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Institut National de la Santé et de la Recherche Médicale U.339 [C. d. L-V., W. R., A. G-G.], Service de Médecine Nucléaire [C. d. L-V., S. A.], and Faculté de Médecine [G. M.], Hôpital Saint-Antoine, 75012 Paris; Service de Chirurgie [P. C., E. S.], Service de Médecine Nucléaire [C. B.], and Service d’Anatomopathologie [C. B., A. d. R.], Hôpital Saint-Louis, Paris; Service de Médecine Nucléaire, Hôpital Trousseau, Paris [M. W.]; and Immunotech SA, Marseille [E. R., J. B.], France
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Circulating CEA fails to correlate with tumor burden and is often within normal range. Rising CEA, however, has been associated with poor prognosis (3 , 14) . In most cases, MTC cells express high levels of CEA at their surface and technetium-99m- or indium-111-labeled monoclonal antibody or antibody fragments specific for CEA have been used in clinical studies for radioimmunodetection and radioimmunotherapy (15 , 16) . We used indium-111-labeled bivalent haptens targeted to cancer cells by means of bispecific antibodies, a technique referred to as the AES, which has been shown to be very effective in the detection of CEA-expressing tumors and particularly MTC (17, 18, 19) . The present study establishes the usefulness of AES immunoscintigraphy and RIGS in the surgical removal of very small tumor metastases in patients with suspected occult metastases. Accuracy of immunoscintigraphy and RIGS results was checked by histology and immunostaining of tissue samples removed by the surgeon.
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PATIENTS AND METHODS |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. CEA level was elevated for each
patient (range, 7–131 ng/ml; normal, <5 ng/ml), except for patient 5.
The selection criterion was the suspicion of occult metastases of MTC.
Patients were considered eligible when no tumor site was found by
preoperative check-up, which included: physical examination; chest
X-ray; cervical and hepatic ultrasonography; cervical, thoracic, and
hepatic CT scan; or when the tumor sites known before
immunoscintigraphy were too small to account for the calcitonin level.
Patients had had different previous treatment at various hospitals in
France and Italy as indicated in Table 1
. They were included because of
a suspicion of occult metastases (high plasma calcitonin). Circulating
calcitonin and serum CEA levels were measured by immunoradiometric
assays (ELSA HCT and ELSA2 ACE, respectively; Cis Biointernational, Gif
s/Yvette, France). Because the ELSA-HCT test detects picogram amounts
of basal calcitonin, the pentagastrin-stimulated calcitonin level was
determined for patients with normal CT levels only. Stimulated
calcitonin was measured 5 min before and 0, 3, and 5 min after an
infusion of pentagastrin (0.5 µg/kg; normal, peak <30 pg/ml).
Circulating calcitonin and CEA follow-up after immunoscintigraphy and
RIGS was terminated after any additional treatment (chemotherapy or
radiotherapy) was performed. Preexisting circulating HAMA was
undetectable in all patients, as assessed by a specific
immunoradiometric assay (Ref. 19
; sensitivity, 0.06
µg/ml).
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The immunoreactivity of the tracer was determined by incubating trace amounts of 111In-labeled di-DTPA-TL in anti-DTPA-indium antibody-coated tubes. Consistently high immunoreactivity (96.5 ± 2.8%) permitted us to use the labeled hapten without purification.
Antibodies and Bispecific Antibody.
The anti-CEA x anti-DTPA-indium bispecific antibody was prepared
by chemical coupling of the Fab fragment of the 734.19.22 antibody (a
monoclonal IgG1,
with specificity to the
DTPA-indium complex) to the reduced Fab' of the F6 antibody (a mouse
IgG1,
, specific for human CEA; Ref.
20
). This antibody is not cross-reactive with
NCA.4
Injection Protocol.
di-DTPA-TL and bispecific antibody were provided by Immunotech Pharma
S.A. (Marseille, France). The bispecific antibody dose (0.1 mg/kg of
body weight) was administered to patients as a 30-min i.v. infusion at
4 ± 1 days before tracer injection. At day 0, the
111In-labeled di-DTPA-TL tracer (118–370 MBq)
was delivered i.v.
