Myeloid cells from Langerhans cell histiocytosis patients exhibit increased vesicle trafficking and an altered secretome capable of activating NK cells.

Langerhans cell histiocytosis (LCH) is a potentially life-threatening inflammatory myeloid neoplasia linked to pediatric neurodegeneration, whereby transformed LCH cells form agglomerated lesions in various organs. Although MAP-kinase pathway mutations have been identified in LCH cells, the functional consequences of these mutations and the mechanisms that cause the pathogenic behavior of LCH cells are not well understood. In our study, we used an in vitro differentiation system and RNA-sequencing to compare monocyte-derived dendritic cells from LCH patients to those derived from healthy controls or patients with Crohn's disease, a non-histiocytic inflammatory disease. We observed that interferon-γ treatment exacerbated intrinsic differences between LCH patient and control cells, including strikingly increased endo- and exocytosis gene activity in LCH patients. We validated these transcriptional patterns in lesions and functionally confirmed that LCH cells exhibited increased endo- and exocytosis. Furthermore, RNA-sequencing of extracellular vesicles revealed the enrichment of pathological transcripts involved in cell adhesion, MAP-kinase pathway, vesicle trafficking and T-cell activation in LCH patients. Thus, we tested the effect of the LCH secretome on lymphocyte activity and found significant activation of NK cells. These findings implicate extracellular vesicles in the pathology of LCH for the first time, in line with their established roles in the formation of various other tumor niches. Thus, we describe novel traits of LCH patient cells and suggest a pathogenic mechanism of potential therapeutic and diagnostic importance.

Comparison of immunohistochemical mesothelial biomarkers in paired biopsies and effusion cytology cell blocks from pleural mesothelioma.

OBJECTIVE: Traditionally, the diagnosis of pleural mesothelioma is based on histological material. Minimally invasive effusion cytology specimens are an alternative that, like biopsies, require ancillary analyses. Validation of immunohistochemical (IHC) analyses on cytology, including the surrogate markers for molecular alterations BAP1 and MTAP, is of interest. METHODS: IHC for eight different markers was performed on 59 paired formalin-fixed, paraffin-embedded pleural biopsies and pleural effusion cell blocks with mesothelioma. Immunoreactivity in ≥10% of tumour cells was considered positive/preserved. The concordance between histological and cytological materials was assessed. RESULTS: The overall percentage of agreement between the histological epithelioid component in 58 biopsies and paired cell blocks was 93% for calretinin, 98% for CK5, 97% for podoplanin, 90% for WT1, 86% for EMA, 100% for desmin, 91% for BAP1, and 72% for MTAP. For 11 cases with biphasic or sarcomatoid histology, the concordance between cytology and the histological sarcomatoid component was low for calretinin, CK5, and WT1 (all ≤45%). For the whole cohort, loss of both BAP1 and MTAP was seen in 40% while both markers were preserved in 11% of the biopsies for epithelioid histology. The corresponding numbers were 54% and 8%, respectively, for the paired cell blocks. CONCLUSIONS: Generally, a high concordance for IHC staining was seen between paired biopsies and pleural effusion cell blocks from mesotheliomas, but the somewhat lower agreement for WT1, EMA, and especially MTAP calls for further investigation and local quality assurance. The lower concordance for the sarcomatoid subtype for some markers may indicate biological differences.

PD-L1 Expression in Non-Small Cell Lung Cancer Specimens: Association with Clinicopathological Factors and Molecular Alterations.

