COVID-19. Biology, pathophysiology, and immunology: a pathologist view.

Even if the SARS-CoV-2 pandemic has been declared over, several risks and clinical problems remain to be faced, including long-COVID sequelae and possible outbreaks of pathogenic variants. Intense research on COVID-19 has provided in these few years a striking amount of data covering different fields and disciplines, which can help to provide a knowledge shield against new potential infective spreads, and may also potentially be applied to other fields of medicine, including oncology and neurology. Nevertheless, areas of uncertainty still remain regarding the pathogenic mechanisms that subtend the multifaceted manifestations of the disease. To better clarify the pathogenesis of the disease, a systematic multidisciplinary evaluation of the many mechanisms involved in COVID-19 is mandatory, including clinical, physiological, radiological, immunological and pathological studies. In COVID-19 syndrome the pathological studies have been mainly performed on autopsy cases, and only a few studies are available on biopsies. Nevertheless, these studies have provided relevant information that can substantially contribute to decipher the complex scenario characterizing the different forms of COVID-19 and long-COVID-19. In this review the data provided by pathological investigations are recapitulated and discussed, in the light of different hypothesis and data provided by clinical, physiological and immunological data.

Clinical, radiological and pathological findings in patients with persistent lung disease following SARS-CoV-2 infection.

Some patients experience pulmonary sequelae after SARS-CoV-2 infection, ranging from self-limited abnormalities to major lung diseases. Morphological analysis of lung tissue may help our understanding of pathogenic mechanisms and help to provide consistent personalised management. The aim of this study was to ascertain morphological and immunomolecular features of lung tissue. Transbronchial lung cryobiopsy was carried out in patients with persistent symptoms and computed tomography suggestive of residual lung disease after recovery from SARS-CoV-2 infection. 164 patients were referred for suspected pulmonary sequelae after COVID-19; 10 patients with >5% parenchymal lung disease underwent lung biopsy. The histological pattern of lung disease was not homogeneous and three different case clusters could be identified, which was mirrored by their clinical and radiological features. Cluster 1 ("chronic fibrosing") was characterised by post-infection progression of pre-existing interstitial pneumonias. Cluster 2 ("acute/subacute injury") was characterised by different types and grades of lung injury, ranging from organising pneumonia and fibrosing nonspecific interstitial pneumonia to diffuse alveolar damage. Cluster 3 ("vascular changes") was characterised by diffuse vascular increase, dilatation and distortion (capillaries and venules) within otherwise normal parenchyma. Clusters 2 and 3 had immunophenotypical changes similar to those observed in early/mild COVID-19 pneumonias (abnormal expression of STAT3 in hyperplastic pneumocytes and PD-L1, IDO and STAT3 in endothelial cells). This is the first study correlating histological/immunohistochemical patterns with clinical and radiological pictures of patients with post-COVID lung disease. Different phenotypes with potentially different underlying pathogenic mechanisms have been identified.

The pathogenic role of epithelial and endothelial cells in early-phase COVID-19 pneumonia: victims and partners in crime.

Current understanding of the complex pathogenesis of COVID-19 interstitial pneumonia pathogenesis in the light of biopsies carried out in early/moderate phase and histology data obtained at postmortem analysis is discussed. In autopsies the most observed pattern is diffuse alveolar damage with alveolar-epithelial type-II cell hyperplasia, hyaline membranes, and frequent thromboembolic disease. However, these observations cannot explain some clinical, radiological and physiopathological features observed in SARS-CoV-2 interstitial pneumonia, including the occurrence of vascular enlargement on CT and preserved lung compliance in subjects even presenting with or developing respiratory failure. Histological investigation on early-phase pneumonia on perioperative samples and lung biopsies revealed peculiar morphological and morpho-phenotypical changes including hyper-expression of phosphorylated STAT3 and immune checkpoint molecules (PD-L1 and IDO) in alveolar-epithelial and endothelial cells. These features might explain in part these discrepancies.

Covid-19 Interstitial Pneumonia: Histological and Immunohistochemical Features on Cryobiopsies.

