Matrix-comparative genomic hybridization from multicenter formalin-fixed paraffin-embedded colorectal cancer tissue blocks

Heiko Fensterer, Bernhard Radlwimmer, Jörn Sträter, Malte Buchholz, Daniela E Aust, Catherine Julié, François Radvanyi, Bernard Nordlinger, Claudio Belluco, Eric Van Cutsem, Claus-Henning Köhne, Hans A Kestler, Carsten Schwaenen, Michelle Nessling, Manfred P Lutz, Peter Lichter, Thomas M Gress, EORTC Gastrointestinal (GI) Group, Heiko Fensterer, Bernhard Radlwimmer, Jörn Sträter, Malte Buchholz, Daniela E Aust, Catherine Julié, François Radvanyi, Bernard Nordlinger, Claudio Belluco, Eric Van Cutsem, Claus-Henning Köhne, Hans A Kestler, Carsten Schwaenen, Michelle Nessling, Manfred P Lutz, Peter Lichter, Thomas M Gress, EORTC Gastrointestinal (GI) Group

Abstract

Background: The identification of genomic signatures of colorectal cancer for risk stratification requires the study of large series of cancer patients with an extensive clinical follow-up. Multicentric clinical studies represent an ideal source of well documented archived material for this type of analyses.

Methods: To verify if this material is technically suitable to perform matrix-CGH, we performed a pilot study using macrodissected 29 formalin-fixed, paraffin-embedded tissue samples collected within the framework of the EORTC-GI/PETACC-2 trial for colorectal cancer. The scientific aim was to identify prognostic genomic signatures differentiating locally restricted (UICC stages II-III) from systemically advanced (UICC stage IV) colorectal tumours.

Results: The majority of archived tissue samples collected in the different centers was suitable to perform matrix-CGH. 5/7 advanced tumours displayed 13q-gain and 18q-loss. In locally restricted tumours, only 6/12 tumours showed a gain on 13q and 7/12 tumours showed a loss on 18q. Interphase-FISH and high-resolution array-mapping of the gain on 13q confirmed the validity of the array-data and narrowed the chromosomal interval containing potential oncogenes.

Conclusion: Archival, paraffin-embedded tissue samples collected in multicentric clinical trials are suitable for matrix-CGH analyses and allow the identification of prognostic signatures and aberrations harbouring potential new oncogenes.

Figures

Figure 1
Figure 1
Comparison of different DNA samples after extraction: Lane 1: DNA-ladder, 2–7 and 9: DNA from paraffin-embedded colorectal cancer samples, lane 8: no DNA loaded, lane 10+11: DNA from cultured cells.
Figure 2
Figure 2
Comparison of the different matrix-CGH-profiles from DNA showing moderate (figure 2a) and higher degree of degradation (figure 2b). Log2 ratios for the different BAC clones (y-axis) are displayed according to their chromosomal position (x-axis).
Figure 3
Figure 3
Representative figures of two-colour FISH on 2 μm-sections of a formalin-fixed tumour sample from no. 29 was performed with the Rhodamin-labeled BAC-clones RP11-89P22 (figure 3a) and RP11-8C15 (figure 3b) and compared with the FITC-labeled BAC-clone RP11-9F13 used as control in figure 3a and 3b. The Rhodamin-labeled signal appears in red and the FITC labeled signal in green (original magnification 630×). It showed the typical pattern of a high level amplification for the clones RP11-8C15 and RP11-89P22, whereas the apparently not involved clone RP11-9F13 was confirmed to be localized outside of the amplified area.
Figure 4
Figure 4
High resolution-mapping of the gain on 13q in tumour 29 using the 6 k array (figure 4b) as compared to the profile obtained with the 0.64 k genomic array (figure 4a). Log2 ratios for the different BAC clones (y-axis) are displayed according to their chromosomal position (x-axis).

