Molecular Markers of Carcinogenesis

Pharmacol. Ther. Vol. 77, No. 2, pp. 135–148, 1998 Copyright © 1998 Elsevier Science Inc.

ISSN 0163-7258/98 $19.00 PII S0163-7258(97)00111-3

Associate Editor: S. Pestka

Molecular Markers of Carcinogenesis Paul W. Brandt-Rauf*‡ and Matthew R. Pincus† *DIVISION OF ENVIRONMENTAL HEALTH SCIENCES, SCHOOL OF PUBLIC HEALTH, COLUMBIA UNIVERSITY, 60 HAVEN AVENUE, B-1, NEW YORK, NY 10032, USA †DEPARTMENT OF PATHOLOGY AND LABORATORY MEDICINE, VA MEDICAL CENTER, 800 POLY PLACE, BROOKLYN, NY 11209, USA

ABSTRACT. The protein products of oncogenes and tumor suppressor genes play critical roles in the development of many cancers. The expression of a number of these proteins can be detected in extracellular fluids such as blood. This article reviews the literature on the application of methods for the detection of the proteins of oncogenes and tumor suppressor genes in the blood of humans with cancer or at risk for the development of cancer. The detection of these proteins in blood may be useful molecular markers of carcinogenesis that could play an important part in cancer diagnosis, prognosis, and prevention. pharmacol. ther. 77(2):135–148, 1998. © 1998 Elsevier Science, Inc. KEY WORDS. Growth factors, oncogene proteins, tumor suppressor gene proteins, cancer. CONTENTS 1. INTRODUCTION . . . . . . . . . . . 2. GROWTH FACTORS . . . . . . . . . 2.1. PLATELET-DERIVED GROWTH FACTOR . . . . . . . . 2.2. TRANSFORMING GROWTH FACTOR-a . . . . . . . . . . . . 2.3. TRANSFORMING GROWTH FACTOR-b . . . . . . . . . . . . 2.4. OTHER GROWTH FACTORS . . . 3. TRANSMEMBRANE GROWTH FACTOR RECEPTORS . . . . . . . . . 3.1. EPIDERMAL GROWTH FACTOR RECEPTOR . . . . . . . . . . . . 3.2. THE C-ERBB-2 RECEPTOR . . . 3.2.1. THE C-ERBB-2 RECEPTOR

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IN BREAST CANCER . . . 3.2.2. THE C-ERBB-2 RECEPTOR IN OTHER CANCERS . . . 4. MEMBRANE-ASSOCIATED SIGNAL TRANSDUCERS . . . . . . . . . . . . 4.1. RAS-RELATED PROTEINS . . . . 4.2. RAS ANTIBODIES . . . . . . . . 5. NUCLEAR ONCOPROTEINS . . . . . . 5.1. MYC-RELATED PROTEINS . . . . 5.2. MYC ANTIBODIES . . . . . . . . 5.3. P53 PROTEIN . . . . . . . . . . 5.4. P53 ANTIBODIES . . . . . . . . 6. CONCLUSIONS . . . . . . . . . . . . ACKNOWLEDGEMENTS . . . . . . . . . . REFERENCES . . . . . . . . . . . . . . .

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ABBREVIATIONS. ASL, angiosarcoma of the liver; bFGF, basic fibroblast growth factor; ECD, extracellular domain; EGFR, epidermal growth factor receptor; PDGF, platelet-derived growth factor; RIA, radioimmunoassay; TGF, transforming growth factor; VC, vinyl chloride.

1. INTRODUCTION In recent years, numerous studies have documented that alterations (e.g., point mutations or overexpression) of oncogenes and tumor suppressor genes can be identified in human tumor tissues from various sites. In many instances, these alterations are believed to occur early in the process of carcinogenesis and to be causally related to the subsequent development of cancer. If correct, the detection of such alterations could be useful molecular markers for following the process of carcinogenesis in vivo for purposes of diagnosis, surveillance and early tumor identification and intervention. Direct detection of such alterations requires access to DNA or mRNA from target tissue, which is normally difficult to obtain for routine surveillance purposes from most target sites. However, in many cases, the protein products of these oncogenes and tumor suppressor genes gain access to the extracellular environment and thus, are ‡Corresponding

author.

amenable to detection in easily obtainable bodily fluids such as blood (Pincus et al., 1996). Therefore, the detection of mutant forms or increased amounts of these oncoproteins in blood may serve as convenient molecular markers of carcinogenesis for human studies of cancer diagnosis, prognosis and prevention. The focus of this review will be on the identification in blood of selected examples of these oncoproteins, including growth factors, transmembrane growth factor receptors, membrane-associated signal transducers, and nuclear DNA-binding proteins of oncogenes and tumor suppressor genes in patients with cancer or at risk for the development of cancer. 2. GROWTH FACTORS Many different growth factors have been identified as stimulating cellular proliferation during oncogenesis. Some growth factors are encoded by oncogenes [e.g., the B chain of the platelet-derived growth factor (PDGF) is encoded by the sis oncogene], and others interact with oncogene pro-

