Systemic Human ILC Precursors Provide a Substrate for Tissue ILC Differentiation.

Innate lymphoid cells (ILCs) represent innate versions of T helper and cytotoxic T cells that differentiate from committed ILC precursors (ILCPs). How ILCPs give rise to mature tissue-resident ILCs remains unclear. Here, we identify circulating and tissue ILCPs in humans that fail to express the transcription factors and cytokine outputs of mature ILCs but have these signature loci in an epigenetically poised configuration. Human ILCPs robustly generate all ILC subsets in vitro and in vivo. While human ILCPs express low levels of retinoic acid receptor (RAR)-related orphan receptor C (RORC) transcripts, these cells are found in RORC-deficient patients and retain potential for EOMES+ natural killer (NK) cells, interferon gamma-positive (IFN-γ+) ILC1s, interleukin (IL)-13+ ILC2s, and for IL-22+, but not for IL-17A+ ILC3s. Our results support a model of tissue ILC differentiation ("ILC-poiesis"), whereby diverse ILC subsets are generated in situ from systemically distributed ILCPs in response to local environmental signals.

Pulmonary pathology in vasculitis.

Pulmonary involvement is frequent in vasculitis, particularly in ANCA-associated small vessel vasculitis. Laboratory and radiological data alone are often sufficient to confirm the clinical hypothesis, but sometimes the pathologist plays a crucial role in the differential diagnosis and the patient's management. In this review, the pathologic features of pulmonary vasculitis and the pathologist's role in this field are illustrated.

Abdominal Lymphadenopathies: Lymphoma, Brucellosis or Tuberculosis? Multidisciplinary Approach-Case Report and Review of the Literature.

Abdominal pain represents a frequent symptom for referral to emergency departments and/or internal medicine outpatient setting. Similarly, fever, fatigue and weight loss are non-specific manifestations of disease. The present case describes the diagnostic process in a patient with abdominal pain and a palpable abdominal mass. Abdominal ultrasonography confirmed the presence of a mass in the mesogastrium. Computed Tomography (CT) and Magnetic Resonance Imaging (MRI) scans oriented toward calcific lymphadenopathies with increased metabolism in the positron emission tomography-computed tomography (PET-CT) scan. Laboratory examinations were inconclusive, although serology for Brucella and the Quantiferon test were positive. After multidisciplinary discussion, the patient underwent surgical excision of the abdominal mass. Histological examination excluded malignancies and oriented toward brucellosis in a patient with latent tuberculosis. The patient was treated with rifampin 600 mg qd and doxycycline 100 mg bid for 6 weeks with resolution of the symptoms. In addition, rifampin was continued for a total of 6 months in order to treat latent tuberculosis. This case underlines the need for a multidisciplinary approach in the diagnostic approach to abdominal lymphadenopathies.

Full cost of diagnostic pathology for lung carcinoma in Italy: results from four Pathology Units.

Objective: To calculate the full cost of diagnostic pathology tests for Non-Small Cell Lung Cancer (NSCLC) across four Italian Pathology Units. Methods: Pathology Units were located in private (2) and public (2) hospitals distributed across the Italian territory (North: 2; Centre: 1; South: 1). Pathologists provided via questionnaire data on tests on NSCLC samples along with the identification and quantification of the necessary healthcare resources (diagnostic technologies, laboratory instruments and personnel). Resources were valued according to hospital-specific unit, yearly and hourly costs (disposables; technologies; professional clusters). Results: The full cost per NSCLC tissue sample included histopathological immunophenotypic and required molecular analysis. Overall, it reached € 659.77 and it was mainly composed of direct costs (77.69%). The processing of a NSCLC tissue sample was labour intensive, as a relevant share of the full cost (44.98%) was actually due to personnel costs, with laboratory technicians, biologists and pathologist driving this finding (17.09%,12.43% and 10.81%, respectively). Conclusions: The results of this research can facilitate the negotiation of new dedicated tariffs for NSCLC sample processing with the national or local third party-payers.

