Maria Raffaella Ambrosio

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.

Diagnosis of Burkitt lymphoma using an algorithmic approach – applicable in both resource‐poor and resource‐rich countries

Distinguishing Burkitt lymphoma (BL) from B cell lymphoma, unclassifiable with features intermediate between diffuse large B‐cell lymphoma (DLBCL) and BL (DLBCL/BL), and DLBCL is challenging. We propose an immunohistochemistry and fluorescent in situ hybridization (FISH) based scoring system that is employed in three phases – Phase 1 (morphology with CD10 and BCL2 immunostains), Phase 2 (CD38, CD44 and Ki‐67 immunostains) and Phase 3 (FISH on paraffin sections for MYC, BCL2, BCL6 and immunoglobulin family genes). The system was evaluated on 252 aggressive B‐cell lymphomas from Europe and from sub‐Saharan Africa. Using the algorithm, we determined a specific diagnosis of BL or not‐BL in 82%, 92% and 95% cases at Phases 1, 2 and 3, respectively. In 3·4% cases, the algorithm was not completely applicable due to technical reasons. Overall, this approach led to a specific diagnosis of BL in 122 cases and to a specific diagnosis of either DLBCL or DLBCL/BL in 94% of cases that were not diagnosed as BL. We also evaluated the scoring system on 27 cases of BL confirmed on gene expression/microRNA expression profiling. Phase 1 of our scoring system led to a diagnosis of BL in 100% of these cases.

Notch3 is activated by chronic hypoxia and contributes to the progression of human prostate cancer

Prostate cancer (PC) is still the second cause of cancer‐related death among men. Although patients with metastatic presentation have an ominous outcome, the vast majority of PCs are diagnosed at an early stage. Nonetheless, even among patients with clinically localized disease the outcome may vary considerably. Other than androgen sensitivity, little is known about which other signaling pathways are deranged in aggressive, localized cancers. The elucidation of such pathways may help to develop innovative therapies aimed at specific molecular targets. We report that in a hormone‐sensitive PC cell line, LNCaP, Notch3 was activated by hypoxia and sustained cell proliferation and colony formation in soft agar. Hypoxia also modulated cellular cholesterol content and the number and size of lipid rafts, causing a coalescence of small rafts into bigger clusters; under this experimental condition, Notch3 migrated from the non‐raft into the raft compartment where it colocalized with the γ‐secretase complex. We also looked at human PC biopsies and found that expression of Notch3 positively correlated with Gleason score and with expression of carbonic anhydrase IX, a marker of hypoxia. In conclusion, hypoxia triggers the activation of Notch3, which, in turn, sustains proliferation of PC cells. Notch3 pathway represents a promising target for adjuvant therapy in patients with PC.

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.

The Epstein Barr-encoded BART-6-3p microRNA affects regulation of cell growth and immuno response in Burkitt lymphoma

BackgroundBurkitt lymphoma is an aggressive B-cell lymphoma presenting in three clinical forms: endemic, sporadic and immunodeficiency-associated. More than 90% of endemic Burkitt lymphoma carry latent Epstein-Barr virus, whereas only 20% of sporadic Burkitt lymphoma are associated with Epstein-Barr infection. Although the Epstein-Barr virus is highly related with the endemic form, how and whether the virus participates in its pathogenesis remains to be fully elucidated. In particular, the virus may impair cellular gene expression by its own encoded microRNAs.MethodsUsing microRNA profiling we compared Epstein-Barr-positive and Epstein-Barr-negative Burkitt lymphoma cases for both cellular and viral microRNAs. The array results were validated by qRT-PCR, and potential targets of viral microRNAs were then searched by bioinformatic predictions, and classified in functional categories, according to the Gene Ontology. Our findings were validated by in vitro functional studies and by immunohistochemistry on a larger series of cases.ResultsWe showed that a few cellular microRNAs are differentially expressed between Epstein-Barr-positive and Epstein-Barr-negative Burkitt lymphoma cases, and identified a subset of viral microRNAs expressed in Epstein-Barr-positive Burkitt lymphomas. Of these, we characterized the effects of viral BART6-3p on regulation of cellular genes. In particular, we analyzed the IL-6 receptor genes (IL-6Rα and IL-6ST), PTEN and WT1 expression for their possible relevance to Burkitt lymphoma. By means of immunohistochemistry, we observed a down-regulation of the IL-6 receptor and PTEN specifically in Epstein-Barr-positive Burkitt lymphoma cases, which may result in the impairment of key cellular pathways and may contribute to malignant transformation. On the contrary, no differences were observed between Epstein-Barr-positive and Epstein-Barr-negative Burkitt lymphoma cases for WT1 expression.ConclusionsOur preliminary results point at an active role for the Epstein-Barr virus in Burkitt lymphomagenesis and suggest new possible mechanisms used by the virus in determining dysregulation of the host cell physiology.

The alteration of lipid metabolism in Burkitt lymphoma identifies a novel marker: adipophilin.

