Pathologica - Journal of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology

Interventional cytopathologist perspective on the Milan System Classification: a study on 929 consecutive salivary gland fine-needle aspirations with a focus on challenging diagnostic categories

Anna Maria Carillo — 2024-12-18

Background. Although the Milan System for Reporting Salivary Gland Cytopathology (MSRSGC) has improved the diagnosis and management of salivary gland lesions, determining the risk of malignancy (ROM) for AUS and SUMP categories remains challenging. We investigated the role of interventional cytopathologists in refining the differential diagnosis of these categories.

Methods. We searched for salivary gland fine-needle aspirations (FNAs) performed at our Institution since the publication of the first edition of MSRSGC. In our Institution, salivary gland FNAs are performed by interventional cytopathologists only. We checked for the availability of histopathology reports to calculate the risk of neoplasm (RON) and ROM. Sensitivity, specificity, negative predictive value, and positive predictive values of our FNAs were assessed by focusing on the contribution of the AUS and SUMP categories to our diagnostic accuracy.

Results. 929 salivary gland FNA diagnoses were retrieved. 37.02% FNAs had an available surgical follow-up. The ROM for each category was: 6% (ND); 0 (NN); 15.15% (AUS); 1.14% (NB); 24.4% (SUMP); 66.7% (SFM); and 94.74% (M). We observed a high level of concordance between our ROM data and the values proposed by the MSRSGC; higher accuracy (93.17%) and sensitivity (97%) were obtained when the AUS category was considered as a positive index for detecting salivary neoplasms; the best diagnostic accuracy (93.33%) was obtained when the SUMP category was considered as a negative index for malignancy.

Conclusion. Interventional cytopathologists play an important role in salivary gland cytopathology, as demonstrated by the overt concordance between our ROM rates and those recommended by the MSRSGC.

DNA methylation analysis from oral brushing reveals a field cancerization effect in proliferative verrucous leukoplakia

Andrea Gabusi — 2024-12-18

Objectives. The aim of the present study was to analyze the methylation status in patients who presented with an Oral Squamous Cell Carcinoma (OSCC) concomitantly with multifocal Proliferative Verrucous Leukoplakia (PVL)(PVL-OSCC).

Methods. Nine patients with OSCC and concomitant PVL lesions were selected. Two brushing samples were collected simultaneously from OSCC and PVL lesions in contralateral mucosa from each patient. 15 genes (272 CpGs) were used to compare methylation profiles of PVL-OSCC and paired OSCC. CpGs with a methylation level superimposable between PVL-OSCC and contralateral OSCC were selected for a comparative analysis between PVL-OSCC, 8 PVL patients with no history of OSCC (PVL) and 23 healthy donors. Samples were also tested using an algorithm that was recently validated for epigenetic alterations in OSCC.

Results. 220/272 CpGs islands (80%) showed a superimposable methylation level in OSCC and in PVL-OSCC. 10 genes (88 CpGs) and in particular PARP15 and ITGA4 (100% of the studied CpGs) were able to stratify PVL-OSCC from PVL and healthy donors. 3/4 (75%) PVL-OSCC patients with a “positive” algorithm score developed second neoplastic events compared to only 1/5 (20%) patients with a “negative” score.

Conclusions. The present study provides evidence that PVL shares an aberrant methylation profile with contralateral OSCC. In agreement with the theory of field cancerization, our data point towards the potential role of epigenetics in patients at risk of developing multiple neoplastic events.

A machine learning approach to predict HPV positivity of oropharyngeal squamous cell carcinoma

Silvia Varricchio — 2024-12-18

HPV status is an important prognostic factor in oropharyngeal squamous cell carcinoma (OPSCC), with HPV-positive tumors associated with better overall survival. To determine HPV status, we rely on the immunohistochemical investigation for expression of the P16^INK4a protein, which must be associated with molecular investigation for the presence of viral DNA. We aim to define a criterion based on image analysis and machine learning to predict HPV status from hematoxylin/eosin stain.

We extracted a pool of 41 morphometric and colorimetric features from each tumor cell identified from two different cohorts of tumor tissues obtained from the Cancer Genome Atlas and the archives of the Pathological Anatomy of Federico II of Naples. On this data, we built a random Forest classifier. Our model showed a 90% accuracy. We also studied the variable importance to define a criterion useful for the explainability of the model. Prediction of the molecular state of a neoplastic cell based on digitally extracted morphometric features is fascinating and promises to revolutionize histopathology. We have built a classifier capable of anticipating the result of p16-immunohistochemistry and molecular test to assess the HPV status of squamous carcinomas of the oropharynx by analyzing the hematoxylin/eosin staining.

