Sequential inverse dysregulation of the RNA helicases DDX3X and DDX3Y facilitates MYC-driven lymphomagenesis.

DDX3X is a ubiquitously expressed RNA helicase involved in multiple stages of RNA biogenesis. DDX3X is frequently mutated in Burkitt lymphoma, but the functional basis for this is unknown. Here, we show that loss-of-function DDX3X mutations are also enriched in MYC-translocated diffuse large B cell lymphoma and reveal functional cooperation between mutant DDX3X and MYC. DDX3X promotes the translation of mRNA encoding components of the core translational machinery, thereby driving global protein synthesis. Loss-of-function DDX3X mutations moderate MYC-driven global protein synthesis, thereby buffering MYC-induced proteotoxic stress during early lymphomagenesis. Established lymphoma cells restore full protein synthetic capacity by aberrant expression of DDX3Y, a Y chromosome homolog, the expression of which is normally restricted to the testis. These findings show that DDX3X loss of function can buffer MYC-driven proteotoxic stress and highlight the capacity of male B cell lymphomas to then compensate for this loss by ectopic DDX3Y expression.

A genomic-augmented multivariate prognostic model for the survival of natural-killer/T-cell lymphoma patients from an international cohort.

With lowering costs of sequencing and genetic profiling techniques, genetic drivers can now be detected readily in tumors but current prognostic models for Natural-killer/T cell lymphoma (NKTCL) have yet to fully leverage on them for prognosticating patients. Here, we used next-generation sequencing to sequence 260 NKTCL tumors, and trained a genomic prognostic model (GPM) with the genomic mutations and survival data from this retrospective cohort of patients using LASSO Cox regression. The GPM is defined by the mutational status of 13 prognostic genes and is weakly correlated with the risk-features in International Prognostic Index (IPI), Prognostic Index for Natural-Killer cell lymphoma (PINK), and PINK-Epstein-Barr virus (PINK-E). Cox-proportional hazard multivariate regression also showed that the new GPM is independent and significant for both progression-free survival (PFS, HR: 3.73, 95% CI 2.07-6.73; p < .001) and overall survival (OS, HR: 5.23, 95% CI 2.57-10.65; p = .001) with known risk-features of these indices. When we assign an additional risk-score to samples, which are mutant for the GPM, the Harrell's C-indices of GPM-augmented IPI, PINK, and PINK-E improved significantly (p < .001, χ2 test) for both PFS and OS. Thus, we report on how genomic mutational information could steer toward better prognostication of NKTCL patients.

Advanced Hodgkin lymphoma in the East of England: a 10-year comparative analysis of outcomes for real-world patients treated with ABVD or escalated-BEACOPP, aged less than 60 years, compared with 5-year extended follow-up from the RATHL trial.

Treatment with ABVD (doxorubicin, bleomycin, vinblastine, and dacarbazine) or escalated(e)-BEACOPP (bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine, and prednisolone) remains the international standard of care for advanced-stage classical Hodgkin lymphoma (HL). We performed a retrospective, multicentre analysis of 221 non-trial ("real-world") patients, aged 16-59 years, diagnosed with advanced-stage HL in the Anglia Cancer Network between 2004 and 2014, treated with ABVD or eBEACOPP, and compared outcomes with 1088 patients in the Response-Adjusted Therapy for Advanced Hodgkin Lymphoma (RATHL) trial, aged 18-59 years, with median follow-up of 87.0 and 69.5 months, respectively. Real-world ABVD patients (n=177) had highly similar 5-year progression-free survival (PFS) and overall survival (OS) compared with RATHL (PFS 79.2% vs 81.4%; OS 92.9% vs 95.2%), despite interim positron-emission tomography-computed tomography (PET/CT)-guided dose-escalation being predominantly restricted to trial patients. Real-world eBEACOPP patients (n=44) had superior PFS (95.5%) compared with real-world ABVD (HR 0.20, p=0.027) and RATHL (HR 0.21, p=0.015), and superior OS for higher-risk (international prognostic score ≥3 [IPS 3+]) patients compared with real-world IPS 3+ ABVD (100% vs 84.5%, p=0.045), but not IPS 3+ RATHL patients. Our data support a PFS, but not OS, advantage for patients with advanced-stage HL treated with eBEACOPP compared with ABVD and suggest higher-risk patients may benefit disproportionately from more intensive therapy. However, increased access to effective salvage therapies might minimise any OS benefit from reduced relapse rates after frontline therapy.

