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Cardiac amyloidosis has been strongly associated with postoperative intractable circulatory failure, and intestinal amyloidosis could lead to intestinal pseudo-obstruction. The latter can be treated with neostigmine, which is notorious for its brief bradyarrhythmic complications. The amyloidosis patient presented herein, suffered an iatrogenic left main dissection, failure of bailout stenting and finally underwent urgent surgery. Meticulous fluid and drug management was key to keeping this patient stable. Postoperative atrial fibrillation was treated with amiodarone. The postoperative course was complicated with intestinal pseudo-obstruction, which was ultimately resolved with neostigmine. This short-lived cholinesterase inhibitor interacted with amiodarone and caused a previously undocumented prolonged complete atrioventricular block that resolved 48 hours after both drugs’ discontinuation. The neostigmine amiodarone interaction warrants clinical vigilance and is speculated to be due to their partially shared second messenger pathway involving cyclic adenosine monophosphate. Patients with cardiac amyloidosis could maintain hemodynamic stability perioperatively.

A key unknown of the functional space in tumor immunity is whether CD4 T cells depend on intratumoral MHCII cancer antigen recognition. MHCII-expressing, antigen-presenting cancer-associated fibroblasts (apCAFs) have been found in breast and pancreatic tumors and are considered to be immunosuppressive. This analysis shows that antigen-presenting fibroblasts are frequent in human lung non-small cell carcinomas, where they seem to actively promote rather than suppress MHCII immunity. Lung apCAFs directly activated the TCRs of effector CD4 T cells and at the same time produced C1q, which acted on T cell C1qbp to rescue them from apoptosis. Fibroblast-specific MHCII or C1q deletion impaired CD4 T cell immunity and accelerated tumor growth, while inducing C1qbp in adoptively transferred CD4 T cells expanded their numbers and reduced tumors. Collectively, we have characterized in the lungs a subset of antigen-presenting fibroblasts with tumor-suppressive properties and propose that cancer immunotherapies might be strongly dependent on in situ MHCII antigen presentation.

Background PD-L1 expression on tumor and immune cells plays an important role in many cancers, including breast cancer (BC), however, discordance has been demonstrated between primary tumors and matched distant metastases. PD-L1 is expressed on circulating tumor cells (CTCs) in BC, however limited data exist on PD-L1 expression on the respective peripheral blood mononuclear cells (PBMCs). In the current study we aimed to investigate tumor and immune PD-L1 expression in primary tumors, and matched peripheral blood (PB) and metastatic sites from CTC-positive patients with BC. Methodology PB was collected from 96 BC patients (early: n=72; de novo metastatic: n=24) and PBMCs were enriched by ficoll-density gradient centrifugation. PBMC cytospins were immunofluorescently stained using antibodies for cytokeratins (Clones: AE1/AE3 & C11) to detect CTCs and PD-L1 (Clone: E1L3N). Matched primary tumor samples from CTC-positive patients were evaluated for PD-L1 expression on tumor cells and TILs by immunocytochemistry (positivity cut-off: ≥1% positive tumor cells or immune cells, respectively). In 7 patients, matched metastatic tumor tissue was also available for analysis. Results CK+ CTCs were identified in 18 out of 96 BC patients (early disease: 11/72, de novo metastatic: 7/24). Overall, PD-L1+ CTCs were detected in 22% of CTC+ patients and PDL1+ tumor cells in 28% of the respective primary tumors. However, there was no concordance for PD-L1 expression positivity between CTCs and corresponding primary tumor cells. PD-L1+ PBMCs were identified in 23% of patients, whereas PD-L1+ TILs were detected in 65% of primary tumor samples (positive concordance 17.6%). In addition, there was no concordance for PD-L1-positivity between PB samples and metastatic tumor samples, regarding either tumor cells (PD-L1+ CTCs identified in 3/7 patients and PD-L1+ tumor cells in 1/7 metastatic tumor samples), or immune cells (PD-L1+ PBMCs were detected in 1/6 and PD-L1-positive TILs in 3/6 metastatic tumors). Finally, when primary and corresponding metastatic tumor samples were compared, PD-L1+ tumor cells were detected in 3 versus 1 of 7 patients, respectively (positive concordance 14.3%), whereas, PD-L1+ TILs were observed in 6 versus 4 patients, respectively (positive concordance, 42.9%). Conclusions Discrepancies in PD-L1 positivity of tumor and immune cells are for the first time demonstrated between PB, primary and metastatic tissue samples in BC. In general, PD-L1 positivity is lower in metastatic compared to primary tumors. In addition, despite that similar rates of PD-L1 expression are observed in primary tumors and the corresponding CTCs, no positive concordance exists. PD-L1 expression on CTCs and PBMCs should be further explored to identify their potential value as prognostic and/or predictive biomarkers in BC patients treated with immunotherapy. Citation Format: Maria A Papadaki, Anastasios V Koutsopoulos, Eleni Lagoudaki, Alexia Monastirioti, Konstantina Thomopoulou, Kostas Kalbakis, Chara Papadaki, Sofia Agelaki, Dimitrios Mavroudis. Comparative analysis of PD-L1 expression on matched primary tumors, metastasis and peripheral blood of patients with breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-05-03.

Objectives The aim of the study was to characterize and evaluate the presence of DLL3-positive Circulating Tumor Cells (CTCs) in SCLC patients receiving front-line chemotherapy and assess their clinical relevance. Materials and methods Peripheral blood was obtained from treatment-naive patients with SCLC (n = 108 patients), after one etoposide/platinum cycle (n = 68 patients) and on disease progression (n = 48 patients). Immunofluorescence staining using antibodies against the DLL3, cytokeratins (CK), CD45 and vimentin (Vim) was used for the detection and characterization of CTCs. Results Before treatment, 74.1% of patients had detectable DLL3+/CD45− CTCs. One-treatment cycle significantly decreased both the detection rate (p Conclusions These findings demonstrate an important heterogeneity of CTCs, based on the expression of CK, Vim and DLL3, in patients with SCLC and the changes of DLL3+/CD45− CTCs during treatment seem to be a dynamic biomarker associated with patients’ clinical outcome.

KRAS mutations are found in approximately one third of non-small cell lung cancer (NSCLC) patients. In this study, we aim to investigate whether KRAS G12/G13 mutant allele fraction (MAF) in cell-free DNA (cfDNA) can provide meaningful prognostic information in NSCLC. Multiplex droplet-digital PCR was used to quantitatively assess KRAS G12/G13 MAF in cfDNA from 114 pre-treated advanced disease NSCLC patients. In 14 patients, changes in KRAS G12/G13 MAF were longitudinally monitored during treatment. Plasma KRAS G12/G13 status was associated with poor patients&rsquo, outcome in terms of progression-free survival (PFS) (p &lt, 0.001) and overall survival (OS) (p &lt, 0.001). In multivariate analysis, the detection of plasma KRAS mutations was an independent predictor of adverse PFS (HR = 3.12, p &lt, 0.001) and OS (HR = 2.53, p = 0.002). KRAS G12/G13 MAF at first treatment evaluation (T1) was higher (p = 0.013) among patients experiencing progressive disease compared to those with disease control, and increased KRAS MAF at T1 was associated (p = 0.005) with shorter PFS. On the contrary, no association was observed between tissue KRAS mutation status and patients&rsquo, prognosis. Our results show that ddPCR-based detection of KRAS G12/G13 mutations in plasma could serve as an independent biomarker of unfavorable prognosis in NSCLC patients. Changes in KRAS MAF can provide valuable information for monitoring patient outcome during treatment.

