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Molecular Oncology, Markers, Clinical Correlates |
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
| ABSTRACT |
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| INTRODUCTION |
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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.
| MATERIALS AND METHODS |
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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 manufacturers 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.
| RESULTS |
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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.
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| DISCUSSION |
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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.
| FOOTNOTES |
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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.
| REFERENCES |
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B. Vahid and P. E. Marik Pulmonary Complications of Novel Antineoplastic Agents for Solid Tumors Chest, February 1, 2008; 133(2): 528 - 538. [Abstract] [Full Text] [PDF] |
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H. Sato, M.E.J. Callister, S. Mumby, G.J. Quinlan, K.I. Welsh, R.M. duBois, and T.W. Evans KL-6 levels are elevated in plasma from patients with acute respiratory distress syndrome Eur. Respir. J., January 1, 2004; 23(1): 142 - 145. [Abstract] [Full Text] [PDF] |
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