Peptide Horm one Precursors, Subunits, and Fragm ents as Hum an Tumor Markers*

Transcription

1 ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 15, No. 5 Copyright 1985, Institute for Clinical Science, Inc. Peptide Horm one Precursors, Subunits, and Fragm ents as Hum an Tumor Markers* EN RIQUE PIM EN TEL, M.D. National Center of Genetics, Institute of Experimental Medicine, Central University of Venezuela, Caracas, Venezuela ABSTRACT H orm one-related peptides (HRPs) are more frequently and abundantly produced in ectopic horm one-secreting tum ors than in tum ors of the respective endocrine glands. M ost HRPs may have little functional activity. Therefore, tumors producing even high amounts of HRPs are either clinically silent or produce m inor endocrine symptoms. The potential use of HRPs as tum or m arkers has several limitations. The difference in pro duction and secretion b etw een the tum or tissues and the respective normal endocrine glands is usually only a quantitative one. In cancer patients, different patterns of structural heterogeneity for different forms of ho r m one precursors and fragm ents may occur. M arked variation in the production and secretion of HRPs may occur depending on the physiological conditions; in some patients the amounts of HRPs may be about the same as in norm al persons. F u rth erm o re, the relative am ounts of these substances, eith er in blood or in tum or tissues, may not be useful for prognosis. Introduction The p o ten tial use of horm ones as tu m o r m arkers has b een discussed recently. 68 Tumors of endocrine organs can secrete abnorm al am ounts of h o r mones (eutopic horm one secretion), and this fact can be applied for the diagnosis and/or the following-up of patients b earing such tum ors. T he property of some * Address reprint requests to: Enrique Pimentel, M.D., Centro Nacional de G enetica, Instituto de M edicina E xperim ental, Apartado Postal 50587, Caracas 1050-A, Venezuela. tum ors from n o n en d o crin e organs to secrete increased amounts of hormones (ectopic hormone secretion) is also useful for clinical purposes in selected cases. Furtherm ore, the study of mechanisms rela te d to ectopic horm one secretion m ay c o n trib u te to a m ore accurate knowledge of some biological phenom ena, including genom e regulation and neoplastic cell transform ation. In addition to th e topological and quantitative aspects of horm one secretion by neoplastic tissues, th e re is at p resent m uch interest in studying possible qualitative changes. Tumor tissues /85/ $02.00 Institute for Clinical Science, Inc.

2 382 PIM ENTEL can produce and secrete, either eutopically or ectopically, p e p tid e horm one p recu rso rs, su b u n its, and fragm ents, i.e., horm one-related peptides (HRPs), w hich are d ifferen t from th e norm al, intact horm one. T hese q u alitativ e changes may lead to the circulation of HRPs th at w ould not be p re se n t, or would be present in small amounts, in normal body fluids. However, many of these qualitative changes are not exclusively found in tumors. In the last few years very specific and sensitive m ethods have b een d e v elo p ed for th e study of polypeptide products, and they are being applied for th e detection and characterization of HRPs. The clinical use of such m ethods has indicated that most, if not all, HRPs described in cancer patients are also present in normal subjects. It is only their presence in abnorm al concentrations or abnorm al ratios in th e neoplastic tissues and/or body flu id s th at brings them to a tte n tio n, especially because they can be exploited for both basic and clinical purposes. In fact, tum or tissues have been very useful for discovering horm one precursors and for the elucidation of horm one biosynthetic pathways. In this review, brief reference will be made to the m ethods that can be applied to the study of HRPs in tum or tissues and body fluids from cancer patients. Selected examples of reported cases will be m entioned, and th e potential m echanisms of production of these substances will be discussed. Methodological Aspects Different types of m ethods are potentially useful for the study of HRPs, not only at the peptide level, but also at the deoxyribonucleic acid (DNA) and rib o nucleic acid (RNA) levels. H ow ever, most studies have been carried out at the peptide level. S t u d i e s a t t h e P e p t i d e L e v e l Im m unological m ethods have been extensively applied to the study of H R P s. 114 P recip itatio n w ith im m une sera, radioimmunoassay, and im m unohistochemical procedures are very useful for the detection of HRPs. However, the validity of these m ethods is lim ited by the possibility of cross-reactions, which may be difficult to recognize. By usual im m unological m ethods alone, som e H RPs cannot be easily d istin g u ish ed from their normal, intact counterparts. However, m ethods have been developed for the specific determ ination of glycop ro te in tropic horm one a and (3 su b u n its, and th e use of m onoclonal an tib o d ies 82 can help to increase the specificity of immunological procedures. Synthetic peptides of the highest grade of purity, stru ctu rally rela te d to h o r mones, may also be very useful for the detection of horm one precursors, subunits and fragments in immunochemical studies The synthetic peptides may serve not only as excellent immunogens for the production of region-specific antisera, but also as valuable substrates for the ch aracterization of a n tisera to be used in highly specific and sensitive im m unoassays. Physicochem ical p ro ced u res w ith increasing resolving pow er for th e separation of polypeptides differing only in m inor characteristics have been developed in recent years. These procedures, including gel filtration, different types of chromatographic techniques (especially, affinity chrom atography using specific antibodies), and isoelectric focusing have been applied for the extraction, separation, concentration, and purification of many HRPs. A powerful m ethod for the analysis of proteins in biological systems, including neoplastic cells, is high-resolution tw o-dim ensional e le c tro p h o re sis. 28 This m ethod selects the electrical charge of a protein for separation in one

3 HORMONE-RELATED PEPTIDES AS TUMOR MARKERS 383 directio n by iso electric focusing, followed by separation in a second, perpendicular direction based upon molecular w eight differences using a gradient polyacrylamide gel. It has perm itted the separation of m ore than 1,300 different polypeptides in some transform ed cells, and its application to the study of HRPs may yield in terestin g results. How ever, for the com parison of tw o-dim ension electrophoresis gels containing such a large num ber of proteins from control and neoplastic tissues, it is essential to have a good reproducibility of absolute and relative p ro te in positions, w hich may be achieved by using com puter-delineated relative coordinate system s for the biophysical description of the different proteins separated by this m ethod. 27 After its purification, the polypeptide can be fu rth er characterized by d e te r mination of its molecular weight and its amino acid com position and sequence. Eventually, the tertiary and quaternary structures of the protein can be d e te r m ined and com p ared to those of the intact horm one. However, the possibility of artifacts introduced by some procedures m ust always be kept in m ind.32 Studies on inco rp o ratio n of radiolabeled amino acids by neoplastic cells or tissues, under either in vivo or in vitro conditions, are useful for th e identification of different compounds involved in hormone biosynthetic pathways, as well as for recognition of specific horm one degradation products. The biological activity of HRPs can be studied by means of bioassays in intact anim als in vivo or in cell system s in vitro.47 It is im portant to determ ine the characteristics of binding of such substances to cellular receptors, especially in radioreceptor assays. 41 These characteristics (affinity constant, capacity, specificity, regulatory phenom ena) may be com pared w ith those of th e norm al, intact hormone. A dissociation between im m unoreactive horm one concentration and concentration m easured by radioreceptor assay, or other biological assay, is suggestive of the p resen ce of H R Ps, since many HRPs are cross-reactive in radioim m unoassay b u t may have dim inished biological activity. P o strecep to r biochemical and physiological effects of HRPs can be studied in appropriate test systems, according to the known actions of the respective hormone. In general, HRPs have little or no biological effects when compared with the effects of the intact hormone, but there are som e exceptions to this rule. For exam ple, th re e different m olecular weight forms of prolactin circulating in the blood of patients with prolactinomas show a good correlation b e tw een radioimmunoassay and a bioassay system determ ining stimulation of growth in a rat lymphoma cell line. 110 Four different fragm ents of th e hum an grow th h o r m one-releasing factor (hgrf) isolated from hum an pancreatic tumors have similar or identical biological potency in com parison w ith the intact horm one w hen assayed in conscious freely-moving rats and in anesthetized rats, where they are equipotent on a molecular basis to stim ulate growth horm one secretion. 108 S t u d i e s a t t h e N u c l e i c A c i d L e v e l Studies at the RNA or DNA levels have been little exploited in relation to H RPs produced in cancer patients. M essen g er RNA (mrna, p o lyadenylated RNA = poly (A)RNA) can be extracted from tum or tissues by chrom atography on oligo(d T)-cellulose. T hereafter, e i th e r total mrnas or aliquots from fractions of them obtained by sedim entation in sucrose gradients can be translated in cell-free extracts containing radiolabeled am ino acids. T he lab eled p o ly p ep tid e translation products can be subjected to some other procedures in order to identify the putative HRPs. This type of procedure has b een successfully applied to

