IGFBP7-AS1 is a p53-responsive long noncoding RNA downregulated by Epstein-Barr virus that contributes to viral tumorigenesis.

Epstein-Barr virus (EBV) is closely related to the development of several malignancies, such as B-cell lymphoma (B-CL), by the mechanism through which these malignancies develop remains largely unknown. We previously observed downregulation of the long noncoding RNA (lncRNA) IGFBP7-AS1 in response to EBV infection. However, the role of IGFBP7-AS1 in EBV-associated cancers has not been clarified. Here, we found that expression of IGFBP7-AS1, as well as its sense gene IGFBP7, is decreased in EBV-positive B-CL cells and clinical tissues. IGFBP7-AS1 stabilizes IGFBP7 mRNA by forming a duplex based on their overlapping regions. The tumour suppressor p53 transcriptionally activates IGFBP7-AS1 expression by binding to the promoter region of the lncRNA gene. The IGFBP7-AS1 expression is able to be rescued in EBV-positive cells in wild-type (wt) p53-dependent manner. IGFBP7-AS1 inhibits the proliferation and promotes the apoptosis of B-CL cells. Moreover, tumorigenic properties due to the depletion of IGFBP7-AS1 were restored by exogenous expression of IGFBP7 or wt-p53. Furthermore, the functional p53/IGFBP7-AS1/IGFBP7 axis facilitates apoptosis by suppressing the production and secretion of the NPPB signal peptide and further regulating the cGMP-PKG signalling pathway. This study demonstrates that EBV promotes tumorigenesis, particularly in B-CL progression, by downregulating the novel p53-responsive lncRNA IGFBP7-AS1.

Calcium-dependent signalling in B-cell lymphomas.

Induced waves of calcium fluxes initiate multiple signalling pathways that play an important role in the differentiation and maturation of B-cells. Finely tuned transient Ca+2 fluxes from the endoplasmic reticulum in response to B-cell receptor (BCR) or chemokine receptor activation are followed by more sustained calcium influxes from the extracellular environment and contribute to the mechanisms responsible for the proliferation of B-cells, their migration within lymphoid organs and their differentiation. Dysregulation of these well-balanced mechanisms in B-cell lymphomas results in uncontrolled cell proliferation and resistance to apoptosis. Consequently, several cytotoxic drugs (and anti-proliferative compounds) used in standard chemotherapy regimens for the treatment of people with lymphoma target calcium-dependent pathways. Furthermore, ~10% of lymphoma associated mutations are found in genes with functions in calcium-dependent signalling, including those affecting B-cell receptor signalling pathways. In this review, we provide an overview of the Ca2+-dependent signalling network and outline the contribution of its key components to B cell lymphomagenesis. We also consider how the oncogenic Epstein-Barr virus, which is causally linked to the pathogenesis of a number of B-cell lymphomas, can modify Ca2+-dependent signalling.

A STING inhibitor suppresses EBV-induced B cell transformation and lymphomagenesis.

Epstein-Barr virus-associated lymphoproliferative disease (EBV-LPD) is frequently fatal. Innate immunity plays a key role in protecting against pathogens and cancers. The stimulator of interferon genes (STING) is regarded as a key adaptor protein allowing DNA sensors recognizing exogenous cytosolic DNA to activate the type I interferon signaling cascade. In terms of EBV tumorigenicity, the role of STING remains elusive. Here we showed that treatment with the STING inhibitor, C-176, suppressed EBV-induced transformation in peripheral blood mononuclear cells. In an EBV-LPD mouse model, C-176 treatment also inhibited tumor formation and prolonged survival. Treatment with B cells alone did not affect EBV transformation, but suppression of EBV-induced transformation was observed in the presence of T cells. Even without direct B cell-T cell contact in a transwell system, the inhibitor reduced the transformation activity, indicating that intercellular communication by humoral factors was critical to prevent EBV-induced transformation. These findings suggest that inhibition of STING signaling pathway with C-176 could be a new therapeutic target of EBV-LPD.

SARS-CoV-2 Infection and Antibody Response in a Symptomatic Cat from Italy with Intestinal B-Cell Lymphoma.

