monitoring of p53 autoantibodies in lung cancer during ...p53 gene is the most common target for...

Vol. 4, 1359-1366, June 1998 Clinical Cancer Research 1359

Advances in Brief

Monitoring of p53 Autoantibodies in Lung Cancer during Therapy:

Relationship to Response to Treatment’

Gerard Zalcman, Beata Schlichtholz,

Jean Tr#{233}daniel, Thierry Urban, Richard Lubin,2

Isabelle Dubois, Bernard Milleron, Albert Hirsch,

and Thierry Soussi3Service de Pneumologie, H#{244}pitalSaint-Louis, 75010 Paris, France

[0. Z., J. 1., A. H.J; U301 Institut National de la Sante et de Ia

Recherche M#{233}dicale,1GM 75010 Paris, France [B. S., R. L., 1. D.,1. S.]; Service de Pneumologie, H#{244}pitalSaint-Antoine, 75012 Paris,France [1. U.]; and Service de Pneumologie, H#{244}pitalTenon, 75020Paris, France [B. M.]

AbstractAlteration of the p53 gene is the most frequent ge-

netic alteration in human cancer, and it leads to the

accumulation of mutant p53 in the nucleus of tumor cells.

In addition, it has been shown that patients with various

types of neoplasias have p53 antibodies in their sera.

ELISA was used to detect anti-p53 antibodies in the sera

of 167 patients with lung cancer. Among these, 32 mdi-

viduals (16 positive for p53 antibodies and 16 negative)

were monitored over a period of 30 months for p53 anti-

bodies. Twelve of 16 antibody positive patients had re-

duced titers during chemotherapy that led to partial or

complete remissions of disease. The specificity of these

antibodies was confirmed by two different ELISA proce-

dures and by immunoprecipitatmon. The very rapid, spe-

cific decrease in these antibodies during therapy suggests

that a constant level of tumoral cells with nuclear accu-

mulating p53 protein is necessary for a detectable hu-

moral anti-p53 response. The good correlation found be-

tween the specific evolution of the p53 antibody titer and

the response to therapy suggests that p53 antibodies could

represent a useful tool for checking the response to ther-

apy and for monitoring some relapses before they are

clinically detectable.

Received 1/7/98; revised 4/13/98; accepted 4/13/98.

The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby marked

advertisement in accordance with 18 U.S.C. Section 1734 solely to

indicate this fact.

I This work was supported by grants from the Ligue Nationale contre le

Cancer (Comit#{233}de Paris and Comit#{233}Nationale), the Association pour IaRecherche contre Ic Cancer, the Mutuelle G#{233}n#{233}ralede l’EducationNationale, the Fondation pour Ia Recherche M#{233}dicale,and the Directionde Ia Recherche Clinique de l’Assistance Publique-H#{244}pitaux de Paris

(Grant P940509).2 Present address: Pharmacell, H#{244}pitalSaint-Louis, 1 avenue Claude

Vellefaux, 75010 Paris, France.3 Present address and to whom requests for reprints should be addressed,at UMR 218 CNRS, Institut Curie, 26 rue d’Ulm, 75248 Paris, France.Phone:33-l-42-34-65- II ;Fax:33-l-42-34-67-25;E-mail:thierry.soussi@

curie.fr.

Introduction

In 1979, DeLeo et a!. (1) showed that the humoral response

of mice to some methylcholanthrene-induced tumor cells was

directed to the p53 protein. It was later found that animals

bearing several types of tumors elicited an immune response

specific for p53 (2-4). In 1982, Crawford et a!. (5) first de-

scribed p53-Abs4 in 9% of breast cancer patient sera. Caron de

Fromentel et a!. (6) 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

lymphoma.

