TP53 and NRAS are the Most Frequently Mutated Genes in Stool DNA of CRC Patients from Central Part of Iran

TP53 and NRAS are the Most Frequently Mutated Genes in Stool DNA of CRC Patients from Central Part of Iran | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article TP53 and NRAS are the Most Frequently Mutated Genes in Stool DNA of CRC Patients from Central Part of Iran Farideh Saberi, Virinder Sarhadi, Omar Youssef, Arto Kokkola, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-792966/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Colorectal cancer (CRC) rated among the three most diagnosed cancers and the fourth main cause of death worldwide. CRC is a curable cancer provided to be diagnosed at its early-stage. Colonoscopy, stool and blood-based tests are in use for CRC diagnosis/screening. Due to low patient compliance, low specificity and high rate of false results, more reliable methods with desired level of detection accuracy and high patients’ compliance are highly demanded. Detecting hotspot mutations in stool DNA emerged as a robust noninvasive alternative, but due to the different genetic background of various populations and hence varied mutation spectrum/prevalence, prior assessment of CRC mutations in the population is essential. Here, we have evaluated stool DNAs from CRC patients and controls using a NGS based 22 genes panel. Hotspot mutations in NRAS , FGFR3, SMAD4 and TP53 genes had higher prevalence among the CRC patients compare to normal controls. Patients were followed up in their post-surgical period. Six of them (12%) with TP53 mutations (2 patients had NRAS mutation as well) were died of cancer. Those harboring mutations in TP53 and/or NRAS would be regarded as high risk and should be provided with special care. Scientific Communication Pathology Stool DNA colorectal cancer mutations next generation sequencing Figures Figure 1 Figure 2 Introduction Colorectal cancer (CRC) is one of the common malignancies worldwide. Incidence and mortality rates are still high and the number of deaths due to CRC are expected to increase to 1.1 million by 2030 1 . Usually precursors of CRC are benign adenomatous colonic polyps (adenomas) 2 . Burden of adenomas are depending on factors like their size, location and malignant grade. Transformation from adenoma to CRC is a decade long stepwise and gradual process 3 with its early detection and resection considerably reduces CRC incidence and mortality 4 , 5 . The gradual nature and accumulation of different genetic alterations during a comparatively long period of time creates excellent opportunity for screening through colonoscopic surveillance. Therefore, CRC is well compatible with the World Health Organization criteria for a diseases best fitted for screening 6 . If high risk patients for CRC undergo regular surveillance colonoscopy and affected individuals have access to improved surgical procedures and adjuvant therapies, CRC mortality would reduce considerably 7 . The five-year CRC survival rate is as high as 90% for earlier stages localized cancer but decrease to even less than 5% while approaching to late stage metastatic disease 8 . In spite of CRC screening proved beneficial for early detection of CRC 8 , 9 and resulted in reduction of CRC mortality and incidence, but still participants in screening programs remains too low; 65% in the US 10 and from 1.9–54% across Europe OECD: Screening, survival and mortality for colorectal cancer 11 . The situation is even more disappointing when considering CRC screening rates in underdeveloped countries. Analysis of 10 years data from Germany regarding individuals’ attitude to undertake colonoscopy for CRC screening was 23% for men and 26% for women 12 . Further analysis of the German patients revealed that in addition to colonoscopy, inclusion of noninvasive tests in screening and availability of broader range of screening choices, compliance and interest to undertaking CRC screening increased from around 25% to as high as 98% 13 . Gradual accumulation of driver mutations over the time will ultimately manifested as a recognizable tumor. Therefor molecular alterations in the colonic epithelium remained undetectable by colonoscopy unless the accumulated genome mutations represent as a distinct morphological abnormality which is late and shows limitation of colonoscopy 14 . Therefore, normal colonoscopy outcome for an individual doesn’t exclude molecular alterations in the colon epithelium. Finding driver mutations in stool sample of CRC patients or those diagnosed as normal individuals in colonoscopy examination would be helpful to further recognize those at higher CRC risk based on their mutation status 14 . It is demonstrated that stool DNA assay is a simple, non-invasive method for analyzing genetic alterations in patients with gastrointestinal neoplasms 15 . Tumors shed more cells in the large-bowel lumen than normal epithelium lining and shedding occurs at all stages of tumor initiation and progression, making it an appropriate target for analyzing pathological in the DNA and can be a reliable approach for early detection of malignancies 16 , 17 . A multi target stool DNA assay for CRC screening was approved by the FDA in 2014 and its application has also increased rapidly in other countries variations 18 – 20 . Variability exists between different populations regarding underlying molecular etiology of CRC. Comparison of Middle East and Western populations revealed the aspects of inconsistency in CRC pathways and mutated genes 21 . Iran, like many other countries especially in Asian continent, is inhabited by a multi ethnic genetically heterogeneous population. Comparison of mutation spectrum of beta thalassemia as a most prevalent single gene disorder in Iran, and many other countries, confirmed the extreme genetic heterogeneity of Iranian population 22 . Therefore, need to study extensively and comparative studies regarding molecular drivers of CRC between Iranian patients and those with different genetics background, like Western Europeans would be beneficial. For this reason we compared mutation spectrum of Iranian CRC patients and controls with Finnish CRC patients as a standard European population performed in the same laboratory at Helsinki university 23 . In the present study, we performed mutation analysis using a 22 gene targeted NGS panel on DNA extracted from stool samples of early stages of CRC patients and their healthy counterparts from central region of Iran, then the patients and control group were followed up. The outcome of this study may facilitate the development of stool-based modality for population specific early-stage CRC risk prediction. Beside colonoscopy which is a gold standard diagnostic/surveillance method, availability of other noninvasive with desired level of detection sensitivity for screening approaches would encourage at risk individuals to undertake regular screening tests. Results The success rate of amplicon based NGS analysis from stool samples was 96% and 98% for patients and controls respectively. Two CRC samples and one normal sample were removed from NGS assay because of poor DNA quality. Among the CRC patients, 18 patients diagnosed with stage I and rest of them categorized as stage II CRC. Average age of CRC patients and controls was 62.8 years (range 43–87) and 64.6 years (range 49–88) respectively. The majority of participants were male (62%). The location of the tumor was more frequent in colon. The characteristics of all 99 individuals included in the study are presented in Table 1 . Table 1 Characteristics of patients and healthy controls included in the study CRC Iranian Normal Iranian CRC Finnish Normal Finnish Male N = 31 N = 28 N = 21 N = 4 Female N = 19 N = 21 N = 12 N = 9 Average age (years) 62.8 (range 43–87) 64.6 (range 49–88) 72 (range 53–85) 44 (range 19–65) Stage I = 18 II = 32 Rectal cancer N = 10 N = 22 Colon cancer N = 40 N = 11 Total 50 49 33 13 Among the CRC patients, mutations were seen in 24/50 patients (48%) and in the 13/49 (26.5%) control samples. Hotspot mutations were noted in 16/50 patients and 7/49 controls. Four of the patients and two of the control samples had more than three mutations. Rest of the samples detected with no mutation with the NGS gene panel we used and need to be evaluated by whole exome sequencing (WES) in order to pinpoint the underlying molecular defects. Out of 22 genes in the Ion AmpliSeq panel, mutations were seen in 19 genes in patients and 17 genes in control samples; mutations in MAP2K and NRAS were seen only in patients and not in controls. A total of 34 hotspot mutations and 37 novel mutations were detected in patient samples, while 13 hotspots and 34 novel mutations were detected in control