Development and validation of a clinical decision support system based on PSA, microRNAs, and MRI for the detection of prostate cancer.

OBJECTIVES: The aims of this study are to develop and validate a clinical decision support system based on demographics, prostate-specific antigen (PSA), microRNA (miRNA), and MRI for the detection of prostate cancer (PCa) and clinical significant (cs) PCa, and to assess if this system performs better compared to MRI alone. METHODS: This retrospective, multicenter, observational study included 222 patients (mean age 66, range 46-75 years) who underwent prostate MRI, miRNA (let-7a-5p and miR-103a-3p) assessment, and biopsy. Monoparametric and multiparametric models including age, PSA, miRNA, and MRI outcome were trained on 65% of the data and then validated on the remaining 35% to predict both PCa (any Gleason grade [GG]) and csPCa (GG ≥ 2 vs GG = 1/negative). Accuracy, sensitivity, specificity, positive and negative predictive value (NPV), and area under the receiver operating characteristic curve were calculated. RESULTS: MRI outcome was the best predictor in the monoparametric model for both detection of PCa, with sensitivity of 90% (95%CI 73-98%) and NPV of 93% (95%CI 82-98%), and for csPCa identification, with sensitivity of 91% (95%CI 72-99%) and NPV of 95% (95%CI 84-99%). Sensitivity and NPV of PSA + miRNA for the detection of csPCa were not statistically different from the other models including MRI alone. CONCLUSION: MRI stand-alone yielded the best prediction models for both PCa and csPCa detection in biopsy-naïve patients. The use of miRNAs let-7a-5p and miR-103a-3p did not improve classification performances compared to MRI stand-alone results. CLINICAL RELEVANCE STATEMENT: The use of miRNA (let-7a-5p and miR-103a-3p), PSA, and MRI in a clinical decision support system (CDSS) does not improve MRI stand-alone performance in the detection of PCa and csPCa. KEY POINTS: • Clinical decision support systems including MRI improve the detection of both prostate cancer and clinically significant prostate cancer with respect to PSA test and/or microRNA. • The use of miRNAs let-7a-5p and miR-103a-3p did not significantly improve MRI stand-alone performance. • Results of this study were in line with previous works on MRI and microRNA.

The protease-inhibitor SerpinB3 as a critical modulator of the stem-like subset in human cholangiocarcinoma.

BACKGROUND AND AIMS: Cholangiocarcinoma (CCA) is a form of primary liver cancer with limited therapeutic options. Recently, cancer stem cells (CSCs) have been proposed as a driving force of tumour initiation and dissemination, thus representing a crucial therapeutic target. The protease inhibitor SerpinB3 (SB3) has been identified in several malignancies including hepatocellular carcinoma. SB3 has been involved in the early events of hepatocarcinogenesis and is highly expressed in hepatic progenitor cells and in a mouse model of liver progenitor cell activation. However, only limited information on the possible role of SB3 in CCA stem-like compartment is available. METHODS: Enrichment of CCA stem-like subset was performed by sphere culture (SPH) in CCA cell lines (CCLP1, HUCCT1, MTCHC01 and SG231). Quantitative RT-PCR and Western blotting were used to detect SB3 in both SPH and parental monolayer (MON) cells. Acquired CSC-like features were analysed using an endogenous and a paracrine in vitro model, with transfection of SB3 gene or addition of recombinant SB3 to cell medium respectively. SB3 tumorigenic role was explored in an in vivo mouse model of CCA by subcutaneous injection of SB3-transfected MON (MONSB3+ ) cells in immune-deficient NOD-SCID/IL2Rgnull  (NSG) mice. SB3 expression in human CCA sections was investigated by immunohistochemistry. Overall survival (OS) and time to recurrence (TTR) analyses were carried out from a transcriptome database of 104 CCA patients. RESULTS: SB3, barely detected in parental MON cells, was overexpressed in the same CCA cells grown as 3D SPH. Notably, MONSB3+ showed significant overexpression of genes associated with stemness (CD24, CD44, CD133), pluripotency (c-MYC, NOTCH1, STAT3, YAP, NANOG, BMI1, KLF4, OCT4, SOX2), epithelial mesenchymal transition (ß-catenin, SLUG) and extracellular matrix remodelling (MMP1, MMP7, MMP9, ADAM9, ADAM10, ADAM17, ITGB3). SB3-overexpressing cells showed superior spherogenic capacity and invasion ability compared to control. Importantly, MONSB3+ exhibited activation of MAP kinases (ERK1/2, p38, JNK) as well as phosphorylation of NFκB (p65) in addition to up-regulation of the proto-oncogene ß-catenin. All these effects were reversed after transient silencing of SB3. According to the in vitro finding, MONSB3+ cells retained high tumorigenic potential in NSG mice. SB3 overexpression was observed in human CCA tissues and analysis of OS as well as TTR indicated a worse prognosis in SB3+ CCA patients. CONCLUSION: These findings indicate a SB3 role in mediating malignant phenotype of CCA and identify a new therapeutic target.

