Poor Prognosis In Diffuse Large B-cell Lymphoma Research Articles

Gene regulatory mechanisms of T cell exhaustion in diffuse large B cell lymphoma based on single-cell transcriptome data

Diffuse large B cell lymphoma (DLBCL) is a heterogeneous and aggressive B cell malignancy that accounts for about 30% of non-Hodgkin lymphomas. The current standard treatment for DLBCL is rituximab plus chemotherapy, but many patients are refractory or relapse, indicating the need for improved understanding of its molecular pathology. T cell exhaustion is a state of dysfunction or impairment that occurs in chronic infections or cancers, and is associated with poor prognosis in DLBCL. However, the molecular mechanisms of T cell exhaustion in DLBCL are poorly understood. In this study, we performed a comprehensive analysis of T cell exhaustion in DLBCL using public single-cell transcriptome data. We identified different subtypes of T cells and characterized their gene expression features. We found that DLBCL had a significantly higher proportion of exhausted T cells than normal tonsil, and that exhausted T cells had distinct gene expression signatures from non-exhausted T cells. These signatures included genes related to inhibitory receptors, cytokines, transcription factors and metabolic enzymes. We also found that ID3 gene was significantly upregulated in exhausted T cells in DLBCL, which may play a key role in T cell exhaustion. We constructed a protein-protein interaction network, identifying major hub proteins involved in T cell exhaustion or migration. We also performed KEGG and GO enrichment analysis for the differentially expressed genes between exhausted and non-exhausted T cells, and found important signaling pathways related to T cell exhaustion in DLBCL. Our results provide new insights into the molecular mechanisms underlying T cell exhaustion and offer novel therapeutic targets for this complex disease.

Prognostic Significance of Double-Expresser Status in Diffuse Large B-cell lymphoma: Experience from a Tertiary Care Cancer Centre in India

Background: Diffuse large B-cell lymphoma (DLBCL) is a neoplasm of medium or large B lymphoid cells with a diffuse histopathologic growth pattern. Co-expression of MYC and BCL2 proteins in DLBCL by immunohistochemistry (IHC) is referred to as the double-expresser (DE) phenotype and is associated with inferior survival. Objectives of this study were to assess the DE status in DLBCL and to assess its utility in predicting the prognosis in DLBCL. Methods: This was a retrospective study conducted in a tertiary care cancer centre. Study population included all the cases of DLBCL, NOS diagnosed in the centre in 2012. MYC and BCL2 protein expression were analysed by immunohistochemistry. The prognostic significance of double-expressers was analysed by comparing progression free survival (PFS) and overall survival (OS) of double-expressers and non-double expressers using appropriate statistical methods. All the data were analysed using SPSS 11 software. Kaplan-Meier method was used to estimate the survival probability. A significant difference in survival between various prognostic factors was tested using the log-rank test. Prognostic factors were assessed using univariate and multivariate Cox-regression model. A P- value < 0.05 was considered to be statistically significant. Results: Double-Expresser lymphoma (DEL) constituted 22.2% (n=18) of all cases of DLBCL, NOS. DE status was associated with shorter PFS (P-value = 0.049) and OS (P-value = 0.015). Conclusion: DE status is associated with poor prognosis in DLBCL, NOS. Assessment of MYC and BCL2 by IHC represents a rapid and inexpensive approach to risk-stratify patients with DLBCL at the time of diagnosis.

S100A8 is a prognostic signature and associated with immune response in diffuse large B-cell lymphoma.

S100A8, a calcium-binding protein belonging to the S100 family, is involved in immune responses and multiple tumor pathogens. Diffuse large B-cell lymphoma (DLBCL) is one of the most common types of B-cell lymphoma and remains incurable in 40% of patients. However, the role of S100A8 and its regulation of the immune response in DLBCL remain unclear. The differential expression of S100A8 was identified via the GEO and TCGA databases. The prognostic role of S100A8 in DLBCL was calculated using the Kaplan-Meier curve. The function enrichment of differentially expressed genes (DEGs) was explored through GO, KEGG, GSEA, and PPI analysis. In our cohort, the expression of S100A8 was verified. Meanwhile, the biological function of S100A8 was applied after the inhibition of S100A8 in an in vitro experiment. The association between S100A8 and immune cell infiltration and treatment response in DLBCL was analyzed. S100A8 was significantly overexpressed and related to a poor prognosis in DLBCL patients. Function enrichment analysis revealed that DEGs were mainly enriched in the IL-17 signaling pathway. Our cohort also verified this point. In vitro experiments suggested that inhibition of S100A8 should promote cell apoptosis and suppress tumor growth. Single-cell RNA sequence analysis indicated that S100A8 might be associated with features of the tumor microenvironment (TME), and immune infiltration analyses discovered that S100A8 expression was involved in TME. In terms of drug screening, we predicted that many drugs were associated with preferable sensitivity. Elevated S100A8 expression is associated with a poor prognosis and immune infiltration in DLBCL. Inhibition of S100A8 could promote cell apoptosis and suppress tumor growth. Meanwhile, S100A8 has the potential to be a promising immunotherapeutic target for patients with DLBCL.

Unraveling the enigma of B cells in diffuse large B-cell lymphoma: unveiling cancer stem cell-like B cell subpopulation at single-cell resolution.

