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Biochemical Procedures in Different Forms of Cancer
Symposium on Medical Aspects of Cancer
Biochemical Procedures in Different Forms of Cancer Morton K. Schwartz, PhD.
For more than 75 years investigators have been searching for biochemical defects in cancer, and numerous theories have been proposed to account biochemically for uncontrolled growth in malignant tissue. 21 Despite these long continuing studies, only during the past few years have specific biochemical procedures been used in the diagnosis of particular forms of cancer and in following the course of disease. 4 The increasing use of clinical biochemistry determinations in the care of cancer patients is indicated by an examination of the statistics of the Department of Biochemistry of Memorial Hospital. In 1949 the Department performed 42,043 tests, and 20 years later, in 1969, 365,944 procedures. This remarkable growth is related to the development of new surgical techniques and effective chemotherapeutic agents with the resulting necessity for extensive laboratory support following major cancer surgery, and to the need for careful monitoring of possible toxic effects in patients on chemotherapeutic regimens. In addition, many cancer patients have exaggerated medical problems which require extensive laboratory evaluation. The purpose of this report is to review some of the newer biochemical procedures which are useful in primary cancer diagnosis or in following therapeutic response and in monitoring progression or regression of disease.
NEUROBLASTOMA, PHEOCHROMOCYTOMA, AND OTHER NEURAL CREST TUMORS The biochemistry of neuroblastoma and the development of biochemical tests for its diagnosis have received a great deal of attention in the 13 years since Mason and his associates reported elevated excretion of epinephrine-like material in the urine and tumor tissue of an infant with neuroblastoma. 18 Chromaffin cells of the adrenal medulla and cells This work was supported in part by Grant CA-08747 from the National Cancer Institute, National Institutes of Health, and Grant CI-24 from the American Cancer Society. Medical Clinics of North America- Vo!. 55, No. 3, May 1971
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of the sympathetic nervous system, from which tumors such as neuroblastoma arise, synthesize pressor substances from the amino acid tyrosine through a well-defined series of enzymatic reactions. In both tissues the reaction (Fig. 1) proceeds as follows: tyrosine, dopa, dopamine, and norepinephrine. Sympathetic nervous tissue does not carry the reaction any further, but in the adrenal medulla norepinephrine is converted to epinephrine. All these compounds are subject to many metabolic steps in their degradation, and dozens of catechol metabolites have been reported in the urine of patients with neural crest tumors.12, 13,34 Von Studnitz concluded, however, that analysis of a large group of metabolites did not allow differentiation of different tumor types. 34 BelP divided neuroblastoma patients into two basic groups. One group excreted increased amounts of total catecholamines, total metanephrine and vanillylmandelic acid (VMA). In the second group, vanillylmandelic acid and metanephrine were normal or slightly elevated, but dopamine and homovanillic acid (RV A) were markedly elevated. Williams and Greer4° summarized data of 67 patients reported in nine different studies and found that, whereas 93 per cent of patients had an elevation in urinary vanillylmandelic acid and 82 per cent an elevation in homovanillic acid, 99 per cent of the patients (66 of 67 patients) exhibited an elevation of one or the other or both. In the case
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Figure 1.
Biosynthetic and metabolic pathways of norepinephrine and epinephrine.
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with normal excretions of both vanillylmandelic and homovanillic acid, special tissue stains indicated that the tumor was not a typical neuroblastoma. Williams 39 has stated, "Any case of a frankly malignant tumor in which excretion of homovanillic acid and vanillylmandelic acid is normal should be accepted as neuroblastoma only with considerable reservation. " In a series of 44 patients with histologically proven neuroblastoma seen at Memorial Hospital, 33 had elevations in urinary catecholamines, vanillylmandelic acid, or both at the time of initial diagnosis and the collection of the first urine specimen. 16 Among 11 patients with normal values at the first examination, 8 developed abnormal urinary concentration 1 to 10 months after the initial diagnosis. Thus, 41 of 43 patients (93 per cent) had elevated values at some time in the course of their disease. Seven of the 11 patients with normal values at the first examination were older than 5 years and represented 70 per cent of the children of that age group in the study. In addition to initial diagnosis, catecholamine and vanillylmandelic acid assays provide a very sensitive measure of therapeutic response. Williams38 reported a three month old child who, on laparotomy, was found to have a massively enlarged liver infiltrated with tumor, together with an independent nodular tumor mass of indefinite origin in the right retrohepatic area. Radiotherapy and nitrogen mustard treatment were instituted, with marked regression of tumor tissue and reduction of liver size. This clinical response was accompanied by a precipitous fall in urinary vanillylmandelic acid from over 500 mg. per gm. of creatinine to values near zero. After about 2 weeks at this low level, vanillylmandelic acid excretion began to increase, despite the fact that the child remained asymptomatic, and in 4 months reached a level of about 50 mg. per gm. of creatinine. At this time nitrogen mustard was again administered and the levels fell toward zero.
Cystathionine 'Excretion Cystathionine is an intermediate in the biosynthesis of cysteine from methionine. This substance is not found normally in urine, but has been observed in urine of children with congenital cystathionuria and in cases of hepatoblastoma, argentaffinoma, and hepatoma. 13 Gjessing reported urinary excretion of cystathionine in each of 6 cases of neuroblastomal l and observed more pronounced cystathionuria in patients with liver metastases. Geiser and Efron lO found no measureable cystathionine in urine of 34 patients with other forms of cancer and in 15 successfully treated neuroblastoma patients. Fourteen of 28 patients with active neuroblastoma had cystathionuria. Urinary vanillylmandelic acid was elevated in 61 per cent of these patients. Cystathionine assays permitted a diagnosis of neuroblastoma in 2 of the patients; this would not have been possible if only the vanillylmandelic acid assay had been carried out. Other tumors of neural crest origin also demonstrate changes in pressor amine excretion. In pheochromocytoma, the diagnosis can be established in more than 90 per cent of the cases by analysis of urinary
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catecholamines, the metanephrines and vanillylmandelic acid. 8 Since there appears to be no defect in dopamine metabolism in pheochromocytoma, urinary homovanillic acid is not elevated. 8 The subject of the biochemical changes in pheochromocytoma has been adequately reviewed by Bodansky,4 who reported that 70 per cent of patients with pheochromocytoma excreted more than 0.50 mg. of catecholamines per day (normal levels up to 0.2 mg. per day) and that vanillylmandelic acid excretion was abnormal in 77 per cent of the patients and markedly elevated (7300 mg. per day) in only 8 per cent of the subjects. In a review of 62 patients with pheochromocytoma seen at the Clinical Center of the National Institutes of Health during the decade 1956 to 1966, elevated values of vanillylmandelic acid were observed in 59 patients, of metanephrine in 60 cases and of catecholamines in 60 cases.:lO In every patient there was increased excretion of at least one of the three parameters, allowing a clear biochemical diagnosis of pheochromocytoma. There are relatively few studies of excretion of catecholamines and their metabolites in ganglioneuroma. l :l It appears that dopamine and its metabolites constitute the major portion of catecholamine derivatives found in the urine of the individuals with this disease. However, it does not appear to be possible to differentiate ganglioneuroma from neuroblastoma on the basis of biochemical assays.
CARCINOID Malignant carcinoid is a gastrointestinal tumor whose diagnosis is based to a great extent on biochemical findings. In these tumors there is an overproduction of serotonin (5-hydrotryptamine) with abnormal urinary excretion of the metabolic degradation product, 5-hydroxyindole acetic acid (5-HIAA). It has been reported that 29 of 30 patients with active carcinoid demonstrated elevations of 5-hydroxyindole acetic acid to levels as high as 75 times the upper limit of normal. 4 After successful surgical extirpation of the carcinoid, the 5-hydroxyindole acetic acid levels became lower and often returned to normal.
