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Objective To profile clinically non-aggressive and aggressive pituitary adenomas (PAs)/pituitary neuroendocrine tumours (PitNETs) and pituitary carcinomas for somatic mutations and epigenetic alterations of genes involved in cell proliferation/differentiation, microRNAs (miRNA)/long noncoding RNA (LncRNA)-post-transcriptional regulators and therapy targets. Design Retrospective observational study. Patients and Measurements A total of 64 non-aggressive and 41 aggressive PAs/PitNETs and 6 pituitary carcinomas treated by endoscopic surgery with >= 1-year follow-up were included. Somatic mutations of 17 genes and DNA methylation of 22 genes were assessed. Ten normal pituitaries were used as control. Results We found at least one mutation in 17 tumours, including 6/64 non-aggressive, 10/41 aggressive PAs/PitNETs, and 1/6 pituitary carcinoma. AIP (N = 6) was the most frequently mutated gene, followed by NOTCH (4), and TP53 (3). Hypermethylation of PARP15, LINC00599, ZAP70 was more common in aggressive than non-aggressive PAs/PITNETs (p < .05). Lower levels of methylation of AIP, GNAS and PDCD1 were detected in aggressive PAs/PITNETs than non-aggressive ones (p < .05). For X-linked genes, males presented higher level of methylation of FLNA, UXT and MAGE family (MAGEA11, MAGEA1, MAGEC2) genes in aggressive vs. non-aggressive PAs/PITNETs (p < .05). In pituitary carcinomas, methylation of autosomal genes PARP15, LINC00599, MIR193 and ZAP70 was higher than in PAs/PITNETs, while X-linked genes methylation level was lower. Conclusions Somatic mutations and methylation levels of genes involved in cell proliferation/differentiation, miRNA/LncRNA-post-transcriptional regulators and targets of antineoplastic therapies are different in non-aggressive and in aggressive PAs/PitNETs. Methylation profile also varies according to gender. Combined genetic-epigenetic analysis, in association with clinico-radiological-pathological data, may be of help in predicting PA/PitNET behaviour.

PRAME (PReferentially expressed Antigen in MElanoma), a cancer-testis antigen expressed in normal and neoplastic tissues with several functions, proved to be a useful diagnostic tool in the differential diagnosis between benign and malignant melanocytic lesions. The current study aims to perform PRAME stain on a retrospective case series of mucosal melanocytic tumors of the head and neck region to compare 3 different scores and evaluate the most reliable one in this diagnostic set. Immunohistochemical analysis for PRAME was performed in 54 benign and malignant mucosal melanocytic tumors of the head and neck region collected from 41 patients. The best-performing cutoff of PRAME-positive cells (nuclear stain) to differentiate benign and malignant mucosal melanocytic tumors of the head and neck region is that proposed by Raghavan and colleagues (

Extranodal T-lymphoproliferative disorders or T-cell lymphomas (TLPD) are classified according to the WHO Classification (4th and upcoming 5th editions) (Swerdlow et al., IARC Press 1; Alaggio et al., Leukemia 36(7):1720-1748, 2) and to the International Consensus Classification Update (Campo et al., Blood 140(11):1229-1253, 3) upon several morphologic, phenotypic, and genetic features. None of those at present included has been characterized by primary pulmonary onset. We herein present two such cases which, to the best of our knowledge, have not been previously reported and that might represent another variant of T-cell proliferation at mucosal sites. The two cases share similar histological and phenotypic features, suggesting an origin from CD4 + effector memory T cells with the expression of a CD279/PD-1 antigen. They are both monoclonal, harbor few mutations, and show no disease progression outside the lung. They only differ concerning the local extension of the process and clinical setting. The two cases are examples of so far unreported primary pulmonary TLDP, with limited stage and low proliferative index. A possible relationship with a local yet unknown inflammatory trigger that might have favored the development of the T-cell clone cannot be ruled out.

In recent years, several studies investigated the complex process called "reprogramming" of seminoma (S) cells. The accepted pathogenetic model is a complex network including SOX2, SOX17, OCT3/4 and PRAME, which modulates the epigenetic transcription of numerous downstream genes and drives a divergent gene expression profile resulting in the transition from pure S (P-S) to S component (S-C) of mixed germ cell tumors of the testis (M-GCTT), and finally to embryonal carcinoma (EC). Herein, we tested a large cohort of GCTT with SOX2 and PRAME to evaluate their expression in the evolutionary steps of GCTT and verify if the modulation in the expression of these two molecules could be relevant for the fate of GCTT.We tested 43, 19 and 17 consecutive and retrospectively enrolled cases of GCTT, germ cell neoplasia in situ (GCNIS) and uninvolved background testes (UBT), respectively. SOX2 and PRAME expressions have been evaluated with H-score and compared by adopting the appropriate statistic tests (Student's t-test and Mann-Whitney U test).We found that SOX2 was more expressed by nonseminomatous-GCTT (NS-GCTT) (p 0.001) and EC (p 0.001) rather than S; by contrast, PRAME showed an opposite expression profile being expressed by S but not by NS-GCTT (p 0.001) and EC (p 0.001). S-C showed different expressions of SOX2 and PRAME compared to both P-S (p = 0.002 and0.001, respectively) and EC (p 0.001 and 0.042, respectively), with intermediate values between these latter two categories. GCNIS and UBT showed no expression of SOX2 (scattered positive Leydig cells) but high H-score levels of PRAME.SOX2 and PRAME are differentially expressed and specularly modulated during the "reprogramming" of S cells [P-S (high levels of PRAME, no expression/low levels of SOX2) → S-C (intermediate levels of PRAME, intermediate levels of SOX2) → EC (no expression/low levels of PRAME, high levels of SOX2)], therefore supporting a complex pathogenetic model where the interactions between these two molecules are crucial in determining the fate of GCTT.