Diffuse large B-cell lymphoma (DLBCL) represents the most prevalent form of aggressive non-Hodgkin lymphoma. Despite receiving standard treatment, a subset of patients undergoes refractory or recurrent cases, wherein the involvement of cancer stem cells (CSCs) could be significant. We comprehensively characterized B cell subpopulations using single-cell RNA sequencing data from three DLBCL samples and one normal lymph tissue. The CopyKat R package was employed to assess the malignancy of B cell subpopulations based on chromosomal copy number variations. CIBERSORTx software was utilized to estimate the proportions of B cell subpopulations in 230 DLBCL tissues. Furthermore, we employed the pySCENIC to identify key transcription factors that regulate the functionality of B cell subpopulations. By employing CellphoneDB, we elucidated the interplay among tumor microenvironment components within the B cell subpopulations. Finally, we validated our findings through immunofluorescence experiments. Our analysis revealed a specific cancer stem cell-like B cell subpopulation exhibiting self-renewal and multilineage differentiation capabilities based on the exploration of B cell subpopulations in DLBCL and normal lymph tissues at the single-cell level. Notably, a high infiltration of cancer stem cell-like B cells correlated with a poor prognosis, potentially due to immune evasion mediated by low expression of major histocompatibility complex molecules. Furthermore, we identified key transcription factor regulatory networks regulated by HMGB3, SAP30, and E2F8, which likely played crucial roles in the functional characterization of the cancer stem cell-like B cell subpopulation. The existence of cancer stem cell-like B cells in DLBCL was validated through immunofluorescent staining. Finally, cell communication between B cells and tumor-infiltrating T cell subgroups provided further insights into the functional characterization of the cancer stem cell-like B cell subpopulation. Our research provides a systematic description of a specific cancer stem cell-like B cell subpopulation associated with a poor prognosis in DLBCL. This study enhances our understanding of CSCs and identifies potential therapeutic targets for refractory or recurrent DLBCL patients.

Molecular determinants of clinical outcomes in a real-world diffuse large B-cell lymphoma population

AbstractMolecular heterogeneity of diffuse large B-cell lymphoma (DLBCL) underlies the variable outcomes achieved with immunochemotherapy. However, outcomes of gene expression profiling (GEP)–defined molecular subgroups in a real-world DLBCL population remain unknown. Here we examined the prevalence and outcomes of molecular subgroups in an unselected population of 1149 patients with de novo DLBCL in British Columbia, Canada. Evaluable biopsies were profiled by fluorescence in situ hybridization (FISH), immunohistochemistry, and digital GEP to assign cell-of-origin and the so-called “double-hit signature” (DHITsig)—a signature originally described as being characteristic for high-grade B-cell lymphoma with MYC and BCL2 rearrangements (HGBCL-DH-BCL2). DHITsig was expressed in 21% of 431 germinal center B-cell-like (GCB)–DLBCL and all 55 Burkitt lymphomas examined. Reflecting this latter finding, DHITsig has been renamed the “dark zone signature” (DZsig). DZsigpos-DLBCL, non-DZsigpos GCB-DLBCL and activated B-cell-like (ABC)–DLBCL were associated with a 2 year overall survival of 57%, 89%, and 71%, respectively. 62% of DZsigpos tumors were negative for HGBCL-DH-BCL2 by FISH, but were associated with outcomes similar to HGBCL-DH-BCL2. A small group of HGBCL-DH-BCL2 that lacked DZsig expression had different molecular features compared with DZsig-expressing HGBCL-DH-BCL2 and were associated with favorable outcomes comparable to DLBCL, not otherwise specified. DZsigpos and ABC-DLBCL had a shorter diagnosis-to-treatment interval (DTI) than GCB-DLBCL, with this metric being associated with outcome. In conclusion, DZsig expression extends beyond HGBCL-DH-BCL2 and captures a poor-prognosis DLBCL subgroup with short DTI, including patients unidentifiable by routine FISH testing, that should be considered for treatment intensification or novel therapies in prospective trials.

Identification and Assessment of Necroptosis-Related Genes in Clinical Prognosis and Immune Cells in Diffuse Large B-Cell Lymphoma.

