Alpha-lactalbumin and GCDFP-15 as breast tumor markers

oB83-2897/87$3.00+0.00

Nucl. Med. Biol. V01.14, No.4, pp.385-395,1987

PergamonJournalsLtd

Int. J. Radiat. Appl. Instrum.Part B Printed in Great Britain

ALPHA-LACTALBUMIN AND GCOFP-15 AS BREAST TUMOR MARKERS (An opportunity to study breast tumors functionally) * Julien COLLETTEl, Jean-Remy VAN CAIJWENBERGE', Viviane LE 00USSAL3,Michel SERILAS2, Jean HUSTINl, Agnes CARLISII, Laurent OEJAROINl, and Paul FRANCHIMONTI 2 1 Gynecology Department Laboratory of Radioimmunoassay, and Liege, C.H.U., B. 23, B-4000 Liege 3Rene Huguenin

, University of

, Belgium

Cancer Research Centre, 92215 Saint-Cloud

, France

INTRODUCTION

The normal function of mammary gland is expressed by synthesis of proteins, fats, and

carbohydrates that

appear in the milk during lactation.

Secretory products of a differentiated glandular tissue, the milk proteins represent molecular

entities that

are a normal property of a cell from which a tumor can derive.

There are not "tumor specific markers" but they can be of value as parameters to identify mammary tumors functionally,

to assess their hormone dependency, and potential degree of

differentiation. Moreover, if they are released at significant levels into the body fluids, in contrast to the normal tissue, they can have a potential utility if not in the diagnostic at least in the monitoring of a neoplastic process. It is within this conceptual approach that our attention has been addressed of the use of two milk proteins (a-lactalbumin and GCOFP-15) as markers in breast cancer. Alpha-lactalbumin functions

is a major

protein of human milk (? 3.5 mg/ml) in which it

as lactose synthetase "specifier protein" (Brodbeck and Ebner, 1966). This

protein is essentially produced by the mammary gland alveolar epithelium under hormonal induction during pregnancy and lactation (Martin et al., 1980). GCOFP-15 is one 15,000 daltons monomer of "Gross Cystic Disease Fluid Proteins" which was identified

and characterized by Haagensen et al., in 1979, as a major component

of breast cyst fluid. This glycoprotein seems

have a predominantly apocrine epithelial

cell distribution in the body (Mazoujian et al., 1983), and is a minor constituent of human milk (+ 16 Pg/ml). We have previously studdied a-lactalbumin and GCOFP-15 by immunohistochemistry in breast lesions and in breast cyst aspirates (Le Ooussal et al., 1984, 1985

; Hustin et

al., 1986). More recently these two Proteins as also EGF (Epidermal Growth factor) were analyzed by radioimnunoassay

(RIA) in breast cyst fluids (Collette et al., 1986).

In the present paper we will describe essentially the results obtained with RIA for GCOFP-15 and a-lactalbumin in breast carcinoma cytosols , and serum of breast cancer patients. _______~____________~~~~~~~~~~~~~_~~~~_~~~___~~~~~~~~~~~~~~~~~~~~~_~~~_~_~~___~____________ $:

Supported by Grant 3.4526.86 from FRSM and by Cancer Foundation of CGER - Belgium

385

386

J. Colletteet al.

MATERIALS AND METHODS

Alpha-lactalbumin was separated from human whey milk using the method of Schultz and Ebner (1977), and the purity of the final product was assessed by disc electrophoresis in 1 % SDS polyacrylamide gel as described by Fairbanks et al., in 1970. GCDFP-15 was isolated

and purified from human breast cyst fluid and generously

provided by Dr D.E. Haagensen, Jr., New England Deaconess Hospital, Boston, M.A., USA. Antisera against these two proteins were raised in rabbits following the immunisation schedule of Vaitukaitis et a1.(1971), and tracers were obtained by iodination of antigens TM with free 125 I according to the medthod of Salacinski et a1.(1979) using the Iodo-Gen reagent (Pierce-Eurochemie, Rotterdam, Holland). Radioimmunoassays

(RIA) for human a-lactalbumin, and GCDFP-15 were developed and

characterized (Collette et al., 1986). However, to be

performed in human serum, the

previously described a-lactalbumin RIA must be modified to avoid the interference of endogenous antibodies to bovine a-lactalbumin (Woods and Heath, 1978). To circumvent this problem

