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Journal of Clinical Oncology, Vol 18, No 21S (November 1 Supplement), 2000: 47s-53s
© 2000 American Society for Clinical Oncology

INTEGRATED SYMPOSIUM

Enhanced Apoptosis With Combination C225/Radiation Treatment Serves as the Impetus for Clinical Investigation in Head and Neck Cancers

By James A. Bonner, Kevin P. Raisch, Hoa Q. Trummell, Francisco Robert, Ruby F. Meredith, Sharon A. Spencer, Donald J. Buchsbaum, Mansoor N. Saleh, Murray A. Stackhouse, Albert F. LoBuglio, Glenn E. Peters, William R. Carroll, Harlan W. Waksal

From the University of Alabama at Birmingham, Comprehensive Cancer Center (Experimental Therapeutics Program), Birmingham, AL, and ImClone Systems, Inc, Somerville, NJ.

Address reprint requests to James A. Bonner, MD, Department of Radiation Oncology, University of Alabama at Birmingham, 1530 3rd Ave South, WTI 105, Birmingham, AL 35294-3300.


   ABSTRACT
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 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION AND REVIEW OF...
 REFERENCES
 
PURPOSE: Epidermal growth factor receptor (EGFr) is overexpressed in a majority of head and neck squamous cell carcinomas, and this overexpression is associated with a poor prognosis. Therefore, EGFr has become the target of investigations aimed at disabling the receptor to determine whether this process leads to improved tumor kill with conventional treatment.

MATERIALS AND METHODS: C225 is an anti-EGFr monoclonal antibody that inhibits receptor activity by blocking the ligand binding site. A panel of human head and neck squamous cell carcinoma cell lines was used to study the combination of C225 and radiation.

RESULTS: It was determined that the combination of C225 (5 µg/mL) delivered simultaneously with radiation (3 Gy) resulted in a greater decrement in cellular proliferation than either treatment alone. This reduction in proliferation correlated with reduced EGFr tyrosine phosphorylation and a reduction in phosphorylated signal transducer and activator of transcription-3 (STAT-3) protein (known to protect cells from apoptosis). Also, the decrement in proliferation correlated with increased apoptotic events, thereby indirectly linking C225/radiation–induced regulation of STAT-3 protein to apoptosis.

CONCLUSION: This preclinical work serves as important support for the ongoing clinical investigation of C225 and radiotherapy for patients with head and neck carcinomas. The initial results of these clinical studies have been promising.


   INTRODUCTION
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 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION AND REVIEW OF...
 REFERENCES
 
TO EFFECTIVELY target a membrane protein for anticancer therapy, there are several characteristics that are advantageous for this process to be fruitful. First, it is advantageous if the protein is overexpressed in tumor cells compared with normal cells. Second, overexpression of the protein should be associated with prognosis, which suggests that manipulation of the protein may result in manipulation of prognosis. Third, preclinical studies should suggest that specific manipulations of the protein can be undertaken to inhibit tumor growth or augment other anticancer therapies. Finally, the effective preclinical manipulations need to be tested in patients for safety and efficacy. The above characteristics are being studied for the membrane protein epidermal growth factor receptor (EGFr).

EGFr is expressed on the cell membrane of a variety of malignant cells1-14 (Table 1). The characteristics of EGFr are compatible with the qualities of a target receptor outlined above. In tumors of head and neck origin, EGFr is overexpressed in a majority of cases. Dassonville et al14 reported the results of 109 consecutive biopsies of tumors from patients with head and neck malignancies. In this study, control biopsy specimens of adjacent normal tissue were obtained in 94 of the 109 cases. EGFr levels were assessed by a radiolabeled ligand-binding assay, and levels of EGFr were expressed as femtomoles per milligram of membrane protein. It was discovered that the mean levels of EGFr in tumors and normal tissue were 165.6 fmol/mg and 12.2 fmol/mg, respectively. However, the ranges of EGFr levels were large in tumors and normal tissue (2 to 2302 fmol/mg and 0 to 98 fmol/mg, respectively). Additionally, it was determined that patients with high levels of EGFr (> 120 fmol/mg) demonstrated decreased relapse-free survival and overall survival compared with patients with values lower than 120 fmol/mg. These results and the work of others8 demonstrate that head and neck tumors are frequently associated with a high level of EGFr expression (compared with normal tissue), and high levels of expression correlate with a reduction in prognosis. Therefore, preclinical studies have been performed to determine whether inhibition of EGFr enhances the outcome of treatments that are currently used for tumors that express EGFr.15-21