In situ antigen presentation is required to sustain active proliferating CD4+ T cells in tumours and to help form memory CD8+ T cells, but the antigen presenting cells (APCs) and pathways involved remain elusive. Cancer associated fibroblasts (CAFs) are prominent stromal constituent of solid tumors. Current immunological dogma considers that CAFs facilitate tumour immune escape. A new subset of MHCII antigen presenting cancer-associated fibroblasts (apCAFs) has been described, but their function remains unknown. Here we report a previously unrecognized function of apCAFs in sustaining CD4+ T cells in primary human and murine lung tumours. In response to IFNγ and oxidative stress in tumours CAFs up-regulated MHCII. Fibroblast-specific targeted ablation of MHCII induced a hypometabolic hypoproliferative state in CD4+ T cells, which impacted MHCII and MHCI immunity, accelerating tumor growth. apCAFs directly presented MHCII-peptide (MCHIIp) complexes to activate the TCRs of CD4+ T cell. Highthrough-put profiling and blocking assays unveiled a novel CAF to T cell communication pathway via complement 1q binding on membrane C1qbp. Thus, apCAFs sustain anti-tumor CD4+ T cells via MHCIIp-TCR and C1q-C1qbp binding. Our studies pave the way to the design of novel immunotherapeutic strategies that will harness apCAFs to help sustain T cells inside solid tumours. Statement of significance This work challenges the immunological dogma that dominant physiological significance in tumour specific immunity comes only through professional APCs, leading to the design of novel MHCII fibroblast-directed strategies to sustain endogenous or adoptively transferred CD4+ T cells within solid tumors.

The role of CD47 and PD-L1 expression on circulating tumor cells (CTCs) remains unclear, and it is currently unknown whether their distribution varies between the blood and tumor tissue in breast cancer (BC). In this study, CD47 and PD-L1 expression was investigated a) on peripheral blood mononuclear cell (PBMC) cytospins from early (n = 100) and metastatic (n = 98) BC patients, by triple immunofluorescence for CD47/PD-L1/Cytokeratins, and b) on matched primary and/or metastatic tumor tissue from CTC-positive patients using immunohistochemistry. CD47+and/orPD-L1+ CTCs were detected in 11%, 16.9%, and 29.6% of early, recurrent, and de novo metastatic patients (p = 0.016). In metastatic disease, CD47highand/orPD-L1high CTCs were associated with disease progression (p = 0.005) and shorter progression-free survival (PFS) (p = 0.010), and independently predicted for an increased risk of relapse (HR: 2.719, p = 0.008) and death (HR: 2.398, p = 0.034). PD-L1 expression rates differed between CTCs and tissue tumor cells and between peripheral blood mononuclear cells (PBMCs) and tumor-infiltrating lymphocytes (TILs) (positive concordance of 3.8% and 4%, respectively). CD47 expression also differed between CTCs and tumor cells (positive concordance of 11.5%). In conclusion, CTCs expressing CD47 and PD-L1 have independent poor prognostic implications in metastatic BC, indicating a potential role of innate and adaptive immune evasion mechanisms in their metastatic potential. The clinical value of the parallel assessment of the peripheral and local immune response merits further evaluation in BC.

Bladder leiomyomas (BLs) are extremely rare benign tumors of mesenchymal origin. The exact pathophysiological mechanisms that lead to their appearance remain unclear including hormonal disorders, chromosomal abnormalities, and fetal remnants in the bladder. They usually remain asymptomatic for a long period of time. Solitary fibrous tumors (SFTs) are also rare neoplasms of mesenchymal origin with malignant potential usually affecting the pleura. The pathogenesis of SFTs remains unclear. We report the case of a 28-year-old male presenting with SFT of the pleura and synchronous BL. The patient presented with persistent cough as a sole symptom. Computed tomography (CT) of the thorax revealed a pleural mass, which was surgically removed and proved to be a SFT. At an early follow-up, abdominal CT scan revealed a bladder wall mass that proved to be a BL. This is the first report of BL with synchronous SFT of the pleura. Synchronous BLs and SFTs may be incidental, but the coexistence of two mesenchymal tumors at different sites, in a young patient, may raise the suspicion of a new clinical syndrome that warrants further investigation.

Introduction To investigate the presence of Bcl-2+CTCs in chemotherapy-naive SCLC patients and their clinical relevance during front-line treatment. Methods Peripheral blood was obtained from 66 consecutive-patients before chemotherapy administration, after one-cycle and at relapse. CTCs were detected by CellSearch and immunofluorescence using anti-Bcl-2, anti-M30, anti-cytokeratins(CK), anti-CD45 and anti-vimentin(Vim) antibodies. Results Before treatment, CTCs were detected in 62.1% and 72.7% of patients using the CellSearch and immunofluorescence (Bcl-2+/CD45−), respectively. One-treatment cycle significantly decreased both CTCs’ detection rate(p Conclusions These results demonstrate an important phenotypic CTCs heterogeneity based on the co-expression of Bcl-2, CK, Vim and M30 in SCLC patients. The changes of Bcl-2+/CD45− CTCs during treatment seem to be a dynamic biomarker associated with treatment efficacy and patients’ clinical outcome.

Erlotinib has been approved for the management of NSCLC patients after failure of the first or subsequent line of chemotherapy. Although the efficacy of erlotinib is clearly associated with the presence of EGFR mutations, there is a subset of patients with EGFR wild-type (EGFRwt) tumors who impressively respond. Patients with EGFRwt NSCLC who received salvage (≥2nd line) treatment with erlotinib for a prolonged period (>6 months), were sought from the database of the Hellenic Oncology Research Group. We retrospectively analyzed the clinical, pathological and molecular characteristics of the patients with available tumor material. Forty-four patients that received erlotinib for >6 months (median 10.1 months) were enrolled in the study. The majority of them were male, never-smokers with adenocarcinoma histology and a good performance status. KRAS and PIK3CA mutations were detected in 21% (9/42 tested) and 13% (4/30 tested) of the patients, respectively. The ALK-EML4 translocation was found in 10% (2/20 tested); there was no patient with HER2 or BRAF mutated tumor. Twelve (54.5%) tumor specimens were considered positive for EGFR-overexpression. Eleven patients experienced a partial response (objective response rate 25%; 95% CI 12–38%) and the remaining 33 had stable disease. The median progression-free survival and overall survival were 10.1 (95% CI 8.6–11.6 months) and 24.1 (95% CI 11.2–37 months), respectively. Treatment with erlotinib significantly improves the clinical outcome in a subset of NSCLC patients with EGFRwt tumors. Further molecular analysis of such tumor specimens could provide a more comprehensive characterization of this particular group of patients. Nevertheless, the presence of other mutations should not prevent the treating physician from using erlotinib at later lines of salvage therapy for NSCLC patients.

