[CANCER RESEARCH 39, 2057-2061 , June 1979]0008-5472/79/0039-0000$02.0O

Frequency of Association of Mammary Tumor Glycoprotein Antigen andOther Markers with Human Breast Tumor&

Julia P. Leung,2 Gerald M. Bordin, Robert M. Nakamura, David H. DeHeer, and Thomas S. Edgington

Department of Molecular Immunology, The Research Institute of Scripps Clinic, and Department of Pathology, The Hospital of Scripps Clinic, La Jolla, California92037

ABSTRACT

Human mammary tumor glycoprotein (MTGP) antigen hasrecently been defined and purified from human breast tumors.In the present study, the incidence and concentrations ofMTGP in breast tumors were analyzed. By quantitative imrnunoeiectrophoresis with a sensitivity of 100 units/mI (approximately 200 ng/mI), MTGP was demonstrated in the ultracentnifugally soluble fraction in all seven metastatic breast tumorstested at concentrations of 480 to 3125 units/mg glycoprotein.When the ultracentnifugally sedimentable fractions of threebreast tumors were solubilized in sodium dodecyl sulfate,MTGP was demonstrated at 140 to 323 units/mg total protein.In the breast carcinoma cell line HS-578T, soluble MTGP wasquantified and estimated to be present at an average of 80,000molecules/cell. Soluble MTGP was not demonstrable in 76normal tissue samples or tumors of other than breast origin(‘<20units/mg protein) but was present in a normal distributionat concentrations from 50 to 695 units/mg soluble protein in66 of 83 (79.5%) histologically confirmed biopsies of ductalcarcinomas of the breast and was present in 76.2% of 101breast carcinomas of all types studied to date. The concentration of MTGP did not significantly correlate with the presenceor concentration of carcmnoembryonic antigen, nonspecificcross-reacting antigen, or estrogen receptor. The presenceand concentration of MTGP were independent of (a) the histological pattern and differentiation of ductal carcinomas of thebreast, (b) the degree of anaplasia or nuclear atypia, and (C)the histological features of the host response. Furthermore,three of five lobular carcinomas of the breast contained MTGP,indicating that it is not restricted to cells of ductal differentiation. Our studies support the hypothesis that MTGP is anindependent marker specific for the neoplastic cells of themammary gland and can be quantitated with currently availabletechniques. Further studies are indicated to establish the relationship of MTGP to the basic neoplastic process and todetermine whether MTGP may function as a tumor-specificantigen for the host immune response.

INTRODUCTION

In the search for molecules uniquely restricted to neoplasticcells of the breast, i.e. , candidate tumor-specific antigens, wehave recently identified, isolated, and characterized a newglycoprotemnpresent in trace quantities in human breast can

I This is Publication 1 652. These collaborative studies were supported by

research grants from Becton-Dickinson and from the National Cancer Institute(CA-i 4346 and CA-i 6600).

2 Recipient of a Becton-Dickinson postdoctoral fellowship in the Immunochem

istry of Cancer.Received November 13, 1978; accepted March 2, 1979.

cinomas (11, 12). This MTGP3of molecular weight circa 19,500was identified in both ultmacentnifugallysoluble and sedimentable forms. In addition, the soluble MTGP occurs in 2 distincttypes that consistently differ in isoelectnic point, buoyant density, carbohydrate composition, and an apparent amino acidsubstitution. Antisera to MTGP have been observed to reactwith a cross-reacting antigen in some spontaneous muninemammary tumors; however, the human MTGP molecules areantigenically unrelated to the glycoprotemn with a molecularweight of 52,000 of muninemammary tumor virus (12) and thusdo not appear to be the same human breast tumor-associatedantigen described by Keydamet a!. (9), Mesa-Tejada et a!. (17),and Temamotoet a!. (22). In view of the association of MTGPwith breast tumors, we have now examined (a) the frequencywith which this candidate marker is present or associated withthe histological type, differentiation, and other features ofbreast tumors; (b) the association between MTGP and otherselected tumor markers; and (C) the degree of specificity fortumors of breast origin.