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teins [e.g., transforming growth factor (TGF)-a binds to the epidermal growth factor receptor (EGFR) encoded by the c-erbB-1 oncogene]. These growth factors are actively secreted by cells into the extracellular environment and thus, represent potential molecular markers for detection in blood during cancer development, progression, or recurrence. The most frequently studied growth factors in this regard are PDGF, TGF-a, and TGF-b. 2.1. Platelet-Derived Growth Factor Plasma PDGF B chain levels initially were identified as elevated (i.e., greater than the highest value of 0.69 ng/mL among 72 noncancer controls) in 19 of 131 (15%) patients with a range of cancers, including carcinomas, sarcomas and lymphomas, using an ELISA (Leitzel et al., 1989, 1991a). Plasma PDGF B chain levels determined by radioimmunoassay (RIA) were also found to be elevated (i.e., twice the lower limit of detection of the assay of 1.56 fmol/ 100 mL) in 2 of 17 (12%) Stage 2 breast cancer patients and 13 of 41 (32%) Stage 4 breast cancer patients compared with 0 of 9 (0%) normal female controls, and seropositive patients had more metastatic involvement and a significantly shorter survival time (Ariad et al., 1991). In another study, plasma PDGF B chain levels were measured by RIA in the plasma of 17 healthy controls (mean 6 SD 5 523 6 157 pg/mL) and 55 brain tumor patients (mean 6 SD 5 881 6 854 pg/mL) (Kurimoto et al., 1995). Correcting for platelet release of PDGF and using 95% confidence limits as a cut-off for seropositivity, 6 (11%) of the cases were identified as having elevated levels, and these levels decreased after treatment of the tumors and in 3 patients, became elevated again with recurrence of the disease. In a recent 2-year follow-up study, several oncogene-related proteins, including sis oncogene-related proteins (sis encodes the B chain of PDGF), were determined by immunoblotting in the serum of 37 patients who subsequently developed fatal cancers, 59 patients who subsequently developed nonfatal cancers, 58 patients who subsequently developed benign tumors, and 94 healthy controls (Weissfeld et al., 1996). Detectable levels of sis-related proteins were statistically significantly more common in the patients who subsequently developed fatal malignancies (16.2%), including lung, gastrointestinal, brain, prostate, ovary, cervical cancers, and lymphomas and leukemias, compared with controls (3.2%) (odds ratio 5 5.9, 95% confidence intervals 5 1.4–24.9). 2.2. Transforming Growth Factor-a Plasma TGF-a levels initially were identified as elevated by ELISA in 71 patients with solid tumors (mean 6 SD 5 346 6 155 pg/mL) compared with 66 controls (mean 6 SD 5 187 6 29 pg/mL), but the difference was not statistically significant (Wolf et al., 1990). In another study based on pooled plasma samples, levels determined by ELISA were 0.051 ng/mL in stomach, colon, and liver cancer patients compared with 0.028 ng/mL in 15 healthy controls (Katoh

P. W. Brandt-Rauf and M. R. Pincus

et al., 1990). As determined by RIA, serum TGF-a levels have been found to be elevated in 83 breast cancer patients (mean 6 SD 5 353 6 98 pg/mL; range 5 210–740 pg/mL) compared with 74 healthy controls (mean 6 SD 5 144 6 17 pg/mL; range 5 nondetectable 207 pg/mL), a difference that was statistically significant (P ,0.001) (Chakrabarty et al., 1994). In another study by the same group, as determined by RIA, serum TGF-a levels were found to be elevated in 100 patients with gastrointestinal cancers (esophageal, gastric, pancreatic, colonic, and rectal cancers; mean 6 SD 5 269 6 102 pg/mL; range 5 119–760 pg/mL) compared with the same controls, and again this difference was statistically significant (P , 0.001), with elevated levels identified in early-, as well as late-, stage disease, suggesting that this might be an early indicator of tumor occurrence (Moskal et al., 1995). In another study, TGF-a levels were determined by ELISA in the banked serum samples from 36 asbestosis cases who subsequently developed cancer, 71 agesex-race-smoking-asbestos exposure-matched asbestosis controls without cancer, and 10 age-sex-race-smoking-matched nonasbestosis noncancer controls (Partanen et al., 1995). The mean serum levels were higher in the asbestosis cases with cancer (mean 6 SD 5 78 6 84 ng/L) or without cancer (mean 6 SD 5 76 6 69 ng/L) than in the nonasbestosis-noncancer controls (mean 6 SD 5 58 6 14 ng/L), although the differences were not statistically significant. Defining a positive elevation of serum TGF-a to be any value greater than two standard deviations above the nonasbestosis control mean, none (0%) of the controls were seropositive compared with 13 (36%) of the asbestosis cases with cancer and 27 (38%) of the asbestosis cases without cancer. Among the cancer cases, serum TGF-a was particularly elevated in adenocarcinomas (mean 6 SD 5 135 6 158 ng/L; 3 of 6, or 50% seropositive) and squamous cell carcinomas (mean 6 SD 5 91 6 86 ng/L; 3 of 5, or 60% seropositive) of the lung. All but one of the seropositive cancer cases had positive banked serum samples prior to the time of disease diagnosis (average 5 6.1 years; range 5 0 –12 years). In another recent study, serum TGF-a levels were determined by ELISA in 80 patients with cirrhosis and hepatocellular carcinoma, 44 patients with cirrhosis without cancer, and 182 healthy controls (Tomiya and Fujiwara, 1996). Levels were significantly higher in the cancer cases (mean 6 SD 5 45 6 40 pg/mL) compared with the cirrhosis patients (mean 6 SD 5 21 6 15 pg/mL), and the levels decreased after successful treatment in 60% of the cancer cases. 2.3. Transforming Growth Factor-b Plasma levels of TGF-b initially were determined by a biological activity assay (growth inhibition of mink lung epithelial cells) and later by ELISA in 26 patients with hepatocellular carcinoma, 12 patients with chronic hepatitis, 11 patients with cirrhosis, and 20 normal controls, and the levels were statistically significantly elevated in the cancer cases (mean 6 SD 5 19.3 6 19.5 ng/mL) compared with

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the other groups (chronic hepatitis mean 6 SD 5 3.7 6 2.1 ng/mL; control mean 6 SD 5 1.4 6 0.8 ng/mL; P , 0.01) (Shirai et al., 1992, 1994). After therapy by embolization and/or resection in 7 of the cases, plasma levels decreased significantly from 22.6 6 16.7 ng/mL to 10.2 6 6.5 ng/mL (P , 0.05). In another study, as determined by biological activity assay and ELISA, plasma TGF-b levels were elevated in 6 chronic myelogenous leukemia patients in accelerated or blast phase (mean 6 SD 5 4.3 6 3.8 ng/mL) compared with 12 normal controls (mean 6 SD 5 2.6 6 1.1 ng/mL), a difference that was statistically significant (P 5 0.01) (Murase et al., 1994). As determined by ELISA, a study of serum TGF-b2 levels in 25 bladder cancer patients and 5 healthy controls found that patients with superficial cancers had values in the normal range (,62 pg/ mL), but the 9 patients with invasive cancer had elevated levels (69–155 pg/mL) (Klocker et al., 1994). Another study examined plasma levels of TGF-b1, TGF-b2, and TGF-b3 by ELISA in 28 breast cancer patients and 42 controls, and found elevated levels (defined as more than two standard deviations above the control mean or .8.1 ng/ mL) in 2 (7%) of the cancers (Wakefield et al., 1995). In another study, plasma TGF-b1 levels were determined by ELISA in 7 normal controls (mean 6 SD 5 3.99 6 0.77 ng/mL; range 5 0.76–6.28 ng/mL), 9 patients with benign prostatic hypertrophy (mean 6 SD 5 2.47 6 0.64 ng/mL; range 5 0.10–4.80 ng/mL), 6 Stage II prostate cancer cases (mean 6 SD 5 6.31 6 2.61 ng/mL; range 5 1.62–18.36 ng/mL), and 6 Stage III/IV prostate cancer cases (mean 6 SD 5 12.80 6 2.03 ng/mL; range 5 7.34–21.50 ng/mL), the latter being statistically significantly elevated compared with the controls (P 5 0.001) (Ivanovic et al., 1995). In a follow-up study of lung cancer patients, plasma TGF-b1 levels determined by ELISA were found to be significantly higher (P , 0.001) at baseline in the 54 cancer cases (mean 6 SD 5 13.0 6 2.5 ng/mL) compared with 20 normal controls (mean 6 SD 5 4.4 6 0.3 ng/mL), with elevated levels (defined as more than two standard deviations above the control mean) identified in 27 (50%) cases compared with no (0%) controls (Kong et al., 1996). During radiation therapy, plasma levels in those patients with elevations declined, and at last follow-up, the TGF-b1 levels correlated significantly with disease status (3 of 4 patients with no evidence of cancer had normal levels compared with 2 of 16 patients with residual or recurrent disease; P 5 0.02). Recently, elevated TGF-b1 plasma levels have also been identified in patients with gastric cancer (Niki et al., 1996).

