CLINICAL CHEMISTRY,. Vol. 40, No. 11, 1994. Creatine Kinase lsoenzyme. BB Increased in Serum and Tumor Tissue of Patients with Giant Cell Tumor of Bone.
CLIN. CHEM. 40/11,
2064-2065
(1994)
Enzymes
#{149}
and Protein
Markers
Creatine Kinase lsoenzyme BB Increased with Giant Cell Tumor of Bone Jun and
Fukuda,”5 Saburou Hitoshi Murayama4
Yagishita,2
Keiko
Yamaoka,2
in Serum Tokiji
Increased creatine kinase isoenzyme BB (CK-BB) has been observed in sera from patients with brain injuries and occasionally in sera from patients with malignancy. We report here that, in two patients with giant cell tumor of bone (GCT), preoperative serum CK-BB increased to -20 and 90 U/L, but in postoperative serum the CK-BB decreased to normal values. That the tumors contained CK-BB was indicated by electrophoretic analysis and immunohistochemical staining. Furthermore, serum CK-BB was detectable in five additional cases of GCT and in cultured tumor cells from a patient with GCT by an electrophoretic method. These results suggest that CK-BB may be a marker for GCT. Indexing
Terms: tumor marker/electrophoresis/cancer
Giant cell tumor of bone (GCT), recognized as a distinct cinicopathological entity, has been morphologically characterized by the presence of three different types of tumor cells: spindle cells, mononuclear round cells (MC), and muitinucleated giant cells (GC).6 However, the origin of these cells is stifi unknown (1). Creatinine kinase isoenzyme BB (CK-BB) is found predominantly in the brain, kidney, stomach, thyroid, urinary bladder, lung, and prostate (2), but rarely in normal serum. We report here that serum CK-BB is increased in patients with GCT, and that many tumor cells in GCT show the presence of CK-BB when subjected to immunohistochemical staining with antiserum to CK-BB.
Materials
and Methods
Serum. Serum CK activity and CK-BB concentration were measured in two patients with typical GCT: a 45-year-old man and 42-year-old woman. The procedures we used were in accordance with the ethical standards of the Kanagawa Rehabilitation Center. Sera were obtained preoperatively and 14 days after the removal of the tumors. A 10-g portion of the tumor tissue from each patient was soaked in 10 mL of saline solution for 12 h at 4#{176}C; afterwards, the CK activity and CK-BB concentration of the supernates were measured. Departments of’ Orthopedic Surgery,2 Pathology, and3 Psychiatry, The Kanagawa Rehabilitation Center, 516, Nanasawa, Atugi, Kanagawa, 243-01, Japan. 4Department of Orthopedic Surgery, The Kanagawa Cancer Center, 54-2, Nakao-tyou, Asahi-ku, Yokohama, Kanagawa, 241,
Japan.
Author for correspondence. Fax mt + 81-462-49-2502. #{176}Nonstandard abbreviations: CK-BB, creatine kinase zyme BB; GCT, giant cell tumor of bone; MC, mononuclear cells; and GC, multinucleated giant cells. Received June 6, 1994; accepted August 9, 1994. #{176}
2064
CLINICAL CHEMISTRY,
Vol. 40, No. 11, 1994
isoenround
and Tumor
Hanihara,3
Kazuyoshi
Tissue
of Patients
Kushida,4
CK activity. Total serum CK activity was measun by an automated chemistry analyzer (Shimadzu ci 730 Shimadzu, Kyoto, Japan). Isoenzymes CK-MM, CK-M and CK-BB were measured electrophoretically (Aut mated Isodot; Helena Labs., Beaumont, TX) as scanned by a densitometer (Helena edc) to quanti their percentages of total CK activity. Immunohistochemistry. An immunohistochemic study was performed on tumors from five patients wi had typical GCT. Each specimen was obtained by su gical procedure in our hospital from 1977 to 1992. Pa of each tumor was impregnated with 100 mi/L buffen formalin solution and embedded in paraffin wax. Th sections were immunostained by reacting with antibo to CK-BB (polyclonal, dilution 1:100; Ultra Clon Wellow, UK) and staining with 3,3’-diaminobenzidin Serum CK-BB was measured in only one of the five case and the concentration was very high. Osteosarcoma, m lignant fibrous histiocytoma, foreign body granulom and
tuberculoma tumors were used as controls. Cell culture. The tumor cells from one subject we cultured for 50 passages over the course of 1 year, RPMI-1640 culture medium. However, only the spind cells continued to grow. CK-BB, both in the superna and the cell homogenate of the cultured cells, was me sured by electrophoresis and was made visible with flu orescent stains. Alveolar soft part sarcoma served as ti control.
