Colorectal cancer (CRC) is the third most common cancer worldwide after lung and prostate carcinoma in men and the second most common after mammary carcinoma in women. Almost 60% of the cases occur in Western, industrialized countries. In terms of mortality, CRC is the second leading cause of cancer-related deaths in both sexes (Ferlay et al., 2010).
Epidemiologically, CRC in developed countries is different from that in developing countries including the Middle East (Kemp et al., 2004). In developing countries, its incidence is lower, with a characteristic young age at onset (≤40 years), left-sided location, poor differentiation, and lack of precursor adenomas in the majority of cases (Abou-Zeid et al., 2002). In Egypt, CRC accounts for 4.5% of all cancers, 4.7% of male malignancies, and 4.2% of female malignancies (Elatar, 2002).
The family of ras gene codes for closely related small monomeric proteins with a molecular weight of 21 kDa that switch between active and inactive states by binding to guanosine triphosphates (GTP) and hydrolyzing them into guanosine diphosphates (GDP), respectively. These proteins (Harvey-[H-], Kirsten-[K-], and neuroblastoma [N-] ras) act in transduction of extracellular signals to the cytoplasm and nucleus, thus affecting cell growth, differentiation, and apoptosis (Malumbres and Barbacid, 2003). Mutations in the ras gene render the protein constitutively active in signaling (even in the absence of extracellular stimuli) by eliminating the GTPase activity (Campbell et al., 1998).
Oncogenic mutations of the K-ras gene (Kirsten rat sarcoma viral oncogene homolog) are most prevalent in pancreatic (72–90%; Kubrusly et al., 2002), thyroid (55%); Bongarzone and Pierotti, 2003), colorectal (11–65%; Bazan et al., 2002), and lung cancers (15–50%; Urban et al., 2000). In CRCs, most of the activating mutations (90%) involve codons 12 (wild-type GGT) and 13 (wild-type GGC) of exon 1 and around 5% affect codon 61 (wild-type CAA) located in exon 2 (Dieterle et al., 2004). The relationship between the presence of K-ras mutations in the primary tumor and poor patient prognosis has been shown by some studies (Font et al., 2001; Bazan et al., 2002), but not by others (Bouzourene et al., 2000; Akkiprik et al., 2007).
The epidermal growth factor receptor (EGFR) signaling pathway is one of the most important pathways that regulate growth, proliferation, differentiation, and survival in mammalian cells. K-ras protein plays an important role in the EGFR signaling cascade by regulating other proteins downstream in the EGFR pathway (Heinemann et al., 2009).
Recent studies strongly suggest that K-ras mutations are predictive of resistance to EGFR inhibitor treatment in CRCs. The mechanisms by which K-ras-mutated CRCs are resistant to EGFR inhibitors are still not known; however, authors have suggested that activating mutation of K-ras may replace the dependence of CRCs on increased signaling from EGFR or decrease apoptosis (Benvenuti et al., 2007).
The aim of this research is to identify the incidence of K-ras gene mutation in Egyptian patients with CRC using the PCR/restriction fragment length polymorphism (PCR/RFLP) method and to determine whether it is linked to clinicopathological parameters of prognostic significance.
Materials and methods
Fifty-six consecutive patients with colorectal carcinoma undergoing elective surgery at the National Cancer Institute, Cairo University, between January 2010 and June 2011 were enrolled in this study after they provided informed consent. The diagnosis had been made by a consultant pathologist on endoscopic-resected tumor specimens. Tumors were classified according to the WHO criteria as adenocarcinoma, mucinous, or signet ring carcinoma and staged according to the tumor, node, and metastasis (TNM) classification of the Union for International Cancer Control (UICC) (Hamilton et al., 2000). Tumors were assigned a single grade of differentiation (well, moderate, or poor). The worst grade of tumor observed was used for the overall grade, unless the worst area was smaller than 10% of the tumor volume and at the advancing margin of the tumor. Tumor tissues were obtained from the surgically excised specimens (viable areas away from hemorrhage and necrosis). Part of each tumor specimen was snap frozen in liquid nitrogen immediately after collection and stored at −80°C until further processing. The predominance of tumor cells (>75%) was checked on the frozen section. The other part was fixed in 10% neutral-buffered formalin, embedded in paraffin, and processed for histopathological evaluation.
