Low-risk Diffuse Large B-cell Lymphoma Research Articles

Using Deauville Scoring to Guide Consolidative Radiotherapy in Diffuse Large B-Cell Lymphoma

Background: The most common aggressive lymphoma in adults is diffuse large B-cell lymphoma (DLBCL). Consolidative radiotherapy (RT) is often administered to DLBCL patients but guidelines remain unclear, which could lead to unnecessary RT. We aimed to evaluate the value of end-of-treatment PET-CT scans, interpreted using the Deauville score (DV), to guide the utilization of consolidative RT, which may help spare low-risk DLBCL patients from unnecessary RT. Methods: We included all DLBCL patients diagnosed between 2010 and 2022 at the National Cancer Centre Singapore with DV measured at the end of the first-line chemoimmunotherapy. The outcome measure was time-to-progression (TTP). The predictive value of DV for RT was assessed based on the interaction effect between the receipt of RT and DV in Cox regression models. Results: The data of 349 patients were analyzed. The median follow-up time was 38.1 months (interquartile range 34.0–42.3 months). RT was associated with a significant improvement in TTP amongst the DV4-5 patients (HR 0.33; 95%CI 0.13–0.88; p = 0.027) but not the DV1-3 patients (HR 0.85; 95%CI 0.40–1.81; p = 0.671) (interaction’s p = 0.133). Multivariable analysis reported that RT was again significantly associated with improved TTP among the DV4-5 patients (adjusted HR 0.29; 95%CI 0.10–0.80; p = 0.017) but not the DV1-3 group (HR 0.86; 95%CI 0.40–1.86; p = 0.707) (interaction’s p = 0.087). Conclusion: Our results suggests that DLBCL patients with end-of-treatment PET-CT DV1-3 may not need consolidative RT. Longer follow-up and prospective randomized trials are still necessary to investigate long-term outcomes.

Identification of lactate regulation pattern on tumor immune infiltration, therapy response, and DNA methylation in diffuse large B-cell lymphoma.

Lactate, produced through glycolytic metabolism in the tumor microenvironment (TME), is implicated in tumorigenesis and progression in diverse cancers. However, the impact of lactate on the remodeling of the TME in diffuse large B-cell lymphoma (DLBCL) and its implications for therapy options remain unclear. A lactate-related (LAR) scoring model was constructed in DLBCL patients using bioinformatic methods. CIBERSORT, XCELL, and ssGSEA algorithms were used to determine the correlation between LAR score and immune cell infiltration. Tumor Immune Dysfunction and Exclusion (TIDE), rituximab, cyclophosphamide, adriamycin, vincristine, and prednisone (R-CHOP) cohorts, and Genomics of Drug Sensitivity in Cancer (GDSC) were utilized to predict the therapeutic response of DLBCL patients. The impact of the hub gene STAT4 on tumor biological behavior and DNA methylation was experimentally validated or accessed by the TSIDE database. The LAR scoring model was developed based on 20 prognosis-related lactate genes, which enabled the division of DLBCL patients into high- and low-risk groups based on the median LAR score. Patients with high-risk DLBCL exhibited significantly worse survival outcomes in both the training cohorts (GSE181063) and the validation cohorts (GSE10846, GSE32918, and GSE69053), as indicated by statistically significant differences (all P<0.05) and area under the curve (AUC) values exceeding 0.6. Immune analyses revealed that low-risk DLBCL patients had higher levels of immune cell infiltration and antitumor immune activation compared to high-risk DLBCL patients. Furthermore, DLBCL patients with high LAR scores were associated with a lower TIDE value and poor therapeutic efficacy of the R-CHOP regimen. GDSC analysis identified 18 drugs that exhibited significant response sensitivity in low-risk DLBCL patients. Moreover, in vitro experiments demonstrated that overexpression of the lactate key gene STAT4 could suppress proliferation and migration, induce cell cycle arrest, and promote cell apoptosis in DLBCL cells. Transcriptional expression and methylation of the STAT4 gene were found to be associated with immunomodulators and chemokines. The lactate-based gene signature effectively predicts the prognosis and regulates TME in DLBCL. Our study underscores the role of lactate gene, STAT4, as an important tumor suppressor in DLBCL. Modulating STAT4 could be a promising strategy for DLBCL in clinical practice.

Positron emission tomography-adapted therapy in low-risk diffuse large B-cell lymphoma: results of a randomized, phase III, non-inferiority trial.

