Development Of B-cell Lymphoma Research Articles

Richter Transformation: Clinical Manifestations, Evaluation, and Management.

Richter transformation is a devastating and rare but not uncommon development of an aggressive B-cell lymphoma in patients with chronic lymphocytic leukemia or small lymphocytic lymphoma. Prognosis is dismal, with survival generally in the realm of months to a couple of years. Many patients progress quickly after traditional first-line immunochemotherapy. Nonetheless, novel therapies are on the horizon. It is important that the advanced practitioner have awareness and knowledge of this condition in order to furnish a crucial timely diagnosis and to provide appropriate treatment.

The m6A methyltransferase WTAP plays a key role in the development of diffuse large B-cell lymphoma via regulating the m6A modification of catenin beta 1

BackgroundDiffuse large B-cell lymphoma (DLBCL) is the most frequently occurring subtype of lymphoma. Unfortunately, the fundamental processes underlying the pathogenesis of DLBCL remain little understood. N6-methyladenosine (m6A) methylation has been shown to be the most common internal alteration of mRNAs found in eukaryotes, and it is thought to play a key role in cancer pathogenesis. However, the precise relationship between m6A mRNA methylation and DLBCL pathogenesis remains to be fully elucidated.MethodsThe mRNA and protein expression of Wilms tumor 1-associating protein (WTAP) were determined using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis in lymphoma cells lines. The effects of WTAP expression on human lymphoma cells lines were assessed using cell proliferation assays, colony formation assays, and CCK8 assays. The Gene Expression Profiling Interactive Analysis (GEPIA) database was used to screen candidate gene targets of WTAP. Finally, the regulatory mechanisms of WTAP in DLBCL were investigated using methylated RNA immunoprecipitation (MeRIP) assays.ResultsThis study investigated the precise function of WTAP in DLBCL formation. The results demonstrated that the levels of m6A RNA methylation and WTAP expression were both elevated in DLBCL cell lines and tissues. Downregulation of WTAP expression in DLBCL cells caused a reduction in cell growth in a functional sense. WTAP knockdown reduced catenin beta 1 (CTNNB1) m6A methylation and CTNNB1 total mRNA levels. Furthermore, CTNNB1 overexpression eliminated the WTAP-induced reduction of cell growth in DLBCL cells.ConclusionsIn conclusion, these findings demonstrated that WTAP promotes DLBCL development via modulation of m6A methylation in CTNNB1.

Identification of LINC00654-NINL Regulatory Axis in Diffuse Large B-Cell Lymphoma In Silico Analysis.

BackgroundThe long non-coding RNA (lncRNA)-mRNA regulation network plays an important role in the development of diffuse large B-cell lymphoma (DLBCL). This study uses bioinformatics to find an innovative regulation axis in DLBCL that will provide a positive reference for defining the mechanism of disease progression.MethodsBatch Cox regression was used to screen prognosis-related lncRNAs, and a random forest model was used to identify hub lncRNA. The clinical value of the lncRNA was evaluated and Spearman correlation analysis was used to predict the candidate target genes. Gene Oncology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were used to define the biological function of the lncRNA. A batch Cox regression model, expression validation, and Spearman correlation analysis were used to select the best downstream target genes. The expression and prognostic value validation of this gene was conducted using public data. Gene Set Enrichment Analysis (GSEA) was performed to explore potential mechanisms for this gene in DLBCL.ResultsLINC00654 was identified as the hub lncRNA and 1443 mRNAs were selected as downstream target genes of the lncRNA. The target genes were enriched in the regulation of GTPase and Notch signaling pathways. After validation, the ninein-like (NINL) gene was selected as the potential target of LINC00654 and the LINC00654-NINL axis was constructed. Patients with better responses to therapy were shown to have high NINL gene expression (p-value = 0.036). NINL also had high expression in the DB cell line and low expression in the OCILY3 cell line. Survival analysis showed that high NINL expression was a risk factor for overall survival (OS) and disease-specific survival (DSS) within older patients and those with advanced-stage cancer. GSEA results showed that NINL may be involved in neutrophil-mediated immunity and NF-κB signaling.ConclusionThis study identified a novel LncRNA00654-NINL regulatory axis in DLBCL, which could provide a favorable reference for exploring the possible mechanisms of disease progression.

Identification of novel integration sites for bovine leukemia virus proviral DNA in cancer driver genes in cattle with persistent lymphocytosis

Enzootic bovine leukosis is one of the unsolved problems of cattle breeding in many countries. The etiological agent of the disease is the bovine leukemia virus (BLV) – an oncogenic retrovirus, that infects B-lymphocytes in cattle. The number and genetic content of BLV provirus integration sites in the bovine genome were reported to can be used as an early diagnostic sign of leukemogenesis in the infected cattle, but patterns of BLV provirus integration into the bovine genome and associations between genomic features of the integration sites and development of lymphocytosis and B-cell lymphomas remain poorly elucidated. Here we present data on five novel BLV provirus integration sites in the genome of cattle with persistent lymphocytosis. Two of these sites were located in introns of scfd2 and pgpep1 genes, which have been recognized as cancer driver genes. Three of the rest integration sites were found in the intergenic spaces between ctps1 and cited4, nampt and ccdc71, skp2 and lmbrd2 genes, from which cited4 and skp2 also possess oncogenic properties. These data support previous findings of the association between localization of BLV proviral DNA near cancer driver genes and leukemogenesis in the BLV-infected cattle.

