Distribution of Radiolabeled Monoclonal Antibody MX35 F(ab ... · fresh, frozen tumor specimens...

Vol. 3, 1433-1442, August 1997 Clinical Cancer Research 1433

Distribution of Radiolabeled Monoclonal Antibody MX35 F(ab’)2 in

Tissue Samples by Storage Phosphor Screen Image Analysis:

Evaluation of Antibody Localization to Micrometastatic

Disease in Epithelial Ovarian Cancer1

Connie L. Finstad,2 Kenneth 0. Lloyd,

Mark G. Federici, Chaitanya Divgi,

Ennapadam Venkatraman, Richard R. Barakat,

Ronald D. Finn, Steven M. Larson,

William J. Hoskins,3 and John L. HummGynecology Service, Department of Surgery: Immunology Program:Nuclear Medicine Service, Department of Medical Imaging:

Department of Epidemiology and Biostatistics: and Department of

Medical Physics, Memorial Sloan-Kettering Cancer Center, New

York, New York 10021

ABSTRACTOur objective was to quantify the targeting of the

monocbonal antibody (mAb) MX35 F(ab’)2 to micrometa-static epithelial ovarian cancer. This mAb detects a Mr

95,000 glycoprotein with homogeneous distribution on 80%of ovarian tumor specimens. Six patients with minimal re-sidual disease from an imaging trial were injected with 2 or

10 mg of ‘�‘I- and ‘25I-labebed mAb MX35 F(ab’)2. Biopsiedsamples were removed at second-look laparotomy 1-5 dayspost-i.v. or -i.p. infusion of antibody. Serial cryostat sections

were stained by indirect immunoperoxidase method for an-

tigen distribution and exposed to storage phosphor screens

for quantitative autoradiography. Coregistration of tumor

histology, antigen expression, and radionuclide distribution

demonstrated specific localization in micrometastatic tumorfoci (50 �im to 1 mm) found within tissue stroma. Theradiolabeled antibody uptake determined by well scintilla-

tion counts ranged between 5.2 and 223.5 x iO� percentageof injected dose/g of tumor tissue for ‘�‘I. Specific bocaliza-tion of mAb in tumor was determined by tumor:normab

tissue (fat) ratios ranging from 0.9:1 to 35.9:1 for ‘�‘I. The

high resolution and linear response of the storage phosphor

Received 8/20/96; revised 1/16/97: accepted 5/7/97.

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 in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

I Supported by the Avon Program for Ovarian Cancer, Memorial Sloan-Kettering Cancer Center, and National Cancer Institute Grants CA-

52477 and CA-08478. This work was presented in part at the 87thAnnual Meeting of the American Association for Cancer Research,Washington, D.C., April 20-24, 1996.2 Present address: United Biomedical, Inc., 25 Davids Drive,

Hauppauge, NY 11788.

3 To whom requests for reprints should be addressed, at MemorialSloan-Kettering Cancer Center, Gynecology Service Academic Office,1275 York Avenue, New York, NY 10021. Fax: (212) 717-3095.

screen imager was used to estimate the radionuclide activity

localized in each micrometastatic site. Quantitation of phos-phor screen response revealed �iCi/g values of 0.026-0.341for normal tissue and 0.184-6.092 for tumor biopsies, eval-uated 4 or 5 days post-antibody injection. The tumor:normal

tissue (adjacent to tumor) ratios were between 1 and 4 timesgreater using the phosphor screen method than well counter

measurements, but even larger variations of ratios up to

20: 1 were observed between tumor cell foci and stromal cellswithin the same tissue section. This study has demonstrated

that mAb MX35 F(ab’)2 localizes to the micrometastaticovarian carcinoma deposits within the peritoneal cavity. The

dosimetry results suggest a therapeutic potential for this

antibody in patients with minimal residual disease (<5 mm).

INTRODUCTION

In the treatment of advanced epithelial ovarian cancer, the

combined effect of surgery and chemotherapy has resulted in a

complete response rate of 45% as confirmed by reassessment

surgery ( I ). However, the risk of recurrence remains high in

patients with advanced disease (stages III and IV), with 50%

recurring within a median of 14 months after negative second-

book laparotomy (2). Patients with residual disease detected at

second-look surgery or recurrent disease after completion of

initial chemotherapy have a poor prognosis, and few, if any, are

cured by currently available salvage therapy. The potential of

radiolabcled mAbs4 for the detection of metastatic spread offers

significant benefits for the subsequent management of these

patients, as well as the possibility to actually treat micrometa-

static disease with antibody carrying the appropriate therapeutic

radionuclide, toxin, or drug.

The application of radiolabeled antibodies for both radio-

immunodiagnosis and treatment of ovarian carcinoma has been

ongoing for more than 10 years (3-10). Epenetos et a!. (4) and

Pateisky et a!. (5) used ‘�‘I and ‘23I-labeled antibodies

(HMFGI and HMFG2) against peptide epitopes of human milk

fat globule. Using gamma camera scintigraphy, they success-

fully demonstrated that >75% of patients having metastatic

spread into the peritoneum imaged positively. Negative scans

were attributed to the absence of disease or the presence of

unresolvable microscopic foci only. The lack of solid tumor

nodules > I .5 cm in diameter would render insufficient image

4 The abbreviations used are: mAb, monocbonab antibody: ckID/g, per-centage of injected dose per gram of tissue: cpd, counts pen day; Tb,

biological half-life: T�. physical half-life: T�, effective half-life: PET,

positron emission tomography.

Research. on June 12, 2020. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

http://clincancerres.aacrjournals.org/

1434 mAb Localization of Micrometastatic Ovarian Cancer

Table 1 Summary of 35 biop. sied specimens analyzed from six patient s with epithelial ovarian canc en”

Number of Number of Tumor cells

Case Diagnosisbiopsies from

normal tissuebiopsies with

tumor cellsTumor cells per

biopsy (%)6

immunostained

(intensity)�

1 Endometrioid 3 6 <20% clusters 90% (+ +)2 Senous 3 1 >80% clusters 100% (+ + +)

3 Endometnioid with clean 2 2 <20% clusters 75% (+ to + +)

cell features and individual cells4 Endometnioid 2 1 >50% clusters 100% (+ + +)

5 Carcinoma (poorly differentiated) 6 1 < 10% clusters 100% ( + + +)

6 Endometrioid (peritoneal) 3 5 <10% clusters 100% (+ + +)

(1 Tumor and normal tissue biopsied specimens were examined from all six cases by well scintillation counting and film autoradiography; biopsiesfrom cases 2-6 were analyzed by storage phosphor screen autoradiography.

b The percentage of tumor cells within the stroma of a tissue section was estimated by indirect immunoperoxidase analysis using anticytokeratinantibody to detect carcinoma cells. Cases I , 4, 5, and 6 showed discrete foci of tumor cells, whereas cases 2 and 3 had tumor cell clusters dispersedthroughout the stromal tissue.

( Frozen tissue sections were analyzed by indirect immunoperoxidase analysis using mAb MX3S to evaluate intensity and heterogeneity ofantigen expression; + + + , strong; + to + + , weak and variable intensity.

contrast to enable specific antibody binding to be detected

against a nonspecific background ( 1 1). Neither gamma camera

imaging nor hand-held surgical radioactivity gamma probes (8,

12) exhibited the sensitivity required to detect micromctastatic

disease (< I cm in diameter) due to insufficient contrast (rarely

> 10: 1 ) of radionuclide activity accumulation within the tumor

relative to the peritumor region ( I I).

Micrometastatic disease may, therefore, remain undetected

by conventional nuclear medicine procedures. Moreover, in

biodistribution studies using biopsied specimens, the presenta-

tion of radiolabelcd antibody uptake and dosimetry as an activ-

ity per unit gram of tissue can be in significant error. This is

because of the small size of the biopsy and the presence of only

clusters of tumor cells within a large region of stromab tissue,

endothelium, and hematopoietic cells. Including nontumor cells

in the activity per unit gram calculations can greatly dilute the

tumor-specific activity.

