ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 13, No. 2 Copyright © 1983, Institute for Clinical Science, Inc.

Alkaline Phosphatase as Tumor Marker SHESHADRI NARAYANAN, Ph.D. Department of Pathology, New York Medical College, Metropolitan Hospital Center, New York, NY 10029

ABSTRACT Applications and limitations of alkaline phosphatase as a tumor marker are discussed. This review focuses on three characteristic isoenzymes that have been found in the serum of cancer patients: (1) the Regan isoenzyme in bronchogenic carcinoma; (2) the Nagao isoenzyme in pleuritis car- cinomatosa; and (3) the hepatoma alkaline phosphatase. Tumor variants of alkaline phosphatase are classified based on their heat stability properties resembling or differing from the placental isoenzyme. They can also be differentiated in their behavior to specific inhibitors. The presence of electrophoretic variants and enzyme-immunoglobulin complexes in other cancers are also described.

Introduction in malignancies such as prostataic car­ cinoma.1 Since the identification and characteri­ zation of a unique isoenzyme of alkaline Methods For Studying Tumor phosphatase, indistinguishable from the Alkaline Phosphatase placental isoenzyme, in the serum of a patient with bronchogenic carcinoma Heat Treatment 4,13 (the Regan isoenzyme5), numerous re­ ports have appeared on the existence of Tumor alkaline phosphatase can be characteristic isoenzymes of alkaline distinguished from the placental isoen­ phosphatase in the sera of patients with zyme in its sensitivity to heat, the latter various malignancies.8,13,20 Unique isoen­ being heat-stable at 65°C for 10 minutes. zymes have been broadly characterized in relation to the placental isoenzyme. Electrophoresis 4,8,11,13,17 In addition to the Regan isoenzyme, the Nagao isoenzyme13,14 and hepatoma Several types of zone media have been alkaline phosphatase isoenzyme have utilized including polyacrylamide, starch been well characterized.7,8,17,19 and agar gels. Subsequent to electropho­ Enzymes may exist in the circulation resis alkaline phosphatase activity is vis­ complexed to immunoglobulins, and this ualized by staining the gel with a sub­ has led to the identification of alkaline strate disodium a-naphthyl phosphate, a phosphatase-immunoglobulin complexes dye such as Fast blue BB salt in Tris or 133 0091-7370/83/0300-0133 $00.60 © Institute for Clinical Science, Inc. 134 NARAYANAN carbonate buffer containing a magnesium the inhibition may be as high as 70 to 80 salt. percent.4,13 Inhibition by L-phenylal­ anine is of the noncompetitive type. Immunoelectrophoresis 1 Serum containing Regan isoenzyme can be precipitated by antibody to placental This procedure followed by staining alkaline phosphatase. Following incuba­ for alkaline phosphatase is used for iden­ tion at 65°C for 10 minutes, both the pla­ tification of immunoglobulin-enzyme cental and Regan isoenzymes retain vir­ complexes. tually all the original activity. The Regan isoenzyme is more sensitive to urea than Gel Permeation Chromatography 3 the placental isoenzyme suggesting dif­ ferences in three-dimensional structure.4 Chromatography of sera on an appro­ priate gel permeation column (such as Nagao Isoenzyme Sephadex-G-200) is useful for the sep­ aration of high molecular weight alkaline This isoenzyme, first reported in the phosphatases complexed to an immuno­ serum of a patient named Nagao who had globulin or abnormal lipid, Lipopro- pleuritis carcinomatosa, resembles the tine-X. Regan isoenzyme in that it is stable after incubation at 65°C for 10 minutes, has a S p e c if ic I n h ib it o r s similar electrophoretic mobility, and is precipitated by antibody to the placental Measurement of alkaline phosphatase enzyme.13,14 However, it is more sensitive activity, following incubation with organ to L-phenylalanine at a concentration of 1 specific inhibitors of alkaline phos­ mM, in that 59 percent of the enzyme phatase, L-phenylalanine, and L-homo- activity is inhibited as compared to 23 per­ arginine, is used to distinguish placental cent for the Regan isoenzyme.13 Its sen­ and non-placental types of tumor alka­ sitivity to L-leucine and EDTA is pro­ line phosphatase. Incubation with L-leu- nounced as compared to the Regan cine is used to identify the Nagao iso­ isoenzyme which is only minimally in­ enzyme.6,13,14 hibited. In table I, the behavior of Nagao Other inhibitors that can be employed and Regan isoenzymes to L-leucine and include ethylenediamine tetraacetic acid EDTA is compared.13 The Nagao isoen­ (EDTA) and urea.8 