LncRNA FIRRE stimulates PTBP1-induced Smurf2 decay, stabilizes B-cell receptor, and promotes the development of diffuse large B-cell lymphoma.

Sustained expression of B-cell receptor (BCR) critically contributes to the development of diffuse large B-cell lymphoma (DLBCL). However, little is known on the mechanism regulating BCR expression. In the present study, we explored the biological significance of functional intergenic repeating RNA element (FIRRE) in DLBCL and its regulation on BCR. Functional impacts of FIRRE on cell viability, transformation, and apoptosis were examined by MTT, colony formation, and flow cytometry, respectively. The interaction between FIRRE and polypyrimidine tract binding protein 1 (PTBP1) was identified by RNA pull-down and verified using RNA immunoprecipitation (RIP) assays. The effects of FIRRE and PTBP1 on Smurf2 mRNA were examined by RIP, RNA pull-down, and mRNA stability assays. Smurf2-mediated BCR ubiquitination was investigated using co-immunoprecipitation, ubiquitination, and protein stability assays. In vivo, xenograft models were used to assess the impacts of targeting FIRRE on DLBCL growth. FIRRE was specifically up-regulated in and essentially maintained multiple malignant behaviors of BCR-dependent DLBCL cells. Through the interaction with PTBP1, FIRRE promoted the mRNA decay of Smurf2, a ubiquitin ligase for the degradation BCR protein. Targeting FIRRE was sufficient toregulat Smurf2 and BCR expressions and inhibit DLBCL malignancy both in vivo and in vitro. FIRRE-PTBP1 interaction, by simulating Smurf2 mRNA decayand stabilizing BCR, promotes the development of DLBCL. Consequently,targeting this signaling mechanism may provide therapeutic benefits for DLBCL.

No evidence of Borrelia in cutaneous infiltrates of B-cell lymphomas with a highly sensitive, semi-nested real-time polymerase chain reaction targeting the 5S-23S intergenic spacer region.

The role of Borrelia in the development of skin lymphomas has been under discussion for decades. A similar association has been shown for Helicobacter pylori and gastric lymphomas (MALT type). Nevertheless, few molecular studies investigated Borrelia in skin lymphomas and the results are controversial. We analysed 46 formalin-fixed, paraffin-embedded skin specimens of clincopathologically confirmed B-cell lymphomas (15 marginal zone lymphomas; 20 follicular lymphomas; three diffuse large B-cell lymphomas; eight secondary cutaneous infiltrates) taken from 36 patients from Northern Germany, an endemic area for Borrelia. Fifteen pseudolymphomatous lesions of cutaneous Borreliosis served as the control. Both groups were examined with a real-time (rt) PCR and a semi-nested PCR targeting the 5S-23S intergenic spacer region (IGS). A multiplex PCR was used to investigate B-cell clonality in all lymphomatous infiltrates (Biomed Primers). With both assays no Borrelia burgdorferi-specific DNA was identified in any of the B-cell lymphomas, while all 15 Borreliosis specimens gave a positive PCR result in the semi-nested PCR protocol, 12 were also positive in the rt PCR (P < 0.01). All B-cell lymphomas showed monoclonal IgH-Rearrangement. Analysis of cutaneous B-cell lymphomas from available studies including ours (n = 334) reveals an odds ratio <1. While some previous studies suggested an association between B. burgdorferi and the development of cutaneous B-cell lymphomas in endemic areas, we were unable to confirm this in our patients, despite a highly sensitive Borrelia PCR assay. Our results including meta-analysis of previous studies question the need for antibiotic therapy in patients with cutaneous B-cell lymphomas.

Vav1 Promotes B-Cell Lymphoma Development.

Vav1 is normally and exclusively expressed in the hematopoietic system where it functions as a specific GDP/GTP nucleotide exchange factor (GEF), firmly regulated by tyrosine phosphorylation. Mutations and overexpression of Vav1 in hematopoietic malignancies, and in human cancers of various histologic origins, are well documented. To reveal whether overexpression of Vav1 in different tissues suffices for promoting the development of malignant lesions, we expressed Vav1 in transgenic mice by using the ubiquitous ROSA26 promoter (Rosa Vav1). We detected Vav1 expression in epithelial tissues of various organs including pancreas, liver, and lung. While carcinomas did not develop in these organs, surprisingly, we noticed the development of B-cell lymphomas. Rac1-GTP levels did not change in tissues from Rosa Vav1 mice expressing the transgenic Vav1, while ERK phosphorylation increased in the lymphomas, suggesting that signaling pathways are evoked. One of the growth factors analyzed by us as a suspect candidate to mediate paracrine stimulation in the lymphocytes was CSF-1, which was highly expressed in the epithelial compartment of Rosa Vav1 mice. The expression of its specific receptor, CSF-1R, was found to be highly expressed in the B-cell lymphomas. Taken together, our results suggest a potential cross-talk between epithelial cells expressing Vav1, that secrete CSF-1, and the lymphocytes that express CSF-1R, thus leading to the generation of B-cell lymphomas. Our findings provide a novel mechanism by which Vav1 contributes to tumor propagation.

