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Research Articles: Therapeutics, Targets, and Development
Novel in vivo imaging shows up-regulation of death receptors by paclitaxel and correlates with enhanced antitumor effects of receptor agonist antibodies
Departments of 1 Experimental Therapeutics, 2 Experimental Diagnostic Imaging, and 3 Biostatistics and 4 Division of Cancer Medicine Phase I Program, University of Texas M.D. Anderson Cancer Center, Houston, Texas and 5 Human Genome Sciences, Inc., Rockville, Maryland
Requests for reprints: Razelle Kurzrock, Division of Cancer Medicine Phase I Program, University of Texas M.D. Anderson Cancer Center, Box 4221515, Holcombe Boulevard, Houston, TX 77230-1402. Phone: 713-794-1226; Fax: 713-563-0236. E-mail: rkurzroc{at}mdanderson.org
Abstract
Susceptibility to apoptosis by tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) is mediated through cognate death receptor signaling. We hypothesized that auto-amplification of this apparatus would enhance antitumor effects in vivo and could be optimized using the results obtained from novel imaging techniques. We therefore imaged mice bearing human colorectal cancer (Colo205) tumor xenografts with HGS-ETR1 and HGS-ETR2 agonist antibodies to TRAIL receptor-1 (TRAIL-R1) and TRAIL-R2, respectively, after radiolabeling the antibodies. Paclitaxel significantly increased in vivo expression of TRAIL-R1 and TRAIL-R2 in a time-dependent manner. The imaging results were confirmed by immunoblots for steady-state protein levels (>20-fold increase in TRAIL-R1 and TRAIL-R2 levels in tumor xenografts by 48 h after paclitaxel administration). TRAIL-R1 and TRAIL-R2 mRNA expression did not change, suggesting that these effects were posttranscriptional. Sequential treatment with paclitaxel followed by HGS-ETR1 or HGS-ETR2 after 48 h resulted in markedly enhanced antitumor activity against Colo205 mouse xenografts. Our experiments suggest that sequential taxane treatment followed by TRAIL-R agonist antibodies could be applied in the clinic, and that novel imaging techniques using radiolabeled receptor antibodies may be exploitable to optimize sequence timing and patient selection. [Mol Cancer Ther 2006;5(12):2991–3000]
Introduction
Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL), also known as Apo2L, is a potent promoter of programmed cell death in diverse tumor types (1). TRAIL binds to a family of receptors, including death receptors 4 and 5 [TRAIL receptor-1 and -2 (TRAIL-R1 and TRAILR2)]. Ligand/receptor interaction leads to apoptosis via a conserved cytoplasmic death-signaling module. The pronounced antitumor activity of the death receptors and their ligand has raised significant interest in these molecules for the treatment of cancer patients. Recently, clinical trials have been initiated with a recombinant TRAIL and with HGS-ETR1 (mapatumumab) and HGS-ETR2 (lexatumumab) and TRAIL-R1– and TRAIL-R2–specific agonist monoclonal antibodies (mAb) that mimic the activity of native TRAIL (2–5). Clinical experience suggests that combinations of anticancer agents are often markedly more potent than single agents. This is not surprising because most cancer cells have numerous aberrant survival pathways, and it is unlikely that any signal agent will completely eradicate advanced tumors. However, the myriad of agents available makes it imperative that combinations be based on a solid biological rationale for optimization of antitumor effects. Drugs, such as taxanes, are often used in combination chemotherapy. These compounds include paclitaxel and docetaxel and are among the most commonly given and most potent agents for a variety of malignancies. Although they have several mechanisms of cytotoxic action, the primary one involves direct binding to ß-tubulin and inhibition of microtubule depolymerization, the latter being a requirement for mitosis and cell proliferation (6).
In the current study, we show that the antitumor activity of the TRAIL-R agonist antibodies HGS-ETR1 and HGS-ETR2 against human colorectal cancer xenografts can be remarkably enhanced by prior administration of paclitaxel. The mechanism by which this occurs seems to be due to a striking, time-dependent up-regulation of TRAIL-R1 and TRAIL-R2, as shown by in vivo tumor imaging using radiolabeled HGS-ETR1 and HGS-ETR2 and by analysis of steady-state protein levels from the colorectal tumor xenografts. These results have important clinical implications in that they suggest that pretreatment with taxanes could substantially augment the antitumor activity of TRAIL/TRAIL-R machinery–derived molecules, and that new imaging techniques may be exploitable to gain important data on agent uptake, tumor pharmacokinetics, and optimal timing of these sequential anticancer compounds in patients.
