Original Paper Gynecol Obstet Invest 1992;33:177-182
Department of Gynaecology and Obstetrics, Herlev Hospital, Department of Gynaecology and Obstetrics and Department of Pathology, Rigshospitalet. Department of Gynaecology, Bispebjerg Hospital, Department of Oncology B, The Finsen Institute, Department of Gynaecology and Obstetrics, Hvidovre Hospital, Biostatistical Department and Hormone Department, Statens Seruminstitut. Copenhagen, Denmark
KeyWords
Ovarian carcinoma Tumour markers Preoperative diagnosis CA-125 Tissue polypeptide antigen Placental alkaline phosphatase
C A -125, Placental Alkaline Phosphatase and Tissue Polypeptide Antigen as Preoperative Serum Markers in Ovarian Carcinoma
Abstract
Blood samples were drawn before laparotomy in 42 cases of benign, 17 cases of borderline, and 53 cases of malignant epithelial ovarian neoplasms. The concentrations of CA-125, tissue polypeptide antigen, and placental alkaline phosphatase (PLAP) were determined. No significant difference was found between the levels of CA-125 and TPA. No significant correlation was seen between tumour type and these two markers; however, both were significantly correlated to tumour malignancy and clinical stage, and CA-125 was also cor related to tumour grade. No correlation was found between PLAP and the other markers or any of the above-mentioned parameters. If at least one of two or three markers was requested to be positive, a moderate increase in the ‘de tection rate’ of malignant tumours was found. However, the rate of positive benign samples increased to as much as 41 %. Requesting at least two markers out of two or three to be positive certainly reduced the number of ‘false positive’ benign tumours, but the sensitivity for malignant tumours was reduced concomitantly to levels where marker determinations would be of little use. The study demonstrated a possible, but limited role for preoperative determinations of the markers. A combination of two or three markers was not superior to single markers. The results indicate that neither of these mark ers will be of significant value in a screening context.
Introduction
The insidious course of epithelial ovarian tumours, not only prior to diagnosis, but also during chemotherapy and prior to second-look operation, has encouraged the search for suitable serum markers. Several markers have been suggested for the monitoring of these tumours, which make up 90% of all malignant neoplasms of the ovary.
Received: September 10,1991 Accepted: October 9.1991
The oncofetal antigen CA-125 first described by Bast et al. [1] has gained widespread use and is now well described. However, a considerable proportion of malig nant tumours are marker negative, and a significant num ber of patients with benign disease or no disease at all have been found positive [2, 3], Placental alkaline phosphatase (PLAP) was originally described by Fishman et al. [4], who found elevated levels
K. Toftager-Larsen, MD Department of Obstetrics and Gynecology Rigshospitalet DK-2100 Copenhagen 0 (Denmark)
© 1992 S. Karger AG. Basel 0378-7346/92/0333-0177 $2.75/0
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Kim Toftager-Larsena Vila Hording'0 Anne Dreislerc Soren Daugaardd Birthe Lunde Johannes Bockh Finn LundvalF Kirsten Frederiksen8 Bent Norgaard-Pedersenh
Blood samples were centrifuged, and the scrum frozen at -2 0 °C until analysis.
Table 1. Histology of tumours and age of patients Type
Benign
Serous Mucinous Endometrioid Mesonephroid Brenner Mixed Undifferentiated Unclassified
22 17 1 -
Borderline Malignant Total
Assays 7 8
-
1 5 1
62 29 6 4 1 4 5 1
42
17
53
112
58 21-84
58 22-86
68 25-90
-
2
1 1
-
-
-
Total Patient age, years Median Range
-
33 4 5 4 -
in serum from 43% of patients with ovarian cancer. Recently, monoclonal antibodies to PLAP have been raised [5, 6], and evidence for a clinical value was pre sented [7], Antibody to tissue polypeptide antigen (TPA) was raised by Bjorklund et al. [8] by immunization of a horse with an extract from 56 different malignant tumours. It may be of particular value during follow-up of ovarian carcinoma [9], and in large series a close relation between increasing TPA levels and progressive disease was found [ 10,
11].
In the present study we investigated the possible role for preoperative determinations of CA-125, PLAP and TPA. Particular attention was paid to a possible value of a combination of marker determinations.
CA-125 was analyzed by an enzyme immunoassay according to the manufacturers’ instructions (Abbott Laboratories, Chicago, 111.. USA) using peroxidase-labelled OC-125 monoclonal antibody to CA-125, with o-phenylenediaminc as substrate. Absorbance was read in a Quantum II dual wavelength photometer (Abbott Laboratories, Chicago, 111., USA) at 492 nm and 600 nm, and concentrations were calculated from the calibration curve obtained. The coefficient of variation for the assay was 6.7 % at 175 kU/1. PLAP was analyzed by an enzyme-linked immunoassay accord ing to the manufacturers’ instructions (Sangtec Medical, Bromma, Sweden) with monoclonal antibody to the PLAP-epitope H7, with p-nitrophenylphosphate in 1 M diethanolamine/HCl as substrate. Absorbance was read at 405 nm. and concentrations calculated from the calibration curve obtained. The coefficient of variation for the assay was 7.9% at 1.1 pg/1. TPA was analyzed by a radioimmunoassay according to the man ufacturers’ instructions (Sangtec Medical, Bromma. Sweden). Horse antibody to TPA was incubated in excess with samples and calibra tors, and l25I-labclled TPA was added. Antigen-antibody complexes were precipitated with rabbit antibody to horse IgG. Precipitates were washed with 11.5% polyethylene glycol 6000. centrifuged, and measured in an LKB Multi gamma counter (LKB, Turku, Finland). Concentrations were determined from the calibration curve ob tained. The coefficient of variation for the assay was 9.4% at 80 U/l.