Those studies, performed in the early l980s, were virtually

ignored for more than 10 years due to the 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. Alteration of the p53 gene occurs

predominantly by missense mutations occurring nonrandomly

throughout the central region of the protein, thereby inactivating

its DNA binding properties (7). The wild-type p53 protein is

regulated by rapid turnover, and the level present in the nuclei

of normal cells is below the sensitivity of immunohistochemical

analysis detection systems. In tumoral cells, changes in the p53

conformation and/or cellular environment lead to metabolic

stabilization of the mutant p53 and its accumulation in the nuclei

(8, 9). Several analyses have shown that there is a close corre-

lation between accumulation of p53 protein and mutation of the

p53 gene ( 10). These findings have shed new light on the

presence of serum p53-Abs. More recent works have indicated

that anti-p53 antibodies can be found in most human cancers

(1 1-15). There is generally a good correlation between their

frequency and that of p13 gene alterations (16, 17). Several

multifactorial studies showed a very good correlation between

the presence of p53-Abs. accumulation of the mutant protein in

the tumor, and the presence of a mutation in the gene ( I 3, 18,

19).Detailed analysis of these antibodies indicates that accu-

mulation of the p53 protein in tumor cells is responsible for the

appearance of autoantibodies with epitopes directed toward the

immunodominant region of the p53 protein (12, 16, 20, 21).

Although the detection of p53-Abs cannot replace molecular or

immunohistochemical analysis of p53 alteration, it has some

specific applications. First, it is now well documented that

p53-Abs can be detected in patients with a high risk of cancer

several years before the clinical manifestation of the disease (22,

23). A second application, which has not been well documented

thus far, concerns the use of p53-Abs for follow-up of patients

during their treatment (15, 24).

4 The abbreviations used are: p53-Ab, autoantibody to p53; SCLC, small

cell lung cancer; NSCLC, non-SCLC; HAV, hepatitis A virus; CT,chemotherapy; PCI, prophylactic cranial irradiation.

Research. on August 7, 2020. © 1998 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

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1360 Monitoring of p53-Abs in Lung Cancer

In the present study, from an original group of 167 pro-

spectively studied patients, we describe the results of a 25-

month follow-up of 32 patients. Of these patients, 16 were

seropositive for p53 antibodies and showed a good correlation

between evolution of the p53-Ab titer and response to therapy.

Control analysis demonstrated the specificity of the p53-Abs

variation.

Patients and MethodsPatients. The initial cohort included 167 consecutive pa-

tients with histologically proven lung cancer during a 25-month

period from April 1993 to May 1995 in three university hospital

in Paris, France (Saint-Louis, Saint-Antoine, and Tenon hospi-

tals). The primary goal of this prospective study was to evaluate

the prognostic value of p53 antibodies in lung cancer. This

protocol has been approved by the Consultative Committee for

the Protection of Persons in Biomedical Research. SCLC pa-

tients (n = 91) were treated in these three institutions with

equivalent therapeutic regimens: six cycles of platinum and

etoposide-based CT. After the second course of CT, additional

concomitant mediastinal radiotherapy was proposed to patients

with localized disease (patients with tumors confined to one

hemithorax). In these patients with limited disease (n = 50),

prophylactic cranial irradiation was proposed to clinically com-

plete responders. Only NSCLC patients (n = 76) from Saint-

Louis Hospital were included in the study and monitored for p53

antibodies because of the heterogeneity of treatments proposed

to NSCLC patients in the three participating centers. The char-

acteristics of the whole cohort of patients and the results of the

prognostic impact study will be described elsewhere?

Among this group of patients, 16 fulfilled the following

eligibility criteria: (a) they were inoperable; (b) they were

treated by CT alone or by chemoradiotherapy; (c) they were p53

seropositive; (d) they had a sufficient number of p53 antibody

determinations during CT (i.e. , at least three serum samples,

with a survival of at least 4 months); and (e) they had evaluable

lesions. Among p53 seronegative patients, 16 were selected for

study and matched to the p53-seropositive patients on the basis

of their response to therapy and their survival status.

