Higher Dose and Dose-Rate in Smaller Tumors Result in Improved Tumor Control

ORIGINAL ARTICLE

Higher Dose and Dose-Rate in Smaller Tumors Resultin Improved Tumor Control

A. Mayer, M.D.,* E. Tsiompanou, M.D., A. A. Flynn, Ph.D.,

R. B. Pedley, Ph.D., J. Dearling, Ph.D.,

R. Boden, and R. H. J. Begent, M.D., F.R.C.P., F.Med.Sci.

Cancer Research UK Targeting and Imaging Group, Department of Oncology, Royal

Free Campus, Royal Free and University College Medical School, University College

London, London, UK

ABSTRACT

Small tumors are more sensitive to radioimmunotherapy (RIT) than larger ones.

A greater proportion of viable radiosensitive areas in small tumors, higher antibody

uptake, and radiation dose may be responsible. Six groups of mice with small (median

tumor size 0.06 cm3) or large LoVo xenografts (median tumor size 0.38 cm3) received

either RIT using a 131I-labeled anti-CEA antibody A5B7, 5-fluorouracil (5-FU)

modulated with folinic acid (FA), or no treatment. The % injected activity/gram,

antibody distribution in viable and necrotic areas, and dose distribution were

determined. High-power microscopy images of the original section were reconstructed

to estimate the proportion of viable areas. Mice with small and large tumors grew

significantly less rapidly when treated with RIT compared to the control group

(p , 0.0004 and p , 0.003, respectively), while 5-FU was ineffective. Small tumors

treated with RIT grew less than large tumors (p , 0.02). A higher amount of % injected

activity/gram of tumor (median 26.6% vs. 8.1%, p ¼ 0:0007Þ and a higher dose-rate

were found in small tumors at 24 hours post injection (viable areas: 56.2 ^ 23.7 vs.

13.3 ^ 7 cGy/h, necrosis 19.2 ^ 16.3 vs. 4.9 ^ 4.7 cGy/h, p ¼ 0:0007Þ: It appears that

as viable tumor masses grow the access to them decreases and this has a fourfold effect

on dose delivered for RIT in this example. These data support the consideration of use

of RIT for adjuvant treatment in colon cancer.

Key Words: LoVo xenografts; Radioimmunotherapy; Radioluminography; Dose-rate.

382

DOI: 10.1081/CNV-120018229 0735-7907 (Print); 1532-4192 (Online)

Copyright q 2003 by Marcel Dekker, Inc. www.dekker.com

*Correspondence: Astrid Mayer, Department of Oncology, Royal Free Campus, Royal Free and University College Medical School,

University College London, Rowland Hill Street, London NW3 2PF, United Kingdom; Fax: þ44 207 794 3341; E-mail:

[email protected].

CANCER INVESTIGATION

Vol. 21, No. 3, pp. 382–388, 2003

MARCEL DEKKER, INC. • 270 MADISON AVENUE • NEW YORK, NY 10016

©2003 Marcel Dekker, Inc. All rights reserved. This material may not be used or reproduced in any form without the express written permission of Marcel Dekker, Inc.

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INTRODUCTION

Colorectal cancer remains a leading cause of cancer

death.[1] Fifty percent of patients with Duke’s stage C

and 20% of patients with Duke’s stage B colon cancer

relapse within 5 years. Adjuvant treatment with modu-

lated 5-fluorouracil(5-FU) has lead to a 30% improve-

ment of 5-year survival in patients with Duke’s stage C

cancer.[2] Research into new and more effective

treatment strategies include targeted therapy that is

based on tumor-specific antibodies. These are either

linked with therapeutic moieties such as radionuclides in

radioimmunotherapy (RIT) or are effective in unconju-

gated form due to immunological mechanisms such as

antibody-dependent cellular cytotoxicity (ADCC). There

are currently two antibodies licensed for clinical use, i.e.,

the humanized monoclonal anti-HER2 antibody trastu-

zumab[3] in breast cancer and the chimaeric anti-CD20

antibody rituximab[4] in lymphoma. In spite of initially

promising results in colon cancer with the unconjugated

antibody 17-1 A,[5] adjuvant treatment with 17-1 A

proved inferior to 5-FU in a recently reported study.[6]