4 384 PIM ENTEL the identification of hum an calcitonin precursor mrna produced in a m edullary thyroid carcinom a. 14 In patients with non-parathyroid tumors associated with hypercalcem ia, an absence of parathyroid horm one (PTH) m RNA has b een o b se rv e d, 89 thus in d icatin g th at such hypercalcemias are not related to ectopic PTH production. M olecular h y b rid izatio n tech n iq u es with either total cellular RNA or poly(a)- rich RNA can be perform ed after denaturation followed by electrophoresis in agarose slab gels and transfer to hybridization m em branes. T h ese tech n iq u es are excellent for the detection of mrnas corresponding to th e precursors of polyp e p tid e horm ones. Two im p o rtant advantages of m ethods operating at the RNA level over those operating at the protein level are (a) the absence of crosshybridization betw een unrelated mrnas (w hereas cross-reactions are difficult problem s w hen im m unological te c h niques are used), and (b) the assurance that the tissue is responsible for the synthesis of the respective peptide product and that the presence of this product cannot be attrib u ted to a sim ple phenom e non of nonspecific adsorption. This type of procedure has b e e n used to dem onstrate, for exam ple, th at w hereas all medullary thyroid carcinomas from eight patients contained calcitonin mrna, the proopiomelanocortin (POMC) gene was expressed only in the metastases of two sporadic cases of the tum or b ut not in the primary tum or tissue of six other patients w ith th e fam ilial ty p e of th e sam e tum or. 92 Interestingly, the m etastases of the two m edullary thyroid carcinom as ectopically expressing the POM C gene contained an mrna about 100 nucleotides longer than the POM C mrna norm ally p re se n t in th e hum an p itu itary gland. Studies at the DNA level can be used for detecting genom ic changes occurring in neoplastic cells producing HRPs. D eoxyribonucleic acid technology may be applied, including the production of cdna copies by reverse transcription, as w ell as p ro ced u res for gene isolation, cloning and sequen ce analysis. T hese p ro ced u res have b een applied to the study of genes involved in the production of horm one precursors. For example, the mrna corresponding to a m et- and leuenkephalin precursor was isolated from a hum an pheochrom ocytom a, and it was used to synthesize a cdna copy of the gene, w hose com plete nucleo tid e sequence was then determ ined. 19 The gene encoding the a subunit of the hcg contains at least two polymorphic sites in its 3' flanking region detected by restriction enzymes H in d lll and EcoRI. The most prevalent genotype for hcg a in normal hum an placenta is associated w ith the absense of the H in d lll polymorphic site and the presence of the EcoRI sites on both chromosomes. In contrast, in the m ajority of p a tie n ts w ith choriocarcinoma, a lack of the EcoRI site and homozygous presence of the H indlll site has been observed. 43 Thus, a particular DNA arrangem ent at the hcg a locus may be associated w ith increased susceptibility to developm ent of choriocarcinom a. The polym orphic site could lie within the hcg a gene or some nearby gene. Hormone Precursors P ep tid e horm ones are synthesized w ithin the secretory cells as large p re cursors called prohorm ones. 15 Hormone p recu rso rs are p otentially useful as human tum or markers under the following conditions: (a) presence of altered ratios of different horm one precursors in tumors of endocrine glands; (b) presence of increased levels of horm one p recu r sors in tumors of nonendocrine tissues, or (c) presence of increased levels of hor

5 HORMONE-RELATED PEPTIDES AS TUMOR MARKERS 385 m one precursors in the body fluids of cancer patients. H o r m o n e P r e c u r s o r s i n T u m o r s o f t h e E n d o c r i n e G l a n d s The presence of altered ratios of different m olecular forms of horm one p re cursors in extracts of tum ors of the endocrine glands has b een su g g ested as a possible aid for the diagnosis of certian n eoplasm s. 49 In addition, ap p aren tly a b erran t m echanism s of processing of horm one p recu rso rs have b e e n d e scribed in isolated cases of these tumors. In a pheochrom ocytom a the tetradecap e p tid e form of som atostatin (som atostatin 14) was generated directly from a large precursor form of somatostatin (6 K IRS) and n o t via th e octacosapeptide form of the horm one (somatostatin 28).113 C-peptide im m unoreactivity w ith different molecular weights has been detected by chrom atography in extracts of human insulinom as. 65 C -p e p tid e -lik e com pounds with both low and high molecular weights were found in about half of the insulinomas from seven patients, but the precise structure of these fractions was not d e te rm in e d. H e te ro g e n e ity of C- p e p tid e circu latin g in th e blood of healthy subjects has been described p reviously. 51 In general, no consistent p attern s of a lte re d pro h o rm o n e ratios or characteristically altered patterns of pro cessing of horm one precursors have been d escrib ed in any type of endocrine tumor. Moreover, high molecular weight (HM W ) form s of horm ones (pro-hormones) may be present in large amounts not only in neoplastic tissues but also in benign diseases of the respective tissues. For example, cells producing big gastrin (G34) are p re se n t in increased amounts in the antrum of patients with hyperacidic d u o d en al u lcer associated with hypergastrinem ia and parietal cell hyperplasia. 46 H o r m o n e P r e c u r s o r s i n T u m o r s o f N o n e n d o c r i n e T i s s u e s H orm ones and th eir precursors may be produced ectopically, som etim es in large amounts, by nonendocrine tum or tissues. In such cases the respective hormone precursors can be dem onstrated by several procedures in the extracts of the tum or tissues. In lung carcinomas, m ore than 50 percent of the adrenocorticotropic horm one (ACTH) im m unoreactivity present in tum ore extracts corresponds to proa CTH and little or no im m unoreactiv ity consists of norm al ACTH (ACTH 1-39). 111 In some patients with th e ectom pic ACTH syndrom e, large m olecular forms of precursors for both ACTH and m et-enkephalin are observed in the tum or extracts. 70 Several species of calcitonin precu rso rs have been detected in the tum or tissue of patients w ith m edullary thyroid carcinom a and small-cell lung carcinoma, and molecular species of high molecular weight, which are probably calcitonin precursors, p re dom inate in these tum or tissues. 4 In isolated cases of tum ors w ith ectopic hormone production an apparently com plete blockade in the biosynthetic pathway of th e horm one has b een found. Am ong three cases of malignant carcinoid of the stomach synthesizing ACTH precursors, in one case the precursor (proacth) was the sole form of the hormone produced by the tum or, and was not being converted to the normal horm one. 39 H o r m o n e P r e c u r s o r s i n t h e B l o o d o f C a n c e r P a t i e n t s The presence of protein hormone precursors in the blood of p atien ts w ith tum ors capable of synthesizing hormones has been known for a long time. Virtually all of the protein hormones have been found to circulate in variable amounts as prohorm ones in the blood of such

6 386 PIM ENTEL patients. The phenom enon is observed in tumors that produce horm ones either eutopically or ectopically. Especially in patients w ith ectopic horm one production, the presence of horm one precursors may predom inate in the blood, and even may be almost the only form of the horm one p re se n t in th e blood. T he sam e precursors, although usually in reduced amounts, can be detected in the blood of norm al subjects, especially u n d e r the influence of endogenous or exogenous factors stim ulating horm one secretion. Examples of prohorm ones circulating in patients w ith tum ors of th e endocrine glands appear in table I. The precursors of glycoprotein hormones (thyrotropin, pituitary gonadotropins, and chorionic gonadotropin) will be discussed in a following section (H orm one Subunits). In insulinoma patients, the presence in the blood of high levels of proinsulin and C-peptide, in addition to insulin, has been extensively used for diagnostic p u r poses. 35,57,66,85,88 The proportion of proinsulin-like m aterial accounting for total serum im m unoreactive insulin (IRI) in insulinoma patients is highly variable. In a study of 19 patients with benign and m alignant insulim om a, proinsulin-like m aterial accounted for eight to 78 p e r cent of total serum im m unoreactive insulin (IRI), and the am ount of that m aterial was abnorm ally in creased in 14 TABLE I Hormone Precursors in the Blood o f P atien ts with Tumors o f the Endocrine Glands Hormone P r e c u r s o r Tumor R e fe r e n c e Progastrin Gastrinoma 115 Proinsulin Insulinoma 116 ProACTH or POMC Pituitary adenoma 117 Pro-growth hormone Pituitary adenoma 33 Pro-prolactin Pituitary adenoma 78 Procalcitonin Medullary thyroid carcinoma 21 Proglucagon Glucagonoma 109 Prosomatostatin S oma tos tatinoma 49 Thyroglobulin Thyroid carcinoma 105 patien ts. 88 In norm al persons, the proportion of proinsulin-like m aterial in blood is also highly variable. Up to 30 percent of the total IRI in fasting samples may correspond to proinsulin-like m aterial, and this portion depends on the age and the physiological condition of the subject. 77 Proinsulin-like m aterial may account for up to 50 percent of IRI in blood samples obtained two hours after oral glucose administration. In rare cases of insulinoma, total serum IRI may be within the normal range and a high proportion of IRI may correspond to proinsulin-like m aterial, 1 which indicates the potential value of proinsulin-like material as a useful m arker for detection of some cases of insulinoma. The proinsulin-like material present in the blood of insulinoma patients has not been characterized but it could consist, at least partially, not of the proinsulin m olecule itself b u t of proinsulin conversion interm ediates. Such interm ediates are formed during the normal processes of insulin synthesis in the beta cells of the pancreatic islets, and they consist of tw o-chained com pounds w here the C- peptide is attached to either insulin A- chain (type II interm ediate) or to insulin B-chain (type I intermediate). A type II interm ediate with a molecular weight of approximately 9,000 was present in two different cohorts w ith fam ilial hyperproinsulinem ia. 76 Unlike the type I in term ediate th at has a biological activity nearly as high as that of insulin itself, the 9,000 m olecular w eight (MW) m aterial has low biological activity and a slow metabolic clearance, which may account for its accumulation in the plasma and its poor hypoglycem ic action. In general, the systemic symptoms observed in insulinoma patients would be determ ined by the absolute am ounts and the proportions of proinsulin, proinsulin type I and type II interm ediates, and insulin p resent in the blood under different physiological conditions.

