BACKGROUND Colorectal cancer(CRC)is a major global health burden.The current diagnostic tests have shortcomings of being invasive and low accuracy.AIM To explore the combination of intestinal microbiome composition an...BACKGROUND Colorectal cancer(CRC)is a major global health burden.The current diagnostic tests have shortcomings of being invasive and low accuracy.AIM To explore the combination of intestinal microbiome composition and multi-target stool DNA(MT-sDNA)test in the diagnosis of CRC.METHODS We assessed the performance of the MT-sDNA test based on a hospital clinical trial.The intestinal microbiota was tested using 16S rRNA gene sequencing.This case-control study enrolled 54 CRC patients and 51 healthy controls.We identified biomarkers of bacterial structure,analyzed the relationship between different tumor markers and the relative abundance of related flora components,and distinguished CRC patients from healthy subjects by the linear discriminant analysis effect size,redundancy analysis,and random forest analysis.RESULTS MT-sDNA was associated with Bacteroides.MT-sDNA and carcinoembryonic antigen(CEA)were positively correlated with the existence of Parabacteroides,and alpha-fetoprotein(AFP)was positively associated with Faecalibacterium and Megamonas.In the random forest model,the existence of Streptococcus,Escherichia,Chitinophaga,Parasutterella,Lachnospira,and Romboutsia can distinguish CRC from health controls.The diagnostic accuracy of MT-sDNA combined with the six genera and CEA in the diagnosis of CRC was 97.1%,with a sensitivity and specificity of 98.1%and 92.3%,respectively.CONCLUSION There is a positive correlation of MT-sDNA,CEA,and AFP with intestinal microbiome.Eight biomarkers including six genera of gut microbiota,MT-sDNA,and CEA showed a prominent sensitivity and specificity for CRC prediction,which could be used as a non-invasive method for improving the diagnostic accuracy for this malignancy.展开更多
Multitarget stool DNA(mt-sDNA) testing was approved for average risk colorectal cancer(CRC) screening by the United States Food and Drug Administration and thereafter reimbursed for use by the Medicare program(2014).T...Multitarget stool DNA(mt-sDNA) testing was approved for average risk colorectal cancer(CRC) screening by the United States Food and Drug Administration and thereafter reimbursed for use by the Medicare program(2014).The United States Preventive Services Task Force(USPSTF) October 2015 draft recommendation for CRC screening included mt-s DNA as an "alternative" screening test that "may be useful in select clinical circumstances",despite its very high sensitivity for early stage CRC.The evidence supporting mt-s DNA for routine screening use is robust.The clinical efficacy of mt-s DNA as measured by sensitivity,specificity,life-years gained(LYG),and CRC deaths averted is similar to or exceeds that of the other more specifically recommended screening options included in the draft document,especially those requiring annual testing adherence.In a population with primarily irregular screening participation,tests with the highest point sensitivity and reasonable specificity are more likely to favorably impact CRC related morbidity and mortality than those depending on annual adherence.This paper reviews the evidence supporting mt-s DNA for routine screening and demonstrates,using USPSTF's modeling data,that mt-s DNA at three-year intervals provides significant clinical net benefits and fewer complications per LYG than annual fecal immunochemical testing,high sensitivity guaiac based fecal occult blood testing and 10-year colonoscopy screening.展开更多