Materials and Methods
Chemicals and Analysis
Sulfo-N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl were purchased from Pierce (Radford, IL). All other chemicals were purchased from Aldrich (Milwaukee, WI). 99mTc-pertechnetate was obtained from a commercial 99mTc generator (Mallinckrodt Diagnostica, Houston, TX), and 111In was purchased from DuPont NEN (Boston, MA).
Cell Lines
Colo205 and PA-1 were purchased from the American Type Culture Collection (Rockville, MD). PA-1 is a human ovarian cancer line, which is maintained in MEM with 10% heat-inactivated fetal bovine serum (Gemini Bio-Products, Woodland, CA). Colo205 is a human colorectal tumor cell line maintained in RPMI 1640 with 10% heat-inactivated fetal bovine serum.
Antibodies
HGS-ETR1 (mapatumumab) and HGS-ETR2 (lexatumumab) are agonistic human mAbs specific for TRAIL-R1 or TRAIL-R2 (Human Genome Science, Inc., Rockville, MD; refs. 2–5). Isotype mAb was used as a control (Human Genome Science). Anti-ß-actin antibody (Sigma, St. Louis, MO) was used to ensure equal protein loading. Anti–poly(ADP-ribose) polymerase antibody (anti-PARP; Calbiochem, San Diego, CA) recognizes full-length PARP and the cleaved fragment during apoptosis. Horseradish peroxide–conjugated secondary antibody (Amersham Pharmacia Biotech, Freiburg, Germany) was used to detect signals. For Western blotting, monoclonal anti–TRAIL-R1 (Imgenex, San Diego, CA) and rabbit polyclonal anti–TRAIL-R2 (Abcam, Cambridge, MA) were used. For cell surface TRAIL-R, monoclonal anti–TRAIL-R1, anti–TRAIL-R2 (Imgenex), mouse IgG1 (Southern Biotech, Birmingham, AL), and goat anti-mouse immunoglobulin FTIC (BD Biosciences, San Jose, CA) were used. Other antibodies included anti-p53, anti–phospho-p53 (Ser15), anti–extracellular signal-regulated kinase 1/2 (anti-Erk1/2), and anti–phospho-Erk1/2 (Thr202/Tyr204; Cell Signaling, Beverly, MA).
3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide Assay to Determine Cell Survival
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (CellTiter 96 Non-Radioactive Cell Proliferation Assay; Promega, Madison, WI) was used for determining cell survival. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay is a colorimetric method based on the cellular conversion of tetrazolium salt into a formazan product that is easily detected using a 96-well plate reader. Assays are done according to the manufacturer's instructions.
Immunoblots for Steady-state Levels of TRAIL-R and Fluorescence-Activated Cell Sorting for Cell Surface TRAIL-R
To verify the steady-state protein level of TRAIL-R1 and TRAIL-R2 in Colo205 and PA-1 cell lines, immunoblots were done. Cells were lysed in immunoprecipitation assay buffer (200 mmol/L Tris-HCl, 130 mmol/L NaCl, 10% glycerol, 0.5% sodium deoxycholate, 0.1% SDS, 1% Triton X-100). Bio-Rad protein assay (Hercules, CA) was done to quantitate protein. Samples were denatured in 5x sample buffer [0.225 mol/L Tris-HCl (pH 6.8), 50% glycerol, 5% SDS, 0.05% bromophenol blue, 0.25 mol/L DTT] for 5 min at 95°C; 50 µg of sample per lane was loaded on an 8% denaturing polyacrylamide gel. The gel was then run for 2 h at room temperature and then transferred to a nitrocellulose membrane (Bio-Rad) for 1 h at 100 V, 4°C. After transfer, the membrane was blocked with TBS-Tween 0.2% plus 5% nonfat dry milk overnight at 4°C. The membrane was incubated with mouse monoclonal anti–TRAIL-R1 or rabbit polyclonal anti–TRAIL-R2 and ß-actin antibody for 1 h at room temperature. The membrane was washed and then incubated for 1 h at room temperature with goat anti-mouse or goat anti-abbit IgG horseradish peroxidase–conjugated secondary antibody as appropriate. The membrane was developed using the ECL chemiluminescence kit (Amersham, Little Chalfont, Buckinghamshire, United Kingdom) according to the manufacturer's protocol and exposed to autoradiographic film and developed. To determine the cell surface TRAIL-R expression, approximately one million Colo205 or PA-1 cells were suspended in 200 µL PBS and incubated with antibodies for 30 min at room temperature, washed twice in PBS, and pelleted by centrifugation. The cells were then incubated with 20 µL goat anti-mouse immunoglobulin FTIC at dark for 30 min, washed twice, and resuspended in 500 µL of PBS. Labeled cells were quantitated by flow cytometry (Coulter Epics XL, Miami, FL).
