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Background Sentinel lymph node biopsy (SNB) still remains a key procedure to appropriately stage melanoma patients and to select those who are candidate to novel treatments with immunotherapy and targeted therapy in the adjuvant setting. The impact of timing of SNB on disease-free survival (DFS) and overall survival (OS) is still unclear. Material and methods The study was conducted at 6 Italian Melanoma Intergroup (IMI) centres and included 8953 consecutive clinical stage I-II melanoma patients who were diagnosed, treated, and followed up between November 1997 and March 2018. All patients were prospectively included in dedicated IMI database. Multivariable Cox regression analyses were performed to investigate how baseline characteristics and time interval until SNB are related to DFS and OS. Results Considering the whole population, at multivariable analysis, after adjusting for age, gender, Breslow thickness, site, ulceration, and the SNB status, a delay in the timing of SNB was associated with a better DFS (adjusted hazard ratio [aHR, delayed versus early SNB] 0.98, 95% confidence interval [CI] 0.97–0.99, p, Highlights • Sentinel lymph node biopsy (SNB) is a key procedure to stage melanoma patients (MPs). • In our series, the time interval of SNB is irrelevant in MPs with positive sentinel lymph node. • A delayed SNB biopsy is not detrimental in clinically stage I-II MPs.

Sentinel lymph node biopsy has been demonstrated to be an effective staging procedure since its introduction in 1992. The new American Joint Committee on Cancer (AJCC) classification did not consider the lack of information that would result from the less usage of the complete lymph node dissection as for a diagnostic purpose. Thus, this makes it difficult the correct staging and would leave about 20% of the further positive non-sentinel lymph nodes in the lymph node basin. In this paper, we aim to describe a new surgical technique that, combined with single-photon emission computed tomography-computed tomography (SPECT-CT), allows for better staging of melanoma patients. This is a prospective study that includes 104 patients with cutaneous melanoma. Sentinel lymph node biopsy was offered according to the AJCC guideline. Planar lymphoscintigraphy was performed in association with SPECT-CT, identifying and removing all non-biologically “excluded” lymph nodes, guiding the surgeon’s hand in detection and removal of lymph nodes. Even if identification and removal of non-sentinel lymph nodes is unable to increase overall survival, it definitely gives better disease control in the basin. With a “classic” setting, the risk of leaving further lymph nodes out of the sentinel lymph node procedure is around 20%, thus, basically, the surgical sentinel lymph node of first and second lymph nodes would have therapeutic value and complete lymph node dissection classically performed.

Human T-cell lymphotropic virus type 1 (HTLV-1) is a retrovirus endemic in many parts of the world. Because of migration, cases of HTLV-1 in non-HTLV-1 endemic countries have been increasingly reported. Clinical presentation of HTLV-1 infection is highly variable, with a significant risk of diagnostic delays. Skin can be the first site affected by HTLV-1-related manifestations such as cutaneous involvement of adult T-cell leukemia/lymphoma (ATLL) and infective dermatitis associated with HTLV-1. A 32-year-old Nigerian man was admitted to the infectious disease department for high fever, asthenia, lymphocytosis, and vesicular bullous lesions on both hand palms and lower limbs. After clinical work-up was performed, bacterial superinfected herpes simplex viurs-2 ulcers were the presenting sign of HTLV-1-related chronic type ATLL. Standard treatment based on interferon-α plus zidovudine was started, but it was poorly tolerated; therefore, switching to an off-label dual antiretroviral regimen was attempted. The increasing prevalence of HTLV-1 in nonendemic areas may enhance the development of alternative treatments with better efficacy and tolerability profiles.

Cancers of unknown primary (CUPs), featuring metastatic dissemination in the absence of a primary tumor, are a biological enigma and a fatal disease. We propose that CUPs are a distinct, yet unrecognized, pathological entity originating from stem-like cells endowed with peculiar and shared properties. These cells can be isolated in vitro (agnospheres) and propagated in vivo by serial transplantation, displaying high tumorigenicity. After subcutaneous engraftment, agnospheres recapitulate the CUP phenotype, by spontaneously and quickly disseminating, and forming widespread established metastases. Regardless of different genetic backgrounds, agnospheres invariably display cell-autonomous proliferation and self-renewal, mostly relying on unrestrained activation of the MAP kinase/MYC axis, which confers sensitivity to MEK inhibitors in vitro and in vivo. Such sensitivity is associated with a transcriptomic signature predicting that more than 70% of CUP patients could be eligible to MEK inhibition. These data shed light on CUP biology and unveil an opportunity for therapeutic intervention., Cancer of unknown primary (CUP) is a mysterious malignancy featuring metastatic dissemination in the absence of a recognizable primary tumor. By characterizing CUP cancer stem cells we show that self-sustained long-term propagation and sensitivity to MEK inhibition are CUP common features.

Sezary syndrome (SS) is a primary cutaneous T-cell lymphoma characterized by measurable levels of malignant lymphocytes in the blood, and progressive impairment of the immune response. Among the mechanisms of immune modulation, adenosine can impair anti-tumor immunity, through the attenuation of protective effector cells, including T and NK cells, and by enhancing the suppressive capacity of T regulatory cells. CD39 and CD73 nucleotide-metabolizing enzymes are involved in the adenosine-generating pathway: CD39 cleaves ATP and ADP down into AMP, which is converted into adenosine by CD73. The extracellular adenosine deaminase/CD26 complex catalyzes the deamination of adenosine to inosine, thus reducing the adenosine levels. The objective of this study were: to investigate the expression of the of CD39 and CD73 nucleotide-metabolizing ectoenzymes in peripheral blood from SS patients; to define the contribution of the CD39/CD73 adenosinergic immunosuppressive pathway to tumor escape from immune response and immune dysfunctions in patients with SS. CD39 and CD73 expression analyzed in whole blood with multiparametric flow-cytometry from SS patients (n=10) and healthy donors (HD, n=11) revealed altered expression of CD39 and CD73 in SS patients compared to HD. Two subgroups of patients can be identified based on the mutually exclusive overexpression of CD39 or CD73 in CD4+ T cells. Indeed, 7 out of 10 patients had high CD39, while 3 had high CD73. CD4+T cells with high CD39 have low CD73, vice versa those with high CD73 have low CD39. To define whether CD39 and CD73 were biologically active, we compared the ability of peripheral blood CD4+T cells from SS patients and HD to hydrolyze ATP and to convert AMP into adenosine, in vitro. Briefly, CD4+ Tcells from SS/HD were seeded in 48-well plates in HBSS, pretreated or not with specific inhibitors for 1h and then incubated with exogenous (e) eATP (patients with high CD39) or eAMP (patients with high CD73) at 37°C. After 1 h incubation, analyses of the supernatant were performed with an RP-HPLC. Results indicated that CD4+ T cells from SS patients with high levels of CD39 showed an increased ability to hydrolyze ATP with increased generation of AMP compared to normal control cells. In parallel, CD4+ T cells with high levels of CD73 showed increased conversion of AMP into ADO, respect to normal control cells. The aberrant expression of CD39 and CD73 along with loss of CD26 expression in circulating Sezary cells suggest that the sequential activity of CD39 and CD73 ectoenzymes scavenges ATP and generates immunosuppressive adenosine in the tumor microenvironment contributing to tumor immune escape. The results inferred from this study are the starting point for more comprehensive studies towards the development of new therapies targeting the CD39/CD73 adenosinergic axis in order to overcome tumor immunosuppression, allowing the induction of effective anti-tumor immune response.

