Aissi S, Buisine MP, Zerimech F, Kourda N, Moussa A, Manai M, Porchet N. Somatic molecular changes and histo-pathological features of colorectal cancer in Tunisia. World J Gastroenterol 2013; 19(32): 5286-5294 [PMID: 23983431 DOI: 10.3748/wjg.v19.i32.5286]
Corresponding Author of This Article
Sana Aissi, PhD, Centre de Recherche Jean-Pierre Aubert, INSERM U837, 1 place de Verdun, 59000 Lille cedex, France. sana.aissi@hotmail.com
Research Domain of This Article
Gastroenterology & Hepatology
Article-Type of This Article
Brief Article
Open-Access Policy of This Article
This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Sana Aissi, Mohamed Manai, Laboratory of Biochemistry and Molecular Biology, Science University of Tunis, 2092 El Manar Tunis, Tunisia
Sana Aissi, Marie Pierre Buisine, Nicole Porchet, Centre de Recherche Jean-Pierre Aubert, INSERM U837, 59000 Lille, France
Marie Pierre Buisine, Farid Zerimech, Nicole Porchet, Laboratory of Biochemistry and Molecular Biology, CHRU de Lille, 59000 Lille, France
Marie Pierre Buisine, Nicole Porchet, Medicine University of H Warembourg, University of Lille Nord de France, 59045 Lille, France
Nadia Kourda, Anatomopathology Department, Charles Nicolle Hospital of Tunis, 1006 Tunis, Tunisia
Amel Moussa, Gastro-Enterology Department, Charles Nicolle Hospital of Tunis, 1006 Tunis, Tunisia
ORCID number: $[AuthorORCIDs]
Author contributions: Aissi S performed and designed of the study, genetics consultation and samples collection, acquisition and interpretation of the familial data, sample database management, analysis and interpretation of the data, and co-writing of the paper; Buisine MP contributed to the conception, design, and supervision of the study, tumor samples selection, analysis and interpretation of the data, and co-writing of the paper; Zerimech F contributed to the statistical analyses and critical revision of the paper; Kourda N contributed to the pathological diagnosis, tumor samples selection; Moussa A contributed to the conception and design of the study, treatment and follow-up of the patients, and clinical data collection; Manai M contributed to the oversight of the work; Porchet N contributed to the critical revision of the paper.
Supported by In part by a grant from the Tunisian Government (to Aissi S)
Correspondence to: Sana Aissi, PhD, Centre de Recherche Jean-Pierre Aubert, INSERM U837, 1 place de Verdun, 59000 Lille cedex, France. sana.aissi@hotmail.com
Telephone: +33-3-20298850 Fax: +33-3-20538562
Received: March 18, 2013 Revised: May 14, 2013 Accepted: July 4, 2013 Published online: August 28, 2013 Processing time: 162 Days and 1.6 Hours
Abstract
AIM: To determine correlations between family history, clinical features and mutational status of genes involved in the progression of colorectal cancer (CRC).
METHODS: Histo-pathological features and molecular changes [KRAS, BRAF and CTNNB1 genes mutations, microsatellite instability (MSI) phenotype, expression of mismatch repair (MMR) and mucin (MUC) 5AC proteins, mutation and expression analysis of TP53, MLH1 promoter hypermethylation analysis] were examined in a series of 51 unselected Tunisian CRC patients, 10 of them had a proven or probable hereditary disease, on the track of new tumoral markers for CRC susceptibility in Tunisian patients.
RESULTS: As expected, MSI and MMR expression loss were associated to the presence of familial CRC (75% vs 9%, P < 0.001). However, no significant associations have been detected between personal or familial cancer history and KRAS (codons 12 and 13) or TP53 (exons 4-9) alterations. A significant inverse relationship has been observed between the presence of MSI and TP53 accumulation (10.0% vs 48.8%, P = 0.0335) in CRC tumors, suggesting different molecular pathways to CRC that in turn may reflect different environmental exposures. Interestingly, MUC5AC expression was significantly associated to the presence of MSI (46.7% vs 8.3%, P = 0.0039), MMR expression loss (46.7% vs 8.3%, P = 0.0039) and the presence of familial CRC (63% vs 23%, P = 0.039).
CONCLUSION: These findings suggest that MUC5AC expression analysis may be useful in the screening of Tunisian patients with high risk of CRC.
