• Identify checkpoints in the cell division cycle that are critical for regulated cell proliferation.

• List molecular targets that are useful for diagnosisng and monitoring solid tumors.

• Explain how microsatellite instability is detected.

• Describe loss of heterozygosity and its detection.

• Contrast cell-specific and tumor-specific molecular targets.

• Show how clonality is detected using antibody and T-cell receptor gene rearrangements.

• Describe translocations associated with hematological malignancies that can be used for molecular testing.

• Interpret data obtained from the molecular analysis of patients' cells, and determine if a tumor population is present.

Oncology is the study of tumors. A tumor, or neoplasm, is a growth of tissue that exceeds and is not coordinated with normal tissue. Tumors are either benign (not recurrent) or malignant (invasive and tending to recur at multiple sites). Cancer is a term that includes all malignant tumors. Molecular oncology is the study of cancer at the molecular level, using techniques that allow direct detection of genetic alterations, down to single base pair changes.

Classification of Neoplasms

Cancer is generally divided into two broad groups, solid tumors and hematological malignancies. Solid tumors are designated according to the tissue of origin as carcinomas (epithelial) or sarcomas (bone, cartilage, muscle, blood vessels, fat). Teratocarcinomas consist of multiple cell types. Benign tumors are named by adding the suffix "-oma" directly to the tissue of origin. For example, an adenoma is a benign glandular growth. An adeno-carcinoma is malignant.

Metastasis is the movement of dislodged tumor cells from the original (primary) site to other locations. Only malignant tumors are metastatic. No one characteristic of the primary tumor predicts the likelihood of metastasis. Both tumor and normal cell factors are involved. The presence of metastasis increases the difficulty of treatment. Clinical analysis may be performed to detect the presence of relocated or circulating metastasized cells to aid in treatment strategy.

With regard to hematological malignancies, tumors arising from white blood cells are referred to as leukemias and lymphomas. Leukemia is a neoplastic disease of blood-forming tissue in which large numbers of white blood cells populate the bone marrow and peripheral blood. Lymphoma is a neoplasm of lymphocytes that forms discrete tissue masses. The difference between these diseases is not clear, as lymphocytic leukemias and lymphomas can display bone marrow and blood symptoms similar to those of leukemias. Furthermore, chronic lymphomas can progress to leukemia. Conversely, leuke-mias can display lymphomatous masses without overpopulation of cells in the bone marrow.

Within lymphomas, Hodgkin's disease is a histologi-cally and clinically different disease than all other types of lymphoma, termed non-Hodgkin's lymphoma (NHL). Plasma cell neoplasms, which arise from terminally differentiated B cells, are also classified in a separate cate-

Advanced Concepts

As imaging technology advanced, several efforts were made for classification of NHL. The earliest was the Rappaport classification in 1966, developed at the Armed Forces Institute of Pathology. The Keil classification, used in Europe, and the Lukes and Collins classification, used in the United States, were proposed in 1974. In 1982 an international group of hematopathologists proposed the Working Formulation for Clinical Usage for classification of NHL.139 The Working Formulation was revised in 1994 by the International Lymphoma Study Group, which proposed the World Health Organization/ Revised European-American Classification of Lym-phoid Neoplasms (REAL). The REAL classification included genetic characteristics in addition to morphological tissue architecture. With increasing ability to detect molecular characteristics of cells, including patterns of gene expression, classification will continue to evolve.

gory. Some of the physiological symptoms of plasma cell tumors are related to the secretion of immunoglobulin fragments by these tumors.

Molecular Basis of Cancer

Cancer is caused by nonlethal mutations in DNA. The mutations affect two types of genes: oncogenes and tumor suppressor genes. These genes control the cell division cycle (Fig. 14-1).

Oncogenes promote cell division. Oncogenes include cell membrane receptors that are bound by growth factors, hormones, and other extracellular signals. These receptors transduce signals through the cell membrane into the cytoplasm through a series of protein modifications that ultimately reach the nucleus and activate factors that initiate DNA synthesis (move the cell from G1 to S phase of the cell cycle) or mitosis (move from G2 to M). Oncogenes also support cell survival by inhibiting apoptosis, or self-directed cell death. More than 100 oncogenes have been identified in the human genome.

Tumor suppressors slow down or stop cell division. Tumor suppressors include factors that control transcrip

■ Figure 14-1 The cell division cycle. After mitosis (M), there are two haploid (one diploid) complements of chromosomes (46 chromosomes) in the G1 phase of the cell division cycle. DNA is replicated during the S phase, resulting in four haploid (two diploid) complements in the G2 phase. The chromosomes are distributed to two daughter cells at mitosis each receiving 46 chromosomes. Cancer results when the cell division cycle proceeds from G1 to S or G2 to M phase inappropriately.

tion, or translation of genes required for cell division. Tumor suppressors also participate in repairing DNA damage and in promoting apoptosis. Tumor suppressors counteract the movement of the cell from G1 to S or G2 to M phase. These two points are therefore referred to as the G1 checkpoint and G2 checkpoint in the cell division cycle. More than 30 tumor suppressor genes have been identified.