Think "HER2" different: integrative diagnostic approaches for HER2-low breast cancer.

This work explores the complex field of HER2 testing in the HER2-low breast cancer era, with a focus on methodological aspects. We aim to propose clear positions to scientific societies, institutions, pathologists, and oncologists to guide and shape the appropriate diagnostic strategies for HER2-low breast cancer. The fundamental question at hand is whether the necessary tools to effectively translate our knowledge about HER2 into practical diagnostic schemes for the lower spectrum of expression are available. Our investigation is centered on the significance of distinguishing between an immunohistochemistry (IHC) score 0 and score 1+ in light of the clinical implications now apparent, as patients with HER2-low breast cancer become eligible for trastuzumab-deruxtecan treatment. Furthermore, we discuss the definition of HER2-low beyond its conventional boundaries and assess the reliability of established diagnostic procedures designed at a time when therapeutic perspectives were non-existent for these cases. In this regard, we examine potential complementary technologies, such as gene expression analysis and liquid biopsy. Ultimately, we consider the potential role of artificial intelligence (AI) in the field of digital pathology and its integration into HER2 testing, with a particular emphasis on its application in the context of HER2-low breast cancer.

Pattern-based Histologic Approach in Very Early Onset IBD: Main Features and Differential Diagnosis.

Very early onset inflammatory bowel disease (VEO-IBD) is a clinical umbrella classification composed of IBD-like diseases encompassing both classic IBD (Crohn's disease and ulcerative colitis) and monogenic disorder, both arising before 6 years of age. VEO-IBD patients present significant clinical differences from IBD occurring in older children and in adults, including more severe disease, often unresponsive to conventional IBD therapy and a greater proportion of cases featuring an underlying genetic alteration. Histologic findings of gastrointestinal biopsies can show an IBD-like pattern (both Crohn's disease-like and ulcerative colitis-like pattern), an apoptotic-like and enterocolitis-like pattern. Findings of specific morphologic alterations, such as villous blunting, apoptosis, dense eosinophilic infiltrates, lack of plasma cells and severe glandular atrophy, can suggest a monogenic disorder. Moreover, individuals with monogenic disorders may develop significant problems such as primary immunodeficiency, impacting treatment options. Finally, IBD histology in childhood can differ from that in older patients and adults. This complexity makes a differential diagnosis between IBD and other pediatric diseases involving the gastrointestinal tract difficult, especially considering that histologic features can be similar between different diseases. Without an appropriate diagnosis, the clinical course of VEO-IBD has greater potential for escalated treatment regimens involving extensive surgery and more intensive medical therapies rather than specific therapy directed toward the underlying defect. For these reasons, a pattern-based histologic approach correlated with clinical and laboratory findings with a multidisciplinary approach is fundamental to reach a correct diagnosis in an adequate clinical context.

B7-H3/CD276 Inhibitors: Is There Room for the Treatment of Metastatic Non-Small Cell Lung Cancer?

The striking clinical outcomes of antibody-based immunotherapy, through the inhibitors of cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) and the programmed cell death protein-1 (PD-1) and its ligand (PD-L1) axis, have driven research aimed at identifying further clinically relevant tumor antigens that can serve as targets in solid tumors. B7 homolog 3 protein (B7-H3, also known as CD276) is a member of the B7 family overexpressed in tumor tissues, including non-small cell lung cancer (NSCLC), while showing limited expression in normal tissues, becoming an attractive and promising target for cancer immunotherapy. B7-H3 expression in tumors has been demonstrated to be associated with poor prognosis. In addition to its role in immune modulation, B7-H3 also promotes pro-tumorigenic functions such as tumor migration, invasion, metastases, resistance, and metabolism. In this review, we will provide an overview of this newly characterized immune checkpoint molecule and its development in the management of metastatic NSCLC.

Extremely late-onset pulmonary metastasis from uterine PEComa.