BACKGROUND: The pathogenetic steps leading to Covid-19 interstitial pneumonia remain to be clarified. Most postmortem studies to date reveal diffuse alveolar damage as the most relevant histologic pattern. Antemortem lung biopsy may however provide more precise data regarding the earlier stages of the disease, providing a basis for novel treatment approaches. OBJECTIVES: To ascertain the morphological and immunohistochemical features of lung samples obtained in patients with moderate Covid-19 pneumonia. METHODS: Transbronchial lung cryobiopsy was carried out in 12 Covid-19 patients within 20 days of symptom onset. RESULTS: Histopathologic changes included spots of patchy acute lung injury with alveolar type II cell hyperplasia, with no evidence of hyaline membranes. Strong nuclear expression of phosphorylated STAT3 was observed in >50% of AECII. Interalveolar capillaries showed enlarged lumen and were in part arranged in superposed rows. Pulmonary venules were characterized by luminal enlargement, thickened walls, and perivascular CD4+ T-cell infiltration. A strong nuclear expression of phosphorylated STAT3, associated with PD-L1 and IDO expression, was observed in endothelial cells of venules and interstitial capillaries. Alveolar spaces macrophages exhibited a peculiar phenotype (CD68, CD11c, CD14, CD205, CD206, CD123/IL3AR, and PD-L1). CONCLUSIONS: Morphologically distinct features were identified in early stages of Covid-19 pneumonia, with epithelial and endothelial cell abnormalities different from either classical interstitial lung diseases or diffuse alveolar damage. Alveolar type II cell hyperplasia was a prominent event in the majority of cases. Inflammatory cells expressed peculiar phenotypes. No evidence of hyaline membranes and endothelial changes characterized by IDO expression might in part explain the compliance and the characteristic pulmonary vasoplegia observed in less-advanced Covid-19 pneumonia.

CAL2 monoclonal antibody is a rapid and sensitive assay for the detection of calreticulin mutations in essential thrombocythemia patients.

Calreticulin (CALR) mutations are detected in the majority of JAK2 wild type patients with essential thrombocythemia (ET). Unlike JAK2V617F and MPL point mutations, CALR mutations are highly heterogeneous, with several types of indels being reported so far. CAL2 is a monoclonal antibody specifically recognizing the C-neoterminal peptide derived from all the frameshift mutations of CALR. We retrospectively analysed 172 ET patients diagnosed at our Institution from 1980 to 2015. In JAK2V617F- and MPLW515K/L-wild type patients CALR mutations were searched on peripheral blood and CAL2 immunostaining was performed on bone marrow. In addition, bone marrow biopsies were histologically reviewed for megakaryocytic features. Thirty-one patients (18%) were CALR-mutated. Concordance between molecular and immunohistological detection of CALR mutations was near complete, albeit a single patient was found to be positive by molecular tests only. Two patterns were defined in CAL2-positive bone marrow samples, characterized by staining of almost only megakaryocytes (pattern A: 41%) or staining of megakaryocytes and ≥ 2% small non megakaryocytic elements (pattern B: 59%), at least partially being myeloid precursors. Pattern B biopsies had higher cellularity and number of megakaryocytes compared to pattern A samples. In this series, CAL2 allowed rapid and cost-efficient identification of CALR-mutated ET patients. The biological significance of different staining pattern should be confirmed in wider and independent series.

Lung neuroendocrine tumours: deep sequencing of the four World Health Organization histotypes reveals chromatin-remodelling genes as major players and a prognostic role for TERT, RB1, MEN1 and KMT2D.

