Correlation of UDP-galactose glycoprotein: Galactosy-transferase levels in the sera with the clinical status of ovarian cancer patients

Cancer Letters, .~ (!97,~) 239--244 © Elsevier/North-HollandScientific Publishers Ltd.

239

CORRELATION OF UDP-GALACTOSE GLYCOPROTEIN:GALACTOSYLTRAN,~FERASE LEVELS IN TIqiES E R A WITH Tile CLLNICAL S T A T U S OF OVAR.~AN CANCER PATIENTS

SUNIL K CHATTERJEE,MALAYAt;HATTACHARYAand JOSEPH J. BARLOW Departmel~t o f Gynecology, Roswell Park Memorial In.~titute, Buffa.lo, New York J~4263 (U.S.A.)

(l~.eceived ]~4 July 1978)

(Accepted 2'7 July 1978)

SUMMARY' Uridine diphosphate-galactose : glycoprotein galactosylt~'ansferase (EC 2.4.1.22) wa.~ measured serially prior to and after surgery in 4 patients with ovarian epi~helial cancer. The levels of this enzyme in the sera correlated we~t with the clinical status of the patients. In 2 other patients, the follow-up was designed to, dete,ct reeurzence, and the enzyme assay was s~a~:~ed when the patients were clinically disease free. Elevation of ga',aci~osyltransferase prec~;ded the clinical appea~,~mce of disease by 3--7 months. Serial determination of glycoprotein galactosylLtansferase in serum may be useful for evaluating the effectiveness of therapeutic programs.

INTRODUCTION

The specffid activity of UDP-galaetose : glyc:oprotdn galactosyltransferase (EC 2.4.1.22) in homogenates prepared from ovarian epithelial tumors is 2--15fold higher than that from n o r m ~ ovaries. The levels of this enzyme in 'the sera of these patiem~ are also ,eltevated in comparison with the age .and blood group matched controls [2,4-]. In a group of 30 patients with ovarian adenocarcinoma, 6 differem~ glycosyltransfe:rases have been measured; among these, only galactosyltransferase a,~:tivitywas consistently elevated in all 30 patients. T h u s g~actosyltr~msferase appeared to be the best enzyme marker for ovarian epithelial carcinoma [4]. ]:n order to study the usefulness of this assay for following the progression and ;egression of ~he. di,~ease, galactosyltransferase wa.~ serially de~ermired in sera of ovvjdan cancer patients during therapy.. Results obtained from 6 such patients are presented in this communication. Address all correspon~deace to: Su~il K. Chatterjee, Ph.D., Department of Gynecology,

RosweH Park Merao~ialInstitute, Buffalo, New York 14263, U.S.A.

240 MATERIALS AND METHODS

UDP-[ ~4C] gaJactose (301 Ci/mol) was obtaiLned from New England Nuclear, Boston, Mass. Other chemicah were the pure:~t avmlable from comraerci~d sources.

Assay o f galactosyltransferase Our earlier assay procedure [2] was slightly modified as follows. The reaction volume was reduced ~o 20 #1, which contained 50 mM 1;ris-maleate (pH 6.2), 5 #! servm, 20 mM NInOl:, 0.5 mM ATP, 2.5 mM DTT, 0.5% ~riton X-100, 80 ug (Lowry protein) of accepto:r (fetuin from which termimtt sialic acid and penultimate galactose were removed), 1 M UDP-[~4C]galactose together with 2 nmol o:f unlabeled UDP-galacto,~e. Incubation was for 1 h at 37°C with continuous shaking. All subsequent, steps were similar to those described earlier [2]. RESULTS

Linearity and other optimum conditions o[ assay Conditions for linear reaction rate and other optimum conditions for the assay were maintained throughout the experiments. No significant effect of age, blood group, sex or ethnic origSn of the blood donors were. noted on t-heir serum galactosyltransferase activity. Serial determination o f galacto~~yltransferase in t'he sere o f ovarign adenocarcinoma patients and their cEnicat status Galactosyltransferase w ~ assayed in the sera of ovarian cance'.r patients before surge~y and at varie,us time intervals the:reafte.~-. The re~ml~Lsare shown in Fig. 1. Patient OS-85 had poorly differentiated ovari~ln adenocarcinoma, Stage III. She had had progression of the disease with Cyl~oxan, 5-flurouracil ~nd CParvum when her first s e ~ m galactosyltrmlsferase'level was determined. Tumor reductive surgery (first t ) removed approx. 75% of the tumor.., and chenaotherapy was changed to high dose: Methotrexate and Cytoxan. When the enzyme was assayed just after surgery, it vvt~sfound to be e~evated, but. in ',Shetallowing 2 months, the level dropped again. During the next 2-monthly visits, the levels were about 2 times that of the noz~aaalmean. Ex~minatioJ~ at the point of the second ~ showed that ~,lmosl; the entire peritoneal cavity was replaced by tumor. Her chemotherapy was again changed, bu~ her condition deteriorated and she exph:ed about 2 monLhs after the last g.'~lactosyltransferase ass;.ay. Patient OS-18 had Stage IV moderately differentiated serous adenocarcinoma of the ovary with metastasis to the anterior abdominal wall. She was receiving high dose Methotrexate and Cytoxan, but her disease was progressing slowly when we startx.d to measure the enzyme level in her serum. A~fter tumor reducfive surgery (first 4,), followed by treatment with c/s-platinum, the level

