ß2-AR inhibition enhances EGFR antibody efficacy hampering the oxidative stress response machinery.

The ß2-Adrenergic receptor (ß2-ARs) is a cell membrane-spanning G protein-coupled receptors (GPCRs) physiologically involved in stress-related response. In many cancers, the ß2-ARs signaling drives the tumor development and transformation, also promoting the resistance to the treatments. In HNSCC cell lines, the ß2-AR selective inhibition synergistically amplifies the cytotoxic effect of the MEK 1/2 by affecting the p38/NF-kB oncogenic pathway and contemporary reducing the NRF-2 mediated antioxidant cell response. In this study, we aimed to validate the anti-tumor effect of ß2-AR blockade and the synergism with MEK/ERK and EGFR pathway inhibition in a pre-clinical orthotopic mouse model of HNSCC. Interestingly, we found a strong ß2-ARs expression in the tumors that were significantly reduced after prolonged treatment with ß2-Ars inhibitor (ICI) and EGFR mAb Cetuximab (CTX) in combination. The ß2-ARs down-regulation correlated in mice with a significant tumor growth delay, together with the MAPK signaling switch-off caused by the blockade of the MEK/ERK phosphorylation. We also demonstrated that the administration of ICI and CTX in combination unbalanced the cell ROS homeostasis by blocking the NRF-2 nuclear translocation with the relative down-regulation of the antioxidant enzyme expression. Our findings highlighted for the first time, in a pre-clinical in vivo model, the efficacy of the ß2-ARs inhibition in the treatment of the HNSCC, remarkably in combination with CTX, which is the standard of care for unresectable HNSCC.

Analysis of microsatellite instability (MSI) in pediatric gonadal and extra-gonadal germ cell tumors.

Gonadal and extragonadal pediatric germ cell tumors (GCTs) are rare neoplasms with different clinical behavior. Although surgery and cisplatin-based chemotherapy are resolutive in most cases, some patients do not respond to chemotherapy and have a worse outcome. Microsatellite instability (MSI) was correlated to resistance to chemotherapy and sensitivity to immunotherapy in different neoplasms. A series of 21 pediatric GCTs were tested by immuno-histochemistry and PCR to evaluate MSI status. Next generation sequencing was applied to further evaluate cases with discordant results between immunohistochemistry and PCR. Twenty-one cases of pediatric GCT were included in the series. The mean age ranged between 1 and 10 years. Nine cases were gonadal GCTs and the remaining 12 were extra-gonadal GCTs. By immunohistochemistry, one case showed a deficit of Mismatch repair (MMR) proteins. This case was a 1-year-old children affected by gonadal yolk sac tumor. However, all cases resulted microsatellite stable (MSS) by PCR and NGS. MSI was not detected in our series of pediatric GCTs, as well as the data present in literature about adult patients with GCTs. Molecular techniques could have a role to confirm the MSI status in case of dMMR by immunohistochemistry.

Combined blockade of mTOR and p21-activated kinases pathways prevents tumour growth in KRAS-mutated colorectal cancer.

BACKGROUND: The identification of novel therapeutic strategies for metastatic colorectal cancer (mCRC) patients harbouring KRAS mutations represents an unmet clinical need. In this study, we aimed to clarify the role of p21-activated kinases (Paks) as therapeutic target for KRAS-mutated CRC. METHODS: Paks expression and activation levels were evaluated in a cohort of KRAS-WT or -mutated CRC patients by immunohistochemistry. The effects of Paks inhibition on tumour cell proliferation and signal transduction were assayed by RNAi and by the use of three pan-Paks inhibitors (PF-3758309, FRAX1036, GNE-2861), evaluating CRC cells, spheroids and tumour xenografts' growth. RESULTS: Paks activation positively correlated with KRAS mutational status in both patients and cell lines. Moreover, genetic modulation or pharmacological inhibition of Paks led to a robust impairment of KRAS-mut CRC cell proliferation. However, Paks prolonged blockade induced a rapid tumour adaptation through the hyper-activation of the mTOR/p70S6K pathway. The addition of everolimus (mTOR inhibitor) prevented the growth of KRAS-mut CRC tumours in vitro and in vivo, reverting the adaptive tumour resistance to Paks targeting. CONCLUSIONS: In conclusion, our results suggest the simultaneous blockade of mTOR and Pak pathways as a promising alternative therapeutic strategy for patients affected by KRAS-mut colorectal cancer.