Survival was evaluated from the date of histological diag-

nosis to the date of death or of the last follow-up visit. Response

was assessed according to Eastern Cooperative Oncology Group

standard guidelines (25). Pathological diagnosis was obtained

mainly from bronchial biopsy samples using WHO histological

criteria (26).

Sera were obtained after diagnosis, but prior to any treat-

ment; then at each follow-up evaluation visit (every 1-3

months), 7 ml of whole blood were centrifuged at 3000 rpm for

15 mm, and the supernatant was stored at -80#{176}Cuntil use. All

analyses were done in duplicate. Sera were analyzed with the

observer blind to patient status.

Survival data were updated in October 1997. Overall sur-

vival was analyzed using the Kaplan-Meier method. Curves

� G. Zalcman, J. Tr#{233}daniel, and T. Soussi. Serum p53 antibodies inpatients with limited small cell lung cancer correlates with poor prog-

nosis, manuscript in preparation.

were compared with the log rank test using the statistical pack-

age Statview 4.02 (Abacus Concepts, Berkeley, CA). The asso-

ciation between a 50% decrease in the p53-Ab titer and partial

or complete response was tested by Fischer’s exact due to the

small sample size.

ELISA for p53 Antibodies. ELISA and its characteris-

tics have been fully described elsewhere ( 17). We devised a

highly specific ELISA by testing all sera with two antigen

preparations; the first contained the relevant antigen, p53,

whereas in the second preparation, this antigen was omitted.

Polystyrene flat-bottomed microtiter plates (Fl6 Maxisorp,

Nunc) were coated with either p53 or control extract in carbon-

ate buffer. Plates were dried for 24 h at 37#{176}C.After saturation,

they were sealed in a polypropylene bag and stored at 4#{176}Cuntil

use. Such plates give reproducible results over a period of 6

months. Before use, plates were washed five times with PBS

containing 0.05% Tween 20; 100 p.1 of blocking buffer (PBS

0.2% Tween 20, 5.0% dried nonfat milk) were added per well.

After 1 h at 37#{176}C,the wells were washed as described above.

Then, 100 jii of sera (diluted 1:100 in PBS, 5.0% dried nonfat

milk) were tested in duplicate. The plates were incubated for I h

at room temperature on an ELISA plate shaker. After five

washings in PBS with 0.05% Tween 20, 100 p.1 of an antihuman

IgG peroxidase conjugate diluted 1 :8 in PBS 5% nonfat milk

were added and incubated for 1 h at 37#{176}C.Preliminary experi-

ments were performed with an antihuman immunoglobulin an-

tibody, but because we had demonstrated that all these p53-Abs

include IgG, we used an antihuman IgG antibody. Plates were

then washed five times and developed using a 3,3’S,S’-tetra-

methylbenzidine substrate. The reaction was stopped after 10

mm by adding I M sulfuric acid. Plates were then read at 450 nm

using a MR 5000 ELISA reader (Dynatech Laboratories). All of

these manipulations were performed simultaneously on a plate

coated either with p53 protein as described above or with the

control extract. The sensitivity and the specificity of this assay

have already been described (17). For the purpose of routine

screening, each serum was diluted 1 : lO(). For the quantitative

measurement of p53-Abs in positive patients during their fol-

low-up, sera were diluted such that there was no saturation

during color development. For a given patient, the same dilution

was used for each sample, and all samples were processed in the

same experiment. Most of these sera were also tested by immu-

noprecipitation as described previously ( I 7).

