|Year : 2014 | Volume
| Issue : 3 | Page : 591-596
Effect of β-catenin alterations in the prognosis of patients with sporadic colorectal cancer
Sara Rafael, Silvia Veganzones, Marta Vidaurreta, Virginia de la Orden, Maria Luisa Maestro
Department of Genomics, Hospital Clínico San Carlos, Madrid, Spain
|Date of Web Publication||14-Oct-2014|
Maria Luisa Maestro
Department of Genomics, Hospital Clínico San Carlos, Martin Lagos s/n. Madrid
Source of Support: This study was supported by Fundación Mutua Madrileña, Spain, Conflict of Interest: None
Context: Wnt pathway activation represents a critical step in the etiology of most of colorectal cancer (CRC) and it is commonly due to mutations in the APC gene, which originates the loss of β-catenin regulatory function. It has been suggested that APC inactivation or β-catenin alteration have similar effects in tumor progression in CRC tumorigenesis.
Aims: The aim of this study was to analyze the frequency of β-catenin gene mutation in patients with sporadic CRC and to determine its effect in prognosis.
Materials and Methods: This was a prospective cohort study, which included 345 patients with sporadic CRC. β-Catenin gene mutations in exon 3 were detected by single strand conformation polymorphism (SSCP). Exon 3 deletion was studied by identifying differences in fragment length of specific amplification products. All the altered samples were confirmed by direct sequencing.
Results: In our population, point mutations were detected in 1.8% of the samples and 4.9% of the samples showed deletion. We observed association between exon 3 mutations and increased levels of Carcinoenbryonic Antigen (CEA). In these patients, clinically relevant improvement in overall survival was also observed.
Conclusion: Frequency of point mutations in exon 3 β-catenin gene is low in our population. It would be interesting to increase the population size to test the clinically relevant influence in the prognosis found, and to test the relation of these events with Microsatellite Instabillity (MSI) pathway. If these findings were confirmed, β-catenin determination would help in the selection of patients with different prognosis.
Keywords: β-Catenin, colorectal cancer, mutations, prognosis
|How to cite this article:|
Rafael S, Veganzones S, Vidaurreta M, Orden Vd, Maestro ML. Effect of β-catenin alterations in the prognosis of patients with sporadic colorectal cancer. J Can Res Ther 2014;10:591-6
|How to cite this URL:|
Rafael S, Veganzones S, Vidaurreta M, Orden Vd, Maestro ML. Effect of β-catenin alterations in the prognosis of patients with sporadic colorectal cancer. J Can Res Ther [serial online] 2014 [cited 2020 Feb 26];10:591-6. Available from: http://www.cancerjournal.net/text.asp?2014/10/3/591/139161
| > Introduction|| |
Carcinogenesis is originated as a consequence of the accumulation of multiple genetic alterations. A sequence of alterations in different oncogenes and tumor suppressor genes has been described as the genetic model in the tumorogenesis in colorectal cancer (CRC).
Wnt pathway activation represents a critical step in the etiology of most of CRC and it is commonly due to mutations in the APC gene, which originates the loss of β-catenin regulatory function. 
APC protein interacts with a serine/threonine kinase (GSK-3β) to regulate β-catenin by phosphorylation in exon 3. This mechanism regulates the degradation and inactivation of β-catenin. ,,
Alterations in APC or β-catenin genes lead to β-catenin protein accumulation and loss of its regulatory activity. β-catenin protein has two main functions, regulation of cellular adhesion after binding to cadherin  and transcriptional activation of Wnt signaling pathway through key gene activation such as c-myc and MMP-7. ,
The main β-catenin gene alterations described are: Exon 3 deletions and serine and threonine residue mutations in this exon (codon 45, 41, 33, and 37). These residues are phosphorylated by the APC complex, and when β-catenin is mutated, the protein included in the APC complex is not degraded by the proteasome. β-Catenin mutations and APC gene mutations are mutually exclusive. Both mutations have been described in different CRC tumorogenic pathways. 
