Research letters
`
`RESEARCH LETTERS
`
`Methylation changes in faecal DNA: a marker for colorectal cancer
`screening?
`Hannes M Müller, Michael Oberwalder, Heidi Fiegl, Maria Morandell, Georg Goebel, Matthias Zitt, Markus Mühlthaler, Dietmar Öfner,
`Raimund Margreiter, Martin Widschwendter
`
`in
`is a common molecular alteration
`DNA methylation
`colorectal cancer cells. We report an assessment of faecal
`DNA from patients with colorectal cancer and controls to
`determine the feasibility, sensitivity, and specificity of this
`approach. By use of MethyLight analysis of faecal DNA from
`three independent sets of patients, we identified SFRP2
`methylation as a sensitive single DNA-based marker for
`identification of colorectal cancer in stool samples (sensitivity
`90% [CI 56–100] and specificity 77% [46–95] in the training
`set [n=23]; sensitivity 77% [46–95] and specificity 77%
`[46–95] in an independent test set [n=26]). Whether a
`combination of genetic and epigenetic markers will identify
`colorectal cancer at an early stage remains to be shown.
`
`Lancet 2004; 363: 1283–85
`Colorectal cancer is among the most frequently diagnosed
`cancers in the industrialised world. Early detection seems to
`be a key factor in reducing rates of death from this disease.1
`Several methods of detection are available, including faecal
`occult blood test, flexible sigmoidoscopy, barium enema, and
`colonoscopy. However, none of these approaches has yet been
`established as a screening method, either because the
`uncomfortable and unpleasant preparation procedures are
`unacceptable to patients, or because of low sensitivity or
`specificity. Faecal DNA analysis opens up a new field for early
`this widespread neoplasia.2 Tagore and
`detection of
`colleagues3 reported a multitarget assay panel in faecal DNA
`analysis consisting of 21 specific mutations in the APC, TP53,
`and KRAS genes, a microsatellite instability marker (BAT-
`26), and a DNA integrity assay, reflecting abnormal apoptosis,
`with a sensitivity of 64%. In addition to these genetic
`alterations, changes in the status of DNA methylation, known
`as epigenetic alterations, count among the most common
`molecular alterations in human neoplasia, including colorectal
`cancer.4 Moreover, it is now widely known that methylated
`DNA can be detected in various body fluids.
`In this proof-of-principle study, we aimed to clarify
`whether methylation changes in faecal DNA isolated from
`stool samples could be used to screen for colorectal cancer.
`We designed a three-step prospective study, aiming to assess
`the most promising epigenetic markers for colorectal cancer
`out of a long list of candidate genes (gene evaluation set) and
`to test these genes in two independent sets of patients
`(training and test set).
`DNA from stool samples was isolated by means of the
`QIAamp DNA Stool Kit (Qiagen, Hilden, Germany; data not
`shown). DNA methylation was assessed by use of MethyLight,
`a fluorescence-based, real-time PCR assay, as described
`elsewhere.5 Patients who underwent colonoscopy for various
`reasons between August, 2003, and January, 2004, who gave
`written informed consent and provided sufficient stool samples
`for DNA isolation, were included in this study. The study was
`approved by the local institutional review board.
`Initially, a gene evaluation set was used to determine the
`most promising epigenetic markers to identify patients with
`
`colorectal cancer. To prevent a collection bias, we used either
`bowel lavage fluid obtained during colonoscopy or mucus
`and bowel content obtained intraoperatively from nine
`patients with colorectal cancer and ten control patients
`without the disease. Next, the methylation status of 44 genes
`in the DNA isolated from these samples was assessed. Most
`of the genes assessed had been studied in serum from patients
`with breast cancer.5 Additionally, four genes (SFRP1, SFRP2,
`SFRP4, and SFRP5) reported to be commonly methylated in
`colorectal cancer tissue specimens4 and OPCML were
`assessed. The four SFRP genes are reported to have the
`potential to detect virtually all colorectal cancers, because 123
`of 124 colorectal cancer tissue specimens showed methylation
`at one or more of these four gene loci.4 Using three different
`statistical methods (Mann-Whitney U test using PMR
`fully methylated reference] values, ␹2
`[percentage of
`contingency test, and prediction analysis for microarrays) we
`found ten genes showing the greatest potential for identifying
`colorectal cancer patients (data not shown).
