Comprehensive insights into AML relapse: genetic mutations, clonal evolution, and clinical outcomes.

INTRODUCTION: Acute myeloid leukemia (AML) is a complex hematologic malignancy characterized by uncontrolled proliferation of myeloid precursor cells within bone marrow. Despite advances in understanding of its molecular underpinnings, AML remains a therapeutic challenge due to its high relapse rate and clonal evolution. METHODS: In this retrospective study, we analyzed data from 24 AML patients diagnosed at a single institution between January 2017 and August 2023. Comprehensive genetic analyses, including chromosomal karyotyping, next-generation sequencing, and gene fusion assays, were performed on bone marrow samples obtained at initial diagnosis and relapse. Clinical data, treatment regimens, and patient outcomes were also documented. RESULTS: Mutations in core genes of FLT3, NPM1, DNMT3A, and IDH2 were frequently discovered in diagnostic sample and remained in relapse sample. FLT3-ITD, TP53, KIT, RUNX1, and WT1 mutation were acquired at relapse in one patient each. Gene fusion assays revealed stable patterns, while chromosomal karyotype analyses indicated a greater diversity of mutations in relapsed patients. Clonal evolution patterns varied, with some cases showing linear or branching evolution and others exhibiting no substantial change in core mutations between diagnosis and relapse. CONCLUSIONS: Our study integrates karyotype, gene rearrangements, and gene mutation results to provide a further understanding of AML heterogeneity and evolution. We demonstrate the clinical relevance of specific mutations and clonal evolution patterns, emphasizing the need for personalized therapies and measurable residual disease monitoring in AML management. By bridging the gap between genetics and clinical outcome, we move closer to tailored AML therapies and improved patient prognoses.

Clinical outcomes of etoposide and cytarabine as consolidation in elderly patients with primary CNS lymphoma.

BACKGROUND: A consolidation strategy has not been established for transplant-ineligible elderly patients with primary central nervous system lymphoma (PCNSL). In this study, we aimed to retrospectively evaluate the clinical outcomes of etoposide and cytarabine (EA) as consolidation chemotherapy for transplant-ineligible patients with PCNSL following high-dose methotrexate (MTX)-based induction chemotherapy. MATERIALS AND METHODS: Between 2015 and 2021, newly diagnosed transplant-ineligible patients with PCNSL with diffuse large B-cell lymphoma were consecutively enrolled. All enrolled patients were over 60 years old and received EA consolidation after achieving a complete or partial response following induction chemotherapy. RESULTS: Of the 85 patients who achieved a complete or partial response to MTX-based induction chemotherapy, 51 received EA consolidation chemotherapy. Among the 25 (49.0%, 25/51) patients in partial remission before EA consolidation, 56% (n = 14) achieved complete remission after EA consolidation. The median overall survival and progression-free survival were 43 and 13 months, respectively. Hematological toxicities were most common, and all patients experienced grade 4 neutropenia and thrombocytopenia. Forty-eight patients experienced febrile neutropenia during consolidation chemotherapy, and 4 patients died owing to treatment-related complications. CONCLUSION: EA consolidation chemotherapy for transplant-ineligible, elderly patients with PCNSL improved response rates but showed a high relapse rate and short progression-free survival. The incidences of treatment-related mortality caused by hematologic toxicities and severe infections were very high, even after dose modification. Therefore, the use of EA consolidation should be reconsidered in elderly patients with PCNSL.

3D Amplified Single-Cell RNA and Protein Imaging Identifies Oncogenic Transcript Subtypes in B-Cell Acute Lymphoblastic Leukemia.