CDK12 activates MYC to repress miR-28-5p/EZH2 and amplifies tonic BCR signaling to promote the development of diffuse large B-cell lymphoma.

Cyclin-dependent kinase 12 (CDK12) is a transcription-associated kinase that participates in various cellular processes. However, its regulatory role in the progression of diffuse large B-cell lymphoma (DLBCL), which is the most prevalent subtype of non-Hodgkin lymphoma (NHL), is still elusive and controversial.The expression of CDK12 was detected by immunohistochemistry (IHC), RT-qPCR was performed to detect miR-28-5p expression of OCI-LY3 and SU-DHL-4 cells. MTT and soft agarose colony formation assays were used to detect cell proliferation. The cell apoptosis was determined by flow cytometry. The protein expressions changes of MYC, EZH2 and the biomarkers of BCR signaling were also detected. A subcutaneous transplantation tumor model of OCI-LY3 cells in nude mice was established to evaluate anticarcinogenic activities of CDK12 knockdown. Elevated expression of CDK12 was observed while miR-28-5p was downregulated in DLBCL tissues. CDK12 knockdown or miR-28-5p overexpression could inhibit proliferation and promote apoptosis of DLBCL cells. miR-28-5p inhibition could reverse the effect of CDK12 knockdown on proliferation and apoptosis of DLBCL cells. In addition, CDK12 knockdown could inhibit DLBCL tumor growth in the mice model. CDK12 activated MYC to repress miR-28-5p/EZH2 and amplified tonic BCR signaling to promote the development of DLBCL, which might provide potential therapeutic targets for future therapeutic intervention in DLBCL.

Helicobacter pylori cagA status and gastric mucosa-associated lymphoid tissue lymphoma: a systematic review and meta-analysis

BackgroundRecent studies have investigated the role of Helicobacter pylori infection in the development of gastric mucosa-associated lymphoid tissue (MALT) lymphoma. It is estimated that approximately 0.1% of people infected with H. pylori develop gastric MALT lymphoma. However, the role of the CagA antigen, the highest causative agent of H. pylori, in increasing the risk of gastric MALT lymphoma remains unclear and controversial. A systematic review and meta-analysis were conducted to evaluate the effect of cagA status on the development of gastric MALT lymphoma.MethodsAll articles evaluating the status of the cagA gene in the development of gastric MALT lymphoma were collected using systematic searches in online databases, including PubMed, Scopus, Embase, and Google Scholar, regardless of publication date. The association between cagA and gastric MALT lymphoma was assessed using the odds ratio (OR) summary. In addition, a random-effects model was used in cases with significant heterogeneity.ResultsA total of 10 studies met our inclusion criteria, among which 1860 patients participated. No association between cagA status and the development of MALT lymphoma (extranodal marginal zone B-cell lymphoma) was found in this study (OR 1.30; 0.906–1.866 with 95% CIs; I2: 45.83; Q-value: 12.92). Surprisingly, a meaningful association was observed between cagA status and diffuse large B-cell lymphoma (OR 6.43; 2.45–16.84 with 95% CIs). We also observed an inverse association between vacA and gastric MALT lymphoma risk (OR 0.92; 0.57–1.50 with 95% CIs).ConclusionsIt seems that the infection with cagA-positive H. pylori strains does not have a meaningful effect on the gastric MALT lymphoma formation, while translocated CagA antigen into the B cells plays a crucial role in the development of diffuse large B-cell lymphoma.

Sequential development of B-cell lymphoma of alternative lineage after treatment for mantle cell lymphoma in a dog

Sequential development of B-cell lymphoma of alternative lineage after treatment for mantle cell lymphoma in a dog

Long non-coding RNA HAGLROS promotes the development of diffuse large B-cell lymphoma via suppressing miR-100.

