanism of AV25R, including cellular and molecular biologic effects. Additionally, this predicted the target binding of the molecule, revealing a high affinity to cancer-related proteins and, thus, classified AVR25 for targeted intervention approaches.

Cyclin-dependent kinase inhibitors in head and neck cancer and glioblastoma-backbone or add-on in immune-oncology?

Cyclin-dependent kinases (CDK) control the cell cycle and play a crucial role in oncogenesis. Pharmacologic inhibition of CDK has contributed to the recent clinical approval of dual CDK4/6 inhibitors for the treatment of breast and small cell lung cancer. While the anticancer cell effects of CDK inhibitors are well-established, preclinical and early clinical studies describe additional mechanisms of action such as chemo- and radiosensitization or immune stimulation. The latter offers great potential to incorporate CDK inhibitors in immune-based treatments. However, dosing schedules and accurate timing of each combination partner need to be respected to prevent immune escape and resistance. In this review, we provide a detailed summary of CDK inhibitors in the two solid cancer types head and neck cancer and glioblastoma multiforme; it describes the molecular mechanisms of response vs. resistance and covers strategies to avoid resistance by the combination of immunotherapy or targeted therapy.

Treosulfan compared with reduced-intensity busulfan improves allogeneic hematopoietic cell transplantation outcomes of older acute myeloid leukemia and myelodysplastic syndrome patients: Final analysis of a prospective randomized trial.

The phase III study was designed to compare event-free survival (EFS) after treosulfan-based conditioning with a widely applied reduced-intensity conditioning (RIC) busulfan regimen in older or comorbid patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) undergoing allogeneic hematopoietic cell transplantation (HCT). A previously reported confirmatory interim analysis of the randomized clinical study including 476 patients demonstrated statistically significant noninferiority for treosulfan with clinically meaningful improvement in EFS. Here, the final study results and pre-specified subgroup analyses of all 570 randomized patients with completed longer-term follow-up are presented. Patients presenting HCT-specific comorbidity index >2 or aged ≥50 years were randomly assigned (1:1) to intravenous (IV) fludarabine with either treosulfan (30 g/m2 IV) or busulfan (6.4 mg/kg IV) after stratification by disease risk group, donor type, and participating institution. The primary endpoint was EFS with disease recurrence, graft failure, or death from any cause as events. EFS of patients (median age 60 years) was superior after treosulfan compared to RIC busulfan: 36-months-EFS rate 59.5% (95% CI, 52.2-66.1) vs. 49.7% (95% CI, 43.3-55.7) with a hazard ratio (HR) of 0.64 (95% CI, 0.49-0.84), p = 0.0006. Likewise, overall survival (OS) with treosulfan was superior compared to busulfan: 36-month-OS rate 66.8% vs. 56.3%; HR 0.64 (95% CI, 0.48-0.87), p = 0.0037. Post hoc analyses revealed that these differences were consistent with the confirmatory interim analysis, and thereby the treosulfan regimen appears particularly suitable for older AML and MDS patients.

COVID-19: Challenges and solutions for the provision of care to seriously ill and dying people and their relatives during SARS-CoV-2 pandemic - perspectives of pandemic response team members: A qualitative study on the basis of expert interviews (part of PallPan).

BACKGROUND: During the SARS-CoV-2 pandemic's initial waves, bans on visiting and isolation measures placed limits on providing services for seriously ill and dying people and their relatives. Pandemic response teams at governmental level (macro), at federal state and municipal level (meso) and in healthcare facilities (micro) played their role in pandemic management procedures. AIM: To explore pandemic-related challenges and solutions of pandemic response teams regarding the provision of care to seriously ill and dying people and their relatives. Findings were to be integrated into a national strategy (PallPan). DESIGN: Semi-structured expert interviews (10/2020-2/2021) analysed via structured content analysis. SETTINGPARTICIPANTS: We interviewed 41 members, who discussed the work of 43 German pandemic response teams (micro n = 23; meso n = 20; no members were available at macro level) from 14 German federal states. RESULTS: Twenty-nine of 43 teams took account of the needs of seriously ill and dying. Their main challenges resulted from pandemic-related legal requirements in hospitals and long-term care facilities. The implementation of such was in the remits of the meso level. Dysfunctional or non-existent communication between the levels was reported to be challenging. To foster patient-related solutions the micro level pandemic response teams supported individual decisions to enable patient-relative contact for example, visiting and saying goodbye outside, meeting via digital solutions. CONCLUSIONS: Pandemic response teams evidently struggled to find appropriate solutions to ease pandemic-related impact on the care of seriously ill and dying patients and their relatives. We recommend bringing palliative care expertise on board.

