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Supplementary Figure 1. Fagan Nomogram analysis. A line is drawn connecting the pre-test probability and the point on the middle vertical line corresponding to the likelihood ratio for the test result, represented by a range of test results (boxes). This line is extended to intersect with the right-hand vertical line, which gives the post-test probability. This point is the new estimate of probability that the patient has the disease. A) Nomogram for post-test probability of having a tumor. B) Nomogram for post-test probability of having a tumor or high-risk adenoma.

Background: DNA integrity analysis could represent an alternative approach to the early detection of colorectal cancer. Previously, fluorescence long DNA (FL-DNA) in stools was extracted using a manual approach and analyzed by capillary electrophoresis assay (CE FL-DNA). We aimed to improve diagnostic accuracy using a simpler and more standardized method [Real Time PCR FL-DNA (RT FL-DNA)] for the detection of early malignant lesions in a population undergoing colorectal cancer screening.Methods: From 241 stool samples, DNA was extracted using manual and semiautomatic extraction systems and analyzed using FL-DNA tests by CE and RT assays. The RT FL-DNA approach showed slightly higher sensitivity and specificity compared with the CE FL-DNA method. Furthermore, we compared the RT FL-DNA approach with the iFOBT report.Results: Nonparametric ranking statistics were used to analyze the relationship between the median values of RT FL-DNA and the clinicohistopathologic characteristics. The median values of both variables were significantly higher in patients with cancer than in patients with noncancerous lesions. According to the Fagan nomogram results, the iFOBT and FL-DNA methods provided more accurate diagnostic information and were able to identify subgroups at varying risks of cancer.Conclusions: The combination of the semiautomatic extraction system and RT FL-DNA analysis improved the quality of DNA extracted from stool samples.Impact: RT FL-DNA shows great potential for colorectal cancer diagnosis as it is a reliable and relatively easy analysis to perform on routinely processed stool samples in combination with iFOBT. Cancer Epidemiol Biomarkers Prev; 23(11); 2553–60. ©2014 AACR.

Supplementary Table 1. CRC and CRC+HRA prevalence as a function of different FL-DNA cut offs in negative-positive iFOBTs separated by the median value of all positive iFOBTs

Advanced prostate cancers, initially sensitive to androgen deprivation therapy, frequently progress to the castration-resistant prostate cancer phenotype (CRPC) through mechanisms not yet fully understood. In this study we investigated mitochondrial involvement in the establishment of refractoriness to hormone therapy. Two human prostate cancer cell lines were used, the parental LNCaP and the resistant LNCaP-Rbic, the latter generated after continuous exposure to 20 μM of (R)-bicalutamide, the active enantiomer of Casodex®. We observed a significant decrease in mtDNA content and a lower expression of 8 mitochondria-encoded gene transcripts involved in respiratory chain complexes in both cell lines. We also found that (R)-bicalutamide differentially modulated dynamin-related protein (Drp-1) expression in LNCaP and LNCaP-Rbic cells. These data seem to indicate that the androgen-independent phenotype in our experimental model was due, at least in part, to alterations in mitochondrial dynamics and to a breakdown in the Drp-1-mediated mitochondrial network.

Cell counting is one of the basic needs of most biological experiments. Numerous methods and systems have been studied to improve the reliability of counting. However, at present, manual cell counting performed with a hemocytometer still represents the gold standard, despite several problems limiting reproducibility and repeatability of the counts and, at the end, jeopardizing their reliability in general. We present our own approach based on image processing techniques to improve counting reliability. It works in two stages: first building a high-resolution image of the hemocytometer's grid, then counting the live and dead cells by tagging the image with flags of different colours. In particular, we introduce GridMos (http://sourceforge.net/p/gridmos), a fully-automated mosaicing method to obtain a mosaic representing the whole hemocytometer's grid. In addition to offering more significant statistics, the mosaic "freezes" the culture status, thus permitting analysis by more than one operator. Finally, the mosaic achieved can thus be tagged by using an image editor, thus markedly improving counting reliability. The experiments performed confirm the improvements brought about by the proposed counting approach in terms of both reproducibility and repeatability, also suggesting the use of a mosaic of an entire hemocytometer's grid, then labelled trough an image editor, as the best likely candidate for the new gold standard method in cell counting.

Background We evaluated the role of single nucleotide polymorphisms in the CYP17A1 gene for predicting clinical outcome in castration-resistant prostate cancer (CRPC) patients treated with abiraterone. Methods Sixty-four patients were genotyped for the selected polymorphisms (rs743572, rs10883783, rs17115100 and rs284849) in CYP17A1. We hypothesized that different genotypes could be associated with progression-free survival (PFS) and overall survival (OS). Results Statistical analyses highlighted no significant associations between these polymorphisms and clinical outcome. However, individuals with the most common TT genotype for rs10883783 had a 3 months’ longer PFS than individuals with the TA + AA genotype. Conclusions With the limitation of the retrospective study design and the small sample size, the analyzed polymorphisms do not seem to be correlated with clinical outcome of CRPC patients treated with abiraterone.

