(P 94) THE ROLE OF CMYC AMPLIFICATION VERSUS CMYC PROTEIN EXPRESSION AS A PROGNOSTIC BIOMARKER IN OSTEOSARCOMA

Objective: Osteosarcoma (OS) is the most common primary bone tumor in children and adolescents. Beyond the presence of metastatic disease and tumor necrosis, prognostic markers to classify patients for risk stratified therapy are limited. The gene cMYC is abnormally overexpressed in >50% of solid cancers, and cMYC is being explored as a prognostic biomarker in OS. Efforts to molecularly subtype OS are ongoing; however, OS is a karyotypically complex cancer which complicates standards for quantifying genomic aberrations, e.g. cMYC amplification. Our objective was to quantify cMYC amplification and cMYC protein expression in OS cell lines and primary patient OS samples and evaluate each as a prognostic biomarker for overall survival. Additionally, we aim to define the tumor immune microenvironment in high versus low cMYC protein expressing specimens.

Methods: Western Blot was performed on U2OS, Saos-2,143B, MG63, and SJSA-1 cell lines to quantify relative cMYC protein expression with a β-actin control in each cell line for comparison. Interphase FISH analysis was performed using DNA probes targeted to the cMYC locus at 8q24 with a chromosome 8 centromere (CEP8) probe as an internal control. Amplification was defined as a ratio of cMYC to CEP8 >2 in greater than 25% of tumor cells. Retrospective review of OS cases at Children’s Hospital Colorado identified132 pediatric cases with available archival tissue. After pathologist review for adequacy, 79 samples were selected for downstream testing. All samples were clinically annotated for age and gender, presence of metastasis, tumor grade, treatment received, tumor necrosis after chemotherapy, rates of progression and relapse, and overall survival at the time of data acquisition. Samples were analyzed for cMYC copy number using FISH as described above. To quantitate cMYC protein expression as well as define the spatial biology in the OS tissue specimens we used multiplex immunohistochemistry. A Vectra Polaris microscope and inForm image analysis software were used to quantify and phenotype tumor-infiltrating immune cells. Targets include cMYC, CD4, CD8, CD 19, CD68, CD163/CD206, CD31, CD45, FOXP3, and PD-1/PDL1.

Results: FISH analysis of cMYC amplification status (Figure 1) and Western Blot (Figure 2) analysis of cMYC protein expression of each cell line yielded coordinate and discordant results. SJSA-1, MG63, and Saos-2 had gene amplification status correlating with protein expression. However, the cell line 143B, often used to model metastatic osteosarcoma, expressed high levels of the cMYC protein but was cMYC non-amplified. Similarly, the cell line U2OS was also high protein-expressing but revealed very low-level cMYC amplification (cMYC/CEP8 ratio 2.1). Of the 79 FFPE OS patient samples selected for downstream testing, FISH testing was technically successful in 53 of the samples (67%). cMYC-amplification was identified in 12 samples (23%), and 41 (77%) were identified as wild-type. The mean age of patients was similar across both groups, 12.8 years (cMYC-amplified) and 12.3 years (wild-type). cMYC-amplified tumors were associated with higher rates of metastasis, increased rate of poor histologic response to neoadjuvant therapy (defined as tumor necrosis < 90% at the time of resection), higher rates of relapse and death, and a shorter time to death (Figure 3). cMYC protein expression using multiplex immunohistochemistry is ongoing. Interestingly, preliminary evaluation of cMYC protein expression has yielded cases with discordant cMYC amplification status as well (Figure 4).

Conclusion: In our cohort of 53 evaluable OS tissue specimens, amplification of cMYC correlated with poor patient outcomes, including higher rates of metastasis, relapse, and death. However, due to the genomic complexity of OS, quantification of cMYC amplification using CEP8 probe for normalization may not accurately capture the full impact of cMYC activity in OS. Our cell line data suggests that cMYC protein expression is an additional method for quantifying cMYC activity in OS, which may not be confounded by the high genomic complexity of OS. Additional IHC evaluation of cMYC protein expression in OS tissue specimens as well as its influence on the tumor immune microenvironment are currently underway.