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[CANCER RESEARCH 60, 1777–1788, April 1, 2000

]

Review

p53 Antibodies in the Sera of Patients with Various Types of Cancer: A Review1
Thierry Soussi2
Laboratoire de ge´notoxicologie des tumeurs, Institut Curie, 75248 Paris, France

Abstract
p53 antibodies (p53-Abs) were discovered 20 years ago during the
course of tumor-associated antigens screening. The discovery of p53 mutation and accumulation of p53 in human tumors shed new light on the
p53 humoral response. In the present review, we have compiled more than
130 papers published in this specific field since 1992. We demonstrate that
p53-Abs are found predominantly in human cancer patients with a specificity of 96%. Such antibodies are predominantly associated with p53
gene missense mutations and p53 accumulation in the tumor, but the
sensitivity of such detection is only 30%. It has been demonstrated that
this immune response is due to a self-immunization process linked to the
strong immunogenicity of the p53 protein. The clinical value of these
antibodies remains subject to debate, but consistent results have been
observed in breast, colon, oral, and gastric cancers, in which they have
been associated with high-grade tumors and poor survival. The finding of
p53-Abs in the sera of individuals who are at high risk of cancer, such as
exposed workers or heavy smokers, indicates that they have promising
potential in the early detection of cancer.

Introduction
The discovery of the p53 protein was the culmination of two types
of studies: (a) the very well known virological approach, with the
binding of p53 protein to oncoviral proteins (1–5); and (b) the more
discrete serological approach with the study of TAAs3 (6, 7). In 1979,
DeLeo et al. (7) showed that the humoral response of mice to some
methylcholanthrene-induced tumor cells such as MethA was directed
to the p53 protein. This protein was found to accumulate in tumor
cells of different origin but was undetectable in normal cells. It was
also found that animals bearing SV40 tumors elicited an immune
response specific for p53 (1, 5, 6). In 1982, Crawford et al. (8) first
described antibodies against human p53 protein in 9% of breast cancer
patient sera. No significant clinical correlation was reported, and, at
that time, no information was available concerning mutations of the
p53 gene. Caron de Fromentel et al. (8) later found that such antibodies were present in sera of children with a wide variety of cancers.
The average frequency was 12%, but this figure increased to 20% in
Burkitt’s lymphoma.
Those studies, performed in the early 1980s, were virtually ignored
for more than 10 years because of a lack of interest in p53 during that
period. In the early 1990s, it was discovered that the p53 gene is the
most common target for molecular alteration in every type of human
cancer (10). This provoked considerable interest in the study of the
p53 protein and its function in normal and transformed cells. It also
Received 10/25/99; accepted 2/16/00.
The costs of publication of this article were defrayed in part by the payment of page
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1
Supported by Ligue Nationale contre le Cancer (Comite´ de Paris), the Association
pour la Recherche contre le Cancer (ARC), the Mutuelle Ge´ne´rale de l’E´ducation
Nationale (MGEN), Direction de la Recherche Clinique (DRC) de l’Assistance PubliqueHoˆpitaux de Paris Contract Grant AOA94084.
2
To whom requests for reprints should be addressed, at Laboratoire de ge´notoxicologie des tumeurs, Institut Curie, 26 rue d’Ulm, 75248 Paris, France. Phone: 33-1-44-3241-60; Fax: 33-1-44-32-42-32; E-mail: thierry.soussi@curie.fr.
3
The abbreviations used are: p53-Ab, p53 antibody; TAA, tumor-associated
antigen; LSH, loop-sheet-helix (motif); SCLC, small cell lung cancer; NSCLC,
non-SCLC; ADC, adenocarcinoma.

led to the rediscovery of this humoral response, which had been found
in cancer patients. Since 1992, more than 150 papers have been
published on p53-Abs. This review will summarize this literature and
focus on the future applications of this assay.
For a comprehensive view of the p53 discovery, the reader is
referred to the excellent review published by Crawford (11). More
reviews on p53 have recently been published (12–15).
The p53 Protein
The tumor suppressor p53 is a phosphoprotein barely detectable in
the nucleus of normal cells (16). On cellular stress, particularly that
induced by DNA damage, p53 can arrest cell cycle progression, thus,
allowing the DNA to be repaired (17) or it can lead to apoptosis (18).
These functions are achieved, in part, by the transactivational properties of p53, which activate a series of genes involved in cell cycle
regulation. In cancer cells that bear a mutant p53, this protein is no
longer able to control cell proliferation, which results in inefficient
DNA repair and the emergence of genetically unstable cells (12–15).
The most common changes of p53 in human cancers are point missense mutations within the coding sequences of the gene (10, 19).
Such mutations are found in all of the major histogenetic groups,
including cancers of the colon, stomach, breast, lung, brain, and
esophagus (20). It is estimated that p53 mutations is the most frequent
genetic event in human cancers and accounts for more than 50% of
cases. More than 90% of the point mutations reported thus far are
clustered between exons 4 and 10 and are localized in the DNA
binding domain of the p53 protein (21). One of the most striking
features of the inactive mutant p53 protein is its increased stability
(half-life of several hours compared with 20 min for wild-type p53)
and its accumulation in the nucleus of neoplastic cells. Positive
immunostaining is usually indicative of abnormalities of the p53 gene
and its product, but it is highly dependent on the type of p53 mutation
for review (22, 23).
The Specificity of p53 Antibodies in Cancer Patients
Dosage of p53 Antibodies. The initial work on p53-Abs used
either immunoprecipitation or Western blot as the detection method
(8, 9). Later, several ELISAs were developed to handle large numbers
of specimens (24, 25), and some of them are now commercially
available. The diversity of all of these assays could account for the
variation in the frequency of p53-Abs observed in the literature. One
of the most important parameters seems to be the antigen used for
these assays. It has been shown that p53-Ab recognized immunodominant epitopes localized in the NH2 and COOH termini of the protein
(see below). It is thus essential to use the entire p53 protein as antigen.
Several attempts to develop an ELISA with synthetic peptides corresponding to these immunodominant epitopes have been unsuccessful,
because they lead to a high level of false-negative results (26).
p53 is heavily phosphorylated at the NH2 and COOH termini. Such
phosphorylation can have an important influence on the reactivity of
p53-Abs toward the protein, which suggests that p53 expressed in
mammalian cell is a better antigen than those expressed in Escherichia coli. Recently, it has been shown that IgA p53-Abs are found

