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J Clin. Biochem. Nutr., 19, 97-105, 1995

Role of Serum in Assessing the Efficacy of Radiation Therapy for Human Carcinoma of Uterine Cervix

Natarajan BALASUBRAMANIYAN,Sorimuthu SUBRAMANIAN, and Saminathan GOVINDASAMY*

Departmentof ,University of Madras, Guindy Campus, Madras-600 025, India

(ReceivedMarch 29, 1995)

Summary In different stages of carcinoma of the uterine cervix, the activities of serum 5'-, lactate (LDH), phos-

phohexose isomerase (PHI), ƒÁ-glutamyl transpeptidase (GGT), trans- aminases, and were studied before and after radiation

treatment. Glycolytic enzymes (LDH, PHI) were found to be increased from stage I onwards, whereas GGT, glutamate oxaloacetate trans-

aminase, glutamate pyruvate , and and alkaline phos-

phatases increased only in the advanced stages (stages III, IV), when compared with normal values. 5'-Nucleotidase was found to be increased significantly from stage II onwards when compared with the normal. The

increased levels of these enzymes were reversed to near normal levels after

radiotherapy. The increased levels of serum , phosphatases,

and GGT in the advanced stages of carcinoma of uterine cervix may be

explained on the basis of liver involvement and bone metastasis. The degree of increase in the activities of PHI and LDH may reflect the status

of the cancer. The activity of 5'-nucleotidase in serum may be used as an

additional marker to assess the status of carcinoma of uterine cervix under circumstances when other markers fail to provide clear indications.

Key Wards: carcinoma, glycolytic enzymes, 5'-nucleotidase, trans-

aminases, phosphatases

The measurement of biochemical markers is being increasingly used for early diagnosis and monitoring of the progress of cancer. Derangement in the metabolic

*To whom correspondence should be addressed .

97 98 N. BALASUBRAMANIYAN, S. SUBRAMANIAN, and S. GOVINDASAMY processes are often reflected in the changes of enzymatic activities. Extensive research has been conducted by numerous workers over the past few decades to find activity differences between normal and tumor cells . Many types of tumor cells secrete much larger quantities of glycolytic enzymes into the blood- stream, as a result of increased glycolytis in neoplastic cells. As a single enzyme may exhibit a similar type of changes under some non-malignant conditions, a combination of different enzyme markers is more significant and valuable than any single enzyme marker. Rao et al. [ 1 ] reported the usefulness of multiple enzyme assays in the diagnosis of various cancer cases. Phosphohexose isomerase (PHI) catalyzes the conversion of -6-phos- phate to -6- in the glycolytic cycle. (LDH), a tetrameric enzyme composed two immunologically distinct subunits (M and H) regulates the interconversion of pyruvate and lactate, the last step of the glycolytic pathway; and this enzyme has been extensively studied and found useful in the recognition of neoplastic disease [2]. Kim et al. [3] concluded LDH activity to be a very useful marker in the diagnosis and prognosis of cancer . Roguljic et al. [4] reported a study showing that PHI, LDH, and the isoenzyme pattern of LDH in combination were very useful in distinguishing fibroadenoma from breast carcinoma. y-Glutamyl transpeptidase (GGT) is a membrane-bound glycoprotein that catalyzes the transfer of y-glutamyl groups between amino or peptides [5]. The physiological function of GGT is thought to be the mediation of translocation across the cell membrane [6]. Although the highest concentration of GGT is found in the brush borders of the epithelial cells that line the proximal convoluted tubules of the kidney, the enzyme has also been demonstrated in a variety of human tissues and body fluids [7]. Transaminases are an important class of enzymes linking and amino acid metabolism, and the study of these enzymes has established the relationships among the intermediates of the citric acid cycle. 5'-Nucleotidase is an established plasma membrane marker in many mammalian cells where the enzyme is present as an ectoenzyme [8]. Boone et al: [9] found that serum 5'-nucleotidase is almost as useful as GGT in indicating hepatobiliary disease in patients with granuloma of the liver, acute pancreatitis, congestive heart failure, rheumatoid arthritis, and carcinoma metastatic of the liver. Kim et al. [3] assessed the diagnostic value of measuring serum alkaline , 5'-nucleotidase, GGT, and activities as an aid to the detection of liver metas- tasis. Hence, in the present study we sought to correlate the activities of these enzymes in the sera of uterine cervical cancer patients with their disease status to understand their validity in diagnosis and to reassess the prognosis during the course of radiotherapy.

