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Although there is still no standard treatment for recurrent glioblastoma multiforme (rGBM), re-irradiation could be a therapeutic option. We retrospectively evaluated the efficacy and safety of re-irradiation using helical TomoTherapy (HT) with a simultaneous integrated boost (SIB) technique in patients with rGBM. 24 patients with rGBM underwent HT-SIB. A total dose of 20 Gy was prescribed to the Flair (fluid-attenuated inversion recovery) planning tumor volume (PTV) and 25 Gy to the PTV-boost (T1 MRI contrast enhanced area) in 5 daily fractions to the isodose of 67% (maximum dose within the PTV-boost was 37.5 Gy). Toxicity was evaluated by converting the 3D-dose distribution to the equivalent dose in 2 Gy fractions on a voxel-by-voxel basis. Median follow-up after re-irradiation was 27.8 months (range 1.6–88.5 months). Median progression-free survival (PFS) was 4 months (95% CI 2.0–7.9 months), while 6-month PFS was 41.7% (95% CI 22.2–60.1 months). Median overall survival following re-irradiation was 10.7 months (95% CI 7.4–16.1 months). There were no cases of re-operation due to early or late toxicity. Our preliminary results suggest that helical TomoTherapy with the proposed SIB technique is a safe and feasible treatment option for patients with rGBM, including those large disease volumes, reducing toxicity.

Subcellular spatial location is an essential descriptor of molecules biological function. Presently, super-resolution microscopy techniques enable quantification of subcellular objects distribution in fluorescence images, but they rely on instrumentation, tools and expertise not constituting a default for most of laboratories. We propose a method that allows resolving subcellular structures location by reinforcing each single pixel position with the information from surroundings. Although designed for entry-level laboratory equipment with common resolution powers, our method is independent from imaging device resolution, and thus can benefit also super-resolution microscopy. The approach permits to generate density distribution maps (DDMs) informative of both objects’ absolute location and self-relative displacement, thus practically reducing location uncertainty and increasing the accuracy of signal mapping. This work proves the capability of the DDMs to: (a) improve the informativeness of spatial distributions, (b) empower subcellular molecules distributions analysis, (c) extend their applicability beyond mere spatial object mapping. Finally, the possibility of enhancing or even disclosing latent distributions can concretely speed-up routine, large-scale and follow-up experiments, besides representing a benefit for all spatial distribution studies, independently of the image acquisition resolution. DDMaker, a Software endowed with a user-friendly Graphical User Interface (GUI), is also provided to support users in DDMs creation.

Background Concurrent chemotherapy and radiation using conventional fractionation is the standard treatment for inoperable, locally advanced non-small-cell lung cancer (NSCLC). We tested accelerated hypofractionated radiotherapy (AHR) and chemotherapy for the treatment of locally advanced NSCLC. Methods Eligible patients with locally advanced NSCLC were treated with induction chemotherapy (cisplatin and docetaxel), followed by AHR using tomotherapy and consolidation chemotherapy. The prescribed doses were 30 Gy/5 daily fractions at the reference isodose (60–70%) to the tumor, and 25 Gy/5 daily fractions to the clinically involved lymph nodes. The primary end-point was response rate (RR); the secondary end-points were acute and late side-effects, local progression-free survival (PFS), metastasis-free survival (MFS) and overall survival (OS). This trial closed before the first planned interim analysis due to poor accrual. Results From January 2009 to January 2012, 17 of the 23 enrolled patients were evaluable. Treatment yielded an overall RR of 82%. Median follow-up was 87 months (range: 6–87), local PFS was 19.8 months (95% CI 9.7 - not reached), MFS was 9.7 months (95% CI 5.8–46.0) and OS was 23 months (95% CI 8.4–48.4). 70% of patients experienced acute G4 neutropenia, 24% G4 leukopenia, 24% G3 paresthesia, 4% G3 cardiac arrythmia, 4% underwent death after chemotherapy. Late toxicity was represented by 24% dyspnea G3. Conclusions AHR combined with chemotherapy is feasible with no severe side-effects, and it appears highly acceptable by patients. Trial registration This study is registered with the EudractCT registration 2008-006525-14. Registered on 9 December 2008.

BackgroundThe presence of hypoxic cells in high-grade glioma (HGG) is one of major reasons for failure of local tumour control with radiotherapy (RT). The use of hyperbaric oxygen therapy (HBO) could help to overcome the problem of oxygen deficiency in poorly oxygenated regions of the tumour. We propose an innovative approach to improve the efficacy of hypofractionated stereotactic radiotherapy (HSRT) after HBO (HBO-RT) for the treatment of recurrent HGG (rHGG) and herein report the results of an ad interim analysis.MethodsWe enrolled a preliminary cohort of 9 adult patients (aged >18 years) with a diagnosis of rHGG. HSRT was administered in daily 5-Gy fractions for 3-5 consecutive days a week. Each fraction was delivered up to maximum of 60 minutes after HBO.ResultsMedian follow-up from re-irradiation was 11.6 months (range: 3.2-11.6 months). The disease control rate (DCR) 3 months after HBO-RT was 55.5% (5 patients). Median progression-free survival (mPFS) for all patients was 5.2 months (95%CI: 1.34-NE), while 3-month and 6-month PFS was 55.5% (95%CI: 20.4-80.4) and 27.7% (95%CI: 4.4-59.1), respectively. Median overall survival (mOS) of HBO-RT was 10.7 months (95% CI: 7.7-NE). No acute or late neurologic toxicity >grade (G)2 was observed in 88.88% of patients. One patient developed G3 radionecrosis.ConclusionsHSRT delivered after HBO appears to be effective for the treatment of rHGG, it could represent an alternative, with low toxicity, to systemic therapies for patients who cannot or refuse to undergo such treatments.Clinical Trial Registrationwww.ClinicalTrials.gov, identifier NCT 03411408.

In this Special Issue, we have invited leading groups to contribute review and original research articles on the role of androgen receptor in breast cancer. Three reviews within this Research Topic address some of the questions still pending and identify challenges in the field, with the aim of re-examining the intricate molecular landscape of AR and offering new clues as to its prognostic and therapeutic value in breast cancers.

Hypofractionation is currently considered a valid alternative to conventional radiotherapy for the treatment of patients with organ-confined prostate cancer. Recent data have demonstrated that extreme hypofractionation, which involves the use of a high radiation dose per delivered fraction and concomitant reduction of sessions, is a safe and effective treatment, even though its radiobiological rationale is still lacking. The present work aims to investigate the biological basis sustaining this approach and to evaluate the potential of a hypofractionated regimen in combination with androgen deprivation therapy, one of the major standards of care for prostate cancer. Findings show that androgen receptor (AR) modulation, by use of androgens and antiandrogens, has a significant impact on cell survival, especially in hypoxic conditions (4% O2). Subsequent experiments have revealed that AR activity as a transcription factor is involved in the onset of malignant senescence-associated secretory phenotype (SASP) and activation of DNA repair cascade. In particular, we found that AR stimulation in hypoxic conditions promotes the enhanced transcription of ATM gene, the cornerstone kinase of the DNA damage repair genes. Together, these data provide new potential insights to justify the use of androgen deprivation therapy, in particular with second-generation anti-androgens such as enzalutamide, in combination with radiotherapy.