1777

this finding was confirmed by numerous studies. such information was not available.p53 ANTIBODIES IN THE SERA OF PATIENTS WITH CANCER as the only isotype in some patients with head and neck cancers. cancers such as esophageal carcinoma and oral squamous cell carcinoma. Nevertheless. manuscript in preparation. nor can we exclude the possibility that the tumor is composed of heterogeneous tissue and that the fragment analyzed does not bear p53 alteration. The only exception is glioma. Analysis of more than 200 sera from various types of cancer unambiguously demonstrated that these antibodies recognize immunodominant epitopes localized in the NH2 terminus and. 36) despite a high frequency of p53 mutations (37). mutant p53 accumulation is an important component of this humoral response. were 4 M. These observations demonstrate that other factors contribute to this humoral response. Monoclonal antibodies specific for the central region of the protein 1778 . Furthermore. Rainov et al. Only a few antibodies recognize the central region of the p53 protein that harbors the mutations. the pleural effusions of patients with pancreatic. Control experiments were performed in 36 studies. again arguing that p53 mutations are involved in the appearance of these antibodies. 48). we should bear in mind that it only works with antibodies that recognize small linear epitopes. and p53 Accumulation There is generally a very good correlation between the presence of p53-Abs and p53 accumulation and/or mutation in the tumor. 1. melanoma. As shown in Table 1. in the COOH terminus of human p53 (26. which have a high rate of p53 mutations. are also negative for p53-Abs. and p53 accumulation. 40). Apart from the healthy control. These analyses. Relationship between p53 Antibodies. Tavassoli and T. 46). As discussed below. but we cannot exclude the fact that modified processing of the mutated protein can also lead to such a response. There is a very strong correlation between the two rates. We should also keep in mind that serological analysis is a global assay that does not depend on sampling. or if no immune response can be elicited in the brain. Accumulation may be an important component in the development of this immune response. in one series. also have a high frequency of p53-Abs. well-known to be devoid of p53 mutations. precise mapping of the epitopes of p53 protein recognized by p53-Abs was performed (47). testicular carcinoma (31. a compilation of all of the serological analyses performed in conjunction with molecular analyses indicates that the repartition of p53 mutations in patients with p53-Abs is similar to that in patients without antibodies (Fig. An undetectable metastasis with p53 alteration might be associated with a p53-negative primary tumor. 32). identical p53 mutation. As shown in Fig. and lung tumors. or human wild-type p53 led to the production of monoclonal antibodies directed to linear epitopes localized in the NH2 and COOH termini of p53 (49 –52). Such a finding is totally in accordance with the work performed in mice on the production of p53 monoclonal antibodies. Studies involving 9489 patients with various types of cancer were analyzed. despite similar types of cancer. Fig. xenopus. Follow-up of patients who were devoid of p53Abs at the time of diagnosis failed to detect any de novo antibody production after therapy failure or relapse despite p53 accumulation in the tumor and a long follow-up. Immunization of mice with murine.4 p53-Abs have also been found in other body fluids such as the ascites of women with ovarian cancer. with a very low rate of p53-Abs (35. In a complete study involving both molecular and serological analyses. 47. However. it is striking to observe that three cancers. Using a set of overlapping synthetic peptides corresponding to human p53. Several studies have shown that. to a lesser extent. Eighty serological analyses of p53 status in 18 cancer types that were published before August 1999 have been compiled (Table 1). Several explanations can account for the lack of a p53 immune response in this type of cancer. This method is very powerful and very rapid for the analysis of a large number of sample. These results are not due to a lack of sensitivity of the current methods of detection but to a real absence of p53-Abs. 70% of the patients were treated with dexamethasone before serum collection (35). In the other studies. 2). We know that the identification of such epitopes is not due to a technical bias based on the use of short synthetic peptides because immunoprecipitation and immunoblot analysis of truncated p53 have led to the same observation (45. 48). the mechanism that leads to the formation of these antibodies is poorly understood. 45. 34). the immune response is dependent on the specific combination of MHC class I and II molecules expressed by each individual. Expression of p53 in oral squamous cell carcinoma is associated with the presence of p53 autoantibodies in sera and saliva. Technical failure cannot be neglected and is difficult to assess. Examination of p53-Abs during follow-up of patients during therapy indicates that the level of p53-Abs can vary depending on the tumor burden (see below). Statistical analysis demonstrated that p53 antibodies are a specific marker of patients with neoplasia (P ⬍ 10-4. It is also possible that there is inefficient antigen presentation if p53 cannot cross the brain barrier. Such antibodies are correlated with serum p53Abs (27. The fact that a majority of ELISAs use a secondary antibody specific for IgG may eventually lead to false-negative sera. 1 shows the correlation between the frequency of p53-Abs and the frequency of p53 mutations published in the literature. 28). The analysis of mouse sera immunized with these proteins indicates that it is due to a specific immune response of the mouse toward this region of the protein and not to a bias in the selection of the hybridoma (49). (35) found p53 mutations in 24 of 60 glioblastomas. Specificity of p53 Antibodies As stated above. in which the frequency of p53-Abs was very similar to that of the control group (Table 2). (38) have shown that only missense mutations can lead to a p53 humoral response. It had been suggested that only p53 mutations that are localized in exons 5 and 6 with an altered protein conformation and that bind to hsp 70 are associated with p53-Abs (39. Only a subset of patients with p53 mutations will have p53 antibodies in their tumor. we should keep in mind that. involving 2404 individuals who were either healthy or had nonmalignant diseases. and glioma. It is possible that the immune privilege of the brain does not allow the induction of this humoral response. Winter et al. p53 Antibodies and p53 Mutation Frequency. Thus. Soussi. and melanoma (33. it is possible that such antibodies are specific to mutant p53 or at least to a mutant p53 conformation. hepatoma. not confirmed in larger series of patients. but none of these patients had p53-Abs. and the saliva of patients with oral cancer (27–29). some patients could be either positive or negative for p53-Abs (41– 44). performed on a small number of patients. for an identical mutation. about 20 – 40% of patients with a p53 mutation will have p53-Abs in their sera. Table 2). p53 Mutations. colon. It is possible that. An analysis of p53-Abs in each type of cancer also showed a significant correlation with malignant diseases for most types of cancer except for testicular carcinoma. This suggests that the capacity to elicit this humoral response is linked to the biological background of the patients. Several works have shown that these antibodies recognize both wildtype and mutant p53 (38. There may be several explanations for the presence of p53-Abs in tumors with a wild-type p53. which can induce perturbation in the immune response. hepatoma (30). Indeed.

