The use of platelets as a clinical tool in oncology: opportunities and challenges.

Platelets are small circulating anucleated cells mainly involved in thrombosis and hemostasis processes. Moreover, platelets play an active role in tumorigenesis and cancer progression, stimulating angiogenesis and vascular remodelling, and protecting circulating cancer cells from shear forces and immune surveillance. Several reports indicate that platelet number in the blood circulation of cancer patients is associated with prognosis and response to treatment. However, the mechanisms of platelets "education" by cancer cells and the crosstalk between platelets and tumor are still unclear, and the role of "tumor educated platelets" (TEPs) is achieving growing interest in cancer research. TEPs are a biological source of cancer-derived biomarkers, especially RNAs that are protected by platelets membrane from circulating RNases, and could serve as a non-invasive tool for tumor detection, molecular profiling and evolution during therapy in clinical practice. Moreover, short platelet lifespan offers the possibility to get a snapshot assessment of cancer molecular profile, providing a real-time tool. We review and discuss the potential and the clinical utility, in terms of cancer diagnosis and monitoring, of platelet count together with other morphological parameters and of the more recent and innovative TEP profiling.

p53/TP53 Status Assessment in Gastroesophageal Adenocarcinoma.

Chromosomal instability (CIN) is very frequent in gastroesophageal adenocarcinoma (GEA) and it is characterized by TP53 deletions/mutations resulting in p53 nuclear accumulation, as revealed by immunohistochemistry (IHC), which considers the cases with "high" staining levels to be positive. Aiming to improve aberrant TP53 detection, droplet digital PCR (ddPCR) was used to evaluate TP53 deletion in formalin-fixed, paraffin-embedded DNA (FFPE-DNA) and cell-free DNA (cfDNA). To further investigate the mutational TP53 profile, next-generation sequencing (NGS) was performed in a subset of FFPE samples. After combining "low" and "high" IHC staining level groups, the proportion of deletion events was significantly higher compared to the "intermediate" group (72.9% vs. 47.5%, p-value = 0.002). The ddPCR TP53 deletion assay was feasible for cfDNA but only had good agreement (72.7%, Cohen's kappa = 0.48) with the assay performed with FFPE-DNA of the "low-level" group. NGS analysis confirmed that, in the "low-level" group, a high percentage (66.7%) of cases were aberrant, with disruptive mutations that probably led to p53 loss. Data suggested that p53 IHC alone underestimates the CIN phenotype in GEA and that molecular analysis in both solid and liquid biopsies could be integrated with it; in particular, in cases of completely negative staining.

Putative Clinical Potential of ERBB2 Amplification Assessment by ddPCR in FFPE-DNA and cfDNA of Gastroesophageal Adenocarcinoma Patients.

Anti-HER2 monoclonal antibody trastuzumab improves the survival of those patients with advanced gastroesophageal adenocarcinoma (GEA) exhibiting HER2/ERBB2 overexpression/amplification. The current gold standard methods used to diagnose the HER2 status in GEA are immunohistochemistry (IHC) and silver or fluorescence in situ hybridization (SISH or FISH). However, they do not permit spatial and temporal tumor monitoring, nor do they overcome intra-cancer heterogeneity. Droplet digital PCR (ddPCR) was used to implement the assessment of HER2 status in formalin-fixed paraffin-embedded (FFPE) tumor DNA from a retrospective cohort (86 patients) and in cell-free DNA (cfDNA) samples from a prospective cohort (28 patients). In comparison to IHC/SISH, ddPCR assay revealed ERBB2 amplification in a larger patient fraction, including HER2 2+ and 0-1+ of the retrospective cohort (45.3% vs. 15.1%). In addition, a considerable number of HER2 2+ and 0-1+ prospective patients who were negative in FFPE by both IHC/SISH and ddPCR, showed ERBB2 amplification in the cfDNA collected just before surgery. cfDNA analysis in a few longitudinal cases revealed an increasing ERBB2 trend at progression. In conclusion, ddPCR in liquid biopsy may improve the detection rate of HER2 positive patients, preventing those patients who could benefit from targeted therapy from being incorrectly excluded.

Mechanisms Involved in the Promoting Activity of Fibroblasts in HTLV-1-Mediated Lymphomagenesis: Insights into the Plasticity of Lymphomatous Cells.

