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The study of miRNAs in PTC has shown that these molecules can help in the diagnosis, prognosis and also as therapeutic candidates [1]. miRNAs are known to be differentially expressed in PTC compared to non-cancerous tissue and a few studies have shown association with some clinico-pathological features. However, little is known regarding their expression in relation to risk of disease progression. In this study, we examined the expression of miRNAs in patients diagnosed with PTC in association with risk of disease recurrence and/or persistence. Patients were classified by three endocrinologists to either high (H) or low (L) risk according to ATA Risk Stratification. PTC tissue was micro-dissected from Formalin Fixed Paraffin Embedded (FFPE) tissue for analysis. OPenArray analysis showed that 21 miRNAs were differentially expressed in H-L groups (n=4/grp). qRT-PCR was used to confirm these differences in a larger cohort of PTC (H=46; L=58). This comparison also included comparisons between cancer and normal tissue and investigation of miRNAs known to be differentially expressed (i.e miRNAs 222, 221 and 146b). All analysis was performed in Rstudio using ΔΔCt relative to expression of miR-16 which was not altered by PTC. By qRT-PCR, only 3 of the 21 miRNAs identified by OpenArray analysis, were differentially expressed in H vs L risk (each P In this study, we identified 3 miRNAs with potential utility for the stratification of patients into those with H or L risk disease recurrence/persistence. As the current ATA 3-tiered system used is still a reflection of the continuum of risk that patients have, whether these 3 miRNAs may have the utility to further stratify those in the intermediate group remains to be investigated. Reference: 1. Ramírez-Moya, J. and P. Santisteban, miRNA-Directed Regulation of the Main Signaling Pathways in Thyroid Cancer. Frontiers in Endocrinology, 2019. 10: p. 430.

There is limited data on prevalence of PD-L1 expression in papillary thyroid cancer (PTC). Anti- PD-L1 therapy is now being trialled for head and neck cancers including thyroid cancer. Whether presence of PD-L1 staining in PTC might result in a better response to anti-PD-L1 therapy is uncertain. We evaluated the frequency of tumoral PD-L1 expression using standard antibody (SP263) and clinicopathologic factors including mutation status - BRAF, NRAS, KRAS using OncoFocus Mass Array System in an Australian PTC cohort with low or high risk of recurrence /treatment response as per 2015 ATA guidelines. The cohort had 128 patients; 66% were females; 40% had both multifocal and bilateral PTC; 47% had low recurrence risk, of whom 92% had excellent treatment response; 34% had high recurrence risk, of whom 37% had structural incomplete treatment response. There was nil (0%) PD-L1 staining in 59%, 1% staining in 20%, 5-10% staining in 11%, >10% staining in rest of the cohort; 30%, 40% and 70% staining was seen in 2% each. PD-L1 in immune cells was positive in 62%. There was no significant association between PD-L1 tumour status and age, gender, size of tumour, multifocality, bilaterality, both multifocality and bilaterality, lymphovascular invasion, perineural invasion, extrathyroidal extension (ETE), histological variants, risk stratification, treatment response, metastases or mutation status. Among PD-L1 negative vs positive patients, BRAFV600E was present in 64.9% vs 67.5%, NRAS in 8.8% vs 0% and nil mutation in 26 % vs 33 % p=0.15. The staining thresholds were chosen according to existing literature. When analysed according to the percentage of PD-L1 staining (0%, 1-10% and >=10% thresholds), there was no significant association between PD-L1 staining category and other features, except for ETE (35% vs 65% vs 27%; p=0.007), a relationship that remained significant in a multivariable-adjusted model (OR 9.9, p=0.04). PD-L1 staining thresholds of =5% were significantly associated with the size of tumour (24.7±15.4 vs 33.3±21.8; p=0.02) and histological variants (57 % vs 61% for classic variants and 27% vs 11% for follicular variants, p=0.03). This is the first study to our knowledge that analysed PD-L1 staining based on the risk stratification categories. The majority of PTC showed negative or low staining intensity for PD-L1 expression. There was no significant association between PD-L1 staining, clinicopathologic characteristics and mutation status of the PTC. Relatively less ETE was seen in the group that showed >=10% and also >=5% PD-L1 staining as compared to the group that showed 1%

PTC and melanoma are known to harbour common mutations, but this has not been extensively investigated. Targeted therapies for BRAF and PD-L1 have been used for melanoma and there are ongoing clinical trials for use of PD-L1 inhibitors in PTC but its utility is uncertain. Additionally, many of these patients have multiple cancers, so, whether they have a tumour predisposition syndrome is also unclear. Both germline and somatic mutations in BRCA1-associated protein 1 (BAP1) are associated with a wide spectrum of tumours. We hypothesized that a common genetic link may be present in our cohort of patients who have both PTC and melanoma. The aim of this study was to elucidate molecular genetics, specifically BRAF, NRAS, KRAS, KIT using OncoFocus Mass Array System as well as expression of PD-L1 and BAP1, using a standard antibody (SP263) and C-4 respectively, in an Australian cohort with concurrent PTC and melanoma. In our cohort of 21 patients (43% females, all Caucasian), melanoma was diagnosed about 8 years prior to PTC (50.3 ± 18.3 vs. 58.6 ± 12.8 years). The most common mutation was BRAFV600E seen in 88% of PTC, followed by NRAS mutation in 12% of PTC. Majority of the PTC (68%) stained negative for PD-L1. There was no significant association between PD-L1 tumour status and clinicopathologic outcomes. Interestingly, majority of multifocal, bilateral and both bilateral and multifocal PTC were PD-L1 negative (85%,69% and 69% respectively, P25%, a much higher degree of staining compared to PTC group. Among 7 patients where data were available for both tissues, concordant mutations were found in only 2 patients (both BRAFV600E). In addition, 11 of the 21 patients had at least one other cancer apart from PTC and melanoma. Nine of the 11 patients who had more than one cancer were BRAF positive. BAP1 staining was retained in the majority of PTCs and melanoma tissues, indicating no loss of BAP1 protein. PTC and melanoma both share molecular markers including BRAF, NRAS, PD-L1 as shown in our cohort. This is the largest study describing the mutation status of both PTC and melanoma. It is also the only study describing the PD-L1 and BAP1 expression in PTC and melanoma. BRAFV600E was the most common mutation. Majority of the PTC and melanoma stained negative for PD-L1. BAP1 expression was retained in both either PTC and melanoma tissues thus making presence of BAP1 tumour predisposition syndrome unlikely.