Gut 1995; 36: 299-302

CASE REPORTS

A variant alkaline phosphatase found in a case ofgastric carcinoma with super bone scan

F Kobayashi, T Ikeda, S Tozuka, 0 Noguchi, T Fukuma, S Sakamoto, F Marumo,T Komoda, Y Sakagishi, C Sato

Department ofInternal Medicine,Yokosuka KyosaiHospital, Kanagawa,JapanF KobayashiT IkedaS Tozuka0 NoguchiT FukumaS Sakamoto

Second Department ofInternal Medicine andDivision ofHealthScience, Faculty ofMedicine, TokyoMedical and DentalUniversity, Tokyo,JapanF MarumoC Sato

First Department ofBiochemistry, SaitamaMedical School,Saitama, JapanT KomodaY Sakagishi

Correspondence to:Dr C Sato, SecondDepartment of InternalMedicine, Faculty ofMedicine, Tokyo Medicaland Dental University, 5-45,Yushima 1-chome,Bunkyo-ku Tokyo 113,Japan.Accepted for publication29 April 1994

AbstractA rare case ofgastric carcinoma associatedwith increased serum variant alkalinephosphatase activities is presented. A 54year old man had extremely high serumalkaline phosphatase activity (18 607 U/1)with normal calcium and phosphate con-centrations. His bone scintigram showedabnormal findings, 'super bone scan'. Hewas diagnosed as having Borrmann type 4gastric carcinoma with diffuse bone meta-stases by examinations of the uppergastrointestinal tract and iliac bone biopsy.The alkaline phosphatase isozyme of thispatient was of the bone type as measuredby cellulose acetate membrane electro-phoresis and the placentalbone type byagarose gel electrophoresis, respectively.Immunoelectrophoresis and the immuno-precipitation method using monoclonalantibodies against various alkaline phos-phatase isozymes, however, showed thathis serum alkaline phosphatase had theliver type antigenicity. Furthermore, it hada larger molecular size and dilferent sugarchains compared with the common livertype alkaline phosphatase. These findingssuggest that a unique variant alkalinephosphatase was produced by gastriccancer cells, which is possibly an explana-tion for the high serum alkaline phos-phatase activities in this patient.(Gut 1995; 36: 299-302)

Keywords: alkaline phosphatase, gastric carcinoma,super bone scan.

Localised bone metastasis is occasionally seenin patients with gastric carcinoma.' 2 Diffusemetastases to the entire skeletal system, how-ever, in which bone scintigraphy showsabnormal findings, for example super bonescan, is uncommon.3 4 In gastric carcinomawith super bone scan, several clinical featuresincluding high serum alkaline phosphatase(ALP) activities have been recognised.5 Highserum ALP activities in those patients arethought to arise from the bone, although ALPproducing gastric carcinoma has also beenreported.6 We describe a patient with gastriccarcinoma with super bone scan, in whomserum ALP was recognised as the bone type by

conventional electrophoresis, but after furtherinvestigation, was identified to be the liver typelike ALP with a larger molecular size and dif-ferent sugar chain structures compared withthe common liver type ALP. It is suggestedthat high serum ALP activities in this patientwere derived from the liver type like ALP ingastric carcinoma.

Case reportOn 22 May 1991, a 54 year old man presentedto our hospital because of increasing lumbagoand back pain since late April. He had alsolost 8 kg in weight a month. Physical examina-tion showed significant anaemia, gingivalbleeding, systolic heart murmur, and hepato-megaly, and he was admitted for furtherexaminations.

Laboratory examinations showed: anaemia(haemoglobin 4.46 mmol/l), thrombocytopenia(42 000/,ul), the appearance of immaturegranulocytes and erythroblasts in the peripheralblood, increased serum activities of lactatedehydrogenase (823 UlI), y glutamyltransferase(348 U/1), aspartate aminotransferase (90 U/1),and alanine aminotransferase (50 U/1). Theserum ALP value was extremely high (18 607U/l, reference range: 140-248 U/1). Renal func-tion and serum electrolytes including calciumand phosphate were normal. Prothrombin timewas normal, but partial prothrombin time wasslightly prolonged and the fibrin degenerativeproduct was increased (11-5 pug/dl). Serum car-cinoembryonic antigen and ferritin concentra-tions were within normal ranges. Aspiration ofbone marrow of the sternum showed a dry tap.A barium meal study and gastrofibrescopy

showed Borrmann type 4 gastric carcinoma.Apart from a compression fracture of the 8ththoracic vertebra, no other abnormalities suchas lytic or blastic bone lesions were detected bymagnetic resonance images and bone tomo-graphy.The whole body scan was performed three

hours after intravenous administration of 0.4mCi/kg technetium-99m (94mTc)-methylenediphosphate, using a Hitachi Gamma View-FRC-1C-1635LF camera equipped with a lowenergy and high resolution collimater. Thewindow centre setting and the window widthwere adjusted to 141 KeV and 20%, respec-tively, and the scan was performed from

