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Clinical Cancer Research Vol. 9, 6381-6388, December 15, 2003
© 2003 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

Interleukin-13 Receptor {alpha}2 Chain in Human Head and Neck Cancer Serves as a Unique Diagnostic Marker

Mariko Kawakami1, Koji Kawakami1, Jan L. Kasperbauer2, Lucinda L. Hinkley2, Mamoru Tsukuda3, Scott E. Strome2 and Raj K. Puri1

1 Laboratory of Molecular Tumor Biology, Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland;
2 Departments of Otolaryngology-Head and Neck Surgery, Mayo Clinic, Rochester, Minnesota; and
3 Department of Otolaryngology, Yokohama City University School of Medicine, Yokohama, Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Previous studies have demonstrated that approximately 30% of squamous cell carcinoma of the head and neck (SCCHN) cell lines express high levels of interleukin-13 receptor(s) (IL-13R). However, the incidence, expression level, and significance of IL-13R expression in human tumor specimens is not known. In addition, it is not known whether normal head and neck tissues express IL-13R. In this study, we evaluated the expression of IL-13R subunits (IL-13R{alpha}1, IL-13R{alpha}2, and IL-4R{alpha}) in 337 surgically excised specimens of SCCHN and normal head and neck tissues. Specimens were obtained from 139 patients with SCCHN and 16 patients with benign tonsil disorders from two centers in the United States and Japan and evaluated with immunohistochemistry and in situ hybridization. Extensive analysis demonstrated that the majority of SCCHN tumors uniformly expressed low levels of IL-13R{alpha}1 chain; however, 77% of the tumors expressed moderate to high levels of IL-4R{alpha}, which forms a signaling complex with IL-13R{alpha}1 chain. On the other hand, 33% of SCCHN tumors expressed moderate to high levels of IL-13R{alpha}2 chain. Using tissue array from 99 patients, we observed that the expression levels of IL-13R{alpha}2 and IL-4R{alpha} were significantly higher in SCCHN than in normal head and neck tissues (P < 0.005). Detailed analysis of clinicopathological features demonstrated a positive statistically significant correlation between IL-13R{alpha}2 expression and clinically advanced primary SCCHN tumor (T4; Tumor-Node-Metastasis classification; P < 0.05). However, there was no correlation among IL-13R expression and sex, age of patients, stage of lymph node metastasis, squamous cell carcinoma grade, or allergic history. Taken together, this study suggests that IL-13R may be involved in SCCHN tumor progression, and 33% of IL-13R{alpha}2-positive SCCHN cases may be targeted by IL-13 cytotoxin and IL-13R-targeted agent.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Advanced squamous cell carcinoma of the head and neck (SCCHN) is a difficult disease to treat by conventional therapies because of its aggressive behavior. Therefore, preventive measures and new treatment modalities are required. Targeted immunotoxins or cytotoxins may offer an alternative approach, in which tumor cell surface molecules (e.g., antigents or receptors) may be targeted by these specific agents for specific cytotoxicity (1) . Identification of unique or overexpressed antigens or receptors is critical for the development of this form of cancer therapy (1) .

Previously, our group has identified a Th2 cytokine receptor, interleukin-13 receptor (IL-13R), which is overexpressed on a variety of human tumor cell lines (2, 3, 4) . Approximately 30% of SCCHN tumor cell lines expressed high levels of IL-13R (5) . SCCHN tumor cell lines express two known chains of IL-13R (IL-13R{alpha}1 and IL-13R{alpha}2) and a primary IL-4-binding protein IL-4R{alpha} chain (5 , 6) . IL-13R{alpha}1 chain forms a signaling complex with IL-4R{alpha} chain (7) . To target IL-13R-expressing tumors, we have produced a recombinant fusion protein comprised of IL-13 and a bacterial toxin (Pseudomonas exotoxin), which has shown specific cytotoxicity to SCCHN tumor cell lines in vitro and in animal studies (5 , 8, 9, 10, 11) . The cytotoxic activity of IL-13 cytotoxin has been primarily mediated by binding to IL-13R{alpha}2 chain, which is shown to be internalized after binding to the ligand (12) . Because IL-13R{alpha}2 is important for immunotoxin targeting, and IL-13 cytotoxin is in clinical trial for glioma therapy, it is of great interest to investigate whether IL-13R{alpha}2 chain is overexpressed in head and neck tumor tissues. In addition, it is of interest to determine the significance of IL-13R{alpha}2 chain expression in tumor biology.