Fluorescent nanodiamonds as innovative delivery systems for MiR-34a replacement in breast cancer.

Nanodiamonds are innovative nanocrystalline carbon particles able to deliver chemically conjugated miRNAs. In oncology, the use of miRNA-based therapies may represent an advantage, based on their ability to simultaneously target multiple intracellular oncogenic targets. Here, nanodiamonds were tested and optimized to deliver miR-34a, a miRNA playing a key role in inhibiting tumor development and progression in many cancers. The physical-chemical properties of nanodiamonds were investigated suggesting electrical stability and uniformity of structure and size. Moreover, we evaluated nanodiamond cytotoxicity on two breast cancer cell models and confirmed their excellent biocompatibility. Subsequently, nanodiamonds were conjugated with miR-34a, using the chemical crosslinker polyethyleneimine; real-time PCR analysis revealed a higher level of miR-34a in cancer cells treated with the different formulations of nanodiamonds than with commercial transfectant. A significant and early nanodiamond-miR-34a uptake was recorded by FACS and fluorescence microscopy analysis in MCF7 and MDA-MB-231 cells. Moreover, nanodiamond-miR-34a significantly inhibited both cell proliferation and migration. Finally, a remarkable anti-tumor effect of miR-34a-conjugated nanodiamonds was observed in both heterotopic and orthotopic murine xenograft models. In conclusion, this study provides a rationale for the development of new therapeutic strategies based on use of miR-34a delivered by nanodiamonds to improve the clinical treatment of neoplasms.

The urotensin-II receptor: A marker for staging and steroid outcome prediction in ulcerative colitis.

BACKGROUND: Urotensin-II receptor- (UTR) related pathway exerts a key-role in promoting inflammation. The aim was to assess the relationship between UTR expression and clinical, endoscopic and biochemical severity of ulcerative colitis (UC), exploring its predictivity of intravenous (iv) steroid administration therapeutic outcome. METHODS: One-hundred patients with first diagnosis of UC and 44 healthy subjects were enrolled. UTR expression was assessed by qPCR, Western Blot (WB) and immunohistochemistry (IHC). Clinical, endoscopic and histological activity of UC were evaluated by using Truelove and Witts (T&W) severity index, Mayo Endoscopic Score (MES), and Truelove and Richards Index (TRI). The partial and full Mayo scores (PMS and FMS) were assessed to stage the disease. RESULTS: The UTR expression, resulted higher in the lesioned mucosa of UC patients in comparison to healthy subjects (p < .0001 all). Direct relationship between UTR (mRNA and protein) expression and disease severity assessment (T&W, PMS, MES and TRI) was highlighted (p < .0001 all). UTR expression resulted also higher in the 72 patients requiring iv steroids administration compared to those who underwent alternative medications, (p < .0001). The 32 steroid-non-responders showed an increased UTR expression (WB, IHC and qPCR from lesioned mucosa), compared to 40 steroid-responders (p: .0002, .0001, p < .0001 respectively). The predictive role of UTR expression (p < .05) on the negative iv steroids administration therapeutic outcome was highlighted and ROC curves identified the thresholds expressing the better predictive performance. CONCLUSIONS: UTR represents a promising inflammatory marker related to clinical, endoscopic, and histological disease activity as well as a predictive marker of steroid administration therapeutic outcome in the UC context.

How to Diagnose Anaplastic Large Cell Lymphoma on Cytological Samples? A Series with Emphasis on Diagnostic Clue and Pitfalls.