Proteomic analysis identifies deregulated metabolic and oxidative-associated proteins in Italian intrahepatic cholangiocarcinoma patients.

BACKGROUND: Cholangiocarcinoma (CCA) is an aggressive disease with poor prognosis. A molecular classification based on mutational, methylation and transcriptomic features could allow identifying tailored therapies to improve CCA patient outcome. Proteomic remains partially unexplored; here, we analyzed the proteomic profile of five intrahepatic cholangiocarcinoma (ICC) derived from Italian patients undergone surgery and one normal bile duct cell line. METHODS: Proteome profile was investigated by using 2D electrophoresis followed by Mass Spectrometry (MS). To validate proteomic data, the expression of four overexpressed proteins (CAT, SOD, PRDX6, DBI/ACBP) was evaluated by immunohistochemistry in an independent cohort of formalin fixed, paraffin-embedded (FFPE) ICC tissues. We also compared proteomic data with those obtained by transcriptomic profile evaluated by microarray analysis of the same tissues. RESULTS: We identified 19 differentially expressed protein spots, which were further characterized by MS; 13 of them were up- and 6 were down-regulated in ICC. These proteins are mainly involved in redox processes (CAT, SODM, PRDX2, PRDX6), in metabolism (ACBP, ACY1, UCRI, FTCD, HCMS2), and cell structure and organization (TUB2, ACTB). CAT is overexpressed in 86% of patients, PRDX6 in 73%, SODM in 100%, and DBI/ACBP in 81% compared to normal adjacent tissues. A concordance of 50% between proteomic and transcriptomic data was observed. CONCLUSIONS: This study pointed out that the impairment of the metabolic and antioxidant systems, with a subsequent accumulation of free radicals, might be a key step in CCA development and progression.

Antitumor Activity of a Novel Fibroblast Growth Factor Receptor Inhibitor for Intrahepatic Cholangiocarcinoma.

Fibroblast growth factor receptor 2 (FGFR2) might have an important role in the pathogenesis and biology of cholangiocarcinoma (CCA). We examined FGFR expression in CCA tumor specimens obtained from patients and CCA cell lines, and then determined the effects of the novel FGFR inhibitor, derazantinib (DZB; formally, ARQ 087), which is currently in clinical phase 2 trials for intrahepatic CCA. DZB inhibited the growth of CCA cell lines in a dose-dependent manner, and extracellular signal-regulated kinase 1/2 and AKT. It also activated apoptotic and cell growth arrest signaling. DZB reduced the in vitro invasiveness and the expression of key epithelial-mesenchymal transition genes. The in vitro data correlated with the expression of FGFRs in human CCA specimens by immunohistochemistry (FGFR1, 30% positive; and FGFR2, 65% positive) and the CCA cell lines assayed by Western blot analysis. These correlated in vitro studies suggest that FGFR may play an important role in the pathogenesis and biology of CCA. Our findings support the notion that FGFR inhibitors, like DZB, should be further evaluated at the clinical stage as targeted therapy for CCA treatment.

Gene Expression Signature Predictive of Neuroendocrine Transformation in Prostate Adenocarcinoma.

Neuroendocrine prostate cancer (NEPC) can arise de novo, but much more commonly occurs as a consequence of a selective pressure from androgen deprivation therapy or androgen receptor antagonists used for prostate cancer (PCa) treatment. The process is known as neuroendocrine transdifferentiation. There is little molecular characterization of NEPCs and consequently there is no standard treatment for this kind of tumors, characterized by highly metastases rates and poor survival. For this purpose, we profiled 54 PCa samples with more than 10-years follow-up for gene and miRNA expression. We divided samples into two groups (NE-like vs. AdenoPCa), according to their clinical and molecular features. NE-like tumors were characterized by a neuroendocrine fingerprint made of known neuroendocrine markers and novel molecules, including long non-coding RNAs and components of the estrogen receptor signaling. A gene expression signature able to predict NEPC was built and tested on independently published datasets. This study identified molecular features (protein-coding, long non-coding, and microRNAs), at the time of surgery, that may anticipate the NE transformation process of prostate adenocarcinoma. Our results may contribute to improving the diagnosis and treatment of this subgroup of tumors for which traditional therapy regimens do not show beneficial effects.

