The patients in the study were classified into a mutant group including 31/100 patients (31%) who showed lost expression of hMLH1 and/or hMSH2, and a nonmutant group including 69/100 patients (69%) who showed preserved expression of both hMLH1 and hMSH2.
As regards the clinicopathological criteria, the age of patients belonging to the mutant group ranged from 18 to 49 years, with a mean age of 40.2 years. This group included 16 men (51.6%) and 15 women (48.4%) with a male-to-female ratio of about 1.1 : 1. Positive family history of CRC was recorded in 6/31 patients (19.4%). Regarding the anatomical location, tumors were found in the right colon in 16/31 patients (51.6%), in the rectum in 9/31 patients (29%), in the rectosigmoid colon in 3/31 patients (9.7%), and in the left colon in 3/31 patients (29%).
Regarding the tumor type 15/31 tumors (48.38%) were conventional adenocarcinomas, 14/31 tumors (45.16%) were mucinous adenocarcinomas, and 2/31 tumors (6.45%) were signet ring cell carcinomas. As regards the tumor grade, 2/31 tumors (6.45%) belonged to grade II, all of them were conventional adenocarcinoma cases, and 15/31 tumors (48.38%) belonged to grade III, including 13/15 adenocarcinoma cases (86.66%) and two signet ring cell carcinoma cases (100%).
Lymph node metastases (stage C2) were detected in 19/31 patients (61.29%) and were absent (stage B2) in 12/31 patients (38.7%).
Synchronous polyps were encountered in 6/31 patients (19.35%) and synchronous cancer in the ascending colon was also found in 1/31 patient (3.2%; Table 1).
Correlation of the clinicopathological features of patients in mutant and nonmutant groups revealed statistically significant correlations in the mutant group with a positive family history, right-sided tumors, poorly differentiated tumors (grade III; P<0.5) and mucinous tumors (P<0.01). The rest of features did not show a significant difference between the two groups (Table 1).
The mutant group (31 patients) was further subdivided into three subgroups, lost hMLH1 subgroup (16/31 patients; 51.6%), lost hMSH2 subgroup (9/31 patients; 29%), and combined loss of hMLH1 and hMSH2 subgroup (6/31 patients; 19.35%).
The clinicopathological criteria of the patients belonging to the three subgroups are presented in Table 2.
Correlation of the clinicopathological features of patients in hMLH1 and hMSH2 mutant subgroups revealed statistically highly significant correlations in male patients of the hMLH1 mutant subgroup, in whom lymph node metastasis was absent (P<0.01). The rest of features did not show a significant difference between the two subgroups (Table 3).
MMR genes are called ‘caretaker’ genes because of their important role in policing the integrity of the genome and correcting DNA replication errors. MMR genes that undergo a loss of function contribute to carcinogenesis by accelerating tumor progression. Mutations in MMR genes, mainly hMLH1 and hMSH2, result in the HNPCC syndrome (Ponz De Leon et al., 2004). Mutations in MMR genes produce microsatellite instability, which exists in 10–15% of sporadic CRCs and in 95% of CRCs in HNPCC (Mitchell et al., 2002).
Proteins associated with the MMR genes can be detected in tumor tissue sections by immunohistochemical analysis. The expression of these proteins is lost in adenocarcinoma, which is associated with loss or inactivation of the relevant MMR gene. Immunohistochemical analysis carried out on paraffin embedded tissue sections has been shown to be a sensitive (77–100%) and specific (98–100%) method for the detection of MMR gene proteins as compared with molecular microsatellite instability testing (Shia, 2008).
In the present study, we tried to identify the MMR genes hMLH1 and hMSH2 in young Egyptian CRC patients using immunohistochemistry, and determine whether their loss contributed to the high prevalence of CRC in young Egyptian patients, as Abou-Zeid et al. (2002) reported that more than 30% of CRC patients in Egypt were younger than 40 years.
Pai et al. (2008) claimed that the loss of expression of hMSH2, hMSH6 or hPMS2 in isolation or in combination provides reasonably strong evidence for a germ-line mutation in the respective gene and, therefore, is highly suggestive of HNPCC; thus, in the current study, CRC patients with a loss of immunohistochemical expression of hMLH1 and/or hMSH2 were considered as the mutant group with HNPCC.
The mutant group included 31% of CRC patients enrolled in this study as they showed loss of hMLH1and hMSH2 protein expression in isolation (16 and 9%, respectively) or in combination (6%). Lower frequencies were reported by other investigators such as Thibodeau et al. (1996) and Kruschewski et al. (2002), who found mutations of these genes in 28 and 12% of Western CRC patients, respectively, and Kaur et al. (2011), who found mutations in 18.7% of Malaysian CRC patients.
