Diagnostic utility of oncogenes and their products in human cancer

Biochimica et Biophysica Acta, 1072 (1991) 193-214 © 1991 Elsevier Science Publishers B.V. All rights reserved tJ304-419X/91/$03.50

193

BBACAN 87241

Diagnostic utility of oncogenes and their products in human cancer Sara J. McKenzie Applied bioTechnology, hw., Cambridge, AL4 (U.S.A.) (Received 16 May 1991)

Contents !.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

193

!!. Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

194

!il. Specific oncogenes in cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. c-ab! chronic myelogenob~ leukemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Neu in breast and ovarian cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Epidermal growth factor receptor in breast and other cancers . . . . . . . . . . . . . . . . . . . . . . . . D. Myc in neuroblastomas, Burkitt's iymphoma and other cancers . . . . . . . . . . . . . . . . . . . . . . . . l. N-myc in neuroblastomas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. c-myc in Burkitt's lymphoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. L-myc in and others lung ~arcinomas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. c-myc in other cancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. nyc summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Ras in breast, colon and other cancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i. ms mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. overexpression of p21 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Ras in serum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. ras summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. p53 as an oncogene? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. Other oncogenes and their associations with cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

194 194 106 199 200 201 202 202 202 203 203 203 204 204 205 205 206

IV. Tumo',' suppressor genes and cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

207

V. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

207

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

208

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

208

I. Introduction Abbreviations: EGFR, epidermal growth factor receptor; CML, chronic myelogenous leukemia; ALL, acute lymphocyte leukemia; PCR, polymerase chain reaction: BL, Burkitt's iymphoma; SCLC, small cell lung cancer; HCC, hepatocellular carcinoma; Rb, retinoblastoma protein. Correspt,,,::nce: S.J. McKenzie, Applied bioTechnology, 80 Rogers Street, Cambridge, MA 02142, US.A.

One c o m m o n i e a t u r e o f all t y p e s o f c a n c e r s , r e g a J d less o f t i s s u e o f o r i g i n , is a loss o f n o r m a l g l o w t h c o n t r o l m e c h a n i s l n s . T h e r e s u l t is a n u n c h e c k e d p r o l i f eration

of neoplastic

cells w h i c h ,

along with other

f a c t o r s , c o n t r i b u t e s to t u m o r f o r m a t i o n . E v i d e n c e g e n e r a t e d in r e c e n t y e a r s c l e a r l y d e m o n s t r a t e s t h a t a c t i -

194 rated oncogenes, and their normal cellular precursors, proto-oncogenes, encode molecules that are involved at various stages of £rowth signaling pathways. Data further suggest that the expression of activated oncegenes may directly contribute to the process of malignant transformation rather than result from it. Consequently, oncogenes and oncoproteins may be appropriate markers for the diagnosis of particular cancers if, of course, the given oncogene is expressed in that cancer in a statistically significant fashion. This article will review those oncogenes and oncoproteins that have already demonstrated a promising level of significant correlation with certain malignancies: abl, in chronic myelogenous leukemia, neu in breast and ovarian cancer, epidermal growth factor receptor (EGFR) in breast and other cancers, myc and ras in various neoplasms, and p53 in a variety of tumor types. (The convention followed in this review is that of using italicized type to represent the genes, and a capitalized notation to indicate the protein product.) !!. Backsround

It was Peyton Rous who hypothesized in 1911 the existence of an infectious agent which could cause tumor formation in chickens, leading to the description of Rous sarcoma virus, a transforming retrovirus [1]. Much of what has since been learned about oncogenes and cancer has its basis in the studies of transforming retroviruses. During the integration process of the retrovirus life cycle, these agents incorporate genetic material from the host into their own genome, in some cases modifying the gene and creating an oncogene. The viruses then undergo replication and infect additional host cells, into which they integrate the newly created oncogene. The progeny of these secondarily infected cells continues to carry the oncogene introduced by the retrovirL,s and suffers the consequenceg of the now unregulated gene. Compelling evidence has led to the conclusion that the precursors to the oncegenes (proto-oncogenes) produce molecules which are intimately involved in the control of cellular proliferation or differentiation; they can be growth factors, growth factor receptors, signal transducers or nuclear factors (Fig. 1). A listing of oncogenes which have been identified is shown in Table l, where they are arranged by class of proto-oncogene function. Oncogenes are derived from their precursors by one of a number of possible mechanisms: (1) via translocation of the gene from its normal site of expression to a new site of expression, resulting in an altered protein produc,, with aberrant function; (2) through deletion of portions of the proto-oncogene, putatively yielding a protein product with an active, growth-inducing configuration in the absence of any normally required stimulus; (3) by mutation, again presumably yielding a pro-

Normal Proto-Oncogene Functions

® Growth Factors

Fig. I. A diagrammatic representation of the possible functiow: ascribed to the normal proto-oncogenes found in cells.

tein product in the active state; or (4)via amplification of the gene, resulting in inappropriate expression or overexpression of the oncoprotein. Thus, the oncegenes encode oncoproteins which are similar to the normal proto-oncogene products, but have lost important regulatory constraints on their activity. The common result of all of the alterations is the activation of the oncogene, resulting in growth signals which contribute to the malignant process. IlL Specific oncogenes in cancer lil-A, c-abl chronic myelogenous leukemia

The ebl oncogene was one of the first cellular oncogenes whose identification resulted from the study of retroviruses. It's viral counterpart, v-abl, was identified in Abelson murine leukemia virus, a virus resulting from genetic recombination of the Maloney murine leukemia virus and the cellular abi proto-oncogene (c-abl) [2]. The activity of the tyrosine kinase domain of TABLE I Partial list of known oncogenes

Categorized according to cell growth functions. Growth factors

Growth factor receptors

Signal transducers

sis int-2

neu EGFR met fms ros trk kit sea ret

abl H-ras K-ras N-ras pim- 1 lck hck fps

Nuclear factors src mos raf pks yes fgr lyn

c-myc N-myc L-myc R-myc jun fos myb

195

c-abl gene is spliced in-frame to a 5' portion of the phi

the oncoprotein is known to mediate the transforming ability of v-abl [3-6]. This observation suggests that the cellular abl oncogene is also involved in signalin/~ normal cell growth through tyrosine kinase activity. The activation of the proto-oneogene occurs through a reciprocal translocation between chromosomes 9 a~,d 22 [7] in which c-abl (9q34) is joined at the breakpoint cluster region (bcr) of the phi gene on chromosome 22 ( q l l ) [8] (see Fig. 2A). The phi gene encodes a protein of unknown function unrelated to any published sequence [9-14]. In the translocation, a 3' portion of the

gene [12,15] resulting in an altered chromosome 22 which is referred to as the Philadelphia (Ph') chromosome [16] (see Fig. 2B). The protein product of the spliced genes in the Ph' chromosome is a molecule 210 kDa in size which has increased tyrosine kinase activity as compared to the normal c-abl proto-oncogene product of 145 kDa [17,18]. Furthermore, the structure of p210 is similar to the gag-abl p160 of the Abelson murine leuLemia virus. In both p160 and p210 there is a change in the amino-terminus of the abl protein

A m

~A

2

s

4

s

e 7s

e

m

.

c-abl

ltan~oosttonALL ~: ~ phl

,~~d

~'~<.., .~ ,r;.~. "~.-,.

GML uml~ocmlon ~"1 234 5

e '

\

1

~////I///~,~'~ p190 {Ph'+/bcr-)

Normal

p210 (Ph'+/bcr+)

Transiocat®d Altered

Chromosom~a 9

Chromosome 9 Chromosom,~ 22

Q

Philadelphia Chromosome (Ph')

O

q34

i

Fig. 2. c-abl translocations. (A) Diagrammatic representation of the recombination event which occurs between the c-abl locus and the phi gene to create either the b c r - rearrangement which yields the p190 product, (on the left) or the bcr + rearrangement which creates p210 (on the right). (B) Representation on the chromosomal level of the same recombination event between c-abl, found on chromosome 9, and the phi gene found on chromosome 22. A reciprocal translocation between chromosome 9, q34 and chromosome 22, ql 1.2 leads to an altered chromosome 9 and the shortened chromosome 22 which is known as the Philadelphia chromosome.

196 product and a resulting increase in tyrosine kinase activity [ 18-21]. The Ph' chromosome occurs in more titan 90% of individuals with chronic myelogenous leukemia (CML) and represents the first description of a specific genetic translocation associated with a human malignancy [16,17,19-22]. A similar disease which also can exhibit the Ph' chromosome, acute lymphocytic leukemia (ALL), is characterized by an increased number of immature lymphoid cells, whereas CML is identified by a rapid proliferation of mature myeloid cells of various lineages. However, in 15-25% of adult ALL and 5% of pediatric ALL cases, patients exhibit a slightly different Ph' rearrangement [23,24]. This results in a protein product 190 kDa in size that lacks a portion of phi found near the bcr.abl junction in p210 [25]. In these instances the breaks in chromosome 22 occur in the first intron of the phi gene, rather than introns 2 or 3 of phi, as is the case in CML [26]. Thus, pig0 lacks approx. 200 amino acids as compared with p210. This difference may be critical with respect to substrate specificity or tntracellular location in vivo, and hence may help to describe the differences between CML and ALL [25]. The first FDA approved product based on analysis of an oncogene detects the Ph' translocation which gives rise to the 210 kDa phl/abl fusion protein. The product, Transprobe-I TM (Oncogene Sciences Inc.) is a DNA probe which can be used in standard Southern blot analysis [27] to detect the rearrangement in chromosome 22. The only other procedure available to detect the Ph' translocation is standard karyotyping ef blood cells; here the defect is noted as a shortening of the long arm of chromosome 22 [16]. Although Southern blot analysis is a much more specific method for detecting the translocation, it is still a cumbersome technique and generally is performed using 3-'P-labeled nucleic acids. It is difficult to predict at this time how well such nucleic acid-based diagnostics will be received by practicing clinicians; certainly, it is unrealistic to believe that all hospitalbased pathology laboratories wiU incorporate such elaborate techniques into their renertoire. However, the specificity and sensitivity of te ts such as this may eventually outweigh the technical difficulties. In general DNA-based tests will be perceived as much more viable products for routine diagr~osis and use if the techniques are supported through automation and nucleic acid probes are coupled with non-radioactive indicators, One such automated DNA-based approach i~ the polymerase chain reaction, also known a~ PCR. This is a powerful technique which utilizes a thermo-stable DNA-s~cnthesizing enzyme, the polymerase of Thermus aquaticus, to perform in vitro synthesis of a specific segment of DNA. Two oligonucleotide primers are

t~sed which flank the sequences of interest and hyI~fidize to opposite strands. With repetitive cycles of qetaaturation of the DNA, primer annealing and enzyr~atic extension of the primers, the specific fragment of it;terest is synthesized and at the same time amplified, ~s the product of each cycle can act as a template in the next cycle. In this way a single copy gene segment ~ata be multiplied to become the major proportion of bNA present in a complex mixture. The DNA can then be hybridized to specific oligonucleotide probes to ~xaxtine the fragment generated by the PCR treatMeat. This method overall allows the examination of a large number of satnples at a level of sensitivity not ~chieveci by other methods. The PCR method should be a viable alternative for the diagnosis of ALL and CML on a routine basis, in this case, however, the RNA transcript must be utilized lbr PCR alnplification as the heterogeneity of the ~reakpoints found in phi will not allow the use of Ol~A as the substrate. This method has already been tlenlonstrated to readily detect the chimeric phl-abl MIgNA, and to detect the aberrant message indepentlent of the presence or absence of an easily identifiable Philadelphia chromosome [28]. With the autoMated systems available, this procedure could allow ~asy identification of the abl transiocation once the ~ppropriate oligonucleotide primers were identified ~tnd the conditions optimized. Another possible technique for diagnosis of genetic tlefects such as that seen in CML and AI..[, is in situ }lyt~ridization. This method is performed on tissue sections m~ch in the same way as immunohistochemical ~taining is performed, with tb,e exception that a nucleic ~tcid is used as the probe. This probe can be either DI~A or RNA, and can be labeled with radioactive molecules, enzymes or light sensitive compounds for detection of hybridization by deposition of radioactive ~rains, color from a precipitating substrate, or by fluorescence, respectively. Thus, the presence of particular ~enes (normal or altered) can be visualized in cells wlaose morphology remains more or less intact. The drawbacks to the technique arc also similar to those for iror,,mo-staining, namely that care must be taken during tissue preparation in order to preserve the integrity tff the target molecule, and that such preparation can vary widely from laboratory to laboratory, rendering direct comparison of results somewhat difficult. Again, hoxvever, the advent of automated technology along ~,it~ computer-aided image analysis may make this technique more attractive for general use in the future.