Establishment and characterization of a human intrahepatic cholangiocarcinoma cell line derived from an Italian patient.

Biliary tract carcinoma is a rare malignancy with multiple causes, which underlie the different genetic and molecular profiles. Cancer cell lines are affordable models, reflecting the characteristics of the tumor of origin. They represent useful tools to identify molecular targets for treatment. Here, we established and characterized from biological, molecular, and genetic point of view, an Italian intrahepatic cholangiocarcinoma cell line (ICC), the MT-CHC01. MT-CHC01 cells were isolated from a tumor-derived xenograft. Immunophenotypical characterization was evaluated both at early and after stabilization passages. In vitro biological, genetic, and molecular features were also investigated. In vivo tumorigenicity was assessed in NOD/SCID mice. MT-CHC01cells retain epithelial cell markers, EPCAM, CK7, and CK19, and some stemness and pluripotency markers, i.e., SOX2, Nanog, CD49f/integrin-α6, CD24, PDX1, FOXA2, and CD133. They grow as a monolayer, with a population double time of about 40 h; they show a low migration and invasion potential. In low attachment conditions, they are able to form spheres and to growth in anchorage-independent manner. After subcutaneous injection, they retain in vivo tumorigenicity; the expression of biliary markers as CA19-9 and CEA were maintained from primary tumor. The karyotype is highly complex, with a hypotriploid to hypertriploid modal number (3n+/-) (52 to 77 chromosomes); low level of HER2 gene amplification, TP53 deletion, gain of AURKA were identified; K-RAS G12D mutation were maintained from primary tumor to MT-CHC01 cells. We established the first ICC cell line derived from an Italian patient. It will help to study either the biology of this tumor or to test drugs both in vitro and in vivo.

Establishment of a patient-derived intrahepatic cholangiocarcinoma xenograft model with KRAS mutation.

BACKGROUND: Intrahepatic cholangiocarcinoma (ICC) is an aggressive, highly lethal tumors and lacks of effective chemo and targeted therapies. Cell lines and animal models, even partially reflecting tumor characteristics, have limits to study ICC biology and drug response. In this work, we created and characterized a novel ICC patient-derived xenograft (PDX) model of Italian origin. METHODS: Seventeen primary ICC tumors derived from Italian patients were implanted into NOD (Non-Obese Diabetic)/Shi-SCID (severe combined immunodeficient) mice. To verify if the original tumor characteristics were maintained in PDX, immunohistochemical (cytokeratin 7, 17, 19, and epithelial membrane antigen) molecular (gene and microRNA expression profiling) and genetic analyses (comparative genomic hybridization array, and mutational analysis of the kinase domain of EGFR coding sequence, from exons 18 to 21, exons 2 to 4 of K-RAS, exons 2 to 4 of N-RAS, exons 9 and 20 of PI3KCA, and exon 15 of B-RAF) were performed after tumor stabilization. RESULTS: One out of 17 (5.8%) tumors successfully engrafted in mice. A high molecular and genetic concordance between primary tumor (PR) and PDX was confirmed by the evaluation of biliary epithelial markers, tissue architecture, genetic aberrations (including K-RAS G12D mutation), and transcriptomic and microRNA profiles. CONCLUSIONS: For the first time, we established a new ICC PDX model which reflects the histology and genetic characteristics of the primary tumor; this model could represent a valuable tool to understand the tumor biology and the progression of ICC as well as to develop novel therapies for ICC patients.

Screening for the FIG-ROS1 fusion in biliary tract carcinomas by nested PCR.