Background Circulating tumor cells (CTCs) are important for metastatic dissemination of cancer. They can provide useful information, regarding biological features and tumor heterogeneity; however, their detection and characterization are difficult due to their limited number in the bloodstream and their mesenchymal characteristics. Therefore, new biomarkers are needed to address these questions. Methods Bioinformatics functional enrichment analysis revealed a subgroup of 24 genes, potentially overexpressed in CTCs. Among these genes, the chemokine receptor CXCR4 plays a central role. After prioritization according to the CXCR4 corresponding pathways, five molecules (JUNB, YWHAB, TYROBP, NFYA, and PRDX1) were selected for further analysis in biological samples. The SKBR3, MDA-MB231, and MCF7 cell lines, as well as PBMCs from normal (n = 10) blood donors, were used as controls to define the expression pattern of all the examined molecules. Consequently, 100 previously untreated metastatic breast cancer (mBC) patients (n = 100) were analyzed using the following combinations of antibodies: CK (cytokeratin)/CXCR4/JUNB, CK/NFYA/ΥWHΑΒ (14-3-3), and CK/TYROBP/PRDX1. A threshold value for every molecule was considered the mean expression in normal PBMCs. Results Quantification of CXCR4 revealed overexpression of the receptor in SKBR3 and in CTCs, following the subsequent scale (SKBR3>CTCs>Hela>MCF7>MDA-MB231). JUNB was also overexpressed in CTCs (SKBR3>CTCs>MCF7>MDA-MB231>Hela). According to the defined threshold for each molecule, CXCR4-positive CTCs were identified in 90% of the patients with detectable tumor cells in their blood. In addition, 65%, 75%, 14.3%, and 12.5% of the patients harbored JUNB-, TYROBP-, NFYA-, and PRDX-positive CTCs, respectively. Conversely, none of the patients revealed YWHAB-positive CTCs. Interestingly, JUNB expression in CTCs was phenotypically and statistically enhanced compared to patients’ blood cells (p = 0.002) providing a possible new biomarker for CTCs. Furthermore, the detection of JUNB-positive CTCs in patients was associated with poorer PFS (p = 0.015) and OS (p = 0.002). Moreover, JUNB staining of 11 primary and 4 metastatic tumors from the same cohort of patients revealed a dramatic increase of JUNB expression in metastasis. Conclusions CXCR4, JUNB, and TYROBP were overexpressed in CTCs, but only the expression of JUNB was associated with poor prognosis, providing a new biomarker and a potential therapeutic target for the elimination of CTCs. Electronic supplementary material The online version of this article (10.1186/s13058-019-1166-4) contains supplementary material, which is available to authorized users.

Despite the development of new treatment options based on the molecular characterization of colorectal cancer, 20% of patients present de novo metastatic disease, whereas 30-40% of patients who receive curative treatment relapse during follow up. Herein, we report 2 cases with rectal cancer that developed uncommon sites of metastasis; the first patient had an isolated breast metastasis, while the second patient developed bone marrow infiltration with synchronous brain metastases. In order to evaluate the uncommon metastatic pattern of rectal cancer, we detected and enumerated circulating tumor cells (CTCs) using both immunofluorescence and real-time reverse transcriptase polymerase chain reaction in these patients’ peripheral blood. The procedure revealed the presence of CTCs, positive for CEACAM5 but negative for epithelial phenotype (EpCAM-), that might explain the patients’ metastatic potential and survival.

Differences in gene expression levels between the primary tumors (PTs) and matched regional lymph nodal metastases (LNs) in patients with totally excised non-small cell lung cancer (NSCLC) were explored. Microdissected formalin-fixed paraffin-embedded (FFPE) samples from (PT) and their matched infiltrated LNs, from 239 patients [183 (with matched PT and LNs samples)-case and 56 PT only samples-control cohorts] were analyzed for BRCA1, ERCC1, RAP80, PKM2, RRM1, RRM2, TS, TSP1, and TXR1 mRNA expression by quantitative real-time polymerase-chain reaction (PCR). Moderately positive correlation between the expression of each gene in the PT and the matched LNs was observed. Concordance rates between the PT and the LNs were: BRCA1 (67.7%), ERCC1 (68.4%), PKM2 (63.4%), RAP80 (68.8%), RRM1 (70.9%), RRM2 (69%), TS (72.9%), TSP1 (69.8%), TXR1 (63.7%). Expression levels and their differences were correlated with Relapse-Free Survival (RFS) and Overall Survival (OS). High BRCA1 PT in patients with squamous histology was associated with increased OS (p = 0.036). High TSP1 PT levels were shown to be the only independent prognostic factor for OS and RFS (p = 0.023 and p = 0.007). PKM2 low levels in both PT and matched LNs were associated with better OS irrespective of the underlying histology (p = 0.031). RRM1 discordant levels between PT and matched LNs were associated with worse OS in squamous tumors (p = 0.019) compared to patients with both low expression in PT and LN.TXR1 high levels in both PT and matched LNs were associated with better OS in patients with squamous tumors (p = 0.007).These findings indicate that there is different gene expression between PT and matched LNs which may affect the outcome in early NSCLC and therefore PT’s molecular biology should not be the sole determinant for prognostication.

// Ismini Lasithiotaki 1, 2 , Eliza Tsitoura 2, 3 , Anastasios Koutsopoulos 4 , Eleni Lagoudaki 4 , Chara Koutoulaki 2 , George Pitsidianakis 1 , Demetrios A. Spandidos 3 , Nikolaos M. Siafakas 1 , George Sourvinos 3, * , Katerina M. Antoniou 1, 2, * 1 Department of Thoracic Medicine, University Hospital, Medical School, University of Crete, Heraklion 71110, Greece 2 Laboratory of Cellular and Molecular Pneumonology, Medical School, University of Crete, Heraklion Crete 71110, Greece 3 Laboratory of Clinical Virology, Medical School, University of Crete, Heraklion Crete 71110, Greece 4 Department of Pathology, Medical School, University of Crete, Heraklion Crete 71110, Greece * These authors have contributed equally to this work Correspondence to: George Sourvinos, email: sourvino@med.uoc.gr Keywords: merkel cell polyomavirus, non-small cell lung cancer, microRNAs, signalling pathways Received: April 23, 2016 Accepted: July 19, 2016 Published: August 11, 2016 ABSTRACT Merkel Cell Polyoma Virus (MCPyV) infection has been associated with non-small cell lung cancer (NSCLC). Viruses can manipulate cellular miRNAs or have a profound impact on cellular miRNA expression to control host regulatory pathways. In this study, we evaluated the expression profiles of cancer-associated and virally affected host microRNAs miR-21, miR-145, miR-146a, miR-155, miR-302c, miR-367 and miR-376c in a series of NSCLC tissue samples as well as in samples from “healthy” sites, distant from the tumour region that were either positive or negative for MCPyV DNA. miR-21 and miR-376c were significantly upregulated whereas miR-145 was significantly downregulated in the MCPyV+ve samples compared to the MCPyV-ve tumour samples. Overall, miR-21 and miR-376c expression was higher in tumour compared to healthy tissue samples. No association was observed between the miR-155, miR-146a, miR-302c and miR-367 levels and the presence of MCPyV. The expression of miR-21 target genes ( Pten, Bcl-2, Daxx, Pkr, Timp3) , miR-376c ( Grb2, Alk7 , Mmp9 ) and miR-145 ( Oct-4, Sox2 , Fascin1 ) and their associated pathways ( Braf, Akt-1, Akt-2, Bax, Hif1a, p53 ) was altered between MCPyV+ve tumor samples and their corresponding controls. These results show a novel association between miR-21, miR-376c and miR-145 and their host target genes with the presence of MCPyV, suggesting a mechanism of virus-specific microRNA signature in NSCLC.

