Sustained hyperammonemia induces TNF-a IN Purkinje neurons by activating the TNFR1-NF-κB pathway.

BACKGROUND: Patients with liver cirrhosis may develop hepatic encephalopathy. Rats with chronic hyperammonemia exhibit neurological alterations mediated by peripheral inflammation and neuroinflammation. Motor incoordination is due to increased TNF-a levels and activation of its receptor TNFR1 in the cerebellum. The aims were to assess (a) whether peripheral inflammation is responsible for TNF-a induction in hyperammonemic rats, (b) the cell type(s) in which TNF-a is increased, (c) whether this increase is associated with increased nuclear NF-κB and TNFR1 activation, (d) the time course of TNF-a induction, and (e) if TNF-a is induced in the Purkinje neurons of patients who die with liver cirrhosis. METHODS: We analyzed the level of TNF-a mRNA and NF-κB in microglia, astrocytes, and Purkinje neurons in the cerebellum after 1, 2, and 4 weeks of hyperammonemia. We assessed whether preventing peripheral inflammation by administering an anti-TNF-a antibody prevents TNF-a induction. We tested whether TNF-a induction is reversed by R7050, which inhibits the TNFR1-NF-κB pathway, in ex vivo cerebellar slices. RESULTS: Hyperammonemia induced microglial and astrocyte activation at 1 week. This was followed by TNF-a induction in both glial cell types at 2 weeks and in Purkinje neurons at 4 weeks. The level of TNF-a mRNA increased in parallel with the TNF-a protein level, indicating that TNF-a was synthesized in Purkinje cells. This increase was associated with increased NF-κB nuclear translocation. The nuclear translocation of NF-κB and the increase in TNF-a were reversed by R7050, indicating that they were mediated by the activation of TNFR1. Preventing peripheral inflammation with an anti-TNF-a antibody prevents TNF-a induction. CONCLUSION: Sustained (4 weeks) but not short-term hyperammonemia induces TNF-a in Purkinje neurons in rats. This is mediated by peripheral inflammation. TNF-a is also increased in the Purkinje neurons of patients who die with liver cirrhosis. The results suggest that hyperammonemia induces TNF-a in glial cells and that TNF-a released by glial cells activates TNFR1 in Purkinje neurons, leading to NF-κB nuclear translocation and the induction of TNF-a expression, which may contribute to the neurological alterations observed in hyperammonemia and hepatic encephalopathy.

Chronic hyperammonemia induces peripheral inflammation that leads to cognitive impairment in rats: Reversed by anti-TNF-α treatment.

BACKGROUND & AIMS: Chronic hyperammonemia induces neuroinflammation which mediates cognitive impairment. How hyperammonemia induces neuroinflammation remains unclear. We aimed to assess whether: chronic hyperammonemia induces peripheral inflammation, and whether this then contributes to neuroinflammation, altered neurotransmission and impaired spatial learning - before assessing whether this neuroinflammation and impairment is reversible following hyperammonemia elimination or treatment of peripheral inflammation with anti-TNF-α. METHODS: Chronic hyperammonemia was induced by feeding rats an ammonia-containing diet. Peripheral inflammation was analyzed by measuring PGE2, TNF-α, IL-6 and IL-10. We tested whether chronic anti-TNF-α treatment improves peripheral inflammation, neuroinflammation, membrane expression of glutamate receptors in the hippocampus and spatial learning. RESULTS: Hyperammonemic rats show a rapid and reversible induction of peripheral inflammation, with increased pro-inflammatory PGE2, TNF-α and IL-6, followed at around 10 days by reduced anti-inflammatory IL-10. Peripheral anti-TNF-α treatment prevents peripheral inflammation induction and the increase in IL-1b and TNF-α and microglia activation in hippocampus of the rats, which remain hyperammonemic. This is associated with prevention of the altered membrane expression of glutamate receptors and of the impairment of spatial memory assessed in the radial and Morris water mazes. CONCLUSIONS: This report unveils a new mechanism by which chronic hyperammonemia induces neurological alterations: induction of peripheral inflammation. This suggests that reducing peripheral inflammation by safe procedures would improve cognitive function in patients with minimal hepatic encephalopathy. LAY SUMMARY: This article unveils a new mechanism by which chronic hyperammonemia induces cognitive impairment in rats: chronic hyperammonemia per se induces peripheral inflammation, which mediates many of its effects on the brain, including induction of neuroinflammation, which alters neurotransmission, leading to cognitive impairment. It is also shown that reducing peripheral inflammation by treating rats with anti-TNF-α, which does not cross the blood-brain barrier, prevents hyperammonemia-induced neuroinflammation, alterations in neurotransmission and cognitive impairment.