Serum samples were collected before bispecific antibody injection and 0.08, 0.5, 1, 5, 24, and 48 h after tracer injection. Urine was collected from 0 to 5, 5 to 24, and 24 to 48 h.
Imaging Protocol.
Whole-body scan and anterior, posterior, and lateral neck and thorax
imaging were performed at 5 and 24 h after tracer injection (late
images were recorded when necessary) using a large field of view
tomographic camera (ADAC; Genesys) tuned to the 173 and 247 keV 
ray
peaks of 111In with a 20% window on each peak.
The scanning speed was 12 cm·mn-1 for
whole-body scans, and a preset count of 400,000 counts was used for
views of the neck and thorax. If necessary, single photon emission
computed tomography was performed with the same camera in stepwise mode
(64 frames of 45 s over 360°). For three patients, control
images were recorded after surgery.
Scan interpretation was performed by two independent experienced observers unaware of conventional imaging results. Interpretations were then compared with pathological results on surgical or biopsy specimens or for one patient (no. 7) to circulating calcitonin determination in samples obtained by selective venous catheterization.
Radioimmunoguided Surgery.
Surgery was performed 3 ± 1 days after tracer injection. The
surgeon explored the operative field with a sterile hand-held gamma
probe (Scintiflex, Novelec, France; Refs. 21
and
22
). For each measurement, normal tissue was counted (five
times, 5–10 s) in the vicinity as a reference. The signal was
considered positive when the mean radioactivity counts
(n = 5) were greater than mean reference counts plus
twice the SD (i.e., the square root of the reference
counts). The location of positive signals originating from small lymph
nodes embedded in large negative tissue areas was marked with a thread
to correlate precisely the pathological results with the radioactive
signals. Each lymph node and surrounding tissue were removed and
analyzed by the pathologist as described below. The hand-held gamma
probe counts were recorded and compared with the results of
pathological examination. Ex vivo gamma probe counts were
performed on 37 locations.
Pathological Examination.
For each tissue specimen, the tumor size was measured by the
pathologist, enabling us to calculate the volume and to estimate tumor
weight. Every specimen was evaluated histologically by multiple
sections. Histological stains used included H&E. Immunohistochemical
analysis included use of monoclonal antibodies to cytokeratin (KL1;
Immunotech), to calcitonin (Dako), to CEA (Dako; CEA specific), and to
CD68 (Kp1; Dako) to detect histiocytes-macrophages.
Pharmacokinetics and Biodistribution.
Plasma and surgical sample radioactivity was measured and
corrected for the physical decay of the isotope. Biexponential curves
of the general formula [(1/V1 -
1/V2)2-t/T
+(1/V2)2-t/Tß]
were fitted to individual plasma radioactivity data by nonlinear
least-square regression. After weighing the surgical samples, the
fraction of injected dose accumulated per gram of tissue was
determined.
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RESULTS |
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Pharmacokinetics
For all patients who underwent a first immunoscintigraphy,
111In-labeled di-DTPA-TL serum pharmacokinetics
fitted a biexponential curve. The first half-life T was
0.48 ± 0.16 h, and the second half-life Tß was
21.4 ± 6.3 h (n = 10). Distribution volumes
were, respectively: V1, 4.3 ± 1.0 liters; and
V2, 8.8 ± 2.0 liters (n = 10). The
excretion of excess activity was fast; the fraction of injected dose
recovered in the urine amounted to 32 ± 7% within 5 h,
51 ± 6% within 24 h (n = 11), and 57 ± 3% within 48 h (n = 5).
Biodistribution
The amount of radioactivity targeted to tumors was assessed in
samples containing 66–100% tumor cells and expressed as a percentage
of the injected dose/kg (% ID/kg) of tumor after correction for
radioactive decay. The mean value for 16 samples from six patients was
28 ± 16% ID/kg; individual values ranged from 9 to 70% ID/kg.
Tumor:normal tissue contrast ratios at the time of surgery were 13 ± 7 (tumor:blood), 31 ± 13 (tumor:muscle), and 28 ± 15
(tumor:normal lymph node).
