Cetuximab-Based Immunotherapy and Radioimmunotherapy of Head and Neck Squamous Cell Carcinoma

Published OnlineFirst March 9, 2010; DOI: 10.1158/1078-0432.CCR-09-2495

Cancer Therapy: Preclinical Clinical Cancer Research Cetuximab-Based Immunotherapy and Radioimmunotherapy of Head and Neck Squamous Cell Carcinoma

Gang Niu1,2, Xilin Sun2,3, Qizhen Cao4, Donald Courter5, Albert Koong5, Quynh-Thu Le5, Sanjiv Sam Gambhir4, and Xiaoyuan Chen2,4

Abstract Purpose: To show the relationship between antibody delivery and therapeutic efficacy in head and neck cancers, in this study we evaluated the pharmacokinetics and pharmacodynamics of epidermal growth factor receptor (EGFR)–targeted immunotherapy and radioimmunotherapy by quantitative positron emission tomography (PET) imaging. Experimental Design: EGFR expression on UM-SCC-22B and SCC1 human head and neck squamous cell cancer (HNSCC) cells were determined by flow cytometry and immunostaining. Tumor delivery and distribution of cetuximab in tumor-bearing nude mice were evaluated with small animal PET using 64Cu-DOTA-cetuximab. The in vitro toxicity of cetuximab to HNSCC cells was evaluated by MTT assay. The tumor-bearing mice were then treated with four doses of cetuximab at 10 mg/kg per dose, and tumor growth was evaluated by caliper measurement. FDG PET was done after the third dose of antibody administration to evaluate tumor response. Apoptosis and tumor cell proliferation after cetuximab treatment were analyzed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and Ki-67 staining. Radioimmunotherapy was done with 90Y-DOTA-cetuximab. Results: EGFR expression on UM-SCC-22B cells is lower than that on SCC1 cells. However, the UM- SCC-22B tumors showed much higher 64Cu-DOTA-cetuximab accumulation than the SCC1 tumors. Cetuximab-induced apoptosis in SCC1 tumors and tumor growth was significantly inhibited, whereas an agonistic effect of cetuximab on UM-SCC-22B tumor growth was observed. After cetuximab treatment, the SCC1 tumors showed decreased FDG uptake, and the UM-SCC-22B tumors had increased FDG uptake. UM-SCC-22B tumors are more responsive to 90Y-DOTA-cetuximab treatment than SCC1 tumors, partially due to the high tumor accumulation of the injected antibody. Conclusion: Cetuximab has an agonistic effect on the growth of UM-SCC-22B tumors, indicating that tumor response to cetuximab treatment is not necessarily related to EGFR expression and antibody delivery efficiency, as determined by PET imaging. Although PET imaging with antibodies as tracers has limited function in patient screening, it can provide guidance for targeted therapy using antibodies as delivery vehicles. Clin Cancer Res; 16(7); 2095–105. ©2010 AACR.

Authors' Affiliations: 1Imaging Sciences Training Program, Radiology Head and neck squamous cell carcinoma (HNSCC) is and Imaging Sciences, Clinical Center and National Institute of theeighthmostcommoncancerintheUnitedStates, Biomedical Imaging and Bioengineering and 2Laboratory for Molecular and ∼47,000 new cases of HNSCC were diagnosed in Imaging and Nanomedicine, National Institute of Biomedical Imaging and Bioengineering, NIH, Bethesda, Maryland; 3Department of Medical 2008 (1). HNSCC comprises ∼80% to 90% of cancers aris- Imaging and Nuclear Medicine, the 4th Affiliated Hospital, Harbin ing from the head and neck region. Patients with HNSCC Medical University, Harbin, China; and 4The Molecular Imaging Program at Stanford, Department of Radiology and Bio-X Program and are treated with surgery, radiotherapy alone, or surgery 5Department of Radiation Oncology, Stanford University School of combined with postoperative adjuvant radiotherapy or Medicine, Stanford, California chemotherapy (2). Recently, concomitant chemoradiation Note: Supplementary data for this article are available at Clinical Cancer has become increasingly popular for advanced resectable Research Online (http://clincancerres.aacrjournals.org/). cancers, although there seems to be no survival advantage Corresponding Author: Xiaoyuan Chen, Laboratory for Molecular when compared with other forms of therapy (3). How- Imaging and Nanomedicine, National Institute of Biomedical Imaging and Bioengineering, NIH, 31 Center Drive, 31/1C22, Bethesda, MD ever, for patients with unresectable advanced disease, even 20892. Phone: 301-451-4246; Fax: 301-480-1613; E-mail: shawn.chen@ the combined therapy only achieved suboptimal disease nih.gov. control with a 5-year survival rate of <10% (4). Even doi: 10.1158/1078-0432.CCR-09-2495 worse, patients with recurrent or metastatic disease have ©2010 American Association for Cancer Research. a median survival of merely 6 to 9 months (5). There is