Simultaneous in situ detection of transcript and protein markers at the single-cell level is essential for gaining a better understanding of tumor heterogeneity and for predicting and monitoring treatment responses. However, the limited accessibility to advanced 3D imaging techniques has hindered their rapid implementation. Here, we present a 3D single-cell imaging technique, termed 3D digital rolling circle amplification (4DRCA), capable of the multiplexed and amplified simultaneous digital quantification of single-cell RNAs and proteins using standard fluorescence microscopy and off-the-shelf reagents. We generated spectrally distinguishable DNA amplicons from molecular markers through an integrative protocol combining single-cell RNA and protein assays and directly enumerated the amplicons by leveraging an open-source algorithm for 3D deconvolution with a custom-built automatic gating algorithm. With 4DRCA, we were able to simultaneously quantify surface protein markers and cytokine transcripts in T-lymphocytes. We also show that 4DRCA can distinguish BCR-ABL1 fusion transcript positive B-cell acute lymphoblastic leukemia cells with or without CD19 protein expression. The accessibility and extensibility of 4DRCA render it broadly applicable to other cell-based diagnostic workflows, enabling sensitive and accurate single-cell RNA and protein profiling.

Clearing soluble MIC reverses the impaired function of natural killer cells from patients with multiple myeloma.

BACKGROUND: Major histocompatibility complex (MHC) class I chain-related protein (MIC) is a stress-induced ligand released from multiple myeloma (MM) cells during progression, and soluble MIC impairs natural killer group 2D (NKG2D) activating receptor-mediated recognition and function of natural killer (NK) cells. However, whether clearing soluble MIC with a monoclonal antibody (mAb) can restore NK cell activity of MM patients remains undetermined. METHODS: We analyzed The Cancer Genome Atlas (TCGA) Multiple Myeloma Research Foundation (MMRF) CoMMpass data set to examine the prognostic significance of MIC expression in MM. We examined the level of soluble MIC in paired peripheral blood (PB) and bone marrow (BM) plasma of patients with MM at diagnosis by ELISA. We evaluated the correlation between the level of soluble MIC and immunophenotype of NK cells from MM patients by multicolor flow cytometry. We also generated MIC-overexpressing MM cell line and characterized the cytotoxic function of patient NK cells in the presence of soluble MIC, and examined the impact of clearing soluble MIC with a humanized mAb (huB10G5). RESULTS: We characterize the importance of MICA in MM by revealing the significantly better overall survival of patients with high MICA expression from TCGA MMRF CoMMpass data set. The level of soluble MICA is more highly elevated in MM than in precursor stages, and the concentration of soluble MICA is higher in BM plasma than in PB. The concentration of soluble MICA in BM was correlated with myeloma burden, while it was negatively correlated with the frequency of NKG2D+ NK cells in diagnostic BM aspirates of MM patients. Soluble MICA downregulated NKG2D expression and decreased cytotoxicity of MM patient NK cells ex vivo, which were reversed by a humanized soluble MIC-clearing mAb (huB10G5) with enhanced degranulation of NK cells. CONCLUSIONS: Our findings indicate targeting soluble MIC with huB10G5 might be a viable therapeutic approach to promote NKG2D-dependent cellular immunotherapy outcome in MM.

Recent advances in cellular immunotherapy for lymphoid malignancies.

Cellular immunotherapy with chimeric antigen receptor (CAR) T-cells has revolutionized the treatment of lymphoid malignancies. This review addresses the need for CAR expression in our endogenous T-cells to kill tumor cells with a focus on the basic principles of T-cell receptor recognition of major histocompatibility complex-peptide complexes. We review the factors associated with CAR T-cell outcomes and recent efforts to employ CAR T-cells in earlier lines of therapy. We also discuss the value of bispecific T-cell engagers as off-the-shelf products with better toxicity profiles. Finally, natural killer cells are discussed as an important cellular immunotherapy platform with the potential to broaden immunotherapeutic applications beyond lymphoid malignancies.

Utility of Plasma Microbial Cell-Free DNA Whole-Genome Sequencing for Diagnosis of Invasive Aspergillosis in Patients With Hematologic Malignancy or COVID-19.

