0883-2897/91$3.00+ 0.00 Copyright 0 1991Pergamon Press plc

Nucl. Med. Biol. Vol. 18, No. 4, pp. 413-424, 1991 hr. 1. Radiat. &II. Instrum. Part B Printed in Great Britain. All rights reserved

Diagnostic Evaluation of ‘“In and 99mTc Radiolabelled Monoclonal Antibodies in Ovarian and Colorectal Cancer: Correlations with Surgery MARIA

GRANOWSKA*,

STEPHEN J. MATHER

and KEITH

E. BRITTON

St Bartholomew’s Hospital and Medical College and St Mark’s Hospital, London ECI, England The results of radioimmunoscintigraphy, RIS, in colorectal and gynaecological cancers were related to the surgical findings and the analysis of the surgical specimens. The improvement in the accuracy of RIS with the progression from “‘In through 123Ito *Tc as radiolabel is demonstrated. The convenience, low cost and low radiation dose of 99”Tc-labelled antibodies brings the technique of RIS into routine use in the management of patients with these cancers.

Introduction Radioimmunoscintigraphy, RIS, is a complex imaging technique and emphasis has been placed mainly on the nature and specificity of the antibody and on the properties of the radiolabel (Goldenberg et al., 1980; Epenetos et al., 1982; Fairweather et al., 1983; Wahl et al., 1983; Siccardi et al., 1986; Granowska et al., 1986; Britton and Buraggi, 1989). While there is a wealth of data on implanted human tumours in the nude mouse, the relevance of these findings to man is in doubt, because of the disparate relationship of tumour size to the size of the host, the unusual nature of the tumour’s blood supply, and the absence of tumour-associated antigens in the host among other things. There is relatively little work on the biological factors that affect the uptake of radiolabelled antibody by tumours in man. The undertaking of RIS shortly before surgery enables the examination of surgical specimens by imaging to give the distribution of the antibody uptake in tumour, mucosa and lymph nodes and to obtain the essential histological correlates with RIS. Counting weighted samples of known histology provides objective quantitative evidence of the efficacy of RIS. We have approached this problem by serial imaging and the examination of surgical specimens taken after the administration of radiolabelled monoclonal antibodies in two clinical situations: ovarian cancer and colorectal cancer. The following biological factors were examined; the relation between the amount of antibody administered and uptake by the *All correspondence and reprint requests should be addressed to: Dr M. Granowska, Department of Nuclear Medicine, St Bartholomew’s Hospital, West Smithfield, London EClA 7BE, England. NMB

Im--0

tumour and the surrounding mucosa; the relation between tumour weight and uptake; between tumour histology, whether well, moderately or poorly differentiated and the uptake; the stage of the tumour in colorectal cancer by the Dukes’ classification and the uptake of antibody in tumour involved and in tumour free lymph nodes, the heterogeneity of uptake macro and microscopically, the effect of coexisting disease and the relation to serum markers. The release of carcinoembryonic antigen (CEA) by colorectal cancer lead to the early application of RIS in its management (Goldenberg et al., 1980; Mach et al., 1980; Chatal et al., 1984). While radioimmunoscintigraphy, RIS, is effective in primary diagnosis, conventional radiology and endoscopy are the established and reliable methods of diagnosis, with biopsy to confirm the malignant change. The main clinical application of RIS is therefore in the identification of subclinical and subradiological recurrences. The Dukes’ classification (1932) has been related to prognosis: Dukes’ A 95%, Dukes’ B 60%, Dukes’ C 30% 5 year survivals. Dukes’ C is the commonest stage found at primary surgery and has a 50% chance of recurrence at 1 year. Conventionally serial serum CEA is used to follow up such patients, but the nonspecific rises and falls of serum CEA require that there is an increasing serum level during 3 successive months. RIS with anti-CEA antibody has been shown to be more sensitive and more specific than serum CEA as a marker of colorectal cancer both in primary and in recurrent or metastatic disease (Granowska et al., 1989a; Lind et al., 1990). Another RIS finding with anti-CEA is that, although CEA is a dedifferentiation antigen, it is poorly produced by undifferentiated tumour, uptake being much lower than in moderately differentiated tumour 413

414

MARIAGRANOWSKA et al.