References

    1. Kallioniemi A, Kallioniemi OP, Sudar D, Rutovitz D, Gray JW, Waldman F, Pinkel D. Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors. Science. 1992;258:818–821. doi: 10.1126/science.1359641.
    1. Bentz M, Plesch A, Stilgenbauer S, Dohner H, Lichter P. Minimal sizes of deletions detected by comparative genomic hybridization. Genes Chromosomes Cancer. 1998;21:172–175. doi: 10.1002/(SICI)1098-2264(199802)21:2<172::AID-GCC14>;2-T.
    1. Kirchhoff M, Gerdes T, Maahr J, Rose H, Bentz M, Dohner H, Lundsteen C. Deletions below 10 megabasepairs are detected in comparative genomic hybridization by standard reference intervals. Genes Chromosomes Cancer. 1999;25:410–413. doi: 10.1002/(SICI)1098-2264(199908)25:4<410::AID-GCC17>;2-J.
    1. Solinas-Toldo S, Lampel S, Stilgenbauer S, Nickolenko J, Benner A, Dohner H, Cremer T, Lichter P. Matrix-based comparative genomic hybridization: biochips to screen for genomic imbalances. Genes Chromosomes Cancer. 1997;20:399–407. doi: 10.1002/(SICI)1098-2264(199712)20:4<399::AID-GCC12>;2-I.
    1. Pinkel D, Segraves R, Sudar D, Clark S, Poole I, Kowbel D, Collins C, Kuo WL, Chen C, Zhai Y, Dairkee SH, Ljung BM, Gray JW, Albertson DG. High resolution analysis of DNA copy number variation using comparative genomic hybridization to microarrays. Nat Genet. 1998;20:207–211. doi: 10.1038/2524.
    1. Mendrzyk F, Radlwimmer B, Joos S, Kokocinski F, Benner A, Stange DE, Neben K, Fiegler H, Carter N, Reifenberger G, Korshunov A, Lichter P. Genomic and Protein Expression Profiling Identifies CDK6 As Novel Independent Prognostic Marker in Medulloblastoma. J Clin Oncol. 2005;23:8853–8862. doi: 10.1200/JCO.2005.02.8589.
    1. Van Cutsem E, Labianca R, Hossfeld D, Bodoky G, Roth A, Aranda E, Nordlinger B, Assadourian S, Wang K, Cunningham D. Randomized phase III trial comparing infused irinotecan/5-fluorouracil (5-FU)/folinic acid (IF) versus 5-FU/FA (F) in stage III colon cancer patients (pts). (PETACC 3) J Clin Oncol (Meeting Abstracts) 2005;23:LBA8.
    1. Paris PL, Albertson DG, Alers JC, Andaya A, Carroll P, Fridlyand J, Jain AN, Kamkar S, Kowbel D, Krijtenburg PJ, Pinkel D, Schroder FH, Vissers KJ, Watson VJE, Wildhagen MF, Collins C, van Dekken H. High-Resolution Analysis of Paraffin-Embedded and Formalin-Fixed Prostate Tumors Using Comparative Genomic Hybridization to Genomic Microarrays. Am J Pathol. 2003;162:763–770.
    1. Nessling M, Richter K, Schwaenen C, Roerig P, Wrobel G, Wessendorf S, Fritz B, Bentz M, Sinn HP, Radlwimmer B, Lichter P. Candidate Genes in Breast Cancer Revealed by Microarray-Based Comparative Genomic Hybridization of Archived Tissue. Cancer Res. 2005;65:439–447.
    1. DeVries S, Nyante S, Korkola J, Segraves R, Nakao K, Moore D, Bae H, Wilhelm M, Hwang S, Waldmann F. Array-Based Comparative Genomic Hybridization from Formalin-Fixed, Paraffin-Embedded Breast Tumors. J Mol Diagn. 2005;7:65–71.
    1. Sambrook J, Fitsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, Cold Spring Harbor Press; 1989.
    1. Cappellen D, Gil Diez dM, Chopin D, Thiery JP, Radvanyi F. Frequent loss of heterozygosity on chromosome 10q in muscle-invasive transitional cell carcinomas of the bladder. Oncogene. 1997;14:3059–3066. doi: 10.1038/sj.onc.1201154.