J. Clin. Biochem. Nutr. SERUM ENZYMES IN HUMAN CARCINOMA OF UTERINE CERVIX 99

MATERIALSAND METHODS

This study was conducted on patients suffering from carcinoma of the uterine cervix admitted to the Cancer Institute, Adyar, Madras, India, and the Institute of Gynecology and Obstetrics, Egmore, Madras, India. In this study, blood samples were obtained from a total number of 238 patients (30 to 60 years old) in different stages of carcinoma of the uterine cervix (stage I [group II], N=35; stage II [group III], N=70; stage III [group IV], N=115; and stage IV [group V], N=18). A total number of 125 healthy females (group I) studied were professionals and employees of different institutions without any gynecological disorders. All the patients in the present study were confirmed to have cervical cancer by cytological screening, and the stages of carcinoma were assessed by oncologists. Blood samples were also collected from groups II, III, and IV after radiation treatment. Group V patients were not included for investigation after radiation therapy as they did not respond to radiation treatment. Venous blood samples were drawn from all the subjects, and serum was separated for further analysis. The activities of glutamate oxaloacetate transaminase (SGOT) (EC. 2.6,1.6) and glutamate pyruvate trans- aminase (SGPT) (EC. 2.6.1.2) were assayed by the method of Mohur and Cook [10]. PHI (EC. 5.3.1.9) was assayed following the method of Horrocks et al [11]. The activity of serum LDH (EC. 1.1.1.27) was assayed by the method of King [12]. The activities of serum acid (EC. 3.1.3.2) and alkaline (EC. 3.1.3.1) phosphatases were assayed by the method of Moog [13] as modified by King [12]. The activity of serum GGT (EC. 2.3.2.1) was assayed according to the method of Jacobs [14]. 5'-Nucleotidase was assayed following the method of Campbell [15].

STATISTICALANALYSIS

Serum LDH, PHI, GGT, SCOT, SGPT, 5'-nucleotidase, and acid and alka- line phosphatases values were expressed as the mean +SD for patients and the healthy population separately. The statistical differences were analyzed by Stu- dent's t-test, and p-value were expressed. Analysis of variance (ANOVA) was done to compare the mean levels of the parameters at different stages of carcinoma of the uterine cervix before and after radiation treatment.

RESULTS

The activities of serum LDH, PHI, SCOT, and SGPT, in normal and patients with carcinoma of uterine cervix before and after radiotherapy are presented in Table 1. LDH and PHI activities were found to be increased significantly (p < 0.001) in the group-II, -III, -IV, and -V patients, whereas the activities of SGOT and SGPT were increased in group-IV and -V patients only, when compared with the normal values. Group-II and -III patients showed no significant change from

Vol. 19, No. 2, 1995 100 N. BALASUBRAMANIYAN, S. SUBRAMANIAN, and S. GOVINDASAMY

Table 1. Activities of serum lactate dehydrogenase (LDH), phosphohexose isomerase (PHI), glutamate oxaloacetate transaminase (SCOT), and glutamate pyruvate transaminase (SGPT) in normal individuals and in patients with carcinoma of uterine cervix before and after radiotherapy.

The values are expressed as IU/liter, mean±SD, with the number of individuals indicated in parentheses. p-Value: * * * <0.001, normals versus different stages of carcinoma , p-Value: +++<0 .001, before treatment versus after treatment. aSignificant difference from other groups (p<0.001).

Table 2. Activities of serum acid and alkaline phosphatases, y-glutamyl transpeptidase (GGT), and 5'-nucleotidase in normal individuals and in patients with carcinoma of uterine cervix before and after radiotherapy.