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Kobayashi, Ikeda, Tozuka, Noguchi, Fukuma, Sakamoto, Marumo, Komoda, Sakagishi, Sato

Figure 1: 99mTc methylenediphosphate bonescintigram showing so

called super bone scan.Note the considerablyincreased uptake in axialskeletons and the absenceof renal images.

Anterior Posterior

anterior and posterior projections. The imageshowed a homogeneously and symmetricallyincreased uptake of the bone tracer withoutrenal images, that is to say, super bone scan (Fig1). The biopsy specimen from the iliac boneshowed the infiltration of poorly differentiatedadenocarcinoma cells, which were similar tothose seen in biopsy gastric specimens.Abdominal ultrasonography and computed

tomography showed hepatosplenomegaly, butspace occupying lesions or other abnormalitieswere not detected.The diagnosis of advanced gastric carci-

noma with diffuse bone metastases was madeand chemotherapy with cisplatin and UFT(Taiho, Tokyo, Japan: an oral drug containingtegafur and uracil) and radiation therapy forthe thoracic and lumbar vertebrae were

started. As the serum ALP activity decreasedto 6100 U/1 and the pain was well controlled bymorphine, he was discharged from the hospitalon 17 August.

Chemotherapy with mitomycin C and UFTwas continued but serum ALP and fibrindegenerative product values gradually increasedagain, bleeding tendency appeared, and he was

readmitted on 11 October. Although he was

treated with cisplatin and UFT again and treat-ment for disseminated intravascular coagula-tion were performed, his status deteriorated,and he died on 6 December.

MethodsThe characteristics of the patient's serum ALPwere investigated biochemically and immuno-logically.

For the agarose gel electrophoresis, Gel 8 forALP isozyme and a high resolution ALPstaining kit were obtained from Ciba CorningDiagnostics Co (Palo Alto, CA, USA).Electrophoresis and the staining of the gelplate were performed according to the manu-

facturer's instructions. To remove the glycan-phosphatidylinositol-anchor moiety or sialicacid in the ALP molecule, the ALP sample was

treated at room temperature for three hours

with 01 U/mI phosphatidylinositol specificphospholipase C (PIPLC, EC 3.1.4. 10,Funakoshi Pharmaceutical Co, Tokyo, Japan)and 0 1 U/ml sialidase (Vibrio cholerae, Koch-Light Ltd, UK), respectively, as reportedearlier.7 8 The resulting aliquot was used forthe electrophoresis. The liver, bone, placental,and intestinal ALP controls were prepared asdescribed previously.9To determine the ALP antigenicity, the ALP

sample (9 pAl) was preincubated for one hour atroom temperature with 1 ,l of monoclonal anti-bodies against human liver (LAP 1/9 clone),bone (BAP 1/9 clone), term placental (H7clone), placental like (Fl1 -clone), and intestinal(D 98 clone) ALPs.7 8 The monoclonal anti-bodies of BAP and LAPIO were gifts fromDrs E M Bailyes and J P Luzio, University ofCambridge, UK, those ofH7 and F11 1 12 werefrom Dr J L Mill'an, La Jolla Cancer ResearchFoundation, CA, USA, and the monoclonalantibody against intestinal ALP13 was from DrH Harris, University of Pennsylvania, PA, USA.In our experience, each monoclonal antibodyhad no significant cross reactions with othertypes of ALP except for antibone ALP, whichhad a little but negligible interaction with liverALP. Subsequently, the resulting ALP isozymeanti-ALP antibody complex in the incubationmixture was electrophoretically isolated with apolyacrylamide disc gel (Alkphor, Johkoh Co,Tokyo).Twenty ,l of the ALP sample was incubated

overnight at 40C with 2 plI of monoclonal anti-bodies against human liver, bone, term placen-tal, and adult intestinal ALPs. The mixturewas then incubated with protein A (BDHChemicals Ltd, Dorset, UK) and left to standfor one hour at room temperature, and cen-trifuged at 2000xg for five minutes. Thesupernatant was assayed for ALP activity.7 8Control assay was carried out with saline inplace of the monoclonal antibodies.The sugar chain heterogeneity of ALP