Background Many oncologists debate if lobular neoplasia (LN) is a risk factor or an obligatory precursor of more aggressive disease. This study has three aims: (i) describe the different treatment options (surgical resection vs observation), (ii) investigate the upgrade rate in surgically treated patients, and (iii) evaluate the long-term occurrences of aggressive disease in both operated and unoperated patients. Methods A series of 122 patients with LN bioptic diagnosis and follow-up information were selected. Clinical, radiological, and pathological data were collected from medical charts. At definitive histology, either invasive or ductal carcinoma in situ was considered upgraded lesions. Results Atypical lobular hyperplasia (ALH), lobular carcinoma in situ (LCIS), and high-grade LN (HG-LN) were diagnosed in 44, 63, and 15 patients, respectively. The median follow-up was 9.5 years. Ninety-nine patients were surgically treated, while 23 underwent clinical-radiological follow-up. An upgrade was observed in 28/99 (28.3%). Age ≥ 54 years (OR 4.01, CI 1.42–11.29, p = 0.009), Breast Imaging-Reporting and Data System (BI-RADS) categories 4–5 (OR 3.76, CI 1.37–10.1, p = 0.010), and preoperatory HG-LN diagnosis (OR 8.76, 1.82–42.27, p = 0.007) were related to upgraded/aggressive disease. During follow-up, 8 patients developed an ipsilateral malignant lesion, four of whom were not initially operated (4/23, 17%). Conclusions BI-RADS categories 4–5, HG-LN diagnosis, and age ≥ 54 years were features associated with an upgrade at definitive surgery. Moreover, 17% of unoperated cases developed an aggressive disease, emphasizing that LN patients need close surveillance due to the long-term risk of breast cancer.

Naevi typically involute after the fourth decade of life and are rare in the elderly. However, the rate at which naevi disappear with age varies greatly with some individuals still having large numbers of naevi in late middle age. A small subset of compound naevi in adolescence exhibits a peripheral rim of small brown clods. These naevi enlarge symmetrically with time, accompanied by a progressive development of reticular pattern until the disappearance of the peripheral clods that indicates their growth stabilization. On the other hand, a melanocytic lesion that shows a growth after the age of 30 should raise the doubt of being a melanoma and should thereafter be considered as candidate for excision. In a clinical setting, common behaviour is to excise a melanocytic lesion that shows growing parameters (like a peripheral rim of clods) after 35 years old.

Cancer of unknown primary (CUP) is an obscure disease characterized by multiple metastases in the absence of a primary tumor. No consensus has been reached whether CUPs are simply generated from cancers that cannot be detected or whether they are the manifestation of a still unknown nosological entity. Here, we report the complete expression and genetic analysis of multiple synchronous metastases harvested at warm autopsy of a patient with CUP. The expression profiles were remarkably similar and astonishingly singular. The whole exome analysis yielded a high number of mutations present in all metastases (fully shared), additional mutations (partially shared) accumulated one after another in a series, and few private mutations were unique to each metastasis. Surprisingly, the phylogenetic trajectory linking CUP metastases was atypical, depicting a common “stream”, sprouting a series of linear “brooks”, at variance from the extensive branched evolution observed in metastases from most cancers of known origin. The distinctive genetic and evolutionary features depicted suggest that CUP is a novel nosological entity., Cancer of unknown primary (CUP) is a heterogeneous clinical syndrome characterized by metastases without primary tumor, an elusive biology and lack of effective treatments. We describe the genetic and evolutionary features of 15 synchronous and spatially distinct metastases from a CUP patient.

Specific subsets of T-helper lymphocytes have been identified as associated with anergy and impairment of CD8+ immune response against cancer cells. In particular, the immune infiltrate of regressed melanoma has been proven to have lower counts of CD25+/CD4+, FOXP3+/CD4+ and PD1+/CD4+ lymphocytes compared to the non-regressed ones.

The presence of ulceration has been recognized as an adverse prognostic factor in primary cutaneous melanoma (PCM).To investigate whether the extent of ulceration (EoU) predicts relapse-free survival (RFS) and overall survival (OS) in PCM.We retrieved data for 477 patients with ulcerated PCM from databases of the Italian Melanoma Intergroup. Univariate and multivariable Cox proportional hazard models were used to assess the independent prognostic impact of EoU.A significant interaction emerged between Breslow thickness (BT) and EoU, considering both RFS (P0·0001) and OS (P = 0·0006). At multivariable analysis, a significant negative impact of EoU on RFS [hazard ratio (HR) (1-mm increase) 1·26, 95% confidence interval (CI) 1·08-1·48, P = 0·0047] and OS [HR (1-mm increase) 1·25, 95% CI 1·05-1·48, P = 0·0120] was found in patients with BT ≤ 2 mm, after adjusting for BT, age, tumour-infiltrating lymphocytes, sentinel lymph node status and mitotic rate. No impact of EoU was found in patients with 2·01-4 mm and4 mm BT.This study demonstrates that EoU has an independent prognostic impact in PCM and should be recorded as a required element in pathology reports.

Background Central nervous system (CNS) spreading from epithelial ovarian carcinoma (EOC) is an uncommon but increasing phenomenon. We previously reported in a small series of 11 patients a correlation between Androgen Receptor (AR) loss and localization to CNS. Aims of this study were: to confirm a predictive role of AR loss in an independent validation cohort; to evaluate if AR status impacts on EOC survival. Results We collected an additional 29 cases and 19 controls as validation cohort. In this independent cohort at univariate analysis, cases exhibited lower expression of AR, considered both as continuous (p p p p = 0.005 for Progression Free Survival (PFS) and p = 0.002 for brain PFS (bPFS) respectively). Comparison of AR expression between primary tissue and paired brain metastases in the combined dataset did not show any statistically significant difference. Conclusions We confirmed AR loss as predictive role for CNS involvement from EOC in an independent cohort of cases and controls. Early assessment of AR status could improve clinical management and patients’ prognosis.