241 SERIAL DETERMINATION OF GAL~CTOSYL TRANSFEI~ASE LEVELS IN SERUM 6C t[~

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dropped[ to almost normal. By clinical exan0ination, no evidence of disease was apparent until the position of the second ~. At this point Lt was suspected that she had recurrence which w ~ confirmed later. She was switched to 2 new drugs but progression continued. She had more tu mo r reductive surgery at the position of the third ,;, emd is now doing well on oral 5-fluorouraci] and Cyl;oxan. In this patient, the elevation o.f galactosyltransferase p:receded the clinic~ detection of recu~ence by 4--5 months. Patient OS-14 had Stage III poorly differentiated sezous, cystadenocarcinoma of the ovary. Her first galactosyltransferase level was meas,ared about 1 week before surgery. After sul:gery (~) she received 5-flu orouracil and Cytoxaa. On this treatraent, her disease has been stationary. Her sen~m enzyme leve't dropped after surgeD' and has remained constant until new, although this tevet is s~fll higher than the upper limit of the :ac,lznal range (normal mean +2 S.D.) reflecting residual dfts(!ase. Patient OS-12 had a Stage IIl, serous cystadenocarcinoma of the ovacj, Surgery removed 85~ of the t u m o r (at the point of the first f }. Following surgery, she was treated with a combination of high dose Methotrexate:,

242 C y t o x a n and C-Parvum. Pier serur/1 e n z y m e level ,was elevated transiently after surgery but d r o p p e d considerably afterwards. SI~e continues to have stationary disease, although her e n z y m e level rose slightly d a ~ l g the 9--11th m o n t h of follow-up. The reason for this t e m p o r a r y elevati[on is uncertain.

Detection o f recurrence by serial determi'nation o f galactosyltransferase in serum. It has been already n o t e d t h a t in patient OS-ZS, elevation o f serum e n z y m e p:receded clinical d e t e c t i o n of" recurrence by 4--5 months. In experiments designed to detect recurrence, s e r i a / d e t e ~ d n a t i o n s of galactosyltransferase &re being d o n e on a n u m b e r of patients w h o have been disease h'ee for at ]east 6--12 months. Results with 2 patients w h o recently had recurrence are presented in Fig. 2. Patient OS-74 had a Stage Ill p o o r l y differenl;iated papillary serous cysta d e n o c a m i n o m a o f t h e ovalsr in 1975. A f t e r second l o o k surgery, she was considered to be cu~ed and her che~aotherapy was stopped. No eviden.ce o f disez~e was £ound until (NED $). At the t i m e of ($) it was suspected that she had recurrence. It was confirmed later by sux'gery that she had an inoperable recurrence. In this patient, the se~am level o f ~alactosyltransferase was already elevated when the serial assays were begun, approx. 7--9 m o n t h s before the recurrence was clinically coJa~Srmed.

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cancer patients. See legen,]s ~o Fig. 1 for de~ails. NED, r o evidlence of disease; Prog., progre&sion of disease.

Patient OS..4 had Stage HI poorly differenl~;iated adenocarcinoma o f the ovary in 1973 which was tree~ed b y surgery f~c,llowed by radiation. She had a recurrence in ].976. When we first measured her serum enzyme level, she already had had 6 courses of M,flph~an and was respond:ing to this drug. Her clinical status before (prog 4) was no evidence of disease. When the disease progression was col~firm.ed, h,l~r c h e m o t h e r a p y protocol was changed. Here ag;tin, at least 3 months befor~ clinical confi~'~ation o f progression, the seru~m galactosylfransferase level starl|ed rising. DISCUSSION

