A simple method of detecting K-ras point mutations in stool samples for colorectal cancer screening using one-step polymerase chain reaction/restriction fragment length polymorphism analysis

Nishikawa, Takashi; Maemura, Kentaro; Hirata, Ichiro; Matsuse, Ryouichi; Morikawa, Hiroshi; Toshina, Ken; Murano, Mitsuyuki; Hashimoto, Kazuhito; Nakagawa, Yoshihito; Saitoh, Osamu; Uchida, Kazuo; Katsu, Ken‐ichi

doi:10.1016/s0009-8981(01)00806-3

Cited by 35 publications

(28 citation statements)

References 23 publications

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“…HIF-1 is stabilised by p53 mutations (Kress et al, 1998;Ravi et al, 2000). Indeed, mutations in the ras oncogene and p53 antioncogene are detectable in 40 -60% of colorectal cancers (Bos, 1989;Kinzler and Vogelstein, 1996;Dong et al, 2001;Rengucci et al, 2001;Nishikawa et al, 2002;Smith et al, 2002).…”

Section: Discussionmentioning

confidence: 99%

“…For example, mutations of K-ras, p53 or within the adenomatous polyposis coli (APC) gene were not detected in any stool samples of healthy controls (Rengucci et al, 2001;Nishikawa et al, 2002;Traverso et al, 2002). However, due to the genetic heterogeneity within colorectal cancer, a multitarget assay panel (e.g.…”

Section: Discussionmentioning

confidence: 99%

“…Samples are stable and the sensitivity of the Tumour M2-PK test is higher than the detection of mutations in p53, ras or APC (Rengucci et al, 2001;Nishikawa et al, 2002;Traverso et al, 2002). Therefore, the detection of Tumour M2-PK levels in the stool might provide an interesting screening tool for colorectal cancer.…”

Section: Discussionmentioning

confidence: 99%

“…The tetramer : dimer ratio of M2-PK can be quantified by gel permeation, isoelectric focusing or by ELISA (Cerwenka et al, 1999;Schulze, 2000;Mazurek et al, 1997Mazurek et al, , 2001Mazurek and Eigenbrodt, 2003). The upregulation of the M2-PK protein is under the control of HIF-1 and ras, which are both consistently altered in gastrointestinal tumours (Kress et al, 1998;Mazurek et al, 2001;Nishikawa et al, 2002;Mazurek and Eigenbrodt, 2003). The tetramer : dimer ratio of M2-PK is under the control of several oncoproteins, such as pp60 vÀsrc kinase, HPV-16 E7 and A-Raf ( Figure 1B) (Eigenbrodt et al, 1998b;Zwerschke et al, 1999;Le Mellay et al, 2002).…”

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confidence: 99%

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Faecal tumour M2 pyruvate kinase: a new, sensitive screening tool for colorectal cancer

et al. 2004

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Proliferating cells, especially tumour cells, express a special isoenzyme of pyruvate kinase, termed M2-PK, which can occur in a tetrameric form with a high affinity to its substrate, phosphoenolpyruvate (PEP), and in a dimeric form with a low PEP affinity. In tumour cells, the dimeric form is usually predominant and is therefore termed Tumour M2-PK. The levels of Tumour M2-PK within tumours and in EDTA-plasma correlate with staging and the ability of the tumour cells to metastasise. Since most colorectal tumours grow intraluminally, it appeared interesting to determine whether Tumour M2-PK is detectable in the faeces of tumour patients. Stool samples were tested by ELISA from controls without colorectal cancer and colorectal cancer patients. Whereas Tumour M2-PK levels were low in the control group (mean value7s.e.m.: 3.370.4, n ¼ 144), they were high in the case of colorectal cancer (56.1715.3, n ¼ 60). At a cutoff value of 4 U ml À1 , the sensitivity was 73%. TNM and Dukes' classification of the tumours revealed a strong correlation between faecal Tumour M2-PK levels and staging. The determination of Tumour M2-PK in faeces provides a new promising screening tool for colorectal tumours.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Faecal tumour M2 pyruvate kinase: a new, sensitive screening tool for colorectal cancer

et al. 2004

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“…[8][9][10] However, the proportion of genes derived from cancer cells in stools is as low as 1%. 11 At this concentration, mutations in stool DNA will not be readily detected using the most currently available gene analyzing methods, 12 such as restriction fragment length polymorphism (RFLP), 13 single-strand conformation polymorphism (SSCP), 14 and microarray chips. 15 The major drawbacks of these assays are that they lack sufficient sensitivity and high stability, and they are rather time and sample consuming and expensive for use in screening for molecular diagnostic purposes.…”

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confidence: 99%

Rapid detection of low-abundance K-ras mutation in stools of colorectal cancer patients using chip-based temperature gradient capillary electrophoresis

Zhang

Wang

et al. 2011

Laboratory Investigation

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Mutant K-ras provides an independent negative predictive marker for epidermal growth factor receptor (EGFR)-targeted therapy in colorectal cancers (CRCs). Rapid, sensitive, and cost-effective screening for K-ras status will overarch rational personalized medicine. Stool-based DNA testing offers unique advantages for CRC screening such as noninvasiveness, high specificity, and patient compliance, whereas complicated procedures and the low sensitivity of the present approaches have hampered its application on a wide scale. In this study, a chip-based temperature gradient capillary electrophoresis (TGCE) technique was applied to detect low-abundance K-ras mutations under a pooled experiment and analyze K-ras mutations in 30 paired stool samples and cancer tissues of CRC patients and 15 stool samples of healthy volunteers. The chip-based TGCE results showed that the successful analysis of K-ras status could be achieved within 6 min with an extremely low sample consumption of 14 nl. Detection is sensitive enough to reliably report 0.2% mutant CRC cells in a wild-type background, and 0.5 ng of template DNA was sufficient for chip-based TGCE. Of the 30 stool samples of CRC patients analyzed, 17 (57%) harbored K-ras mutations, and the lowest percentage of the detectable mutant K-ras in stool samples was 2%. The coincidence rate for K-ras mutations between stools and tissues obtained by the chip-based method reached 97% (29/30). One of the 15 stool samples of normal controls carried K-ras mutations, producing a specificity of 93%. Clone sequencing data entirely confirmed the results obtained by chip-based TGCE. The study demonstrates that chip-based TGCE is capable of rapidly screening low-abundance K-ras mutations with high sensitivity, reproducibility, simplicity, and significant savings of time and sample. Application of this method to genotype the K-ras gene in stools would provide a potential means for predicting the effectiveness of EGFR-targeted therapy in CRC patients using noninvasive approaches.

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Molecular stool screening for colorectal cancer

Mak

Lalloo

Evans

et al. 2004

British Journal of Surgery

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A simple method of detecting K-ras point mutations in stool samples for colorectal cancer screening using one-step polymerase chain reaction/restriction fragment length polymorphism analysis

Cited by 35 publications

References 23 publications

Faecal tumour M2 pyruvate kinase: a new, sensitive screening tool for colorectal cancer

Faecal tumour M2 pyruvate kinase: a new, sensitive screening tool for colorectal cancer

Rapid detection of low-abundance K-ras mutation in stools of colorectal cancer patients using chip-based temperature gradient capillary electrophoresis

Molecular stool screening for colorectal cancer

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