Range for Normal Values For CA-125, the level for definitely elevated values was chosen at 65 kU/1. Concentrations between 35 and 65 kU/1 were considered possibly elevated [ 1]. For PLAP, the levels for definitely and possibly elevated samples were chosen at 3.5 pg/1 and between 2.0 and 3.5 pg/1, respectively. In a previous study, PLAP/H7 was expressed in U/l by a slightly different method [13], according to NorgaardPedersen et al. [ 14]. The conversion factor was found to be 4.5 pg/1 to 1 U/l. For TPA, the levels for definitely and possibly elevated sam ples were chosen at 100 U/l and between 80 and 100 U/l, respectively [
10].
Statistics
During an 18-month period, blood samples were drawn prior to laparotomy from all patients with adnexal masses suspected to be ovarian neoplasms at the Departments of Gynaecology of four uni versity hospitals in Copenhagen: Rigshospitalet, Hvidovre, Bispebjerg, and Herlev hospitals. Forty-two benign, 17 borderline and 53 malignant epithelial ovarian neoplasms were found (table 1). O f the 53 malignant tumours, 21 were stage I, 8 were stage II, 18 were stage III, and 6 were stage IV tumours. Eight tumours were of histological grade 1,18 were of grade 2, and 27 were of grade 3. For comparative purposes, we also examined samples from 22 patients with dermoid cysts and from 15 patients with endometriosis cysts found during that period. The age distribution of the patients is shown in table 1. Histopathological specimens from all patients were reviewed and classified according to the WHO nomenclature [ 12] by one patholo gist (S.D.).
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Group levels of serum concentrations were compared using an ordinary one-way analysis of variance. All calculations were per formed on base-ten logarithms in order to stabilize the variation and to obtain Gaussian-distributed variables. A p value below 0.05 was considered significant.
Results
Tumour Malignancy (Benign, Borderline and Malignant) Highly significant differences in the serum marker concentrations between patients with malignant, border line and benign epithelial tumours were found for CA-125 (p < 0.0001) and for TPA (p < 0.0001) with higher con-
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Materials and Methods
Table 2. Percent positive samples at high or low cut-off limits combining two or three tests
Marker
Epithelial tumors malignant borderline benign
Dermoid cysts
Endome triosis
Test positive, % CA-125
high low
70.4 74.1
29.4 47.1
9.1 18.2
9.1 9.1
12.5 43.8
TPA
high low
68.5 83.3
41.2 47.1
20.5 45.5
0.0 22.7
6.5 18.8
high low
38.9 51.9
5.9 23.5
22.7 31.8
4.5 18.2
18.8 25.0
PLAP
At least one test positive, % CA-125 + TPA
high low
79.6 92.6
58.8 58.8
27.3 50.0
9.1 31.8
18.8 62.5
CA-125 + PLAP
high low
77.8 83.3
29.4 52.9
29.5 43.2
13.6 27.3
31.3 68.8
TPA + PLAP
high low
85.2 96.3
41.2 52.9
38.6 65.9
4.5 31.8
6.3 12.5
CA-125 + TPA + PLAP
high low
85.2 96.3
52.9 64.7
40.9 68.2
13.6 40.9
31.3 75.0
Both/at least two tests positive, % CA-125 + TPA
high low
59.3 64.8
17.6 35.3
2.3 11.4
0.0 0.0
0.0 0.0
CA-125 + PLAP
high low
29.6 42.6
5.9 23.5
2.3 2.3
0.0 0.0
0.0 0.0
TPA + PLAP
high low
22.2 38.9
5.9 11.8
4.5 9.1
0.0
6.3 12.5
high low
74.1 79.6
17.6 41.2
9.1 22.7
0.0
CA-125 + TPA + PLAP
9.1 9.1
6.3 12.5
High limits: CA-125: > 65; TPA: > 100; PLAP: > 3.5. Low limits: CA-125: > 35; TPA: > 80; PLAP: > 2.0.
Histological Type No significant differences were found between the marker concentrations in the five histological types of car cinoma regarding TPA or PLAP. A slightly significant dif ference was found for CA-125 (p = 0.04), with lower val
ues for mucinous and mesonephroid tumours. However, these tumours were few and in an early stage of disease. The concentration of CA-125 was high in the only patient with stage III disease. In 15 serous and mucinous borderline tumours, mar ker concentrations above the cut-off limit were found in 7, 6 and 1 case for CA-125, TPA and PLAP, respectively. No significant differences in marker levels between the two histological borderline types were seen for any of the markers, or between the four types of benign tumours. Histological Grade Significantly higher concentrations of CA-125 were found (p = 0.0007) with increasing grade. An apparently
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centrations found in malignant tumours. For PLAP, no significant difference was found. However, wide ranges in concentrations were seen for all three markers, and in malignant tumours, marker concentrations were below the definite cut-off limits in 30% (CA-125 and TPA) to 60% (PLAP) (table 2). Also, levels were elevated in 923% of the patients with benign epithelial tumours (ta ble 2).