Oncogenic activation of the STAT3 pathway drives PD-L1 expression in natural killer/T-cell lymphoma.

Mature T-cell lymphomas, including peripheral T-cell lymphoma (PTCL) and extranodal NK/T-cell lymphoma (NKTL), represent a heterogeneous group of non-Hodgkin lymphomas with dismal outcomes and limited treatment options. To determine the extent of involvement of the JAK/STAT pathway in this malignancy, we performed targeted capture sequencing of 188 genes in this pathway in 171 PTCL and NKTL cases. A total of 272 nonsynonymous somatic mutations in 101 genes were identified in 73% of the samples, including 258 single-nucleotide variants and 14 insertions or deletions. Recurrent mutations were most frequently located in STAT3 and TP53 (15%), followed by JAK3 and JAK1 (6%) and SOCS1 (4%). A high prevalence of STAT3 mutation (21%) was observed specifically in NKTL. Novel STAT3 mutations (p.D427H, E616G, p.E616K, and p.E696K) were shown to increase STAT3 phosphorylation and transcriptional activity of STAT3 in the absence of cytokine, in which p.E616K induced programmed cell death-ligand 1 (PD-L1) expression by robust binding of activated STAT3 to the PD-L1 gene promoter. Consistent with these findings, PD-L1 was overexpressed in NKTL cell lines harboring hotspot STAT3 mutations, and similar findings were observed by the overexpression of p.E616K and p.E616G in the STAT3 wild-type NKTL cell line. Conversely, STAT3 silencing and inhibition decreased PD-L1 expression in STAT3 mutant NKTL cell lines. In NKTL tumors, STAT3 activation correlated significantly with PD-L1 expression. We demonstrated that STAT3 activation confers high PD-L1 expression, which may promote tumor immune evasion. The combination of PD-1/PD-L1 antibodies and STAT3 inhibitors might be a promising therapeutic approach for NKTL, and possibly PTCL.

A Pre-Leukemic DNA Methylation Signature in Healthy Individuals at Higher Risk for Developing Myeloid Malignancy.

PURPOSE: DNA methylation alterations are widespread in acute myelogenous leukemia (AML) and myelodysplastic syndrome (MDS), some of which appear to have evolved independently of somatic mutations in epigenetic regulators. Although the presence of somatic mutations in peripheral blood can predict the risk of development of AML and MDS, its accuracy remains unsatisfactory. EXPERIMENTAL DESIGN: We performed global DNA methylation profiling in a case control study nested within the Singapore Chinese Health Study to evaluate whether DNA methylation alterations were associated with AML/MDS development. Targeted deep sequencing and methylated DNA immunoprecipitation sequencing (MeDIP-seq) were performed on peripheral blood collected a median of 9.9 years before diagnosis of AML or MDS, together with age-matched still-healthy individuals as controls. RESULTS: Sixty-six individuals who developed AML or MDS displayed significant DNA methylation changes in the peripheral blood compared with 167 age- and gender-matched controls who did not develop AML/MDS during the follow-up period. Alterations in methylation in the differentially methylation regions were associated with increased odds of developing AML/MDS. CONCLUSIONS: The epigenetic changes may be acquired independently and before somatic mutations that are relevant for AML/MDS development. The association between methylation changes and the risk of pre-AML/MDS in these individuals was considerably stronger than somatic mutations, suggesting that methylation changes could be used as biomarkers for pre-AML/MDS screening.

