Resistance to Fas-Mediated Apoptosis Does Not Correlate to Structural Alterations or Expression Changes of the Death Receptor in Papillary Thyroid Carcinomas.

BACKGROUND: Malignant cells exhibit significant resistance to FAS-mediated cell death, through different processes, including FAS mutations, soluble FAS expression, or FAS transcriptional dysregulation by P53, eventually escaping from immune surveillance. Since thyroid carcinomas were shown to be resistant to FAS-mediated apoptosis, we investigated the above mechanisms in thyroid carcinoma samples. METHODS: Thirty-seven thyroid carcinoma samples were analyzed for mutations in FAS exon 9 and TP53 exons 5-8 and protein expression by means of immunohistochemistry. Moreover, thyroid carcinoma mRNA samples were subjected to reverse transcription - PCR, to evaluate the relative expression of transmembrane FAS versus its soluble form. RESULTS: Analysis revealed indications for TP53 mutations in the anaplastic carcinomas, but not in the other thyroid specimens examined for TP53 or FAS exon 9 mutations. FAS receptor expression was observed in almost all thyroid specimens (97%) with significant up-regulation in papillary carcinomas. P53 nuclear staining was observed only in anaplastic carcinomas. Full-length FAS mRNA was detected in all specimens examined, with soluble FAS mRNA being either absent or present in very low amounts. CONCLUSIONS: Our results denote that FAS death domain or TP53 DNA-binding domain mutations, down-regulation of FAS receptor expression, or expression of FAS soluble isoform are not responsible for the seeming inhibition of FAS-mediated apoptosis in papillary thyroid carcinoma cells.

Expression and prognostic significance of VEGF and mTOR pathway proteins in metastatic renal cell carcinoma patients: a prognostic immunohistochemical profile for kidney cancer patients.

PURPOSE: To identify prognostic molecular profiles in patients with mRCC treated with sunitinib, we performed immunohistochemical analysis for VEGF and PI3K/Akt/mTOR pathway components. METHODS: The immunohistochemical expression of VEGF, p85α, p110γ, PTEN, p-Akt, p-mTOR, p-4E-BP1 and p-p70S6K was studied in 79 patients with mRCC who received first-line treatment with sunitinib. Expression was correlated with clinicopathological features and survival. RESULTS: VEGF was highly expressed (median H-Score 150), while positivity for the markers of the PI3K/Akt/mTOR pathway was: p85α 43/66 (65 %), p110γ41/60 (68 %), PTEN 32/64 (50 %), p-Akt57/63 (90 %), p-mTOR48/64 (75 %), p-4E-BP1 58/64 (90 %) and p-p70S6K 60/65 (92 %). No single immunohistochemical marker was found to have prognostic significance. Instead, the combination of increased p-mTOR and low VEGF expression was adversely correlated with overall survival (OS) (3.2 vs. 16.9 months, P = 0.001). CONCLUSION: Immunohistochemistry for VEGF and p-mTOR proteins may discriminate patients refractory to first-line sunitinib with poor prognosis. Prospective validation of our findings is needed.

Clinical significance of AGE-RAGE axis in colorectal cancer: associations with glyoxalase-I, adiponectin receptor expression and prognosis.

BACKGROUND: Advanced glycation end products (AGEs) and their receptor RAGE emerge as important pathogenic contributors in colorectal carcinogenesis. However, their relationship to the detoxification enzyme Glyoxalase (GLO)-I and Adiponectin receptors (AdipoR1, AdipoR2) in colorectal carcinoma (CRC) is currently understudied. In the present study, we investigated the expression levels of the above molecules in CRC compared to adjacent non-tumoral tissue and their potential correlation with clinicopathological characteristics and patients' survival. METHODS: We analyzed the immunohistochemical expression of AGE, RAGE, GLO-1, AdipoR1 and AdipoR2 in 133 primary CRC cases, focusing on GLO-I. The tumour MSI status was further assessed in mucinous carcinomas. Western immunoblotting was employed for validation of immunohistochemical data in normal and tumoral tissues as well in three CRC cell lines. An independent set of 55 patients was also used to validate the results of univariate survival analysis regarding GLO-I. RESULTS: CRC tissue showed higher intensity of both AGE and RAGE expression compared with normal colonic mucosa which was negative for GLO-I in most cases (78 %). Western immunoblotting confirmed AGE, RAGE and GLO-I overexpression in tumoral tissue. GLO-I expression was directly related to RAGE and inversely related to AGE immunolabeling. There was a trend towards higher expression of all markers (except for RAGE) in the subgroup of mucinous carcinomas which, although of borderline significance, seemed to be more prominent for AdipoR1 and AGE. Additionally, AGE, AdipoR1 and Adipo R2 expression was related to tumor grade, whereas GLO-1 and AdipoR1 to T-category. In survival analysis, AdipoR2 and GLO-I overexpression predicted shortened survival in the entire cohort and in early stage cases, an effect which for GLO-I was reproduced in the validation cohort. Moreover, GLO-I emerged as an independent prognosticator of adverse significance in the patients' cohort. CONCLUSIONS: We herein provide novel evidence regarding the possible interactions between the components of AGE-RAGE axis, GLO-I and adiponectin receptors in CRC. AGE and AdipoR1 are possibly involved in colorectal carcinogenesis, whereas AdipoR2 and GLO-I emerged as novel independent prognostic biomarkers of adverse significance for patients with early disease stage. Further studies are warranted to extend our observations and investigate their potential therapeutic significance.

