Breast Cancer Research and Treatment 17: 15-21, 1990. © 1990 Kluwer Academic Publishers. Printed in the Netherlands.

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Tissue polypeptide antigen in tumor cytosol: a new prognostic indicator in primary breast cancer

Massimo Gion, Riccardo Mione, Carlo Gatti, Ruggero Dittadi, Antonette Leon, Cristina Castiglioni a, Ottorino Nascimben2 and Giuliano Bruscagnin

Division of Radiotherapy and Nuclear Medicine, Oncology Center, Regional General Hospital, 30122 Venice, Italy; 20ncology Center and 12nd Division of Surgery, Regional General Hospital, 30172 Mestre (Ve), Italy

Key words: breast cancer, cytosol, prognosis, tissue polypeptide antigen, tumor markers Summary The assessment of the risk of relapse is a critical need in the management strategy of breast cancer patients. To date, the most reliable prognostic factor is axillary nodal status. Several other pathological and biological parameters are currently under evaluation. Since 1982 we have been studying the prognostic role of several tumor markers in breast cancer cytosol. Elevated cytosol concentrations of tissue polypeptide antigen (TPA) have been found to have a highly significant direct correlation with both prolonged relapse-free interval (RFI) and higher survival rate. The information provided by cytosol TPA was independent of both axillary nodal status and steroid receptor content. In patients with a low risk of relapse (no axillary metastases, estrogen and progesterone receptor positive), cytosol TPA was still a significant prognostic indicator.

Introduction The availability of effective adjuvant therapies for primary breast cancer [1, 2] stresses the need for the selection of patients with a higher risk of relapse. The prognostic value of several clinical, pathological and biological parameters has been evaluated. The axillary nodal status is considered the most effective prognostic indicator [3]. Several other prognostic factors have been proposed: histologic grade [4], estrogen (ER) and progesterone receptors (PgR) [5, 6], the labelling index (LI) [7], DNA ploidy [8], the presence of EGF receptor (EGFr) [9], and the expression of the HER2/neu oncogene [10] are currently under evaluation. Serum levels of tumor markers are useful prognostic

indicators in advanced disease [11]; however, in primary breast cancer they are related to tumor mass, so that their prognostic value is dependent upon tumor stage [11]. The determination of tumor markers in the tissue provides information which should be independent of the tumor bulk, since (1) their concentration is not affected by dilution in body fluids nor by metabolism and secretion rates, and (2) the bias due to cross reactant molecules produced by different organs is probably avoided. Since 1982 we have been measuring several tumor markers in breast cancer tissue with the goal of better characterizing the phenotypic pattern of the tumor [12-14]. Among the tumor markers evaluated, tissue polypeptide antigen (TPA) [15] showed higher cytosol levels in E R + P g R + than in E R P g R - tumors [12], being a possible marker of

Address for offprints: M. Gion, Division of Radiotherapy, Regional General Hospital, 30122 Venice, Italy

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M Gion et al.

more differentiated and probably less aggressive tumors [16, 17]. The information provided by cytosol TPA determination should be independent of receptor status, since TPA, which is a member of the cytokeratin family, is not an estrogen-induced protein [15, 18]. On the basis of these findings we were prompted to evaluate the prognostic value of cytosol TPA determination in breast cancer. The present paper reports preliminary data on the ongoing study.

Materials and methods

From 1982 to 1985, 205 patients with primary breast cancer were evaluated. Patients were staged using UICC criteria [19]. Inclusion criteria were as follows: (1) infiltrating ductal type tumors, (2) no irradiation or chemotherapy before mastectomy, (3) no previous or concomitant malignancies of different organs, (4) availability of complete information about menopausal status, clinical stage, tumor size, and number of positive lymph nodes, and (5) minimum follow-up time of 24 months. To date, 104 patients (32 premenopausal, 72 postmenopausal), fulfilled the above criteria and entered the study; 26 were in Stage I, 58 in Stage II, and 20 in Stage III. Stage I patients had no further therapy after surgery. Stage II and III patients had radiotherapy on the axilla as well as adjuvant systemic chemotherapy (CHT) (CMF, 6 cycles) in premenopausal and tamoxifen (Tam) (30 mg daily for one year) in postmenopausal. Tissue collection and storage, as well as high

