Colorectal cancer (CRC) is a heterogeneous disease and the third most common cancer in the western countries [1]. Its incidence rate is lower in North Africa [2] but has significantly increased these last two decades. In Algeria, CRC is the second most common cancer after lung cancer in men and breast cancer in women [3]. More than half of the patients were staged III and IV at the diagnosis and are younger than the patients in the western countries [4]. The possible causes of this include genetic, environmental and lifestyle factors [5]. Diet, obesity and comorbidities such as diabetes increase the risk of developing a cancer. Several factors such as socioeconomic status, screening, diagnosis and differences in treatment can explain the different health outcomes among patients [6]. Despite progress in recent years in the treatment of CRC with the use of the anti- EGFR and anti-vascular epidermal growth factor (anti-VEGF) agents which have significantly improved the survival of CRC patients [7], mortality remains high in Algeria. Molecular biomarkers that are clinically used have become important and may provide treatment with anti-EGFR agents [8]. It has been reported that patients with mutated RAS, do not benefit from anti-EGFR therapy [9] [10] . The aim of the current study was to detect some molecular alterations MSI, BRAF, KRAS which proved to be significant prognosis and /or predictive markers in the daily clinical practice and which may help define a better management of the Algerian patients with CRC. The development of platforms for detection of molecular alterations including BRAF and RAS hot spot mutations in tumors will facilitate the prescription of target therapies in Algeria.

Clinical data was collected from 102 Algerian patients with advanced CRC histopathologically proven and who were being treated at the medical oncology department in Pierre and Mary Curie Center, a specialized University hospital in Algiers, Algeria, between 2006–2009 and after radical surgical resection. All participants signed an informed consent and the study was approved by the ethical committee of our institution. Clinicopathological information including age at diagnosis, sex, tumor location, stage, pathological tumor staging system (p TNM/UICC), were available for all patients.

TISSUE SELECTION
Primary tumors paraffin included were cut in 4 µm sections and stained with Hematoxylin and Eosin (H&E) for histopathological examination. Formalin fixed and paraffin embedded (FFPE) tumor sections were reviewed by pathologist to confirm diagnosis and define tumor areas containing 50 to 70% tumor cellularity and areas of adjacent normal tissue (25 to 30%) which were immersed in xylene and ethanol. When necessary, the proportion of tumoral cells was maximed by macrodissection.

DNA ISOLATION
Genomic DNA was extracted using the Qiamp miniKit Qiagen following the manufacturer's recommendations (Qiagen courtaboeuf, France) and quantified by spectrophotometry with nanodrop (Thermo Fisher scientific).

MOLECULAR ANALYSIS

Microsatellite instability (MSI) status
Microsatellite markers (Bat25, Bat 26, NR 21, NR 22 and NR24) a pentaplex of mononucleotide repeat were used to evaluate MSI status by polymerase chain reaction (PCR). Instability at only one of the five markers tested was labeled Microsatellite Low (MSL). Instability for two for more was MSI-High (MSI-H) and no instability at any of the five markers tested was labeled Micro-satellite Stable (MSS). In this study MSI-L and MSS are combined into one group which is non MSI-H.

KRAS and BRAF status: Genomic DNA was analyzed by direct sequencing of amplified PCR products. (Applied Biosystem).

STATISTICAL ANALYSIS
Different variables were compared using chi-square test and Fisher student test. P-value < 0.05 was considered statistically significant.

In this study, we analyzed 102 advanced CRC tumors from Algerian patients. Clinicopathological features are summarized in (Table 1). The gender distribution was 58 males (56.8%) and 44 females (43.2%) (p = 830) with 48 patients (47.1%) < 50 years and 54 > 50 years (52.9%) (p = 0.830) range (18–83 years). Tumors location was distributed to the proximal (28.4%), distal (36.3%) and rectum (35.3%) (p = 0.130). Histological analysis demonstrated 41 (40.2%) well differentiated 39 (38.2%) moderately differentiated and 22 (21.6%) poorly differentiated adenocarcinomas (p = 0.130). Of the 102 tumors analyzed, KRAS mutations in exon 2 codons 12–13 were detected in 32/102 (31. 4%). The most prevalent mutation observed in codon 12 was G12D (43.8%), G12A (25%), followed by G12V (9.3%), G12C and G12S (6.3%) respectively and in codon 13 we found G13D (9.3%) (Table 2). Analysis of KRAS mutations showed 40.6%, in the proximal colon, 25% in the distal colon and 34.4% in the rectum. Correlation of KRAS mutations with gender showed that KRAS mutations were more frequently observed in women (62.5%) than in men (37.5%), and no significant difference was found in other variables. MSI and BRAF status were determined of all colorectal tumors cases and MSI-High (MSI-H) was detected in 19 of 102 (18.6%) tumors patients analyzed. 17 of 19 (89.5%) tumors showed instability in all 5 markers used in this study. BRAF mutation was observed in 5 cases (4.9%) analyzed with a wild type (Wt) profile in KRAS. The majority of tumors MSI-H, 15/19 (79%) were BRAF Wild Type (Wt). Molecular markers included MSI status, KRAS and BRAF mutations in 102 tumors of Algerian patients analyzed are regrouped in (Table 3). As shown in (Table 4) correlation of tumor location and molecular markers shown KRAS mutation at the right colon 40.6% (13/32) versuss 25% (8/32) in the left colon and 34.4% (11/32) in the rectum. 3 of 5 tumors (60%) with v600E BRAF mutation were located significantly in proximal colon and found in patients older of 50 years.

