Quantitative Analysis of Keratin 18 in the Urine of Patients with Bladder Cancer

0022-534 7/88/1402-0436$02.00/0 Vol. 140, August

THE JOURNAL OF UROLOGY

Copyright © 1988 by The Williams & Wilkins Co.

Printed in U.S.A.

QUANTITATIVE ANALYSIS OF KERATIN 18 IN THE URINE OF PATIENTS WITH BLADDER CANCER WILLIAM C. BAKER, RALPH DEVERE WHITE, PAUL V. ROSSITTO, BYUNG HEE MIN ROBERT D. CARDIFF*

AND

From the Department of Patlwlogy, University of California Sclwol of Medicine, Davis, California and the Department of Urology, University of California, Davis Medical Center, Sacramento, California

ABSTRACT

The level of urinary keratin was compared in patients with and without bladder cancer using the newly developed IRMAK-18. Patients with bladder cancer had higher levels of keratin (74.6 ± 146.9) than normals (2.5 ± 4.7). When the upper limit of normal was set at keratin <20 ng./ml., the test was highly specific (98%) for bladder cancer but had a sensitivity of only 46%. Adjusting to keratin <10 ng./ml. increased the sensitivity to 57% but decreased the specificity to 91 %. Elevated keratins were associated with stage and grade, aneuploidy and disease history. The IRMAK-18 assay for keratin may be useful for monitoring the clinical progress of some patients. (J. Ural., 140: 436-439, 1988) Keratins are an essential part of the protein cytoskeleton of epithelial cells. 1 • 2 Nineteen distinct human keratins have been described in acidic and basic groups with molecular weights ranging from 40 kd to 67 kd. 1 Four keratins, numbers 7, 8, 18 and 19 have been detected in normal and neoplastic urothelium. 3 The antigen known as tissue polypeptide antigen (TPA) has recently been found to contain keratins.4 Elevated levels of TPA have been found in the serum and urine of patients with bladder cancer. 5 However, elevated levels of TPA have also been registered in patients with sarcomas and lymphomas which are not expected to produce keratin. 6 • 7 Therefore, the specificity of the assay is open to question. The issue could be resolved by evaluating keratins in such patients using an assay which is more specific for keratin. Recently, we developed an immunoradiometric assay for keratin that primarily detects extracellular keratin 18 (IRMAK-18) in the culture media from human breast cancer cell cultures. 8 The preceding article reports the application of this assay to the detection of keratin in voided urine of healthy volunteers. 13 The present study reports the quantitative comparison of keratin in the voided urine of normal volunteers, patients with nonmalignant urological disease and patients with transitional cell carcinoma of the urinary bladder. Since many of the patients were subjects in an ongoing DNA flow cytometry study, the results from both arms of the study could be correlated. MATERIALS AND METHODS

Urine samples. Three hundred and seven voided urine specimens from 268 subjects were received and assigned a number so that analysis could be carried out in an unbiased fashion. One hundred thirty separate specimens from freshman medical students and other healthy volunteers were submitted for IRMAK-18 analysis alone. 13 Their average age was 26 with a range of 22 to 38 years. One hundred seventy-seven patient specimens were submitted for both IRMAK-18 and flow cytometric evaluation. The 1 77 patient samples were from patients of the Department of Urology. Group I contained samples from 26 patients whose age ranged Accepted for publication March 15, 1988. * Requests for reprints: Dept. of Pathology, School of Medicine, University of California, Davis, CA 95616. Supported by a grant from Triton Biosciences Inc., Alameda, CA, and NCI grant U01CA41034. 436