ROS1 rearrangements have been detected in a variety of tumors and are considered as suitable targets of anticancer therapies. We developed a new, quick, specific, and sensitive PCR test to screen for the FIG-ROS1 fusion and applied it to a series of Italian patients with bile duct carcinoma (BTC). Formalin-fixed, paraffin-embedded tissues, derived from 65 Italian BTC patients, and six cell lines were analyzed by nested PCR to investigate the prevalence of a previously reported FIG-ROS1 fusion. The specificity and sensitivity of nested PCR were investigated in FIG-ROS1 positive U118MG cells in reconstitution experiments with peripheral blood mononuclear cells. We found that six out of 65 (9%) BTC patients were positive for the FIG-ROS1 fusion, comprising two out of 14 (14%) gallbladder carcinoma (GBC) patients and four out of 25 (16%) extrahepatic cholangiocarcinoma (ECC) patients. None of the 26 intrahepatic cholangiocarcinoma cases harbored the FIG-ROS1 fusion. All the cell lines were negative for this variant. In conclusion, 14-16% of GBC and ECC were positive for FIG-ROS1. This may have clinical implications, since these patients will potentially benefit from the treatment with specific ROS1 inhibitors.

Anti-cancer effect and gene modulation of ET-743 in human biliary tract carcinoma preclinical models.

BACKGROUND: Standard chemotherapy in unresectable biliary tract carcinoma (BTC) patients is based on gemcitabine combined with platinum derivatives. However, primary or acquired resistance is inevitable and no second-line chemotherapy is demonstrated to be effective. Thus, there is an urgent need to identify new alternative (chemo)therapy approaches. METHODS: We evaluated the mechanism of action of ET-743 in preclinical models of BTC. Six BTC cell lines (TFK-1, EGI-1, TGBC1, WITT, KMCH, HuH28), two primary cell cultures derived from BTC patients, the EGI-1 and a new established BTC patient-derived xenografts, were used as preclinical models to investigate the anti-tumor activity of ET-743 in vitro and in vivo. Gene expression profiling was also analyzed upon ET-743 treatment in in vivo models. RESULTS: We found that ET-743 inhibited cell growth of BTC cell lines and primary cultures (IC50 ranging from 0.37 to 3.08 nM) preferentially inducing apoptosis and activation of the complex DNA damage-repair proteins (p-ATM, p-p53 and p-Histone H2A.x) in vitro. In EGI-1 and patient-derived xenografts, ET-743 induced tumor growth delay and reduction of vasculogenesis. In vivo ET-743 induced a deregulation of genes involved in cell adhesion, stress-related response, and in pathways involved in cholangiocarcinogenesis, such as the IL-6, Sonic Hedgehog and Wnt signaling pathways. CONCLUSIONS: These results suggest that ET-743 could represent an alternative chemotherapy for BTC treatment and encourage the development of clinical trials in BTC patients resistant to standard chemotherapy.

Structure-guided optimization of 3-hydroxybenzoisoxazole derivatives as inhibitors of Aldo-keto reductase 1C3 (AKR1C3) to target prostate cancer.

AKR1C3 is an enzyme that is overexpressed in several types of radiotherapy- and chemotherapy-resistant cancers. Despite AKR1C3 is a validated target for drug development, no inhibitor has been approved for clinical use. In this manuscript, we describe our study of a new series of potent AKR1C3-targeting 3-hydroxybenzoisoxazole based inhibitors that display high selectivity over the AKR1C2 isoform and low micromolar activity in inhibiting 22Rv1 prostate cancer cell proliferation. In silico studies suggested proper substituents to increase compound potency and provided with a mechanistic explanation that could clarify their different activity, later confirmed by X-ray crystallography. Both the in-silico studies and the crystallographic data highlight the importance of 90° rotation around the single bond of the biphenyl group, in ensuring that the inhibitor can adopt the optimal binding mode within the active pocket. The p-biphenyls that bear the meta-methoxy, and the ortho- and meta-trifluoromethyl substituents (in compounds 6a, 6e and 6f respectively) proved to be the best contributors to cellular potency as they provided the best IC50 values in series (2.3, 2.0 and 2.4 µM respectively) and showed no toxicity towards human MRC-5 cells. Co-treatment with scalar dilutions of either compound 6 or 6e and the clinically used drug abiraterone led to a significant reduction in cell proliferation, and thus confirmed that treatment with both CYP171A1-and AKR1C3-targeting compounds possess the potential to intervene in key steps in the steroidogenic pathway. Taken together, the novel compounds display desirable biochemical potency and cellular target inhibition as well as good in-vitro ADME properties, which highlight their potential for further preclinical studies.

Fluoropyrimidine­induced cardiotoxicity in colorectal cancer patients: a prospective observational trial (CHECKPOINT).