Polycystin-1 and polycystin-2 are involved in the acquisition of aggressive phenotypes in colorectal cancer.

The polycystins PC1 and PC2 are emerging as major players in mechanotransduction, a process that influences all steps of the invasion/metastasis cascade. We hypothesized that PC1 and PC2 facilitate cancer aggressiveness. Immunoblotting, RT-PCR, semi-quantitative and quantitative real-time PCR and FACS analyses were employed to investigate the effect of polycystin overexpression in colorectal cancer (CRC) cells. The impact of PC1 inhibition on cancer-cell proliferation was evaluated through an MTT assay. In vitro data were analyzed by Student's t-test. HT29 human xenografts were treated with anti-PC1 (extracellular domain) inhibitory antibody and analyzed via immunohistochemistry to determine the in vivo role of PC1 in CRC. Clinical significance was assessed by examining PC1 and PC2 protein expression in CRC patients (immunohistochemistry). In vivo and clinical data were analyzed by non-parametric tests, Kaplan-Meier curves, log-rank test and Cox model. All statistical tests were two-sided. PC1 overexpression promotes epithelial-to-mesenchymal transition (EMT) in HCT116 cells, while PC2 overexpression results in upregulation of the mTOR pathway in SW480 cells. PC1 inhibition causes reduced cell proliferation in CRC cells inducing tumor necrosis and suppressing EMT in HT29 tumor xenografts. In clinical study, PC1 and PC2 overexpression associates with adverse pathological parameters, including invasiveness and mucinous carcinomas. Moreover, PC1 overexpression appears as an independent prognostic factor of reduced recurrence-free survival (HR = 1.016, p = 0.03) and lowers overall survival probability, while aberrant PC2 expression predicts poor overall survival (p = 0.0468). These results support, for the first time, a direct link between mechanosensing polycystins (PC1 and PC2) and CRC progression.

In situ assessment of PI3K and PTEN alterations in mycosis fungoides: correlation with clinicopathological features.

Deregulated signalling through phosphatidylinositol 3-kinase (PI3K) pathway plays a critical role in tumour initiation and progression. We have already shown that AKT is activated in skin lesions in Mycosis Fungoides (MF) and we herein further investigate the frequency and clinical significance of PTEN and PI3K at the protein and at the DNA level as well as the presence of AKT1 mutations in skin lesions from 50 patients with MF clinical stages I-IV in relation to clinicopathological features. Increased p-AKT expression correlated with poor prognosis in plaques (P = 0.0198), whereas p-AKT was an independent predictor of poor survival in the entire cohort (P = 0.017, HR = 1.012). PTEN cytoplasmic expression was found low or absent in all 77.3% of cases and inversely correlated with advanced clinical stages (P = 0.0744). Molecular analysis showed no AKT1 mutation, no PI3KCA copy number gain, only 1 case with PI3KCA mutation in exon 9 and 3 cases with PTEN mutations (7%) in exons 7, 8 and 5. The latter correlated with disease (P = 0.0253) and progression (P < 0.0001) free survival in tumour stage. Although activation of PI3K/AKT signalling pathway due to PTEN alterations is rarely attributed to abnormalities in PTEN, PI3K, and AKT1 genes, PTEN mutations exert a negative effect on patients' prognosis with tumours.

Nationwide Real-World Data of Microsatellite Instability and/or Mismatch Repair Deficiency in Cancer: Prevalence and Testing Patterns.

