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Non-small-cell-lung cancer accounts for 80-85% of all forms of lung cancer as leading cause of cancer-related death in human. Despite remarkable advances in the diagnosis and therapy of lung cancer, no significant improvements have thus far been achieved in terms of patients' prognosis. Here, we investigated the role of INSL4 - a member of the relaxin-family - in NSCLC. We overexpressed INSL4 in NSCLC cells to analyse in vitro the growth rate and the tumourigenic features. We investigated the signalling pathways engaged in INSL4 overexpressing cells and the tumour growth ability by studying the tumour development in a patient derived tumour xenograft mouse model. We found an INSL4 cell growth promoting effect in vitro in H1299 cells and in vivo in NOD/SCID mice. Surprisingly, in NSCLC-A549 cells, INSL4 overexpression has not similar effect, despite huge basal INSL4-mRNA expression respect to H1299. The INSL4-mRNA analysis of eight different NSCLC-derived cell lines, revealed highly difference in the INSL4-mRNA amount. Transfection of NSCLC lines with INSL4-Myc showed huge level of INSL4-mRNA with a very low amount of protein expressed. Notably, similar discrepancy has been observed in NSCLC patients. However, in a cohort of NSCLC patients analysing a database, we found a significant inverse correlation between INSL4 expression and Overall Survival. By combining the in vitro and in vivo results, suggest that in patients whose NSCLC adenocarcinoma spontaneously expressed high levels of INSL4 post-transcriptional modifications affecting INSL4 do not allow to assess precision therapy in selected patients without consider protein INSL4 amount.

Hemoglobinuria, clinically revealing as gross hematuria associated with anemia, increased hemolysis indices, acute kidney injury (AKI), can all be caused by mechanical intravascular hemolysis following mitral valve surgery. It can result from factors related to the surgical procedure or acquired later, such as paravalvular leak (PL), whose definite diagnosis is based on transesophageal echocardiography. We report the case of a patient who experienced macrohematuria and AKI, initially attributed to acute glomerulonephritis, two months after mitral valve surgery. Careful microscopic examination of the urinary sediment was a diriment diagnostic tool to differentiate acute renal failure caused by hemoglobinuria from hematuria in the course of acute glomerulonephritis, directing clinicians to investigate post-operative valvular dysfunction. From the literature review we can deduce that, notwithstanding new technologies in cardiac surgery, this rare form of AKI from intravascular hemolysis requires immediate nephrological attention and that the use of microscopic urinary sediment is decisive.

Pediatric primary lung carcinomas are extremely rare. Apart from known associations with congenital adenomatoid malformations, cases of primary lung adenocarcinomas after prolonged treatments of pediatric malignancy have been reported. We describe the morphological and molecular features of three cases of lung adenocarcinoma developed in adolescents aged 8 to 17 years during progression of their bone osteosarcoma or Ewing sarcomas. The morphological features overlapped those of adult lung adenocarcinoma including in situ, minimally invasive, and invasive forms. EGFR gene mutations were found in all three cases by targeted next-generation sequencing. The two patients with Ewing sarcoma had no progression of their lung cancer and no further progression of the metastatic bone tumor after additional chemo- and radio-therapy. Conversely, the osteosarcoma patient refused further treatments after thoracic surgery for metastatic osteosarcoma and locally advanced adenocarcinoma and died 2 years later of widespread distant metastases. Our results indicate that primary lung cancer might originate in pediatric patients during prolonged adjuvant therapies for primary bone neoplasm, and this possibility should be considered in the presence of suspected lung disease progression to correctly monitor the primary tumor evolution and define the appropriate therapeutic strategy at each time point. If appropriately treated, second primary lung cancer may not affect the patients' prognosis. The pathogenetic mechanisms of these rare lung adenocarcinomas are not clear, but the presence of EGFR mutations in all three cases indicates an oncogene addiction of the lung tumor, rather than a direct cancerogenic effect of the sarcoma-related treatment.

Purpose Decades of quality control efforts have raised the standards of immunohistochemistry (IHC), the principle method used for biomarker testing in breast cancer; however, computational pathology and reverse transcription quantitative PCR (RT-qPCR) may also hold promise for additional substantial improvements. Methods Herein, we investigated discrepancies in the assessment of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) and marker of proliferation Ki67 comparing routinely obtained IHC (and FISH) data (ORI) with the results of manual (REV) and semi-automated (DIA) re-evaluation of the original IHC slides and then with RNA expression data from the same tissue block using the MammaTyper® (MT) gene expression assay. Results Correlation for ER and PR was high between ORI IHC and the other three study methods (REV, DIA and RT-qPCR). For HER2, 10 out of 96 discrepant cases can be detected between ORI and REV that involved at least one call in the equivocal category (except for one case). For Ki67, 22 (29.1%) cases were categorized differently by either REV alone (n = 17), DIA alone (n = 15) or both (n = 10) and 28 cases (29.2%) for RT-qPCR. Most of the discrepant Ki67 cases changed from low to high between the original and following assessment and belonged to the intermediate Ki67 expression range (between 9 and 30%). Conclusions Determination of the breast cancer biomarkers ER, PR, HER2 and Ki67 at the mRNA level shows high degree of correlation with IHC and compares well with correlations between original with subsequent independent manual or semi-automated IHC assessments. The use of methods with wider dynamic range and higher reproducibility such as RT-qPCR may offer more precise assessment of endocrine responsiveness, improve Ki67 standardization and help resolve HER2 cases that remain equivocal or ambiguous by IHC/FISH. In summary, our findings seem to configure RT-qPCR as a complementary method to be used in cases of either equivocal results or presenting, at the traditional determination assays, biomarkers expressions close to the cut-off values.

