Medical Brief: Osteosarcoma Molecular Profiling Results

Medical Brief: Osteosarcoma Molecular Profiling Results

FidoCure’s Lucas Rodrigues DVM, MS. PhD discusses the challenges of osteosarcoma (OSA), the most common primary bone tumor in dogs, with over 10,000 cases diagnosed yearly. He also discusses the genomic landscape of this cancer based upon the literature and FidoCure’s own dataset.

Osteosarcoma (OSA) is the most common primary bone tumor in dogs, with over 10,000 cases diagnosed yearly. This tumor most frequently occurs in large and giant breed dogs such as Rottweilers, German Shepherd dogs, Boxers, Doberman Pinschers and Great Danes. OSA most commonly occurs in either young or middle-age to older dogs. It is a very aggressive disease with a poor prognosis due to the fact that at diagnosis 90% of patients already have micrometastatic disease (tumors that are distant to the primary tumor)1,2.

The etiology of OSA is commonly associated as a result of replicative DNA mutations, with discrete contributions of heritable and environmental factors3. Since sex hormones like testosterone and estrogen contributes to the skeletal maturity, an early gonadectomy might increase adiposity and body condition, cause delayed physeal closure and longer lifespan in dogs, contributing to increased OSA risk in the middle age 3–6. Males and females are equally affected according to the FidoCure® database, and the median of mutation per each dog is similar in males and females groups (Figure 1).

OSA can be observed in both the appendicular and axial skeleton, with 64% and 33% of cases seen in these sites respectively (Figure 2). Less commonly seen are extraskeletal OSAs which account for  5-7% of the cases. Clinical signs of this disease are associated with the tumor location. Dogs with appendicular OSA commonly develop progressive lameness, pain and local swelling. The diagnosis is based on radiographs of the tumor, fine needle aspiration/cytology and/or tumor biopsy.  Clinical staging is recommended to include: 3 view chest radiographs, CBC and biochemical profile. CT scans and abdominal ultrasonography may also be recommended to evaluate if metastatic disease is present 1 ,2 ,7, 8.

The standard treatment for canine OSA consists of surgical amputation followed by cytotoxic chemotherapy. The goal of this treatment is to address both the local and systemic complications of this disease. Removing the tumor helps dogs by controlling or eliminating the source of the pain. Chemotherapy can help increase the disease free interval and overall survival time as well as decrease the likelihood of developing metastatic lesions in the lungs 1, 9.


Figure 2. Appendicular osteosarcoma in dogs. a) Dorso-ventral radiographic view of an osteosarcoma in the left distal ulna; b) Lateral radiograph of a distal tibia of affected dog; c) chest radiographic of a dog with metastatic osteosarcoma in the lungs  (Dr. Lucas Rodrigues).

Currently, the most common chemotherapeutic agents used in dogs are doxorubicin and carboplatin. Although treatment with carboplatin or doxorubicin has increased median survival times in dogs with adequate primary tumor control, approximately 80% of patients develop distant metastatic disease and are subsequently euthanized 10 to 14 months after the diagnosis, and just 25% of dogs will survival for longer than 2 years.

Radiation therapy is another treatment option for dogs with OSA. Radiation therapy can be used as a way to control the pain from an OSA lesion if surgical resection is not an option1, 9–12.

Despite all of these therapies, the median survival time for canine patients is still quite short ranging from 5 months with amputation alone to 10-14 months when combined with cytotoxic chemotherapy1. Genomic evaluation and the personalized medicine approach which allows a specific treatment tailored to the individual genomic characteristics of each patient, is a newer strategy to treat cancer in humans and dogs, and this approach may benefit dogs  with OSA 13,14.

Whole exome sequencing (WES) had been conducted on OSA from dogs and identified mutations similar to human pediatric tumors. TP53 was the most common mutation identified in dogs, and the majority of them were localized in the DNA-binding domain. As in humans, TP53 mutation may disrupt checkpoint responses to DNA damaging-drug and cause genomic instability. TP53 mutation is identified in 95% of canine OSA when whole genome sequencing (WGS) is performed, and in 81% of samples when WES was performed 15.

SETD2 is the second most commonly mutated gene in canine OSA  with 21% of all canine osteosarcomas showing this mutation. The percentage of OSAs with this mutation varies by breed: Golden Retrievers (32%), Rottweiler (19%) and Greyhounds (13%)16. In the FidoCure® database, 181 somatic mutations previously published or identified based on variant allele frequency analysis were identified in 42 dogs with OSA. These dogs are distributed in 25 different breeds, so no correlation was established between breed and mutation in this data set (Figure 3). In canine OSA samples analyzed by WGS, somatic point mutations, deletions, and chromosomal translocations in SETD2 gene were identified in 42%15. SETD2 mutations can inactivate the function of the encoded protein and contribute to epigenetic modification of cells 17.

In the FidoCure® database, the genes PARP1 and CREBBP are mutated in 19% and 14.2% of OSA in dogs, respectively (Figure 4). PARP1 encodes a molecule related to DNA repair activity that prevents apoptotic cell death by recruiting BRCA1/2. PARP1 activity can also confer a survival advantage to cancer cells, even more when BRCA1/2 are defected and can not prevent the cell proliferation with damaged DNA. In human OSA patients, PARP1 and BRCA1/2 mutations are related to shorter survival, being inhibitors of PARP1, such as olaparib, a new option to therapeutic strategies18–22. The common capacity of human OSA cells to resist therapy is correlated to PARP1 and BRCA1/2 function alterations. When olaparib is combined with doxorubicin to treat OSA cell lines in vitro, there is an increase in the apoptotic rate compared to drugs used alone19. The CREB-binding protein, a histone acetyltransferase, encoded by CREBBP gene functions as a co-activator of transcription, allowing chromatin accessibility and regulation of suppressor tumour such as p53, RB1 and BRCA1. This gene is commonly studied in hematological malignancies, but is also observed that high-level amplification of CREBBP was associated with aggressive bone tumors such as OSA in humans 23–26.

