SERUM TUMOR MARKERS FOR DETECTION OF BONE METASTASIS IN BREAST CANCER PATIENTS

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1 Acta Oncologcu Vol. 33, No. 2, pp , 1994 SERUM TUMOR MARKERS FOR DETECTON OF BONE METASTASS N BREAST CANCER PATENTS ADNAN AYDNER, ERKAN Topuz, RAN D~q, VLDAN YASASEVER, MAKTAV D~NCER, KORAY DNCoL and NJAD BLGE Acta Oncol Downloaded from nformahealthcare.com by on 5/2/14 For the dagnoss of bone metastass n breast cancer patents durng systemc treatment serum tumor markers, ncludng carbohydrate antgens 15-3 (CA 15-3) and 19-9 (CA 19-9), cancer antgen 125 (CA 125), alpha-fetoproten ( AFP), carcnoembryonc antgen (CEA), beta-2 mcroglobuln (BMG), ferrtn, and tssue polypeptde antgen (determned by the M3 monocolonal antbody, TPS) were measured n 22 patents wth known bone metastases and n 3 patents wthout documented metastases. The most useful sngle marker was CA By stepwse dscrmnant analyss, t was found that 9% of the patents could be dagnosed truly by usng the markers CA 15-3, BMG and ferrtn. t s concluded that montorng wth combnatons of tumor markers at regular ntervals ncreases the dagnostc effcency. Bone metastases are common n breast cancer and develop n approxmately 7% of patents wth metastatc breast cancer (). At autopsy bone metastases are found n more than half of the patents who de of breast cancer (2-4). Radologc examnaton of the bones and radonuclde bone scan have for years been the standard methods for detecton of bone metastases (5-7). Some studes suggest that serum tumor markers could play some role n the dagnoss of metastases (8). Osseous metastases often cause mportant morbdty as fractures (9, 1). The goal of treatment for these patents s bascally pallaton to mprove the qualty of lfe (1 1). An mportant practcal queston s whether and when patents durng chemotherapy should be examned by magng methods for detecton of possble non-symptomatc bone metastases. Ths may be especally mportant n postmenopausal patents who often Receved 14 May Accepted 9 December From the nsttute of Oncology, Dept. of Medcal Oncology (A. Aydner, E. Topuz, K. DnGol), stanbul Faculty of Medcne, Dept. of Publc Health (n Bostatstcs and demography secton) (R. DeG), nsttute of Oncology, Dept. of Basc Scences (V. Yasasever), nsttute of Oncology, Dept. of Radaton Oncology (M. DnGer, N. Blge), Unversty of stanbul, Turkey. Correspondence to: Dr Adnan Aydner, Bagdat Cad. Nur Apt. 65/5, Kzltoprak-Kadkoy, stanbul, Turkey. receve adjuvant endocrne treatment for 2 to 5 years. Serum tumor markers may be useful n these crcumstances. Relatvely lttle has been publshed regardng the choce of nvestgatve methods for dagnoss of osseous metastases durng systemc treatment ( 12). The present report s a study on the effcency of sngle tumor markers and combnatons of such markers n the dagnoss of bone metastases n breast cancer durng systemc treatment. Materal and Methods ncluded n the study were 22 breast cancer patents wth known bone metastases and 3 breast cancer patents wthout documented metastases. The characterstcs of the patents are shown n Table 1. Patents wthout documented metastases were elgble for ncluson n ths study f they had at least 2 courses of chemotherapy after the surgery. n patents wth bone metastases the samples were also taken after at least two courses of.v. chemotherapy (). Blood samples were taken between the thrd and sxth course of chemotherapy at one tme only, and the sample was taken one day before the chemotherapy course. Tamoxfen (2 mg/day) was admnstered to the postmenopausal patents, and durng the study all of the postmenopausal patents had receved tamoxfen for more than 12 weeks (14). C> Scandnavan Unversty Press SSN X 181

2 82 A AYDNER ET AL Acta Oncol Downloaded from nformahealthcare.com by on 5/2/14 n all cases blood samples were separated nto plan and EDTA tubes and spun at rpm for 1-15 mn wthn one hour after removal. The plasma or serum was then mmedately placed nto contaners and stored at -35 C and was studed wthn two months. Whenever blood samples were taken, a full clncal examnaton was carred out and chest x-rays and abdomnal ultrasonography were performed. n patents wth abnormal lver functon tests but wth normal abdomnal ultrasonograpy, CT of the abdomen was performed. Before chemotherapy, bone scannng was performed n all patents. n patents wth 99mTc pyrophosphate bone scan