The results of this preliminary study show that 70/102 (68.6%) Algerian patients with CRC could benefit from anti-EGFR therapy. EGFR has become an important target for treatment decisions making of CRC. So it has become important to multiply the platforms for determination of molecular alterations in tumors in Algeria, which would facilitate the prescription of target therapy in the country. Activating KRAS mutations in codons 12 and 13 have a clinical impact on patients with CRC [11] and predict resistance to anti-EGFR antibodies, cetuximab a human-mouse chimeric IgG1 and panitumumab a human IgG2 monoclonal antibodies which have been entered in the personalized treatment in patients with CRC [12]. KRAS is part of the EGFR signaling pathway downstream to EGFR. Activation of the pathway leads to the modulation of angiogenesis, cell migration, proliferation and metastasis formation. In this study, we identified 31.4% of tumors with KRAS codons 12, 13 mutations and a higher frequency in women, 20/32, (62.5%) versus 12/32 (37.5%) in males. The most prevalent mutations were observed in codon 12 with (90.7 %) than in codon 13 (9.3%). We found that the most frequent location was in the proximal colon with 40.6% versus 25% in the distal colon. No significant age difference was found in our study between patients with KRAS mutated tumors and Wt KRAS tumors and no association has been found between KRAS mutations and MSI phenotype, this is in accordance with results of other research studies [13][14]. Recently, prospective and retrospective analyses demonstrated that patients with tumors KRAS and NRAS Wt in exons 2, 3 and 4 predict benefit from anti-EGFR therapy associated with chemotherapy [15] [16]. The KRAS mutations are also detected in lung [17], pancreatic and cervical cancers and the anti-EGFR therapies have shown to be effective in patients with CRC. The prognostic role of KRAS mutations is more debated, and has been associated with a worse prognosis in some studies [14]. The BRAF Wt is also required for response to cetuximab or panitumumab [18] suggesting that BRAF analysis should be used with KRAS for the selection of the patients [19]. We observed that activating mutation of BRAF was (5%) in this study whereas it is about 10–20% in the majority of studies performed on sporadic CRC in western countries [20]. This mutation was found to be more frequent in the right colon and old age at presentation 3/5 (60%). However, no activating mutation has been observed at codon 600 of BRAF in 88 cases analyzed in western Africa (Ghana) with a high frequency of MSI-H [21] and the highest frequency is reported in the United States (21%). A low incidence was observed in Taiwan 1% [22], in Morocco 1.6% [23] and 5.4% [24], in Tunisia 2% [25], in Saudi Arabia 2.5% [26], in China, 3.8% [27], Japan, 6.5% [28], Korea and 9.6% [29]. These variations could be attributed to ethnic differences and the effect of other environmental and genetic factors. The implication of genetic factors in a population where the overall incidence of CRC is low, would suggest a greater proportion of familial versus sporadic cases. More studies are needed to confirm these differences. The BRAF mutation detection could have been also influenced by the mutation analysis methodology. Variety of methods including Sanger sequencing, pyrosequencing, high resolution melting, allele -specific PCR and new generation sequencing have been used and may have contributed to the wide variations in the prevalence of those mutations. The BRAF mutation was proposed as a marker to discriminate between sporadic cancer and HNPCC labeled also Lynch syndrome [30]. Its incidence which is 3–5% of CRC cases in western countries is higher in Algeria (7–10%) [4]. BRAF mutation is associated with MSI-H through its relationship to high-level CpG island methylator phénotype (CIMP) and with worse prognosis [31][32]. The prognostic value of MSI is influenced by the BRAF status which is a genetic consequence of a MisMatch Repair genes (MMR) defect [33]. Other studies suggested that the prognostic effects of BRAF mutations depended on the MSI status [34][35]. A negative prognostic effect of BRAF mutations was reported only for MSS patients but not for patients with MSI [36] and a predictive effect of BRAF for response to anti-EGFR therapy in metastatic colorectal cancer is not required for treatment decision but it may be useful as a prognostic factor and could be used for better management of patients with CRC because of its implication on microsatellite instability [14]. Our results showed approximately 80% tumors MSI-H / BRAF Wt and suggest the possible presence of HNPCC syndrome. MSI-H tumors in CRCs may be sporadic or associated with Lynch syndrome and germline mutation analysis is required for tumors MSI-H that are BRAF wild type because mutations in the BRAF gene was found in sporadic MSI-H tumors but not in HNPCC syndrome [37][38].

In conclusion, we have found that the clinicopathologic characteristics in Algerian patients with CRC are similar to those reported in other studies. As a result, a total of 70% Algerian patients could benefit from anti-EGFR therapy. RAS testing had an impact on therapeutic strategy and must be realized in all oncology departments in Algeria. In order to reduce the time of the process and to prescribe targeted therapy for the patients with CRC in the daily clinical practice. A limitation of this study is the absence of data of KRAS exons 3–4 and NRAS exons 2-3-4 which have been established as predictive markers of the response to EGFR-targeted therapy. Our study suggests also the possibility of the presence of the HNPCC syndrome and use of BRAF molecular analysis is only a step before germline genetic testing. Screening program should be set up to determine the real incidence rate of the HNPCC which tends to be more frequent in Algeria than in western countries. Further studies including a large number of patients are needed to confirm our results.

This work was supported by the Direction Générale de la Recherche Scientifique et du Développement Technologique in Algeria, (DGRSDT), Ministére de l'Enseignement Supérieur et de la Recherche Scientifique (MESRS) and Agence Thématique de la Recherche Scientifique Algéria (ATRS).

We thank, Helene Blons , Pierre Laurent Puig: European Hospital Georges Pompidou (HEGP), Paris, France and Jaqueline Lehmann, Hughes De Thé: Saint Louis Hospital, Paris, France for initiation of analysis molecular biology tests.

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