tumor receptor imaging: proceedings of the national cancer ...vol. 1, 921-932, august /995 clinical...

Vol. 1, 921-932, August /995 Clinical Cancer Research 921

Meeting Report

Tumor Receptor Imaging: Proceedings of the National Cancer

Institute Workshop, Review of Current Work, and

Prospective for Further Investigations1

John A. tzbo34

R. Edward Coleman,2 Randall A. kins23

Kenneth A. Kr23 Steven M. Larson,2

John Mendelsohn, C. Kent Osborne,

David Piwnica-Worms, Richard C. �

Barry A. Siegel,3 Michael J. �

and Faina Shtern3Department of Chemistry, University of Illinois, Urbana, Illinois

61801 [J. A. K.]; Department of Radiology, Duke University MedicalCenter, Durham, North Carolina 27710 [R. E. C.]; Department ofRadiology, University of California School of Medicine, San

Francisco, California 94143 [R. A. H.]; Department of Radiology,University of Washington School of Medicine, Seattle, Washington

98195 [K. A. K.]; Departments of Medicinal Imaging [S. M. L.] and

Medicine [J. M.], Memorial Sloan-Kettering Cancer Center, NewYork, New York 10021; Department of Medicine, The University ofTexas Health Science Center, San Antonio, Texas 78284 [C. K. 0.];Divisions of Radiation Sciences [D. P-W., M. J. W.] and Nuclear

Medicine [B. A. S.], Malbinckrodt Institute of Radiology, Washington

University School of Medicine, St. Louis, Missouri 63110;

Department of Radiology, University of Chicago, Chicago, Illinois

60637 [R. C. R.]; and Diagnostic Imaging Research Branch, National

Cancer Institute, Bethesda, Maryland 20852 [F. S.]

AbstractIn February 1994, the National Cancer Institute held a

workshop to evaluate the current and future role of emission

tomographic imaging methods, positron emission tomogra-

phy and single-photon emission computed tomography, in

improving the accuracy of cancer diagnosis and the efTec-

tiveness of treatment and in elucidating basic aspects of

human cancer biology. Reviews covered many of the recep-

tor and transport systems for hormones and growth factors,

as well as metabolic changes important in human cancer,

and topical presentations reviewed the current status of

receptor-based imaging in the most well-characterized sys-

tems: somatostatin receptor imaging of neuroendocrine tu-

mors, estrogen receptor imaging of breast cancer, and epi-

dermal growth factor receptor and tumor metabolic

imaging. A critical analysis was made of the current re-

search and of new directions for the future development and

use of receptor-imaging methods in oncology. In each area,

Received 1/19/95; revised 5/1/95; accepted 5/2/95.I This workshop also received support from the Department of Energy,

Office of Health and Environmental Research.2 Member of the Organizing Committee.3 Workshop Panel Moderator.

4 To whom requests for reprints should be addressed, at Department ofChemistry, University of Illinois, 461 Roger Adams Lab, Box 37, 600

South Mathews Avenue, Urbana, IL 61801.

recommendations were made for further investigation,

where emerging understanding of tumor cell biology and

defined molecular targets might be combined with the meth-

ods of radiopharmaceutical design and evaluation, to de-

vebop new approaches to critical issues in the diagnosis,

staging, and treatment of cancer through tumor receptor

imaging.

IntroductionCancer is now the second leading cause of death in the

United States and, by the next century, is predicted to emerge as

the leading cause ofdeath (1). Although significant progress has

been made in cancer diagnosis and treatment, the annual mom-

tality statistics attest to both the magnitude of the problem and

the challenge it poses to medical science.

Major advances in understanding the molecular biology of

cancer are now making possible new diagnostic and treatment

strategies linked to the fundamental molecular characteristics of

cancer (2). Imaging methods based on radiophammaceuticals

targeted to receptor systems and metabolic characteristics that

are specific to cancer offer intriguing opportunities for the

development of a new generation of diagnostic methods. Some

of these are already beginning to be realized in areas of neu-

noendocnine tumor imaging with somatostatin analogues and

breast tumor imaging with steroids and metabolic probes, and

promising developments are under way in the area of tumor

growth factor receptors and transport systems. Although still at

an early stage of development, these imaging methods may

facilitate both improved cancer diagnosis and staging, and early

assessment of the effectiveness of treatment, by providing a

more accurate in vito characterization of tumor receptor status

and metabolic activity.

In February 1994, the Diagnostic Imaging Research Branch

of the Division of Cancer Treatment of the National Cancer

Institute (Rockville, MD) convened a workshop on ‘ ‘Tumor

Receptor Imaging’ ‘ in Bethesda, MD. Organized as a series of

formal presentations that covered the major areas of ongoing

research, followed by topical panels and open discussion in

which each area was subject to critical analysis, this workshop

covered many of the new advances in cancer biology involving

receptors for hormones and growth factors and certain biochem-

ical changes in the malignant state that may serve as targets for

radiopharmaceutical design for tumor imaging.

This report presents a summary of the workshop presenta-

tions and discussions covering ongoing work in three areas:

somatostatin receptor imaging of neunoendocnine tumors, ste-

noid receptor imaging of hormone-responsive cancers, and im-

aging based on tumor growth factor receptors and certain tnans-

port systems. It also provides an analysis of current research and

new possibilities for using tumor receptor imaging to address

important issues in cancer diagnosis and therapy, and it con-

Research. on June 4, 2020. © 1995 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

http://clincancerres.aacrjournals.org/

Natural Ligand Imaging Agentor Substrate

A-G-C-K-N-F-F�,,5

5 KC-S-T-F-T0’

Somatostatin (identified, 1973)

dF-C-Y�sd�V5 K

HO-T-C-T �

[tTh1�Tyr]�Octmeotide

��DTP�dF�C-F �

HO-T-C-T�

1111n-Pentetmeotide (OctmeoScan#{174})

Estradiol 16a-[18F]Fluomoestradiol (FES)

Glucose

HO

2-[18F]Fluomo-2-deoxyglucose (FDG)

922 Tumor Receptor Imaging: A Workshop Report

eludes each area with a series of recommendations for further

investigation.

