Detection of frameshift mutations of RIZ in gastric cancers with microsatellite instability

AIM: To study the frameshift mutations of the retinoblastoma protein-interacting zinc finger gene RIZ in gastric cancer with microsatellite instability, and to identify two coding polyadenosine tracts of RIZ. METHODS: Frameshift mutations at (A)8 and (A)9 tracts of RIZ were detected in 70 human gastric cancer (HGC) specimens by DHPLC and DNA sequencing. Microsatellite instability (MSI) status was assessed by two mononucleotide markers, BAT26 and BAT25, by means of denaturing high-performance liquid chromatography (DHPLC). RESULTS: In 70 HGC samples, 8 (11.4%) were found positive for instabilities at BAT26 and BAT25. In 7 of the 8 cases with instabilities at both BAT26 and BAT25 (MSI-H), 1 was unstable at BAT26 but stable at BAT25. Frameshift mutations were identified in 4 (57.1%) of the 7 samples with MSI-H in the (A)9 tract of RIZ without mutations in the (A)8 tract. In contrast, frameshift mutations were found in neither of the polyadenosine tracts in 63 samples of MSI-L or MSI stable tumors. Pro704 LOH detection in 4 cases with frameshift mutations did not find LOH in these cases. CONCLUSION: Frameshift mutations of RIZ may play an important role in gastric cancers with MSI.     

BRAF, K-ras and BAT26 mutations in colorectal polyps and stool

AIM: To assess the feasibility of using BRAF, K-ras and BAT26 genes as stool-based molecular markers for detection of colorectal adenomas and hyperplastic polyps (HPs). METHODS: We applied PCR-SSCP and direct sequencing to detect BRAF mutations of polyps and paired stool samples. Primer-mediated restriction fragment length polymorphism (RFLP) analysis and mutant-enriched PCR were used in detection of K-ras mutations of polyp tissues and paired stool samples respectively. BAT26, a microsatellite instability marker was examined by detection of small unstable alleles in a poly (A) repeat. RESULTS: No genetic alterations were detected in the 36 colonoscopically normal patients in either tissues or stools. BRAF, K-ras and BAT26 mutations were found in 4 (16%), 10 (40%) and 3 (12%) of 25 adenoma tissues and among them, 75%, 80% and 100% of patients were observed to contain the same mutations in their corresponding stool samples. In HPs, mutations of BRAF and K-ras were detected in the tumor DNA of 2 (11.1%) and 8 (33.3%) of 18 patients respectively, all of whom had identical alterations in their stools. Taken together, the three genetic markers detected 15 (60%) of 25 adenomas and 8 (44.4%) of 18 HPs. The sensitivity of stool detection was 80% for adenomas and 100% for HPs with an overall specificity of 92% for adenomas and 100% for HPs. CONCLUSION: BRAF, K-ras and BAT26 genes have the potential to be molecular markers for colorectal adenomas and HPs, and can be used as non-invasive screening markers for colorectal polyps.     

Detection of hypermethylated DNA or cyclooxygenase-2 messenger RNA in fecal samples of patients with colorectal cancer or polyps

BACKGROUND: Detection of fecal DNA is a promising approach to colorectal cancer screening. However, the sensitivity of current fecal DNA tests for colorectal polyps is low. We evaluated the feasibility of detecting aberrantly methylated DNA or cyclooxygenase-2 (COX-2) mRNA in feces of patients with colorectal cancer or polyps. METHODS: Fecal samples were collected prior to colonoscopy from 20 patients with colorectal cancer, 30 patients with colorectal polyps, and 30 subjects with normal colonic examination. Presence of hypermethylated DNA in 7 tumor-related genes (APC, ATM, hMLH1, sFRP2, HLTF, MGMT, and GSTP1) in stool was analyzed by methylation-specific PCR. COX-2 mRNA in fecal samples was detected by RT-PCR. RESULTS: With the use of this panel of methylation markers, the sensitivity of detecting colorectal cancer and adenoma was 75% (95% CI 50.9-91.3%) and 68% (95% CI 46.5-85.1%), respectively. Three normal subjects also had methylated DNA detected in stool, which gives a specificity of 90% (95% CI 73.5-97.9%). The mean number of genes methylated in DNA from the stool of patients with colorectal cancer and adenoma was 1.4 and 0.9, respectively. In contrast, COX-2 mRNA was detected in the stool samples of 10 (50%) cancer patients and one (4%) patient with advanced adenoma only. Two (6.7%) stool samples from normal subjects also had COX-2 mRNA detected. CONCLUSION: Detection of aberrantly methylated DNA in fecal samples is more sensitive than COX-2 mRNA for detection of colorectal cancer and adenoma.     

