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Articles |
Departments of
1
Obstetrics and Gynaecology,
2
Clinical Chemistry,
3
Surgery, and
4
Medical Oncology, Academic Hospital Vrije Universiteit, De Boelelaan 1117, NL-1081 HV Amsterdam, The Netherlands.
a Author for correspondence. Fax +31-20-4444811; e-mail kenemans{at}azvu.nl
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Key Words: indexing terms: polymorphic epithelial mucin • mucin glycoproteins • tumor markers • chemiluminescence immunoassay • monoclonal antibodies • method comparisons • ROC curve analysis
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The first commercially available PEM assay is the Centocor (Malvern, PA) CA 15-3TM RIA (5). The assay uses two MAbs, directed to epitopes located on the tandem repeat forming the peptide core of the extracellular domain of the PEM molecule. One MAb, DF3 (6), is directed to an epitope in the DTRPAGS region of the tandem repeat; the other MAb, 115D8 (7), is directed to a peptide carbohydrate epitope on the same repeat (8). To improve the analytical performance of the Centocor CA 15-3 RIA, researchers developed automated assays that were based on ELISA or microparticle enzyme immunoassay (MEIA) technologies.
The Chiron Diagnostics (formerly Ciba Corning, E. Walpole, MA)
ACSTM BR, a single-determinant assay, is based on MAb
B27.29 (9), which recognizes the 8-amino-acid sequence
(SAPDTRPA) within the 20-amino-acid tandem repetitive sequence of the
mucin core (10). This epitope overlaps in part with that
recognized by MAb DF-3 incorporated in the CA 15-3 assays (Fig. 1
).
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In the current study we assessed the clinical and technical performance of the newly developed fully automated ACS BR chemiluminescence serum immunoassay and compared this assay with the original Centocor CA 15-3 RIA and with two automated PEM assay systems of different design: the Abbott (Abbott Park, IL) IMx® CA 15-3 MEIA and the Boehringer Mannheim (Tutzing, Germany) Enzymun-Test® CA 15-3.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All clinical charts were reviewed for diagnosis, histology, and staging; only patients who were thoroughly documented were included in the current study. Procedures followed were in accordance with the Helsinki declaration of 1975, as revised in 1983, and in accordance with the guidelines for research of our institute.
pem assays
ACS BR.
The ACS BR assay is a competitive,
chemiluminescence immunoassay for detecting PEM in serum. Purified
antigen, isolated from ZR-75-1 breast cancer cells and immobilized on
paramagnetic microparticles, competes for binding to MAb B27.29 with
the MUC1 gene-derived antigen present in calibrators and
samples (9). The MAb is conjugated to acridinium ester as
a tracer (BR Lite reagent), and the signal is measured by a
luminometer. The assay is run on the Chiron ACS:180®
system, a fully automated, random-access immunoassay system, for which
no predilution of samples is required. The ACS BR assay is calibrated
against a master curve. The concentrations of the seven calibrators
used in the master curve range from 0 to 475 kU/L (U = arbitrary
units); the range is extended to 2250 kU/L by utilizing the autodilute
feature of the ACS:180. The bar-coded master curve information is
supplied with each new lot number of solid phase and Lite reagent. The
system requires a two-point recalibration every 7 days to adjust it to
the master curve. For this purpose, purified breast cancer antigen at
low (~29 kU/L) and high (~250 kU/L) concentration in a matrix of
equine serum is used.
The ACS BR assay requires 25 µL of sample, which is dispensed into a reaction cuvette simultaneously with the paramagnetic particles and Lite reagent. After incubation at 37 °C for 7.5 min, during which the competition reaction takes place, the microparticles are separated and washed twice. Starter reagent is then added and the emitted light is measured with a luminometer. The relative light units measured are in inverse correlation to the concentration of PEM in the tested sample. The ACS:180 system can be operated either in a batch-processing mode, if large numbers of samples are to be assessed, or in a random-access mode, for single analyses. In both cases, the analyzer is capable of testing 180 samples per hour.
