Clinica Chimica Acta 318 (2002) 107 – 112
`
`www.elsevier.com/locate/clinchim
`
`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
`
`Takashi Nishikawa a,*, Kentaro Maemura a, Ichiro Hirata a, Ryouichi Matsuse b,
`Hiroshi Morikawa a, Ken Toshina a, Mitsuyuki Murano a, Keiichi Hashimoto a,
`Yoshihito Nakagawa a, Osamu Saitoh a, Kazuo Uchida b, Kenichi Katsu a
`
`aSecond Department of Internal Medicine, Osaka Medical College, Takatsuki, Japan
`bKyoto Medical Science Laboratory, Kyoto, Japan
`
`Received 17 July 2001; received in revised form 20 November 2001; accepted 10 December 2001
`
`Abstract
`
`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  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. D 2002 Elsevier Science B.V. All rights reserved.
`
`Keywords: K-ras; Colorectal cancer; Stool; PCR/RFLP
`
`1. Introduction
`
`Recent advances in research into the molecular bio-
`logy of colorectal cancer have identified a close rela-
`tionship between oncogenes, abnormalities of tumor
`
`* Corresponding author. 7676 Phoenix Drive, #1509 Houston,
`TX 77030, USA. Tel.: +1-713-796-8680.
`E-mail address: tnishika@mdanderson.org (T. Nishikawa).
`
`suppressor genes (such as APC, K-ras, p53, and DCC),
`and the development and progression of colorectal
`cancer. K-ras is an oncogene that exists on chromo-
`some 12p12.1 and is activated by point mutations at
`codons 12, 13, and 61 [1]. The products of K-ras are
`GTP/GDP-binding proteins that have a molecular
`weight of approximately 21 kDa and are found on the
`inner side of the cell membrane where they act as trans-
`mitters for cell proliferation and differentiation signals
`
`0009-8981/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.
`PII: S 0 0 0 9 - 8 9 8 1 ( 0 1 ) 0 0 8 0 6 - 3
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`
`[1]. Activation of K-ras by point mutations appears to
`play a role in carcinogenesis by increasing the active
`GTP-bound form, which increases the activity of tran-
`scription factors through binding to target proteins
`(Raf, PI3kinase, and RalGDS) [2 – 5]. K-ras mutations
`are common in various cancers, with a prevalence of
`40 – 50% in colorectal cancer [6 – 9], 75 – 93% in pan-
`creatic cancer [9], 22 – 33% in pulmonary adenocarci-
`noma [9]. In colorectal cancer, most (70 – 80%) of these
`mutations occur at codon 12 [6 – 9]. In colorectal can-
`cer, K-ras point mutations occur early in the adenoma –
`carcinoma sequence and are believed to contribute to
`the growth and increased atypia of adenomas [8,10].
`At present, fecal occult blood testing is commonly
`used in mass screening for colorectal cancer. However,
`this method is not specific for cancer because it detects
`the secondary phenomenon of bleeding and is therefore
`also positive for bleeding caused by inflammation or
`hemorrhoids. For a diagnostic tool to be specific for
`cancer, it must be capable of the direct detection of ge-
`netic alterations in cancer cells within stool samples.
`Since Sidransky et al. [11] first reported that K-ras
`point mutations were detectable in stool samples, se-
`veral groups have analyzed specimens using modified
`techniques such as mutant allele-specific amplification
`[12,13] and mutant-enriched PCR [14]. However, these
`methods cannot be readily applied to mass screening
`because of contamination and low reproducibility due
`to the complexity of the procedures. In an attempt to
`overcome these problems, we used one-step polymer-
`ase chain reaction/restriction fragment length polymor-
`phism (PCR/RFLP) to detect K-ras point mutations in
`stool samples, and we investigated the specificity and
`the mutation detection limit of this method.
`
`2. Materials and methods
`
`2.1. Cell culture
`
`A human colon cancer cell line (HT29) was main-
`tained at 37 °C in McCoy’s 5A medium (Dai-Nippon
`Pharmaceutical, Japan) supplemented with 10% fetal
`bovine serum and penicillin/streptomycin.
