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Cancer Epidemiology, Biomarkers & Prevention
Biomarker Modulation in a Nonhuman Rhesus Primate Model for Molly Brewer,2 Urs Utzinger, William Satterfield,
spectroscopy was the most sensitive marker for drug
Lori Hill, David Gershenson, Robert Bast,
activity and the apparent increase in NAD and FAD in
J. Taylor Wharton, Rebecca Richards-Kortum, and
the 4HPR group is consistent with the effect of 4HPR
Michele Follen
observed in cell culture. The differences between the
Departments of Gynecologic Oncology [M. B., D. G., J. T. W., M. F.], OCP and the 4HPR groups suggest a different
Veterinary Sciences Science Park [W. S., L. H.], and Medicine [R. B.], mechanism of activity of these drugs.
University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030;Department of Obstetrics, Gynecology and Reproductive Science, Universityof Texas Medical School, Houston, Texas 77030 [M. B., M. F.]; and Introduction
Biomedical Engineering Program, University of Texas at Austin, Austin, Texas Epithelial ovarian cancer has the highest mortality rate of any of the gynecological cancers, with a 5-year survival rate of 30%or less, despite aggressive treatment. Seventy percent of these Abstract
cancers are diagnosed with widespread intra-abdominal diseaseor distant metastases, which accounts for the dismal prognosis Objective: The objective of this study was to explore
for ovarian cancer. Even ovarian cancer limited to the pelvis has whether a nonhuman primate model could be developed to a 5-year survival rate of only 50% (1). Thus, cancer prevention test drugs for the prevention of ovarian cancer. merits at least as much attention as does treatment of disease Methods: Nineteen adult female Rhesus macaques
that has already occurred. Cancer chemoprevention is a rapidly were given fenretinide (4HPR), oral contraceptives
growing area of research because of the difficulties in treating (OCP), the combination (4HPR ؉ OCP), or no
advanced cancers. Chemoprevention refers to the administra- medication for 3 months. Exploratory laparotomy was
tion of chemical agents that prevent or delay cancer in normal done pre- and postdrug to assess intermediary
or undiseased people. Necessary elements for chemoprevention biomarkers of neoplastic phenotype, proliferation,
studies include appropriate agents that are safe and which have response pathways, and growth-regulatory and metabolic
both epidemiological and mechanistic data that support their markers. Fluorescence emission spectra were plotted for
use; a suitable cohort with adequate incidence of disease and in each group pre- and postdrug and means were overlaid
which the risk:benefit ratio is acceptable; and measurable bi- on these plots and normalized. Fluorescence intensities
omarkers that are likely to be affected by the agent and whose were compared using the 2-tailed Student t test, (P ؍
modulation supports the postulated chemopreventive activity 0.1– 0.01).
(2). Biomarkers are important because they are relevant to the Results: All monkeys tolerated drugs and surgeries
development of neoplasia, either phenotypically (proliferation, without difficulty. Histochemical markers showed no
angiogenesis, or nuclear morphometry) or mechanistically (mo- significant trend. However, fluorescence spectroscopy
lecular markers), they are likely to be required for a response to showed increased intensity at 450 nm excitation, 550 nm
the chemopreventive agent, or they are relevant to carcinogen- emission correlating with increased FAD presence. The
4HPR group (P ؍ 0.01) showed higher intensity than the
Two drugs that have received attention as agents that can OCP group (P ؍ 0.05– 0.07) when compared with the
prevent ovarian cancer are OCPs3 and retinoids. There are controls. Decreased emission was seen at 350 nm
multiple epidemiological studies that show that OCP use of at excitation, 450 nm emission correlating with decreased
least 5 years’ duration is associated with a 50% or greater NAD(P)H presence. The OCP group showed the largest
reduction in the odds of developing ovarian cancer (4 – 8). The change (P < 0.01), and the control group showed the
mechanism of this prevention is unclear; ovulation suppression smallest change.
may be one factor, but other mechanisms are hypothesized. A Conclusions: The nonhuman primate is an excellent
single prospective study showed that OCP and progesterone model to test drug effect on the ovarian surface
increased the rate of apoptosis of ovarian surface epithelial cells epithelium and merits additional study. Fluorescence
in primates (9). Premenopausal women with a family history ofbreast cancer, ovarian cancer, or both are excellent candidatesfor prevention strategies; however, in premenopausal women Received 12/1/00; revised 5/25/01; accepted 5/31/01.
