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Clinicopathological and immunohistochemical study of mismatch repair gene (hMLH1 and hMSH2) expression in colorectal carcinoma of young Egyptian patients

Salman, Manal I.a; Elhefnawy, Nadia G.a; Abou-Zeid, Ahmed A.b; Nada, Mohamed A.b

Egyptian Journal of Pathology: July 2012 - Volume 32 - Issue 1 - p 52–58
doi: 10.1097/01.XEJ.0000416358.48186.92
ORIGINAL ARTICLES
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Background Mismatch repair gene (MMR) mutations result in microsatellite instability. Microsatellite instability is seen in virtually all cases of hereditary nonpolyposis colorectal cancer (HNPCC), ∼15% of sporadic cases, and in more than 70% of colorectal carcinoma (CRC) cases in young patients.

Objective The present study aimed at evaluating the immunohistochemical expression of human mutL homolg 1 (hMLH1) and human mutS homolog 2 (hMSH2) in young Egyptian patients with CRC in an attempt to identify the frequency of MMR gene loss as well as the associated clinicopathological criteria and to try and map out some genetic errors in those patients.

Patients and methods The study included 100 Egyptian patients younger than 50 years with CRC, recruited from Ain Shams University Hospitals in the period between March 2006 and June 2010. All patients were scheduled for surgical resections of their tumors. All colectomy specimens were subjected to routine pathological examination as well as immunohistochemical staining for the protein products of hMLH1 and hMSH2.

Results Among the 100 patients, 7% had a positive family history of CRC, 34% had right-sided tumors, 70% had conventional adenocarcinomas, 5% had signet ring cell carcinomas, and 25% had mucinous adenocarcinomas. In total, 47% of tumors belonged to grade III, including 42 cases of adenocarcinoma and five cases of signet ring cell carcinoma. Lymph node metastases were found in 67% of patients. Synchronous polyps were encountered in 10% of patients, and synchronous cancer in the ascending colon was also found in one patient (1%). Loss of expression of hMLH1 and/or hMSH2 was encountered in 31% of patients and they constituted the mutant group. Statistically significant correlations were observed in the mutant group with a positive family history, right-sided tumors, grade III tumors (P<0.05), and mucinous tumors (P<0.01). Moreover, statistically significant correlations were observed in male patients of the hMLH1 mutant group, in whom lymph node metastasis (P<0.01) was absent.

Conclusion From this study we can 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.
  • The main pathological features of HNPCC in Egyptian patients include the increased incidence of mucinous carcinoma, right-sided tumors, synchronous polyps, and a high tumor grade, along with decreased incidence of lymph node metastasis.

Departments of aPathology

bGeneral Surgery, Faculty of Medicine, Ain Shams University, Cairo, Egypt

Correspondence to Manal I. Salman, Department of Pathology, Faculty of Medicine, Ain Shams University, Cairo, Egypt Tel: +20 100 604 9309; e-mail: manalsalman@live.com

Received November 5, 2011

Accepted November 20, 2011

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Introduction

Colorectal carcinoma (CRC) is a common cancer worldwide; it is the third most common cancer both in the USA (Nease et al., 2004) and in Egypt (Mokhtar et al., 2007). The highest incidence for CRC is seen at ages of 60–79 years; fewer than 20% of the cases occur before the age of 50 years (Pensotti et al., 1997). In Egypt and other developing countries, however, there is a peculiar age distribution for CRC, with almost one-third of the patients being younger than 40 years (Abou-Zeid et al., 2002). The age adjusted mortality rates in young Egyptians are likewise high (Soliman et al., 2001).

It is known that CRC can occur in hereditary or sporadic forms. The hereditary form is characterized by a positive family history, young age of onset, and the presence of other specific tumors and defects (Nease et al., 2004). Diagnosis of hereditary CRC is based on family history, pathologic criteria, and, recently, on genetic criteria. It is now accepted that 80% of the cases of young onset CRC are hereditary (Nease et al., 2004).