Among the mechanisms leading to progression to Adult T-cell Leukaemia/Lymphoma in Human T-cell Leukaemia Virus type 1 (HTLV-1)-infected subjects, the contribution of stromal components remains poorly understood. To dissect the role of fibroblasts in HTLV-1-mediated lymphomagenesis, transcriptome studies, cytofluorimetric and qRT-PCR analyses of surface and intracellular markers linked to plasticity and stemness in coculture, and in vivo experiments were performed. A transcriptomic comparison between a more lymphomagenic (C91/III) and the parental (C91/PL) cell line evidenced hyperactivation of the PI3K/Akt pathway, confirmed by phospho-ELISA and 2-DE and WB analyses. C91/III cells also showed higher expression of mesenchymal and stemness genes. Short-term coculture with human foreskin fibroblasts (HFF) induced these features in C91/PL cells, and significantly increased not only the cancer stem cells (CSCs)-supporting CD10+GPR77+ HFF subpopulation, but also the percentage of ALDH1bright C91/PL cells. A non-cytotoxic acetylsalicylic acid treatment decreased HFF-induced ALDH1bright C91/PL cells, downregulated mesenchymal and stemness genes in cocultured cells, and delayed lymphoma growth in immunosuppressed mice, thus hindering the supportive activity of HFF on CSCs. These data suggest that crosstalk with HFF significantly intensifies the aggressiveness and plasticity of C91/PL cells, leading to the enrichment in lymphoma-initiating cells. Additional research is needed to better characterize these preliminary findings.

MSI Analysis in Solid and Liquid Biopsies of Gastroesophageal Adenocarcinoma Patients: A Molecular Approach.

Gastroesophageal adenocarcinoma (GEA) patients with the microsatellite instability (MSI) subtype emerged as optimal candidates for immunotherapy. To date, immunohistochemistry (IHC) is the gold standard for MSI assessment in formalin-fixed paraffin-embedded (FFPE) specimens. However, IHC, although useful for diagnostic typing, cannot be used to analyze cell-free DNA (cfDNA) in liquid biopsy, a tool that could overcome tumor heterogeneity and enable longitudinal monitoring. In order to find an alternative diagnostic method to IHC, we analyzed 86 retrospective GEAs FFPE samples with multiplex PCR. Moreover, to verify the feasibility of MSI detection in liquid biopsy, cfDNA samples of five patients that resulted in having MSI in a prospective cohort of 35 patients were evaluated by multiplex PCR, real-time PCR and droplet digital PCR (ddPCR). Analysis of FFPE showed 100% concordance between multiplex PCR and IHC (Cohen's Kappa agreement = 1). On the contrary, only ddPCR was able to detect MSI in cfDNAs of T3/T4 GEA patients. In conclusion, data highlight the molecular analysis as an optimal alternative to IHC for the diagnostic typing and suggest that the ddPCR assay can be considered as the most reliable and promising molecular approach to detect MSI in the cfDNA of GEA patients.

Periostin and Epithelial-Mesenchymal Transition Score as Novel Prognostic Markers for Leiomyosarcoma, Myxofibrosarcoma, and Undifferentiated Pleomorphic Sarcoma.

PURPOSE: Interpatient clinical variability in soft-tissue sarcomas (STS) highlights the need for novel prognostic markers supporting patient risk stratification. As sarcomas might exhibit a more mesenchymal or a more epithelial state, we focused on epithelial-mesenchymal and mesenchymal-epithelial transitions (EMT/MET) for prognostic clues, and selected three histotypes with variable aggressiveness. EXPERIMENTAL DESIGN: The expression of EMT/MET-related factors was measured by qRT-PCR in 55 tumor samples from patients with leiomyosarcoma, myxofibrosarcoma, or undifferentiated pleomorphic sarcoma. The identified marker was further evaluated by IHC in 31 leiomyosarcomas and by measuring its circulating levels in 67 patients. The prognostic value of a sarcoma-tailored EMT score was analyzed. Epirubicin chemosensitivity and migration were studied in primary STS cultures. Associations with overall survival (OS) were assessed using Kaplan-Meier and Cox regression methods. RESULTS: High expression of periostin, a mesenchymal matricellular protein, in sarcoma tissues (P = 0.0024), its high stromal accumulation in leiomyosarcomas (P = 0.0075), and increased circulation (>20 ng/mL, P = 0.0008) were associated with reduced OS. High periostin expression [HR 2.9; 95% confidence interval (CI), 1.3-6.9; P = 0.0134] and circulation (HR 2.6; 95% CI, 1.3-5.1; P = 0.0086), and a mesenchymal EMT score (mesenchymal vs. transitioning; HR, 5.2; 95% CI, 2.1-13.0, P = 0.0005) were associated with increased risk in multivariable models. An intrinsic or induced mesenchymal state enhanced chemoresistance and migration in sarcoma cell lines. CONCLUSIONS: Although limited to a pilot study, these findings suggest that periostin might contribute prognostic information in the three studied STS histotypes. Moreover, a transitioning EMT score measured in the tumor might predict a less active and a more chemosensitive disease.

