1707
`
`Mutations of the p53 Gene in the Stool of Patients
`with Resectable Colorectal Cancer
`
`Susumu Eguchi, M.D.
`Norihiro Kohara, M.D.
`Koh Komuta, M.D.
`Takashi Kanematsu, M.D.
`
`Department of Surgery 11, Nagasaki University
`School of Medicine, Nagasaki, Japan.
`
`Presented at the Seventh Annual Meeting of the
`Japanese Research Society for Gastroentero-
`logical Carcinogenesis, Oita, Japan, September
`8-9, 1995.
`
`The authors thank Professor H. Shiku and M.
`Ohara for their helpful comments.
`
`Address for reprints: Takashi Kanematsu, M.D.,
`Department of Surgery 11, Nagasaki University
`School of Medicine, 1-7-1 Sakamoto, Nagasaki
`852, Japan.
`
`Received December 4, 1995, accepted Decem-
`ber 8, 1995.
`
`0 1996 American Cancer Society
`
`BACKGROUND. This study was undertaken to evaluate whether genetic analysis in
`the stool can be useful for detecting malignant tumors in the colon and rectum.
`We searched for the possible presence of mutations in the p53 gene in the stool
`of patients with resectable colorectal cancer. Alterations in the p53 gene are the
`most frequent among mutant genes related to colorectal cancer.
`METHODS. Surgically resected tumor specimens and stool samples from 25 patients
`with colorectal cancer were examined for mutations of exons 5-8 of the p53 gene
`by polymerase chain reaction single-strand conformation polymorphism (PCR-
`SSCP). Results were compared with those achieved by fecal occult blood testing.
`RESULTS. Mutations of the p53 gene were found in the tumor tissues in 11 of 25
`patients (44%). Of these 11 patients, 7 (64%) had evidence of alterations in the p53
`gene within the stool. Of five patients who were negative for fecal occult blood
`testing, p53 mutations in the stool were evident in three patients.
`CONCLUSIONS. This method of stool DNA analysis for tumor-specific mutations is
`expected to have a wide application in clinical screening for colorectal cancer.
`Cancer 1996; 77:1707-10. 0 1996 American Cuncer Society.
`KEYWORDS p53 gene, DNA analysis, stool, colorectal cancer.
`
`reviously, p53 gene mutations have been identified in the urine sam-
`
`P ples of patients with a high proportion of primary urinary bladder
`
`cancer,' and rm oncogene mutations have been detected in DNA purified
`from the stool of nine patients with curable colorectal tumors.' Alterations
`in the p53 gene are the most frequent among mutant genes related to
`colorectal cancer and mutations have been observed in 50-86% of colo-
`rectal cancer^.^-^
`We searched for the possible presence of the same mutations in the
`p53 gene in resected colorectal specimens and stool samples using the
`polymerase chain reaction single-strand conformation polymorphism
`analysis,' with the objective of establishing a noninvasive and sensitive
`screening method for colorectal cancer.
`MATERIALS AND METHODS
`Sixteen Japanese men and 9 women (average age, 64 years) with histologi-
`cally proven carcinoma of the colon and rectum were studied. One patient
`(Patient 8, Table 1) had a concomitant adenoma. Prior to surgery, the
`colon was cleansed with a laxative and kanamycin and metronidazole
`were given for 2 days. Stool samples were collected one day prior to
`surgery. Tumor samples were obtained from the surgically resected speci-
`mens. All samples were stored at a temperature of -80 "C.
