Background & Aims: Current models of colorectal adenoma to carcinoma progression do not fully reflect the genetic heterogeneity and complexity of the disease. The aim of the present study was to identify genetic changes discriminating adenomas that have progressed to carcinoma from adenomas that have not progressed, and to refine the current genetic models of colorectal adenoma to carcinoma progression, based on a genome-wide analysis of chromosomal aberrations. Methods: Sixty-six nonprogressed colorectal adenomas, 46 progressed adenomas (malignant polyps), and 36 colorectal carcinomas were screened for chromosomal aberrations by comparative genomic hybridization, and for mutations in the adenomatous polyposis coli (APC) and K-ras gene. Data analysis focused on cancer-associated genetic changes in adenomas. Results: Accumulation of losses in 8p21-pter, 15q11-q21, 17p12-13, and 18q12-21, and gains in 8q23-qter, 13q14-31, and 20q13 were strongly associated with adenoma-to-carcinoma progression, independent of the degree of dysplasia. Hierarchic cluster analysis demonstrated the presence of 3 distinct subgroups of adenomas, characterized by unique combinations of genetic aberrations in the adenomas (17p loss and K-ras mutation, 8q and 13q gain, and 18q loss and 20q gain, respectively). Conclusions: The presence of 2 or more of the aforementioned 7 chromosomal changes was associated with progressed colorectal adenomas and colorectal cancer. In addition, evidence was found that these chromosomal abnormalities occurred in specific combinations of a few abnormalities rather than as a mere accumulation of events, indicating the existence of multiple independent chromosomal instability pathways of colorectal cancer progression.

Background: Kirsten ras (Ki-ras) gene mutations occur early in the progression of colorectal adenoma to carcinoma. The aim of this collaborative study was to clarify the association between Ki-ras mutations, patient outcome, and tumor characteristics by use of data from colorectal cancer patients worldwide, Methods: Investigators who had published data on Ki-ras and colorectal cancer were invited to complete a questionnaire for each patient entered into a database. Two-sided statistical tests were used to analyze data. Results: Patients (n = 2721) were recruited from 22 groups in 13 countries. Mutations of Ki-ras codon 12 (wild type = GGT = glycine) or codon 13 (wild type = GGC = glycine) were detected in 37.7% of the tumors; 80.8% (584 of 723) of all the specified mutations occurred in codon 12, and 78.1% (565 of 723) of all the specified mutations were at the second base of either codon. Mutations were not associated with sex, age, tumor site, or Dukes' stage. Mutation rates seen in patients with sporadic tumors were comparable to those observed in patients with a predisposing cause for their cancer. Poorly differentiated tumors were less frequently mutated (P = .002), Multivariate analysis suggested that the presence of a mutation increased risk of recurrence (P<.001) and death (P =.004), In particular, any mutation of guanine (G) to thymine (T) but not to adenine (A) or to cytosine (C) increased the risk of recurrence (P =.006) and death (P<.001). When individual, specific mutations were evaluated, only valine codon 12 was found to convey an independent, increased risk of recurrence (P =.007) and death (P =.004), Conclusions: Ki-ras mutations are associated with increased risk of relapse and death, but some mutations are more aggressive than others.

Mutant ras genes occur frequently in human neoplasia and, in particular, in pancreatic, colorectal, and lung adenocarcinomas, Recent evidence suggests that G-->T and G-->C transversions of the Ki-ras gene in codon 12 may lead to biological effects in vitro and in vivo that may be associated with an abnormal cell cycle and increased tumour aggressiveness. The role of Ki-ras activation (a G-->C transversion in codon 12, arginine for glycine) in the cell cycle and apoptosis was investigated using control and permanently transfected NIH3T3 mouse fibroblasts. Flow cytometry was used to evaluate the G1-, S- and G2M-phase transit times, the potential doubling time, the growth fraction, and the cell loss factor during asynchronous exponential growth. Apoptosis was induced in both cell lines by absence of growth factors for an extended period of time (72 h) and quantitatively evaluated using the TUNEL method coupled with flow cytometry, It was found that codon 12 G-C Ki-ras transfected cells compared with controls, had a significant prolongation of G1 by about 50%, a reduction of the G2M transit time by 30%, and a decrease of the cell loss factor by about 90%. Apoptotic cells were about 10% in control and less than 0.5% in Ki-ras transfected cells after 72 h starvation-confluency. These data suggest that codon 12 G-->C Ki-ras activation in mouse NIH3T3 fibroblasts is associated with deregulation of checkpoint controls in the GI and G2M phases of the cell cycle and inhibition of apoptosis. It appears plausible that these cell mechanisms are related to a proliferative advantage and that they may also be important in the progression of human tumours characterized by specific Ki-ras mutations. Copyright (C) 2000 John Wiley & Sons, Ltd.

