伺机性筛查新模式在结直肠肿瘤患者一级亲属中的探索性研究

目的探究结直肠病房筛查新模式在结直肠肿瘤患者一级亲属筛查的有效性。 方法采用结直肠肿瘤风险问卷评分、粪便潜血免疫化学检测（FIT）以及粪便多靶点FIT-DNA检测对2019年10月至2021年7月在中国医学科学院肿瘤医院结直肠外科就诊的结直肠癌及进展期腺瘤患者的一级亲属进行检测，根据检测结果将一级亲属进行筛查风险分层以及肠镜检查推荐分类，分析不同分层分类后一级亲属的肠镜依从率与病变检出率。 结果共250名受试者被纳入本研究。总体人群肠镜依从率为38.0%（95/250），肠镜病变检出率为9.5%（9/95）；高风险人群（A类推荐人群）肠镜依从率为78.9%（15/19），肠镜病变检出率为26.7%（4/15）；中风险人群（B类推荐人群）肠镜依从率为61.2%（30/49），肠镜病变检出率为16.7%（5/30）；低风险人群（C类推荐人群）肠镜依从率为27.5%（50/182），肠镜病变检出率为0（0/50）。 结论三种筛查方法联合使用可以高效精准地区分一级亲属的筛查风险，此方案是一个可以在病房开展的有效可行的结直肠肿瘤患者一级亲属人群的伺机性筛查新模式。     

伺机性筛查新模式在结直肠肿瘤患者配偶的探索性研究

目的探究结直肠病房筛查新模式在结直肠肿瘤患者配偶筛查的有效性。 方法采用结直肠肿瘤风险问卷调查、粪便潜血免疫化学检测（FIT）以及粪便多靶点FIT-DNA检测对2019年10月至2021年7月在中国医学科学院肿瘤医院结直肠外科就诊的结直肠癌及进展期腺瘤患者的配偶进行检测,根据检测结果将配偶的筛查风险以及肠镜检查推荐分为A、B、C、D四类,分析不同分类后配偶肠镜依从率与病变检出率。 结果共206名受试者被纳入本研究。总体配偶人群肠镜依从率为29.6%（61/206）,肠镜病变检出率为9.8%（6/61）;A类至D类推荐人群肠镜依从率分别为90.9%（10/11）、53.5%（23/43）、20.5%（23/112）和12.5%（5/40）,肠镜病变检出率分别为30.0%（3/10）、8.7%（2/23）、4.3%（1/23）和0（0/5）。 结论三种筛查方法联合使用可以高效精准地区分配偶的筛查风险,此方案是一个可以在病房开展的有效可行的结直肠肿瘤患者配偶人群的伺机性筛查新模式。     

多靶点粪便FIT-DNA检测技术在结直肠癌患者、配偶及一级亲属筛查中的应用研究

目的 初步探究多靶点粪便FIT-DNA检测技术对结直肠癌筛查的有效性,同时探索结直肠癌患者配偶及其一级亲属的结直肠癌及癌前病变发病风险.方法 采用新型多靶点粪便FIT-DNA联合检测技术对于2017年4月至2019年3月全国八家医院的结直肠外科或普外科就诊的结直肠癌患者、患者配偶及一级亲属进行检测,并结合肠镜结果评价其...     

联合检测粪便APC、K-ras基因突变及BAT-26位点不稳定在结直肠癌早期诊断中的应用

目的探讨联合检测粪便APC、K-ras基因突变及BAT-26微卫星位点在结直肠癌早期诊断中的价值。方法以52例结直肠癌患者、35例大肠良性病变患者和24例健康对照者为研究对象,以其粪便为材料,应用聚合酶链反应单链构象多态性分析（PCR-SSCP）技术,检测BAT-26位点微卫星不稳定性、K-ras第一外显子和APC第15外显子突变密集区的变异。结果52例结直肠癌患者,其粪便APC、K-ras基因突变及BAT-26微卫星位点检出率分别为48.1%（25/52）、42.3%（22/52）和17.3%（9/52）;联合检测这三个靶点对结直肠癌和腺瘤的检出率为分别为76.9%、28.6%。结论联合检测粪便APC、K-ras基因突变及BAT-26微卫星位点是一种敏感性较高的结直肠癌早期诊断方法。     