Prostate cancer represents the major cause of cancer-related death in men and patients frequently develop drug-resistance and metastatic disease. Most studies focus on hormone-resistance mechanisms related to androgen receptor mutations or to the acquired property of prostate cancer cells to over-activate signaling pathways. Tumor microenvironment plays a critical role in prostate cancer progression. However, the mechanism involving androgen/androgen receptor signaling in cancer associated fibroblasts and consequences for prostate cancer progression still remains elusive. We now report that prostate cancer associated fibroblasts express a transcriptional-incompetent androgen receptor. Upon androgen challenging, the receptor co-localizes with the scaffold protein filamin A in the extra-nuclear compartment of fibroblasts, thus mediating their migration and invasiveness. Cancer-associated fibroblasts move towards epithelial prostate cancer cells in 2D and 3D cultures, thereby inducing an increase of the prostate cancer organoid size. Androgen enhances both these effects through androgen receptor/filamin A complex assembly in cancer-associated fibroblasts. An androgen receptor-derived stapled peptide, which disrupts the androgen receptor/filamin A complex assembly, abolishes the androgen-dependent migration and invasiveness of cancer associated fibroblasts. Notably, the peptide impairs the androgen-induced invasiveness of CAFs in 2D models and reduces the overall tumor area in androgen-treated 3D co-culture. The androgen receptor in association with β1 integrin and membrane type-matrix metalloproteinase 1 activates a protease cascade triggering extracellular matrix remodeling. The peptide also impairs the androgen activation of this cascade. This study offers a potential new marker, the androgen receptor/filamin A complex, and a new therapeutic approach targeting intracellular pathways activated by the androgen/androgen receptor axis in prostate cancer-associated fibroblasts. Such a strategy, alone or in combination with conventional therapies, may allow a more efficient treatment of prostate cancer.

Cancer is a complex disease in which both genetic defects and microenvironmental components contribute to the development, progression, and metastasization of disease, representing major hurdles in the identification of more effective and safer treatment regimens for patients. Three-dimensional (3D) models are changing the paradigm of preclinical cancer research as they more closely resemble the complex tissue environment and architecture found in clinical tumors than in bidimensional (2D) cell cultures. Among 3D models, spheroids and organoids represent the most versatile and promising models in that they are capable of recapitulating the heterogeneity and pathophysiology of human cancers and of filling the gap between conventional 2D in vitro testing and animal models. Such 3D systems represent a powerful tool for studying cancer biology, enabling us to model the dynamic evolution of neoplastic disease from the early stages to metastatic dissemination and the interactions with the microenvironment. Spheroids and organoids have recently been used in the field of drug discovery and personalized medicine. The combined use of 3D models could potentially improve the robustness and reliability of preclinical research data, reducing the need for animal testing and favoring their transition to clinical practice. In this review, we summarize the recent advances in the use of these 3D systems for cancer modeling, focusing on their innovative translational applications, looking at future challenges, and comparing them with most widely used animal models.

Purpose The role played by radiation therapy after pleurectomy/decortication or surgical biopsy in malignant pleural mesothelioma is uncertain. We treated patients with accelerated hypofractionated radiotherapy using helical tomotherapy and intensity-modulated arc therapy in an attempt to keep lung toxicity to a minimum. The present study reports the feasibility and toxicity of this approach. Material and Methods Between 2008 and 2012, 36 patients with malignant pleural mesothelioma underwent accelerated hypofractionated radiotherapy to the hemithorax after pleurectomy/decortication (19 patients) or biopsy (17 patients). The prescription dose was 25 Gy in five fractions over 5 consecutive days. Results We observed three patients with G3 pneumonitis, five cases of grade 2 dyspnea and six cases of grade 2 cough. The median follow-up was 37 months (range: 3–54 months). The median overall survival for patients who underwent pleurectomy/decortication followed by radiotherapy was 21.6 months [95% confidence interval (95% CI): 15.5–24.1] compared to 19.4 months for patients not submitted to surgery. Conclusion Treatment of intact lung with pleural intensity-modulated arc irradiation in malignant pleural mesothelioma patients with malignant pleural mesothelioma proved safe and feasible, with an acceptable rate of pneumonitis. Survival rates were encouraging for both biopsy-only and pleurectomy/decortication groups. We are currently conducting a phase II dose escalation trial in a similar patient setting to prospectively evaluate the impact of radiotherapy on toxicity, disease-free survival and overall survival.

Silvia Carloni,1 Giulia Gallerani,1 Anna Tesei,1 Emanuela Scarpi,2 Giorgio Maria Verdecchia,3 Salvatore Virzì,4 Francesco Fabbri,1 Chiara Arienti1 1Biosciences Laboratory, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, 2Unit of Biostatistics and Clinical Trials, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, 3Department of Surgery and Advanced Cancer Therapies, Morgagni-Pierantoni Hospital, Forlì, 4Department of Surgery, Bentivoglio Hospital, Bologna, Italy Objective: We investigated the correlation between ploidy or S-phase fraction (SPF) and the clinical pathological characteristics of patients with peritoneal carcinomatosis from ovarian cancer. We also assessed their relation with the in vivo and in vitro response to several chemotherapeutic agents. Patients and methods: Fifty-three patients with peritoneal carcinomatosis from ovarian cancer were enrolled. Frozen tumor tissue was dissociated by a detergent–trypsin method, and the resulting cell suspension was stained with RNase A and propidium iodide. Samples were then analyzed for ploidy and SPF by flow cytometry. Fresh tumor tissue was dissociated by enzymatic digestion, and cells were exposed to different concentrations of cisplatin, adriamycin, carboplatin, gemcitabine and taxol for 72 hours. In vitro drug sensitivity was then measured using the sulforhodamine B assay.Results: No significant correlation was found between ploidy or SPF and patient characteristics, even though primary carcinomas were mainly hyperdiploid and more proliferative than recurrent tumors. SPF differed significantly among ploidy categories (P=0.01), and high SPF was associated with short-term survival (P=0.48). Patients with multiploid tumors were the most resistant to platinum-based chemotherapy, whereas those with hyperdiploid tumors were the most responsive. In vitro multiploid tumors were the least sensitive, while hypodiploid samples showed the highest sensitivity to the tested drugs. Sensitivity to adriamycin was significantly correlated with ploidy (P=0.03), whereas sensitivity to taxol was correlated with SPF (P=0.04).Conclusion: Our results indicate that ploidy and SPF could facilitate the choice of therapy for patients with peritoneal carcinomatosis. Keywords: DNA index, aneuploidy, SPF, chemotherapeutic agent, in vitro sensitivity, in vivo response&nbsp