BackgroundLong non‐coding RNAs (lncRNAs), a vital component of functional regulators, are involved in various human cancers development, including diffuse large B‐cell lymphoma (DLBCL). In particular, lncRNA HAGLROS has been reported to be associated with several types of cancer in humans. Nevertheless, the role of HAGLROS in DLBCL has yet to be described.MethodsThe HAGLROS expression patterns and its relationship with clinicopathological features and survival were investigated in DLBCL patients. CCK‐8 and transwell assays were used to analyze the cell proliferation, migration, and invasion capacities. AGO2‐RIP, dual‐luciferase assay, RT‐qPCR, and rescue experiments were fulfilled to measure the physical interaction between HAGLROS and miR‐100. Xenograft assay was conducted to test tumor growth ability.ResultsHAGLROS was upregulated in DLBCL tissues and cells, and closely associated with advanced clinicopathological features. Upregulation of HAGLROS resulted in poor survival outcomes in DLBCL patients. In addition, HAGLROS knockdown inhibited the proliferation, migration, and invasion of DLBCL cells in vitro. Further experiments revealed that HAGLROS negatively regulated the expression of miR‐100 in DLBCL, and the expression of miR‐100 and HAGLROS showed an inverse correlation in DLBCL tissues. HAGLROS functioned as a competing endogenous RNA for miR‐100 in DLBCL cells, and miR‐100 overexpression abolished the oncogenic effects of HAGLROS upregulation on DLBCL progression. Besides, in‐vivo assays revealed that HAGLROS knockdown suppressed tumor growth in nude mice.ConclusionHAGLROS overexpression contributes to DLBCL development and poor prognosis via targeting miR‐100, which could be a potential prognostic biomarker and therapeutic target for DLBCL patients.

SEPT6_TRIM33 Gene Fusion and Mutated TP53 Pathway Associate With Unfavorable Prognosis in Patients With B-Cell Lymphomas.

BackgroundMounting studies have sought to identify novel mutation biomarkers having diagnostic and prognostic potentials. Nevertheless, the understanding of the mutated pathways related to development and prognosis of B-cell lymphoma is still lacking. We aimed to comprehensively analyze the mutation alterations in genes of canonical signaling pathways and their impacts on the clinic outcomes of patients with B-cell lymphoma.MethodsCirculating cell-free DNA (cfDNA) samples from 79 patients with B-cell lymphomas were used for targeted sequencing with a 560-gene panel for depicting mutation landscapes and identifying gene fusion events. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analyses of mutated genes were performed. The associations of mutation status of genes and seven canonical oncogenic pathways with progression-free survival (PFS) were assessed using Kaplan-Meier test and multivariate Cox analysis. The variant allele frequencies (VAFs) of genes in TP53 and Hippo pathways in paired baseline and post-treatment samples from 18 B-cell lymphoma patients were compared. Finally, the associations of identified fusion genes, mutated genes, and pathways with treatment response were evaluated based on objective response rates (ORRs) comparisons of groups.ResultsWe identified 666 mutations from 262 genes in baseline cfDNAs from 79 B-cell lymphoma patients, and found some genes were preferentially mutated in our cohort such as GNAQ, GNAS, H3F3A, DNMT3A, HLA-A, and HLA-B. These frequently mutated genes were significantly associated with negative “regulation of gene expression, epigenetic” and virus infections such as cytomegalovirus, Epstein-Barr virus, human immunodeficiency virus 1 infections. We detected five fusion genes in at least two patients with B-cell lymphoma, and among them, TCF7L2_WT1 gene fusion was most frequently detected in 30.4% of patients (24 of 79 cases). SEPT6_TRIM33 gene fusion, mutated TP53 and Hippo pathways were significantly associated with poor PFS, and SEPT6_TRIM33 fusion gene and mutated TP53 pathway were independent prognostic factors for B-cell lymphoma. A decreased VAF of TP53 p.Y88C and LATS2 p.F972L was detected in patients with complete response to treatments. Moreover, a significant difference in ORR was observed in patients with NPM1_NR4A3 and SEPT6_TRIM33 fusions.Conclusions SEPT6_TRIM33 gene fusion and mutated TP53 and Hippo pathways may serve as prognostic makers for B-cell lymphoma patients.

IRAK1-regulated IFN-\u03b3 signaling induces MDSC to facilitate immune evasion in FGFR1-driven hematological malignancies

