Many human tumors show significant changes in their signal transduction pathways and, thus, the way the cells interact with their environment. Often caused by chromosomal rearrangements, including gene amplifications, translocations or deletions, the altered levels of gene expression may provide a tumor-specific signature that can be exploited for diagnostic or therapeutic purposes. We investigated the utility of multiplexed fluorescence in situ hybridization (FISH) using non-isotopically labeled cDNA probes detected by Spectral Imaging as a sensitive and rapid procedure to measure tumor-specific gene expression signatures. We used a commercially available system to acquire and analyze multicolor FISH images. Initial investigations used panels of fluorescent calibration standards to evaluate the system. These experiments were followed by hybridization of five-to-six differently labeled cDNA probes, which target the transcripts of tyrosine kinase genes known to be differently expressed in normal cells and tumors of the breast or thyroid gland. The relatively simple, yet efficient, molecular cytogenetic method presented here may find many applications in characterization of solid tumors or disseminated tumor cells. Addressing tumor heterogeneity by means of multi-parameter single cell analyses is expected to enable a wide range of investigations in the areas of tumor stem cells, tumor clonality and disease progression.
References
[1]
Lersch, R.A.; Fung, J.; Hsieh, H.B.; Smida, J.; Weier, H.U. Monitoring signal transduction in cancer: from chips to fish. J. Histochem. Cytochem.？2001, 49, 925–926, doi:10.1177/002215540104900715.
[2]
Weier, H.; Munne, S.; Lersch, R.A.; Hsieh, H.; Smida, J.; Chen, X.; Korenberg, J.R.; Pedersen, R.A.; Fung, J. Towards a full karyotype screening of interphase cells: 'FISH and chip' technology. Mol. Cell. Endocrinol.？2001, 183(Suppl. 1), S41–S45.
Trakhtenbrot, L.; Cohen, N.; Rosner, E.; Gipsh, N.; Brok-Simoni, F.; Mandel, M.; Amariglio, N.; Rechavi, G. Coexistence of several unbalanced translocations in a case of neuroblastoma: the contribution of multicolor spectral karyotyping. Cancer Genet. Cytogenet.？1999, 112, 119–123, doi:10.1016/S0165-4608(98)00263-5.
[7]
Yurov, Y.B.; Iourov, I.Y.; Monakhov, V.V.; Soloviev, I.V.; Vostrikov, V.M.; Vorsanova, S.G. The variation of aneuploidy frequency in the developing and adult human brain revealed by an interphase FISH study. J. Histochem. Cytochem.？2005, 53, 385–390, doi:10.1369/jhc.4A6430.2005.
Nikiforov, Y.E. Genetic alterations involved in the transition from well-differentiated to poorly differentiated and anaplastic thyroid carcinomas. Endocr. Pathol.？2004, 15, 319–327.
[12]
Aktas, D.; Utine, E.G.; Mrasek, K.; Weise, A.; von Eggeling, F.; Yalaz, K.; Posorski, N.; Akarsu, N.; Alikasifoglu, M.; Liehr, T.; Tuncbilek, E. Derivative chromosome 1 and GLUT1 deficiency syndrome in a sibling pair. Mol. Cytogenet.？2010, 3, 10.
[13]
Lee, T.K. The value of imprint cytology in tumor diagnosis: a retrospective study of 522 cases in northern China. Acta Cytol.？1982, 26, 169–171.
Wang, J.Q. Analysis of mRNA expression using double in situ hybridization labeling with isotopic and nonisotopic probes. Methods Mol. Med.？2003, 79, 153–159.
[16]
Williams, S.V.; Platt, F.M.; Hurst, C.D.; Aveyard, J.S.; Taylor, C.F.; Pole, J.C.; Garcia, M.J.; Knowles, M.A. High-resolution analysis of genomic alteration on chromosome arm 8p in urothelial carcinoma. Genes Chromosomes Cancer？2010, 49, 642–659.
[17]
Fabien, N.; Paulin, C.; Santoro, M.; Berger, N.; Grieco, M.; Galvain, D.; Barbier, Y.; Dubois, P.M.; Fusco, A. Detection of RET oncogene activation in human papillary thyroid carcinomas by in situ hybridisation. Br. J. Cancer？1992, 66, 1094–1098.