, bovine a-lactalbumin (Sigma L-5385) was added in each assay and the rabbit anti-

human a-lactalbumin antiserum was preliminarily treated with a bovine a-lactalbumin-CNBractivated Sepharose conjugate to eliminate the antibodies binding the bovine protein. So, each 400 ~1 incubation volume contained

: 150 ~1 of incubation buffer (phosphate

buffer 0.05 M, pH 7.5, 0.5 "6 BSA, 0.05 % NaN3) containing 1 Pg of bovine a-lactalbumin; 50 Pl of incubation buffer containing varying quantities of unlabeled human a-lactalbumin (0 to 50 ng) or 50 Pl of human serum or 50 ~1 of tissue cytosol

; 100 ~1 of tracer ; and

100 ~1 of treated rabbit antiserum diluted to 1:20,000 in the incubation buffer. After 48 h. incubation at 2O"C, the separation of bound from free human a-lactalbumin was accomplished as previously reported (Collette et al., 1986). The sensitivity of this modified human a-lactalbumin RIA was 30 pg/tube; no cross-reaction was observed with bovine a-lactalbumin while the other assay characteristics were unchanged. Human EGF isolated from urine (Gregory and Willshire EGF were generously provided by Dr H. Gregory

, 1975) and rabbit anti-human

, ICI Ltd., Macclesfield, U.K. A sensitive

radioinznunoassay was developed by Jaspar and Franchimont (1985). Prolactin RIA used in our laboratory was previously described by Reuter et a1.(1976). Progesterone and 17 B- estradiol radioimmunoassay kits were purchased from Sorin Biomedica

, Salluggia , Italy.

CEA determinations were performed with the Abbott CEA-RIA Monoclonal kit (Abbott Laboratories

, North Chicago, IL , USA).

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387

Breast carcinoma cytosol preparations and the assays for estrogen (ER) and progesterone (PR) receptors were carried out according to the method described by EORTC Breast Cooperative Group, in 1980. The limit of significant detection was 10 fmol/mg of cytosol protein (C.P.), as measured by the method of Bradford (1976). Presence of serum albumin in breast tumor cytosols was determined by imnunodiffusion method using "LC Partigen" plates (Behringwerke AG, Marburg, West Germany). The concentration of proteins originating from breast in a cytosol was estimated by deducting its serum albumin content multiplied by loo/60 from its total protein content. The GCDFP-15 and a-lactalbumin cytosol levels wereexpressed in terms of this estimated cytosol protein concentration. The histopathological grade of breast tumors was established according to the method of Bloom and Richardson (1957).

RESULTS

1. Alpha-lactalbumin and GCOFP-15 in breast carcinoma cytosols We have performed breast carcinoma cytosol analysis for a-lactalbumin and GCDFP-15 contents on 158 biopsy specimens which

were obtained from 52 premenopausal

, and 106 post-

menopausal women. These specimens included different breast carcinoma pathological sub-types

:

122 ductal, 13 lobular, 8 medullary, 8 mutinous, 3 papillary and 4 comedo- carcinomas. They were stratified for estrogen and progesterone receptors content and for histopathological grade (I

: well, II : moderately, III

( < 10 and > 10 fmol/mg C.P.), : poorly differentiated).

All breast carcinoma specimens contained GCDFP-15 with a large range in cytosol content (6 to 8, 140 ng/mg

C.P.). When the 158 breast carcinoma samples were separated relative to

histopathological .grade, the GCOFP-15 cytosol levels were significantly higher in grade I than in grade III

tumors (p < 0.02).

Alpha-lactalbumin was only detected in 70 breast carcinoma cytosols

: 58 ductal,

7 lobular and 5 polymorphic (ductal and lobular) carcinomas originating from 25 premenopausal (age

: 43 + 6 years) and 45 post-menopausal (age : 67 + 10 years) women. The a-lactalbumin

levels were ranged between 0.4 and 24 ng/mg C.P. In these 70 breast carcinoma cytosols, there was no correlation between the a-lactalbumin and GCDFP-15 levels. If the levels of the two proteins were higher in post-menopausal women samples, they were not significantly influenced by the patient menopausal status. When all 70 breast carcinoma cytosols wereseparatedrelative

to histopathological grade,

the two proteins cytosols levels were significantly higher in the more differentiated (grade I) than in the poorly differentiated (grade III)

tumors (p < 0.03). However, with regard to

menopausal status, this finding was only confirmed in the post-menopausal women samples (Figure 1).

J. Colletteet al.

PREMENOPAUSAL M~E.S.M.

