The Epstein–Barr virus (EBV) is linked to various B-cell lymphomas, including Burkitt lymphoma (BL), classical Hodgkin lymphoma (cHL) and diffuse large B-cell lymphoma (DLBCL) at frequencies ranging, by routine techniques, from 5 to 10% of cases in DLBCL to >95% in endemic BL. Using higher-sensitivity methods, we recently detected EBV traces in a few EBV-negative BL cases, possibly suggesting a “hit-and-run” mechanism. Here, we used routine and higher-sensitivity methods (qPCR and ddPCR for conserved EBV genomic regions and miRNAs on microdissected tumor cells; EBNA1 mRNA In situ detection by RNAscope) to assess EBV infection in a larger lymphoma cohort [19 BL, 34 DLBCL, 44 cHL, 50 follicular lymphomas (FL), 10 T-lymphoblastic lymphomas (T-LL), 20 hairy cell leukemias (HCL), 10 mantle cell lymphomas (MCL)], as well as in several lymphoma cell lines (9 cHL and 6 BL). qPCR, ddPCR, and RNAscope consistently documented the presence of multiple EBV nucleic acids in rare tumor cells of several cases EBV-negative by conventional methods that all belonged to lymphoma entities clearly related to EBV (BL, 6/9 cases; cHL, 16/32 cases; DLBCL, 11/30 cases), in contrast to fewer cases (3/47 cases) of FL (where the role of EBV is more elusive) and no cases (0/40) of control lymphomas unrelated to EBV (HCL, T-LL, MCL). Similarly, we revealed traces of EBV infection in 4/5 BL and 6/7 HL cell lines otherwise conventionally classified as EBV negative. Interestingly, additional EBV-positive cases (1 DLBCL, 2 cHL) relapsed as EBV-negative by routine methods while showing EBNA1 expression in rare tumor cells by RNAscope. The relapse specimens were clonally identical to their onset biopsies, indicating that the lymphoma clone can largely loose the EBV genome over time but traces of EBV infection are still detectable by high-sensitivity methods. We suggest EBV may contribute to lymphoma pathogenesis more widely than currently acknowledged.

Background: The Tumor Microenviroment (TME) is a complex milieu that is increasingly recognized as a key factor in multiple stages of disease progression and responses to therapy as well as escape from immune surveillance. However, the precise contribution of specific immune effector and immune suppressor components of the TME in Burkitt lymphoma (BL) remains poorly understood. Methods: In this paper, we applied the computational algorithm CIBERSORT to Gene Expression Profiling (GEP) datasets of 40 BL samples to draw a map of immune and stromal components of TME. Furthermore, by multiple immunohistochemistry (IHC) and multispectral immunofluorescence (IF), we investigated the TME of additional series of 40 BL cases to evaluate the role of the Programmed Death-1 and Programmed Death Ligand-1 (PD-1/PD-L1) immune checkpoint axis. Results: Our results indicate that M2 polarized macrophages are the most prominent TME component in BL. In addition, we investigated the correlation between PD-L1 and latent membrane protein-2A (LMP2A) expression on tumour cells, highlighting a subgroup of BL cases characterized by a non-canonical latency program of EBV with an activated PD-L1 pathway. Conclusion: In conclusion, our study analysed the TME in BL and identified a tolerogenic immune signature highlighting new potential therapeutic targets.

We recently examined the reproducibility of MYC and BCL-2 immunohistochemical (IHC) scoring and the impact of high expression of MYC and BCL-2 (double expresser status, DE) on survival and progression in a large retrospective cohort of aggressive B-cell lymphoma patients treated with rituximab plus cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP) or R-CHOP-like regimens. 1 We found that IHC scoring for MYC and BCL-2 was highly reproducible when cut-off values of ≥70% for MYC and ≥50% for BCL-2 were used. This threshold also predicted the presence of gene rearrangements identifying MYC translocations in 88% of cases. Patients with dual MYC expression of ≥70% and BCL-2 expression of ≥50% showed a significantly inferior clinical course and, therefore, represent candidates for novel treatment modalities. 1 We have now validated these findings in an independent cohort of 461 patients enrolled in prospective clinical trials of the German High-Grade Non-Hodgkin Lymphoma Study Group (DSHNHL). 2,3 In these trials, patients underwent R-CHOP-14 if >60 years of age and R-CHOEP/R-MegaCHOEP if ≤60 years

