[CANCER RESEARCH 55, 3687-3691,
September 1, 1995]
Advances in Brief
Incorporation of 5-Bromo-2'-deoxyuridine into Colorectal Liver Métastasesand
Liver in Patients Receiving a 7-Day Hepatic Arterial Infusion1
James A. Knol,2 Suzette C. Walker, John M. Robertson, Zhaomin Yang, Susan DeRemer, Philip L. Stetson,
William D. Ensminger, and Theodore S. Lawrence
Departments of Surgery []. A. K.I, Internal Medicine ¡W.D. E.. S. C. W.¡,Radiation Oncology ¡T.S. L, J. M. R.I, and Pharmacology
University of Michigan Medical School, Ann Arbor, Michigan 48109
Abstract
Preclinical and clinical data suggest that the combination of hepatic
arterial bromodeoxyuridine
(BrdUrd), a thymidine analogue radiation
sensitizer, and high-dose three-dimensional conformai radiation therapy
offer a high potential for improving the local control of intrahepatic
cancers. A key step in the design of a successful protocol is to determine
in patients the conditions for BrdUrd administration that would be ex
pected to produce selective radiosensitization of the tumor. Therefore, we
designed a clinical trial to assess BrdUrd incorporation into the DNA of
hepatic colorectal métastasesand normal liver after a 7-day continuous
BrdUrd infusion at a dose rate of 25 mg/kg/day (the maximal tolerated
dose for a 14-day infusion) for patients undergoing laparotomy for either
¡P.L. S., S. D.. Z. Y.. W. D. E.¡.
for the treatment of intrahepatic malignancies for two reasons: (a) our
experiments with nude mice bearing human colon cancer xenografts
demonstrated that, under equally toxic conditions, BrdUrd is signifi
cantly better incorporated into tumors than is IdUrd (6); and (b)
BrdUrd demonstrates a greater regional advantage than does IdUrd
when delivered as an hepatic arterial infusion (7). These factors
appeared to outweigh the fact that, for the same extent of incorpora
tion, IdUrd probably produces slightly greater radiosensitization than
BrdUrd does (1-4).
Although these data suggested that a clinical trial of hepatic artery
BrdUrd and high-dose conformai radiation might be effective, clinical
success depends on whether adequate incorporation occurs in tumors
resection of liver métastasesor hepatic arterial catheter and pump place
(and not the normal liver) in patients. To define the conditions to make
ment. Thirteen patients were entered into this study. We found that the
this determination, we used our preclinical and clinical studies. First,
average replacement of thymidine by BrdUrd in the tumor and normal
we
have shown that incorporation of BrdUrd into tumors implanted in
liver were 11.6 ± 1.2% and 1.1 ±0.2%, respectively. This extent of
nude mice plateaued after a continuous infusion of between 4 and 7
incorporation would be expected to produce a single fraction radiation en
days (6). In addition, our Phase I study of hepatic arterial BrdUrd
hancement of 1.5 in the tumor without detectable sensi t¡/¡it
ion of the normal
defined the maximum tolerated dose of BrdUrd (administered as a
liver. Immunohistochemical staining for BrdUrd revealed heterogeneity of
incorporation with a range of approximately 60-80% of the cells labeled in
continuous hepatic arterial infusion over 14 days) of 25 mg/kg/day,
different regions of the specimens. These findings suggest that hepatic arterial
with thrombocytopenia as the dose-limiting toxicity (8). These data
BrdUrd given at this dose and schedule has a high likelihood of producing
suggested that a therapeutic clinical trial might use a 14-day infusion
clinically significant radiosensitization for patients with hepatic métastases of BrdUrd, with radiation beginning after day 7. Therefore, we carried
from colorectal cancer. Furthermore, the demonstrated selectivity of tumor
out a study to measure the incorporation of BrdUrd in colorectal liver
perfusion that can be obtained with hepatic arterial infusion combined with
métastasesand normal liver in patients receiving a 7-day hepatic
the high proliferative rate of colorectal métastases(versus normal liver)
arterial infusion of BrdUrd at a dose rate level equal to the maximum
suggests that these patients may be good candidates for tumor-directed gene
tolerated dose for a 14-day course.
transfer therapy by using regionally delivered retroviral vectors.