especially for SCC NSCLC. association with advanced stage and p53 accumulation in the tumor. association with better survival after radiotherapy NSCLC. 1979 –1999 Methodology Control studiesa IPb IP Wb Wb ELISAc Wb ELISA ELISA ELISA ELISA Wb ELISA ⫹ Wb ELISA ELISA ELISA ELISA ELISA ⫹ Wb ⫹ IP ELISA ⫹ Wb ⫹ IP Wb ELISA ELISA ELISA ELISA ELISA ELISA ELISA ELISA ELISA Wb ELISA ⫹ Wb ELISA ⫹ Wb ELISA ⫹ Wb ⫹ IP ELISA ⫹ IP ELISA ELISA ELISA ELISA Wb ELISA Breast IP IP Wb IP ⫹ Wb ELISA ELISA ELISA Wb ELISA IF ELISA ELISA ⫹ Wb ELISA ELISA ELISA Lung Wb ELISA ELISA ELISA ELISA ELISA ⫹ IP ⫹ Wb ELISA ELISA ELISA ⫹ Wb Wb Wb ELISA ELISA Wb ELISA ELISA Colon cancer IP ELISA ELISA ELISA ELISA ELISA Positive Total 0 1 0 0 2 1 1 0 0 0 0 3 0 2 4 0 164 88 51 67 206 73 76 49 17 15 48 330 40 36 63 41 0 1 0 30 19 24 0 0 0 0 0 1 0 0 2 0 0 0 0 0 0 0 0 0 0 17d 195 20 9 40 10 69 140 41 50 10 11 60 50 51 24 9 41 17 50 70 14 14 7 15 3 12 10 45 48 21 42 15 2 9 39 155 122 60 100 105 93 290 176 182 50 353 165 12 61 82 6 10 6 10 16 9 18 13 38 9 1 19 17 27 54 20 46 42 73 42 136 107 67 62 188 111 14 84 140 170 231 97 2 13 63 59 42 10 16 82 249 184 235 41 Comment Healthy women Children with nonmalignant disease Healthy control Acute and chronic liver diseases Healthy control Healthy women Healthy control Nonmalignant lung disease Healthy control Healthy control Healthy control Healthy control Healthy control Nonmalignant liver disease Healthy control Healthy control (n ⫽ 29) Nonmalignant lung disease (n ⫽ 12) Healthy control Nonmalignant esophageal disease Healthy control (n ⫽ 10) Nonmalignant lung disease (n ⫽ 14) Healthy control Healthy control Gastric polyps Nonmalignant liver disease Healthy control Healthy women Chronic hepatitis C Healthy control Healthy control Healthy control Healthy control (n ⫽ 6) ⫹ HBV-infected patients (n ⫽ 5) Healthy control Healthy control Healthy women Nonmalignant digestive disease Healthy control Healthy control Chronic inflammatory diseases of head and neck Healthy control Nonmalignant oral diseases Association Association Association Association with with with with high grade high grade specific p53 mutation binding to hsp70 high grade and with tumor negative for steroid hormone receptor Association Association Association Association Association Association with with with with with with tumor negative for steroid hormone receptor p53 accumulation in the tumor high histological grade and p53 accumulation in the tumor good survival short survival short survival Association with tumor size No clinical correlation detected Correlation with missense mutation and p53 accumulation Association with p53 accumulation in the tumor NSCLC.p53 ANTIBODIES IN THE SERA OF PATIENTS WITH CANCER Table 1 Frequency of p53-Abs in various types of cancer: a survey of the literature. association with p53 accumulation in the tumor NSCLC. association with poor prognosis for limited-stage SCLC patients Association with poor differentiation and short survival Association with p53 accumulation and short survival Association with p53 accumulation in the tumor 1779 Reference (8) (9) (38) (110) (24) (111) (65) (112) (113) (69) (70) (66) (84) (84) (92) (114) (91) (91) (76) (115) (93) (71) (116) (85) (117) (118) (119) (120) (121) (122) (123) (124) (26) (82) (125) (81) (81) (79) (126) (8) (127) (39) (45) (128) (47) (24) (111) (65) (68) (64) (66) (120) (129) (67) (38) (47) (24) (48) (112) (130) (131) (114) (132) (76) (76) (75) (77) (79) e (78) (127) (24) (73) (72) (115) (85) . association with p53 accumulation in the tumor and short survival for SCC only SCLC. no association with clinical parameters SCLC SCLC. no association with survival NSCLC. association with poor survival SCLC NSCLC. association with poor survival.