(ll, B, Meu in breast a,va' or,arian cancer t h e neu oncogene (HER-2 or c-erbB-2) was first described in association with a neuroblastoma after treatment of rats with the chemical carcinogen ethylni-

197 trosourea [29]. The human homologue, residing on chromosome 17, has also been isolated [30,31] and is known to encode a glycoprotein of 185 kDa (p185) which is closely related to, but distinct from. the human epidermal growth factor receptor (EGFR). N e u is one of multiple erbB-iike oncogenes, a family identified through homology with v - e r b B, the retroviral oncogene from avian erythroblastosis virus which produces a truncated version of EGFR [32,33]. This family of genes encodes receptor tyrosine kinases possessing a cysteine-rich extracellular domain, a transmembrane domain and an intracellular domain where the enzymatic activity is localized. In spite of homology between the two receptors, Neu and EGFR, epidermal growth factor does not bind to Neu. However, one ligand has been identified which putatively interacts with both EGFR and human Neu [34]. The rat n e u oncogene is activated via a single point mutation in the transmembrane domain. This mutation presumably alters the entire protein conformation in such a way as to achieve a constitutively active state of the tyrosine kinase domain [35,36]. A similar mutation has not been observed for the human n e u oncogene. Rather, association of n e u with human cancers is via gene amplification a n d / o r overexpression of the protein product. The first report of a correlation between n e u gene amplification and a specific cancer was made by Slamen et al. [37]. This report indicated that the n e u gene was amplified in 30% of 189 patients with primary breast cancel Other investigators have since generated data which also indicate 16% to 20% of breast cancer

TABLE II Neu in breast and ocarian cancer

Alteration

Observa t,ion

Reference

Gone amplification in breast cancer

16-20% of tumors

37-42

in node-positive patients, correlates with: - poor short term prognosis - spread of cancer - clinical course of disease Overexpression of p185 occurs in 17-30% of tt~mols; ~ot in normal or in breast cancer benign correlates with gene amplification is an independent predictor of survival in node-positive patients Overexpression of p185 26-32% of tumors examined; correlates with in ovarian cancer poor survival three breast cancer patients Detectable levels of with high tumor levels of neu in serum p185

37. 38, 42 41) 41 43-45

46-48 49-51

45, 52

56

W~ors studied had muh~ple copies of the n e u gene [38-42]. Furthermore, these studies indicated a statistically significant correlation between amplification of n e u and: relapse and survival in node positive patients [37,42]; poor short-term prognosis [38]; spread of cancer [40]; or overall clinical course of the disease [41]. Other studies have shown an association between overexpression of the Neu oncoprotein with breast cancer, i,~dependent of gene amplification. One such report indicated 30% of 37 breast carcinomas overexpressed Neu, whereas none of 24 benign tissues overexpressed the oncoprotein [43] and in another, 17% of 191 primary breast tumors overexpressed the Neu oncoprotein [44]. A second study by Slamon et al. contained a much larger sampling of patients with breast cancer (526 with clinical follow-up) as well as 120 primary ovarian cancers [45]. In a comprehensive examination of gone and expression at both the RNA and protein levels, this group verified the association between n e u and clinical outcome in both types of malignancies. Other investigators have since confirmed correlation of n e u gene amplification with overexpression of the protein [46-48]. Additional evidence supports the findings that Neu oncoprotein overexpression is of prognostic significance in breast cancer. One study of 350 node-positive breast cancer patients indicated shorter disease-free and overall survival in patients with higher Neu oncoprotein levels [49], and further analysis of the data showed that overexpression of Neu oncoprotein is an independent predictor of survival. In the second study of 292 primary breast cancers, patients with Neu oncoprotein overexpression also had significantly worse overall survival with twice the mortality rate [50]. A third study of 290 breast cancer patients indicated that in late stage tumors, Neu overexpression correlated with shortened disease-free survival and overall survival [51]. These results all suggest that Neu overexpression may be an independent prognostic indicator for patient s,rvival. Overexpression of Neu in ovarian cancer is also associated with poor survival. Berchuk et ai. reported that 32% of 73 ovarian cancers had overexpression of the oncoprotein, and that survival of 23 of these patients was significantly worse than that of 50 patients with normal Neu expression [52]. And othe, investigators have since associated Neu overexpression with poor short-term survival in patients with adenocarcinomas of the lung [53] and in patients with gastric cancer [54]. See Table II for a summary of the data concerning n e u expression in breast and ovarian cancers. Recent reports have indicated that a portion of the Neu oncoprotein (presumably the extracellular domain) is shed by breast tumor cell lines in vitro [55,56] and is present in the serum of some breast cancer patients [56,57]. Sera from 3 patients (of 12 examined)

198 demonstrated high reactivity in an ELISA using two monoclonal antibodies which recognize the extracellular domain of the Neu oncoprotein [56]. Tumor specimens from two of these patients also had heavy immunohistological staining of Neu; the third was unavailable for study. In the second report, the plasma of 145 breast cancer patients were examined~ ~iong with plasma from 81 individuals either considered normal, or with benign breast disease, or in lactation. These samples were measured for levels of Neu-related protein by an ELISA developed from another pair of antibodies specific for the extracellular domain ~f ti,~ Neu oncoprotein [58]. The results of this study showed a striking correlation between high levels of Neu, as measured by the ELISA, and patients with metastatic disease. Although it is clear that Neu overexpression can be observed in approx. 20-30% of breast cancer patients, it is unclear whether the marker will actually be of value diagnostically, or if its role will be in the prognosis of breast cancer. A diagnostic tool based on a marker such as Neu may not contribute new information to the diagnosis of breast cancer considering the effectiveness of currently available methods. However, data do support the claim that the level of Neu overexpression in tumors is a significant prognostic indicator in node-positive patients, and hence may be of great value to physicians in determining appropriate treatments. Other studies are underway to determine the value of measuring Neu oncoprotein levels in nodenegative breast cancer patients; a marker for prognosis in these patients would be of great value. Paterson et al. recently reported that quantitation of n e u oncogene copy number is indeed a strong and independent prognostic indicator in this node-negative patient population [59]. In this study, a population of 115 women with node-negative breast cancer and recurrent disease were matched with a population of node-negative patients who had not recurred during long-term follow-up. The results showed that 18% of the patients who recur~'ed had a high copy number of the n e u oncogene, whereas only 5% of the patients who remained in remission had multiple copies of the n e u oncogene in their primary tumor. These data were statistically significant in predicting poor prognosis, especially disease-free survival. However, this interpretation must be balanced with a recently reported observation by Clark and McGuire that n e u gene amplification is not a significant indicator in patients with node-negative breast cancer [60]. In this study, 185 node-positive patients were compared with 177 node-negative patients but no comment was made as to matching of these patients with respect to treatment modalities or other prognostic indicators. In any case, n e u gene amplification was not associated with either disease-free survival or overall survival. These contradictory reports clearly show that many

more patients must be examined to determine the ultimate use of n e u as a prognostic indicator for nodenegative breast disease. The limited number of studies to date examining the overexpression of Neu in ovarian cancer have shown the same type of correlation with disease and survival as in breast cancer patients. Additional studies must be performed to determine how early Neu overexpression occurs during the course of ovarian disease, and hence how useful measuring levels of the Neu oncoprotein will be for the diagnosis of ovarian cancer. As there are ao speclhc symptoms of early ovarian cancer identifying one or m~re reliable markers such as Neu in serum would be of great value. An additional possible antibody-based methodology for the diagnosis of ovarian or breast cancer is that of in vivo imaging. The Neu oncoprotein is an example of an appropriate target for this method, as it is an overexprc,'~sed protein found on the surface of the cell. A specific monoclonal antibody which recognizes the cell surface antigen, coupled to a radioisotope such as technetium-99 or indium-Ill could target tumor expressing the antigen, thus enhancing the physician's ability to detect occttlt lesions, and potentially contributing to monitoring a n d / o r staging of the disease. The procedures in general have made great strides in the last decade, and continue to improve as coupling agents and computer aided image analysis become m~r~, refined. ~' One of the ELISAs to quantitate Neu in tissue and serum mentiored above is commercially available (Applied bioTechnology, a subsidiary of Oncogene Science, Inc.)./The assay is cap~t~!e of measuring what appears to be the extracdlular domain of the Neu oncoprotein in the serum or plasma of breast cancer patients, as well as the full-length oncoprotein in cell lines or tumor specimens. The ELISA is a relatively simple and straightforward assay, and as configured, does not use radioactive materials. It provides quantio tative data, requires little sample, and in the case of serum or plasma, uses a readily obtainable sample from cancer patients. One drawback to the assay is its inherent inability to define the cellular component of the sample harvested for testing. Results are reported as units per microgram of total protein for tissue testing, but it is unknown how much of that protein is due to cells versus connective tissue, and if due to cells, what type of cell. Thus, an obvious improvement is to be able to measure against some common internal control, such as an epithelial cell antigen when examining a tumor of epithelial origin. However, the advantages to even semi-quantitation of a marker with an easily performed, reproducible assay that can be compared across laboratories should compensate until these drawbacks can be corrected. The availability of an assay such as this should aid the medical community in

199 generating the data which can address the issues presented above, and tile routine application of the assay to the diagnosis or prognosis of breast, ovarian or other cancers can then be made clear.

III-C. Epidermal growt!: factor receptor in breast and other cancers The oncogenic potential of epidermal growth factor receptor (EGFR) was only uncovered by the discovery of its shared sequence homology with v-erb-B [33]. It, like Neu, is a transmembrane tyrosine kinase receptor protein (reviewed in Ref. 61), and shares significant homology with Neu, as described above. The gene is located on chromosome 7 and encodes a glycoprotein of 170 kDa in size which binds to several other ligands besides the epidermal growth factor, EGF. EGF-receptor is intimately involved with growth signalling in epidermal cells. Binding of epidermal growth factor (EGF) induces autophosphorylation of E G F R as well as tyrosine phosphorylation of other intracellular substrates [62]. The activation of the ki,aase domain following ligand binding is apparently induced by receptor oligomerization [63] which in the case of E G F R results from a conformational change induced by the binding of EGF [64]. In general, oligomerized growth factor receptors demonstrate an increased tyrosine kinase activity [65-67]. In addition, it has been shown that E G F can induce tyrosine phosphorylation of phospholipase C-T in tissue culture cells [68-70]. While it is not yet clear whether this plays a role in the transformation process, elucidation of the specific substrate molecules such as phospholipase C-7 may in turn lead to the next step in the cascade, as well

as rational targets for approaching therapies through inhibition of the downstream signaling steps. Overexpression of EGF-receptor has bee~a implicated in a number of neoplasms, including breast cancer (see below), squamous cell carcinomas [71-74], glioma [75], bladder carcinoma [76-80], lung cancer [79, 81-84], ovarian carcinoma [85,86], and esophageal cancer [87,88]. A summary of the data concerning overexpression of E G F R is shown in Table III. Overexpression of E G F R correlated with poor prognosis in 47% of esophageal cancers tested by Yano et al. [88]. In this study it was demonstrated that patients with strongly expressed E G F R had lymph node metastases more frequently, and subsequently poorer prognosis. Ozawa et al. reported that in a population of 32 patients with esophageal squamous cell cancer, the survival rate was lower for patients with high levels of E G F R on their tumors (P < 0.05) [87]. There is also an apparent correlation between overexpression of EGFR and survival in ovarian cancer patients. Berchuk and colleagues [86] t0und that among 87 ovarian cancers studied, 67% overexpressed EGFR, and this was significantly correlated with poor survival (P < 0.05). Another study reported that 35 of 55 tumors tested positive for EGF-receptor overexpression and this also significantly correlated with progression of disease (P < 0.05) [85]. Among the different types of lung cancer, EGF-receptor overexpression is noted most often in non-small cell lung carcinomas: it has been observed in anywhere from 52% to 80% of specimens examined. Hendler et al. described a correlation between overexpression and poorly differentiated tumors as well as poor survival [83]. No obvious correlation existed in the study by

TABLE III EGFR in breast cancer and other neoplasms

Tumor type Breast cancer Glioma Bladder carcinoma

Lung cancer

Ovarian carcinoma Esophageal cancer

Observation 21-83% overexpression; correlation with low ER overexpression correlates with early relapse and shortened smvival overexpression correlates with short-term, disease-free survival overexpre~sion associated with gene amplification receptor truncated; in some cases in the region of EGF binding 87% invasivetumors positive for overexpression correlation between tumor stage and overexpression,versus no correlation with tumor stage 48% overexpress;correlates with invasivenessand time to recurrence 52% to 80% non-smallcell overexpress overexpressmn correlates with poor survival no obviouscorrelation with poor survival,but overexpression in 66% non-smallcell 49% to 64% overexpre~s;correlates with poor survival 38% overexpression 47% overexpression;correlates with poor prognosis

Reference 94-104 100 105 89 90-.93 76 78 vs. 80 79 81, 82, 84 83 79 85, 86 87 88

200 Harris et al., although they did observe that 66% of non-small cell lung cancers had higher levels of expression than did normal lung tissue [79]. EGFR overexpression has been noted in 29% to 48% of bladder cancers tested. Neal et al. observed 87% of invasive tumors were positive, whereas only 29% of superficial tumors had high levels of the receptor [76]. In a later study, the same group demonstrated a correlation between overexpression of EGFR and tumor stage [78]. However, Moriyama et al. suggested that expression was independent of tumor stage [80]. Another report indicated that 48 of 101 bladder tumors tested had high levels of EGFR, and that this correlated with poorly differentiated invasive tumors and time to recurrence [79]. Only in gliomas does the overexpression of EGFR appear to be associated with amplification of the gene [89]. More recent studies suggest that the EGF-receptots expressed in a number of gliomas are actually truncated molecules [90-92] and in some instances the deleted regions overlap the EGF binding domains [93,]. Overexpression of EGF-receptor has been most studied in breast carcinoma. The majority of studies have demonstrated a correlation between higher than normal levels of EGFR and low levels of estrogen receptor. These studies have reported that 21% to 83% of the tumors studied showed overexpression, with most rest, Its in the 25% to 35% range [94-104]. Additionally, N~cholson et al. reported a correlation with early relapse and shorter survival when tumors showed overexpression of EGFR [100], and Lewis et al. reported a correlation with short-term, disease-free survival [105]. It is clear that EGF-receptor plays a s~gnificant role in a number of neoplasms of epithelial cell origins, as well as in gliomas. Competitive ligand-binding assays which utilize radiolabeled EGF have been developed and are used in nlany research laboratories [85,87,106]. However, these assays are not standardized and are not commercially produced rendering comparisons between laboratories difficult. It should be straightforward to develop additional reagents and/or kits for clinical use which can quantitate the level of the protein in tumor tissue, such as those for the Neu oncoprotein. It will be of great interest to examine the serum of cancer patients with high levels of EGFR on their tumors, to determine whether, like Neu, all or part of the protein is shed from the cells in detectable amounts. Since EGFR is a cell surface molecule, in vivo imaging should be a viable alternative as a diagnostic strategy. In fact, one recent report describes the use of and iridium-Ill-labeled anti-EGFR antibody in phase I clinical trials for imaging lung tumors [107]. During the course of these studies, it was found that antibody localized predominantly in the tumor and not in nor-

mal tissues. A second report by Kalofonos et al. demonstrated that imaging with an anti-EGFR antibody which did not bird to the EGF-binding site resulted in antibody uptake by glial tumors but not by skin or other normal epithelial tissues [108]. These studies demonstrate the great promise in store not only for in vivo diagnosis of tumors with overexpressed EGFR, but also for other oncoproteins expressed on the surface of the tumor ce!ls. III-D. My: in neuroblastomas, Burkitt's lymphoma and other cancers The myc oncogene was first identificd by homology to an avian leukemia virus [109,110]. The v-myc form is the only known oncogene which can induce tumors in all three major tissues: epithelial, mesenchymal and hematopoietic [111]. The c-myc oncogene represents one of many related mammalian genomic sequences that constitute the myc gene family, the other most common being N-myc and L-myc. The c-myc and N-myc genes have a similar structure consisting of three exons, with the first being untranslated. The gene encoding L-myc also has three exons, but has a smaller noncoding first exon and a large 3' untranslated region (see Fig. 3A). The resulting proteins are 439 residues (c-myc), 456 residues (N-myc) and 364 residues (L-myc) in length. The protein products themselves are posttranslationally phosphorylated and are locali',zed in the nucleus. Although Myc has been avidly studied the function of the molecule is still not completely understood. Studies of other proteins with structural similarities to Myc are providing some clues as to the possible function of Myc in the cell. One such structure, inferred from amino acid sequence, is referred to as the helixloop-helix (H-L-H) motif; other proteins possessing this structure have been shown to be important regulators of transcription [112,113]. The second structural similarity with other regulatory proteins is the 'leucine zipper'. It is believed that both structures are involved in protein-protein interaction and that this interaction appears to be coupled to DNA binding [114-119]. The postulated presence of these specific structures in Myc has led to speculation that it must be involved in the same kinds of activities as proteins with homologous sequences. Recent evidence from two laboratories provides support for this speculation by identifying a DNA binding site for c-Myc homodimers. Blackwell et al. demonstrated that the carboxy-terminal portion of Myc binds to the DNA sequence CACGTG [120], and Prendergast and Ziff confirmed binding of Myc dimers to oligonucleotides with the C A - T G consensus [121]. This is of course only a first step towards elucidating the function of Myc, and further work is required to

201 A

c-m¥c

N-myc

L.myc

o. ,c

Translocation ~o Immunoglobulln lot;us

Rearranged

Ig ONA

myc exon 2

myc °xon 3

oec~utatea Transcription normal

c-myc ~

,

. . . . . . . . . . .