    1. Schwaenen C, Nessling M, Wessendorf S, Salvi T, Wrobel G, Radlwimmer B, Kestler HA, Haslinger C, Stilgenbauer S, Dohner H, Bentz M, Lichter P. Automated array-based genomic profiling in chronic lymphocytic leukemia: development of a clinical tool and discovery of recurrent genomic alterations. Proc Natl Acad Sci USA. 2004;101:1039–1044. doi: 10.1073/pnas.0304717101.
    1. Hoang JM, Cottu PH, Thuille B, Salmon RJ, Thomas G, Hamelin R. BAT-26, an indicator of the replication error phenotype in colorectal cancers and cell lines. Cancer Res. 1997;57:300–303.
    1. Loukola A, Salovaara R, Kristo P, Moisio AL, Kaariainen H, Ahtola H, Eskelinen M, Harkonen N, Julkunen R, Kangas E, Ojala S, Tulikoura J, Valkamo E, Jarvinen H, Mecklin JP, de la Chapelle A, Aaltonen LA. Microsatellite instability in adenomas as a marker for hereditary nonpolyposis colorectal cancer. Am J Pathol. 1999;155:1849–1853.
    1. Lichter P, Bentz M, Joos S. Detection of chromosomal aberrations by means of molecular cytogenetics: painting of chromosomes and chromosomal subregions and comparative genomic hybridization. Methods Enzymol. 1995;254:334–359.
    1. Bubendorf L, Kononen J, Koivisto P, Schraml P, Moch H, Gasser TC, Willi N, Mihatsch MJ, Sauter G, Kallioniemi OP. Survey of gene amplifications during prostate cancer progression by high-throughout fluorescence in situ hybridization on tissue microarrays. Cancer Res. 1999;59:803–806.
    1. Korn WM, Yasutake T, Kuo WL, Warren RS, Collins C, Tomita M, Gray J, Waldmann FM. Chromosome arm 20q gains and other genomic alterations in colorectal cancer metastatic to liver, as analyzed by comparative genomic hybridization and fluorescence in situ hybridization. Genes Chromosomes Cancer. 1999;25:82–90. doi: 10.1002/(SICI)1098-2264(199906)25:2<82::AID-GCC2>;2-6.
    1. Nakao K, Mehta KR, Fridlyand J, Moore DH, Jain AN, Lafuente A, Wiencke JW, Terdiman JP, Waldmann FM. High-resolution analysis of DNA copy number alterations in colorectal cancer by array-based comparative genomic hybridization. Carcinogenesis. 2004;25:1345–1357. doi: 10.1093/carcin/bgh134.
    1. Douglas EJ, Fiegler H, Rowan A, Halford S, Bicknell DC, Bodmer W, Tomlinson IPM, Carter NP. Array Comparative Genomic Hybridization Analysis of Colorectal Cancer Cell Lines and Primary Carcinomas. Cancer Res. 2004;64:4817–4825. doi: 10.1158/0008-5472.CAN-04-0328.
    1. Jones AM, Douglas EJ, Halford SE, Fiegler H, Gorman PA, Roylance RR, Carter NP, Tomlinson IPM. Array-CGH analysis of microsatellite-stable, near-diploid bowel cancers and comparison with other types of colorectal carcinoma. Oncogene. 2004;24:118–129. doi: 10.1038/sj.onc.1208194.
    1. Moertel CG, Fleming TR, Macdonald JS, Haller DG, Laurie JA, Tangen CM, Ungerleider JS, Emerson WA, Tormey DC, Glick JH. Fluorouracil plus levamisole as effective adjuvant therapy after resection of stage III colon carcinoma: a final report. Ann Intern Med. 1995;122:321–326.
    1. Rooney PH, Boonsong A, McKay JA, Marsh S, Stevenson DA, Murray GI, Curran S, Haites NE, Cassidy J, McLeod HL. Colorectal cancer genomics: evidence for multiple genotypes which influence survival. Br J Cancer. 2001;85:1492–1498. doi: 10.1054/bjoc.2001.2095.