`Next, we obtained stool samples from patients with
`colorectal
`cancer
`and healthy
`controls undergoing
`colonoscopy for various reasons at the Department of
`General and Transplant Surgery, Innsbruck Medical
`University, and the Department of General Surgery, Saint
`Vinzenz Hospital in Zams, Tyrol. The stool was collected by
`the patients themselves, either the day before colonoscopy
`(first stool during bowel preparation) or at hospital the day
`before surgical intervention. Stool samples were obtained
`from 53
`endoscopically diagnosed healthy controls,
`12 patients with histologically diagnosed adenomas,
`11 patients undergoing control endoscopy during colorectal
`cancer follow-up, and 31 patients with colorectal cancer.
`Before starting the DNA isolation procedure we excluded the
`patients diagnosed with adenomas and those having had
`colorectal cancer to ensure clearly defined groups of patients.
`Next, we determined two independent age-matched sets of
`patients (training and test set). Furthermore, because of the
`different amounts and consistency of the stool collected by
`the patients and possible degradation of DNA during the self-
`collection procedure, we checked all samples for their DNA
`content.
`We were able to isolate DNA from 26 endoscopically
`diagnosed healthy controls and 23 patients with colorectal
`cancer (table). All people undertaking
`the
`isolation
`procedure and MethyLight analyses were masked to the
`disease status of patients. Methylation status of the ten genes
`identified in the gene evaluation set (SFRP1, SFRP2,
`SFRP5, TFF1, PGR, IGFBP2, CALCA, CDH13, TWIST1,
`MYOD1) was assessed in the faecal DNA of the patients
`(n=10) and controls (n=13), representing the predetermined
`training set (table). We found significant differences in DNA
`methylation at a specified gene locus for SFRP2, SFRP5,
`PGR, CALCA, and IGFBP2 (p=0·003, 0·04, 0·03, 0·019,
`and 0·015, respectively; Mann-Whitney U test) in faecal
`DNA of colorectal cancer patients compared with healthy
`
`THE LANCET • Vol 363 • April 17, 2004 • www.thelancet.com
`
`1283
`
`Geneoscopy Exhibit 1037, Page 1
`
`

`

`RESEARCH LETTERS
`
`Indication Colonoscopy result Histology Locality Stage PMR values
`
`SFRP2
`
`SFRP5
`
`PGR
`
`CALCA
`
`IGFBP2
`
`Patient
`age*, sex
`TrS/C
`ABE
`63, F
`39, M CHBH
`66, M OTHER
`42, F
`PEP
`60, M OTHER
`56, M PEP
`44, M PFOBT
`69, F
`CHBH
`18, M CHBH
`46, F
`CHBH
`67, F
`CHBH
`32, M CHBH
`46, F
`OTHER
`TrS/CRC
`80, M UNKN.