A 79-year-old woman underwent surgical resection of a peripheral, solitary, pulmonary lesion that was diagnosed as malignant PEComa. Her clinical history was positive for uterine leiomyosarcoma, excised 20 years before. Re-evaluation of the primary uterine lesion led to the final diagnosis of lung metastasis from uterine PEComa. While long latency between primary tumour and metastasis is a known and characteristic feature of PEComas, a 20-year interval is unprecedented in the literature.

DOG-1 positive primary acinic cell carcinoma of the lung and investigation of molecular status.

Primary acinic cell carcinoma (ACC) of the lung is an extremely rare neoplasm that more often arises near to a right bronchus. It is characterized by two populations of clear and dark eosinophilic cells, arranged in a glandular acinar pattern. Mitosis are rare and tumor cells show small and eccentric nuclei. Positive stain for PAS, PAS-D, cytokeratin, A1AT and A1ACT is reported, while TTF1, p40, synaptophysin, SMA, and S100 are substantially negative. DOG-1 positive stain was observed in ACC of the salivary glands and its negativity was proposed to distinguish between primary and metastatic ACC of the lung. Here, we report the 30th case of primary ACC of the lung, describing the immunohistochemical positivity for DOG-1 and the molecular status of the neoplasm for the first time.

Immunohistochemistry with 3 different clones in anaplastic lymphoma kinase fluorescence in situ hybridization positive non-small-cell lung cancer with thymidylate synthase expression analysis: a multicentre, retrospective, Italian study.

Introduction: ALK rearrangement is the only druggable oncogenic driver detectable by immunohistochemistry (IHC) not requiring further confirmation of positivity in accessing first-line specific inhibitors. ALK-positive patients experience clinical benefit from pemetrexed-based chemotherapy possibly due to lower thymidylate synthase (TS) levels. This study assesses agreement with three different ALK IHC clones in 37 FISH-positive NSCLC. TS expression by real time (RT)-PCR was compared with ALK FISH-negative cases. Materials and methods: 37 ALK FISH-positive NSCLC cases diagnosed between 2010 and 2015 in 7 Italian centres were investigated with ICH using three different anti-ALK antibodies (ALK1, 5A4 and D5F3). Staining for ALK1 and 5A4 was graded as 0+,1+,2+, and 3+, while the scoring for D5F3 was recorded as negative or positive. Proportion agreement analysis was done using Cohen's unweighted kappa (k). TS and ß-actin expression levels were analysed by quantitative RT-PCR. Comparison between TS expression in ALK FISH-positive specimens and a control cohort of ALK FISH-negative ones was performed with the Mann-Whitney and Kruskal-Wallis tests. Results: Considering 2+ and 3+ as positive, the proportion of IHC agreement was 0.1691 (95% CI 0-0.4595) for ALK1/5A4, 0.1691 (95% CI 0-0.4595) for ALK1/D5F3, and 1 for D5F3/5A4. Considering 3+ as positive, it was 0.1543 (95% CI 0-0.4665) for ALK1/ 5A4, 0.0212 (95% CI 0-0.1736) for ALK1/D5F3, and 0.2269 (95% CI 0-0.5462) for 5A4/D5F3. Median TS expression was 6.07 (1.28-14.94) and ALK-positive cases had a significant lower TS expression than ALK-negative tumours (p = 0.002). Conclusions: IHC proved to be a reliable tool for the diagnosis of ALK-rearranged NSCLC. D5F3 and 5A4 clones have the highest percentage of agreement. TS levels are significantly lower in FISH-positive patients.

Accuracy and inter-observer agreement of the nice and kudo classifications of superficial colonic lesions: a comparative study.