Despite the gradual decrease in incidence, gastric cancer is still the third leading cause of cancer death worldwide. Although chemotherapy enhances overall survival and quality of life in advanced disease, the median overall survival is < 12 months. In recent years, the human epidermal growth factor receptor (ErbB) family has been extensively investigated in gastric cancer. The ErbB family is composed of four closely-related members: ErbB-1 (HER1 or epidermal growth factor receptor, EGFR), ErbB-2 (HER2), ErbB-3 (HER3), and ErbB-4 (HER4), all of which play a critical role in regulating cell growth, proliferation and migration of tumors. It is well known that gastric cancer overexpresses HER in a heterogeneous pattern, especially EGFR, and HER2. HER3 is another important member of the ErbB family that preferentially activates the phosphatidylinositol 3-kinase (PI3K) pathway. Furthermore, its heterodimerization with HER2 seems fundamental for steering HER2-overexpressing breast cancer tumor growth. Less is known about the impact of HER4 on gastric cancer. Improved survival from the use of trastuzumab has paved the way for ErbB receptor family-targeted treatments in gastric cancer. However, unlike trastuzumab, ErbB receptor-targeted drugs have not consistently maintained the encouraging results obtained in preclinical and early clinical trials. This may be attributable to the intrinsic heterogeneity of gastric cancer and/or to the lack of standardized test quality for established biomarkers used to evaluate these biological targets. This review presents an overview of the most recent clinical studies on agents targeting the ErbB family in gastric cancer.

Despite the key role played by androgen receptor (AR) in tumor cell aggressiveness and prostate cancer (PCa) progression, its function in the tumor microenvironment (TME) is still controversial. Increasing studies highlight the crucial role played by TME modulation in treatment outcome and tumor cell spreading. In this context, targeting specific constituents of the TME could be considered an alternative approach to classic treatments directed against cancer cells. Currently, androgen deprivation therapy (ADT) is a routinely adopted strategy in the management of PCa, with initial success, and consecutive fail. A possible justification to this is the fact that ADT aims to target all the transcription/translation-related activities of AR, which are typical of tumor epithelial cells. Less is still known about side effects of ADT on TME. Cancer Associated Fibroblasts (CAFs), for example, express a classic AR, mostly confined in the extra-nuclear portion of the cell. In CAFs ADT exerts a plethora of non-transcriptional effects, depending by the protein partner linked to AR, leading to cell migration, proliferation, and differentiation. In recent years, substantial progress in the structure-function relationships of AR, identification of its binding partners and function of protein complexes including AR have improved our knowledge of its signaling axis. Important AR non-genomic effects and lots of its cytoplasmatic binding partners have been described, pointing out a fine control of AR non-genomic pathways. Accordingly, new AR inhibitors have been designed and are currently under investigation. Prompt development of new approaches to target AR or block recruitment of its signaling effectors, or co-activators, is urgently needed. The present review takes an in-depth look at current literature, furnishing an exhaustive state-of-the-art overview of the non-genomic role of AR in PCa, with particular emphasis on its involvement in TME biology.

Introduction: Despite the increasing financial outlay on cancer research and drug discovery, many advanced cancers remain incurable. One possible strategy for increasing the approval rate of new anticancer drugs for use in clinical practice could be represented by three-dimensional (3D) tumor models on which to perform in vitro drug screening. There is a general consensus among the scientific community that 3D tumor models more closely recapitulate the complexity of tumor tissue architecture and biology than bi-dimensional cell cultures. In a 3D context, cells are connected to each other through tissue junctions and show proliferative and metabolic gradients that resemble the intricate milieu of organs and tumors. Areas covered: The present review focuses on available techniques for generating tumor spheroids and discusses current and future applications in the field of drug discovery. The article is based on literature obtained from PubMed. Expert opinion: Given the relative simplicity of spheroid models with respect to clinical tumors, we must be careful not to overestimate the reliability of their drug-response prediction capacity. The next challenge is to combine our knowledge of co-culture methodologies with high-content imaging and advanced microfluidic technologies to improve the readout and biomimetic potential of spheroid-based models.

Introduction: Pancreatic cancer (PC) is one of the most lethal tumor worldwide, with no prognosis improvement over the past 20-years. The silent progressive nature of this neoplasia hampers the early diagnosis, and the surgical resection of the tumor, thus chemotherapy remains the only available therapeutic option. Sigma receptors (SRs) are a class of receptors proposed as new cancer therapeutic targets due to their over-expression in tumor cells and their involvement in cancer biology. The main localization of these receptors strongly suggests their potential role in ER unfolded protein response (ER-UPR), a condition frequently occurring in several pathological settings, including cancer. Our group has recently identified RC-106, a novel pan-SR modulator with good in vitro antiproliferative activities toward a panel of different cancer cell lines. In the present study, we investigated the in vitro properties and pharmacological profile of RC-106 in PC cell lines with the aim to identify a potential lead candidate for the treatment of this tumor. Methods: Pancreatic cancer cell lines Panc-1, Capan-1, and Capan-2 have been used in all experiments. S1R and TMEM97/S2R expression in PC cell lines was quantified by Real-Time qRT-PCR and Western Blot experiments. MTS assay was used to assess the antiproliferative effect of RC-106. The apoptotic properties of RC-106 was evaluated by TUNEL and caspase activation assays. GRP78/BiP, ATF4, and CHOP was quantified to evaluate ER-UPR. Proteasome activity was investigated by a specific fluorescent-based assay. Scratch wound healing assay was used to asses RC-106 effect on cell migration. In addition, we delineated the in vivo pharmacokinetic profile and pancreas distribution of RC-106 in male CD-1 mice. Results: Panc-1, Capan-1, and Capan-2 express both SRs. RC-106 exerts an antiproliferative and pro-apoptotic effect in all examined cell lines. Cells exposure to RC-106 induces the increase of the expression of ER-UPR related proteins, and the inhibition of proteasome activity. Moreover, RC-106 is able to decrease PC cell lines motility. The in vivo results show that RC-106 is more concentrated in pancreas than plasma. Conclusion: Overall, our data evidenced that the pan-SR modulator RC-106 is an optimal candidate for in vivo studies in animal models of PC.

A Correction to this paper has been published: https://doi.org/10.1038/s41419-021-03483-4

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.