`65, M CHBH
`46, M ABE
`85, F
`UNKN
`68, F
`CA
`84, M CA
`67, M CA
`57, F
`UNKN
`55, M ABE
`74, M PFOBT
`TS/C
`CHBH
`40, F
`40, M CHBH
`36, F
`CHBH
`65, F
`CHBH
`48, M ABE
`47, M OTHER
`58, M ABE
`54, F
`PFOBT
`47, F
`UNKN
`26, M CHBH
`42, M OTHER
`74, F
`OTHER
`63, M OTHER
`TS/CRC
`84, M UNKN
`64, F
`UNKN
`63, M UNKN
`90, M PFOBT
`34, F
`UNKN
`44, M CA
`63, M CA
`56, M CA
`59, M CA
`62, M CA
`81, F
`CA
`68, M CA
`69, F
`UNKN
`
`NM
`NM
`NM
`NM
`SP
`SP
`MP
`NM
`INFL
`DIV
`MP
`MP
`INFL
`
`CA
`CA
`CA
`CA
`CA
`CA
`CA
`CA
`CA
`CA
`
`NM
`SP
`DIV
`NM
`SP
`MP
`NM
`NM
`SP
`NM
`DIV
`DIV
`MP
`
`CA
`CA
`CA
`CA
`CA
`CA
`CA
`CA
`CA
`CA
`CA
`CA
`CA
`
`··
`··
`··
`··
`··
`··
`··
`··
`SHP/ND R
`SHP/ND RC
`NM
`R
`··
`··
`INFL
`··
`NM
`··
`MHP/ND R
`MHP/ND R
`NM
`··
`
`AC
`AC
`AC
`AC
`AC
`AC
`AC
`AC
`AC
`AC
`
`LC
`R
`RC
`RC
`R
`R
`LC
`LC
`LC
`R
`
`··
`··
`SHP/ND LC
`··
`··
`··
`··
`SHP/ND LC
`MHP/ND LC
`··
`··
`··
`··
`INFL
`LC
`NM
`··
`NM
`··
`··
`··
`MHP/ND LC
`
`AC
`AC
`AC
`UNKN
`AC
`AC
`AC
`AC
`AC
`AC
`AC
`AC
`AC
`
`LC
`LC
`R
`R
`R
`RC
`LC
`LC
`R
`R
`LC
`RC
`LC
`
`··
`··
`··
`··
`··
`··
`··
`··
`··
`··
`··
`··
`··
`
`pT2
`pT3
`pT3
`pT4
`pT3
`pT3
`pT3
`pT2
`pT3
`pT3
`
`··
`··
`··
`··
`··
`··
`··
`··
`··
`··
`··
`··
`··
`
`pT3
`pT3
`pT3
`pT3
`pT3
`pT3
`pT4
`pT3
`pT3
`pT3
`pT2
`pT2
`pT3
`
`0·00
`0·00
`1·67
`1·84
`0·00
`0·00
`0·00
`0·00
`0·00
`0·00
`367·53
`0·00
`0·00
`
`37·05
`0·09
`5·28
`2·41
`87·22
`2·67
`1·88
`5·77
`0·00
`4·51
`
`0·00
`0·00
`0·00
`0·00
`0·00
`19·19
`7·02
`0·00
`0·00
`0·00
`0·00
`31·28
`0·00
`
`8·85
`0·00
`3·39
`3·81
`0·00
`2·00
`39·44
`28·33
`9·00
`14·48
`38·76
`0·00
`9·88
`
`0·00
`0·00
`0·10
`0·06
`0·00
`0·00
`0·00
`0·18
`0·07
`0·00
`8·19
`0·09
`0·00
`
`4·16
`0·06
`0·28
`0·16
`3·45
`2·06
`1·51
`0·05
`0·00
`nd
`
`0·00
`0·00
`0·00
`0·00
`0·81
`0·00
`0·02
`0·00
`0·00
`0·00
`3·46
`0·00
`0·00
`
`0·21
`0·00
`1·09
`2·47
`0·00
`1·03
`75·64
`5·33
`156·95
`0·78
`0·22
`0·00
`10·59
`
`0·00
`0·00
`7·06
`0·36
`0·00
`0·00
`0·00
`0·00
`0·00
`0·00
`10·77
`0·00
`0·58
`
`12·15
`0·07
`0·47
`0·73
`0·00
`1·01
`0·73
`12·38
`0·00
`2·73
`
`0·00
`19·85
`0·00
`0·00
`0·09
`0·51
`0·29
`0·00
`0·00
`0·00
`0·00
`0·00
`0·00
`
`7·10
`1·89
`0·90
`2·26
`0·00
`0·64
`24·98
`2·56
`0·00
`0·65
`3·98
`0·00
`0·34
`
`0·00
`0·00
`0·00
`0·00
`0·00
`0·00
`0·00
`0·00
`0·00
`0·00
`0·00
`0·00
`nd
`
`0·00
`0·00
`0·03
`3·47
`0·00
`2·15
`0·25
`0·00
`0·00
`0·00
`
`0·00
`0·00
`0·00
`0·00
`0·00
`0·00
`0·00
`0·00
`0·00
`0·00
`0·00
`0·00
`0·00
`
`0·32
`0·00
`2·49
`0·61
`0·00
`0·00
`15·10
`0·00
`0·00
`0·00
`0·00
`0·00
`0·00
`
`0·00
`0·00
`0·00
`0·00
`0·00
`0·00
`0·00
`0·00
`0·00
`0·00
`0·00
`0·00
`0·00
`
`0·00
`0·00
`0·00
`0·00
`0·24
`0·00
`0·07
`0·00
`0·00
`0·00
`
`0·00
`0·00
`0·00
`0·00
`0·00
`8·09
`0·00
`0·00
`0·00
`0·00
`0·00
`0·00
`0·00
`
`0·00
`0·00
`0·00
`0·00
`0·00
`0·00
`0·00
`0·02
`0·00
`0·00
`0·00