showng more than two hot spots and wthout non-malgnant dsorders accountng for these lesons skeletal radologcal surveys were performed to confrm possble bone metastases. Complete blood count, serum calcum, total proten, albumn, alkalne phosphatase, blrubn, gammaglutamyl transpeptdase, aspartate amnotransferase, and creatnne were determned n all patents. Serum tumor markers ncludng carbohydrate antgen 19-9 (CA 19-9), carbohydrate antgen 15-3 (CA 15-3). cancer antgen 125 (CA 125), alpha-fetoproten (AFP), carcnoembryonc antgen (CEA), beta-2 mcroglobuln (BMG). and ferrtn were measured wth commercally avalable RA kts, and tssue polypeptde antgen was determned usng the M3 monoclonal antbody (TPS) (CA 15-3 and CA 19-9: CS Bo nternatonal, Gf-Sur-Yuette Cedex, France; CA 12-5, AFP, ferrtn: Abbott Laboratores, USA; CEA: Bo Mereux, Marcy- Etole, France; BMG: Behrngwerke AG, Merburg, Germany; TPS: Bek Dagnostcs AB, Bromma, Sweden). All measurements were performed n duplcate, and the results were expressed n arbtrary unts. The cut-off values of the markers were CA 19-9: 37 U/ml; CA 15-3: 3 U/ml; CA 125: 35 U/ml; AFP: 1 U/ml; CEA: 1 ng/ml; BMG: 2.5 mg/l; ferrtn: 12 ng/ml; TPS 8 U/l. Statstcal analyss. Before statstcal analyss logarthmc transformaton was performed for all tumor marker values to normalze the frequency dstrbuton. For comparson of mean values Student s t-test, for medan values Mann-Whtney U test, and for ratos Yates % -test were used. Although dscrmnant analyss s most often employed to explan or descrbe factors that dscrmnate among groups of nterest, the procedure can also be used to classfy future subjects (15, 16). Classfcaton nvolves the determnaton of a separate predcton equaton correspondng to each group that gves the probablty of belongng to that group. For classfcaton of a future subject, a predcton s calculated for each group and the ndvdual s classfed as belongng to the group he or she most closely resembles (15, 16). To detect the combnaton of markers that could best dfferentate the patents wth and wthout bone metastases dscrmnant analyss was used. Due to the lmted sample of patents we dd not use the dscrmnant analyss n preand postmenopausal patents separately. Stepwse dscrmnant analyss was performed by usng SPSS/PC package program. Results The results are shown n Tables 1-5 and Fgs. 1 and 2. As renal functon tests were normal n all patents (creatnne < 12 mg/l), correctons for serum BMG level were not performed (17). Except one case wth lver metastass, alkalne phosphatase and lver enzyme levels were wthn the normal ranges n all patents. n one patent wth lver metastass gammaglutamyl transpeptdase level was elevated. There was no sgnfcant dfferences between the metastatc and non-metastatc cases as regards blood counts, lver functon tests, renal functon tests, total proten, albumn and calcum levels (p >.5). Varable Number of patents Age Mean 2 SD Premenopausal/ postmenopausal Prevous treatment Surgery (breast) RT (breast) Systemc treatment Chemotherapy Endocrne treatment (tamoxfen) Therapy durng the study Surgery (breast) Surgery (skeleton) RT (breast) RT (skeleton) Systemc treatment Chemotherapy Endocrne treatment ( tamoxfen) Tme to begnnng of chemotherapy (mean weeks 2 SD) Tme to begnnng of endocrne treatment (medan weeks) Stes of metastatc nvolvement Lung Lung and bran Bran Lver and bran Table 1 Patents characterstcs Bone metastases Present / f Absent / f

3 ~ TUMOR MARKERS FOR BONE METASTASES 83 Table 2 Analyss oj" tumor marker levels wth respect to bone metastases d l Markers Non-metastatc Metastatc p-value Specfcty Senstvty AE bj patents patents % '% '% Medan (n) Medan (n) CA (3) 43.5 (22) U/ml BMG 2.75 (3) 3.95 (22) mg/l Ferrtn 34 (3) 115 (22) ng/ml TPS U/l 69 (3) 1 (2) ') The levels are medan values. The analyss was performed wth the Mann-Whtney U-test. The non-sgnfcant markers are not wrtten. b, Actual effcency. Acta Oncol Downloaded from nformahealthcare.com by on 5/2/14 Table 3 Relaton between tumor marker levels und occurrence of bone metustusr.