The current standard of care for non-bulky diffuse large B-cell lymphoma (DLBCL) patients with an International Prognostic Index (IPI) of 0 is four cycles of rituximab plus cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP) but whether the same efficacy can be achieved with reduced chemotherapy regimen of four cycles for non-bulky DLBCL patients with an IPI of 1 remains unclear. This study compared four cycles versus six cycles of chemotherapy in non-bulky low-risk DLBCL patients with negative interim positron emission tomography with computed tomography (PET-CT, Deauville 1-3), irrespective of age and other IPI risk factors (IPI 0-1). This was an open-label, randomized, phase III, non-inferiority trial. Patients aged 14-75 years with newly diagnosed low-risk DLBCL, according to IPI, achieving PET-CT confirmed complete response (CR) after four cycles of R-CHOP were randomized (1:1) between four cycles of rituximab (4R-CHOP+4R arm) or two cycles of R-CHOP plus two cycles of rituximab (6R-CHOP+2R arm). The primary endpoint was 2-year progression-free survival (PFS), conducted in the intention-to-treat population. Safety was assessed in patients with at least one cycle of assigned treatment. The non-inferiority margin was -8%. A total of 287 patients were included in the intention-to-treat analysis, the median follow-up was 47.3 months, and the 2-year PFS rate was 95% (95% confidence interval [CI], 92% to 99%) and 94% (95% CI, 91% to 98%) for the 4R-CHOP+4R and 6R-CHOP+2R arm. The absolute difference in 2-year PFS between the two arms was 1% (95% CI, -5% to 7%), supporting the non-inferiority of 4R-CHOP+4R. Grade 3-4 neutropenia was lower in the last four cycles of rituximab alone in the 4R-CHOP+4R arm (16.7% versus 76.9%), with decreased risk of febrile neutropenia (0.0% versus 8.4%) and infection (2.1% versus 14.0%). For newly diagnosed low-risk DLBCL patients, interim PET-CT after four cycles of R-CHOP was effective in identifying patients with Deauville 1-3 who would have a good response and Deauville 4-5 patients who might have high-risk biological features or develop resistance. Reducing the standard six cycles to four cycles of chemotherapy had comparable clinical efficacy and fewer adverse events in low-risk, non-bulky DLBCL with interim PET-CT confirmed CR.

17th International Conference on Malignant Lymphoma, Palazzo dei Congressi, Lugano, Switzerland, 13 - 17 June, 2023.

Background: Previous researches have mainly focused on the discussion of whether cancer stem cells exist in diffuse large B-cell lymphoma (DLBCL). However, some subgroups with dismal prognosis that exhibit stem cell-like characteristics have been overlooked. Methods: Using an innovative one-class logistic regression (OCLR) machine learning algorithm, we calculated stemness indices and systematically assessed their ability to reflect the oncogenic dedifferentiation-like characteristics of DLBCL. Next, we identified signatures associated with oncogenic dedifferentiation-like features and prognosis using LASSO and SVM-RFE algorithms and developed a proportional hazards model (Riskscore) for DLBCL through Cox regression. Then we stratified the risk of DLBCL (n = 2133) and evaluated the prognostic value of the risk model across known clinical and molecular subgroups. We further compared the characteristics of high- and low-risk DLBCL with Burkitt lymphoma. Finally, we investigated the mechanisms of poor prognosis in high-risk DLBCL using transcriptomics, genomics (n = 576) and single-cell RNA sequencing (n = 19) data, as well as cell experiments and internal validation cohorts. Results: In this study, we identified and validated a DLBCL subgroup (25.6% of DLBCL) with stem cell-like characteristics and dismal prognosis. This high-risk group was defined as polyamine metabolism-cold immune tumor with upregulated polyamine metabolic key enzyme (ODC1) and desert-like immune infiltration, and had a poor prognosis with lower 3-year OS rate (54.3% vs. 83.6%, p < 0.0001) and PFS rate (42.8% vs. 75.2%, p < 0.0001) compared to the low-risk group. We found that some patients with MYC rearrangement, double-hit, double-expresser, or complete remission may have either favorable or poor prognosis, which can be accurately identified by our risk stratification model. Additionally, the high-risk group exhibited malignant proliferative phenotypes similar to Burkitt lymphoma. Genomic analysis revealed widespread copy number losses in the chemokine and interferon coding regions 8p23.1 and 9p21.3 in the high-risk group. Bulk and single-cell transcriptome analysis indicated that the upregulation of ODC1 might mediate the cold immune microenvironment of DLBCL, and knocking down ODC1 effectively inhibited DLBCL cell proliferation. Promisingly, our model effectively identified patients who were insensitive to immunotherapy (CAR-T and immune checkpoint antibodies). The research was funded by: This work was supported by grants from the National Natural Science Foundation of China (grant no. 81873450, 82170181), Beijing Hospitals Authority Youth Programme (code: QML20200201), and Beijing Natural Science Foundation (No. 7222027) to Liang Wang. Keywords: Bioinformatics, Computational and Systems Biology, Microenvironment, Risk Models No conflicts of interests pertinent to the abstract.

PET-Driven Radiotherapy (RT) in Patients with Low Risk Diffuse Large B-Cell Lymphoma (DLBCL): 5-Year Results of the DLCL10 Multicenter Phase 2 Trial By Fondazione Italiana Linfomi (FIL)

