Single-Step IGHV Next-Generation Sequencing Detects Clonality and Somatic Hypermutation in Lymphoid Malignancies: A Phase III Diagnostic Accuracy Study.

BACKGROUND: Multiplex PCR based on consensus primers followed by capillary electrophoresis and Sanger sequencing are considered as the gold standard method for the evaluation of clonality and somatic hypermutation in lymphoid malignancies. As an alternative, the next-generation sequencing (NGS) of immune receptor genes has recently been proposed as a solution, due to being highly effective and sensitive. Here, we designed a phase III diagnostic accuracy study intended to compare the current gold standard methods versus the first commercially available NGS approaches for testing immunoglobulin heavy chain gene rearrangements. METHODS: We assessed IGH rearrangements in 68 samples by means of both the NGS approach (LymphoTrack® IGH assay, and LymphoTrack® IGH somatic hypermutation assay, run on Illumina MiSeq) and capillary electrophoresis/Sanger sequencing to assess clonality and somatic hypermutations (SHM). RESULTS: In comparison to the routine capillary-based analysis, the NGS clonality assay had an overall diagnostic accuracy of 96% (63/66 cases). Other studied criteria included sensitivity (95%), specificity (100%), positive predictive value (100%) and negative predictive value (75%). In discrepant cases, the NGS results were confirmed by a different set of primers that provided coverage of the IGH leader sequence. Furthermore, there was excellent agreement of the SHM determination with both the LymphoTrack® FR1 and leader assays when compared to the Sanger sequencing analysis (84%), with NGS able to assess the SHM rate even in cases where the conventional approach failed. CONCLUSION: Overall, conventional Sanger sequencing and next-generation-sequencing-based clonality and somatic hypermutation analyses gave comparable results. For future use in a routine diagnostic workflow, NGS-based approaches should be evaluated prospectively and an analysis of cost-effectiveness should be performed.

Effects of Ocimum basilicum L. Extract on Hippocampal Oxidative Stress, Inflammation, and BDNF Expression in Amnesic Aged Rats.

The present study was conducted to investigate the effects of Ocimum basilicum L. (OB) extract on learning and memory impairment in aged rats. Male rats were divided into the following experimental groups: Group 1 (control): including 2 months old rats, Group 2 (aged) including 2 years old rats, Groups 3-5 (aged-OB): including 2 years old rats received 50, 100, and 150 mg/kg OB for 8 weeks by oral gavage. Aging increased the delay to find the platform but, however, decreased the time spent in the target quadrant when tested by Morris water maze (MWM). Aging also reduced the latency to enter the dark chamber in the passive avoidance (PA) test compared to the control group. Moreover, interleukin-6 (IL-6) and malondialdehyde (MDA) levels were raised in the hippocampus and cortex of aged rats. In contrast, thiol levels and enzymatic activity of superoxide dismutase (SOD) and catalase (CAT) significantly reduced. In addition, aging significantly reduced BDNF expression. Finally, OB administration reversed the mentioned effects. The current research showed that OB administration improves learning/memory impairment induced by aging. It also found that this plant extract protects the brain tissues from oxidative damage and neuroinflammation.

Use of Next Generation Sequencing to Define the Origin of Primary Myelofibrosis.

Primary myelofibrosis (PMF) is a chronic myeloproliferative neoplasm (MPN) characterized by progressive bone marrow sclerosis, extra-medullary hematopoiesis, and possible transformation to acute leukemia. In the last decade, the molecular pathogenesis of the disease has been largely uncovered. Particularly, genetic and genomic studies have provided evidence of deregulated oncogenes in PMF as well as in other MPNs. However, the mechanisms through which transformation to either the myeloid or lymphoid blastic phase remain obscure. Particularly, it is still debated whether the disease has origins in a multi-potent hematopoietic stem cells or instead in a commissioned myeloid progenitor. In this study, we aimed to shed light upon this issue by using next generation sequencing (NGS) to study both myeloid and lymphoid cells as well as matched non-neoplastic DNA of PMF patients. Whole exome sequencing revealed that most somatic mutations were the same between myeloid and lymphoid cells, such findings being confirmed by Sanger sequencing. Particularly, we found 126/146 SNVs to be the e same (including JAK2V617F), indicating that most genetic events likely to contribute to disease pathogenesis occurred in a non-commissioned precursor. In contrast, only 9/27 InDels were similar, suggesting that this type of lesion contributed instead to disease progression, occurring at more differentiated stages, or maybe just represented "passenger" lesions, not contributing at all to disease pathogenesis. In conclusion, we showed for the first time that genetic lesions characteristic of PMF occur at an early stage of hematopoietic stem cell differentiation, this being in line with the possible transformation of the disease in either myeloid or lymphoid acute leukemia.