HORMONE· DEPENDENT TUMORS For more than 70 years the role of hormones in tumor growth and the possible use of hormones in the treatment of cancer have been of great interest. The elaboration of hormones or hormone-like substances by nonendocrine tumors and the relationship of hormones to the growth of cancer of the breast, prostate, thyroid, kidney, endometrium, and seminal vesicle, as well as lymphosarcoma and leukemia, have also been of concern, and many efforts have been made to use biochemical parameters to predict the response of these tumors to hormonal therapy.
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The use of individual steroid analyses in predicting responsiveness to adrenalectomy, hypophysectomy, or chemotherapeutic hormonal therapy has not been very successful. This lack of success is undoubtedly due in part to the effect of age, weight, emotional stress of hospitalization, and extent of the illness upon hormone excretion, as well as the relatively large analytical error in steroid analysis. During the past decade some workers have used a grouping of steroid analyses to predict hormone responsiveness. Bulbrook and his associates in England found that urinary etiocholanolone levels tended to be high in patients who subsequently had positive clinical responses to adrenalectomy or hypophysectomy, and that the 17-hydroxycorticosteroids (17-0HCS) tended to be low in successful cases. 2 • 15 By means of a discriminant function of these parameters, it was possible to predict the success or failure of adrenalectomy or hypophysectomy in oreast cancer. The Bulbrook discriminant is: D = 80 - 80 (17-0HCS) + Etiocholanolone (mg/24 hr. urine) (jJ-g/24 hr. urine) The more positive the discriminant, the greater is the chance of palliative success following ablative surgery. In 35 patients with positive discriminants, hypophysectomy was considered successful in 46 per cent of the cases and a failure in 31 per cent of the cases. In 30 patients with negative discriminants, adrenalectomy was a failure in 67 per cent of the patients and successful in only 9 per cent. Patients chosen at random for adrenalectomy exhibit a failure rate of 45 per cent and a success rate of 28 per cent,15 In a more recent report, hypophysectomy was successful in 67 per cent of patients with positive discriminants and in only 15 per cent of patients with negative values. 2 Wade and his associates evaluated the etiocholanolone-17-hydroxycorticosteroids discriminant in normal women, hospitalized women without cancer, and 26 patients with early breast cancer.36 Five of 32 normal women, 9 of 33 women admitted for operations other than for cancer, and only 2 of 26 women with breast cancer had negative discriminants. The authors concluded that discriminants cannot be used in patients with early breast cancer. In a commentary on this report, Bulbrook and Hayward stated that their original statements may have been oversimplifications of a very complicated situation, and that the age and menopausal status of the patient, as well as the stage and grade of cancer, are important.6 Sarfaty and Tallis developed a discriminant including urinary 11deoxy-17-ketosteroids and 17-hydroxycorticosteroids, and established probabilities of remission based on the numerical value of the discriminant. 24 For example, a discriminant of +2 has a 55 per cent probability probability of remission and a discriminant of -2 a probability of 15 per cent. Rather than a discriminant, a simple ratio between urinary 11-deoxy-17-ketosteroids (mg. per 24 hours) and 17-hydroxycortico-
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steroids (mg. per 24 hours) has been used to obtain data similar to that observed with the Bulbrook discriminant. 32 Ratio values less than 0.17 were equivalent to a negative discriminant, and those above to a positive. In 210 breast cancer patients adrenalectomy or hypophysectomy was a failure in 59 per cent of the patients with a negative discriminant and in 59 per cent of the patients with a ratio less than 0.17. 32 Marmorston and his associates have devised discriminants for the differentiation between benign and malignant breast and prostatic diseaseP For breast disease the discriminant is: D
= -
3.8 etiocholanolone + 2.8 estriol + 2.4 age
Fourteen of 18 benign breast tumor patients had negative scores, and each of 7 cancer patients had positive values. The discriminant for prostatic disease is a complicated one involving estrone, estriol, 17ketogenic steroids, Porter-Silber chromogens, and the beta-fraction. With this discriminant, 17 of 21 patients with prostatic cancer had a positive value and 16 of 18 patients with benign prostate hypertrophy had negative values. The discriminant described by Moore and associates utilizes a ratio between urinary 17-hydroxycorticosteroids and 17-ketosteroids, each expressed as mg. per gm. of creatinine in the 48 hour urine collection just before and during the intravenous administration of 40 units of adrenocorticotropic hormone over a 6 to 8 hour period each day.19 The discriminant also uses the free period (the time between mastectomy and the first recurrence of disease). With this discriminant it was possible to identify 12 out of 15 responders and 12 of 16 nonresponders to adrenalectomy. It was emphasized that the denial of ablative surgery based on biochemical discriminants alone should be considered with great caution and that palliative surgery should be denied only if both the biochemical and the clinical discriminants were unfavorable. 19 Rao has devised a steroid discriminant for evaluation of patients with lung cancer which requires urinary androsterone, etiocholanolone, 17-ketosteroids and 17-hydroxycorticosteroids.22 Seventy-five of 84 patients with inoperable lung cancer had a negative discriminant. Ninety-two of 100 nonhospitalized and 35 of 42 hospitalized controls had a positive discriminant.
CHORIONIC GONADOTROPIN In trophoblastic neoplasms the quantitative measurement of urinary chorionic gonadotropin (HCG) is essential for initial diagnosis and then for following the course of a disease in which clinical evidence of response to treatment may appear after there is a significant change in chorionic gonadotropin titre. Despite the development of immunochemical and radioimmune assays for human chorionic gonadotropin, at the present time the most sensitive quantitative assay is concentration
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of urinary hormone by physical chemical methods and bioassay by the mouse uterine weight method. In choriocarcinoma the response to chemotherapy is related to the human chorionic gonadotropin titre, and the remission rate has been reported to be 95 per cent if the titre is less than 100,000 International Units per 24 hours and the patients are treated within four months of the onset of the disease. 23 If the titre is greater than 100,000 International Units per 24 hours, and the duration of the disease is longer than 4 months, the remission rate is 36 per cent. Complete remission is defined as disappearance of clinical evidence of disease and a decrease of human chorionic gonadotropin to the level of pituitary gonadotropin.
SERUM AND URINARY ENZYMES Serum and urinary enzyme assays are routine procedures in the evaluation of many diseases. There has been extensive study in attempts to define tissue specific enzymes which could be of assistance in the diagnosis of a primary carcinoma or in helping to define the tissue of origin of metastatic tumor.