(Granowska et al., 1989a). Thus the poorly differentiated tumour which is the more likely to metastasize will be less easy to detect than the well differentiated tumour. In our study 80% of poorly differentiated tumours were staged as Dukes’ C whereas 93% of the moderately or well differentiated tumours were staged as Dukes’ A or B. The staging of colorectal cancer cannot be performed using anti-CEA for RIS, since CEA is trapped by normal nodes more effectively than by involved nodes. This is because once the carcinoma has broken through the basement membrane the shed antigen travels in the local lymphatics and is held by the sinusoid lining cells of the lymph nodes (Beatty et al. 1986; Granowska er al., 1989a). This is one of the disadvantages of using an antigen that is shed. An epithelial surface membrane antigen such as B72.3 does not appear to have this problem (Colcher et al., 1987). We have examined the use of new monoclonal antibody, PRlA3, Class IgGl, developed by the Imperial Cancer Research Fund Laboratories. It is against an antigen present in the cell membrane of the apical cytoplasmic region and microvillous brush border of the surface and upper crypt columnar absorptive cells of normal colon (Richman and Bodmer, 1987). PRlA3 does not react with mucus cells or cytoplasmic constituants. There is a slight reaction with some cells of the stomach, ileum and trachea, but not with any other tissue. It binds strongly to both well, moderately well and poorly differentiated colorectal carcinoma and to all stages of colorectal carcinoma. Testing showed that 59 out of 60 colorectal tumours react with PRlA3. It has advantages over anti-CEA monoclonal antibody since its antigen is fixed. When imaging the surgical specimen after RIS, radiolabelled PRlA3 does not appear in normal lymph nodes draining a tumour as does CEA (Granowska et al., 1989b). The colonic basement membrane prevents access of PRlA3 to normal mucosa in contrast to CEA which is released. The signal from the radiolabel is the basis of the detection of the sites of specific uptake of the monoclonal antibody. Although 13’1 was used initially because of its convenience, the low count rate obtained meant that several days had to elapse for tissue background activity to fall to enable the poor signal to be detected. When the same antibody is labelled with lz31of the same activity which gives 20 times the count rate of 13’1,the same tumour was detectable within 4 h (Britton et al., 1987). “‘In using the bifunctional chelate method of Hnatowich et al. (1983) to label antibody has been successfully used for imaging colorectal cancer, but imaging may still take 72 h to complete (Granowska et al., 1989a). “‘In has two y-ray energies, 171 and 247 keV, the higher requires that the y camera is used with a heavier collimator which reduces sensitivity and resolution. “‘In has to be ordered regularly, is expensive and gives a high radiation absorbed dose to the patient

Table I. Radionuclides for radioimmunoscintigraphy

%Tc 12JI “‘In

Half life, T,,z

Gamma ray energy (MeV) (abundance %)

Amount injected (MRq)

Thyroid block

6.0 13.2 6’7.4

0.14(100) 0.159 (97) 0.171(89) 0.247 (100)

600 120 120

NO Yes No

mSv Whole body’ equivalent 2 4.8 24

*Use of F(ab’), fragments reduces these effective dose equivalents for whole labelled antibody by half for “‘In or a third for ‘%

(Table 1). When “‘In-labelled antibodies are metabolized in the reticuloendothelial system, the “‘In is deposited in the tissue giving a high liver and bone marrow background and there is often a high large bowel activity. 99mTcis the radiolabel that is continuously available in any nuclear medicine department. It is cheap, gives a low radiation absorbed dose and is ideally suited to the modern y camera with a low energy collimator (Table 1). The problem of labelling a y globulin with *Tc has been solved by the Schwarz technique (Schwarz and Steinstrasser, 1987). The S-S bonds linking the heavy chains near the hinge region of the antibody are opened using 2-mercaptoethanol as a reducing agent. The antibody retains its immunoreactivity and may be frozen or stored. When required the antibody is thawed and a bone scanning methylene diphosphonate kit is added to provide the tin reducing agent followed by 99mTcgenerator eluate. The 99mTc monoclonal antibodies labelled in this way are stable in oivo for 24 h. There is no detectable uptake in the thyroid when imaging patients who have received no thyroid blocking agent, either 30 min or 24 h after the intravenous injection of the 99mTc-labelled antibody. Excellent results have been obtained using 99mTclabelled anti-CEA antibody by Baum et al. (1989) and Lind et al. (1990). We report the use of 99”Tc-labelled PRlA3 in colorectal carcinoma and *“‘Tc-SM3 in ovarian carcinoma. These are compared with their “‘In-1abelled counterparts.