Comparison between Classical Preoperative Check-Up and
Immunoscintigraphy Results
The 13 patients were referred for AES immunoscintigraphy with a
delay of 4 ± 1 days between bispecific antibody and tracer
injection. Whole-body scan and anterior, posterior, and lateral neck
and thorax planar imaging were performed at 5 and 24 h after
tracer injection (late images were recorded when necessary). The sites
of activity uptake and the sites detected by the preoperative check-up
are summarized in Table 2
. All sites but
two (as indicated in Table 2
) were checked by histology and
immunostaining of calcitonin (as described in "Patients and
Methods"). A typical whole-body scan (patient 3) is shown on Fig. 1
. Each detected site was visualized on
planar images; when necessary, the spatial location was assessed by
single photon emission computed tomography.
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). In patient 13,
echography and CT scan detected one false-positive site in the thyroid
area, as assessed by the pathological examination. It was a
true-negative site with AES immunoscinti-graphy.
Occult Tumors Revealed by AES Immunoscintigraphy.
AES immunoscintigraphy revealed 22 occult metastases that had escaped
conventional imaging (seven patients, nos. 3, 4, 6, 7, and 9–11). For
patients 3 and 4, no tumor had been in evidence in any site by the
preoperative conventional imaging evaluation; AES immunoscintigraphy
detected two metastases in patient 3 and four in patient 4 (because
images of the thorax at 24 h were doubtful for patient 4,
additional images were recorded at 72 h, which confirmed one of
the four radioactivity accumulation sites corresponding to a
metastasis). The recurrence detected by conventional imaging in patient
6 (right neck) corresponded to five distinct metastases detected by
immunoscintigraphy: four were true positives, and one could not be
found by the surgeon. Because circulating calcitonin was not restored
to normal after surgery for this patient, we were unable to determine
whether this location was a true or a false positive.
Immunoscintigraphy disclosed two occult iliac bone metastases in
patient 7, one mediastinal metastasis in patient 9, and in patient 10,
there were two metastases at the right neck site: 1 pretracheal and 1
in the thorax. In the liver of patient 11, ultrasonography detected two
metastases, and AES immunoscintigraphy disclosed more than eight foci
accumulating activity; coelioscopy revealed the presence of numerous
small metastatic nodules. Weak signals recorded in patients 6
(inflammatory site) and 12 corresponded to false-positive results.
Weight of the Detected Metastases.
The smallest radioactive site detected by AES immunoscintigraphy
corresponded to a tissue sample, weighing 360 mg. This sample contained
a small tumor lymph node surrounded by connective tissue partially
infiltrated with tumor cells.
RIGS
RIGS was performed at 3 ± 1 days after tracer injection, as
described in "Patients and Methods." For each measurement, normal
vicinal tissue was counted as a reference. The results of in
situ gamma probe exploration for 208 locations (11 patients) are
summarized in Table 3
: 22.1% true
positive, 64.4% true negative, 7.2% false negative, and 6.2% false
positive. The accuracy was 86%, the sensitivity was 75%, and the
specificity was 90%. Taking into account only the results concerning
the lymph nodes >0.5 mm in diameter (n = 45), the
accuracy was 98%, the sensitivity was 100%, and the specificity was
94%.
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The smallest detected metastatic lymph nodes were 0.2–0.3 cm in
diameter (5–15 mg). Some were only partially infiltrated with tumor
cells. A 0.1-mm-diameter metastasis located in a blood vessel was
detected by RIGS (Fig. 2)
. In the
surrounding tissue, an inflammatory reaction with numerous giant cells
around a foreign body was detected by the pathologist. The giant cell
macrophages present in the specimen were negative for CEA (in contrast
to histiocytes-macrophages present in the three inflammatory
false-positive lymph nodes described above). Because the tumor cells
were located in a blood vessel, their accessibility to the bispecific
antibody and the labeled hapten was high, and their radioactivity
uptake might have been much higher than in less accessible tumor cells.
We nevertheless cannot exclude that the observed accumulation of the
radioactivity, allowing the detection of this very tiny tumor, was due
to nonspecific uptake by macrophages.