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  		Table 1. EGFr Overexpression in Various Tumors
 
Under physiologic conditions, the binding of EGF to EGFr leads to receptor tyrosine kinase activity and subsequently a complex cascade of events that can lead to cellular proliferation. This proliferation process is enhanced by antiapoptotic effects22-26 (Fig 1). C225 is a chimeric monoclonal antibody that binds to EGFr and results in the inhibition of cellular proliferation and apoptotic events, as recently demonstrated by our group in A431 squamous cell carcinoma cells.16 This study explores the mechanism of interaction between C225 and radiation in a panel of human squamous cell carcinoma cell lines. These studies enhance the knowledge gained in prior preclinical investigations by our group,16,17 examining the interaction of radiation and the inhibition of EGFr. The studies reported herein build on the prior preclinical investigations and give further preclinical rationale for the ongoing clinical investigations reviewed later in this article.



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  		Fig 1. Schematic of a portion of the EGF-EGFr signal transduction process. EGF exposure leads to mitosis, and it is believed that the mitogenic response is facilitated by protection from apoptosis. C225 leads to inhibition of cellular proliferation and apoptosis. Abbreviations: MAPK, mitogen-activated protein kinase; STAT-3, signal transducer and activator of transcription-3.

 

   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION AND REVIEW OF...
 REFERENCES
 
Tumor Lines
A panel of human squamous cell carcinoma cells was used for these studies. One gynecologic squamous cell carcinoma (A431 cells) was used because these cells are known to overexpress EGFr, with more than one million receptors per cell. The remaining squamous cells (UM-SCC-1, UM-SCC-5, UM-SCC-6, UM-SCC-22A, and UM-SCC-29) were all human head and neck malignancies obtained from Thomas Carey, MD, at the University of Michigan’s Department of Otolaryngology (Ann Arbor, MI). The A431 cells were grown in Dulbecco’s modified Eagle’s medium and F12 (50:50) with 7% fetal bovine serum, and the head and neck carcinomas were grown in Dulbecco’s modified Eagle’s medium with 15% fetal bovine serum. The medium was supplemented with L-glutamine, penicillin, and streptomycin, and the cells were cultured at 37°C in 5% CO2.

Cellular Proliferation Assay
This assay was performed as previously described.17 Briefly, cells were plated and allowed to enter an exponential growth phase, before treatment (48 hours). At this point, treatment with the C225 anti-EGFr antibody (ImClone Systems, Inc, Somerville, NJ) alone or with C225 and radiation (3 Gy) was initiated. Also, cells were treated with radiation alone, and control cells received no treatments. Four days after treatment initiation, cells were removed from plates with papain/trypsin/EDTA (papain 0.1 mg/mL, 0.025% trypsin, and 0.1% EDTA). Cells were counted with a cell counter (Beckman Coulter, Fullerton, CA).