Genomic DNA extraction from the frozen CRC specimens and control nonlesional colorectal tissue was performed using the ISOLATE Genomic DNA Mini Kit (Bioline, USA) according to the manufacturer’s recommendations.
Polymerase chain reaction amplification
Detection of the K-ras gene point mutation at codon 12 was performed using the PCR/RELP method according to Hadžija et al. (2007). The primer sequences (Metabion, Germany) are shown in Table 1. Genomic DNA from tumors was amplified by a PCR in a final volume of 25 µl containing 1× GoTaq master buffer (Promega, USA), 50 µmol/l of each dNTP (Thermo Scientific/Fermentas, USA), 5 pmol of forward and reverse primers, and 1 U GoTaq (Promega) and 2.5 µl of extracted DNA (50–100 ng).
Two PCR amplification rounds were performed under conditions of an initial denaturation at 94°C for 5 min, 30 cycles of denaturation (30 s at 94°C), annealing (30 s at 56°C), and an elongation step (30 s at 72°C), and a final extension step at 72°C for 10 min. Reactions were performed in a Perkin Elmers (480 machine) thermocycler. PCR products were digested with the BstN1 (BseB1) restriction enzyme (Bioron GmbH, Germany) according to the manufacturer’s instructions and visualized in an ethidium bromide-stained 3% agarose gel under ultraviolet light.
Tumor DNA positive for codon 12 K-ras mutation (documented previously by sequencing analysis) was used as a positive control. In order to avoid false-positive results, negative controls containing no template DNA were subjected to the same procedure in each run.
The K-ras mutation status was compared with the clinical and pathologic parameters, including the age and sex of the patients, tumor location, TNM stage, histological type and grade of carcinoma, and nodal metastasis.
The study data were summarized as frequency and percentage (qualitative data) or as mean±SD (numerical data). Data were analyzed using SPSS win statistical package version 12. The χ 2-test (Fisher’s test) was used to examine the relationship between qualitative variables, whereas for quantitative data, the Mann–Whitney test was used for variables that were not normally distributed. P value of 0.05 or less was considered as statistically significant.
Clinical and pathological findings
The current study population included 33 men and 23 women, with a male : female ratio of about 1.43. Their mean age at first diagnosis was 50.32±14.3 years (minimum 24 years and maximum 76). In terms of site distribution, generally, there were more colonic tumors (55.4%) than rectal tumors (44.6%). Primary sites were most commonly identified in the rectum including rectosigmoid and the anal canal (44.6%), followed by the left colon, including splenic flexure (23.3%), the right colon including hepatic flexure (19.6%), and the transverse colon (12.5%). Tumors were classified according to the TNM staging system as stage II (66%), stage III (30.4%), and stage IV (3.6%) tumors. Data are presented in Table 2.
The size of colonic malignant tumors included ranged from 2 to 16 cm, with a median of 6.5 cm. Most of the CRC (82.1%) were histologically classified as gland-forming adenocarcinoma and only 17.9% were mucinous. Only one case of gland-forming adenocarcinoma developed on top of villous adenoma. Most of the patients (78.6%) had low and moderately differentiated tumors, whereas 21.4% had high-grade tumors (Table 2). In terms of lymph node metastasis, 23 CRC (41%) showed positive nodal metastasis and 32 (59%) were negative. There was one case with no detectable lymph nodes dissected. Twelve CRC (21.4%) showed adjacent organ infiltration, most commonly the small intestine followed by the urinary bladder.
Molecular results and clinicopathologic correlations
Mutation analysis of codon 12 of the K-ras gene was performed with the PCR/RFLP method using genomic DNA extracted from primary CRC of 56 patients. In this method, two rounds of PCR amplifications for the K-ras gene were performed, each followed by BstN1 restriction enzyme digestion. Agarose gel electrophoresis of the second (final) BstN1 digestion products indicated a single 128 bp fragment in tumors with the wild-type K-ras gene. In CRC with a mutated K-ras gene, a 157 bp band (representing mutant type) was found either alone or with a 128 bp band (representing wild type). PCR results were validated by the mutation-positive DNA control.