Next-generation sequencing (NGS) was applied to 148 lung neuroendocrine tumours (LNETs) comprising the four World Health Organization classification categories: 53 typical carcinoid (TCs), 35 atypical carcinoid (ACs), 27 large-cell neuroendocrine carcinomas, and 33 small-cell lung carcinomas. A discovery screen was conducted on 46 samples by the use of whole-exome sequencing and high-coverage targeted sequencing of 418 genes. Eighty-eight recurrently mutated genes from both the discovery screen and current literature were verified in the 46 cases of the discovery screen, and validated on additional 102 LNETs by targeted NGS; their prevalence was then evaluated on the whole series. Thirteen of these 88 genes were also evaluated for copy number alterations (CNAs). Carcinoids and carcinomas shared most of the altered genes but with different prevalence rates. When mutations and copy number changes were combined, MEN1 alterations were almost exclusive to carcinoids, whereas alterations of TP53 and RB1 cell cycle regulation genes and PI3K/AKT/mTOR pathway genes were significantly enriched in carcinomas. Conversely, mutations in chromatin-remodelling genes, including those encoding histone modifiers and members of SWI-SNF complexes, were found at similar rates in carcinoids (45.5%) and carcinomas (55.0%), suggesting a major role in LNET pathogenesis. One AC and one TC showed a hypermutated profile associated with a POLQ damaging mutation. There were fewer CNAs in carcinoids than in carcinomas; however ACs showed a hybrid pattern, whereby gains of TERT, SDHA, RICTOR, PIK3CA, MYCL and SRC were found at rates similar to those in carcinomas, whereas the MEN1 loss rate mirrored that of TCs. Multivariate survival analysis revealed RB1 mutation (p = 0.0005) and TERT copy gain (p = 0.016) as independent predictors of poorer prognosis. MEN1 mutation was associated with poor prognosis in AC (p = 0.0045), whereas KMT2D mutation correlated with longer survival in SCLC (p = 0.0022). In conclusion, molecular profiling may complement histology for better diagnostic definition and prognostic stratification of LNETs. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Transbronchial Cryobiopsies for the Diagnosis of Diffuse Parenchymal Lung Diseases: Expert Statement from the Cryobiopsy Working Group on Safety and Utility and a Call for Standardization of the Procedure.

Transbronchial cryobiopsies (TBCB) have recently been introduced as a promising and safer alternative to surgical lung biopsy in the diagnostic approach to diffuse parenchymal lung diseases (DPLD). Despite a substantial and expanding body of literature, the technique has not yet been standardized and its place in the diagnostic algorithm of DPLD remains to be defined. In part, this reflects concerns over the diagnostic yield and safety of the procedure, together with the rapid spread of the technique without competency and safety standards; furthermore, there is a substantial procedural variability among centers and interventional pulmonologists. We report this expert statement proposed during the third international conference on "Transbronchial Cryobiopsy in Diffuse Parenchymal Lung Disease" (Ravenna, October 27-28, 2016), which formulates evidence- and expert-based suggestions on the indications, contraindications, patient selection, and procedural aspects of the procedure. The following 5 domains were reviewed: (1) what is the role of TBCB in the diagnostic evaluation of DPLD: patient selection; (2) pathological considerations; (3) contraindications and safety considerations; (4) how should TBCB be performed and in what procedural environment; and (5) who should perform TBCB. Finally, the existence of white paper recommendations may also reassure local hospital credentialing committees tasked with endorsing an adoption of the technique.

Epithelial to mesenchymal transition-related proteins ZEB1, ß-catenin, and ß-tubulin-III in idiopathic pulmonary fibrosis.

Epithelial to mesenchymal transition has been suggested as a relevant contributor to pulmonary fibrosis, but how and where this complex process is triggered in idiopathic pulmonary fibrosis is not fully understood. Beta-tubulin-III (Tubß3), ZEB1, and ß-catenin are partially under the negative control of miR-200, a family of micro-RNAs playing a major role in epithelial to mesenchymal transition, that are reduced in experimental lung fibrosis and idiopathic pulmonary fibrosis. We wonder whether in situ expression of these proteins is increased in idiopathic pulmonary fibrosis, to better understand the significance of miR-200 feedback loop and epithelial to mesenchymal transition. We investigated the immunohistochemical and immunofluorescent expression and precise location of ZEB1, Tubß3, and ß-catenin in tissue samples from 34 idiopathic pulmonary fibrosis cases and 21 controls (5 normal lungs and 16 other interstitial lung diseases). In 100% idiopathic pulmonary fibrosis samples, the three proteins were concurrently expressed in fibroblastic foci, as well in damaged epithelial cells overlying these lesions and in pericytes within neo-angiogenesis areas. These results were also confirmed by immunofluorescence assay. In controls the abnormal expression of the three proteins was absent or limited. This is the first study that relates concurrent expression of Tubß3, ZEB1, and ß-catenin to abnormal epithelial and myofibroblast differentiation in idiopathic pulmonary fibrosis, providing indirect but robust evidence of miR-200 deregulation and epithelial to mesenchymal transition activation in idiopathic pulmonary fibrosis. The abnormal expression and localization of these proteins in bronchiolar fibro-proliferative lesions are unique for idiopathic pulmonary fibrosis, and might represent a disease-specific marker in challenging lung biopsies.