7 HORMONE-RELATED PEPTIDES AS TUMOR MARKERS 387 The C-peptide has no significant biological activity but it may be clinically useful because infusion of exogenous insulin lowers serum C -peptide levels in norm al subjects, w hereas this su p pression is abnorm al in insulinom a p a tie n ts. 37,87,101 In any case, elev ated concentrations of b o th p ro insulin-like material and C -peptide are not specific markers of insulinoma because they can also be observed in patients, including children, with various causes of hyperinsulinism and/or hypoglycem ia. 3 In six patients with oat cell carcinoma of the lung associated w ith a typical clinical picture of the syndrom e of inapprop riate an tid iu re tic h o rm o n e secretio n (SIADH), high amounts of propressophysin (a common precursor of vasopressin and its neurophysin) w ere d etected circulating in the p lasm a. 120 In contrast, propressophysin was not found in the circulation of patients with SIADH owing to central nervous system diseases, in whom the oversecretion of vasopressin probably originated in the posterior pituitary gland, which indicates the possible value of p ro p resso p h y sin as a tum or m arker for p a tie n ts w ith ectopic vasopressin production. Proopiom elanocortin (POM C) is the precursor molecule of various peptides with hormonal activity, including ACTH, a- and (3-lipotropin, a- and [3-melanocyte-stimulating horm one (MSH) and (3- endorphin. 50 These pep tid es are generated by complex processing mechanisms of the precursor m olecule that occur with several variations in d ifferen t tissues such as the a n te rio r p itu ita ry and the neurointerm ediate lobe of the pituitary. These mechanisms are frequently altered in cases of ectopic production by nonpitu itary tum ors, and also in cases of eutopic p ro d u ctio n, i.e., in p itu itary tum ors, 30 which may result in the circulation of abnormal amounts of different m olecular w eight form s of A CTH and other related peptides. In some patients with gastric carcinoid and with tum ors of th e lung, pancreas and o th er organs, proacth may be the only form of circulating ACTH im m unoreactivity. 39,72 The biological activity of proacth is less than four p ercen t of its im m unoreactivity, w hich explains th e m ild and p ro tracted clinical course of the Cushing s syndrom e observ ed in som e of th ese p atien ts. Plasm a proa C TH has been found to be elevated in more than 60 p erc en t of p a tien ts w ith lung carcinom as w ithout sym ptom s of C ushing s d isease. 10 Similar considerations are valid for o th er horm one p recu rso rs, w hich may circulate in large amounts in cancer patients without producing rem arkable symptoms. In some patients with ectopic ACTH-secreting tumors, the blood may contain heterogeneous forms of ACTH precursors, which are apparently p ro duced by variation of the cleavage patterns of the pro-opiocortin precursor. 71 A hum an am ino-term inal segm ent of proopiomelanocortin has been suggested as a potential m arker for lung carcinoma. 11 At least three immunoreactive molecular weight variants have been described for human serum prolactin. A monomer (MW 23,000) constitutes 80 to 85 percent of circulating immunoreactive prolactin, w hereas a presum ed dim er (MW about ) comprises approximately nine to 2 0 percent of circulating prolactin, and a polymeric big-big prolactin constitutes the remaining small proportion (zero to five percent) of the same activity. In the sera of patients with prolactinomas, these prolactin variants are not proportionally different from that found in norm al subjects. 110 Consequently, the study of high m olecular w eight form s of prolactin would not add useful information for the diagnosis of hum an prolactinomas. In patients w ith glucagonoma, a glucagon p recu rso r (approxim ate M W = 9.000) has been found in the blood, 109 but the same com ponent is also present in the blood of normal subjects. 42 F u rth er

8 388 PIM ENTEL more, the changes in blood concentration of the precursor m olecule and the biologically active glucagon m olecule (MW = 3,500) after various stimulation and suppression tests are similar38 and the m easurem ent of the precursor concentration is also not useful for the localization of the tum or site (pancreatic versus extrapancreatic). 42 Thyroglobulin, although m ore a storage protein for thyroid hormones than a hormone precursor, is a reliable tum or marker when its blood level is m easured for th e surveillance of p a tie n ts w ith tre a te d d iffe re n tia te d thyro id carcinoma, and synthesis and secretion of 19S thyroglobulin may be observed in hum an u n d iffe re n tia te d th yroid carcinoma. 58 Unfortunately high molecular weight (HMW) com pounds reacting w ith antibodies to th e norm al horm one can be produced by aggregation or noncovalent binding of polypeptide horm one m onom ers. Such a m echanism has b een suggested to explain the presence of different forms of im m unoreactive calcitonin in a patient w ith thyroid m edullary carcinom a. 9 Sim ilar h e te ro g en e ity of im m unoreactive calcitonin, w ith a p re dom inance of H M W form s, has been o bserved in th e sera~of lung cancer p atients, 6 and cultures of small-cell carcinomas of the lung. 5 Some of these are converted to low molecular weight forms (LMW) in serum at room tem perature. In sera from acrom egalic patients, the longer the sam ples w ere frozen, the m ore im m u n o reactiv ity of big -b ig hum an grow th horm one (hgh) was detected, suggesting that big-big hgh may be an artifact produced by long-term storage in a frozen condition. 99 Rechrom atography of big-big hg H showed that it can be converted to the big hgh and little hg H. Up to 70 percent of big hg H may be co n v e rted to little hg H during the usual extraction procedures. 94 Big hg H is a dim er linked by a disulfide bond and shows decreased activity in the radioreceptor and lym phom a cell bioassay, whereas another interm ediate form, m edium -big hg H, a te tra m e r of little hg H, can be co n v erted to little hg H after m ercaptoethanol treatm ent. Thus, only fresh samples of serum, tum or biopsies, or cu ltu re m edia of tu m o r cells should be used for these studies. In summary, although the secretion and circulation of hormone precursors in m any cancer p atien ts is a w ell docu m en ted phenom enon, its potential clinical use is limited by the presence of the same precursors in the blood of normal subjects and, m ost im portant, by th e highly variable concentrations of such precursors in different patients, and even in the same patient under different physiological conditions. Hormone Subunits The glycoprotein horm ones (thyrotropin, p itu ita ry gonadotropins, and chorionic gonadotropin) are composed of a and (3 subunits. W hereas all of them have com m on a subunits, each one has a unique (B subunit. U nder norm al conditions, a delicate balance exists betw een the synthesis and assembly of a and (3 subunits, b u t the mechanism s responsible for the m aintenance of this balance rem ain poorly understood. W hereas m ost investigators accept that the synthesis of 3 subunits is limiting to the production of hum an chorionic gonadotropin (hcg ), 13 recent findings support the concept of independent regulation of a and (3 subunit synthesis and secretion. 17 The regulation of luteinizing horm one (LH) and thyrotropin synthesis by estradiol in the anterior pituitary gland are exerted at the transcriptional level for both the a and (3 subunits of the horm ones. 20,53,64 The genes encoding the a and 3 subunits of glycoprotein hormones have b e e n assigned to hum an crom o- som es 6 and 19, respectively. 45,62 T he

9 HORMONE-RELATED PEPTIDES AS TUMOR MARKERS 389 human genome contains seven genes for the 3 subunit of hcg but only one for the (3 subunit of L H. 96 The genes coding for the 3 subunits of hcg and hlh are linked and organized in the form of a complex m ultigene family, with four of the genes arranged in tandem pairs and four in inverted pairs The nucleotide sequences of at least some of the seven hcg 3 subunit genes are not identical, resulting in amino acid variations in the 3 subunit chain. 69 T he biological m eaning of the multiplicity of hcg 3 subunit genes is obscure, and it is not known if all of these genes are transcribed sim ultaneously or if they are switched on and off at various stages of developm ent in a m an n er sim ilar to globin genes. O nly th re e of th e seven hum an chorionic gonadotropin 3 ~sub u n it genes can be expressed in the placenta. 97 The behavior of these m ultiple genes in different types of neoplastic cells rem ains also uncharacterized. Specific radioimmunoassays have been developed for the determ ination of these subunits in the blood. 48,81 A monoclonal antibody against a synthetic pep tid e is specific for the free native hc G 3 subu n it. 8 A synthetic m olecule corresponding to the 118 to 145 peptide sequence present in the 3 subunit carboxyl term i nus of hcg but absent on hum an LH was used as an antigen in the developm ent of antibodies by the hybridom a technique. T hese antibodies recognize th e w hole hcg whereas antibodies directed to the 109 to 118 amino acid sequence of hcg may specifically recognize th e free 3 subunit of hcg. These results suggest that residues 109 to 118 are in part or totally exposed in free 3 hcg but are masked in whole hc G. 8 F ree subunits of the glycoprotein ho r m ones are p resent in small concentrations in th e blood of norm al persons, especially u n d e r certain physiological conditions, when the horm one secretion is increased by endogenous or exogenous stimuli. Free subunits of gonadotropins are p resent in relatively high concentrations in the blood of pregnant and postm enopausal w om en. 29,52 An in creased secretion of a subunits is observed in normal adults after i.v. injection of gonadotropin-releasing hormone (LH RH). 25 D isturbances of subunit balance are frequently observed in pituitary tumors, which may result in increased secretion and circulation of free subunits. A p re dom inant secretion of free subunits is observed under basal conditions in some patients with pituitary tumors. Patients with hyperthyroidism owing to thyrotropin (TSH)-secreting tum ors have an a subunit/t S H m olar ratio g re a te r than unity. 87,106 In a woman with follicle stim ulating horm one (FSH ) and a su b u n it secretin g p itu itary adenom a, th e su p pression of FSH following administration of estrogen, brom o cry p tin e, or dexam ethasone was consistently m ore marked than the suppression of free a subunit. 12 In rare cases, pure glycoprotein hormone subunits are secreted by pituitary tum ors. Pure a subunit secretion has been d etected in patients classified as having nonfunctioning c h ro mophobe pituitary adenomas. 75 Since free subunits have no known biological activity, such tumors are not characterized by sym ptom s rela te d to excessive hormone secretion and, on the contrary, p an hypopituitarism may be produced by the tum or mass. However, the presence of high levels of a subunits in the blood may be valuable as a m arker for the d etectio n of such p itu ita ry tum ors. A p u re a su b u n it-secretin g tum or has also b een observ ed in th e m ouse after serial transplantation of a pituitary thyrotropic tum or for several years. 7 The mechanisms involved in this conversion rem ain to be characterized, but they could consist of structural or regulatory changes at the DNA or RNA levels or at some translational or posttranslational events.

