Effects of Therapy on Carcinoembryonic Antigen ...

Effects of Therapy on Carcinoembryonic Antigen Activity in the Urines of Patients with Cancer of the Bladder KAREN JAMES, M.S., M.T.(ASCP), JOSEPH ALROY, D.V.M., ALEXANDER W. MILLER, M.D. AND MALACHI FLANAGAN, M.D.

Departments of Immunology and Pathology, Rush-Presbyterian St. Luke's Medical Center and Rush Medical College, Chicago, Illinois

In the current study we quantitated the levels of urinary CEA of patients with invasive transitional-cell carcinoma. CEA levels in urines from patients who had received radiotherapy or therapy with the radiomimetic agent thiotepa, are compared with CEA levels found in urines of patients with similar carcinomas, but who were not treated. Materials and Methods Patients

CARCINOEMBRYONIC ANTIGEN (CEA) is a glycoprotein 9 associated with the cell membrane surface coat (i.e., glycocalyx) of normal21-23 and neoplastic epithelium. 1016,20 Elevation of serum or plasma CEA is associated with carcinomas of gastrointestinal origin,9,29 but is also found in carcinomas of other origins. 27,29 In transitional-cell carcinoma of the urinary bladder, CEA is elevated in both serum 8,11,12,14,31,33 and 4.8.12-14.22.25.30.32,3.1,36 Though many investigators urine. agree that with urinary bladder carcinomas, urinary CEA determinations may be of some clinical importance, the value of plasma CEA determinations in management of these patients is debatable. 812,17,25,30,31,36 CEA levels in urines obtained from patients with urinary bladder carcinomas may be influenced by the amount of CEA synthesized by the malignant cells, the tumor grade, 8,25 stage, 8,13,17,25,30,36 tumor mass, and surface area. 13 Urinary CEA quantitation has been found to be useful during patient follow-up25,33; however, certain courses of therapy may interfere with the usefulness of urinary CEA quantitation 11 in evaluation of these patients.

Sixteen patients with invasive transitional-cell carcinoma of the urinary bladder were evaluated. Specimens of urine for CEA determination were either collected overnight prior to cystoscopy or obtained during cystoscopy. The tumors were excised; graded histologically according to the recent Armed Forces institute of Pathology Fascicle 26 and staged according to the classification of Jewett. 24 Carcinoembryonic

Antigen

Assay

The collected urine was centrifuged to remove any sediment and stored at - 2 0 C until assayed. The CEA levels were determined by the Hansen Z-gel method 15 of Roche Diagnostics.* The urine samples were extracted with perchloric acid and processed as previously described for plasma samples. 28,29 Results

Received June 5, 1979; accepted for publication June 18, 1979. Supported in part by the Department of Health, Education and Welfare Public Health Service Grant of the National Cancer Institute #5 P30 CA 17086-03. Current address of Dr. Alroy: Department of Pathology, School of Medicine and Veterinary Medicine, Tufts University, Boston, Massachusetts. Address reprint requests to Ms. James: Rush-Presbyterian St. Luke's Medical Center, 1753 West Congress Parkway, Chicago, Illinois 60612.

Tumor sizes, grades, stages, methods of sample collection, and levels of urinary CEA for these 16 patients are listed in Table 1. Figure 1 illustrates the level of urinary CEA found for each patient, with mean and standard error given for each group. Eight patients who had not received either radiotherapy or thiotepa therapy prior to this study had markedly elevated CEA levels, 20.3 ± 8.7 ng/ml urine (mean ± SD), whereas eight patients treated with either x-irradiation or thio* Division of Hoffman-La Roche, Inc., Nutley, New Jersey.

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James, Karen, Alroy, Joseph, Miller, Alexander VV., and Flanagan, Malachi: Effects of therapy on carcinoembryonic antigen activity in the urines of patients with cancer of the bladder. Am J Clin Pathol 73: 250-253, 1980. Carcinoembryonic antigen (CEA) levels were quantitated in specimens of urine for 16 patients with invasive transitional-cell carcinoma of the urinary bladder. Four of the patients had received x-irradiation to the bladder and four had been treated with the radiomimetic agent, thiotepa. Urinary CEA levels in these patients (3.7 ± 2.4 ng/ml) were significantly lower (t = 6.17, P < .001) than levels from a similar group of eight patients who had not been treated with radiotherapy (20.3 ± 8.7 ng/ml). These results suggest that "false-negative" urinary CEA levels may, in some cases, be associated with previous radiotherapy. (Key words: Urinary CEA; Bladder cancer; Thiotepa; Radiation therapy.)