Stage Grade
l 1
Grades 1 -3
« s a 11 mMm 2
Fig. 1. The percentage of preoperative samples with marker con centrations in serum above the upper cut-off limits (table 2) in clini cal stages I-IV, and in histological grades 1-3.
similar difference was found for TPA, but this difference was not significant. Accordingly, the ratio of marker-posi tive cases rose from 38% and 50% in grade 1 disease to 81 % and 78% in grade 3 disease for CA-125 and TPA, respectively (fig. 1). Although the ratio of PLAP-positive cases decreased with increasing grade, this decrease was not significant. Clinical Stage o f Tumour Highly significant differences between the four clinical stages of disease were found for CA-125 (p = 0.003) and for TPA (p = 0.002) with increasing concentrations in higher stages, whereas no consistent differences were found for PLAP. Accordingly, the ratio of marker-posi tive cases rose from 48% (CA-125) and 43% (TPA) in stage I disease to 100% in stage IV disease (fig. 1). Correlation between Marker Concentrations A consistently significant correlation between the marker concentrations of CA-125 and TPA was found comparing the marker concentrations (a) between malig nant, borderline, and benign tumours, (b) in different stages, and (c) in different grades of disease, with coeffi
180
cients of correlation of 0.44 (a), 0.39 (b), and 0.50 (c), whereas no consistent correlation existed between PLAP and the two other markers in any of these entities. Combination o f Markers The proportion of patients with malignant tumours being positive for at least one of two or three markers was slightly greater than the values found for single markers (table 2). Simultaneously, however, the false-positive rate (positive benign tumours) increased considerably. Using single markers only (and using high limits), as many as 70% of malignant tumours were positive, whereas 9-13% of benign tumours were positive (CA-125). Using CA-125 in combination with the two other markers (high limits), the proportion of positive malignant tumours rose to at most 85%, but positive benign tumours rose to 31 % (ta ble 2). A considerably lower false-positive rate was seen if a demand of both/at least two positive markers was made. CA-125 and either TPA or PLAP were not positive in any case of dermoid cysts or endometriosis, and only in very few benign epithelial tumours (high or low limits) (ta ble 2). However, the proportion of positive malignant tumours decreased to 59% or lower, unless all three mark ers were included (table 2). Using the low limits generally increased the proportion of marker-positive malignant tumours significantly, but simultaneously the proportion of positive benign tumours invariably increased, in most cases to unacceptable values.
Discussion
In agreement with Panza et al. [ 11 ] we found no signifi cant difference between the concentrations of CA-125 and TPA comparing benign and malignant epithelial tumours, although the relative differences in marker con centrations between malignant and other tumours were considerably higher for CA-125 compared to TPA. We did not find any significant difference between the concentrations of PLAP in benign and malignant tu mours in spite of the use of a specific assay with mono clonal antibody. Similarly, with a different monoclonal antibody, Fisken et al. [ 15] found the sensitivity and spec ificity of PLAP insufficient for clinical use. The extreme specificity of PLAP for ovarian carcinomas stated by Eerdekens et al. [ 16] could thus not be confirmed. Initial reports on CA-125 suggested that this marker would be of little value in the management of mucinous and mesonephroid tumours [1, 11, 17], However, during
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Stages I- IV
[ 10].
Brioschi et al. [19] and Cruickshank et al. [18] found no significant relationship between the concentration of CA-125 and the histological grade of the tumour, whereas Zanaboni et al. [22] found an inverse relationship. In the present study all pathological specimens were examined by one pathologist to ensure a uniform grading, and a highly significant increase in the marker concentration of CA-125 was found with increasing grade, possibly indi cating the higher malignant potential with increased shed ding of antigen. A similar - though not statistically signifi cant - increase was found for TPA. Interestingly, the con centration of PLAP decreased with increasing malignan cy, possibly indicating that PLAP is a highly specialized enzyme not being expressed by dedifferentiated tumour cells. As did others, we found a highly significant correlation between the marker concentration and the stage of disease for TPA [10, 11] and CA-125 [18, 19, 21,22], This could have an impact on the preoperative management of patients with suspected ovarian malignancy, but simulta neously it considerably reduces the usefulness of the markers in a screening context. The ultimate goal of a screening program for ovarian cancer is to detect the tumour at a stage where a radical operation can be per formed. More than half of our stage I tumours remained undetected by CA-125 and by TPA, and even one fourth (TPA) to one third (CA-125) of stage II tumours were missed. Furthermore, the samples in this study were drawn from patients where medical assistance already had been requested. At an earlier time in the course, fig ures for stage I disease possibly would be even lower. Also, 10-20% of benign tumours were marker positive. With the lower cut-off limits, the sensitivity increased slightly, but simultaneously, the false-positive rate more than dou bled, for TPA to a hardly acceptable 46%. A combination of two or more markers has been sug gested to increase the sensitivity for malignant tumours
[7, 11, 23, 24], We did find an increase in the ‘detection rate’ of malignant tumours to at most 85% if at least one of two or three markers were requested to be positive, but again the rate of positive benign samples increased to as much as 41%. Although no normal patients were exam ined, a considerable - and unacceptable - number of patients, up to 31 %, with endometriosis or dermoid cysts was positive. Using lower cut-off limits for the marker concentrations only worsened these figures. Requesting both or at least two markers out of two or three to be positive definitely reduced the number of ‘false-positive’ benign tumours and endometriosis to in deed very low levels. However, the sensitivity for malig nant tumours was reduced concomitantly to levels where marker determinations would be of little use. In all, a combined use of two or three markers demand ing just one marker to be positive seems to confuse the interpretation more than it adds useful information to the preoperative management of ovarian tumours. The per centage of marker-positive patients with benign disease makes this concept unacceptable in a screening context. A concept of (at least) two markers being positive leads to a considerable reduction in the percentage of positive be nign cases; however, an unacceptable decrease in the sen sitivity for malignant tumours is also found. This combi nation, therefore, can be recommended neither for preop erative use nor for screening purposes. The use of one marker - either CA-125 or TPA, but not PLAP - appears to give a less confusing and probably more effective information preoperatively. The role of the markers in a screening context remains to be seen.