INTRODUCTION: Anaplastic large cell lymphoma (ALCL) is a rare mature T-cell non-Hodgkin's lymphoma characterized by large and pleomorphic neoplastic CD30-positive T cells. ALCL includes different subtypes with different clinical and biological features: systemic ALCL, primary cutaneous ALCL, breast implant-associated ALCL (BIA-ALCL). Anaplastic lymphoma kinase (ALK) is overexpressed and rearranged in some systemic cases. Diagnosis of ALCL may be challenging on cytological samples, but the correct diagnosis is mandatory for the management of the patient. METHODS: A retrospective series of 12 ALCLs diagnosed by cytology is reported. Cytological samples included lymph nodes and skin lesions fine needle aspiration cytology, peritoneal effusion, and periprosthetic fluid. Microscopic evaluation was performed on direct smears, cell-block sections, and cytocentrifugated slides. Immunocytochemistry was performed on cell-block sections, direct smears, and cytocentrifugated slides. Molecular evaluation by fluorescent in-situ hybridization (FISH) was performed on cell-block sections. RESULTS: The series included 4 ALK+ ALCLs, 5 ALK- ALCLs, and 3 BIA-ALCLs. FNAC was performed on lymph nodes in 8 cases and on skin lesion in 1 case. In this last case, a peritoneal effusion was also evaluated. Breast periprosthetic fluids were evaluated in 3 cases. A large immunocytochemical panel was performed in each case, and FISH in 3 cases, demonstrating ALK rearrangement in a case of ALK+ ALCL. A final diagnosis was rendered in all cases. In the case of skin lesion, the differential diagnosis between systemic ALCL and primary cutaneous ALCL was possible. CONCLUSION: The cytological diagnosis of ALCL may be challenging, and the proper management of the collected sample is mandatory. The rapid on-site evaluation and the realization of a cell block are strongly recommended. Immunocytochemistry is mandatory for the diagnosis and a large antibodies panel is needed as differential diagnosis includes many different neoplasms. FISH may be useful to evaluate ALK rearrangements. When properly managed, cytology can lead to a reliable final diagnosis of ALCL.

PD-L1 Overexpression in the Lungs of Subjects Who Died from COVID-19: Are We on the Way to Understanding the Immune System Exhaustion Induced by SARS-CoV-2?

Knowledge of the pathogenic mechanisms of severe acute respiratory syndrome-associated coronavirus 2 (SARS-CoV-2) is certainly a priority for the scientific community. Two main elements are involved in the biology of the most severe forms of coronavirus disease 2019 (COVID-19): the direct cytopathic effect of the virus against the host tissues, and a dysfunction of the immune system, characterized by the exhaustion of T lymphocytes. The exhaustion of T cells in COVID-19 is poorly understand, but some data could suggest a possible role of PD-1/PD-L1 axis. The aim of this study was to evaluate the possible role of PD-L1 expression in the pulmonary tissue in subjects affected by COVID-19. The presence of SARS-CoV-2 in the pulmonary tissue, and its exact location, was indagated by in situ hybridization; the expression of PD-L1 and CD8 in the same tissue was indagated by immunohistochemistry. Overall, PD-L1 resulted diffusely expressed in 70% of the cases, and an intense expression was observed in 43.5% of cases. Diffuse and intense presence of SARS-CoV-2 by in situ hybridization significantly correlated with an intense PD-L1 expression, and with expression of PD-L1 by pneumocytes. PD-L1 is overexpressed in the pulmonary tissue of subjects died from COVID-19, and mainly in subjects with a high viral load. These data suggest a possible role of PD-L1 in the immune system exhaustion at the basis of the severe forms of the disease.

Glycated ACE2 receptor in diabetes: open door for SARS-COV-2 entry in cardiomyocyte.

RATIONALE: About 50% of hospitalized coronavirus disease 2019 (COVID-19) patients with diabetes mellitus (DM) developed myocardial damage. The mechanisms of direct SARS-CoV-2 cardiomyocyte infection include viral invasion via ACE2-Spike glycoprotein-binding. In DM patients, the impact of glycation of ACE2 on cardiomyocyte invasion by SARS-CoV-2 can be of high importance. OBJECTIVE: To evaluate the presence of SARS-CoV-2 in cardiomyocytes from heart autopsy of DM cases compared to Non-DM; to investigate the role of DM in SARS-COV-2 entry in cardiomyocytes. METHODS AND RESULTS: We evaluated consecutive autopsy cases, deceased for COVID-19, from Italy between Apr 30, 2020 and Jan 18, 2021. We evaluated SARS-CoV-2 in cardiomyocytes, expression of ACE2 (total and glycosylated form), and transmembrane protease serine protease-2 (TMPRSS2) protein. In order to study the role of diabetes on cardiomyocyte alterations, independently of COVID-19, we investigated ACE2, glycosylated ACE2, and TMPRSS2 proteins in cardiomyocytes from DM and Non-DM explanted-hearts. Finally, to investigate the effects of DM on ACE2 protein modification, an in vitro glycation study of recombinant human ACE2 (hACE2) was performed to evaluate the effects on binding to SARS-CoV-2 Spike protein. The authors included cardiac tissue from 97 autopsies. DM was diagnosed in 37 patients (38%). Fourth-seven out of 97 autopsies (48%) had SARS-CoV-2 RNA in cardiomyocytes. Thirty out of 37 DM autopsy cases (81%) and 17 out of 60 Non-DM autopsy cases (28%) had SARS-CoV-2 RNA in cardiomyocytes. Total ACE2, glycosylated ACE2, and TMPRSS2 protein expressions were higher in cardiomyocytes from autopsied and explanted hearts of DM than Non-DM. In vitro exposure of monomeric hACE2 to 120 mM glucose for 12 days led to non-enzymatic glycation of four lysine residues in the neck domain affecting the protein oligomerization. CONCLUSIONS: The upregulation of ACE2 expression (total and glycosylated forms) in DM cardiomyocytes, along with non-enzymatic glycation, could increase the susceptibility to COVID-19 infection in DM patients by favouring the cellular entry of SARS-CoV2.