Overview: Nuclear Medicine and Cancer-New

Opportunities from Tumor-Receptor ImagingIn a comprehensive review entitled, ‘ ‘Tumor Imaging:

Current Status, Advances and Limitation of Nuclear Medicine”,

Steven M. Larson (Memorial Sloan-Kettering Cancer Center,

New York, NY) noted that general nuclear medicine procedures

contribute significantly to the practice of oncology. For exam-

ple, common procedures such as bone scans and gallium scans

are used to detect and stage a variety of musculoskeleta! and soft

tissue tumors, while nuclear medicine cardiac wall motion stud-

ies are helpful in monitoring potential cardiotoxic effects of

some forms of chemotherapy (3).

In the last decade, nuclear medicine has benefited greatly

from advances in technology of imaging; in particular, improved

instrumentation such as modern PET5 and SPECT, and comput-

enized image processing, especially methods for ‘ ‘fusion’ ‘ of

emission tomographic images with CT or MRI images. The

preparation of new radiopharmaceuticals and the development

of quantitative modeling methods enable the study of hitherto

inaccessible chemical processes occurring at nanomolar concen-

trations in living tissues in vivo. These methodological tools are

now available for fundamental studies on tumors and practical

applications in clinical oncology. The ‘ ‘new cancer cell biol-

ogy,’ ‘ i.e., the explosion of basic knowledge about cancer cau-

sation and prevention, can be used as a road map for radiophar-

maceutical development (2). These radiopharmaceuticals may

provide in return, through images based on interactions with

specific molecular targets, detailed information on receptor 1ev-

els and metabolism in vivo. This information about individual

patients can be used to address critical issues in cancer diagnosis

and effectiveness of therapy.

Metabolic imaging of tumors by PET is already a clinical

reality, and some aspects of this method have been reviewed

recently (4). This method depends on accelerated metabolism in

tumor versus normal tissue: increased glucose utilization, DNA

synthesis, and protein synthesis are hallmarks of the transformed

cell, with the rate of increase roughly corresponding to the rate

of growth of these tumors, and positron-labeled sugars, amino

acids, and nucleotides have been successfully used for tumor

imaging based on their increased uptake by tumors. Metabolic

imaging with the glucose analogue FDG (Fig. 1) appears to have

considerable potential in lung, breast, and colon carcinoma,

lymphoma, thyroid carcinoma, and primary brain tumors. An-

ticipated benefits include improvements in distinguishing be-

nign from malignant tumors, staging of cancers, detecting re-

5 The abbreviations used are: PET, positron emission tomography; AR,andmogen receptor; CT, computed tomography; EGF, epidermal growthfactor; ER, estrogen receptor; �‘#{176}Tc,technetium-99m; FDG, 2-[’8F]-fluomo-2-deoxy-D-glucose; FES, 16a-[18F]fluonoestradiol-17�3; GR, glu-

coconticoid receptor; MDR, multidnug resistance (expression of mdr gene);

PR, progesterone receptor; SPED’, single-photon emission computed to-mography; TGF-a, transforming growth factor a.

dF-C-F �5 dWsk

HO-T-C-T�’

Octmeotide (Sandostatin#{174} SMS 201-995)

IBREAST TUMOR IMAGING 1

GLUCOSE METABOLiC IMAGING 1

Fig. 1 Natural ligands on substrates for tumor receptors or metabolic

systems and analogues used for imaging. Left, natural ligand for the

somatostatin receptor and the improved ligand (octreotide), natural

ligand for the ER, and normal substrate by the glucose transporter.Right, ligand and substrate analogues used for imaging these receptors

or metabolic systems.

currence, and predicting and monitoring the response of tumors

to anticancer treatment.

The proliferation of cancer cells is regulated by a number

of hormones and growth factors that bind to membrane and

intracellular receptors. These hormone growth factor receptor

systems activate signal transduction pathways that are important

in controlling transcription and cell growth, and are potential

targets for imaging agents that could be used to identify specific

tumors and that might provide an assessment of tumor receptor

levels. Examples of several hormone growth factor receptors

and transporter proteins of potential use in imaging specific

tumors are shown in Table 1, along with ligands on antibodies

that bind to these receptors. In addition to the specific tumor

receptor and transporter systems shown in Table 1, there are

many tumor-associated antigens which are being investigated

for tumor-specific imaging with antibodies (16-19).

Agents used to image these specific tumor receptor systems

may be ligands for these receptors (e.g., somatostatin derivatives

for the somatostatin receptor and estrogens for the ER), sub-

strates (e.g., sestamibi for the MDR transporten P-glycoprotein),

or antibodies for several of the growth factor receptors.

mAbs (20) have played an important role in detecting

tumor-associated antigens that eventually are shown to have

important biological functions, and they can serve as archetypi-

cal ligands for these antigen receptors. They are often highly

specific and have been used to map the receptor distribution in



Clinical Cancer Research 923

Table I Examples of tmansmembrane growth factor receptors, intracellular steroid receptors, and transport proteins of potential use in specific

tumor imaging

Imaging agent/System Ligand Tumor Function antibody Reference

Receptors

EGF receptor

ER

EGF, TGF-a

Estradiol

Non-small cell lungcarcinoma

Breast cancer

Growthregulation

Growth

Ab225, 528

FES

(5)

(6)

HER-2/neu Heregulin Breast, ovaryregulation

Growth Ab4D5 (7)

Gastrin-releasing Bombesin GRP Small cell lung

regulation

Growth Anti-GRP (8)

peptide receptor carcinoma regulation

Interleukin 2 Interleukin 2 Lymphoma T cells Activation Anti-TAC (9)receptor

Somatostatin Somatostatin Neuroendocnine Growth ‘ I ‘In-labeled (10)receptor regulation pentetreotide

Transporterp-Glycoprotein Biologically

derived

drug/toxins

LeukemiaColon carcinoma

Renal carcinoma

Detoxify MDR Sestamibi;AbMRK-l6

(1 1, 12)

Glucose transporter Monosaccharides Glioma

Lung carcinomaBreast carcinoma

Glucose

uptakeFDG; anti-GT1 (13, 14)

Transferrin Transferrin Lymphoma Lung Iron and AbD6S.30 (15)receptor carcinoma trace metal A27. 15

transport

normal tissues and in tumors. mAbs themselves can be used to

develop novel targeting madioligands, but optimization of their

behavior as madiopharmaceuticals may require protein engineer-

ing.

The technology of nuclear medicine has advanced to the

point where the stage is set for important advances in our

understanding of the biology of human tumors based on nonin-

vasive imaging approaches. The recent advances in the molec-

ubam and cell biology of cancer now point the way toward

development of nadiopharmaceuticabs targeted to specific recep-

tons or metabolic characteristics of tumors, and imaging based

on these agents may provide new insights into the cause and

prevention of cancer.