PURPOSE: For superficial colonic lesions, the NICE and Kudo classifications are used in the in vivo prediction of histology and as guide to therapy. The NICE system derives information from unmagnified NBI endoscopic images. The Kudo one necessitates a magnification, but, as this tool is not commonly available, it is applied also to characterize unmagnified pictures to compare their diagnostic performances. METHODS: We conducted a prospective comparison of the NICE versus the Kudo classification for the differential diagnosis of colonic polyps taking histology as the gold standard. The inter-observer agreement for both classifications among 11 colonoscopists was also evaluated. Short unmagnified NBI videoclips of 64 colonic polyps were sent twice to the participants. In the first round, they classified the lesions according to the NICE classification; 4 months later, the same videos were assessed with the Kudo system. The diagnosis provided by the participants was grouped in non-neoplastic, non-invasive neoplasia, invasive neoplasia. RESULTS: Overall, the diagnostic accuracy was 82% (95%CI: 79-85) with the NICE system and 81% (95%CI: 78-84) with the Kudo one (ρ = 0.78). The accuracy of the NICE classification for non-neoplastic lesions was greater compared with the Kudo's (ρ = 0.03). Sensitivity sub-analyses revealed a higher ability of the NICE in distinguishing between neoplastic vs. non-neoplastic lesions (ρ = 0.01). The overall inter-rater agreement did not differ when the classifications were compared. CONCLUSION: The NICE and the Kudo classifications might be considered comparable. Our data could allow the use of the NBI Kudo classification even in those centers where magnification is not available.

A New Intraepithelial γδ T-Lymphocyte Marker for Celiac Disease Classification in Formalin-Fixed Paraffin-Embedded (FFPE) Duodenal Biopsies.

BACKGROUND: The histopathologic diagnosis of celiac disease (CD) may be challenging when the duodenal biopsies mucosal injury is limited. Intraepithelial T-lymphocytes (IELs) can be useful to characterize the degree of mucosal inflammation. A small fraction of IELs expresses the γδ T-cell receptor (named γδ-IELs), whose density, determined by flow cytometry or frozen section immunohistochemistry (IHC), is a specific marker for CD. AIM: To establish a new IHC assay for γδ-IELs applicable to formalin-fixed paraffin-embedded (FFPE) duodenal biopsies. METHODS: We analyzed γδ-IELs using IHC in 138 duodenal biopsies using a standard IHC staining protocol with a new monoclonal antibody H-41. IELs were quantitated with digital image analysis. RESULTS: Compared to those in non-celiac controls (n = 51), γδ-IEL density was significantly increased in newly diagnosed celiac disease patients (n = 22, p < 0.0001). In ROC-curve analysis, the cutoff of 6.5 γδ-IELs/100 enterocytes distinguished optimally active CD patients from non-celiac controls (sensitivity 96%, specificity 95%). γδ-IEL density in CD patients on a gluten-free diet (n = 53) were also higher than in controls (p < 0.0001), but lower than those in newly diagnosed CD (p < 0.0001). The diagnostic value of γδ-IELs outperformed that of CD3 + IELs in both patient groups. γδ-IELs were better than CD3 + IELs distinguishing between celiac disease and conditions histologically mimicking celiac disease (n = 12). CONCLUSIONS: Intraepithelial γδ T-lymphocytes can be stained and quantitated reliably in FFPE duodenal biopsies. The results showed excellent specificity and sensitivity for celiac disease. The new IHC method of detection of γδ-IELs is a promising addition to the routine histopathologic assessment methodology of celiac disease.

Intestinal adenosquamous carcinoma with a synchronous skin metastasis: a immunohistochemical and molecular analysis.

INTRODUCTION: Intestinal adenosquamous carcinoma (ASC) is a rare colorectal neoplasm frequently occurring at onset as a locally advanced disease with distant metastases. The liver is the most common site of metastasis, followed by the peritoneum and the lung. Cutaneous metastases from usual colorectal adenocarcinoma occur in about 3% of cases, both at the time of diagnosis in advanced disease and during the follow-up. To the best of our knowledge, skin metastasis from ASC has never been described, and no biological landscape of ASC has ever been investigated. METHODS: We report a case of synchronous intestinal ASC and cutaneous single facial metastasis in a 70-year-old man with morphological, immunohistochemical, and molecular analysis of primary and metastatic lesions. RESULTS: Primary and metastatic ASC showed the same morphological and immunohistochemical features. Target sequencing analysis revealed, both in primary tumor and metastasis, a pathogenic KRAS gene missense mutation c.38G > A p.(Gly13Asp) and a likely pathogenic CTNNB1 gene missense mutation c.94G > A p.(Asp32Asn). A nuclear localization of ß-catenin protein in adenocarcinomatous component of primary and metastatic lesions was observed on immunohistochemistry. CONCLUSION: We describe a case of single synchronous facial cutaneous metastasis from intestinal ASC showing KRAS and CTNN1B mutations both on primary and metastatic lesions.