Lung tumorspheres reveal cancer stem cell-like properties and a score with prognostic impact in resected non-small-cell lung cancer.

The high resistance against current therapies found in non-small-cell lung cancer (NSCLC) has been associated to cancer stem-like cells (CSCs), a population for which the identification of targets and biomarkers is still under development. In this study, primary cultures from early-stage NSCLC patients were established, using sphere-forming assays for CSC enrichment and adherent conditions for the control counterparts. Patient-derived tumorspheres showed self-renewal and unlimited exponential growth potentials, resistance against chemotherapeutic agents, invasion and differentiation capacities in vitro, and superior tumorigenic potential in vivo. Using quantitative PCR, gene expression profiles were analyzed and NANOG, NOTCH3, CD44, CDKN1A, SNAI1, and ITGA6 were selected to distinguish tumorspheres from adherent cells. Immunoblot and immunofluorescence analyses confirmed that proteins encoded by these genes were consistently increased in tumorspheres from adenocarcinoma patients and showed differential localization and expression patterns. The prognostic role of genes significantly overexpressed in tumorspheres was evaluated in a NSCLC cohort (N = 661) from The Cancer Genome Atlas. Based on a Cox regression analysis, CDKN1A, SNAI1, and ITGA6 were found to be associated with prognosis and used to calculate a gene expression score, named CSC score. Kaplan-Meier survival analysis showed that patients with high CSC score have shorter overall survival (OS) in the entire cohort [37.7 vs. 60.4 months (mo), p = 0.001] and the adenocarcinoma subcohort [36.6 vs. 53.5 mo, p = 0.003], but not in the squamous cell carcinoma one. Multivariate analysis indicated that this gene expression score is an independent biomarker of prognosis for OS in both the entire cohort [hazard ratio (HR): 1.498; 95% confidence interval (CI), 1.167-1.922; p = 0.001] and the adenocarcinoma subcohort [HR: 1.869; 95% CI, 1.275-2.738; p = 0.001]. This score was also analyzed in an independent cohort of 114 adenocarcinoma patients, confirming its prognostic value [42.90 vs. not reached (NR) mo, p = 0.020]. In conclusion, our findings provide relevant prognostic information for lung adenocarcinoma patients and the basis for developing novel therapies. Further studies are required to identify suitable markers and targets for lung squamous cell carcinoma patients.

Characterization of triple-negative breast cancer preclinical models provides functional evidence of metastatic progression.

Triple-negative breast cancer (TNBC), an aggressive, metastatic and recurrent breast cancer (BC) subtype, currently suffers from a lack of adequately described spontaneously metastatic preclinical models that faithfully reproduce the clinical scenario. We describe two preclinical spontaneously metastatic TNBC orthotopic murine models for the development of advanced therapeutics: an immunodeficient human MDA-MB-231-Luc model and an immunocompetent mouse 4T1 model. Furthermore, we provide a broad range of multifactorial analysis for both models that could provide relevant information for the development of new therapies and diagnostic tools. Our comparisons uncovered differential growth rates, stromal arrangements and metabolic profiles in primary tumors, and the presence of cancer-associated adipocyte infiltration in the MDA-MB-231-Luc model. Histopathological studies highlighted the more rapid metastatic spread to the lungs in the 4T1 model following a lymphatic route, while we observed both homogeneous (MDA-MB-231-Luc) and heterogeneous (4T1) metastatic spread to axillary lymph nodes. We encountered unique metabolomic signatures in each model, including crucial amino acids and cell membrane components. Hematological analysis demonstrated severe leukemoid and lymphoid reactions in the 4T1 model with the partial reestablishment of immune responses in the immunocompromised MDA-MB-231-Luc model. Additionally, we discovered ß-immunoglobulinemia and increased basal levels of G-CSF correlating with a metastatic switch, with G-CSF also promoting extramedullary hematopoiesis (both models) and causing hepatosplenomegaly (4T1 model). Overall, we believe that the characterization of these preclinical models will foster the development of advanced therapeutic strategies for TNBC treatment, especially for the treatment of patients presenting both, primary tumors and metastatic spread.