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.

Pulmonary adenocarcinoma with enteric differentiation: Dissecting oncogenic genes alterations with DNA sequencing and FISH analysis.

BACKGROUND: Pulmonary Adenocarcinoma with Enteric Differentiation (PAED) is a rare subtype of adenocarcinoma of emerging interest, recently introduced in the 2015 WHO classification. However, little is known about major molecular signatures of this class of adenocarcinomas and information about new biomarkers totally lack. METHODS: We examined the NRAS, PIK3CA, EGFR, KRAS and BRAF status through mass spectrometry sequencing and ALK rearrangement by FISH in a series of 8 PAEDs. RESULTS: 1/8 (12.5%) case had a simultaneous PIK3CA mutation (E545K) and an EML4-ALK translocation. KRAS gene showed a mutation in the codon 12 in 4/8 of PAED (50%), NRAS, BRAF and EGFR genes were wild type in all tumor samples. CONCLUSIONS: We concluded that PIK3CA mutations and ALK rearrangement occur also in PAEDs, while NRAS mutations might be a very rare event similarly to pulmonary adenocarcinomas of conventional type. KRAS is the prevailing gene mutated in this class of adenocarcinoma.

Transbronchial Lung Cryobiopsy in Diffuse Parenchymal Lung Disease: Comparison between Biopsy from 1 Segment and Biopsy from 2 Segments - Diagnostic Yield and Complications.

BACKGROUND: Transbronchial lung cryobiopsy is an innovative method of obtaining samples from the parenchyma of patients with diffuse parenchymal lung diseases. However, the technique is not yet standardized, and uncertainty exists about the optimal protocol, including the number of samples, the biopsy size, and the choice of the biopsy site. OBJECTIVES: To compare the diagnostic yield and complications of cryobiopsy with different strategies adopted to sample lung tissue (number of samples, biopsy site, and sample size). METHODS: We prospectively enrolled 46 patients with suspected diffuse parenchymal lung diseases for the diagnosis of which a biopsy was deemed useful. All patients underwent transbronchial lung cryobiopsy, and they were randomly assigned to group A (4 samples obtained from the same segment) or group B (2 samples obtained from one segment and 2 samples obtained from a different segment of the same lobe). Analysis of the samples was performed sequentially (from the first to the last sample), and pathologists reformulated their histopathologic diagnosis with the addition of each sample. RESULTS: The mean diagnostic yield of the procedure combining the 2 groups and performing only the first sampling was 69%. When a second biopsy was performed as well, the mean diagnostic yield improved, but this increase was significant only when the 2 samples were obtained from 2 different segments (96%, group B). CONCLUSIONS: This study suggests that the strategy of performing 2 biopsies with a cryoprobe may be associated with an increased diagnostic yield in diffuse parenchymal lung diseases if these samples are obtained from 2 different segments within the same lobe.

Middle age has a significant impact on gene expression during skin wound healing in male mice.

The vast majority of research on the impact of age on skin wound healing (WH) compares old animals to young ones. The middle age is often ignored in biogerontological research despite the fact that many functions that decline in an age-dependent manner have starting points in mid-life. With this in mind, we examined gene expression patterns during skin WH in late middle-aged versus young adult male mice, using the head and back punch models. The rationale behind this study was that the impact of age would first be detectable at the transcriptional level. We pinpointed several pathways which were over-activated in the middle-aged mice, both in the intact skin and during WH. Among them were various metabolic, immune-inflammatory and growth-promoting pathways. These transcriptional changes were much more pronounced in the head than in the back. In summary, the middle age has a significant impact on gene expression in intact and healing skin. It seems that the head punch model is more sensitive to the effect of age than the back model, and we suggest that it should be more widely applied in aging research on wound healing.