AIM To determine the uptake of noninvasive multitarget stool DNA (mt-s DNA) in a cohort of colorectal cancer(CRC) screening non-compliant average-risk Medicare patients.METHODS This cross sectional primary care office...AIM To determine the uptake of noninvasive multitarget stool DNA (mt-s DNA) in a cohort of colorectal cancer(CRC) screening non-compliant average-risk Medicare patients.METHODS This cross sectional primary care office-based study examined mt-s DNA uptake in routine clinical practice among 393 colorectal cancer screening non-compliant Medicare patients ages 50-85 ordered by 77 physicians in a multispecialty group practice (USMD Physician Services, Dallas, TX) from October, 2014-September, 2015. Investigators performed a Health Insurance Portability and Accountability Act compliant retrospective review of electronic health records to identify mt-s DNA use in patients who were either > 10 years since last colonoscopy and/or > 1 year since last fecal occult blood test. Test positive patients were advised to get diagnostic colonoscopy and thereafter patients were characterized by the most clinically significant lesion documented on histopathology of biopsies or excisional tissue. Descriptive statistics were employed. Key outcome measures included mt-s DNA compliance and diagnostic colonoscopy compliance on positive cases.RESULTS Over 12 mo, 77 providers ordered 393 mt-sD NA studies with 347 completed (88.3% compliance). Patient mean age was 69.8 (50-85) and patients were 64% female. Mt-sD NA was negative in 85.3%(296/347) and positive in 14.7%(51/347). Follow-up colonoscopy was performed in 49 positive patients (96.1% colonoscopy compliance) with two patients lost to follow up. Index findings included: colon cancer(4/49, 8.2%), advanced adenomas (21/49, 42.9%), non-advanced adenomas(15/49, 30.6%), and negative results (9/49, 18.4%). The positive predictive value for advanced colorectal lesions was 51.0% and for any colorectal neoplasia was 81.6%. The mean age of patients with colorectal cancer was 70.3 and all CRC's were localized Stage Ⅰ(2) and Stage Ⅱ (2), three were located in the proximal colon and one was located in the distal colon.CONCLUSION Mt-s DNA provided medical benefit to screening noncompliant Medicare population. High compliance with mt-s DNA and subsequent follow-up diagnostic colonoscopy identified patients with clinically critical advanced colorectal neoplasia.展开更多
AIM： To detect tumor-associated DNA changes in stool samples among Iranian patients with colorectal cancer （CRC） compared to healthy individuals using BAT-26, p16 hypermethylation and long DNA markers. METHODS： St...AIM： To detect tumor-associated DNA changes in stool samples among Iranian patients with colorectal cancer （CRC） compared to healthy individuals using BAT-26, p16 hypermethylation and long DNA markers. METHODS： Stool DNA was isolated from 45 subjects including 25 CRC patients and 20 healthy individuals using a new, fast and easy extraction method. Long DNA associated with tumor was detected using polymerase chain reaction method. Microsatellite studies were performed utilizing denaturating polyacrylamide gel to determine the instability of BAT-26. Methylation status of p16 promoter was analyzed using methylation-specific PCR （MSP）. RESULTS： The results showed a significant difference in existence of long DNA （16 in patients vs 1 in controls, P 〈 0.001） and p16 （5 in patients vs none in controls, P = 0.043） in the stool samples of two groups. Long DNA was detected in 64% of CRC patients; whereas just one of the healthy individuals was positive for Long DNA. p16 methylation was found in 20% of patients and in none of healthy individuals. Instability of BATo26 was not detected in any of stool samples. CONCLUSION： We could detect colorectal cancer related genetic alterations by analyzing stool DNA with a sensitivity of 64% and 20% and a specificity of 95% and 100% for Long DNA and p16 respectively. A non- invasive molecular stool-based DNA testing can provide a screening strategy in high-risk individuals. However, additional testing on more samples is necessary from Iranian subjects to determine the exact specificity and sensitivity of these markers.展开更多