Workshop Summary, Analysis of Current Research

and New Directions, and Recommendations for

Further Investigation

Somatostatin Receptor Imaging of Neuroendocrine Tumors

Workshop Summary (Presentations by Thomas M.

0 ‘Dorisio and Larry K. Kvols). ‘ ‘Somatostatin Receptor Im-

aging’ ‘ was presented by Thomas M. O’Donisio (Ohio State

University Hospitals, Columbus, OH), who reviewed the use of

this receptor system in tumor imaging, utilizing long-acting

somatostatin analogues (21, 22). Various neuroendocnine tu-

moms, such as carcinoids, vasoactive intestinal peptide-secreting

tumors, insulinomas, glucagonomas, and gastninomas, contain

somatostatin receptors, and somatostatin and its analogues have

been used in the treatment of clinical endocrine syndromes that

result from these tumors. The usefulness of somatostatin itself (a

tetnadecapeptide) as a therapeutic agent on in vivo probe, how-

ever, has been limited by its very short plasma half-life.

Sandoz Inc. developed a bong-acting somatostatin ana-

bogue, the modified octapeptide octmeotide (SMS 201-995, San-

dostatin; Fig. 1), which is far more resistant to enzymatic

degradation than somatostatin; its plasma half-life is 60 mm

compared with 2-3 mm for somatostatin. Octreotide has been

shown to be efficacious in the treatment of gut endocrine tu-

moms, and Lamberts et a!. (10, 23) have utilized a modified form

of octreotide (in which the phenylalanine residue at position 3 is

replaced with a tyrosine residue radiolabeled with 1251 or 1231;

Fig. 1) to detect somatostatin receptors in human endocrine

tumors. [‘ 23I-Tyn�Joctmeotide scintigmaphy localized primary

pancreatic tumors, gastninomas, and many other endocrine tu-

moms, and identified previously unknown metastases (Fig. 2, A

and B).

The group at Ohio State (22) has used [‘251-Tyr1]octreotide

in conjunction with a hand-held probe to measure the activity of

areas of suspected tumor mass for the intmaoperative localization

of neunoendocnine tumors. In seven of seven patients with

known or suspected neuroendocnine tumors (localized by CT

scanning), the tumor mass accumulated labeled octreotide at a

ratio of 2: 1 compared with adjacent normal tissue, whereas in

four of the five patients with clinically suspected tumors, no

tumor was found. The Ohio State group suggests that intmaop-

erative localization utilizing iodinated octreotide can be useful

for both tumor detection and for determining which patients

may benefit from octreotide therapy.

In a related presentation, Larry K. Kvols (Mallinckmodt

Medical, Inc., St. Louis, MO; formerly Mayo Clinic and Foun-

dation, Rochester, MN) discussed the experience at Mayo Clinic

utilizing somatostatin. In 77% of the patients suffering from

malignant cancinoid syndrome, administration of octreotide me-

sulted in clinical improvement. A subsequent study showed a



Ii’

- #{149}

c� c�i

-� � . �.-�


have been performed in many institutions using labeled oct-

M

r:iL:j ,�-.

:-:�

Fig. 2 Radionuclide images of tumors based on receptor binding or metabolism of a radiopharmaceutical tracer. A, somatostatin receptor imaging

24 h after injection of ‘ ‘ ‘In-labeled pentetreotide in a 55-year-old man with multiple paragangliomas. The anterior image of the head and neck

demonstrates a right glomus tympanicum tumor (7), a left gbomus jugulare tumor (1), bilateral carotid body tumors (CB), and a left mediastinal tumor(Al) (Courtesy of T. M. O’Dorsio, unpublished data). B, anterior 24-h “tn-labeled pentetmeotide image of the abdomen in a 74-year-old woman witha 1.5-cm insulinoma (arrow) in the head of the pancreas. Normal uptake of the tracer in the liver, spleen, and kidneys is evident, as is normal excretionof the tracer into the bowel (Courtesy of T. M. O’Donisio, unpublished data). C and D, PET images obtained with FES (C) and EDO (D) in a

55-year-old woman with a 3- x 3-cm ER-positive carcinoma of the might breast. The images shown are anterior maximum-pixel-intensityvolume-rendered reprojection views compiled from a series of transa.xial PET scans. Both images show tracer uptake in the primary tumor (long

arrow) and in ipsilateral palpable axillary nodes (short arrow). The FES image shows normal intense tracer uptake in the liver (L); the FDG imageshows normal activity in the myocardium (Al) and the liver (L), and excreted tracer in the left renal collecting system (K) (Courtesy of F. Dehdashtiand B. Siegel, unpublished data).

correlation between the presence of tumor somatostatin recep-

ton, assayed by autoradiography with [‘25I-Tyr�]octreotide, and

the response to octreotide in patients with advanced, metastatic,

neuroendocrine tumors. Receptors were detected in cancinoid

tumors in 16 of 20 patients, and all but 1 of these 16 patients

improved clinically after treatment with octreotide. In contrast,

only one of the four patients with receptor-negative carcinoid

tumor showed clinical improvement. All eight patients with

metastatic islet cell carcinomas were positive for somatostatin

receptors and showed improvement with octreotide treatment,

while three patients with medullary carcinoma of the thyroid

were receptor negative and showed no response. Thus, the

presence of somatostatin receptors in malignant neuroendocrine

tumor tissue appears to correlate with the response to octreotide

therapy.

The Mayo group evaluated [‘23I-Tyr�’]octreotide in vivo to

detect known carcinoid and islet cell tumors and to correlate

tumor uptake with the presence or absence of somatostatin

receptors. Tumors were best seen on scans obtained 1-4 h after

injection. Of the 28 patients, 22 had positive scans with uptake

in tumors, 3 showed photon-deficient uptake in regions of

known tumors, and 3 had negative scans; 17 patients in whom

results of the tumor biopsy were positive for somatostatin me-

ceptors had positive scans, and 1 patient in whom results of the

biopsy were negative for somatostatin receptors had a negative

scan. Previously unsuspected lesions were detected on the 123I

labeled octreotide scans in 4 of the 28 patients. Thus, 1231..

labeled octmeotide appears to be a useful tracer for the bocaliza-

tion of neuroendocnine tumors and, most likely, other soft tissue

tumors as well.

