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In patients with advanced triple-negative breast cancer (TNBC), translational research efforts are needed to improve the clinical efficacy of immunotherapy with checkpoint inhibitors. Here, we report on the immunological characterization of an exceptional, long-lasting, tumor complete response in a patient with metastatic TNBC treated with dual PD-1 and LAG-3 blockade within the phase I/II study CLAG525X2101C (NCT02460224) The pre-treatment tumor biopsy revealed the presence of a CD3+ and CD8+ cell infiltrate, with few PD1+ cells, rare CD4+ cells, and an absence of both NK cells and LAG3 expression. Conversely, tumor cells exhibited positive staining for the three primary LAG-3 ligands (HLA-DR, FGL-1, and galectin-3), while being negative for PD-L1. In peripheral blood, baseline expression of LAG-3 and PD-1 was observed in circulating immune cells. Following treatment initiation, there was a rapid increase in proliferating granzyme-B+ NK and T cells, including CD4+ T cells, alongside a reduction in myeloid-derived suppressor cells. The role of LAG-3 expression on circulating NK cells, as well as the expression of LAG-3 ligands on tumor cells and the early modulation of circulating cytotoxic CD4+ T cells warrant further investigation as exploitable predictive biomarkers for dual PD-1 and LAG-3 blockade. Trial registration: NCT02460224. Registered 02/06/2015.

Background: Mucosal Melanomas (MM) are highly aggressive neoplasms arising from mucosal melanocytes. Current treatments offer a limited survival benefit for patients with advanced MM; moreover, the lack of pre-clinical cellular systems has significantly limited the understanding of their immunobiology. Methods: Five novel cell lines were obtained from patient-derived biopsies of MM arising in the sino-nasal mucosa and designated as SN-MM1-5. The morphology, ultrastructure and melanocytic identity of SN-MM cell lines were validated by transmission electron microscopy and immunohistochemistry. Moreover, in vivo tumorigenicity of SN-MM1-5 was tested by subcutaneous injection in NOD/SCID mice. Molecular characterization of SN-MM cell lines was performed by a mass-spectrometry proteomic approach, and their sensitivity to PI3K chemical inhibitor LY294002 was validated by Akt activation, measured by pAkt(Ser473) and pAkt(Thr308) in immunoblots, and MTS assay. Results: This study reports the validation and functional characterization of five newly generated SN-MM cell lines. Compared to the normal counterpart, the proteomic profile of SN-MM is consistent with transformed melanocytes showing a heterogeneous degree of melanocytic differentiation and activation of cancer-related pathways. All SN-MM cell lines resulted tumorigenic in vivo and display recurrent structural variants according to aCGH analysis. Of relevance, the microscopic analysis of the corresponding xenotransplants allowed the identification of clusters of MITF-/CDH1-/CDH2 + /ZEB1 + /CD271 + cells, supporting the existence of melanoma-initiating cells also in MM, as confirmed in clinical samples. In vitro, SN-MM cell lines were sensitive to cisplatin, but not to temozolomide. Moreover, the proteomic analysis of SN-MM cell lines revealed that RICTOR, a subunit of mTORC2 complex, is the most significantly activated upstream regulator, suggesting a relevant role for the PI3K-Akt-mTOR pathway in these neoplasms. Co

In the context of cervical cancer prevention, where human papillomavirus (HPV) infection is pivotal, HPV testing is replacing Pap Smear in primary screening. This transition offers an opportunity for integrating self-sampling to enhance coverage. We evaluated the accuracy of HPV testing using self-collected urine and vaginal samples, comparing them to physician-collected cervical swabs. From a cohort of 245 women with abnormal cytology, we collected self-sampled vaginal, urine, and clinician-administered cervical specimens. Employing AnyplexTMII HPV28 assay, outcomes revealed HPV positivity rates of 75.1% (cervical), 78.4% (vaginal), and 77.1% (urine). Significant, hr-HPV detection concordance was observed between self-taken cervical samples and clinical counterparts (k = 0.898 for vaginal; k = 0.715 for urine). This study extends beyond accuracy, highlighting self-collected sample efficacy in detecting high-grade cervical lesions. The insight underscores self-sampling’s role in bolstering participation and aligns with WHO’s goal to eliminate cervical cancer by 2030.

