[CANCER RESEARCH 37, 2557-2559, August 1977] Variation of Levels of Plasma Guanosine Diphosphate L.Fucose:fl-D-galactosyl a-2-L- in Acute Adult Leukemia1

Prem Khilanani,2 Ta-Hsu C.hou, Pavel L. Lomen, and David Kessel

Department of Oncology, Wayne State University School of Medicine, Detroit, Michigan 48201

SUMMARY MATERIALS AND METHODS

We have measured plasma levels of an a-2-L-fucosyltnans GDP-L-['4C]fucose (174 Ci/mole) was purchased from fenase in 18 patients with acute adult leukemia at various New England Nuclear, Boston, Mass., in 50% ethanol. The clinical stages along with simultaneous bone marrow aspi solvent was removed before use. Fetuin (Calbiochem, Los nations and biopsies. Patients in remission had significantly Angeles, Calif.) was treated with mild acid to remove tenmi lower levels of this than did nonmesponding on nal siahic acid as described by Spiro (11). Other chemicals, relapsing patients. Furthermore, plasma levels were come of reagent grade or better, were provided by Sigma Chemi lated with percentage of marrow blast cells. This enzyme calCo.,St.Louis,Mo. may serve as a biological marker for monitoring patients Plasma samples were obtained from normal donors and with acute leukemia receiving chemotherapy. It is particu leukemia patients, using EDTA as anticoagulant. RBC larly helpful as a guide to chemotherapy in patients on were removed by low-speed centnifugation, platelets were whom bone marrow aspirates are technically difficult to removed by a subsequent centnifugation at 10,000 x g for obtain. 10 mm, and the plasmas were then stored at —70°. Enzyme assays were carried out in a 200-pi system con taming50 j.dplasma;0.5mg desialatedfetuin;50mM caco INTRODUCTION dylate buffer, pH 7.0; 10 mM [ethylene-bis(oxyethylene nitrolo)]tetraacetic acid; 10 mM MgCI2; and 1 @Mundiluted The presence of 2 types of fucosyltransferase activity has [14C]GDP-fucose. N-Ethylmaleimide, a specific inhibitor of been demonstrated in human plasma. One enzyme cata a-2-L-fucosyltransferase (3), was added to duplicate sam lyzes transfer of fucose from the donor GDP-fucose onto pIes. The level of this could therefore be calcu low- on high-molecular-weight acceptors with terminal ga hated by difference, thereby correcting for the action of lactose residues (2, 8, 10); this enzyme is specified by the other on endogenous acceptors. human blood group H . Another type of enzyme uti After incubation at 37°for 60 mm, the reaction mixtures lizes, as the fucose acceptor, N-acetylglucosamine residues were washed through 0.5- x 1-cm columns of Dowex 1 (OH) of appropriate glycoproteins (8, 10). We had previously with 2 column-volumes of water. This procedure resulted in reported an elevation in total fucosyltransferase activity in the selective elution of glycoprotein but not of fucose phos plasma samples obtained from cancer patients (6). How phate on GDP-fucose, as outlined in Ref. 3. Fucose ap ever, the presence of endogenous acceptors for both types peared in the eluate, but substantially later than did gly of greatly complicated these determinations and coprotein. The madioactive product was measured by liquid prevented precise delineation of levels of each type of en scintillation counting. zyme. In initial experiments, we verified the presence of madioac Using a new procedure (3) where, in plasma, a-2-L-fuCOs tive fucose in the product by acid hydrolysis and chroma yltransfenase activity was totally inhibited and GDP-L-fu tography (8). Results are reported here in terms of activity cose:N-acetylglucosammnide fucosyltransfemase activity was units = cpm of radioactivity incorporated into product per not inhibited by a sulfhydryl reagent, N-ethylmaieimide, we 50 @lofplasma during 60-mm incubations. could unambiguously measure levels of the H gene-speci fied plasma fucosyltransfemase. The data indicate a marked correlation between plasma levels of this enzyme versus RESULTS clinicalstatusofadultswithacuteleukemia. Levels of a-2-L-fucosyltransfenase were measured on 21 occasions on 18 patients with acute leukemia. The age, sex, type of leukemia, disease status, and results of bone mar 1 Supported in part by Gnant CA 7177 from the USPHS, NIH. 2 To whom requests for reprints should be addressed. row aspiration (percentage of marrow myeloblasts) ameme ReceivedJanuary31,1977;acceptedMay 6,1977. ported in Table 1, along with plasma fucosyltransfenase

