Biochemical Procedures As Aids in Diagnosis of Different Forms of Cancer

Home , Homovanillic acid, Vanillylmandelic acid

Biochemical Procedures as Aids in Diagnosis of Different Forms of Cancer

MORTON K. SCHWARTZ, Ph.D.

Memorial Sloan-Kettering Cancer Center, New York, NY 10021

ABSTRACT The application of biochemical analyses as aids in the diagnosis of cancer is discussed with emphasis on the fact that biochemical testing is more useful in following the regression and progression of disease than in early initial diagnosis. The uses of biochemical analyses of metabolic degradation products, lipids, hormones and their receptors, enzymes, including isoenzymes, and trace metals are included.

Introduction indicated in table I these include neuro Diagnostic procedures in cancer must be blastoma, pheochromocytoma, carcinoid, designed to achieve several purposes. They hepatocellular carcinoma, multiple mye must define the disease, describe its extent loma, osteogenic sarcoma and other osteo and be useful in following its progression blastic bone tumors. In these diseases, the or regression. For more than 75 years, in assay of the listed components is essential vestigators have searched for biochemical for confirmation of the diagnosis and in defects in cancer cells that could be ex following the response to therapy. ploited in diagnosis. Despite these long and continuing studies, few biochemical proce Serum Enzymes dures have been developed for early diag Historically, the biochemical procedure nosis of specific forms of cancer. The most used for the longest time as a diagnostic useful application of biochemical assays aid in cancer is acid phosphatase. This has been in following the regression and serum enzyme has been used for 35 years progression of disease and in establishing in the evaluation of carcinoma of the pros the presence of métastasés.35’36 tate. The enzyme activity is elevated in From the standpoint of general use, the only 24 percent of patients with non most important tests would be those useful metastatic prostate carcinoma and in 81 in cancers with the highest population inci percent of patients with skeletal metas- dence. In the United States these are can tases.39 Efforts have been made to increase cer of the colon and rectum, female breast, the sensitivity of the acid phosphatase as lung, bronchus and uterus. However, most say in prostatic disease by utilizing specific well defined biochemical procedures are tartrate inhibition of prostatic acid phos those related to rarer forms of cancer. As phatase and assay of the “prostatic” frac-

95 9 6 SCHWARTZ

TA BLE I

B i o c h e m i c a l A s s a y s U s e f u l i n I n i t i a l D i a g n o s i s a n d F o l l o w -U p o f R a r e C a n c e r s

Disease Biochemical Constituent

Neuroblas toma Catecholamines, vanillylmandelic acid, metanephrine, cystathionine, dopamine 3-hydrolase, dopamine, homovanillic acid

Pheo chromo cy toma Catecholamines, metanephrine, vanillylmandelic acid

Carcinoid 5-Hydroxyindoleacetic acid

Trophoblastic neoplasms Chorionic gonadotrophin (choriocarcinoma, testicular tumors)

