Reduced Intensity Conditioning Prior Autologous Stem Cell Transplantation in Elderly DLBCL Patients.

High-dose chemotherapy (HDCT) followed by autologous stem cell transplantation (ASCT) is widely used in patients with diffuse large B-cell lymphoma. HDCT/ASCT is associated with increased morbidity in elderly/unfit patients. We retrospectively evaluated the use of reduced intensity conditioning in DLBCL patients. Our study included 146 patients aged 60 years and older treated at our institution between 2005 and 2019; 86 patients received standard intensity conditioning (SI group) with BEAM or TEAM (BCNU or thiotepa, etoposide, cytarabine, melphalan). Sixty patients received reduced intensity high-dose conditioning (RI group) with BM (BCNU, melphalan, 43.3%), TM (thiotepa, melphalan, 16.7%), BCNU or busulfan thiotepa (38.4%), or bendamustine melphalan (1.7%). Median follow-up was 62.4 months. We observed comparable toxicities in the SI and RI groups. The cumulative incidence of relapse at 3 years was higher in the RI group (30.8% vs. 23.4%, p = 0.034). There was no difference in nonrelapse mortality (NRM). In univariate analyses, SI vs. RI conditioning resulted in superior progression-free survival (PFS) (HR 1.80 CI 1.11-2.92, p = 0.017) but not in superior overall survival (OS) (HR 1.48 CI 0.86-2.56, p = 0.152). On multivariate analysis, we observed no difference in PFS (HR 0.74 CI 0.40-1.38, p = 0.345) and a trend toward better OS with RI conditioning (HR 0.45 CI 0.22-0.94, p = 0.032). Age 60-69 versus ≥ 70 years and remission prior to ASCT were the only factors predicting better PFS. Factors associated with better OS were RI conditioning, age 60-69 versus ≥ 70 years, ECOG 0 versus ≥ 1 performance status, bulky disease, and prior lines 1 versus ≥ 2. In conclusion, RI conditioning prior to ASCT may be feasible in elderly patients and led to a comparable outcome when corrected for several significant confounders.

Evaluation of risk for bronchiolitis obliterans syndrome after allogeneic hematopoietic cell transplantation with myeloablative conditioning regimens.

Bronchiolitis obliterans syndrome (BOS), as chronic manifestation of graft-versus-host disease (GVHD), is a debilitating complication leading to lung function deterioration in patients after allogeneic hematopoietic cell transplantation (allo-HCT). In the present study, we evaluated BOS development risk in patients after receiving myeloablative conditioning (MAC) regimens. We performed a retrospective analysis of patients undergoing allo-HCT, who received MAC with busulfan/cyclophosphamid (BuCy, n = 175) busulfan/fludarabin (FluBu4, n = 29) or thiotepa/busulfan/fludarabine (TBF MAC, n = 37). The prevalence of lung disease prior allo-HCT, smoking status, GvHD prophylaxis, HCT-CI score, EBMT risk score and GvHD incidence varied across the groups. The cumulative incidence of BOS using the NIH diagnosis consensus criteria at 2 years after allo-HCT was 8% in FluBu4, 23% in BuCy and 19% in TBF MAC (p = 0.07). In the multivariate analysis, we identified associated factors for time to BOS such as FEV1

Comprehensive genetic profiling and molecularly guided treatment for patients with primary CNS tumors.

Despite major advances in molecular profiling and classification of primary brain tumors, personalized treatment remains limited for most patients. Here, we explored the feasibility of individual molecular profiling and the efficacy of biomarker-guided therapy for adult patients with primary brain cancers in the real-world setting within the molecular tumor board Freiburg, Germany. We analyzed genetic profiles, personalized treatment recommendations, and clinical outcomes of 102 patients with 21 brain tumor types. Alterations in the cell cycle, BRAF, and mTOR pathways most frequently led to personalized treatment recommendations. Molecularly informed therapies were recommended in 71% and implemented in 32% of patients with completed molecular diagnostics. The disease control rate following targeted treatment was 50% and the overall response rate was 30%, with a progression-free survival 2/1 ratio of at least 1.3 in 31% of patients. This study highlights the efficacy of molecularly guided treatment and the need for biomarker-stratified trials in brain cancers.