speed cytosol preparation, were carried out as previously described [12]. ER and PgR were measured with a standard saturation analysis [20]. Cytosol protein concentration was assayed with the Bradford colorimetric assay [21]. TPA was measured in the cytosol with a commercially available immunoradiometric assay (IRMA) (Sangtec Medical, Bromma, Sweden). Cytosol samples were diluted using phosphate buffer containing bovine serum albumin 3 mg/ml, which was demonstrated to improve the performance characteristics of the method in a low protein milieu, such as cytosol (data not shown). The precision of the method, expressed as coefficient of variation between 15 replicates, was below 7.5% within assay and below 8.5% between assay. Accuracy was evaluated using the dilution test [22], which showed a high correlation between the calculated (x) and the measured (y) concentrations of the antigen (n = 6, y = 40 + 1.0x, coefficient of correlation 0.999, p < 0.0001). TPA concentration was expressed as units per mg of cytosol protein (U/mg c.p.).

Results

The follow-up time ranged between 24 and 72 months (median 41 months). At the last clinical examination, 78 patients were relapse-free and 26 had recurrence; 22 patients died from the disease. The prognostic value of all the available variables was evaluated using the Cox multivariate analysis (Table 1) [23]. The most reliable prognostic indicator for both survival and RFI was the number

Table 1. Prognostic indicators in primary breast cancer (Cox multivariate analysis; 104 evaluable cases)

Variable

Number positive lymph nodes Stage Tumor size ER PgR Cytosol TPA

Relapse-free interval

Survival

chiz

P

chiz

P

17.8 6.2 0.2 2.9 4.3 13.1

0.0005 0.013 0.690 0.090 0.040 0.0005

17,4 4,1 0.2 4,5 4.4 14.3

0.0005 0.042 0.680 0.034 0.036 0.0005

Cytosol TPA and breast cancer prognosis

I00

17

---t

~L~

. . . . . . ~. . . . . . . . . . . . . . .

', ,,

BO,

I

.......

(n=431

PERCENTAGEOF 60PATIENTS FREE OF DISEASE

I

TPA +

p = 0.022 TPA -

40-

(n=61)

20-

0 0

I 10

I 20

I 30

I 40

I 50

I 60

I 70

I 80

MONTHS Fig. 1. Relapse-free survival curves stratified by cytosol T P A .

of positive lymph nodes; the relation between prognosis and clinical stage, ER, and PgR was weak. TPA cytosol levels showed a highly significant, direct correlation with both RFI and survival. The prognostic information of TPA was independent of clinical stage, tumor size, axillary metastases, and receptor status. TPA was also measured in the cytosol of 62 histologically proven normal breast tissue samples selected among 96 samples in which glandular components were well represented. TPA was higher in cytosol of carcinoma (median 321 U/mg c.p., interquartile range 185-594) than in normal tissue (median 58 U/mg c.p., interquartile range 22-122, p < 0.0001). TPA levels were higher in carcinoma than in normal tissue collected from the same paTable 2. T P A levels in cytosol samples of T P A + and T P A t u m o r samples

median interquartile range 10-90th percentile range

TPA +

TPA -

646 558-1086 497-1483

219 73-286 30-361

tient in 88% of cases. The distribution of TPA concentration in normal breast samples was not gaussian (Kolmogorov-Smirnov test, p = 0.003). Therefore, normal samples were evaluated using non parametric statistics, and the 95th percentile value of the concentrations found in normal breast tissue cytosol (443 U/mg c.p.) was used as a cutoff level to categorize tumor samples as T P A + or T P A - . TPA concentrations in T P A + and T P A tumor samples are summarized in Table 2. RFI (Fig. 1) as well as survival (Fig. 2) was significantly longer in T P A + than in T P A - cases (using the product limit method) [24]. The prognostic value of cytosol TPA was conserved when patients with and without axillary metastases were examined separately (Fig. 3) as well as in E R + PgR+ cases (p = 0.005). In E R - P g R - cases the evaluation could not be carried out due to the low number of T P A + cases in the E R - P g R - group. In patients having two favorable prognostic indicators (no axillary metastases, E R + P g R + ) , cytosol TPA was still an effective additional prognostic indicator, with RFI and survival being respectively 67% and 70% in T P A - , and both 100% in T P A + cases (p = 0.022 for RFI, p = 0.037 for survival).

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M Gion et al.

I00

~---'

L_~

.....................

(n=4S)

TPA

+

'I

80.

p =

I

60-

CUMULATIVE SURVIVAL (Z)

0.015

TPA (n=69)

4O.

20.