Background Recent evidence suggests that lipid pathway is altered in many human tumours. In Burkitt lymphoma this is reflected by the presence of lipid droplets which are visible in the cytoplasm of neoplastic cells in cytological preparations. These vacuoles are not identifiable in biopsy section as lipids are “lost” during tissue processing. Methods and Results In this study we investigated the expression of genes involved in lipid metabolism, at both RNA and protein level in Burkitt lymphoma and in other B-cell aggressive lymphoma cases. Gene expression profile indicated a significant over-expression of the adipophilin gene and marked up-regulation of other genes involved in lipid metabolism in Burkitt lymphoma. These findings were confirmed by immunohistochemistry on a series od additional histological samples: 45 out of 47 BL cases showed strong adipophilin expression, while only 3 cases of the 33 of the not-Burkitt lymphoma category showed weak adipophilin expression (p<0.05). Conclusions Our preliminary results suggest that lipid metabolism is altered in BL, and this leads to the accumulation of lipid vacuoles. These vacuoles may be specifically recognized by a monoclonal antibody against adipophilin, which may therefore be a useful marker for Burkitt lymphoma because of its peculiar expression pattern. Moreover this peptide might represent an interesting candidate for interventional strategies.

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.

Clonality Analysis of Immunoglobulin Gene Rearrangement by Next-Generation Sequencing in Endemic Burkitt Lymphoma Suggests Antigen Drive Activation of BCR as Opposed to Sporadic Burkitt Lymphoma

Objectives: Recent studies using next-generation sequencing (NGS) analysis disclosed the importance of the intrinsic activation of the B-cell receptor (BCR) pathway in the pathogenesis of sporadic Burkitt lymphoma (sBL) due to mutations of TCF3/ID3 genes. Since no definitive data are available on the genetic landscape of endemic Burkitt (eBL), we first assessed the mutation frequency of TCF3/ID3 in eBL compared with sBL and subsequently the somatic hypermutation status of the BCR to answer whether an extrinsic activation of BCR signaling could also be demonstrated in Burkitt lymphoma. Methods: We assessed the mutations of TCF3/ID3 by RNAseq and the BCR status by NGS analysis of the immunoglobulin genes (IGs). Results: We detected mutations of TCF3/ID3 in about 30% of the eBL cases. This rate is significantly lower than that detected in sBL (64%). The NGS analysis of IGs revealed intraclonal diversity, suggesting an active targeted somatic hypermutation process in eBL compared with sBL. Conclusions: These findings support the view that the antigenic pressure plays a key role in the pathogenetic pathways of eBL, which may be partially distinct from those driving sBL development.

Plasmablastic transformation of a pre-existing plasmacytoma: a possible role for reactivation of Epstein Barr virus infection

Background: Juvenile myelomonocytic leukemia (JMML) is an aggressive clonal myeloid neoplasm of early childhood associated with mutations in Ras pathway genes (PTPN11, KRAS, NRAS, CBL and NF1). Elevated fetal hemoglobin (HbF) levels and monosomy 7 are frequently observed. Stem cell transplantation is the only available curative treatment option but only provides an event-free survival of about 50%. Aims: Gain insight in the molecular networks involved in JMML pathogenesis based on mRNA, microRNA and long non-coding RNA transcriptome analysis of JMML samples. Methods: Expression of 27958 mRNA probes and 23042 lncRNA probes was assessed in diagnostic bone marrow or peripheral blood mononuclear cells of 63 JMML patients and 5 healthy donors, using a custom designed Agilent array. In addition, cDNA of 768 microRNAs was pre-amplified and quantified using miRNA specific Taqman probes. Results: Unsupervised clustering of an initial cohort of 14 patients generated two subgroups with let-7e and RNA-binding protein LIN28B amongst the most significantly differentially expressed genes. In the final cohort, relative higher LIN28B expression was observed in 35 of 63 cases (55.6%) and was defined as the average of the healthy donors plus three standard deviations. Univariable Cox regression showed that logarithmic LIN28B expression as a dichotomous variable can predict overall survival (p=0.035, exp(B) = 4.227, CI(95%) = 1.108 – 16.125). Patients with higher LIN28B mRNA levels experience a significant worse overall survival (Kaplan-Meier plot, p=0.022). HbF and platelet count were also significant prognostic factors, as described previously (p=0.023 and 0.027 respectively). There was no association between LIN28B expression and Ras pathway mutation status. We observed the strongest miRNA anti-correlation between LIN28B and five let-7 family members (d, b, g, e and a), and the second highest positive mRNA correlation between LIN28B and HMGA2. Recently, it was shown that the LIN28B – let-7 – HMGA2 axis determines higher self-renewal of fetal hematopoietic stem cells (Copley, 2013). This indicates that LIN28B confers augmented self- renewal to leukemic hematopoietic stem cells in JMML and – since this is an early childhood disease – this is potentially already initiated during embryogenesis. JMML patients frequently show elevated HbF levels at diagnosis. A positive correlation was found between LIN28B expression and HbF levels (rs=0.64, p<0.001, N = 41). Interestingly, our gene expression profiling data showed that both probes corresponding to HBG1 (encoding the human gamma globin chain) and HBBP1 (encoding a lncRNA-affiliated hemoglobin beta pseudogene) were strongly correlated with LIN28B expression in our patient series. This emphasizes the central role for LIN28B in the fetal (leukemic) hematopoietic stem cell system. Strikingly, patients with monosomy 7 (n=7/56) never displayed increased LIN28B expression (Chi-square p = 0.0017), suggesting the presence of a LIN28B activating transcription factor on chromosome 7. We identified MNX1 (HLXB9) as a possible activator of LIN28B based on a very strong correlation and siRNA knockdown.