the potential teratogenicity of retinoids is a concern (10). Epi- The costs of publication of this article were defrayed in part by the payment of demiological and laboratory data suggest that retinoids may page charges. This article must therefore be hereby marked advertisement in have a role as preventive or therapeutic agents for ovarian accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 cancer (11–12). 4-HPR has the fewest side effects of the vita- Supported by the Sandra G. Davis Ovarian Cancer Research Program and Dr.
J. Taylor Wharton.
2 To whom requests for reprints should be addressed, at Department of Gyneco-logic Oncology, The University of Texas M. D. Anderson Cancer Center, Hous-ton, 3 The abbreviations used are: OCP, oral contraceptive; 4-HPR, fenretinide; EEM Nonhuman Primate Model for Ovarian Cancer Prevention
min-A derivatives and is currently being used in chemopreven- Bastrop, Texas, an Association for the Assessment and Accred- tion studies in other organ sites including lung, head and neck, itation of Laboratory Animal Care International-accredited fa- cervix, and bladder. Preliminary results from the 2972 women cility, in accordance with the Guide for the Care and Use of in the Italian randomized chemoprevention trial for prevention Laboratory Animals. Numbers were chosen based on cost and of secondary breast cancers suggest that retinoids have preven- availability of animals: five animals were initially placed in tive activity against ovarian cancer (12). Ovarian cancer devel- each drug group, and four were placed in the control group.
oped in six women in the placebo arm, but there were no This study was a pilot study designed to generate measurements cancers in the 4HPR arm. Although this response was not so that future studies could be designed with adequate numbers.
durable, it suggested retinoid activity in the ovary. Experimen- The animals were given 4HPR (five monkeys), OCP (five tal studies have demonstrated that retinoids can affect human monkeys), the combination of 4HPRϩOCP (five monkeys), or ovarian cancer cell growth by inhibiting proliferation and in- no medication (four monkeys) daily for 3 months. Doses of ducing apoptosis (13–15). Preliminary data from our laboratory 4HPR and OCP were calculated by allometric scaling, which shows that 4HPR induces a rate of 40 –95% apoptosis in im- calculates a dose based on both weight and basal metabolic rate mortalized ovarian surface epithelial cells and 30 –90% in nor- (22). This is derived from the equation Y ϩ K(W 0.75), where mal ovarian surface epithelial cells,4 compared with the base- Y is the resting animal’s energy output in kcal/24 h (also termed line rate of 1–3% in untreated cells. The probability of the minimum energy cost), K is a constant based on core developing a neoplasm will decrease with increasing apoptosis.
temperature, which is specific for each species, and W is mass Substantial research has been done to develop optical in kilograms. Monkeys ranged in weight from 6 – 8 kg, but methods as early diagnostic tools for cancers in the last decade doses were calculated for a 7 kg monkey. The OCP used was (16 –19). Optical spectroscopy has the potential to decrease the Ortho-Novum 1/35, a medium-dose OCP known to suppress necessity for pathological diagnosis; to aid in early, near-real- cyst formation, which has 1 mg of norethindrone and 35 ␮g of time diagnosis; and to identify abnormal areas that may not ethinyl estradiol in each pill. The 4-HPR dose was calculated in have a visible lesion for localization of biopsy. Fluorescence the same manner from the human dose of 200 mg daily. The spectroscopy uses energy in the form of a light photon to OCP dose was 0.2 mg norethindrone/0.07 mg of ethinyl estra- activate certain molecules within a cell; the subsequent radia- tive relaxation of the molecule and the release of a reemission Before starting medication and after 90 days of medica- photon is the process called fluorescence. It is an optical tion, monkeys underwent laparotomy and ovarian biopsies.
