Pancreatic elastase 1 in stool: variations within one stool passage and individual changes from day to day

Concentration of fecal pancreatic elastase 1 has been claimed to be a highly sensitive and specific noninvasive test for exocrine pancreatic function. The aim of our study was to investigate variations in elastase concentration within one stool passage and from day to day. For the analysis of the variation of fecal elastase within one stool passage, we utilized 3 different samples collected from 8 patients. Further, we assessed the individual day to day variation of fecal elastase using stools collected on 3 consecutive days from 40 patients. For the determination of pancreatic elastase 1 in stool we used an ELISA kit. We found a relatively considerable variation of fecal elastase concentration within one stool passage (n = 8, mean CV = 22%, range 4.6-83.1%) and from day to day (n = 40; mean CV = 26%, range 2.4-61.1%). Therefore, we recommend routine analysis of more than 1 stool sample collected on different days for fecal elastase and to use a borderline area of +/- 25% of the recommended cut off of 200 micrograms/g stool for the diagnosis of pancreatic insufficiency.     

肿瘤型M2-PK、APC、K-ras检测在结直肠癌诊断中的意义

目的检测结直肠癌患者粪便和血清的M2型丙酮酸激酶(M2-PK)、APC、K-ras水平,并分析M2-PK、APC、K-ras的水平与结直肠癌病理T分期的关系。方法将2015年1-10月在该院手术治疗的结直肠癌患者200例纳入该研究作为患者组,另外选取健康体检者100例作为对照组。采用酶联免疫吸附测定(ELISA)检测结直肠癌患者血清和粪便的M2-PK、APC、Ras水平,并结合患者的病理资料对M2-PK、APC、K-ras的水平与结直肠癌病理T分期的关系进行分析。结果结直肠癌患者血清和粪便的M2-PK、APC、K-ras的表达水平分别高于对照组(P0.05)。M2-PK、APC、K-ras的表达与结直肠癌的病理T分期有密切关系,且临床病理T分期较晚的患者M2-PK、APC、K-ras的表达水平更高(P0.05)。结论结直肠癌患者血清和粪便的M2-PK、APC、K-ras水平较高且与肿瘤的临床病理T分期有关。M2-PK、APC、K-ras可作为早期诊断结直肠癌的分子标志物。     

Stool testing for the early detection of pancreatic cancer: rationale and current evidence

The development of effective tools for the early detection of pancreatic cancer, or its precursors, in high-risk subjects could play a key role in reducing the burden of this disease, which is the most lethal among solid gastrointestinal tumors. Given the poor accessibility of the pancreas due to its anatomic site, and given the limitations of imaging modalities, biomarker screening might be a promising diagnostic option. This review focuses on the rationale of using stool markers for the early detection of pancreatic cancer, and systematically summarizes current evidence. Despite several potential advantages of stool testing for pancreatic cancer and its biological plausibility, only six studies investigating two genetic markers in stool (the K-ras and the p53 gene) could be identified. Even though these studies were limited in size and could hardly approximate the screening setting, both markers appear to lack sensitivity and, in particular, specificity. The investigation of further marker candidates (e.g., epigenetic markers) in adequately designed studies represents an important next step to explore the potential of stool testing for pancreatic cancer. Pertinent studies could greatly benefit from recent methodological advances gained in connection with stool testing for colorectal cancer.     

Single LDL apheresis improves serum remnant-like particle-cholesterol,C-reactive protein,and malondialdehyde-modified-low-density lipoprotein concentrations in Japanese hypercholesterolemic subjects

