Frequent activation of EGFR in advanced chordomas

Abstract

Background: Chordomas are rare neoplasms, arising from notochordal remnants in the midline skeletal axis, for which the current treatment is limited to surgery and radiotherapy. Recent reports suggest that receptor tyrosine kinases (RTK) might be essential for the survival or proliferation of chordoma cells, providing a rationale for RTK targeted therapy. Nevertheless, the reported data are conflicting, most likely due to the assorted tumor specimens used for the studies and the heterogeneous methodological approaches. In the present study, we performed a comprehensive characterization of this rare entity using a wide range of assays in search for relevant therapeutic targets.

Methods: Histopathological features of 42 chordoma specimens, 21 primary and 21 advanced, were assessed by immunohistochemistry and fluorescent in situ hybridization (FISH) using PDGFRB, CSF1R, and EGFR probes. Twenty-two of these cases, for which frozen material was available (nine primary and 13 advanced tumors), were selectively analyzed using the whole-genome 4.3K TK-CGH-array, phospho-kinase antibody array or Western immunoblotting. The study was supplemented by direct sequencing of KIT, PDGFRB, CSF1R and EGFR.

Results: We demonstrated that EGFR is frequently and the most significantly activated RTK in chordomas. Furthermore, concurrent to EGFR activation, the tumors commonly reveal co-activation of alternative RTK. The consistent activation of AKT, the frequent loss of the tumor suppressor PTEN allele, the recurrent activation of upstream RTK and of downstream effectors like p70S6K and mTOR, all indicate the PI3K/AKT pathway as an important mediator of transformation in chordomas.

Conclusions: Given the complexity of the signaling in chordomas, combined treatment regimens targeting multiple RTK and downstream effectors are likely to be the most effective in these tumors. Personalized therapy with careful selection of the patients, based on the molecular profile of the specific tumor, is anticipated.

Figure 1

Histology and EGFR protein expression in chordomas. A and B/ Examples of histologic appearance of chordomas stained with hematoxylin and eosin (H&E). C - F/ Illustration of chordoma cases with heterogeneous type of positive EGFR immunostaining. F/ The typical physaliphorous cells with abundant vacuolated cytoplasm, showing EGFR membrane staining.

Figure 2

Frequency (%) of gained and lost regions detected by 4.3K TK aCGH in chordomas. Gains are shown in grey, losses in blue and amplification in black. Important recurrent gains and losses are circled in red. No rearrangements or high level amplification of genes encoding TK were detected.

Figure 3

Representative examples of dual-color interphase FISH images on paraffin sections in chordomas. Detected by the co-hybridization of SpectrumOrange labeled EGFR DNA probe (red signals) and SpectrumGreen labeled chromosome 7 CEP probe (green signals). (A) Case 1a, showing EGFR disomy. (B) Case 10a reveals EGFR polysomy. (C) Case 20 shows low level amplification in < 10% of nuclei. Of note, this amplification is not detected by aCGH. (D) Case 12b, showing high level amplification of EGFR in > 40% of nuclei.

Figure 4

Representative images from phospho-RTK (left panel) and phospho-kinase (right panel) arrays from chordoma cases 18, 17b, 20 and 21. The EGFR and EPHB2 TK are frequently activated and downstream RTK signaling intermediates are activated consistently in chordomas. Each kinase is spotted in duplicate. The pairs of dots in each corner are positive controls. Each pair of the most positive kinase dots is denoted by a numeral, with the identity of the corresponding kinases listed as follows: 1) EGFR, 2) CSF1R, 3) MSPR, 4) PDGFRB, 5) FGFR3, 6) EPHB2, 7) HER2, 8) TOR, 9) AKT, 10) TP53, 11) RSK1/2/3, 12) S6K, 13) CREB, 14) YES, 15) MSK1/2, 16) RSK1/2, 17) eNOS.

Figure 5

Western immunoblot of nine chordoma cases. The immunoblot confirms the frequent expression of EGFR and PDGFRB, and frequent activation of EGFR, but not of PDGFRB and KIT proteins. Equal amounts of total protein extracts from nine tumors were separated on a gel, immunoblotted and then probed with the indicated antibodies.