individuals (Fig. 1 ). TP53 was the most frequently mutated gene in patients with a total of 13 mutations detected, while SMAD4 and DDR2 were the most frequently mutated genes in controls, with 7 mutations in each gene. All mutations in TP53 were hotspot mutations. Among the patients, hotspot mutations were most common in TP53 , followed by NRAS , FGFR3 and SMAD4 (Table 2 ). Most of the mutations in controls were novel and only seven individuals had hotspot mutations. The AKT , CTNNB1 and NOTCH1 genes had no mutation in both groups. The recurrently occurring hotspot mutations among patients were codon 273 of TP53 , seen in 4 patients (2 with R273C, 2 with R273H) and NRAS codon 12 (3 patients) and codon 61 mutations (2 patients). The entire list of mutations (COSMIC and novel) in patients and normal individuals can be found as Supplementary Table S1 online. Table 2 Comparison of hotspot mutations detected in stool DNA of Iranian and Finnish colorectal cancer patients and controls Finnish CRC Iranian CRC Iranian Controls Gene Patient Mutation Patient Mutation Individual Mutation AKT1 28 E17K ALK C16 E1197K KRAS 55 G12V T15 G13V C9 D119G 28 G12D BRAF T1 G606R C16 T599I CTNNB1 ERBB2 EGFR C43 Q486* ERBB4 T1 E317K C9 S239L T9 C304F FBXW7 T45 H379P NRAS T13 G13V T4 Q61K T5 Q61K T40 G12V T9 G12S T43 G12V PIK3CA T9 A1066T C7 A1020V PTEN C16 R234Q SMAD4 T43 R361H T9 R135* T9 R420H STK11 22 F354L C9 A205T FGFR1 T9 A299V C9 D165DD FGFR2 FGFR3 T1 V392M T27 S373G T23 F386L MET T9 S349G T23 M1268T NOTCH1 TP53 12 P72A T1 E358K C23 T230A 31 Y205D T1 G279E C33 R273C 55 T172fs T10 P151S C9 E285K 68 Y163H T27 Y205H C10 M169T T3 R273C T5 E286K T4 R248W T6 R273H T40 R248W T27 N235S T52 R273C T44 G245S T43 R273H Mutations in Finnish cohort: Among the Finnish CRCs patients, hotspot mutations were seen in 6/33 patients (18%) and two novel mutations were seen in the control group. All mutations in CRC group were hotspot and two patients had more than one mutation, whereas no hotspot mutation was seen in the control group. Out of the 22 genes in the Ion AmpliSeq panel, eight mutations were seen in four genes, AKT1 , KRAS , STK11 and TP53 in six patients. TP53 was the most commonly mutated gene followed by KRAS (Table 2 ). Comparison of hotspot mutations in Iranian and Finnish CRC patients and controls Although data related to Finnish patients was published already but for the sack of comparison of two typical ethnic representatives of Middle-East and Europe, the results of stool DNA analysis of the Finnish cohort which have been worked out by the same gene panel, we review and included their results here. Overall, higher proportion of Iranian patients showed mutations in their stool DNA compared to Finnish patients (32% and 18% respectively), with 34 mutations detected in 32% Iranian patients compared to eight mutations in 18% Finnish patients. TP53 was the most commonly mutated gene in both the Iranian and Finnish cohorts. The second most commonly mutated gene was a RAS gene, being NRAS in Iranian patients and KRAS in Finnish patients. The frequency of NRAS mutations was significantly higher in Iranian patients than that in Finnish patients. Mutations in AKT1 were noted only in Finnish patients while mutations in MET , FGFR3 , SMAD4 , NRAS , FBXW7 , were seen only in Iranian patients (Fig. 2 ). Similar to the pattern seen for patients, mutations were more frequent in Iranian control group, with 7 out of 49 healthy Iranian individuals showed mutations, while no hotspot mutations were seen among Finnish healthy individuals. Iranian patient’s treatment From the CRC group, 29 patients (18 patients with stage I and 11 patients from 32 patients with stage II of cancer) were in group A and only did surgery according to their pathology reports, while 21 patients in group B received adjuvant treatment in addition to surgery. From group A one patients died of independent cancer (DOIC) with no hotspot mutation and two patients died of cancer (DOC), one patient had hotspot mutation in TP53 and one patient with novel mutations in ERBB2 , FGFR3 and MET genes. From group B, 6 patients (12%) were died. DOC (one patient with COSMIC mutations in NRAS, ERBB4, MET, PIK3CA, FGFR1 and SMAD4 genes and numerous novel mutations in other genes, one patient with COSMIC mutations in BRAF, ERBB4, FGFR3 and TP53 genes in addition to numerous novel mutations, one patient with COSMIC mutations in SMAD4 , TP53 and NRAS genes, two patients only with TP53 mutation and one patient with mutations in TP53 and NRAS genes). From this group one patient was DOIC with no mutation (Table 3 ). Table 3 Following up data of the CRC patients and characteristics of those how died due to cancer Code DOIC/DOC* Stage/TNM Grade^ Histological type P53 COSMIC mutation NRAS COSMIC mutation Other COSMIC mutations Group A (29 Patients) T10 T30 T2 DOC DOC DOIC I/T2N0MX I/T1N0MX I/T1N0MX p w w Adenocarcinoma // // yes no no no no no no no no Group B (21 Patients) T1 T3 T9 T40 T43 T44 T48 DOC DOC DOC DOC DOC DOC DOIC II/T4N0MX II/T3N0MX II/T3N0MX II/T3N0MX II/T3N0MX II/T4aN0MX II/T3N0MX p p w p p p w // // // // // // // yes yes no yes yes yes no no no yes yes yes no no FGFR3,ERBB4,BRAF no ERBB4,MET,FGFR1,SMAD4, PIK3CA no SMAD4 no no *DOI: Died Of Cancer, DOIC: Died of Independent Cancer, ^ Poorly (p) or well (w) differentiated Also, the follow up results showed that three normal individuals (more than 6%) encountered gastrointestinal complications such as bleeding and abdominal pain after 33 months. All these three individuals were detected with mutations (one patient in BRAF, PTEN and ALK genes, one patient in KRAS, ERBB4, STK11, FGFR1 and TP53 genes and one patient only in TP53 gene). One of the controls detected with mutation in five genes showed polyps (4mm) in his recent colonoscopy examination. Also, one of the control samples detected with two novel mutations in FGFR3 gene and three mutations in SMAD4 and other novel mutations, had gastrointestinal complications in follow up. Discussion 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. Materials And Methods Patients and stools collection and follow up Iranian samples: The stool samples collected from 52 patients diagnosed with CRC by colonoscopy before starting any cancer treatment and 50 healthy individuals with normal colonoscopy results. Samples were collected from July 2016 to December 2017 at Alzahra university hospital, Isfahan Iran. The study was approved by Isfahan University of Medical Sciences Ethics Committee and informed written consent was obtained from all participants recruited to this study. Stool samples were stored at -80 o C until DNA extraction. Patients followed up since their first surgery. The median follow-up period was 30 months (range 23–39 months), for this aim patients were classified in two groups based on their pathology report (stage I and II). This study was approved by ir.mui.rec.1394.3.936 code at Isfahan University of medical science ethnical committee. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all individual participants included in the study. Finnish samples: The stool samples from Finnish CRC patients were collected between April 2015 to May 2017 from patients referred to Kirugi, Meilahti and Jorvi hospitals in Finland. The sequencing results of stool DNA samples collected from 33 CRC patients and 13 healthy individuals (Table 1 ) of Finnish origin published previously 24 . Mutation analysis by NGS DNA isolation QIAamp DNA stool mini kit (Qiagen GmbH, Hidden, Germany) was used for DNA extraction from approximately 200 mg stool samples according to the manufacturer instructions. Extracted DNAs were quantified by Qubit 2.0 Fluorimeter (Life Technologies, Carlsbad, CA, United States) using the Qubit dsDNA BR Assay Kit and stored at -20 o C. Library preparation DNA libraries were prepared from 20 ng of DNA per sample using Ion AmpliSeq Library Kit 2.0 (Life Technologies, California, US) following the kit guidelines. Ion AmpliSeq Colon and Lung Cancer panel v2 (Life Technologies, California, United States) consisting of a primer pool for analyzing 504 mutational hotspots from 92 amplicons was used to amplify the stool DNA. The genes included in this panel are: AKT1 , ALK , KRAS, BRAF, CTNNB1, DDR2, ERBB2, EGFR, ERBB4, FBXW7, NRAS, PIK3CA, PTEN, SMAD4, STK11, FGFR1, FGFR2, FGFR3, MAP2K1, MET, NOTCH1 and TP53 that are commonly mutated in colorectal cancer. Amplified libraries were purified by Agincourt AM Pure XP beads (Beckman Coulter Genomics, High Wycombe, UK). Qubit 2.0 Fluorimeter and the Qubit dsDNA HS assay kit were used to quantify the barcoded libraries. Sequencing Before sequencing, templates were prepared using the Ion OneTouch TM 2 system (Life Technologies, California, United States); this