Multiple myeloma Bence-Jones protein, immuno globulin

Hepatocellular et-Fetoprotein carcinoma

Osteogenic sarcoma Alkaline phosphatase

tion.13 In one study, elevations of tartrate- serum from a patient with prostatic carci sensitive acid phosphatase were found in noma (total serum acid phosphatase of 290 14 of 34 patients with prostatic carcinoma, units) or of a homogenate of prostatic tis 2 of 90 patients with benign prostatic sue demonstrated additional isoenzymes in hypertrophy, 15 of 76 patients with other the post albumin area. This technique re forms of cancer, and 23 patients without quires further evaluation as a diagnostic cancer.16 It has been our experience that aid in cancer of the prostate.31 Assays of the prostatic acid phosphatase as deter bone marrow serum acid phosphatase have mined by tartrate inhibition is not useful as been proposed as a technique for detecting a clinical laboratory tool.8 early metastasis to bone in adenocarcinoma Disc electrophoresis on polyacrylamide of the prostate and in staging of the dis gel has indicated there are three to five ease.30 acid phosphatase isoenzymes in the serum Many other serum enzymes have been of healthy adults.31 Electrophoresis of the suggested as useful parameters in the di BIOCHEMICAL DIAGNOSIS 9 7 agnosis of cancer.36 Serum amylase is ele pared with placental alkaline phospha vated in a small percentage of patients with tase.12’40 In two studies of 913 patients with cancer of the pancreas,38 and alkaline phos a variety of cancers, the Regan isoenzyme phatase is elevated in primary tumors of was observed in the serum in 66 instances bone.39 Alkaline phosphatase is more useful or 7.2 percent.22’41 The highest incidence in evaluating metastatic disease to bone was in patients with cancer of the ovary and liver. The enzyme is elevated predom and lung. With a sensitive immunochemical inantly in osteoblastic bone lesions. In assay, Regan isoenzyme has been detected breast carcinoma, most cases of bony me- in serum of 89 of 91 normal adults and 106 tastases are osteolytic, and alkaline phos of 112 serums from patients with malignant phatase levels are usually in the normal disease.45 In the cancer patients, only 11 range or slightly elevated. In prostatic car showed elevated activities to an extent of cinoma, most of the metastatic lesions are 3 to 300 times the average normal value. An osteoblastic, and the serum alkaline phos alkaline phosphatase variant different from phatase is elevated to levels 4 to 10 times the Regan isoenzyme has been observed in the upper limit of normal.39 serum of patients with hepatoma. In one During successful treatment, there may study, 6 of 21 patients with hepatoma ex be an elevation in the serum alkaline phos hibited the alkaline phosphatase variant.28 phatase presumably related to the repair of All of these patients had «-fetoprotein in bone. These elevations have been referred their serum. It was concluded that the alka to as the “paradoxical rise” in serum alka line phosphatase variant was not useful in line phosphatase.39 In liver disease, eleva hepatoma. tions of alkaline phosphatase may be ob An important demand made of the clin served in patients with extrahepatic ob ical biochemistry laboratory is to aid the struction of the biliary tract and in patients clinician in establishing whether or not an with intrahepatic metastases. In infiltrative elevated serum alkaline phosphatase is due disease of the liver such as leukemia, retic to liver or bone metastases or to a combi ulum cell sarcoma and Hodgkin’s disease, nation of both. Numerous attempts have alkaline phosphatase activity is markedly been made to use electrophoresis to sepa elevated, the activity being directly related rate liver from bone alkaline phosphatase. to the extent of liver involvement.36 However, in our experience, there is too Recently, there has been great interest in much overlap in the electrophoretic migra the isoenzymes of serum alkaline phospha tion of these isoenzymes to permit their tase and the so-called “Regan isoenzyme” routine clinical use. We have successfully has been used in studies of patients with used serum 5'-nucleotidase for this pur cancer. In 1968, Fishman and his associates pose.39 5'-Nucleotidase is a substrate spe identified an alkaline phosphatase in the cific phosphatase present in liver tissue but serum and tissue of a patient with carci not in bone. When it is elevated in the noma of the lung.10 This enzyme was serum, it is indicative of liver involvement. named the Regan isoenzyme after the pa Two other enzymes, y-glutamyl trans tient in whom it was found.11 Investigations peptidase and leucine aminopeptidase have have indicated that the isoenzyme is iden also been used as specific indicators of liver tical to the heat stable, L-phenylalanine disease. In a comparison of these enzymes sensitive alkaline phosphatase of the hu in a series of 96 patients in whom the man placenta, that it demonstrates iden clinician suspected liver involvement, the tical electrophoretic mobility to that from three enzyme activities were observed placenta before and after neuramidase within normal limits in 26 individuals and treatment and reacts with antiserum pre all elevated in 39 others. In seven patients, 9 8 SCHWARTZ

TA BLE II

E l e v a t i o n s o f “ U b i q u i t o u s E n z y m e s ” i n S e r u m o f P a t i e n t s w i t h