..n2

I o.n3I--

mRNA

Fig. 3. The rnyc gene family. (A) Diagrammatic representation of the three most common members of the myc family, c-, N- and L-myc. Non-coding regions are indicated by the shaded areas, coding regions by the open boxes, and the highly conserved sequences found in all tnyc genes are indicated by the cross-hatched regions. (Modified from LeGouy et al. (1987) in Nuclear Oncogenes (Air, F.W, Hariow, E. and Ziff, E.B., eds.) Cold Sping Harbor Laboratory.) (B) Schematic of the translocation of c-myc which occurs in Burkitt's iymphoma. The myc exons 2 and 3 are translocated to the immunoglobulin locus on chromosome 2, 14 or 22, where its expression becomes deregulated. The result is a normal c-myc mRNA which is expressed at inappropriate times.

provide direct evidence for the involvement of Myc in transcription. Data are available which support the suggestion that two members of the gene family contribute to the development and/or progression of certain cancers. This includes amplification of N-myc in neuroblastomas and translocation of c-myc in Burkitt's lymphoma. Overexpression of c-myc has also been noted in a variety of malignancies and overexpressed L-myc and other members of the myc family has been noted in small cell lung carcinoma. See Table IV for a summary of myc expression in various neoplasms. III-D. 1. N-myc in neuroblastomas Schwab et al. originally discovered that human neuroblastoma cell lines contained multiple copies of a DNA sequence related to v-myc and c-myc, and called the new sequence N.myc [122]. A later study by Seeger et al. established the link between this proto-oncogene and the biological behavior of the tumor in vivo [123].

TABLE IV Summary of myc alterations in various neoplasms

Tumor type

Observation

N-myc gene amplification is a prognostic indicator of disease Burkitt's lymphoma c-myc geJle translocation observed in all confirmed cases c-myc gene amplification in 6Breast cancer -57% of tumors examined elevated levels of c-myc mRNA correlate with poor prognosis c.myc gene amplification in 10 Colorectal cahcer to 20% of cases; not statistically significant aggressive subtypes have modest amplification of c-myc c-myc gene amplification Squamous cell associated with advanced carcinoma stages L-myc overexpression Small cell lung carcinoma

Neuroblastomas

Reference 123-128 138-;43 38, 161-163 38, 163 15~!,!64-169

17() 172-175

151-154,157

202 Studies prior to this had demonstrated that the N-myc gene was amplified in 20% of neuroblastomas, amplification is associated with more aggressive variants of the tumor [124,125] and genomic amplification results in production of high levels of mRNA [124,126-128]. The pivotal study of N-myc in neuroblastomas demonstrated that the number of gene copies is a prognostic factor independent of stage of disease [123]. Generally, stage I, II, III or IV neuroblastomas with only one copy respond well to conventional therapy, but patients who have stage II, III or IV tumors with multiple copies of N-myc have progressive disease soon after diagnosis. Furthermore, this relationship between genomic amplification and outcome of the disease was shown to be significant among patients with stage IV disease. Overall, the data sugges: that genomic amplification of N.myc directly contributes to the aggressive nature of the neuroblastoma. N-myc amplification has also been noted in retinoblastoma [129,130], glioblastomas [131-133] and leukemias [134-136], and elevated levels of N.myc mRNA have been noted in colon carcinomas as well [137]. However, in none of these diseases has the correlation with prognosis yet been shown to be as striking as for neuroblastomas. III.D.2. c-myc in Burkitt's lymphoma Rearrangement of the c-myc gene has been routinely observed in Burkitt's lymphoma (BL). In fact, in all confirmed eases of this disease, whether or not the affected lymphocytes carry Epstein-Barr virus, one of three characteristic chromosomal markers is observed: the translocation of chromosome 8 to one of three immunoglobulin gene-carrying chromosomes: chromosome 14 (the heavy chain locus), or less frequently to chromosome 2 (the • light chain locus), or to chromosome 22 (the a light chain locus) [138]. And it has been documented that the translocated portion of chromosome 8 does harbor the c-myc oncogene [13%140]. The second and third exons of c-myc are transposed either head-to-head with the heavy chain gene or head-to-tail with the light chain genes and always remain intact [141-143] (see Fig. 3B). How do these translocations affect the function of Myc? Theories include: a loss of regulatory control resulting from constitutive expression of c-Myc at inappropriate times in the cell cycle [144,145]; overexpression of functional c-Myc protein as a result of constitutive gene expression [142,146148]; and/¢,r increased stability of the mRNA leading to overexpression of the protein product [149,150]. llI.D.3. L.myc in lung ca~inomas The L-myc member of the fa,.dly was first noted in small cell lung cancer (SCLC): the gene was found to be amplified in four SCLC cell lines and in one tumor specimen [151]. Later studies by the same group indicated that 6 of 31 independently derived cell lines from

SCLC tumors had amplified N-mvc seq~lences ~ - ' 1 Johnson et al. showed that myc gene family DNA was amplified in 13 of 44 SCLC specimens from patients, and that relapsed patients with amplified myc genes survived a shorter period of time than did those with no amplification [153]. In subsequent studies Johnson and colleagues found that cell lines with amplified myc gene sequences often were derived from tumors of patients who had received chemotherapy [154,155]. Other groups have independently verified the amplification of one or more of the myc genes in SCLC [156,157]. III-D.4. c-myc in other cancers Amplification of the c-myc oncogene and/or overexpression of the protein has been noted in a variety of other neoplasms, including five of the most common solid tumors, namely stomach [158,159], breast, colon, cervix and head and neck cancers (see below and Ref. 160 for a complete review). Unfortunately, the data collected to date are not yet definitive as to the degree of association of c-myc expression with many of these malignancies. The majority of studies of c-myc have been in breast cancer; over 500 specimens have been investigated with regards to c-myc gene alteration or overexpression. Amplification has been detected in anywhere from 6% to 57% of tumors examined [38,161-163]. In one ,:vch study of 121 patients, there was a significant correlation of c-myc amplification or genetic rearrangement with menopausal status [161]. In contrast, a smaller investigation of 41 patients discovered no association between c-myc gene alterations and age, whereas there did appear to be a significant correlation between altered c-myc genes and poor short term prognosis

[38]. Counter to these discrepant results of gene alteration, it seems that elevated levels of e-myc RNA do correlate with prognosis in breast cancer. The Varley et al. study mentioned above found that half of those patients with elevated e-myc RNA have since had recurrence or died [38], and a study by Guerin et al. demonstrated a statistically significant correlation between high levels of c-myc mRNA and lymph node involvement [163]. Only a limited number of studies have examined c-myc gene amplification or overexpression in colon, cervical or head and neck cancers. To date there is no statistical correlation in colorectal cancer [158,164169], although a recent report which utilized a more ~ensitive method for detecting gene amplification suggested a correlation with more aggressive subtypes of colorect~.l cancer [170]. There does appear to be an association between c-Myc overexpression and poor prognosis in cervical cancer [171], and further, amplification of c-myc is apparently associated with advanced

203 stages of a variety of squamous cell carcinomas [172175]. III-D.5. m y c s u m m a r y

Clearly a great deal of work remains to be done to better understand the role of c-myc in various neoplasms. A tool to directly examine patient lymphocytes would be of great use to physicians presented with a patient who exhibits symptoms associated with BL. One such as that created for the abl translocation in ci~ronic myelogenous leukemia might obviate the need for bone marrow aspiration which is frequently used now to establish diagnosis of CML. In situ hybridization on metaphase chromosomes would also allow for the detection of the transloeated gene, although may be cumbersome if large ..ambers of cells must be examined at this level. A PCR-based methodology for the detection of N - m y c gene amplification would clearly be an asset for establishing treatment regimens for patients with neuroblastoma. In this instance, however, PCR will not be as useful until a relia01e and reprodueible method is developed for quantitating gene copy number following PCR amplification. More data must be accumulated to help establish the importance of c-myc gene amplification or overexpression of the protein in solid tumors. It could be that current controversies in the contribution of this oncogene to the course of various diseases is at least in part due to the disparate reagents and methodologies used to examine th,~ oncogene. A common set of reagents and procedures would control for the methods aspect of the investigations and ieave to physicians the interpretation of the contribution of other factors such as staging, age and treatment regimes, to name only a few. Although c-myc may not as yet be useful as a single diagnostic tool for certain cancers, further controlled studies are necessary to establish its potential use.

III-E. Ras in breast, colon a n d other cancers

Transfection experiments led to the identification of a family of activated genes which are homologous to those of the Harvey and Kirsten murine sarcoma viruses [176,177] which were subsequently dubbed c-Ha- and e-FO-ras (or H- and K-ras). Another member of this multigene family, N-ras, was originally identified in a human neuroblastoma, but no viral counterpart has been found [177]. Since c-ras was discovered it has been shown to be present in all vertebrate species studied, as well as in invertebrates and in yeast [1781841. The ras genes in mammalian cells encode a 21 kDa intracellular membrane protein called p21 [185] which can be detected in normal, untransformed cells as well as in malignant cells [178]. The third amino acid from

the C-terminal end of p21 is a cysteine residue, to which a poly-isoprenoid group is attached, thus offering an archor for intracellu!ar plasma membrane adhesion [186,187]. It is mutant forms of p21 with point mutations resulting in specific amino acid substitutions at positions 12, 13 or 61 which possess the transforming ability of Ras [188-191]. The ras p21 is known to bind guanine nucleotides [192-194] and shares sequence homology and similar predicted structure with a family of other guanine nucleotide-binding proteins [195]. In fact the crystal structure of the nucleotide binding region of Ras is known [196,197]. Despite extensive studies of the structure of ras p21 and investigation of its biochemical ~at.are, the exact mechanism of action of p21 is not known. In yeast it is well established that ras proteins are primary regulators of adenylate cyclase ectivity [49,198]; unfortunately, this does not apr~ear to be the case for mammalian Ras. The norraal mammalian p21 protein has intrinsic GTPase activity [199-202] which in the presence of GAP ((JTPase activating protein) is increased 500-fold over mutant forms of p21 [203]. Mutant p21 incapable of hydrolizing GTP to GDP, and as a consequence remain,~ bound with GTP. It has been proposed that GAP maintains normal p21 in its inactive, GDPbound state. However, the oncogenic p21 remains in the constitutive GTP-bound state, and escapes normal GAP control. It is believed this GTP-bound state actively transduces growth signals downstream in the cell [195]. Theoretically, then, the oncogenic p21 is constantly 'turned-on' and transmitting positive signals for growth in the absence of a normal growth stimulus. Closely following the experiments which demonstrated ras transformation of murine cells [204-206] came experiments identifying the presence of mutated ras genes in tumors and cells lines derived from tumors [205-209]. In addition, investigators observed overexpression of the ras gene by hybridization studies [210,21!] and of p21 by immunohistochemistry [212216]. Unfortunately, some studies have been performed with reagents such as the RAP-5 monoclonal antibody, which have since been shown to bind to other proteins in addition to Ras [217,218]. Thus, one must examine carefully the data which have been gathered before trying to assess the significance of ras expression. See Table V for a summary of ras expression in various tumors.

III-E.1. ras mutations

A broad range of studies has implicated involvement of ras in stomach [219-221], lung [222-224], pancreatic [225-227], head and neck [228], and prostatic cancers [229-231], in myeloid leukemias [232-236], and in breast and colon cancers (see below, and Refs. 160 and 237 for a complete review).

204

TABLE V Summary of ras alterations in various neoplasms

Tumor t y p e Breast cancer

Observation elevatedH-ras mRNA levels correlate with advanced types increased levelsof p21 in malignant tissue Colorectai cancer 50% of colon tumors h a v e mutations in K-ras gene ras gene mutations in adenoma and carcinoma tissue point mutations in ras genes in Lung carcinoma 20-30% of tumors K-ras mutations identify subgroup with poorer prognosis Pancreaticcancer K.ras point mutations in 90% of cases examined p21 expressed in malignant Stomach cancer tissue at higher levels than normal tissues codon 12 mutations in Ha.ras correlates with metastasisand survival M,~,eloid leukemias N.ras mutations detected in 10-50% of cases Nude mouse model p21 in serum of tumor-bearing

Reference 211,243 245. 247, 248 239-241 239,241,242 222 224

III-E.2. overexpression o f p21

225-227 219-221 238 232-236 249,250

mice

Investigators have found activated ras genes in 2030% of lung carcinomas [222], as well as in 90% of pancreatic carcinomas [225]. K-ras mutations seem to predominate in pancreatic tumors [226,227], and K-ras point mutations also appear to identify a subgroup of lung cancer patients with poorer prognosis and shorter disease-free survival times [224]. In stomach carcinomas expression has been found to be significantly higher in malignant tissue than in normal tissue [219,220] and expression of both TGF-a and H-ras p21 indicated a poor prognosis as compared to t h o s e stomach tumors with low levels of expression [221]. Deng et al. further TABLE V! Summary of p53 alterations in various neoplasms

Tumor type

Observation

Breast cancer

elevated levels in 40% 271 ~ 3 in serum of 9% of patients 272 50-86% show accumulation 273, 274 of p53

Colorectal cancer

Lung carcinomas Esophageal cancer

elevatedin 45-70% 5/14 tumors had point mutations in p53 genes Sarcomas 6% have gene mutations Livercarcinoma 50% of tumors have p53 mutations Bladder cancer 11 of 18 invasivetumors showed genetic alterations Li-Fraumenisyndrome germlinep53 mutations

suggest that mutations of Ha-ras correlate with metastasis and sun, ival in gastric cancer patients [238]. There is a great deal of evidence which indicates that mutation of ras genes contr~bhtes to the developmeant _of colon carcinoma. Using nucleic acid-based probes, 50% of colon tumors examined by a number ot groups wer~ shown to have mutations in the ras gene [239-241]. Data further suggest that mutations are an early occurrence in the development of colon carcinoma; in many tumors the same mutation was observed in adenomatous as well as carcinomatous colon tissue. However, in some cases the mutation was found in carcinoma tissue only [239,241.242].

