Diapositiva 1

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XVIII Congreso Chileno de Cancerología
Nueva Generación de Biomarcadores en Cáncer
Dr. Alejandro Corvalán R. Departamento
Hematología-Oncología Pontificia Universidad
Católica de Chile
Hotel de la Bahi,a, Coquimico, Chile 21 Octubre
2010
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Marcadores Tumorales
http//www.medindia.net/patients/patientinfo/tumor
-markers-for-cancer-diagnosis-and-prognosis.htm
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genética
Histone acetylation DNA
methylation microRNA
epigenética
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Metilación del DNA
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Metilación del DNA
Esteller 2002 J Pathol 196 17.
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DNA METHYLATION
Esteller 2002 J Pathol 196 17.
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Bisulfite Conversion for Analysis of Methylated
Cytosines
CH3
CH3
H
H
5-methylcytosine
H
thymidine
Thymidine
5-methylcytosine
Treatment of DNA with bisulfite converts cytosine
residues to uracil, but leaves 5-methylcytosine
residues unaffected.
Clark et al., 1994 Nucleic Acids Research
222990-2997
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Methylation specific PCR (MSP) for Analysis of
Methylated Cytosines
CAAAAAATCTCAACAAACTCACACCA AAATCTCAACGAACTCA
CGCCG
5-methylcytosine
cytosine
Herman et al., 1996 Proc Natl Acad Sci U S A
939821-9826
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Balch, C., F. Fang, et al. (2009). "Minireview
epigenetic changes in ovarian cancer."
Endocrinology 150(9) 4003-4011. Castelli, T., S.
Cimino, et al. (2010). "Molecular markers for
prostatic cancer." Front Biosci (Elite Ed) 2
641-656. Goel, A. (2010). "DNA methylation-based
fecal biomarkers for the noninvasive screening of
GI cancers." Future Oncol 6(3) 333-336. Gossage,
L. and T. Eisen (2010). "Alterations in VHL as
potential biomarkers in renal-cell carcinoma."
Nat Rev Clin Oncol 7(5) 277-288. Hoque, M. O.
(2009). "DNA methylation changes in prostate
cancer current developments and future clinical
implementation." Expert Rev Mol Diagn 9(3)
243-257. Kim, M. S., J. Lee, et al. (2010). "DNA
methylation markers in colorectal cancer." Cancer
Metastasis Rev 29(1) 181-206. Kim, W. J. and Y.
J. Kim (2009). "Epigenetic biomarkers in
urothelial bladder cancer." Expert Rev Mol Diagn
9(3) 259-269. Martens, J. W., A. L. Margossian,
et al. (2009). "DNA methylation as a biomarker in
breast cancer." Future Oncol 5(8)
1245-1256. Muraki, Y., K. Banno, et al. (2009).
"Epigenetic DNA hypermethylation clinical
applications in endometrial cancer (Review)."
Oncol Rep 22(5) 967-972. Nagarajan, R. P. and J.
F. Costello (2009). "Epigenetic mechanisms in
glioblastoma multiforme." Semin Cancer Biol
19(3) 188-197. Nagarajan, R. P. and J. F.
Costello (2009). "Molecular epigenetics and
genetics in neuro-oncology." Neurotherapeutics
6(3) 436-446. Pfeifer, G. P. and T. A. Rauch
(2009). "DNA methylation patterns in lung
carcinomas." Semin Cancer Biol 19(3)
181-187. Szalmas, A. and J. Konya (2009).
"Epigenetic alterations in cervical
carcinogenesis." Semin Cancer Biol 19(3)
144-152. van der Kwast, T. H. and B. Bapat
(2009). "Predicting favourable prognosis of
urothelial carcinoma gene expression and genome
profiling." Curr Opin Urol 19(5) 516-521. Xu,
L., B. G. Czito, et al. (2010). "Epigenetic
markers in rectal cancer." Clin Cancer Res
16(10) 2699-2701.
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MGMT - predicting benefit from alkylating agents
in patients with glioblastomas
PITX2 - predicting outcome in lymph node-negative
breast cancer patients
GSTP1 - early diagnosis of prostate cancer
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MGMT - predicting benefit from alkylating agents
in patients with glioblastomas
PITX2 - predicting outcome in lymph node-negative
breast cancer patients
Reprimo early diagnosis of gastric carcinoma
GSTP1 - early diagnosis of prostate cancer
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Hegi ME, Diserens AC, Gorlia T, et al. MGMT gene
silencing and benefit from temozolomide in
glioblastoma. New Engl J Med 20053529971003.
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Duffy, M. J., R. Napieralski, et al. (2009).
"Methylated genes as new cancer biomarkers." Eur
J Cancer 45(3) 335-346.
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Unresolved issues in the use of methylated genes
as cancer markers