BACKGROUND: We examined a technique for detecting point mutations of K-ras codon 12 in stool samples using one-step polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) analysis, in order to determine whether it could be used to screen for colorectal cancer. METHODS: DNA was extracted from 200-mg stool specimens of 5 healthy controls and 31 colorectal cancer patients. A 107-base-pair fragment of exon 1 of K-ras was amplified by PCR using mismatched primers. PCR products were digested with Bst NI and analyzed by gel electrophoresis followed by silver staining. Specificity of one-step PCR/RFLP was examined by using synthetic oligonucleotides. The detection limit of K-ras codon 12 mutations was determined by using SW480 and HT29 cells. RESULTS: The K-ras gene was successfully amplified from all healthy controls and colorectal cancer patients studied. Mutations of K-ras codon 12 were not detected in any of the healthy controls, but were identified in 13 (41.9%) of the 31 patients with colorectal cancer. Mutations were detectable in all six synthetic mutant DNAs, while none were detected among the wild type. The detection limit of this method was > or = 0.1%. CONCLUSIONS: PCR/RFLP analysis could be used in mass screening for colorectal cancer, because it is highly specific, has a low detection limit, and is simpler than conventional methods for detecting genetic abnormalities.     

Recent developments in colorectal cancer screening and prevention

Colorectal cancer is a significant contributor to morbidity and mortality in the United States. Studies published in the early 1990s, showing that screening for colorectal cancer can reduce colorectal cancer-related mortality, led many organizations to recommend screening in asymptomatic, average-risk adults older than 50 years. Since then, however, national screening rates remain low. Several important studies published over the past four years have refined our understanding of existing screening tools and explored novel means of screening and prevention. The most important new developments, which are reviewed in this article, include the following: Additional trial results support the effectiveness of fecal occult blood testing in reducing the incidence of, and mortality from, colorectal cancer. New studies document the sensitivity of fecal occult blood testing, sigmoidoscopy, and double-contrast barium enema compared with colonoscopy. Cost-effectiveness models show that screening by any of several methods is cost-effective compared to no screening. Randomized trials show that calcium is effective but fiber is not effective in preventing reoccurrence of adenomatous polyps. Preliminary data suggest that nonsteroidal anti-inflammatory drugs may prevent adenomatous polyps and that DNA stool tests and virtual colonoscopy may show promise as screening tools. This new information provides further support for efforts to increase the use of colorectal cancer screening and prevention services in adults older than 50 years.     

A comparison of three stool tests for colorectal cancer screening.

The annual guaiac or immunochemical fecal occult blood test (FOBT) is one of the five colorectal cancer (CRC) screening regimens recommended by the American Cancer Society (Smith, Cokkinides, & Eyre, 2005). Stool-based deoxyribonucleic acid (DNA) testing for CRC is considered a promising technology (Smith, Cokkinides, & Eyre, 2003). Numerous features of three noninvasive stool tests for CRC are compared.     

Human urine contains small, 150 to 250 nucleotide-sized, soluble DNA derived from the circulation and may be useful in the detection of colorectal cancer

Human urine has been shown to possess submicrogram per milliliter amounts of DNA. We show here that DNA isolated from human urine resolves into two size categories: the large species, greater than 1 kb, being predominantly cell associated and heterogeneous in size, and the smaller, between 150 to 250 bp, being mostly non-cell associated. We showed that the low molecular weight class of urine DNA is derived from the circulation, by comparing the mutated K-ras sequences present in DNA isolated from tumor, blood, and urine derived from an individual with a colorectal carcinoma (CRC) containing a mutation in codon 12 of the K-ras proto-oncogene. In the urine, mutated K-ras sequences were abundant in the low molecular weight species, but far less abundant in the large molecular weight-derived DNA. Finally, the possibility that detection of mutant K-ras sequences in DNA derived from the urine correlates with the occurrence of a diagnosis of CRC and polyps that contain mutant K-ras was explored in a blinded study. There was an 83% concurrence of mutated DNA detected in urine and its corresponding disease tissue from the same individuals, when paired urine and tissue sections from 20 subjects with either CRC or adenomatous polyps were analyzed for K-ras mutation. The possibility that the source of the trans renal DNA is apoptotic cells, and the potential use of this finding for cancer detection and monitoring is discussed.     

Melting temperature analysis as quantitative method for detection of point mutations.

