Functional categories of TP53 mutation in colorectal cancer: results of an International Collaborative Study.

BACKGROUND: Loss of TP53 function through gene mutation is a critical event in the development and progression of many tumour types including colorectal cancer (CRC). In vitro studies have found considerable heterogeneity amongst different TP53 mutants in terms of their transactivating abilities. The aim of this work was to evaluate whether TP53 mutations classified as functionally inactive (< or=20% of wildtype transactivation ability) had different prognostic and predictive values in CRC compared with mutations that retained significant activity. MATERIALS AND METHODS: TP53 mutations within a large, international database of CRC (n = 3583) were classified according to functional status for transactivation. RESULTS: Inactive TP53 mutations were found in 29% of all CRCs and were more frequent in rectal (32%) than proximal colon (22%) tumours (P < 0.001). Higher frequencies of inactive TP53 mutations were also seen in advanced stage tumours (P = 0.0003) and in tumours with the poor prognostic features of vascular (P = 0.006) and lymphatic invasion (P = 0.002). Inactive TP53 mutations were associated with significantly worse outcome only in patients with Dukes' stage D tumours (RR = 1.71, 95%CI 1.25-2.33, P < 0.001). Patients with Dukes' C stage tumours appeared to gain a survival benefit from 5-fluorouracil-based chemotherapy regardless of TP53 functional status for transactivation ability. CONCLUSIONS: Mutations that inactivate the transactivational ability of TP53 are more frequent in advanced CRC and are associated with worse prognosis in this stage of disease.

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.

Kirsten ras mutations in patients with colorectal cancer: the 'RASCAL II' study.

Researchers worldwide with information about the Kirsten ras (Ki-ras) tumour genotype and outcome of patients with colorectal cancer were invited to provide that data in a schematized format for inclusion in a collaborative database called RASCAL (The Kirsten ras in-colorectal-cancer collaborative group). Our results from 2721 such patients have been presented previously and for the first time in any common cancer, showed conclusively that different gene mutations have different impacts on outcome, even when the mutations occur at the same site on the genome. To explore the effect of Ki-ras mutations at different stages of colorectal cancer, more patients were recruited to the database, which was reanalysed when information on 4268 patients from 42 centres in 21 countries had been entered. After predetermined exclusion criteria were applied, data on 3439 patients were entered into a multivariate analysis. This found that of the 12 possible mutations on codons 12 and 13 of Kirsten ras, only one mutation on codon 12, glycine to valine, found in 8.6% of all patients, had a statistically significant impact on failure-free survival (P = 0.004, HR 1.3) and overall survival (P = 0.008, HR 1.29). This mutation appeared to have a greater impact on outcome in Dukes' C cancers (failure-free survival, P = 0.008, HR 1.5; overall survival P = 0.02, HR 1.45) than in Dukes' B tumours (failure-free survival, P = 0.46, HR 1.12; overall survival P = 0.36, HR 1.15). Ki-ras mutations may occur early in the development of pre-cancerous adenomas in the colon and rectum. However, this collaborative study suggests that not only is the presence of a codon 12 glycine to valine mutation important for cancer progression but also that it may predispose to more aggressive biological behaviour in patients with advanced colorectal cancer.

Variation in mutation rate and direction between tetranucleotide STR loci in human colorectal carcinomas.

Replication error (RER) is defined as mutation events in repetitive DNA segments. To investigate further the RER phenomenon and reveal any differences in mutation outcome between different short tandem repeat (STR) loci, we have investigated the somatic mutation rate and the size distribution of new tumour alleles in four tetranucleotide STRs in a large material of unselected colorectal adenocarcinomas. DNA was extracted from the blood and carcinomas of 217 patients. All blood/tumour pairs were analysed using the STRs HUMTHO1, HUMFES/FPS, HUMVWA31/A and HUMF13A1. Mutations are detected at all four loci. There are substantial differences in mutation rate and mutation direction (i.e. expansion versus contraction) between different STR loci. In all four STRs, the majority of events represent gain or loss of a single repeat. Almost all new tumour alleles correspond to known alleles in a population database, indicating that these are also composed of integers of the four base pair repeat. There is no statistically significant size bias in the mutating alleles as compared to the allelic distribution in the population database.