Analysis of promoter methylation in stool: a novel method for the detection of colorectal cancer.

BACKGROUND & AIMS: Detection of tumor-derived DNA alterations in stool is an intriguing new approach with high potential for the noninvasive detection of colorectal cancer (CRC). Because of heterogeneity of tumors, usually multiple markers distributed throughout the human genome need to be analyzed. This is labor intensive and does not allow for high through-put screening. Therefore, markers with high sensitivity and good specificity are needed. We explored the potential of a single epigenetic marker in comparison with fecal occult blood testing (FOBT) for the discrimination of patients with CRCs and adenomas from those without. METHODS: Methylation-specific polymerase chain reaction (PCR) was performed to analyze hypermethylated in cancer 1 (HIC1) promoter methylation status in a blinded fashion in stool samples from 26 patients with CRC, 13 with adenoma > or =1 cm, 9 with hyperplastic polyps, 9 with chronic inflammatory bowel disease, and 32 with endoscopically normal colon. RESULTS: Ninety-seven percent of the stool samples contained amplifiable DNA. Forty-two percent of the samples from patients with CRC and 31% of the samples from patients with colorectal adenoma > or =1 cm were positive for HIC1 promoter methylation. No methylated HIC1 promoter DNA was detected in the fecal DNA from patients with endoscopically normal colon or hyperplastic polyps. CONCLUSIONS: The epigenetic marker HIC1 promoter methylation carries high potential for the remote detection of CRCs. We postulate that a panel of merely a few genetic and epigenetic markers will be required for the highly sensitive and specific detection of CRCs and adenomas in fecal samples from affected patients.     

Approaches to identification of HNPCC suspected patients in Slovak population

Patients with hereditary non-polyposis colorectal cancer (HNPCC) have a DNA mismatch repair defect (MMR) in their tumor tissue that results in instability of microsatellite DNA sequences (MSI). Thus, MSI analysis may effectively indicate this form of cancer that should be then proved by analysis of germline mutations in MMR genes. The aim of this study was to identify HNPCC suspected patients in the Slovak population by investigating microsatellite instability in colorectal tumor tissues. MSI was studied at 5-11 loci in matched tumor and normal DNA using radioactively labeled PCR products separated on sequencing gels. High microsatellite instability (MSI-H) was present only in patients younger than 50 years, in 100% of patients having two affected relatives by colorectal cancer and in 67% of patients with only one affected relative. In both groups of patients colorectal cancer was present in two successive generations. No MSI-H was found in the group of patients older than 50 years, even if they had positive family history for colorectal cancer. Among all markers used, the BAT26 mononucleotide repeat (100%), DI0S197 and D13S175 (62.5%) dinucleotide repeats were the most frequently altered in the tumor tissues. Retrospective analysis revealed that some of the patients having MSI-H tumors have had clinicopathological characteristics frequently reported to HNPCC. The family members of those patients with MSI-H are enrolled in preventive health care program until mutational analyses will enable to select carriers from non-carriers of mutated MMR genes.     