`0·00
`0·00
`
`TrS/C=training set, controls. TrS/CRC=training set, patients with colorectal cancer. TS/C=test set, controls. TS/CRC=test set, patients with colorectal cancer.
`F=female. M=male. ABE=abnormal barium enema. CHBH=changes in bowel habits. OTHER=other reasons. PEP=pre-existing polyp. PFOBT=positive faecal occult blood
`test. UNKN=unknown. CA=carcinoma. CRC=colorectal cancer. NM=normal mucosa. SP=single polyp. MP=multiple polyps. INFL=inflammation. DIV=diverticulosis.
`SHP/ND=single hyperplastic polyp without dysplasia. MHP/ND=multiple hyperplastic polyps without dysplasia. AC=adenocarcinoma. nd=not determined. R=rectum.
`RC=right colon. LC=left colon. *Years.
`Clinicopathological features of patients
`
`controls; nine of ten patients with colorectal cancer and three
`of 13 without the disease had methylated SFRP2 in their
`faecal DNA (sensitivity 90% [CI 56–100] and specificity
`77% [46–95]).
`These findings were then assessed in the faecal DNA of an
`independent test set (13 patients with colorectal cancer and
`13 controls; table). Analysis using PMR values showed that
`three genes were still differentially methylated between patients
`with and without colorectal cancer (p=0·017, 0·017, and
`0·047 for SFRP2, SFRP5, and PGR, respectively; Mann-
`Whitney U test); SFRP2 was shown to be methylated in the
`faecal DNA in ten of 13 patients with colorectal cancer and
`three of 13 without the disease (sensitivity 77% [CI 46–95]
`and specificity 77% [46–95]).
`
`The findings of this proof-of-principle study show that a
`sole DNA-based marker (SFRP2), assessed in independent
`sets of patients, has a sensitivity of 77–90% for identifying
`patients with colorectal cancer. Since most of our control
`patients were symptomatic due to various diseases (table), the
`specificity of 77% might be higher when asymptomatic
`healthy controls are investigated. We established this self-
`collection approach as an easy procedure for patients in order
`to increase their willingness to participate in this screening
`test, which besides sensitivity and specificity is an important
`criterion for introducing a screening test into clinical routine.
`We are aware that this self-collection approach might
`represent a possible shortcoming, which should be addressed
`in further studies by more standardised collection procedures.
`
`1284
`
`THE LANCET • Vol 363 • April 17, 2004 • www.thelancet.com
`
`Geneoscopy Exhibit 1037, Page 2
`
`

`

`Nevertheless, to our knowledge SFRP2 methylation
`represents one of the most sensitive markers for identifying
`colorectal cancer, besides mutation analysis3 and protein
`analysis,6 in stool samples. Whether a panel of genetic and
`epigenetic markers in stool could be used to identify
`colorectal cancer at an early stage remains to be shown.