The neurotrophic tropomyosin receptor kinase (NTRK) genes (NTRK1, NTRK2, and NTRK3) code for three transmembrane high-affinity tyrosine-kinase receptors for nerve growth factors (TRK-A, TRK-B, and TRK-C) which are mainly involved in nervous system development. Loss of function alterations in these genes can lead to nervous system development problems; conversely, activating alterations harbor oncogenic potential, promoting cell proliferation/survival and tumorigenesis. Chromosomal rearrangements are the most clinically relevant alterations of pathological NTRK activation, leading to constitutionally active chimeric receptors. NTRK fusions have been detected with extremely variable frequencies in many pediatric and adult cancer types, including central nervous system (CNS) tumors. These alterations can be detected by different laboratory assays (e.g., immunohistochemistry, FISH, sequencing), but each of these approaches has specific advantages and limitations which must be taken into account for an appropriate use in diagnostics or research. Moreover, therapeutic targeting of this molecular marker recently showed extreme efficacy. Considering the overall lack of effective treatments for brain neoplasms, it is expected that detection of NTRK fusions will soon become a mainstay in the diagnostic assessment of CNS tumors, and thus in-depth knowledge regarding this topic is warranted.

Breslow thickness (BT) is the most important histopathologic factor for primary melanoma staging. BT determines the margins for wide local excision whether sentinel lymph node biopsy should be performed and subsequent melanoma staging, and patient management. The correct determination of a 0.8-mm cutoff in melanoma is important for pathologists because discrepancies leading to a change in stage can have significant clinical implications, including incorrect and/or inappropriate prognostic information, investigation, management, and follow-up. Difficulties in BT determination are mostly represented by the presence of regression or melanoma associated with a pre-existing nevus. This study aimed at investigating a molecular parameter, namely microRNA (miRNA) expression, in reference to BT assessment. Melanoma cell proliferation is influenced by miRNA dysregulation. Indeed, some miRNAs sustain and induce proliferative signals or repress growth-suppressive pathways, thereby promoting melanoma carcinogenesis. To identify the miRNAs correlating with BT, we analyzed our global miRNA expression data of 20 thin melanomas and identified two potential candidates, miR-21-5p and miR-146a-5p. We assessed the expression of these two specific miRNAs in 90 archive formalin-fixed and paraffin-embedded samples of superficially spreading melanomas (SSMs) and 25 nodular melanomas from two independent cohorts and correlated the individual and combined miRNA expression with BT and other tumor characteristics. The individually normalized expression of miR-21-5p and miR-146a-5p showed a highly significant and linear correlation with BT in SSM, and their combined expression value was more strongly correlated (Pearson’s r = 0.799, 95% CI = 0.71–0.86) than their individual expressions. This correlation was not significant in nodular melanoma. In SSM, we observed that the combined miRNA expression above or below 1.5 was significantly associated with overall survival and successfully identified all patients with relapsing SSM. We concluded that the combined assessment of miR-21-5p and miR-146a-5p expression in superficially spreading melanoma, in association with BT measurement, could aid pathologists in SSM staging.

Background: Dp71 is the most abundant dystrophin (DMD) gene product in the nervous system. Mutation in the Dp71 coding region is associated with cognitive disturbances in Duchenne muscular dystrophy (DMD) patients, but the function of dystrophin Dp71 in tumor progression remains to be established. This study investigated Dp71 expression in glioblastoma, the most common and aggressive primary tumor of the central nervous system (CNS). Methods: Dp71 expression was analyzed by immunofluorescence, immunohistochemistry, RT-PCR, and immunoblotting in glioblastoma cell lines and cells isolated from human glioblastoma multiforme (GBM) bioptic specimens. Results: Dp71 isoform was expressed in normal human astrocytes (NHA) cell lines and decreased in glioblastoma cell lines and cells isolated from human glioblastoma multiforme bioptic specimens. Moreover, Dp71 was localized in the nucleus in normal cells, while it was localized into the cytoplasm of glioblastoma cells organized in clusters. We have shown, by double labeling, that Dp71 colocalizes with lamin B in normal astrocytes cells, confirming the roles of Dp71 and lamin B in maintaining nuclear architecture. Finally, we demonstrated that decreased Dp71 protein in cells isolated from human bioptic specimens was inversely correlated with the Ki-67 tumor proliferative index. Conclusion: A decreased Dp71 expression is associated with cancer proliferation and poor prognosis in glioblastoma.

Rectal carcinoma, representing about a third of all newly diagnosed colorectal cancers, is one of the most common malignant tumors. The standard of care for locally advanced rectal cancer (LARC), consisting of neoadjuvant chemo/radiotherapy prior to surgical resection, is poorly effective, leading to complete tumor regression only in 10-30% of the cases. Approximately 40% of LARC harbor activating KRAS mutations, which have been extensively associated with primary resistance to targeted EGFR therapy and with radioresistance as well. Previous work showed that the MET receptor tyrosine kinase supports radioresistance and can be inhibited to radiosensitize tumor cell subpopulations retaining stem-like properties. Here, we show that LARC often express high levels of MET and can be successfully radiosensitized by MET inhibition. This was assessed in rectal stem-like cells isolated from human tumors (rectospheres), and transplanted in the mouse to regenerate tumors that faithfully reproduce the phenotype, the genotype and the therapeutic response of the original tumor. Mechanistically, we found that radioresistant KRAS-mutant rectospheres display significantly higher basal levels of RAD51, a master regulator of DNA homologous recombination repair, and increased RAD51 recruitment to irradiation-induced DNA double-strand breaks, as compared with KRAS-wild type rectospheres. Importantly, we showed that MET pharmacological inhibition by small-molecule kinase inhibitors, combined with radiotherapy, impairs RAD51 expression and function. This leads to DNA damage accumulation and results in effective radiosensitization of K-RAS-mutant rectal stem-like cells in vitro and in vivo, and significant inhibition of experimental tumors. Therefore, preclinical evidence is provided that MET can be exploited as a therapeutic target to radiosensitize KRAS-mutant rectal cancer at stem-like cell level. Citation Format: Antonio D'Ambrosio, Federica Verginelli, Francesca Orzan, Raffaella Albano, Elena Casanova, Paolo Luraghi, Francesca De Bacco, Andrea Bertotti, Livio Trusolino, Rebecca Senetta, Anna Sapino, Elena Del Mastro, Marco Gatti, Paolo M. Comoglio, Carla Boccaccio. MET inhibition radiosensitizes KRAS-mutant rectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1387.

