Maryam Etebari

Distinct Viral and Mutational Spectrum of Endemic Burkitt Lymphoma

Endemic Burkitt lymphoma (eBL) is primarily found in children in equatorial regions and represents the first historical example of a virus-associated human malignancy. Although Epstein-Barr virus (EBV) infection and MYC translocations are hallmarks of the disease, it is unclear whether other factors may contribute to its development. We performed RNA-Seq on 20 eBL cases from Uganda and showed that the mutational and viral landscape of eBL is more complex than previously reported. First, we found the presence of other herpesviridae family members in 8 cases (40%), in particular human herpesvirus 5 and human herpesvirus 8 and confirmed their presence by immunohistochemistry in the adjacent non-neoplastic tissue. Second, we identified a distinct latency program in EBV involving lytic genes in association with TCF3 activity. Third, by comparing the eBL mutational landscape with published data on sporadic Burkitt lymphoma (sBL), we detected lower frequencies of mutations in MYC, ID3, TCF3 and TP53, and a higher frequency of mutation in ARID1A in eBL samples. Recurrent mutations in two genes not previously associated with eBL were identified in 20% of tumors: RHOA and cyclin F (CCNF). We also observed that polyviral samples showed lower numbers of somatic mutations in common altered genes in comparison to sBL specimens, suggesting dual mechanisms of transformation, mutation versus virus driven in sBL and eBL respectively.

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.

Molecular signature of Epstein Barr virus-positive Burkitt lymphoma and post-transplant lymphoproliferative disorder suggest different roles for Epstein Barr virus.

Epstein Barr virus (EBV) infection is commonly associated with human cancer and, in particular, with lymphoid malignancies. Although the precise role of the virus in the pathogenesis of different lymphomas is largely unknown, it is well recognized that the expression of viral latent proteins and miRNA can contribute to its pathogenetic role. In this study, we compared the gene and miRNA expression profile of two EBV-associated aggressive B non-Hodgkin lymphomas known to be characterized by differential expression of the viral latent proteins aiming to dissect the possible different contribution of such proteins and EBV-encoded miRNAs. By applying extensive bioinformatic inferring and an experimental model, we found that EBV+ Burkitt lymphoma presented with significant over-expression of EBV-encoded miRNAs that were likely to contribute to its global molecular profile. On the other hand, EBV+ post-transplant diffuse large B-cell lymphomas presented a significant enrichment in genes regulated by the viral latent proteins. Based on these different viral and cellular gene expression patterns, a clear distinction between EBV+ Burkitt lymphoma and post-transplant diffuse large B-cell lymphomas was made. In this regard, the different viral and cellular expression patterns seemed to depend on each other, at least partially, and the latency type most probably played a significant role in their regulation. In conclusion, our data indicate that EBV influence over B-cell malignant clones may act through different mechanisms of transcriptional regulation and suggest that potentially different pathogenetic mechanisms may depend upon the conditions of the interaction between EBV and the host that finally determine the latency pattern.

The evolution of clonality testing in the diagnosis and monitoring of hematological malignancies

Currently, distinguishing between benign and malignant lymphoid proliferations is based on a combination of clinical characteristics, cyto/histomorphology, immunophenotype and the identification of well-defined chromosomal aberrations. However, such diagnoses remain challenging in 10–15% of cases of lymphoproliferative disorders, and clonality assessments are often required to confirm diagnostic suspicions. In recent years, the development of new techniques for clonality detection has allowed researchers to better characterize, classify and monitor hematological neoplasms. In the past, clonality was primarily studied by performing Southern blotting analyses to characterize rearrangements in segments of the IG and TCR genes. Currently, the most commonly used method in the clinical molecular diagnostic laboratory is polymerase chain reaction (PCR), which is an extremely sensitive technique for detecting nucleic acids. This technique is rapid, accurate, specific, and sensitive, and it can be used to analyze small biopsies as well as formalin-fixed paraffin-embedded samples. These advantages make PCR-based approaches the current gold standard for IG/TCR clonality testing. Since the completion of the first human genome sequence, there has been a rapid development of technologies to facilitate high-throughput sequencing of DNA. These techniques have been applied to the deep characterization and classification of various diseases, patient stratification, and the monitoring of minimal residual disease. Furthermore, these novel approaches have the potential to significantly improve the sensitivity and cost of clonality assays and post-treatment monitoring of B- and T-cell malignancies. However, more studies will be required to demonstrate the utility, sensitivity, and benefits of these methods in order to warrant their adoption into clinical practice. In this review, recent developments in clonality testing are examined with an emphasis on highly sensitive systems for improving diagnostic workups and minimal residual disease assessments.

