Erlotinib inhibits growth of a patient-derived chordoma xenograft

Abstract

Chordomas are rare primary bone tumors that occur along the neuraxis. Primary treatment is surgery, often followed by radiotherapy. Treatment options for patients with recurrence are limited and, notably, there are no FDA approved therapeutic agents. Development of therapeutic options has been limited by the paucity of preclinical model systems. We have established and previously reported the initial characterization of the first patient-derived chordoma xenograft model. In this study, we further characterize this model and demonstrate that it continues to resemble the original patient tumor histologically and immunohistochemically, maintains nuclear expression of brachyury, and is highly concordant with the original patient tumor by whole genome genotyping. Pathway analysis of this xenograft demonstrates activation of epidermal growth factor receptor (EGFR). In vitro studies demonstrate that two small molecule inhibitors of EGFR, erlotinib and gefitinib, inhibit proliferation of the chordoma cell line U-CH 1. We further demonstrate that erlotinib significantly inhibits chordoma growth in vivo. Evaluation of tumors post-treatment reveals that erlotinib reduces phosphorylation of EGFR. This is the first demonstration of antitumor activity in a patient-derived chordoma xenograft model and these findings support further evaluation of EGFR inhibitors in this disease.

Figure 1. The chordoma PDX maintains the histological and immunohistochemical profile of the original patient tumor.

A and B. The patient’s clival tumor (A) and PDX, passage 8, (B) retained the classic chordoma features, including physaliphorous cells (inset). Both the patient's tumor (C) and PDX, passage 8 (D), were immunoreactive for brachyury. Magnifications: A and B, 100x; inset 260x; C and D, 160x. E. Karyotype of chordoma genomes. Estimation of copy number gains and losses in the original patient sample (left most bar) and PDX passages 1, 2, 3 and 4 (left to right). Green bar = gain; red bar = loss. Chromosome 7 has one copy number gain including the EGFR locus.

Figure 2. EGFR is activated in the chordoma PDX.

A. RTK phosphorylation array of the PDX demonstrated EGFR activation (box 1). Other activated kinases include EphB4 (2), Fgr (3), JAK2 (4), Lyn (5), SRMS (6), and TNK1 (7). The first six columns of the first row and last two columns of the last row contain positive controls. B and C. Analytical flow cytometry of the chordoma PDX (B) and A549 (C) for EGFR. Red line: anti-EGFR antibody; blue line: isotype control antibody. D and E. EGFR staining of the original patient tumor demonstrated surface staining of scattered tumor cells. Magnifications: D, 160x; E, 260x.

Figure 3. Erlotinib and gefitinib inhibit growth of U-CH1 in vitro.

Proliferation assays were performed following treatment of U-CH1 cells with control and increasing concentrations of erlotinib (A) and gefitinib (B). Data shown is mean relative cell proliferation (percent of control) + standard deviation. Experiment was repeated at least 3 times with quantitatively similar results.

Figure 4. Erlotinib inhibits growth of the chordoma PDX.

A. Growth curves of animals treated with vehicle (red line) or erlotinib (50 mg/kg, blue line) (p = 0.002). The growth curve of vehicle treated animals was censored at 37 days as tumors in two animals in this group reached 2,000 mm³ at this time point and were euthanized. B. Representative mice bearing flank xenografts treated with vehicle (left) and erlotinib (right).

Figure 5. Pathological changes in the PDX following treatment with erlotinib.

A. The control xenograft was composed largely of compact tissue with only a minority of loose, less cellular tumor. B. Loose, discohesive, less cellular areas were more common in erlotinib-treated xenografts. C and D. Nuclei in the control (C) and erlotinib (D) treated xenografts were immunoreactive for brachyury. E. Control treated xenografts had a brisk Ki-67 index. F. Ki-67 indices were generally lower in erlotinib-treated xenografts. Magnifications: A and B, 64X; C and D, 160X; E and F, 100x.

Figure 6. Phosphorylation of EGFR is reduced following treatment with erlotinib.

Representative EGFR phosphorylation arrays from control (left) and erlotinib treated tumors (right) demonstrated reduced phosphorylation of the Tyr845 site of EGFR (box 1) following treatment with erlotinib. The Erb2 phosphorylation sites Tyr 1112 and Tyr 1248 were also reduced (boxes 2 and 3). The first 3 columns of the first two rows and the last column of the last two rows contain positive controls.