Molecular Profiling of Kenyan Acute Myeloid Leukemia Patients.

Acute myeloid leukemia (AML) is an infrequent disease, and it is associated with high morbidity and mortality. It harbors a unique configuration of cytogenetic abnormalities and molecular mutations that can be detected using microscopic and molecular methods respectively. These genetic tests are core elements of diagnosis and prognostication in high-income countries. They are routinely incorporated in clinical decision making, allowing for the individualization of therapy. However, these tests are largely inaccessible to most patients in Kenya and therefore no data has been reported on this group of patients. The main purpose of this study is to describe the cytogenetic and molecular abnormalities of acute myeloid leukemia patients seen at the hemato-oncology unit of Kenyatta National Hospital. A cross-sectional descriptive study was carried out over a 3-month period on ten patients with a diagnosis of AML. Social demographics and clinical data were collected through a study proforma. A peripheral blood sample was collected for conventional metaphase G-banding technique and next generation sequencing. Particularly, targeted DNA sequencing (Illumina myeloid panel) and whole exome sequencing (WES) were performed. Cytogenetic analysis failed in 10/10 cases. Targeted sequencing was successfully obtained in 8 cases, whereas WES in 7. Cytogenetic studies yielded no results. There were 20 mutations detected across 10 commonly mutated genes. All patients had at least one clinically relevant mutation. Based on ELN criteria, NGS identified three patients with high-risk mutations, affecting TP53 (n = 2) and RUNX1 (n = 1). One patient was classified as favorable (PML-RARA) while 4 were standard risk. However, WT1 mutations associated with unfavorable prognosis were recorded in additional 2 cases. WES showed concordant results with targeted sequencing while unveiling more mutations that warrant further attention. In conclusion, we provide the first molecular profiling study of AML patients in Kenya including application of advanced next generation sequencing technologies, highlighting current limitations of AML diagnostics and treatment while confirming the relevance of NGS in AML characterization.

Association between interleukin-1 receptor antagonist (IL-1ra) VNTR, gene polymorphism and breast cancer susceptibility in Iranian population: Experimental and web-based analysis.

Breast cancer is one of the leading causes of cancer mortality. Growing evidence indicates that interleukins and its polymorphisms are involved in the pathogenesis of breast cancer. Variable number of tandem repeat (VNTR) polymorphism can affect transcription rate, mRNA stability and also the resulting protein expression and activity. Hence, present study aimed to assess the possible association between interleukin-1 receptor antagonist (IL-1Ra) VNTR polymorphism, and breast cancer susceptibility in Iranian population. A total of 300 Iranian individuals, 150 breast cancer patients and 150 age-matched healthy women, were included in this study. DNA extracted by salting out method and genotyping was done using the polymerase chain reaction. The frequency of the allele 2(5% vs. 22%) and the 2/2 genotype (22% vs. 46%) of IL-1Ra VNTR polymorphism was significantly higher in healthy control compared to breast cancer patient: therefore, A2 allele may play a protective role against breast cancer and its progression (p = .0001 and OR = 0.105, 95% CI: [0.044-0.248]). The allele 2 and 2/2 genotype of the IL-Ra VNTR polymorphism can be a protective factor against breast cancer susceptibility.

COVID-19 Vaccine: Between Myth and Truth.