This study was conducted since it has been suggested that APC inactivation or β-catenin alteration have similar effects as Wnt pathway activation in tumor progression in CRC tumorigenesis. The objective of this study was to determine the β-catenin gene alterations in a wide series of sporadic CRC patients and to evaluate its prognostic significance.
| > MaterialS AND METHODS|| |
A minimum population size of 300 patients was calculated in accordance with previous literature frequencies and we established a recruiting period of 8 years. A total of 345 patients were recruited. Patients were consecutively operated on for primary CRC at the Surgery Department of our hospital between March 1995 and April 2003. It is a prospective cohort study. All patients were operated on by the same surgeon, performing a radical oncological surgery according to the tumor location. The surgery was defined as curative, when after re-section, there was no evidence of residual macroscopic tumor. According to this criterion, curative resection was performed in 285 patients (82.6%) and palliative resection in 60 patients (17.4%). Patients with metachronic tumors, hereditary polyposis, hereditary non-polyposis colorectal cancer, and bowel inflammatory disease were excluded. None of our patients had received neoadjuvant treatment. Informed consent of all patients was obtained prior to inclusion. This project was favorably evaluated by the hospital ethics and clinical research committee. The clinical follow-up of all patients was approved by the ethics and clinical research committee. CRC was stratified according to Dukes' stage. It was considered proximal colon when tumor was located in the right side and transverse colon and distal colon when the left colon and sigma were involved. Prognostic subgroups have been made in CRC patients with metastasis according to Kφhne classification. 
Prognosis factors stage II: 41.5% of our population received adjuvant chemotherapy and 1.2% adjuvant radiotherapy. 94.7% of the chemotherapy regime administered was based in 5-fuoruracyl and 5.3% in oxaliplatin.
Immediately after resection, the tumor and non-tumor tissue samples were immersed in liquid nitrogen and maintained at − 80°C. Anatomopathological analysis was performed by two independent pathologists especially for this study. All tumor samples had over 80% tumoral cells.
For DNA extraction, tumor tissue samples were incubated overnight at 50°C in lysis buffer (10 mM Tris-HCl, 1 mM EDTA, 100 mM NaCl, 1% SDS, 500 ug/mL proteinase K). DNA was isolated with phenol-chloroform and precipitated with ethanol.
β-Catenin gene exon 3 mutations were detected by single strand conformation polymorphism (SSCP). DNA was amplified in a 25-μL final volume which included 10 pmol of each primer (sense 5΄-CCAATCTACTAATGCTAATACTG-3΄, antisense 5΄-CTGCATTCTGACTTTCAGTAAGG-3΄),  dNTP 200 μM, Taq Polymerase 2 units (Roche) and 100 ng of DNA. Master mix was amplified, 40 cycles: 60 s at 94°C, 60 s at each primer annealing temperature and 45 s at 72°C. The amplified products were denaturalized and run in polyacrylamide gel 10%, 140 V for 50 min.
All the samples which showed different migration in the SSCP bands were sequenced with 3100-Avant Genetic Analyzer (Applier Biosystems, Foster City, CA, USA).
For exon 3 deletions screening, region between exon 2 and 4 was amplified using 100 ng of DNA in a final volume of 25 μL. The reaction mix had 10 pmol of each primer (sense 5΄-CCAGCGTGGACAATGGCTAC-3΄, antisense 5΄-TGAGCTCGAGTCATTGCATAC-3΄), [ 10 ] dNTP 200 μM and 2 units of Taq Polymerase (Roche). Amplification conditions were: One cycle of denaturation at 95°C for 5 min followed by 40 cycles of amplification: 30 s at 94°C, 45 s at 60°C and 1 min at 72°C. The amplified products were run in agarose gel 2% and stained with ethidium bromide.
