Gabriella Mattei

Displayed correlation between gene expression profiles and submicroscopic alterations in response to cetuximab, gefitinib and EGF in human colon cancer cell lines.

BackgroundEGFR is frequently overexpressed in colon cancer. We characterized HT-29 and Caco-2, human colon cancer cell lines, untreated and treated with cetuximab or gefitinib alone and in combination with EGF.MethodsCell growth was determined using a variation on the MTT assay. Cell-cycle analysis was conducted by flow cytometry. Immunohistochemistry was performed to evaluate EGFR expression and scanning electron microscopy (SEM) evidenced the ultrastructural morphology. Gene expression profiling was performed using hybridization of the microarray Ocimum Pan Human 40 K array A.ResultsCaco-2 and HT-29 were respectively 66.25 and 59.24 % in G0/G1. They maintained this level of cell cycle distribution after treatment, suggesting a predominantly differentiated state. Treatment of Caco-2 with EGF or the two EGFR inhibitors produced a significant reduction in their viability. SEM clearly showed morphological cellular transformations in the direction of cellular death in both cell lines treated with EGFR inhibitors. HT-29 and Caco-2 displayed an important reduction of the microvilli (which also lose their erect position in Caco-2), possibly invalidating microvilli absorption function. HT-29 treated with cetuximab lost their boundary contacts and showed filipodi; when treated with gefitinib, they showed some vesicles: generally membrane reshaping is evident. Both cell lines showed a similar behavior in terms of on/off switched genes upon treatment with cetuximab. The gefitinib global gene expression pattern was different for the 2 cell lines; gefitinib treatment induced more changes, but directly correlated with EGF treatment.In cetuximab or gefitinib plus EGF treatments there was possible summation of the morphological effects: cells seemed more weakly affected by the transformation towards apoptosis. The genes appeared to be less stimulated than for single drug cases.ConclusionThis is the first study to have systematically investigated the effect of cetuximab or gefitinib, alone and in combination with EGF, on human colon cancer cell lines. The EGFR inhibitors have a weaker effect in the presence of EGF that binds EGFR. Cetuximab treatment showed an expression pattern that inversely correlates with EGF treatment. We found interesting cyto-morphological features closely relating to gene expression profile. Both drugs have an effect on differentiation towards cellular death.

A role for epidermal growth factor receptor in idiopathic pulmonary fibrosis onset

In idiopathic pulmonary fibrosis (IPF) patients the presence of missense polymorphisms (SNP) in members of the epidermal growth factor receptor (EGFR) family or their genetic association could influence the binding affinity of natural ligands, modifying the expression and the behavior of the correlated genes. EGFR family members are particularly involved in the epithelial injury and fibrotic process in IPF. Genetic variations in HER family of receptors may alter the possible therapeutic efficacy of EGFR inhibitors. This study aimed to analyze the relationships between IPF and specific EGF receptor family functional polymorphisms. We tested the presence of common EGFR, HER2 and HER3 non-synonymous SNPs in the peripheral blood of 20 Italian IPF patients and their association with the disease. Our data indicated that the HER2 variant allele frequency was significantly lower in patients than in controls, with an odds ratio of 0.31 (95% CI 0.080, 0.98). Our finding suggests that HER2 variant could be a protective factor against IPF onset.

A candidate gene study of one-carbon metabolism pathway genes and colorectal cancer risk.

The risk of colorectal cancer (CRC) may be influenced by aberrant DNA methylation and altered nucleotide synthesis and repair, possibly caused by impaired dietary folate intake as well as by polymorphic variants in one-carbon metabolism genes. A case-control study using seventy-one CRC patients and eighty unrelated healthy controls was carried out to assess the genetic association of fifteen SNP and one insertion in nine genes belonging to the folate pathway. Polymorphism selection was based on literature data, and included those which have a known or suspected functional impact on cancer and missense polymorphisms that are most likely to alter protein function. Genotyping was performed by real-time PCR and PCR followed by restriction analysis. The likelihood ratio statistic indicated that most of the polymorphisms were not associated with the risk of CRC. However, an increased risk of CRC was observed for two variant alleles of SNP mapping on the transcobalamin 2 gene (TCN2): C776G (rs1801198) and c.1026-394T>G (rs7286680). Considering the crucial biological function played by one-carbon metabolism genes, further investigations with larger cohorts of CRC patients are needed in order to confirm our preliminary results. These preliminary results indicate that TCN2 polymorphisms can be a susceptibility factor for CRC.

