Molecular genetic changes in benign colorectal tumors synchronous with microsatellite unstable carcinomas do not support a field defect.

BACKGROUND: A colorectal cancer may develop through a particular molecular genetic pathway, raising the question of whether the particular molecular changes are random, or are unique to the particular segment of colon. We wanted to determine whether molecular changes found within a colorectal cancer might also be detected in separate adenomas and polyps removed from the same area of colon at surgery. Microsatellite instability was chosen as a marker for a pathway of colon carcinogenesis. METHODS: We studied a total of 46 primary colorectal cancers with microsatellite instability and 77 synchronous adenomas and polyps. All tumors were evaluated for microsatellite instability, BRAF and KRAS mutations, and methylation using standard polymerase chain reaction based methods. RESULTS: Forty-nine benign tumors did not follow a pathway similar to that of their 31 synchronous primary cancers. For two distinct subsets of the microsatellite unstable colorectal cancers, those with acquired methylation and BRAF mutation, and those without methylation suggestive of an underlying germ line mutation, the molecular changes in the majority of their synchronous benign tumors were different from the colorectal cancer. CONCLUSIONS: These differences suggest a stochastic process within the colon regarding the particular molecular carcinogenic pathways followed by the synchronous tumors, rather than a 'field defect' within the colon segments. Variability in molecular findings was present for colorectal cancers arising from acquired methylation, as well as those cancers suggestive of a germ line origin.

GNAS gene mutation may be present only transiently during colorectal tumorigenesis.

Mutations of the gene GNAS have been shown to activate the adenylate cyclase gene and lead to constitutive cAMP signaling. Several preliminary reports have suggested a role for GNAS gene mutations during colorectal carcinogenesis, particularly mucinous carcinomas. The aim of this study was to clarify the incidence of GNAS mutations in adenomas (tubular, tubulovillous, and villous), carcinomas with residual adenoma, and carcinomas, and to relate these findings to mutations of the KRAS gene and to the mucinous status of the tumors. We used standard PCR techniques and direct gene sequencing to evaluate tumors for gene mutations. No GNAS mutations were identified in 25 tubular adenomas, but were present in 6.4% of tubulovillous adenomas and 11.2% of villous adenomas. A GNAS mutation was found in 9.7% of the benign portion of carcinoma with residual adenoma, but in none of 86 carcinomas. A similar trend was seen for KRAS mutation across the five groups of tumors. GNAS mutations may function as an important driver mutation during certain phases of colorectal carcinogenesis, but may then be lost once the biological advantage gained by the mutated gene is no longer necessary to sustain or advance tumor development.

Copy number of the Adenomatous Polyposis Coli gene is not always neutral in sporadic colorectal cancers with loss of heterozygosity for the gene.

BACKGROUND: Changes in the number of alleles of a chromosome may have an impact upon gene expression. Loss of heterozygosity (LOH) indicates that one allele of a gene has been lost, and knowing the exact copy number of the gene would indicate whether duplication of the remaining allele has occurred. We were interested to determine the copy number of the Adenomatous Polyposis Coli (APC) gene in sporadic colorectal cancers with LOH. METHODS: We selected 38 carcinomas with LOH for the APC gene region of chromosome 5, as determined by amplification of the CA repeat region within the D5S346 loci. The copy number status of APC was ascertained using the SALSA® MLPA® P043-B1 APC Kit. LOH for the DCC gene, KRAS gene mutation, and microsatellite instability were also evaluated for each tumor, utilizing standard polymerase chain reaction methods. RESULTS: No tumor demonstrated microsatellite instability. LOH of the DCC gene was also present in 33 of 36 (91.7%) informative tumors. A KRAS gene mutation was present in 16 of the 38 (42.1%) tumors. Twenty-four (63.2%) of the tumors were copy number neutral, 10 (26.3%) tumors demonstrated major loss, while two (5.3%) showed partial loss. Two tumors (5.3%) had copy number gain. CONCLUSIONS: Results of APC and DCC LOH, KRAS and microsatellite instability indicate our colorectal cancer cases were typical of sporadic cancers following the 'chromosomal instability' pathway. The majority of our colorectal carcinomas with LOH for APC gene are copy number neutral. However, one-third of our cases showed copy number loss, suggesting that duplication of the remaining allele is not required for the development of a colorectal carcinoma.

Colorectal Cancers with the Uncommon Findings of KRAS Mutation and Microsatellite Instability.