Different methods have been devised to detect point mutations. Some are very sensitive, detecting mutations even in a background of normal tissue, but none provide information about the percentage of cells with mutant DNA. Here we describe an easy, fast and reliable method, melting temperature analysis, which not only detects point mutations but also provides quantitative information on the percentage of cells with mutant DNA. By this method we detected a G-A transition in codon 12 of the K-ras gene in DNA of subjects with colorectal cancer. The K-ras mutation was found in 9/10 bowel cancers and 8/10 normal adjacent samples. It was also detected in 4/7 stool samples from the same patients. In colorectal cancers, the proportion of K-ras mutant cells was variable: in two the mutant/wild-type DNA ratio was 30/70, in three 50/50, and in four 70/30. Melting temperature analysis was sensitive for the detection of point mutations in bowel cancer and also in apparently normal tissue, providing quantitative information about the percentage of cells with mutant DNA.     

DNA integrity as a potential marker for stool-based detection of colorectal cancer

BACKGROUND: Molecular genetic analysis of DNA in patient stools has been proposed for screening of colorectal cancer (CRC). Because nonapoptotic cells shed from tumors may contain DNA that is less degraded than DNA fragments from healthy colonic mucosa, our aim was to show that DNA fragments isolated from stools of patients with CRC had higher integrity than DNA isolated from stools of patients with healthy colonic mucosa. METHODS: We purified DNA from the stools of a colonoscopy-negative control group and patients with CRC and examined the relationship between long DNA fragments and clinical status by determining stool DNA integrity, using oligonucleotide-based hybrid captures with specific target sequences in increasingly long PCR reactions (200 bp, 400 bp, 800 bp, 1.3 kb, 1.8 kb, 24 kb). DNA fragments obtained from CRC patients were compared with fragments obtained from colonoscopy-negative individuals for length and/or integrity. RESULTS: DNA fragments isolated from CRC patients were of higher molecular weight (>18 bands detected of a total of 24 possible bands) than fragments isolated from fecal DNA of the colonoscopy-negative control group. CONCLUSIONS: The presence of long DNA fragments in stool is associated with CRC and may be related to disease-associated differences in the regulation of proliferation and apoptosis. An assay of fecal DNA integrity may be a useful biomarker for the detection of CRC.     

Identification of predictive markers of response to the MEK1/2 inhibitor selumetinib (AZD6244) in K-ras-mutated colorectal cancer

Mutant K-ras activity leads to the activation of the RAS/RAF/MEK/ERK pathway in approximately 44% of colorectal cancer (CRC) tumors. Accordingly, several inhibitors of the MEK pathway are under clinical evaluation in several malignancies including CRC. The aim of this study was to develop and characterize predictive biomarkers of response to the MEK1/2 inhibitor AZD6244 in CRC in order to maximize the clinical utility of this agent. Twenty-seven human CRC cell lines were exposed to AZD6244 and classified according to the IC(50) value as sensitive (≤ 0.1 μmol/L) or resistant (>1 μmol/L). All cell lines were subjected to immunoblotting for effector proteins, K-ras/BRAF mutation status, and baseline gene array analysis. Further testing was done in cell line xenografts and K-ras mutant CRC human explants models to develop a predictive genomic classifier for AZD6244. The most sensitive and resistant cell lines were subjected to differential gene array and pathway analyses. Members of the Wnt signaling pathway were highly overexpressed in cell lines resistant to AZD6244 and seem to be functionally involved in mediating resistance by shRNA knockdown studies. Baseline gene array data from CRC cell lines and xenografts were used to develop a k-top scoring pair (k-TSP) classifier, which predicted with 71% accuracy which of a test set of patient-derived K-ras mutant CRC explants would respond to AZD6244, providing the basis for a patient-selective clinical trial. These results also indicate that resistance to AZD6244 may be mediated, in part, by the upregulation of the Wnt pathway, suggesting potential rational combination partners for AZD6244 in CRC.     

