identification and measurement of calcitonin …...irma ct07-ct08) was used for the specific...

(CANCER RESEARCH 49. 6845-6851. December I. 1989]

Identification and Measurement of Calcitonin Precursors in Serum of Patients withMalignant Diseases'

Pascale P. Chilian!, Philippe Motte, FrÃ©dÃ©ricTroalen, Annick Jullienne, Paule Gardet, Thierry Le Chevalier,Philippe Rougier, Martin Schlumberger, Claude Bohuon, and Dominique Bellet2

l'ailÃ©d'Ãmmunoehimie IP. P. (i., P. M., F. T., C. B., D. B.], Â¡heDepartement de MÃ©decine\ucleaireÂ¡P. G., M. S./, the Seirice de MÃ©decineB Â¡T.L. C.J. and the Sen-icede Gastroenterologie Â¡P.R.], Institut (instare Roussy, rue Camille Desmoulins, 94805 l'illejuif Cedex, France, and L' 113 INSERM Â¡A.J.] and l'A 163 CNRS, HÃ´pitalST Antoine, 27 rue de Chaligny, 75012 Paris, France

ABSTRACT

Previous studies have suggested that molecular species larger than themature calcitonin Â«I ) are produced by tumors of different origin. Inorder to study these species, we developed a monoclonal immunoradi-ometric assay for calcitonin precursors (CT-pr). This assay was based onboth monoclonal antibody KC01 directed to the 1-11 region of katacalcinand monoclonal antibody CT08 directed to the 11-17 portion of CT. Thesensitivity of this monoclonal immunoradiometric assay for CT-pr was<100 pg/ml. Only one of 131 healthy subjects had CT-pr serum levels>100 pg/ml; this value was therefore selected as the standard serumvalue in healthy individuals. CT-pr was present in the serum of seven often patients with advanced renal failure and in that of 11 of 52 patients(40%) with benign liver disease but was undetectable in sera of patientswith other benign diseases. The serum ( ' I -pr level was correlated with

that of mature CT in patients with medullary carcinoma of the thyroid.In contrast, the serum CT-pr level was frequently elevated in the absenceof a detectable CT level in patients with various malignant tumors and,particularly, in those with either tumors of the neuroendocrine system(60%) or hepatocellular carcinomas (62%). CT-pr was detected in tumorextract from a patient with a hepatocellular carcinoma. Moreover, hybridization experiments with total RNA extracted from this tumor demonstrated the presence of RNAs hybridizing with complementary DNAencoding for common region, calcitonin, and katacalcine sequences. Theseresults show that CT precursors are excreted by numerous cancers andmight well be useful biological markers for the follow-up of productivetumors.

INTRODUCTIONThe hypocalcÃ©miehormone CT3 is a 32-amino-acid-long

polypeptide in its mature form which derives from the post-translational processing of a larger precursor in thyroid C-cells(1). The complete sequence of this precursor, designated pre-procalcitonin, has been deduced on the basis of the nucleotidesequence of cloned cDNAs encoding the peptide (2). Withinthe preprocalcitonin, CT is linked to the KC sequence andpreceded by an 84-amino-acid-long N-terminal region that terminates in a Lys-Arg cleavage signal. The biosynthesis of thehormone involves consecutive proteolytic events affecting preprocalcitonin: the signal peptide is removed as preprocalcitoninenters the endoplasmic reticulum to produce procalcitonin andthe N-terminal region is then cleaved. The resulting 57-amino-acid-long polypeptide is composed of the CT sequence separated from the KC sequence (21 residues) by the Gly-Lys-Lys-Arg cleavage amidation site (3, 4). Finally, calcitonin is releasedby proteolysis, and the proline C-terminal residue is simultaneously amidated (5).

Received 1/30/89; revised 7/21/89; accepted 9/1/89.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 17.14 solely to indicate this fact.

' This work was supported by a grant from "Association pour la Recherchesur le cancer," Villejuif and grant 88 D29 from Institut Gustave-Roussy.

2To whom requests for reprints should be addressed."*The abbreviations used are: CT. calcitonin; m-IRMA, monoclonal immuno

radiometric assay; CT-pr. calcitonin precursor; mAb, monoclonal antibody; MCT.medullary carcinoma of the thyroid: cDNA. complementary DNA; KC. katacalcin; SCLC. small cell lung carcinoma; NSE. neuron-specific enolase.