system performs emulsion PCR for template amplification and its further purification and enrichment. Eventually, sequencing was accomplished using the Ion PGM™ Sequencing Hi-Q View Kit V2 and the amplified and enriched libraries loaded on Ion 316S chips and sequenced on the Ion Personal Genome Machine System (PGM™; Life Technologies, California, United States). Data analysis To analyze data and to evaluate run performance, Torrent suite software version 5.2.2 (Life Technologies) was used. Integrative Genomics Viewer (IGV v 2.2; Broad Institute, Cambridge, MA, United State) used for visual inspection of the aligned reads. All variants identified were further filtered by quality score (> 15) and mutant allele frequency (> 5%). We used SIFT and PROVEAN in-silico tools to identify all those variants which resulted in non-synonymous, stop codon or frameshift amino acid changes and those that are predicted to have deleterious protein product. Previously reported somatic mutations and novel mutations were checked in COSMIC and dbSNP databases. Variants that are not recorded in either of the databases are labeled as ‘Novel’. Kaplan-Meier analysis was performed to estimate the survival curve of the patients with different rate of mutations. Conclusion Early-stage stool DNA mutation analysis from CRC stage I/II patients would be a suitable, non-invasive approach for population specific screening. Our result shows TP53 and NRAS are the most frequently mutated genes in stool DNA of CRC patients from central part of Iran. Also comparing CRC and control group mutations can help to improve our understanding of mutations involved in cancer development mutation spectrum in other genes included in the NGS targeted panel that we used for this study is relatively different comparing to the previously reported mutation spectrum using the same NGS panel on ethnically different Iranian population as well as Finish CRC patients and controls. Our present data would help in development of stool-based CRC screening tools as well as more precisely selection of those who may benefit from anti EGFR monoclonal antibody therapy. Declarations DATA AVAILABILITY All data generated or analyzed during this study are included in this published article and its Supplementary Information file. Acknowledgments Research reported in this publication was supported by Elite Researcher Grant Committee under award number 971202 from the National Institute for Medical Research Development (NIMAD), Tehran, Iran provided to Rasoul Salehi. The study was funded by the Sigrid Juselius Foundation grant to SK. Author Contributions FS, VS and OY contributed to acquisition and analysis of data and drafting the manuscript, AK, PP contributed to the design and execution of the work, SK, and RS contributed to the concept development, manuscript revision, data interpretation and approval. Corresponding Author Correspondence to Rasoul Salehi. Additional Information Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Ethics Declarations Competing interests The authors declare no potential conflicts of interest. References Ferlay, J. et al. Cancer incidence and mortality worldwide: IARC CancerBase. GLOBOCAN 2012 , v10 , 11 (2012). Morson, B. The evolution of colorectal carcinoma. Clinical radiology , 35 , 425–431 (1984). Winawer, S. et al. 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Nature , 538 , 260 (2016). Additional Declarations No competing interests reported. Supplementary Files SupplementaryDataset1.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-792966","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":50248860,"identity":"0d13d701-3537-494f-9776-2d82592ba2f5","order_by":0,"name":"Farideh Saberi","email":"","orcid":"","institution":"Isfahan University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Farideh","middleName":"","lastName":"Saberi","suffix":""},{"id":50248861,"identity":"a94e4e60-4787-4656-91e2-9cdf237c3072","order_by":1,"name":"Virinder Sarhadi","email":"","orcid":"","institution":"University of Helsinki","correspondingAuthor":false,"prefix":"","firstName":"Virinder","middleName":"","lastName":"Sarhadi","suffix":""},{"id":50248862,"identity":"be1806ef-c91e-48ad-a9ff-4f977fefcae6","order_by":2,"name":"Omar Youssef","email":"","orcid":"","institution":"University of Helsinki","correspondingAuthor":false,"prefix":"","firstName":"Omar","middleName":"","lastName":"Youssef","suffix":""},{"id":50248864,"identity":"4ccf5cec-2d82-4973-a081-5f32fc43eac0","order_by":3,"name":"Arto Kokkola","email":"","orcid":"","institution":"University Central Hospital of Helsinki","correspondingAuthor":false,"prefix":"","firstName":"Arto","middleName":"","lastName":"Kokkola","suffix":""},{"id":50248866,"identity":"dab3eaac-45f5-4007-bd1c-9164e7a4b653","order_by":4,"name":"Pauli Puolakkainen","email":"","orcid":"","institution":"University Central Hospital of Helsinki","correspondingAuthor":false,"prefix":"","firstName":"Pauli","middleName":"","lastName":"Puolakkainen","suffix":""},{"id":50248867,"identity":"587d2b10-6941-437b-9e26-7ecbd6a0b3ca","order_by":5,"name":"Sakari Knuutila","email":"","orcid":"","institution":"University of Helsinki","correspondingAuthor":false,"prefix":"","firstName":"Sakari","middleName":"","lastName":"Knuutila","suffix":""},{"id":50248869,"identity":"9545933e-d671-4a94-b52d-0bf738e5ca86","order_by":6,"name":"Rasoul Salehi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4UlEQVRIie3OsarCMBSA4ePSLgeyHrHgKxQKteDgq6QUepeC6x3u0MmX8D06OUQOODlLQYcGodMddBEnMdjFxUY3h/xwCAl85AC4XN9aAwqFOckMPl6kjUhDhuWnBELVEXuT0tcn+XsIoh2v60HBAfjcgF69JoHCiOS2xXifZ8mgYgTMQ5Dta0JmeUoXbEgR04NAYVZVfcQ/XtMbY7ScXzoi/m0EYkpLxnBUeB0h6y8YJ3LDSPs8StLqBz1qQ2VbrD798UwsM12fq+lMiEzraw+BcfN0kWY8M33A5XK5XG90B+tOSwnSj6sdAAAAAElFTkSuQmCC","orcid":"","institution":"Isfahan University of Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Rasoul","middleName":"","lastName":"Salehi","suffix":""}],"badges":[],"createdAt":"2021-08-08 08:14:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-792966/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-792966/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":13715383,"identity":"d8840daf-123a-4b28-91c8-8a9ade8e284b","added_by":"auto","created_at":"2021-09-17 14:47:25","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":444153,"visible":true,"origin":"","legend":"Genes with novel and COSMIC mutations in stool DNA of Iranian colorectal cancer patients and controls.\n\n","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-792966/v1/8d1e6357aad93fefbc20160c.jpg"},{"id":13715385,"identity":"4db4c8f2-a059-41d9-b92c-06f787f3ae5b","added_by":"auto","created_at":"2021-09-17 14:47:25","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":264865,"visible":true,"origin":"","legend":"Comparison of hotspot mutations seen in stool DNA of colorectal cancer patients and controls of Iranian and Finnish origin","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-792966/v1/b0cb7552407bd8eca022024b.jpg"},{"id":13715921,"identity":"2ca51b2e-c98d-4ee7-b6ef-4ad5a948e334","added_by":"auto","created_at":"2021-09-17 14:50:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":585128,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-792966/v1/fbadf7a9-8300-4b18-9124-8aa46334cb55.pdf"},{"id":13715920,"identity":"7c58f660-1938-43a1-8cbd-4588327f9311","added_by":"auto","created_at":"2021-09-17 14:50:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":579347,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-792966/v1/169af5e0-955b-4a24-9a67-17fc2041b1a0.pdf"},{"id":13715919,"identity":"1d143ee1-0e05-4f0b-8d1c-ef6275e1dfd4","added_by":"auto","created_at":"2021-09-17 14:50:25","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":291698,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryDataset1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-792966/v1/0766362026104ff49de0a4e5.