USE OF 30-DEOXY-30-[18F]FLUOROTHYMIDINE PET/CT FOR EVALUATING

RESPONSE TO CYTOTOXIC CHEMOTHERAPY IN DOGS WITH

NON-HODGKIN’S LYMPHOMA

JESSICA LAWRENCE, MATTHEW VANDERHOEK, DAVID BARBEE, ROBERT JERAJ, DANIEL B. TUMAS, DAVID M. VAIL

Imaging and measurement of proliferation with computed tomography (CT) and positron emission tomography

(PET) provide a noninvasive method for improved staging and monitoring of response to cancer treatment. We

evaluated prospectively the proliferation marker 30-deoxy-30[18F] fluorothymidine (FLT) in the context of FLT-

PET/CT for detection of early response, confirmation of posttreatment response, and prediction of relapse in

dogs with non-Hodgkin’s lymphoma. Nine dogs with non-Hodgkin’s lymphoma who were scheduled to receive

five cycles of an investigational cytotoxic chemotherapy agent were included. All dogs received baseline FLT-

PET/CT imaging immediately before chemotherapy. Intent was to repeat imaging with FLT-PET/CT at

various time points: group 1 (n¼ 4), 5 days after initiation of chemotherapy and 3 weeks following the last

chemotherapy administration; group 2 (n¼ 5), before the fourth cycle of chemotherapy and 3 weeks following

the last administration. Two dogs in group 2 did not undergo repeat PET/CT. Body mass standardized uptake

values (SUV) for FLT were calculated for each dog. Eight dogs had initially increased FLT uptake (mean

SUVmax¼ 9.8 [2.6–22.3]). Mean SUV decreased significantly for the seven dogs that underwent follow-up

PET/CT following chemotherapy (mean SUVmax¼ 3.5 [1.1–7.9], Po0.016). Increased uptake preceded clin-

ical and cytological evidence of relapse in two dogs. Ki-67 immunohistochemistry confirmed decreased pro-

liferation corresponding to decreased SUV in three canine lymph node samples. FLT-PET/CT functional and

anatomical imaging shows promise for the evaluation of response to cytotoxic chemotherapy in dogs with non-

Hodgkin’s lymphoma and for predicting relapse before standard clinical and clinicopathologic confirmation.

Veterinary Radiology & Ultrasound, Vol. 50, No. 6, 2009, pp 660–668.

Key words: 30-deoxy-30[18F] fluorothymidine, canine, non-Hodgkin’s lymphoma, positron emission

tomography.

Introduction

CANINE NON-HODGKIN’S LYMPHOMA is a spontaneous,

rapidly proliferative neoplasia that is similar in bio-

logic behavior to high-grade non-Hodgkin’s lymphoma in

humans. Standard therapy for lymphoma involves multi-

agent chemotherapy administered systemically. Initial

treatment of lymphoma is often rewarding, however, re-

lapse is common, and only 25% of dogs survive 2 years

or longer with standard protocols.1 Relapse is most often

detected by reenlargement of lymph nodes or return of

clinical signs, at which point significant tumor volume has

developed. Early detection of relapse permits earlier inter-

vention, which may result in longer response duration and

improved outcome. Methods of detecting early response

and relapse are of interest in the management of canine

lymphoma, among other tumors. In addition, accurate

monitoring of therapy is extremely important as new cyto-

toxic or molecular agents are investigated.

There is a tremendous interest in molecular imaging

for the diagnosis, staging, and evaluation of response

for individuals with cancer. Modalities such as photon

emission computed tomography (SPECT), positron emis-

sion tomography (PET), magnetic resonance imaging,

and computed tomography (CT) allow precise anatomic

localization and offer a unique method of analyzing

tissue differences in response to therapy. Nuclear medicine

imaging methods such as PET use tracers to target

specific mechanisms that typically differ between normal

and cancer cells, often preceding changes in anatomic

structure identified with conventional imaging.2–10 Func-

tional imaging has garnered much attention due to its po-

tential ability to detect early response to therapy and

differentiation of tumor from inflammation, necrosis, or

fibrosis.2,11–14

Funding: Provided by Gilead Sciences Inc.Material presented in part: Veterinary Cancer Society 2007 (Ft. Lau-

derdale, FL) and American College of Veterinary Radiology 2008 (SanAntonio, TX).Address correspondence and reprint requests to David M. Vail, at the

above address. E-mail: vaild@svm.vetmed.wisc.eduReceived May 17, 2009; accepted for publication June 24, 2009.doi: 10.1111/j.1740-8261.2009.01612.x