Core tip: This study reports, for the first time in Tunisia, the value of various histo-pathologic features and somatic molecular changes [BRAF, KRAS, CTNNB1, TP53, mismatch repair (MMR) expression, microsatellite instability (MSI), MLH1 promoter methylation] in distinguishing patients with hereditary non polyposis colorectal cancer. Our results revealed that MUC5AC expression was significantly associated with the presence of MSI (46.7% vs 8.3%, P = 0.0039), MMR expression loss (46.7% vs 8.3%, P = 0.0039) and the presence of familial colorectal cancer (63% vs 23%, P = 0.039). These findings suggest that mucin 5AC expression analysis may be useful in the screening of Tunisian patients with high risk of colorectal cancer.
Citation: Aissi S, Buisine MP, Zerimech F, Kourda N, Moussa A, Manai M, Porchet N. Somatic molecular changes and histo-pathological features of colorectal cancer in Tunisia. World J Gastroenterol 2013; 19(32): 5286-5294
Colorectal cancer (CRC) is a complex biological process involving many genes. Intensive screening for genetic alteration in CRC led to the identification of at least two different molecular mechanisms implicated in CRC carcinogenesis: chromosomal (CIN) and microsatellite instabilities (MSI). The CIN pathway is found in about 80% of sporadic CRC and in familial adenomatous polyposis[1]. It involves chromosomal allelic losses[2,3]. The MSI pathway is found in most cases of hereditary non-polyposis colorectal cancer (HNPCC) and in 12% of sporadic CRC. It involves inactivation of DNA mismatch repair (MMR) genes. The presence of MMR deficiency leads to the accumulation of mutations in mononuclear tracts in the coding region of genes controlling cell cycle[4]. Although CRC shows genetic heterogeneity, the same four different signalling pathways could be implicated in tumor progression. The WNT/Wingless pathway could be activated through an APC mutation in CIN tumors or through a CTNNB1 stabilizing mutation in MSI tumors[5]. CTNNB1 and APC mutations were observed as early as the adenomatous stage of CRC neoplasia. The transforming growth factor beta (TGFβ) pathway is driven by SMAD2 or SMAD4 inactivating mutation in CIN tumors[6] or by a frame-shift mutation in the TGFβ type II receptor in MSI tumors[7]. The RAS-MAP kinase pathway is activated by KRAS mutations in CIN[8] or by BRAF mutations in sporadic MSI tumors. Alteration of these genes correlated closely with the progression of the adenoma to cancer. The TP53 pathway is inactivated by TP53 mutations in CIN tumors or by BAX inactivating mutation in MSI tumors. These alterations contribute to the adenoma-carcinoma transition. More recently, the existence of a third phenotype was suggested. The main alteration associated with this group of tumors is the hypermethylation of the promoter region of numerous genes, leading to their inactivation[9,10]. Activating somatic mutation of BRAF gene has been reported in 15% of sporadic tumors with MSI due to MLH1 hypermethylation and never in tumors from HNPCC families with MLH1 and MSH2 germline mutations[11]. MMR germline mutations detections is an important supplement to HNPCC clinical diagnosis. It enables at-risk and mutation-positive relatives to be informed about their cancer risks and to benefit from intensive surveillance programs that have been proven to reduce the incidence of CRC[12]. However, germline tests are time-consuming and costly due to heterogeneity of mutations. In addition, MMR germline mutations are not always detected in Amsterdam positive families (sensibility, 50%-78%)[13]. The difference in somatic mutation status between sporadic CRC and HNPCC-related cancers may prove helpful in distinguishing HNPCC patients. In this study, we analysed for the first time in Tunisia the value of various histo-pathologic features and somatic mutations of 51 CRC cases in predicting CRC susceptibility.
MATERIALS AND METHODS
Patients and tissue specimens
Fifty-one formalin-fixed, paraffin embedded primary colorectal carcinomas and paired normal bowel of 51 different patients who had undergone colonic resection for the treatment of CRC were retrieved by retrospective review of the pathology archives. Ten of these patients were previously characterized for MMR germline mutations associated to Lynch syndrome[14]. Patients were evaluated according the revised Bethesda guidelines for the identification of HNPCC patients[15]. MSI testing, immunohistochemistry and somatic mutational analysis were performed in all patients regardless of age, personal or family history of cancer, and tumor characteristics.
DNA preparation
DNA was extracted from paraffin-embedded tissue samples of primary CRC and paired normal bowel using the DNeasy® tissue kit (Qiagen, Courtaboeuf, France).
MSI analysis
MSI was assessed using a set of five mononucleotide markers (BAT25, BAT26, NR21, NR22, NR24)[15,16].
