(CANCER RESEARCH 49, 1600-1608, March 15, 1989]

Immunolymphoscintigraphy for the Detection of Lymph Node Métastasesfrom

Breast Cancer

Joe J. Tjandra, Ian S. Russell, John P. Collins, John T. Andrews, Meir Lichtenstein, David Binns, andIan F. C. McKenzie1

Department of Pathology, Research Centre for Cancer and Transplantation, University of Melbourne [J, J. T., I, F. C. M.] and Royal Melbourne Hospital[J. J. T., I. S. R., J. P. C., J. T. A., M. L., D. B., I. F. C. M.J, Parkville, Victoria, Australia

ABSTRACT

The presence of métastases in the regional lymph nodes is the majorprognostic factor in breast cancer in the absence of overt distant métastases and is also an important indicator of the need for adjuvant therapyin "early'1 breast cancer. Currently, the accurate assessment of axillary

lymph node status requires axillary dissection which has an associatedmorbidity. An alternative method of identifying patients who are "nodepositive'" has been developed by means of immunolymphoscintigraphy

with s.c. administered radioiodinated monoclonal antibody. The '"I-

labeled anti-breast cancer antibody (RCC-1; 400 ng) and cold iodine-labeled "blocking" antibody (Ly-2.1; 2 mg which is nonreactive with

breast cancer) were injected s.c. into both arms and scintigraphy imageswere obtained 16-18 h after the injection, using the axilla contralateral

to the side of the breast cancer as the control. Studies were reported aspositive (and therefore indicative of lymph node métastases)if the amountof background-subtracted radioactive count in the axilla of interest ex

ceeded the normal side by a ratio equal to or greater than 1.5:1.0 asassessed by computer analysis. In 38 of 40 patients the findings onscintigraphy were correlated with operative and histopathological findings on the axillary dissection specimen or cytological findings of fineneedle aspiration of axillary lymph nodes. In a prospective study of 26patients, the method is more sensitive (86%) and specific (92%) thanpreoperative clinical assessment (57% sensitivity, 58% specificity) in thedetection of axillary lymph node métastases; and by combining bothmodalities of assessment, there was an improvement in the sensitivity(100%) but a deterioration in the specificity (50%). There was no significant complication from this essentially outpatient procedure and only 1of 40 patients developed a human anti-mouse antibody response. This

novel and safe method of imaging may become a most useful adjunct inthe surgical management of breast cancer.

INTRODUCTION

Carcinoma of the breast remains the leading cause of cancermortality in women in Western countries and its incidence isrising (1). At present, a number of parameters (tumor size,histológica! type, histological and nuclear grade, the presenceof lymph node métastases,and estrogen receptor status) areused to predict probable patient outcome in breast cancer. Thedisease-free interval and overall survival prospects of womenwho present with localized breast cancer are influenced stronglyby the extent of the cancer at diagnosis; tumor size and thepresence of métastasesin the regional lymph nodes are themajor prognostic factors in the absence of overt distant métastases (2-4); the 10-year survival of node-positive patients is 15-35% compared with 75% 10-year survival of node-negativepatients. Nonetheless, 25-35% of patients with Stage I diseaseeventually die from metastatic cancer (5). Clinical studies suggest that these occult métastasesat the time of presentation arenot affected by the initial surgical treatment (6). This suggeststhat there are two types of "early" breast cancer: one remains

Received 5/23/88; revised 9/16/88; accepted 11/22/88.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1To whom requests for reprints should be addressed.

local and rarely spreads, while the other has occult microme-tastases when the patient is first seen clinically. For the lattergroup, local therapy alone has no effect on the distant métastases already present, but patients may benefit from adjuvantsystemic therapy (7). Currently the best predictor of microme-tastases is the involvement of the ipsilateral axillary lymphnodes, the accurate detection of which requires axillary dissection which has an associated morbidity. Clinical assessment ofaxillae is unreliable and of patients with breast cancer who haveno palpable lymph nodes in the axillae, about one-third havehistological evidence of lymph node invasion (8). Alternativemethods of detecting axillary lymph node métastaseshave beensought but not with great success; isotope-colloid preparationshave not been successful (9) despite an earlier report of the useof 99mTc-antimony su Hide colloid injected periareolarly (10).

Since the description of monoclonal antibodies with a preferential activity for cancer tissue, radiolabeled antibodies havebeen used for the radioimmunodetection of cancer and métastases. Administration i.v. of radiolabeled monoclonal antibodies[using whole immunoglobulin, F(ab')2 or Fab] combined with

computerized nuclear scintigraphy has allowed detection oftumors in different sites (11-14). However, such scintigraphicprocedures, using i.v. administered radiolabeled antibodies, arelimited by background radioactivity in the blood and extravas-cular spaces and, furthermore, antibodies may be catabolizedbefore reaching their target, resulting in only a very small tumoruptake.

This concept of immunoscintigraphy has been extended inexperimental (15, 16) and clinical studies (17, 18), using s.c.injected radiolabeled tumor-associated antibodies to delineatemetastatic deposits in regional lymph nodes. The detection oflymph node métastasesfrom breast cancer was previously reported in a preliminary study of eight women with breast cancerby using s.c. administered (interdigital injection) I31l-labeledanti-breast cancer antibody 3E1.2 (IgM: 100 /ig) (18). However,further study of a larger number of patients (N = 40) with asimilar technique has shown that nonspecific uptake by normallymph glands and other tissues is a major problem,2 and this

phenomenon of nonspecific uptake of radiolabeled specific antibody was also found by others (19).

We have further assessed this technique of using s.c. injectedradiolabeled antitumor antibodies by altering some parameters,namely, site (arm and breast instead of hand), dose (400 insteadof 100 í¿g)>and report a new technique with substantiallyimproved results which entails the addition to the I3'l-anti-breast cancer antibody of an excess of cold iodine-labeled antibody, which does not react with breast cancer.

MATERIALS AND METHODSMonoclonal Antibodies. RCC-1 (formerly called 17.1) is a mouse

IgG2a MoAb3 raised by immunizing inbred Biozzi mice with the MCF-

2 Unpublished observations.3The abbreviations used are: MoAb(s), monoclonal antibody(ies); PBS, phos

phate-buffered saline.

1600

Research. on August 25, 2020. © 1989 American Association for Cancercancerres.aacrjournals.org Downloaded from

IMMUNOLYMPHOSCINTIGRAPHY FOR DETECTION OF LYMPH NODE METASTASES

7 breast cell line (20); it reacts with most breast cell lines and breastcarcinoma but has no reaction with other tissues of relevance to thisstudy (e.g., muscle, fat, blood vessel, erythrocytes). The Ly-2.1 MoAb(IgG2a) was reactive with the murine Ly-2.1 specificity but not with

human breast cancer (21). The murine MoAb 30.6 (IgG), directedagainst an antigen present on human colon secretory epithelium andreactive against a number of colon carcinoma cell lines, nonreactivewith either the breast cancer cell lines or human breast cancer tissue(22) was used as the nonreactive negative control in immunoperoxidasestaining. All the MoAbs were isolated from ascites fluid by precipitationwith 40% ammonium sulfate, dissolution in PBS, and dialysis with thesame buffer and further purified on Protein A-Sepharose (Pharmacia,