Neoplastic C-cells secrete large amounts of calcitonin inserum, and CT is used as a tumor-associated marker of MCT.Several groups have reported the heterogeneity of circulatingforms of calcitonin and the presence of immunoreactive molecular species of higher molecular weight than that of monomericCT, but the precise nature of these forms has not been clearlydetermined (6). However, immunoprecipitable calcitonin-spe-cific precursor molecules have been demonstrated followingcell-free translation from either rat and human tissues or celllines (7-9). These data suggested that the heterogeneity ofimmunoreactive forms of serum CT as well as the ectopiesecretion of calcitonin described with radioimmunoassays basedon polyclonal antibodies might be due to the presence of circulating CT-pr.

This study was aimed at both identifying the presence anddetermining the level of CT precursors in serum from healthyindividuals and patients with either benign or malignant diseases. We used a library of mAbs directed against distinctepitopes of the calcitonin or katacalcin molecule to develop twom-IRMAs for the specific identification and quantification ofeither mature calcitonin or biosynthetic CT precursors. Wereport here the simultaneous presence of both CT and CT-prin sera of patients with MCT. In contrast, the presence of CT-pr, usually in the absence of a detectable CT level, was foundin sera of patients with malignant tumors of other origin.

MATERIALS AND METHODS

Subjects. We studied serum samples collected from a total of 876individuals. The first time, 131 healthy individuals aged 20 to 60 yrand 30 pregnant women were evaluated as well as 101 patients withvarious benign diseases and 27 patients with MCT. The latter weredivided into three different groups. A first group included untreatedpatients whose diagnosis of MCT was later confirmed (n = 5). A secondgroup was composed of patients with no evidence of relapse andconsidered to be in complete remission (n = 19). This group was dividedinto two subgroups depending upon whether patients had (N+; n = 10)or did not have (N -, n = 9) lymph node involvement. Patients withrecurrence of the disease (local neck recurrence or distant mÃ©tastases;n = 7) were included in the third group. Patients whose sera were testedboth before and after treatment or recurrence could be classified intotwo different groups. Moreover, pentagastrin stimulation tests werecarried out in five patients with MCT and one healthy subject. Afterinformed consent from each individual was obtained, the stimulationof CT secretion was performed by i.v. injection of pentagastrin (0.5 jig/kg; Peptavlon; I.C.I. Pharma, Cergy, France) and serum samples werecollected before and 2 and 5 min after infusion.

Furthermore, serum samples from 587 patients with non-MCT malignant tumors of various origin were also studied. The presence ofliver mÃ©tastaseswas assessed by echographic examination. Finally,serial studies were carried out on samples collected from 3 subjectswith SCLC.

After drawing, blood samples were rapidly separated by centrifuga-tion for 15 min at 1500 x g, and sera were rapidly stored frozen untilanalysis. After thawing, samples were heat inactivated (30 min at 56Â°C)

to prevent enzymatic degradation of calcitonin in the serum (10) and6845

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SERUM CALCITONIN PRECURSOR MEASUREMENT AND MALIGNANT DISEASE

assayed using different m-IRMAs. We have previously determined thatheating the samples had no effect on immunoreactivity of either calci-tonin or its precursors (data not shown).

Production and Characterization of Monoclonal Antibodies to Calci-tonin or to Katacalcin. The production and characterization of monoclonal antibodies directed to either mature CT or KC have already beendescribed (11, 12). Briefly, these antibodies were obtained after fusionof splenocytes from mice immunized with either CT or KC, bothconjugated to tetanus toxoid. Two monoclonal anti-CT antibodiesdesignated CT07 and CT08 were directed against distinct and separateepitopes. (a) mAb CT07 (IgG2, A',sn= 0.9 x 10'Â°M~') recognized an

epitope located in the 26-32 region of mature CT and did not bind toprocalcitonin (11). It was previously shown that CT07 bound to the26-32 sequence bearing the C-terminal carboxamide group present onmature CT and did not bind to a similar sequence bearing a C-terminalcarboxyl group, (h) mAb CT08 (IgGl, A'â€žâ€ž= 3.0 x 10'Â°M~') recognized

an epitope present in the 11-17 portion of CT and which was alsoexpressed on pcptidcs mimicking the sequence of procalcitonin. Amonoclonal anti-katacalcin antibody identified as KC01 has also beencharacterized (12). mAbKCOl (IgGl, A'a.n= 1.5 x 109M~') recognized

an epitope localized to the 1-11 region of katacalcin and also boundits epitope within the precursor molecule. The antibody binding sitesof these three antibodies arc presented in Fig. 1.

