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Current pathology practice is being shaped by the increasing complexity of modern medicine, in particular of precision oncology, and major technological advances. In the "next-generation technologies era", the pathologist has become the person responsible for the integration and interpretation of morphologic and molecular information and for the delivery of critical answers to diagnostic, prognostic and predictive queries, acquiring a prominent position in the molecular tumor boards.

Gastroesophageal adenocarcinoma (GEA) is a global health issue with a high fatality-to-case ratio and a 5-year overall survival that has only slightly improved. High-throughput molecular profiling has uncovered a profound complexity and heterogeneity in GEA biology, which limits considerably the treatment advances. Liquid biopsy with circulating tumor (ct)DNA analysis could elucidate GEA molecular heterogeneity and provide diagnostic, prognostic and predictive information to guide clinical decision-making. However, only a handful of studies have shown positive results for the application of ctDNA analysis in GEA clinical management. As a result, no comprehensive information is available to date on this continuously evolving topic. Here, we discuss the current state of knowledge, along with promises and challenges related to ctDNA analysis in GEA.

Immune checkpoint inhibitors for blocking the programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) axis are now available for squamous cell carcinoma of the head and neck (HNSCC) in relapsing and/or metastatic settings. In this work, we compared the resulting combined positive score (CPS) of PD-L1 using alternative methods adopted in routine clinical practice and determined the level of diagnostic agreement and inter-observer reliability in this setting. The study applied 5 different protocols on 40 tissue microarrays from HNSCC. The error rate of the individual protocols ranged from a minimum of 7% to a maximum of 21%, the sensitivity from 79% to 96%, and the specificity from 50% to 100%. In the intermediate group (1 ≤ CPS < 20), the majority of errors consisted of an underestimation of PD-L1 expression. In strong expressors, 5 out of 14 samples (36%) were correctly evaluated by all the protocols, but no protocol was able to correctly identify all the "strong expressors". The overall inter-observer agreement in PD-L1 CPS reached 87%. The inter-observer reliability was moderate, with an ICC of 0.774 (95% CI (0.651; 0.871)). In conclusion, our study showed moderate interobserver reliability among different protocols. In order to improve the performances, adequate specific training to evaluate PD-L1 by CPS in the HNSCC setting should be coordinated.

The e-learning event “PD-L1 Key learning in HNSCC” was organized by Global Studio SRL over several days (December 2019 to May 2020) and ended with a final webinar on June 12, 2020. There were 3 learning days with overall 28 sessions comprising both traditional lectures and interactive slide viewing sessions for overall 6 h of learning activity, with involvement of a mean of six lecturers of the faculty for each event. The final meeting was supported by Zoom videoconferencing software (Zoom Video Communications, Inc., San Jose, CA, USA). After subscription, all participants received a permanent link with meeting ID and password for all online material. The faculty included Albino Eccher (University and Hospital Trust of Verona, Verona), Gabriella Fontanini (University of Pisa, Pisa), Nicola Fusco (European Institute of Oncology, Milan), Paolo Graziano (Foundation IRCCS “Casa Sollievo della Sofferenza,” San Giovanni Rotondo, Foggia), Elena Guerini Rocco (European Institute of Oncology, Milan), Maurizio Martini (Catholic University-Fondazione Policlinico Universitario “A. Gemelli” IRCCS, Rome), Patrizia Morbini (University of Pavia and IRCCS Policlinico S. Matteo, Pavia and Chief of Head and Neck Pathology Study Group for Italian Society for Anatomic Pathology and Cytology [SIAPEC]), Giancarlo Troncone (University of Naples Federico II, Naples), and Elena Vigliar (University of Naples Federico II, Naples). Attendees were 86 pathologists from all over Italy. All of the events were structured with lectures and interactive practical sessions involving case discussions.

A meeting among expert pathologists was held in 2019 in Rome to verify the results of the previous harmonization efforts on the PD-L1 immunohistochemical testing by scoring a representative series of non-small cell lung cancer (NSCLC) digital slides. The current paper shows the results of this digital experimental meeting and the expertise achieved by the community of Italian pathologists. PD-L1 protein expression was determined using tumor proportion score (TPS), i.e., the percentage of viable tumor cells showing partial or complete membrane staining at any intensity. The gold standard was defined as the final PD-L1 score formulated by a panel of seven lung committed pathologists. PD-L1 status was clustered in three categories, namely negative (TPS < 1), low (TPS 1-49%), and high (TPS ≥ 50%). In 23 cases (71.9%) PD-L1 staining was performed using the companion diagnostic 22C3 pharmDx kit on Dako Autostainer, while in nine (28.1%) cases it was performed using the SP263 Ventana kit on BenchMark platform. A complete PD-L1 scoring agreement between the panel of experts and the participants was reached in 57.1% of cases, whereas a minor disagreement in 16.1% of cases was recorded. Italian pathologists performed best in strong positive cases (i.e., tumor proportion score TPS > 50%), whereas only 10.8% of disagreement with the gold standard was observed, and 55.6% regarded a single challenging case. The worst performance was achieved in the negative cases, with 32.0% disagreement. A significant difference resulted from the analysis of the data separated by the different clones used: 22.3% and 38.1% disagreement (p = 0.01) was found in the group of cases analyzed by 22C3 and SP263 antibody clones, respectively. In conclusion, this workshop record proposed the application of a digital pathology platform to share controversial cases in educational meetings as an alternative possibility for improving the interpretation and reporting of specific histological tools. Due t