[be values are expressed as IU/liter, mean±SD, with the number of individuals indicated in parentheses. p-Value: * * * <0.001, normals versus different stages of carcinoma, p-Value: +++<0 .001, before treatment versus after treatment. aSignificant difference from other groups (p<0.001).

normal in the level of activity of SGOT and SGPT. The activities of LDH and PHI were found to be decreased after radiation treatment from their elevated levels in stage-I, -II, and -III patients. The activities of SGOT and SGPT decreased significantly after radiation treatment in group-IV patients when compared with their pretreatment values.

J. Clin. Biochem. Nutr. SERUM ENZYMES IN HUMAN CARCINOMA OF UTERINE CERVIX 101 Table 2 shows the activities of serum GGT, 5'-nucleotidase, and acid and alkaline phosphatases in normal and patients with carcinoma of uterine cervix before and after radiotherapy. The activities of GGT and acid and alkaline phosphatases were increased significantly (p<0.001) in group-IV and -V patients, but there was no significant change in their values of groups II and III when compared with normal levels. Radiotherapy resulted in the return of all these enzyme activities to near normal in group-IV patients. Radiation treatment did not produce any change in the activities of these enzymes in group-II and -III patients. The activity of 5'-nucleotidase was significantly elevated in group-III, -IV, and -V patients. After radiotherapy the enzyme activity was found to be normal in the sera of these cervical cancer patients. The group-V patients were not subjected to radiation treatment.

DISCUSSION

The activities of LDH and PHI in serum of group-II, -III, -IV, and -V patients were elevated when compared with those of age-matched normals. It is interesting to note that the activities of these enzymes were increased significantly even in the early stages of the disease. After radiation treatment the enzyme activities reduced back to near normal levels in group-II, -III, and -IV patients. Maity and Burma [16] reported elevated levels of LDH and PHI in primary and metastatic cancer. Also, the activity of LDH was found to be increased significantly in the sera of ovarian and brain cancer patients when compared with the normal activity [17, 18]. Furthermore, this activity was found to be increased predominantly in the sera of gastric cancer patients compared with that of duodenal ulcer patients [19]. Giannoulaki et al. [20] reported the presence of an abnormal LDH isoenzyme, LDH-lex, located anodally to the LDH-1 isoenzyme and associated with malignant states. Several investigators have reported that serum PHI activity is often elevated in cancer of the head, neck, esophagus, lungs, and breast [21, 22]. A possible factor contributing to the elevated levels of PHI and LDH in the sera of patients with carcinoma of uterine cervix may be the enhanced during the growth of the cancer mass. The elevated activities of these enzymes may also be due to the differences in the rates of synthesis, degradation, or excretion of these enzymes in cancer. The significant decrease in enzyme activities after radia- tion treatment may be ascribed to selective killing of the malignant cells by the radiation. The activities of the transaminases (SCOT and SGPT) in the serum were found to be increased significantly (p<0.001) in the group-IV and -V patients, when compared with those of non-cancerous age-matched controls. After the radiation treatment the activities of these two transaminases reverted back to near normal in the group-IV patients. A rise in SGPT activity is almost always due to hepatocellular damage and is usually accompanied by an increase in SGOT. Clyde

