We compared mutations detected in DNA from stool of CRC patients and healthy individuals. Applying NGS analysis on stool DNA from CRC patients and healthy individuals using the Ion AmpliSeq Colon and Lung Cancer panel we were able to successfully get results in 96% of patients, thus overcoming the problem of low DNA quantity/quality associated with stool analysis 24,25.
From the DOC patients, who had only TP53 mutations, two patients were DOC and one patient had metastases approximately 24 months after their therapy. Moreover, %18 of patients had double mutations and two of them had mutation in both NRAS and TP53 genes who were DOC. It can show that mutation in TP53 gene or both TP53 and NRAS genes could reduce patient’s survival compare to patients with no mutation or mutation in other genes. A study in 2014 showed that patients with mutation in KRAS, NRAS, TP53 and PIK3CA genes had dramatically shorter survival than patients with any other mutations 26. These results also showed that patients with more than one mutation had lower survival rate than patients with one mutation or no mutation 26.
Interestingly NRAS mutations seen in 6 patients (12%), four females with codon 12 (p.G12S, p.G12V) or 13 (p.G13V) (mean age 66 years) in stage II of primary tumor located in left colon. A female and a male patient detected with codon 61 (p.Q61K) mutations. Cercek et al found that patients with NRAS mutations, particularly in exon 3, comprised aggressive subset of metastatic colorectal cancer with overall reduced survival than patients with KRAS mutations or RAS wild-type tumors. They also showed that African Americans CRC patients with NRAS mutations are mostly located at left side colon 27. In a large scale study on Iranian metastatic CRC specimens from 1000 patients using pyrosequencing and high resolution melting (HRM) analysis, KRAS mutations detected in 33.6% cases (codon 12 and 13) 28. Another study from west of Iran analyzed the KRAS, BRAF and NRAS genes on formalin fixed paraffin embedded (FFPE) tissues from 33 CRC patients showed KRAS mutations (codon 12 and 13) in 12 patients (27.3%) and no mutations in NRAS and BRAF 29. Similarly, another study from Northeastern region of Iran showed that none of the 87 tissue samples from CRC patients had mutation in NRAS gene which is similar to the results from West part of Iran 30. Data from 7 European countries showed that mutations in NRAS is predictors of lower response to anti-EGFR MoAbs treatment in CRC 31. Jouini et al showed that NRAS gene mutated in 6.9% of the 129 Tunisian colorectal cancer specimens 32. In Finnish stool DNAs, only one patient with rectum adenoma showed NRAS Q61R mutation 24, while in another cohort of stool samples from Iranian CRC patients, none of them showed NRAS mutation in codon 12, 13 or 61 25. All the three studies just mentioned, performed by the same NGS gene panel in the same laboratory at Helsinki. The exact reason for the higher frequency of NRAS mutations in the present Iranian cohort is not clear. The samples for this study were collected mainly from Isfahan province located at the central part of Iran while samples for earlier Iranian cohort were mainly collected from Tehran region 25. A study on RAS mutations in 353 Chinese CRC patients showed that NRAS alterations in tumor tissues were more frequent in female CRC patients than in male patients (75.0% vs 41.1%), which is quite similar to the observation in our study, with 5 out of 6 Iranian patients with NRAS mutations were female 33. NRAS mutations reported to be associated with poor prognosis and resistance to anti-EGFR monoclonal antibodies in CRC patients. A retrospective European Consortium analysis of 1022 tumor DNA samples of metastatic CRC found mutations in 40% of KRAS, in 2.6% of NRAS, in 4.7% of BRAF and in 14.5% of PIK3CA. In wild type KRAS patients, the presence of any mutations in NRAS, BRAF or PIK3CA was associated with lower response rates, disease control rates and shorter overall survival. These findings suggested that up to 60–65% of patients with wild type KRAS tumors are resistant to anti-EGFR agents, which is accounted for presence of other mutations 31,34. An NGS study on 91 Brazilian CRC patients demonstrated that driver mutation in the APC, TP53 and KRAS genes were significantly high in CRC patients 35. They also showed that at least one gene involved in the MAPK-ERK pathway (KRAS, BRAF, and NRAS) had mutation in 68% of cases. Another study on 145 CRC patients by targeted deep sequencing showed that the TP53 gene was most commonly mutated gene and was significantly linked to lower disease-free survival which is similar to our study 36. However, in TCGA datasets APC mutation is mostly common followed by TP53 and KRAS mutations.
In our study, detection of relatively high percentage of NRAS mutation (12%) among CRC samples from central part of Iran would emphasis the necessity of paying more attention to NRAS mutation analysis at least for patients from this particular ethnicity. Our stool DNA mutation analysis showed some aspects of uniqueness which would be helpful for more accurate personalized treatment decision and development of early population specific noninvasive CRC screening tests.
To find out the likelihood of developing CRC in control individuals with normal colonoscopy results but detected with mutations in their stool DNA we organized a following up program. As mentioned earlier, hotspot mutations detected in 7 non-affected control individuals (Table 2). Although based on the colonoscopy examination they categorized as unaffected normal individuals but later in follow up period 3 of them developed polyps and experienced polypectomy. It is obviously showing the importance of genetic analysis for those who are undergoing colonoscopy even though emerging with normal colonoscopy results. Recent studies analyzing the mutations in normal individuals have shown that mutations increased with age in colon tissue 37,38. Since colonoscopic report for these 7 individuals reflect a polyp free condition, presence of some particular hotspot mutations in their stool DNA analysis emphasis the necessity of surveillance colonoscopy at regular time intervals for timely detection of any probable polyp development.
Some mutations reported to be an early event in tumor development 14. Presence of tumor driver mutations in normal individuals related to tumor initiation and progression like KRAS, ERBB gene family, TP53 and so, are important criteria based on which we can categorize normal individuals as high-risk group for developing CRC in future and regular colonoscopy surveillance should be recommended.