[DNVF Memorandum: Health Services Research in the Last Year of Life]. / DNVF-Memorandum Versorgungsforschung im letzten Lebensjahr.

This memorandum outlines current issues concerning health services research on seriously ill and dying people in the last year of their lives as well as support available for their relatives. Patients in the last phase of life can belong to different disease groups, they may have special characteristics (e. g., people with cognitive and complex impairments, economic disadvantage or migration background) and be in certain phases of life (e. g., parents of minor children, (old) age). The need for a designated memorandum on health services research in the last year of life results from the special situation of those affected and from the special features of health services in this phase of life. With reference to these special features, this memorandum describes methodological and ethical specifics as well as current issues in health services research and how these can be adequately addressed using quantitative, qualitative and mixed methods. It has been developed by the palliative medicine section of the German Network for Health Services Research (DNVF) according to the guidelines for DNVF memoranda.

The Inhibitory Response to PI3K/AKT Pathway Inhibitors MK-2206 and Buparlisib Is Related to Genetic Differences in Pancreatic Ductal Adenocarcinoma Cell Lines.

The aberrant activation of the phosphoinositide 3-kinase (PI3K)/ protein kinase B (AKT) pathway is common in pancreatic ductal adenocarcinomas (PDAC). The application of inhibitors against PI3K and AKT has been considered as a therapeutic option. We investigated PDAC cell lines exposed to increasing concentrations of MK-2206 (an AKT1/2/3 inhibitor) and Buparlisib (a pan-PI3K inhibitor). Cell proliferation, metabolic activity, biomass, and apoptosis/necrosis were evaluated. Further, whole-exome sequencing (WES) and RNA sequencing (RNA-seq) were performed to analyze the recurrent aberrations and expression profiles of the inhibitor target genes and the genes frequently mutated in PDAC (Kirsten rat sarcoma virus (KRAS), Tumor protein p53 (TP53)). MK-2206 and Buparlisib demonstrated pronounced cytotoxic effects and limited cell-line-specific effects in cell death induction. WES revealed two sequence variants within the direct target genes (PIK3CA c.1143C > G in Colo357 and PIK3CD c.2480C > G in Capan-1), but a direct link to the Buparlisib response was not observed. RNA-seq demonstrated that the expression level of the inhibitor target genes did not affect the efficacy of the corresponding inhibitors. Moreover, increased resistance to MK-2206 was observed in the analyzed cell lines carrying a KRAS variant. Further, increased resistance to both inhibitors was observed in SU.86.86 carrying two TP53 missense variants. Additionally, the presence of the PIK3CA c.1143C > G in KRAS-variant-carrying cell lines was observed to correlate with increased sensitivity to Buparlisib. In conclusion, the present study reveals the distinct antitumor effects of PI3K/AKT pathway inhibitors against PDAC cell lines. Aberrations in specific target genes, as well as KRAS and TP53, individually or together, affect the efficacy of the two PI3K/AKT pathway inhibitors.

Inhibitory Response to CK II Inhibitor Silmitasertib and CDKs Inhibitor Dinaciclib Is Related to Genetic Differences in Pancreatic Ductal Adenocarcinoma Cell Lines.

Casein kinase II (CK2) and cyclin-dependent kinases (CDKs) frequently interact within multiple pathways in pancreatic ductal adenocarcinoma (PDAC). Application of CK2- and CDK-inhibitors have been considered as a therapeutic option, but are currently not part of routine chemotherapy regimens. We investigated ten PDAC cell lines exposed to increasing concentrations of silmitasertib and dinaciclib. Cell proliferation, metabolic activity, biomass, and apoptosis/necrosis were evaluated, and bioinformatic clustering was used to classify cell lines into sensitive groups based on their response to inhibitors. Furthermore, whole exome sequencing (WES) and RNA sequencing (RNA-Seq) was conducted to assess recurrent mutations and the expression profile of inhibitor targets and genes frequently mutated in PDAC, respectively. Dinaciclib and silmitasertib demonstrated pronounced and limited cell line specific effects in cell death induction, respectively. WES revealed no genomic variants causing changes in the primary structure of the corresponding inhibitor target proteins. RNA-Seq demonstrated that the expression of all inhibitor target genes was higher in the PDAC cell lines compared to non-neoplastic pancreatic tissue. The observed differences in PDAC cell line sensitivity to silmitasertib or dinaciclib did not depend on target gene expression or the identified gene variants. For the PDAC hotspot genes kirsten rat sarcoma virus (KRAS) and tumor protein p53 (TP53), three and eight variants were identified, respectively. In conclusion, both inhibitors demonstrated in vitro efficacy on the PDAC cell lines. However, aberrations and expression of inhibitor target genes did not appear to affect the efficacy of the corresponding inhibitors. In addition, specific aberrations in TP53 and KRAS affected the efficacy of both inhibitors.