To explore ways around this problem, we have examined

the use of storage phosphor screen technology to determine the

distribution of radioactivity in surgical specimens obtained from

an antibody-imaging trial on the use of radiolabeled murine

mAb MX3S F(ab’), fragment in patients with ovarian carci-

noma having minimal residual disease. Digital images from

scanned storage phosphor screens were compared with autora-

diographic images obtained using film techniques and MX35

antigen localization determined by indirect immunohistochem-

istry to confirm the specific uptake of radiolabeled mAb MX35

F(ab’), to tumor cell foci. The data from phosphor digital

images were used to evaluate the radionuclide distribution and

to estimate accumulation in micrometastatic tumors relative to

adjacent nontumor tissue. These estimates of tumor-specific

activity were compared with traditional estimates of the %ID/g

determined by well scintillation counting.

MATERIALS AND METHODS

Patient Selection. Patients in this study had undergone

prior surgery for epithelial ovarian cancer and had completed a

prescribed course of platinum-based chemotherapy. Eligibility

criteria included known or suspected carcinoma of the ovary, a

Karnofsky performance status greater than 60, no prior admin-

istration of murine mAb or fragment, and/or a negative human

antimurine antibody titer. Informed consent was obtained from

all patients before participation in the study; the study and

consent forms were approved by the Institutional Review Board

of Memorial Sloan-Kettering Cancer Center. Prior to participat-

ing in this trial, either paraffin-embedded tumor specimens or

fresh, frozen tumor specimens from an earlier surgery were

examined by immunohistochemistry for expression of the

MX35 antigen on at least 75% of the carcinoma cells. Tissue

specimens from six patients who participated in the mAb im-

aging trial prior to a second-look surgery are summarized in

Table 1 . Control antibody was not injected into patients for this

study.

Preparation of Radiolabeled mAb MX35 F(ab’)2.mAb MX3S, a murine IgGl, was generated from the hybridoma

fusion of NS- I murine myeboma cells with splenocytes from a

mouse immunized with four fresh ovarian carcinoma specimens

(13) and purified as described previously (14). For fragmenta-

tion, purified mAb MX35 IgG (2 mg in 400 jil) was dialysed

overnight in 0. 1 M citric acid buffer, pH 4.5. Pepsin (25 jib of 1.5

mg/ml; Sigma Chemical Co., St. Louis, MO) was then added to

the antibody and digested for 3 h at 37#{176}Cwith agitation. F(ab’)2

fragments were isolated using an Avid Chrom F(ab’)2 kit (Uni-

Syn Technologies Inc., Tustin, CA). High-yield binding buffer

(250 pA) containing 30 jil of antipepsin was added to the

antibody-pepsin combination. The entire sample was diluted

with 220 pA of high-yield binding buffer and centrifuged to 2000

rpm for 15 mm, and the supcrnatant was placed on a protein

A-Avid Chrom cartridge. The unadsorbed fraction was concen-

trated using a Centricon 30 unit (Amicon, Beverly, MA) at

1075 X g at 4#{176}C.The final antibody concentration obtained was

9.3-13.6 mg/mb. The identity ofthe fragments was confirmed by

SDS-PAGE under reducing conditions; staining of the gel with

Coomassie blue revealed bands of Mr 23,000 (light chains) and

Mr 25,000 (cleaved heavy chains).

The mAb MX35 F(ab’)2 fragments were radiolabeled with

iodine radionuclides using the chboramine-T method as follows:

two mg of antibody fragments were added to 0.5 ml of 0. 10 si

phosphate buffer, pH 7.4. To the radionuclides, 131J and 125J

were added 100 jib of phosphate buffer, and this solution was



Clinical Cancer Research 1435

added to the antibody fragment solution. Chboraminc-T (2 mg/

ml) in phosphate buffer was added, and after 2 mm, the reaction

was quenched by the addition of 50 jib of sodium metabisulfite

(10 mg/mb). The protein was separated by passage through a

Biogel P6 column (10 ml; Bio-Rad Laboratories, Melville, NY)

using 1 % human serum albumin in 0. 1 5 M NaCl as eluant.

Terminal sterilization was achieved by filtering through a 0.22

jim filter. Immunoreactivity of the labeled product was deter-

mined by sequential absorptions with an antigen-expressing cell

line (OVCAR-3). Between 50 and 65% of the radioactivity was

adsorbed using the method described by Mattes et a!. (14).

Percentage of labeled protein was determined by TLC, and

incorporation ofradiolabeled iodine into protein was >95%. All

procedures were performed aseptically with pyrogen-free

material.

Administration of Radiolabeled mAb MX35 F(ab’)2.

Beginning at least 24 h prior to antibody administration and

continuing to the time of surgery, patients were treated p.o. with

10 drops of a saturated solution of potassium iodide three times

daily. ‘31I1’25I-labeled mAb MX35 F(ab’)2 was administered by

an iv. route in a 0.9% sodium chloride solution containing 5%

human serum albumin (total volume, 100 ml) through a 0.2 jim

Millex G-V filter (Millipore, Bedford, MA) over a period of 1 h.

Radiolabeled mAb MX35 F(ab’)2 was administrated by an i.p.

route as follows: 500 ml of 0.9% sodium chloride were dcliv-

ered using a catheter or an existing i.p. port into the peritoneal

cavity to facilitate antibody distribution, 100 ml of radiolabeled

antibody were added in the same solution as the i.v. route, and

an additional 500 ml of 0.9% sodium chloride were delivered.

Five patients were entered at the 2-mg antibody dose labeled

with both ‘�‘i and 1251 Three patients were injected by an iv.

route and two patients by an i.p. route. One patient was entered

at the 10-mg antibody dose [2 mg of radiolabebed antibody plus

8 mg of unlabeled mAb MX35 F(ab’)2] and injected by an iv.

route.

Blood Samples and Tissue Biopsies. Blood was drawn

prior to radiobabeled mAb infusion, 1-4 h after infusion, during

surgery, and 4-7 days after surgery. Whole blood was centri-

fuged at <2000 rpm for 10 mm, and serum was aspirated and

stored at -20#{176}C.One ml of “surgery” serum was weighed and

then counted in a Packard Cobra well scintillation counter

(Packard Instrument Co., Douners Grove, IL) to compare radio-

labeled antibody levels in the blood with those in the biopsied

material.

Multiple biopsied specimens, including adjacent normal

tissue (fat, muscle, and peritoneal wall) were retrieved from six

patients during second-look surgery as summarized in Table 1.

Fresh surgical biopsies were divided as follows: one-half of

each specimen was paraffin embedded and used for routine

histological evaluation in our Department of Pathology. The

other half of each biopsy was weighed and counted in a Packard

Cobra well scintillation counter and then snap frozen in liquid

nitrogen, embedded in OCT compound (Miles Laboratory Inc.,

Elkhart, IN), and stored at -80#{176}C.A proportion of the frozen

surgical biopsies from each case were cut using a motorized

cryostat (Bright Instrument Co., Huntingdon, England) and air

dried onto microscope slides. Adjacent tissue sections (6-jim

thickness) from each biopsy were then analyzed for MX35

antigen localization using an indirect immunoperoxidase proce-

dure and for radionuclide distribution by autoradiography using

film and storage phosphor screens.

The number of cpm was obtained in two windows centered

at 25 keV for 1251 and 364 keV for ‘�‘I. The cpm was converted

into activity by measuring 1251 and ‘�‘I standards alongside the

tissue specimens. The %ID/g for each radionuclide was deter-

mined for the serum and each tissue biopsy by dividing the

specific activity (i.e., jiCi/g) by the activity administered to the

patient and multiplying by 100.