Radioimmunotherapy is potentially more effective

than an unconjugated antibody. It has shown only limited

response in advanced colon cancer,[7] while a response

rate of 26% was reported in small-volume (less than

2.5 cm) colorectal cancer.[8] This is in agreement with

experimental data, which indicated 10 times more

efficient localization of antibody in tumors of less

than 100 mg when compared with larger tumors.[9]

Blumenthal et al.[10] confirmed these data by demon-

strating that tumor nodule size affected the therapeutic

outcome of RIT using GW-39 colonic tumor xenografts

even at the microscopic level. As a result, very small

tumors can be eradicated while larger tumors exhibit a

growth reduction but not a cure.

As colorectal carcinomas progress they outgrow

their blood supply and heterogeneity of tumor masses

results. Central areas tend to be necrotic, while the well-

perfused periphery contains viable, radiosensitive cells.

Specific antibodies preferentially localize in the viable,

radiosensitive part of the tumor where the radiation dose

is most effective.[11] Penetration into the tumor is further

restricted by antibody size.[12] By contrast, cytostatic

drugs have a much smaller molecular weight (5-FU,

130.1 Da) and no affinity for tumor-associated antigens

and so penetration should be a relatively minor obstacle.

Indeed, a larger percentage of the tumor volume is

accessible for small molecules.[13] One explanation for

greater efficacy of RIT in small tumors would be a

greater proportion of radiosensitive viable cells, alter-

natively, uptake may decrease throughout the tumor

mass with increasing tumor size. It is important to know

the extent of any effect, and this can be studied by

making estimates of radiation dose delivered to the

viable and necrotic areas. Comparison of the effects of

RIT and modulated 5-FU make it possible to assess the

extent to which the size effect is specific for antibodies

compared with conventional cytotoxic chemotherapy.

MATERIALS AND METHODS

LoVo Xenograft and Treatment

The moderately well-differentiated carcinoembryo-

nic antigen (CEA)-expressing human colonic tumor cell

line LoVo[14] was used to develop a xenograft in the

flanks of female nude (nu/nu) mice. Passaging was done

by subcutaneous implantation of small tumor pieces.

Xenografts were grown in one group until they reached a

median size of 0.06 cm3 (range 0.02–0.11 cm3) and in

the second group until a median size of 0.38 cm3 (range

0.16–0.57 cm3). Mice weighed 20–25 g at the beginning

of the experiment and were weighed on the day of

treatment and on every subsequent third or fourth day.

Tumors were measured in three dimensions (length,

width, and height) and the volume estimated as

length £ width £ height/2. Mice with tumors exceeding

2 cm3 were killed. White blood cell (WBC) counts were

performed before the experiment and every subsequent

week to evaluate treatment toxicity.

Six mice of each group received 40mg of 131I

(Amersham, Little Chalfont, UK)-labeled A5B7, a

monoclonal anti-CEA antibody.[7] The A5B7, an IgG1

molecule, was raised against heat-treated CEA and

purified by protein A affinity chromatography. The

antibody was labeled by the Iodogen method[15] to a

specific activity of 370 MBq/ mg protein. Another six

mice per group were treated with 90 mg/kg folinic acid

(FA) (David Bull Laboratories, Warwick, UK) i.p.

followed by 27.5 mg/kg 5-FU (David Bull Laboratories,

Warwick, UK) i.v. 2 hours later for 5 days. Six mice of

each group were kept as controls.

Laboratory Gamma Counting

and Radioluminography

Tumors were weighed after removal and radioac-

tivity was measured in a laboratory gamma counter

(1470 Wizard, Wallac, Milton Keynes, UK). The

percentage injected activity/gram of tumor was

calculated.

