Pathogenesis and Treatment of Canine Thyroid Tumors

Campos, Miguel (2014). Pathogenesis and Treatment of Canine Thyroid Tumors. (Dissertation, Ghent University, Faculty of Veterinary Medicine)

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In the past decade, major advances have been made in unveiling the molecular pathogenesis of human thyroid cancer. The PI3K/Akt pathway is the major signaling pathway involved in human follicular thyroid carcinoma which is remarkably similar in terms of histology and biological behavior to canine differentiated follicular cell thyroid carcinoma (dFTC). The discovery of the main genetic events involved in thyroid gland tumorigenesis in humans led to the discovery of new molecular targets and to the development of innovative treatments. Furthermore, stratification of patient risk with prognostic markers, TSH-suppressive therapy and increased 131I uptake with rhTSH, have allowed a significant improvement of the treatment of human dFTC and provide an interesting perspective for optimization of treatment of canine dFTC.
The molecular pathogenesis and expression of therapeutic targets in canine thyroid cancer are largely unknown and, to present date, research on prognostic markers and treatment optimization remains scarce. Up to 38% of dogs with thyroid cancer have metastases at the time of diagnosis and almost half of dogs treated with thyroidectomy develop recurrent or metastatic disease within 2 years of surgery. Given the modest results of chemotherapy, it is imperative to investigate new ways to improve treatment. The general aim of this research was to provide new insights into the pathogenesis and treatment of canine thyroid cancer.
In our 2 first studies we focused on the pathogenesis of canine thyroid cancer investigating genetic alterations and prognostic markers. In our first study (chapter 3) we try to unveil the molecular pathogenesis of canine thyroid cancer by investigating mutational hotspots and mRNA expression of candidate genes in 43 canine follicular cell thyroid carcinomas (FTCs) and 16 canine medullary thyroid carcinomas (MTCs). Mutation analysis of known hotspots of RAS (H, K, H), PIK3CA, BRAF, RET and of the entire coding region of PTEN, revealed 2 activating missense mutations in K-RAS, also described in human thyroid cancer. A G12R substitution was present in 1 FTC and an E63K substitution was present in 1 MTC. No functional mutations were found in the sequenced regions of H-RAS, N-RAS, PIK3CA, BRAF, RET and PTEN demonstrating that the mutations most frequently associated with human thyroid neoplasia are rare in canine thyroid cancer.
Quantitative RT-PCR was performed for selected receptor tyrosine kinases (RTKs) (VEGFR-1, VEGFR-2, EGFR) and PI3K/Akt pathway members (PIK3CA, PIK3CB, PDPK1, PTEN, AKT1, AKT2, COX-2) known to be commonly amplified in human thyroid cancer. The mRNA expression levels of VEGFR-1, VEGFR-2, PDPK1, AKT1, and AKT2 were increased in FTC, and those of EGFR, VEGFR-1, and PIK3CA were increased in MTC when compared to normal thyroid. The increased mRNA expression of these genes indicates the involvement of the PI3K/Akt signaling pathway in the pathogenesis of canine thyroid cancer, particularly in FTC. Further research is necessary to investigate if gene amplification is responsible for the increased mRNA expression of these genes.
Given the lack of prognostic factors for dogs with operable thyroid tumors, in our second study (chapter 4) we investigated clinical, pathological and immunohistochemical prognostic factors in 50 dogs with dFTC and 20 dogs with MTC. In this retrospective study, IHC for calcitonin, Ki-67 and E-cadherin was performed in all tumor samples and tumor features (diameter, volume, localization, scintigraphic uptake, thyroid function, IHC) were correlated with local invasiveness and metastatic disease at diagnosis. Furthermore, 44 dogs (28 dFTCs, 16 MTCs; stage I-III) treated by thyroidectomy were included in a survival analysis. In agreement with a previous report, we found that MTC was significantly less likely to be locally invasive at diagnosis. However, we found no difference in the incidence of metastatic disease at the time of diagnosis and, more importantly, following thyroidectomy outcome was comparable between dogs with dFTC and MTC. Macroscopic and histologic vascular invasion were independent negative predictors for disease-free survival. In contrast with human reports, E-cadherin expression was not associated with outcome.

