Prevalence of USP8, USP48 and BRAF mutations
The prevalence of USP8 mutations in CD reported thus far ranges from 21-62% (Supplementary Table 3).[1–6,13,14] Our study, the first from South Asia, found a prevalence of 37%. The USP8 mutations found in our patients have been previously described and no novel mutations were discovered, however, we found a hitherto unreported USP48 splice-site mutation - p.Pro433IVS+2 T>A.
Somatic mutations of both USP48 and BRAF genes in patients with CD were reported only in 2018.[9,10] The pooled incidence of USP48 and BRAF mutations in these two studies was 41/308 (13.3%) and 16/227 (7%).[15] In our study, the prevalence of USP48 mutations in USP8 wild-type adenomas was 13.8%, almost identical to that in the literature. Of interest is that almost all USP48- and BRAF-variants were noted only in patients with USP8 wild-type tumours, although they themselves were not mutually exclusive.
Another important finding in our study was that not a single BRAF mutation was detected in the 29 patients with USP8 wild-type status. The absence of BRAF mutations in our patients with CD is in stark contrast to the 16.5% prevalence reported from China[9] but is similar to 1% prevalence amongst Caucasians.[10] Ethnicity may also have a bearing on the presence of USP8-variants,[1,2,5,7,10,14] as is evident from the higher prevalence of USP8 mutations (62%) in the Chinese population[3] as compared to that in the European population (21-48%).
Genotype-phenotype correlation of USP8-variants
Several studies have shown a significantly higher, if not exclusive occurrence of USP8-variants in women.[2,3,6,10,13,16] This was however not the case in the series by Albani et al.,[14] who found that there was no significant gender association (USP8 mutations in 39% of females vs. 30% of males). In our series, only 1 male out of 8 (12.5%) had a USP8 mutation while 16/38 (42.1%) females carried USP8-variants. No male patient out of the 4 with USP8 wild-type CD harboured USP48 mutations. While this difference did not attain statistical significance due to the relatively small sample size, there seems to be a clear tendency towards female preponderance and might explain, at least partially, the well-known female predilection in CD which so far has not been adequately understood. Perez-Rivas et al.[2] first proposed that oestrogens could demonstrate a growth-stimulating effect on USP8-mutated corticotrophs, a hypothesis supported by the fact while a balanced sex-ratio exists amongst paediatric patients with CD[17] in adults there is a distinct female predominance of the disease.11 In addition, the demonstration of oestrogen receptors on corticotroph cells along with the stimulatory effect of oestradiol on murine corticotroph cell proliferation via EGFR signalling might be a reason for a higher incidence of CD in females.[18,19]
Some reports indicate that USP8-mutated tumours occur in younger adults [2,7,14] and older children,[13] however, we and others[3–6] found no significant age difference between carriers of USP8-mutant and wild-type adenomas. BMI was found to be significantly higher in patients harbouring USP8-mutant tumours in two studies,[2,10] while another found that BMI was lower amongst patients with USP8-mutant tumours.[13] We could not demonstrate any statistically significant difference in BMI between the 2 groups.
We found that the USP8-mutants in our series tended to be larger than their wild-type counterparts, although this did not achieve statistical significance (p=0.09). Findings on the issue of tumour size vary considerably, with variant-carrying tumours being reportedly smaller and less invasive in some studies,[3,6] while others have observed a greater size in USP8-mutant microadenomas compared with wild-type microadenomas.[2,5] This difference may be at least partially attributed to a selection bias in some studies. For example, Hayashi and colleagues[6] oversampled Crooke’s cell adenomas, while the Chinese series[3] included a relatively large proportion of invasive tumours (>20%) and giant adenomas. Also worth noting is that none of the series reported so far mirror the ratio between micro- and macroadenomas normally found in CD, i.e. less than 10-20% macroadenomas, probably a consequence of the need for adequate pathological specimens to perform DNA or RNA sequencing.
With regards to the biochemical characteristics of these patients, one study[2] found that patients with USP8 mutations demonstrate increased suppression of cortisol after 8 mg dexamethasone while another found significantly lower plasma ACTH levels in these patients.[6] Ma et al.[3] found that ACTH secretion was higher in patients carrying USP8 mutations. We failed to establish any significant difference between wild-type and mutated tumours in the biochemical parameters studied, a finding corroborated by others.[2,5,13,14]
However, despite revealing no significant difference between the ACTH, UFC and serum cortisol in USP8-variant and USP8 wild-type we did find that the proportion of patients with Crooke’s hyaline change in the adjoining adenohypophysis was significantly higher in the USP8-variant group. Crooke’s hyaline change is typically seen with higher levels of hypercortisolism,[20] so it may be conjectured that patients with USP8-mutant adenomas may have experienced severe hypercortisolism at some point of their illness.
The remission rates of CD was similar in the USP8-variant and wild type tumors as reported by others.[2,3,14] Some authors[5,6] note a higher remission rate in patients with USP8 mutations while others[13,14] report higher recurrence rates in mutated tumours. Another study found parallel recurrence rates, but a shorter mean time to recurrence in the USP8-variant adenomas.[3] We too found a higher recurrence rate amongst our patients with a USP8 mutation (33% vs. 18%), although it was not statistically significant due to the small sample size.
Genotype-phenotype correlation of USP48-variants
The study by Chen et al.[9] could not demonstrate any significant clinical, biochemical or radiological differences between patients with USP48-variants and USP48 wild-type corticotroph adenomas. However a subsequent study reported that USP48-mutant adenomas were smaller than wild-type adenomas.[10] In our series, the occurrence of cavernous sinus invasion was significantly higher in the 4 patients with USP48-mutant adenomas (50% vs. 4%, p=0.042). This difference has not been reported by any other study, however further research is required on these less common mutations before any meaningful conclusions can be drawn. The literature available on the genotype-phenotype correlation of CD patients with USP8 and USP48 mutations is summarised in Table 6.
Strengths and limitations of this study
This single-centre, prospective study from the Indian subcontinent, determines the prevalence of USP8, USP48 and BRAF mutations in patients with CD using fresh frozen tumour tissue that provided uniformity in the quality of tissue used for mutational analysis. The main limitation of the study was its relatively small sample size that precluded the establishment of statistically significant phenotypic differences between patients with USP8-/ USP48-variant tumours and their wild-type counterparts. Moreover, only mutations in the previously described mutational hotspots of the USP8, USP48 and BRAF genes were tested for. Next-generation sequencing (NGS) may have identified additional mutations in our series, as was seen in the study by Ballmann and colleagues,[4] where NGS detected a few USP8 mutations that were missed on Sanger sequencing.