**4. Discussion**

Human studies have found that 75% of samples with SDHA mutations lacked immunoreactivity for SDHA and SDHB [9] and that 90% of samples with SDHB, C, D, or SDHAF2 mutations had immunoreactivity for SDHA and lacked immunoreactivity for SDHB [9,12,13]. When this is applied to the samples in our study, these patterns sugges<sup>t</sup> that 29 out of the 35 (82.8%) samples have a mutation in at least one of the SDH family genes (Figure 2).

The predominance of samples with an invasion lacking SDHB immunoreactivity agrees with several human studies [5,12,14,15], which show that a lack of SDHB immunoreactivity is associated with more aggressive tumor behavior.

Only one sample lacked SDHA immunoreactivity but maintained SDHB immunoreactivity (Figure 1); this sample showed signs of invasion and metastasis to the renal vein. However, as inactivation of SDHA does not lead to tumorigenesis [16], this may represent a somatic hypermethylation of the promoter region [17], leading to an accumulation of succinate and later an inhibition of demethylase enzymes. This can lead to promoter hypermethylation and tumor suppressor gene inactivation [18]; in humans, this is known as Leigh syndrome.

The lack of significant association between the immunohistochemical findings and tissue invasion may be due to limited numbers of cases with both SDHA and SDHB immunoreactivity.

Ultimately, while these data are encouraging, sequencing is needed to fully determine the role of SDH family genes in pheochromocytoma in canine pheochromocytomas. These findings do sugges<sup>t</sup> that IHC has a similar utility in narrowing candidate mutations in pheochromocytomas in dogs; given, in particular, the relatively higher cost of sequencing, restricting the set of possible candidate genes using immunohistochemistry would be helpful in future sequencing studies. However, determining the true utility of SDH immunohistochemistry would require sequencing information and additional

case information; if immunohistochemistry is associated with specific mutations and/or mutations in specific genes are closely associated with clinical behavior, immunohistochemistry would become an important tool in case management.