Acknowledgements This study was supported by grants from the Danish Cancer Soci ety. Appreciation is expressed to the departments of pathology at the hospitals involved for supplying us with specimens for revision. Kits for determination of TPA and PLAP were kindly supplied by Sangtec Medical, and kits for determination of CA-125 were kindly supplied by Abbott Diagnostics.
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recent years several authors have found that high values ofCA-125 are found in advanced stages of these tumours [18,19-21 ], and Cruickshank et al. [ 18] reported high val ues also in nonepithelial malignant ovarian tumours. We did not find any significant differences in marker concen trations of CA-125 in the different types of tumours inves tigated. Although the number of mucinous and mesonephroid tumours was small in our study, CA-125 was well above the chosen cut-off limits in the case of advanced stage. No differences between the concentra tions of TPA in the different types of epithelial tumours were found in this study, as in the study by Inoue et al.
1 Bast RC, Klug TL, St. John E, Jenison E, Niloff JM. Lazarus H, Berkowitz RS. Leavitt T, Grif fiths CT, Parker L, Zurawski VR, Knapp RC: A radioimmunoassay using a monoclonal anti body to monitor the course of epithelial ovar ian cancer. N Engl J Med 1983;309:883-887. 2 Soper JT, Hunter VJ, Daly L, Tanner M, Creasman WT, Bast RC: Preoperative serum tumor-associated antigen levels in women with pelvic masses. Obstet Gynecol 1990:75:249— 254. 3 Lundqvist EÂ, Nordstrom L, Sjövall K, Eneroth P: Evaluation of seven different tumour markers for the establishment of tumour marker panels in gynecologic malignancies. EurJ Gynaecol Oncol 1989;10:395-405. 4 Fishman WH, Inglis NR, Vaitukaitis J, Stolbach LL: Regan isoenzyme and human cho rionic gonadotropin in ovarian cancer. Natl Cancer Inst Monogr 1975:42:63-73. 5 Millan JL, Stigbrand T. Ruoslahti E, Fishman WH: Characterization and use of an allotypespecific monoclonal antibody to placental alka line phosphatase in the study of cancer-related phosphatase polymorphism. Cancer Res 1982; 42:2444-2449. 6 Travers P, Bodmer W: Preparation and charac terisation of monoclonal antibodies against placental alkaline phosphatase and other hu man trophoblast-associated determinants. Int J Cancer 1984;33:633-641. 7 Ward BG. Cruickshank DJ, Tucker DF, Love S: Independent expression in serum of three tumour-associated antigens: CA-125, placental alkaline phosphatase and HMFG2 in ovarian carcinoma. Br J Obstet Gynaecol 1987;94: 696-698. 8 Björklund B, Björklund V, Wiklund B, Lundstrom R. Ekdahl PH, Hagbard L, Kaijser K, Eklund G, Lüning B: A human tissue polypep tide related to cancer and placenta. 1. Prepara tion and properties. II. Assay technique. III. Clinical studies of 1,483 individuals with can cer and other conditions; in Björklund B (ed): Immunological Techniques for Detection of Cancer. Stockholm, Bonniers, 1973. pp 133— 187.