Targeted therapy of metastatic colorectal cancer (mCRC) with monoclonal antibody anti-epidermal growth factor receptor (EGFR) agents, such as cetuximab (CTX) or panitumumab, is the treatment strategy of choice in patients characterised by a wild-type (wt) RAS gene status. However, despite selection based on RAS status, a high proportion of patients do not respond to therapy. EGFR methylation has been reported to have a role in predicting the response to anti-EGFR agents. The present study aimed to evaluate the role of EGFR methylation in association with the clinical outcome of patients with mCRC treated with CTX. In total, 64 patients with mCRC were assessed in the present study. Genomic DNA was extracted from tumoral tissue and EGFR methylation and mutation of the KRAS, BRAF and PIK3CA genes were analysed by pyrosequencing. EGFR expression was assessed by immunohistochemistry. The various alterations were analysed by assessing the objective response rate (ORR), progression free survival (PFS) and overall survival (OS) rates. In total, 42 cases (66%) exhibited >10% EGFR methylation and there was no correlation with EGFR expression. Mean EGFR methylation of 41 and 9% was observed in KRAS-mutated and -wt patients, respectively (P=0.05). Conversely, a high EGFR methylation was observed in BRAF-wt patients with compared with patients possessing the mutated gene (18 vs. 3%, respectively; P=0.07). EGFR methylation was significantly correlated with the OS rate [hazard ratio, 0.98; 95% confidence interval (CI), 0.96–1.00; P=0.019], but not PFS rate. In patients with a methylation rate 10%, the median OS rate was 7.5 months (95% CI, 4.4–9.4 months) and 12.0 months (95% CI, 8.7–13.9 months), respectively (P=0.034). In conclusion, the present study revealed a correlation between EGFR methylation and improved OS rate in patients treated with CTX-based chemotherapy. The presence of EGFR methylation is inversely correlated with BRAF and PIK3CA mutations, indicating that the prognostic value of gene methylation may be worth verifying in further studies.

Single nucleotide polymorphisms (SNPs) may be associated with the response or toxicity to different types of treatment. Although SNP analysis is usually performed on DNA from peripheral blood, formalin fixed paraffin-embedded (FFPE) tissue is often used for retrospective studies. We analyzed VEGF (−2578C>A, −1498C>T, −1154G>A, −634C>G, +936C>T) and eNOS (+894G>T, −786T>C, VNTR (variable number of tandem repeats) 27bp intron 4) polymorphisms by direct sequencing or Real Time PCR in 237 patients with advanced colorectal cancer. Peripheral blood was used for 153 patients, whereas only FFPE tumor tissue was available for 84 patients. All SNP frequencies were in Hardy-Weinberg Equilibrium (HWE), with the exception of VEGF −1154, which was only in HWE in peripheral blood specimens. We therefore analyzed this SNP in DNA extracted from FFPE tumor tissue compared to FFPE healthy tissue and peripheral blood from 20 patients. Numerous heterozygous patients in peripheral blood DNA were homozygous for the A-allele in both tumor and healthy FFPE tissues. Our findings indicate that, although FFPE tissue might be a suitable specimen for genotyping, VEGF −1154 does not give reliable results on this type of material. As other SNPs may also have this limitation, genotype concordance should first be confirmed by comparing results obtained from FFPE and fresh sample analyses.

Although combination chemotherapy and radiotherapy have become the standard of care in numerous tumors, the mechanisms of interaction are often still unclear. The purpose of this study was to analyze the efficacy of radiation treatment and cisplatin sequences and to investigate their mechanisms of interaction. Three melanoma cell lines were used to evaluate in vitro radiation-induced cytotoxicity before and after cisplatin treatment. Expression levels of a panel of genes were determined by real-time RT-PCR. Cytotoxic effect was evaluated by flow cytometry analysis and Comet assay. We also used normal human dermal fibroblasts (HUDE) to evaluate the cytotoxicity of the two treatments by clonogenic assay. Radiation and cisplatin used singly were not particularly effective in reducing proliferation in melanoma cells. Conversely, radiation treatment followed by cisplatin showed a strong synergistic interaction in all cell lines, with a ratio index ranging from 16 to >100. The synergistic effect was accompanied by apoptosis induction (up to 40%) and an increase in the percentage of comet-shaped nucleoids from 85% to 99%. In parallel, our results also showed that radiation treatment of HUDE fibroblasts followed by cisplatin only induced weak cytotoxicity. Our findings highlight the efficacy of the sequence radiation cisplatin in reducing cell proliferation and in inducing apoptosis in melanoma cell lines. This sequence also modulated a network of proteins involved in DNA damage repair. J. Cell. Physiol. 229: 1548–1556, 2014. © 2014 Wiley Periodicals, Inc.