Inc., Piscataway, NJ) (23).Antibody activity was determined by resetting with sheep anti-mouse

immunoglobulin (24) and for RCC-1 also by immunoperoxidasemethod on snap-frozen sections of fresh breast cancer (20). The prepared antibodies were retested for activity after all procedures, filteredthrough a 0.22-/im Millex-GV filter (Millipore, Bedford, Ann Arbor,MI) and batch tested for pyrogens and sterility before and after radio-labeling (Pharmacology Department, Melbourne University, and SigmaPharmaceuticals, Clayton, Victoria, Australia); for purity by sodiumdodecyl sulfate polyacrylamide gel electrophoresis (>98% pure).

lodination of Monoclonal Antibodies. The purified RCC-1 antibody(400 Mg) was labeled with 2 mCi of 13II (200 mCi/ml, AmershamInternational, United Kingdom) to a specific activity of 1 mCi of ml/mg of RCC-1 antibody, 131I:RCC-1 molar ratio, 0.1, using an Enzy-mobead reagent (Bio-Rad, Richmond, CA; 25) and the final radioiodi-

nated antibody was suspended in a 1% human serum albumin solution(Commonwealth Serum Laboratories, Melbourne, Australia). The purified murine MoAb Ly-2.1 (2 mg) was labeled with cold sodium iodide(nonradioactive Nal) by using the chloramine-T method (26). To studythe biodistribution of iodinated MoAb Ly-2.1 alone, 400 ¿tgor 2 mg ofthe antibody was labeled with '"I to a specific activity of 1 mCi/mg or0.25 mCi/mg, respectively, by the chloramine-T method. Iodinatedantibody was separated from free I31Iby using a Sephadex G-25 column

(PD10, Pharmacia, Sweden) which has previously been equilibratedwith 3 ml of 25% human serum albumin and 30 ml of sterile normalsaline (Travenol, New South Wales, Australia). The radioactivity of theiodinated antibody was measured in both a gamma counter (LKBWallac 1260, Finland) and a radioisotope calibrator (Capintec CRC-2Ni Capintec, NY). The sample was then centrifuged at 100,000 x gfor 60 min to remove immunoglobulin aggregates and filtered througha 0.22-i/m Millipore filter in a sterile laminar flow hood. To comparethe effect of different iodination methods on antibody activity, theantibody RCC-1 (200 Mg)was also labeled with 1 mCi of I3'I (200 mCi/

ml, Amersham International, United Kingdom) to a specific activity of1 mCi of 131I/mg of RCC-1 antibody with the chloramine-T (26),iodobead (27), or enzymobead methods (25). The chloramine-T andiodobead methods resulted in a greater loss of immunoreactivity thanwith the enzymobead method when assessed by the titer of antibodyand percentage of rosette-forming cells on resetting assay and in anenzyme-linked immunosorbent assay system with a membrane preparation from a breast cancer cell line (T47D-RCC-1*). Immunoreactivityof MoAb RCC-1 after labeling was also tested in a direct radioimmuno-assay by competitive assay with unlabeled RCC-1; labeled antibodycompeted well with unlabeled antibody for antigen binding on T47Dmembrane (data not shown). Again, the Enzymobead method resultedin minimal loss of reactivity (~5%), while the chloramine-T methodresulted in greatest loss of reactivity (30%); the Enzymobead methodwas, therefore, used as the method of choice for labeling RCC-1.Chromatographie analysis showed that 86-96% (mean, 92%) of theadministered 131Iactivity was bound to the antibody RCC-1. A final

yield of 90% of the antibody was obtained.Patients. Forty patients with histologically proven or cytological

features suggestive of breast cancer were studied (Table 1): subject tohistológica! confirmation, 2 of 40 patients had benign breast disease.Of the remaining 38 patients, all except 3 (Stage IV) had Stage I or IIbreast cancer by using the standard Union Internationale Contre leCancer classification (28). The clinical assessment of axillary lymphnode status was performed independently by two experienced breast

surgeons. All patients with Stage I or II breast cancer had axillarydissection either as part of a Patey mastectomy, or in addition to partialmastectomy. Two patients with benign breast lumps (sclerosing ade-nosis and fibroadenoma) were studied: initial fine needle aspirationcytology suggested the presence of malignant cells but subsequentexcision biopsy failed to find any malignancy. Two patients had breastcancer proven by fine needle aspiration cytology but were subsequentlyfound to have Stage IV breast cancer with bone métastases;neither hadaxillary dissection but both had malignant axillary lymph nodes onclinical examination. Patient 36 previously had a total mastectomy andaxillary dissection and then developed disseminated disease as well aslocal recurrence and had malignant lymph nodes in left (ipsilateral)axilla, proven by cytology. Of the patients studied, 4 of 40 receivedradioiodinated RCC-1 alone injected s.c. between digits or into thearms; 1 of 40 had periareolar injection of radioiodinated RCC-1 intothe breast; 29 of 40 received radioiodinated RCC-1 (reactive with breastcancer) together with cold iodine-labeled Ly-2.1 (nonreactive withbreast cancer) administered s.c. into both arms; 3 of 40 receivedradioiodinated RCC-1 together with unlabeled Ly-2.1 injected s.c. intoboth arms; and 3 of 40 received radioiodinated Ly-2.1 alone injecteds.c. between digits. One patient (Patient 36) who received arm injectionof radioiodinated RCC-1 alone had daily scans performed up to thethird day after injection. To inhibit thyroid uptake of radioactive iodine,each patient received potassium iodide (5 ml of 16.54%, w/v) andsodium perchlorate (400 mg) p.o., l h before the s.c. injection ofradioiodinated monoclonal antibody; the potassium iodide was continued for 5 days after the injection of 13'I. Blood samples were obtainedfrom all patients immediately before, and weekly for 4-6 weeks afterinjection of radioiodinated MoAbs for determining human anti-mouseantibody response.

Scintigraphy. Scintillation camera images were recorded 16-18 hafter injection of the radiolabeled antibody. Anterior axillary and upperbody views were recorded with a Toshiba GC A402 gamma camera byusing a high-energy collimator and computerized acquisition with anInformatek Simis 4 computer (Informatek, Sydney, Australia), using awindow setting of 360 keV with a 20% window; images were obtainedover 600 s and then digitally recorded into a matrix of 128 x 128words. Regions of interest on the images were defined by two independent investigators by manual drawing over the axillary lymph noderegions on both sides by using anatomical landmarks (Fig. 1, upper) aswell as over the adjacent background, and was found to be reproducible.The fraction of radiolabeled antibody localized in the axillary nodes (F)was estimated by measurement of nodal uptake (A ) with the gammacamera, and compared with uptake in the other regions of interest andthe amount of radiolabeled antibody injected (/). Nodal uptake wasadjusted for background activity (b), camera response, and attenuationthrough the anterior axillary fold by using an attenuation factor (A)calculated with the use of a known source placed in the axilla. Allcounts were corrected for isotope decay and the following formulaewere used:

F(%) = 100

N2(cpm) = x camera sensitivity

lbPn\\ An - - x —¿�VP"

(A)

(B)

(C)

Where F is the fraction of antibody localized in the nodes; N is thenodal uptake (A', in ¿iCi,/V2in cpm); n is the total gamma camera

counts (cpm) over lymph node regions; 1 is the actual injected dose(pC'i);b is the background activity (cpm); P„is the number of pixels in

region of interest (lymph node region); P/, is the number of pixels inbackground; A is the attenuation factor; e~" is the factor for isotopedecay, 0.94 for I3'I at 18 h; camera sensitivity (122.4 cpm/VCi) was

determined by counting the amount of radioactivity as cpm with knownamount (1 MCi)of ml.