Materials and Methods
Introduction
The thymidine analogue radiation sensitizers offer significant
promise of improving the outcome of treatment for patients with
intrahepatic malignancies. Human colon cancer cell culture studies
demonstrate that radiosensitization is detectable after replacement of
as little as 2-5% of the thymidine by analogue, and substitution in the
range of 10% produces an enhancement ratio of 1.5 (1-4). If this level
of radiation enhancement could be combined with up to approxi
mately 7000 cGy of external beam irradiation, which can now be
safely administered to parts of the liver by using three-dimensional
conformai radiation therapy (5), it might be possible to improve
significantly the local control of unresectable intrahepatic cancers.
We have focused our efforts on the thymidine analogue BrdUrd3
Received 7/5/95; accepted 7/21/95.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1 These investigations were supported in part by NIH Grants MOI RR 00042, POI CA
42761. and an American Society of Clinical Oncology Clinical Research Career Devel
opment Award (J. M. R.).
2 To whom requests for reprints should be addressed, at Department of Surgery,
University of Michigan, 1500 Medical Center Drive. Ann Arbor, Ml 48109-0331.
3 The abbreviations used are: BrdUrd, 5-bromo-2'-deoxyuridine;
IdUrd, 5-iodo-2'-
Patient Population. Patients with colorectal cancer metastatic to the liver
who were scheduled to undergo attempted liver resection or placement of an
hepatic artery catheter and infusion pump were evaluated for entry into this
study. Eligibility criteria included, age >18 years, life expectancy >4 months,
Karnofsky score a60%, adequate organ function, no recent anticancer therapy
or surgery, no previous upper abdominal radiation, and no serious intercurrent
illness. All patients signed a consent form approved by the University of
Michigan Institutional Review Board describing the experimental nature of the
study.
BrdUrd Infusion and Tissue Acquisition. Patients were admitted to the
General Clinical Research Center of the University of Michigan. A percuta
neous catheter was positioned (via the left brachial artery) in the hepatic artery
to perfuse the entire liver while minimizing extrahepatic perfusion (requiring
embolization of the gastroduodenal artery in some patients). Patients were
administered dicloxacillin and aspirin or heparin to decrease the chance of
infection and arterial thrombosis. BrdUrd, obtained from the Investigational
Drug Branch of the National Cancer Institute, was infused via the arterial
catheter for 7 days at a dose rate of 25 mg/kg/day, which is the maximum
tolerated dose for a 2-week hepatic artery infusion of BrdUrd (8). Shortly after
beginning the infusion, a macroaggregated albumin scan was performed to
verify that the hepatic arterial catheter was completely perfusing the liver (and,
thus, all tumor-bearing areas) and not delivering extrahepatic perfusion.
Plasma BrdUrd levels were measured two to three times during an infusion,
according to methods published previously (9). At the end of the infusion, the
deoxyuridine.
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BrdUrd INCORPORATION
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hepatic artery catheter was removed, and the patient was scheduled to undergo
the planned operation within the next 4 h, at which time biopsies (either
excisional or incisional) of the tumor and normal liver were obtained. In some
patients, portal lymph nodes were also sampled.
Assessment of BrdUrd Incorporation. When possible (based on tumor
size and the proportion of tumor needed for medical pathological analysis), the
center of the tumor was separated from the periphery. Biopsies were then
assessed for overall replacement of thymine by bromouracil as described
previously (10, 11). Specimens were immediately homogenized, the cells were
lysed, and the suspension was sequentially treated with collagenase, proteinase, and RNase. Aliquots of the resulting lysates were stored at —
20°Cuntil
assay. For analysis, DNA was precipitated, hydrolyzed to its bases, and
converted to trimethysilyl derivatives. Bromouracil replacement of thymine
was determined by gas chromatography/mass spectroscopy with selective ion
monitoring. Incorporation of BrdUrd is expressed as the fraction of thymidine
replaced.
Determination of Labeling Index. We determined labeling index using
immunohistochemistry,
as described previously (12). Tissues were fixed in
cold 70% ethanol, dehydrated, and embedded in paraffin. Microtome sections
were deparaffinized, treated with HCI and Triton X-100, boiled, and then
exposed to B44 mouse anti-BrdUrd antibody, followed by exposure to an
antimouse IgG secondary antibody coupled to peroxidase. Sections were
exposed to diaminobenzidine and hydrogen peroxide and subsequently stained
lightly with hematoxylin.
