LT and SOX9 expression are associated with gene sets that distinguish Merkel cell polyomavirus (MCPyV)-positive and MCPyV-negative Merkel cell carcinoma.

BACKGROUND: Merkel cell carcinoma (MCC) is an aggressive malignant neuroendocrine tumour. There are two subsets of MCC, one related to Merkel cell polyomavirus (MCPyV) and the other to ultraviolet radiation (UVR). MCPyV-positive and MCPyV-negative MCCs have been considered to be different tumours, as the former harbour few DNA mutations and are not related to UVR, and the latter usually arise in sun-exposed areas and may be found in conjunction with other keratinocytic tumours, mostly squamous cell carcinomas. Two viral oncoproteins, large T antigen (LT; coded by MCPyV_gp3) and small T antigen (sT; coded by MCPyV_gp4), promote different carcinogenic pathways. OBJECTIVES: To determine which genes are differentially expressed in MCPyV-positive and MCPyV-negative MCC; to describe the mutational burden and the most frequently mutated genes in both MCC subtypes; and to identify the clinical and molecular factors that may be related to patient survival. METHODS: Ninety-two patients with a diagnosis of MCC were identified from the medical databases of participating centres. To study gene expression, a customized panel of 172 genes was developed. Gene expression profiling was performed with nCounter technology. For mutational studies, a customized panel of 26 genes was designed. Somatic single nucleotide variants (SNVs) were identified following the GATK Best Practices workflow for somatic mutations. RESULTS: The expression of LT enabled the series to be divided into two groups (LT positive, n = 55; LT negative, n = 37). Genes differentially expressed in LT-negative patients were related to epithelial differentiation, especially SOX9, or proliferation and the cell cycle (MYC, CDK6), among others. Congruently, LT displayed lower expression in SOX9-positive patients, and differentially expressed genes in SOX9-positive patients were related to epithelial/squamous differentiation. In LT-positive patients, the mean SNV frequency was 4.3; in LT-negative patients it was 10 (P = 0.03). On multivariate survival analysis, the expression of SNAI1 [hazard ratio (HR) 1.046, 95% confidence interval (CI) 1.007-1.086; P = 0.02] and CDK6 (HR 1.049, 95% CI 1.020-1.080; P = 0.001) were identified as risk factors. CONCLUSIONS: Tumours with weak LT expression tend to co-express genes related to squamous differentiation and the cell cycle, and to have a higher mutational burden. These findings are congruent with those of earlier studies.

Merkel cell carcinoma (MCC) is an aggressive form of skin tumour. There are two subtypes of MCC: one of them is related to a virus called Merkel cell polyomavirus (MCPyV); the other one is related to persistent exposure to sunlight. The aim of this research was to find differences between these subtypes in their molecular behaviour (the genes that are expressed and the mutations that may be found). To do this, we carried out two studies, one to investigate gene expression (the process cells use to convert the instructions in our DNA into a functional product such as a protein) and one to look at gene mutations (changes in the DNA sequence). We found that the tumours that were not related to MCPyV expressed genes related to epithelial differentiation (the process by which unspecialized cells gain features characteristics of epithelial cells, which, among other things, make up the outer surface of the body), which means that the origin of both MCC subtypes may be different. We also found that MCPyV-related tumours had fewer mutations. Our findings are important because they help us to understand the biology of the MCC subtypes and could help with the development of new treatments for people diagnosed with skin tumours.

Immune and stromal transcriptional patterns that influence the outcome of classic Hodgkin lymphoma.

Classic Hodgkin lymphoma (cHL) is characterized by a rich immune microenvironment as the main tumor component. It involves a broad range of cell populations, which are largely unexplored, even though they are known to be essential for growth and survival of Hodgkin and Reed-Sternberg cells. We profiled the gene expression of 25 FFPE cHL samples using NanoString technology and resolved their microenvironment compositions using cell-deconvolution tools, thereby generating patient-specific signatures. The results confirm individual immune fingerprints and recognize multiple clusters enriched in refractory patients, highlighting the relevance of: (1) the composition of immune cells and their functional status, including myeloid cell populations (M1-like, M2-like, plasmacytoid dendritic cells, myeloid-derived suppressor cells, etc.), CD4-positive T cells (exhausted, regulatory, Th17, etc.), cytotoxic CD8 T and natural killer cells; (2) the balance between inflammatory signatures (such as IL6, TNF, IFN-γ/TGF-ß) and MHC-I/MHC-II molecules; and (3) several cells, pathways and genes related to the stroma and extracellular matrix remodeling. A validation model combining relevant immune and stromal signatures identifies patients with unfavorable outcomes, producing the same results in an independent cHL series. Our results reveal the heterogeneity of immune responses among patients, confirm previous findings, and identify new functional phenotypes of prognostic and predictive utility.

Extranodal natural killer/T-cell lymphoma nasal type in a western population: Molecular profiling identifies new therapeutic targets.