Materials and Methods “‘In anti-CEA and PRlA3 The anti-CEA monoclonal antibody C46, a y globulin class IgG2A was provided by Amersham International. It is free of activity with non-specific cross reacting antigen, NCA and has high reactivity with colorectal cancer. The monoclonal antibody PRlA3 was provided by the Imperial Cancer Research Fund and is described before. Labelling with “‘In was by the bifunctional chelate technique using the cyclic anhydride of diethylenetriaminepentaacetic acid, ADTPA (Hnatowich et al., 1983) at an average of one chelating group per antibody molecule. Metal free vessels and reagents are essential. The antibody is derivatized with ADTPA using an anhydride antibody molar ratio of between 2 and 4: 1. The unbound ADTPA is separated from the protein bound DTPA by chromatography using a Superose-12 FPLC gel filtration

%Tc antibodies and surgery column. The derivatized antibody is collected and aliquoted into 1 mg doses .and frozen for future use. Sterility and apyrogenicity tests are undertaken. Radiolabelling is performed as follows. The vial of “‘In chloride is buffered with 5 M acetate solution to pH 5.5 so that the concentration of acetate is 0.5 M. The antibody is thawed and added drop by drop to the buffered “‘In and the mixture is allowed to react for 20 min. Column chromatography is used to separate the free indium salts and the purity of the labelled antibody is tested using thin layer chromatography. Immunoreactivity was confirmed by ELISA testing to be over 90%. *Tc

PRIA3

PRlA3 was labelled with 99mT~as follows: the S-S bonds holding the heavy chains together in the hinge region of the y globulin are opened using 2-mercaptoethanol. The antibody is concentrated by ultrafiltration to approx. lOmg/mL using a centricon system. It is transferred to a sterile plastic vial. Then to a stirred solution of antibody, sufficient 2-mercaptoethanol, ME, is added to provide a molar ratio of 1000: 1 (0.47 FL ME/l mg antibody). The mixture is incubated at room temperature for 30 min with continuous rotation. The reduced antibody is purified by gel filtration on a 20mL Sephadex-GSO column and eluted using phosphate-buffered saline as the mobile phase. The antibody fractions are collated and pooled into relevant fractions measuring O.D. at 280 nm. These are filtered into sterile multidose vials, the concentrations are measured. 0.5 mg aliquots are dispensed into sterile plastic vials in a clean cabinet and frozen immediately at -20°C and stored ready for use. When imaging is required the antibody aliquot is thawed and reconstituted using an Amerscan methylene diphosphonate, MDP, bone imaging kit with 5 mL 0.9% sterile saline. 35 PL of MDP solution, containing 35 gg MDP and 2.4 pg SnF,, are added to antibody aliquot and mixed well. Then the required amount of w”Tc-pertechnetate (700 MBq approx.) is added to antibody/MDP mixture, which is gently shaken for 5 min. The labelling efficiency is assessed by thin layer chromatography (TLC) developed in 0.9% saline. 99”Tc-MDP runs at the solvent front and 9vmTc-PR1A3 remains at the origin and should be over 95%. If below this, the labelled antibody can be further purified by gel filtration on Sephadex G-50 using 1% human serum albumin in phosphate buffered saline and the relevant fractions are pooled. The labelled antibody is filtered into sterile multidose vials and is stable for some hours in vitro after preparation. This 99mTc-labelled antibody is stable in vivo. There is no thyroid uptake of 99mTc even after 24 h in patients who have received no thyroid blocking medication. This technique has been successfully used for five different antibodies in clinical use in this department to date (Mather and Ellison, 1991).