Since the coronavirus disease (COVID-19) pandemic was first identified in early 2020, rare cases of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection in pet cats have been reported worldwide. Some reports of cats with SARS-CoV-2 showed self-limiting respiratory or gastrointestinal disease after suspected human-to-feline transmission via close contact with humans with SARS-CoV-2. In the present study, we investigated a cat with SARS-CoV-2 that was presented to a private animal clinic in Northern Italy in May 2020 in a weak clinical condition due to an underlying intestinal B-cell lymphoma. The cat developed signs of respiratory tract disease, including a sneeze, a cough and ocular discharge, three days after an oropharyngeal swab tested positive for SARS-CoV-2 viral RNA using two real-time reverse transcriptase polymerase chain reaction (RT-qPCR) assays for the envelope (E) and RNA-dependent RNA polymerase (RdRp) gene. Thus, SARS-CoV-2 viral RNA was detectable prior to the onset of clinical signs. Five and six months after positive molecular results, the serological testing substantiated the presence of a SARS-CoV-2 infection in the cat with the detection of anti-SARS-CoV-2 receptor binding domain (RBD) immunoglobulin (IgG) antibodies and neutralizing activity in a surrogate virus neutralization assay (sVNT). To the best of our knowledge, this extends the known duration of seropositivity of SARS-CoV-2 in a cat. Our study provides further evidence that cats are susceptible to SARS-CoV-2 under natural conditions and strengthens the assumption that comorbidities may play a role in the development of clinical disease.

Pathologically Relevant Mouse Models for Epstein-Barr Virus-Associated B Cell Lymphoma.

The Epstein-Barr virus (EBV) is endemic in humans and can efficiently transform infected B cells under some circumstances. If an EBV carrier experiences immune suppression, EBV+ B cells can turn into lymphoblasts and exhibit growth expansion that may cause lymphoproliferative diseases which often develop into lymphoma. Our immune system conducts surveillance for EBV+ B cells in order to block spontaneous tumor formation. Here, we summarize the EBV products involved in tumorigenesis, EBV-associated lymphomas, and pathologically relevant mouse models. Preclinical mouse models for a range of EBV-associated diseases not only clear the path to new therapeutic approaches but also aid in our understanding of the nature of lymphomagenesis and immune surveillance.

Oncolytic virotherapy-mediated anti-tumor response: a single-cell perspective.

Talimogene laherparepvec (T-VEC) is a genetically modified herpes simplex 1 virus (HSV-1) approved for cancer therapy. We investigate its effect on the clinical, histological, single-cell transcriptomic, and immune repertoire level using repeated fine-needle aspirates (FNAs) of injected and noninjected lesions in primary cutaneous B cell lymphoma (pCBCL). Thirteen patients received intralesional T-VEC, 11 of which demonstrate tumor response in the injected lesions. Using single-cell sequencing of the FNAs, we identify the malignant population and separate three pCBCL subtypes. Twenty-four hours after the injection, we detect HSV-1T-VEC transcripts in malignant and nonmalignant cells of the injected lesion but not of the noninjected lesion. Oncolytic virotherapy results in a rapid eradication of malignant cells. It also leads to interferon pathway activation and early influx of natural killer cells, monocytes, and dendritic cells. These events are followed by enrichment in cytotoxic T cells and a decrease of regulatory T cells in injected and noninjected lesions.

Usefulness of Plasma SARS-CoV-2 RNA Quantification by Droplet-based Digital PCR to Monitor Treatment Against COVID-19 in a B-cell Lymphoma Patient.

We report the case of an HIV-1-infected patient, treated with anti-CD20 monoclonal antibody for a B-cell lymphoma previously treated by autologous stem cell transplant. He suffered from chronic COVID19 and we monitored by plasma SARS-CoV-2 RNA by highly sensitive droplet-based digital PCR technology (ddPCR). Under tocilizumab therapy and despite a first clinical improvement biologically associated with decreasing inflammatory markers, a slight increase of SARS-CoV-2 RNAaemia quantified by ddPCR was highlighted, confirming the absence of viral efficacy of this treatment and predicting the subsequent observed deterioration. As expected, his complete recovery, finally achieved after COVID-19 convalescent plasmatherapy, strictly paralleled plasma SARS-CoV-2 RNA clearance. With these results, we confirmed the interest of SARS-CoV-2 RNAaemia monitoring by ddPCR in COVID-19 patients, particularly during treatment, and firstly showed that this new and specific biomarker could be helpful to select eligible patient for anti-IL6 receptors therapy considering the variable levels of efficacy recently observed with such therapy.