Circulating miRNA-375 as a potential novel biomarker for active Kaposi's sarcoma in AIDS patients.

The aim of this study was to identify circulating microRNAs (miRNAs) that could be used as biomarkers in patients at risk for or affected by AIDS-Kaposi's sarcoma (KS). Screening of 377 miRNAs was performed using low-density arrays in pooled plasma samples of 10 HIV/human herpesvirus 8 (HHV8)-infected asymptomatic and 10 AIDS-KS patients before and after successful combined antiretroviral therapy (cART). MiR-375 was identified as a potential marker of active KS, being the most down-regulated in AIDS-KS patients after cART and the most up-regulated in naïve AIDS-KS patients compared to naïve asymptomatic subjects. Validation on individual plasma samples confirmed that miR-375 levels were higher in AIDS-KS compared to asymptomatic patients, decreased after cART-induced remission in most AIDS-KS patients and increased in patients with active KS. In asymptomatic patients miR-375 was up-regulated after cART in both screening and validation. Statistical analyses revealed an association between miR-375 changes and CD4 cell counts, which could explain the discordant cases and the opposite trend between asymptomatic and AIDS-KS patients. These data suggest that circulating miR-375 might be a good indicator of active AIDS-KS. Moreover, changes in miR-375 levels may have a prognostic value in HIV/HHV8-infected patients undergoing treatment. Further large-scale validation is needed.

A Preclinical Model for the ATLL Lymphoma Subtype With Insights Into the Role of Microenvironment in HTLV-1-Mediated Lymphomagenesis.

Adult T cell Leukemia/Lymphoma (ATLL) is a mature T cell malignancy associated with Human T cell Leukemia Virus type 1 (HTLV-1) infection. Among its four main clinical subtypes, the prognosis of acute and lymphoma variants remains poor. The long latency (3-6 decades) and low incidence (3-5%) of ATLL imply the involvement of viral and host factors in full-blown malignancy. Despite multiple preclinical and clinical studies, the contribution of the stromal microenvironment in ATLL development is not yet completely unraveled. The aims of this study were to investigate the role of the host microenvironment, and specifically fibroblasts, in ATLL pathogenesis and to propose a murine model for the lymphoma subtype. Here we present evidence that the oncogenic capacity of HTLV-1-immortalized C91/PL cells is enhanced when they are xenotransplanted together with human foreskin fibroblasts (HFF) in immunocompromised BALB/c Rag2-/-γc-/- mice. Moreover, cell lines derived from a developed lymphoma and their subsequent in vivo passages acquired the stable property to induce aggressive T cell lymphomas. In particular, one of these cell lines, C91/III cells, consistently induced aggressive lymphomas also in NOD/SCID/IL2Rγc KO (NSG) mice. To dissect the mechanisms linked to this enhanced tumorigenic ability, we quantified 45 soluble factors released by these cell lines and found that 21 of them, mainly pro-inflammatory cytokines and chemokines, were significantly increased in C91/III cells compared to the parental C91/PL cells. Moreover, many of the increased factors were also released by human fibroblasts and belonged to the known secretory pattern of ATLL cells. C91/PL cells co-cultured with HFF showed features reminiscent of those observed in C91/III cells, including a similar secretory pattern and a more aggressive behavior in vivo. On the whole, our data provide evidence that fibroblasts, one of the major stromal components, might enhance tumorigenesis of HTLV-1-infected and immortalized T cells, thus throwing light on the role of microenvironment contribution in ATLL pathogenesis. We also propose that the lymphoma induced in NSG mice by injection with C91/III cells represents a new murine preclinical ATLL model that could be adopted to test novel therapeutic interventions for the aggressive lymphoma subtype.

