Long-term follow-up after R-High CHOP/CHASER/LEED with Auto-PBSCT in untreated mantle cell lymphoma-Final analysis of JCOG0406.

Progression-free survival after R-High CHOP/CHASER/LEED with auto-PBSCT in untreated mantle cell lymphoma in JCOG0406 study. A continuous pattern of relapse was observed.

Postazacitidine clone size predicts long-term outcome of patients with myelodysplastic syndromes and related myeloid neoplasms.

Azacitidine is a mainstay of therapy for myelodysplastic syndrome (MDS)-related diseases. The purpose of our study is to elucidate the effect of gene mutations on hematological response and overall survival (OS), particularly focusing on their posttreatment clone size. We enrolled a total of 449 patients with MDS or related myeloid neoplasms. They were analyzed for gene mutations in pretreatment (n = 449) and posttreatment (n = 289) bone marrow samples using targeted-capture sequencing to assess the impact of gene mutations and their posttreatment clone size on treatment outcomes. In Cox proportional hazard modeling, multihit TP53 mutation (hazard ratio [HR], 2.03; 95% confidence interval [CI], 1.42-2.91; P < .001), EZH2 mutation (HR, 1.71; 95% CI, 1.14-2.54; P = .009), and DDX41 mutation (HR, 0.33; 95% CI, 0.17-0.62; P < .001), together with age, high-risk karyotypes, low platelets, and high blast counts, independently predicted OS. Posttreatment clone size accounting for all drivers significantly correlated with International Working Group (IWG) response (P < .001, using trend test), except for that of DDX41-mutated clones, which did not predict IWG response. Combined, IWG response and posttreatment clone size further improved the prediction of the original model and even that of a recently proposed molecular prediction model, the molecular International Prognostic Scoring System (IPSS-M; c-index, 0.653 vs 0.688; P < .001, using likelihood ratio test). In conclusion, evaluation of posttreatment clone size, together with the pretreatment mutational profile as well as the IWG response play a role in better prognostication of azacitidine-treated patients with myelodysplasia.

Hypoxia-induced paclitaxel resistance in cervical cancer modulated by miR-100 targeting of USP15.

Objective: Hypoxia, which occurs during the development of cervical cancer, confers chemotherapy resistance. MicroRNA expression is regulated by hypoxia and is associated with the onset and progression of certain types of cancer. MicroRNA-100 (miR-100) is a microRNA, associated with nasopharyngeal and oral squamous cell carcinomas, whose expression is decreased in cervical cancer. This study aims to ascertain the effect of hypoxia on expression levels of both miR-100 and its target genes, as well as exploring the sensitivity to paclitaxel under hypoxic conditions. Methods: We investigated the effect of hypoxia on miR-100 expression. We also explored the regulators of paclitaxel response under hypoxic conditions of cervical cancer. Results: Using RT-qPCR, we found that expression of miR-100 in cervical cancer cell lines SiHa and HeLa is significantly higher under hypoxic conditions (1% O2). We also confirmed that human ubiquitin-specific protease 15 (USP15) is the one of the target proteins of miR-100. Hypoxia and overexpression of miR-100 both reduced the activity of the luciferase reporter containing the 3'-untranslated region of USP15, which contains the miR-100 binding site. Furthermore, a western blot analysis showed that hypoxia suppresses the expression of the USP15 protein, while RT-qPCR showed hypoxia-induced downregulation of USP15 mRNA levels. We also discovered that overexpression of miR-100 induces paclitaxel resistance, thereby reducing the drug's therapeutic effect on cell death. Conclusions: Our results are consistent with the hypothesis that cervical cancer cells overexpress miR-100 in response to hypoxia and that miR-100 is a facilitator of USP15 downregulation and inactivation.

Germ line DDX41 mutations define a unique subtype of myeloid neoplasms.