10 390 PIM ENTEL The dimeric form of hcg, as well as variable amounts of free a and (3 subunits of hcg, are present in the sera of pregnant wom en and in patients w ith gestational trophoblastic disease. F re e hcg subunits seem to be biologically inactive since they have no apparent affinity for the hcg receptor at the cell surface and no physiological role has been assigned to them. In pregnancy, hc G serum levels are highest at around 1 0 weeks after th e last m en stru al perio d, d eclining thereafter. F ree a subunit serum levels rise as hcg levels decline, accounting for 30 to 40 percent of total serum a subunits in the th ird trim e ste r of pregnancy, whereas free hcg 3 subunits are at the highest levels in the first weeks of pregnancy, w hen an average of 16 percent free (3 subunits can be detected. 18 At the time of the hcg peak during pregnancy, free (3 subunits account for only a small pro p o rtio n (2.4 to 3.6 p ercen t) of the total amount of 3 subunits. The levels of free 3 su b u n its found in th e sera of patients w ith gestational trophoblastic neoplasms are similar to those of early pregnancy, w hich suggests th a t th ese neoplasms may develop at this stage of pregnancy. 18 The (3 subunit of hcg has been widely used as a m arker for tro p h o b lastic tum ors. 81,103,104 The determ ination of this subunit in blood has been used for m onitoring of chem otherapy in patients with g estational tro p h o b lastic n eo p lasm s. 44 Furtherm ore, the (3 subunit of hcg has been successfully applied as a m arker for the im m unochem ical dem onstration of hcg synthesis in trophoblastic tum ors. 31 Both hc G and its free a su b u n it may coexist in th e blood of p a tie n ts w ith hydatiform m ole and choriocarcinom a, but this is not seen in all cases, and no isolated a su b u n it secretio n has b een observed in p a tie n ts w ith th ese d iseases. 102 Furtherm ore, in patients with choriocarcinom a the presence of circulating free a subunits is not clearly associated with a bad prognosis. Ectopic secretion of either a or (3 subunits of glycoprotein horm ones, especially of hc G, has b e e n d escrib ed in som e p a tie n ts w ith nontro p h o b lastic tum ors. 80,81,107 However, the secretion of free subunits is observ ed in isolated cases, which severely limits its potential use for tum or detection. In patients with d ifferent kinds of n o ntrophoblastic tumors, the (3 subunit of hcg is elevated only in a fraction of the total cases, and the degree of elevation is usually small. 23 In one study, none of 101 patients with n o ntrophoblastic gynecological cancer had elevated levels of immunoreactive a subunits of hcg in the blood. 86 Immunohistochem ical techniques have been used to detect ectopic synthesis of hcg (3 subunits in nontrophoblastic tum ors; only seven out of 93 patients had positive results. 7 Among 750 patients with breast disease, only 14 percent of patients with b re a st cancer w ere found to have e le vated serum hcg levels as determ ined by a double-antibody radioimmunoassay using antisera to the (3 subunit of hc G. 60 M oreover, raised hcg-(3 titers w ere not stage- or tu m o r-ty p e -re la ted, b ut o c cu rred only in postm enopausal su b jects, and the study showed that those patients with elevated hcg levels also had raised levels of hlh, and assay crossreactivity was shown to account for the sp u rio u s hc G ele v a tio n s. 60 In addition, an im m unocytochem ical study failed to find hcg as an ectopic breast tu m o r co n stitu e n t and/or pro d u ct. In another study, serum concentrations of hcg 3 subunit were found elevated in 23 percent of 53 patients with nonseminomatous testicular tumors, but free subunit secretion with the dimeric hormone was n ev er d e te c te d. 56 It m ay be concluded that hcg subunits are not useful markers for the study of nontrophoblastic tum ors.

11 HORMONE-RELATED PEPTIDES AS TUMOR MARKERS 391 An ectopic production of a subunits of glycoprotein horm ones has b e e n observed in some cultured cells in vitro. HeLa cell lines, which are derived from a carcinoma of the uterine cervix, secrete free a su b u n its of glycopro tein h o r m ones, 54 and expression of the a subunit of hc G seem s to b e specifically correlated with tum origenicity in HeLa-fibroblast hybrid cell populations m aintained in culture. 91 H um an lung cells (ChaGo) derived from a bronchogenic carcinoma produce predom inatly the a subunit of hcg under culture conditions and treatm ent of these cells w ith polycyclic aromatic hydrocarbons (benzo(a)pyrene or dim ethylbenzanthracene); at concentrations which do not affect cell growth or macromolecular synthesis, it stimulates the production of a hcg-specific mrna in these cells. 112 Apparently, these effects are associated with hypom ethylation of specific sequences in the hc G gene. Horm one Fragments Peptide fragm ents from several h o r mones have been found in some cancer patients, either in tum or tissue or in cells cultured from tum ors in blood samples. The fragments correspond to degradation products of the respective hormones and/ or their precursors. Some fragments may give immunological cross-reactions with the respective intact horm one and, consequently, their detection is difficult by usual radioim m unoassays tech n iq u es. H ow ever, th e p rese n c e of such fragm ents may be suggested by double-antibody p recip ita tio n tech n iq u es, w hich would indicate the existence in the sample of polypeptides w ith different b in d ing characteristics to different antisera. O ther techniques (gel filtration, chrom atography, electro p h o resis) are also helpful for the detection and identification of horm one fragments. H orm one fragm ents are m ore fre q u en tly found in hum an tum ors w ith ectopic horm one p roduction th at in tum ors of the resp ectiv e e n d o crin e glands. In cultured cervical cancer cells, two forms of desialylated ectopic 3 subunits of hcg were detected, and one of them, although longer than the normal (3 subunit, paradoxically lacked the characteristic carboxyl-terminal peptide. 16 In a m alignant thym ic carcinoid tum or, a polypeptide corresponding to the 2 to 38 sequence of normal pituitary ACTH has been d e te c te d. 55 A m ino-term inal fragm ents of progastrin have been isolated from hum an gastrinom a. 73 Isolated reports have appeared on the occurrence of horm one fragm ents circulating in the blood of cancer patients. Am ino-term inal gastrin fragm ents, p robably corresponding to amino acids 1 to 13 of the normal gastrin heptadecapeptide (G 1 to 17), have been found in the serum of some patients with gastrinoma (Zollinger-Ellison syndrome), 22 but the fragm ents are absent in other patients with the syndrom e. 119 Similar fragments have been found in patients w ith nonneoplastic diseases, such as duodenal ulcer and acute gastritis. 67 Medullary thyroid carcinomas (MTC) may elab o rate and se cre te a noncalcitonin 2 1 -am ino acid p e p tid e fragm ent (CCAP) corresponding to the carboxylterm in al region of p rep ro c a lc ito n in. 79 C oncentrations of CCAP can be d e te r m ined in plasm a by a specific radioim m unoassay. W hereas norm al persons have undetectable levels of CCAP (less than 0.25 ng p e r ml), 11 o u t of 17 patients w ith MTC had levels ranging from 0.25 to more than 7000 ng per ml. The levels of CCAP and calcitonin imm unoreactivity varied from p a tie n t to p a tie n t in nearly a parallel m an n er so th at the calcitonin to CCAP ratio rem ained close to unity. After stim ulation by p en tag astrin injection, both calcitonin and CCAP plasm a levels