Immunoblots
After treatment of cells with various combinations of C225 and radiation, cells were spiked with EGF (10 nmol/L) for 5 minutes and assessed for EGFr, phosphorylated EGFr, mitogen-activated protein kinase (MAPK), and phosphorylated signal transducer and activator of transcription-3 (STAT-3) by previously described methods.17 The cells were then lysed in lysis buffer (0.025 M Tris HCl [pH 7.5], 0.25 M NaCl, 0.005 M EDTA, 1% (v/v) NP-40, 0.001 M phenylmethyl sulfonyl fluoride, aprotinin 15 µg/mL, leupeptin 10 µg/mL, 0.001 M sodium orthovanadate, 0.05 M sodium fluoride, and 0.03 M sodium pyrophosphate). After the gel lanes were loaded with equal amounts of protein, separation was performed by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to Immobilon-P membrane (Millipore Corp, Bedford, MA). The immunoblots were blocked in 10% milk–Tris-buffered saline with Tween-20 (TBS-T) (20 mmol/L Tris HCl [pH 7.5], NaCl 137 mmol/L, and 0.05% Tween-20) for 1 hour at room temperature. The primary antibodies anti-EGFr (ICN Biomedicals, Inc, Costa Mesa, CA), antiphosphotyrosine (Santa Cruz Biotechnology, Inc, Santa Cruz, CA), antiphosphorylated MAPK (New England Biolabs, Beverly, MA), and antiphosphorylated STAT-3 (Cell Signaling Technology, Beverly, MA) were incubated with 2% milk–Tris-buffered saline with Tween-20 overnight at 4°C at the appropriate dilution. The appropriate secondary antibody of either anti-mouse IgG–horseradish peroxidase antibody (Sigma Chemical Co, St Louis, MO) or anti-rabbit IgG–horseradish peroxidase (Sigma) was incubated at room temperature for 1 hour. The blots were developed by chemiluminescence (Amersham Life Sciences, Inc, Arlington Heights, IL).

Measurement of Apoptotic Cell Death
Apoptotic events were explored as a mechanism to account for decrements in cellular proliferation. Apoptosis was assessed using an annexin V–fluorescein isothiocyanate apoptosis detection kit (Oncogene Research Products, Cambridge, MA). UM-SCC-6 cells were treated with C225 antibody, radiation, or the combination of the two at various time points. The cells were collected according to the manufacturer’s protocol. The data were collected using a Becton Dickinson (San Jose, CA) fluorescence-activated cell sorter Calibur system and analyzed using CellQuest version 3.1 software (Becton Dickinson). Samples were assessed in triplicate and expressed as the mean ± SD of the mean.27


   RESULTS
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION AND REVIEW OF...
 REFERENCES
 
A panel of human head and neck squamous cell carcinoma cell lines was tested for EGFr expression by an immunoblot technique. A431 human gynecologic squamous carcinoma cells were included as a positive control because these cells express over 1 million receptors per cell. Cells were assessed for EGFr with or without a 24-hour exposure to the anti-EGFr monoclonal antibody, C225 (5 µg/mL). All cells were also treated with a 5-minute exposure to EGF (10 nmol/L) at the end of the 24-hour antibody exposure. (Control cells received the 5-minute exposure to EGF alone.) This assessment revealed that the selected panel of human squamous cell carcinoma cells showed substantial variability in EGFr expression (with or without the C225 exposure); however, all of the cell lines demonstrated EGFr expression (Fig 2A). All of the cell lines showed a decrease in EGF-induced EGFr receptor tyrosine phosphorylation (RTP) after the C225 exposure compared with treatment with EGF alone (Fig 2B).



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  		Fig 2. Immunoblot analysis of EGFr (A) and tyrosine phosphorylated EGFr (B). Cells were incubated without (a) or with C225 antibody (5 µg/mL) for 24 hours (b), then pulsed with EGF (10 nmol/L) for 5 minutes. Cell lysates (5 µg/lane) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a nylon membrane. The following cell lines were examined: lane 1, A431; lane 2, UM-SCC-1; lane 3, UM-SCC-5; lane 4, UM-SCC-6; lane 5, UM-SCC-22A, and lane 6, UM-SCC-29. Immunoblots were developed using chemiluminescence. Abbreviation: p-tyr, receptor tyrosine phosphorylation.