Immune checkpoint inhibitors (ICI) targeting programmed cell death-1 or its ligand (PD-L1) have improved outcomes in non-small cell lung cancer (NSCLC). High tumor PD-L1 expression, detected by immunohistochemistry (IHC) typically on formalin-fixed paraffin-embedded (FFPE) histological specimens, is linked to better response. Following our previous investigation on PD-L1 in cytological samples, the aim of this study was to further explore the potential impacts of various clinicopathological and molecular factors on PD-L1 expression. Two retrospective NSCLC cohorts of 1131 and 651 specimens, respectively, were investigated for PD-L1 expression (<1%/1−49%/≥50%), sample type, sample site, histological type, and oncogenic driver status. In both cohorts, PD-L1 was positive (≥1%) in 55% of the cases. Adenocarcinomas exhibited lower PD-L1 expression than squamous cell carcinomas (p < 0.0001), while there was no difference between sample types, tumor locations, or between the two cohorts in multivariate analysis (all p ≥ 0.28). Mutational status correlated significantly with PD-L1 expression (p < 0.0001), with the highest expression for KRAS-mutated cases, the lowest for EGFR-mutated, and the KRAS/EGFR wild-type cases in between. There was no difference in PD-L1 levels between different prevalent KRAS mutations (all p ≥ 0.44), while mucinous KRAS-mutated adenocarcinomas exhibited much lower PD-L1 expression than non-mucinous (p < 0.0001). Our data indicate that cytological and histological specimens are comparable for PD-L1 evaluation. Given the impact of KRAS mutations and the mucinous growth pattern on PD-L1 expression, these factors should be further investigated in studies on ICI response.

Diagnostic performance of a stepwise cytological algorithm for biliary malignancy in primary sclerosing cholangitis.

BACKGROUND AND AIMS: Detection of early cholangiocarcinoma (CCA) in primary sclerosing cholangitis (PSC) is challenging. The aim of this study was to evaluate the diagnostic accuracy of a stepwise approach to biliary brush cytology with sequential use of fluorescence in-situ hybridization (FISH) for the detection of biliary malignancy in PSC. METHOD: We retrospectively studied consecutive patients with PSC who underwent biliary brushings at Karolinska University Hospital between 2009 and 2015 (n = 208). Brush samples were categorized as benign, equivocal (atypical or suspicious) and malignant. Equivocal cases were further analysed with FISH. Samples with a malignant cytology or positive FISH were considered positive. The diagnosis was determined after 12 months of follow-up. RESULTS: The diagnosis CCA was confirmed in 15 patients (7%), high-grade dysplasia in three patients, and low-grade dysplasia in five patients at follow-up. Using the diagnostic algorithm, the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) for a diagnosis of CCA were 80% (95%CI 52%-96%), 96% (95%CI 92%-98%), 60% (95%CI 36%-81%) and 98% (95% CI 95%-100%). In patients with equivocal cytology (n = 61), the sensitivity for CCA diagnosis increased to 100% (95%CI 72%-100%) with a lower PPV of 58% (95%CI 34%-78%). The diagnostic accuracy for detection of CCA in all patients was 95% (95%CI 91%-97%). CONCLUSION: Biliary brush cytology with sequential use of FISH in equivocal cases seems to be a highly predictive diagnostic test for CCA in PSC. These results support the use of FISH when cytology is equivocal for detection of biliary malignancy in PSC.

Management of cytological material, pre-analytical procedures and bio-banking in effusion cytopathology.

Serous effusion fluid is one of the most commonly encountered specimens in routine cytopathology practice. It provides invaluable information about the patient and the clinical status; but to get the most of it, specimen handling and processing must be carried out properly. Cytomorphology is the basis of a successful analysis which should complemented by ancillary tests when needed. A wide spectrum of ancillary techniques - ranging from immunocytochemistry and flow cytometry to different assays of molecular pathology - can be applied to serous effusions. This article describes the acquisition and management of serous effusion fluids, methods for preservation and transportation, different techniques of cytopreparation, application of immunocytochemistry, flow cytometry, and fluorescence in-situ hybridization (FISH), as well as DNA extraction for polymerase chain reaction (PCR) and next generation sequencing (NGS). Principles of bio-banking of effusion samples are also discussed which is getting more important in correlation with the developments in personalized medicine.

Molecular targets and signaling pathways regulated by nuclear translocation of syndecan-1.