The enzyme indoleamine 2,3-dioxygenase 1 (IDO1) degrade tryptophan (Trp) into kynurenine (Kyn) at the initial step of an enzymatic pathway affecting T cell proliferation. IDO1 is highly expressed in various cancer types and associated with poor prognosis. Nevertheless, the serum Kyn/Trp concentration ratio has been suggested as a marker of cancer-associated immune suppression. We measured Kyn and Trp in blood samples of a wide cohort of non-small-cell lung cancer (NSCLC) patients, before they underwent surgery, and analyzed possible correlations of the Kyn/Trp ratio with either IDO1 expression or clinical–pathological parameters. Low Kyn/Trp significantly correlated with low IDO1 expression and never-smoker patients, while high Kyn/Trp was significantly associated with older (≥68 years) patients, advanced tumor stage, and squamous cell carcinoma (Sqcc), rather than the adenocarcinoma (Adc) histotype. Moreover, high Kyn/Trp was associated, among the Adc group, with higher tumor stages (II and III), and, among the Sqcc group, with a high density of tumor-infiltrating lymphocytes. A trend correlating the high Kyn/Trp ratio with the probability of recurrences from NSCLC was also found. In conclusion, high serum Kyn/Trp ratio, associated with clinical and histopathological parameters, may serve as a serum biomarker to optimize risk stratification and therapy of NSCLC patients.

Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of lung cancer. However, their clinical benefit is limited to a minority of patients. To unravel immune-related factors that are predictive of sensitivity or resistance to immunotherapy, we performed a gene expression analysis by RNA-Seq using the Oncomine Immuno Response Assay (OIRRA) on a total of 33 advanced NSCLC patients treated with ICI evaluating the expression levels of 365 immune-related genes. We found four genes (CD1C, HLA-DPA1, MMP2, and TLR7) downregulated (p &lt, 0.05) and two genes (IFNB1 and MKI67) upregulated (p &lt, 0.05) in ICI-Responders compared to ICI-Non-Responders. The Bayesian enrichment computational analysis showed a more complex interaction network that involved 10 other genes (IFNA1, TLR4, CD40, TLR2, IL12A, IL12B, TLR9, CD1E, IFNG, and HLA-DPB1) correlated with different functional groups. Five main pathways were identified (FDR &lt, 0.0001). High TLR7 expression levels were significantly associated with a lack of response to immunotherapy (p &lt, 0.0001) and worse outcome in terms of both PFS (p &lt, 0.001) and OS (p = 0.03). The multivariate analysis confirmed TLR7 RNA expression as an independent predictor for both poor PFS (HR = 2.97, 95% CI, 1.16–7.6, p = 0.023) and OS (HR = 2.2, 95% CI, 1–5.08, p = 0.049). In conclusion, a high TLR7 gene expression level was identified as an independent predictor for poor clinical benefits from ICI. These data could have important implications for the development of novel single/combinatorial strategies TLR-mediated for an efficient selection of “individualized” treatments for NSCLC in the era of immunotherapy.

In recent years, new treatments with novel action mechanisms have been explored for advanced non-small cell lung cancer (NSCLC). Retinoids promote cancer cell differentiation and death and their trafficking and action is mediated from specific cytoplasmic and nuclear receptors, respectively. The purpose of this study was to investigate the effect of Cellular retinol binding protein-1 (CRBP-1) transfection in H460 human NSCLC cell line, normally not expressing CRBP-1. H460 cells were transfected by using a vector pTargeT Mammalian expression system carrying the whole sequence of CRBP-1 gene. For proliferation and apoptosis studies, cells were treated with different concentrations of all-trans Retinoic Acid (atRA) and retinol. AKT-related gene expression was analyzed by using western blot and Signosis array and results analysed by one-way analysis of variance (ANOVA) or by t-student test. CRBP-1+ showed reduced proliferation and viability in basal condition and after atRA treatment when compared to empty-transfected H460 cells. Reduced proliferation in CRBP-1+ H460 cells associated to the down-regulation of pAKT/pERK/pEGFR-related genes. In particular, gene array documented the down-regulation of AKT and Stat-3-related genes, including M-Tor, Akt1, Akt2, Akt3, Foxo1, p27, Jun. Restoration of CRBP-1 expression in H460 cells reduced proliferation and viability in both basal condition and after atRA treatment, likely by down-regulating AKT-related gene level. Further studies are needed to better clarify how those CRBP-1-related intracellular pathways contribute to counteract NSCLC progression in order to suggest a potential tool to improve efficacy of retinoid anti lung cancer adjuvant therapy.