Determination of microsatellite instability (MSI)/mismatch repair (MMR) status in cancer has several clinical implications. Our aim was to integrate MSI/MMR status from patients tested in Greece to assess the prevalence of MSI-high (MSI-H)/deficient MMR (dMMR) per tumor type, testing patterns over time and concordance between MSI and MMR status. We retrospectively recorded MSI/MMR testing data of patients with diverse tumor types performed in pathology and molecular diagnostics laboratories across Greece. Overall, 18 of 22 pathology and/or molecular diagnostics laboratories accepted our invitation to participate. In the 18 laboratories located across the country, 7916 tumor samples were evaluated for MSI/MMR status. MSI/MMR testing significantly increased in patients with colorectal cancer (CRC) and other tumor types overtime (p < 0.05). The highest prevalence was reported in endometrial cancer (47 of 225 patients, 20.9%). MSI-H/dMMR was observed in most tumor types, even in low proportions. Among 904 tumors assessed both for MSI and MMR status, 21 had discordant results (overall discordance rate, 2.3%). We reported MSI-H/dMMR prevalence rates in patients with diverse cancers, while demonstrating increasing referral patterns from medical oncologists in the country overtime. The anticipated high rate of concordance between MSI and MMR status in paired analysis was confirmed.

Phosphorylated 4E-binding protein 1 (p-4E-BP1): a novel prognostic marker in human astrocytomas.

AIMS: To investigate the significance of the mammalian target of rapamycin (mTOR) pathway in astrocytic tumours, published information in this context being limited, especially regarding phosphorylated 4E-binding protein (p-4E-BP) 1. METHODS AND RESULTS: Paraffin-embedded tissue from 111 patients with astroglial tumours (grades II-IV) was investigated for the association of phosphorylated mTOR (p-mTOR) signalling components with phosphorylated extracellular signal-related kinase 1/2 (p-ERK1/2) and phosphorylated AKT (p-AKT) expression, clinicopathological features, angiogenesis, isocitrate dehydrogenase 1 (IDH1)-R132H, and survival. Expression was also quantified by western blot analysis in 12 cases and in three primary glioma cell cultures following rapamycin treatment. p-mTOR expression correlated with p-4E-BP1 expression and marginally with p-p70S6K expression. p-4E-BP1 expression increased with tumour grade. Rapamycin induced a decline in phosphorylation levels of all three proteins. Nuclear p-AKT and cytoplasmic p-ERK1/2 immunoexpression correlated with p-4E-BP1 expression, whereas cytoplasmic p-AKT expression correlated with p-p70S6K expression. All three proteins were associated with increased angiogenesis but not with IDH1-R132H expression status. p-mTOR adversely affected overall and disease-free survival in univariate analysis. In multivariate survival analysis, the presence of p-4E-BP1 predicted shortened overall survival in the entire cohort and glioblastomas. CONCLUSIONS: mTOR signalling components are differentially involved in the acquisition of a more aggressive and angiogenic phenotype in astrocytic tumours. Moreover, p-4E-BP1 emerges as a novel prognostic marker, which might aid in the selection of patients who are more likely to benefit from therapy with mTOR inhibitors.

A comprehensive immunohistochemical and molecular approach to the PI3K/AKT/mTOR (phosphoinositide 3-kinase/v-akt murine thymoma viral oncogene/mammalian target of rapamycin) pathway in bladder urothelial carcinoma.