Amylase Amylase determinations are apparently of little use in the early diagnosis of carcinoma of the pancreas. In a study of 83 patients with histologically proven carcinoma of the pancreas, Ansari and Burch found no elevations of serum amylase in any of the 30 patients in which this determination was done.' Other workers have reported elevations in 8 to 40 per cent of their patients. 29 Amylase can also be elevated in nonpancreatic conditions such as perforated duodenal ulcer, intestinal obstruction, and renal disease. 29
Acid Phosphatase Acid phosphatase is elevated in the serum of only 24 per cent of patients with nonmetastatic prostate carcinoma, but in 81 per cent of these patients with skeletal metastases. 28 The specificity of acid phosphatase in prostate carcinoma is related to substrate, and elevations are observed in many other diseases when orthophosphate esters other than beta-glycerophosphate are used. However, with beta-glycerophosphate as substrate, elevations are rarely observed other than in prostatic disease or in certain myeloproliferative disease where modest elevations are related to the white cell or platelet count. 28 Efforts have been made to increase the sensitivity of the acid phosphatase assay in prostatic disease by utilizing specific tartrate inhibition of prostatic acid phosphatase and assay of the "prostatic" fraction. Green and his associates evaluated "prostatic" acid phosphatase in 454 patients. Elevat~ons of tartrate-sensitive acid phosphatase were found in 14 of 34 patients with prostatic carcinoma, in 2 of 90 patients with benign prostatic hypertrophy, in 15 of 76 patients with other forms of cancer, and in 23 patients without cancer.28
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Alkaline Phosphatase In primary tumors of bone, as well as in metastatic disease of bone, alkaline phosphatase is elevated when the tumor is predominantly osteoblastic. 28 In breast carcinoma, most cases of bony metastases are osteolytic, and alkaline phosphatase levels are usually in the normal range or slightly elevated. In prostatic carcinoma most of the metastatic lesions are osteoblastic, and the serum alkaline phosphatase is elevated to levels 4 to 10 times the upper limit of normal. Modest elevations of alkaline phosphatase are observed in about one third of patients with multiple myeloma, and more extensive elevations are seen in over 95 per cent of patients with adenomas or carcinoma of the parathyroid. 28 In liver disease, elevations of alkaline phosphatase are observed in patients with extrahepatic obstruction of the biliary tract and in patients with intrahepatic metastases. Recently, there has been great interest in the isoenzymes of serum alkaline phosphatase, and electrophoretic and kinetic techniques have been used for their separation. Although these methods permit clear differentiation of kidney or intestine phosphatase from bone or liver phosphatase, it has not been possible until recently to precisely separate the bone from the liver enzyme. Fennelly and his associates, by use of gel filtration, differential thermostability, and differential urea inhibition, differentiated bone from liver phosphatase and feel that the serum patterns of patients with skeletal and hepatic disease can be identified with a high degree of confidence.9 A heat stabile, L-phenylalanine inhibited alkaline phosphatase similar to placental enzyme has been found in the serum of some patients with cancer.31 In 520 cancer patients Stolbach identified this placental enzyme-like alkaline phosphatase in 15 cases and in malignant effusion from 2 patients. 31 Stolbach concluded that the cancer specific alkaline phosphatase isoenzyme (Regan isoenzyme) is useful in following the course of disease, identifying malignant pleural effusion, in clarifying the nature of unexplained elevations of serum alkaline phosphatase activity.
5' -Nucleotidase Serum 5 '-nucleotidase is useful in determining the presence or absence of liver disease in a patient with an elevated serum alkaline phosphatase activity, and in following the progression of liver metastases. Schwartz and Bodansky reported elevated 5 ' -nucleotidase in 27 of 31 patients with cancer and varying degrees of liver involvement. 26 The classical method of utilizing serum 5 '-nucleotidase in evaluating liver and bone disease is illustrated by examining data in a patient with metastatic cancer of the colon. Upon skeletal survey this patient had widespread bony metastases. Bromsulphalein retention was 7 per cent; total bilirubin, 0.4 mg. per 100 ml.; alkaline phosphatase, 24 Bodansky units; the liver was not palpable. However, his serum 5 ' -nucleotidase was elevated to 68 Campbell units, or six times the upper limit of normal. At autopsy, he was found to have liver metastases. 28 It is of interest that in a study of 80 patients with an elevation of
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5/ -nucleotidase or alkaline phosphatase, 30 per cent (23 patients) had an elevation of 5/ -nucleotidase when the alkaline phosphatase was normal. 28 There have been reports that elevations of serum 5/ -nucleotidase and alkaline phosphatase parallel each other in hepatic disease and that alkaline phosphatase is more frequently elevated. Our data indicate that in a cancer patient population the 5/-nucleotidase is elevated more frequently than alkaline phosphatase in hepatic disease. Van der Slik and his associates have concluded that consecutive assays of serum 5/ -nucleotidase furnish indispensable data in patients with liver disease. 33 They stated that in metastatic liver disease 5/nucleotidase may be elevated at a stage when alkaline phosphatase is still within normal limits; that high serum alkaline phosphatase with normal 5/-nucleotidase is strong evidence for the presence of osteoblastic bone disease, and finally that serum 5/-nucleotidase, rather than alkaline phosphatase, is the enzyme of choice in evaluating liver disease in children or pregnant women.
Lactic Dehydrogenase Lactic dehydrogenase (LDH) is one of a group of ubiquitous enzymes whose activity is elevated in a variety of diseases. Total serum lactic dehydrogenase is modestly elevated in most patients with extensive hepatic metastases. In one study, total serum lactic dehydrogenase was elevated to three times the upper limit of normal in 91 per cent of the patients with liver metastasesP The enzyme is more consistently elevated, and to a greater extent, in serum of patients with various types of leukemia. Lactic dehydrogenase activity in pleural and peritoneal effusions containing malignant cells is reported to be higher than the serum lactic dehydrogenase, whereas the lactic dehydrogenase of benign effusions is lower.z7 It has been claimed that urinary lactic dehydrogenase is useful in the recognition of cancer of the bladder or kidney. In a recent study of urinary lactic dehydrogenase in 263 Memorial Hospital patients, it was found that elevations of the urinary enzymes were more related to pyuria, hematuria, and bacteriuria than the presence of a tumor. 14 Only rarely was an elevated enzyme level measured in a patient with a normal urinalysis. It was concluded that urinary lactic dehydrogenase is not a useful routine diagnostic procedure in the study and follow-up of patients with malignant disease of the genitourinary tract. In cancer, the increase in serum lactic dehydrogenase is usually associated with a minor change in the isoenzyme pattern, particularly an increase in LDH3 and LDH5 (the faster moving isoenzymes).37 Wilkinson has reported extra isoenzyme bands in a patient with inoperable carcinoma of the esophagus with metastasis to the liver. Additional bands of activity were found between LDHl and LDHz and between LDHz and LDH3. The appearance of extra lactic dehydrogenase bands occurs rarely.37
Phosphohexose Isomerase Phosphohexose isomerase (PHI) is a ubiquitous glycolytic enzyme found in most tissues. Of the various ubiquitous enzymes that have been
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studied in neoplastic disease, phosphohexose isomerase is elevated more frequently than any other serum enzyme. 5 Thus, of 119 patients with carcinoma of the gastrointestinal tract, phosphohexose isomerase was elevated in 74 per cent of the cases, in contrast to frequencies of 70 per cent for elevations of aldolase, of 53 per cent for lactic dehydrogenase and 35 per cent for glutamic oxalacetic transaminase. In a study of 284 patients with liver metastases, the phosphohexose isomerase was elevated in 84 per cent of the cases, compared to only 51 per cent elevations of glutamic oxalacetic transaminase and 69 per cent lactic dehydrogenase. 5 Serum phosphohexose isomerase is useful in following the progression or regression of metastatic disease. In a study of sequential assays in 6 patients with cancer, it was possible to define with some degree of certainty, on the basis of changes in serum phosphohexose isomerase, a total of 21 distinct episodes of clinical worsening. 25 Phosphohexose isomerase is an indicator of metastatic growth, and the claim has been made that it is the serum enzyme which provides the most sensitive index of clinical status. 7 Muir has attempted to use vaginal fluid phosphohexose isomerase activity in screening for cervical carcinoma. 2o The phosphohexose isomerase was markedly elevated in the vaginal fluid of women with gynecological disorders. The author concludes that although the phosphohexose isomerase determination could not replace vaginal and cervical cytology, it could become a simple screening tool. Based on estimates of gynecological disorders it was calculated that the cytologist would be required to examine 42 of every 100 cases screened by the enzyme assay. The cytologist, however, would have the assurance that every specimen with elevated phosphohexose isomerase activity came from a patient with a gynecologicallesion. SUMMARY An attempt has been made to review some of the biochemical procedures useful in the diagnosis of cancer and in following the progression of the disease. Because of limitations of space it has not been possible to cover all areas. Omissions were not made on the basis of relative importance and have included serum proteins in myeloma and other forms of cancer, hormones such as parathyroid hormone, calcitonin, trace metals, and the general effects of carcinoma on carbohydrate, protein, and calcium and phosphorus metabolism. REFERENCES 1. Ansari, A., and Burch, G. E.: A correlation study of proven carcinoma of pancreas in 83 patients. Amer. J. Gastroenterol., 50:456-475, 1968. 2. Atkins, H., Bulbrook, R. D., Falconer, M. A., Hayward, J. L., MacLean, K. S., and Schurr, P. H.: Ten years' experience of steroid assays in the management of breast cancer. Urinary steroids in the prediction of response to adrenalectomy or hypophysectomy. Lancet, 2: 1255-1263,1968.