(A) Correlation matrix of unsupervised co-regulated genes, based on the 208 genes included in the NanoString platform. Some of the clusters of co-regulated genes corresponded to the following: Inflammatory cells; Epstein-Barr virus; B-cells; Cytotoxic T-cells; T-cells; and Proliferation. (B) Analysis of genomic alterations by targeted sequencing. Distribution of mutations in the 62 analyzed genes. Rows correspond to sequenced genes, columns represent individual patients. Color coding: green, missense; blue, synonymous; pink, frameshift; violet, Indel; red, stop gained; yellow, UTR.

An integrated prognostic model for diffuse large B-cell lymphoma treated with immunochemotherapy.

Diffuse large B-cell lymphoma (DLBCL), the most frequent non-Hodgkin's lymphoma subtype, is characterized by strong biological, morphological, and clinical heterogeneity, but patients are treated with immunochemotherapy in a relatively homogeneous way. Here, we have used a customized NanoString platform to analyze a series of 197 homogeneously treated DLBCL cases. The platform includes the most relevant genes or signatures known to be useful for predicting response to R-CHOP (Rituximab, Cyclophosphamide, Doxorubicin, Vincristine, and Prednisone) in DLBCL cases. We generated a risk score that combines the International Prognostic Index with cell of origin and double expression of MYC/BCL2, and stratified the series into three groups, yielding hazard ratios from 0.15 to 5.49 for overall survival, and from 0.17 to 5.04 for progression-free survival. Group differences were highly significant (p < 0.0001), and the scoring system was applicable to younger patients (<60 years of age) and patients with advanced or localized stages of the disease. Results were validated in an independent dataset from 166 DLBCL patients treated in two distinct clinical trials. This risk score combines clinical and biological data in a model that can be used to integrate biological variables into the prognostic models for DLBCL cases.

PLCγ1/PKCθ Downstream Signaling Controls Cutaneous T-Cell Lymphoma Development and Progression.

Developing mechanistic rationales can improve the clinical management of cutaneous T-cell lymphomas. There is considerable genetic and biological evidence of a malignant network of signaling mechanisms, highly influenced by deregulated TCR/PLCγ1 activity, controlling the biology of these lesions. In addition, activated signal transducer and activator of transcription 3 is associated with clinical progression, although the alterations responsible for this have not been fully elucidated. Here, we studied PLCγ1-dependent mechanisms that can mediate STAT3 activation and control tumor growth and progression. Downstream of PLCγ1, the pharmacological inhibition and genetic knockdown of protein kinase C theta (PKCθ) inhibited signal transducer and activator of transcription 3 activation, impaired proliferation, and promoted apoptosis in cutaneous T-cell lymphoma cells. A PKCθ-dependent transcriptome in mycosis fungoides/Sézary syndrome cells revealed potential effector genes controlling cytokine signaling, TP53, and actin cytoskeleton dynamics. Consistently, an in vivo chicken embryo model xenografted with mycosis fungoides cells showed that PKCθ blockage abrogates tumor growth and spread to distant organs. Finally, the expression of a number of PKCθ target genes found in mycosis fungoides cells significantly correlated with that of PRKCQ (PKCθ) in 81 human mycosis fungoides samples. In summary, PKCθ can play a central role in the activation of malignant cutaneous T-cell lymphoma mechanisms via multiple routes, including, but not restricted to, STAT3. These mechanisms may, in turn, serve as targets for specific therapies.

Peripheral T-cell lymphoma: molecular profiling recognizes subclasses and identifies prognostic markers.

Peripheral T-cell lymphoma (PTCL) is a clinically aggressive disease, with a poor response to therapy and a low overall survival rate of approximately 30% after 5 years. We have analyzed a series of 105 cases with a diagnosis of PTCL using a customized NanoString platform (NanoString Technologies, Seattle, WA) that includes 208 genes associated with T-cell differentiation, oncogenes and tumor suppressor genes, deregulated pathways, and stromal cell subpopulations. A comparative analysis of the various histological types of PTCL (angioimmunoblastic T-cell lymphoma [AITL]; PTCL with T follicular helper [TFH] phenotype; PTCL not otherwise specified [NOS]) showed that specific sets of genes were associated with each of the diagnoses. These included TFH markers, cytotoxic markers, and genes whose expression was a surrogate for specific cellular subpopulations, including follicular dendritic cells, mast cells, and genes belonging to precise survival (NF-κB) and other pathways. Furthermore, the mutational profile was analyzed using a custom panel that targeted 62 genes in 76 cases distributed in AITL, PTCL-TFH, and PTCL-NOS. The main differences among the 3 nodal PTCL classes involved the RHOAG17V mutations (P < .0001), which were approximately twice as frequent in AITL (34.09%) as in PTCL-TFH (16.66%) cases but were not detected in PTCL-NOS. A multivariate analysis identified gene sets that allowed the series of cases to be stratified into different risk groups. This study supports and validates the current division of PTCL into these 3 categories, identifies sets of markers that can be used for a more precise diagnosis, and recognizes the expression of B-cell genes as an IPI-independent prognostic factor for AITL.

Subcutaneous panniculitis-like T-cell lymphoma, lupus erythematosus profundus, and overlapping cases: molecular characterization through the study of 208 genes.

