Clinical Cancer Research Vol. 6, 4069-4072, October 2000
© 2000 American Association for Cancer Research
Molecular Oncology, Markers, Clinical Correlates |
Evaluation of Serum KL-6, a Mucin-like Glycoprotein, as a Tumor Marker for Breast Cancer
Yoshinari Ogawa1,
Tetsuro Ishikawa,
Katsumi Ikeda,
Bunzo Nakata,
Tetsuji Sawada,
Kana Ogisawa,
Yasuyuki Kato and
Kosei Hirakawa
First Department of Surgery [Y. O., T. I., K. I., B. N., T. S., K. O., K. H.] and Department of Oncology, Institute of Geriatrics and Medical Science [Y. K.], Osaka City University Medical School, Osaka 545-8585, Japan
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ABSTRACT
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The utility of serum
KL-6 as a tumor marker for breast cancer was evaluated in this study.
The sera from 146 patients with breast cancer, 13 with benign breast
disease, and 108 healthy individuals were measured for KL-6 titer using
a sandwich enzyme immunoassay method. Carcinoembryonic antigen (CEA)
and carbohydrate antigen 15-3 (CA15-3) titers were also tested in the
same sera from the patients. The mean KL-6 titer of patients with
primary breast cancer was 673 units/ml, which was significantly higher
than that of benign and healthy individuals (P =
0.037 and P < 0.0001, respectively). The titer of
patients with relapsed breast cancer was 1964 units/ml, which was also
higher than that of primary cancer (P = 0.013).
KL-6 titer was related to tumor stage, distant metastasis, and relapse
site (P = 0.0053, P < 0.0001,
and P = 0.0251, respectively). Using the cutoff
value of 467 units/ml, the sensitivity of KL-6 was 31% for primary
breast cancer (16% for stage I and 29% for stage II) and 73% for
relapsed breast cancer (50% for local relapse and 89% for distant
relapse). The specificity was 92%. The sensitivity of KL-6 was higher
than that of CA15-3 and CEA. Combination of the three markers, followed
by KL-6 and CEA, raised the sensitivity for primary breast cancer.
Single use of KL-6 demonstrated a higher sensitivity than in each
combination for relapsed breast cancer. In conclusion, serum
KL-6 may be helpful for clinical use as a tumor marker for breast
cancer, and it may play an important role, especially in the
surveillance of disease relapse.
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INTRODUCTION
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In various markers, the best single and established marker for
breast cancer is
CA15-3,2
followed
by CEA (1)
. Nevertheless, the American Society of Clinical
Oncology (2)
has stated in the Clinical Practice
Guidelines for the Use of Tumor Markers that neither CA15-3 nor
CEA is recommended for routine use for diagnosing breast cancer, and
new powerful markers for breast cancer are needed.
KL-6 is a high molecular weight, mucin-like glycoprotein that was
originally discovered as a circulating pulmonary
adenocarcinoma-associated antigen (3
, 4)
. Expression of
KL-6 has been observed in both various carcinomas and normal cells
(3)
, and it is thought to be released into the serum in
cases of cell damage (5)
. Biochemical properties of KL-6
are similar to those of other MUC-1 mucins (6)
. KL-6 does
not directly reflect events in the process of tumorigenesis. The
clinical value of serum KL-6 has been recognized as a marker for the
disease activity of interstitial pneumonitis (5
, 7)
. Kohno
et al. (3)
has reported that serum KL-6 levels
were also elevated in breast and pancreatic cancers.
In this study, we analyzed the serum KL-6 titer in breast cancer and
evaluated the clinical value of KL-6 as a tumor marker for breast
cancer.
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MATERIALS AND METHODS
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Clinical Materials.
Sera from 146 female patients with breast cancer treated at the First
Department of Surgery, Osaka City University Hospital, were analyzed.
Sera were obtained before therapy. Among the patients, 131 had primary
invasive breast cancer, and 15 had relapsed cancer. The mean age of the
patients was 54 years (range, 28–81 years). None of the patients had
other malignancies or active pulmonary disease. The characteristics of
the patients are summarized in Table 1
.
Clinicopathological factors were abstracted from the initial surgical
pathology reports. Staging was determined according to the TNM
Classification of Malignant Tumors set by the Union International
Contre Cancer (8)
. Histological grade was analyzed
according to the Scarff-Bloom-Richardson histological grading system
(9)
. The survival period was defined as the interval
between the time point at which the serum sample was obtained and the
date of first relapse or death.
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Table 1 %Correlation of serum KL-6 titer to
clinicopathological factors in primary breast cancer and relapse sites
in cases of relapsed breast cancer
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Serum samples from 108 healthy females, who had voluntarily cooperated
in a blood donation campaign in Osaka, with a mean age of 31 years
(range, 18–63 years) served as healthy controls. Sera from 13 patients
with benign breast disease (2 phyllodes tumors, 4 intraductal
papillomas, 4 mastopathies, 1 fibroadenoma, 1 adenoma, and 1 mastitis)
treated in our hospital were also analyzed for KL-6. Additionally, the
titers of CEA and CA15-3 were measured in the same sera from patients.
