Serum Lactate Dehydrogenase Isoenzyme 1 in ...

Serum Lactate Dehydrogenase Isoenzyme 1 in Patients with Advanced Testicular Cancer FRANK LIU, M.D., HERBERT A. FRITSCHE, PH.D., JOSE M. TRUJILLO, M.D., AND MELVIN L. SAMUELS, M.D.

IT IS WELL KNOWN that for patients with germcell tumors of the testes the tumor marker profile consisting of serum alpha-fetoprotein (AFP) and human chorionic gonadotropin (B-hCG) has significantly improved clinical management.5,9"12,21,26 Recently, total serum lactic dehydrogenase (LD) activity has been suggested as a potential tumor marker for testicular cancer and a significant correlation was demonstrated between the total serum LD activity and the tumor burden.2,9,15'29 LD activity appears in human serum as five distinct isoenzymes that are tetramers of two different subReceived September 14, 1981; received revised manuscript and accepted for publication October 29, 1981. Address reprint requests to Dr. Liu: Laboratory Medicine, The University of Texas System Cancer Center, M. D. Anderson Hospital, Houston, Texas 77030.

Departments of Laboratory Medicine and Medicine, The University of Texas, M. D. Anderson Hospital and Tumor Institute at Houston, Houston, Texas

units.13,17 These five isoenzymes are represented by LD1, LD-2, LD-3, LD-4, and LD-5, and these can be separated by electrophoretic technics on the basis of their electrophoretic mobility.13,17 LD-1 is the fastest anodally migrating isoenzyme and LD-5 is the slowest.13,17 Several investigators have reported that the LD-1 isoenzyme is frequently elevated in the serum of patients with germ-cell tumors of the testes.1,30,32,34In those studies, an elevation of LD-1 was defined as an increased LD-1 fraction with either an elevated or normal total LD activity. The objective of this paper is to confirm that the serum level of the LD-1 isoenzyme is elevated in the circulation of patients with advanced testicular cancer and to illustrate the clinical usefulness of LD1 as a tumor marker for monitoring therapy and detecting recurrent disease. In addition, we describe more comprehensive criteria for assessing LD-1 abnormality than has been previously reported. This new interpretive criteria improves the sensitivity of the test for testicular cancer. Materials and Methods Patients Forty-two patients admitted to the University of Texas M.D. Anderson Hospital and Tumor Institute at Houston with stage III testicular cancer were included in this study. The pathological classification of Dixon and Moore 4 was used to group the histopathologic types of the disease, which were classified into five groups: I seminoma, pure; II embryonal carcinoma, pure, or with seminoma; III teratoma, pure, or with seminoma; IV teratoma with either embryonal carcinoma or choriocarcinoma, or both, and with or without seminoma; V choriocarcinoma, pure or with embryonal carcinoma or seminoma, or both. Clinical staging of the patients was performed according to the method of Samuels and associates.25 Stage III-B-1 patients had gynecomastia with or with-

0002-9173/82/0800/0178 $01.10 © American Society of Clinical Pathologists

178

Downloaded from http://ajcp.oxfordjournals.org/ by guest on April 10, 2016

Abnormal levels of serum lactic dehydrogenase-1 (LD-1) activity have been observed in 81% (34/42) of patients with stage III germ cell malignancy of the testis. The criteria for evaluating the electrophoretic isoenzyme patterns of these patients were, as follows: For criterion 1 elevations the LD-1 value in absolute units was greater than 52.0 U/l with the LD-1/Total LD ratio greater than 37.2%. Criterion 2 elevations had absolute values of LD-1 less than 52.0 U/l, but the LD-1/Total LD ratio was greater than 37.2%. For criterion 3 elevations of LD-1, the absolute value was greater than 52 U/l and the LD-1/Total LD ratio was less than 37.2% activity with the LD-5/LD-1 ratio less than 0.5; or when the LD-5/LD-1 ratio was greater than 0.5 but the LD-1 is equal to or greater than the LD-2. The frequency of LD-1 elevation correlated well with the extent of the disease (stage III-B-1 and 2, 50%; stage IIIB-3, 86%; stage III-B-4, 91%; stage III-B-5, 93%). LD-1 elevation occurred in groups I, II, IV and V histopathologic cell types (Dixon and Moore Classification) and there did not appear to be any correlation between the histologic cell type and the frequency of elevation of LD-1. Interpretation of LD-1 activity only on the basis of its relative ratio to the total LD value (criterion 1 and 2) identified a total of 28 patients (67%). A criterion 3 elevation was demonstrated in 6 (14%) additional patients. All patients with persistent elevations or recurrent elevations of LD-1 have shown progressive or recurrent disease and patients with no clinical evidence of disease have demonstrated normal LD-1 values. In those patients with elevated LD-1 activity, serial measurements of serum LD-1 isoenzyme reflect the response of the patient to therapy. (Key words: Tumor marker; AFP (alpha-fetoprotein); B-hCG (beta subunit of human chorionic gonadotropin); LD-1 isoenzyme; Germ cell malignancy of the testis; Advanced testicular cancer; LD isoenzyme pattern) Am J Clin Pathol 1982; 78: 178-183