Pathogenetic and diagnostic significance of microRNA deregulation in peripheral T-cell lymphoma not otherwise specified

Peripheral T-cell lymphomas not otherwise specified (PTCLs/NOS) are rare and aggressive tumours whose molecular pathogenesis and diagnosis are still challenging. The microRNA (miRNA) profile of 23 PTCLs/NOS was generated and compared with that of normal T-lymphocytes (CD4+, CD8+, naive, activated). The differentially expressed miRNA signature was compared with the gene expression profile (GEP) of the same neoplasms. The obtained gene patterns were tested in an independent cohort of PTCLs/NOS. The miRNA profile of PTCLs/NOS then was compared with that of 10 angioimmunoblastic T-cell lymphomas (AITLs), 6 anaplastic large-cell lymphomas (ALCLs)/ALK+ and 6 ALCLs/ALK−. Differentially expressed miRNAs were validated in an independent set of 20 PTCLs/NOS, 20 AITLs, 19 ALCLs/ALK− and 15 ALCLs/ALK+. Two hundred and thirty-six miRNAs were found to differentiate PTCLs/NOS from activated T-lymphocytes. To assess which miRNAs impacted on GEP, a multistep analysis was performed, which identified all miRNAs inversely correlated to different potential target genes. One of the most discriminant miRNAs was selected and its expression was found to affect the global GEP of the tumours. Moreover, two sets of miRNAs were identified distinguishing PTCL/NOS from AITL and ALCL/ALK−, respectively. The diagnostic accuracy of this tool was very high (83.54%) and its prognostic value validated.

The presence of Epstein-Barr virus significantly impacts the transcriptional profile in immunodeficiency-associated Burkitt lymphoma

Burkitt lymphoma (BL) is an aggressive neoplasm derived from mature, antigen-experienced B-lymphocytes. Three clinical/epidemiological variants have been recognized, named sporadic, endemic and immunodeficiency-associated BL (ID-BL). Although they are listed within a unique entity in the current WHO Classification, recent evidence indicated genetic and transcriptional differences among the three sub-groups. Further, the presence of latently persisting Epstein-Barr virus (EBV) has been associated with specific features in endemic and sporadic cases. In this study, we explored for the first time whether EBV infection could be related with a specific molecular profile in immunodeficiency-associated cases. We studied 30 BL cases, including nine occurring in HIV-positive patients (5 EBV-positive and 4 EBV-negative) by gene and microRNA (miRNA) expression profiling. We found that ID-BL presented with different profiles based on EBV presence. Specifically, 252 genes were differentially expressed, some of them being involved in intracellular signaling and apoptosis regulation. Furthermore, 28 miRNAs including both EBV-encoded (N = 18) and cellular (N = 10) ones were differentially regulated. Of note, genes previously demonstrated to be targeted by such miRNA were consistently found among differentially expressed genes, indicating the relevant contribution of miRNA to the molecular profile of the examined cases. Grippingly, 17 out of the 252 differentially expressed genes turned out to be potentially targeted by both cellular and EBV-encoded miRNA, suggesting a complex interaction and not excluding a potential synergism. In conclusion, we documented transcriptional differences based on the presence of EBV in ID-BL, and suggested a complex interaction between cellular and viral molecules in the determination of the global molecular profile of the tumor.

Burkitt lymphoma beyond MYC translocation: N-MYC and DNA methyltransferases dysregulation.

BackgroundThe oncogenic transcription factor MYC is pathologically activated in many human malignancies. A paradigm for MYC dysregulation is offered by Burkitt lymphoma, where chromosomal translocations leading to Immunoglobulin gene-MYC fusion are the crucial initiating oncogenic events. However, Burkitt lymphoma cases with no detectable MYC rearrangement but maintaining MYC expression have been identified and alternative mechanisms can be involved in MYC dysregulation in these cases.MethodsWe studied the microRNA profile of MYC translocation-positive and MYC translocation-negative Burkitt lymphoma cases in order to uncover possible differences at the molecular level. Data was validated at the mRNA and protein level by quantitative Real-Time polymerase chain reaction and immunohistochemistry, respectively.ResultsWe identified four microRNAs differentially expressed between the two groups. The impact of these microRNAs on the expression of selected genes was then investigated. Interestingly, in MYC translocation-negative cases we found over-expression of DNA-methyl transferase family members, consistent to hypo-expression of the hsa-miR-29 family. This finding suggests an alternative way for the activation of lymphomagenesis in these cases, based on global changes in methylation landscape, aberrant DNA hypermethylation, lack of epigenetic control on transcription of targeted genes, and increase of genomic instability. In addition, we observed an over-expression of another MYC family gene member, MYCN that may therefore represent a cooperating mechanism of MYC in driving the malignant transformation in those cases lacking an identifiable MYC translocation but expressing the gene at the mRNA and protein levels.ConclusionsCollectively, our results showed that MYC translocation-positive and MYC translocation-negative Burkitt lymphoma cases are slightly different in terms of microRNA and gene expression. MYC translocation-negative Burkitt lymphoma, similarly to other aggressive B-cell non Hodgkin’s lymphomas, may represent a model to understand the intricate molecular pathway responsible for MYC dysregulation in cancer.