C a r c i n o m a M e t a s t a t i c t o L i v e r 42

No. of No. with Percent Enzyme Patients Elevations Elevated

Phosphohexose isomerase 72 60 84 Aldolase 100 75 75 Lactate dehydrogenase 1 5 6 108 69 Malate dehydrogenase 70 43 62 Isocitrate dehydrogenase 65 34 53 Aspartate aminotransferase 188 94 50 Glutathione reductase 68 32 47 Alanine aminotransferase 179 60 33

y-glutamyl transpeptidase was the only course of disease in patients with liver elevation; in two patients, this enzyme was metastases. Of all these enzymes, serum normal when the others were elevated. In phosphohexose isomerase appears to be each of two other cases, either 5'-nucleo- elevated in the greatest number of patients tidase or leucine aminopeptidase was ele (table II).42 vated when the other activities were within normal limits. In 11 cases, the leucine aminopeptidase was normal when the Body Fluid Enzymes other two were elevated; in seven cases, Attempts have been made to use the the 5'-nucleotidase was normal when leu ubiquitous enzymes in fluids other than cine aminopeptidase and y-glutamyl trans blood in cancer diagnosis and follow-up. peptidase were at supranormal activities.37 Urinary lactic dehydrogenase has been The extreme sensitivity of y-glutamyl trans extensively studied as a diagnostic aid in peptidase and its elevation in a large vari cancers of the bladder and kidney. It has ety of situations, particularly alcoholism, been concluded from our study of 263 pa may mitigate against its use in establishing tients with various diseases of the genito the presence of liver metastases in cancer. urinary tract that these assays are not use The main points suggested by these data ful as a routine diagnostic procedure in the are the need for multiple enzyme assays evaluation of patients with genito-urinary and the fact that no single determination symptoms.13 Elevations of urinary LDH is universally applicable to the evaluation seem to reflect more closely pyuria, hema of liver diseases. turia or bacteriuria than the presence of a The ubiquitous enzymes are a large specific proved tumor. group of metabolically involved enzymes The activities in vaginal washings of 6- present in all tissues. They include lactic phosphogluconate dehydrogenase, phos dehydrogenase, phosphohexose isomerase, phohexose isomerase and the lysosomal aldolase, transaminases, isocitric dehydrog enzyme /3-glucuronidase have been used in enase, etc. They are not useful in primary the evaluation of cervical cancer.6’21’9 Ele diagnosis but are helpful in following the vations of these enzymes were observed in BIOCHEMICAL DIAGNOSIS 9 9 washings from a majority of women with sons suffering from muscular dystrophy. uterine cancers and also from many women Gliomas contain aldolase C as does normal with benign disease. Although these assays brain tissue; in meningiomas, aldolase C flag patients with gynecological disorders, is not detected and only aldolase A is the high false positive rate led to a con present.33 Aldolase A is found in fetal brain clusion that it is unlikely that vaginal fluid and liver and in tumors. Cervical carci enzymology can provide a screening test noma and carcinoma of the uterine corpus for cervical carcinoma. These findings must exhibit an electrophoretically distinct hexo- be reevaluated and extended to studies of kinase isoenzyme not found in normal these and other enzymes in tissue washings tissue.34 or brushings from the lung, stomach and Tissue enzymes other than the ubiqui colon as well as the cervix. tous ones may also be useful in evaluation of cancer. A glutaminase with a low affinity Tissue Enzymes for phosphate appears to be present in can The metabolically involved enzymes cerous lung with activity inversely propor have also been evaluated in tissue biopsy tional to the mitotic rates and degrees of material as aids to the pathologist in es differentiation of the tumors.39 Two sulfate tablishing the presence of cancer and the activating enzymes, sulfate adenyltrans- tissue of origin of a metastatic lesion. A ferase and adenylsulfate kinase are stated recrudescence of interest in tissue LDH to be absent in hormone non-responsive isoenzymes has focused particularly on the breast tissue. The assay of the sulfurylation ratio of LDHg and LDIJ,. A shift to LDH3 enzymes has been proposed as a useful tool (the muscle variant) has been observed in in conjunction with estrogen receptor pro human cancerous breast, uterus, brain, tein in evaluation of patients prior to abla lung, stomach and kidney. The most im tive endocrine surgery or hormonal ther pressive study is that of 619 surgical pros- apy.7 state specimens. In these cases the LDHB/ A discussion of enzymes in cancer would LDHi ratio was greater than one in 90 of be incomplete if mention were not made 116 (77.6 percent) specimens with histo of muramidase and its role in leukemia.26 logically verified carcinoma of the prostate This enzyme has been found only in the and in only 70 of 503 (13.9 percent) of urine of patients with granulocytic leuke tissue specimens from patients with benign mia who do not have a Philadelphia chro disease.23 mosome.27 The elevations in these cases are The aldolase isoenzymes differ in some presumably due to release of the enzyme human cancers and tend to revert to a from monocytes and from more mature fetal form. Aldolase exists as three families members of the granulocyte series. Serum of isoenzymes; aldolase A in muscle, aldo muramidase has been found elevated in lase B in liver and aldolase C (the most patients with acute myelogenous and anodic form on electrophoresis) in brain chronic granulocytic leukemia but not in and nerve tissue. Primary tumors of the acute lymphatic leukemia.29 The elevations liver have been observed to contain aldo in patients with chronic granulocytic leu lase A and not the B form found in normal kemia are up to 10 times the upper limit liver.24 Serum from patients with cancer of normal. contains a greater proportion of muscle variant aldolase than serum from normal Steroid Discriminants persons.44 Other than patients with cancer, The use of individual steroid analyses in this has been found only in serum of per primary tumor diagnosis or predicting re 1 0 0 SCHWARTZ