Fluoropyrimidines (FP) are the backbone chemotherapy in colorectal cancer (CRC) treatment; however, their use is associated with cardiotoxicity, which is underreported. In the present study, it was aimed to prospectively determine the incidence rates and related risk factors of FP­induced cardiotoxicity (FIC) in CRC patients and at identifying predictive biomarkers. A total of 129 consecutive previously untreated CRC patients underwent active cardiological monitoring, including 5­items simplified questionnaire on symptoms, electrocardiogram (ECG) and plasma sample collection during FP chemotherapy. FIC was defined as the presence of ECG alterations and/or the arising of at least one symptom of chest pain, dyspnoea, palpitations or syncope. The primary objective was the evaluation of FIC incidence. Secondary objectives were the correlation of FIC with well­known cardiological risk factors and the identification of circulating biomarkers (serum levels of troponin I, pro hormone BNP; miRNA analysis) as predictors of FIC. A total of 20 out of 129 (15.5%) patients experienced FIC. The most common symptoms were dyspnoea (60%) and chest pain (40%), while only 15% of patients presented ECG alterations, including one acute myocardial infarction. Retreatment with FP was attempted in 90% of patients with a favourable outcome. Despite 48% of patients having cardiological comorbidities, an increased FIC was not observed in this subgroup. Only the subgroup of females with the habit of alcohol consumption showed an increased risk of FIC. None of the circulating biomarkers evaluated demonstrated a clinical utility as FIC predictors. FIC can be an unexpected, life­threatening adverse event that can limit the subsequent treatment choices in patients with CRC. In this prospective study, well­known cardiological comorbidities were not related to higher FIC risk and circulating biomarkers predictive of toxicity could not be found. With careful monitoring, mainly based on symptoms, almost all patients completed the FP treatment.

Paclitaxel Restores Sensitivity to Chemotherapy in Preclinical Models of Multidrug-Resistant Intrahepatic Cholangiocarcinoma.

The treatment of unresectable cholangiocarcinoma (CCA) is limited by the development of resistance to conventional first-line chemotherapy based on gemcitabine (GEM). In addition, a prior treatment with GEM frequently induces cross-resistance to other drugs employed in the second-line. Paclitaxel (PTX) is now emerging as an alternative option for the management of advanced/metastatic CCA. In the present work, we evaluate the antitumor activity of PTX in preclinical models of multidrug-resistant intrahepatic cholangiocarcinoma (iCCA). In vitro, PTX decreases tumor cell viability by affecting the cell cycle and inducing apoptosis and impairs the stem cell compartment. In vivo, a therapeutic regimen containing albumin-bound nanoparticle (Nab)-PTX overcomes drug resistance resulting in delayed tumor growth, impaired organization of the tumor vasculature, and reduced glucose uptake. Together, our results provide a rationale to consider PTX-based regimens in patients with iCCA who became refractory to conventional therapies.

HER2-positive breast cancer cells resistant to trastuzumab and lapatinib lose reliance upon HER2 and are sensitive to the multitargeted kinase inhibitor sorafenib.

Trastuzumab has changed the prognosis of HER2 positive breast cancers. Despite this progress, resistance to trastuzumab occurs in most patients. Newer anti-HER2 therapies, like the dual tyrosine-kinase inhibitor (TKI) lapatinib, show significant antitumor activity, indicating that HER2 can be still exploited as a target after trastuzumab failure. However, since a high proportion of patients fail to respond to these alternative strategies, it is possible that cell escape from HER2 targeting may rely on HER2 independent pathways. The knowledge of these pathways deserve to be exploited to develop new therapies. We characterized two human HER2 overexpressing breast cancer cell lines resistant to trastuzumab and lapatinib (T100 and JIMT-1) from a molecular and biological point of view. Indeed, we assessed both in vitro and in vivo the activity of the multitarget inhibitor sorafenib. In both cell lines, the previously proposed mechanisms did not explain resistance to HER2 inhibitors. Notably, silencing HER2 by shRNA did not affect the growth of our cells, suggesting loss of reliance upon HER2. Moreover, we identified alterations in two antiapoptotic proteins Mcl-1 and Survivin which are known to be targets of the multikinase inhibitor sorafenib. Moreover, sorafenib, strongly inhibited the in vitro growth of T100 and JIMT-1 cells, through the downregulation of both Mcl-1 and Survivin. Similar results were obtained in JIMT-1 xenografts subcutaneously injected in NOD SCID mice. We provide preclinical evidence that tumor cells resistant to trastuzumab and lapatinib may rely on HER2 independent pathways that can be efficiently inhibited by sorafenib.