UNLABELLED: What's known on the subject? and What does the study add? A few published studies investigating single or various PI3K/AKT/mTOR signalling components have produced inconsistent results. Moreover, PI3K regulatory subunit p85a and activated p70S6K expression levels have not been previously examined in urothelial carcinoma (UC). The present study addresses simultaneously all key members of PI3K/AKT/mTOR signalling cascade supporting a differential implication of PI3K/AKT/mTOR pathway components in urothelial tumorigenesis. Furthermore, we propose p-4E-BP1 as a potential prognostic marker in UC, which might assist the selection of patients more likely to benefit from chemotherapy regimens based on PI3K/AKT/mTOR pathway inhibition. Finally, the present study indicates PIK3CA/AKT1 mutational status as a potential predictive marker for time-to-recurrence. OBJECTIVE: • To perform a comprehensive simultaneous assessment of all key members of phosphoinositide 3-kinase/v-akt murine thymoma viral oncogene/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway along with AKT homolog 1 (AKT1) and PIK3 catalytic alpha polypeptide (PIK3CA) mutations in bladder urothelial carcinoma (UC). • Published information is limited to a few studies looking into single or various combinations of members of this pathway with inconsistent results. In particular the expression status of phosphorylated (p-)p70S6 kinase (p70S6K) and p85a subunit of PI3K has not been tested in UC. PATIENTS AND METHODS: • Paraffin-embedded transurethral resection tissue from 113 patients with UC was investigated for the association of p85aPI3K, p-AKT, p-mTOR, p-p70S6K and p-4E-BP1 (eukaryotic initiation factor 4E-binding protein 1) expression status, as well as PIK3CA and AKT1 mutations with p-extracellular signal-regulated kinase 1/2 (ERK1/2), fibroblast growth factor receptor 3 (FGFR3), pathological features, recurrence and cancer-specific survival. RESULTS: • With the exception of p-p70S6K, all others components of the PI3K/AKT/mTOR pathway were upregulated in UCs as compared with normal urothelium. • p-mTOR expression strongly correlated with its upstream p-AKT and marginally with its downstream p-p70S6K. p85aPI3K and p-ERK1/2 levels were also marginally correlated. • PIK3CA and AKT1 mutations were distinctly uncommon and mutually exclusive, without any association with pathological features. However, the presence of AKT1 mutations was associated with increased FGFR3 levels and was restricted to p85aPI3K immunonegative cases, whereas PIK3CA mutant cases had marginally lower p85aPI3K levels. • The presence of PIK3CA single or combined with AKT1 mutations was associated with shorter recurrence-free survival in univariate survival analysis. An inverse relationship was established between p-4E-BP1 immunopositivity and histological grade or T category, as well as between p-p70S6K levels and T category, the latter relationship being of marginal significance. • p-4E-BP1 nuclear expression was marginally associated with the presence of lymphovascular invasion and adversely affected survival in multivariate, but not in univariate analysis. CONCLUSIONS: • PI3K/AKT/mTOR signalling components appear to be differentially implicated in urothelial tumorigenesis and, with the exception of p85aPI3K, are unrelated to the PIK3CA or AKT1 mutational status. • Our findings propose p-4E-BP1 as a potential prognostic marker in UC independent of its association with pathological features, which might assist the selection of patients more likely to benefit from PI3K/AKT/mTOR axis inhibition. • PIK3CA/AKT1 mutational status may have a place in the prediction of time-to-recurrence.

ERK/pERK expression and B-raf mutations in colon adenocarcinomas: correlation with clinicopathological characteristics.

BACKGROUND: Colorectal (CRC) carcinogenesis through various morphological stages has been linked to several genetic and epigenetic changes. The Raf/MEK/ERK (MAPK) signal transduction cascade is an important mediator of a number of cellular fates. METHODS: In this study, we investigated the presence of B-raf and K-ras mutations in 94 consecutive cases of primary colon adenocarcinoma in correlation with the immunohistochemical expression of total and activated ERK and the expression of mismatch repair proteins (MMR) hMLH1 and hMSH2 as well as their correlations with standard clinicopathological parameters. RESULTS: The immunostaining pattern for total and activated ERK was nuclear and cytoplasmic. hMLH1 and hMSH2 proteins were preserved in 45/63 (71.43%) cases and 35/53 (66.04%) cases respectively. Total ERK nuclear expression, was positively correlated with tumor stage (p = 0.049), whereas nuclear pERK expression was positively correlated with histological grade (p = 0.0113) and tumor stage (p = 0.0952), although the latter relationship was of marginal significance. DNA sequencing showed that 12 samples (12.7%) had a mutation in B-RAF Exon 15 and none in Exon 11, whereas 22 (23.4%) had a K-ras mutation. Disruption of the MAP kinase pathway-either through K-ras or B-raf mutation-was detected in 37% of all the examined cases, although the overexpression of total and activated ERK1/2 was not correlated with the mutational status of K-ras or B-raf genes. Finally, the preservation of hMLH1 or hMSH2 immunoexpression was not correlated with the presence of B-raf and/or K-ras mutations. CONCLUSIONS: In this study, we present evidence that ERK activation occurs in a K-ras or B-raf -independent manner in the majority of primary colon cancer cases. Moreover, B-raf mutations are not associated with mismatch-repair deficiency through loss of hMLH1 or hMSH2 expression. Activated ERK could possibly be implicated in tumor invasiveness as well as in the acquisition of a more aggressive phenotype.

Methylation analysis of APC, AXIN2, DACT1, RASSF1A and MGMT gene promoters in non-small cell lung cancer.