Background Recent data show that RT on PET-negative sites after chemo-immunotherapy in early stage DLBCL can be avoided. Nevertheless, bulky sites at diagnosis are still irradiated after rituximab-chemotherapy (R-CT), whereas residual uptake area (RUA at other sites are considered as failure. As PET negativity is mandatory to define complete remission (CR), we hypothesized that PET-neg areas after R-CT do not need consolidation RT independently from their size at onset Aims: To assess the role of RT in PET-neg and in PET-pos low-risk DLBCL patients after R-CT Methods: The DLCL10 protocol was a phase II study of patients (pts) >=18 years with low risk DLBCL according to the MiNT trial, (aa IPI 0 and bulky, aa IPI 1 +/- bulky) conducted in 19 FIL centers. Pts were treated with 6 courses of RCHOP and final response was evaluated with FDG-PET. Both pre and post treatment PET scans were centrally reviewed through the Widen web platform by a panel of 5 nuclear medicine experts. Positive scans were those centrally classified with Deauville score 3-4-5 by the first 2 concordant reviewers. Pts with one RUA received RT, 36 Gy involved-site, regardless of bulky disease at onset, while those with multiple RUA were shifted to salvage systemic therapy. Primary aim was to obtain a 2-year PFS of at least 85% for post R-CT PET-neg pts. Secondary endpoints were OS and response. Results: From January 2012 to December 2017, 115 consecutive pts were screened, and 110 were evaluable. Median age 58 years (47-65); M:F 61 /49; DLBCL de novo 90%, aa IPI 0 16 , aa IPI 1 94 (20% with bulky mass), bulky disease in the whole series 35 pts ; RCHOP-14 /RCHOP-21 73/37. At the time of the present analysis median follow-up was 63 months and 13 pts died (3 lymphoma, 2 acute toxicitiy, 2 late toxicity, 2 secondary neoplasm, 2 complication from allo-transplant, 1 other causes, 1 unknown). A total of 105 pts completed the R-CT program, while five were discontinuated for lymphoma progression (1), toxicity (2, both died), histological review (1) and patient dispersion (1). At end of treatment, 83 patients had PET-neg, whereas 17 had single RUA and received involved -site RT. In PET-neg patients, PFS was 90.6% (95% CI 81.1-95.4) at 2 years and 82.48% (95% CI 78.4-94.3) at 5 years. OS was 96.37% (95% CI 89.17-98.81) at 2 years and 87.81% (95% CI 77.62-93.54) at 5 years. After RT, 15 pts reached CR, one PR and one was not evaluable. None of them relapsed. Thus, all patients with positive focal RUA after R-CT were cured with involved-site RT. Concerning the 35 pts with bulky disease, 21 reached PET-neg and 14 had single RUA after R-CT and were thus irradiated (1 PD). There were two relapses in the PET-neg/not irradiated group, but only one in previously bulky site. In the PET-pos /RT group no relapse occurred. In the total population, 5 -year PFS and OS are 80.9% (95%, CI 79.28-92.18) and 87.1% (95%, CI 78.66- 92.38), respectively. Conclusion: Our data suggest that irradiating only sites of unique PET RUA, regardless of bulky at onset, can be considered as a reasonable strategy for low risk DLBCL pts. In cases with bulky disease, PET-driven RT allowed RT sparing in approximately half of patients in this small series. Moreover, consolidation RT in those with focal residual PET positivity, guaranteed excellent prognosis (17/17 cured) and can be considered as a valid option Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The prognostic value of end-of-treatment FDG-PET/CT in diffuse large B cell lymphoma: comparison of visual Deauville criteria and a lesion-to-liver SUVmax ratio-based evaluation system.

The aim of this study was to determine a better criterion for end-of-treatment PET (EoT-PET) assessment and prognostic evaluation of patients with diffuse large B cell lymphoma (DLBCL). EoT-PET scans were assessed using the visual Deauville 5-point scale (5PS) and LLR, the maximum standard uptake value ratio between the lesion and the liver. The cutoff value of LLR was obtained by receiver operator characteristic curve analysis. Patient outcomes were compared using Kaplan-Meier survival analysis. Prognostic indexes of different criteria were compared. Multivariate Cox regression analysis was performed to evaluate the prognostic factors. Four hundred forty-nine newly diagnosed DLBCL patients who received rituximab-based immunochemotherapy were included, and the median follow-up duration was 41.4months. Patients with Deauville score (DS) 4 displayed significantly longer PFS and OS compared with patients with DS 5 (both p < 0.001), and they had significantly shorter PFS (p < 0.01) but similar OS (p = 0.057) compared with patients with DS 1-3. The differences in PFS and OS between groups were all significant whether positive EoT-PET was defined as DS 4-5 or DS 5 (all p < 0.001). The optimal cutoff of LLR was 1.83, and both PFS and OS were significantly different between EoT-PET-positive and EoT-PET-negative patients as defined by the cutoff (both p < 0.001). LLR-based criterion displayed higher specificity, positive predictive value, and accuracy than 5PS-based criterion in the prediction of disease progression and death events. In the multivariate analysis, positive EoT-PET (as defined by LLR) was related to unfavorable PFS and OS (both p < 0.001). Additional treatment was not correlated with outcomes of EoT-PET-negative patients either defined by LLR or 5PS or EoT-PET-positive patients classified by 5PS, but it was the only beneficial factor for OS (p < 0.05) in EoT-PET-positive patients with LLR ≥ 1.83. The optimal cutoff of LLR may be superior to Deauville criteria in identifying low-risk DLBCL patients with negative EoT-PET after the first-line immunochemotherapy and sparing them the cost and toxicity of additional treatment.

Risk stratification model based on VEGF and International Prognostic Index accurately identifies low-risk diffuse large B-cell lymphoma patients in the rituximab era.