A non-invasive test to facilitate the diagnosis of non-small cell lung cancer (NSCLC) and idiopathic pulmonary fibrosis (IPF) is still not available and represents an important goal. Forty-eight patients with stage I NSCLC, 45 with IPF, 30 with other idiopathic interstitial pneumonias (IIPs) including idiopathic non-specific interstitial pneumonia (NSIP) and chronic hypersensitivity pneumonitis (HP), 35 with diffuse non-malignant disease and 30 healthy donors were enrolled onto the study. Free circulating (fc)DNA and MMP-7 levels were evaluated by Real Time PCR and ELISA, respectively. Median fcDNA levels were similar in NSCLC (127 ng/mL, range 23.6–345 ng/mL) and IPF (106 ng/mL, range 22–224 ng/mL) patients, and significantly lower in IIPs patients, in individuals with other diseases and in healthy donors (p &lt, 0.05). Conversely, median MMP-7 values were significantly higher in IPF patients (9.10 ng/mL, range 3.88–19.72 ng/mL) than in those with NSCLC (6.31 ng/mL, range 3.38–16.36 ng/mL, p &lt, 0.0001), NSIP (6.50 ng/mL, range 1.50–22.47 ng/mL, p = 0.007), other diseases (5.41 ng/mL, range 1.78–15.91, p &lt, 0.0001) or healthy donors (4.35 ng/mL, range 2.45–7.23, 0.0001). Serum MMP-7 levels seem to be capable of distinguishing IPF patients from those with any other lung disease. fcDNA levels were similar in NSCLC and IPF patients, confirming its potential role as a biomarker, albeit non-specific, for the differential diagnosis of NSCLC.

Although numerous studies have focused on the link between CpG island methylator phenotypes and the development of colorectal cancer, few studies have dealt specifically with methylation profiling in rectal cancer and its role in predicting response to neoadjuvant chemoradiotherapy (NCRT). We characterized methylation profiles in normal and neoplastic tissue samples from patients with rectal cancer and assessed the role of this molecular profile in predicting chemoradioactivity. We evaluated 74 pretreatment tumor samples and 16 apparently normal tissue biopsies from rectal cancer patients submitted to NCRT. The methylation profile of 24 different tumor suppressor genes was analyzed from FFPE samples by methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA). Methylation status was studied in relation to tissue type and clinical pathological parameters, in particular, pathological response evaluated by tumor regression grade (TRG). ESR1, CDH13, RARB, IGSF4, and APC genes showed high methylation levels in tumor samples (range 18.92–49.77) with respect to normal tissue. Methylation levels of the remaining genes were low and similar in both normal (range 1.91–14.56) and tumor tissue (range 1.84–11). Analysis of the association between methylation and response to therapy in tumor samples showed that only TIMP3 methylation status differed significantly within the four TRG classes (ANOVA, P

The characterization of circulating tumor cells (CTCs) could substantially improve the management of cancer patients. However, their study is still a matter of debate, often due to lymphocyte contamination. In the present paper, an investigation of CTCs was carried out for the first time using DEPArray, a dielectrophoresis-based platform able to detect and sort pure CTCs. Analyses were conducted on peripheral blood (PB) samples from patients with metastatic colon cancer. After 100% pure cell recovery and whole genome amplification, KRAS gene mutation of CTCs was screened and compared to gene status in the primary tumor tissue. CTCs were found in 21 colon cancer patients (52.5%), with more than three tumor cells per 7.5 ml. KRAS gene mutation analysis, showed a mutational concordance between CTCs and primary tumor in 50% of matched cases. The present study demonstrates for the first time the feasibility of analyzing at the molecular level pure CTCs avoiding lymphocyte contamination using an innovative instrumentation, and a KRAS discordance between CTCs and primary tissue. Our results present dielectrophoresis-based procedures as a new standard in single cell analysis and recovery and invite careful reflection on the value of CTCs characterization.

