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. 2008 Oct 1;72(2):534-42.
doi: 10.1016/j.ijrobp.2008.06.1490.

Augmentation of radiation response by panitumumab in models of upper aerodigestive tract cancer

Tim J Kruser  1 , Eric A Armstrong, Amol J Ghia, Shyhmin Huang, Deric L Wheeler, Robert Radinsky, Daniel J Freeman, Paul M Harari
Affiliations

Augmentation of radiation response by panitumumab in models of upper aerodigestive tract cancer

Tim J Kruser et al. Int J Radiat Oncol Biol Phys. .

Abstract

Purpose: To examine the interaction between panitumumab, a fully human anti-epidermal growth factor receptor monoclonal antibody, and radiation in head-and-neck squamous cell carcinoma and non-small-cell lung cancer cell lines and xenografts.

Methods and materials: The head-and-neck squamous cell carcinoma lines UM-SCC1 and SCC-1483, as well as the non-small-cell lung cancer line H226, were studied. Tumor xenografts in athymic nude mice were used to assess the in vivo activity of panitumumab alone and combined with radiation. In vitro assays were performed to assess the effect of panitumumab on radiation-induced cell signaling, apoptosis, and DNA damage.

Results: Panitumumab increased the radiosensitivity as measured by the clonogenic survival assay. Radiation-induced epidermal growth factor receptor phosphorylation and downstream signaling through mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) was inhibited by panitumumab. Panitumumab augmented radiation-induced DNA damage by 1.2-1.6-fold in each of the cell lines studied as assessed by residual gamma-H(2)AX foci after radiation. Radiation-induced apoptosis was increased 1.4-1.9-fold by panitumumab, as evidenced by Annexin V-fluorescein isothiocyanate staining and flow cytometry. In vivo, the combination therapy of panitumumab and radiation was superior to panitumumab or radiation alone in the H226 xenografts (p = 0.01) and showed a similar trend in the SCC-1483 xenografts (p = 0.08). In vivo, immunohistochemistry demonstrated the ability of panitumumab to augment the antiproliferative and antiangiogenic effects of radiation.

Conclusion: These studies have identified a favorable interaction in the combination of radiation and panitumumab in upper aerodigestive tract tumor models, both in vitro and in vivo. These data suggest that clinical investigations examining the combination of radiation and panitumumab in the treatment of epithelial tumors warrant additional pursuit.

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Figures

Figure 1
Figure 1
In vivo efficacy of panitumumab (PNT) in HNSCC (A, B) and NSCLC (C) xenograft models. 2 × 106 cells were inoculated into the flank of aythmic mice (Day 0) as described in Materials and Methods. Mice were treated with indicated doses of PNT twice per week by intra-peritoneal injection (vertical arrows). Points, mean tumor size in mm3 (n=8 per group in H226 & UMSCC-1, n=10 per group in SCC-1483); bars, SEM.
Figure 2
Figure 2
Impact of panitumumab (PNT) on radiation response. Clonogenic survival was assessed in H226 (A) and UM-SCC1 (B) cells after exposure to radiation as described in Materials and Methods. Cells were exposed to either panitumumab (25 nM) or non-specific IgG for 72 hours before irradiation. Points, mean values from 6 seeded wells; bars, SEM. (C) PNT blocks radiation-induced EGFR phosphorylation and downstream signaling. UM-SCC1 cells were exposed to either PNT (25 nM) or human IgG for 6 hours before radiation, and then harvested 24 hours after radiation.
Figure 3
Figure 3
Panitumumab (PNT) augments radiation-induced apoptosis. H226 (A) and SCC-1483 cells (B) were either treated with 25 nM PNT or IgG control 24 hours before radiation. 48 hours after radiation cells were stained with Annexin V-FITC and analyzed as described in the methods. Plotted values are the mean % of cells undergoing early apoptosis (Annexin V positive, PI negative) in 3 replicates for each condition; bars, SEM. Asterisks indicate sets in which PNT augmented response (p <0.05, student’s t-test). (C) UMSCC-1 cells were exposed to either 25 nM PNT or human IgG for 6 hours before radiation (0–9 Gy), then 48 hours after radiation harvested for cleaved PARP analysis as described in the methods. Western blot analysis shows that PNT enhances radiation-induced PARP cleavage.
Figure 4
Figure 4
Panitumumab augments radiation-induced DNA damage. Cells from HNSCC lines UM-SCC1 (A) and SCC-1483 (B) and NSCLC line H226 (C) were exposed to either human IgG or panitumumab (PNT, 25 nM) for 1 hour before radiation. Cells were then incubated for 24 hours and analyzed for residual H2AX foci as described in the Materials and Methods. Foci were counted in >100 cells for each condition; bars, SEM. Asterisks indicate sets in which PNT resulted in significantly more residual foci (p <0.05, student’s t-test).
Figure 5
Figure 5
Panitumumab blocks radiation-induced EGFR nuclear translocation in SCC-1 cells. Panitumumab (PNT, 25 nM) or non-specific IgG was adminstered 1 hour before radiation (3 Gy). (A) Cells were lysed 20 minutes after radiation and cytosolic and nuclear fractions analyzed by Western blot. EGFR bands were quantitated using NIH ImageJ software, and relative amounts to IgG control bands calculated. Histone H3 blotting demonstrates adequate separation of nuclear and cytosolic fractions. (B) The relative ratio of nuclear to cytoplasmic EGFR for each condition demonstrates that EGFR translocates to the nucleus following radiation, and PNT abrogates this response.
Figure 6
Figure 6
In vivo activity of panitumumab (PNT) +/− radiation in tumor xenografts. (A) SCC-1483 (2 × 106) or (B) H226 (2×106) cells were inoculated subcutaneously into the flank of athymic mice as described in Materials and Methods. Mice were treated with PNT (intra-peritoneal injection), radiation (XRT), or the combination twice weekly (vertical arrows). Points, mean tumor size (mm3, n=10/group); bars, SEM. (C), (D) demonstrate the impact of PNT and XRT on PCNA expression in SCC-1483 and H226 xenografts, respectively. Positive staining (brown) in the nucleus is pronounced in control tumors, while tumors treated with the combination of PNT and XRT demonstrate little positive nuclear staining for PCNA (all micrographs taken at 200x).
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