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eTP53 and NRAS are the Most Frequently Mutated Genes in Stool DNA of CRC Patients from Central Part of Iran\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eColorectal cancer (CRC) is one of the common malignancies worldwide. Incidence and mortality rates are still high and the number of deaths due to CRC are expected to increase to 1.1\u0026nbsp;million by 2030 \u003csup\u003e1\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eUsually precursors of CRC are benign adenomatous colonic polyps (adenomas) \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Burden of adenomas are depending on factors like their size, location and malignant grade. Transformation from adenoma to CRC is a decade long stepwise and gradual process \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e with its early detection and resection considerably reduces CRC incidence and mortality \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. The gradual nature and accumulation of different genetic alterations during a comparatively long period of time creates excellent opportunity for screening through colonoscopic surveillance. Therefore, CRC is well compatible with the World Health Organization criteria for a diseases best fitted for screening \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. If high risk patients for CRC undergo regular surveillance colonoscopy and affected individuals have access to improved surgical procedures and adjuvant therapies, CRC mortality would reduce considerably \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe five-year CRC survival rate is as high as 90% for earlier stages localized cancer but decrease to even less than 5% while approaching to late stage metastatic disease \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. In spite of CRC screening proved beneficial for early detection of CRC \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e and resulted in reduction of CRC mortality and incidence, but still participants in screening programs remains too low; 65% in the US \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e and from 1.9\u0026ndash;54% across Europe OECD: Screening, survival and mortality for colorectal cancer \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. The situation is even more disappointing when considering CRC screening rates in underdeveloped countries. Analysis of 10 years data from Germany regarding individuals\u0026rsquo; attitude to undertake colonoscopy for CRC screening was 23% for men and 26% for women \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Further analysis of the German patients revealed that in addition to colonoscopy, inclusion of noninvasive tests in screening and availability of broader range of screening choices, compliance and interest to undertaking CRC screening increased from around 25% to as high as 98% \u003csup\u003e13\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eGradual accumulation of driver mutations over the time will ultimately manifested as a recognizable tumor. Therefor molecular alterations in the colonic epithelium remained undetectable by colonoscopy unless the accumulated genome mutations represent as a distinct morphological abnormality which is late and shows limitation of colonoscopy \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Therefore, normal colonoscopy outcome for an individual doesn\u0026rsquo;t exclude molecular alterations in the colon epithelium. Finding driver mutations in stool sample of CRC patients or those diagnosed as normal individuals in colonoscopy examination would be helpful to further recognize those at higher CRC risk based on their mutation status \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIt is demonstrated that stool DNA assay is a simple, non-invasive method for analyzing genetic alterations in patients with gastrointestinal neoplasms \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Tumors shed more cells in the large-bowel lumen than normal epithelium lining and shedding occurs at all stages of tumor initiation and progression, making it an appropriate target for analyzing pathological in the DNA and can be a reliable approach for early detection of malignancies \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. A multi target stool DNA assay for CRC screening was approved by the FDA in 2014 and its application has also increased rapidly in other countries variations \u003csup\u003e\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eVariability exists between different populations regarding underlying molecular etiology of CRC. Comparison of Middle East and Western populations revealed the aspects of inconsistency in CRC pathways and mutated genes \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Iran, like many other countries especially in Asian continent, is inhabited by a multi ethnic genetically heterogeneous population. Comparison of mutation spectrum of beta thalassemia as a most prevalent single gene disorder in Iran, and many other countries, confirmed the extreme genetic heterogeneity of Iranian population \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Therefore, need to study extensively and comparative studies regarding molecular drivers of CRC between Iranian patients and those with different genetics background, like Western Europeans would be beneficial. For this reason we compared mutation spectrum of Iranian CRC patients and controls with Finnish CRC patients as a standard European population performed in the same laboratory at Helsinki university \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e In the present study, we performed mutation analysis using a 22 gene targeted NGS panel on DNA extracted from stool samples of early stages of CRC patients and their healthy counterparts from central region of Iran, then the patients and control group were followed up. The outcome of this study may facilitate the development of stool-based modality for population specific early-stage CRC risk prediction. Beside colonoscopy which is a gold standard diagnostic/surveillance method, availability of other noninvasive with desired level of detection sensitivity for screening approaches would encourage at risk individuals to undertake regular screening tests.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThe success rate of amplicon based NGS analysis from stool samples was 96% and 98% for patients and controls respectively. Two CRC samples and one normal sample were removed from NGS assay because of poor DNA quality. Among the CRC patients, 18 patients diagnosed with stage I and rest of them categorized as stage II CRC. Average age of CRC patients and controls was 62.8 years (range 43\u0026ndash;87) and 64.6 years (range 49\u0026ndash;88) respectively. The majority of participants were male (62%). The location of the tumor was more frequent in colon. The characteristics of all 99 individuals included in the study are presented in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u0026nbsp;\u003c/p\u003e\u0026nbsp;\u003ctable border=\"1\" id=\"Tab1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eCharacteristics of patients and healthy controls included in the study\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCRC\u003c/p\u003e\n \u003cp\u003eIranian\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003cp\u003eIranian\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCRC\u003c/p\u003e\n \u003cp\u003eFinnish\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003cp\u003eFinnish\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eMale\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN\u0026thinsp;=\u0026thinsp;31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN\u0026thinsp;=\u0026thinsp;28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN\u0026thinsp;=\u0026thinsp;21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN\u0026thinsp;=\u0026thinsp;4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eFemale\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN\u0026thinsp;=\u0026thinsp;19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN\u0026thinsp;=\u0026thinsp;21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN\u0026thinsp;=\u0026thinsp;12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN\u0026thinsp;=\u0026thinsp;9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eAverage age (years)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e62.8 (range 43\u0026ndash;87)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e64.6 (range 49\u0026ndash;88)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e72 (range 53\u0026ndash;85)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e44 (range 19\u0026ndash;65)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eStage\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eI\u0026thinsp;=\u0026thinsp;18\u003c/p\u003e\n \u003cp\u003eII\u0026thinsp;=\u0026thinsp;32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eRectal cancer\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN\u0026thinsp;=\u0026thinsp;10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN\u0026thinsp;=\u0026thinsp;22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eColon cancer\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN\u0026thinsp;=\u0026thinsp;40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN\u0026thinsp;=\u0026thinsp;11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e50\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e49\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e33\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e13\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003eAmong the CRC patients, mutations were seen in 24/50 patients (48%) and in the 13/49 (26.5%) control samples. Hotspot mutations were noted in 16/50 patients and 7/49 controls. Four of the patients and two of the control samples had more than three mutations. Rest of the samples detected with no mutation with the NGS gene panel we used and need to be evaluated by whole exome sequencing (WES) in order to pinpoint the underlying molecular defects.