It has recently been accepted that there are at least two completely different pathogenetic pathways for CRC. The most common genetic alterations, occurring in ∼85% of the cases, are allelic losses (so-called loss of heterozygosity), chromosomal amplifications, and translocations that affect mainly tumor-suppressor genes (APC, p53, SMAD4) and some oncogenes (k-ras). These alterations are characteristic of the chromosomal instability pathway (Lindor et al., 2002). The remaining 15% have frame-shift mutations and base-pair substitutions in microsatellites, which are repetitive genetic loci with one to five base pairs repeated 15–30 times. Microsatellites are prone to slippage during DNA replication, resulting in small loops in the DNA strand. These errors are normally controlled and repaired by the DNA mismatch repair (MMR) genes such as human mutL homolg 1 (hMLH1), human mutS homolog 2 (hMSH2), hMSH6, hPMS2, and hMSH3 (Scartozzi et al., 2002). This form of genetic destabilization is caused mainly by the loss of function of MMR genes, and is referred to as the microsatellite instability pathway. Microsatellite instability is seen in virtually all cases of hereditary nonpolyposis colorectal cancer (HNPCC), ∼15% of sporadic cases, and in more than 70% of CRC in young patients (Durno et al., 2005).

Mutations in hMLH1 or hMSH2 account for over 90% of the identifiable mutations in patients with HNPCC (Ruszkiewicz et al., 2002; Kariola et al., 2003).

Most DNA MMR gene mutations associated with high-frequency microsatellite instability cancers predict protein truncations. These are usually associated with absent protein expression in the tumor due to either message or protein instability and degradation. As a result, absence of hMLH1 or hMSH2 protein expression on immunohistochemical analysis is a valuable adjunct to characterize MMR deficiency (Müller and Fishel, 2002).

Jass (2004) proposed that age at onset of colorectal cancer, family history, and tumor morphology are important data that aid in distinguishing sporadic CRC from HNPCC. Moreover, Christensen et al. (2002) and Shia et al. (2004) concluded that in clinically suspected HNPCC patients, immunohistochemistry is often used as the first line screening tool, which is followed up by microsatellite instability testing if there is no immunohistochemical abnormality. Cancers showing lack of any MMR gene proteins will be confirmed by the germ-line mutation studies for that specific gene.

The present study aimed at evaluating the immunohistochemical expression of hMLH1 and hMSH2 in young Egyptian patients with CRC in an attempt to identify the frequency of MMR gene loss as well as the associated clinicopathological criteria and to try and map out some genetic errors in those patients.

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Patients and methods

The present study was conducted on 100 Egyptian patients younger than 50 years of age suffering from CRC. All patients were recruited from the Ain Shams University Hospitals in the period between March 2006 and June 2010. All patients were scheduled for surgical resections of their tumors at the Department of General Surgery, and all surgical specimens were subjected to routine pathological examination as well as immunohistochemical staining for the protein products of hMLH1 and hMSH2 at the Department of Pathology.

Patients with CRC along with familial polyposis or ulcerative colitis, as well as those with unresectable tumors, were excluded from the study.

Informed consent was obtained from all patients.

Patients included in the study were subjected to history taking, clinical examination, and routine laboratory and radiological workup. Special diagnostic investigations were carried out according to the merits of each case.

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Pathological examination

All surgical specimens were subjected to gross examined, following which they were processed and sections were taken from the tumors as well as from the nearby mucosa, surgical margins, any associated polyps, and lymph nodes. Sections taken were routinely processed and embedded in paraffin blocks.

Histopathological examination was carried out on hematoxylin and eosin stained sections to evaluate the tumor histological type, grade, depth of invasion, lymph node affection, and modified Duke’s stage.

