Aims The clinical implications of the programmed cell death 1 (PD1)/programmed cell death‐ligand 1 (PD‐L1) axis in patients with post‐transplant lymphoproliferative disorders are largely unknown, and its association with Epstein–Barr virus (EBV) status and PD‐L1 copy number alterations (CNAs) has not been thoroughly studied. Methods and results PD1/PD‐L1 expression was studied in 50 adult post‐transplant lymphoproliferative disorders, and the correlations with PD‐L1 CNAs, EBV, clinicopathological features and outcome were evaluated. Thirty‐seven (74%) cases were classified as diffuse large B‐cell lymphoma (DLBCL), nine (18%) cases were classified as polymorphic, and four (8%) cases were classified as classic Hodgkin lymphoma. Thirty‐four cases were EBV‐positive, with 29 of 34 (85%) having latency II or III, and 15 of 34 (44%) having viral replication. PD‐L1 expression in tumour cells and tumour‐associated macrophages was observed in 30 (60%) and 37 (74%) cases, respectively. PD1 positivity was seen in 16 (32%) cases. PD‐L1 expression was associated with EBV with latency II or III (P = 0.001) and organ rejection (P = 0.04), and, in DLBCL, with non‐germinal centre type DLBCL (P < 0.001). Cases with PD‐L1‐positive tumour cells showed a higher number of PD‐L1 CNAs than PD‐L1‐negative cases (P = 0.001). Patients with EBV/latency III/replication and simultaneous PD‐L1 expression showed the worst overall survival (P < 0.001). Conclusions The PD1/PD‐L1 axis is deregulated in post‐transplant lymphoproliferative disorders, with frequent PD‐L1 expression and PD1 negativity. PD‐L1 expression is associated with EBV latency II or III and PD‐L1 CNAs, and probably reflects a proinflammatory tumour microenvironment. The combined analysis of EBV status and PD‐L1 expression may help to identify deeply immunosuppressed patients who can benefit from immune reconstitution approaches.

Background: Desmoplastic melanoma (DM) is a rare subtype of spindle cell malignant melanoma characterized by frequent local recurrences and hematogenous spread, but without molecular classification. The aim of the study was to investigate in a DM series the incidence of relevant gene alterations in cancer, the programmed death-ligand 1 (PD-L1) expression status and the association with clinicopathological features and melanoma progression. Methods: A total of 38 patients were included. Clinical follow-up and the histopathological features of all cases were retrospectively collected. PD-L1 expression by immunohistochemistry (IHC) and BRAF genomic alterations by real-time PCR were determined in 34 samples. Additionally, a molecular analysis by nextgeneration sequencing was performed in 25 DMs. Results: Tumors occurred predominantly in men (76%) and in the head and neck region (50%). Most tumors were pure DMs (66%), containing less than 10% of conventional melanoma. Overall, 48% of our cohort harbored TP53 mutations, most of them showing a molecular signature associated with ultraviolet (UV)-oncogenesis, and 29%, BRAF mutations. A positive correlation between TP53 with depth of invasion (P=0.005) and presence of elastosis (P=0.002) was found. High-expression of PD-L1 in tumor cells was observed in 38% of cases and correlated with depth of tumoral infiltration (P=0.003), TP53 (P=0.016), PD-1 (P<0.001) and tumor-infiltrating lymphocytes (TILS) (P<0.001). PD-L1 expression in immune cells correlated with PD-1 (P=0.006), tumoral PD-L1 expression (P=0.029) and TP53 mutation (P=0.002). Survival correlated with depth of invasion (P=0.003), stage of tumors (P=0.015), positive sentinel lymph node (P=0.004), lymph node metastasis (P=0.024) and distant metastasis (P<0.001). Conclusions: Our results suggest that progressed DMs with deep tumoral infiltration frequently harbor TP53 mutations, PD-L1 expression and present a high inflammatory response, probably related to adaptive immune resistance in this tumor-type.

Human herpesvirus type 8 (HHV8) is a gamma herpesvirus known for its role in lymphoid neoplasms, especially in immunosuppressed patients. We describe the case of a 64-year-old male, without known immunodeficiency, with 1-year-long clinical history of mediastinal and abdominal lymphadenopathies and recurrent pulmonary infections. Histopathological evaluation of a mediastinal lymph node revealed the presence of scattered atypical large cells with Hodgkin and Reed-Sternberg morphology in a background of lymphocytes and extensive areas of fibrosis. The large cells were positive for HHV8 and Epstein-Barr virus (EBV), with a clonal pattern of IGH gene rearrangement. A descriptive diagnosis of "HHV8-positive, EBV-positive Hodgkin lymphoma-like large B-cell lymphoma" was rendered. Interestingly, the retrospective evaluation of a previous biopsy, diagnosed as reactive lymphadenitis, revealed the presence of HHV8-and EBV-positive cells, with a polyclonal pattern and a small peak corresponding to that of the most recent biopsy. This case presents diagnostic challenges due to the presence of particular features not clearly related to current HHV8-associated entities, and also suggests the possibility for disease progression in the spectrum of HHV8-and EBV-associated lymphoproliferative disorders.

Aim: Single biomarker diagnostic test of BRAFV600 locus in metastatic melanoma is mandatory for treatment decision; however, multiple-gene based techniques, such as targeted next-generation sequencing (NGS) are being used to maximize the number of patients that can benefit from a targeted therapy. The main objective of this study is to investigate whether an NGS panel could be adopted in routine clinical care for advanced melanoma. Methods: Patients diagnosed with advanced melanoma at our center from 2017 to 2019 were included. Presence of genetic alterations was performed using two methods: real-time polymerase chain reaction-based Idylla test (Biocartis) and NGS with the oncomine solid tumor DNA kit (Thermo Fisher Scientific). Total genomic DNA was extracted from formalin-fixed and paraffin embedded samples for sequencing. Results: A total of 155 samples were evaluated for molecular analysis but 40 samples (25.8%) were inadequate for sequencing. The clinical utility of BRAFV600 real-time polymerase chain reaction and targeted-NGS was compared in 29 samples and a very good concordance was observed (Kappa = 0.89, 95% confidence interval 0.68 ± 1.05). An oncogenic mutation by NGS was found in 75 samples (65%) – 53% of whom were candidates for personalized therapies. The most prevalent mutated genes were BRAF (39%), TP53 (23%), and NRAS (14%). Other genes identified at lower incidence (< 5%) were: PIK3CA, ERBB4, CTNNB1, STK11, FGFR1, SMAD4, KRAS, FGFR3, PTEN and AKT. Co-occurrence of oncogenic mutations was detected in 40% of the samples. Among the mutations identified, TP53 was significantly more prevalent in men (men 31.8% versus women 12.2%, P = 0.03) and NRAS in women (men 9.1% versus women 24.4%, P = 0.03). Conclusions: Targeted-NGS testing is a feasible technique to implement in the routine clinical practice. Based on our results, NGS has provided more information on target-genes than RT-PCR technique, maximizing the benefit for patients with advanced melanoma.