The possible role of K‐ras2 mutations and aneuploidy toward increase of proliferation and adenoma size in Familial Adenomatous Polyposis (FAP) adenomas is not known. The present study addresses these issues by investigating 147 colorectal adenomas obtained from four FAP patients. The majority of adenomas had size lower than or equal to 10 mm (86%), low grade dysplasia (63%), and were preferentially located in the right colon (60%). Normal mucosa samples were obtained from 19 healthy donors. Three synchronous adenocarcinomas were also investigated. K‐ras2 mutation spectrum was analysed by PCR and Sequence Specific Oligonucleotide (SSO) hybridization, while flow cytometry (FCM) was used for evaluating degree of DNA ploidy and S‐phase fraction. Overall, incidences of K‐ras2 mutations, DNA aneuploidy and high S‐phase values (>7.2%) were 6.6%, 5.4% and 10.5%, respectively. In particular, among the adenomas with size lower than 5 mm, K‐ras2 mutation and DNA aneuploidy frequencies were only slightly above 1%. Statistically significant correlations were found between K‐ras2 and size, DNA ploidy and size and K‐ras2 and S‐phase (p). In particular, among the wild type K‐ras2 adenomas, high S‐phase values were detected in 8% of the cases versus 57% among the K‐ras2 mutated adenomas (p=0.0005). The present series of FAP adenomas indicates that K‐ras2 activation and gross genomic changes play a role toward a proliferative gain and tumour growth in size.

Cell cycle variations and DNA aneuploidy, were investigated in different phases of azoxymethane (AOM)-induced colon carcinogenesis in rats by flow cytometry. K-ras gene mutations (transitions Gright curved arrow A) were frequently detected in aberrant crypt foci (ACF) initial pre-neoplastic lesions. The fraction of cells in the G2M-phase of the cell cycle was higher in ACF compared to the normal mucosa of control rats. A similar modification of the cell cycle was found in adenomas and adenocarcinomas but, unexpectedly, also in morphologically normal mucosa from AOM-treated animals indicating that AOM treatment permanently modifies cell cycle control in rat colon mucosa. These alterations, however, were not associated with DNA aneuploidy as reported in human sporadic colorectal cancer, suggesting that tumour development in AOM-treated rats is less dependent on aneuploidy.

Recent studies indicate that p21ras proteins mediate their multiple cell functions through interactions with multiple effectors and that the number of new effectors is growing. We recently reported that K-ras2 mutations in human colorectal adenomas were associated with chromosome instability and proliferation changes. In the present study, we extend these previous observations. Hereditary and multiple (n > or = 5) adenomas and adenomas with early cancer were excluded. Dysplasia was moderate in 91 cases and high in 25, and the median adenoma size was 1.5 cm. K-ras2 spectrum analysis was done by sequence-specific oligonucleotide hybridization using nuclear suspensions provided by analysis and sorting of multiparameter flow cytometry. In particular, tissue inflammatory cells were separated for DNA diploid tumors, whereas DNA aneuploid epithelial subclones were analyzed separately. K-ras2 mutations and DNA aneuploidy were both detected in 29 of 116 (25%) cases. DNA aneuploid index was in the near-diploid region in the majority of cases. DNA aneuploidy was strongly associated with G-->C/T transversions. An association was also found between low S-phase values and G-->A transitions. These findings were confirmed using multivariate logistic regression analysis to account for the effects of size, dysplasia, site, type, age, and sex. These data suggest that specific K-ras2 mutations in a subgroup of human sporadic colorectal adenomas play a role in chromosome instability and, contrary to expectations, are associated with inhibition of proliferation.