Germ line DDX41 variants have been implicated in late-onset myeloid neoplasms (MNs). Despite an increasing number of publications, many important features of DDX41-mutated MNs remain to be elucidated. Here we performed a comprehensive characterization of DDX41-mutated MNs, enrolling a total of 346 patients with DDX41 pathogenic/likely-pathogenic (P/LP) germ line variants and/or somatic mutations from 9082 MN patients, together with 525 first-degree relatives of DDX41-mutated and wild-type (WT) patients. P/LP DDX41 germ line variants explained ∼80% of known germ line predisposition to MNs in adults. These risk variants were 10-fold more enriched in Japanese MN cases (n = 4461) compared with the general population of Japan (n = 20 238). This enrichment of DDX41 risk alleles was much more prominent in male than female (20.7 vs 5.0). P/LP DDX41 variants conferred a large risk of developing MNs, which was negligible until 40 years of age but rapidly increased to 49% by 90 years of age. Patients with myelodysplastic syndromes (MDS) along with a DDX41-mutation rapidly progressed to acute myeloid leukemia (AML), which was however, confined to those having truncating variants. Comutation patterns at diagnosis and at progression to AML were substantially different between DDX41-mutated and WT cases, in which none of the comutations affected clinical outcomes. Even TP53 mutations made no exceptions and their dismal effect, including multihit allelic status, on survival was almost completely mitigated by the presence of DDX41 mutations. Finally, outcomes were not affected by the conventional risk stratifications including the revised/molecular International Prognostic Scoring System. Our findings establish that MDS with DDX41-mutation defines a unique subtype of MNs that is distinct from other MNs.

Amplified EPOR/JAK2 Genes Define a Unique Subtype of Acute Erythroid Leukemia.

Acute erythroid leukemia (AEL) is a unique subtype of acute myeloid leukemia characterized by prominent erythroid proliferation whose molecular basis is poorly understood. To elucidate the underlying mechanism of erythroid proliferation, we analyzed 121 AEL using whole-genome, whole-exome, and/or targeted-capture sequencing, together with transcriptome analysis of 21 AEL samples. Combining publicly available sequencing data, we found a high frequency of gains and amplifications involving EPOR/JAK2 in TP53-mutated cases, particularly those having >80% erythroblasts designated as pure erythroid leukemia (10/13). These cases were frequently accompanied by gains and amplifications of ERG/ETS2 and associated with a very poor prognosis, even compared with other TP53-mutated AEL. In addition to activation of the STAT5 pathway, a common feature across all AEL cases, these AEL cases exhibited enhanced cell proliferation and heme metabolism and often showed high sensitivity to ruxolitinib in vitro and in xenograft models, highlighting a potential role of JAK2 inhibition in therapeutics of AEL. SIGNIFICANCE: This study reveals the major role of gains, amplifications, and mutations of EPOR and JAK2 in the pathogenesis of pure erythroleukemia. Their frequent response to ruxolitinib in patient-derived xenograft and cell culture models highlights a possible therapeutic role of JAK2 inhibition for erythroleukemia with EPOR/JAK2-involving lesions. This article is highlighted in the In This Issue feature, p. 369.

Potential of a sphingosine 1-phosphate receptor antagonist and sphingosine kinase inhibitors as targets for multiple myeloma treatment.

Sphingosine 1-phosphate (S1P) is a bioactive lipid involved in cancer progression through its binding to S1P receptors (S1PRs). However, the association between multiple myeloma (MM) and S1P is unclear. The current study aimed to investigate the potential anti-cancer effects of fingolimod and sphingosine kinase (SK) inhibitors in myeloma cells and the effects of S1P-induced chemoresistance and neovascularization on MM cell proliferation. MM cell lines were treated with the S1PR1 antagonist fingolimod and the SK inhibitors ABC294640 and SK1-I, after which cell proliferation was measured. Protein expression was also assessed under each condition using immunoblotting. Serum S1P levels in patients with MM, monoclonal gammopathy of undetermined significance and healthy volunteers were assessed. Human umbilical vessel cells (HUVECs) were co-cultured with anti-S1P agents to assess the effect on cell migration. All treatments suppressed myeloma cell proliferation and caspase-3-mediated apoptosis by suppressing S1P activity. These findings suggest that S1P activation is associated with proliferation and survival for MM cells. S1P attenuated the proteosome inhibitor (PI) effect, while the anti-S1P agents recovered the effect. In addition, S1P promoted the migration and proliferation of HUVECs, whereas the S1P inhibitors reduced the influence of S1P. This study highlights the therapeutic potential of anti-S1P agents for MM treatment. Inhibition of S1P function may overcome resistance to PI developed by myeloma cells and inhibit the changes to the bone marrow microenvironment via neovascularization.