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(21) resulted in significantly longer median survival when Translational Relevance compared with chemotherapy alone in patients with recurrent or metastatic HNSCC. Due to the high expression of epidermal growth fac- Although clinical results for EGFR targeting with specific torreceptor(EGFR)inheadandneckcancerandits antibodies are promising, most studies indicate that only a critical role in supporting aggressive growth in cancer, subgroup of patients receiving the monoclonal antibodies EGFR is a valid target for the treatment of cancer benefit from the drug (22, 23). No correlation has been patients, especially when combined with radiation found between the efficacy of cetuximab and EGFR tumor- therapy. In this study, we found that the efficacy of al staining intensity by immunohistochemistry (24, 25). EGFR-targeted therapy with cetuximab in selected head In addition, cetuximab response has been observed in pa- and neck squamous cell carcinoma is not related to an- tients with EGFR-negative tumors (26). Although it has tibody delivery, as measured by small animal positron been reported that EGFR gene copy number may predict emission tomography imaging. In addition, we ob- response to cetuximab, there are concerns regarding repro- served that cetuximab has an agonistic effect on the ducibility of such assays (27). Using 64Cu-labeled panitu- growth of UM-SCC-22B tumors, emphasizing the mumab, we have done small animal positron emission importance of patient selection before providing this tomography (PET) in several HNSCC tumor models therapeutic regimen. Although positron emission to- (28). Although no correlation with tumor EGFR expres- mography imaging with antibodies as tracers has lim- sion was observed, quantitative PET imaging with radiola- ited function in patient screening, it can provide beled antibody enabled visualization and quantification guidance for targeted therapy using antibodies as de- of a number of parameters, including tumor-specific bind- livery vehicles. ing, perfusion, vascularity, vascular permeability, and plasma half-life. It provided more comprehensive information about antibody delivery than immunohistochemistry or fluorescence in situ hybridization (29). To explore the po- thus an urgent need for both early detection of HNSCC tential of immuno-PET to assess EGFR-targeted therapy, and development of new therapeutic regimens and drugs. we next investigated the therapeutic efficacy of cetuximab Accumulating evidence suggests that targeted biological and 90Y-cetuximab in two HNSCC tumor models. Our therapies selectively interfering with cancer cell and/or data showed that with very high local accumulation of endothelial cell growth signaling may improve HNSCC antibody, UM-SCC-22B tumors are resistant to cetuximab patient survival by enhancing radiation and chemotherapy and sensitive to 90Y-cetuximab. efficacy without increasing treatment-related toxicity (6, 7). Currently, epidermal growth factor receptor (EGFR) is a Materials and Methods valid target for the treatment of cancer patients (8). As a member of the structurally related erbB family of receptor Chemicals and reagents. 1,4,7,10-Tetraazadodecane-N,N′, tyrosine kinases (9), EGFR promotes tumor progression in N′′,N′′′-tetraacetic acid (DOTA) was purchased from several solid cancers (10). Dysregulation of EGFR is asso- Macrocyclics, Inc. FITC and Chelex 100 resin (50–100 ciated with several key features of cancer, such as autono- mesh) were purchased from Sigma-Aldrich. Water mous cell growth, inhibition of apoptosis, angiogenic and all buffers were passed though Chelex 100 columns potential, invasion, and metastasis (11, 12). Elevated (1 × 15 cm) before use in radiolabeling procedures to en- EGFR level has been reported in >95% of HNSCCs com- sure that the aqueous buffers were free of heavy metals. pared with normal mucosa (13). In addition, elevated PD-10 desalting columns were purchased from GE Health- EGFR expression is an independent indicator of poor care. 64Cu was provided by the University of Wisconsin- prognosis and lower survivals in HNSCC patients (14). Madison. 90Y was obtained from Perkin-Elmer. Cetuximab EGFR-targeted therapies include monoclonal antibodies, was purchased from ImClone Systems, Inc. such as cetuximab (IMC-C225, Erbitux) and panitumu- Cell lines and tumor models. The human head and neck mab (ABX-EGF, Vectibix), that block the extracellular squamous carcinoma cell lines SCC1 and UM-SCC-22B ligand-binding domain of the receptor and tyrosine kinase were obtained from the University of Michigan. They were inhibitors that prevent the activation of the cytoplasmic maintained in DMEM supplemented with 10% fetal bo- kinase portion (15–17). These targeting approaches have vine serum, 1% glutamine, 100 units/mL penicillin, and shown great promise in preclinical studies (18, 19). It 100 mg/mL streptomycin (Invitrogen). All animal experi- has been reported that radiation activates EGFR signaling, ments were done under a protocol approved by the Stan- leading to radio resistance by inducing cell proliferation ford University Administrative Panel on Laboratory and enhanced DNA repair (20). In patients with locore- Animal Care. Subcutaneous HNSCC tumor models were gionally advanced HNSCC, the combination of cetuximab established in 4-wk-old to 6-wk-old female athymic nude and high-dose radiation was found to yield superior sur- mice obtained from Harlan. Typically, 5 × 106 cells sus- vival than radiation alone (6). Similarly, the addition of pended in 50 μL of PBS were injected, and the mice were cetuximab to chemotherapy in a large randomized study subjected to therapeutic or small animal PET studies when

