Analysis of somatic molecular changes, clinicopathological features, family history, and germline mutations in colorectal cancer families: evidence for efficient diagnosis of HNPCC and for the existence of distinct groups of non-HNPCC families

Objective: To analyse somatic molecular changes, clinicopathological features, family history, and germline mutations in families with colorectal cancer (CRC). Methods: Molecular changes (K-ras and ß-catenin mutations, chromosome 18q allele loss (LOH), APC LOH, microsatellite instability (MSI), and expression of ß-catenin and p53) were examined in four series of CRC patients with proven or probable hereditary disease: hereditary non-polyposis colon cancer (HNPCC); MYH associated polyposis (MAP); multiple (>5) colorectal adenomas without familial adenomatous polyposis (FAP); and other families/cases referred to family cancer clinics (FCC series). HNPCC was diagnosed using a combination of germline mutation screening and tumour studies. A series of unselected CRC patients was also studied. Results: There was overlap between genetic pathways followed by each type of CRC, but significant differences included: increased frequency of K-ras mutation and reduced frequency of APC LOH in cancers from MAP, but not from multiple adenoma patients; reduced frequency of LOH in HNPCC CRCs; and increased MSI in CRCs from HNPCC, but not from FCC or multiple adenoma patients. HNPCC was apparently detected efficiently by combined germline and somatic analysis. Cancers from the FCC, unselected, and multiple adenoma series shared similar molecular characteristics. In the FCC and multiple adenoma series, hierarchical cluster analysis using the molecular features of the cancers consistently identified two distinct groups, distinguished by presence or absence of K-ras mutation. Conclusions: While K-ras mutation status is known to differentiate hereditary bowel cancer syndromes such as MAP and FAP, it may also distinguish groups of non-HNPCC, FCC patients whose disease has different, as yet unknown, genetic origins.     

Mutations of the APC adenomatous polyposis coli) gene

Several investigators have reported germline mutations of the APC gene in patients with familial adenomatous polyposis (FAP) as well as somatic mutations in tumors developed in digestive organs (stomach, pancreas, colon, and rectum). Those results provide evidence that inactivation of the APC gene plays a significant role in FAP and in sporadic tumors of these tissues. APC mutations have led to some interesting observations. First, the great majority of the mutations found to date would result in truncation of the APC product. Second, almost all the mutations have occurred within the first half of the coding sequence, and somatic mutations in colorectal tumors are further clustered in a particular region called MCR (mutation cluster region). Third, most identified point mutations in the APC gene are transitions from cytosine to other nucleotides. Fourth, the location of germ-line mutations tends to correlate with the number of colorectal polyps in FAP patients. Furthermore, inactivation of both alleles of the APC gene seems to be required as an early event to develop most of adenomas and carcinomas in the colon and rectum as well as some of those in the stomach. © 1993 Wiley-Liss, Inc.     

Detection of APC mutations by a yeast-based protein truncation test (YPTT)

APC gene mutations play a role in the initiation step of colorectal carcinogenesis in both familial adenomatous polyposis (FAP) and non-FAP patients. Almost all of the APC mutations are nonsense or frameshift mutations, which truncate the APC protein and are thought to inactivate normal APC function. We show a novel method for detecting nonsense and frameshift APC gene mutations by using Saccharomyces cerevisiae. Polymerase chain reaction (PCR)-amplified APC fragments are cloned directly into yeast expression vectors in vivo, and the yeast expresses a hemagglutinin epitope (HA)-tagged APC peptide. When an APC fragment contains a nonsense or frameshift mutation, HA-tagged truncating APC peptide can be detected by Western blotting using an anti-HA antibody. We identified both germ-line and somatic APC mutations in patients with FAP and non-FAP colorectal tumors, respectively. This method, called the yeast-based protein truncation test (YPTT), is simple and fairly cheap, and it can be applied to any genes that are inactivated by protein truncating mutations. Genes Chromosomes Cancer 21:290–297, 1998. © 1998 Wiley-Liss, Inc.     