References

257, 275, 276 277 278 258, 279 280 270

In breast cancer it appears that overexpression of p21, rather than alteration through mutation of the gene, may be a contributing factor to the development of the disease. Spandidos and colleagues correlated elevation of H-ras mRNA with advanced histological types in breast tumors [211,243]. Another group reported normal levels of H-ras but increased levels of N-ras and K-ras [244]. In an immunochemical examination of breast tissue, Tanaka et al. found elevated K-ras and N-ras p21, but no increased amounts of H-ras [245], whereas another group using a different antibody did not observe any significant staining of malignant tissue [246]. In contrast, two studies performed using Western blot analysis found Ras to be elevated in malignant versus normal tissue [247,248] and further, a correlation existed between high levels of p21 and shortened disease-free survival [248]. The significance of elevated levels of ras, if indeed the elevation is a real phenomenon, remains to be be demonstrated. Standardized methods a n d / o r reagents may help to confirm or deny the observation. III-E.3. Ras in serum

Finally, as with p185 he', it appears as though p21 may be found in the circulation of patients with certain cancers. Kakkanas and Spandidos reported detection of p21 in the sera of mice bearing a Harvey murine sarcoma virus-induced tumor, as well as in certain human patient sera [249]. The amount of p21 detected both by ELISA and by Western blot in the sera of tumor-bearing mice correlated with the size of the tumor. In human patients, 3 of 13 cases (all of which had stomach adenocarcinomas and were receiving chemotherapy) presented higher p21 le"cls hi serum as compared to other cancer patients or normal individuals. A more quantitative double-determinant ELISA was reported by Hamer et al. to be capable of detecting mutant p21 in cell culture fluid and in plasma of tumor bearing mice [250]. Here again, Western blot assays were used to confirm the presence of the p21. In both studies, immunohistochemical analysis of the tu-

205 mor material corroborated the expression of the Ras oncoprot~,~n. It is unclear how the antigen1 may be gaining entry into the circulation, other than by necrosis of the tumor or immunocytotoxicity mechanisms. But once more only further study will bear out the significance of the observation. I I I - E . 4 . ras s w n m a r y

The ras oncogene product p21 appears to be involved in a variety of cancers through a number of mechanisms. Specific mutations in the ras gene occur in lung, pancreatic, colorectal, prostatic cancers, in leukemias and head and neck cancers, whereas overexpression of ras p21 oncoprotein appears primarily in breast and stomach cancers. It should be noted that where Ras is a normal cellular protein, mutated Ras is found associated or~ly with cancers and never with normal tissues. In this way mutant, activated p21 can be considered to be a true tumor-specific antigen. Obviously, further studies are required to unambiguously establish 'the significance of either activated or overexpressed ras in various tumors. It may be that through further investigation it will be apparent that specific mutations can be associated with specific neoplasms. It has already been demonstrated that in adenocarcinoma of the lung, pancreas, and colon, mutational activation occurs primarily in K-ras [224,226, 227,239-241,251]; whereas N - r a s mutations predominate in myeloid leukemias [232-236]. Quality monoclonal antibodies are available from certain commercial vendors; these include unique, mutation-specific monoclonals which recognize certain position 12 mutations (Applied bioTechnology, Cambridge, MA; Cetus Corp., San Fransisco, CA). Antibodies which recognize the family members H-, K- and N- are also available (Oncogene Sciences, Inc.). These types of reagent, along with immunoassays which incorporate them, will prove extre,'~ldy useful in defining the nature of mutations found in specific cancers. At the same time, assays which quantitate the levels of p21 in tissue will help define the correlation between overexpression and outcome in certain neoplasms. Given the preliminary results that activated p21 may even be found in sera, it seems that z quantitative assay, especially one for n~:~;tant forms of p21, would also be of great value. But if a large number of sera from additional patients were examined with a stan,4ardized, sensitive ELISA, data would become clear more rapidly as to the significance of Ras in serum. The same types of assay could also be used to assess tissue samples in order to supplement observations made by immunoh~stechemical analysis. The PCR/RNAase mismatch method could also be used to determine whether there were mutations in the ras gene of any given tumor. In this method, the DNA amplified through the PCR process is hybridized to

non-mutated RNA specific for ras. When this duplex is treated with RNAase, the mismatched R N A will be cleaved, and will appear as two bands when electrophoresed in an agarose or polyacrylamide gel. RNA which matches completely will not be cleaved, will appear as one band upon electrophoresis, and hence indicate the presence of a normal ras gene. One advantage to this method is that it would provide a rapid, simple way to determine the presence of a mutation. However, the method would require convincing proof that it could be useful for specific alterations in order to overcome the drawback that currently only 50% of mutations can be detected successfully. Additionally, the PCR technique would not be predicted to be of use on serum samples, as the DNA from the tumor would not expected to be found in significant amounts in the circulation. I l I . F . p 5 3 as an o n c o g e n e ?

-~,

Although p53 was originally identified as an oncogene [252] later experimentation indicated that all of the cloned p53 genes actually contained point mutations. Experiments using the mutated forms of p53 yielded cells with transformed phenotypes, whereas studies with the wild type p53 gene resulted in growth inhibition of cells transformed by the m y c or ras oncogenes [253-255]. Thus, it is suggested that the wild type p53 molecule acts as a tumor suppressor gene, performing an as yet unidentified function in maintaining normal cell growth (see discussion below), whereas mutated forms of p53 act as oncoproteins. Additionally, it has been shown by a number of investigators that the commonly observec~ deletions in the short arm of chromosome 17 observed in many cancers results in the loss of one p53 allele [256]. It is not surprising then that the phenotype of many cancers includes a deletion in chromosome 17, resulting in the loss of one p53 allele, together with mutations in the remaining p53 gene [256-258]. Unlike the ras p21 oncoprotein which has exhibited specific mutations in three codons, mutations in p53 occur in four different conserved regions of the protein [259]. Any mutation in one of these regions apparently alters the conformation of the protein to the degree that it can no longer function normally [260]. The change also leads to an increased half-life of the p53 ill the cell, with the consequent appearance that p53 is overexpressed by the tumor cell. Both the wild type p53 and p53 oncoprotein oligomerize in cells [261] and approximately half of the mutant r,xoteins studied to date have been shown to bind to ~ellular heat shock proteins [262-264]. Wild type p5 3 is knowa to bind to the transforming proteins of SV40 [265-267] and adenovirus [268] as well e.s with the E6 protein of human papilloma virus

206 [269]. It is postulated that the mechanism of action o[ the transforming viruses is at least in part due to binding and inactivation of a cell's normal growth regulatory protein, p53. Mutations in p53 have been discovered in a wide variety of cancers. This is exemplified by the studies summarized below on breast, colon and lung cancer, in addition to studies in carcinomas of the liver, esophagus and bladder, and one report on sarcomas. A recent report also describes germ-line mutations in p53 which o¢~:l " in a familial syndrome known as Li-Fraumeni syndrome, in which family members are predisposed to a number of diverse neoplasms including breast cancer and sarcomas [270]. See Table VI for a summary. Increased levels of p53, due to the accumulation of mutant p53 in the tumor cells, was reported by Crawford et al. in 40% of breast tumors examined [271]. The same group also identified the presence of antibodies in the serum of 9% of patients with breast tumors [272]. Mutant p53 oncoprotein has also been detected in 50% to 86% of colon tumors examined [273,274], and in 45% to 70% of lung cancers [257,275,276]. Studies of esophageal cancer have shown that 2 of 4 cell lines examined and 5 of 14 tumors had point mutations in p53 genes [277] and 6% of sarcomas examined had p53 mutations as reported by Suatton et al. [278]. Two recent studies of liver cancer reported that 50% of tumors had p53 mutations [258,279]. Both groups of investigators independently found that these mutations clustered at codon 249, and were characterized as a 13 ~ T substitution. Bressac ct al. [258] made this observation through the study of hepato. ¢~Uular ca~inoma (HCC) samples from southern Africa; the second group reached the same conclusions through the study of HCC from patients in China [279]. Not only is HCC prevalent in both of these regions of the world, but also, aflato~n B~, the main food-contaminatin$ aflatoxin in Africa and China, is known to be a risk factor for HCC. Additionally, in experimental s~tems, aflatoxin is known to induce G ~ T substitutions (see Ref. 258 and references therein). This raises the very interesting postulation that the distinct p53 mutation in HCC is actually a marker for the presence of a specific environmental carcinogen. Recently, Sidransky and colleagues were able to demonstrate the presence of p53 gene alterations by using PCR techniques on cells obtained from the urine of bladder cancer patients [280]. Of 18 invasive tumor specimens examined, II contained p53 mutations a,,~ determined by PCR of exons 5 to 9. The urine s~diment from three patients with identified p53 mutations was then tested by cloning the resulting PCR fragmenlts into a bacteriophage vector and hybridizing resulting bacteriophage clones to radiolabeled oligonucleotide probes specific for the p53 mutations. These investiga-

tors were thus able to demonstrate the presence of exactly the same mutations in cells in the urine as were detected in cells in the tumor tissue, in spite of an excess amount of wild type p53 sequences present in the urine sediment. Any one of a number of methods may be useful for the analysis of p53 mutations and their contributions to different cancers. Just as has been done in the research phase, investigators will be able to use PCR techniques to examine the mutations in the gene, either at the germiine level or the somatic level. This has already been demonstrated by the work on invasive bladder cancer by Sidransky and colleagues [280], and in the study on Li-Fraumeni syndrome [270]. PCR may prove to be an extremely powerful technique, as analysis of a readily available sample such as urine will undoubtedly contribute to the frequency of this type of analysis. This approach may also prove useful for analysis of cells in stool samples of patients with polyps, adenomas or carcinomas of the colon or rectum. In addition, since the mutant protein accumulates to levels 100 to 1000-fold greater than those seen in normal tissues, quantitation of p53 in tumor tissues by either immunohistochemical methods or ELISA should prove a viable alternative. Finally, more data may also demonstrate the presence of antibodies to p53 in a higher t~ercentage of patients with various cancels. This will allow analysis of an easily obtainable sample (serum) and will lend itself more readily to routine, repeated examination. It is enticing to speculate on the proposition of p53 as a general screen for cancer. Since another intracellular oncoprotein, ras p21, has been detected in serum (see above) it is not farfetched to think that a mutated p53 oncoprotein may also be detectable. A screen for p53 mutants, especially if used on families with predispositions to certain types of cancer, or even on the general population past a certain age, could potentially notify a physician of the presence of an otherwise undetected tumor. With this information in hand, one could justify a more rigorous, costly and invasive series of tests to identify exactly the nature of the neoplasm. 111-(7. Other oncogenes and their associations with cancer

A number of other oncogenes have been analyzed with respect to their association with a variety of diseases, although no large studies have as yet demonstrated strong correlations for any one of them. Some of these studies include the implication of e-s/s (the platelet derived growth factor/3 chain) with asbestosl'~l~ted di.~ease [281] and with lung cancer [282]; int-2 (a fibroblast growth factor-like oncoprotein) in breast cancer [283-285] and head and neck cancer [286]; and

207 fms (the receptor for macrophage colony stimulating factor) in gynecologic tumors [287] and in leukemias [2881. The be! gene is another whose translocation is associated at high frequency with certain B cell malignancies. In particular, 90% of follicular lymphomas have the t(14;18Xq32;q21)reciprocal translocation associated with this gene [289,290]. Neither the normal function of the gene product nor the effects of its deregulation are yet known, thus it remains in the ranks of the putative oncogenes. A recent report however has suggested that the protein product may be involved in programmed cell death [291]. Recently, the product of the trk gene, originally isolated from a colon carcinoma [292] was identified as the receptor for nerve growth factor in neurons [293,294]. This may represent an example of a normal growth factor receptor for one tissue becoming activated and possibly adding to the malignant transformation of another tissue. The diagnostic potential of any of these oncogenes or putative oncogenes awaits further definition. IV. Tumor suppressor" genes and cancer

In the years since retroviral oncogenes and their cellular counterparts were discovered gi-eat strides have been made towards identifying their potential role in the carcinogenic process. As most proto-oncogene products appear to be involved in normal pathways of growth signaling, it is logical that any change which causes their deregulation or constitutive activation would result in rampant cell growth. However, there are cell processes which control growth signaling under normal situations; why should they not be capable of controlling the activated oncogene products? The answer to these questions may lie in part with the more recently discovered class of molecules: the products of tumor suppressor genes, as represented by the prototype retinoblastoma gene [29-297]. As the products of oncogenes appear to activate cell growth at a number of stages in the signaling pathway, so might tumor ~uppressor gene products inhibit cell growth at various steps. It is thought that the retinoblastoma protein (Rb) may be involved in regulation of the cell cycle; unphosphorylated Rb is postulated to suppress cell growth and promote differentiation, whereas phosphorylatJon inactivates Rb and allows the cell to enter S phase [298]. Some other possible roles of tumor suppressor gene products may be: (1) contact inhibition receptors, (2) secreted factors that are negative regulators of the cell's own growth, or (3) secreted factors which induce end-stage differentiation of other cells [299]. Recent studies further suggest that some tumor suppressor gene products may take the form of enzymes such as gJrosine phosphatases

which have the reverse enzymatic action of the many tyrosine kinase receptor oncoproteins, such as ~cu [300-302]. Tumor suppressor genes may soon come to be considered more important contributors to the process of tumorigenesis than oncogenes, and as such, important targets for diagnosis and treatment of cancer. In fact, in the short time since the retinoblastoma gene was originally discovered, reagents have been developed to study the gene and its protein product, and Rb now appears to be involved in not only retinoblastoma, but also in osteosarcomas [295,303], small cell lung carcinoma [304], and bladder [305] and breast cancers [306). Similarly, as described above, p53 mutations have been detected in a wide variety of cancers and may be present in most types of cancers. It is possible that with continued investigation, this will also be the case :for other tumor suppressor genes. V. Summary The first clear cut association of an oncogene with a specific cancer is the c-abl translocation in chronic myelogenous leukemia and acute lymphocytic leukemia; it has been observed in 90% of CML cases examined. This is the major contributing factor to its being the target of the first oncogene-based FDA-approved diagnostic test. Although the role of the abl translocation in the tumorigenic process is not yet understood, it is clear that somehow it must be causally related to the disease, and thus is an ideal target for a diagnostic test. The association of this oncogene with a specific cancer is the model on which all others may be based in the future. Second generation tests could easily include PCR on mRNA, a n d / o r in situ hybridization, both of which could be performed using blood samples. Both methods would provide a faster means of testing a large number of cells, however, the methodologies must bt~ improved through automation and computer-aided image analysis, respectively, in order to become useful routine tests. Both neu and epidermal growth factor receptor (EGFR) appear to have a close correlation between overexpression of the gene product and outcome of disease in breast cancer: valuable informatien for prognosis of the disease. And again, although the actual mechanism of action of these molecules and how this relates to the tumorigenic process is not yet known, it is believed from the very nature of the molecules that they must in some way contribute to the progression of the disease. In both cases, the protein products are overexpressed in tissue, and in the case of Neu, it appears as though at least some of the patients have a Neu-related protein in their serum. These molecules present relatively easy targets for the development of diagnostic/prognostic assays, as antibodies are easily