 
Next, assessments were made to determine the antiproliferative effects of C225, radiation, and the combination of these treatments. Cells were plated and allowed to enter an exponential growth phase. Cells were then treated with C225 alone (5 µg/mL), 3 Gy of radiation alone, or C225 and radiation. The cells were assessed for growth under these designated conditions at 96 hours (Fig 3). In all cases, a greater decrement in proliferation was noted for cells exposed to the combined treatment compared with either treatment given alone. When C225 was given as a single agent, cellular proliferation was inhibited by approximately 35% for the lowest EGFr-overexpressing cell line, UM-SCC-1 cells, and by 70% for the highest overexpressing cell line, UM-SCC-6 cells. These cell lines also illustrated the range in sensitivity to 3-Gy radiation. The UM-SCC-1 cells demonstrated the greatest resistance and the UM-SCC-6 cells the greatest sensitivity. It was shown that greater growth inhibition was found for the combination treatment, compared with either of the individual treatments, regardless of the inherent EGFr expression of the cell line (Fig 2A).



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  		Fig 3. Cell proliferation assay of A431 and five head and neck squamous cell carcinoma cell lines. Cells were treated with C225 antibody (5 µg/mL), 3-Gy radiation, or a combination of both agents on day 0. The antibody remained for the duration of the experiment, and cells were counted on day 4. All treated cells were normalized to their respective untreated control (100%). All samples were done in quadruplicate and prepared as described in the Materials and Methods. Abbreviation: RT, radiotherapy.

 
A representative cell line (UM-SCC-6) was used to test downstream events (relative to EGFr) that may be associated with the antiproliferative effects of the individual and combination treatments observed above (Fig 4). Initial experiments were performed to test the effects of C225, radiation, and the combined treatment on EGF-induced RTP. It was determined that C225 treatment (48 hours) resulted in a decrement in RTP in a dose-dependent manner (Fig 4). The exposure of cells to C225 (0.075 µg/mL) for 48 hours resulted in approximately a 50% reduction in RTP, whereas exposure of cells to C225 (1 µg/mL) for 48 hours resulted in a more than 90% reduction in RTP. Treatment with radiation alone did not significantly alter EGFr RTP (48 hours after radiotherapy) at low doses (2 to 4 Gy); however, there was a small decrease in RTP after treatment with 8 Gy (approximately 25% by densitometry). There was an increased decrement in RTP for the combination of low concentrations of C225 (0.075 and 0.1 µg/mL) and radiation (2 to 8 Gy) compared with either C225 alone or radiation alone; however, combined treatment resulted in similar decrements in RTP compared with C225 alone at higher concentrations of C225 (>= 0.25 µg/mL). Therefore, C225 caused decrements in RTP at all concentrations that were tested (0.075 to 1 µg/mL). Radiation treatment alone caused a small decrement in RTP only at the highest dose (8 Gy). The combination treatment showed a greater decrement in RTP at low concentrations of C225 compared with C225 or radiation alone, but this effect was not discernible at higher concentrations of C225 (Fig 4).



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  		Fig 4. Immunoblot (UM-SCC-6 cells) of the dose response of C225 exposures (48 hours). The effects of various concentrations of C225 in combination with various doses of radiation (0 to 8 Gy) on EGFr and EGF-induced EGFr RTP levels were investigated. All cells were treated with EGF (10 nmol/L) for 5 minutes before processing. Cells were processed as described in the Materials and Methods. Abbreviation: mAb, monoclonal antibody.

 
To further investigate the effects of C225, radiation, and the combined treatment on RTP, a time course study was performed. Cells were treated with radiation (4 Gy) alone, C225 (1 µg/mL) alone, or radiation followed by C225 for various times before a 5-minute exposure to ligand (EGF at 10 nmol/L). The time course studies produced similar results at all time points (0 to 24 hours), except for the immediate (0-hour) time point (Fig 5). When cells were exposed to C225 and immediately treated with EGF (0-hour condition), there was less decrement in RTP, compared with the longer exposures to C225 (1 to 24 hours [Fig 5]). Additionally, the combined treatment of radiation followed by various times of exposure to C225 produced similar RTP results compared with C225 alone for the conditions that were studied. Also, the various treatments did not affect levels of activated MAPK (Fig 5).