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Vol. 73 • No. 2

tepa had only 3.7 ± 2.4 ng/ml CEA in their urine. This difference is statistically significant using the Students T test with separate variance estimate (t = 5.21), P < .005). Statistical analysis of the treated vs. untreated groups for all other parameters detected no differences between the groups (grade: t = 0.48; mass: t = 0.28; method of collection: chi square = 1.0). The log transformation of the CEA level was tested to stabilize the variances. This also indicated a significant difference^ = 6.17,/* < .001). An analysis of variance on the log transformation also detected a significant difference between the patients who had previously received therapy and those who had not (f = 38.04, P < .001). There was no significant difference between the two treatment groups (f = 0.46, df = 2,13).

X-lrrodiotion TH10-TEPA

Discussion

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WITHOUT THERAPY

WITH THERAPY

FIG. I. Levels of CEA found in urines of individual patients. White box at extreme right of each group indicates mean; the bar shows standard error.

tract infection and malignancy had urinary CEA levels less than 2.5 ng/ml, which was considered by us and others11-13 to be normal. In patients with similar invasive carcinomas—that is, similar in type, grade, stage and size—who had been treated earlier either by radiotherapy or with thiotepa, the levels of urinary CEA were closer to normal than were those of their nontreated counterparts. In exfoliated cancerous bladder cells, the amounts of CEA found varied among cells within one tumor-cell population. The amount of CEA may relate to the proportion of cells that are in a specific phase of their cycle.37 It has been

Table I. Urinary CEA of Patients with Invasive Transitional-cell Carcinoma Treated Patients

Patients Without Treatment Tumor Stag e

Tumor Size (cu mm)

Urinary CEA (ng/ml)

III II 11 II III II/III III 111

C A B A C A B D

2.0 0.61 4.7 6.75 0.2 73.1 8.0 3.4

19.0 13.6 12.5 12.2 32.0 18.0 35.0 20.0

* O. Overnight urine collection. t C. Urine collected at cystoscopy.

Method of Collection

Tumor Grade

Tumor Stage

Tumor Size (cu mm)

Urinary CEA (ng/ml)

Method of Collection

0* Ct

nit

A A A C A D B C

0.3 1.0 9.0 6.7 48.62 1.62 2.25 6.0

0.8 3.0 2.3 7.6 1.5 4.8 3.0 6.3

O O C C C C C C

c c c c0 c

lit II/III* -H-

Tumor Grade

II§ III§ HIS III§ t X-irradiation. § Thiotepa.


The glycoprotein CEA wasfirstdescribed as a tumorassociated antigen found in the cell membranes of carcinomas from entodermally derived digestive system tissue9,27 and later in nonentodermally derived carcinomas.27,29 Furthermore, CEA was found in smaller amounts in various normal tissues.21,23,34 CEA is thought to be associated with the cell membrane surface coats of both neoplastic10,16,20 and normal cells.21,23 It is shed from the cell surface into the surrounding environment {i.e., plasma or urine) and can thereby be detected in either environment.811,27,29 The levels of urinary CEA obtained from patients clinically proven with urinary bladder carcinomas may be useful during patient follow-up.25,33 In these patients, several factors may influence the levels of urinary CEA, e.g., the amount of CEA synthesized by the malignant cells, tumor grade,8,25 stage,8,13,17,25,30,36 size and surface area,13 and possibly therapy.11 In the current study we have documented high levels of urinary CEA in patients with invasive transitionalcell carcinomas. Patients who were free of urinary

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References 1. Alroy J. Pauli BU. Weinstein RS et al: Association of symmetric unit membrane plaque formation in the urinary bladder of adult humans with therapeutic radiation. Experientia 33:1645-1647, 1977 2. Alroy J, Teramura K. Miller A. et al: Isoantigens A,B and H in urinary bladder carcinomas following radiotherapy. Cancer 41:1739-2745, 1978 3. Caldwell WL: Radiotherapy: definitive, integrated and palliative therapy. Urol Clin N Am 3:129-148, 1975 4. Coombs GB, Hall RR, Laurence DJR et al: Urinary carcinoembryonic antigen (CEA)-like molecules and urothelial malignancy: a clinical appraisal. Br J Cancer, 31:135-142, 1975 5. Decenzo JM, Howard R, Irish CE: Antigenic deletion and prognosis of patients with Stage A transitional cell bladder carcinoma. J Urol 114:878, 1975