208 made and can be incorporated into a variety of assay fermats. Current assays available, an ELISA for Neu and a radio-ligand binding assay for EGFR, are highly sensitive, reproducible and relatively easy to perform. Only the ELISA is commercially available, however, and hence allows for easy comparison between laboratories. An obvious step towards the routine measurement of EGFR then is the development of a comparable commercially available test. An improvement for both types of assay would be the incorporation of an internal control to gauge the cellular component of the tissue samples that are tested. The outcome of the applications of myc and ras to cancer diagno~,ics is not so easily predictable, with a couple of exceptions. In the case of N-myc, gene amplification is a significant prognostic indicator: patients with stage II disease and multiple gene copies should be treated as if they have stage 4 cancers. Clearly this is an important piece of informattt, a for thos~ treating patients. A prognostic assay in this case must take the form of a DNA-based assay which is capable of quantitating gene copy number, unless data become available which suggest that the protein is also overexpressed. The case of c-myc translocations in Burkitt's l~m. phoma is the second clear application of potential diagnostic/prognostic applications of myc detection. In this case, a DNA-based test, either using conventional probe technology and Southern blottil~g, or in situ hybridization, could be used to confirm diagnosis on an easily obtained blood sample. Other applications of ras and m y c oncogene measurement to cancer diagnosis and prognosis await further information. With the advent of appropriate immunoassays, DNA-based tests, and new tests not yet envisioned, the data will be developed which will aid in determining the relevance of these oncogenes in particular cancers. As for p53, mutations in this molecule appear to occur in a wide variety of cancers. Although this speaks to the importance of its normal cellular role, it remains unclear at this point in time how it may be utilized as a distinguishing factor for diagnosis or prognosis. It may be that specific mutations will be prevalent in certain cancers, as appears to be the case for ras, or that those p53 mutants which bind heat shock proteins provide a specific prognosis. Certain p53 mutations may also be indicative of environmental carcinogens, as appears to be the case with aflatoxin and hepatocellular carcinoma. And more study must be done to fully appreciate the implications of the germ-line mutations observed in Li-Fraumeni syndrome. Again, only by continued investigation into the association of p53 with various neoplasms will its usefulness in diagnosis, and the most appropriate method(s) of study, become understood. In the 1950s, Foulds first postulated that inoependent multiple events led to the neoplastic phenotype,

and consequently that the same transformed phenotype can be reached through many possible pathways [307,308]. Certainly, this postulate is being borne out by the evidence generated concerning oncogenes and tumor suppressoc genes. The most recent information generated by Vogelstein and colleagues points to exactly this multi-step process in colorectal carcinogenesis. With the discovery of yet another chromosomal deletion associated with this disease, and cloning of the gene named 'DCC' ('deleted in colorectal cancer') [309] these investigators added another step in the development scheme, which previously included mutations in a gene (MCC; 'mutated in colorectal cancer') located on chromosome 5 [310], ras gene mutations, loss of the DCC gene from chromosome 18 and alteration in chromosome 17 resulting in mutated p53. It is likely that colorectal cancer is a paradigm for other cancers in this respect. Thus, it may be that detection of the activation of any one oncogene, or the loss of any one tumor suppressor gene, will not provide the definitive diagnosis for any one cancer. It is more likely that analysis of a single tumor biopsy would indicate at best only a snapshot in time of the genetic changes occurring in that malignancy. More significant information may be gained by searching for multipk elements at many time points in the same neoplasm, ~'~nddetermining risk of development or of progression of disease based on the accumulation of these multiple events. It is obvious that our knowledge continues to grow as investigation into the mechanisms of tumorigenesis proceeds. We must continue to define the events which are involved, the inherited and acquired genetic changes that lead to the development of cancer, as well as later somatic changes which are involved in tumor progression. With continued research, we will be able to gain insight into the biochemical signaling pathways involved and through that insight we will be able invent new ways to search for the genetic damage which today remains undetected. Gains in our basic knowledge will have significant impact on identifying those oncogene tumor markers which will be useful for diagnosis, predicting susceptibility to specific cancers, and devising new strategies for treatment of the disease.

Acknowledgements I would like to thank Dr. Arthur Bruskin for his review and valuable commentary on the manuscript during its preparation, and for the use of Fig. I. Thanks also to Dr. Walter Carney for his assistance on the ras section of the article.

References 1 Rous, P. (1967) Science 157, 24-28. 2 Goff, S.P., Gilboa, E., Witte, O.N. and Baltimore, D. (1980) Cell 22, 777-785.

209 3 Witte, O.N., Golf, S., Rosenberg, N. and Baltimore, D. (19801 Proc. Natl. Acad. Sci USA 77, 4493-4997. 4 Prywes, R., Foulkes, J.G., Rosenberg, M. and Baltimore, D. (1983) Cell 34, 569-579. 5 Reynolds, R.K., Van de Ven, W.J.M. and Stephenson, J.R. (19811) J. Virol. 36, 374-386. 6 Rosenberg, N.E., Clark, D.R. and Witte, O.N. 119801 J. ViroL 36, 766-774. 7 Nowell, P.C. and Hungerford, D.A. (1961)J. Natl. Canc. Inst. 27, 1013-1035. 8 Stam, K., Heisterkamp, N., Reynolds, F.H. and Groffen, J. (1987) J. Mol. Cell. Biol. 7, 1955-1960. 9 De Klein, A., Van Kessel, A.G., Grosveld, G., Bartram, C.R., Hagemeijer, A., Bootsma, D., Spurr, N.K., Heisterkamp, N., Groffen, J. and Stephenson, J.R. (19821 Nature 300, 765-767. l0 Bartram, C.R., De Klein, A., Hagemeijer, A., Van Agthoven, T., Van Kessel, A.G., Bootsma, D., Grosveid, G., Ferguson-Smith, M.A., Davies, T., Stone, M., Heisterkamp, N., Stephenson, J.R. and Groffen, J. (1983) Nature 3116, 277-281~. 11 Heisterkamp, N., Stephenson, J.R., Groffcn, J., Hanscn, P.F., De Klein, A., Bartram, C.R. and Grosve~d, tll. (19831 Nature 306, 239-242 12 Heisterkamp, N., Stare, K., Groffen, J., Dc Klein, A. and Grosveld, G. 119851 Nature 315, 758-761. 13 Groft~n, J., Stephcnson, J.R., ltcisterkamp, N., De Klein, A., Bartram, C.R. and Grosveld, G. (1984) Cell 36, 93-99. 14 Hariharan, I.K. and Adams, J.M. (19871 EMBO J. 6, 115-119. 15 Shtivelman, E., Lifshitz, B., Gale, R.P and Canaan,, E. (1985) Nature 315, 550-554. 16 Nowell, i~'.C, and Hungerford, D.A. 119601 Science 132, 14971499. 17 Konopka J.B. Watanabe, S.M., Singer, J.W., Collins, S.J. and Witte, O.iq. (19851 Proc. Natl. Acad. Sci. USA 82. 1810-1814. 18 Davis, R.L., Konopka, J.B. and Witte, O.N. 119851 Mol. Cell. Biol. 5, 2114-213. 19 Konopka, J.B., Watanabe, S.M. and Witte, O.N. 11984) Cell 37, !1135- i 042. 20 Konopka, J.B. and Witte, O.N. (1t185) Mol. Cell. Biol. 5, 31163123. 21 Ben-Neriah, Y., Daley, G.te., Mes-Masson, A.-M., Witte, O.N. and Baltimore, D. 119861 Science 233, 212-214. 22 Rowley, J.D. 11973) Nature 243, 290-293. 23 Catovsky, D. (19791 Br. J. Haematol. 42, 493-498 24 Kurzrock, R., Shtalrid, M., Romero, P., Kloetzer, W.S., Talpas, M., Trujillo, J.M., Blick, M., Beran, M. and Gutterman, J.U. (1987) Nature 325, 631-635. 25 Walker, L.C., Ganesan, T.S., Dhut, S., Gibbons, B., Lister, T.A., Rothbard, J. and Young, B.D. 119871 Nature 329, g51-1,,53. 26 Hermans, A., Heisterkamp, N., Von Lindem, M., Van Baal, S., Meijer, D., Van der Plas, D., Wiedemann, L.M., Groffen, J., Bootsma, D. and Grosveld, G. 11987) Cell 51, 33-40. 27 Southern, E.M. 119751 J. Mol. Biol. 98, 503-517. 28 Kawasaki, E.S., Clark, S.S., Coyne, M.Y., Smith, S.D., Chaml~lin, R., Witte, O.N. and McCormick, F.P. (1988) Proc. Natl. Acad. Sci. USA 85, 5698-5702. 29 Schecter, A.L., Stern, D.F., Vaidyanathan, L., Decker, S.I., Drebin, J.A., Green, M.I. and Weinberg, R.A. 11984) Nature 312, 513-516. 30 Sehecter, A.L., Hung, M.-C., Vaidyanathan, L., Weinberg, R.A., Yang-Feng, T.L., Franke, U., UIIrich, A. and Coussens, L. 119851 Science 229, 976-978. 31 Yamamoto, T., Ikawa, S., Akiyama, T., Semba, K., Nomura, N., Miyajima, N., Saito, T. and Toyoshima, K. 11986) Nature 319, 230-234. 32 Ulirich, A., Coussens, L., Hayflick, J.S., Dull, T.J., Gray, A., Tam, A.W., Lee, J., Yarden, Y., Libermann, T.A., Schlessinger,

J., Downward, J., Mayes, E.L.V., Whittle, N., Waterfield, M.D. and Seeburg, P.H. (19841 Nature 309, 418-425. 33 Downward, J., Yarden. Y., Mayes, E., Scrace, G., Totty, N., Stockwell, P., Ullrich, A., Schlessinger, J. and Waterfieid, M.D. 119841 Nature 307, 521-527. 34 Lupu, R., Colomer, R., Zugmaier, G., Sarup, J., Shepard, M., Siamon, D. and Lippman, M.E. (19901 Science 249, 1552-1555. 35 Bargman, C.I., Hung, M.-C. and Weinberg, R.A. 119861 Cell 45, 649-657. 36 Bargman, C.I. and Weinberg, R.A. 119881 EMBO Jo 7, 20432052. 37 Slamon, D.J., Ciark, G.M.. Wong, S.G., Levin, W.J., Uilrich, A. and McGuire, W.L. 119871 Science 235, 177-182. 38 Varley, J.M., Swallow, J.W., Breammar, W.J., Whittaker, J.L. and Walker, R.A. 119871 Oncogene 1,423-430. 39 Van de Vijver, M., Van de Bersselaar, R., Devilee, P., Cornelisse, C., Peterse, J. and Nusse, R. 119871 Mol. Cell. Biol. 7, 21119-2023. 411 Zhou, D., Battifora, it., Yokota, J., Yamamoto, T. and Clhle, M.J. 11987) Cane. Res. 47, 6123-6125. 41 Cline, M,J., Battifora, |I. and Yokota, J. 119871J. Clin. Oncol. 5, 999-1006. 42 Borg, A., Baidetorp, B., ~Terno, M., Killander, D., Olsson, H. and Sigurdssen, H. 119911 Oncogene 6, 137-143. 43 Tsutsunli, Y., Naber, S . P DeLeilis, R.A., Wolfe, |-l.J., Marks, P.J., McKenzie, S.J .... d "a'i~,S. ~19901 Hum. Pathol. 21,750-758. 44 McCann, A., Dervan, P.A., Johnston, P.A., Gullick, W.J. and Carney, D.N. 11990) Cancer 65, 88-92. 45 Slamon, D.J., Godolphin, W., Jones, L.A., Holt, J.A., Wong, S.G., Keith, D.E., Levin, W.J., Stuart, S.G., Udove, J., UIIrich, A. and Press, M.F. 119891 Science 244, 707-712. 46 Venter, D.J., Tuzi, N.L., Kumar, S. and Gullick, W.J. 11987) Lancel 69--72. 47 Borg, A., Tandon, A.K., Sigurdsson, H., Clark, G.M., Ferno, M., Fuqua, S.A.W., Killander, D. and McGuire, W.L. (1990'~ Cancer Res. 50 4332-4337. 48 Bcrger, M.S., Locher, ti.W., 5aurer, 5, £Juillt:K, W.J., Waterfield, M.D., Groner, B. and Hynes, N.E. 11988) Cancer Res. 48, 1238-1243. 49 Tandon, A.K., Clark,.G.M, Chamness, G.C., UIIrich, A. and McGui'e, W.L. (1989)J. Clin. Oncol. 7, 1120-1128. 51) Paik, S., Hazan, R., Fisher, E.R., Sass, R.E., Fisher, B., Redmond, C., Schlessinger, J., Lippman, M.E. and King, C.R. 11990) J Clin. Oncol. 8, 103-112. 51 Thor, A.D., Schwartz, L.H., Koerner, F.C., Edgerton, S.M., Skates, S.J., Yin, S., McKenzie, S.J., Panicali, D.L., Marks, P.J., Fingert, H.J. and Wood, W.C. 119891 Cancer Res. 49, 7147-7152. 52 Berchuk, A., Kamel, A., Wbitaker, R., Kerns, B., OIt, G., Kinney, R., Soper, J.T., Dodge, R., Clarke-Pearson, D.L., Marks, P., McKenzie, S., Yin, S. and Bast, R.C., Jr. 119901 Cancer Res. 50, 4087-4091. 53 Kern, J.A., Schwartz, D.A., Nordberg, J.E., Weiner, D.B., Greene, M.I.. Torney, L. and Robinson, R.A. 11990) Cancer Res. 50, 5184-5191. 54 Yonemura, Y., Ninomiya, I., Yamaguchi, A., Fushida, S., Kimura, H., Ohoyama, S., Miyazaki, I., Endou, Y., Tanaka, M. and Sasaki, T. 119911 Cancer Res. 51, 1034-1038. 55 Zabr,'cky, J.A., Lain, T., McKenzie, S.J. and Carney, W. (1991) J. Biol. Chem. 266, 1716-1720. fr~ Mori, S., Mori, Y., Mukaiyama, T., Yamada, Y., Sonobe, Y,, Matsushita, H., Sakamoto, G., Akiyama, T., Ogawa, M., Shiraishi, M., Toyoshima, K. and Yamamoto, T. (19901 Jpn. J. Cancer Res. 81,489-494. 57 Hayes, D.F., Carney, W., Tondini, C., Petit, D,, Henderson, I.C. and Kufe, D.W. 119891 Breast Cancer Treat. 14, 135.