HER2-positive advanced gastric cancer patients frequently develop resistance to trastuzumab through mechanisms still poorly understood. In breast cancer, other members of the HER-family are known to be involved in trastuzumab-resistance, as is overexpression of the scaffold protein IQGAP1. In the present work, we investigated acquired resistance to trastuzumab in gastric cancer experimental models. Trastuzumab-resistant (HR) subclones derived from 3 HER2-overexpressing gastric cancer cells were generated and characterized for alterations in HER2-signaling mechanisms by next-generation sequencing, immunohistochemical, western blot and qRT-PCR techniques, and molecular modeling analysis. All subclones showed a reduced growth rate with respect to parental cell lines but each had a different resistance mechanism. In NCI N87 HR cells, characterized by a marked increase in HER2-signaling pathways with respect to the parental cell line, trastuzumab sensitivity was restored when IQGAP1 expression was silenced. AKG HR subclone showed higher HER3 protein expression than the parental line. High nuclear HER4 levels were observed in KKP HR cells. In conclusion, our study revealed that high IQGAP1 expression leads to resistance to trastuzumab in gastric cancer. Furthermore, 2 new mutations of the HER2 gene that may be involved in acquired resistance were identified in AKG HR and KKP HR subclones.

Despite the interest aroused by sigma receptors (SRs) in the area of oncology, their role in tumor biology remains enigmatic. The predominant subcellular localization and main site of activity of SRs are the endoplasmic reticulum (ER). Current literature data, including recent findings on the sigma 2 receptor subtype (S2R) identity, suggest that SRs may play a role as ER stress gatekeepers. Although SR endogenous ligands are still unknown, a wide series of structurally unrelated compounds able to bind SRs have been identified. Currently, the identification of novel antiproliferative molecules acting via SR interaction is a challenging task for both academia and industry, as shown by the fact that novel anticancer drugs targeting SRs are in the preclinical-stage pipeline of pharmaceutical companies (i.e., Anavex Corp. and Accuronix). So far, no clinically available anticancer drugs targeting SRs are still available. The present review focuses literature advancements and provides a state-of-the-art overview of SRs, with emphasis on their involvement in cancer biology and on the role of SR modulators as anticancer agents. Findings from preclinical studies on novel anticancer drugs targeting SRs are presented in brief.

Concerns, challenges and promises of high-content analysis of 3D cellular models There is intense excitement in the scientific community about 3D cellular model systems because they promise to resemble and recapitulate the in vivo tissue environment more faithfully than 2D systems1. A rapidly expanding offering of commercially available in vitro technologies for high-throughput 3D cell-based disease models, combined with advances in material sciences, are enabling widespread application and adoption of these models across academic and industrial research groups. However, there is a lack of conclusive evidence that such models accurately recapitulate in vivo tissue physiology and disease pathophysiology, and thereby provide sufficiently quantitative and reproducible data to replace current models and improve the clinical success rates of drug candidates. Prominent cellular high-content screening (HCS) and bioimage informatics societies are therefore calling for further debate to discuss the value of these emerging 3D model systems in an effort to establish more transparent and standardized guidelines in the field. Specifically, scientific community representatives highlight the lack of validated methodologies and software tools that enable robust quantitative analysis of the vast number of newer 3D cellular models.

The past two decades have seen a shift from cytotoxic drugs to targeted therapy in medical oncology. Although targeted therapeutic agents have shown more impressive clinical efficacy and minimized adverse effects than traditional treatments, drug resistance has become the main limitation to their benefits. Several preclinical in vitro/in vivo models of acquired resistance to targeted agents in clinical practice have been developed mainly by using two strategies: i) genetic manipulation for modeling genotypes of acquired resistance, and ii) in vitro/in vivo selection of resistant models. In the present work, we propose a unifying framework, for investigating the underlying mechanisms responsible for acquired resistance to targeted therapeutic agents, starting from the generation of drug-resistant cellular subclones to the description of silencing procedures used for restoring the sensitivity to the inhibitor. This simple time- and cost-effective approach is widely applicable, and could be easily extended to investigate resistance mechanisms to other targeted therapeutic drugs in different tumor histotypes.

Aim: HPV DNA has never been investigated in nipple discharges (ND) and serum–derived extracellular vesicles, although its presence has been reported in ductal lavage fluids and blood specimens. Materials & methods: We analyzed 50 ND, 22 serum-derived extracellular vesicles as well as 51 pathologic breast tissues for the presence of 16 HPV DNA types. Results: We show that the presence of HPV DNA in the ND is predictive of HPV DNA-positive breast lesions and that HPV DNA is more represented in intraductal papillomas. We also show the presence of HPV DNA in the serum-derived extracellular vesicles. Conclusion: Our data supports the use of liquid biopsy to detect HPV DNA in breast pathology.

Although several molecular targets against cancer have been identified, there is a continuous need for new therapeutic strategies. Sigma Receptors (SRs) overexpression has been recently associated with different cancer conditions. Therefore, novel anticancer agents targeting SRs may increase the specificity of therapies, overcoming some of the common drawbacks of conventional chemotherapy. Areas covered: The present review focuses on patent documents disclosing SR modulators with possible application in cancer therapy and diagnosis. The analysis reviews patents of the last two decades (1996-2016); patents were grouped according to target subtypes (S1R, S2R, pan-SRs) and relevant Applicants. The literature was searched through Espacenet, ISI Web, PatentScope and PubMed databases. Expert opinion: The number of patents related to SRs and cancer has increased in the last twenty years, confirming the importance of this receptor family as valuable target against neoplasias. Despite their short history in the cancer scenario, many SR modulators are at pre-clinical stage and one is undergoing a phase II clinical trial. SRs ligands may represent a powerful source of innovative antitumor therapeutics. Further investigation is needed for validating SR modulators as anti-cancer drugs. We strongly hope that this review could stimulate the interest of both Academia and pharmaceutical companies.

Radiation therapy is one of the most important treatment modalities for thoracic tumors. Despite significant advances in radiation techniques, radiation-induced lung injury (RILI) still occurs in up to 30% of patients undergoing thoracic radiotherapy, and therefore remains the main dose-limiting obstacle. RILI is a potentially lethal clinical complication of radiotherapy that has 2 main stages: an acute stage defined as radiation pneumonitis, and a late stage defined as radiation-induced lung fibrosis. Patients who develop lung fibrosis have a reduced quality of life with progressive and irreversible organ malfunction. Currently, the most effective intervention for the treatment of lung fibrosis is lung transplantation, but the lack of available lungs and transplantation-related complications severely limits the success of this procedure. Over the last few decades, advances have been reported in the use of mesenchymal stem cells (MSCs) for lung tissue repair and regeneration. MSCs not only replace damaged lung epithelial cells but also promote tissue repair through the secretion of anti-inflammatory and anti-fibrotic factors. Here, we present an overview of MSC-based therapy for radiation-induced lung fibrosis, focusing in particular on the molecular mechanisms involved and describing the most recent preclinical and clinical studies carried out in the field.

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.

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.

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.