Vol. 78 • No. 2

SERUM LD-1 IN TESTICULAR CANCER

Table 1. LD-1 Isoenzyme Fraction in Patients with Stage III Testicular Cancer Number of Patients Stage of Disease

Number of Patients

Criterion 1 Elevation



Total No. of Patients with Abnormal LD-1

III B-l & 2 III B-3 III B-4 II B-5

10 7 11 _1£

2 (20%) 3(43%) 8(73%) 11 (79%)

3 (30%) 1(14%) 0(0%) 0 (0%)

0 (0%) 2(29%) 2(18%) 2 (14%)

5 (50%) 6(86%) 10(91%) 13 (93%)

Total

42

24(57%)

4(10%)

6(14%)

34(81%)

Determination of LD-1 Values by

Electrophoresis

The LD isoenzyme values were determined with the Pol-E-film agarose system (Pfizer, Inc., New York, NY). Visualization of the isoenzymes was accomplished with a mixture of P-nitroblue tetrazolium dye, nicotinamide adenine dinucleotide, and phenazine methosulfate. The stained agarose film was scanned at 575 nni with a Beckman R-112 Densitometer (Beckman Instruments, Inc., Clinical Instruments, Fullerton, CA). The normal ranges for the LD isoenzymes were LD1 = 21.5% to 37.2%, LD-2 = 30.2% to 46.1%, LD-3 = 14.3% to 28.6%, LD-4 = 4.8% to 10.5% and LD-5 = 2.7% to 10.9%. The upper limit of normalcy for the LD-5/LD-1 ratio calculated from these percentages was 0.5, (10.9%/21.5%) and any LD-5/LD-1 ratio greater than 0.5 was indicative of increased LD isoenzyme 5 and, therefore, considered positive for liver cell or skeletal muscle damage.

Sample

Collection

Blood samples were collected by venipuncture and assayed on the day of sample collection. All serum samples were kept at room temperature until assayed. Hemolysed serum specimens were not accepted for assay. Criteria for Interpretation of LD-1 Values In this study of LD-1 as a potential tumor marker for testicular cancer, we assessed the LD-1 activity in terms of both its absolute and relative values. The upper limit of normalcy for the LD-1 fraction in absolute units was 37.2% of 139 U / l or 52 U/l. A criterion 1 elevation of LD-1 consisted of an LD-1 value in absolute units greater than 52 U / l with LD-1/Total LD ratio greater than 37.2%. A criterion 2 elevation consisted of an absolute value of LD-1 less than 52 U / l but a relative ratio greater than 37.2%. A criterion 3 elevation consisted of either an absolute value of LD-1 greater than 52 U / l with the relative ratio LD-1/Total LD less than 37.2% and the LD-5/LD-1 ratio less than 0.5, or the LD-5/LD-1 ratiogreaterthan 0.5 but LD-l/LD-2 ratio greater than or equal to 1. Serum samples with an LD1 value of less than 52 U / l and a LD-1/Total ratio less than 37.2% of total LD activity were considered to be normal. Results Table 1 shows the incidence of elevated LD-1 isoenzyme as defined by the three criteria in patients with stage III testicular cancer. Table 2 summarizes the frequency of elevation of the LD-1 isoenzyme activity in patients with stage III testicular cancer. Approximately 81% (34/42) of the patients showed an elevation of the LD-1 fraction by one of the three criteria. LD-1 elevation occurred in 4 of 6 (67%) patients with seminoma, 17 of 21 (81%) patients with embryonal carcinoma, pure, or with seminoma, 8 of 10 (80%) patients with teratoma with either embryonal carcinoma or choriocarcinoma or both and all the 5 (100%) patients with choriocarcinoma, pure or with embryonal carcinoma or seminoma, or both.