MATERIALS AND METHODS

Cytosols. The aqueous soluble and nonsedimentable fractions (cytosols) of biopsies from breast carcinomas were prepared by homogenization of 1.0 g of frozen tissue in 5 ml of 10mM Tmis-HCI-1.5 mM EDTA-1 mM dithiothmeitol, pH 7.4, at 4°according to the method of Johnson at a!. (8). The homogenatewas centrifuged at 100,000 x g@4,4at 4°for 30 mm to obtainthe clear lipid-free supemnatant.The samples were adjusted to2 mg protein per ml by Lowry assay (13).

PCA Extracts. Two-g samples of neoplastic or normal tissueswere homogenized at a volume of 12 ml in 0.6 N PCA at 4°,and the supernatants were recovered. After centrifugation for30 mm at 100,000 x g@84(12), the supemnatantscontainingthe PCA-soluble glycoproteins were extensively dialyzed, lyophilized, redissolved, and adjusted to 5 mg glycoprotein perml by the assay of Lowry et a!. (13).

Particulate Fractions. Tissues were homogenized as aboveand centrifuged at 100,000 x g at 4°for 30 mm. The pnecipitate was washed 3 times in 5 ml of 10 mM Tmis-HCI,1.5 mMEDTA, and 1 mM dithiothreitol, pH 7.4, at 4°and then solubilized by incubating the pellets overnight at 37°with equalvolumes of 0.5% 505 (12). After centnifugation at 10,000 x gfor 30 mm at 20°,the supemnatantwas dialyzed at 22—24°against 0.05% SOS, concentrated, and adjusted to 2.5 mgprotein per ml by modified Lowry assay (14).

Antisera. Antisera were produced by immunizing goats andrabbits with 150 @gpartially purified MTGP or with 10 @tghighly

3 The abbreviations used are: MTGP, mammary tumor glycoprotein; PCA,

perchloric acid; SDS, sodium dodecyl sulfate; CEA-S, carcinoembryonic antigenspecies; NCA, nonspecific cross-reacting antigen.

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J. P. Leung eta!.

purified MTGP in complete adjuvant (12). The animals wereboosted s.c. each month with I 0 @gMTGP to maintain the titerof antibody as tested by gel double diffusion. Goat antiserum(G-300) was absorbed with lyophilized homogenates of a panelof normal tissues, after which 4-mi aliquots were passed at 4°through 200 ;L9of immobilized PCA-soluble glycoprotein fromnormal bowel and lung coupled to glutamaldehyde-activatedBiogel P-6 (Bio-Rad Laboratories, Richmond, Calif.) (23) orcyanogen bromide-activated Sepharose 4B (2, 12). The freeprotein peak was eluted with 0.14 M NaCI solution, the ‘yglobulin was precipitated at 45% ammonium sulfate saturationat 4°,and the precipitate was dissolved and dialyzed against0.14 M NaCI, 0.01 M sodium phosphate, pH 7.2.

Radioassays. The estrogen receptor content of tumor cytosols was analyzed by the simplified Scatchard plot assaydescribed by Johnson et a!. (8). CEA-S and NCA were assayedby equilibrium competitive inhibition radioimmunoassays (3,18).

Quantitative Electroimmunodiffusion. The concentration ofMTGP in the cytosol fractions oftissues and in the PCA-solubleglycoprotein fractions was estimated by quantitative electroimmunodiffusion in agamoseat 1.5 V/cm for 16 hr as describedby Zimmerman et a!. (25) and adapted for MTGP (1 2). For

MTGP solubilized in SOS, the procedure was modified asdescribed by Converse and Papermaster (1) using electmophoretic migration through Lubrol to displace SDS from the sampleand permit effective immunoprecipitation. A PCA-soluble fraction of a metastatic ductal cell carcinoma of the breast (T-1 24)at 218 @.tgprotein per ml was assigned an arbitrary value of1000 units/mI and used at serial dilutions in all assays as a

secondary standard. Selected assays were performed withpurified MTGP also present as a primary standard and toprovide gravimetmic quantitation of the secondary standard(Chart 1). One unit was estimated as approximately 2.0 ng forboth type I and type II MTGP with the current reagents. Quantitation of MTGP in the standard PCA-soluble extract of tumorTi 24 was linear from 1 25 to 1000 units MTGP per ml. Prepa

rations of purified MTGP were linear over a similar unit mangecorresponding to 250 to 2000 ng/ml except for some deviationof type I at low concentrations. Unknown samples were assayed and calculated as units penmg total protein or glycoprotein, since the amount of material was usually insufficient fortyping and assignment of MTGP mass values.