This difference may be attributed to the inclusion of young age as a selection criterion in our study but not in other studies.
In contrast, a higher frequency was reported by Stormorken et al. (2005), who observed that 45% of young Western CRC patients had mutations in hMLH1 and/or hMSH2 genes.
As regards the demographic data, there was an even distribution between men and women among the mutation group, though men represented 67% of the nonmutation group; this difference between the two groups was statistically insignificant (P>0.05). Similar results were reported by Soliman et al. (2001) and Abou-Zeid et al. (2002) in two separate studies on Egyptian CRC patients, by Liu et al. (2004) in a study on Chinese HNPCC patients, and by Kaur et al. (2011) in a study on Malaysian HNPCC patients.
A positive family history was observed in 7% of the cases in the present study, in 19.4% of the cases in the mutant group, and in only 1.4% of the cases in the nonmutant group, with a statistically significant difference (P<0.05); thus, we speculate that a positive family history carries great significance, and as most patients with family history belonged to the mutant group, it should be considered as a highly positive factor for suspecting the diagnosis of HNPCC. However, as more than 80% of the mutant group had negative family history, its absence does not exclude the presence of MMR gene mutations in young Egyptian CRC patients.
Similarly, a low prevalence of family history was also observed in other Egyptian studies (Soliman et al., 2001; Abou-Zeid et al., 2002), as well as in Far East studies (Liu et al., 2004; Tomita et al., 2004) and Western studies (Grady, 2005).
This low prevalence of family history could be attributed to spontaneous germ-line mutations (Durno et al., 2005) or could reflect the presence of exogenous environmental factors that predispose an individual to germ-line mutations (Mahdavinia et al., 2005).
Right-sided tumors represented 34% of the tumors in the current study with a significantly higher prevalence in the mutant group (51.6%) as compared with the nonmutant group (26%) (P<0.05). A similar result was reported by Kaur et al. (2011), who found that 45% of HNPCC tumors were on the right side; however, a higher percentage of right-sided tumors in HNPCC patients (70%) was reported by Liu et al. (2004) and Mueller-Koch et al. (2005).
Rectal tumors represented a high percentage in the current study (39%) as well as in the mutant group (29%). Lower percentages were reported by Western and Far East studies (Liu et al., 2004; Mueller-Koch et al., 2005; Kaur et al., 2011); however, Soliman et al. (2001) claimed that rectal carcinoma showed a higher prevalence among Egyptian CRC patients compared with Western CRC patients and suggested that this high prevalence could be attributed to the presence of exogenous factors that affected the rectum and were responsible for carcinogenesis.
As regards the pathological features of the tumors, in concordance with studies by Jass (2004) and Kaur et al. (2011), a significant correlation was found between mutations of hMLH1and/or hMSH2 and poorly differentiated (grade III) tumors (P<0.05), as well as mucinous carcinomas (P<0.05). These findings may be attributed to the high prevalence of poorly differentiated and mucinous tumors among HNPCC patients (Redston, 2001; Stormorken et al., 2005) or their high prevalence among Egyptian CRC patients (Soliman et al., 2001; Mokhtar et al., 2007). On the contrary, Kruschewski et al. (2002) found no correlation between HNPCC and sporadic CRC in Western CRC patients with regard to grading.
Lymph node metastasis was reported in 67% of all cases studied. A lower prevalence of lymph node metastasis was reported by Western studies such as that by Kruschewski et al. (2002), who found lymph node involvement only in 32% of their CRC patients.
The higher percentage in the current study might be attributed to the late presentation and diagnosis of CRC in Egyptian patients.
The mutant group in our study showed a lower incidence of lymph node metastasis and hence a lower tumor stage (61.3%) compared with the nonmutant group (69.6%), but it did not reach statistical significance. Although Redston (2001) noticed a high prevalence of lymph node involvement among Western HNPCC patients compared with Western sporadic CRC patients, Kruschewski et al. (2002) found no correlation between HNPCC and sporadic CRC in Western patients as regards staging or lymph node involvement.
Regarding the presence of synchronous polyps, we noticed that 10% of the total cases had synchronous polyps, which increased to 19.4% in the mutant group compared with 5.8% in the nonmutant group; however, the difference did not reach a statistical significance (P>0.05). A similar incidence was reported by Mueller-Koch et al. (2005). A higher percentage, however, was reported by Stormorken et al. (2005), who proposed that polyps in HNPCC patients may have an increased propensity to become malignant in MMR mutation carriers.
