A retrospective search in the authors’ registries was conducted to identify MECs characterized by remarkable keloid-like sclerotic stroma and dense inflammatory infiltrate. In total, 25 cases of SMEC were retrieved from the consultation files of the Tumor Registry at the Department of Pathology, Faculty of Medicine in Pilsen and Bioptic Laboratory, Ltd. in Pilsen, Czech Republic, and tumor registries of the co-authors. All cases were reviewed by the senior author (AS) and three other head and neck pathologists (RHWS, BK, and MB), and it was confirmed that they met the diagnostic criteria of SMEC, in particular, keloid-like sclerosis in more than 50% of the total tumor volume and abundant variable inflammatory infiltrate, in particular, rich on lymphocytes, plasma cells, and/or eosinophils.

Clinical features and outcomes (e.g., age, sex, site of primary tumor, follow-up period, recurrence, and distant metastasis) were recorded. Clinical information on the cases was collected from hospital records and the referring pathologists. The study was approved by the institutional review board.

Histology and immunohistochemistry

For conventional microscopy, tissues were fixed in formalin, processed routinely, embedded in paraffin (FFPE), cut, and stained with hematoxylin and eosin.

For immunohistochemistry, 4-μm-thick sections were cut from the paraffin blocks and mounted on positively charged slides (TOMO, Matsunami Glass INC, Osaka, Japan). Sections were processed on a BenchMark ULTRA (Ventana Medical Systems, Tucson, AZ), deparaffinized, and subjected to heat-induced epitope retrieval by immersion in CC1 solution (pH 8.6) at 95 °C. All primary antibodies used in this study are summarized in Table 1. Visualization was performed using the ultraView Universal DAB Detection Kit (Roche, Tucson, AZ) and the ultraView Universal Alkaline Phosphatase Red Detection Kit (Roche, Tucson, AZ). The slides were counterstained with Mayer’s hematoxylin. Appropriate positive and negative controls were employed.

Table 1 Antibodies used for immunohistochemical studyMolecular genetic studiesNext-generation sequencing

The in-house customized version of Archer FusionPlex Solid Kit was used to construct a cDNA library for detecting fusion transcripts in 118 genes (including AKT1, AKT3, ALK, AR, ARHGAP26, AXL, BCOR, BRAF, BRD3, BRD4, CALCA, CAMTA1, CCNB3, CCND1, CD274, CIC, CSF1, CSF1R, DNAJB1, EGFR, EPC1, ERBB2, ERBB4, ERG, ESR1, ESRRA, ETV1, ETV4, ETV5, ETV6, EWSR1, FGFR1, FGFR2, FGFR3, FGR, FOXO1, FOXO4, FUS, GLI1, GNAS, GPI, GRB7, HMGA2, CHMP2A, INSR, JAK2, JAK3, JAZF1, KRT20, KRT7, MAML2, MAP3K3, MAP3K8, MAST1, MAST2, MEAF6, MET, MGEA5, MKL2, MN1, MSMB, MUSK, MYB, MYBL1, MYOD1, NCOA1, NCOA2, NOTCH1, NOTCH2, NR4A3, NRG1, NTRK1, NTRK2, NTRK3, NUMBL, NUTM1, PAX3, PDGFB, PDGFRA, PDGFRB, PHF1, PIK3CA, PKN1, PLAG1, PPARG, PRKACA, PRKACB, PRKCA, PRKCB, PRKD1, PRKD2, PRKD3, PTH, PTPN1, RAB7A, RAF1, RELA, RET, ROS1, RSPO2, RSPO3, SLC5A5, SS18, STAT6, TAF15, TCF12, TERT, TFE3, TFEB, TFG, THADA, TMPRSS2, TTF1, USP6, VCP, VGLL2, YAP1, and YWHAE) and point mutations in 4 genes (including BRAF, EGFR, MET, PDGFRA). All steps were performed according to the manufacturer’s instructions, and the library was sequenced on an Illumina platform as described previously [26].

FISH and RT-PCR methodologies were described in detail in our earlier report [2]. For RT-PCR analysis, we used the primers listed in Table 2.

Table 2 Polymerase chain reaction primer descriptionsTruSight oncology 500 kit (TS500)

Mutation analysis and fusion transcript detection were performed using TruSight Oncology 500 Kit (Illumina, San Diego, CA). RNA was extracted using the Maxwell RSC DNA FFPE Kit and the Maxwell RSC Instrument (Promega, Madison, WI) according to the manufacturer’s instructions and quantified using the Qubit HS RNA Assay Kit (Thermo Fisher Scientific, Waltham, MA). DNA was extracted using the QIAsymphony DSP DNA mini (Qiagen, Hilden, Germany) and quantified using the Qubit BR DNA Assay Kit (Thermo Fisher Scientific, Waltham, MA). The quality of DNA was assessed using the FFPE QC kit (Illumina) and the quality of RNA using Agilent RNA ScreenTape Assay (Agilent, Santa Clara, CA). DNA samples with Cq < 5 and RNA samples with DV200 ≥ 20 were used for further analysis. After enzymatic fragmentation of DNA with KAPAFrag Kit (KAPA Biosystems, Wilmington, MA), DNA and RNA libraries were prepared with the TruSight Oncology 500 Kit (Illumina) according to the manufacturer’s protocol. Sequencing was performed on the NovaSeq 6000 sequencer (Illumina) following the manufacturer´s recommendations. Data analysis was performed using the TruSight Oncology 500 v2.2 Local App (Illumina) as described earlier [27].

Review of the literature

The study intended to examine previously reported cases of SMEC with inflammation. We examined whether SMECs contain typical neoplastic components commonly seen in MEC and whether SMECs demonstrate charcteristic clinicopathological behavior that would require their classification separately from MEC as was proposed for SMECE of the thyroid.

The inclusion criteria for this systematic review were as follows:

1.

All research papers must be original research articles that report clinical cases of SMEC.

2.

All articles must be published in English.

3.

All articles must describe the histologic appearance of SMEC in text and images.

4.

All articles must report a confirmation of the diagnosis of MEC using histochemical, immunohistochemical, and/or molecular techniques.

The exclusion criteria were as follows:

1.

Studies reviewing previous works without reporting any new cases

2.

Studies that investigated non-salivary MECs

3.

Studies that published duplicate cases

4.

Epidemiological studies and consultation cases

5.

Cases published by a predatory journal or non-indexed journal

We conducted an electronic search in PubMed, Science Direct, Web of Science, Scopus, Scielo, Google Scholar, EMBASE (Ovid), Europe PMC, ProQuest, Crossref, and Medline databases. The searched medical subject headings included “mucoepidermoid carcinoma”, AND “salivary gland”, AND “sclerosis*.” The search excluded “thyroid” AND/OR “breast” AND/OR “mammary” AND/OR “lung” AND/OR “pulmonary” AND/OR “pancreas*” AND/OR “skin” AND/OR “cutaneous.”

The time range was customized from 1981 to 2022. Two hundred and eighty articles were found. Duplicated articles were deleted using Mendeley software. There were 88 unique articles. After excluding non-English articles, the number of remaining articles was 53. We then screened the titles and abstracts of all of them and excluded publications not meeting the inclusion criteria. After implementing all the above criteria, the final number of remaining articles was 32 [15,16,17,18,19,20,21, 28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52] (the process is described in Supplementary File 1).