Maria Grazia Narducci

Sézary syndrome (SS) is a rare and aggressive type of cutaneous T-cell lymphoma (CTCL). Due to its rarity and marked biological heterogeneity, SS diagnosis is frequently delayed, as it requires the integration of multiple complex diagnostic tests interpreted by highly specialized biomedical figures. In this context, we investigated the expression of CXCL13—increasingly implicated in several inflammatory and neoplastic skin disorders—as a potential screening biomarker to discriminate SS from mycosis fungoides (MF), the most common CTCL subtype, and from clinically overlapping inflammatory skin diseases, i.e. atopic dermatitis (AD), psoriasis (PS) and eczema (EC). Real-time PCR analysis of CXCL13 mRNA expression in 51 samples showed a significant upregulation in peripheral blood mononuclear cells (PBMCs) from SS patients compared with MF, AD and healthy donors (HD). Protein-level analysis in a larger cohort (n = 142), including allergic and atopic EC patients, revealed significantly higher plasma CXCL13 concentrations in SS than in all other conditions and HD, as assessed by ELISA. Receiver operating characteristic (ROC) analysis confirmed the excellent performance of plasma CXCL13 as a differential marker (AUC = 93%). In contrast, CXCL13 immunohistochemical expression in skin biopsies was broadly detected across all conditions, limiting its diagnostic value. In conclusion, quantification of CXCL13 expression in PBMCs and, more robustly, measurement of its plasma levels by widely available techniques such as RT-qPCR and ELISA may represent practical screening tools to identify patients with suspected SS. These assays could facilitate earlier referral to specialized centres for confirmatory testing and prompt initiation of appropriate therapy.

Background: Sézary syndrome (SS) is an aggressive and leukemic variant of Cutaneous T-cell Lymphoma (CTCL) with an incidence of 1 case per million people per year. It is characterized by a complex and heterogeneous profile of genetic alteration ns that has so far precluded the development of a specific and definitive therapeutic intervention.Methods: Deep-RNA-sequencing (RNA-seq) data were used to analyze the single nucleotide variants (SNVs) carried by 128 putative CTCL-driver genes, previously identified as mutated in genomic studies, in longitudinal SS samples collected from 17 patients subjected to extracorporeal photopheresis (ECP) with Interferon-α. Results obtained were integrated with Whole Exome Sequencing (WES) data. SNVs were validated using the Sanger method. Pathway analysis was performed with g:Profiler web server (https://biit.cs.ut.ee/gprofiler/gost). Statistical analyses were performed with GraphPad PRISM 8 software.Results: Nonsynonymous SNVs were identified in 56 genes. Integration of RNA-seq with WES data revealed that about half of these genes contained somatic mutations. Among them, the most frequently transcribed mutated genes were TET2, JAK3, NCOR1, PDCD11, RHOA, and TP53. Nearly all the remaining genes had germline-restricted mutations, and included ARID1A, ATM, ATR, CREBBP, POLD1, and POT1 genes, which are involved in DNA repair, homologous recombination, and chromatin remodeling, and the CROCC gene, implicated in centrosome cohesion. Monitoring of the mutated genes, identified within an enlarged panel of CTCL associated genes, revealed their reduction in almost 70% of SS patients as well as a significant decline of total number of mutations (SNVs) during ECP treatment. Several mutated genes persisted post-therapy, representing novel candidates associated with ECP resistance that could also have a potential prognostic relevance. Notably, these genes mainly converge on pathways related to DNA repair (ATR, ATRIP, POLD1, TP53, TP53BP1/2) which might represent novel targets to be explored in combination with ECP.Conclusions: This is the first evaluation in SS of expressed mutations in a large panel of CTCL-driver genes. Also innovative is the monitoring of mutated genes in patients’ malignant lymphocytes during ECP, a first-line treatment of CTCL, which highlights novel candidates associated with ECP resistance that might unmask novel pharmacological vulnerabilities to be exploited during ECP for a personalized treatment.

Background: The neutrophil/lymphocyte ratio (NLR) at baseline has been demonstrated to correlate with higher stages of disease and to be a prognostic factor in numerous cancers. However, its function as a prognostic factor for mycosis fungoides (MF) has not been yet clarified. Objective: Our work aimed to assess the association of the NLR with different stages of MF and to outline whether higher values of this marker are related to a more aggressive MF. Methods: We retrospectively calculated the NLRs in 302 MF patients at the moment of diagnosis. The NLR was obtained using the complete blood count values. Results: The median NLR among patients with early stage disease (low-grade IA-IB-IIA) was 1.88, while the median NLR for patients with high-grade MF (IIB-IIIA-IIIB) was 2.64. Statistical analysis showed positive associations of advanced MF stages with NLRs higher than 2.3. Conclusions: Our analysis demonstrates that the NLR represents a cheap and easily available parameter functioning as a marker for advanced MF. This might guide physicians in recognizing patients with advanced stages of disease requiring a strict follow-up or an early treatment.

