Genetic and protein markers related to in situ growth and multiplicity in small sporadic colorectal adenomas.

BACKGROUND: Some early genetic events in the development of colorectal adenomas are known, but their relationship to in vivo growth characteristics is uncertain. This study compared in situ size changes and other clinicopathological variables with selected genetic and protein markers. METHODS: 56 adenomas (< or = 10 mm) from 39 patients were analysed for APC, CTNNB1 and K-ras mutations, allelic imbalance on 1p and 18q, microsatellite instability and immunohistochemical expression of HLA-DR, BAX, BCL-2 and Ki-67. For 42 of the adenomas, in situ growth was measured over 3 years. The total number of polyps in each patient was recorded. RESULTS: K-ras was mutated in 8/56 adenomas. None of the regressing adenomas revealed such mutations, compared to 20% in those that maintained or increased their size. Multivariate linear regression analysis showed that tumour growth was higher in females compared to males, and was even higher in the presence of a K-ras mutation. APC mutations were found in 37/56 adenomas. CTNNB1 mutations were found in 2/19 adenomas without APC mutation. Deletions of 1p were found in 12/56 adenomas and, seemingly, most frequent in patients with few tumours. The most frequently expressed protein was BAX (33/41), but neither this nor the other proteins showed associations with an in situ growth pattern. CONCLUSION: The multivariate linear regression model showed that patient gender and the presence of K-ras mutation had significant effects on tumour growth. The lack of the proliferative stimulus resulting from a K-ras mutation may contribute to the process of adenoma regression.

APC and CTNNB1 mutations in a large series of sporadic colorectal carcinomas stratified by the microsatellite instability status.

BACKGROUND: Adenomatous polyposis coli (APC) and beta-catenin (encoded by CTNNB1) are important components in the WNT signalling pathway, a pathway altered in nearly all colorectal tumours. Conflicting results are reported on whether APC mutations are less common in tumours with a high degree of microsatellite instability (MSI-H) than in microsatellite stable (MSS) ones, and whether mutations in the regulatory domain of CTNNB1 substitute for APC mutations in the MSI-H tumours. METHODS: A consecutive series of 218 primary colorectal carcinomas, stratified by MSI status, were analysed for mutations in the APC gene (by the protein truncation test) and in the CTNNB1 gene (by single-strand conformation polymorphism). RESULTS: APC mutations detected in 66% of the patients were significantly more frequent in the MSS and MSI-L (low) tumours than in the MSI-H tumour group (P < 0.001). The MSI-H tumours tended to have more frameshift mutations than the MSS/MSI-L tumours. The majority of the APC mutations were located in the mutation cluster region (MCR). Patients that had lost all beta-catenin binding sites of the APC gene showed a shorter survival time than patients who retained some or all of these binding sites (P = 0.045). Two mutations were found in the CTNNB1 gene, but neither of them was located in the regulatory domain in exon 3. CONCLUSION: This study confirms that APC mutations are less frequent in MSI-H tumours than in MSS and MSI-L tumours. However, CTNNB1 mutations do not substitute for APC mutations in MSI-H tumours in these Norwegian patients.

Strong HLA-DR expression in microsatellite stable carcinomas of the large bowel is associated with good prognosis.

Progression of colorectal cancer may follow either of two main genetic routes: the chromosome- or microsatellite-instability pathways. Association between the patients' prognosis and microsatellite instability has been questioned. Improved survival has previously been found in patients with expression of HLA-DR antigens on their tumour cells. In this study, the expression of HLA-DR antigen was investigated by immunohistochemistry in 357 large bowel carcinomas stratified by microsatellite instability status. Sixteen per cent of the tumours showed strong HLA-DR expression and 35% had weak DR expression. We confirmed that patients with strong positive HLA-DR staining had improved survival (P<0.001) compared to patients with no HLA-DR expression. Strong epithelial HLA-DR staining was significantly associated with high level of microsatellite instability (P<0.001). In the subgroup of tumours with characteristics typical of the chromosomal instability phenotype, i.e. in microsatellite-stable tumours, the patients positive for the HLA-DR determinants showed better survival than those without HLA-DR expression. The protective effect of HLA-DR expression on survival was confirmed by multivariate analysis, both in the whole patient group and in the microsatellite-stable/microsatellite instability-low group. This might be explained by enhanced T-cell mediated anti-tumour immune responses against tumour cells in the HLA-DR positive tumours. The finding of better patient survival in the subgroup of strong HLA-DR positive microsatellite-stable tumours may have clinical implications for these patients.

WNT1 inducible signaling pathway protein 3, WISP-3, a novel target gene in colorectal carcinomas with microsatellite instability.