from 38 to 89 years with an average of 69 years. They had active transitional cell carcinoma, undergoing either transurethral resection or radical cystectomy for their disease. Voided urine specimens were collected prior to surgery. Catheterized specimens were also obtained at the time of surgery from seven patients. No specimens were collected by bladder barbitage or bladder wash. Group II consisted of 97 voided urine specimens from 58 patients with a past history of bladder cancer. Their ages ranged from 40 to 88 years with an average of 68 years. Group III consisted of 54 patients who did not have transitional cell carcinoma. This group consisted of seven patients with urinary tract infections, 10 patients with benign prostatic hypertrophy, nine patients with upper tract stones, seven patients with non-urologic cancer, 19 patients with prostatic adenocarcinoma and two patients with renal cell carcinoma. Group III had an average age of 63 years with a range of 34 to 85 years. Flow cytometry. The urine for flow analysis was centrifuged at 1100 rpm for 10 minutes. The supernatant was refrigerated for IRMAK analysis. The cell pellet was resuspended in PBS without Ca++ or Mg++ and recentrifuged. The resulting suspension was the placed in propidium iodide according to the technique of Deitch and deVere White. 10 The suspension was stored at 4C overnight and run on a Coulter DNA flow cytometer the next day. The DNA histograms generated were interpreted using standard criterion developed for evaluating the degree ofploidy. 11 They were interpreted as either abnormal or normal histograms. Urine samples for cytokeratin analysis. Urine samples used in the IRMAK-18 were processed for flow cytometry by centrifuging out the cells. The clarified urine was adjusted to pH 7 and stored for no more than one week at 4C before being assayed by the IRMAK-18. IRMAK-18. The IRMAK-18 for cytokeratin 18 is a sandwichtype radioimmunoassay in which the antigen in the sample mixture links 125I-UCD/AB 6.11 to UCD/PR 10.11 bound to the plastic well. 8 • 13 The production of the monoclonal antibodies UCD/AB 6.11 and UCD/PR 10.11 has been described. 8 • 12 Both MABs used in the IRMAK-18 were purified from ascites fluid by using an ABX ion exchange column (J.T. Baker, Co., Phillipsburg, NJ) and a Beckman 334 HPLC system (Beckman Instruments, Palo Alto, CA). UCD/AB 6.11 was iodinated using the iodogen technique as described elsewhere. 13

URH~ARY KERATil\f

The assay involved coating "u" bottom 96-well chloride microtiter plates (Dynatech Inc., Catalog #0010102401, Alexandria,V A) with UCD/PR 10.11 (20 ng./50 µl. in 0.1 NaHC0 3 , pH 9, 50 µL/well.) and incubating for 24 hr. at room temperature. The plates were than washed three times with saline (0.9% NaCl, and 0.01 % NaN 3 ) and then blocked for one hour with 150 of IRMAK-buffer (0.15 M NaCl, 0.5% bovine serum albumin, 0.01 % NaN2, and 50 mM sodium phosphate, pH 8). After blocking, 50 of standards diluted in IRMAK buffer or whole urine was then added in triplicate, followed by 50 µL of I' 25 -UCD/AB 6.11 (20 ng./50 µl., 1 x 107 cpm/µg.). Plates were incubated at 4C or 72 hours, washed three times with 0.9% NaCl, dried, and counted using a LKB 1260 gamma counter (LKB Instruments, Gaithersberg, MD). Immunohistochemistry. Twenty-one paraffin embedded, formalin fixed tissues from the 26 cases of patients with active transitional cell carcinoma (TCC) were acquired from the surgical pathology files. Two pathologists (Byung Hee Min and Robert D. Cardiff) verified the presence of cancer on H&E sections and reviewed them for accuracy of grade and stage determinations. Tissue sections were incubated with a 1:5000 dilution of UCD/PR 10.11, followed by biotinylated horse anti-mouse IgG and avidin-horseradish peroxidase (Vector Labs, Burlingame, CA). The antigen-antibody complexes were localized with the substrate diaminobenzidine/H202 followed a hematoxylin counterstain. Normal mouse serum or PBS was substituted for antibody on one slide of each specimen as a negative control. Tissue reactivity was scored based on the frequency of epithelial cells staining as well as the intensity of the stain (1weak, 2-fair, 3-strong). The results of the immunohistochemistry were compared to the grade and stage of the tumor, as well as the IRMAK-18 level, and the DNA histogram. RESULTS