Since December 2019, a pandemic caused by the newly identified SARS-CoV-2 spread across the entire globe, causing 364,191,494 confirmed cases of COVID-19 to date. SARS-CoV-2 is a betacoronavirus, a positive-sense, single-stranded RNA virus with four structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N). The S protein plays a crucial role both in cell binding and in the induction of a strong immune response during COVID-19 infection. The clinical impact of SARS-CoV-2 and its spread led to the urgent need for vaccine development to prevent viral transmission and to reduce the morbidity and mortality associated with the disease. Multiple platforms have been involved in the rapid development of vaccine candidates, with the S protein representing a major target because it can stimulate the immune system, yielding neutralizing antibodies (NAbs), blocking viral entry into host cells, and evoking T-cell immune responses. To date, 178 SARS-CoV-2 vaccine candidates have been challenged in clinical trials, of which 33 were approved by various national regulatory agencies. In this review, we discuss the FDA- and/or EMA-authorized vaccines that are mostly based on mRNA or viral vector platforms. Furthermore, we debunk false myths about the COVID-19 vaccine as well as discuss the impact of viral variants and the possible future developments.

Metabolic Switch and Cytotoxic Effect of Metformin on Burkitt Lymphoma.

Altered cellular energetic metabolism has recently emerged as important feature of neoplastic cells. Indeed, interfering with cancer cell metabolism might represent a suitable therapeutic strategy. In this study, we aimed to assess glucose metabolism activation in human lymphomas and evaluate how metformin can exert its action on lymphoma cells. We studied a large series of human lymphomas (N = 252) and an in vitro model of Burkitt lymphoma (BL) cells. We combined molecular biology techniques, including global gene expression profiling (GEP) analysis, quantitative PCR (qPCR) and Western blotting, and biochemical assays, aimed to assess pentose phosphate pathway, tricarboxylic acid (TCA) cycle, and aerobic glycolysis rates. We found that glucose metabolism is overall enhanced in most lymphoma subtypes, based on gene expression profiling (GEP), with general shift to aerobic glycolysis. By contrast, normal B cells only showed an overall increase in glucose usage during germinal center transition. Interestingly, not only highly proliferating aggressive lymphomas but also indolent ones, like marginal zone lymphomas, showed the phenomenon. Consistently, genes involved in glycolysis were confirmed to be overexpressed in BL cells by qPCR. Biochemical assays showed that while aerobic glycolysis is increased, TCA cycle is reduced. Finally, we showed that metformin can induce cell death in BL cells by stressing cellular metabolism through the induction of GLUT1, PKM2, and LDHA. In conclusion, we unveiled glucose metabolism abnormalities in human lymphomas and characterized the mechanism of action of metformin in Burkitt lymphoma model.

Constitutive PSGL-1 Correlates with CD30 and TCR Pathways and Represents a Potential Target for Immunotherapy in Anaplastic Large T-Cell Lymphoma.

Due to the high expression of P-selectin glycoprotein ligand-1 (PSGL-1) in lymphoproliferative disorders and in multiple myeloma, it has been considered as a potential target for humoral immunotherapy, as well as an immune checkpoint inhibitor in T-cells. By investigating the expression of SELPLG in 678 T- and B-cell samples by gene expression profiling (GEP), further supported by tissue microarray and immunohistochemical analysis, we identified anaplastic large T-cell lymphoma (ALCL) as constitutively expressing SELPLG at high levels. Moreover, GEP analysis in CD30+ ALCLs highlighted a positive correlation of SELPLG with TNFRSF8 (CD30-coding gene) and T-cell receptor (TCR)-signaling genes (LCK, LAT, SYK and JUN), suggesting that the common dysregulation of TCR expression in ALCLs may be bypassed by the involvement of PSGL-1 in T-cell activation and survival. Finally, we evaluated the effects elicited by in vitro treatment with two anti-PSGL-1 antibodies (KPL-1 and TB5) on the activation of the complement system and induction of apoptosis in human ALCL cell lines. In conclusion, our data demonstrated that PSGL-1 is specifically enriched in ALCLs, altering cell motility and viability due to its involvement in CD30 and TCR signaling, and it might be considered as a promising candidate for novel immunotherapeutic approaches in ALCLs.

Co-administration of 2'3'-cGAMP STING activator and CpG-C adjuvants with a mutated form of HPV 16 E7 protein leads to tumor growth inhibition in the mouse model.