BACKGROUND: We evaluated the clinical accuracy and utility of whole-genome sequencing (WGS) of plasma microbial cell-free DNA (cfDNA) as a novel noninvasive method in diagnosing invasive aspergillosis (IA) in patients with hematologic malignancy (HM) or coronavirus disease 2019 (COVID-19). METHODS: Adults with HM or COVID-19 and suspected IA were recruited. IA cases were retrospectively diagnosed according to EORTC/MSG definitions and ECMM/ISHAM criteria for HM and COVID-19 patients, respectively. The results of cfDNA WGS were compared with the conventional diagnosis. RESULTS: Microbial cfDNA WGS was performed 53 times from 41 participants (19 from HM, 16 from COVID-19, and 7 from the control group). In participants with HM, Aspergillus cfDNA was detected in 100% of proven IA and 91.7% of probable IA cases. In participants with COVID-19, 50.0% of probable IA were positive for Aspergillus in cfDNA WGS. Concordance between Aspergillus cfDNA detection and proven/probable IA conventional diagnosis was significantly higher in participants with HM than in those with COVID-19. IA diagnosed using EORTC/MGS definitions showed significantly high concordance between Aspergillus cfDNA detection and proven/probable IA. CONCLUSIONS: Aspergillus cfDNA detection strongly correlated with proven/probable IA diagnosed using EORTC/MSG definitions and could be used as an additional diagnostic tool for IA.

DRP1 Inhibition Enhances Venetoclax-Induced Mitochondrial Apoptosis in TP53-Mutated Acute Myeloid Leukemia Cells through BAX/BAK Activation.

Although TP53 mutations in acute myeloid leukemia (AML) are associated with poor response to venetoclax, the underlying resistance mechanism remains unclear. Herein, we investigated the functional role of dynamin-related protein 1 (DRP1) in venetoclax sensitivity in AML cells with respect to TP53 mutation status. Effects of DRP1 inhibition on venetoclax-induced cell death were compared in TP53-mutated (THP-1 and Kasumi-1) and TP53 wild-type leukemia cell lines (MOLM-13 and MV4-11), as well as in primary AML cells obtained from patients. Venetoclax induced apoptosis in TP53 wild-type AML cells but had limited effects in TP53-mutated AML cells. DRP1 expression was downregulated in MOLM-13 cells after venetoclax treatment but was unaffected in THP-1 cells. Cotreatment of THP-1 cells with venetoclax and a TP53 activator NSC59984 downregulated DRP1 expression and increased apoptosis. Combination treatment with the DRP1 inhibitor Mdivi-1 and venetoclax significantly increased mitochondria-mediated apoptosis in TP53-mutated AML cells. The combination of Mdivi-1 and venetoclax resulted in noticeable downregulation of MCL-1 and BCL-xL, accompanied by the upregulation of NOXA, PUMA, BAK, and BAX. These findings suggest that DRP1 is functionally associated with venetoclax sensitivity in TP53-mutated AML cells. Targeting DRP1 may represent an effective therapeutic strategy for overcoming venetoclax resistance in TP53-mutated AML.

Real-world data on prognostic value of measurable residual disease assessment by fragment analysis or next-generation sequencing in multiple myeloma.

Measurable residual disease (MRD) negativity is a strong prognostic indicator in multiple myeloma (MM). However, the optimal use of MRD in daily clinical practice has been hampered by the limited feasibility of MRD testing. Therefore, we examined the clinical relevance of commercially available MRD modalities based on clonality assays by fragment analysis with IdentiClone® (n = 73 patients) and next-generation sequencing (NGS) with LymphoTrack® (n = 116 patients) in newly diagnosed patients with MM who received autologous stem cell transplantation (ASCT). MRD was assessed at the end of induction (pre-ASCT) and/or at 100 days after ASCT (post-ASCT). MRD could not predict survival when assessed by fragment analysis. However, NGS-based MRD negativity at pre- or post-ASCT was beneficial in terms of progression-free and overall survival. Moreover, NGS-based MRD negativity was independently associated with improved progression-free and overall survival, and MRD-positive patients both pre- and post-ASCT had worst outcome. Indeed, initial adverse prognostic features by high-risk cytogenetics could be mitigated upon achieving MRD negativity by NGS. We demonstrate the feasibility and clinical benefit of achieving MRD negativity by commercially available clonality-based MRD assays in MM and support incorporating NGS, but not fragment analysis, to tailor therapeutic strategies in real-world practice.