Hypermethylated SFRP2 gene in fecal DNA is a high potential biomarker for colorectal cancer noninvasive screening

AIM： To investigate the feasibility of detecting hypermethylated secreted frizzled-related protein 2 （SFRP2） gene in fecal DNA as a non-invasive screening tool for colorectal cancer （CRC）. METHODS： Fluorescence-based real-time PCR assay （MethyLight） was performed to analyze SFRP2 gene promoter methylation status in a blinded fashion in tumor tissues and in stool samples taken from 69 CRC patients preoperatively and at the 9th postoperative day, 34 patients with adenoma ≥1 cm, 26 with hyperplastic polyp, and 30 endoscopically normal subjects. Simultaneously the relationship between hypermethylation of SFRP2 gene and clinicopathological features was analyzed. RESULTS： SFRP2 gene was hypermethylated in 91.3% （63/69） CRC, 79.4% （27/34） and 53.8% （14/26） adenoma and hyperplastic polyp tissues, and in 87.0% （60/69）, 61.8% （21/34） and 42.3% （11/26） of corresponding fecal samples, respectively. In contrast, no methylated SFRP2 gene was detected in mucosal tissues of normal controls, while two cases of matched fecal samples from normal controls were detected with hypermethylated SFRP2. A significant decrease （P 〈 0.001） in the rate of hypermethylated SFRP2 gene was detected in the postoperative （8.7%, 6/69） fecal samples as compared with the preoperative fecal samples （87%, 60/69） of CRC patients. Moreover, no significant associations were observed between SFRP2 hypermethylation and clinicopathological features including sex, age, tumor stage, site, lymph node status and histological grade, etc.CONCLUSION： Hypermethylation of SFRP2 gene in fecal DNA is a novel molecular biomarker of CRC and carries a high potential for the remote detection of CRC and premalignant lesions as noninvasive screening method.     

Low rate of microsatellite instability in young patients with adenomas: reassessing the Bethesda guidelines

BACKGROUND AND AIM: Screening adenomas for microsatellite instability (MSI) in patients younger than 40 yr of age has been recommended by the Bethesda Guidelines as a means of identifying patients at risk for hereditary nonpolyposis colorectal cancer (HNPCC). We sought to determine the rate of MSI in adenomas removed from individuals under 40 yr of age over a 5-yr period in a university general gastroenterology practice. METHODS: We identified patients between 18 and 39 yr of age with endoscopically removed adenomatous colorectal polyps. Patients with polyposis syndromes, inflammatory bowel disease, or colorectal carcinoma were excluded. A three-generation family history was obtained via telephone interview. Endoscopic and histology reports were reviewed. Adenomas were tested for MSI using the BAT26 and BAT40 microsatellite markers, and expression of the MSH2 and MLH1 proteins was assessed by immunostaining. RESULTS: A total of 34 patients had 46 adenomas removed endoscopically. Out of 34 patients, 14 (41%) had a family history of colorectal cancer and 3 were from Amsterdam criteria positive families. A total of 28 of 46 adenomas (61%) were distal to the splenic flexure. Polyps ranged in size from 2 to 20 mm and averaged 6.6 mm. Five polyps (11%) were tubulovillous adenomas, and the remainder were tubular adenomas. None of the polyps were serrated adenomas and none demonstrated high-grade dysplasia. Among the 40 adenomas available for testing, none demonstrated MSI using either BAT26 (0/40) or BAT40 (0/21), nor did any of the polyps tested demonstrate loss of either MSH2 or MLH1 expression (0/16). CONCLUSION: Screening adenomas from patients younger than 40 yr of age for MSI was ineffective in identifying potentially new cases of HNPCC. New strategies that improve on the current clinical and molecular screening methods should be developed so that at-risk individuals can be identified and referred for germline testing before developing their first cancer.     