Objective:Stool DNA(sDNA)tests offer a noninvasive form of colon cancer screening for pa-tients,and although the test is expected to increase uptake of colon cancer screening,it is unknown if patients’perceptions of ...Objective:Stool DNA(sDNA)tests offer a noninvasive form of colon cancer screening for pa-tients,and although the test is expected to increase uptake of colon cancer screening,it is unknown if patients’perceptions of the sDNA test differ according to race and other patient characteristics.Methods:We conducted a self-administered survey of patients undergoing both a colonoscopy and an sDNA test to evaluate perceptions of sDNA testing.Results:Of the 613 participants who were sent surveys,423 responded(69%response rate).Respondents self-identified as African American(n=127,30%),Caucasian(n=284,67%),and other ethnicity(n=12,3%).In general,participants found the sDNA test more suitable than a colonos-copy(n=309,75%).In univariate analyses,a higher percentage of Caucasians as compared with African Americans found the sDNA test more suitable than a colonoscopy(89%vs.76%,p<0.01),and more Caucasians than African Americans preferred the sDNA test(43%vs.32%,p<0.05).Ad-justment for covariates reduced these racial differences to no significance.A family history of colo-rectal cancer remains a significant factor for patient’s preferences for screening regardless of race.Conclusions:Our study shows no racial differences in the perception of and preference for sDNA testing for colon screening.Intervention to increase the uptake of sDNA testing may help reduce racial disparities in colorectal cancer.展开更多
Colorectal cancer(CRC)is the third most commonly diagnosed cancer and the second leading cause of cancer death worldwide.The leading risk factors for CRC include male gender,age over 50,family history,obesity,tobacco ...Colorectal cancer(CRC)is the third most commonly diagnosed cancer and the second leading cause of cancer death worldwide.The leading risk factors for CRC include male gender,age over 50,family history,obesity,tobacco smoking,alco-hol consumption,and unhealthy diet.CRC screening methods vary considerably between countries and depend on incidence,economic resources and healthcare structure.Important aspects of screening include adherence,which can vary signi-ficantly across ethnic and socioeconomic groups.Basic concepts of CRC screening include pre-stratification of patients by identifying risk factors and then using fecal immunochemical test or guaiac-based fecal occult blood test and/or colono-scopy or radiologic imaging techniques.Technological capabilities for CRC scree-ning are rapidly evolving and include stool DNA test,liquid biopsy,virtual colo-nography,and the use of artificial intelligence.A CRC prevention strategy should be comprehensive and include active patient education along with targeted imple-mentation of screening.展开更多
AIM To study cancer hotspot mutations by next-generation sequencing(NGS) in stool DNA from patients with different gastrointestinal tract(GIT) neoplasms. METHODS Stool samples were collected from 87 Finnish patients d...AIM To study cancer hotspot mutations by next-generation sequencing(NGS) in stool DNA from patients with different gastrointestinal tract(GIT) neoplasms. METHODS Stool samples were collected from 87 Finnish patients diagnosed with various gastric and colorectal neoplasms, including benign tumors, and from 14 healthy controls. DNA was isolated from stools by usingthe PSP~? Spin Stool DNA Plus Kit. For each sample, 20 ng of DNA was used to construct sequencing libraries