Validation of an LC-MS/MS Method for the Quantification of the CK2 Inhibitor Silmitasertib (CX-4945) in Human Plasma.

Silmitasertib (CX-4945) is currently being investigated in clinical trials against various types of cancer. The U.S. Food and Drug Administration (FDA) has already granted orphan drug designation to the compound for the treatment of advanced cholangiocarcinoma, medulloblastoma, and biliary tract cancer. Silmitasertib inhibits the serine/threonine protein kinase CK2, which exerts a proliferation-promoting and anti-apoptotic effect on cancer cells. In view of current and future applications, the measurement of silmitasertib levels in plasma is expected to play an important role in the evaluation of therapeutic and toxic concentrations in cancer patients. In the present work, we therefore present an LC-MS/MS method for the quantification of silmitasertib in human plasma. Using a simple liquid-liquid extraction with ethyl acetate and a mixture of n-hexane and ethyl acetate, this method can be performed in any laboratory with mass spectrometry. The validation was carried out according to the FDA guideline.

A Simple LC-MS/MS Method for the Quantification of PDA-66 in Human Plasma.

The treatment of cancer is one of the most important pharmacotherapeutic challenges. To this end, chemotherapy has for some time been complemented by targeted therapies against specific structures. PDA-66, a structural analogue of the inhibitor of serine-threonine kinase glycogen synthase kinase 3ß SB216763, has shown preclinical antitumour effects in various cell lines, with the key pathways of its anticancer activity being cell cycle modulation, DNA replication and p53 signalling. For the monitoring of anticancer drug treatment in the context of therapeutic drug monitoring, the determination of plasma concentrations is essential, for which an LC-MS/MS method is particularly suitable. In the present study, a sensitive LC-MS/MS method for the quantification of the potential anticancer drug PDA-66 in human plasma with a lower limit of quantification of 2.5 nM is presented. The method was successfully validated and tested for the determination of PDA-66 in mouse plasma and sera.

[COVID-19 pandemic response teams: organization, competencies, and challenges-understanding and using structural realities]. / COVID-19-Pandemiekrisenstäbe: Organisation, Befugnisse und Herausforderungen ­ Strukturelle Gegebenheiten verstehen und nutzen.

BACKGROUND AND AIM: Germany has a federal state system. Pandemic response teams are key instruments of pandemic management. The aim of this article is to describe the structures and powers of pandemic response teams that were explored during a study on the care of the critically ill and dying in times of a pandemic (PallPan). The focus is on health-related pandemic response teams on the national state level (macrolevel) and federal and community level (mesolevel) as well as pandemic response teams in healthcare facilities (microlevel). METHODS: Members of pandemic response teams took part in qualitative semi-structured interviews (October 2020-February 2021). The evaluation was carried out by means of qualitative structuring content analysis. RESULTS: Forty-two persons reported on 43 crisis teams from 14 federal states. Response teams in healthcare facilities and public administration differ primarily with regard to their competencies. Officially predetermined regulations regarding the initiation, personal composition, tasks, responsibilities, and competencies of pandemic response teams are not predefined in Germany. The macrolevel defined the legal and financial conditions for pandemic management. Meso- and microlevel pandemic response teams bear responsibility for maintaining the provision of healthcare. The defaults of local public health authorities are decisive for the pandemic response team's work. Main tasks and measures were the provision of information and the procurement and distribution of resources. DISCUSSION: In terms of preparing for future pandemic situations, the knowledge gained will help to address concerns about maintaining healthcare for specific population groups, such as seriously ill and dying people, to the locally differing responsible bodies, even under pandemic conditions.

Combined vaccine-immune-checkpoint inhibition constitutes a promising strategy for treatment of dMMR tumors.

