ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 9, No. 4 Copyright © 1979, Institute for Clinical Science, Inc.

The Significance of Creatine Kinase (CKBB) in Metastatic Cancer of the Prostate

DONALD T. FORMAN, Ph .D.

Departments o f Hospital Laboratories, Pathology and Biochemistry,

North Carolina Memorial Hospital and University o f North Carolina,

Chapel Hill, NC 27514

ABSTRACT

Alterations of serum creatine kinase isoenzymes were observed in five cases of prostatic carcinoma. Creatine kinase isoenzyme BB was found in the serum of two of three cases with métastasés. Its presence in serum does not seem to be related to acid phosphatase activity but seems associated w ith extension of the tumor to other tissues. Prelim inary studies on effusions from patients with malignant and non-malignant prostates showed that CK-BB was detectable only in cytology positive effusions. This finding suggests that CK-BB may be a tumor product rather than a result of a host response. The observation of CK-BB in a significant percentage of patients (two of three) with metastatic carcinoma of the prostate is of in terest and suggests that CK-BB isoenzymes may have some predictive value in following patients with malignant disease.

Introduction

D uring the past several years, it has becom e apparen t that certain cancers synthesize special protein moieties which circulate in the serum and which may be p a rticu la rly h e lp fu l in d e te c tin g and staging certain tum ors11 (table I). These biologic markers may also help identify early recurrence of the tum or and monitor the efficacy of various therapeutic mo­dalities.19,20 The recent developm ent of sensitive and specific methods to measure these proteins in the serum of patients w ith certain cancers makes it possible to use these tumor products as markers for the p resence of neoplasm .17 The very

recen t report of an electrophoretically d istinct form of serum galactosyltrans- ferase (GT-II), predom inately in patients with colorectal carcinoma, and a correla­tion w ith overall extent o f tum or is of great interest.13

In 1964, the enzym e creatine kinase (ATP: creatine phosphotransferase, EC 2.7.3.2) was add ed to the rap id ly in ­creasing group of enzym es w hich can appear in m ultiple m olecular form.10 The soluble fractions obtained from different t is su e s g en e ra lly show one to th re e electrophoretically distinguishable crea­tine kinase isoenzymes. In addition to the cytoplasmic creatine kinases, one addi­tional form exists in m itochondria from

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334

Tumor A sso c ia te d Markers in Serum

FORMAN

TABLE I

O n c o - F e t a l A n t i g e n s (S e ru m ) M o s t Common A s s o c i a t e d C a r c in o m a s

Alpha-fetoprotein (AFP) carcinoembryonic antigen (CEA)

F e t o - P l a c e n t a l P r o t e i n s

Human placental lactogen (HPL) Placental alkaline phosphatase (PAP) Human chorionic gonadotropin (B-HCG) Creatine kinase (CK-BB)

Hepatocellular, testicular, pancreatic (liver involvement) Colorectal, gastric, lung, cahcer of cervix, corpus

uteri, ovary

Gonadal, testicular, breast Lung, breastTesticular, breast, pancreatic, gastric Gastric, prostatic, oat-cell

E c t o p i c H o rm o n es

Adrenocorticotrophic hormone, ADH, hypercalcemic agents,

Lung, pancreas

several tissues.4 These isoenzymes rep­resen t various enzym atic forms which catalyze, in a given tissue, the same main reaction, bu t which differ in one or more characteristics, for example, kinetic con­stan ts , th e rm o sta b ility , re sp o n se to stimulators and inhibitors, or even the action on a secondary substrate.

It is well known that these molecular forms may vary according to tissues, age and pathological conditions. Our working hypothesis, suggested by previous studies on breast tum or lactate dehydrogenase and hepatom a aldolase,16 was the analogy betw een cancerous tissue and their en­zymic forms at an initial stage of onto­genesis.