Histopathology. A representative section was removed fromthe center of each of the tumors fixed in 10% neutral bufferedformalin, embedded in paraffin, and stained with hematoxylinand eosin. The histological classification of the breast camcinoma biopsies was based on criteria presented by Fisher et a!.(4). Six histological features were systematically analyzed,including (a) histological pattern of growth and (b) degree ofdifferentiation. Invasive ductal tumors were designated asGrade 3, poorly differentiated, if they consisted of solid cordsand/or solid islands, sheets of cells with no ductal structures.Grade II, moderately differentiated tumors were composed ofmixed solid and ductal areas. Grade I were well-differentiatedtumors with predominant ductal differentiation. (C) Nuclearatypia was noted. Grade I nuclei were regular and vesicularwith a delicate nuclear membrane, chromatin was finely distnibuted, and mitoses were frequent. Grade II nuclei were intermediate between Grade I and Grade Ill nuclei, which werehypenchromatic, pleomomphic,and sometimes giant or multiple.

MTGP(Units/mIonng/ml)

CC

.@.

Chart 1. Representative standard curves for electroimmunodiffusion (E.l.O)analysis of MTGP. The standard (Std.) is a PCA-soluble fraction of tumor 124,and MTGP is expressed in units/mI (•).Purified MTGP-l (0) and purified MTGPII (0) are given in ng/ml. Shaded area, limit of sensitivity.

Nucleoli were prominent and mitoses were frequent. (d) Cellulamitywas estimated from the relative proportion of the tissueoccupied by tumor cells. (e) Necrosis was scored, as was theamount of fibrous or hyalinized stroma. (f) The inflammatoryinfiltrate was graded as to relative extent, and the compositionwas scored as predominantly lymphocytic, plasmacytic, ormixed.

Cell Culture. Human breast carcinoma line HS-578T [previously (12) designated AY-726] was grown in minimum essentialmedium supplemented with 20% fetal calf serum, 50 unitspenicillin-streptomycmnper ml, 0.2 mt@iL-glutamine, and 20 m@4-(2-hydroxyethyl)-i -pipemazineethanesulfonic acid (Grand Island Biological Co., Grand Island, N. V.) in stationary cultures.Cells were harvested by scraping.

Statistical Analysis. The Pearson product-moment correlation (6) was used for parametric analyses comparing the concentrations of estrogen receptor, NCA, CEA-S, and MTGP inthe 83 breast carcinoma biopsies. The significance (p) andcorrelation (r) were obtained from Simpson et a!. (21). TheSpearman's rank correlation was used for nonpamametmicanalysis of the concentration of MTGP in breast tumor biopsycytosols in relation to the ranking of tissue histopathology (20).Student's t test was used to evaluate the significance of thecorrelation (21). The sampling distribution of MTGP concentrations was determined from a standardized normal distribution(20), and the probability that MTGP concentrations of <50units occurred in the distribution was calculated by a 1-tailedtest of significance (20). Distribution was also verified by probitanalysis (5) using the log10of MTGP concentration.

RESULTS

MTGP was identified and quantitated in 7 large metastaticductal carcinomas of the breast in which relatively homogeneous areas of tumor tissue, devoid of inflammation and necrosis,could be confidently sampled (Table 1). Serial titmationsof thePCA-soluble glycopmoteinfractions of these tumors gave dilutional slopes parallel to that of the standard (data not shown).The mean concentration of MTGP was 1044 ±927 (S.D.)(range, 480 to 3120) units/mg PCA-soluble glycoprotein,which is equivalent to a mean of 1.36 to 3.97 j.tgMTGP per mgglycoprotein, depending on whether the MTGP is of type I orII (12). The ultracentrifugally sedimentable (i.e. , particulate)fractions of 3 tumors contained MTGP at 140 to 323 units/mg

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MTGP antigen in 7 carcinomas of the breast metastatic to theliverTumorMTG°antigen(units/mgprotein)SDS-solubilized

particuSoluble glycoprotein latefractioni

234567

Mean±S.D.3120

323790 140675762 ND560 ND480 ND920 175

1044±927213±97a

ND, not determined.