Centocor CA 15-3.
The CA 15-3 RIA is a nonautomated
heterologous double-determinant RIA with the capture antibody MAb 115D8
(raised against human milk fat globule membranes) coated onto a solid
phase of plastic beads and the tracer 125I-labeled MAb DF3
(raised against a membrane-enriched fraction of a human breast
carcinoma (5)). In a first step, a specimen is prediluted
1:50 in kit diluent and then is added to the solid phase. After a 2-h
incubation, unbound material is removed by washing. In a second step,
125I-labeled MAb DF3 is added and, after a 3-h incubation
and washing, the bound radioactivity is quantified with a gamma
counter. The signal is proportional to the concentration of PEM in the
specimen.
Enzymun-Test CA 15-3.
The Enzymun-Test CA 15-3 assay
(Boehringer Mannheim) is a heterologous enzyme-linked sandwich
immunoassay using streptavidin-coated tubes to which antigen is bound
after capture by biotinylated MAb 115D8. Peroxidase-labeled DF3 is used
as tracer antibody. The assay was performed on the fully automated
Enzymun-Test system ES 300 (11).
IMx CA 15-3.
The Abbott IMx CA 15-3 assay is a
heterologous MEIA in which MAb 115D8-coated microparticles are mixed
and incubated with prediluted patients' sera. Alkaline
phosphatase-conjugated DF3 tracer antibody is added, which then
complexes with the antigen bound to MAb 115D8. The assay is run on an
automated IMx test system (12).
All assays were performed in one laboratory according to the manufacturers' instructions. The laboratory staff had no knowledge of the corresponding clinical data while performing this analysis.
technical evaluation
The analytical performance of the ACS BR was assessed by
evaluating (a) the within-run imprecision (three samples, at
low, middle, and high PEM concentrations, run 20 times in duplicate);
(b) the between-run imprecision (the same three samples run
on 23 consecutive working days); and (c) the linearity on
dilution (analysis of seven samples with concentrations of 222-1012
kU/L, diluted stepwise as many as eight times).
statistical considerations and data analysis
The agreement between the four assays was evaluated by
least-squares linear regression analysis (13) with SPSS-PC
software (SPSS, Chicago, IL). The relative difference between the
results obtained by the ACS BR assay and by each of the three CA 15-3
assays taken individually was calculated as [(CA 15-3 assay value
- ACS BR value)/CA 15-3 assay value] x 100%. The upper limit of the
reference interval for healthy individuals was determined, and the
diagnostic value of each assay was also evaluated by receiver-operating
characteristic curve analysis (14). Monitoring graphs
obtained from individual patients with all four assays were compared.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Within-assay imprecision was lowest in the high-concentration
range (CV 2.2%) and highest in the median range (CV 3.9%). Interassay
precision experiments gave CVs ranging from 5.6% to 3.9% for the low-
and high-concentration control samples, respectively.
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ACS BR linearity on dilution.
The correlation
coefficients for the seven serially diluted serum samples ranged from
0.8615 to 0.9893, with recoveries ranging from 95% to 105% (Fig. 2
).
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Correlation between the assays.
As Table 2
shows, the linear regression analysis correlation coefficients
(r) for ACS BR vs Centocor CA 15-3 range from 0.8567 in
healthy controls to 0.9845 in all cancer patients. For Enzymun-Test CA
15-3 vs ACS BR, r ranged from 0.8763 to 0.9781 in healthy
controls and in all cancer patients, respectively. For IMx CA 15-3 vs
ACS BR, r ranged between 0.9070 in healthy controls to
0.9818 in pretreatment breast cancer patients.