Evolving therapeutic landscape of diffuse large B-cell lymphoma: challenges and aspirations.

Diffuse large B-cell lymphoma (DLBCL) represents the commonest subtype of non-Hodgkin lymphoma and encompasses a group of diverse disease entities, each harboring unique molecular and clinico-pathological features. The understanding of the molecular landscape of DLBCL has improved significantly over the past decade, highlighting unique genomic subtypes with implications on targeted therapy. At the same time, several new treatment modalities have been recently approved both in the frontline and relapsed settings, ending a dearth of negative clinical trials that plagued the past decade. Despite that, in the real-world setting, issues like drug accessibility, reimbursement policies, physician and patient preference, as well as questions regarding optimal sequencing of treatment options present difficulties and challenges in day-to-day oncology practice. Here, we review the recent advances in the therapeutic armamentarium of DLBCL and discuss implications on the practice landscape, with a particular emphasis on the context of the healthcare system in Singapore.

Aberrant JAK-STAT signaling-mediated chromatin remodeling impairs the sensitivity of NK/T-cell lymphoma to chidamide.

BACKGROUND: Natural killer/T-cell lymphoma (NKTL) is a rare type of aggressive and heterogeneous non-Hodgkin's lymphoma (NHL) with a poor prognosis and limited therapeutic options. Therefore, there is an urgent need to exploit potential novel therapeutic targets for the treatment of NKTL. Histone deacetylase (HDAC) inhibitor chidamide was recently approved for treating relapsed/refractory peripheral T-cell lymphoma (PTCL) patients. However, its therapeutic efficacy in NKTL remains unclear. METHODS: We performed a phase II clinical trial to evaluate the efficacy of chidamide in 28 relapsed/refractory NKTL patients. Integrative transcriptomic, chromatin profiling analysis and functional studies were performed to identify potential predictive biomarkers and unravel the mechanisms of resistance to chidamide. Immunohistochemistry (IHC) was used to validate the predictive biomarkers in tumors from the clinical trial. RESULTS: We demonstrated that chidamide is effective in treating relapsed/refractory NKTL patients, achieving an overall response and complete response rate of 39 and 18%, respectively. In vitro studies showed that hyperactivity of JAK-STAT signaling in NKTL cell lines was associated with the resistance to chidamide. Mechanistically, our results revealed that aberrant JAK-STAT signaling remodels the chromatin and confers resistance to chidamide. Subsequently, inhibition of JAK-STAT activity could overcome resistance to chidamide by reprogramming the chromatin from a resistant to sensitive state, leading to synergistic anti-tumor effect in vitro and in vivo. More importantly, our clinical data demonstrated that combinatorial therapy with chidamide and JAK inhibitor ruxolitinib is effective against chidamide-resistant NKTL. In addition, we identified TNFRSF8 (CD30), a downstream target of the JAK-STAT pathway, as a potential biomarker that could predict NKTL sensitivity to chidamide. CONCLUSIONS: Our study suggests that chidamide, in combination with JAK-STAT inhibitors, can be a novel targeted therapy in the standard of care for NKTL. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02878278. Registered 25 August 2016, https://clinicaltrials.gov/ct2/show/NCT02878278.

Patterns of Oncogene Coexpression at Single-Cell Resolution Influence Survival in Lymphoma.