`
`DNA Preparation
`DNA was first purified from the stool and tumor tissues using Sepagenew
`(Sanko Junyaku, Co. Ltd., Tokyo, Japan). Briefly, approximately 100 mg
`
`Geneoscopy Exhibit 1053, Page 1
`
`

`

`1708
`
`CANCER Supplement April 15, 1996 / Volume 77 / Number 8
`
`TABLE 1
`Clinical Characteristics
`
`Patient
`
`Age
`
`Sex
`
`Location’
`
`Size (cm)
`
`DukesIb
`
`Histologf
`
`F.0.B.d
`
`Tumor
`
`Stool
`
`Tumor
`
`Stool
`
`Tumor
`
`DNA (pdg
`
`Mutant ~ 5 3 ~
`
`1
`2
`3
`4
`5
`6
`7 a
`
`9
`10
`11
`12
`13
`14
`15
`16
`17
`la
`19
`20
`21
`22
`23
`24
`25
`
`76
`79
`73
`a1
`40
`73
`71
`52
`
`66
`67
`45
`68
`65
`56
`62
`69
`72
`65
`51
`65
`51
`72
`50
`6a
`58
`
`M
`M
`M
`F
`M
`M
`M
`M
`
`M
`M
`F
`F
`F
`F
`M
`F
`F
`F
`M
`M
`M
`M
`M
`F
`M
`
`TI
`R
`TI
`R
`As
`Rs
`R
`R
`
`TI
`R
`R
`R
`R
`Total
`R
`S h
`S k
`R
`SIg
`R
`SIg
`P
`R
`P
`R
`
`1.4 X 0.9
`6.3 X 7.5
`4.7 X 6.2
`2.0 x 1.5
`4.5 x 4.5
`2.8 x 2.2
`2.4 X 2.3
`12 X 5.0
`7.5 x 3.5
`4.0 x 2.8
`8.0 X 6.5
`2.8 x 2.1
`10.0 X 4.0
`9.0 X 7.0
`F.P.‘
`7.0 X 5.0
`6.0 x 3.9
`3.0 x 2.8
`5.0 X 3.5
`4.0 X 4.0
`6.0 X 5.2
`3.7 x 3.5
`6.5 X 4.2
`5.3 x 4.3
`6.3 X 5.5
`7.5 X 3.1
`
`A
`-
`B
`A
`C
`C
`A
`C
`-
`B
`B
`C
`C
`B
`C
`C
`C
`C
`C
`B
`C
`C
`C
`B
`B
`C
`
`Pap.
`Mod.
`Well
`Mod.
`Mod.
`Mod.
`Mod.
`Mod.
`Adenoma
`Well
`Mod.
`Mod.
`Poor
`Mod.
`Mod.
`Mod.
`Mod.
`Mod.
`Mod.
`Well
`Mod.
`Mod.
`Mod.
`Mod.
`Well
`Mod.
`
`-
`t
`t
`t
`t
`t
`t
`t
`
`-
`t
`-
`t
`t
`t
`t
`t
`-
`t
`t
`t
`t
`t
`-
`t
`t
`
`283
`176
`365
`72
`66
`154
`144
`530
`340
`126
`62
`332
`120
`60
`2900
`1418
`1900
`526
`780
`342
`748
`la4
`aa
`984
`304
`258
`
`2a
`X‘
`400 (PCRX)‘
`974
`X‘
`200
`22
`324
`
`9a
`246
`410
`146
`178
`164
`152
`108
`aa
`aoo
`50
`4700
`16
`228
`50
`1536
`110
`
`t [Exon 5)
`
`t (Exon 7)
`
`t (Exon 7)
`
`t (Exon 5)
`t (Exon a)
`
`t (Exon 6)
`t (Exon 6)
`
`t (Exon 7)
`t [Exon 6)
`
`t [Exon 7)
`
`t [Exon 5)
`
`’As.: ascending colon; Tr.: transverse colon; Sig.: sigmoid colon; R.: rectum; P.: anal canal; Total: a case of familial polyposis. Numerous polyp were present all over the colon.
`Dukes’ classification.
`‘ Histological type. Pap.: papillary adenocarcinoma; WeU well differentiated adenocarcinoma; Mod.: moderately differentiated adenocarcinoma; Poor poorly differentiated adenocarcinoma.
`F.O.B.: Fecal occult testing.
`‘DNA was extractable but not amplifiable.
`‘DNAs were not extractable.
`Concentration of the DNAs at 260 nm.
`Detection by polymerase chain reaction-single-strand conformation polymorphism analysis of exons 5, 6, 7, and 8 of the p53 gene.
`’ F.P.: a case of familial polyposis with evidence of malignancy.
`
`of the stool frozen at -80 “C was diluted with 100 pL of
`Tris HC1. Proteins were digested with 100 pL of guanidine
`thiocyanate and extracted with 700 pL of bentonite chlo-
`roform and 400 pL of sodium acetate without phenol.