Peptide ELISA for p53 Antibodies. The major sites of

recognition by p53-Abs are localized in the amino and carboxyl

termini of the protein. This has been repeatedly demonstrated by

testing the binding of sera against a series of 77 peptides

overlapping the complete p53 protein. The majority (98%) of

positive sera reacted with an amino-terminal peptide, and 48%

reacted with a carboxyl-terminal peptide (20). We then devised

a new ELISA with six peptides (18). Five peptides correspond

to p53 peptides: peptide 1 , EPPLSQESDLWKLLPENNVLSPL,

corresponds to the first immunodominant epitope localized in

the amino terminus; peptide 2, DDLMLSPDDIEQWFT, and

peptide 3, SPDDIEQWFI’EDPGP, correspond to the second

immunodominant epitope localized in the amino terminus; and

peptide 4, EALELKDAQAGKEPGGSRAHSSHLK, and pep-

tide 5, GSRAHSSHLKSKKGQSTSRHKKLMF, correspond to

two immunodominant peptides localized in the carboxyl termi-



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Fig. 1 Variations in p53 antibodies. For all but two patients (GAT and BRE), p53 antibodies were monitored using either entire p53 (left row) orwith synthetic peptides (rig/it row). For ELISA using the entire p53, results are expressed as the ratio between p53 and the control antigen as describedin “Patients and Methods.” For ELISA using peptides, absorbance is given for each assay. Values of the control peptide have been deduced for eachmeasurement.

Clinical Cancer Research 1361

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Table

Patient Cancer Therapy Response

I Characteristics

Status atlast follow-up

of the

Survival(days)

patients studied

Variation ofCause of death p53-Ab p53-Ab” Peptides

AYO 5CC lilA CT-RI CR Alive 499 Yes + + 1,2,3,5BAL LCC 1V5 CT NR Deceased 224 Encephalic metastasis Yes - 1, 2, 3, 5BEA SCLC, localized CT-RT CR Deceased 447 Hepatic metastasis Yes + + NoneBEL SCC lIlA CT-RI CR Alive 95 1 Yes + + 1,2,3,4,5

BEN SCLC, localized CT-RI CR Deceased 369 Encephalic metastasis Yes + + I, 2, 3, 4, 5BOU SCLC, localized CI CR Deceased 237 Local progression

(superior vena cavasyndrome)

Yes + 1

BRE Adeno IIIB CT-RI PR Deceased 405 Encephalic metastasis Yes + + 1CAG Adeno IIIB CT PR Deceased 440 Local progression

(pleural)Yes + + 1, 2, 3, 5

DEB SCLC, localized CT-RI PR Deceased 3 15 Cerebral stroke Yes + + I , 2, 3DEJ SCLC, localized Cl-RI CR Deceased 303 Encephalic metastasis Yes + + 1, 2, 3, 5FRO SCLC, extensive CT CR Deceased 600 Local progression +

adrenal metastasisYes + + I, 2, 3

GAl SCLC, localized CT PR Alive 7 1 5 Yes + + 1 , 2, 3, 5HUC SCLC, localized CT-RI PR Deceased 173 Local progression

(superior vena cava

syndrome)

Yes + + 1, 2, 3

MEU SCLC, localized CI PR Deceased 970 Encephalic metastasis Yes - 1, 2PEC Adeno IIIB Cl-RI CR Alive 672 Yes + + I , 2, 3, 5RAM Adeno lilA CT NR Deceased 191 Local progression

(pulmonary

carcinomatous

lymphangitis)

Yes - 1, 2, 3

AKA SCLC, localized Cl-RI CR Deceased 793 Hepatic metastasis NoARN SCLC, extensive CI PR Deceased 306 Local progression No

LEB SCLC. localized CT-RI PR Deceased 442 Hepatic & pancreatic

metastasis

No

BOD SCLC, extensive CI PR Deceased 510 Disease progression

(hemoptysis)

No

CAC SCLC, extensive CI PR Deceased 342 Encephalic metastasis No

LAN Adeno IV Cl NR Deceased 1262 Hepatic metastasis No

LOl LCC IV Cl NR Deceased 246 Local progression No

VAN 5CC IlIB Cl NR Deceased 473 Hepatic, bone, & localprogression(pulmonarycarcinomatous

lymphangitis)

No

ZAI SCLC, localized Cl-RI NR Deceased 198 Hepatic metastasis No

LAS SCLC, localized Cl-RI CR Alive 979 NoHEB SCC IIIB Cl-RI CR Alive 1604 No

LES SCLC, localized Cl-RI CR Deceased 796 Hepatic metastasis NoDEl SCLC, localized Cl-RI CR Alive I 162 No