• Aging and benign diseases
• Age RARß2, RASSF1A and GSTP1 in prostatic
  cancer and MYOD1 in colorectal cancer
• Benign tumours, may also exhibit altered gene
  methylation

• Analytical issues
• Bisulphite sequencing detection of CpG island
  methylation of new genes
• MethyLight quantitative methylation-specific
  PCR (MSP) for sensitive detection of methylated
  alleles
• Pyrosequencing quantitation of individual CpG
  sites

• Requirement for clinical validation
• Measurement of methylated genes must provide
  additional information to that available from
  existing diagnostic, prognostic and predictive
  modalities and should be clinically relevant.
• Preliminary findings from small scale /
  retrospective design must be confirmed in
  high-level evidence studies (prospective trials
  /meta-pooled analysis of individual data from
  smaller-scale studies).
• Multicenter collaboration will be necessary to
  accelerate clinical validation,

Kagan et al (2007). "Towards Clinical
Application of Methylated DNA Sequences as Cancer
Biomarkers A Joint NCI's EDRN and NIST Workshop
on Standards, Methods, Assays, Reagents and
Tools." Cancer Res 67(10) 4545-4549.
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Reprimo a potential biomarker for early
detection of gastric carcinoma
Lee et al., Int J Cancer, 2006. 118(9)2315-21.
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Llorens P. Semin Surg Oncol. 19917(6)339-43.
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The Hallmarks of Cancer
SHP1 ER
E-cadherin APC Reprimo SEMA3B
RUNX3FHIT DAPK P73 BLU PTEN
SOCS COX2
TIMP3 RAR-beta RIZ1
In Silico
BRCA1 MGMT hMLH1 GSTp1
3OST2 P14 P15 P16
PubMed
Hanahan Weinberg Cell 1005770,2000
Corvalán, A. FONDECYT 1030130 1080563
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Retrospective gastric cancer clinical samples (32
cases) Analysis of 24 genes
Carolina Bernal
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Mucinous/signet ring cell type association by
unsupervised hierarchical cluster analysis
cases
genes
methylation positive methylation negative no data
Francisco Ossandon Cabrera
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Prospective gastric cancer clinical samples (43)
and healthy donors (31) Analysis of 7 genes
Figure 1. Histogram representing the percentage
of positive cases for Reprimo and other genes
(APC, SHP1, CDH-1, ER, SEMA3B and 3OST2) in 43
prospectively collected gastric cancer cases and
31 asymptomatic age- and gender-matched controls.
Only Reprimo shows significative differences in
plasma between gastric cancer and asymptomatic
controls (from and Bernal C, Aguayo F, Villarroel
C, Vargas M, Díaz I, Ossandon FJ, Santibáñez E,
Palma M, Aravena E, Barrientos C, Corvalan AH.
Clin Cancer Res. 2008146264-9 patent
200909028174 INAPI).
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DNA methylation of Reprimo
N P M 1 2 3 4 5 6
7
plasma tumor patients
plasma asymptomatic controls
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
20 21 22 P N
plasma healthy donors
Santibanez E 31 Ene 2006
Eudocia Santibanez 31 Ene 2006
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Cynthia Villarroel
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65 RPRM pos 12 endoscopias
Dr.. Alfonso Calvo Dr. Edmundo Aravena Dr. Carlos
Barrientos
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Gastric cancer
Healthy donors
Healthy donors Blood Bank Hospital Sotero del
Rio 0.000677 copies/mL Gastric cancer patients
CRS San Rafael 4.23 copies/mL
Maria Jose Maturana
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Absolute quantification for Repimo detection in
plasma
promoter region of Rperimo
RPRM 350 pb
Maria Jose Maturana
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Absolute Quantification of DNA Methylation by
MethyLight technology
Hospital Sotero del Rio
23 Gastric Cancer cases 42 Healthy donors (Blood
bank)