using the Ion AmpliS eq Cancer Hotspot Panel v2 or Ion AmpliS eq Colon and Lung Cancer panel v2. Sequencing was performed on Ion PGM. Torrent Suite Software v.5.2.2 was used for variant calling and data analysis.RESULTS NGS was successful in assaying 72 GIT samples and 13 healthy controls, with success rates of the assay being78% for stomach neoplasia and 87% for colorectal tumors. In stool specimens from patients with gastric neoplasia, five hotspot mutations were found in APC,CDKN2 A and EGFR genes, in addition to seven novel mutations. From colorectal patients, 20 mutations were detected in AKT1, APC, ERBB2, FBXW7, KIT, KRAS,NRAS, SMARCB1, SMO, STK11 and TP53. Healthy controls did not exhibit any hotspot mutations, except for two novel ones. APC and TP53 were the most frequently mutated genes in colorectal neoplasms, with five mutations, followed by KRAS with two mutations.APC was the most commonly mutated gene in stools of patients with premalignant/benign GIT lesions.CONCLUSION Our results show that in addition to colorectal neoplasms,mutations can also be assayed from stool specimens of patients with gastric neoplasms.展开更多
目的:考察在不同温度下储存时间和反复冻融对粪便中人基因组DNA含量的影响。方法:1.将粪便样本在室温、4℃、-40℃和-70℃条件下分别放置不同时间和-40℃条件下保存并反复冻融后,使用QIAamp DNA Stool Mini Kit试剂盒提取得到粪便DNA,...目的:考察在不同温度下储存时间和反复冻融对粪便中人基因组DNA含量的影响。方法:1.将粪便样本在室温、4℃、-40℃和-70℃条件下分别放置不同时间和-40℃条件下保存并反复冻融后,使用QIAamp DNA Stool Mini Kit试剂盒提取得到粪便DNA,通过实时荧光定量PCR体系对人KRAS基因定量确定人基因组DNA含量,评价粪便样本不同冻存条件对其中人基因组DNA含量的影响。2.将粪便DNA样本在4℃和-40℃条件下分别放置不同时间和-40℃条件下保存并反复冻融后,评价粪便DNA样本不同冻存条件下对其中人基因组DNA含量的影响。结果:1).粪便样本常温放置2小时,其中人基因组DNA即发生明显降解(P<0.01),4℃可保存3天左右,-40℃可保存4周,-70℃可保存3个月以上,粪便反复冻融第3次,其中人基因组DNA降解具有统计学意义(P<0.05)。2).粪便DNA 4℃可保存3天,-40℃可保存4周,粪便DNA反复冻融第4次,其中人基因组DNA降解具有统计学意义(P<0.05)。结论:符合大肠癌早期无创分子诊断要求的粪便DNA贮存条件:粪便样本室温收集后尽快保存;短期可处理的粪便样本存放在4℃条件下(3天内);暂无法处理则存于-40℃(1个月内);粪便样本长期保存在-70℃条件下,可保存3个月。展开更多
目的观察改变细菌裂解温度对提取粪便细菌基因组DNA量和纯度的影响。方法收集10例肝硬化患者粪便,每例患者粪便等分成两份,再随机分成两组。采用QIAamp DNA Stool Mini Kit试剂盒(国际通用粪便细菌基因组DNA提取试剂盒)进行抽提,一组(A...目的观察改变细菌裂解温度对提取粪便细菌基因组DNA量和纯度的影响。方法收集10例肝硬化患者粪便,每例患者粪便等分成两份,再随机分成两组。采用QIAamp DNA Stool Mini Kit试剂盒(国际通用粪便细菌基因组DNA提取试剂盒)进行抽提,一组(A组)按照试剂盒说明书温度要求进行粪便细菌基因组DNA提取,一组(B组)对试剂盒细菌裂解温度改良后再进行提取。结果与按照试剂盒说明书温度进行提取组相比,改变细菌裂解温度后粪便基因组DNA提取量明显增高(P<0.05),而粪便基因组DNA提取纯度(OD260/280、OD260/230)无明显变化(P>0.05)。结论改变细菌裂解温度能明显提高粪便细菌基因组DNA提取量,而不改变粪便细菌基因组DNA的提取纯度。该方法可以解决因粪便采样量少导致粪便基因组DNA提取量不足的问题。展开更多
Colorectal carcinoma(CRC) is a common cause of morbidity and mortality worldwide. Two pathogenic pathways are involved in the development of adenoma to CRC. The first pathway involves APC/β-catenin characterized by c...Colorectal carcinoma(CRC) is a common cause of morbidity and mortality worldwide. Two pathogenic pathways are involved in the development of adenoma to CRC. The first pathway involves APC/β-catenin characterized by chromosomal instability resulting in the accumulation of mutations. The second pathway is characterized by lesions in DNA mismatch repair genes.Aberrant DNA methylation in selected gene promoters has emerged as a new epigenetic pathway in CRC development. CRC screening is the most efficient strategy to reduce death. Specific DNA methylation events occur in multistep carcinogenesis.Epigenetic gene silencing is a causative factor of CRC development. DNA methylations have been extensively examined in stool from CRC and precursor lesions. Many methylated genes have been described in CRC and adenoma, although no definite DNA methylation biomarkers panel has been established. Multiple DNA methylation biomarkers, including secreted frizzled-related protein 2, secreted frizzled-related protein 1, tissue factor pathway inhibitor 2, vimentin, and methylguanine DNA methyltransferase, have been further investigated, and observations have revealed that DNA methylation biomarkers exhibit with high sensitivity and specificity. These markers may also be used to diagnose CRC and adenoma in early stages. Real time polymerase chain reaction(q PCR) is sensitive, scalable, specific, reliable, time saving, and cost effective. Stool exfoliated markers provide advantages, including sensitivity and specificity. A stool q PCR methylation test may also be an enhanced tool for CRC and adenoma screening.展开更多