Neoplasms (n —22) Nonneoplastic tissues (n —54)Carcinomas:

Pancreas (3). Bowel (5), pancreas (7), salivary glands(5).liver(2), lung (4), stomach placenta (3), breast milk (3), uterus

(3), tongue (1), kidney (2), endometrium (2), urinary bladdermucosacolon(4), testis (1) (2), testis (1), gallbladder (1), ovary(1),Sarcomas

(2) thyroid (1), adrenals (2), kIdneys(4),skeletalmuscle(2), spleen(3), lung(3),liver

(2), braIn (1), heart (1), plasma(3),seminalvesicle (1), prostate(1)MTGPERCU-SNCA

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Mammary Tumor Glycoprotein

total protein. In contrast, in 54 samples of normal tissues andin 22 tumors of sites other than the breast (Table 2), no MTGPwas demonstrable in the current assay with a limit of sensitivityof 20 units/mg glycoprotein.

To estimate the concentration of this molecule in breastcarcinoma cells, we assayed tumor cell line HS-578T (7),previously shown by immunohistochemical techniques to contam MTGP within the cytoplasm and at the surface of viablecells (12). The concentration in the PCA-soluble fraction was192 units/ 1O@cells. This cell line produced MTGP type I byreference to isoelectric point (p15.35). Since MTGP type I hasa conversion of 1.3 ng/unit, the cells were estimated to contain2.5 ng soluble MTGP per 106cells or about 80,000 molecules/cell in the cytoplasmic pool. MTGP has been undetectable incontrol cell lines.

The concentration of MTGP in the cytosol preparation of 83histologically characterized biopsies of breast ductal carcinomas was analyzed. Of these, 66 (79.5%) contained more than50 units MTGP per mg total protein [mean, 195 ±114 (S.D.)units/mg total protein; range, 50 to 695] as illustrated in Chart2. The concentrations of MTGP described a normal Gaussiandistribution; the probability that samples with <50 units were

a distinct subpopulation and were excluded from the normaldistribution was p < 0.1 5. A number of these latter samplescould have contained trace quantities of MTGP, and this probability was statistically supported by probit analysis.

Two other glycoprotein markers, i.e. , CEA-S and NCA, aswell as the estrogen receptor, were also assayed in the 83biopsy-derived cytosols of the ductal carcinomas of the breast(Chart 2). Of the 83 biopsies, 71 (85.5%) were positive forestrogen receptor with a mean of 43.1 ±115 (S.D.) (range, Ito 901 ) fmol/mg total protein. The CEA-S content was greaterthan 14.5 units/mg total protein in 54 of the 83 biopsy cytosols(65.5%) with a mean of 40.8 ±66.5 (S.D.) (range, 14.5 to364) units/mg total protein. NCA was demonstrable in allbiopsy cytosols varying from 34.4 to 166,000 units (0.24 to1160 @Lg)NCA per rng total protein with a mean concentrationof 3941 units (37.4 ;Lg) and a S.D. of ±6088 units/mg totalprotein.

The presence and concentration of the 4 markers, i.e.,MTGP, estrogen receptor, NCA, and CEA-S, were comparedwhen positive (Table 3). The presence and concentration ofeach of the 4 segregated independently, and absence of MTGPdid not correlate with the presence, absence, or concentration

Table2Table1

Tissues devoid of detectab.d MTGPThesetissuescontainedlessthan20 unitsMTGPper mgglycopro

tein in thePCA-solublefractionof 2 g of tissueanalyzedbyquantitativeelectroimmunodiffusion.

io2

101,

=..,

r4,@

=

Chart 2. ConcentratIon of 4 markers In the cytosols of 83biopsies of breast carcinomas. Mean concentration (i): MTGP,195 ±114 units/mg protein;estrogenreceptor(ER), was 43.1± 1 1 5 fmol/mg protein; CEA-S, 40.8 ± 66.5 units/mg protein;and NCA, 3941 ±6088 units/mg protein (mean ±S.D.).

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Statisticalcorrelationsof MTGP,EstrogenReceptor,CEA-S,andNCAin cytosolsof biopsiesof ductalcarcinomasof thebreastMarkers

No.@aMTGP

vs. ERe@ 0. 139 >0.1MTGPvs.CEA-S 45 0.145 >0.1MTGP vs. NCA 67 —0.051 >0.1ER vs. NCA 71 0.236 <0.1ER vs.CEA-S 48 0.012 >0.1NCA vs. CEA-S 54 0.266 <0.1

Association of MTGP with biopsies from different types ofcarcinomasofthe breastPositive

forMTGP@Histological

type Total no. No. Mean ±S.D.Ductal

83 66 195 ±l14@'Lobular5 3 112 ±85Tubular10Medullary1 1188Mucinous10a

Concentration of >50 units/mg total cytosol protein.