210 58 McKenzie, S.J., Marks, P.J., Lain, T., Morgan, J., Panicali, D.L., Trimpe, K.L and Carney, W.P. (1989) Oncogene 4, 543-548. 59 Paterson, M.C., Dietrich, K.D., Danyluk, J., Paterson, A.H.G., Lees, A.W., Jamii, N., Hanson, J., Jenkins, H., Krause, B.E., McBlain, W.A., Siamon, D.J. and Fourney, R.M. (1991) Cancer Res. 51, 556-567. 60 Clark, G.M. and McGuire, W.L. (1991) Cancer Res. 51,944-948. 61 Carpenter, G. (1987) Annu. Rev. Biochem. 56, 881-914. 62 Betrics, PJ. and Gill, G. (1985) J. Biol. Chem. 260, 14642-14647. 63 Sehlessinger, J. (1988) Trends Biochem. Sci. 13, 443-447. 64 Greenfield, C., Hils, I., Waterfield, M.D., Federwisch, W., Wollmer, A., Blundell, T.L and McDonald, N. (1989) EMBO J. 8, 4115-4124. 65 Yarden, Y. and Schlessinger, J. (1987) Biochemistry 26, 14431451. 66 Yarden, Y. and Schlessinger, J. (1987) Biochemistry 26, 14341442, 67 Boni.Schnetzler, M. and Pilch, P,F. (1987) Proc. Natl, Acad. Sc. USA 84, 7832-7836. 68 Margolis, B., Rhee, S.G, Felder, S., Metric, M., Lyall, R., Levitzki, A., Ullrich, A., Zilberstein, A. and Schlessinger, J. (1989) Cell 57, 1101-1107. 69 Meisenhelder, J., Suh, P.-G., Rhee, S.¢3 and Hunter, T. (1989) Cell 57, 1109-1122. 70 Wahl, M., Nishibe, S., Sub, P.-G., Rhee, S.G. and Carpenter, G. (1989) Prec. ',~t|. Acad. Sci. USA 86, 1568-1572. 71 Guilick, W.J, Marsden, J.J., Whittle, H., Ward, B., Bobrow, L. and Waterfield, M.D. (1985) Cancer Res. 46, 285-292. 72 Hunts, J., Ueda, M., Ozawa, S., Abe, O., Pastan, I. and Shimuzu, N. (1985) J. Cancer Res. 76, 663-666. 73 Richards, C.S., Hendler, F., Shum, A., Burns, D., Gusterson, B., Cowley, G., Smith, J.A. and Ozanne, B. (1986) J. Pathol. 149, 9-14. 74 Yamamoto, T., Kamata, N., Kawano, H., Shimuzu, S., Kuroki, T., Toyoshima, K., Rikimaru, K., Nomura, N., lshizaki, R., Pastan, I., Gamou, S. and Shimuzu, N. (1986) Cancer Res. 46, 414-416. 75 Liberman, T.A., Nusbaum, H.R., Razon, N., Kris, R., Lax, I., Soreq, H., Whittle, N., Waterfield, M.D., Ullrich, A. and Schlessinge~, J.D. (1985) Nature 313, 144-147. 76 Neal, !~., Marsh, C,, Bennet, M.K., Abel, P.D., Hall, R.R., Sainsbury, J.R.C. and Harris, A.L (1985) Lancet i, 366-368. 77 BerBer, M~S., Greenfield, C., Gullick, W.J., Haley, J., Downward, J,, Neal, D.E,, Harris, A.L. and Waterfield, M.D. (1987) Br. J. Cancer 56, 533-537. 78 Neai, D.E. Smith, K,, Fennelly, J.A,, Bennett, M.K., Hall, R.R. and Harris, A,L, (1989) J. Urol. 141, 517-521. 79 Harris, A. (1990) Prec. Am. Assoc. Cancer Res. 31, 458-460. 80 Morb'ama, M., Akiyama, I"., Kawamoto, T., Kato, T, Sato, K., Watanuki, T., Katsuta, N. and Mori, S. (1991) J. Urol. 145, 423-42"7. 81 Cerny, 3"., Barnes, D.M., Haselton, P, Barber, P.V., Healy, IL, Gullick, W. and Thatcher, N. (1986) Bt'. J. Cancer 54, 265-269. 82 Veale, D, Ashcroft, T., Marsh, C., Gibson, G.J. and Harris, A.L. (1987) Br. J. Cancer 55, 513-516. 83 Hendler, F., Shum-Siu, A., Oechsli, M., Nanu, L., Richards, C.S. and Ozanne, B.W. (1989) Cancer Cells 7, 347-351. 84 Veale, D, Kerr, N., Gibson, G.J. and Harris, A.L (1989) Cancer Res. 49, 1313-1317. 85 Foekens, J.A., Van Putten, W.L, Portengen, H, Rodenburg, C.J., Reubi, J.C, Berns, P.M., Henzen-Logmans, S.C., Van der Berg, M.E, Alexieva-Figusch, J. and Klijn, J.G. (1990) J. Steroid Bioehem. Mol. Biol. 37, 815-821. 86 Berchuk, A., Rodriguez, G.C., Kamel, A., Dodge, R.K., Soper, J.T., ClarkeoPe~son, D.L and Bast, R.C, Jr. (1991) Am. J. Obstet. GynecoL 164, 669-674.

87 Ozawa, S., Ueda, M., Ando, N., Shimuzu, N. and Abe, O. (1989) Cancer 63, 2169-2173. 88 Yano, H., Shiozaki, H., Kohayashi, K., Yano, T., Tahara, H., Tamura, S. and Mori, T. (1991) Cancer 67, 91-98. 89 Wong, A.J., Bigner, S.H., Bigner, D.D., Kinzler, K.W., Hamilton, S.R. and Vogelstein, B. (1987) Prec. Natl. Acad. Sci. USA 84, 6899-6903. 90 Humphrey, P.A, Wong, A.J., Vogeistein, B., Friedman, H.S., Werner, M.H., Bigner, D.D. and Bigner, S.H. (1988) Cancer Res. 48, 2231-2238. 91 Malden, L.T, Novak, U., Kaye, A.H. and Burgess, A.W. (1988) Cancer Res. 48, 2711-2714. 92 Yamazaki, H., Fukui, Y., Ueyama, Y, Tamaoki, N., Kawamoto, T., Taniguchi, S. and Shibuya, M. (1988) Mol. Cell. Biol. 8, 1816-1820. 93 Lax, I., Beilot, F., Howk, R., Ullrich, A., Givol, D. and Schiessinger, J. (1989) EMBO J. 8, 421-427. 94 Perez, R., Pascual, M., Macian, A. and Lage, A. (1984) Breast Cancer Res. Treat. 4, 189-193. 95 Fitzpatrick, S.L., Brightweli, J., Witdiff, J.L., Barrows, G.H. and Schultz, G.S. (1984) Cancer Res. 44, 3448-3451. 96 Sainsbury, J.R.C., Farndon, J.R., Sherbet, G.V. and Harris, A.L. (1985) Lancet i, 364-366. 97 Sainsbury, J.R.C., Farndon, J.R., Harris, A,L. and Sherbet, G.V. (1985) Br. J. Surg. 72, 186-188. 98 Llorens, M.A., Bermejo, M.J., Salcedo, M.C, Charro, A.L. and Puente, M. (1989) J. Steroid Biochem. 34, 505-509. 99 Harris, A.L., Nicholson, S., Sainsbury, R.C., Farndon, J. and Wright, C. (1989) J. Steroid Biochem. 34. 123-131. 100 Nicholson, S., Richard, J., Sainsbury, ~., :~alcrow, P., Kelly, P., Angus, B., Wright, C., Henry, J., Farna~.% J.R. and Harris, A.L. (1990) Br. J. Cancer 63, 146-150. 101 To, M., Nakamura, T., Mukaida, H., Wada, T., Osaki, A., Yamada, H., Toge, T., Niimoto, M. and Hattori, T. (1990) Cancer 65, 1980-1984. 102 Bevilacqua, P., Gasparini, G., Dal Fief, S. and Corradi, G. (1990) Oncology 47, 313-317. 103 Dotzlaw, H, Miller, T., Karvelas, J. and Murphy, L.C. (1990) Cancer Res. 50, 4204-4208. 104 Bolufer, P., Mirailes, F., Rodriguez, A., Vazquez, C., Liuch, A., Garcia-Conde, J. and Oimos, T. (1990) Eur. J. Cancer 26, 283-290. 105 Lewis, S., Locker, A., Todd, J.H., Bell, J.A., Nichoison, R., Eiston, C.W., Blarney, R.W. and Ellis, !.O. (1990) J. Clin. Pathol. 43, 385-389. 106 Wiehle, R.D., Johnson, W.T., Doug, J. and Wittliff, J.L. (1990) FASEB J. 4, A2142. 107 Mcndelsohn, J. (1990) Proc. Am. Assoc. Cancer Res. 31, 460461. 108 Kalofonos,H.P, Pawlikowska,T.R., Hemingway,A., CourtenayLuck, N, Dhokia, B., Snook, D., Sivolapenko, G.B., Hooker, G.R., McKenzie, C.G., Lavender, P.J., Thomas, D.G.T. and Epenetos, A.A. (1989) J. Nucl. Med. 30, 1636-1645. 109 Duesberg, P.H., Blister, K. and Vogt, P.K. (1977) Proc. Natl. Acad. Sci. USA 74, 4320-4324. 110 Sheiness, D. and Bishop, J.M. (1979) J. Virol. 31, 514-521. 111 Klein, G. and Klein, E. (1985) Nature 315, 190-195. 112 Jones, N. (1 990) Cell 61, 9-11. 113 Olson, E.N. (1990) Genes Dev. 4, 1454-1461. 114 Lassar, A.B., Buskin, J.N., Lockshon, D., Davis, R.L., Apone, S., Hauschka, S.D. and Weintraub, H. (1989) Cell 58, 823-831. 115 Murre, C., McCaw, P.S. and Baltimore, D. (1989) Cell 56, 777-783. 116 Murre, C., McCaw, P.S., Vaessin, H., Caudy, M., Jan, L.Y., Jan, Y.N., Cabrera, C.V., Buskin, J.N., Hauschka, S.D., Lassar, A.B., Weintraub, H. and Baltimore, D. (1989) Cell 58, 537-544.

211 117 O'Shea, E.K., Rutkowski, R.H., Stafford, W.S. and Kim, P.S. (1989) Science 245, 646-648. 118 Vinson, C.R., Sigler, P.B. and McKnight, S.L. (1989) Science 246, 911-916. 119 Davis, R.L., Chen, P.-F., Lassar, A.B. and Weintraub, H. (1990) Cell 60, 733-746. 120 Blackwell, T.K., Kretzner, L., Blackwood, E.M., Eisenman, R.N. and Weintraub, H. (1990) Science 250, 1149-1151. 121 Luscher, B. and Eisenman, R.N. (1990) Genes Dev. 4, 20252035. 122 Schwab, M., Alitalo, K., Klempnauer, K.-H., Varmus, H., Bishop, J., Gilbert, F., Brodeur, G., Goldstein, M. and Trent, J. (1983) Nature 305, 245-247. 123 Seeger, R.C., Brodeur, G.M., Sather, H., Dalton, A., Siegel, S.E., Wong,, K.Y. and Hammond, D. (1985) N. Engl. J. Med. 313, 1111-1116. 124 Schwab, M., EIiison, J., Buscil, M., Rost:nau, W., Varmus, H.E. and Bishop, J.M. (1984) Proc. Natl. Acad. Sci. USA 81, 49404944. 125 Brodeur, G.M., Seeger, R.C., Schwab, M., Varmus, H.E. and Bishop, J.M. (1984) Science 224, 1121-1124. 126 Kohl, N.E., Kanda, N., Schreck, R.R., Burns, G., Latt, S.A., Gilbert, F. and AIt, F.W. (1983) Cell 35, 359-367. 127 Kohl, N.E., Gee, C.E. and Air, F.W. (1984) Science 226, 13351337. 128 Michitsch, R.W., Montgomery, K.T. and Me,era, P.W. (1984) Mol. Cell. Biol. 4, 2370-2380. 129 Lieberman, T.A., Nusbaum, H.R., Razor, } , KAs, R., Lax, I., Soreq, H., Whittle, N., Waterfield, M.D. L ~ricb A. and Schlessinger, J. (1985) Nature 3i3, 144-147. 130 Trent, J., Meltzer, P., Rusenblum, M., :-larsh, G., Kinzler, K., Mashal, R., Feinberg, A. and Vogelsteil,, L (1986) Proc. Natl. Acad. ScL USA 83, 470-473. 131 Collins, S. and Groudine, M. (1982) Natul~~. 298, 679-681. 132 Dalla-Favera, R., Wong-Staal, F. and Gaik" R.C. (1982) Nature 299, 61-63. 133 McCarthy, D.M., Rassool, F.V., Goldman, J.M., Graham, S.V. and Birnie, G.D. (1984) Lancet ii, 1362-1355. 134 Alitalo, K., Schwab, M., Lin, C.C., Varnms, H.E. and Bishop, J.M. (1983) Proc. Natl. Acad. Sci. USA 80, 1707-1711. 135 Lin, C.R., Chen, W.S., Kruiget, W., Stolarsky, L.S., Weber, W., Evans, R.M., Verma, I.M., Gill, G.N. and Rosenfcld, M.G. (1984) Science 224, 843-848. 136 Merlino, G.T., Xu, Y.-H., Ishii, S., Clark, A.J.L., Semba, K., Toycshima, K., Yamamoto, T. and Pastan, I. (1984) Science 224, 417-419. 137 Finley, G.G., Schuitz, N.T., Hill, S.A., Geiser, J.R., Pipas, J.M. and Meisler, A.I. (1989) Oncogene 4, 963-971. 138 Klein, G. (1 983) Cell 32, 311-315. 139 Dalla-Favera, R., Bregni, M., Erickson, J., Patterson, D., Gallo, R.C. and Croce, C.M. (1982) Proc. Natl. Acad. Sci. USA 79, 7824-7827. 140 Taub, R., Klrsch, I., Morton, C., Lenoir, G., Swan, D., Tronick, S., Aaronsen, S. and Leder, P. (1982) Proc. Natl. Acad. Sci. USA 79, 7837-7841. 141 Croce, C.M., Thierfelder, W., Erikson, J., Nishikura, K., Finan, J., Lenoir, G.M. and Nowell, P.C. (1983) Proc. Nat. Acad. Sci. USA 80, 6922-6926. 142 Erikson, J., Nishikura, K., Ar-Rushdi, A., Finan, J., Emanuei, B., Lenoir, G., Nowell, P.C. and Croce, C.M. (1983) Proc. Natl. Acad. Sci. USA 80, 7581-7585. 143 Emanuel, B.S., Seldon, J.R, Chaganti, R.S.K., Jhanwar, S., Nowdl, P.C. and Croce, C.M. (1984) Proc. Natl. Acad. Sci. USA 81, 2444-2446. 144 Taub, R., Moulding, C., Battey, J., Murphy, W., Vasicek, T., Lenoir, G.M. and Leder, P. (1984) Cell 36, 339-348. 145 Cesarman, E., Dalla-Favera, R., Bentley, D. and Groudine, M. (1987) Science 238, 1272-1275.