Cytogenetic testing by fluorescence in situ hybridization is improved by plasma cell sorting in multiple myeloma.

Accurate detection of cytogenetic abnormalities has become more important for improving risk-adapted treatment strategies in multiple myeloma (MM). However, precise cytogenetic testing by fluorescence in situ hybridization (FISH) is challenged by the dilution effect of bone marrow specimens and poor growth of plasma cells ex vivo. It has been suggested that FISH should be performed in combination with plasma cell enrichment strategies. We examined cytogenetic abnormalities in newly diagnosed MM and compared the efficacy of three different enrichment modalities for FISH: direct FISH (n = 137), fluorescence immunophenotyping and interphase cytogenetics as a tool for the investigation of neoplasms (FICTION) technique (n = 224), and a plasma cell sorting FISH with fluorescence-activated cell sorter (FACS) (n = 132). FISH disclosed cytogenetic abnormalities in 38.0% of samples by direct FISH, 56.3% by FICTION, and 95.5% by FACS-FISH, and the percentage of cells with abnormal signals detected by FISH was significantly higher by FACS-FISH than direct FISH or FICTION. Our results suggest that the efficacy of FISH is dependent on the plasma cell enrichment modalities and reveal that plasma cell sorting FISH with FACS enables better detection of cytogenetic abnormalities in diagnostic MM samples.

Ig Gene Clonality Analysis Using Next-Generation Sequencing for Improved Minimal Residual Disease Detection with Significant Prognostic Value in Multiple Myeloma Patients.

Next-generation sequencing (NGS) of rearranged Ig genes is an effective technology for identifying pathologic clonal cells in multiple myeloma (MM) and tracking minimal residual disease. The clinical effect of implementing NGS in Ig gene clonality analysis was evaluated via a retrospective chart review. A total of 312 patients diagnosed with MM were enrolled in the study. Ig gene clonality was determined by fragment analysis using BIOMED-2 multiplex PCR assays and by NGS using the LymphoTrack IGH FR1 Assay and LymphoTrack IGK Assay. The clonality detection rates in diagnostic samples obtained using fragment analysis and NGS were 96.7% and 95.4%, respectively (statistically nonsignificant difference; P = 0.772). Among samples of patients in complete remission, the clonality detection rates obtained using fragment analysis and NGS were 33.3% and 60.3%, respectively (statistically significant difference; P = 0.034). Progression-free survival was significantly longer in negative than positive patients by NGS analysis (P = 0.03). Clonality detection by NGS-based methods using IGH FR1 and IGK assays in routine clinical practice is feasible, provides good clonality detection rates in diagnostic samples, and allows monitoring of samples in MM patients with significant prognostic value.

Predictive Factors of Event-Free Survival at 24 Months in Patients with Peripheral T-Cell Lymphoma: A Retrospective Study.

PURPOSE: Event-free survival at 24 months (EFS24) is known to be a surrogate marker for overall survival (OS) for patients with peripheral T-cell lymphoma (PTCL). We examined the role of EFS24 in PTCL compared to diffuse large B-cell lymphoma (DLBCL), and then assessed the clinical predictive factors of achieving EFS24. MATERIALS AND METHODS: Patients with newly diagnosed PTCL treated with anthracycline-based chemotherapy were included. Subsequent OS was defined as the time elapsed from 24 months after diagnosis until death from any cause in those who achieved EFS24. RESULTS: Overall, 153 patients were evaluated, and 51 patients (33.3%) achieved EFS24. Patients who achieved EFS24 showed superior OS compared to patients who did not (p < 0.001). EFS24 could stratify the subsequent OS although it did not reach to that of the general population. After matching the PTCL group to the DLBCL group based on the international prognostic index, the subsequent OS in patients who achieved EFS24 was similar between the two groups (p=0.094). Advanced stage was a significant factor to predict the failing EFS24 by multivariable analysis (p < 0.001). CONCLUSION: Patients with PTCL who achieve EFS24 could have a favorable subsequent OS. Since advanced disease stage is a predictor of EFS24 failure, future efforts should focus on developing novel therapeutic strategies for PTCL patients presenting with advanced disease.