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.

SNPs Array Karyotyping in Non-Hodgkin Lymphoma

The traditional methods for detection of chromosomal aberrations, which included cytogenetic or gene candidate solutions, suffered from low sensitivity or the need for previous knowledge of the target regions of the genome. With the advent of single nucleotide polymorphism (SNP) arrays, genome screening at global level in order to find chromosomal aberrations like copy number variants, DNA amplifications, deletions, and also loss of heterozygosity became feasible. In this review, we present an update of the knowledge, gained by SNPs arrays, of the genomic complexity of the most important subtypes of non-Hodgkin lymphomas.

Abstract 2362: Identification and characterization of pathogenetic pathways and potential therapeutic targets in tumors derived from histiocytes and follicular dendritic cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Tumors derived from histiocytes and follicular dendritic cells (FDC) are extremely rare, constituting less than 1% of hematopoietic tumors. Among others, follicular dendritic cell sarcoma (FDC-S), histiocytic sarcoma (HS) , and Langerhans histiocytosis (LH) are the commonest types. The clinical course is variable from indolent to aggressive; however, the disease is more often incurable. Further, probably due to their rarity and complexity, the pathobiology of these tumor is nearly unknown at present, with only scattered studies being reported. In this study, we investigated by gene expression profiling (GEP) and extensive immunohistochemistry (IHC) a relatively large series of cases, aiming 1) to identify and characterize functional pathways possibly involved in the pathogenesis of the disease, and 2) to unveil potential targets for novel treatments. We studied by GEP FDC-S (N=30), LH (N=9), and HS (N=4) as well as non neoplastic cells including tissue microdissected FDC (N=5), CD1c+ myeloid dendritic cells (N=4), peripheral blood monocytes (N=8), and human cultured fibroblasts (N=3). All cases were studied with the Illumina DASL whole genome microarray. The same cases as well as and independent cohort was then used for IHC validation studies. First, unsupervised approaches (principal component analysis and hierarchical clustering) indicated that tissue samples were distinct from cellular ones, while the different entities could not be clearly separated. Second, we found by supervised comparison (ANOVA, p<0.05, fold change ≥2, FDR according to Benjamini-Hockeberg), the 3 tumor types could be discriminated. Particularly, the tumors differed for genes related to immune response (consistent with their histogenesis) and to intracellular signaling. In this regard, we found several oncogenic pathways to be differentially activated while others were consistently deregulated including those related to cellular adhesion, chemotaxis, angiogenesis and response to growth factors. We then compared each tumor type with the specific supposed counterpart (i.e. FDC-s vs. FDC; LH vs. myeloid DC; HS vs. monocytes) and identified genes and cellular programs deregulated in each of them. Interestingly, all tumors presented with consistent deregulation of previously unknown potential therapeutic targets, including PDGF/PDGFRs, PTEN, JAK/STAT, mTOR/AKT, and EGFR. Importantly, IHC confirmed GEP data and confirmed the activation of these selected pathways. We defined for the first time the cellular programs deregulated in tumors derived from histiocytes and FDC and identified remarkable potential therapeutic targets. As appropriate experimental models (i.e. cell lines, animal models) are not available at present, international collaboration would be advisable to confirm these observation in large series before eventually moving to early phase clinical trials. Citation Format: Pier-Paolo Piccaluga, Maura Rossi, Giovanna Motta, Sylvia Hartmann, Claudia Doering, Fabio Fuligni, Claudio Agostinelli, Maria Rosaria Sapienza, Maria Antonella Laginestra, Federica Melle, Maryam Etebari, Mohsen Navari, Anna Gazzola, Claudia Mannu, Clara Bertuzzi, Claudio Tripodo, Martin L. Hansmann, Fabio Facchetti, Stefano A Pileri. Identification and characterization of pathogenetic pathways and potential therapeutic targets in tumors derived from histiocytes and follicular dendritic cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2362. doi:10.1158/1538-7445.AM2014-2362