The APC gene I1307K variant is rare in Norwegian patients with familial and sporadic colorectal or breast cancer.

Recently, a T-to-A transversion creating an 8-base mononucleotide tract in the APC gene, resulting in substitution of lysine for isoleucine at codon 1307 (I1307K), was found in a subset of Ashkenazi Jews. This sequence variant was most frequent in colorectal cancer patients with a positive family history of colorectal cancer. To determine whether the I1307K variant plays a role in colorectal or breast cancer predisposition in the Norwegian population, we have analyzed blood samples from 210 colorectal cancer patients and 183 breast cancer patients by PCR and direct sequencing. Thirty-seven of the colorectal cancer patients had a positive family history of cancer. Among the breast cancer patients, 24 had a family history of colorectal cancer and 75 a family history of breast and/or ovarian cancer. Only one colorectal cancer patient who belonged to a Jewish family was found to carry the A variant. Our data show that the I1307K variant is rare in the Norwegian population and should not be viewed as a candidate for susceptibility testing for colorectal cancer.

TP53 and long-term prognosis in colorectal cancer: mutations in the L3 zinc-binding domain predict poor survival.

In a consecutive series of 222 colorectal carcinomas from patients with a median follow-up time of 56.8 months (range, 0.5-92.2) treated with surgery, the TP53 gene was screened for mutations. Exons 5-8 were analyzed using constant denaturant gel electrophoresis followed by sequencing, and mutations were found in 102 cases (45.9%). Mutations were found more frequently in rectal tumors versus other locations (P = 0.029) and in aneuploid compared to diploid tumors (P < 0.001). Presence of a TP53 mutation was also significantly associated with absence of microsatellite instability (P = 0.028), as well as with loss of heterozygosity at 17p13 (P < 0.001). The TP53 mutations in the left-sided and rectal tumors were more often transversions than transitions, indicating a different etiology in the development of these tumors. The tendency for shorter cancer-related survival among patients with mutations in their tumors was only statistically significant for patients with left-sided tumors (P = 0.003). All patients with mutations affecting the L3 domain of the protein involved in zinc binding had a significantly shorter cancer-related survival (P = 0.036), indicating that mutations affecting this domain have biological relevance in terms of colorectal cancer disease course. These results suggest that knowledge of a patient's TP53 status, with respect to both the presence and the localization of the mutation, may be important in prognosis evaluation, particularly when selecting patients for more aggressive postoperative therapeutic intervention.

Association of p53 accumulation with TP53 mutations, loss of heterozygosity at 17p13, and DNA ploidy status in 273 colorectal carcinomas.

The aim of this study was to establish an experimentally based cutoff level for assessing p53 immunoreactivity in colorectal tumors. The accumulation of p53 protein in 273 colorectal tumors was correlated with previously obtained data on TP53 mutation and loss of heterozygosity at two 17p13 loci in the same tumors. The monoclonal antibody PAb 1801 was used for p53 staining, and the results obtained by immunohistochemistry and immunoblotting were similar. Mutation analyses of exons 5-8 were performed using constant denaturant gel electrophoresis followed by sequencing. There were no statistically significant differences for any measured TP53 gene alteration between the group of tumors without p53-positive nuclei (n = 83) and the group with <5% positive nuclei (n = 58). The majority of mutations within these groups were deletions/insertions and nonsense mutations without p53 accumulation. Therefore, we assume that 5% p53-positive nuclei is the relevant cutoff level to assess TP53 damage in colorectal tumors. A prerequisite for this recommendation is optimal conditions for p53 protein detection. The parameters for p53 dysfunction were correlated to DNA aneuploidy measured by flow cytometry. TP53 mutations were significantly associated with DNA aneuploidy (P < 0.00001), and a nonrandom distribution of TP53 gene alterations among diploid (DI = 1), hyperdiploid (1.0 < DI < 1.3), and highly aneuploid (DI > 1.3) tumors indicates that DNA hyperdiploid tumors constitute a separate developmental entity different from tumors with gross aneuploidy.

K-ras mutations and prognosis in large-bowel carcinomas.