12 392 PIM ENTEL increased in 11 out of 13 patients, but in two patients the increase of CCAP was only about 2 0 percent of the calcitonin increase. 79 The relative value of CCAP as a tum or marker, as compared to calcitonin, is limited by the following facts: (a) the levels of CCAP are under the limit of detection by radioimmunoassay not only in normal persons, but also in about one third of patients with MTC; (b) after stimulation with pentagastrin, both calcitonin and CCAP increase fre q u e n tly in parallel, but in some patients with MTC the levels of CCAP increase to a lesser extent than those of calcitonin, and (c) it seems likely that CCAP is also se c re te d in sm all am ounts by norm al perso n s, even in basal conditions. 79 The origin of CCAP has not been determ ined; recently, a 37- amino acid peptide, term ed calcitonin gene-related peptide (CGRP), has been found in several hum an tissues, including m edullary th y ro id carcinom a and norm al thyroid, pituitary and b rain. 100 Apparently, CGRP is produced as a nonorgan-specific p e p tid e by a lte rn a tiv e splicing of the hum an calcitonin gene primary RNA transcript. 83 However, mean levels of CGRP may be up to 1,500 times higher in m edullary thyroid carcinom a than in normal thyroid glands, and it is possible that CGRP and calcitonin are encoded by different genes. It is also possible th at calcitonin, CCAP, and CG RP rep resen t products from differential translational processing events of a com m on p o ly p ep tid e p r e cursor. T he study of clones D N A and genomic restriction maps indicate that, probably, th e m essen g er RNAs for human calcitonin and CGRP originate by alternative processing of the transcript from a simple gene. 63,93 The CGRP-calcitonin gene was found to be transcribed in all of ten hum an lung tum or cell lines examined, including six small cell, one large cell, two adenocarcinom as, and one squamous cell carcinoma. All ten of these cell lines contained CGRP mrna, and most of them also contained detectable calcitonin mrna. Interestingly, the ratio of CGRP to calcitonin was higher in most of the lung carcinomas cell lines than in a m edullary thyroid carcinoma cell line. 63 The hum an p arath y ro id horm one (PTH) is normally secreted as a peptide chain containing 84 amino acids and is cleaved in th e liver into two p o ly p ep tides, an amino-terminal fragment of 34 amino acids (PTH 1-34) with all the biological activity of the intact hormone, and a biologically inactive carboxyl-terminal fragment that circulates in the blood and is ex tracted by the kidney and the liv e r PTH 1-34 is usually not d e tected in normal hum an blood but may be p resen t in small am ounts in the venous effluent from some patients with hyperparathyroidism. Fragm ents of PTH have b een found in cell cultures established from a p a tie n t w ith h y p e rn e phrom a. 34 G astrin-releasing p e p tid e (GRP), a mammalian analogue of amphibian bom besin, has b een considered as an excellent m arker for small-cell carcinoma of the lung. 61 However, the same substance is p resent in o ther types of lung carcinom a as w ell as in fetal lu n g s. 118 Two peaks of im m unoreactive GRP w ere observed in tum or tissues and fetal lungs but, whereas the proportion of them was almost identical in the fetal lungs, in tu mors it was different from case to case. The results suggested that the ratio of (GRP) peak 1 to (GRP) peak 2 depends on the enzymatic activity that converts peak 1 to peak 2, and that the enzyme activity is constant in the neuroendocrine cells of the fetal lung but not in tum or cells. 118 G astrin-releasing peptide, as well as several amino-terminal fragments and a carboxy-terminal fragment of GRP, have been detected in a hum an malignant lung carcinoid tum or. 84 These re

13 HORMONE-RELATED PEPTIDES AS TUMOR MARKERS 393 suits agree with the general phenom enon of p eptid e horm one heterogeneity observed frequently in tum or tissues. Finally, some horm one fragments may have specific biological activity. For exam ple, a region of th e hum an grow th hormone containing amino acid residues 7 to 13 (hgh 7-13) is associated with insulin-like activity. 2 H ow ever, it is not known w hether the insulin-like effects of hg H observ ed u n d e r certain p h y s iological conditions are p ro d u ced by degradation fragm ents of the horm one containing this active sequence, and hyperglycemia, rather than hypoglycemia, is commonly observed in acromegalic patients. The same considerations are also valid for fragments of other hormones. Synthetic horm one fragments containing defined am ino acid seq u en ces can be used as reliable tools to explore different functional sites of protein horm one m olecules. In spite of some isolated reports and inconclusive data, 58 no definitive, clear fragm ent-specific physiological effects have b een d e m o n stra ted for any natural peptide horm one fragm ent under either normal or abnormal conditions. M echanisms of Production of H orm one-r elated Peptides by Tum ors D ifferen t types of m olecular m ech a nisms may be related to the production of p e p tid e horm one p recu rso rs, su b u n its, and fragm ents in tu m o r cells 24 (table II). H ow ever, th e available ev i dence suggests that in most or perhaps all cases, the mechanisms responsible for the production of these compounds by tum or cells are the same as those operating in the respective normal cells, and that the differences observed betw een norm al cells and tu m o r cells are only quantitative ones. No consistent qualitative differences have b e e n found betw een normal and tum or cells in relation to the synthesis and secretion of hor- TABLE I I P oten tial Mechanisms fo r the Production o f Hormone-related P eptides (HRPs) by Tumors 1. Presence o f multiple d iffe r e n t copies o f the gene. 2. Structural changes of the genes or their flan king regions (mutations, d eletions, in s e r tio n s, rearrangem ents). 3. Abnormal tran scription (in it ia t io n or termination at abnormal s i t e s ). 4. Abnormal RNA s p lic in g {use of alternative forms of sp lic in g of the primary t r a n s c r ip t ). 5. Abnormal translation (errors in the tran slational m achinery). 6. Abnormal post- translational phenomena (alterations in the enzymatic processing of hormone p recursors, m odification of polypeptide c hain s, aggregation o f disaggregation of monomers, coupling of protein s u b u n it s ). 7. Abnormal degradation (altered patterns o f polypeptide chain c le a v a g e ). m one-related peptides. These considerations are valid not only for eutopic p ro d u ctio n of th ese com pounds by tum ors, b u t also for ectopic horm one production. Conclusions At present, the possible clinical use of HRPs for tum or m arker purposes is difficult to evaluate. In general, these p e p tides are m ore freq u en tly and a b u n dantly p ro d u ced in tum or tissues, especially in ectopic hormone-producing tum ors, than in norm al tissues. M ost horm one precursors, subunits, and fragm ents are nonfunctional or have only little functional activity. C onsequently, tum ors producing even very large am ounts of th ese substances (w ithout secreting significantly increased amounts of the respective normal hormones) are either clinically silent from the endocrine poin t of view or th ey p ro d u ce m inor endocrine sym ptom s. A dissociation betw een immunoactivity and bioactivity in horm one assays is suggestive of ectopic, ra th e r than eutopic horm one secretion. The determ ination of subunits of the glycoprotein horm ones m ay con

14 394 PIM ENTEL tribute to an increase in the specificity of detections and, in some cases, may also be im portant for the identification of rare cases of p u re subunit secretion. The potential use of horm one p rec u r sors, subunits, and fragments as tum or markers is lim ited by the following facts: (a) The procedures for the detection and characterization of HRPs are fre q u e n tly cum bersom e, e x p en sive, and tim e consum ing, being available in only a few laboratories. (b) Both in production and secretion, the differences observed betw een the tum or tissues and th e respective normal endocrine glands are almost always, or perhaps always, quantitative in character. The same HRPs p rese n t in tum ors are usually p resen t in the respective normal endocrine gland, and this is valid not only for tumors producing hormones eutopically, but also for th e ectopic p ro d u ctio n of h o r m ones and HRPs. (c) In cancer patients, different p atterns of structural heterogeneity for different forms of horm one p recursors and fragments may occur, d e p en d in g on variations in the general physiological conditions of the individual or on the action of specific factors capable o f modifying tu m o r horm one pro d u ctio n. The patterns of production o f horm one p recu rso rs, su b u n its and fragm ents in a given p a tie n t are frequently not constant but show marked changes on different days and even at different times of the same day. (d) M arked variations are usually observed from patient to patient in th e p ro d u ctio n and secretio n of H R Ps. In som e p a tie n ts, th e am ounts of these substances, pro duced and/or secreted by tum ors, are about the same as in normal persons. (e) Dynam ic tests, especially stim ulation tests, usually do not contribute to a m ore accurate diagnosis w hen HRPs are used as criteria, com pared w ith th e use of the respective intact horm one. (f) The relative amounts of HRPs in either blood or tum or tissues is not clearly associated w ith a good or bad prognosis in particular patients w ith cancer. References 1. A l s e v e r, R. N., R o b e r t s, J. P., G e r b e r, J. G., M a k o, M. E., and R u b e n s t e i n, A. H.: Insulinoma with low circulating insulin levels: The diagnostic value o f proinsulin measurem ents. Ann. Int. Med. 2: , A r m s t r o n g, J. M., B o r n s t e i n, J., B r o m l e y, J. B., M a c a u l a y, S. L., and N g, F. M.: Parallel insulin-like actions of human growth hormone and its part sequence hcg Acta Endocrinol. 102: , A y n s l e y - G r e e n, A., J e n k i n s, P., T r o n ie r, B., and H e d i n g, L. G. : Plasma proinsulin and C- peptide concentrations in children with hyperinsu lin aem ic hyp oglycaem ia. Acta Pediat. Scand. 73: , B a y l in, S. B., W ie m a n, K. C., O 'N e i l, J. A., and R oos, B. A.: M ultiple forms of human tumors calcitonin demonstrated by denaturing polyacrylamide gel electrophoresis and lectin affinity chromatography. J. Clin. Endocrinol. Metab. 53: , B e c k e r, K. L., G a z d a r, A. F., C a r n e y, D. N., S n i d e r, R. H., M o o r e, C. F., and S il v a, O. L.: Calcitonin secretion by continuous cultures of small cell carcinoma of the lung: Incidence and immunoheterogeneity studies. Cancer Letters is: , B e c k e r, K. L., S n i d e r, R. H., S il v a, O. L., and M o o r e, C. F.: Calcitonin heterogeneity in lung cancer and medullary thyroid cancer. Acta Endocrinol. 9:89-99, B e l l e t, D., A r r a n g, J. M., C o n t e s s o, G., C a il l a u d, J. M., and B o h u o n, C.: Localization of the beta subunit of human chorionic gonadotrophin on various tum ors. Eur. J. Cancer 76: , B e l l e t, D., B id a r t, J. M., J o l iv e t, M., T a r t a r, A., C a il l a u d, J. M., O z t u r k, M., S t r u g o, M. C., A u d ib e r t, F., G r a s- M a s s e, H., A s s ic o t, M., and B o h u o n, C.: A monoclonal antibody