Immunohistochemical Analysis and MX35 Antibody

Localization. Frozen tissue sections were fixed with cold

acetone and analyzed for reactivity with mAb MX35 as de-

scribed previously (15). Antibody staining patterns were scored

in a semiquantitative fashion. Specimens were classified as

showing strong ( + + + ) antigen expression when 75% or more

of the tumor cells were stained; heterogeneous ( + to + +)expression with variable intensity when 10-75% of the tumor

cells stained; and no expression when negative or less than 10%

of the tumor stained.

In a xenograft murine model, nonspecific, control antibody

L6 was injected for comparison with mAb MX35, both intact

and F(ab’)2. In the animal studies, mAb MX35 (and not mAb

L6) targeted specifically to MX35-positive OVCAR-3 human

ovarian cancer cells, growing as tumors in the mice. In this

clinical trial, a nonspecific, control antibody was not included

for ethical reasons.

Film Autoradiography for Distribution of Radionuclide

Activity. Autoradiography was performed using Kodak X-

OMAT AR film (Eastman KOdak, Rochester, NY). Tissue sec-tions were covered with Saran wrap and overlaid with film alone

or with an enhancer screen for exposure durations of between 1

and 14 days. Films were developed in a Kodak RP X-OMAT

processor (Eastman Kodak). Film images were digitized using

the Nikon Coobscan (Nikon Electronic Imaging, Melville, NY)

and compared to the digitized 35-mm Ektachrome film images

of adjacent tissue sections immunostained with mAb MX35.

Coregistration of the serial sections permitted visualization of

the radiolabeled antibody activity over regions of tumor cells

and nontumor tissue.

Autoradiography Using Storage Phosphor Screens forDistribution of Radionuclide Activity. For the analysis of

storage phosphor screen images, either a model GS-250

(100-jim pixel resolution) or model GS-350 (85-jim pixel res-

olution) Molecular Imager system (Genetics Systems Division,

Bio-Rad Laboratories Inc., Hercules, CA) connected to a Macin-

tosh Quadra 840AV computer (Apple Computers Inc., Cuper-

tino, CA) was used. The storage phosphor screen (type BI) is

fabricated from strontium sulfide commingled with elemental

cerium and samarium (16). The interaction of ionizing radiation

with the storage phosphor screen excites electrons into the

conduction band, from which they fall into electron traps. Quan-

titation of the number of filled traps is proportional to the

amount of energy deposited in the screen. The detector signal is

read out using an externally applied scanning laser (pulsed IR

diode at 910 nm), which scans the screen, releasing the electrons

from their traps pixel by pixel. The nominal resolution is de-

limited by the focal spot of the laser. The scan itself possesses

a greater intrinsic resolution. The process of electron de-excita-

tion results in the emission of fluorescent photons, which are




collected in a fiber optic pipe and counted using a photomulti-

plier tube. The resultant signal is processed using an analogue-

to-digital converter, which provides the final 10-16-megabyte

image at 16 bits/pixel. The phosphor image data were analyzed

on a Power Macintosh 7100/66 (Apple Computers, Inc.) using

Molecular Analyst/Macintosh (version 2.0) data analysis soft-

ware (Bio-Rad Laboratories, Inc.) and the public domain NIH

Image (Version 1.55) program (written by Wayne Rasband,

NIH), which is available from the Internet.

Tissue sections were covered with Saran wrap and then

clamped against a storage phosphor screen in a light-tight cas-

sette for a preliminary exposure duration of 24 h and a second

exposure duration of 4-16 days. The storage phosphor screen

was erased after image readout by exposure to IR light for 15

mm and erased a second time immediately before exposure to

the samples.

Calibration of Storage Phosphor Screen Using Radio-

nuclide Standards. A storage phosphor screen image consists

of a two-dimensional array of intensities. The conversion of

these intensities into specific activity units requires calibration

of the storage phosphor screen response. The screen response

was analyzed with three sets of radiolabeled standard sources.

Strips containing graded standards of ‘4C (RPA.504 and

RPA.5 1 1) and 251 (RPA.523) for autoradiographic calibration

were purchased (Amersham Laboratories, Buckinghamshire,

England). The commercial 1251 standards embedded in polymer

were quoted at 20-jim tissue equivalent thickness. A set of 1311

standards was made by dilution of a stock solution containing a

known activity of ‘�‘1 as follows. Eosin Y (1% alcoholic solu-

tion; Polysciences, Inc. Warrington, PA) was added to the

radioactive solution, which was then mixed with the OCT com-

pound until a uniform coloration was achieved. The samples

were weighed and counted, and the relative specific activities of

the two blocks were determined to be 0.536 jiCilg and 4.680

jiCi/g, respectively. The two 311 standards were sectioned at

6-jim thickness and dried onto microscope slides in the same

way as the tissue sections prior to exposure to the screen for

24 h. The response of the phosphor screen was determined to be

1814, 5352, and 1003 cpd per pixel for 1311 1251 and 14C,

respectively, for a source of I jiCi/g specific activity. The

higher sensitivity of the storage phosphor screen means that

exposure times are typically between 5 and 10 times shorter than

film for the same image quality (17). The ‘4C standards were

placed alongside the iodine standards to evaluate the constancy

of the storage phosphor screen over a long period.

The phosphor screen images were analyzed retrospectively

for all patients after ascertaining the phosphor screen response,

i.e., cpd per pixel per unit specific activity, as a function of

exposure duration from standards. 1311 251 and ‘4C standards

were exposed alongside sections of both normal and tumor

tissues for patients S and 6. This allowed the unknown activity

distribution in the biopsied specimen to be determined by direct

scaling from the known activity of the standards and the ratio of

the radionuclide response. The validity of our approach to

patients 2, 3, and 4, who were studied prior to the simultaneous

exposure to standards, was verified by estimating the specific

activity for patients 5 and 6 using both methods. The ratios

between the values obtained by the new method (involving

simultaneous exposure to standards) to the previous method

(applying the known response and fade characteristics of the

phosphor screen) were 0.86 and 0.95 for cases 5 and 6, respec-

tively.

The storage phosphor screen exhibits a slow signal fade

during the signal acquisition time. Thus, for each sample expo-

sure time, it was required to convolute the signal accumulation

with the signal fade. The fade characteristics were determined

by repeatedly exposing the screen to the standards for the same

1-h duration and varying the interval before reading the screen,

from immediate up to 14 days. The correction factor to account

for signal fade (F) for a specimen exposure of duration t� is

given by the convolution integral f�e�’ . e5’��dT, where XF

is the rate of signal loss attributed to fade and X� is the physical

decay constant for the radionuclide. This integral accounts for

the variable amount of fade during the continuous phosphor

screen exposure; i.e. , for an 8-day exposure, the counts on day

I fade for 7 days, those on day 2 fade for 6 days, and so on.

Solving the integral, one obtains the following:

F = eXPt�[I _e_��j/(XF _X�)

The rate constant XF for phosphor screen fade was found to

be 0.0967/day. Inserting the decay constant X� for 1311 the

above equation becomes

F = e#{176}#{176}862“[1 - e#{176}#{176}’#{176}5��]/0 0105,

where t0 is the time in days the phosphor screen is exposed to an

I 3 ‘I-labeled specimen.