Clinical usefulness of microsatellite instability test in Korean young patients with high-risk features associated with adenoma

The aim of this study was to determine the correlation between microsatellite instability (MSI) and young age in patients with advanced colorectal adenomas. We retrospectively analyzed young patients (≤40years of age) with advanced adenomas (n=84) between January 1996 and December 2006. We randomly selected the control group as patients≥50years of age with advanced adenomas (n=84) during the same time period. Of these patients, the MSI test and MLH1 immunohistochemistry were performed in the available tissue samples from patients with advanced adenomas. The number of patients who had the two tests was 52 in the young group and 49 in the old group. The monomorphic nature of the BAT26 panel for MSI analysis was used without comparison of normal tissue. MSI was detected in three young patients (n=52) and none of the old patients (n=49). There was no statistical difference between the two groups (P=0.243). All three young patients with MSI had a strong family history of colorectal cancer. MSI analysis was not a useful method of screening for HNPCC in young patients with advanced colorectal adenoma, at least in cases without a family history of colorectal cancer.     

结直肠锯齿状病变的微卫星状态研究

目的 比较结直肠锯齿状病变与传统腺瘤、腺癌的微卫星状态的差异,以期间接验证传统型锯齿状成瘤通路的存在.方法 收集北京军区总医院病理科保存的75例大肠息肉及肿瘤组织蜡块标本,其中锯齿状腺癌(Sca)15例,非锯齿状腺癌(N-Sca)20例,传统型锯齿状腺瘤(TSA)20例,普通腺瘤20例.抽提基因组DNA,采用荧光标记引物扩增BAT25、BAT26两个位点,随后使用DNA自动测序仪检测其微卫星状态,并对实验结果进行统计学分析.结果部分标本扩增失败,对于成功扩增的68例标本:18例TSA中6例为高度微卫星不稳定型(MSI-H),12例为低度微卫星不稳定型(MSI-L)/微卫星稳定型(MSS);18例普通腺瘤均为MSS;13例Sca中3例为MSI-H,10例为MSI-L/MSS;19例N-Sca中仅1例为MSI-H,18例为MSI-L/MSS.统计学分析表明,普通腺瘤组、N-Sca组MSI-H发生率明显低于TSA组和Sca组(P<0.05),而后两组间差异无统计学意义(P>0.05).结论 与普通腺瘤、N-Sca相比,MSI-H更多见于TSA、Sca,由此推断存在一条有别于传统"腺瘤-癌"发生模式的传统型锯齿状成瘤通路,但尚需大规模前瞻性研究确认.     

Testing of multiple samples increases the sensitivity of stool decay-accelerating factor test for the detection of colorectal cancer

OBJECTIVES: We previously reported that the measurements of stool decay-accelerating factor (DAF), a membrane-bound, complement regulatory protein, may be valuable for the detection of colorectal cancer. Recently we have refined the immunoassay for stool DAF. In the present study, using the refined assay, we measured stool DAF concentrations in multiple samples from patients with colorectal cancer and in healthy controls to determine whether testing of multiple samples would increase the sensitivity of the stool DAF test. METHODS: DAF was measured in three spontaneously passed stool samples from each of 100 patients with colorectal cancer and 100 control subjects without apparent colorectal disease. RESULTS: The stool DAF concentrations in the patients with colorectal cancer (median 11.1 ng/g stool; interquartile range 2.9-32.7 ng/g) were significantly higher than concentrations in the subjects without colorectal diseases (median 1.6 ng/g stool; interquartile range 0.4-3.4 ng/g) (p<0.0001). Testing of two samples from each patient significantly increased the sensitivity (72%) of the stool DAF test without significantly decreasing its specificity (92%). The stool DAF test was positive in more than one half of patients with colorectal cancer at a relatively early TNM stage or with negative fecal occult blood test. CONCLUSIONS: These findings suggest that stool DAF is a marker of colorectal cancer independent of fecal occult blood and testing of two samples increases the sensitivity of the stool DAF test. Measurement of stool DAF now seems worthy of further consideration as a noninvasive method for the detection of colorectal cancer.     