[18]
Garini, Y.; Macville, M.; du Manoir, S.; Buckwald, R.A.; Lavi, M.; Katzir, N.; Wine, D.; Bar-Am, I.; Schr？ck, E.; Cabib, D.; Ried, T. Spectral karyotyping. Bioimaging？1996, 4, 65–72.
[19]
Garini, Y.; Gil, A.; Bar-Am, I.; Cabib, D.; Katzir, N. Signal to noise analysis of multiple color fluorescence imaging microscopy. Cytometry？1999, 35, 214–226, doi:10.1002/(SICI)1097-0320(19990301)35:3<214::AID-CYTO4>3.0.CO;2-D.
Macville, M.V.; Van der Laak, J.A.; Speel, E.J.; Katzir, N.; Garini, Y.; Soenksen, D.; McNamara, G.; de Wilde, P.C.; Hanselaar, A.G.; Hopman, A.H.; Ried, T. Spectral imaging of multi-color chromogenic dyes in pathological specimens. Anal. Cell. Pathol.？2001, 22, 133–142.
[22]
Dickinson, M.E.; Bearman, G.; Tille, S.; Lansford, R.; Fraser, S.E. Multi-spectral imaging and linear unmixing add a whole new dimension to laser scanning fluorescence microscopy. BioTechniques？2001, 31, 1272, 1274–1276, 1278.
[23]
Levenson, R.M.; Lynch, D.T.; Kobayashi, H.; Backer, J. M.; Backer, M.V. Multiplexing with multispectral imaging: from mice to microscopy. ILAR J.？2008, 49, 78–88, doi:10.1093/ilar.49.1.78.
Garini, Y.; Katzir, N.; Cabib, D.; Buckwald, R.A.; Soenksen, D.G.; Malik, Z. Spectral bio-imaging. In Fluorescence Imaging Spectroscopy and Microscopy; Wang, X.F., Herman, B., Eds.; John Whiley & Sons: New York, USA, 1996; Vol. 137, pp. 87–124.
[26]
Schroeck, E.; du Manoir, S.; Veldman, T.; Schoell, B.; Wienberg, J.; Ferguson-Smith, M.A.; Ning, Y.; Ledbetter, D.H.; Bar-Am, I.; Soenksen, D.; Garini, Y.; Ried, T. Multicolor spectral karyotyping of human chromosomes. Science？1996, 273, 494–497.
[27]
Veldman, T.; Vignon, C.; Schroeck, E.; Rowley, J.D.; Ried, T. Hidden chromosome abnormalities in haematological malignancies detected by multicolour spectral karyotyping. Nat. Genet.？1997, 15, 406–410.
[28]
Fung, J.; Hyun, W.; Dandekar, P.; Pedersen, R.A.; Weier, H.U. Spectral imaging in preconception/preimplantation genetic diagnosis of aneuploidy: multicolor, multichromosome screening of single cells. J. Assist. Reprod. Genet.？1998, 15, 323–330, doi:10.1023/A:1022508930762.
[29]
Marquez, C.; Cohen, J.; Munne, S. Chromosome identification in human oocytes and polar bodies by spectral karyotyping. Cytogenet. Cell Genet.？1998, 81, 254–258, doi:10.1159/000015040.
[30]
Zitzelsberger, H.; Lehmann, L.; Hieber, L.; Weier, H.U.; Janish, C.; Fung, J.; Negele, T.; Spelsberg, F.; Lengfelder, E.; Demidchik, E.P.; Salassidis, K.; Kellerer, A.M.; Werner, M.; Bauchinger, M. Cytogenetic changes in radiation-induced tumors of the thyroid. Cancer Res.？1999, 59, 135–140.
[31]
Ning, Y.; Laundon, C. H.; Schroeck, E.; Buchanan, P.; Ried, T. Prenatal diagnosis of a mosaic extra structurally abnormal chromosome by spectral karyotyping. Prenat. Diagn.？1999, 19, 480–482, doi:10.1002/(SICI)1097-0223(199905)19:5<480::AID-PD547>3.0.CO;2-H.