isozymes'4 15 was determined by lectin affinitychromatographies according to the methodsdescribed previously.8 The following lectinswere purchased from E Y Lab (San Mateo,CA, USA): concanavalin A, phytohaemagglu-tinin E, pea lectin, and wheat germ agglutinin.Briefly, the ALP preparation with a specificactivity of 1 pumol/min was applied on a con-canavalin A column, and allowed to stand atroom temperature for three hours. The threefractions were obtained using two differentconcentrations (0.01 and 0.5 mol/l) ofa-methylmannoside: an unbound fraction(fraction I), a weakly bound fraction (fractionII), and a strongly bound fraction (fraction III).These fractions were further applied on thephytohaemagglutinin E, pea lectin, and wheatgerm agglutinin columns. The unbound andbound fractions were separated on the respec-tive columns using N-acetyl-D-glucosamine at0-5 mol/1, a-metylmannoside at 0.5 mol/l as theelution buffer. Each value shown represents theenzyme activity ofone of the following fractionsas a percentage of the total activity: Ia, the frac-tion passing through both the concanavalin Aand phytohaemagglutinin E column; Ib, the

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A variant alkaline phosphatase found in a case ofgastric carcinoma with super bone scan

Figure 2: ALP isozymepatterns with agarose gelelectrophoresis.1-3; patient'sALP (1, untreated;2, sialidase treated;3, phosphatidylinositolspecific phospholipase Ctreated);4, liverALP;5, bone ALP;6, placental ALP;7, intestinal ALP.

+.

1

2

3

kDa168

166154152

5

fraction passing through the concanavalin Aand binding to the phytohaemagglutinin Ecolurmn; IIa, the fraction binding weakly toconcanavalin A column and passing throughthe pea lectin column; IIb, the fraction bindingweakly to concanavalin A and binding tothe pea lectin colum: IIa, the fraction bind-ing strongly to concanavalin A and passingthrough the wheat germ agglutinin column;IIIb, the fraction binding strongly to both theconcanavalin A and wheat germ agglutinincolumn. The yield of the enzyme activityfrom respective lectin columns was more than90%.The enzyme sample was treated for three

hours with 0.1 U/ml PIPLC and 0.1 U/mlsialidase for apparent molecular size deter-minations. The resulting aliquot was run on50/l polyacrylamide slab gel with Od%sodium dodecylsulphate under the non-reduc-ing condition.8 i6 The enzyme active bandwas detected by 5-bromo-3-indolylphosphatep-toluidine salt containing 1t0p rmol ofZn(CH3Co0)2 and 1 MMObIMgC12. Thecomparative molecular size was estimated fromthe mobility using standard molecular sizemarkers (Pharmacia, Uppsala, Sweden).

Figure 3: ALP isozymepatterns of the patient'sserum incubated with orwithout the monoclonalantibodies against variousALP isozymes. Thesupporting medium usedwas 7.7% polyacrylamidedisc gel. 1, untreated;2, with antihuman liverALP (LAP 1/9 clone);3, antihuman bone ALP(BAP 1/9 clone);4, antihuman placentalALP (H7 clone);5, antihuman placental likeALP (F 1I clone);6, antihuman intestinalALP (D98 clone).

+1 2 3 4

Figure 4: Molecular sizes of various alkaline phosphataseisoenzymes. 1, bone ALP; 2, liverALP; 3, patient's ALP;4, intestinal ALP. All the ALPs were pretreated withsialidase. The comparative molecular size ofALP isozymeswas determined by 5.0% polyacrylamide slab gelelectrophoresis with 0 1% sodium dodecylsulphate undernon-reducing conditions.

ResultsWith conventional electrophoresis on a celluloseacetate membrane, the patient's ALP isozymewas shown to be the bone type. By electro-phoresis on an agarose gel, his ALP was foundat the placenta/bone ALP region (Fig 2). Inaddition, the molecule of the patient's ALP hadsialic acid, but no glycan-phosphatidylinositol-anchor moiety (Fig 2). Using polyacrylamidedisc gel electrophoresis, his ALP was found atthe intestinal ALP region (data not shown).As Fig 3 shows, the ALP reacted mainly

with the monoclonal antibody against humanliver ALP isozyme (LAP 1/9 clone). By animmunoprecipitation method, the amount ofthe patient's serum ALP isozyme wasestimated as follows: the liver type 92%, thebone type 6%, the intestinal type 2%. Theapparent molecular size of his ALP (168 kDa)determined by sodium dodecylsulphate-polyacrylamide gel electrophoresis was largerthan that of common liver type (154 kDa) orthe bone type (165 kDa) (Fig 4).As the Table shows, the sugar chain moiety

of the patient's ALP had more fractions Ia(multiantennary complex type sugar chain)and Ib (complex sugar chain bearing bisectingGlcNAc) than those of healthy adult serum,human bone, and human liver ALPs.