In this study, we performed an extensive analysis of IL-13R subunit expression in 337 biopsy and/or surgically excised SCCHN tumor sections and surrounding normal tissue samples obtained from 139 patients with SCCHN and normal tissues from 16 patients with benign tonsil disorders from two centers in the United States and Japan. Immunohistochemistry (IHC) and in situ hybridization (ISH) assays were performed for receptor expression. Any correlation between receptor expression and clinical parameters was carefully examined.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients, Tissue Specimens, and Tissue Array.
Human head and neck tumor and corresponding normal tissue specimens were obtained from newly diagnosed patients at the Mayo Clinic (Rochester, MN; n = 327 specimens from 129 patients) and the Yokohama City University School of Medicine, Yokohama, Japan (n = 10 from 10 patients). Information was accumulated regarding the biopsy and surgical pathology reports [location and squamous cell carcinoma (SCC) grade]; demographic information such as patient age, sex, Tumor-Node-Metastasis (TNM) classification as defined by the American Joint Commission on Cancer; and any history of allergic diseases. Tissue sections were prepared from 10% formalin-fixed and paraffin-embedded specimens. For tissue arrays, each slide was mounted with approximately 60 samples representing 2–3 replicate sections of each tissue. Tissue array slides and associated information was obtained from the Mayo Clinic (13) . Approximately 300 SCCHN specimens originating from tonsils, metastatic lymph nodes, and normal adjacent tonsil tissues from the same patient or normal donors were analyzed. All histopathological and immunohistochemical assessments were performed by two independent investigators in a blinded fashion. The protocol to obtain tissue samples for this study was approved by Institutional Review Board of the Mayo Clinic and Yokohama City University School of Medicine.

ISH Analysis.
SCCHN and normal tissue samples were also analyzed for mRNA expression of various receptor chains using ISH as described previously (14 , 15) . Briefly, 5-µm sections mounted on positively charged slides (HistoServe Inc., Gaithersburg, MD) were rehydrated by serial incubation with decreasing percentages of ethanol and RNase-free water, followed by enzymatic treatment with 10 µg/ml proteinase K (Sigma, St. Louis, MO) for 15 min at 37°C. Sections were then acetylated with freshly prepared 0.25% acetic anhydride, 0.1 M triethanolamine, buffer (pH 8.0), for 10 min. Tissue sections were then hybridized overnight at 55°C with 20 µl of heat-denatured antisense or sense digoxigenin-labeled RNA probes at a concentration of 100 ng/ml in hybridization solution (10 mM Tris/HCl (pH 7.4), 600 mM NaCl, 1 mM EDTA, 50% deionized-formamide, 1x Denhardt’s solution, 10 mg/ml salmon sperm DNA, 10 mg/ml yeast tRNA, 0.25% SDS, and 10x dextran sulfate) in a humidified chamber (all reagents from Sigma), and subsequently washed in 2 to 0.1 x SSC. Digoxigenin-labeled antisense or sense RNA probes were generated by using SP6 or T7 RNA polymerase and an in vitro transcription system (Roche, Indianapolis, IN). Hybridization reactions were detected by immunofluorescent staining with FITC- or Rhodamine-conjugated anti-digoxigenin antibodies (Roche). After washing, sections were dried and layered with ProLong Antifade mounting medium (Molecular Probes, Eugene, OR) and a coverslip. The slides were viewed on an Olympus IX70 fluorescence microscope using appropriate filters (Olympus Optical Co., Tokyo, Japan). Images were compiled from sets of three consecutive single optical sections using Spot Insight version 3.2 software (Diagnostic Instruments, Sterling Heights, MI). The positive fluorescence density was grouped into 1+, 2+, and 3+.

