Salivary mucoepidermoid carcinoma: histological variants, grading systems, CRTC1/3-MAML2 fusions, and clinicopathological features.

AIMS: To investigate the histological diversity of salivary mucoepidermoid carcinoma (MEC), its clinicopathological features, and its associations with CRTC1/3-MAML2 fusions. METHODS AND RESULTS: Salivary MEC cases (n = 177) were examined for CRTC1/3-MAML2 fusions, histological variants were classified, and tumours were graded according to four different grading systems. Adverse histological features considered to be unusual in MEC were also investigated. Of the 177 MEC cases, 110 were positive for CRTC1/3-MAML2 fusions. The classical variant was the most frequent in the fusion-positive case group, the fusion-negative case group, and the total case group. The clear/oncocytic variant was the second most frequent in the fusion-positive and total case groups. Oncocytic, Warthin-like and spindle variants were seen in the fusion-positive case group only. Clear cell, sclerosing, mucinous and central variants were seen in both the fusion-positive case group and the fusion-negative case group. No case was classified as a ciliated variant, as a mucoacinar variant, or as a high-grade transformation. As compared with the classical variant, non-classical variants were characterised by frequent CRTC1/3-MAML2 fusions and a low clinical stage in all cases. Of the four histological features considered to be unusual in MEC, marked nuclear atypia, frequent mitoses (>10/10 high-power fields) and extensive necrosis were found independently of the fusion status, and were present in 3-5% of all cases. However, none of the cases showed overt keratinisation. On comparison, the Armed Forces Institute of Pathology and modified Healey grading systems downgraded tumours, the Brandwein system upgraded tumours, and the Memorial Sloan Kettering system provided a moderate means of assessment. CONCLUSION: Recognition of the histological diversity of MEC, its clinicopathological features and its associations with CRTC1/3-MAML2 fusions is helpful for an accurate diagnosis of this carcinoma.

Pleomorphic adenoma: detection of PLAG1 rearrangement-positive tumor components using whole-slide fluorescence in situ hybridization.

Pleomorphic adenoma (PA) consists of heterogeneous histological architecture mixed with epithelioid and mesenchymal forms. Various types of epithelial or myoepithelial malignancies arise from PA, but sarcomas are extremely rare. A human androgen receptor gene (HUMARA) clonality assay has suggested that PA is clonal in nature. However, clonality of various tumor components of PA would be difficult to determine with this assay. In addition, the results obtained should be carefully interpreted. PLAG1 rearrangements are considered a good molecular marker for neoplasticity in PA. We aimed to clarify the neoplasticity of the various tumor components present in PA using whole-slide fluorescence in situ hybridization (FISH). Five PA cases positive for PLAG1 rearrangements were examined. Using an immunohistochemistry panel, cell components in PA were classified into eight cell types. To precisely localize PLAG1 rearrangement-positive cell components at the cellular level, sequential retrieval of whole-slide imaging (WSI) data of HE histology and FISH for PLAG1 rearrangement was carried out. PLAG1 rearrangements were detected in ductal cells, myoepithelial spindle cells, myoepithelial oncocytic cells, myoepithelial plasmacytoid cells, and mesenchymal chondroid cells, but not in mesenchymal lipid cells, mesenchymal fibrous cells, or vascular endothelial cells. Immunohistochemical PLAG1 expression was restricted to cell components harboring PLAG1 rearrangements.The results of the present study indicate that ductal and myoepithelial, chondroid cells are neoplastic but lipid, fibrous, and endothelial cells are not. PLAG1 immunohistochemistry is useful in discriminating neoplastic from non-neoplastic cell components. These findings may be important for elucidating tumorigenesis and the process of malignant transformation in PA.

Next-generation sequencing assay in salivary gland cytology: A pilot study.