BackgroundWith the unveiling of new mechanisms and the advent of new drugs, the prognosis of diffuse large B-cell lymphoma (DLBCL) becomes promising, but some patients still progress to the relapse or refractory stage. Necroptosis, as a relatively novel programmed cell death, is involved in the development of multiple tumors. There are no relevant studies on the prognostic significance of necroptosis in DLBCL to date.MethodsWe identified the differential necroptosis-related genes (NRGs) by comparing the DLBCL and normal control in GSE12195 and GSE56315 datasets. TCGA DLBC and GSE10846 containing clinical information and microarray expression profiling were merged as the entire cohort. We performed consensus clusters based on NRGs and two clusters were obtained. Kaplan–Meier (K-M) survival analysis, GSVA, GO, KEGG, and ssGSEA were used to analyze the survival, function, and immune microenvironment between two clusters. With LASSO and proportional hazard model construction, we identified differentially expressed genes (DEGs) between NRG clusters, calculated the risk score, established a prognostic model, and validated its value by calibration and ROC curves. The entire cohort was divided into the training and test cohort, and GSE87371 was included as an external validation cohort. K-M, copy number variation, tumor mutation burden, and drug sensitivity were also analyzed.ResultsWe found significant differences in prognosis between the two NRG clusters. Cluster A with a poor prognosis had a decreased expression of NRGs and a relatively suppressed immune microenvironment. GSVA analysis indicated that cluster A was related to the downregulation of the TGF-β signaling pathway and the activation of the Notch signaling pathway. The risk score had an accurate predictive ability. The nomogram could help predict the survival probability of DLBCL patients in the entire cohort and the external validation cohort. The area under the curve (AUC) of the nomogram, risk score, and International Prognostic Index was 0.723, 0.712, and 0.537, respectively. γ/δ T cells and Macrophage 1 cells decreased while Macrophage 2 cells and Natural Killer resting cells increased in the high-risk group. In addition, the high-risk group was more sensitive to the PI3K inhibitor and the PDK inhibitor.ConclusionWe explored the potential role of necroptosis in DLBCL from multiple perspectives and provided a prognostic nomogram for the survival prediction of DLBCL. Necroptosis was downregulated and was correlated with an immunosuppressed tumor microenvironment and poor prognosis in DLBCL. Our study may deepen the understanding and facilitate the development of new therapy targets for DLBCL.

The Expression of the RUVBL1 Component of the R2TP Complex Correlates with Poor Prognosis in DLBCL

Introduction: Diffuse large B-cell lymphoma (DLBCL) is the most prevalent subtype of non-Hodgkin’s lymphoma (NHL) accounting for 30% of adult NHL worldwide and 50% in developing countries like India. DNA damage and Myc-induced transformation are well-known contributing factors towards development of DLBCL. A recently identified HSP90 co-chaperone complex R2TP has been shown to contribute towards DNA damage and Myc-induced transformation. This study aimed to analyse the immunohistochemical (IHC) expression of R2TP complex components RUVBL1, PIH1D1, and RPAP3 in DLBCL patients and correlate with prognosis. Methods: DLBCL (n = 54) histological slides were retrieved from archives, and detailed histomorphological and clinical features were noted. IHC staining of R2TP complex components RUVBL1, PIH1D1, and RPAP3 was performed on 54 cases (FFPE) of DLBCL. Expression data were correlated with survival and clinical features. Results: Out of the 54 DLBCL cases, 59.26% (n = 32) stained positive for RUVBL1. The RUVBL1 expression was associated with poor prognosis in both progression-free survival (PFS) (p = 0.0146) and overall survival (OS) (p = 0.0328). The expression was positively correlated with bone marrow involvement (p = 0.0525). The expression of PIH1D1 was observed in 68.51% (n = 32) of DLBCL cases, and positive correlation was observed with international prognostic index score (p = 0.0246); however, no correlation was observed with PFS or OS. Finally, RPAP3 was found immunopositive in only 1 case of DLBCL. Conclusions: Immunopositivity for RUVBL1 is associated with poor prognosis along with a higher relapse rate amongst the DLBCL patients. PIH1D1 immunopositivity correlated with a higher IPI score.

High PTX3 expression is associated with a poor prognosis in diffuse large B-cell lymphoma.

Tumor‐associated macrophages (TAMs) are associated with a poor prognosis of diffuse large B‐cell lymphoma (DLBCL). As macrophages are heterogeneous, the immune polarization and their pathological role warrant further study. We characterized the microenvironment of DLBCL by immunohistochemistry in a training set of 132 cases, which included 10 Epstein–Barr virus‐encoded small RNA (EBER)‐positive and five high‐grade B‐cell lymphomas, with gene expression profiling in a representative subset of 37 cases. Diffuse large B‐cell lymphoma had a differential infiltration of TAMs. The high infiltration of CD68 (pan‐macrophages), CD16 (M1‐like), CD163, pentraxin 3 (PTX3), and interleukin (IL)‐10‐positive macrophages (M2c‐like) and low infiltration of FOXP3‐positive regulatory T lymphocytes (Tregs) correlated with poor survival. Activated B cell‐like DLBCL was associated with high CD16, CD163, PTX3, and IL‐10, and EBER‐positive DLBCL with high CD163 and PTX3. Programmed cell death‐ligand 1 positively correlated with CD16, CD163, IL‐10, and RGS1. In a multivariate analysis of overall survival, PTX3 and International Prognostic Index were identified as the most relevant variables. The gene expression analysis showed upregulation of genes involved in innate and adaptive immune responses and macrophage and Toll‐like receptor pathways in high PTX3 cases. The prognostic relevance of PTX3 was confirmed in a validation set of 159 cases. Finally, in a series from Europe and North America (GSE10846, R‐CHOP‐like treatment, n = 233) high gene expression of PTX3 correlated with poor survival, and moderately with CSF1R, CD16, MITF, CD163, MYC, and RGS1. Therefore, the high infiltration of M2c‐like immune regulatory macrophages and low infiltration of FOXP3‐positive Tregs is associated with a poor prognosis in DLBCL, for which PTX3 is a new prognostic biomarker.

Prognostic Impact of Muscle Quantity and Quality and Fat Distribution in Diffuse Large B-Cell Lymphoma Patients.