BackgroundStem Cell leukemia/lymphoma syndrome (SCLL) presents as a myeloproliferative disease which can progress to acute myeloid leukemia and is associated with the coincident development of B-cell and T-cell lymphomas. SCLL is driven by the constitutive activation of fibroblast growth factor receptor-1 (FGFR1) as a result of chromosome translocations with poor outcome. Mouse models have been developed which faithfully recapitulate the human disease and have been used to characterize the molecular genetic events that are associated with development and progression of the disease.MethodsCRISPR/Cas9 approaches were used to generate SCLL cells null for Interleukin receptor associated kinase 1 (IRAK1) and interferon gamma (IFNG) which were introduced into syngeneic hosts through tail vein injection. Development of the disease and changes in immune cell composition and activity were monitored using flow cytometry. Bead-based immunoassays were used to compare the cytokine and chemokine profiles of control and knock out (KO) cells. Antibody mediated, targeted depletion of T cell and MDSCs were performed to evaluate their role in antitumor immune responses.ResultsIn SCLL, FGFR1 activation silences miR-146b-5p through DNMT1-mediated promoter methylation, which derepresses the downstream target IRAK1. IRAK1 KO SCLL cells were xenografted into immunocompetent syngeneic mice where the typical rapid progression of disease was lost and the mice remained disease free. IRAK1 in this system has no effect on cell cycle progression or apoptosis and robust growth of the KO cells in immunodeficient mice suggested an effect on immune surveillance. Depletion of T-cells in immunocompetent mice restored leukemogenesis of the KO cells, and tumor killing assays confirmed the role of T cells in tumor clearance. Analysis of the immune cell profile in mice transplanted with the IRAK1 expressing mock control (MC) cells shows that there is an increase in levels of myeloid-derived suppressor cells (MDSCs) with a concomitant decrease in CD4+/CD8+ T-cell levels. MDSC suppression assays and depletion experiments showed that these MDSCs were responsible for suppression of the T cell mediated leukemia cell elimination. Immuno-profiling of a panel of secreted cytokines and chemokines showed that activation of IFN-γ is specifically impaired in the KO cells. In vitro and in vivo expression assays and engraftment with interferon gamma receptor-1 (IFNGR1) null mice and IFNG KO SCLL cells, showed the leukemia cells produced IFN-γ directly participating in the induction of MDSCs to establish immune evasion. Inhibition of IRAK1 using pacritinib suppresses leukemogenesis with impaired induction of MDSCs and attenuated suppression of CD4+/CD8+ T-cells.ConclusionsIRAK1 orchestrates a previously unknown FGFR1-directed immune escape mechanism in SCLL, through induction of MDSCs via regulation of IFN-γ signaling from leukemia cells, and targeting IRAK1 may provide a means of suppressing tumor growth in this syndrome by restoring immune surveillance.

LINC00908 Promotes Diffuse Large B-Cell Lymphoma Development by Down-Regulating miR-671-5p [Retraction

[This retracts the article DOI: 10.2147/CMAR.S299715.].

P26 A case of B-cell lymphoma affecting the eye in a patient with rheumatoid arthritis following a reduction in the frequency of rituximab therapy

P26 A case of B-cell lymphoma affecting the eye in a patient with rheumatoid arthritis following a reduction in the frequency of rituximab therapy

PLK1-dependent phosphorylation restrains EBNA2 activity and lymphomagenesis in EBV-infected mice

While Epstein–Barr virus (EBV) establishes a life-long latent infection in apparently healthy human immunocompetent hosts, immunodeficient individuals are at particular risk to develop lymphoproliferative B-cell malignancies caused by EBV. A key EBV protein is the transcription factor EBV nuclear antigen 2 (EBNA2), which initiates B-cell proliferation. Here, we combine biochemical, cellular, and in vivo experiments demonstrating that the mitotic polo-like kinase 1 (PLK1) binds to EBNA2, phosphorylates its transactivation domain, and thereby inhibits its biological activity. EBNA2 mutants that impair PLK1 binding or prevent EBNA2 phosphorylation are gain-of-function mutants. They exhibit enhanced transactivation capacities, accelerate the proliferation of infected B cells, and promote the development of monoclonal B-cell lymphomas in infected mice. Thus, PLK1 coordinates the activity of EBNA2 to attenuate the risk of tumor incidences in favor of the establishment of latency in the infected but healthy host.

MPN-053: Tumor-Intrinsic IRAKI-Dependent Inflammation Signaling Induces MDSC to Establish Immune Evasion in FGFRI-Driven Hematological Malignancies

Context: Stem cell leukemia/lymphoma syndrome (SCLL) presents as a myeloproliferative disease that can progress to acute myeloid leukemia and is associated with the coincident development of B-cell and T-cell lymphomas. SCLL is driven by the constitutive activation of FGFR1, as a result of chromosome translocations, with poor outcome. Objective: In this study, we use this model to investigate how FGFR1 drives IRAK1-dependent inflammatory signaling in the modulation of antitumor immunity. Design: CRISPR/Cas9 approaches were used to generate SCLL cells null for IRAK1, which were introduced into syngeneic hosts through tail vein injection. Development of the disease and changes in immune cell composition were monitored using flow cytometry. Results: In SCLL, FGFR1 activation silences miR-146b-5p through DNMT1-mediated promoter methylation, which derepresses the downstream target IRAK1. IRAK1 knockout (KO) in SCLL cells leads to the loss of typical rapid progression when xenografted into immunocompetent mice, and the mice remained disease-free. IRAK1 in this system has no effect on cell cycle progression or apoptosis, and robust growth of the KO cells in immunodeficient mice suggested an effect on immune surveillance. Depletion of T-cells in immunocompetent mice restored leukemogenesis of the KO cells. Analysis of the immune cell profile in mice transplanted with the IRAK1-null cells shows that there is a reduction in levels of myeloid-derived suppressor cells (MDSCs) with a concomitant increase in CD4+/CD8+ T-cell levels. Immunoprofiling of a panel of secreted cytokines and chemokines showed that activation of INF-γ is specifically impaired in the KO cells. In vitro and in vivo expression assays and engraftment into IFNGR1-null mice showed SCLL leukemia cells produced INF-γ, not only promoting intrinsic PD-L1 expression but also participating in the induction of MDSCs to establish immune evasion. Inhibition of IRAK1 using pacritinib suppresses leukemogenesis with impaired induction of MDSCs and attenuated suppression of CD4+/CD8+ T-cells. Conclusions: IRAK1 orchestrates a previously unknown FGFR1-directed immune escape mechanism in SCLL, through induction of IFN-γ, and targeting IRAK1 may provide a means of suppressing tumor growth in this syndrome by restoring immune surveillance. This work was supported by grant CA076167 from the National Institutes of Health.