Molecular International Prognostic Scoring System for Myelodysplastic Syndromes.

BACKGROUND: Risk stratification and therapeutic decision-making for myelodysplastic syndromes (MDS) are based on the International Prognostic Scoring System­Revised (IPSS-R), which considers hematologic parameters and cytogenetic abnormalities. Somatic gene mutations are not yet used in the risk stratification of patients with MDS. METHODS: To develop a clinical-molecular prognostic model (IPSS-Molecular [IPSS-M]), pretreatment diagnostic or peridiagnostic samples from 2957 patients with MDS were profiled for mutations in 152 genes. Clinical and molecular variables were evaluated for associations with leukemia-free survival, leukemic transformation, and overall survival. Feature selection was applied to determine the set of independent IPSS-M prognostic variables. The relative weights of the selected variables were estimated using a robust Cox multivariable model adjusted for confounders. The IPSS-M was validated in an external cohort of 754 Japanese patients with MDS. RESULTS: We mapped at least one oncogenic genomic alteration in 94% of patients with MDS. Multivariable analysis identified TP53multihit, FLT3 mutations, and MLLPTD as top genetic predictors of adverse outcomes. Conversely, SF3B1 mutations were associated with favorable outcomes, but this was modulated by patterns of comutation. Using hematologic parameters, cytogenetic abnormalities, and somatic mutations of 31 genes, the IPSS-M resulted in a unique risk score for individual patients. We further derived six IPSS-M risk categories with prognostic differences. Compared with the IPSS-R, the IPSS-M improved prognostic discrimination across all clinical end points and restratified 46% of patients. The IPSS-M was applicable in primary and secondary/therapy-related MDS. To simplify clinical use of the IPSS-M, we developed an open-access Web calculator that accounts for missing values. CONCLUSIONS: Combining genomic profiling with hematologic and cytogenetic parameters, the IPSS-M improves the risk stratification of patients with MDS and represents a valuable tool for clinical decision-making. (Funded by Celgene Corporation through the MDS Foundation, the Josie Robertson Investigators Program, the Edward P. Evans Foundation, the Projects of National Relevance of the Italian Ministry of University and Research, Associazione Italiana per la Ricerca sul Cancro, the Japan Agency for Medical Research and Development, Cancer Research UK, the Austrian Science Fund, the MEXT [Japanese Ministry of Education, Culture, Sports, Science and Technology] Program for Promoting Research on the Supercomputer Fugaku, the Japan Society for the Promotion of Science, the Taiwan Department of Health, and Celgene Corporation through the MDS Foundation.)

A multicenter, phase II study of R-THP-COP therapy for elderly patients with newly diagnosed, advanced-stage, indolent B-cell lymphoma.

