Clinical applications of tumor suppressor genes and oncogenes in cancer

ELSEVIER

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Clinical applications of tumor suppressor genes and oncogenes in cancer Eleftherios P. D i a m a n d i s ~ Department qf Clinical Biochemistry, University of Toronto, 100 College Street, Toronto, Ontario, Canada M5G IL5 Received 27 October 1995: revised 22 January 1996; accepted l0 February 1996

Abstract

In the search for new ways to better diagnose and monitor cancer, scientists have turned to oncogenes and tumor suppressor genes. These genes are involved in cell differentiation, communication and proliferation and their alteration is frequently associated with cancer. Such alterations include mutations, translocations, amplifications and deletions. In this review, I give examples of using the detection of such alterations for patient diagnosis and monitoring. The practical examples are restricted to a few cancer types, but the identification of new tumor suppressor genes, like BRCA-1 and BRCA-2, is creating new possibilities for determining cancer risk of individual family members. There is no doubt that the cloning of new genes which predispose to sporadic cancer will lead to the introduction of widespread testing to assess risk and to the application of preventive measures. Copyright © 1997 Elsevier Science B.V. Keywords: Oncogenes; T u m o r suppressors; Cancer diagnosis; Tumor markers: BRCA-I: BRCA-2

1. Introduction

Cancer is one of the leading causes of mortality and morbidity. Early detection of cancer is believed to contribute to better treatment and thereby to the prolongation of a patient's life. One of the goals of physicians and t Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, 600 University Ave., Toronto, Ontario, Canada M5G 1X5. Tel.: + 1 416 5868443; fax: + 1 416 5868628. 0009-8981/97/$17.00 Copyright © 1997 Elsevier Science B.V. All rights reserved PII S0009-8981 (96)06442-X

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scientists is to identify new biochemical testing for cancer screening, diagnosis, monitoring and prognosis. The investigation of oncogenes and tumor suppressor genes has enjoyed explosive growth over the last 10 years. In this presentation I will examine how oncogenes and tumor suppressor genes may constitute the basis of new biochemical testing for cancer. Cancer is now regarded as a genetic disease [1]. Many genes have been found to be defective in cancer. Genetic alterations include translocations, amplifications, mutations, deletions or abnormal gene regulation. These alterations usually involve genes that are classified as oncogenes or tumor suppressor genes. Do oncogenes and tumor suppressor genes have the potential to become the tumor markers of the future? In the following pages I review the current use and propose the future potential of selected oncogenes and tumor suppressor genes.

2. Oncogenes and tumor suppressor genes

Normal cells grow, divide, communicate and differentiate in a coordinated fashion, all accomplished by the programmed expression of many different genes. It is currently believed that tumor formation arises as a consequence of alterations in genes that are involved in the control of cell proliferation. By definition, an oncogene is a gene whose abnormal expression or altered gene product leads to malignant transformation. In the healthy state, such genes should not exist in the human genome. Oncogenes represent either normal genes that have been altered or exogenous genetic material [2]. Normal cellular genes that can give rise to oncogenes are called protooncogenes. A proto-oncogene can be transformed to an oncogene by a genetic process called 'oncogene activation'. More recently, sets of genes have been found that are negative regulators of cell growth. These genes are now collectively called 'tumor suppressor genes' or 'antioncogenes', but the terms are not precisely descriptive of their functions. It is believed that loss or inactivation of tumor suppressor genes will free cells from negative growth signals and may also lead to malignant transformation. There is, however, an important distinction between the activation process of oncogenes and tumor suppressor genes. Each cell has not one but two genes of any kind. Oncogenes are considered 'dominant' genes because they can transform cells despite the simultaneous expression of the other normal allele. An oncogene is thus a promoter of cell growth deregulation and this promotion is not affected by the presence of the normal allele.

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Tumor suppressor genes are considered 'recessive' genes because the loss or inactivation of one allele does not, at least in theory, lead to any change in function of the cell. The other normal allele will still produce the physiological product that negatively regulates the growth of the cell. Whereas the recessive nature of tumor suppressor genes is widely accepted, there are some important exceptions. When the heterozygous tumor suppressor gene locus is involved in carcinogenesis, the mutation is called 'dominant negative'. Dominant negative mutations have been explained by different mechanisms. For example, the mutant protein may inactivate the wild-type protein through complexation, the wild-type protein may be produced at levels below a threshold level necessary for negative regulation, or the mutant product may acquire oncogenic properties and act like an oncogene. It was known for many years that acutely transforming retroviruses (ATR) are able to transform cells in culture within days and induce tumors in animals with latency periods of 2-8 weeks. Intensive studies with these RNA tumor viruses revealed that they carry specific genes that cause tumorigenesis. These genes were called v-onc, standing for viral oncogenes. A major breakthrough occurred in 1976 when it was found that v-onc share extensive homology to host genes, the cellular proto-oncogenes. These observations led to the concept that normal cells contain genes responsible for cell growth and differentiation (proto-oncogenes). These proto-oncogenes can be activated to oncogenes (c-onc) by various mechanisms including mutation and translocation. Proto-oncogenes can also be taken up by retroviruses and become transforming oncogenes (v-onc) after extensive mutations. The realization that the retroviral oncogenes have cellular homologs led to the identification of many cellular proto-oncogenes by studying the retroviral genes. The genes responsible for the tumorigenic potential of DNA tumor viruses have no apparent relation to cellular genes. Gene products of DNA tumor viruses are also believed to be involved in the control of cell proliferation. The emerging picture related to the pathogenesis of cancer is that this disease is the outcome of multiple genetic abnormalities occurring and accumulating over long periods. This process includes activation of oncogenes and inactivation of tumor suppressor genes.