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the tumor volume reached 100 to 200 mm3 (2–3 wk after per milliliter per minute by using a conversion factor. As- inoculation). suming a tissue density of 1 g/mL, the counts per milliliter Flow cytometry. HNSCC cells were harvested and per minute were converted to counts per gram per minute washed with PBS containing 0.5% bovine serum albumin. and then divided by the injected dose (ID) to obtain an After blocking by 2% bovine serum albumin in PBS, the imaging ROI-derived % ID/g. cells were incubated with cetuximab (10 μg/mL in PBS For FDG imaging, the animals were fasted overnight containing 2% bovine serum albumin). FITC-conjugated before scanning. After a tail vein injection of 3.7 MBq of donkey anti-human IgG (1:200) was then added and 18F-FDG in 150 μL of PBS, a 7-min prone acquisition scan allowed to incubate for 1 h at room temperature. After was done ∼60 min after injection. Mice were maintained washing, the cells were analyzed using an LSR flow cyt- under isoflurane anesthesia during the injection, accumu- ometer (Beckman Coulter). The FITC signal intensity was lation, and scanning periods. PET images were analyzed analyzed using Cell-Quest software (version 3.3, Becton and quantified as described above. Dickinson). Immunotherapy and radioimmunotherapy. To assess the MTT assay. The toxicity of cetuximab to SCC1 and therapeutic efficacy of cetuximab to HNSCC tumors, inoc- UM-SCC-22B cells was determined by MTT assay. All stud- ulated mice with a uniform tumor size approaching ies were done with triplicate samples and repeated at least ∼100 mm3 at 2 to 4 wk after tumor cell inoculation were thrice. Briefly, cells were harvested by trypsinization, resus- chosen for study. For SCC1 tumor, four doses of cetuxi- pended in DMEM, and plated in a 96-well plate at 4,000 mab (10 mg/kg) were given by i.v. injection every 4 d. or 2,000 cells per well. At 72 or 120 h after treatment with The same dose of cetuximab was given to UM-SCC-22B– different doses of cetuximab (ranging from 0.1 nmol/L to bearing tumor at an interval of every 2 d, because the tu- 0.5 μmol/L), the culture medium was replaced and 50 μL mor growth of UM-SCC-22B is much faster than that of of 1.0 mg/mL sterile filtered MTT (Sigma) were added to SCC1. To characterize the dose response of 90Y-cetuximab, each well. The unreacted dye was removed after 4 h, and animals were randomly divided into three groups and the insoluble formazan crystals were dissolved in 150 μL injected with a single dose of 3.7 MBq 90Y-cetuximab, of DMSO. The absorbance at 570 nm (reference wave- 3.7 MBq 90Y-IgG, or saline (n = 7 per group). Tumor length, 630 nm) was measured with a Tecan microplate growth was monitored by caliper measurement, and tu- reader (Tecan). mor volume (assuming an ellipsoidal shape) was approx- Antibody labeling. Detailed procedures for FITC and imated by the equation: tumor volume (mm3)=long DOTA conjugation of cetuximab have been reported earli- diameter × (short diameter)2 /2. 64 64 er (28). For Cu labeling, CuCl2 (74 MBq) was diluted Immunofluorescence staining. Frozen HNSCC tumor in 300 μL of 0.1 mol/L sodium acetate buffer (pH 6.5) and slices (5-μm thickness) were fixed with cold acetone for added to 50 μg of DOTA-cetuximab. The reaction mixture 10 min and dried in air for 30 min. The slices were rinsed was incubated for 1 h at 40°C with constant shaking. with PBS for 2 min and blocked with 10% donkey serum 64Cu-DOTA-cetuximab was then purified by PD-10 column for 30 min at room temperature. The slices were then in- using PBS as the mobile phase. The labeling yield was cubated with cetuximab and rat anti-mouse CD31 anti- calculated by dividing the radioactivity of 64Cu-DOTA- body for 1 h at room temperature and visualized using cetuximab by the total input radioactivity. FITC-conjugated donkey anti-human secondary antibody For radioimmunotherapy, 90Y was added to the DOTA- (1:200; Jackson ImmunoResearch Laboratories, Inc.) and cetuximab conjugates at 0.68 μg of DOTA-cetuximab per Cy3-conjugated rat anti-mouse IgG (1:200; Jackson Immu- MBq of 90Y. The reaction mixture was incubated for 1 h at noResearch Laboratories, Inc.). The slides were observed 40°C with constant shaking. The 90Y-DOTA-cetuximab under a microscope (Axiovert 200M, Carl Zeiss USA), conjugate was purified by PD-10 column using PBS as and images were acquired under the same conditions the eluent. The radioactive fractions containing 90Y- and displayed at the same scale for comparison. DOTA-cetuximab were collected and passed through a For in vivo staining, tumor samples were collected at 0.2-μm syringe filter for further in vivo experiments. 24 h after 50 μg FITC-cetuximab was injected through the Small animal PET and image analysis. PET imaging of tail vein. The tumor sections were fixed and costained with tumor-bearing mice was done on a microPET R4 rodent CD31 using a procedure similar to that described above. model scanner (Siemens Medical Solutions) as described Microvascular density measurement. After CD31 staining, earlier (28). The mice were i.v. injected with 64Cu- 10 random views in both the center and the periphery of DOTA-cetuximab or 64Cu-DOTA-IgG (Jackson Immuno- the tumor slices were selected for microvascular density Research Laboratories; 7–8MBq/6μg/mouse) and static (MVD) analysis using an observer-set threshold to distin- scans were acquired at 4, 20, 30, and 48 h postinjection. guish vascular elements from surrounding tissue paren- For each microPET scan, three-dimensional regions of chyma. Any vessel that contained branching points was interest (ROI) were drawn over the tumor, liver, heart, counted as a single vessel. The number of vessels counted and muscle on decay-corrected whole-body coronal was divided by the field of view to yield the MVD (as images. The average radioactivity concentration within a vessels/mm2). tumor or an organ was obtained from mean pixel values Ki-67 index measurement. HNSCC tumor sections within the ROI volume, which were converted to counts were stained with Ki-67–specific SP6 rabbit monoclonal