Cancers often overexpress multiple clinically relevant oncogenes, but it is not known if combinations of oncogenes in cellular subpopulations within a cancer influence clinical outcomes. Using quantitative multispectral imaging of the prognostically relevant oncogenes MYC, BCL2, and BCL6 in diffuse large B-cell lymphoma (DLBCL), we show that the percentage of cells with a unique combination MYC+BCL2+BCL6- (M+2+6-) consistently predicts survival across four independent cohorts (n = 449), an effect not observed with other combinations including M+2+6+. We show that the M+2+6- percentage can be mathematically derived from quantitative measurements of the individual oncogenes and correlates with survival in IHC (n = 316) and gene expression (n = 2,521) datasets. Comparative bulk/single-cell transcriptomic analyses of DLBCL samples and MYC/BCL2/BCL6-transformed primary B cells identify molecular features, including cyclin D2 and PI3K/AKT as candidate regulators of M+2+6- unfavorable biology. Similar analyses evaluating oncogenic combinations at single-cell resolution in other cancers may facilitate an understanding of cancer evolution and therapy resistance. SIGNIFICANCE: Using single-cell-resolved multiplexed imaging, we show that selected subpopulations of cells expressing specific combinations of oncogenes influence clinical outcomes in lymphoma. We describe a probabilistic metric for the estimation of cellular oncogenic coexpression from IHC or bulk transcriptomes, with possible implications for prognostication and therapeutic target discovery in cancer. This article is highlighted in the In This Issue feature, p. 1027.

Role of PET imaging in peritoneal involvement of subcutaneous panniculitis-like T-cell lymphoma.

Subcutaneous panniculitis-like T-cell lymphoma is a rare subtype of cutaneous T-cell lymphomas and represents less than 1% of non-Hodgkin's lymphomas. Currently, the diagnosis is based on clinical and histological findings although clinical features may be nonspecific. Often, it is localised to subcutaneous tissue without lymph node involvement. The literature is sparse but unusual presentations have been described to involve mesentery, breast and even eyelids. Fluorine-18 fluorodeoxyglucose positron-emission tomography/computed tomography has been reported to be useful in assessing disease activity, extent and treatment response in subcutaneous panniculitis-like T-cell lymphoma but we find that it can also be a diagnostic aid for atypical presentations. In our case report, we describe a patient who presented with a neck lump but did not have any other obvious cutaneous lesions. This was biopsied and had histological features in keeping with subcutaneous panniculitis-like T-cell lymphoma. Due to the atypical presentation, positron-emission tomography was crucial for detecting the extracutaneous and likely primary site of disease in the peritoneum, which hence guided the subsequent biopsy to this affected area and confirmed the diagnosis.

Obstacles for T-lymphocytes in the tumour microenvironment: Therapeutic challenges, advances and opportunities beyond immune checkpoint.

The tumour microenvironment (TME) imposes a major obstacle to infiltrating T-lymphocytes and suppresses their function. Several immune checkpoint proteins that interfere with ligand/receptor interactions and impede T-cell anti-tumour responses have been identified. Immunotherapies that block immune checkpoints have revolutionized the treatment paradigm for many patients with advanced-stage tumours. However, metabolic constraints and soluble factors that exist within the TME exacerbate the functional exhaustion of tumour-infiltrating T-cells. Here we review these multifactorial constraints and mechanisms - elevated immunosuppressive metabolites and enzymes, nutrient insufficiency, hypoxia, increased acidity, immense amounts of extracellular ATP and adenosine, dysregulated bioenergetic and purinergic signalling, and ionic imbalance - that operate in the TME and collectively suppress T-cell function. We discuss how scientific advances could help overcome the complex TME obstacles for tumour-infiltrating T-lymphocytes, aiming to stimulate further research for developing new therapeutic strategies by harnessing the full potential of the immune system in combating cancer.

Treatment outcomes of T and natural-killer/T-cell lymphoma with ifosfamide, carboplatin and etoposide chemotherapy.

