Multiple putative oncogenes at the chromosome 20q amplicon contribute to colorectal adenoma to carcinoma progression

Objective: This study aimed to identify the oncogenes at 20q involved in colorectal adenoma to carcinoma progression by measuring the effect of 20q gain on mRNA expression of genes in this amplicon. Methods: Segmentation of DNA copy number changes on 20q was performed by array CGH (comparative genomic hybridisation) in 34 non-progressed colorectal adenomas, 41 progressed adenomas (ie, adenomas that present a focus of cancer) and 33 adenocarcinomas. Moreover, a robust analysis of altered expression of genes in these segments was performed by microarray analysis in 37 adenomas and 31 adenocarcinomas. Protein expression was evaluated by immunohistochemistry on tissue microarrays. Results: The genes C20orf24, AURKA, RNPC1, TH1L, ADRM1, C20orf20 and TCFL5, mapping at 20q, were significantly overexpressed in carcinomas compared with adenomas as a consequence of copy number gain of 20q. Conclusion: This approach revealed C20orf24, AURKA, RNPC1, TH1L, ADRM1, C20orf20 and TCFL5 genes to be important in chromosomal instability-related adenoma to carcinoma progression. These genes therefore may serve as highly specific biomarkers for colorectal cancer with potential clinical applications

Evolution of the hypoxia-sensitive cells involved in amniote respiratory reflexes

The evolutionary origins of the hypoxia-sensitive cells that trigger amniote respiratory reflexes – carotid body glomus cells, and ‘pulmonary neuroendocrine cells’ (PNECs) - are obscure. Homology has been proposed between glomus cells, which are neural crest-derived, and the hypoxia-sensitive ‘neuroepithelial cells’ (NECs) of fish gills, whose embryonic origin is unknown. NECs have also been likened to PNECs, which differentiate in situ within lung airway epithelia. Using genetic lineage-tracing and neural crest-deficient mutants in zebrafish, and physical fate-mapping in frog and lamprey, we find that NECs are not neural crest-derived, but endoderm-derived, like PNECs, whose endodermal origin we confirm. We discover neural crest-derived catecholaminergic cells associated with zebrafish pharyngeal arch blood vessels, and propose a new model for amniote hypoxia-sensitive cell evolution: endoderm-derived NECs were retained as PNECs, while the carotid body evolved via the aggregation of neural crest-derived catecholaminergic (chromaffin) cells already associated with blood vessels in anamniote pharyngeal arches

Multitarget Stool DNA Test Performance in an Average-Risk Colorectal Cancer Screening Population

INTRODUCTION: We set out to evaluate the performance of a multitarget stool DNA (MT-sDNA) in an average-risk colonoscopy-controlled colorectal cancer (CRC) screening population. MT-sDNA stool test results were evaluated against fecal immunochemical test (FIT) results for the detection of different lesions, including molecularly defined high-risk adenomas and several other tumor characteristics. METHODS: Whole stool samples (n = 1,047) were prospectively collected and subjected to an MT-sDNA test, which tests for KRAS mutations, NDRG4 and BMP3 promoter methylation, and hemoglobin. Results for detecting CRC (n = 7), advanced precancerous lesions (advanced adenoma [AA] and advanced serrated polyps; n = 119), and non-AAs (n = 191) were compared with those of FIT alone (thresholds of 50, 75, and 100 hemoglobin/mL). AAs with high risk of progression were defined by the presence of specific DNA copy number events as measured by low-pass whole genome sequencing. RESULTS: The MT-sDNA test was more sensitive than FIT alone in detecting advanced precancerous lesions (46% (55/119) vs 27% (32/119), respectively, P < 0.001). Specificities among individuals with nonadvanced or negative findings (controls) were 89% (791/888) and 93% (828/888) for MT-sDNA and FIT testing, respectively. A positive MT-sDNA test was associated with multiple lesions (P = 0.005), larger lesions (P = 0.03), and lesions with tubulovillous architecture (P = 0.04). The sensitivity of the MT-sDNA test or FIT in detecting individuals with high-risk AAs (n = 19) from individuals with low-risk AAs (n = 52) was not significantly different. DISCUSSION: In an average-risk screening population, the MT-sDNA test has an increased sensitivity for detecting advanced precancerous lesions compared with FIT alone. AAs with a high risk of progression were not detected with significantly higher sensitivity by MT-sDNA or FIT

Proteins in stool as biomarkers for non-invasive detection of colorectal adenomas with high risk of progression