Background: Since tumor cells may escape from immune surveillance through the programmed cell death 1 (PD-1)/programmed death ligand (PD-L)1 axis, this study was designed in order to evaluate whether there is a correlation between the levels of PD-1+ and PD-L1+-expressing immune cells (ICs) and circulating tumor cells (CTCs) in patients with non-small cell lung cancer (NSCLC). Patients and methods: Peripheral blood was obtained from 37 chemotherapy-naïve patients with metastatic NSCLC before treatment. PD-1 and PD-L1 expression was evaluated (1) on ICs with anti-tumor function (CD4+ and CD8+ T-cells, B-cells, monocytes/dendritic cells) using flow cytometry, (2) on CTCs by immunofluorescence and (3) on cells from tumor tissues by immunohistochemistry. The levels of PD-1+ and PD-L1+-expressing ICs were correlated with progression-free survival (PFS). Results: The presence of PD-1+ CD8+ cells, with reduced interferon (IFN)-γ expression, but not other ICs, were positively correlated with PD-L1+ CTCs (p < 0.04). Increased percentages of PD-1+ CD8+ T-cells, were associated with a worse response to treatment (p = 0.032) and shorter PFS (p = 0.023) which, in multivariate analysis, was revealed as an independent predictor for decreased PFS [hazard ratio (HR): 4.1, p = 0.0007]. Conclusion: The results of the current study, for first time, provide evidence for a possible interaction between ICs and CTCs in NSCLC patients via the PD-1/PD-L1 axis and strongly support that the levels of PD-1+ CD8+ in these patients may be of clinical relevance.

Liquid biopsy analysis, mainly based on circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA), provides an extremely powerful tool for the molecular profiling of cancer patients in real time. In this study, we directly compared PIK3CA hotspot mutations (E545K, H1047R) in EpCAM‐positive CTCs and paired plasma‐ctDNA in breast cancer (BrCa). PIK3CA hotspot mutations in CTCs and ctDNA were analyzed using our previously developed highly sensitive (0.05%), specific, and validated assay in plasma‐ctDNA from 77 early and 73 metastatic BrCa patients and 40 healthy donors. We further analyzed and directly compared PIK3CA hotspot mutations in DNAs isolated from CellSearch® cartridges (CTCs) and paired plasma‐ctDNA, in 56 cases of early and 27 cases of metastatic breast cancer, and 16 corresponding primary tumors. In plasma‐ctDNA,PIK3CA hotspot mutations were identified in 30/77(39.0%) early and 35/73(47.9%) metastatic BrCa cases; none (0/40, 0%) of the healthy donors’ plasma‐ctDNA samples were positive. Our direct comparison study in DNAs isolated from CellSearch® cartridges (CTCs) and paired plasma‐ctDNA from the same blood draws has shown a lack of concordance in early BrCa (27/56, 48.2%), while the concordance in the metastatic setting was higher (18/27, 66.6%). Our results were validated by ddPCR methodology, and the concordance between our assay and ddPCR for PIK3CA E545K hotspot mutation was 30/37 (81.1%). In many cases, PIK3CA hotspot mutations were detected in samples found to be negative for CTCs in CellSearch®. Our data demonstrated for the first time that (a) PIK3CA hotspot mutations are present at high frequencies in CTCs isolated from CellSearch® cartridges and paired plasma‐ctDNA both in early and metastatic BrCa, (b) the detection and concordance of PIK3CA hotspot mutations between plasma‐ctDNA and CTCs are higher in the metastatic setting, (c) PIK3CA mutational status significantly changes after therapeutic intervention, and (d) PIK3CA mutation detection in CTCs and plasma‐ctDNA provides complementary information., Our direct comparison study on the presence of PIK3CA hotspot mutations (E545K, H1047R) in EpCAM‐positive CTCs and paired plasma‐ctDNA demonstrated for the first time that PIK3CA mutations are present at high frequencies in both CTCs and paired plasma‐ctDNA and that there is a high concordance in the metastatic setting. PIK3CA mutational status significantly changes after therapeutic intervention.

Background: Circulating tumor cells (CTCs) could escape from the immune system through the programmed death-ligand 1 (PD-L1)/programmed cell death protein 1 (PD-1) axis leading to the development of metastasis. The current study investigated the expression of PD-1/PD-L1 on CTCs isolated from non-small cell lung cancer (NSCLC) patients treated with chemotherapy. Patients and methods: CTCs were isolated from 30 chemo-naïve stage IV NSCLC patients before and after front-line chemotherapy using the ISET filtration platform. CTCs were detected by Giemsa and immunofluorescence (IF) staining. Samples were analyzed with the ARIOL system. Results: Giemsa staining revealed that 28 (93.3%) out of 30 and 9 (81.8%) out of 11 patients had detectable CTCs at baseline and after the third chemotherapy cycle, respectively. Cytokeratin (CK)+/CD45- CTCs by IF could be detected in 17 of 30 (56.7%) patients at baseline and in 8 of 11 (72.7%) after the third chemotherapy cycle. Spearman analysis revealed a significant correlation ( p = 0.001) between Giemsa-positive and IF-positive (CK+/CD45-) CTCs. At baseline, PD-1 and PD-L1 expression was observed in 53% and in 47% CK-positive patients, respectively. After the third treatment cycle the corresponding numbers were 13% and 63% respectively. Median progression-free survival (PFS) was significantly shorter in patients with >3 PD-1(+) CTCs at baseline compared with those with 1 Giemsa-positive tumor cells ( p = 0.025). Conclusion: PD-1(+) and PD-L1(+) CTCs could be detected before and after front-line chemotherapy in patients with metastatic NSCLC. The presence of high PD-1(+) CTC numbers before treatment is associated with a poor patient clinical outcome.