Based on our previous preclinical data, a phase I study was designed to investigate the tolerability of a novel sequence, docetaxel (DOC)-gemcitabine (GEM), in the treatment of non small cell lung cancer (NSCLC). Preclinical study: We evaluated the cytotoxicity of DOC and GEM on NSCLC cell lines and assessed the type of interaction between drug activities following different treatment schemes. Clinical study: Fifteen patients with stage IIIB-IV NSCLC received DOC (day 1) and GEM (days 3 and 8) every 21 days. Dose escalation of both agents was used to identify the maximum tolerated dose. The study was closed at the fifth dose level due to the occurrence of three dose-limiting toxicities: grade 4 febrile neutropoenia, persistent grade 2 fever and grade 3 diarrhoea. The most frequent toxicity was neutropoenia. Non haematological toxicities were diarrhoea, nausea and vomiting, mucositis and alopoecia. Of the 14 evaluable patients, 1 complete response, 4 partial responses, 4 stable diseases and 5 disease progressions were observed. Based on the results of the present study, a phase II trial is ongoing using the fourth dose levels.

Cell invasiveness quantification is of paramount importance in cancer research and is often evaluated in vitro through scratch wound healing assays that determine the rate at which a population of cells fills a gap created in a confluent 2D culture. The quantification of the results of this experiment, however, lacks standardization and is often highly time consuming and user dependent. To overcome these limitations, we have developed AIM (Automatic Invasiveness Measure), a freely-available software tool for the automatic quantification of the cell-free region in scratch wound healing assays. This study will completely describe AIM and will show its equivalence to three analysis methods commonly used for the quantification of the scratch area and the measure of true wound extension. Furthermore, the analysis time and the dependency of the results of these techniques on the structure of the time course (total duration and number of points) will be studied. To the best of our knowledge, AIM is the first entirely-automated analysis method for scratch wound healing assays and represents a significant improvement of this technique both in terms of results' quality and reliability.

In solid tumors, metastasis occurs through the dissemination of tumor cells in the bloodstream and the lymphatic system. In particular, lymph node infiltration gives useful prognostic information and represents one of the most important factors for selecting the type of clinical treatment in disease management. Furthermore, the analysis of lymph node infiltration has become important for identifying patients with breast cancer or malignant melanoma who may be candidates for regional lymph node dissection. Tumor cells in lymph nodes are currently identified in tissue sections using morphological and immunohistochemical analyses, but these approaches are time-consuming, and micrometastases may escape detection. The aim of the present study was to define the potential of a flow cytometric (FCM) determination based on cell size and autofluorescence to shorten the time required for lymph node analysis. The sensitivity of the FCM approach, defined on mixtures of tumor cells from established cell lines and peripheral blood lymphocytes (PBLs) at different concentrations, was 1 tumor cell/1,000 PBLs. FCM analysis was performed on 89 lymph nodes, 29 from breast, 41 from lung and 19 from colon cancer patients. Agreement between FCM and morphological results, used as gold standard, was observed in 83% of the cases, and there was a 90% sensitivity to the FCM approach for each tumor type. Disagreement was observed for 15 lymph nodes and was due, in the majority of cases (80%), to FCM-positive and morphologically negative results. A large number of patients and a more accurate pathological examination of consecutive histological sections of lymph nodes are needed to further evaluate the validity of the FCM approach.

Shape and volume are two of the most important features of a tumour. In particular, the treatment choices of a tumour are often driven by its volume. Accordingly, the correct estimation of the volume is fundamental to assess the effectiveness of therapy. Very few methods have been proposed in the literature to accurately estimate the tumour’s volume arising from one 2D projection and even less provide a 3D reconstruction of the tumour shape, that is important for the clinical evaluation. In this work, we address the morphology of tumour spheroids typically employed for testing drugs, chemotherapy and radiotherapy treatments in pre-clinical studies. In particular, we propose a new 3D volume rendering method based on the assumption of spherical symmetry of the object around the principal axis. By using real-world, home-made 3D models, mimicking the spheroid morphology, we proved the effectiveness of our method for both 3D reconstruction and volume assessment.

Summary Background: Vinorelbine and ifosfamide are active drugs against breast cancer, but the best treatment schedule has yet to be defined by preclinical or clinical studies. The antitumor activity of 4-hydroxy-ifosfamide (4-OH-IF), the active form of ifosfamide, and vinorelbine (VNB) and their interaction were investigated in two established breast cancer cell lines (MCF-7 and BRC-230) and in 10 primary breast cancer cultures. Materials and methods: Cytotoxic activity was evaluated by a highly efficient clonogenic assay (HECA). The median-effect principle was applied to evaluate synergistic and antagonistic interactions and the corresponding combination index values were calculated. Cell cycle perturbations were analysed by flow cytometry. Results: In MCF-7 and BRC-230 cell lines the sequence VNB for 4 hours followed by 4-OH-IF for 24 hours produced an antagonistic effect. Conversely, the inverse sequential scheme, 4-OH-IF -> VNB provided synergistic effects on both cell lines. The synergism was associated with a strong block in the G2-M phase. Synergistic activity of 4-OH-IF -> VNB sequence was confirmed in 7 of 10 primary breast cancer cultures. Conclusions: In conclusion, the sequence 4-OH-IF -> VNB appeared to be the most effective scheme both in established cell lines and in primary breast cancer cultures.

The activity of the following drugs was investigated in two established NSCLC cell lines: docetaxel, gemcitabine, vinorelbine, paclitaxel, doxorubicin (0.01, 0.1, 1 μg ml−1), cisplatin, ifosfamide (1, 2, 3 μg ml−1) and carboplatin (2, 4, 6 μg ml−1). The cytotoxic activity was evaluated by the sulphorhodamine B assay. The two most active drugs, docetaxel and gemcitabine, used singly and in association, were investigated as a function of treatment schedule. The sequence docetaxel→gemcitabine produced only a weak synergistic interaction in RAL but a strong synergism in CAEP cells. The synergistic interaction increased in both cell lines after a 48-h washout between the drug administrations. Flow cytometric analysis showed that in docetaxel→gemcitabine sequence, docetaxel produced a block in G2/M phase and, after 48 h, provided gemcitabine with a large fraction of recovered synchronized cells in the G1/S boundary, which is the specific target phase for gemcitabine. Conversely, simultaneous treatment induced an antagonistic effect in both cell lines, and the sequential scheme gemcitabine→docetaxel produced a weak synergistic effect only in RAL cells. Moreover, the synergistic interaction disappeared when washout periods of 24 or 48 h between two drug administrations were adopted. The synergistic activity of docetaxel→ 48-h washout→gemcitabine was confirmed in 11 of 14 primary cultures, which represents an important means of validating experimental results before translating them into clinical practice. © 1999 Cancer Research Campaign