Synchronous tumors were reported in cases of HNPCC in 18% of Western patients (Box et al., 1999), 3.1% of Chinese patients (Liu et al., 2004), 3.1% of Egyptian patients (Abou-Zeid et al., 2002), and 7.6% of Malaysian patients (Kaur et al., 2011). Similarly, we reported one patient in our study representing 1% of the total cases and 3.2% of the mutant group. Different incidences of synchronous tumors in different countries may be related to other carcinogenic factors unique to each country or may be related to the fact that carcinomas may occur on top of polyps that may be multifocal.
Predominance of isolated hMLH1 mutations (16%) over isolated hMSH2 mutations (9%) was observed in our study. Similar results were reported in many other studies (Kruschewski et al., 2002; Mueller-Koch et al., 2005; Kaur et al., 2011).
A difference was observed in the current study between the hMLH1 and hMSH2 mutant subgroups, as loss of hMLH1 was significantly correlated with male sex and absent lymph node involvement (P<0.01), although Kruschewski et al. (2002) found no significant difference between hMLH1 and hMSH2 subgroups among Western HNPCC patients as regards tumor stage or grade.
From this study we conclude the following:
- There is a lower prevalence of HNPCC among young Egyptian CRC patients (31%) compared with Western CRC patients (45%) of the same age group.
- There is a high prevalence of loss of MMR genes in patients with a positive family history of CRC.
- CRC of the HNPCC type in Egyptian patients is characterized by increased incidence of mucinous carcinoma, right-sided tumors, synchronous polyps, and high tumor grade, along with a reduced incidence of lymph node metastasis.
Conflicts of interest
There are no conflicts of interest.
Abou-Zeid AA, Khafagy W, Marzouk DM, Alaa A, Mostafa I, Ela MA. Colorectal cancer in Egypt. Dis Colon Rectum. 2002;45:1255–1260
Box JC, Rodriguez-Bigas MA, Weber TK, Petrelli NJ. Clinical implications of multiple colorectal carcinomas in hereditary nonpolyposis colorectal carcinoma. Dis Colon Rectum. 1999;42:717–721
Christensen M, Katballe N, Wikman F, Primdahl H, Sørensen FB, Laurberg S, et al. Antibody-based screening for hereditary nonpolyposis colorectal carcinoma compared with microsatellite analysis and sequencing. Cancer. 2002;95:2422–2430
Durno C, Aronson M, Bapat B, Cohen Z, Gallinger S. Family history and molecular features of children, adolescents and young adults with colorectal carcinoma. Gut. 2005;54:1146–1150
Grady WM. Molecular basis for subdividing hereditary colon cancer? Gut. 2005;54:1676–1678
Hsu S, Raine L, Fanger H. Comparative study of the peroxidase antiperoxidase method and avidin biotin complex method for studying polypeptide hormone with radioimmunoassay antibodies. Am J Clin Pathol. 1981;75:734–738
Jarvinen HJ, Aranio M. Surveillance of mutation carriers of DNA mismatch repair genes. Ann Chir et Gyn. 2000;89:207–210
Jass JR. HNPCC and sporadic MSI-H colorectal cancer: a review of the morphological similarities and differences. Fam Cancer. 2004;3:93–100
Kariola R, Otway R, Lönnqvist KE, Raevaara TE, Macrae F, Vos YJ, et al. Two mismatch repair gene mutations found in a colon cancer patient - Which one is pathogenic? Hum Genet. 2003;112:105–109
Kaur G, Masoud A, Raihan N, Radzid M, Khamizar W, Kam LS. Mismatch repair genes expression defects & association with clinicopathological characteristics in colorectal carcinoma. Indian J Med Res. 2011;134:186–192
Kruschewski M, Noske A, Haier J, Runkel N, Anagnostopoulos Y, Buhr HJ. Is reduced expression of mismatch repair genes MLH1 and MSH2 in patients with sporadic colorectal cancer related to their prognosis? Clin Exp Metastasis. 2002;19:71–77
Lindor NM, Burgart LJ, Leontovich O, Goldberg RM, Cunningham JM, Sargent DJ, et al. Immunohistochemistry versus microsatellite instability testing in phenotyping colorectal tumors. J Clin Oncol. 2002;20:1043–1048
Liu SR, Zhao B, Wang ZJ, Wan YL, Huang YT. Clinical features and mismatch repair gene mutation screening in Chinese patients with hereditary nonpolyposis colorectal carcinoma. World J Gastroenterol. 2004;10:2647–2651