AUGUST 1977 2557

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1977 American Association for Cancer Research. P. Khilanani et a!. Table1 Plasma fucosyltransferase and disease status of patients with acute leukemias @ Data are given in terms of cpm of radioactivity incorporated per 50 of plasma during a 60-mm incubation at 37° man Serial now myelo no.PatientAgeSexFucosyl(%)“1W. transferaseStatusBone blasts remission02L. T.25M289Complete J.35F420Remission03D. remission04P.K.22F383Complete remission15J.C.21M285Complete remission165.M.53F98Complete nemission17P.W.50M509Complete remission38C.H.28F250Complete M.26M574Nonnesponding79V. D.66M499Responding7105. H.34M1047Nonnesponding1211J. H.52M1095Nonresponding1312P. M.35F943Nonresponding22130. K.22F294Relapsing2214I. response5015C.D.28M1433No R.61F720Nonresponding5016FT.61M1714Nonresponding5017M.

S.35M1458Nonnesponding6018P. L.48F1656Nonresponding6219V. treatment8420N.D.―66M1017No B.31F876Nonresponding8521L.J.b35F1077Notreatment93

‘SPercentage of myeloblasts measured by microscopy. b All patients except these 2 were receiving combination chemotherapy [adniamycmn, oncovin (vincristine), arabinosylcytosine , prednisone].

levels. In mixing experiments, we found no evidence for the presence of activators or inhibitors in any plasma prepana tionexamined. Chart 1 is a scattengram relating plasma enzyme level to percentage of marrow myeloblasts. Three clusters of en U) I- zyme values were found to correspond to disease status: M1 U) (0 to 5% myeloblasts), M2 (5 to 25% blasts), and M3 (>25% blasts). The corresponding levels of fucosyltransferase are: M1, <450 units; M2, 450 to 1000 units; and M3, >1000 units. Statistical evaluation of these data showed a correlation between percentage of blasts in marrow versus plasma enzyme level: p < 0.01 ; x2 = 16. Mean enzyme levels ±S.D. were 320 ±130 units for responders and 1030 ±430 units for nonnesponders. Mean enzyme levels (units) ±S.D. were M1status patients, 320 ± 130; M2status patients, 740 ±330; and M3 status patients, 1240 ±370. Levels of this plasma fucosyltnansferase in normal donors were 315 ±75 units (10 values).

oc:—2—L—FUCOSYLTRANSFERASE(UNITS) DISCUSSION Chart 1. Correlation between plasma levels of fucosyltransfarase and per centaga of marrow blasts in patients with acute myaloganous leukamias. Previous studies showed elevated levels of activity of Boxes show values for patients in 3 different categories: M = 0 to 5% marrow several glycosyltransfenases in plasma of the cancer pa blasts; M2, 5 to 25%; M3, 25 to 100%. r = 0.67; p < 0.01. tient. The list now includes sialyltransferase (4, 5, 12), fu cosyltransferase (6), and (1). In an plasma glycosyltransfenases in patients with neoplastic dis other study, Podolsky and Weisen (9) found that total ease. The availability of a new procedure for delineating plasma galactosyltransfenase was only slightly elevated in fucosyltransferases (3) allowed us to extend a previous patients with stomach, colon, and pancreatic tumors, but study (6) which indicated that elevated plasma fucosyltrans that a galactosyltnansferase isoenzyme appeared in plasma ferase was often associated with neoplasia. Since the acute of these patients. adult leukemias generally permit a quantitative assessment In a study designed to seek specific determinants of tu of total tumor burden, i.e. , by the measurement of percent momburden, we have compared levels of activity of several age of marrow myeloblasts, we compared these clinical

