Laser Microdissection for Gene Expression Study of Hepatocellular Carcinomas Arising in Cirrhotic and Non-Cirrhotic Livers

  • Maria TretiakovaEmail author
  • John Hart
Part of the Methods in Molecular Biology book series (MIMB, volume 755)


Laser microdissection (LMD) is a robust well-established technology for the isolation of chosen cell populations from surrounding tissues and cells. This technique is particularly useful to minimize bias inherent in the molecular analysis of highly heterogeneous whole tissue sections. The aim of this study was to identify the pattern of mRNA expression in hepatocellular carcinoma (HCC) arising in cirrhotic liver and compare it to the pattern of expression in HCC arising from non-cirrhotic liver. The expression profiles of the tumors were also compared to that of the surrounding liver (either cirrhotic or non-cirrhotic) from the same patient. In addition, the expression pattern of each of the four tissues were compared to normal hepatic tissue. Samples of HCC tissue and surrounding cirrhotic or non-cirrhotic parenchyma were collected at the time of resection or liver transplantation. The samples were snap frozen and stored at −80°C. The snap frozen samples were then cryosectioned and stained with hematoxylin and eosin for LMD. Hepatocytes from each sample were collected using the Leica LMD instrument. The RNA was extracted according to standard methodology and amplified. Microarray analysis was performed using the Affymetrix human genome array platform. The resulting microarray data were analyzed using Affymetrix Microarray Suite 5.0 (MAS 5.0). Results were displayed using Genespring, dChip, SAM, and GenMapp/MAPP Finder software. Validation studies on selected genes and proteins were performed utilizing RT-PCR and immunohistologic techniques.

Key words

Hepatocellular carcinoma Cirrhotic Non-cirrhotic Laser microdissection Gene expression 



We cordially thank Dr. Xinmin Li and Jian Zhou from the Functional Genomics Facility (University of Chicago) for their help in performing the Affymetrix Microarray experiments.


  1. 1.
    Parkin, D. M., Bray, F., Ferlay, J., and Pisani, P. (2005) Global cancer statistics, 2002. CA Cancer. J. Clin. 55, 74–108.Google Scholar
  2. 2.
    Coleman, W. B. (2003) Mechanisms of human hepatocarcinogenesis. Curr. Mol. Med. 3, 573–588.CrossRefGoogle Scholar
  3. 3.
    Feitelson, M. A., Sun, B., Satiroglu Tufan, N. L., Liu, J., Pan, J., and Lian, Z. (2002) Genetic mechanisms of hepatocarcinogenesis. Oncogene. 21, 2593–2604.CrossRefGoogle Scholar
  4. 4.
    Chang, C. H., Chau, G. Y., Lui, W. Y., Tsay, S. H., King, K. L., and Wu, C. W. (2004) Long-term results of hepatic resection for hepatocellular carcinoma originating from the noncirrhotic liver. Arch. Surg. 139, 320–5; discussion 326.Google Scholar
  5. 5.
    Chen, M. F., Tsai, H. P., Jeng, L. B., Lee, W. C., Yeh, C. N., Yu, M. C., and Hung, C. M. (2003) Prognostic factors after resection for hepatocellular carcinoma in noncirrhotic livers: univariate and multivariate analysis. World J. Surg. 27, 443–447.CrossRefGoogle Scholar
  6. 6.
    Nzeako, U. C., Goodman, Z. D., and Ishak, K. G. (1995) Comparison of tumor pathology with duration of survival of North American patients with hepatocellular carcinoma. Cancer. 76, 579–588.CrossRefGoogle Scholar
  7. 7.
    Nzeako, U. C., Goodman, Z. D., and Ishak, K. G. (1996) Hepatocellular carcinoma in cirrhotic and noncirrhotic livers. a clinico-histopathologic study of 804 North American patients. Am. J. Clin. Pathol. 105, 65–75.Google Scholar
  8. 8.
    Ho, M. K., Lee, J. M., Chan, C. K., and Ng, I. O. (2003) Allelic alterations in nontumorous liver tissues and corresponding hepatocellular carcinomas from Chinese patients. Hum. Pathol. 34, 699–705.CrossRefGoogle Scholar
  9. 9.
    Kim, G. J., Cho, S. J., Won, N. H., Sung, J. M., Kim, H., Chun, Y. H., and Park, S. H. (2003) Genomic Imbalances in Korean hepatocellular carcinoma. Cancer Genet. Cytogenet. 142, 129–133.CrossRefGoogle Scholar
  10. 10.
    Wong, N., Lai, P., Lee, S. W., Fan, S., Pang, E., Liew, C. T., Sheng, Z., Lau, J. W., and Johnson, P. J. (1999) Assessment of genetic changes in hepatocellular carcinoma by comparative genomic hybridization analysis: relationship to disease stage, tumor size, and cirrhosis. Am. J. Pathol. 154, 37–43.CrossRefGoogle Scholar
  11. 11.
    Iizuka, N., Oka, M., Yamada-Okabe, H., Mori, N., Tamesa, T., Okada, T., Takemoto, N., Hashimoto, K., Tangoku, A., Hamada, K., Nakayama, H., Miyamoto, T., Uchimura, S., and Hamamoto, Y. (2003) Differential gene expression in distinct virologic types of hepatocellular carcinoma: association with liver cirrhosis. Oncogene. 22, 3007–3014.CrossRefGoogle Scholar
  12. 12.
    Irizarry, R. A., Hobbs, B., Collin, F., Beazer-Barclay, Y. D., Antonellis, K. J., Scherf, U., and Speed, T. P. (2003) Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics. 4, 249–264.CrossRefGoogle Scholar
  13. 13.
    Bolstad, B. M., Irizarry, R. A., Astrand, M., and Speed, T. P. (2003) A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics. 19, 185–193.CrossRefGoogle Scholar
  14. 14.
    Tretiakova, M. S., Hart, J., Shabani-Rad, M. T., Zhang, J., and Gao, Z. H. (2009) Distinction of hepatocellular adenoma from hepatocellular carcinoma with and without cirrhosis using e-cadherin and matrix metalloproteinase immunohistochemistry. Mod. Pathol. 22, 1113–1120.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Department of PathologyUniversity of ChicagoChicagoUSA

Personalised recommendations