BACKGROUND & AIMS: Microsatellite instability (MSI) is the phenotype of colorectal carcinomas with defect mismatch repair. Genes with repetitive sequences within their coding regions are targets for mutations in these tumors. We have evaluated 2 novel candidate genes for potential involvement in development of MSI colorectal carcinomas and compared them with alterations in known target genes. METHODS: The MSI status was determined by multiplex polymerase chain reactions (PCRs) of 5-17 markers in a Norwegian series of 275 colorectal carcinomas. All MSI tumors were analyzed for gene mutations using fluorescence PCR followed by capillary electrophoresis. Two novel candidate genes, WNT1-inducible signaling pathway protein 3 (WISP-3) and caspase-1, and 9 known target genes were analyzed. RESULTS: Thirteen percent of the tumors were MSI-high (H) and 12% were MSI-low (L). Thirty-three of 37 MSI-H vs. 1 of 34 MSI-L tumors showed mutations in the target genes (P < 0.001). WISP-3 was mutated in 31% of the MSI-H tumors. The frequencies of frameshift mutations in the known target genes were comparable with other studies. CONCLUSIONS: The relative high frequency of mutation, higher than those seen for other known target genes, the predicted truncation of the protein product, and the homology with WISP-1 and WISP-2, 2 proteins induced downstream of WNT1 signaling, strongly suggest WISP-3 as a novel target in development of MSI-H colorectal carcinomas.

Identification of two potential suppressor gene regions on chromosome arm 14q that are commonly lost in advanced colorectal carcinomas.

BACKGROUND: Remarkably little is known about the molecular alterations contributing to the establishment of a distant metastasis from a primary colorectal carcinoma. Previous studies on primary colorectal carcinomas have suggested an association between loss of chromosome 14 sequences and cancer progression. METHODS: In the present study, we analyzed 20 distant metastases and peripheral blood samples from 18 patients using 24 microsatellite markers spanning chromosome arm 14q. In addition, DNA from microdissected corresponding primary tumors (formalin-fixed and paraffin-embedded) was analyzed at selected 14q loci. RESULTS: Sixty-five percent (13/20) of the metastases, from 11/18 patients, showed loss of one or more markers at 14q, and the majority (94%) of the primary carcinomas showed identical 14q genotypes to those found in the metastasis. Two minimal common deleted regions were delineated in the metastases, one between markers D14S288-D14S52 at 14q13-21 and the other between D14S284-D14S81 at 14q24-31, pinpointing two previously unrecognized map positions for potential target genes. The genotype pattern of five tumors was consistent with monosomy or large chromosomal deletions spanning both potential suppressor regions. The reasons for monosomy in cancer remain unknown, but our data support the hypothesis that deletions of several tumor suppressor genes are more readily obtained by one chromosome loss than by several molecular events, and through this unison loss a growth advantage may be provided. CONCLUSION: Our data suggest that 14q loss is not a rate-limiting event in colorectal metastasis formation, but the high frequency of this alteration in primary tumors with metastatic ability, as well as in the metastases themselves, suggests it is part of the tumor clone with selective growth capacity.

Evaluation of 1p losses in primary carcinomas, local recurrences and peripheral metastases from colorectal cancer patients.

Cytogenetic and molecular genetic analyses of colorectal adenomas and carcinomas have shown that loss of the distal part of chromosome arm 1p is common, particularly in tumors of the left colon. Because the importance of 1p loss in colorectal cancer metastases is unknown, we compared the frequency, exact site and extent of 1p deletions in primary carcinomas (n=28), local recurrences (n=19) and metastases (n=33) from 67 colorectal cancer patients using 14 markers in an allelic imbalance study. Loss of 1p was found in 50% of the primary carcinomas, 33% of the local recurrences, and 64% of the metastases, revealing a significant difference between the local recurrences and the metastases (P=.04). The smallest region of 1p deletion overlap (SRO) defined separately for each group of lesions had the region between markers D1S2647 and D1S2644, at 1p35-36, in common. The genes PLA2G2A (1p35.1-36) and TP73 (1p36.3) were shown to lie outside this consistently lost region, suggesting that neither of them are targets for the 1p loss. In the second part of the study, microdissected primary carcinomas and distant metastases from the same colorectal cancer patients (n=18) were analyzed, and the same 1p genotype was found in the majority of patients (12/18, 67%). The finding that primary carcinoma cells with metastatic ability usually contain 1p deletions, and that some cases lacking 1p alterations in the primary tumor acquire such changes during growth of a metastatic lesion, supports the notion that 1p loss may be important both early and late in colorectal carcinogenesis, with the apparent exception of local recurrences.

Apoptosis and expression of Bax, Bcl-x, and Bcl-2 apoptotic regulatory proteins in colorectal carcinomas, and association with p53 genotype/phenotype.