Clinical Characteristics of Hemophagocytic Lymphohistiocytosis Associated with Non-Hodgkin B-Cell Lymphoma: A Multicenter Retrospective Study.

BACKGROUND: Hemophagocytic lymphohistiocytosis (HLH) associated with B-cell lymphoma is a highly aggressive disease with unclear clinical features and has no standard treatment. PATIENTS AND METHODS: We analyzed the clinical characteristics of 31 patients from two individual centers. RESULTS: The median overall survival was only 1.5 months. Both univariate and multivariate analyses, based on lymphoma or HLH-related characteristics, revealed that patients with high Epstein-Barr virus (EBV) DNA load and ≥ 2 extranodal lesions, or hypofibrinogenemia, respectively, showed significantly poorer overall survival. Interestingly, some patients with high EBV DNA load had EBV-positive natural killer (NK) and/or T cells, which may be related to the coexistence of immunodeficiency and/or chronic active EBV infection. Molecular genetics examination confirmed that 47.4% (9/19) of patients had complex karyotypes, 37.5% (3/8) of patients had TP53 deletions, and 21.34% (3/14) of patients had TP53 mutation or alteration of malignancy-related pathways, including BCR/NF-κB, JAK-STAT, and epigenetic regulatory pathways, which may provide clues to choose targets for therapy. Treatment regimens containing etoposide, anti-CD20 monoclonal antibodies, or anthracyclines improved patient prognosis (P = .0183, .025, and .0436, respectively). Patients with infections had significantly shorter survival than those without infections (P = .00019). CONCLUSION: The patients' performance status, number of extranodal lesions, high EBV DNA load, and hypofibrinogenemia are poor prognostic factors for HLH associated with B-cell lymphoma. Molecular genetic high-risk factors are of particular importance because these factors can provide information for prognosis prediction, treatment decisions, and disease surveillance.

Development of an in situ hybridization assay using an AS1 probe for detection of bovine leukemia virus in BLV-induced lymphoma tissues.

Enzootic bovine leukosis (EBL) is a malignant B cell lymphoma caused by infection with bovine leukemia virus (BLV). Histopathological examination is commonly used for diagnosis of the disease, but observation of lymphoma alone does not confirm EBL because cattle may be affected by sporadic forms of lymphoma that are not associated with BLV. Detection of BLV in tumor cells can be definitive evidence of EBL, but currently, there is no technique available for such a purpose. In this study, we focused on a viral non-coding RNA, AS1, and developed a novel in situ hybridization assay for the detection of BLV from formalin-fixed paraffin-embedded (FFPE) tissues. RNA-seq analysis revealed that all examined B lymphocytes derived from clinical EBL abundantly expressed AS1 RNA, indicating a possible target for detection. The in situ hybridization assay using an AS1 probe clearly detected AS1 RNA in fetal lamb kidney cells persistently infected with BLV. The utility of this assay in clinical samples was assessed using three EBL-derived lymph node specimens and one BLV-negative specimen, and AS1 RNA was detected specifically in the EBL-derived tissues. These results suggest that AS1 RNA is a useful target for the detection of BLV from FFPE specimens of tumor tissues. This technique is expected to become a powerful tool for EBL diagnosis.

High-grade B-Cell Lymphoma With MYC, BCL2, and/or BCL6 Translocations/Rearrangements: Clinicopathologic Features of 51 Cases in a Single Institution of South China.