method of illuminating tissue with monochromatic light and Two to three 2-mm samples were taken from the left ovary at exciting the natural fluorophores within tissue, which then the time of the first surgery and from the right ovary at the time emits fluorescent light when a paired electron in an excited state of the second surgery. Fluorescence measurements were taken returns to a lower or ground state. Natural fluorophores in tissue from each site before biopsy. Biopsy and healing of the ovary include NADH; FAD; structural proteins such as collagen, was hypothesized to interfere with the spectroscopic signature elastin, and their cross-links; and the aromatic amino acid because of the increased collagen associated with healing as tryptophan, each of which has a characteristic wavelength for well as with histochemical assessment. Thus, to avoid this bias, excitation with an associated characteristic emission. Fluoro- one ovary was biopsied on the first surgery and the other ovary phore concentrations change as tissue progresses from normal to a preneoplastic state to cancer (20). Theoretically, differentchanges in the fluorescent signature should occur in response to Biomarkers. Biomarkers chosen for this trial include markers
an agent that induces quiescence or apoptosis.
of neoplastic phenotype (p53), proliferative markers (Ki67), It is difficult to study the ovary because its intraperitoneal markers of intact response pathways (estrogen, progesterone, location makes access difficult; therefore, the development of and nuclear retinoid receptors), or inducible growth-regulatory an animal model to study these drugs is appealing. Small molecules (TGF-␤ and receptors HER-2, BAX, and BCLx).
animal models (mice, chicken, and guinea pigs) are being Apoptosis was evaluated with Apotag. The remainder of the developed for ovarian cancer that are useful for understanding markers were either mouse or rabbit antibodies that were eval- basic mechanisms, but these animals differ from the human uated by immunohistochemistry in the standard manner. The reproductively. Primates, however, are much closer to humans choice of these markers was based on previous studies (apo- in reproductive function and anatomy, hormonal and menstrual ptotic, proliferative, neoplastic, and retinoid markers), known patterns (9, 21), and histochemistry and will be a better model pathways (retinoid markers), and hypothesized mechanisms with which to develop strategies for prevention of ovarian cancer in the future. This study describes the use of the Macaca Complete characterization of the fluorescence properties mulatta as a nonhuman primate model to test drugs for the of an unknown sample requires measurement of a fluorescence EEM, with the fluorescence intensity recorded as a function ofboth excitation and emission wavelengths (23). Our custom-made system records EEMs in Ͻ1.5 min and consists of a Materials and Methods
xenon arc lamp coupled to a scanning spectrometer that pro- Initially, a pilot study was done with three animals to test drug vides excitation light between 300 – 480 nm. A fiber optic probe tolerance, tolerance for two surgical procedures, and the feasi- directs excitation light to the tissue and collects emitted fluo- bility of the model. After this pilot, 19 female adult M. mulatta rescence light, which it delivers to an imaging spectrograph and were brought together for this preliminary exploratory study.
CCD camera. Fig. 1 shows the probe placed on a monkey This protocol was approved by the Animal Care Use Commit- ovary. Fluorescence emission spectra ranging from 320 – 850 tee at The University of Texas M.D. Anderson Cancer Center nm were collected sequentially at 19 excitation wavelengths and was conducted at the Department of Veterinary Sciences in ranging from 300 – 480 nm. Before assembling the data intofluorescence EEMs, system-dependent response and back-ground signals were removed. Tissue exposure to broadband UV radiation from this device is below the total exposure limits Cancer Epidemiology, Biomarkers & Prevention
in the other two dimensions (see Fig. 1). At the time of the
second surgery, the scar from the previous biopsy was com-
pletely healed and there was a small visible area Ͻ1 mm where
the defect had filled in. In the larger study, there was one death
of a monkey in the 4HPR group that was not related to this
study. She was a 9-year-old obese macaque with severe amy-
loidosis on necropsy.
Fluorescence Measurements. There were consistent differ-
ences in the absolute fluorescence intensities and relative con-
tributions noted between the pre- and postdrug measurements in
each drug group as well as in the controls. However, the
differences noted in the control group that were attributable to
a time effect were much smaller than those seen in the three
drug groups. Fig. 2 shows areas of significant differences
between the pre- and postdrug measurements for all 4 groups.