BACKGROUND: The cell-surface heparan sulfate proteoglycan syndecan-1 is important for tumor cell proliferation, migration, and cell cycle regulation in a broad spectrum of malignancies. Syndecan-1, however, also translocates to the cell nucleus, where it might regulate various molecular functions. RESULTS: We used a fibrosarcoma model to dissect the functions of syndecan-1 related to the nucleus and separate them from functions related to the cell-surface. Nuclear translocation of syndecan-1 hampered the proliferation of fibrosarcoma cells compared to the mutant lacking nuclear localization signal. The growth inhibitory effect of nuclear syndecan-1 was accompanied by significant accumulation of cells in the G0/G1 phase, which indicated a possible G1/S phase arrest. We implemented multiple, unsupervised global transcriptome and proteome profiling approaches and combined them with functional assays to disclose the molecular mechanisms that governed nuclear translocation and its related functions. We identified genes and pathways related to the nuclear compartment with network enrichment analysis of the transcriptome and proteome. The TGF-ß pathway was activated by nuclear syndecan-1, and three genes were significantly altered with the deletion of nuclear localization signal: EGR-1 (early growth response 1), NEK11 (never-in-mitosis gene a-related kinase 11), and DOCK8 (dedicator of cytokinesis 8). These candidate genes were coupled to growth and cell-cycle regulation. Nuclear translocation of syndecan-1 influenced the activity of several other transcription factors, including E2F, NFκß, and OCT-1. The transcripts and proteins affected by syndecan-1 showed a striking overlap in their corresponding biological processes. These processes were dominated by protein phosphorylation and post-translation modifications, indicative of alterations in intracellular signaling. In addition, we identified molecules involved in the known functions of syndecan-1, including extracellular matrix organization and transmembrane transport. CONCLUSION: Collectively, abrogation of nuclear translocation of syndecan-1 resulted in a set of changes clustering in distinct patterns, which highlighted the functional importance of nuclear syndecan-1 in hampering cell proliferation and the cell cycle. This study emphasizes the importance of the localization of syndecan-1 when considering its effects on tumor cell fate.

Excerpts from the 1st international NTNU symposium on current and future clinical biomarkers of cancer: innovation and implementation, June 16th and 17th 2016, Trondheim, Norway.

The goal of biomarker research is to identify clinically valid markers. Despite decades of research there has been disappointingly few molecules or techniques that are in use today. The "1st International NTNU Symposium on Current and Future Clinical Biomarkers of Cancer: Innovation and Implementation", was held June 16th and 17th 2016, at the Knowledge Center of the St. Olavs Hospital in Trondheim, Norway, under the auspices of the Norwegian University of Science and Technology (NTNU) and the HUNT biobank and research center. The Symposium attracted approximately 100 attendees and invited speakers from 12 countries and 4 continents. In this Symposium original research and overviews on diagnostic, predictive and prognostic cancer biomarkers in serum, plasma, urine, pleural fluid and tumor, circulating tumor cells and bioinformatics as well as how to implement biomarkers in clinical trials were presented. Senior researchers and young investigators presented, reviewed and vividly discussed important new developments in the field of clinical biomarkers of cancer, with the goal of accelerating biomarker research and implementation. The excerpts of this symposium aim to give a cutting-edge overview and insight on some highly important aspects of clinical cancer biomarkers to-date to connect molecular innovation with clinical implementation to eventually improve patient care.

Proteome screening of pleural effusions identifies galectin 1 as a diagnostic biomarker and highlights several prognostic biomarkers for malignant mesothelioma.