The prognostic role of family history of cancer (FHC) in resected colorectal cancer (CRC) is controversial. The aim of the current study was to evaluate its impact in a monoinstitutional series of stage III CRC patients. This single institution retrospective analysis is aimed at evaluating whether FHC affects overall survival (OS) and disease-free survival (DFS) in stage III CRC patients. Moreover, the role of both colorectal FHC (FHCRC, in patients with at least one relative with CRC) and FHC “burden” have been investigated; patients were classified according to FHC in FHC negative, FHC-low (one “familial cluster” among parents/children/grandparents, brothers/sisters, uncles/cousins), and FHC-high (at least two clusters of those above mentioned). From October 2000 to March 2019, 112 consecutive stage III CRC patients have been evaluated. Median age was 67 years (range 24–89); male/female ratio was 64/48. Fifty-three (47.3%) patients were FHC-negative while 59 (52.7%) patients were FHC-positive, 18 (16.1%) of whom were FHCRC-positive. Thirty-three (29.5%) patients were FHC-low, and 10 (8.9%) were FHC-high. At a median follow-up of 41.9 months, no statistically significant differences in DFS were found. FHC-positive patients had a significantly longer OS than FHC-negative (HR = 0.32 [95% CI 0.12–0.84], p = 0.0210), and a significant trend towards improved OS according to the FHC burden was found (p = 0.0255). No statistically significant differences were found in DFS and OS according to FHCRC. In this retrospective analysis, FHC-positive stage III CRC patients had a significantly longer OS compared to FHC-negative. Moreover, this survival benefit seems to increase according to the FHC burden. Further prospective studies, with longer follow-up and larger sample size, are necessary to confirm FHC as prognostic factor in this setting.

Indoleamine 2,3-dioxygenase 2 (IDO2) is an analog of the tryptophan degrading and immunomodulating enzyme indoleamine 2,3-dioxygenase 1 (IDO1). Although the role of IDO1 is largely understood, the function of IDO2 is not yet well-elucidated. IDO2 overexpression was documented in some human tumors, but the linkage between IDO2 expression and cancer progression is still unclear, in particular in non-small cell lung cancer (NSCLC). Immunohistochemical expression and cellular localization of IDO2 was evaluated on 191 formalin-fixed and paraffin-embedded resected NSCLC. Correlations between IDO2 expression, clinical-pathological data, tumor-infiltrating lymphocytes (TILs), immunosuppressive tumor molecules (IDO1 and programmed cell death ligand-1 – PD-L1 –) and patients' prognosis were evaluated. IDO2 high expression is strictly related to high PD-L1 level among squamous cell carcinomas group (p = 0.012), to either intratumoral or mixed localization of TILs (p < 0.001) and to adenocarcinoma histotype (p < 0.001). Furthermore, a significant correlation between IDO2 high expression and poor non-small cell lung cancer prognosis was detected (p = 0.011). The current study reaches interesting knowledge about IDO2 in non-small cell lung cancer. The close relationship between IDO2 expression, PD-L1 increased levels, TILs localization and NSCLC poor prognosis, assumed IDO2 as a potential prognostic biomarker to be exploited for optimizing innovative combined therapies with immune checkpoint inhibitors.

Circulating autoantibodies to phospholipase A2 receptor (PLA2R-Ab) are detected in >70% of patients with primary membranous glomerulonephritis (MGN). Detection of PLA2R antigen in renal tissue, with immunohistochemistry (PLA2R IHC), strongly correlates with serum PLA2R-Ab, although it is more sensitive. As PLA2R IHC in literature has no univocal interpretation, we suggest reliable criteria for a standard approach for the assessment of immunostaining for differential diagnosis between primary and secondary MGN. We analyzed PLA2R IHC expression in 40 biopsies of patients with MGN and serum PLA2R-Ab titer at the time of biopsy. We carefully evaluated, at high magnification, the immunostaining pattern and distribution, regardless of intensity, in capillary loops, mesangium, and podocytes of all glomeruli.We defined, adopting this approach, positive stain when a granular pattern, coarse and/or fine, diffuse or focal, and global or segmental were observed. Negative stain was defined by mesangial staining, when there was a dirty pattern, or a peripheral staining of capillary loops with a smoky linear pattern. Podocytes showed homogenous cytoplasmatic stain both in positive and negative cases and in external negative controls. We found PLA2R IHC and serum PLA2R-Ab positivity in early-middle stage MGN compared with advanced stage more frequently. Correct stratification of patients with MGN needs PLA2R-Ab detection in serum and renal tissue. PLA2R IHC test, although a challenging stain, can be an easy diagnostic tool but requires reliable interpretation keys for a standard approach to the assessment of immunostaining.