Cancers of Unknown Primary (CUPs), featuring metastatic dissemination in the absence of a primary tumor, anatomically or histologically recognizable through a standardized work-up, are a fatal disease and still a biological enigma. Here, we propose CUPs as a distinct, yet unrecognized, pathological entity originating from stem-like cells endowed with unique properties, irrespective of their different genetic backgrounds. These cells were isolated and long-term propagated in vitro in highly stringent conditions as ‘agnospheres', and serially transplanted in vivo, displaying an extremely high tumorigenic potential and reproducing the undifferentiated histology of the original tumors. Early after subcutaneous engraftment, agnospheres recapitulated the CUP clinical presentation, as they spontaneously and quickly disseminated, establishing widespread metastases and retracing the whole metastatic cascade. Agnospheres invariably displayed cell-autonomous proliferation and self-renewal, mostly relying on unrestrained activation of the MAP kinase/MYC axis. This feature conferred sensitivity to MEK inhibitors, which induced apoptosis in vitro and impaired in vivo growth and dissemination. We generated and validated a transcriptional signature that, applied to original tumors, predicts eligibility to MEK inhibition in 75% of CUP patients. Altogether, these findings shed light on CUP biology, unveiling an opportunity for a targeted therapeutic intervention and, concomitantly, provide a novel in vivo model, suitable for assessing molecular determinants of the metastatic cascade. Citation Format: Federica Verginelli, Alberto Pisacane, Gennaro Gambardella, Antonio D'Ambrosio, Ermes Candiello, Marco Ferrio, Mara Panero, Laura Casorzo, Silvia Benvenuti, Eliano Cascardi, Rebecca Senetta, Elena Geuna, Andrea Ballabio, Filippo Montemurro, Anna Sapino, Paolo M. Comoglio, Carla Boccaccio. Hypermetastatic stem-like cells from 'cancer of unknown primary' (CUP) model multi-organ dissemination and unveil liability to MEK inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2871.

Sentinel lymph node biopsy (SNB) still remains a key procedure to appropriately stage melanoma patients and to select those who are candidate to novel treatments with immunotherapy and targeted therapy in the adjuvant setting. The impact of timing of SNB on disease-free survival (DFS) and overall survival (OS) is still unclear.The study was conducted at 6 Italian Melanoma Intergroup (IMI) centres and included 8953 consecutive clinical stage I-II melanoma patients who were diagnosed, treated, and followed up between November 1997 and March 2018. All patients were prospectively included in dedicated IMI database. Multivariable Cox regression analyses were performed to investigate how baseline characteristics and time interval until SNB are related to DFS and OS.Considering the whole population, at multivariable analysis, after adjusting for age, gender, Breslow thickness, site, ulceration, and the SNB status, a delay in the timing of SNB was associated with a better DFS (adjusted hazard ratio [aHR, delayed versus early SNB] 0.98, 95% confidence interval [CI] 0.97-0.99, p 0.001) and OS (aHR 0.98, 95% CI 0.97-0.99, p = 0.001). Specifically, in patients with a negative SNB status, a beneficial impact of delayed SNB (i.e. at least 32 days after primary excision) was confirmed for DFS (aHR 0.70, 95%CI 0.63-0.79, p 0.001) and OS (aHR 0.69, 95%CI 0.61-0.78, p 0.001), whereas in those with a positive SNB status, DFS (aHR 0.96, 95%CI 0.84-1.09, p = 0.534) and OS (aHR 0.94 95%CI 0.81-1.08, p = 0.374) were not significantly different in patients with early or delayed SNB.Our study does not support a strict time interval for SNB. These results may be useful for national guidelines, for counselling patients and reducing the number of high urgency referrals.

Melanoma is one of the most aggressive skin cancers. The 5-year survival rate of stage III melanoma patients ranges from 93% (IIIA) to 32% (IIID) with a high risk of recurrence after complete surgery. The introduction of target and immune therapies has dramatically improved the overall survival, but the identification of patients with a high risk of relapse who will benefit from adjuvant therapy and the determination of the best treatment choice remain crucial. Currently, patient prognosis is based on clinico-pathological features, highlighting the urgent need of predictive and prognostic markers to improve patient management. In recent years, many groups have focused their attention on identifying molecular biomarkers with prognostic and predictive potential. In this review, we examined the main candidate biomarkers reported in the literature.

Purpose: The MHC-unrestricted activity of cytokine-induced killer (CIK) cells against chemo-surviving melanoma cancer stem cells (mCSC) was explored, as CSCs are considered responsible for chemoresistance and relapses. Experimental Design: Putative mCSCs were visualized by engineering patient-derived melanoma cells (MC) with a lentiviral vector encoding eGFP under expression control by stemness gene promoter oct4. Their stemness potential was confirmed in vivo by limiting dilution assays. We explored the sensitivity of eGFP+ mCSCs to chemotherapy (CHT), BRAF inhibitor (BRAFi) or CIK cells, as single agents or in sequence, in vitro. First, we treated MCs in vitro with fotemustine or dabrafenib (BRAF-mutated cases); then, surviving MCs, enriched in mCSCs, were challenged with autologous CIK cells. CIK cell activity against chemoresistant mCSCs was confirmed in vivo in two distinct immunodeficient murine models. Results: We visualized eGFP+ mCSCs (14% ± 2.1%) in 11 MCs. The tumorigenic precursor rate in vivo was higher within eGFP+ MCs (1/42) compared with the eGFP− counterpart (1/4,870). In vitro mCSCs were relatively resistant to CHT and BRAFi, but killed by CIK cells (n = 11, 8/11 autologous), with specific lysis ranging from 95% [effector:tumor ratio (E:T), 40:1] to 20% (E:T 1:3). In vivo infusion of autologous CIK cells into mice bearing xenografts from three distinct melanomas demonstrated significant tumor responses involving CHT-spared eGFP+ mCSCs (P = 0.001). Sequential CHT–immunotherapy treatment retained antitumor activity (n = 12, P = 0.001) reducing mCSC rates (P = 0.01). Conclusions: These findings are the first demonstration that immunotherapy with CIK cells is active against autologous mCSCs surviving CHT or BRAFi. An experimental platform for mCSC study and rationale for CIK cells in melanoma clinical study is provided. Clin Cancer Res; 23(9); 2277–88. ©2016 AACR.