Results Preoperative total CK activity was slightly increas and CK-BB concentration was significantly above no ma! in the two patients (CK, U/L: male 204, female 15 CK-BB: 45% and 13.5%, respectively). By 14 days afti tumor removal, both CK activity and CK-BB concentr tion had decreased to normal values in each patiei (CK, 59 and 69 U/L; CK-BB, 1% and 0%, respectivel) In contrast, CK activity and CK-BB concentration i the tumor tissue were very high (CK, 23 900 and 45] U/L; CK-BB, 87% and 77.7%, respectively). Most
CK-BB; negative.
of the
GC
showed
positive
a few GO with an atrophic Immunoreactivity was
immunostaining
nucleus were demonstrated
fi
usual in bol
the cytoplasm and nuclei (Fig. 1). Some MC were in munoreactive to CK-BB; these cells had larger nucl and more prominent nucleoli than did the CK-B] negative MC. The stainability of MC was virtually ide: tical to that of GO. A few spindle cells showed immi noreactivity in their cytoplasm and nucleus. All fi’ patients examined showed similar immunocytocheni cal findings.
a tumor sponse.
product
CK-BB
Ordinarily,
,
,,
.._
-
1. Light micrograph of giant cell tumor of bone immunostained :h CK-BB antibody (x200). st of the multinucleated giant cells are immunostained positively with -BB in both the cytoplasm and nuclei. .
The ?re ese ant ma
GO in osteosarcoma and macrophages in MFH very rarely positive for CK-BB; most of the cells in tumors were CK-BB-negative. Foreign-body-type cells and Langhans-type giant cells in tubercualso gave negative results. Although CK-BB was detected by the visual fluoresnce method, the results could not be scanned by a unsitometer to obtain a percentage, given the very low K-BB veolar
concentration. soft
part
No sarcoma
CK-BB
was
detected
in the
control.
scussion Adenylate kinase is known to interfere with OK derminations, particularly when cell culture or tissue pernates are used (3). The normal inhibitors for adenate kinase in blood do not overcome the increased nount of this enzyme in some tissues and cell cultures. In this report, we confirm the presence of CK-BB in .e serum of patients with GOT as well as in the superLtes from tumor tissue (being remarkably high in the tter). Furthermore, the presence of CK-BB was clearly ,monstrated in all the cases examined by immunohischemistry. The fact that CK-BB is detectable in culred GCT tissue suggests that the tumor itself has me ability to produce CK-BB. Thus perhaps CK-BB tould be detected in the serum of patients with GCT as
rather
than
as a result
catalyzes
the
of a host
reversible
re-
transfer
of high-energr phosphate from phosphocreatine to adenosine diphosphate to regenerate adenosine triphosphate. In this respect, CK-BB may play an important role in the metabolism of GCT, just as it does in the cartilage during skeletal growth (4-6). CK-BB is rarely detected in sera from healthy subjects. Increased serum CK-BB has been observed mostly in patients with brain injuries (7) and occasionally in patients with certain malignancies, such as gastric cancer (8) and prostatic cancer (9). Among primary bone diseases, increased serum CK-BB has been reported only in autosomal osteopetrosis type 11(10). We measured serwn CK-BB in patients with tumors or with conditions clinically difficult to distinguish from GCT (e.g., osteosarcoma, malignant fibrous histiocytoma, and aneurysmal bone cyst), and found its concentrations to be normal in all the cases examined. Thus, CK-BB may be a useful marker for characterizing GOT, pathologically as well as clinically. References 1. Dahlin DC. Giant cell tumor (osteoclastoma). Bone tumors. Springfield, IL: CC Thomas, 1978:99-115. 2. Smith AF. Separation of tissue and serum creatine kinase isoenzyme on polyacrylamide gel slabs. Clin Chim Acta 1972;39: 351-9. 3. Silverman LM. Creatine kinase BB isoenzyme activity in bonemarrow serum [Letter]. Clin Chem 1978;24:1423-5. 4. Funanage VL, Carango P, Shapiro IM, Tokuoka T. Creatine kinase
activity
synthesis
is required
in endochondral
for mineral
growth
deposition
cartilage.
and
Bone Miner
matrix
1992;17:
228-36.
5. Katoh age-related
R, lyoda
K, Oohira
difference
in the
A, Kato amounts
K, Nogami H. Zonal and of creatine kinase subunits
in cartilage. Cliii Orthop 1991;271:283-7. 6. Somjen D, Schluter K]), Wingender E, Mayer H. Stimulation of cell proliferation in skeletal tissues of the rat by defined parathyroid hormone fragments. Biochem J 1991;277:863-8. 7. Somer H, Kaste M, Troupp H, Konttinen A. Brain creatine kinase in blood after acute brain injury. J Neurol Neuroaurg
Psychiatry 8. Lederer
1985;38:572-6.
WH, Gerstbrein HL. Creatine in serum of a patient with gastric Clin Chem 1976;22:1748-9. 9. Feld 1W, Witte DL. Presence of creatine in some patients with prostatic carcinoma.
activity
kinase
isoenzynie
cancer
[Case
kinase Clin
BB Report].
BB isoenzyme Chem 1977;23:
1930-2. 10. Yoneyama T, Fowler HL, Pendleton 1W, Lui CY, Eldridge TH. Elevated serum BB in autosomal dominant osteopetrosis Cliii Genet 1992;42:39-42.
CLINICAL
CHEMISTRY,
JW, Sforza PP, levels of creatine type Il-a family
Vol. 40, No. 11,
1994
Gerard kinase study.
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