EVA SCLC, localized Cl-RI CR Alive I 108 NoMER SCLC, localized Cl-RI CR Deceased 1 137 Encephalic and bone

metasasis

No

CHA Adeno IV CT PR Deceased 312 Pneumocystis carmniipneumonia

No

(1 Ihree levels of variation of p53-Abs are shown: -, no decrease; +, decrease of less than 50%; + +, decrease of more than 80% ordisappearance.

5 LCC, large cell carcinoma; RI, radiotherapy; CR, complete response; NR, no response; PR, partial response.

nus of p53. Peptide 6 was a human serum albumin peptide used

as an internal negative control in every experiment. This assay

was conducted as described previously with streptavidine-

coated plates, biotin-labeled peptides, and direct binding of

human antibodies diluted 1 : 100 for assay (18). Results were

expressed as a ratio between binding to p53 peptides and con-

trol.

HAV ELISA. The detection of total antibodies to HAV

was performed with a commercially available ELISA (HAV

Total, Sanofi Diagnostic Pasteur, Paris, France). The assay was

performed according to the manufacturer’s instructions and had

the following specifications: 1 :5 to 1 : 100 diluted patient serum

and peroxidase-conjugated anti-HAV antibodies were added for

4 h at 40#{176}Cto microplate wells coated with HAV antigen. If any

anti-HAV antibodies were present in the patient serum, this

resulted in competition with the conjugate. After washing, the

substrate 3,5,3,5-tetramethylbenzidine solution was added for

30 mm at room temperature. The enzymatic reaction was

stopped by adding I N sulfuric acid. The absorption was meas-

ured at 450 nm on a spectrophotometer (Dynatech MRS000). A




M 1 35 41 42 61 101 130190 199 230 259 DEJ (23). Pulmonary function was a contraindication to surgical

I � � - �-, � Hp53t� � � � .� --

upper right lobectomy. The patient was therefore treated with

monthly chemo-radiotherapy. He experienced a good partial

radiological response with normalization of endoscopic bron-

chial exploration and negativation of bronchial biopsies. He is

+ . 1 124 252 543 574 589 620 662 725 937 BEL

��ais .� Hp53

�

currently alive and well (December 1997), with no sign of

progression. It is clear that in this case the level of p53-Abs

increased dramatically during disease progression (Figs. I and

2). Only after CT were we able to observe a sharp decrease in

M - 1 49 72 90 RAM

I �__� .� Hp53, � � - -

p53-Ab. A similar follow-up was performed in patients who

were initially negative for p53-Abs. Some of these patients were

followed for more than 2 years. In none of these patients have

we been able to detect p53-Abs during the course of their

therapy.

Fig. 2 Monitoring of p53 antibodies using immunoprecipitation of in These observations indicate that the level of p53-Ab de-vitro translated p53. +. positive control (HR23I monoclonal antibody):- , negative control (sera from a blood donor): M, molecular weight

markers. The second band below p53 corresponds to a truncated p53

creases during the therapy but do not give any information.

concerning the specificity of this variation.

produced during in vitro translation. The Variation in p53 Antibodies Is Specific. Most of

these patients received CT as part of their treatment. It is widely

known that such treatment can eventually lead to partial immu-

sample was considered positive for anti-HAV antibodies if its

absorbance value was less than or equal to the cutoff value. The

cutoff value was calculated as the average of the negative and

positive control mean values. For the purpose of routine screen-

ing, each serum was diluted 1 : I 00. For the quantitative meas-

urement of anti-HAV antibodies in positive patients during their

follow-up, sera were diluted so that there was no saturation

during color development. For a given patient, the same dilution

was used for each sample, and all samples were processed in the

same experiment.

nosuppression or other immunological disorders that can induce

nonspecific disappearance of immunoglobulins. The level of

total serum immunoglobulins did not change significantly dur-

ing the course of the follow-up (data not shown). Nevertheless,

we cannot exclude the possibility that there was a general loss of

circulating antibodies. To control that only p53-Abs varied

during the course of therapy, we checked the humoral response

directed toward another antigen not related to neoplasia. We

chose to test HAV antibodies that are frequently present in the

sera of a normal population. In our series, all patients were

found to carry HAV antibodies at the time of diagnosis and were

Results

p53 Antibodies in Sera of Patients with Lung Cancer.