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Fig. 1. A, flow cytometric analysis of EGFR expression on HNSCC cells. Cetuximab was used as the primary antibody, and FITC-conjugated donkey anti-human IgG was used as secondary antibody. The mean values of FITC signal intensity (MFI) of the three measurements are shown. Columns, mean; bars, SD. 22B, UM-SCC-22B. B and C, immunofluorescence examination of EGFR expression. Images were obtained under the same conditions and are displayed at the same magnification and scale. Tumor sections were directly stained with cetuximab as primary antibody and FITC-conjugated donkey anti-human IgG as secondary antibody. Murine CD31 was stained with Cy3-conjugated IgG to visualize tumor vasculature (B). Thirty hours after DOTA-cetuximab injection, tumors were harvested, and tumor sections were stained with FITC-conjugated donkey anti-human IgG (C). Red color from Cy3 for CD31, green color from FITC for EGFR and cetuximab, blue color from DAPI for nuclei visualization. Representative photos were taken under ×200 magnification. Scale bar, 50 μm. MicroPET images of HNSCC UM-SCC-22B (D) and SCC1 (E) tumor-bearing nude mice at different time points after i.v. injection of 64Cu-DOTA-cetuximab (n = 4 per group). Decay-corrected coronal images at different time points are shown, and the tumors are indicated by white arrows.

antibody (Abcam, Inc.) and FITC-conjugated goat anti- on the samples. The slides were then incubated in a hu- rabbit secondary antibody (1:200; Jackson Immuno- midified atmosphere for 60 min at 37°C in the dark. After Research Laboratories, Inc.) as described above. Nuclear rinsing with PBS and mounting with DAPI containing staining of Ki-67 was considered positive. The Ki-67 stain- mounting medium, the samples were observed under a ing index (SI) was defined as the percentage of positive fluorescence microscope using DsRed channel (excita- nuclei within the total number of nuclei in 10 random tion/emission maxima, 558 nm:583 nm). views, as indicated by 4′,6-diamidino-2-phenylindole Statistical analysis. Quantitative data were expressed as (DAPI) staining. The positive nuclei were counted by mean ± SD. Means were compared using one-way ANOVA one investigator (X. S.) without prior knowledge of the and Student's t test. P values of <0.05 were considered sta- treatment information. tistically significant. Terminal dUTP nick-end labeling assay. Apoptosis induced by cetuximab treatment was detected by terminal dUTP nick-end labeling (TUNEL) assay using an in situ cell Results death detection kit (Roche Applied Science) according to the manufacturer's instructions. After fixation and permea- High expression of EGFR in both SCC1 and UM-SCC-22B bilization, 50 μL of TUNEL reaction mixture were added cells. As determined by flow cytometry, both SCC1 and