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Influence of HAMA on Immunoscintigraphy and RIGS in a Repeated
Investigation
In one patient (no. 10), the control image obtained after the
first surgery indicated the persistence of some radioactive sites. Two
months later, another surgery was planned, and a second
immunoscintigraphy was performed, although this patient had developed
HAMA. No adverse effect was experienced by the patient. No site of
activity uptake was imaged; nevertheless, RIGS detected six tumor lymph
nodes of eight (tumor:muscle ratio, 
1.7).
Calcitonin and CEA Serum Level Follow-Up
Postsurgical calcitonin levels were normal (<10 pg/ml) or
restored to normal for four patients (nos. 1, 2, 3, and 5) and remained
normal for 30–42 months, whereas their serum calcitonin levels had
ranged from 59 to 1135 pg/ml before surgery (Table 4)
. In two patients, the pentagastrin
stimulation tests were normal 30 months after surgery (patients 1 and
3; test refused by patient 2). For one patient (no. 5), the calcitonin
level was 33 pg/ml at t = 3 min, in agreement with the
presence of a few residual tumor cells. CEA serum levels remained
normal (<5 ng/ml) for these patients during the follow-up.
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. For five of
these patients, circulating calcitonin levels after 22–58 months of
follow-up were lower than before surgery (nos. 4, 6, and 9) or similar
(nos. 10 and 12). Follow-up of CEA serum level was performed for its
elevation is indicative of poor prognosis (3
, 14)
; it was
restored to normal after surgery for patients 4, 6, and 10 (presurgery
levels, 10.3, 6.9, and 26.4, respectively; Table 5
). It remained normal for patient 6 and
became slightly elevated for patients 4 and 10 at 38–58 months of
follow-up. Patient 9’s CEA level was considerably lowered after 22
months of follow-up (17.7 ng/ml, whereas the presurgery level was 131
ng/ml). Patient 13 had a tumor resection judged adequate by the surgeon
but without significant decrease of calcitonin serum level. For patient
7, surgery was not performed because of the impossibility of removing
the iliac bone metastases. For two patients (nos. 8 and 11), a total
resection of the known metastases was not feasible.
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. The detection of occult metastases by
immunoscintigraphy had the following consequences: decision to operate
(no. 10) or to perform bilateral instead of monolateral surgery (no.
3), or to perform sternotomy (nos. 4 and 9). For one patient (no. 7),
repeatedly operated before, whole-body immunoscintigraphy showed two
unknown metastases on the iliac bone, confirmed by magnetic resonance
imaging and histology on biopsy samples. Because the resection was not
possible, the surgery planned to remove a neck metastasis was canceled.
For two patients (nos. 6 and 12), AES immunoscintigraphy visualized
every metastases, but an additional signal was a false positive.
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. For four of these patients (nos. 4, 6, 9, and
10), surgery was considered complete according to RIGS, but the
calcitonin serum levels were not restored to normal, indicating the
persistence of residual disease.
For two patients (nos. 3 and 5), CEA and basal CT levels were
normalized. Immunoscintigraphy detected two occult metastases in
patient no. 3 (Fig. 1)
; one, which escaped bilateral lymphadenectomy,
was removed thanks to RIGS. The pentagastrin test was still negative 30
months after surgery. In patient 5, the RIGS signal led to the removal
of a 2-mm tumor lymph node. Basal serum calcitonin of this patient
remained normal 42 months after surgery.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although the hapten circulated as a complex with the bispecific antibody, its initial decline in the circulation was fast because of the rapid clearance of the unbound fraction in the urine. As a consequence, tumor:normal tissue ratios increased rapidly, 24 h after hapten administration being the optimal time for immunoscintigraphy. Tumor uptake was stable, and thus tumor:nontumor contrast ratios kept increasing over time, and this has been useful in a few cases where imaging at later times has allowed us to detect additional tumor sites. It also made RIGS possible up to 7 days after hapten administration.
The results presented here have been confirmed by pathological examination of surgical or biopsy samples or selective venous catheterization. The technique is very sensitive given that, of 34 metastases detected by AES immunoscintigraphy, 22 escaped classical preoperative imaging. High-contrast images were obtained in most cases and indicated that two successive injections 4 days apart (one of the bispecific antibody, the other of the labeled bivalent hapten) greatly enhanced diagnostic reliability. One patient received a second administration of the AES reagents while she was HAMA positive. The presence of HAMA did not cause any clinical problem but definitely decreased targeting efficiency (25) .