Arsenic trioxide synergistically promotes the antileukaemic activity of venetoclax by downregulating Mcl-1 in acute myeloid leukaemia cells.

BACKGROUND: The evasion of apoptosis through dysregulated Bcl-2 family members is a hallmark of leukaemia stem cells (LSCs) in acute myeloid leukaemia (AML). Therefore, targeting Bcl-2 with venetoclax has been suggested as an attractive strategy for inducing apoptosis in AML LSCs. However, the selective inhibition of Bcl-2 in AML often leads to upregulation of Mcl-1, another dominant anti-apoptotic Bcl-2 family protein conferring venetoclax resistance. METHODS: We assessed the combined effect of venetoclax and arsenic trioxide (ATO) on leukaemic cell viability, apoptosis, combination index, and cell cycle in the human LSC-like KG1 and KG1a cells. The synergistic effect of venetoclax and ATO on apoptosis was also examined in primary CD34+ and CD34+CD38- LSCs from the bone marrow (BM) of AML patients, and compared with those from healthy donors. RESULTS: Venetoclax efficiently impaired cell viability and dose-dependently promoted apoptosis when combined with ATO; their synergism was aptly represented by the combination index. The combination of venetoclax and ATO impaired cell cycle progression by restricting cells within the sub-G1 phase and facilitating caspase-dependent apoptotic cell death associated with the loss of mitochondrial membrane potential, while sparing healthy BM haematopoietic stem cells. Mechanistically, ATO mitigated venetoclax-induced upregulation of Mcl-1 by the inhibition of AKT and ERK, along with activation of GSK-3ß. This led to the Mcl-1 destabilisation, triggering Noxa and Bim to facilitate apoptosis and the consequent activation of the apoptosis executioner protein Bak. Moreover, the combination promoted phosphorylation of ATM, Chk2, p38, and H2AX, indicating an active DNA damage response. CONCLUSIONS: Our findings demonstrate the synergistic, preferential antileukaemic effects of venetoclax and ATO on LSCs, providing a rationale for preclinical and clinical trials by combining these agents already being used in clinical practice to treat acute leukaemia.

Adaptive Natural Killer Cells Facilitate Effector Functions of Daratumumab in Multiple Myeloma.

PURPOSE: To investigate the different roles of heterogeneous natural killer (NK)-cell subpopulations in multiple myeloma and to identify NK-cell subsets that support the robust anti-myeloma activity of daratumumab via antibody-dependent cellular cytotoxicity (ADCC). EXPERIMENTAL DESIGN: We performed single-cell RNA sequencing of NK cells from patients with newly diagnosed multiple myeloma (NDMM) and delineated adaptive NK cells in their bone marrow (BM). We further characterized the distinct immunophenotypic features and functions of adaptive NK cells by multicolor flow cytometry in 157 patients with NDMM. RESULTS: Adaptive NK cells exhibit a significantly lower level of CD38 expression compared with conventional NK cells, suggesting that they may evade daratumumab-induced fratricide. Moreover, adaptive NK cells exert robust daratumumab-mediated effector functions ex vivo, including cytokine production and degranulation, compared with conventional NK cells. The composition of adaptive NK cells in BM determines the daratumumab-mediated ex vivo functional activity of BM NK cells in patients with NDMM. Unlike conventional NK cells, sorted adaptive NK cells from the BM of patients with NDMM exert substantial cytotoxic activity against myeloma cells in the presence of daratumumab. CONCLUSIONS: Our findings indicate that adaptive NK cells are an important mediator of ADCC in multiple myeloma and support direct future efforts to better predict and improve the treatment outcome of daratumumab by selectively employing adaptive NK cells.