Evaluation of tumor microsatellite instability using five quasimonomorphic mononucleotide repeats and pentaplex PCR

BACKGROUND & AIMS: The microsatellite instability (MSI) phenotype is a characteristic of the hereditary nonpolyposis colorectal cancer syndrome as well as approximately 15% of sporadic colon and gastric tumors. It is a valuable diagnostic marker for the identification of hereditary nonpolyposis colorectal cancer cases and may be a molecular predictive marker for the identification of colon cancer patients who benefit from chemotherapy. To evaluate MSI, a reference panel was proposed at an international consensus meeting, comprised of 2 mononucleotide (BAT-25, BAT-26) and 3 dinucleotide repeats. Analysis of BAT-26 is sufficient for detecting the MSI phenotype in most, but not all, cases. Additional results with dinucleotide markers can sometimes lead to incorrect classification of MSI tumors. METHODS: We describe here a single fluorescent multiplex system comprising 5 quasimonomorphic mononucleotide repeats for the detection of MSI tumors. RESULTS: None of 184 germline DNA samples, including 56 from African subjects, was found to contain allelic size variations in more than 2 of these markers. In contrast, all MSI tumors showed allelic size variations in 3 or more of the microsatellites. Using this assay, we confirmed (or reclassified in 6 cases) the MSI status of 124 colon and 50 gastric primary tumors and 16 colon cell lines. CONCLUSIONS: We propose that using a pentaplex polymerase chain reaction system allows accurate evaluation of tumor MSI status of DNA with 100% sensitivity and specificity without the need to match normal DNA. This assay is simpler to use than those involving dinucleotides and is more specific than using BAT-26 alone.     

粪便DNA中MGMT、XAF1基因启动子甲基化联合检测在结直肠癌筛查中的应用研究

目的使用甲基化特异性PCR(MSP)方法检测粪便DNA中O6-甲基鸟嘌呤-DNA甲基转移酶(MGMT)、X染色体连锁凋亡抑制蛋白相关因子1(XAF1)基因启动子甲基化情况,并探讨其在结直肠肿瘤诊断中的意义。方法收集40例结直肠腺癌、40例腺瘤性息肉、52例正常对照住院患者粪便标本,使用试剂盒提取其粪便中肠道脱离细胞DNA,通过MSP方法检测其MGMT、XAF1基因启动子甲基化情况。结果 MGMT、XAF1基因启动子甲基化在结直肠腺癌中的阳性率分别为50.0%、55.9%;在腺瘤性息肉中的阳性率分别为42.1%、52.6%;二者联合检测在结直肠腺癌及腺瘤性息肉中的阳性率分别为73.5%、68.4%;特异性为52.0%。结直肠腺癌患者粪便中粪便潜血阳性率为35.3%,CEA阳性率为35.3%。结论通过试剂盒提取粪便DNA具有较高成功率;粪便DNA中MGMT、XAF1基因启动子甲基化状态用于检测结直肠腺癌及腺瘤性息肉具有较高的敏感性。检测粪便基因甲基化有望成为CRC高风险人群筛查的一个重要途径。     