15 HORMONE-RELATED PEPTIDES AS TUMOR MARKERS 395 against a synthetic peptide is specific for the free native human chorionic gonadotropin (3- subunit. Endocrinology 225: , B e r t a g n a, X. Y., B l o o m g a r d e n, Z. T., R a b in, D., R o b e r t s, L. J., and O r t h, D. N.: M olecular weight forms of immunoreactive calcitonin in a patient with medullary carcinoma of the thyroid: Dynamic studies with calcium, pentagastrin and somatostatin. Clin. Endocrinol. 13: , B o n d y, P. K. : The pattern of ectopic hormone production in lung cancer. Yale J. Biol. Med. 54: , C h a n, J. S. D., S ie d a h, N. G., and C h r é t i e n, M.: Human NH 2 terminal of proopiomelanocortin as potential marker for pulmonary carcinoma. Cancer Res. 43: , C h a p m a n, A. J., M a c f a r l a n e, I. A., S h a l e t, S. M., B e a r d w e l l, C. G., D u t t o n, J., and S u t t o n, M. L.: Discordant serum a-subunit and F S H concentrations in a woman with a pituitary tumor. Clin. Endocrinol. 21: , C h a t t e r je e, M., and M u n r o, H. N.: Structure and biosynthesis of human placental peptide hormones. Vitam. Horm. 35: , C h i n, W. W., G o o d m a n, R. H., J a c o b s, J. W., W o l f e, H. J., D a n ie l s, G. H., and H a b e n e r, J. F. : M edullary thyroid carcinoma identified by cell-free translation o f tumor m essenger ribonucleic acid in a patient with a neck mass and the syndrome of ectopic adrenocorticotropin. J. Clin. Endocrinol. Metab. 52: , C h r é t i e n, M. and S e i d a h, N. G.: Precursor polyproteins in endocrine and neuroendocrine systems. Int. J. Peptide Protein Res. 23: , C o l e, L. A., B ir k e n, S., S u t p h e n, S., H u s s a, R. O., and P a t t i l l o, R. A.: Absence o f the C O O H -term inal peptide on ectop ic human chorionic gonadotropin beta-subunit (hcg). Endocrinology 110: , C o l e, L. A., H u s s a, R. O., and R a o, R. V: D iscordant synthesis and secretion of human chorionic gonadotropin and subunits by cervical carcinoma cells. Cancer Res. 42: , C o l e, L. A., K r o l l, T. G., R u d d o n, R. W., and H u s s a, R. O.: Differential occurrence of free beta and alpha subunits of human chorionic gonadotropin (hcg) in pregnancy sera. J. Clin. Endocrinol. Metab. 58: , C o m b, M., S e e b u r g, P. H., A d e l m a n, J., E i d e n, L., and H e r b e r t, E.: Primary structure of the human met- and leu-enkephalin precursor and its mrna. Nature 295: , C r o y l e, M. L. and M a u r e r, R. A.: Thyroid hormone decreases thyrotropin subunit mrna levels in rat anterior pituitary. DNA 3: , D e f t o s, L. J., R o o s, B. A., B r o n z e r t, D., and P a h i i i e m o r e, J. G.: Im m unochem ical heterogeneity of calcitonin in plasma. J. Clin. Endocrinol. Metab. 40: , D ockray, G. J. and W a lsh, J. H. : Amino terminal gastrin fragment in serum of Zollinger- Ellison syndrome patients. G astroenterology 68: , D o s o g n e - G u e r i n, M., V t o l a r c a y k, A., and B o r k o w s k y, A.: Prospective study of the alpha and beta subunits of human chorionic gonadotrophin in the blood of patients with various benign and malignant conditions. Eur. J. Cancer 14: , D o u g l a s s, J., C iv e l l i, D., and H e r b e r t, E.: Polyprotein gene expression: Generation of diversity of neuroendocrine peptides. Ann. Rev. Biochem. 53: , E d m o n d s, M., M o l i t c h, M., P i e r c e, J. G., and O d e l l, W. D.: Secretion of alpha subunits of luteinizing hormone ( L H ) by the anterior pituitary. J. Clin. Endocrinol. Metab. 42: , F o c h -A n d e r s e n, N., L a d e f o g e d, J., M O l l e r - P e d e r s e n, ].: Renal and hepatic extraction of carboxyl-terminal immunoreactive parathyrin in norm al man. J. C lin. C hem. B iochem 22: , F o s s l ie n, E., P r a s a d, R., and S t a s t n y, J.: Computer-aided standardization of two-dimensional electrophoresis: Im provem ent of biophysical description of proteins in relative versus absolu te coord in ate sy stem s. E lectro p h o resis 5: , F o s s l i e n, E., S t a st n y, J., and Y a m a s h ir o y a, H.: Advances in protein analysis of biological systems. Surv. Synth. Pathol. Res. 2 : , F r a n c h im o n t, P., G a s p a r d, V., R e u t e r, A., and H e y n e n, G.: Polymorphism of protein and polypeptide hormones. Clin. Endocrinol. 2: , F u l l e r, P. J., L i m, A. T. W., B a r l o w, J. W., W h i t e, E. L., K h a l i d, B. A. K., C o p o l o v, D. L., L o l a i t, J. W., F u n d e r, J. W., and S t o c k i g t, J. R. : A pituitary tumor producing high molecular w eight adrenocorticotropinrelated peptides: Clinical and cell culture studies. J. Clin. Endocrinol. Metab. 58: , G a r t n e r, A., L a r s s o n, L. I., and S j o b e r g, N. L.: Immunohistochemical demonstration of chorionic gonadotropin in trophoblastic tumors. Acta Obstet. Gynaecol. Scand. 54: , G e o r k a s, M. C., Y a l o w, R. S., S t r a u s, E. W., and G o l d, E. M.: Peptide radioimmunoassays in clinical medicine. Ann. Int. Med. 97: , G o r d e n, P., H e n d r ic k s, C. M., and R o t h, J.: Evidence for big and little components of human plasma and pituitary growth hormone. J. Clin. Endocrinol. Metab. 36: , G r e e n b e r g, P. B., M a r t in, T. J., a n d S u t c l i f f e, H. S.: S y n th e s is a n d r e le a s e of p a r a th y