Persistent infection with high-risk genotypes of human papillomavirus (HPV) is the leading cause of cervical cancer. The HPV oncoprotein E7 is constitutively expressed in cervical cancer and considered as an essential target for tumor-specific immunity. The goal of this study was to develop a candidate therapeutic vaccine based on the mutated E7 protein that had possibly reduced transformation capacity while was able to elicit a robust immune response. Therefore, the mutant type of HPV 16 E7 (E7GRG) protein was recombinantly expressed in E. coli. The protein was then purified and formulated with 2'-3'cGAMP CDN and/or CpG-C ODN adjuvants and subcutaneously injected to female C57BL/6 mice. To evaluate the immunogenic response, lymphocyte proliferation, secretion levels of IFN-γ and IL-4 cytokines, granzyme B level, and total IgG and subclasses of IgG antibody were measured. The anti-tumor activity was evaluated in tumor-harboring C57BL/6 mice. The highest rate of cell proliferation, IFN-γ and granzyme B levels, and amount of IgG antibody were found in mice group that were injected by E7GRG + 2'-3'cGAMP + CpG-C. Therapeutic immunization with E7GRG + 2'-3'cGAMP + CpG-C also significantly suppressed TC-1 tumor growth in mice. In conclusion, the results demonstrated that E7GRG + 2'-3'cGAMP + CpG-C induced strong cell-mediated and humoral immune responses that resulted in inhibition of tumor in mouse model.

Gene expression profile predicts response to the combination of tosedostat and low-dose cytarabine in elderly AML.

Tosedostat is an orally administered metalloenzyme inhibitor with antiproliferative and antiangiogenic activity against hematological and solid human cancers. Clinical activity has been demonstrated in relapsed acute myeloid leukemia (AML). Thirty-three elderly patients with AML (median age, 75 years) received 120 mg tosedostat orally once daily combined with subcutaneous low-dose cytarabine (20 mg twice per day for 10 days, up to 8 cycles), until disease progression. Induction mortality was 12%. According to an intention-to-treat analysis, the complete remission (CR) rate was 48.5%, and thus the primary end point of the study was reached (expected CR, 25%). The partial remission rate was 6.1%, with an overall response rate of 54.5%. Furthermore, 4 of 33 patients had stable disease (median: 286 days). The median progression-free survival and overall survival (OS) were 203 days and 222 days, respectively. Responding patients had a longer median OS than nonresponding patients (P = .001). A microarray analysis performed in 29 of 33 patients identified 188 genes associated with clinical response (CR vs no CR). Three of them (CD93, GORASP1, CXCL16) were validated by quantitative polymerase chain reaction, which correctly classified 83% of the patients. Specifically, CR achievement was efficiently predicted by the gene expression patterns, with an overall accuracy exceeding 90%. Finally, a negative predictive value of 100% was validated in an independent series, thus representing the first molecular predictor for clinical response to a specific combination drug treatment for AML. This trial has been registered at the European Medicines Agency and on the European Clinical Trials Database (https://www.clinicaltrialsregister.eu) as #2012-000334-19.

Cross-Immunization Against Respiratory Coronaviruses May Protect Children From SARS-CoV2: More Than a Simple Hypothesis?

In January 2020, a new coronavirus was identified as responsible for a pandemic acute respiratory syndrome. The virus demonstrated a high infectious capability and not-neglectable mortality in humans. However, similarly to previous SARS and MERS, the new disease COVID-19 caused by SARS-CoV-2 seemed to relatively spare children and younger adults. Some hypotheses have been proposed to explain the phenomenon, including lower ACE2 expression in children, cross-immunization from measles/rubella/mumps and BCG-vaccination, as well as the integrity of respiratory mucosa. Herein, we hypothesize that an additional mechanism might contribute to children's relative protection from SARS-CoV-2, the cross-immunization conferred by previous exposures to other common respiratory coronaviruses. To support our hypothesis, we show a statistically significant similarity in genomic and protein sequences, including epitopes for B- and T-cell immunity, of SARS-CoV-2 and the other beta coronaviruses. Since these coronaviruses are highly diffused across pediatric populations, cross-reactive immunity might reasonably induce an at least partial protection from SARS-CoV-2 in children.

Interferon gamma inducible protein 16 (IFI16) expression is reduced in mantle cell lymphoma.