Pro-Fibrotic Phenotype in a Patient with Segmental Stiff Skin Syndrome via TGF-ß Signaling Overactivation.

Transforming growth factor ß (TGF-ß) superfamily signaling pathways are ubiquitous and essential for several cellular and physiological processes. The overexpression of TGF-ß results in excessive fibrosis in multiple human disorders. Among them, stiff skin syndrome (SSS) is an ultrarare and untreatable condition characterized by the progressive thickening and hardening of the dermis, and acquired joint limitations. SSS is distinct in a widespread form, caused by recurrent germline variants of FBN1 encoding a key molecule of the TGF-ß signaling, and a segmental form with unknown molecular basis. Here, we report a 12-year-old female with segmental SSS, affecting the right upper limb with acquired thickening of the dermis evident at the magnetic resonance imaging, and progressive limitation of the elbow and shoulder. To better explore the molecular and cellular mechanisms that drive segmental SSS, several functional studies on patient's fibroblasts were employed. We hypothesized an impairment of TGF-ß signaling and, consequently, a dysregulation of the associated downstream signaling. Lesional fibroblast studies showed a higher phosphorylation level of extracellular signal-regulated kinase 1/2 (ERK1/2), increased levels of nuclear factor-kB (NFkB), and a nuclear accumulation of phosphorylated Smad2 via Western blot and microscopy analyses. Quantitative PCR expression analysis of genes encoding key extracellular matrix proteins revealed increased levels of COL1A1, COL3A1, AGT, LTBP and ITGB1, while zymography assay reported a reduced metalloproteinase 2 enzymatic activity. In vitro exposure of patient's fibroblasts to losartan led to the partial restoration of normal transforming growth factor ß (TGF-ß) marker protein levels. Taken together, these data demonstrate that in our patient, segmental SSS is characterized by the overactivation of multiple TGF-ß signaling pathways, which likely results in altered extracellular matrix composition and fibroblast homeostasis. Our results for the first time reported that aberrant TGF-ß signaling may drive the pathogenesis of segmental SSS and might open the way to novel therapeutic approaches.

Methylation Density Pattern of KEAP1 Gene in Lung Cancer Cell Lines Detected by Quantitative Methylation Specific PCR and Pyrosequencing.

BACKGROUND: The KEAP1/NRF2 pathway is the key regulator of antioxidants and cellular stress responses, and is implicated in neoplastic progression and resistance of tumors to treatment. KEAP1 silencing by promoter methylation is widely reported in solid tumors as part of the complex regulation of the KEAP1/NRF2 axis, but its prognostic role remains to be addressed in lung cancer. METHODS: We performed a detailed methylation density map of 13 CpGs located into the KEAP1 promoter region by analyzing a set of 25 cell lines from different histologies of lung cancer. The methylation status was assessed using quantitative methylation specific PCR (QMSP) and pyrosequencing, and the performance of the two assays was compared. RESULTS: Hypermethylation at the promoter region of the KEAP1 was detected in one third of cell lines and its effect on the modulation KEAP1 mRNA levels was also confirmed by in vitro 5-Azacytidine treatment on lung carcinoid, small lung cancer and adenocarcinoma cell lines. QMSP and pyrosequencing showed a high rate of concordant results, even if pyrosequencing revealed two different promoter CpGs sub-islands (P1a and P1b) with a different methylation density pattern. CONCLUSIONS: Our results confirm the effect of methylation on KEAP1 transcription control across multiple histologies of lung cancer and suggest pyrosequencing as the best approach to investigate the pattern of CpGs methylation in the promoter region of KEAP1. The validation of this approach on lung cancer patient cohorts is mandatory to clarify the prognostic value of the epigenetic deregulation of KEAP1 in lung tumors.