Patients with solid cancer frequently develop bone metastases (BM). Zoledronic acid (Zometa®, ZA), routinely used to treat patients with BM, acts on osteoclasts and also has antitumor properties. We aimed to assess the effect of ZA over time in novel bone turnover markers (RANK/receptor activator of nuclear factor-k B ligand (RANK-L)/ Osteoprotegerin (OPG)) and to correlate these with serum N-terminal telopeptide (NTX). The study prospectively evaluated levels of RANK, RANK-L and OPG transcripts by real-time PCR and NTX expression by ELISA in the peripheral blood of 49 consecutive patients with advanced breast, lung or prostate cancer. All patients received the standard ZA schedule and were monitored for 12 months. Median baseline values of RANK, RANK-L and OPG were 78.28 (range 7.34–620.64), 319.06 (21.42–1884.41) and 1.52 (0.10–58.02), respectively. At 12 months, the median RANK-L value had decreased by 22% with respect to the baseline, whereas median OPG levels had increased by about 96%. Consequently, the RANK-L/OPG ratio decreased by 56% from the baseline. Median serum NTX levels decreased over the 12-month period, reaching statistical significance (p < 0.0001). Our results would seem to indicate that ZA modulates RANK, RANK-L and OPG expression, thus decreasing osteoclast activity.

Five-year survival rate for lung cancer is limited to 10% to 15%. Therefore, the identification of novel therapeutic prognostic factors is an urgent requirement. The aim of this study is thus to highlight specific biomarkers in chemoresistant non-small cell lung cancer cell lines. Therefore, we checked—in the control condition as well as after short-term pharmacological treatment with either docetaxel or gemcitabine—the expression of genes such as tumor suppressor genes (CDKN2A, DAPK, FHIT, GSTP1, MGMT, RARβ2, RASSF1A, and TIMP3), genes associated with drug resistance (BRCA1, COX2, ERCC1, IGFBP3, RRM1, and TUBB3), and stemness-related genes (CD133, OCT4, and SLUG) in two cellular models of squamous carcinoma (CAEP) and adenocarcinoma (RAL) of the lung originally established. Their promoter methylation profile was also evaluated. Drug-related genes were upregulated. Cisplatin resistance matched with high levels of BRCA1 and ERCC1 in both cell lines; docetaxel sensitivity of CAEP cells was associated to levels of TUBB3 lower than RAL cells. Although CAEP cells were more sensitive to gemcitabine, both cell lines showed high levels of RRM1. Stemness-related genes were downregulated in the control condition but became upregulated in docetaxel-resistant cells, indicating the selection of a population with stemness features. We did not find an unequivocal correspondence between gene expression and respective DNA promoter methylation status, suggesting the involvement of additional mechanisms of gene expression regulation. These results highlight specific biomarkers consistent with the different responses of the two cell lines to standard pharmacological treatments and indicate specific molecular traits for their chemoresistance.

Multicellular cancer spheroids are solid 3D tumours widely employed in pre-clinical studies for testing drugs, chemotherapy and radiotherapy treatments. The volume is considered one of the most relevant features and its changes are typically correlated to the efficacy of the tested treatment. Thereby, the correct estimation of the volume is fundamental for the whole analysis. Nevertheless, very few methods have been proposed in the literature to accurately estimate the tumour’s volume arising from one 2D projection, such as that represented by an image acquired with a standard widefield microscope. In this work, we proposed a 3D rendering method to reconstruct the surface and estimate the volume of spheroids. In particular, to assess the effectiveness of our method, we proposed a new comparison approach based on the analysis of real-world, home-made 3D objects, mimicking the spheroid morphology. The experiments performed proved the effectiveness of our method for the volume assessment, meanwhile providing a 3D reconstruction of the spheroid’s shape.

Patients with non-muscle invasive bladder cancer (NMIBC) generally have a high risk of relapsing locally after primary tumor resection. The search for new predictive markers of local recurrence thus represents an important goal for the management of this disease. We studied the copy number variations (CNVs) of 24 oncogenes (MDM4, MYCN, ALK, PDGFRA, KIT, KDR, DHFR, EGFR, MET, SMO, FGFR1, MYC, ABL1, RET, CCND1, CCND2, CDK4, MDM2, AURKB, ERBB2, TOP2A, AURKA, AR and BRAF) using multiplex ligation probe amplification technique to verify their role as predictive markers of recurrence. Formalin-fixed paraffin-embedded tissue samples from 43 patients who underwent transurethral resection of the bladder (TURB) were used; 23 patients had relapsed and 20 were disease-free after 5 years. Amplification frequencies were analyzed for all genes and MDM4 was the only gene that showed significantly higher amplification in non recurrent patients than in recurrent ones (0.65 vs. 0.3; Fisher's test p=0.023). Recurrence-free survival analysis confirmed the predictive role of MDM4 (log-rank test p=0.041). Our preliminary results indicate a putative role for the MDM4 gene in predicting local recurrence of bladder cancer. Confirmation of this hypothesis is needed in a larger cohort of NMIBC patients.