Expression of MMR proteins
MMR was assessed by immunohistochemistry as previously described[16]. Immunohistochemistry for mucin (MUC) 5AC and TP53. Tumor sections were analysed using mouse monoclonal antibody against p53 (clone DO-7, Dakocytomation) and MUC5AC (clone CLH2, Novocastra). For TP53, a tumor was scored as TP53 overexpression-positive if nuclear staining was seen in more than 20% of the neoplastic cells in the absence of staining in the tumor adjacent cells. For MUC5AC, which is never expressed in normal colon mucosa[17], expression was interpreted as positive if more than 10% of tumor cells displayed cytoplasmic staining in the absence of staining in the tumor adjacent cells.
TP53 mutations screening
Primers were designated for the coding regions and exon-intron boundaries of exons 5 to 8. Exons 4 and 9 were only analysed on those samples negative for mutations in exons 5-8. Primer sequences and polymerase chain reaction (PCR) conditions are available on request.
Mutation screening for KRAS, CTNNB1 and BRAF genes
KRAS (codon 12, 13), BRAF (exon 15) and CTNNB1 (β-catenin) (exon 3) were screened in each CRC cancer using direct sequencing in forward and reverse orientations. Primer sequences and PCR conditions are available on request.
MLH1 promoter methylation assay
Genomic DNA obtained from paraffin-embedded tissue section was modified with sodium bisulfite using the EZ DNA Methylation kit (Zymo Research) according to the specifications of the manufacturer. Primer sequences for methylation-specific PCR were modified from Grady et al[18].
Statistical analysis
Continuous variables are described as mean and range (min-max) and categorical variables as frequencies and percentages. The association between the different measured parameters was tested using non parametric tests. The difference between two independent groups was determinate by Mann-Whitney U-test and the significance of differences between more than two groups was calculated using Kruskal-Wallis test. Categorical data were compared by χ2 appropriate or Fisher exact tests. A P-value < 0.05 was considered as statistically significant. Statistical analyses were performed with SPSS version 15.0 (SPSS, Chicago, IL, United States).
RESULTS
Clinical and pathological features
Demographic, clinical and tumor-related characteristics of the study group are summarised in Table 1. Twenty-five (49.0%) probands were under 50 years of age, including 12 (23.5%) under 40 years of age and 5 (9.8%) under 30 years of age; 26 (51%) aged more than 50 years, including 20 (39.2%) aged more than 60 years. Six patients (11.8%) had a personal history of synchronous/metachronous CRC tumors (4 cases) or previous primary CRC and HNPCC-related extracolonic tumors (2 cases). In 8 cases (16%), the proband was found to have at least one first-degree relative with CRC and/or HNPCC-related extracolonic cancers. In total, 25 (49%) of the 51 CRC patients belonged to families fulfilling the Amsterdam Criteria[19] for the clinical definition of HNPCC or fulfilled at least one criterion of the revised Bethesda criteria for the identification of HNPCC patients[15]. Criterion 1 was the most commonly satisfied Bethesda criterion (22/51, 43.1%). Clinical data analysis revealed that CRC was essentially right sided for patients having at least one first- or second-degree relative with CRC; whereas cancer was more frequently left sided or rectal for patients without a familial history of CRC (P = 0.039) (Table 2). However, no significant difference in tumor site was seen when Bethesda criteria where considered. The Bethesda-positives CRC tumors same to be associated to a more advanced stage of the disease (P = 0.050) (Table 2). However, no statistical difference has been seen when familial history was considered (Table 2).
Table 1 Clinical and histo-pathological characteristics of the 51 colorectal cancer patients n (%).
Characteristic
Patient
Age of onset of the first cancer (range) (yr)
51 (17-85)
≤ 50
25 (49.0)
> 50
26 (51.0)
Sex
Male
30 (58.8)
Female
21(41.2)
Site of the first CRC
Right colon
14 (27.5)
Left colon
16 (31.4)
Rectum
21 (41.2)
TNM tumor stage
I
3 (5.9)
II
24 (47.1)
III
20 (39.2)
IV
3 (5.9)
Others
1 (2.0)
Degree of differentiation
Well
33 (64.7)
Moderate
14 (27.5)
Poor
2 (3.9)
Mucinous CRC
2 (3.9)
Mucinous carcinoma type
≥ 50%
14 (27.5)
≤ 50%
37 (72.5)
Signet ring cell carcinoma
2 (3.9)
Tumor infiltrating lymphocyte
Crohn’s–like reaction
2 (3.9)
Intra epithelial lymphocytes
1 (2.0)
Lymphoïde peritumoral reaction
10 (19.6)
Synchronous CRC
3 (5.9)
Metachronous CRC and HNPCC related cancer
3 (5.9)
Fulfillment of guidelines
Amsterdam
3 (5.9)
Revised Bethesda
22 (43.1)
B1
22
B2
3
B3
11
B4
1
B5
1
Table 2 Statistical analysis of clinicopathological parameters of the 51 colorectal cancer studied tumors as a function of tumoral phenotype n (%).