Nonspecific Interstitial Pneumonia: What Is the Optimal Approach to Management?

We reviewed current aspects of the clinical and pathogenic profile of nonspecific interstitial pneumonia (NSIP), to better elucidate the complex issue of management and treatment options for NSIP patients. Recent findings suggest that idiopathic NSIP is a complex clinical entity with a disease spectrum that includes at least three different phenotypes: NSIP associated with autoimmune features, emphysema, and familial interstitial lung disease. This distinction, based mainly on clinical findings, may be of critical importance when it comes to making a decision on patients' management. This hypothesis warrants further studies. Currently, two major radiologic-pathologic different profiles have been well established. First, the "inflammatory type" characterized by prominent lymphocytic inflammation both on biopsy and bronchoalveolar lavage (BAL), and high-resolution computed tomography (HRCT) with mixed NSIP/organizing pneumonia pattern that tends to have a better response to corticosteroid and immunosuppressive treatment. Second, the "highly fibrotic" subgroup that shows prominent reticular changes and traction bronchiectasis by HRCT, high fibrotic background on biopsy, and no lymphocytosis on BAL. The latter fibrotic NSIP is the subgroup with less potential to respond to immunosuppressive treatment and a marginal risk to evolve into "full-blown idiopathic pulmonary fibrosis." The management of patients with fibrotic, progressive, and immunosuppressive treatment, refractory NSIP remains uncertain, and further studies are needed to address the role of antifibrotic drug in this settings. Oxygen therapy, pulmonary rehabilitation, and lung transplantation are of importance in the current management of severe, progressive, and refractory NSIP patients.

Invasive diagnostic techniques in idiopathic interstitial pneumonias.

Fibrosing interstitial lung diseases (f-ILDs) represent a heterogeneous group of disorders in which the aetiology may be identified or, not infrequently, remain unknown. Establishing a correct diagnosis of a distinct f-ILD requires a multidisciplinary approach, integrating clinical profile, physiological and laboratory data, radiological appearance and, when appropriate, histological findings. Surgical lung biopsy is still considered the most important diagnostic tool as it is able to provide lung samples large enough for identification of complex patterns such as usual interstitial pneumonitis (UIP) and nonspecific interstitial pneumonitis. However, this procedure is accompanied by significant morbidity and mortality. Bronchoalveolar lavage is still a popular diagnostic tool allowing identification of alternative diagnoses in patients with suspected idiopathic pulmonary fibrosis (IPF) when an increase in lymphocytes is detected. Conventional transbronchial lung biopsy has a very low sensitivity in detecting the UIP pattern and its role in this clinical-radiological context is marginal. The introduction of less invasive methods such as transbronchial cryobiopsy show great promise to clinical practice as they can be used to obtain samples large enough to morphologically support a diagnosis of IPF or other idiopathic interstitial pneumonias, along with fewer complications. Recent advances in the field suggest that less invasive methods of lung sampling, without significant side effects, in combination with other diagnostic methods could replace the need for surgical lung biopsy in the future. Indeed, these new multidisciplinary procedures may become the main diagnostic work-up method for patients with suspected idiopathic interstitial pneumonia.

Safety and Diagnostic Yield of Transbronchial Lung Cryobiopsy in Diffuse Parenchymal Lung Diseases: A Comparative Study versus Video-Assisted Thoracoscopic Lung Biopsy and a Systematic Review of the Literature.