9 Hagbard L, Sorbe B: Preliminary experiences of TPA (tissue polypeptide antigen) in cancer of the ovary. Laekartidningen 1978;75:3433— 3435. 10 Inoue M. Inoue Y, Hiramatsu K, Ueda G: The clinical value of tissue polypeptide antigen in patients with gynecologic tumors. Cancer 1985;55:2618-2623. 11 Panza N, Pacilio G, Campanella L, Pcluso G, Battista C, Amoriello A, Utech W, Vacca C, Lombardi G: Cancer antigen 125, tissue poly peptide antigen, and beta-chain human cho rionic gonadotropin as serum markers of epi thelial ovarian carcinoma. Cancer 1988;61:76— 83. 12 Dallenbach-Hallweg G: On the histogenesis and morphology of ovarian carcinomas. Can cer Res Clin Oncol 1984; 107:71 -80. 13 Hording U, Toftagcr-Larsen K, Drcisler A, Lund B, Daugaard S, Lundvall F, Arends J, Winkel P, Rorth M: CA 125, placental alkaline phosphatase and tissue polypeptide antigen in the monitoring of ovarian carcinoma: A com parative study of three different tumor mark ers. Gynecol Obstet Invest 1990;30:178-183. 14 Millan JL, Nustad K, Norgaard-Pedersen B: Highly sensitive solid-phase immunoenzymometric assay for placental and placental-like alkaline phosphatases with a monoclonal anti body and monodisperse polymer particles. Clin Chem 1985;31:54-59. 15 Fisken J. Leonard RCF, Shaw G, Bowman A, Roulston JE: Serum placental-like alkaline phosphatase (PLAP): A novel combined en zyme linked immunoassay for monitoring ovarian cancer. J Clin Pathol 1989;42:40-45. 16 Eerdekens MW, Nouwen EJ, Pollet DE, Briers TW, de Broe ME: Placental alkaline phospha tase and cancer antigen 125 in sera of patients with benign and malignant diseases. Clin Chem 1985;35:687-690.
17 Einhorn N, Bast RC. Knapp RC, Tjernberg B, Zurawski VR: Preoperative evaluation of se rum CA 125 levels in patients with primary epithelial ovarian cancer. Obstet Gynecol 1986;67:414-416. 18 Cruickshank DJ, Fullerton WT, Klopper A: The clinical significance of pre-operative se rum CA 125 in ovarian cancer. Br J Obstet Gynaecol 1987;94:692-695. 19 Brioschi PA, Irion O, Bischof P, Bader M, Forni M, Krauer F: Serum CA 125 in epithelial ovarian cancer. A longitudinal study. Br J Ob stet Gynaecol 1987;94:196-201. 20 Canney PA, Moore M, Wilkinson PM, James RD: Ovarian cancer antigen CA-125: A pro spective clinical assessment of its role as a tumour marker. Br J Cancer 1984;50:765— 769. 21 Alvarez RD, To A, Boots LR, Shingleton HM, Hatch KD, Hubbard J, Soong SJ, Potter ME: CA 125 as a serum marker for poor prognosis in ovarian malignancies. Gynecol Oncol 1987; 26:284-289. 22 Zanaboni F, Vergadoro F, Prcsti M, Gallotti P, Lombardi F, Bolis G: Tumor antigen CA 125 as a marker of ovarian epithelial carcinoma. Gynecol Oncol 1987;28:61-67. 23 Negishi Y, Furukawa T, Oka T, Sakamoto M, Hirata T, Okabe K, Matayoshi K, Akiya K, Soma H: Clinical use of CA 125 and its combi nation assay with other tumor marker in pa tients with ovarian carcinoma. Gynecol Obstet Invest 1987;23:200-207. 24 Lahousen M, Stettner H, Pickel H, Urdl W, Purstner P: The predictive value of a combina tion of tumor markers in monitoring patients with ovarian cancer. Cancer 1987;60:22282232.
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References
Department of Gynaecology and Obstetrics, Herlev Hospital, Department of Gynaecology and Obstetrics and Department of Pathology, Rigshospitalet. Department of Gynaecology, Bispebjerg Hospital, Department of Oncology B, The Finsen Institute, Department of Gynaecology and Obstetrics, Hvidovre Hospital, Biostatistical Department and Hormone Department, Statens Seruminstitut. Copenhagen, Denmark
KeyWords
Ovarian carcinoma Tumour markers Preoperative diagnosis CA-125 Tissue polypeptide antigen Placental alkaline phosphatase
C A -125, Placental Alkaline Phosphatase and Tissue Polypeptide Antigen as Preoperative Serum Markers in Ovarian Carcinoma
Abstract
Blood samples were drawn before laparotomy in 42 cases of benign, 17 cases of borderline, and 53 cases of malignant epithelial ovarian neoplasms. The concentrations of CA-125, tissue polypeptide antigen, and placental alkaline phosphatase (PLAP) were determined. No significant difference was found between the levels of CA-125 and TPA. No significant correlation was seen between tumour type and these two markers; however, both were significantly correlated to tumour malignancy and clinical stage, and CA-125 was also cor related to tumour grade. No correlation was found between PLAP and the other markers or any of the above-mentioned parameters. If at least one of two or three markers was requested to be positive, a moderate increase in the ‘de tection rate’ of malignant tumours was found. However, the rate of positive benign samples increased to as much as 41 %. Requesting at least two markers out of two or three to be positive certainly reduced the number of ‘false positive’ benign tumours, but the sensitivity for malignant tumours was reduced concomitantly to levels where marker determinations would be of little use. The study demonstrated a possible, but limited role for preoperative determinations of the markers. A combination of two or three markers was not superior to single markers. The results indicate that neither of these mark ers will be of significant value in a screening context.
Introduction
The insidious course of epithelial ovarian tumours, not only prior to diagnosis, but also during chemotherapy and prior to second-look operation, has encouraged the search for suitable serum markers. Several markers have been suggested for the monitoring of these tumours, which make up 90% of all malignant neoplasms of the ovary.