Vascular endothelial growth factor affects the invasiveness of solid tumors by regulating angiogenesis. However, it is not clear whether VEGF could be used to predict the prognosis of DLBCL in the era of rituximab-based immunotherapy. We conducted a retrospective study to explore response to therapy and the prognostic value of VEGF on DLBCL in the rituximab era. The subjects were 65 patients with a histological diagnosis of DLBCL from the Affiliated Hospital of Xuzhou Medical University. Kaplan-Meier analysis was performed to estimate the cumulative survival rate of patients with different VEGF and IPI levels, and comparisons between groups were made using the log-rank test. DLBCL patients with elevated VEGF were more likely to have extranodal involvement, advanced stage, Myc/Bcl-2 double expression, and a higher Ki-67 score. Elevated VEGF was associated with poor therapeutic response and survival. When patients were divided into low, low-intermediate, high-intermediate and high-risk groups using the V-IPI model based on VEGF and IPI, PFS rates were 94.4, 74.1, 40.6 and 14.8%, respectively. This model better identified low-risk patients than IPI (85.9, 88.9, 37 and 7.8%). Our results demonstrate that VEGF predicts therapeutic response in DLBCL and the V-IPI model accurately predicts PFS of low-risk DLBCL in the rituximab era.

A prognostic gene signature for predicting survival outcome in diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) is an heterogenous cancer that can have profound differences in survival outcomes. Molecular profiling has allowed for the identification of DLBCL subclasses, and together with clinical prognostic factors, such as the international prognostic index, have improved clinical care and survival. Despite these advances, a gene signature that is associated with overall survival (OS) and is reproducible across different DLBCL studies could better classify risk and predict OS. Here, we have identified genes that are associated with OS in DLBCL using data from the Lymphoma/Leukemia Molecular Profiling Project and developed a prognostic gene signature consisting of 33 genes that — when transformed into a risk score — can stratify individuals into high or low risk groups that have significantly different OS. The prognostic gene signature was associated with OS in multiple clinical studies, and when used in conjunction with DLBCL molecular subtype and IPI score, significantly predicted OS. Thus, we identified a potential prognostic gene signature that can discriminate high-risk from low-risk DLBCL patients.

Response-adapted therapy with infusional EPOCH chemotherapy plus rituximab in HIV-associated, B-cell non-Hodgkin's lymphoma.

Four cycles of rituximab plus CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) chemotherapy is as effective as six cycles in low-risk diffuse large B-cell lymphoma (DLBCL). Here we report a post-hoc analysis of a prospective clinical trial in patients with human immunodeficiency virus-associated DLBCL and high-grade lymphoma treated with four to six cycles of EPOCH plus rituximab based on a response-adapted treatment strategy. One hundred and six evaluable patients with human immunodeficiency virus-associated DLBCL or highgrade CD20+ non-Hodgkin lymphoma were randomized to receive rituximab (375 mg/m2) given either concurrently prior to each infusional EPOCH cycle, or sequentially (weekly for 6 weeks) following completion of EPOCH. EPOCH consisted of a 96-hour intravenous infusion of etoposide, doxorubicin, and vincristine plus oral prednisone followed by an intravenous bolus of cyclophosphamide every 21 days for four to six cycles. Patients received two additional cycles of therapy after documentation of a complete response by computed tomography after cycles 2 and 4. Sixty-four of 106 evaluable patients (60%; 95% confidence interval [95% CI]: 50%-70%) in both treatment arms had a complete response. The 2-year event-free survival rates were similar in the 24 patients with complete response who received four or fewer cycles of EPOCH (78%; 95% CI: 55%-90%) due to having achieved a complete response after two cycles, compared with those who received five or six cycles of EPOCH (85%; 95% CI: 70%-93%) because a complete response was first documented after cycle 4. A response-adapted strategy may permit a shorter treatment duration without compromising therapeutic efficacy in patients with human immunodeficiency virus-associated lymphoma treated with EPOCH plus rituximab, which merits further evaluation in additional prospective trials. Clinical Trials.gov identifier NCT00049036.

Chemoimmunotherapy for localised low-risk diffuse large B-cell lymphoma

Chemoimmunotherapy for localised low-risk diffuse large B-cell lymphoma

Response-Adapted Therapy with Infusional EPOCH Chemotherapy Plus Rituximab in HIV-Associated, B-Cell Non-Hodgkin's Lymphoma