Sporadic colorectal cancers with microsatellite instability (MSI) frequently contain a mutation of the BRAF gene. Additionally, it has been shown that BRAF mutations in colorectal cancers are mutually exclusive of KRAS mutation. We evaluated 14 cases of colorectal cancer with MSI that were BRAF wild type but demonstrated a KRAS mutation. The codon 12/13 region in exon 2 of the KRAS oncogene and the codon 600 region in exon 15 of the BRAF gene were analyzed with standard PCR methods. MSI was evaluated by using the Bethesda panel of markers. The methylation status of the mismatch repair system was ascertained using the SALSA(®) MS-MLPA(®) methylation-specific DNA detection. The mismatch repair proteins MLH1, MSH2, MSH6, and PMS2 were evaluated by immunohistochemical staining. A total of 530 colorectal cancers were studied for MSI and KRAS gene mutation. Fourteen (2.6%) cancers with both MSI and a KRAS mutation were identified, and all cancers were BRAF wild type. Methylation was present in 7 (50%), 5 demonstrated methylation of MLH1, 1 showed methylation of MGMT, and 1 showed methylation of MSH2. Four patients had simultaneous cancers, some of which showed different genetic changes. Immunohistochemical staining suggested a germ line mutation for 4 of 10 cases with complete staining information. KRAS mutation may occur with MSI in colorectal cancers with wild-type BRAF. If a mutation in KRAS co-exists with MSI, then strong methylation of the MLH1 gene is unlikely. These tumors demonstrate that a small number of colorectal cancers will develop with atypical patterns of molecular genetic changes, suggesting that a specific pattern of genetic changes may not be as crucial as the overall accumulation of changes, consistent with the 'unique tumor principle'.

Strong correlation between molecular changes in endometrial carcinomas and concomitant hyperplasia.

OBJECTIVE: Endometrial cancer (EC) results from the accumulation of numerous genetic abnormalities contributing to the progression from hyperplasia to EC. Information on these various genetic changes has been primarily derived from studying groups of either hyperplasias or cancers.We evaluated both hyperplastic and EC tissue obtained from the same surgical specimens for KRAS mutations, microsatellite instability (MSI), and mismatch repair gene methylation, and results were correlated between the paired hyperplastic tissue and EC. The aim was to determine if molecular alterations appearing in ECs might also be present in the premalignant (hyperplastic) region of the tumor. METHODS: One hundred ninety-seven cases of EC with associated hyperplasia were evaluated. DNA samples were studied using primer sets for KRAS gene codons 12/13 and for MSI utilizing the Bethesda panel. Methylation testing was performed on specimens that were microsatellite unstable using the MRC Holland SALSA MS-MLPA methylation-specific DNA detection kit. RESULTS: Forty-one (20.8%) of 197 cancers demonstrated a KRAS mutation, with 35 (85.4%) of 41 accompanying hyperplasias also containing a KRAS mutation. Forty-five cancers (22.8%) were microsatellite unstable, with 38 (84.4%) of 45 accompanying hyperplasias also demonstrating instability. Of the 45 microsatellite unstable cancers, 28 (62.2%) demonstrated methylation in both the cancer and the accompanying hyperplasia, whereas 9 pairs (20%) showed no methylation for either the cancer or hyperplasia. CONCLUSIONS: Approximately 95% of endometrial specimens demonstrated identical molecular findings regarding KRAS mutation and microsatellite stability in the paired cancer and hyperplastic tissue. The same methylation pattern was found in 82.2% of the studied paired samples. Our findings strongly suggest that the molecular changes of KRAS mutation, MSI, and methylation occur early in the neoplastic process. We propose that endometrial biopsies revealing only hyperplasia should be studied for these molecular alterations as an indicator of possible early carcinogenesis.

Colorectal tumors from APC*I1307K carriers principally harbor somatic APC mutations outside the A8 tract.