Qualitative variables were described with their corresponding frequency distribution. Quantitative variables were expressed as mean, standard deviation (SD), and range. The age variable was recoded into two groups according to the median (71 years old). Association between qualitative variables was analyzed with χ2 test and in the case that more than 25% of the expected frequencies were less than five, with Fisher's exact test.
In overall survival (OS), an event was defined as death occurring as a consequence of the tumor, excluding live patients and deaths due to another cause. OS was calculated as the time elapsed between the surgery date and the death or last follow-up. In disease-free survival (DFS) an event was defined as the diagnosis of locoregional recurrent disease or distant recurrent disease in previously disease-free patients, only patients who underwent curative surgery were considered. DFS was calculated as the time elapsed between the surgery date and the first diagnosis of recurrence. OS and DFS functions were estimated using Kaplan-Meier method. Comparison of survival functions was performed with the Breslow exact test considering the median follow-up time as the reference point. It was adjusted to a Cox regression model of proportional risks. Relative risk (RR) was also calculated with a confidence interval of 95% (CI 95%). Variables included in this model using biological criteria were: Sex, age, Dukes' stage, tumor location, differentiation grade, histologic type, preoperative CEA, adjuvant treatment, prognostic factors in stages II and IV, and β-catenin gene alterations. Association between risks proportionality and presence of interactions were evaluated.
In all hypothesis contrasts, the null hypothesis was rejected with a type I error less than 0.05. The software used for this analysis was SSPS for Windows version 11.5.
| > Results|| |
Of the 345 patients included in this study, 53.3% were male and 46.7% females. Mean age was 69.9 years, SD 11.2, median 71.0 years, and range between 30 and 95 years old. With the cut-off point of 71 years, 48.1% of the patients were younger. Clinico-pathological variables of our population are shown in [Table 1]. 59.7% of tumors were located in the colon (104 in proximal colon and 102 in distal colon) and 40.3% (139 patients) in rectum. 8.1% of samples were mucinous adenocarcinomas and 91.9% adenocarcinomas. Differentiation grade could not be determined in 36 patients.
|Table 1: Clinico-pathological variables of 345 patients with colorectal cancer. Relationships of these variables with β-catenin alterations |
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In the study of β-catenin gene alterations, 1.8% of samples showed point mutation in exon 3 (6/335) 3 out 6 altered samples had codon 45 mutation. Exon 3 deletion was detected in 4.9% (17/345) of patients, in 70.6% of these cases, deletion was observed in both alleles (12/17). In our cohort, 6.3% of the patients had β-catenin gene alteration.
β-Catenin genetic alterations, exon 3 mutations and deletions, were not related to any of the clinic-pathological variables studied [Table 1], but they were associated with high CEA serum levels (P = 0.05).
Postsurgical evolution: Overall survival study
Median follow-up time in our study was 73 months (6 years), with a P 25 -P 75 interquartile range between 57.5 and 89.1 months. OS in our study population at 73 months was 62.1%. All survival analyses were referred to our median follow-up. During follow-up, 115 patients died as a consequence of the neoplasia. One patient was lost to follow-up.
OS univariate analysis is shown in [Table 2]. Patients with β-catenin exon 3 point mutation had 83% OS and those without any alteration had 61% (P = 0.29) OS. The RR of patients without mutations was 2.7 times higher (CI 95% =0.38-19.71). Exon 3 deletion did not have any relationship with survival (69% vs. 61%).
|Table 2: 73 months overall survival univariant analysis in relation with clinico-pathological variables of 345 patients with colorectal cancer |
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OS stratified analysis was performed to test whether β-catenin gene alterations influenced OS according to the different clinic-pathological subgroups. We observed that, although the relationship was not statistically significant, there was a better prognosis, with a clinically relevant tendency, in patients with mutation and adenocarcinoma histological type, colon tumors, stage C or moderately differentiated [Table 3].
|Table 3: 73 months overall survival stratified analysis of β -catenin gene alteration in relation to clinico-pathological variables of 345 patients with colorectal cancer |
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Regarding adjuvant treatment, when this study was stratified by stages, there was not a statistically significant difference between the group of patients who received adjuvant treatment and the group of patients who did not (data not included).
| > Discussion|| |
Nowadays, the aim of oncological research is to identify and characterize different genetic alterations involved in tumor development and its relationship with CRC patients' clinical evolution.