The optimal combined chemotherapy regimen with rituximab has yet to be established for elderly patients with advanced-stage indolent B-cell lymphoma (B-NHL). A multicenter study was performed to evaluate the efficacy and toxicity of R-THP-COP therapy in elderly patients (aged 70-79 years) with newly diagnosed advanced-stage indolent B-NHL using the complete response rate (%CR) as the primary endpoint. Patients with newly diagnosed, clinical stage III/IV, indolent B-NHL, aged 70-79 years, with a performance status of 0-2 were eligible for this study. R-THP-COP consists of 375 mg/m2 of rituximab, 50 mg/m2 of pirarubicin, 750 mg/m2 of cyclophosphamide, 1.4 mg/m2 of vincristine, and 100 mg/day of oral prednisolone for 5 days. This study was discontinued due to poor accrual after the enrollment of 18 patients, although the planned sample size was 40 patients. The numbers of patients with follicular lymphoma, mucosa-associated lymphoid tissue lymphoma, and mantle cell lymphoma were 16, 1, and 1, respectively. The median age was 73 (range, 70 to 79) years. The %CR including unconfirmed CR was 45% (95% confidence interval: 25-66%) and the overall response rate was 72%. The estimated 5-year overall survival and progression-free survival rates were 55% and 28%, respectively. The major toxicity observed was grade 4 neutropenia (94%). Grade 4 non-hematological toxicities were not observed and no patients developed grade 3/4 cardiac toxicities. This phase II study provides useful information regarding the efficacy and toxicity of R-THP-COP therapy for patients aged 70 years or older with newly diagnosed, advanced-stage, indolent B-NHL, although the sample size was small.

Clonal evolution and clinical implications of genetic abnormalities in blastic transformation of chronic myeloid leukaemia.

Blast crisis (BC) predicts dismal outcomes in patients with chronic myeloid leukaemia (CML). Although additional genetic alterations play a central role in BC, the landscape and prognostic impact of these alterations remain elusive. Here, we comprehensively investigate genetic abnormalities in 136 BC and 148 chronic phase (CP) samples obtained from 216 CML patients using exome and targeted sequencing. One or more genetic abnormalities are found in 126 (92.6%) out of the 136 BC patients, including the RUNX1-ETS2 fusion and NBEAL2 mutations. The number of genetic alterations increase during the transition from CP to BC, which is markedly suppressed by tyrosine kinase inhibitors (TKIs). The lineage of the BC and prior use of TKIs correlate with distinct molecular profiles. Notably, genetic alterations, rather than clinical variables, contribute to a better prediction of BC prognosis. In conclusion, genetic abnormalities can help predict clinical outcomes and can guide clinical decisions in CML.

Overexpression of RUNX3 Represses RUNX1 to Drive Transformation of Myelodysplastic Syndrome.

RUNX3, a RUNX family transcription factor, regulates normal hematopoiesis and functions as a tumor suppressor in various tumors in humans and mice. However, emerging studies have documented increased expression of RUNX3 in hematopoietic stem/progenitor cells (HSPC) of a subset of patients with myelodysplastic syndrome (MDS) showing a worse outcome, suggesting an oncogenic function for RUNX3 in the pathogenesis of hematologic malignancies. To elucidate the oncogenic function of RUNX3 in the pathogenesis of MDS in vivo, we generated a RUNX3-expressing, Tet2-deficient mouse model with the pancytopenia and dysplastic blood cells characteristic of MDS in patients. RUNX3-expressing cells markedly suppressed the expression levels of Runx1, a critical regulator of hemaotpoiesis in normal and malignant cells, as well as its target genes, which included crucial tumor suppressors such as Cebpa and Csf1r. RUNX3 bound these genes and remodeled their Runx1-binding regions in Tet2-deficient cells. Overexpression of RUNX3 inhibited the transcriptional function of Runx1 and compromised hematopoiesis to facilitate the development of MDS in the absence of Tet2, indicating that RUNX3 is an oncogene. Furthermore, overexpression of RUNX3 activated the transcription of Myc target genes and rendered cells sensitive to inhibition of Myc-Max heterodimerization. Collectively, these results reveal the mechanism by which RUNX3 overexpression exerts oncogenic effects on the cellular function of and transcriptional program in Tet2-deficient stem cells to drive the transformation of MDS. SIGNIFICANCE: This study defines the oncogenic effects of transcription factor RUNX3 in driving the transformation of myelodysplastic syndrome, highlighting RUNX3 as a potential target for therapeutic intervention.

Effect of the extracellular component of bone marrow mesenchymal stromal cells from healthy donors on hematologic neoplasms and their angiogenesis.