tions above the control mean) were also reported in 9% of liver cancers, 56% of brain cancers, 70% of renal cancers, and 79% of lung cancers compared with 0% of controls (Ii et al., 1993). Mean serum bFGF levels have also been reported to be significantly elevated in cases of esophageal, stomach, colon, liver, breast, and pancreatic cancer (Kurobe et al., 1993). Serum levels of bFGF have also been reported in several other studies of breast cancer (Li et al., 1993; Takai et al., 1994; Sliutz et al., 1995a) with elevated levels declining significantly following surgery (Takai et al., 1994) and with increasing levels following therapy presaging relapses in 37.5% of cases (Sluitz et al., 1995a). In a similar study of cervical cancer patients, increasing serum bFGF levels following complete remission presaged relapse in 50% of cases (Sliutz et al., 1995b). Elevated plasma levels of bFGF have also been reported in patients with multiple endocrine neoplasia Type I (Zimering et al., 1993) and B-cell chronic lymphocytic leukemia (Duensing and Atzpodien, 1995), and elevated serum levels have been reported in patients with prostate cancer (Meyer et al., 1995) and renal cell carcinoma patients with pulmonary metastases (Duensing et al., 1995). Elevated serum levels of epidermal growth factor have been reported in stomach cancer (Pawlikowski et al., 1989), cancer of the tongue (Bhatavdekar et al., 1993), and ovarian cancer (Shah et al., 1994). Elevated plasma levels of insulin-like growth factors have been reported in ovarian cancer (Shah et al., 1994) and breast cancer (Peyrat et al., 1990), and elevated serum levels of hepatocyte growth factor have been reported in hepatocellular carcinoma (Hioki et al., 1993).

2.4. Other Growth Factors

3.1. Epidermal Growth Factor Receptor

Basic fibroblast growth factor (bFGF) initially was identified by ELISA in the serum of renal cell carcinoma patients (Fujimoto et al., 1991), and a subsequent study found detectable levels (730 pg/mL) in 54% of renal cell carcinoma patients, 27% of urothelial cancer patients, and 29% prostatic cancer patients (Fujimoto et al., 1995). Elevated serum bFGF levels (defined as more than three standard devia-

In one study, the banked serum samples from 38 asbestosis cases who subsequently developed cancer, 72 age-sex-racesmoking-asbestos exposure-matched asbestosis controls without cancer, and 20 age-sex-race-smoking-matched nonasbestosis noncancer controls were examined for the EGFR ECD by ELISA (Partanen et al., 1994a,b). The serum EGFR ECD levels in the cancer cases (mean 6 SD 5 636 6

3. TRANSMEMBRANE GROWTH FACTOR RECEPTORS Many of the transmembrane growth factor receptors [particularly c-erbB-1, which encodes the EGFR and c-erbB-2 (neu, Her-2)] are commonly overexpressed in human cancers. The growth signal transduction process for these receptors appears to involve dimerization with increased intracellular tyrosine kinase activity accompanied by proteolytic cleavage of the extracellular domain (ECD) of the receptor, with accumulation of the ECD in the extracellular environment (Brandt-Rauf et al., 1994a). Therefore, detection of increased ECD of EGFR or the c-erbB-2 receptor in blood becomes a potential molecular marker for following cancer development, progression, or recurrence.

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299 fmol/mL) were statistically significantly elevated compared with the asbestosis controls (mean 6 SD 5 546 6 147 fmol/mL) or the nonasbestosis controls (mean 6 SD 5 336 6 228 fmol/mL) (P , 0.05). Defining a positive elevation as any value greater than two standard deviations above the nonasbestosis control mean, 4 (6%) of the asbestosis controls and 1 (5%) of the nonasbestosis controls were seropositive compared with 7 (18%) of the cancer cases. All of the seropositive cancers cases (including lung cancer, mesothelioma, non-Hodgkin’s lymphoma, kidney cancer, and pancreatic cancer) had positive banked serum samples prior to the time of disease diagnosis (average 5 5.1 years). In another study, serum EGFR ECD levels were found to be significantly elevated (P 5 0.007) in 22 former uranium miners with lung cancer compared with 7 healthy controls (Braun et al., 1995). 3.2. The c-erbB-2 Receptor Many studies have been done examining the c-erbB-2 receptor ECD in the blood of cancer patients. The vast majority of these studies have focused on breast cancer. 3.2.1 The c-erbB-2 receptor in breast cancer. Mori et al. (1990) first reported elevated serum erbB-2 ECD levels by ELISA (40- to 190-fold higher than controls) in 3 of 12 breast cancer patients compared with 35 controls. Furthermore, tumor tissue was available from 9 of these cases, and the erbB-2 receptor was found to be immunohistochemically elevated in 2 of these, both from patients with elevated serum ECD levels. In another study, 3 of 42 (7%) normal women had elevated serum ECD levels by ELISA (defined as greater than two standard deviations above the mean) compared with 5 of 33 (15%) women with untreated primary breast cancer and 24 of 105 (23%) women with metastatic breast cancer, a statistically significant difference compared with controls (P , 0.001) (Carney et al., 1991). In a third study, ELISA detected elevated serum ECD levels in 0 to 30 (0%) cases of benign breast disease, 2 of 64 (3%) cases of Stage I/II primary breast cancer, 5 of 17 (29%) cases of Stage III/IV primary breast cancer, 3 of 12 (33%) cases of locally recurrent breast cancer, 26 of 51 (51%) cases of recurrent metastatic disease, but 0 of 57 (0%) cases with no evidence of recurrence (Narita et al., 1992a,b). In this study, there was also a close association between serum elevation and tissue overexpression, and in several cases, changes in serum levels reflected the clinical status of disease. In a study using RIA, elevated serum ECD levels were found in 12 of 53 (23%) patients with metastatic or locally advanced disease compared with 0 of 69 (0%) controls (Hosono et al., 1993). In two of those cases, changes in serum ECD levels correlated with disease status during therapy. In another study, 0 of 19 (0%) controls and 0 of 35 (0%) patients without metastatic disease following removal of the primary breast tumor had elevated serum ECD levels by ELISA compared with 9 of 26 (35%) patients with residual metastatic disease, and in 3 of the se-