Mutation MMR -
Mutation MMR +
Family history of colorectal cancer
P
Ams - and Beth -
Ams - and Beth +
P
Ams (- or +) and Beth +
P
(n = 6)
(n = 4)
Yes (n = 8)
No (n = 43)
(n = 26)
(n = 22)
(n = 25)
Mutation geminale
MMR+
4
0
0
2
4
MMR-
3
3
0
5
6
Site of tumor (CCR)
NS
NS
NS
Right colon
1
3
5 (63)
10 (23)
5 (19)
9 (41)
10 (40)
Left colon
2
0
1 (13)
14 (33)
8 (31)
7 (32)
7 (28)
Rectum
3
1
2 (25)
19 (44)
13 (50)
6 (27)
8 (32)
Left colon + rectum
5
1
3 (38)
33 (77)
0.039
21 (81)
13 (59)
NS
15 (60)
NS
Right + left colon
3
3
6 (75)
24 (56)
NS
13 (50)
16 (73)
NS
17 (68)
NS
TNM Stage
NS
NS
NS
I
0
1
1 (14)
2 (5)
2 (8)
0 (0)
1 (4)
II
1
0
2 (29)
23 (53)
16 (62)
9 (41)
9 (38)
III
4
3
4 (57)
15 (35)
7 (27)
11 (50)
12 (50)
IV
0
0
0 (0)
3 (7)
1 (4)
2 (9)
2 (8)
I/II
1
1
3 (43)
25 (58)
NS
18 (69)
9 (41)
10 (42)
III/IV
4
3
4 (57)
18 (42)
8 (31)
13 (59)
NS
14 (58)
0.050
Microsatellite instability
< 0.001
NS
0.038
MSI (MSI-L or MSI-H)
1
4
6 (75)
4 (9)
2 (8)
6 (27)
8 (32)
MSS (MSI-L or MSS)
5
0
2 (25)
39 (91)
24 (92)
16 (73)
17 (68)
Somatic mutations
TP53
6
0
3 (38)
25 (64)
NS
14 (61)
13 (62)
NS
14 (58)
NS
KRAS
1
2
2 (25)
14 (33)
NS
9 (35)
5 (23)
NS
7 (28)
BRAF
0
0
0 (0)
1 (2)
1 (4)
0 (5)
0 (0)
CTNNB1
0
0
0 (0)
1 (2)
0 (0)
1 (5)
1 (4)
Immunohistochemistry
Loss of MMR
1
4
5 (50)
5 (50)
NS
2 (8)
6 (27)
NS
8 (32)
0.038
Overexpression of p53
5
0
2 (25)
19 (44)
NS
9 (35)
11 (50)
NS
12 (48)
NS
Overexpression of MUC5AC
2
3
5 (63)
10 (23)
0.039
6 (23)
8 (36)
NS
9 (36)
NS
Pattern and frequency of MSI
MSI-high (MSI-H) phenotype was detected in 10 (19.6%) of the 51 tested tumors. All the MSI tumors showed instability in all 5 analysed markers. Eight patients (8/25; 32%) were Bethesda-positives and only 2 (2/26; 8%) were Bethesda-negatives (Table 2). For the remaining 41 cases, the tumors were microsatellite stable (MSS) including 1 (1/3, 33%) Amsterdam I-positive patient and 16 (16/25, 64%) Bethesda-positives patients. Six (27%) of the 22 Bethesda-positives Amsterdam-negatives patients showed MSI in tumor tissue. Hence, the sensitivity and the specificity of the Bethesda criteria in the prediction of MSI were 80% and 60%, respectively. MSI was also observed in 3 sporadic CRC cases (3/49, 6%). Other laboratories have demonstrated a frequency of MSI between 10% and 20% amongst sporadic CRC cases. Therefore, our results are comparable with results from other series[20,21]. CRC was diagnosed before 50 years of age in 80% (8/10) of the patients with MSI (Table 3). The mean age at tumor diagnosis in MSS patients was higher than in MSI patients [56.2 years (range 17-85 years) vs 42.4 years (range 18-72 years)] (Table 3). Two of the 10 MSI patients (20%) had synchronous/metachronous colorectal cancer and no one had additional extracolonic cancer. Mucinous colloid component was significantly more important in MSI-H tumors (P = 0.0178) (Table 3). No significant associations were observed between MSI phenotype and sex, tumor site and tumor node metastasis (TNM) stage (Table 3). However, MSI-H tumors have been reported to be more frequent in the proximal colon[22]. A KRAS somatic mutation was detected in 4 (4/10, 40%) MSI-H tumors (Table 4): 3 were located at codon 13 (p.Gly13Asp) and 1 was at codon 12 (p.Gly12Asp). No significant association has been detected between MSI and KRAS alterations (Table 4). However, an inverse correlation came to exist between MSI-H phenotype and TP53 overexpression (P = 0.0335) (Table 4). On the other hand, MUC5AC abnormal expression was significantly more frequent in MSI tumors compared to MSS tumors (P = 0.0039) (Table 4). This result was in accordance with data reported by Biemer-Hüttmann et al[23].