Immunotherapy has become the standard-of-care in many solid tumors. Despite the significant recent achievements in the diagnosis and treatment of cancer, several issues related to patients' selection for immunotherapy remain unsolved. Multiple lines of evidence suggest that, in this setting, the vision of a single biomarker is somewhat naïve and imprecise, given that immunotherapy does not follow the rules that we have experienced in the past for targeted therapies. On the other hand, additional immune-related biomarkers that are reliable in real-life clinical practice remain to be identified. Recently, the immune-checkpoint blockade has been approved in the US irrespective of the tumor site of origin. Further histology-agnostic approvals, coupled with with tumor-specific companion diagnostics and guidelines, are expected in this field. In addition, immune-related biomarkers can also have a significant prognostic value. In this review, we provide an overview of the role of these biomarkers and their characterization in the management of lung cancer, melanoma, colorectal cancer, gastric cancer, head and neck cancer, renal cell carcinoma, urothelial cancers, and breast cancer

An important harmonization effort was produced by the scientific community to standardize both the preanalytical and interpretative phases of programmed death-ligand 1 (PD-L1) immunohistochemical (IHC) testing in non-small-cell lung cancer (NSCLC). This analysis is crucial for the selection of patients with advanced-stage tumors eligible for treatment with pembrolizumab and potentially with other anti-PD-1/PD-L1 checkpoint inhibitors. This multicentric retrospective study evaluated the reproducibility of PD-L1 testing in the Italian scenario both for closed and open platforms. In the evaluation of the well-known gold-standard combinations (Agilent 22C3 PharmDx on Dako Autostainer versus Roche's Ventana SP263 on BenchMark), the results confirmed the literature data and showed complete overlapping between the two methods. With regard to the performances by using open platforms, the combination of 22C3 with Dako Omnis or Benchmark obtained good results basically, while the 28,8 clone seemed to be associated with worse scores.

Aims Very recent papers proposed a possible role for the expression of terminal deoxynucleotidyl transferase (TdT) in the tumourigenesis of gonadal and extragonadal germ cell-derived tumours (GCTs). Our multicentric study evaluated the magnitude of the immunoreactivity for TdT in GCTs, encompassing seminoma, dysgerminoma, mature teratoma and mixed GCTs. Methods and results The histological series was stained with both monoclonal and polyclonal antibodies, yielding a positivity of 80% of cases with well-defined nuclear reactivity. A significant difference in staining intensity between monoclonal and polyclonal antibodies was observed (p=0.005). However, exploiting western blot and more innovative proteomic approaches, no clear-cut evidence of the TdT protein was observed in the neoplastic tissues of the series. Conclusions Alternatively to the pathogenetic link between TdT expression and GCTs tumourigenesis, we hypothesised the occurrence of a spurious immunohistochemical nuclear cross-reaction, a well-known phenomenon with important implications and a possible source of diagnostic pitfalls in routine practice for pathologists.

Background: The need to integrate the classification of cancer with information on the genetic pattern has emerged in recent years for several tumors. Methods: The genomic background of a large series of 208 papillary thyroid cancers (PTC) followed at a single center was analyzed by a custom MassARRAY genotyping platform, which allows the simultaneous detection of 19 common genetic alterations, including point mutations and fusions. Results: Of the PTCs investigated, 71% were found to have pathognomonic genetic findings, with BRAF V600E and TERT promoter mutations being the most frequent monoallelic alterations (42% and 23.5%, respectively), followed by RET/PTC fusions. In 19.2% of cases, two or more point mutations were found, and the co-occurrence of a fusion with one or more point mutation(s) was also observed. Coexisting BRAF V600E and TERT promoter mutations were detected in a subgroup of aggressive PTCs (12%). A correlation between several aggressive features and mutation density was found, regardless of the type of association (i.e., only point mutations, or point mutations and fusions). Importantly, Kaplan-Meier curves demonstrated that mutation density significantly correlated with a higher risk of persistent disease. In most cases, the evaluation of the allelic frequencies normalized for the cancer cell content indicated the presence of the monoallelic mutation in virtually all tumor cells. A minority of cases was found to harbor low allelic frequencies, consistent with the presence of the mutations in a small subset of cancer cells, thus indicating tumor heterogeneity. Consistently, the presence of coexisting genetic alterations with different allelic frequencies in some tumors suggests that PTC can be formed by clones/subclones with different mutational profiles. Conclusions: A large mono-institutional series of PTCs was fully genotyped by means of a cost- and time-effective customized panel, revealing a strong impact of mutation density and genetic hete