IRMAK-18. The IRMAK-18 assay detected one ng. of keratin/ml. The binding curve was linear between 20 and 200 ng./ ml. The binding of the "signal" antibody occurred primarily in the presence of keratin 18. 13 Other intermediate filaments did not result in significant binding of the signal. The detailed characterization of the assay may be found in the companion article. 13 Urinary keratin. The urines of the 130 medical students and other healthy volunteers ranged from undetectable to 46 ng./ ml. The average of the 51 positive urines was 2.5 ± 4.7 ng./ml. (figure The urines of the 37 of 54 """v'""" without bladder cancer had detectable keratin with an average and standard deviation of 4.1 ± 8 ng./ml. In contrast, 24 of 26 (82%) with active bladder cancer had IRMAK-18 uecec:~wme keratin The keratin ranged from undetectable to 630 ng./ml. with an average of 74.6 ± 146.9 This group was different from all other at the p >0.001 level using the chi square test (figure Group H patients with a past history of transitional cell carcinoma but without currently detectable disease also had a high proportion of patients with detectable urinary keratins (87%). The maximum keratin value in this group was 291 ng./ ml. This group averaged 20.4 ± 66 ng./mL (figure Six patients in this group have keratin >20 ng./ml. Two standard deviations beyond the average in the urines from urological patients without bladder cancer is 20.1 ng./ml. keratin and two standard deviations above the average in the urines from the healthy volunteers is 11.4. Therefore, the four groups were compared using the values of 10 and 20 ng. keratin/ ml. to define the upper limit of normal (table 2). The sensitivity of the IRMAK-18 using 10 ng./ml. and 20 ng./ml. as criteria, was 57% and 46% respectively. The specificity was 91 % and 98% respectively. Increasing the upper limit

437

AND BLADDER CANCER

600

500

-e .......

400

Oi

C

~

300

:z

~

a:: ~

200

100

0

DISEASE FREE SUBJECTS

UROLOGICAL DISORDERS NON TCC

PATIENTS WITH PAST HISTORY OF TCC

N • 130 2.46±4.7

N • 54 4.1±8

N ° 58 20.4±66

x

O

x•

x•

PATIENTS WITH ACTIVE TCC N • 26 7 4.6±146.9

x•

FIG. 1. Histogram illustrating level of keratin 18 in voided urines of 268 patients in four groups as measured by IRMAK-18. Line indicates 20 ng./ml. outer limit of normal as defined in text. N refers to number of patients in each group. TABLE

1. Keratin 18, stage, grade, and flow cytometry of TCC patients

Patient Number BUlOOO BU4052 BU3103 BU2020 BU2023 BU2146 BU3095 BU3070 BU2141 BU2029 BU2031 BU3152

BU2039 BU3060 BU2042 BU2137 BU2037 BU2117 BU2133 BU4072 BU3089 BU2136 BU2099 BU4006 BU3002 BU2091

Keratin 18 >20 ng./

DNA

Stage

Grade

A

B B

3 3 3 2,CIS l,CIS 3,CIS

rnl.

630.0 412.0 363.0 160.0 120.0 108.0 74.0 60.0 48.0 44.0 32.0 31.0 Kei'atin 18 <20 ng./ml 16.0 13.0 iO.O 8.6 8.0 5.4

4.6 3.0 2.0 1.4 1.0 1.0 .0 .0

A N A

A A H A A A A A

N N N A A H H A A A H N A A

A

0 0 B 0 B B B 0 A

3

2 3,CIS 3 1

2

0

2

0

2,CIS

0 0 0 0

2

2

0 0

1 2

A 0 0 0 0 0

2 2,CIS 1 2

2 2

2 3

DNA: A= aneuploid, H = hyperdiploid, N = normal, DNA. Stage: 0 = superficial A = invading laminopropria B = invading muscle. Grade: Grades are standard WHO nomenclature, CIS = Carcinoma in situ.

of normal from 10 ng./ml. to 20 ng./ml. increased the predictive value of a positive result from 48% to 75%. The overall accuracy of the IRMAK-18 was not significantly increased by changing the upper limits. In view of the predictive values of the assay, group II,

BAKER AND ASSOCIATES

438 TABLE

2. Sensitivity and specificity calculations for IRMAK-18

TABLE

Keratin 18 >10 ng./ml. >20 ng./ml. 12% 46% 98% 2% 54% 75% 93% 91%

12% 57% 91% 9% 43% 48% 93% 87%

Prevalence Sensitivity Specificity False positive False negative Positive Predictor Negative Predictor Accuracy