The newly identified prognostic factors provide relevant information for owners and clinicians and may help to adapt follow-up and adjunctive therapy to the patients’ risk. However, to present date no single treatment modality has been shown to be effective for adjunctive therapy. Given the need to investigate new ways to improve treatment our following studies focused on treatment optimization, namely on new therapeutic targets (chapter 5), effect of levothyroxine therapy on patient survival (chapter 6) and on the safety and value of rhTSH to optimize 123I uptake in dogs with thyroid tumors (chapters 7, 8, 9).
In the study reported in chapter 5 we investigated the expression of potential therapeutic targets in 54 canine FTCs and 20 canine MTCs. For this purpose, we performed IHC for vascular endothelial growth factor (VEGF), p53, cyclooxygenase-2 (Cox-2) and P-glycoprotein (P-gp) in all tumor samples. 80% of FTCs and all MTCs had a high percentage (76-100%) of neoplastic cells immunopositive for VEGF, suggesting it may play an important role in the pathogenesis of these highly vascularized tumors. Consequently, the VEGF system seems to be an attractive target for the treatment of both FTC and MTC in the dog. 13% of FTCs and 50% of MTCs expressed Cox-2, and 7% of FTCs and 70% of MTCs expressed P-gp, which suggests these could be interesting molecular targets for the treatment of canine MTC. No tumor was immunopositive for p53 expression.
Pursuing our research on treatment optimization (chapter 6), we investigated the effect of levothyroxine therapy and TSH suppression (TSH < 0.1 ng/mL) on survival of 42 dogs with thyroid tumors undergoing different treatment modalities. In this retrospective study, dogs were grouped according to treatment as follows: thyroidectomy with or without levothyroxine therapy (n=17); radioactive iodine-131 with or without levothyroxine therapy (n=11); no treatment or levothyroxine therapy alone (n=14). Although we could not demonstrate that levothyroxine therapy or TSH suppression improve patient survival, randomized clinical studies are needed.
rhTSH may allow a significant optimization of 131I therapy in dogs with thyroid cancer. However, if as in humans rhTSH leads to an increase in thyroid gland volume it must be used carefully in dogs with large thyroid tumors or distant metastases to avoid compression of key anatomical structures. Therefore, we first evaluated the short-term effect of rhTSH on thyroid gland volume and echogenicity, measured by ultrasonography, in 7 healthy Beagles (chapter 7). In this prospective blinded cross-over study, a single observer evaluated thyroid echogenicity, homogeneity, shape, capsule delineation, and measurement of thyroid length, width and height at baseline, and at 6, 24 and 48h after injection of rhTSH (100 μg IV) or placebo. rhTSH had no significant effect on thyroid gland volume, echogenicity, homogeneity or capsule delineation and no adverse effects were noticed. Although these results could not be extrapolated to dogs with thyroid tumors, this preliminary data did not suggest that rhTSH induced swelling or edema of the canine thyroid gland at the dosage used.
After concluding a pilot study (chapter 8) on the effect of rhTSH on thyroid scintigraphy in healthy Beagles we investigated the effect of rhTSH on the uptake of 123I in 9 dogs with thyroid tumors (chapter 9). In this prospective cross-over study, rhTSH (100 μg) administered IV 24 h before 123I (37 MBq IV) caused no significant change on thyroid tumor radioactive iodine uptake (RAIU) at 8 h or at 24 h. Interestingly, a significant positive correlation was found between the effect of rhTSH on tumor 8h-RAIU and rhTSH serum concentrations 6 h, 12 h and 24 h after rhTSH administration, suggesting that higher dosages of rhTSH may be necessary. Further studies are needed to determine the best protocol of rhTSH administration to optimize thyroid tumor RAIU.
Our research starts to uncover the pathogenesis of canine thyroid cancer identifying 2 activating mutations in K-RAS and showing the involvement of the PI3K/Akt pathway, particularly in FTC. The prognostic markers found in our research provide relevant information to owners and clinicians, and could be used to adapt follow-up and adjunctive therapy to each patient’s risk. The newly identified therapeutic targets are easily detectable with immunohistochemistry and open the possibility of personalized medicine. Although we could not demonstrate an improved survival with levothyroxine therapy or an enhanced tumor 123I uptake with rhTSH, further research is necessary. Future challenges include pursuing the main genetic events leading to canine thyroid cancer, performing clinical trials to further explore the molecular targets identified in our research, and continuing to search for ways to optimize 131I therapy and improve patient outcome.

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