From the School of Veterinary Medicine (Lawrence), Department ofMedical Physics (Vanderhoek, Barbee, Jeraj), Paul P Carbone Compre-hensive Cancer Center (Jeraj, Vail), University of Wisconsin-Madison,2015 Linden Drive, Madison, WI 53706, and the Department of DrugSafety Evaluation, Gilead Sciences Inc., 333 Lakeside Drive, Foster City,CA 94404 (Tumas).

660

PET using the glucose analogue 2-[18F]fluoro-2-deoxy-D-

glucose (FDG) is the most commonly utilized imaging

metabolic biomarker.15 FDG significantly enhances the

sensitivity of detection of lymphoma and residual uptake is

an accurate and independent predictor of progression-free

survival.16–22 Consequently, FDG-PET is now considered

a standard component to the routine workup of human

lymphoma patients.15,23,24 FDG-PET imaging has recently

been evaluated in canine lymphoma and mast cell tumor

and was useful in staging both diseases.25

FDG is not tumor specific, however, and can also ac-

cumulate in inflammatory tissues, which can confound on-

cologic imaging.26–28 Much attention has been directed

toward other tracers that can increase the specificity for

malignant lesions to complement the information currently

obtained with FDG functional studies. For example, pro-

liferative activity was more specific for malignant tumors

when compared with an increase of glucose metabolism.29

Measurement of tumor growth and DNA synthesis in vivo

may therefore be more appropriate for staging and assess-

ment of response to therapy for neoplasia.

The thymidine-analogue 30-deoxy-30[18F]fluorothymi-

dine (FLT) is a PET tracer that accumulates in prolifer-

ating tissues, including malignant tumors.30 FLT appears

to accurately reflect DNA synthesis, is taken up via passive

diffusion as well as facilitated transport and is subsequently

phosphorylated by thymidine kinase 1 (TK1) and trapped

intracellularly as [18F]FLT-monophosphate.13 FLT is not

incorporated into DNA, but rather acts as a chain termi-

nator due to the absence of 30-hydroxyl necessary for in-

tegration in the existing chain.12,31 TK1 activity is minimal

in quiescent cells whereas it may be three- to fourfold

higher in malignant cells.32,33 In addition, tumor cells may

harbor mutations in the carboxyl terminus of TK1, dis-

rupting normal degradation at mitosis, thereby leading to

deregulation of enzyme activity.34 Alterations in TK1 reg-

ulation and increased activity render FLT an attractive

molecular tracer for detection of malignant cells. In hu-

mans, physiologic FLT uptake occurs in bone marrow,

liver and gallbladder, and the urinary tract. In contrast to

FDG-PET, however, no uptake is evident in the brain,

skeletal muscles, or myocardium. In humans there is sig-

nificant correlation between tumor proliferation measured

using Ki-67 immunohistochemistry and FLT uptake in

lymphoma and other solid tumors.35–37 Also, FLT-PET

imaging has promise for early evaluation of remission sta-

tus in human non-Hodgkin’s lymphoma patients treated

with standard chemotherapy regimens.38 We theorized that

similar results could be obtained in dogs with non-Hodg-

kin’s lymphoma. In this study, we report a pilot study of

nine dogs with non-Hodgkin’s lymphoma that were eval-

uated with FLT-PET/CT imaging. Our hypotheses were

threefold: that FLT-PET/CT would be useful in dogs with

lymphoma for detection of early response to chemother-

apy, for confirmation of response following chemotherapy

completion, and for prediction of relapse.