Assay for Serum KL-6, CEA, and CA15-3.
The serum samples were stored at -40°C until use. The samples were
assayed for KL-6 with a sandwich-type enzyme-linked immunoassay using
the Eitest KL-6 kit (Sanko Jyunyaku Co. Ltd., Tokyo, Japan) according
to the manufacturer’s instructions. The kit was composed of mouse
monoclonal antibody against KL-6 antigen. The coefficient of variation
of the kit was <10% in each of four examinations using the 2.5 and 10
units/ml KL-6 control samples. In brief, 20 µl of serum samples
diluted to 1:201 using Tris-buffer with bovine albumin or 20 µl of
known concentration KL-6-controls, with an addition to 100 µl of Tris
buffer with normal rabbit serum, were pipetted into the wells of the
microplate, which had been precoated with a mouse monoclonal antibody
for KL-6. After mixing, the plate was covered with plate sealer and
incubated at room temperature for 2 h. Each well was washed
thoroughly three times with 0.85% NaCl. One hundred µl of
appropriately diluted antibody to KL-6 conjugated to horseradish
peroxidase was pipetted into each well. After incubation at room
temperature for 1 h, wells were washed, and 100 µl of substrate
with 2,2'-azino-bis-3-ethyl-benzo-thiazoline-6-sulfonic acid were added
for the color development. After incubation at room temperature for 30
min, the color reaction was terminated by the addition of 100 µl of
stop solution. The absorbance of each well was determined by a MTP-120
micro plate reader (Corona Electric Co., Ibaragi, Japan) set to 405 nm.
The concentration of each serum sample was determined from the standard
curve using the KL-6controls.
CEA was measured by a counting immunoassay using a commercially
available kit (Ranream CEA; TOA Medical Electronics, Kobe, Japan) in
conjunction with an automated PAMTA-100 analyzer (TOA Medical
Electronics). CA15-3 was measured by a two-step sandwich
immunoradiometric assay using a commercially available kit [CA15-3 RIA
kit (TFB), Fujirebio Diagnostics, Inc., Malvern, PA).
Statistical Analysis.
The assays were read without knowledge of case and control status. The
correlation coefficient was assessed by simple linear regression
analysis. The Mann-Whitney U test and Kruskal Wallis one-way
analysis were used for the comparison. Survival analysis was estimated
by the Kaplan-Meier method and examined by the log-rank test.
P < 0.05 was considered to be statistically
significant.
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RESULTS
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Analyses of KL-6 Titers.
The linear regression analysis of all sera from the cases and controls
did not show a correlation between KL-6 titer and age
(r = 0.142; P = 0.024), although there
was a sizeable difference between the ages of the cases and controls.
The mean ± SD of serum KL-6 titer in healthy controls was
267 ± 200 units/ml (range, 0–1291 units/ml), and that of
patients with benign disease was 297 ± 58 units/ml (range,
200–395 units/ml). The titer in patients with breast cancer ranged
from 10 to 10297 units/ml. It was 673 ± 1310 units/ml in primary
cancer and 1964 ± 2915 units/ml in relapsed cancer (Fig. 1)
. The titers in patients with primary
cancer were significantly higher than those of healthy controls and
patients with benign disease (P < 0.0001 and
P = 0.037, respectively), and the difference in KL-6
titers between patients with primary and relapsed cancer was
significant (P = 0.013).
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Fig. 1. Distribution of individual serum KL-6 values in
healthy controls, patients with benign breast tumors, patients with
primary breast cancer, and patients with relapsed breast cancer. Data
are presented as upper and lower quartile and range
(box), median value (vertical line), and
the middle 90% distribution (whisker line). The
differences were analyzed by the Mann-Whitney U test.
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The correlation of serum KL-6 titer to clinicopathological factors in
primary breast cancer and relapse sites in relapsed cancer is shown in
Table 1
. The titer of KL-6 increased according to tumor size
(P = 0.0053). The titers were significantly higher in
patients with distant metastasis or stage IV disease (P < 0.0001, both), and the titers in patients relapsed at distant sites
were significantly higher than those in patients who had relapsed at
loco-regional sites (P = 0.0251).
One hundred thirteen curatively operated patients were divided into
high and low KL-6 groups using a serum value of 673 units/ml, which was
the mean titer in patients with primary breast cancer. There was no
difference of disease-free and overall survival rates between the two
groups in the median follow-up term of 74 months (P =
0.70 and P = 0.45, respectively).
Comparison of Serum KL-6 with CEA and CA15-3.
The cutoff value of KL-6 was determined as 467 units/ml, which was the
mean of healthy individuals plus the SD. The cutoff values of CEA and
CA15-3 recommended by the manufacturers are 6.5 ng/ml and 30 units/ml,
respectively. Cases and controls were divided into positive and
negative groups using these cutoff values.