3. Functions of proto-oncogenes, oncogenes and tumor suppressor genes

Proto-oncogene products are involved in pathways related to cell division and differentiation. Many proto-oncogenes have been found to be highly conserved through evolution and must play a central role in cell life. Most

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of the currently known proto-oncogenes and oncogenes have been sequenced, and their respective mRNA and protein products have been characterized. It is now clear that proto-oncogene products are part of the cellular signalling network acting at multiple levels. For example, they act on the cell surface as ligands and growth factors, on the cell membrane as receptors or signal transducers, in the cytoplasm as communication links between the cell membrane and nucleus and in the nucleus itself where they are involved in gene transcription. Oncogene products presumably affect these same proto-oncogene pathways but in an aberrant fashion. Oncogenes are usually classified on the basis of the function of their products. These include growth factors (e.g. v-sis which is homologous to platelet derived growth factor), receptor and non-receptor protein-tyrosine kinases (e.g. the epidermal growth factor receptor and bcr/abl oncogene, respectively), receptors lacking protein kinase activity (e.g. mas, the angiotensin receptor), membrane associated G proteins (e.g. the ras family), cytoplasmic proteinserine kinases (e.g. the raf/mil family) cytoplasmic regulators (e.g. the crk oncogene) and nuclear transcription factors (e.g. myc, fos, jun, ets and many others). For more information please see reference [3]. Among the best characterized tumor suppressor genes are Rb (retinoblastoma), p53, DCC (deleted in colon carcinoma), NF-1 (neurofibromatosis), WT (Wilm's tumor) and BRCA-1 (breast/ovarian cancer gene). Most of them are putative transcription factors. Their protein products are repressers of cellular proliferation. Once these genes are lost, usually through mutations and deletions, cellular proliferation escapes the negative control and this may lead to tumorigenesis. More details in the functions of some clinically important tumor suppressor genes are given in specific paragraphs of this review.

4. Genetic changes that lead to oncogene activation or loss of tumor suppression

4.1. Chromosomal rearrangements

Movement of genetic material from its normal position to another (translocation) has been consistently observed in a number of human malignancies (Table 1). In 80% of Burkitt's lymphoma patients, the c-myc locus on chromosomes 8 (8q24) is translocated to a locus on chromosome 14 (14q32). This translocation repositions c-myc close to the immunoglobulin heavy-chain gene locus. In the remaining 20% of patients, the c-myc gene remains on chromosome 8, but the loci of k-light chain gene (5% of cases) or 2-light chain gene (15% of cases) translocate from chromosomes 2 and

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22, respectively, to a locus adjacent to c - m y c on chromosome 8. The involvement o f c - m y c in the pathogenesis of Burkitt's l y m p h o m a is very likely, although the exact mechanisms of c - m y c activation due to the translocations is not fully understood. It appears that the translocation triggers the constitutive, uncontrolled expression of c-myc, which gives rise to the lymphoma. The Philadelphia chromosome (Ph) is found in the karyotype of malignant cells in at least 90% of patients with chronic myelogenous leukemia (CML) and in 10-15% o f patients with acute lymphocytic leukemia (ALL). Ph is formed from a translocation of genetic material between chromosomes 9 and 22, which causes chromosome 22 to become shorter (this is the Ph chromosome) and chromosome 9 to become longer [4]. The translocation brings the proto-oncogene c-abl from its normal position on chromosome 9 to an area on chromosome 22 that is called bcr (breakpoint cluster region). The juxtaposed bcr and abl genes produce an 8.5 kb bcr-abl hybrid m R N A that encodes a novel protein having a molecular weight of 210 kDa. This protein is considered the etiologic factor of CML. Diagnostic tests for the Ph-positive C M L (and ALL, see later in this review) are based on either D N A rearrangement studies at the bcr locus, detection of the 8.5-kb bcr-abl m R N A , or detection of the 210-kDa protein.