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  		Fig 5. Immunoblot (UM-SCC-6 cells) of the time course (0 to 24 hours) of C225 (1 µg/mL) exposure alone or in combination with radiation (4 Gy). All cells were treated with EGF (10 nmol/L) for the last 5 minutes before processing. Cells were assessed for EGFr, EGFr RTP (p-tyr), phosphorylated MAPK, and phosphorylated STAT-3 after exposure to C225 with or without radiation, as stipulated above the gel. Cells were processed as described in the Materials and Methods.

 
Since treatment with C225 alone or radiation followed by C225 reduced cellular proliferation and RTP, studies were performed to assess whether apoptotic events played a role in these decrements in cellular growth. It was determined that the combined treatment of C225 (1 µg/mL) and radiation (3 Gy) produced a greater level of apoptosis at 48 hours after the initiation of treatment, compared with either treatment alone (Fig 6).



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  		Fig 6. The percentage of UM-SCC-6 cells undergoing apoptosis after exposure to C225 (1 µg/mL) for 48 hours with or without 3-Gy radiation before the C225 exposure. Cells were processed as described in the Materials and Methods. Abbreviation: FITC, fluorescein isothiocyanate.

 
Recently, EGFr signaling has been linked to apoptosis through a STAT-3 pathway.23,25 Therefore, studies were performed to assess the role of STAT-3 in the apoptotic events observed above. It has been shown that activated STAT-3 (phosphorylated STAT-3) results in a protective function with respect to apoptosis.28,29 It was determined that treatments with either C225 (1 µg/mL) alone or C225 preceded by radiation (4 Gy) resulted in dramatic reductions in phosphorylated STAT-3 (Fig 5). Therefore, these results suggest a link between the effects of C225/radiation at the membrane level and phosphorylated STAT-3–induced control of apoptosis, as depicted in Fig 1.


   DISCUSSION AND REVIEW OF CLINICAL TRIALS INVOLVING C225 AND RADIATION THERAPY FOR HEAD AND NECK CARCINOMAS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION AND REVIEW OF...
REFERENCES
 
In previous work from our group, we studied the effects of combined C225/radiation treatment in vitro and in vivo for A431 human gynecologic squamous cell carcinomas.16 In both the in vitro and in vivo models, the combined treatment (C225/radiation) resulted in a greater decrement in cellular proliferation than either individual treatment. Although, the combination treatment substantially decreased cellular proliferation in vitro, this effect was more prominent in vivo. This effect correlated with apoptotic events in vitro and in vivo.16 The current findings have expanded this previous work to a panel of human head and neck squamous cell carcinoma lines, and the results suggest a link between C225/radiation–induced apoptosis and a decrease in phosphorylated STAT-3.

The results reported herein are consistent with the recent work of others. Grandis et al28 found that human head and neck squamous cell carcinomas (overexpressing EGFr) showed activation of STAT-3 after EGFr stimulation. By using a gene therapy approach to deliver a STAT-3 antisense plasmid, they inhibited STAT-3 activation, and increased tumor cell apoptosis was observed. In a separate report, Grandis et al29 studied human squamous cell carcinoma xenografts. The tumors were transfected with a dominant negative mutant STAT-3 that stimulated apoptosis and resulted in growth inhibition. Further work will be necessary to determine whether combinations of C225/radiation and gene therapy approaches with an aim to inactivate STAT-3 are feasible and potentially synergistic.

The mechanism of linkage between EGF-induced stimulation of EGFr and the activation of STAT-3 is controversial.25,30,31 It has been suggested that STAT-3 activation may be dependent on an intermediate step involving the JAK family of tyrosine kinases.31 However, this JAK-STAT sequence may not be required for activation of STAT-3 through the EGFr pathway.24,25 David et al25 recently suggested that STAT-3 activation may occur directly by EGFr stimulation (Fig 1). They concluded that EGFr tyrosine kinase is necessary and sufficient for the activation of many members of the STAT family of transcription factors. However, they did not rule out the possibility that JAK-like kinases may be involved in the process. Future work will be necessary to decipher the interplay of these signal transduction events in order to potentially augment our ability to exploit these processes therapeutically.