Received: September 10,1991 Accepted: October 9.1991
The oncofetal antigen CA-125 first described by Bast et al. [1] has gained widespread use and is now well described. However, a considerable proportion of malig nant tumours are marker negative, and a significant num ber of patients with benign disease or no disease at all have been found positive [2, 3], Placental alkaline phosphatase (PLAP) was originally described by Fishman et al. [4], who found elevated levels
K. Toftager-Larsen, MD Department of Obstetrics and Gynecology Rigshospitalet DK-2100 Copenhagen 0 (Denmark)
© 1992 S. Karger AG. Basel 0378-7346/92/0333-0177 $2.75/0
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 11:45:39 AM
Kim Toftager-Larsena Vila Hording'0 Anne Dreislerc Soren Daugaardd Birthe Lunde Johannes Bockh Finn LundvalF Kirsten Frederiksen8 Bent Norgaard-Pedersenh
Blood samples were centrifuged, and the scrum frozen at -2 0 °C until analysis.
Table 1. Histology of tumours and age of patients Type
Benign
Serous Mucinous Endometrioid Mesonephroid Brenner Mixed Undifferentiated Unclassified
22 17 1 -
Borderline Malignant Total
Assays 7 8
-
1 5 1
62 29 6 4 1 4 5 1
42
17
53
112
58 21-84
58 22-86
68 25-90
-
2
1 1
-
-
-
Total Patient age, years Median Range
-
33 4 5 4 -
in serum from 43% of patients with ovarian cancer. Recently, monoclonal antibodies to PLAP have been raised [5, 6], and evidence for a clinical value was pre sented [7], Antibody to tissue polypeptide antigen (TPA) was raised by Bjorklund et al. [8] by immunization of a horse with an extract from 56 different malignant tumours. It may be of particular value during follow-up of ovarian carcinoma [9], and in large series a close relation between increasing TPA levels and progressive disease was found [ 10,
11].
In the present study we investigated the possible role for preoperative determinations of CA-125, PLAP and TPA. Particular attention was paid to a possible value of a combination of marker determinations.
CA-125 was analyzed by an enzyme immunoassay according to the manufacturers’ instructions (Abbott Laboratories, Chicago, 111.. USA) using peroxidase-labelled OC-125 monoclonal antibody to CA-125, with o-phenylenediaminc as substrate. Absorbance was read in a Quantum II dual wavelength photometer (Abbott Laboratories, Chicago, 111., USA) at 492 nm and 600 nm, and concentrations were calculated from the calibration curve obtained. The coefficient of variation for the assay was 6.7 % at 175 kU/1. PLAP was analyzed by an enzyme-linked immunoassay accord ing to the manufacturers’ instructions (Sangtec Medical, Bromma, Sweden) with monoclonal antibody to the PLAP-epitope H7, with p-nitrophenylphosphate in 1 M diethanolamine/HCl as substrate. Absorbance was read at 405 nm. and concentrations calculated from the calibration curve obtained. The coefficient of variation for the assay was 7.9% at 1.1 pg/1. TPA was analyzed by a radioimmunoassay according to the man ufacturers’ instructions (Sangtec Medical, Bromma. Sweden). Horse antibody to TPA was incubated in excess with samples and calibra tors, and l25I-labclled TPA was added. Antigen-antibody complexes were precipitated with rabbit antibody to horse IgG. Precipitates were washed with 11.5% polyethylene glycol 6000. centrifuged, and measured in an LKB Multi gamma counter (LKB, Turku, Finland). Concentrations were determined from the calibration curve ob tained. The coefficient of variation for the assay was 9.4% at 80 U/l.
Range for Normal Values For CA-125, the level for definitely elevated values was chosen at 65 kU/1. Concentrations between 35 and 65 kU/1 were considered possibly elevated [ 1]. For PLAP, the levels for definitely and possibly elevated samples were chosen at 3.5 pg/1 and between 2.0 and 3.5 pg/1, respectively. In a previous study, PLAP/H7 was expressed in U/l by a slightly different method [13], according to NorgaardPedersen et al. [ 14]. The conversion factor was found to be 4.5 pg/1 to 1 U/l. For TPA, the levels for definitely and possibly elevated sam ples were chosen at 100 U/l and between 80 and 100 U/l, respectively [
10].
Statistics
During an 18-month period, blood samples were drawn prior to laparotomy from all patients with adnexal masses suspected to be ovarian neoplasms at the Departments of Gynaecology of four uni versity hospitals in Copenhagen: Rigshospitalet, Hvidovre, Bispebjerg, and Herlev hospitals. Forty-two benign, 17 borderline and 53 malignant epithelial ovarian neoplasms were found (table 1). O f the 53 malignant tumours, 21 were stage I, 8 were stage II, 18 were stage III, and 6 were stage IV tumours. Eight tumours were of histological grade 1,18 were of grade 2, and 27 were of grade 3. For comparative purposes, we also examined samples from 22 patients with dermoid cysts and from 15 patients with endometriosis cysts found during that period. The age distribution of the patients is shown in table 1. Histopathological specimens from all patients were reviewed and classified according to the WHO nomenclature [ 12] by one patholo gist (S.D.).