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The relative difference between the results obtained in benign breast
disease and in cancer patients with the ACS BR assay and each of the
three other PEM assays taken individually is illustrated in Fig. 3
. The trend of relative differences was closest to zero over the
whole range of values when comparing ACS BR with Centocor CA 15-3,
whereas IMx CA 15-3 gave lower PEM values than ACS BR throughout this
range. In the low-concentrations range, ACS BR gave higher results than
Enzymun-Test CA 15-3, whereas for high PEM concentrations ACS BR
results were lower than Enzymun-Test CA 15-3 results (Fig. 3
).
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clinical evaluation
Cutoff values.
According to the 95th
percentile of results for healthy women, the cutoff values (kU/L) for
each assay were as follows: ACS BR, 39; Centocor CA 15-3, 35;
Enzymun-Test CA 15-3, 30; IMx CA 15-3, 26. These values were calculated
from the results obtained from 250 healthy women, after exclusion of 3
samples with outlier ACS BR values, which were reanalyzed separately
(Table 3
). ACS BR assay results for these three samples were discordant
with those obtained by the CA 15-3 assays and were therefore not
included in the calculations. Analysis performed on samples obtained
from the same women in two subsequent years again gave discordant
above-normal ACS BR test results. All samples were rerun by ACS BR at
Chiron Diagnostics, which show less-discordant ACS BR results in all
three series. Western blot analysis revealed low concentrations of PEM
in all samples. Sample 1 (Table 3
), first year, was serially diluted to
test for linearity on dilution and for percentage of recovery. Recovery
in the 1:2 dilution was 96%, but results for further step-wise
dilutions were not linear and showed increasingly lower recoveries
(i.e., 65%, 57%, 33%, and 8%, respectively). Possible interfering
factors such as high concentrations of protein or triglycerides were
not found in the samples.
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Benign breast diseases.
Measurements in sera obtained
before treatment from patients with benign breast disease (Table 4
) revealed median assay values ranging from 15 kU/L for IMx CA
15-3 to 19 kU/L for ACS BR. At the cutoff value of 30 kU/L advised by
the manufacturers, the lowest number of false-positive test results was
obtained with the IMx CA 15-3 assay (1.5%), whereas the highest number
of false positives (6.1%) was found with both the Centocor CA 15-3 and
the Enzymun-Test CA 15-3 assays. The number of false-positive test
results with ACS BR was 4.5%. At the cutoff values based on the
95th percentile of results for healthy controls determined
in this study, the ACS BR assay produced no false-positive test
results, whereas in the other assays the rate of false positivity
ranged between 3.0% for Centocor CA 15-3 and 7.6% for Enzymun-Test CA
15-3.
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Malignant breast disease.
In sera obtained before
treatment from patients with breast cancer, the highest median PEM
serum concentration was found with Centocor CA 15-3 (24 kU/L) and the
lowest with IMx CA 15-3 (16 kU/L) (Table 4
). At a cutoff of 30 kU/L,
the highest true-positive rate was reached with the Centocor CA 15-3
assay (37%), the lowest with the IMx CA 15-3 (7.1%). The
corresponding values for ACS BR and Enzymun-Test CA 15-3 were 18.9%
and 12.6%, respectively. Utilizing the 95th percentile of healthy
controls obtained in the current study, however, we obtained
true-positive rates ranging between 26% for the Centocor CA 15-3 and
7.9% for the ACS BR. The diagnostic value of the assays for detecting
breast cancer, with a cutoff value based on the 95th percentile of
results for healthy subjects, is given in Table 5
.
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Receiver-operating characteristic curves comparing all four assays for
breast cancer patients vs healthy controls and patients with benign
breast disease are given in Fig. 4
. The greatest area under the curve was found with the Centocor
CA 15-3 assay (0.64); all three automated assays had essentially
similar areas (~0.5), but these were not significantly different from
that for the Centocor assay. Table 4
lists results according to stage
for breast cancer patients sampled before treatment (n = 127).