Table 1 Chromosomal translocations in human malignancies Gene locus Human neoplasm

c-myc

Burkitt's lymphoma

bcr-abl

Chronic myelogenous leukemia Acute lymphocytic leukemia Chronic lymphocytic leukemia of B cell type Follicular lymphoma

bcl- 1 bcl-2

Percentage of tumors with translocation of gene rearrangement

Chromosome translocation

80 15 5 90-95

t(8; 14)(q24;q32) t(8;22)q34;ql 1) t(2;8)(q 11;q24) t(9;22)(q34;q 11)

10-20

t(11;14)(q13;q32)

85-95

t(14;18)(q32;q21)

From Park M, Vande Woude GF. Cancer. Principles and Practice of Oncology. Pp 45 6. Philadellphia: J.B. Lippincott, 1989. With permission.

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Table 2 Cellular oncogenes amplified in human tumors Tumor

Oncogene

Amplification

Small-cell lung cancer

c-myc N-myc L-myc N-myc c-erb-B (EGFR)

Up Up Up Up Up Up

Neuroblastoma Glioblastoma Mammary carcinoma

c-erb-B2 (HER2)

to to to to to to

80X 50X 20X 250X 50X 30X

From Park M, Vande Woude GF. Cancer. Principles and Practice of Oncology. Pp. 45-66. Philadelphia: J.B. Lippincott, 1989. With permission.

4.2. Gene amplifications

Cytogenetically visible differences between normal and cancer cells, known as double minutes (DM), and homogeneously staining regions (HSR) have been known for many years. It is now known that DM and HSR contain multiple copies of genes (amplified genes) that are represented by only one copy in the normal human genome. Gene amplification is a common finding in cell lines derived from tumors. The amplified genes are frequently oncogenes (Table 2). Gene amplification is usually a predictor of poor clinical prognosis. It is postulated that the multiple gene copies cause abnormally increased levels of m R N A and protein, leading to the initiation and progression of the malignant phenotype. In many cases, the sequences of normal and amplified genes have been determined. In general, differences were present, suggesting that the oncogene activation in these cases is associated with overproduction of a normal protein. 4.3. Mutations and deletions

A single point mutation at specific codons of the ras gene family is enough to activate this oncogene. It has been shown that such mutations can markedly decrease the GTPase activity of ras, which is then left in the activated form. This condition may induce tumorigenesis. In the case of other genes, a point mutation may lead to loss of the physiological function. For example, a gene may acquire a point mutation which blocks its ability to produce a normal protein (e.g. by premature termination of transcription). Alternatively, the gene may encode a mutant protein which is physiologically inactive. The loss of a normal gene product may be critical in initiating tumorigenesis especially when the encoded protein is a negative regulator of the cell cycle. Loss of function leading to tumorigenesis is

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characteristic of genes known as tumor suppressors [5-8]. In order for these genes to be completely inactivated (homozygous state) it is necessary for the cell to lose both alleles either by a process involving two mutations or by mutation of one allele and deletion of the other. Other reasons for complete loss of a gene function may be due to one mutant allele product forming inactive complexes with the normal allele product, or a normal allele product forming biologically inactive complexes with other amplified oncogene products. Gene deletions are very c o m m o n in cancers and sometimes these are cytogenetically visible giving hints that tumor suppressor genes may be encoded in the deleted areas. In general, oncogenes can be activated by alteration of only one allele, whereas tumor suppressor genes are fully inactivated only after the loss of function of both alleles.

4.4. General diagnostic" strategies Oncogenes and tumor suppressor genes can be studied at the level of DNA, m R N A or protein. Once a cancer cell is generated it is assumed to contain altered genes which will give rise to altered m R N A and to altered proteins (Fig. 1). Alternatively, cancer cells lose regulation of gene expression and produce proteins of normal structure but in inappropriate amounts or at ectopic sites, or proteins which are abnormally glycosylated, or expressed only during embryonal life. How could we use such alterations for diagnosis and other clinical applications? Traditionally, we have used the

NormalCell I Genetic abnormalities in DNA

CancerCell I

I

Genetically altered mRNA Mutant proteins/Loss of physiological proteins

DIAGNOSTIC STRATEGIES Detection of cancer cells in the general circulation, lymph nodes, urine, stool, sputum, exudates, smears, etc. Detection of secreted tumor-associated proteins (e.g. oncogene or tumor suppressor gene products) in biological fluids. Detection of specific autoantibodies against tumor proteins in biological fluids. Fig. 1. G e n e r a l d i a g n o s t i c strategies in cancer.