Estimation of Dosimetry using Storage Phosphor

Screens. The radiation dose is directly proportional to the

cumulative specific activity of the radiolabelcd antibody in the

tumor. Autoradiographic images provide information about the

activity in the tissue at only a single time. Because these patients

only had minimal residual disease, it was not possible to obtain

tumor clearance data from nuclear medicine gamma camera

scans. Therefore, the storage phosphor screens were only able to

accurately measure the specific activity of the radiolabel in the

tumor microdeposits relative to the surrounding normal tissue or

nontumor specimens. Parallel studies, performed with the same

radiolabeled ‘31I-labeled mAb MX35 F(ab’), in a human ovar-

ian cancer xenograft model (OVCAR-3), showed a biological

half-life (Tb) of 15.5 h for both i.v. and i.p. routes of injection

( 18). This half-life is reasonable for F(ab’), antibodies directed

against secreted antigens, as evidenced by biological half-lives

reported in patient trials after i.p. injection with other radiola-

beled mAbs; e.g., Tb = 21 h for ‘ ‘ ‘In-mAb OCl25 (19), and

Tb 14 h for ‘86Re-mAb NR-CO-02 (20). The mAb MX35

binds to a cell-surface antigen, and it may be that the short

biological half-life observed in animal studies is the result of

dciodination. Our clinical data suggest a similar clearance in

patients. One patient (case 4) was biopsied after only IS h,

compared to the other five patients. On the basis of the ratio of

the %ID/g for case 4 relative to cases I , 3, and 6, which were

biopsied at 4 days, we calculated an approximate Tb of 18.4 h

for the patients studied. The similarity of this value with data

reported by other investigators shows internal consistancy with

our patient data. Unfortunately, true clearance data from micro-

scopic tumor cell foci are not currently possible in situ. Assum-




locally.

Table 2 Summary of %ID/g in tumor, normal tissue specimens, and serum for radiolabeled mAb MX35 F(ab’)2 by well scintillation counting ofwhole biopsies”

%ID/g for ‘31b-labeled mAbMX35 F(ab’),’

%ID/g for ‘25I-labebed mMX35 F(ab’),’

Ab

Tumor:Serum Tumor:Serum Tumor:Fat’ Tumor:Fat”Normal tissue Serum”(Xl03) (fat) (XlO3) I3l1 1251 ‘�‘I 1251

0.21-0.75 (0.21) 2.86 0.28-0.70 0.29-0.64 3.82-8.96 3.92-8.78

0.34-1.02 (0.34) 2.05 2.75 2.89 18.21 17.42

0.28-0.32 (0.32) 1.95 0.15-0.39 0.15-0.38 0.87-2.49 0.88-2.28

l.13-6.90(l.l3) 3.76 5.05 5.61 17.69 18.73

0.16-1.33 (0.16) 1.63 3.73 3.91 35.89 38.96

0.38-0.58 (0.47) 1.75 0.37-0.58 0.37-0.63 1.40-2.20 1.40-2.36

Normal tissue Serum” Tumor(XlO3) (fat) (XlO3) (XlO3)

0.35-1.26 (0.35) 4.82 0.82-1.83

0.54-1.71 (0.54) 3.17 5.89

0.49-0.53 (0.53) 3.41 0.29-0.74

1.27-7.27(1.26) 4.42 21.09

0.22-1.72 (0.22) 2.14 6.36

0.52-0.70 (0.59) 2.25 0.65-1.10

(‘ Two mg of mAb MX3S F(ab’) were labeledwith 31I and 1251 (cases 1-5). In case 6, radiolabeled mAb was mixed with 8 mg of cold mAb

MX3S F(ab’),.h mAb was delivered by iv. route (cases 1-3 and 6) or by i.p. route (cases 4 and 5) between 15 hours to 5 days prior to surgery.

‘ The amount of radionuclide activity administered per patient ranged from 2 to 15 mCi for ‘�‘I and 2 to 5 mCi for 251,I Whole blood removed during surgery was centrifuged, serum was aspirated, and I ml of serum was weighed and counted in a well scintillation

counter... Tumor:fat ratios are presented because fat (values in parentheses in columns 4 and 7) was the only tissue consistently available for all six cases

examined. The range of normal tissue data could be used to calculate the tumor:average normal tissue ratios.

CaseTime of

surgery”Tumor

(X l0�)

I Day4 1.35-3.18

2 DayS 8.71

3 Day 4 0.52-1.334 15 h 22.35

5 Day 5 7.986 Day4 0.83-1.30

ing the same biological half-life for patients and animals would

result in a T� [Te TbTp/(Tb + Tn)] of 14.3 h for 131i and 15.3 h

for 1251 and the physical half-life (Tn) is 8.04 days for ‘�‘I and

60 days for 1251.

In this clinical trial, the principal radionuclide delivered

was either 1251 (cases 1 and 2) or 1311 (cases 3-6), and the

calculations that follow estimated the specific activity in micro-

metastases for the appropriate radiolabeled mAb MX3S F(ab’)2.

To estimate radiation dose to micrometastatic foci required acalculation of the cumulative specific activity (area under ac-

tivity-time curve). Therefore, the activity versus time must be

back extrapolated from measurements of the whole biopsy, to a

time t,�. Using the assumption of a monoexponentiab clearance

rate C0 = C(t)e�”, where C0 and C(t) are the specific activity

in jiCilg at the time of mAb infusion and surgery, respectively,

t is the time interval, and X = 0.693/Te. In this study, we

estimated the doses based on an assumed Tb of 1 5.5 h, corre-

sponding to the murine data (18), and a Tb at 24 h, a simplified

average from other clinical trials (4-8). These values were to

bracket the range of possible doses and to show the strong

dependence of these estimates upon the assumed Tb. The radi-

ation absorbed dose is given by the integral of the specific

activity Cofe�T� over time multiplied by 2.13 �n1E141. The

second term consists of the product between the total energy

(In1E1) released by radionuclide emission, the fraction of emis-

sion energy absorbed within the tumor (4), and 2.13, which is a

unit conversion factor. For 131j, the sum of the 13-ray energy

emitted per decay (�n1E1 = 0.187 MeV) multiplied by 2.13

equals 0.398 g-cGy/jiCi-h. For nonpenetrating radiations, such

as 13-particles, it is recommended by the MIRD committee (21)

to use unity for the absorbed fraction 4. For a micrometastatic

deposit containing less than I g of tumor cells, the value of 4 is

less than 1 . The absorbed fractions for several radionuclides,

including ‘�‘I are published by Humm (22) and Goddu et a!.

(23). For a 100-jim micrometastatic lesion, � is equal to 0.17;

i.e., 17% of the energy emitted within the lesion is deposited

RESULTS

From a study evaluating the localization of radiolabebed

mAb MX35 F(ab’)2 in patients with ovarian carcinoma, speci-

mens were taken during second-look surgery after antibody

administration 1-5 days earlier. The activity determined by well

scintillation counting of the whole-tissue specimens was com-

pared with storage phosphor screen autoradiography of tissue

sections (24). Biopsied specimens from six patients were ana-

lyzed (Table 1). All tumors expressed MX35 antigen as deter-

mined by immunohistochemical analysis. In total, 19 normal

tissue biopsies and 16 biopsies containing tumor cell foci were

evaluated in the laboratory.

Determination of Specific Activity of Radiolabeled An-tibody in Whole-Tissue Biopsies Using Well Scintillation

Counting. The %ID/g was calculated for the whole biopsyspecimens and serum sample for each case (Table 2). The

%ID/g for biopsies with tumor ranged from 0.5 to 8.7 X l0��

(for 1311 calculations) and from 0.3 to 6.4 X l0� (for 1251

calculations) for samples analyzed immediately after surgery,

i.e., between 4 and S days post-antibody administration. The

%ID/g for a single tumor sample studied 15 h post-antibody

infusion was 22 x l0�. The tumor:scrum ratios ranged from

0.2: 1 to 2.8: 1 in the patients receiving antibody by the iv. route

and from 3.7:1 to 5.6:1 by the i.p. route. The tumor:normal

tissue (fat) ratios ranged from 0.9: 1 to 39: 1 . The tumor:normal

tissue ratios were greater in the two patients (cases 4 and 5)

receiving antibody via the i.p. route. The percentage of tumor

cells within the biopsy specimens was variable, ranging from

<10% to >75% ofa sample (Table 1). In two ofsix cases (cases

2 and 4), greater than 50% of the biopsy consisted of tumor foci,

and in these cases, the tumor:normal tissue ratios were signifi-

cant (18:1). In three of six cases, less than 20% of the biopsy

consisted of tumor foci, and the tumor:normal tissue ratios were

in the range between 0.9: 1 and 8.9: 1 . One specimen (case 5)

with < I 0% tumor cells in the biopsy had the highest tumor:

normal tissue ratio, 36: 1.