基金Supported by the Medical and Health Research Project of Zhejiang Province,No.2021KY1048 and 2022KY1142Ningbo Health Young Technical Backbone Talents Training Program,No.2020SWSQNGG-02the Key Science and Technology Project of Ningbo City,No.2021Z133.
文摘BACKGROUND Colorectal cancer(CRC)is a major global health burden.The current diagnostic tests have shortcomings of being invasive and low accuracy.AIM To explore the combination of intestinal microbiome composition and multi-target stool DNA(MT-sDNA)test in the diagnosis of CRC.METHODS We assessed the performance of the MT-sDNA test based on a hospital clinical trial.The intestinal microbiota was tested using 16S rRNA gene sequencing.This case-control study enrolled 54 CRC patients and 51 healthy controls.We identified biomarkers of bacterial structure,analyzed the relationship between different tumor markers and the relative abundance of related flora components,and distinguished CRC patients from healthy subjects by the linear discriminant analysis effect size,redundancy analysis,and random forest analysis.RESULTS MT-sDNA was associated with Bacteroides.MT-sDNA and carcinoembryonic antigen(CEA)were positively correlated with the existence of Parabacteroides,and alpha-fetoprotein(AFP)was positively associated with Faecalibacterium and Megamonas.In the random forest model,the existence of Streptococcus,Escherichia,Chitinophaga,Parasutterella,Lachnospira,and Romboutsia can distinguish CRC from health controls.The diagnostic accuracy of MT-sDNA combined with the six genera and CEA in the diagnosis of CRC was 97.1%,with a sensitivity and specificity of 98.1%and 92.3%,respectively.CONCLUSION There is a positive correlation of MT-sDNA,CEA,and AFP with intestinal microbiome.Eight biomarkers including six genera of gut microbiota,MT-sDNA,and CEA showed a prominent sensitivity and specificity for CRC prediction,which could be used as a non-invasive method for improving the diagnostic accuracy for this malignancy.
文摘Multitarget stool DNA(mt-sDNA) testing was approved for average risk colorectal cancer(CRC) screening by the United States Food and Drug Administration and thereafter reimbursed for use by the Medicare program(2014).The United States Preventive Services Task Force(USPSTF) October 2015 draft recommendation for CRC screening included mt-s DNA as an "alternative" screening test that "may be useful in select clinical circumstances",despite its very high sensitivity for early stage CRC.The evidence supporting mt-s DNA for routine screening use is robust.The clinical efficacy of mt-s DNA as measured by sensitivity,specificity,life-years gained(LYG),and CRC deaths averted is similar to or exceeds that of the other more specifically recommended screening options included in the draft document,especially those requiring annual testing adherence.In a population with primarily irregular screening participation,tests with the highest point sensitivity and reasonable specificity are more likely to favorably impact CRC related morbidity and mortality than those depending on annual adherence.This paper reviews the evidence supporting mt-s DNA for routine screening and demonstrates,using USPSTF's modeling data,that mt-s DNA at three-year intervals provides significant clinical net benefits and fewer complications per LYG than annual fecal immunochemical testing,high sensitivity guaiac based fecal occult blood testing and 10-year colonoscopy screening.