Immunoblots for PARP Cleavage to Detect Apoptosis
To determine the effect of HGS-ETR antibodies on Colo205 cell line, several concentrations (300, 600, and 1,200 ng/mL) and time point (24 h) were tested. Cells were isolated by centrifugation and lysed in radioimmunoprecipitation assay buffer. Immunoblots were done as above. Antibodies used were anti-PARP (Calbiochem).
Annexin V/Propidium Iodide and Fluorescence-Activated Cell Sorting Analysis to Determine Apoptosis
Cells were tested for apoptosis by using the Annexin V-Fluos Staining kit (Roche Diagnostics GmbH, Mannheim, Germany). To determine the effect of HGS-ETR antibodies, several concentrations (75, 150, 300, and 600 ng/mL) and time point (24 h) were tested. Cells (1 x 106) were then centrifuged, washed twice with cold PBS, and incubated with Annexin V-Fluos in the dark at room temperature for 15 min. Labeled cells were quantitated by flow cytometry (Coulter Epics XL).
Radiosynthesis of 99mTc-EC-HGS-ETR and 111In-EC-HGS-ETR Antibodies
The antibodies were labeled with technetium (99mTc) or indium (111In). 99mTc and 111In have half-lives of 6.01 h and 2.805 days, respectively. Ethylenedicysteine (EC) was selected as a chelator because EC-drug conjugates could be labeled with 99mTc or 111In easily and efficiently with high radiochemical purity and stability (7–9). Synthesis of EC was prepared in a two-step manner according to a method described (10–12). EC was conjugated to HGS-ETR1 or HGS-ETR2 or isotype control mAb or bovine serum albumin (BSA) using sulfo-N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl as coupling agents. HGS-ETR1 and HGS-ETR2 and an isotype control mAb obtained from Human Genome Sciences or BSA was stirred with EC, sulfo-N-hydroxysuccinimide, and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl at room temperature for 17 h. After dialysis, 2.3 to 3.4 mg of EC antibody was obtained. The Na99mTcO4 was added into a vial containing EC antibody and SnCl2 to yield 99mTc-EC-HGS-ETR1, 99mTc-EC-HGS-ETR2, 99mTc-EC-isotype control mAb, or 99mTc-EC-BSA. The procedures for radiosynthesis of 111In-labeled antibodies were the same as above, except without adding SnCl2, and 111In was substituted for 99Tc. Radiochemical purity for EC antibodies (Rf = 0.1) were >95% as determined by using radio-TLC (Bioscan, Washington, DC) eluted with saline or acetone.