\u003c/p\u003e\n\u003cp\u003eOut of 22 genes in the Ion AmpliSeq panel, mutations were seen in 19 genes in patients and 17 genes in control samples; mutations in \u003cem\u003eMAP2K\u003c/em\u003e and \u003cem\u003eNRAS\u003c/em\u003e were seen only in patients and not in controls. A total of 34 hotspot mutations and 37 novel mutations were detected in patient samples, while 13 hotspots and 34 novel mutations were detected in control individuals (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTP53\u003c/em\u003e was the most frequently mutated gene in patients with a total of 13 mutations detected, while \u003cem\u003eSMAD4\u003c/em\u003e and \u003cem\u003eDDR2\u003c/em\u003e were the most frequently mutated genes in controls, with 7 mutations in each gene. All mutations in \u003cem\u003eTP53\u003c/em\u003e were hotspot mutations. Among the patients, hotspot mutations were most common in \u003cem\u003eTP53\u003c/em\u003e, followed by \u003cem\u003eNRAS\u003c/em\u003e, \u003cem\u003eFGFR3\u003c/em\u003e and \u003cem\u003eSMAD4\u003c/em\u003e (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Most of the mutations in controls were novel and only seven individuals had hotspot mutations. The \u003cem\u003eAKT\u003c/em\u003e, \u003cem\u003eCTNNB1\u003c/em\u003e and \u003cem\u003eNOTCH1\u003c/em\u003e genes had no mutation in both groups.\u003c/p\u003e\n\u003cp\u003eThe recurrently occurring hotspot mutations among patients were codon 273 of \u003cem\u003eTP53\u003c/em\u003e, seen in 4 patients (2 with R273C, 2 with R273H) and \u003cem\u003eNRAS\u003c/em\u003e codon 12 (3 patients) and codon 61 mutations (2 patients).\u003c/p\u003e\n\u003cp\u003eThe entire list of mutations (COSMIC and novel) in patients and normal individuals can be found as Supplementary Table S1 online.\u0026nbsp;\u003c/p\u003e\u0026nbsp;\u003ctable border=\"1\" id=\"Tab2\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eComparison of hotspot mutations detected in stool DNA of Iranian and Finnish colorectal cancer patients and controls\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eFinnish CRC\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eIranian CRC\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eIranian Controls\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eGene\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ePatient\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eMutation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ePatient\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eMutation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eIndividual\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eMutation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cspan class=\"BoldItalic\" name=\"Emphasis\" type=\"BoldItalic\"\u003eAKT1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eE17K\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cspan class=\"BoldItalic\" name=\"Emphasis\" type=\"BoldItalic\"\u003eALK\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eE1197K\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cspan class=\"BoldItalic\" name=\"Emphasis\" type=\"BoldItalic\"\u003eKRAS\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eG12V\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eG13V\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD119G\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eG12D\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cspan class=\"BoldItalic\" name=\"Emphasis\" type=\"BoldItalic\"\u003eBRAF\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eG606R\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT599I\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cspan class=\"BoldItalic\" name=\"Emphasis\" type=\"BoldItalic\"\u003eCTNNB1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cspan class=\"BoldItalic\" name=\"Emphasis\" type=\"BoldItalic\"\u003eERBB2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cspan class=\"BoldItalic\" name=\"Emphasis\" type=\"BoldItalic\"\u003eEGFR\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQ486*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cspan class=\"BoldItalic\" name=\"Emphasis\" type=\"BoldItalic\"\u003eERBB4\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eE317K\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS239L\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC304F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cspan class=\"BoldItalic\" name=\"Emphasis\" type=\"BoldItalic\"\u003eFBXW7\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH379P\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cspan class=\"BoldItalic\" name=\"Emphasis\" type=\"BoldItalic\"\u003eNRAS\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eG13V\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQ61K\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQ61K\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eG12V\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eG12S\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eG12V\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cspan class=\"BoldItalic\" name=\"Emphasis\" type=\"BoldItalic\"\u003ePIK3CA\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eA1066T\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eA1020V\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cspan class=\"BoldItalic\" name=\"Emphasis\" type=\"BoldItalic\"\u003ePTEN\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR234Q\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cspan class=\"BoldItalic\" name=\"Emphasis\" type=\"BoldItalic\"\u003eSMAD4\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR361H\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR135*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR420H\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cspan class=\"BoldItalic\" name=\"Emphasis\" type=\"BoldItalic\"\u003eSTK11\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF354L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eA205T\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cspan class=\"BoldItalic\" name=\"Emphasis\" type=\"BoldItalic\"\u003eFGFR1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eA299V\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD165DD\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cspan class=\"BoldItalic\" name=\"Emphasis\" type=\"BoldItalic\"\u003eFGFR2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cspan class=\"BoldItalic\" name=\"Emphasis\" type=\"BoldItalic\"\u003eFGFR3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eV392M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS373G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF386L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cspan class=\"BoldItalic\" name=\"Emphasis\" type=\"BoldItalic\"\u003eMET\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS349G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM1268T\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cspan class=\"BoldItalic\" name=\"Emphasis\" type=\"BoldItalic\"\u003eNOTCH1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cspan class=\"BoldItalic\" name=\"Emphasis\" type=\"BoldItalic\"\u003eTP53\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eP72A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eE358K\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT230A\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eY205D\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eG279E\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR273C\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT172fs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eP151S\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eE285K\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eY163H\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eY205H\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM169T\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR273C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eE286K\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR248W\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR273H\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR248W\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN235S\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eT52\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR273C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eG245S\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR273H\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cdiv