and poor survival No clinical correlation detected (69) (70) (71) (134) 7 24 15 32 18 23 15 9 39 7 37 11 47 32 14 70 50 74 82 117 80 39 143 30 97 30 177 73 11 3 6 16 33 36 20 65 ELISA ELISA Liver cancer (HCC) Wb 16 33 63 57 20 80 ELISA ELISA ELISA ELISA ELISA ⫹ Wb Ovarian cancer ELISA ELISA ELISA ELISA ELISA ELISA ELISA ELISA 9 5 28 3 14 130 39 86 7 38 10 11 12 3 15 18 8 11 72 86 33 17 40 86 30 38 10 41 4 30 174 46 2 14 2 5 8 5 23 46 73 46 78 29 96 145 6 33 2 2 83 65 (128) (24) 4 108 (47) 8 29 (47) 4 1 1 2 3 3 88 107 50 50 50 83 (47) (24) (149) (149) (149) (150) ELISA ELISA ELISA ⫹ IP Pancreatic cancer ELISA ELISA ELISA ELISA ⫹ Wb ELISA ELISA ⫹ Wb ELISA ⫹ Wb ELISA Prostate cancer ELISA ELISA Thyroid cancer ELISA Bladder cancer ELISA Leukemia ELISA ELISA Wb Wb Wb ELISA Comment Association with p53 accumulation in the tumor No clinical correlation detected Leukoplatia Association with poor differentiation and p53 accumulation in the tumor No clinical correlation detected Association with p53 accumulation in the tumor Association with p53 accumulation in the tumor and short survival Association with therapy failure Associated with tumors with p53 complexed to Hsp70 Patient with recurrence Association with tumor size Association with p53 accumulation in the tumor Esophageal cancer. poor differentiation and short survival Association with poor differentiation. high stage. no relationship with clinical parameter Not done European patients Advanced disease Association with p53 accumulation in the tumor and better survival Association with poor overall survival Association with p53 accumulation in the tumor Association with poor survival Benign ovarian tumors No clinical correlation detected Association with poor histological differentiation and p53 accumulation in the tumor Association with poor histological differentiation and short survival Chronic pancreatitis Association with high grading (grade III). and p53 accumulation in the tumor 1/19 acute pancreatitis 4/33 chronic pancreatitis HTLV-1 asymptomatic carrier ATL HAM/TSP Association with p53 mutation 1780 Reference (120) (121) (124) (82) (74) (135) (92) (92) (136) (137) (119) (80) (125) (81) (40) (126) (126) (138) (139) (91) (91) (93) (44) (140) (141) (110) (84) (142) (116) (118) (122) (128) (24) (113) (113) (86) (143) (28) (117) (144) (145) (26) (128) (47) (24) (146) (147) (123) (123) (148) . short survival. association with p53 accumulation in the tumor Barrett’s metaplasia Association with p53 accumulation in the tumor and p53 mutation in the core domain Association with p53 mutation in the tumor.p53 ANTIBODIES IN THE SERA OF PATIENTS WITH CANCER Table 1 Continued Methodology Positive Total ELISA Wb IP ⫹ Wb ⫹ ELISA ELISA ELISA Lymphoma IP ELISA ELISA Gastric cancer ELISA Wb ELISA ELISA Oral cancer Wb ELISA ELISA ELISA ELISA ELISA ELISA ELISA ELISA Wb ELISA ELISA ELISA ELISA Esophageal cancer ELISA ⫹ Wb ⫹ IP ELISA ⫹ Wb ⫹ IP ELISA IP ⫹ Wb ⫹ ELISA 10 32 9 14 53 42 47 65 54 229 14 3 2 119 115 14 High prevalence in Burkitt1 lymphoma and T-cell lymphoma From patients with Sjo¨gren syndrome (9) (24) (133) 23 61 8 13 120 501 25 81 Association with p53 accumulation in the tumor and short survival Association with large tumor.