`Contributors
`Experimental work was done by H Fiegl, and M Morandell. H M Müller,
`M Oberwalder, M Zitt, M Mühlthaler, and D Öfner recruited patients and
`collected samples. All authors were involved in data analysis. Statistical
`analysis and study design were done by G Goebel. R Margreiter had
`overall responsibility for patient care. H M Müller and M Widschwendter
`designed the study, did data analysis and interpretation, and wrote the
`manuscript. M Widschwendter initiated and supervised the entire study.
`H M Müller, M Oberwalder, and H Fiegl contributed equally to this work.
`
`Conflict of interest statement
`None declared.
`
`Acknowledgments
`We thank Inge Gaugg for excellent technical assistance. This work was
`funded by grants from Fonds zur Förderung der wissenschaftlichen
`Forschung, P15995-B05 and P16159-B05.
`
`1 Etzioni R, Urban N, Ramsey S, et al. The case for early detection.
`Nat Rev Cancer 2003; 3: 243–52.
`2 Traverso G, Shuber A, Levin B, et al. Detection of APC mutations in
`faecal DNA from patients with colorectal tumors. N Engl J Med 2002;
`346: 311–20.
`3 Tagore KS, Lawson MJ, Yucaitis JA, et al. Sensitivity and specificity
`of a stool DNA multitarget assay panel for the detection of advanced
`colorectal neoplasia. Clin Colorectal Cancer 2003; 3: 47–53.
`4 Suzuki H, Gabrielson E, Chen W, et al. A genomic screen for genes
`upregulated by demethylation and histone deacetylase inhibition in
`human colorectal cancer. Nat Genet 2002; 31: 141–49.
`5 Müller HM, Widschwendter A, Fiegl H, et al. DNA methylation in
`serum of breast cancer patients: an independent prognostic marker.
`Cancer Res 2003; 63: 7641–45.
`6 Davies RJ, Freeman A, Morris LS, et al. Analysis of minichromosome
`maintenance proteins as a novel method for detection of colorectal
`cancer in stool. Lancet 2002; 359: 1917–19.
`
`Departments of General and Transplant Surgery (H M Müller MD,
`M Oberwalder MD, M Zitt MD, D Öfner MD, R Margreiter MD), Obstetrics
`and Gynecology (H Fiegl PhD, M Morandell MD, M Widschwendter MD),
`and Biostatistics and Documentation (G Goebel PhD), Medical
`University Innsbruck, A-6020 Innsbruck, Austria; and Department of
`General Surgery, St Vinzenz Hospital, Zams, Austria (M Mühlthaler MD)
`
`Correspondence to: Dr Martin Widschwendter, Department of
`Obstetrics and Gynecology, Medical University Innsbruck,
`Anichstrasse 35, A-6020 Innsbruck, Austria
`(e-mail: martin.widschwendter@uibk.ac.at)
`
`Isolation of Salmonella enterica
`serotype choleraesuis resistant to
`ceftriaxone and ciprofloxacin
`C H Chiu, L H Su, C Chu, J H Chia, T L Wu, T Y Lin, Y S Lee, J T Ou
`
`Salmonella enterica serotype choleraesuis (S choleraesuis)
`usually causes systemic
`infections
`in man that need
`antimicrobial treatment. We isolated a strain of S choleraesuis
`that was resistant to ceftriaxone and ciprofloxacin from a
`patient with sepsis. Ciprofloxacin resistance was associated
`with mutations in gyrA and parC, whereas the ampC gene
`(blaCMY-2), responsible for ceftriaxone resistance, was carried by
`a transposon-like mobile element. This element was found
`inserted into finQ of a potentially transmissible 140 kb plasmid,
`with an 8 bp direct repeat flanking the junction regions. The
`appearance of this resistant S choleraesuis is a serious threat
`to public health, and thus constant surveillance is warranted.