文摘AIM To determine the uptake of noninvasive multitarget stool DNA (mt-s DNA) in a cohort of colorectal cancer(CRC) screening non-compliant average-risk Medicare patients.METHODS This cross sectional primary care office-based study examined mt-s DNA uptake in routine clinical practice among 393 colorectal cancer screening non-compliant Medicare patients ages 50-85 ordered by 77 physicians in a multispecialty group practice (USMD Physician Services, Dallas, TX) from October, 2014-September, 2015. Investigators performed a Health Insurance Portability and Accountability Act compliant retrospective review of electronic health records to identify mt-s DNA use in patients who were either > 10 years since last colonoscopy and/or > 1 year since last fecal occult blood test. Test positive patients were advised to get diagnostic colonoscopy and thereafter patients were characterized by the most clinically significant lesion documented on histopathology of biopsies or excisional tissue. Descriptive statistics were employed. Key outcome measures included mt-s DNA compliance and diagnostic colonoscopy compliance on positive cases.RESULTS Over 12 mo, 77 providers ordered 393 mt-sD NA studies with 347 completed (88.3% compliance). Patient mean age was 69.8 (50-85) and patients were 64% female. Mt-sD NA was negative in 85.3%(296/347) and positive in 14.7%(51/347). Follow-up colonoscopy was performed in 49 positive patients (96.1% colonoscopy compliance) with two patients lost to follow up. Index findings included: colon cancer(4/49, 8.2%), advanced adenomas (21/49, 42.9%), non-advanced adenomas(15/49, 30.6%), and negative results (9/49, 18.4%). The positive predictive value for advanced colorectal lesions was 51.0% and for any colorectal neoplasia was 81.6%. The mean age of patients with colorectal cancer was 70.3 and all CRC's were localized Stage Ⅰ(2) and Stage Ⅱ (2), three were located in the proximal colon and one was located in the distal colon.CONCLUSION Mt-s DNA provided medical benefit to screening noncompliant Medicare population. High compliance with mt-s DNA and subsequent follow-up diagnostic colonoscopy identified patients with clinically critical advanced colorectal neoplasia.
基金Supported by a grant from the vice chancellor for research at Mashhad University of Medical Sciences,NO. 84082
文摘AIM： To detect tumor-associated DNA changes in stool samples among Iranian patients with colorectal cancer （CRC） compared to healthy individuals using BAT-26, p16 hypermethylation and long DNA markers. METHODS： Stool DNA was isolated from 45 subjects including 25 CRC patients and 20 healthy individuals using a new, fast and easy extraction method. Long DNA associated with tumor was detected using polymerase chain reaction method. Microsatellite studies were performed utilizing denaturating polyacrylamide gel to determine the instability of BAT-26. Methylation status of p16 promoter was analyzed using methylation-specific PCR （MSP）. RESULTS： The results showed a significant difference in existence of long DNA （16 in patients vs 1 in controls, P 〈 0.001） and p16 （5 in patients vs none in controls, P = 0.043） in the stool samples of two groups. Long DNA was detected in 64% of CRC patients; whereas just one of the healthy individuals was positive for Long DNA. p16 methylation was found in 20% of patients and in none of healthy individuals. Instability of BATo26 was not detected in any of stool samples. CONCLUSION： We could detect colorectal cancer related genetic alterations by analyzing stool DNA with a sensitivity of 64% and 20% and a specificity of 95% and 100% for Long DNA and p16 respectively. A non- invasive molecular stool-based DNA testing can provide a screening strategy in high-risk individuals. However, additional testing on more samples is necessary from Iranian subjects to determine the exact specificity and sensitivity of these markers.