Immunohistochemical Analysis.
IHC was performed using the Vector avidin-biotin complex method peroxidase kit according to the manufacturer’s instructions (Vector Laboratories, Inc., Burlingame, CA). Briefly, paraffin-embedded tissue sections were deparaffinized by xylene treatment and washed with an alcohol gradient (from 100% to 50%) and PBS. Sections were incubated with monoclonal antibodies against human IL-4R{alpha} [M57; kindly supplied by Immunex Corp. (Seattle, WA); 20 µg/ml], IL-13R{alpha}1 (Diaclone, Besancon, France; 10 µg/ml), IL-13R{alpha}2 (Diaclone; 10 µg/ml), or isotype control (mouse IgG1) for 18 h at 4°C. Slides were developed using 3,3'-diaminobenzidine substrate biotinylated peroxidase reagent (Vector Laboratories, Inc.) and then lightly counter stained with hematoxylin (Sigma). The intensity of positive staining was grouped into -, 1+, 2+, and 3+. The percentage of positive field in the receptor-positive sections was assessed by viewing the entire tumor field under the same magnification and by counting stained tumor cells. Because of manual evaluation and counting positive fields, assessment of the percentage of positive field was only done in individual tissue samples but not in tissue array slides. Tissue arrays were evaluated for the intensity of staining for different chains.

Statistical Analyses.
The statistical difference in staining intensity was evaluated by Student’s t test or {chi}2 test. The difference in expression level was evaluated between tumor tissues and tissues without cancer. The {chi}2 test was used to evaluate significant differences in the expression level of IL-4R{alpha} and IL-13R{alpha}2 chains in each clinicopathological sample and their correlation with and between these clinical parameters.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In Situ mRNA Expression of IL-13R Chains by ISH.
We performed ISH to detect mRNA for various IL-13-binding proteins in SCCHN tumors. As shown in Fig. 1Citation , two randomly selected SCCHN tissue samples expressed mRNA for the IL-4R{alpha} chain with moderate to strong fluorescent staining intensity, whereas mRNA for IL-13R{alpha}1 was of moderate fluorescent intensity. However, the fluorescent staining intensity of mRNA for the IL-13R{alpha}2 chain was variable. Sample 014 showed strong density of mRNA for IL-13R{alpha}2, whereas sample 003 did not show staining or showed low staining density for IL-13R{alpha}2. Interestingly, connective tissue surrounding the tumor, which most likely consisted of fibroblasts, expressed IL-13R{alpha}2 mRNA in mild density compared with the tumor tissue island (clearly seen in sample 003). When images for IL-13R{alpha}1 and IL-13R{alpha}2 mRNA staining were merged, we found that IL-13R{alpha}2 mRNA co-localized with IL-13R{alpha}1 mRNA in sample 014 but not in sample 003 because this sample did not show strong staining for IL-13R{alpha}2 mRNA. The incidence and intensity of various receptor chains in SCCHN from the United States and Japan showed similar expression. The results of ISH staining were confirmed by IHC staining in the same samples.



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  		Fig. 1. In situ hybridization (ISH) analysis of interleukin-13 receptor (IL-13R) chains in squamous cell carcinoma of the head and neck (SCCHN). IL-13R{alpha}2 receptor-positive or -negative tumor expresses various levels of IL-13R chains. The lowest panel is superimposed image of IL-13R{alpha}1 and IL-13R{alpha}2 (original magnification, 400x).