c ELISA. Soussi. f Unpublished observations. manuscript in preparation. not significant. e P. This question is difficult to answer because there is no normal situation of wild-type p53 accumulation in humans that could be used to test this hypothesis. but thus far. adult T-cell leukemia. although some patients exhibit a predominant IgA response (25). which suggests very weak (if any) tolerance to endogenous p53. No IgG3 or IgG4 was detected.p53 ANTIBODIES IN THE SERA OF PATIENTS WITH CANCER Table 1 Continued Methodology Testicular cancer ELISA Hepatoma ELISA Melanoma ELISA Multiple myeloma ELISA ELISA ELISA Glioma ELISA ELISA ELISA Endometrial cancer ELISA Positive Total Comment Reference 0 144 (24) 1 150 (24) 0 58 (24) 5 18 0 165 119 80 (24) (151) (150) 2 0 4 12 70 107 (36) (35) 2 10 (144) b a We have included all individuals both healthy and with nonmalignant disease. p53-Abs have been discovered in the sera of animals that bear tumors induced by SV40 (1. As stated above. d This single study represented 50% of positive cases in healthy controls. the level of p53 proteins in a normal organism is very low. It is not clear whether p53 mutation is really required for the production of p53-Abs or whether the sole accumulation of p53 protein can lead to this humoral response. respectively. c HCC. regardless of the location of their epitopes. NA. Western blot. It is possible that it is released after cell necrosis. (c) an L2 loop containing a small helix. p53 has not been found reproducibly in human sera (see below. Murray. wild-type p53 is stabilized through its interaction with SV40 large T antigen. they were all able to recognize p53 mutants that had undergone conformational changes (53). In such tumors. 56). These two loops are held together by a zinc atom tetracoordinated to Cys176 and His179 on the L2 loop and to Cys278 and Cys242 on the L3 loop. and M. human T-cell lymphotrophic virus. On the other hand. the conformational changes in p53 were dissected by a new battery of monoclonal antibodies directed against the central region of the protein (53). an ␣-helix. (b) the correlation between p53 accumulation (and p53 gene mutation) in tumor cells and p53-Abs responses. not applicable. Again. The L2 loop is presumed to provide stabilization by associating with the L3 loop. This result indicates 1781 . T. The mechanism by which p53 is presented to the immune system is unknown. such a situation occurs in animals. a this result strengthens the hypothesis of an active humoral response to p53. HAM/TSP.05. and the L1 loop. Furthermore. or else other mechanisms of p53 presentation are involved. WB. These observations indicate that p53 accumulation alone is the main component of this autoimmunization. It is quite remarkable to note the striking correspondence between these various structural elements and the four evolutionarily conserved blocks (II to V). None of these antibodies was able to recognize native. and (d) an L3 loop composed mainly of turns. one study contributed to 17 (50%) positives in healthy controls. ATL. The LSH motif and the L3 helix are involved in direct DNA interaction (LSH with the major groove and L3 with the minor groove). hepatocellular carcinoma. 5). and (d) the similarity of antigenic site profiles in patients and hyperimmunized animals—all suggest that p53 accumulation is the major component of the humoral response in patients with cancer. Taken together (a) the presence of immunodominant epitopes outside the hot spot region of the p53 mutation. These two sheets form a kind of compact sandwich that holds the other elements. NS. Tavassoli. b IP. Some patients also had IgM. (c) the similarity of humoral responses in patients independent of the cancer type. type 1. 20% of the assays were homemade. immunofluorescence.015. b As shown in Table 1. This accumulation could lead to a self-immunization process culminating in the appearance of p53-Abs. immunoprecipitation. On the other hand. This core region has been shown to include the following motifs: (a) two antiparallel ␤ sheets composed of four and five ␤-strands. prostate carcinoma reaches significance with a P of 0. Either the p53 protein is very rapidly eliminated from sera. All of the statistical analyses including this study were performed emphasizing the strength of these trends.03 NS NA NA NA 1600/9489 ⬍10⫺4 Correlation between cancer patients and healthy individuals were tested by the ␹2 test. IF. HTLV-1. Isotyping of p53-Abs has shown that they correspond mainly to IgG1 and IgG2 subclasses.005 0. Table 2 p53-Ab frequency in various types of cancer: statistical evaluation Status Healthy Esophageal cancer Oral cancer Bladder cancer Colon cancer HCCc Ovarian cancer Lung cancer Breast cancer Gastric cancer Pancreatic cancer Multiple myeloma Lymphoma Leukemia Glioma Prostate cancer Testicular cancer Melanoma Hepatoma Total cancers Frequency of p53-Abs 35/2404 Pa b 85/274 309/1062 8/29 307/1244 82/387 140/635 219/1282 296/2006 105/727 60/650 23/364 19/248 14/428 6/144 4/148 0/144 0/58 0/150 ⬍10⫺4 ⬍10⫺4 ⬍10⫺4 ⬍10⫺4 ⬍10⫺4 ⬍10⫺4 ⬍10⫺4 ⬍10⫺4 ⬍10⫺4 ⬍10⫺4 ⬍10⫺4 ⬍10⫺4 0. If this study is omitted (18 positive controls). 54). wild-type p53. (b) a LSH containing three ␤-strands. although none had p53 IgM as the only isotype. HTLV-associated myelopathy/tropical spastic paraparesis. The levels of significance were set at P ⬍ 0. whereas 80% were commercial (2 main distributors). 55. HCC. were obtained only after a special immunization or selection procedure (53. The central region (amino acids 102–292) was crystallized in the form of a protein-DNA complex (57). Serum p53 antibodies: predictors of survival in small-cell lung cancer?. X-ray crystallography of human p53 was an important step in the understanding of the structure of this protein. hepatocellular carcinoma.