Cff;gl !3i lDiscussion

Super bone scan is defined as a homo-geneously and symmetrically increased uptakeof bone tracers in the skeleton compared withsoft tissues. It may be misinterpreted asnormal, and it is important for differentialdiagnosis that there is no visualisation of renal

~r~y .~., images despite normal renal functions.3Although these scans are most commonly seenin patients with diffuse metastases of prostatic

+ cancer or breast cancer,7 i8 some cases ofgastric carcinoma have also been reported.2-5

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302 Kobayashi, Ikeda, Tozuka, Noguchi, Fukuma, Sakamoto, Marumo, Komoda, Sakagishi, Sato

Comparative amounts of the six fractions ofALP isozymes by serial lectin affinitychromatographies

% Of respective fractions ofALP activity

Ia lb Ia IIb IIIa IIIb

Patient's serum 20 11 28 12 1 28Healthy adult serum 0 1 35 60 2 2Human liver 1 0 31 35 1 32Human bone 0 1 4 18 19 58

Percentages of the fraction ofALP activity obtained by serial lectin chromatography. SeeMethods section.

Therefore, gastric carcinoma should also beconsidered when super bone scan is seen.

In diffuse bone metastases of gastric carci-noma, specific clinical features are recognised.5Common symptoms are anaemia, lumbagoand back pain, and a bleeding tendency, andcommon laboratory findings are leukoblasticanaemia, data suggesting disseminatedintravascular coagulation, high serum lactatedehydrogenase activities, and extremely highserum ALP values.The origin of the ALP in diffuse bone meta-

stases of gastric carcinoma has not been clari-fied. Uchida et al 6 reported a necropsy case ofoccult gastric carcinoma with diffuse bonemetastases, and extremely high serum ALPactivities. They stained carcinoma cellsimmunohistochemically and assumed thatsome of the serum ALP might have originatedfrom the carcinoma cells, although it mighthave been partially derived from the bone.They did not analyse serum ALP isozymes orexamined the bone by scintigraphy.ALP and its isozymes have been investigated

in gastrointestinal malignancies as tumourmarkers.19-23 It has been reported that theALP produced by gastric carcinoma is theplacental type, the placenta like type, or ahybrid form consisting of subunits of theintestinal type and the placental type.21 Wecharacterised ALP isozymes in the patient bio-chemically and immunologically. The conven-tional electrophoresis on a cellulose acetatemembrane showed that the patient's ALP wasthe bone type and the result of agarose gelelectrophoresis was also the placenta/bonetype. The result of polyacrylamide disc gelwas, however, different, and the patient'sALP seemed to be the intestinal type.Immunoelectrophoresis and immunoprecipita-tion with monoclonal antibodies againstvarious ALP isozymes showed that thepatient's serum ALP had an antigenicity of theliver type ALP. Moreover, it is suggested thatthis antigenetically liver type ALP is a uniquevariant ALP because of its larger molecular sizeand the different sugar chains. The liver typeor liver type like ALP producing gastric carci-noma has not been reported before. Highserum ALP activities may result from thetumour production in our patient. Studies ofthe enzyme in the patient tissues would havebeen very helpful. The mechanism of the pro-duction of the variant liver ALP by cancerouscells remains to be investigated.The prognosis of gastric carcinoma with

super bone scan is usually poor. The mean

survival time varies from about one month toabout five months.5 In this patient, treatmentswith cisplatin and UFT before overt dissemi-nated intravascular coagulation were effective,and the patient survived more than six monthsdespite the advanced carcinoma. Of particularimportance is the correlation between theserum activity of ALP and the efficacy of thechemotherapy.

1 Pagani JJ, Libshitz HI. Imaging bone metastases. Radiol ClinNorth Am 1982; 20: 545-60.

2 Abe H, Nakamura M, Sakamoto K. The bone metastasis ofgastric cancer-evaluated by bone scintigraphy. JMed Imag1990; 10: 992-7 (in Japanese with an abstract in English).

3 Fukuda T, Inoue Y, Ochi H, Nakazima H, Sawa H,Onoyama Y. Abnormally high diffuse activity on bonescintigram. The importance of exposure time for itsrecognition. Eurj Nucl Med 1982; 7: 275-7.

