Presence of Epstein-Barr virus (EBV) antigens detected by sensitive methods has no influence on local immune environment in diffuse large B cell lymphoma.

EBV+ diffuse large B cell lymphoma (DLBCL) not otherwise specified (NOS) is a new entity confirmed by the World Health Organization (WHO) in 2017. In this new entity, the virus may contribute to a tolerogenic microenvironment. Traces of the virus have been described in DLBCL with more sensitive methods, in cases that were originally diagnosed as negative. The aim of this study was to analyze the expression of immune response genes in the tumor microenvironment to disclose the role of the virus and its traces in DLBCL. In 48 DLBCL cases, the expression of immune response genes and the presence of molecules that induce tolerance, such as TIM3, LAG3 and PDL1 by immunohistochemistry (IHC), were studied. To broaden the study of the microenvironment, tumor-associated macrophages (TMAs) were also explored. No significant differences were observed in the expression of immune response genes in the EBV+ DLBCL and those cases that were EBV- DLBCL but that exhibited viral traces, assessed by ViewRNA assay. Only the EBV+ DLBCL cases displayed a significantly higher increase in the expression of CD8 and cytotoxic T cells detected by gene expression analysis, and of PDL1 in tumor cells and in the expression of CD68 in the tumor microenvironment detected by IHC, not observed in those cases with viral traces. The increase in CD8 and cytotoxic T cells, PDL1 and CD68 markers only in EBV+ DLBCL may indicate that traces of viral infection might not have influence in immune response markers.

Different Mutational Profiles of Subcutaneous Panniculitis-like T-cell Lymphoma and Lupus Panniculitis: An Additional Case Series.

BACKGROUND: Subcutaneous panniculitis-like T-cell lymphoma (SPTCL) is a rare cytotoxic T-cell lymphoma with indolent behavior, mostly present in women and associated with immunological diseases whose pathogenic background is still poorly understood. SPTCL is associated with lupus erythematosus panniculitis (LEP) and histologically misdiagnosed. OBJECTIVES: The aim of our study was to identify mutations affecting the pathogenesis of both SPTCL and LEP. MATERIALS AND METHODS: We studied a total of 10 SPTCL and 10 LEP patients using targeted next-generation sequencing and pyrosequencing. Differences in gene expression between molecular subgroups were investigated using NanoString technology. Clinical data were collected, and correlations sought with the molecular data obtained. RESULTS: The mutational profile of SPTCL and LEP is different. We identified fewer pathogenic mutations than previously reported in SPTCL, noting a single HAVCR2-mutated SPTCL case. Interestingly, 40% of our SPTCL cases showed the pathogenic TP53 (p.Pro72Arg) (P72R) variant. Although cases showing HAVCR2 mutations or the TP53 (P72R) variant had more severe symptomatic disease, none developed hemophagocytic syndrome (HPS). Furthermore, TP53 (P72R)-positive cases were characterized by a lower metabolic signaling pathway and higher levels of CD28 expression and Treg signaling genes. In addition, 30% of our cases featured the same mutation (T735C) of the epigenetic modificatory gene DNMT3A. None of the LEP cases showed mutations in any of the studied genes. CONCLUSIONS: The mutational landscape of SPTCL is broader than previously anticipated. We describe, for the first time, the involvement of the TP53 (P72R) pathogenic variant in this subgroup of tumors, consider the possible role of different genetic backgrounds in the development of SPTCL, and conclude that LEP does not follow the same pathogenic pathway as SPTCL.

Advanced-stage mycosis fungoides: role of the signal transducer and activator of transcription 3, nuclear factor-κB and nuclear factor of activated T cells pathways.