Double-hit/triple-hit lymphomas (DH/THLs) are high-grade B-cell lymphomas with MYC and BCL2 rearrangements and/or BCL6 rearrangements, which have poor outcomes after standard chemoimmunotherapy. This retrospective study analyzed 51 patients (range, 19 to 82 y) diagnosed from 2016 to 2019 and treated for DH/THL (n=34 MYC/BCL6 DHL, n=14 MYC/BCL2 DHL, n=3 THL) at one institution in South China. Extranodal lesions occurred in 32 patients (62.7%), more frequently in MYC/BCL6 DHL (22/34, 64.7%) than in MYC/BCL2 DHL (7/14, 50%). The most common extranodal sites were the stomach (8/32, 25.0%) and intestine (5/32, 15.6%). Most patients (33/45, 73.3%) presented with Ann Arbor stage III/IV. Interestingly, 14.3% (4/28) of MYC/BCL6 DHL tumors showed diffuse, medium-intensity CD30 expression. Epstein-Barr virus-encoded RNA was positive in 3 cases, all MYC/BCL6 DHL. Among 48 patients (94.1%) with follow-up data, 18 (37.5%) died owing to the disease, and the median survival was 5.5 months. Germinal center B cells were observed more frequently in MYC/BCL2 DHL (14/14, 100.0%) than in MYC/BCL6 DHL (15/34, 44.1%; P<0.001). Bone marrow involvement tended to lower overall survival (OS) (P=0.033). No association was observed between stage, B symptoms, lactate dehydrogenase levels, and central nervous system involvement and OS. A total of 25 patients (25/47, 53.2%) with previous hepatitis B virus (HBV) infections had significantly poorer OS (P=0.014). Chronic HBV infection was positively correlated with MYC/BCL6 DHL (r=0.317, P=0.030). Compared with DH/THL in western countries, the disease in South China has distinct characteristics with a higher prevalence of MYC/BCL6 DHL. We speculate that HBV is important in DH/THL tumorigenesis. These findings might provide clues for novel treatment strategies.

Functional interplay of Epstein-Barr virus oncoproteins in a mouse model of B cell lymphomagenesis.

Epstein-Barr virus (EBV) is a B cell transforming virus that causes B cell malignancies under conditions of immune suppression. EBV orchestrates B cell transformation through its latent membrane proteins (LMPs) and Epstein-Barr nuclear antigens (EBNAs). We here identify secondary mutations in mouse B cell lymphomas induced by LMP1, to predict and identify key functions of other EBV genes during transformation. We find aberrant activation of early B cell factor 1 (EBF1) to promote transformation of LMP1-expressing B cells by inhibiting their differentiation to plasma cells. EBV EBNA3A phenocopies EBF1 activities in LMP1-expressing B cells, promoting transformation while inhibiting differentiation. In cells expressing LMP1 together with LMP2A, EBNA3A only promotes lymphomagenesis when the EBNA2 target Myc is also overexpressed. Collectively, our data support a model where proproliferative activities of LMP1, LMP2A, and EBNA2 in combination with EBNA3A-mediated inhibition of terminal plasma cell differentiation critically control EBV-mediated B cell lymphomagenesis.

Intrasinusoidal HHV8-EBV-Positive Large B-Cell Lymphoma With Features of Germinotropic Lymphoproliferative Disorder.

Germinotropic lymphoproliferative disorder (GLPD) is a poorly characterized lymphoproliferative entity, recently included in the World Health Organization classification of hematolymphoid neoplasms. The histological pattern of this disease comprises monotypic plasmablasts that involve the germinal centers of the lymphoid follicles (germinotrophism), forming confluent aggregates positive for both human herpes virus type 8 (HHV8) and Epstein-Barr virus. Currently, after 17 years of its first description, only 18 cases have been reported. In this article, we describe a case of a GLPD presenting in an immunocompetent 79-year-old woman with localized axillary lymphadenopathy, showing a prominent sinusoidal growth pattern, with no evidence of germinotrophism or extrasinusoidal spread. Stinking pleomorphism in tumor cells was also noted. An extension study has not revealed involvement of other groups of lymph nodes or extralymphoid sites. The patient is alive and has shown no relapse after 8 years follow-up (the longest follow-up reported period for this entity). This previously unrecognized pure sinusoidal growth pattern along with the striking pleomorphism in neoplastic cells closely mimics the appearance of an anaplastic large cell lymphoma. GLPD is not usually considered in such a setting, but it should be included in the differential diagnosis of sinusoidal proliferations. Our findings contribute to the expansion of the morphological spectrum of HHV8-associated lymphoproliferative lesions and aids in the characterization of the very infrequent GLPD entity.

In situ hybridization of feline leukemia virus in a primary neural B-cell lymphoma.

An 8-y-old castrated male, outdoor European shorthair cat was presented with a history of hindlimb weakness and paralysis. Disease progression was continuous from the onset; deep algesia disappeared at the final stage. Radiography of the vertebral column was unremarkable; along with patient history and physical examination results, magnetic resonance imaging suggested inflammatory lesions in the spinal cord, although neoplasia could not be ruled out. Feline leukemia virus (FeLV) positivity was confirmed by a serum ELISA prior to euthanasia. Upon postmortem examination, hemorrhages were present in the spinal cord at the level of vertebrae T7-8. Histologic and immunohistochemical analysis revealed primary diffuse large B-cell lymphoma of the spinal cord with multifocal myelomalacia and hemorrhages. To determine the presence of a pathogen within the lesion, we developed a novel in situ hybridization protocol for FeLV (RNAscope). The reaction revealed large amounts of FeLV viral RNA in the tumor cells.

