Cristina Albuquerque

BRAF , KRAS and PIK3CA mutations in colorectal serrated polyps and cancer: Primary or secondary genetic events in colorectal carcinogenesis?

BackgroundBRAF, KRAS and PIK3CA mutations are frequently found in sporadic colorectal cancer (CRC). In contrast to KRAS and PIK3CA mutations, BRAF mutations are associated with tumours harbouring CpG Island methylation phenotype (CIMP), MLH1 methylation and microsatellite instability (MSI). We aimed at determine the frequency of KRAS, BRAF and PIK3CA mutations in the process of colorectal tumourigenesis using a series of colorectal polyps and carcinomas. In the series of polyps CIMP, MLH1 methylation and MSI were also studied.MethodsMutation analyses were performed by PCR/sequencing. Bisulfite treated DNA was used to study CIMP and MLH1 methylation. MSI was detected by pentaplex PCR and Genescan analysis of quasimonomorphic mononucleotide repeats. Chi Square test and Fishers Exact test were used to perform association studies.ResultsKRAS, PIK3CA or BRAF occur in 71% of polyps and were mutually exclusive. KRAS mutations occur in 35% of polyps. PIK3CA was found in one of the polyps. V600E BRAF mutations occur in 29% of cases, all of them classified as serrated adenoma. CIMP phenotype occurred in 25% of the polyps and all were mutated for BRAF. MLH1 methylation was not detected and all the polyps were microsatellite stable. The comparison between the frequency of oncogenic mutations in polyps and CRC (MSI and MSS) lead us to demonstrate that KRAS and PIK3CA are likely to precede both types of CRC. BRAF mutations are likely to precede MSI carcinomas since the frequency found in serrated polyps is similar to what is found in MSI CRC (P = 0.9112), but statistically different from what is found in microsatellite stable (MSS) tumours (P = 0.0191).ConclusionOur results show that BRAF, KRAS and PIK3CA mutations occur prior to malignant transformation demonstrating that these oncogenic alterations are primary genetic events in colorectal carcinogenesis. Further, we show that BRAF mutations occur in association with CIMP phenotype in colorectal serrated polyps and verified that colorectal serrated polyps and MSI CRC show a similar frequency of BRAF mutations. These results support that BRAF mutations harbour a mild oncogenic effect in comparison to KRAS and suggest that BRAF mutant colorectal cells need to accumulate extra epigenetic alterations in order to acquire full transformation and evolve to MSI CRC.

SomaticApc mutations are selected upon their capacity to inactivate the ?-catenin downregulating activity

The APC gene, originally identified as the gene for familial adenomatous polyposis (FAP), is now considered as the true “gatekeeper” of colonic epithelial proliferation. Its main tumor suppressing activity seems to reside in the capacity to properly regulate intracellular β‐catenin signaling. Most somatic APC mutations are detected between codons 1286 and 1513, the mutation cluster region (MCR). This clustering can be explained either by the presence of mutation‐prone sequences within the MCR, or by the selective advantage provided by the resulting truncated polypeptides. Here, a Msh2‐deficient mouse model (Msh2Δ7N ) was generated and bred with Apc1638N and ApcMin that allowed the comparison of the somatic mutation spectra along the Apc gene in the different allelic combinations. Mutations identified in Msh2Δ7N/Δ7N tumors are predominantly dinucleotide deletions at simple sequence repeats leading to truncated Apc polypeptides that partially retain the 20 a.a. β‐catenin downregulating motifs. In contrast, the somatic mutations identified in the wild type Apc allele of Msh2Δ7N/Δ7N /Apc+/1638N and Msh2Δ7N/Δ7N /Apc+/Min tumors are clustered more to the 5′ end, thereby completely inactivating the β‐catenin downregulating activity of APC. These results indicate that somatic Apc mutations are selected during intestinal tumorigenesis and that inactivation of the β‐catenin downregulating function of APC is likely to represent the main selective factor.