A novel method to capture methylated human DNA from stool: implications for colorectal cancer screening

BACKGROUND: Assay of methylated DNA markers in stool is a promising approach for colorectal cancer (CRC) screening. A method to capture hypermethylated CpG islands from stool would enrich target analyte and allow optimal assay sensitivity. METHODS: Methyl-binding domain (MBD) protein was produced using a pET6HMBD plasmid with MBD DNA sequence cloned from rat MeCP2 gene and bound to a column of nickel-agarose resin. We first established the feasibility of using the MBD column to extract methylated human DNA in a high background of fecal bacterial DNA. To explore the impact of MBD enrichment on detection sensitivity, the tumor-associated methylated vimentin gene was assayed with methylation-specific PCR from stools to which low amounts of cancer cell DNA (0-50 ng) were added and from stools from CRC patients and healthy individuals. Stools from cancer patients were selected with low amounts of human DNA (median 7 ng, range 0.5-832 ng). RESULTS: With MBD enrichment, methylated vimentin was detected in stools enriched with >/=10 ng of cancer cell DNA and in CRC stool with a range of native human DNA amounts from 4 to 832 ng. Without MBD enrichment, methylated vimentin was not detected in the enriched stools and was detected in only 1 cancer stool with high human DNA (832 ng). In stools from healthy individuals methylated vimentin was not detected, with or without MBD enrichment. CONCLUSIONS: MBD capture increases assay sensitivity for detecting methylated DNA markers in stool. Applied clinical studies for stool cancer screening are indicated.     

粪便SDC2基因甲基化检测联合结肠镜在早期结直肠癌筛查中的意义

目的探讨粪便SDC2基因甲基化检测联合结肠镜在早期结直肠癌(CRC)筛查中的意义。方法选择2018年1月-2019年10月在深圳市宝安区中心医院体检的1 000例体检者作为研究对象,使用试剂盒分别检测粪便SDC2基因甲基化和血浆SEPT9基因甲基化,对两者任一结果为阳性者再行结肠镜检查。比较SDC2和SEPT9基因甲基化检测的阳性率以及两者联合结肠镜对进展性腺瘤和CRC的检出率。结果在1 000例筛查对象中,粪便SDC2基因甲基化检测阳性率明显高于血浆SEPT9基因甲基化〔18.10%(181/1 000)比9.80%(98/1 000)〕,差异有统计学意义(P<0.05);粪便SDC2基因甲基化检测联合结肠镜对进展性腺瘤和CRC的检出率均明显高于血浆SEPT9基因甲基化检测联合结肠镜筛查〔进展性腺瘤检出率:2.50%(25/1 000)比1.00%(10/1 000),CRC检出率:1.50%(15/1 000)比0.50%(5/1 000)〕,差异均有统计学意义(均P<0.05)。结论粪便SDC2基因甲基化检测是一种简单无创的CRC筛查新技术,患者接受程度更高,能够避免大规模肠镜筛查带来的弊端,联合结肠镜检测可作为CRC早期筛查的首选策略。     

Increased expression of annexin A1 is correlated with K-ras mutation in colorectal cancer

The activation of K-ras gene and expression of annexin A1 play an important role in colorectal tumorigenesis. We initiated this study to analyze the possible relationship between the annexin A1 expression and the K-ras mutation status in colorectal cancer. K-ras mutations are present in one fourth to one half of colorectal cancers. Annexin A1, a 37-kDa calcium- and phospholipid-binding protein, is over-expressed in colorectal cancers and may be involved in invasive tumor growth and metastasis. Here, we examined twenty paired specimens of colorectal cancer and adjacent normal tissues for K-ras mutations and annexin A1 expression. Sequencing analysis of codons 12 and 13 of K-ras revealed the presence of K-ras mutations in six colorectal cancer tissue specimens (30%). RT-PCR and immunoblotting studies further found that the expression levels of annexin A1 mRNA and protein were increased (2.9-fold and 1.7-fold, respectively) in colorectal cancers harboring K-ras codon 12 or codon 13 mutation compared with adjacent normal tissues (P < 0.05). In colorectal cancer tissues with wild-type K-ras, 12 (85.7%) specimens showed reduced expression of annexin A1 (0.48-fold and 0.81-fold, respectively). No significant association was found between K-ras mutations or annexin A1 over-expression and demographic or other clinicopathological parameters such as gender, differentiation or metastasis. However, a significant and positive correlation was identified between K-ras mutations and annexin A1 over-expression. Our findings indicate that annexin A1 could be implicated in colorectal cancer development and progression and could be of potential use as a predictive marker for guiding targeted therapy for colorectal cancer.     