WOMEN IN = 25) 500t M2E.S.M.

Figure 1

: a-lactalbumin and GCOFP-

15 levels (M + ESM) in 70 primary breast carcinoma cytosols which have been stratified for histopathological grade and for the menopausal status of patients. The values were not normally distributed and were conseqently analyzed by the MannWhitney nonparametric rank test. ______ POST-MENOPAUSAL WOMEN (n&51

When the 70 breast carcinoma cytosols were stratified relative to the presence (> 10 fmol/mg C.P. or the absence

(< 10 fmol/mg C.P.

of ER and/or PR, we observed that the a-lactalbumin and GCOFP-15 levels were significantly higher when ER and PR were present together (p < 0.03 and p < 0.004 respectively) Moreover the GCOFP-15 cytosol contents were significantly higher P: MANN-WHITNEY

TEST

when ER were present (p = 0.006) or when PR were present (p = 0.01). However these findings were not observed in the 25 premenopausal women samples and were only confirmed in the 45 post-menopausal women breast cytosols. 2. GCOFP-15 in human breast cancer cells (MCF-7) culture Recently we have begun to investigate the hormonal modulation of GCOFP-15 secretion by breast cancer cells "in vitro". Our preliminary results established that androgens induced rapidly a production of GCOFP-15 by MCF-7 cells. These

human breast cancer cells were

cultured in DMEM culture medium complemented with 10 % FCS (Fetal Calf Serum) in the presence of 0.5 PM of androstenedione (ASD), or 0.5 FM dihydrotestosterone (OHT), or 0.5 CM testosterone (T), or no added hormones during 6 hours. In the presence of no added hormones there was no production of GCDFP-15 in the culture medium, while the androgens induced a GCOFP-15 pnoduction by MCF-7 cells (0.7 to 1.3 ng of GCOFP-15 per pg DNA after 6 hours). OHT and T were found to have a same degree of stimulation capacity on GCOFP-15 production while the action of AS0 was significantly lower (p < 0.005). 3. Alpha-lactalbumin and GCOFP-15 in breast cyst fluids The results were previously described and analyzed (Collette et al., 1986) but are interesting to recall.

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Breast cyst fluids samples were centrifuged to remove gross particulate matter and the assayswere performed in the supernatants by RIA. Alpha-lactalbumin was detected in 14 % of 253 breast cyst fluids with a concentration range of 0.4 to 956 ng/ml. GCDFP-15 was detected in all breast cyst fluids analyzed (n = 266) with an average

level of 11,l c 10,2 mg/ml

( M ? SD).

We have found EGF in all breast cyst fluids with levels ranged between 5 ng/ml and 1.95 pg/ml. It was interesting to note that EGF and GCDFP-15 levels were respectively and significantly lower when a-lactalbumin was detected (p < 0.001 for EGF and p < 0.03 for GCDFP-15). Moreover a strong, significant relationship between GCDFP-15 and EGF levels was observed in this medium (p < 0.0005). 4. Alpha-lactalbumin

in serum

Serum from 158 (non pregnant and non lactating) healthly women (age range

: 18 to 65

years) were analyzed by a-lactalbumin RIA. As described by table I

, a-lactalbumin was significantly detected (serum level > 0.6

ng/ml) in 43 % of samples with levels ranged between 0.7 and 160 ng/ml.

N

CLINICAL STATUS

ALPHA-LACTALBUMIN

( ng/ml ) SO.6

SO.6 158

90

( 57 % )

68

( 43 % )

- Benign

93

68

( 27 % )

31

27

( 73 % ) ( 87 % )

25

- Malignant

HEALTHY WOMEN (NP and NG) NON BREAST DISEASES

4(13%)

BENIGN BREAST DISEASES - Fibroadenoma

10

- Fibrocystic disease

32

14

2(20%)

38

23

( 44 % )

8(80%) 18

( 56 % )

BREAST CANCERS - Before mastectomy - After

$:

mastectomy

* *

59

( 61 % ) 40 ( 68 % )

15 ( 39 % ) 19 ( 32 %

)

Before chemotherapy TABLE I

: DISTRIBUTION OF ALPHA-LACTALBUMIN SERUM LEVELS IN WOMEN

The incidence of a-lactalbumin serum levels higher than 0.6 ng/ml seen in different age groups did

not vary significantly. However the proportion of women with detectable serum

a-lactalbumin decreased above the age of 40 to reach 13 % in those aged 55 or over.