BACKGROUND: Mlh1-knock-out-driven mismatch-repair-deficient (dMMR) tumors can be targeted immunologically. By applying therapeutic tumor vaccination, tumor growth is delayed but escape mechanisms evolve, including upregulation of immune-checkpoint molecules (LAG-3, PD-L1). To counteract immune escape, we investigated the therapeutic activity of a combined tumor vaccine-immune-checkpoint inhibitor therapy using α-PD-L1. DESIGN: In this trial, Mlh1-knock-out mice with established gastrointestinal tumors received single or thrice injections of α-PD-L1 monoclonal antibody clone 6E11 (2.5 mg/kg bw, q2w, i.v.) either alone or in combination with the vaccine. Longitudinal flow cytometry and PET/CT imaging studies were followed by ex vivo functional immunological and gene expression assays. RESULTS: 6E11 monotherapy slightly increased median overall survival (mOS: 6.0 weeks vs. control 4.0 weeks). Increasing the number of injections (n = 3) improved therapy outcome (mOS: 9.2 weeks) and was significantly boosted by combining 6E11 with the vaccine (mOS: 19.4 weeks vs. 10.2 weeks vaccine monotherapy). Accompanying PET/CT imaging confirmed treatment-induced tumor growth control, with the strongest inhibition in the combination group. Three mice (30%) achieved a complete remission and showed long-term survival. Decreased levels of circulating splenic and intratumoral myeloid-derived suppressor cells (MDSC) and decreased numbers of immune-checkpoint-expressing splenic T cells (LAG-3, CTLA-4) accompanied therapeutic effects. Gene expression and protein analysis of residual tumors revealed downregulation of PI3K/Akt/Wnt-and TGF-signaling, leading to T cell infiltration, reduced numbers of macrophages, neutrophils and MDSC. CONCLUSIONS: By successful uncoupling of the PD-1/PD-L1 axis, we provide further evidence for the safe and successful application of immunotherapies to combat dMMR-driven malignancies that warrants further investigation.

Allogeneic hematopoietic stem cell transplantation improves long-term outcome for relapsed AML patients across all ages: results from two East German Study Group Hematology and Oncology (OSHO) trials.

Relapse of acute leukemia is a frequent complication with uncertain outcome and poorly defined risk factors. From 1621 patients entered into two prospective clinical trials (AML02; n = 740 and AML04; n = 881), 74.2% reached complete remission (CR) 1 after induction(s) and 59 patients after additional induction ± hematopoietic cell transplantation (HCT). Of the non-refractory patients, 48.4% with a median age of 63 (range 17-85) years relapsed. Relapses occurred within 6 months after CR in 46.5%, between 7 and 18 months in 38.7%, and after 18 months in 14.8% of patients. Relapse treatment resulted in CR2 in 39% of patients depending upon age (54.5% of ≤ 60 and 28.6% of > 60 years), duration of CR1, and treatment of relapse. Overall survival (OS) was 10.9 (7.4-16.2) %, but OS after HCT ± intensive chemotherapy (ICT) was 39.3% (31.8-48.6) at 5 years and not different in younger and older patients. Donor lymphocyte infusion ± chemotherapy and ICT alone resulted only in OS of 15.4% and of 5%, respectively. Independent favorable factors for OS were long CR1 duration, and HCT, while non-monosomal disease was beneficial for OS in elderly patients. Leukemia-free survival [LFS; 24.9 (19.5-31.7) % at 10 years] was affected by similar risk factors. In a competing risk model, the relapse incidence at 5 years was 53.5 ± 3.5% and the non-relapse mortality rate 21.7 ± 2.9%. Lower relapse incidence was observed in patents with HCT, long CR1 duration, and female gender. Risk factors for non-relapse mortality were HCT in younger and type of AML in elderly patients. In conclusion, allogeneic HCT ± IC improved the results in relapsed AML in younger and elderly patients. Increasing CR2 rates and HCT frequency will be the challenge for the next years. Relapse of the disease remains the major problem.

Precursor B-ALL Cell Lines Differentially Respond to SYK Inhibition by Entospletinib.