P. W. Brandt-Rauf and M. R. Pincus

ropositive cases, correspondingly elevated expression in the tumor tissue was identified (Kynast et al., 1993). In another study using RIA, elevated serum ECD levels were reported in 0 of 50 (0%) healthy controls and 0 of 25 (0%) breast cancer cases with Stage I/II disease compared with 6 of 40 (15%) cases with Stage III/IV disease, and the correlation between tumor overexpression and serum elevation was statistically significant (P , 0.01) (Pupa et al., 1993b). Similarly, elevated serum ECD levels by ELISA were reported in 26 of 61 (43%) patients with metastatic breast cancer, and there was reasonably good correlation between serum and tissue levels and with the clinical course of disease (Kath et al., 1992, 1993). Likewise, elevated serum ECD levels by ELISA were found in 9 of 36 (25%) cases of newly diagnosed primary breast cancer compared with 1 of 25 (4%) matched controls, a statistically significant difference (P 5 0.03) (Breuer et al., 1993, 1994). Two of the seropositive cases had tumor tissue overexpression and two cases with elevated preoperative levels had normal postoperative levels. Furthermore, there were 7 cases of in situ carcinoma without evidence of invasion in this study, and 3 of these (43%) had elevated serum ECD levels. Another study reported elevated serum ECD levels in 3 of 66 (5%) controls, 0 of 12 (0%) cases of benign breast disease, 1 of 13 (8%) cases of preoperative cancer, 2 of 62 (3%) cases of postoperative cancer without recurrent disease, and 55 of 93 (59%) cases with recurrent disease (Anderson et al., 1995). Also, elevated serum level was statistically significantly associated with tumor overexpression (P 5 0.044). Similar results were obtained in a follow-up study of 200 patients with primary breast cancer and no evidence of residual disease after therapy, of whom 89 subsequently developed metastases (Molina et al., 1996). In this case, 25 of the 89 (28%) patients with recurrence had elevated serum ECD levels prior to diagnosis with a lead time of 4.5 6 2.4 months. Sensitivity for early diagnosis of recurrence was significantly higher when restricted to patients with overexpression in their tumor tissue (80%) compared with those without overexpression (3.3%) (P 5 0.0001). More recent studied have also focused on ECD status in relation to response to therapy, although the results have been less encouraging. For example, in a study of plasma ECD levels in 33 patients with advanced breast cancer receiving chemotherapy, 10 (30%) were seropositive, but there was no significant difference in response to chemotherapy between seropositive (4 of 10 responders) versus seronegative (10 of 23 responders) cases (Revillion et al., 1996). In another study of serum ECD levels, 58 of 300 (19.3%) cases were seropositive, but serial ECD levels did not show a high overall correlation with the clinical course for patients with metastatic disease treated with hormonal therapy (Volas et al., 1996). On the other hand, a more recent study of 94 patients with metastatic breast cancer undergoing hormonal therapy, of whom 32 (34%) were ECD seropositive, found that low pretreatment ECD levels were the most powerful predictor of response to therapy (odds ratio 5 22.4; P 5 0.0001) (Yamauchi et al., 1997). Many other smaller studies of ECD levels in breast

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cancer have also been reported (Leitzel et al., 1991b; Ohuchi et al., 1991; Yu et al., 1991; Estabrook et al., 1992; Hayden et al., 1992; Isola et al., 1992; Hayes et al., 1993; Montero et al., 1994). Finally, as noted below for other oncoproteins, in some cases, an immune response develops in cancer patients as a result of the altered expression of oncoproteins in their tumor and blood. This apparently can occur with erbB-2 as well. For example, an inducible immune response to the erbB-2 oncoprotein, as determined by the production of antibodies against the protein from circulating lymphocytes isolated and transformed by Epstein-Barr virus, has been identified in some breast cancer patients (Pupa et al., 1993a; Disis et al., 1994).

(Brandt-Rauf et al., 1994a). In this study, the average serum ECD level in the cases was statistically significantly elevated (P , 0.001) compared with the control groups, and, in 3 of the 7 seropositive cases, serum samples were also positive prior to the diagnosis of disease (average 5 35 months). However, in a larger follow-up study of 39 pneumoconiosis patients who developed cancer (primarily lung cancer) and 73 pneumoconiosis patients without cancer, there was no significant difference in serum ECD levels between the patients with and without cancer (Partanen et al., 1994a).

