Cytometry and DNA Ploidy: Clinical Uses and Molecular Perspective in Gastric and Lung Cancer VITTORIO D’URSO, 1,2 ANGELO COLLODORO, 3 ELISEO MATTIOLI, 4 ANTONIO GIORDANO, 2,4,5 AND LUIGI BAGELLA 1,5 * 1 Division of Biochemistry and Biophysics, Department of Biomedical Sciences, National Institute of Biostructures and Biosystems, University of Sassari, Sassari, Italy 2 Flow Cytometry Laboratory, CROM Cancer Research Center, Mercogliano (AV), Italy 3 Institute of Respiratory Disease, University of Siena, Siena, Italy 4 Department of Human Pathology and Oncology, University of Siena, Siena, Italy 5 Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, Temple University, Philadelphia Flow cytometry is one of the most powerful and specific methods used for the integrated study of the molecular and morphological events occurring during cell proliferation. Many methods have been described for investigating this process. Several cell cycle regulators controlling the correct entry and progression through the cell cycle are altered in tumors. In fact, in most, if not all, human cancers there is a deregulated control of G1 phase progression, the period when cells decide if they will start proliferation or stay quiescent. Cytometry (flow and image) is able to analyze DNA content thanks to the use of the same ‘‘molecule’’ conjugates with a fluorochrome that permits to identify DNA content of single cell in a sample. Most important results of studies on DNA ploidy have been reviewed during the last years and as a result the analyses of DNA ploidy in cancer may provide clinically useful information on diagnostic, therapeutic and prognostic aspects. In fact, aneuploid cancer has a high proliferative activity and a metastatic or invasive potential, markers of a poor prognosis. Multiparametric flow cytometry should allow the simultaneous determination of morphology, phenotype, intracellular protein expression, and status of chromatin and DNA. Evaluating if a particular protein is responsible for the aggressiveness of cancer, or the alteration of DNA content, or if the activation of its state is the cause of rapid growth of cancer cells, is very important and it can facilitate the clinical treatment of patients. J. Cell. Physiol. 222: 532–539, 2010. ß 2009 Wiley-Liss, Inc. The use of fluorescence activated cell sorting (FACS) instruments and methods for clinical purposes started almost at the same time of its introduction (Tung et al., 2007). However, the widespread application of FACS in clinical research and practice really began with the development of monoclonal antibodies that recognized surface proteins or other markers able to identify and differentiate functional subsets of peripheral blood lymphocytes (Tung et al., 2007). FACS analysis has become a standard tool used in the analysis of cell cycle distributions in populations of cells (Schorl and Sedivy, 2007). DNA content is the most significant cellular parameter able to monitor the cell proliferation and the neoplastic transformation. Flow cytometry contributed in the quantitative measurement of DNA. DNA histogram distribution of thousands of cells gives significative information on both S-phase fraction and presence of aneuploid subpopulations. Uncontrolled proliferation is a hallmark of cancer cells’ presence (Fig. 1). Many cell cycle regulators controlling the correct entry and progression through the cell cycle are altered in tumors. In fact, in most, if not all, human cancers a deregulated control of G1 phase progression it is shown, when cells choose between proliferation and quiescence (Golias et al., 2004). Clinical and biological events of cancer growth are results of mutations; in particular, DNA mutations are frequent in solid tumors. In cancer cells of solid tumor is often visible an alteration of DNA content, first cause of genetic alteration (chromosomic and/or sub-chromosomic), fundamental for the development of the disease. There are many clinical evidences on the correlation between DNA content alteration and poor prognosis. Moreover, recent clinical studies focused on the rapid growth of tumors, determined by fraction of proliferant cancer cells relative to normal cells. These facts are less evident in ematologic neoplasm, where cytogenetic alterations are less widespread, in comparison to solid tumors: this is the reason why, in the last years, DNA analysis by FCM has been used for the study of solid neoplasm. Deviation from diploidy is caused by an alteration in number or in structure of the chromosome. Alterations in chromosome’s number are the aneuploidy with loss (ipodiploidy) or with more genetic material (hyperdiploidy), which creates monosomic or polisomic forms. The nuclear DNA content of cells can be measured at high speed by flow cytometry. The principle is that the stained material has incorporated an amount of dye (such as propidium *Correspondence to: Luigi Bagella, Division of Biochemistry and Biophysics, Department of Biomedical Sciences, Medical School, University of Sassari, Viale San Pietro, 43/b, 07100 Sassari, Italy. E-mail: lbagella@uniss.it Received 20 October 2009; Accepted 21 October 2009 Published online in Wiley InterScience (www.interscience.wiley.com.), 17 December 2009. DOI: 10.1002/jcp.21991 REVIEW ARTICLE 532 Journal of Journal of Cellular Physiology Cellular Physiology ß 2009 WILEY-LISS, INC.