mTc-HMFGI

415 and *T&M3

The monoclonal antibody HMFGl is against a human milk fat globule glycoprotein antigen found on the epithelial surface of the lactiferous duct of the breast and the lining of the ovarian follicle as well as at other sites (Taylor-Papadimitriou et af., 1981). It is an IgGl class y globulin produced by the Imperial Cancer Research Fund Laboratories. The HMFG antigen has the amino acid sequence: Asp-Thr-Arg in its epitope. SM3 is against a stripped mucin antigen and was devised when it was shown that cancer cells have a reduced ability to glycosylate glycoprotein in their surface membrane. The mucin core protein was stripped of its glycosyl groups with hydrogen fluoride and used as an immunogen. SM3 was obtained and found to react with the longer amino acid sequence: Pro-Asp-Thr-Arg-Pro. This is thought to be accessible in the cancer cell glycoprotein since there are fewer glycosyl branches concealing it from the antibody, whereas in the benign cell the more extensive glycosyl branching tends to cover this larger sequence. SM3 has been shown to be more specific than HMFG for breast cancer as compared to benign breast tumours (Burchell et al., 1987, 1989). It has high selectivity for breast and ovarian cancer, low selectivity for other cancers and for normal tissues. Both antibodies were labelled with %Tc using the method described above. SM3 was labelled with “‘In as for “‘In anti-CEA. In some patients a new macrocycle chelate (9N3 Celltec) was used. The labelling method, pharmacokinetics and results will be described in detail elsewhere. SM3 was also labelled with ‘23I using the iodogen technique (Granowska et al., 1986). Radioimmunoscintigraphy for ovarian cancer

Patients with suspected or known ovarian cancer who were between 30 and 75 years of age were selected for radioimmunoscintigraphy by the gynaecological oncology surgeons. The studies were approved by the City and Hackney District Health Authority Ethical Committee and licensed by the Administration of Radioactive Substances Advisory Committee of the Department of Health and Social Services. The procedure was explained to each patient and signed informed consent was obtained. For those receiving lz31-SM3, potassium iodide 60mg twice daily was given orally the day before and for 3 days during and after the study, which was completed within 24 h. No special preparation was given for the patient receiving “‘In-SM3 or 99mTc-SM3. NO skin testing was performed so as to avoid sensitization to mouse y globulin. Patients were questioned closely as to their history of allergy to foreign protein or significant atopy. Such patients would be excluded from study but no such patient was submitted. The large field of view y camera (Siemens ZLC) is set up with a general purpose parallel hole collimator designed for energies up to 200 keV for 12’1ot *Tc;

M.uu~ GRANOWSKA et al.

416

or designed for energies up to 300 keV for “‘In studies. The y camera is peaked for the appropriate energies with a 20% window and for “‘In the counts in the two energy windows are summed. Uniformity and quality control is performed daily to ensure its optimal performance. The data are recorded on line to a Nodecrest Micas 3 computer for further image analysis. The patient lies supine on the scanning couch and a y camera is placed over the pelvis anteriorly. The injection of lOO-150MBq (2.5-3.5mCi) of ‘231labelled monoclonal antibody containing 0.5 mg of antibody is given intravenously to the patient. Static images, each containing 800,000 counts are taken at 10 min, 4 h and 22 h anteriorly and posteriorly of the whole of the abdomen. For each planar image, a similar image with radioactive “‘Ba markers over the prominent bony landmarks were taken to facilitate the subsequent repositioning of the patient (Granowska et al., 1988). Similar procedures occur at 4 and 22 h. For “‘InSM3, an injection of 80-120 MBq (2-3 mCi), images were taken at lOmin, 24, 48 or 72 h. The final imaging was usually performed on the morning of surgery. For %Tc-SM3 an injection of 600 MBq (15 mCi) was used and images were taken at 10 rnin, 3,6 and 22 h. In each study between 0.5 and 1 mg of monoclonal antibody was administered. All image data are transformed directly to the computer as well as to the microdot transparent film processor unit. Radioimmunoscintigraphy for colorectal cancer

Patients were selected by the surgeons of St Mark’s Hospital. Three types of patients were studied: those thought to have a primary colorectal cancer; those thought to have a recurrence of cancer on follow up; and those who were symptom free 1 year after resection of a Dukes’ C colorectal cancer, at which time there is a 50% likelihood of recurrence. The study was accepted by the City and Hackney District Ethical Committee and informed signed consent was obtained from each patient. A history of allergy to foreign protein or other atopy was sought from each patient but was obtained in none. No skin test was made with the antibody to avoid sensitization. The Table

2. Colorectal

Primary rectal cancer Primary colonic cBnccr Pelvic recurrence

cancer radioimmunoscintigraphy “‘In-anti-CEA Image positive

Histology positive

12 S 3

12 S 3 Histology negative

Racto sigmoid adenoma Pelvic recurrence

Para aortic nodes

1 2

1 Uncongrmcd

Image negative

Histology positive

1

flensitivity for colorectal cancer: 23/24, 96%.