Materials and Methods

Nine dogs were evaluated prospectively as part of

a clinical trial evaluating the efficacy of a novel nucleoside

analogue chemotherapy agent, GS-9219.39,40 Exclusion

criteria were dogs assessed as clinical substage b, dogs

weighing o10kg, concurrent cytotoxic therapy, and pres-

ence of concurrent illnesses that might affect drug tolerance

or short-term survival. Dogs were of various breeds, in-

cluding Golden Retriever (n¼ 2), Beagle (n¼ 1), Grey-

hound (n¼ 1), German Shepherd (n¼ 1), Bernese

Mountain Dog (n¼ 1), Bassett Hound (n¼ 1), Dachshund

(n¼ 1), and Hound Cross (n¼ 1). The median age was

8 years. Four dogs had naı̈ve lymphoma, four had relapsed

following previous treatment with a standard cyclophosph-

amide–doxorubicin–vincristine–prednisone (CHOP) con-

taining chemotherapy protocol and one dog had relapsed

following prednisone therapy. Three dogs were classified as

stage III non-Hodgkin’s lymphoma, four with stage IV

non-Hodgkin’s lymphoma, and two with stage V non-

Hodgkin’s lymphoma. Two dogs had T-cell lymphoma

while the remaining dogs had B-cell lymphoma following

immunochemical analysis.

A baseline FLT-PET/CT examination was performed

within 24h preceding the first administration of chemo-

therapy. FLT-PET/CT imaging was planned to be re-

peated at day 5 (n¼ 4; group 1) or before the fourth dose of

chemotherapy (n¼ 5; group 2). All dogs were scheduled to

undergo a third FLT-PET/CT scan 3 weeks following

completion of chemotherapy. Six of the nine dogs com-

pleted the protocol as outlined (Table 1).

Chemotherapy was administered intravenously at vari-

ous intervals, as determined by randomization at enroll-

ment, for a total of 15 weeks of planned therapy.40 Dogs

were evaluated with bloodwork, physical examination, and

lymph node aspirates at each visit. Clinical remission status

was documented for each dog using the most current cri-

teria applied to humans with lymphoma that predates

the inclusion of PET/CT assessment (international work-

shop to standardize response criteria for non-Hodgkin’s

lymphoma).41

FLT was synthesized as described42 and obtained from

the cyclotron and radiopharmaceutical laboratory at the

Department of Medical Physics at the University of Wis-

consin-Madison. Dogs were sedated and subsequently

anesthetized with propofol induction and isoflurane main-

tenance. Dogs were in sternal recumbency in the PET/CT

gantry and imaging was performed with a clinical GE

Discovery LS PET/CT scanner.� Noncontrast whole body

�General Electric, Waukesha, WI.

661CANINE LYMPHOMA MONITORING WITH FLT-PET/CTVol. 50, No. 6

Table1.ClinicalOutcomeandFLT-PET/C

TResponse

Dog

Diagnosis

Stage

Planned

PET/C

TSchedule

(Group)

Baseline

FLT-PET/C

TScan

Clinical

Response

toChem

o-Therapy

Day5

FLT-PET/C

TResponse:

Day5

Clinical

Response

toChem

o-Therapy

ThirdCycle

FLT-PET/C

TResponse:

ThirdCycle

Clinical

Response

Postchem

o-

therap

y

FLT-PET/C

TResponse

Post-FLT

Outcomefrom

Initial

PET/C

T

1B-cellLSA

StageIIIa

2Low

uptakein

lymphnodes

(LN)

CR

NA

CR

Nouptake

CR

Nouptake

Relap

seat9months

2B-cellLSA

StageIV

a2

Widespread

LN

uptake

CR

NA

CR

Nouptake

CR

Nouptake

Relap

seat9months

3B-cellLSA

StageIIIa

1Widespread

LN

uptake

PR

CR

CR

NA

CR

HighuptakeR

tonsilan

dR

retropharyngeal

LN

Relap

seat6months

4T-cellLSA

StageIIIa

2Widespread

uptake

PR

NA

NA

NA

NA

NA

Euthanized

at18days

dueto

lymphoma

5T-cellLSA

StageIV

a2

Widespread

LN

uptake

NA

NA

NA

NA

NA

NA

Acute

tetraparesis

2daysafter

treatm

ent.Died

3daysafter

treatm

ent;

widespread

lymphomapresent

atnecropsy

6B-cellLSA

StageIV

a1

Widespread

LN

uptake

CR

Decreased

uptake

CR

NA

CR

Highuptakeleft

mandibularand

retro-pharyngeal

LN

Outofremissionat

4.5

months

cytologically

(1month

post

final

PET/C

T)