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Group levels of serum concentrations were compared using an ordinary one-way analysis of variance. All calculations were per formed on base-ten logarithms in order to stabilize the variation and to obtain Gaussian-distributed variables. A p value below 0.05 was considered significant.
Results
Tumour Malignancy (Benign, Borderline and Malignant) Highly significant differences in the serum marker concentrations between patients with malignant, border line and benign epithelial tumours were found for CA-125 (p < 0.0001) and for TPA (p < 0.0001) with higher con-
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Materials and Methods
Table 2. Percent positive samples at high or low cut-off limits combining two or three tests
Marker
Epithelial tumors malignant borderline benign
Dermoid cysts
Endome triosis
Test positive, % CA-125
high low
70.4 74.1
29.4 47.1
9.1 18.2
9.1 9.1
12.5 43.8
TPA
high low
68.5 83.3
41.2 47.1
20.5 45.5
0.0 22.7
6.5 18.8
high low
38.9 51.9
5.9 23.5
22.7 31.8
4.5 18.2
18.8 25.0
PLAP
At least one test positive, % CA-125 + TPA
high low
79.6 92.6
58.8 58.8
27.3 50.0
9.1 31.8
18.8 62.5
CA-125 + PLAP
high low
77.8 83.3
29.4 52.9
29.5 43.2
13.6 27.3
31.3 68.8
TPA + PLAP
high low
85.2 96.3
41.2 52.9
38.6 65.9
4.5 31.8
6.3 12.5
CA-125 + TPA + PLAP
high low
85.2 96.3
52.9 64.7
40.9 68.2
13.6 40.9
31.3 75.0
Both/at least two tests positive, % CA-125 + TPA
high low
59.3 64.8
17.6 35.3
2.3 11.4
0.0 0.0
0.0 0.0
CA-125 + PLAP
high low
29.6 42.6
5.9 23.5
2.3 2.3
0.0 0.0
0.0 0.0
TPA + PLAP
high low
22.2 38.9
5.9 11.8
4.5 9.1
0.0
6.3 12.5
high low
74.1 79.6
17.6 41.2
9.1 22.7
0.0
CA-125 + TPA + PLAP
9.1 9.1
6.3 12.5
High limits: CA-125: > 65; TPA: > 100; PLAP: > 3.5. Low limits: CA-125: > 35; TPA: > 80; PLAP: > 2.0.
Histological Type No significant differences were found between the marker concentrations in the five histological types of car cinoma regarding TPA or PLAP. A slightly significant dif ference was found for CA-125 (p = 0.04), with lower val
ues for mucinous and mesonephroid tumours. However, these tumours were few and in an early stage of disease. The concentration of CA-125 was high in the only patient with stage III disease. In 15 serous and mucinous borderline tumours, mar ker concentrations above the cut-off limit were found in 7, 6 and 1 case for CA-125, TPA and PLAP, respectively. No significant differences in marker levels between the two histological borderline types were seen for any of the markers, or between the four types of benign tumours. Histological Grade Significantly higher concentrations of CA-125 were found (p = 0.0007) with increasing grade. An apparently
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centrations found in malignant tumours. For PLAP, no significant difference was found. However, wide ranges in concentrations were seen for all three markers, and in malignant tumours, marker concentrations were below the definite cut-off limits in 30% (CA-125 and TPA) to 60% (PLAP) (table 2). Also, levels were elevated in 923% of the patients with benign epithelial tumours (ta ble 2).
Stage Grade
l 1
Grades 1 -3
« s a 11 mMm 2
Fig. 1. The percentage of preoperative samples with marker con centrations in serum above the upper cut-off limits (table 2) in clini cal stages I-IV, and in histological grades 1-3.
similar difference was found for TPA, but this difference was not significant. Accordingly, the ratio of marker-posi tive cases rose from 38% and 50% in grade 1 disease to 81 % and 78% in grade 3 disease for CA-125 and TPA, respectively (fig. 1). Although the ratio of PLAP-positive cases decreased with increasing grade, this decrease was not significant. Clinical Stage o f Tumour Highly significant differences between the four clinical stages of disease were found for CA-125 (p = 0.003) and for TPA (p = 0.002) with increasing concentrations in higher stages, whereas no consistent differences were found for PLAP. Accordingly, the ratio of marker-posi tive cases rose from 48% (CA-125) and 43% (TPA) in stage I disease to 100% in stage IV disease (fig. 1). Correlation between Marker Concentrations A consistently significant correlation between the marker concentrations of CA-125 and TPA was found comparing the marker concentrations (a) between malig nant, borderline, and benign tumours, (b) in different stages, and (c) in different grades of disease, with coeffi
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cients of correlation of 0.44 (a), 0.39 (b), and 0.50 (c), whereas no consistent correlation existed between PLAP and the two other markers in any of these entities. Combination o f Markers The proportion of patients with malignant tumours being positive for at least one of two or three markers was slightly greater than the values found for single markers (table 2). Simultaneously, however, the false-positive rate (positive benign tumours) increased considerably. Using single markers only (and using high limits), as many as 70% of malignant tumours were positive, whereas 9-13% of benign tumours were positive (CA-125). Using CA-125 in combination with the two other markers (high limits), the proportion of positive malignant tumours rose to at most 85%, but positive benign tumours rose to 31 % (ta ble 2). A considerably lower false-positive rate was seen if a demand of both/at least two positive markers was made. CA-125 and either TPA or PLAP were not positive in any case of dermoid cysts or endometriosis, and only in very few benign epithelial tumours (high or low limits) (ta ble 2). However, the proportion of positive malignant tumours decreased to 59% or lower, unless all three mark ers were included (table 2). Using the low limits generally increased the proportion of marker-positive malignant tumours significantly, but simultaneously the proportion of positive benign tumours invariably increased, in most cases to unacceptable values.