Different roles of surveillance positron emission tomography according to the histologic subtype of non-Hodgkin's lymphoma.

BACKGROUND/AIMS: Although the use of surveillance 18F-fluorodeoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT) is discouraged in patients with diffuse large B-cell lymphoma, its usefulness in different subtypes has not been thoroughly investigated. METHODS: We retrospectively evaluated 157 patients who showed positive results on surveillance FDG-PET/CT every 6 months following complete response for up to 5 years. All of the patients also underwent biopsies. RESULTS: Seventy-eight (49.6%) of 157 patients had true positive results; the remaining 79 (50.3%), including eight (5.1%) with secondary malignancies, were confirmed to yield false positive results. Among the 78 patients with true positive results, the disease in seven (8.9%) had transformed to a different subtype. The positive predictive value (PPV) of FDG-PET/CT for aggressive B-cell non-Hodgkin's lymphoma (NHL) was lower than that for indolent B-cell or aggressive T-cell NHL (p = 0.003 and p = 0.018, respectively), especially in patients with a low/low-intermediate international prognostic index (IPI) upon a positive PET/CT finding. On the other hand, indolent B-cell and aggressive T-cell NHL patients showed PPVs of > 60%, including those with low/low-intermediate secondary IPIs. CONCLUSION: The role of FDG-PET/CT surveillance is limited, and differs according to the lymphoma subtype. FDG-PET/CT may be useful in detecting early relapse in patients with aggressive T-cell NHL, including those with low/low-intermediate risk secondary IPI; as already known, FDG-PET/CT has no role in aggressive B-cell NHL. Repeat biopsy should be performed to discriminate relapse or transformation from false positive findings in patients with positive surveillance FDG-PET/CT results.

Immunosuppressive role of CD11b+ CD33+ HLA-DR- myeloid-derived suppressor cells-like blast subpopulation in acute myeloid leukemia.

OBJECTIVE: Myeloid-derived suppressor cells (MDSCs) facilitate tumor growth and development by suppressing T cell function; however, their role in acute myeloid leukemia (AML) remains unclear. Here, we investigated the immunosuppressive role and prognostic value of blasts with an MDSC-like phenotype. METHODS: CD11b+ CD33+ HLA-DR- MDSC-like blasts from bone marrow mononuclear cells of patients with AML were analyzed. To investigate their T cell-suppressing function, MDSC-like blasts were isolated using flow cytometry and co-cultured with CD8+ cytotoxic T cells and NB4 leukemic cells. Treatment outcomes were then compared between the MDSC-like blasts low (≤9.76%) and high (>9.76%) groups to identify clinical significance. RESULTS: MDSC-like blasts showed higher expression of arginase-1 and inducible nitric oxide synthase. Isolated MDSC-like blasts significantly suppressed CD8+ T cell proliferation induced by phytohemagglutinin A. NB4 cell proliferation was significantly suppressed upon co-culture with CD8+ cytotoxic T cells and partially restored upon co-culture with MDSC-like blasts. Patients with high MDSC-like blasts at diagnosis showed substantially shorter overall survival and leukemia-free survival relative to low MDSC-like blasts patients, with subgroup analysis showing statistically significant differences in patients not receiving allogeneic hematopoietic stem cell transplantation. CONCLUSION: We demonstrated that MDSC-like blasts drive AML-specific immune-escape mechanisms by suppressing T cell proliferation and restoring T cell-suppressed NB4 cell proliferation, with clinically higher fractions of MDSC-like blasts at diagnosis resulting in poor prognosis.

Renal intravascular large B cell lymphoma: the first case report in Korea and a review of the literature.