Baseline CT scans of 116 patients (48% female, median 64 years) with diffuse large B-cell lymphoma (DLBCL) were retrospectively reviewed to investigate the prognostic role of sarcopenia and fat compartment distributions on overall survival (OS), progression-free survival (PFS), and early therapy termination. Skeletal muscle index (SMI), skeletal muscle density (SMD), and intermuscular adipose tissue (IMAT) were quantified at the level of the third lumbar vertebra (L3) and proximal thigh (PT). Low L3-SMD, but not low L3-SMI, was associated with early therapy termination (p = 0.028), shorter OS (HR = 6.29; 95% CI = 2.17–18.26; p < 0.001), and shorter PFS (HR = 2.42; 95% CI = 1.26–4.65; p = 0.008). After correction for sex, International Prognostic Index (IPI), BMI, and R-CHOP therapy (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone), low L3-SMD remained associated with poor OS (HR = 3.54; 95% CI = 1.10–11.40; p = 0.034) but not with PFS. Increased PT-IMAT was prognostic for poor OS and PFS after correction for sex, IPI, BMI, and R-CHOP therapy (HR = 1.35; CI = 1.03–1.7; p = 0.03, and HR = 1.30; CI = 1.04–1.64; p = 0.024, respectively). Reduced muscle quality (SMD) and increased intermuscular fat (IMAT), rather than low muscle quantity (SMI), are associated with poor prognosis in DLBCL, when measured at the L3 level, and particularly at the level of the proximal thigh. The proximal thigh represents a novel radiological landmark to study body composition.

Expression Patterns and Prognostic Values of ORMDL1 in Different Cancers

The mammalian orosomucoid-like gene family (ORMDL), containing ORMDL1, ORMDL2, and ORMDL3, is the important regulator of sphingolipid metabolism, which is relevant to cell growth, proliferation, migration, and invasion. Since the role of ORMDL1 in cancers remained unclear, the main purpose of our study was to explore the expression patterns and prognostic values of ORMDL1 in different tumors, especially in cholangiocarcinoma (CHOL), lymphoid neoplasm diffuse large B cell lymphoma (DLBCL), acute myeloid leukemia (LAML), and thymoma (THYM). Bioinformatics tools including GEPIA, CCLE, LinkedOmics, cBioPortal, and TIMER databases were used. As a result, the expression levels of ORMDL1 in tumor tissues and normal tissues varied in different cancers, especially significantly upregulated in CHOL, DLBCL, LAML, and THYM. Moreover, ORMDL1 mRNA was also highly expressed in cell lines of DLBCL and LAML. Further studies showed that ORMDL1 overexpression was associated with poor prognosis in DLBCL, but not significant in CHOL, LAML, and THYM. Consistently, there were genetic alterations of ORMDL1 in DLBCL, and patients with genetic alterations indicated worse survival. Coexpressed genes and related biological events with ORMDL1 in DLBCL were found via LinkedOmics, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. The relationship between ORMDL1 and cancer immune cells was investigated, and ORMDL1 expression was positively correlated with infiltrating levels of B cells. In conclusion, ORMDL1 is suggested to be a tumorigenic factor and considered as the potential therapeutic target and prognostic biomarker in DLBCL.

Plasma cell-free DNA is a prognostic biomarker for survival in patients with aggressive non-Hodgkin lymphomas.

Cell-free DNA (cfDNA) can be released from tumor cells during proliferation and apoptosis; thus, a fraction of the cfDNA in patients with cancer is tumor-derived. However, the prognostic value of cfDNA in aggressive non-Hodgkin lymphoma (NHL) has not been determined. Between March 2017 and April 2019, plasma cfDNA was obtained from 158 patients with aggressive NHL who were registered in a prospective Samsung Medical Center lymphoma cohort (diffuse large B cell lymphoma (DLBCL), n = 51; T cell lymphoma (TCL), n = 51; NK/T cell lymphoma (NKTCL), n = 56). The concentration of cfDNA was estimated in longitudinal samples collected from patients with NHL before and during various chemotherapy regimens. In pretreatment samples, the median cfDNA concentration of all patients with aggressive lymphoma was 13.7ng/dl (range 1.7-1792), which was significantly higher than that of healthy volunteers (median 7.4ng, range 3.7-14.4, p < 0.001), and advanced stages showed a higher cfDNA level than earlier stages. Multivariate analysis identified high cfDNA as an independent factor for event-free survival that predicted poor prognosis in DLBCL (hazard ratio [HR] = 5.33, 95% confidence interval [CI] = 1.72-16.52, p = 0.003) and TCL (HR = 2.82, 95% CI = 1.10-7.20, p = 0.030). NKTCL patients with a high level of cfDNA had worse overall survival (HR = 4.71, 95% CI = 1.09-20.35, p = 0.037) compared with those with a low level of cfDNA. In this study, our results suggest the usefulness of pretreatment cfDNA as a prognostic marker for patients with DLBCL, TCL, and NKTCL.