Effects of KEAP1 Silencing on the Regulation of NRF2 Activity in Neuroendocrine Lung Tumors.

BACKGROUND: The KEAP1/NRF2 pathway has been widely investigated in tumors since it was implicated in cancer cells survival and therapies resistance. In lung tumors the deregulation of this pathway is mainly related to point mutations of KEAP1 and NFE2L2 genes and KEAP1 promoter hypermethylation, but these two genes have been rarely investigated in low/intermediate grade neuroendocrine tumors of the lung. METHODS: The effects of KEAP1 silencing on NRF2 activity was investigated in H720 and H727 carcinoid cell lines and results were compared with those obtained by molecular profiling of KEAP1 and NFE2L2 in a collection of 47 lung carcinoids. The correlation between methylation and transcript levels was assessed by 5-aza-dC treatment. RESULTS: We demonstrated that in carcinoid cell lines, the KEAP1 silencing induces an upregulation of NRF2 and some of its targets and that there is a direct correlation between KEAP1 methylation and its mRNA levels. A KEAP1 hypermethylation and Loss of Heterozygosity at KEAP1 gene locus was also observed in nearly half of lung carcinoids. CONCLUSIONS: This is the first study that has described the effects of KEAP1 silencing on the regulation of NRF2 activity in lung carcinoids cells. The epigenetic deregulation of the KEAP1/NRF2 by a KEAP1 promoter hypermethylation system appears to be a frequent event in lung carcinoids.

Increased frequency of bronchiolar histotypes in lung carcinomas associated with idiopathic pulmonary fibrosis.

AIMS: The association between lung cancer and idiopathic pulmonary fibrosis (IPF) is well known, but the significance of this association is poorly understood. Bronchiolar honeycomb cysts have been proposed as possible precursors for the development of carcinoma, but limited evidence in support of this hypothesis is available. The aim of this study was to investigate this hypothesis analysing a series of carcinomas arising in IPF by immunohistochemistry. METHODS AND RESULTS: Thirty-three lung carcinomas arising in patients with IPF were analysed with a panel of immunohistochemical markers. The antibodies included those against pneumocyte markers [thyroid transcription factor 1 (TTF1), napsin-A, and surfactant protein A], the goblet cell marker mucin 5AC, markers of basal/squamous cell differentiation [cytokeratin (CK) 5/6 and ΔN-p63], and markers related to enteric differentiation (CDX2, mucin 2, CK20, and villin). A series of 100 consecutive lung adenocarcinomas arising in smokers without IPF were investigated as controls. All carcinomas arising in IPF patients were peripherally located on imaging analysis. The diagnoses were: eight squamous cell carcinomas, 20 adenocarcinomas, three small-cell carcinomas (including one composite small-cell carcinoma and adenocarcinoma), and two large-cell carcinomas. Among adenocarcinomas, a 'pneumocyte' profile (TTF1/napsin-A/SPA1-triple-positive) was observed in seven of 20 (35% versus 84% in non-IPF controls, P = 0.0001). The remaining 13 adenocarcinomas (65%) showed rare histotypes: four invasive mucinous adenocarcinomas (20% in IPF patients versus 1% in non-IPF controls, P = 0.002), seven tumours (35%) that were characterized by variable expression of markers of enteric differentiation, and two tumours (10%) that showed a peculiar basaloid component. CONCLUSIONS: The immunohistochemical characterization of carcinomas arising in IPF patients shows striking divergence from that in non-IPF smokers. The prevalence of rare entities showing bronchiole-related markers is in line with the hypothesis that these tumours arise from transformed small airways in honeycomb lung areas where abnormal bronchiolar proliferation takes place.