Diagnostic biochip array for fast and sensitive detection of K-ras mutations in stool

BACKGROUND: Tumor cells that shed into stool are attractive targets for molecular screening and early detection of colon or pancreatic malignancies. We developed a diagnostic test to screen for 10 of the most common mutations of codons 12 and 13 of the K-ras gene by hybridization to a new biochip array. METHODS: DNA was isolated from 26 stool samples by column-based extraction from 9 cell lines. Peptide nucleic acid (PNA)-mediated PCR clamping was used for mutant-specific amplification. We used a biochip, consisting of a small plastic support with covalently immobilized 13mer oligonucleotides. The read out of the biochip was done by confocal time-resolved laser scanning. Hybridization, scanning, and data evaluation could be performed in <2 h. RESULTS: Approximately 80 ng of DNA was obtained from 200-mg stool samples. No inhibition of the PCR by remaining impurities from stool was observed. Mutation detection was possible in 1000-fold excess of wild-type sequence. Discrimination ratios between the mutations were >19 as demonstrated by hybridization with tumor cell line DNA. Stool samples (n = 26) were analyzed in parallel with PNA-PCR, restriction assay for K-ras codon 12 mutations, sequencing, and hybridization to the biochip. Nine mutations were found by hybridization, all confirmed by sequencing. PNA-PCR alone leads to an overestimation of mutations because suppression of the wild type is not effective enough with high concentrations of wild-type DNA. The restriction assay found only four mutations. CONCLUSIONS: The K-ras biochip is well suited for fast mutation detection from stool in colorectal cancer screening.     

Adapting Champion's Breast Cancer Fear Scale to colorectal cancer: Psychometric testing in a sample of older Chinese adults

PurposeColorectal cancer (CRC) is the most common type of cancer in both men and women, and older adults are more susceptible to this disease. Previous studies suggest that cancer fear may be a key predictor of participation in cancer screening. Yet there is a lack of validated measuring tools of fear relating to CRC for the Chinese older adult population. This study aims to test the psychometric properties of the Chinese version of the Colorectal Cancer Fear Scale (CRCFS), adapting from the Champion's Breast Cancer Fear Scale.MethodsThe CRCFS was developed by altering the wording ‘breast cancer’ to ‘colorectal cancer’. Interviewer-administered surveys were carried out with a convenience sample of 250 community-dwelling adults aged at least 60 years old without a history of cancer. A subsample of 40 participants completed the scale again at one-month.ResultsConfirmatory factor analysis revealed that the one-factor model provided excellent fits to the overall data, and two randomly split samples. Cronbach's alpha of the scale was 0.95 and test-retest reliability was 0.52. Positive and significant correlations of CRC Cancer Fear with CRC-related susceptibility, severity and barriers were observed. A non-linear relationship with benefits was found.ConclusionsThe findings provide support for the psychometric properties of a Chinese version of the Champion Cancer Fear with an adaption to CRC in a sample of community dwelling older Chinese adults. The scale provides a useful tool to assess CRC-related fear, which interventions should address in order to improve screening rates among older Chinese adults.     

Gut microbiome: New biomarkers in early screening of colorectal cancer

BackgroundCertain “star intestinal bacteria” have been found to act as a contributor to the development of colorectal cancer (CRC). Besides, given that the gut microbiome can be detected in a diverse range of samples (stool, tissue, blood, etc), it is categorized into fecal microbiome, blood microbiome, and tissue microbiome.MethodsTo provide an overview of the recent research progress, this review summarizes the characteristics of the gut microbiome in different samples at each stage of CRC and their screening efficiency.ResultsThe screening models constructed from different sample microbiomes (healthy/colorectal adenoma, healthy/CRC, and colorectal adenoma/CRC) have both strengths and constraints in terms of biomarker reproducibility and area under the receiver‐operating characteristic curve (AUC) of the screening models. Many bacteria, such as Bifidobacteria, Fusobacterium nucleatum (F. n), Geotrichum candidum, Porphyromonas asaccharolytica, Escherichia coli, Rhodococcus, Anaerostipes caccae, Enhydrobacter, Lachnoclostridiumsp. m3, Bacteroides clarus, Clostridium hathewayi, Ruminococcaceae, Bacteroides thetaiotaomicron, Culinariside, and enterotoxigenic Bacteroides fragilis (ETBF), show favorable diagnostic efficacy in early screening of colorectal cancer.ConclusionsThis review highlights stool, blood, tissue, and bowel fluid are the main sample sources for biomarkers, each with its own advantages and limitations. Moreover, other samples such as extracellular vesicles and biofilms also should been deserved further attention.     