In lung cancer. and adjuvant is not necessary to obtain a high titer of antibodies. This question has never been thoroughly discussed or experimentally tested. one should bear in mind one question that has not been assessed thus far. controversies exist concerning the clinical value of p53-Abs (Table 1). it is conceivable that p53-Abs could either cross-react with such proteins or were produced toward one of the homologues. Extensive assays of sera with or without p53-Abs indicate that most sera are specific for p53. any clinical correlation with p53-Abs would be due to the presence of p53 inactivation via mutation. Nevertheless. two of three studies also found an association between p53-Abs and short survival (72–74). the role of such antibodies in the neoplastic process. Such conformation and the observation that this region is highly hydrophobic explain its poor immunogenicity. As for p53 mutation and p53 immunohistochemical analysis.e. Unzal. 1. In breast cancer. several p53 homologues have been identified (59 – 62). In that case. several studies indicate that p53-Abs are found in patients with tumors that have high grades and/or that are negative for steroid hormone receptors (8. Relationship between the frequency of p53 mutation and p53 antibodies in various types of cancer. and T.p53 ANTIBODIES IN THE SERA OF PATIENTS WITH CANCER Fig. 45. Tominaga. This can explain why such a region is at the surface of the molecule. which indicated that the immunological response was not dependent on any particular p53 mutation and that immunization with this live virus vaccine works effectively against mutant p53 protein expressed in a tumor cell (63). It is plausible that early p53 accumulation. it is quite possible that we experience several unknown precancers that arise after a genetic variation in an oncogene or a tumor suppressor gene. 65). 6 Fig. Such preneoplastic cancers could be quickly eliminated either because the mechanisms that control cellular proliferation have been able to overcome these tumoral cells or because they have been eliminated from the organism through various surveillance mechanisms including the immune system. In NSCLC. (63).5 Recently. as for p53 mutations. is an incredibly potent antigen. It should also be mentioned that the p53 protein. these studies have reported contradictory results. the conservation of the sequence is only partial. and another study (50 patients) found an association with good survival (68). 1782 . manuscript in preparation. O. 24.6 p53 Antibodies and Clinical Parameters Numerous studies have attempted to evaluate the clinical value of p53-Abs (Table 1). on 353 and 165 patients. Soussi. This tumor protection was equally effective regardless of whether wild-type or mutant p53 was used for the immunization. Two studies. can lead to the production of a humoral and cellular response that participates in the elimination of the tumor. Before examining them. and the frequency of p53-Abs is taken from Table 1. Immunization with canarypox virus recombinants that expressed human or murine p53 protected BALB/c mice from a challenge with a highly tumorigenic mouse fibroblast tumor cell line that express high levels of mutant p53. D. In colon cancer. 5 Y. The frequency of p53 mutations is taken from the literature. that the central region of wild-type p53 protein has a very compact structure that is held in place by the two antiparallel B sheets. such as that seen in lung or oral cancer. it is quite possible that such an immune response is totally neutral toward the organism and is simply the consequence of self-immunization toward a self-protein. Legros and T. whatever its phylogenetic origin. Immunization of mice with p53 requires only a small amount of protein. unpublished observation. Lane. However. K.. personal communication. 2. During our lifetime. two clinical parameters already known to be associated with p53 mutations and bad prognosis. 64. found an association between p53-Abs and short survival (64. Although there is no formal proof that a natural response toward p53 can protect from precancerous tumor. In gastric cancer. whereas one study (82 patients) did not find any association (67). A recent study (58) indicates that critical residues of epitopes recognized by several anti-p53 monoclonal antibodies correspond to key residues of p53 involved in interaction of the mdm2 protein with the NH2 terminus of the p53 protein. 66). Distribution of p53 mutations and p53-Abs in patients in whom both serological and molecular analyses were performed. p73 antibodies are found in the sera of patients with various types of cancer. i. three of four studies found an association between p53-Abs and poorly differentiated tumors and short survival (69 –71). Although. Such a hypothesis is supported by the work of Roth et al. it is not possible to exclude such a hypothesis. specific antibodies toward p73 have been detected. Soussi.

82– 87). WB Selected Individuals/ Possible Carcinogen Heavy smokers/tobacco residues Clinical Status at the Time of p53Ab Detection Description No detectable malignant or premalignant lesion Two individuals with p53-Abs Patient 90: no follow-up. This increase in p53-Abs has been detected 3 months before the detection of a relapse (5). p53-Abs could be a useful tool for controlling the response to therapy and for monitoring certain early relapses before they are clinically detectable. in such tumor types. Such studies can only be performed using a quantitative assay. 88) IHC⫹b Patient 24 with ASL: p53-Abs 10 yr before diagnosis Patient 23 with ASL: p53-Abs 3 mo before diagnosis Patient 19 with Raynaud’s syndrome: p53-Abs 4 yr before diagnosis Worker 22 without diagnosed cancer: p53-Abs 4 yr before assay Worker 21 without diagnosed cancer: p53-Abs 6 yr before assay ND Patient 12 with lung cancer. clinical data were not available. and (c) a decrease of p53-Abs can occur in patients who have been treated by surgery without any chemo.p53 ANTIBODIES IN THE SERA OF PATIENTS WITH CANCER Table 3 Frequency of p53-Abs in high-risk individuals: a survey of the literature 1979 –1999 Methodology ELISA. WB Heavy smokers with COPD/tobacco residue ND ELISA ELISA. not done. c These four normal subjects were heavy consumers of tobacco and betel quid. In oral cancer. nearly as rapid as the half-life of human IgG (83. it is reasonable to presume that such p53-Abs could be used as an early indicator of p53 mutations in tumors in which such alterations occur early during tumoral progression (Table 3). it is possible to detect the reappearance of p53-Abs 2 years after initial therapy. IP. 83. Several studies have addressed this question in various types of cancer (24. Thus. The value of p53-Abs in terms of survival is promising. p53-Abs 7 mo before diagnosis Patient 10 with lung cancer. (83) showed that there is a good correlation between the specific evolution of the p53-Abs titer and the response to therapy in patients with lung cancer. Thus. p53-Abs 11 mo before diagnosis ND ND ND ND ND Barrett’s metaplasia Retrospective study (90) Mutant p53 IHC⫹ IHC⫹ Exposed individuals: 4/63c Leukoplakia Retrospective study (42) Prospective study (92) Premalignant (leukoplakia): 15/50 Cancer: 24/70 No follow-up of these patients ND Patient 88 with Barrett’s esophagus. In several patients. Taken together. 81). b p53-Abs seem to be associated with poor survival. Removing the tumor would prevent such stimulation. Several arguments demonstrate the specificity of p53-Abs variation during therapy: (a) there is no variation in total serum immunoglobulins. there is a trend toward an association between p53-Abs and tumors with poor differentiation. In breast cancer. WB ELISA. One good model for testing this hypothesis is that of 1783 . p53-Abs 1 mo before detection of ADC Patient 2 with ADC. IP. tumor was detected before clinical manifestation of the cancer. 86). WB Betel nut chewer/tobacco and copper in betel nut Barrett’s metaplasia p53 Alteration Type of Study (Reference) NDa Prospective with follow-up (48. These antibodies are usually IgG indicating a secondary response after a prolonged immunization before the diagnosis of the disease (25). p53 accumulation is the major component in the appearance of these p53-Abs. two studies have also demonstrated an association between p53-Abs and short survival (80. Zalcman et al. p53-Abs 7 mo before diagnosis Patient 11 with prostate cancer. the disappearance of p53-Abs was very rapid.or radiotherapy. especially in squamous cell carcinoma (75–77). IHC ⫹. angiosarcoma of the liver. p53 and Follow-Up of Patients during Therapy Because p53 accumulation is the main trigger of this humoral response. 28. immunohistochemistry. As indicated above. patient still alive after therapy Factory workers in industry/vinyl chloride ND ELISA. (b) there is no variation in the amount of antibodies directed toward other antigens. but additional studies are necessary before this can be clearly established. several studies have demonstrated that sera that score negative at the time of diagnosis never turn positive during follow-up (82. p53 Antibodies and Populations at High Risk of Cancer As demonstrated in the previous section. IP. died 8 months later from lung cancer Patient 37: effective follow-up. p53-Abs 1 mo before diagnosis ND Retrospective study (91) Mutant p53 a ND. Using immunoprecipitation and two different ELISA formats. whereas in SCLC. a feature already observed with p53 mutations. IHC. In other studies. but this has not been taken into account in many reports. All of the studies described up until now used sera taken at the time of diagnosis before any treatment. A similar situation was described in colorectal (82) and ovarian cancer (24). 88). ASL. developed a lung cancer 2 years later. p53 accumulation in the tumor. p53-Abs 6 mo before diagnosis Patient 8 with breast cancer. it was of interest to examine the behavior of these p53-Abs during therapy to see whether there was a relationship between tumor disappearance and a decrease in p53-Abs. IP. the studies are very divergent (78. All of these observations indicate that constant stimulation of the immune system is necessary to maintain a high level of p53-Abs. 79). in all of the these studies.