Silencing of tumour-suppressor genes through promoter methylation is frequently observed in carcinogenesis. In this study, we determined the methylation status of RASSF1A, MGMT, APC, AXIN2 and DACT1 genes in 73 cases of non-small cell lung cancer. Methylation-sensitive high-resolution melting analysis (MS-HRM) was used to analyse the promoter methylation, which was further validated with Bisulfite pyrosequencing or Sanger sequencing. Promoter methylation of RASSF1A and APC was frequently found (56% and 49% of cases, respectively), while methylation of MGMT, AXIN2, DACT1 was observed in 30%, 19% and 16%, respectively. Concurrent gene methylation of at least two genes was observed in 55% of the examined cases, with a total of 89% of samples displaying methylation in one or more of the investigated genes. Further analysis of concurrent methylation revealed a positive correlation between AXIN2-DACT1 and an inverse correlation of APC-MGMT. Associations of methylated genes and clinicopathological features were emerged. In more detail, APC promoter methylation was correlated with smoking status (p= 0.020) and non-metastatic cases (p= 0.003). Moreover, MGMT methylation was preferentially found in TTF1-negative cases (p= 0.049). Interestingly, correlation occurred between AXIN2/DACT1 methylation and smoking status (p= 0.009) as well as tumour grade (p= 0.013), as none of these genes was methylated in the majority of smokers and one of the genes was methylated in high-grade tumours. We conclude that aberrant promoter methylation was observed in our cohort while concurrent methylation patterns were also determined. APC, MGMT and AXIN2/DACT1 methylation are potentially of clinical importance regarding prognosis and histological subtyping of NSCLC.

Prognostic significance of replication protein A (RPA) expression levels in bladder urothelial carcinoma.

OBJECTIVE: To elucidate the role of replication protein A (RPA) in both superficial (Ta-T1) and muscle-invasive (T2-T4) urothelial carcinomas (UCs), investigating its potential prognostic usefulness. PATIENTS AND METHODS: Paraffin-embedded tissue from 156 patients with bladder UC was immunostained for RPA1 and RPA2. RESULTS: RPA1 and RPA2 labelling indexes (LIs) decreased with increasing histological grade (both P < 0.001) and T-category in the entire cohort (P = 0.008 and P < 0.001, respectively) and in muscle-invasive carcinomas (P = 0.014 and P = 0.012, respectively). RPA1 expression was positively correlated with RPA2 (Spearman's correlation coefficient ρ = 0.309, P < 0.001). Both RPA1 and RPA2 LIs were positively correlated with cyclin D1 expression (ρ = 0.354, P < 0.001 and ρ = 0.934, P < 0.001). In survival analysis of the entire cohort decreased RPA2 and RPA1 correlated with a lesser probability of survival (P < 0.001 and P = 0.018). In non-muscle-invasive tumours (Ta-T1) only lower RPA2 (P < 0.001) was correlated with shortened survival, whereas in muscle-invasive tumours (T2-T4) decreased RPA2 and RPA1 expression levels were associated with adverse prognosis (P = 0.035 and P = 0.042, respectively). In multivariate survival analysis of the entire cohort and in non-muscle-invasive cases RPA2 expression remained significant, even when adjustment for cyclin D1 expression was applied. CONCLUSIONS: RPA1 and RPA2 overexpression seems to be more important during early T-categories of bladder carcinogenesis. Showing similar kinetics with cyclin D1. RPA2 expression emerges as a valuable marker of favourable prognosis in the entire cohort and in non-muscle-invasive tumours, supplementing the information obtained by standard clinicopathological prognosticators.

Discerning the ancestry of European Americans in genetic association studies.

European Americans are often treated as a homogeneous group, but in fact form a structured population due to historical immigration of diverse source populations. Discerning the ancestry of European Americans genotyped in association studies is important in order to prevent false-positive or false-negative associations due to population stratification and to identify genetic variants whose contribution to disease risk differs across European ancestries. Here, we investigate empirical patterns of population structure in European Americans, analyzing 4,198 samples from four genome-wide association studies to show that components roughly corresponding to northwest European, southeast European, and Ashkenazi Jewish ancestry are the main sources of European American population structure. Building on this insight, we constructed a panel of 300 validated markers that are highly informative for distinguishing these ancestries. We demonstrate that this panel of markers can be used to correct for stratification in association studies that do not generate dense genotype data.

Evidence for frequent concurrent DCUN1D1, FGFR1, BCL9 gene copy number amplification in squamous cell lung cancer.