Due to depth of focus constraints, the acquisition of a single 2-D completely in-focus image of 3-D objects characterized by a relevant depth dimension is not possible with a standard light microscope. Since the Seventies numerous methods have been proposed to overcome this problem, mainly through different fusion processing techniques to extend the microscope's depth of focus. However, given a specific application, it is very difficult to know which method yields the best results because there are no validated approaches or tested metrics that are suitable for real world cases typically lacking in a reference ground truth. Although the Universal Quality Index (UQI) is widely used to evaluate output quality in image processing, it requires a reference ground truth. Some UQI extensions have been proposed to evaluate the output of fusion methods without a ground truth, but sufficient analyses have not been carried out to confirm their equivalence to the standard UQI in terms of (evaluation) performance. We propose a new method to extend the microscope's depth of focus and, using synthetic stacks of images with ground truth attached, show that it is superior to state-of-the-art methods. We also demonstrate that the output of metrics proposed as UQI extensions is different from that of the UQI. Finally, we validate a new approach to evaluate extended depth of focus methods using real world stacks of slices, as per the UQI, but without the need for a reference ground truth.

The phosphatidylinositol 3'-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway contributes to prostate cancer progression and transition to androgen-independent disease. Furthermore, recent microarray analysis demonstrates that this pathway is often deregulated during prostate cancer progression. Thus, targeting of PI3K/AKT/mTOR may present a promising therapy for castration-refractory prostate cancer (CRPC). In recent years, several interesting strategies have been developed that interfere with distinct components of the PI3K/AKT/mTOR cascade. This article discusses many of the mechanisms involved, specifically in the context of prostate cancer. In addition, we present an overview of preliminary data on the activity of mTOR inhibitors and on the key steps to evaluate which of these compounds are most suitable for the treatment of prostate cancer. Particular emphasis is also placed on the development of novel perspectives to improve the poor prognosis of patients with CRPC.

Lung cancer is the leading cause of cancer mortality worldwide and despite efforts made to improve clinical results, continuing poor survival rates indicate that novel therapeutic approaches are needed. Valproic acid (VPA), a short-chain branched fatty acid used mainly for the treatment of epilepsy and bipolar disorder, has been shown to inhibit class I histone deacetylases (HDAC-I), a group of enzymes involved in chromatin remodeling and which are thought to play a role in tumor development. Although evidence of VPA's therapeutic efficacy has also been observed in patients with solid tumors, the very high concentration required to induce antitumor activity limits its clinical usefulness. We used a panel of NSCLC cell lines to evaluate the activity and mechanisms of action of organosulfur valproic acid derivatives, a promising new class of compounds designed to improve the safety and efficacy of the valproic acid molecule and created by coupling it with a hydrogen sulfide (H(2) S)-releasing moiety. Our results highlighted the increased cytotoxic activity of the novel organosulfur derivatives, ACS33 and ACS2, with respect to VPA, starting from low concentrations. In particular, ACS2 exhibited important pro-apoptotic activity triggered by the mitochondrial pathway and also showed anti-invasion potential. Furthermore, our in vitro results identified a highly effective combination schedule of ACS2 and cisplatin capable of inducing a synergistic interaction even when the two drugs were used at low concentrations, which could prove a valid alternative to traditional chemotherapeutic regimens used for advanced lung cancer. Further studies are needed to confirm these preliminary findings.

Bladder cancer diagnosis and patient surveillance present a wide range of diagnostic methods but essentially only instrumental approaches are available in the clinical setting. Although numerous new noninvasive biomarkers have been proposed in the last 10 years, few are US FDA-approved for clinical purposes, and none are widely used in routine clinical practice. In this review, we summarize the tests developed for early diagnosis and patient surveillance and verify whether, for any, there is some level of evidence to suggest a real usefulness in a clinical setting.

BACKGROUND The efficacy of current therapy for hormone-refractory prostate cancer is still unsatisfactory and new agents and therapeutic modalities are needed. The aims of the present work were to examine the in vitro activity and mechanisms of action of different antitumor drug combinations in hormone-resistant prostate cancer (HRPC) cell lines. METHODS The activity of docetaxel (Doc), cisplatin (Cis), oxaliplatin (Oxa), SN-38 and ST1481, singly or in combination, was assessed in different HRPC cell lines (PC3, parental DU145 and taxane-resistant DU145-R) by SRB test. Apoptosis was evaluated by TUNEL and ANN-V assays. Extrusion pump activity was studied by Hoechst 33342 assay, while gene expression related to drug efflux mechanisms and DNA damage repair was analyzed by RT-PCR. RESULTS Doc induced a high cytocidal effect in the HRPC cells, whereas Cis, Oxa, SN-38 and ST1481 exerted prevalently cytostatic activity. Doc followed by ST1481 proved to be the most effective drug sequence among those investigated, producing an important synergistic effect (R.I. from 2.0 to 5.2) in all the tested cell lines. Moreover, this sequence induced a significant downregulation of xenobiotic extrusion pump and DNA damage repair gene expression. ST1481 synergistically increased the cytocidal effect of Doc, probably through a downregulation of extrusion pump activity and DNA damage repair-related genes. CONCLUSIONS Our results show that the Doc ST1481 sequence effectively reduces the cancer cell population and restores Doc activity in taxane-resistant HRPC, indicating its potential usefulness as first- or second-line treatment of hormone-refractory prostate cancer. Prostate 70: 219–227, 2010. ©2009 Wiley-Liss, Inc.