Scintigraphic Imaging and Autoradiography Studies
The animal experiments were approved by the University of Texas M.D. Anderson Institutional Animal Care and Use Committee. All experiments involved four or five animals per subgroup. Six- to 8-week-old female nude mice (National Cancer Institute, Washington, DC) were inoculated i.m. into the hind legs with 0.1 mL of tumor cell suspensions (1 x 107 per mouse). Animals were divided into four subgroups (group 1 = 111In-EC-BSA, group 2 = 111In-EC-isotype control mAb, group 3 = 111In-EC-HGS-ETR1, and group 4 = 111In-EC-HGS-ETR2). The imaging studies were done 12 days after inoculation when tumor had grown to 1 cm in greatest diameter. Each group of animal was injected i.v. with 100 µCi 111In-labeled compounds as above. In other experiments, 1 mCi 99mTc-labeled compounds were used. At 2, 24, and 48 h following administration of the radiotracers, the scintigraphic images, using a 
camera (Siemens, Hoffman, IL) equipped with a medium-energy (111In) or low-energy (99mTc), parallel-hole collimator, were obtained. Animals were then treated with paclitaxel (60 mg/kg i.v.) and, 24 h later, were injected with 100 µCi 111In-labeled compounds or 1 mCi 99mTc-labeled compounds as above. Again, the scintigraphic images were obtained 2, 24, and 48 h following administration of the radiotracer. Computer-outlined regions of interest (counts per pixel) of the tumor lesion site and symmetrical normal muscle site were used to determine tumor/muscle count density ratios. The ratios were used to compare dynamic tumor uptake pretreatment and posttreatment of paclitaxel. Whole-body autoradiograms were also obtained by a quantitative analyzer (Cyclone Storage Phosphor System, Meridian, CT). Following the imaging, one animal from each group was sacrificed, and the body was fixed in carboxymethyl cellulose (4%) as described (13, 14). The frozen body was mounted on to a cryostat and cut into 100-µm coronal sections. Each section was thawed and mounted on a slide. The slide was then place in contact with the multipurpose phosphor storage system screen and exposed for 16 h.
Immunoblots to Detect TRAIL-R, p53, and Erk1/2 Expression and Phosphorylation Level in Mouse Xenograft Tumors after Paclitaxel Treatment
Six- to 8-week-old nude female mice (National Cancer Institute) were inoculated i.m. with Colo205 cell suspensions (1 x 107 per mouse). Paclitaxel treatment (60 mg/kg i.v. in tail vein) was done 10 to 14 days after inoculation when tumor had grown to 1 cm in greatest diameter. Tumors were incised 24 or 48 h after paclitaxel treatment. Tissue was diced into very small pieces using a clean razor blade and thawed in immunoprecipitation assay buffer. Further disruption and homogenization of tissue was done with a dunce homogenizer. Immunoblots were done as described previously with antibodies as appropriate: anti–TRAIL-R1, anti–TRAIL-R2, anti-p53, anti–phospho-p53 (Ser15), anti-Erk1/2, and anti–phospho-Erk1/2 (Thr202/Tyr204).
Real-time Reverse Transcription-PCR to Quantitate TRAIL-R Transcripts
RNA was extracted from Colo205 tumor established in the mouse model using RNeasy Mini kit from Qiagen (Valencia, CA). Random hexamer primer was used to amplify cDNA sequence using Transcriptor First-Strand cDNA Synthesis kit from Roche Diagnostics. Transcript quantification was done with ABI 7500 Sequence Detection System using the Taqman Universal PCR Master Mix according to the manufacturer's protocol (Applied Biosystems, Foster City, CA). The relative efficiency of amplification of each cDNA sample (TRAIL-R1 and TRAIL-R2) was monitored in parallel, separate wells by analysis of glyceraldehyde-3-phosphate dehydrogenase cDNA using primer for TRAIL-R1 (Hs00269492_m1), TRAIL-R2 (Hs00366272_m1), and glyceraldehyde-3-phosphate dehydrogenase (Hs99999905_m1) purchased from Applied Biosystems.
In vivo Evaluation of the Antitumor Effect of Antibody to TRAIL-R after Paclitaxel Treatment
Six- to 8-week-old nude female mice (National Cancer Institute) were injected with Colo205 cell suspensions (5 x 106 per mouse) at the right flank area 
1 cm craniad to the hind limb (n = 5 per group). Body weight and tumor growth were monitored thrice per week. Palpable tumors were measured using a vernier caliper. Tumor volume (mm3) was calculated using the formula: volume = 0.5 (L x W2). When the tumors grew to 8 to 10 mm in average diameter, the mice were randomized according to tumor size (small, medium, and large) into treatment groups. Mice were treated for 3 weeks i.v. (tail vein) injection with either single-agent treatment or paclitaxel followed 2 days later by antibodies HGS-ETR1 or HGS-ETR2 or isotype control mAb. Animal studies were conducted in the veterinary facilities of the M.D. Anderson Cancer Center in accordance with institutional guidelines.