`After ethanol precipitation, the DNAs were dried using a
`decompression drier and diluted with Tris-ethylenedi-
`amine tetraacetic acid (EDTA). The concentrations of
`DNAs were measured at 260 nm using a spectrophotome-
`ter. and are listed in Table 1.
`
`Polymerase Chain Reaction
`Exons 5-8 of p53 gene were then amplified from each
`DNA by polymerase chain reaction (PCR), and subjected
`to polyacrylamide gel electrophoresis. Oligonucleotide
`primers for PCR amplification were synthesized in a 380B
`
`DNA synthesizer. The following four sets of primers, 5U-
`5D, 6U-6D, 7U-7D, and EU-ED, were used to amplify ex-
`ons 5, 6, 7, and 8, respectively, of the p53 gene. Sense
`primers used were: 5U, 5’-GGGGATCCTCTTCCTGCA-
`GTACTC-3’; 6U, 5’-GTCTGGCCCCTCCTCAGCAT-3‘; 7U,
`5’-GGGGATCCTAGGTTGGCTCTGACT-3’; and 8U, 5‘-
`GGGGATCCTATCCTGAGTAGTGGT-3’ (each primer con-
`tained a benzamide HI site). The antisense primers were:
`5D, 5’-GCAAATTTCCTTCCACTCGG-3; 6D, 5’-GGAATT-
`TGCAAACCAGACCTC-3‘; 7D, 5’-GGGAATTCAAGTGGC-
`TCCTGACCT-3’; and ED, 5’-GTCCTGCTTGCTTACCTCG-
`3‘ (each primer contained an Eco RI site). PCR was per-
`formed using a thermal cycler (PC-70, Astec, Fukuoka,
`Japan) with 2 pL of genomic DNA in a total volume of 20
`FL containing 8 p L of ialpha-p3’1yATP. Thirty-nine cycles
`
` 10970142, 1996, 8, Downloaded from https://acsjournals.onlinelibrary.wiley.com/doi/10.1002/(SICI)1097-0142(19960415)77:8<1707::AID-CNCR43>3.0.CO;2-0, Wiley Online Library on [29/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
`
`Geneoscopy Exhibit 1053, Page 2
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`

`

`p53 in the Stool of Golorectal Cancer PatientdEguchi et al.
`
`1709
`
`comprised of 90 seconds at 92 "C for denaturation, 90
`seconds at 55 "C for annealing, and 1 minute at 72 "C for
`extension were run.
`
`Single-Strand Conformation Polymorphism
`The PCR products were examined by Single-Strand Con-
`formation Polymorphism analysis to identify mutations
`in the p53 gene. The PCR mixture was heated at 90 "C
`with 7 pL of a formamide dye mixture (95% formamide,
`20 mM EDTA, 0.05% xylene cyanol, and 0.05% bromphe-
`no1 blue), and 1.5 pL of the mixture was applied to a 6%
`polyacrylamide gel containing 50 mM Tris-borate. Glyc-
`erol (5%) was also added. Electrophoresis was performed
`at 1800 volts with cooling by a fan for 2 hours at 4 "C and
`for 1.5 hours at room temperature. The gel was dried on
`filter paper and exposed to X-ray film for 24 hours at
`room temperature. Polymerase chain reaction-single-
`strand conformation polymorphism analysis was per-
`formed in duplicate.
`
`Fecal Occult Blood Testing
`To search for occult blood in the stool, the gold colloid
`immunochromato method which reacts on human he-
`moglobin, was used."
`
`RESULTS
`DNA was extracted from all tumor tissues, but in two
`patients (Patients 2 and 5), DNA could not be extracted
`from the stool (Table 1). Mean concentrations of ex-
`tracted DNA in the tumor were 510 ? 558 pg/mL (range,
`60-2900 pg/mL), and 561 2 1168 pglmL (range, 16-4
`700 pgrlmL) in the stool. Amplification of exons 5-8 was
`successful in the tumor specimens of all patients, whereas
`it was unsuccessful in the stool sample in one patient
`(Patient 3; Table 1).