Tumor microenvironment-targeted poly-L-glutamic acid-based combination conjugate for enhanced triple negative breast cancer treatment.

The intrinsic characteristics of the tumor microenvironment (TME), including acidic pH and overexpression of hydrolytic enzymes, offer an exciting opportunity for the rational design of TME-drug delivery systems (DDS). We developed and characterized a pH-responsive biodegradable poly-L-glutamic acid (PGA)-based combination conjugate family with the aim of optimizing anticancer effects. We obtained combination conjugates bearing Doxorubicin (Dox) and aminoglutethimide (AGM) with two Dox loadings and two different hydrazone pH-sensitive linkers that promote the specific release of Dox from the polymeric backbone within the TME. Low Dox loading coupled with a short hydrazone linker yielded optimal effects on primary tumor growth, lung metastasis (∼90% reduction), and toxicological profile in a preclinical metastatic triple-negative breast cancer (TNBC) murine model. The use of transcriptomic analysis helped us to identify the molecular mechanisms responsible for such results including a differential immunomodulation and cell death pathways among the conjugates. This data highlights the advantages of targeting the TME, the therapeutic value of polymer-based combination approaches, and the utility of -omics-based analysis to accelerate anticancer DDS.

Combined plasma rich in growth factors and adipose-derived mesenchymal stem cells promotes the cutaneous wound healing in rabbits.

BACKGROUND: The use of Plasma Rich in Growth Factors (PRGF) and Adipose Derived Mesenchymal Stem Cells (ASCs) are today extensively studied in the field of regenerative medicine. In recent years, human and veterinary medicine prefer to avoid using traumatic techniques and choose low or non-invasive procedures. The objective of this study was to evaluate the efficacy of PRGF, ASCs and the combination of both in wound healing of full-thickness skin defects in rabbits. With this purpose, a total of 144 rabbits were used for this study. The animals were divided in three study groups of 48 rabbits each depending on the administered treatment: PRGF, ASCs, and PGRF+ASCs. Two wounds of 8 mm of diameter and separated from each other by 20 mm were created on the back of each rabbit: the first was treated with saline solution, and the second with the treatment assigned for each group. Macroscopic and microscopic evolution of wounds was assessed at 1, 2, 3, 5, 7 and 10 days post-surgery. With this aim, 8 animals from each treatment group and at each study time were euthanized to collect wounds for histopathological study. RESULTS: Wounds treated with PRGF, ASCs and PRGF+ASCs showed significant higher wound healing and epithelialization rates, more natural aesthetic appearance, significant lower inflammatory response, significant higher collagen deposition and angiogenesis compared with control wounds. The combined treatment PRGF+ASCs showed a significant faster cutaneous wound healing process. CONCLUSIONS: The combined treatment PRGF+ASCs showed the best results, suggesting this is the best choice to enhance wound healing and improve aesthetic results in acute wounds.

Selective targeting of collagen IV in the cancer cell microenvironment reduces tumor burden.

Goodpasture antigen-binding protein (GPBP) is an exportable1 Ser/Thr kinase that induces collagen IV expansion and has been associated with chemoresistance following epithelial-to-mesenchymal transition (EMT). Here we demonstrate that cancer EMT phenotypes secrete GPBP (mesenchymal GPBP) which displays a predominant multimeric oligomerization and directs the formation of previously unrecognized mesh collagen IV networks (mesenchymal collagen IV). Yeast two-hybrid (YTH) system was used to identify a 260SHCIE264 motif critical for multimeric GPBP assembly which then facilitated design of a series of potential peptidomimetics. The compound 3-[4''-methoxy-3,2'-dimethyl-(1,1';4',1'')terphenyl-2''-yl]propionic acid, or T12, specifically targets mesenchymal GPBP and disturbs its multimerization without affecting kinase catalytic site. Importantly, T12 reduces growth and metastases of tumors populated by EMT phenotypes. Moreover, low-dose doxorubicin sensitizes epithelial cancer precursor cells to T12, thereby further reducing tumor load. Given that T12 targets the pathogenic mesenchymal GPBP, it does not bind significantly to normal tissues and therapeutic dosing was not associated with toxicity. T12 is a first-in-class drug candidate to treat cancer by selectively targeting the collagen IV of the tumor cell microenvironment.