Background Increased protein citrullination and peptidylarginine deiminases (PADIs), which catalyze the citrullination process, are central in Rheumatoid arthritis pathogenesis and probably involved in the initial steps towards autoimmunity. Approximately, 10% of RA patients develop clinically significantly ILD. A possible shared role of protein citrullination in rheumatoid arthritis associated interstitial lung disease (RA-ILD), and idiopathic pulmonary fibrosis (IPF) pathogenesis remains unclear. Methods We evaluated PADI2 and PADI4 mRNA expression in bronchoalveolar lavage fluid (BALF) cells of 59 patients with IPF, 27 patients RA-ILD and 10 healthy controls. PADI 2 and 4 expression was analyzed by western blot and immunohistochemistry. Citrullinated protein levels were also quantified. Results PADI4 mRNA and protein levels were higher in RA-ILD and IPF than controls. Furthermore, PADI4 mRNA levels showed an increase among smokers in RA-ILD. PADI4 expression was detected in granulocytes and macrophages in all groups, with the strongest cytoplasmic expression observed in granulocytes in RA-ILD and IPF. PADI2 mRNA and immunostaining of BAL cells, were similar in all groups among smokers. Overall, stronger staining was observed in current smokers. Citrullinated peptides were significantly increased in IPF compared to RA-ILD and controls. In RA-ILD, protein citrullination strongly correlated with PADI4 expression and anti-citrullinated protein antibodies (ACPAs). Conclusions These results suggest that the citrullination pathway is upregulated in IPF and in RA-ILD. Electronic supplementary material The online version of this article (10.1186/s12931-017-0692-9) contains supplementary material, which is available to authorized users.

Objectives: Acute lower limb ischemia caused by tumor embolization is rare, despite the fact that cancer is a common cause of hypercoagulability predisposing to venous thrombosis. Arterial embolization is mostly associated with intracardiac tumors while lung malignancies are the second most common cause of tumor embolism. Methods: In this report, we present a male patient who developed acute bilateral lower limb ischemia in the immediate postoperative period after a thoracotomy for attempted left upper lobe resection for lung cancer. Results: The patient was treated with a subclavian-bifemoral bypass, since an initial attempted embolectomy was unsuccessful. Histopathological characteristics of thrombus obtained during embolectomy were consistent with the histopathology of the primary tumor. Conclusion: Acute ischemia is an emergency that should be diagnosed and treated immediately. Interestingly, in this case, the presence of epidural anesthesia masked the initial symptoms and delayed diagnosis. Preoperative assessment with transesophageal echocardiography may reveal patients at high risk for tumor embolism.

Background To evaluate the phenotypic heterogeneity of circulating tumor cells (CTCs) based on the expression of proliferative, apoptotic and Epithelial-to-Mesenchymal Transmission (EMT) markers during front-line treatment in patients with small cell lung cancer (SCLC) and to evaluate their clinical relevance. Methods CTCs from 108 chemotherapy-naive patients with SCLC were analyzed by double immunofluorescence staining using anti-Ki67, anti-M30, anti-Vimentin along with anti-CKs antibodies. In 83 patients CTCs were also enumerated using the CellSearch. Results Sequential samples were available from 76 and 48 patients after one-treatment cycle and on disease progression (PD), respectively, for immunofluorescence and from 50 and 36 patients after one-cycle and on PD, respectively, for CellSearch. At baseline, 60.2% of the patients had detectable CTCs by either method. Both proliferative (CK67+) and non-proliferative (Ki67-), apoptotic (M30+) and non-apoptotic (M30-) as well as EMT (Vim+) CTCs were present in the same patient. Among 22 patients without detectable CTCs by CellSearch, CK+/Ki67+ and CK+/Vim+ CTCs could be detected in 6 (27.3%) and 6 (27.3%) patients, respectively. One-chemotherapy cycle reduced both the incidence of detection (p

Background: Tumour cells exclusively express the embryonic M2 isoform of pyruvate kinase (PKM2). PKM2 expression levels have been correlated with the effect of platinum compounds in cancer cell lines and xenograft models. The potential predictive role of PKM2 in patients with metastatic/advanced non-small-cell lung cancer (NSCLC) receiving platinum-based chemotherapy as first-line was investigated. Methods: Quantitative real-time PCR was used to assess the expression of PKM2 in tumour samples from 148 and 157 NSCLC patients in the training and the validation set, respectively. All patients received front-line platinum-based chemotherapy. PKM2 mRNA expression was also analysed in a control group of 85 NSCLC patients treated with non-platinum containing regimens. Results: In the training set, high PKM2 mRNA levels were associated with decreased progression-free survival (PFS; 4.9 months vs 6.4, P=0.006), overall survival (OS; 10.1 vs 17.0 months, P=0.01) and disease control rate (DCR; 57.7% vs 74.3% P=0.021) compared to patients with low PKM2 levels. In the validation set, high PKM2 mRNA levels were also associated with deceased PFS (3.7 vs 5.9 months, P=0.006), OS (8.3 vs 16.8 months, P=0.003) and DCR (57.7% vs 70.9% P=0.049) compared to those with low PKM2 mRNA levels. There was no correlation between the PKM2 mRNA levels and the PFS (5.6 vs 5.9, P=0.43) or the OS (9.8 vs 10.1, P=0.51) in the control group. Multivariate analysis revealed high PKM2 mRNA expression as an independent predictive factor for the poor patients' outcome. Conclusions: PKM2 expression may be a predictive biomarker of platinum sensitivity in advanced NSCLC patients treated with platinum-based chemotherapy.

The aim of the study was to evaluate the phenotypic CTCs heterogeneity (TTF-1+ and/or CD56+) in SCLC patients and correlate it with the CellSearch. Peripheral blood was obtained from 108 consecutive patients. CTCs were detected by CellSearch and double-immunofluorescence using anti-CD45, anti-TTF-1 and anti-CD56 antibodies. Before chemotherapy TTF-1+/CD45−, CD56+/CD45− and TTF-1+/CD56+ CTCs were detected in 66(61.1%), 55(50.9%) and 46(42.6%) patients, respectively; 60.2% of patients were CellSearch+. Among the 22 patients with 0 CTCs/7.5 ml on CellSearch, TTF-1+/CD45−, CD56+/CD45− and TTF-1+/CD56+ CTCs were detected in 8(36.4%), 6(27.3) and 6(27.3%) patients, respectively; no CK+/EpCAM+ or TTF1+/EpCAM+ CTCs were detected in these patients. One-chemotherapy cycle decreased both the number of positive patients (p p p = 0.048) and OS (p = 0.041), respectively. There is an important CTC heterogeneity in such patients according to the expression of TTF-1 and CD56 which could detect EpCAM− CTC subpopulations and, thus, undetectable by CellSearch. These CTC subpopulations are dynamically correlated with treatment efficacy and disease-progression.