BACKGROUND: Contemporary data of peripheral T-cell lymphoma (PTCL) and natural-killer/T-cell lymphoma (NKTL) patients treated with ifosfamide, carboplatin and etoposide (ICE) are limited. AIMS: We performed a retrospective analysis to estimate outcomes of ICE-treated PTCL and NKTL patients at three tertiary cancer centres in Singapore. METHODS AND RESULTS: Patients were identified through lymphoma databases from National Cancer Centre Singapore (NCCS), National University Hospital, Singapore (NUHS), and Singapore General Hospital (SGH). Responses and survival outcomes were determined from electronic medical records. A total of 75 patients with a median age of 50 were included. ICE was used as first-line treatment in 14 patients (19%) and as subsequent lines of treatment in 61 patients (81%). The overall response rates (ORR) for all patients was 63% (40% complete response [CR]). The ORR and CR in the first line were 86% and 64% respectively. At a median follow-up duration of 71.0 months, the median progression-free (PFS) and overall survival (OS) for all patients were 4.4 months (95%CI, 2.7-6.0) and 16 months (95%CI, 8.3-45.4) respectively. CONCLUSION: In summary, ICE showed high ORR but poor PFS in relapsed/refractory PTCL and NKTL. ORR of ICE in the first line setting appears better than real-world CHOP data and warrants further study.

High-dose methotrexate is effective for prevention of isolated CNS relapse in diffuse large B cell lymphoma.

The role of central nervous system (CNS) prophylaxis with high-dose methotrexate (HDMTX) in DLBCL is controversial. In this retrospective study, we evaluated the efficacy of prophylactic HDMTX on isolated CNS relapse, concomitant CNS and systemic relapse, systemic relapse, and survival outcomes in 226 patients with newly diagnosed DLBCL and high-risk CNS International Prognostic Index (CNS-IPI) score treated with RCHOP. The three-year risk of isolated CNS relapse was significantly lower in patients who received HDMTX, at 3.1% compared to 14.6% (P = 0.032) in those who did not. However, neither concomitant CNS-systemic relapse rates, systemic relapse rates, nor three-year PFS and OS were significantly different between treatment groups in multivariable analysis. Among propensity score-matched patients (N = 102), HDMTX was also associated with significantly lower isolated CNS relapse rates (HR 0.06, 95% CI 0.004-0.946, P = 0.046). HDMTX was well tolerated with manageable toxicities when given at a dose of 3 g/m2 by day 3 of RCHOP chemotherapy. Using propensity score matching and multivariable regression to yield treatment groups with well-balanced covariates, we showed that prophylactic HDMTX improved isolated CNS relapse rates but did not decrease concomitant CNS-systemic relapse rates, systemic relapse rates, or improve survival outcomes.

HEMATOPOIETIC STEM CELL TRANSPLANT IN AGGRESSIVE T AND NK/T CELL LYMPHOMA - ROLE OF UPFRONT AUTOLOGOUS TRANSPLANT IN NODAL PERIPHERAL T-CELL LYMPHOMA.

Aggressive T and NK/T-cell lymphoma are known to have a high risk of relapse and poor long-term prognosis. Hematopoietic stem cell transplantation has been performed as part of consolidation or salvage treatment. We retrospectively studied the outcomes of autologous (A) and allogeneic (allo) hematopoietic stem cell transplantation (SCT) in aggressive T and NK/T-cell lymphoma at our center between 2010 to 2020. Patients with nodal peripheral T-cell lymphoma (PTCL) that were younger than 65 years old who did not receive upfront autologous SCT (ASCT) at first complete remission were selected from our registry data for further comparison. Thirty-six patients underwent ASCT, and 16 patients underwent alloSCT. In the ASCT cohort, 18 patients with nodal PTCL who underwent upfront ASCT at first complete remission (upfront ASCT) were compared with 15 patients with nodal PTCL who were in first complete remission after single-line induction but did not receive ASCT. The two-year progression-free survival (PFS) and overall survival (OS) rates for the ASCT cohort were 58% and 73%, respectively. The two-year PFS and OS for the alloSCT cohort were 47% (P=0.35, P=0.02, respectively). Twenty-four patients who received SCT at first remission (21 ASCT and three alloSCT) had a two-year PFS and OS of 75% and 89%, respectively. In comparison, 28 patients who received SCT at relapse/refractory (15 ASCT and 13 alloSCT) had a two-year PFS and OS of 40% and 50%, respectively (P=0.047, P=0.024, respectively). Patients in complete remission prior to transplantation (n=42) had a two-year PFS and OS of 59% and 73%, respectively. In contrast, patients in partial remission prior to transplantation (n=10) had a two-year PFS and OS of 40% and 48%, respectively (p>0.05). Non-relapse mortality occurred in 6% and 43% of ASCT and AlloSCT, respectively. Multivariate analysis revealed that EBV-positivity at diagnosis indicated poorer PFS. EBV-positivity at diagnosis and more than two prior lines of treatment at transplant were associated with poorer OS. For nodal PTCL, the two-year PFS and OS were 79% and 100% for the upfront ASCT cohort and 78% and 92% for the non-upfront ASCT cohort, respectively (p>0.05). Hematopoietic SCT is a feasible treatment option for aggressive T and NK/T-cell lymphoma. Patients who underwent SCT at first remission had better survival rates than those who underwent SCT at relapse/refractory. Nevertheless, due to the limited sample size of the current study, the role of upfront ASCT in patients with nodal PTCL who achieved first complete remission remains unclear.