An immunohistochemical score to predict the outcome for oral squamous cell carcinoma.

BACKGROUND: Oral cancer is a major public health problem worldwide, with a poor survival. Our aim was to evaluate several protein markers in oral squamous cell carcinomas (OSCC) and analyse their prognostic value on patient's survival. METHODS: We analysed the expression of EGFR, p53, p27, p16, cyclin D1, cyclin A2, COX-2, Ki-67, Bcl-2, VEGFR-1 and VEGFR-2, by immunohistochemistry on 67 primary OSCC. Cancer-specific survival (CSS) analysis was evaluated by the Cox regression model. RESULTS: Markers showed variable expression between 27.9% and 95.2%. In univariate analysis for CSS, we found that four of the tested markers, namely high expression of p53 (P = .001), EGFR (P = .003), cyclin A2 (P = .005) and low expression of p16 (P = .019), along with clinical stage (P < .001), tumour size (P < .001), presence of nodal metastasis (P < .001) and perineural permeation (P = .039) were related to decreased survival. On the basis of these results, we constructed an immunohistochemical score hinging on the possibility that any tumour could express none of these four markers (score 0), one or two markers (score 1) and three or more markers (score 2). In multivariable analysis, this immunohistochemical score revealed an independent prognostic value on cancer-specific survival (P = .001; HR: 3.7: 95%CI 1.7-7.9). Moreover, we confirmed that in early-stage tumours (stage I or II) this score maintained its independent prognostic value (P = .025; HR: 7.9, 95%CI 1.3-49.1) on CSS. CONCLUSION: The expression of the markers p53, p16, EGFR and cyclin A in OSCC, combined to give an immunohistochemical score, may identify high-risk subgroups for decreased survival and to further guide therapeutic decisions.

Local and systemic diffusion of antineoplastic drugs following vertebroplasty using acrylic cement mixed with cisplatin or methotrexate: experimental study in pigs.

PURPOSE: To determine the efficacy of cisplatin- or methotrexate-containing acrylic cement for local and systemic antineoplastic drug diffusion. Among the uses of acrylic cement or Polymethylmethacrylate (PMMA), there is the possibility to employ it as vehicle for drug diffusion. This capability is of interest in the treatment of pathological fractures: The curative effects of the cement (cytotoxicity of the monomer and increased temperature) are added to the antineoplastic effect of the drugs. METHODS: In the experimental study, two groups of ten pigs underwent vertebroplasty using cement mixed with 500 mg of powder cisplatin or 1000 mg of powder methotrexate. Vertebroplasty was performed in two non-consecutive lumbar vertebrae with bipedicular cement injection. Transpedicular bone biopsy was performed weekly to measure levels of antineoplastic agent in bone tissue and blood plasma. Cisplatin was studied by atomic absorption spectrometry and methotrexate by fluorescence polarization immunoassay. Renal and hepatic function and blood analysis were performed weekly. RESULTS: Cisplatin and methotrexate levels were found in bone tissue at more than 5 weeks following surgery. The cisplatin peak occurred at week 3 (mean 1269 µg/g bone) and the methotrexate peak at week 1 (mean 862.76 µg/g bone). Plasma drug levels were found 72 h after surgery, with a peak at 24 h for cisplatin (mean 0.23 µmol/L) and at 30 min for methotrexate (mean 0.92 µmol/L). None of the animals died during the study. Animals with intracanal cement leaks showed no neurological involvement. Renal, hepatic and hemogram studies remained within normal limits. CONCLUSIONS: There is local diffusion of antineoplastic agents from the cement to bone and plasma. We found methotrexate and cisplatin levels in bone at up to 5 weeks, comparable to previous in vitro reports. At the doses administered, there were no cases of myelosuppression, hepatotoxicity, or nephrotoxicity.

Distinct Osteomimetic Response of Androgen-Dependent and Independent Human Prostate Cancer Cells to Mechanical Action of Fluid Flow: Prometastatic Implications.