    1. Eppert K, Scherer SW, Ozcelik H, Pirone R, Hoodless P, Kim H, Tsui LC, Bapat B, Gallinger S, Andrulis IL, Thomsen GH, Wrana JL, Attisano L. MADR2 maps to 18q21 and encodes a TGFbeta-regulated MAD-related protein that is functionally mutated in colorectal carcinoma. Cell. 1996;86:543–552. doi: 10.1016/S0092-8674(00)80128-2.
    1. Hahn SA, Schutte M, Hoque AT, Moskaluk CA, da Costa LT, Rozenblum E, Weinstein CL, Fischer A, Yeo CJ, Hruban RH, Kern SE. DPC4, a candidate tumor suppressor gene at human chromosome 18q21.1. Science. 1996;271:350–353. doi: 10.1126/science.271.5247.350.
    1. Woodford-Richens KL, Rowan AJ, Gorman P, Halford S, Bicknell DC, Wasan HS, Roylance RR, Bodmer WF, Tomlinson IPM. SMAD4 mutations in colorectal cancer probably occur before chromosomal instability, but after divergence of the microsatellite instability pathway. PNAS. 2001;98:9719–9723. doi: 10.1073/pnas.171321498.
    1. Jen J, Kim H, Piantadosi S, Liu ZF, Levitt RC, Sistonen P, Kinzler KW, Vogelstein B, Hamilton SR. Allelic loss of chromosome 18q and prognosis in colorectal cancer. N Engl J Med. 1994;331:213–221. doi: 10.1056/NEJM199407283310401.
    1. Alazzouzi H, Alhopuro P, Salovaara R, Sammalkorpi H, Jarvinen H, Mecklin JP, Hemminki A, Schwartz S, Aaltonen LA, Arango D. SMAD4 as a Prognostic Marker in Colorectal Cancer. Clin Cancer Res. 2005;11:2606–2611. doi: 10.1158/1078-0432.CCR-04-1458.
    1. Boulay JL, Mild G, Lowy A, Reuter J, Lagrange M, Terracciano L, Laffer U, Herrmann R, Rochlitz C. SMAD4 is a predictive marker for 5-fluorouracil-based chemotherapy in patients with colorectal cancer. Br J Cancer. 2002;87:630–634. doi: 10.1038/sj.bjc.6600511.
    1. Reilly JT. FLT3 and its role in the pathogenesis of acute myeloid leukaemia. Leuk Lymphoma. 2003;44:1–7. doi: 10.1080/1042819021000040233.
    1. Zhang H, Wu J, Meng L, Shou CC. Expression of vascular endothelial growth factor and its receptors KDR and Flt-1 in gastric cancer cells. World J Gastroenterol. 2002;8:994–998.
    1. Wing LY, Chen HM, Chuang PC, Wu MH, Tsai SJ. The Mammalian Target of Rapamycin-p70 Ribosomal S6 Kinase but Not Phosphatidylinositol 3-Kinase-Akt Signaling Is Responsible for Fibroblast Growth Factor-9-induced Cell Proliferation. J Biol Chem. 2005;280:19937–19947. doi: 10.1074/jbc.M411865200.
    1. Jin C, Wang F, Wu X, Yu C, Luo Y, McKeehan WL. Directionally Specific Paracrine Communication Mediated by Epithelial FGF9 to Stromal FGFR3 in Two-Compartment Premalignant Prostate Tumors. Cancer Res. 2004;64:4555–4562. doi: 10.1158/0008-5472.CAN-03-3752.
    1. Schaefer KL, Wai DH, Poremba C, Korsching E, van Valen F, Ozaki T, Boecker W, Dockhorn-Dworniczak B. Characterization of the malignant melanoma of soft-parts cell line GG-62 by expression analysis using DNA microarrays. Virchows Arch. 2002;440:476–484. doi: 10.1007/s00428-001-0558-9.
    1. Todo T, Kondo T, Kirino T, Asai A, Adams EF, Nakamura S, Ikeda K, Kurokowa T. Expression and growth stimulatory effect of fibroblast growth factor 9 in human brain tumors. Neurosurgery. 1998;43:337–346. doi: 10.1097/00006123-199808000-00098.
    1. Penault-Llorca F, Bertucci F, Adelaide J, Parc P, Coulier F, Jacquemier J, Birnbaum D, deLapeyriere O. Expression of FGF and FGF receptor genes in human breast cancer. Int J Cancer. 1995;61:170–176. doi: 10.1002/ijc.2910610205.

Source: PubMed

赞助者和合作者

医疗条件

药物干预

3
订阅