外周血SEPT9基因甲基化检测在老年结直肠癌筛查中的意义

目的探讨外周血SEPT9基因甲基化检测在老年结直肠癌筛查中的意义。方法筛查对象为1 407例军队退休干部体检者。IFOBT、TF、SEPT9基因甲基化检测三者任一结果为阳性,通知受试者行结肠镜检查。分别计算IFOBT、TF、IFOBT+TF(双联)、SEPT9基因甲基化检测和三者联合检测在老年结直肠癌筛查的性能指标。结果 1 407例军队退休干部体检者中1 369例体检者均进行了TF、IFOBT和SEPT9基因甲基化检测,至少1项结果为阳性者有176例(12.9%),其中96例(54.5%)接受了结肠镜检查,共检出9例结直肠癌和15例进展性腺瘤。使用IFOBT、TF、IFOBT+TF(双联)检测、SEPT9基因甲基化和三种方法联合检测对结直肠癌和进展期腺瘤的检出率分别为0.4%、0.1%、0.5%、1.2%、1.8%;SEPT9基因甲基化检测对结直肠癌和进展期腺瘤的检出率优于IFOBT+TF(双联)(1.2%vs 0.5%,P=0.04),但三者联合检出率较单纯SEPT9基因甲基化检测,差异无统计学意义(1.8%vs 1.2%,P=0.27),对筛查对象进行性别和年龄分层分析显示,各项检测指标男女差别不大,但SEPT9基因甲基化检测在70岁以上患者中筛查优势明显。结论 SEPT9基因甲基化是一种简单无创的结直肠癌筛查新技术,单独使用对结直肠癌和腺瘤的检出率显著高于IFOBT和TF联合检测,因此SEPT9基因甲基化检测可作为老年人结直肠癌早期筛查的首选策略。     

粪便隐血试验联合结肠镜序贯筛查社区结直肠癌的流行病学分析

背景:近年随着人们生活、饮食、环境等改变,结直肠癌的发病率在我国呈明显升高的趋势,部分地区已开展结直肠癌筛查。目的:探讨粪便隐血试验联合结肠镜的序贯检查对筛查社区结直肠癌的作用。方法:选择2014年6月—2014年10月奉贤区多个社区40~79岁、连续2次粪便隐血试验阳性并行结肠镜筛查的无症状人群作为筛查对象,分析结直肠息肉、结直肠癌的流行病学情况。结果:共2 251名无症状人群完成粪便隐血试验和结肠镜的序贯筛查,结直肠息肉检出率为19.5%,结直肠癌检出率为0.8%。不同性别和年龄者的结直肠息肉检出率相比差异均有统计学意义(P0.05),而结直肠癌检出率无明显差异(P0.05)。结直肠息肉和结直肠癌多位于直肠和乙状结肠,息肉病理类型以腺瘤性息肉为主。结论:社区粪便隐血试验阳性无症状人群的结直肠息肉检出率高,可能与其性别、年龄、部位有关。50岁以上需重视结直肠癌筛查,可早期发现和治疗癌前疾病和结直肠癌。     

粪便钙卫蛋白检测诊断结直肠癌87例

目的:比较粪便钙卫蛋白、便潜血试验及血清CEA在结直肠癌诊断中的应用价值.方法:研究组共纳入肠镜证实的87例结直肠癌初诊患者,对照组60例接受肠镜检查的健康成人.所有研究对象在接受肠镜检查时留取大便标本,检测便潜血及粪便钙卫蛋白,肠镜检查后抽取静脉血4 mL,用于检测血清CEA.随访患者手术记录及手术病理.结果:粪便钙卫蛋白、便潜血试验及血清CEA诊断结直肠癌的敏感性分别为88.51%、83.91%和44.83%,特异性分别为88.33%、96.67%和93.33%.Dukes分期为A和B期患者便潜血试验及血清CEA的阳性率明显低于C和D期患者(P=0.0173,0.0059),Dukes不同分期的患者中,粪便钙卫蛋白的阳性率无明显差异:不同部位的结直肠癌与粪便钙卫蛋白、大便潜血试验及血清CEA的阳性率无显著差异:钙卫蛋白联合应用FOBT或/和CEA检测结直肠癌可以一定限度地提高其敏感性,但三种联合检测组合较单用粪便钙卫蛋白检测均无显著性差异.结论:粪便钙卫蛋白检测结直肠癌有较高的敏感性,且不受肿瘤分期的影响,可以作为门诊筛查结直肠癌的标志物.     