Aneuploidy is a common feature of many cancers. The aberrant centrosome numbers are common in many types of cancers including breast, lung, bone, pancreas, colorectal, prostate, head, and neck.  An alternative mechanism for tumorigenesis is based on the transformation and cellular progression model from adenoma to colorectal carcinoma proposed by Vogelstein. A cumulative number of molecular and genetic alterations such as APC, k-ras, and p53 are responsible for this progression.  It has been suggested that APC inactivation or β-catenin alteration have similar effects in tumor progression in CRC tumorigenesis. 
β-Catenin gene alterations frequency range between 0 and 16% in CRC according to the different published studies.  In our study, 6.3% of our patients had exon 3 alteration, 1.8% had point mutations and 4.9% had deletion. Deletion of the β-catenin gene is more common than point mutations.
Iwao et al. analyzed β-catenin gene alterations and found no somatic point mutation. However, they found exon 3 total or partial interstitial deletions in 7 tumors (12%).  They observed that short sequences of nucleotides in both extremes of each deletion are identical or complementary, indicating that these sequences can be involved in somatic reorganization. Kitaeva et al. observed 2% mutations (2/92 patients) and association with right colon and stage III. Both patients were alive and disease free 5 years later. They also determined MSI, finding association between mutations and RER phenotype.  These authors suggest that β-catenin mutations occurring in colon tumors can be associated with repairing gene defects.
Differences in the frequency of mutation may be due to the use of different techniques. , We selected a screening method more feasible for its use in patients' handling as a routine. This method has good proficiency in the detection of mutation in this gene  and in other genes.  Other important source of discrepancies may be the population composition, we only included sporadic tumor and we worked in a Mediterranean population. Some authors performed the β-catenin analysis in APC non-mutated tumors, a subpopulation with an expected increased mutational frequency in our gene.  Differences in the mutations involved in tumorigenesis have been demonstrated according to environmental factors. 
β-Catenin gene exon 3 codifies for the regulatory domain NH 2 -terminal of the protein (codons 29-45) where the substrate for GSK-3 β is located, and where it has been described activating mutations have been described previously. , In CRC tumor samples, mutations are preferentially located in codon 45, which blocks β-catenin degradation and produces nuclear protein accumulation.  In our study, 50% of point mutations were located in codon 45, which matches with the results published by other authors.
The aim of this work was also to assess the effect of β-catenin in the prognosis of CRC patients. We did not observe any relationship between β-catenin gene alterations and CRC patient evolution. Deletion of exon 3 does not imply differences in patient survival. However, we observed clinically relevant and better survival in those patients with exon 3 point mutation. Even though our study population is large, exon 3 mutations have low frequency and it is difficult to get statistically significant results.
Point mutations can be caused by the defects in the mismatch repair system genes, as suggested by Kitaeva et al. Previous studies have demonstrated that β-catenin mutations are more common in MSI-H tumors, related with DNA mismatch repair gene alterations. ,, According to Kim et al., all β-catenin point mutations are more frequent in proximal colon tumors, which are more frequently developed through MSI pathway.  Unlike point mutations, deletions may occur through another pathway and this would explain the differences in the behavior of tumors carrying different types of β-catenin alteration. 
Frequency of point mutations in exon 3 β-catenin gene is low in our population. It would be interesting to increase the population size to test the clinically relevant influence in the prognosis found and to test the relation of these events with MSI pathway. If these findings are confirmed, β-catenin would help to discriminate patients with different prognosis.
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[Table 1], [Table 2], [Table 3]