Subcutaneous panniculitis-like T-cell lymphoma (SPTCL) is a rare cytotoxic cutaneous lymphoma. Differential diagnosis with lupus erythematosus panniculitis (LEP) can be challenging and overlapping cases have been described. In this study, we investigate whether gene expression profiling may or not identify markers that can be used to improve our understanding of the disease and to make a precise differential diagnosis. SPTCL, LEP, and overlapping cases were analyzed using a customized NanoString platform including 208 genes related to T-cell differentiation, stromal signatures, oncogenes, and tumor suppressor genes. Gene expression unsupervised analysis of the samples differentiated SPTCL from LEP samples. Most overlapping cases were clustered with LEP cases. Differentially expressed genes were observed when comparing SPTCL with LEP cases; and overlapping with LEP cases. Gene set enrichment analysis recognized gene sets defining each group. In conclusion, SPTCL and LEP have distinctive molecular profiles and the molecular background of overlapping cases more closely resembles LEP.

An analysis of genetic targets for guiding clinical management of follicular lymphoma.

Introduction: Follicular lymphoma (FL) is one of the most common non-Hodgkin lymphoma (NHL) types, where genomic studies have accumulated potentially useful information about frequently mutated genes and deregulated pathways, which has allowed to a better understanding of the molecular pathogenesis of this tumor and the complex interrelationship between the tumoral cells and the stroma. Areas covered: The results of the molecular studies performed on Follicular Lymphoma have been here reviewed, summarizing the results of the clinical trials so far developed on this basis and discussing the reasons for the successes and failures. Searches were performed on June 1st, 2020, in PubMed and ClinicalTrials.gov. Expert opinion: Targeted therapy for follicular lymphoma has multiple opportunities including the use of epigenetic drugs, PI3K inhibitors, modifiers of the immune stroma and others. Data currently known on FL pathogenesis suggest that combining these treatments with immunotherapy should be explored in clinical trials, mainly for patients with clinical progression or adverse prognostic markers. Association of targeted trials with dynamic molecular studies of the tumor and serum samples is advised. Chemotherapy-free approaches should also be explored as first-line therapy for FL patients.

Identification of tipifarnib sensitivity biomarkers in T-cell acute lymphoblastic leukemia and T-cell lymphoma.

Patients diagnosed with T-cell leukemias and T-cell lymphomas (TCLs) still have a poor prognosis and an inadequate response to current therapies, highlighting the need for targeted treatments. We have analyzed the potential therapeutic value of the farnesyltransferase inhibitor, tipifarnib, in 25 TCL cell lines through the identification of genomic and/or immunohistochemical markers of tipifarnib sensitivity. More than half of the cell lines (60%) were considered to be sensitive. Tipifarnib reduced cell viability in these T-cell leukemia and TCL cell lines, induced apoptosis and modified the cell cycle. A mutational study showed TP53, NOTCH1 and DNMT3 to be mutated in 84.6%, 69.2% and 30.0% of sensitive cell lines, and in 62.5%, 0% and 0% of resistant cell lines, respectively. An immunohistochemistry study showed that p-ERK and RelB were associated as potential biomarkers of tipifarnib sensitivity and resistance, respectively. Data from RNA-seq show that tipifarnib at IC50 after 72 h downregulated a great variety of pathways, including those controlling cell cycle, metabolism, and ribosomal and mitochondrial activity. This study establishes tipifarnib as a potential therapeutic option in T-cell leukemia and TCL. The mutational state of NOTCH1, p-ERK and RelB could serve as potential biomarkers of tipifarnib sensitivity and resistance.

Update on peripheral T-cell lymphomas with T-helper phenotype: Are there too many subtypes?

Follicular helper T (TFH) cells are the providers of T-cell help to B-cells in the development of germinal centers and for the generation of most class-switched antibodies. The markers most commonly associated with TFH activity are IL21, IL4, CD40L, BCL6, SAP, CXCR5/CXCL13, and ICOS. T-cell lymphoma genomic studies have shown that different T-cell lymphoma types express signatures typical for TFH cells, this including angioimmunoblastic T-cell lymphoma (AITL), a related condition termed peripheral T-cell lymphoma with TFH phenotype and primary cutaneous CD4+ small/medium T-cell lymphoproliferative disorder. Angioimmunoblastic T-cell lymphoma is a well-established entity, a clinically aggressive disease with a survival of 30% OS after 5 years. Molecular and clinical studies have confirmed this as a well-established clinicopathological entity with relatively specific gene mutations, including mutations found in hematopoietic precursor cells and others. Peripheral T-cell lymphoma with TFH phenotype is an associated disorder with histology of PTCL but a TFH phenotype, as defined by the expression of 2-3 immunohistochemical markers. Molecular studies on this entity are showing a partial overlap with AITL. Primary cutaneous CD4+ small/medium lymphoproliferative disorder is an entirely different process that takes place in the skin, showing frank cytologic atypia, monoclonal TCR rearrangement and TFH phenotype in the context of a clinically benign lesion. Here we review the main clinical, molecular and diagnostic features of these three lymphoproliferative processes.