The main aim of the study was to assess the feasibility of matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) in the pathological investigation of Medullary Thyroid Carcinoma (MTC). Formalin-fixed paraffin-embedded (FFPE) samples from seven MTC patients were analysed by MALDI-MSI in order to detect proteomic alterations within tumour lesions and to define the molecular profiles of specific findings, such as amyloid deposition and C cell hyperplasia (CCH). nLC-ESI MS/MS was employed for the identification of amyloid components and to select alternative proteomic markers of MTC pathogenesis. Results highlighted the potential of MALDI-MSI to confirm the classic immunohistochemical methods employed for the diagnosis of MTC, with good sensitivity and specificity. Intratumoural amyloid components were also detected and identified, and were characterised by calcitonin, apolipoprotein E, apolipoprotein IV, and vitronectin. The tryptic peptide profiles representative of MTC and CCH were distinctly different, with four alternative markers for MTC being detected; K1C18, and three histones (H2A, H3C, and H4). Finally, a further 115 proteins were identified through the nLC-ESI-MS/MS analysis alone, with moesin, veriscan, and lumican being selected due to their potential involvement in MTC pathogenesis. This approach represents a complimentary strategy that could be employed to detect new proteomic markers of MTC. Statement of significance: Medullary thyroid carcinoma (MTC) is a rare endocrine malignancy that originates from the parafollicular C-cells of the thyroid. The diagnosis is typically established using a combination of fine-needle aspiration biopsy (FNAB) of a suspicious nodule along with the demonstrable elevation of serum biomarkers, such as calcitonin and carcinoembryonic antigen (CEA). Unfortunately, this combination is often associated with a high degree of false-positive results and this can lead to misdiagnosis and avoidable total th

The authors present an unusual case of a patient suffering from visual deficit due to pituitary granulomatosis with polyangiitis (GPA) associated with Rathke's cleft cyst (RCC). The patient was referred to our Neurosurgery Department presenting right eye amaurosis, third cranial nerve palsy, and left temporal hemianopsia. Magnetic resonance imaging documented a sellar or suprasellar lesion with solid and cystic components. The dura mater of the skull base was also strongly enhanced. The patient underwent surgery. Histologic examination revealed RCC associated with pituitary GPA. To our knowledge, this is the first reported case of concomitant pituitary GPA and RCC. Pituitary involvement in GPA is rare, usually diagnosed in hormonal dysfunctions. The patient in case first presented optic chiasm compression, probably due to inflammation of both the pituitary gland and the previously asymptomatic RCC. We focus on the symptoms that led us to diagnose GPA pituitary involvement and on the peculiar and unusual Magnetic resonance imaging of the case presented.

Purpose: Neuroendocrine tumors of the lung (LNETs) encompass a heterogeneous group of lesions, including tumors with no or low metastatic potential, such as typical (TCs) and atypical (ACs) carcinoids, and highly aggressive neuroendocrine carcinomas. To date, only a few biomarkers with prognostic impact have been identified in LNETs. Previous experimental studies have suggested that the cytokine CXCL12 might have a role in stratifying the outcome of lung cancer as well as LNET patients. However, the reliability of immunohistochemical (IHC) tissue expression of CXCL12 in evaluating the prognosis of resected LNETs is currently not known. Methods: Here, we subjected a cohort of 112 resected LNETs specifically enriched for ACs to IHC for CXCL12 and Ki67 using routine procedures. The clinical value of CXCL12 was assessed by applying the Cox proportional-hazards model to overall and disease-free survival rates. Results: We found that CXCL12 was expressed in 8.3 to 38% of LNETs, depending on the different diagnostic categories. Upon survival analysis, when considering the whole cohort, we found that CXCL12-positive cases exhibited shorter disease-free survival rates compared to CXCL12-negative cases. Among ACs, tumors overexpressing CXCL12 showed significantly shorter disease-free survival rates. Finally, we found that the Ki67 index in ACs was higher in the CXCL12-positive cases. Conclusion: CXCL12 immunohistochemistry may serve as a potentially useful tool to better stratify LNETs, and more specifically ACs, in clinical practice.