Herein, we describe the first case of renal intravascular large B cell lymphoma in Korea occurring in a 66-year-old female. She presented with mild fever and dyspnea. On physical and laboratory evaluations, hemophagocytic lymphohistiocytosis was suspected, but the bone marrow biopsy results were unremarkable. During the work-up, massive proteinuria developed, which led to a renal biopsy. The renal architecture was relatively well-preserved, but the glomeruli were hypercellular with the infiltration of atypical, large lymphoid cells with increased nucleus-cytoplasm ratio and clumped chromatin. Similar cells were also present in the peritubular capillaries. The tumor cells exhibited membranous staining for CD20 and CD79a. After the diagnosis of intravascular large B cell lymphoma, the patient received rituximab-based chemotherapy under close follow-up.

ULK1 inhibition as a targeted therapeutic strategy for FLT3-ITD-mutated acute myeloid leukemia.

BACKGROUND: In acute myeloid leukemia (AML), internal tandem duplication mutations in the FLT3 tyrosine kinase receptor (FLT3-ITD) are associated with a dismal outcome. Although uncoordinated 51-like kinase 1 (ULK1), which plays a central role in the autophagy pathway, has emerged as a novel therapeutic target for various cancers, its role in FLT3-ITD AML remains elusive. In this study, we evaluated the effects of ULK1 inhibition on leukemia cell death in FLT3-ITD AML. METHOD: We evaluated ULK1 expression and the levels of apoptosis and autophagy following ULK1 inhibition in FLT3-ITD AML cell lines and investigated the mechanism underlying apoptosis induced by ULK1 inhibition. Statistical analysis was performed using GraphPad Prism 4.0 (GraphPad Software Inc). RESULTS: FLT3-ITD AML cells showed significantly higher ULK1 expression than FLT3-wild-type (WT) AML cells. Two ULK1 inhibitors, MRT 68921 and SBI-0206965, induced apoptosis in FLT3-ITD AML cells, with relatively minimal effects on FLT3-WT AML cells and normal CD34-positive cells. Apoptosis induction by ULK1 inhibition was associated with caspase pathway activation. Interestingly, ULK1 inhibition paradoxically also induced autophagy, showing synergistic interaction with autophagy inhibitors. Hence, autophagy may act as a prosurvival mechanism in FLT3-ITD AML cells. FLT3-ITD protein degradation and inhibition of the ERK, AKT, and STAT5 pathways were also observed in FLT3-ITD AML cells following treatment with ULK1 inhibitors. CONCLUSION: ULK1 is a viable drug target and ULK1 inhibition may represent a promising therapeutic strategy against FLT3-ITD AML.

Upfront autologous hematopoietic stem cell transplantation for high-risk patients with double-expressor diffuse large B cell lymphoma.

Although MYC and BCL2 co-expression in diffuse large B cell lymphoma (DLBCL) is associated with inferior prognosis, it remains uncertain whether upfront autologous hematopoietic stem cell transplantation (ASCT) is beneficial in this lymphoma. This study aimed to investigate whether ASCT consolidation could have a positive role for patients with MYC and BCL2 co-expression (double-expressor lymphoma, DEL). We retrospectively evaluated 67 DLBCL patients who underwent upfront ASCT following rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone chemotherapy. The 5-year overall survival (OS) and progression-free survival (PFS) were 82.3% and 79.2%, respectively. There were 23 (34.3%) patients with DEL and 51 (76.1%) patients with non-germinal center B cell (GCB) subtype. The 5-year OS and PFS of patients with DEL were not different from those with non-DEL (P = 0.429 and P = 0.614, respectively). No survival difference for OS and PFS was also observed between GCB and non-GCB subtypes (P = 0.950 and P = 0.901, respectively). The OS and PFS were comparable for patients with DEL and non-DEL and both GCB and non-GCB subtypes. In conclusion, MYC and BCL2 co-expression did not have a poor prognostic impact among high-risk patients with DLBCL treated with upfront ASCT regardless of molecular classification. This preliminary study suggested that the role of consolidative ASCT is needed to be evaluated in a prospective randomized clinical trial.

PERK/NRF2 and autophagy form a resistance mechanism against G9a inhibition in leukemia stem cells.

