**4. Somatic mutations in canine thyroid cancer**

Most of thyroid carcinomas in dogs are sporadic, the same as it is in humans. Sporadic cancers are caused by somatic mutations that occur in somatic cells, which are different from germline causal mutations in hereditary cancers. These somatic mutations are not inherited from parents of the individual, but are acquired by random DNA replication error during cell divisions that occurred by chance or due to exogenous or endogenous carcinogens that can increase the risk for the cancer. These exogenous carcinogens include smoking and X-ray. Endogenous carcinogen includes reactive oxygen species produced during metabolism [15]. When these mutations occur in proto-oncogene or tumor-suppressor gene, then a cancerous cell may arise.

Identification of somatic mutation at a genome-wide scale becomes possible with the development of next-generation sequencing technologies. The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) projects have profiled tens of thousands of human cancers of different types and origins [16, 17]. Normally, whole-genome or whole-exome sequences of tumor tissue and matched normal tissue (blood or healthy tissue adjacent to tumor tissue) are generated and compared to identify those somatic mutations that are unique to tumor cells. Among somatic mutations, according to their role in tumorigenesis, driver and passenger mutations are defined. Driver mutations are somatic mutations that are important to the tumor initiation and growth, and passenger mutations are those neutral mutations that do not contribute to tumorigenesis. Identification of driver mutation is one of the major tasks in oncogenic research. Identification of driver mutations sheds light on molecular mechanisms underlying tumor initiation and development. Those driver mutations have potential value to be used to develop targeted treatment to kill cancerous cells.

Somatic mutations of human thyroid carcinoma have been extensively investigated at a genome-wide scale. Somatic mutations of canine thyroid carcinoma are still poorly studied. A somatic mutation in *P53* gene has been identified in canine FTC [18]. We profiled the somatic mutations of the hereditary FCC identified in Dutch GLPs at a genome-wide scale [19]. As far as we know, there was no genome-wide profile of somatic mutations in canine thyroid carcinoma before our study. In our study, a missense somatic mutation in the *GNAS* gene, p.A204D, stands up where it was identified in four of seven FCC samples that were whole-genome sequenced and validated in 20 out of the 32 affected dogs' thyroid tumor samples [19]. This

high prevalence of the somatic mutation is a strong evidence of the driver role of this mutation in these canine thyroid carcinomas.

The *GNAS* gene encodes the alpha-subunit of stimulatory G-protein (Gαs) that can activate adenylyl cyclase downstream of G-protein-coupled receptors (GPCRs). Activated adenylyl cyclase increases cellular cyclic adenosine monophosphate (cAMP). cAMP is an important second messenger that can upregulate many downstream molecular signaling cascades, including pathways involved in cell proliferation, such as the PKA signaling pathway [20].

The *GNAS* gene is a known proto-oncogene. Somatic mutations in the *GNAS* gene have been identified in many different types of tumors in humans. It is known that activating mutations in the *GNAS* gene can result in increased cell division in humans. The most common activating mutations in the *GNAS* identified in human tumors are p.R201C/H/S and p.Q227R/L [21]. According to an investigation in 274,694 human tumors, appendiceal adenocarcinoma has highest frequency of *GNAS* activating mutation (35.9%). Ovarian carcinosarcoma, rectum adenocarcinoma, gastroesophageal junction adenocarcinoma, stomach adenocarcinoma diffuse type, small intestine adenocarcinoma, stomach adenocarcinoma, esophagus adenocarcinoma, breast carcinoma, colon adenocarcinoma, breast invasive ductal carcinoma, and duodenal adenocarcinoma have prevalence of *GNAS* somatic mutation in the range between 5% and 7% [21]. However, prevalence of somatic mutation in the *GNAS* gene in human thyroid cancer seems to be low where only 13 of 1,837 human thyroid neoplasms capture *GNAS* somatic mutations. Likewise, somatic mutations in the *GNAS* gene were identified in only two out of 496 PTC samples that were included in the TCGA project [22].

Besides our genome-wide study, Campos et al. investigated somatic mutation landscape of 43 canine FCCs and 16 canine MTCs by targeted sequencing of some driver genes identified in human thyroid carcinoma [23]. Those genes include *HRAS*, *KRAS*, *PIK3CA*, *BRAF*, *RET*, and *PTEN* genes. However, they only identified two missense mutations in the *KRAS* gene that are homologous to mutations identified in human thyroid carcinoma. No somatic mutation in other genes under investigation was identified. This seems to suggest that canine thyroid carcinoma uses different driver mutations compared with human thyroid carcinoma. In our study, *GNAS* p.A204D somatic mutation was observed in FCC neoplasms of 62.5% of affected GLPs. However, in human PTCs, *GNAS* somatic mutation is rarely observed. This suggests the potential difference in driver events of thyroid carcinoma between humans and GLPs. We suggest that the prevalence of the *GNAS* somatic mutation in more canine thyroid tumors should be investigated because it might be a major driver mutation of canine thyroid tumor according to our study. Meanwhile, we also suggest investigating driver mutations in sporadic canine thyroid carcinomas at a genomewide scale to elucidate the molecular mechanisms underlying canine sporadic thyroid tumor initiation and development and to investigate the potential value of dogs with sporadic thyroid carcinoma to be used as disease models.

### **5. Pathways involved in thyroid carcinoma in dogs**

In humans, molecular signaling pathways that are involved in thyroid carcinoma are extensively investigated. The most dominant molecular signaling pathways are the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3K)/ Akt pathways [24]. Activation of these pathways as a result of activating mutations

in proto-oncogenes (such as *BRAF* and *RAS* genes) involved in the pathway leads to cancerous cells. In dogs, PI3K/AKT pathway is also involved in the pathogenesis of thyroid carcinoma with the evidence of increased expression of several genes associated with the pathway [23]. However, the involvement of MAPK pathway in canine thyroid carcinoma development needs to be investigated. Besides the evidence of increased expression of genes, to confirm the role of these pathways in the canine thyroid carcinoma development, somatic mutations in those genes should also be further investigated.