e20086 Background: The aim of the study was to characterize and evaluate the presence of DLL3-positive Circulating Tumor Cells (CTCs) in SCLC patients receiving front-line chemotherapy and assess their clinical relevance. Methods: Peripheral blood was obtained from treatment-naïve patients with SCLC (n = 108 patients), after one-etoposide/platinum cycle (n = 68 patients) and on disease progression (n = 48 patients). CTCs were detected after immunofluorescence staining using antibodies against the DLL3, cytokeratins (CK), CD45 and vimentin (Vim). Results: Before treatment, 74.1% of patients had detectable CTCs (DLL3+/CD45-). One-treatment cycle significantly decreased both the detection rate of DLL3+/CD45- CTCs ( p< 0.001) and their absolute number ( p< 0.001). Triple immunofluorescence staining using anti-CK, anti-Vim and anti-DLL3 antibodies revealed an important CTC heterogeneity since DLL3 could be detected in Vim+, Vim-, CK+ and CK- CTCs. On disease progression, both the detection rate of DLL3+/CD45- CTCs as well as their number were significantly increased compared to post-1st cycle values ( p< 0.001 and p= 0.002, respectively). In addition, 22.7% of patients had detectable DLL3+/CD45- cells which could not be captured by the CellSearch assay. In multivariate analysis, the detection of DLL3+/CD45- CTCs at baseline was significantly associated with decreased progression-free survival (HR = 10.8; p= 0.005) whereas their detection on disease progression was associated with decreased overall survival (HR: 28.2; p= 0.016). Conclusions: These findings demonstrate an important heterogeneity of CTCs, based on the expression of CK, Vim and DLL3, in patients with SCLC and the changes of DLL3+/CD45- CTCs during treatment seem to be a dynamic biomarker associated with treatment efficacy and patients’ clinical outcome.

Although the role of human papillomavirus (HPV) in the development of uterine cervical cancer is well established, the role of HPV in lung carcinogenesis remains controversial. The detection rates of HPV DNA are subject to a wide variation from 0 to 100%. This is partly influenced by the detection techniques employed. To elucidate the impact of HPV infection on lung parenchyma, we analyzed 100 non-small cell lung cancer (NSCLC) specimens (39 squamous cell carcinomas, 50 adenocarcinomas, 5 samples with characteristics of both squamous cell and adenocarcinoma, 5 undifferentiated and 1 large cell carcinoma) from the region of Crete, Greece. Sixteen non-cancerous samples served as the negative controls. DNA was extracted from 100 paraffin-embedded tissue sections obtained from NSCLC patients. The specimens were examined for the detection of HPV DNA by Real-Time PCR using GP5+/GP6+ primers. Furthermore, the HPV-positive samples were subjected to genotyping. In contrast to the absence of viral genomes in the control samples, HPV DNA was detected in 19 NSCLC specimens (19%). In particular, 4 squamous cell carcinomas, 12 adenocarcinomas, 1 sample with characteristics of both squamous cell and adenocarcinoma, and 2 undifferentiated samples were HPV-positive. The distribution of HPV genotypes was as follows: HPV 16: eight cases (42.1%); HPV 11: three cases (15.8%); HPV 6: one case (5.2%); HPV 59: one case (5.2%); HPV 33: two cases (10.5%); HPV 31: two cases (10.5%) and HPV 18: two cases (10.5%). The presence of HPV in the tumor samples provides evidence of the potential role of HPV in NSCLC and strongly argues for additional research on this issue.

To investigate the prognostic role of Beclin 1 in endometrial adenocarcinomas of the endometrioid cell type. Beclin 1 is a known tumor suppressor gene, but its function may be altered under conditions of an accelerated autophagic activity, which provides additional energy to proliferating cells by recycling defective organelles and long-lived cytoplasmic proteins.One hundred and fifty-five endometrioid adenocarcinomas were investigated for their autophagic activity using the monoclonal antibody Beclin 1 and an automated immunohistochemical technique. The extent of Beclin 1 expression was evaluated on a three-tier scale as follows: low (10% positive tumor cells), intermediate (between 10% and 50% positive tumor cells), and high (50% positive tumor cells). The results were correlated with the degree of tumor differentiation, the depth of myometrial invasion and the overall 5-year survival. In addition, the endometrial tumors were immunostained with the hypoxia inducible factor 1α (HIF1α) and their expression was related to Beclin 1.A high Beclin 1 reactivity occurred in 18.1% of endometrial adenocarcinomas studied and was associated with high tumor grade, high myometrial invasion and a poor 5-year survival. It was also correlated positively with HIF1α. Of the remaining adenocarcinomas 29.7% were of intermediate Beclin 1 reactivity and 52.2% of low, but correlations with prognostic factors were insignificant.An increased Beclin 1 expression is connected with the most aggressive endometrioid adenocarcinomas, probably as a result of its strong association with tumor hypoxia.

Introduction: Circulating tumor cells (CTCs) are considered as a “liquid biopsy” that allows the assessment of tumor changes over time. Tumor cells may escape from the immune system through the activation of PD-1/PD-L1 axis. Targeting these molecules with monoclonal antibodies has shown encouraging results at many types of cancers, including NSCLC. In the current study we investigated the expression of PD-1/PD-L1 molecules on the CTCs isolated from NSCLC patients treated with Nivolumab. Methods: CTCs were isolated based on their size using the ISET platform from 27 patients before treatment, after 1 cycle and 3 cycles. CTCs were detected with Giemsa staining and immunofluorescence (IF) experiments, using either pancytokeratin (A45-B/B3) (CK7)/PD-1/CD45 or (A45-B/B3)(CK7)/PD-L1/CD45 combination of antibodies and analysis with the ARIOL system. Spiking experiments using the NSCLC cell lines: H460, H1299, HCC827 and SKMES in normal blood were used to evaluate the detection method. Results: Giemsa evaluation in Nivolumab-treated patients at baseline (25 evaluable samples), after the 1st (9 evaluable samples) and the 3rd (8 evaluable patients) cycle of treatment showed that CTCs could be detected in 48% (12/25), 33.3% (3/9) and 50% (4/8) of patients, respectively. IF could also reveal the presence of CK-positive cells in 44.4% (12/27), 22% (2/9) and 75% (6/8) patients, respectively. PD-1 (+) CTCs were detected in 33.3% (4/12) of patients at baseline, in, 0% after the 1 and 16.7% (1/6) of patients after the 3rd cycle. The same percentages were identified for PD-L1 expression in the same cohort of patients. The expression of PD-1 at baseline was associated with poorer OS (p=0.022) and PFS (p=0.011), while the expression of PD-L1 was associated with shorter PFS (p=0.011). Multivariate analysis revealed that the presence of CK-positive cells is an independent prognostic factor for OS (p=0.028) Conclusion: Nivolumab reduced the number of PD-1- and PD-L1-expressing CTCs in advanced NSCLC patients. Furthermore the expression of both markers at baseline is associated with the clinical outcome. Citation Format: Galatea Kallergi, Anastasios Koutsopoulos, Eleni Lagoudaki, Despoina Agouraki, Eleni-Kiriaki Vetsika, Anastasia Voumvouraki, Stuart S. Martin, Filippos Koinis, Christos Stournaras, Vassilis Georgoulias, Athanasios Kotsakis. Detection of PD-L1 and PD-1 positive circulating tumor cells (CTCs) in non-small cell lung cancer (NSCLC) patients treated with nivolumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1587.