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measurement as tumor secreted proteins which can be found in the general circulation. More recently, investigators have expanded the search to fluids like urine, exudates or CSF, and other biological specimens including feces and sputum. One major limitation of these approaches is that circulating proteins which are absolutely specific for tumors are not known. An alternative approach would be to measure antitumor antibodies which circulate in blood. A number of reports have suggested that antitumor antibodies are generated early during tumorigenesis. A third and probably the most general approach is to search for tumor cells. Obviously, for this approach to be successful we need methods which can detect one or a few tumor cells intermixed with vast amounts of normal cells. To do this, one can examine tumor-specific RNAs or altered genomic sequences. In the relatively small number of cases where cancer is familial, genetic alterations can be studied in any cell; in sporadic cancers the alterations are present only in tumor cells. Fig. 1 summarizes the possible biochemical molecular diagnostic strategies for cancer.

5. Oncogenes and tumor suppressor genes in clinical practice 5.1. The bcr-abl translocation

More than 90% of patients with chronic myelogenous leukemia (CML) carry the Ph chromosome in their leukemic cell population. The translocation brings most of the c-abl proto-oncogene from chromosome 9 to the bcr locus on chromosome 22 [9]. Expression of the fused gene gives rise to an 8.5-kb chimeric mRNA, which in turn is translated to a 210-kDa protein (p210) [10-17]. Variants that produce fusion mRNAs of a different size also exist. The normal c-abl proto-oncogene is transcribed into a 6.0 and 7.0-kb mRNA, which gives rise to a 150-kDa protein (p150). About 15(/o of patients with acute lymphocytic leukemia (ALL) also carry the Ph chromosome. In ALL, however, the fusion m R N A is shorter (7.0 kb) and gives rise to a 190-kDa protein (p190). Diagnostic tests for CML and ALL are based on the fusion of bcr and abl loci and can be performed at the level of DNA, m R N A or protein. Each of these approaches has merits and limitations. The detection limit of the test is of primary importance because it is used in patients who have undergone bone marrow transplantation or patients in remission or early stages of relapse and in these cases, the percentage of leukemic cells may be very low. At the D N A level, the translocation can be studied with probes that hybridize inside the breakpoint region of chromosome 22. This region is approximately 5.8 kb long. When the translocated gene from chromosome

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9 is inserted, changes in restriction enzyme sites create new DNA fragments which hybridize with the probe [18]. These new bands (rearrangements) can be seen on Southern blots along with the normal bands from the unaffected chromosome. Although the breakpoint regions on chromosomes 9 and 22 are variable, they occur only in introns and therefore produce only a small number of mature mRNAs. These contain junctions between exon II of abl and either exon 3, exon 2, or the 5'-exon of bcr, and give rise to proteins of varying molecular weight, between 210 and 190 kDa. The normal abl encodes a protein of 140 to 150 kDa. Normal and abnormal proteins can be quantified by using metabolic labeling with 32p-phosphate followed by immunoprecipitation with antibodies, agarose gel electrophoresis and autoradiography. Alternatively, labeling with 32p can be achieved by using the kinase activity of the protein and 32P-ATP. The availability of specific antibodies against the various protein products of the fusion genes can also permit the assay of the proteins using immunoassay. The analysis of specific mRNAs can also be used as diagnostic tests for CML and AML. Northern blot analysis reveals the abnormal mRNAs in CML (8.5 kb) and ALL (7.4 kb). However, more elaborate procedures for detecting the aberrant forms of m R N A in CML and ALL have been described that are based on PCR amplification [19,20]. The mRNAs are reverse-transcribed with reverse transcriptase to the corresponding cDNAs. They are then amplified with PCR using the appropriate primers. The fragments are then analyzed by agarose gel electrophoresis, Southern-transferred, and hybridized to probes that recognize only the junction sequences of bcr and abl. This method is highly specific for the chimeric mRNAs. The various possible strategies for the diagnosis of CML or ALL at the DNA, mRNA, or protein level are simple. Which method is the most advantageous? Methods based on direct assays of DNA or m R N A (Southern or Northern analysis) are straightforward. However, the PCR amplification step used in the study of mRNAs gives this method the extreme sensitivity needed for the investigation of those patients who have undergone bone marrow transplantation, or patients who are in remission or early stages of the disease. The PCR method can detect aberrant m R N A from one leukemic cell. In contrast 104 cells are needed to detect the aberrant protein product. Since both the mRNAs and the proteins originating from the fusion of bcr and abl are unique substances, not found in any normal state, they are 'true tumor markers.'