• Sensibilidad 87
• Especificidad 97.6
• Criterio gt6.05/0.4 ml
• (15 copias/mL)
• Area under the ROC curve 0,923
• Standard error  0,040
• 95 CI 0,829 to 0,974
• p value 0,0001

Oslando Padilla Agosto 2010
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Proyecto D09i1137 TEST DE TAMIZAJE PARA LA
DETECCIÓN PRECOZ DE CÁNCER GÁSTRICO  XVII
Concurso de Proyectos FONDEF de ID 2009
.
Director, Alejandro Corvalán Médico-cirujano/pat
ólogo molecular, investigador principal/co-investi
gador proyectos FONDECYT y FONIS. Capacidad de
producción científica. Especialista en estudios
de metilación. Directora alterna, Catterina
Ferreccio Médico-cirujano/epidemióloga,
investigador principal/co-investigadora proyectos
FONDECYT y FONIS. Capacidad de producción
científica. Especialista en trabajos de
investigación a nivel poblacional Investigadores
principales. Alfonso Calvo Médico-cirujano/ciruja
no/endoscopista. Especialista y asesor Ministerio
de Salud en estrategias detección precoz cáncer
gástrico. Co-investigador proyectos FONDECYT.
Capacidad de producción científica. Ignacio
Duarte Medico-cirujano/anatomo-patólogo,
investigador principal/co-investigador proyectos
FONDECYT. Especialista en patología del cáncer
gástrico. Francisco Aguayo Doctor en Biología
Molecular, investigador principal y
coinvestigador proyectos FONDECYT. Capacidad de
producción científica. Especialista en estudios
de metilación. Investigadores Asistentes Pablo
Cortes Medico-cirujano/gastroenterólogo
/endoscopista. Sonia Lagos Médico-cirujano,
genetista molecular, co-investigadora proyectos
FONDECYT. Oslando Padilla Estadístico,
co-investigador proyectos FONDECYT. MaríaJosé
Maturana Bioquímica, experiencia en estudios de
metilación. Profesional transferencia
tecnológica y gestión del proyecto. Oscar Solar
Ingeniero Civil Bioquímico. Especialista en
evaluación y control técnico-financiera proyectos
CORFO, CONICYT, Unión Europea y privados.
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• Puente Alto
• 1000 hombres
• gt40 a.
• Endoscopia
• Suero H.pylori Atrofia
• Plasma Reprimo
• Desarrollo Triage

• Molina
• 3000 Poblacion General
• gt40 a.
• Validacion Triage
• (H.pylori AtrofiaReprimo)
• Endoscopia