High performance of multiplex fluorescence in situ hybridization to simultaneous detection of BCL2 and BCL6 rearrangements: useful application in the characterization of DLBCLs.

Chromosomal rearrangements involving BCL2, BCL6 and MYC are commonly found in the most frequent B cell lymphomas, namely follicular lymphomas (FLs) and diffuse large B-cell lymphomas (DLBCLs). Particularly, BCL2-rearrangement represents a diagnostic hallmark in FLs, whereas MYC translocation can occur simultaneously with BCL2 and/or BCL6 rearrangements, defining a specific category of DLBCLs with a poorer prognosis. In this study, we aim to validate the diagnostic performance of multiplex BCL2/BCL6 FISH approach in formalin-fixed paraffin-embedded FLs and DBCLs and cytological samples of DLBCL comparing to the classic set of single break-apart probes. We collected a series of lymphomas, including 85 DLBCLs, 45 FLs and 36 other B-cell lymphoma histotypes and 16 cytological samples of DLBCLs. MYC, BCL2 and BCL6 rearrangements were previously assessed by a classic FISH test using single break-apart probes. All samples were analysed by a multiplex FISH assay. In the FL series, 38 cases showed BCL2-R; in the DLBCLs series, MYC-R was detected in 21 out of 85 DLBCL patients, BCL2-R in 10 out of 85 and BCL6-R in 33 out of 85. In the DLBCL cytological series, MYC-R was detected in 4 out of 16, BCL2-R in 4 out of 16 and BCL6-R in 1 out of 16. Notably, in FFPE, 13 double-hit lymphomas (DHLs) and 3 triple-hit lymphomas (THLs) were detected; in the cytological series, only 3 DHL cases were observed. The dual BCL2/BCL6 FISH probe test results were fully concordant with the results obtained using classic BCL2 and BCL6 single break apart. Particularly, multiplex FISH to simultaneously detect BCL2-R and BCL6-R on a single slide could find a wide application in the characterisation of double- and triple-hit DLBCLs.

Dual inhibition of TGFß and AXL as a novel therapy for human colorectal adenocarcinoma with mesenchymal phenotype.

A subset of colorectal cancer (CRC) with a mesenchymal phenotype (CMS4) displays an aggressive disease, with an increased risk of recurrence after surgery, reduced survival, and resistance to standard treatments. It has been shown that the AXL and TGFß signaling pathways are involved in epithelial-to-mesenchymal transition, migration, metastatic spread, and unresponsiveness to targeted therapies. However, the prognostic role of the combination of these biomarkers and the anti-tumor effect of AXL and TGFß inhibition in CRC still has to be assessed. To evaluate the role of AXL and TGFß as negative biomarker in CRC, we conducted an in-depth in silico analysis of CRC samples derived from the Gene Expression Omnibus. We found that AXL and TGFß receptors are upregulated in CMS4 tumors and are correlated with an increased risk of recurrence after surgery in stage II/III CRC and a reduced overall survival. Moreover, we showed that AXL receptor is differently expressed in human CRC cell lines. Dual treatment with the TGFß galunisertib and the AXL inhibitor, bemcentinib, significantly reduced colony formation and migration capabilities of tumor cells and displayed a strong anti-tumor activity in 3D spheroid cultures derived from patients with advanced CRC. Our work shows that AXL and TGFß receptors identify a subgroup of CRC with a mesenchymal phenotype and correlate with poor prognosis. Dual inhibition of AXL and TGFß could represent a novel therapeutic strategy for patients with this aggressive disease.