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5.2. T h e r a s o n c o g e n e s

The r a s proto-oncogenes are activated by point mutations at codons 12, 13, or 61. Of the three r a s genes, the K - r a s is mutated more frequently than the H - r a s and N - r a s . The activated oncogene has reduced GTPase activity, a property that is thought to be linked to its tumorigenic potential. R a s gene mutations are found with variable frequency in several malignancies [21-25]. The highest incidences are found in adenocarcinoma of the pancreas (90%), colon (50%), lung (30%) and in leukemias (30%). The diagnostic and prognostic value of the detection of r a s mutations is currently unclear. In most cases, the presence of the mutation does not seem to correlate with the anatomical location, depth of invasion and level of differentiation of the tumor nor does it relate to the age, or sex of the patient. The notion that the presence of the mutation is an indicator of a more invasive tumor has not been generally confirmed but does seem to hold true for lung adenocarcinoma [26]. The r a s gene mutations have been studied extensively in colon carcinoma in efforts to develop models of the initiation and progression of the disease [24,25]. Historically, r a s gene mutations were identified with biological assays using DNA from tumors, which was used to transfect NIH/3T3 cells. Although very useful, these assays are not suitable for screening large numbers of samples because they are cumbersome and slow. Currently, PCR is used to amplify specific segments of the r a s genes that contain a mutation at codon 12 or 61. The PCR product is then used as a target in dot-blot hybridizations with a labeled probe that recognizes only the mutant PCR product [27]. Specific hybridization probes have to be used to cover all the possible mutations. For example, the normal codon 12 of K - r a s is GGT (glycine). Thus, point mutations will result in the following possible combinations: GGA (gly), GGT (gly), GGC (gly), AGT (ser), TGT (cys), CGT (arg), GCT (ala), GAT (asp) and GTT (val). Probes detecting the mutations of the third nucleotide of the codon are not used because the codons GGA, GGT and GGC all code for glycine (degeneracy of genetic code). Thus, six probes are needed to cover all of the known point mutations of the K - r a s codon 12. A newly developed technique based on exponential amplification, the ligase chain reaction, may also be applicable. The necessary probes for the study of r a s point mutations are now commercially available. Mutant r a s proteins can also be detected with monoclonal antibodies [28,29]. Recently, it became possible to identify r a s oncogene mutations in the stools of patients with colorectal tumors [30]. Cancer cells carrying the mutations are shed into the stool, where they can be identified by PCR amplification and Southern or dot-blot analysis. These findings can be used for diagnosis. This approach works whenever the tumor cells carry r a s

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mutations, and this is true for 40-50% of patients. When the mutation is present, its correct identification in stool occurs with a frequency of about 90%. It is important to note that the test detects patients with adenomas which can be treated successfully if detected at an early stage in their development. In a more recent study, Tobi et al. [31] used enriched PCR in colonic effluent samples to study K - r a s gene mutations in patients who were at high risk for colorectal cancer. They identified mutations in codon 12 of K - r a s in 7/39 (40%) of subjects with either a family history of colon cancer, in patients with adenomatous polyps and in patients with previously removed colon cancer. One patient was shown to have the mutation 4 years prior to the diagnosis of colon cancer. Since 75-100% of pancreatic carcinomas contain mutated ras genes [32], investigators have sought tumor cells in the stool of pancreatic carcinoma patients. Caldas et al. [33] found K - r a s gene mutations in 6/11 patients with pancreatic adenocarcinoma and in 1/3 patients with chronic pancreatitis. Others have found ras gene mutations in the DNA isolated from cells in pancreatic juice, in 6/6 patients with pancreatic adenocarcinoma and in 2/6 patients the mutations were found in DNA from peripheral blood [34]. Unfortunately, ras gene mutations also seem to be present in DNA from non-malignant diseases such as ductal cyst adenomas [35], mucus cell hyperplasia and other benign hyperplasia [36]. Whether all these diseases are precursors of malignant disease is unclear. Mao et al. [37] examined ras and p53 gene mutations in the DNA isolated from sputum. They found mutations of at least one of the two genes in 10/15 primary lung cancer patients. In 80% of the patients the same mutation was found in the sputum and in the lung tumor. In one patient the mutation in the sputum was found 1 year prior to clinical diagnosis. Others have attempted to diagnose bladder cancer by examining H - r a s gene mutations in DNA from urine [38,39]. They found such mutations in 48% of the cases. 5.3. The m y c oncogene

In Burkitt's lymphoma, virtually 100% of the patients have translocations that involve the c - m y c proto-oncogene locus. In 80% of patients, the c - m y c is translocated from chromosome 8 to 14, in an area that is in close proximity to the immunoglobulin heavy-chain locus. The other 20% of cases involve translocations that juxtapose the K and 2 light-chain genes with c - m y c on chromosome 8 [40]. The breakpoints in the c - m y c locus are quite variable. The mechanism by which the translocations cause Burkitt's lymphoma is unknown. A close relationship seems to exist between tumorigenesis and partial deletions within regulatory elements of the first exon of