Contextual fear conditioning (CFC) paradigm is routinely used to study fear-based learning in animals and it provides a useful model for understanding fear and anxiety in human. In the present study, such model was used following the previously established CFC protocol, and immunohistochemistry, enzymatic activity and western blotting analysis approaches were used to identify the expression of acid sphingomyelinase (aSMase) and vitamin D receptor (VDR) in prefrontal region brain of rat. Results revealed an increase of aSMase activity in conditioned rats, suggesting an apoptotic condition in such animals. In addition, an increase of density and organization of axonal neurofilaments and of VDR expression has been observed in brain of conditioned rats, supporting an induction of growth and organization of new neurons in prefrontal regions, whose contribution to various aspects of contextual fear learning is still largely unknown.

Proliferation biomarkers such as MIB-1 are strong predictors of clinical outcome and response to therapy in patients with non-small-cell lung cancer, but they require histological examination. In this work, we present a classification model to predict MIB-1 expression based on clinical parameters from positron emission tomography.We retrospectively evaluated 78 patients with histology-proven non-small-cell lung cancer (NSCLC) who underwentThe proposed model showed ability to predict the correct proliferation group with overall accuracy82% (78% and 86% for the low- and high-proliferation group, respectively).Our results indicate that radiotracer activity evaluated via SUV

Background Non-Small Cell Lung Cancer accounts for 80–85% of all forms of Lung Cancer as leading cause of cancer-related death in human. Despite remarkable advances in the diagnosis and therapy of Lung Cancer, no significant improvements have thus far been achieved in terms of patients’ prognosis. Here, we investigated the role of INSL4 – a member of the relaxin family –in NSCLC.Methods We permanently overexpressed INSL4 in NSCLC cells in vitro to analyse the growth rate and the tumourigenic features. We further investigated the signalling pathways engaged in INSL4 overexpressing cells and the tumour growth ability by studying the tumour development in a patient derived tumour xenograft mouse model. Results We found a cell growth promoting effect by INSL4 overexpression in vitro in H1299 cells and in vivo in NOD/SCID mice. Surprisingly, in NSCLC-A549 cells, stable INSL4 overexpression has not showed similar effect, despite has an INSL4-mRNA expressed up to 22.000 fold more respect H1299. The INSL4-mRNA analysis of eight different NSCLC-derived cell lines, has revealed a great discrepancy between the amount of INSL4-mRNA and specific protein. Notably, similar result has been observed in studied NSCLC patients analysing and comparing INSL4 mRNA and protein expression. However, in a cohort of NSCLC patients, we found a significant inverse correlation between INSL4 expression and Overall Survival.Conclusions By combining the results from the in vitro and in vivo models and in silico analysis in patients whose NSCLCs adenocarcinoma spontaneously expressed high levels of INSL4 our results suggest that epigenetic modifications that affect INSL4 does not allow to assess precision therapy in selected patients without consider protein INSL4 amount.

A correlation between inflammation and cancer has been identified in the case of nasal cancer, however a specific connection between nasal inflammation and extramedullary nasal plasmacytoma (ENP), to the best of our knowledge, has not yet been determined. The present case report describes a patient affected by ENP, in who the tumor arose in the same area, from which a nasal polyp was previously surgically removed, five months after the polyp excision. The patient underwent surgical endoscopic tumor asportation without being treated with radio-chemotherapy. ENP was totally removed via surgery and no signs of recurrence were identified by endoscopy or magnetic resonance imaging during the last check-up 1 year after tumor asportation. It was hypothesized that in this elderly patient, who was exposed to viral infections and pollution for several years, ENP may be correlated to the inflammatory process that occurred after surgery, and this likely contributed to a neoplastic mutation in B-cells.

Garcinoic acid (GA or δ-T3-13'COOH), is a natural vitamin E metabolite that has preliminarily been identified as a modulator of nuclear receptors involved in β-amyloid (Aβ) metabolism and progression of Alzheimer's disease (AD). In this study, we investigated GA's effects on Aβ oligomer formation and deposition. Specifically, we compared them with those of other vitamin E analogs and the soy isoflavone genistein, a natural agonist of peroxisome proliferator–activated receptor γ (PPARγ) that has therapeutic potential for managing AD. GA significantly reduced Aβ aggregation and accumulation in mouse cortical astrocytes. Similarly to genistein, GA up-regulated PPARγ expression and apolipoprotein E (ApoE) efflux in these cells with an efficacy that was comparable with that of its metabolic precursor δ-tocotrienol and higher than those of α-tocopherol metabolites. Unlike for genistein and the other vitamin E compounds, the GA-induced restoration of ApoE efflux was not affected by pharmacological inhibition of PPARγ activity, and specific activation of pregnane X receptor (PXR) was observed together with ApoE and multidrug resistance protein 1 (MDR1) membrane transporter up-regulation in both the mouse astrocytes and brain tissue. These effects of GA were associated with reduced Aβ deposition in the brain of TgCRND8 mice, a transgenic AD model. In conclusion, GA holds potential for preventing Aβ oligomerization and deposition in the brain. The mechanistic aspects of GA's properties appear to be distinct from those of other vitamin E metabolites and of genistein.