1

with

Table 3. Colorectal cancer uptake with “‘In-anti-CEA

Mean SEM Range Tumour :mucosa Ratio

Differentiated (% ID x 10-‘/g)

Undifferentiated 1% ID x 10-‘/g)

2.13 (1 I) *1.12 0.22-13.3

0.21 (4) 50.08 0.04-0.39

s.01: 1 2.0-9.6

Range

3.33: 1 IS-S.9

patient lies supine on the imaging couch with the y camera set over the pelvis. The y camera (Siemens ZLC 75 tube Digitrac System) is set for “‘In or %Tc as described for ovarian cancer. The patients received 110-170 MBq (3-4.5 mCi) of “‘In anti-CEA or “‘In-PRlA3 monoclonal antibody by intravenous injection over 30 s. Imaging was undertaken at 10 min, 24,48 and or 72 h after injection. The more recent patients received 600 MBq (15 mCi) of 99mTc-PR1A3 intravenously over 30 s. Imaging was performed at 5-10 min, 2-3, 5-6 and at 22-24 h. In all patients anterior and posterior views of the lower chest and upper abdomen, and lower abdomen and pelvis were obtained together with images of six radioactive markers sources set on the bone landmarks to check repositioning of the patient and the image at each time point. Single photon emission computed tomography, SPECT, was performed at 24 h for “‘In and at 5 h and occasionally at 24 h for *Tc-labelled antibody. No untoward reactions occurred in any patient. Gamma camera images were also made of the excised surgical specimen where possible. The histological staging and grading of the tumour was undertaken. Specimens of the tumour, nearby mucosa and lymph nodes known to be involved or not involved with tumour were selected and counted, together with standards and appropriate background samples. These values were decay corrected for the time since injection and expressed as a percentage of the injected dose per gram of specimen. Serial blood and urine samples were also obtained and assayed in some cases. A similar approach has been made by Chetanneau et al. (1989).

Colorectal Cancer: Results and Discussion Results of studies with “‘In anti-CEA are summarized in Tables 2 and 3 and with “‘In PRlA3 in Table

4. Colorectal

cancer radioimmunoscintigraphy “‘In-PRlA3

Primary rectal cancer Primary colonic cancer Pelvic recurrence

with

Image positive

Histology positive

18 11 2

18 11 2

Image negative Ca colon (splenic flexure) Ca rectum (inadequate study) Stnsitivity for colorectal cancer: 31/33, 94%.

1

1

1

I

Fig. 1.Moderately differentiated adenocarcinoma of the rectum imaged with “‘In-PRlA3. Posterior pelvis images: left, IO min; centre, 24 h; right, 48 h. High uptake increasing with time is seen. Tumour was stage Dukes’ A, weight, 7.2 g. Tumour:mucosa ratio 46: 1. Fig. 2. Moderately differentiated adenocarcinoma of the rectum imaged with WmTc-PR1A3. Posterior pelvic images: left, 5 min; centre, 6 h; right, 22 h, show increasing focal uptake with time in the low pelvis, clearing of vascular activity, low marrow uptake and high renal concentration of activity increasing with time. Anterior and SPECT views showed no bladder activity. Rectal tumour was confirmed surgically (courtesy of the British Journal of Cancer).

417

(bl

Fig. 3. Poorly differentiated mutinous adenocarcinoma of the transverse colon imaged with 9A”Tc-PRIA3. (a) Anterior abdominal images: left, 5 min; centre, 6 h; right, 22 h. Note the increasing tumour uptake with time at the top of the image, the fading blood pool, the “empty” pelvis and the absence of large bowel and urinary activity. The liver has less uptake than the tumour. (b) Image of the tumour specimen shortly after surgery on the same day as the 22 h image. Note no lymph node uptake, Dukes’ B tumour 7.8 g. The tumour:mucosa ratio was 63: 1 and 0.017% of the injected dose was in the tumour. (c) Pathological specimen. (d) Diagram of pathological specimen Dukes’ B. No lymph nodes were involved (courtesy of the British Journal of Cancer).