AIMS: Spontaneous apoptosis and expression of the apoptotic regulatory proteins Bax, Bcl-x, and Bcl-2 were investigated in 50 colorectal carcinomas. The p53 genotypes/phenotypes and BAX genotypes were also determined, and possible associations of these with apoptosis and/or with expression of the different apoptotic regulatory proteins were studied. METHODS: Terminal deoxynucleotidyl transferase (TdT) mediated dUTP labelling of DNA fragments was used to detect apoptotic tumour cells in sections and peroxidase immunohistochemistry was used to assess protein expression. p53 genotype/phenotype was determined using constant denaturant gel electrophoresis/immunoblotting and bax genotype was determined using polymerase chain reaction based methods. RESULTS: The distribution of tumour apoptotic indices was bimodal with a natural cut off at 1.0% (range, 0.0-5.4%); the median fraction of apoptotic tumour cells was 0.8%. Tumour apoptosis was not associated significantly with tumour DNA ploidy status. Normal mucosal tissue had less than 0.1% apoptotic cells. Staining intensities for Bax, Bcl-x, and Bcl-2 were strong; that is, equivalent to or greater than positive normal mucosal cells, in 11 of 50, 20 of 49, and 20 of 48 carcinomas. Frameshift mutations in the bax gene were detected in three of 42 tumours analysed, all of which were DNA diploid, and Bax protein expression in these tumours was absent or very low. Bax, Bcl-x, and Bcl-2 protein expression were not correlated with tumour apoptosis or tumour DNA ploidy status. p53 was expressed in 34 of 50 tumours and p53 gene mutations were detected in 22 of 29 p53 positive tumours analysed. Apoptosis was significantly lower in a greater number of p53 positive tumours than p53 negative tumours. In addition, Bcl-2 protein expression was significantly higher in a greater number of p53 positive tumours compared with p53 negative tumours. Bax and Bcl-x protein expression were not significantly associated with p53 phenotype/genotype. CONCLUSIONS: The results indicate that acquisition of a p53 phenotype is associated with lower spontaneous apoptosis and higher expression of Bcl-2. The results also suggest that p53 is not a major determinant for Bax expression in colorectal carcinomas in vivo.

Comparison of genetic changes in frozen biopsies and microdissected archival material from the same colorectal liver metastases.

Microdissection of tissue sections from formalin-fixed, paraffin-embedded tumor material allows separation of microscopic sites within a sample. DNA can easily be extracted, and polymerase chain reaction (PCR) technology makes it possible to perform different molecular biologic analyses on small cell populations. The presence of normal cells or tumor heterogeneity may cause false negatives in allelic imbalance (AI) studies. Microdissected well-defined cell populations from a tumor section are assumed to increase the sensitivity of AI analyses. The present study has evaluated this in colorectal liver metastases by comparing genotypes in frozen biopsies with genotypes in microdissected archival samples from the same patients. Constitutional genotypes were obtained from corresponding peripheral blood leukocytes as well as normal liver tissue. Archival samples (n = 43) from 16 patients were analyzed after microdissection with 2-5 of 10 selected microsatellite markers. Frozen biopsies from one metastasis of each patient had previously been investigated at numerous microsatellite loci. From those results we selected, for the comparable analysis of archival samples, 41 tumor genotypes at 10 loci representing 11 heterozygotes, 13 AI, 7 losses of heterozygosity (LOHs), 8 homozygotes, and 2 microsatellite unstable cases. The microdissected samples revealed AI or a complete loss of one allele (LOH) in 5 of 11 (45%) genotypes that were previously evaluated as unchanged (retained heterozygosity) in the frozen biopsies, and LOH in 8 of 13 (62%) genotypes at loci known to exhibit AI in the frozen biopsies. Microsatellite instability, LOH, and homozygosity found in the frozen samples were all confirmed by analyses of the archival material. Intertumoral genetic heterogeneity was found in samples from two patients. The same allelic intensities were seen in DNA from tumor-close liver tissue as in blood DNA from the same patient except in one sample. The present study shows a 54% increase in sensitivity of genetic alterations if pure tumor cell components are used (five "new" AIs and LOHs and eight "new" LOHs among previously scored heterozygotes [n = 11] and AI [n = 13], respectively). In total, a 93% success rate (108/115 analyses) was obtained using standard PCR conditions for the 10 selected markers. The fact that standard PCR conditions and 5-micron tumor sections are used shows how easy these analyses are to perform, and that only minor amounts of valuable archival material is used.

Allelotype profiles of local recurrences and distant metastases from colorectal-cancer patients.

Several genetic alterations have been described in benign and malignant primary tumors of the colorectum, but few such associations have been made with the progression of these tumors. This study compares genetic changes found in distant metastases (n = 22) with local recurrences (n = 15) as well as with primary carcinomas (n = 12). Complete allelotypes of the tumors were obtained by analyzing 43 microsatellite loci, representing all non-acrocentric chromosome arms and mapping to the mid-portion of the arms. Allelic imbalances in the tumor DNA were evaluated by comparison with the patient's constitutional pattern in blood DNA. The allelotype profile of the distant metastases was different from those found in the local recurrences and in the primary carcinomas. More than 20% of the distant metastases exhibited allelic imbalances at loci representing 20 chromosome arms. The majority of these regions were less frequently changed in the local recurrences and in the primary tumors. The markers that most often were altered in the metastasis (>40%) represented chromosome arms 14q, 17p, 18p and 18q. Only two regions, 10p and 19p, were unaltered in all tumors analyzed. We found that the median value of fractional allelic imbalance was twice as high in the distant metastases as in the recurrent tumors. Novel alleles at microsatellite loci were observed in all 3 tumor types, but in the advanced tumors this phenotype was characterized by only a single novel allele seen at less than 10% of the analyzed loci.