In women with fibroadenoma and fibrocystic disease of the breast, circulating a-lactalbumin was detected in 80 % and 56%of significant

cases respectively. However, there was no

difference between these two groups and the healthy women group according to the

distribution of a-lactalbumin serum levels. Moreover it was impossible to discriminate between

J. Collette det al

390

breast cancer patients before mastectomy and those after mastectomy with an a-lactalbumin serum assay. It was interesting to note that when circulating a-lactalbumin was detected in the healthy women, the concomitant prolactin serum levels were significantly higher (p = 0.0017). This finding was also observed in women with benign non breast diseases (p = 0.008) and in breast cancer patients after mastectomy (p = 0.017), while it did not find in women with benign breast diseases, and breast cancer patients before mastectomy. We did not observe a similar relationship between

circulating a-lactalbumin and

concomitant progesterone or 17 s-estradiol srerum levels. 5. GCDFP-15 in serum Serum samples of above-mentioned 158 healthy women were also analyzed to define a normal serum range of GCDFP-15. The mean serum level of GCDFP-15 in these women was 3.6 ? 1.4 ng/ml (M f SD)

, with 98 % having levels below 10 ng/ml as described by Table II.

The

GCDFP-15 serum levels seen in different age groups did not vary significantly.

CLINICAL STATUS

GCDFP-15

N

HEALTHY WOMEN (NP and NL)

158

( ng/ml )

4 10

>I0

155 ( 98 % )

3('2%)

?

NON BREAST DISEASES - BENIGN Kidney (Before hemodialysis)

20

Others

91

76

( 84 % )

- MALIGNANT

30

25

( 83 % )

7(35X)

13

( 65 % )

15

( 16 % )

5(17%)

BENIGN BREAST DISEASES - Fibroadenoma

10

- Fibrocystic disease

49

9(90%)

l(lO%)

29 ( 59 % )

20 ( 41 % )

38

20

18

60

41

BREAST CANCERS - Before mastectomy

*

- After mastectomy* I:

( 53 % ) ( 68 % )

( 47 % ) )

19 ( 32 %

Before chemotherapy TABLE II

: DISTRIBUTION OF GCDFP-15 SERUM LEVELS IN WOMEN

Different groups of women with diseases other than breast have been studied for GCDFP-15 serum levels. It is interesting to note that of 20 patients with kidney failure 65 % had GCDFP-15 serum levels higher than 10 ng/ml, while in samples from 91 patients with a variety of other benign diseases, 16 % only had GCDFP-15 levels above 10 ng/ml. This incidence was the same (17 %) in a group of 30 patients with malignant diseases other than breast.

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Two groups of women with benign breast diseases have been analyzed group consisted of 10 women with fibroadenoma, 10 % had serum levels

. In the first

above 10 ng/ml and in

the second group consisted of 49 patients with fibrocystic disease, 41 4:had GCDFP-15 values above 10 ng/ml. Two groups of breast cancer patients have been analyzed for GCDFP-15 serum levels and concomitant measurements of CEA have been made. In 38 patients with operable breast carcinoma 47 % had GCDFP-15 levels above 10 ng/ml and 16 % had CEA levels above 5 ng/ml before the initiation of a treatment. Elevated

serum

levels of CEA and GCDFP-15 occured concomitantly in 13 % of patients. In 60 patients who had undergone an operation for breast carcinoma and before the initiation of a postmastectomy treatment, 32 % had GCDFP-15 values above 10 ng/ml and 3 % had CEA levels higher than 5 ng/ml. Both markers were elevated in 2 % of cases. The breast cancer patients were treated for their disease with chemical therapy before and after mastectomy. Of 18 patients with operable breast carcinoma and GCDFP-15 serum levels above 10 ng/ml, 9 (50 %) had a decrease in GCDFP-15 levels greater than 50 % and 9 had decreases less than 50 % during

the 2 months of pre-mastectomy therapy. Three patients had decreases of greater

than 80 4:and a significant reduction of tumor mass (at least 50 % of the two diameters of the tumor) was observed in these 3 cases while no correlation could be established between the tumoral opacity regression and CEA serum levels. Of 20 patients with operable breast carcinoma and GCDFP-15 serum levels below 10 ng/ml, 2 had a decrease in GCDFP-15 levels greater than 50 % and 18 had stable levels (in the normal range) during pre-mastectomy therapy. We have followed the group of 60 postmastectomy patients by serial measurements of GCDFP-15. Table III

describe the serum GCDFP-15 profile alterations that occurred in these

patients during treatment versus their clinical status. The serum GCDFP-IS level changes were evaluated with the following criteria treatment)