`Mutations of the p53 gene were detected in exons 5-
`8 in the tumors of 11 of 25 patients (44%). Mutations
`were evident in exons 5 and 6 in three patients each, exon
`7 in four patients, and exon 8 in one patient. Among the
`11 patients, mutations of the p53 gene were identified in
`the stool of 7 patients (64%) (Fig. 1). However, there were
`no patients who showed the presence of mutations in the
`p53 gene in the stool of the abscence of the mutation in
`the tissue (Table 1). No significant difference in clinico-
`pathologic variables, including location and size of tu-
`mors and stage of the disease, was noted between groups
`with or without evidence of the p53 gene mutations in
`the tumor or stool samples.
`Among the 25 patients, occult blood was positive in
`the stool of 20 patients (80%). In the remaining five pa-
`tients who were negative for occult blood, mutations of
`the p53 gene were identified in three (Patients 11, 17, and
`23, Table 1).
`
`FIGURE 1. Detection of p53 mutations by polymerase chain reaction
`single-strand conformation polymorphism analysis. hp represents normal
`human placenta DNA. Arrowheads indicate bands that migrated at different
`positions from the normal ones. DNA from the stool sample was the same
`migration as DNA from the tumor in 7 of 11 patients (e.g., 8, Patient 21).
`T: tumor with mutant p53 genes; S: stool from the patient whose tumor
`was with mutant p53 genes.
`
`DISCUSSION
`There were 156,000 new cases of colorectal cancer diag-
`nosed annually in the United States in 1992, and 58,200
`patients died of the malignant disease,8 although a slow
`decline in the mortality of patients with this disease was
`r e p ~ r t e d . ~ Early detection of colorectal cancer favorably
`alters the clinical course. Fecal occult blood testing has
`been the technique used to detect neoplasms in the large
`bowel. However, the measurement of occult blood in the
`stool using immunochemical methods is unsatisfactory
`because many tumors do not bleed."
`A common cancer-related gene change that can oc-
`cur at the gene level in the colon is a p53 m ~ t a t i o n , ~ ' ~
`and nearly all existing tumors can be detected by analysis
`of the four mutational hot spots, exons 5, 6, 7, and 8.".'*
`In the present study, the p53 mutations were detected in
`the tumor tissues of 11 of 25 patients (44%); among these
`11 patients, genetic alterations in the stool were found in
`7 patients (64%). In addition, p53 mutations were de-
`tected in the stool of three patients with negative fecal
`occult blood testing.
`Thus, this genetic approach can be useful for de-
`tecting a malignant tumor in the colon and rectum. More-
`over, it also may be useful for detecting colorectal cancer
`in patients with false-negative fecal occult blood testing.
`However, although the present study illustrates the prin-
`
` 10970142, 1996, 8, Downloaded from https://acsjournals.onlinelibrary.wiley.com/doi/10.1002/(SICI)1097-0142(19960415)77:8<1707::AID-CNCR43>3.0.CO;2-0, Wiley Online Library on [29/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
`
`Geneoscopy Exhibit 1053, Page 3
`
`

`

`1710
`
`CANCER Supplement April 15,1996 / Volume 77 / Number 8
`
`ciple that specific gene changes can be detected in the
`stool, molecular genetic approaches are not readily
`adaptable for screening for colorectal cancer. These anal-
`yses should provide criteria for a screening test because
`the simplicity, economic, feasibility, sensitivity, noninva-
`siveness, and overall accuracy of molecular genetic ap-
`proaches are substantially better than those of fecal oc-
`cult blood te~ting.’~ Nevertheless, the results show that
`this strategy may serve as a pertinent method for screen-
`ing individuals who are at high risk for colorectal cancer,
`particularly those with inherited syndromes, a personal
`history of adenoma or colorectal cancer, and inflamma-
`tory bowel disease.
`
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` 10970142, 1996, 8, Downloaded from https://acsjournals.onlinelibrary.wiley.com/doi/10.1002/(SICI)1097-0142(19960415)77:8<1707::AID-CNCR43>3.0.CO;2-0, Wiley Online Library on [29/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
`
`Geneoscopy Exhibit 1053, Page 4
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