Epidermal Growth Factor Receptor (EGFR) mutation analysis, gene expression profiling and EGFR protein expression in primary prostate cancer.

BACKGROUND: Activating mutations of the epidermal growth factor receptor (EGFR) confer sensitivity to the tyrosine kinase inhibitors (TKi), gefitinib and erlotinib. We analysed EGFR expression, EGFR mutation status and gene expression profiles of prostate cancer (PC) to supply a rationale for EGFR targeted therapies in this disease. METHODS: Mutational analysis of EGFR TK domain (exons from 18 to 21) and immunohistochemistry for EGFR were performed on tumour tissues derived from radical prostatectomy from 100 PC patients. Gene expression profiling using oligo-microarrays was also carried out in 51 of the PC samples. RESULTS: EGFR protein overexpression (EGFRhigh) was found in 36% of the tumour samples, and mutations were found in 13% of samples. Patients with EGFRhigh tumours experienced a significantly increased risk of biochemical relapse (hazard ratio-HR 2.52, p=0.02) compared with patients with tumours expressing low levels of EGFR (EGFRlow). Microarray analysis did not reveal any differences in gene expression between EGFRhigh and EGFRlow tumours. Conversely, in EGFRhigh tumours, we were able to identify a 79 gene signature distinguishing mutated from non-mutated tumours. Additionally, 29 genes were found to be differentially expressed between mutated/EGFRhigh (n=3) and mutated/EGFRlow tumours (n=5). Four of the down-regulated genes, U19/EAF2, ABCC4, KLK3 and ANXA3 and one of the up-regulated genes, FOXC1, are involved in PC progression. CONCLUSIONS: Based on our findings, we hypothesize that accurate definition of the EGFR status could improve prognostic stratification and we suggest a possible role for EGFR-directed therapies in PC patients. Having been generated in a relatively small sample of patients, our results warrant confirmation in larger series.

Two Novel Ceramide-Like Molecules and miR-5100 Levels as Biomarkers Improve Prediction of Prostate Cancer in Gray-Zone PSA.

Reliable liquid biopsy-based tools able to accurately discriminate prostate cancer (PCa) from benign prostatic hyperplasia (BPH), when PSA is within the "gray zone" (PSA 4-10), are still urgent. We analyzed plasma samples from a cohort of 102 consecutively recruited patients with PSA levels between 4 and 16 ng/ml, using the SANIST-Cloud Ion Mobility Metabolomic Mass Spectrometry platform, combined with the analysis of a panel of circulating microRNAs (miR). By coupling CIMS ion mobility technology with SANIST, we were able to reveal three new structures among the most differentially expressed metabolites in PCa vs. BPH. In particular, two were classified as polyunsaturated ceramide ester-like and one as polysaturated glycerol ester-like. Penalized logistic regression was applied to build a model to predict PCa, using six circulating miR, seven circulating metabolites, and demographic/clinical variables, as covariates. Four circulating metabolites, miR-5100, and age were selected by the model, and the corresponding prediction score gave an AUC of 0.76 (C.I. = 0.66-0.85). At a specified cut-off, no high-risk tumor was misclassified, and 22 out of 53 BPH were correctly identified, reducing by 40% the false positives of PSA. We developed and applied a novel, minimally invasive, liquid biopsy-based powerful tool to characterize novel metabolites and identified new potential non-invasive biomarkers to better predict PCa, when PSA is uninformative as a tool for precision medicine in genitourinary cancers.

A Novel Multidrug-Resistant Cell Line from an Italian Intrahepatic Cholangiocarcinoma Patient.