3.2.2. The c-erbB-2 receptor in other cancers. Wu et al. (1993) have reported elevated serum erbB-2 ECD levels not only in breast cancer patients, but also in patients with colorectal, pancreatic, prostatic, hepatic, and ovarian cancers. Subsequent studies have examined the ECD levels in several of these cancers, as well as in gastric and lung cancers. For example, McKenzie et al. (1993) reported elevated serum ECD levels in 7 of 48 (15%) cases of ovarian cancer with a good correlation between serum and tissue overexpression. In a study of serum ECD levels in hepatocellular carcinoma, average levels in 23 male Taiwanese who subsequently developed liver cancer were found to be statistically significantly elevated (P 5 0.008) compared with 23 matched controls who did not develop cancer, and increasing serum ECD levels showed a significant linear trend in relation to the subsequent development of cancer (Luo et al., 1993; Yu et al., 1994). In this study, those individuals with elevated levels averaged 26.4 months between the time of serum collection and disease diagnosis. In another study, the mean plasma ECD level in 54 patients with colonic adenomas was found to be statistically significantly elevated (P , 0.05) compared with the mean level in 55 controls, with patients with large adenomas (>10 mm) having higher levels than patients with small adenomas (,10 mm) Brandt-Rauf et al., 1994b). In a subsequent study, elevated serum ECD levels were found in 8 of 22 (36%) patients with colorectal carcinomas, and all seropositive patients also had overexpression in their tumor tissue (Vogel et al., 1996). In this study, two other patients with advanced abdominal adenocarcinoma of unknown primary were also seropositive, but all patients with other gastrointestinal cancers were seronegative, including cases of small intestine, esophageal, and gastric carcinomas. However, in another study, elevated serum ECD levels have been reported in patients with gastric cancer, again with good correlation between serum and tissue overexpression (Kaetsu et al., 1991; Kaetsu, 1992; Nakai et al., 1992). Elevated serum ECD levels have also been described in patients with lung cancer (Ohsaki et al., 1993). In another study, serum ECD levels were measured in multiple banked serum samples from 11 pneumoconiosis patients who subsequently developed lung cancer, 11 matched controls, and 55 unmatched controls

In many instances, growth signal transduction in the cell subsequent to activation of growth factor receptors involves multiple intermolecular protein interactions resulting in the activation of membrane-associated G-proteins that bind guanine nucleotide and have GTPase activity. These membrane-associated G-proteins play a critical role in further transduction of the growth signal to cytoplasmic kinases and eventually to the nucleus. The most important example is the 21-kDa protein encoded by the ras oncogenes (p21-ras). Qualitative (i.e., point mutations) and quantitative (i.e., overexpression) changes in p21-ras have been identified as contributing to human carcinogenesis. By as yet unclearly defined mechanisms that may involve membrane shedding, p21-ras proteins gain access to the extracellular environment. Thus, the detection of increased amounts or point-mutated forms of p21-ras in blood can represent potential molecular markers of cancer development, progression, or recurrence.

4. MEMBRANE-ASSOCIATED SIGNAL TRANSDUCERS

4.1. Ras-Related Proteins Overexpression of p21-ras-related proteins in blood have been noted in a number of studies. Elevated levels (5-fold over controls) of p21 in serum initially were detected by immunoblotting in 3 of 16 (19%) workers with heavy occupational carcinogen exposure (Brandt-Rauf and Niman, 1988). In 18 months of follow-up, one of these seropositive workers developed a premalignant colonic tumor (villous adenoma), and upon removal of the tumor, the patient’s serum p21 returned to normal, suggesting that the adenoma was the source of the serum elevation and that detection prior to clinical disease occurrence was possible (BrandtRauf et al., 1990a). Elevated serum p21 has also been detected by ELISA in 5 of 34 (15%) patients with early-stage malignancies and 26 of 59 (44%) patients with advanced malignancies compared with 1 of 58 (2%) controls, with the highest levels in cases of lymphoma, breast, and urogenital cancers (Epelbaum et al., 1989a,b). Kakkanas and Spandidos (1990) reported increased serum p21 by ELISA in 3 of 13 (23%) patients with stomach cancer compared with 0 of 3 (0%) normal controls. In another study, elevated levels (5-fold over controls) of p21 in serum by immunoblotting were found in 15 of 18 (83%) lung cancer patients com-

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pared with 2 of 18 (11%) normal controls (Brandt-Rauf, 1991). Other studies have found elevated serum p21 by immunoblotting in individuals with lung and other cancers (Perera et al., 1990) or in individuals at risk for the development of lung cancer due to their workplace or environmental exposures to carcinogens (Brandt-Rauf et al., 1989, 1990b; Perera et al., 1992). A study of multiple banked serum samples from 46 pneumoconiosis patients demonstrated elevated serum p21 levels by immunoblotting in 7 of 18 (39%) patients who developed cancer compared with 2 of 28 (7%) patients who did not develop cancer, a statistically significant difference (P 5 0.012) (Brandt-Rauf et al., 1992). Furthermore, in this study, 6 of the 7 seropositive cancer cases had elevated p21 prior to the clinical detection of disease (average 5 16.3 months, range 5 3–26 months; positive predictive value 5 0.78, negative predictive value 5 0.70), suggesting that serum p21 may be a molecular marker of early malignant disease in some cases. Another study examined serum p21 by immunoblotting in 80 cancer cases (including breast, prostate, colon, lung, and liver cancer) and 188 noncancer controls (Weissfeld et al., 1994). Elevations were identified in up to 67.5% of the cancer cases compared with 15.7% of the controls, a statistically significant difference (odds ratio 5 11.1, 95% confidence intervals 5 6.0–20.6). Elevated p21 in plasma has also been reported by immunoblotting in 4 of 47 (8.5%) controls, 10 of 54 (18.5%) colonic adenoma patients, and 9 of 22 (40.9%) colonic carcinoma patients, a statistically significant difference between cancer cases and controls (P 5 0.003) (Luo et al., 1996). Plasma p21 overexpression was found to increase with increasing size of adenoma and increasing stage of carcinoma, and there was a statistically significant correlation between overexpression in the plasma and in the corresponding tumor tissue. In a recent 2-year follow-up study, ras-related proteins were determined by immunoblotting in the serum of 37 patients who subsequently developed fatal cancers, 59 patients who subsequently developed nonfatal cancers, 58 patients who subsequently developed benign tumors, and 94 healthy controls (Weissfeld et al., 1996). Detectable serum levels of ras-related proteins were statistically significantly more common in patients who subsequently developed fatal malignancies (10.8%), including lung, gastrointestinal, brain, prostate, ovary, cervical cancers, and lymphomas and leukemias, compared with controls (1.1%) (odds ratio 5 11.3, 95% confidence intervals 5 1.2–104). Point-mutated p21-ras protein has also been detected in blood. For example, one study identified Asp 13 mutant c-Ki-ras p21 protein by immunoblotting in the serum of patients with angiosarcoma of the liver (ASL) and in individuals with heavy vinyl chloride (VC) exposure who are at risk for the development of ASL (DeVivo et al., 1994; Brandt-Rauf et al., 1995). This study found 4 of 5 (80%) VC-induced ASLs to contain the Asp 13 mutant c-Ki-ras gene in the tumor tissue and the Asp 13 mutant p21 protein in the tumor tissue and in the serum, but one case of ASL and a case of hepatocellular carcinoma, both without