`Another human colonic cancer cell line (SW480)
`was maintained at 37 °C in L-15 medium (Dai-
`Nippon Pharmaceutical) supplemented with 10% fetal
`bovine serum and penicillin/streptomycin.
`
`2.2. Patients and specimens
`
`Stool and tissue specimens were collected from 31
`patients with colorectal cancer. As a normal control,
`stool specimens were obtained from five patients who
`had no evidence of malignant tumors on colonoscopy,
`gastroduodenoscopy, and upper abdominal ultraso-
`nography. All of these patients gave informed consent
`for our study. The stool specimens were immediately
`frozen at 80 °C until use.
`Tissue samples were obtained either from formalin-
`fixed, paraffin-embedded tissue blocks or from biopsy
`materials. Paraffin-embedded tissue samples were
`serially cut into 10-mm sections. All specimens were
`reviewed by a pathologist in our department.
`
`2.3. DNA extraction from stool specimens
`
`Approximately 200 mg of stool was resuspended in
`5 ml of TEN buffer (500 mmol/l Tris buffer, pH 9.0,
`containing 29 mmol/l EDTA and 10 mmol/l NaCl) and
`incubated for 1 h at room temperature. The supernatant
`obtained by centrifugation at 3000 rpm for 10 min was
`digested with 1 mg/ml proteinase K at 56 °C overnight
`in the presence of 1% sodium dodecylsulfate (SDS).
`Then, extraction was performed with phenol/chloro-
`form/isoamyl alcohol (25:24:1) and chloroform/iso-
`amyl alcohol (24:1), and DNA was precipitated with
`isopropyl alcohol. The DNA pellet was obtained by
`centrifugation at 15,000 rpm for 5 min and dried under
`a vacuum. For cetyltrimethylammonium bromide
`(CTAB) treatment, the DNA pellet was dissolved in
`500 ml of TE buffer (pH 8.0) containing 0.35 mol/l
`NaCl and 1% (w/v) CTAB, and then the reaction
`mixture was incubated at 65 °C for 15 min. After
`extraction with chloroform/isoamyl alcohol (24:1),
`DNA was precipitated with isopropyl alcohol and
`dissolved in 30 ml of distilled sterilized H2O.
`
`2.4. DNA extraction from tissue sections
`
`Paraffin-embedded tissue sections were deparaffi-
`nized with xylene and dehydrated in a graded ethanol
`series. Subsequently, cancerous regions were separated
`from noncancerous regions using a razor blade.
`The samples thus obtained were treated with SDS/
`proteinase K and DNA was extracted and purified as
`described above, except for CTAB treatment.
`
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`109
`
`2.5. PCR/RFLP analysis
`
`To detect K-ras gene alterations, PCR/RFLP anal-
`ysis was performed according to the protocol described
`elsewhere with several modifications. The oligonu-
`cleotide primers used for PCR amplification were
`
`5V-GACTGAATATAAACTTGTGGTAGTTG-
`GACCT-3V, (sense) and
`5V-CTATTGTTGGATCATATTCGTCC-3V (anti-
`sense).
`
`The sense primer was designed to introduce a base
`substitution that created a Bst NI recognition site only
`for the wild-type codon 12. A 107-base-pair (bp)
`fragment of exon 1 of K-ras was amplified. The
`PCR mixture consisted of 5 ml of DNA and 45 ml of
`10 mmol/l Tris – HCl buffer (pH 8.3) containing 3
`mmol/l MgC12, 50 mmol/l KCl, 0.01% (w/v) gelatine,
`20 mmol/l of each dNTP, 1.25 U of Taq DNA
`polymerase (AmpliTaq Goldk; Perkin Elmer, USA),
`and 0.5 mmol/l of each primer. PCR was carried out
`in a Thermal Cycler (TaKaRa TP3000, Japan), with
`preheating at 95 °C for 10 min, followed by 40
`cycles of 94 °C for 30 s, 56 °C for 1 min, and 72 °C
`for 30 s. An aliquot (10 ml) of PCR products was
`digested with 30 units of Bst NI at 60 °C for 3 h,
`and analyzed by polyacrylamide gel electrophoresis
`followed by silver staining. If there was no mutation
`of K-ras codon 12, the 107-bp fragment was cleaved
`into 77- and 30-bp fragments.