Screening to detect colorectal cancer (CRC) in an early or premalignant state is an effective method to reduce CRC mortality rates. Current stool-based screening tests, e.g. fecal immunochemical test (FIT), have a suboptimal sensitivity for colorectal adenomas and difficulty distinguishing adenomas at high risk of progressing to cancer from those at lower risk. We aimed to identify stool protein biomarker panels that can be used for the early detection of high-risk adenomas and CRC. Proteomics data (LC–MS/MS) were collected on stool samples from adenoma (n = 71) and CRC patients (n = 81) as well as controls (n = 129). Colorectal adenoma tissue samples were characterized by low-coverage whole-genome sequencing to determine their risk of progression based on specific DNA copy number changes. Proteomics data were used for logistic regression modeling to establish protein biomarker panels. In total, 15 of the adenomas (15.8%) were defined as high risk of progressing to cancer. A protein panel, consisting of haptoglobin (Hp), LAMP1, SYNE2, and ANXA6, was identified for the detection of high-risk adenomas (sensitivity of 53% at specificity of 95%). Two panels, one consisting of Hp and LRG1 and one of Hp, LRG1, RBP4, and FN1, were identified for high-risk adenomas and CRCs detection (sensitivity of 66% and 62%, respectively, at specificity of 95%). Validation of Hp as a biomarker for high-risk adenomas and CRCs was performed using an antibody-based assay in FIT samples from a subset of individuals from the discovery series (n = 158) and an independent validation series (n = 795). Hp protein was significantly more abundant in high-risk adenoma FIT samples compared to controls in the discovery (p = 0.036) and the validation series (p = 9e-5). We conclude that Hp, LAMP1, SYNE2, LRG1, RBP4, FN1, and ANXA6 may be of value as stool biomarkers for early detection of high-risk adenomas and CRCs

Evolution of the hypoxia-sensitive cells involved in amniote respiratory reflexes

The evolutionary origins of the hypoxia-sensitive cells that trigger amniote respiratory reflexes - carotid body glomus cells, and 'pulmonary neuroendocrine cells' (PNECs) - are obscure. Homology has been proposed between glomus cells, which are neural crest-derived, and the hypoxia-sensitive 'neuroepithelial cells' (NECs) of fish gills, whose embryonic origin is unknown. NECs have also been likened to PNECs, which differentiate in situ within lung airway epithelia. Using genetic lineage-tracing and neural crest-deficient mutants in zebrafish, and physical fate-mapping in frog and lamprey, we find that NECs are not neural crest-derived, but endoderm-derived, like PNECs, whose endodermal origin we confirm. We discover neural crest-derived catecholaminergic cells associated with zebrafish pharyngeal arch blood vessels, and propose a new model for amniote hypoxia-sensitive cell evolution: endoderm-derived NECs were retained as PNECs, while the carotid body evolved via the aggregation of neural crest-derived catecholaminergic (chromaffin) cells already associated with blood vessels in anamniote pharyngeal arches.This work was funded by the Wellcome Trust (Ph.D. Studentship 086804/Z/08/Z to DH; Senior Investigator Award 102889/Z/13/Z to AST), the NIDCR/NIH (R21-DE021509 to SF; R01-DE018477 to EWK), the NIDDK/NIH (1DP2DK098092 to PDSD), the NIH (R01-HL092217 to EWK), the Zebrafish Initiative of the Vanderbilt University Academic Venture Capital Fund (to EWK), the Vanderbilt International Scholar Program (to GU), the HFSP (Long-Term Fellowship to CM) and the Swiss National Science Foundation (Advanced Postdoctoral Fellowship and Professorship to CM). For further information, please visit the publisher's website

MiR-17-92 cluster is associated with 13q gain and c-myc expression during colorectal adenoma to adenocarcinoma progression

Background:MicroRNAs are small non-coding RNA molecules, which regulate central mechanisms of tumorigenesis. In colorectal tumours, the combination of gain of 8q and 13q is one of the major factors associated with colorectal adenoma to adenocarcinoma progression. Functional studies on the miR-17-92 cluster localised on 13q31 have shown that its transcription is activated by c-myc, located on 8q, and that it has oncogenic activities. We investigated the contribution of the miR-17-92 cluster during colorectal adenoma to adenocarcinoma progression.Methods:Expression levels of the miR-17-92 cluster were determined in 55 colorectal tumours and in 10 controls by real-time RT-PCR. Messenger RNA c-myc expression was also determined by real-time RT-PCR in 48 tumours with array comparative genomic hybridisation (aCGH) data available.Results:From the six members of the miR-17-92 cluster, all except miR-18a, showed significant increased expression in colorectal tumours with miR-17-92 locus gain compared with tumours without miR-17-92 locus gain. Unsupervised cluster analysis clustered the tumours based on the presence of miR-17-92 locus gain. Significant correlation between the expression of c-myc and the six miRNAs was also found.Conclusion:Increased expression of miR-17-92 cluster during colorectal adenoma to adenocarcinoma progression is associated to DNA copy number gain of miR17-92 locus on 13q31 and c-myc expression. © 2009 Cancer Research UK

Promoter methylation of Wnt-antagonists in polypoid and nonpolypoid colorectal adenomas.

