Understanding and overcoming the pitfalls in the diagnosis of pleomorphic and carcinoma ex‐pleomorphic adenoma of salivary glands

The study illustrates a recurrent pitfall in the diagnosis of pleomorphic adenoma and carcinoma ex pleomorphic adenoma. Salivary gland neoplasms often present significant diagnostic challenges due to their overlapping histological features, particularly in cases of mucoepidermoid carcinoma (MEC). MEC is traditionally identified by the presence of three cell types, including mucocytes, but certain cases deviate from this norm, demonstrating authentic MAML2 gene fusions without mucocytes. This overlap becomes especially complex as other neoplasms, like pleomorphic adenoma (PA), and carcinoma ex-pleomorphic adenoma (CXPA), may also exhibit mucocytes, albeit less frequently. The diagnostic complexity is increased by PA and CXPA's biphasic proliferation pattern, which contains an admixture of bilayered ducts, myoepithelial cells, stroma, and various forms of metaplasia.1, 2 These appearances underscore the importance of careful histological analysis to avoid misdiagnosing PA or CXPA as MEC based solely on the presence of mucocytes. In this series, six cases of salivary gland tumours were initially diagnosed as MEC, CXPA, or PA, involving patients aged 55–81 years, with tumour sizes ranging from 14 to 50 mm. The tumours predominantly affected the parotid gland, with single cases involving the tongue and palate. The histological features were reevaluated, revealing critical diagnostic pitfalls. Among the cases, three were initially diagnosed as MEC, with one showing squamous differentiation and two displaying oncocytic proliferation. Further molecular analysis reclassified these as PA or CXPA, emphasizing the potential for diagnostic misinterpretation based on histology alone. Histological examination showed a biphasic proliferation pattern in all cases, characterized by epithelial and myoepithelial cell differentiation associated with ductal structures (Figure 1A), sometimes showing dispersed myoepithelial cells within a myxoid stroma (Figure 1B). These cells varied in form, often appearing as large eosinophilic cells with distinct cytoplasm and centrally located nuclei (Figure 1C). In three cases, oncocytic metaplasia was observed, presenting cells with granular cytoplasm and prominent nucleoli (Figure 1D). Squamous metaplasia was also noted, with cells showing a range of atypia (Figure 1E–G), sometimes containing mucocytes (Figure 1H). Immunohistochemically, all cases were positive for SOX10 (Figure 2A), CK7 (Figure 2B), p40, and p63 (Figure 2C), with EMA variably expressed in ductal cells (Figure 2D), and a negativity for androgen receptors. Five cases showed positivity for PLAG1 (Figure 2E), while one for HMGA2 (Figure 2F). Importantly, molecular analyses by RNA sequencing confirmed PLAG1 rearrangement in five cases with different partners including TNS4, CPQ, ACTA2, or CTNNB1, while HMGA2 rearrangement was found in one with RB1 as the gene partner. Fluorescence in situ hybridization (FISH) analysis validated these findings, with all MAML2 probes testing negative. This series highlights significant diagnostic challenges in distinguishing PA and CXPA from MEC in salivary gland tumours, due to their overlapping mucoid features. Although mucocytes are classically associated with MEC, they are also present in other salivary malignancies like PA and CXPA, complicating diagnoses based on morphology alone. Their occurrence seems more frequent than previously reported, with mucinous proliferation in PA and CXPA appearing in diverse forms, ranging from isolated cells to extensive cystic clusters that closely mimic MEC. Immunohistochemistry has emerged as a valuable tool, particularly with the use of SOX10 and p63 markers, which aid in differentiating MEC from the oncocytic subtype of PA.3 However, since MECs can occasionally test positive for SOX10 (weak to moderate, and more focally), using a combination of markers such as PLAG1 and HMGA2 is recommended to improve diagnostic specificity.4, 5 Molecular testing, including FISH or RNA sequencing, is essential in ambiguous cases, as the presence of PLAG1 or HMGA2 fusions supports PA or CXPA diagnoses,6 while MAML2 rearrangement supports MEC. This integrated approach of histology, immunohistochemistry, and molecular diagnostics helps avoid misdiagnoses that could alter patient management, especially among general pathologists who may be less familiar with these histological pitfalls. N.B. contributed to the study design. Z.A. and N.B. contributed to analysing the results and writing the article. D.P., F.D., and J.L. contributed to the RNAseq analysis. C.L., J.R., E.U.C., A.S., V.C.M., A.C., and N.B. independently reviewed slides. All authors contributed to the writing and reviewing of the article. The authors thank Centre de Ressources Biologiques-HCL Lyon Sud (BB-0033-00046) for its help and DRS (Direction de la Recherche en Santé-HCL) for language editing and critical suggestions. The authors do not declare any conflict of interest. The data that support the findings of this study are available from the corresponding author upon reasonable request.