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3. Bell, M.: Observations on the biochemical diagnosis of neuroblastoma. In Bohuon, C., ed.: Recent Results in Cancer Research, Neuroblastoma Biochemical Studies. Berlin, Springer Verlag, 1966, pp. 42-51. 4. Bodansky, 0.: Biochemical changes of clinical significance in cancer. In Proceedings of the Fifth National Cancer Conference, Philadelphia, J. B. Lippincott and Co., 1965, pp. 687-703. 5. Bodansky, 0., and Schwartz, M. K.: Phosphohexose isomerase: Clinical aspects. In Colowick, S., Kaplan, N. and Wood, W. A., eds.: Methods of Enzymology. New York, Academic Press, 1966, vol. IX, pp. 568-575. 6. Bulbrook, R D., and Hayward, J. L.: Discriminants and breast cancer. Lancet, 1:1161, 1969. 7. Campbell, D. M., and King, K J.: Serum phosphatase and glycolytic enzymes in cancer of the breast. Biochem. J., 82 :23p, 1962. 8. Crout, R J.: Pheochromocytoma. Pharm. Rev., 18:651-657, 1966. 9. Fennelly, J. J., Dunne, J., McGeeney, K., Chong, L., and Fitzgerald, M.: The importance of varying molecular size, differential heat and urea inactivation of phosphatase in the identification of disease patterns. Ann. N .Y. Acad. Sci., 166: 794-810, 1969. 10. Geiser, C. F., and Efron, M. L.: Cystathioninuria in patients with neuroblastoma or ganglioneuroblastoma. Cancer, 22 :856-860, 1968. 11. Gjessing, L. R: Studies of functional neural tumors. n. Cystathioninuria. Scand. J. Clin. Invest., 15:475-478, 1963. 12. Gjessing, L. R, and Rorud, 0.: Studies of functional neural tumors. VII. Urinary excretion of phenolic pyruvic acids. Scand. J. Clin. Lab. Invest., 17 :80-84, 1965. 13. Gjessing, L. R: Biochemistry of functional neural crest tumors. Adv. Clin. Chem., 11 :81131,1968. 14. Grabstald, H., and Schwartz, M. K.: Urinary lactic dehydrogenase in genitourinary tract disease. J.A.M.A., 207:2062-2066,1969. 15. Hayward, J. L., and Bulbrook, R D.: The value of urinary steroid estimations in the prediction of response to adrenalectomy or hypophysectomy. Cancer Res., 25:11291139,1965. 16. Helson, L., Murphy, M. L., and Schwartz, M. K.: Neuroblastoma survival and serial catecholamine excretion. (Manuscript in preparation.) 17. Marmorston, J., Geller, P. J., and Weiner, J. M.: Pretreatment urinary hormone patterns and survival in patients with breast cancer, prostate cancer, or lung cancer. Ann. N.Y. Acad. Sci., 164:483-493,1969. 18. Mason, G. A., Hart-Mercer, J., Millar, K J., Strang, L. B., and Wynne, N.: Adrenalinesecreting neuroblastoma in an infant. Lancet, 2 :322-325,1957. 19. Moore, F. D., Woodrow, S. I., Aliapoulios, M. A., and Wilson, R: Carcinoma of the Breast. Boston, Little, Brown and Co., 1968, pp. 46-50. 20. Muir, G. G.: Possible use of phosphohexose isomerase as a preliminary to exfoliative cytology in screening for cervical carcinoma. J. Clin. Path., 19: 3 78-383, 1966. 21. Potter, Van R: Recent trends in cancer biochemistry, The importance of studies on fetal tissue. Cancer Res., 24:1085-1095, 1965. 22. Rao, L. G. S.: Discriminant function based on steroid abnormalities in patients with lung cancer. Lancet, 2:441-447,1970. 23. Ross, G. T., Goldstein, D. P., Hertz, R, Lipsett, M. D., and Ode1l, W. D.: Sequential use of methotrexate and actinomycin D in the treatment of metastatic choriocarcinoma and related trophoblastic diseases in women. Amer. J. Obstet. Gynec., 93:223-229,1965. 24. Sarfaty, G., and Tallis, M.: Probability of a woman with advanced breast cancer responding to adrenalectomy or hypophysectomy. Lancet, 2 :685-689,1970. 25. Schwartz, M. K., Greenberg, K, and Bodansky, 0.: Comparative values of phosphatases and other serum enzymes in following patients with prostatic carcinoma: Consideration of phosphohexose isomerase, glutamic oxaloacetic transaminase, isocitric dehydrogenase and acid and alkaline phosphatase. Cancer, 16:583-594, 1963. 26. Schwartz, M. K., and Bodansky, 0.: Serum 5'-nucleotidase in patients with cancer. Cancer, 18:886-892,1965. 27. Schwartz, M. K., and Bodansky, 0.: Lactic dehydrogenase (clinical analysis). In Colowick, S., Kaplan, N., and Wood, W. A., eds.: Methods in Enzymology. New York, Academic Press, 1966, vol. IX, pp. 294-302. 28. Schwartz, M. K., Fleisher, M., and Bodansky, 0.: Clinical application of phosphohydrolase measurements in cancer. Ann. N. Y. Acad. Sci., 166:775-793,1969. 29. Schwartz, M. K., and Fleisher, M.: Diagnostic biochemical methods in pancreatic disease. Adv. Clin. Chem., 11 : 113-159, 1970. 30. Sjoerdsma, A., Engelman, K., Waldmann, T. A., Cooperman, L. H., and Hammond, W. G.: Pheochromocytoma: current concepts of diagnosis and treatment. Ann. Int. Med., 65: 1302-1326,1966. 31. Stolbach, L. L.: Clinical application of alkaline phosphatase isoenzyme analysis. Ann. N.Y. Acad. Sci., 166:760-774, 1969.
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32. Thomas, B. S., Bulbrook, R. D., and Hayward, J. L.: Urinary steroid assays and response to endocrine ablation. Brit. Med. J., 3:523-524,1967. 33. Van der Slik, W., Persijn, J. P., Engelsman, E., and Riethorst, A.: Serum 5'-nuc1eotidase. Clin. Biochem., 3:59-80,1970. 34. Von Studnitz, W., Kaiser, H., and Sjoerdsma, A.: Spectrum of catecholamine biochemistry in patients with neuroblastoma. New Eng. J. Med., 269:232-235,1963. 35. Vorhess, M. L., and Gardner, L. I.: Neuroblastoma and catecholamine excretion. Lancet, 1 :1288, 1961. 36. Wade, A. P., Davis, J. C., Tweedie, M. C. K., Clarke, C. A., and Haggart, B.: The discriminant function in early carcinoma of the breast. Lancet, 1 :853-858, 1969. 37. Wilkinson, J. H.: Clinical applications of isoenzymes. Clin. Chem., 16: 733-739, 1970. 38. Williams, C. M.: Homovanillic acid and vanilmandelic acid in diagnosis of neuroblastoma. J.A.M.A., 183:134-138, 1963. 39. Williams, C. M.: Biochemical diagnosis of neuroblastoma. Postgrad. Med., 36:A95-A99, 1964. 40. Williams, C. M., and Greer, M.: IV. Estimation by gas chromatography of urinary homovanillic acid and vanilmandelic acid in neuroblastoma. Methods Med. Res., 12: 106-114, 1970.
