Ambrogio Fassina

Introduction: High inter- and intraobserver variability is acknowledged in negative for intraepithelial lesion or malignancy (NILM), atypical squamous cells of undetermined significance (ASC-US), and low-grade squamous intraepithelial lesion (LSIL) diagnostic categories in cervical cytology. As digital cytology has emerged, whole-slide images (WSIs) have been compared to conventional light microscopy (LM) to evaluate its feasibility in routine practice. Methods: Six cytopathologists evaluated 20 liquid-based cervical samples diagnosed as ASC-US, LSIL, or NILM with known hrHPV status, both by conventional LM and WSI, equalling 240 evaluations. Interobserver agreement was measured using Fleiss’s kappa, Kendall´s coefficient and interclass correlation (ICC). Results: Most of the samples (93.75%) were assigned to the same original range of categories: NILM 5 (25%), ASC-US 10 (50%), and LSIL 5 (25%). WSI versus LM interobserver agreement varied with Fleiss’s kappa (0.324 versus 0.319), Kendall’s W (0.670 versus 0.572), and ICC (0.585 versus 0.414). Interobserver agreement for the ASC-US category was higher in Kendall’s W (0.435 for WSI versus 0.329 for LM) and in ICC (0.394 versus 0.118). Fleiss’s kappa showed lower interobserver agreement for ASC-US in WSI than LM (0.000 versus 0.039). Conclusion: This study illustrated that despite overall better agreement in WSI, ASC-US results were less favourable in WSI than LM, according to Fleiss’s kappa test, but not other tests.

Background: High‐risk human papilloma virus (HR HPV) testing and liquid‐based cytology are used for primary cervical screening. Digital cytology, based on whole‐slide scanned samples, is a promising technique for teaching and diagnostic purposes. The aim of our study was to evaluate the interobserver and intraobserver variation in low‐grade squamous lesions, HR HPV status bias, and the use of whole‐slide scanned digital cervical cytology slides.Methods: Fifteen expert cytopathologists evaluated 71 digitalized ThinPrep slides (31 atypical squamous cells of undetermined significance [ASC‐US], 21 negative for intraepithelial lesion or malignancy, and 19 low‐grade squamous intraepithelial lesion cases). HR HPV data were accessible only in the second round.Results: In interobserver analysis, Kendall’s coefficient of concordance was 0.52 in the first round and 0.58 in the second round. Fleiss’ kappa values were 0.29 in the first round and 0.31 in the second round. In the ASC‐US category, Fleiss kappa increased from 0.19 to 0.22 in the second round and the increase was even higher expressed by Kendall’s coefficient: from 0.42 to 0.52. In intraobserver analysis, personal scores were higher in the second round.Conclusions: The interobserver and intraobserver variability in low‐grade squamous lesions was within fair agreement values in the present study, in line with previous works. The comparison of two rounds showed that expert cytopathologists are generally unbiased by the knowledge of HR HPV data, but that being informed of the HR HPV status leads to a better agreement. Stain quality and back discomfort were highlighted as factors affecting digital cytopathology use.

Background: Hepatocellular carcinoma (HCC) is a highly lethal cancer and the second leading cause of cancer-related deaths worldwide. As demonstrated in other solid neoplasms and HCC, infiltrating CD8⁺ T cells seem to be related to a better prognosis, but the mechanisms affecting the immune landscape in HCC are still mostly unknown. Necroptosis is a programmed, caspase-independent cell death that, unlike apoptosis, evokes immune response by releasing damage-associated molecular factors. However, in HCC, the relationship between the necroptotic machinery and the tumor-infiltrating lymphocytes has not been fully investigated so far. Methods: We investigated the association between the main necroptosis-related genes, that is, RIPK1, RIPK3, MLKL-p, and CD3⁺/CD8⁺ tumor-infiltrating T cell by RNA-seq data analysis in 371 patients with primary HCC from The Cancer Genome Atlas and then by immunohistochemistry in two independent cohorts of HCC patients from Italy (82) and Japan (86). Results: Our findings highlighted the immunogenetic role of necroptosis and its potential prognostic role in HCC: RIPK1, RIPK3 and MLKL-p were found significantly associated with intratumoral CD3⁺ and CD8⁺ T cells. In addition, multivariate survival analysis showed that the expression of RIPK1, RIPK3 and MLKL-p was associated with better overall survival in the two independent cohorts. Conclusions: Our results confirmed the immunogenetic properties of necroptosis (NCP) in human HCC, showing that tumor-infiltrating lymphocytes (TILs) and, specifically, CD8+ T cells accumulate in tumors with higher expression of the necroptosis-related genes. These results suggest the importance of further studies to better assess the specific composition, as well as the functional features of the immune environment associated with a necroptotic signature in order to explore new possible diagnostic and immunotherapeutic scenarios.

Malignant melanoma (MM) is the most lethal skin cancer. AXL is a tyrosine kinase receptor involved in several oncogenic processes and might play a role in blocking necroptosis (a regulated cell death mechanism) in MM through the downregulation of the necroptotic-related driver RIPK3. The aim of this study was to evaluate the clinical impact of the expression of AXL and RIPK3 in 108 primary cutaneous MMs. Association between AXL and RIPK3 immunoreactivity and clinical–pathological variables, sentinel lymph node status, and tumor-infiltrating lymphocytes (TILs) was assessed. Immunoreaction in tumor cells was detected in 30 cases (28%; range, 5%–80%) and in 17 cases (16%; range, 5%–50%) for AXL and RIPK3, respectively. Metastases in the sentinel lymph nodes were detected in 14 out of 61 patients, and these were associated with AXL-positive immunoreactivity in the primary tumor (p < 0.0001). No association between AXL and TILs was found. RIPK3 immunoreactivity was not associated with any variables. A final logistic regression analysis showed Breslow and AXL-positive immunoreactivity as the stronger predictor for positive sentinel node status [area under the receiver operating characteristic curve (AUC) of 0.96]. AXL could be a potential new biomarker for MM risk assessment, and it deserves to be further investigated in larger studies.