`
`2.6. Specificity of one-step PCR/RFLP analysis
`
`We examined whether mutations could be detected
`by the PCR/RFLP method using six synthetic oligonu-
`cleotides containing different mutations of K-ras
`codon 12. The sequences of the mutated codon were
`AGT, TGT, CGT, GAT, GTT, or GCT. In addition,
`oligonucleotides containing wild-type codon 12 (GGT)
`were synthesized.
`
`2.7. Detection limit of one-step PCR/RFLP analysis
`
`To establish the detection limit of our method,
`SW480 cells (which have two mutant alleles at codon
`12 of the K-ras oncogene) were mixed with HT29
`cells (which have the wild-type K-ras oncogene).
`
`SW480 cells were added at dilutions from 1:1 to
`1:1,000,000. Subsequently, DNA was extracted and
`subjected to PCR/RFLP analysis.
`
`3. Results
`
`3.1. Detection of K-ras gene mutations in stool
`specimens from colon cancer patients
`
`In all cases, an intense band for the 107-base-pair
`fragment was detected with ethidium bromide staining
`after the one-step PCR (data not shown). In normal
`controls, no extra band indicating K-ras mutation was
`obtained by Bst NI digestion (Fig. 1a). On the other
`hand, an extra band at 107 bp was clearly detected in
`some colon cancer patients (Fig. 1b).
`
`Fig. 1. Detection of K-ras codon 12 point mutation. One-step PCR/
`RFLP products were electrophoresed in a 12% polyacrylamide gel
`followed by silver staining. (a) In all normal controls, only a 77-bp
`fragment was detected. (b) In some colorectal cancer patients, a
`107-bp fragment as well as a 77-bp fragment were detected (lanes 1,
`4, 6). M: molecular weight marker.
`
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`110
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`T. Nishikawa et al. / Clinica Chimica Acta 318 (2002) 107–112
`
`3.2. K-ras codon 12 mutations in stool or tissue
`specimens from colon cancer patients
`
`The results of PCR/RFLP analysis of K-ras codon
`12 mutations in stool and tissue specimens are sum-
`marized in Table 1. Among the 31 cancer patients
`examined, we found somatic K-ras mutations in stool
`from 13 patients (41.9%) and 13 patients had muta-
`tions in their cancer tissues. Eleven of these patients
`had mutations in both their stool specimen and the
`tumor. In 16 patients, no mutations were observed in
`both the stool specimen and the tumor. However, two
`
`Table 1
`Summary of K-ras codon 12 mutations in stool or tissue samples of
`colon cancer patients
`
`Case Sex Age Tumor
`
`K- ras mutation
`
`Stool
`
`Tissue
`
`Site Size (mm) Stage
`52  42
`15  9
`20  19
`33  29
`29  27
`70  55
`11  9
`30  27
`14  8
`10  10
`20  20
`92  81
`60  55
`51  42
`52  34
`40  31
`32  23
`20  19
`60  60
`60  58
`20  18
`60  45
`55  51
`57  55
`54  50
`27  25
`25  25
`42  40
`35  30
`20  20
`32  30
`A — ascending colon; D — descending colon; S — sigmoid colon;
`R — rectum; T — transverse colon.
`
`Fig. 2. Specificity of one-step PCR/RFLP analysis. Digested syn-
`thetic oligonucleotide containing wild type K-ras codon 12 (lane 1:
`GGT) shows a 77-bp fragment. Synthetic oligonucleotides contain-
`ing mutant type K-ras codon 12 (lane 2: CGT, lane 3: AGT, lane 4:
`GAT, lane 5: GTT, lane 6: TGT, lane 7: GCT) show a 107-bp fragment
`even after Bst NI digestion. M: molecular weight marker.
`
`patients only had mutations in the stool specimen and
`another two patients only had mutations in the tumor.