class=\"Section2\" id=\"Sec3\"\u003e\n \u003ch2\u003eMutations in Finnish cohort:\u003c/h2\u003e\n \u003cp\u003eAmong the Finnish CRCs patients, hotspot mutations were seen in 6/33 patients (18%) and two novel mutations were seen in the control group. All mutations in CRC group were hotspot and two patients had more than one mutation, whereas no hotspot mutation was seen in the control group. Out of the 22 genes in the Ion AmpliSeq panel, eight mutations were seen in four genes, \u003cem\u003eAKT1\u003c/em\u003e, \u003cem\u003eKRAS\u003c/em\u003e, \u003cem\u003eSTK11\u003c/em\u003e and \u003cem\u003eTP53\u003c/em\u003e in six patients. \u003cem\u003eTP53\u003c/em\u003e was the most commonly mutated gene followed by \u003cem\u003eKRAS\u003c/em\u003e (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec4\"\u003e\n \u003ch2\u003eComparison of hotspot mutations in Iranian and Finnish CRC patients and controls\u003c/h2\u003e\n \u003cp\u003eAlthough data related to Finnish patients was published already but for the sack of comparison of two typical ethnic representatives of Middle-East and Europe, the results of stool DNA analysis of the Finnish cohort which have been worked out by the same gene panel, we review and included their results here. Overall, higher proportion of Iranian patients showed mutations in their stool DNA compared to Finnish patients (32% and 18% respectively), with 34 mutations detected in 32% Iranian patients compared to eight mutations in 18% Finnish patients. \u003cem\u003eTP53\u003c/em\u003e was the most commonly mutated gene in both the Iranian and Finnish cohorts. The second most commonly mutated gene was a \u003cem\u003eRAS\u003c/em\u003e gene, being \u003cem\u003eNRAS\u003c/em\u003e in Iranian patients and \u003cem\u003eKRAS\u003c/em\u003e in Finnish patients. The frequency of \u003cem\u003eNRAS\u003c/em\u003e mutations was significantly higher in Iranian patients than that in Finnish patients. Mutations in \u003cem\u003eAKT1\u003c/em\u003e were noted only in Finnish patients while mutations in \u003cem\u003eMET\u003c/em\u003e, \u003cem\u003eFGFR3\u003c/em\u003e, \u003cem\u003eSMAD4\u003c/em\u003e, \u003cem\u003eNRAS\u003c/em\u003e, \u003cem\u003eFBXW7\u003c/em\u003e, were seen only in Iranian patients (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eSimilar to the pattern seen for patients, mutations were more frequent in Iranian control group, with 7 out of 49 healthy Iranian individuals showed mutations, while no hotspot mutations were seen among Finnish healthy individuals.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec5\"\u003e\n \u003ch2\u003eIranian patient\u0026rsquo;s treatment\u003c/h2\u003e\n \u003cp\u003eFrom the CRC group, 29 patients (18 patients with stage I and 11 patients from 32 patients with stage II of cancer) were in group A and only did surgery according to their pathology reports, while 21 patients in group B received adjuvant treatment in addition to surgery. From group A one patients died of independent cancer (DOIC) with no hotspot mutation and two patients died of cancer (DOC), one patient had hotspot mutation in \u003cem\u003eTP53\u003c/em\u003e and one patient with novel mutations in \u003cem\u003eERBB2\u003c/em\u003e, \u003cem\u003eFGFR3\u003c/em\u003e and \u003cem\u003eMET\u003c/em\u003e genes. From group B, 6 patients (12%) were died. DOC (one patient with COSMIC mutations in \u003cem\u003eNRAS, ERBB4, MET, PIK3CA, FGFR1\u003c/em\u003e and \u003cem\u003eSMAD4\u003c/em\u003e genes and numerous novel mutations in other genes, one patient with COSMIC mutations in \u003cem\u003eBRAF, ERBB4, FGFR3\u003c/em\u003e and \u003cem\u003eTP53\u003c/em\u003e genes in addition to numerous novel mutations, one patient with COSMIC mutations in \u003cem\u003eSMAD4\u003c/em\u003e, \u003cem\u003eTP53\u003c/em\u003e and \u003cem\u003eNRAS\u003c/em\u003e genes, two patients only with \u003cem\u003eTP53\u003c/em\u003e mutation and one patient with mutations in \u003cem\u003eTP53\u003c/em\u003e and \u003cem\u003eNRAS\u003c/em\u003e genes). From this group one patient was DOIC with no mutation (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable border=\"1\" id=\"Tab3\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eFollowing up data of the CRC patients and characteristics of those how died due to cancer\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"9\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCode\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDOIC/DOC*\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eStage/TNM\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGrade^\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eHistological type\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP53\u003c/em\u003e COSMIC mutation\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eNRAS\u003c/em\u003e COSMIC mutation\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eOther COSMIC mutations\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eGroup A (29 Patients)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT10\u003c/p\u003e\n \u003cp\u003eT30\u003c/p\u003e\n \u003cp\u003eT2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDOC\u003c/p\u003e\n \u003cp\u003eDOC\u003c/p\u003e\n \u003cp\u003eDOIC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eI/T2N0MX\u003c/p\u003e\n \u003cp\u003eI/T1N0MX\u003c/p\u003e\n \u003cp\u003eI/T1N0MX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ep\u003c/p\u003e\n \u003cp\u003ew\u003c/p\u003e\n \u003cp\u003ew\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAdenocarcinoma\u003c/p\u003e\n \u003cp\u003e//\u003c/p\u003e\n \u003cp\u003e//\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eyes\u003c/p\u003e\n \u003cp\u003eno\u003c/p\u003e\n \u003cp\u003eno\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eno\u003c/p\u003e\n \u003cp\u003eno\u003c/p\u003e\n \u003cp\u003eno\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eno\u003c/p\u003e\n \u003cp\u003eno\u003c/p\u003e\n \u003cp\u003eno\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eGroup B (21 Patients)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT1\u003c/p\u003e\n \u003cp\u003eT3\u003c/p\u003e\n \u003cp\u003eT9\u003c/p\u003e\n \u003cp\u003eT40\u003c/p\u003e\n \u003cp\u003eT43\u003c/p\u003e\n \u003cp\u003eT44\u003c/p\u003e\n \u003cp\u003eT48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDOC\u003c/p\u003e\n \u003cp\u003eDOC\u003c/p\u003e\n \u003cp\u003eDOC\u003c/p\u003e\n \u003cp\u003eDOC\u003c/p\u003e\n \u003cp\u003eDOC\u003c/p\u003e\n \u003cp\u003eDOC\u003c/p\u003e\n \u003cp\u003eDOIC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eII/T4N0MX\u003c/p\u003e\n \u003cp\u003eII/T3N0MX\u003c/p\u003e\n \u003cp\u003eII/T3N0MX\u003c/p\u003e\n \u003cp\u003eII/T3N0MX\u003c/p\u003e\n \u003cp\u003eII/T3N0MX\u003c/p\u003e\n \u003cp\u003eII/T4aN0MX\u003c/p\u003e\n \u003cp\u003eII/T3N0MX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ep\u003c/p\u003e\n \u003cp\u003ep\u003c/p\u003e\n \u003cp\u003ew\u003c/p\u003e\n \u003cp\u003ep\u003c/p\u003e\n \u003cp\u003ep\u003c/p\u003e\n \u003cp\u003ep\u003c/p\u003e\n \u003cp\u003ew\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e//\u003c/p\u003e\n \u003cp\u003e//\u003c/p\u003e\n \u003cp\u003e//\u003c/p\u003e\n \u003cp\u003e//\u003c/p\u003e\n \u003cp\u003e//\u003c/p\u003e\n \u003cp\u003e//\u003c/p\u003e\n \u003cp\u003e//\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eyes\u003c/p\u003e\n \u003cp\u003eyes\u003c/p\u003e\n \u003cp\u003eno\u003c/p\u003e\n \u003cp\u003eyes\u003c/p\u003e\n \u003cp\u003eyes\u003c/p\u003e\n \u003cp\u003eyes\u003c/p\u003e\n \u003cp\u003eno\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eno\u003c/p\u003e\n \u003cp\u003eno\u003c/p\u003e\n \u003cp\u003eyes\u003c/p\u003e\n \u003cp\u003eyes\u003c/p\u003e\n \u003cp\u003eyes\u003c/p\u003e\n \u003cp\u003eno\u003c/p\u003e\n \u003cp\u003eno\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFGFR3,ERBB4,BRAF\u003c/p\u003e\n \u003cp\u003eno\u003c/p\u003e\n \u003cp\u003eERBB4,MET,FGFR1,SMAD4, PIK3CA\u003c/p\u003e\n \u003cp\u003eno\u003c/p\u003e\n \u003cp\u003eSMAD4\u003c/p\u003e\n \u003cp\u003eno\u003c/p\u003e\n \u003cp\u003eno\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"9\"\u003e*DOI: Died Of Cancer, DOIC: Died of Independent Cancer, ^ Poorly (p) or well (w) differentiated\u003c/td\u003e\n \u003c/tr\u003e\n \n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eAlso, the follow up results showed that three normal individuals (more than 6%) encountered gastrointestinal complications such as bleeding and abdominal pain after 33 months. All these three individuals were detected with mutations (one patient in \u003cem\u003eBRAF\u003c/em\u003e, \u003cem\u003ePTEN\u003c/em\u003e and \u003cem\u003eALK\u003c/em\u003e genes, one patient in \u003cem\u003eKRAS\u003c/em\u003e, \u003cem\u003eERBB4\u003c/em\u003e, \u003cem\u003eSTK11\u003c/em\u003e, \u003cem\u003eFGFR1\u003c/em\u003e and \u003cem\u003eTP53\u003c/em\u003e genes and one patient only in \u003cem\u003eTP53\u003c/em\u003e gene). One of the controls detected with mutation in five genes showed polyps (4mm) in his recent colonoscopy examination. Also, one of the control samples detected with two novel mutations in \u003cem\u003eFGFR3\u003c/em\u003e gene and three mutations in \u003cem\u003eSMAD4\u003c/em\u003e and other novel mutations, had gastrointestinal complications in follow up.