Sorafenib is a multikinase inhibitor that has shown promising therapeutic results in different tumor histotypes, both as a single agent or in combination with other treatments. We analyzed the in vitro activity of sorafenib in pancreatic cancer, one of the most lethal and chemo-radio-resistant tumors, using four human pancreatic cancer cell lines (t3m4, Capan 1, Capan 2, and MiaPaca 2), characterized by different K-ras gene status and RAF/MEK/ERK profile. Sorafenib exerted a strong anti-proliferative effect independently of RAS/RAF/MEK/ERK and induced various degrees of apoptosis in the cell lines. The mechanisms involved were explored in detail in t3m4 and Capan 1, in which sorafenib induced the highest and lowest levels of apoptosis, respectively. In t3m4, the RAF/AKT/STAT-3 rather than the RAF/MEK/ERK pathway was involved, whereas in Capan 1 cells there was a strong decrease in pMEK and pERK which was not accompanied by an important reduction in RAF, AKT, and STAT-3 proteins or in their phosphorylation. Moreover, U0126-induced MEK inhibition did not induce apoptosis in any cell line, reinforcing the hypothesis of a MEK/ERK-independent mechanism of sorafenib activity. Mcl-1 appears to play a crucial role in sorafenib-induced apoptosis. In fact, both protein and mRNA were downregulated in t3m4 and upregulated in Capan 1, in which siRNA-induced silencing resulted in the same level of apoptosis as observed in t3m4. Our results show that sorafenib exerts anti-proliferative and pro-apoptotic activity in pancreatic cancer cells. Used singly or in combination with other drugs, it could therefore represent valid treatment for pancreatic cancer.

Studies performed in different experimental and clinical settings have shown that Docetaxel (Doc) is effective in a wide range of tumors and that it exerts its activity through multiple mechanisms of action. However, the sequence of events induced by Doc which leads to cell death is still not fully understood. Moreover, it is not completely clear how Doc induces mitotic catastrophe and whether this process is an end event or followed by apoptosis or necrosis. We investigated the mechanisms by which Doc triggers cell death in hormone-refractory prostate cancer cells by analyzing cell cycle perturbations, apoptosis-related marker expression, and morphologic cell alterations. Doc induced a transient increase in G2/M phase followed by the appearance of G0/1 hypo- and hyperdiploid cells and increased p21 expression. Time- and concentration-dependent apoptosis was induced in up to 70% of cells, in concomitance with Bcl-2 phosphorylation, which was followed by caspase-2 and -3 activation. In conclusion, Doc would seem to trigger apoptosis in hormone-refractory prostate cancer cells via mitotic catastrophe through two forms of mitotic exit, in concomitance with increased p21 expression and caspase-2 activation.

Non-steroidal anti-inflammatory drugs (NSAIDs) have repeatedly shown to be effective in tumor prevention, but important side-effects limit their wide clinical use. Nitric oxide-releasing derivatives (NO-NSAIDs) are a promising class of compounds synthesized by combining a classic NSAID molecule with an NO-releasing moiety to counteract side-effects. These new chemical entities exhibit a significantly higher activity and much lower toxicity with respect to the parental drug. In the present paper, we report the results obtained from in in vitro experimental systems aimed to evaluate the activity and mechanisms of action of the novel NO-releasing aspirin derivative, NCX 4040. The in vitro studies were carried out on a panel of human colon (LoVo, LoVo Dx, WiDr, LRWZ), bladder (HT1376, MCR), and pancreatic (Capan-2, MIA PaCa-2, T3M4) cancer cell lines. With regard to colon cancer, NCX 4040 activity was also investigated in vitro in combination with drugs currently used in clinical practice and was validated in vivo on tumor-bearing mice xenografted with the aforementioned colon cancer cell lines. The in vitro studies showed a high cytotoxic activity of NCX 4040 in all tumor histotypes and demonstrated the pivotal role of the NO component in drug activity. It was also observed that NCX 4040 exerts a pro-apoptotic activity via a mitochondria-dependent pathway. Moreover, the in vivo studies on xenografted mice further confirmed the antitumor efficacy and low toxicity of NCX 4040 in colon cancer and highlighted its role as sensitizing agent of oxaliplatin cytotoxicity.