The global outbreak of human monkeypox virus (hMPXV1) in 2022 highlighted the usefulness of dermatological manifestations for its diagnosis. Infection by the human monkeypox virus thus necessitated inclusion in the diagnostic repertoire of dermatopathology. To assess the histopathological and microscopical findings of cutaneous lesions related to hMPXV infection, we analyzed skin biopsies from patients with positive MPXV DNA polymerase chain reaction presenting with a typical course of hMPXV1 infection. The most prominent histopathological findings were ascribable to a pustular stage in which epidermal necrosis with areas of non-viable keratinocytes and a “shadow cell” appearance were evident; in some cases, the deep portion of the hair follicle and the acrosyringial epithelium were affected. The main cytopathic modifications included ballooning keratinocytes, followed by Guarnieri bodies and a ground glass appearance of the keratinocytes’ nuclei, together with a dense mixed inflammatory cell infiltrate with prominent neutrophil exocytosis. Transmission electron microscopy analysis demonstrated viral particle aggregates in the cytoplasm of keratinocytes, without any involvement of the nucleus. Interestingly, we also found the presence of viral particles in infected mesenchymal cells, although to a lesser extent than in epithelial cells. Through this study, we contributed to expanding the histological and microscopic knowledge of the human mpox virus, a key step to understanding current and potential future trends of the disease, as well as of other Orthopoxvirus infections.

Objective: Trained immunity (TI) is a de facto memory program of innate immune cells, characterized by immunometabolic and epigenetic changes sustaining enhanced production of cytokines. TI evolved as a protective mechanism against infections; however, inappropriate activation can cause detrimental inflammation and might be implicated in the pathogenesis of chronic inflammatory diseases. In this study, we investigated the role of TI in the pathogenesis of giant cell arteritis (GCA), a large-vessel vasculitis characterized by aberrant macrophage activation and excess cytokine production. Methods: Monocytes from GCA patients and from age- and sex-matched healthy donors were subjected to polyfunctional studies, including cytokine production assays at baseline and following stimulation, intracellular metabolomics, chromatin immunoprecipitation-qPCR, and combined ATAC/RNA sequencing. Immunometabolic activation (i.e. glycolysis) was assessed in inflamed vessels of GCA patients with FDG-PET and immunohistochemistry (IHC), and the role of this pathway in sustaining cytokine production was confirmed with selective pharmacologic inhibition in GCA monocytes. Results: GCA monocytes exhibited hallmark molecular features of TI. Specifically, these included enhanced IL-6 production upon stimulation, typical immunometabolic changes (e.g. increased glycolysis and glutaminolysis) and epigenetic changes promoting enhanced transcription of genes governing pro-inflammatory activation. Immunometabolic changes of TI (i.e. glycolysis) were a feature of myelomonocytic cells in GCA lesions and were required for enhanced cytokine production. Conclusions: Myelomonocytic cells in GCA activate TI programs sustaining enhanced inflammatory activation with excess cytokine production.

HPV testing in cervical cancer screening programs offers the possibility of introducing molecular standardized biomarkers for the triage of HPV-positive women. This study aimed to evaluate the role of HPV genotyping and viral load as possible diagnostic biomarkers of high-grade cervical lesions (CIN2+) by performing a preliminary evaluation of a new HPV test. Cervical specimens were obtained from 200 women referred for a colposcopy. Samples were tested using both AnyplexTM II HR-HPV as well as OncoPredict HPV® Screening (SCR) and quantitative typing (QT). Using a cycle threshold cutoff (Ct) of 36.8 for the SCR assay and 1.27 log10 (viral copies/104 cells) for the QT assay, relative clinical sensitivity for CIN2+ and relative clinical specificity for CIN2− as compared to AnyplexTM II HR-HPV were, respectively, 0.92 and 1.00 for SCR and 1.35 and 1.24 for QT. The distribution of high-risk HPV (HR-HPV) genotypes (p = 0.009) as well as the viral copy numbers (CIN2−: 3.7 log10 (viral copies/104 human cells); CIN2+: 4.3 log10 (viral copies/104 human cells); p = 0.047) were found to differ in women with high- and low-grade cervical lesions, suggesting a possible role of HPV genotyping and normalized viral load as potential biomarkers to identify women at increased risk of cervical lesions.

BACKGROUND: Tumor microenvironment (TME) is a dynamic setting and changes in TILs and their subpopulations are potential candidates to influence the metastatic process. Aim of this pilot study is to describe the changes occurring between primary breast cancers and their paired metastases in terms of TILs composition. To assess if these changes influence the process of metastasis development, we used a control group of patients.METHODS: We retrospectively identified 18 Luminal patients, for whom primary and metastatic tissue were available (cases) and 18 paired-matched patients (controls), not relapsed after at least 9years of follow-up, and we quantified TILs and their composition (i.e. T CD8+ and CD4+/FOXP3+). The presence of TILs was defined as ≥10%.RESULTS: Our results showed that the microenvironment composition of relapsed patients was poor of TILs (median=5%, I-III quartiles=0.6-5%), CD8+ (2.5%, 0-5%) and CD4+/FOXP3+(0%, 0-0.6%) in the primary tumor. Comparable results were observed in their related metastases (TILs 3.8%, 0.6-5%; CD8+0%, 0-1.3%; CD4+/FOXP3+0%,0-1.9%). On the contrary, the microenvironment in the control group was richer of TILs (5%, 5-17.5%) in comparison to cases, both in primary tumor (p=0.035) and related metastases (p=0.018). Although CD8+ in controls were similar to cases at primary tumor (p=0.6498), but not at metastasis (p=0.0223), they expressed only one part on the TILs subpopulations (p=0.0060), while TILs in the cases at primary tumor were almost completely CD8+ (p=0.5034).CONCLUSIONS: These findings suggest that the lack of activation of immune system in the primary tumor might influence the multifactor process of cancer progression.