Studies were reported as positive and therefore indicative of lymphnode métastasesif the number of counts in the axilla on the tumor sideexceeded the normal side by a ratio equal to or greater than 1.5:1.0,

1601

Research. on August 25, 2020. © 1989 American Association for Cancercancerres.aacrjournals.org Downloaded from

IMMUNOLYMPHOSCINTIGRAPHY FOR DETECTION OF LYMPH NODE METASTASES

after adjustment for background activity as indicated above. Theamount of nodal uptake of radioactivity in //Ci (,N,) was also calculatedfor each axilla with the above formulae and the mean nodal uptake ineach axilla was expressed as the percentage of the injected dose. Acomparison of the mean nodal uptake between axillae with and withoutlymph node métastasescould therefore be made. All images wereinterpreted without knowledge of the side of breast lesion or the lymphnode status.

In 2 patients [Patient 1 given an injection of 131I-RCC-1MoAb (400Mg)and cold iodine-labeled Ly-2.1 MoAb; Patient 34 given an injectionof mI-RCC-l MoAb (400 Mg)alone], axillary fat, malignant and normal

lymph nodes were sampled and the amount of radioactivity per g ofindividual tissue was determined. In this way, the amount of radioactivity localized in each tissue could be expressed as the percentage ofthe injected dose per g of various tissues studied (axillary fat, malignantand normal lymph nodes).

Analysis of Excised Lymph Nodes and Breast Cancer Tissue. In mostcases (35 of 40), the patients had surgery ~36-40 h after the injectionof radioiodinated antibody. At surgery (total or partial mastectomywith axillary dissection) the nodes were removed in a tissue block andpinned onto a foam board to facilitate orientation. After formalinfixation at room temperature overnight, each node was dissected freeof fat; and gamma camera images, radioactive count, and the weightsof individual nodes were obtained in some cases. Each node wasprocessed, stained, and 6-^m sections were examined histologically fortumor. To assess binding of the antibody to any tumor cells present inthe lymph nodes, immunoperoxidase staining of snap-frozen sectionsfrom some patients was performed (11). In addition, fresh primarybreast cancer tissue was also obtained from some patients, snap-frozen,and tested for its reactivity with RCC-1 antibody by immunoperoxidasestaining. A nonreactive negative control was processed for each lymphnode section by incubating with MoAb 30.6 (IgG), as the primaryantibody. The sections were then assessed by light microscopy toestimate the percentage of carcinoma cells stained with the antibodyand the result was expressed semiquantitatively as 0-4 according towhether there was no staining (0), up to 25% (1), 26-50% (2), 51-75%(3), or more than 75% (4) of carcinoma cells stained.

Human Anti-Mouse Antibody Response. Human IgM and IgG antibodies against the murine MoAbs were measured by an enzyme-linkedimmunosorbent assay modified from that previously described (29).Ninety-six-well flexible polyvinyl chloride plates (Costar, Cambridge,MA) were coated with 50 ^I/well of administered MoAb (10 Mg/nil ofpurified RCC-1 or Ly-2.1 MoAbs) in 0.1 M carbonate buffer, pH 9.6,and incubated at 37°Cfor 2 h. The plates were then washed with PBS/

0.05% Tween-20 and nonspecific binding was blocked with 1% bovineserum albumin/PBS pH 7.6 for 2 h at 37°C.Serial dilutions of patients'

sera (50 ^I/well) were added to the coated wells and left for overnightincubation at 4°C.Plates were then washed and then reacted with a

1:400 (50 M'/well) dilution of phosphatase-labeled affinity-purified goatanti-human IgM or IgG (Kirkegaard & Perry, Gaithersburg, MD) at37°Cfor 3 h, washed, 50 Ml/wellof alkaline phosphatase substrate were

added, and the color reaction was read with an enzyme-linked immunosorbent assay plate reader (Titertek, Multiscan, MC) at a wavelengthof 405 nm. A comparison was made between the test sample and pooledsamples of normal human serum which served as the control, resultsbeing expressed as the absorbance value of patient serum comparedwith pooled normal human serum, and a positive test was consideredto be a value at least twice the control.

Statistical Analysis. For statistical inference about the difference inaccuracy of immunolymphoscintigraphy and clinical assessment,McNemar's test for correlated proportions was applied (30). The "exact," binomial version of the test was used.

RESULTS

Optimal Method for Immunolymphoscintigraphy

mI-RCC-l (400 /ig) plus Cold Iodine-labeled Ly-2.1 (2 mg):

Arm Injection. Patients 1-26 in Table 1 received radioiodinatedRCC-1 antibody (reactive with breast cancer) with a specific

activity of 1 mCi 13ll/mg of RCC-1 (by Enzymobead method),

together with cold iodine-labeled Ly-2.1 (nonreactive withbreast cancer) injected s.c. into both arms just above the ante-cubital fossae. As the purpose of cold iodine-labeled Ly-2.1

antibody was to saturate nonspecific binding sites by cold"damaged" antibodies, the chloramine-T method (shown above

to damage antibodies) was used to iodinate Ly-2.1 antibody.Twenty-six patients were investigated by this method and correct prediction (true positive, true negative) of lymph nodestatus was achieved in 88% (23 of 26 patients). Fourteen of 26patients had histologically or cytologically proven axillarylymph node métastasesand 12 of 14 (86%) of these patientswere detected by scintigraphy, while 12 of 26 patients did nothave lymph node métastasesand a negative scintigraphy wasobtained in 11 of 12 (92%) of these patients. This method gavethe best results in immunolymphoscintigraphy among variousdifferent techniques tested (see below).

Some representative examples are described in more detailbelow. Patient 1 had left primary breast cancer and was considered clinically not to have axillary lymph node métastases;although the preoperative axillary immunolymphoscintigraphy(Fig. 1, lower) showed a preferential localization of 13'I-RCC-1

MoAb in left axilla (L:R ratio, 2.0:1.0) suggestive of left axillarylymph node métastases.A left Patey mastectomy was performed and tumor deposits were found by histology in 12 ofthe 16 lymph nodes recovered from the axillary dissectionspecimen.

Patient 2 was considered to have axillary lymph node métastases on clinical examination but scintigraphy showed equaluptake of '"I-RCC-1 in both axillae with a ratio of background-

subtracted count of 1.1:1.0 (R:L) between the 2 axillae (Fig. 2).Histology of the 14 lymph nodes recovered showed reactivehyperplasia with no métastases.The immunoperoxidase staining of regional lymph nodes with RCC-1 antibody also did not

identify any tumor deposits.Patient 3 had right breast cancer and clinically noninvolved

axillary lymph nodes. A right Patey mastectomy was performedand a single focus of micrometastases was identified in 1 of the12 axillary lymph nodes recovered on histology. The preoperative immunolymphoscintigraphy showed an increased localization of I3II-RCC-1 in the right axillary lymph node region with

a ratio between axilla of interest (R) and contralateral axilla (L)of 1.5:1 (Fig. 3). The uptake of radioactivity by the liver wasalso noted.

Other Parameters Examined

In this study, the MoAb used was the IgG class (RCC-1),used in the belief that the smaller size of IgG (as compared toIgM) would reduce the nonspecific uptake of radioiodinatedMoAb. Various pilot studies (Table 1) were carried out todetermine the effect of various parameters, namely site (arm,breast, and hand), dose (400 /¿gand 1 mg instead of 50-100Mg)on the results of immunolymphoscintigraphy. The biodis-tribution of l31I-Ly-2.1 alone was also investigated.