Intravesical gemcitabine (Gem) has shown promising activity against transitional cell carcinomas (TCC) of the bladder, with moderate urinary toxicity and low systemic absorption. The present phase II study evaluated the activity of biweekly intravesical treatment with Gem using a scheme directly derived from in vitro preclinical studies. Patients with Bacille Calmette-Guerin (BCG) -refractory Ta G3, T1 G1-3 TCC underwent transurethral bladder resection and then intravesical instillation with 2000 mg Gem diluted in 50 ml saline solution on days 1 and 3 for 6 consecutive weeks. Thirty-eight (95%) of the 40 patients showed persistent negative post-treatment cystoscopy and cytology 6 months after Gem treatment, while the remaining 2 patients relapsed at 5 and 6 months. At a median follow-up of 28 months, recurrences had occurred in 14 patients. Among these, four had downstaged (T) disease, three had a lower grade (G) lesion and three had a reduction in both T and G. Urinary and systemic toxicity was very low, with no alterations in biochemical profiles. In conclusion, biweekly instillation of Gem proved active in BCG-refractory Ta G3, T1 G1-3 TCC. Our results highlight the importance of preclinical studies using in vitro systems that adequately reproduce the conditions of intravesical clinical treatment to define the best therapeutic schedule.

Objective: Taxanes and fluoropyrimidines are active in metastatic breast cancer (MBC), and their combination has proven effective in anthracycline-refractory patients. We conducted a phase I study to determine the maximum tolerated dose (MTD) of uracil plus tegafur (UFT) given in combination with leucovorin (LV) and paclitaxel (Pacl) in patients with refractory MBC. Methods: Pacl was infused at a fixed dose of 150 mg/m2 on day 1. UFT, at doses escalated by 50 mg/m2 starting from 200 mg/m2 · day, and LV, at a fixed dose of 90 mg/day, were given orally every 8 h for 11 days (days 3–13). Cohorts of at least 3 patients were treated at each dose level, and if 1 experienced dose-limiting toxicity (DLT), a maximum of 3 additional patients were added at the same dose level. MTD was reached if 2 out of the 6 patients experienced DLT. Results: Sixteen patients were enrolled in the study. The most important toxicity observed was hematological. Nonhematological toxicities were paresthesia and myalgia, asthenia, nausea, and mucositis. DLT occurred in only 1 patient (grade 3 hepatic toxicity). Conclusions: The recommended dose for a subsequent phase II trial is Pacl 150 mg/m2 on day 1, and UFT 300 mg/m2 and LV 90 mg on days 3–13, every 2 weeks.

Background Bevacizumab plus chemotherapy is a widely used therapeutic option for first-line treatment of metastatic colorectal cancer (mCRC). However, molecular predictors of bevacizumab efficacy have not yet been identified. We analyzed vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) polymorphisms in relation to response to bevacizumab. Methods Two hundred and thirty-seven patients with mCRC enrolled onto the phase III prospective multicentre randomized “Italian Trial in Advanced Colorectal Cancer (ITACa)” trial were evaluated. One hundred fourteen patients received chemotherapy plus bevacizumab (CT + B) and 123 received chemotherapy (CT) alone. Five single nucleotide polymorphisms (SNPs) (−2578, −1498, −1154, −634 and +936) for VEGF and 2 SNPs (−786, +894) and one variable number tandem repeat in intron 4 for eNOS were analyzed for each patient. The polymorphisms were assessed in relation to progression-free survival (PFS), objective response rate (ORR) and overall survival (OS). Results VEGF 936C/T, eNOS +894 G/T and VNTR were significantly correlated with outcome in CT + B patients, but not in CT-only patients. In particular, patients with a specific haplotype combination of the 2 eNOS polymorphisms (defined eNOS Haplo1/Haplo1 and eNOS Haplo 2/Haplo2) showed significantly longer PFS (15.0 vs 9.1 months, P = 0.001) and OS (34.5 vs 20.5 months P = 0.002), and a higher ORR (71 vs 45.9%, P = 0.013) than those with the other genotypes, respectively. Conclusions Specific eNOS polymorphisms may be capable of identifying a subset of mCRC patients who are more responsive to bevacizumab-based chemotherapy. If confirmed, these results would permit individually tailored treatment with bevacizumab. Electronic supplementary material The online version of this article (doi:10.1186/s12967-015-0619-5) contains supplementary material, which is available to authorized users.