Development of Monoclonal Immunoradiometric Assays for EitherMature Calcitonin or Calcitonin Precursors. Monoclonal antibodiesCT07, CT08, and KC01 were utilized to construct two different monoclonal immunoradiometric assays developed in a "one-step" (simulta

neous) format. One assay based on CT07 and CT08 antibodies (m-IRMA CT07-CT08) was used for the specific measurement of matureCT, while the other, based on KC01 and CT08 antibodies (m-IRMAKC01-CT08) was constructed for the specific quantification of CT-pr(12). Briefly, either CT07 or KC01 served as "capture antibody" and

was linked to a solid phase support by incubating polystyrene beads(Polystyrene Plastic Balls, Chicago, IL) overnight at room temperaturewith a 500-fold dilution of the mAb-containing ascitic fluid in 0.1 Mphosphate-buffered saline, pH 7.4. After extensive washing, the antibody-coated beads were incubated with sample or standard (200 n\),and mAb CT08( 100^1) was labeled with I25Iusing the lodogen method

(13) and utilized as tracer (100,000 cpm). After overnight incubationat room temperature, the beads were washed with distilled water, andbound radioactivity was measured in a y scintillation counter. Syntheticcalcitonin and a 47-amino-acid-long polypeptide analogous to the car-boxyl-terminal part of procalcitonin (PTN47) were used as the standardin m-IRMA CT07-CT08 and m-IRMA KC01-CT08, respectively; thelimit of detection of both assays was determined as previously described(12).

Preparation of Tissue Extract. Liver tumor and normal liver tissueswere placed in liquid nitrogen immediately after resection and storedat -80Â°C until time of extraction. The tissues were pulverized while

still frozen and then homogenized with Polytron in phosphate-bufferedsaline containing 0.1% Aprotinin. After ccntrifugation (40,000 x g for30 min at 4Â°C),the supernatant was collected and assayed by both m-

IRMAs.Affinity Chromatographies. Monoclonal antibody CT07 or KC01

(ascitic fluid) was coupled to CNBr-activated Sepharose 4B (Pharmacia,

Serum samplefrom patient with MCT

IRMA CT (CT07-CT08)

IRMACT-pr(KC01-CT08)

- PREPROCALCITONIN -

â€¢PROCALCITONIN -

sequence N-terminal region Calcitonin KatacalcinÂ« X > 4 1 <

T T TCTOa CT07 KC01

Fig. I. Binding site of monoclonal antibodies to either the calcitonin orkatacalcin molecule. MAbCTOS recognizes the CT [11-17] sequence. mAbCT07recognizes the CT [26-32] amide sequence, and mAb KC01 recognizes the KC[1-11 ] sequence.

m-IRMA CT (A)

m-IRMA CT-pr -Â»fa)

m-IRMA CT-pr

m-IRMA CT m-IRMA CT-pr

Elution withacetic acid 2.5%

m-IRMACT

(H)

m-IRMA CT-pr

Fig. 2. Sequence of affinity Chromatographies followed by m-IRMAs for thecharacterization of CT and CT-pr in serum of a patient wilh MCT.

Uppsala, Sweden) according to the manufacturer's instructions. Then,

both gels were equilibrated with a Na2HPO4 0.05 Mstarting buffer (pH8.5). Serum sample was incubated with the gel coupled with mAbKC01. After a rotating agitation for 18 h at 4Â°C,the gel was packed

into a column; nonadsorbed material was eluted by extensive washingwith distilled water, and fractions were collected every 5 min. Theunbound fractions were then incubated with the gel coupled with mAbCT07. This latter gel was also stirred 18 h at 4Â°Cbefore packing into

a column and collection of nonadsorbed material, as previously described. Bound material was eluted from both columns with 2.5% aceticacid, and fractions were collected every 5 min. After neutralization, 200ÃŸ\of all fractions diluted 1:1 in normal human serum were assayed byboth CT07-CT08 and KC01-CT08 m-IRMAs. Fig. 2 shows the sequence of affinity Chromatographies followed by specific assays ofbound and unbound fractions.

Neuron-specific Enolase Measurement. NSE serum levels were determined using a commercial radioimmunoassay (Pharmacia, Uppsala,Sweden). This assay was performed according to the manufacturer's

instructions. The normal range of NSE serum levels observed by thistechnique is <12.5 ng/ml.