13II-RCC-1 (1 mg) plus Cold Iodine-labeled Ly-2.1 (2 mg):

Arm Injection. Three patients (Patients 27-29) received thehigher dose of specific MoAb (RCC-1,1 mg) while maintainingthe same amount of I31I (2 mCi) to a specific activity of 0.5mCi of 13ll/mg of antibody, and the same amount of coldiodine-labeled Ly-2.1 MoAb was given. Correct prediction of

lymph node status was achieved in 2 of 3 patients, but theamount of radioactive uptake in either axilla was less and a

1602

Research. on August 25, 2020. © 1989 American Association for Cancercancerres.aacrjournals.org Downloaded from

IMMUNOLYMPHOSCINTIGRAPHYFOR DETECTION OF LYMPH NODE METASTASES

Table 1 Summary of axillary immunolymphoscintigraphy results in 40 patients with suspected breast cancer

Patient"12345678910111213141516171819202122232425262728293031323334353637383940Side/histologyof breast

tumor*Left/ductalRight/ductalRight/ductalLeft/ductalLeft/lobularLeft/ductalRight/mucinousRight/ductal

(Stage4)Left/ductal(Stage4)Right/ductalRight/ductalRight/ductalLeft/ductalRight/ductalLeft/ductalRight/ductalRight/ductalLeft/ductalRight/ductalLeft/ductalLeft/ductalRight/ductalLeft/mucinousRight/ductalRight/ductalLeft/ductalRight/ductalLeft/carcinoma

insituLeft/sclerosingadenosisLeft/ductalRight/ductalLeft/carcinoma

insituLeft/fibroadenomaLeft/ductalRight/ductalLeft/ductal

(Stage4)L«ft/ductalLeft/ductalLeft/ductalRight/ductalNodal

me-tastasesr12/160/91/9^12/200/250/201/12++19/190/90/40/1012/1518/290/90/140/197/110/297/93/30/91/112/250/151/100/8—0/117/210/18—1/143/13+0/120/220/201/10Axillary

lymph nodeclinically in

volved Scanratio''No

2.0:1.0Yes.1:1.0No.5:1.0Yes.0:1.0No.0:1.0Yes.0:1.0No2.3:1.0Yes4.4:1.0Yes6.0:1.0Yes2.1:1.0NoNoNoNoYesYesNo

<NoNoYesYesYesNo

<NoYesYesNo.0:1.0.0:1.0.1:1.0.9:1.0.6:1.0.0:1.0.0:1.0.7:1.0.6:1.0.0:1.0.3:1.0.8:1.0.0:1.0.6:1.0.6:1.0.0:1.0.0:1.0No

1.0:1.0No1.0:1.0No<1.0:1.0No1.0:1.0No2.0:1.0No2.0:1.0No2.0:1.0No<1.0:1.0Yes1.5:1.0Yes

No uptakeineitheraxillaNo1.0:1.0No1.0:1.0No

1.1:1.0Scan

resultTP'TNTPFNTNTNTPTPTPTPTNTNTNTPTPTNTNFPTPTNFNTPTNTPTPTNFNTNTNTNFNFPFPTPFNTP

°Patients 1-26 had arm injection of I3II-RCC-1 (400 Mg)and cold iodine-labeled Ly-2.1 (2 mg); Patients 27-29 had arm injection of '"I-RCC-1 (1 mg) and coldiodine-labeled Ly-2.1 (2 mg); Patients 30-32 had arm injection of '"I K( <' I (400 UK)and unlabeled Ly-2.1 (2 mg); Patients 33 and 34 had interdigital injection of'"I-RCC-1 (400 Mg);Patients 35 and 36 had arm injection of 13'I-RCC-1 (400 jig); Patient 37 had periareolar injection of 13'I-RCC-1 (400 Mg) into the breasts;Patients 38 and 39 had interdigital injection of 131I-Ly2.1(2 mg); and Patient 40 had interdigital injection of 131I-Ly2.1(400 Mg).

b All patients had breast carcinoma except Patients 29 and 33 who had benign breast disease.' Nodal métastasesexpressed as number of involved nodes/total number of nodes recovered from operative specimen. +, nodal métastasesdetermined by clinical

examination and fine needle aspiration cytology in patients with Stage 4 breast cancer; —¿�,axillary dissection was not performed and clinically the axilla was free ofdisease.

d Scan ratio is expressed as the ratio of background-subtracted radioactive count of the axilla of interest (insilatemi to breast tumor), to that of contralateral axilla.

A ratio > 1.5:1.0 was regarded as significant.' TP, true positive; FP, false positive; TN, true negative; FN, false negative.'Patient 3 had only a single focus of micrometastases in one lymph node.

poorer quality image was obtained.131I-RCC-1(400 /ig) plus Unlabeled Ly-2.1 (2 mg): Arm Injec

tion. As a control, 3 patients (Patients 30-32) were giveninjections of radioiodinated RCC-1 (400 ng), together withunlabeled Ly-2.1 (2 mg). Wrong prediction was obtained in 2

of 3 patients (1 false positive, 1 false negative)."'I-RCC-1 (400 /ig): Interdigital or Arm Injection. Four pa

tients (Patients 33-36) received radioiodinated RCC-1 alonewithout the "blocking" cold iodine-labeled antibody, and 1 of

the 4 patients had fibroadenoma, a benign condition, while theother 3 patients had breast cancer. Correct prediction of lymphnode métastaseswas obtained in 2 of 4 patients. The fourthpatient (Patient 36), with advanced breast cancer (Stage IV)and malignant left axillary lymph nodes on clinical examination(confirmed by fine needle aspiration cytology) was given aninjection of 13ll-labeled RCC-1 (400 ^g) into both arms s.c.

Serial studies were obtained at 16, 40, and 64 h after injectionand showed increased uptake in the left axilla (L:R ratio,1.5:1.0). The differential uptake between abnormal and normal

axillae did not change with time and appears to be optimal at16 h after injection. It is of interest that this patient previouslyhad a left axillary dissection.

I31I-RCC-1 (400 fig): Intramammary Injection. One patient(Patient 37) had periareolar injection of radioiodinated RCC-1

in an attempt to simulate natural lymphatic drainage of thebreast. However the injected radioiodinated RCC-1 failed to

drain to either the axilla or the internal mammary chain oflymph nodes and remained localized in the breast for at least48 h. This patient did not have any métastasesin the axillarylymph nodes on histológica! examination.

l31I-Ly-2.1: Interdigital Injection. Three patients (Patients 38-

40) received radioiodinated Ly-2.1 alone. Two patients withoutaxillary lymph node métastases(Patients 38 and 39) had 13II-

Ly-2.1 with a specific activity of 0.25 mCi/mg (total amount ofantibody injected, 2 mg) and one patient with right axillarylymph node métastases(Patient 40) received 13lI-Ly-2.1 with aspecific activity of 1 mCi/mg (total amount of Ly-2.1 injected,400 Mg)- In each patient, the amount of radioactive uptake

1603

Research. on August 25, 2020. © 1989 American Association for Cancercancerres.aacrjournals.org Downloaded from

IMMUNOLYMPHOSCINTIGRAPHY FOR DETECTION OF LYMPH NODE METASTASES

Fig. 2. Immunolymphoscintigraphic image of anterior chest in a patient withright breast cancer and palpably enlarged right axillary lymph nodes; none of thelymph nodes contained tumor deposits on histology. Scan ratio (left:right),1.1:1.0. A, scatter of radioactivity from the site of arm injection.