Identification of epigenetic modifiers essential for growth and survival of AML1/ETO-positive leukemia.

Aberrant gene expression patterns in acute myeloid leukemia (AML) with balanced chromosomal translocations are often associated with dysregulation of epigenetic modifiers. The AML1/ETO (RUNX1/MTG8) fusion protein, caused by the translocation (8;21)(q22;q22), leads to the epigenetic repression of its target genes. We aimed in this work to identify critical epigenetic modifiers, on which AML1/ETO-positive AML cells depend on for proliferation and survival using shRNA library screens and global transcriptomics approaches. Using shRNA library screens, we identified 41 commonly depleted genes in two AML1/ETO-positive cell lines Kasumi-1 and SKNO-1. We validated, genetically and pharmacologically, DNMT1 and ATR using several AML1/ETO-positive and negative cell lines. We also demonstrated in vivo differentiation of myeloblasts after treatment with the DNMT1 inhibitor decitabine in a patient with an AML1/ETO-positive AML. Bioinformatic analysis of global transcriptomics after AML1/ETO induction in 9/14/18-U937 cells identified 973 differentially expressed genes (DEGs). Three genes (PARP2, PRKCD, and SMARCA4) were both downregulated after AML1/ETO induction, and identified in shRNA screens. In conclusion, using unbiased shRNA library screens and global transcriptomics, we have identified several driver epigenetic regulators for proliferation in AML1/ETO-positive AML. DNMT1 and ATR were validated and are susceptible to pharmacological inhibition by small molecules showing promising preclinical and clinical efficacy.

Early-life infection depletes preleukemic cells in a mouse model of hyperdiploid B-cell acute lymphoblastic leukemia.

ABSTRACT: Epidemiological studies report opposing influences of infection on childhood B-cell acute lymphoblastic leukemia (B-ALL). Although infections in the first year of life appear to exert the largest impact on leukemia risk, the effect of early pathogen exposure on the fetal preleukemia cells (PLC) that lead to B-ALL has yet to be reported. Using cytomegalovirus (CMV) infection as a model early-life infection, we show that virus exposure within 1 week of birth induces profound depletion of transplanted E2A-PBX1 and hyperdiploid B-ALL cells in wild-type recipients and in situ-generated PLC in Eµ-ret mice. The age-dependent depletion of PLC results from an elevated STAT4-mediated cytokine response in neonates, with high levels of interleukin (IL)-12p40-driven interferon (IFN)-γ production inducing PLC death. Similar PLC depletion can be achieved in adult mice by impairing viral clearance. These findings provide mechanistic support for potential inhibitory effects of early-life infection on B-ALL progression and could inform novel therapeutic or preventive strategies.

Development of combination therapies with BTK inhibitors and dasatinib to treat CNS-infiltrating E2A-PBX1+/preBCR+ ALL.

ABSTRACT: The t(1;19) translocation, encoding the oncogenic fusion protein E2A (TCF3)-PBX1, is involved in acute lymphoblastic leukemia (ALL) and associated with a pre-B-cell receptor (preBCR+) phenotype. Relapse in patients with E2A-PBX1+ ALL frequently occurs in the central nervous system (CNS). Therefore, there is a medical need for the identification of CNS active regimens for the treatment of E2A-PBX1+/preBCR+ ALL. Using unbiased short hairpin RNA (shRNA) library screening approaches, we identified Bruton tyrosine kinase (BTK) as a key gene involved in both proliferation and dasatinib sensitivity of E2A-PBX1+/preBCR+ ALL. Depletion of BTK by shRNAs resulted in decreased proliferation of dasatinib-treated E2A-PBX1+/preBCR+ cells compared with control-transduced cells. Moreover, the combination of dasatinib with BTK inhibitors (BTKi; ibrutinib, acalabrutinib, or zanubrutinib) significantly decreased E2A-PBX1+/preBCR+ human and murine cell proliferation, reduced phospholipase C gamma 2 (PLCG2) and BTK phosphorylation and total protein levels and increased disease-free survival of mice in secondary transplantation assays, particularly reducing CNS-leukemic infiltration. Hence, dasatinib with ibrutinib reduced pPLCG2 and pBTK in primary ALL patient samples, including E2A-PBX1+ ALLs. In summary, genetic depletion and pharmacological inhibition of BTK increase dasatinib effects in human and mouse with E2A-PBX1+/preBCR+ ALL across most of performed assays, with the combination of dasatinib and BTKi proving effective in reducing CNS infiltration of E2A-PBX1+/preBCR+ ALL cells in vivo.