out elevations of AFP and B-hCG, and had no gross tumor detectable; stage III-B-2 patients had minimal pulmonary disease, i.e., up to five metastatic masses in each lung, with the largest diameter of any single lesion no larger than 2.0 cm; stage III-B-3 patients had advanced pulmonary disease with any mediastinal or hilar mass, neoplastic pleural effusion, or intrapulmonary mass greater than 2.0 cm in diameter; stage III-B-4 patients had advanced abdominal disease with any palpable abdominal mass, ureteral displacement or obstructive uropathy; and stage III-B-5 patients had visceral disease (excluding lung) with metastases to visceral organs such as liver, gastrointestinal tract, brain and inferior vena cava.25 A careful review of each patient's medical chart was performed to identify any disease condition known to cause serum elevations of the LD-1 isoenzyme. Assay for total serum LD activity. The total serum LD activity was determined by a modified Wacker method31 using reagents obtained from CalbiochemBehring (10933 North Torrey Pines Rd., LaJolla, CA 92037). The normal range for total serum LD in our laboratory was 49-139 U/l.

LIU ET AL.

180

A.J.C.P. • August 1982

Table 2. Frequency of Elevation of Serum LD-1 Isoenzymes in Patients with Stage III Advanced Testicular Cancer* Histopatho|ogical Type (Dixon and Moore Classification) I II IV V Total

Stage of Disease III-B-1 & 2

III-B-3

0/1 5/8 0/1 0/0

0/0 3/4 3/3 0/0

5/10(50%)

6/7 (86%)

III-B-4 3/3 4/4 1/2 2/2 10/11 (91%)

III-B-5 1/2 5/5 4/4 3/3 13/14(93%)

Total 4/6 (67%) 17/21 (81%) 8/10(80%) 5/5 (100%) 34/42(81%)

* Ratio represents number of patients with initial elevated LD-1 isoenzyme/total number of patients in each group.

Discussion The LD isoenzymes are present in various distributions in many human tissues. Heart, kidney, brain, and erythrocytes have abundant LD-1, whereas liver, skeletal muscle, and skin show the highest activity of LD5.6,7,22,28 An elevation of serum LD-1 is observed in patients following myocardial infarction,3,6,23 renal infarction, 3,616 and hemolytic disorders.18,20 An increase in serum LD-5 is observed with disease associated with hepatic cell or skeletal muscle damage. 6,7,23 Three different types of LD isoenzyme patterns have been observed in a variety of malignancies. An elevated LD-5 to LD-1 ratio has been noted in malignant prostatic

tumors, l9,24 prostatic fluids,8 and in the serum of patients with prostatic carcinoma. 24 A general increase in LD2, LD-3, and LD-4 isoenzymes has been observed in the serum of patients with leukemia, malignant lymphoma, metastatic carcinoma, 34 neuroblastoma, 14 and oral cancers.27 Up to the present time, serum LD-1 isoenzymes have been found elevated in the serum of patients with osteosarcoma27 and germ-celj tumors of the ovaries and testes.1,30,32,34 Our data substantiate the preliminary reports on the usefulness of the LD-1 isoenzyme as a tumor marker for germ cell malignancy of the testis. LD-1 elevation occurred in cell type groups, I, II, IV, and V, with a frequency ranging from 67% (group I) to 100% (group V). Although all five cases of group V patients had LD1 elevations, they all had extensive disease of stage IIIB-4 (2 cases) and stage III-B-5 (3 cases). Therefore, there did not appear to be any correlation between the histologic cell type and the frequency of elevation of LD-1. A significant relationship appeared to exist between the stage of testicular cancer and the frequency of LD-1 elevation: only 50%, or 5 of the 10 patients with stage III-B-1 and 2 disease, had elevations of LD1, whereas 86% (6/7) with stage III-B-3 disease, 91% (10/11) with stage III-B-4 disease, and 93% (13/14)

1 stage III-B-1 patient responded to chemotherapy, is free of disease with normal LD-1 level. 2 patients with initial criterion 1 elevation of LD-1 \ 1 stage III-B-2 oatient resoonded to treatment, is free of disease with return of LD-1 to normal. 3 patients with initial criterion 2 elevation of LD-1

All 3 patients were successfully treated and are in complete remission with subsequent normal LD-1.

5 patients with initial normal serum LD-1 level

All 5 patients responded to chemotherapy, have no evidence of disease and have no elevations of LD-1.