A novel multitarget stool DNA test for colorectal cancer screening

Review of: Imperiale TF, Ransohoff DF, Itzkowitz SH, Levin TR, Lavin P, Lidgard GP, Ahlquist DA, Berger BM. Multitarget stool DNA testing for colorectal-cancer screening. N Engl J Med 2014;370(14):1287–97.

This Practice Pearl reviews the results of a prospective, multicenter, cross-sectional clinical study that evaluated the performance of a new multitarget stool DNA (or mt-sDNA) screening test for colorectal cancer (CRC) and compared it with a fecal immunochemical test (FIT) in individuals at average risk for CRC. The potential impact of this test on the future of CRC screening is also discussed in a brief commentary. mt-sDNA testing is a noninvasive screening test designed to detect DNA biomarkers associated with colorectal neoplasia and occult hemoglobin in the stool. The sensitivity of mt-sDNA testing for detection of CRC was 92.3%, compared with 73.8% for FIT (p = 0.002). Sensitivity for detecting advanced precancerous lesions was 42.4% for mt-sDNA testing and 23.8% for FIT (p < 0.001). The specificities of mt-sDNA testing and FIT were 86.6% and 94.9%, respectively (p < 0.001). mt-sDNA testing thus may be a first-line screening option for asymptomatic individuals at average risk for CRC who do not want to have a colonoscopy.     

Elastase 1 and chymotrypsin B in pancreatic juice and feces

A chymotrypsin-like protease was detected along with elastase 1 in pancreatic secretion and stool. This enzyme was isolated from necrobiotic human pancreas, purified, partially characterized and designated as chymotrypsin B. Quantitative studies by rocket immunoelectrophoresis indicated that neither elastase 1 nor chymotrypsin B was degraded during intestinal passage. On the basis of a clinical study, both enzymes were found to reflect pancreatic function.     

New fecal occult blood tests may improve adherence and mortality rates

Several new fecal occult blood tests have advantages over older ones when used for colorectal cancer screening. Fecal immunochemical tests can detect antibodies to human globin in the stool and can be used without the dietary restrictions needed with traditional guaiac tests. Although colonoscopy is often considered the gold standard, we hope that these new tests will allow more people to be screened and more cases of colorectal cancer to be detected early.     

焦磷酸测序法快速检测胰腺癌K-ras基因点突变

目的 建立基于焦磷酸测序技术的胰腺癌K-ras基因点突变的检测方法,并与Sanger测序法作一比较.方法 用焦磷酸测序法(Pyrosequencing)和Sanger测序法(Sanger sequencing)分别在10名正常胰腺组织、49例胰腺癌、11例慢性胰腺炎、18例胰腺良性囊肿、7例胰岛素癌、9例壶腹癌、7例胆管癌及7例十二指肠乳头癌石蜡包埋组织的DNA中检测K-rag基因12密码子点突变.结果 采用上述两种方法在所有正常胰腺组织、慢性胰腺炎、胰腺良性囊肿、胰岛素癌、壶腹癌、胆管癌及十二指肠乳头癌均未见K-ras基因点突变,而采用焦磷酸测序法发现胰腺癌石蜡包埋组织K-ras基因点突变率为71.4%(35/49),显著高于采用Sanger测序法发现胰腺癌石蜡包埋组织K-rag基因点突变率(61.2%,30/49).结论 焦磷酸测序法较Sanger测序法更为敏感,且焦磷酸测序法准确、快速、高通量,适合临床标本的批量测定.