Clinical Outcome and Prognostic Factors of Patients with Richter's Syndrome: Retrospective Multicenter Study of the Spanish Chronic Lymphocytic Leukemia (CLL) Study Group (GELLC)

Richter syndrome (RS), the development of diffuse large B-cell lymphoma (DLBCL) in the context of CLL, is an uncommon complication with a dismal prognosis. Currently, data on biological and clinical features, and factors predicting the outcome of patients are scarce, and there is still no consensus on the best therapeutic approach for these patients (pts). The aim of this study was to analyze the presenting characteristics, treatment outcomes, and factors predicting for survival in a series of pts with RS from a large population-based cohort of the Spanish CLL Study Group (GELLC).Pts diagnosed with RS during 1988 - 2017 were retrospectively identified in eleven centers of the GELLC group. Only cases with confirmed biopsy-proven RS during follow-up were included. Clinical-biological features at diagnosis of RS, including cytogenetics, clonal relation with the pre-existing CLL, Epstein-Barr virus (EBV) status detected by in situ hybridization, cell of origin (COO) analyzed by immunohistochemistry, International Prognostic Index score (IPI) and the RS score (Tsimberidou AM et al, J Clin Oncol, 2006), as well as treatment and outcomes, were analyzed.One hundred and twenty-three pts with transformation of CLL to high-grade lymphoma were identified, including 108 classical DLBCL RS, 14 pts with Hodgkin lymphoma (HL), and one case of plasmablastic lymphoma. Median age of the whole series was 66 years (range, 31 to 92 years), and 64% were men. For the DLBCL RS, median time from the diagnosis of CLL to RS development was of 4.7 years (range, 0-24.8 years, with 9 simultaneous diagnosis of CLL and RS), and the median number of prior therapies for CLL/SLL was 1 (range, 0-7). The main clinical characteristics of the pts are shown in table 1. EBV was detected in 12/36 (33%) cases with classical RS and in 9/11 (82%) of patients with HL variant. According to COO the majority of RS tested (20 of 24, 83%) had a non-germinal center phenotype, whereas 19/23 (83%) cases were clonally related to the preceding CLL. Twelve out of 43 pts had 17p deletion or mutation of TP53 in the CLL clone at the time of RS diagnosis.Among the 108 patients with DLBCL RS, data on the treatment for RS was available in 99 patients. Twenty-one patients did not received treatment due to frailty, comorbidities, or poor performance status. Of the 78 patients that were treated for RS, 46 patients received chemoimmunotherapy, mainly R-CHOP (n=44), 29 patients chemotherapy (most commonly CHOP and purine analogue-based therapy), and others (n=3). The overall response rate for the 99 treated patients was 44% (chemotherapy, 24%; chemoimmunotherapy, 60%; P=0.003). A total of 13 patients underwent stem-cell transplantation (SCT), allogeneic SCT (n=7) or autologous SCT (n=6).After a median follow-up for surviving patients of 36 months (range 0.8-160 months), median overall survival (OS) from the time of transformation of CLL to high grade lymphoma was 7.3 months for the entire cohort, 5.7 months for DLBCL RS patients, and 30.8 months for patients with HL variant (P=0.07). Factors associated with shorter OS in the DLBCL RS series included, ECOG PS >1, Hb level < 10 g/dL, platelet count < 100x109/L, LDH > 1.5 UNL, β2-microglobulin > 3mg/dL, prior CLL therapies >1, p53 positive determined by IHQ, and 17p deletion or mutation of TP53 determined in the CLL clone. RS score was also predictive for OS (median OS of 59.5 months [0-1 points], 5.4 months [2 points], 2 months [3 points], and 3 months [4-5 points], p= 0.006); on the contrary, IPI was not discriminative for OS in this series. In addition, RS clonally related presented a median OS of 5.9 months in comparison with 74.8 months for the clonally unrelated RS (p= 0.071). Finally, patients treated with chemoimmunotherapy combinations presented a median OS of 35.4 months, and the 13 patients that could consolidated the response obtained with SCT (autologous or allogeneic SCT) obtained a median OS of 59.5 months.Although clinical outcome of patients with RS is generally poor, chemoimmunotherapy combinations are effective and obtain durable responses in a proportion of patients. RS score and TP53 disruption are predictive of survival. Finally, pts attaining a response should be considered for SCT. Further clinical development with novel promising treatment options is warranted. [Display omitted] DisclosuresDelgado:Abbvie, Jansen, Gilead, Roche: Consultancy, Speakers Bureau.