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Immunohistochemical examination

Five-micrometer-thick sections were cut from paraffin blocks of all tumor tissue specimens of the nearby mucosa and were mounted on positively charged slides. Immunohistochemical staining was performed using the hMLH1 rabbit polyclonal antibody and hMSH2 rabbit polyclonal antibody (Santa Cruz Biotechnology Inc., California, USA) and the supersensitive detection kit (Biogenex, California, USA) following the avidin-biotin peroxidase technique (Hsu et al., 1981). The deparaffinized tissue sections were subjected to microwave antigen retrieval in 10 mmol/l citrate buffer (pH 6.0) for 15 min. After washing, the endogenous peroxidase activity was blocked using 3% hydrogen peroxide. Thereafter, the tissue sections were incubated with hMLH1 and hMSH2 antibodies (dilution 1 : 40) overnight at 4°C. Then, the biotinylated goat anti-mouse IgG was added for 30 min at room temperature, followed by the avidin—biotin peroxidase complex for 3 min. The site immune reaction was visualized with diaminobenzidine tetrachloride. Normal nearby mucosa was used as an internal positive control. The negative control consisted of tested tissue sections after replacing the primary antibody with PBS. Positive cases showed brown nuclear staining. Cytoplasmic staining was considered nonspecific (Kruschewski et al., 2002).

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Statistical analysis

Analysis of data was carried out on IBM computer using SPSS (SPSS Inc., Chicago, Illinois, USA) as follows.

Descriptive data including quantitative and qualitative variables were analyzed.

Comparisons between data were made using the χ2-test and Fisher’s exact test.

The P-value was considered insignificant if it was greater than 0.05, significant if it was less than 0.05, and highly significant if it was less than 0.01.

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Results

Hundred young Egyptian patients with CRC were subjects in the present study, 62 men (62%) and 38 women (38%); their ages ranged from 18 to 49 years with a mean age of 41.5 years. A positive family history of CRC was detected in seven of 100 patients (7%), all of them were consistent with Park criteria for HNPCC (Park et al., 1999), but none of them were consistent with Amsterdam criteria (Jarvinen and Aarnio, 2000).

Regarding the tumor location, 34/100 patients (34%) had right-sided tumors (12 cecum, 14 ascending colon, five hepatic flexure, and three transverse colon), 39/100 patients (39%) had rectal tumors, 12/100 patients (12%) had rectosigmoid tumors, 9/100 patients (9%) had sigmoid tumors, and 6/100 patients (6%) had left colonic tumors.

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Pathological results

Regarding the tumor type 70/100 tumors (70%) were conventional adenocarcinomas, 25/100 tumors (25%) were mucinous adenocarcinomas, and 5/100 tumors (5%) were signet ring cell carcinomas. As regards the tumor grade, 28/100 tumors (28%) belonged to grade II, all of them were conventional adenocarcinoma cases representing 40% of adenocarcinoma cases, and 47/100 tumors (47%) belonged to grade III including 42/70 adenocarcinoma cases (60%) and five signet ring cell carcinoma cases (100%). Mucinous carcinoma cases were not included in the grade.

Lymph node metastases were detected in 67/100 patients (67%) who belonged to the modified Duke’s stage C2 group and were absent in 33/100 patients (33%) who belonged to the modified Duke’s stage B2 group. No cases belonged to stage A or D.

Synchronous polyps were observed in 10/100 patients (10%) and synchronous cancer in the ascending colon was also found in 1/100 patients (1%) (Table 1).

Table 1

Table 1

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Immunohistochemical results

In total, 69/100 tumors (69%) showed positive expression of both hMLH1 and hMSH2, 16/100 tumors (16%) showed lost expression of hMLH1 with preserved expression of hMSH2 (Figs 1 and 2), 9/100 tumors (9%) showed lost expression of hMSH2 with preserved expression of hMLH1 (Figs 3 and 4), and 6/100 tumors (6%) showed lost expression of both hMLH1 and hMSH2.

Fig. 1

Fig. 1

Fig. 2

Fig. 2

Fig. 3

Fig. 3

Fig. 4

Fig. 4

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.

Table 2

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).

Table 3

Table 3

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Discussion

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.

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Conclusion

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.
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Acknowledgements

Conflicts of interest

There are no conflicts of interest.

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