BACKGROUND: Colorectal carcinogenesis is regarded as a multistep process involving several genetic alterations, with mutation in the K-ras gene in about half of the tumours. We aimed at clarifying the role of this genetic alteration related to survival and clinicopathologic variables. METHODS: One hundred large-bowel carcinomas operated on between 1978 and 1982 were studied for the presence of point mutations in codons 12 and 13 of the K-ras gene, using enriched polymerase chain reaction amplification, restriction fragment length polymorphism analysis, and direct sequencing. RESULTS: Forty mutations were found (40%): 31 in codon 12 and 9 in codon 13, 7 different types. There was no relationship between tumours with and without K-ras mutations with regard to Dukes' stages, age or sex of the patient, tumour localization, histologic grade, DNA ploidy pattern, HLA-DR staining pattern, or survival. Samples from 5 different localizations in 7 carcinomas showed identical K-ras mutation pattern, as did 19 recurrences/ metastases originating from 11 carcinomas. CONCLUSIONS: When present, the primary tumour shows homogeneous distribution of K-ras mutation, and the mutation follows the carcinoma in the secondary deposit, regardless of lymphogenous or hematogenous spread. The presence of K-ras mutation does not seem to have prognostic significance for the patient, and the precise nucleotide change is furthermore not predictive of tumour behaviour.

Different genetic pathways to proximal and distal colorectal cancer influenced by sex-related factors.

Mutations in the k-ras and TP53 genes, as well as microsatellite instability (MIN), are frequent genetic alterations in colorectal carcinomas and represent 3 different mechanisms in the carcinogenic process. Both the incidence of colorectal cancer and the frequency of genetic alterations in such tumours have been related to different clinico-pathological variables, including age and gender of the patient and location of the tumour. A number of studies have also reported associations between different types of genetic alterations. We therefore wanted to explore the relationship between these genetic and clinico-pathological variables using multivariate analysis on material from 282 colorectal carcinomas. Three logistic regression models were constructed: 1) the presence of K-ras mutations was dependent on MIN and age and gender of patient, with an especially low frequency among younger males and in tumours with MIN (overall p = 0.0003); 2) the presence of TP53 mutations was only dependent on tumour location, with a positive association to cancers occurring distally (p = 0.002); and 3) the presence of MIN was dependent on age, gender and K-ras and TP53 mutations, as well as on tumour location. MIN was most frequent among younger male and older female patients, was rare in tumours with K-ras or TP53 mutations and was found almost exclusively in the proximal colon (overall p < 0.0001). Our data confirm that different genetic pathways to colorectal cancer dominate in the proximal and distal segments of the bowel and suggest that the K-ras- and MIN-dependent pathways are influenced by different sex-related factors.

K-ras mutations and HLA-DR expression in large bowel adenomas.

A total of 72 sporadic colorectal adenomas in 56 patients were studied for the presence of point mutations in codons 12 and 13 of the K-ras gene and for HLA-DR antigen expression related to clinicopathological variables. Forty K-ras mutations in 39 adenomas were found (54%): 31 (77%) in codon 12 and nine (23%) in codon 13. There was a strong relationship between the incidence of K-ras mutations and adenoma type, degree of dysplasia and sex. The highest frequency of K-ras mutations was seen in large adenomas of the villous type with high-grade dysplasia. Fourteen out of 15 adenomas obtained from 14 women above 65 years of age carried mutations. HLA-DR positivity was found in 38% of the adenomas, large tumours and those with high-grade dysplasia having the strongest staining. Coexpression of K-ras mutations and HLA-DR was found significantly more frequently in large and highly dysplastic adenomas, although two-way analysis of variance showing size and grade of dysplasia to be the most important variable. None of the adenomas with low-grade dysplasia showed both K-ras mutation and HLA-DR positivity (P = 0.004). K-ras mutation is recognised as an early event in colorectal carcinogenesis. The mutation might give rise to peptides that may be presented on the tumour cell surface by class II molecules, and thereby induce immune responses against neoplastic cells.

Secretory component mRNA and protein expression in colorectal adenomas and carcinomas.