Biochemical Procedures in Different Forms of Cancer Morton K. Schwartz, PhD.
For more than 75 years investigators have been searching for biochemical defects in cancer, and numerous theories have been proposed to account biochemically for uncontrolled growth in malignant tissue. 21 Despite these long continuing studies, only during the past few years have specific biochemical procedures been used in the diagnosis of particular forms of cancer and in following the course of disease. 4 The increasing use of clinical biochemistry determinations in the care of cancer patients is indicated by an examination of the statistics of the Department of Biochemistry of Memorial Hospital. In 1949 the Department performed 42,043 tests, and 20 years later, in 1969, 365,944 procedures. This remarkable growth is related to the development of new surgical techniques and effective chemotherapeutic agents with the resulting necessity for extensive laboratory support following major cancer surgery, and to the need for careful monitoring of possible toxic effects in patients on chemotherapeutic regimens. In addition, many cancer patients have exaggerated medical problems which require extensive laboratory evaluation. The purpose of this report is to review some of the newer biochemical procedures which are useful in primary cancer diagnosis or in following therapeutic response and in monitoring progression or regression of disease.
NEUROBLASTOMA, PHEOCHROMOCYTOMA, AND OTHER NEURAL CREST TUMORS The biochemistry of neuroblastoma and the development of biochemical tests for its diagnosis have received a great deal of attention in the 13 years since Mason and his associates reported elevated excretion of epinephrine-like material in the urine and tumor tissue of an infant with neuroblastoma. 18 Chromaffin cells of the adrenal medulla and cells This work was supported in part by Grant CA-08747 from the National Cancer Institute, National Institutes of Health, and Grant CI-24 from the American Cancer Society. Medical Clinics of North America- Vo!. 55, No. 3, May 1971
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of the sympathetic nervous system, from which tumors such as neuroblastoma arise, synthesize pressor substances from the amino acid tyrosine through a well-defined series of enzymatic reactions. In both tissues the reaction (Fig. 1) proceeds as follows: tyrosine, dopa, dopamine, and norepinephrine. Sympathetic nervous tissue does not carry the reaction any further, but in the adrenal medulla norepinephrine is converted to epinephrine. All these compounds are subject to many metabolic steps in their degradation, and dozens of catechol metabolites have been reported in the urine of patients with neural crest tumors.12, 13,34 Von Studnitz concluded, however, that analysis of a large group of metabolites did not allow differentiation of different tumor types. 34 BelP divided neuroblastoma patients into two basic groups. One group excreted increased amounts of total catecholamines, total metanephrine and vanillylmandelic acid (VMA). In the second group, vanillylmandelic acid and metanephrine were normal or slightly elevated, but dopamine and homovanillic acid (RV A) were markedly elevated. Williams and Greer4° summarized data of 67 patients reported in nine different studies and found that, whereas 93 per cent of patients had an elevation in urinary vanillylmandelic acid and 82 per cent an elevation in homovanillic acid, 99 per cent of the patients (66 of 67 patients) exhibited an elevation of one or the other or both. In the case
O ,:?
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""TYROSINE "-
O ?'
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I
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~O
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~-~-NH,
""DOPAMINE
~-~-NH,
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~
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OH
3,4DIHYDROXYMANDELIC ACID
HH
"- OH H HOO,
OH
I
H H
HOOC-C-COOH H NH, HO ~
OCH, HOMOVANILlC ACID (HVAI
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HH , C-C-COOH H NH,
HOOC-C-NH H H I HO CH,
~,
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CH,OO' HO
OH I H CH,OOC-C-NH, HH HO ~ NORMETANEPHRINE (NMI
I
?HHH CH o?' C-C-N-CH , O , H H ' HO ~ METANEPHRINE (MI
t_c~O H OH
~
VANILMANDELlC ACID (VMAI
Figure 1.
Biosynthetic and metabolic pathways of norepinephrine and epinephrine.
BIOCHEMICAL PROCEDURES IN DIFFERENT FORMS OF CANCER
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with normal excretions of both vanillylmandelic and homovanillic acid, special tissue stains indicated that the tumor was not a typical neuroblastoma. Williams 39 has stated, "Any case of a frankly malignant tumor in which excretion of homovanillic acid and vanillylmandelic acid is normal should be accepted as neuroblastoma only with considerable reservation. " In a series of 44 patients with histologically proven neuroblastoma seen at Memorial Hospital, 33 had elevations in urinary catecholamines, vanillylmandelic acid, or both at the time of initial diagnosis and the collection of the first urine specimen. 16 Among 11 patients with normal values at the first examination, 8 developed abnormal urinary concentration 1 to 10 months after the initial diagnosis. Thus, 41 of 43 patients (93 per cent) had elevated values at some time in the course of their disease. Seven of the 11 patients with normal values at the first examination were older than 5 years and represented 70 per cent of the children of that age group in the study. In addition to initial diagnosis, catecholamine and vanillylmandelic acid assays provide a very sensitive measure of therapeutic response. Williams38 reported a three month old child who, on laparotomy, was found to have a massively enlarged liver infiltrated with tumor, together with an independent nodular tumor mass of indefinite origin in the right retrohepatic area. Radiotherapy and nitrogen mustard treatment were instituted, with marked regression of tumor tissue and reduction of liver size. This clinical response was accompanied by a precipitous fall in urinary vanillylmandelic acid from over 500 mg. per gm. of creatinine to values near zero. After about 2 weeks at this low level, vanillylmandelic acid excretion began to increase, despite the fact that the child remained asymptomatic, and in 4 months reached a level of about 50 mg. per gm. of creatinine. At this time nitrogen mustard was again administered and the levels fell toward zero.
Cystathionine 'Excretion Cystathionine is an intermediate in the biosynthesis of cysteine from methionine. This substance is not found normally in urine, but has been observed in urine of children with congenital cystathionuria and in cases of hepatoblastoma, argentaffinoma, and hepatoma. 13 Gjessing reported urinary excretion of cystathionine in each of 6 cases of neuroblastomal l and observed more pronounced cystathionuria in patients with liver metastases. Geiser and Efron lO found no measureable cystathionine in urine of 34 patients with other forms of cancer and in 15 successfully treated neuroblastoma patients. Fourteen of 28 patients with active neuroblastoma had cystathionuria. Urinary vanillylmandelic acid was elevated in 61 per cent of these patients. Cystathionine assays permitted a diagnosis of neuroblastoma in 2 of the patients; this would not have been possible if only the vanillylmandelic acid assay had been carried out. Other tumors of neural crest origin also demonstrate changes in pressor amine excretion. In pheochromocytoma, the diagnosis can be established in more than 90 per cent of the cases by analysis of urinary
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catecholamines, the metanephrines and vanillylmandelic acid. 8 Since there appears to be no defect in dopamine metabolism in pheochromocytoma, urinary homovanillic acid is not elevated. 8 The subject of the biochemical changes in pheochromocytoma has been adequately reviewed by Bodansky,4 who reported that 70 per cent of patients with pheochromocytoma excreted more than 0.50 mg. of catecholamines per day (normal levels up to 0.2 mg. per day) and that vanillylmandelic acid excretion was abnormal in 77 per cent of the patients and markedly elevated (7300 mg. per day) in only 8 per cent of the subjects. In a review of 62 patients with pheochromocytoma seen at the Clinical Center of the National Institutes of Health during the decade 1956 to 1966, elevated values of vanillylmandelic acid were observed in 59 patients, of metanephrine in 60 cases and of catecholamines in 60 cases.:lO In every patient there was increased excretion of at least one of the three parameters, allowing a clear biochemical diagnosis of pheochromocytoma. There are relatively few studies of excretion of catecholamines and their metabolites in ganglioneuroma. l :l It appears that dopamine and its metabolites constitute the major portion of catecholamine derivatives found in the urine of the individuals with this disease. However, it does not appear to be possible to differentiate ganglioneuroma from neuroblastoma on the basis of biochemical assays.