Systematic large-scale meta-analysis identifies a panel of two mRNAs as blood biomarkers for colorectal cancer detection

Colorectal cancer (CRC) is the third most common cancer in the world. A significant survival rate is achieved if it is detected at an early stage. A whole blood screening test, without any attempt to isolate blood fractions, could be an important tool to improve early detection of colorectal cancer. We searched for candidate markers with a novel approach based on the Transcriptome Mapper (TRAM), aimed at identifying specific RNAs with the highest differential expression ratio between colorectal cancer tissue and normal blood samples. This tool permits a large-scale systematic meta-analysis of all available data obtained by microarray experiments. The targeting of RNA took into consideration that tumour phenotypic variation is associated with changes in the mRNA levels of genes regulating or affecting this variation. A real time quantitative reverse transcription polymerase chain reaction (qRT- PCR) was applied to the validation of candidate markers in the blood of 67 patients and 67 healthy controls. The expression of genes: TSPAN8, LGALS4, COL1A2 and CEACAM6 resulted as being statistically different. In particular ROC curves attested for TSPAN8 an AUC of 0.751 with a sensitivity of 83.6% and a specificity of 58.2% at a cut off of 10.85, while the panel of the two best genes showed an AUC of 0.861 and a sensitivity of 92.5% with a specificity of 67.2%. Our preliminary study on a total of 134 subjects showed promising results for a blood screening test to be validated in a larger cohort with the staging stratification and in patients with other gastrointestinal diseases.

Gene expression profile of human colon cancer cells treated with cross-reacting material 197, a diphtheria toxin non-toxic mutant.

Cross-Reacting Material 197 (CRM197) is a diphtheria toxin non-toxic mutant that has shown antitumor activity in mice and humans. It is still unclear whether this anti-tumorigenic effect depends on its strong inflammatory-immunological property, its ability to inhibit heparin-binding epidermal growth factor (HB-EGF), or even its possible weak toxicity. CRM197 is utilized as a specific inhibitor of HB-EGF that competes for the epidermal growth factor receptor (EGFR), overexpressed in colorectal cancer and implicated in its progression. In this study we evaluate the effects of CRM197 on HT-29 human colon cancer cell line behaviour and, for CRM197 recognized ability to inhibit HB-EGF, its possible influence on EGFR activation. In particular, while HT-29 does not show any reduction of viability after CRM197 treatment (MTT modified assay), or changes in cell cycle distribution (flow cytometry), in EGFR localization, phospho-EGFR detected signals (immunohistochemistry) or in morphology (scanning electron microscopy, SEM) they show a change in the gene expression profile by microarray analysis (cDNA microarray SS-H19k8). The overexpression of genes like protein phosphatase 2, catalytic subunit, alpha isozyme (PPP2CA), guanine nucleotide-binding protein G subunit alpha-1(GNAIl) and butyrophilin, subfamily 2, member A1 (BTN2A1) has been confirmed with real-time-qPCR. This is the first study where the CRM197 treatment on HT-29 shows a possible scarce implication of endogenous HB-EGF on EGFR expression and cancer cell development. At the same time, our results show the alteration of a specific and selected number of genes.

The EGFR R521K polymorphism influences the risk to develop colorectal cancer.

Epidermal growth factor receptor (EGFR) family members (EGFR, HER2, HER3 and HER4) have been extensively investigated for its possible involvement in cancer development and progression. In colorectal cancer (CRC) EGFR family has been found frequently over-expressed, thus therapy targeting EGFR has been developed. Interestingly, it has been observed that genetic variants in these receptors may alter the therapeutic efficacy of EGFR inhibitors. Polymorphic variants in members of the EGFR family could influence different biologic activities, such as ligands affinity, dimerization efficiency, kinase activity, expression levels, with a consequent impact in signalling pathways and cell behaviour. This study aimed to verify whether single nucleotide polymorphisms (SNPs) of EGFR family members could represent susceptibility factors able to influence the risk to develop CRC. Peripheral blood of 70 Italian colon cancer patients and 72 healthy controls was used as a source of genomic DNA to investigate EGFR, HER2 and HER3 common non-synonymous SNPs. Genetic association tests were performed to verify a possible relationship with CRC. Evidence of genotype association was found for the R521K EGFR polymorphism under a dominant mode of inheritance (Mid-P=0.031). Genotypes with the variant allele of EGFR R521K SNP confer a risk reduction to develop CRC.

Possible Gender-Related Modulation by the ROCK1 Gene in Colorectal Cancer Susceptibility

Aim: In view of accumulating evidence supporting a pivotal role of the Rho/ROCK pathway in cancer, we investigated Rho-kinase polymorphisms as potential susceptibility factors in colorectal cancer (CRC) in a representative sample of the Italian population. Methods: DNA obtained from the peripheral blood samples of 137 CRC patients and 141 healthy controls was genotyped for four ROCK1 (rs35996865; rs73963110; rs2127958; rs288980) and five ROCK2 (rs12692437; rs7563468; rs35768389; rs17463896; rs16857265) selected single nucleotide polymorphisms. Results: None of the allelic variants of the nine selected markers was associated with the occurrence of CRC or with the development of regional lymph node metastasis. By contrast, the ROCK1 rs35996865 G variant allele was significantly more frequent in male patients (p = 0.028) than in the control group. Conclusion: This finding is, at present, the first that points to a possible gender-related modulation by the ROCK1 gene in CRC susceptibility.

