Victor Coutinho Bastos

Purpose: Polymorphous adenocarcinoma of the salivary gland is characterized by cellular uniformity associated with a variety of morphological growth patterns, a fact that makes its diagnosis challenging. Therefore, the identification of genetic alterations and signaling pathways emerges as a tool for elucidation of the pathogenesis of this tumor and accurate differential diagnosis. The aim of this study was to assess mutations in the PRKD1 gene and in protein components of the HH pathway ( SHH , IHH , SMO , and GLI ‐1) in cases of polymorphous adenocarcinoma of the salivary gland. Methods: Sanger sequencing was used to interrogate hotspot mutations in PRKD1 exon 15 and immunohistochemistry to analyze the protein expression of PRKD1 , SHH , IHH , SMO , and GLI ‐1. Results: The PRKD1 c. 2130A >C/T hotspot mutation was detected in 50% of the sequenced samples. A previously unreported variant, c. 2110C >T resulting in p. His704Tyr , was identified in one case, while 100% of the samples carried the intronic variation rs2273813, regardless of tissue invasion (perineural, lymphovascular, fat, bone, muscle, and mucous acini). Immunostaining revealed significant results for several associations between PRKD1 , IHH , SMO , and GLI ‐1. In contrast, SHH immunoexpression did not correlate with the expression of the other proteins. Conclusion: The PRKD1 E710D hotspot mutation and protein expression of PRKD1 and HH components ( SHH , IHH , SMO , and GLI ‐1) were detected in PAC regardless of tissue invasion, although HH proteins contributed to the morphogenesis of this rare oral cancer. Additionally, the positive correlation between the expression of PRKD1 and HH pathway components ( IHH , SMO , and GLI ‐1) suggests a possible interaction between these proteins, independent of the HH pathway ligand.

Understanding the early molecular events driving oral carcinogenesis is vital for diagnosing oral squamous cell carcinoma (OSCC) promptly. While metabolic differences between oral leukoplakia (OLK), OSCC, and healthy oral mucosa have been reported, the metabolic changes distinguishing malignant transformed OLKs (MT-OLK) from non-transformed OLKs (NT-OLK) remain unexplored. Here, we examine the metabolomic profiles of 5 cases of MT-OLK and 15 of NT-OLK to identify key predictive molecules using untargeted high-performance liquid chromatography-mass spectrometry. The potentially discriminant compounds were highlighted through a robust statistical analysis workflow, and the dysregulated metabolic pathways were illustrated by enrichment analysis. Seventeen molecular features, primarily lipids—including phospholipids, oxidised lipids, cholesteryl esters, and fatty acids—were identified as discriminants between MT-OLK and NT-OLK across statistical and bioinformatic approaches. Pathway enrichment analysis revealed alterations in lipid metabolism, particularly fatty acid synthesis and degradation, steroid hormone biosynthesis, and glycerophospholipid metabolism. Predictive models showed high accuracy (AUC = 0.88) in distinguishing the two groups. This study suggests that metabolomics has the potential to differentiate between MT-OLK and NT-OLK by identifying candidate biomarkers that may contribute to the understanding of malignant transformation. Validation in larger cohorts is warranted to translate these findings into clinical practice.

Background: Aspergillosis is a fungal disease caused by the inhalation of fungal spores of the genus Aspergillus spp. This fungus mainly affects the lungs but can spread and infect the maxillofacial region through the bloodstream or inoculation of the fungus after extraction or endodontic treatment, especially in the upper posterior teeth. The disease has nonspecific clinical manifestations that hinder its early diagnosis. Although the Polymerase Chain Reaction (PCR) technique holds promise as a diagnostic tool for aspergillosis, anatomopathological analysis services do not routinely adopt this method. Objectives: Therefore, the present study aimed to evaluate the applicability of PCR and standardise the techniques of preparation of biological samples for the detection of the three species: Aspergillus niger, Aspergillus fumigatus and Aspergillus flavus. Methods: Six samples of formalin-fixed, paraffin-embedded tissue (FFPE) with a histopathological diagnosis suggestive of aspergillosis were investigated using PCR. As a positive control for the PCR reaction, morphologically and genetically characterized cultures were used, with their sequences deposited at NCBI under accession codes MW837777 (A. fumigatus) and MW837779 (A. niger). The A. flavus culture used is reference RC 2053. Results: Four of the six samples evaluated were positive for Aspergillus spp., of which one was co-infected with A. fumigatus and A. flavus species, while two others were positive only for A. flavus, and one sample was positive only for A. fumigatus. Conclusions: These findings suggest that PCR can be used as an auxiliary method for diagnosing aspergillosis. However, this was a pilot study, and expansion of the sample size and the evaluation of PCR in comparison with other diagnostic tests for aspergillosis are essential to determine the accuracy of the method.