BACKGROUND: Preoperative diagnosis of salivary gland tumors (SGTs) by fine-needle aspiration (FNA) cytology is challenging. Next-generation sequencing (NGS)-based assays for somatic mutations have a great advantage in that a large number of genes can be analyzed simultaneously. Although NGS may have an enormous diagnostic potential in cytology, to our knowledge, the significance of NGS in SGT cytology remains to be clarified. METHODS: In this pilot study, we retrospectively examined 32 frozen SGT samples obtained at surgery (14 malignant and 18 benign). After the stored frozen tumor tissues were thawed, aspirate samples were obtained using 22-gauge needles and subjected to smear tumor samples and to DNA extraction for an NGS assay employing the Illumina AmpliSeq Cancer Hotspot Panel v2. The results were correlated to preoperative cytological diagnosis. RESULTS: The preoperative diagnoses obtained by FNA cytology included 23 negative lesions (no malignancy in 6 and benign tumor in 17) and nine positive lesions (suspicious for malignancy in 4 and malignancy in five), providing a sensitivity and a specificity of 9/14 (64%) and 18/18 (100%), respectively. The NGS assay detected somatic mutations in 10/14 malignant and 1/18 benign SGT cases, providing a sensitivity and a specificity of 71% and 94%, respectively. CONCLUSION: The NGS assay may be helpful for detecting the malignant potential in SGT cases and can be used as an ancillary test for SGT cytology.

Prognostic impact of CRTC1/3-MAML2 fusions in salivary gland mucoepidermoid carcinoma: A multiinstitutional retrospective study.

Mucoepidermoid carcinoma (MEC) is rare, but the most common primary malignancy of the salivary gland and not infrequent in young individuals. CRTC1/3-MAML2 fusions are frequently detected in MEC and are useful as a diagnostic biomarker. However, there has been debate as to whether the fusions have prognostic significance. In this study, we retrospectively collected 153 salivary gland MEC cases from 11 tertiary hospitals in Japan. As inclusion criteria, the MEC patients in this study had curative surgery as the initial treatment, received no preoperative treatment, and had no distant metastasis at the time of the initial surgery. The MEC diagnosis was validated by a central pathology review by five expert salivary gland pathologists. The CRTC1/3-MAML2 fusions were detected using FISH and RT-PCR. In 153 MEC cases, 90 (58.8%) were positive for CRTC1/3-MAML2 fusions. During the follow-up period, 28 (18.3%) patients showed tumor recurrence and 12 (7.8%) patients died. The presence of the fusions was associated with favorable tumor features. Of note, none of the fusion-positive patients died during the follow-up period. Statistical analysis showed that the presence of the fusions was a prognostic indicator of a better overall survival in the total and advanced-stage MEC cohorts, but not in the early-stage MEC cohort. In conclusion, CRTC1/3-MAML2 fusions are an excellent biomarker for favorable overall survival of patients with salivary gland MEC.

Clinicopathological significance of EGFR pathway gene mutations and CRTC1/3-MAML2 fusions in salivary gland mucoepidermoid carcinoma.

AIMS: Mucoepidermoid carcinoma (MEC) is one of the most common salivary gland carcinomas. Epidermal growth factor receptor (EGFR) signalling pathway gene mutations are important in predicting a patient's prognosis, selecting molecularly targeted drugs and estimating the efficacy of a molecular therapy. However, their significance in MEC have been poorly clarified. CRTC1/3-MAML2 fusions are specific to MEC and may be associated with favourable characteristics in these patients. METHODS AND RESULTS: We looked for CRTC1/3-MAML2 fusions and gene alterations in the EGFR, RAS family (KRAS, HRAS and NRAS), PIK3CA, BRAF and AKT1 in 101 MEC cases. We also examined mutations in TP53. CRTC1/3-MAML2 fusions were found in 62.4% of the cases. KRAS, HRAS and PIK3CA mutations were detected in 6.9%, 2.0% and 6.9%, respectively, but other EGFR pathway genes were not mutated. In total, gene mutations (RAS/PIK3CA) in the EGFR pathway were detected in 14.9% of the cases. TP53 mutations were found in 20.8%. CRTC1/3-MAML2 fusions were associated with a better prognosis and RAS/PIK3CA mutations a worse prognosis of the patients, respectively, and both were selected as independent prognostic factors for the overall survival of the patients. TP53 mutations had no prognostic impact. CRTC1/3-MAML2 fusion-positive rates were inversely associated with the patients' age and the fusions were found in 82% of patients aged < 30 years. CONCLUSIONS: RAS/PIK3CA mutations were frequently detected, and may be a biomarker for a poorer prognosis in MEC patients. CTRC1/3-MAML2 fusions were positive in most of the young MEC patients.