PURPOSE: APC*I1307K (c.3920T>A) is an inherited variant associated with colorectal tumour risk found almost exclusively in those of Ashkenazi Jewish ancestry. A single nucleotide substitution creates an oligo-adenine tract (A8) that appears to be inherently prone to further mis-pairing and slippage. The reported multiple tumor phenotype of carriers is not easily reconciled with molecular and population genetics data. We postulated that some c.3920T>A carriers with multiple adenomas have other unidentified APC germ line or somatic mutations. METHODS: DNA from 82 colonic tumours and accompanying normal tissue collected from 29 carriers with multiple colorectal tumors was directly sequenced between codons 716 and 1604. We also assessed APC gene loss of heterozygosity. RESULTS: One patient (3.4%) was found to have an additional APC germ line mutation. Twenty-five of the tumours showed no significant somatic molecular change, 36 showed one change, 20 showed two, and one tumour showed more than 2 changes. Our data suggest a correlation between advancing histology and fewer beta-catenin binding sites remaining in the mutant proteins. CONCLUSIONS: There were no other common germ line variants identified within the region of the APC gene examined, suggesting that any effect from this region on tumour production is attributable to the c.3920T>A allele. Our findings further suggest the only somatic genetic change clearly attributable to the c.3920T>A mutation is the c.3924_3925insA.

Similarities of molecular genetic changes in synchronous and metachronous colorectal cancers are limited and related to the cancers' proximities to each other.

Synchronous and metachronous colorectal cancers are distinct primary neoplasms diagnosed either simultaneously or sequentially in the same patient. Because they arise in a common genetic and environmental background, they offer a unique opportunity to study molecular genetic changes occurring during carcinogenesis. We evaluated tumors from 50 patients with synchronous and five additional patients with metachronous cancers for loss of heterozygosity of the genes APC and DCC, KRAS and BRAF gene mutations, and microsatellite instability and methylation. Standard PCR methods were used. Approximately two thirds of the synchronous tumors that were informative for each of the five primary molecular genetic changes showed the same results when located in the same colon segment. However, there was less consistency of molecular findings for the synchronous pairs separated by one or more colonic segments, with half or more of these pairs showing different molecular results. Metachronous tumors also showed variation of molecular genetic findings, but this was less when the subsequent tumor was close to the segment of the first tumor. Molecular genetic findings between synchronous and metachronous tumors can be different, even for tumors sharing the same microenvironment of the same colon segment. These findings support the concept that a mutagen might produce different genetic pathways in two proximate tumors.

Detailed molecular genetics of the APC*E1317Q mutation in tumor tissue suggest it may not be pathologically significant.

APC*E1317Q is a low-penetrance variant of the APC gene suggested as a risk for the development of colorectal adenomas and carcinomas. There is very little in the literature describing the molecular details of APC*E1317Q in tumor tissue. We provide information about the molecular genetics of 3 patients with APC*E1317Q. For 1 patient, we show linkage to a specific APC allele. We further show that loss of heterozygosity of the APC gene in tumors from carriers of the APC*E1317Q mutation may involve the mutated allele, not just the wild-type allele, suggesting the APC*E1317Q missense mutation may not be pathologically significant in the development of colorectal tumors.

KRAS gene mutations are more common in colorectal villous adenomas and in situ carcinomas than in carcinomas.

We have evaluated the frequency of KRAS gene mutations during the critical transition from villous adenoma to colorectal carcinoma to assess whether the adenomas contain a KRAS mutation more frequently than carcinomas. We analyzed sporadic villous and tubulovillous adenomas, in situ carcinomas, and primary colorectal carcinomas from multiple patients. The cancers were further evaluated for mucinous status and microsatellite instability. Standard PCR molecular techniques were used for KRAS and microsatellite analyses. A KRAS mutation was found in 61.9% of 134 adenomas, 67.8% of 84 in situ carcinomas, and just 31.6% of 171 carcinomas. Our study clearly demonstrates that tubulovillous and villous adenomas, as well as both the benign and malignant parts of in situ carcinomas, are statistically more likely to contain a somatic KRAS gene mutation than colorectal carcinomas. This difference is confined to the non-mucinous and the microsatellite stable tumors. Our data support the possibility that non-mucinous and microsatellite stable carcinomas with wild-type KRAS gene may have had a mutation in the KRAS gene during their earlier stages, with the mutation lost during further growth.

Ki-ras gene mutations are invariably present in low-grade mucinous tumors of the vermiform appendix.