Garcinoic acid (GA or delta-T3-13'COOH), is a natural vitamin E metabolite that has preliminarily been identified as a modulator of nuclear receptors involved in beta-amyloid (Abeta) metabolism and progression of Alzheimer's disease (AD). In this study, we investigated GA's effects on Abeta oligomer formation and deposition. Specifically, we compared them with those of other vitamin E analogs and the soy isoflavone genistein, a natural agonist of peroxisome proliferator-activated receptor gamma (PPARgamma) that has therapeutic potential for managing AD. GA significantly reduced Abeta aggregation and accumulation in mouse cortical astrocytes. Similarly to genistein, GA up-regulated PPARgamma expression and apolipoprotein E (ApoE) efflux in these cells with an efficacy that was comparable to that of its metabolic precursor delta-tocotrienol and higher than those of alpha-tocopherol metabolites. Unlike for genistein and the other vitamin E compounds, the GA-induced restoration of ApoE efflux was not affected by pharmacological inhibition of PPARgamma activity, and specific activation of pregnane X receptor (PXR) was observed together with ApoE and multidrug resistance protein 1 (MDR1) membrane transporter up-regulation in both the mouse astrocytes and brain tissue. These effects of GA were associated with reduced Abeta deposition in the brain of TgCRND8 mice, a transgenic AD model. In conclusion, GA holds potential for preventing Abeta oligomerization and deposition in the brain. The mechanistic aspects of GA's properties appear to be distinct from those of other vitamin E metabolites and of genistein. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

To assess whether the use of digital image analysis (DIA) in primary Sjögren's syndrome (pSS) for the calculation of the total area of the salivary gland (SG), focus score (FS) and SG area occupied by the inflammatory infiltrate (area fraction, AF), was able to generate reproducible readings among different raters, reducing disagreement.Haematoxylin and Eosin digital slides from pSS and non-specific chronic sialadenitis (NSCS) patients were analysed blindly by 4 independent raters among 3 centres. Using an open-source software (QuPath) raters were asked to provide the total area of the gland i) using a grid-based method and ii) a software-based area-calculation tool, iii) the number of inflammatory foci and iv) the total area of the inflammatory infiltrate. Collected data was used to calculate the inter-rater agreement.For the calculation of the total SG area, DIA generated higher agreement among raters than grid-based calculation (inter-class correlation coefficient ICC=0.85 vs 0.98). Agreement for calculated total area of the inflammatory infiltrate (ICC=0.94) and for AF (ICC=0.94) was higher than infiltrates count number (ICC=0.54) and FS (ICC=0.56). AF achieved a 30% improvement over the FS at generating consensus among raters when used as a diagnostic cut-off.A digital approach achieved a far superior inter-rater agreement when calculating the total area compared to a grid-based approach. The calculation of AF proved superior to FS in correctly classifying pSS vs NSCS biopsies. We suggest that digitally calculated AF should be used alongside FS for large multi-centre studies to improve data harmonisation.

Background/aim Proliferation biomarkers such as MIB-1 are strong predictors of clinical outcome and response to therapy in patients with non-small-cell lung cancer, but they require histological examination. In this work, we present a classification model to predict MIB-1 expression based on clinical parameters from positron emission tomography. Patients and methods We retrospectively evaluated 78 patients with histology-proven non-small-cell lung cancer (NSCLC) who underwent 18F-FDG-PET/CT for clinical examination. We stratified the population into a low and high proliferation group using MIB-1=25% as cut-off value. We built a predictive model based on binary classification trees to estimate the group label from the maximum standardized uptake value (SUVmax) and lesion diameter. Results The proposed model showed ability to predict the correct proliferation group with overall accuracy >82% (78% and 86% for the low- and high-proliferation group, respectively). Conclusion Our results indicate that radiotracer activity evaluated via SUVmax and lesion diameter are correlated with tumour proliferation index MIB-1.

Pregnane X receptor (PXR) is a master xenobiotic-sensing transcription factor with a key role in drug metabolism and disposition. Its activity regulates a number of physiological processes in the liver and intestine, and it is now a validated target for human diseases associated with inflammation and dysregulation of the immune system. The identification of chemical probes to investigate the therapeutic relevance of the receptor is still highly desired. In fact, currently available PXR ligands are not highly selective and can exhibit toxicity and/or potential off-target effects. In this study, we have identified the naturally-occurring garcinoic acid as a selective and efficient PXR agonist. The properties of garcinoic acid as a specific PXR agonist was demonstrated using different approaches - screening on a panel of nuclear receptors, physical and thermodynamic evaluation of binding affinity, and co-crystallization study. Cytotoxicity assays, transcriptional and functional experiments were carried out in human liver cells, in mouse liver and gut tissue to prove compound activity and target engagement. Taken together, these data support the conclusion that garcinoic acid efficiently activates PXR and may prove to be an amenable lead toward the development of differentially acting PXR regulating compounds.