()
Ferreccio et al., Cancer Epidemiol Biomarkers
Prev, 2007. 16(4)662-7.
Ferreccio 2007
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Niveles plasmáticos de Reprimo en pacientes
tratados por cáncer gástrico
t0 t1
t0 t1
J.P.H.H. O.R.C.C. M.E.A.V.
Dr. Marcelo Garrido Dept Hematologia-Oncologia Dr.
Ricardo Funkel Dept. Cirugia Digestiva
N copias de Reprimo/ml
Pacientes Cancer Gastrico Metastasicos Confirmacio
n histologica (ICD-9-151.0) Respuesta radiologica
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María José Maturana 8 Oct 2010
(Metodología Methylight, cuantificación absoluta)
Maria Jose Maturana
t0 t1
t0 t1
t0 t1
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Bisulfite sequence analysis of Reprimo in gastric
carcinoma cell lines
Veronica Torres
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RT-PCR (-) (-) () ()
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Reprimo (RPRM)
Promoter region
GTTTTAGAAGAGTTTAGTTGTTGCGCGCGTCGGAGAGGTTTTTGGGAAAT
TTTTACGGTTTA GGGATTTTCGAAAGTAGAGCGAGGAGTTTTCGTACG
CGTTAGTTTGCGAGTGAGCGTTTAG TTCGGTATTTGTTTTTTTAGCGT
CGTCGTTTTTTTATTTTTCGGATTCGCGTCGTTCGCGGCGT TCGTTCG
TTTTTGCGATGAATTCGGTTTTAGGTAATTAGACGGACGTGGCGGGTTTG
TTTTTG GTTAATAGTA
261 pb 30 CpG
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CpG Unmethylated Cytosine CpG
Methylated Cytosine
DNA analysis
MSP Methylated and unmethylated primers
Bisulfite sequencing
Línea celular
N de clones
AGS
7
MKN 45
6
NCI-N87
10
MKN28
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Imagen referencia Modificada de Takahashi, 2005
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5-azacytidine Treatment
AGS 5 uM
MKN45 5 uM
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NCI87 1 uM
MKN28 1 uM
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Sequence Analyzer Tool
Bisulfite Sequence Analyzer
BISA
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Development of a new software for bisulfite
sequence analysis
Ricardo Droguett Marcelo Mourgues
Sequence AnaLyzer Tool
Bisulfite Sequence Analyzer (BISA)
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CCGGGAACGAGCACCACCAGGGCTGGAGCGGACGGCTTTAGAAGAGCCTA
GCTGCTGCGCGCGTCGGAGAGGCTCCTGGGAAACTCCCACGGCCCAGGGA
CTTTCGAAAGCAGAGCGAGGAGCCCTCGCACGCGCTAGTCTGCGAGTGAG
CGCTCAGCCCGGCACCTGTTCCTCCAGCGCCGCCGCCTTCCCACCCCTCG
GACCCGCGCCGCTCGCGGCGCCCGCCCGTTCCTGCGATGAATCCGGCCCT
AGGCAACCAGACGGACGTGGCGGGCCTGTTCCTGGCCAACAGCAGCGAGG
CGCTGGAGCGAGCCGTGCGCTGCTGCACCCAGGCGTCCGTGGTGACCGAC
GACGGCTTCGCGGAGGGAGGCCCGGACGAGCGTAGCCTGTACATAATGCG
CGTGGTGCAGATCGCGGTCATGTGCGTGCTCTCACTCACCGTGGTCTTCG
GCATCTTCTTCCTCGGCTGCAATCTGCTCATCAAGTCCGAGGGCATGATC
AACTTCCTCGTGAGGCATGATCAACTTCCTCGTGAAGGACCGGAGGCCGT
CTAAGGAGGTGGAGGCGGTGGTCGTGGGGCCCTACTGACCCGCCCTCTGC
CCCCGCGGCAACCGCTCCCACGCCTGCCCACTTTGCTAGCCCGGCTGTGC
CCCTCACTATCAGAGACTGGGCGAAGCAAACCTGTCGGAGTCAATTATTT
CTCTCGATTCGGCCTTTCGGAAAGAAGCGACCGGTTTCTCCCTCGCCCTC
TGAAAGTCCTCATGCCTGGCAGTCGGAGGAGAGCGCCCAGACTCTGAACT
CAGCAGAAAGTGGCAAGAAGAGGGCGATTAGGGCGCAGAACTTTGGAAGC
TGCTACTTACTTGGAATGCGGGGAGACCGACGGTGCGAAGGCCCTTCTCC