Analysis of Current Research and New Directions

(Moderators: Michael J. Welch and Larry K. Kvols; Contribu-

tions by Mario Maggi, Edward Deutsch, and J. Lister-James).

The presentations and discussions in this panel focused on the

identification and characterization of the somatostatin receptors

in tumors, as well as approaches that have been taken to image

these receptors with novel radiopharmaceuticals. As was me-

viewed above, somatostatin-based tumor-imaging agents are the

most developed members of this class. Clinical studies, which




reotide (Sandostatin), have demonstrated the utility of this me-

cepton probe in identifying tumors and predicting their respon-

siveness to therapy with somatostatin analogues (23, 24).

A Sandostatin analogue labeled with ‘ ‘ ‘In via a diethyl-

enetetramine pentaacetic acid bifunctional chebate, ‘ ‘ ‘In-labeled

pentetreotide, initially developed in collaboration between San-

doz and investigators at the University Hospital Rotterdam (25),

is being commercialized by Mallinckrodt Medical, Inc. under

the trade name OctreoScan. As discussed by Edward Deutsch

(Mallinckrodt Medical, Inc.), this agent is being evaluated in

several European centers where clinical trials are being carried

out, aimed at establishing its efficacy in diagnosing a variety of

diseased states, including neuroendocnine tumors, lymphomas

(both Hodgkin’s and non-Hodgkin’s), breast cancer, small cell

lung cancer, and various other cancers (Fig. 2, A and B). The use

of ‘ ‘ ‘In-labeled pentetreotide in detecting neunoendocnine tu-

moms was approved by the U.S. Food and Drug Administration

in June 1994, and U.S. trials to establish the utility of this agent

in detecting lymphomas are under way. Dr. Deutsch also

pointed out that other nadiolabeled forms of octreotide are being

developed in academia and industry; these include ‘8F, 68Ga,

and 64Cu compounds for PET imaging and 90Y, ‘86Re, MCu,

and 67Cu forms for radiotherapy.

The development of new 99mTclabeled somatostatin ana-

bogues was discussed by J. Listen-James (Diatech, Inc., Lon-

donderry, NH). 99mTc has advantages over other radionuclides

due to its ready availability and the fact that, in many cases,

madionuclides of rhenium can be substituted for technetium to

develop agents for radiotherapy. A lead compound that has a

high affinity for the receptor (K, = 0.15 n�t as the rhenium

complex) and shows high uptake in rat tumors will soon be

ready to undergo clinical trials.

The presentations in this panel showed the current interest

in the studies of somatostatin and nonmetabolizabbe peptide

analogues, both for diagnosis and for therapy, utilizing both the

native peptide and the peptide labeled with various madionu-

clides. These many evident successes nothwithstanding, theme

are fundamental questions regarding the basic biology of soma-

tostatin receptors that remain unknown. For example, as dis-

cussed by M. Maggi (University of Florence, Florence, Italy), at

least five types of somatostatin receptors have been identified,

all of which have been cloned, and somatostatin and octreotide

display different binding affinities to these five sites (24, 26,

27). SomatostatinRl receptors, present in three of eight neuno-

blastoma cell lines, bind with low capacity and very high affin-

ity, whereas somatostatinR2 receptors have a high capacity and

were widely distributed in all of the cell lines investigated. The

active interplay between clinical studies utilizing labeled oct-

neotide in imaging various tumors and basic studies on the

nature and distribution of somatostatin receptor subtypes may

further improve both the diagnostic and therapeutic uses of these

receptor probes, and advance our fundamental understanding of

the receptors for this growth-regulating peptide.

Recommendations for Further Investigation. The nec-ommendations are:

Compare further diagnostic results with therapeutic out-

come utilizing somatostatin analogues in various tumor systems.

Evaluate whether somatostatin receptor imaging is a cost-

effective alternative to other imaging modalities.

Investigate whether it is possible to quantify the number of

receptors from radiolabeled somatostatin analogue uptake and

thereby predict whether patients would benefit from therapy

with a somatostatin receptor-directed �3-emitting, Auger-emit-

ting, on a-emitting nadionuclide.

Develop agents with potential for therapeutic applications

that have less accumulation in normal tissue, particularly in the

organs involved in their metabolism and excretion.

Examine the effect of various treatment regimens, such as

chemotherapy, immunotherapy, and hepatic deartenialization,

on the expression of somatostatin receptors through sequential

imaging with a somatostatin analogue.

Correlate receptor subtype with tumor type to see whether

the content of the five known subtypes of receptor vary with the

type of tumor known to contain somatostatin receptors.

Develop agents specific for the various receptor subtypes.

Steroid Receptors and Imaging Hormone-resnonsive

Cancers

Workshop Summary (Presentations by Farrokh Deh-

dashti and Richard Wahi). The use of a clinically refined

receptor system for PET imaging was presented by Farrokh

Dehdashti (Mallinckrodt Institute of Radiology), who summa-

nized the Saint Louis experience with ‘ ‘Estrogen Receptor Im-

aging in Breast Cancer.’ ‘ ERs are found at quite high bevels in

biopsy samples of certain breast cancers, and the tumor response

to hormone therapy often correlates quite well with ER levels.

Using FES (Fig. 1) as a probe for ERs (28), this group has

shown the following: (a) it is possible to identify the ER-

positive breast tumors by PET with FES (29); (b) FES bocaliza-

tion in ER-positive distant metastases from breast carcinoma

(e.g. , bone and soft tissue) is receptor mediated, as the uptake is

significantly blocked by antiestrogen treatment (6); (c) FES

uptake is quantitatively related to the ER concentration (Fig. 3),

but not to cell metabolism measured by FDG uptake; (6) FES

uptake is selective and occurs only in ER-positive malignant

tumors, whereas FDG uptake is increased in both ER-positive

and ER-negative malignant tumors (Fig. 2, C and D), and

abnormal uptake of either probe does not occur in benign breast

tumors; and (e) FES uptake indicates hormone status and, there-

fore, potentially predicts hormone responsiveness, while FDG

metabolism may predict response to chemotherapy (30). These

studies appear to provide a ‘ ‘proof of concept’ ‘ that quantitative

tumor receptor imaging has the potential to contribute useful

clinical information regarding prognosis and selective therapeu-

tic response in patients with breast cancer.