Neck Kaposiform haemangioendothelioma in a Fischer's lovebird (Agapornis fischeri).

A six-year-old female Fischer's lovebird (Agapornis fischeri) presented at necropsy with a cutaneous mass on the neck, 3.5cm in diameter, yielding and with blood content. Histopathological findings showed a neoplasm characterized by proliferation of vascular endothelial cells. The histology of the mass revealed a multinodular, focally infiltrating tumor. Deeper dermal nodules were made of spindle cells forming vascular slits reminiscent of the histology seen in Kaposi's sarcoma (KS). More superficially located dermal nodules consisted of small blood vessels, with histology resembling capillary hemangioma. The spindle cells and capillaries were strongly positive for Vimentin, endothelial cell marker CD31, and negative for sarcomeric α-smooth muscle actin (α-SMA). Intravascular platelet trapping and Periodic acid-Schiff (PAS)-positive hyaline globules were also observed. Differential diagnosis included Kaposi's sarcoma, capillary haemangioma, spindle cell haemangioendothelioma, and epithelioid haemangioendothelioma. Based on morphological and immunohistochemical findings, the tumor was diagnosed as a cutaneous Kaposiform haemangioendothelioma (KHE), a rare, low-grade malignant vascular neoplasm. Other organs showed no abnormalities. PCR amplifications, conducted using Kaposi's sarcoma-associated herpesvirus (KSHV)-specific primers and degenerate sets of primers designed to detect and characterize members of the Herpesviridae, on DNA extracted from tumor tissue and from whole blood failed to amplify any KSHV-related sequence. Moreover, no specific signal was obtained using primers for detection of psittacine herpesvirus, known to be linked to Pacheco's disease in parrots. To the best of our knowledge, this unusual case is the third report of KHE in a non-human animal species, the first described in a bird.

Predictors of immune reconstitution inflammatory syndrome associated with Kaposi's sarcoma: a case report.

We present here a case of immune reconstitution inflammatory syndrome associated with Kaposi's sarcoma (KS-IRIS) developed in an AIDS patient two months after initiation of antiretroviral therapy (ART). Baseline characteristics of this IRIS-KS case, within a cohort of 12 naïve AIDS-KS patients, were analyzed. No statistically significant differences in CD4 cell counts, plasma HIV RNA load, KS clinical staging, human herpesvirus 8 (HHV8) antibody titers and HHV8 load in peripheral blood mononuclear cells and saliva were evidenced. HHV8 load in plasma was found to be significantly higher in the KS-IRIS patient (> 6 log10 genome equivalents/ml, p = 0.01, t-test) compared to the 11 patients with KS regression. This case highlights that measurement of HHV8 load in plasma may be useful to identify patients at risk for KS-IRIS, and that this parameter should be included in the design of larger studies to define KS-IRIS risk predictors.

Telomere shortening in mucosa surrounding the tumor: biosensor of field cancerization and prognostic marker of mucosal failure in head and neck squamous cell carcinoma.

OBJECTIVES: The aim of the present study was to investigate the pattern of telomere length and telomerase expression in cancer tissues and the surrounding mucosa (SM), as markers of field cancerization and clinical outcome in patients successfully treated for with head and neck squamous cell carcinoma (HNSCC). MATERIALS AND METHODS: This investigation was a prospective cohort study. Telomere length and levels of telomerase reverse transcriptase (TERT) transcripts were quantified by real-time PCR in cancer tissues and SM from 139 and 90 patients with HNSCC, respectively. RESULTS: No correlation was found between age and telomere length in SM. Patients with short telomeres in SM had a higher risk of mucosal failure (adjusted HR=4.29). Patients with high TERT levels in cancer tissues had a higher risk of regional failure (HR=2.88), distant failure (HR=7.27), worse disease-specific survival (HR for related death=2.62) but not mucosal failure. High-risk patients having both short telomeres in SM and high levels of TERT in cancer showed a significantly lower overall survival (HR=2.46). CONCLUSIONS: Overall these findings suggest that telomere shortening in SM is a marker of field cancerization and may precede reactivation of TERT. Short telomeres in SM are strongly prognostic of mucosal failure, whereas TERT levels in cancer tissues increase with the aggressiveness of the disease and are prognostic of tumor spread.