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the mutation, did not have detectable mutant p21 in the tumor tissue or serum. In one of the seropositive ASLs with multiple serum samples over time, relative levels of the Asp 13 p21 in serum correlated with the clinical status of the disease and were detectable prior to clinical recurrence. Furthermore, 8 of 9 (89%) individuals with potentially premalignant angiomatous lesions of the liver and 22 of 45 (49%) individuals with heavy VC exposure, but no tumors, were also seropositive, whereas 0 of 28 (0%) age-sex-racematched controls were seropositive. Stratification of this cohort by degree of VC exposure yielded a highly statistically significant linear trend (P 5 0.00001) for seropositivity with increasing exposure. A follow-up study of a larger cohort of VC-exposed individuals found 65 of 172 (37.8%) seropositive for Asp 13 mutant p21 among the exposed compared with 0 of 43 (0%) among the age-sex-race-smoking-drinking-matched unexposed controls, with a similar strong relationship between exposure and seropositivity (P , 0.001) (Li et al., 1997). 4.2. Ras Antibodies As noted in Section 3. 2. 1 for erbB-2, in some cases an immune response may develop in cancer patients as a result of the altered expression of oncoproteins in their tumor or blood. This has been noted for ras also. For example, circulating antibodies against Asp 12 mutant p21 (a very common mutation in colonic cancers) were detected by ELISA and immunoblotting in 51 of 160 (32%) colon carcinoma patients compared with 1 of 40 (2.5%) normal controls (Takahashi et al., 1995). 5. NUCLEAR ONCOPROTEINS Two important nuclear DNA-binding proteins that appear to play critical roles in the regulation of cell growth and division are the p62/64 protein of the c-myc oncogene and the p53 tumor suppressor gene protein. The c-myc oncoprotein is activated to cause cell transformation by overexpression, resulting in intracellular accumulation. The p53 tumor suppressor gene protein is subject to many different point mutations that result in a loss of its normal growth inhibitory function and a considerably increased half-life, with resultant intracellular accumulation. Thus, for both p62/64-myc and mutant p53, increased levels of the proteins can be detected in human tumors, and although the mechanisms as yet are unknown, these proteins can also accumulate in the extracellular environment. Thus, the detection of the myc oncoprotein or mutant p53 protein in blood represents other potential molecular markers of cancer development, progression, or recurrence. 5.1. Myc-Related Proteins A myc-related protein was initially identified as increased by immunoblotting in the serum of 51 patients with a wide variety of solid tumors in comparison with 16 controls with nonmalignant disease and 17 healthy controls (Chan et al., 1986, 1987). Localization of the production of myc proteins

Molecular Markers of Carcinogenesis

to the tumor was demonstrated in vivo by radioimmunoscintigraphy in 12 of the cancer patients. Furthermore, serial measurements of myc-related proteins in serum in patients with resected colorectal carcinomas showed a gradual return to normal levels following surgery. Increased levels of the myc protein have also been demonstrated by serum immunoblotting in 2 of 10 (20%) breast cancer cases compared with 0 of 10 (0%) matched controls, and both seropositive cases had increased myc protein in the tumor tissue; a postsurgical serum sample from one of these cases showed normal levels of the protein following removal of the tumor (DeVivo et al., 1993). In a follow-up study, increased myc protein was identified in 7 of 36 (19%) breast cancer cases compared with 0 of 25 (0%) matched controls, a statistically significant difference (P 5 0.02), although in this study, several cases whose tumors had increased protein did not have elevated serum levels (Breuer et al., 1994). On the other hand, one case of intraductal carcinoma without evidence of invasion was seropositive for myc protein in this study, suggesting that in certain cases, this may be a molecular marker for early malignant disease. 5.2. Myc Antibodies As in the case of ras proteins, circulating antibodies to the myc protein have also been detected in cancer patients. Serum antibodies to myc initially were described in 4 of 6 (67%) colon cancer patients, 12 of 125 (10%) breast cancer patients, 1 of 2 (50%) osteosarcoma patients, 1 of 9 (11%) ovarian cancer patients, and 3 of 3 (100%) patients with cancers of unknown origin (Ben-Mahrez et al., 1988). A subsequent study identified myc antibodies in serum in 25 of 44 (57%) cases of colorectal cancer compare with 8 of 46 (17%) normal controls, a statistically significant difference (P 5 0.001) (Ben-Mahrez et al., 1990). Serum antibodies to myc have also been detected in patients with myeloid leukemia and lymphoma, including Burkitt’s lymphoma (Lafond et al., 1992). 5.3. P53 Protein Increased levels of mutant p53 protein in serum initially were detected by ELISA in 15 of 82 (18%) breast cancer patients compared with 0 of 20 (0%) normal controls (Micelli et al., 1992). Virji et al. (1992) reported increased levels of mutant p53 protein in serum by ELISA (i.e., greater than 0.3 ng/mL, the upper limit of 100 normals) in 11 of 54 (20%) patients with hepatocellular carcinoma, as well as 30% of patients with cirrhosis, a group known to be at increased risk for the development of hepatocellular carcinoma. Increased serum mutant p53 detected by ELISA and immunoblotting have been reported in 3 of 23 (13%) lung cancer patients compared with 0 of 23 (0%) age-sexrace-matched controls and 2 of 58 (3%) unmatched controls, and increased tissue p53 and/or p53 mutations were found in the 3 serum-positive cancer cases (Luo et al., 1994). A larger study of lung cancer cases reported mutant