In 1993, we set up a prospective study in the respiratory depart-

ments of the Saint Louis, Saint-Antoine, and Tenon Hospitals.

p53 antibodies were evaluated in the sera of every patient with

diagnosed lung cancer. The results of that study will be pub-

lished elsewhere. In the present work, 32 patients were eligible

for detailed analysis. Sixteen patients had p53-Abs at the time of

diagnosis (7 NSCL and 9 SCLC), whereas 16 others were

negative (5 NSCLC and 1 1 SCLC). All these patients had a

follow-up of at least 6 months, with 4-10 sera taken during

therapy.

Monitoring of p53 Antibodies during Follow-up of

Treated Lung Cancer Patients. The level of p53-Abs was

thus suitable for this follow-up analysis. Fig. 3 clearly shows

that the level of HAV antibodies did not change during the

course of therapy, whatever the evolution of p53-Abs. For each

patient, the level of variation in HAV antibodies never attained

more than 15% (Fig. 3 and data not shown). Similar studies in

patients without any p53-Abs showed similar results (Fig. 3 and

data not shown).

Variation in p53 Antibodies Specific for Various

Epitopes. We previously showed that these antibodies recog-

nized specific immunodominant epitopes localized in the amino

and carboxyl termini of human p53 (20). Using the peptide

ELISA described in “Patients and Methods,” we performed a

detailed analysis of the epitopes recognized by the serum p53-

Abs and their variation during therapy. Five sera were specific

monitored during treatment of the various patients. The use of for the amino terminus, whereas seven sera also recognized the

an internal control in ELISA led to a quantitative assay as shown carboxyl terminus of p53. No serum was specific for the car-

in Fig. 1 and summarized in Table 1 . Of the 16 patients analyzed boxyl terminus of p53, as we had previously demonstrated.

here, 12 had a substantial decrease in p53-Abs (over 50% None of the sera showed any cross-reactions with the control

compared to their initial level), and 4 had no change in the level peptide. Analysis of the behavior of p53-Abs during therapy is

of p53-Abs. or else had a decrease of less than 50% (RAM, shown in Fig. I . For all sera, variations in the different sub-

MEU, BAL, and BOU). Patient BEL is a perfect example of the populations of immunoglobulins directed toward various

variation in p53-Abs. This patient (BEL, previously designated epitopes of the protein were strictly identical to those detected

PT37) has already been described (23). Initially referred to our using the entire p53 protein (Fig. 3 and data not shown). Fur-

department for a benign tracheal chondroma, he was found to be thermore, comparison of the variation in p53-Abs with the two

positive for p53-Abs. 15 months before he was diagnosed with ELISAs emphasized that true p53-Abs are detected by both

a squamous cell lung cancer of the upper right lobe of the lung ELISAs.




>4I

DaysDays _____________________________

�:�vAN �:: � RAM � HAV1.6� -e-- HAV 1.6 1.6 1,61.4i .-.-- p53 1.4 1.4 1.4

� 1.0�1.01.2 1.20.8 I �O.8

0.60.40.20.0

0.60.40.2

Fig. 3 Variations in anti-p53 and anti-HAVantibodies during follow-up of four patients

(FRO, BEN, VAN, and RAM). Ihe percent-

age of anti-p53 and anti-HAV antibodies isexpressed as a function of elapsing time (1).

For the sake of comparison. the level of anti-

p53 and anti-HAV antibodies at the time ofdiagnosis (��) was considered to be 100%.