Aryl hydrocarbon receptor (AhR) is expected to promote initiation, progression and invasion of cancer cells regulating proliferation, differentiation, gene expression, inflammation, cell motility and migration. Furthermore, an immunosuppressant function of AhR has been recognized. This study evaluated AhR expression and its role in thyroid cancer progression. AhR expression was assessed by qPCR in 107 thyroid cancer samples (90 PTCs, 11 MTCs, 6 ATCs), and by immunohistochemistry in 41 PTCs. To estimate receptor activation, the expression of target genes CYP1A1 and CYP1B1 was measured. AhR functional effects were evaluated in kynurenine-stimulated FTC-133 and BcPap cell lines by analyzing the expression of genes involved in EMT and cell motility. AhR mRNA expression resulted significantly higher in all the analyzed thyroid cancer samples compared to normal thyroid and a statistically significant correlation with CYP1B1 was detected. Kynurenine-stimulated FTC-133 and BcPap showed the activation of a specific AhR-driven EMT program characterized by E-cadherin decrease and SLUG, N-cadherin and fibronectin increase, resulting in boost of cell motility and invasion. This study confirmed the importance of the IDO1-Kyn-AhR pathway in thyroid cancer tumorigenesis, suggesting an AhR pivotal role in mediating an immunosuppressive microenvironment and favoring the acquisition of a mesenchymal phenotype that could promote invasiveness and metastasis.

// Valentina Cocciolone 1, 2, * , Katia Cannita 2, * , Alessandra Tessitore 1 , Valentina Mastroiaco 1 , Lucia Rinaldi 2 , Stefania Paradisi 2 , Azzurra Irelli 2 , Paola Lanfiuti Baldi 2 , Tina Sidoni 2 , Enrico Ricevuto 1, 3 , Antonella Dal Mas 4 , Giuseppe Calvisi 4 , Gino Coletti 4 , Antonietta Ciccozzi 5 , Laura Pizzorno 6 , Valter Resta 6 , Alberto Bafile 6 , Edoardo Alesse 1 and Corrado Ficorella 1, 2 1 Department of Biotechnological and Applied Clinical Sciences, University of L’Aquila, L’Aquila, Italy 2 Medical Oncology Department, S. Salvatore Hospital, University of L’Aquila, L’Aquila, Italy 3 Oncology Network ASL1 Abruzzo, UOSD Oncology Territorial Care, S. Salvatore Hospital, University of L’Aquila, L’Aquila, Italy 4 Pathology Department, S. Salvatore Hospital, L’Aquila, L’Aquila, Italy 5 Radiology Department, S. Salvatore Hospital, L’Aquila, L’Aquila, Italy 6 Breast Unit, S. Salvatore Hospital, L’Aquila, L’Aquila, Italy * These authors have contributed equally to this work Correspondence to: Valentina Cocciolone, email: vale.cocciolone@tiscali.it Keywords: neaodjuvant; dose-dense; PIK3CA; real life Received: July 27, 2017 Accepted: April 06, 2018 Published: June 08, 2018 ABSTRACT Background : Dose-dense chemotherapy is one of the treatments of choice for neoadjuvant therapy in breast cancer (BC). Activating mutations in PIK3CA gene predict worse response to neoadjuvant chemotherapy for HER2-positive patients, while their role is less clearly defined for HER2-negative tumors. Methods : We conducted a phase I/II study of neoadjuvant, sequential, dose-dense anthracycline/taxane chemotherapy, plus trastuzumab in HER2-positive patients and investigated the correlation of pre-treatment PIK3CA mutation status with pathologic complete response (pCR) and long-term outcome in a real-life setting. Results : we established a dose-dense docetaxel recommended dose of 60 mg/m 2 and 65 mg/m 2 , with or without trastuzumab, respectively, according to HER2-status, following dose-dense epirubicin-cyclophosphamide (90/600 mg/m 2 ), every 2 weeks. The overall pCR rate was 21.4%; median disease-free survival (DFS) was 52 months and median overall survival (OS) was not yet reached. PIK3CA mutation status was not significantly associated with the pCR rate: 18% for both mutated and wild-type patients. The pCR rate was: 25% in the mutated and 24% in the wild-type (p 0.560) cohort of the HER2-positive subgroup; 33% both in the mutant and wild-type cohort of the triple-negative subgroup; no pCR neither in the mutant nor in the wild-type cohort of the HR-positive/HER2-negative subgroup. Among the HER2-positive population, a trend toward worse DFS was observed in case of mutation, as opposed to the triple negative population. Conclusions : This study proposes an effective and safe neoadjuvant dose-dense anthracycline/taxane schedule and suggests that PIK3CA mutation analysis can be usefully performed in real-life clinical practice.