Non-small cell lung cancer (NSCLC) targeted therapies are mostly based on activating mutations and rearrangements which are rare events in Lung Squamous Cell Carcinomas (LUSC). Recently advances in immunotherapy have improved the therapeutic repository for LUSC, but there is still an urgent need for novel targets and biomarkers. We examined 73 cases of LUSC for relative copy number amplification of DCUN1D1, BCL9, FGFR1 and ERBB2 genes and searched for correlations with molecular alterations and clinicopathological characteristics. In our cohort BCL9 gene was amplified in 57.5 % of the cases, followed by DCUN1D1 in 37 %, FGFR1 in 19 % whereas none of the cases were amplified in ERBB2 gene. The majority of the samples exhibited amplification in at least one gene while half of them displayed concurrent amplification of two/three genes. Interestingly, 93 % of the FGFR1 amplified cases were also found co amplified with DCUN1D1 and/or BCL9 genes. Linear correlations were found between BCL9 and DCUN1D1 as well as BCL9 and FGFR1 gene amplification. BCL9 and DCUN1D1 genes' amplification was correlated with poorly differentiated tumors (p = 0.035 and p = 0.056 respectively), implying their possible role in tumor aggressiveness. This is the first study, to the best of our knowledge that examines the correlation of DCUN1D1 and BCL9 genes relative copy number amplification with molecular alterations and clinicopathologic characteristics of squamous cell lung cancer tissue samples. Our findings show concurrent amplification of genes in different chromosomes, with possible involvement in tumor aggressiveness. These results support the complexity of LUSC tumorigenesis and imply the necessity of multiple biomarkers / targets for a more effective therapeutic result in LUSC.

mRNA coexpression patterns of Wnt pathway components and their clinicopathological associations in breast and colorectal cancer.

Aberrant Wnt signaling is implicated in carcinogenesis triggering efforts for the development of new therapeutic agents, many of which have entered clinical trials. We extend our previous analysis of WNT3, FZD7, LEF1 expression levels in breast and colorectal cancer including WNT2, FZD4 and ß-catenin expression, in an effort to delineate their relative expression levels along with concurrent expression patterns and possible prognostic value. We analyzed 82 breast and 102 colorectal carcinomas for relative mRNA expression levels of the investigated genes by RT-PCR relative quantification with the ΔΔCt method. Statistical analysis was performed in order to determine associations of relative mRNA expression and linear correlations. ß-catenin expression was determined by immunochemistry. Regarding breast carcinomas, decreased relative mRNA expression levels of WNT2, FZD4 were found frequently and WNT2 expression was correlated with ER/ PR status (p = 0.045/p = 0.028), whereas ß-catenin with grade (p = 0.026). In colorectal carcinomas, increased relative mRNA expression levels of WNT2 and FZD4 were found in 59% and 32% of cases respectively, whereas ß-catenin showed decreased mRNA expression levels in 57% of cases and a correlation with pN-category (p = 0.037). Linear correlations were observed between WNT2/FZD4 (R=0.542, p < 0.001), WNT2/ß-catenin (R=0.254, p = 0.010), FZD4/ß-catenin (R=0.406, p < 0.001) expression and a correlation between mRNA expression and membranous/cytoplasmic ß-catenin emerged (p = 0.039/0.046). Our results suggest a possible clinical significance for Wnt pathway gene expression levels in both tumour types. The concurrent expression of the investigated genes as well as the different expression profiles, underlines the complexity of this pathway and the necessity of patient selection in order to maximize the efficacy of drugs targeting Wnt pathway.

MTOR/4EBP1 signaling and MMR status in colorectal cancer: New correlations and arising perspectives.

This is the first study aiming to investigate mTOR signaling and its relation to mismatch repair status (MMR status) in colorectal cancer (CRC). MMR status and the phosphorylated proteins, pmTOR and p4EBP1, have been immunohistochemically analyzed in 108 formalin-fixed, paraffin-embedded CRC specimens. The correlations between them and with clinicopathological data, MAPK pathway (KRAS, NRAS, BRAF) as well as their impact on patients' overall survival have been statistically analyzed. Our results indicated that positive pmTOR expression was significantly associated with KRAS mutations (p = 0.004). From multivariate survival analysis, only p4EBP1 expression emerged as independent adverse prognostic factor for overall survival (HR, 3.322; 95%CI, 1.110-9.945; p = 0.032). Furthermore, MMR deficient carcinomas tend to express low p4EBP1 protein levels (p = 0.002). A survival analysis stratified by MMR status and p4EBP1 expression, showed that MMR proficient tumours with high p4EBP1 expression had the worst overall survival compared with the other examined subgroups (p = 0.019). In conclusion, MAPK and PI3k/Akt pathways seem to be simultaneously overactivated in CRC. P4EBP1 could be used as a prognostic biomarker. By further analyzing the significant association between MMR status and p4EBP1 expression, we suggest that MMR deficient tumours could represent a subpopulation most likely to derive treatment benefit from mTOR inhibition.