CARCINOID Malignant carcinoid is a gastrointestinal tumor whose diagnosis is based to a great extent on biochemical findings. In these tumors there is an overproduction of serotonin (5-hydrotryptamine) with abnormal urinary excretion of the metabolic degradation product, 5-hydroxyindole acetic acid (5-HIAA). It has been reported that 29 of 30 patients with active carcinoid demonstrated elevations of 5-hydroxyindole acetic acid to levels as high as 75 times the upper limit of normal. 4 After successful surgical extirpation of the carcinoid, the 5-hydroxyindole acetic acid levels became lower and often returned to normal.
HORMONE· DEPENDENT TUMORS For more than 70 years the role of hormones in tumor growth and the possible use of hormones in the treatment of cancer have been of great interest. The elaboration of hormones or hormone-like substances by nonendocrine tumors and the relationship of hormones to the growth of cancer of the breast, prostate, thyroid, kidney, endometrium, and seminal vesicle, as well as lymphosarcoma and leukemia, have also been of concern, and many efforts have been made to use biochemical parameters to predict the response of these tumors to hormonal therapy.
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The use of individual steroid analyses in predicting responsiveness to adrenalectomy, hypophysectomy, or chemotherapeutic hormonal therapy has not been very successful. This lack of success is undoubtedly due in part to the effect of age, weight, emotional stress of hospitalization, and extent of the illness upon hormone excretion, as well as the relatively large analytical error in steroid analysis. During the past decade some workers have used a grouping of steroid analyses to predict hormone responsiveness. Bulbrook and his associates in England found that urinary etiocholanolone levels tended to be high in patients who subsequently had positive clinical responses to adrenalectomy or hypophysectomy, and that the 17-hydroxycorticosteroids (17-0HCS) tended to be low in successful cases. 2 • 15 By means of a discriminant function of these parameters, it was possible to predict the success or failure of adrenalectomy or hypophysectomy in oreast cancer. The Bulbrook discriminant is: D = 80 - 80 (17-0HCS) + Etiocholanolone (mg/24 hr. urine) (jJ-g/24 hr. urine) The more positive the discriminant, the greater is the chance of palliative success following ablative surgery. In 35 patients with positive discriminants, hypophysectomy was considered successful in 46 per cent of the cases and a failure in 31 per cent of the cases. In 30 patients with negative discriminants, adrenalectomy was a failure in 67 per cent of the patients and successful in only 9 per cent. Patients chosen at random for adrenalectomy exhibit a failure rate of 45 per cent and a success rate of 28 per cent,15 In a more recent report, hypophysectomy was successful in 67 per cent of patients with positive discriminants and in only 15 per cent of patients with negative values. 2 Wade and his associates evaluated the etiocholanolone-17-hydroxycorticosteroids discriminant in normal women, hospitalized women without cancer, and 26 patients with early breast cancer.36 Five of 32 normal women, 9 of 33 women admitted for operations other than for cancer, and only 2 of 26 women with breast cancer had negative discriminants. The authors concluded that discriminants cannot be used in patients with early breast cancer. In a commentary on this report, Bulbrook and Hayward stated that their original statements may have been oversimplifications of a very complicated situation, and that the age and menopausal status of the patient, as well as the stage and grade of cancer, are important.6 Sarfaty and Tallis developed a discriminant including urinary 11deoxy-17-ketosteroids and 17-hydroxycorticosteroids, and established probabilities of remission based on the numerical value of the discriminant. 24 For example, a discriminant of +2 has a 55 per cent probability probability of remission and a discriminant of -2 a probability of 15 per cent. Rather than a discriminant, a simple ratio between urinary 11-deoxy-17-ketosteroids (mg. per 24 hours) and 17-hydroxycortico-
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steroids (mg. per 24 hours) has been used to obtain data similar to that observed with the Bulbrook discriminant. 32 Ratio values less than 0.17 were equivalent to a negative discriminant, and those above to a positive. In 210 breast cancer patients adrenalectomy or hypophysectomy was a failure in 59 per cent of the patients with a negative discriminant and in 59 per cent of the patients with a ratio less than 0.17. 32 Marmorston and his associates have devised discriminants for the differentiation between benign and malignant breast and prostatic diseaseP For breast disease the discriminant is: D
= -
3.8 etiocholanolone + 2.8 estriol + 2.4 age
Fourteen of 18 benign breast tumor patients had negative scores, and each of 7 cancer patients had positive values. The discriminant for prostatic disease is a complicated one involving estrone, estriol, 17ketogenic steroids, Porter-Silber chromogens, and the beta-fraction. With this discriminant, 17 of 21 patients with prostatic cancer had a positive value and 16 of 18 patients with benign prostate hypertrophy had negative values. The discriminant described by Moore and associates utilizes a ratio between urinary 17-hydroxycorticosteroids and 17-ketosteroids, each expressed as mg. per gm. of creatinine in the 48 hour urine collection just before and during the intravenous administration of 40 units of adrenocorticotropic hormone over a 6 to 8 hour period each day.19 The discriminant also uses the free period (the time between mastectomy and the first recurrence of disease). With this discriminant it was possible to identify 12 out of 15 responders and 12 of 16 nonresponders to adrenalectomy. It was emphasized that the denial of ablative surgery based on biochemical discriminants alone should be considered with great caution and that palliative surgery should be denied only if both the biochemical and the clinical discriminants were unfavorable. 19 Rao has devised a steroid discriminant for evaluation of patients with lung cancer which requires urinary androsterone, etiocholanolone, 17-ketosteroids and 17-hydroxycorticosteroids.22 Seventy-five of 84 patients with inoperable lung cancer had a negative discriminant. Ninety-two of 100 nonhospitalized and 35 of 42 hospitalized controls had a positive discriminant.
CHORIONIC GONADOTROPIN In trophoblastic neoplasms the quantitative measurement of urinary chorionic gonadotropin (HCG) is essential for initial diagnosis and then for following the course of a disease in which clinical evidence of response to treatment may appear after there is a significant change in chorionic gonadotropin titre. Despite the development of immunochemical and radioimmune assays for human chorionic gonadotropin, at the present time the most sensitive quantitative assay is concentration
BIOCHEMICAL PROCEDURES IN DIFFERENT FORMS OF CANCER
619
of urinary hormone by physical chemical methods and bioassay by the mouse uterine weight method. In choriocarcinoma the response to chemotherapy is related to the human chorionic gonadotropin titre, and the remission rate has been reported to be 95 per cent if the titre is less than 100,000 International Units per 24 hours and the patients are treated within four months of the onset of the disease. 23 If the titre is greater than 100,000 International Units per 24 hours, and the duration of the disease is longer than 4 months, the remission rate is 36 per cent. Complete remission is defined as disappearance of clinical evidence of disease and a decrease of human chorionic gonadotropin to the level of pituitary gonadotropin.
SERUM AND URINARY ENZYMES Serum and urinary enzyme assays are routine procedures in the evaluation of many diseases. There has been extensive study in attempts to define tissue specific enzymes which could be of assistance in the diagnosis of a primary carcinoma or in helping to define the tissue of origin of metastatic tumor.