7B-cellLSA

StageVa

1Widespread

LN

uptake

PR

Decreased

uptake

CR

NA

CR

Nouptake

Outofremissionat

4.75months

(3weekspost

final

PET/C

T)

8B-cellLSA

StageVa

2Widespread

LN

uptake

PR

NA

CR

Nouptake

CR

Nouptake

Outofremissionat

6.8

months

9B-cellLSA

StageIV

a1

Widespread

LN

uptake

SD

Widespread

uptake

NA

NA

NA

NA

Progressivelymphoma

at15days

CT,computedtomography;FLT,fluorothym

idine;

PET,positronem

issiontomography.

662 LAWRENCE ET AL. 2009

spiral CT scans were obtained, followed immediately by

FLT-PET imaging which was automatically coregistered to

the CT images. Acquisition of the whole body CT was

obtained in approximatley 50–60 s with a multislice helical

technique. As the table moved forward from CT to PET

acquisition, table height and cantilever point were con-

stant. Both scans were acquired using the same cranial and

caudal borders, field of view, and slice thickness, thus per-

mitting automatic coregistration of the images by the

hardware. Whole body images were acquired over six to

eight bed positions (205–273 slices) with 10-min acquisi-

tions per bed position 50–60min following injection of ap-

proximately 220MBq FLT (range, 185–260MBq). The

PET scanner contains 12,096 individual crystals arranged

into 18 rings of 672 crystals, and permits the simultaneous

acquisition of 35 transaxial PET emission images over an

axial field of view (FOV) with a 4.25mm slice thickness

(matrix size 128 � 128 � 35).43 The PET scanner permitted

evaluation of large breed dogs with imaging FOV of

50.0 cm and bore size of 60.0 cm. The total system sensi-

tivity for true events is 223kcps/(microCi/cc) with septa in

and 1200kcps/(microCi/cc) with septa out.43 The GE Dis-

covery LS PET scanner contains two rotating 68Ge rod

sources for transmission scanning at a speed of 20 rpm.

Each rod is 15.3mm long and 4.0mm in diameter over an

axial FOV of 15.3 cm, with a maximum activity of

370MBq per rod and a total activity 740MBq. Emission

data were corrected for attenuation, and subsequently re-

constructed using an iterative ordered-subset expectation

maximization (OSEM) algorithm. Image pixel counts were

calibrated to activity concentrations (Becquerel/ ml) and

decay corrected using the scan start time as a reference

point.

All PET/CT images were assessed by two individuals.

Lymph nodes were contoured on all CT images and cir-

cular regions of interest with a diameter of 0.5 cm were

placed within each lymph node in the area with highest

FLT uptake, as described previously.38,43 The maximum

body mass standardized uptake values (SUVmax) used in

analysis were calculated from each region of interest using

the formula

SUV ¼ Imaged activity concentration ðBq=gÞ � bodyweight ðgÞInjected activity ðBqÞ

For definition of regions of interest and data analysis,

AMIRA software was used.w Statistical analyses were per-

formed using a commercial statistical software package.zNonparametric two-tailed paired T tests were done to

compare maximum SUV values at various time-points.

Differences were considered significant in all analyses if a

P value o0.05 was obtained.

Ki-67 immunohistochemistry was performed using stan-

dard techniques on tumor tissues in three dogs in group 1

by a commercial laboratoryy using a rabbit Ki-67mono-

clonal antibody zpreviously validated in the dog. Popliteal

or prescapular lymph node samples were obtained from

anesthetized dogs several hours before the PET/CT scan.