OBJECTIVE: Low-grade mucinous tumors of the appendix appear to have a simple histological structure. Paradoxically, reports have suggested a greater frequency of Ki-ras gene mutation in these lesions than in more complex lesions such as benign colonic adenomas and carcinomas. We assessed several molecular genetic changes, including Ki-ras gene mutations, in a large series of low-grade mucinous tumors of the appendix. MATERIAL AND METHODS: We retrospectively ascertained low-grade mucinous tumors of the appendix from computerized pathology records. Extracted DNA was analyzed for APC and DCC gene loss of heterozygosity, microsatellite instability and for the presence of Ki-ras gene mutation using standard molecular techniques. Controls consisted of normal appendices, other appendiceal neoplasms, and ovarian mucinous cystadenomas. RESULTS: A total of 31 low-grade appendiceal mucinous tumors were identified. All were microsatellite stable and none demonstrated loss of heterozygosity for the APC or DCC genes. By contrast, all 31 lesions contained a Ki-ras gene mutation. CONCLUSIONS: The presence of a Ki-ras gene mutation in all lesions, with no other molecular changes identified, strongly suggests a possible etiological role of the Ki-ras mutation in the development of this particular lesion of the appendix. Based on other work regarding intestinal bacteria, we hypothesize a relationship between chronic inflammation of the appendix from bacterial overgrowth and Ki-ras gene mutation.

Microsatellite instability and DNA methylation of endometrial tumors and clinical features in young women compared with older women.

INTRODUCTION: Molecular genetic changes in endometrial cancers are important to identify possible family cancer syndromes and thus, to facilitate appropriate screening. Most studies in this regard have focused primarily on young women. We have assayed cancers for microsatellite instability (MSI) and DNA methylation from a large group of patients younger than 50 years and a comparable group of older women. We obtained personal and medical histories of the patients and their family cancer histories. METHODS: The Bethesda panel of markers was used for the detection of MSI. Methylation status of mismatch repair genes was ascertained using the methylation-specific DNA detection kit SALSA MS-MLPA No. ME011. RESULTS: There were 101 patients younger than 50 years and 112 older women. The 2 age groups did not differ in the percentage of patients who were obese, carried a diagnosis of diabetes, or previously had another cancer. The younger patients were more likely to be nulliparous, whereas the older patients were more likely to have hypertension. Among the younger group, 21 (20.8%) tumors revealed MSI, and 13 (61.9%) of these were unmethylated. For the older women, 35 (31.2%) had MSI tumors, and only 6 (17.1%) of these were unmethylated. Young women with a family history of a hereditary nonpolyposis colorectal cancer-related cancer were more likely to have a tumor revealing MSI and no methylation, but family history was less helpful in older women in this regard. CONCLUSION: We did not find personal risk factors or a history of an additional cancer to be different between the 2 age groups. The combination of MSI testing and DNA methylation studies resulted in the identification of presumptive hereditary nonpolyposis colorectal cancer syndrome in approximately 13% of women with endometrial cancer presenting at age younger than 50 years and in approximately 5% of older women. Family history was more helpful with younger women than with older women.

Adequacy of colonoscopic biopsy specimens for molecular analysis: a comparative study with colectomy tissue.

Molecular analyses of tumors are increasingly useful for prognosis and for guiding therapy. Colonoscopic biopsy provides the first source of tissue for most cases of colorectal carcinoma and therefore might become an important source for molecular analyses. We have addressed the question whether molecular analyses of colonoscopic biopsy yield results similar to the findings from the surgical specimen. Further, we analyzed 2 separate areas of the colectomy specimen to assess tumor heterogeneity. We evaluated 3 samples from each of 67 patients for point mutations in the KRAS gene, loss of heterozygosity (LOH) at the Adenomatous Polyposis Coli (APC) and Deleted in Colon Cancer (DCC) genes and for microsatellite instability (MSI) using polymerase chain reaction based techniques. The average time interval between biopsy and surgery was 2.2+/-0.15 weeks. Lesions were from all colon segments and all surgical stages. The degree of agreement between the biopsy and surgical sites was high for APC LOH, MSI, and KRAS mutations (kappa=0.85, 1.00, and 0.93, respectively) but less so for DCC LOH (kappa=0.62). Colonoscopic biopsies are an acceptable source of neoplastic DNA for studies of KRAS, APC LOH, and MSI, but less so for DCC LOH, primarily resulting from technical considerations.

Clinical and genetic findings in an Ashkenazi Jewish population with colorectal neoplasms.