HCV infection and non-Hodgkin lymphomas: an evolving story.

Hepatitis C virus infection represents a global health problem with 3% of population infected worldwide. Several epidemiological studies have shown an increased risk of B cell non-Hodgkin lymphomas in HCV-infected subjects with a wide geographic variability. The observation that HCV eradication by antiviral treatment is associated with successful lymphoma response provided the most convincing evidence for the causal role of HCV in lymphoma's development. According to the most accepted model, HCV-driven chronic antigenic stimulation may represent the major stimulus for lymphoma growth. Several evidences have led to recommend antiviral therapy (in the past interferon-based, now the new direct-acting antiviral agents) in the setting of asymptomatic indolent B cell lymphomas not requiring an immediate systemic treatment. The favourable profile of direct-acting antiviral agents supports the HCV eradication also in the setting of HCV-positive diffuse large B cell lymphoma; however, further studies are needed to assess the appropriate timing of these drugs in the treatment of aggressive lymphomas. Multidisciplinary management involving expert hepatologists is highly warranted.

Atypical presentation and pathogenesis of a macaque lymphocryptoviral-associated B-cell lymphoma in a cynomolgus monkey.

We report the unique pathogenesis and presentation of a rapidly progressive B-cell lymphoma in a 3-year-old female cynomolgus monkey on day 50 of a 13-week toxicity study. Clinical pathology evaluation revealed a marked leukocytosis with bicytopenia. A serum protein electrophoresis was consistent with monoclonal gammopathy. The architecture of the lymph node, spleen, and thymus were variably effaced by neoplastic cells, which also infiltrated other tissues. Immunohistochemistry of the affected tissues confirmed a predominant population of CD20+, CD79a+, CD3-, CD68-, and CD34-neoplastic cells. The full data best support a diagnosis of Stage V lymphoma. Nextgen sequencing and negative prestudy serology results suggested a recent infection by macaque lymphocryptovirus (mLCV) with a unique transcriptional profile comparable with a rarely observed direct LCV infection model. This infection model might be associated with a temporary lack of an LCV antigen-specific cytotoxic T-cell adaptive immune response. Consistent with the established mechanisms of LCV-related lymphoproliferation, MYC and BCL2L11 gene expression were increased and decreased, respectively. While there was no overt immunosuppression, immunophenotyping revealed the index animal had a relatively low NK cell count, which further decreased by >50% on day 24 of the study. In addition to the temporary lack of adaptive immunity, the low NK cell counts were suggestive of an impaired innate immunity to control the virally-transformed cells and the subsequent unchecked lymphoproliferation. To our knowledge, this is the first report of a Stage V lymphoma with a unique pathogenesis in an otherwise immunocompetent cynomolgus monkey.

T-Cell Receptor Stimulation Enhances the Expansion and Function of CD19 Chimeric Antigen Receptor-Expressing T Cells.

PURPOSE: Current protocols for CD19 chimeric antigen receptor-expressing T cells (CD19.CAR-T cells) require recipients to tolerate preinfusion cytoreductive chemotherapy, and the presence of sufficient target antigen on normal or malignant B cells. PATIENTS AND METHODS: We investigated whether additional stimulation of CD19.CAR-T cells through their native receptors can substitute for cytoreductive chemotherapy, inducing expansion and functional persistence of CD19.CAR-T even in patients in remission of B-cell acute lymphocytic leukemia. We infused a low dose of CD19.CAR-modified virus-specific T cells (CD19.CAR-VST) without prior cytoreductive chemotherapy into 8 patients after allogeneic stem cell transplant. RESULTS: Absent virus reactivation, we saw no CD19.CAR-VST expansion. In contrast, in patients with viral reactivation, up to 30,000-fold expansion of CD19.CAR-VSTs was observed, with depletion of CD19+ B cells. Five patients remain in remission at 42-60+ months. CONCLUSIONS: Dual T-cell receptor and CAR stimulation can thus potentiate effector cell expansion and CAR-target cell killing, even when infusing low numbers of effector cells without cytoreduction.