Diffuse Large B Cell Lymphoma (DLBCL)-Released NM23-H1 Promotes Monocyte Survival and Inflammatory Cytokine Release: A Mechanistic Link between the Dual Impacts of NM23-H1 Expression and Reduced Lymphocyte:Monocyte Ratio in DLBCL Prognosis

Elevated circulating levels of NM23-H1 have been associated with poor prognosis in haematological malignancies including AML and non-Hodgkin's lymphomas. In diffuse large B-cell lymphoma (DLBCL), several studies have demonstrated that both elevated circulating plasma levels and intra-tumoural levels of NM23-H1 correlate with poorer prognosis. These observations bring into question whether the mechanistic link of NM23-H1 expression with DLBCL prognosis depends an intrinsic intracellular mechanisms or is mediated by release of NM23-H1. We have shown that DLBCL lymphoma cell lines (Farage, HT, OCI-LY1, OCI-LY3, OCI-LY7, SUDHL4, SUDHL5 SUDHL6, U2932) express both NM23-H1 protein and the highly related protein NM23-H2. We also demonstrated that DLBCL cell lines release significant levels of NM23-H1 into their extracellular environment but not NM23-H2. Release of NM23-H1 was highly variable between cell lines with some releasing very high levels and some releasing much lower levels. Thus, DLBCL cell lines represent appropriate models to investigate the role of high and low levels of NM23-H1 on prognosis. The DLBCL cell lines HT and OCI-LY1 where selected for further study as representative of high and low level releasers of NM23-H1, respectively. CRISPR-Cas9 knockout of NM23-H1 (KO-NM23-H1) in HT and OCI-LY1 DLBCL cells had no impact on cell growth, viability or immuno-phenotype either in normoxic or hypoxic cultures. Thus NM23-H1 appears to not be required intrinsically by DLBCL cell lines. We therefore considered that the link between higher expression and release of NM23-H1 with prognosis is mediated via tumour-host environment interactions. We used an NSG mouse model transplanted subcutaneously with 1x106 CRISPR control (CTRL)-HT, CTRL-OCIL-Y1, KO-NM23-H1-HT or KO-NM23-H1-OCI-LY1 cell lines. We did not observe significant differences in tumour growth between CTRL and KO-NM23-H1 cells in the low NM23-H1 expressing OCI-LY1 DLBCL cell line. However, the high-expressing KO-NM23-H1-HT cells had significantly slower tumour progression and increased host survival when compared to CTRL-HT cells. We interpret to indicate that NM23-H1 release above a certain threshold provides a tumour growth advantage. Reduced lymphocyte:monocyte ratios (LMR) are also associated with poor prognosis in DLBCL. To investigate a potential functional link between NM23-H1 release and monocyte behaviour we first exposed peripheral blood leucocytes to Alexa 647 labelled fluorescent recombinant-NM23-H1 and found that monocytes but not neutrophils, T-cells or B-cells bound NM23-H1. We also observed that monocyte viability and survival were elevated in serum free cultures when supplemented with rNM23-H1, an observation that might indicate that elevated circulating NM23-H1 and reduced LMR in poor prognosis DLBCL may be mechanistically linked. We next co- cultured CTRL-HT or koNM23-H1-HT in a 1:1 ratios with purified monocytes from healthy human donors. Principle component analyses of 27 cytokines simultaneously measured by luminex assays identified that IL-1β, IL-6, IL-8, MIP-1α, MIP-1β and TNF-α were elevated when monocytes were co-cultured with CTRL-HT cells compared to co-culture with koNm23-H1-HT cells. In contrast, IP-10 was found elevated in the co-culture with koNm23-H1-HT cells. These observations indicate potentially important cross talk between the malignant DLBCL cells and innate immune cells of the host. Consistent with this, IL-6 and IL-8 serum levels have been shown to be elevated in pretreatment DLBCL patients compared to control subjects and MIP-1α, IL-6, and IL-8 serum levels have a greater association with DLBCL than follicular lymphoma. In conclusion ours is the first study to investigate potential mechanistic links between the associations of elevated NM23-H1 and poor prognosis in DLBCL and indicate a role for cross-talk between tumour cells and innate immune cells. Our data also indicate that NM23-H1 release from DLBCL cells may be a driving component in driving reduced LMRs that are also associated with poor prognosis. Whether reduced LMR and NM23-H1 release are casually related or not, the possibility that patients with both elevated monocytes and elevated circulating NM23-H1 levels are likely to represent a group with particularly poor prognosis requires urgent investigation. Disclosures Drayson: Abingdon Health: Consultancy, Equity Ownership. Rushworth:Abbvie: Research Funding; Janssen: Research Funding.

System-Level Disease-Driven Immune Signatures in Patients with Diffuse Large B-Cell Lymphoma Associated with Poor Survival