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c-myc. These deletions can now be studied by PCR. The deletions will cause the disappearance of the normal PCR product. Genetic alterations of the myc oncogene have been found in many other malignancies. For example, in breast carcinomas the c-myc was found to be amplified 2- to 15-fold in 30% of patients. Amplification and overexpression were also seen in multiple myeloma, colon and lung carcinomas and rhabdomyosarcomas [41-45]. N-myc, a gene homologous to c-myc, is amplified in tissues and cell lines from patients with neuroblastoma. About 40% of patients show amplification, sometimes up to 300-fold [45]. It has been found that myc gene amplification is a bad prognostic indicator and patients with the amplification have shorter survival rates than patients with single-copy myc genes. Gene amplification is usually associated with an increased amount of m R N A and protein product, but the protein is not mutated. Thus, the oncogenic consequences of amplification must be due to the overproduction of the normal protein molecule [46]. A recent review on myc amplification has been published [47]. 5.4. The c-erbB-2 proto-oncogene

The c-erbB-2 proto-oncogene, also known as Her-2 or neu, is a transmembrane protein that shows homology to the epidermal growth factor receptor. A number of studies have shown that this proto-oncogene is amplified in about 15-40% of primary breast and ovarian carcinomas [48-51]. The amplification leads to elevated levels of m R N A and protein. It has been suggested that amplification of this gene is associated with positive lymph node involvement and poor patient outcome (even in patients without nodal involvement) [52]. Other studies failed to confirm this association [49]. However, there is reasonable agreement that c-erbB-2 may be involved in the pathogenesis of the disease and that it may contribute to the development of distinct histologic types. The link between c-erbB-2 and pathogenesis has prompted the development of antibodies against the protein product that can be injected therapeutically in an effort to inactivate it [53]. The assessment of the c-erbB-2 status at the DNA, m R N A and protein levels seems to be important because in some tumors increased m R N A and protein can be seen without the presence of D N A amplification [48]. Amplification studies with D N A and m R N A are performed with Southern or Northern blot techniques, respectively. Protein levels are assessed with Western blotting or immunohistochemistry. More recently, an ELISA assay for c-erbB-2 protein has been developed and used to measure levels of the protein in tumors [54]. Recently, Inaji et al. [55] examined the levels of c-erbB-2 protein in nipple discharge from patients with benign and malig-

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nant lesions of the breast. Although 6/9 patients had elevated levels of the protein, 1/19 patients with fibrocystic disease also had elevated levels. This result serves to demonstrate the limitations of oncogenes as absolutely specific tumor markers. Although c-erbB-2 amplification was described almost a decade ago, the interest on this gene has not diminished [56-63].

5.5. The retinoblastoma gene

The tumor suppressor genes, one of which is related to retinoblastoma, Rb, are referred to as recessive genes because the presence of one normal allele is usually sufficient to prevent the expression of the malignant phenotype. Loss of function of the remaining normal allele, a process known as 'loss of heterozygosity,' has been observed in many tumor types, including retinoblastoma, osteosarcoma, Wilm's tumor, hepatoblastoma and rhabdomyosarcoma [8,64]. In hereditary retinoblastomas and osteosarcomas, the patient's germ cells are heterozygous for the Rb locus [65]. One somatic mutational event is needed to produce homozygosity. However, in sporadic retinoblastoma and in all other tumors in which the Rb locus is found to be homozygously alerted, the Rb inactivation at both loci is due to purely somatic events. This holds true for tumors like small-cell lung carcinoma and bladder and breast carcinomas [66,67]. Rb gene inactivation can occur at one allele as a result of loss of a chromosome, a chromosome arm, or a small band. Some of these changes may be cytogenetically visible. The loss of the other allele can occur through point mutation, insertion, translocation, or deletion. Although alterations of the Rb gene have been observed in many tumor cell lines, e.g. small-cell lung, bladder, breast carcinomas and in leukemias, studies on actual tumors are limited. In one study involving 77 breast carcinoma tumors, about 19% had structural abnormalities of the gene [67]. The gene product, a l l0-kDa nuclear phosphoprotein, was found to be absent from the cells that had the abnormal gene. Although the structural changes observed in the Rb gene have been implicated in the etiology of tumor development, no prognostic value has been attached to these findings. Simple tests for the detection of heterozygosity could aid in the identification of individuals who are predisposed to the development of retinoblastoma and other malignancies. Since most breast tumors continue to express Rb it has been suggested that Rb alteration is not an initiating event but a progressive event in breast cancer and some studies have shown that Rb is lost most frequently in larger tumors.