IFI16, member of the IFN-inducible PYHIN-200 gene family, modulates proliferation, survival and differentiation of different cell lineages. In particular, IFI16 expression, which is regulated during the differentiation of B cells, was recently studied in B-CLL as well. Here, we compared IFI16 expression in several lymphomas including Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, marginal zone lymphoma and mantle cell lymphoma with respect to normal cell counterparts. We observed that IFI16 expression was significantly deregulated only in mantle cell lymphoma (p < 0.05). Notably, IFI16 was associated with the expression of genes involved in interferon response, cell cycle, cell death and proliferation and, interestingly, lipid and glucose metabolism, suggesting that IFI16 deregulation might be associated with relevant changes in cell biology. In our group of mantle cell lymphoma samples a correlation between patient survival and IFI16 expression was not detected even though mantle cell lymphoma prognosis is known to be associated with cell proliferation. Altogether, these results suggest a complex relationship between IFI16 expression and MCL which needs to be analyzed in further studies.

Molecular switch from MYC to MYCN expression in MYC protein negative Burkitt lymphoma cases.

MYC is the most altered oncogene in human cancer, and belongs to a large family of genes, including MYCN and MYCL. Recently, while assessing the degree of correlation between MYC gene rearrangement and MYC protein expression in aggressive B-cell lymphomas, we observed few Burkitt lymphoma (BL) cases lacking MYC protein expression despite the translocation involving the MYC gene. Therefore, in the present study we aimed to better characterize such cases. Our results identified two sub-groups of MYC protein negative BL: one lacking detectable MYC protein expression but presenting MYCN mRNA and protein expression; the second characterized by the lack of both MYC and MYCN proteins but showing MYC mRNA. Interestingly, the two sub-groups presented a different pattern of SNVs affecting MYC gene family members that may induce the switch from MYC to MYCN. Particulary, MYCN-expressing cases show MYCN SNVs at interaction interface that stabilize the protein associated with loss-of-function of MYC. This finding highlights MYCN as a reliable diagnostic marker in such cases. Nevertheless, due to the overlapping clinic, morphology and immunohistochemistry (apart for MYC versus MYCN protein expression) of both sub-groups, the described cases represent bona fide BL according to the current criteria of the World Health Organization.

Transcriptional Analysis of Lennert Lymphoma Reveals a Unique Profile and Identifies Novel Therapeutic Targets.

Lennert lymphoma (LL) is a lymphoepithelioid morphological variant of peripheral T-cell lymphoma-not otherwise specified (PTCL/NOS), clinically characterized by better prognosis if compared with other PTCL/NOS. Although well characterized as far as morphology and phenotype are concerned, very little is known regarding its molecular features. In this study, we investigated the transcriptional profile of this tumor aiming 1) to identify its cellular counterparts; 2) to better define its relation with other PTCLs-and, therefore, its possible position in lymphoma classification; and 3) to define pathogenetic mechanisms, possibly unveiling novel therapeutic targets. To address these issues, we performed gene and microRNA expression profiling on LL and other PTCL/NOS cases; we identified different genes and microRNAs that discriminated LL from other PTCL/NOS. Particularly, LL revealed a molecular signature significantly enriched in helper function and clearly distinguishable from other PTCL/NOS. Furthermore, PI3K/Akt/mTOR pathway emerged as novel potential therapeutic target. In conclusion, based on the already known particular morphological and clinical features, the new molecular findings support the hypothesis that LL might be classified as a separate entity. Preclinical and clinical studies testing the efficacy of PI3K/MTOR inhibitors in this setting are warranted.

Transcriptional analysis distinguishes breast implant-associated anaplastic large cell lymphoma from other peripheral T-cell lymphomas.