Correlation of MTGP with the histological features of ductal carcinomasof thebreastMTGPMean

±S.D. ofRange of posiHistological features No. No. positivepositivetiverapaDifferentiation

83660.036>0.2Intraductal1 1231Invasive

8265Welldifferentiated 7 4 131 ±4675—203Moderately

differentiated 18 15 196 ±14875-695Poorlydifferentiated 57 45 200 ±10853—510Cellularity

83 66—0.1 14>0.2<25%of biopsy 19 11 223 ±11575—42625—75%ofbiopsy 60 52 186±10953—695>75%ofbiopsy

4 3260±22571-510Nuclearatypia 8366—0.057>0.2Mild

5 5 171 ±8975-280Moderate69 53 179 ±8553-695Marked9 8250±15495-510a

Derived from Spearman's rank correlation analysis (19).

J. P. Leung et a!.

of any other marker. For example, MTGP was found in bothestrogen receptor-positive and -negative breast carcinomas.Marginally weak correlations were observed between NCA andCEA-S (r = 0.266, p < 0.1 ) for comparison of estrogenreceptor and NCA (r = 0.236, p < 0.1).

When the concentration of MTGP was compared with thehistological degree of differentiation, themewas no significantcorrelation (Table 4). A similar lack of correlation was observedfor the degree of cellularity and for the degree of nuclearatypia. Additional data (not shown) indicated that there was nocorrelation with the extent of the inflammatory infiltrates, thetype of inflammatory cells present, on the stromal proliferationand degree of fibrosis.

In a series of 94 biopsy cytosols from various types of breastcarcinomas, 70 were positive (Table 5). Three of 5 lobularcarcinomas were positive, indicating that MTGP is not mestricted to cells of ductal differentiation. Numerous other histological types were inadequate, other than to note that amedullary carcinoma was also positive. Of all breast tumorsstudied to date, the 94 biopsies and 7 metastatic ductal carcinomas, 76.2% contained quantifiable amounts of MTGP.

DISCUSSION

The newly described glycoprotein, MTGP, may be a specificmarker for breast carcinoma, since in the present study wehave found this marker in most breast tumors studied to datebut in no other tumor or benign tissue. It is present in most,though apparently not all, carcinomas of the breast. At thecurrent level of assay sensitivity, MTGP was found in breasttumor of both ductal and lobular cell origin. As noted previously

Table3

(I 2), MTGP was not demonstrated in normal or dysplasticmammary glands by sensitive immunohistochemical means norin whole lactating breast milk or its glycoprotein fraction byquantitative electroimmunodiffusion assay. In contrast to CEAS in colonic tumors (3), the quantity of MTGP present in breasttumors did not correlate with the degree of differentiation,cytological features of anaplasia, or invasion. If MTGP were anormal differentiation product, one might anticipate higherconcentrations in more highly differentiated tumors, whereasif it were an embryonic protein most characteristic of undiffer

entiated cells it should be present at higher concentrations inthe least-differentiated tumors. Neither is suggested from theanalyses in Table 4; rather MTGP is the frequent though minorproduct of the majority of these tumors.

It is not known whether MTGP is really absent from somebreast tumors or whether the number of tumor cells present insome of the biopsies was too small to permit detection ofMTGP. However, all 7 tumors with homogeneous tumor tissuecontained significant concentrations of MTGP. Considering thesmall quantities of this glycoprotein per cell in many tumors(Table 1 and Chart 2) and in other breast carcinoma cell lines,4it is possible that MTGP might be present in the negative breast

tumors. Statistical analysis suggests a normal Gaussian distnibution of the concentration of MTGP and a significant possibility that MTGP concentrations in breast tumors may extend

below the current detection threshold.We suggest that the MTGP quantitated in this study was of

cytoplasmic origin and was representative of the total MTGPcontent of the tumor. This is suggested from prior observations

4 Unpublished observation.

Table5

a Pearson product-moment analysis of values greater than threshold.

b Estrogen receptor.