Although epidermal growth factor receptor (EGFR) and v-Ki-ras2 Kirsten rat sarcoma viral oncogene (KRAS) mutations were thought to be mutually exclusive in patients with non-small cell lung cancer (NSCLC), the development of high sensitive large-scale mutation analysis, has increasingly shown that activating EGFR mutations occasionally coexist with other dominant genetic alterations. Herein, we discuss the case of a patient with advanced NSCLC harboring both the uncommon EGFR G719S and the KRAS G12C mutations, who was treated for 9 years with erlotinib achieving a long-term survival. In light of their rarity, multiple mutations are very challenging for the decision of tyrosine kinase inhibitors (TKIs) treatment, especially when EGFR mutations occur together with mutations known to provide resistance to EGFR TKIs, such as KRAS.

The Feline Immunodeficiency Virus (FIV) is a lentivirus that affects cats worldwide. The detection ofthe virus’ 24 Kda capsid protein (p24) antibody is the most common way to diagnose FIV infections.In Brazil, the most used commercial assay for FIV diagnosis is a rapid ELISA test based on the lateralflow principle, with high sensitivity and specificity (SNAP®test). However, the high cost of this importedassay undermines not only the diagnosis of this infection but also its epidemiology. The objective of thisstudy was to optimize and validate an indirect ELISA using the protein p24 recombinant antigen (r-p24)with national technology and maintaining sensitivity and specificity comparable to those of availablecommercial rapid ELISA tests. Twenty-six reference cats’ sera samples (13 negative and 13 positives for FIV),previously analyzed by PCR and the SNAP® test, were used as a reference to optimize the ELISA. Serumsamples of 226 cats from private owners and from shelters of the metropolitan area of Rio de Janeirowere used to validate the assay. The sensitivity and specificity of the r-p24 ELISA were 95.4% and 99.4%,respectively. When compared to the SNAP® Combo Test, the accuracy of r-p24 ELISA was 99%, with aKappa index of 0.96. Our results indicate that the national technology-based ELISA r-p24 has sensibilityand specificity values that are comparable to rapid ELISA kits. This test is therefore recommendable, usingnational technology for FIV routine diagnosis, research, and epidemiological studies., O Vírus da Imunodeficiência Felina (FIV) é um lentivírus que afeta gatos em todo o mundo. A detecçãodo anticorpo para proteína do capsídeo viral de 24 kDa (p24) é a forma mais comum para diagnosticarinfecções pelo FIV. No Brasil, o ensaio comercial mais utilizado no diagnóstico FIV é um teste rápidode ELISA (SNAP®test), baseado no princípio de fluxo lateral, com alta sensibilidade e especificidade.No entanto, o alto custo deste ensaio importado inviabiliza não só o diagnóstico da infecção, como tambémsua epidemiologia. O objetivo deste trabalho foi aperfeiçoar e validar um teste de ELISA indireto, comtecnologia nacional, utilizando o antígeno recombinante da proteína p24 (r-p24), com sensibilidade eespecificidade comparáveis às dos testes rápidos, comercialmente disponíveis. Vinte e seis amostras desoro gatos (13 negativos e 13 positivos para FIV), previamente analisados por PCR e Snap®, foram utilizadascomo amostras de referência para aperfeiçoar o ELISA e 226 amostras de soro de gatos, de proprietáriosprivados ou de abrigos da área metropolitana Rio de Janeiro foram utilizadas para validar o ensaio. O ELISAr-p24 apresentou uma sensibilidade de 95,4% e 99,4% de especificidade quando comparadas às do testeSNAP Combo, uma precisão de 99% e um índice Kappa de 0.96. Nossos resultados indicam que o ELISAr-p24 apresentou sensibilidade e especificidade comparáveis às dos testes rápidos, utilizando tecnologianacional para diagnóstico, pesquisa e estudos epidemiológicos do FIV.

Summary Candida albicans is implicated in intestinal diseases. Identifying host signatures that discriminate between the pathogenic versus commensal nature of this human commensal is clinically relevant. In the present study, we identify IL-9 and mast cells (MCs) as key players of Candida commensalism and pathogenicity. By inducing TGF-β in stromal MCs, IL-9 pivotally contributes to mucosal immune tolerance via the indoleamine 2,3-dioxygenase enzyme. However, Candida-driven IL-9 and mucosal MCs also contribute to barrier function loss, dissemination, and inflammation in experimental leaky gut models and are upregulated in patients with celiac disease. Inflammatory dysbiosis occurs with IL-9 and MC deficiency, indicating that the activity of IL-9 and MCs may go beyond host immunity to include regulation of the microbiota. Thus, the output of the IL-9/MC axis is highly contextual during Candida colonization and reveals how host immunity and the microbiota finely tune Candida behavior in the gut., Graphical Abstract, Highlights • IL-9/IL-9R signaling affects MC function in mucosal candidiasis • IL-9 and mucosal MCs contribute to barrier function loss in leaky gut models • IL-9 and stromal MCs induce local protective tolerance in infection via IDO1 • IL-9 and mucosal MCs expand and IDO1 decreases in human celiac disease, Deciphering the mechanisms by which Candida albicans promotes either pathology or protective tolerance in the gut could be clinically relevant. Renga et al. show a key role for IL-9 and mast cells in promoting either inflammatory dysbiosis and pathology or tolerance in leaky gut models and human celiac disease.