Ovarian microcystic stromal tumor: A novel extracolonic tumor in familial adenomatous polyposis

Ovarian microcystic stromal tumor (MCST) is a very rare neoplasm; hence, its nomenclature was recently designated as “Distinctive morphologic and immunohistochemical features” in 2009. Its exact origin, etiological genetic alterations, and background are not yet clearly known. Familial adenomatous polyposis (FAP) is an autosomal dominant disease that leads to development of colorectal polyps via germ‐line mutations of the APC gene on chromosome 5q21～22. In this study, we report a 40‐year‐old female patient who had ovarian MCST and FAP. On sequencing the APC gene in ovarian MSCT, we detected a novel somatic mutation of the APC gene in exon 11, with a heterozygous deletion at nucleotide position c.1540delG (p.Ala514 Profs*9). Mutations of β‐catenin (CTNNB1) and FOXL2 were not detected. Although one case demonstrating involvement of Wnt/β‐catenin in ovarian MCST associated with FAP has been presented previously, no detailed information was provided. Thus, this is the ovarian MCST with a somatic mutation of APC in a patient with FAP. © 2015 Wiley Periodicals, Inc.     

A β-catenin mutation in a sporadic colorectal tumor of the RER phenotype and absence of β-catenin germline mutations in FAP patients

As a signaling protein in the Wnt pathway β-catenin plays a crucial role in the regulation of cellular proliferation. Recently, oncogenic β-catenin mutations were described in human colorectal cancer and melanoma cell lines. Since activating mutations in the β-catenin gene have similar effects on the biochemical level as inactivating mutations in the tumor suppressor gene APC, it is speculated that β-catenin mutations may substitute APC gene inactivation in carcinogenesis. To address this question we analyzed twenty-three sporadic colorectal tumors of different progression states for mutations in the β-catenin gene. Eighteen of these tumors showed the wildtype APC gene sequence. In only one of the tumors with wildtype APC a β-catenin gene mutation was found. This tumor was of the RER (replication error) phenotype which may explain the finding that the mutation occurred in a sequential repeat motif of the β-catenin gene. The second aim of this study was to investigate whether differences in the phenotypic variability in FAP (familial adenomatous polyposis coli) might be due to inherited alterations in the β-catenin gene. For this we analyzed DNA from fourteen FAP patients from eight different families for germline mutations in the β-catenin gene. We did not find any β-catenin gene alteration in these samples. Our results indicate that somatic β-catenin activating mutations contribute only to a minor part of human colorectal tumors and that germline β-catenin mutations do not play a role in the variability of symptoms in FAP. Genes Chromosomes Cancer 22:37–41, 1998. © 1998 Wiley-Liss, Inc.     

APC promoter 1B deletion in seven American families with familial adenomatous polyposis

Familial adenomatous polyposis (FAP) is a colorectal cancer predisposition syndrome caused by mutations in the adenomatous polyposis coli (APC) gene. Clinical genetic testing fails to identify disease causing mutations in up to 20% of clinically apparent FAP cases. Following the inclusion of multiplex ligation‐dependent probe amplification (MLPA) probes specific for APC promoter 1B, seven probands were identified with a deletion of promoter 1B. Using haplotype analysis spanning the APC locus, the seven families appear to be identical by descent from a common founder. The clinical phenotype of 19 mutation carriers is classical FAP with colectomy at an average age of 24. The majority of cases had a large number of duodenal and gastric polyps. Measurements of allele‐specific expression of APC mRNA using TaqMan assay confirmed that relative expression in the allele containing the promoter 1B deletion was reduced 42–98%, depending on tissue type. This study confirms the importance of APC promoter deletions as a cause of FAP and identifies a founder mutation in FAP patients from the United States.     

MUTYH‐associated polyposis – variability of the clinical phenotype in patients with biallelic and monoallelic MUTYH mutations and report on novel mutations

Morak M, Laner A, Bacher U, Keiling C, Holinski‐Feder E. MUTYH‐associated polyposis – variability of the clinical phenotype in patients with biallelic and monoallelic MUTYH mutations and report on novel mutations. To further characterize 215 APC mutation‐negative patients with colorectal neoplasias classified in classical, attenuated, or atypical familial adenomatous polyposis (FAP) coli we performed mutation screening in the Mut Y homologue (MUTYH) gene. The incidence was 15% for biallelic and 3.7% for monoallelic MUTYH mutations. We describe six novel MUTYH mutations in biallelic constellation and two novel monoallelic missense mutations. Of 33 MUTYH‐associated polyposis coli (MAP) patients 57% were attenuated familial adenomatous polyposis (AFAP) patients, 10% display early‐onset classical FAP and 18% had only few adenomas at higher age. Biallelic cases had a high incidence of extracolonic polyposis in 32% and colorectal cancer (CRC) in 33% of the cases. The clinical picture of MAP ranged from classical FAP or synchronous CRC at age 30 years to few adenomas at age 54 years without evidence of CRC, initially suspected for hereditary non‐polyposis colorectal cancer (HNPCC). The mean age of onset was 43 years, with 11 (33%) patients being younger than 40 years of age, indicating that the clinical manifestation can be earlier than so far reported. Monoallelic MUTYH mutation carriers had a positive family history in seven of eight cases allowing the hypothesis of a disease‐causing synergism of MUTYH mutations with other genes.     