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A mouse model of Rb deletion has recently been created with transgenic mice. Homozygous Rb-deficient embryos are not viable and die before the 16th day of gestation with major defects in the hematopoietic and neuronal systems. Heterozygotes did not develop retinoblastomas but very frequently developed pituitary tumors which lost the wild-type allele. 5.6. The p53 gene The p53 gene is located on the short arm of chromosome 17 and encodes a 53-kDa nuclear phosphoprotein (p53). This protein binds viral oncoproteins, including the SV40 large T antigen, with very high affinity. It is thought that the wild-type p53 may bind to a cellular homolog of SV40 large T antigen as part of its role in the control of the cell cycle. The mutant p53 loses its binding ability, an event that is thought to be critical in deregulating the cell cycle and thus initiating the malignant transformation. The mode of action of p53 has been repeatedly revised [68,69]. p53 regulates the expression of two other genes, p21 and GADD45 (growtharrest and D N A damage-inducible), p21 binds and inactivates cyclindependent kinases, enzymes that regulate cyclins and p21 expression leads to cell cycle arrest. The expression of GADD45 leads to an increase in D N A repair, a function in which PCNA (proliferating cell nuclear antigen) is also involved. These new hypotheses are now being studied in more detail in order to further understand the interrelationships between p53, p21, GADD45, PCNA and other genes involved in DNA repair e.g. ERCC3. Mutations of the p53 gene are the most frequent genetic alterations found in human malignancies [70-72]. Usually, one of the p53 alleles is lost through a deletion, and the other is mutated. Thus, the tumor cells are practically homozygous for the loss of the p53 normal locus. The point mutations of the p53 gene are not localized in one or a few codons. However, they do cluster in exons 5 to 9, the exons that are highly conserved among species. The mutant p53 protein has a much longer half-life in comparison to the wild-type protein, which leads to the accumulation of mutant p53 in cells and aids in the quantification of the protein [731. The high frequency of mutation of the p53 gene offers diagnostic possibilities. Studies can be conducted at the DNA, mRNA, or protein levels. Because the point mutations are not localized, they can be studied by sequencing the gene. Methods that use either D N A or cDNA generated from m R N A start with an initial amplification step with PCR. After sequencing, the exact mutational event can be identified [74]. Mutations can also be studied by PCR amplification coupled to newer techniques for detecting mutations, e.g. RNAse protection assay, single-strand conforma-

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tional polymorphism, or denaturing gel electrophoresis, with the hydroxylamine-osmium tetroxide method or with constant denaturant gel electrophoresis [75,76]. The p53 protein can also be studied with panels of monoclonal and polyclonal antibodies that recognize wild-type and/or mutant p53 [73,7780]. Immunometric-type assays for measuring mutant p53 have been developed [81-85]. These assays are very effective in quantifying mutated p53 in tumor cell line lysates and tumor tissue homogenates. In a recent study involving 212 human malignant tumors, 76% had accumulated mutant p53 as demonstrated by immunohistochemistry [86]. Tumor types were breast, colon, stomach, bladder and testicular, as well as sarcomas and melanomas. The pattern of p53 expression in breast cancers, as judged by immunohistochemistry may have some value for tumor subclassification. All the studies conducted so far that have examined the value of p53 as a prognostic indicator have concluded that tumors tend to be more aggressive when they are p53 positive. The prognostic value of p53 has been confirmed for breast cancer [87 91], ovarian cancer [92,93] and cancers of the lung, stomach, prostate, cervix, endometrium and bladder [94-99]. Other applications of p53 measurements such as genetic screening for Li-Fraumeni syndrome have also been reported [100-103]. The presence of mutant p53 protein in such a high percentage of diverse tumors [86,104] led to the notion that p53 may be a tumor marker if it appears in serum. This possibility is currently under investigation. Alternatively, it has been proposed that mutant p53 is immunogenic for the host, which would mean that the host would develop antibodies against mutant p53 that, in term, could be measured in serum. Such antibodies were found to exist in about 10-20% of patients with breast carcinoma [105-108]. More extensive studies have revealed that colon, ovarian and lung cancers are very immunogenic with positivity rates of about 10-20% [109-117]. Recently, it has been reported that p53 gene mutations can be detected in tumor cells found in urine sediment from patients with bladder carcinoma [118]. This finding could, in theory, be used as a diagnostic tool and as an aid in monitoring patients who have undergone surgery, for early detection of relapse.