e22056 Background: Cell-free tumor DNA (cftDNA) is released into the circulation and its recovery from plasma is a non-invasive alternative to tumor biopsy for applications related to molecular profiling. A potential use in therapeutic interventions is the periodic monitoring of cftDNA for the identification of molecular changes associated with resistance to target-specific treatments or when the mutational status in tumor tissue is not available. Methods: Samples (6 ml) of peripheral blood were drawn from patients with colorectal (CRC, n= 19) and thyroid (n= 1) cancers and melanoma (n= 8). CRC tumors were KRAS wild-type (n= 3) or carried the BRAF V600E (n= 6), KRAS G12D (n= 9) and G12V (n= 4) mutations. All melanomas and the thyroid cancer carried the BRAF V600E mutation. DNA was extracted from plasma with QIAamp Circulating Nucleic Acid Kit to recover DNA fragments of ≤1000 bp. PCR amplification was carried out with a QX100 ddPCR System (Bio-Rad) on 20 μL-samples containing cftDNA and TaqMan probes for BRAF V600E (1799T>A), KRAS G12D (35G>A) and G12V (35G>T) labeled with FAM/VIC. Samples were then loaded into a droplet reader, which discriminates the difference in fluorescence amplitudes on the basis of target gene amplification. Results: The concordance between mutations in tumors (T) and plasma (P) for BRAF V600E was: melanomas 8 T vs. 6 P; thyroid cancer 1 T vs. 0 P; CRC 6 T vs 3 P. Concerning KRAS in CRC the results were: G12D 9 T vs. 7 P, G12V 4 T vs. 0 P. Overall, the percentage of concordant samples were: V600E 60%, G12D 77,8%, G12V 0%. Interestingly, in 2 patients with wild-type KRAS tumor, the G12D and G12V mutations were found in plasma. Conclusions: ddPCR is a third-generation PCR technique for highly sensitive detection of DNA fragments. Detection of mutations in cftDNA by advanced technological platforms has important applications in the monitoring of patients for the occurrence of secondary mutations, amplifications and expansion of cell clones that render their tumors resistant to target-specific anticancer agents, unraveling the resistant phenotype before the clinical progression of the disease. Acknowledgments: this study was funded in part by MIUR/PRIN 2011-2012 (Rome, Italy).

Peritoneal carcinomatosis (PC) is observed in approximately 10% of patients with colorectal cancer at the time of primary cancer resection. Most of these patients receive 5-fluorouracil (5-FU)- or oxaliplatin-containing chemotherapy regimens as first-, second-, or third-line treatment. In the present study, sensitivity and resistance to drugs used to treat PC were better defined by a conventional chemosensitivity test than by biomarker expression.5-Fluorouracil- or oxaliplatin-based regimens are the treatments of choice in patients with PC from colon cancer. There are currently no useful preclinical evaluations to guide the decision-making process for tailored therapy. The aim of the present study was to compare the advantages and limits of a conventional in vitro chemosensitivity test with those of a panel of biomolecular markers in predicting clinical response to different drugs used to treat colon cancer-derived PC.Fresh surgical biopsy specimens were obtained from 28 patients with peritoneal carcinomatosis from colon cancer. TS, TP, DPD, MDR1, MRP-1, MGMT, BRCA1, ERCC1, GSTP1, and XPD gene expression levels were determined by real-time reverse transcription polymerase chain reaction. An in vitro chemosensitivity test was used to define a sensitivity or resistance profile to the drugs used to treat each patient.Expression levels of the genes analyzed were generally poorly related to each other. TS and ERCC1 expression was inversely related to response to 5-FU-and/or oxaliplatin-containing regimens. Significant predictivity in terms of sensitivity but poor predictivity of resistance (56.2%) (P=.037) were observed for ERCC1 expression (90%), and high predictivity of resistance (100%) but very low predictivity of sensitivity (40%) (P=.014) were registered for TS. The best overall and significant predictivity was observed for chemosensitivity test results (62.5% sensitivity and 89% resistance; P=.005).Sensitivity and resistance to drugs used in vivo was better defined by the chemosensitivity test than by biomarker expression.

In non-small cell lung cancer (NSCLC) patients, somatic EGFR and K-ras mutations predict therapeutic effectiveness and resistance, respectively, to EGFR tyrosine kinase inhibitors (TKIs). Transesophageal ultrasound-guided fine needle aspiration (EUS-FNA) is a validated technique for diagnosis and staging of NSCLC. In the present study, we compared the feasibility and reliability of EGFR and K-ras gene mutation analysis in fixed and fresh mediastinal lymph nodes and extra-lymph nodal samples obtained by EUS-FNA in patients suspicious for NSCLC. Thirty-six patients were enrolled into the study. For each patient, DNA was extracted from both fresh samples and fixed cytological smears. Exons 18-21 of EGFR and exon 2 of K-ras were amplified by PCR and mutation status was determined by direct sequencing and pyrosequencing. All cases were eligible for analysis. NSCLC was diagnosed in 32 patients (25 adenocarcinomas and 7 squamous cell carcinomas) and 4 patients were free of malignancy. Of the 25 patients with adenocarcinoma, EGFR mutations were detected in 2 (8%) fresh tumor samples and in 3 (12%) fixed cytological smears. K-ras mutations were detected in 8 (32%) fresh samples, and in 9 (36%) fixed cytological smears. Fixed and stained cytological samples seem to be more reliable than fresh material for molecular analysis.

Background Platinum-based regimens are the treatments of choice in ovarian cancer, which remains the leading cause of death from gynecological malignancies in the Western world. The aim of the present study was to compare the advantages and limits of a conventional chemosensitivity test with those of new biomolecular markers in predicting response to platinum regimens in a series of patients with peritoneal carcinomatosis from ovarian cancer. Methods Fresh surgical biopsy specimens were obtained from 30 patients with primary or recurrent peritoneal carcinomatosis from ovarian cancer. ERCC1, GSTP1, MGMT, XPD, and BRCA1 gene expression levels were determined by Real-Time RT-PCR. An in vitro chemosensitivity test was used to define a sensitivity or resistance profile to the drugs used to treat each patient. Results MGMT and XPD expression was directly and significantly related to resistance to platinum-containing treatment (p = 0.036 and p = 0.043, respectively). Significant predictivity in terms of sensitivity and resistance was observed for MGMT expression (75.0% and 72.5%, respectively; p = 0.03), while high predictivity of resistance (90.9%) but very low predictivity of sensitivity (37.5%) (p = 0.06) were observed for XPD. The best overall and significant predictivity was observed for chemosensitivity test results (85.7% sensitivity and 91.3% resistance; p = 0.0003). Conclusions The in vitro assay showed a consistency with results observed in vivo in 27 out of the 30 patients analyzed. Sensitivity and resistance profiles of different drugs used in vivo would therefore seem to be better defined by the in vitro chemosensitivity test than by expression levels of markers.