P53 immunoexpression as a prognostic marker for human astrocytomas: a meta-analysis and review of the literature.

During the past few decades, researchers have been looking for parameters with an impact on the prognosis of patients with astrocytic tumors. p53 is one of the most widely investigated molecules in human gliomas. We aimed to comprehensively review the evidence for the prognostic usefulness of p53 immunohistochemical expression in paraffin-embedded tissue specimens from diffusely infiltrating astrocytomas. We conducted a systematic review of the PubMed database through December 2007 to identify cohort studies that evaluated p53 immunohistochemical expression as a prognostic marker for human astrocytomas. Estimates of significance were extracted from association tests and hazard ratios with 95% CI from actuarial curves and Cox regression analyses. A meta-analysis was performed on the studies that applied Cox models and had adjusted the hazard ratio of p53 expression with tumor grade and patients' age. We reviewed 44 publications (including 3,627 patients), 14 of which included the estimates (HR and 95% CI) derived from Cox regression. Descriptive analysis showed that most of the published articles did not contain information on important variables such as sex and age (missing from 25% and 11% of studies, respectively), previous treatment, tissue-retrieval and follow-up period (56%). The quantitative synthesis showed that p53 expression is not a significant prognostic factor (combined HR = 1.034, P = 0.531). There was no significant between-study heterogeneity and publication bias. A second meta-analysis performed only on glioblastomas showed that the overall risk of mortality in these tumors was -0.123 (-0.346 to 0.100) and was not statistically significant. After almost 20 years of research, published evidence does not substantiate the usefulness of p53 immunohistochemical expression as a prognostic marker in patients with astrocytic neoplasms.

Concurrent Wnt pathway component expression in breast and colorectal cancer.

Wnt signaling pathway regulates important cell functions such as proliferation and migration and is frequently deregulated in colorectal and breast cancer. Thus, it constitutes an attractive therapeutic target with many drugs being investigated in clinical trials. Eighty-two breast and 102 colorectal carcinomas were analyzed for: relative mRNA expression levels of Wnt pathway components namely Wnt3 ligand, Frizzled 7 receptor and LEF1 transcriptional factor, their concurrent expression patterns and their correlation with clinicopathological features. Regarding breast carcinomas, increased relative mRNA expression levels of WNT3 were found in 54 % of cases whereas decreased relative mRNA expression levels were observed in FZD7 and LEF1 in 82 % and 43 % of cases, respectively. Expression levels of WNT3 were significantly correlated with tumour grade (p = 0.021) in breast cancer. As far as colorectal carcinomas are concerned, increased relative mRNA expression levels of WNT3, FZD7 and LEF1 were found in 60 %, 37 % and 48 % of cases respectively. A statistically significant correlation emerged between LEF1expression levels and pT-category (p = 0.027), suggesting a possible association with tumour aggressiveness in colorectal carcinomas. Statistically significant linear correlations were observed between the expression of WNT3/LEF1 (R = 0.233, p = 0.035) and FZD7/LEF1 (R = 0.359, p = 0.001) in breast carcinomas as well as in colorectal carcinomas (R = 0.536, p < 0.01 and R = 0.210, p = 0.034) respectively. Our results demonstrate a possible clinical significance of Wnt pathway gene expression levels in both tumour types. The distinct expression patterns and simultaneous expression of the investigated genes underscore the complexity of this pathway in breast and colorectal carcinogenesis and highlights the necessity of patient selection with regard to the effectiveness of Wnt pathway inhibitors.

Association between PD-L1 expression and driver gene mutations in non-small cell lung cancer patients: correlation with clinical data.