The treatment for hepatocellular carcinoma (HCC) relies on liver resection, which is, however, burdened by a high rate of recurrence after surgery, up to 60% at 5 years. No pre-operative tools are currently available to assess the recurrence risk tailored to every single patient. Recently liquid biopsy has shown interesting results in diagnosis, prognosis and treatment allocation strategies in other types of cancers, since its ability to identify circulating tumor cells (CTCs) derived from the primary tumor. Those cells were advocated to be responsible for the majority of cases of recurrence and cancer-related deaths for HCC. In fact, after being modified by the epithelial-mesenchymal transition, CTCs circulate as "seeds " in peripheral blood, then reach the target organ as dormant cells which could be subsequently "awakened " and activated, and then initiate metastasis. Their presence may justify the disagreement registered in terms of efficacy of anatomic vs non-anatomic resections, particularly in the case of microvascular invasion, which has been recently pointed as a histological sign of the spread of those cells. Thus, their presence, also in the early stages, may justify the recurrence event also in the contest of liver transplant. Understanding the mechanism behind the tumor progression may allow improving the treatment selection according to the biological patient-based characteristics. Moreover, it may drive the development of novel biological tailored tests which could address a specific patient to neoadjuvant or adjuvant strategies, and in perspective, it could also become a new method to allocate organs for transplantation, according to the risk of relapse after liver transplant. The present paper will describe the most recent evidence on the role of CTCs in determining the relapse of HCC, highlighting their potential clinical implication as novel tumor behavior biomarkers able to influence the surgical choice.

Objectives: The aim of the present study was too investigate prevalence and persistence of human papilloma virus (HPV) and cytological abnormalities (CAs) in the anal swabs of people living with HIV (PLWH): men who have sex with men (MSM), men who have sex with women (MSW) and women (W). Methods: Between March 2010 and January 2019, an anal swab for cytological and HPV genotyping tests was offered to all PLWH attending our clinic. Logistic regression analysis was conducted to identify predictors of infection. Results: In all, 354 PLWH were screened: 174 MSM, 90 MSW and 61 W. Prevalence of at least one high-risk (HR) HPV was higher in MSM (91%) and W (85%) than in MSW (77%) (P < 0.05). Cytological abnormalities were found in 21.1% of the entire population. At multivariable regression analysis a lower risk for HPV infection was found for W than for MSM [odds ratio = 0.24 (95% confidence interval: 0.115–0.513)] and for MSW than for MSM [0.37 (0.180–0.773)] and there was a significantly higher risk of CAs in PLWH with HPV 16 and 18 [3.3 (1.04–10.49)]. A total of 175 PLWH (103 MSM, 33 MSW and 26 W) had at least one follow-up visit (T1) after a median (interquartile range) follow-up of 3.6 (2.1–5.7) years. The acquisition rate of HR-HPV was high, with 66.7% of PLWH negative for HR-HPV at T0 who became positive at T1 (P < 0.001). The prevalence of CAs was stable (20.6%). A significant association between CAs at T1 and persistence of HPV-16 and/or 18 was found (P < 0.05). Conclusions: HPV 16 and 18 are associated with the presence and development of CAs irrespective of sexual orientation.

Colorectal cancer (CRC) is the fourth cause of death from cancer worldwide mainly due to the high incidence of drug-resistance. During a screen for new actionable targets in drug-resistant tumours we recently identified p65BTK – a novel oncogenic isoform of Bruton's tyrosine kinase. Studying three different cohorts of patients here we show that p65BTK expression correlates with histotype and cancer progression. Using drug-resistant TP53-null colon cancer cells as a model we demonstrated that p65BTK silencing or chemical inhibition overcame the 5-fluorouracil resistance of CRC cell lines and patient-derived organoids and significantly reduced the growth of xenografted tumours. Mechanistically, we show that blocking p65BTK in drug-resistant cells abolished a 5-FU-elicited TGFB1 protective response and triggered E2F-dependent apoptosis. Taken together, our data demonstrated that targeting p65BTK restores the apoptotic response to chemotherapy of drug-resistant CRCs and gives a proof-of-concept for suggesting the use of BTK inhibitors in combination with 5-FU as a novel therapeutic approach in CRC patients. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.