结直肠癌错配修复蛋白和微卫星不稳定检测的对比分析

目的目前最常用的筛查结直肠癌DNA错配修复基因缺失的方法是免疫组化检测错配修复（MMR）基因相关蛋白的表达,以及基于PCR检测多个微卫星位点判断有否微卫星不稳定（MSI）这2种方法,本研究主要目的是比较这二种检测之间的一致性,并对分子病理室作室内质量控制。 方法收集2014年8月至2015年10月湖北省肿瘤医院结直肠癌368例手术切除标本,免疫组化常规检测癌组织MLH1,PMS2,MSH2及MSH6这4种蛋白的表达。免疫组化显示任一蛋白完全缺失,判读为MMR蛋白缺失（dMMR）;如癌细胞4个MMR有多少不等的核着色,判读为MMR无缺失（pMMR）。选取其中的65例行PCR-毛细管电泳法检测MSI,其中28例为pMMR,37例为dMMR。然后对这65例组织用PCR毛细管电泳法检测Bethesda推荐的5个微卫星位点。比对这65例患者上述二种检测结果之间的一致性,并分析、整理其临床病理特征。 结果368例结直肠癌中有37例免疫组化结果为dMMR中,其余331例为pMMR。37例中剔除2例后对其中35例行毛细管PCR法检测,显示高频MSI者32例,微卫星稳定（MSS）者3例。选取331例中的28例行PCR检测,显示MSS者27例,MSI-H者1例。免疫组化法检测的敏感度和特异性分别为97.0%和90.0%,PCR检测结果的敏感度和特异性分别为91.4%和96.4%;二者总的一致性为93.7%。伴MSI的结直肠癌原发灶以右半结肠最多见（占48.6%）,病理形态以低分化腺癌伴淋巴细胞浸润和粘液分泌最常见,病理TNM分期以Ⅱ期和Ⅲ期为主。 结论免疫组化检测MMR蛋白和基于PCR的毛细管电泳法检测MSI二者的一致性高,其中免疫组化法可以作为临床初筛结直肠癌微卫星不稳定性的一种经济而便捷的方法,值得推广。     

Stool-based DNA testing, a new noninvasive method for colorectal cancer screening, the first report from Iran

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 BAT-26 was not detected in any of stool samples.CONCLUSION:We could detect colorectal cancer related genetic alterations by analyzing stool DNA witha 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.     

Role of detection of microsatellite instability in Chinese with hereditary nonpolyposis colorectal cancer or ordinary hereditary colorectal cancer

AIM: To detect microsatellite instability (MSI) in patients with hereditary nonpolyposis colorectal cancer or ordinary hereditary colorectal cancer and to provide criteria for screening the kindreds with hereditary nonpolyposis colorectal cancer at molecular level. METHODS: MSI was detected in the specimens from 20 cases with HNPCC, 20 cases with ordinary hereditary colorectal cancer and 20 cases with sporadic colorectal cancer by means of polymerase chain reaction-single strand conformation polymorphism. RESULTS: The positive rate of MSI was 85% (17/20) in HNPCC group, 40% (8/20) in ordinary hereditary colorectal cancer group and 10% (2/20) in the sporadic colorectal cancer group respectively. The differences were significant. The mean ages of the three groups were 43.6, 52.2, and 61.8 years respectively, which increased gradually. The incidence of right hemicolon cancer was 64.7%, 37.5%, and 0% respectively, which decreased gradually and had significant difference. The expression ratio of BAT26 and BAT25 was 94.1% respectively, which was highest in the 5 gene sites studied. The incidence of poorly differentiated adenocarcinoma was 70.6% in HNPCC group among high frequency microsatellite instability (MSI-H), which was higher than the other two groups, which had 50% and 50% respectively. CONCLUSION: The incidence of MSI-H is higher in HNPCC group. The detection of MSI is simple and economical and has high correlation with the clinicopathologic feature of HNPCC and can be used as a screening method to detect the germ line mutation of the mismatch repair gene.     