4. Sensitivity and specificity of IRMAK-18 and DNA flow cytometry with keratin 18 > 20 ng.jml.

Prevalence Sensitivity Specificity False positive False negative Positive Predictor Negative Predictor Accuracy

N= 209

IRMAK 18

Flow

Flow or IRMAK

Flow and IRMAK

32% 46% 96% 4% 54% 86% 79% 80%

32% 81% 81% 19% 19% 68% 90% 81%

32% 81% 77% 23% 19% 63% 89% 78%

32% 46% 100% 0% 54% 100% 79% 83%

N=80. TABLE 3.

2

2 comparison of IRMAK-18 and flow cytometry data on urine from patients with urological disorders

X

IRMAK-18 Patients with active transitional cell carcinoma at the time of urine collection.

8±E

<20 ng./ml.

2

>20 ng./ml.

Normal DNA 20

<20 ng./ml.

4

>20 ng./ml.

Normal DNA 43

IRMAK-18 Patients with non-TCC urological disorders.

>20 ng./ml.

35 Q

E .____ 0)

s co ,... z

~ ex:

w ~

12

53 5

Abnormal DNA 11

B

<20 ng./ml.

14

Abnormal DNA 38

rn

IRMAK-18 Patients with a past history of transitional cell carcinoma.

Abnormal DNA 21

Normal DNA 5

54 0

0

s

30 25 20 15 10 5 10

20

30

40

50

60

70

80

90

100

TIME (days) FIG. 2. Graph illustrates levels of one patient followed with serial urine samples analyzed using IRMAK-18. Letter S indicates day of surgery. Letter C indicates initiation of chemotherapy.

consisting of patients with a past history of bladder cancer, was the most interesting. Since six patients have elevated keratins, Group II is being followed with repeated studies and for the development of clinically observable disease (table 3). The history and values of one such patient are illustrated in figure 2. Correlation of IRMAK-18 with DNA histograms. A comparison of IRMAK-18 and DNA histograms was performed using the patients whose urine were analyzed with both IRMAK-18

and flow cytometry (table 1, 3 and 4). The prevalence of cancer in this group was 32%. Of the 26 patients with bladder cancer, 81 % (21/26) had abnormal DNA histograms, but only 12 had keratin >20 ng./ml. Interestingly, five of the bladder cancer patients had both normal keratin and DNA. Three of these patients had small grade I lesions while the other two had more advanced lesions (table 1). When using an abnormal DNA histogram and an elevated IRMAK-18 as a criterion for cancer, the specificity and predictive value of a positive result were 100% (table 4). However, the sensitivity was only 46%, and the overall accuracy was not significantly different from using either test alone. Conversely using an abnormal histogram or elevated keratins as criteria for positivity, the sensitivity continued to be the same as that of the flow cytometer alone, and predictive value of a positive result dropped to 63%. The accuracy of either format for combining the tests was not different from the accuracy of using either test alone. A high proportion (64%) of the patients with a past history of transitional cell carcinoma had abnormal histograms, but only five of those patients had keratin >20 ng./ml. (table 3). One patient had keratin >20 ng./ml. with a normal histogram. The significance of these results will not be known until long term follow-up data is available. Abnormal DNA histograms were found in 20% of the patients without bladder cancer but none of these patients had keratin >20 ng./ml. (table 3). Correlation with grade and stage. Analysis of the clinical data shows that 66% (8/12) of the group I patients with keratin >20 ng.fml. had invasive disease. Most patients (13/14) with keratin <20 ng./ml. had stage O disease. Furthermore, most patients (11/12) with either grade 2 or 3 lesions, had keratins >20 ng./ ml. (table 1). Immunohistochemistry. Twenty-one cases of transitional cell carcinoma were subjected to immunohistochemical staining with a battery of MABs. The immunoperoxidase verified that all the cancers had keratin detected by UCD/PR 10.11 as well as being positive with a variety of other anti-keratin MABs. No significant correlation of 10.11 reactivity with grade, stage, IRMAK-18, or DNA histograms was identified (data not shown). DISCUSSION