Today, drug combinations are frequently used in the treatment of cancer to increase therapeutic efficacy. Currently used clinical protocols for cancer combination therapies are mainly obtained empirically or on the basis of results from previous clinical trials. Information obtained from clinical protocols is invaluable, but it is time-consuming, expensive and does not provide data on the biochemical and molecular mechanisms of interaction of the drugs used in combination treatments at cellular level. Therefore, in vitro drug combination studies on established cell lines or primary cell cultures play an important role in designing and optimising combination protocols. A variety of in vitro assays and different mathematics models have been developed to investigate cytotoxic effects and to analyse the type of drug interactions. Increased knowledge of the cellular targets of traditional and new drugs and the development of new technologies have resulted in a new role for the in vitro tests which are no longer used only to evaluate the cytotoxic effects of drugs, but also to investigate the interference on cell cycle, induction of apoptosis and molecular or biochemical interactions. A review on in vitro preclinical tests used to evaluate the effects of drug combinations and to design the rationale of combined chemotherapy protocols is presented.

Background Zoledronic acid is used to treat bone metastases and has been shown to reduce skeletal-related events and exert antitumor activity. The present in vitro study investigates the mechanism of action of Zoledronic Acid on breast cancer cell lines with different hormonal and HER2 patterns. Furthermore, we investigated the efficacy of repeated versus non-repeated treatments. Methods The study was performed on 4 breast cancer cell lines (BRC-230, SkBr3, MCF-7 and MDA-MB-231). Non-repeated treatment (single exposure of 168 hrs’ duration) with zoledronic acid was compared with repeated treatment (separate exposures, each of 48 hrs’ duration, for a total of 168 hrs) at different dosages. A dose–response profile was generated using sulforhodamine B assay. Apoptosis was evaluated by TUNEL assay and biomolecular characteristics were analyzed by western blot. Results Zoledronic acid produced a dose-dependent inhibition of proliferation in all cell lines. Anti-proliferative activity was enhanced with the repeated treatment, proving to be statistically significant in the triple-negative lines. In these lines repeated treatment showed a cytocidal effect, with apoptotic cell death caused by caspase 3, 8 and 9 activation and decreased RAS and pMAPK expression. Apoptosis was not observed in estrogen receptor-positive line: p21 overexpression suggested a slowing down of cell cycle. A decrease in RAS and pMAPK expression was seen in HER2-overexpressing line after treatment. Conclusions The study suggests that zoledronic acid has an antitumor activity in breast cancer cell lines. Its mechanism of action involves the decrease of RAS and RHO, as in osteoclasts. Repeated treatment enhances antitumor activity compared to non-repeated treatment. Repeated treatment has a killing effect on triple-negative lines due to apoptosis activation. Further research is warranted especially in the treatment of triple-negative breast cancer.

Background: Epithelial ovarian cancers represent 80 to 90% of all ovarian malignancies and are the most common cause of death from gynecological tumors in the Western world. The absence of specific symptoms in the early stages of disease means that around two-thirds of patients are initially diagnosed with advanced disease and/or peritoneal dissemination. Although standard primary treatment now exists for advanced ovarian cancer, there is still no single accepted therapeutic strategy for those who develop recurrent intra-abdominal disease more than 6 months after a complete response. The present study aimed to identify a panel of genes whose expression is correlated with sensitivity or resistance to platinum compounds. Material and methods: Fresh surgical biopsy specimens were obtained from 22 patients with advanced epithelial ovarian cancer during intra-abdominal cytoreductive surgery. Expression levels of ERCC1, GSTP1, MGMT, XPD, BRCA1 were determined by Real Time RT-PCR. Relative gene expression was quantified according to the comparative threshold cycle method (2-ΔΔCτ) using β2-microglobulin and HPRT as endogenous controls and commercial RNA controls derived from normal ovarian tissue mRNA as calibrators. The correlation between pre- and post-surgery expression levels of these markers and response to platinum treatment was examined. Results: Time to progression (TTP) was calculated from the first day of treatment to the date on which objective disease progression was first documented, or death in the absence of progressive disease. At univariate analysis, estimated hazard ratio (HR) and 95% confidence intervals (95% CI) were calculated using the Cox proportional hazard model. In particular, the risk of progression increased significantly as ERCC1 (HR=12.00, 95% CI 1.08-133.35, p=0.04), GSTP1 (HR=6.01, 95% CI 1.09-33.01, p=0.004), XPD (HR=10.37, 95% CI 1.26-85.52, p=0.003), MGMT (HR=10.26, 95% CI 1.18-88.94, p=0.035) and BRCA1 (HR= 7.52, 95% CI 1.32-42.74, p=0.023) levels increased. Conclusions: Our results indicate that these biomarkers could be useful predictors of clinical response to platinum compounds in patients with advanced ovarian cancer who develop recurrent intra-abdominal disease. It is now planned to compare tailored and empiric therapy in a well-designed randomized trial. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2709.

Nitric oxide-releasing nonsteroidal antiinflammatory drugs (NO-NSAIDs) are reported to be safer than NSAIDs because of their lower gastric toxicity. We compared the effect of a novel NO-releasing derivate, NCX 4040, with that of aspirin and its denitrated analog, NCX 4042, in in vitro and in vivo human colon cancer models and investigated the mechanisms of action underlying its antitumor activity. In vitro cytotoxicity was evaluated on a panel of colon cancer lines (LoVo, LoVo Dx, WiDr and LRWZ) by sulforhodamine B assay. Cell cycle perturbations and apoptosis were evaluated by flow cytometry. Protein expression was detected by Western blot. In the in vivo experiments, tumor-bearing mice were treated with NCX 4040, five times a week, for six consecutive weeks. In the in vitro studies, aspirin and NCX 4042 did not induce an effect on any of the cell lines, whereas NCX 4040 produced a marked cytostatic dose-related effect, indicating a pivotal role of the -NO2 group. Furthermore, in LoVo and LRWZ cell lines, we observed caspase-9 and -3-mediated apoptosis, whereas no apoptotic effect was observed after drug exposure in WiDr or LoVo Dx cell lines. In in vivo studies, both NCX 4040 and its parental compound were administered per os. NCX 4040 induced a 40% reduction in tumor weight. Conversely, aspirin did not influence tumor growth at all. NCX 4040, but not its parental compound, aspirin, showed an in vitro and in vivo antiproliferative activity, indicating its potential usefulness to treat colon cancer.