基金the National Cancer Institute(R01 CA136726 and U01CA181770 to L.Li,and P50CA50964 to L.Li and G.Cooper).
文摘Objective:Stool DNA(sDNA)tests offer a noninvasive form of colon cancer screening for pa-tients,and although the test is expected to increase uptake of colon cancer screening,it is unknown if patients’perceptions of the sDNA test differ according to race and other patient characteristics.Methods:We conducted a self-administered survey of patients undergoing both a colonoscopy and an sDNA test to evaluate perceptions of sDNA testing.Results:Of the 613 participants who were sent surveys,423 responded(69%response rate).Respondents self-identified as African American(n=127,30%),Caucasian(n=284,67%),and other ethnicity(n=12,3%).In general,participants found the sDNA test more suitable than a colonos-copy(n=309,75%).In univariate analyses,a higher percentage of Caucasians as compared with African Americans found the sDNA test more suitable than a colonoscopy(89%vs.76%,p<0.01),and more Caucasians than African Americans preferred the sDNA test(43%vs.32%,p<0.05).Ad-justment for covariates reduced these racial differences to no significance.A family history of colo-rectal cancer remains a significant factor for patient’s preferences for screening regardless of race.Conclusions:Our study shows no racial differences in the perception of and preference for sDNA testing for colon screening.Intervention to increase the uptake of sDNA testing may help reduce racial disparities in colorectal cancer.
文摘Colorectal cancer(CRC)is the third most commonly diagnosed cancer and the second leading cause of cancer death worldwide.The leading risk factors for CRC include male gender,age over 50,family history,obesity,tobacco smoking,alco-hol consumption,and unhealthy diet.CRC screening methods vary considerably between countries and depend on incidence,economic resources and healthcare structure.Important aspects of screening include adherence,which can vary signi-ficantly across ethnic and socioeconomic groups.Basic concepts of CRC screening include pre-stratification of patients by identifying risk factors and then using fecal immunochemical test or guaiac-based fecal occult blood test and/or colono-scopy or radiologic imaging techniques.Technological capabilities for CRC scree-ning are rapidly evolving and include stool DNA test,liquid biopsy,virtual colo-nography,and the use of artificial intelligence.A CRC prevention strategy should be comprehensive and include active patient education along with targeted imple-mentation of screening.
文摘AIM To study cancer hotspot mutations by next-generation sequencing(NGS) in stool DNA from patients with different gastrointestinal tract(GIT) neoplasms. METHODS Stool samples were collected from 87 Finnish patients diagnosed with various gastric and colorectal neoplasms, including benign tumors, and from 14 healthy controls. DNA was isolated from stools by usingthe PSP~? Spin Stool DNA Plus Kit. For each sample, 20 ng of DNA was used to construct sequencing libraries using the Ion AmpliS eq Cancer Hotspot Panel v2 or Ion AmpliS eq Colon and Lung Cancer panel v2. Sequencing was performed on Ion PGM. Torrent Suite Software v.5.2.2 was used for variant calling and data analysis.RESULTS NGS was successful in assaying 72 GIT samples and 13 healthy controls, with success rates of the assay being78% for stomach neoplasia and 87% for colorectal tumors. In stool specimens from patients with gastric neoplasia, five hotspot mutations were found in APC,CDKN2 A and EGFR genes, in addition to seven novel mutations. From colorectal patients, 20 mutations were detected in AKT1, APC, ERBB2, FBXW7, KIT, KRAS,NRAS, SMARCB1, SMO, STK11 and TP53. Healthy controls did not exhibit any hotspot mutations, except for two novel ones. APC and TP53 were the most frequently mutated genes in colorectal neoplasms, with five mutations, followed by KRAS with two mutations.APC was the most commonly mutated gene in stools of patients with premalignant/benign GIT lesions.CONCLUSION Our results show that in addition to colorectal neoplasms,mutations can also be assayed from stool specimens of patients with gastric neoplasms.