Background: Lymphovascular invasion (LVI) is considered an unfavorable prognostic factor in cutaneous melanoma (CM). However, its detection by hematoxylin and eosin (H&E) is challenging, with discordant data about its association with clinical-pathological features and no previous studies investigating the inter- (IrOA) and intra-observer (IaOA) agreement. Herein, we tested H&E and double staining (DS) for CD34/SOX10 to detect the LVI in a cohort of 92 CMs, evaluating the IrOA, the IaOA, and the association with the other clinical-pathological features. Methods: Five authors independently evaluated 92 consecutive and retrospectively enrolled cases of CMs. We assessed the IrOA (Fleiss's Kappa/FK and intraclass correlation coefficient/ICC) and the IaOA (Cohen's Kappa/CK) with both H&E and CD34/SOX10. Furthermore, we compared the LVI assessment with the two stains and analyzed the association with other clinical-pathological features [χ2 tests for dichotomous and categorical data; Student t-test (normal distribution) and Mann-Whitney U-test (non-normal distribution) for continuous data]. Results: The IrOA was almost identical with H&E (FK=0.446; ICC=0.805) and CD34/SOX10 (FK=0.454; ICC=0.810); by contrast, the IaOA was higher with H&E for one pathologist (CK: 0.809) and with CD34/SOX10 for the other one (CK: 0.563). Applying previously defined criteria, LVI was detected in 10 (9.2%) and 11 (10.1%) cases with H&E and CD34/SOX10, respectively (p = 1.000). Both H&E and CD34/SOX10 were significantly associated with vertical growth phase (H&E, p: 0.014; CD34/SOX10, p: 0.010), mitosis ≥ 1/mm2 (H&E, p: 0.000; CD34/SOX10, p: 0.004), pT (H&E, p: 0.000; CD34/SOX10, p: 0.001), Breslow thickness (H&E, p: 0.000; CD34/SOX10, p: 0.001), and lymph node and/or distant metastasis (H&E, p: 0.005; CD34/SOX10, p: 0.000); only H&E was associated with ulceration (p: 0.002) and distant metastasis (p: 0.000), conversely, only CD34/SOX10 was associated with lymph node metastasis (p: 0.003). Conclusions: CD34/SOX10 does not improve the IrOA and the IaOA of the LVI assessment in CM; furthermore, H&E and CD34/SOX10 show a similar profile of association with the other unfavorable clinical-pathological features of CM. As result, CD34/SOX10 could be a redundant diagnostic tool if applied for the prognostic characterization of not-selected CM in a routine diagnostic scenario.

Background: The diagnosis of nodal nevi (NN) is challenging as they mimic melanoma metastases (MM), with a detection rate mostly ranging between 1% and 11% in sentinel lymph node biopsy (SLNB). Herein, we assessed the incidence of NN and the association with the clinical-pathological features of primary melanoma, adopting the updated European Organisation for Research and Treatment of Cancer (EORTC) protocol for SLNB. Methods: All cases of paired melanoma and SLNB were retrospectively evaluated (April 2019-May 2020). Appropriate statistical tests were adopted, with significant variables included in the logistic regression model. Results: 81 patients and a total of 186 lymph nodes (LNs) were included. Eleven patients had only NN and 4 had both NN and MM (18.5%); 29 LNs (15.6%) showed at least one NN and 12 (6.5%) showed more than one NN (a total amount of 43 NN was detected). All NN and none MM stained for p16. NN were associated with age < 60 years (p: 0.042), no ulceration (p: 0.025) and nevus-associated melanoma (NAM) (p: 0.018), with this latter being the only predictor at the logistic regression model (p: 0.022). Conclusions: The updated EORTC protocol shows a high number of NN and highlights a strong association with NAM.