Lymph node biopsies were performed the same day as the

PET/CT scans immediately before the initial cytotoxic

treatment and subsequently 5 days later. Lymph nodes

from normal beagle dogs removed before and following

treatment with GS-9219 were used as positive controls.39

Briefly, paraffin imbedded tissues were deparaffinized, air

dried out of alcohol, and antigens retrieved using heat-

induced steam. Slides underwent protein/enzymes blockade

followed by incubation with antibody, then developed and

counterstained with hematoxylin.

Results

Increased baseline FLT uptake was documented in the

lymph nodes of eight of nine dogs. One dog had low FLT

uptake before, during and following chemotherapy. Two

dogs in group 2 died shortly after the baseline scan was

performed, thus subsequent scans were not performed.

Mean SUVmax before chemotherapy for all dogs was 9.8

(range, 2.6–22.3) and 3.5 following chemotherapy (range,

1.1–7.9). These differences were statistically significant

(Po0.016) (Fig. 1).

FLT-PET/CT images indicated evidence of early re-

sponse to chemotherapy in both group 1 and group 2 dogs.

One dog in group 1 was considered to be in complete clinical

remission on the basis of physical examination and lymph

node aspirates at the time of the second FLT-PET/CT

scan (day 5), two dogs were considered to have achieved

partial remission, and the final dog had stable disease as

defined by International Workship response criteria. In

one of the dogs achieving partial remission at day 5, a

Fig. 1. Mean maximum body mass standardized uptake value (SUVmax)for seven dogs in which prechemotherapy and postchemotherapy flu-orothymidine (FLT)-positron emission tomography (PET)/computed to-mography (CT) scans were performed. Mean SUVmax before chemotherapyfor all dogs was significantly higher (9.8; range 2.6–22.3) compared withmean SUVmax following cytotoxic chemotherapy (3.5; 1.1–7.9; Po0.016).

wAMIRA 4.1.1, Visage Imaging Inc., Carlsbad, CA.zPrism 4.0b; GraphPad Software, San Diego, CA.

yIHCtech, Aurora, CO.zClone SP6, Lab Vision, Fremont, CA.

663CANINE LYMPHOMA MONITORING WITH FLT-PET/CTVol. 50, No. 6

firm left prescapular lymph node was noted on physical

examination that was cytologically consistent with lym-

phoma on fine needle aspirate. FLT-PET/CT confirmed

persistent increased FLT uptake within the left prescapular

lymph node, however, other lymph nodes displayed low

tracer uptake, corroborating the physical examination

findings. PET/CT scan indicated decreased FLT uptake

in the peripheral nodes compared with baseline, although

increased SUV remained present compared with surround-

ing tissue. The dog classified as having stable disease had

palpably smaller lymph nodes that did not as yet meet

International Workship criteria for a response; FLT-PET/

CT scan indicated decreased FLT uptake in peripheral

lymph nodes, although the lymph nodes remained enlarged

on physical exam and CT images. This dog developed

progressive lymphoma and was euthanized 2 weeks later.

One dog achieved complete remission in group 1 and had

low FLT uptake on FLT-PET/CT scans 3 weeks follow-

ing completion of chemotherapy (Fig. 2A and B). This

dog was in partial remission on day 5 based on enlarged

lymph nodes and presence of lymphoma on cytological

evaluation.

Three dogs in group 2 were considered to be in complete

clinical remission based on physical examination and

lymph node aspirates. FLT-PET/CT scans performed be-

fore fourth cycle of chemotherapy were characterized by

low FLT uptake compared with initial scans. The two re-

maining dogs in group 2 died before subsequent scans.

One dog was euthanized 3 days following the initial FLT-

PET/CT scan as a result of diffuse swelling and degener-

ation of the caudal spinal cord and widespread lymphoma.

The second dog was euthanized due to progressive lym-

phoma 3 weeks following the initial FLT-PET/CT scan.

The three dogs that were in complete remission from their

lymphoma at the first repeat scan remained in complete

remission following completion of chemotherapy. FLT-

PET/CT scans indicated minimal lymph node uptake in

these dogs, which corroborated examination and lymph

node aspirate findings.

FLT-PET/CT scans were successful in detecting early

lymphoma recrudescence in two dogs, both in group 1.