A lterations o f serum creatine kinase isoenzyme patterns in neoplastic states have recently been reported in gastric cancer, oat cell carcinoma and prostatic carcinom a.3,6,13 Several pa tien ts have been observed w ith metastatic carcinoma of the prostate, whose serum contained m arkedly increased amounts of CK-BB isoenzyme. This report discusses the po­tential value of this tum or m arker in the diagnosis and follow-up of patients with prostatic cancer.

mer. Each protom er may be of the muscle type (M) which is electrophoretically slow (at an alkaline pH) and moves on cellulose ace ta te e le c tro p h o re s is tow ard the cathode or a brain type (B) w hich is the fast fraction and moves toward the anode. T he h y b rid MB iso en zy m e exh ib its electrophoretic m igration on cellu lose acetate corresponding to an interm ediate position (figure 1). A fter sep ara tio n , creatine kinase isoenzymes are located by a tetrazolium dye (NBT) after coupling the CK reaction w ith hexokinase and glucose-6-phosphate reactions. The hy­drogen ions necessary for reduction of the NBT dye come from the reduction of NA DP, in the p re se n c e o f g lucose , creatine phosphate, ADP and Mg2+; AMP is added in order to inhibit the adenylate kinase reaction.

An e lec tro p h o res is appara tus* and cellulose acetate strips were used with the latter system according to the manufac­turer’s instructions. Relative isoenzyme activities were then quantitated.f A con­trol specim en! containing all three CK isoenzymes was determ ined with each electrophoretic run. Total creatine kinase activity was m easured and assayed with a

Materials and M ethods * Beckman Model 100 Microzone.. r _ , 15 , f Beckman Model 111 Microzone Computing

I he work ot Eppenberger et al3 has Densitometer.shown that creatine kinase (CK) is a di- t Ortho Diagnostics.

CREATINE KINASE IN METASTATIC CANCER OF PROSTATE 335

Gilford Model 3400 D iscrete Analyzer? u sin g CK rea g en ts (cy s te in e hydro- ch lo rid e-ac tiv a ted ).11 T otal acid phos­phatase and prostatic acid phosphatase activity were determ ined using a p-nitro- phenol phosphate substrateK and a fast parallel analyzer,** both in the presence and in the absence of 0.04 mol tartrate ion per 1.

Results

In table II are shown the results of CK isoenzyme assays on serum from patients w ith biopsy proven prostatic cancer. Of the five patients studied, one (JK) had m ark ed ly e le v a te d CK-BB fractions (figure 2) and demonstrated widespread metastases (lung, bone) at autopsy. Two other patients (CB and EL) dem onstrated increased total CK (normal, 4 to 50 U per liter) and the absence of a definitive BB band. The rem aining subjects (AA and RK) had borderline elevated total CK and in the case of patient (AA) the CK-BB fraction approxim ated 17 percent. The latter patient on autopsy showed exten­sion o f the carcinom a to b ladder and rectum. Patients JK and AA did not have th e ir b ra in s ex am in ed for m etasta tic tumor. All o f the subjects studied had elevated total acid phosphatase (normal, Oto 9 U per liter) as well as prostatic acid phosphatase (normal, 0 to 4 U per liter). T hese findings w ere dem onstrated in consecutive assays on all patients.

Discussion

Evidence is accumulating that serum CK-BB is detectable or increased in a w ide variety of clinical circumstances, even with techniques that are less sensi­tive than RIA, CK-BB has been reported

§ Gilford Instrument Laboratories, Inc., Oberlin, OH 44074.

11 Worthington Diagnostics, Freehold, NJ 07726. H Smith Kline Instruments, Inc., Sunnyvale,

CA 94086.** Union Carbide Centrifichem 400.

