Jonathan Lopez

Work flow analysis for the multiplexed immunofluorescence staining.

Description of the MELPREDICT cohort and design of experiments performed in the study.

Clinical features of MELPREDICT patients at baseline prior immunotherapy onset

Optimized immunofluorescence staining conditions for the multiplex immunofluorescence panel

Composition of DC aggregates and frequency in R and NR patients.

Epithelial-myoepithelial carcinoma (EMC) is a rare malignant tumor of the salivary glands, often characterized by HRAS mutations. The present study aimed to better define the molecular and transcriptomic landscape of EMC and explore its relationship with other head and neck neoplasms, particularly basal cell adenoma (BCA). We retrospectively analyzed 14 EMC cases using histology, immunohistochemistry, and whole-exome capture RNA sequencing. HRAS mutations were identified in 57.0% (8/14) of EMC, predominantly at p.Gly61Arg. Additional mutations identified included PIK3CA , STAT5B , and NOTCH1 , as well as gene fusions such as HMGA2 :: WIF1 and FBXO32 :: PLAG1 . A comparative analysis with 54 RAS -mutated head and neck tumors revealed that HRAS mutations were not exclusive to EMC, as they were also found in benign entities such as BCAs. Transcriptomic profiling found that EMC and BCA shared significant gene expression similarities, clustering closely. Differential expression analysis found upregulation of GATA3 , CHI3L1 , GRIA2 , and MME in EMC, while BCAs had enrichment of Wnt-beta-catenin signaling. In contrast, EMCs had activation of the Ras-Raf-MAPK and PI3K-Akt pathways, supported by upregulation of E2F and G2-M checkpoint targets. Immunohistochemistry confirmed GATA3 positivity in HRAS -mutated EMCs. The frequent RAS mutations and overlapping transcriptomic profiles raise important diagnostic considerations and highlight the need for integrated molecular analyses to differentiate these entities. The EMCs displayed a recurrent HRAS mutation, also observed in other head and neck tumors, and were partially clustered with the cases of BCA, suggesting potential biological similarities.

Since the histogenesis of heterologous elements within Sertoli-Leydig cell tumors (SLCTs) is poorly understood, we aimed to study the molecular relationship between Sertoli cells and the heterologous elements in 16 ovarian SLCTs. We performed a comprehensive molecular study on both SLCT and heterologous components, separately. Eleven tumors (68.7%) had one heterologous element and 5/16 (31.3%) had 2. Heterologous elements were epithelial (7/21 [81%]) (benign mucinous epithelium [9/21, 42.9%], borderline mucinous tumor [1/21, 4.8%], infiltrative mucinous adenocarcinoma [3/21, 14.3%], carcinoid tumor [3/21, 14.3%], and hepatocytes [1/21,4.8%]) or mesenchymal (4/21, 19%) (rhabdomyosarcoma [3/21,14.3%] and chondrosarcoma [1/21, 4.8%]). A DICER1 pathogenic variant was shared between SLCT and the heterologous elements in all cases with interpretable results (15/15), and other common likely-pathogenic/pathogenic variants were shared between SLCTs and heterologous components (3/16, 18.75%), favoring a clonal relationship. In contrast, the identification of distinct variants between components favored a different evolution. The molecular profile of heterologous elements differed from that of their ovarian counterparts occurring without SLCT (eg, mucinous heterologous elements were KRAS wild-type). Chromosome 8 gains, TERT and NRAS/KRAS variants, and absence of fusion transcript, were the hallmark of rhabdomyosarcoma components (3/3, 100%). The progression-free survival rate was significantly shorter for patients with TERT pathogenic variant ( P =0.0029). One patient had pleomorphic Sertoli cells associated with TP53 variants and very poor prognosis with early recurrence after complete initial surgery of a stage IA tumor. These data highlight the biological relationship between SLCTs and their heterologous elements, and the clinical usefulness of identifying pathogenic variants (ie, TERT and TP53 ), although this last point needs to be confirmed in a larger series.

Work flow analysis for the multiplexed immunofluorescence staining.

Composition of DC aggregates and frequency in R and NR patients.