Ten of 108 healthy controls had positive KL-6 levels. Among the 13
patients with benign disease, none had a positive KL-6 level, whereas
one had both positive CEA and CA15-3. The specificity of KL-6 for
breast cancer was 92%. Table 2
shows the
sensitivity of each marker. For primary breast cancer, the sensitivity
of KL-6 was 31%. It was higher than that of CEA and CA15-3. In staging
analysis, the sensitivity of KL-6 was next to that of CEA in stage I,
and it was higher than those of CEA and CA15-3 in other stages. The
combination of three markers had the highest sensitivity in each stage,
and it was followed by the combination of KL-6 and CEA. For relapsed
breast cancer, the sensitivity of KL-6 was 73%. It was 50% in
patients who had relapsed at the loco-regional site and 89% in
patients who had relapsed at distant sites. None of the combinations
was more sensitive than KL-6 alone.
|
DISCUSSION
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Our results revealed that serum KL-6 titer was elevated in
patients with breast cancer. These results support the report by Kohno
et al. (3)
. The difference between the age of
the cases and controls should not affect the elevation of KL-6 titer in
breast cancer, because our results revealed that age did not correlate
with the titer. KL-6 titer was elevated according to tumor growth,
distant metastasis, and staging, and the titer was markedly elevated in
patients with relapsed disease, especially in patients who had relapsed
at distant sites. These results suggest that an increase in the bulk of
a tumor might raise the serum KL-6 level.
Tumor markers are expected to play a role in the differential
diagnoses, early detection of cancer, prognostic predictions,
monitoring of treatment efficacy, and surveillance of disease relapse
(1)
. For differential diagnosis, the sensitivity and the
specificity of KL-6, which were 31 and 92%, may not be satisfactory.
Several markers occasionally increase in healthy subjects, most often
in individuals with benign disease of the liver (10)
. KL-6
has been reported to increase in patients with active pulmonary
tuberculosis and interstitial pneumonitis, although it did not in
patients with hepatic disorders or other inflammatory diseases
(3
, 5
, 7) . These effects must be taken account to avoid
false-positive diagnoses. Of interest was the combination of markers to
improve the diagnostic potential of tumor markers (1
, 11)
.
Our results revealed that a combination of three markers, followed by
KL-6 and CEA, efficiently raised the sensitivity for primary breast
cancer. For a marker to be valuable in screening for cancer, it would
have to detect early stages of cancer in asymptomatic populations.
Summaries of multiple studies have shown that the sensitivity of CEA in
patients with stages I and II breast cancer was 10 and 19%, and that
of CA15-3 was 9 and 19%, respectively (2)
. The
sensitivity of KL-6 was 16% for stage I disease and 29% for stage II.
Although it was higher than those summarized for CEA and CA15-3, it was
not sufficient to use for early diagnosis in breast cancer. The
differential diagnosis and detection of breast cancer in its early
stages are comparatively easy, because most breast tumors are palpable
and can be approached, and for nonpalpable small tumors, aspiration
biopsy combined with ultrasonography or mammography (12)
is useful. The present results did not reveal a prognostic
predictive value of serum KL-6. Tumor markers reflect tumor volume;
increasing marker levels may be consequently related to poor prognosis.
However, the main approach for achieving a positive clinical outcome
remains an appropriate treatment response. Therefore, the first-point
titer may not always predict prognosis. There are many useful
prognostic predictors, including lymph node status, histological grade,
and molecular biological markers, in breast cancer
(13, 14, 15, 16)
. The role of prognostic prediction may be limited
to such factors instead of tumor markers. The issue of monitoring
treatment efficacy could not be addressed in this study. It must be
examined in future studies. As indicators of disease relapse, the
sensitivities of CEA and CA15-3 in other reports were summarized as 50
and 67%, respectively (2)
. Low sensitivities of CEA and
CA15-3 in our results may not reveal their actual potential because the
number of relapsed patients was small in our study. However, the
sensitivity of KL-6 was higher than those of CEA and CA15-3, and KL-6
can detect the relapsed cases with negative CEA and CA15-3 in our
study. The sensitivity of KL-6 was up to 89% in patients who had
relapsed at distant sites. Compared with cases of relapse at
loco-regional sites, those who had relapsed at visceral organs are
difficult to screen and diagnosis. Therefore, KL-6 could play an
important role for surveillance of disease relapse in these cases.
Among the five roles expected for tumor markers, monitoring treatment
efficacy and surveillance of disease relapse may be most important for
breast cancer. From our results, serum KL-6 might be appropriate for
clinical usage as a marker for breast cancer.
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FOOTNOTES
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 To whom requests for reprints should be
addressed, at First Department of Surgery, Osaka City University
Medical School, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585, Japan.
Phone: 6-6645-3838; Fax: 6-6646-6450. 
2 The abbreviations used are: CA15-3, carbohydrate
antigen 15-3; CEA, carcinoembryonic antigen.
Received 5/15/00;
revised 7/31/00;
accepted 8/ 8/00.
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ABSTRACT
INTRODUCTION