Genetic Characterization and Clinical Impact of Loss of MHC Class I and II Expression in De Novo Diffuse Large B-Cell Lymphoma

Introduction: Immune recognition by T cells requires the presentation of processed peptides in association with major histocompatibility complex class I (MHC-I) and class II (MHC-II) molecules. Among the tumor immune escape mechanisms described to date, alterations in the expression of MHC molecules play a crucial role in the development of diffuse large B-cell lymphoma (DLBCL). Loss of MHC-I or -II expression in DLBCL can occur through multiple mechanisms, including deletion of the MHC-I and -II locus, loss of function mutations of B2M, and genetic alterations of CIITA ; however, the role of additional genetic and epigenetic mechanisms remains largely unknown. With regard to clinical significance, a number of studies have reported that low MHC-II expression on tumor cells is associated with shorter survival in DLBCL, while the prognostic effect of MHC-I expression has not been systematically evaluated in a large, homogeneously treated cohort. In addition, although the frequency of loss of MHC-II expression differs between ABC- and GCB-DLBCL cell of origin (COO) subtypes, the prognostic impact remains ill-defined. Here, we conducted a comprehensive phenotypic and genetic analysis in a population-based DLBCL cohort to investigate the genetic background and clinical relevance of loss of MHC-I and -II expression.Method: We analyzed biopsies from 347 patients newly diagnosed with de novo DLBCL and uniformly treated with R-CHOP at the BC Cancer Agency. High-resolution copy number analysis was performed using Affymetrix SNP 6.0 arrays. Mutation status was determined using deep targeted re-sequencing of the coding exons of 57 genes with a Truseq Custom Amplicon assay (Illumina) and/or Fluidigm Access Array chips, and RNAseq. Immunohistochemical staining of MHC-I and -II was performed on tissue microarrays (n=332). COO was assigned by gene expression (Lymph2Cx assay) in 323 cases (183 GCB, 104 ABC and 36 unclassifiable).Results: We found the following MHC-I and -II staining patterns:membrane-positive (n=188 (57%) and n=233 (72%), respectively), cytoplasmic only-positive / membrane-negative (n=86 (26%) and 31 (10%), respectively) and negative staining for both membrane and cytoplasmic (n=55 (17%) and n=59 (18%), respectively). Overall, membranous MHC-I and -II expression was not detected in 141/329 (43%) and 90/323 (28%) cases, respectively. While the frequency of loss of MHC-I expression was not different between COO subtypes, loss of MHC-II expression occurred more often in ABC-DLBCL (36% vs 22%, p=0.026). This is consistent with previous observations showing that MHC-II expression is reduced during plasmacytic differentiation and in ABC-DLBCL. Loss of MHC-I expression was associated with B2M mutation and deletion as well as with HLA-C deletions (all p <0.001, Fisher's exact test). Interestingly, mutations of EZH2 and GNA13 were detected more frequently in cases that had loss of MHC-I expression (p <0.001 and p=0.004, respectively). Moreover, mutations of CIITA and RFXAP (components of the MHC-II enhanceosome), and mutations of PRDM1 and TNFAIP3, ABC-DLBCL-specific mutations, were significantly enriched in MHC-II negative cases. Of note, mutations of B2M and EZH2 were significantly associated with loss of MHC-II expression, as well (p=0.001 and p=0.016, respectively). The associations between EZH2 mutation and MHC-I and -II expression were also observed when the analysis was restricted to GCB-DLBCL (both p <0.0001).With a median follow up of 6.5 years for living patients, the loss of MHC-I expression was not associated with survival in the entire DLBCL cohort, as well as within each COO subtype. Although there was also no significant survival difference according to the MHC-II expression status in the entire DLBCL cohort, loss of MHC-II expression was associated with an inferior 5y-time to progression (TTP, 59% vs 79%, p=0.007) in the GCB subtype. Interestingly, in the ABC subtype, loss of MHC-II expression was associated with a superior 5y-TTP (64% vs 40%, p=0.03).Conclusion: Our approach using next generation sequencing and high resolution SNP arrays uncovered the genetic background, as well as the clinical impact of loss of MHC-I and-II expression in a uniformly R-CHOP-treated large population-based cohort of DLBCL patients. Our results warrant further studies investigating a potential mechanism of EZH2 mutations regulating MHC-I and -II expression in DLBCL. DisclosuresSavage:Bristol-Myers Squibb: Honoraria; Seattle Genetics: Consultancy, Honoraria; Roche: Research Funding; Celgene: Consultancy; Merck: Honoraria. Sehn:Celgene: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Roche/Genentech: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria. Morin:Epizyme, Inc: Consultancy. Connors:Genentech: Research Funding; Bayer Healthcare: Research Funding; Lilly: Research Funding; Merck: Research Funding; Seattle Genetics: Research Funding; Amgen: Research Funding; Janssen: Research Funding; F Hoffmann-La Roche: Research Funding; NanoString Technologies: Research Funding; Bristol-Myers Squibb: Research Funding; Cephalon: Research Funding; Takeda: Research Funding; NanoString Technologies, Amgen, Bayer, BMS, Cephalon, Roche, Genentech, Janssen, Lilly, Merck, Seattle Genetics, Takeda,: Research Funding. Scott:Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; BCCA: Patents & Royalties: Patent describing molecular subtyping of DLBCL licensed to NanoString Technologies. Patent describing measurement of the proliferation signature in MCL.. Steidl:Affimed Therapeutics: Consultancy; Juno Therapeutics: Consultancy.