4 Saphner T, Love RR, Perlman S. Super bone scan inmetastatic stomach cancer. Wis Med J 1990; 89: 161-3.

5 Yasutake K, Matushita K, Imamura Y, Oya M, Hozumi T,Kato J, et al. A case of early gastric carcinoma with'disseminated carcinomatosis of bone marrow'. J7pn JfGastroenterol 1989; 86: 1304-9 (in Japanese with anabstract in English).

6 Uchida T, Shikata T, Shimizu S, Takimoto Y, Iino S,Suzuki H. Gonadotropin and alkaline phosphataseproducing occult gastric carcinoma with widespreadmetastasis of generalized bone. Cancer 1989; 48: 140-50.

7 Miura M, Matsuzaki H, Bailyes EM, Koyama I, SakagishiY, Sekine T, et al. Difference between human liver- andbone-type alkaline phosphatases. Clin Chim Acta 1989;168: 177-88.

8 Komoda T, Sato M, Furiya K, Sakagishi Y, Sekine T. Alleicand ectopic polymorphisms in human placental alkalinephosphatases: sugar chain heterogeneities. Clin Chim Acta1989; 186: 203-10.

9 Koyama I, Miura M, Matsuzaki H, Sakagishi Y, KomodaT. Suger-chain heterogeneity of human alkaline phos-phatases: differences between normal and tumor-associated isozymes. J Chromatogr 1987; 413: 65-78.

10 Bailyes EM, Seabrook RN, Calvin J, Maguire GA, PriceCP, Siddle K, et al. The preparation of monoclonal anti-bodies to human bone and liver alkaline phosphatase andtheir use in immunoaffinity purification and in studyingthese enzymes when present in serum. Biochem J 1987;244: 725-33.

11 Jemmerson R, Shah N, Fishman WH. Evidence for homologyof normal and neoplastic human placental alkaline phos-phatases as determined by monoclonal antibodies to thecancer-associated enzyme. Cancer Res 1985; 45: 3268-73.

12 Jemmerson R, Fishman WH. Convenient selection ofmonoclonal antibodies to isoenzymes of placental alkalinephosphatase using the catalytic activity of the antigen inenzyme-antigen immunoassay. Anal Biochem 1982; 124:286-92.

13 Wray LK, Harris H. Demonstration using monoclonal anti-bodies of inter-locus heteromeric isozymes ofhuman alka-line phosphatase. J Immunol Methods 1982; 55: 13-8.

14 Cummings RD, Kornfeld S. Fractionation of asparaginelinked oligosaccharides by serial lectin agarose affinitychromatography. J Biol Chem 1982; 257: 11235-40.

15 Osawa T, Tsuji T. Fractionation and structural assessmentof oligosaccharides and glycopeptides by use of immo-bilized lectins. Ann Rev Biochem 1987; 56: 21-42.

16 Komoda T, Koyama I, Fujimori-Arai Y, Yamagishi F,Sakagishi Y, Hirota N. Lectin affinity chromatography asa useful tool for determining the heterogeneity of alkalinephosphatases - intestinal-like enzymes. In: Miyai K,Kanno T, Ishikawa, eds. Progress in clinical biochemistry.Shannon, Ireland: Elsevier, 1992: 345-9.

17 Witherspoon LR, Blonde L, Shuler SE. Bone scan patternsof patients with diffuse metastatic carcinoma of the axialskeleton. J NuclMed 1975; 17: 253-7.

18 Thrupkaew AK, Henkin RE, Quinn JL. False negative bonescans in disseminated metastatic disease. Radiology 1974;113: 383-6.

19 Skinner JM, Whitehead R. Carcinoplacental alkaline phos-phatase in malignant and premalignant conditions of thehuman digestive tract. Virchows Arch (Pathol Anat) 1981 ;394: 109-18.

20 Imanishi H, Hada T, Muratani K, Hirano K, Higashino K.An alkaline phosphatase reacting with monoclonal anti-bodies to intestinal and placental isoenzymes. Clin ChimActa 1989; 186: 309-14.

21 Imanishi H, Hada T, Muratani K, Hirano K, Higashino K.Expression of a hybrid form of alkaline phosphataseisoenzyme in a newly established cell line (HuG-i) from agastric cancer patient. Cancer Res 1990; 50: 3408-12.

22 Aizawa K, Motoyama T, Watanabe H. Placental alkalinephosphatase-like isoenzymes produced by human gastriccancer cells. Acta PatholtJpn 1989; 39: 630-7.

23 Watanabe H, Tokuyama H, Ohta H, Sotomura Y, Okai T,Ooi A, et at. Expression of placental alkaline phosphatasein gastric cancers. An immunohistochemical study usingthe prepared monoclonal antibody. Cancer 1990; 66:2575-82.

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