Table 3 Comparison of the somatic phenotype and genotype as a function of the patient’s clinical characteristics.
Forty-one of the 51 (80.4%) analysed CRCs exhibited normal MMR protein expression. Of the remaining 10 (19.6%) CRCs, 8 (80%) showed a combined MLH1 and PMS2 proteins expression loss suggesting an MLH1 deleterious mutation, 1 (10%) showed a combined MSH2 and MSH6 proteins expression loss, hardly suggesting an MSH2 deleterious mutation (or MSH6, eventually), while just 1 (10%) demonstrated loss of only MSH6 protein, suggesting an MSH6 deleterious mutation. Two (2/10, 20%) of these patients were Amsterdam-positives whereas 8 (8/10, 80%) where Amsterdam-negatives (Table 2). Five patients with MMR proteins expression loss had a family history of cancer. Of the 8 cases with MLH1 expression loss, 2 (2/8, 25%) had an Amsterdam-positive family history. For MSH2 and/or MSH6 none of the 2 cases with expression loss had a cancer family history. The 10 tumors with MMR expression loss corresponded to the 10 MSI-H tumors. All the MSS tumors showed normal MMR proteins expression. Hence, immunohistochemical analysis had 100% sensitivity for the detection of tumors with high MSI. Of note, germline deleterious mutations in MMR genes had been reported in our previous studies[14,24] in 4 patients with MSI-H CRC tumors and MMR protein expression loss.
TP53 protein expression analysis
TP53 positive nuclear immunostaining was observed in the CRC tumors from 21 (21/51, 41%) patients. No significant association has been detected between TP53 overexpression and selection criteria (Amsterdam or Bethesda) or the presence of relatives with CRC (Table 2). Overall, no association has been detected between TP53 overexpression and the different clinical parameters, including age at tumor diagnosis, gender, TNM stage or tumor location (Table 3). All but one (20/21, 95.2%) of the CRC tumors with TP53 overexpression were MSS and showed normal MMR proteins expression. The remaining tumor was of MSI-H phenotype associated to a combined MLH1 and PMS2 proteins expression loss, suggesting an MMR deficiency. On other hand, we have noted a close correlation between TP53 mutations and TP53 protein level (P = 0.0090) (Table 5), as previously reported[25,26]. The absence of mutation in the 4 tumors overexpressing TP53 may be due to a lack of sensibility of the utilized sequencing technique, which requires greater than 15%-20% of neoplastic cells burden in the analysed specimens. In addition, mutations may be located outside the screened exons (exons 4-9), which represent less than 5% of the TP53 detected mutations[27].
Table 5 Comparison of TP53 somatic mutations as a function of tumoral parameters.
Abnormal MUC5AC expression was identified in 15 CRC tumors (15/51, 29.41%), 6 of them showed mucinous colloid component ≥ 50%. In 3 tumors, the stained area was limited to the focal glands. MUC5AC expression was significantly associated to the presence of personal and family history of CRC (P = 0.039) (Table 2). It is very interesting to note that abundant MUC5AC expression was seen in the tumor of 3 HNPCC subjects with deleterious germline MMR mutations[14]. However, we didn’t detect any other significant association between MUC5AC expression and clinico-pathological characteristics (Table 6). Interestingly, MUC5AC expression was significantly associated to MSI phenotype and MMR proteins expression loss (P = 0.0039) (Table 6). In contrast, no significant association was detected between MUC5AC expression and TP53 or KRAS genes mutations (Table 6).
Table 6 Comparison of mucin 5AC expression as a function of tumoral parameters.