BACKGROUND: The authors evaluated the frequency of the carrier status of three ancestral colorectal neoplasm-associated mutations (APC:I1307K, BLM(Ash), and MSH2*1906G>C) found in the Jewish population among a case series with documented colorectal neoplasms. They further compared family and personal histories plus environmental exposures of the carriers and noncarriers of the I1307K mutation and examined clinical differences with regard to the colorectal neoplasms and the specific molecular genetic changes in these lesions. METHODS: Analyses were performed on tissue from stored paraffin-embedded blocks for the three germline mutations plus the KRAS mutation and APC loss of heterozygosity (LOH) and APC gene sequencing. RESULTS: Fifty-four of the 429 individuals (12.6%) were found to carry the APC:I1307K mutation, whereas 4 (0.9%) were found to be heterozygous for the BLM(Ash) mutation and 3 (0.7%) were carriers of the MSH21906G>C* mutation. Carriers of the I1307K mutation did not appear to differ from noncarriers with regard to the number of neoplasms, patient age at detection, or tumor location within the colon. There was no significant difference noted between I1307K carriers and noncarriers with regard to the percentage of patients with first-degree relatives with colorectal carcinoma. A significant risk for APC LOH was found in lesions from carriers who smoked cigarettes compared with nonsmokers. The I1307K mutation was found to be clearly associated with a somatic additional adenine insertion in the region of codons 1306-1309, but other mutations in the region of codons 1277-1348 were found to be no more prevalent in carriers than in noncarriers. CONCLUSIONS: In Jewish individuals previously diagnosed with a colorectal neoplasm, MSH2*1906G>C is uncommon but has been associated with carcinoma occurring at a young age. The BLM(Ash) mutation is uncommon and appears to be of little effect. The I1307K mutation is common among Jews who have had colorectal neoplasms, but overall it was found to have little effect clinically in the current study group. There may be a gene-environment interaction between the I1307K mutation and cigarette use.

The Adenomatous Polyposis Coli (APC) gene microsatellite marker D5S1385 is equally informative for loss of heterozygosity as the marker D5S346.

Molecular analyses for loss of heterozygosity (LOH) at a gene locus are used to identify genomic imbalance in tumor tissue. A frequently utilized microsatellite marker for the Adenomatous Polyposis Coli (APC) gene is denoted D5S346. However, when an individual has two identical forms of this microsatellite, then a second microsatellite, also near the APC gene, is needed. We present data on an APC microsatellite marker designated D5S1385, located on the 5' end of the APC gene, and we compare these to data utilizing the marker D5S346. Polymerase chain reaction (PCR) was used, followed by gel electrophoresis. Homozygosity for the marker D5S1385 was present in 55 individuals, or 28%. Thirty-seven carcinomas and 32 adenomas were assessed for LOH from individuals informative with both microsatellite markers. D5S1385 detected LOH in 39 of 41 lesions, or 95%, for which D5S346 detected LOH. D5S1385 is moderately polymorphic and is informative at least as often as D5S346.

The characterization of somatic APC mutations in colonic adenomas and carcinomas in Ashkenazi Jews with the APC I1307K variant using linkage disequilibrium.

Mutations of the adenomatous polyposis coli (APC) gene play a critical role in the development of colorectal neoplasms. A novel mechanism involves a germline variant, at codon 1307 of the APC gene. The mutation is thought to create an unstable segment of DNA, which facilitates the development of somatic mutations. I1307K has been shown to be ancestral in Middle Eastern populations. The aim of the present study was to confirm this observation for a Western population and to utilize this information in the characterization of the somatic changes in colorectal neoplasia in carriers of I1307K. DNA from 182 US Ashkenazim was screened for the I1307K variant, which was found in 22 (12.1%), and the ancestral nature of this variant was confirmed in this population by showing that all of those with the I1307K variant carried a specific allele at the D5S346 locus, while the majority shared a D5S1385 allele. Subsequently, 79 neoplasms were analysed from 15 I1307K carriers for loss of heterozygosity (LOH) at the D5S346 locus. LOH was detected in 18 neoplasms (23%). Of these 18, four neoplasms showed loss of the I1307K-associated allele, while 14 neoplasms had retained the I1307K-associated allele and, by implication, the I1307K variant. PCR products were also cloned into a plasmid vector to isolate individual APC alleles. For those neoplasms with LOH, 13 of the 18 neoplasms (72%) had a somatic mutation, of which 12 involved the I307K-bearing chromosome. This study is consistent with two previous studies in showing that additional somatic mutations close to codon 1307 are almost always on the I1307K-bearing chromosome, but either allele may demonstrate LOH. Further evidence for the interpretation of the action of I1307K as producing DNA instability is provided by analysing multiple neoplasms from the same person and by showing that these neoplasms have differing patterns of LOH and associated somatic mutations.