ACCCGCAGGTGGGCCAAGCTCTGGGGGCAGGTGGAGAGGGCGGGCAGGGG
AGAGACCCAGCGGCACTGATCGCCTTGTGACCGGAAGAGTGACCTGTTAA
AAGCCACGCAGCAGACTCATGGGGTCTCACAAATCCGTGTCCGGGTGCGC
TCCCACTCTTCTCCTGCTCCCCCCCTGCCCTGGAGGGGAGGGGCGATAAA
TACCTTTGATTGTAACGTGCCGTTTTAAGAGGTTTTGTGTTTGTTTGCTT
GAATACAAATGTTTGATAAGTCTTTTTCTGCCCCAGTGGCCTGTTTGCCT
GCCTGAGGAGTTACAGTTTTGTCATTGTGGGAGAAGGGGTGGGGGGAGGG
GGAGCCTGCGAATTTGAACGGGGTGAGTTGTTTCTTTTAGTGCATTTCCC
ACTGGGTCTTTTGGGAGGCGTCTAGCGTTCCTGCTGGCCCTGGGACAAAG
ACCCAGAATAGAACTCGTAGCTCGTGACTGCACGGTTTACGCCACAAAAG
TGCTCTTGACATCCGTGACACCGTTTTGACTTTTTGTTTTTTTCTTATTT
AACATTTCCTTAATAAATGCAACATTTAAGCGTTT
5 UTR 26 CpG? 30 CpG
Coding region 1 exon 109 Aa
2q23.3
3 UTR miR-335 miR-597 SNPs
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Transfección de línea celular AGS
Análisis de expresión de RPRM en línea celular
transfectada con el vector pcDNA3.1 con la
región codificante (cDNA) de Reprimo.
303 pb 1 exon
RPRM 350 pb
AGS Control
AGS Transfectada
Cynthia Villarroel Veronica Torres
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A soft agar colony formation assay after stable
transfection of full coding sequence of Reprimo
AGS_vector
AGS_Reprimo
Francisco Aguayo Veronica Torres
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Role of Reprimo, a p53-dependent G2 arrest
mediator candidate, in the pathogenesis of human
gastric carcinoma
Role of Reprimo, a p53-dependent G2 arrest
mediator candidate, in the pathogenesis of human
gastric carcinoma
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Centro de Referencia de Salud San Rafael, La
Florida Servicio de Salud Metropolitano Sur
Oriente. Dr. Alfonso Calvo Aux Patricia
Verdugo Instituto Chileno-Japones de Enfermedades
Digestivas Hospital Clínico San
Borja-Arriarán Dr. Carlos Barrientos Dr. Edmundo
Aravena Dr. Ricardo Estela Departamento Salud
Publica P. Universidad Catolica de Chile Dra.
Catterina Ferreccio Oslando Padilla
Ph.D. Departamento Anatomía Patológica
P.Universidad Católica de Chile Dr. Gonzalo
Carrasco Dr. Jose Valbuena Dr. Ignacio
Durate Departamento de Patologia Hospital Max
Peralta Cartago, Costa Rica Dr. Fernando Mena
Laboratorio de Patologia Molecular y
Epidemiologia, Departamento Hematologia-Oncologia
P.Universidad Católica de Chile María Jose
Maturana, BQ,Lab Manager Wilda Olivares, Ph.D.
Student Cs Biologicas PUC Verónica Torres,
tesista Bioquimica PUCV Rodrigo Droguett, tesita
Bioinformatica U.Talca Marcelo Mourgues, tesista
Bioinformatica, U.Talca Francisco Aguayo,
Ph.D. Viviana Montecinos, Ph.D. Bq. Carolina
Bernal Bq. Macarena Vargas Bq. Cynthia
Villarroel Bq. Francisco Ossandon Bq. Pamela
Gonzalez Lic. Biol. Ignacio Díaz Bq. Eudocia
Santibáñez
Centro de Investigacion Oncologia Traslacional
(CIOT) Dr. Marcelo Garrido (Dept.
Hematologia-Oncologia) Dr. Ricadrdo Funkel
(Dept.Cirugia Digestiva) Enf. Lidia Medina
FONDECYT-1030130 FONDECYT-1080563 FONIS-SA06i20019
FONDEF-D09i1137
FONDECYT 1030130, FONIS SA06I20019
PG07/06 PUENTE-2006/09 INTERDEPARTAMENTAL 2010
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Thanks!!