Despite the significant improvements in advanced melanoma therapy, there is still a pressing need for biomarkers that can predict patient response and prognosis, and therefore support rational treatment decisions. Here, we investigated whether circulating miRNAs could be biomarkers of clinical outcomes in patients treated with targeted therapy. Using next-generation sequencing, we profiled plasma miRNAs at baseline and at progression in patients treated with BRAF inhibitors (BRAFi) or BRAFi + MEKi. Selected miRNAs associated with response to therapy were subjected to validation by real-time quantitative RT-PCR. Receiver Operating Characteristics (ROC), Kaplan–Meier and univariate and multivariate Cox regression analyses were performed on the validated miR-1246 and miR-485-3p baseline levels. The median baseline levels of miR-1246 and miR-485-3p were significantly higher and lower, respectively, in the group of patients not responding to therapy (NRs) as compared with the group of responding patients (Rs). In Rs, a trend toward an increase in miR-1246 and a decrease in miR-485-3p was observed at progression. Baseline miR-1246 level and the miR-1246/miR-485-3p ratio showed a good ability to discriminate between Rs and NRs. Poorer PFS and OS were observed in patients with unfavorable levels of at least one miRNA. In multivariate analysis, a low level of miR-485-3p and a high miR-1246/miR-485-3p ratio remained independent negative prognostic factors for PFS, while a high miR-1246/miR-485-3p ratio was associated with an increased risk of mortality, although statistical significance was not reached. Evaluation of miR-1246 and miR-485-3p baseline plasma levels might help clinicians to identify melanoma patients most likely to be unresponsive to targeted therapy or at higher risk for short-term PFS and mortality, thus improving their management.

Sézary syndrome (SS) is an aggressive variant of cutaneous t-cell lymphoma characterized by the accumulation of neoplastic CD4+ lymphocytes—the SS cells—mainly in blood, lymph nodes, and skin. The tumor spread pattern of SS makes this lymphoma a unique model of disease that allows a concurrent blood and skin sampling for analysis. This review summarizes the recent studies highlighting the transcriptional programs triggered by the crosstalk between SS cells and blood–skin microenvironments. Emerging data proved that skin-derived SS cells show consistently higher activation/proliferation rates, mainly driven by T-cell receptor signaling with respect to matched blood SS cells that instead appear quiescent. Biochemical analyses also demonstrated an hyperactivation of PI3K/AKT/mTOR, a targetable pathway by multiple inhibitors currently in clinical trials, in skin SS cells compared with a paired blood counterpart. These results indicated that active and quiescent SS cells coexist in this lymphoma, and that they could be respectively treated with different therapeutics. Finally, this review underlines the more recent discoveries into the heterogeneity of circulating SS cells, highlighting a series of novel markers that could improve the diagnosis and that represent novel therapeutic targets (GPR15, PTPN13, KLRB1, and ITGB1) as well as new genetic markers (PD-1 and CD39) able to stratify SS patients for disease aggressiveness.

Activation of Akt/Protein Kinase B (PKB) by phosphatidylinositol‐3‐kinase (PI3K) controls several cellular functions largely studied in mammalian cells, including preimplantation embryos. We previously showed that early mouse embryos inherit active Akt from oocytes and that the intracellular localization of this enzyme at the two‐cell stage depends on the T‐cell leukemia/lymphoma 1 oncogenic protein, Tcl1. We have now investigated whether Akt isoforms, namely Akt1, Akt2 and Akt3, exert a specific role in blastomere proliferation during preimplantation embryo development. We show that, in contrast to other Akt family members, Akt2 enters male and female pronuclei of mouse preimplantation embryos at the late one‐cell stage and thereafter maintains a nuclear localization during later embryo cleavage stages. Depleting one‐cell embryos of single Akt family members by microinjecting Akt isoform‐specific antibodies into wild‐type zygotes, we observed that: (a) Akt2 is necessary for normal embryo progression through cleavage stages; and (b) the specific nuclear targeting of Akt2 in two‐cell embryos depends on Tcl1. Our results indicate that preimplantation mouse embryos have a peculiar regulation of blastomere proliferation based on the activity of the Akt/PKB family member Akt2, which is mediated by the oncogenic protein Tcl1. Both Akt2 and Tcl1 are essential for early blastomere proliferation and embryo development.

Sezary Syndrome is an aggressive T-cell Lymphoma involving blood, skin and lymphonodes Involvement of the CXCR4-SDF1 has been previously shown. We here present evidence also of the involvement of B-arrestin a downstream regulator of CXCR4, that is depleted and downregulated as well as a potential functional role for this depletion.