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2.1 INTRODUCTION

Advances in understanding the biology of cancer have transformed the field of oncology. Although genetic analysis was limited previously to gross chromosomal abnormalities in karyotypes, DNA and RNA in cells can now be analyzed at the individual base-pair level, and there is increasing knowledge about the structure and interactions of proteins. This intricate knowledge of the molecular properties of cancer increases the possibility that personalized treatment for individual cancers might be achieved. To appreciate the relevance and nature of these technological advances, as well as their implications for function, an understanding of the modern tools of molecular biology is essential. This chapter reviews the cytogenetic, nucleic, proteomic, and bioinformatics methods used to study the molecular basis of cancer, and highlights methods that are likely to affect its future management.

2.2 PRINCIPAL TECHNIQUES FOR NUCLEIC ACID ANALYSIS

2.2.1 Cytogenetics and Karyotyping

Cancer is thought to arise from a stepwise accumulation of genetic changes that confer a selective growth advantage to the involved cells. These changes may consist of abnormalities in specific genes (such as amplification of oncogenes or deletion of tumor-suppressor genes). Although molecular techniques can identify specific DNA mutations, cytogenetics provides an overall description of chromosome number, structure, and the extent and nature of chromosomal abnormalities.

Several techniques can be used to obtain tumor cells for cytogenetic analysis. Biopsies of leukemias and lymphomas from peripheral blood, bone marrow, or lymph node are easily dispersed into single cells suitable for chromosomal analysis. Cytogenetic analysis of solid tumors is more challenging. The cells are tightly adherent and must be dispersed by mechanical means and/or by digestion with proteolytic enzymes (eg, trypsin, collagenase), which can damage cells. Second, the mitotic index in solid tumors is often low, making it difficult to find enough metaphase cells to obtain good-quality cytogenetic preparations. Finally, lymphoid, myeloid, and other (normal) cells infiltrate solid tumors and may be confused with the malignant cell population.

Chromosomes are usually examined in metaphase, when they become condensed and appear as 2 identical sister chromatids held together at the centromere, as DNA replication has already occurred at that stage of mitosis. Exposure of the tumor cells to agents such as colcemid arrests them in metaphase by disrupting the mitotic spindle fibers that normally separate the chromatids. The cells are then swollen in a hypotonic solution, fixed in methanol-acetic acid, and metaphase “spreads” are prepared by physically dropping the fixed cells onto glass microscope slides.

Chromosomes can be recognized by their size and shape and by the pattern of light and dark “bands” observed after specific staining. The usual way of generating banded chromosomes is proteolytic digestion with trypsin, followed by a Giemsa stain. A typical metaphase spread prepared using conventional methods has approximately 550 bands, whereas cells spread at prophase can have more than 800 bands; these bands can be ...

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