Clonal overexpression of metallothionein is induced by somatic mutation in morphologically normal colonic mucosa

Metallothionein (MT) overexpression occurs frequently in human tumours but the underlying mechanism is unknown. Morphologically normal-appearing mucosa from human colorectal carcinoma resection specimens and of the colons of ageing laboratory mice contains scattered single crypts whose cells show uniformly increased MT immunostaining, suggesting that MT overexpression arises directly from random crypt stem cell somatic mutation, followed by colonization of the clonal unit by the mutated progeny. This hypothesis has now been tested by quantifying the frequency of immunocytochemically detectable monocryptal colorectal MT overexpression, 5 and 30 days after injection of 8-week-old mice with a single dose of the mutagen dimethylhydrazine (DMH, 30 mg/kg subcutaneous). Otherwise normal-appearing MT-positive crypts were recorded as either wholly or partially involved by the overexpressing phenotype. Five days after DMH injection, the median frequency of partially involved MT-positive crypts was 11·7×10declining significantly to 1·8×10⁻⁴ at 30 days (Mann–Whitney U, P<0·01). In contrast, the median frequency of wholly involved crypts was 0·2×10⁻⁴ at 5 days, increasing significantly (P<0·005) to 12·9×10⁻⁴ at 30 days. The frequency of MT-positive crypts and the time course of evolution of partially involved to wholly involved forms were similar to those described for mutation-induced crypt-restricted loss of glucose-6-phosphate dehydrogenase activity in mice treated with an identical DMH regimen. The findings indicate that cellular MT overexpression can occur as a direct consequence of somatic mutation, either cis-activating mutation(s) of the MT gene itself, or trans-activating mutation(s) of other genes involved in controlling MT expression. This is likely to be an important mechanism underlying MT overexpression in neoplasia. Such mutation-induced aberrant MT expression may be involved in the acquisition of selective cellular growth or survival advantage during tumour progression. © 1998 John Wiley & Sons, Ltd.     

Defects in mismatch repair occur after APC mutations in the pathogenesis of sporadic colorectal tumours

The roles of the intrinsic mutation rate and genomic instability in tumorigenesis are currently controversial. In most colorectal tumours, it is generally supposed that the first mutations occur at the adenomatous polyposis coli (APC) locus; APC mutations are thought to provide cells with a selective advantage but have no known effect on the mutation rate. It has also been suggested that genomic instability is the initiating event in colorectal tumorigenesis and, if this is true, mutations of DNA mismatch repair (MMR) genes (or at similar loci) are the most likely candidates. If defective MMR precedes APC mutations, the APC mutations of colon tumours with defective MMR and hence replication errors (RER+) should differ from those of RER- tumours, in at least three specific ways: (1) a higher frequency of allele loss at APC in RER- tumours; (2) more frameshift than nonsense mutations in RER+ tumours; and (3) APC mutations in simple repeat sequences [(N)_n, (N₁N₂)_n, or (N₁N₂N₃)_n] in RER+ tumours. We found no evidence that sporadic RER+ and RER- colon cancers (including cell lines) differ in any of these three ways. Although it remains possible that MMR is abnormal in tumours from HNPCC families before APC mutations occur, it is likely that in sporadic colon tumours, APC mutations, rather than genomic instability, are the initiating events in tumorigenesis. Hum Mutat 11:114–120, 1998. © 1998 Wiley-Liss, Inc.     

Adenoma development in familial adenomatous polyposis and MUTYH‐associated polyposis: somatic landscape and driver genes

Familial adenomatous polyposis (FAP) and MUTYH‐associated polyposis (MAP) are inherited disorders associated with multiple colorectal adenomas that lead to a very high risk of colorectal cancer. The somatic mutations that drive adenoma development in these conditions have not been investigated comprehensively. In this study we performed analysis of paired colorectal adenoma and normal tissue DNA from individuals with FAP or MAP, sequencing 14 adenoma whole exomes (eight MAP, six FAP), 55 adenoma targeted exomes (33 MAP, 22 FAP) and germline DNA from each patient, and a further 63 adenomas by capillary sequencing (41 FAP, 22 MAP). With these data we examined the profile of mutated genes, the mutational signatures and the somatic mutation rates, observing significant diversity in the constellations of mutated driver genes in different adenomas, and loss‐of‐function mutations in WTX (9%; p < 9.99e‐06), a gene implicated in regulation of the WNT pathway and p53 acetylation. These data extend our understanding of the early events in colorectal tumourigenesis in the polyposis syndromes. © 2015 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Colorectal cancers show distinct mutation spectra in members of the canonical WNT signaling pathway according to their anatomical location and type of genetic instability