0.60.4

0.2

0 20 40 60 80 100 o 100

Days Days

12 >.4

1.0 I0.8

0.60.40.2

200


0) R. Lubin and I. Soussi, unpublished results.

Detection of p53 Antibodies by Immunoprecipitation.

It has been suggested that ELISA can lead to false positive

results due to cross-reactions with other proteins that may be

present in the cell extract. Although the use of a control extract

should alleviate such false-positive results, we controlled the

presence of p53-Abs by immunoprecipitation of native p53

obtained by in vitro transcription and translation. In this assay,

p53 was the only labeled protein. All sera except one (BEA) that

were positive by ELISA could also immunoprecipitate p53 (Fig.

2 and data not shown). The remaining serum was repeatedly

found to be weakly positive for the ELISA procedure, showing

the higher sensitivity of ELISA compared to immunoprecipita-

tion. None of the sera that were negative by ELISA could bring

down p53 in this immunoprecipitation assay. Such an assay

could also be quantitative, as shown in Fig. 2. The variation in

immunoprecipitated p53 on the gel was similar to the signal

shown in Fig. 1 by ELISA.

All results described here indicate that the variation in

p53-Abs detected in sera of patients was indeed specific.

p53 Antibody Variations and Clinical Response to

Therapy. A decrease in p53-Ab was observed in 12 patients

during therapy. There was a trend toward an association be-

tween a decrease in p53-Ab titers and overall response to

treatment, i.e., partial or complete response (P = 0.0571 ; Fis-

cher’s exact test). When survival curves of patients with a 50%

decrease in their p53-Ab titers and of patients without a signif-

icant decrease in these titers were plotted, there was a trend

toward an association between better survival and decreased

p53-Ab titers of more than 50%. However, the difference of

survival between the two groups did not reach statistical signif-

icance (P = 0.163). presumably because of the small sample

size. All three patients still alive at the time of updating expe-

rienced a substantial decrease in their p53-Ab titers, with two of

them becoming p53 seronegative (BEL and PEC).

and its role in human cancers (5). Since 1991, a body of

evidence has demonstrated that these p53-Abs are linked to the

presence of p53 accumulation in human tumors (27). p53-Abs

are not present in the normal population, and the frequency of

cancer patients with such Abs is proportional to the frequency of

p53 alterations (17). Although most patients with p53-Abs har-

bor a p53 mutation, the opposite is not true, and recent multi-

factorial analysis indicates that only 30% of patients with p53

alterations developed a humoral response toward p53.

In the present study, we address the question of the fate of

these p53-Abs during therapy. Thus far, only a few works have

dealt with this question ( I 5, 24). In the present prospective

study, 32 lung cancer patients were chosen for a detailed anal-

ysis of the behavior of p53-Abs during therapy. Of the 16

patients without p53-Abs at the time of diagnosis, none under-

went a p53 humoral response during treatment, whatever the

fate of the disease. This observation has been extended to more

than 100 other patients with various types of cancer.6 These

results confirm the observation that each patient might have an

inherent capacity to develop such p53 humoral response. Such

behavior could be linked to the position of the mutation in the

p53 protein, as previously suggested (13), and/or to some spe-

cific genetic background. Indeed, immunodominant p53

epitopes might need specific histocompatibility complex class I

molecules to be properly presented to the immune system and

therefore lead to the development of p53 antibodies.

A decrease in p53-Abs was observed in 12 patients during

therapy. The finding that there is a true disappearance of p53-

Abs is emphasized by the observation that such a decrease can

be seen both on the whole native p53 by two immunological

techniques and with various peptides corresponding to different

parts of the p53 protein. Nevertheless, it was essential to show

that such a decrease of p53-Abs from sera is specific because the

overall serum antibodies level could be affected during therapy.