Purpose: In vitro data suggest that down-regulation of thrombospondin 1 (TSP1) expression from TXR1 is associated with resistance to taxane-based chemotherapy. The prognostic and predictive value of tumoral expression of both genes was evaluated in patients with lung adenocarcinoma treated with first-line docetaxel and gemcitabine. Experimental Design: Tumor samples from 96 patients, with stage IIIB (with pleural effusion) or IV lung adenocarcinomas, were analyzed for TXR1 and TSP1 mRNA levels by quantitative real-time PCR, from microdissected cells derived from patients' primary tumors. Results: The mRNA levels of the two genes were inversely correlated (Spearman's test = -0.49; P < 0.0001). Patients with low TXR1 mRNA levels experienced a longer median time to tumor progression (TTP; P < 0.0001) and median overall survival (mOS; P = 0.001) when compared with patients with high TXR1 expression. Patients with high TSP1 expression presented longer TTP (P = 0.002) and mOS (P < 0.0001) when compared with patients with low TSP1 expression. Moreover, patients with high TSP1 and low TXR1 expression (n = 36) presented higher prolonged TTP (P = 0.009) and mOS (P < 0.0001) compared with patients with high TXR1 and low TSP1 expression. Multivariate analysis showed that high TXR1/low TSP1 expression was an independent prognostic factor for decreased TTP (hazard ratio, 1.7; 95% confidence interval, 1.1-3.27; P = 0.016) and mOS (hazard ratio, 2.55; 95% confidence interval, 1.57-4.15; P < 0.0001). Conclusion: These data confirm the in vitro model of TSP1 and TXR1 effect on taxane resistance in lung adenocarcinomas and merit further evaluation.

Basaloid squamous cell carcinoma (BSCC) is a histologically distinct variant of squamous cell carcinoma. It occurs in various sites of the head and neck region and is believed to carry a dismal prognosis. The palate is a very rare site of BSCC development and only three cases have been reported in the international literature. In this report, we present a case of basaloid squamous cell carcinoma of the soft palate. The therapeutic strategy and histological findings are described in detail, including immunohistochemistry with the use of involucrin, an agent used for the first time for BSCC diagnosis. In addition, a brief review of the literature is presented.

In non-small-cell lung cancer (NSCLC), epidermal growth factor receptor (EGFR) and K-RAS mutations of the primary tumour are associated with responsiveness and resistance to tyrosine kinase inhibitors (TKIs), respectively. However, the EGFR and K-RAS mutation status in metastases is not well studied. We compared the mutation status of these genes between the primary tumours and the corresponding metastases of 25 patients. Epidermal growth factor receptor and K-RAS mutation status was different between primary tumours and corresponding metastases in 7 (28%) and 6 (24%) of the 25 patients, respectively. Among the 25 primary tumours, three 'hotspot' and two non-classical EGFR mutations were found; none of the corresponding metastases had the same mutation pattern. Among the five (20%) K-RAS mutations detected in the primary tumours, two were maintained in the corresponding metastasis. Epidermal growth factor receptor and K-RAS mutations were detected in the metastatic tumours of three (12%) and five (20%) patients, respectively. The expressions of EGFR and phosphorylated EGFR showed I 0 and 50% discordance, in that order. We conclude that there is substantial discordance in EGFR and K-RAS mutational status between the primary tumours and corresponding metastases in patients with NSCLC and this might have therapeutic implications when treatment with TKIs is considered.

Ribonucleotide reductase subunits M1 (RRM1) and M2 (RRM2) are involved in the metabolism of gemcitabine (2′,2′-difluorodeoxycytidine), which is used for the treatment of nonsmall cell lung cancer. The mRNA expression of RRM1 and RRM2 in tumours from lung adenocarcinoma patients treated with docetaxel/gemcitabine was assessed and the results correlated with clinical outcome. RMM1 and RMM2 mRNA levels were determined by quantitative real-time PCR in primary tumours of previously untreated patients with advanced lung adenocarcinoma who were subsequently treated with docetaxel/gemcitabine. Amplification was successful in 42 (79%) of 53 enrolled patients. Low levels of RRM2 mRNA were associated with response to treatment (P< 0.001). Patients with the lowest expression levels of RRM1 had a significantly longer time to progression (P=0.044) and overall survival (P=0.02) than patients with the highest levels. Patients with low levels of both RRM1 and RRM2 had a significantly higher response rate (60 vs 14.2%; P=0.049), time to progression (9.9 vs 2.3 months; P=0.003) and overall survival (15.4 vs 3.6; P=0.031) than patients with high levels of both RRM1 and RRM2. Ribonucleotide reductase subunit M1 and RRM2 mRNA expression in lung adenocarcinoma tumours is associated with clinical outcome to docetaxel/gemcitabine. Prospective studies are warranted to evaluate the role of these markers in tailoring chemotherapy.

Lung cancer, formerly known to be solely a smoker9s disease, may now be considered the outcome of environmental and genetic interactions, including viral infections. Merkel Cell Polyoma Virus (MCPyV) has already been associated with human lung cancer. Although the molecular pathways are yet to be elucidated, microRNA epigenetic aberrations could cause altered regulation of gene expression. The current study intended to investigate the expression profile of microRNAs (miR-21, miR-367, miR-155,miR-146a, miR-302c, miR-376c and miR-145) in paraffin embedded tissue samples in a primary non-small cell lung cancer (NSCLC) patients cohort. The presence of MCPyV DNA, microRNA expression profiles and target genes9 expression profiles were determined via Real-Time PCR. MiR-21 and miR-376c were significantly upregulated in all McPyV positive tumor samples compared to controls, while their target genes (Bcl-2, PTEN, EIF4A2, TIMP3, AKT, Myc, RB1, BRAF, RKIP andALK7, MMP9 respectively) showed significant dysregulation in all McPyV positive NSCLC samples compared to controls, with significant microRNA-target gene correlation. These results implicate for the first time miR-21 and miR-376c with MCPyV and human NSCLC. MiR-21 –already linked to poor NSCLC survival- targets various cell cycle genes, including Bcl-2 , previously implicated in the MCPyV-NSCLC interaction. Events originating from the deregulation of miR-21 and miR-376c and their targets in MCPyV-positive NSCLC samples could be cardinal in tumorigenesis, however their role is yet to be elucidated.

Polymorphisms in ERCC1, XPD, and XRCC1 were examined for (a) association with the clinical outcome of 107 non-small cell lung cancer patients receiving front-line platinum-based chemotherapy, and (b) correlation with the ERCC1 mRNA levels of 176 chemo-naive primary tumors. The ERCC1-C8092 allele and the number of ERCC1 polymorphic variants (C8092A and Asn118Asn) were associated with progression-free survival. In non-squamous histology, tumoral ERCC1 mRNA levels were lower in patients homozygous for ERCC1-C8092 as compared with the patients carrying the A allele (p = .024). These findings merit investigation in larger cohorts of patients treated with uniform regimens.