A �

.�‘.

a C d

g0 #{149}_-� f

t:�...

b

I

C

4�

d

*

r’1�

:L1.�#{248}, .!�1_b7

C

t,

�

t��i;

d

-�

.�., ‘:‘i.., .� �%

� �

:�i � �

� � : .�

;-.� � #{163}m��

� ____I

.‘�*


B�: �-.: �

‘� �, 1141�8

. � ,� �. .P � . � 4

� .; � .“ � , � .:‘ �

e.t�

� .� .. - �

�

C

,.

:: � � � �. � ‘ -#{246}�.

� ..

Fig. I A, tumor biopsy from patient 2 with a serous ovarian carcinoma following administration of radiolabebed mAb MX35 F(ab’)2 (5 days earlier)

by an iv. route. Indirect immunoperoxidase staining with mAb MX35 of carcinoma at high power (a: bar, 100 jim) and bow power (b; bar, 2 mm).

Adjacent tumor sections of digitized film image at low power (c) and storage phosphor screen image at low power (6). Note the strongimmunoperoxidase staining of tumor cell clusters and colocabization of radiolabebed mAb to the tumor. In contrast, immunoperoxidase staining of thenormal penitoneal wall biopsy at low power (e; bar, 2 mm) and uptake of radiolabeled mAb on the corresponding digitized film image (f) and storagephosphor screen image (g) were not detected. B, para-aortic lymph node from patient 5 with a poorly differentiated ovarian carcinoma following

administration of radiobabebed mAb MX35 F(ab’), (5 days earlier) by an i.p. route. Indirect immunoperoxidase staining with mAb MX35 at highpower (a; bar, 100 p.m) and low power (b; bar, 2 mm). Adjacent tumor sections of digitized film image (saturated) at low power (c) and storagephosphor screen image at low power (d). Note the distribution of immunostained tumor cells between the hematopoietic cells and stromal tissue (b,

arrowhead) and localization of radiolabebed mAb to the tumor cell area specifically. C, tumor cell clusters in ovary (a-d) and fallopian tube (e-h)



4’

Phosphor screen Image

12 13 14 15 16

distance (mm)


0

0U,C00.‘I,0

Fig. 2 Determination of specific activity of radionuclide within tumor

specimens from patient 4 with an endometrioid ovarian carcinoma

following administration of radiolabeled mAb MX3S F(ab’)2 (I day

earlier) by an i.p. route. Three line profiles were drawn to traverse

regions of micrometastatic tumor foci that impregnate the regions of

normal stromab tissue (left, a-c). The locations of the profiles areindicated by lines in the tumor biopsy (right, a-c). The ordinate is in

units of cpd, which can be converted to p.Ci/g by dividing by the

conversion factor 1841 cpd/p.Ci/g. Bar, 2 mm.

Immunohistochemical Delineation of Areas of TumorCells in Tissue Sections and Comparison with Film and

Storage Phosphor Screen Autoradiography. The autoradio-

graphs from X-ray film and storage phosphor screens provide an

image of the distribution of radionuclide devoid of its relation to

the histology of the tissue section. Coregistration of phosphor

images and/or digitized film images with the digitized 35-mm

Ektachrome film images of tissue sections, stained by immu-

noperoxidase using mAb MX35 for antigen localization, al-

bowed visual assessment of the efficacy of radiolabebed mAb

targeting. Film images of tumor and normal tissue sections were

available for six cases. Clusters of tumor cells were detected

clearly by film visualization in five of six cases; in the other

case, single tumor cells and small clusters of < 10 tumor cells

were detected weakly by film (case 3). Phosphor screen images

were analyzed for tumor and normal tissue sections in five

cases; single tumor cells and small tumor cell islets did not

image clearly in case 3.

Analysis of adjacent tissue sections confirmed the coinci-

dence of the radiolabeled mAb MX3S F(ab’), to regions of

MX3S-positive tumor cells. Fig. I illustrates the results from

three cases comparing film with storage phosphor screen im-

ages. Fig. IA illustrates a small laparoscopy specimen (case 2)

with >80% of the specimen having strong MX3S antigen cx-

pression on the scrous-type ovarian carcinoma cells at high

power (Fig. lA, ci). The unstained regions represent stromal

tissue in which tumor cell foci are embedded. Coregistration of

the image from a tumor section immunostained for antigen

localization (Fig. IA. b) with both the film autoradiographic

image (Fig. 1A, c) and the phosphor screen image (Fig. 1A, d)

shows specific targeting in the areas of radiolabeled mAb ac-

cumulation. The immunoperoxidasc-stained antigen localization

shows a variable intensity within the tumor cell foci, and this

may account for an apparent heterogeneity of the autora-

diograpic images. Sections of the adjacent peritoneal wall bi-

opsy are negative (Fig. IA, e-g).

Fig. lB illustrates a para-aortic lymph node specimen (case

5) with a micrometastatic tumor cell cluster from a poorly

differentiated ovarian carcinoma detected by immunostaining at

high power (Fig. IB, a) and low power (Fig. lB. b, arrowhead�

surrounded by hematopoietic cells. The film image has saturated

at the 7-day exposure (Fig. lB, c). The phosphor screen image

has a greater dynamic range and therefore does not saturate (Fig.

lB, d). The 1-day film and phosphor screen images were less

intense and showed radionuclide localization only to the specific

clusters of tumor cells as detected by immunostaining in Fig.

IB, b (data not shown).

Fig. 1C illustrates an endometrioid-type carcinoma of the

peritoneum (case 6). A micrometastatic tumor cell cluster was

found near the surface of the ovary (Fig. I C, a-d), and multiple

clusters of tumor cells were detected adjacent to the fallopian

tube epithelium (Fig. 1C, e-h). The small lesion in the ovary

(Fig. 1 C, b, arrowhead) is targeted specifically by radiolabeled

mAb MX35 F(ab’)2, as seen in the autoradiographic film and

phosphor screen images (Fig. I C. c and d). Note that the

fallopian tube epithelium expresses MX35 antigen along the

apical surface (Fig. IC, e andf), but the radiolabeled antibody

localizes preferentially to the tumor cell clusters (Fig. I C. g

and Ii).

Determination of Specific Activity of Radiolabeled An-tibody within Tissue Sections Using Storage Phosphor

Screen Analysis. Line profiles were drawn so as to traverse

regions of micrometastatic tumor cell foci, which impregnate

the regions of normal stromal tissue. The variation in cpd per

pixel was relatively uniform throughout normal tissue and in

areas of stromal tissue in biopsies containing tumor but exhib-

ited “peak” values at spatial positions corresponding to tumor

cell foci, as confirmed by immunoperoxidase staining. For small

tumor cell clusters, the peak values are averaged, and for biop-

sies with multiple, discrete tumor cell foci, a range of values is

quoted corresponding to different regions or different tissue

sections. Average pixel values are quoted for normal tissues.

Ratios of the “peak” response (converted to cpd) overlying the

MX35-positive tumor deposits relative to the normal stromal

tissue background can differ significantly, as illustrated in Fig.

from patient 6 with an endometnioid-type carcinoma of the peritoneum following administration of nadiolabebed mAb MX3S F(ab’), (4 days earlier)

by an iv. route. Indirect immunoperoxidase staining with mAb MX35 at high power (a and e; bars, 100 �j.m) and low power [h (bar, 5 mm) andf

(bar. 2 mm)]. Adjacent tumor sections of digitized film images at low power (c and g) and storage phosphor screen images at low power (d and h).

The location of high-power photographs (a and e) is indicated in low-power photographs (b and ft arrowheads). Note that both the fallopian tube

epithelium and tumor cell clusters show strong immunopenoxidase staining. However, the radiobabeled mAb localizes to the tumor cell clusters on

the film and phosphor screen images and not the adjacent normal tube epithelium.