Secretary component (SC) is expressed basolaterally as a transmembrane protein (pIg receptor) on secretory epithelial cells. As pIg receptor it plays a central role in humoral immunity by mediating the external translocation of dimeric IgA and pentameric IgM. A few case reports have suggested that reduced or absent SC protein expression is associated with diarrhoeal disease, but there is no convincing evidence that a primary pIg receptor deficiency can occur. In this study the relative presence of SC mRNA was determined by Northern blot analysis and related to immunohistochemically determined SC protein expression in 33 colorectal adenomas (31 patients) with increased risk of developing sporadic colorectal cancer, as well as in 19 colorectal carcinomas from 19 patients with such sporadic tumours. In the adenomas, SC mRNA levels were positively related to SC protein expression; both mRNA and SC protein were negatively related to histological grade. Similarly, SC mRNA levels tended to be related to the SC protein expression in the carcinomas. SC mRNA was detected in all adenomas, and only two of ten carcinomas (10.5%) deemed to be SC deficient by immunohistochemistry also lacked SC mRNA expression, suggesting diallelic alterations in the SC-encoding gene (locus PIGR). This possibility agreed with Southern blot analysis performed on a separate sample of 32 other colonic carcinomas in which the diallelic loss of D1S58 (which exhibits a close linkage centromerically to PIGR) was calculated to be 6.4%. Together these findings suggested that reduced SC protein expression in colorectal adenomas might be a transcriptional defect reflecting the degree of cellular dysplasia, whereas absent SC protein expression in colorectal carcinomas might also involve post-transcriptional defects and occasional diallelic gene deletions representing late events in carcinogenesis.

[Extrapulmonary tuberculosis. An important differential diagnosis in immigrants with suspected malignancy]. / Ekstrapulmonal tuberkulose. Viktig differensialdiagnose ved malignitetsmistanke hos innvandrere.

The incidence of tuberculous disease is increasing all over the world, mostly in the poor, developing countries, but also in some industrialized countries. In Norway, extrapulmonary tuberculosis is a rare phenomenon. It is found mostly among older Norwegians and in younger immigrants from the third world. Since the disease is rare, it may be overlooked or confused with malignant disease. We describe two patients with unusual forms of extrapulmonary tuberculosis, both mimicking neoplastic disease. The first patient was a 27-year-old woman from South-East Asia, who was operated on for suspected intraductal comedo-type carcinoma of the breast, but histological examination showed tuberculous mastitis. The second patient was a 26-year-old man from East Africa with a medical history indicating intra-abdominal lymphoma. The final diagnosis, however, was mesenteric tuberculous lymphadenitis. Both patients were treated successfully with isoniazid, rifampicin and pyrazinamide.

Somatic mutations in the hMSH2 gene in microsatellite unstable colorectal carcinomas.

Microsatellite instability is frequently seen in tumors from patients with hereditary nonpolyposis colorectal cancer (HNPCC). Germline mutations in the mismatch repair gene hMSH2 account for approximately 50% of these cases. Tumors from sporadic cases also exhibit this microsatellite instability phenotype, although at a lower frequency, and very few somatically derived mutations have so far been reported in such tumors. In this study DNA from 23 primary colorectal carcinomas (four familial and 19 sporadic cases) exhibiting microsatellite instability were screened for mutations in the hMSH2 gene using constant denaturant gel electrophoresis (CDGE). Among the sporadic cases, five (26%) were found to have somatically derived mutations. One tumor revealed two different mutations, possibly leading to a homozygous inactivation of the gene. One of the four familial cases was classified as having HNPCC, and a germline as well as a somatic mutation were found in this tumor. These results demonstrate that a considerable proportion of sporadic colorectal cancers with microsatellite instability, have somatic mutations in the hMSH2 gene.

Deletion of 1p loci and microsatellite instability in colorectal polyps.

