Serum levels of the soluble epidermal growth factor receptor (sEGFR) and its ligands epidermal growth factor (EGF), transforming growth factor-α (TGF-α) and amphiregulin (AR) were measured in healthy donors and patients with non-small cell lung cancer (NSCLC) and head and neck carcinoma (HNC). In NSCLC, we found sEGFR and EGF levels significantly lowered in patients with respect to healthy donors. In HNC patients, significantly diminished levels were found in the case of sEGFR, EGF and also AR. In both malignancies, no significant association was found between the serum levels of the molecules and the patients' gender, age or smoking habit. Only a significant association was found between the decrease of sEGFR and the absence of distant metastasis in NSCLC and the tumour stage in HNC. The most interesting result was that combining sEGFR and EGF, sensitivities of 88% in NSCLC and 100% in HNC were reached without losing specificity (97.8% in both cases). The use of discriminant analysis and logistic regression improved the sensitivity for NSCLC and the specificity for HNC. These data demonstrate a potentially interesting value of the serum levels of sEGFR and EGF, especially when combined, as markers for NSCLC and HNC.

Colorectal cancer is the fourth cause of cancer-related deaths worldwide, though detection at early stages associates with good prognosis. Thus, there is a clear demand for novel non-invasive tests for the early detection of colorectal cancer and premalignant advanced adenomas, to be used in population-wide screening programs. Aberrant DNA methylation detected in liquid biopsies, such as serum circulating cell-free DNA (cfDNA), is a promising source of non-invasive biomarkers. This study aimed to assess the feasibility of using cfDNA pooled samples to identify potential serum methylation biomarkers for the detection of advanced colorectal neoplasia (colorectal cancer or advanced adenomas) using microarray-based technology. cfDNA was extracted from serum samples from 20 individuals with no colorectal findings, 20 patients with advanced adenomas, and 20 patients with colorectal cancer (stages I and II). Two pooled samples were prepared for each pathological group using equal amounts of cfDNA from 10 individuals, sex-, age-, and recruitment hospital-matched. We measured the methylation levels of 866,836 CpG positions across the genome using the MethylationEPIC array. Pooled serum cfDNA methylation data meets the quality requirements. The proportion of detected CpG in all pools (> 99% with detection p value < 0.01) exceeded Illumina Infinium methylation data quality metrics of the number of sites detected. The differential methylation analysis revealed 1384 CpG sites (5% false discovery rate) with at least 10% difference in the methylation level between no colorectal findings controls and advanced neoplasia, the majority of which were hypomethylated. Unsupervised clustering showed that cfDNA methylation patterns can distinguish advanced neoplasia from healthy controls, as well as separate tumor tissue from healthy mucosa in an independent dataset. We also observed that advanced adenomas and stage I/II colorectal cancer methylation profiles, grouped as advanced neoplasia, are largely homogenous and clustered close together. This preliminary study shows the viability of microarray-based methylation biomarker discovery using pooled serum cfDNA samples as an alternative approach to tissue specimens. Our strategy sets an open door for deciphering new non-invasive biomarkers not only for colorectal cancer detection, but also for other types of cancers.

The need for novel biomarkers that could aid in non-small cell lung cancer (NSCLC) detection, together with the relevance of Matrix Metalloproteases (MMPs) -1, -2, -7, -9 and -10 in lung tumorigenesis, prompted us to assess the diagnostic usefulness of these MMPs and the Tissue Inhibitor of Metalloproteinase (TIMP) -1 in NSCLC patients. Markers were evaluated in an initial study cohort (19 NSCLC cases and 19 healthy controls). Those that better performed were analyzed in a larger sample including patients with benign lung diseases. Serum MMPs and TIMP-1 were determined by multiplexed immunoassays. Logistic regression was employed for multivariate analysis of biomarker combinations. MMPs and TIMP-1 were elevated in the serum of NSCLC patients compared to healthy controls. MMP-1, -7 and -9 performed at best and were further evaluated in the sample including benign pathologies, corroborating the superiority of MMP-9 in NSCLC discrimination, also at early-stage NSCLC. The optimal diagnostic value was obtained with the model including MMP-9, gender, age and smoking history, that demonstrated an AUC of 0.787, 85.54% sensitivity and 64.89% specificity. Our results suggest that MMP-9 is a potential biomarker for NSCLC diagnosis and its combined measurement with other biomarkers could improve NSCLC detection.

