Cytopathology in focus: Reflections on use of Milan System, edition 1: Areas to be explored for edition 2

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● Molecular markers and antibody detection of gene rearrangements. The molecular features of SGNs are a rapidly evolving field, holding promise not only for specific diagnostic markers but also as potential targets for therapeutic precision medicine. Table 1 illustrates the most up to date molecular features of SGNs.¹⁰ These molecular alterations can be detected by using fluorescence in situ hybridization and greatly enhance diagnostic specificity and accuracy.

Currently, however, sophisticated molecular techniques like FISH and next-generation sequencing are not widely available outside of major academic medical centers. More importantly, the low cellularity common in FNA specimens often makes molecular analysis ineffective. A practical solution would be the development of immunohistochemical surrogates for the diagnostic genetic tests. Earlier attempts at using such immunostains in SGN cytopathology yielded disappointing results. MYB protein is consistently expressed in adenoid cystic carcinoma, but it is also commonly detected in diagnostic mimickers. PLAG1 protein is usually seen in pleomorphic adenomas, but it is also expressed in various carcinomas such as ex-pleomorphic adenoma.

But a promising finding was reported recently. In 2021, Skaugen and colleagues¹¹ demonstrated that NR4A3 immunostaining is highly successful in diagnosing salivary gland acinic cell carcinoma on cell block material retrieved from FNA, outperforming not only DOG1 immunostaining but also NR4A3 FISH. The diagnosis of acinic cell carcinoma is often challenging in FNAs because the routinely used acinar markers DOG1 and SOX10 do not help with the differential diagnosis between tumor and normal salivary acini. The authors demonstrate that because normal acini are negative, NR4A3 immunostaining solves this classic diagnostic dilemma with ease. NR4A3 immunostaining has also shown to be effective in samples with low cellularity that are insufficient for molecular analysis.¹² Thus, this immunohistochemical marker appears to make the FNA diagnosis of acinic cell carcinoma straightforward in the cases with adequate material for cell block.

Another exciting immunostain showing potential is the immunostain for Amphiregulin (AREG), an epidermal growth factor receptor ligand. AREG has been shown to be a downstream target of CRTC1-MAML2 fusion. Detection of AREG expression using immunohistochemistry helps identify fusion-positive MECs.¹³ Ideally, additional immunohistochemical surrogates of genetic signatures could be developed and applied to salivary gland cytopathology to aid in difficult cases.

● In view of recently approved immune checkpoint inhibitors (e.g. nivolumab, atezolizumab, and pembrolizumab), testing for PD-L1 expression on tumor cells at the time of diagnosis has been required in pulmonary, gastric, urothelial, and head and neck squamous cell carcinoma. Guidelines for SGNs have not been established, but clinical studies are ongoing with positive results after PD-L1 inhibitor treatment.¹⁴ PD-L1 expression is traditionally determined by IHC testing in histologic samples. Given that FNA is usually the first-line diagnostic modality for SGN, it is surprising that reports evaluating salivary gland FNA as an adequate substrate for PD-L1 expression measurement are not found in the literature. Ongoing research studies by the authors will soon aid in providing answers to this important question.

Toper MH, Sarioglu S. Molecular pathology of salivary gland neoplasms: diagnostic, prognostic, and predictive perspective. Adv Anat Pathol. 2021;28(2):81–93. Reprinted with permission.

Significant progress has been made in the diagnosis and characterization of salivary gland lesions after the widespread application of the MSRSGC in 2018, coupled with several important clinical factors that are pertinent to patient management. It is exciting to learn that the second edition of the MSRSGC reporting guidelines is expected to be published in the later part of 2022 and will feature updated risks of malignancy based on new evidence in the literature and other significant advances in salivary gland cytopathology.

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Dr. Razzano is a cytopathology fellow and Dr. Wang is associate professor of pathology—both at Yale University School of Medicine. Dr. Wang is a member of the CAP Cytopathology Committee.