BACKGROUND AND METHODS: Prostate cancer frequently expresses an osteomimetic phenotype, but it is unclear how it is regulated and what biological and clinical implications it confers. Because mechanical forces physiologically regulate bone-remodeling activity in osteocytes, we hypothesized that mechanical action of fluid flow (MAFF) at the cancer microenvironment may similarly foster prostate cancer cell osteomimicry. RESULTS: We showed that in vitro MAFF on androgen-dependent (LNCap) and androgen-independent (PC3) prostate cancer cells remarkably increased OPG, VEGF, RunX2, PTH1R, and PTHrP gene expression in both cell lines irrespective of their androgen dependency. MAFF also altered the cytokine secretion pattern of prostate cancer cells, including Ang2, SCF, and TNFα increase with TRAIL decrease in the supernatant of both cell lines; preferential increase of Leptin and PDGF-BB in LnCap and of VEGF, IL-8, and G-CSF in PC3; and exclusive increase of FGFß, MIF, and PECAM-1 with HGF decrease in LnCap, and of TGBß1, HGF, M-CSF, CXCL1, and CCL7 with NGF decrease in PC3. Murine MLO-Y4 osteocyte-conditioned medium (CM) abrogated M-CSF, G-CSG, IL-8, TNFα, and FGFß secretion-stimulating activity of mechanical stimulation on PC3 cells, and did the opposite effect on LnCap cells. However, MAFF fostered osteomimetic gene expression response of PC3 cells, but not of LnCap cells, to mechanically stimulated osteocyte-CM. Moreover, it abrogated TNFα and IL-8 secretion inhibitory effect of osteocyte-CM on mechanically stimulated PC3 cells and G-CSF, TNFα, and FGFß-stimulating effect on mechanically stimulated LnCap cells. CONCLUSIONS: MAFF activated osteoblast-like phenotype of prostate cancer cells and altered their responses to osteocyte soluble factors. It also induced osteocyte production of osteomimetic gene expression- and cytokine secretion-stimulating factors for prostate cancer cells, particularly, when they were mechanically stimulated. Importantly, MAFF induced a prometastatic response in androgen-independent prostate cancer cells, suggesting the interest of mechanical stimulation-dependent transcription and secretion patterns as diagnostic biomarkers, and as therapeutic targets for the screening of bone-metastasizing phenotype inhibitors upregulated during prostate cancer cell response to MAFF at the cancer microenvironment. Prostate 77:321-333, 2017. © 2016 Wiley Periodicals, Inc.

Analysis of the immune microenvironment in resected non-small cell lung cancer: the prognostic value of different T lymphocyte markers.

The prognosis of non-small cell lung cancer (NSCLC) remains poor and heterogeneous and new biomarkers are needed. As the immune system plays a pivotal role in cancer, the study of immune-related markers may provide valuable prognostic information of NSCLC. In 122 formalin-fixed, paraffin-embedded tumor tissue samples from early-stage NSCLC, tumor and tumor-near stromal areas were microdissected and gene expression levels of conventional and regulatory T cell markers were assessed by quantitative polymerase chain reaction. Also, the presence of infiltrating CD4+, CD8+, and FOXP3+ cells in tumor samples was assessed by immunohistochemistry. The relative proportion of conventional and regulatory T cells present in the tumor environment was assessed and found to be key to understand the importance that the immune system analysis has in the prognostics of NSCLC patients. The presence of CD8+ cells in the tumor compartment was associated with better outcome, whereas the presence of FOXP3+ cells was associated with worse overall survival. The negative prognostic value of combined biomarkers, indicating high levels of FOXP3 in the stroma and low levels of CD4 or CD8 in tumors, was observed at mRNA level and was validated by immunohistochemistry.In conclusion, the proportion of T helper and cytotoxic cells vs. regulatory T cells in different locations of the tumor microenvironment have opposite prognostic impacts in resected NSCLC.

Acute cellular rejection versus recurrent hepatitis C after liver transplantation: Clinical and pathological features driving a rational diagnostic approach.