sponsiveness to adrenalectomy, hypophy- trolled factors that can affect the metab sectomy or chemotherapeutic hormonal olism and excretion of hormones. therapy has not been very successful. This Jensen and his colleagues found that the lack of success is undoubtedly due in part estrogen receptor was in an 8S sucrose to the effect of age, weight, the emotional density gradient peak.18 Using in vitro stress of hospitalization and the extent of techniques, these workers observed that 19 the illness on hormone excretion, as well patients without binding ability failed to as the relatively large analytical error in respond to adrenalectomy and 5 of 11 pa steroid analysis.35 tients with binding protein had remissions. During the past decade, numerous work Wittliff et al also found the estrogen bind ers have used a grouping of steroid anal ing protein in the 8S to 9S fraction.46 Of yses to predict hormone responsiveness in 75 tumor specimens, 29 had positive bind breast, prostate and lung cancer. Bulbrook ing (43.0 ±5.3 fmoles of receptor per mg and his associates in England found that protein), 36 were negative and 10 were urinary etiocholanolone levels tended to be borderline. One of 21 normal and non- high in breast patients who subsequently cancerous breast tissue specimens demon had positive clinical responses to adrenal strated specific estrogen binding. The bind ectomy or hypophysectomy and that the 17- ing in tumor tissue was not related to the hydroxycorticosteroids (17-OHCS) tended presence of metastases or the number of to be low in successful cases.3’17 By means carcinoma cells in the studied specimen. of a discriminant function of these param Wittliff reviewed the literature and re eters, it was possible to predict the success ported that of 329 primary breast tumors or failure of adrenalectomy or hypophysec examined, 158 or 48 percent contained spe tomy in breast cancer. The more positive cific estrogen receptors. Long term studies the discriminant, the greater the chance of are required to correlate studies of hor palliative success following ablative sur mone binding proteins and the clinical re gery. Other workers have designed other sponse of patients to therapy. discriminants using other steroid analyses (estriol, 11-oxysteroids, 17-ketosteroids) P olyam ines and other factors, including age and the Several biochemical procedures have free period (time between primary diag been described which require further clin nosis and recurrence). Their findings have ical evaluation to establish their true role been similar to those of the English group.7 in cancer diagnosis. Polyamine synthesis is accelerated in rapidly growing normal or Estrogen Binding malignant tissue, presumably owing to in The binding of estrogen to macromolec- creases in ornithine decarboxylase activity ular components (receptor protein) has re in stimulated growing tissues. These effects ceived wide attention as a possible indi appear to be hormone dependent. Russell cator of response to ablative hormone and her associates demonstrated elevated surgery. In vivo and in vitro studies have urinary excretion of polyamines in cancer shown that human breast cancer often ac patients.32 In 50 normal persons excretions cumulates more estrogen than normal were: spermine 3.4 ± 0.67 mg per 24 breast tissue. In vivo studies are limited hours; spermidine, 3.1 ± 0.56 mg per 24 because of possible radiation hazards, the hours and putrescine, 2.7 ± 0.53 mg per expense of the large amount of needed 24 hours. In cancer patients, excretions radioactive hormone and the many uncon were increased 5 to 10 fold, and surgical BIOCHEMICAL DIAGNOSIS 1 0 1 removal of a tumor mass always led to a and cholesterol were markedly decreased decrease in urinary polyamine concentra and the lipoprotein decreases were not tions. In 24 patients with acute