Zitzelsberger, H.; Bruch, J.; Smida, J.; Hieber, L.; Peddie, C.M.; Bryant, P.E.; Riches, A.C.; Fung, J.; Weier, H.U.; Bauchinger, M. Clonal chromosomal aberrations in simian virus 40-transfected human thyroid cells and in derived tumors developed after in vitro irradiation. Int. J. Cancer？2001, 96, 166–177, doi:10.1002/ijc.1015.
[34]
Liehr, T.; Ewers, E.; Kosyakova, N.; Klaschka, V.; Rietz, F.; Wagner, R.; Weise, A. Handling small supernumerary marker chromosomes in prenatal diagnostics. Expert Rev. Mol. Diagn.？2009, 9, 317–324, doi:10.1586/erm.09.17.
[35]
Nederlof, P.M.; van der Flier, S.; Wiegant, J.; Raap, A.K.; Tanke, H.J.; Ploem, J.S.; van der Ploeg, M. Multiple fluorescence in situ hybridization. Cytometry？1990, 11, 126–131, doi:10.1002/cyto.990110115.
[36]
Fan, Y.S.; Siu, V.M.; Jung, J.H.; Xu, J. Sensitivity of multiple color spectral karyotyping in detecting small interchromosomal rearrangements. Genet. Test.？2000, 4, 9–14, doi:10.1089/109065700316417.
[37]
Greulich, K.M.; Kreja, L.; Heinze, B.; Rhein, A.P.; Weier, H.G.; Bruckner, M.; Fuchs, P.; Molls, M. Rapid detection of radiation-induced chromosomal aberrations in lymphocytes and hematopoietic progenitor cells by mFISH. Mutat. Res.？2000, 452, 73–81, doi:10.1016/S0027-5107(00)00057-9.
[38]
Machikhin, A.S.; Pozhar, V.E. Method for correcting spectral distortions for spectrometer images. Instr. Exp. Techn.？2009, 6, 92–98.
[39]
Lee, C.; Gisselsson, D.; Jin, C.; Nordgren, A.; Ferguson, D.O.; Blennow, E.; Fletcher, J.A.; Morton, C.C. Limitations of chromosome classification by multicolor karyotyping. Am. J. Hum. Genet.？2001, 68, 1043–1047, doi:10.1086/319503.
[40]
Jossart, G.H.; Greulich, K.M.; Siperstein, A.E.; Duh, Q.; Clark, O.H.; Weier, H.U. Molecular and cytogenetic characterization of a t(1;10;21) translocation in the human papillary thyroid cancer cell line TPC-1 expressing the ret/H4 chimeric transcript. Surgery？1995, 118, 1018–1023, doi:10.1016/S0039-6060(05)80108-4.
[41]
Stampfer, M.R.; Bartley, J.C. Induction of transformation and continuous cell lines from normal human mammary epithelial cells after exposure to benzo[a]pyrene. Proc. Natl. Acad. Sci. USA？1985, 82, 2394–2398, doi:10.1073/pnas.82.8.2394.
[42]
Ishizaka, Y.; Itoh, F.; Tahira, T.; Ikeda, I.; Ogura, T.; Sugimura, T.; Nagao, M. Presence of aberrant transcripts of ret proto-oncogene in a human papillary thyroid carcinoma cell line. Jpn. J. Cancer Res.？1989, 80, 1149–1152, doi:10.1111/j.1349-7006.1989.tb01645.x.
[43]
Jossart, G. H.; O'Brien, B.; Cheng, J.F.; Tong, Q.; Jhiang, S.M.; Duh, Q.; Clark, O.H.; Weier, H.U. A novel multicolor hybridization scheme applied to localization of a transcribed sequence (D10S170/H4) and deletion mapping in the thyroid cancer cell line TPC-1. Cytogenet. Cell Genet.？1996, 75, 254–257, doi:10.1159/000134495.
[44]
Zitzelsberger, H.F.; O’Brien, B.; Weier, H.-U.G. Multicolor FISH techniques for the detection of inter- and intrachromosomal rearrangements. In FISH Technology; Rautenstrauss, B., Liehr, T., Eds.; Springer Verlag: Heidelberg, Germany, 2002; pp. 408–424.