`In the ascending colon, 3 out of 4 (75%) tumors, as
`well as 1 out of 1 (100%) in transverse colon, 1 out of
`4 (25%) in the descending colon, 8 out of 15 (53.3%)
`in the sigmoid colon, and 2 out of 7 (28.6%) in the
`rectum were found to have K-ras mutations. Muta-
`tions were found in 1 out of 4 (25%) tumors that
`measured 1 – 2 cm in diameter, as well as in 4 out of 8
`(50%) tumors measuring 2 – 3 cm, and 10 out of 19
`(52.6%) tumors measuring > 3 cm. The following
`were found to have mutations: 4 out of 10 (40%)
`
`Fig. 3. Detection limit of one-step PCR/RFLP analysis. SW480 cells
`were mixed with HT29 cells in the following ratios. Lane 1—1:1.
`Lane 2 —1:10. Lane 3 —1:100. Lane 4 —1:1000. Lane 5—
`1:10,000. Lane 6 —1:100,000. Lane 7 —1:1,000,000. Detection
`limit of this method was 1:1000 mutant type cells in wild type cells.
`Mutations were detected when at least 0.1% of the cells were
`SW480 cells.
`
`1
`2
`3
`4
`5
`6
`7
`8
`9
`10
`11
`12
`13
`14
`15
`16
`17
`18
`19
`20
`21
`22
`23
`24
`25
`26
`27
`28
`29
`30
`31
`
`M 42
`M 66
`F
`64
`M 70
`M 42
`F
`41
`M 57
`M 64
`M 66
`M 59
`M 65
`F
`73
`F
`58
`M 64
`M 69
`F
`69
`F
`72
`F
`70
`F
`59
`M 67
`F
`51
`M 70
`F
`80
`F
`52
`M 75
`M 71
`M 40
`M 57
`F
`48
`M 61
`F
`74
`
`A
`D
`S
`S
`R
`S
`S
`S
`S
`S
`D
`A
`S
`S
`A
`S
`S
`S
`R
`R
`S
`R
`R
`R
`A
`D
`R
`S
`D
`S
`T
`
`Dukes C +
`Dukes A
`Dukes A
`Dukes B +
`Dukes C
`Dukes C +
`Dukes A
`Dukes C
`Dukes A +
`Dukes A
`Dukes B
`Dukes C +
`Dukes B
`Dukes C +
`Dukes C +
`Dukes A
`Dukes A +
`Dukes A +
`Dukes C
`Dukes C +
`Dukes B +
`Dukes B
`Dukes C
`Dukes C
`Dukes C
`Dukes C +
`Dukes A
`Dukes C
`Dukes C
`Dukes A
`Dukes C +
`
`+
`
`
`+
`
`
`
`
`+
`
`
`+
`
`+
`+
`
`+
`+
`+
`+
`+
`
`
`
`
`
`
`
`
`+
`+
`
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`111
`
`Dukes A tumors, 2 out of 5 (40%) Dukes B tumors,
`and 9 out of 16 (56.3%) Dukes C tumors.
`
`3.3. Specificity of one-step PCR/RFLP analysis
`
`Six synthetic oligonucleotides containing K-ras
`codon 12 mutations were not digested by Bst NI,
`although synthetic oligonucleotides having the wild-
`type sequence were digested and yielded smaller
`fragments (Fig. 2).
`
`3.4. Detection limit of one-step PCR/RFLP analysis
`
`Mutations were detected when at least 0.l% of the
`cells were SW480 cells (Fig. 3).
`
`4. Discussion
`
`The detection rate of K-ras mutations in stool
`specimens from our patients with colorectal cancer
`was 41.9% (13:31). Among patients who were pos-
`itive for mutations in tumor tissue, the detection rate
`in stool specimens was high (84.6%, 11:13). This
`positive rate is well compatible to results reported
`previously [11,14,15]. Mutations were identified only
`in the stool specimens of two patients (Nos. 6 and 26)
`and only in the tissue specimens of other two patients
`(Nos. 19 and 30). This was probably because K-ras
`mutations are not uniform (heterogenous) within the
`same tumor. In addition, K-ras mutations are reported
`to be detected with high rate in aberrant crypt foci
`[16,17] and have also been detected in some hyper-
`plastic polyps [18,19] and normal-appearing tissue
`[18,20], which may account for the false positives in
`some stool specimens. Contamination by large
`amounts of enteric bacteria, food residue, and hemo-
`globin may also have interfered with DNA extraction
`and may have led to dilution of cells shed by the
`tumor, thereby causing false negative results.