One dog was in complete remission based on physical ex-

amination, lymph node aspirate, and FLT-PET/CT scan

on day 5 following initiation of chemotherapy. This dog

was considered to remain in remission at recheck evalua-

tion 3 weeks following all chemotherapy cycles based on

physical examination. Mandibular lymph node aspirate

was consistent cytologically with a normal lymph node.

Fig. 2. (A) Left—fluorothymidine (FLT)-positron emission tomography (PET)/computed tomography (CT) image of a 3-year-old dog illustrating FLTuptake in the peripheral nodes, bone marrow, kidneys bladder, and spleen. (B) Right—FLT-PET/CT image of the same dog 3 weeks following the final dose ofchemotherapy. The lymph nodes were small on CT images with minimal FLT uptake on PET images. Note the persistent uptake in the bone marrow, kidneys,and bladder.

664 LAWRENCE ET AL. 2009

However, FLT-PET/CT scan indicated increased FLT up-

take within the right tonsil and right retropharyngeal

lymph node (Fig. 3A and B). Fine needle aspirate of the

right tonsil was performed following the scan and was cy-

tologically consistent with lymphoma.

The second dog in group 1 was in partial remission

based on physical examination characteristics at reevalu-

ation on day 5 following initiation of chemotherapy. Fine

needle aspirate of his left mandibular lymph node was

consistent cytologically with reactive lymphoid hyperpla-

sia. FLT-PET/CT findings were consistent with reduced

FLT uptake compared with baseline scan. The lymph

nodes became smaller over time and the dog was consid-

ered to have achieved complete remission within 3 weeks of

starting cytotoxic therapy. Three weeks following comple-

tion of chemotherapy, the dog was considered on physical

examination to be in complete remission. It was difficult to

obtain representative samples of lymph node to confirm

remission cytologically due to their small size. However, on

the PET/CT scan there was marked increase in FLT up-

take within the left mandibular lymph node. Aspirates of

the left mandibular lymph node were obtained under an-

esthesia but cytologically were not diagnostic of lym-

phoma. The dog was monitored and subsequently came

out of remission in the left mandibular lymph node within

30 days of the FLT-PET/CT scan.

Ki-67 IHC on lymph node tissue from normal beagle

dogs was characterized by typical proliferative activity in

the germinal centers of lymph node follicles before treat-

ment (Fig. 4A) and significant suppression of proliferation

5 days following cytotoxic chemotherapy (Fig. 4B). Similar

decrease in proliferative activity, as measured by Ki-67,

was also found in pet dogs with non-Hodgkin’s lymphoma

before and after cytotoxic chemotherapy (Fig. 4C and D)

at similar time periods. The decrease in Ki-67 labeling

fraction mimicked the reduction in FLT-PET/CT SUV at

similar time periods of analysis.

Discussion

Based on this trial, our hypotheses that FLT-PET/CT is

useful in dogs with lymphoma for detection of early re-

sponse and confirmation of response to chemotherapy,

were correct. Importantly, FLT-PET/CT was also able to

predict early lymphoma recrudescence in two patients be-

fore clinical detection. These features could result in real

time modification of chemotherapy protocols, timing and

intensity, ultimately translating into improvements in dis-

ease-free interval and overall survival in dogs with non-

Hodgkin’s lymphoma.

There are few published data regarding clinical use of

PET/CT scanning in staging or treatment monitoring of

Fig. 3. (A) Left—fluorothymidine (FLT)-positron emission tomography (PET)/computed tomography (CT) image of a 10-year-old dog with lymphoma,illustrating lack of FLT uptake in the peripheral lymph nodes 5 days following initiation of chemotherapy. There is increased FLT uptake surrounding theoriginal intravenous catheter site in the left saphenous region. (B) Right—FLT-PET/CT image of the same dog, illustrating increased FLT uptake within theright tonsil and right retropharyngeal lymph node. Note the lack of FLT uptake in the area in (A); cytologically, the tonsil was consistent with lymphoma.