418

Fig. 4. Endometrial adenocarcinoma of the uterus imaged with “‘InSM3. Anterior images of the pelvis: top left, 10 min; top right, 24 h; bottom left, 48 h; bottom right, 72 h. Increasing focal uptake in the uterine cavity is seen. Uterus was distended with blood and tumour was confirmed at operation. Fig. 5. Undifferentiated endometrioid ovarian cancer imaged with WmTc-SM3. Images of the anterior pelvis: left, 10 min; centre, 6 h; right, 24 h. Focal increasing uptake with time is seen in the centre of the pelvis with a central defect due to cystic necrosis (courtesy of the Inrernational Journal of Biologicnl Markers).

419

421

%Tc antibodies and surgery Table

Table 5. Colorectal cancer uptake with “‘In-PRlA3 Well differentiated (% ID x IO-*/g)

Moderately differentiated (% ID x 1O-2/g)

1.09 (05) +0.35 0.37-2.19

1.94 (10) +0.87 0.3-9.56

13.8: 1 3.3-50.7

9.3: 1 2.2-37.6

Mean SEM Range Tumour :mucosa Ratio Range

Tables 4 and 5 and compared in Table 6. A typical image of rectal carcinoma with “‘In-PRlA3 is shown in Fig. 1. Analysis of the surgical specimens showed no relationship between tumour weight and uptake, but there was a relationship between the degree of differentiation and tumour uptake. Both anti-CEA and PRlA3 have some uptake in colorectal adenoma and polyps but only CEA is found in imageable amounts in normal lymph nodes in the drainage area of a tumour. Anti-CEA was taken up by Crohn’s disease. Twenty-five patients were studied with %TcPRIA3 (Table 7) and typical images are shown in Figs 2 and 3. All the patients with primary rectal cancer or cancer in the ascending, transverse or sigmoid colon were correctly identified. In 7 patients with suspected recurrent disease, two showed positive uptake and liver metastases and three showed positive uptake in pelvis recurrences. Two of these patients with detected pelvic recurrence were Dukes’ C patients imaged a year after primary surgery, one with a rising CEA and one with a normal CEA. Two other Dukes’ C patients were negative on RIS and are being followed up. Primary villous adenoma was detected in one patient and recurrent villous adenoma in another, both requiring surgery. Two patients had normal images: one having mild inflammatory bowel disease; and the other having a recurrence suspect on colonoscopy but confirmed negative on biopsy. This gives a sensitivity for colorectal cancer of 19/19, lOO%, specificity 4/6,67%, although villous adenoma is considered premalignant by some, and an accuracy of 23125, 92%. Seven patients coming to primary surgery, had the surgical specimen imaged and counted. The results are given in Table 8. The range of tumour uptake was between 0.3 and 1.69 x lo-* % of the injected dose per gram. Specimens of blood and urine were analysed and the mean blood clearance half life of 99”Tc-PR1A3 was 24 h. Urinary activity was between 10 and 12% of the injected does per day. Table 6. “‘In-anti-CEA

and “‘In-PRIA3

Tumour uptake (% ID x IO-‘/g) Well and moderately digerentiated Poorly ditferentiated Mean tumour:mucosa

ratio

PRIA3/CEA ratio

CEA

PRIA3

2.73(15)

1.66(15)

0.6: 1

0.21 (4) 4.5: 1

0.59 (4) 10.8: 1

2.8:1 2.4

7. Colorectal

cancer radioimmunoscintigraphy @“‘Tc.PRI A?

Primary colorectal cancer Villous adenoma Pelvic recurrence Liver metastases

Dukes’ C follow up Inflammatory bowel disease Colonoscopy abnormality

Image positive

Histology positive

14 2 3 2

14 2 1

Image negative

Histology negative

2 1 1

with

Confirmed

2 2 Follow up negative 2

1 1

Sensitivity for colorectal cancer: 19/19, 100%; accuracy: 23/35,92%.