6. Dimopoulos J. Hascheck H, Vedrilla D: Radiotherapie des blasenkarzinoms: inditcation and ergebnisse. Helv Chir Acta 43:321-325, 1976 7. Drewinko B, Yang LY: Restriction of CEA synthesis to the stationery phase of growth of cultured human colon carcinoma cells. Exp Cell Res 101:414-416. 1976 8. Fraser RA, Ravey MJ, Segura JW et al: Clinical evaluation of urinary and serum carcinoembryonic antigen in bladder cancer. J Urol 114:226-229, 1975 9. Gold P, Freedman SO: Specific carcinoembryonic antigens of the human digestive system. J Exp Med 122:467-481, 1965 10. Gold P, Krupey J, Ansari H: Position of carcinoembryonic antigen of the human digestive system in ultrastructure of tumor cell surfaces. J Natl Cancer Inst 45:219-225, 1970 11. Guinan P, Ablin RJ, Barakat H, et al: Carcinoembryonic antigen in patients with urologic cancers. Urol Res 1:101-105, 1972 12. Guinan P, Ablin RJ, Sadoughi N, et al: Carcinoembryoniclike antigen in the urine of patients with carcinoma of the bladder and normal controls. J Surg Oncol 7:127-131, 1974 13. Guinan P, John T, Sadoughi N, et al: Urinary carcinoembryonic-like antigen levels in patients with bladder carcinomas. J Urol 111:350-352, 1975 14. Guinan PD, McKiel C, Sundar B, et al: The carcinoembryonic antigen test in urologic cancer. Natl Cancer Inst Monogr 49:225-229, 1978 15. Hansen, HJ, Lance XP, Krupey J: Demonstration of an ion sensitive site on carcinoembryonic antigen using zirconyl phosphate. Clin Res 19:143, 1971 16. Heberman RB, Aoki T, Cannon G. et al: Location by immunoelectron microscopy of carcinoembryonic antigen on cultured adenocarcinoma cells. J Natl Cancer Inst 55:797798, 1975 17. Hering H, Hering FJ, Weidner W: Plasma and urinary CEA assay in patients with urological tumors. Urol IA] 15:300303, 1976 18. Hicks RM: Discussion of morphological markers of early neoplastic changes in the urinary bladder. Cancer Res 37: 2822-2823, 1977 19. Hicks RM, Wakefield J St J: Membrane changes during urothelial hyperplasia and neoplasia. Cancer Res 35:25022507, 1976 20. Huitric E: An ultrastructural study of the localization of carcinoembryonic antigen in adenocarcinomas of the human colon. Ann Immunol 129c:603-608, 1973 21. Huitric E, Laumoneir R, Burtin P, et al: Ultrastructural study of the localization of carcinoembryonic antigen (CEA) in normal and cancerous human rectocolonic mucosa. Lab Invest 34:97-107, 1976 22. Ionescu G, Romas NA, lonescu L et al: Carcinoembryonic antigen and bladder carcinoma. J Urol 115:46-48, 1976 23. Issacson P, Judd MA: Carcinoembryonic antigen (CEA) in normal human small intestine. A light and electron microscopic study. Gut, 18:786-791, 1977 24. Jewett HJ: Cancer of the bladder. Diagnosis and staging. Cancer 32:1072-1074, 1973 25. Korsten CB, Persijn JP, Renaud J, et al: Carcino-embryonic antigen activity in urine of patients with bladder carcinoma. Clinical evaluation of carcino-embryonic antigen II. J Clin Chem Clin Biochem 14:389-393, 1976 26. Koss LG: Tumors of the urinary bladder, Atlas of Tumor Pathology. Second Series fascicle. II. Washington, D. C . Armed Forces Institute of Pathology, 1975 27. LoGerfo P, Krupey J, Hansen H: Demonstration of an antigen common to several varieties of neoplasia. Assay using ziconyl phosphate gel. N Engl J Med 285:138-141, 1973 28. Martin EW, James KK, Hurtubise PE, et al: The use of CEA as an early indicator for gastrointestinol tumor recurrence and second-look procedures. Cancer 39:440-446, 1977


reported that stationary cells or cells in the G1 phase of their cycle synthesize the largest amount of CEA, whereas little synthesis is found in exponentially growing cells. 7 The proliferation rate of transitional cells appears to accelerate following x-irradiation. 35 The accelerated rate may shorten the Gl and stationary phases of these cells, resulting in smaller amounts of CEA being synthesized. This may explain the earlier report of normal values of urinary CEA found in patients with urinary bladder carcinomas who had received more than 6,000 rads to their bladders." Our study confirms the above observation on the influence of radiotherapy and radiomimetic drugs such as thiotepa on the levels of urinary CEA in these patients. Radiotherapy of patients with urinary bladder carcinomas is a common form of localized therapy used under different clinical circumstances as part of various therapeutic courses. Details concerning procedures, indication, and results were recently discussed. 3,6 Radiotherapy acts primarily at the nuclear level; however, in some cells the plasma membrane is also radiosensitive. 38 Alterations in certain cell membrane components, such as the replacement of asymmetric unit membrane by glycocalyx-rich symmetric membrane 1819 and the deletion of ABH isoantigens from urinary bladder epithelium,5-39 are thought to be potential markers for malignancy and behavior of this epithelium. However, in both instances, the presence of asymmetric unit membrane in bladder epithelium of older persons and the presence of ABH isoantigens in invasive uninary bladder carcinomas were associated with radiation therapy.1-2 The results of our study have direct bearing on the interpretation of normal levels of urinary CEA patients with urinary bladder carcinomas who have been treated with either therapy. More study is needed to identify the time span and the dose of therapy needed in order to obtain normal or near-normal levels of urinary CEA in patients with invasive transitional-cell carcinomas of the urinary bladder.