Proton therapy for non-squamous cell carcinoma of the head and neck: planning comparison and toxicity.

To investigate optimal treatment planning using proton beams for non-squamous cell carcinoma of the head and neck (NSCHN), the dose distributions of plans involving pencil beam scanning (PBS) with or without a patient-specific aperture system (PSAS), passive-scattering proton therapy (PSPT) and X-ray intensity-modulated radiotherapy (IMRT) were compared. As clinical results, toxicities of PBS with PSAS, including changes in quality of life, were reported. Between April 2014 and August 2016, a total of 30 patients were treated using PBS with PSAS. In 20 patients selected at random, the dose distributions of PBS with or without the PSAS, PSPT and IMRT plans were compared. Neutron exposure by proton therapy was calculated using a Monte Carlo simulation. Toxicities were scored according to CTCAE ver. 4.0. Patients completed EORTC quality of life survey forms (QLQ-C30 and QLQ-HN35) before and 0-12 months after proton therapy. The 95% conformity number of PBS with the PSAS plan was the best, and significant differences were detected among the four plans (P < 0.05, Bonferroni tests). Neutron generation by PSAS was ~1.1-fold higher, but was within an acceptable level. No grade 3 or higher acute dermatitis was observed. Pain, appetite loss and increased weight loss were more likely at the end of treatment, but recovered by the 3 month follow-up and returned to the pretreatment level at the 12 month follow-up. PBS with PSAS reduced the penumbra and improved dose conformity in the planning target volume. PBS with PSAS was tolerated well for NSCHN.

Postoperative radiotherapy for T1/2N0M0 mucoepidermoid carcinoma positive for CRTC1/3-MAML2 fusions.

BACKGROUND: The National Comprehensive Cancer Network (NCCN) guidelines recommend considering postoperative radiotherapy (PORT) for completely resected T1/2N0M0 salivary mucoepidermoid carcinomas when they show tumor spillage, perineural invasion, or intermediate/high-grade histology. CRTC1/3-MAML2 fusions have been associated with a favorable clinical outcome. METHODS: Forty-seven T1/2N0M0 mucoepidermoid carcinoma cases positive for CRTC1/3-MAML2 fusions were completely resected and were not treated with PORT. RESULTS: Pathologically, none of the cases showed tumor spillage or perineural invasion. Cases with intermediate/high-grade histology numbered 9 (19%) to 26 (55%) with the currently used 3 different grading systems. During the follow-up (median 60 months), locoregional tumor recurrence occurred in 4 cases, which were treated with surgery alone. At the last follow-up (median 60 months; 7-160), all patients were alive with no evidence of disease. CONCLUSION: An excellent prognosis may be achieved without PORT in T1/2N0M0 mucoepidermoid carcinoma patients positive for CRTC1/3-MAML2 fusions when the tumors are completely resected without tumor spillage.

Striated duct adenoma presenting with intra-tumoral hematoma and papillary thyroid carcinoma-like histology.