BACKGROUND: Impaired B-cell receptor (BCR) function has been associated with the progress of several B-cell malignancies. The spleen tyrosine kinase (SYK) represents a potential therapeutic target in a subset of B-cell neoplasias. In precursor B-acute lymphoblastic leukemia (B-ALL), the pathogenic role and therapeutic potential of SYK is still controversially discussed. We evaluate the application of the SYK inhibitor entospletinib (Ento) in pre- and pro-B-ALL cell lines, characterizing the biologic and molecular effects. METHODS: SYK expression was characterized in pre-B-ALL (NALM-6) and pro-B-ALL cell lines (SEM and RS4;11). The cell lines were exposed to different Ento concentrations and the cell biological response analyzed by proliferation, metabolic activity, apoptosis induction, cell-cycle distribution and morphology. BCR pathway gene expression and protein modulations were further characterized. RESULTS: Ento significantly induced anti-proliferative and pro-apoptotic effects in NALM-6 and SEM, while barely affecting RS4;11. Targeted RNAseq revealed pronounced gene expression modulation only in NALM-6, while Western Blot analyses demonstrated that vital downstream effector proteins, such as pAKT, pERK, pGSK3ß, p53 and BCL-6, were affected by Ento exposure in the inhibitor-sensitive cell lines. CONCLUSION: Different acting modes of Ento, independent of pre-BCR dependency, were characterized, unexpected in SEM. Accordingly, SYK classifies as a potential target structure in a subset of pro-B-ALLs.

Combined Gemcitabine and Immune-Checkpoint Inhibition Conquers Anti-PD-L1 Resistance in Low-Immunogenic Mismatch Repair-Deficient Tumors.

Tumors arising in the context of Lynch Syndrome or constitutional mismatch repair deficiency are hypermutated and have a good response towards immune-checkpoint inhibitors (ICIs), including α-PD-L1 antibodies. However, in most cases, resistance mechanisms evolve. To improve outcomes and prevent resistance development, combination approaches are warranted. Herein, we applied a combined regimen with an α-PD-L1 antibody and gemcitabine in a preclinical tumor model to activate endogenous antitumor immune responses. Mlh1-/- mice with established gastrointestinal tumors received the α-PD-L1 antibody (clone 6E11; 2.5 mg/kg bw, i.v., q2wx3) and gemcitabine (100 mg/kg bw, i.p., q4wx3) in mono- or combination therapy. Survival and tumor growth were recorded. Immunological changes in the blood were routinely examined via multi-color flow cytometry and complemented by ex vivo frameshift mutation analysis to identify alterations in Mlh1-/--tumor-associated target genes. The combined therapy of α-PD-L1 and gemcitabine prolonged median overall survival of Mlh1-/- mice from four weeks in the untreated control group to 12 weeks, accompanied by therapy-induced tumor growth inhibition, as measured by [18F]-FDG PET/CT. Plasma cytokine levels of IL13, TNFα, and MIP1ß were increased and also higher than in mice receiving either monotherapy. Circulating splenic and intratumoral myeloid-derived suppressor cells (MDSCs), as well as M2 macrophages, were markedly reduced. Besides, residual tumor specimens from combi-treated mice had increased numbers of infiltrating cytotoxic T-cells. Frameshift mutations in APC, Tmem60, and Casc3 were no longer detectable upon treatment, likely because of the successful eradication of single mutated cell clones. By contrast, novel mutations appeared. Collectively, we herein confirm the safe application of combined chemo-immunotherapy by long-term tumor growth control to prevent the development of resistance mechanisms.

Combined Application of Pan-AKT Inhibitor MK-2206 and BCL-2 Antagonist Venetoclax in B-Cell Precursor Acute Lymphoblastic Leukemia.

Aberrant PI3K/AKT signaling is a hallmark of acute B-lymphoblastic leukemia (B-ALL) resulting in increased tumor cell proliferation and apoptosis deficiency. While previous AKT inhibitors struggled with selectivity, MK-2206 promises meticulous pan-AKT targeting with proven anti-tumor activity. We herein, characterize the effect of MK-2206 on B-ALL cell lines and primary samples and investigate potential synergistic effects with BCL-2 inhibitor venetoclax to overcome limitations in apoptosis induction. MK-2206 incubation reduced AKT phosphorylation and influenced downstream signaling activity. Interestingly, after MK-2206 mono application tumor cell proliferation and metabolic activity were diminished significantly independently of basal AKT phosphorylation. Morphological changes but no induction of apoptosis was detected in the observed cell lines. In contrast, primary samples cultivated in a protective microenvironment showed a decrease in vital cells. Combined MK-2206 and venetoclax incubation resulted in partially synergistic anti-proliferative effects independently of application sequence in SEM and RS4;11 cell lines. Venetoclax-mediated apoptosis was not intensified by addition of MK-2206. Functional assessment of BCL-2 inhibition via Bax translocation assay revealed slightly increased pro-apoptotic signaling after combined MK-2206 and venetoclax incubation. In summary, we demonstrate that the pan-AKT inhibitor MK-2206 potently blocks B-ALL cell proliferation and for the first time characterize the synergistic effect of combined MK-2206 and venetoclax treatment in B-ALL.