Ectopic Production of Β-hCG and Loss of P 16 as a Predictor of Outcome in Patients with Newly Diagnosed Osteosarcoma

Osteosarcoma is the most common malignant bone tumor in children and young adults and is associated with high mortality. We investigated the expression of βhCG and P16 in osteosarcoma and correlated with outcomes. Methods: Immunohistochemistry (IHC) for β-hCG was performed on diagnostic osteosarcoma specimens and post-treatment specimens. IHC for P16 was performed on diagnostic specimens. Results: Median age was 32. Median progression free survival (PFS) was 11.5 months. Median overall survival (OS) was 38.0 months. Positive β-hCG staining on diagnostic specimens did not correlate with percent tumor necrosis, 2 year PFS or OS. Patients with a post-treatment β-hCG staining of ≥ 50% had a median PFS of 6.1 months vs 19.2 months in patients with β-hCG less than 50% (p = 0.03). Patients with a post-treatment β-hCG staining ≥ 50% also demonstrated a trend toward shorter median OS (17.1 months vs 19.2). There was no statistically significant relationship between P16 staining on diagnostic osteosarcoma specimens and posttreatment percent tumor necrosis. Patients with negative P16 staining on diagnostic specimens had a lower 2 year PFS compared to positive P16 staining (2 year PFS 0% vs 71%), p=0.022. There was a trend toward worse 2 year OS in patients with P16 negative diagnostic specimens compared to patients with P16 positive tumors, 22% vs 86%. Discussion: We have demonstrated feasibility and utility in examining P16 and β-hCG in osteosarcoma. We found that post-treatment β-hCG expression correlated with poorer outcomes, specifically worsened PFS. In congruence with previous reports, negative P16 staining confers worse outcomes. Nye L, Yeldandi A, Peabody T, Attar S, Salamon MA, Gandhi M, Gursel D, Rademaker A, Hayes JP and Agulnik M* Division of Hematology/ Oncology, Robert H. Lurie Comprehensive Cancer Center Northwestern University Feinberg School of Medicine, USA Agulnik M, et al. Clinics in Oncology Sarcoma Remedy Publications LLC. , | http://clinicsinoncology.com/ 2016 | Volume 1 | Article 1061 2 patients and demographic, clinical, pathological, radiological and laboratory data was collected. Previously stored formalin fixed and paraffin embedded blocks from available pretreatment diagnostic specimens and the designated study pathologist reviewed posttreatment surgical specimens of included patients, and the most representative block was chosen for sectioning. The slides with the greatest proportion of tumor were then selected for staining. Two representative slides from the diagnostic specimen (one for β-hCG and one for P16 staining) and one representative slide from the post treatment resection specimen (only for β-hCG ) were selected per subject. Immunohistochemistry staining for β-hCG was performed on the formalin fixed, paraffin embedded tissue sections of the diagnostic osteosarcoma specimens and post treatment surgical resection specimens. Rabbit polyclonal anti-human β-hCG antibody (Dako, Carpinteria, California) was utilized with pH adjusted (pH 6.0) antigen retrieval. Placenta was used as positive control. Stained slides were examined by the study pathologist and analyzed for cytoplasmic staining intensity (absent or present) and frequency of expression (categorized as 0%, <10%, 10-50% or ≥ 50%) was reported. Both intensity and frequency of β-hCG expression was analyzed, as there is no established grading system for β-hCG staining pattern in osteosarcoma (Figure 1). Immunohistochemistry staining for P16 was performed on the formalin fixed, paraffin embedded tissue sections of the diagnostic osteosarcoma specimens. Mouse monoclonal anti-human P16INK4a antibody (Biocare Medical, LLC, Concord, California) was utilized with pH adjusted (pH 9.0) antigen retrieval. The positive control was cervical cancer specimens. Tonsil tissue microarrays were used for negative control. Both intensity and frequency of P16 expression was analyzed and reported. Specimens were classified as negative (absent) or positive (present) for P16 nuclear expression (Figure 1). Positive staining for P16 included specimens with ≥ 30% nuclear staining. This threshold was chosen based on prior reports of P16 staining in osteosarcoma [10,11]. Assessment of tumor necrosis is reported as a percentage and was obtained from post-treatment surgical specimen pathology report on chart review. Progression-free and overall survival was analyzed using Kaplan-Meier curves. Subgroups were compared using the log rank test. Comparisons of biomarkers between p16 positive and negative subgroups were done using the Wilcoxon rank sum test.