It is unclear whether the mutation spectra in WNT genes vary among distinct types of colorectal tumors. We have analyzed mutations in specific WNT genes in a cohort of 52 colorectal tumors and performed a meta‐analysis of previous studies. Notably, significant differences were found among the mutation spectra. We have previously shown that in familial adenomatous polyposis, APC somatic mutations are selected to provide the “just‐right” level of WNT signaling for tumor formation. Here, we found that APC mutations encompassing at least two β‐catenin down‐regulating motifs (20 a.a. repeats) are significantly more frequent in microsatellite unstable (MSI‐H) than in microsatellite stable (MSS) tumors where truncations retaining less than two repeats are more frequent (P = 0.0009). Moreover, in cases where both APC hits are detected, selection for mutations retaining a cumulative number of two 20 a.a. repeats became apparent in MSI‐H tumors (P = 0.001). This type of mutations were also more frequent in proximal versus distal colonic tumors, regardless of MSI status (P = 0.0008). Among MSI‐H tumors, CTNNB1 mutations were significantly more frequent in HNPCC than in sporadic lesions (28% versus 6%, P < 10‐6) and were preferentially detected in the proximal colon, independently of MSI status (P = 0.017). In conclusion, the observed spectra of WNT gene mutations in colorectal tumors are likely the result from selection of specific levels of β‐catenin signaling, optimal for tumor formation in the context of specific anatomical locations and forms of genetic instability. We suggest that this may underlie the preferential location of MMR deficient tumors in the proximal colon. © 2010 Wiley‐Liss, Inc.     

No difference in stem cell somatic mutation between the background mucosa of right- and left-sided sporadic colorectal carcinomas

Epidemiological, morphological, and molecular differences exist between carcinomas of the right and left sides of the large bowel. To investigate whether this is reflected in differences in somatic mutation frequency in the background mucosa, mutation of the neutral O-acetyltransferase gene (oat) was quantified in histologically normal resection margins from 20 informative (heterozygous) patients with caecal or ascending colon cancer (11 males, median age 75 years) and 20 with sigmoid colon or rectal cancer (10 males, median age 70 years). Mutant discordant crypts lacking O-acetyltransferase activity were visualized by mPAS staining and classified as wholly or partially involved by the mutant phenotype; median frequencies (×10⁻⁴) were compared (Mann–Whitney U-test) after assessing a sample of more than 10 000 crypts per case. No significant difference was found between the frequencies of wholly involved mPAS-positive crypts in background mucosa of left- and right-sided cancers (p=0·4569), indicating that tumours on both sides of the colon are associated with similar levels of lifetime-accumulated stem cell mutational load. However, partially involved mPAS-positive crypts were significantly more frequent in mucosa from left-sided cancers (p<0·04), indicating increased mutational activity during the previous 12 months. Analysis of mucosa proximal and distal to left-sided cancers showed that this increase was due to a statistically higher frequency of partially involved crypts in proximal mucosa, which probably resulted from the obstructive effects of the tumour causing increased exposure of the proximal mucosa to luminal carcinogens and/or epithelial regeneration in response to low-grade inflammation or ischaemia. The findings indicate that although left-sided colonic cancer is commoner than right-sided cancer in the British population, carcinomas on both sides of the large bowel arise in a background of similar levels of stem cell mutational activity.     