BACKGROUND: Nonpolypoid adenomas are a subgroup of colorectal adenomas that have been associated with a more aggressive clinical behaviour compared to their polypoid counterparts. A substantial proportion of nonpolypoid and polypoid adenomas lack APC mutations, APC methylation or chromosomal loss of the APC locus on chromosome 5q, suggesting the involvement of other Wnt-pathway genes. The present study investigated promoter methylation of several Wnt-pathway antagonists in both nonpolypoid and polypoid adenomas. METHODS: Quantitative methylation-specific PCR (qMSP) was used to evaluate methylation of four Wnt-antagonists, SFRP2, WIF-1, DKK3 and SOX17 in 18 normal colorectal mucosa samples, 9 colorectal cancer cell lines, 18 carcinomas, 44 nonpolypoid and 44 polypoid adenomas. Results were integrated with previously obtained data on APC mutation, methylation and chromosome 5q status from the same samples. RESULTS: Increased methylation of all genes was found in the majority of cell lines, adenomas and carcinomas compared to normal controls. WIF-1 and DKK3 showed a significantly lower level of methylation in nonpolypoid compared to polypoid adenomas (p < 0.01). Combining both adenoma types, a positive trend between APC mutation and both WIF-1 and DKK3 methylation was observed (p < 0.05). CONCLUSIONS: Methylation of Wnt-pathway antagonists represents an additional mechanism of constitutive Wnt-pathway activation in colorectal adenomas. Current results further substantiate the existence of partially alternative Wnt-pathway disruption mechanisms in nonpolypoid compared to polypoid adenomas, in line with previous observations

Promoter methylation of Wnt-antagonists in polypoid and nonpolypoid colorectal adenomas

BACKGROUND: Nonpolypoid adenomas are a subgroup of colorectal adenomas that have been associated with a more aggressive clinical behaviour compared to their polypoid counterparts. A substantial proportion of nonpolypoid and polypoid adenomas lack APC mutations, APC methylation or chromosomal loss of the APC locus on chromosome 5q, suggesting the involvement of other Wnt-pathway genes. The present study investigated promoter methylation of several Wnt-pathway antagonists in both nonpolypoid and polypoid adenomas. METHODS: Quantitative methylation-specific PCR (qMSP) was used to evaluate methylation of four Wnt-antagonists, SFRP2, WIF-1, DKK3 and SOX17 in 18 normal colorectal mucosa samples, 9 colorectal cancer cell lines, 18 carcinomas, 44 nonpolypoid and 44 polypoid adenomas. Results were integrated with previously obtained data on APC mutation, methylation and chromosome 5q status from the same samples. RESULTS: Increased methylation of all genes was found in the majority of cell lines, adenomas and carcinomas compared to normal controls. WIF-1 and DKK3 showed a significantly lower level of methylation in nonpolypoid compared to polypoid adenomas (p < 0.01). Combining both adenoma types, a positive trend between APC mutation and both WIF-1 and DKK3 methylation was observed (p < 0.05). CONCLUSIONS: Methylation of Wnt-pathway antagonists represents an additional mechanism of constitutive Wnt-pathway activation in colorectal adenomas. Current results further substantiate the existence of partially alternative Wnt-pathway disruption mechanisms in nonpolypoid compared to polypoid adenomas, in line with previous observations

Identification of key genes for carcinogenic pathways associated with colorectal adenoma-to-carcinoma progression

Colorectal adenomas form a biologically and clinically distinct intermediate stage in development of colorectal cancer (CRC) from normal colon epithelium. Only 5% of adenomas progress into adenocarcinomas, indicating that malignant transformation requires other biological alterations than those involved in adenoma formation. The present study aimed to explore which cancer-related biological processes are affected during colorectal adenoma-to-carcinoma progression and to identify key genes within these pathways that can serve as tumor markers for malignant transformation. The activity of 12 cancer-related biological processes was compared between 37 colorectal adenomas and 31 adenocarcinomas, using the pathway analysis tool Gene Set Enrichment Analysis. Expression of six gene sets was significantly increased in CRCs compared to adenomas, representing chromosomal instability, proliferation, differentiation, invasion, stroma activation, and angiogenesis. In addition, 18 key genes were identified for these processes based on their significantly increased expression levels. For AURKA and PDGFRB, increased mRNA expression levels were verified at the protein level by immunohistochemical analysis of a series of adenomas and CRCs. This study revealed cancer-related biological processes whose activities are increased during malignant transformation and identified key genes which may be used as tumor markers to improve molecular characterization of colorectal tumors