Lung cancer is the leading cause of cancer death worldwide. Recently, promising therapies have emerged based on PD-1/PD-L1 immune checkpoint inhibitors, which have been approved even as frontline treatment for patients with non-small cell lung cancer (NSCLC). We examined the association between PD-L1 expression and clinicopathological parameters as well as overall survival in 220 NSCLC patients. PD-L1 expression was estimated by immunohistochemistry using 22C3 PharmDx Dako assay and was defined as high, if TPS was ≥ 50%, low, if TPS was 1%-49%, and absent, if TPS was < 1%. EGFR mutations were detected by COBAS while KRAS and BRAF mutations by pyrosequencing. ROS1 and ALK rearrangements were estimated by immunohistochemistry with positive cases being confirmed by CISH and FISH, respectively. Data analysis was performed using SPSS v25.0. PD-L1 expression was positively correlated with KRAS mutations. Anti-PD-1 therapy (pembrolizumab) prolonged overall survival compared to any other treatment. This effect was more pronounced in KRAS-mutated cases compared to KRAS wild-type ones. Patients with positive PD-L1 expression - high or low - who had been treated with pembrolizumab, showed significant survival benefit compared to positive or negative PD-L1 expressors who did not receive immunotherapy. In multivariate analysis, PD-L1 status, stage and pembrolizumab treatment were independent variables for overall survival. PD-L1 expression (TPS ≥ 1%) by itself emerged as a poor prognostic factor, while treatment with pembrolizumab prolonged overall survival. KRAS mutations may affect tumour microenvironment and patient's response to immunotherapy. Immune checkpoint inhibitors could represent an alternative therapeutic option particularly for KRAS-mutated NSCLC patients. Further investigation into this notion is warranted in order to validate this observation.

ATSC transplantation contributes to liver regeneration following paracetamol-induced acute liver injury through differentiation into hepatic-like cells.

INTRODUCTION: Drug-induced liver injury (DILI) is a leading cause of acute liver injury (ALI). Acetaminophen (also termed paracetamol), can often be found in drugs that may be abused (i.e., prescription for pain relief). Animal experiments have shown that mesenchymal stem cell transplantation can ameliorate or even reverse hepatic injury. MATERIAL AND METHODS: ALI was induced in Wistar rats using paracetamol. ATSCs were transplanted via the intravenous, portal vein, or intrahepatic route directly onto the liver parenchyma. Histological evaluation was conducted to assess drug-induced injury following transplantation. Fluorescence in situ hybridization (FISH) was used to verify the location of stem cells on the liver parenchyma. The effect of those cells on liver regeneration was tested by immunohistochemistry for hepatic growth factor (HGF). In addition, reverse transcription-quantitative PCR (qRT-PCR) was used to assess hepatic growth factor (HGF), hepatic nuclear factor 4α (HNF4α), cytochrome P450 1A2 (CYP1A2) and α-fetoprotein (AFP) mRNA expression. RESULTS: Immunohistochemical staining for HGF was stronger in the transplanted groups than that in the control group (P<0.001). HNF4α and HGF mRNA levels were increased on day 7 following transplantation (P<0.001 and P=0.009, respectively). CYP1A2 mRNA levels were also increased (P=0.013) in the intravenous groups, while AFP levels were higher in the intrahepatic groups (P=0.006). ATSC transplantation attenuates ALI injury and promotes liver regeneration. Furthermore, expression of specific hepatic enzymes points to ATSC hepatic differentiation. CONCLUSION: The study showed the positive effects of transplanted adipose tissue stem cells (ATSCs) on liver regeneration (LG) through hepatotrophic factors. Furthermore, increased expression of hepatic specific proteins was recorded in ATSC transplanted groups that indicate stem cells differentiation into hepatic cells.

Microsatellite Instability in Greek Colorectal Carcinoma Patients: Clinicopathological and Molecular Correlations.

BACKGROUND/AIM: In the present retrospective study, we assessed the molecular profile and clinicopathological correlations of Greek colorectal carcinoma (CRC) patients. PATIENTS AND METHODS: Data from 157 CRC patients were collected. High Resolution Melting Analysis and Pyrosequencing/Sanger sequencing were applied to identify KRAS, BRAF, NRAS mutations and microsatellite instability (MSI) status. Immunohistochemistry was performed to characterize the associated Mismatch Repair Protein loss. Statistical calculations were performed using the statistical package SPSS v21.0. RESULTS: KRAS mutations were detected in 39.3% of cases, BRAF in 10.9% and NRAS in 4.9%. MSI status was recognized in 11.5% of CRC patients and was associated with right colon tumors. MSI phenotype was inversely correlated with stage, N status and KRAS mutations and positively correlated with BRAF mutations. CONCLUSION: MSI positive CRCs in the Greek population are more often right-sided, free of metastasis, KRAS wild type and BRAF mutated. Providing more detailed clinicopathological and molecular data for specific populations will enable better clinical management and individualized therapy in the future.