BACKGROUND: The malignant mechanisms that control the development of cutaneous T-cell lymphoma (CTCL) are beginning to be identified. Recent evidence suggests that disturbances in specific intracellular signalling pathways, such as RAS-mitogen-activated protein kinase, T-cell receptor (TCR)-phospholipase C gamma 1 (PLCG1)-nuclear factor of activated T cells (NFAT) and Janus kinase (JAK)-signal transducer and activator of transcription (STAT), may play an essential role in the pathogenesis of CTCL. OBJECTIVES: To investigate the mechanisms controlling disease development and progression in mycosis fungoides (MF), the most common form of CTCL. METHODS: We collected 100 samples that were submitted for diagnosis of, or a second opinion regarding, MF between 2001 and 2018, 80% of which were in the early clinical stages of the disease. Formalin-fixed paraffin-embedded tissues were used for histological review and to measure the expression by immunohistochemistry of surrogate markers of activation of the TCR-PLCG1-NFAT, JAK-STAT and NF-κB pathways. Folliculotropism and large-cell transformation were also examined. RESULTS: NFAT and nuclear factor kappa B (NF-κB) markers showed a comparable activation status in early and advanced stages, while STAT3 activation was more frequent in advanced stages and was associated with large-cell transformation. Consistently with this observation, STAT3 activation occurred in parallel with MF progression in two initially MF-negative cases. A significant association of NFAT with NF-κB markers was also found, reflecting a common mechanism of activation in the two pathways. Genomic studies identified nine mutations in seven genes known to play a potential role in tumorigenesis in T-cell leukaemia/lymphoma, including PLCG1, JAK3 and STAT3, which underlies the activation of these key cell-survival pathways. A higher mutational allele frequency was detected in advanced stages. CONCLUSIONS: Our results show that STAT3 is activated in advanced cases and is associated with large-cell transformation, while the activation of NFAT and NF-κB is maintained throughout the disease. These findings could have important diagnostic and therapeutic implications. What's already known about this topic? Mycosis fungoides is characterized by a clonal expansion of T cells in the skin. The mechanisms controlling disease development and progression are not fully understood. What does this study add? An association of the nuclear factor of activated T cells and nuclear factor kappa B pathways was found, which could reflect a common mechanism of activation. These pathways were activated in early and advanced stages at the same level. Signal transducer and activator of transcription 3 activation was associated with large-cell transformation and was more frequent in advanced stages. A genomic analysis of cutaneous T-cell lymphoma-associated genes was performed. Nine mutations were detected. What is the translational message? These results could have important implications for the treatment of MF in the near future.

MicroRNA-based molecular classification of non-BRCA1/2 hereditary breast tumours.

BACKGROUND: Hereditary breast cancer comprises 5-10% of all breast cancers. Mutations in two high-risk susceptibility genes, BRCA1 and BRCA2, along with rare intermediate-risk genes and common low-penetrance alleles identified, altogether explain no more than 45% of the high-risk breast cancer families, although the majority of cases are unaccounted for and are designated as BRCAX tumours. Micro RNAs have called great attention for classification of different cancer types and have been implicated in a range of important biological processes and are deregulated in cancer pathogenesis. METHODS: Here we have performed an exploratory hypothesis-generating study of miRNA expression profiles in a large series of 66 primary hereditary breast tumours by microarray analysis. RESULTS: Unsupervised clustering analysis of miRNA molecular profiles revealed distinct subgroups of BRCAX tumours, 'normal-like' BRCAX-A, 'proliferative' BRCAX-B, 'BRCA1/2-like' BRCAX-C and 'undefined' BRCAX-D subgroup. These findings introduce a new insight in the biology of hereditary breast cancer, defining specific BRCAX subgroups, which could help in the search for novel susceptibility pathways in hereditary breast cancer. CONCLUSION: Our data demonstrate that BRCAX hereditary breast tumours can be sub-classified into four previously unknown homogenous groups characterised by specific miRNA expression signatures and histopathological features.

WT 1 expression in nevi and melanomas: a marker of melanocytic invasion into the dermis.

BACKGROUND: WT1, first recognized as a tumor suppressor gene involved in the development of Wilms' tumor, may have apparently contradictory findings and functions. As WT1 has been identified as a molecular target for cancer immunotherapy, immunodetection of WT1 in tumor cells has become an essential step in cancer studies. METHODS: We compare the expression of this protein among different types of melanocytic nevi and among stages in primary melanoma progression. Tissue microarrays containing normal tissues and 271 primary melanocytic lesion samples (163 primary melanomas and 108 nevi) were studied by immunohistochemistry using monoclonal antibody against WT1. RESULTS AND DISCUSSION: The present study shows these: 1. WT1 protein is predominantly expressed in the cytoplasm of the neoplastic cells. 2. A higher rate of WT1 staining in melanocytic nevi against melanomas has been observed. 3. WT1 expression is increased in advanced stages of melanoma progression: a significant (p < 0.05) increase of expression of WT1 was detected in vertical cases 46.5% vs. radial cases 16.0%, in high levels of Clark (IV, V) 57.4% vs. low levels (I, II, III) 33.0% and when comparing depth of invasion within thickness subgroups. 4. Finally, this study establishes an association of WT1 protein expression with shorter overall survival in melanoma.

The genomic profile of HER2-amplified breast cancers: the influence of ER status.