BAT-26 identifies sporadic colorectal cancers with mutator phenotype: a correlative study with clinico-pathological features and mutations in mismatch repair genes

Microsatellite instability (MSI) is present in most colorectal cancers (CRC) associated with hereditary nonpolyposis colorectal cancer (HNPCC). MSI testing in so‐called sporadic forms of CRC may become a useful tool in identifying new HNPCC kindred. The aim of this study was to analyse the utility of BAT‐26 as a marker to identify CRCs with MSI and to investigate whether sporadic CRCs with MSI have a phenotypic expression similar to HNPCC cases. MSI was detected using two methods, an association of 7 poly(CA) repeats and a poly(A) repeat alone, BAT‐26, in a series of 62 patients with apparently sporadic forms of CRC. Germ‐line and somatic mutations in the hMSH2, hMLH1, and hMSH6 genes were analysed in patients with MSI+ tumours. Patients with MSI+ at poly(CA) loci and at BAT‐26 were younger (p = 0·024 and p = 0·002), had tumours more frequently right sided (p = 0·017 and p = 0·0001) and more often mucinous (p = 0·037 and p = 0·005, respectively) than patients with MSI negative tumours. Mutation analysis allowed the identification of two patients carrying germ‐line mutations in the hMLH1 gene (both were BAT‐26+) and two other patients who had somatic mutation in the hMSH2 and in hMLH1 genes. In conclusion, the detection of MSI using poly(CA) repeats or BAT‐26 alone allowed the identification of a subset of patients with clinico‐pathological characteristics similar to those associated to HNPCC. BAT‐26 has the advantage of being a simple and less expensive method that might be used as a screening procedure before mutation analysis. Copyright

APC or MUTYH mutations account for the majority of clinically well-characterized families with FAP and AFAP phenotype and patients with more than 30 adenomas

Patients presenting familial adenomatous polyposis (FAP), attenuated familial adenomatous polyposis (AFAP) or multiple colorectal adenomas (MCRAs) phenotype are clinically difficult to distinguish. We aimed to genetically characterize 107 clinically well‐characterized patients with FAP‐like phenotype, and stratified according to the recent guidelines for the clinical management of FAP: FAP, AFAP, MCRA (10–99 colorectal adenomas) without family history of colorectal cancer or few adenomas (FH), MCRA (10–99) with FH, MCRA (3–9) with FH. Overall, APC or MUTYH mutations were detected in 42/48 (88%), 14/20 (70%) and 10/38 (26%) of FAP, AFAP and MCRA patients, respectively. APC and MUTYH mutations accounted for 81% and 7% of FAP patients and for 30% and 40% of AFAP patients, respectively. Notably, MCRA patients did not present APC mutations. In 26% of these patients, an MUTYH mutation was identified and the detection rate increased with the number of adenomas, irrespectively of family history, being significantly higher in MCRA patients presenting more than 30 adenomas [7/12 (58%) vs 2/14 (14%), p = 0.023]. We validate the recently proposed guidelines in our patients cohort and show that APC or MUTYH germline defects are responsible for the majority of clinically well‐characterized patients with FAP and AFAP phenotype, and patients with more than 30 colorectal adenomas. The different mutation frequencies according to family history and to the number of adenomas underscore the importance of an adequate familial characterization, both clinically and by colonoscopy, in the management of FAP‐like phenotypes. The phenotypes of the mutation‐negative patients suggest distinct etiologies in these cases.

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.

Colorectal cancers choosing sides

In contrast to the majority of sporadic colorectal cancer which predominantly occur in the distal colon, most mismatch repair deficient tumours arise at the proximal side. At present, these regional preferences have not been explained properly. Recently, we have screened colorectal tumours for mutations in Wnt-related genes focusing specifically on colorectal location. Combining this analysis with published data, we propose a mechanism underlying the side-related preferences of colorectal cancers, based on the specific acquired genetic defects in β-catenin signalling.