The studies reported here using a newly developed assay for keratin, IRMAK-18, detected keratin in the voided urines of healthy volunteers and urological patients. However, most urinary keratins >20 ng./ml. were found in patients with either a past history of TCC or with active bladder cancer. These results suggest that, under appropriate circumstances, urinary keratins levels may be useful in the evaluation of bladder cancer, in the monitoring of patients with bladder cancer and in the evaluation of quantitative DNA flow cytometry. Keratin or keratin-like proteins have been previously identified in the urine of bladder cancer patients. 14 The antigen most extensively studied, tissue polypeptide antigen (TPA), is a tissue extract which contains keratins 8, 18 and 19.4 TP A has

439 been found in the u.:riT1e cf bladder cancer and 1101T.nal patients."'· 16 Some studies repo:rt that cancer and noncancer urologic i,o.cn,u,," have similar levels of TP A. 14 However, the assay systems also revealed elevated levels of TP A in patients with tumors which should not contain keratin. 6 • 7 Further, the current TP A assay is based on intermediate filament extract of HeLa cells which contains both keratin and vimentin raising qu:estw,ns concerning the specificity of the assay. 17 The IRMAK-18 test described here uses MABs which are specific for keratin. 13 The assay is relatively specific for keratin 18, but does show minor cross reactivity with keratin 8. 13 The assay readily detected keratins released from cultured epithelial cells,8 encouraging us to apply the IRMAK-18 to urinary cancer. The IRMAK-18 detected keratin >20 ng./mL almost exclusively in the voided urines from our patients with active bladder cancer or a history of transitional cell carcinoma. Analysis of the clinical stage suggests that elevated keratin correlates with the extent of the disease, The specificity when 20 ng./ml. was used as the cutoff was 96%. This suggests that the assay can be specific for bladder cancer. However, we have observed keratin >20 ng./mL in urines from patients with ileal loop diversions, and from a patient passing a kidney stone. Further, two female medical students had urinary keratins >20 ng./ml. 13 One was ptegnant and the other had a family history of bladdet disease. Neither sample was a clean catch. The age of the patient does not appear to effect the keratin level. Therefore, the keratin >20 ng./mL in six patients with past history of cancer is provocative, but without ~""""'.-"''J apparent current disease, long term will be required before we can ~h,=~~~ the data. The source of the keratin in urine is not understood. Our data suggest that elevated m-inary keratins may be related to increased stage, grade or, more probably, volume of intravesical cancer. The release of keratin has been attributed to loss during cell proliferation,1 8 to cell death 8 or possibly due to normal metabolic process. 8 Vve suspect that the urinary keratins are associated with cell turnover. A malignant epithelium is more likely to have a high turnover rate and more likely to produce elevated keratins. The IRMAK-18 may have utility in monitoring patients who have been treated for bladder cancer. Bladder cancer frequently recurs and an assay which helps assess these ,J~'"'~'''"~ would be valuable. We are following a number of patients with histories of bladder cancer to determine whether the urinary keratin level will correlate with the appearance of recurrent disease. As with ""'t,tc; hu, DNA 1

We gratefully acknowledge the support of

Dr. B.F. Edwards and Dr. F.J. Meyers during this project. REFERENCES 1. Cooper, D., Schermer A. and Sun, T. T.: Biology of disease: clas-

2.

3.

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5. 6.

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0

"~,.u,w.,,u~ has resulted in a concomitant increase in "ab9 As a normal" a second parameter, such as could be useful in uu,s.,u;;mc,u,uM abnormal ceHs, In the current study, 11 of the 54 urological patients without bladder cancer had abnormal histograms. The 20% figure agrees 11 However, all eleven patients had keratin with earlier <20 ng./ml. this specific circumstance, if keratin >20 ng./ ml. were used as a second selection parameter to aid in the interpretation of the histogram, the rate of "false positives" would fall to zero. In this sense, urinary keratin may prove to be a useful independent variable and merits further study.

14.

15.

16. 170

18. 19.

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