Identification of a biomarker panel for colorectal cancer diagnosis

Background Malignancies arising in the large bowel cause the second largest number of deaths from cancer in the Western World. Despite progresses made during the last decades, colorectal cancer remains one of the most frequent and deadly neoplasias in the western countries. Methods A genomic study of human colorectal cancer has been carried out on a total of 31 tumoral samples, corresponding to different stages of the disease, and 33 non-tumoral samples. The study was carried out by hybridisation of the tumour samples against a reference pool of non-tumoral samples using Agilent Human 1A 60-mer oligo microarrays. The results obtained were validated by qRT-PCR. In the subsequent bioinformatics analysis, gene networks by means of Bayesian classifiers, variable selection and bootstrap resampling were built. The consensus among all the induced models produced a hierarchy of dependences and, thus, of variables. Results After an exhaustive process of pre-processing to ensure data quality--lost values imputation, probes quality, data smoothing and intraclass variability filtering--the final dataset comprised a total of 8, 104 probes. Next, a supervised classification approach and data analysis was carried out to obtain the most relevant genes. Two of them are directly involved in cancer progression and in particular in colorectal cancer. Finally, a supervised classifier was induced to classify new unseen samples. Conclusions We have developed a tentative model for the diagnosis of colorectal cancer based on a biomarker panel. Our results indicate that the gene profile described herein can discriminate between non-cancerous and cancerous samples with 94.45% accuracy using different supervised classifiers (AUC values in the range of 0.997 and 0.955)

Identification of a biomarker panel for colorectal cancer diagnosis

Background:Malignancies arising in the large bowel cause the second largest number of deaths from cancer in the Western World. Despite progresses made during the last decades, colorectal cancer remains one of the most frequent and deadly neoplasias in the western countries. Methods: A genomic study of human colorectal cancer has been carried out on a total of 31 tumoral samples, corresponding to different stages of the disease, and 33 non-tumoral samples. The study was carried out by hybridisation of the tumour samples against a reference pool of non-tumoral samples using Agilent Human 1A 60-mer oligo microarrays. The results obtained were validated by qRT-PCR. In the subsequent bioinformatics analysis, gene networks by means of Bayesian classifiers, variable selection and bootstrap resampling were built. The consensus among all the induced models produced a hierarchy of dependences and, thus, of variables. Results: After an exhaustive process of pre-processing to ensure data quality--lost values imputation, probes quality, data smoothing and intraclass variability filtering--the final dataset comprised a total of 8, 104 probes. Next, a supervised classification approach and data analysis was carried out to obtain the most relevant genes. Two of them are directly involved in cancer progression and in particular in colorectal cancer. Finally, a supervised classifier was induced to classify new unseen samples. Conclusions: We have developed a tentative model for the diagnosis of colorectal cancer based on a biomarker panel. Our results indicate that the gene profile described herein can discriminate between non-cancerous and cancerous samples with 94.45% accuracy using different supervised classifiers (AUC values in the range of 0.997 and 0.955).

On-line breath analysis of volatile organic compounds as a method for colorectal cancer detection

Background: Analysis of exhaled volatile organic compounds (VOCs) in breath is an emerging approach for cancer diagnosis, but little is known about its potential use as a biomarker for colorectal cancer (CRC). We investigated whether a combination of VOCs could distinct CRC patients from healthy volunteers. Methods: In a pilot study, we prospectively analyzed breath exhalations of 38 CRC patient and 43 healthy controls all scheduled for colonoscopy, older than 50 in the average-risk category. The samples were ionized and analyzed using a Secondary ElectroSpray Ionization (SESI) coupled with a Time-of-Flight Mass Spectrometer (SESI-MS). After a minimum of 2 hours fasting, volunteers deeply exhaled into the system. Each test requires three soft exhalations and takes less than ten minutes. No breath condensate or collection are required and VOCs masses are detected in real time, also allowing for a spirometric profile to be analyzed along with the VOCs. A new sampling system precludes ambient air from entering the system, so background contamination is reduced by an overall factor of ten. Potential confounding variables from the patient or the environment that could interfere with results were analyzed. Results: 255 VOCs, with masses ranging from 30 to 431 Dalton have been identified in the exhaled breath. Using a classification technique based on the ROC curve for each VOC, a set of 9 biomarkers discriminating the presence of CRC from healthy volunteers was obtained, showing an average recognition rate of 81.94%, a sensitivity of 87.04% and specificity of 76.85%. Conclusions: A combination of cualitative and cuantitative analysis of VOCs in the exhaled breath could be a powerful diagnostic tool for average-risk CRC population. These results should be taken with precaution, as many endogenous or exogenous contaminants could interfere as confounding variables. On-line analysis with SESI-MS is less time-consuming and doesn’t need sample preparation. We are recruiting in a new pilot study including breath cleaning procedures and spirometric analysis incorporated into the postprocessing algorithms, to better control for confounding variables.

Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma

Inactivation of the genes involved in DNA mismatch repair is associated with microsatellite instability (MSI) in colorectal cancer. We report that hypermethylation of the 5′ CpG island of hMLH1 is found in the majority of sporadic primary colorectal cancers with MSI, and that this methylation was often, but not invariably, associated with loss of hMLH1 protein expression. Such methylation also occurred, but was less common, in MSI− tumors, as well as in MSI+ tumors with known mutations of a mismatch repair gene (MMR). No hypermethylation of hMSH2 was found. Hypermethylation of colorectal cancer cell lines with MSI also was frequently observed, and in such cases, reversal of the methylation with 5-aza-2′-deoxycytidine not only resulted in reexpression of hMLH1 protein, but also in restoration of the MMR capacity in MMR-deficient cell lines. Our results suggest that microsatellite instability in sporadic colorectal cancer often results from epigenetic inactivation of hMLH1 in association with DNA methylation.

β-Catenin mutations in cell lines established from human colorectal cancers

β-catenin has functions as both an adhesion and a signaling molecule. Disruption of these functions through mutations of the β-catenin gene (CTNNB1) may be important in the development of colorectal tumors. We examined the entire coding sequence of β-catenin by reverse transcriptase–PCR (RT-PCR) and direct sequencing of 23 human colorectal cancer cell lines from 21 patients. In two cell lines, there was apparent instability of the β-catenin mRNA. Five different mutations (26%) were found in the remaining 21cell lines (from 19 patients). A three-base deletion (codon 45) was identified in the cell line HCT 116, whereas cell lines SW 48, HCA 46, CACO 2, and Colo 201 each contained single-base missense mutations (codons 33, 183, 245, and 287, respectively). All 23 cell lines had full-length β-catenin protein that was detectable by Western blotting and that coprecipitated with E-cadherin. In three of the cell lines with CTNNB1 mutations, complexes of β-catenin with α-catenin and APC were detectable. In SW48 and HCA 46, however, we did not detect complexes of β-catenin protein with α-catenin and APC, respectively. These results show that selection of CTNNB1 mutations occurs in up to 26% of colorectal cancers from which cell lines are derived. In these cases, mutation selection is probably for altered β-catenin function, which may significantly alter intracellular signaling and intercellular adhesion and may serve as a complement to APC mutations in the early stages of tumorigenesis.

Alternative genetic pathways in colorectal carcinogenesis

The comparative typing of matched tumor and blood DNAs at dinucleotide repeat (microsatellite) loci has revealed in tumor DNA the presence of alleles that are not observed in normal DNA. The occurrence of these additional alleles is possibly due to replication errors (RERs). Although this observation has led to the recognition of a subtype of colorectal cancer with a high incidence of RERs (caused by a deficiency in DNA mismatch repair), a thorough analysis of the RER frequency in a consecutive series of colorectal cancers had not been reported. It is shown here that the extensive typing of 88 colorectal tumors reveals a bimodal distribution for the frequency of RER at microsatellite loci. Within the major mode (75 tumors, RER− subtype), the probability that a locus exhibited instability did not differ significantly among loci and tumors, being 0.02. The subsequent development of a statistical test for an operational discrimination between the RER− and RER+ subtypes indicated that the probability of misclassification did not exceed 0.001 in this series. The frequency of K-ras mutation was found to be equivalent in the two subtypes. However, in the RER+ tumors, the p53 gene mutation was less frequently detected, the adenomatous polyposis coli (APC) mutation was rare, and the biallelic inactivation of either of these genes was not observed. Furthermore, the concomitant occurrence of APC and tumor growth factor β receptor type II gene alterations was found only once. These data suggest that the repertoires of genes that are frequently altered in RER+ and RER− tumors may be more different than previously thought.

The onset and extent of genomic instability in sporadic colorectal tumor progression

Cancer cell genomes contain alterations beyond known etiologic events, but their total number has been unknown at even the order of magnitude level. By sampling colorectal premalignant polyp and carcinoma cell genomes through use of the technique inter-(simple sequence repeat) PCR, we have found genomic alterations to be considerably more abundant than expected, with the mean number of genomic events per carcinoma cell totaling approximately 11,000. Colonic polyps early in the tumor progression pathway showed similar numbers of events. These results indicate that, as with certain hereditary cancer syndromes, genomic destabilization is an early step in sporadic tumor development. Together these results support the model of genomic instability being a cause rather than an effect of malignancy, facilitating vastly accelerated somatic cell evolution, with the observed orderly steps of the colon cancer progression pathway reflecting the consequences of natural selection.