Vol. 19, No. 2, 1995 102 N. BALASUBRAMANIYAN, S. SUBRAMANIAN, and S. GOVINDASAMY and Frances [23] found a correlation between SGOT and SGPT and the measure- ments of tumor mass during therapy. Dang et al. [24] observed increased SGPT levels in patients with hepatic malignancy. In the present investigation the raised serum levels of transaminases in the advanced stages of carcinoma of uterine cervix may be due to the secondary implications of cervical cancer, as these enzymes are elevated only in the advanced stages of the disease. The activity of acid and alkaline phosphatases were found to be elevated significantly in the sera of group-IV and -V patients when compared with that of age-matched non-cancerous healthy controls. The group-II and -III patients did not show any remarkable alteration in the activity of these enzymes. The activities of both acid and alkaline phosphatases in group IV were found to be near normal at the end of the radiation therapy. An increase in the activity of was reported in carcinoma of the gastrointestinal tract with liver metastases [25, 26]. Early detection of liver metastasis is an essential element in establishing the prognosis and therapeutic strategy for cancer patients. Generally speaking, alkaline phosphatase is described in the literature as being the enzyme that varies most markedly during the course of liver metastasis [27]. The activity of has been reported to be elevated in the serum of patients with metastatic carcinoma of the prostate [28]. Increased activity of alkaline phosphatase has also been reported in breast cancer [29]. Thus the observed increase in the activities of phosphatases in sera of patients suffering from carcinoma of uterine cervix also provides additional information about the nature and behavior of serum phosphatases in the advanced stages of cervical cancer and its response to radiation treatment. Numerous investigations have shown that increased GGT activity is associat- ed with animal tumors both in vivo and in vitro [7, 30, 31]. Murray et al. have suggested that increased plasma levels of GGT might correlate with both tumor recurrence and disease progression [32]. In the present investigation, the activity of GGT was found to be elevated in the sera of group-IV and -V patients when compared with that value for healthy controls. There was no alteration in the activity of the enzyme in group-II and -III patients. Radiotherapy resulted in a decrease of the enzyme activity to near normal in the group-IV patients. Radiation treatment did not reveal any change in the activity of this enzyme in group-Il and -III patients. An increase in the activity of GGT was reported in patients with liver diseases [33]. This enzyme was found to be a reliable indicator for the diagnosis of primary or metastatic carcinoma of the liver. Increased activity of GGT in the sera of cancer patients is a good marker of metastases in the liver of patients with primary tumors of the lung, breast, and digestive tract [21, 26, 30, 34]. Therefore, the patients in this study who had elevated serum GGT values (groups IV and V) could have had cancer that metastasized to the liver, whereas the patients with GGT values within the control range (groups II and III) may not have suffered metastasis of the cancer to the liver. Further, it has been suggested that the increased serum level of GGT

J. Clin. Biochem. Nutr. SERUM ENZYMES IN HUMAN CARCINOMA OF UTERINE CERVIX 103 in cancer patients may be due to an increased tumor burden. The finding that radiation treatment normalized the activity of GGT in group-IV patients may be due to regressive influence on the tumor mass. 5'-Nucleotidase is a plasma membrane marker enzyme. The increase in activity of this enzyme in the circulation would presumably indicate the accelerat- ed breakdown and release of cell-surface constituents. In sera, the activity of 5'-nucleotidase was found to be significantly elevated (p<0.001) in group-III, -IV, and -V patients. After radiotherapy the enzyme activity returned to normal in the sera of the cervical cancer patients. The group-V patients were not subjected to radiation treatment. Schwartz and Bodansky [35] determined the serum 5'-nucleotidase activity in control and patients with various types of neoplastic diseases and found that the activity of the enzyme was elevated in the serum of cancer patients. Kim et al. [3] reported the diagnostic value of measuring serum 5'-nucleotidase activity as an aid to the detection of liver metastasis. Increased activity of 5'-nucleotidase in the sera of ovarian tumors was reported by Chatterjee et al. [36]. Dao et al. [37] reported that 5'-nucleotidase activity fell within the normal range in breast cancer patients having objective evidence of disease regression following surgical treatment. Therefore it is clear that the removal of a tumor mass can cause a reduction in serum 5'-nucleotidase activity to the normal level in breast cancer. Similarly regression of a tumor mass by radiation treatment, as in the present study, is reflected by a drop in the activity of 5'-nucleotidase to the normal level, thereby providing positive indication to assess the extent of tumor regression. In conclusion, the activities of PHI and LDH were found to be increased in cervical cancer patients, and the increase in their activities reflected the status of the cancer. Further regression of the tumor mass by radiation reduced their levels to a certain extent, thereby providing positive indication of a tumor-regression response to radiation treatment. The raised serum GGT, transaminases, and phosphatases in the advanced stages of carcinoma of uterine cervix may be due to the secondary implications of cervical cancer, as these enzymes were elevated only in the advanced stages of the disease. The activity of 5'-nucleotidase in serum may be used as an additional marker to assess the status of carcinoma of uterine cervix under circumstances when other markers fail to provide clear indications.

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Vol. 19, No. 2, 1995