AIM: The aim of our study was develop and validate an algorithm system based on morphological features for finding the differences between recurrent hepatitis C virus (HCV) and acute cellular rejection (ACR) in liver biopsies of HCV-transplanted patients. METHODS: Two hundred and eighty-eight liver biopsies were analyzed from 121 patients transplanted for HCV. A diagnostic consensus was reached between clinicians and pathologists in 214 biopsies for the diagnosis of recurrent HCV or ACR. A random sample of 114 liver biopsies (derivation cohort) was taken to generate the diagnostic tree and was subsequently evaluated using the validation cohort in 100 liver biopsies by recursive partitioning analysis of morphological variables and time since transplantation. RESULTS: The presence of endotheliitis together with a time of less than 6 weeks since LT definitely excluded recurrent HCV. After obtaining the regression tree, diagnostic accuracy was 96% and 93% in the derivation and validation cohort, respectively. Both cases surpassed the pathologist's original diagnosis, which had a diagnostic accuracy of 91% (P < 0.05, for both comparisons). CONCLUSION: A recursive partitioning analysis of the morphological features in liver biopsies from HCV-transplanted patients may be useful for easily distinguishing between recurrent HCV and ACR.

Microarray phosphatome profiling of breast cancer patients unveils a complex phosphatase regulatory role of the MAPK and PI3K pathways in estrogen receptor-negative breast cancers.

Phosphatases are proteins with the ability to dephosphorylate different substrates and are involved in critical cellular processes such as proliferation, tumor suppression, motility and survival. Little is known about their role in the different breast cancer (BC) phenotypes. We carried out microarray phosphatome profiling in 41 estrogen receptor-negative (ER-) BC patients, as determined by immunohistochemistry (IHC), containing both ERBB2+ and ERBB2- in order to characterize the differences between these two groups. We characterized and confirmed the distinct phosphatome of the two main ER- BC subgroups (in two independent microarrays series) and that of ER+ BC (in three large independent series). Our findings point to the importance of the MAPK and PI3K pathways in ER- BCs as some of the most differentially expressed phosphatases (like DUSP4 and DUSP6) sharing ERK as substrate, or regulating the PI3K pathway (INPP4B, PTEN). It was possible to identify a selective group of phosphatases upregulated only in the ER- ERBB2+ subgroup and not in ER+ (like DUSP6, DUSP10 and PPAPDC1A among others), suggesting a role of these phosphatases in specific BC subtypes, unlike other differentially expressed phosphatases (DUSP4 and ENPP1) that seemed to have a role in multiple BC subtypes. Significant correlation was found at the protein level by IHC between the expression of DUSP6 and phospho-ERK (p=0.04) but not of phospho-ERK with DUSP4. To show the potential prognostic relevance of phosphatases as a functional group of genes, we derived and validated in two large independent BC microarray series a multiphosphatase signature enriched in differentially expressed phosphatases, to predict distant metastasis-free survival (DMFS). ER- ERBB2+, ER- ERBB2- and ER+ BC patients have a distinct pattern of phosphatase RNA expression with a potential prognostic relevance. Further studies of the most relevant phosphatases found in this study are warranted.

HER2 status determination using RNA-ISH--a rapid and simple technique showing high correlation with FISH and IHC in 141 cases of breast cancer.

AIMS: the assessment of the human epidermic growth factor receptor 2 (HER2) is currently performed in most laboratories using two techniques: Fluorescence in situ hybridisation (FISH) and immunohistochemistry (IHC), and novel methodology is being investigated continuously in the assessment of HER2, such as SISH, CISH, DNA chips, ELISA or real time PCR to make assessment easier, faster or more accurate. RNA-ISH (RNA in Situ Hybridisation) is a new technique designed to detect mRNA expression levels, conducted by light microscope without the need for counting or grading systems in a total processing time of 4 hours. This study aims to determine if RNA-ISH is a viable and effective technique and a possible alternative to the currently used techniques by analysing and comparing genetic amplification (FISH) and protein levels (IHC) with mRNA over-expression (RNA-ISH) in 141 cases of breast cancer. RESULTS: This study demonstrated a 96.5% concordance between over-expression of HER2 as determined by RNA-ISH and gene amplification as determined by FISH. The relationship between RNA-ISH-evaluated and IHC-evaluated over-expression was equally well reflected with a 95.2% concordance. Importantly, a considerable reduction in processing and evaluation time was achieved of only 4 hours. CONCLUSIONS: We conclude that the probe developed for RNA-ISH represents a viable, effective possible alternative to FISH and IHC for analysing HER2 status in primary breast tumours.

Combined cytoplasmic and membranous EGFR and p53 overexpression is a poor prognostic marker in early stage oral squamous cell carcinoma.