Discussion

The first description of p53-Abs in 1984 was not fully

understood due to the lack of knowledge on the p53 mutation




This was demonstrated by examining the level of another hu-

moral response. HAV was chosen because it is known that more

than 90% of the population over 60 years old has such antibod-

ies. There was no variation in these antibodies in any patients

whether they were positive or negative for p53-Abs. thus con-

firming the specificity of p53-Ab variation. Furthermore, such a

correlation with the p53-Ab titer and tumor growth is well

demonstrated in patient BEL. p53-Abs were detected in this

patient 2 years before the detection of the tumor. During those

2 years, this patient was carefully monitored, but no treatment

was administrated because there was no clue at that time that

p53-Ab could be an early indicator of cancer. During the 2-year

period, the level of p53-Abs increased concomitantly with the

development of the tumor that was clinically detected 2 years

after the initial detection of p53-Abs. The decrease in p53-Abs

was only seen after the beginning of treatment. There was no

variation in HAV antibodies during the entire follow-up of this

patient before or after the detection of the tumor (data not

shown). The behavior of the tumor in this patient indicates that

p53-Abs are linked to the presence of tumor growth. This

finding raises interesting questions concerning the immunoge-

nicity of the p53 protein. We previously demonstrated that the

p53 protein is highly immunogenic, with a specific humoral

response directed toward immunodominant epitopes localized in

the amino and carboxyl termini of the protein (12, 16, 2 1). The

present work shows that high levels of p53-Abs require the

presence of tumoral cells; otherwise, there is a very rapid

decrease in these antibodies during and after therapy. In some

patients, this decrease corresponds to the half-life of human

serum IgG, suggesting that continuous exposure to p53 is nec-

essary to maintain a steady-state level of p53-Abs.

Twelve of 16 patients with p53-Abs had a decrease in the

p53-Ab level of more than 50% compared to the initial titer.

Among these patients, eight underwent a complete response to

therapy, whereas four obtained only a partial response. Of the

five patients without any variation in their p53-Ab levels, two

showed a partial response, whereas no response was observed in

the three others. Although the number of patients was too small

for statistical analysis, no patient with a complete response had

a stable level of p53-Abs, whereas patients with no response

always maintained an unvarying level of p53.

One of the benefits of using p53-Abs for follow-up of

patients could be the detection of the reappearance of such

antibodies after therapy. In lung cancer, the survival of patients

is usually rather short and can preclude such studies. In this

study, three complete responder patients relapsed without any

rise in their p53 antibody titers (BEA, BEN, and DEJ). Two of

these patients had cerebral metastasis as the unique site of

relapse. The remaining patient had an initial weakly positive

ELISA test and died only 6 weeks after relapse was diagnosed,

a period presumably too brief to elicit a detectable p53 humoral

response. Brain was long considered as an immune “sanctuary.”

For instance, no p53 antibody was detected in a series of patients

with tumors of the central nervous system, whereas p53 muta-

tions were actually detected in tumoral tissue (28). An alterna-

tive explanation would be that relapse was due to a tumoral

clone retaining metastasizing or invasive potential but without

any p53 mutation. In a recent work, we showed that temporal

changes in the level of p53-Abs could be closely correlated with

disease progression or regression in colon cancer, where a

longer follow-up is possible (24). In breast cancer, we were able

to detect the reappearance of p53-Abs 2 years after the initial

therapy. This increase in p53-Ab was detected 3 months before

the detection of a relapse.7 Thus, in such tumor types, monitor-

ing p53-Ab positive patients revealed a potential for evaluating

response to CT/radiotherapy or detecting infraclinical relapse.

p53-Ab could be a useful tool for oncologists in their attempt to

analyze the patient response to therapy in addition with standard

clinical and radiological evaluation.

Acknowledgments

We are grateful to J. Bram for reading the manuscript.

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1998;4:1359-1366. Clin Cancer Res G Zalcman, B Schlichtholz, J Trédaniel, et al. relationship to response to treatment.Monitoring of p53 autoantibodies in lung cancer during therapy:

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