Bone marrow mesenchymal stromal cells (BM-MSCs) from healthy donors are a promising source of cell therapy. However, their effectiveness in cancer remains less known. This study is the first to evaluate the quality of BM-MSCs obtained from young and elderly healthy volunteers (KNT cells). The KNT cells had normal karyotypes and were positive for MSC markers (CD90, CD73, CD105). When cultured under appropriate conditions, they showed adipogenic or osteogenic potential. Hence, the anti-neoplastic effects of secretory factors [supernatant or extracellular vesicles (EV)] from KNT cells were verified using several neoplastic cells (three multiple myeloma, three myeloid leukemia, and three lymphoma cell lines). The conditioned medium (CM), but not EV, of KNT cells derived from young healthy donors significantly inhibited myeloma and lymphoma cell proliferation, but enhanced myeloid leukemia proliferation. Anti-angiogenesis effect of CM and EV derived from young KNT against hematologic neoplasia-induced angiogenesis was evident and more prominent in CM than in EV but not evident in elderly KNT-derived EV. These findings indicate that the anti-tumor effect of KNT cells depends on the types of hematologic neoplasia, with elements existing in the supernatant and not in EVs. Therefore, BM-MSC may produce soluble factors that affect cell proliferation of neoplasia, causing cell-to-cell communication. The anti-angiogenesis effect of KNT cells depends on the age of BM-MSC donors.

Downregulation of extracellular vesicle microRNA-101 derived from bone marrow mesenchymal stromal cells in myelodysplastic syndrome with disease progression.

To evaluate the mechanism underlying the communication between myeloid malignant and bone marrow (BM) microenvironment cells in disease progression, the current study established BM mesenchymal stromal cells (MSCs) and assessed extracellular vesicle (EV) microRNA (miR) expression in 22 patients with myelodysplastic syndrome (MDS) and 7 patients with acute myeloid leukemia and myelodysplasia-related changes (AML/MRC). Patients with MDS were separated into two categories based on the revised International Prognostic Scoring System (IPSS-R), and EV-miR expression in BM-MSCs was evaluated using a TaqMan low-density array. The selected miRs were evaluated using reverse transcription-quantitative PCR. The current study demonstrated that the expression of BM-MSC-derived EV-miR was heterogenous and based on MDS severity, the expression of EV-miR-101 was lower in high-risk group and patients with AML/MRC compared with the control and low-risk groups. This reversibly correlated with BM blast percentage, with which the cellular miR-101 from BM-MSCs or serum EV-miR-101 expression exhibited no association. Database analyses indicated that miR-101 negatively regulated cell proliferation and epigenetic gene expression. The downregulation of BM-MSC-derived EV-miR-101 may be associated with cell-to-cell communication and may accelerate the malignant process in MDS cells.

Treatment-free remission after first-line dasatinib treatment in patients with chronic myeloid leukemia in the chronic phase: the D-NewS Study of the Kanto CML Study Group.

Treatment outcomes for chronic myeloid leukemia (CML) have dramatically improved with the development of tyrosine kinase inhibitors (TKI). However, due to the improved prognosis for CML, problems have arisen from long-term administration of TKI. The present study sought to verify whether more patients could achieve treatment-free remission (TFR) after stopping the administration of dasatinib using dasatinib as frontline treatment. Treatment-naïve chronic phase CML cases were treated with dasatinib as frontline treatment. Dasatinib treatment was stopped for 26 patients who achieved deep molecular response (DMR) within 24 months and were able to maintain DMR for an additional 2 years. Ten patients (38.5%) achieved DMR maintenance after 12 months. Recurrence was confirmed in 16 patients, and the median recurrence-free survival time was 5.1 months. The cumulative DMR rates at six and 12 months after restarting treatment were 84.6% and 100%, respectively. The results of this study demonstrated that the DMR maintenance rate after 12 months was 38.5%, which was not significantly different from previous TKI stop trials. The 2-year dasatinib administration period after reaching DMR did not contribute to improve TFR rates. These results suggest that the type of TKI is not associated with better TFR rates.