Introduction. The detection of tumor-specific markers in urine has paved the way for new early noninvasive diagnostic approaches for prostate cancer. We evaluated the DNA integrity in urine supernatant to verify its capacity to discriminate between prostate cancer and benign diseases of the urogenital tract.Patients and Methods. A total of 131 individuals were enrolled: 67 prostate cancer patients and 64 patients with benign diseases of the urogenital tract (control group). Prostate-specific antigen (PSA) levels were determined. Urine cell-free (UCF) DNA was isolated and sequences longer than 250 bp corresponding to 3 genes (c-MYC,HER2, andAR) were quantified by Real-Time PCR to assess UCF-DNA integrity.Results. UCF-DNA was quantifiable in all samples, while UCF-DNA integrity was evaluable in all but 16 samples. Receiver operating characteristic analysis showed an area under the curve of 0.5048 for UCF-DNA integrity and 0.8423 for PSA. Sensitivity was 0.58 and 0.95 for UCF-DNA integrity and PSA, respectively. Specificity was 0.44 and 0.69, respectively.Conclusions. UCF-DNA integrity showed lower accuracy than PSA and would not seem to be a reliable marker for early prostate cancer diagnosis. Despite this, we believe that UCF-DNA could represent a source of other biomarkers and could detect gene alterations.

Circulating cell-free DNA has been recognized as an accurate marker for the diagnosis of prostate cancer, whereas the role of urine cell-free DNA (UCF DNA) has never been evaluated in this setting. It is known that normal apoptotic cells produce highly fragmented DNA while cancer cells release longer DNA. We thus verified the potential role of UCF DNA integrity for early prostate cancer diagnosis. UCF DNA was isolated from 29 prostate cancer patients and 25 healthy volunteers. Sequences longer than 250 bp (c-Myc,BCAS1, andHER2) were quantified by real-time PCR to verify UCF DNA integrity. Receiver operating characteristic (ROC) curve analysis revealed an area under the curve of 0.7959 (95% CI 0.6729–0.9188). At the best cut-off value of 0.04 ng/μL, UCF DNA integrity analysis showed a sensitivity of 0.79 (95% CI 0.62–0.90) and a specificity of 0.84 (95% CI 0.65–0.94). These preliminary findings indicate that UCF DNA integrity could be a promising noninvasive marker for the early diagnosis of prostate cancer and pave the way for further research into this area.

Primer sequences for VEGF and eNOS SNPs.

Correlation between eNOS haplotypes and ORR.

Genotype distribution of VEGF and eNOS polymorphisms.

The present study deals with the preparation of albumin nanocapsules containing fenretinide and their evaluation in experimental models of human non-small cell lung cancer. These nanocapsules showed enhanced antitumor activity with respect to free fenretinide due to the solubilization effect of albumin on the hydrophobic drug, known to improve bioavailability. The high expression of caveolin-1 on the A549 cell surface further enhanced the antitumor activity of the nanoencapsulated fenretinide. Caveolin-1 favored albumin uptake and improved the efficacy of the fenretinide-loaded albumin nanocapsules, especially in 3-D cultures where the densely packed 3-D structures impaired drug diffusibility and severely reduced the activity of the free drug. The efficacy of the fenretinide albumin nanocapsules was further confirmed in tumor xenograft models of A549 by the significant delay in tumor progression observed with respect to control after intravenous administration of the novel formulation. From the Clinical Editor This study describes the preparation of fenretinide containing albumin nanocapsules and their evaluation in experimental models of non-small cell lung cancer, showing enhanced antitumor activity compared to free fenretinide.

Purpose: The identification of new molecular markers for the early detection of colorectal cancer has become an important objective. We compared the sensitivity and specificity of free circulating DNA with that of the more conventional carcinoembryonic antigen (CEA) and evaluated the two markers in combination. Experimental Design: The study was carried out on 75 healthy donors and 75 colorectal cancer patients. Free DNA was determined in serum with quantitative PCR analysis. The diagnostic accuracy of each assay was calculated using receiver operating characteristic (ROC) curves. The diagnostic relevance of the two-marker combination was analyzed by the logistic regression model. Results: Median free DNA concentration was ∼5-fold higher in patients than in healthy donors (P < 0.001). The area under the ROC curve was 0.86, and when 12.5 ng/mL was used as cutoff, 81.3% sensitivity and 73.3% specificity were observed for the overall series. As CEA and free DNA provided independent diagnostic information, they were also considered in combination. ROC curve analysis of the combined CEA and free DNA algorithms showed a higher diagnostic capacity (area under the ROC curve, 0.92) than that of markers considered singly, with 84% sensitivity and 88% specificity. Conclusions: Free circulating DNA, especially when used in combination with CEA, represents a potentially useful tool for the diagnosis of early-stage colorectal cancer.