PD-1 blockade delays tumor growth by inhibiting an intrinsic SHP2/Ras/MAPK signalling in thyroid cancer cells.

BACKGROUND: The programmed cell death-1 (PD-1) receptor and its ligands PD-L1 and PD-L2 are immune checkpoints that suppress anti-cancer immunity. Typically, cancer cells express the PD-Ls that bind PD-1 on immune cells, inhibiting their activity. Recently, PD-1 expression has also been found in cancer cells. Here, we analysed expression and functions of PD-1 in thyroid cancer (TC). METHODS: PD-1 expression was evaluated by immunohistochemistry on human TC samples and by RT-PCR, western blot and FACS on TC cell lines. Proliferation and migration of TC cells in culture were assessed by BrdU incorporation and Boyden chamber assays. Biochemical studies were performed by western blot, immunoprecipitation, pull-down and phosphatase assays. TC cell tumorigenicity was assessed by xenotransplants in nude mice. RESULTS: Human TC specimens (47%), but not normal thyroids, displayed PD-1 expression in epithelial cells, which significantly correlated with tumour stage and lymph-node metastasis. PD-1 was also constitutively expressed on TC cell lines. PD-1 overexpression/stimulation promoted TC cell proliferation and migration. Accordingly, PD-1 genetic/pharmacologic inhibition caused the opposite effects. Mechanistically, PD-1 recruited the SHP2 phosphatase to the plasma membrane and potentiated its phosphatase activity. SHP2 enhanced Ras activation by dephosphorylating its inhibitory tyrosine 32, thus triggering the MAPK cascade. SHP2, BRAF and MEK were necessary for PD-1-mediated biologic functions. PD-1 inhibition decreased, while PD-1 enforced expression facilitated, TC cell xenograft growth in mice by affecting tumour cell proliferation. CONCLUSIONS: PD-1 circuit blockade in TC, besides restoring anti-cancer immunity, could also directly impair TC cell growth by inhibiting the SHP2/Ras/MAPK signalling pathway.

Lymph node fine needle aspiration cytology (FNAC) in paediatric patients: Why not? Diagnostic accuracy of FNAC in a series of heterogeneous paediatric lymphadenopathies.

Fine needle aspiration cytology (FNAC) can be a precious tool for the evaluation of lymphadenopathies in children and adolescents. The purpose of this study was to analyse the diagnostic accuracy of FNAC in a paediatric lymph node series. We report a series of 76 patients, aged up to 19 years, who underwent lymph node FNAC. In our series, 57 cases were diagnosed as non-neoplastic, including benign reactive hyperplasia and other inflammatory lesions, 18 cases were diagnosed as malignant and 1 case was diagnosed as suspicious for lymphoproliferative process, not otherwise specified. Sensitivity, specificity, positive predictive value and negative predictive value were 93 %, 100 %, 100 % and 98 %, respectively. Diagnostic accuracy resulted 98.6 %. FNAC is an accurate, minimally invasive method with minimal complications that allows evaluation of paediatric lymphadenopathies and a correct triage of reactive/inflammatory and neoplastic lymphadenopathies. The application of rapid on-site evaluation, the realization of a cell block and the application of ancillary diagnostic tests, including at least immunocytochemistry and flow cytometry, allows to achieve an excellent diagnostic performance.

Adequacy of Cytologic Samples by Ultrasound-Guided Percutaneous Transthoracic Fine-Needle Aspiration Cytology of Peripheral Pulmonary Nodules for Morphologic Diagnosis and Molecular Evaluations: Comparison With Computed Tomography-Guided Percutaneous Transthoracic Fine-Needle Aspiration Cytology.

