The Value of PRKD1 E710D Sequencing of May-Grünwald-Giemsa Stained Cytology Specimens in Separating Polymorphous Adenocarcinoma from Adenoid Cystic Carcinoma and Pleomorphic Adenoma Andreasen S1,2, Grauslund M3, Melchior LC3, Wessel I1, Kiss K3, Agander TK3 1Department of Otolaryngology Head & Neck Surgery and Audiology, Copenhagen University Hospital Rigshospitalet, Copenhagen, Denmark 2Department of Otorhinolarlyngology and Maxillofacial Surgery, Zealand University Hospital, Køge, Denmark, 3Department of Pathology, Copenhagen University Hospital Rigshospitalet, Copenhagen, Denmark ABSTRACT METHODS RESULTS Polymorphous low-grade adenocarcinoma, termed polymorphous adenocarcinoma (PAC) in the 2017 WHO classification, is an indolent tumor of salivary gland origin most frequently arising in the minor salivary glands of the palate. It has a significant morphological overlap with adenoid cystic carcinoma (ACC) and pleomorphic adenoma (PA), especially on fine needle aspiration cytology (FNAC). However, the prognosis and clinical management differs dramatically between these three tumor types. Many types of salivary gland tumors are characterized by fusion oncogenes, including common as well as rare entities.1,2,3 The recent discovery of the PRKD1 E710D point mutation found exclusively in PLGA could help in making an accurate preoperative diagnosis on FNAC material.4 Here, we investigate the value of the PRKD1 E710D point mutation in May-Grünwald-Giemsa (MGG)-stained FNAC smears, and their corresponding surgical specimens, in a series of PAC, ACC, and PA. The exact diagnosis could be valuable in the pre-operative surgical planning. Archival FNACs from 16 PACs, 18 ACCs, and 24 PAs originating from the palate were retrieved along with corresponding FFPE surgical specimens. DNA was extracted from scraped-off FNAC material and surgical specimens. Dideoxynucleotide sequencing of PRKD1 exon 15, encoding E710D, was performed in nested PCR reactions and assessed blinded to diagnosis. PAC FNACs contained E710D point mutations in 8 cases (c.2130 A>T and c.2130 A>C in 3 and 5 cases, respectively) with 7 cases being wild type and 1 case being inconclusive. All FNACs from ACCs and PAs were wild-type. Sequencing of the surgical specimens identified identical E710D or wild-type mutations as the FNAC in all cases of all three tumor categories. The specificity of PRKD1 E710D point mutation in separating PAC from ACC and PA was 100% (95%CI:91.6-100%), the sensitivity in diagnosing PAC was 53.3% (26.6-78.7%), the positive predictive value 100% (63-100%), and the negative predictive value 85.7% (72.7-94%). CONCLUSIONS PRKD1 E710D sequencing in MGG FNAC is feasible and highly specific in separating PAC from ACC and PA, whereas the sensitivity in doing so is only moderate. This can be highly useful in the diagnostic work-up and planning of surgery in patients with these most frequent salivary gland tumors of the palate. OBJECTIVES - To determine the feasibility of PRKD1 E710D hotspot sequencing of MGG-stained cytology specimens in separating PAC, ACC, and PA of the palatal minor salivary glands. Figure 2. May-Grünwald-Giemsa (MGG) stained smear from a polymorphous adenocarcinoma from the palate. Clusters of basaloid, epithelial tumor cells with oval nuclei with numerous intermingled hyaline globules (arrows) resembling those seen in adenoid cystic carcinoma. Scarce areas of fibrillar stroma resembling that of pleomorphic adenoma is seen in the background (asterisk). REFERENCES A B Recurrent fusion of MYB and NFIB transcription factor genes in carcinomas of the breast and head and neck. Proc Natl Acad Sci USA 106:18740-4. NUT carcinoma of the sublingual gland. Head Neck Pathol 10:362-6. Promoter swapping between the genes for a novel zinc finger protein and beta catenin in pleiomorphic adenomas with t(3;8)(p21;q12) translocations. Nat Genet 15: 170-4. Hotspot activating PRKD1 somatic mutations in polymorphous low-grade adenocarcinomas of the salivary glands Nat Genet 46:1166-9. Figure 3. Dideoxynucleotide sequencing of PRKD1 exon 15 encoding amino acid 710. Nucleotide 2130 in the third position of this codon is substituted with (A) T or (B) C, both resulting in an amino acid change from glutamic acid to aspartic acid characteristic of polymorphous adenocaricnoma. Figure 1. (A) Clinical photograph of a patient with an ulcerating mass of the posterior part of the hard palate. (B) H&E from the same patient with a predominantly cribriform polymorphous adenocarcinoma. USCAP 2017; San Antonio