Extraction of Total RNA and Dot-Blot Hybridization. Total RNA wasprepared by a modification of the method of Chomczynski and Sacchi(14). in which tissue was homogenized in guanidinium thiocyanatc andthen extracted with phenol and chloroform-isoamyl alcohol mixture,before isopropanol precipitations. Purification of RNA was achievedby LiCl precipitation. RNA was quantified by measurement of absorb-ance at 260 nm. Purified total RNA denatured in formaldehyde for 15min at 60Â°Cwas spotted onto GeneScreen (New England Nuclear,Boston, MA). The paper was air dried and then baked at 80Â°Cfor 2 h.The paper was then prehybridized in a buffer containing 50% form-amide for 3 h, after which the nick-translated radiolabeled probe wasadded for 18 h at 42Â°C.In this experiment, we have used a cDNA

encoding for common region, calcitonin and katacalcine sequences.After incubation, the paper was twice washed in a buffer containing0.15 M NaCI, 0.015 M sodium citrate, and 0.1% sodium dodecyl sulfateat 55Â°Cfor 30 min each. The filter was then autoradiographed.

RESULTS

All serum samples were tested in two different assays. In one,we measured CT levels with the CT07-CT08 m-IRMA, which

6846




is specific for mature calcitonin and displays a limit of detectionof 10 pg/ml. In the other, we measured CT-pr levels with KC01-CT08 m-IRMA, which is specific for biosynthetic CT precursors and has a limit of detection of 100 pg/ml.

In an attempt to estimate the normal range of serum CT-prlevels, we measured CT-pr values in serum samples collectedfrom healthy individuals. We found that all but one subject hadundetectable CT-pr serum levels (Fig. 3). The normal range wastherefore defined as < 100 pg/ml. We also studied CT-pr levelsin sera of patients with various benign diseases. Seven of 10patients with advanced renal failure and 40% (21 of 52) ofthose with benign liver disease had elevated CT-pr serum values,while CT-pr was undetectable in sera of other patients (Fig. 3).It was noteworthy that 2 of 10 patients with renal failure hadsimultaneous elevation of both CT and CT-pr values. In contrast, CT was undetectable in sera of those with benign liverdisease. Indeed, we had previously found an elevation of serumCT in 33% of patients with renal failure, but none in those withbenign liver disease (15). Thus, the elevation of CT-pr in seraof patients with benign liver disease appears to be independentof secretion of mature calcitonin.

Identification and Measurement of CT-pr in Patients withMCT. We measured both CT and CT-pr serum levels in MCT

patients either untreated or with a residual tumor burden. Thesepatients had CT levels in the range of 12 to 220,000 pg/ml. Inthose sera, CT-pr serum levels were consistently detectable andranged from 112 pg/ml to 6.8 ng/m\. As shown by a regressioncurve, CT and CT-pr levels were correlated (r = 0.97), and itwas estimated from this curve that CT-pr values were 11 timesas high as CT values (Fig. 4). MCT patients in complete clinicalremission, with no residual tumor burden and a serum CT level<10 pg/ml, had undetectable CT-pr levels, as did patients whounderwent total thyroidectomy. Taken together, these resultsstrongly suggested that neoplastic C-cells secrete calcitoninprecursors as well as mature calcitonin.

Furthermore, we investigated the effect of pentagastrin onCT and CT-pr secretion. For this purpose, sera from onehealthy subject and from 5 MCT patients were evaluated beforeand 2 and 5 min after injection of pentagastrin. The healthyindividual had undetectable CT and CT-pr levels during thestimulation test. In contrast, the five MCT patients had anelevated basal serum CT level (range, 19 to 1,180 pg/ml) anddisplayed a positive response to pentagastrin stimulation. The

peak CT values ranged from 207 to 35,700 pg/ml and the peak/basal ratios were higher than 2 (mean Â±SEM = 14.4 Â±7.4).Interestingly, the peak CT-pr values ranged from 189 to 91,800pg/ml, but the peak/basal ratios of CT-pr were consistentlylower than those of calcitonin (mean Â±SEM 3.6 Â±1.7) (Table1).