To our knowledge. and that used such assays for clinical management of the patient. Two years later. The finding of p53-Abs in patients with Barrett’s esophagus may be promising if confirmed in a larger population because it may predate clinical diagnosis of esophageal ADC (91). Indeed. and such mutations usually occur early in the transforming process (89).p53 ANTIBODIES IN THE SERA OF PATIENTS WITH CANCER Table 4 Quantification of p53 in human sera Assay Patients Amount of p53 ELISAa/PAb240 71 controls 40 colon cancers 12 polyps 58 controls 23 lung cancers 100 controls 13 nonmalignant lung diseases 114 patients with lung cancers 47 controls 61 “normal” with history of colon cancer 54 adenomas 22 carcinomas 10 controls 36 asbestos controls 27 lung cancers No controls 15 esophageal cancer 800 cancer patients 67 lung cancer patients 144 controls 184 colon cancers 0.44 ng/ml 0.04 ng/mlb 0. This work is of importance because it is known that p53 mutations are frequent in individuals exposed to various carcinogens.6 mg/ml 1. A similar situation occurs in individuals with premalignant oral lesions (leukoplakia) due to tobacco or betel nut chewing. 105) have been detected in sera of patients with various types of cancers. Due to the high frequency of this type of cancer in countries such as India or Pakistan.33 ng/ml 0.22–0. c-myb (100). Similarly. Unfortunately. Therefore. 88). There exist certain clinical situations in which nonmalignant lesions can predate their progression toward cancer.55 ng/ml 0. Such individuals are at high risk of developing oral cancer (5–10%). in carefully controlled experiments. 42). Antibodies to ras (99).84 ⫾ 0. b lung cancer and heavy smokers. Two sera were shown to be false positive after checking for heterophilic antibodies. Mutations and accumulation of p53 are found mainly in the transition from low. mdm2 (103) or HER2-neu (104. high-performance liquid chromatography. 90).09 ng/ml 0. P ⬍ 0. c HPLC. lung cancer was detected in this patient before any clinical manifestations of the disease (88). and a recent check-up indicated that neither the tumor nor p53-Abs had reappeared. The patient showed good response to therapy that paralleled the total disappearance of p53-Abs (83.55 ng/ml 0. who have been exposed to several carcinogens such as vinyl chloride. several studies have demonstrated that p53-Abs can be found in the sera of high-risk individuals. 93–98). with regular assay for p53-Abs and thoracic X-rays. The presence of antibodies to HER-2/neu were detected in 12 1784 .41 ng/ml 0. a variety of molecular changes have been characterized and correlated with tumor initiation and progression.6 ng/ml Negative Negative 0. which can lead to high background and false-positives. Ref. Sera have always been tested undiluted. c-myc (102). Antibodies toward Other Oncogenes and Tumor Suppressor Genes Only a few published studies have addressed this question. no follow-up has been performed on these patients. Levesque et al.6 mg/ml Should be taken with caution (56) (38) (98) a All of these ELISA were performed with the same commercial kit using a specific monoclonal antibody as a capture antibody.6–8. Since that work. which suggests that such antibodies could be used for early detection of cancer (Table 3. PT37.07 ng/ml ELISAa/PAb240 ELISA ELISAa/PAb240 ELISAa/PAb1801 ELISAa/PAb1801 ELISA ELISA HPLCc Comment References No statistical difference between controls and cancers (97) No statistical difference between controls and cancers (95) No p53 protein No p53 protein No p53 protein Statistical difference between controls and cancer. in 1994. The recent discovery that p53-Abs can be found in saliva indicates that easy screening could be organized to verify the value of these antibodies (27). In Barrett’s esophagus.44 ng/ml 0. Refs. it is found in individuals. p53-Abs have been detected in the sera of patients with chronic obstructive pulmonary disease and in heavy smokers (Table 3. Due to the lack of reliability of the various assays used. Angiosarcoma of the liver is an extremely rare cancer in humans.02 (55) No statistical difference between controls and cancer (94) (96) (93) 0. Indeed.31 ⫾ 0. 55. this is the only prospective study that addressed the importance of p53Abs in individuals at high risk for cancer. Ref. this patient has been tumor-free.04 ng/ml ⬍0. Ref.33 ⫾ 0. including workers in several types of industries.31 ⫾ 0. It should be pointed out that most assays use a commercial ELISA kit that was developed for the detection of p53 protein in cell or tumor extracts but that has not been fully verified on serum samples. was placed under surveillance. p53-Abs were found in two heavy smoker who were negative for any detectable lung cancer (48).04 ng/ml ⬍0. The second patient. Since 1996. this kind of diagnosis could be of importance.to high-grade dysplasia and are associated with an in- creased risk of cancer. this assay could be useful for early identification of cancer in individuals occupationally exposed to carcinogens.16 ng/ml ⬍0.8–5. L-myc (101). It is well established that p53 accumulation is an early event in lung cancer and that such cancer is strongly associated with tobacco smoking. p53 Protein in Sera This question continues to be a subject of debate with highly divergent results (Table 4. serum p53 protein should not be considered as valid as long as the protein has not been formally identified in sera using a reliable assay. p53Abs have been found at high frequency in patients with premalignant and malignant lesions.55 ⫾ 1.44 ng/ml 0. This is the case in Barrett’s esophagus. (55) demonstrated that some false-positive sera were caused either by the presence of human antibodies with broad antispecies specificities that can cross-react with some antibodies used in the assay or by other nonspecific reactants that interfere with the assay. The histopathological sequence for (Barrett’s) metaplasia.61 ⫾ 0. One of the patients could not be followed but died 8 months later from a rapidly growing lung tumor.37 ⫾ 0. p53-Abs were detected in the sera of individuals several years before the diagnosis of the tumors (Table 3.92). 56.46 ng/ml 0.29 ng/ml 0. which develops—as a consequence of chronic reflux—to dysplasia and then to carcinoma is well established for these tumors.