免疫法粪便潜血试验在结直肠癌筛查中的价值

目的 探讨免疫法粪便潜血试验(IFOBT)在大系列健康体检人群中筛查结直肠癌及其癌前病变的临床价值.方法 对2006年7月至2007年6月间在上海交通大学医学院附属仁济医院行健康体检的5919例采用IFOBT筛查结直肠癌及其癌前病变,对阳性病例进行结直肠镜检查或x线钡剂灌肠检查,结合临床及病理资料进行分析.结果 5919例体检人群中IFOBT阳性者314例,阳性率为5.30%,其中241例(76.75%)接受了结直肠镜检查,23例(7.32%)接受了X线钡剂灌肠检查,总随访率达84.08%,失访50例.剔除失访病例后,共发现结直肠癌16例,检出率为2.73‰(16/5869),其中Dukes A期8例(50.00%),Dukes B期7例(43.75%),Dukes C期1例(6.25%),IFOBT阳性者中的结直肠癌检出率为6.06%(16/264).共发现结直肠腺瘤样息肉94例,检出率为16.01‰(94/5869),包括管状腺瘤55例(58.51%),绒毛状-管状腺瘤23例(24.47%),绒毛状腺瘤16例(17.02%),其中单发者55例(58.51%),多发者39例(41.49%);另见活动期溃疡性结肠炎6例.IFOBT阳性者中共检出116例结直肠癌或癌前病变,检出率为43.94%(116/264).结论 IFOBT适合于大系列人群结直肠癌及其癌前病变的筛查,能发现较早期结直肠癌和癌前病变,使疾病在可治愈阶段得到根治,从而有望减少结直肠癌的发病率和死亡率.     

人粪便中OSMR和TFPI2基因甲基化在结直肠癌诊断中的意义

目的:探讨检测人粪便中抑瘤素M型受体(OSMR)基因和组织因子途径抑制子2(TFPI2)基因甲基化对于结直肠癌及腺瘤诊断的可行性及临床意义.方法:从60例结直肠癌患者和17例腺瘤患者及30名正常对照者的粪便中分别提取DNA,应用甲基化特异性PCR方法检测人粪便中OSMR和TFPI2基因的甲基化状态.结果:结直肠癌患者粪便中OSMR及TFPI2基因甲基化检出率分别高于腺瘤患者和正常对照者[OSMR:35%(21/60)vs12%(2/17),7%(2/30);TFPI2:70%(42/60)vs18%(3/17),3%(1/30);均P<0.01].二者联合检测甲基化检出率为81.7%(49/60),特异性90%.结论:检测粪便中OSMR和TFPI2基因甲基化在结直肠癌诊断和筛查中有潜在的应用价值.     

粪便Vimentin和SFRP2甲基化在结直肠癌筛查中的作用

目的探讨粪便中Vimentin和SFRP2甲基化状态在结直肠癌筛查中的价值。方法收集患者合格的粪便标本69例,其中结直肠癌23例、进展期腺瘤24例和健康人群22名,采用甲基化特异性PCR技术分析Vimentin和SFRP2甲基化状态,并与单个基因甲基化和粪便免疫隐血试验(FIT)的检测性能相比较,评价其在结直肠癌筛查中的灵敏度和特异度。结果结直肠癌组中单个Vimentin和SFRP2甲基化检出率分别为82.6%和69.6%,进展期腺瘤组为62.5%和41.7%,正常对照组为13.6%和13.6%。Vimentin和SFRP2联合检测在结直肠癌组的灵敏度为87.0%,高于FIT的56.5%(χ~2=5.25,P0.05),与单基因检测比较,差异无统计学意义(P0.05)。进展期腺瘤组中,联合检测的灵敏度为70.8%,高于SFRP2的41.7%(χ~2=4.15,P0.05)和FIT的29.2%(χ~2=8.33,P0.01),与Vimentin检测比较差异无统计学意义(P0.05)。正常对照组联合检测的特异度为86.4%,与单基因检测相同,与FIT(72.7%)比较,差异无统计学意义(P0.05)。联合检测在管状腺瘤中检出率为92.3%,高于SFRP2的53.8%(χ~2=4.9,P0.05),与Vimentin(76.9%)比较,差异无统计学意义(P0.05),在绒毛状管状腺瘤和管状腺瘤/绒毛状管状腺瘤中检出率与Vimentin相同,与SFRP2比较,差异无统计学意义(P0.05)。联合检测在伴有上皮内瘤变中的检出率与单基因检测差异均无统计学意义(P0.05)。结论粪便中联合Vimentin和SFRP2检测优于单基因及FIT检测,在结直肠癌筛查中具有潜在的应用价值。     