Background: The PD-L1 assessment is mandatory for the selection of patients affected by advanced non-small-cell lung cancer (NSCLC) who can benefit from the PD-1/PD-L1 checkpoint inhibitors therapy. Previous studies tested PD-L1 on cytological smears to evaluate this sample as an alternative to formalin-fixed paraffin-embedded (FFPE) ones, but several critical issues needed to be clarified. Aim: We evaluated the cyto-histological agreement (CHA) and the PD-L1 interobserver agreement (IrOA) among three different pathologists (Path1, Path2, Path3) on 160 paired cytological smears and histological samples of advanced NSCLC. Results: With the cut-off of < 50%/≥ 50%, CHA resulted good for Path1 (Cohen's k: 0.702) and Path3 (Cohen's k: 0.731), moderate for Path2 (Cohen's k: 0.576) adopting the same cut-off, the IrOA was moderate (ICC 0.72 [95% CI: 0.63–0.78]) for smears and good for histological samples (ICC 0.85 [95% CI: 0.80–0.85]). Conclusion: With a cut-off system of < 50%/≥ 50%, PD-L1 assessment shows moderate to good CHA and exhibited moderate IrOA on smears and good IrOA on FFPE. As result, PD-L1 assessment should be improved on cytological smears as well as could be a suitable alternative for patients without FFPE samples and not eligible for pembrolizumab, adopting a cut-off of < 50%/≥ 50%; presumably, an appropriate pathologist training could further improve the reproducibility.

Cutaneous melanoma (cM) is the deadliest of all primary skin cancers. Its prognosis is strongly influenced by the stage at diagnosis, with early stages having a good prognosis and being potentially treatable with surgery alone; advanced stages display a much worse prognosis, with a high rate of recurrence and metastasis. For this reason, the accurate and early diagnosis of cM is crucial—misdiagnosis may have extremely dangerous consequences for the patient and drastically reduce their chances of survival. Although the histological exam remains the “gold standard” for the diagnosis of cM, a continuously increasing number of immunohistochemical markers that could help in diagnosis, prognostic characterization, and appropriate therapeutical choices are identified every day, with some of them becoming part of routine practice. This review aims to discuss and summarize all the data related to the immunohistochemical analyses that are potentially useful for the diagnosis of cM, thus rendering it easier to appropriately applicate to routine practice. We will discuss these topics, as well as the role of these molecules in the biology of cM and potential impact on diagnosis and treatment, integrating the literature data with the experience of our surgical pathology department.

The recently described SWI/SNF complex-deficient sinonasal carcinoma (SMARCB1SMARCA4) may exhibit a yolk sac-like morphology. Tumors with similar features (yolk sac-like histology combined with the immunohistochemical loss of SMARCB1/INI1 and/or SMARCA4/BRG1) have also been described in other sites, such as the female genital tract. In this study, we immunohistochemically assessed SMARCB1/INI1 and SMARCA4/BRG1 expression to evaluate if these proteins could be involved in the pathogenesis of testicular yolk sac tumors of postpubertal type (YSTpt). Specifically, we analyzed a retrospective case series comprising pure YSTpt and mixed germ cell tumors of the testis (GCTT) with YSTpt components. In the present study, no testicular YSTpt showed loss of SMARCB1/INI1 (0/24, 0%) or SMARCA4/BRG1 (0/24, 0%). However, testicular choriocarcinoma (CHC) and isolated syncytiotrophoblast cells (iSTCs) demonstrated abnormal staining patterns for SMARCA4/BRG1 [CHC: 4/4 (100%); iSTCs: 12/12 (100%), respectively], including focal or diffuse loss of expression in a subset of cases. The results of our study suggest that functional loss of SMARCA4/BRG1 represents a recurrent event that may be relevant for the pathogenesis of a subset of testicular CHC.

Background: The differential diagnosis between flat urothelial lesions [reactive urothelial atypia (RUA), atypia of unknown significance (AUS), urothelial dysplasia (UD) and carcinoma in situ (CIS)] has relevant prognostic and therapeutic implications. This crucial distinction could be very challenging but it is currently performed on hematoxylin and eosin (H&E) slides, with a great amount of partially discordant and/or not conclusive findings of the potential adjunctive role of immunohistochemistry. Herein, we tested double staining (DS) for p53/CK20 to verify if p53(+) cells, CK20(+) cells and double-positive cells (DPCs) are differentially expressed among these lesions and if p53/CK20 could be a useful tool in this diagnostic setting. Methods: We tested 50, 9, 36 and 29 consecutive and retrospectively enrolled cases of RUA, AUS, UD and CIS, respectively. p53(+) cells, CK20(+) cells and DPCs were evaluated and compared by adopting the appropriate statistic tests (Mann-Whitney U and Kruskal-Wallis tests). Results: We found that p53(+) cells (p = 0.000), CK20(+) cells (p = 0.000) and DPCs (p = 0.000) showed statistically significant differences among the different flat urothelial lesions. Besides, when dichotomized, both CIS and RUA are easily differentiable from their histological mimickers adopting all these markers; by contrast, AUS and UD did not reach statistically significant differences able to differentiate them from each other [p53(+) cells, p = 0.123; CK20(+) cells, p = 0.567; DPCs, p = 0.409], except if compared to CIS [AUS VS CIS: p53(+) cells, p = 0.013; CK20(+) cells, p = 0.000; DPCs, p = 0.000; UD vs CIS: p53(+) cells, p = 0.000; CK20(+) cells, p = 0.000; DPCs, p = 0.000]. Conclusions: p53(+) cells, CK20(+) cells and DPCs are differently expressed by flat urothelial lesions and p53/CK20 could be a time- and money-saving tool for the appropriate management of these lesions if applied to a routine scenario.