Statistical Analysis
All statistical analyses are done by our biostatistician L.B. (see author list). The change in tumor volume from baseline across time was computed. Because the baseline tumor volumes were significantly different, a one-way analysis of covariance was done to test for differences in the average change in tumor volume between the five groups, adjusting for the baseline tumor volume. In addition, a Lowess plot was produced to graph the change in tumor volume (relative to background) for the five groups.
Results
TRAIL-R Are Expressed in Colo205 Cells and to a Lesser Extent in PA-1 Cells
Steady-state protein levels of TRAIL-R1 and TRAIL-R2 in Colo205 and PA-1 cell lines were studied by Western blot analysis (Fig. 1A
). Colo205 cancer cells expressed higher levels of TRAIL-R1 and TRAIL-R2 than PA-1 cells. Cell surface TRAIL-R expressions were also measured on Colo205 and PA-1 cells using flow cytometry (Fig. 1B). Colo205 cells express TRAIL-R twice as high as PA-1 cells. TRAIL-R2 is expressed more than TRAIL-R1 in both of cell lines.
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60% of cells to undergo apoptosis at the same concentrations. To confirm the presence of programmed cell death in HGS-ETR1– and HGS-ETR2–treated Colo205 cells, we analyzed the cleavage of PARP (Fig. 4
), a major substrate of the effector caspase-3 during apoptosis (16). As shown in Fig. 4, in Colo205 cells, HGS-ETR1 and HGS-ETR2 caused PARP cleavage after 24 h of treatment.
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Discussion
In the present study, we determined the effect of the anti-microtubule taxane (paclitaxel) on TRAIL-R1 and TRAIL-R2 expression and on receptor agonist antibody (HGS-ETR1 and HGS-ETR2)–induced regression of human colorectal (Colo205) xenograft tumors. We show that treatment with tolerable doses of paclitaxel increased TRAIL-R1 and TRAIL-R2 protein levels substantially in a time-dependent manner. This was shown both by in vivo imaging using a novel radiolabeling technique that allowed imaging of HGS-ETR1 and HGS-ETR2 binding to tumor in vivo (Fig. 9) and by analysis of steady-state protein levels of TRAIL-R1 and TRAIL-R2 derived from the treated tumors (Fig. 10). Quantitative real-time reverse transcription-PCR showed that treatment of Colo205 xenografts with paclitaxel did not induce TRAIL-R1 and TRAIL-R2 transcripts, suggesting that the in vivo induction of TRAIL-R1 and TRAIL-R2 protein levels by paclitaxel is posttranscriptional.
HGS-ETR1 has completed phase 1 and 2 single-agent studies; HGS-ETR2 is currently being examined in phase 1 studies. Preliminary reports indicate that these antibodies are well tolerated, and, whereas evidence of tumor regression has been seen with HGS-ETR1 as a single agent in patients with non-Hodgkin's lymphoma, their administration has primarily been associated with stabilization of disease (20, 21). This finding, however, is not unexpected considering the fact that the treated patients had advanced metastatic disease and had generally failed to respond to multiple prior therapies (22). Even so, because many if not most patients with metastatic tumors are resistant to single-agent therapy, strategies to potentiate the antitumor properties of individual drugs are very appealing. Indeed, curative regimens for diseases such as lymphomas depend on the use of multiple anti-neoplastic drugs given together.
Deciding which drugs to combine in the management of cancer is a complex problem, and the number of possible combinations is daunting. Clinical strategies often involve using two or more drugs with proven salutary effects and distinct toxicity profiles. Mechanism-based combinations are also highly desirable and may be sensitive to sequencing and other factors. Of interest, taxanes, such as paclitaxel, are among the most active antitumor compounds available, and their use in a broad range of tumors, including but not limited to breast, lung, and ovarian cancer, is well established (6). Despite their potency, however, these drugs are rarely curative.
Our experiments showing marked antitumor synergy in vivo when paclitaxel treatment is followed by HGS-ETR1 or HGS-ETR2 is therefore of substantial clinical relevance. When this sequence was given, tumor regressions were marked or complete (Figs. 12 and 13). The basis of this synergy seems to be a striking up-regulation of HGS-ETR1 and HGS-ETR2 expression by paclitaxel in the tumor but not normal tissue in vivo. The sequence of paclitaxel followed by ETR1 and ETR2 showed antitumor synergy without enhanced toxicity. As discussed, this is probably related to specific up-regulation of TRAIL-R of tumor cell but not normal cell, as detected by imaging. This specificity indicates that antitumor synergy without enhanced toxicity may be seen in patents.