Chemotherapy resistance is a relevant clinical issue in tumor treatment, in particular in biliary tract carcinoma (BTC), for which there are no effective therapies, neither in the first nor in the second line. The development of chemoresistant cell lines as experimental models to investigate the mechanisms of resistance and identify alternative druggable pathways is mandatory. In BTC, in which genetics and biological behavior depend on the etiology, ethnicity, and anatomical site of origin, the creation of models that better recapitulate these characteristics is even more crucial. Here we have established and characterized an intrahepatic cholangiocarcinoma (iCCA) cell line derived from an Italian patient, called 82.3. Cells were isolated from a patient-derived xenograft (PDX) and, after establishment, immunophenotypic, biological, genetic, molecular characteristics, and tumorigenicity in vivo in NOD/SCID mice were investigated. 82.3 cells exhibited epithelial morphology and cell markers (EPCAM, CK7, and CK19); they also expressed different cancer stem markers (CD44, CD133, CD49b, CD24, Stro1, PAX6, FOXA2, OCT3/4), α-fetoprotein and under anchorage-independent and serum-free conditions were capable of originating cholangiospheres. The population doubling time was approximately 53 h. In vitro, they demonstrated a poor ability to migrate; in vivo, 82.3 cells retained their tumorigenicity, with a long latency period (16 weeks). Genetic identity using DNA fingerprinting analysis revealed 16 different loci, and the cell line was characterized by a complex hyperdiploid karyotype. Furthermore, 82.3 cells showed cross-resistance to gemcitabine, 5-fluorouracil, carboplatin, and oxaliplatin; in fact, their genetic profile showed that 60% of genes (n = 168), specific for drug resistance and related to the epithelial-mesenchymal transition, were deregulated in 82.3 cells compared to a control iCCA cell line sensitive to chemotherapeutics. RNA sequencing analysis revealed the enrichment for genes associated with epithelial to mesenchymal transition (EMT), vasculature development, and extracellular matrix (ECM) remodeling, underlining an aggressive phenotype. In conclusion, we have created a new iCCA cell line of Caucasian origin: this could be exploited as a preclinical model to study drug resistance mechanisms and to identify alternative therapies to improve the prognosis of this tumor type.

Identification of novel circulating microRNAs in advanced heart failure by next-generation sequencing.

AIMS: Risk stratification in patients with advanced chronic heart failure (HF) is an unmet need. Circulating microRNA (miRNA) levels have been proposed as diagnostic and prognostic biomarkers in several diseases including HF. The aims of the present study were to characterize HF-specific miRNA expression profiles and to identify miRNAs with prognostic value in HF patients. METHODS AND RESULTS: We performed a global miRNome analysis using next-generation sequencing in the plasma of 30 advanced chronic HF patients and of matched healthy controls. A small subset of miRNAs was validated by real-time PCR (P < 0.0008). Pearson's correlation analysis was computed between miRNA expression levels and common HF markers. Multivariate prediction models were exploited to evaluate miRNA profiles' prognostic role. Thirty-two miRNAs were found to be dysregulated between the two groups. Six miRNAs (miR-210-3p, miR-22-5p, miR-22-3p, miR-21-3p, miR-339-3p, and miR-125a-5p) significantly correlated with HF biomarkers, among which N-terminal prohormone of brain natriuretic peptide. Inside the cohort of advanced HF population, we identified three miRNAs (miR-125a-5p, miR-10b-5p, and miR-9-5p) altered in HF patients experiencing the primary endpoint of cardiac death, heart transplantation, or mechanical circulatory support implantation when compared with those without clinical events. The three miRNAs added substantial prognostic power to Barcelona Bio-HF score, a multiparametric and validated risk stratification tool for HF (from area under the curve = 0.72 to area under the curve = 0.82). CONCLUSIONS: This discovery study has characterized, for the first time, the advanced chronic HF-specific miRNA expression pattern. We identified a few miRNAs able to improve the prognostic stratification of HF patients based on common clinical and laboratory values. Further studies are needed to validate our results in larger populations.

AKR1C3 is a biomarker and druggable target for oropharyngeal tumors.

PURPOSE: Oropharynx squamous cell carcinoma (OPSCC) is a subtype of head and neck squamous cell carcinoma (HNSCC) arising from the base of the tongue, lingual tonsils, tonsils, oropharynx or pharynx. The majority of HPV-positive OPSCCs has a good prognosis, but a fraction of them has a poor prognosis, similar to HPV-negative OPSCCs. An in-depth understanding of the molecular mechanisms underlying OPSCC is mandatory for the identification of novel prognostic biomarkers and/or novel therapeutic targets. METHODS: 14 HPV-positive and 15 HPV-negative OPSCCs with 5-year follow-up information were subjected to gene expression profiling and, subsequently, compared to three extensive published OPSCC cohorts to define robust biomarkers for HPV-negative lesions. Validation of Aldo-keto-reductases 1C3 (AKR1C3) by qRT-PCR was carried out on an independent cohort (n = 111) of OPSCC cases. In addition, OPSCC cell lines Fadu and Cal-27 were treated with Cisplatin and/or specific AKR1C3 inhibitors to assess their (combined) therapeutic effects. RESULTS: Gene set enrichment analysis (GSEA) on the four datasets revealed that the genes down-regulated in HPV-negative samples were mainly involved in immune system, whereas those up-regulated mainly in glutathione derivative biosynthetic and xenobiotic metabolic processes. A panel of 30 robust HPV-associated transcripts was identified, with AKR1C3 as top-overexpressed transcript in HPV-negative samples. AKR1C3 expression in 111 independent OPSCC cases positively correlated with a worse survival, both in the entire cohort and in HPV-positive samples. Pretreatment with a selective AKR1C3 inhibitor potentiated the effect of Cisplatin in OPSCC cells exhibiting higher basal AKR1C3 expression levels. CONCLUSIONS: We identified AKR1C3 as a potential prognostic biomarker in OPSCC and as a potential drug target whose inhibition can potentiate the effect of Cisplatin.