Idiopathic pulmonary fibrosis and polymorphisms of the folate pathway genes

OBJECTIVES This study aims to determine the possible association between folate pathway gene polymorphisms and idiopathic pulmonary fibrosis. This represents the first study carried out on folate pathway gene polymorphisms as possible risk factors in this kind of pathology. The premise is that several polymorphisms mapping on genes responsible for folate uptake are associated with the risk of numerous diseases occurring between pregnancy and old age, and that too little is currently known about idiopathic pulmonary fibrosis. DESIGN AND METHODS We genotyped 9 single nucleotide polymorphisms and 1 polymorphic insertion in 7 essential genes belonging to the folate pathway in 32 Italian idiopathic pulmonary fibrosis patients and 81 control subjects. This was done by PCR and restriction analysis. RESULTS Allelic and genotypic association tests indicated that for all the analysed polymorphisms there were no significant differences between patients and controls. Nevertheless, the haplotype association analysis revealed a significant association between idiopathic pulmonary fibrosis and transcobalamin II gene polymorphisms: specifically the haplotype 776G (rs1801198)-c.1026-394G (rs7286680)-444C (rs10418) (OR=2.84; 95% C.I. 1.36-5.93, P value=0.004). CONCLUSIONS This small-scale preliminary study would suggest the importance of further research focusing on the role of folate in the onset of idiopathic pulmonary fibrosis.

LGALS4, CEACAM6, TSPAN8, and COL1A2: Blood Markers for Colorectal Cancer—Validation in a Cohort of Subjects With Positive Fecal Immunochemical Test Result

Background A noninvasive blood test for the early detection of colorectal cancer (CRC) is highly required. We evaluated a panel of 4 mRNAs as putative markers of CRC. Materials and Methods We tested LGALS4, CEACAM6, TSPAN8, and COL1A2, referred to as the CELTiC panel, using quantitative reverse transcription polymerase chain reaction, on subjects with positive fecal immunochemical test (FIT) results and undergoing colonoscopy. Using a nonparametric test and multinomial logistic model, FIT‐positive subjects were compared with CRC patients and healthy individuals. Results All the genes of the CELTiC panel displayed statistically significant differences between the healthy subjects (n = 67), both low‐risk (n = 36) and high‐risk/CRC (n = 92) subjects, and those in the negative‐colonoscopy, FIT‐positive group (n = 36). The multinomial logistic model revealed LGALS4 was the most powerful marker discriminating the 4 groups. When assessing the diagnostic values by analysis of the areas under the receiver operating characteristic curves (AUCs), the CELTiC panel reached an AUC of 0.91 (sensitivity, 79%; specificity, 94%) comparing normal subjects to low‐risk subjects, and 0.88 (sensitivity, 75%; specificity, 87%) comparing normal and high‐risk/CRC subjects. The comparison between the normal subjects and the negative‐colonoscopy, FIT‐positive group revealed an AUC of 0.93 (sensitivity, 82%; specificity, 97%). Conclusion The CELTiC panel could represent a useful tool for discriminating subjects with positive FIT findings and for the early detection of precancerous adenomatous lesions and CRC. Micro‐Abstract To implement the effectiveness of early detection for colorectal cancer, we searched for mRNA blood markers (CELTiC panel) in a total of 231 blood samples from healthy subjects, colorectal cancer patients (CRC), and subjects with negative colonoscopy but positive fecal immunochemical test (FIT) results. Our data suggest that the CELTiC panel might represent a useful tool for discriminating subjects with positive FIT results and for early detection of precancerous adenomatous lesions and CRC.

Contents Vol. 82, 2015

Founded 1938 as ‘Schweizerische Zeitschrift für allgemeine Pathologie und Bakteriologie’ by A. v. Albertini, A. Grumbach and H. Mooser, continued as ‘Pathologia et Microbiologia’ (1960–1975) and ‘Experimental Cell Biology’ (1976–1989); incorporating ‘Pathology and Immunopathology Research’, founded 1982 as ‘Survey and Synthesis of Pathology Research’ by J.M. Cruse and R.E. Lewis, continued as ‘Pathobiology’, edited by J.M. Cruse and R.E. Lewis (1990–1998) Continued by Ch. Wittekind (1999–2004)