Author ORCID Identifier

0000-0002-1337-3282

Date of Graduation

8-2017

Document Type

Thesis (MS)

Program Affiliation

Clinical and Translational Sciences

Degree Name

Masters of Science (MS)

Advisor/Committee Chair

Eugenie S. Kleinerman

Committee Member

Patrick Hwu

Committee Member

Robert Bast Jr.

Committee Member

Shreyaskumar Patel

Committee Member

Alexander Lazar

Abstract

Survival for patients with osteosarcoma has not improved for > 30 years. Despite aggressive multi-agent chemotherapy combined with surgical resection, a significant fraction of patients with localized disease relapse after optimal treatment. We evaluated the occurrence of cytoplasmic LC3B (light chain 3B)-positive puncta (a marker of autophagy) and presence of HSP27 (heat shock protein 27) in cancer cells within pre-treatment biopsy, post-treatment surgical resection, and metastatic osteosarcoma specimens by immunohistochemistry in 260 patients. LCB3+ puncta expression was seen in 34% of pre-treatment. 50% of resection, and 67% of metastasis samples. Sixty-six percent of all specimens were scored positive for HSP27 (85% of pre-treatment. 52% of resection, and 50% of metastasis samples). Among 215 patients with localized disease, pre-treatment HSP27 expression was associated with inferior overall survival (adjusted HR 26.7, p=0.0263) as well as at resection following chemotherapy (adjusted HR 1.85, p=0.039). Lack of LC3B-puncta expression was an independent poor prognostic marker at resection (adjusted HR 1.75, p=0.045). Patients with LC3B+/HSP27- tumors at resection had the best prognosis whereas patients with LC3B-/HSP27+ osteosarcoma had the worst long-term survival. Neither HSP27 nor LC3B expression correlated with tumor necrosis. These findings indicate that HSP27 expression is a negative prognostic biomarker in osteosarcoma. Conversely, presence of autophagy following neoadjuvant chemotherapy, as measured by LC3B-puncta, predicts longer overall survival in osteosarcoma patients with localized disease.

We additionally evaluated the significance of chemotherapy-induced autophagy in 2 human osteosarcoma cell lines: LM7 and CCH-OS-D. Both doxorubicin (DOX) and cisplatin (CDDP) were found to induce autophagy. In LM7 cells, autophagy inhibition with hydroxychloroquine (HCQ) prior to chemotherapy resulted in a trend towards decreased viability consistent with a cytoprotective role of autophagy. In CCH-OS-D cells, autophagy inhibition prior to DOX significantly decreased chemosensitivity suggesting a cytotoxic role of autophagy in this setting. The post-treatment expression of phosphorylated HSP27 was increased in LM7 and decreased in CCH-OS-D following DOX or CDDP. These findings support a dual role of chemotherapy-induced autophagy and potential application of pHSP27 as a predictive biomarker of autophagy inhibitors in osteosarcoma.

Keywords

Osteosarcoma, autophagy, heat shock protein, biomarkers, LC3B, HSP27

Available for download on Friday, February 09, 2018

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