Introduction: Six cycles of rituximab plus infusional EPOCH is considered a preferred regimen for first-line treatment of HIV-associated diffuse large B-cell lymphoma (DLBCL), HHV8-positive DLBCL, and primary effusion lymphoma, and is among the preferred regimens for HIV-associated Burkitt's lymphoma in the 2019 NCCN guidelines. A phase III trial demonstrated non-inferiority of 4 cycles of R-CHOP (followed by 2 additional doses of rituximab) compared with 6 cycles of R-CHOP in immunocompetent patients with low-risk DLBCL (stage I-II, age 18-60 years, and an age-adjusted International Prognostic Index score of 0), indicating that de-escalation of treatment duration may be safely achieved without compromising curability in an appropriately selected patient population. Here we report the outcomes for patients with HIV-associated DLBCL and high-grade non-Hodgkin lymphoma (NHL) treated with 4-6 cycles of EPOCH plus rituximab based a response-adapted treatment strategy under a completed prospective clinical trial (AMC-034). Methods: One hundred-six patients with HIV-associated DLBCL or high-grade CD20-positive NHL enrolled at multiple centers across the U.S. were randomized to receive rituximab (375 mg/m2) given either concurrently prior to each infusional EPOCH cycle, or sequentially (weekly for 6 weeks) following completion of dose-adjusted EPOCH regimen tailored for HIV+ patients. EPOCH consisted of a 96-hour IV infusion of etoposide, doxorubicin, and vincristine plus oral prednisone followed by IV bolus cyclophosphamide every 21 days for 4 to 6 cycles. Patients received 2 additional cycles of therapy after documentation of a complete response (CR) by computerized tomography after cycles 2 and 4. Results: Sixty-four of 106 patients (60%, 95% CI 50%, 70%) had a CR in both treatment arms; characteristics of entire population and CR proportions stratified by number of EPOCH treatment cycles were similar (Table 1).The 2-year event-free survival (EFS) rates were similar in the 24 patients with CR who received 4 or fewer EPOCH cycles (78%, 95% confidence intervals [55%, 90%]) due to achieving a CR after 2 cycles, compared with those who received 5-6 cycles of EPOCH (85%, 95% CI 70%, 93%) due to failure to achieve a CR after 2 cycles (Table 2 and Figure 1). Time to disease progression and overall survival wer also similar for those treated with 4 or fewer cycles compared with 5-6 cycles (91% vs. 87%, and 78% vs. 90%, respectively) (Table 2 and Figure 1). Conclusion: A response-adapted strategy may permit a shorter treatment duration without compromising therapeutic efficacy in an appropriately selected population of patients with HIV-associated NHL, which merits further evaluation in additional prospective trials. Disclosures Noy: Janssen: Consultancy; Medscape: Honoraria; Prime Oncology: Honoraria; NIH: Research Funding; Pharamcyclics: Research Funding; Raphael Pharma: Research Funding.

Pirna-30473 Contributes to Tumorigenesis By Regulating RNA m6A Methylation in DLBCL

N6-methyladenosine (m6A), the most abundant modification on eukaryote messenger RNA (mRNA), functions in various fundamental bioprocesses. However, the role of m6A in diffuse large B-cell lymphoma (DLBCL) remains poorly understood. Here, we reported that piRNA-30473, which expression supported the aggressive phenotype of DLBCL and was correlated with poor prognosis, was a potent regulator of m6a modification through targeting m6a methyltransferase WTAP (Figure 1).Piwi-interacting RNAs (piRNAs), a novel class of small non-coding RNAs, have been documented to be involved in the epigenetic regulation of cancer. A cohort of 42 patients with newly diagnosed DLBCL, uniformly treated and followed, was studied. We show that piRNA-30473 was significantly upregulated in high-risk DLBCL patients compared to low-risk DLBCL patients by microarray assay on 6 samples, further confirmed by qPCR analysis on 42 samples (Figure 2). Moreover, silencing of piRNA-30473 expression reduced proliferation and induced cell cycle arrest, but not apoptosis in SU-DHL-8 and Toledo lymphoma cells. In “human-in-mouse” xenograft DLBCL models, injection of antagomir-30473 into mice led to a significant reduction in tumor volume compared with control(Figure 3).Our data further indicated that the combination of piRNA-30473 signature with the National Comprehensive Cancer Network International Prognostic Index (NCCN-IPI) and cytogenetics status had a better prediction for PFS and OS than those without the piRNA-30473 signature in the univariate and multivariate analyses by an AUC analysis with cross-validation.Silencing of piRNA-30473 diminished global m6A level in SU-DHL-8 and Toledo cells and decreased m6a methyltransferase WTAP in mRNA and protein levels. We further identified that piRNA-30473 enhanced WTAP expression through direct binding to its 3' UTR. Consistently, WTAP knockdown decreased the global m6A level and induced cell cycle arrest and growth inhibition in SU-DHL-8 and Toledo cells. Moreover, Silencing of piRNA-30473 displayed decreased cell proliferation, which could be abrogated by WTAP overexpression. Kaplan-Meier analysis showed that high WTAP levels in DLBCL patients were correlated with OS by analyzing the gene expression profiling of DLBCL patients from GEO database (GSE10846) (Figure 4).Next, we mapped the m6A methylomes of siCtrl and siWTAP SU-DHL-8 cells by m6A sequencing (m6A-seq) with independent biological replicates. The RGACH motif (R = G/A; H = A/C/U) were identified to be highly enriched within m6A sites in the SU-DHL-8 cells. M6A peaks in siCtrl and siWTAP SU-DHL-8 cells were abundant in coding sequences (CDSs), 3′ untranslated regions (UTRs), and near stop codons. Remarkably, 85% m6A-Hyper transcripts identified from siCtrl SU-DHL-8 cells turned into m6A-Hypo in siWTAP SU-DHL-8 cells, with approximately 66% of the Hypo-down and 65% of Hypo-up transcripts became Hyperup and Hyper-down, respectively, which might be genuine targets of WTAP. Eleven genes listed in Table 2 showed a significant change between siCtrl and siWTAP SU-DHL-8 cells in m6A peak levels, and abundance of the corresponding mRNA transcript, and were also significantly positively or negatively correlated with WTAP in expression in four datasets of large cohorts of DLBCL. Collectively, our data demonstrate that an enzyme hexokinase II (HK2), which was reported to be a key metabolic driver of the DLBCL phenotype, were functionally important targets of WTAP. WTAP-mediated regulation of HK2 depended on its m6A demethylase activity and the m6A modifications in the target mRNA transcripts (Figure 5).In summary, our finding demonstrate that piRNA-30473 were significantly associated with both PFS and OS in the univariate analysis, and were still statistically significant after adjusting for NCCN-IPI, and adverse cytogenetics in the multivariate analysis. Moreover, we provide compelling evidence demonstrating that WTAP, which was downregulated by piRNA-30473, played a critical oncogenic role in DLBCL, through enhancing m6A levels in mRNA transcripts of its critical target gene HK2 and thereby triggering corresponding signaling cascades. Our study highlights the functional importance of the m6A modification machinery in DLBCL, and provides profound insights into the molecular mechanisms underlying tumorigenesis by revealing a previously unrecognized mechanism of gene regulation in DLBCL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Expression and phosphorylation of translation regulatory protein 4E-binding protein (BP)-1 in low-risk diffuse large B cell lymphoma