CONTEXT.­: Fine-needle aspiration cytology (FNAC) of pulmonary nodules is usually guided by computed tomography (CT), whereas ultrasonography (US) is generally considered not applicable for such purposes. OBJECTIVE.­: To evaluate the clinical applicability and diagnostic utility of US-guided transthoracic FNAC of peripheral pulmonary nodules. DESIGN.­: Ultrasonography-guided transthoracic FNAC was obtained from 40 selected patients with peripheral, subpleural, and paravertebral pulmonary nodules. Air-dried and Diff-Quik-stained smears were used for rapid on-site evaluation; additional smears were alcohol fixed for Papanicolaou staining. Cell blocks were set up for immunocytochemical and molecular studies; in 2 cases, a flow cytometry evaluation was also performed. The series was compared to 40 CT-guided pulmonary FNAC samples from patients with pleural, peripheral, and paravertebral pulmonary nodules, to evaluate differences in terms of diagnostic rate, time of execution, safety, and cost. RESULTS.­: The US-guided FNAC samples had results that were adequate and representative in 95% of cases. No significant differences were observed between the 2 groups in terms of diagnostic rate, number of passes, and cellularity of both smears and cell blocks. The mean time needed for the execution of US-guided FNAC was 13.1 minutes, whereas the mean time for CT-guided FNAC was 23.6 minutes. Thus, US-guided FNAC was significantly more rapid than CT-guided pulmonary FNAC. Because pneumothorax occurred in 1 individual who underwent US-guided FNAC and in 9 who underwent CT-guided FNAC, we might conclude that US-guided FNAC is a significantly safer procedure. Finally, comparing the costs of both procedures, US-guided FNAC is less expensive. CONCLUSIONS.­: Our experience showed an elevated clinical applicability and diagnostic utility of US-guided transthoracic FNAC for selected pulmonary nodules.

Structural analysis of human SEPHS2 protein, a selenocysteine machinery component, over-expressed in triple negative breast cancer.

Selenophosphate synthetase 2 (SEPHS2) synthesizes selenide and ATP into selenophosphate, the selenium donor for selenocysteine (Sec), which is cotranslationally incorporated into selenoproteins. The action and regulatory mechanisms of SEPHS2 as well as its role in carcinogenesis (especially breast cancer) remain ambiguous and need further clarification. Therefore, lacking an experimentally determined structure for SEPHS2, we first analyzed the physicochemical properties of its sequence, modeled its three-dimensional structure and studied its conformational behavior to identify the key residues (named HUB nodes) responsible for protein stability and to clarify the molecular mechanisms by which it induced its function. Bioinformatics analysis evidenced higher amplification frequencies of SEPHS2 in breast cancer than in other cancer types. Therefore, because triple negative breast cancer (TNBC) is biologically the most aggressive breast cancer subtype and its treatment represents a challenge due to the absence of well-defined molecular targets, we evaluated SEPHS2 expression in two TNBC cell lines and patient samples. We demonstrated mRNA and protein overexpression to be correlated with aggressiveness and malignant tumor grade, suggesting that this protein could potentially be considered a prognostic marker and/or therapeutic target for TNBC.

Simultaneous detection of lung fusions using a multiplex RT-PCR next generation sequencing-based approach: a multi-institutional research study.

BACKGROUND: Gene fusion events resulting from chromosomal rearrangements play an important role in initiation of lung adenocarcinoma. The recent association of four oncogenic driver genes, ALK, ROS1, RET, and NTRK1, as lung tumor predictive biomarkers has increased the need for development of up-to-date technologies for detection of these biomarkers in limited amounts of material. METHODS: We describe here a multi-institutional study using the Ion AmpliSeq™ RNA Fusion Lung Cancer Research Panel to interrogate previously characterized lung tumor samples. RESULTS: Reproducibility between laboratories using diluted fusion-positive cell lines was 100%. A cohort of lung clinical research samples from different origins (tissue biopsies, tissue resections, lymph nodes and pleural fluid samples) were used to evaluate the panel. We observed 97% concordance for ALK (28/30 positive; 71/70 negative samples), 95% for ROS1 (3/4 positive; 19/18 negative samples), and 93% for RET (2/1 positive; 13/14 negative samples) between the AmpliSeq assay and other methodologies. CONCLUSION: This methodology enables simultaneous detection of multiple ALK, ROS1, RET, and NTRK1 gene fusion transcripts in a single panel, enhanced by an integrated analysis solution. The assay performs well on limited amounts of input RNA (10 ng) and offers an integrated single assay solution for detection of actionable fusions in lung adenocarcinoma, with potential savings in both cost and turn-around-time compared to the combination of all four assays by other methods.

Diffuse Idiopathic Pulmonary Neuroendocrine Cell Hyperplasia (DIPNECH) Syndrome and Carcinoid Tumors With/Without NECH: A Clinicopathologic, Radiologic, and Immunomolecular Comparison Study.