BACKGROUND: A diagnosis of interstitial lung diseases (ILDs) may include surgical lung biopsy (SLB), which is associated with significant morbidity and mortality and also appreciable costs. Transbronchial lung cryobiopsy (TBLC) is adopting an important role. OBJECTIVES: The aim of this study was to compare the diagnostic yield (DY) and safety of TBLC and SLB in a large cohort of patients and to perform a systematic review of the literature as well as a meta-analysis. METHODS: We performed a retrospective analysis of 447 cases with ILD undergoing TBLC and/or SLB and a systematic review of the literature (MEDLINE and Embase for all original articles on the DY and safety of TBLC in ILDs up to July 2015). RESULTS: A total of 150 patients underwent SLB and 297 underwent TBLC. The median time of hospitalization was 6.1 days (SLB) and 2.6 days (TBLC; p < 0.0001). Mortality due to adverse events was observed for 2.7% (SLB) and 0.3% (TBLC) of the patients. Pneumothorax was the most common complication after TBLC (20.2%). No severe bleeding was observed. TBLC was diagnostic for 246 patients (82.8%), SLB for 148 patients (98.7%, p = 0.013). A meta-analysis of 15 investigations including 781 patients revealed an overall DY of 0.81 (0.75-0.87); the overall pooled probability of developing a pneumothorax, as retrieved from 15 studies including 994 patients, was 0.06 (95% CI 0.02-0.11). CONCLUSION: Cryobiopsy is safe and has lower complication and mortality rates compared to SLB. TBLC might, therefore, be considered the first diagnostic approach for obtaining tissue in ILDs, reserving the surgical approach for cases in which TBLC is not diagnostic.

From "traction bronchiectasis" to honeycombing in idiopathic pulmonary fibrosis: A spectrum of bronchiolar remodeling also in radiology?

BACKGROUND: The diagnostic and prognostic impact of traction bronchiectasis on high resolution CT scan (HRCT) in patients suspected to have idiopathic pulmonary fibrosis (IPF) is increasing significantly. MAIN BODY: Recent data demonstrated that cysts in honeycombing areas are covered by epithelium expressing bronchiolar markers. In IPF bronchiolization is the final consequence of a variety of pathogenic events starting from alveolar stem cell exhaustion, and ending in a abnormal/dysplastic proliferation of bronchiolar epithelium. CT scan features of traction bronchiectasis and honeycombing should be interpreted under the light of these new pathogenetic and morphologic considerations. SHORT CONCLUSION: We suggest that in IPF subjects traction bronchiectasis and honeycombing -now defined as distinct entities on HRCT scan- are actually diverse aspects of a continuous spectrum of lung remodeling.

Regulation of IL-8 gene expression in gliomas by microRNA miR-93.

BACKGROUND: Different strategies have been proposed to target neoangiogenesis in gliomas, besides those targeting Vascular Endothelial Growth Factor (VEGF). The chemokine Interleukin-8 (IL-8) has been shown to possess both tumorigenic and proangiogenic properties. Although different pathways of induction of IL-8 gene expression have been already elucidated, few data are available on its post-transcriptional regulation in gliomas. METHODS: Here we investigated the role of the microRNA miR-93 on the expression levels of IL-8 and other pro-inflammatory genes by RT-qPCR and Bio-Plex analysis. We used different disease model systems, including clinical samples from glioma patients and two glioma cell lines, U251 and T98G. RESULTS: IL-8 and VEGF transcripts are highly expressed in low and high grade gliomas in respect to reference healthy brain; miR-93 expression is also increased and inversely correlated with transcription of IL-8 and VEGF genes. Computational analysis showed the presence of miR-93 consensus sequences in the 3'UTR region of both VEGF and IL-8 mRNAs, predicting possible interaction with miR-93 and suggesting a potential regulatory role of this microRNA. In vitro transfection with pre-miR-93 and antagomiR-93 inversely modulated VEGF and IL-8 gene expression and protein release when the glioma cell line U251 was considered. Similar data were obtained on IL-8 gene regulation in the other glioma cell line analyzed, T98G. The effect of pre-miR-93 and antagomiR-93 in U251 cells has been extended to the secretion of a panel of cytokines, chemokines and growth factors, which consolidated the concept of a role of miR-93 in IL-8 and VEGF gene expression and evidenced a potential regulatory role also for MCP-1 and PDGF (also involved in angiogenesis). CONCLUSION: In conclusion, our results suggest an increasing role of miR-93 in regulating the level of expression of several genes involved in the angiogenesis of gliomas.