The fractions of labeled tumor and normal cells were determining by
scoring color photomicrographs (X400) of the specimens. Because initial
review of tumor sections revealed heterogeneity of labeling, separate determi
nations were made of regions, which under low power light microscopy
showed high, intermediate, and low labeling density (Fig. 1). For the liver
specimens (which were grossly homogenous), we separately determined the
fraction of labeled hepatocytes versus sinusoidal-lining cells. The labeling
index was determined by averaging the scores of two independent examiners.
Toxicity. Treatment toxicity was evaluated during the week of infusion and
at 3 and 5 weeks after completing the infusion by physical exam, chemistries,
complete blood count, and platelet count by using the National Cancer Insti
tute/Cancer Therapy Evaluation Program Common Toxicity Criteria.
Statistics. All averages are presented as the mean ±SEM. Groups were
compared using Student's paired I test. Comparisons of series data (i.e.,
plasma) were evaluated by ANOVA. Significance
P < 0.05 (two-tailed) unless otherwise specified.
is defined at the level of
Results
Patients. Thirteen patients, 7 men and 6 women, ages 37-71 years
(median 62) were entered in the study. Nine of the patients had been
treated with systemic chemotherapy (5-fluorouracil), and one patient
had received a multidrug regimen for lymphoma. Five patients had a
Karnofsky score of 100, four had a score of 90, and four had a score
of 80. All patients completed the infusion. Twelve of 13 patients were
evaluated for toxicity through the entire follow-up period; 1 patient
transferred his care elsewhere before the 5-week follow-up, but the
history taken 6 months later showed no toxicity.
Incorporation of BrdUrd into Tumor and Normal Liver. We
measured BrdUrd incorporation into the DNA of the tumor periphery
in all patients (Table 1). BrdUrd incorporation into the tumor pcriph-
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Fig. 1. Heterogeneity of BrdUrd labeling of tumor cells in a colon cancer liver metastasis after a 7-day hepatic arterial infusion of BrdUrd at 25 mg/kg/day. The tissue was prepared
for BrdUrd ¡mmunohistochcmistry as described in "Materials and Methods." A low power view (X 40) of the specimen from patient 7 is shown, with representative areas defined as
"intermediate" (A), "low" (ß)and "high" (C) labeling (X 400).
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BrdUrd Plasma Concentrations
during Infusion. Plasma
BrdUrd concentrations averaged ~0.20 JJ.Mwith no significant trend
Table 1 BrdUrd incorporation into the DNA of colorectal liver métastasesand normal
liver after a 7-day hepatic arterial infusion of BrdUrd at 25 mg/kg/day
over the course of the infusion.
Toxicity. There was some toxicity associated with catheter place
(%)1.1ND*ND*2.61.20.80.91.60.80.90.80.60.7
Patient1°2345C6''r8910111213"Diameter
(cm)1.16.01.21.21.31.56.09.40.63.53.61.52.08.0TumorPeriphery
(%)10.37.111.99.314.714.46.214.413.710.021.111.45.82.6Central
(%)10.68.00.99.16.815.212.4Liver
ment. One patient experienced temporary left arm paralysis related to
vasospasm upon the withdrawal of the left brachial artery catheter,
and the symptoms resolved without sequelae with the use of a vaso
dilator. One patient had prolonged postoperative dysesthesia in the left
hand, possibly due to the catheter, but also possibly due to the
positioning at operation. Three patients who developed partial or
complete hepatic artery thrombosis underwent thrombectomy, arterial
repair, and reestablishment of hepatic arterial flow at operation.
Two patients appeared to have grade three toxicity (nausea and
hypertension) related to the BrdUrd infusion. Eleven of 13 patients
experienced grade one thrombocytopenia; the other 2 patients exhib
ited no thrombocytopenia. The lowest platelet count in any patient
" Catheter thrombosed at operation.
*ND,not determinedbecauseof technicaldifficulties.
was 108,000/|j.l.
1 Catheter repositioned on day 2.
' Sampling of tissues delayed for 2 days after infusion completed.
e Sampling of tissues delayed for 16 h after infusion completed.
Discussion
We have shown that a 7-day hepatic arterial infusion with 25 mg/
kg/day of BrdUrd, which is the maximum tolerated dose for a 14-day
ery, presumably the actively replicating area of the tumor, was
11.6 ±1.2%.