The diagnostic criteria of diffuse idiopathic pulmonary neuroendocrine cell hyperplasia (DIPNECH) are not well defined, and DIPNECH can be mistaken for carcinoids associated with neuroendocrine cell hyperplasia (NECH). In this study, we compared clinical, radiologic, histologic, immunohistochemical, and molecular features of DIPNECH and isolated carcinoids with/without NECH. The study population included 151 cases (77 female patients and 74 male patients), 19 with DIPNECH and 132 with carcinoids with/without NECH. None of the cases displayed molecular alterations or anaplastic lymphoma kinase expression. Compared with individuals with carcinoids with/without NECH, patients with DIPNECH were more likely to be female individuals (P<0.0001), nonsmokers (P=0.021), and symptomatic, and to have an obstructive/mixed respiratory defect, peripheral location of the lesions, and air trapping (P<0.0001) on chest computed tomography, and constrictive bronchiolitis on histology (P<0.0001). Among immunohistochemical markers, DIPNECH was associated with higher expression of thyroid transcription factor-1, CD10, and gastrin-releasing peptide/bombesin-like peptide (P<0.0001). Yet, when a purely histopathologic definition of DIPNECH was applied, 40% of isolated carcinoids also met the diagnostic criteria for DIPNECH, even in the absence of symptoms and/or radiologic abnormalities. Therefore, as DIPNECH represents a distinct clinical syndrome, we suggest the term DIPNECH be limited to cases presenting with respiratory symptoms, functional and/or radiologic abnormalities, and constrictive bronchiolitis on histology.

Immunohistochemistry for Cancer Stem Cells Detection: Principles and Methods.

Cancer stem cells (CSCs) are rare immortal cells within a tumor that can self-renew and drive tumorigenesis. CSCs play a pivotal role in the tumor development, progression and relapse, as well as in the resistance of anticancer therapy. Different tools could help in the analysis of CSCs, especially Immunohistochemistry (IHC) represents a useful technique able to identify several specific CSC markers. The main aims of this chapter are the description of the explain immunohistochemical methods used in the characterization of CSCs. Furthermore, focus on the most common troubleshooting in CSCs IHC is provided, especially the pitfalls of the CSCs markers IHC on tissue microarrays.

Effects of the single and combined treatment with dopamine agonist, somatostatin analog and mTOR inhibitors in a human lung carcinoid cell line: an in vitro study.

Somatostatin analogues and mTOR inhibitors have been used as medical therapy in lung carcinoids with variable results. No data are available on dopamine agonists as treatment for lung carcinoids. The main aim of the current study was to evaluate the effect of the combined treatment of somatostatin analogue octreotide and the dopamine agonist cabergoline with mTOR inhibitors in an in vitro model of typical lung carcinoids: the NCI-H727 cell line. In NCI-H727 cell line, reverse transcriptase-quantitative polymerase chain reaction and immunofluorescence were assessed to characterize the expression of the somatostatin receptor 2 and 5, dopamine receptor 2 and mTOR pathway components. Fifteen typical lung carcinoids tissue samples have been used for somatostatin receptor 2, dopamine receptor 2, and the main mTOR pathway component p70S6K expression and localization by immunohistochemistry. Cell viability, fluorescence-activated cell sorting analysis and western blot have been assessed to test the pharmacological effects of octreotide, cabergoline and mTOR inhibitors, and to evaluate the activation of specific cell signaling pathways in NCI-H727 cell line. NCI-H727 cell line expressed somatostatin receptor 2, somatostatin receptor 5 and dopamine receptor 2 and all mTOR pathway components at messenger and protein levels. Somatostatin receptor 2, dopamine receptor 2, and p70S6K (non phosphorylated and phosphorylated) proteins were expressed in most typical lung carcinoids tissue samples. Octreotide and cabergoline did not reduce cell viability as single agents but, when combined with mTOR inhibitors, they potentiate mTOR inhibitors effect after long-term exposure, reducing Akt and ERK phosphorylation, mTOR escape mechanisms, and increasing the expression DNA-damage-inducible transcript 4, an mTOR suppressor. In conclusion, the single use of octreotide and cabergoline is not sufficient to block cell viability but the combined approach of these agents with mTOR inhibitors might reduce the mTOR inhibitors-induced escape mechanisms and/or activate the endogenous mTOR suppressor, potentiating the effect of the mTOR inhibitors in an in vitro model of typical lung carcinoids.