The benefit of AES immunoscintigraphy for the patients was illustrated
by the influence of immunoscintigraphy on the efficacy of surgery. It
was clearly demonstrated in one of the four patients (nos. 3, 4, 9, and
10), operated on because immunoscintigraphy detected occult tumors;
patient 3 had his serum calcitonin and CEA restored to normal. In
addition, the time to progression may have been delayed in the other
cases (nos. 4, 9, and 10), in which a large decrease in circulating
CEA, an important prognostic factor (3
, 14)
, was achieved.
CEA was restored to normal for two of these patients after surgery and
remained below presurgery level 22–58 months later for the three
(Table 5)
. Finally, whole-body scanning allowed the detection of
distant metastases, which represents a major advantage of the present
approach. In one patient, two occult iliac bone foci, out of surgical
reach, were visualized by immunoscintigraphy. Cancellation of a useless
surgery was an appreciated benefit for this patient, in agreement with
the earlier observation of Juweid et al. (29)
.
Improved sensitivity and specificity of tumor imaging translated into better clinical management. Surgery may often be only palliative in MTC recurrences, when tumor resection is not complete, as shown by the absence of normalization of serum calcitonin (4 , 6) . This explains the decision to operate, even with marginally significant immunoscintigraphy indications. False-positive immunoscintigraphy interpretation could probably be minimized with a longer practice of the technique. In addition, we cannot exclude that some of these marginally significant images were attributable to very small positive lymph nodes not found by the surgeon.
Localization of tumors by their activity uptake provided a major
surgical opportunity. RIGS allowed us to detect very small tumors,
including those too small to be detected by immunoscintigraphy. The
efficacy of the present technique was established because 16 detected
pathological lymph nodes were 0.2–0.3 cm in diameter. In six cases,
RIGS allowed resection of tumor sites that otherwise would have escaped
surgery, and serum calcitonin was restored to normal (
10 pg/ml) in
two patients (patients 3 and 5).
MTC is a rare disease; the number of patients investigated thus far ruled out statistical treatment. AES immunoscintigraphy, followed by RIGS, enabled us to remove occult metastases with a benefit for six patients, including two patients in which basal calcitonin was restored to normal (follow-up, 30 and 52 months), and for one of these, the normalization of the pentagastrin-stimulated calcitonin. In the absence of RIGS, only very extensive surgery ("microsurgery") achieves calcitonin normalization in a significant number of occult metastatic MTC (2 , 30, 31, 32) , but the surgical technique is more difficult, and complications are greater than conventional surgery (3) . Therefore, the approach of most experienced centers is a more conservative palliative treatment for recurrent MTC (3) .
In conclusion, AES immunoscintigraphy and RIGS allow the resection of very small unanticipated tumors. They are new and powerful tools for the conventional surgical management of patients in recurrent MTC, primarily for occult recurrent MTC. In addition they might be of valuable help for the "microdissection" surgical approach. Only very small lesions may escape both. In such cases, AES radioimmunotherapy after RIGS may become the treatment of choice.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by Grant 6073 from the
Association pour la Recherche contre le Cancer. 
2 To whom requests for reprints should be
addressed, at Faculté de Médecine Saint-Antoine, INSERM
U.339, 27 Rue Chaligny, 75012 Paris, France. Phone: (33)-1-40-01-14-66;
Fax: (33)-1-43-43-89-46; E-mail: gruaz{at}adr.st-antoine.inserm.fr
3 The abbreviations used are: MTC, medullary
thyroid carcinoma; CEA, carcinoembryonic antigen; AES, Affinity
Enhancement System; RIGS, radioimmunoguided surgery; CT, computed
tomography; HAMA, human antimouse antibody; di-DTPA-TL,
N--DTPA-tyrosyl-N-
-DTPA-lysine; %
ID, percentage of injected dose.
Received 5/25/99; revised 10/25/99; accepted 11/ 9/99.
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REFERENCES |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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