Lack of expression of TUBB3 characterizes both BCL2-positive and BCL2-negative follicular lymphoma.

Follicular lymphoma is characterized by aberrant BCL2 expression, a feature that is exploited for diagnostic purposes. However, a certain percentage of follicular lymphomas might be BCL2-negative by immunohistochemistry, increasing the difficulties in differentiating them from follicular hyperplasia. The expression of TUBB3 has been recently reported as negative in a small series of follicular lymphomas. We have therefore tested a larger series, including 61 BCL2-positive and 25 BCL2-negative cases, and compared them with 61 reactive lymphoid tissues. First, a subjective score of TUBB3 staining was applied, showing that it was consistently positive in reactive germinal centers, while most follicular lymphomas were negative; in fact, only 10/61 (16%) BCL2-positive and 1/25 (4%) BCL2-negative cases showed a positive staining for TUBB3, while 58/61 (95%) of tissues with follicular hyperplasia were positive. The application of a standardized scoring system to a large number of follicles, based on virtual slides, demonstrated that reactive lymphoid tissues had a significantly higher number of TUBB3-positive follicles both compared with BCL2-positive cases and to BCL2-negative cases. Our data support the use of TUBB3 staining in differentiating follicular lymphoma, including BCL2-negative cases, from follicular hyperplasia.

Renal cell carcinoma with smooth muscle stroma lacks chromosome 3p and VHL alterations.

Renal cell carcinoma with prominent smooth muscle stroma is a rare neoplasm composed of an admixture of epithelial cell with clear cytoplasm arranged in small nest and tubular structures and a stroma composed of smooth muscle. In the epithelial component, loss of chromosome 3p detected by fluorescence in situ hybridization (FISH) has been reported and on this basis these neoplasms have been viewed as variants of clear cell renal cell carcinoma. To test the validity of this classification, we have evaluated the chromosome 3 and VHL status of three of these tumors using FISH, array comparative genomic hybridization, gene sequencing, and methylation-specific multiplex ligation-dependent probe amplification analysis. None of the tumors showed deletion of chromosome 3p, VHL mutation, a significant VHL methylation, or changes in VHL copy number and all three tumors demonstrated a flat profile in the comparative genomic hybridization analysis. We conclude that renal cell carcinoma with smooth muscle stroma should be considered as an entity distinct from clear cell renal cell carcinoma.

High-throughput mutation profiling identifies novel molecular dysregulation in high-grade intraepithelial neoplasia and early gastric cancers.

BACKGROUND: There is still no widely accepted molecular marker available to distinguish between gastric high-grade intraepithelial neoplasia (HG-IEN) and invasive early gastric cancer (EGC). METHODS: HG-IEN and EGC lesions coexisting in the same patient were manually microdissected from a series of 15 gastrectomies for EGC; 40 ng DNA was used for multiplex PCR amplification using the Ion AmpliSeq Cancer Panel, which explores the mutational status of hotspot regions in 50 cancer-associated genes. RESULTS: Of the 15 EGCs, 12 presented at least one somatic mutation among the 50 investigated genes, and 6 of these showed multiple driver gene somatic mutations. TP53 mutations were observed in 9 cases; APC mutations were identified in 3 cases; and ATM and STK11 were mutated in 2 cases. Seven HG-IEN lesions shared an identical mutational profile with the EGC from the same patient; 13 mutations observed in APC, ATM, FGFR3, PIK3CA, RB1, STK11, and TP53 genes were shared by both HG-IEN and ECG lesions. CDKN2A, IDH2, MET, and RET mutations were observed only in EGC. TP53 deregulation was further investigated in an independent series of 75 biopsies corresponding to all the phenotypic lesions occurring in the EGC carcinogenetic cascade. p53 nuclear immunoreaction progressively increased along with the dedifferentiation of the lesions (P < 0.001). Overall, 18 of 20 p53-positive lesions showed a TP53 mutated gene. DISCUSSION: Our results support the molecular similarity between HG-IEN and EGC and suggest a relevant role for TP53 in the progression to the invasive phenotype and the use of immunohistochemistry as a surrogate to detect TP53 gene mutations.