1 alpha,25-dihydroxyvitamin D3 (1,23(OH)2 D3) is known to play a dual role in cancer, by promoting or inhibiting carcinogenesis via 1,23(OH)2 D3 receptor (VDR) and phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Fok I polymorphism of VDR may indirectly influence the receptor levels through autoregulation. The involvement of neutral sphingomyelinase in the non-classic VDR-mediated genomic pathway response to 1,23(OH)2 D3 treatment has been reported. Until now no information were reported about Fok I polymorphism of VDR in NCI-N87 human gastric cancer cells and the relation between acid sphingomyelinase and 1,23(OH)2 D3. Herein, we showed that NCI-N87 human gastric cancer cells are homozygous for the Fok I 'C' allele; resulting in a three amino acid-truncated protein form of the VDR. Surprisingly 1,23(OH)2 D3 treatments strongly down-regulated the expression of VDR whereas acid sphingomyelinase and PTEN expression were upregulated. No changes of neutral sphingomyelinase expression were observed after 1,23(OH)2 D3 treatment, whereas acid sphingomyelinase activity increased. Furthermore 1,23(OH)2 D3 induced over-expression of caspase 8, CDKN2B, MAP3K5, cytochrome C apoptotic genes. Morphological analysis highlighted some very large round or oval cells and small cells with angular or fusiform extensions, confirmed by MIB-1 immunodetection and Hercep test. Taken together our results indicated that the action of 1,23(OH)2 D3 in gastric cancer cells was independent on 1,23(OH)2 D3 receptor and suggested the acid sphingomyelinase as a possible target to induce molecular events.

BackgroundAnti-programmed cell death protein 1 and programmed cell death ligand 1 (PD-L1) agents are broadly used in first-line and second-line treatment across different tumor types. While immunohistochemistry-based assays are routinely used to assess PD-L1 expression, their clinical utility remains controversial due to the partial predictive value and lack of standardized cut-offs across antibody clones. Using a high throughput immunoassay, the reverse phase protein microarray (RPPA), coupled with a fluorescence-based detection system, this study compared the performance of six anti-PD-L1 antibody clones on 666 tumor samples.MethodsPD-L1 expression was measured using five antibody clones (22C3, 28–8, CAL10, E1L3N and SP142) and the therapeutic antibody atezolizumab on 222 lung, 71 ovarian, 52 prostate and 267 breast cancers, and 54 metastatic lesions. To capture clinically relevant variables, our cohort included frozen and formalin-fixed paraffin-embedded samples, surgical specimens and core needle biopsies. Pure tumor epithelia were isolated using laser capture microdissection from 602 samples. Correlation coefficients were calculated to assess concordance between antibody clones. For two independent cohorts of patients with lung cancer treated with nivolumab, RPPA-based PD-L1 measurements were examined along with response to treatment.ResultsMedian-center PD-L1 dynamic ranged from 0.01 to 39.37 across antibody clones. Correlation coefficients between the six antibody clones were heterogeneous (range: −0.48 to 0.95) and below 0.50 in 61% of the comparisons. In nivolumab-treated patients, RPPA-based measurement identified a subgroup of tumors, where low PD-L1 expression equated to lack of response.ConclusionsContinuous RPPA-based measurements capture a broad dynamic range of PD-L1 expression in human specimens and heterogeneous concordance levels between antibody clones. This high throughput immunoassay can potentially identify subgroups of tumors in which low expression of PD-L1 equates to lack of response to treatment.

ObjectivesCounting intraepithelial lymphocytes (IEL) is central to the histological diagnosis of coeliac disease (CD), but no definitive ‘normal’ IEL range has ever been published. In this multicentre study, receiver operating characteristic (ROC) curve analysis was used to determine the optimal cut-off between normal and CD (Marsh III lesion) duodenal mucosa, based on IEL counts on >400 mucosal biopsy specimens.DesignThe study was designed at the International Meeting on Digestive Pathology, Bucharest 2015. Investigators from 19 centres, eight countries of three continents, recruited 198 patients with Marsh III histology and 203 controls and used one agreed protocol to count IEL/100 enterocytes in well-oriented duodenal biopsies. Demographic and serological data were also collected.ResultsThe mean ages of CD and control groups were 45.5 (neonate to 82) and 38.3 (2–88) years. Mean IEL count was 54±18/100 enterocytes in CD and 13±8 in normal controls (p=0.0001). ROC analysis indicated an optimal cut-off point of 25 IEL/100 enterocytes, with 99% sensitivity, 92% specificity and 99.5% area under the curve. Other cut-offs between 20 and 40 IEL were less discriminatory. Additionally, there was a sufficiently high number of biopsies to explore IEL counts across the subclassification of the Marsh III lesion.ConclusionOur ROC curve analyses demonstrate that for Marsh III lesions, a cut-off of 25 IEL/100 enterocytes optimises discrimination between normal control and CD biopsies. No differences in IEL counts were found between Marsh III a, b and c lesions. There was an indication of a continuously graded dose–response by IEL to environmental (gluten) antigenic influence.