Using sequential affinity chromatography with either mAbKC01 or mAb CT07 linked to a CNBr-activated Sepharosecolumn, we separated biosynthetic CT precursors from CT inserum of a given MCT patient whose initial serum levels of CTand CT-pr, determined by specific m-IRMAs, were 10,130 pg/

ml and 3,000 ng/ml, respectively. The serum was first laid ontothe KC01 affinity column. After elution of unbound and thenbound material, m-IRMAs performed on fractions demonstrated that the unbound material showed CT immunoreactivity(Fig. 5, A and B). while the bound material collected afterFraction 10 displayed the peak of CT-pr immunoreactivity (Fig.5, C and D). CT immunoreactive fractions (A) were then laidonto the CT07 affinity column. Assays performed on bothunbound and bound fractions after elution demonstrated boththe absence of significant CT-pr immunoreactivity in thosefractions and the presence of a CT immunoreactive peak in thebound fractions collected after elution (Fig. 5, E to //).

Measurement of CT-pr in Various Non-MCT Malignant Tumors. After identification and measurement of biosyntheticcalcitonin precursors in sera of patients with MCT, we extendedthe clinical study to non-MCT malignant tumors. First, weperformed assays in sera of patients with tumors deriving fromthe neuroendocrine system, as does MCT. For this purpose,and in line with the Amine Precursor Uptake and Decarbox-ylation concept (16, 17), we tested sera from patients witheither insulinoma, neuroblastoma, carcinoid tumors, malignantpheochromocytoma, small cell lung carcinoma, or nonspecificapudoma. These tumors are known for their neural crest originand their potentiality for multiple peptide secretion (18). Inparticular, such malignancies have been described as producingneuropeptides (19, 20). We found that 7% of such patients hadsimultaneous elevation of CT and CT-pr levels, whereas 53%of patients had a detectable CT-pr level in the absence of adetectable CT value (Fig. 6). As 60% of patients with SCLChad elevated CT-pr serum levels, while only 6% had simultaneous elevation of the CT level, we studied whether or not theCT-pr level was increased in lung cancers of other histolÃ³gica!

Fig. 3. CT-pr serum levels in healthy subjects, pregnantwomen, and patients with benign diseases. â€¢.10 cases; â€¢.O, lcase; O. patient with detectable CT. Numbers in parentheses.serum level of CT. The percentage represents the incidence ofdetectable CT-pr serum level in each group of patients.

J}# "3" .?

70%

$ml

r

6847




01

3Q.I

Ãœ ,

CT (pg/ml)Fig. 4. CT and CT-pr serum levels in patients with medullary carcinoma of

the thyroid.

Table I Effect ofpentagastrin stimulation on CT and CT-pr secretion

Pentagastrin stimulation tests were carried out in one healthy subject (PatientA) and in five patients (B to F) as described in "Materials and Methods."

CT (pg/ml) at the followingtimes (min)

CT-pr (pg/ml) at the following times (min)

Patients

ABCnEF<10*1930904861.180<102075041,6901,77535,700<101434339732,25111,600<1001001811.1001.91012,000<1001234515,6003.40068,000<1001892723,8004.30091.000

" Serum levels measured before and 2 and 5 min after pentagastrin injection.

m-IRMA CTCT07-CT08

m-IRMA CT-prKC01-CT08

10Â»

IO4

10s

IO4

IO3

10s

IO-

IO"

10-

(A) (B)

.A.

(D)

(H)

5 10 15 20 25 5 10 15 20 25

Elution fractions

Fig. 5. Pattern of CT and CT-pr immunoreactivities in serum samples from apatient with MCT, as revealed by specific assays for either CT or CT-pr after asequence of affinity chromatographies. [, beginning of elution with 2.5% aceticacid. A lo H refer to the fractions shown in Fig. 2.

10'

10

io-

IO1

...o"Â«'Â«119

75% 60%

OBÂ»Â»

.\

V

60%

// â€¢' JFig. 6. CT-pr serum levels in patients with tumors of the neuroendocrine

system. â€¢10 cases; â€¢.O. +. 1 case: O. patient with detectable CT level (valueindicated in parentheses); +. patient with liver mÃ©tastases.The percentage represents the incidence of detectable CT-pr serum level in each group of patients.

10*

10

I

Sfami

* In 1*31

""8,,

Â¡!