Identification of clinical factors impacting outcome in patients undergoing autologous hematopoietic cell transplantation after BEAM and TEAM conditioning.

Organ dysfunction, including pulmonary function impairment, plays a key role in the choice of conditioning chemotherapy before autologous hematopoietic stem cell transplantation (auto-HSCT). Replacement of BCNU/carmustine as part of BEAM (BCNU/carmustine, etoposide, cytarabine, and melphalan) conditioning protocol by thiotepa (TEAM) reduces pulmonary toxicity while maintaining efficacy. We retrospectively analyzed the association of clinical characteristics, comorbidities, and organ function with outcomes after conditioning with BEAM or TEAM. Three hundred ninety-six patients undergoing auto-HSCT (n = 333 with BEAM; n = 63 with TEAM) at our institution between 2008 and 2021 were included in this study. In the multivariate analysis, CO-diffusion capacity corrected for hemoglobin (DLCOcSB) ≤ 60% of predicted, progressive disease (PD) before auto-HSCT, Karnofsky performance score (KPS) ≤ 80%, HCT-CI score ≥ 4, and cardiac disease before auto-HSCT were associated with decreased overall survival (OS) in patients treated with BEAM. In contrast, only PD before auto-HSCT was identified in patients treated with TEAM. Patients conditioned with BEAM and DLCOcSB ≤ 60% had higher non-relapse mortality, including pulmonary cause of death. In summary, we have identified clinical and pulmonary risk factors associated with worse outcomes in patients conditioned with BEAM compared to TEAM. Our data suggest TEAM conditioning as a valid alternative for patients with comorbidities, including pulmonary dysfunction and/or poorer performance scores, before auto-HSCT.

Comparison of fludarabine/melphalan (FluMel) with fludarabine/melphalan/BCNU or thiotepa (FBM/FTM) in patients with AML in first complete remission undergoing allogeneic hematopoietic stem cell transplantation - a registry study on behalf of the EBMT Acute Leukemia Working Party.

Conditioning protocols for patients undergoing allogeneic hematopoietic cell transplantation (allo-HCT) are being developed continuously to improve their anti-leukemic efficacy and reduce their toxicity. In this study, we compared the conditioning protocol of fludarabine with melphalan 140 mg/m2 (FluMel) with conditioning protocols based on this same backbone but with an additional alkylating agent i.e., either fludarabine/BCNU (also known as carmustine)/melphalan (FBM), or fludarabine/thiotepa/melphalan (FTM) 110 mg/m2. We included 1272 adult patients (FluMel, n = 1002; FBM/FTM, n = 270) with acute myeloid leukemia (AML) with intermediate/poor cytogenetic risk in first complete remission (CR) from the registry of the EBMT Acute Leukemia Working Party. Despite patients in the FBM/FTM group were older (64.1 years vs. 59.8 years, p < 0.001) and had a worse Karnofsky performance score (KPS < 90, 33% vs. 24%, p = 0.003), they showed a better overall survival (OS) (2 y OS: 68.3% vs. 58.1%, p = 0.02) and less non-relapse mortality (NRM) (2 y NRM: 15.8% vs. 22.2%, p = 0.009) compared to patients treated with FluMel. No significant differences were observed in relapse incidence (RI) (2 y RI: 24.9% vs. 23.7%, p = 0.62). In conclusion, the addition of a second alkylating agent (BCNU/carmustine or thiotepa) to FluMel as FBM/FTM conditioning, improves OS in AML patients in first CR with intermediate/poor risk cytogenetics after allo-HCT.

Long-term follow-up of patients with acute myeloid leukemia undergoing allogeneic hematopoietic stem cell transplantation after primary induction failure.