146 Erikson, J., Ar-Rushdi, A., Drwinga, H.L., Nowell, P.C. and Croce, C.M. (1983) Proc. Natl. Acad. Sci. USA 80, 820-824. 147 Erikson, J., Finger, L., Sun, L., Ar-Rushdi, A., Nishikura, K., Minowada, J., Finan, J., Emanuel, B.S., Nowell, P.C. and Croce, C.M. (1986) Science 232, 884-886. 148 Hayday, A.C., Gillies, S.D., Saito, H., Wood, C., Wiman, K., Hayward, W.S. and Tonegawa, S. (1984) Nature 307, 334-340. 149 Eick, D., Piechaczyk, M., Henglein, B., Blanchard, J.-M., Traub, B., Kofler, E., Wiest, S., Leaoir, G.M. and Bornkamm, G.W. (1985) EMBO J. 4, 3717-3725. 150 Rabbitts, P.H., Forster, A., Stinson, M.A. and Rabbitts, T.H. (1985) EMBG 1 4, 3727-3733. 151 Nau, M.M., Brooks, B.L, Battey, J., Sausviile, E., Gazdar, A.F., Kirsch, I.R., Wesley McBride, O., Bertness, V., Hollis, G.F. and Minna, J.D. (1985) Nature 318, 69-73. 152 Nau, M.M., Brooks, B.J., Carney, D.N., Gazdar, J.F., Bat:eL J.F., Sausville, E.A. and Minn,:, J.D. (1986) Proc. Natl. Acad. Sci. USA 83, 1092-1096. 153 Johnson, B.E., lhde, D.C., Makuch, R.W., Gazdar, A.F.~ Carney, D.N., Oie, H., Russell, E., Nau, M.M. and Minna, J.D. (1987) J. Clin. Invest. 79, 1629-1634. 154 Johnson, B.E., Makuch, R.W, Simmons, A.D., Gazdar, A.F., Burch, D. and Cashell, A.W. (1988) Cancer Res. 48, 5163-5~66, ~55 Brennan, J., O'Connor, T, Makuch, R.W., Simmons, A.I~'~ Russell, E., Linnoila, R.I., Phelps, R.M., Gazdar, A.F., lhde, D.C. and Johnson, B.E. (19~'1) Cancer Res. 51, 1708-1712. 156 Wong, A.J., Ruppert, J.M., EggL'ston, J., Hamilton, S.R., B: ylirh S.B. and Vogelstein, B. (1986) Science 233, 461-464. 157 Kiefer, P.E., Bepler, G., Kubasch, M. and Haremann, K. (1987) Cancer Res. 47, 6236-6242. 158 Yokota, J., Tsuwetsugu-Yokota, V.. Battifora, H,, Le Fevre, C. and Ciine, J.J. (1986) Science 231, 261-267. 159 Allum, W.H., Newbold, K.M., MacDonald, F., Russell, B. and Stokes, H. (1987) Br. J. Cancer 56, 785-786. 160 Field, J.K. and Spandidos, D.A. (1990) Anticancer Res. 10, 1-22.

161 Escot, C., Theillet, C., Lidereau, R., Spyratos, F., Campeme, M., Gest, J. and Callahan, R. (1986) Proc. Nat. Acad. Sci. USA 83, 4834-4838. 162 Bonilla, M., Ramirez, M., Lopez-Cueto, J. and Gariglio, P. (1988) J. Natl. Cancer. Inst. 80, 66~-671. 163 Guerin, M., Barrois, M., Terrier, M.J., Sp¢ilmann, M. and Riou, G. (1988) Oncogene Res. 3, 21-31. 164 Erisman, M.D., Litwin, S., Keidan, R.D., Comis, R.L. and Astrin, S.M. (1988) Cancer Res. 48, 1350-1355. 165 Monnat, M., Tardy, S., Saraga, P., Diggelman, H. and Costa, J. (1987) Int. J. Cancer 40, 293-299. 166 Meltzer, S.J., Aheen, D.J., Battifora, H., Yokota, J. and Cline, J.J. (1987) Gastroenterology 92, 1174-1180. 167 Erisman, M.D., Rothberg, P.G., Diehl, R.E., Morse, C.C., Spandoffer, J.M. and Astrin, S.M. (1985) Mol. Cell. Biol. 5, 19691976. 168 Rothberg, P.G., Spandorfer, J.M., Erisma~, M., Staroscik, R.W., Sears, H.F., Peterson, R.O. and Astrin, S.M. (1985) Br. J. Cancer 52, 629-632. 169 Jones, D.J., UhosL A.K., Moore, M. and Schofield, P.F. (1987) Br. J. Cancer 56, 779-783. I70 Heerdt, B.G., Molinas, S.. Deitch, D. and Augenlicht, L.H. (1o91) Oncogene 6, 125-129. 171 R~ou, G., Barrois, M., Le, M.G., George, M., Le Doussal, V. and Hale, C. (1987) Lancet ii, 761-763. 172 Field, J.K. and Spandidos, D.A. (1987) J. Oral Pathol. 16, 97-107. 173 Spandidos, D.A., Lamothe, A. and Field, J.K. (1985) Anticancer Res. 5, 221-224. 174 Field, J.K., Lamothe, A. a~id Spandidos, D.A. (1986) Anticancer Res. 6, 595-600. 175 Saranath, D., Panchal, R.G., Nair, R., Mehta, A.R., Sanghari,

212 V., Sumegi, J., Klein, G. and Doe, M.G. 119891 Jpn. J. Cancer Res. 80, 430-437 176 Harvey, J.J. (1964)Nature 104, 1104-1105. 177 Barbacid, M. 119871 Annu. Rev. Biochem. 3o, 779-827. 178 Langbeheim, H., Shih, T.Y. and Scolnick, E.M. 119801 Virology 106, 292-300. 179 Shilo, B. and Weinberg, R.A. 119811 Proc. Natl. Acad. Sci. USA 78, 6789-6792. 180 Ellis, R.W., DeFeo, D., Shih, T.Y., Gonda, M.A., Young, H.A., Tsuchida, N., Lowy, D.R. and Scoinick, E.M. (1981) Nature 292, 506-51i. 181 DeFeo-Jones, D., Scolnick, E.M., Koiler, R. and Dhar, R. 119831 Nature 306, 707-709. 182 Neuman-Silberberg, F.S., Schejter, E., Hoffman, F.M. and Shilo, B.-Z. (19841 Cell 37, 1027-1033. 183 Tgmeles, G.L, DeFeo-Jones, D., Tatchell, K., Elliagcr, M.S. and Scolnick, E,M. (1984) Mol. Cell. Biol. 4 2298-2305. 184 Fukui, Y. and Kaziro, Y. 119851 EMBO J. 4, 687-691. 185 Willingham, M.C., Pastan, I., Shih, T.Y. and Scoinick, E.M. (19801 Cell 19, 10115-1014. 186 Hancock, J.F., Magcc, A.l., Childs, J.E. and Marshall, C.J. (1989) Cell 57, 1167-1177. 187 Willamsen, B,, Christensen, A., Hubbcrt, N.L., Papageorge, A.G. and Lowry, D.R. 119841 Nature 310, 583-586. 188 Tabin, C.J., Bradley, S.M., Bargmann, C.I., Weinberg, R.A., Papageorge, A,G., Scolnick, E.M., Dhar, R., Lowy, D.R. and Chang, E.H. (19821 Nature 300, 143-149. 189 Reddy, E.P., Reynolds, R.K., Santos, E. and Barbacid, M. (19821 Nature 300, 149-152. 1911 Yuasa, Y,, Srivastava, S.K., Dunn, C.Y., Rhim, J.S., Reddy, E.P. and Aaronsen, S.A. (1983) Nature 303, 775-779. 191 Bos, J.L., Toksoz, D., Marshall, C.J., Verlaan-De Vries, M., Veeneman, G.H., Van der Eb, A.J., Van Boom, J.H., Janssen, J.W.G. and Steenvoorden, A.C.M. (19851 Nature 315, 716-730. 192 Scolnick, E.M., Papageorge, A.G. and Shih, T.Y. (1979) Proc. Nat. Acad. Sci. USA 76 5355-5359. 193 Papageorge, A., L~wy, D. and Scolnick, E.M. (1982) J. Virol. 44, 509-519. 194 Finkei, T., Der, C.J. and Cooper, G.M. 11984) Cell 37, 15! -158. 195 McCormick, F. (19891 in Oncogenes and the Molecular Crigias of Cancer (Weinberg, R.A., e.d.), pp. 125-145, Cold Spring Harbor Laborator~ Press, Cold Spring Harbor. 196 De Vos, A.M., Tong, L, Milburn, M.V., Matias, P.M., Jancarik, J., Noguchi, S., Nishima, S., Miura, K., Ohtsuka, E. and Kim, S,-H, (1988) Science 239, 888-893. 197 Pal, E.F., Kabsch, W., Krengel, U., Holmes, K.C., John, J. and Wittinghofer, A. 119891 Nature 341, 209-214. t98 Toda, 1"., Uno, I., Ishikawa, 1"., Powers, S., Kataoka, T., Broek, D., Cameron, S., Broach, J., Matsumoto, K. and Wiglet, M. 119851 Cell 40, 27-36. 109 McGrath, J.P., Capon, D.J., Goeddel, D.V. and Levinson, A.D. 119841 Nature 310, 644-649. 200 Sweet, R.W., Yokoyama, S., Kamata, T., Feramisco, J., Rosenberg, M. and Gross, M. 119841 Nature 311,273-275. 201 Gibbs, J.B., Sigal, l.S., Poe, M. and Scolnick, E.M. 119841 Proc. Natl. Acad, SCi, USA 81, 5704-5708, 202 Manne, V., Bekesi, E. and Kung, H.-F. (19851 Proc. Natl. Acad. Sci. USA 82, 376-380. 203 Trahey, M, and McCormick, F. 119871 Science 238, 542-545. 204 Shih, C. and Weinberg, R.A. 119821 Cell 29, 161-169. 205 Perucho, M., Goldfarb, M., Shimizu, K., Lama, C., Fogh, J. and W~ler, M. 119811 Cell 27, 467-476. 206 Krontiris, T. and Cooper, G.M. 119811 Proc. Natl. Acad. Sci. USA 78, 1181-1184. 207 Parada, LF., Tabin, CJ., Shih, C. and Weinberg, R.A. 119821 Nature 297, 474-478.

208 Santos, E~, Tronick, S.R., Aaronsen, S.A., Pulciani, S. and Barbacid, M. 0982) Nature 298, 343-347. 209 Der, C.J., Krontis, T.G. and Cooper, G.M. 0982) Proc. Natl. Acad. Sci. USA 79, 3637-3640. 210 Spandidos, D.A. and Kerr, I.B. 0984) Br. J. Cancer 49, 681-688. 211 Spandidos, D.A. and Agnantis, N.J. (1984)Anticancer Res. 4, 269-272. 212 Williams, A.R.W., Piris, J., Spandidos, D,A. and Wyllie, A.H. (1985) Br. J. Cancer 52, 687-693. 213 Agnantis, N.J., Petraki, C., Markoulatos, P. and Spandidos, D.A. (1985) Antieancer Res. 6, 1157-1160. 214 Papadimitriou, K.. Yiagnisis, M., Tolls, G. and Spandidos, D.A. (1988) Anticancer Res. 8, 1223-!227. 215 Spandidos, D.A., Yiagnisis, M., Papadimitriou, K. and Field, J.K. 11989) Anticancer Res. 9, 1385-1394. 21O Tiniakos, D., Spandidos, D.A., Kakkanas, A., Pintzas, A., Pollice, L. and Tiniakos, G. (19891 Anticancer Res. 9, 715-722. 217 Samowitz W.S., Paull, G. and Hamilton, S.R. 11988) Hum. Pathol. 19, 127-132. 218 Robinson, A., Williams, A.R.W., Piris, J., Spandidos, D.A. and Wyllie, A.H. (1986) Br. J. Cancer 54, 877-883. 219 DiBiasi, F., Del Sal, G. and Horan-Hand, P. 119891 Int. ,I. Cancer 43. 431-435. 2211 Yoshida, K., Hamatani, K., Koide, H., Ikeda, H., Nakamura, N., Akiyama, M., Tsuchiyama, H., Nakayama, E. and Shiku, H. (1988) Cancer Res. 48, 5503-5509. 221 Yamamoto, T. and Hattori, T. (19881 Path. Res. Pr,tct. 183, 663-669. 222 Rodenhuis, S., Slebos, R.J.C., Boot, A.J.M., Evans, S.G., Mori, W.J., Wagenaar, S.S., Van Bodegom, P.C. and Bos, J.L. (1988) Cancer Res. 48, 5738-5741. 223 Kurzrock, R.. Gallick, G.E. and Gutterman, J.U. (1986) Cancer Res. 46, 1530-1534. 224 Slebos, R.J.C., Kibbelaar, R.E., Dalesio, O., Kooistra, A., Stam, J., Meijer, C.J.L.M., Wagenaar, S.S., Vanderschueren, R.GJ.R.A., Van Zandwijk, N., Mooi, W.J., Bos, J.L. and Rodenhuis, S. (1990) New Engl. J. Med. 323, 561-565. 225 AImoguera. C., Shibata, D., Forrester, K., Martin, J., Arnheim, N. and Perucho, M. (1988) Cell 53, 549-554. 226 Grunewald, K., Lyons, J., Frohlich, A., Feichtinger, H.,. Weger, R.A., Schwab, G., Janssen, J.W.G. and Bartram, C.R. (19891 int. J. Cancer 43, 1037-1041. 227 Smit, V.T.H.B., Boot, A.J.M., Stairs, A.M.M., Fieuren, G.J., Cornelisse, C.J. and Bos, J.L. (19881 Nucleic Acids Res. 16, 7773-7782. 228 Azuma, M., Furumoto, N., Kawamata, H., Yoshide, H., Yanagawa, T., Yura, Y., Hiyashi, Y., Takegawa, Y. and Sato, M. (1987) Cancer J. 1,375-380. 229 Peehl, D.M., Weber, N. and Stamey, T.A. (19871 Prostate 10, 281-289. 230 Carter, B.S., Epstein, J.l. and Isaacs, W.B. 11990) Cancer Res. 50, 6830-6832. 231 Thompson, T.C. (1990) Cancer Cells 2, 345-354. 232 Bos, J.L., Verlaan-De Vries, M., Van der Eb, A.J., Janssen J.W.G., Delwel, R., L,,,venberg, B. and Coily, L.P. 11987) Blood 69, 1237--1241. 233 Needleman, S.W., Kraus, M.H., Srivastava, S.K., Levine, P.H. and Aaronsen, S.A. (19861 Blood 67, 753-757. 234 Janssen, J.W,G., Steenvoorden, A.C.M., Lyons, J., Anger, B., Bohlke, J.U., Bos, J.L., Seliger, H. and Bartram, C.R. 119871 Proc. Natl. Acad. Sci. USA 84, 9228-9232. 235 Senn, H.P., Tran-Thang, C., Wodnar-Filipowicz, A., Jiriney, J., Popp, M., Gratwohl, A., Signer, E., Weber, W. and Moroni, C. 119881 Int. J. Cancer 41, 59-69. 236 Hirai, H., Kohoyashi, Y., Mano, H., Hagiwara, K., Maru, Y.,