Fecal occult blood and flexible sigmoidoscopy screening for colorectal cancer: modeling the impact on colonoscopy requirements and cancer detection rates

AiM: The aim of this study was to estimate the colonoscopy requirements and the likely impact of fecal occult blood and flexible sigmoidoscopy screening on the detection of colorectal cancer by using previously published data. METHODS: Fecal occult blood and flexible sigmoidoscopy screening programs were applied to the 2.04 million subjects aged 50-65 years, at a participation rate of 40%. The following strategies were evaluated: Fecal occult blood testing with colonoscopy follow up of all positive tests; flexible sigmoidoscopy with colonoscopy follow up of all adenomatous polyps; and flexible sigmoidoscopy with colonoscopy follow up of all adenomatous polyps > 10 mm in size. RESULTS: The fecal occult blood program detected 5.6% of all colorectal cancer cases at a rate of 2,914 colonoscopies/percentage of detection of colorectal cancer. The flexible sigmoidoscopy program detected 14% of all colorectal cancer cases at a rate of 8,160 colonoscopies/percentage of detection of colorectal cancer. The flexible sigmoidoscopy program with follow up of adenomatous polyps > 10 mm in size detected 13% of all colorectal cancer cases at a rate of 1,230 colonoscopies/percentage of detection of colorectal cancer. CONCLUSIONS: Flexible sigmoidoscopy screening followed by colonoscopic follow up of adenomatous polyps > 10 mm in size is the most efficient screening strategy in terms of colonoscopies generated and cases of colorectal cancer detected.     

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.     

Diagnostic value of occult fecal blood testing for colorectal cancer screening

AIM: To evaluate the diagnostic value of occult fecal blood testing in mass colorectal cancer screening.METHODS: A reverse passive hemagglutination reaction fecal occult blood test (RPHA-FOBT) and colorectal cancer risk factor quantitative method were used as preliminary screening for colorectal cancer. A 60-cm fiber optic colonoscopy was used to validate the preliminary screen and was used to detect colorectal cancer in a community of 75813 subjects.RESULTS: Compared to the 60-cm fiber optic colonoscopy as a standard reference, FOBT has a sensitivity of 41.9%, specificity of 95.8%, Youden′s index of 0.38, and positive predictive value of 0.68%. These results increased with subject age from the first detection. A 3-year follow up in the target mass showed that all new cases had initially been FOBT-negative.CONCLUSION: The value of FOBT as an indicator of colorectal cancer in mass screening is limited.     

Immunochemical Fecal Occult Blood Test Is Inadequate for Screening Test of Stomach Cancer

The present study was carried out to investigate the diagnostic accuracy of the immunochemical fecal occult blood test in the screening for stomach cancer. In the hospital-based case–control study, the test was positive in 6 (14.3%) subjects with stomach cancer, in 32 (76.2%) subjects with colorectal cancer, and in 10 (7.9%) healthy subjects, respectively, showing a significant difference in detection rate between the subjects with stomach cancer and colorectal cancer (p < 0.01). In the population-based cross-sectional study, detection rate for stomach cancer was 0.13% and 0.15% for negative and positive groups decided by immunochemical fecal occult blood test, indicating no significant difference. These results reveal that the immunochemical fecal occult blood test is inadequate as the screening test for stomach cancer and suggest that examination of the upper digestive tract is unnecessary in cases where the result of fecal occult blood test is positive with no sign of colorectal diseases.     

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.     

Colorectal cancer screening in Health Examination Centers

OBJECTIVES: The aim of this study was to describe the results of colorectal cancer screening performed in the Health Examination Centers of the French general health insurance system. METHODS: The population consisted of 1,262,833 subjects (52.6% men) aged 50 to 74 years old who attended periodic health consultations from 1998 to 2003 in 89 Health Examination Centers in France. Subjects with increased risk for colorectal cancer and subjects with a positive fecal occult blood test (Hemoccult II') were invited to undergo colonoscopy. Subsequent follow-up and diagnostic data were collected. RESULTS: Prior screening practices for colorectal cancer (recent colonoscopy or fecal occult blood test, local screening campaign) were noted in 18% of the subjects attending Health Examination Center consultations. High risk for colorectal cancer (familial or personal factor) without ongoing surveillance or prior screening was observed in 3% of the study population. A fecal occult blood test was proposed to 79% of the population and of them, 89% effectively performed the test: 3.2% of tests were positive. A follow-up protocol was initiated for 63,357 subjects. A colonic exploration was performed in 69% of high-risk subjects and enabled detection of cancer in 85 and adenomas in 1683. A colonic exploration was performed in 88% of subjects with a positive fecal occult blood test and enabled detection of 674 cancers (positive predictive value of fecal occult blood test (PPV)=4.7%) including 174 Dukes A, and 2618 adenomas (PPV=18%) including 776 adenomas measuring more than 10 mm (PPV=5.4%). CONCLUSION: This study confirms the importance of implementing organized screening practices within Health Examination Centers before undertaking a generalized screen campaign targeting the entire French population.     