Elevation of circulating glycosyltransferase~; i~l patients with various neoplastic diseases have been repol~led from sever~l laboratories. Sialyltransferase levels have been found to be eh~vated in large percentage o f patients with vadous malignancies ~6--8]. Henderson and K~ssei [6] have found a correlation begween sialyltransferase le~'els in plasma m d course o~ disease in 46 of 51' patients studied serially. Fucc,syltran~sferases :in sera and plasma of patients have also been reported to be go4,d markers o f malignancy [1,9]. Weiser et td. [ 13 ] found that on the average the ito~a][serum gatactosyltransferase activits~ J~ cancer patients was only slightly higher that. t h a t o f ~he control subjects. However, by discontinuous poly~Lcrylamide gel electrophoresis they detected an isoenzyme o f gaJac~osyltransfcrase in the sera of 43 of 58 patients with v~rious types of malignancies. ,~nong that gro:tp o f patients, only 1 had ovarian cancer, eua
244 o f t h e cell surface (~m e c t o e n z y r a e ) . O n e m e c h a u i s m of a p p e a r a n c e of this e n z y m e m ~ne s e r u m may: be t h e s h e d d i n g o f p l a s m a ]ne:mbrane c o n s ; i t u e n t s i n t o t h e s y s t e m i c c i r c u l a t i o n [ 5 ] , as we p r o p o s e d f o r c y t i d i n e 5 ' - m o n o p h o s pho-N-acetyI-neuram~n~c acid h y d r o l a s e [ 3 ] . S~udies are n o w ir~ progress fox t]he e l u c i d a t i o n o f t h e m e c h a n i s m o f galactosyltransferase release, i n t o the, c i r c u l a t i o n . F u r t h e r know~edge a b o u t this enzyme:, m a y h e l p u s devel<)p a )~aethod for t h e early d e t e c t i o n o f ovarian cancer. ACK~[OWLEDGEMENTS

We t h a n k Angela D i L o r o ~nd X o o n - S h e n O u f o r t h e i r t e c h n i c a l assistauce. This w o r k was s u p p o r t e d in p a r t b y t h e N a t i o n a l C a n c e r I n s t i t u t e C o n t r a c t s NO1-CB-64013 and NO1-CM-67t17. REFEI:,~ENCES

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2 Bhattacharya, M., Chatterjee, S.K. and Barlow, J.J. C:.976) Uridine 5'-diphosphategalae.tose : glycop~otein galactosyltransferase activity in the ovarian cancer patient. Can~.'er IRes., 36, 2096~210Z. 3 Chal~terjee, S.K., Bhattacharya, M. and Barlow, J.J. (1978') Elevated activity of cy'~idine 5'-m~nophospho-N-acetyl neurarr.dnic acid hydrolase i~, selmm of ovarian cancer patients as a possible indicator of malignancy. Bioehem. Biophys. Res. Commun., ~0,826--832. 4 Chatz;erjee, S.K., Bhattaeharya, M. and Barlow, J.J. (1978) Glycoprotcin ~Ilycosyltransferasc (GT) and glycosidase (G,~) activities in the ovarian c.'mcer patient, Prec. Am. Argot. Cancer Res., 19, 165. 5 Chatterjee, S.K. and Kim, U. (1977) Galactosyltransferase acti,rity in rnet~tasizing and nonmvtasta~izing rat mammary caxcinomas and its possible zelationshilc~with tumor cell surface antigen shedding. J. Natl. Cancer Inst., 58, 273--280. 6 Henderson, M. and Kessel, D. (1977) Alterations in plasma sialylt~armferase levels in patienLs with neoplastic disease. Cancer, 39, 1129--113 ~. 7 Ip, C..rod Dao, T. (1978) Alterations in serum glycosy]lltransfera~es .~md 5'-nucleotida~.e in breast cancer patients. Cancer Res., 38,723~728. 8 Kesse], D. and Allen, J. (1975) Elevated plasma sialyltre~sfer~e in ~he cancer ~k~ent. Cancer Res., 35, 670--672. 9 KhilamLni, P., Chou, T.H., R~tanatharathorn, V. and Ke~.~sel,Y). (1978) E~aluation of two phsma fucosyltransferases as marker enzymes in n,Dnhodgkin's lymphoma. Cancer, 41,701--705. :[0 Kips, Y.S., Whitehead, fi[.S. ~nd Curtis, K.J. (1972) Glyeosyltransferazes in human blood. IL Study of serum galactosyltransferase ~md N-aeetylgalactns~minyltransferase in patients with ]~ver diseases. J. Clan. Invest. 51, 20~3--2(}39. t 1 Porter, C.W. and Bernaeki, R.J. (197,5) Ultra~¢rtmtural evidence for ectoglycosyttransferase s:~stems.Nature, 256,648--'650, 12 Roth, S. and White, D. (1972)]ntraeelD/]~:contact and =e]l~urfaeeg~Jaetosyltransfer~ e acti~,ty.Prec. Na~l. Acad. SeL US.A, 69,485--4S9. 13 Weiser, M., Podolsky , D.K. and ~elhaeher, K.J. (1976 ) Caaeer.a~.s~,eiatedisoenzyme of serum ga!actosyltransfervse.Prec. Natl. Acad. ScL USA, 73, 1319--i~22.