Manuel Valladares Ayerbes

Expression of Wnt gene family and frizzled receptors in head and neck squamous cell carcinomas

Genes of the Wnt and Frizzled class, expressed in HNSCC tissue and cell lines, have an established role in cell morphogenesis and differentiation, and also they have oncogenic properties. We studied Wnt and Fz genes as potential tumor-associated markers in HNSCC by qPCR. Expression levels of Wnt and Fz genes in 22 unique frozen samples from HNSCC were measured. We also assessed possible correlation between the expression levels obtained in cancer samples in relation to clinicopathologic outcome. Wnt-1 was not expressed in the majority of the HNSCC studied, whereas Wnt-5A was the most strongly expressed by the malignant tumors. Wnt-10B expression levels were related with higher grade of undifferentiation. Related to Fz genes, Fz-5 showed more expression levels in no-affectation of regional lymph nodes. Kaplan–Meier survival analyses suggest a reduced time of survival for low and high expression of Wnt-7A and Fz-5 mRNA, respectively. qPCR demonstrated that HNSCC express Wnt and Fz members, and suggested that Wnt and Fz signaling is activated in HNSCC cells.Genes of the Wnt and Frizzled class, expressed in HNSCC tissue and cell lines, have an established role in cell morphogenesis and differentiation, and also they have oncogenic properties. We studied Wnt and Fz genes as potential tumor-associated markers in HNSCC by qPCR. Expression levels of Wnt and Fz genes in 22 unique frozen samples from HNSCC were measured. We also assessed possible correlation between the expression levels obtained in cancer samples in relation to clinicopathologic outcome. Wnt-1 was not expressed in the majority of the HNSCC studied, whereas Wnt-5A was the most strongly expressed by the malignant tumors. Wnt-10B expression levels were related with higher grade of undifferentiation. Related to Fz genes, Fz-5 showed more expression levels in no-affectation of regional lymph nodes. Kaplan–Meier survival analyses suggest a reduced time of survival for low and high expression of Wnt-7A and Fz-5 mRNA, respectively. qPCR demonstrated that HNSCC express Wnt and Fz members, and suggested that Wnt and Fz signaling is activated in HNSCC cells.

Evaluation of COX-2, EGFR, and p53 as biomarkers of non-dysplastic oral leukoplakias.

OBJECTIVE Identify candidate SEBs (surrogate endpoint biomarkers) for premalignant trends in head and neck mucosa. STUDY DESIGN Study, by qPCR (quantitative real-time polymerase chain reaction), the expression of COX-2, EGFR and p53 in 24 biopsies of non-dysplastic oral leukoplakia and contra-lateral normal-appearing mucosa. RESULTS COX-2 was up-regulated in leukoplakia (79.2%); whereas EGFR and p53 were up-regulated (p>0.05) in oral contra-lateral normal-appearing mucosa (60% and 46% respectively). Also, p53 expression was correlated with tobacco smoke habits and Spearmans rank correlation coefficient showed a positive linear correlation between p53 and EGFR mRNA expression levels. CONCLUSIONS COX-2 would serve as SEB of oral leukoplakia. The results suggest that p53 appears to be one of the molecular targets of tobacco-related carcinogens in leukoplakia and that the co-expression of p53 and EGFR may play a role in this kind of oral pre-cancerous lesion. More detailed studies of EGFR and p53 should be continued in the future.

Origin of renal cell carcinomas.

Cancer is a heritable disorder of somatic cells: environment and heredity are both important in the carcinogenic process. The primal force is the “two hits” of Knudson’s hypothesis, which has proved true for many tumours, including renal cell carcinoma. Knudson et al. [1, 2] recognised that familial forms of cancer might hold the key to the identification of important regulatory elements known as tumour-suppressor genes. Their observations (i.e., that retinoblastoma tend to be multifocal in familial cases and unifocal in sporadic presentation) led them to propose a two-hit theory of carcinogenesis. Furthermore, Knudson postulated that patients with the familial form of the cancer would be born with one mutant allele and that all cells in that organ or tissue would be at risk, accounting for early onset and the multifocal nature of the disease. In contrast, sporadic tumours would develop only if a mutation occurred in both alleles within the same cell, and, as each event would be expected to occur with low frequency, most tumours would develop late in life and in a unifocal manner [3, 4]. The kidney is affected in a variety of inherited cancer syndromes. For most of them, both the oncogene/tumour-suppressor gene involved and the respective germline mutations have been identified. Each of the inherited syndromes predisposes to distinct types of renal carcinoma. Families with hereditary predisposition to cancer continue to provide a unique opportunity for the identification and characterisation of genes involved in carcinogenesis. A surprising number of genetic syndromes predispose to the development of renal cell carcinoma, and genes associated with five of these syndromes have been already identified: VHL, MET, FH, BHD and HRPT2. Few cancers have as many different types of genetic predisposition as renal cancer, although to date only a small proportion of renal cell cancers can be explained by genetic predisposition.