SFRP2 methylation in fecal DNA—a marker for colorectal polyps

Introduction DNA methylation of secreted frizzled-related proteins (SFRPs) can be detected in colorectal cancer (CRC) tissue, in tissue of adenomas, and in aberrant crypt foci, whereas in normal colorectal mucosa tissue, SFRP genes are unmethylated. Recently, our study group was able to demonstrate SFRP2 methylation as the most sensitive single DNA-based marker in stool for identification of CRC. The purpose of this study was to clarify whether SFRP2 methylation in fecal DNA can be found in stool of individuals with hyperplastic and adenomatous colorectal polyps. Materials and methods Patients who were diagnosed with colorectal polyps or showed negative colonoscopy were included in this study. DNA from stool samples was isolated. SFRP2 methylation was assessed by means of MethyLight. Results Stool samples from 68 individuals were checked for DNA content; 23% of the samples (6 of 26) from healthy controls, 46% of the samples (6 of 13) from patients with hyperplastic polyps, and 45% of the samples (13 of 29) from patients with adenomas were positive for human DNA. SFRP2 methylation in stool samples was found in none of the healthy controls, in 33% (2 of 6) patients with hyperplastic polyps, and in 46% (6 of 13) patients with adenomas. Statistical analysis revealed that the frequency of SFRP2 methylation increased significantly (P = 0.028) from healthy controls to patients with hyperplastic polyps and to patients with adenomas. Conclusions In the current study, we report for the first time that SFRP2 methylation in fecal DNA increases significantly from healthy controls to patients with hyperplastic polyps and to patients with adenomas. SFRP2 methylation may serve as a marker for molecular stool-based adenoma and CRC screening.     

Fecal excretion of calprotectin in colorectal cancer: relationship to tumor characteristics

BACKGROUND: The aim of this study was to evaluate fecal calprotectin in patients treated for colorectal cancer. Furthermore, the changes in fecal calprotectin concentration from before to after surgery were investigated. METHODS: In 155 patients with newly diagnosed colorectal cancer, two spot samples were taken from the same feces on two consecutive days. RESULTS: Three ways of evaluating calprotectin excretion were compared, (1st spot 1st stool; maximum of 1st spot 1st stool and 2nd spot 1st stool; maximum of 1st spot 1st stool and 1st spot 2nd stool) and gave similar results with median fecal calprotectin values 47 mg/l, 52 mg/l and 54 mg/l, respectively. Median calprotectin concentration did not differ significantly between different tumor stages, although the levels were slightly lower in Dukes stage A tumor than in the rest of the stages. Neither were there any differences in the concentrations related to the localization, size or the histological grading of the carcinoma. As the currently used cut-off level for fecal calprotectin is 10 mg/l, 87% of all patients had elevated fecal calprotectin. Seventy-nine percent of the patients had levels above 15 mg/l and 74% had levels above 20 mg/l (1st spot 1st stool). In patients who delivered fecal samples after the operation the calprotectin value fell significantly from a preoperative median value of 45 mg/l to 14 mg/l after the resection. CONCLUSIONS: The majority of patients with colorectal cancer have increased fecal concentration of calprotectin. One single fecal spot seems to be sufficient for determination of the calprotectin level. Measurement of fecal calprotectin may possibly become of value as a marker for colorectal cancer, although calprotectin, similar to fecal occult blood (FOB) tests, is a non-specific test for colorectal pathology, also being elevated in inflammatory bowel diseases. Further investigation of its specificity is therefore needed.     

几种粪便脱落细胞检查方法的大肠癌筛检效率比较

目的 评价不同粪便脱落细胞检查方法的大肠癌筛检效率.方法 对814例结肠镜检查者分别经清肠液法、二次自然粪便法、磁选法、自然排便联合清肠液液基法提取粪便脱落细胞,进行细胞学诊断.部分经清肠液法受检者提取细胞后行DNA核定量分析(清肠液+DNA核定量分析法).部分二次自然粪便法提取的粪便行基因检测.二次自然粪便法和自然排便联合清肠液液基法提取的粪便同时进行粪隐血检测.结果 粪便脱落细胞检出大肠癌的敏感性为66.27%(112/169),特异性为99.56%(225/226).大肠癌患者粪便中提取异犁细胞的阳件率与大肠癌分化程度和Duke分期无关(P＞0.05).二次自然排便法、自然排便联合清肠液液基法、清肠液+DNA核定量分析法筛检大肠癌的敏感性(分别为76.09%、68.29%、76.92%)优于单纯清肠液法和磁选法(分别为26.31%、43.24%).粪便脱落细胞p53、K-ras、腺瘤性息肉病(APC)基因检测阳性率与癌组织相应基因检测的一致率为83.33%(25/30)、9/10、9/10.基因检测的阳性率低于粪便脱落细胞学检测.联合进行粪隐血和粪脱落细胞学检测检出大肠癌的敏感性(93.10%)高于单独进行粪隐血试验(80.46%)和粪便脱落细胞学检测(73.56%).结论 粪便脱落细胞学检查是大肠癌筛检的有效手段.利用液基薄层技术对患者自然排便或清肠液进行两次筛查并联合粪隐血检测是最佳筛检方案.     