The quality of images obtained using *Tc-PRlA3 were usually excellent. Primary and recurrent colorectal tumours were often evident on the 6 h image and confnmed on the 22 h image. SPECT helped to separate the anterior bladder activity for the posterior placed pelvic recurrence. Tumour uptake at 22 h was usually as strong or stronger than that of the liver and kidneys which were similar. Liver, bowel and marrow uptake appear less with *Tc-PRlA3 than with “‘In-PRlA3 (Granowska et al., 1989b). The improved image quality is due to the high count rates obtained and the consequent reduction of the intrinsic noise in the signal so that it is readily demonstrable over the tissue and blood background activity (B&ton and Granowska, 1987). The specific uptake of the antibody increases with time. It is the comparison of the later images with sites of uptake with the first image template without specific uptake that gives confidence in demonstrating and interpreting the presence of small tumours on the serial images. The initial small series shows high sensitivity and accuracy in the detection of primary and recurrent colorectal cancer. It also demonstrates the relatively higher uptake of %Tc-PRlA3 in poorly differentiated tumours, the most likely to spread, over moderately differentiated tumour (Table 8).

Ovarian Cancer: Results and Discussion The images from RIS were reported jointly by two nuclear medicine physicians in the absence of clinical and radiological information. The site of a primary tumour was described and for the serial changes in uptake, a decision as to whether it was benign or malignant was made. Specific uptake in a malignant tumour typically is low or absent on the 10 min image and uptake increases with time. A benign vascular tumour such as a fibroid has a high initial nonspecific uptake decreasing with time as the blood activity falls. A benign cyst appears as a defect without a change in intensity in its wall whereas a malignant cyst shows increasing uptake peripherally often with some focal uptake. Recurrences may be identified at pelvic, paracolic, paraaortic, omental, hepatic or pulmonary sites. Malignant ascites typically show a horseshoe of negative uptake in the

MARIAGRANOWSKA et al.

422

Table 8. *Tc-PRIA3 uptake in surgical specimens Tumour histolow Moderately differentiated Poorly differentiated

No.

Uptake % D/g *SEM

Tumour:mucosa ratio

4 3

0.0059* 0.0021 0.0149f 0.001

2.5: 1 26.6:1

10 min image, which has increased uptake with time, often with sites of focal uptake, for example in the omentum. The results of RIS were compared with diagnostic surgical and histological findings. radiological, Thirty-five patients were studied with “‘In-SM3: 29 using “‘In-DTPA chelate and 6 using an “‘In-9N3 macrocycle chelate (Celltec). Positive uptake was reported on RIS in the following benign ovarian tumours: some serous cystadenoma, a ruptured dermoid cyst, a mutinous cystadenoma and an ovarian fibroma. Where tested, SM3 uptake was shown on immunoperoxidise staining. Lack of uptake was noted in endometriosis. Uptake was also seen in uterine cystic glandular endometrial hyperplasia. All but one malignant ovarian and uterine primary or recurrent tumour was reported correctly including serous papillary and mutinous cystadenocarcinomas, endometroid and clear cell carcinomas, mixed mesodermal tumour, undifferentiated and endometrial carcinoma. One large cyst was called malignant but was benign on histology. This gave for “‘In-SM3 an overall sensitivity for gynaecological malignancy of 96% but a specificity of only 33%, accuracy 75%. However, the results using “‘In macrocycle SM3 were better with an accuracy of 83% compared to “‘InDTPA chelated SM3, accuracy of 69%. The results with ‘231-SM3 gave a sensitivity of loo%, specificity of 67% and accuracy of 86%, but with *“T&M3 all the malignant and benign tumours were correctly identified (Table 9, Figs 4 and 5). In ovarian cancer, previous studies showed that the typical uptake of HMFGl was between 0.001 and 0.004% injected does per gram. These studies showed the uptake of SM3 was between 0.001 and 0.01% ID/g, with some overlap between benign and malignant ovarian tumours. This accounts for the visualization of some benign tumours with these antibodies and their relatively lower specificity in spite of high sensitivity. With “‘In as the radiolabel the high Table 9. Number of studies Number of patients Positive images Malignant Non-malignant Negative images Malignant Non-malignant Sensitivity for malignancy

“‘In 36* 35 31 23 8 5

q

5 4

I

23;4 96% Specificityfor malignancy 4/12 25% Accuracy 75% *Six “‘In macrocycle 9N3 (Celltec).