Table 3 Comparison of p.Ci/g values and tu mor:normal tissue (T:N) ratios from biopsied sample

storage phosphor screen response

s analyzed by well scintillation counts versus

- Case Biopsy site”

. Well counts”Timeofsurgery jiCi/g T:N

Phosphor screen response’-_____________________

jiCi/g T:N

1 Omentum (T) Day 4 0.090 3.4:1 NA NA

Muscle (N) 0.027 NA

2 Diaphragm (T) Day 5 0.309 13.4:1 0.265 10.2:1

Abdominal wall (N) 0.023 0.026

3 Omentum (T) Day 4 0.142 2.7:1 0.184 2.0:1

Muscle (N) 0.052 0.0924 Cub de sac (T) 15 h 2.429 3.1:1 23.116 8.7:1

Apex (N) 0.790 2.6705 Lymph node (T) Day 5 1.197 5.5:1 4.179-6.082 17.8-19.7:1

Normal node (N) 0.2 18 0.212-0.341

6 Penitoneum (T) Day 4 0.228 1.9:1 1.645-1.849 7.2-8.0:1

Muscle (N) 0.123 0.206-0.258

(‘ The same biopsied samples were evaluated by well scintillation counts (whole specimen) and storage phosphor screen response (tissuesections). T, biopsy site containing tumor: N, normal tissue biopsy adjacent to tumor.

I, Well scintillation counter measurements represent total activity per gram from the entire biopsy specimen.‘ Storage phosphor screen responses disassociate counts arising from nadiolabebed antibody localized in tumor cell foci from stroma. The

variation in counts pen pixel was relatively uniform throughout stromal tissue. Average pixel values are quoted for the normal tissues. Peak pixelvalues are given for small tumor cell clusters. For some tumor specimens. a range of values could be quoted corresponding to different regions or

different biopsy specimens. NA, not available.

2 from case 4. Each profile in counts per pixel was converted to

local specific activity in jiCi/g from the phosphor screen re-

sponse determined by standards. Table 3 compares the specific

activity (expressed as jiCi/g) for mAb MX35 F(ab’), in the

same tumor biopsy samples and adjacent normal tissues, meas-

ured by storage phosphor screens (tissue section) and well

scintillation counter (whole specimen). Estimates of the tumor-

specific activity, expressed as jiCi/g from the storage phosphor

screens, were compared with measurements of the whole biop-

sied specimen from a well scintillation counter. The jiCi/g

values for normal tissue ranged from 0.023 to 0.218 by well

counts and from 0.026 to 0.341 by phosphor screen response for

samples evaluated 4 or 5 days post-antibody injection. In con-

trast, jiCi/g values for corresponding tumor biopsies ranged

from 0. 142 to I . 197 by well counts and from 0. 184 to 6.092 by

phosphor screen response. Similar jiCi/g values for tumor bi-

opsies were estimated by both well counts and phosphor re-

sponse methods for case 2 with >80% tumor cells in the sample

and for case 3 with small clusters and individual tumor cells

dispersed throughout the sample. However, jiCi/g values were

significantly higher by the phosphor screen response method in

samples with distinct tumor cell foci (cases 5 and 6), as illus-

trated in Fig. I , B and C. The tumor:normal tissue ratios were

between I and 4 times greater using the storage phosphor screen

method than well counter measurements (Table 3), but even

larger variations of ratios up to 20: 1 were observed between

tumor cell foci and hematopoictic cells from within the same

tissue section (Fig. IB). This is a consequence of the ability of

the phosphor screen to disassociate counts arising from radio-

labeled antibody localized in tumor cell foci from the adjacent

stroma and to accurately measure high activity tumor foci within

a specimen rather than to use a single average value on the

whole specimen from well counter measurements.

Estimation of Dosimetry within Tissue Sections Using

Storage Phosphor Screen Analysis. Radiation dose esti-

mates for mAb MX35 F(ab’)2 in the tumor were made assuming

hypothetical Ths of 15.5 h (from animal data) and 24 h, respec-

tively. The estimated dosimetry using a Tb of 15.5 h would

result in radiation absorbed doses of 465, 1 10, 554, 10663, and

I 109 cGy/mCi injected for cases 2, 3, 4, 5, and 6, respectively,

assuming the usual absorbed fraction of 1 for the 13-particle

emissions of 1311 However, for isolated microscopic volumes,

these doses can be significantly reduced due to the boss of

13-particle flux from the tumor cell cluster. Also note that the

projected tumor doses are very sensitive to small changes in the

assumed biological half-life. For example, when Tb = 24 h, the

absorbed doses would be 104, 72, 564, 2375, and 361, respec-

tively, for the same patients. These dose estimates appear cx-

cessively high compared to values reported from other radiola-

beled mAb clinical trials (25, 26). Although these values could

be the consequence of an incorrect assumption for Tb in esti-

mating the cumulative specific activity, this study differs from

others, because we are quantifying the activity per gram in

microscopic tumor cell volumes. Furthermore, the dose depos-

ited in small microscopic foci of disease depends on the ab-

sorbed fraction of energy emanating from the high-energy

13-particles of ‘�‘I, which is deposited locally (22). The fraction

of 1311 13-ray energy absorbed locally within a 100 jim-diameter

lesion is 0. 1 7 (23). This would reduce the dose per mCi esti-

mates to the micrometastatic foci to 0. 17 times the values

presented above. The jiCi/g determined by the well counter and

storage phosphor screen techniques arc summarized in Table 3.

DISCUSSION

In this study, we evaluated the radiolabeled antibody up-

take ofthe murine mAb MX3S F(ab’)2 in biopsied samples from

patients with epithelial ovarian cancer by well scintillation

counting and by autoradiography using storage phosphor

screens. Specific localization of mAb in tumor was demon-

strated by coregistration of the immunohistochemical staining in

areas of tumor cell clusters with autoradiographic film and




phosphor screen images. In all specimens with micrometastatic

spread, radiolabeled mAb uptake showed specific localization to

the carcinoma cells (Fig. I ). Factors other than antigen distri-

bution are involved in the localization of radiolabeled antibody

in tissues. In this study, we noted that antibody localized to

tumor cell foci but did not accumulate in the antigen-positive,

adjacent normal fallopian tube epithelium (Fig. 1C) in the tissue

sample. In this case, accessibility of the antibody to the luminal

side of the ducts may be limited.

The radiolabeled antibody uptake determined by well scm-

tillation counting (1-5 days post-mAb infusion) ranged between

5.2 and 223.5 x l0-� %ID/g of tumor tissues for ‘�I and

between 2.9 and 210.9 X I0� %ID/g for 251 There was a

general relationship between the radiolabeled mAb uptake in the

tumor biopsy and both the level and the intensity of the immu-

nohistochemical expression of the MX35 antigen in the corre-

sponding tumor tissue section. Specific localization of mAb in

tumor was demonstrated by tumor:normal tissue (fat) ratios

rangingfrom0.9:l to35.9:I for ‘3’Iandfrom0.9:l to39.0:l for

251 Significantly higher tumor:normal tissue ratios were cal-

culatcd for the two patients given radiolabeled antibody by the

i.p. route (e.g., 17.7:1 and 35.9:1 for 1311) These results can be

compared to an earlier clinical trial in which mAb MX35 whole

IgG was used (8). In that study. tumor samples obtained atsurgery (7-20 days post-mAb infusion) showed a mAb accu-

mulation of between 0.3 and 67.0 X l0�� %ID/g of tissue, and

the tumor:normal tissue (fat) ratios ranged from 2.3: 1 to 34.4:1.

The tumor:normal tissue ratios were not related significantly to

mAb dose, the level of immunohistochemical antigen expres-

sion, or the interval between mAb infusion and surgery (8).

Also, in contrast to the present study, tumor:serum ratios rarely

exceeded 1.0 (8).