Protein translation initiation is controlled by eukaryotic initiation factor 4E (eIF-4E) binding protein 1 (4E-BP1). Phosphorylated 4E-BP1 dissociates from eIF-4E, allowing translation of transcripts mediating cell cycling, survival, and angiogenesis. The expression and phosphorylation of 4E-BP1, and influence on prognosis of diffuse large B cell lymphoma (DLBCL), is unknown. Because at least some patients with low-risk (International Prognostic Index score 0–2) DLBCL treated with standard anthracycline-based chemotherapy experience a short survival, we examined if 4E-BP1 expression and phosphorylation may provide additional prognostic information in 35 patients with low-risk DLBCL. We examined their initial diagnostic pathology specimens for 4E-BP1 expression and phosphorylation using immunohistochemistry, and we correlated these with clinical outcomes. While 4E-BP1 was uniformly expressed, there was wide distribution in its level of phosphorylation (biological activity). 4E-BP1 phosphorylation correlated strongly with overall survival (OS; P = 0.007) and progression-free survival (PFS; P = 0.02) and was the most significant independent variable for both OS and PFS on multivariable analysis. Our findings suggest that immunohistochemical evaluation of 4E-BP1 phosphorylation may help refine the prognostication of patients with low-risk DLBCL. Prospective studies in an independent cohort of patients are warranted.

Phase III study of ACVBP versus ACVBP plus rituximab for patients with localized low-risk diffuse large B-cell lymphoma (LNH03-1B)

BackgroundThe superiority of a chemotherapy with doxorubicin, cyclophosphamide, vindesine, bleomycin and prednisone (ACVBP) in comparison with cyclophosphamide, doxorubicin, vincristin and prednisone plus radiotherapy for young patients with localized diffuse large B-cell lymphoma (DLBCL) was previously demonstrated. We report the results of a trial which evaluates the role of rituximab combined with ACVBP (R-ACVBP) in these patients. Patients and methodsUntreated patients younger than 66 years with stage I or II DLBCL and no adverse prognostic factors of the age-adjusted International Prognostic Index were randomly assigned to receive three cycles of ACVBP plus sequential consolidation with or without the addition of four infusions of rituximab. ResultsA total of 223 patients were randomly allocated to the study, 110 in the R-ACVBP group and 113 in the ACVBP group. After a median follow-up of 43 months, our 3-year estimate of event-free survival was 93% in the R-ACVBP group and 82% in the ACVBP group (P = 0.0487). Three-year estimate of progression-free survival was increased in the R-ACVBP group (95% versus 83%, P = 0.0205). Overall survival did not differ between the two groups with a 3-year estimates of 98% and 97%, respectively (P = 0.686). ConclusionIn young patients with low-risk localized DLBCL, rituximab combined with three cycles of ACVBP plus consolidation is significantly superior to ACVBP plus consolidation alone.

Current treatment strategy of diffuse large B-cell lymphomas

Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin's lymphoma, accounting for approximately 25-30% of all new patients. Rituximab, a genetically engineered chimeric monoclonal antibody that specifically binds to CD20, is the first monoclonal antibody approved for the treatment of B-cell lymphoma. Rituximab significantly improves treatment outcome in DLBCL. A large-phase III study demonstrated improved overall survival (OS) in patients with DLBCL treated with R-CHOP therapy. The Groupe d'Etude des Lymphomes de l'Adulte (GELA), Eastern Cooperative Oncology Group (ECOG) and German High-Grade Non-Hodgkin Lymphoma Study Group (DSHNHL) studies designed for patients older than 60 years have clearly shown prolonged event-free survival (EFS) and OS among patients who received rituximab and chemotherapy. For patients under 60 years, the MabThera International Trial Group (MInT) study demonstrated improved EFS and OS after the addition of rituximab to CHOP therapy. However, the R-CHOP therapy does not provide a satisfactory treatment outcome in the high-risk group according to the international prognostic index. Therefore, R-CHOP therapy is the new standard therapy in elderly and young, low-risk DLBCL patients. For young, high-risk DLBCL patients, treatment that incorporates rituximab and hematopoietic stem cell transplantation has been administered in clinical studies.