Malignant mesothelioma is an aggressive asbestos-induced cancer, and affected patients have a median survival of approximately one year after diagnosis. It is often difficult to reach a conclusive diagnosis, and ancillary measurements of soluble biomarkers could increase diagnostic accuracy. Unfortunately, few soluble mesothelioma biomarkers are suitable for clinical application. Here we screened the effusion proteomes of mesothelioma and lung adenocarcinoma patients to identify novel soluble mesothelioma biomarkers. We performed quantitative mass-spectrometry-based proteomics using isobaric tags for quantification and used narrow-range immobilized pH gradient/high-resolution isoelectric focusing (pH 4-4.25) prior to analysis by means of nano liquid chromatography coupled to MS/MS. More than 1,300 proteins were identified in pleural effusions from patients with malignant mesothelioma (n = 6), lung adenocarcinoma (n = 6), or benign mesotheliosis (n = 7). Data are available via ProteomeXchange with identifier PXD000531. The identified proteins included a set of known mesothelioma markers and proteins that regulate hallmarks of cancer such as invasion, angiogenesis, and immune evasion, plus several new candidate proteins. Seven candidates (aldo-keto reductase 1B10, apolipoprotein C-I, galectin 1, myosin-VIIb, superoxide dismutase 2, tenascin C, and thrombospondin 1) were validated by enzyme-linked immunosorbent assays in a larger group of patients with mesothelioma (n = 37) or metastatic carcinomas (n = 25) and in effusions from patients with benign, reactive conditions (n = 16). Galectin 1 was identified as overexpressed in effusions from lung adenocarcinoma relative to mesothelioma and was validated as an excellent predictor for metastatic carcinomas against malignant mesothelioma. Galectin 1, aldo-keto reductase 1B10, and apolipoprotein C-I were all identified as potential prognostic biomarkers for malignant mesothelioma. This analysis of the effusion proteome furthers our understanding of malignant mesothelioma, identified galectin 1 as a potential diagnostic biomarker, and highlighted several possible prognostic biomarkers of this disease.

Nuclear translocation of heparan sulfate proteoglycans and their functional significance.

BACKGROUND: Heparan sulfate proteoglycans (HSPGs) are important constituents of the cell membrane and they act as co-receptors for cellular signaling. Syndecan-1, glypican and perlecan also translocate to the nucleus in a regulated manner. Similar nuclear transport of growth factors and heparanase indicate a possible co-regulation and functional significance. SCOPE OF REVIEW: In this review we dissect the structural requirement for the nuclear translocation of HSPGs and their functional implications.s MAJOR CONCLUSIONS: The functions of the nuclear HSPGs are still incompletely understood. Evidence point to possible functions in hampering cell proliferation, inhibition of DNA topoisomerase I activity and inhibition of gene transcription. GENERAL SIGNIFICANCE: HSPGs influence the behavior of malignant tumors in many different ways. Modulating their functions may offer powerful tools to control fundamental biological processes and provide the basis for subsequent targeted therapies in cancer. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.

Guidelines for the cytopathologic diagnosis of epithelioid and mixed-type malignant mesothelioma. Complementary statement from the International Mesothelioma Interest Group, also endorsed by the International Academy of Cytology and the Papanicolaou Society of Cytopathology.

OBJECTIVE: To provide practical guidelines for the cytopathologic diagnosis of malignant mesothelioma. DATA SOURCES: Cytopathologists with an interest in the field involved in the International Mesothelioma Interest Group (IMIG) and the International Academy of Cytology (IAC) contributed to this update. Reference material includes peer-reviewed publications and textbooks. RATIONALE: This article is the result of discussions during and after the IMIG 2012 conference in Boston, followed by thorough discussions during the 2013 IAC meeting in Paris. Additional contributions have been obtained from cytopathologists and scientists who could not attend these meetings, with final discussions and input during the IMIG 2014 conference in Cape Town.

Characterization and drug sensitivity profiling of primary malignant mesothelioma cells from pleural effusions.

BACKGROUND: Patients with malignant mesothelioma have a poor prognosis and only 40% respond to first line treatment; a combination of pemetrexed and cisplatin or carboplatin. We used primary malignant mesothelioma cells and an ex vivo chemosensitivity assay with future purpose to predict best choice of treatment. The clinical outcome of these patients might be predicted by measuring drug sensitivity. METHODS: Pleural effusions containing primary malignant mesothelioma cells were received from the diagnostic routine. We characterized and tested the chemosensitivity of 18 malignant samples and four benign samples from 16 different patients with pleural effusions. Cells were seeded in a 384-well plate for a robotized ex vivo testing of drug sensitivity to 32 different drugs. The primary cells were further characterized by immunocytochemistry to evaluate the proportion of malignant cells and to study the RRM1 and ERCC1 reactivity, two proteins associated with drug resistance. RESULTS: We observed great individual variability in the drug sensitivity. Primary cell isolates were affected by between one and ten drugs, and resistant to the remaining tested drugs. Actinomycin D and daunorubicin were the two drugs effective in most cases. Adjusting efficiency of individual drugs for varying proportion of tumor cells and to the average effect on benign cells correlated with effect of pemetrexed, cisplatin and survival time. General drug sensitivity, proportion of malignant cells and reactivity to RRM1 correlated to each other and to survival time of the patients. CONCLUSIONS: The proportion of malignant cells and RRM1 reactivity in the pleural effusions correlate to drug sensitivity and survival time. The variability in response to the commonly used chemotherapies emphasizes the need for tests that indicate best individual choice of cytotoxic drugs. The efficiency of the obtained results should preferably be corrected for admixture of benign cells and effects of given drugs on benign cells.