Prospective evaluation of fecal calprotectin as a screening biomarker for colorectal neoplasia

OBJECTIVES: Stool testing is a well established method of screening for colorectal neoplasia. Emerging data suggest that novel biomarkers may offer performance advantages over fecal occult blood. In this large, prospective study, we assessed fecal calprotectin (a leukocyte-derived protein) as a screening biomarker for colorectal neoplasia. Fecal calprotectin was directly compared to fecal hemoglobin (Hb) and colonoscopy as the existing criterion standards for stool screening and structural evaluation, respectively. METHODS: Subjects included colonoscopy patients with a personal history of colorectal neoplasia, family history of colorectal cancer, or iron deficiency anemia. Stool specimens were collected before purgation, processed appropriately, and quantitatively analyzed for calprotectin (Nycomed Pharma, Oslo, Norway) and for Hb (Mayo Medical Laboratories, Rochester, MN) by masked technicians. Colonoscopies were performed by experienced endoscopists without prior knowledge of the fecal assay results. RESULTS: Among 412 subjects, 97 (24%) subjects had one or more colorectal neoplasms (including three with adenocarcinomas). Fecal calprotectin levels did not differ significantly between subjects with versus subjects without colorectal neoplasms (p = 0.33). Neither tumor number (p = 0.85) nor tumor size (p = 0.86) significantly influenced the observed fecal calprotectin concentrations. Estimates of the sensitivity, specificity, and positive and negative predictive values of fecal calprotectin for any colorectal neoplasms were 37%, 63%, 23%, and 76%, respectively. Comparable performance estimates for fecal Hb were 3%, 97%, 27%, and 77%, respectively. CONCLUSIONS: In this cohort of colonoscopy patients at above average risk, fecal calprotectin was a poor screening biomarker for colorectal neoplasia. Further investigation of tumor-derived, rather than blood-based, biomarkers may be a more rewarding approach to stool screening for colorectal neoplasia.     

Immunochemical Fecal Occult Blood Test Is Not Suitable for Diagnosis of Hemorrhoids

PURPOSE: This study was conducted to clarify the diagnostic value of an immunochemical fecal occult blood test for hemorrhoids.PATIENTS AND METHODS: In a case-control study, an immunochemical fecal occult blood test with a 2-day method was carried out on 82 subjects with hemorrhoids, on 82 subjects with colorectal cancer, and on 82 healthy subjects. In a population-based cross-sectional study, 29,714 subjects who received an immunochemical occult blood screening with a 2-day method were divided into two groups, according to the results of a questionnaire on hemorrhoids, and the positivity rate of an immunochemical test as well as the predictive value for colorectal cancer were compared in the two groups. Moreover, both an immunochemical occult blood test with a 2-day method and colonoscopy were conducted at the same time on asymptomatic subjects during a medical checkup.RESULTS: In the case-control study, the test was positive in 13.4% subjects with hemorrhoids, in 84.1% subjects with colorectal cancer, and in 4.9% healthy subjects, respectively, showing a significant difference in the detection rate between the two diseases (P <0.001). In the population screening program, the test was positive in 6.9% subjects with hemorrhoids and in 6.5% subjects without hemorrhoids, and the predictive value was 3.2% in subjects with and without hemorrhoids, respectively, indicating no significant difference in the positivity rate as well as the predictive value between the two groups. Among 232 subjects in a medical checkup, 28 patients with hemorrhoids and 21 patients with colorectal polyp 1 cm or larger were diagnosed by colonoscopy, and the occult blood test was positive in 16.7% patients with hemorrhoids and in 52.4% patients with colorectal polyp, respectively. There was a significant difference in the sensitivity between the two disease groups (P <0.05).CONCLUSIONS: These findings indicate that the immunochemical fecal occult blood is unsuitable for the diagnosis of the patients with hemorrhoids and an examination of the colorectum is necessary in cases where the occult blood test is positive but there is a sign of hemorrhoids.