The use of metabolic imaging in cancer diagnosis was

reported by Richard Wahl (University of Michigan Medical

Center, Ann Arbor, MI) in his presentation ‘ ‘Tumor Imaging

With PET.’ ‘ PET imaging using FDG (Fig. 1) has demonstrated

the potential for noninvasive tumor staging, including the de-

tection of lymph node metastases from breast and lung cancers

as well as from melanoma and head and neck carcinoma, based

on the increased glucose utilization in these malignant tumors

(31). For tumor staging and assessment of treatment response,

PET appears to have advantages over purely anatomical imag-

ing methods in that it can separate, in many cases, scam and other

nontumorous masses from viable tumor. Similarly, detection of



.,t�

6

4

2

0

TUMOR UPTAKE VS ER CONCENTRATION


-2 � � � � � � . I . � . I . I

0 25 50 75 100 125 150 175 200

RECEPTOR CONCENTRATION (fmol/mg protein)

Fig. 3 Relationship between breast tumor uptake of FES and ERcontent of the tumor. Tumor uptake is corrected for nonspecific bindingand partial volume effect, and expressed as percentage of injecteddose/mb X i0-� (Adapted from Ref. 6).

small tumors also appears to be possible with PET in some

settings in which CT and other morphological methods are

insensitive. Dr. Wahl believes that primary uses for the FDG

probe in cancer will be to differentiate a benign from a malig-

nant mass, to determine the tumor growth mate, to differentiate

necrosis from recurrent or residual tumor, and to monitor re-

sponse to therapy in individual patients (13, 32).


[Moderators: John A. Katzenellenbogen and Barry A. Siegel:

Contributions by C. Kent Osborne (University of Texas Health

Science Center, San Antonio, TX), Eugene DeSombre (Univer-

sity of Chicago, Chicago, IL), Donald Tindall (Mayo Research

Foundation, Rochester, MN), Mark Schoenberg (Johns Hopkins

University, Baltimore, MD), E. Brad Thompson (University of

Texas, Galveston, TX), John Katzenellenbogen (University of

Illinois, Urbana, IL), and Michael Welch (Mallinckrodt Institute

ofRadiology)]. In the area of steroid hormone-responsive can-

cers, the dual potential of radiodiagnostic imaging (a) to assess

on characterize function by quantification of tumor receptor

levels or metabolic activity, and (b) to locate tumors based on

these functions or activities is beginning to be realized. Two

general applications can be envisioned: (a) imaging of tumor

function on location to assist in patient staging or prognosis in

order to make a more appropriate selection of alternative treat-

ments, and (b) imaging of tumor function to make an early

assessment of the effectiveness of therapy.

Breast Cancer. ER and PR are well established as markers

that are useful as both predictive (response to hormonal therapy)

and prognostic (tumor aggressiveness) factors (33, 34). Patients

with receptor-negative tumors have a more rapidly progressive

clinical course and more frequent visceral metastases; they show

a low, but still significant response rate to tamoxifen treatment.

Patients with receptor-positive tumors have a more indolent

course and more frequently benefit from endocrine therapy such

as tamoxifen (especially tumors with high receptor content;

Refs. 35 and 36). Since drugs like tamoxifen have little toxicity,

many patients are treated after primary surgery even if there is

only a small chance of benefit; yet, the drug is costly and does

have side effects, as is becoming increasingly evident in major

recent tamoxifen trials (37), so that prospective identification of

patients with no real chance of benefit from tamoxifen treatment

would be useful clinically.

In their overview presentations, Dns. Wahl and Dehdashti

demonstrated in preliminary clinical studies that primary and

metastatic tumors (that included axillany and mediastinal lymph

nodes and distant lesions) could be cleanly imaged based on ER

uptake of FES and/or elevated FDG uptake (13, 30, 31). The

latter method enabled a reliable distinction between benign and

malignant disease (31, 32), and the former, a reliable assessment

of ER positivity (6, 29, 30). In addition, a good correlation was

found between tumor FES uptake and ER levels by quantitative

assay (Fig. 3; Ref. 29). FES uptake decreased after tamoxifen

therapy (6), and FDG uptake was reduced after chemotherapy

(13). More extensive studies are needed to investigate the nela-

tionship between tumor FES and FDG uptake and the clinical

outcome of hormonal or chemotherapy.

Other potential applications include the following: (a) The

concurrent assessment of tumor metabolic activity (with FDG)

and ER content (with FES) in multiple metastases in a single

breast cancer patient might provide an internally controlled

assessment of tumor heterogeneity; this might be particularly

important in recurrent disease or in situations where tumor sites

are inaccessible for ER assay. (b) To the extent that FES on FDG

imaging can provide a reliable assessment of the involvement of

lymph nodes, these noninvasive methods might obviate diag-

nostic lymph node dissection in some patients; the specificity of

imaging in providing a negative assessment of lymph node

involvement would need to be established carefully, since mi-

croscopic metastases may be important pnognostically. (c) These

imaging methods might enable a rapid assessment of the poten-

tial effectiveness of hormonal therapy prior to surgery or other

treatment. In a ‘ ‘preoperative tamoxifen challenge test, ‘ ‘ the

effectiveness of hormonal therapy might be evident after even a

brief (1-3-week) course of tamoxifen treatment through an

alteration in tumor metabolic activity. The transient agonist

activity that may precede the onset of effective tamoxifen an-

tagonism, that is manifest in some patients as a tamoxifen flare

(38), could result in a transient elevation of tumor FDG uptake

followed by an eventual suppression as tamoxifen antagonism

begins to dominate. The results of such a test might provide a

more reliable means for selecting hormonal therapy in the

primary or adjuvant setting, and could reduce the cost and risk

of ineffective or superfluous treatment.

In the ER and other steroid receptor systems, efforts are

being made to develop SPECT imaging agents, and theme are

published clinical SPECF studies with two iodoestrogens in

breast cancer (39-41). These agents, as well as ones labeled

with 99mTc (42, 43), could take advantage of the greaten avail-

ability associated with SPECT compared to PET imaging. The

potential for ER-directed therapy (44) based on Auger electrons

emitted from t23I is being explored as an approach to localized

radiotherapy of ER-positive cancers.

PR is also a potentially useful target in breast cancer. While

PR-based imaging agents have been prepared (45), none has yet




proved useful in humans (46). If a good agent was available, it

could be used for tumor receptor imaging in patients on tamox-

ifen therapy (that would result in full occupancy of ER), and the

rise in PR levels reported early after the start of tamoxifen

treatment (47) might offer another means for establishing the

effectiveness of hormone therapy.