Fecal Excretion of Calprotectin in Colorectal Cancer Relationship to Tumor Characteristics

Background: The aim of this study was to evaluate fecal calprotectin in patients treated for colorectal cancer. Furthermore, the changes in fecal calprotectin concentration from before to after surgery were investigated. Methods: In 155 patients with newly diagnosed colorectal cancer, two spot samples were taken from the same feces on two consecutive days. Results: Three ways of evaluating calprotectin excretion were compared, (1st spot 1st stool; maximum of 1st spot 1st stool and 2nd spot 1st stool; maximum of 1st spot 1st stool and 1st spot 2nd stool) and gave similar results with median fecal calprotectin values 47 mg/l, 52 mg/l and 54 mg/l, respectively. Median calprotectin concentration did not differ significantly between different tumor stages, although the levels were slightly lower in Dukes stage A tumor than in the rest of the stages. Neither were there any differences in the concentrations related to the localization, size or the histological grading of the carcinoma. As the currently used cut-off level for fecal calprotectin is 10 mg/l, 87% of all patients had elevated fecal calprotectin. Seventy-nine percent of the patients had levels above 15 mg/l and 74% had levels above 20 mg/l (1st spot 1st stool). In patients who delivered fecal samples after the operation the calprotectin value fell significantly from a preoperative median value of 45 mg/l to 14 mg/l after the resection. Conclusions: The majority of patients with colorectal cancer have increased fecal concentration of calprotectin. One single fecal spot seems to be sufficient for determination of the calprotectin level. Measurement of fecal calprotectin may possibly become of value as a marker for colorectal cancer, although calprotectin, similar to fecal occult blood (FOB) tests, is a non-specific test for colorectal pathology, also being elevated in inflammatory bowel diseases. Further investigation of its specificity is therefore needed.     

遗传性非息肉性结直肠癌患者腺瘤和癌组织微卫星基因型分析

背景：遗传性非息肉性结直肠癌（HNPCC）是一种由错配修复基因种系突变引起的常染色体显性遗传病，高度微卫星不稳定（MSI—H）为其分子生物学特征之一。目的：利用5个微卫星位点建立正常结直肠黏膜、结直肠腺瘤和癌组织的微卫星基因型，探讨HNPCC的MSI发生情况和MSI检测的临床意义。方法：纳入源自33个HNPCC家系的腺瘤28例和腺癌14例，其中4例为同步腺瘤-癌；以32例散发性结直肠腺瘤和24例散发性结直肠癌作为对照。选用BAT25、BAT26、D2S123、D5S346、D17S250五个微卫星位点行荧光标记聚合酶链反应（PCR），以GeneMapper软件分析PCR产物。通过与正常黏膜微卫星序列PCR片段长度进行比较，判定腺瘤和癌组织的MSI情况。结果：HNPCC腺瘤和癌组织MSI-H发生率分别显著高于散发性结直肠腺瘤和结直肠癌（64-3％对3．1％，71.4％对12．5％，P〈0．05）。4例同步腺瘤-癌均表现为MSI—H．其腺瘤和癌组织的MSI类型不同。结论：HNPCC腺瘤和癌组织MSI—H发生率高。同步腺瘤一癌来源于不同克隆。MSI检测可作为HNPCC的临床初筛方法。