Serum concentrations for all four assays in node-negative and
node-positive patients are also given. Comparison is made between serum
PEM concentrations in breast cancer patients with recurrent or
progressive disease and a high tumor load, and those in breast cancer
patients with no evidence of disease. The lowest median pretreatment
concentrations determined by ACS BR were found in those patients having
carcinoma in situ of the breast (median 14 kU/L); the highest were in
the patients with metastatic breast disease (median 111 kU/L). No
significant difference in ACS BR-determined PEM serum concentrations
was seen in relation to the presence or absence of nodal metastasis.
However, the difference in ACS BR results for patients with a high
tumor load (median 294 kU/L) and those having no evidence of disease
(median 25 kU/L) was significant (P <0.0001). Similar
results were obtained with the CA 15-3 assays.
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Other malignancies.
The highest median serum
concentrations for all four assays were obtained in patients with
ovarian cancer, for whom values ranged between 38 kU/L by ACS BR and 26
kU/L by IMx CA 15-3 (Table 6
). The lowest serum concentrations were found in patients with
cervical cancer, the median values ranging between 15 kU/L by ACS BR
and 12 kU/L by Enzymun-Test CA 15-3 and IMx CA 15-3.
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Breast cancer follow-up.
Serial serum samples were
obtained during follow-up in 30 patients with breast cancer. PEM serum
concentrations measured during the course of disease revealed a highly
similar pattern for all four assays. Some examples are shown in Fig. 5
. Increasing concentrations of this marker correlated with
recurrent or progressive disease, whereas decreasing values indicated
regressive disease. In none of the patients with no evidence of disease
were assay results above normal.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Among the PEM assays included in this study, the one evaluated most extensively in pretreatment sera from patients with breast tumors is the Centocor CA 15-3 RIA. Pons-Anicet et al. (15) found above-normal results by Centocor CA 15-3 in 27% of patients with local disease, compared with 54% in patients presenting with positive lymph nodes and 91% in those with metastatic disease. In patients with metastatic carcinoma, the highest values reported by Centocor CA 15-3 were seen in patients with liver and visceral metastases; the lowest were those for patients with skin and soft tissue metastases (16)(17)(18)(19). High pretreatment serum concentrations of PEM are strongly indicative of the presence of distant metastases and can, therefore, be an important tool in the choice of primary therapy, supporting confirmation or exclusion of distant metastases before therapy mode decisions are made (20).
As a double-determinant, nonautomated RIA, the Centocor CA 15-3 assay is time- and sample-consuming and has environmental side effects. Therefore, faster and easier to handle CA 15-3 assays, run on automated systems, have been developed. Further, PEM-based serum immunoassays, which detect other epitopes on the PEM molecule, have also been developed in an attempt to improve the clinical performance of the tumor marker by increasing its sensitivity for breast cancer. Examples of such assays are assays for mucin-like carcinoma-associated antigen (21), based on MAb b-12, and CA M29 and CA M26 (22), which use MAbs M38, M29, and M26 for detection of epitopes on the same PEM molecule. The recently developed ACS BR serum immunoassay uses a single MAb, B27.29 (9), which recognizes a specific 8-amino-acid sequence within the 20-amino-acid tandem repeating sequence of the core protein of PEM (10).
In the present study comparing ACS BR with the original, manual
Centocor CA 15-3 RIA and two established automated CA 15-3 assays, the
analytical performance of the ACS BR assay appears to be excellent,
especially in the higher PEM concentration range (within-assay CV 2.2%
and between-assay CV 3.9%). Dilution experiments showed recoveries of
between 95% and 105% for concentrations ranging from 222 to 1012
kU/L. ACS BR results for patients with breast cancer correlated best
with the original Centocor CA 15-3 RIA results, especially in the high
concentration range (r = 0.9845). The trend of relative
differences was closest to zero over the whole range of values for the
comparison of ACS BR with Centocor CA 15-3 (Fig. 3
).
The reference value for Centocor CA 15-3 as obtained in healthy women
is higher than that recommended by the manufacturer, confirming results
of a previous study (23). Among 253 healthy women, 3
showed discordant ACS BR serum results for which we found no
explanation (Western blot analysis revealed normal PEM concentrations
in these sera). The discord seems to be specific to the subject rather
than to sample, because results obtained in the samples taken from the
same women 2 and 3 years later were again discordant (Table 3
).