// Gloria Mittica 1, 2, * , Rebecca Senetta 3, * , Giulia Scotto 1 , Massimo Aglietta 1, 2 , Furio Maggiorotto 4 , Eleonora Ghisoni 1, 2 , Sofia Genta 1, 2 , Renzo Boldorini 5 , Claudia Manini 6 , Isabella Morra 7 , Roberta Buosi 8 , Anna Sapino 3, 9 , Paola Cassoni 9, * and Giorgio Valabrega 1, 2, * 1 Department of Oncology, University of Turin, Turin, Italy 2 Division of Medical Oncology-1, Candiolo Cancer Institute-FPO- IRCCS, Candiolo, Italy 3 Unit of Pathology Candiolo Cancer Institute-FPO- IRCCS, Candiolo, Italy 4 Division of Gynecologic Oncology, Candiolo Cancer Institute-FPO- IRCCS, Candiolo, Italy 5 Department of Health Science, University of Eastern Piedmont “Amedeo Avogadro”, Novara, Italy 6 Unit of Pathology, Giovanni Bosco Hospital, Turin, Italy 7 Unit of Pathology, Citta della Salute e della Scienza, Turin, Italy 8 Division of Oncology, Santo Spirito Hospital, Casale Monferrato, Italy 9 Department of Medical Sciences, University of Turin, Turin, Italy * These authors have contributed equally to this work Correspondence to: Giorgio Valabrega, email: giorgio.valabrega@ircc.it Keywords: androgen receptor, ovarian cancer, brain metastases Received: November 24, 2016 Accepted: March 17, 2017 Published: April 12, 2017 ABSTRACT Brain metastases are uncommon localizations in epithelial ovarian cancer (EOC), their reported incidence is increasing and no predictive biomarkers have been identified yet. Goals of this study were: i) to define a possible association between Estrogen Receptor (ER), Progesterone Receptor (PR), Androgen Receptor (AR),human EGF receptor 2 (HER2) and brain progression in EOC patients, and ii) to identify differences in ER, PR, AR and HER2 protein expression from primary EOC and its matched resected brain metastasis. A retrospective series of 11 EOC with matched brain metastasis surgically removed was collected. For comparison, a “Control dataset” of 22 patients, without evidence of brain involvement after an adequate follow up was matched. ER, PR, AR and HER2 status were analyzed by means of immunohistochemistry forCases (both primary and metastatic lesions) and Controls. Univariate analysis showed that AR status was significantly associated with brain localization, both considered as discrete variable (cut-off: 10%, p =0.013) and as continuous one ( p =0.035). Multivariate analysis confirmed this trend ( p =0.053). When considered as continuous variables, ER and AR showed greater expression in primary tumors in comparison with brain metastases ( p =0.013 and p =0.032, respectively). In our series, AR predicts brain involvement, with a 9.5 times higher propensity for AR-negative EOC. Moreover, brain dissemination is probably the result of progressive dedifferentiation of primary tumor, shown by reduction of ER and AR expression in metastases. Further studies are required, in order to anticipate and improve multimodal treatment of brain metastases.

// Simona Ruggieri 1 , Roberto Tamma 1, 5 , Nicoletta Resta 2 , Francesco Albano 3 , Nicoletta Coccaro 3 , Daria Loconte 2 , Tiziana Annese 1 , Mariella Errede 1 , Giorgina Specchia 3 , Rebecca Senetta 4 , Paola Cassoni 4 , Domenico Ribatti 1, 5 , Beatrice Nico 1 1 Department of Basic Medical Sciences, Neurosciences and Sensory Organs, Section of Human Anatomy and Histology, University of Bari Medical School, Bari, Italy 2 Division of Medical Genetics, Department of Biomedical Sciences and Human Oncology (DIMO), University of Bari Medical School, Bari, Italy 3 Department of Emergency and Transplantation, Section of Hematology, University of Bari Medical School, Bari, Italy 4 Department of Biomedical Sciences and Human Oncology, University of Turin Medical School, Turin, Italy 5 National Cancer Institute “Giovanni Paolo II”, Bari, Italy Correspondence to: Beatrice Nico, email: beatrice.nico@uniba.it Domenico Ribatti, email: domenico.ribatti@uniba.it Keywords: angiogenesis, cancer stem cells, endothelium, lymphoma, Stat3 Received: January 12, 2017 Accepted: March 01, 2017 Published: March 10, 2017 ABSTRACT With the aim of elucidating the relationship between Stat3 expression and tumor vessels abnormalities in the PCNLs, in this study we evaluated Stat3 and pStat3 expression by Real-time PCR and by immunohistochemistry in biopsy sections from PCNSL patients. Correlations of the expression levels with the presence of aberrant vessels were analyzed by confocal laser microscopy analysis, using FVIII as endothelial cell marker, CD133 and nestin as cancer stem cell (CSC) marker, CD20 as tumor cell marker, and Stat3. In addition, we investigated Stat3 mutations in lymphoma cells to clarify the role of the constitutive expression of Stat3 and of its phosphorylated forms. Results showed that in PCNSL, putative endothelial cells lining the vessels are heterogeneous, expressing FVIII/ pStat3/CD133 (presumably originally they are vascular progenitor cells), as well as FVIII/CD20/CD133 (presumably originally they are tumor cells). Finally, we detected a fraction of the FVIII + endothelial cell that co-expressed Stat3 bearing a tetraploid karyotype, while no amplification signal for the Stat3 gene was detected.

The diagnosis of 206 low and high grade adult gliomas, including 40 oligoastrocytomas, was revised based on the immunohistochemical reactivity for the ATRX protein, IDH1/2 mutation status and 1p/19q chromosomal status. All oligodendrogliomas kept the initial diagnosis. Astrocytomas did not change diagnosis in 30 of 36 cases (83.3 %); four of 36 (11.1 %) cases were reclassified as oligodendroglioma, one (2.8 %) as DNT and the other (2.8 %) as reactive gliosis. Oligoastrocytomas changed diagnosis in 35 of 40 (87.5 %) cases, being reclassified 22 of 40 (55 %) as astrocytoma, 11 of 40 (27.5 %) as oligodendroglioma and two of 40 (5 %) as reactive gliosis. Four (10 %) remained unclassifiable. In one case only (2.5 %), the diagnosis of oligoastrocytoma could not be excluded since tumor astrocytes and tumor oligodendrocytes coexisted in mixed tumor areas. In the GBM tumor subgroup, GBMO disappeared because they were not substantiated by molecular genetics. Pilocytic astrocytomas retained ATRX expression. Loss of nuclear ATRX protein expression was strongly associated to IDH1/2 mutations (p = 0.0001) and mutually exclusive with total 1p/19q co-deletion (p = 0.0001). In astrocytic tumors, loss of immunoreactivity for the ATRX protein was significantly associated to the ALT phenotype (p = 0.0003). The constitutive ATRX expression in microglia/macrophages may be misleading, especially in the identification of an oligodendroglial tumor infiltration. Of paramount importance in the recognition of oligodendroglial and astrocytic tumor cells were the double immunostainings for ATRX/GFAP, ATRX/IDH1R132H, ATRX/Iba-1 and ATRX/CD68.