In Silico and In Vitro Analysis of Small Breast Epithelial Mucin as a Marker for Bone Marrow Micrometastasis in Breast Cancer

Molecular signatures associated with malignant phenotype would be useful for detection of micrometastatic carcinoma cells. The small breast epithelial mucin (SBEM) gene is predicted to code for a low molecular weight glycoprotein. To evaluate its potential role as a marker for bone marrow (BM) micrometastasis in breast cancer (BC) patients, we have studied in silico and in vitro expression profiles of SBEM gene. Digital SBEM expression in libraries obtained from normal and neoplastic tissues and cell-lines (CL) were displayed and counted on the SAGE Anatomic Viewer. Profiles for cytokeratin-19 and mammaglobin (hMAM), commonly targets used for detection of disseminated BC cells were obtained and compared with SBEM data. Human breast and haematopoietic cancer CL and normal BM were examined by RT-PCR for SBEM and hMAM. Bioinformatics tools were used to gain further insights about the biological role of SBEM in normal breast and BC. Genes with expression patterns in breast libraries correlating with SBEM were identified using two-dimensional display. SBEM tag was detected in 40 libraries (21 BC; 8 non-cancerous breast tissues). Intermediate to high expression was found on 15/21 BC libraries and 7/8 non-tumor breast tissue. SBEM tag count was correlated with ERBB2 (0.662), hMAM (0.409), and RRM2 (-0.379). A model system based on RT-PCR for SBEM mRNA was highly sensitive and specific in order to detect isolated tumor cells. Our results demonstrate that SBEM mRNA may be an imp ortant marker for targeting BC micrometastasis.

Prognostic and predictive markers of response to treatment in patients with locally advanced unresectable and metastatic pancreatic adenocarcinoma treated with gemcitabine/nab-paclitaxel: Results of a retrospective analysis

Background: Recent studies support the use of gemcitabine and nab-paclitaxel in adults with locally advanced unresectable or metastatic pancreatic adenocarcinoma although insufficient data are available on prognostic and predictive markers of response to treatment. Objective: The objective of this study is to identify treatment response markers in patients with locally advanced unresectable or metastatic pancreatic adenocarcinoma. Materials and Methods: This is an observational, retrospective, and multicenter study. Sociodemographic, clinical, and therapeutic data were collected. Cox regression models were applied to determine associations. Results: In total, 39 patients were included; 23.1% presented locally advanced pancreatic cancer and 76.9% metastatic disease. They received a mean of 6 ± 3 treatment cycles; 59% required dose reduction, 59% treatment delay, and 20.5% switched to a biweekly regimen. The overall response rate was 23% and the disease control rate was 81%. Median progression-free survival was 9 months and median overall survival (OS) was 15 months. A higher neutrophil/lymphocyte ratio (NLR) was significantly associated with lower OS. We reported Grades 1–4 nonhematological and hematological toxicities. Conclusion: NLR is a useful prognostic factor for OS in patients with locally advanced unresectable or metastatic pancreatic adenocarcinoma treated with gemcitabine and nab-paclitaxel. Moreover, we suggest that a biweekly regimen is an option for certain groups of patients.

Molecular expression profiling and pathway analysis of formalin-fixed paraffin-embedded primary renal tumor specimens.

448 Background: Many studies have demonstrated genetic and environmental factors lead to renal cell carcinoma (RCC) occurring during a protracted period of tumorigenesis. It seemed suitable identify and characterize potential molecular markers which might provide rapid and effective possibilities for early detection of RCC. The purpose from this analysis was to derive predictive models which could predict more accurately than any one factor alone. METHODS We assessed using quantitative real-time PCR (qPCR) with SYBR Green the profile of 32 predictive markers involved in the cascade of events leading to the formation and progression of this disease to evaluate their involvement in oncogenesis. RNA of quality was obtained in 90% of samples (N=80, 52/80 clear RCC, 6/80 papillary RCC and 14/80 chromophobe RCC) to carry out the study of gene expression. GenEx software was used for qPCR data processing and analysis. The potential correlation between mRNA expression and the pathological features of the study subjects was assessed by Pearson Chi-squared. Linear rather than logistic regression models were used (SPSS statistics 21.0 software). Additionally, a knowledgebases of biological pathways, Reactome and Snow (Babelomics), were used to superimpose our quantitative expression data. RESULTS The best gene predictors related to different pathological variables (histological type, tumor size, Fuhrman grade, number of involved nodes, renal pelvis invasion, lymphatic vessels invasion, and rupture of the renal capsule): Hif1-α (p < 0.001), Hif1-β (p = 0.035), DLL1 (p = 0 .012), DLL3 (p< 0.001), c-Kit (p = 0.002), PDGFR-β (p =0.002), PDGFR-α (p = 0.026), Bax-β (p = 0.005), Survivin (p = 0.028), Notch1 (p = 0.006), Notch2 (p = 0.007), Notch3 (p = 0.016), Notch4 (p = 0.005), EGFR (p = 0.029), Glut3 (p = 0.026), Glut5 (p = 0.036), FH (p = 0.018), CA9 (p = 0.030), and VHL-1(p = 0.015). CONCLUSIONS Our data suggested that altered expression of certain members involved in the main pathways which feed RCC and their downstream targets analyzed together could improve the diagnostic accuracy for renal cancer by combining determinations of mRNA markers.