6. Familial breast cancer genes

Recent reports have described the identification of two genes (BRCA-1 and BRCA-2) that predispose to familial breast cancer [119-124]. These two genes probably account for about 60-70% of familial breast cancer, or roughly 5% of all breast cancer cases. BRCA-1 is also strongly associated

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with a predisposition to familial ovarian cancer and loss of heterozygosity was found in some prostate cancer patients as well [125]. Women who inherit a defective BRCA-1 gene have a 60% risk of breast cancer by the age 50 and a 90% lifetime risk. BRCA-1 is a very large gene spanning more than 100 Kb of D N A and consists of 21 exons. The protein has 1863 amino acids. It contains a zinc finger domain similar to those found in DNA-binding proteins and it is probably a transcription factor. BRCA-1 has been found to be mutated in familial breast/ovarian cancer patients. Mutations seem to cause loss of function and BRCA-1 can be classified as a tumor suppressor. However, the examination of 32 sporadic breast cancers and 12 sporadic ovarian cancers, showed that BRCA-1 was not frequently mutated which suggests that this gene is not important in the development of sporadic breast cancer. The cloning of BRCA-1 has raised questions as to how it could be used clinically. Current knowledge suggests that screening of the general population for BRCA-1 mutations is not warranted at this point for the following reasons: (1) The gene is very large and the mutations seem to be scattered meaning that mutation detection will be difficult and costly. (2) A negative test does not exclude breast cancer risk since women who develop sporadic breast cancer do not have a mutated BRCA-1. (3) Even in women who are predisposed to familial breast cancer, a negative test would not be absolutely reassuring since genes other than BRCA-I are involved in about 60-70% of these familial cancers. The test will be useful however, if germline mutations in BRCA-1 are identified during screening. In these patients, risk of developing breast cancer will be 60% by age 50 and 90% at lifetime. Here, the important question is what type of intervention will be advisable, an issue that has not as yet been satisfactorily answered. Also important is the finding of the wild-type BRCA-1 gene in women from families with known cosegregation of BRCA-1 mutations, since these women can be advised that their risk for developing breast cancer is not significantly different from the risk of the general population. BRCA-2 resides on chromosome 13 and is also large, encoding a protein of more than 2329 amino acids. No known proteins show homology to BRCA-2 protein and its function is still obscure. Patients with familial breast cancer not linked to BRCA-1 were found to have mutations in BRCA-2 which disrupt the coding sequence, leading to premature stops. These mutations are deletions of only a few bases. These deletions are scattered and their detection may prove to be expensive when screening will be initiated. Mutations of the BRCA-2 gene do not predispose to ovarian cancer but may predispose to prostate, laryngeal and other cancers.

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Although this may happen in the future, the identification of BRCA-1 and BRCA-2 did not as yet help us in understanding the pathogenesis of sporadic breast cancer.

7. Conclusions

Cancer is a leading cause of death, and research in this field is intensive. It has been postulated for years that tumor markers, substances that are released by a tumor into the circulation, must exist and that their identification could be used for cancer diagnosis, monitoring, treatment and prognosis. The tumor markers that have been identified during the last 20-25 years suffer from a lack of specificity and are not suitable for screening or diagnosis. These tumor markers are either oncofetal antigens (normal constituents during embryonal life) or physiological substances that are present in biological fluids in the normal state but at low concentrations. Abnormal control of gene expression in cancer cells can cause their reappearance in serum (for oncofetal antigens) or their increased, inappropriate production. However, these biochemical changes are now considered late epiphenomena and not to be involved in the pathogenesis of cancer. Oncogenes and tumor suppressor genes or their products could be used as specific tumor markers for the following reasons: (1) The alterations in many of these genes are considered pathogenetic and must be present at the initiation stage of cancer. (2) Alterations in some genes are found only in specific cancer types, whereas alterations of other genes may be found in many cancer types, offering possibilities for both type-specific and non-specific diagnosis. (3) In some instances, these genetic alterations produce unique products (mRNA, protein) that do not exist in the normal state and are thus truly specific indicators of malignancy. Currently, however, with some exceptions, it is not clear whether the abnormal genes or gene products can be assayed in easily obtained material such as blood, urine, sputum or feces. If this is not the case, the usefulness of a marker will be restricted to studies on tumor tissue for subclassification and prognosis, but not for diagnosis. With the exception of the relatively rare hereditary forms of cancer, the genetic alterations in cancer are somatic, which means that they are found only in tumor cells but not in normal cells, again necessitating tumor sampling for study. Additionally, specific genetic abnormalities often exist only in a percentage of tumors. For good sensitivity the use of panels rather than individual tests will be required. The best genetic markers are those present in malignant or even in premalignant cells at a very early stage. Studying such

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cells is required to ensure that the tumor is diagnosed when there are good prospects for successful therapy. Some of the genetic alterations identified in oncogenes and tumor suppressor genes are already in use for tumor subclassification and prognosis. With the exception of chromosomal translocations, these genetic markers are not absolutely specific and have relatively low sensitivity. But this is only the beginning. In the future, with the use of already existing powerful detection, sequencing and amplification techniques, the detection of minute amounts of tumor-specific genetic markers (DNA, mRNA) or their protein products in the circulation may be possible and the laboratory scientist should be ready to transfer those advances from the bench to the bedside.

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