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Table 1. Biodistribution of 64Cu activity in HNSCC tumor models

22B (% ID/g) SCC1 (% ID/g) 4 h 20 h 30 h 48 h 4 h 20 h 30 h 48 h

Blood 14.64 ± 1.00 6.71 ± 2.17 5.75 ± 2.40 5.50 ± 2.10 21.60 ± 3.24 11.47 ± 1.22 9.39 ± 0.78 8.67 ± 0.15 Liver 14.86 ± 2.82 11.17 ± 1.78 10.70 ± 2.02 10.21 ± 2.34 17.71 ± 0.56 13.28 ± 2.72 12.78 ± 2.20 12.02 ± 3.01 Muscle 3.43 ± 0.99 3.19 ± 0.83 2.02 ± 0.28 2.34 ± 0.55 3.16 ± 0.45 2.44 ± 0.22 2.25 ± 0.41 1.22 ± 0.33 Tumors 2.12 ± 0.13 18.92 ± 1.14 19.07 ± 0.26 21.90 ± 0.98 2.12 ± 0.13 6.20 ± 0.55 7.35 ± 0.75 10.98 ± 2.97

NOTE: Data are given as % ID/g and represent mean ± SD of three mice.

UM-SCC-22B cells expressed relatively high levels of EGFR However, the antibody distribution pattern was very dif- using cetuximab as primary antibody (Fig. 1A). EGFR ex- ferent in these two tumor types. For UM-SCC-22B tumors, pression on SCC1 cells is significantly higher than on the antibody showed a more diffusive and homogenous UM-SCC-22B cells. Immunofluorescence staining of tu- distribution within the whole tumor region, as indicated mor sections derived from these two cell lines also showed by the green fluorescence signals. By contrast, in SCC1 tu- that there is higher EGFR expression on SCC1 tumor cells mors, the antibody only diffused around the perivascular than on UM-SCC-22B tumor cells (P < 0.01; Fig. 1B). regions and showed more patchy distribution (Fig. 1C).

Fig. 2. Antitumor activity of cetuximab in established HNSCC xenografts. A and B, the cytotoxic effect of cetuximab on SCC1 (A) and UM-SCC-22B (B) cells. Cells were treated with serial concentrations of cetuximab. At 72 and 120 h after inoculation, the cell proliferation was determined by MTT assay. C and D, comparison of SCC1 (C) and UM-SCC-22B (D) tumor growth in nude mice treated with cetuximab versus control animals. E and F, comparison of body mass of nude mice bearing SCC1 (E) and UM-SCC-22B (F) treated with cetuximab versus control animals. Animals were injected through tail vein with 50 mg/kg cetuximab for four doses. *, P < 0.05; **, P < 0.01.

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Fig. 3. Small animal PET images of SCC1 (A) or UM-SCC-22B (B) tumor-bearing nude mice with 18F-FDG at day 5 after treatment with cetuximab (n =4–7). Decay-corrected whole-body coronal images that contain the tumor were shown, and the tumors are indicated by white arrows. SCC1 (C) and UM-SCC-22B (D) tumor uptake of 18F-FDG as quantified from small animal FDG PET scans (n =4–7).