Fig. 7. CT-pr serum levels in patients with lung tumors. â€¢.10 cases; â€¢,O, +.1 case; O. patient with detectable CT level (value indicated in parentheses); +,patient with liver mÃ©tastases.The percentage represents the incidence of detectable CT-pr serum level in each group of patients.

types, namely, squamous cell carcinoma, large cell carcinoma,adenocarcinoma, and undifferentiated carcinoma. Results presented in Fig. 7 show that the percentage of those patients withan elevated CT-pr level ranged from 17.5% (squamous cellcarcinoma) to 53% (large cell carcinoma) and that only 2% ofpatients with non-SCLC lung tumors had simultaneous elevation of the serum CT level. We also determined CT-pr serumlevels in patients with various malignancies (Fig. 8). While CT-pr was undetectable in differentiated carcinoma of the thyroid,the percentage of positivity in the sera of other patients varied

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Fig. 8. CT-pr scrum levels in patients with non-MCT malignant tumors. â€¢ 10 cases; â€¢.O, 1 case; O. patient withdetectable CT level (value indicated in parentheses). The percentage represents the incidence of detectable CT-pr scrumlevel in each group of patients.

widely, ranging from 7% (breast tumor) to 62% (hepatocellularcarcinoma) depending on the cell type and stage of the tumor.Moreover, it was noteworthy that only 2 of these 298 patientshad detectable levels of CT. Finally, we tested samples ofpatients with MCT and non-MCT malignant tumors at multipledilutions to determine whether reactivity in the m-IRMAKC01-CT08 was comparable (parallel) to that of standard. Fig.9 shows that the dose-response curve of the standard (PTN47)was parallel to those displayed by various serum samples ofpatients with MCT and non-MCT tumors taken as representative.

Measurement of CI' and CT-pr Gene Transcript in a Hepato

cellular Carcinoma. To confirm the production of CT-pr by anon-MCT tumor, we studied the presence of CT-pr at the tumorlevel. We prepared tumor extracts from both a normal livertissue and a liver carcinoma of a patient displaying a serumCT-pr level of 11,000 pg/ml. Using the m-IRMA KC01-CT08,we did not find any significant binding in the extract of normalliver tissue, while we found a level of 28.2 pg/mg of tissue inthe extract of liver carcinoma. Moreover, we performed hybridization experiments on the same tumor to detect CT-relatedRNAs. As a positive control in this experiment, we spotted anequivalent amount (25 Â¿ig)of total RNA isolated from a humanMCT. As a negative control, we spotted 25 /ug of yeast RNA.We saw more hybridization of the radiolabeled CT probe totumor than to normal liver RNAs (data not shown).

10s

p 10'

10'

10* -

io-3 io-1 io-1 i

Serum dilutions

Fig. 9. Dose-response curves of standards (50.000 pg/ml) (â€¢)and sera ofpatients with medullary carcinoma of the thyroid (x). leukemia (*). large celllung carcinoma (A), small cell lung carcinoma (â€¢).hepatocellular carcinoma (A).and neuroblastoma (*) at multiple dilutions.

Serial Studies in Patients with Small Cell Lung Carcinoma.As patients with SCLC were those with the highest incidenceof detectable CT-pr levels among patients with lung cancers(60%), we performed serial studies of CT-pr levels to evaluatethe usefulness of CT precursors for the follow-up of thesetumors. Simultaneously, we determined the serum levels ofboth CT and NSE, since the latter parameter has been describedas a useful marker for follow-up of patients with SCLC (21).These studies were carried out in 3 patients during the courseof their disease. In one patient, CT-pr levels remained consistently elevated, whereas both CT and NSE levels were undetect-able during the clinical disease-free period. In fact, a slightdecrease in CT-pr levels during the first 2 mo of treatment wasfollowed by a regular increase in levels up until evidence of livermÃ©tastasesby echographic examination. Similar results wereobserved in the other two patients, in whom elevation of CT-pr levels preceded clinical or echographic evidence of recurringdisease by 3 to 10 mo. However, in one of these patients, CTlevels remained undetectable, while NSE and CT-pr levelsincreased simultaneously; in the other patient, both NSE andCT remained undetectable, while CT-pr levels increased.