Histological regression and tumour infiltrating lymphocytes represent an early sign of activation of the immune system against primary melanoma. The first phenomenon has been especially discussed in the literature because of its prognostic role, but no clear agreement on its evaluation has been reached. Immunotherapy of advanced stage melanoma has recently shown promising results; an improved understanding of the initial interplay between melanoma cells and the immune system would potentially help tailor treatment for patients. Seventy consecutive melanomas with regression were analysed to identify a prognostic cut-off value of regression extension. Then, we compared the immune infiltrate between regressed and not regressed areas of these regressed melanomas, assessing CD3, CD4, CD8, CD20, CD123, PD1 and FOXP3/CD25 expression. The immune infiltrate of these cases was further compared with 28 control melanomas without regression. A regression extension of 10% represented a reliable cut-off to distinguish two different risk categories in regressed melanomas. Regressed areas were less infiltrated by CD4/CD25, FOXP3/CD4 or PD1/CD4 compared to not regressed areas of each sample. These lymphocyte subsets are associated with anergy and hamper the immune CD8+ response towards the cancer cells. Moreover, the relevance of these findings was further supported by the observation that not regressed controls were significantly more infiltrated by these anergic immune cell subsets compared to the regressed cases. These results help understand the real meaning of regression in melanoma. Moreover, the association here identified between specific immunomodulatory immune cell subsets and regression could help in developing new therapeutic strategies.

Despite several molecular signatures for “lower grade diffuse gliomas” (LGG) have been identified, WHO grade still remains a cornerstone of treatment guidelines. Mitotic count bears a crucial role in its definition, although limited by the poor reproducibility of standard Hematoxylin & Eosin (H&E) evaluation. Phospho-histone-H3 (PHH3) and Ki-67 have been proposed as alternative assays of cellular proliferation. Therefore in the present series of 141 LGG, the molecular characterization (namely IDH status, 1p/19q co-deletion and MGMT promoter methylation) was integrated with the tumor “proliferative trait” (conventional H&E or PHH3-guided mitotic count and Ki-67 index) in term of prognosis definition. Exclusively high PHH3 and Ki-67 values were predictor of poor prognosis (log rank test, P = 0.0281 for PHH3 and P = 0.032 for Ki-67), unlike standard mitotic count. Based on Cox proportional hazard regression analyses, among all clinical (age), pathological (PHH3 and Ki-67) and molecular variables (IDH, 1p/19q codeletion and MGMT methylation) with a prognostic relevance at univariate survival analysis, only IDH expression (P = 0.001) and Ki-67 proliferation index (P = 0.027) proved to be independent prognostic factors. In addition, stratifying by IDH expression status, high Ki-67 retained its prognostic relevance uniquely in the IDH negative patient (P = 0.029) doubling their risk of death (hazard ratio = 2.27). Overall, PHH3 immunostaining is the sole reliable method with a prognostic value to highlight mitotic figures in LGG. Ki-67 proliferation index exceeds PHH3 mitotic count as a predictor of patient's prognosis, and should be integrated with molecular markers in a comprehensive grading system for LGG.

Aims Granular-cell astrocytomas (GCAs) are morphologically characterized by a prominent component of granular periodic acid-Schiff-positive cells, and show increased aggressiveness as compared with 'ordinary' astrocytomas. The aim of this study was to investigate, in a small series of three GCAs, the expression of mesenchymal/radioresistance-associated biomarkers [such as chitinase-3-like protein 1 (YKL-40), hepatocyte growth factor receptor (c-Met), and caveolin 1 (Cav1)] that could contribute to the poor outcome associated with this glioma subgroup. Methods and results Our results show that GCAs, according to the new molecular glioma classifications, consistently show a prognostically negative molecular trait (IDH1wt-ATRX noloss-1p/19q nocodeletion). Furthermore, GCAs significantly differed from a control series of 33 'conventional' astrocytomas, because of diffuse and strong immunohistochemical coexpression of YKL-40, c-Met, and Cav1. Conclusions Our findings show that specific morphological traits, such as a granular-cell component, could represent useful features in guiding the search for prognostic and predictive biomarkers that could eventually be therapy-targetable (e.g. Met inhibitors aimed at reducing radioresistance).

Brain metastases develop in one-third of patients with non-small-cell lung cancer and are associated with a dismal prognosis, irrespective of surgery or chemo-radiotherapy. Pathological markers for predicting outcomes after surgical resection and radiotherapy responsiveness are still lacking. Caveolin 1 has been associated with chemo- and radioresistance in various tumors, including non-small-cell lung cancer. Here, caveolin 1 expression was assessed in a series of 69 brain metastases from non-small-cell lung cancer and matched primary tumors to determine its role in predicting survival and radiotherapy responsiveness. Only caveolin 1 expression in brain metastasis was associated with poor prognosis and an increased risk of death (log rank test, p = 0.015). Moreover, in the younger patients (median age of

Bcl-6 translocation is a genetic alteration that is commonly detected in Primary Central Nervous System Lymphoma. The role of this protein in cerebral tumors is unclear. In this study we investigated Bcl-6 translocation and its transcriptional and translational levels in formalin-fixed, paraffin-embedded cerebral tissue sections from glioblastoma (GBM), low-grade glioma (Astrocytoma grade II and III), and meningioma patients, and correlated them with apoptotic processes and p53 and caspase-3 expression. The results showed a frequency of 36.6% of Bcl-6 translocation in GBM patients and a decreased expression in low-grade glioma patients, correlated with the severity of the disease. Bcl-6 translocation induced an overexpression of both Bcl-6 protein and messenger in GBM, inhibiting apoptotic processes and caspases 3 expression. On the contrary, in low-grade gliomas and meningiomas Bcl-6 expression was reduced, resulting in an increase of apoptotic processes. Finally, p53 expression levels in brain tumors were comparable to Bcl-6 levels. Overall, these data demonstrate, for the first time, that the Bcl-6 gene translocates in GBM patients and that its translocation and expression are correlated with apoptosis inhibition, indicating a key role for this gene in the control of cellular proliferation. This study offers further insights into glioblastoma biology, and supports Bcl-6 as a new diagnostic marker to evaluate the disease severity.