myelocytic related to extent of disease or type of treat leukemia in relapse, the values were as ment.25 Another interesting lipid observa follows: Putrescine, 5.5 ±1.1 mg per 24 tion is elevated urinary excretion of non- hours; spermidine 26.4 ± 3.4 mg per 24 esterified cholesterol in women with carci hours; and spermine 38.1 ± 4.2 mg per noma of the steroid producing glands and 24 hours. In 5 patients with ovarian car in men with testicular and prostate can cinoma, the 24 hours values were putre cers.1’2 Abnormal excretions (above 1.20 scine, 29.9 ± 5.7 mg, spermidine 43.5 ± 6.7 mg per 24 hours) were observed in 29 of mg and spermine 38.7 ± 6.1 mg. 32 patients with adenocarcinoma of the In a study of blood polyamines, putre prostate and in each of eight patients with scine and spermine were not detected and choriocarcinoma, teratocarcinoma and em spermidine was found at a concentration bryonal cell carcinoma of the testis. Excre of 0.32 ± 0.07 nM per ml in 10 normal tions were within normal limits in 19 persons.20 In cancer patients, putrescine was patients with testicular seminomas. The not detected and spermine was found in abnormal values seemed to correlate with 3 of 18 patients at levels between 0.14 the clinical course of the disease. and 0.28 nmol per ml. However, spermi dine was observed in the serum of all 18 Tryptophane Metabolites cancer patients at levels between 0.44 and Tryptophane metabolites appear to be 3.30 nmol per ml. The highest value was excreted in abnormal amounts in patients in a case of mediastinal choriocarcinoma. with breast cancer.5 Exploiting this obser Sixteen of the 18 patients had values vation a tolerance test has been described greater than two standard deviations above for use in cancer diagnosis and follow-up the mean value in the control group. in patients with Hodgkin’s disease, breast and bladder cancer. In this test, urinary Trace Elements tryptophane metabolites are measured fol Trace elements may play an important lowing an oial dose of tryptophane. In a role in cancer. Their use as diagnostic aids study of 36 patients with bladder cancer, has not been outlined. Serum copper con increased urinary kynurenine was found in centrations have been reported to be ab 12 subjects, 3-hydroxy-kynurenine in 25, normally high in untreated cases of Hodg kynurenic acid in 9, xanthurenic acid in kin’s disease.43 In one study, the high levels 4 and acetylkynurenine in 10.14 The supra- returned toward normal when the disease normal excretions reverted toward normal was successfully treated and became ab levels when there was a favorable response normal during relapse, usually before clin to therapy. The rationale for the test is the ical indications were noted. finding of increased activity of tryptophane pyrrolase and decreased kynureninase in L ipid s the liver of patients with cancer. Lipids also play an important role in the molecular biochemistry of the cancer cell.4 Conclusions Serum lipoproteins are reported present in It is not possible to review all of the bio decreased amounts in patients with ad chemical tests that have been proposed as vanced breast cancer and gynecological tu diagnostic aids in cancer. Most of these mors. In a study of 122 patients with malig have not had the specificity necessary to nant tumors, lipoproteins, phospholipids make them useful in cancer screening and 1 0 2 SCHWARTZ cancer control. It is to be hoped that as 10. F i s h m a n , W. H . , I n g l i s , N. J., S t o l b a c h , new and more sensitive biochemical tech L. L., a n d K r a n t , M . J.: A serum alkaline phosphatase isoenzyme of human neoplastic niques are developed, a diagnostic proce cell origin. Cancer Res. 28:150-154, 1968. dure of a general nature will be found. In 11. F i s h m a n , W. H . , I n g l i s , N . 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