A monoclonal antibody (AS-2) raised by using isolated nuclei from a human erythroleukemia cell line as immunogen is described.AS-2 was of IgM type and recognized proteins present in both isolated cytoplasms and nuclei. The molecular weight of the AS-2 recognized proteins in the cytoplasm was 200 kDa and 70 and 60 kDa in the nucleus. The relative amount of these proteins were measured simultaneously with DNA content by flow cytometry. We found the highest protein content (or stainability) for both cells and nuclei in late-G1, S and G2, at approximately the same level, and the lowest content in M and early-G1. Sorting based on DNA content and AS-2 associated fluorescence helped identifying the staining pattern of cells and nuclei. Interphase isolated nuclei and cell cytoplasms were characterized by interdispersed staining over the entire surfaces while mitoses showed two dots only. The present preliminary data indicate that the proteins recognized by the AS-2 monoclonal are cell cycle related and suggest that in mitoses they are associated with the centrosomes.

The p53 tumour suppressor gene has an important role in the the maintenance of genome stability and its mutational inactivation may be at the origin of aneuploidy in cancer cells. The aim of this study was to determine whether p53 mutations were associated to DNA aneuploidy, as assessed by flow cytometry, in colorectal adenocarcinomas. Analysis of p53 mutations spectrum of the sorted nuclei was done by Denaturing Gradient Gel Electrophoresis (DGGE) and DNA sequencing. Overall, we studied 20 adenocarcinomas, the corresponding control mucosa, and 7 lymph node metastases. Five tumours (25%) were DNA diploid, while 15 tumours (75%) were composed of DNA aneuploid and diploid subpopulations. DNA diploid control mucosa and adenocarcinomas showed no p53 mutations, while 60% of the tumours with DNA aneuploidy had p53 mutations. Therefore, p53 mutations occurred significantly more often in DNA aneuploid than in DNA diploid tumours (p < 0.04, Fisher’s exact test). Incidences of DNA aneuploidy and p53 mutations in lymph node metastases were 60 and 86%, respectively. In all tumours showing a p53 mutation, the wild-type allele was not or only bearly visible in DNA aneuploid cells suggesting that, in such cells, aneuploidy is accompanied by complete p53 functional inactivation. The present observations suggest that p53 mutations may have a role in the origin of aneuploidy at late stages of colorectal carcinogenesis.

Detailed information about intratumor K-ras2 mutations in colorectal adenocarcinomas and a possible association with DNA content heterogeneity is still lacking. DNA diploid and aneuploid subclones, detected among multiple histologically selected primary sectors (57 superficial and 40 deep) and 9 lymph node metastases, were flow cytometrically sorted and separately submitted to codons 12-13 K-ras2 mutation spectrum analysis. DNA aneuploidy was absent among 20 near and 20 distant mucosa sites and present in 7/9 lymph node metastases and in 17/19 primary tumors (90%). Primary intratumor DNA multiclonality was approximately 50%. Degree of DNA aneuploidy (DNA Index) distribution was nonrandom and showed peaks at approximate mean DNA Index values 1.2, 1.5, and 1.8. K-ras2 mutations were detected in 0/20 mucosa cases, in 2/9 lymph node metastases, and in 9/19 adenocarcinomas (47%). No more than one mutation type per tumor was detected. Intratumor distribution of K-ras2 mutations was homogeneous in 6 and heterogeneous in 3 cases. Homogeneous distribution was associated with DNA near-diploid aneuploidy. K-ras2 mutations were strongly associated with DNA Index in the near-diploid region (83%) and almost absent (5%) among DNA near-triploid subclones (P = 0.0001). K-ras2 mutation intratumor heterogeneity indicates that sampling of the tumor may be a critical step and suggests that K-ras2 activation may be a late event in a subgroup of tumors. Our data also suggest the existence of an early process of the colorectal carcinogenesis that favors both K-ras2 mutations and DNA near-diploid aneuploidy. Onset of DNA near-triploid subclones appears, instead, to be independent from K-ras2 activation.