Table4

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Mammary Tumor Glycoprotein

3. Edgington, T. S., Plow, E. F., Go, W., Herberman, A., Burtin, P., Jordan, I.,Chavkin, C., DeHeer, D. H., and Nakamura, A. M. A comparison of CEA-Sand CEA concentrations in sera and the independence of CEA-S, NCA, andblood group antigens. Bull. Cancer (Paris), 63: 613—621, 1976.

4. Fisher, ER., Gregorio, A. M., and Fischer, B. The pathology of invasivebreast cancer. A syllabus derived from findings of the national surgicaladjuvant breast project(protocol number 4). Cancer(Phil.), 36: 1—85,1975.

5. Fisher, A. A., and Yates, F. Statistical Tables for Biological, Agricultural andMedical Research. Ed. 4, Oliver and Boyd Ltd., Edinburgh, 1963.

6. Goldstein, A. Correlation. In: Blostatistics, an Introductory Text, Ed. 1, pp.129-1 87. New York: Macmillan Publishing Co., 1964.

7, Hackett, A., Smith, H. S., Springer, E. L., Owens, A. B., Nelson-Rees, W. A.,Riggs, J. L., and Gardner, M. B. Two syngeneic cell lines from human breasttissue: the anueploid mammary epithelial (HS 578T) and the diplold myoepithelial(HS 578 Bst)cell lines. J. NatI. Cancer Inst., 58: 1795—1806, 1977.

8. Johnson, A. B., Nakamura, A. M., and Libby, R. M. Simplified Scatchardplot assay for estrogen receptor in human breast tumor. Clin. Chem., 21:1925—1730,1965.

9. Keydar, I., Mesa-Tejada, R., Ramanarayanan, M., Ohno, T., Fenogllo, C.,and Spiegleman, S. Detection of viral proteins in mouse mammary tumorsby immunoperoxidase staining of paraffin sections. Proc. Nati. Acad. Sci. U.S. A., 75: 1524-1528, 1978.

10. Leung, B. S., Mandigh, L. C., and Wood, 0. C. Estradiol receptors in benignand malignant disease of the breast. Clin. Chim. Acta, 46: 69-76, 1973.

11. Leung, J. P., Plow, E. F., Nakamura, A. M., and Edgington, T. S. Identificationof a new human mammary tumor glycoprotein. Fed. Proc., 37: 1485, 1978.

12. Leung, J. P.. Plow, E. F., Nakamura, A. M., and Edgington, T. S. Aglycoprotein set specifically associated with the surface and cytosol ofhuman breast carcinoma cells. J. Immunol., 12 1: 1287—1296, 1978.

13. Lowry, 0. H., Rosebrough, N. J., Fair, A. L., and Randall, A. J. Proteinmeasurement with the Folin phenol reagent. J. Biol. Chem., 193: 265—275,1951.

14. Markwell, M. K., Haas, S. M., Bieber, L. L., and Tolbert, N. E. A modificationof the Lowry procedure to simplify protein determination in membrane andIlpoprotein samples. Anal. Biochem., 71: 206-210, 1978.

15. McClendon, J. E., Appleby, P., Clandon, D. B., Donegan, W. L., andDeCossa, J. J. Colon neoplasms: Tissue estrogen receptor and carcinoembryonic antigen. Arch. Surg., 112: 240—241, 1977.

16. Menendez-Botet,C. J., Nisselbaum,J. S.. Fleisher,M., Rosen,P. P.,Fracchia, A., Robbins, G., Urban, J. A., and Schwartz, M. K. Correlationbetween estrogen receptor protein and CEA in normal and carcinomatoushumor in breast tissue. Clin. Chem., 22: 1366—1371, 1976.

17. Mesa-Tejada,A., Keydar,I., Ramamarayanan,M., Ohno,T., Fenglio,C.,and Splegelman, 5. Detection In human breast carcinomas of an antigenimmunologically related to a group-specific antigen of mouse mammarytumor virus. Proc. Nafi. Aced. Sci. U. S. A., 75: 1529-1533, 1978.

18. Plow,E. F., andEdgington,T. S. Isolationandcharacterizationof a homogeneous isomeric species of carcinoembryonic antigen, CEA-S. Int. J.Cancer, 15: 748—761,1975.

19. Ratajczak, T., and Hahnel. R. The protection, stabilization and reactivationof estradiol receptors in human myometrial cytosol. Biochim. Blophys. Acta,338: 104-107, 1974.