Mahdavinia M, Bishehsari F, Ansari R, Norouzbeigi N, Khaleghinejad A, Hormazdi M, et al. Family history of colorectal cancer in Iran. BMC Cancer. 2005;5:112–123
Mitchell RJ, Farrington SM, Dunlop MG, Campbell H. Mismatch repair genes hMLH1 and hMSH2 and colorectal cancer: a HuGE review. Am J Epidemiol. 2002;156:885–902
Mokhtar N, Gouda I, Adel I Cancer pathology registry 2003–2004 and time trend analysis. 2007 Cairo Department of Pathology, National Cancer Institute, Cairo University
Mueller-Koch Y, Vogelsang H, Kopp R, Lohse P, Keller G, Aust D, et al. Hereditary non-polyposis colorectal cancer: Clinical and molecular evidence for a new entity of hereditary colorectal cancer. Gut. 2005;54:1733–1740
Müller A, Fishel R. Mismatch repair and the hereditary non-polyposis colorectal cancer syndrome (HNPCC). Cancer Invest. 2002;20:102–109
Nease DE Jr., Stoffel E, Turgeon DK, Ruffin MT IV. Colorectal cancer screening. Clinics Family Prac. 2004;6:693–707
Pai RK, Wilcox R, Hart JTubbs RR, Stoler MH. Molecular gastrointestinal, liver and pancreatic pathology. Cell and tissue based molecular pathology: a volume in the foundations in diagnostic pathology series. 20081st ed. Philadeplphia Churchill Livingstone:269–293
Park JG, Vasen HFA, Park KJ, Peltomaki P, Ponz De Leon M, Rodriguez-Bigas MA, et al. Suspected hereditary nonpolyposis colorectal cancer: International Collaborative Group on Hereditary Nonpolyposis Colorectal Cancer (ICG-HNPC) criteria and results of genetic diagnosis. Dis Colon Rectum. 1999;42:710–716
Pensotti V, Radice P, Presciuttini S, Calistri D, Gazzoli I, Perez APG, et al. Mean age of tumor onset in hereditary non-polyposis colorectal cancer (HNPCC) families correlates with the presence of mutations in DNA mismatch repair genes. Genes Chromosomes Cancer. 1997;19:135–142
Ponz De Leon M, Benatti P, Borghi F, Pedroni M, Scarselli A, Di Gregorio C, et al. Aetiology of colorectal cancer and relevance of monogenic inheritance. Gut. 2004;53:115–122
Redston M. Carcinogenesis in the GI tract: from morphology to genetics and back again. Modern Pathol. 2001;14:236–245
Ruszkiewicz A, Bennett G, Moore J, Manavis J, Rudzki B, Shen L, et al. Correlation of mismatch repair genes immunohistochemistry and microsatellite instability status in HNPCC-associated tumours. Pathology. 2002;34:541–547
Scartozzi M, Bianchi F, Rosati S, Galizia E, Antolini A, Loretelli C, et al. Mutations of hMLH1 and hMSH2 in patients with suspected hereditary nonpolyposis colorectal cancer: correlation with microsatellite instability and abnormalities of mismatch repair protein expression. J Clin Oncol. 2002;20:1203–1208
Shia J. Immunohistochemistry versus microsatellite instability testing for screening colorectal cancer patients at risk for hereditary nonpolyposis colorectal cancer syndrome: Part I. The utility of immunohistochemistry. J Mol Diagn. 2008;10:293–300
Shia J, Ellis NA, Klimstra DS. The utility of immunohistochemical detection of DNA mismatch repair gene proteins. Virchows Arch. 2004;445:431–441
Soliman AS, Bondy ML, El-Badawy SA, Mokhtar N, Eissa S, Bayoumy S, et al. Contrasting molecular pathology of colorectal carcinoma in Egyptian and Western patients. B J Cancer. 2001;85:1037–1046
Stormorken AT, Bowitz-Lothe IM, Norèn T, Kure E, Aase S, Wijnen J, et al. Immunohistochemistry identifies carriers of mismatch repair gene defects causing hereditary nonpolyposis colorectal cancer. J Clin Oncol. 2005;23:4705–4712
Thibodeau SN, French AJ, Roche PC, Cunningham JM, Tester DJ, Lindor NM, et al. Altered expression of hMSH2 and hMLH1 in tumors with microsatellite instability and genetic alterations in mismatch repair genes. Cancer Res. 1996;56:4836–4840
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Tomita N, Fukunaga M, Okamura S, Nakata K. The novel germline mutation of the hMLH1 gene in a case of suspected hereditary non-polyposis colorectal cancer (HNPCC) in a patient with no family history of cancer. Jpn J Clin Oncol. 2004;34:556–560