Modeling Lymphoplasmacytic Lymphoma/Waldenström Macroglobulinemia in Transgenic Mice Using Human MYD88L256P Mutated Protein

Lymphoplasmacytic lymphoma (LPL)/Waldenström macroglobulinemia (WM) is a B-cell disorder resulting from the accumulation of cloncal lymphoplasmacytic cells in the bone marrow (BM) along with excessive secretion of immunoglobulin M paraprotein. Despite recent advances in treatment LPL/WM remains incurable. Generating a refined LPL/WM mouse model that would facilitate studying this disease as well as enable stringent in vivo validation of potential therapeutic approaches has been a long-sought goal for the filed. Interestingly, among the most common mutations identified by recent high-throughput studies there was a single gain of function point mutation in the MYD88 gene, with a predicted non-synonymous amino acid (L265P) change, found in over 91% of patients. Therefore, we hypothesized that generation of transgenic mice overexpressing MYD88L256Pcould recapitulate the human setting of LPL/WM.To overcome problems inherently related to embryonic lethality, we generated MYD88WT and MYD88L256P conditional transgenic mice expressing human proteins. To remove a transcriptional stop cassette and activate MYD88 expression in vivo, we mated the conditional mice with mice expressing Cre recombinase under the control of Activation-Induced Cytidine Deaminase (AID) gene promoter to generate AIDCre/-;MYD88WT/- and AIDCre/-;MYD88L256P/- double-mutant mice. Recombinase activity driven by the AID gene promoter caused removal of the stop cassette and expression of MYD88 in germinal center (GC) and post-GC B cells, the appropriate B-cell compartment from which LPL/WM clones are thought to originate in humans. Young (up to 30 weeks of age) AIDCre/-;MYD88WT/- and AIDCre/-;MYD88L256P/- mice where indistinguishable from AIDCre/- mice as assessed by external physical examination. Moreover, obvious histologic and IHC differences in spleens, lymph nodes, and bone marrows between the mutant genotypes and the AIDCre/- control mice were not found. No clonal bands for the IgH gene, nor M-spikes were detected in Southern blots and serum protein electrophoresis, respectively. However, after 30 weeks of age, and at variable times thereafter, some of the AIDCre/-; MYD88L256P/- aging cohort (n=27) mice developed skin rash and excoriation as well as loss of hair in the submandibular areas. A fraction of AIDCre/-; MYD88L256P/- animals that weren't sacrificed because of the severe dermatitis had enlarged lymph nodes and appeared sick. To assess whether MYD88 transgenic mice develop B-cell lymphomas or major pathologic alterations consistent with the diagnosis of LPL/WM, animals were euthanized at different ages and lymphoid organs were rigorously examined. In spleens and lymph nodes of 8 of the AIDCre/-; MYD88L256P/- animals, a nodular lymphoid infiltrate composed predominantly of small atypical cells varyingly infiltrated with CD138+ plasma cells was observed. Lymphoid cells infiltrated other organs including the BM. Enhanced expression of MYD88 and activation of NF-κB activity within atypical lymphoid nodules in diseased transgenic mice was documented by IHC staining for MYD88 and p65. However, no clonal bands of the IgH gene were detected in Southern blots, suggesting that overexpression of MYD88L256P/- mutant protein in activated B-cells gave rise to a polyclonal lymphoproliferative disorder. Consistently, serum protein electrophoresis analysis showed diffuse, polyclonal increase in γ-globulins. Moreover, stains of peripheral blood smears revealed rouleaux formation suggesting increased concentrations of plasma proteins. In addition, one of AIDCre/-; MYD88L256P/- animals developed a more aggressive monoclonal disease resembling human diffuse large B-cell lymphoma. However, large atypical lymphoid cells in this mouse lacked expression of MYD88 and nuclear p65. All AIDCre/- controls and AIDCre/-; MYD88WT/- mice lacked described phenotypes and were histologically unremarkable.Taken together, these results demonstrate that although MYD88L256P/- alone might not be sufficient to induce a malignant transformation in the mature B-cell compartment, it gave rise to a low-grade mature non-clonal B-cell lymphoproliferative disorder with some of the characteristic futures of LPL/WM. Therefore, the MYD88L256P/- mutation might be necessary but not sufficient for LPL/WM development and must be present with concomitant “second hit” genetic alterations for a neoplasm to develop. DisclosuresTreon:Pharmacyclics: Consultancy, Research Funding.