\ unulyse5 by stepwse dscrmnant analyss (n = 52) Markers Wlks' lambda Sgnfcance Specfcty Senstvty AE p-value '% %, '% CA <.1 BMG.46 <.1 Ferrtn.42 <.1 The non-sgnfcant markers are not ncluded. Table 4 Fsher's lnear dscrmnant junctons Markers Non-metastatc Metastatc A B M1: CA 15-3 al: 14.7 bl: 2.4 M2: BMG a2: 29.5 h2: 4.1 M3: Ferrtn a3: 7.1 h3: 9.9 (constant) C1: C2: Functon A=(al xlogm)(a2 xlogm2)(a3 xlogm3)(c) Functon B = (bl x log M) (b2 x log M2) (b3 x log M3) (C2) An example of a case: The real group s the 'non-metastatc group'. Marker Level LogM Functon A M,: CA U/ml 1.14 M,: BMG 2.9 mg/l.46 M,: Ferrtn 34.2 ng/ml Functon A > As functon A s larger than functon B, ths Functon B 15.5 Functon B case s n group A (non-metastatc group). Analyss of the tumor marker levels wth respect to bone metastases s shown n Table 2. Concernng CEA, CA 125, CA 19-9 and AFP there were no statstcally sgnfcant dfferences between the metastatc and non-metastatc cases. CEA levels were elevated n 1 patents (46%) wth metastases and n 1 1 patents (37%) wthout metastases. The medan n the former group was 8.95ng/ml and the latter 6.8 ng/ml (p >.21). CA 125 was elevated n 6 patents (27%) wth metastases and n one of the patents (.3%) wthout metastases. The medan n the former group was U/ml and n the latter group 12.4 U/ml (p >.26). CA 19-9 levels were elevated n 1 patents (46%) wth metastases and n 8 patents (27%) wthout metastases. The medan of the former was 35.4 U/ml and of the latter 23.5 U/ml (p >.). AFP levels were elevated n 2 patents (9%) wth metastases and n none of the patents wthout metastases. The medan of the former was 4. 5 U/ ml and of the latter 2.3 U/ml (p >.32) (Fgs. 1 and 2).

4 184 A. AYDNER ET AL Table 5 The dagnostc value of tumor murkers alone or n combnaton n relaton lo occurrence of bone metastases CA 15-3 BMG Ferrtn Specfcty % Senstvty o/o AE >3 U/ml >2.5 mg/l > 12 ng/ml / / / / / / / / / / /3 9. 7/ / / AE: Actual effcency; and -: ndcate whether the mode of nvestgaton n queston was taken nto account n calculatng the predctve values. Acta Oncol Downloaded from nformahealthcare.com by on 5/2/14 1 1: 1: f 1Ok 1 CA 15-3 (p(o.1) Ferrtn (~(.1) BMG (~(.2) $ f 1: 1: E 1: t 1: t TPS (~(.2) 3 Fg.. Cut-off levels (long horzontal lnes), medans (short horzontal lnes) and serum levels of the markers CA 15-3, BMG, ferrtn and TPS (statstcal analyss was performed wth Mann-Whtney U test). When stepwse dscrmnant analyss was used for detecton of the most sgnfcant tumor markers for dagnoss of bone metastases, CA 15-3, BMG and ferrtn were found to be the most useful markers when they were combned (p <.1). When three markers were used together, senstvty, specfcty and actual effcency (AE: overall percentages of cases that were correctly classfed) were 82%, 97% and 9% respectvely (Table 3 and 4). The dagnostc value of CA 15-3, BMG and ferrtn alone or n combnaton wth respect to ther cut-off values, n relaton to occurrence of bone metastases s shown n Table 5. n 22 patents (1%) wth bone metastases and n 18 (6%) of the patents wthout metastases one or both of the CA 15-3 and BMG levels were elevated (AE: 65%). n 17 patents (77%) wth bone metastases and n 6 patents (ZOO/,) wthout metastases, any two of these three marker levels were hgher than the cut-off values (AE: 79/). n 22 patents (1%) wth bone metastases and n 21 patents (7%) wthout metastases one, two or three of these marker levels were elevated (AE: 6%). When the patents were grouped as premenopausal (n = 26) and postmenopausal (n = 26) and analyzed by the

5 TUMOR MARKERS FOR BONE METASTASES U/ml CA 125 (p).26) 1 1: 1; : r 1oL 1: c t.1 ' 1' Acta Oncol Downloaded from nformahealthcare.com by on 5/2/14 1 o l 1 U/ml CA 19-9 (p).) * 1 lo/ U/ml t l t- AFP (p)k3t 4.1'.1' METASTATC NON-METASTATC METASTATC NON-METASTATC Fg. 2. Cut-off levels (long horzontal lnes), medans (short horzontal lnes) and serum levels of the markers CEA, CA 125, CA 19-9 and AFP (statstcal analyss was performed wth Mann-Whtney U test). Mann-Whtney U test, only ferrtn levels were found to be sgnfcantly hgher n postmenopausal patents (p <.2). n postmenopausal patents only CA 15-3 showed hgher levels n patents wth bone metastases than n nonmetastatc patents (p <,1). Wth respect to the cut-off values specfcty, senstvty and AE were 69%, 85% and 77% respectvely. n premenopausal patents the serum levels of ferrtn, BMG, TPS and CA 15-3 levels were hgher n patents wth bone metastases (p <.1, p <.4, p <.15, p <.21; AE: 85%, 69%, 69%1 and 69% respectvely). Dscusson Detecton of metastatc dsease n patents wth breast cancer mght be mportant for choce of adequate therapy and smplfed detecton of metastases could facltate the care of the patents. Serum tumor