Lung neuroendocrine neoplasms (lung NENs) are categorised by morphology, defining a classification sometimes unable to reflect ultimate clinical outcome. Subjectivity and poor reproducibility characterise diagnosis and prognosis assessment of all NENs. Here, we propose a machine learning framework for tumour prognosis assessment based on a quantitative, automated and repeatable evaluation of the spatial distribution of cells immunohistochemically positive for the proliferation marker Ki-67, performed on the entire extent of high-resolution whole slide images. Combining features from the fields of graph theory, fractality analysis, stochastic geometry and information theory, we describe the topology of replicating cells and predict prognosis in a histology-independent way. We demonstrate how our approach outperforms the well-recognised prognostic role of Ki-67 Labelling Index on a multi-centre dataset comprising the most controversial lung NENs. Moreover, we show that our system identifies arrangement patterns in the cells positive for Ki-67 that appear independently of tumour subtyping. Strikingly, the subset of these features whose presence is also independent of the value of the Labelling Index and the density of Ki-67-positive cells prove to be especially relevant in discerning prognostic classes. These findings disclose a possible path for the future of grading and classification of NENs.

Chordoma, a neoplasm of notochordal differentiation, is a rare subtype of bone sarcoma typically occurring along the axial skeleton. The Authors report the case of a 63-yrs M with a history of progressive nasal obstruction.FNA demonstrated a characteristic cytological pattern for this peculiar entity.

The mechanism of epithelial–mesenchymal transition (EMT) is fundamental for carcinogenesis, tumor progression, cancer cell invasion, metastasis, recurrence, and therapy resistance, comprising important events, such as cellular junction degradation, downregulation of epithelial phenotype markers, overexpression of mesenchymal markers, and increase in cellular motility. The same factors that drive epithelial cells toward a mesenchymal phenotype may also drive endothelial cells toward a proangiogenic phenotype. The aim of this exploratory study was to investigate a potential interplay between EMT and angiogenesis (quantified through CD105 expression) in laryngeal carcinoma (LSCC). CD105-assessed microvessel density (MVD) and EMT markers (E-cadherin, N-cadherin, Snail, Slug, Zeb1, and Zeb2) were assessed on 37 consecutive LSCC cases. The univariate Cox regression model identified pN+ status (p = 0.0343) and Slug expression (p = 0.0268) as predictive of disease-free survival (DFS). A trend toward significance emerged for CD105-assessed MVD (p = 0.0869) and N-cadherin expression (p = 0.0911). In the multivariate Cox model, pN-status, Slug, and N-cadherin expressions retained their significant values in predicting DFS (p = 0.0346, p = 0.0430, and p = 0.0214, respectively). Our data support the hypothesis of a mutual concurrence of EMT and angiogenesis in driving LSCC cells toward an aggressive phenotype. To better characterize the predictive performance of prognostic models based on EMT and angiogenesis, further large-scale prospective studies are required.

The spontaneous adrenal hematoma is a rare event. An 83-year-old male patient presented a 26-cm asymptomatic retroperitoneal mass of doubtful renal–adrenal origin. He had been evaluated 10 years before for an adrenal incidentaloma of 2.3 cm and had refused surgery when it had reached 7 cm. Later, the mass enlarged to 26 cm and was surgically removed through an open anterior approach. The histopathology showed a solid 4 kg mass of fibrinoid–hemorrhagic material, partially necrotic, mixed with adrenal tissue, with a well-vascularized capsule. No relapse is present at 6-month follow-up. This is the largest case described of spontaneous intra-adrenal hematoma in a case with previous non-secreting adrenal adenoma. The hematoma (a 4 kg mass) developed 10 years after the first diagnosis and exposed the patient to potential damage of the surrounding organs and to high-risk abdominal surgery. Long-term follow-up of non-secreting adrenal adenomas should be recommended.

Breast cancer–associated protein 1 (BAP1) gene is a broad-spectrum tumor suppressor. Indeed, its loss of expression, due to biallelic inactivating mutations or deletions, has been described in several types of tumors including melanoma, malignant mesothelioma, renal cell carcinoma, and others. There are so far only two reports of BAP1-mutated paraganglioma, suggesting the possible involvement of this gene in paraganglioma (PGL) and pheochromocytoma (PCC) pathogenesis. We assessed BAP1 expression by immunohistochemistry (IHC) in a cohort of 56 PCC/PGL patients (and corresponding metastases, when available). Confirmatory Sanger sequencing (exons 1–17) of BAP1 has been performed in those samples which resulted negative by IHC. BAP1 nuclear expression was lost in 2/22 (9.1%) PGLs and in 12/34 (35.3%) PCCs, five of which harboring a germline mutation predisposing the development of such tumors (MENIN, MAX, SDHB, SDHD, and RET gene). Confirmatory Sanger sequencing revealed the wild-type BAP1 status of all the analyzed samples. No heterogeneity between primary and metastatic tissue was observed. This study documents that the loss of BAP1 nuclear expression is quite a frequent finding in PCC/PGL, suggesting a possible role of BAP1 in the pathogenesis of these tumors. Gene mutations do not seem to be involved in this loss of expression, at least in most cases. Other genetic and epigenetic mechanisms need to be further investigated.