Prostate Cancer. Although AR is detectable in most prostate

cancers, it has not proven to be a reliable predictor of response to

hormone therapy (48, 49). Nevertheless, imaging based on the

uptake of a suitable radiolabeled AR ligand on elevated metabolic

activity by the primary tumor on metastatic sites might provide an

effective means for staging the cancer. In particular, a more defin-

itive determination of disease dissemination or pelvic lymph node

involvement would assist in the decision to undertake radical

prostatectomy. The need for improved staging is becoming more

acute, because the sensitive detection methods of transrectal ultra-

sonography, magnetic resonance imaging, and prostate-specific

antigen are resulting in increasing numbers of patients identified at

early stages (50). In advanced prostate cancer, an AR imaging

probe could also be of value to assess the likelihood of response to

a second endocrine manipulation after failure of initial andnogen

deprivation (onchiectomy). Although they have not yet been studied

in humans, some ‘8Flabeled andnogens have been used to image

the prostate in baboons with PET (51); a SPECT agent is under

investigation (52).

Ovarian Cancer. This cancer commonly spreads i.p., and

present imaging techniques are inaccurate in identifying the pres-

ence of the metastases. Although diagnostic imaging of penitoneal

metastases using nadiolabeled steroids might be limited by the

presence of gut activity from hepatobiliary excretion of the metab-

olites, a hand-held detector might aid the surgeon in locating small

receptor-positive lesions. Furthermore, metabolic imaging with

FDG appears to be promising in detecting ovarian metastatic dis-

ease. Receptor-directed radiotherapy with Auger electron-emitting

nadionuclides might provide a more effective therapy, especially

for micrometastases, than is currently available (44).

Leukemias and Lymphomas. While the application of

tumor imaging based on GR must await the development of

effective nadiopharmaceuticals (53), the correlation of GR levels

with response to therapy could make diagnostic imaging a

useful prognostic tool (54, 55). GR-based imaging on metabolic

imaging using FDG could assist in the location of lymphoid

tumors, and GR imaging in certain nonlymphoid tumors where

glucocorticoids are of benefit could also be of value, as would

receptor imaging methods that can surmount the difficulties in

GR evaluation in sequestered sites (e.g. , central nervous system,

bone marrow, and pancreas).

Recommendations for Further Investigation. The nec-

ommendations are:

Breast Cancer. Establish clinical correlations between the

uptake of FES and FDG and the outcome of hormone therapy

and chemotherapy.

Determine whether tumor heterogeneity in patients with

multiple metastases can be determined by combined FES and

FDG imaging and related to the success of therapy.

Develop an effective PR-based imaging agent.

Identify optimal ligands and imaging times to assess the

potential of SPECT imaging for breast cancer.

Establish whether the assessment of lymph node involve-

ment by madiolabeled estrogens or metabolic markers is suffi-

ciently reliable to reduce the need for axillary node dissection.

Investigate whether the potential success of hormone treat-

ment can be assessed preopenatively with a tamoxifen challenge

test that would involve a rapid, functional assessment of tumor

response based on changes in the uptake of FDG or a PR ligand.

Investigate the possibility of developing imaging agents for

other markers of steroid-induced responses (e.g. , growth factors

on growth factor receptors, other steroid-induced specific gene

products, as well as general metabolic markers) that might be

used in functional assessment of endocrine therapy. Such a

predictive test would also be useful in deciding between che-

mothenapy and hormonal therapy in patients with advanced

metastatic breast cancer.

Prostate Cancer. Determine whether an AR-based imaging

agent on FDG can provide (a) a definitive assessment of disease

dissemination on pelvic lymph node involvement to assist in the

selection of therapy, and (b) an estimation of the likelihood of

response to a second endocrine manipulation after failure of

initial androgen deprivation.

Ovarian Cancer. Determine whether novel methods for

receptor-directed radiotherapy can be developed based on the

selective distribution of suitable receptor ligands.

Leukemias and Lymphomas. Develop GR-based agents

suitable for imaging of lymphoid tumors.

Assess the potential for GR- on FDG-based imaging for the

detection of lymphoid tumors in nodes and the brain, and

develop correlates between imaging and treatment.

Ligand Development and Methodology. Develop effective

ligands for PR- and OR-based imaging.

Develop and evaluate receptor-based imaging agents la-

beled with more readily available radionuclides useful for

SPECT, such as 670a, “In, �23I and 9mTc

Improve an understanding of the relationship between ste-

moid structure and in vitro measures of binding affinity and in

vivo uptake characteristics.

Develop reliable methods to assess the comparative metab-

olism characteristics of imaging agents in animals and humans

to improve the value of animal studies in predicting radiopham-

maceutical behavior in humans.

Develop methods suitable for receptor quantification in

tumors from in vivo imaging data that address the particular

problems in tumor systems.

Tumor Growth Factor Receptors and Cancer Biology:

New Targets for Imaging

Workshop Summary (Presentations by John Mendelsohn

and David Piwnica-Worms). In his keynote address, ‘ ‘Tumor

Receptors: Biology and Cancer Therapy,’ ‘ John Mendelsohn

(Memorial Sloan-Kettering Cancer Center) described the ad-

vances his laboratory has made in developing antibodies as

antitumom therapeutic drugs. TOF-ce and EGF are required

growth factors for many types of cells, stimulating cell cycle

passage from 0, into the S-phase. TOF-a is produced by the

tumor cells and stimulates cell growth through binding to EGF

receptors, either on the same tumor cell (autocnine action) or

near neighbor tumor cells (panacnine action). EGF receptors




have been identified in nearly all renal, squamous lung, and

head and neck cancers, and in many breast cancers as well as

other cancers. Expression of high levels of EGF receptor and its

ligands, EGF and TOF-ot, in some tumors is associated with a

poor prognosis (56). Although normal cells have a concentration

of approximately 10,000 EGF receptors/cell, there are as many

2 X 106 EGF receptors/malignant cell; this concentration dif-

fenential is a key factor in the potential of this receptor as a

target for diagnostic imaging and radiotherapy.

Dr. Mendelsohn’s group has generated anti-EOF receptor

mAbs, mAb 225 and 528, that bind to the EGF receptor with an

affinity comparable to the natural ligand (Kd = 2 nM), compete

with EGF binding, down-regulate the receptor, and block the

activation of receptor tynosine kinase by EGF or TOF-a (57).