16 396 P IM E N T E L roid hormone by a renal carcinoma in cell culture. Clin. Sci. Mol. Med. 45: , G u t m a n, R., L a z a r u s, N. R., P e n h o s, J. C., F a j a n s, S., and R e c a n, L. : Circulating proinsulin-like materials in patients with functioning insulinomas. N ew Engl. J. M ed. 284: , H a b e n e r, J. F.: Recent advances in parathyroid hormone research. Clin. Biochem. 14: , H e d i n c, L., T u r n e r, R. C., and H a r r is, E.: C- peptide, proinsulin and insulin responses to fish-insulin induced hypoglycemia in the diagnosis of insulinoma. Diabetes 24 (Suppl.2):412, H e n d r i k s, T., J a n s e n, J. B. J. M., and v a n T o g e r e n, T. H. M.: The glucagonom a syndrome: Stimulus-induced plasma responses of circulating glucagon com ponents IRG9000 and IRG3500. Acta Endocrinol. 105: , H ir a t a, Y., S a k a m o t o, N., Y a m a m o t o, H., M a t - s u k u r a, S., I m u r a, H., and I k a d a, S.: Gastric carcinoid with ectopic production of ACTH and beta-msh. Cancer 37: , H ir a t a, Y., Y a m a m o t o, H., M a t s u k u r a, S., and I m u r a, H. : In vitro release and biosynthesis of tum or ACTH in ecto p ic ACTH producing tumors. J. Clin. Endocrinol. Metab. 4 i: , H o l d a w a y, I.: Principles of radioreceptorassays. Principles of Competitive Protein-building Assays. Odell, W. D., and Franchimont, P., eds. New York, John W iley and Sons, 1983, pp H o l s t, J. J.: Molecular heterogeneity of glucagon in normal subjects and in patients with glucagon-producing tumors. Diabetologia 24: , H o s h i n a, M., B o o t h b y, M. R., H u s s a, R. D., P a t t i l l o, R. A., C a m e l, H. M., and B o i m e, I.: Segregation patterns of polymorphic restriction sites of the gene encoding the subunit of human chorionic gonadotropin in trophoblastic disease. Proc. Natl. Acad. Sci. (USA) 8-Z: , J o n e s, W. B., L e w i s, J. L., and L e h r, M.: Monitor of chemotherapy in gestational trophoblastic neoplasms by RIA of beta-subunits of hcg. Am. J. Obstet. Gynecol. 140: , J u l i e r, C., W e i l, D., C o u i l l i n, P., C o t e, J. C., V a n C o n c, N., F o u b e r t, C., T h i r i o n, J. P., K a p l a n, J. C., and J u n i e n, C. : The beta chorionic gonadotropin-beta luteinizing gene cluster maps to human chromosome 19. Human G enet 67: , K i s h i m o t o, S., S h i m i z u, S., K o g, H., Y a m a m o t o, M., T a m a r u, T., S a n o, K., K a j iy a m a, G., M i y o s h i, A., a n d Y a n a h a i r a, N.: B i g g a s t r i n p r o d u c i n g c e l l s i n d u o d e n a l u l c e r. G u t P e p t i d e s a n d U l c e r. M i y o s h i, A., e d. T o k y o, B i o m e d i c a l R e s e a r c h F o u n d a t i o n, 1983, p p K l e e, G. G.: Bioassay of hormones. Human Pathol. i5: , K o u r i d e s, I. A., W e i n t r a u b, B. D., L e v k o, M. A., and A l o o f, F.: Alpha and beta subunits of human thyrotropin: purification and development of specific radioimmunoassays. Endocrinology 94: , K r e j s, G. J., O r c i, L., C o n l o n, J. M., R a v a z - z o l a, M., D a v is, G. R., R a s k i n, P., C o l l i n s, S. M., M c C a r t h y, D. M., B a e t e n s, D., R u - b e n s t e i n, A., A l d o r, T. A. M., and U n g e r, R. H.: Somatostatinoma syndrome: biochemical, morphologic and clinical features. New Engl. J. Med. 301: , K r i e g e r, D. T.: The multiple faces of proopiomelanocortin, a prototype precursor molecule. Clin. Res. 31: , K u z u y a, H., B l i x, P. M., H o r w i t z, D. L., R u - b e n s t e i n, A. H., S t e i n e r, D. F.,» B i n d e r, C., and F a b e r, O. K. : Heterogeneity of circulating C-peptide. J. Clin. Endocrinol. Metab 44: , L a b u r t h e, M. C., D o l l a i s, J. R., and R o s s e - l i n, G. E.: Evidence for circulating alpha subunits of pituitary gonadotropins in plasma. J. Clin. Endocrinol. Metab. 37: , L a n d e f e l d, T. and K e p a, J.: Regulation of LH beta subunit mrna in the sh eep pituitary gland during different feedback states of estradiol. B ioch em. B iophys. Res. Com m. 722: , L i e b l i c h, J. M., W e i n t r a u b, B. D., R o s e n, S. W., C h o u, J. Y., and R o b i n s o n, J. C. : HeLa C ells secrete alpha subunit o f glycoprotein tropic hormones. Nature 260: , L o w r y, P. J., R e e s, L. H., T o m l i n, S., B u i l - l i e s, G., and L a n d o n, J.: Chemical characterization of ectopic ACTH purified from a malignant thym ic carcinoid tum or. J. Clin. Endocrinol. Metab. 43: , M a n n, K., and K a r l, H - J. : Molecular heterogeneity of human chorionic gonadotropin and its subunits in testicular cancer. Cancer 52: , M e l a n i, F., R y a n, W. G., R u b e n s t e i n, A. H., and S t e i n e r, D. F.: Proinsulin secretion by a pancreatic beta-cell adenoma: proinsulin and C - peptide secretion. New Engl. J. Med. 283: , M it t r a, I.: Som atom edins and proteolytic bioactivation of prolactin and growth hormone. Cell 38: , M o n a c o, F., C a r d u c c i, C., D e L u c a, M., A n d r e o l i, M., and D i m i n i c i, R. : Human undifferentiated thyroid carcinoma synthesizes and secretes 19S throglobulin. Cancer 54:79-83, M o n t e i r o, J. C. M. P., F e r g u s o n, K. M., M c K i n n a, J. A., G r e e n i n g, W. P., and N e v i l l e, A. M. : Ectopic production of human chorionic gonadotropin-like material by breast cancer. Cancer 53: , M o o d y, T. W., P e r t, C. B., G a z d a r, A. F., C a r n e y, D. N., and M i n n a, J. D.: High levels of intracellular bom besin characterize human small-cell lung carcinoma. Science 214: , N a y l o r, S. L., C h i n W. W., G o o d m a n, H. M.,

17 H O R M O N E -R E L A T E D P E P T ID E S AS T U M O R M A R K E R S 397 L a l l e y, P. A., G r z e s c h i k, K-H., a n d S a k a c u - c h i, A. Y.: C h r o m o s o m e a s s i g n m e n t o f g e n e s e n c o d i n g t h e a a n d 3 s u b u n i t s o f g l y c o p r o t e i n h o r m o n e s i n m a n a n d m o u s e. S o m a t. C e l l G e n e t. 9: , N e l k i n, B. D., R o s e n f e l d, K. I., d e B u s t r o s, A., L e o n g, S. S., Roos, B. A., and B a y u n, S. B. : Structure and expression o f a gen e encoding human calcitonin and calcitonin gene related p ep tid e. B ioch em. B iophys. Res. Comm. 723: , N i l s o n, J. H., N e j e d l i k, M. T., V i r g i n, J. B., C r o w d e r, M. E., and N e t t, T. M.: Expression o f subunit and luteinizing hormone (3 genes in the ovine anterior pituitary. Estradiol suppresses accumulation of mrnas for both a subunit and luteinizing hormone 3. J. Biol. Chem. 258: , O h n e d a, A., S a k a i, T., K o b a y a s h i, T., and N i h e i, J.: Insulin and C-peptide in plasma and tumor of insulinoma patients. Tohoku J. Exp. Med. 239:73-81, P e r m u t t, M., B i e s b r o e c k, J., and C h y n, R.: Characteristics of high molecular weight insulins in insulinoma patients. J. Clin. Endocrinol. Metab. 44: , P e t e r s e n, B., and A n d e r s e n, B. N.: Increased concentrations of the NH2-terminal fragment of gastrin-17 in acute duodenal ulcer and acute gastritis. Scand. J. Gastroenterol. 28: , P i m e n t e l, E.: H orm ones as tumor markers. Cancer D etect. Prev. 6:86 93, Po l i c a s t r o, P., O v i t t, C. E., H o s h i n a, M., F u k o u k a, H., B o o t h b y, M. R., and B o i m e, I.: The 3 subunit of human chorionic gonadotropin is encoded by multiple genes. J. Biol. Chem. 25S: , P u l l a n, P. T., C l e m e n t - J o n e s, V., C o r d e r, R., L o w r y, P. J., B e s s e r, G. M., and R e e s, L. H.: ACTH, L P H and related p e p tid e s in the ectopic ACTH syndrome. Clin. Endocrinol. 23: , R a t t e r, S. J., G i l l i e s, G., H o p e, J., H a l e, A. C., G r o s s m a n, A., G a i l l a r d, R., C o o k, D., E d w a r d s, C. R. W., and R e e s, L. H. : Proopiocortin related peptides in human pituitary and ectopic ACTH secreting tum ors. Clin. Endocrinol. 18: , R a t t e r, S. J., L o w r y, P. J., B e s s e r, G. M., and R e e s, L. H.: Chromatographic characterization of adrenocorticotrophin in human plasma. J. Endocrinol. 55: , R e e v e, J. R., W a l s h, J. H., T o m p k i n s, R. K., H a w k e, D., and S h i v e l y, J. E.: Amino terminal fragments of human progastrin from gastrinom a. B ioch em. B iophys. Res. Com m. 223: , R i d g w a y, E. C., K i e f f e r, J. D., Ross, D. S., D o w n i n g, M., M o v e r, H., and C h i n, W. W.: Mouse pituitary tumor line secreting only the alpha-subunits o f the glycoprotein hormones: developm ent from a thyrotropic tumor. Endocrinology 223: , R id g w a y, E. C., K l i b a n s k i, A., L a n d e n s o n, P. W., C l e m m o n s, D., B e i t i n s, I. Z., M e A r t h u r, J. W., M a r t o r a n a, M. A., and Z e r v a s, N. T. : Pure alpha-secreting pituitary adenomas. N ew Engl. J. Med. 304: , R o b b in s, D. C., S h o e l s o n, S. E., R u b e n s t e in, A. H., and T a g e r, H. S.: Familial hyperproinsulinemia. Two cohorts secreting indistinguishable type II intermediates of proinsulin conversion. J. Clin. Invest. 73: , R o b b in s, D. C., T a g e r, H. S., and R u b e n s t e in, A. H. : B iologic and clinical im portance o f proinsulin. New Engl. J. Med. 320: , R o g o l, A. D. and R o s e n, S. W.: Prolactin of apparent large molecular size: the major immunoactive prolactin com ponent in plasma of a patient with a pituitary tumor. J. Clin. Endocrinol. Metab. 38: , Roos, B. A., H u b e r, M. B., B ir n b a u m, R. S., A r o n, D. C., L i n d a l l, A. W., L i p s, K., and B a y l in, S. B.: Medullary thyroid carcinomas secrete a noncalcitonin peptide corresponding to the carboxyl-terminal region of preprocalcitonin. J. Clin. Endocrinol. Metab. 56: , R o s e n, S. W., and W e in t r a u b, B. D.: Ectopic production of the isolated alpha subunit of the glycoprotein hormones: a quantitative marker in certain cases of cancer. New Engl. J. Med. 290: , R o s e n, S. W., W e in t r a u b, B. D., V a it u k a it is, J. L., S u s s m a n, H. H., H e r s h m a n, J. M., and M u g g ia, F. M.: Placental proteins and their subunits as tumor markers. Ann. Int. Med. 82: , R o s e n, S. W., W i n t e r, J. N., and E p s t e i n, A. L.: Application of monoclonal antibodies to tumor diagnosis and therapy. Ann. Clin. Lab. Sei. 23: , R o s e n f e l d, M. G., M e r m o d, J-J, A m a r a, S. G., S w a n s o n, L. W., S a w c h e n k o, P. E., R iv ie r, J. E., V a l e, W. W., and E v a n s, R. M.: Production of a novel neuropeptide encoded by the calcitonin gene via tissue specific RNA processing. Nature 304: , R o t h, K. A., E v a n s, C. J., W e b e r, E., B a r - c h a s, J. D., B o s t w i c k, D. G., and B e n s c h, K. G.: Gastrin-releasing peptide-related peptides in a human m alignant lung carcinoid tumor. Cancer Res. 43: , R u b e n s t e in, A. H., K u z u y a, H., and H o r w it s, D. L.: Clinical significance of circulating C- peptide in diabetes mellitus and hypoglycemic disorders. Arch. Int. Med. 137: , R u t a n e n, E. M.: The circulating alpha subunit of human chorionic gonadotrophin in gyneocologic tumors. Int. J. Cancer 22: , S e r v i c e, F. J., H o r w i t z, D. L., R u b e n s t e i n, A. H., K a z u y a, H., M a k o, M. E., R e y n o l d s, C., and M o l n a r, G. O.: C-peptide suppression test for insulinoma. J. Lab. Clin. Med. 90: , S h e r m a n, B. K., Pek, S., F a ja n s, S. S., F l o y d, J. C., and C o n n, J. W.: Plasma proinsulin in