In the present study, the PCR/RFLP analysis method showed a point mutation at codon 12 of the K-ras oncogene in 39.3% (22/56) of analyzed CRC, whereas 60.7% (34/56) had the wild-type K-ras gene (Figs 1–5).
When the clinicopathologic features of CRCs with mutated K-ras and wild-type K-ras were compared, the K-ras gene mutation did not show an association with patient age, sex, tumor site (Fig. 6), tumor size, or lymph node metastasis. In contrast, a highly statistically significant correlation (P<0.04) was found between the K-ras gene mutation and both histologic type and grade. Seventy percent (7/10) of the mucinous adenocarcinomas analyzed showed the K-ras gene mutation versus 32.6% (15/46) of gland-forming tumors. In terms of histologic grade, the mutated K-ras gene was identified in 66.7% of poorly differentiated CRC versus 34.1% of moderately differentiated tumors, whereas none of the well-differentiated tumors showed oncogene mutation. The association between tumor TNM stage and K-ras gene mutation was evident as 32.4% of stage II CRC, 47.1% of stage III, and all stage IV tumors had the mutated gene. However, statistical analysis indicated only borderline significance (P=0.12) owing to the small number of stage IV tumors. Similarly, the correlation between the K-ras gene mutation and adjacent organ infiltration indicated a borderline statistical significance (P=0.18). Data are presented in Table 3.
Survival data were available in only 30 CRC patients. After 6–24 months of follow-up, 27 patients were free of tumor, whereas three patients developed distant metastasis. All three patients had the K-ras gene mutation in the primary tumor.
The K-ras gene encodes a 21 kDa small guanosine triphosphate (GTP)-binding protein. Wild-type K-ras protein is activated transiently in response to certain stimuli such as EGFR signaling. Point mutations at codon 12, 13, or 61 of the K-ras gene result in stabilization of the K-ras protein in the GTP-bound conformation, rendering it constitutively active (Malumbres and Barbacid, 2003). Mutational activation of K-ras protein contributes to oncogenic transformation by providing molecular signals that promote cell proliferation, inhibit apoptosis and differentiation, and induce angiogenesis (Downward, 2003).
The mutational analysis of the K-ras gene has been established recently as a complementary in-vitro diagnostic test for the identification of patients with CRC who will not benefit from anti-EGFR treatment (Benvenuti et al., 2007).
In developed countries, CRC is considered as a disease that occurs in the elderly. Most CRC occur in patients older than 50 years of age and affect a young population, with an incidence of 2–6%. Most CRC arise in the colon (70–75%), whereas 25–30% affect the rectum (Kemp et al., 2004).
In the present study, it was found that the clinicopathological features of the Egyptian CRC cohort were similar to those found in developing countries. Egyptian CRC patients were relatively young (28.6%, <40 years), with a peak incidence between 40 and 60 years, the male to female ratio was 1.43, there were no precursor adenomas (except in one tumor), a large proportion of the tumors were located in the rectum (44.6%), and mucinous histology was observed in about 18% of cases.
Our findings agree with and reinforce former studies on Egyptian CRC patients. Soliman et al. (2001) showed that Egyptian CRC patients (n=59) were relatively younger than their Western counterparts (44%, <40 years), male to female ratio of 1.36, about half of the tumors were located in the rectum (51%), and 37% of the tumors were mucinous.
Similarly, Abou-Zeid et al. (2002) showed in their research that CRC in Egypt shares the epidemiological characteristics of developing countries, which are a higher incidence in younger patients and a predominance of rectal carcinoma. The authors carried out a 7-years review on 177 CRC patients and observed that more than one-third of the tumors affected a young population (<40 years), the disease frequently presented at an advanced stage, and predisposing adenomas were rare.
In the present research, 55.4% of CRC were colonic and 44.6% were rectal. The size of the carcinomas ranged from 2 to 16 cm, with a median of 6.5 cm. Most of the CRC (82.1%) were classified as gland-forming adenocarcinoma and 17.9% were mucinous. Most of the patients (78.6%) had low and moderately differentiated tumors, whereas high-grade tumors were found in 21.4% of the patients.