665CANINE LYMPHOMA MONITORING WITH FLT-PET/CTVol. 50, No. 6

dogs with cancer. 18FDG-PET imaging was used in dogs

with lymphoma and with mast cell tumor.25 With regard to

the dogs with non-Hodgkin’s lymphoma in that report,18FDG-PET imaging was helpful for staging and assessing

response to therapy.25 In the dog with small cell lymphoma

in the prior report, persistent increased 18FDG uptake was

observed in one lymphocenter during chemotherapy, how-

ever, differentiation of residual disease from inflammation

or lymphoid hyperplasia was not possible.25 Clearly, it

would be of benefit to distinguish reactive lymphadenopathy

from malignant disease.

Our results suggest that FLT-PET is highly sensitive in

detecting lymphoma, as previously shown in humans and

rodents.36,38 Two dogs with relapsed lymphoma were iden-

tified on FLT-PET/CT before detection of recrudescence

on physical examination. Interestingly, the dog with lym-

phoma relapse within the right tonsil and right retropha-

ryngeal lymph node was cytologically free of lymphoma in

the right mandibular lymph node. This highlights the abil-

ity of lymphoma to relapse in a locoregional manner,

which may be missed on routine examination. Lymphoma

within peripheral lymph nodes was detected in all patients,

similar to human patients with lymphoma.38 In dogs, pe-

ripheral lymph node enlargement is the most common sign

of relapse, and this study indicates that FLT-PET/CT

imaging may provide earlier detection. Theoretically, ear-

lier detection would allow quicker reinstitution of cytotoxic

therapy, which may favorably affect remission duration

with rescue protocols. It may also aid in the detection of

dogs that are not in remission following induction chemo-

therapy, allowing earlier commencement to alternative,

more effective protocols.

It is interesting to note that one dog in our study did not

have appreciable FLT uptake within his lymph nodes on

any PET/CT scan. This dog still had decreased mean

lymph node SUV postchemotherapy compared with pre-

chemotherapy (mean SUV 11.0 vs. 3.1). This biopsy sam-

ple from this dog was not available for review. However,

the clinical presentation was characteristic of high-grade

malignant lymphoma, with rapidly increasing peripheral

lymphadenopathy and thrombocytopenia, presumably im-

mune mediated. The dog responded to chemotherapy

within 14 days of administration, suggesting a proliferative

neoplasia. The reason for the poor FLT uptake is unclear.

However, a similar experience has been found in a human

with anaplastic large cell lymphoma, in which FLT-PET

scan indicated low FLT uptake within the tumor despite

high tumor cell Ki-67 positivity.36 Review of this patient’s

tumor indicated marked fibrosis that accounted for430%

of the mass and it was concluded that the low cellularity

Fig. 4. Ki-67 Immunohistochemistry of lymphoid tissues and lymphoma. (A) Normal peripheral lymph node from a beagle dog 5 days after receivingplacebo drug vehicle. Note the significant Ki-67 immunoreactivity of cells in the highly proliferative germinal center (� 100) characterized by amber staining.(B) Normal peripheral lymph node from a beagle dog 5 days after receiving GS-9219 cytotoxic chemotherapy. Note the lack of Ki-67 immunoreactivity anddepletion of cells in the germinal center (� 100). Lymphoma tissue from the prescapular lymph node of a dog in study before initiation of GS-9219 cytotoxicchemotherapy (C) compared with the contralateral prescapular lymph node in the same dog biopsied 4 days following GS-9219 cytotoxic chemotherapy (D)(� 600). Note the significant decrease in Ki-67 immunoreactivity following therapy indicating an antiproliferative effect.

666 LAWRENCE ET AL. 2009

and desmoplastic reaction secondary to lymphoma may

have explained the low FLT uptake.36 Additionally, mu-

tations or alterations in TK1 activity in an individual can-

cer could result in decreased FLT trapping.