Detailed information about intratumor K-ras2 mutations in colorectal adenocarcinomas and a possible association with DNA content heterogeneity is still lacking. DNA diploid and aneuploid subclones, detected among multiple histologically selected primary sectors (57 superficial and 40 deep) and 9 lymph node metastases, were flow cytometrically sorted and separately submitted to codons 12-13 K-ras2 mutation spectrum analysis. DNA aneuploidy was absent among 20 near and 20 distant mucosa sites and present in 7/9 lymph node metastases and in 17/19 primary tumors (90%). Primary intratumor DNA multiclonality was approximately 50%. Degree of DNA aneuploidy (DNA Index) distribution was nonrandom and showed peaks at approximate mean DNA index values of 1.2, 1.5, and 1.8. K-ras2 mutations were detected in 0/20 mucosa cases, in 2/9 lymph node metastases, and in 9/19 adenocarcinomas (47%). No more than one mutation type per tumor was detected. Intratumor distribution of K-ras2 mutations was homogeneous in 6 and heterogeneous in 3 cases. Homogeneous distribution was associated with DNA near-diploid aneuploidy. K-ras2 mutations were strongly associated with DNA Index in the near-diploid region (83%) and almost absent (5%) among DNA near-triploid subclones (P = 0.0001). K-ras2 mutation intratumor heterogeneity indicates that sampling of the tumor may be a critical step and suggests that K-ras2 activation may be a late event in a subgroup of tumors. Our data also suggest the existence of an early process of the colorectal carcinogenesis that favors both K-ras2 mutations and DNA near-diploid aneuploidy. Onset of DNA near-triploid subclones appears, instead, to be independent from K-ras2 activation.

Background/Aims: K-ras-2 mutations and DNA content heterogeneity represent early events of human colorectal tumor progression. The aim of the study was to investigate if specific K-ras-2 mutations in 58 human sporadic adenomas were correlated with DNA aneuploidization and cell proliferation. Methods: Multiparameter flow cytometry, based on scatter parameters and DNA content, was performed using 4,6-diamidino-2-phenilindole-2-hydrochloride-stained nuclei obtained from adenoma fragments with either mild-moderate or severe dysplasia. K-ras-2 polymerase chain reaction and spectrum analysis were performed using sorted DNA specific epithelial subclones. Results: We detected six G-A transitions, and four G-C and two G-T transversions. The DNA aneuploid subclones were 25 with DNA index values in the near diploid region (DNA index < 1.3) for the vast majority of cases (80%). DNA aneuploidy among the mutated adenomas with G-A transitions was 1 of 6 (17%) and 6 of 6 (100%) among G-C and G-T transversions. Although DNA aneuploidy and high S-phase values were also present among K-ras-2 wild-type adenomas, their statistical associations with K-ras-2 status were P < 0.005 and P < 0.05, respectively. Conclusions: The present series of sporadic colorectal adenomas indicates that codon 12 G-C and G-T K-ras-2 transversion mutations and DNA aneuploidy are correlated. The underlying mechanisms that explain such association remain to be investigated.

Annals of the New York Academy of SciencesVolume 768, Issue 1 p. 261-263 K-ras2 Mutation Spectrum, DNA Aneuploidy, and Epithelial Cell Proliferation in Colorectal Adenomas† W. GIARETTI, W. GIARETTI Istituto Nazionale per la Ricerca sul Cancro 16132 Genoa, ItalySearch for more papers by this authorA. RAPALLO, A. RAPALLO Istituto Nazionale per la Ricerca sul Cancro 16132 Genoa, ItalySearch for more papers by this authorN. PUJIC, N. PUJIC Istituto Nazionale per la Ricerca sul Cancro 16132 Genoa, ItalySearch for more papers by this authorS. NIGRO, S. NIGRO Istituto Nazionale per la Ricerca sul Cancro 16132 Genoa, ItalySearch for more papers by this authorE. GEIDO, E. GEIDO Istituto Nazionale per la Ricerca sul Cancro 16132 Genoa, ItalySearch for more papers by this authorM. RISIO, M. RISIO Ospedale S. Giovanni Vecchio Torino, ItalySearch for more papers by this authorA. DI VINCI, A. DI VINCI Istituto Nazionale per la Ricerca sul Cancro 16132 Genoa, ItalySearch for more papers by this author W. GIARETTI, W. GIARETTI Istituto Nazionale per la Ricerca sul Cancro 16132 Genoa, ItalySearch for more papers by this authorA. RAPALLO, A. RAPALLO Istituto Nazionale per la Ricerca sul Cancro 16132 Genoa, ItalySearch for more papers by this authorN. PUJIC, N. PUJIC Istituto Nazionale per la Ricerca sul Cancro 16132 Genoa, ItalySearch for more papers by this authorS. NIGRO, S. NIGRO Istituto Nazionale per la Ricerca sul Cancro 16132 Genoa, ItalySearch for more papers by this authorE. GEIDO, E. GEIDO Istituto Nazionale per la Ricerca sul Cancro 16132 Genoa, ItalySearch for more papers by this authorM. RISIO, M. RISIO Ospedale S. Giovanni Vecchio Torino, ItalySearch for more papers by this authorA. DI VINCI, A. DI VINCI Istituto Nazionale per la Ricerca sul Cancro 16132 Genoa, ItalySearch for more papers by this author First published: September 1995 https://doi.org/10.1111/j.1749-6632.1995.tb12136.xCitations: 1 † This work was supported by the Consiglio Nazionale delle Ricerche (Grant No. 94.02331.PF39) and the Associazione Italiana per la Ricerca sul Cancro (AIRC). AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article.Citing Literature Volume768, Issue1Cancer Prevention: From the Laboratory to the Clinic: Implications of Genetic, Molecular, and Preventive ResearchSeptember 1995Pages 261-263 RelatedInformation