文摘目的:考察在不同温度下储存时间和反复冻融对粪便中人基因组DNA含量的影响。方法:1.将粪便样本在室温、4℃、-40℃和-70℃条件下分别放置不同时间和-40℃条件下保存并反复冻融后,使用QIAamp DNA Stool Mini Kit试剂盒提取得到粪便DNA,通过实时荧光定量PCR体系对人KRAS基因定量确定人基因组DNA含量,评价粪便样本不同冻存条件对其中人基因组DNA含量的影响。2.将粪便DNA样本在4℃和-40℃条件下分别放置不同时间和-40℃条件下保存并反复冻融后,评价粪便DNA样本不同冻存条件下对其中人基因组DNA含量的影响。结果:1).粪便样本常温放置2小时,其中人基因组DNA即发生明显降解(P<0.01),4℃可保存3天左右,-40℃可保存4周,-70℃可保存3个月以上,粪便反复冻融第3次,其中人基因组DNA降解具有统计学意义(P<0.05)。2).粪便DNA 4℃可保存3天,-40℃可保存4周,粪便DNA反复冻融第4次,其中人基因组DNA降解具有统计学意义(P<0.05)。结论:符合大肠癌早期无创分子诊断要求的粪便DNA贮存条件:粪便样本室温收集后尽快保存;短期可处理的粪便样本存放在4℃条件下(3天内);暂无法处理则存于-40℃(1个月内);粪便样本长期保存在-70℃条件下,可保存3个月。
文摘目的观察改变细菌裂解温度对提取粪便细菌基因组DNA量和纯度的影响。方法收集10例肝硬化患者粪便,每例患者粪便等分成两份,再随机分成两组。采用QIAamp DNA Stool Mini Kit试剂盒(国际通用粪便细菌基因组DNA提取试剂盒)进行抽提,一组(A组)按照试剂盒说明书温度要求进行粪便细菌基因组DNA提取,一组(B组)对试剂盒细菌裂解温度改良后再进行提取。结果与按照试剂盒说明书温度进行提取组相比,改变细菌裂解温度后粪便基因组DNA提取量明显增高(P<0.05),而粪便基因组DNA提取纯度(OD260/280、OD260/230)无明显变化(P>0.05)。结论改变细菌裂解温度能明显提高粪便细菌基因组DNA提取量,而不改变粪便细菌基因组DNA的提取纯度。该方法可以解决因粪便采样量少导致粪便基因组DNA提取量不足的问题。
文摘Colorectal carcinoma(CRC) is a common cause of morbidity and mortality worldwide. Two pathogenic pathways are involved in the development of adenoma to CRC. The first pathway involves APC/β-catenin characterized by chromosomal instability resulting in the accumulation of mutations. The second pathway is characterized by lesions in DNA mismatch repair genes.Aberrant DNA methylation in selected gene promoters has emerged as a new epigenetic pathway in CRC development. CRC screening is the most efficient strategy to reduce death. Specific DNA methylation events occur in multistep carcinogenesis.Epigenetic gene silencing is a causative factor of CRC development. DNA methylations have been extensively examined in stool from CRC and precursor lesions. Many methylated genes have been described in CRC and adenoma, although no definite DNA methylation biomarkers panel has been established. Multiple DNA methylation biomarkers, including secreted frizzled-related protein 2, secreted frizzled-related protein 1, tissue factor pathway inhibitor 2, vimentin, and methylguanine DNA methyltransferase, have been further investigated, and observations have revealed that DNA methylation biomarkers exhibit with high sensitivity and specificity. These markers may also be used to diagnose CRC and adenoma in early stages. Real time polymerase chain reaction(q PCR) is sensitive, scalable, specific, reliable, time saving, and cost effective. Stool exfoliated markers provide advantages, including sensitivity and specificity. A stool q PCR methylation test may also be an enhanced tool for CRC and adenoma screening.