Of interest, this up-regulation is apparent at 24 h (Fig. 8) but is considerably further enhanced by 48 h and reaches levels of >20-fold (Fig. 10). Such results emphasize the importance of finding ways to ascertain and to up-regulate expression of the death receptors in tumors in vivo in the clinic to select patients for treatment and to augment activity of TRAIL-related compounds.
Our current results confirm and extend those of Nimmanapalli et al. (23) who showed up-regulation of TRAIL-R1 and TRAIL-R2 by paclitaxel in vitro in prostate cancer cell lines, accompanied by enhanced apoptosis when these cell lines were pretreated with paclitaxel before exposure to TRAIL. Our studies suggest that these in vitro findings can be extrapolated to the in vivo setting and thus support bringing this sequential therapy to the patient care setting. Similarly, it has been reported that treatment with a different class of anticancer drugs (the DNA-damaging agents: etoposide, doxorubicin, and CPT-11) and radiation can induce p53 and/or nuclear factor-
B, which can up-regulate TRAIL-R1 and TRAIL-R2 expression, hence enhancing TRAIL-mediated apoptosis (24–29). The mechanism of death receptor up-regulation by DNA- damaging agents seems to be distinct from that of the microtubule inhibitor paclitaxel because the former increase death receptor transcripts, whereas paclitaxel increases protein levels without a change in mRNA expression (ref. 26; data not shown). Of interest in this regard is the observation that paclitaxel administration was associated with an increase in phospho-p53 and phospho-Erk1/2 in our colorectal tumor xenografts. The role of p53 activation in taxane-induced apoptosis is not clear-cut, and both p53-dependent and p53-independent pathways have been shown (30, 31). Taxane-induced microtubule damage also triggers signaling cascades that involve Erk1/2 (30, 31). Erk1/2 acts both upstream and downstream of TRAIL-R; therefore, it is also possible that activation of Erk1/2 occurs as a downstream effect of up-regulation of the TRAIL-R (17, 18). Although p53 is known to induce the death receptors, it is unlikely that this mechanism is operative here as p53 up-regulation of these receptors occurs at the transcriptional level (19), and our experiments showed that paclitaxel induced TRAIL-R via a posttranscriptional mechanism (because mRNA levels were not increased).
In summary, the presence of death receptors is a necessary (although not always sufficient) prerequisite for antitumor activity induced by TRAIL and related molecules. Using novel imaging techniques, we show a profound, time-dependent in vivo up-regulation of death receptors after paclitaxel treatment associated with marked antitumor synergy. In the clinic, imaging-based ascertainment of binding of receptor agonist antibodies HGS-ETR1 or HGS-ETR2 to tumor could conceivably be used to determine if cancers with poor binding are less likely to respond to these antibodies. Although a correlation between up-regulation of TRAIL-R and antitumor activity is implicated by our experiments, it has been reported that up-regulation of TRAIL-R is not required for enhanced cytotoxicity in other systems (32). The effect of up-regulation of TRAIL-R in patients' tumors, therefore, needs investigation. Optimization of timing of drug sequence in patients may be necessary in the clinic and could be elicited with the use of imaging of radiolabeled HGS-ETR1 and HGS-ETR2, as done in the current animal studies (Fig. 9). These imaging techniques could be a noninvasive alternative to the use of tumor biopsies, which are painful and risky, and also often inaccurate because of i.t. variability. Ultimately, a wealth of clinical experience supports the bench-side observations of the complexity of survival pathways in human malignancies and indicates that mechanism-based combinations of treatments, such as those described herein, need to be applied in the clinic to eradicate a malignancy.
Acknowledgments
We thank Noelise Cornelius for her secretarial support.
Footnotes
Grant support: M.D. Anderson Cancer Center CORE grant NIH CA-16672.
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.
Received 4/ 5/06; revised 9/ 8/06; accepted 10/10/06.
References
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