Inasmuch as CA 15-3 and ACS BR are not suited for screening purpose,
this problem is predominantly of scientific interest. Further research
on the subject is ongoing at Chiron Diagnostics and at our institute.
No circulating antibodies to MUC-1 were present in these samples.
The clinical performance of the ACS BR assay, which uses a MAb
detecting an epitope on the PEM molecule that overlaps with the
epitopes recognized in the other assays, was equal to that of the CA
15-3 assays. In sera from patients with malignancies other than breast
cancer, the highest values were found in patients with ovarian cancer,
which is in concordance with earlier reports (24). High
results were obtained in breast cancer patients with advanced stage of
disease and high tumor load, low results in breast cancer patients with
an early stage of disease or no evidence of disease and in patients
with benign breast lesions. Our findings confirm the study by Dnistrian
et al. (25), in which BR27.29, a competitive inhibition
RIA using MAb B27.29, was evaluated in breast cancer patients; those
authors concluded that increased concentrations determined with the
BR27.29 assay, as with CA 15-3 (20), are associated
predominantly with late-stage disease. Although the Centocor CA 15-3
assay showed a slightly better performance than the automated assays,
the clinical value of PEM-detecting serum immunoassays for the
diagnosis of breast cancer is limited (Table 5
, Fig. 4
).
In the breast cancer patients followed during the course of disease, we observed similar marker patterns for all four assays. As we saw when monitoring patients during the course of disease, the trend of the values reflects disease activity more accurately than does the absolute value of the marker; we therefore recommend that one and the same assay be used in follow-up of individual patients. Further prospective studies should reveal the clinical significance of ACS BR and CA 15-3 assays in the early detection of recurrent disease in breast cancer patients.
In summary, ACS BR is a fast, reliable, and reproducible assay for quantifying PEM in serum of breast cancer patients. It has an excellent analytical performance and the advantage of an automated easy-to-handle assay system. Although based on a single MAb directed to an overlapping epitope on the PEM molecule, ACS BR correlates better with the original Centocor CA 15-3 RIA for samples from breast cancer patients than do the automated CA 15-3 assays included in this study.
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Acknowledgments |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following articles in journals at HighWire Press have cited this article:
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  B. Handy The Clinical Utility of Tumor Markers Lab Med, February 1, 2009; 40(2): 99 - 103. [Abstract] [Full Text] [PDF]
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 S. P. Treon, P. Maimonis, D. Bua, G. Young, N. Raje, J. Mollick, D. Chauhan, Y.-T. Tai, T. Hideshima, Y. Shima, et al. Elevated soluble MUC1 levels and decreased anti-MUC1 antibody levels in patients with multiple myeloma Blood, November 1, 2000; 96(9): 3147 - 3153. [Abstract] [Full Text] [PDF]
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 S. von Mensdorff-Pouilly, A. A. Verstraeten, P. Kenemans, F. G. M. Snijdewint, A. Kok, G. J. Van Kamp, M. A. Paul, P. J. Van Diest, S. Meijer, and J. Hilgers Survival in Early Breast Cancer Patients Is Favorably Influenced by a Natural Humoral Immune Response to Polymorphic Epithelial Mucin J. Clin. Oncol., February 1, 2000; 18(3): 574 - 574. [Abstract] [Full Text] [PDF]
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 M. Gion, R. Mione, A. E. Leon, and R. Dittadi Comparison of the Diagnostic Accuracy of CA27.29 and CA15.3 in Primary Breast Cancer Clin. Chem., May 1, 1999; 45(5): 630 - 637. [Abstract] [Full Text] [PDF]
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, ACS BR;
*, Enzymun-Test CA 15-3; +, IMx CA 15-3; 
, Centocor CA 15-3) as
measured during the course of disease in four breast cancer patients.