zyme has an increased affinity for the substrate phenylphosphate as compared Regan Isoenzyme to the Regan isoenzyme. The Km for this substrate using the Nagao isoenzyme was This isoenzyme first reported in a pa­ tient named Peter Regan with bron­ chogenic carcinoma is virtually indistin­ TABLE I guishable from the placental enzyme in Inhibitor Differences Between its electrophoretic mobility and sensitiv­ Nago and Regan Isoenzymes ity to heat.5 Like the placental enzyme, it P erc e n t is also sensitive to L-phenylalanine. The Concentra ti on I n h ib i tio n degree of inhibition is dependent on the Inhibitor (mM) Regan Nagao concentration of L-phenylalanine. At 1 L-leucine 0.4 3 50 mM L-phenylalanine concentration, both EDTA* 10 7 90 the placental and the Regan isoenzyme are inhibited 23 percent whereas at 5 mM ♦Ethylenediamine tetraacetic acid ALKALINE PHOSPHATE AS TUMOR MARKER 135 0.26 mM as compared to a Km of 2.2 mM Other Malignancies obtained with the Regan isoenzyme.13 The Nagao isoenzyme has been re­ The presence of alkaline phosphatase ported to be a variant of placental al­ with altered electrophoretic mobility and kaline phosphatase, a hybrid, perhaps, of properties in various malignancies such liver and placental alkaline phosphatase, as carcinoma of ovary,16 bile duct,10 the D-variant.9 However, the identity stomach,15 pancreas,18 colon and rectum2 between the Nagao isoenzyme and the have been reported. However, definitive D-variant has been questioned.13 studies are needed before these isoen­ zymes can be regarded as specific tumor Hepatoma Alkaline Phosphatase markers. Recently an alkaline phosphatase vari­ Sera of patients with hepatoma have ant was described in the serum of a pa­ circulating alkaline phosphatase which is tient with renal cell carcinoma.20 The heat sensitive. Whereas the Regan and variant appeared to be an altered form of Nagao isoenzymes are virtually stable normal renal alkaline phosphatase. It is after incubation at 65°C for 10 minutes, as more resistant to heat than liver alkaline much as 79 percent of the hepatoma al­ phosphatase, the half-inactivation time at kaline phosphatase activity is inhibited.8 56°C being 184 seconds for the variant as Inhibition studies using 1 mM L- compared to 112 and 456 seconds for phenylalanine and 0.5 mM L-leucine bone and liver alkaline phosphatases, re­ suggest that it is more akin to the Regan spectively. The variant also migrates as compared to the Nagao isoenzyme. differently from liver, intestinal, and kid­ Similarly, it is not particularly sensitive ney alkaline phosphatase on polyacryl­ to L-homoarginine, an inhibitor of liver amide gel electrophoresis. The migration alkaline phosphatase, thus resembling is closer to that of the bone isoenzyme, Regan isoenzyme. However, like the although not identical to it. Nagao isoenzyme the hepatoma alkaline The presence of serum alkaline phos­ phosphatase is highly sensitive to EDTA. phatase complexed to IgG has been re­ In the presence of urea it is more stable ported in a patient with carcinoma of than liver alkaline phosphatase but less prostate.1 The complex had a reduced so than placental alkaline phosphatase.17 mobility on cellulose acetate electropho­ In table II is summarized the sensitivity resis distinct from liver, bone, placental, of hepatoma alkaline phosphatase to and intestinal bands. Evidence for an various inhibitors.8 IgG-alkaline phosphatase complex was demonstrated by the presence of enzyme activity in the immunoprecipitin arc and by dissociation of the complex with tryp­ TABLE II sin. The inhibitor characteristics and heat Sensitivity of Hepatoma Alkaline Phosphatase to Inhibitors sensitivity of the complex were inter­ mediate between that of liver and bone Concentration P ercent alkaline phosphatase. In h ib ito r (mM) In h ib itio n

L-phenylalanine 1.0 41 Summary L-leucine 0.5 13 EDTA* 0.5 83 L-homoarginine 10.0 28 The usefulness of alkaline phosphatase Urea 4.0 66 as a tumor marker will require more de­ finitive studies on the variants that have *Ethylenediamine tetraacetic acid been described. Limitations in present 136 NARAYANAN methodologies include accurate resolu­ phenylalanine, L-tryptophan and L-homoar- tion and identification of isoenzyme ginine. Enzymologica 41:141 —167, 1971. 