Fig. 1. Upper, scintigraphic image of the anterior chest of a patient and therespective regions of interest were drawn manually.. I. right axilla; B, backgroundregion adjacent to right axillary region; C, left axilla; and D, background regionadjacent to left axillary region. Lower, immunolymphoscintigraphic image of thesame patient who had left axillary lymph node métastasesfrom breast cancer.Scan ratio (left:right), 2.0:1.0.

Fig. 3. Immunolymphoscintigraphic image showing an increased uptake ofradiolabeled antibody in right axilla (scan ratio, right:left, 1.5:1.0) which corresponded to the micrometastases in one of the right axillary lymph nodes excised.

1604

Research. on August 25, 2020. © 1989 American Association for Cancercancerres.aacrjournals.org Downloaded from

IMMUNOLYMPHOSCINTIGRAPHY FOR DETECTION OF LYMPH NODE METASTASES

between the two axillae was the same, even in the presence of chronic asthma on steroids had an exacerbation of her asthmaaxillary lymph node métastases(Table 1). Thus it appears thatthe uptake of the radioiodinated nonspecific antibody (Ly-2.1)in node-positive axilla is not higher than that in node-negativeaxilla, in contrast to that found with radioiodinated specificantibody (RCC-1) when injected together with the blockingcold iodinated Ly-2.1.

Test Parameters

Tables 2 and 3 summarize the results of preoperative clinicalassessment and axillary immunolymphoscintigraphy in 26 patients who received I31I-RCC-1 (400 ^g) and cold iodine-labeledLy-2.1 (2 mg). This new method of immunolymphoscintigraphywith the use of two antibodies is more sensitive (86%) andspecific (92%) than preoperative clinical assessment (57% sensitivity, 58% specificity) but still not as accurate as axillarydissection and histological assessment which is the standardagainst which it is compared. By combining immunolymphoscintigraphy with preoperative clinical assessment, there wasan improvement in the sensitivity (100%) but it resulted in adeterioration in the specificity (50%). Overall accuracy of thisnew method of immunolymphoscintigraphy (88%) was superiorto preoperative clinical assessment (58%) and the difference isstatistically significant (P < 0.001).

Complications

The procedure was performed in 33 of 40 patients as anoutpatient procedure, and in the remaining 7 patients it wasperformed during the patients' hospital stay for other medical

and social reasons, unrelated to the procedure itself. The patients tolerated the procedures well and there were no allergicreactions or sepsis locally or generalized. One patient who had

Table 2 Comparison of immunolymphoscintigraphy by using '"I-RCC-1 MoAb(400 iig) and cold iodine-labeled (blocking) Ly-2.1 MoAb with clinical and

pathological assessment of the axillae in 26 patients with breast cancer (Patients1-26)

Scan ResultPositiveNegative

Clinical assessment'

PositiveNegativePathological

node positive12

286Assessment"

node negative1

1157

" Node positive implies -\ nodal métastases;node negative implies no nodal

métastases.'' Positive, palpable axillary lymph nodes felt to contain tumor deposits; neg

ative, nodes not palpable, or if palpable, felt not to contain tumor deposits.

Table 3 Comparison of immunolymphoscintigraphy and clinical examination inpredicting axillary lymph node métastases

Immunolymphoscintigraphy

Parameter"(%)Sensitivity

SpecificityAccuracy86

9288a

conc\Clinical

ex- Immunolymphoscin-aminations tigraphy -I-clinical ex-

(%) amination(%)57

5858TP100

5077

Specificity = TNTP + FP '

Accuracy =TP + TN

Total no. of patients tested

in which TP is true positive; TN is true negative; FP is false positive; FN is falsenegative.

which was easily controlled by increasing the dose of the steroid.Human anti-mouse antibody response was detected in theserum of only 1 of 40 patients at 3 weeks after injection.

Biodistribution Data

In 36 patients (Patients 1-36), the fraction of RCC-1 MoAbretained in the nodes (/•").calculated as a percentage of the

actual injected dose was determined and compared among thevarious techniques studied (Table 4). In the patients who received 13II-RCC-1 (400 Mg)and cold iodine-labeled Ly-2.1 (2

mg), the node-positive (on histology or cytology) axillae had amean nodal uptake of 3.68% of the injected dose, which washigher than the mean nodal uptake (1.31%) of node-negativeaxillae calculated from the gamma camera count rates usingEquations A, B, and C. This particular method had the optimaldifferential ratio between node-positive and node-negative axillae when compared with other methods of immunolymphoscintigraphy (Table 4). In addition, the fraction of MoAb Ly-2.1 retained in the nodes after injection of l3'I-Ly-2.1 (400 Mg)alone was the same (1.21% of the injected dose) between node-positive and node-negative axillae.

In 2 patients (Patients 7 and 34), the axillary dissectionspecimens were examined to sample the axillary fat and recoverall the lymph nodes. The amount of radioactivity uptake inaxillary fat, metastatic and normal lymph nodes in Patient 1[who had 131I-RCC-I (400 Mg)and cold iodine-labeled Ly-2.1(2 mg)] and in Patient 34 [who had '"I-RCC-1 (400 Mg)alone]

is shown in Table 5. Correction was made to account for thephysical half-life of I31I and to obtain a relative count with

regard to the injected amount. The localization index refers tothe ratio of radiolabeled antibody present in the tissues (metastatic lymph node versus normal lymph node versus axillaryfat; Table 5) and it was higher in the involved nodes than thenormal nodes in the patient who received I31I-RCC-1 and coldiodine-labeled Ly-2.1 than the patient who had I31I-RCC-1

alone. By contrast, the background counts from the surroundingaxillary fat were low with either method. As the in vitro radioactivity measurements on the lymph nodes were done in only a

Table 4 Comparison of nodal uptake (%) ofn'I-RCC-l in axillae with and

without lymph node métastases,using different methods ofimmunolymphoscintigraphy

Mean nodal uptake" (% of injected dose)

Pathology Method 1* Method 2 Method 3 Method 4A Method 4B

Positive axilla'

Negative axilla

3.68(13)''

1.31 (39)2.90(1)1.82(5)

0.8(1)1.7(5)

14.0(1)14.2(3)

4.6 (2)5.2(2)

" Mean nodal uptake as calculated by using Equations A, B, and C in the text.'Method 1: 131I-RCC-1 (400 >ig) plus cold iodine-labeled Ly-2.1 (2 mg);

Method 2: '"I-RCC-1 (1 mg) + cold iodine-labeled Ly-2.1 (2 mg); Method 3:"'I-RCC-1 (400 Mg)+ unlabeled Ly-2.1 (2 mg); Method 4: 131I-RCC-1 (400 ng);

.l. interdigital injection; B, arm injection.c Axilla is positive if there were >1 lymph node métastases,negative if there

were no lymph node métastases.'' Numbers in parentheses, number of axillae studied.

Table 5 Mean concentration (% ofdose/g tissue) ofl3lI-RCC-l MoAb in different

tissues by using two different methods of immunolymphoscintigraphy

TissuesAxillary

fatMetastaticLN*

Normal LNLocalization Indices

Metastatic LN/normal LNMetastatic LN/fatMethod

1°l.Ox

IO"32.8 x 10-'3.3 xIO'28.5

280.0Method

24.0x IO"3

6.4 x 10-'4.2 xIO''1.3

160.0" Method 1: '"I-RCC-1 (400 ^g) + cold iodine-labeled Ly-2.1 (2 mg); Method

2:'"I-RCC-1 (400 Mg).* LN, axillary lymph node(s).