Amylase Amylase determinations are apparently of little use in the early diagnosis of carcinoma of the pancreas. In a study of 83 patients with histologically proven carcinoma of the pancreas, Ansari and Burch found no elevations of serum amylase in any of the 30 patients in which this determination was done.' Other workers have reported elevations in 8 to 40 per cent of their patients. 29 Amylase can also be elevated in nonpancreatic conditions such as perforated duodenal ulcer, intestinal obstruction, and renal disease. 29
Acid Phosphatase Acid phosphatase is elevated in the serum of only 24 per cent of patients with nonmetastatic prostate carcinoma, but in 81 per cent of these patients with skeletal metastases. 28 The specificity of acid phosphatase in prostate carcinoma is related to substrate, and elevations are observed in many other diseases when orthophosphate esters other than beta-glycerophosphate are used. However, with beta-glycerophosphate as substrate, elevations are rarely observed other than in prostatic disease or in certain myeloproliferative disease where modest elevations are related to the white cell or platelet count. 28 Efforts have been made to increase the sensitivity of the acid phosphatase assay in prostatic disease by utilizing specific tartrate inhibition of prostatic acid phosphatase and assay of the "prostatic" fraction. Green and his associates evaluated "prostatic" acid phosphatase in 454 patients. Elevat~ons of tartrate-sensitive acid phosphatase were found in 14 of 34 patients with prostatic carcinoma, in 2 of 90 patients with benign prostatic hypertrophy, in 15 of 76 patients with other forms of cancer, and in 23 patients without cancer.28
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Alkaline Phosphatase In primary tumors of bone, as well as in metastatic disease of bone, alkaline phosphatase is elevated when the tumor is predominantly osteoblastic. 28 In breast carcinoma, most cases of bony metastases are osteolytic, and alkaline phosphatase levels are usually in the normal range or slightly elevated. In prostatic carcinoma most of the metastatic lesions are osteoblastic, and the serum alkaline phosphatase is elevated to levels 4 to 10 times the upper limit of normal. Modest elevations of alkaline phosphatase are observed in about one third of patients with multiple myeloma, and more extensive elevations are seen in over 95 per cent of patients with adenomas or carcinoma of the parathyroid. 28 In liver disease, elevations of alkaline phosphatase are observed in patients with extrahepatic obstruction of the biliary tract and in patients with intrahepatic metastases. Recently, there has been great interest in the isoenzymes of serum alkaline phosphatase, and electrophoretic and kinetic techniques have been used for their separation. Although these methods permit clear differentiation of kidney or intestine phosphatase from bone or liver phosphatase, it has not been possible until recently to precisely separate the bone from the liver enzyme. Fennelly and his associates, by use of gel filtration, differential thermostability, and differential urea inhibition, differentiated bone from liver phosphatase and feel that the serum patterns of patients with skeletal and hepatic disease can be identified with a high degree of confidence.9 A heat stabile, L-phenylalanine inhibited alkaline phosphatase similar to placental enzyme has been found in the serum of some patients with cancer.31 In 520 cancer patients Stolbach identified this placental enzyme-like alkaline phosphatase in 15 cases and in malignant effusion from 2 patients. 31 Stolbach concluded that the cancer specific alkaline phosphatase isoenzyme (Regan isoenzyme) is useful in following the course of disease, identifying malignant pleural effusion, in clarifying the nature of unexplained elevations of serum alkaline phosphatase activity.
5' -Nucleotidase Serum 5 '-nucleotidase is useful in determining the presence or absence of liver disease in a patient with an elevated serum alkaline phosphatase activity, and in following the progression of liver metastases. Schwartz and Bodansky reported elevated 5 ' -nucleotidase in 27 of 31 patients with cancer and varying degrees of liver involvement. 26 The classical method of utilizing serum 5 '-nucleotidase in evaluating liver and bone disease is illustrated by examining data in a patient with metastatic cancer of the colon. Upon skeletal survey this patient had widespread bony metastases. Bromsulphalein retention was 7 per cent; total bilirubin, 0.4 mg. per 100 ml.; alkaline phosphatase, 24 Bodansky units; the liver was not palpable. However, his serum 5 ' -nucleotidase was elevated to 68 Campbell units, or six times the upper limit of normal. At autopsy, he was found to have liver metastases. 28 It is of interest that in a study of 80 patients with an elevation of
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5/ -nucleotidase or alkaline phosphatase, 30 per cent (23 patients) had an elevation of 5/ -nucleotidase when the alkaline phosphatase was normal. 28 There have been reports that elevations of serum 5/ -nucleotidase and alkaline phosphatase parallel each other in hepatic disease and that alkaline phosphatase is more frequently elevated. Our data indicate that in a cancer patient population the 5/-nucleotidase is elevated more frequently than alkaline phosphatase in hepatic disease. Van der Slik and his associates have concluded that consecutive assays of serum 5/ -nucleotidase furnish indispensable data in patients with liver disease. 33 They stated that in metastatic liver disease 5/nucleotidase may be elevated at a stage when alkaline phosphatase is still within normal limits; that high serum alkaline phosphatase with normal 5/-nucleotidase is strong evidence for the presence of osteoblastic bone disease, and finally that serum 5/-nucleotidase, rather than alkaline phosphatase, is the enzyme of choice in evaluating liver disease in children or pregnant women.
Lactic Dehydrogenase Lactic dehydrogenase (LDH) is one of a group of ubiquitous enzymes whose activity is elevated in a variety of diseases. Total serum lactic dehydrogenase is modestly elevated in most patients with extensive hepatic metastases. In one study, total serum lactic dehydrogenase was elevated to three times the upper limit of normal in 91 per cent of the patients with liver metastasesP The enzyme is more consistently elevated, and to a greater extent, in serum of patients with various types of leukemia. Lactic dehydrogenase activity in pleural and peritoneal effusions containing malignant cells is reported to be higher than the serum lactic dehydrogenase, whereas the lactic dehydrogenase of benign effusions is lower.z7 It has been claimed that urinary lactic dehydrogenase is useful in the recognition of cancer of the bladder or kidney. In a recent study of urinary lactic dehydrogenase in 263 Memorial Hospital patients, it was found that elevations of the urinary enzymes were more related to pyuria, hematuria, and bacteriuria than the presence of a tumor. 14 Only rarely was an elevated enzyme level measured in a patient with a normal urinalysis. It was concluded that urinary lactic dehydrogenase is not a useful routine diagnostic procedure in the study and follow-up of patients with malignant disease of the genitourinary tract. In cancer, the increase in serum lactic dehydrogenase is usually associated with a minor change in the isoenzyme pattern, particularly an increase in LDH3 and LDH5 (the faster moving isoenzymes).37 Wilkinson has reported extra isoenzyme bands in a patient with inoperable carcinoma of the esophagus with metastasis to the liver. Additional bands of activity were found between LDHl and LDHz and between LDHz and LDH3. The appearance of extra lactic dehydrogenase bands occurs rarely.37
Phosphohexose Isomerase Phosphohexose isomerase (PHI) is a ubiquitous glycolytic enzyme found in most tissues. Of the various ubiquitous enzymes that have been
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studied in neoplastic disease, phosphohexose isomerase is elevated more frequently than any other serum enzyme. 5 Thus, of 119 patients with carcinoma of the gastrointestinal tract, phosphohexose isomerase was elevated in 74 per cent of the cases, in contrast to frequencies of 70 per cent for elevations of aldolase, of 53 per cent for lactic dehydrogenase and 35 per cent for glutamic oxalacetic transaminase. In a study of 284 patients with liver metastases, the phosphohexose isomerase was elevated in 84 per cent of the cases, compared to only 51 per cent elevations of glutamic oxalacetic transaminase and 69 per cent lactic dehydrogenase. 5 Serum phosphohexose isomerase is useful in following the progression or regression of metastatic disease. In a study of sequential assays in 6 patients with cancer, it was possible to define with some degree of certainty, on the basis of changes in serum phosphohexose isomerase, a total of 21 distinct episodes of clinical worsening. 25 Phosphohexose isomerase is an indicator of metastatic growth, and the claim has been made that it is the serum enzyme which provides the most sensitive index of clinical status. 7 Muir has attempted to use vaginal fluid phosphohexose isomerase activity in screening for cervical carcinoma. 2o The phosphohexose isomerase was markedly elevated in the vaginal fluid of women with gynecological disorders. The author concludes that although the phosphohexose isomerase determination could not replace vaginal and cervical cytology, it could become a simple screening tool. Based on estimates of gynecological disorders it was calculated that the cytologist would be required to examine 42 of every 100 cases screened by the enzyme assay. The cytologist, however, would have the assurance that every specimen with elevated phosphohexose isomerase activity came from a patient with a gynecologicallesion. SUMMARY An attempt has been made to review some of the biochemical procedures useful in the diagnosis of cancer and in following the progression of the disease. Because of limitations of space it has not been possible to cover all areas. Omissions were not made on the basis of relative importance and have included serum proteins in myeloma and other forms of cancer, hormones such as parathyroid hormone, calcitonin, trace metals, and the general effects of carcinoma on carbohydrate, protein, and calcium and phosphorus metabolism. REFERENCES 1. Ansari, A., and Burch, G. E.: A correlation study of proven carcinoma of pancreas in 83 patients. Amer. J. Gastroenterol., 50:456-475, 1968. 2. Atkins, H., Bulbrook, R. D., Falconer, M. A., Hayward, J. L., MacLean, K. S., and Schurr, P. H.: Ten years' experience of steroid assays in the management of breast cancer. Urinary steroids in the prediction of response to adrenalectomy or hypophysectomy. Lancet, 2: 1255-1263,1968.