Small animal PET imaging of 64Cu-cetuximab tumor 86% compared with control cells. Almost no proliferation delivery. To further characterize the whole body biodistri- inhibition effect was observed in UM-SCC-22B cells even bution and tumor targeting efficiency of the therapeutic at 120 hours after addition of 50 μmol/L of cetuximab antibody in vivo, we did small animal imaging with (Fig. 2B). In contrast, cetuximab showed marked inhibi- 64Cu-DOTA-cetuximab on HNSCC tumor-bearing mice. tion in tumor growth after i.v. injection of four doses of The decay-corrected whole-body coronal images contain- 10 mg/kg body weight in SCC1 tumor-bearing mice ing the tumors are shown in Fig. 1D for UM-SCC-22B (Fig.2C).AsshowninFig.2D,however,anagonistic tumors and Fig. 1E for SCC1 tumors. The accumulation effect on the tumor growth was found in cetuximab- of 64Cu-DOTA-cetuximab in UM-SCC-22B tumors was treated UM-SCC-22B–bearing animals. The treatment of much higher than that in SCC1 tumors at all time points cetuximab was well tolerated in both SCC1 and UM- examined after tracer injection. Quantitative data based on SCC-22B–bearing animals, and no apparent body weight ROI analysis are shown in Table 1. At 30 hours postinjec- loss was observed (Fig. 2E and F). tion, the UM-SCC-22B tumor uptake of 64Cu-DOTA- Monitoring therapeutic effect by FDG PET imaging. By cetuximab was 19.07 ± 0.26% ID/g, whereas SCC1 tumor measuring glucose metabolism, 18F-FDG PET can be used uptake was 7.35 ± 0.75% ID/g. The liver also had to evaluate the efficacy of therapeutic intervention (30). prominent radioactivity accumulation, with an uptake of We carried out 18F-FDG small animal PET scans on 10.70 ± 2.02% ID/g in UM-SCC-22B tumor-bearing mice SCC1 and UM-SCC-22B tumor models after the third dose and 12.78 ± 2.20% ID/g in SCC1 tumor-bearing mice at of cetuximab treatment. For SCC1 tumors, cetuximab 30 hours postinjection, respectively. Blood activity concen- treatment resulted in a significant decrease of FDG uptake tration was 5.75 ± 2.40% ID/g and 9.39 ± 0.78% ID/g at within the tumor region, indicating slower tumor growth 30 hours postinjection, indicating the long circulation life- after treatment (P < 0.01; Fig. 3A and C). By contrast, FDG time of the antibody. uptake in UM-SCC-22B tumors showed a significant in- Therapeutic effect of cetuximab on HNSCC tumors. As crease after cetuximab treatment (P < 0.05; Fig. 3B and shown in Fig. 2A, treatment with cetuximab produced on- D), which is consistent with the accelerated tumor growth. ly modest inhibition of cell proliferation on SCC1 cells Cetuximab-induced apoptosis and proliferation. To further in vitro as determined by MTT assay. With 50 μmol/L ce- investigate the mechanism of sensitivity and resistance of tuximab, SCC1 cells still kept a survival fraction around HNSCC tumors to cetuximab, we did TUNEL assays to

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evaluate cell apoptosis induced by cetuximab in both 33.02 ± 2.18% at 48 hours and to 33.34 ± 8.15% at SCC1 and UM-SCC-22B tumors. In SCC1 tumors, apopto- 72 hours (P < 0.05). sis was observed as early as 24 hours after cetuximab treat- Radioimmunotherapy with 90Y-cetuximab. The PET imag- ment and apoptotic cells were found distributed along ing data showed that UM-SCC-22B tumors had very high vasculature. At 48 hours, more apoptotic cells were visual- uptake of cetuximab. To our surprise, cetuximab did not ized within the whole tumor region (Fig. 4A). However, show growth inhibition of UM-SCC-22B tumors but in- for UM-SCC-22B tumors, no apoptosis was observed in stead showed a proliferation-stimulating effect. We sur- nonnecrotic tumor tissue regions, even at 72 hours after mised that radioimmunotherapy, by contrast, might be cetuximab treatment (Fig. 4B). Based on CD31 staining, effective in tumor control, due to the high radioactivity we also quantified MVD in cetuximab-treated and control dose deposited in the tumor region. In fact, as shown in tumors. As shown in Fig. 4C and D, MVD decreased sig- Fig. 6, in UM-SCC-22B tumor-bearing mice that received nificantly even at 24 hours after treatment (P < 0.01). an i.v. injection of 3.7 MBq of 90Y-cetuximab achieved sig- However, there is no significant change of MVD in UM- nificant tumor growth inhibition. As described above, SCC-22B tumors at 48 hours after treatment with cetuxi- there is significant passive accumulation of antibody in mab, and an increased MVD was observed at 72 hours UM-SCC-22B tumor due to high tumor vasculature and (P < 0.05). vasculature permeability. Consequently, tumor growth in- UM-SCC-22B is a fast growing cell line, and inoculated hibition was also observed after i.v. injection of 3.7 MBq tumors also showed high growth rate. It is not surprising 90Y-IgG. For SCC1 tumors, 90Y-cetuximab only produced to observe more Ki-67–positive cells in UM-SCC-22B tu- moderate tumor growth inhibition. At the same time, mors than in SCC1 tumors (SI = 24.01 ± 5.01% versus 3.7 MBq 90Y-IgG showed no therapeutic effect on SCC1 11.44 ± 3.93%, P < 0.01; Fig. 5). One dose of cetuximab tumor growth. treatment decreased the Ki-67 SI from 11.44 ± 3.93% to 4.73 ± 1.78% and 3.19 ± 1.72% at 24 and 48 hours after Discussion therapy, respectively (P < 0.01). To the contrary, after a single-dose cetuximab treatment, the Ki-67 SI in UM- In this study, we have imaged 64Cu-cetuximab distribu- SCC-22B tumors increased from 24.01 ± 5.01% to tion and tumor delivery in HNSCC tumor-bearing mice