Expression profiling studies have suggested that HER2-amplified breast cancers constitute a heterogeneous group that may be subdivided according to their ER status: HER2-amplified ER-positive breast carcinomas that fall into the luminal B cluster; and HER2-amplified ER-negative cancers which form a distinct molecular subgroup, known as the erbB2 or HER2 subgroup. ER-negative breast cancer differs significantly from ER-positive disease in the pattern, type, and complexity of genetic aberrations. Here we have compared the genomic profiles of ER-positive and ER-negative HER2-amplified cancers using tiling path microarray-based comparative genomic hybridization (aCGH). Validation of the differentially amplified regions was performed in an independent series of 70 HER2-amplified breast cancers. Although HER2-amplified cancers had remarkably complex patterns of molecular genetic aberrations, ER-positive and ER-negative HER2-amplified breast carcinomas shared most molecular genetic features as defined by aCGH. Genome-wide Fisher's exact test analysis revealed that less than 1.5% of the genome was significantly differentially gained or lost in ER-positive versus ER-negative HER2-amplified cancers. However, two regions of amplification were significantly associated with ER-positive carcinomas, one of which mapped to 17q21.2 and encompassed GJC1, IGFBP4, TNS4, and TOP2A. Chromogenic in situ hybridization analysis of an independent validation series confirmed the association between ER status and TOP2A amplification. In conclusion, although hormone receptor status does not determine the overall genetic profile of HER2-amplified breast cancers, specific genetic aberrations may be characteristic of subgroups of HER2 breast cancers.

Contribution of JAK2 mutations to T-cell lymphoblastic lymphoma development.

The JAK-STAT pathway has a substantial role in lymphoid precursor cell proliferation, survival and differentiation. Nonetheless, the contribution of JAK2 to T-cell lymphoblastic lymphoma (T-LBL) development remains poorly understood. We have identified one activating TEL-JAK2 translocation and four missense mutations accumulated in 2 out of 16 T-LBL samples. Two of them are novel JAK2 mutations and the other two are reported for the first time in T-LBL. Notably, R683G and I682T might have arisen owing to RNA editing. Mutated samples showed different mutated transcripts suggesting sub-clonal heterogeneity. Functional approaches revealed that two JAK2 mutations (H574R and R683G) constitutively activate JAK-STAT signaling in γ2A cells and can drive the proliferation of BaF3-EpoR cytokine-dependent cell line. In addition, aberrant hypermethylation of SOCS3 might contribute to enhance the activation of JAK-STAT signaling. Of utmost interest is that primary T-LBL samples harboring JAK2 mutations exhibited increased expression of LMO2, suggesting a mechanistic link between JAK2 mutations and the expression of LMO2, which was confirmed for the four missense mutations in transfected γ2A cells. We therefore propose that active JAK2 contribute to T-LBL development by two different mechanisms, and that the use of pan-JAK inhibitors in combination with epigenetic drugs should be considered in future treatments.

Loss of TCR-beta F1 and/or EZRIN expression is associated with unfavorable prognosis in nodal peripheral T-cell lymphomas.

Nodal peripheral T-cell lymphoma (nodal PTCL) has an unfavorable prognosis, and specific pathogenic alterations have not been fully identified. The biological and clinical relevance of the expression of CD30/T-cell receptor (TCR) genes is a topic under active investigation. One-hundred and ninety-three consecutive nodal PTCLs (89 angioimmunoblastic T-cell lymphomas (AITL) and 104 PTCL-unspecified (PTCL-not otherwise specified (NOS)) cases) were analyzed for the immunohistochemical expression of 19 molecules, involving TCR/CD30 pathways and the associations with standard prognostic indices. Mutually exclusive expression was found between CD3 and TCR-beta F1 with CD30 expression. Taking all PTCL cases together, logistic regression identified a biological score (BS) including TCR molecules (TCR-beta F1 and EZRIN) that separates two subgroups of patients with a median survival of 34.57 and 5.20 months (P<0.001). Multivariate analysis identified BS and the prognostic index for PTCL (PIT) score as independent prognostic factors. This BS maintained its significance in multivariate analysis only for the PTCL-NOS subgroup of tumors. In AITL cases, only a high level of ki67 expression was related to prognosis. A BS including molecules involved in the TCR signaling pathway proved to be an independent prognostic factor of poor outcome in a multivariate analysis, specifically in PTCL-NOS patients. Nevertheless, validation in an independent series of homogeneously treated PTCL patients is required to confirm these data.