The storage phosphor screen enabled radiolabeled antibody

to be quantified directly relative to tissue histology and immu-

nohistochemistry. The radionuclide uptake evaluated 4 or 5 days

post-antibody injection ranged from 0.03 to 0.34 jiCi/g for

normal tissues, from 0.18 to 0.27 jiCi/g in small tumor cell

clusters dispersed throughout the tissue (cases 2 and 3), and

from I .65 to 6.08 jiCi/g in discrete tumor cell foci by the

phosphor screen technique. In contrast, a single average value

ranged from 0.02 to 0.22 jiCi/g for normal tissues and from 0.09

to 1 .20 jiCi/g in whole, biopsied specimens containing tumor by

well scintillation counts. The tumor:normal tissue ratios were

between I and 4 times greater using the phosphor screen method

than well counter measurements, but even larger variations of

ratios were observed between tumor cell foci and stromal cells

from within the same tissue section. In this study, the ovarian

tumors grew as micromctastatic clusters of cells within loose

connective tissue or lymphatic spaces, making them accessible

targets for antibody uptake. In an earlier study using radiola-

beled-mAb MX3S IgG (8), solid ovarian tumors showed auto-

radiographic film images with localization confined to the outer

rim of viable tumor or within areas of necrotic tumor tissue,

indicating a problem with antibody penetration. In sum, these

observations suggest that the specific activity may be more

accurately measured in specimens with microscopic disease.

Our analysis provided a measure of the specific activity of

biopsied specimens at one single time point, namely, at the time

the specimen was removed and frozen. Because there was no

means to determine the specific activity and microdistribution of

the radiolabeled antibody before this time, we performed hypo-

thetical calculations of the radiation dose based on assumed

effective half-lives for the ‘311-labcbed mAb MX35 F(ab’), of

15.5 h (18) and 24 h. Absorbed dose estimates to microscopic

tumor cell clusters must also correct for the fraction of local

energy absorption. Such corrections to the absorbed dose are

possible with the storage phosphor screen approach because it

can account for microscopic variations in local energy deposi-

tion, which are averaged by well scintillation counting methods.

To improve the radiation dose estimates presented here, it

would be necessary to determine the specific activity of the

radiolabeled antibody in the tumor at multiple time points. This

information is not available for microscopic disease. Griffith et

a!. (27) proposed a method to implant thermoluminescent do-

simeters, mounted at the tip of a catheter into tissue, to directly

measure the radiation dose in situ. However, this method would

not be readily applicable to microscopic disease, due to the

uncertainty of the location of the tumor cells. A method using

PET to quantitatively assess mAb localization in suit has been

developed and tested with ‘241-babeled mAb 3F8 in a patient

with glioma (28). High-resolution PET has been used to localize

human ovarian cancer in nude rats using 24I�labeled mAb

MX35 (29). The high sensitivity of PET may allow the detection

of microscopic disease provided the tumor:background ratio is

sufficient to produce an adequate image contrast. However, the

resolution of current PET scanners in the abdomen is not better

than 4 mm (General Electric Advance PET Scanner, Mibwau-

kee, WI).

When an antibody can be shown convincingly to localize to

micrometastatic tumors, cpithebial ovarian cancer affords an

ideal opportunity to use the antibody or antibody conjugates for

therapy. The specific targeting of mAb MX35 F(ab’)-, to micro-

metastatic disease as shown in this study demonstrates the

potential of this radiolabebed antibody conjugate for such a

therapeutic trial. The ability to target minimal residual disease

embedded in tissue stroma may be a significant rationale for

treating patients with refractory or recurrent ovarian cancer with

radioimmunotherapy. Small tumors may be more uniformly

accessible to mAb and will require bower doses of radiation to

eradicate than bulky disease. Ovarian cancer often spreads su-

perficially on the surface of the peritoneum, where it forms

small tumor foci within the peritoneal cavity. Extraperitoneal

metastases, other than spread to local lymph nodes, are rare.

Administration of antibody through an i.p. route, therefore,

provides an optimal mode for the treatment of this disease. An

additional advantage of i.p. administration is that, although

small tumors may be undervascubarized, they will still be ac-

cessible to radiolabeled antibody by diffusion from the penito-

neal fluid.

This study has demonstrated (Figs. I and 2) that mAb

MX35, in its F(ab’)., form, localizes avidly to the micrometa-

static ovarian carcinoma deposits within the peritoncal cavity.

Well scintillation counter techniques may underestimate the

radiation dose to microscopic tumor cell clusters, because the

activity per gram is averaged uniformly over tumor and stromal

cells. Storage phosphor screens offer a means of measuring the

accumulation of radiolabeled antibody activity within small

tumor cell foci more accurately. Preliminary dose estimates




performed in this study suggest a therapeutic potential for the

antibody in patients with minimal residual disease.

ACKNOWLEDGMENTS

We thank Dr. Lloyd J. Old, Elizabeth C. Richards, and Mary John

of the Ludwig Institute for Cancer Research, New York Branch, at

Memorial Sloan-Kettering Cancer Center for preparing the clinical

grade mAb MX35 used for this antibody-imaging trial.

REFERENCES

1 . Rubin, S. C. Second-look baparotomy in ovarian cancer. In: M.Markman and W. J. Hoskins (eds.), Cancer of the Ovary, pp. 175-185.New York: Raven Press, 1993.

2. Rubin. S. C.. Hoskins, W. J., Hakes, T. B., Markman, M., Cain, J. M.,

and Lewis, J. L., Jr. Recurrence after negative second-book laparotomy

for ovarian cancer: analysis of risk factors. Am. J. Obstet. Gynecol.,159: 1094-1098, 1988.

3. Granowska, M., Shepard, J., Britton, K. E., Ward, B., Mather, S.,Taybon-Papadimitriou, J., Epenetos. A. A.. Carroll, M. J., Nimmon,C. C., Hawkins, L. A., Slevin, M., Flatman, W., Home, T., Burchell, J.,Durbin, H.. and Bodmer, W. Ovarian cancer: diagnosis using 23!

monocbonal antibody in comparison with surgical findings. Nucb. Med.

Commun., 5: 485-499, 1984.

4. Epenetos, A., Shephard, J., Britton, K. E., Mather, S., Taybor-Papad-imitnou, J., Granowska, M., Durbin, H., Nimmon, C. C., Hawkins,

L. R., Malpas, J. S., and Bodmer, W. F. 231 radioiodinated antibody

imaging of occult ovarian cancer. Cancer (Phila.), 55: 984-987, 1985.

5. Pateisky. N., Philipp, K., Skodler, W. D., Czerwenka, K., Hamilton,

G., and Burchell, J. Radioimmunodetection in patients with suspectedovarian cancer. J. Nucb. Med., 26: 1369-1376, 1985.

6. Chatab, J. F., Fumoleau, P., Saccavini, J. C., Thedrez, P., Curtet, C.,Biano-Arco, A., Chetanneau, A., Peltier, P., Knemer, M., and Guillard,

Y. Immunoscintigraphy of recurrences of gynecobogic carcinomas.J. NucI. Med., 28: 1807-1819, 1987.

7. Stewart, J. S. W., Hind, V., Snook, D., Sullivan, M., Hooker, G.,

Coutenay-Luck. N., Sivolapenko, G., Griffiths, M., Myers, M. J., Lam-bent, H. E., Munro, A. J., and Epenetos, A. A. Intraperitoneal radioim-

munotherapy for ovarian cancer: pharmacokinetics, toxicity, and effi-cacy of I- I 3 1 -labeled monocbonab antibodies. Int. J. Radiat. Oncol. Biol.

Phys., 16: 405-413, 1989.

8. Rubin, S. C., Kostakoglu, L., Divgi. C., Federici, M. G., Finstad, C. L.,Lloyd, K. 0., Larson, S. M., and Hoskins, W. J. Biodistnibution andintraoperative evaluation of nadiolabeled monocbonal antibody MX3S in

patients with epithebial ovarian cancer. Gynecol. Oncol., 51: 61-66, 1993.