Induction of multiple myeloma bone marrow stromal cell apoptosis by inhibiting extracellular vesicle miR-10a secretion.

Bone marrow stromal cells (BMSCs) interact with multiple myeloma (MM) cells in the bone marrow and create a permissive microenvironment for MM cell proliferation and survival. In this study, we investigated the role of extracellular vesicles (EVs) from BMSCs derived from patients with MM (MM-BMSCs). EV-encapsulated miR-10a expression was high while intracellular miR-10a was low in MM-BMSCs. We therefore hypothesized that miR-10a was packaged into EVs that were actively released into the extracellular space. Inhibition of EV release resulted in accumulation of intracellular miR-10a, inhibition of cell proliferation, and induction of apoptosis in MM-BMSCs. In contrast, proliferation and apoptosis of BMSCs derived from healthy individuals were unaffected by inhibition of EV release. Furthermore, miR-10a derived from MM-BMSCs was transferred into MM cells via EVs and enhanced their proliferation. These results suggest that inhibition of EV release induced apoptosis in MM-BMSCs and inhibited MM cell proliferation, indicating a possible role for MM-BMSC-targeted therapy.

Author Correction: Lineage-specific RUNX2 super-enhancer activates MYC and promotes the development of blastic plasmacytoid dendritic cell neoplasm.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A novel non-invasive monitoring assay of 5-azacitidine efficacy using global DNA methylation of peripheral blood in myelodysplastic syndrome.

Purpose: Monitoring response and resistance to 5-azacitidine (AZA) is essential when treating patients with myelodysplastic syndrome (MDS). To quantify methylated DNA not only in the promoter region but also in the gene body, we established a single-molecule methylation assay (SMMA). Patients and methods: We first investigated the methylation extent (expressed as methylation index [MI]) by SMMA among 28 MDS and 6 post-MDS acute myeloid leukemia patients. We then analyzed the MI in 13 AZA-treated patients. Results: Whole-blood DNA from all 34 patients had low MI values compared with healthy volunteers (P<0.0001). DNA hypomethylation in MDS patients was more evident in neutrophils (P=0.0008) than in peripheral mononuclear cells (P=0.0713). No consistent pattern of genome-wide DNA hypomethylation was found among MDS subtypes or revised International Prognostic Scoring System (IPSS-R) categories; however, we found that the MI was significantly increased for patients at very high risk who were separated by the new cytogenetic scoring system for IPSS-R (P=0.0398). There was no significant difference in MI before AZA, regardless of the response to AZA (P=0.8689); however, sequential measurement of MI in peripheral blood demonstrated that AZA non-responders did not have normalized MI at the time of next course of AZA (P=0.0352). Conclusion: Our results suggest that sequential SMMA of peripheral blood after AZA may represent a non-invasive monitoring marker for AZA efficacy in MDS patients.

Lineage-specific RUNX2 super-enhancer activates MYC and promotes the development of blastic plasmacytoid dendritic cell neoplasm.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive subtype of acute leukemia, the cell of origin of which is considered to be precursors of plasmacytoid dendritic cells (pDCs). Since translocation (6;8)(p21;q24) is a recurrent anomaly for BPDCN, we demonstrate that a pDC-specific super-enhancer of RUNX2 is associated with the MYC promoter due to t(6;8). RUNX2 ensures the expression of pDC-signature genes in leukemic cells, but also confers survival and proliferative properties in BPDCN cells. Furthermore, the pDC-specific RUNX2 super-enhancer is hijacked to activate MYC in addition to RUNX2 expression, thereby promoting the proliferation of BPDCN. We also demonstrate that the transduction of MYC and RUNX2 is sufficient to initiate the transformation of BPDCN in mice lacking Tet2 and Tp53, providing a model that accurately recapitulates the aggressive human disease and gives an insight into the molecular mechanisms underlying the pathogenesis of BPDCN.