Forty-three 8-week-old male Wistar rats were studied to evaluate temporal changes of transforming growth factor beta1, (TGF-beta1) mRNA levels in thyroid tissue during pharmacologically induced goiter. Four rats were treated with purified bovine thyrotropin (TSH; Ambinon, 2 mU/day sc) for 7 days before being sacrificed. Thirty-one were treated with propylthiouracil (PTU), added to their drinking water at a concentration of 0.2 g%, and subsequently were sacrificed as follows: five after 1 week (PTU-1): five after 2 weeks (PTU-2); five after 4 weeks (PTU-4); five after 8 weeks (PTU-8); five after 12 weeks (PTU-12). In six rats, after 12 weeks of treatment. PTU was withdrawn for 2 months and subsequently started again in three rats which were sacrificed after 2 weeks (PTU-R); the remaining three rats were sacrificed without any further treatment (PTU-R control). Eight rats (control rats) were never treated and served as controls. After sacrifice, blood was drawn for determination of total thyroxine and the thyroid was excised and subdivided into two lobes. Northern analysis for TGF-beta1 was performed in one lobe. while histological and immunohistochemical studies were performed in the other lobe. Gene expression of TGF-beta1 was induced in TSH- and PTU-treated rats. In TSH-treated rats TGF-beta1 gene expression was less detectable than in PTU-treated rats, where it became evident after 2 weeks and remained through weeks 4-8. Gene expression of TGF-beta1 wits also seen in PTU-R rats, but not in the control and in the PTU-R control. Immunohistochemical analysis showed a different presence and location for the TGF-beta1 protein, which appears to be dependent on the time of exposure to mitogenic stimulus. In conclusion, TGF-beta1 is produced in response to both a direct (TSH by itself) and indirect (TSH induced by PTU-induced hypothyroidism) cellular proliferative stimulus and is not linked to an adaptative phenomenon secondary to hypothyroidism. The immunohistochemical location of TGF-beta1 within the thyrocytes is influenced by mitogen exposure time. A TGF-beta1 immunohistochemical evaluation may be important to define exposure time and activity of goitrogenic stimuli.

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.

Aims Burkitt lymphoma (BL) is an aggressive B‐cell lymphoma that, in some instances, may show a granulomatous reaction associated with a favourable prognosis and occasional spontaneous regression. In the present study, we aimed to define the tumour microenvironment (TME) in four such cases, two of which regressed spontaneously. Methods and results All cases showed aggregates of tumour cells with the typical morphology, molecular cytogenetics and immunophenotype of BL surrounded by a florid epithelioid granulomatous reaction. All four cases were Epstein–Barr virus (EBV)‐positive with type I latency. Investigation of the TME showed similar features in all four cases. The analysis revealed a proinflammatory response triggered by Th1 lymphocytes and M1 polarised macrophages encircling the neoplastic cells with a peculiar topographic distribution. Conclusions Our data provide an in‐vivo picture of the role that specific immune cell subsets might play during the early phase of BL, which may be capable of maintaining the tumour in a self‐limited state or inducing its regression. These novel results may provide insights into new potential therapeutic avenues in EBV‐positive BL patients in the era of cellular immunotherapy.

BACKGROUND: The Tumor Microenviroment (TME) is a complex milieu that is increasingly recognized as a key factor in multiple stages of disease progression and responses to therapy as well as escape from immune surveillance. However, the precise contribution of specific immune effector and immune suppressor components of the TME in Burkitt lymphoma (BL) remains poorly understood.METHODS: In this paper, we applied the computational algorithm CIBERSORT to Gene Expression Profiling (GEP) datasets of 40 BL samples to draw a map of immune and stromal components of TME. Furthermore, by multiple immunohistochemistry (IHC) and multispectral immunofluorescence (IF), we investigated the TME of additional series of 40 BL cases to evaluate the role of the Programmed Death-1 and Programmed Death Ligand-1 (PD-1/PD-L1) immune checkpoint axis. RESULTS: Our results indicate that M2 polarized macrophages are the most prominent TME component in BL. In addition, we investigated the correlation between PD-L1 and latent membrane protein-2A (LMP2A) expression on tumour cells, highlighting a subgroup of BL cases characterized by a non-canonical latency program of EBV with an activated PD-L1 pathway. CONCLUSION: In conclusion, our study analysed the TME in BL and identified a tolerogenic immune signature highlighting new potential therapeutic targets.

No abstract