1605

Research. on August 25, 2020. © 1989 American Association for Cancercancerres.aacrjournals.org Downloaded from

IMMUNOLYMPHOSCINTIGRAPHY FOR DETECTION OF LYMPH NODE METASTASES

small number of cases, the results have to be interpreted withcaution.

Correlation with Immunoperoxidase Staining

To assess the reactivity of the breast cancer with the MoAband the binding of RCC-1 to tumor cells present in the axillarylymph nodes, immunoperoxidase staining of snap-frozen sections of primary breast cancer and lymph node métastaseswasperformed. Fresh sections were obtained from 10 patients, allof whom had I31I-RCC-1and cold iodine-labeled Ly-2.1, and 6

of 10 patients had lymph node métastases.Five of six patientshad primary breast cancer and lymph node métastaseswhichreacted strongly (staining score, 3-4) with RCC-1. These fivepatients had preoperative scintigraphy images indicative oflymph node métastases(scan ratio > 1.8:1.0). By contrast, 1 of6 patients (Patient 4) had primary breast cancer and lymphnode métastaseswhich reacted weakly (staining score, 1) withRCC-1 on immunoperoxidase staining and the immunolym-phoscintigraphy failed to detect the lymph node métastasesinthis case, suggesting that failure of the methods (13II-RCC-1plus cold iodine-labeled Ly-2.1) to localize lymph node métastases in some cases may be contributed, in part, by the poorreactivity of the RCC-1 MoAb with that particular tumor.

DISCUSSION

Previous study with s.c. administration of 50-200 /¿gof 3E1.2(IgM) or RCC-1 (IgG) alone failed to delineate secondarydeposits in axillary lymph nodes from breast cancer in a substantial proportion of patients.2 A major problem was the high

nonspecific uptake of radiolabeled MoAb by normal lymphnodes, a finding also noted by other groups (19, 31). We havefurther evaluated immunolymphoscintigraphy as a means ofdetecting axillary lymph node métastasesfrom breast cancer ina prospective study of 40 patients, 38 of whom had breastcancer and 2 patients had benign breast disease, confirmed byhistological or cytological examination. Attempts to improvethe results were made in this study by using MoAb of the IgGclass only (RCC-1) in the belief that the smaller size of IgGthan IgM will reduce the nonspecific uptake of radioiodinatedMoAb. A higher dose (400 Mg)of anti-breast cancer RCC-1MoAb (IgG) was injected more proximally into the arms withimproved scintigraphy images from a higher dose of radioiodinated MoAb reaching the axilla, but the sensitivity in thedetection of lymph node métastaseswith this modificationalone has not improved. Studies with delayed views up to 3days in one patient given I31I-RCC-1alone also failed to show

any advantage; optimal images and count rates were obtainedat 16-18 h after injection. However, the addition of a second,cold iodine-labeled antibody (Ly-2.1), of the same isotype specifically chosen because of its nonreactivity with breast cancer,improved the accuracy of the immunolymphoscintigraphy; presumably, as it was able to block the nonspecific binding of thelabeled antibody. The second blocking antibody was iodinatedwith nonradioactive sodium iodide so that it is chemicallymodified and this was done by using the chloramine-T method,as it was established that it caused more damage to the MoAbsthan did the Enzymobead method, it being considered that thedamaged antibody should bind to the normal lymph nodes inpreference to radioiodinated RCC-1 and hence block the nonspecificuptake of radiolabeled anti-breast cancer antibody. Thisstudy shows that immunolymphoscintigraphy by using twoantibodies can successfully localize lymph node métastasesinpatients with breast cancer in 86% of cases. The procedure is

safe and easy to perform; image subtraction and enhancementof visualization was not necessary. The nonspecific uptake ofradiolabeled antibody by normal lymph nodes is reduced withthe addition of the second blocking antibody, resulting in agreater differential uptake between malignant and normallymph nodes (Fig. 1, lower) with improved localization indices(Table 5). This approach of immunolymphoscintigraphy alsoproved superior to preoperative clinical assessment in detectingaxillary lymph node métastases(Tables 2 and 3). The overallaccuracy of a positive scintigraphy result was 88% in comparison with that of clinical assessment which was 58%, and thedifference is statistically significant (P < 0.001). The false-positive and false-negative rates in the clinical assessment ofthe axillary lymph node status in this study is in keeping withpreviously reported studies (32-34). Although the results obtained with immunolymphoscintigraphy are still not as good astotal axillary clearance and histology, the accuracy in relationto lesser surgical procedures such as axillary sampling for thedetection of lymph node métastasesis unknown. Lower axillarylymph node sampling was introduced as an alternative to moreradical surgical procedures in determining axillary lymph nodestatus in patients with primary breast cancer, and while therewere reports which support its application (35, 36), severalstudies have challenged its accuracy (37, 38). It would be ofinterest to compare the accuracy of immunolymphoscintigraphy with that of lower axillary node sampling in defining theproportion of patients who are qualitatively node positive. Inaddition, it has been shown that careful reexamination of he-matoxylin and eosin-stained sections of lymph nodes can detectadditional number of micrometastases which otherwise was notapparent (39, 40). It is possible that the patient (Patient 18)who had false-positive scintigraphy images may have micrometastases in the axillary lymph nodes not apparent on routinehistological examination, but this is not proven. If immunolymphoscintigraphy and clinical examination were combined in theassessment of axillary lymph nodes, and to regard an abnormality of either assessment as significant, there would be animprovement in the sensitivity from 86% for immunolymphoscintigraphy alone to 100% for combination assessment, butwith a decrease in the specificity (from 92 to 50%).

The failure of intramammary injection of radioiodinatedMoAb to drain to the axillary lymph nodes even by the thirdday in a single case is of interest and is probably related to theslow lymphatic drainage in the breast compared with the upperlimbs, where muscular pump action improves lymphatic drainage. Delayed scintigraphic images performed beyond the thirdday may have revealed eventual localization. In addition, thisroute of injection has sound anatomical and physiological basisas it simulates the natural lymphatic drainage in the breast andmay theoretically image internal mammary chain of lymphnodes.

As the involvement of axillary lymph nodes is of such importance as a prognostic factor, and as an indicator for adjuvanttherapy (41), further improvement in the diagnostic accuracy isneeded before axillary dissection and histology could be replaced by immunolymphoscintigraphic scanning of the axillae.It has previously been established that MoAb RCC-1 reactedwith only 90% of fresh breast cancer sections examined (20),presumably due to the phenomenon of tumor heterogeneityboth within and between different tumor deposits. Indeed, in apatient (Patient 4) whose breast cancer reacted poorly withRCC-1, the immunoscintigraphic scan failed to identify thelymph node métastases.Thus the false-negative rate in thisstudy (14% or 2 of 14) could be accounted for by the lack of

1606

Research. on August 25, 2020. © 1989 American Association for Cancercancerres.aacrjournals.org Downloaded from

IMMUNOLYMPHOSCINTIGRAPHY FOR DETECTION OF LYMPH NODE METASTASES

immunoreactivity of the antibody with a proportion of thetumors, but this was not proven. It is possible that furtherimprovement may result from using a mixture of different anti-breast cancer antibodies to overcome the problems of tumorheterogeneity (42).