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe 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 \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFrom the DOC patients, who had only \u003cem\u003eTP53\u003c/em\u003e 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 \u003cem\u003eNRAS\u003c/em\u003e and \u003cem\u003eTP53\u003c/em\u003e genes who were DOC. It can show that mutation in \u003cem\u003eTP53\u003c/em\u003e gene or both \u003cem\u003eTP53\u003c/em\u003e and \u003cem\u003eNRAS\u003c/em\u003e genes could reduce patient\u0026rsquo;s survival compare to patients with no mutation or mutation in other genes. A study in 2014 showed that patients with mutation in \u003cem\u003eKRAS\u003c/em\u003e, \u003cem\u003eNRAS\u003c/em\u003e, \u003cem\u003eTP53\u003c/em\u003e and \u003cem\u003ePIK3CA\u003c/em\u003e genes had dramatically shorter survival than patients with any other mutations \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. These results also showed that patients with more than one mutation had lower survival rate than patients with one mutation or no mutation \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eInterestingly \u003cem\u003eNRAS\u003c/em\u003e 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 \u003cem\u003eNRAS\u003c/em\u003e mutations, particularly in exon 3, comprised aggressive subset of metastatic colorectal cancer with overall reduced survival than patients with \u003cem\u003eKRAS\u003c/em\u003e mutations or \u003cem\u003eRAS\u003c/em\u003e wild-type tumors. They also showed that African Americans CRC patients with \u003cem\u003eNRAS\u003c/em\u003e mutations are mostly located at left side colon \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. In a large scale study on Iranian metastatic CRC specimens from 1000 patients using pyrosequencing and high resolution melting (HRM) analysis, \u003cem\u003eKRAS\u003c/em\u003e mutations detected in 33.6% cases (codon 12 and 13) \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Another study from west of Iran analyzed the \u003cem\u003eKRAS\u003c/em\u003e, \u003cem\u003eBRAF\u003c/em\u003e and \u003cem\u003eNRAS\u003c/em\u003e genes on formalin fixed paraffin embedded (FFPE) tissues from 33 CRC patients showed \u003cem\u003eKRAS\u003c/em\u003e mutations (codon 12 and 13) in 12 patients (27.3%) and no mutations in \u003cem\u003eNRAS\u003c/em\u003e and \u003cem\u003eBRAF\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Similarly, another study from Northeastern region of Iran showed that none of the 87 tissue samples from CRC patients had mutation in \u003cem\u003eNRAS\u003c/em\u003e gene which is similar to the results from West part of Iran \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Data from 7 European countries showed that mutations in \u003cem\u003eNRAS\u003c/em\u003e is predictors of lower response to anti-EGFR MoAbs treatment in CRC \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Jouini et al showed that \u003cem\u003eNRAS\u003c/em\u003e gene mutated in 6.9% of the 129 Tunisian colorectal cancer specimens \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. In Finnish stool DNAs, only one patient with rectum adenoma showed \u003cem\u003eNRAS\u003c/em\u003e Q61R mutation \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, while in another cohort of stool samples from Iranian CRC patients, none of them showed \u003cem\u003eNRAS\u003c/em\u003e mutation in codon 12, 13 or 61 \u003csup\u003e25\u003c/sup\u003e. 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 \u003cem\u003eNRAS\u003c/em\u003e 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 \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. A study on \u003cem\u003eRAS\u003c/em\u003e mutations in 353 Chinese CRC patients showed that \u003cem\u003eNRAS\u003c/em\u003e 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 \u003cem\u003eNRAS\u003c/em\u003e mutations were female \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eNRAS\u003c/em\u003e 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 \u003cem\u003eKRAS\u003c/em\u003e, in 2.6% of \u003cem\u003eNRAS\u003c/em\u003e, in 4.7% of \u003cem\u003eBRAF\u003c/em\u003e and in 14.5% of \u003cem\u003ePIK3CA\u003c/em\u003e. In wild type \u003cem\u003eKRAS\u003c/em\u003e patients, the presence of any mutations in \u003cem\u003eNRAS\u003c/em\u003e, \u003cem\u003eBRAF\u003c/em\u003e or \u003cem\u003ePIK3CA\u003c/em\u003e was associated with lower response rates, disease control rates and shorter overall survival. These findings suggested that up to 60\u0026ndash;65% of patients with wild type \u003cem\u003eKRAS\u003c/em\u003e tumors are resistant to anti-EGFR agents, which is accounted for presence of other mutations \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. An NGS study on 91 Brazilian CRC patients demonstrated that driver mutation in the \u003cem\u003eAPC\u003c/em\u003e, \u003cem\u003eTP53\u003c/em\u003e and \u003cem\u003eKRAS\u003c/em\u003e genes were significantly high in CRC patients \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. They also showed that at least one gene involved in the MAPK-ERK pathway (\u003cem\u003eKRAS\u003c/em\u003e, \u003cem\u003eBRAF\u003c/em\u003e, and \u003cem\u003eNRAS\u003c/em\u003e) had mutation in 68% of cases. Another study on 145 CRC patients by targeted deep sequencing showed that the \u003cem\u003eTP53\u003c/em\u003e gene was most commonly mutated gene and was significantly linked to lower disease-free survival which is similar to our study \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. However, in TCGA datasets \u003cem\u003eAPC\u003c/em\u003e mutation is mostly common followed by \u003cem\u003eTP53\u003c/em\u003e and \u003cem\u003eKRAS\u003c/em\u003e mutations.\u003c/p\u003e \u003cp\u003eIn our study, detection of relatively high percentage of \u003cem\u003eNRAS\u003c/em\u003e mutation (12%) among CRC samples from central part of Iran would emphasis the necessity of paying more attention to \u003cem\u003eNRAS\u003c/em\u003e 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.\u003c/p\u003e \u003cp\u003eTo 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\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). 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 \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. 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.\u003c/p\u003e \u003cp\u003eSome mutations reported to be an early event in tumor development \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Presence of tumor driver mutations in normal individuals related to tumor initiation and progression like \u003cem\u003eKRAS\u003c/em\u003e, \u003cem\u003eERBB\u003c/em\u003e gene family, \u003cem\u003eTP53\u003c/em\u003e 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.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePatients and stools collection and follow up\u003c/h2\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003eIranian samples:\u003c/h2\u003e \u003cp\u003eThe stool samples collected from 52 patients diagnosed with CRC by colonoscopy before starting any cancer treatment and 50 healthy individuals with normal colonoscopy results. Samples were collected from July 2016 to December 2017 at Alzahra university hospital, Isfahan Iran. The study was approved by Isfahan University of Medical Sciences Ethics Committee and informed written consent was obtained from all participants recruited to this study. Stool samples were stored at -80\u003csup\u003eo\u003c/sup\u003eC until DNA extraction. Patients followed up since their first surgery. The median follow-up period was 30 months (range 23\u0026ndash;39 months), for this aim patients were classified in two groups based on their pathology report (stage I and II). This study was approved by ir.mui.rec.1394.3.936 code at Isfahan University of medical science ethnical committee. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all individual participants included in the study.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eFinnish samples:\u003c/h2\u003e \u003cp\u003eThe stool samples from Finnish CRC patients were collected between April 2015 to May 2017 from patients referred to Kirugi, Meilahti and Jorvi hospitals in Finland. The sequencing results of stool DNA samples collected from 33 CRC patients and 13 healthy individuals (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) of Finnish origin published previously \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMutation analysis by NGS\u003c/h2\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003eDNA isolation\u003c/h2\u003e \u003cp\u003eQIAamp DNA stool mini kit (Qiagen GmbH, Hidden, Germany) was used for DNA extraction from approximately 200 mg stool samples according to the manufacturer instructions. Extracted DNAs were quantified by Qubit 2.0 Fluorimeter (Life Technologies, Carlsbad, CA, United States) using the Qubit dsDNA BR Assay Kit and stored at -20\u003csup\u003eo\u003c/sup\u003eC.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003eLibrary preparation\u003c/h2\u003e \u003cp\u003e DNA libraries were prepared from 20 ng of DNA per sample using Ion AmpliSeq Library Kit 2.0 (Life Technologies, California, US) following the kit guidelines. Ion AmpliSeq Colon and Lung Cancer panel v2 (Life Technologies, California, United States) consisting of a primer pool for analyzing 504 mutational hotspots from 92 amplicons was used to amplify the stool DNA. The genes included in this panel are: \u003cem\u003eAKT1\u003c/em\u003e, \u003cem\u003eALK\u003c/em\u003e, \u003cem\u003eKRAS, BRAF, CTNNB1, DDR2, ERBB2, EGFR, ERBB4, FBXW7, NRAS, PIK3CA, PTEN, SMAD4, STK11, FGFR1, FGFR2, FGFR3, MAP2K1, MET, NOTCH1\u003c/em\u003e and \u003cem\u003eTP53\u003c/em\u003e that are commonly mutated in colorectal cancer.\u003c/p\u003e \u003cp\u003eAmplified libraries were purified by Agincourt AM Pure XP beads (Beckman Coulter Genomics, High Wycombe, UK). Qubit 2.0 Fluorimeter and the Qubit dsDNA HS assay kit were used to quantify the barcoded libraries.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eSequencing\u003c/h2\u003e \u003cp\u003eBefore sequencing, templates were prepared using the Ion OneTouch\u003csup\u003eTM\u003c/sup\u003e2 system (Life Technologies, California, United States); this system performs emulsion PCR for template amplification and its further purification and enrichment. Eventually, sequencing was accomplished using the Ion PGM\u0026trade; Sequencing Hi-Q View Kit V2 and the amplified and enriched libraries loaded on Ion 316S chips and sequenced on the Ion Personal Genome Machine System (PGM\u0026trade;; Life Technologies, California, United States).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eTo analyze data and to evaluate run performance, Torrent suite software version 5.2.2 (Life Technologies) was used. Integrative Genomics Viewer (IGV v 2.2; Broad Institute, Cambridge, MA, United State) used for visual inspection of the aligned reads. All variants identified were further filtered by quality score (\u0026gt;\u0026thinsp;15) and mutant allele frequency (\u0026gt;\u0026thinsp;5%). We used SIFT and PROVEAN in-silico tools to identify all those variants which resulted in non-synonymous, stop codon or frameshift amino acid changes and those that are predicted to have deleterious protein product. Previously reported somatic mutations and novel mutations were checked in COSMIC and dbSNP databases. Variants that are not recorded in either of the databases are labeled as \u0026lsquo;Novel\u0026rsquo;. Kaplan-Meier analysis was performed to estimate the survival curve of the patients with different rate of mutations.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eEarly-stage stool DNA mutation analysis from CRC stage I/II patients would be a suitable, non-invasive approach for population specific screening. Our result shows \u003cem\u003eTP53\u003c/em\u003e and \u003cem\u003eNRAS\u003c/em\u003e are the most frequently mutated genes in stool DNA of CRC patients from central part of Iran. Also comparing CRC and control group mutations can help to improve our understanding of mutations involved in cancer development mutation spectrum in other genes included in the NGS targeted panel that we used for this study is relatively different comparing to the previously reported mutation spectrum using the same NGS panel on ethnically different Iranian population as well as Finish CRC patients and controls. Our present data would help in development of stool-based CRC screening tools as well as more precisely selection of those who may benefit from anti EGFR monoclonal antibody therapy.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eDATA AVAILABILITY\u003c/h2\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article and its Supplementary Information file.\u003c/p\u003e\n\u003ch2\u003eAcknowledgments\u003c/h2\u003e\n\u003cp\u003eResearch reported in this publication was supported by Elite Researcher Grant Committee under award number 971202 from the National Institute for Medical Research Development (NIMAD), Tehran, Iran provided to Rasoul Salehi. The study was funded by the Sigrid Juselius Foundation grant to SK.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eAuthor Contributions\u003c/h2\u003e\n\u003cp\u003eFS, VS and OY contributed to acquisition and analysis of data and drafting the manuscript, AK, PP contributed to the design and execution of the work, SK, and RS contributed to the concept development, manuscript revision, data interpretation and approval.\u003c/p\u003e\n\u003ch2\u003eCorresponding Author\u003c/h2\u003e\n\u003cp\u003eCorrespondence to Rasoul Salehi.\u003c/p\u003e\n\u003ch2\u003eAdditional Information\u003c/h2\u003e\n\u003ch2\u003ePublisher\u0026apos;s note\u003c/h2\u003e\n\u003cp\u003eSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.\u003c/p\u003e\n\u003ch2\u003eEthics Declarations\u003c/h2\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare no potential conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFerlay, J. \u003cem\u003eet al.\u003c/em\u003e Cancer incidence and mortality worldwide: IARC CancerBase. \u003cem\u003eGLOBOCAN 2012\u003c/em\u003e, \u003cb\u003ev10\u003c/b\u003e, 11 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorson, B. 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CRC is a curable cancer provided to be diagnosed at its early-stage. Colonoscopy, stool and blood-based tests are in use for CRC diagnosis/screening. Due to low patient compliance, low specificity and high rate of false results, more reliable methods with desired level of detection accuracy and high patients\u0026rsquo; compliance are highly demanded. Detecting hotspot mutations in stool DNA emerged as a robust noninvasive alternative, but due to the different genetic background of various populations and hence varied mutation spectrum/prevalence, prior assessment of CRC mutations in the population is essential. Here, we have evaluated stool DNAs from CRC patients and controls using a NGS based 22 genes panel. Hotspot mutations in \u003cem\u003eNRAS\u003c/em\u003e, \u003cem\u003eFGFR3, SMAD4\u003c/em\u003e and \u003cem\u003eTP53\u003c/em\u003e genes had higher prevalence among the CRC patients compare to normal controls. Patients were followed up in their post-surgical period. Six of them (12%) with \u003cem\u003eTP53\u003c/em\u003e mutations (2 patients had \u003cem\u003eNRAS\u003c/em\u003e mutation as well) were died of cancer. Those harboring mutations in \u003cem\u003eTP53\u003c/em\u003e and/or \u003cem\u003eNRAS\u003c/em\u003e would be regarded as high risk and should be provided with special care.\u003c/p\u003e","manuscriptTitle":"TP53 and NRAS are the Most Frequently Mutated Genes in Stool DNA of CRC Patients from Central Part of Iran","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-09-17 14:47:22","doi":"10.21203/rs.3.rs-792966/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d8b31cb0-fa90-4d92-ae17-e5aafafad48a","owner":[],"postedDate":"September 17th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":7255343,"name":"Scientific Communication"},{"id":7255344,"name":"Pathology"}],"tags":[],"updatedAt":"2021-09-17T14:47:23+00:00","versionOfRecord":[],"versionCreatedAt":"2021-09-17 14:47:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-792966","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-792966","identity":"rs-792966","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}