OBJECTIVE: Our aim was to evaluate the expression of several molecules that regulate growth, the cell cycle and signalling pathways including EGFR, p53, p16 and p27 in oral squamous cell carcinomas (OSCC). We examined their utility as prognostic markers by relating to clinicopathological characteristics and the clinical outcome. PATIENTS AND METHODS: Using tissue microarray technology, we analysed 67 primary OSCC and examined immunohistochemical expression of EGFR, p53, p16 and p27. Multivariate analysis was conducted to examine their role in survival. RESULTS: Many of the markers were highly expressed in these cancers. Membranous EGFR expression in 95.2%, both membrane and cytoplasm expression in 35%, p53 expression in 61.6%, p27 expression in 89.5% and p16 expression in 27.9% of cases. In the multivariate analysis, independent prognostic influence of a lower overall survival was determined only for advanced tumour stage (P < 0.001), p53 overexpression (P = 0.004), EGFR cytoplasm and membrane co-expression location (P = 0.002) and p16 reduced expression (P = 0.002). When considering a subgroup of early stage tumours, p53 overexpression (P = 0.028) and combined membranous and cytoplasm EGFR co-expression (P = 0.039) were indicators of a lower overall survival. For disease-free survival, in addition to these three factors, the histological grade (P = 0.011) showed independent prognostic values. CONCLUSION: The independent value of EGFR subcellular location (cytoplasm and membrane) and p53 overexpression in overall survival even in early stages of OSCC suggests that these markers may serve as reliable biological markers to identify high-risk subgroups and to guide therapy.

High-risk endometrial carcinoma profiling identifies TGF-ß1 as a key factor in the initiation of tumor invasion.

Endometrial cancer is among the three most common cancers in females in industrialized countries. In the majority of cases, the tumor is confined to the uterus at the time of diagnosis and presents a good prognosis. However, after primary surgery, 15% to 20% of these tumors recur and have limited response to systemic therapy. We carried out gene expression profiling of high-risk recurrence endometrial cancers to identify new therapeutic approaches targeting the molecular pathways involved in the acquisition of an aggressive tumor phenotype. A microarray gene-expression analysis on a total of 51 human endometrial carcinomas revealed 77 genes specifically altered in high-risk recurrence tumors (P < 0.001). The bioinformatics analysis of gene-gene interactions and molecular relationships among these genes pointed to a prominent role for TGF-ß1 signaling in the acquisition of an aggressive phenotype. We further showed that TGF-ß1 has a principal role at the initiation of endometrial carcinoma invasion through the promotion of the epithelial to mesenchymal transition that leads to the acquisition of an invasive phenotype in HEC-1A and RL95-2 cells. Impairment of this initial step with SB-431542, a specific TGF-ß1 inhibitor, precluded further persistent endometrial carcinoma invasion. In conclusion, we showed that the characterization of the molecular changes associated with the acquisition of an aggressive phenotype represents a realistic strategy for the rational identification and characterization of new potential therapeutic targets in an effort to improve the clinical management and the outcome of high-risk endometrial cancer patients.

Clinical relevance of epidermal growth factor receptor (EGFR) alterations in human pancreatic tumors.

Pancreatic cancer is a malignant neoplasm with an extremely poor prognosis. The mechanisms of aggressive growth and metastasis are currently not well understood. Expression of epidermal growth factor receptor (EGFR) has been suggested to be associated with the malignant transformation of pancreatic cancer. In this study, we examined the EGFR status of 52 pancreatic tumors by PCR-sequencing (exons 19 and 21), immunohistochemistry and FISH probes. We subsequently investigated the relationship between EGFR status and clinicopathological factors. Somatic alterations in EGFR (R841R, T571T and R831C) were observed only in ductal adenocarcinoma (3/34). In 4 (8%) of the 52 tumors analyzed EGFR was overexpressed, 6 (12%) of the tumors showed moderate expression while 19 (32%) were weakly stained. EGFR overexpression (3+ score) was frequently found in endocrine tumors (29%) followed of ampullary tumors (13%; p < 0.01). No significant correlation was observed between the presence of a somatic EGFR mutation and clinicopathological variables. Fluorescence in situ hybridization (FISH) analysis did not demonstrate amplification in any tumors. Only three somatic mutations in the EGFR gene were detected in pancreatic ductal adenocarcinoma and no association was observed with the clinical variables. Our results suggest that EGFR mutations are rare in pancreatic tumors and not associated with clinical prognosis, and treatment response.