DISCUSSION

Polyclonal antibodies directed to peptide hormones such assomatostatin, vasoactive intestinal polypeptide, and calcitoninappear to recognize heterogeneous immunoreactive moleculesproduced by various malignant tumors (22). For example, 59%of tumor tissues collected at random contain immunoreactiveCT ( 18). Among these immunoreactive molecules are molecularspecies of higher molecular weight than those of native hormones. Previous studies suggested that such species might wellbe precursor forms of these hormones (9). Hitherto, radio-immunoassays based on polyclonal antibodies were not capableof determining the precise nature of these forms. Recently, itwas demonstrated that m-IRMAs enable the distinction ofclosely related gene products as well as the direct measurementof hormone precursors (12, 23). In fact, m-IRMA CT07-CT08is an assay specific for mature CT, since antibody CT07 has theunique property of recognizing the 26-32 portion bearing acarboxamide function on its C-terminal amino acid residue asfound on mature CT. The specificity of this assay was confirmedby the absence of reactivity of both CT-pr and KC in the m-IRMA CT07-CT08. In contrast, the construction of m-IRMAKC01-CT08 based on one mAb directed to KC and the otherone to CT enables the specific recognition of CT-pr. Thespecificity of this assay was confirmed by the lack of reactivityof both CT and KC in the m-IRMA KC01-CT08. We used such

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m-IRMAs to measure CT precursors separately from matureCT. Our results confirm the heterogeneity of immunoreactiveCT in serum and demonstrate the presence of circulating CTprecursors in patients with either benign or malignant diseases.Elevated values of up to 50,000 pg/ml were detected in 40%and 70rr of patients with benign liver disease and advanced

renal failure, respectively, while all except one healthy subjecthad an undetectable serum CT-pr level (<100 pg/ml). Thepresence of CT-pr in the blood of patients with renal failuremight be due to delayed clearance of CT precursors; such amechanism had already been suggested by the finding of matureCT in patients with advanced renal failure (15, 24). In contrast,the isolated presence of CT-pr in 40% of patients with benignliver diseases such as hepatitis or cirrhosis, although CT isconsistently undetectable in such patients, might be due to anectopie production of precursors by hepatic cells as well as to adefect in the hepatic metabolism of these molecules.

Based on sera from patients with MCT, we established thatneoplastic C-cells released incompletely processed CT precursors in addition to mature CT. It was noteworthy that, in thosepatients, CT-pr levels were correlated with CT levels, with theformer value being approximately 11 times higher than thelatter. This observation does not favor the hypothesis of alterations in specific mechanisms of CT precursor processing inthose neoplastic C-cells. Such alterations would lead to markedvariations in the ratios of precursors to mature hormones, asobserved in other tumor tissues containing prohormones andhormones, for example, gastrinomas producing progastrin andgastrin (25). Moreover, it was striking that, after pentagastrininjection, the peak/basal ratio of CT-pr was lower than that ofCT, suggesting that the reserve of precursor forms was smallerthan that of mature CT in secretory granules of neoplastic C-cells. Previous studies performed by gel filtration separationfollowed by m-IRMA KC01-CT08 on sera of patients withMCT demonstrated the presence of immunoreactive productswith molecular weights of 14,000 and 8,000. Molecular weightsof these products were consistent with the hypothesis that theKC01-CT08 combination binds to procalcitonin and to intermediates of biosynthesis which include the CT sequence linkedto the KC sequence (12).

In contrast to MCT tumors which produce both precursorsand mature hormones, non-MCT tumors, in their large majority, appeared to secrete CT precursors in the absence of matureCT. It was noteworthy that the dose-response curves observedwith multiple dilutions of serum of patients with MCT andnon-MCT tumors were parallel to that of standards. Thisobservation strongly suggests that m-IRMA KC01-CT08 detects molecular species in sera comparable to the standard.Rises in serum CT-pr level in non-MCT patients were consistent with previously reported ectopie secretion of immunoreactive CT (18, 26). This production of immunoreactive hormonewas found with radioimmunoassays which measured any speciesreacting with a given polyclonal anti-CT antibody. Thus, it islikely that a large number of tumors previously described asproducing "immunoreactive CT" do, in fact, produce CT pre

cursors. In order to confirm the production of CT-pr by non-

MCT tumors, we performed experiments with a liver carcinomacollected from a patient displaying a detectable serum CT-prlevel in the absence of a detectable CT level. The finding ofimmunoreactivity on the tumor extract with the m-IRMAKC01-CT08 strongly suggests that the material measured inserum of this patient was produced by its tumor. Moreover, wefound CT-related RNAs in this tumor. This observation showsthat the tumor was transcribing the calcitonin gene. All to

gether, these latter results are consistent with the productionand the excretion of CT precursors detected in serum by thetumor of this patient.

