Expression profiling in canine osteosarcoma: identification of biomarkers and pathways associated with outcome
Liza E O'Donoghue
0
Andrey A Ptitsyn
2
Debra A Kamstock
0
Janet Siebert
1
Russell S Thomas
3
Dawn L Duval
0
0
Department of Clinical Sciences, Colorado State University
,
Fort Collins, Colorado
,
USA
1
CytoAnalytics, Analytical Services
,
Denver, Colorado
,
USA
2
Department of Microbiology, Immunology and Pathology, Colorado State University
,
Fort Collins, Colorado
,
USA
3
The Hamner Institutes for Health Sciences
,
Research Triangle Park, North Carolina
,
USA
Background: Osteosarcoma (OSA) spontaneously arises in the appendicular skeleton of large breed dogs and shares many physiological and molecular biological characteristics with human OSA. The standard treatment for OSA in both species is amputation or limb-sparing surgery, followed by chemotherapy. Unfortunately, OSA is an aggressive cancer with a high metastatic rate. Characterization of OSA with regard to its metastatic potential and chemotherapeutic resistance will improve both prognostic capabilities and treatment modalities. Methods: We analyzed archived primary OSA tissue from dogs treated with limb amputation followed by doxorubicin or platinum-based drug chemotherapy. Samples were selected from two groups: dogs with disease free intervals (DFI) of less than 100 days (n = 8) and greater than 300 days (n = 7). Gene expression was assessed with Affymetrix Canine 2.0 microarrays and analyzed with a two-tailed t-test. A subset of genes was confirmed using qRT-PCR and used in classification analysis to predict prognosis. Systems-based gene ontology analysis was conducted on genes selected using a standard J5 metric. The genes identified using this approach were converted to their human homologues and assigned to functional pathways using the GeneGo MetaCore platform. Results: Potential biomarkers were identified using gene expression microarray analysis and 11 differentially expressed (p < 0.05) genes were validated with qRT-PCR (n = 10/group). Statistical classification models using the qRT-PCR profiles predicted patient outcomes with 100% accuracy in the training set and up to 90% accuracy upon stratified cross validation. Pathway analysis revealed alterations in pathways associated with oxidative phosphorylation, hedgehog and parathyroid hormone signaling, cAMP/Protein Kinase A (PKA) signaling, immune responses, cytoskeletal remodeling and focal adhesion. Conclusions: This profiling study has identified potential new biomarkers to predict patient outcome in OSA and new pathways that may be targeted for therapeutic intervention.
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Background
Osteosarcoma (OSA) is the most common malignant
primary bone tumor of children and accounts for roughly
5% of all childhood cancers in the United States.
Characteristically, OSA is found in the metaphyseal regions of
long bones in the appendicular skeleton. More than 15%
of patients present with clinically detectable pulmonary
metastases and it is estimated that 80% or more have
micrometastases at presentation [1]. Advances in
treatment such as multi-agent chemotherapy have improved
prognosis over the last several decades with five-year
survival rates up to 70%. Despite these advances, patients
that present with metastases or those whose tumors are
refractory to neoadjuvant chemotherapy continue to have
a poor prognosis [1]. This suggests that within the same
histologic type of tumor, different genetic mechanisms
may be operating, altering response to chemotherapy and
metastatic capability in some tumors.
Osteosarcoma is also the most common primary bone
malignancy in dogs. The majority of these tumors occur
in the appendicular skeleton of middle-aged large and
giant breeds. Roughly 10,000 new cases of OSA are
identified in dogs annually. Standard treatment involves
amputation or limb-sparing surgery followed by
adjuvant chemotherapy with doxorubicin, a
platinumbased drug, or a combination of the two drug types [2].
Median disease-free interval (DFI) following amputation
has ranged from 165 to 470 days depending on adjuvant
chemotherapy protocol and study size [3-7]. Median
survival time in dogs undergoing amputation alone
ranges from 134 to 175 days [3-7]. Like their human
companions, pulmonary metastases are typically the
cause of terminal morbidity. It has been suggested that
up to 90% of canine patients may present with
microscopic metastases that are undetectable via routine
imaging [2]. The high variability in DFI suggests that canine
OSA exhibits variable metastatic capability, rate and/or
resistance to adjuvant chemotherapy, similar to the
disease in humans.
Canine OSA shares many features with human OSA,
making dogs a valuable comparative model. Pet dogs
develop OSA primarily in the metaphyseal regions of long
bones, as do human patients. The lesions are histologically
identical [2]. The similarities between the molecular
characteristics of human and canine OSA have been
established (see [8] for review). Furthermore, Thomas and
colleagues recently demonstrated that some of the same
genetic abe (...truncated)