Evaluation of Messenger RNA of Pituitary Tumour-transforming Gene-1 (PTTG1) as a Molecular Marker for Micrometastasis

Pituitary tumor-transforming gene-1 (PTTG1) has been implicated as an inhibitor of chromatid separation; its overexpression may lead to chromosomal instability. PTTG1 functions through SH3-mediated signal transduction pathways and activation of growth factors, including basic fibroblast growth factor (bFGF) mediated-angiogenesis. In order to detect micrometastasis in solid tumor patients, we developed a model system based on reverse transcriptase (RT)-PCR amplification of PTTG1 mRNA. We analyzed PTTG1 expression, using RT-PCR, in a panel of human tumors cell-lines (hTCL), including breast, gastrointestinal, small-cell lung cancer, and haematopoietic neoplasms. In addition, to newly hTCL established in our laboratory were investigated: Pancreatic, MBQ-OJC1. Small cell lung carcinoma, JCA-OJC3. Colon, JJPF-OJC4. Specific amplicon for PTTG1 was confirmed by RT-PCR in all the hTCL tested. In breast cell lines expression for PTTG1-mRNA was not related to invasiveness, tumorogenicity, or estrogen receptor (ERα) status. PTTG1-mRNA was examined by PCR amplification of cDNA obtained from normal lymph nodes, bone marrow, and peripheral blood (cellular and plasmatic mRNA). Using a hot-start PCR and different amounts of input RNA, PTTG1 transcript was detected in control bone marrow but not on lymph nodes. PTTG1-mRNA was detected in peripheral blood from 4/14 healthy donors analyzed. Our data confirms that PTTG1 is abundantly expressed in human tumors of different histologic types. PTTG1 qualitative mRNA detection may be useful as a surrogate molecular marker for angiogenic phenotype and invasive tumor, however, it is not useful as a micrometastasis marker due to its transcription in normal bone marrow and peripheral blood.

Biomolecular characterization of formalin-fixed paraffin-embedded primary renal tumor specimens.

421 Background: Renal cell carcinoma (RCC) is the most common type of kidney cancer. It is required a better understanding of signalling pathways involved in renal cells. METHODS We assessed using immunohistochemistry (IHC) and quantitative real-time PCR (qPCR) with SYBR Green the profile of predictive markers involved in the cascade of events leading to the formation and progression: invasiveness, angiogenesis and antiapoptotic mechanisms. 80 RCC tissues were detected by IHC. RNA of quality only was obtained in 72 (80% clear RCC, 10% papillary RCC and 10% chromophobe RCC) to carry out the study of gene expression through qPCR. A pool of normal kidney-derived RNA samples (N=5) was used as healthy control. GAPDH and YWHAZ were used as reference genes. GenEx software was used for qPCR data processing and analysis. Nonparametric tests (Pearsons correlation coefficient test and Spearmans rho test, Kruskal - Wallis and Mann - Whitney) were applied for statistical data analysis (SPSS 19). RESULTS p53 showed higher expression in those cases with Furhman grade I (p=0.036). Moreover p53 showed a positive association with VEGF, GLUT1, GLUT4, VEGFR-2 and VHL (r=0.241, sig. level 0.05; r=0.291, sig. level 0.01; r=0.456, sig. level 0.01; r=0.187, sig. level 0.097 and Spearmans rho=0.269, sig. level 0.05 respectively). Hif1- α, Hif1- β, Notch1 and Notch3 were upregulated in chromophobe RCC (p=0.045, p=0.03, p=0.03 and p=0.02 respectively). Hif1- α and Notch3 were upregulated in clear RCC (p=0.034 and p=0.041 respectively). Spearmans correlation coefficient showed a strong positive correlation between Nocth1-4 members and their receptors and Hif1-α and β genes. Highlight the correlation found between: Notch1 and Hif1-α (Spearmans rho = 0.740, significance level 0.01) and Hif1-β and Jagged1 (Spearmans rho = 0.752, significance level 0.01). CONCLUSIONS The pathway involving the tumor suppressor gene p53 could regulate tumor angiogenesis. Co-expression of Notch receptors, their ligands and Hif-1 α and Hif1- β subunits may play a role in human RCC. Notch cascade may represent a novel and therapeutically accessible pathway in chromophobe and clear RCC. More detailed studies of these crossing pathways are in progress.