0

I

10

I 20

I 30

I

40

I 50

I 60

I

I

70

80

MONTHS

Fig. 2. Overall survival curves stratified by cytosolTPA. Discussion

Adjuvant therapies in primary breast cancer provide almost certainly a prolonged RFI and probably a longer survival [1, 2]. However, several problems are connected with adjuvant therapies. Both early and late toxicity result from CHT [25, 26]; the antiestrogen Tam is well tolerated and has a lower toxicity, but its adjuvant role is not yet established in premenopausal patients, in which CHT is probably more effective [1, 2]. An accurate evaluation of the risk of relapse should therefore be of great value in the choice of patients to whom adjuvant therapies will be administered. The most reliable prognostic factor is the axillary lymph node status [3]. Histologic grading has recently been confirmed to be a very effective prognostic indicator [4]. However, the assessment of histologic grade is subjective and difficult to standardize for routine use [27]. The prognostic usefulness of steroid receptors is currently under debate; the prolonged survival time in receptor-positive cases is at least in part related to the higher response rate to endocrine therapy of patients with advanced receptor-positive breast cancer [28];

moreover, the prognostic advantage of receptorpositive cases was not confirmed after an adequately long [29] follow-up time [30]. LI is a prognostic parameter independent of both steroid receptors and axillary status, but the evaluation of results is subjective and time consuming [7]. The D N A distribution pattern of tumor cells has been related to prognosis of breast cancer, patients with aneuploid tumors having a poorer prognosis than those with euploid ones [8]. The methods for analysis of DNA, as well as the criteria used to define ploidy pattern, have not yet been standardized [8]. The evaluation of the prognostic role of both EGFr and HER2/neu oncogene expression is so far restricted to a limited number of centers. Tumor marker determination is simple and reproducible for routine use; however, the prognostic role of serum tumor markers is limited, their blood level being directly related to tumor burden [11]. The fact that tumor marker concentration in breast cancer cytosol is independent of tumor bulk [12-14] has provided the stimulus for evaluating its prognostic role. Moreover, cytosol is prepared using a standardized, well reproducible procedure

Cytosol TPA and breast cancer prognosis

100

.............................. .......

19

N-TPA+ ,

,

cn.25

80(,.,-zo)

E1 CUMULATIVE

BO-

" .......

(n"33)

N-TPA-

p ,, o. 046

I N+TPA-

SURVIVAL (;I;)

(n=26)

40-

20-

0 0

I 10

I 20

I 30

I 40

I 50

I BO

I 70

I 80

MONTHS Fig. 3. Overall survival curves stratified by axillary nodal status and cytosol TPA. N: axillary nodal status.

[20], currently used for the determination of steroid receptors. The concentration of TPA in tumor cytosol has been shown to be a good prognostic indicator in the present patient series. For the Kaplan and Meier statistic a negative/positive cut off point was preliminarily calculated on the basis of the TPA levels found in a control group (histologically proven normal breast samples), as is currently done when categorizing a biological parameter. However, the epithelial components of normal breast tissue are less represented than in breast cancer. This aspect could be critical when considering TPA, which is a member of the cytokeratin family. Therefore, the cut off point with the best prognostic discriminating value will be recalculated on the basis of the cytosol TPA values of the wider patient series presently under evaluation. The prognostic value of cytosol TPA was independent of axillary status as well as of both E R and PgR. The relationship between cytosol TPA and prognosis is opposite to that previously reported for serum [31], a poorer prognosis being related to higher serum but lower cytosol TPA concentrations. Cytosol TPA, which is not correlated with

serum TPA level [12, 13], was previously demonstrated to have a relationship with receptor status which is also opposite to that found in serum; both higher cytosol and lower serum TPA levels were shown in E R + P g R + than in E R - P g R - cases [12]. This behavior could be due to a lower secretion rate of TPA by the less aggressive tumors, considering that, among the tumor markers evaluated in breast cancer, only TPA could be at least in part related to the proliferative activity rather than to the mass of the malignancy [15, 18]. Apart from any possible interpretation, the determination of TPA in cytosol and in serum provides different information. Moreover, it should be emphasized that the determination of cytosol TPA by IRMA is simple, accurate, and precise. Therefore, from the present findings obtained in a selected group of patients, it seems worthwhile to further investigate the role of cytosol TPA as a new prognostic factor in primary breast cancer.

Acknowledgements The present investigation was financially support-

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M Gion et al.

ed in part by the Regione Veneto, Italy, and by the Italian Association for Cancer Research (A.I.R.C.), Milan, Italy.

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