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p53 detected by ELISA in the serum of 17 of 50 (34%) non-small-cell cancer patients compared with 0 of 15 (0%) controls, and the levels of p53 protein accumulation in the tumor tissue were found to be strongly correlated with the serum levels (P 5 0.007) (Fontanini et al., 1994). In another study of breast cancer, increased serum mutant p53 determined by ELISA was found in 5 of 60 (8%) patients, with levels decreasing following surgical resection of the tumors, but, in this case, tissue levels of p53 correlated poorly with serum levels (Rosanelli et al., 1993). Elevated levels of mutant p53 have also been reported in the sera of 6 of 33 (18%) patients with Hodgkin’s lymphoma (Trumper et al., 1994). In a study of colon tumors, elevated total serum p53 protein (i.e., greater than in 10 controls) was detected in 6 of 16 (38%) patients with colonic adenomas and 18 of 28 (64%) patients with colonic carcinomas (Greco et al., 1994). In another study of colon tumors, plasma levels of mutant p53 were found to be statistically significantly increased among 54 cases of colonic adenomas (mean 5 0.44 ng/mL; 20% seropositive) and 22 cases of colonic carcinomas (mean 5 0.55 ng/mL; 32% seropositive) compared with 47 individuals with normal colonoscopic examinations (mean 5 0.12 ng/mL; 4% seropositive) (P , 0.02), and plasma levels tended to increase with increasing adenoma size and increasing carcinoma stage (Luo et al., 1995). In a follow-up of this study, tumor tissue samples were obtained from 16 of the cases and examined immunohistochemically for accumulations of mutant p53 protein; a statistically significant correlation was found between tissue and plasma levels of mutant p53 (P 5 0.002) (unpublished data). Total serum p53 levels determined by ELISA have also been found to be statistically significantly elevated in 22 former uranium miners with lung cancer compared with 7 healthy controls (P 5 0.003) (Braun et al., 1995). In a study of multiple banked serum samples from asbestosis patients, elevated total and mutant serum p53 was identified by ELISA in up to 6 of 32 (19%) asbestosis patients who subsequently developed cancer (primarily lung cancer and mesothelioma) compared with 2 of 36 (6%) matched asbestosis patients without cancer and 1 of 10 (10%) nonasbestosis–noncancer controls; tumor tissue samples were obtained for 10 of the lung and mesothelioma cases and examined for accumulations of mutant p53 protein and p53 gene mutations, and a statistically significant correlation was found between the tissue and serum results (P 5 0.017) (Partanen et al., 1995; Hemminki et al., 1996; Husgafvel-Pursiainen et al., 1997). Furthermore, in this study, the 6 seropositive cancer cases had elevated mutant p53 prior to the clinical detection of disease (average 5 5.3 years, range 5 2–12 years; positive predictive values 5 0.67, negative predictive value 5 0.83), suggesting that in some cases, serum mutant p53 may be a molecular marker of early malignant disease. In another study of banked serum samples, elevated mutant serum p53 was identified by ELISA in 5 of 6 (83%) individuals who subsequently developed Hodgkin’s lymphoma, 5 of 11 (45%) individuals who subsequently developed non-Hodgkin’s lymphoma, and 3 of

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7 (43%) individuals who subsequently developed multiple myeloma compared with 6 of 63 (10%) matched noncancer controls, indicating a highly significant increased risk for malignancies of lymphoid origin in seropositive individuals (relative risk 5 6.7, 95% confidence intervals 5 1.9–24), with an average lead time to diagnosis of 7 years (Lehtinen et al., 1993, 1996). Serum mutant p53 has also been examined in VC-exposed workers with and without ASL (Brandt-Rauf et al., 1996; Smith et al., 1996; Li et al., 1997). This study found 3 of 5 (60%) VC-induced ASLs to contain p53 gene mutations in the tumor tissue and accumulations of mutant p53 protein in the tumor tissue and serum, but the two cases of ASL and a case of hepatocellular carcinoma without mutations did not have detectable mutant p53 protein in the tumor tissue or serum. In one of the seropositive ASLs with multiple serum samples over time, levels of mutant p53 in serum correlated with the clinical status of the disease and were detectable prior to clinical recurrence. Furthermore, 71 of 172 (41.3%) individuals with heavy VC exposure, but no tumors, were also seropositive, whereas 3 of 43 (7.0%) age-sex-race-smoking-drinkingmatched unexposed controls were seropositive. Stratification of this cohort by degree of VC exposure yielded a highly statistically significant linear trend (P , 0.001) for seropositivity with increasing exposure. A recent study of serum mutant p53 in individuals with colorectal tumors found statistically significantly elevated levels in 50 cancer cases (mean 6 SD 5 0.97 6 0.14 ng/mL, range 5 0.7–1.37 ng/mL) and 13 polyp cases (mean 6 SD 5 0.73 6 0.06 ng/ mL, range 5 0.69–0.83 ng/mL) compared with 41 healthy controls (P , 0.001) (Shim et al., 1997). In this study, serum samples from 26 of the cancer cases were also available 1 week after surgical resection of the tumors, and postoperative levels (mean 6 SD 5 0.82 6 0.07 ng/mL) were statistically significantly lower than preoperative levels (mean 6 SD 5 0.97 6 0.13 ng/mL) (P , 0.001). In another recent study, total serum p53 was examined by ELISA in 31 chromium workers at risk for the development of lung cancer and 10 unexposed controls (Hanaoka et al., 1997). Levels were found to be higher in the exposed workers (mean 6 SD 5 355.7 6 259.5 pg/mL; range 5 116.4–1122.6 pg/mL; 36% seropositive as defined by mean 6 2 SD of controls) than in the controls (mean 6 SD 5 217.1 6 58.0 pg/mL; range 5 117.4–305.8 pg/mL; 0% seropositive). 5.4. P53 Antibodies As in the case of ras and myc proteins, circulating antibodies to p53 have also been detected in individuals with cancer or at risk for the development of cancer. Crawford et al. (1982) first reported p53 antibodies in the serum of 14 of 155 (9%) breast cancer patients compared with 0 of 164 (0%) controls. Fromentel et al. (1987) reported p53 antibodies in the serum of 14 of 119 (12%) children with various types of cancer, including 6 of 28 (21%) cases of B-cell lymphoma, compared with 1 of 88 (1%) controls. Winter et al. (1992) reported p53 antibodies in the serum of 6 of 46