Receptor for the scatter factor HGF MET is a key player in invasive growth, a genetic program which is implicated in the metastatic process. MET mutations affecting brain secondary lesions from NSCLC are significantly associated to radioresistance and poor outcome [[i]]. All the mutations found, affected either the SEMA domain of the receptor or its tyrosine kinase (TK) domain. We here focused on the nucleotide changes identified, namely E168D (SEMA), and V1312I (TK) mutations. Their oncogenic potential was investigated by reproducing the mutations in cDNAs and expressing the mutated MET proteins in T47D cells, as already published [[ii]]. We demonstrated that TK mutant was constitutively phosphorylated, both on the catalytic domain (Y1234 and Y1235) and the docking-site tail (Y1349). As expected, the SEMA mutant was not basally phosphorylated. Both mutations sustained invasive phenotype. The tumorigenic potential of SEMA mutations was somehow unexpected since they do not affect receptor phosphorylation. We hypothesized that the occurrence of SEMA mutations may affect the physical interaction of NSCLC metastatic cells and surrounding microenvironment thus facilitating, their invasive properties. We performed atomic forces microscopy (AFM) measures. We demonstrated that, if compared to T47DTK and T47Dwt cells, T47DSEMA cells displayed lower Young module and activation of interactomic interactions between cell surface and surrounding stroma upon cell deformation. MET SEMA-mutated NSCLC metastases feature: high invasive capacity; specific viscoelasticity providing the property to torsionally deform and cross anatomical obstacles; significant radioresistance. [i] Stella GM, et al Neuro Oncol 2016 [ii] Stella GM et al. Hu Mut 2011

Maspin, a member of the serpin superfamily, is a double-face gene enacting different activities in cancer. Several studies support that in melanoma maspin loss could contribute to disease progression and metastatic dissemination, sustaining its putative tumour-suppressor role; others did not confirm these observations[1-5]. Also, the maspin subcellular localization in immunohistochemistry (nuclear vs. cytoplasm) has been related to divergent effects, such as tumor promotion or inhibition[5]. This article is protected by copyright. All rights reserved.

Some viral proteins expressed by Human Polyomaviruses (HPyV) are known to be able to cause cancer. Besides Merkel-Cell-Polyoma-Virus (MCV), four new human polyomaviruses have been identified in the last years. In 2010, the Sixth and Seventh HPyV (HPyV6 and HPyV7) were discovered2. Similar to MCV, HPyV6 and HPyV7 seem to be constituents of the human skin microbiome. Co-infecting HPyVs may therefore act as a co-factor in the development of cancers, including those induced by other oncoviruses. Despite these observations, their role in cancer remains controversial. This article is protected by copyright. All rights reserved.

Stromal content heavily impacts the transcriptional classification of colorectal cancer (CRC), with clinical and biological implications. Lineage-dependent stromal transcriptional components could therefore dominate over more subtle expression traits inherent to cancer cells. Since in patient-derived xenografts (PDXs) stromal cells of the human tumour are substituted by murine counterparts, here we deploy human-specific expression profiling of CRC PDXs to assess cancer-cell intrinsic transcriptional features. Through this approach, we identify five CRC intrinsic subtypes (CRIS) endowed with distinctive molecular, functional and phenotypic peculiarities: (i) CRIS-A: mucinous, glycolytic, enriched for microsatellite instability or KRAS mutations; (ii) CRIS-B: TGF-β pathway activity, epithelial–mesenchymal transition, poor prognosis; (iii) CRIS-C: elevated EGFR signalling, sensitivity to EGFR inhibitors; (iv) CRIS-D: WNT activation, IGF2 gene overexpression and amplification; and (v) CRIS-E: Paneth cell-like phenotype, TP53 mutations. CRIS subtypes successfully categorize independent sets of primary and metastatic CRCs, with limited overlap on existing transcriptional classes and unprecedented predictive and prognostic performances., Stromal cells contribute to the gene expression profiles based on which colorectal cancer (CRC) molecular subtypes are classified. Here, patient-derived xenografts enable the authors to obtain cancer cell-specific transcriptomes by excluding transcripts from murine stromal cells, based on which they define CRC intrinsic subtypes (CRIS) and evaluate their prognostic and predictive potential.

Supplemental digital content is available in the text., Evaluation of the molecular status of 1p and 19q is a major relevant diagnostic, prognostic, and predictive tool for oligodendroglial brain tumors. Fluorescence in situ hybridization (FISH) is the most commonly used technique for determining 1p and 19q allelic losses, but it lacks fully standardized criteria for analysis. This lack of standardization has led to interinstitutional disagreement in the interpretation of results, thereby contributing to a “gray prognostic zone” that includes codeleted patients with an unexpectedly unfavorable outcome. To optimize the prognostic potential of 1p/19q status determination, we first compared the actual criteria used for FISH reading (i.e. different ratio cutoff values and the percentage of neoplastic nuclei carrying this chromosomal deletion) in a retrospective series of 143 pure and mixed oligodendroglial tumors. We then created a “weighted” FISH reading based on the merged ratio and percentage of neoplastic cells carrying the deletion that was further differentially modulated for 1p and 19q, respectively. This weighted codeletion setting significantly strengthened the favorable prognostic power of 1p/19q losses by reducing the number of poor outcomes from 42% to 12.5% for patients with codeleted tumors. Thus, by identifying as codeleted only those cases with more than 50% of cells having a combined loss of 1p (using 0.7 ratio cutoff) and 19q (using 0.8 ratio cutoff) arms, we created a molecular report that bears higher clinical impact and strengthens the prognostic potential of 1p/19q allelic loss.

Background: Inguinal metastases in patients affected by anal cancer are an independent prognostic factor for local failure and overall mortality. Since 2001, sentinel lymph node biopsy was applied in these patients. This original study reports an update of personal and previous published series, which were compared with Literature to value the incidence of inguinal metastases T-stage related and the overall incidence of false negative inguinal metastases at sentinel node. Methods: In all, 63 patients diagnosed with anal cancer submitted to inguinal sentinel node. Furthermore a research in the Pub Med database was performed to find papers regarding this technique. Results: In our series, detection rate was 98.4%. Inguinal metastases were evidentiated in 13 patients (20.6%). Our median follow-up was 35 months. In our series, no false negative nodes were observed. Conclusion: Sentinel node technique in the detection of inguinal metastases in patients affected by anal cancer should be considered as a standard of care. It is indicated for all T stages in order to select patients to be submitted to inguinal radiotherapy, avoiding related morbidity in negative ones. An overall 3.7% rate of false negative must be considered acceptable.

Background: The new AJCC classification has highlighted some particular risk factors for squamous cell carcinoma (SCC) relevant for prognosis. Incomplete excision is not infrequent in SCC. The aim of this study is to examine features that can predict an incomplete excision on the basis of the new AJCC classification and to review the literature on this topic. Materials and Methods: 81 SCC patients were included. All patients were submitted to excisional biopsy with a margin of at least 4 mm from the clinical edges as recommended. Histological characteristics of the lesions analysed were maximum diameter, grading, site, Breslow thickness, Clark level, deep tissue invasion (neural, bone, muscle), presence of ulceration and positivity of the margins. Results: The average Breslow thickness was 3.93 mm. Out of the 81 patients included, 14 showed involved margins. The 2 parameters that were implicated in predicting involvement of the margins in the multivariable model were Breslow thickness and location of the lesion on the ear or lip. Grading was not associated with involvement of margins. Conclusion: According to the new AJCC classification, this study could be useful to plan the most suitable surgical technique in order to avoid the risk of incomplete surgery.