Pharmacotherapy of B-cell non-Hodgkin Lymphoma:&amp;nbsp; Focus on Rituximab

Rituximab, a genetically engineered chimeric monoclonal antibody that specifically binds to CD20, is the first monoclonal antibody approved for the treatment of B-cell lymphoma. The addition of rituximab to chemotherapy has improved progression-free survival and overall survival for patients with diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma. Rituximab-CHOP therapy is the new standard therapy in elderly and young, low-risk DLBCL patients. For young, high-risk DLBCL patients, treatment that incorporates rituximab and hematopoietic stem cell transplantation has been administered in clinical studies. Rituximab maintenance therapy for DLBCL is not effective at present. On the other hand, when active treatment is required in patients with follicular lymphoma, rituximab-chemotherapy is now the optimal treatment. In follicular lymphoma, randomized controlled trials of rituximab-chemotherapy show lengthened time to progression but, as yet, no improvement in overall survival.

Cyclophosphamide, doxorubicin, vincristine, methotrexate, bleomicin and prednisone plus rituximab in untreated young patients with low-risk (age-adjusted international prognostic index 0–1) diffuse large B-cell lymphoma

Young patients (aged 18–60 years) with good-prognosis diffuse large B-cell lymphoma (DLBCL) [0 and 1 risk factor according to age-adjusted international prognostic index (aa-IPI)] are distinguished from patients with poor-prognosis. Six cycles of cyclophosphamide, doxorubicin, vincristine, prednisone (CHOP) in combination with rituximab achieved best results in young patients with low-risk DLBCL. We retrospectively analyzed the data of 82 patients (18–60 years) with untreated aa-IPI 0–1 DLBCL, from six Italian institutions, who underwent, between January 2002 and January 2007, rituximab-cyclophosphamide, doxorubicin, vincristine, methotrexate, bleomicin and prednisone (R-MACOP-B) therapy followed, in patients with bulky presentation, by 30–36 Gy involved-field radiation therapy (34 patients). An overall response rate was noted in 77 out of 82 (94%) patients (75 patients had a complete response (91%), 2 patients had a partial response). With a median follow-up of 46 months, 7-year progression-free and overall survival were estimated to be 91% and 94%, respectively. R-MACOP-B regimen followed by involved-field radiation on bulky presentation is safe and very effective in the treatment of young patients with low-risk DLBCL.

Rituximab Combined With MACOP-B or VACOP-B and Radiation Therapy in Primary Mediastinal Large B-Cell Lymphoma: A Retrospective Study

BackgroundThird-generation regimens (MACOP-B [methotrexate/leucovorin (LV)/doxorubicin/cyclophosphamide/vincristine/prednisone/bleomycin] or VACOP-B [etoposide/LV/doxorubicin/cyclophosphamide/vincristine/prednisone/bleomycin]) in combination with local radiation therapy seem to improve lymphoma-free survival of primary mediastinal large B-cell lymphoma (PMLBCL). Recently, the superiority of R-CHOP (rituximab plus cyclophosphamide/doxorubicin/vincristine/prednisone) over CHOP-like regimens has been demonstrated in elderly and younger patients with low-risk diffuse large B-cell lymphoma. Patients and MethodsRetrospectively, between February 2002 and July 2006, 45 previously untreated patients with PMLBCL were treated with a combination of a third-generation chemotherapy regimen (MACOP-B or VACOP-B), concurrent rituximab, and mediastinal radiation therapy. ResultsTwenty-six (62%) patients achieved a complete response (CR), and 15 (36%) obtained a partial response after MACOP-B/VACOP-B plus rituximab. After radiation therapy, the CR rate was 80%. At a median follow-up of 28 months, among the 34 patients who obtained a CR, 3 relapsed after 16, 19, and 22 months, respectively. Projected overall survival was 80% at 5 years; the relapse-free survival (RFS) curve of the 34 patients who achieved CR was 88% at 5 years. ConclusionIn this retrospective study, in patients with PMLBCL, combined-modality treatment using the MACOP-B/VACOP-B regimen plus rituximab induces a high remission rate, with patients having a > 80% chance of surviving relapse free at 5 years. In comparison with historical data on MACOP-B/VACOP-B without rituximab, there are no statistically significant differences in terms of CR and RFS rates.