Fig. 4. Double staining of TUNEL and CD31 of SCC1 (A) and UM-SCC-22B (B) tumor section after treatment with 100 mg/kg of cetuximab. Vasculature is shown in green, and apoptotic nuclei are shown in red. Normal cell nuclei are shown in blue, stained by DAPI. Representative photos were taken under ×200 magnification. Scale bar, 50 μm. C and D, MVD measurement in SCC1 (C) and UM-SCC-22B (D) tumors. *, P < 0.05; **, P < 0.01.

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with small animal PET. Consistent with our previous ob- generally believed that anti-EGFR therapy could downre- servation, UM-SCC-22B tumors showed higher tumor gulate VEGF levels (36, 37). However, results from ELISA accumulation of imaging tracer, although the EGFR indicated no significant change in tumoral hVEGF levels or expression was lower than that in SCC1 tumors. SCC1 tu- circulating mVEGF levels upon cetuximab treatment in mors responded very well to cetuximab therapy, which is UM-SCC-22B–bearing mice. In addition, hVEGF levels consistent with studies done by Huang and Harari (31). in cell culture media were found to be downregulated However, treatment with cetuximab did not produce any by cetuximab in UM-SCC-22B cells, but not in SCC1 cells therapeutic effect in UM-SCC-22B tumor growth, but rath- (Supplementary Fig. S2). VEGF level is thus not directly re- er accelerated its growth. As reflected by Ki67 staining, lated to UM-SCC-22B tumor accelerating growth induced by UM-SCC-22B tumor proliferation rate increased signifi- cetuximab treatment. The UM-SCC-22B tumor-stimulating cantly compared with tumors in the control group at 48 effect of cetuximab will need further investigation. hours after cetuximab treatment. Several possible mechan- The 64Cu-cetuximab accumulation in SCC1 tumors is isms have been suggested to explain the tumor resistance lower than that in UM-SCC-22B tumors, although SCC1 to cetuximab treatment. For example, it has been reported cells showed higher EGFR expression. As observed in our that mutant EGFR, especially EGFRvIII, contributes to the previous study (28), besides cell surface antigen level, oth- resistance to EGFR-targeted therapy (32). There is no er factors including tumor-specific binding, perfusion, vas- EGFRvIII mutation in SCC1 or UM-SCC-22B cells (Supple- cularity, vascular permeability, and plasma half-life also mentary Fig. S1), however. KRAS mutation and PTEN loss influence the final PET imaging quantification when radi- have also been reported to be related to the resistance of olabeled antibody is used as an imaging tracer. In vivo colorectal cancers to cetuximab treatment (33, 34). How- immunostaining with FITC-cetuximab also confirmed ever, PCR results revealed abundant PTEN expression in the limited perivasculature distribution of cetuximab in both tumors (data not shown), and KRAS mutations are SCC1 tumor. It has been reported that the patchy and in- rare in HNSCC (35). At 72 hours after cetuximab treat- complete tumor perfusion could result in suboptimal ther- ment, MVD increase was obvious in UM-SCC-22B tumors. apeutic effects when therapeutic efficacy is dependent We speculated that cetuximab might stimulate vascular upon uniform delivery to tumor cells (38, 39). However, endothelial growth factor (VEGF) expression, as it is the patchy and limited distribution of cetuximab in SCC1

Fig. 5. Double staining of Ki67 and CD31 of SCC1 (A) and UM-SCC-22B (B) tumor section after treatment with 100 mg/kg of cetuximab. Vasculature is shown in green, and Ki67-positive nuclei are shown in red. Representative photos were taken under ×200 magnification. Scale bar, 50 μm. Ki-67 SI calculated based on staining for SSC1 (C) and UM-SCC-22B (D). *, P < 0.05; **, P < 0.01.