Higher Dose and Dose-Rate in Small Tumors 383



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Storage phosphor plate technology[11] was used to

assess the distribution of the antibody in sections of

tumor in a separate study. Seven small tumors (median

0.07 cm3, range 0.05–0.11 cm3) and seven large tumors

(median 0.35 cm3, range 0.16–0.61 cm3) were included.

Tumors were fixed in 10% formalin for 48 hours,

embedded in paraffin, and 3mm-thick sections were cut.

After dewaxing, tumor sections were exposed to

phosphor plates for 7 days. The images were digitized

with a phosphor plate reader (Model 425 Phosphor-

imager, Molecular Dynamics, Chesham, UK) and

analyzed using software written in Interactive Data

Language. All sections were stained with haematoxylin

and eosin after scanning for comparison of radiolabeled

antibody distribution and tissue morphology. Necrotic

and viable areas were determined by histological

examination.

High-power microscopy images of sections taken

from the center of small and large tumors were acquired

using a Minolta Rd-175 digital camera (Minolta, UK)

mounted on a microscope. Images were taken of adjacent

areas of the entire section while ensuring there was an

overlap with surrounding areas. These images were

automatically reconstructed to form a mosaic of the

original section by iteratively searching for the

maximum cross-correlation between overlapping areas

of adjacent images.[16] Regions of interest were drawn

around viable tumor areas in the complete section. The

sum of the areas of these regions then gave a measure of

the relative amount of viable areas in the tumor. These

were used to estimate the equivalent thickness of a viable

shell in a spherical model of a tumor.

Low-resolution images of the stained sections were

also obtained using a desk scanner (Hewlett Packard Ltd,

Palo Alto, CA). Registration of radioluminographs and

corresponding histological section was performed

according to the cross-correlation method.[17] Regions

of interest were drawn around the viable and necrotic

areas on the digitized stained histological image and

were copied onto the corresponding radioluminograph

(Fig. 1). The ratio of mean counts per pixel in viable

relative to necrotic regions (X) was calculated and

compared for both large and small tumors. The

percentage injected activity/gram in viable and necrotic

areas was calculated using:

Viable ¼2XP

X þ 1and ð1Þ

Necrotic ¼2P

X þ 1ð2Þ

where P is the percentage injected activity/gram for the

whole tumor.

Dosimetry

Doserate is defined as the amount of radioactivity

deposited per unit of time. Higher dose-rate is known to

be important for the therapeutic effect of RIT as it allows

cells less DNA damage repair.

The mean dose-rate in the tumor was calculated

using the equation

R ¼ PA0e2ltS ð3Þ

where:

A0 is the injected activity, in Bq

l is the radionuclide decay constant (s21)

t is the time of observation, in seconds

S is the mean dose-rate per unit activity in the tumor

and is given by Bardies et al.[18] for each tumor size.

Beta point dose kernels[19] were used to generate the

dose-rate distribution from the images of antibody

distribution. The regions of interest that defined viable

and necrotic areas were then copied onto the image of

dose distribution (Fig. 1). This allowed the calculation of

Figure 1. Image of tumor morphology (A) enables the quantitation of antibody uptake (B) and dose-rate (C) in viable and

necrotic areas. (See color figure at end of issue.)

Mayer et al.384



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the ratio of dose-rate of viable and necrotic cells (X). The

actual dose-rate to viable and necrotic cells was then

estimated by setting P ¼ R in equations 1 and 2.

Data Analysis and Statistics

The natural logarithm of tumor size at each time point

was used to calculate the slope for each mouse based on a

least-square fit on Microsoft Excel 5.0. This allowed

comparison of tumor growth. Comparison between

groups was carried out using the unpaired t-test assuming

equal variances.

RESULTS

Growth of LoVo Xenografts and Toxicity

The growth of LoVo xenografts was measured for

22 days unless tumor size exceeded 2 cm3 prior to that.