Targeting EGFR/HER2 pathways enhances the antiproliferative effect of gemcitabine in biliary tract and gallbladder carcinomas.

BACKGROUND: Advanced biliary tract carcinomas (BTCs) have poor prognosis and limited therapeutic options. Therefore, it is crucial to combine standard therapies with molecular targeting. In this study EGFR, HER2, and their molecular transducers were analysed in terms of mutations, amplifications and over-expression in a BTC case series. Furthermore, we tested the efficacy of drugs targeting these molecules, as single agents or in combination with gemcitabine, the standard therapeutic agent against BTC. METHODS: Immunohistochemistry, FISH and mutational analysis were performed on 49 BTC samples of intrahepatic (ICCs), extrahepatic (ECCs), and gallbladder (GBCs) origin. The effect on cell proliferation of different EGFR/HER2 pathway inhibitors as single agents or in combination with gemcitabine was investigated on BTC cell lines. Western blot analyses were performed to investigate molecular mechanisms of targeted drugs. RESULTS: EGFR is expressed in 100% of ICCs, 52.6% of ECCs, and in 38.5% of GBCs. P-MAPK and p-Akt are highly expressed in ICCs (>58% of samples), and to a lower extent in ECCs and GBCs (<46%), indicating EGFR pathway activation. HER2 is overexpressed in 10% of GBCs (with genomic amplification), and 26.3% of ECCs (half of which has genomic amplification). EGFR or its signal transducers are mutated in 26.5% of cases: 4 samples bear mutations of PI3K (8.2%), 3 cases (6.1%) in K-RAS, 4 (8.2%) in B-RAF, and 2 cases (4.1%) in PTEN, but no loss of PTEN expression is detected. EGI-1 cell line is highly sensitive to gemcitabine, TFK1 and TGBC1-TKB cell lines are responsive and HuH28 cell line is resistant. In EGI-1 cells, combination with gefitinib further increases the antiproliferative effect of gemcitabine. In TFK1 and TGBC1-TKB cells, the efficacy of gemcitabine is increased with addiction of sorafenib and everolimus. In TGBC1-TKB cells, lapatinib also has a synergic effect with gemcitabine. HuH28 becomes responsive if treated in combination with erlotinib. Moreover, HuH28 cells are sensitive to lapatinib as a single agent. Molecular mechanisms were confirmed by western blot analysis. CONCLUSION: These data demonstrate that EGFR and HER2 pathways are suitable therapeutic targets for BTCs. The combination of gemcitabine with drugs targeting these pathways gives encouraging results and further clinical studies could be warranted.

Evolution of the Experimental Models of Cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a rare, aggressive disease with poor overall survival. In advanced cases, surgery is often not possible or fails; in addition, there is a lack of effective and specific therapies. Multidisciplinary approaches and advanced technologies have improved the knowledge of CCA molecular pathogenesis, highlighting its extreme heterogeneity and high frequency of genetic and molecular aberrations. Effective preclinical models, therefore, should be based on a comparable level of complexity. In the past years, there has been a consistent increase in the number of available CCA models. The exploitation of even more complex CCA models is rising. Examples are the use of CRISPR/Cas9 or stabilized organoids for in vitro studies, as well as patient-derived xenografts or transgenic mouse models for in vivo applications. Here, we examine the available preclinical CCA models exploited to investigate: (i) carcinogenesis processes from initiation to progression; and (ii) tools for personalized therapy and innovative therapeutic approaches, including chemotherapy and immune/targeted therapies. For each model, we describe the potential applications, highlighting both its advantages and limits.