To the right of each graph is the P, from 0.01– 0.1. Two main
areas of change can be identified. The center of the first area is
located at 450 nm excitation and 550 nm emission wavelength,
Fast EEM probe placed on the monkey ovary at the time of laparotomy.
consistent with the FAD emission peak. This area of fluores-cence intensity is increased in the three postdrug groups, whichcorrelates with increased FAD presence and decreased FADH.
The 4HPR group (P ϭ 0.01) shows a larger area of difference developed by the American Conference of Governmental In- in pre and postdrug intensity than the OCP group (P ϭ 0.05– dustrial Hygienists (ACGIH) for epithelial tissues.
0.07), whereas the combination group is in between the 4HPR Data Analysis. A visual analytic tool was developed to ex-
and OCP groups. There were no differences attributable to plore fluorescence differences grouped by monkey, drug, and treatment seen in the control group in this area of the EEM. The time (pre- and postdrug), and emission spectra were plotted for second area is located in the 350 nm excitation, 450 nm emis- each group pre- and postdrug at each excitation wavelength sion wavelength, which is consistent with the NAD(P)H emis- measured. Means were calculated and overlaid on these plots to sion peak. All four groups show decreases in these intensities view the relationship. The data were examined in two fashions: that correlate with a decreased NAD(P)H presence and thus an the raw intensities were compared, and emission spectra were increase in NAD(P) (the oxidized form). However, the OCP normalized to a common wavelength to enhance relative spec- group shows the largest change (P Ͻ 0.01), the control group tral contributions. Using this information, we compared all shows the smallest change (very small area of significant fluorescence measurements between the first and second meas- change), followed by the 4HPR and OCP group; and, again, the urements using the two-tailed Student t test. Ps were between drug combination lies in between the 4HPR and OCP groups.
0.1 and 0.01 for this exploratory study. Areas where statistically Other Biomarkers. Markers were evaluated visually in a 2 ϫ
significant differences were observed between the first (pre- 2 table. Missing data in the 4HPR as well as the OCP group drug) and second (postdrug) measurements were identified in precluded any data analysis, inasmuch as there were only two the EEM and plotted as contour lines.
monkeys with histochemical marker data in the 4HPR group Animal Care. Monkeys used in this study were culled from
and four in the OCP group because of difficulties in slide the specific pathogen free rhesus colony because they devel- preparation. The markers did not show consistent changes in oped herpes-B-indeterminate status, they were poor breeders, response to drug group, but there were trends noted in marker or they had chronic diarrhea. The monkeys were prevented expression. EGFR was consistently present, in both pre- and from eating for 12 h before surgery. Anesthesia was induced postdrug measurements; Her-2, p21, and p53 were rarely pres- with an i.m. injection of tiletamine HCl/zolazepam HCl (Tela- ent in either group, whereas BAX was usually present in both zol; Fort Dodge Laboratories, Inc., Fort Dodge, IA). When the groups. Estrogen receptors were absent, whereas progesterone monkey was sedated, she was removed from the cage and receptors were usually present and did not change in response intubated, and anesthesia was maintained on 2–2.5% isoflurane to drug exposure. Two monkeys in the OCP group showed gas with oxygen. After each initial procedure, monkeys were apoptosis and TGF␤ declining after exposure to drug. However, maintained in separate housing and were fed the drug in a there were two monkeys that did not show either marker present flavored treat. Menses were recorded daily, and progesterone before or after drug exposure; However, if trends were actually levels were measured 2 weeks apart to evaluate ovulatory present, they were not large enough, given our numbers, to have adequate power to detect a difference statistically.
Pathology. Biopsy specimens were fixed in 10% neutral buff-
ered formalin, embedded in paraffin, cut into 4-mm sections,
Discussion
and stained with H&E for light microscopic examination. Sec-tions were evaluated for morphological characteristics and The pilot study of three monkeys was done to assess and plan the feasibility of developing a primate model for a chemopre-vention study. After that, we did a larger study to developadditional expertise in administering the drugs for a longer period of time, in multiple surgical procedures on a larger Animal Tolerance. The pilot study showed that the three
number of animals, and in handling and processing the ovary.
animals tolerated the drugs and two surgeries without difficulty.