N. C... P. Virology. there is an increased burden of tumors due to carcinogen exposure. A. P. 19. p50.. T. Radioimmunoassay of the cellular protein p53 in mouse and human cell lines. 1979. Exp. Vernie.. 1994. Hilgers.. A. Genes Chromosomes Cancer. Finally. uncontrolled industrial development... G. 14. Melero. D. and 50% of them have a mutation in their p53 gene. Resnitzky... P... J... L. Such approaches are still under development and remain costly.. J. 36: 547–555.. J. and Craig. 22. Cassingena. P. thanks to the knowledge we have gained about p53. Virol.... Wild-type p53 induces apoptosis of myeloid leukaemic cells that is inhibited by interleukin-6.. Karlan. and Slamon.. 1982. 4: 1–15. R. Nucleic Acids Res. A. Caron de Fromentel. Such an effort.. Cancer Res. 1979. both in academic and private laboratories has never been made for any other single gene thus far. Autoantibodies to p53 in ovarian cancer patients and healthy women: a comparison between whole p53 protein and 18-mer peptides for screening purposes. Plummer. L. May-Levin. B. D. Bussel. Nature (Lond.. Cancer Lett. It is possible that p53 mutations in cancer related to such exposure are high (109). D. Yonish-Rouach. Cell. 17: 43–52. and Schlichtholz. Casey. M. then we can deduce that about 1 million of these patients have p53-Abs. which could lead to the discovery of new cancer genes... R.. Cell.. D.. Cell. Abelson murine leukemia virus-induced tumors elicit antibodies against a host cell protein.. R. Wilson. 1997.. T. 1987. Mol. R. Giaccia. 5. The SV40 A gene product is required for the production of a 54.. S. whose very life has been saved. Witte. 3. and Soussi. C. 1982. ACKNOWLEDGMENTS I am grateful to L... T. J. 1997. P. Y. and Kastan. This is the result of an increase in cigarette consumption.. T. and Old. Pim. 1998. Bennett. Old for reading the manuscript.. I think that this patient is the first to have benefited from significant improvement. L. 1: 1055–1062. Nevertheless. A.. Sachs. TP53 tumor suppressor gene: a model for investigating human mutagenesis. A. P. Cancer Res. Pim. Lane. if we estimate that there are 8 million patients with various types of cancer throughout the world. L. B.. Lane. Diamandis. A.. W.. 54: 2914 –2918. 1998. O. Maltzman. J. M. 76: 2420 –2424. Greenblatt. Crawford. 1: 1463–1469. V. 10. 95: 5– 8.. 1994. 31: 472– 483. Linzer. let me dedicate this review to Patient PT37 (88). Over the past 10 years. J. Caron de Fromentel. and Soussi.p53 ANTIBODIES IN THE SERA OF PATIENTS WITH CANCER (11%) of 107 breast cancer patients versus none (0%) of 200 normal controls (104). Cancer... Acad. . The paucity of these data compared with p53 suggests that p53 immunogenicity is a rather specific situation related to the striking immunogenicity of the p53 protein. J. W. 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T antigen is bound to a host protein in SV40transformed cells. G. 1979. Clinical utility of the immunocytochemical detection of p53 protein in cytological specimens. M. Oren. Levine.. Pathol. Vennegoor.. The presence of antibodies to HER-2/neu was also correlated with the accumulation of HER-2/neu protein in the patient’s primary tumor. 1979. p53 antibodies in patients with various types of cancer: assay. B. M. D. and the absence of regulation in waste evacuation. M.. and Baltimore. E. and Bunting. D. and Levine.. Law. Drijfhout. that enables identification of specific antibodies associated with gene overexpression in tumors (106. Only prospective studies on various types of cancer in which relapses occur several months or years after treatment will enable us to validate this assay. 278: 261–263. The use of a low-cost assay for the detection of p53-Abs could be of public health benefit in such countries. C. De Leo.. J. W. 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