P. W. Brandt-Rauf and M. R. Pincus

(13%) patients with lung cancer compared with 0 of 51 (0%) controls; all antibody-positive cases had p53 gene missense mutations and increased p53 protein in their tumors. Davidoff et al. (1992) reported p53 antibodies in the serum of 7 of 60 (11%) patients with breast cancer compared with 0 of 15 (0%) controls, and all 7 positive cases had p53 mutations and increased p53 protein in their tumors. Schlichtholz et al. (1992) reported p53 antibodies in the serum of 15 of 100 (15%) patients with breast cancer. Labrecque et al. (1993) reported p53 antibodies in the serum of 9 of 175 (5%) patients with various cancers, including colon, breast, lung, and ovary, compared with 0 of 22 (0%) controls. p53 antibodies were also identified in the serum of 20 of 80 (25%) patients with hepatocellular carcinoma compared with 0 of 67 (0%) controls (Volkman et al., 1993). In another study, antibodies to p53 were found in the serum of 12 of 93 (13%) breast cancer patients, 2 of 83 (2%) prostate cancer patients, 4 of 108 (4%) thyroid cancer patients, 10 of 42 (24%) lung cancer patients, 8 of 29 (28%) bladder cancer patients, 4 of 88 (5%) leukemia patients, 14 of 73 (19%) pancreas cancer patients, and one patient each with ovarian cancer, hepatoma, and kidney cancer (Lubin et al., 1993). Angelopolou and Diamandis (1993) reported p53 antibodies in serum determined by two different methods in 3 of 105 (3%) breast cancer patients, 10 of 72 (14%) ovarian cancer patients, 11 of 77 (14%) colon cancer patients, and 2 of 46 (4%) pancreas cancer patients. In an expanded study of p53 antibodies in serum in 1392 patients with various malignancies by two different methods, the highest prevalence of p53 antibodies was found in ovarian and colon cancers (15%), lung cancers (8%), and breast cancers (5%), with lower prevalence in other malignancies (,4%) and controls (,1–2%) (Angelopolou et al., 1994). In a study of lung cancer, p53 antibodies were found in the sera of 10 of 32 (24%) cases (including small-cell, large-cell, squamous, and adenocarcinomas) compared with 2 of 58 (3%) controls with nonmalignant respiratory diseases; one of the seropositive controls had a tracheal chondroma and was found to have lung cancer 14 months later, and the other was diagnosed with lung cancer 5 months later, suggesting that in certain cases, p53 antibodies may be a molecular marker of early malignancy (Schlichtholz et al., 1994; Lubin et al., 1995). Mudenda et al. (1994) reported p53 antibodies in serum in 48 of 182 (26%) breast cancer patients compared with 1 of 76 (1.3%) normal controls, and for 68 patients there was a significant correlation between seropositivity and increased p53 protein in the tumor; in addition, 8 of 23 (35%) patients with ductal carcinoma in situ were seropositive, suggesting once again that in some cases, p53 antibodies may be a molecular marker of early disease. Peyrat et al. (1994, 1995) reported p53 antibodies in 42 of 348 (12%) cases of locoregional primary breast cancer, with relapse-free survival and overall survival being worse in seropositive cases. Trivers et al. (1994b) reported p53 antibodies in 36 female lung cancer patients with good correlation between seropositivity and p53 gene mutations in the tumors. In a study of 9 patients

Molecular Markers of Carcinogenesis

with VC exposure and ASL, serum p53 antibodies were detectable in 3 of the cases prior to the clinical diagnosis of disease (average 5 8 years, range 5 0.3–12 years), once again suggesting that in some cases, this may be an early marker of disease (Trivers et al., 1994a, 1995). Marxsen et al. (1994) reported p53 antibodies in serum as determined by 3 different methods in 5 of 78 (6.4%) pancreatic cancer patients compared with 0 of 82 (2.4%) patients with benign pancreatic diseases. On the other hand, Laurent-Puig et al. (1995) reported p53 antibodies in serum by ELISA in 8 of 29 (28%) patients with pancreatic cancer and 5 of 33 (15%) patients with biliary tract cancer compared with 1 of 33 (3%) patients with benign pancreatic or biliary conditions. Houbiers et al. (1995) reported p53 antibody seropositivity by ELISA (with selected confirmation by immunoblotting) in 65 of 255 (25.5%) cases of colorectal cancer with a statistically significant correlation between seropositivity and p53 accumulation in the tumor tissue (P 5 0.05); in addition, seropositivity was association with poorer prognosis in terms of disease-free and overall survival, particularly in Stage A and Stage B1 patients. Guinee et al. (1995) reported serum p53 antibodies by three different methods in 9 of 107 (8.4%) patients with lung cancer, and 7 of the seropositive patients had p53 gene missense mutations or accumulations of p53 protein in the tumor tissue. Another study reported p53 antibodies in serum by two different ELISAs in 16 of 136 (11.8%) lung cancer patients compared with 0 of 52 (0%) patients with nonmalignant respiratory disease; however, in this case, although 47 of the tumors were found to contain p53 gene mutations, only 7 of those were seropositive, and of 32 tumors with accumulations of p53 protein, only 5 were seropositive (Wild et al., 1995). Willsher et al. (1996) reported serum p53 antibodies in 30 of 82 (48%) patients with breast cancer, but over 5 years of follow-up, seropositivity showed no correlation with disease-free interval or survival. Similarly, Regidor et al. (1996) reported serum p53 antibodies in 9 of 61 (14.5%) breast cancer patients with no correlation between seropositivity and disease-free interval. Ryder et al. (1996) reported serum p53 antibodies in 14 of 33 (42%) patients with hepatocellular carcinoma, and 10 of the seropositive cases had p53 accumulations in the tumor tissue, but 9 tissue-positive cases did not have serum antibodies; in addition, serum antibodies were detectable in 6 of 12 (50%) patients with small tumors (,4 cm). Several recent studies have examined p53 antibodies in patients with head and neck cancer. Bourhis et al. (1996) reported serum p53 antibodies in 15 of 80 (17.8%) head and neck cancer patients, and in 2 years of follow-up, seropositivity was significantly associated with increased risk of relapse (P 5 0.003) and death (P 5 0.002). LaVieille et al. (1996) reported serum p53 antibodies in 32 of 73 (44%) patients with head and neck cancer with a significant correlation between seropositivity and tumor tissue accumulation (P , 0.0001). Werner et al. (1997) reported serum p53 antibodies in 46 of 196 (23.5%) patients with head and neck cancer, and seropositive patients had a significantly worse survival (P , 0.05).

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6. CONCLUSIONS Studies to date suggest that blood levels of growth factors, oncogene proteins, tumor suppressor gene proteins, or antibodies to these proteins may be useful and convenient molecular markers of cancer. In most instances, blood levels seem to reflect reasonably well the expression of these proteins in target tissues. Furthermore, these markers cannot only distinguish individuals with cancer from those without, but they also can predict the risk of developing cancer or the risk of recurrence of cancer in some cases. Thus far, application of these markers has been experimental. Prior to more routine clinical application, further study will be necessary, but in the future, they could play an important role in cancer diagnosis and prevention. Acknowledgements–This work was supported in part by grants from the National Cancer Institute (R01-CA42500 and R01-CA69423) and the Environmental Protection Agency (R-825361).

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