CREBBP cooperates with the cell cycle machinery to attenuate chidamide sensitivity in relapsed/refractory diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) exhibits frequent inactivating mutations of the histone acetyltransferase CREBBP, highlighting the attractiveness of targeting CREBBP deficiency as a therapeutic strategy. In this study, we demonstrate that chidamide, a novel histone deacetylase (HDAC) inhibitor, is effective in treating a subgroup of relapsed/refractory DLBCL patients, achieving an overall response rate (ORR) of 25.0% and a complete response (CR) rate of 15.0%. However, the clinical response to chidamide remains poor, as most patients exhibit resistance, hampering the clinical utility of the drug. Functional in vitro and in vivo studies have shown that CREBBP loss of function is correlated with chidamide sensitivity, which is associated with modulation of the cell cycle machinery. A combinatorial drug screening of 130 kinase inhibitors targeting cell cycle regulators identified AURKA inhibitors, which inhibit the G2/M transition during the cell cycle, as top candidates that synergistically enhanced the antitumor effects of chidamide in CREBBP-proficient DLBCL cells. Our study demonstrates that CREBBP inactivation can serve as a potential biomarker to predict chidamide sensitivity, while combination of an AURKA inhibitor and chidamide is a novel therapeutic strategy for the treatment of relapsed/refractory DLBCL.

The impact of time from diagnosis to treatment in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is a high-grade lymphoma that requires treatment. We retrospectively analyzed the impact of time from diagnosis-to-treatment (TDT) on progression-free survival (PFS) and overall survival (OS) in 581 R-CHOP-treated patients. TDT was defined as the interval between diagnostic biopsy date and day 1 R-CHOP. Cox regression showed stage 3-4 disease (p = .01) and longer TDT (HR 1.13, p =.031) were associated with shorter OS. Eastern Cooperative Oncology Group ≥2 (p = .02), stage 3-4 disease (p < .001), and longer TDT (HR 1.12, p = .028) predicted shorter PFS. The significant interactions between TDT with lactate dehydrogenase (LDH) and with disease stage prompted separate analyses in high versus normal LDH, and stage 3-4 versus 1-2 disease. Longer TDT was associated with shortened PFS and OS only with advanced stage, and, if high LDH was present. Treatment should be started as early as possible for high-tumor burden disease. Delaying treatment in patients with early stage or low LDH does not seem harmful.

Stem cells in cardiac repair.

Advances in the treatment of acute myocardial infarction have led to increased survival but also an increase in those suffering from heart failure. The concept of adult progenitor cell therapy has grown from observations that cardiac function improves after injection of bone marrow-derived cells into infarcted regions in animal models. The mechanism of action is unclear and is probably multifactorial. Different subpopulations of autologous cells have been utilised in humans with acute myocardial infarction and heart failure, as well different routes of delivery. These small Phase I clinical trials suggest that this approach is safe and leads to clinical improvement, and larger scale randomised control studies are underway. We believe that cell therapy heralds a revolutionary new era in the treatment of disease.