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3. Bell, M.: Observations on the biochemical diagnosis of neuroblastoma. In Bohuon, C., ed.: Recent Results in Cancer Research, Neuroblastoma Biochemical Studies. Berlin, Springer Verlag, 1966, pp. 42-51. 4. Bodansky, 0.: Biochemical changes of clinical significance in cancer. In Proceedings of the Fifth National Cancer Conference, Philadelphia, J. B. Lippincott and Co., 1965, pp. 687-703. 5. Bodansky, 0., and Schwartz, M. K.: Phosphohexose isomerase: Clinical aspects. In Colowick, S., Kaplan, N. and Wood, W. A., eds.: Methods of Enzymology. New York, Academic Press, 1966, vol. IX, pp. 568-575. 6. Bulbrook, R D., and Hayward, J. L.: Discriminants and breast cancer. Lancet, 1:1161, 1969. 7. Campbell, D. M., and King, K J.: Serum phosphatase and glycolytic enzymes in cancer of the breast. Biochem. J., 82 :23p, 1962. 8. Crout, R J.: Pheochromocytoma. Pharm. Rev., 18:651-657, 1966. 9. Fennelly, J. J., Dunne, J., McGeeney, K., Chong, L., and Fitzgerald, M.: The importance of varying molecular size, differential heat and urea inactivation of phosphatase in the identification of disease patterns. Ann. N .Y. Acad. Sci., 166: 794-810, 1969. 10. Geiser, C. F., and Efron, M. L.: Cystathioninuria in patients with neuroblastoma or ganglioneuroblastoma. Cancer, 22 :856-860, 1968. 11. Gjessing, L. R: Studies of functional neural tumors. n. Cystathioninuria. Scand. J. Clin. Invest., 15:475-478, 1963. 12. Gjessing, L. R, and Rorud, 0.: Studies of functional neural tumors. VII. Urinary excretion of phenolic pyruvic acids. Scand. J. Clin. Lab. Invest., 17 :80-84, 1965. 13. Gjessing, L. R: Biochemistry of functional neural crest tumors. Adv. Clin. Chem., 11 :81131,1968. 14. Grabstald, H., and Schwartz, M. K.: Urinary lactic dehydrogenase in genitourinary tract disease. J.A.M.A., 207:2062-2066,1969. 15. Hayward, J. L., and Bulbrook, R D.: The value of urinary steroid estimations in the prediction of response to adrenalectomy or hypophysectomy. Cancer Res., 25:11291139,1965. 16. Helson, L., Murphy, M. L., and Schwartz, M. K.: Neuroblastoma survival and serial catecholamine excretion. (Manuscript in preparation.) 17. Marmorston, J., Geller, P. J., and Weiner, J. M.: Pretreatment urinary hormone patterns and survival in patients with breast cancer, prostate cancer, or lung cancer. Ann. N.Y. Acad. Sci., 164:483-493,1969. 18. Mason, G. A., Hart-Mercer, J., Millar, K J., Strang, L. B., and Wynne, N.: Adrenalinesecreting neuroblastoma in an infant. Lancet, 2 :322-325,1957. 19. Moore, F. D., Woodrow, S. I., Aliapoulios, M. A., and Wilson, R: Carcinoma of the Breast. Boston, Little, Brown and Co., 1968, pp. 46-50. 20. Muir, G. G.: Possible use of phosphohexose isomerase as a preliminary to exfoliative cytology in screening for cervical carcinoma. J. Clin. Path., 19: 3 78-383, 1966. 21. Potter, Van R: Recent trends in cancer biochemistry, The importance of studies on fetal tissue. Cancer Res., 24:1085-1095, 1965. 22. Rao, L. G. S.: Discriminant function based on steroid abnormalities in patients with lung cancer. Lancet, 2:441-447,1970. 23. Ross, G. T., Goldstein, D. P., Hertz, R, Lipsett, M. D., and Ode1l, W. D.: Sequential use of methotrexate and actinomycin D in the treatment of metastatic choriocarcinoma and related trophoblastic diseases in women. Amer. J. Obstet. Gynec., 93:223-229,1965. 24. Sarfaty, G., and Tallis, M.: Probability of a woman with advanced breast cancer responding to adrenalectomy or hypophysectomy. Lancet, 2 :685-689,1970. 25. Schwartz, M. K., Greenberg, K, and Bodansky, 0.: Comparative values of phosphatases and other serum enzymes in following patients with prostatic carcinoma: Consideration of phosphohexose isomerase, glutamic oxaloacetic transaminase, isocitric dehydrogenase and acid and alkaline phosphatase. Cancer, 16:583-594, 1963. 26. Schwartz, M. K., and Bodansky, 0.: Serum 5'-nucleotidase in patients with cancer. Cancer, 18:886-892,1965. 27. Schwartz, M. K., and Bodansky, 0.: Lactic dehydrogenase (clinical analysis). In Colowick, S., Kaplan, N., and Wood, W. A., eds.: Methods in Enzymology. New York, Academic Press, 1966, vol. IX, pp. 294-302. 28. Schwartz, M. K., Fleisher, M., and Bodansky, 0.: Clinical application of phosphohydrolase measurements in cancer. Ann. N. Y. Acad. Sci., 166:775-793,1969. 29. Schwartz, M. K., and Fleisher, M.: Diagnostic biochemical methods in pancreatic disease. Adv. Clin. Chem., 11 : 113-159, 1970. 30. Sjoerdsma, A., Engelman, K., Waldmann, T. A., Cooperman, L. H., and Hammond, W. G.: Pheochromocytoma: current concepts of diagnosis and treatment. Ann. Int. Med., 65: 1302-1326,1966. 31. Stolbach, L. L.: Clinical application of alkaline phosphatase isoenzyme analysis. Ann. N.Y. Acad. Sci., 166:760-774, 1969.
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