Aims: Pulmonary adenofibromas are rare benign fibroepithelial tumours of the lung with unknown histogenesis and an indolent clinical behaviour. Their stroma resembles that of solitary fibrous tumours, whereas the glands are composed of respiratory epithelium organized in a phyllodes-like architecture. Differentiation of pulmonary adenofibromas from other more aggressive intrathoracic tumours is clinically relevant. However, their biology is unknown. Here, we sought to characterize pulmonary adenofibromas at a clinicopathological level and to define whether they could be underpinned by a highly recurrent somatic genetic alteration akin to tumours with similar morphology. Methods and results: Seven pulmonary adenofibromas were subjected to immunohistochemical analysis for thyroid transcription factor 1 (TTF1), napsin A, cytokeratin 7, E-cadherin, CD99, CD34, CD31, STAT6, oestrogen receptor (ER), progesterone receptor, androgen receptor, bcl-2, and vimentin, as well as electron microscopy and capillary sequencing on microdissected samples to evaluate the presence of NAB2–STAT6 fusion genes and MED12 exon 2 mutations in their discrete components. A control group comprising pulmonary solitary fibrous tumours, pulmonary hamartomas and breast fibroadenomas was also analysed. We confirmed that the stromal elements of pulmonary adenofibromas pertain to the fibroblastic lineage, and show ER overexpression in 71% of cases, whereas the epithelium consists of TTF1-positive, E-cadherin positive bronchiolar elements. A highly recurrent NAB2–STAT6 fusion variant (exon 4–exon 2) was detected in the stroma but not in the epithelium. No MED12 mutations were identified. Conclusions: Here, we demonstrate that pulmonary adenofibromas are neoplastic lesions harbouring the molecular hallmark of solitary fibrous tumours

Adenoid cystic carcinoma of the breast is a rare histological type of triple-negative breast cancer with an indolent clinical behavior, often driven by the MYB-NFIB fusion gene. Here we sought to define the repertoire of somatic genetic alterations in two adenoid cystic carcinomas associated with high-grade triple-negative breast cancer. The different components of each case were subjected to copy number profiling and massively parallel sequencing targeting all exons and selected regulatory and intronic regions of 488 genes. Reverse transcription PCR and fluorescence in situ hybridization were employed to investigate the presence of the MYB-NFIB translocation. The MYB-NFIB fusion gene was detected in both adenoid cystic carcinomas and their associated high-grade triple-negative breast cancer components. Although the distinct components of both cases displayed similar patterns of gene copy number alterations, massively parallel sequencing analysis revealed intratumor genetic heterogeneity. In case 1, progression from the trabecular adenoid cystic carcinoma to the high-grade triple-negative breast cancer was found to involve clonal shifts with enrichment of mutations affecting EP300, NOTCH1, ERBB2 and FGFR1 in the high-grade triple-negative breast cancer. In case 2, a clonal KMT2C mutation was present in the cribriform adenoid cystic carcinoma, solid adenoid cystic carcinoma and high-grade triple-negative breast cancer components, whereas a mutation affecting MYB was present only in the solid and high-grade triple-negative breast cancer areas and additional three mutations targeting STAG2, KDM6A and CDK12 were restricted to the high-grade triple-negative breast cancer. In conclusion, adenoid cystic carcinomas of the breast with high-grade transformation are underpinned by the MYB-NFIB fusion gene and, akin to other forms of cancer, may be constituted by a mosaic of cancer cell clones at diagnosis. The progression from adenoid cystic carcinoma to high-grade triple-nega

AIMS: Polymorphous low-grade adenocarcinoma (PLGA) is the second most common intra-oral salivary gland malignancy. The vast majority of PLGAs harbour a PRKD1 E710D hot-spot somatic mutation or somatic rearrangements of PRKD1, PRKD2 or PRKD3. Given the kinase domain homology among PRKD1, PRKD2 and PRKD3, we sought to define whether PLGAs lacking PRKD1 somatic mutations or PRKD gene family rearrangements would be driven by somatic mutations affecting the kinase domains of PRKD2 or PRKD3. METHODS AND RESULTS: DNA was extracted from eight microdissected PLGAs lacking PRKD1 somatic mutations or PRKD gene family rearrangements. Samples were thoroughly centrally reviewed, microdissected and subjected to Sanger sequencing of the kinase domains of the PRKD2 and PRKD3 genes. None of the PLGAs lacking PRKD1 somatic mutations or PRKD gene family rearrangements harboured somatic mutations in the kinase domains of the PRKD2 or PRKD3 genes. CONCLUSION: PLGAs lacking PRKD1 somatic mutations or PRKD gene family rearrangements are unlikely to harbour somatic mutations in the kinase domains of PRKD2 or PRKD3. Further studies are warranted to define the driver genetic events in this subgroup of PLGAs.