FIG. 1. Outcome of ten patients with Stage III-B-1 and 2 disease.


Figures 1, 2, 3 and 4 summarize the clinical course of the 42 patients and the nature of the LD-1 abnormality in these patients. These results show that all patients with persistent elevations or re-elevations of LD-1 have shown progressive or recurrent disease and patients with no clinical evidence of disease have demonstrated normal LD1 values. In those patients with elevated LD-1 activity, serial measurements of serum LD-1 isoenzyme reflected the response of the patient to therapy.

Vol. 78 • No. 2


181

2 patients responded to chemotherapy, are in complete remission with return of LD-1 to normal. 3 patients with initial criterion 1 elevation of LD-1 \ 1 patient initially responded to therapy with return of LD-1 to normal. Recurrence of disease to lungs with a criterion 1 elevation of LD-1 occurred, and the patient was treated with high dose chemotherapy with return of LD-1 to normal. Recurrence with metastases to liver and brain was observed with a criterion 1 elevation of LD-1.

1 patient with criterion 2 elevation of LD-1

Responded to therapy with return of LD-1 to normal, free of disease. 1 patient responded to therapy temporarily w i t h return o f LD-1 to normal. Recurrence with re-elevation of LD-1 ( c r i t e r i o n 1 ) . Progressive disease with persistent c r i t e r i o n 1 elevation of LD-1.

/ 2 patients with i n i t i a l criterion 3 elevation of LD-1 1 patient successfully treated, free of disease with normal LD-1. 1 patient with initial normal LD-1

Successfully treated, free of disease with normal LD-1. FIG. 2. Outcome of seven patients with Stage III-B-3 disease.

with stage III-B-5 disease had elevations of LD-1 values. Our study also indicates the usefulness of evaluating the LD-1 activity in terms of both its absolute and relative values. Interpretation of LD-1 activity only on the basis of its relative ratio to the total LD activity (criteria / / 8 patients with i n i t i a l criterion 1 elevation of LD-1

1 and 2) would have identified a total of 28 patients (67%). A criterion 3 elevation was demonstrated in 6 (14%) additional patients. It is important to realize that increased total LD activity may not only originate in tumor cells but also be related to the release of isoenzymes from other tissues; this may result in a conversion

5 patients responded to therapy, free of disease with normal follow-up LD-1 values. 2 patients i n i t i a l l y responded to therapy with temporary remission and return of LD-1 to normal. Both patients had recurrence of disease with reaDpearance of c r i t e r i o n 1 elevation. One of them had l i v e r metastasis.

1 patient who also had advanced pulmonary disease with metastases in lungs, mediastinum and r i g h t pleural cavity responded well to chemotherapy with decreasing LD-1 values. He refused the 4th course of chemotherapy, was discharged against medical advise and had been lost to follow-up f o r 6 months. After 6 months he came back to the hospital with end-stage refractory disease with persistently high c r i t e r i o n 1 elevation. Autopsy revealed massive metastases in lungs, l i v e r , spleen, retroperitoneum, lymph nodes, and bone marrow. ( I n addition to c r i t e r i o n I elevation of LD-1 in his serum, his pleural f l u i d had a t o t a l LD value of 520 IU/L, LD-1 absolute value of 452 IU/L and LD-1/Total LD Ratio of 87%). 1 patient had both advanced pulmonary and abdominal disease, responded to therapy temporarily with return of LD-1 to normal. Recurrent disease with metastasis i n l i v e r and persistently elevated LD-1 values. 2 patients with i n i t i a l criterion 3 elevation of LD-1 1 patient responded to therapy, is free of disease with normal follow-up LD-1 values. 1 patient with i n i t i a l normal LD-1

Has no evidence of disease after therapy with normal follow-up LD-1 values. FIG. 3. Outcome of 11 patients with Stage III-B-4 disease.


\

LIU ET AL.

182

A.J.C.P. • August 1982

4 patients refused to receive therapy or to complete the entire course of therapy, had progression of disease and persistent abnormal LD-1 values. Two of them have liver metastasis.

/

11 patients with criterion 1 5 patients responded to therapy for various periods of partial or complete remission with return of elevation, all LD-1 to normal. Recurrence with progression of disease correlated with re-elevation and persistent have end-stage abnormal levels of LD-1. All have metastasis in the liver refractory -^^ disease 1 patient was unresponsive to therapy with persistent high LD-1 values and liver metastasis. 1 patient with liver metastasis responded to therapy, is in complete remission and has normal LD-1 values. 1 patient with liver metastasis decided not to have chemotherapy, has persistantly elevated LD-1 values.