414 100% 213 61% 86%

*Tc I2 12 IO 10 0 2 0 2 IO/IO 100% 212 100% 100%

Total 55 54 46 37 9

8 31138 97% 8116 50% 83%

marrow and bowel activity may have contributed also to the lower specificity seen in Table 7. In radioimmunoscintigraphy of gynaecological malignancy the move from “‘In and ‘23Ito 99mT~as the radiolabel appears to be justified. From the preliminary results, it also appears that use of an “‘In macrocycle is preferable to the cyclic anhydride of DTPA as the chelating agent. In spite of the theory, no important difference between SM3 and HMFGl has been shown in the differentiation of benign serous and mutinous cystadenoma from malignant. It remains to be determined whether OVTW is more specific although initial results are encouraging (Buist et al., 1990). The importance of this distinction is the urgent need to reduce the number of unnecessary operations on well women whose pelvic ultrasound has found a mass. 5000 screened, 324 operated and only 9 ovarian cancers were found (Campbell et al., 1989). Interpolation of RIS with a specific antibody, which does not react with benign ovarian or uterine masses or with pelvic inflammatory disease, between positive ultrasound and the decision to operate should increase the percentage of “necessary operations”. Many unanswered questions remain. Should ovarian cystadenomas be considered premalignant? Most pathologists argue not. Should they be removed anyhow? What is the outcome if they remain? Should every simple cyst be removed? What is its chance of rupture? The combination of ultrasound and more specific RIS with the 99mT~label giving a low absorbed radiation dose may help to answer these questions, if applied serially to those in whom operation may be considered unnecessary. Radioimmunoscintigraphy is established as an accurate way of evaluating the effects of chemotherapy and in determining whether a residual mass seen by ultrasound or x-ray CT is due to post operative or therapy fibrosis or viable tumour (Granowska et al., 1986). Small recurrences can be demonstrated using kinetic analysis with probability mapping (Granowska et al., 1988). However, with 99mT~ as a radiolabel, sub-clinical and subradiological disease may also he detected such computer-assisted analysis.

without

Conclusions and Summary A monoclonal antibody against a normal colonic columnar cell surface antigen called PRlA3 has high selectivity for colorectal cancer. After initial clinical studies using the “‘In-PRlA3 has been successfully radiolabelled with 99mTc using a modified Schwarz technique. It has been evaluated as an agent for radioimmunoscintigraphy in a pilot study of primary

423

%Tc antibodies and surgery and secondary

colorectal cancer. Poorly differentiated tumour which is the more likely to spread has a (1.49 + 0.2 x 10-2% high uptake of *Tc-PRlA3 ID/g), an advantage over anti-CEA antibodies (0.211 + -0.08 x lo-*% lD/g). A specificity of 100% and accuracy of 92% was found in primary and recurrent colorectal cancer with %Tc-PRlA3. A monoclonal antibody against a mucin core glycoprotein of the epithelial surface lining of breast lactiferous duct and ovarian follicle called SM3 has high sensitivity for ovarian cancer. The improvement in accuracy from 75% with “‘In to 86% with ‘231, and 100% with *Tc as radiolibels confirms the efficacy of using a short lived radionuclide. In all cases the surgical findings and the analysis of surgical specimens provide the essential validation for radioimmunoscintigraphy and help to define the biological factors that affect its use in patients. The great advantage of the ss”‘Tc-labelled monoclonal antibodies: anti-CEA (Baum et al., 1989) PRIA3 and SM3 is that a request for radioimmunoscintigraphy received early in the morning can be proceeded with at once. A provisional result may be given at the end of the same afternoon and confirmed on the 22 h image the next day. This improvement in logistics with %Tc and the reduction in cost and radiation does as compared to “‘In, are helping to overcome the barriers to the widespread application of radioimmunoscintigraphy to colorectal and ovarian cancer. The use of 99mTc-labelled monoclonal antibodies can now become a routine service provided by a general Nuclear Medicine Department. Acknowledgements-We acknowledge the considerable help of our surgical colleagues, J. M. A. Northover, R. K. S. Phillips, T. Jobling, J. H. Shepherd and pathologists J. R. Jass and D. G. Lowe. We are grateful for the support of the Imperial Cancer Research Fund and for the use of the facilities provided by the St Bartholomew’s Hospital Research Trust.

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