Comparison of the growth of small and large tumors

treated with RIT showed that small tumors grew

significantly less compared to large tumors (p , 0.02),

while there was no significant difference of small and

large tumor growth in the group treated with 5-FU and in

the control group. Small (p , 0.0004) and large

(p , 0.003) xenografts treated with RIT grew signifi-

cantly less when compared to the control group. There

was no significant growth delay for mice with large or

small LoVo xenografts treated with 5-FU. However,

small LoVo xenografts treated with RIT also grew

significantly less than small xenografts treated with 5-FU

ðp ¼ 0:00003Þ: Growth curves for small and large

xenografts are shown in Fig. 2.

Toxicity in terms of weight loss was not observed in

any of the groups with small LoVo xenografts, while a

mean drop in weight of 6% on day 7 was measured in

mice with large tumors receiving RIT and of 8% in mice

with large tumors receiving 5-FU/FA. There was no

weight loss in the control group. A drop of the WBC

count to less than 30% of the pretreatment values was

found on day 7 and day 14 in both groups receiving RIT

and in mice bearing large tumor xenografts treated with

5-FU/FA. The drop of WBC count in mice with small

xenografts receiving 5-FU/FA was very moderate

(,10%). The individual WBC counts showed a

considerable variance. The WBC counts recovered by

day 21, the drop in WBC counts is therefore most likely a

treatment-related side effect. One mouse treated with

RIT and bearing a large LoVo xenograft was found dead

on day 10. Death due to toxicity seems unlikely at this

dose level.

Antibody Uptake and Dosimetry

Laboratory gamma counting showed that the

counts/minute for large tumors were 12% higher

compared to small tumors. This was not significantly

different ðp ¼ 0:755Þ: However, the median percentage

injected activity/gram was 26.6% after 24 hours in small

tumors (range 19.9–59.2%) and 8.1% in large tumors

(range 1.6–12.5%), which was significantly different

ðp ¼ 0:0007Þ:

Figure 2. Comparison of tumor growth in mice with small

and large LoVo xenografts (mean values). A significant growth

delay was found in mice bearing small xenografts treated with

RIT when compared with the group receiving 5-FU/FA ðp ¼

0:00003Þ and the untreated control group (p , 0.0004). Large

tumors treated with RIT grew significantly less than the

untreated control group (p , 0.003). There was no significant

difference between the group treated with RIT compared with

5-FU/FA.




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Comparison of the percentage of the tumor volume

that is viable in small and large tumors showed a

significantly higher percentage of viable areas in large

tumors (85%) compared to small tumors (72%, p ¼

0:023Þ: The thickness of the viable shell increased with

tumor size (Fig. 3).

The ratio of counts per pixel in viable versus

necrotic areas was 2.5 in small tumors and 2.8 in large

tumors ðp ¼ 0:42Þ: However, % injected activity/gram in

both viable ðp ¼ 0:0007Þ and necrotic ðp ¼ 0:0006Þ parts

of small tumors was significantly higher than in large

tumors. Details are given in Table 1.

Dose rate at 24 hours post injection was significantly

higher in viable ðp ¼ 0:0007Þ and necrotic ðp ¼ 0:0006Þ

parts of small tumors compared to large tumors.

DISCUSSION

This experiment confirms the enhanced therapeutic

effect of RIT in small tumors. The small tumors used in

our studies had a median radius of 2.5 mm, while the

radius of the larger tumors measured 4.4 mm (median).

Comparison of the extent of viable, radiosensitive parts

in small and large tumors unexpectedly showed a higher

amount of viable areas in the group of the large tumors,

which resulted in rejection of the hypothesis that a higher

proportion of viable, radiosensitive areas in smaller

tumors causes the enhanced therapeutic effect of RIT.

However, our mathematical modeling (Fig. 3) suggests

that the proportional amount of viable areas will decrease

with tumor size above a certain threshold, which is

around 4 mm radius in LoVo tumors. The relationship

beween viable and necrotic areas seems to be more

complex in smaller tumors depending on blood vessel

development and oxygen diffusion.