`Although there was no significant relationship bet-
`ween the mutation rate and the tumor site, the mutation
`rate tended to be higher for large or advanced cancers,
`suggesting that activation of K-ras promote tumor cell
`proliferation.
`In the present study, we were able to detect point
`mutations of K-ras codon 12 in stool samples using a
`one-step PCR/RFLP. Many investigators have used this
`
`method to detect K-ras mutations [21 – 23], but we
`could find only one report on stool samples [15]. This is
`probably because extracting and purifying very small
`quantities of human DNA from stool is difficult, hinder-
`ing subsequent PCR amplification and detection of
`mutations. In a preliminary study aimed at overcoming
`this problem, we were able to detect point mutations of
`codon 12 in stool specimens simply and with high
`sensitivity. In the present study, DNA was successfully
`extracted from all patients using standard SDS/protei-
`nase K treatment, phenol/chloroform/isoamyl alcohol
`extraction, isopropyl alcohol precipitation, and treat-
`ment with CTAB. It has been traditional to use CTAB
`for extracting and purifying DNA from plants and
`bacteria [24], and it appears to facilitate DNA extrac-
`tion by removing polysaccharides derived from enteric
`bacteria and plant residue in the stool. The efficiency of
`analyzing K-ras mutations was also increased by using
`a hot-start PCR employing the DNA polymerase
`AmpliTaq Goldk (Perkin Elmer) to improve specific
`amplification. We also used silver staining to detect
`mutations, because this is more sensitive than ethidium
`bromide staining [25,26]. Through the combined use of
`these techniques, we were able to employ the one-step
`PCR in all of our patients.
`A study with synthetic DNA confirmed the appear-
`ance of bands specific for the six mutant sequences, and
`investigation using cell lines showed a detection limit of
`0.1%. This is higher than is generally reported for mutant
`allele-specific amplification (0.01 – 0.001%) [27] or
`mutant-enriched PCR ( < 0.01%) [28]. However, the
`specific amplification technique aims to specifically
`amplify mutant alleles involving one base located at
`the 3V end of the forward primer, and it is therefore
`possible that amplification of the wild-type allele will
`proceed if there is a slight change in conditions such as
`the annealing temperature [29]. Another problem with
`the PCR itself is that incorrect reading of nucleotides
`cannot be avoided during the amplification process,
`giving rise to mutations. Mutant-enriched PCR has
`certain disadvantages when used to detect genetic muta-
`tions, because there is a high risk that mutations arising
`during the PCR process will be detected and every effort
`must be made to avoid cloning of the PCR product.
`The one-step PCR/RFLP method therefore has a
`higher reproducibility and is simpler than other tech-
`niques. As a result,
`it should be more useful for
`screening.
`
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`112
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`T. Nishikawa et al. / Clinica Chimica Acta 318 (2002) 107–112
`
`In the present study, we successfully extracted and
`purified DNA from the stool samples of all subjects
`tested, and established a method for detecting K-ras
`point mutations using one-step PCR/RFLP that was
`far simpler than conventional techniques.
`Because K-ras point mutations are only associated
`with 40 – 50% of colorectal cancers,
`the detection
`sensitivity of one-step PCR/RFLP analysis for muta-
`tions is low among cancers as a whole. However, the
`technique is highly specific, noninvasive, and cost-
`effective, and should provide a more sensitive and
`specific tool for mass screening of colorectal cancer
`than is currently available, especially if used in combi-
`nation with fecal occult blood testing and other meth-
`ods for detecting genetic abnormalities.
`
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`Geneoscopy Exhibit 1011, Page 6
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