Anode (+)

F i g u r e 1. CK isoenzyme pattern with extracts of voluntary muscle, heart and brain. CK-3 (MM) is the principle band for muscle, CK-2 (MB) for heart and CK-1 (BB) for brain.

in ren a l fa ilu re ,8 R eye’s synd rom e,18 m alignant hypertherm ia ,1 m yopathy,12 neuropathy,12 pregnancy and the peripar- tum period.14

In prosta tic carcinom a, CK-BB was found in the serum of two of five cases. The observation of CK-BB in a significant percentage of patients with highly inva­sive carcinoma of prostate (metastatic) is o f interest and, along with the report of F e ld and W itte,6 suggests that CK-BB

TABLE II

T otal Acid and P r o s ta tic Acid Phosphatase A c t iv i t i e s and CK Isoenzymes in Serum

o f P r o s ta tic Cancer P atien ts

Units per liter PercentPatient ACP-T* ACP-Pi Total CK CKMM CKMB CKBB

JK§ 86 69 480 0 0 100(lung,bone)

CB& 29 22 97 100 0 0(bone)

EL 12 7 129 100 0 0AA§ 33 25 64 83 0 17(bladder,rectum)

RK 14 5 52 100 0 0

*Total acid phosphatase (EC 3.1.3.2) tProstatic acid phosphatase §Distant metastases

3 3 6 FORMAN

T ABLE III

B H M _ Correlation of CK-BB in Serum and Effusion

jk 1 SerumCK-BB EffusionC K Type Percent CK-BB

• ISOENZYMES Benign disease (4)* 0 0

i Cancerjk •Cytology positive (1)* 100 2+Cytology negative (4)* 0 0

♦Number of patients studied.t

H DB H M

F ig u re 2. Electropherogram from sera of a patient (JK) with m etastatic carcinom a o f the prostate. The positions of CK isoenzyme migrating to the anode are labeled B (brain) and H (heart). The position of muscle CK (M) is closest to the cathode. Other unlabeled patterns represent three patients with muscle and cardiac disease.

isoenzymes may have some predictive value in studying patients with neoplastic diseases. These findings suggest a corre­lation of serum CK-BB w ith ex ten t of cancer. Some pa tien ts w ith both c ir­cumscribed and extensive prostatic car­cinoma also demonstrated no detectable serum CK-BB. This finding may be re­lated to differences in the biology of the tumor or response of the host rather than extent of the disease. Alternatively the in­ability to detect CK-BB in some patients with prostatic carcinoma may reflect the p resence of an inhibitor. In d eed , the presence of a CK inhibitor has recently been detected in the serum of a patient with Reye’s Syndrome7 and Bruns2 has reported low apparent creatine kinase ac­tivity and prolonged lag phases in serum of patients with metastatic disease.

H oag9 has s tu d ie d p ro sta tic t umor hom ogenates and shown an increased proportion of CK-BB. Several of these cases dem onstrated CK-BB in the serum. Preliminary studies on effusions from pa­tients with malignant and non-malignant prostates showed that CK-BB is detecta­

ble only in cytology positive effusions (ta­ble III). This finding suggests that CK-BB may be a tumor product rather than a re­sult of host response.

The presence of CK-BB in serum of pa­tients with prostatic cancer and in the tis­sue homogenates and effusions from pa­tients with m alignant disease suggests some degree of specificity that may be of eventual use in diagnosis and treatm ent of patients with metastatic prostatic disease.

Various cancerous tissues and m eta­static malignancies demonstrate unusual isoenzym ic m odifications. A ldolase B (liver type) is decreased in hepatoma; al­dolase A (muscle type) is decreased in spleen reticulosarcoma. The “ M” type lactate dehydrogenase predom inates in cancerous tissues and an “ H” type has been found in some hepatom as.16 Several m etastatic prostatic tum ors have been shown to exhibit an increased serum and tissue level of CK-BB. A common feature of these modifications is their fetal-like pattern which may be an expression of dedifferentiation at a m olecular level, characterized by the repression of synthe­sis of the most specific form of the tissue enzyme.

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CREATINE KINASE IN METASTATIC CANCER OF PROSTATE 337

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