9. Buist, M. R., Kenemans, P., Mobthoff, C. J. M., Roos, J. C., DenHollander, W., Brinkhuis, M., and Baak, J. P. A. Tumor uptake ofintravenously administered radiobabeled antibodies in ovarian carci-

noma patients in relation to antigen expression and other tumor char-

actenistics. Int. J. Cancer, 64: 92-98. 1995.

10. Muto, M. G., and Kassis, A. I. Monocbonab antibodies used in the

detection and treatment of epithelial ovarian cancer. Cancer (Phila.), 76:

2016-2027, 1995.

1 1. Bradwell, A. R., Fairweather, D. S., Dykes, P. W., Keeling, A.,Vaughan, A., and Taylor, J. Limiting factors in the localization of tumours

with radiolabelbed antibodies. Immunol. Today, 6: 163-170, 1985.

12. Barber H. B., Barrett, H. H., Wolfender, J. M., Myers, K. J., andHickernell, T. S. Comparison of in vito scintillation probes and gamma

cameras for detection of small, deep tumors. Phys. Med. Biol., 34:

727-739, 1989.

13. Mattes, M. J., Look, K., Furukawa, K., Pierce, V. K., Old, L. J.,

Lewis, J. L., Jr., and Lloyd, K. 0. Mouse monoclonal antibodies tohuman epithelial differentiation antigen expressed on the surface ofovarian carcinoma ascites cells. Cancer Res., 47: 6741-6750, 1987.

14. Mattes, M. J., Lloyd, K. 0., and Lewis, J. L., Jr. Binding parameters

of monocbonal antibodies reacting with ovarian carcinoma ascites cells.

Cancer Immunob. Immunothen., 28: 199-207, 1989.

15. Rubin, S. C.. Finstad, C. L., Hoskins, W. J., Federici, M. G., Lloyd,

K. 0., and Lewis, J. L., Jr. A longitudinal study of antigen expression inepithelial ovarian cancer. Gynecol. Oncol., 34: 389-394, 1989.

16. Nguyen, Q., and Heffelfinger, D. M. Imaging and quantitation ofchemiluminescence using photoexcitable storage phosphor screen. Anal.

Biochem., 226: 59-67, 1995.

17. Humm, J. L., Chin, L. M., Lanza, R. C., Schaeffer, C. M., Speidel, M.,and Greene, R. E. Digital imaging for autoradiography. In: R. E. Greeneand J. W. Oestmann (eds.), Computer Digital Radiography in Clinical

Practice, pp. 167-172. New York: Thieme Medical Publishers, Inc., 1992.

18. Kostakoglu, L., Rubin, S. C., Federici, M., Finstad, C. L., Larson,S. M.. Hoskins, W. J., and Lloyd, K. 0. A comparative study of intactand F(ab’), fragments of monoclonal antibody MX3S in a xenograft

model. J. NucI. Med., 34: 2l6P, 1993.

19. Hnatowich, D. J., Gionet, M., Rusckowski, M., Siebecker, D. A.,Roche, J., Shealy, D., Mattis, J. A., Wilson, J., McGann, J., Hunter,R. E., Griffin, T., and Doherty, P. W. Pharmacokinetics of ‘ ‘ ‘In-labeled

OC-125 antibody in cancer patients compared with the 19-9 antibody.Cancer Res., 47: 61 1 1-61 17, 1987.

20. Breitz, H. B., Weiden, P. L., Vanderheyden, J. L., Appelbaum,J. W., Bjorn, M. J., Fen, M. F., Wolf, S. B., Ratliff, B. A., Seiber, C. A.,Foisie, D. C., Fisher, D. R., Schroff, R. W., Fritzberg, A. R., and

Abrams, P. G. Clinical experience with rhenium-186-labebed mono-cbonab antibodies for radioimmunotherapy: results of phase I trials.J. Nucl. Med., 33: 1099-1112, 1992.

21. Loevinger, R., Budinger, T. F., and Watson, E. E. MIRD Primer for

Absorbed Dose Calculations. New York: The Society of Nuclear Mcd-

icine, Inc., 1991.

22. Humm, J. L. Dosimetric aspects of nadiolabelled antibodies fortumor therapy. J. NucI. Med., 27: 1490-1497, 1986.

23. Goddu, S. M., Rao, D. V., and Howells, R. W. Multicellulardosimetry for micrometastases: dependence of self-dose versus cross-

dose to cell nuclei on type and energy of radiation and subcelbular

distribution of radionuclides. J. NucI. Med., 35: 521-530, 1994.

24. Finstad, C. L., Humm, J. L., Federici, M. G., Lloyd, K. 0., Barakat,

R., Divgi, C., Larson, S. M., and Hoskins, W. J. Localization andquantitation using phosphor storage plates of radiolabeled monoclonab

antibody MX3S F(ab’), in patients with micrometastatic epithelial ovar-ian cancer. Proc. Am. Assoc. Cancer Res., 37: 61 1, 1996.

25. Oosterwijk, E., Bander, N. H., Divgi, C. R., Welt, S., Wakka, J. C.,

Finn, R. D., Carswell, E. A., Larson, S. M., Warnaar, S. 0., Fleuren,

G. J., Oettgen, H. F., and Old, L. J. Antibody localization in human renalcell carcinoma: a phase I study of monoclonal antibody G250. J. Clin.

Oncol., 11: 738-750, 1993.

26. Welt, S., Divgi, C. R., Kemeny, N., Finn, R. D., Scott, A. M.,Graham, M., St. Germain, J., Carwell, E., Larson, S. M., Oettgen, H. F.,and Old, L. J. Phase 1/11 study of iodine 131-labeled monoclonalantibody A33 in patients with advanced colon cancer. J. Clin. Oncol.,

12: 1561-1571, 1994.

27. Griffith, M. H., Yorke, E. D., Wessels, B. W., Dc Nardo, G. L.. andNeacy, W. P. Direct dose confirmation of quantitative autoradiography

with micro-TLD measurements for radioimmunotherapy. J. Nucl. Med.,

29: 1795-1809, 1988.

28. Daghighian, F., Pentbow, K. S., Larson, S. M., Graham, M. C.,DiResta, G. R., Yeh, S. D., Macapinlac, H., Finn, R. D., Arbit, E., andCheung, N. K. Development of a method to measure kinetics of radio-labelled monocbonal antibody in human tumour with applications tomicrodosimetry: positron emission tomography studies of iodine- 124

labelled 3F8 monocbonal antibody in glioma. Eur. J. Nucl. Med., 20:

402-409, 1993.

29. Rubin, S. C., Kairemo, K. J. A., Brownell, A. L., Daghighian, F.,

Federici, M. G., Pentlow, K. S., Finn, R. D., Lambrecht, R. M., Hoskins,

W. J., Lewis, J. L., Jr., and Larson, S. M. High-resolution positronemission tomography of human ovarian cancer in nude rats using

‘24I-labeled monocbonal antibodies. Gynecol. Oncol., 48: 61-67, 1993.



1997;3:1433-1442. Clin Cancer Res C L Finstad, K O Lloyd, M G Federici, et al. epithelial ovarian cancer.evaluation of antibody localization to micrometastatic disease inin tissue samples by storage phosphor screen image analysis: Distribution of radiolabeled monoclonal antibody MX35 F(ab')2

Updated version

http://clincancerres.aacrjournals.org/content/3/8/1433

Access the most recent version of this article at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://clincancerres.aacrjournals.org/content/3/8/1433To request permission to re-use all or part of this article, use this link



http://clincancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]



Date post:	06-Jun-2020
Category:	Documents
Upload:	others
View:	5 times
Download:	0 times

Distribution of Radiolabeled Monoclonal Antibody MX35 F(ab ... · fresh, frozen tumor specimens...

Documents