Promoter‐specific alterations of APC are a rare cause for mutation‐negative familial adenomatous polyposis

In familial adenomatous polyposis (FAP), 20% of classical and 70% of attenuated/atypical (AFAP) cases remain mutation‐negative after routine testing; yet, allelic expression imbalance may suggest an APC alteration. Our aim was to determine the proportion of families attributable to genetic or epigenetic changes in the APC promoter region. We studied 51 unrelated families/cases (26 with classical FAP and 25 with AFAP) with no point mutations in the exons and exon/intron borders and no rearrangements by multiplex ligation‐dependent probe amplification (MLPA, P043‐B1). Promoter‐specific events of APC were addressed by targeted resequencing, MLPA (P043‐C1), methylation‐specific MLPA, and Sanger sequencing of promoter regions. A novel 132‐kb deletion encompassing the APC promoter 1B and upstream sequence occurred in a classical FAP family with allele‐specific APC expression. No promoter‐specific point mutations or hypermethylation were present in any family. In conclusion, promoter‐specific alterations are a rare cause for mutation‐negative FAP (1/51, 2%). The frequency and clinical correlations of promoter 1B deletions are poorly defined. This investigation provides frequencies of 1/26 (4%) for classical FAP, 0/25 (0%) for AFAP, and 1/7 (14%) for families with allele‐specific expression of APC. Clinically, promoter 1B deletions may associate with classical FAP without extracolonic manifestations. © 2014 Wiley Periodicals, Inc.     

Human colonic stem cell mutation frequency with and without irradiation

Mild periodic acid-Schiff (mPAS) staining distinguishes O-acetylated from non-O-acetylated sialoglycoproteins. In human colonic mucosa, individuals possess one of three phenotypes: uniformly mPAS-positive (non-O-acetylated), uniformly mPAS-negative (O-acetylated), and negative with infrequent scattered positive crypts. This is due to a polymorphism in a single autosomal gene (oat). Discordant crypts have not been found in children's colons, suggesting that they result from somatic mutation in heterozygous individuals. We now present evidence to confirm this based on a study of radiation-induced changes. Comparison of mPAS staining of large intestinal mucosa from patients given radiation 4 weeks before surgery for carcinoma of the rectum with matched controls receiving surgery alone showed a similar phenotype distribution, but informative irradiated patients showed an increased frequency of discordant crypts (irradiated vs. non-irradiated 14·5 ± 8·2 × 10^?4 vs. 6·1 ± 4·2 × 10^?4). When these were classified as wholly or partially involved by the aberrant phenotype, the increase was most marked in partially involved crypts (7·5 ± 4·5 × 10^?4 vs. 0·3 ± 0·5 × 10^?4, Mann-Whitney U, P<0·005). Two patients receiving radiotherapy many years before colectomy showed a very high total discordant crypt frequency but relatively few partially affected crypts. Studies of somatic mutation in colonic or small intestinal crypts following a single dose of mutagen in mice have shown early partial crypt involvement by the mutated phenotype and later complete crypt involvement. The demonstration in man that recent radiation greatly increases the frequency of partially involved discordant crypts while long-term radiation greatly increases the frequency of wholly involved discordant crypts confirms that the discordant crypts result from stem cell mutation in individuals heterozygous for a polymorphic gene controlling O-acetylation of sialoglycoproteins. They show that the mPAS technique can be used to identify and quantify colonic stem cell somatic mutation in man.     

Common colorectal cancer risk alleles contribute to the multiple colorectal adenoma phenotype,but do not influence colonic polyposis in FAP

The presence of multiple (5–100) colorectal adenomas suggests an inherited predisposition, but the genetic aetiology of this phenotype is undetermined if patients test negative for Mendelian polyposis syndromes such as familial adenomatous polyposis (FAP) and MUTYH-associated polyposis (MAP). We investigated whether 18 common colorectal cancer (CRC) predisposition single-nucleotide polymorphisms (SNPs) could help to explain some cases with multiple adenomas who phenocopied FAP or MAP, but had no pathogenic APC or MUTYH variant. No multiple adenoma case had an outlying number of CRC SNP risk alleles, but multiple adenoma patients did have a significantly higher number of risk alleles than population controls (P=5.7 × 10⁻⁷). The association was stronger in those with ≥10 adenomas. The CRC SNPs accounted for 4.3% of the variation in multiple adenoma risk, with three SNPs (rs6983267, rs10795668, rs3802842) explaining 3.0% of the variation. In FAP patients, the CRC risk score did not differ significantly from the controls, as we expected given the overwhelming effect of pathogenic germline APC variants on the phenotype of these cases. More unexpectedly, we found no evidence that the CRC SNPs act as modifier genes for the number of colorectal adenomas in FAP patients. In conclusion, common colorectal tumour risk alleles contribute to the development of multiple adenomas in patients without pathogenic germline APC or MUTYH variants. This phenotype may have ‘polygenic'' or monogenic origins. The risk of CRC in relatives of multiple adenoma cases is probably much lower for cases with polygenic disease, and this should be taken into account when counselling such patients.