Prostacyclin-mediated activation of peroxisome proliferator-activated receptor δ in colorectal cancer

There is evidence from both genetic and pharmacologic studies to suggest that the cyclooxygenase-2 (COX-2) enzyme plays a causal role in the development of colorectal cancer. However, little is known about the identity or role of the eicosanoid receptor pathways activated by COX-derived prostaglandins (PG). We previously have reported that COX-2-derived prostacyclin promotes embryo implantation in the mouse uterus via activation of the nuclear hormone receptor peroxisome proliferator-activated receptor (PPAR) δ. In light of the recent finding that PPARδ is a target of β-catenin transactivation, it is important to determine whether this signaling pathway is operative during the development of colorectal cancer. Analysis of PPARδ mRNA in matched normal and tumor samples revealed that expression of PPARδ, similar to COX-2, is up-regulated in colorectal carcinomas. In situ hybridization studies demonstrate that PPARδ is expressed in normal colon and localized to the epithelial cells at the very tips of the mucosal glands. In contrast, expression of PPARδ mRNA in colorectal tumors was more widespread with increased levels in transformed epithelial cells. Analysis of PPARδ and COX-2 mRNA in serial sections suggested they were colocalized to the same region within a tumor. Finally, transient transfection assays established that endogenously synthesized prostacyclin (PGI2) could serve as a ligand for PPARδ. In addition, the stable PGI2 analog, carbaprostacyclin, and a synthetic PPARδ agonist induced transactivation of endogenous PPARδ in human colon carcinoma cells. We conclude from these observations that PPARδ, similar to COX-2, is aberrantly expressed in colorectal tumors and that endogenous PPARδ is transcriptionally responsive to PGI2. However, the functional consequence of PPARδ activation in colon carcinogenesis still needs to be determined.

The APC variants I1307K and E1317Q are associated with colorectal tumors, but not always with a family history

Classical familial adenomatous polyposis (FAP) is a high-penetrance autosomal dominant disease that predisposes to hundreds or thousands of colorectal adenomas and carcinoma and that results from truncating mutations in the APC gene. A variant of FAP is attenuated adenomatous polyposis coli, which results from germ-line mutations in the 5′ and 3′ regions of the APC gene. Attenuated adenomatous polyposis coli patients have “multiple” colorectal adenomas (typically fewer than 100) without the florid phenotype of classical FAP. Another group of patients with multiple adenomas has no mutations in the APC gene, and their phenotype probably results from variation at a locus, or loci, elsewhere in the genome. Recently, however, a missense variant of APC (I1307K) was described that confers an increased risk of colorectal tumors, including multiple adenomas, in Ashkenazim. We have studied a set of 164 patients with multiple colorectal adenomas and/or carcinoma and analyzed codons 1263–1377 (exon 15G) of the APC gene for germ-line variants. Three patients with the I1307K allele were detected, each of Ashkenazi descent. Four patients had a germ-line E1317Q missense variant of APC that was not present in controls; one of these individuals had an unusually large number of metaplastic polyps of the colorectum. There is increasing evidence that there exist germ-line variants of the APC gene that predispose to the development of multiple colorectal adenomas and carcinoma, but without the florid phenotype of classical FAP, and possibly with importance for colorectal cancer risk in the general population.

A systematic review of the effects of screening for colorectal cancer using the faecal occult blood test, Hemoccult

Abstract

Spectral karyotyping suggests additional subsets of colorectal cancers characterized by pattern of chromosome rearrangement

The abundant chromosome abnormalities in most carcinomas are probably a reflection of genomic instability present in the tumor, so the pattern and variability of chromosome abnormalities will reflect the mechanism of instability combined with the effects of selection. Chromosome rearrangement was investigated in 17 colorectal carcinoma-derived cell lines. Comparative genomic hybridization showed that the chromosome changes were representative of those found in primary tumors. Spectral karyotyping (SKY) showed that translocations were very varied and mostly unbalanced, with no translocation occurring in more than three lines. At least three karyotype patterns could be distinguished. Some lines had few chromosome abnormalities: they all showed microsatellite instability, the replication error (RER)+ phenotype. Most lines had many chromosome abnormalities: at least seven showed a surprisingly consistent pattern, characterized by multiple unbalanced translocations and intermetaphase variation, with chromosome numbers around triploid, 6–16 structural aberrations, and similarities in gains and losses. Almost all of these were RER−, but one, LS411, was RER+. The line HCA7 showed a novel pattern, suggesting a third kind of genomic instability: multiple reciprocal translocations, with little numerical change or variability. This line was also RER+. The coexistence in one tumor of two kinds of genomic instability is to be expected if the underlying defects are selected for in tumor evolution.