Primary induction failure (PIF) in acute myeloid leukemia (AML) patients is associated with poor outcome, with allogeneic hematopoietic stem cell transplantation (HCT) being the sole curative therapeutic option. Here, we retrospectively evaluated long-term outcomes of 220 AML patients undergoing allogeneic HCT after PIF who never achieved remission, and identified clinical and molecular risk factors associated with treatment response and ultimate prognosis. In this high-risk population, disease-free survival was 25.2% after 5 years and 18.7% after 10 years, while overall survival rates were 29.8% and 21.6% after 5 and 10 years of HCT, respectively. 10-year non-relapse mortality was 32.5%, and 48.8% of patients showed disease relapse within 10 years after allogeneic HCT. Adverse molecular risk features determined at initial diagnosis, poor performance status at the time of allogeneic HCT, and long diagnosis-to-HCT intervals were associated with unfavorable prognosis. Collectively, our data suggests that immediate allogeneic HCT after PIF offers long-term survival and cure in a substantial subset of cases and that high-risk AML patients who never achieved complete response during induction might benefit from early donor search.

Age and ligand specificity influence the outcome of pathogen engagement on preleukemic and leukemic B-cell precursor populations.

Common infections have long been proposed to play a role in the development of pediatric B-cell acute lymphoblastic leukemia (B-ALL). However, epidemiologic studies report contradictory effects of infection exposure on subsequent B-ALL risk, and no specific pathogen has been definitively linked to the disease. A unifying mechanism to explain the divergent outcomes could inform disease prevention strategies. We previously reported that the pattern recognition receptor (PRR) ligand Poly(I:C) exerted effects on B-ALL cells that were distinct from those observed with other nucleic acid-based PRR ligands. Here, using multiple double-stranded RNA (dsRNA) moieties, we show that the overall outcome of exposure to Poly(I:C) reflects the balance of opposing responses induced by its ligation to endosomal and cytoplasmic receptors. This PRR response biology is shared between mouse and human B-ALL and can increase leukemia-initiating cell burden in vivo during the preleukemia phase of B-ALL, primarily through tumor necrosis factor α signaling. The age of the responding immune system further influences the impact of dsRNA exposure on B-ALL cells in both mouse and human settings. Overall, our study demonstrates that potentially proleukemic and antileukemic effects can each be generated by the stimulation of pathogen recognition pathways and indicates a mechanistic explanation for the contrasting epidemiologic associations reported for infection exposure and B-ALL.

Functional Characterization of Transforming Growth Factor-ß Signaling in Dasatinib Resistance and Pre-BCR+ Acute Lymphoblastic Leukemia.

The multi-kinase inhibitor dasatinib has been implicated to be effective in pre-B-cell receptor (pre-BCR)-positive acute lymphoblastic leukemia (ALL) expressing the E2A-PBX1 fusion oncoprotein. The TGFß signaling pathway is involved in a wide variety of cellular processes, including embryonic development and cell homeostasis, and it can have dual roles in cancer: suppressing tumor growth at early stages and mediating tumor progression at later stages. In this study, we identified the upregulation of the TGFß signaling pathway in our previously generated human dasatinib-resistant pre-BCR+/E2A-PBX1+ ALL cells using global transcriptomic analysis. We confirm the upregulation of the TGFß pathway member SMAD3 at the transcriptional and translational levels in dasatinib-resistant pre-BCR+/E2A-PBX1+ ALL cells. Hence, dasatinib blocks, at least partially, TGFß-induced SMAD3 phosphorylation in several B-cell precursor (BCP) ALL cell lines as well as in dasatinib-resistant pre-BCR+/E2A-PBX1+ ALL cells. Activation of the TGFß signaling pathway by TGF-ß1 leads to growth inhibition by cell cycle arrest at the G0/G1 stage, increase in apoptosis and transcriptional changes of SMAD-targeted genes, e.g. c-MYC downregulation, in pre-BCR+/E2A-PBX1+ ALL cells. These results provide a better understanding about the role that the TGFß signaling pathway plays in leukemogenesis of BCP-ALL as well as in secondary drug resistance to dasatinib.

Functional characterization of the PI3K/AKT/MTOR signaling pathway for targeted therapy in B-precursor acute lymphoblastic leukemia.