Breast implant-associated anaplastic large cell lymphoma is a new provisional entity in the revised World Health Organization classification of lymphoid malignancies, the pathogenesis and cell of origin of which are still unknown. We performed gene expression profiling of microdissected breast implant-associated anaplastic large cell lymphoma samples and compared their transcriptional profiles with those previously obtained from normal T-cells and other peripheral T-cell lymphomas and validated expression of selected markers by immunohistochemistry. Our results indicate that most breast implant-associated anaplastic large cell lymphomas exhibit an activated CD4+ memory T-cell phenotype, which is associated with CD25 and FoxP3 expression. Gene ontology analyses revealed upregulation of genes involved in cell motility programs (e.g., CCR6, MET, HGF, CXCL14) in breast implant-associated anaplastic large cell lymphomas compared to normal CD4+ T-cells and upregulation of genes involved in myeloid cell differentiation (e.g., PPARg, JAK2, SPI-1, GAB2) and viral gene transcription (e.g., RPS10, RPL17, RPS29, RPL18A) compared to other types of peripheral T-cell lymphomas. Gene set enrichment analyses also revealed shared features between the molecular profiles of breast implant-associated anaplastic large cell lymphomas and other types of anaplastic large cell lymphomas, including downregulation of T-cell receptor signaling and STAT3 activation. Our findings provide novel insights into the biology of this rare disease and further evidence that breast implant-associated anaplastic large cell lymphoma represents a distinct peripheral T-cell lymphoma entity.

Increased angiogenesis seems to correlate with inferior overall survival in myeloid sarcoma patients.

Myeloid sarcomas (MS) are tumors composed by myeloid elements and developing outside bone marrow. The prognosis is overall poor, only stem cell transplantation being consistently reposted as a potentially curative approach. In this study we explored whether microvessel density, a biomarker of angiogenesis, might be relevant in MS. We studied 60 MS, 24 acute myeloid leukemia, 5 normal bone marrow samples and 2 cases of extramedullary hemopoiesis in patients without evidence of hematological malignancy. We used immunohistochemistry (anti-CD34) to identify and quantify micro-vessel density (MVD) and micro-vessel grading (MVG). We found that MS had significantly higher MVD and MVG than normal bone marrow (p = 0.0002 and p < 0.001, respectively). We then found that cases with monocytic morphology had significantly higher MVD than myelo-monocytic and blastic ones (p = 0.005), while no differences were recorded based on extramedullary site. Finally, we found that higher MVD and higher MVG were associated with inferior outcome in terms of overall survival in multivariate analysis (p = 0.05 and p = 0.02, respectively), when censoring for stem cell transplantation was undertaken. In conclusion, we documented for the first time that increased angiogenesis is characteristic of MS and correlates with survival, suggesting that anti-angiogenic approaches might deserve a clinical evaluation in this setting.

Pathobiologic Roles of Epstein-Barr Virus-Encoded MicroRNAs in Human Lymphomas.

Epstein-Barr virus (EBV) is a human γ-herpesvirus implicated in several human malignancies, including a wide range of lymphomas. Several molecules encoded by EBV in its latent state are believed to be related to EBV-induced lymphomagenesis, among which microRNAs-small RNAs with a posttranscriptional regulating role-are of great importance. The genome of EBV encodes 44 mature microRNAs belonging to two different classes, including BamHI-A rightward transcript (BART) and Bam HI fragment H rightward open reading frame 1 (BHRF1), with different expression levels in different EBV latency types. These microRNAs might contribute to the pathogenetic effects exerted by EBV through targeting self mRNAs and host mRNAs and interfering with several important cellular mechanisms such as immunosurveillance, cell proliferation, and apoptosis. In addition, EBV microRNAs can regulate the surrounding microenvironment of the infected cells through exosomal transportation. Moreover, these small molecules could be potentially used as molecular markers. In this review, we try to present an updated and extensive view of the role of EBV-encoded miRNAs in human lymphomas.

Unveiling Another Missing Piece in EBV-Driven Lymphomagenesis: EBV-Encoded MicroRNAs Expression in EBER-Negative Burkitt Lymphoma Cases.