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BRIEF SCIENTIFIC REPORTS

29. Martin EW, Kibbey WF, DiVecchia L. et al: Carcinoembryonic antigen: clinical and historical aspects. Cancer 37:62-81, 1976 30. Murphy WM. Vandevoorde JP, Rao MK: The clinical value of urinary carcinocmbryonic antigen-like substances in urothelial cancer. J Urol 118:806-808. 1977 31. Orjasaetcr H, Fossa SD, Schjolseth SA. Carcinoembryonic antigen (CEA) in plasma of patients with carcinoma of the bladder/urethra. Cancer. 42:287-295. 1978 32. Pcrsijn JP, Korsten CB, Batterman JJ. et al: Clinical significance of urinary carcinoembryonic antigen estimations during the follow-up of patients with bladder carcinoma or previous bladder carcinoma. Clinical evaluation of carcinoembryonic antigen III. J Clin Chem Clin Biochem 14: 395-399. 1976 33. Pcrsijn J P. Korsten CB: The development of a radioimmunoassay for carcinoembryonic antigen with some applications. Clinical evaluation of carcinoembryonic antigen I. J Clin Chem Clin Biochem 14:377-387. 1976

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34. Pusztaszeri G. Mach, JP: Carcinoembryonic antigen (CEA) in non-digestive cancerous and normal tissues. Immunochemistry 10:197-204, 1973 35. Schreiber H. Oehlert W, Kugler K: Regeneration and proliferation kinetics of normal and x-irradiated transitional epithelium in rats. Virchows Archiv [Cell Pathol|4:30-44, 1969 36. Wahren B. Edsmyr F. Zimmerman R: Measurement of urinary CEA-like substance. An aid in management of patients with bladder carcinoma. Cancer 36:1490-1495, 1975 37. Wahren B. Esposti P, Zimmerman R: Characterization of urothelial carcinoma with respect to the content of carcinoembryonic antigen in exfoliated cells. Cancer 40:15111518, 1977 38, Wallach DFH: Membrane biology and cancer therapy. Pathobiol Annu 8:189-216, 1978 39. Weinstein RS, Alroy J, Farrow GM et al: Blood group isoantigen deletion in carcinoma in-situ of the urinary bladder. Cancer 43:661-668, 1979

BERIA CABELLO, M.D., JACK LUBIN, M.D., ARKADI M. RYWLIN, M.D., AND RUTH FRENKEL, B.S., C(ASCP)

Cabello, Beria, Lubin, Jack, Rywlin, Arkadi, and Frenkel, Ruth: Significance of a sixth lactate dehydrogenase isoenzyme (LDH„). Am J Clin Pathol 73: 253-258, 1980. An unusual band on the cathodic side of the membrane was found on lactate dehydrogenase electrophoresis of blood specimens from 18 patients. Fifteen patients died shortly after the finding. No specific cause was found for the unique band, but most of the patients had arteriosclerotic cardiovascular disease with acute passive hyperemia of the liver, and prerenal azotemia. (Key words: Lactate dehydrogenase isoenzymes; Sixth lactate dehydrogenase isoenzyme.)

WE REPORT eighteen patients whose sera had similar spurious lactate dehydrogenase (LDH) bands (LDH6) located on the cathodic side of LDH5. Extra bands in the electrophoretic pattern of LDH have been described previously. 1•2••'1•', They have been attributed to the complexing of various substances with LDH isoenzymes, often using substrates different from the ones we have used. The additional bands have been found in different portions of the strip. Markel and Janich4 described two additional bands between LDH3 and LDH4 which resulted from the binding of LDH with immunoglobulin A Received April 13, 1979; accepted for publication May 17, 1979. Acknowledgement to John Di Bella, M.D. Address reprint requests to Dr. Cabello: Department of Pathology and Laboratory Medicine, Mount Sinai Medical Center, 4300 Alton Road, Miami Beach, Florida 33140.

Department of Pathology and Laboratory Medicine, Mount Sinai Medical Center, Miami Beach, Florida

*

II

tl FIG. 1. Center row shows pattern with LDH6 band (arrow) compared with normal band (below). Electrophoretic pattern with increased LDH, in top row.

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Significance of a Sixth Lactate Dehydrogenase Isoenzyme (LDH6)