Pathogenicity of missense and splice site mutations in hMSH2 and hMLH1 mismatch repair genes: implications for genetic testing

Background: In hereditary non-polyposis colorectal cancer, over 90% of the identified mutations are in two genes, hMSH2 and hMLH1. A large proportion of the mutations detected in these genes are of the missense type which may be either deleterious mutations or harmless polymorphisms. Aim: To investigate whether nine missense and one splice site mutation of hMLH1 and hMSH2, in 10 kindreds with a familial history of colorectal cancer or young age of onset, could be interpreted as pathogenic. Methods: Clinical and genetic characteristics were collected: (i) evolutionary conservation of the codon involved; (ii) type of amino acid change; (iii) occurrence of mutation in healthy controls; (iv) cosegregation of mutation with disease phenotype; (v) functional consequences of gene variant; and (vi) microssatellite instability and immunoexpression of hMSH2 and hMLH1 analysis. Results: Seven different missense and one splice site mutation were identified. Only 1/8 was found in the control group, 2/7 occurred in conserved residues, and 5/7 resulted in non-conservative changes. Functional studies were available for only 2/8 mutations. Segregation of the missense variant with disease phenotype was observed in three kindreds. Conclusion: In the majority of families included, there was no definitive evidence that the missense or splice site alterations were causally associated with an increased risk of developing colorectal cancer. Until further evidence is available, these mutational events should be regarded and interpreted carefully and genetic diagnosis should not be offered to these kindreds.

Validation and simplification of Bethesda guidelines for identifying apparently sporadic forms of colorectal carcinoma with microsatellite instability.

Colorectal tumors with microsatellite instability (MSI) that do not comply with previously defined clinical criteria may be found in recently diagnosed hereditary nonpolyposis colorectal carcinoma families. Until recently, the indications for MSI testing were not clearly established. The objective of the current study was to validate the recently published Bethesda guidelines for MSI testing in a series of patients with apparently sporadic forms of colorectal carcinoma (CRC).

Losses of Heterozygosity on Chromosomes 9p and 17p Are Frequent Events in Barrett’s Metaplasia Not Associated With Dysplasia or Adenocarcinoma

OBJECTIVE:Losses of heterozygosity (LOH) on chromosomes 9p and 17p frequently accompany malignant transformation of Barrett’s esophagus (BE). They have been reported in adenocarcinoma, dysplasia, and adjacent metaplasia of patients with long-segment BE (LSBE). This study aimed to evaluate and compare the frequency of LOH on 9p and 17p in patients with long- and short-segment BE (SSBE) without dysplasia or adenocarcinomaMETHODS:Matched metaplasia and blood DNA were evaluated for LOH on chromosomes 9p and 17p in patients with a previous diagnosis of BE and no dysplasia or cancer.RESULTS:We included 18 patients (12 long-segment BE and six short-segment BE). The overall prevalence of LOH was 61% (10 of 18), with no significant difference between LSBE (58%) and SSBE (50%). The frequencies of LOH on 9p and 17p were similar (35% and 39%, respectively), with 18% of the patients showing losses at both chromosomes.CONCLUSIONS:LOH on 9p and 17p are highly frequent events in BE, even in the absence of dysplasia and adenocarcinoma. The presence of these abnormalities in non-neoplastic epithelium suggests they might be useful markers for risk stratification within endoscopic surveillance programs.

Aggressive phenotype of MYH-associated polyposis with jejunal cancer and intra-abdominal desmoid tumor: report of a case.

ABSTRACT: MYH-associated polyposis is an inherited autosomal recessive disease, linked to biallelic germline MYH mutations, which predisposes to the development of multiple colorectal adenomas and cancer. The colonic and extracolonic phenotype of this syndrome is very heterogeneous. We report the case of a young male patient with an aggressive MYH-associated polyposis phenotype. He presented at aged 30 years with more than 100 colonic polyps and 4 colonic adenocarcinomas. At aged 35 years, Spigelman Stage IV duodenal adenomatosis was detected. When he was 39 years old, he developed three synchronous jejunal adenocarcinomas and a mesenteric desmoid tumor. Based on this report, we believe that screening of the entire small bowel should be recommended in MYH-associated polyposis patients, especially in those with duodenal adenomas. Similar to patients with familial adenomatous polyposis, desmoid tumors also may be part of the clinical spectrum of MYH-associated polyposis and may prove to be a significant clinical problem in patients submitted to prophylactic colectomy.