Previous cytogenetic studies have indicated that a subset of large bowel adenomas have distal 1p deletions. We addressed this question by examining 70 sporadic polyps (63 adenomas, 5 hyperplastic polyps, and 2 polyps of undetermined histology) from 55 patients for alterations at eight loci on the short arm of chromosome 1 and found allelic imbalance (AI) or loss of one allele (LOH) in 14 (20%). The locus most frequently changed was MSI, which maps to 1p33-35. Fluorescence in situ hybridisation with centromeric and telomeric probes for chromosome 1, performed for 11 polyps, did not yield an abnormal number of signals, in accordance with the interpretation that the observed AI and LOH were the result of interstitial deletions in 1p. Whereas allelic imbalance at five other loci (mapping to 5q, 8p, 10p, 11p and 17q) was found less frequently, and then mainly in large (> 2 cm) tumours, the 1p alterations were equally distributed among small (< 1 cm) and large polyps. They were preferentially found in left-side tumours. Instability at microsatellite loci--the mutator phenotype--is demonstrated by shifts in the electrophoretic mobility of normal alleles. The mutator phenotype was first associated with hereditary nonpolyposis colorectal cancer but is also occasionally found in sporadic colorectal carcinomas; however, it is still uncertain when in the adenoma-carcinoma sequence in this type of genomic instability arises. We therefore looked for it at 12 dinucleotide repeat loci and found that seven tumours (six adenomas and one hyperplastic polyp) from seven patients had acquired new alleles not seen in the patients' corresponding normal DNA. Our results suggest that inactivation of a putative suppressor gene distally in chromosome arm 1p is an early event in colorectal tumourigenesis. They also show that microsatellite instability can be detected in large bowel polyps, indicating that this phenomenon, too, probably plays a pathogenic role for some colorectal tumours early in the adenoma-carcinoma sequence.

Antigen levels of urokinase plasminogen activator and its receptor at the tumor-host interface of colorectal adenocarcinomas are related to tumor aggressiveness.

The distributions of urokinase and tissue plasminogen activators (uPA, tPA), uPA receptor (uPAR), and plasminogen activator inhibitors (PAI-1, PAI-2) were studied immunohistochemically in two subsets of colorectal adenocarcinomas with low and high aggressiveness, respectively: nine Dukes' stage A tumors with additional other good prognostic markers and 13 Duke's stage C tumors with also other poor prognostic markers (referred to as Dukes' stage A and Dukes' stage C tumors). The results showed that these components of the tissue destructive plasminogen activation system were accumulated at the invading front of the tumors. Both tumor groups showed accumulations of uPA, uPAR, and PAI-1 at the tumor-host interface compared with the location within the tumor epithelium and the adjacent normal mucosa and muscularis propria (all P < .05). However, the uPA level at the tumor-host interface in the Dukes' stage C tumors was twice the level in the Dukes' stage A tumors (P < .05). The uPAR level was also significantly higher in the Dukes' stage C tumors (P < .05), whereas the PAI-1 level was not significantly higher. This may indicate that uPA in more aggressive tumors exceeds the inhibitory capacity represented by PAIs, resulting in enhanced tissue destructive potential that promotes tumor invasion. uPA and uPAR antigen levels and the uPA/PAI-1 ratio at the tumor-host interface appeared to be related to tumor aggressiveness in colorectal cancer.

Somatic mutations in VNTR-locus D1S7 in human colorectal carcinomas are associated with microsatellite instability.

To elucidate mutation mechanisms in hypervariable VNTR loci, we have studied somatic mutation events with the minisatellite probe MS1 (VNTR locus D1S7) in 224 colorectal carcinomas (CRC). The D1S7 locus consists of a 9-basepair (bp) repeat unit. The copy number varies from about 100 to 2000, and the germline mutation rate is high. Here we demonstrate a high D1S7 somatic mutation rate in CRC (37/224), higher than indicated earlier by others. We also demonstrate that the most frequent mutational event by far (n = 34) involves small reductions in VNTR fragment size (median loss 22 repeat units, range 2-154), furthermore, in one-half of these cases, this event is biallelic. We wanted to test whether these somatic mutations mirror the same genetic instability as seen by RER (replication error), a phenomenon recently described in tumour DNA from both sporadic and familial cases of CRC. All blood/tumour DNA pairs displaying MS1 mutation (n = 37) as well as 37 randomly selected pairs without MS1 mutation were tested with four tetranucleotide short tandem repeats (STRs, microsatellites). There is a strong association between mutations at the D1S7 locus and the occurrence of new STR alleles (P < 0.001). This is the first report of the existence of a minisatellite as a marker for genetic instability/RER in colorectal carcinomas. The findings may also cast light upon the mechanism for somatic mutations in this minisatellite.