The primate was chosen because of the close association with Rhesus monkey ovaries are easily accessible through a midline humans, and the rhesus monkeys were chosen because of their laparotomy incision and are 1–1.5 cm in length and 0.75–1 cm availability, their cost, and their ease of handling. A “before and Nonhuman Primate Model for Ovarian Cancer Prevention
Student t test of pre- and postdrugmeasurements are presented for thecontrol group (top left), the 4HPRgroup (top right), the OCP group(bottom left), and the combinationgroup (bottom right). Each graphillustrates Ps between 0 and 0.1 atall measured fluorescence intensi-ties.
emission wavelength; ordinate, thefluorescence excitation wavelength.
Ps are color coded, and a low P isrepresented by a dark color, indicat-ing an area where the drug treatmenthas affected significantly the aver-age fluorescence emission.
after” drug study has the advantage of controlling for intraspe- we evaluated it as a marker for drug activity in the ovary.
cies variation as well as variations attributable to time—things Redox potential can be calculated by (FAD/(FAD that historical controls or separate control groups cannot ac- NADH) and is a measure of the relative hypoxia of the tissue (20). FAD and NAD(P) are reduced in the citric acid cycle Animal models are developed to reconcile biological phe- (anaerobic glycolysis) to FADH and NAD(P)H, which are nomena between species (24) and to allow for extrapolation of used as coenzymes in the electron transport chain. In tumors, knowledge from one species to another. Humans are difficult to these coenzymes will accumulate in their reduced state use as an experimental model because of ethical limitations, (NADH or FADH) with a unique fluorescence signature cost limitations, and lack of volunteers. Transgenic mice are (NADH, high, and FAD, low) as a result of alterations in commonly used because of their availability and cost, but, in blood flow, decreased pH of the tissue, and abnormal mito- addition to their different reproductive physiology (21) from all chondria as well as abnormal transport of electron carrier primates, their compromised immune systems cannot approx- molecules into the mitochondria (27), where the electron imate a normal host for chemoprevention. The nonhuman pri- transport chain takes place. Although these coenzymes have mate more closely approximates the human in its reproductive a unique signature, there is considerable overlap in tissue physiology and thus remains an important model that needs from other fluorophores, such as structural proteins, and additional development. The cost of procuring and caring for reabsorption of emitted fluorescence by blood. Therefore, rhesus monkeys and their low reproductive rates, long devel- results in living tissue are affected by several factors and not opmental periods, and relative scarcity have limited their use these coenzymes alone. This study shows that the opposite as animal models for human diseases. However, the close occurs in response to the chemopreventive agents. NAD(P)H genetic similarity and, thus, the high probability of producing is reduced consistently by the drugs, and FAD is increased.
results that can be extrapolated to humans more than overcomes 4HPR decreases NAD(P)H less than does OCP or the com- the above limitations (25). Histochemical analysis (26) and bination of the two drugs, and it increases FAD more than hormone activity (21) of the nonhuman primate ovary show either OCP or the combination, suggesting that each agent remarkable similarities to that of humans, and the microanat- has a unique effect on cellular metabolism. These agents omy of all primates, both human and nonhuman, is almost show an increase in the redox potential of the target organ, identical, which is consistent with their close phylogenetic suggesting that less hypoxia is present and that the system is linkage. The fact that all animals in this study were not more quiescent. Although numbers were small in this study, “reproductively healthy” should not affect our results, and in variances were also small, suggesting that there is a consist- fact should make this study population more analogous to the ent effect of these drugs on the fluorescence signature of the diversity seen in women, rather than the selection seen in most ovary. By measuring fluorescence from endogenous fluoro- phores with this system, we were able to identify two areas Fluorescence spectroscopy is being used to noninva- in the EEM that are affected by these drugs. Thus, a system sively detect cancers in many organ systems. In this study, could be constructed easily to interrogate the entire ovary Cancer Epidemiology, Biomarkers & Prevention
and to potentially quantify the drug effect on the ovary as 5. Franceschi, S., Parazzini, F., Negri, E., Booth, M., La Vecchia, C., Beral, V., well as to identify areas of occult cancer, which are thought Tzonou, A., and Trichopoulos, D. Pooled analysis of 3 European case-controlstudies. III. Oral contraceptive use. Int. J. Cancer, 49: 61– 65, 1991.
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