The purpose of this study was to investigate the correlation of flow cytometric (FCM) DNA content and cell cycle characteristics of Barrett's Esophagus (BE) with age and sex of the patients, length, histologic type and dysplasia of BE. Forty-one patients affected by histologically confirmed BE had multiple biopsies taken from the metaplastic epithelium and one biopsy taken from the gastric fundus, as control. The samples were either stored at -80 degrees C or immediately measured. Nuclei suspensions were obtained, stained with DAPI, measured with a high resolution flow cytometer (ICP22A, Ortho Instruments) and analyzed for the evaluation of the relative DNA content and the S- and G2 + M phases of the cell cycle. DNA histograms having two distinct G0/G1 peaks were classified as DNA aneuploid. The degree of DNA aneuploidy (DNA Index, DI), defined as the ratio of abnormal to normal DNA content, was obtained from the mixture of each BE sample with its control sample and trout erythrocytes. We found six patients with DNA aneuploid populations (14.6%), whose DNA Index values were 1.05 (in two), 1.8 (in one), and 2.0 (in three cases). DNA ploidy did not correlate with age and sex of the patients, length, histologic type, and dysplasia of BE. Among the 13 patients with dysplasia (6 indefinite, 4 low grade and 2 high grade with intramucous adenocarcinoma) only two (one indefinite and one low grade) showed DNA aneuploidy (15.4%). In addition, we found that the S-phase and the G2 + M-phase fractions in BE samples were both significantly higher than those of the controls (respectively, p = 0.01 and p = 0.0008).(ABSTRACT TRUNCATED AT 250 WORDS)

In order to better understand the relationship of DNA ploidy, dysplasia, early cancer, and colorectal tumor progression, 11 colorectal adenomas containing carcinoma invading the submucosa were investigated using DNA flow cytometry. Multiple frozen samples were taken from the selected sectors corresponding to adenoma tissue with low-grade dysplasia, high grade dysplasia and early cancer. Sampling accuracy was performed under histologic examination by multiple cryostatic sections. Data were compared with previously reported results in non-cancerous adenomas and advanced carcinomas. Incidence of DNA aneuploidy among the dysplastic regions of the adenomas containing carcinomas resulted higher than that observed in non-cancerous adenomas (p=0.02). Furthermore, among the DNA aneuploid populations, the frequency of clones with high DNA Index (DI>1.3) was slightly higher in adenomas with cancer than in adenomas without cancer (p=0.07). We suggest that differences may exist in DNA aneuploidy evolution between these two types of lesions. In early cancer, the near-diploid clones were 57% with respect to 18% (p=0.01) in advanced cancer since in this latter case the majority of the DNA abnormal clones were in the near-hypertriploid region (82%). Thus, the acquisition of the invasive phenotype appears to be linked with the expansion and stabilization of high DNA aneuploid clones. Further analysis on a larger number of cases of adenomas containing carcinoma are necessary to validate these interpretations.