Because the environment in the center of a tumor might not be
favorable for proliferation (and, therefore, BrdUrd incorporation), we
compared, when possible, the fraction of thymidine replaced in the
tumor periphery to the tumor center (Table 1). We found that incor
poration into the central part of the tumor (9.0 ±1.7%) did not differ
significantly from the periphery.
The average incorporation of BrdUrd into the uninvolved liver was
1.1 ±0.2%. This value was significantly less than what was found in
either the central or peripheral part of the tumor. The ratio of BrdUrd
incorporation in peripheral tumor DNA to normal liver DNA was
calculated for each patient; the average was 13.0 ±2.0.
The average BrdUrd incorporation into DNA of portal lymph nodes
uninvolved with tumor was 1.5 ±0.3% (n = 8). These levels were not
significantly different than those from normal liver.
Fraction of Cells Incorporating BrdUrd. Our initial evaluation
of tumor biopsies showed that there was significant heterogeneity of
incorporation within the samples, which suggested that an overall
labeling index would not adequately describe our results. Therefore,
we assessed each specimen by first finding areas with grossly high,
intermediate, and low fractions of labeled cells and then by scoring
each of these regions independently (see "Materials and Methods";
Fig. 1). The mean labeling indexes for portions of tumor that grossly
appeared to have labeling densities that were high, moderate, and low
were 83 ±3%, 80 ±3%, and 58 ±6%, respectively (Table 2). The
low labeling density areas were typically from the center of the tumor.
The centers of the tumors typically had few viable tumor cells present
in a background of fibroblasts and ground substance and/or necrotic
material. Tumor center as a separate histopathological specimen for
labeling was obtained in only one patient whose tumor was 3.6 cm in
diameter; the labeling index for that center specimen averaged 83%.
In the liver parenchyma, cells were categorized as hepatocytes
(averaging 71% of the counted cells); lining cells (Kupffer, endothelium, and bile duct epithelium; 18% of counted cells); or indetermi
nate cells (11% of counted cells). Among these, the hepatocytes had
a mean labeling index of 8 ±2%. The median hepatocyte labeling
index was 6%; one patient had an hepatocyte labeling index of 23%,
which slightly skewed the mean. Labeling indices for lining cells and
indeterminate cells were 12 ±2% and 22 ±3%, respectively.
BrdUrd labeling indices were significantly greater in tumor than in
hepatocytes for all levels of tumor-labeling density.
infusion (8), can produce a degree of thymidine replacement in tumors
that would be anticipated to lead to a radiation enhancement ratio of
approximately 1.5, although causing virtually no radiosensitization of
the surrounding hepatic parenchyma (1-4). We are currently carrying
out a trial by using hepatic artery BrdUrd at 25 mg/kg/day in combi
nation with high dose external beam radiation. In this trial, patients
receive two 2-week courses of hepatic arterial BrdUrd, with radiation
starting on day 8 of each infusion. Either 4800 or 6600 cGy (in 150
cGy fractions) is administered to patients with focal liver disease.
Treatment is planned by using three-dimensional conformai tech
niques, and the dose is determined by the fraction of normal liver
which is spared from treatment (5). If an enhancement ratio of 1.5
occurs after each fraction of the 32-44 fraction course used in this
trial, it would be expected to produce a highly significant therapeutic
benefit.
Our results are consistent with the study of Speth et al. (13) in
which patients with colorectal cancer metastatic to the liver received
an infusion of IdUrd via the hepatic artery before surgery. They found
that both labeling index and overall incorporation of IdUrd into the
tumor were elevated compared to normal liver. However, the actual
levels of tumor incorporation (approximately 3% thymidine replaced)
and labeling (32%) were far lower than reported here. This is probably
because they used only a 3-day infusion and because of the less
efficient incorporation of IdUrd compared to BrdUrd (6, 14). Our
Table 2 Labeling indices in colorectal liver métastasesand normal liver after 7-day
hepatic arterial infusion of BrdUrd at 25 mg/kg/day
Tumor
High
Moderate
Low
Liver
Patient
1234567g910111213868291ND9379729279918863768365
' ND, not determined because of technical difficulties.
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results also validate our animal model, in which a similar labeling
index to that achieved in this study was found in xenografts after a
4-7-day infusion (6). The higher incorporation of BrdUrd in patient
tumors and the greater tumonnormal liver ratio in patients compared
to our xenografts (approximately 6% thymidine replaced and a 3-4:1
ratio of incorporation in tumor to normal liver) is probably a result of
the difference between the hepatic artery infusion used in this trial and
the s.c. administration used in the athymic mice.