(50% drop, 75% drop, @ 3 cutoffs) was the same in the 2 dose groups. Median survival was 50 months for the entire cohort, with a 2& 5year survival of 88%, 65%. DE-RT pts have not yet achieved median survival. The 2-year DFS was 70% (37 mo median DFS). None of the factors including RT dose, T down staging, MNC, different cut off points for SUV drop were significant for outcomes on DFS, OS. At last follow-up, 17 pts (46%) were free of disease and 14 alive (38%). Conclusions: RT dose escalation resulted in improvement in MNC and correlated with the PET response in our series. Long-term follow-up and larger pt numbers are needed for the DE-RT pts to prove the concept. PET response can be utilized as predictor of outcomes in operable NSCLC pts. Author Disclosure: A. Turaka: None. S. Hasan: None. T. Li: None. R. Mehra: None. W.J. Scott: None. J.Q. Yu: None.

The effect on growth of the long-acting somatostatin analogue lanreotide (LAN), alone or in combination with 5-fluorouracil (5-FU) and mitomycin C (MIT), was investigated in three human colon cancer lines. Cell survival inhibition induced by LAN alone, as evaluated by sulforhodamine B assay, ranged from 20% to 40% as a function of cell line and concentration. The IC50, the concentration inhibiting cell survival by 50%, was never reached. The antiproliferative effect produced by a 48 h exposure to 5-FU or MIT was synergistically enhanced in all cell lines by a subsequent 48 h exposure to LAN. The synergistic interaction was not related to specific cell cycle perturbations or to the somatostatin receptor 2 (sst2) mRNA abundance. In conclusion, our study seems to indicate that LAN is a potentially useful modulating agent for enhancing 5-FU and MIT activity in colorectal cancer patients.

The ability of lonidamine (LND), a derivative of indazole-carboxylic acid, to modulate the cytotoxic activity of anticancer drugs was investigated in two human hepatocarcinoma (HCC) cell lines. The cytotoxicity of drugs used singly, in association or in sequence was evaluated using the Sulforhodamine B (SRB) assay. LND did not appreciably potentiate the effect of antitumor drugs when given before or simultaneously, in either cell line. Conversely, a synergistic interaction was observed in both cell lines when LND was given after conventional drugs. LND produced a moderate decrease in S-phase cell fraction and did not induce apoptosis. Conversely, paclitaxel (TAX) induced an important block in G2 and an increase in apoptosis. Following a 48-h TAX wash out, a progressive passage of cells from G2 to M phase was observed with a corresponding increase in apoptotic cells. Post-treatment with LND increased the cytotoxicity of some antitumor drugs, especially TAX, in hepatocarcinoma cells, possibly by preventing, as an energolytic drug, cell damage repair or by producing an additional effect on microtubule stabilization.

High expression of the epidermal growth factor receptor (EGFR) family confers a growth advantage on malignant cells in various tumor types. Most pancreatic cancers express EGFR, which seems to play an important role in the acquisition of aggressive clinical behaviour and in tumor invasion. Iressa (ZD1839), a quinazoline tyrosine kinase inhibitor selective for the EGF receptor, has shown good anti-tumor activity in both preclinical and clinical studies. Using two pancreatic cancer cell lines that express different EGFR and ErbB-2 levels, we analyzed the activity of Iressa and evaluated its modulation effect on four conventional cytotoxic drugs: gemcitabine, oxaliplatin, docetaxel and SN38. Iressa was tested at scalar doses up to the plasma peak level concentration and showed a similar weak cytostatic effect in both cell lines. Conversely, an additive or weak synergistic effect was observed when the drug was administered simultaneously with or following cytotoxic drugs. Our data show that Iressa has only a weak activity at doses within the plasmatic peak concentration and that its effect is independent of EGFR and p42/p44 expression and phosphorylation levels. This is in agreement with recent literature data that attribute an essential role to a specific EGFR mutation in mediating response to Iressa. This mutation was absent in both pancreatic cell lines tested.

The response of pancreatic cancer to treatments remains unsatisfactory, highlighting the need for more effective therapeutic regimens. Sorafenib, an orally available multikinase inhibitor, is active against different tumors, including pancreatic cancer. We studied the antitumor efficacy of sorafenib in combination with different antitumor drugs currently used in clinical practice in in vitro and in vivo experimental models of human pancreatic cancer. The cytotoxic effect of sorafenib and conventional antitumor drug combinations was evaluated in vitro in human pancreatic cancer cell lines and the efficacy of the most active combination was tested on tumor-bearing mice. Flow cytometric, Western blot and immunohistochemistry analyses were performed to investigate the mechanisms involved in the activity of single drugs and in their interaction when used in combination. Sorafenib showed a strong sequence-dependent synergistic interaction in vitro with docetaxel, which was highly dependent on the drug sequence employed. In vivo, human pancreatic cancer-xenografted mice treated with docetaxel followed by sorafenib reduced and delayed tumor growth, with complete tumor regression observed in half of the mice. This marked antitumor effect resulted in an overall increase in mouse survival of about 70% and in a complete cure in 3 of the 8 treated mice. The strong activity was also accompanied by marked apoptosis induction, inhibition of tumor angiogenesis and downregulation of ERK signalling. Our results show that the docetaxel and sorafenib combination exerts high therapeutic efficacy in experimental models of human pancreatic cancer, indicating a promising antitumor strategy for clinical use.

Background: Although two-dimensional (2-D) monolayer cell cultures provide important information on basic tumor biology and radiobiology, they are not representative of the complexity of three-dimensional (3-D) solid tumors. In particular, new models reproducing clinical conditions as closely as possible are needed for radiobiological studies to provide information that can be translated from bench to bedside. Methods: We developed a novel system for the irradiation, under sterile conditions, of 3-D tumor spheroids, the in vitro model considered as a bridge between the complex architectural organization of in vivo tumors and the very simple one of in vitro monolayer cell cultures. The system exploits the same equipment as that used for patient treatments, without the need for dedicated and highly expensive instruments. To mimic the passage of radiation beams through human tissues before they reach the target tumor mass, 96-multiwell plates containing the multicellular tumor spheroids (MCTS) are inserted into a custom-built phantom made of plexiglass, the material most similar to water, the main component of human tissue. Results: The system was used to irradiate CAEP- and A549-derived MCTS, pre-treated or not with 20 μM cisplatin, with a dose of 20 Gy delivered in one session. We also tested the same treatment schemes on monolayer CAEP and A549 cells. Our preliminary results indicated a significant increment in radiotoxicity 20 days after the end of irradiation in the CAEP spheroids pre-treated with cisplatin compared to those treated with cisplatin or irradiation alone. Conversely, the effect of the radio- chemotherapy combination in A549-derived MCTS was similar to that induced by cisplatin or irradiation alone. Finally, the 20 Gy dose did not affect cell survival in monolayer CAEP and A549 cells, whereas cisplatin or cisplatin plus radiation caused 100% cell death, regardless of the type of cell line used. Conclusions: We set up a system for the irradiation, under sterile conditions, of tumor cells grown in 3-D which allows for the use of the same dose intensities and schedules utilized in clinical practice. This irradiation system, coupled with 3-D cell cultures, has the potential to generate information that could be used to individually tailor radiotherapy