B-cell precursor acute lymphoblastic leukemias (B-ALL) are characterized by the activation of signaling pathways, which are involved in survival and proliferation of leukemia cells. Using an unbiased shRNA library screen enriched for targeting signaling pathways, we identified MTOR as the key gene on which human B-ALL E2A-PBX1+ RCH-ACV cells are dependent. Using genetic and pharmacologic approaches, we investigated whether B-ALL cells depend on MTOR upstream signaling pathways including PI3K/AKT and the complexes MTORC1 or MTORC2 for proliferation and survival in vitro and in vivo. Notably, the combined inhibition of MTOR and AKT shows a synergistic effect on decreased cell proliferation in B-ALL with different karyotypes. Hence, B-ALL cells were more dependent on MTORC2 rather than MTORC1 complex in genetic assays. Using cell metabolomics, we identified changes in mitochondrial fuel oxidation after shRNA-mediated knockdown or pharmacological inhibition of MTOR. Dependence of the cells on fatty acid metabolism for their energy production was increased upon inhibition of MTOR and associated upstream signaling pathways, disclosing a possible target for a combination therapy. In conclusion, B-ALL are dependent on the PI3K/AKT/MTOR signaling pathway and the combination of specific small molecules targeting this pathway appears to be promising for the treatment of B-ALL patients.

Monitoring of Measurable Residual Disease Using Circulating DNA after Allogeneic Hematopoietic Cell Transplantation.

Relapse of the underlying disease is a frequent complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). In this study, we describe the clinical utility of measurable residual disease (MRD) and mixed chimerism (MC) assessment in circulating cell-free DNA (cfDNA) analysis to detect earlier relapse in patients with hematological malignancies after allo-HSCT. A total of 326 plasma and peripheral blood mononuclear cell (PBMCs) samples obtained from 62 patients with myeloid malignancies were analyzed by droplet-digital PCR (median follow-up: 827 days). Comparison of MC in patients at relapse and in complete remission identified an optimal discriminating threshold of 18% of recipient-derived cfDNA. After performing a targeted next-generation sequencing (NGS) panel, 136 mutations in 58 patients were detected. In a total of 119 paired samples, the putative mutations were detected in both cfDNA and PBMCs in 73 samples (61.3%). In 45 samples (37.8%) they were detected only in cfDNA, and in only one patient (0.9%) were they detected solely in DNA from PBMCs. Hence, in 6 out of 23 patients (26%) with relapse after allo-HSCT, MRD positivity was detected earlier in cfDNA (mean 397 days) than in DNA derived from PBMCs (mean 451 days). In summary, monitoring of MRD and MC in cfDNA might be useful for earlier relapse detection in patients with myeloid malignancies after allo-HSCT.

Comparison of fludarabine-melphalan and fludarabine-treosulfan as conditioning prior to allogeneic hematopoietic cell transplantation-a registry study on behalf of the EBMT Acute Leukemia Working Party.

In recent years considerable variations in conditioning protocols for allogeneic hematopoietic cell transplantation (allo-HCT) protocols have been introduced for higher efficacy, lower toxicity, and better outcomes. To overcome the limitations of the classical definition of reduced intensity and myeloablative conditioning, a transplantation conditioning intensity (TCI) score had been developed. In this study, we compared outcome after two frequently used single alkylator-based conditioning protocols from the intermediate TCI score category, fludarabine/melphalan 140 mg/m2 (FluMel) and fludarabine/treosulfan 42 g/m2 (FluTreo) for patients with acute myeloid leukemia (AML) in complete remission (CR). This retrospective analysis from the registry of the Acute Leukemia Working Party (ALWP) of the European Society of Bone Marrow Transplantation (EBMT) database included 1427 adult patients (median age 58.2 years) receiving either Flu/Mel (n = 1005) or Flu/Treo (n = 422). Both groups showed similar 3-year overall survival (OS) (54% vs 51.2%, p value 0.49) for patients conditioned with FluMel and FluTreo, respectively. However, patients treated with FluMel showed a reduced 3-year relapse incidence (32.4% vs. 40.4%, p value < 0.001) and slightly increased non-relapse mortality (NRM) (25.7% vs. 20.2%, p value = 0.06) compared to patients treated with FluTreo. Our data may serve as a basis for further studies examining the role of additional agents/ intensifications in conditioning prior to allo-HCT.

The epigenetics of breast cancer - Opportunities for diagnostics, risk stratification and therapy.