7. H i g a s h i n o , K., H ashinotsume , M., K a n g , K., bands on electrophoresis, since these are T a k a h a s h i , Y., and Y a m a m u r a , Y.: Studies on complicated with closely migrating variant alkaline phosphatase in sera of patients with hepatocellular carcinoma. Clin. Chim. bands. Identification of a variant based Acta 40:67—81, 1972. on its sensitivity to L-phenylalanine is 8. H i g a s h i n o , K ., K u d o , S., O h t a n i e , R., and limited since inhibition also occurs with Y a m a m u r a , Y.: A hepatoma-associated alkaline phosphatase, the kasahara isozyme compared the placental and intestinal isoenzymes. with one of the isozymes of FL amnion cells. The Regan isoenzyme has been reported Ann. N Y , Acad. Sci. 259:337-346, 1975. in some normal subjects, although not at 9. I n g l i s , N. R., K i r l e y , S., S t o l b a c h , L. L., and F i s h m a n , W . H .: Phenotypes of the Regan such high concentrations as found in pa­ isoenzyme and identity between the placental tients with tumors.12 D-Variant and the Nagao isoenzyme. Cancer In spite of these limitations, variant al­ Res. 33:1657-1661, 1973. 10. J a c o b y , B . and B a g s h a w e , K . D.: Placental-type kaline phosphatases offer promise as alkaline phosphatase from human tumour tis­ tumor markers. The discovery of variants sue. Clin. Chim. Acta 35:473—481, 1971. on electrophoresis and characterization 11. M a s u z a w a , M . , K a m a d a , T., S a k o y a m a , Y., O g i t a , Z., and I t o , F.: Detection of carcinopla- of variants or variant complexes formed cental alkaline phosphatase by thin layer poly­ with immunoglobulin or Lipoprotein-X acrylamide gel electrophoresis. Ann. NY Acad. ought to increase our understanding of Sci. 259:321-324, 1975. 12. M c C o m b , R. B ., B o w e r s , G. N., and P o s e n , S.: tumor alkaline phosphatases in cancer. Serum alkaline phosphatase activity in patients Hopefully, the identification of tumor with various malignant neoplasms. Alkaline related variants early in the course of dis­ Phosphatase. New York, Plenum Press, 1979, ease should facilitate treatment before pp. 478-479. 13. N a k a y a m a , T. and K i t a m u r a , M.: L-leucine métastasés occurs. sensitive alkaline phosphatase isozyme from cancer tissue. Ann. N Y Acad. Sci. 259:325—336, 1975. References 14. N a k a y a m a , T., Y o s h i d a , M., and K i t a m u r a , M.: L-leucine sensitive, heat stable alkaline phos­ 1. B u t t e r y , J. E., M i l n e r , C. R., N e n a d o v i c , P., phatase isoenzyme detected in a patient with and P a n n a l , P . R.: Detection of alkaline pleuritis carcinomatosa. Clin. Chim. Acta phosphatase/immunoglobulin complexes. Clin. 30:546-548, 1970. Chem. 26:1620-1621, 1980. 15. R o m s l o , I., B j a r k , P., and S o l b e r g , C. O .: Ec­ 2. F a b r i c i u s -B j e r r e , N., H a n e l , H . K., and topic alkaline phosphatase production in H o l s t -C h r i s t e n s e n , J.: Alkaline phosphatase metastasizing ventricular carcinoma. Scand. J. in colorectal cancer: A quantitative analysis of Clin. Lab. Invest. 28:21-26, 1971. the enzyme content. Scand. J. Gastroenterol. 16. S t o l b a c h , L. L., F i s h m a n , W . H ., and K r a n t , 7:369-373, 1972. M. J.: Ectopic production of an alkaline phos­ 3. Fennelly, J. J., Dunne, J., McGeeney, K., phatase isoenzyme in patients with cancer. Chong, L., and Fitzgerald, M.: The importance New Eng. J. Med. 281:757-762, 1969. of varying molecular size, differential heat and 17. S u z u k i , H . I i n o , S., E n d o , Y., T o r r i , M., urea inactivation of phosphatase in the identifi­ K a z u m a s a , M., and O d a , T .: Tumor-specific al­ cation of disease patterns. Ann. NY Acad. Sci. kaline phosphatase in hepatoma. Ann. NY. i 66:794—810, 1969. Acad. Sci. 259:307-320, 1975. 4. F i s h m a n , W. H.: Immunologic and biochemical 18. W a r n e s , T . W ., T i m p e r l e y , W . R., H i n e , P., and approaches to alkaline phosphatase isoenzyme K a y , G.: Pancreatic alkaline phosphatase and a analysis: The Regan isoenzyme. Ann. NY Acad. tumour variant. Gut 13:513—519, 1972. Sci. 166:745-759, 1969. 19. W a r n o c k , M. L. and R e i s m a n , R.: Variant al­ 5. F i s h m a n , W. H ., I n g l i s , N. K., S t o l b a c k , L. L., kaline phosphatase in human hepatocellular and K r a n t , M. J.: A serum alkaline phosphatase cancers. Clin. Chim. Acta 24:5-11, 1969. isoenzyme of human neoplastic cell origin. 20. W h i t a k e r , K . B ., E c k l a n d , D., H o d g s o n , Cancer Res. 28:150-154, 1968. H . G. F., S a v e r y m u t t u , S., W i l l i a m s , G., and 6. F i s h m a n , W. H. and S i e , H. G.: Organ-specific Moss, D. W .: A variant alkaline phosphatase in inhibition of human alkaline phosphatases of renal cell carcinoma. Clin. Chem. 28:374-377, liver, bone, intestine, and placenta: L- 1982.