The genomic analysis of more than 40 canine OSA using FidoCure® Diagnostics identified several mutations in KMT2C and KMT2D genes. They are epigenetic regulators in cancer involved in histone H3 lysine-specific methylation activity. In humans, KMT2C mutations are present in 87% of OSA samples14. Mutations in KMT2C can interfere in the activity of estrogen by modifying its modulation and role. Estrogen receptors play a fundamental function in the development and production of the bone matrix, since they facilitate the opening of chromatin and the recruitment of transcription factors in osteoblasts 14, 27, 28.

According to the literature, DMD and DLG2 mutations are also common in canine OSA15,29,30. DMD encodes a dystrophin protein that forms a dystrophin-glycoprotein complex and bridges the inner cytoskeleton and the extracellular matrix. Deletions in the DMD gene were associated with increased tumor cell migration, invasion and anchorage-independent growth, acting like a tumour suppressor gene. In humans, mutation in the DMD gene causes Duchenne’s muscular dystrophy and Becker muscular dystrophy or cardiomyopathy, as well the progression of a myogenic tumor to a high grade letal sarcomas. Human patients with Duchenne's muscular dystrophy, a rare neurological disease that carries a mutation in exon 53 of the DMD gene, have been treated with dasatinib with promising results. In canine OSA analyzed by WGS, DMD somatic point mutations were identified in 38% of samples, supporting the potential role of this gene in sarcoma initiation and progression 15, 29, 30.

The high prevalence of DMD and SETD2 mutations in dogs but not in humans OSA, could be considered as a potential major difference between the two species, which may be related to the inbreeding of canine breeds that develop OSA. DLG2 gene is deleted in 42% of humans and 56% of dogs OSA, which is  involved in multiple signal transduction networks and regulation of cell cycle progression as a tumor suppressor gene. In mice, Dgl2 homozygous deletion accelerated tumor development and shortened survival, suggesting the negative regulatory role for DLG2 in the GTPase signaling pathway in human and canine OSA cell migration 31, 32.

Genes such as ATRX, RB1 and MDM2 are also commonly altered in human OSA. The function loss of ATRX and RB1 genes are involved in cancer progression, since they are related to immortalization and cell cycle regulatory pathways33,34. On the other hand, overexpression of MDM2, which encodes a protein that binds and blocks the activity of p53, is related to a disrupted cell cycle regulation and OSA progression, this mechanism has been demonstrated in canine tumor cells 34, 35.

Mutations in the PIK3CA gene were identified in 16.6% of FidoCure® cases. The gene encodes an enzyme subunit p110a also called PI3K. This protein activates AKT and mTOR pathways and regulates glucose metabolism, cell proliferation and survival36,37. Several studies have been evaluating the PI3K/mTOR pathway in human OSA and identifying enhanced anti-tumor effects with the dual inhibition of PI3K and mTOR. The combination of tyrosine kinase inhibitors able to block VEGF receptors and MAPK/ERK pathway, such as sorafenib and toceranib, associated with mTOR inhibitors such as rapamycin (sirolimus), had demonstrated inhibition of cell growth, angiogenesis and metastasis 38, 39. Similarly, tyrosine kinase inhibitors blocking PDGF/PDGFR signaling such as dasatinib, which also inhibits the steroid receptor co-activator (SRC), are involved in PI3K/mTOR pathway and induce apoptosis in human OSA cells in vitro in addition to improve survival by stabilizing or inducing partial remission of pulmonary metastases in dogs with OSA 40–42.

Molecular analysis of human OSA have revealed profound genomic instability and heterogeneity across patients, with nearly universal TP53 aberration, and driver mutational events have not been clearly established39. Similar genomic pattern in canine OSA has been documented in published genomic studies and corroborated with the FidoCure® database. This fact enhances the need for a personalized approach to identify drivers mutations, altered pathways and opportunities for therapeutic intervention for each individual patient.


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In the FidoCure database PARP1 and CREBBP genes are mutated in 19 and 14.2% of OSA in dogs, respectively. The Poly (ADP-ribose) polymerase 1 (PARP1) is a molecule related to DNA repair and it prevents apoptotic cell death by recruiting BRCA1/2, which are tumour suppressor, to repair the DNA. This PARP1 activity can confer a survival advantage to cancer cells, even more when BRCA1/2 are defected and can not prevent the cell proliferation with DNA damage. In human OSA patients, PARP1 and BRCA1/2 mutations are related to shorter survival, being inhibitors of PARP1 a new option to therapeutic strategies22–26. In canine OSA cell lines, the cleaved PARP by loss of ΔNp63, an oncogenic isoform of p63, induced cell death from apoptosis, demonstrating the potential role of PARP in the survival of canine cancer cells27. The CREB-binding protein, a histone acetyltransferase, encoded by CREBBP gene functions as a co-activator of transcription, allowing chromatin accessibility and regulation of suppressor tumour such as p53, RB1 and BRCA1. This gene is commonly studied in hematological malignancies, but is also observed that high-level amplification of CREBBP was associated with aggressive bone tumors such as OSA 28–31.

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