Insulin-like growth factor 1 receptor and response to anti-IGF1R antibody therapy in osteosarcoma

Abstract

Background: Survival outcomes for patients with osteosarcoma (OS) have remained stagnant over the past three decades. Insulin-like growth factor 1 receptor (IGF1R) is over-expressed in a number of malignancies, and anti-IGF1R antibodies have and are currently being studied in clinical trials. Understanding the molecular aberrations which result in increased tumor response to anti-IGF1R therapy could allow for the selection of patients most likely to benefit from IGF1R targeted therapy.

Methods: IGF1R mRNA expression was assessed by RT PCR in OS patient primary tumors, cell lines, and xenograft tumors. IGF1R copy number was assessed by 3 approaches: PCR, FISH, and dot blot analysis. Exons 1-20 of IGF1R were sequenced in xenograft tumors and 87 primary OS tumors, and surface expression of IGF1R was assessed by flow cytometry. Levels of mRNA and protein expression, copy number, and mutation status were compared with tumor response to anti-IGF1R antibody therapy in 4 OS xenograft models.

Results: IGF1R mRNA is expressed in OS. Primary patient samples and xenograft samples had higher mRNA expression and copy number compared with corresponding cell lines. IGF1R mRNA expression, cell surface expression, copy number, and mutation status were not associated with tumor responsiveness to anti-IGF1R antibody therapy.

Conclusions: IGF1R is expressed in OS, however, no clear molecular markers predict response to IGF1R antibody-mediated therapy. Additional pre-clinical studies assessing potential predictive biomarkers and investigating targetable molecular pathways critical to the proliferation of OS cells are needed.

Figure 1. OS Xenograft Model Response to anti-IGF1R Antibody SCH 717454 Treatment.

Three OS xenograft models were treated with an anti-IGF1R antibody SCH 717454 (A) (open square) or vehicle (closed circle), small molecule IGF1R inhibitor BMS 754807 (B) (open square) or vehicle (closed circle) or IMC A12 (C) (open square) or vehicle (closed circle). Mice were treated with 0.5 mg SCH 717454 administered twice weekly via intraperitoneal injection for 4 weeks, BMS 754807, 25 mg/kg administered orally BID for 6 days, repeated for 6 weeks, or IMC A12, 1 mg/mouse administered intraperitoneally twice weekly for 6 weeks. These studies were previously reported; however, the figures for M17 and M31 were not published , , . Ten mice were included in each arm of the experiments. Tumor size was monitored weekly for a maximum of 6 weeks of treatment or until a relative tumor volume increased to 4× baseline.

Figure 2. Relative IGF1-R mRNA Gene Expression in OS Primary Samples and Corresponding Cell Lines.

Quantitative Real Time PCR detecting IGF1R mRNA expression was performed in OS primary samples (A) and OS cell lines (B). Grey bars represent relative quantification of IGF1-R expression using the ΔΔCT method using GAPDH as an endogenous control and MSC as a calibrator. Scatter plot comparing IGF1-R expression fold change in primary OS samples and their corresponding cell lines (C).

Figure 3. IGF1R Copy Number Assessed by PCR.

IGF1R copy number was assessed in OS cell lines (A) and OS primary samples (B). MSCs were set to 2 copies of IGF1R and used as the calibrator. Three different copy number assays (blue, red, and green bars) were performed as described in the methods. Copy number is rounded to the nearest whole number. Blue bars represent results for the assay located at chr15∶99491821 (overlaps Intron 19 - Exon 20). Red bars represent results for the assay located at chr15∶99460087 (overlaps Exon 10 - Intron 10). Green bars represent results for the assay located at chr15∶99251313 (overlaps Exon 2 - Intron 2).(OS) signifies nucleic acids were derived directly from a human sample, (M) signifies cells were derived from a human tumor grown as a mouse xenograft, and (c) signifies the cells were passaged in cell culture.”

Figure 4. IGF1R Cell Surface Expression in OS Xenograft Models.

FACS analysis of IGF1R surface expression in 4 OS xenograft models (M1: high responder, M2: intermediate responder, M17: intermediate responder, M31: high responder) and a positive control (MCF7) (A). The isotype control is represented by a red line and cells stained with IGF1R antibody are represented by a blue line.