The antiproliferative effect of chemotherapy docu-

mented by FLT-PET/CT in our study correlates with Ki-

67 immunoreactivity, an immunohistochemical measure of

proliferation; a correlation also observed in humans with

high-grade non-Hodgkin’s lymphoma.35,36

The use of FLT-PET/CT is not limited to detection of

lymphoma and may be able to more accurately identify

lesion localization for solid tumors. Response assessment

via cellular proliferation has been evaluated in humans with

adenocarcinoma, high-grade sarcoma, small cell lung can-

cer, and breast carcinoma.44–46 In human patients with pri-

mary and metastatic breast cancer, changes in FLT uptake

after one course of chemotherapy were significantly corre-

lated to eventual changes in tumor marker levels.45 In vet-

erinary medicine, specific tumor markers are rarely used for

continued monitoring, thus subsequent PET/CT imaging

may offer a reliable method of tracking remission status.

It is unlikely that FLT-PET/CT will provide an advan-

tage over FDG-PET for staging purposes in lymphoma,

given that there is normally high FLT uptake in the bone

marrow of dogs. However, given the evaluation of larger

numbers of dogs, it is possible that a significant difference

in FLT uptake is detected between normal dogs and those

with bone marrow involvement. We evaluated a dog with

multiple myeloma via FLT-PET/CT imaging and found

the initial scan to show low FLT uptake in bone marrow.

Following a positive response to systemic chemotherapy,

the dog’s bone marrow displayed high FLT uptake, sug-

gesting a return to normal marrow function. We noted

elevated FLT uptake in the liver compared with fat (data

not shown), however, further study of a larger subset of

dogs will be required to definitively comment on the utility

of FLT-PET for detecting lymphoma infiltration within the

liver. FLT-PET/CT offers significant benefit over FDG-

PET in patients that have received chemotherapy or radi-

ation therapy, as it is less likely to accumulate in inflam-

matory cells that often occur following a substantial

amount of tumor cell death. It is likely that information

garnered from both FLT-PET and FDG-PET will be at

least in part complementary.

There are several limitations to our study. Only a

small number of dogs were evaluated, making it difficult to

evaluate differences between histologic subtypes of non-

Hodgkin’s lymphoma (B-cell vs. T-cell), stage at diagnosis,

or body size differences. The two dogs in this study with

T-cell non-Hodgkin’s lymphoma appeared to have extraor-

dinarily high FLT uptake compared with the dogs with B-

cell non-Hodgkin’s lymphoma, however, it was impossible

to draw conclusions with such small numbers. A consensus

does not currently exist as to the optimal method of cal-

culating SUV based on PET scans. Several different meth-

ods have been described, as well as the use of ratios to help

minimize variability among observers. Regardless, the

differences in FLT uptake here were significant when we

assessed individual lymph nodes or total lymph node vol-

ume, as mathematic models were consistent between scan-

ning points. The role of chemotherapy must be addressed

with respect to its affect on FLT uptake as well. The drug

used to treat the dogs that underwent FLT-PET/CT scan-

ning in this trial were treated with a nucleoside analogue,

which upregulates TK1 activities.47,48 In contrast, agents

such as cisplatin decrease FLT uptake.47 The role of che-

motherapy may not have a marked impact on overall re-

sponses identified on PET/CT, however, various agents are

likely to affect how quickly changes may be seen. Dogs

treated with an agent that increases TK1 activity may have

enhanced FLT uptake 24h postadministration of the drug,

which may not accurately reflect response of the tumor

cells to therapy. Optimal timing of sequential PET/CT

scanning will need to be determined with larger numbers of

dogs and are likely to depend on the underlying tumor

histology and the type of chemotherapy applied.

In conclusion, our data suggest that FLT-PET/CT in

dogs with lymphoma is useful for detection of disease

sites, determination of both early and late responses to

chemotherapy, and the recognition of disease recrudes-

cence. Further investigation should continue to evaluate

the utility of FLT-PET/CT in the response monitoring of

lymphoma as well as for lesion localization, staging and

detection of response, or recurrence for other forms of

canine cancer.

ACKNOWLEDGEMENTS

The authors are extremely grateful to MacKenzie Wessel, Drs. Ce-cilia Robat, Angela Kozicki, Timothy Stein, and Kristi Hall for theirassistance and dedication to the dogs in this trial. Special thanks areextended toward the owners of all dogs enrolled in the trial. This studywas supported by research funding from Gilead Sciences Inc. to D.M.V.D.B.T. is an employee of Gilead Sciences Inc.

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