To investigate whether the predominant emphysema type is associated with the high resolution computed tomography (HRCT) pattern of fibrosis in combined pulmonary fibrosis and emphysema (CPFE).Fifty-three smokers with upper lobe emphysema and lower lobe pulmonary fibrosis on - HRCT - were retrospectively evaluated. Patients were stratified into 3 groups according to the predominant type of emphysema: Centrilobular (CLE), paraseptal (PSE), CLE = PSE. Patients were also stratified into 3 other groups according to the predominant type of fibrosis on HRCT: Typical usual interstitial pneumonia (UIP), probable UIP and nonspecific interstitial pneumonia (NSIP). HRCTs were scored at 5 predetermined levels for the coarseness of fibrosis (Coarseness), extent of emphysema (emphysema), extent of interstitial lung disease (TotExtILD), extent of reticular pattern not otherwise specified (RetNOS), extent of ground glass opacity with traction bronchiectasis (extGGOBx), extent of pure ground glass opacity and extent of honeycombing. HRCT mean scores, pulmonary function tests, diffusion capacity (DLCO) and systolic pulmonary arterial pressure were compared among the groups.The predominant type of emphysema was strongly correlated with the predominant type of fibrosis. The centrilobular emphysema group exhibited a significantly higher extent of emphysema (P0.001) and a lower extent of interstitial lung disease (P0.002), reticular pattern not otherwise specified (P0.023), extent of ground glass opacity with traction bronchiectasis (P0.002), extent of honeycombing (P0.001) and coarseness of fibrosis (P0.001) than the paraseptal group. The NSIP group exhibited a significantly higher extent of emphysema (P0.05), total lung capacity (P0.01) and diffusion capacity (DLCO) (P0.05) than the typical UIP group. The typical UIP group exhibited a significantly higher extent of interstitial lung disease, extent of reticular pattern not otherwise specified, extent of ground glass opacity with traction bronchiectasis, extent of honeycombing and coarseness of fibrosis (0.039P0.000). Although the pulmonary arterial pressure was higher in typical UIP group relative to the NSIP group, the difference was not statistically significant.In CPFE patients, paraseptal emphysema is associated more with UIP-HRCT pattern and higher extent of fibrosis than centrilobular emphysema.

CNS tumors are the leading cause of cancer-related death in children. Medulloblastoma is the commonest pediatric CNS malignancy, wherein, despite multimodal therapy with surgery, radiation, and chemotherapy, 5 year survival rates merely approach 60%. Until present, gene expression and cytogenetic studies have produced contradicting findings regarding the molecular background of the specific disease. Through integration of genomics, bioinformatics, and proteomics, the current study aims to shed light at the proteomic-related molecular events responsible for MBL pathophysiology, as well as to provide molecular/protein/pathway answers concerning tumor-onset. Experiments were performed on tissues collected at surgery. With 17p loss being the commonest chromosomal aberrance observed in our sample set, array-CGH were employed to first distinguish for 17p-positive cases. 2-DE coupled to mass spectrometry identification exposed the MBL-specific protein profile. Protein profiles of malignant tissues were compared against profiles of normal cerebellar tissues, and quantitative protein differences were determined. Bioinformatics, functional and database analyses, characterization, and subnetwork profiling generated information on MBL protein interactions. Key molecules of the PI3K/mTOR signaling network were identified via the techniques applied herein. Among the findings IGF2, PI3K, Rictor, MAPKAP1, S6K1, 4EBP1, and ELF4A, as part of the IGF network (implicating PI3K/mTOR), were founded to be deregulated.

Ocular malignant melanoma is characterized by an unpredictable course, and metastases may develop, after a long disease-free interval, anywhere in the body. The mediastinum, however, is a rare site of these metastases, and metastatic melanoma presenting as a large mediastinal mass is quite unusual. We report herein a peculiar case of a solitary, late metastasis of malignant ocular melanoma, manifesting as a sizable posterior mediastinal mass and presenting with paroxysmal atrial fibrillation. Transthoracic echocardiography, computed tomography scan findings, and surgical treatment and pathologic examination are described.

Introduction: Circulating tumour cells (CTCs) are responsible for the metastatic dissemination of the tumor. They have been shown to express Programmed Death-Ligand1 (PD-L1) to escape from the immune system surveillance through its ligation with the PD-1receptor on the surface of effector immune cells. We investigated the expression of PD-1/PD-L1 on CTCs isolated from NSCLC patients treated with chemotherapy. Methods: CTCs were isolated based on their size using the ISET platform from 30 stage IV chemo-naïve NSCLC patients (before and after chemotherapy). CTCs were detected after staining with Giemsa and immunofluorescence (IF). Double and triple staining experiments with different combination of antibodies: [Cytokeratins(CK)/PD-1/CD45 and CK/PD-L1/CD45] were performed and the samples were analyzed with the ARIOL system. Results Giemsa staining showed that twenty-three (77%) out of 30 and six (54.5%) out of 11 patients had detectable CTCs at baseline and after the 3rd cycle of front-line chemotherapy. IF staining revealed seventeen out of 30 (56.7%) patients positive for CTCs at baseline level and 8 out of 11 (72.7%) samples after the 3rd cycle of treatment. PD-1 and PD-L1 expression was observed in 53% (9/17) and in 47% of the CTC-positive patients at baseline; in addition, 13% (1/8) and 63% (5/8) patients had PD-1 and PD-L1, respectively after the 3rd cycle. Among the total number of detected CTCs, 67% were PD-1(+) at baseline and 25% after the 3rd cycle (p=0.069). In addition, 26% and 80% were PD-L1(+) at baseline and after the 3rd cycle, respectively. Patients with more than 3 PD-1 positive CTCs showed shorter PFS (p=0.022). Primary tissue from ten of the examined patients was also available. More than 5% of PD-L1 (+) tumor infiltrating lymphocytes (TILs) were observed in 20% (2/10) of the patients. More than 5% PD-L1 positive cells in the primary tumor were observed in 20%. However the two group of the patients were different. In addition both patients with PD-L1 (+) TILs harvested CTCs with PD-1 expression and one of them had also PD-L1 positive CTCs. Conclusion: PD-1- and PD-L1-positive CTCs could be detected before and during 1st line treatment in metastatic NSCLC. This expression was related to patients’ prognosis, implying that these molecules can be served as targets for metastasis restoration. Furthermore the expression of PD-1 on CTCs suggests a bilateral cross-talk between tumor and immune cells. Citation Format: Galaktea Kallergi, Eleni Kyriaki Vetsika, Despoina Aggouraki, Eleni Lagoudaki, Anastasios Koutsopoulos, Filippos Koinis, Panagiotis Katsarlinos, Maria Trypaki, Christos Stournaras, Vassilis Georgoulias, Athanasios Kotsakis. Evaluation of PD-L1/PD-1 on circulating tumor cells (CTCs) and on primary tumor in advanced non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1726. doi:10.1158/1538-7445.AM2017-1726