 
In Situ Detection of IL-13R Protein by IHC.
Three hundred and thirty-seven SCCHN tumor and normal surrounding head and neck sections obtained from 139 patients were subjected to IHC analyses for the expression of IL-13R subunits. The clinicopathological characteristics of 139 patients with SCCHN are summarized in Table 1Citation . All tumor specimens were graded as SCC originating from the head and neck regions. Most SCCHN tumor samples originated from tonsils, and the majority was grade 3 SCC. As shown in Fig. 2Citation , two SCCHN tumor samples expressed all three IL-13R subunits (IL-4R{alpha}, IL-13R{alpha}1, and IL-13R{alpha}2 chains) at various levels. Sample number 014 showed strong staining intensity for IL-4R{alpha} (2+) and IL-13R{alpha}2 (3+) chain, whereas staining for IL-13R{alpha}1 chain was weak (1+). On the other hand, sample 003 showed moderate staining intensity (2+) for IL-4R{alpha} chain, mild intensity (1+) for IL-13R{alpha}1 chain, and no staining for IL-13R{alpha}2 chain. This sample, however, showed strong staining intensity of IL-13R{alpha}2 chain for connective tissue surrounding the tumor. Furthermore, even in the same tissue section, staining intensity for various receptor chains was different between islands of tumor cells. For example, IL-4R{alpha} chain staining showed pockets of strong positive islands in sample 003. Because of these findings, we evaluated every tissue section for staining intensity (grade of intensity, -, 1+ - 3+) and the percentage of positive area (percentage of positive fields). Table 2Citation summarizes the results of staining intensity for three receptor subunits in SCCHN samples. Seventy-seven percent of SCCHN samples were moderately to strongly positive (2+ and 3+) for IL-4R{alpha} and 23% samples were weakly positive (1+). The majority (93%) of tissue sections was weakly positive for IL-13R{alpha}1 chain, whereas only 7% of samples showed no staining for IL-13R{alpha}1 chain. There was even distribution for IL-13R{alpha}2 chain expression; 33% samples were moderately to strongly positive (2+ and 3+), 33% weakly positive (1+), and 34% were negative for IL-13R{alpha}2 chain. For receptor-positive SCCHN samples, Table 3Citation shows the percentage of positive fields. Between 30% and 90%, fields of tumor tissue were positive for IL-4R{alpha}; between 70% and 100%, fields of tumor tissue were positive for IL-13R{alpha}1; and between 30% and 90%, fields of tumor tissue were positive for IL-13R{alpha}2 chain. These results suggest that the SCCHN tumor samples examined show heterogeneity in IL-13R expression. Each receptor chain showed different distribution. IL-13R{alpha}1 chain seems to be expressed ubiquitously in SCCHN tumors, whereas IL-13R{alpha}2 and IL-4R{alpha} chains are expressed selectively.


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  		Table 1 Demographics of head and neck cancer patients

 


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  		Fig. 2. Immunohistochemical analysis of interleukin-13 receptor (IL-13R) subunits in squamous cell carcinoma of the head and neck (SCCHN). Clinical SCCHN tissue samples were stained with monoclonal antibody for IL-13R chain subunits (IL-4R{alpha}, IL-13R{alpha}1, or IL-13R{alpha}2) and developed with diaminobenzidine tetrahydrochloride substrate. Mouse IgG1 serves as control for each antibody (original magnification, 400x).

 

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  		Table 2 In situ expression of IL-13Ra in human SCCHN tumors

 

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  		Table 3 Percent positive fields of IL-13Ra expression in SCCHN tumors

 
Significant Correlation Between Clinicopathological Parameters and IL-13R Subunit Expression in Patients with SCCHN of Tonsil Origin.
Because IL-13R{alpha}1 chain was uniformly expressed in most SCCHN, additional comparisons with patient characteristics were not performed. However, the expression level of IL-4R{alpha} and IL-13R{alpha}2 chains was significantly higher in SCCHN tumors compared with normal tissues. To further investigate the significance of IL-13R{alpha}2 and IL-4R{alpha} expression in SCCHN, we examined 99 tonsil-derived SCCHN samples in tissue arrays and analyzed the relationship between IL-13R and IL-4R expression levels and their clinicopathological parameters. As shown in Fig. 3Citation , both primary tumor site (sample A011) and lymph node metastasis site (sample A051) expressed IL-13R{alpha}2 and IL-4R{alpha} chains. Forty-four percent of primary SCCHN tumors from tonsil samples expressed IL-4R{alpha} chain whereas 30% expressed strong intensity (2+ and 3+) for IL-13R{alpha}2 chain (see Table 4Citation for IL-13R{alpha}2 chain). Sixty percent of metastatic lymph node samples expressed IL-4R{alpha} chain, and 35% of samples expressed IL-13R{alpha}2 chain.



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  		Fig. 3. Interleukin-13 receptor (IL-13R) expression in tissue array. Tissue array slides were stained with H&E, monoclonal antibody for IL-13R{alpha}2 or IL-4R{alpha} chains (original magnification, 100x).