: (1) initial serum CGDFP-15 level (before

; (2) serial trend with an increase in level greater than 50 % for progression (P) ;

(3) serial trend with a decrease in level greater than 50 % for regression (R)

; and (4) serial

trend with GCDFP-15 level changes less than 50 % considered as stable (S). We observed that of 19 patients with initial GCDFP-15 serum level above 10 ng/ml, 10 (53 %) have developed a metastatic process or a breast carcinoma recurrence. Of 41 patients with initial GCDFP-15 serum level below 10 ng/ml

, 10 (24 %) have developed a metastatic

process or a breast carcinoma recurrence. There was a good correlation between the direction of GCDFP-15 level changes and the clinical evaluation of the disease, as described by Table III.

J. Callette et al.

392

INITIAL GCDFP-15 SERUM LEVEL

$10

DIRECTION OF MARKER CHANGE

41

ng/ml

19

>lO ng/ml TABLE III

Nt

DISEASE STATUS N

GOOD EVOLUTION

Mt or RECURRENCE

R

2

2 (100 %)

1 P

4

1

( 25 %)

3

( 75 %)

S

35

28

( 80 %)

7

( 20 %)

R

10

7

( 70 %)

3

( 30 X)

( 33 %)

(100 %) 78 % 4 ( 67 I) I 3

i

S P

6 3

2

: SERUM GCDFP-35 PROFILE DURING CHEMOTHERAPY VERSUS CLINICAL EVALUATION OF DISEASE STATUS FOR 60 BREAST CANCER PATIENTS AFTER MASTECTOMY.

( R : Regression, P : Progession, S : Stable )

CONCLUSIONS

The results observed with RIA for a-lactalbumin and GCDFP-15 in breast carcinoma cytosols are agreed with our previous results obtained by immunohistochemistry (Le Doussal et al., 1984, 1985). All analyzed breast carcinoma specimens contained GCDFP-15 with a large range of concentrations (6 to 8,140 ng/mg cytosol protein). Some ductal (47 %) and lobular (54 %) breast carcinomas would contain a-lactalbumin in low concentration (< 10 ng/mg cytosol protein). We could not establish if it was native a-lactalbumin or a related peptide (a-lactalbumin-like substance or pre-a-lactalbumin) as suggested by Hall et al., in 1981. Unfortunately the cytosol concentrations

of a-lactalbumin weretooweak

to emphasize its presence using an enzymatic

method. Alpha-lactalbumin and GCDFP-15 breast cytosol levels varied according to the histopathological grading of tumors and were higher in the well-differentiated tumors in postmenopausal women samples. In these patients, the cytosol levels of the two proteins were higher when the ER and PR were present together. So, a-lactalbumin and GCDFP-15 can be considered as "differentiation and hormone-dependency markers" emphasizing the integrity of the hormonereceptor-protein synthesis sequence of which the cancer cell is the seat. As reported by Dilley et a1.(1983) "in vivo" , we observed an induction of GCDFP-15 production by androgens in human breast cancer cells (MCF-7) culture. So, GCDFP-15 can give an opportunity to study breast tumors functionally and to identify breast carcinomas having functional androgen receptors. The presence of a-lactalbumin in breast cyst fluid (14 %) would reflect the persistence of differentiated cells in the cyst epithelium. This presence could be modulated by EGF. In the breast cyst fluid GCDFP-15 would be an "apocrine metaplasia" index.The strong relationship between EGF and GCDFP-15 levels observed in this medium suggests that androgens and EGF could

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play a role in the genesis of cystic disease and breast cancer, a role in which GCDFP-15 would be a biochemical reflection. In serum an a-lactalbumin assay does not allow to discriminate between the presence or absence of a benign or malignant breast disease. In this

medium a-iactalbumin appears

as a "functional marker" of the mammary gland and could be an index of the action that prolactin exert on it. From the evaluation of serum samples from patients with non-breast benign diseases (except kidney diseases in which GCDFP-15 excretion in urine could be decreased) and malignant diseases, it is apparent that GCDFP-15 serum level elevations are more specific for breastrelated pathology, especially breast cystic disease and breast carcinoma. As opposed to a-lactalbumin

, GCDFP-15 serum determinations could be very useful in

the monitoring of breast cancer patients as index of disease regression or progression and to judge the effectiveness of the therapy.

394

J. Collette -. et al

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