The aim of this study has been to investigate the potential of serum biomarkers used in clinical practice (CEA, CYFRA 21–1, SCC) together with the serum epidermal growth factor receptor (EGFR) and its associated ligands (EGF, TGF-α, HB-EGF) as outcome predictors of non-small cell lung cancer (NSCLC) patients treated with the TKI erlotinib. The pretreatment levels of these markers were evaluated through immunoassays carried out in 58 patients. The progression-free survival (PFS) and overall survival (OS) were assessed by the Kaplan-Meier method and differences between groups were compared by means of the Log-Rank test. Association of risk factors with survival was evaluated using the univariate and multivariate Cox modelling procedures. Higher CEA (>5 ng/mL) and sEGFR (>56.87 ng/mL) concentrations associated significantly with a higher overall survival. The pre-treatment sEGFR serum levels constituted an independent prognostic factor. The EGFR gene mutational status and the sEGFR level combination was the single to associate significantly with longer progression-free survival periods, in circumstances in which the EGFR gene was mutated and increased protein serum levels were detected. The overall survival as assessed through a Cox analysis revealed similar death hazards with respect to low sEGFR levels combined both with non-mutated EGFR genotypes and low CEA serum levels. Our results suggest that the pre-treatment CEA and sEGFR serum levels may provide a comparable source of information to that supplied by the EGFR gene mutational status with respect to the prognosis of erlotinib treated NSCLC patients. A combined sEGFR and CEA level appraisal could be of considerable value to select patients to undergo EGFR-TKI treatments.

In previous studies we described a decreased alpha-L-fucosidase activity in colorectal tumors, appearing as a prognostic factor of tumoral recurrence. The aim of this work was to extend the knowledge about tissue alpha-L-fucosidase in colorectal cancer by quantifying the expression of its encoding gene FUCA1 in tumors and healthy mucosa. FUCA1 mRNA levels were measured by RT-qPCR in paired tumor and normal mucosa tissues from 31 patients. For the accuracy of the RT-qPCR results, five candidate reference genes were validated in those samples. In addition, activity and expression of alpha-L-fucosidase in selected matched tumor and healthy mucosa samples were analyzed. According to geNorm and NormFinder algorithms, RPLP0 and HPRT1 were the best reference genes in colorectal tissues. These genes were used for normalization of FUCA1 expression levels. A significant decrease of more than 60% in normalized FUCA1 expression was detected in tumors compared to normal mucosa (p = 0.002). Moreover, a gradual decrease in FUCA1 expression was observed with progression of disease from earlier to advanced stages. These findings were confirmed by Western blot analysis of alpha-L-fucosidase expression. Our results demonstrated diminished FUCA1 mRNA levels in tumors, suggesting that expression of tissue alpha-L-fucosidase could be regulated at transcriptional level in colorectal cancer.

Background: The role of phenotypic plasticity is increasingly being recognized in the field of evolutionary studies. In this paper we look at the role of genetic determinationversusplastic response by comparing the protein expression profiles between two sympatric ecotypes adapted to different shore levels and habitats using two-dimensional protein maps.Results: We compared qualitative and quantitative differences in protein expression between pools of both ecotypes from different environments (field and laboratory conditions). The results suggested that ecotype differences may affect about 7% of the proteome in agreement with previous studies, and moreover these differences are basically insensitive to environmental changes. Thus, observed differences between wild ecotypes can be mainly attributed to genetic factors rather than phenotypic plasticity.Conclusions: These results confirm the mechanism of adaptation already proposed in this species and a minor role of phenotypic plasticity in this ecological speciation process. In addition, this study provides a number of interesting protein spots potentially involved in adaptation, and therefore candidates for a future identification.