The TP53 mutation analysis was possible in the CRC tumors of 47 patients. In total, a deleterious somatic mutation has been detected in 28 patients (28/47, 59.6%). Overall, there were no significant association of TP53 mutations with Bethesda criteria, cancer family history (Table 2) or patient’s clinical and histo-pathological data (Table 3). Particularly, we didn’t detect any association between the presence of TP53 mutations and tumor site (P = 0.0658) (Table 3). This was in contrast with data reported by other groups which showed that TP53 mutations where more frequent in left-sided and rectal tumors[28-30]. A KRAS somatic mutation was identified in 7 (7/28, 25%) of the CRC tumors with TP53 mutations (Table 5). All these mutations were G>A transitions in codon 12 (5 mutations were p.Gly12Asp and 1 was p.Gly12Ser) and no mutation has been detected in codon 13.
KRAS somatic mutations
A KRAS mutations was identified in 16 (16/51, 31.5%) of all the CRC tumors. There was no significant association of KRAS mutations with Bethesda criteria, cancer family history (Table 2) and patient’s clinical and histo-pathological data (Table 3). In addition, no significant association has been detected between KRAS mutations and the other tumoral parameters (Table 7). However, the mutation spectra same to be different between MSS and MSI tumors and more varied mutations have been detected in MSS tumors (Figure 1). Some amino acid changes were detected only in MSS tumors (Figure 1). Whereas, the KRAS mutation p.Gly13Asp have been detected only in MSI-H tumors in the absence of TP53 mutations or TP53 overexpression (Figure 1).
Figure 1 KRAS mutation spectrum as a function of tumoral microsatellite instability phenotype.
A: Microsatellite stable (MSS); B: Microsatellite instability-high (MSI-H).
Table 7 Comparison KRAS somatic mutations as a function of tumoral parameters.
Presence of KRAS mutations (n = 16)
Absence of KRAS mutation (n = 35)
P
MSI
25.00%
17.10%
NS
MMR expression loss
25.00%
17.10%
NS
TP53 mutations
46.70%
65.60%
NS
TP53 overexpression
31.30%
45.70%
NS
MUC5AC overexpression
25.00%
31.40%
NS
BRAF mutations
The BRAF activating mutation c.1796A>T, p.Val600Glu was found in only 1 (1/51, 2%) stage II non-mucinous and non-invasive tumor of the proximal colon of a 79 years old man with no cancer family history. This mutation was shown to be specific to sporadic CRC tumors due to MLH1 promoter hypermethylation and absent in CRC tumors with MSS phenotype[31] and patients with MLH1 or MSH2 germline mutations[11]. Because we didn’t examine the entire BRAF gene, we cannot rule out the presence of other mutations in the remaining CRC tumors. This tumor showed peritumoral lymphatic reaction, MSS phenotype, normal MMR proteins expression and abnormal MUC5AC expression. In addition, no somatic mutations in KRAS or TP53 genes or overexpression of TP53 protein were detected in this tumor. According to some authors, this tumoral phenotype characterized CRC tumors arising from serrated polyps[32].
CTNNB1 mutations
Only 1 putative pathogenic somatic mutation was detected in an MSI-H CRC tumor (1/51, 2%). The change was a typical missense mutation causing alteration of serine at codon 45 (c.134C>T, p.Ser45Phe). This patient was operated of TNM stage II well differentiated and mucinous adenocarcinoma of the sigmoid at 36 years of age and didn’t have any relatives with cancer. Tumor analysis detected a p.Gly13Asp KRAS mutation and a combined MLH1 and PMS2 proteins expression loss. These findings were in accordance with the specific affinity of CTNNB1 mutation to MSI-H CRC tumors[5,33]. In addition, normal TP53 and MUC5AC proteins expression was detected in this tumor in the absence of TP53 or BRAF somatic alterations. According to Young et al[34], this tumoral phenotype characterises CRC in Lynch syndrome, highlighting the presence of this syndrome in this patient.
MLH1 promoter methylation
Further analysis of MLH1 promoter methylation status in the tumor of 4 patients showing MSI-H phenotype and MLH1 protein expression loss in the absence of MLH1 deleterious somatic mutation by MLPA or sequencing, did not detect aberrant methylation discarding the hypothesis of sporadic cancers due to epigenetic inactivation of MLH1 gene.