Other studies suggested that many cancers are associatedwith ectopie hormone elaboration, but only a portion elaboratebiologically active hormones and produce clinically recognizable syndromes (27). Our data demonstrate that, at least in thecase of the calcitonin hormone, many cancers are associatedwith ectopie hormone precursor elaboration. The absence ofboth a detectable circulating CT level and a clinically recognizable ectopie CT syndrome might be due to the lack of enzymescapable of metabolizing CT precursors to bioactive CT. Alternatively, the absence of complete processing of CT precursorsin cancer patients might result from structural differences between CT-pr present in their blood and precursors elaboratedin normal thyroid C-cells; modification in the biochemicalstructure would prevent the proteolytic cleavage of precursormolecules. Such alterations have been shown in the small celllung carcinoma cell line DMS53, which biosynthesizes CTprecursors. In these cells, glycosylation of CT precursors is nota feature of posttranslational processing, and the lack of glycosylation seen in DMS53 cells might explain why apparentlyidentical intracellular and extracellular precursors are detectable and perhaps why mature forms are only rarely present (9).

Among the cancer patients with detectable CT-pr levels intheir blood, the highest incidence (62%) was found in patientswith hepatocellular carcinomas. Interestingly, 80% of patientswith hepatocellular carcinomas, as well as 10 to 40% of patientswith benign liver diseases, produced Â«-fetoprotein (28). Thus,patients with either benign or malignant liver diseases have thecapacity to produce molecules as different as oncofetal proteinor hormone precursors. A variety of substances, including several peptide hormones such as calcitonin, were found to bepresent in serum from patients with lung cancers, particularlySCLC (29, 30). Biosynthesis of high-molecular-weight calci

tonin by human small cell carcinoma cells in tissue cultures wasreported by several groups (9, 31, 32). We tested patients withlung cancers of different histopathological types for the presence of CT and/or CT-pr in their blood and found the highestincidence (60%) of detectable CT-pr levels in patients withSCLC. Surprisingly, 53% of patients with large cell carcinomasalso displayed an elevation in CT-pr serum levels. It is noteworthy that these latter lung tumors do not belong to theneuroendocrine system as do SCLC, MCT, and other tumorsderived from Amine Precursor Uptake and Decarboxylationcells, a significant percentage of which produce CT-pr. However, the histological typing of lung cancers has often beenfound to be difficult, and the histology of such cancers as wellas their capacity to produce CT-pr might change after therapy,mutation, or new oncogene expression (33). Alternatively, theproduction of CT-pr by numerous lung tumors of various typesmight be explained by the existence of a common stem cell forall cell types; the existence of such stem cells has previouslybeen suggested (34), and tumors deriving from these cells mighthave the common capacity to produce CT-pr.

Since 60% of patients with SCLC had detectable CT-pr levels,

we performed serial measurements of CT precursors to evaluatethe usefulness of CT-pr determination for the follow-up ofpatients with SCLC. Our preliminary data showed that CT-prlevels paralleled changes in the clinical status and tumor burdenof these patients. Interestingly, the rise in CT-pr levels precededevidence of tumor recurrence by several months. None of severalbiological markers previously proposed as indicators of eitherthe extent of disease or the clinical response to cytotoxic therapy

6850




appeared sensitive or specific enough for general use in themanagement of patients with SCLC. In light of our data, themeasurement of CT-pr might be useful for the follow-up of

these tumors.In conclusion, our study demonstrates that numerous patients

with malignant tumors and, in particular, patients with tumorsderiving from neuroendocrine cells had detectable levels of CTprecursors. Finally, the development of m-IRMAs for differenthormone precursors is important for measuring molecules thatmight be useful as tumor-associated markers and for understanding the processing of these precursors in cancer cells.

ACKNOWLEDGMENTS

We thank Dr. Jean-Michel Bidart and Dr. Alain Puisieux for theircontinuous help and valuable advice. We are grateful to Dr. Moukhtarfor his thoughtful criticism of the data. We are indebted to MoniqueBenoit, Jean-Louis Bobot, Lionel Fougeat, and Yannick Smith forskillful technical assistance. We wish to thank Dr. Bidet for his help inthe collection of sera from patients with lung tumors.

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1989;49:6845-6851. Cancer Res Pascale P. Ghillani, Philippe Motté, Frédéric Troalen, et al. Serum of Patients with Malignant DiseasesIdentification and Measurement of Calcitonin Precursors in

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