In addition to radiosensitization, chemosensitization by BrdUrd for
certain antineoplastic agents may occur at the levels of tumor DNA
incorporation observed here after hepatic artery infusion. Chemosen
sitization has been demonstrated for cisplatin with 16% BrdUrd DNA
incorporation in Chinese hamster V79 cells (15). The cytotoxicities of
mitomycin C and bleomycin have also shown synergistic enhance
ment in combination with the preadministration of BrdUrd in cell
cultures of human colon cancer and squamous cell cancer, respec
tively (16, 17).
The immunohistochemistry data reveal a potential limitation of the
use of hepatic arterial BrdUrd. Although these tumors tend to be
highly proliferative compared to the surrounding normal liver paren
chyma, the identification of regions with only approximately 60%
labeling confirms that not all cells incorporate BrdUrd during the
7-day infusion. Computer modeling studies suggest that even a small
fraction of unlabeled cells might confer overall tumor resistance (18).
However, it is uncertain if computer modeling based on proliferation
rates before radiation will describe the behavior of tumors after
treatment has been initiated. Furthermore, our current therapy proto
col uses a total of 4 weeks of infusion. It is possible that cells that do
not incorporate BrdUrd during a 1-week infusion would incorporate it
at some later time if treated with a 4-week infusion.
Another limitation of our results is that they are derived from
unirradiated patients; it is possible that the initiation of radiation could
stop cells from cycling (19, 20) and decrease subsequent incorpora
tion. However, the capacity for cells to arrest in G, after irradiation
appears to require wild-type p53, which is mutant in most colorectal
cancers (21); in confirmation of this hypothesis are our studies show
ing that HT29 colon cancer xenografts (which are p53 mutant) avidly
incorporate BrdUrd after radiation.4 However, it is possible that the
minority of tumors that contain wild-type p53 would arrest in G, and
not incorporate BrdUrd after radiation. Thus, it will be relevant to
determine the role of p53 in the outcome of the combination of
radiation therapy and hepatic arterial BrdUrd.
These data also have implications for gene transfer therapy by using
an hepatic artery infusion of a retroviral vector for patients with
colorectal cancer metastatic to the liver. Although potentially effect
ing more durable gene transfer, a relative disadvantage for the use of
retroviral vectors compared to other methods is that retroviral gene
transfer requires a proliferating cell population (for reviews, see Refs.
22 and 23). Our finding that between 60 and 80% of cells undergo
DNA synthesis during one week suggests that colorectal métastases
are a good target for retroviral therapy. In addition, the large differ
ence between the labeling index of the tumor and normal tissue means
that retroviral methods may result in selective delivery to the tumor
compared to other methods. The recent development of a retroviral
vector in which the gene for the bacterial enzyme cytosine deaminase
is placed under the control of the carcinoembryonic antigen promoter
is especially interesting in this regard. The introduction of this gene
into a cell leads to the local conversion of 5-flucytosine, an antifungal
agent, to 5-fluorouracil, a cytotoxic and radiosensitizing drug in the
4 T. S. Lawrence, E. Y. Chang, M. A. Davis, P. L. Stetson, and W. D. Ensminger.
Effect of irradiation on BrdUrd incorporation in human colon cancer xenografts. Int. J.
Radial. Oncol. Biol. Phys.. submitted for publication, 1994.
treatment of colorectal cancer. Tumor regression occurs when only
1-5% of the cells in the tumor express the transduced gene (24, 25).
The efficacy of 5-fluorouracil and the multiple levels of selectivity
made possible by a tumor-selective promoter, hepatic arterial infu
sion, and the high proliferative rate in the tumor compared to the
normal liver appear to make the use of a retroviral vector containing
the carcinoembryonic antigen/cytosine deaminase gene unusually
promising as a treatment for patients with colorectal cancer metastatic
to the liver.
Acknowledgments
The authors wish to thank Jon Maybaum for helpful discussions,
and Tammara Johnson and Marlene Langley for help in preparing the
manuscript.
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Incorporation of 5-Bromo-2′-deoxyuridine into Colorectal Liver
Metastases and Liver in Patients Receiving a 7-Day Hepatic
Arterial Infusion
James A. Knol, Suzette C. Walker, John M. Robertson, et al.
Cancer Res 1995;55:3687-3691.
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