Our results were in agreement with those obtained from a large study carried out by Abd El-Hameed (2005) on 180 CRC cases in Egypt. The author showed that 49.1% of CRC were colonic and 50.9% were rectal. The mean tumor size was 7.3 cm (range 2.5–16 cm). Almost 79.6% were conventional adenocarcinomas, 17.4% were mucinous, and 3% were signet ring. Most of the tumors were grade 2 (79.8%) and 20.2% were grade 3.
In our study population (56 CRC patients), the incidence of codon the 12 K-ras gene mutation was 39.3%. This finding is in agreement with previously published reports (Van den Brandt et al., 2003; Russo et al., 2005; Akkiprik et al., 2007; Liu et al., 2011). K-ras mutations are believed to be an early event in colorectal neoplasia and are observed in 9–10% of small adenomas, 40–50% of large adenomas, and 40–65% of colon carcinomas (Russo et al., 2005).
However, a much lower incidence of the K-ras gene mutation (11%) among Egyptian CRC patients (n=59) was recorded by Soliman et al. (2001). The difference in incidence may be related to two main factors. First, we used fresh CRC samples whereas they used old archived CRC samples that were fixed in unbuffered formalin, a reagent that is known to modify the DNA and occasionally hampers the mutation detection method. Second, their samples were collected between 1996 and 1997, which means that the two analyses (his and ours) are 13 years apart, with drastic changes in life styles and environmental factors.
In a relatively more recent study carried out by El-Serafi et al. (2010), the incidence of the K-ras gene mutation (codon 12) was 32.2% among 80 Egyptian patients with stage II CRC. This slightly lower incidence than ours may be because of the lack of stage III and IV tumors in their study, whereas in our study, both stage III and IV tumors constituted 34% of all cases analyzed.
In the literature, the reported incidence of the K-ras gene mutation in CRC patients ranged from 11 to 50% (Soliman et al., 2001; Castagnola and Giaretti, 2005; Akkiprik et al., 2007; Liu et al., 2011). This broad variation in the reported frequencies of the K-ras mutation can be attributed to different factors such as racial differences, the sensitivity and specificity of mutation detection methods, sample size, sample fixation, and region of the gene analyzed (only codon 12, codon 13, and/or codon 61 (Bazan et al., 2002).
In this study, the K-ras gene mutation did not show an association with patient age, sex, tumor site, tumor size, or lymph node metastasis. Concordant results have been reported by other researchers (Liu et al., 2011).
We found that the point mutation in codon 12 of the K-ras gene was significantly (P<0.04) associated with the mucinous histotype. Several authors have reported an association between the presence of a specific codon 12 K-ras gene mutation and the mucinous histotype in colorectal carcinomas (Nakao et al., 2000; Bazan et al., 2002) and in intestinal-type ovarian carcinomas (Gemignani et al., 2003). This may indicate that activating mutations of codon 12 of the K-ras gene are responsible for the development of most of the mucinous CRC or it is associated with mucinous differentiation.
In the current research, a highly statistically significant correlation (P<0.04) was found between the K-ras gene mutation and the histologic grade of CRC. The K-ras mutation was more frequent in poorly differentiated tumors (66.7%) than moderately differentiated tumors (34.1%), whereas none of the well-differentiated CRC showed the K-ras mutation. There was a trend for an association between codon 12 K-ras gene mutation and both TNM tumor stage (P=0.12) and adjacent organ infiltration (P=0.18).
Several studies support the importance of mutational activation of K-ras in the progression of CRC. Oliveira et al. (2007) reported that the frequency of simultaneous K-ras and BRAF mutations increased with the depth of wall invasion: T2 (2.8%), T3 (3.5%), and T4 (9.4%). Earlier studies have reported that patients showing codon 12 K-ras mutated to GAT (Asp) or GTT (Val) had a worse prognosis (Andreyev et al., 2001). Some studies have related disease progression of CRC to codon 13 mutation. Bazan et al. (2002), analyzing 160 CRC cases, showed that specific codon 13 (but not codon 12) K-ras mutations were significantly associated with advanced Dukes’ stage, lymph node metastases, and a high S-phase fraction, and were independently related to the risk of relapse or death.
Point mutation affecting codon 12 of the K-ras oncogene is a common genetic abnormality (39.3%) among Egyptian patients with CRC and it is significantly associated with mucinous histotype and tumor histologic grade.
Conflicts of interest
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