 

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  		Table 4 In situ IL-13R{alpha}2a expression in SCCHN tumors and normal tissues

 
Normal adjacent tonsil tissue samples from the same patients (n = 65) were also stained for IL-4R{alpha} and IL-13R{alpha}2 chains. Twenty-nine percent of samples did not stain whereas 71% showed weak staining for IL-4R{alpha} chain. On the other hand, 68% of samples did not show staining for IL-13R{alpha}2 chain although 32% showed weak staining (see Table 4Citation for IL-13R{alpha}2 chain). In addition, normal tonsils from other normal donors (n = 16) did not show moderate to strong staining. Thirty-three percent of samples were negative although 67% were weakly positive for IL-4R{alpha} chain. Similarly, 85% of samples were negative although 15% were weakly positive for IL-13R{alpha}2 chain (see Table 4Citation for IL-13R{alpha}2 chain). Although additional normal tissues including gingival, soft palate, and areas of tongue were not examined, these results suggest that tonsil SCCHN tumors of primary and lymph node metastasis express high levels of IL-4R{alpha} and IL-13R{alpha}2 chains compared with normal adjacent tonsil tissue and normal tonsils, and this difference is statistically significant for both chains (P < 0.005). In addition, our data indicate that there is no significant difference in IL-13R expression level between primary and lymph node metastasis tumor or between normal adjacent tonsil tissue and normal tonsils.

We next investigated the possible relationship between the expression level of IL-13 binding chains (IL-4R{alpha} and IL-13R{alpha}2) and clinicopathological parameters in 99 patients with malignant tonsil SCCHN. Various parameters such as (a) age, (b) sex, (c) tumor TNM classification, (d) lymph node TNM classification, (e) SCC pathological grade, and (f) history of allergy were compared with receptor expression. For age, we divided these patients into the following two groups: <60 and >60 years of age. There was a tendency toward higher levels of IL-4R{alpha} expression in SCCHN tumors derived from patients who were <60 years of age compared with those who were >60 year age. However, this difference was not statistically significant (P = 0.1). Similarly, the number of samples from male patients was much higher than that from female patients; however, there was no significant correlation with receptor expression.

Interestingly, when correlated with tumor TNM classification, we found that the level of IL-13R{alpha}2 chain positivity was much higher in T4 stage of tumors derived from primary and lymph node metastasis tumor (P < 0.05) compared with lower tumor TNM classifications. As shown in Table 4Citation , IL-13R{alpha}2 expression level was moderate to high (2+ and 3+ intensity) in 30% of primary tumors (n = 22) and 35% of lymph node metastasis (n = 19). Thirty-nine percent of primary tumors and 38% of lymph node metastasis were weakly positive (1+) for IL-13R{alpha}2 chain. In primary tumor and lymph node metastasis (samples that were positive with 2+ and 3+ staining intensity for IL-13R{alpha}2 chain) the incidence of positivity significantly increased in T4 primary and lymph node metastasis tumor samples (Table 5)Citation . These results suggest that IL-13R{alpha}2 expression levels increase as the invasive and progressive tendency of tumor increases, indicating a role of IL-13R{alpha}2 chain in tumorigenicity. On the other hand, when lymph node TNM classification was compared with IL-4R{alpha} and IL-13R{alpha}2 expression, there was no significant correlation between them. For SCC grade, there was a tendency of higher IL-13R{alpha}2 expression in high N grade tumors; however, this was not statistically significant (P = 0.1; not shown).


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  		Table 5 Correlation of IL-13R{alpha}2a expression with T stage of SCCHN

 
Because IL-13 has been shown to play a major role in inflammatory diseases including allergies, we also examined whether IL-13R expression had any relationship with the state of patients as far as their history of known allergies is concerned (16) . There was no relationship between IL-13R{alpha}2 and IL-4R{alpha} chain expression levels in tumors or normal tissues obtained from allergic or nonallergic patients (data not shown).


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Previous studies have demonstrated that IL-13 cytotoxin is not cytotoxic to normal cells and tumor cells including SCCHN expressing no or low levels of IL-13R in vitro and in vivo (5 , 8 , 9) . In three ongoing phase I/II clinical trials in which IL-13 cytotoxin is being administrated intratumorally to malignant glioma and surrounding normal brain, no toxicity to normal brain has been demonstrated to date because normal brain cells do not express high levels of IL-13R{alpha}2 chain (17) . On the other hand, although these are phase I clinical trials, evidence for tumor necrosis has been obtained in several patients whose tumors seem to express high levels of IL-13R (17, 18, 19) . In addition, IL-13 cytotoxin was administered i.v. to cynomolgus monkeys to assess safety of this molecule. At the dosage up to 50 µg/kg given every alternate day for three times a day, only hepatic toxicity was observed, which was reversible (20) . On the basis of monkey and murine studies, IL-13 cytotoxin was tested in a phase I clinical trial in patients with advanced renal cell carcinoma (20) . Initial results suggest that IL-13 cytotoxin-mediated dose limited renal toxicity. Studies are currently ongoing to determine the possible mechanism of renal toxicity. Based on this experience, IL-13 cytotoxin may be a useful agent for SCCHN patients whose tumors express high levels of IL-13R. In addition, we, as well as others, have demonstrated previously that SCCHN expresses detectable IL-4R; however, normal tissues express no or low levels of IL-4R (21 , 22) . Our current study confirmed these observations and further suggests that IL-4 Pseudomonas fusion toxin may also be useful for the treatment of SCCHN. Indeed in our preclinical studies, we have found that IL-4 Pseudomonasexotoxin mediates significant antitumor activity in animal models of human SCCHN (21) .