The work presented in this article has enhanced our preclinical understanding of the interaction of radiation and the inhibition of EGFr in order to increase radiation-induced tumor kill. This information further supports our previous studies,16,17 which served as preclinical background for clinical investigations involving C225/radiation in human head and neck squamous cell carcinomas. A phase Ib/IIa trial was performed at the University of Alabama at Birmingham to explore the feasibility of delivering weekly C225 (final dose after escalation procedures: 400 mg/m2 loading and 250 mg/m2 maintenance doses) in combination with standard radiotherapy (70 Gy/35 fractions qd or 74.4 Gy/62 fractions bid) for patients with locally advanced and unresectable squamous cell carcinomas of the head and neck. The formal results of this trial are in preparation (Ezekiel et al, manuscript in preparation); however, several recently published abstracts of this work suggest that the regimen is feasible and response rates are encouraging. An abstract presented at the 2000 Annual Meeting of the American Society of Clinical Oncology showed that all 15 assessable patients had a major response to treatment (13 complete responses and two partial responses).32 These results confirmed the encouraging early response results.33 Nine of the 15 patients have maintained locoregional control with more than 2 years of follow-up.32 These results compare favorably with the results of other trials in which radiation alone was used to treat unresectable head and neck tumors.34,35 In general, the locoregional control rates for these advanced tumors have fallen below or well below 50% at 2 years.34,35

Therefore, based on the promising preclinical results and reports of the above-noted phase Ib/IIa trial, a randomized phase III trial was begun (in the summer of 1999) to further investigate the regimen of the University of Alabama at Birmingham phase Ib/IIa trial. The trial was designed to compare the combined treatment of radiation and C225 (as studied in the phase Ib/IIa trial) with radiation alone for locally advanced squamous cell carcinomas of the head and neck (Fig 7). Approximately 150 patients have been entered onto this trial (as of August 2000) and the accrual goal is 410 patients.



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  		Fig 7. Schema for ongoing phase III trial for patients with locally advanced head and neck cancer.

 


   NOTES
 
J.A.B. receives financial reimbursement as a consultant to Imclone Systems, Inc.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION AND REVIEW OF...
 REFERENCES
 
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33. Ezekiel MP, Robert F, Meredith RF, et al: Phase I study of anti-epidermal growth factor receptor (EGFr) antibody C225 in combination with irradiation in patients with advanced squamous cell carcinoma of the head and neck (SCCHN). Proc Am Soc Clin Oncol 17: 395a, 1998 (abstr 1522)

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N. R. Schechter, R. E. Wendt III, D. J. Yang, A. Azhdarinia, W. D. Erwin, A. M. Stachowiak, L. D. Broemeling, E. E. Kim, J. D. Cox, D. A. Podoloff, et al.
Radiation Dosimetry of 99mTc-Labeled C225 in Patients with Squamous Cell Carcinoma of the Head and Neck
J. Nucl. Med., October 1, 2004; 45(10): 1683 - 1687.
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A. Chakravarti, A. Chakladar, M. A. Delaney, D. E. Latham, and J. S. Loeffler
The Epidermal Growth Factor Receptor Pathway Mediates Resistance to Sequential Administration of Radiation and Chemotherapy in Primary Human Glioblastoma Cells in a RAS-dependent Manner
Cancer Res., August 1, 2002; 62(15): 4307 - 4315.
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A. Chakravarti, J. S. Loeffler, and N. J. Dyson
Insulin-like Growth Factor Receptor I Mediates Resistance to Anti-Epidermal Growth Factor Receptor Therapy in Primary Human Glioblastoma Cells through Continued Activation of Phosphoinositide 3-Kinase Signaling
Cancer Res., January 1, 2002; 62(1): 200 - 207.
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