Striated duct adenoma of the salivary gland is a rare benign tumor characterized by unilayered duct epithelium and striations of the tumor cell membranes. To the best of our knowledge, only six cases have been reported in the literature. Here we report an additional case, which was complicated by an intra-tumoral hematoma on clinical presentation and by papillary thyroid carcinoma-like histology on intra-operative frozen section diagnosis. An asymptomatic 78-year-old male presented with a two-year-history of a painless tumor of the left parotid. An intra-tumoral hematoma, which is unusual for a salivary gland tumor, was suspected from results of pre-operative radiology. The patient then underwent a left parotidectomy. The intra-operative frozen section diagnosis indicated a benign tumor, although ectopic papillary thyroid carcinoma was raised as a differential diagnosis since the eosinophilic tumor cells occasionally possessed nuclear grooves and nuclear pseudo-inclusions. By precise histopathological examination using paraffin sections, the tumor was finally diagnosed as striated duct adenoma. This type of tumor has unique features of hypervascular stroma and papillary thyroid carcinoma-like nuclei. In our case, the former feature was associated with the intra-tumoral hematoma and the latter feature, with difficulty in frozen section tumor diagnosis.

Clinicopathological significance of MAML2 gene split in mucoepidermoid carcinoma.

CRTC1-MAML2 and CRTC3-MAML2 fusions have been associated with favorable clinicopathological features of mucoepidermoid carcinomas. However, the significance of the MAML2 gene split has not been fully clarified. In the present study, 95 mucoepidermoid carcinomas (paraffin-embedded materials) were analyzed for CRTC1-MAML2 and CRTC3-MAML2 fusions by RT-PCR and for the MAML2 gene split by FISH. Quantitative RT-PCR for the CRTC1-MAML2 transcript was performed in selected cases. MLL gene involvement, which has been reported in some leukemia cases, was examined by FISH in fusion partner-unknown cases. CRTC1-MAML2 and CRTC3-MAML2 fusions were detected in 37 and 6 cases, respectively. The MAML2 gene split was detected in 62 cases, which included all CRTC1/3-MAML2 fusion-positive cases. The level of CRTC1-MAML2 transcript expression was highly variable, and its clinicopathological impact was unclear. The MLL gene split was not detected. Mucoepidermoid carcinomas negative for CRTC1/3-MAML2 and positive for the MAML2 gene split (n = 19) showed favorable clinicopathological tumor features similar to those positive for CRTC1/3-MAML2 fusions. Compared with negative cases (n = 33), mucoepidermoid carcinomas positive for the MAML2 split (n = 62) were associated with lower patient age, a mild female predilection, a smaller tumor size, less frequent nodal metastasis, a lower clinical stage, a lower histological grade, and longer overall and disease-free survival. The MAML2 gene split emerged as an independent prognostic factor for both overall and disease-free survival in multivariate prognostic analysis. The presence of the MAML2 gene split defines a distinct mucoepidermoid carcinoma subset that is associated clinicopathologically with favorable tumor features.

Impact of CRTC1/3-MAML2 fusions on histological classification and prognosis of mucoepidermoid carcinoma.

AIMS: The aim of study was to evaluate the impact of CRTC1-MAML2 and CRTC3-MAML2 fusions on the histological classification of mucoepidermoid carcinoma (MEC) of the salivary glands and on the prognosis of patients. METHODS AND RESULTS: MEC cases (n = 111) were screened for CRTC1-MAML2 and CRTC3-MAML2 fusions by reverse transcription polymerase chain reaction. We developed a system of 'molecular Armed Forces Institute of Pathology (AFIP) classification' that combined the AFIP histological classification proposed by Goode et al. and the presence of CRTC1-MAML2 or CRTC3-MAML2 fusions. MEC cases positive for CRTC1-MAML2 or CRTC3-MAML2 fusion formed a favourable tumour subset that was distinct from fusion-negative cases. When positive for the fusions, 'high-risk' patients, including those with a higher histological grade or an advanced clinical stage, showed an excellent prognosis. For overall survival, 'molecular AFIP classification' was selected as a powerful independent prognostic factor (P=0.0038), as was the clinical stage (P =0.0032). For disease-free survival, 'molecular AFIP classification' was also selected as an independent prognostic factor (P = 0.0006). CONCLUSIONS: Molecular AFIP classification may be useful in predicting the prognosis of patients with MEC.