They also block EGF on TGF-a stimulation of the growth of

tumor cells, and in mouse xenograft models they have caused

regression of established human tumors when given in combi-

nation with chemotherapy (58). The munine anti-EOF receptor

mAbs have been labeled with ‘ ‘ ‘In and ‘�‘i, and, in collabona-

tion with Dr. Steve Larson and the Nuclear Medicine group at

Memorial Sloan-Kettering Cancer Center, the Mendelsohn

group has studied the biodistnibution of the labeled mAbs by

imaging patients with several types of cancers (5). The group

has now made human chimenic antinecepton mAbs, and they are

poised to initiate studies in humans.

David Piwnica-Worms (Harvard Medical School, Boston,

MA) discussed MDR, a tumor phenotype that correlates with the

expression of a small family of human multidrug resistant

(MDRJ) and multidrug resistant-associated genes. The protein

products encoded by these MDR genes, including the anchetypic

MDR1 P-glycoprotein, are plasma membrane transporters that

function as energy-dependent efflux pumps of many of the most

potent chemotherapeutic drugs used in cancer treatment, includ-

ing anthracyclines, Vinca alkaloids, actinomycin D, and taxol,

with overlapping, but nonidentical substrate specificities. P-

glycoprotein is physiologically expressed in many organs in-

volved in transport, excretion, and detoxification. This protein

or its mRNA has also been detected at significant levels in

specimens derived from all forms of human cancers, including

leukemias, lymphomas, sarcomas, and carcinomas, and the

tnansfection of cloned P-glycoprotein is sufficient to confer

MDR in experimental systems.

Overexpression of this gene is one of several mechanisms

that may be responsible fon resistance to a broad spectrum of

diverse cytotoxic agents (59, 60) in human cancer, and strategies

designed to block expression or to circumvent this form of drug

resistance are being actively sought by many laboratories in

cancer research (61). Particularly desirable are agents that in-

hibit P-glycopnotein transport activity at concentrations that

alone produce little on no cytotoxic effect. Termed MDR-neven-

sal or -modulation agents, these drugs might be useful in oven-

coming MDR when administered in combination with other

anticancer agents during chemotherapy.


[Moderators: Randall A. Hawkins and Kenneth A. Krohn: Con-

tributions by John Mendelsohn and Steven Larson (Memorial

Sloan-Kettering Cancer Center); Napoleone Ferrara (Genen-

tech, Inc., South San Francisco, CA); Lee J. Helman and Carol

J. Thiele (National Cancer Institute, Bethesda, MD); David

Piwnica-Worms (Harvard Medical School); Ira Pastan and Jeffrey

Schlom (National Cancer Institute, NIH)]. Cell surface proteins

represent important targets for cancer therapy and diagnosis (cf

Table 1), and innovative applications for functional and ana-

tomical imaging with radiolabeled antibodies, peptides, organic

biomimetics, organometallics, and metal-chelate ligands repre-

sent a significant new focus for cancer receptor imaging (62).

Growth Factor Receptors and Signal Transduction in

Cancer. Based on his extensive studies on the EGF receptor-

TOF-a system, John Mendelsohn (Memorial Sloan-Kettering

Cancer Center) summarized three important short-term goals for

the applications of growth factor receptor imaging in clinical

oncology: (a) In Phase I/Il clinical trials explore the use of

mAbs to treat tumors, including imaging with labeled mAbs to

determine critical pharmacokinetic issues such as antibody dis-

tnibution, uptake in both small and large tumor masses, and

retention by tumors as well as normal tissues. If labeled receptor

ligands themselves become available, determine the ability of

the mAbs to block ligand-necepton interaction in vivo and con-

relate this with tumor response and normal tissue toxicity. (b) If

anti-EGF receptor niAb therapy is shown to be effective for

tumors expressing high levels of EGF receptor, use imaging

studies with labeled mAbs on the appropriate madioligands to

identify patients who are likely to respond to treatment. Also,

determine heterogeneity of tumor expression from site to site

and correlate this with tumor response. (c) In patients who are

started on antitumon therapy, use metabolic imaging with FDO-

PET to determine at an early time point (e.g., after 72 h) whether

this expensive and prolonged therapy is going to be worthwhile

for individual patients.

The biochemistry and tumor biology of other tynosine

kinase receptors, such as those for the insulin-like growth fac-

tons I and II (63-65), brain-derived neumotmophin factor (66),

and vascular endothelial growth factor (67, 68), are under in-

tense investigation at the basic level at this time. As insight is

gained into their respective contributions to tumor growth and

angiogenesis, opportunities for application of the madiotnacem

method may anise.

Thus fan, the only agents used to image growth factor

receptors have been antibodies, and most mAbs used thus fan

in clinical trials have been of munine origin (20), typically

IgOs with Mr 160,000. Such large proteins diffuse slowly into

tumors and can cross-react with normal tissues via the Fe

receptor. Thus, tumor uptake selectivity is not always high.

Being munine in origin, these mAbs will in most cases induce

a powerful immune response in humans. Improved behavior

may be achieved through genetic engineering. By grafting the

munine complementanity-determining regions (which occupy

less than 5% of the total IgO) onto a human IgO backbone,

humanized mAbs with reduced immunogenicity are produced

(69); by joining the ends (Fv fragments) of the IgO with a

genetically engineered linker of proper length, one can create

a single-chain antigen-binding protein (70) that retains anti-

gen-binding activity but is much smaller than the IgO. These

single-chain antibodies have improved targeting charactenis-

tics in vivo, with more rapid tumor localization and tissue

clearance (71).