Aspergillosis is a fungal disease caused by the inhalation of fungal spores of the genus Aspergillus spp. This fungus mainly affects the lungs but can spread and infect the maxillofacial region through the bloodstream. The disease has nonspecific clinical manifestations, which hinders early diagnosis. Even though the Polymerase Chain Reaction (PCR) technique holds promising potential to aid in diagnosing aspergillosis, anatomopathological analysis services have not yet routinely adopted the method. Therefore, the present study aims to evaluate the applicability and standardize the techniques of preparation of biological samples and PCR for the detection of the species Aspergillus niger, Aspergillus fumigatus and Aspergillus flavus. Six samples of formalin-fixed, paraffin-embedded tissue (FFPE), with histopathological diagnosis suggestive of aspergillosis, were investigated by PCR. Five of the six samples evaluated were positive for the fungus Aspergillus spp, 2/5 of which were co-infected by A. fumigatus and A. flavus species, and the others were positive for A. flavus species. The findings of this study suggest that PCR may be an auxiliary method in diagnosing aspergillosis. However, the expansion of the sample size and the evaluation of PCR in comparison with other diagnostic tests for aspergillosis is essential to determine the accuracy of the method.

Key Clinical Message: Whole‐exome sequencing (WES) analysis of an expansive case florid cemento‐osseus dysplasia were reported for the first time. Also, the new potential candidate genes were reported to expand our knowledge about their molecular pathogenesis. We report a case of expansive florid cemento‐osseus dysplasia in a 32‐year‐old female patient who presented an expansive tumoral lesion in the anterior mandible. As florid cemento‐osseus dysplasia have only been molecularly investigated using targeted‐sequencing, fragments of the lesion were collected and subjected to molecular investigation using WES to assess somatic mutations as well as copy number alterations. No gains and losses of chromosomal arms or segments longer than 1 Mb were detected. Our findings revealed a pathogenic stopgain variant at the KIF5C gene, a stoploss variant at MAPK10, and missense SNV at COL6A2 at DCDC1, suggesting potential candidate genes associated with florid cemento‐osseus dysplasia.

The advances in molecular technologies have allowed a better understanding of the molecular basis of odontogenic cysts and tumours. PTCH1 mutations have been reported in a high proportion of odontogenic keratocyst. BRAF p.V600E are recurrent in ameloblastoma and KRAS p.G12V/R in adenomatoid odontogenic tumour, dysregulating the MAPK/ERK pathway. Notably, BRAF p.V600E is also detected in ameloblastic carcinoma, but at a lower frequency than in its benign counterpart ameloblastoma. Recently, adenoid ameloblastoma has been shown to be BRAF wild‐type and to harbour CTNNB1 (β‐catenin gene) mutations, further suggesting that it is not an ameloblastoma subtype. CTNNB1 mutations also occur in other ghost‐cell‐containing tumours, including calcifying odontogenic cysts, dentinogenic ghost cell tumours and odontogenic carcinoma with dentinoid, but the link between CTNNB1 mutations and ghost cell formation in these lesions remains unclear. Regarding mixed tumours, BRAF p.V600E has been reported in a subset of ameloblastic fibromas, ameloblastic‐fibrodentinomas and fibro‐odontomas, in addition to ameloblastic fibrosarcoma. Such mutation‐positivity in a subset of samples can be helpful in differentiating some of these lesions from odontoma, which is BRAF‐wild‐type. Recently, FOS rearrangements have been reported in cementoblastoma, supporting its relationship with osteoblastoma. Collectively, the identification of recurrent mutations in these aforementioned lesions has helped to clarify their molecular basis and to better understand the interrelationships between some tumours, but none of these genetic abnormalities is diagnostic. Since the functional effect of pathogenic mutations is context and tissue‐dependent, a clear role for the reported mutations in odontogenic cysts and tumours in their pathogenesis remains to be elucidated.

Previously, by employing 3D organotypic tissue culture and patient‐derived xenograft (PDX) model, oral myxoma response to a MAPK/MEK inhibitor was observed. Gross examination of the tumour fragments obtained after 55 days of PDX grafting revealed increased capsule vascularization. Microscopic analyses showed blood capillaries intermixed with myxoma cells, but the origin of these capillaries, from mice or humans, was not established. This study aimed to investigate whether the endothelial cells observed in the myxoma PDX model are derived from the mouse or from the primary human tumour. Immunohistochemistry was performed on five tumour fragments from the PDX of myxoma after 55 days of implantation in mice. Immunopositivity for antibodies against human (HLA‐ABC) and mouse (H2 Db/H2‐D1) major histocompatibility complex class I (MHCI) was assessed in the endothelial cells. The endothelial cells in the PDX fragments revealed a membrane staining for the human MHCI protein in the PDX tumour and adjacent connective tissue capsule, indicating that capillaries were derived from the human tumour fragment. Considering the probable human origin of the endothelial cells from capillary blood vessels in the myxoma PDX, we conclude that this PDX model is an interesting model to study myxoma angiogenesis.