A Digital Workflow for Automated Assessment of Tumor-Infiltrating Lymphocytes in Oral Squamous Cell Carcinoma Using QuPath and a StarDist-Based Model

Angela Crispino — 2024-12-18

The search for reliable prognostic markers in oral squamous cell carcinoma (OSCC) remains a critical need. Tumor-infiltrating lymphocytes (TILs), particularly T lymphocytes, play a pivotal role in the immune response against tumors and are strongly correlated with favorable prognoses. Computational pathology has proven highly effective for histopathological image analysis, automating tasks such as cell detection, classification, and segmentation.

In the present study, we developed a StarDist-based model to automatically detect T lymphocytes in hematoxylin and eosin (H&E)-stained whole-slide images (WSIs) of OSCC, bypassing the need for traditional immunohistochemistry (IHC). Using QuPath, we generated training datasets from annotated slides, employing IHC as the ground truth. Our model was validated on Cancer Genome Atlas-derived OSCC images, and survival analyses demonstrated that higher TIL densities correlated with improved patient outcomes.

This work introduces an efficient, AI-powered workflow for automated immune profiling in OSCC, offering a reproducible and scalable approach for diagnostic and prognostic applications.

Metformin radiosensitizes OSCC in 2D and 3D models: possible involvement of CAF-1

Mariangela Palazzo — 2024-12-18

Objective. This study investigated metformin as a sensitizer for radiotherapy in oral squamous cell carcinoma (OSCC) to reduce the radiation intensity. It evaluated the drug’s effect on Chromatin Assembly Factor-1 (CAF-1) expression, whose high levels correlate with worse prognosis of this cancer.

Methods. The effects of metformin, alone and with radiotherapy, were evaluated on CAL27 (HPV-) and SCC154 (HPV+) OSCC cells. The analyses were performed on cell monolayers by colony-forming assay, motility, and confocal microscopy. In spheroid 3D models, the sensitizing effect of metformin was assessed by measuring areas. CAF-1 expression affected by metformin was evaluated via Western blot, and its role was investigated by siRNAs.

Results. Metformin reduced the cells’ ability to form colonies, migrate and invade, and promoted the acquisition of a less aggressive phenotype by increased E-cadherin and decreased N-cadherin expressions. Moreover, metformin lowered the IC50 of radiotherapy and showed strong effects on spheroid growth. Metformin downmodulated the expression of the major subunits of CAF-1, and the knockdown of this protein by siRNAs elicited a metformin-like effect on cell aggressiveness.

Conclusions. Metformin emerged as a promising adjuvant drug in OSCC because of its effects on cell aggressiveness and radiosensitizing action. These activities could be CAF-1-mediated.

The oral microbiome and its role in oral squamous cell carcinoma: a systematic review of microbial alterations and potential biomarkers

Angela Crispino — 2024-12-18

Background. Oral squamous cell carcinoma (OSCC) is one of the most common cancers worldwide. Despite advances in diagnosis and treatment, the incidence of OSCC is increasing, and the mortality rate remains high. This systematic review aims to examine the potential association between the composition of the oral microbiota and OSCC.

Materials and methods. This study’s protocol was developed according to the PRISMA guidelines. Several search engines, including Medline-PubMed, Scopus (via Elsevier), and Google Scholar, were used to identify original studies that analyzed differences in the oral microbiome between OSCC patients and controls. Twenty-seven studies were identified that reported significant differences in microbial abundance between OSCC and controls.

Results. The systematic review highlights a complex relationship between the oral microbiome and the pathogenesis of OSCC. Significant changes in the microbial composition were identified, with a predominance of phyla such as Bacteroidetes and Fusobacteria, which are associated with inflammatory mechanisms facilitating tumor progression. A remarkable variability in microbial profiles emerged based on the different stages of the disease and the types of samples analyzed, demonstrating the complexity of the oral microbial ecosystem.

Conclusion. Although alterations in the oral cavity microbiome composition are evident in patients with OSCC, identifying a specific pattern remains challenging. However, the integration of advanced analytical techniques, such as artificial intelligence, could overcome this problem, allowing the identification of crucial biomarkers and improving the understanding of the role of the microbiome in carcinogenesis. This approach could transform microbiome analysis into a useful tool for screening and monitoring patients with OSCC.