The quantitative analysis of the immunolymphoscintigraphicscans by obtaining a ratio of background-subtracted radioactive

count of either axilla improved the objectivity of interpretation.A ratio > 1.5:1.0 between the "axilla of interest" and the normal

axilla was regarded as significant in the study and this is selectedbecause at and above that ratio, there was an obvious visualdifference between the scintigraphy images of the 2 axillae, andit gave optimal sensitivity and specificity in the detection oflymph node métastasesin this study when the results wereanalyzed. This study has shown that the amount of radioiodi-nated tumor-associated MoAb reaching its target tissue after

s.c. administration is small (Tables 4 and 5) and is of the sameorder as the amount localized in lymph node métastasesafteri.v. administration of radiolabeled MoAbs (43-45). Conceivablythe localization of the antibody in the lymph nodes couldtheoretically be the result of antibody escaping to the circulationfollowing s.c. administration, but the blocking of nonspecificuptake by cold iodine-labeled Ly-2.1 at a dose of 2 mg tends tosuggest that this was not the case.

Furthermore, despite the many theoretical disadvantages ofthe radiophysical characteristics of I31I (7 energy and in vivo

deiodination), it is by far the most practical and availableradiolabel, certainly in the Southern hemisphere, where I31Iismuch more available than '"In or 123I, and none of these

theoretical disadvantages appears to be a problem in this study,where the primary concern is the localization of axillary lymphnode métastases.The mechanism of uptake of radioactivity byliver and spleen had not been elucidated, but possibilities include in vivo deiodination and attachment of the iodine to otherproteins, nonspecific Fc receptor binding, and catabolism ofimmunoglobulins by organs with high reticuloendothelial system with resultant prolonged retention of '"I. In addition,

MoAb RCC-1 has been known not to cross-react with either

liver parenchyma or spleen (20).Despite the results obtained in successfully demonstrating

lymph node métastaseswith a radioiodinated breast cancer-associated MoAb (RCC-1) and a cold blocking second antibody,

the mechanism of nonspecific uptake by normal lymph nodesand the mechanisms of action of the second blocking antibodyare not clear. It appears from the study, however, that wheninjected alone, the uptake of radioiodinated Ly-2.1 is the samebetween node-positive and node-negative axillae, suggesting

that the blocking of nonspecific uptake was independent of thesize of the lymph nodes or the presence or absence of lymphnode métastases.The phenomenon of nonspecific uptake bynormal lymph nodes in patients with cancer of the cervixundergoing immunolymphoscintigraphy of the ilioinguinal region in combination with standard radiological intralymphaticlymphangiography has been found elsewhere (31) and wasconsidered to be due to binding through the Fc portion of themurine immunoglobulin. The improvement in the results withthe addition of a second antibody could be due to the competition for nonspecific binding to normal lymph nodes, or it maybe related to some special nature of "coldly" labeled Ly-2.1

antibody. Future studies with higher doses of blocking secondantibody and its prior injection may also further improve theresults.

ACKNOWLEDGMENTS

The authors would like to thank Toula Athanasiadis and BeverleyCosgrove for their secretarial assistance. We are also grateful to G. A.Pietersz, P. J. McLaughlin, C. H. Thompson, and Mark Smyth fortheir advice, and to the staff of the Nuclear Medicine and AnatomicalPathology Departments at the Royal Melbourne Hospital for theirassistance.

REFERENCES

1. Kelsey, J. L. A review of the epidemiology of human breast cancer. Epidemici.Rev., /: 74-109, 1979.

2. Bonadonna, G., Valagussa, P., Rossi, A., Tancini, G., Brambilla, C., Zam-betti, M., and Veronesi, U. Ten years' experience with CMF-based adjuvant

chemotherapy in resectable breast cancer. Breast Cancer Res. Treat., 5: 95-115, 1985.

3. Harris, J. R., Canellos, G. P., Hellman, S., and Fisher, B. Cancer of thebreast. In: V. T. De Vita, Jr., S. Hellman, and S. A. Rosenberg (eds.), Cancer.Principles and Practice of Oncology, pp. 1119-1127. Philadelphia: J. B.Lippincott Co., 1985.

4. Tattersall, M. H. N. Breast cancer—fiction, facts and fashion. Med. J. Ausi.,¡47:318-319, 1987.

5. Fisher, B., Bauer, M., Wickerham, D. L., Redmond, C. K., and Fisher, E. R.Relation of number of positive axillary nodes to the prognosis of patientswith primary breast cancer: an NSABP update. Cancer, 52: 1551-1557,1983.

6. Park, W. W., and Lees, V. C. The absolute curability of cancer of the breast.Surg. Gynecol. Obstet., 93: 129-157, 1951.

7. Hellman, S., Beadle, G., Silver, B., and Harris, J. R. Primary breast cancer:the experience of the joint center for radiation therapy. In: V. Veronesi andG. Bonadonna (eds.). Clinical Trials in Cancer Medicine, pp. 137-144. NewYork: Academic Press, 1985.

8. Henderson, C., and Canellos, G. P. Cancer of the breast; the past decade. N.Engl. J. Med., 302:17-30, 1980.

9. (.aspanni. M., Andreoli, C., Rodari, A., Costa, A., and Buraggi, G. L. Lackof efficacy of lymphoscintigraphy in detecting axillary lymph node métastasesfrom breast cancer. Eur. J. Cancer Clin. Oncol., 23:475-480, 1987.

10. Agwunobi, T. C., and Boak, J. L. Diagnosis of malignant breast disease byaxillary lymphoscintigraphy: a preliminary report. Br. J. Surg., 65:379-383,1978.

11. Epenetos, A. A., Britton, K. E., Mather, S., Sheperd, J., Granowska, M.,Taylor-Papadimitriou, J., Nimmon, C. C., Durbin, H., Hawkins, L. R.,Malpas, J. S., and Bodmer, W. F. Targeting of Iodine-123 labelled tumour-associated monoclonal antibodies to ovarian, breast and gastro-intestinaltumours. Lancet, 2: 999-1004, 1982.

12. Rainsbury, R. M., Westwood, J. H., Coombes, R. C., Neville, A. M., Oh, R.J., Kalirai, T. S., McCready, V. R., and Gazet, J. C. Location of metastaticbreast carcinoma by a monoclonal antibody chetate labeled with Indium-111.Lancet, A-934-938, 1983.

13. Smedley, H. M., Finan, P., Lennox, E. S., Ritson, A., Takei, F., Wraight,P., and Sikora, K. Localization of metastatic carcinoma by a radiolabeledmonoclonal antibody. Br. J. Cancer, 47: 253-259, 1983.

14. Fairweather, D. S., Bradwell, A. R., Dykes, P. W., Vaughan, A. T., Watson-James, S. F., and Chandler, S. Improved tumour localization using indium111 labelled antibodies. Br. Med. J., 287: 167-170, 1983.

15. Weinstein, J. N., Parker, R. J., Keenan, A. M., Dower, S. K., Morse, H. C.,and Seiber, S. M. Monoclonal antibodies in the lymphatics: towards thediagnosis and therapy of métastases.Science (Wash. DC), 218: 1334-1337,1982.

16. DeLand, F. H., Kim, E. E., Corgan, R. L., Casper, S., Primus, F. J.,Spremulli, E., Estes, N., and Goldenberg, D. M. Axillary lymphoscintigraphyby radioimmunodetection of carcinoembryonic antigen in breast cancer. J.Nucí.Med., 20: 1243-1250, 1979.