MDR. An important controversy in the MDR field is

whether identification of tissue expression of P-glycopnotein per




se with mAbs or mRNA probes is sufficient, on whether it is

necessary to assay directly the functional transport activity of

this integral membrane protein. It was recently reported that the

lipophilic cationic nadiopharmaceutical uumTc sestamibi is nec-

ognized and transported by the MDR P-glycoprotein (11) and

[3Hjcolchicine, a known P-glycopmotein substrate, has been

shown to differentiate drug-resistant from drug-sensitive tumors

in vivo (72). Intriguing new functional imaging opportunities

enabling direct monitoring of the efflux transport function of

P-glycopnotein may now be feasible with -1’-emitting agents of

these classes. Thus, functional diagnostic imaging approaches

may allow direct assessment of P-glycoprotein expression and

activity in human tumors in vivo. In addition, the effectiveness

of modulating on reversal agents in enhancing tumor delivery of

chemothemapeutic agents could be monitored prior to a full chemo-

therapeutic trial, saving patients the morbidity of a futile cycle of

chemotherapy. Transport substrates like ��mTc sestamibi could

serve as surrogate chemothenapeutic agents, allowing whole-body

and tumor pharmacokinetics to be imaged after modulation them-

apy. Such a “modulator challenge test’ ‘ might provide a means to

select the most effective reversing agent for each tumor type, and

allow patients to be directed to individualized and specific cancer

therapies. Because normal expression of P-glycopnotein in liver,

kidney, and intestine may potentially interfere with abdominal

imaging, these strategies might best be applied to tumors of the

head and neck, breast, lung, and extremities.

Recommendations for Further Investigation. The nec-

ommendations are:

Growth Factor Receptors. Develop nadioligands suitable

for imaging various cancer cell growth factor receptors and

associated signaling systems.

Study the relationships between growth factor receptor

expression (assessed by specific imaging agents), tumor growth

(assessed by FDG metabolic imaging), and prognosis in vivo.

Monitor therapeutic outcome with antigrowth factor mecep-

ton mAbs.

MDR. Develop radiolabeled binding and transport sub-

strates for the MDR P-glycoprotein.

Establish the relationship between tumor expression of

P-glycopmotein, transporter function, and chemotherapeutic out-

come.

Investigate whether successful P-gbycopnotein modula-

tion therapy can be assessed with a modulator challenge test

that would involve the functional assessment of transport

inhibition using changes in tumor uptake kinetics of madiola-

beled P-gbycopnotein substrates as surrogate chemotherapeu-

tic agents.

Issues in the Development of Tumor Receptor Imaging

Radiopharmaceuticals [Moderators: Kenneth A. Krohn and

Carol J. Thiele; Contributions by Stanimir Vuk-Pavlovik (Mayo

Clinic and Foundation, Rochester, MN); Michael J. Welch

(Mallinckrodt Institute of Radiology); John A. Katzenellenbo-

gwN: (University of Illinois); Kenneth A. Krohn (University of

Washington School ofMedicine, Seattle, WA)] While the focus

of the Workshop and this report has been on the application of

radiopharmaceuticals to tumor receptor imaging and not on the

technical aspects of nadiophanmaceutical development per Se, a

number of important points concerning this latter issue were

raised during presentations and discussions.

Although tumor receptor or metabolic imaging (without

quantification) can in many cases provide important infor-

mation on the location of tumors and define certain func-

tional characteristics, the ability to perform tumor receptor

imaging quantitatively may determine how useful this infom-

mation is in making critical diagnostic and therapeutic deci-

sions. The quantification of receptor bevels on metabolic

activity from in vivo uptake studies is a complex issue that

requires the development and validation of phanmacokinetic

models; some of these have been worked out in certain

systems (73-75), but the uncertainty of permeability barriers,

blood flow, and the mix of viable versus necrotic tissue pose

further complications in the development of quantitative me-

ceptor and metabolism models for tumors.

Animal models that are suitable for the qualitative and

quantitative assessment of madiophammaceutical distribution

selectivity and kinetics are critical for the development of

tumor receptor imaging agents for humans. However, one can

encounter marked variation of madiophammaceutical metabo-

lism between animals, nonhuman primates, and humans (76-

78), and even between the sexes (79), such that the behavior

of the agents in humans may be either far worse or consid-

erably better than in rodents (46, 51). Thus, the development

and validation of appropriate animal model systems is im-

pontant. Studies in nonhuman primates can provide an im-

portant middle ground, and in vitro systems for studying

comparative species metabolism may have predictive value

(76-78).

While the mass dose of most tumor imaging nadiophar-

maceuticals is so low as to have no pharmacological effect,

the radiation dose needs to be evaluated carefully. Thus,

administered doses need to be set at levels that will ensure

that the radiation doses to normal tissue in humans are as low

as possible. This is a particular concern for potential radio-

therapy applications. If, for example, an analogue of oct-

reotide were to be used, the route and mate of elimination

would need to be carefully examined, since the nature and the

position of the nadiolabel can markedly affect clearance and

metabolism: � octreotide is retained in the liver,

while ‘ ‘ ‘In-labeled pentetreotide is retained in the kidney

(80), so an analogue with fasten clearance characteristics

would be desirable for therapeutic applications. Although

differences in the metabolism and the routes and rates of

nadiophanmaceutical elimination between experimental ani-

mals and humans can complicate such assessments, reason-

abbe estimates of radiation doses to humans can generally be

made. As noted above, small alterations in radiopharmaceu-

tical design on method of nadiolabeling can have major ef-

fects on the routes and mates of elimination (80, 81); factors

that affect not only the dosimetry, but also the regions of the

body obscured to imaging during the process of elimination.

Conclusions

Recent advances in our understanding of tumor hormone

and growth factor receptor systems and in our knowledge of

biochemical changes that occur during tumor growth have

opened up new opportunities for the use of imaging methods in




clinical oncology and in basic studies of tumor biology. When

targeted appropriately to these receptor systems and metabolic

characteristics, these imaging methods can facilitate cancer di-

agnosis, staging, and treatment. Clinical investigations to date

have been advanced in only a few systems, imaging based on

somatostatin receptors for neuroendocnine tumors, on ERs for

breast cancer, on the EGF receptor in various tumors, and on

glucose utilization as a general tumor metabolic marker, but

there are many opportunities for developing imaging agents

directed at other tumor targets, receptor systems, and metabolic

changes. Some of these possibilities have been presented and

analyzed in some detail in the individual sections of this report.

The areas that appear most fruitful for additional research are

summarized in the ‘ ‘ Recommendations for Further Investiga-

tion. ‘ ‘ The successful development of new tumor receptor im-

aging agents and protocols will continue to demand that careful

attention be paid to aspects of the imaging agent design and

labeling, administration, quantification and modeling, and con-

siderations of safety.

Acknowledgments

We are grateful for helpful comments from William C. Eckelman

and Janet F. Eary.

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1995;1:921-932. Clin Cancer Res J A Katzenellenbogen, R E Coleman, R A Hawkins, et al. further investigations.Institute workshop, review of current work, and prospective for Tumor receptor imaging: proceedings of the National Cancer

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