Global gene expression profiling of the tumor microenvironment in diffuse large B-cell lymphoma (DLBCL) has revealed broad innate immune signatures that distinguish the heterogeneous disease subtypes and correlate with good treatment outcome. However, we still lack tools to identify the relatively large group of patients that are refractory to initial therapy and have a dismal prognosis. Here, we used mass cytometry and serum profiling in a systems-level approach to analyze immune responses in 36 patients with aggressive B cell lymphoma and age- and sex-matched healthy controls. Stochastic neighbor embedding (t-SNE) analysis of protein profiles divided patients into two distinct clusters, with cluster 2 representing patients with a more severe deviation in their protein expression compared to healthy controls. Patients in cluster 2 showed a more dramatic perturbation of their immune cell repertoires with expansion of myeloid-derived suppressor cells (MDSCs), increased T cell differentiation and significantly higher expression of metabolic markers such as GLUT-1 and activation markers, including Ki67, CD38 and PD-1. An extended analysis of serum protein profiles in two independent cohorts (n=69 and n=80 patients, respectively) revealed that that the identified systemic immune signatures were linked to poor progression free survival (PFS) and inferior overall survival (OS). Immune monitoring during chemo-immunotherapy showed that most patients normalized their serum protein profiles. Notably, non-responding patients retained higher than normal expression of several proteins, including PD-L1, CD70, IL-18, granzyme A and CD83. These studies demonstrate distinct patterns of disease-driven alterations in the systemic immune response of DLBCL patients that are associated with poor survival and persist in patients who are refractory to therapy. Figure 1 System-level immune signatures associated with poor prognosis in DLBCL. A) Altered serum profiles in patients compared to healthy controls. Two clusters of patients were identified based on t-SNE analysis of serum profiles. B) Patients in cluster 2 had bulky disease and B symptoms. C) t-SNE map of all patients (n=36) and controls (n=17). Relative abundance of cells from healthy controls and patients in all areas of the t-SNE clustering, highlighting cell subsets that are larger or smaller in patients compared to healthy donors. Colors indicate the difference in kernel density estimation of the t-SNE data for patients and healthy controls. D) Abundance of monocytic myeloid-derived suppressor cells as percentage of all CD45+ cells in healthy donors and the two patient clusters. White, Healthy controls; Blue, Cluster 1; Red, Cluster 2. E) Major phenotypic differences between patient clusters shown as mean mass intensity (MMI) or percent positive cells for selected markers (CD38 and PD-1) across multiple subsets. White, Healthy controls; Blue, Cluster 1; Red, Cluster 2. F-G) Overall survival in patients with serologically defined immune signatures belonging to cluster 1 or 2. H) Abundance of serum proteins in patients that stayed in remission (n=24) compared to those that did not (n=6). Figure 1 Disclosures Olweus: Gilead Kite: Research Funding; Intellia: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Wahlin:Roche and Gilead: Consultancy. Fehniger:Cyto-Sen Therapeutics: Consultancy; Horizon Pharma PLC: Other: Consultancy (Spouse). Holte:Novartis: Honoraria, Other: Advisory board. Kolstad:Nordic Nanovector: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Research Funding. Malmberg:Fate Therapeutics, Inc.: Consultancy, Research Funding; Vycellix: Consultancy, Membership on an entity's Board of Directors or advisory committees.

The anaphase-promoting complex/cyclosome: a new promising target in diffuse large B-cell lymphoma and mantle cell lymphoma

BackgroundThe aggressive B-cell non-Hodgkin lymphomas diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) are characterised by a high proliferation rate. The anaphase-promoting complex/cyclosome (APC/C) and its co-activators Cdc20 and Cdh1 represent an important checkpoint in mitosis. Here, the role of the APC/C and its co-activators is examined in DLBCL and MCL.MethodsThe expression and prognostic value of Cdc20 and Cdh1 was investigated using GEP data and immunohistochemistry. Moreover, the therapeutic potential of APC/C targeting was evaluated using the small-molecule inhibitor proTAME and the underlying mechanisms of action were investigated by western blot.ResultsWe demonstrated that Cdc20 is highly expressed in DLBCL and aggressive MCL, correlating with a poor prognosis in DLBCL. ProTAME induced a prolonged metaphase, resulting in accumulation of the APC/C-Cdc20 substrate cyclin B1, inactivation/degradation of Bcl-2 and Bcl-xL and caspase-dependent apoptosis. In addition, proTAME strongly enhanced the anti-lymphoma effect of the clinically relevant agents doxorubicin and venetoclax.ConclusionWe identified for the first time APC/C as a new, promising target in DLBCL and MCL. Moreover, we provide evidence that Cdc20 might be a novel, independent prognostic factor in DLBCL and MCL.

Targeted Inhibition of PI3K α/δ Is Synergistic with BCL-2 Blockade in Genetically Defined Subtypes of DLBCL

Diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous disease that is transcriptionally classified into germinal center B-cell (GCB) and activated B-cell (ABC) subtypes. A subset of both GCB- and ABC-DLBCLs are dependent on B-cell receptor (BCR) signaling. Previously, we defined distinct BCR/PI3K-mediated survival pathways and subtype-specific apoptotic mechanisms in BCR-dependent DLBCLs (Cancer Cell 2013 23:826). In BCR-dependent DLBCLs with low baseline NF-κB activity (GCB tumors), targeted inhibition or genetic depletion of BCR/PI3K pathway components induced expression of the pro-apoptotic HRK protein. In BCR-dependent DLBCLs with high NF-κB activity (ABC tumors), BCR/PI3K inhibition decreased expression of the anti-apoptotic NF-κB target gene, BFL1.Our recent analyses revealed genetic bases for perturbed BCR/PI3K signaling and defined poor prognosis DLBCL subsets with discrete BCR/PI3K/TLR pathway alterations (Nat Med 2018 24:679). Cluster 3 DLBCLs (largely GCB tumors) exhibited frequent PTEN deletions/mutations and GNA13 mutations. Cluster 5 DLBCLs (largely ABC tumors) had frequent MYD88L265P and CD79B mutations that often occurred together. These DLBCL subtypes also had different genetic mechanisms for deregulated BCL2 expression - BCL2 translocations in Cluster 3 and focal (18q21.33) or arm level (18q) BCL2 copy number gains in Cluster 5. These observations prompted us to explore the activity of PI3K inhibitors and BCL2 blockade in genetically defined DLBCLs.We utilized a panel of 10 well characterized DLBCL cell line models, a subset of which exhibited hallmark genetic features of Cluster 3 and Cluster 5. We first evaluated the cytotoxic activity of isoform-specific, dual PI3Kα/δ and pan-PI3K inhibitors. In in vitro assays, the PI3Kα/δ inhibitor, copanlisib, exhibited the highest cytotoxicity in all BCR-dependent DLBCLs.We next assessed the transcriptional abundance of BCL2 family genes in the DLBCLs following copanlisib treatment. In BCR-dependent GCB-DLBCLs, there was highly significant induction of the pro-apoptotic HRK. In BCR-dependent ABC-DLBCLs, we observed significant down-regulation of the anti-apoptotic BFL1 protein and another NF-κB target gene, BCLxL (the anti-apoptotic partner of HRK).We then used BH3 profiling, to identify dependencies on certain BCL2 family members and to correlate these data with sensitivity to copanlisib. BCLxL dependency significantly correlated with sensitivity to copanlisib. Importantly, the BCLxL dependency was highest in DLBCL cell lines that exhibited either transcriptional up-regulation of HRK or down-regulation of BCLxL following copanlisib treatment.In all our DLBCL cell lines, PI3Kα/δ inhibition did not alter BCL2 expression. Given the genetic bases for BCL-2 deregulation in a subset of these DLBCLs, we next assessed the activity of the single-agent BCL2 inhibitor, venetoclax, in in vitro cytotoxicity assays. A subset of DLBCL cell lines was partially or completely resistant to venetoclax despite having genetic alterations of BCL2. We postulated that BCR-dependent DLBCLs with structural alterations of BCL2 might exhibit increased sensitivity to combined inhibition of PI3Kα/δ and BCL2 and assessed the cytotoxic activity of copanlisib (0-250 nM) and venetoclax (0-250 nM) in the DLBCL cell line panel. The copanlisib/venetoclax combination was highly synergistic (Chou-Talalay CI<1) in BCR-dependent DLBCL cell lines with genetic bases of BCL2 deregulation.We next assessed copanlisib and venetoclax activity in an in vivo xenograft model using a DLBCL cell line with PTENdel and BCL2 translocation (LY1). In this model, single-agent copanlisib did not delay tumor growth or improve survival. Single-agent venetoclax delayed tumor growth and improved median survival (27 vs 51 days, p<0.0001). Most notably, we found that the combination of copanlisib and venetoclax delayed tumor growth significantly longer than single-agent venetoclax (p<0.0001). Additionally, the combined therapy significantly increased survival in comparison with venetoclax alone (median survival 51 days vs not reached, p<0.0013).Taken together, these results provide in vitro and in vivo pre-clinical evidence for the rational combination of PI3Kα/δ and BCL2 blockade and set the stage for clinical evaluation of copanlisib/venetoclax therapy in patients with genetically defined relapsed/refractory DLBCL. DisclosuresLetai:AbbVie: Consultancy, Other: Lab research report; Flash Therapeutics: Equity Ownership; Novartis: Consultancy, Other: Lab research report; Vivid Biosciences: Equity Ownership; AstraZeneca: Consultancy, Other: Lab research report. Shipp:AstraZeneca: Honoraria; Merck: Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Research Funding.

Chromosomal abnormality variation detected by G-banding is associated with prognosis of diffuse large B-cell lymphoma treated by R-CHOP-based therapy.

Diffuse large B‐cell lymphoma (DLBCL), which is the most prevalent disease subtype of non‐Hodgkin lymphoma, is highly heterogeneous in terms of cytogenetic and molecular features. This study retrospectively investigated the clinical impact of G‐banding‐defined chromosomal abnormality on treatment outcomes of DLBCL in the era of rituximab‐containing immunochemotherapy. Of 181 patients who were diagnosed with DLBCL and treated with R‐CHOP or an R‐CHOP‐like regimen between January 2006 and April 2014, metaphase spreads were evaluable for G‐banding in 120. In these 120 patients, 40 were found to harbor a single chromosomal aberration type; 63 showed chromosomal abnormality variations (CAVs), which are defined by the presence of different types of chromosomal abnormalities in G‐banding, including 19 with two CAVs and 44 with ≥3 CAVs; and 17 had normal karyotypes. No specific chromosomal break point or numerical abnormality was associated with overall survival (OS) or progression‐free survival (PFS), but the presence of ≥3 CAVs was significantly associated with inferior OS rates (hazard ratio (HR): 2.222, 95% confidence interval (CI): 1.056–4.677, P = 0.031) and tended to be associated with shorter PFS (HR: 1.796, 95% CI: 0.965–3.344, P = 0.061). In addition, ≥3 CAVs more frequently accumulated in high‐risk patients, as defined by several conventional prognostic indices, such as the revised International Prognostic Index. In conclusion, our results suggest that the emergence of more CAVs, especially ≥3, based on chromosomal instability underlies the development of high‐risk disease features and a poor prognosis in DLBCL.

Favourable outcomes of poor prognosis diffuse large B-cell lymphoma patients treated with dose-dense Rituximab, high-dose Methotrexate and six cycles of CHOP-14 compared to first-line autologous transplantation.