/

2 patients with i n i t i a l criterion 3 elevation, end-stage refractory disease

\

Responded well to therapy, had no evidence of disease with normal follow-up LD-1 values. FIG. 4. Outcome of 14 patients with Stage III-B-5 disease.

of the LD-1/Total LD ratio to a normal value. Consideration of the LD-5/LD-1 ratio may help to identify those cases in which the total LD abnormality is due to hepatic cell damage unrelated to metastatic disease. In the case of metastatic disease to the liver, we observed 14 patients in which the predominant abnormality in the serum LD activity occurred in the LD-1 fraction. Logically, metastatic disease to the liver should result in an increase of the LD-5 fraction secondary to damage of the liver parenchyma. This observation strongly suggests that testicular germ-cell tumors synthesize predominantly the LD-1 isoenzyme and release it into the circulation. Also, the presence of LD1 abnormality in a pleural fluid of a patient with metastatic testicular cancer suggests that the tumor cells are the source of LD-1 isoenzymes. The results of our clinical correlation studies show that patients who responded to therapy demonstrated decreasing LD-1 values that returned to normal during remission. A rise in LD-1 values with persistent elevation of the LD-1 isoenzyme appears to be a useful tumor marker for assessing the response to therapy and monitoring for disease recurrence. However, since LD1 values may also be elevated in other disease conditions it is important to rule put the presence of myocardial injury, hemolytic disorders, and renal infarction. Our study of 42 stage III patients with testicular germ-cell malignancy strongly suggests that the serum LD-1 level is a useful tumor marker for advanced testicular cancer. However, additional studies are required to verify the presence of LD-1 in primary tumor and metastatic tissues of different histopathologic types.

Acknowledgments. The authors sincerely wish to thank Mr. Robert Crawford and Mr. Dan Cook for providing all of the computer data necessary for this study; Ms. Christine Clayton and Ms. Patricia Bunns for the manuscript's preparation.

References 1. Boyle LE, Samuels ML: Serum LDH activity and isozyme patterns in nonseminomatous germinal (NSG) testis tumors (abstract). Proc Am Assoc Cancer Res 1977; 18:278 2. Brindley CO, Frances FL: Serum lactic dehydrogenase and glutamicoxaloacetic transaminase correlations with measurements of tumor masses during therapy. Cancer Res. 1963; 23:112117 3. Cohen L, Djordjevich J, Ormiste V: Serum lactic dehydrogenase isozyme patterns in cardiovascular and other diseases, with particular reference to acute myocardial infarction. J Lab Clin Med 1964; 64:355-374 4. Dixon FJ, Moore RA: Testicular tumors: a clinicopathological study. Cancer 1953; 6:427-454 5. Fraley EE, Lange PH, Williams RD, Ortlip SA: Staging of early nonseminomatous germ-cell testicular cancer. Cancer 1980; 45:1762-1767 6. Galen RS: The enzyme diagnosis of myocardial infarction. Hum Pathol 1975; 6:141-155 7. Goodfriend TL, Kaplan NO: Isoenzymes in clinical diagnosis. Circulation 1965; 32:1010-1019 8. Grayhack JT, Wendel EF, Lee C, Oliver L, Cohen E: Lactate dehydrogenase isoenzymes in human prostatic fluid: an aid in recognition of malignancy? J Urol 1977; 118:204-208 9. Javadpour N: The role of biologic tumor markers in testicular cancer. Cancer 1980; 45:1755-1761 10. Javadpour N: Improved staging for testicular cancer using biologic tumor markers: a prospective study. J Urol 1980; 124:5859 11. Javadpour N, Mclntire K.R, Waldman TA: Human chorionic gonadotropin (HCG) and alpha-fetoprotein (AFP) in sera and tumor cells of patients with testicular seminoma. A prospective study. Cancer 1978; 42:2768-2772 12. Javadpour N: Applications of biologic tumor markers in testicular cancer. Cancer Treat Rep 1979; 63:1643-1647


1 patient with i n i t i a l normal LD-1 with bone metastasis

1 patient with brain metastasis was resistant to therapy, and progression of disease with persistent abnormal serum LD-1.