Laboratory gamma counting showed that there was

no significant difference in the absolute amount of

radioactivity in large and small tumors. However, a

significantly higher percentage of radioactivity per gram

of tumor was found in small tumors, leading to a higher

dose throughout the tumor. Furthermore, radiolumino-

graphy confirmed the higher amount of antibody in

viable and necrotic parts of small tumors. Consequently,

the dose-rate, which gives a measurement of the amount

of radioactivity deposited per unit of time, was

significantly higher in viable and necrotic areas in

small tumors at 24 hours. Therefore, the enhanced

therapeutic effect in small tumors is due to the higher

dose and dose-rate throughout the tumor and to the viable

radiosensitive areas in particular. This leads to the

question: Why is the absolute amount of antibody

delivered to small and large tumors the same, i.e., the

delivery of antibodies less efficient in larger tumors?

We would like to discuss two possible explanations:

The amount of antibody available for localization in the

tumor is primarily determined by the concentration in the

blood and the total blood volume in the tumor. The total

blood volume in the tumor increases relatively less with

tumor size as the blood vessel density decreases, i.e., the

average vascular surface area decreases with tumor

growth, resulting in reduced transvascular exchange in

larger tumors compared to smaller tumors.[20] Therefore,

there is no significant difference in the absolute amount

of antibody in large and small tumors. Consequently, the

percentage injected activity per gram is inversely related

to the tumor volume. Alternatively, increased per-

meability of tumor vasculature may be responsible.

Studies of tumor blood vessels revealed that particularly

mature veins and venules at the tumor-host interface

showed increased permeability for circulating macro-

molecules using 70 and 150 kD fluorescinated dextrans,

while immature interface vessels and tumor-penetrating

vessels did not leak these macromolecular tracers

significantly.[21] Blumenthal et al.[22] found that the

vascular permeability was dependent on tumor size;

small untreated tumors were found to have a higher

vascular permeability than large tumors due to a

comparably lower interstitial pressure.

The extent of the increased radiation dose of

fourfold, as shown here, indicates that a major

therapeutic gain will result if RIT is used in small-

volume disease. The gain in efficacy for 5-FU/FA was

significantly less than for RIT, indicating that the effect is

particularly marked for antibody therapy. Clinical

results, so far, are in agreement with our experimental

data. A higher response rate with RIT was reported in

small-volume disease[8] compared to advanced colon

Figure 3. Variation of the thickness of the viable shell with

tumor radius.

Mayer et al.386



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cancer.[7] Radioimmunotherapy therefore seems favor-

able for adjuvant treatment in colon cancer.

ACKNOWLEDGMENTS

This work was supported by Cancer Research UK,

The European Society for Medical Oncology, and the

Ronald Raven Chair in Clinical Oncology Trust.

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Table 1. Comparison of amount of activity in large and small tumors.

Small tumors Large tumors p-value

Total amount of radioactivity

(counts per minute)

726092 819885 p ¼ 0:755

Percentage activity/gram after

24 hours

26.6% 8.1% p ¼ 0:0007

Mean viable area 71.6% 85.2% p ¼ 0:02

Activity in

Viable areas (mean counts/pixel) 47.7 11.1 p ¼ 0:0007

Necrotic areas (mean counts/pixel) 18.8 3.9 p ¼ 0:0006

Viable/necrotic tissue ratio 2.5 2.8 p ¼ 0:42

Dosimetry at 24 hours in

Viable areas (cGy/hr) 56.24 ^ 23.68 13.32 ^ 6.96 p ¼ 0:0007

Necrotic areas (cGy/hr) 19.24 ^ 16.28 4.88 ^ 4.74 p ¼ 0:0006




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Mayer et al.388



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From “Higher Dose and Dose-Rate in Smaller Tumors Result in Improved Tumor Control,” by A. Mayer et al.,

pp. 382–388.

Figure 1. Image of tumor morphology (A) enables the quantitation of antibody uptake (B) and dose-rate (C) in viable and

necrotic areas.



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Documents

Higher Dose and Dose-Rate in Smaller Tumors Result in Improved Tumor Control