213 Omine, M., Mizoguchi, H., Nishida, J. and Takata, F. (19871 Nature 327, 430-432. 237 Bos, J.L. (1989) Cancer Res. 49, 4682-4689. 238 Deng, G., Liu, X. and Wang, J. (19911 Oncogene Res. 6, 33-38. 239 Bos, J.L., Fearon, E.R., Hamilton, S.R., Verlaan-De Vries, M., Van Boom, J.H., Van der Eb, A.J. and Vogelstein, B. (19871 Nature 327, 293-297. 2411 Vogeistein, B., Fearon, E.R.. Hamilton, S.R., Kern, S.E., Presinger, A.C., Leppert, M., Nakamura, Y., Whyte, R., Smits, A.M.M. and Bos, J.L (19881 New Engl. J. Med. 319, 525-532. 241 Forrester, K., Almoguera, C., Han, K., Grizzle, W.E. and Perucho, M. (19871 Nature 327, 298-303. 242 Burmer, G.C. and Loeb, L.A. (19891 Proc. Nat. Acad. Sci. USA 86, 2403-2407. 243 Agnantis, N.J., Parissi, P., Anagnostakis, D. and Spandidos, D.A. (1986) Oncoiogy 43, 36-39. 244 Whittaker, J.L., Walker, R.A. and Varley, J.M. (19861 Int. J. Cancer 38, 651-655. 245 Tanaka, T., Slamon, D.J., Battifora, It. and Ciine, M.J. (1986) Cancer Res. 46, 1465-1470. 246 Candlish, W., Kerr, I.B. and Simps~n, tt.W. (19861 J. Palhol. 15(1, 163- i 67. 247 De Bortoli, M.E., Abou-lssa, I!., H~,ley, B.E. and Cho-Chung, Y.S. (19851 Biochem. Biophys. Res. Commun. 127, 699-706. 248 Clair, T., Miller, W.R. and Cho-Chung, Y.S. (19871 Cancer Res. 47, 5290-5293. 249 Kakkanas, A. and Spandidos, D.A. (19901 In Vivo 4, 115-120. 250 Hamer, P., La Vecchio, J., Ng, S., DeLellis, R., Wolfe, H. and Carney, W.P. (19911 Oncogene 6, 1609-1615. 251 Farr, C.J., Marshall, C.J., Easty, D.J., Wright, N.A., Poweli, S.C. and Paraskeva, C. (1988) Oncogene 3, 673-678. 252 Crawford, L.V., Pin, D.C., Gurney, E.G., Goodfellow, P. and Taylor-Papadimitriou, J. (1981) Proc. Natl. Acad. Sci. USA 78, 41-45. 253 Hinds, P.W., Finlay, C., Frey, A.B. and Levine, A.J. (1987) Mol. Cell Biol. 7, 2863-2869. 254 Hinds, P.W., Finlay, C.A. and Levine, A.J. (19891 J. Viroi. 63, 739-746. 255 Finlay, C.A., Hinds, P.W. and Levinc, A.J. (19891 Cell 57, 1083-1093. 256 Baker, S.J., Ft~aron, E.R., Nigro, J.M., ttamilton, S.R, Preisinger, A.C., Jessup, J.M., VanTuinen, P., Ledbetter, D.H., Barker, D.F., Nakamura, Y., White, R. and Vt,gelstein, B. (1989) Science 244, 217-221. 257 Takahashi, q., Nau, M.M., Chiba, I., Birrer, M., Rosenburg, K., Vincour, M., Levitt, M., Pass, H., Gazdar, A.F. and Minna, J.D. (1989) Science 246, 491-494. 258 Bressac, B., Kew, M., Wands, J. and Ozturk, M. (1991) Nature 350, 429-431. 259 Nigro, J.M., Baker, S.J., Preisinger, A.C., Jessup, J.M., Hostetter, R., Cleary, K., Bigner, S.H., Davidson, N., Baylin, S., Devlice, P., Glover, T., Collins, F.S., Weston, A., Modali, R., Harris, C.C. and Vogelstein, B. (19891 Nature 342, 705-708. 260 Gannon, J.V., Greaves, R., Iggo, R. and Lane, D.P. (19901 EMBO J. 9, 1595-1602. 261 Kraiss, S., Ouaiser, A., Oren, M. and Montenarh, M. (19881 J. Virol. 62, 4737-4744. 262 Clarke, C.F., Cheng, K., Frey, A.B., Stein, R., Hinds, P.W. and Levine, A.J. (1988) Mol. Cell. Biol. 8, 1206-1215. 263 Finlay, C.A., Hinds, P.W., Tan, T.-H., Eliyahu, D., Oren, M. and Levine, A.J. (1988) Mol. Cell. Biol. 8, 531-539. 264 Sturzbecher, H.-W., Addison. C. and Jenkins, J.R. (1988) Moi. Ceil. Biol. 8, 3740-3747. 265 Lane, D.P. and Crawford, L.V. (1979) Nature 278, 261-263. 266 Linzer, D.I.H. and Levine, A.J. (19791 Cell 17, 43-52. 267 McCormick, F. and Harlow, E. (19801 J. Virol. 34, 213-224.

268 Sarnow, P., Ho, Y.S., Williams, J ;,~,d Lev|ne, A.J. (19821 Co!! 28, 387-394. 269 Werness, B.A., Levine, A.J. and Howley, P.M. (19901 Science 248, 76-79. 270 Malkin, D., Li, F.P., Strong, L.C., Fraumeni, J.J.F., Nelson, C.E., Kim, D.H., Kassel, J., Gryka, M.A., Bischoff, F.Z., Tainsky, M.A. and ~'riend, S.H. (19901 Science 250, 1233-1238. 271 Crawford, L.V., Pim, D.C. and Lamb, P. (19841 Mol. Biol. Med. 2, 261-272. 272 Crawford, L.V., Pim, D.C. and Bulbrook, R.D. (19821 Int. J. Cancer 30, 403-408. 273 Baker, S.J., Markowitz, S., Fearon, E.R., Wilson, J.K.V. and Vogelstein, B. (19901 Science 249, 912-915. 274 Rodriguez, N.R., Rowan, A., Smith, I~,;.E.F., Kerr, I.B., Bodmer, W.F., Gannon, J.V. and Lane, D.P. (19901 Proc. Natl. Acad, Sc. USA 87, 7555-7559. 275 Chiba, I., Tak~hashi, T., Nau, M.M., D'Amico, D., Curiel, D.T., Mitsudomi, T., Buchhagen, D.L., Carbone, D., Piantadosi, S., Koga., i-1,, Reissman, P.T., Slamon, D.J., Holmes, E.C. and Minna, J.D. (19901 Oncogene 5, 1603-~610. 276 lggo, R., Garter, K., Lane, D. and ltarris, A.L. (19901 Lancet 335, 675-679. 277 Hollstein, M.C., Metcalf, R.A., Welsh, J.A., Montesano, R. and Harris, C.C. (19901 Proc. Nat. Acad. Sci. USA 87, 9958-9961. 278 Stratton, M.R., Moss, S., Warren, W., Patterson, H., Clark, J., Fisher, C., Fletcher, C.D.M., Bail, A., Thomass, M., Gusterson, B.A. and Cooper, C.S. (19901 Oncogene 5, 1297-1301. 279 Hsu, I.C., Metcalf, R.A., Sun, T., Welsh, J.A., Wang, N.J. and Harris, C.C. (19911 Nature 350, 427-428. 280 Sidransky, D., Von Eschenbach, A., Tsai, Y.C., Jones, P., Summerhayes, I., Marshall, F., Paul, M., Green, P., ltamilton, S.R., Frost, P. and Vogelstein, t3. (19911 Science 252, 706-709. 281 Rom, W.N., Travis, W.D. and Brody, A.R. (19911 Am. Rev. Resp. Dis. 143, 408-422. 282 Bergh, J.C. (19901 Am. Rev. Resp. Dis. 142, 520-26. 283 A!i, !.U., Lidereau, R. and Callahan, R. (19881 Cancer Treat. Res. 40, 25-48. 284 Ernberg, I.T. (1990) Acta Oncol. 29, 331-334. 285 Borg, A., Sigurdsson, H., Clark, G.M., Ferno, M., Fuqua, S,A., Olsson, H., Killander, D. and McGurie, W.L. (19911 Br. J. Cancer 63, 136-142. 286 Merrit|, W.D., Weissler, M.C., Turk, B.F. and Gilmer, T.M. (19901 Arch. Otolaryngol. Head Neck Surg. 116, 1394-1398. 287 Baioechi, G., Kavanagh, J.J., Talpaz, M., Wharton, J.T., Gutterman, J.IJ. and Kurzrock, R. (19911 Cancer 67, 990-996. 288 Tamura, T., Brost, H., Kabisch, A., Lampert, F., HadwigerFs,ngmeier, A. and Niemann, H. (19891 J. Cancer Re.s. Clin. Oncol. i 15, 235-241. 289 Yunis, J.J., Oken, M.M., Kaplan, M.E., Ens"ud, K.M., Howe, R.R. and Theologides, A. (19821 N. Engi. J. Med. 307, 12311236. 290 Fukuhara, S., Rowley, J.D., Variakojis, D. and Golomb, ll.M. (1979) Cancer Res. 39, ~ i 19-3131. 291 He:kenbery, D., Nunez, G., Milliman, C., Schreiber, R.D. and Korsmeyer, S.J. (1990) Nature 348, 334-336. 292 Martin-Zanca, D., Hughes, S.H. and Barbacid, M. (19861 Nature 319, 743-748. 293 Kaplan, D.R., Hcmpstead, B.L., Martin-Zanca, D., Chap, M.V. and Parada, L.F. (19911 Science 252, 554-558. 294 Klein, R., Jing, S., Nanduri, V., O'Rourke, E. and Barbacid, M. (19911 Cell 65, 189-197. 295 Friend, S.H., Bernards, R., Rogelj, S., Weinberg, R.A., Rapaport, J.M., Albert, D.M. and Dryja, T.P. (19861 Nature 323, 643-646. 296 Lee, W.H., Bookstein, R., Hong, F., Young, L.J., Shew, J.-Y. and Lee, E.Y.-H.P. (19871 Science 235, 1394-1399.

214 297 Fung, Y.-K.T., Murphr¢e, A.L., T'Ang, A., Qian, J., Hinrichs, S.H. and Benedict, W.F. (1987) Science 236, 1657-1661. 298 DeCaprio, J.A., Ludlow, J.W., Lynch, D., Furukawa, Y., Griffin, .~., Piwnica-Worrns, H., Huang, C.-M. and Livingston, D.M. (1989) Cell 58, 1085-1095. 299 Friend, S.H., Dryja, T.P. and Weinberg, R.A. (1988) New Engl. J. Med. 318, 618-622. 300 Charbonneau, H., Tonk.~, N.K., Walsh, K.A. and Fischer, E.H. (1988) Proc. Natl. Acad. Sci. USA 85, 7182-7186. 301 Hunter, T. (1989) Cell 58, 1013-1016. 302 Brown-Shimer, S., Johnson, ILA., Lawrence, J.B., Johnson, C., Bruskin, A.. Green, N.R. and Hill, D.E. (1990) Proc. Nat. Acad. Sci. USA 87, 5148-5152. 303 Hansen, M.F., Koufos, A., Gallie, B.L., Phillips, R.A., Fodstad, O., Brosger, A., Gedde-Dahl, T. and Cavenee, W.K. (1985) Proc. Natl. Acad. Sci. USA 82, 6216-6220. 304 Harbour, J.W., Lai, S.-L., Whang-Peng, J., Gazdar, A.F., Minna, J.D. and Kaye, FJ. (1988) Science 241,353-357.

305 Horowitz, J.M., Yandell, D.W., Park, S.-H., Canning, S., Whyte, P., Buchkovich, K., Harlow, E., Weinberg, R.A. and Dryja, T.P. (1989) Science 243, 937-940. 306 Lee, E.Y.-H.P., To, H., Shew, J.-Y., Bookstein, R., Scully, P. and Lee, W.-H. (1988) Science 241~ 218-22!. 307 Foulds, L~ (1954) Cancer Res. 14, 327-339 308 Foulds, L. (1958) J. Chron. Dis. 8, 2-37. 309 Fearon, E.R., Cho, K.R., Nigro, J.M., Kern, S.E., Simons, J,W., Ruppert, J.M., Hamilton, S.R., Preisinger, A.C., Thomas, G., Kinzler, K.W. and Vogelstein, B. (1990) Science 247, 49-55. 310 Kinzler, K.W., Nilbert, M.C., Vogelstein, B., Bryan, T.M., Levy, D.B., Smith, KJ., Preisinger, A.C., Hamilton, S.R., Hedge, P., Markham, A., Carlson, M., Joslyn, G., Groden, J., White, R., Miki, Y., Miyoshi, Y., Nishisho, I. and Nakarnura, Y. (1991) Science 251, 1366-1370.