Vol. 78 • No. 2


25. Samuels ML, Johnson DE, Brown B, Brachen RB, Moran ME, von Eschenbach A: Velban plus continuous infusion bleomycin (VB-3) in stage III advanced testicular cancer: results in 99 patients with a note on high dose velban and sequential cisplatinum. Cancer of the Genitourinary Tract. Edited by DE Johnson, ML Samuels, New York, Raven Press, 1979; pp. 159-172 26. Scardino PT, Cox HD, Waldmann TA, Mclntire KR, Mittenmeyer B, Javadpour N: The value of serum tumor markers in the staging and prognosis of germ cell tumors of the testis. J Urol 1977; 118:994-999 27. Talageri VR: Evaluation of plasma lactate dehydrogenase (LDH) isoenzymes in cancer patients. II: Qualitative and quantitative assessment in patients with oral cancers and osetosarcoma. Indian J Cancer 1977; 14:50-57 28. Vesell ES, Beam AG: Isoenzymes of lactic dehydrogenase in human tissues. J Clin Invest 1961; 40:586-591 29. von Eyben FE: Biochemical markers in advanced testicular tumors. Serum lactate dehydrogenase, urinary chorionic gonadotropin and total urinary estrogens. Cancer 1978; 41:648-652 30. Vugrin D, Friedman A, Schwartz M, Golbey RB: Serum lactate dehydrogenase (LDH) isoenzyme 1 (LDH-1) and 2 (LDH-2) as tumor marker in germ cell tumor (GCT) (abstract). Proc Am Assoc Cancer Res 1980; 21:177 31. Wacker WEC, Ulmer DD, Vallee BL: Metalloenzymes and myocardial infarction. II. Malic and lactic dehydrogenase activities and zinc concentration in serum. N Engl J Med 1956; 255:449456 32. Wampler GL, Hazra T: Use of LDH isoenzyme 1 as a serum marker for seminoma (abstract). Proc Am Assoc Cancer Res 1977; 18:339 33. Wilkinson JH: Clinical applications of isoenzymes. Clin Chem 1970; 16:733-739 34. Zondag HA, Klein F: Clinical applications of lactate dehydrogenase isoenzymes: alterations in malignancy. Ann NY Acad Sci 1968; 151:578-586


13. Kaplan NO: Lactate dehydrogenase—structure and function. Brookhaven Symp Biol 1964; 17:131-153 >, . 14. Kinumaki H, Takeuchi H, Ohmi K: Serum lactate dehydrogenase isoenzyme pattern in neuroblastoma. Eur J Pediatr 1976; 123:83-87 15. Lieskovsky G, Skinner DG: Significance of serum lactic dehydrogenase in stage B and C non-seminomatous testis tumors. J Urol 1980; 123:516-517 16. London IL, Hoffsten P, Perkoff GT, Pennington TG: Renal infarction: elevation of serum and urinary lactic dehydrogenase (LDH). Arch Intern Med 1968; 121:87-90 17. Markert CL: Lactate dehydrogenase isoenzymes: dissociation and recombination of subunits. Science 1963; 140:1329-1330 18. McCarthy CF, Fraser ID, Read AE: Plasma lactate dehydrogenase in megaloblastic anemia. J Clin Pathol 1966; 19:51-54 19. Oliver JA, ElHilali MM, Belitsky P, MacKinnon KJ: LDH isoenzymes in benign and malignant prostatic tissue. The LDHV/I ratio is an index of malignancy. Cancer 1970; 25:863-866 20. Paloheimo JA, Ikkala E: Serum lactic dehydrogenase activity and isoenzyme patterns in some hemaetological diseases. Acta Med Scand. 1965; 177:115-120 21. Pearson JC, Spaulding JT, Friedman MA: Testicular cancer: role of biological tumor markers. Cancer Treat Rev 1979; 6:217221 22. Plagemann PGW, Gregory KF, Wroblewski F: The electrophoretically distinct forms of mammalian lactic dehydrogenase. J Biol Chem 1960; 235:2282-2293 23. Roe CR, Limbird LE, Wagner GS, Nerenberg ST: Combined isoenzyme analysis in the diagnosis of myocardial injury: application of electrophoretic methods for the detection and quantitation of the creatine phosphokinase MB isoenzyme. J Lab Clin Med 1972; 80:577-590 24. Sandhu RS, Conover RE: Unusually high creatine kinase BB isoenzyme and study of lactate dehydrogenase pattern in metastatic adenocarcinoma of the prostate. Clin Biochem. 1980; 13:30-33

183