Discussion
In agreement with Panza et al. [ 11 ] we found no signifi cant difference between the concentrations of CA-125 and TPA comparing benign and malignant epithelial tumours, although the relative differences in marker con centrations between malignant and other tumours were considerably higher for CA-125 compared to TPA. We did not find any significant difference between the concentrations of PLAP in benign and malignant tu mours in spite of the use of a specific assay with mono clonal antibody. Similarly, with a different monoclonal antibody, Fisken et al. [ 15] found the sensitivity and spec ificity of PLAP insufficient for clinical use. The extreme specificity of PLAP for ovarian carcinomas stated by Eerdekens et al. [ 16] could thus not be confirmed. Initial reports on CA-125 suggested that this marker would be of little value in the management of mucinous and mesonephroid tumours [1, 11, 17], However, during
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Stages I- IV
[ 10].
Brioschi et al. [19] and Cruickshank et al. [18] found no significant relationship between the concentration of CA-125 and the histological grade of the tumour, whereas Zanaboni et al. [22] found an inverse relationship. In the present study all pathological specimens were examined by one pathologist to ensure a uniform grading, and a highly significant increase in the marker concentration of CA-125 was found with increasing grade, possibly indi cating the higher malignant potential with increased shed ding of antigen. A similar - though not statistically signifi cant - increase was found for TPA. Interestingly, the con centration of PLAP decreased with increasing malignan cy, possibly indicating that PLAP is a highly specialized enzyme not being expressed by dedifferentiated tumour cells. As did others, we found a highly significant correlation between the marker concentration and the stage of disease for TPA [10, 11] and CA-125 [18, 19, 21,22], This could have an impact on the preoperative management of patients with suspected ovarian malignancy, but simulta neously it considerably reduces the usefulness of the markers in a screening context. The ultimate goal of a screening program for ovarian cancer is to detect the tumour at a stage where a radical operation can be per formed. More than half of our stage I tumours remained undetected by CA-125 and by TPA, and even one fourth (TPA) to one third (CA-125) of stage II tumours were missed. Furthermore, the samples in this study were drawn from patients where medical assistance already had been requested. At an earlier time in the course, fig ures for stage I disease possibly would be even lower. Also, 10-20% of benign tumours were marker positive. With the lower cut-off limits, the sensitivity increased slightly, but simultaneously, the false-positive rate more than dou bled, for TPA to a hardly acceptable 46%. A combination of two or more markers has been sug gested to increase the sensitivity for malignant tumours
[7, 11, 23, 24], We did find an increase in the ‘detection rate’ of malignant tumours to at most 85% if at least one of two or three markers were requested to be positive, but again the rate of positive benign samples increased to as much as 41%. Although no normal patients were exam ined, a considerable - and unacceptable - number of patients, up to 31 %, with endometriosis or dermoid cysts was positive. Using lower cut-off limits for the marker concentrations only worsened these figures. Requesting both or at least two markers out of two or three to be positive definitely reduced the number of ‘false-positive’ benign tumours and endometriosis to in deed very low levels. However, the sensitivity for malig nant tumours was reduced concomitantly to levels where marker determinations would be of little use. In all, a combined use of two or three markers demand ing just one marker to be positive seems to confuse the interpretation more than it adds useful information to the preoperative management of ovarian tumours. The per centage of marker-positive patients with benign disease makes this concept unacceptable in a screening context. A concept of (at least) two markers being positive leads to a considerable reduction in the percentage of positive be nign cases; however, an unacceptable decrease in the sen sitivity for malignant tumours is also found. This combi nation, therefore, can be recommended neither for preop erative use nor for screening purposes. The use of one marker - either CA-125 or TPA, but not PLAP - appears to give a less confusing and probably more effective information preoperatively. The role of the markers in a screening context remains to be seen.
Acknowledgements This study was supported by grants from the Danish Cancer Soci ety. Appreciation is expressed to the departments of pathology at the hospitals involved for supplying us with specimens for revision. Kits for determination of TPA and PLAP were kindly supplied by Sangtec Medical, and kits for determination of CA-125 were kindly supplied by Abbott Diagnostics.
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recent years several authors have found that high values ofCA-125 are found in advanced stages of these tumours [18,19-21 ], and Cruickshank et al. [ 18] reported high val ues also in nonepithelial malignant ovarian tumours. We did not find any significant differences in marker concen trations of CA-125 in the different types of tumours inves tigated. Although the number of mucinous and mesonephroid tumours was small in our study, CA-125 was well above the chosen cut-off limits in the case of advanced stage. No differences between the concentra tions of TPA in the different types of epithelial tumours were found in this study, as in the study by Inoue et al.
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