The optimal therapeutic approach for young diffuse large B-cell lymphoma (DLBCL) patients with high-intermediate and high-risk age-adjusted international prognostic index (aaIPI) remains unknown. Hereby we report a 10-year single-centre study of 63 consecutively treated patients. To optimize outcome, two approaches were carried out: Cohort 1 patients received four cycles R-CHOP-21 (rituximab, cyclophosphamide, daunorubicin, vincristine, prednisolone over 21days) followed by first-line high-dose chemotherapy with autologous stem-cell support (HDCT-ASCT), resulting in 2-year progression-free (PFS) and overall survival (OS) of 60·6% and 67·9%. 39·4% of those patients were not transplanted upfront, mainly due to early progressive disease (24·2%). Cohort 2 patients received an early intensified protocol of six cycles of CHOP-14 (cyclophosphamide, daunorubicin, vincristine, prednisolone over 14days) with dose-dense rituximab and high-dose methotrexate resulting in promising overall response- (93·3%) and complete remission (90%) rates and sustained survival (2-year PFS and OS: 93·3%). In an intention-to-treat analysis, 2-year PFS (60·6% vs. 93·3%, hazard ratio [HR] 7·2, P=0·009) and OS (69·7% vs. 93·3%, HR 4·95, P=0·038) differed significantly, in favour of the early intensified protocol (Cohort 2). In a multivariate Cox-regression model, PFS (HR 8·12, 95% confidence interval [CI] 1·83-35·9, P=0·006) and OS (HR 5·86, 95% CI 1·28-26·8, P=0·02) remained superior for Cohort 2 when adjusted for aaIPI3 as the most important prognostic factor. Survival of young poor-prognosis DLBCL patients appears superior after early therapy intensification.

Meta analysis of prognosis in diffuse large B-cell lymphoma patients with c-myc gene rearrangement

Objective To investigate the significance of c-myc gene rearrangement on the long term efficacy of diffuse large B-cell lymphoma (DLBCL), and to provide the evidence-based medicine for prognosis of DLBCL. Methods The Chinese and English database were searched to identify all the articles about the DLBCL with c-myc gene rearrangement by computer. Meta-analysis was completed by RevMan 5.2 software. Results A total of eight studies were included. The Meta-analysis results displayed that DLBCL patients with c-myc gene rearrangement had the poor prognosis. Five years progression free survival (PFS) and overall survival (OS) were significantly worse (HR= 2.28, 95% CI 1.64-3.18 and HR= 2.35, 95% CI 1.93-2.85). Conclusion C-myc gene rearrangement is a potential biomarker for the poor prognosis of DLBCL. Key words: Gene, c-myc; Lymphoma, large B-cell, diffuse; Prognosis; Meta analysis

Early Treatment Intensification with R-ICE Chemotherapy Followed By Autologous Stem Cell Transplantation (ASCT) Using Zevalin-BEAM for Patients with Poor Risk Diffuse Large B-Cell Lymphoma (DLBCL) As Identified By Interim PET/CT Scan Performed after Four Cycles of R-CHOP-14: A Multicenter Phase II Study of the Australasian Leukaemia Lymphoma Study Group (ALLG)

Early Treatment Intensification with R-ICE Chemotherapy Followed By Autologous Stem Cell Transplantation (ASCT) Using Zevalin-BEAM for Patients with Poor Risk Diffuse Large B-Cell Lymphoma (DLBCL) As Identified By Interim PET/CT Scan Performed after Four Cycles of R-CHOP-14: A Multicenter Phase II Study of the Australasian Leukaemia Lymphoma Study Group (ALLG)

Predictive value of F-18 FDG PET/CT quantization parameters in diffuse large B cell lymphoma: a meta-analysis with 702 participants.

F-18 fluorodeoxyglucose (FDG) positron emission tomography/computerized tomography (PET/CT) is considered to be the most beneficial imaging method for staging patients with lymphoma. Whether maximum standardized uptake value (SUVmax), metabolic tumor volume (MTV) and total lesion glycolysis (TLG) acquired from F-18 FDG PET/CT are predictors of prognosis of diffuse large B cell lymphoma (DLBCL) is controversial, with some studies concluding that it is and others concluding the opposite. Therefore, a systematic review was performed to explore the relationship of F-18 FDG PET/CT quantization parameters with the prognosis of DLBCL. Seven trials with a total of 703 DLBCL patients were included for analysis. Hazard ratios (HRs) for progression-free survival (PFS) and overall survival (OS), and odds ratios (ORs) for 3-year PFS and OS were pooled using the STATA package. Combined results suggested a strong link between the high SUVmax, MTV and TLG values and the poor 3-year PFS with ORs of 2.59, 3.69 and 2.29, respectively. Similarly, high MTV and TLG values unfavorably influenced the 3-year OS with ORs of 5.40 and 2.19, respectively. The pooled results also showed that high SUVmax and MTV were negative predictors of PFS with HRs of 1.61 (p=0.038) and 2.18 (p=0.000), respectively. The TLG value was not predictive of PFS. And for OS, only high MTV was a strong predictor of poor prognosis in DLBCL with HR 2.99 (p=0.000). Our results suggested that SUVmax and MTV may be significant prognostic markers for PFS and MTV may be the only predictor for OS in DLBCL.