In cancer cells, mutations in oncogenes are usually gain of function mutations, resulting from amplification or translocation of DNA regions containing the genes or activating mutations that cause aberrant activity of the proteins. Mutations in tumor suppressor genes are usually loss of function mutations, resulting in inactivation of the tumor suppressor gene products. These mutations may occur through deletion, translocation, or mutation of the genes. Molecular laboratory testing aids in the diagnosis and treatment strategies for tumors by detecting abnormalities in specific tumor suppressors or oncogenes.

Analytical Targets of Molecular Testing

Although not yet completely standardized, several tests are performed in almost every molecular pathology labo ratory. These include tests that target tissue-specific or tumor-specific genes. Tissue-specific targets are molecular characteristics of the type of tissue from which a tumor arose. The presence of DNA or RNA from these targets in abnormal amounts or locations is used to detect and monitor the presence of the tumor. For example, molecular tests are designed to detect DNA or RNA from cytokeritin genes in gastric cancer, carci-noembryonic antigen in breast cancer, and rearranged immunoglobulin or T-cell receptor genes in lymphoma. Although tissue-specific markers are useful, they are also expressed by normal cells, and their presence does not always prove the presence of cancer.

In contrast, tumor-specific genes are not expressed in normal cells and are, therefore, more definitive with respect to the presence of tumor. Tumor-specific genetic structures result from genome, chromosomal, or gene abnormalities in oncogenes and tumor suppressor genes that are associated with the development of the tumor. Gene mutations and chromosomal translocations are found in solid tumors, leukemias, and lymphomas. Genome mutations, or aneuploidy, result in part from the loss of coordinated DNA synthesis and cell division that occurs when tumor suppressors or oncogenes are dysfunctional. Research is ongoing to find more of these tumor-specific markers to improve molecular oncology testing.

The following sections describe procedures most commonly performed in molecular pathology laboratories; however, due to the rapid advances in this area, the descriptions cannot be all-inclusive. The discussion is divided into solid tumor testing and testing for hemato-logical malignancies. As will be apparent, however, some tests are applicable to both types of malignancies.

Gene and Chromosomal Mutations in Solid Tumors

A number of tests are routinely performed to aid in the diagnosis, characterization, and monitoring of solid tumors. Some of these tests have been part of molecular pathology for many years. Others are relatively new to the clinical laboratory. The methods applied to detect molecular characteristics of tumors are described in Chapters 6-10 of this text.

• Human Epidermal Growth Factor Receptor 2, HER2/neu/erb-b2 1 (17q21.1)

HER2/neu was discovered in rat neuro/glioblastoma cell lines in 1985.1 Later it was found to be the same gene as the avian erythroblastic leukemia viral oncogene homolog 2, or ERBB2, which codes for a 185-kd cell membrane protein that adds phosphate groups to tyrosines on itself and other proteins (tyrosine kinase activity). This receptor is one of several transmembrane proteins with tyrosine kinase activity (Fig. 14-2). It is very similar to a family of epidermal growth factor receptors that are over-expressed in some cancers2 (Fig. 14-3).

In normal cells, this protein is required for cells to grow and divide. HER2/neu is overexpressed in 25%-30% of human breast cancers, in which overexpression of HER2/neu is a predictor of a more aggressive growth and metastasis of the tumor cells. It is also an indication for use of anti-HER2/neu antibody drug, Herceptin (tras-tuzumab) therapy, which works best on tumors overex-pressing HER2/neu. Herceptin therapy is indicated presently for women with HER2/neu-positive (HER2/neu overexpressed) breast cancer that has spread to lymph nodes or other organs.

Of all testing for the overexpression of the HER2/neu oncogene, 80%-95% is performed by immunohisto-chemistry (IHC) using monoclonal and polyclonal antibodies to detect the HER2/neu protein. The HercepTest was developed by Dako and Genentech as a method to define conditions for performance and interpretation of

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■ Figure 14-2 Receptor tyrosine kinases include epidermal growth factor receptor (EGFR), insulin growth factor receptor (IGFR), nerve growth factor receptor (NGFR), platelet-derived growth factor receptor (PDGFR), fibroblast growth factor receptor (FGFR), vascular endothelial growth factor receptor (VEGFR), and ephrin receptor (EPHR). These molecules share similarities in that they include a kinase domain, transmembrane domain, cysteine-rich domain, and immunoglobulin-like domain. The EPH receptor has two fibronectin type III domains.

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