There was a positive correlation between IL-13R{alpha}2 chain expression and high-stage SCCHN, suggesting a role of IL-13R in tumor progression. Although there was not a significant correlation between IL-13R expression and nodal (N) stage disease, the correlation with tumor stage (T) may indicate that IL-13R expression in SCC may be modulated by intratumor physiological microenvironment. We could not carefully dissect viable tumor area surrounding hypoxic or necrotic areas from the edges of the tumor. However, it is possible that receptor expression of these two areas would be different. These possibilities should be tested in the animal model of human SCCHN. The significance of enhanced IL-13R{alpha}2 chain expression in advanced grade SCCHN is not known. It is possible that IL-13R{alpha}2 chain contributes to evading the immune system resulting in tumor development. In this regard, previous studies have demonstrated that neutralization of IL-13 by treatment of the host with the extracellular domain of IL-13R{alpha}2 chain or transfection of IL-13R{alpha}2 in human tumor cells inhibits tumor development in mice, indicating that IL-13/IL-13R complex plays a role in tumor immunosurveillance (23 , 24) .

In contrast to SCCHN tumors, tissue sections derived primarily from tonsils of cancer-bearing hosts and some from normal donors only showed weak staining for IL-13R{alpha}2 chain. It will be useful to examine tissues from multiple sites (such as gingival and soft palate); however, in the current study, normal tonsils were examined for IL-13R expression because most SCCHN were obtained from tonsils. We have demonstrated previously that normal tissues derived from cheek mucosa and tongue epithelium showed only weak staining with IL-4R{alpha} antibody, which is similar to what we observed in the current study.

Finally, the results of the present study suggest that IL-13R{alpha}2 chain may serve as a unique diagnostic marker identifying a unique subset of SCCHN. Additional studies are needed to confirm these conclusions. Because IL-13R{alpha}2 chain serves as a target for IL-13 cytotoxin, a novel receptor targeted agent, our studies suggest that IL-13 cytotoxin may have a role in targeted therapy of IL-13R{alpha}2 chain-positive SCCHN. Moreover, because IL-13R{alpha}2 chain is overexpressed in SCCHN but not in normal tissues, it may also serve as a target for antigen directed immunotherapy.


   ACKNOWLEDGMENTS
 
We thank Pamela Dover, Dr. Syed R. Husain, and other members of the Laboratory of Molecular Tumor Biology, Center for Biologics Evaluation and Research, Food and Drug Administration for support and reading the manuscript; Drs. Fumihiko Takeshita and Koichi Suzuki, Department of Microbiology, Leprosy Research Center, National Institute of Infectious Diseases, Tokyo, Japan; Drs. Saoko Takeshita and Masaaki Shiina, Yokohama City University School of Medicine, Japan for technical advice in establishment of ISH experiments; and Akiko Ishii, Yokohama City University School of Medicine, Yokohama, Japan for sample preparation.


   FOOTNOTES
 
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.

Note: This study was conducted as part of a collaboration between the Food and Drug Administration and NeoPharm Inc. under a Cooperative Research and Development Agreement.

Requests for reprints: Raj K. Puri, Laboratory of Molecular Tumor Biology, Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, NIH Building 29B, Room 2NN10, 29 Lincoln Drive MSC 4555, Bethesda, Maryland 20892. Phone: (301) 827-0471; Fax: (301) 827-0449; E-mail: puri{at}cber.fda.gov

Received 5/27/03; revised 8/25/03; accepted 8/26/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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