markers, ncludng CEA, AFP, CA 15-3, CA 19-9, CA 125 and TPA have been establshed and used for the detecton of carcnomas (18-2). CA 15-3 whch s consdered as a specfc marker of breast cancer has been consdered as partcularly valuable for detecton of bone metastases. t has been suggested that the CA 15-3 levels are related to bulk of the dsease (21). BMG may reflect the tumor burden and durng follow-up, occult tumor cells may actvate the mmune system (22, 23). Ferrtn s an acute phase proten rsng n patents wth advanced dsease and ferrtn concentratons have been found to be sgnfcantly hgher n patents wth advanced breast cancer than n tumor-free control patents (24). TPS seems to ndcate the rate of prolferaton rather than the tumor burden (25, 26). n the present study all these four markers were found to be valuable for the dagnoss of bone metastases. Each tumor marker may play an ndependent bologcal role (27). Changes n tumor marker levels are a reflecton of cytolyss, altered producton, secreton and clearance factors and all these factors may be altered by treatment. Dfferent tumor cells have dfferent propertes to home to specfc stes (28). The stes of the metastases may thus nfluence the alteratons of tumor marker levels. t has prevously been reported that the senstvty of CA 15-3 s greater than that of CEA n patents wth only local metastases and n patents wth only bone metastass (21). n the present study, 4ly of the patents wth bone metastases also had metastases n other organs, whch possbly contrbuted to the tumor marker results. The results suggest that CA 15-3, BMG and ferrtn are markers of tumor burden and that a combnaton of these three markers may be valuable n the dagnoss of bone metastases durng therapy. f only one marker had been used, the hghest AE would be 73%, and f the effcences

6 186 A. AYDNER ET AL Acta Oncol Downloaded from nformahealthcare.com by on 5/2/14 ofcombnatons of these markers wth respect to ther cut-off values were calculated, the hghest AE would be 79%. Ths was the value obtaned when any two or three markers were hgher than ther cut-off levels. By stepwse dscrmnant analyss t was shown that 9% of the patents who had bone metastases could be dentfed by usng these three markers. As ths AE was not dependent on the cut-off values of the markers, t suggests that determnng the serum tumor markers at regular ntervals s especally effcent for the dagnoss of bone metastases. t also suggests that tumor marker values lower than the cut-off levels can be useful for the detecton of metastases (Table 4). When the patents are analyzed wth respect to the menopausal status CA 15-3 was the only sngle marker that showed statstcally sgnfcant dfferences between the metastatc and non-metastatc cases. n the present study, one of the man dfferences between premenopausal and postmenopausal patents was n the systemc treatment, snce tamoxfen had been admnstered to all of the postmenopausal patents. Effects of the tamoxfen on the metastatc cells could be the reason for the dfferent tumor marker levels n the two groups. Ths subject should be studed further. These results suggest that multple factors nfluence the serum marker levels. As each tumor marker probably has an ndependent bologc sgnfcance, combnaton of all the tumor markers at regular ntervals wll ncrease the dagnostc effcency. A useful mathematcal formulaton for the dagnoss of metastases mght be constructed by usng stepwse dscrmnant analyss (Tables 3 and 4). n order to establsh a general formulaton, the normal ranges of tumor marker measurements must be the same n all laboratores. However, the present as well as prevous ones shows that there s a wde overlap of the values between the study groups. Ths causes problems n the use of tumor markers at an ndvdual level. Tumor markers wll certanly not replace the magng methods but mght, by usng some formulaton, be useful as gudance for recommendng magng methods durng the follow-up. REFERENCES. Barry WF, Wells SA, Cox CE, et al. Clncal and radographc correlatons n breast cancer patents wth osseous metastases. Skeletal Rado1 1981; 6: Vadana E, Bross DJ, Pckren JW. An autopsy study of some routes of dssemnaton of cancer of the breast. Br J Cancer 1973; 27: Hegemester FB, Buzdar AU, Luna MA, Blumenschen GR. Causes of death n breast cancer. A clncopathologc study. 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