Background Lymphatic drainage to multiple basins (MLBD) is frequently observed in patients with primary melanoma located in the trunk. Conflicting data regarding the prognostic impact of MLBD are reported. Objective and methods We reviewed our case series of 352 patients with trunk melanoma to evaluate the pattern of basin drainage and to analyse whether different basin drainages may have different significance in negative sentinel lymph node (SLN) patients. The presence of single/multiple basin drainage, the status of SLN, the presence of melanoma regression, Breslow thickness, ulceration and type of melanoma were recorded for each patients and correlated to Disease Free Survival (DFS) and Overall Survival (OS). Results MLBD occurred in 77 patients (21.9%) and single basin lymphatic drainage (SLBD) occurred in 275 patients (79.1%). The presence of metastases in SLN was not significantly different in patients with MLBD compared to those with SLBD (26% vs. 19.6%). No differences in OS and DFS were found in SLBD/MLBD independently from SLN status. However DFS was higher in patients with MLBD and negative SLN (P = 0.0001), in addition, in patients with negative SLN and SLBD disease recurrence was 19% while was only 7% in patients with negative SLN obtained from MLBD (P = 0.03). Multivariate analysis showed that Breslow thickness

Although conventional histopathologic examination is still the undisputable mainstay for the diagnosis of melanocytic skin neoplasms, application of molecular testing has experienced tremendous growth and will continue to expand in the future as the need for more specific diagnoses and new targeted therapies evolve. Ancillary molecular methods, including comparative genomic hybridization and fluorescence in situ hybridization, have the potential to provide important new information to challenging cases, and will help improve diagnostic accuracy, particularly in cases in which morphology is not conclusive. Pathologists are increasingly involved in the prospective genotyping of melanoma, which leads to patient stratification in light of the novel personalized therapeutic approaches in the advanced setting.

Background The incidence of meningeal carcinomatosis appears to be higher than in the past due to advances in neuro-imaging diagnostic techniques and improvements in cancer survival. Among solid tumors, breast cancer is the cancer most commonly associated with meningeal carcinomatosis, with an incidence rate of between 0.8 and 16 %. Aim of this study has been i) to evaluate the incidence of meningeal carcinomatosis in a continuous breast cancer unselected series treated in a dedicated Breast Unit and ii) to define the clinico-pathological and molecular parameters associated with meningeal carcinomatosis development. Methods A retrospective series of 1915 consecutive patients surgically treated for breast cancer between 1998 and 2010 was collected. Clinico-pathological data were recorded from medical charts and pathological reports, including the date of development of symptomatic meningeal carcinomatosis. Meningeal carcinomatosis incidence was determined at both 5- and 10-year follow-ups. Results Three patients in the first 5 years of follow-up and six patients in 10 years of follow-up developed meningeal carcinomatosis. An incidence rate of 5.44 per 10,000 patients (95 % CI: 1.75–16.9) was observed, with a 5-year risk of 0.3 %. At 10-year follow up, the rate increased to 7.55 per 10,000 patients (95 % CI: 3.39–16.8). In a univariate analysis, young age, tumor size larger than 15 mm, histological grade 3, more than three metastatic lymph nodes, negative estrogen receptor, positive HER2 and high proliferative index were significantly associated with meningeal carcinomatosis development. Conclusions In an unselected breast cancer population, meningeal carcinomatosis is a rare event that is associated with adverse prognostic factors. Meningeal carcinomatosis incidence is overestimated when recorded in biased/high-risk selected breast cancer patients and should not be considered to accurately reflect the overall breast cancer population.

Background: MicroRNAs (miRNAs) are small non-coding RNA molecules regulating mRNAs translation. Significant differences in miRNAs expression have been found in glioblastoma compared to normal brain tissue. MiR-124a, miR-381 and miR-637 are involved in regulation of pathways which are commonly deregulated in glioblastoma (MAPK/ERK, WNT/β-catenin and AKT pathways, respectively). AIM: To investigate the expression profile and prognostic/predictive role of miR-124a, miR-381 and miR-637 in primary IDH-wildtype glioblastoma. Materials and Methods: Inclusion criteria were: pathological diagnosis of primary IDH-wildtype glioblastoma (according to WHO 2016 criteria), partial/total surgical resection followed by at least one line of postoperative adjuvant treatment, ≥ 6-months follow-up after diagnosis, Caucasian ethnicity. Histological revision was performed independently by two pathologists to confirm diagnosis. IDH1/IDH2 status was assessed by Sanger sequencing according to standard protocols. Total RNA was extracted using miRNeasy FFPE (QIAGEN), miRNAs were amplified with TaqMan MicroRNA Assays (ThermoFisher) and RNU6B was used as endogenous control, while ten normal brain areas were selected as external controls. Relative quantification miRNA expression values (RQ) were calculated using the ΔΔCt method. X-tile plots and Stata (StataCorp LP) were used to calculate RQ cut-off values and for statistical analyses, respectively. Informed consent was obtained according to local regulations. Results: Here we present preliminary results based on 49 patients, 30 males (61%). Median age was 59 years (range 38–75), median KPS before surgery 80 (range 70–90). Partial resection was achieved in 27/49 (55%), total in 22/49 (45%). Median progression-free survival (PFS) was 9.4 months (range 3.5–26), median overall survival (OS) 16 months (range 3.5–42.7). Total resection and adjuvant treatment according to Stupp regimen were both associated with a better outcome as expected. Glioblastoma samples showed a reduced expression of all three miRNAs compared to normal brain. Significantly better PFS (9.9 vs 6.0 months, p=0.002) was found in patients with up-regulated miR-637 (RQ cut-off: 0.248), whereas OS difference was not significant (16.9 vs 15.6, p=0.161). A specific RQ range of miR-381 (RQ 0.104–0.287) was associated with better PFS (9.8 vs 7.4, p=0.0183), but not with OS (16.4 vs 15.8, p=0.782). No significant associations were found between miR-124a and outcome. Conclusions: A better PFS was significantly associated with miR-637 up-regulation, a finding consistent with its reported inhibitory effect on the AKT pathway. Interestingly, the favorable role of miR-381 seems to be limited to a specific range of RQ comprising the cut-off value. These preliminary findings need to be assessed in a larger series and the study is ongoing.