2558 CANCERRESEARCHVOL. 37

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1977 American Association for Cancer Research. Serum Fucosyltransferase data with laboratory findings. The data shown in Table 1 ACKNOWLEDGMENTS and Chart 1 indicate a marker correlation between clinical status of these patients and plasma levels of the H gene We thank Joanne Blahnik, Melody Sands, Cheryl Murphy, and Jean Devos for technical assistance; Dr. W. J. Kuhns and Prof. W. M. Watkins for helpful specified a-2-L-fucosyltnansferase. We found no such come advice; and Tern Buvia for manuscript preparation. lations when levels of plasma sialyltransfenase and galacto syltnansfenase were examined.3 In another study of the plasma a-2-L-fucosyltnansfenase, but utilizing low-molecular-weight acceptors, Kuhns et a!. REFERENCES (7) reported a result opposite to that found here: an inverse correlation between disease status and plasma fucosyl 1. Bhattacharya, M., Chatterjee, 5. K., and Barlow, J. J. Uridine-5'-Diphos phate Galactose:Glycoprotain Galactosyltransferase Activity in the Oven transfenase activity. Since levels of high-molecular-weight ian Cancer Patient. Cancer Ras., 36: 2096-2101 . 1976. endogenous enzyme acceptors were generally found to in 2. Chester, M. A., Yates, A. D., and Watkins, W. M. Phenyl f3-D-Galactopy ranoside as an Acceptor Substrate for the Blood-Group H Gene Associ crease as the disease progressed,3 we propose that compe ated Guanosina Diphosphata L-fucOse:f@-D-GaIactosyl a-2-L-Fucosyl tition between such endogenous acceptors and exogenous transfarasa. European J. Biochem., 69: 583-592, 1976. low-molecular-weight acceptors in the assay system might 3. Chou, T. H., Murphy, C. , and Kessal, D. Selective Inhibition of a Plasma Fucosyltransfarase by N-athylmaleimide. Biochem. Biophys. Ras. Corn account for this discrepancy. The latter are less efficient mun., 74: 1001-1006, 1977. acceptors of fucose transfer than is the high-molecular 4. Henderson, M., and Kassel, D. Alterations in Plasma Sialyltransfenasa Levels in Patients with Neoplastic Disease. Cancer, 39: 1129-1134, 1977. weight fetuin derivative (8). 5. Kassel, D., and Allan, J. Elevated Plasma Sialyltransferasa in the Cancer Another relevant factor in the measurement of fucosyl Patient. Cancer Ras., 35: 670-672, 1975. transfenases in plasma is the presence of serum a-fucosi 6. Kessel, D., Chou, T. H., and Henderson, M. Determinants of Fucosyl transfarasa Level in Plasma of the Cancer Patient. Proc. Am. Assoc. dase activity which can vary widely, even in controls (13). Cancer Ras., 17: 16, 1976. Under the conditions used here, we found that isolation of 7. Kuhns, W. J.. Oliver, R. T. D., and Watkins, W. M. Alterations of Serum radioactive product, followed by incubation with fresh and Tissue Enzymes in Acute Leukemia (Abstract). Am. Soc. Hamatol., No. 117, 91, 1976. plasma, resulted in <10% conversion of product to fucose@ 8. Munro, J. R., and Schachtar, H. The Presence of Two GDP-fu measured as described in Ref. 8. These experiments will be cose:Glycoprotain Fucosyltnansferasas in Human Sanum. Arch. Bio chem. Biophys.. 156: 534-542, 1973. described in detail, elsewhere. 9. Podolsky, D. K., and Waisar, M. M. Galactosyltransferasa Activities in The disease status of patients with acute leukemias can Human Sara: Detection of a Cancer-Associated Isoenzyme. Biochem. be determined by periodic bone marrow aspirations. We Biophys. Ras. Commun., 65: 545-551 , 1975. 10. Schenkel-Brunnar, H., Chester, M. A., and Watkins, W. M. a-L-Fucosyl propose here an alternative method which may not replace transferasas in Human Serum from Donors of Different ABO, Secretor the bone marrow studies, but which is sufficiently reliable to and Lewis Blood-Group Phenotypes. European J. Biochem., 30: 269- guide the course of chemotherapy in patients for whom 277,1972. 11. Spiro, R. G. Periodata Oxidation of the Glycoprotain Fetuin. J. Biol. bone marrow aspirations are technically difficult. The mea Cham., 239: 567-573, 1964. sunement of this fucosyitnansferase in plasma appears to be 12.. Warren, L., Fuhner, J. P., Buck, C. A., and Walborg, E. F., Jr. Membrane a valid biological marker for monitoring the disease status Glycoproteins in Normal and Virus-Transformed Cells. In: J. Schultz and R. E. Block (ads.), Membrane Transformations in Neoplasia, pp. 1-21. of such patients. New York: Academic Press, Inc., 1974. 13. Wood, S. Plasma Alpha-L-Fucosidasa: Presence of a Low Activity Variant 3 T. H. Chou and D. Kessal, unpublished observations. in Some Normal Individuals. J. Lab. Clin. Med., 88: 469-476, 1976.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1977 American Association for Cancer Research. Variation of Levels of Plasma Guanosine Diphosphate l-Fucose: β-d-galactosyl α-2-l-Fucosyltransferase in Acute Adult Leukemia

Prem Khilanani, Ta-Hsu Chou, Pavel L. Lomen, et al.

Cancer Res 1977;37:2557-2559.

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