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man glioblastoma U87MG tumor model with 90Y-etaracizumab, a monoclonal antibody against human integrin αvβ3. The maximum tolerated dose and dose response analysis in that study revealed that 7.4 MBq per mouse was the optimal therapeutic dose. After comparing the biodistribution data obtained from small animal PET with 64Cu-etaracizumab (42) and or 64Cu-cetuximab (43), we chose a one-time dose of 3.7 MBq of 90Y-labeled cetuximab for radioimmunotherapy in the current study. With this dose, UM-SCC-22B tumor growth was significantly inhibited. Moreover, four of seven treated tumors dis- appeared, that is, showed a complete response. Even 90Y- IgG provided a therapeutic effect, because the passive accumulation of IgG in the tumor region deposited suffi- cient radioactivity. Meanwhile, in SCC1 tumors, we only observed a moderate effect of radioimmunotherapy with 90Y-cetuximab and no therapeutic effect with 90Y-IgG. The antibody amount used in radioimmunotherapy was only around 5 μg/mouse instead of the around 200 μg/mouse used in immunotherapy with cetuximab. We believe that most of the therapeutic power of 90Y- cetuximab came from the radiation dose delivered along with the antibodies, but not the antibodies themselves. The 3.7-MBq dose used in this study showed no observ- able toxicity, as indicated by unchanged mouse body weight (Supplementary Fig. S3). Besides radioisotopes, it should also be promising to conjugate other therapeutic Fig. 6. 90Y-centuximab inhibition of SCC1 (A) and UM-SCC-22B (B) moieties, such as chemotherapeutics, toxins, or molecular tumor growth in vivo. Female athymic nude mice bearing tumors were therapeutics onto the antibody for targeted delivery, injected with a one-time dose of saline and 3.7 MBq of 90Y-cetuximab. given the dominant tumor uptake of the antibody after The growth inhibition of experimental groups was monitored via serial systemic administration, as shown in the case of UM- caliper measurements. SCC-22B tumors. According to the studies presented in this report, the efficacy of EGFR-targeted therapy with cetuximab, as deter- tumors did not hinder therapeutic efficacy. TUNEL assay mined by small animal PET imaging, is not related to revealed that at 24 hours after antibody administration, antibody delivery. Another interesting finding is that cetux- apoptotic cells are localized around vasculature, which is imab therapy has an agonistic effect on the growth of UM- consistent with the antibody distribution. At 48 hours, a SCC-22B tumors. Although the underlying mechanism is more homogenous distribution of apoptotic cells was ob- still unclear, this observation emphasizes the importance served, which might have resulted from antibodies diffus- of patient selection before providing this therapeutic regi- ing to greater distances. Another possibility is that low men. Finally, our study revealed limitations of PET imaging concentrations of antibodies might need a longer time with antibodies as tracers to determine antigen expression to take effect. Increased tumor cell apoptosis, decreased and to predict tumor response to antibody therapy. Howev- proliferation, and MVD have been reported in cetuximab- er, immuno-PET can provide guidance for targeted therapy responding tumors upon receiving treatment (40, 41). In using antibodies as delivery vehicles. this study, we also observed similar therapeutic changes in SCC1 xenografts as early as 24 hours after cetuximab treat- Disclosure of Potential Conflicts of Interest ment. In addition, FDG PET imaging confirmed decreased metabolism in the SCC1 xenografts following cetuximab No potential conflicts of interest were disclosed. treatment. Although high local antibody delivery did not achieve an inhibitory effect on UM-SCC-22B tumors, we speculat- Acknowledgments ed that we may be able to take advantage of the high tu- We thank Dr. Henry S. Eden for proofreading this manuscript. mor uptake of antibodies in UM-SCC-22B tumors. As previously reported, the high tumor uptake comes from Grant Support both specific antibody EGFR binding and passive targeting due to highly vascularized tumor tissue and high vascular National Cancer Institute grants R21 CA102123 (X. Chen), P50 permeability (28). In another study (42), we treated a hu- CA114747 (X. Chen), U54 CA119367 (X. Chen), R24 CA93862

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Niu et al.

(X. Chen), R01 CA118582 (Q.-T. Le), and P01 CA67166 (Q.-T. Le) and The costs of publication of this article were defrayed in part by the payment training grant PHS CA09302 (D. Courter) and Department of Defense of page charges. This article must therefore be hereby marked advertisement Prostate Cancer Postdoctoral Training Award (G. Niu). Dr. G. Niu current- in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ly is an Imaging Sciences Training Fellowship jointly supported by Radi- ology and Imaging Sciences Department, NIH Clinical Center and Intramural Research Program, National Institute of Biomedical Imaging Received 09/13/2009; revised 01/20/2010; accepted 01/21/2010; and Bioengineering, NIH. published OnlineFirst 03/09/2010.

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Cetuximab-Based Immunotherapy and Radioimmunotherapy of Head and Neck Squamous Cell Carcinoma

Gang Niu, Xilin Sun, Qizhen Cao, et al.

Clin Cancer Res 2010;16:2095-2105. Published OnlineFirst March 9, 2010.

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