17. Weinstein, J. N., Steller, M. A., Keenan, A. M., Covell, D. G., Key, M. E.,Seiber, S. M., Oldham, R. K., Hwang, K. M., and Parker, R. J. Monoclonalantibodies in the lymphatics: selective delivery to lymph node métastasesofa solid tumour. Science (Wash. DC), 222: 423-426, 1983.

18. Thompson, C. H., Lichtenstein, M., Stacker, S. A., Leyden, M. J., Salehi,N., Andrews, J. T., and McKenzie, I. F. C. Immunoscintigraphy for detectionof lymph node métastasesfrom breast cancer. Lancet, 2: 1245-1247, 1984.

19. Nelp, W. B., Eary, J. F., and Jones, R. F. Preliminary studies of monoclonalantibody lymphoscintigraphy in malignant melanoma. J. Nucí.Med., 28:34-41, 1987.

20. Thompson, C. H., Jones, S. L., Whitehead. R. H., and McKenzie, I. F. C. Ahuman breast tissue-associated antigen detected by a monoclonal antibody.J. Nati. Cancer Inst., 70:409-419, 1983.

21. Hogarth, P. M., Henning, M. M., and McKenzie, I. F. C. Alloantigenicphenotype of radiation induced thymomas in the mouse. Immunology, 46:135-144, 1982.

22. Thompson, C. H., Jones, S. L., Pihl, E., and McKenzie, I. F. C. Monoclonalantibodies to human colon and colorectal carcinoma. Br. J. Cancer, 47: 595-605, 1983.

23. Eye, P. L., Prowse, S. J., and Jenkin, C. R. Isolation of pure IgG,, IgGz. and

1607

Research. on August 25, 2020. © 1989 American Association for Cancercancerres.aacrjournals.org Downloaded from

IMMUNOLYMPHOSCINTIGRAPHY FOR DETECTION OF LYMPH NODE METASTASES

IgGib immunoglobulins from mouse serum using protein A-Sepharose. Immunochemistry. / 5:429-436, 1978.

24. Parish, C. R.. and McKenzie. I. F. C. A sensitive resetting method fordetecting subpopulations of lymphocytes which react with alloantisera. J.Immunol. Methods, 20: 173-183, 1978.

25. Morrison. M., and Boyse, C. S. Catalysis of iodination by lactoperoxidase.Biochemistry, 9: 2995-3000, 1970.

26. Greenwood, F. C., Innter, W. M., and Glover, J. S. The preparation of '"I-

labeled human growth hormone of high specific radioactivity. Biochem. J.,89: 114-123. 1963.

27. Markwell, M. A. K. A new solid state reagent to iodinate proteins. Ann.Biochem., 125: 427-432, 1982.

28. American Joint Committee on Staging. Manual for Staging of Cancer. Ed.2. Philadelphia: Lippincott, 1983.

29. Schroff, R. W., Foon, K. A., Beatty, S. M., Oldham, R. K., and Morgan, A.C. Human antimurine immunoglobulin responses in patients receiving monoclonal antibody therapy. Cancer Res., 45: 879-885, 1985.

30. Armitage, P. Statistical Methods in Medical Research. Oxford: BlackwellScientific. 1971.

31. Epenetos, A. A. Antibody guided lymphangiography in the staging of cervicalcancer. Br. J. Cancer, 51: 805-808, 1985.

32. Butcher, H. Radical mastectomy for mammary carcinoma. Ann. Surg., 170:883-884, 1969.

33. Haagensen, C. D., and Cooley, E. Radical mastectomy for mammary carcinoma. Ann. Surg., 170: 884-888, 1969.

34. Schottenfeld, D., Nash, A. G., Robbins, G. F., and Beattie, E. T., Jr. Ten-year results of the treatment of primary operable breast carcinoma. Cancer(Phila.),.?«: 1001-1007, 1976.

35. Fisher, B., Wolmark, N., Bauer, M., Redmond, C., and Gebhardt, M. Theaccuracy of clinical nodal staging and of limited axillary dissection as adeterminant of histologie nodal status in carcinoma of the breast. Surg.Gynecol Obstet., 152: 765-772, 1981.

36. Steele, R. J. C., Forrest, A. P. M., Gibson, T., Stewart, H. J., and Chetty, U.The efficiency of lower axillary sampling in obtaining lymph node status inbreast cancer: a controlled randomized trial. Br. J. Surg., 92:368-369, 1985.

37. Davies, G. C., Millis, R. R., and Hayward, J. I . Assessment of axillary lymphnode status. Ann. Surg., 192: 148-151, 1980.

38. Kissin, M. W., Thompson, E. M., Price, A. B., Slavin, G., and Kark, A. E.The inadequacy of axillary sampling in breast cancer. Lancet, /: 1210-1212,1982.

39. Pickren, J. W. Significance of occult métastases.A study of breast cancer.Cancer (Phila.), 14: 1266-1271, 1961.

40. Fisher, E. R., Swamidoss, S., Lee, C. H., Rockette, H., Redmond, C., andFisher, B. Detection and significance of occult axillary node métastasesinpatients with invasive breast cancer. Cancer (Phila.), 42: 2025-2031, 1978.

41. I le 11man, S., and Harris, J. R. The appropriate breast cancer paradigm.Cancer Res., 47: 339-342, 1987.

42. Chatal, J. E., Saccavini, J. C., Fumoleau, P., Douillard, J-Y., Curtet, C.,Kremer, M., Mevel, B. I... and Koprowski, H. Immunoscintigraphy of coloncarcinoma. J. Nucí.Med., 25: 307-314, 1984.

43. Epenetos, A. A., Snook, D., Durbin, H., Johnson, P. M., and Taylor-Papadimitriou, J. Limitations of radiolabeled monoclonal antibodies forlocalization of human neoplasms. Cancer Res., 46: 3183-3191, 1986.

44. Siccardi, A. G., Buraggi, G. L., Callegaro, L., Mariani, G., Natali, P. G.,Abbati, A., Bestagno, M., Caputo, V., Mansi, L., Masi, R., Paganelli, G.,Riva, P., Salvatore, M., Sanguineti, M., Troncone, L., Turco, G. L., Scassettati, G. A., and Ferrone, S. Multicenter study of immunoscintigraphy withradiolabelled monoclonal antibodies in patients with melanoma. Cancer Res.,Â¥6:4817-1822, 1986.

45. Carasquillo, J. D., Bunn, P. A., Keenan, A. M., Reynolds, J. C., Schroff, R.W., Foon, K. A., Ming-Hsu, S., Gazdar, A. F., Mulshine, J. L., Oldham, R.K., Perentesis, P., Horowitz, M., Eddy, J., James, P., and Larson, S. M.Radioimmunodetection of cutaneous T cell lymphoma with 111-In-labelledT101 monoclonal antibody. N. Engl. J. Med., 315:673-680, 1986.

1608

Research. on August 25, 2020. © 1989 American Association for Cancercancerres.aacrjournals.org Downloaded from

1989;49:1600-1608. Cancer Res   Joe J. Tjandra, Ian S. Russell, John P. Collins, et al.   Metastases from Breast CancerImmunolymphoscintigraphy for the Detection of Lymph Node

  Updated version

  http://cancerres.aacrjournals.org/content/49/6/1600

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

  .pubs@aacr.orgDepartment 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://cancerres.aacrjournals.org/content/49/6/1600To request permission to re-use all or part of this article, use this link

Research. on August 25, 2020. © 1989 American Association for Cancercancerres.aacrjournals.org Downloaded from