20. Siegal, S. Non-parametric Statistics. New York: McGraw-Hill Book Co.,1956.

21. Simpson, G., Roc, A., and Leworth, A. Quantitative Zoology, p. 240. NewYork: Harcourt Brace Javanovich, Inc., 1960.

22. Teramoto, Y. A., Kufe, D., and Schlom, J. Multiple antigenic determinantson the major surface glycoprotein of murine mammary tumor viruses. Proc.NatI. Acad. Sci. U. S. A., 74: 3564-3568, 1977.

23. Ternynck T., and Avrameas, S. Polyacrylamide-protein immunoabsorbentsprepared with glutaraldehyde. FEBS Left., 23: 24-28, 1972.

24. Wittliff, J. L., Hilf, A., Brooks, W. F., Savlov, E. D., Hall, T. C.. and Orlando,A. A. Specific estrogen-binding capacity of the cytoplasmic receptor innormal and neoplastic breast tissue of the human. Cancer Res., 32: 1983—1992,1972.

25. Zimmerman, T. S., Hoyer, L. W., Dickson, L., and Edgington, T. S. Determination of von Wlllebrand's disease antigen (Factor VIll-related antigen) inplasma by quantitative immunoelectrophoresis. J. Lab. Clin. Med., 86: 152—159, 1975.

that only 1 to 9% of the total MTGP was recovered in the100,000 x g sedimentable fraction of tumor homogenates(12). Immunofluorescent studies of fixed cells has indicatedthat most MTGP was distributed throughout the cytoplasm(12). Recent studies further indicate that the MTGP in cytosolpreparations behaves on polyacrylamide gel electrophoresisas soluble rather than membrane-associated MTGP.4

Using a newly described breast carcinoma cell line, HS-578T(7), we have not only shown the presence of this marker (11),but have also estimated its concentration at about 80,000molecules/cell. Although modest in number, this marker ispresent in sufficient concentrations to permit cellular studies ofsynthesis by sensitive techniques.

The presence of estrogen receptor has been used widely asa basic for prognosis and for therapeutic management of breastcancer patients (16). However, not only does the incidenceand concentration of the estrogen receptor vary in breastcarcinomas, but also this marker is present in benign breasttissues (1 0, 24), uterine muscle (1 9), and gastrointestinal neoplasms (15). We found no correlation between MTGP andestrogen receptor, i.e. , MTGP occurred in both estrogen meceptor-positive and -negative breast carcinomas. Thus, MTGPdoes not replace the estrogen receptor as a prognostic ortherapeutic marker. Neither NCA nonCEA-S are tissue specific.We found carcinoembryonic antigen-related antigen in themajority (65%) of breast ductal carcinomas, as did MenendezBotet, et a!. (16), but no relationship to MTGP was apparent.

Since most tumors of both ductal and lobular cell origincontain MTGP, it may be of use to identify the tissue of originof tumors by immunohistochemical techniques. In light of therelative tumor specificity of MTGP, it will be important to determine whether the synthesis of this molecule relates to the basicneoplastic change in the cell. Whether MTGP serves as a targetantigen for the immune response of the tumor host also remainsa highly provocative possibility. In this regard, the presence ofhumoral or cellular immune response to MTGP in breast cancerpatients is under study.

ACKNOWLEDGMENTS

The authors are pleased to acknowledge the generous provision of breastcarcinoma cell line HS-578T by Dr. Walter A. Nelson-Rees, Cell Culture Laboratory, School of Public Health, University of Calif., Berkeley. This study wasconducted with the dedicated technical assistance of Nancy O'Aourke andWilliam Garstka as well as the preparation of the manuscript by Mary Gortmakerand Sharon Garland.

REFERENCES

1. Converse, C., and Papermaster, D. Membrane protein analysis by twodimensional immunoelectrophoresis. Science, 189: 469—472,1975.

2. Cuatrecasas, P. ProteIn purification by affinity chromatography derivatizationof agarose and polyacrylamide beads. J. Biol. Chem., 245: 3059-3065,1970.

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1979;39:2057-2061. Cancer Res   Julia P. Leung, Gerald M. Bordin, Robert M. Nakamura, et al.   Antigen and Other Markers with Human Breast TumorsFrequency of Association of Mammary Tumor Glycoprotein

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