DISCUSSION
The detection of subjects at high risk of CRC remains problematic. It was essentially based on the family history of patients. Nevertheless, MSI testing and MMR protein expression analysis still the major screening tool for identifying HNPCC. In the present report, we have studied the phenotype and the genetic characteristics of the CRC tumors of 51 Tunisian non related patients selected according to the revised Bethesda criteria in order to compare the tumor phenotype due to MMR deficiency with somatic alterations in genes implicated in CRC tumorigenesis. Our aim was to define for each tumor the pathway of carcinogenesis and to identify new tumoral markers which may help in the diagnosis of CRC susceptibility and easy to use in medical practice. Clinical data analysis showed that CRC was essentially right sided in patients with first or second degree CRC relatives, whereas CRC was mostly distal (left colon and rectum) in patients without cancer family history. These findings are in accordance with data published[35,36]. As expected, genetic characteristics analysis of the 51 tumors showed that MSI phenotype and MMR expression loss were significantly associated to the presence of a CRC family history (P < 0.001). TP53 mutations have been detected in 59.6% of the analysed patients. This finding was in agreement with previous studies in CRC, which reported TP53 mutation frequencies between 50% and 70%[28-30]. Our study shows statistically inverse relationships between MSI and TP53 alterations in CRC (P = 0.0335). This finding was in accordance with that reported by Samowitz et al[29]. This data highlights the hypothesis that MMR deficient CRC tumors evolve through a pathway that is independent of TP53 gene. KRAS mutations were identified in 31.5% of all CRC tumors. This is consistent with previous reports that have identified KRAS mutations in 30%-45% of CRC tumors[8,29,37]. No significant association had been detected between MSI phenotype and KRAS alterations. However, the mutation spectrum was different between MSS and MSI-H tumors. In spite of our reduced number of tumors this finding was in accordance with that reported[8]. On the other hand, abnormal MUC5AC expression was found to be significantly associated to MSI phenotype (P = 0.0039) and CRC personal and family history (P = 0.039). In contrast, no significant association was detected between MUC5AC expression and KRAS or TP53 genes mutations.
In conclusion, we suggest that MUC5AC expression analysis of CRC tumors may be useful in the screening of patients with high risk of CRC.
ACKNOWLEDGMENTS
This study was the result of collaboration between the Universities of Tunis-El Manar and Lille 2. We are indebted to all family members who agreed to attend our study. We also thank Dr Trabelsi O, Gastroenterologist in Tunis, Tunisia, for providing us with families, Dr Wacrenier A, Department of Pathology of the CHRU of Lille, France, for her help in immunohistochemistry of MMR proteins, and the “Plateau Commun de Biologie Moléculaire du Centre de Biologie-Pathologie” of the CHRU of Lille.
COMMENTS
Background
This paper tend to make correlations between family history, clinical features and mutational status of genes involved in the progression of colorectal cancer (CRC) on the track of new tumoral markers for CRC susceptibility in Tunisian patients.
Research frontiers
Authors have screened 51 CRC tumors containing mixed hereditary nonpolyposis colorectal cancer (HNPCC) and sporadic CRC Tunisian cases for somatic changes in KRAS, BRAF and CTNNB1, for microsatellite instability, for expression of mismatch repair (MMR) and mucin (MUC) 5AC, for mutation and expression of TP53 and for MLH1 promoter hypermethylation. They have also compared these molecular findings with clinical, pedigree and pathological data, regardless of age, personal or family history of cancer, and tumor characteristics.
Innovations and breakthroughs
In this study, authors report for the first time in Tunisia the value of various histo-pathologic features and somatic molecular changes in distinguishing patients with HNPCC from those with sporadic colorectal cancer in the aim to identify new tumoral markers of colorectal cancer susceptibility easy to use in the design of diagnostic, therapeutic and preventive strategies in Tunisia.
Applications
MUC5AC expression was significantly associated to the presence of the presence of familial CRC, microsatellite instability and MMR expression loss. This finding suggests that MUC5AC expression analysis may be useful in the screening of patients with high risk of CRC in Tunisia.
Peer review
Screening the subjects at risk of CRC is important. In this manuscript, the authors retrospectively reviewed the histo-pathological features, molecular changes and family history of 51 Tunisian CRC patients. Among many genetic and clinical variables, MUC5AC expression was concluded to be useful in the screening of patients at high risk of CRC susceptibility.
Footnotes
P- Reviewers Li YY, Rodriguez DC S- Editor Gou SX L- Editor A E- Editor Zhang DN
Johnson V, Volikos E, Halford SE, Eftekhar Sadat ET, Popat S, Talbot I, Truninger K, Martin J, Jass J, Houlston R. Exon 3 beta-catenin mutations are specifically associated with colorectal carcinomas in hereditary non-polyposis colorectal cancer syndrome.Gut. 2005;54:264-267.
[PubMed] [DOI][Cited in This Article: ][Cited by in Crossref: 84][Cited by in F6Publishing: 96][Article Influence: 5.1][Reference Citation Analysis (0)]
Duval A, Hamelin R. Mutations at coding repeat sequences in mismatch repair-deficient human cancers: toward a new concept of target genes for instability.Cancer Res. 2002;62:2447-2454.