The stage and molecular pathology-dependent prognosis of breast cancer, the limited treatment options for triple-negative carcinomas, as well as the development of resistance to therapies illustrate the need for improved early diagnosis and the development of new therapeutic approaches. Increasing data suggests that some answers to these challenges could be found in the area of epigenetics. In this study, we focus on the current research of the epigenetics of breast cancer, especially on the potential of epigenetics for clinical application in diagnostics, risk stratification and therapy. The differential DNA methylation status of specific gene regions has been used in the past to differentiate breast cancer cells from normal tissue. New technologies as detection of circulating nucleic acids including microRNAs to early detect breast cancer are emerging. Pattern of DNA methylation and expression of histone-modifying enzymes have been successfully used for risk stratification. However, all these epigenetic biomarkers should be validated in larger clinical studies. Recent preclinical and clinical studies show a therapeutic benefit of epigenetically active drugs for breast cancer entities that are still difficult to treat (triple negative, UICC stage IV). Remarkably, epigenetic therapies combined with chemotherapies or hormone-based therapies represent the most promising strategy. At the current stage, the integration of epigenetic substances into established breast cancer therapy protocols seems to hold the greatest potential for a clinical application of epigenetic research.

The impact of pulmonary function in patients undergoing autologous stem cell transplantation.

High-dose chemotherapy, followed by autologous hematopoietic stem cell transplantation (auto-HSCT), is an established therapy for patients with hematological malignancies. The age of patients undergoing auto-HSCT and, therefore, the comorbidities, has increased over the last decades. However, the assessment of organ dysfunction prior to auto-HSCT has not been well studied. Therefore, we retrospectively analyzed the association of clinical factors and lung and cardiac function with outcome and complications after conditioning with BEAM (BCNU/carmustine, etoposide, cytarabine, melphalan) or high-dose melphalan in patients undergoing auto-HSCT. This study included 629 patients treated at our institution between 2007 and 2017; 334 and 295 were conditioned with BEAM or high-dose melphalan, respectively. The median follow-up was 52 months (range, 0.2-152) and 50 months (range, 0.5-149), respectively. In the multivariate analysis, we identified that progressive disease, CO-diffusion capacity corrected for hemoglobin (DLCOcSB) ≤ 60% of predicted, Karnofsky Performance Status (KPS) ≤ 80%, Hematopoietic Cell Transplantation Comorbidity Index (HCT-CI) score ≥ 4, and age > 70 years were associated with decreased overall survival (OS) in patients treated with BEAM. Similarly, DLCOcSB ≤ 60% of predicted, HCT-CI score ≥ 4, and age > 60 years were identified in patients treated with high-dose melphalan. Abnormalities in DLCOcSB ≤ 60% of predicted were associated with chemotherapy with lung-toxic substances, mediastinal radiotherapy, KPS ≤ 80%, current/previous smoking, and treatment in the intensive care unit. More often, patients with DLCOcSB ≤ 60% of predicted experienced nonrelapse mortality, including pulmonary causes of death. In summary, we identified DLCOcSB ≤ 60% of predicted as an independent risk factor for decreased OS in patients conditioned with BEAM or high-dose melphalan prior to auto-HSCT.

AML1/ETO and its function as a regulator of gene transcription via epigenetic mechanisms.

The chromosomal translocation t(8;21) and the resulting oncofusion gene AML1/ETO have long served as a prototypical genetic lesion to model and understand leukemogenesis. In this review, we describe the wide-ranging role of AML1/ETO in AML leukemogenesis, with a particular focus on the aberrant epigenetic regulation of gene transcription driven by this AML-defining mutation. We begin by analyzing how structural changes secondary to distinct genomic breakpoints and splice changes, as well as posttranscriptional modifications, influence AML1/ETO protein function. Next, we characterize how AML1/ETO recruits chromatin-modifying enzymes to target genes and how the oncofusion protein alters chromatin marks, transcription factor binding, and gene expression. We explore the specific impact of these global changes in the epigenetic network facilitated by the AML1/ETO oncofusion on cellular processes and leukemic growth. Furthermore, we define the genetic landscape of AML1/ETO-positive AML, presenting the current literature concerning the incidence of cooperating mutations in genes such as KIT, FLT3, and NRAS. Finally, we outline how alterations in transcriptional regulation patterns create potential vulnerabilities that may be exploited by epigenetically active agents and other therapeutics.