Epstein-Barr virus (EBV) is a gammaherpesvirus linked to a number of lymphoid and epithelial malignancies, including Burkitt lymphoma (BL) in which its frequency ranges from 30% in sporadic cases to 100% in the endemic ones. The possible contribution of EBV to BL pathogenesis is largely unknown. It has been suggested that EBV may be associated with all of the cases, including those diagnosed as EBV negative by a mechanism of hit-and-run. Early during oncogenesis, viral genes are essential for initiating disease. Progressively, viral genome is lost to escape the immune system and host mutations accumulate in proto-oncogenic cell. The main problem with the hit-and-run hypothesis is the lack of evidence in primary tumors. The routine methods applied to detect the virus [i.e., immunohistochemistry and EBV-encoded RNAs (EBER) in situ hybridization (ISH)] have a low specificity and accuracy. The aim of this study was to identify the most suitable method to detect EBV infection in pathology samples by applying conventional and non-conventional methods (i.e., EBV-microRNAs detection and EBV viral load measurement). We investigated a total of 10 cases and we found that all the samples (n = 6) diagnosed as EBV negative by immunohistochemistry and EBER-ISH demonstrated the presence of EBV-microRNAs and EBV genome. This points at the possibility that EBV might have contributed to lymphomagenesis in all our patients, and propose microRNAs detection as the most specific and sensitive tool to recognize EBV vestiges. It is worth noting that our data would have considerable implications for EBV-related diseases control. By using anti-EBV vaccines, one could potentially prevent also some cancers less suspected of a viral origin because of viral genome loss.

MicroRNAs sequencing unveils distinct molecular subgroups of plasmablastic lymphoma.

Plasmablastic lymphoma (PBL) is an aggressive lymphoma, often arising in the context of immunodeficiency and associated with Epstein-Barr virus (EBV) infection. The most frequently detected genetic alteration is the deregulation of MYC gene through the translocation - t(8;14)(q24;q32). The diagnosis of PBL is often challenging because it has an overlap in morphology, immunophenotype, cytogenetics and virus association with other lymphomas and plasma cell neoplasms; further, its molecular basis remains elusive. In the present study we aimed to better define the possible contribution of EBV infection as well as miRNA deregulation in PBL pathogenesis. We studied 23 cases of PBL, 19 Burkitt lymphomas (BL), and 17 extra-medullary plasmacytoma (EMPC). We used qPCR and immunohistochemistry to assess EBV latency patterns, while micro-RNA (miRNA) profiling was performed by next generation sequencing (Illumina) and validated by qPCR. Our analysis revealed a non-canonical EBV latency program with the partial expression of some proteins characterizing latency II and the activation of an abortive lytic cycle. Moreover, we identified miRNA signatures discriminating PBL from BL and EMPC. Interestingly, based on the miRNA profile, PBL appeared constituted by two discrete subgroups more similar to either BL or EMPC, respectively. This pattern was confirmed in an independent set of cases studied by qPCR and corresponded to different clinico-pathological features in the two groups, including HIV infection, MYC rearrangement and disease localization. In conclusion, we uncovered for the first time 1) an atypical EBV latency program in PBL; 2) a miRNA signature distinguishing PBL from the closest malignant counterparts; 3) the molecular basis of PBL heterogeneity.

Virus-encoded microRNA contributes to the molecular profile of EBV-positive Burkitt lymphomas.

Burkitt lymphoma (BL) is an aggressive neoplasm characterized by consistent morphology and phenotype, typical clinical behavior and distinctive molecular profile. The latter is mostly driven by the MYC over-expression associated with the characteristic translocation (8;14) (q24; q32) or with variant lesions. Additional genetic events can contribute to Burkitt Lymphoma pathobiology and retain clinical significance. A pathogenetic role for Epstein-Barr virus infection in Burkitt lymphomagenesis has been suggested; however, the exact function of the virus is largely unknown.In this study, we investigated the molecular profiles (genes and microRNAs) of Epstein-Barr virus-positive and -negative BL, to identify specific patterns relying on the differential expression and role of Epstein-Barr virus-encoded microRNAs.First, we found significant differences in the expression of viral microRNAs and in selected target genes. Among others, we identified LIN28B, CGNL1, GCET2, MRAS, PLCD4, SEL1L, SXX1, and the tyrosine kinases encoding STK10/STK33, all provided with potential pathogenetic significance. GCET2, also validated by immunohistochemistry, appeared to be a useful marker for distinguishing EBV-positive and EBV-negative cases. Further, we provided solid evidences that the EBV-encoded microRNAs (e.g. BART6) significantly mold the transcriptional landscape of Burkitt Lymphoma clones.In conclusion, our data indicated significant differences in the transcriptional profiles of EBV-positive and EBV-negative BL and highlight the role of virus encoded miRNA.