`
`Lancet 2004; 363: 1285–86
`
`RESEARCH LETTERS
`
`Salmonella enterica serotype choleraesuis (S choleraesuis)
`usually causes systemic
`infections
`in man that need
`antimicrobial treatment. The emergence of S choleraesuis that
`is resistant to multiple antimicrobial agents—notably,
`fluoroquinolones—has aroused concern about the treatment
`of systemic non-typhoidal salmonellosis.1 Nevertheless,
`ceftriaxone-resistant S choleraesuis has not, until now, been a
`clinical problem. We report a resistant S choleraesuis strain in
`a patient with sepsis, and our investigation into the genetic
`basis of that resistance.
`In January, 2002, a 58-year-old man with a history of
`oesophageal cancer was admitted to hospital because of
`sepsis. From his blood culture, a strain of S choleraesuis was
`isolated and proved resistant to all antimicrobial agents
`commonly used
`to
`treat
`salmonellosis,
`including
`ciprofloxacin-ceftriaxone (table). Treatment with imipenem-
`cilastatin was initiated, but the patient died 7 days after
`admission.
`We examined the S choleraesuis isolate SCB67 from this
`from an unrelated
`patient, and another strain SCB431
`patient with sepsis. We obtained signed consent from the
`family of the patient, and the research was approved by the
`Chang Gung ethics committee. We identified salmonella
`isolates using standard methods: biochemical reactions and
`agglutination tests with specific antisera (Difco Laboratories,
`Detroit, MI, USA). The isolates were serogroup C1
`salmonella unable to use citrate as a sole carbon source.
`H antiserum
`further confirmed
`the
`isolates
`to be
`S choleraesuis in tube-agglutination tests. An S typhi strain,
`NCTC8393, was the control.
`We used a standard broth-microdilution method to check
`the susceptibility of the isolates, and interpreted the results
`according to the criteria of the National Committee for
`Clinical Laboratory Standards. The minimum inhibitory
`concentrations of ceftriaxone and ciprofloxacin against
`SCB67 were both at a resistant value of 16 ␮g/mL.
`Furthermore, a confirmatory test recommended by the
`committee
`showed
`that
`the minimum
`inhibitory
`concentrations of cefotaxime (16 ␮g/mL) and ceftazidime
`(32 ␮g/mL) to SCB67 remained unchanged irrespective of
`the presence or absence of clavulanic acid, and that of
`cefoxitin was at a resistant value (64 ␮g/mL). These findings
`suggest that SCB67 might produce AmpC but not extended-
`spectrum ␤ lactamases.
`PCR assays showed that the 50 kb plasmids of SCB67 and
`SCB43 were both spvC-positive, indicating that they were
`the serotype-specific virulence plasmid of S choleraesuis.1 To
`detect the ampC gene, we next did PCR and sequencing as
`described earlier,2 and amplified a 1143 bp fragment with
`complete homology to the blaCMY-2 gene from SCB67, but not
`from SCB43 or the control (table). ampC is a group of
`includes blaCMY-2. Southern-blot
`resistance genes
`that
`hybridisation with the PCR product as the probe confirmed
`that blaCMY-2 was located on the 140 kb plasmid of SCB67.
`This plasmid was non-conjugative, but it did contain an oriT
`
`Plasmid Antibiotic
`Ceftriaxone Mutations
`resistance
`in DNA gyrase size (kb)
`resistance
`gene
`and
`phenotype*
`topoisomerase
`genes
`
`T
`None
`None
`NCTC8393 None
`ACSSuTGmKTpCip
`90, 50
`gyrA, parC
`SCB43
`None
`SCB67
`140, 50 ACSSuTGmKTpCroCip
`gyrA, parC
`blaCMY-2
`*A=ampicillin. C=chloramphenicol. S=streptomycin. Su=sulfonamide.
`T=tetracycline. Gm=gentamicin. K=kanamycin. Tp=trimethoprim.
`Cro=ceftriaxone. Cip=ciprofloxacin.
`Antimicrobial susceptibility, plasmid profile, and antimicrobial
`resistance phenotype of the clinical isolates of S choleraesuis
`(SCB43 and SCB67) and the control strain (NCTC8393)
`
`THE LANCET • Vol 363 • April 17, 2004 • www.thelancet.com
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`1285
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