[PubMed] [DOI][Cited in This Article: ]
Oliveira C, Westra JL, Arango D, Ollikainen M, Domingo E, Ferreira A, Velho S, Niessen R, Lagerstedt K, Alhopuro P. Distinct patterns of KRAS mutations in colorectal carcinomas according to germline mismatch repair defects and hMLH1 methylation status.Hum Mol Genet. 2004;13:2303-2311.
[PubMed] [DOI][Cited in This Article: ][Cited by in Crossref: 101][Cited by in F6Publishing: 107][Article Influence: 5.4][Reference Citation Analysis (0)]
Buisine MP, Devisme L, Savidge TC, Gespach C, Gosselin B, Porchet N, Aubert JP. Mucin gene expression in human embryonic and fetal intestine.Gut. 1998;43:519-524.
[PubMed] [DOI][Cited in This Article: ]
Grady WM, Rajput A, Lutterbaugh JD, Markowitz SD. Detection of aberrantly methylated hMLH1 promoter DNA in the serum of patients with microsatellite unstable colon cancer.Cancer Res. 2001;61:900-902.
[PubMed] [DOI][Cited in This Article: ]
Aaltonen LA, Salovaara R, Kristo P, Canzian F, Hemminki A, Peltomäki P, Chadwick RB, Kääriäinen H, Eskelinen M, Järvinen H. Incidence of hereditary nonpolyposis colorectal cancer and the feasibility of molecular screening for the disease.N Engl J Med. 1998;338:1481-1487.
[PubMed] [DOI][Cited in This Article: ]
Iino H, Simms L, Young J, Arnold J, Winship IM, Webb SI, Furlong KL, Leggett B, Jass JR. DNA microsatellite instability and mismatch repair protein loss in adenomas presenting in hereditary non-polyposis colorectal cancer.Gut. 2000;47:37-42.
[PubMed] [DOI][Cited in This Article: ][Cited by in Crossref: 117][Cited by in F6Publishing: 112][Article Influence: 4.7][Reference Citation Analysis (0)]
Biemer-Hüttmann AE, Walsh MD, McGuckin MA, Simms LA, Young J, Leggett BA, Jass JR. Mucin core protein expression in colorectal cancers with high levels of microsatellite instability indicates a novel pathway of morphogenesis.Clin Cancer Res. 2000;6:1909-1916.
[PubMed] [DOI][Cited in This Article: ]
Aissi-Ben Moussa S, Moussa A, Lovecchio T, Kourda N, Najjar T, Ben Jilani S, El Gaaied A, Porchet N, Manai M, Buisine MP. Identification and characterization of a novel MLH1 genomic rearrangement as the cause of HNPCC in a Tunisian family: evidence for a homologous Alu-mediated recombination.Fam Cancer. 2009;8:119-126.
[PubMed] [DOI][Cited in This Article: ][Cited by in Crossref: 18][Cited by in F6Publishing: 17][Article Influence: 1.1][Reference Citation Analysis (0)]
Greenblatt MS, Bennett WP, Hollstein M, Harris CC. Mutations in the p53 tumor suppressor gene: clues to cancer etiology and molecular pathogenesis.Cancer Res. 1994;54:4855-4878.
[PubMed] [DOI][Cited in This Article: ]
Samowitz WS, Holden JA, Curtin K, Edwards SL, Walker AR, Lin HA, Robertson MA, Nichols MF, Gruenthal KM, Lynch BJ. Inverse relationship between microsatellite instability and K-ras and p53 gene alterations in colon cancer.Am J Pathol. 2001;158:1517-1524.
[PubMed] [DOI][Cited in This Article: ][Cited by in Crossref: 133][Cited by in F6Publishing: 135][Article Influence: 5.9][Reference Citation Analysis (0)]
Børresen-Dale AL, Lothe RA, Meling GI, Hainaut P, Rognum TO, Skovlund E. TP53 and long-term prognosis in colorectal cancer: mutations in the L3 zinc-binding domain predict poor survival.Clin Cancer Res. 1998;4:203-210.
[PubMed] [DOI][Cited in This Article: ]
Young J, Simms LA, Biden KG, Wynter C, Whitehall V, Karamatic R, George J, Goldblatt J, Walpole I, Robin SA. Features of colorectal cancers with high-level microsatellite instability occurring in familial and sporadic settings: parallel pathways of tumorigenesis.Am J Pathol. 2001;159:2107-2116.
[PubMed] [DOI][Cited in This Article: ][Cited by in Crossref: 270][Cited by in F6Publishing: 284][Article Influence: 12.3][Reference Citation Analysis (0)]