Therapeutic Compliance and Efficacy of Dose-Dense R-CHOP-14 Immunochemotherapy Plus Pegfilgrastim for First-Line Treatment of Low Risk Diffuse Large B-Cell Lymphoma Patients: Preliminary Results of An Open-Label Clinical Trial in Spain

Diffuse large B-cell lymphoma (DLBCL) is the most prevalent form of non-Hodgkin lymphoma. Three-weekly R-CHOP has become the standard treatment for DLBCL. Dose-dense immunochemotherapy could improve the efficacy of treatment; however these regimens are worse tolerated due to mielotoxicity. We are conducting a prospective, open-label, single arm clinical trial of treatment with dose-dense R-CHOP supported by pegfilgrastim in patients with DLBCL older than 65 with IPI 0–5 or younger than 65 with IPI 0–2. The aim of this sub-analysis was to evaluate the efficacy and treatment compliance in patients with low risk DLBCL (IPI 0–2). Patients received R-CHOP administered every 14 days followed by pegfilgrastim (6 mg per cycle) on day 2. Patients with tumor masses > 10 cm were allowed to receive radiotherapy with 30 Gy, and those at risk of central nervous system infiltration receive prophylactic lyposomal cytarabine. Fifty-eight patients with IPI 0–2 were included in this analysis, median age was 58 years old (range 18–82), 22 (38%) patients were older than 65 yr, and 32 (53%) were male. Characteristics of the patients at diagnosis were as follows: stage III–IV: 29 (50%), bulky disease: 13 (22%), extra nodal involvement: 34 (59%), ³2 extranodal sites: 6 (10%), B symptoms: 10 (17%), ECOG 0–1: 55 (95%), elevated LDH 21 (36%), elevated b2microglobulin: 15 (26%), IPI 0–1 28 (48%). Fifty-four patients completed 6 cycles of treatment and 4 patients were dropped out, 1 due to progression and 3 due to exitus: 1 respiratory insufficiency, 1 bilateral pneumonia and 1 septic shock. Overall, 335 chemotherapy cycles were administered and 23 (6.9%) were delayed due to: neutropenia (3), febrile neutropenia (4), hepatotoxicity (1), fever or infection without neutropenia (11) and other adverse events (4). Doses of myelotoxic drugs were reduced in 8 (2.4%) cycles (3 patients). Most cycles (91.9%) were administered as scheduled. Serious adverse events were reported in 23 (39.7%) patients. Among them, febrile neutropenia episodes were observed in 10 patients (17.3%). Out of 54 evaluated patients for efficacy after 130-days follow-up, 45 (83%) achieved complete remission, 5 (9%) partial remission, 3 (5.5%) stable disease and 1 (1.8%) progressed. In an intention-to-treat analysis among the 58 patients, 46 (79.3%) patients achieved CR, 6 (10.3%) PR and 2 (3.4%) were not evaluable. High remission rates suggest that dose-dense immunochemotherapy with pegfilgrastim support is an efficacious treatment for DLBCL in low risk patients. The regimen is well tolerated, treatment compliance is high and most of cycles were administered as scheduled.

Efficacy of Dose-Dense R-CHOP-14 Chemoimmunotherapy Plus Pegfilgrastim for First-Line Treatment of Diffuse Large B-Cell Lymphoma (DLBCL) in Low Risk Patients: An Interim Analysis of an Open-Label Clinical Trial in Spain. On Behalf of GEL/TAMO (Spanish Group of Lymphoma).

Diffuse large B-cell lymphoma (DLBCL) is the most prevalent form of non-Hodgkin lymphoma. The addition of rituximab to the standard treatment with 3-weekly CHOP (R-CHOP) has notably improved the survival in DLBCL. However, it is unclear whether a dose-dense regimen of 2-weekly R-CHOP-14 is efficacious and well tolerated. The aim of the current study is to determine the efficacy and tolerability of 6 cycles of dose dense R-CHOP-14 for low risk DLBCL. This is an ongoing, open-label, multicentre, single-arm clinical trial. Eligible patients are aged 18–65 years, with CD20+ DLBCL, an international prognostic index (IPI) of 0–2 and an Eastern Cooperative Oncology Group (ECOG) performance status of 0–2. Six cycles of R-CHOP-14 are administered with pegfilgrastim 6 mg sc on day 2 of each cycle. Patients with tumor masses > 10 cm are allowed to receive radiotherapy with 30 Gy, and those at risk of central nervous system infiltration receive prophylactic liposomal cytarabine with each chemotherapy cycle. An interim analysis of efficacy was planned when the first 25 patients were included. Patients receiving ≥1 dose of study medication were included in the tolerability analysis, while 130-day follow-up was required for inclusion in the efficacy analysis. The first 25 patients were included between June 2006 and January 2007: mean age was 46.5 years, 56% were male. The characteristics of the disease at diagnosis were as follows: stage III-IV 40%, ECOG 0–1 96%, B-symptoms 12%, bulky disease 28%, extranodal involvement 56% (most frequently spleen and gastrointestinal system), elevated LDH 44%, elevated β2microglobulin 36%, IPI 0–1 60%. All patients completed 6 cycles of chemotherapy (150 cycles). Among the 125 cycles excluding the first one, there were 7 (5.5%) delays, 3 because of neutropenic fever, 2 because neutropenia, and 2 because of infection without neutropenia. Dose modification due to hematological toxicity occurred in 4 cycles (3.2%) all in the same patient. Twenty-two patients were included in the efficacy analysis. The global response rate, the complete remission rate and the partial remission rate were 91%, 81.8% and 9.1%, respectively. No patient exhibited disease progression or relapse, whereas 2 (9.1%) had stable disease. All 22 patients were alive at 130 days follow-up. In this interim analysis, high remission rates suggest that dose dense R-CHOP-14 with pegfilgrastim support is an efficacious treatment for low risk DLBCL in patients younger than 65. The regimen was well tolerated with most patients completing their full treatment schedule as planned.