BACKGROUND: The histone methyltransferase G9a has recently been identified as a potential target for epigenetic therapy of acute myeloid leukemia (AML). However, the effect of G9a inhibition on leukemia stem cells (LSCs), which are responsible for AML drug resistance and recurrence, is unclear. In this study, we investigated the underlying mechanisms of the LSC resistance to G9a inhibition. METHODS: We evaluated the effects of G9a inhibition on the unfolded protein response and autophagy in AML and LSC-like cell lines and in primary CD34+CD38- leukemic blasts from patients with AML and investigated the underlying mechanisms. The effects of treatment on cells were evaluated by flow cytometry, western blotting, confocal microscopy, reactive oxygen species (ROS) production assay. RESULTS: The G9a inhibitor BIX-01294 effectively induced apoptosis in AML cell lines; however, the effect was limited in KG1 LSC-like cells. BIX-01294 treatment or siRNA-mediated G9a knockdown led to the activation of the PERK/NRF2 pathway and HO-1 upregulation in KG1 cells. Phosphorylation of p38 and intracellular generation of reactive oxygen species (ROS) were suppressed. Pharmacological or siRNA-mediated inhibition of the PERK/NRF2 pathway synergistically enhanced BIX-01294-induced apoptosis, with suppressed HO-1 expression, increased p38 phosphorylation, and elevated ROS generation, indicating that activated PERK/NRF2 signaling suppressed ROS-induced apoptosis in KG1 cells. By contrast, cotreatment of normal hematopoietic stem cells with BIX-01294 and a PERK inhibitor had no significant proapoptotic effect. Additionally, G9a inhibition induced autophagy flux in KG1 cells, while autophagy inhibitors significantly increased the BIX-01294-induced apoptosis. This prosurvival autophagy was not abrogated by PERK/NRF2 inhibition. CONCLUSIONS: PERK/NRF2 signaling plays a key role in protecting LSCs against ROS-induced apoptosis, thus conferring resistance to G9a inhibitors. Treatment with PERK/NRF2 or autophagy inhibitors could overcome resistance to G9a inhibition and eliminate LSCs, suggesting the potential clinical utility of these unique targeted therapies against AML.

Serum albumin and C-reactive protein as significant predictors of non-relapse mortality in lower gastrointestinal graft-versus-host disease.

Acute graft-versus-host disease (aGVHD) of the lower gastrointestinal (GI) tract is the major cause of non-relapse mortality (NRM) in allogeneic hematopoietic stem cell transplantation (alloHSCT). This study aimed to identify variables associated with corticosteroid response and NRM in patients who developed lower GI aGVHD. We retrospectively analyzed the clinical data of patients treated at Yonsei University Severance Hospital between 2008 and 2017. Among 244 recipients of alloHSCT, 48 (19.7%) were diagnosed as lower GI aGVHD at a median of 22 days after alloHSCT. In these cases, 20 (41.6%) patients were resistant to corticosteroid therapy. Corticosteroid resistance was associated with advanced stage of lower GI aGVHD (P = 0.019), low serum albumin (P = 0.006), and elevated CRP (P = 0.030) on day 7 after corticosteroid therapy. NRM rate was significantly higher in the corticosteroid-resistant group compared with the sensitive group (HR 5.339, P = 0.003). Multivariate analysis revealed serum albumin (P = 0.046), and CRP levels (P = 0.032) were independent prognostic factors for NRM. When the patients were classified into 3 groups according to Glasgow prognostic score (GPS), the rate of corticosteroid resistance was significantly higher in the high GPS group compared with the intermediate or low GPS group (83.3 vs. 27.2 and 15.3%, respectively, P < 0.001). We demonstrated that low serum albumin and elevated CRP level on day 7 after corticosteroid therapy are objective biomarkers of corticosteroid resistance and a significant predictor for higher NRM. These simple and practical parameters could be valuable information predicting response and prognosis in lower GI aGVHD.