18 398 P IM E N T E L patients with pancreatic islet cell tumors. J. Clin. Endocrinol. Metab. 35: , S i m p s o n, E. L., D S o u z a, S. M., I b b o t s o n, K. L., B o c k m a n, R., and J a c o b s, J. W.: Absence of parathyroid hormone m essenger RNA in nonparathyroid tumors associated with hypercalcemia. New Engl. J. Med. 309: , S m a l l r i d g e, R. C. and S m i t h, C. E.: Hyperthyroidism due to thyrotropin secreting pituitary tumors. Arch. Int. Med. 143: , S t a n b r i d g e, E. J., R o s f. n, S. W., and S u s s m a n, H. H.: E xpression o f the alpha su bunit of human chorionic gonadotropin is specifically correlated w ith tu m origen ic expression in human cell hybrids. Proc. Natl. Acad. Sci. (USA) 79: , S t e e n b e r c h, P. H., H o p p e n e r, J. W. M., Z a n d b e r g, J., Roos, B. A., J a n s z, H. S., and L i p s, C. J. M.: Expression of the proopiomelanocortin gene in human medullary throid carinoma. J. Clin. Endocrinol. M etab. 5 8 : , S t e e n b e r g h, P. H., H o p p e n e r, J. W. M., Z a n - BERG, J., VAN D E V e N, W. J. M., J a NSZ, H. S., and L i p s, C. J. M.: Calcitonin gene related peptide coding sequence is conserved in the human genom e and is expressed in medullary thyroid carcinoma. J. Clin. Endocrinol. Metab. 59: , S t o l a r, M. W., A m b u r n, K., and B a u m a n n, G.: Plasma big and big-big growth hormone (GH) in man: An oligomeric series composed of structurally diverse monomers. J. Clin. Endocrinol. Metab. 59: , S u l m a n, C., G o s s e l i n, P., C a r p e n t i e r, P., and L e m a i r e, B.: Value of the estimation of thyroglobulin levels in the surveillance of the treated d ifferen tiated thyroid carcinom a. J. C lin. Chem. Clin. Biochem. 22: , T a l m a d g e, K., B o o r s t e i n, W. R., and F i d d e s, J. C.: The human genom e contains genes for the (3-subunit of chorionic gonadotropin but only one gene for the (3-subunit of luteinizing hormone. DN A 2: , T a l m a d g e, K., B o o r s t e i n, W. R., V a m v a k o p o u - l o s, N. C., G e t h i n g, M-J., and F i d d e s, J. C.: O nly th ree o f th e se v e n hum an chorion ic gonadotropin beta su bunit g en es can be expressed in the placenta. Nucl. Acid. Res. 22: , T a l m a d g e, K., V a m v a k o p o u l o s, N. C., and F i d d e s, J. C.: Evolution of the genes for the (3 subunits of human chorionic gonadotropin and luteinizing hormone. Nature 307: , T a n a k a, T., A i b a, X, and S h i s h i b a, Y.,: D iscrepancy between immunoreactivity and bioactivity of big-big and big human growth hormone in acromegaly. Endocrinol. Jap,. 32: , T s c h o p p, F. A., T o b l e r, P. H., and F i s c h e r, J. A.: Calcitonin gene-related peptide in the human thyroid, pituitary and brain. Mol. Cell. Endocrinol. 36:53-57, T u r n e r, R. C., and H e d i n g, L. G.: Plasma proinsulin, C-peptide and insulin in diagnostic suppression test for insulinom a. Diabetologia 23: , V a it u k a it is, J. L. : Subunit secretion. Endocrinology. Jam es, V. H. T., ed. A m sterdam, Excerpta Medica, 1977, pp V a i t u k a i t i s, J. L., B r a u n s t e i n, G. D., and Roos, G. X.: A radioimmunoassay which specifically measures human chrionic gonadotropin in the presence of human luteninizing hormone. Amer. J. Obstet. Gvnecol. 223: , V a i t u k a i t i s, J. L., Roos, G. X., B r a u n s t e i n, G. D., and R a y f o r d, P. L. : Gonadotropins and th eir subunits: B asic and clin ical stu d ies. Recent Progr. Horm. Res. 32: , V a n H e r l e, A. J., and U l l e r, R. P.: Elevated serum thyroglobulin: A marker of metastases in d ifferen tiated thyroid carcinom a. J. Clin. Invest. 56: , W e i n t r a u b, B. D., G e r s h e n g o r n, M. C., K o u r i d e s, I. A., and F e i n, H.: Inappropriate secretion of thyroid-stimulating hormone. Ann Int. Med. 95: , W e i n t r a u b, B. D., and R o s e n, S. W.: Ectopic production o f the isola ted b eta su bunit of human chorionic gonadotropin. J. Clin. Invest. 52: , W e h r e n b e r g, W. B., and L i n g, N.: In vivo biological potency of rat and human growth hormone-releasing factor and fragments of human growth horm one-releasing factor. Biochem. Biophys. Res. Comm. 225: , W e i r, G. C., H o r t o n, E. S., A o k i, X. X, S l o - v i k, D., J a s p a n, J., and R u b e n s t e i n, A. H. : Secretion by glucagonomas of a possible glucagon precursor. J. Clin. Invest. 59: , W h i t a k e r, M. D., K l e e, G. G., K a o, P. C., R a n d a l l, R. V., and H e s e r, D. W.: D em onstration of biological activity of prolactin molecular weight variants in human sera. J. Clin. Endocrinol. Metab. 58: , W o l f s e n, A. R. and O d e l l, W. D.:ProACXH : Use for early detection of lung cancer. Amer. J. Med. 66: , W o n g, D. X. W., H a r t ig a n, J. A., and B is w a s, D. K.: Mechanism of induction of human chorionic gonadotropin in lung tumor cells in culture. Increased levels of a-hum an chorionic gonadotropin-specific mrna sequences and benzo(a)pyrene-induced hypom ethylation. J. Biol. Chem. 259: , W u, P., P e n m a n, E., C o y, D. H., and R e e s, L. H.: E vid en ce for direct produ ction of somatostatin-14 from a larger precursor than som atostatin-28 in a phaeochrom ocytom a. Regul. Peptides 5: , Y a l o w, R. S.: Radioimmunoassay in oncology. Cancer 53: , Y a l o w, R. S. and B e r s o n, S. A.: And now, big, big gastrin. Biochem. Biophys, Res. Comm. 48: , 1972.