BRCA1 mutations do not increase prostate cancer risk: results from a meta-analysis including new data.

BACKGROUND: Although in recent years deleterious BRCA1 mutations have been extensively studied as a prostate cancer risk factor, results are inconclusive. To assess the contribution of the BRCA1 Galician founder mutation c.211A>G in prostate cancer morbidity we conducted a case-control study. Moreover, to better elucidate whether deleterious BRCA1 mutations are involved in the development of prostate cancer, we performed a systematic review and a meta-analysis of BRCA1 studies on prostate cancer. METHODS: A total of 905 unselected men diagnosed with adenocarcinoma of the prostate and a control group of 936 unrelated men without history of prostate cancer were evaluated for c.211A>G. Adjusted by age Odds ratios (OR) and 95% confidence intervals (CIs) were estimated using logistic regression. To construct the meta-analysis, genotype-based epidemiological studies reporting BRCA1 founder mutations on prostate cancer were identified by comprehensive and systematic bibliographic search. After extraction of relevant data, main and subgroup analysis by mutation were performed to assess the effect of BRCA1 on prostate cancer risk. RESULTS: Four c.211A>G heterozygous individuals, one patient and three controls, were detected (OR = 0.27; 95% CI: 0.01-2.36; P = 0.28). Meta-analysis results from the integration of our data and other seven studies with BRCA1 genotyping data (5,705 prostate cancer cases and 13,218 controls) did not detect an association with prostate cancer risk (OR = 1.36; 95% CI: 0.87-2.14; P = 0.18). CONCLUSIONS: Our conclusive trial demonstrates the lack of association between Galician splicing mutation c.211A>G in the BRCA1 gene and prostate cancer risk. Moreover, the result of the meta-analysis also discards the involvement of BRCA1 mutations in the development of prostate cancer.

Ductal adenocarcinoma of the pancreas: Expression of growth factor receptors, oncogenes and suppressor genes, and their relationship to pathological features, staging and survival.

Pancreatic ductal adenocarcinoma results in high short-term mortality despite recent advances in diagnostics, surgery and chemotherapy. Modern chemotherapeutic agents directed to specific tumor receptors have higher therapeutic efficacy and lower adverse effects. However, few studies exist that evaluate the clinical impact in pancreatic cancer. The expression of tumor growth factor receptors, oncogenes and tumor suppressor oncogenes in surgical pancreatic cancer specimens as related to pathological characteristics, staging and prognosis was evaluated. Data were recorded for 50 patients who underwent a pancreatic cancer resection and were suitable for immunohistochemical evaluation (32 male, mean age 61 years, range 44-78) with regard to pTN, tumor size and location, histological differentiation grade, vascular and perineural invasion, adjuvant chemotherapy and survival time. Tumor specimens and normal pancreatic tissue were deparaffinized and the expression of vascular epidermal growth factor (VEGF) receptors (R)-1 and -2, epidermal growth factor receptor (EGFR), Her-2/neu, COX-2, p16, p21 and p53 was immunohistochemically evaluated using tissue microarrays. Associations between molecular marker expression and clinicopathological tumor characteristics were evaluated using the Chi-square test (SPSS) and the survival time was defined. The Kaplan-Meier method was utilized to analyze survival curves, verified by the log-rank test. No molecular markers evaluated were expressed in normal tissue. Tumor expression data included VEGF-R1 (74%), EGFR (52%), Her-2/neu (7.84%), COX-2 (21.5%), p16 (29.4%), p21 (21.7%) and p53 (50%). Tumors expressing VEGF-R1, EGFR and/or p53 were larger (p<0.02), frequently poorly differentiated (p<0.05) and more frequently associated with perineural and lymph node invasion (p<0.05). Marker expression did not correlate with pathological tumor characteristics. The median post-surgery survival was 15 months; 60 and 27% patients survived to 12 and 24 months, respectively, with a longer survival time in patients receiving adjuvant chemotherapy (n=20) (median 36 vs. 15 months, p<0.02). Growth factor receptors, oncogenes and tumor suppressor genes were frequently expressed in pancreatic cancer tissue. VEGF-R1, EGFR and p53 expression were associated with poor tissue differentiation and perineural and lymph node infiltration. Only VEGF-R1 expression was associated with a longer survival time and a more favorable response to adjuvant chemotherapy.