Selenite induces posttranscriptional blockade of HLA-E expression and sensitizes tumor cells to CD94/NKG2A-positive NK cells.

CD94/NKG2A is an inhibitory receptor that controls the activity of a large proportion of human NK cells following interactions with the nonclassical HLA class Ib molecule HLA-E expressed on target cells. In this study, we show that selenite (SeO(3)(2-)), an inorganic selenium compound, induces an almost complete loss of cell surface expression of HLA-E on tumor cells of various origins. Selenite abrogated the HLA-E expression at a posttranscriptional level, since selenite exposure led to a dose-dependent decrease in cellular HLA-E protein expression whereas the mRNA levels remained intact. The loss of HLA-E expression following selenite treatment was associated with decreased levels of intracellular free thiols in the tumor cells, suggesting that the reduced HLA-E protein synthesis was caused by oxidative stress. Indeed, HLA-E expression and the level of free thiols remained intact following treatment with selenomethionine, a selenium compound that does not generate oxidative stress. Loss of HLA-E expression, but not of total HLA class I expression, on tumor cells resulted in increased susceptibility to CD94/NK group 2A-positive NK cells. Our results suggest that selenite may be used to potentiate the anti-tumor cytotoxicity in settings of NK cell-based immunotherapies.

Utility of BerEp4/calretinin and desmin/epithelial membrane antigen (EMA) dual immunocytochemical staining in effusion cytology.

BACKGROUND: Pleural mesothelioma (PM) is typically diagnosed late during the disease. Earlier detection can increase the chance of effective therapy. Recurrent pleural effusions are the earliest symptoms displaying an array of cytomorphological changes from reactive atypia to malignancy. Diagnosis is possible on effusion cytology by applying molecular and immunocytochemical markers, the main difficulty being when to suspect PM and to differentiate PM from metastatic adenocarcinoma and reactive mesothelial proliferations. METHODS: We evaluated the diagnostic performance of two immunocytochemical dual stains (BerEp4/Calretinin and Desmin/Epithelial Membrane Antigen (EMA)) on 149 ethanol-fixed cytospin preparation as an initial step to solve the mentioned diagnostic difficulty. The immunocytochemical reactivity pattern was evaluated by two independent investigators. The final diagnosis corresponded to PM (n = 20), metastatic adenocarcinoma (n = 83), and mesotheliosis (n = 46) in these cases. RESULTS: Calretinin had 99% specificity and 98% sensitivity for indicating a mesothelial phenotype, while BerEp4 distinguished the adenocarcinoma cases with 98% specificity and 99% sensitivity. EMA displayed 96% specificity and 99% sensitivity in malignant cases, while Desmin without EMA present showed 99% specificity and 96% sensitivity for indicating benign mesothelial proliferation. CONCLUSIONS: Interpretation of the four immunoreactions is improved when performed as dual stains. The dual staining is a useful tool in the initial handling of atypical effusions and guides the subsequent choice of antibody panels for more detailed subclassification of malignant effusions.

Difficulties in diagnostics of lung tumours in biopsies: an interpathologist concordance study evaluating the international diagnostic guidelines.

AIMS: Accurate and reliable diagnosis is essential for lung cancer treatment. The study aim was to investigate interpathologist diagnostic concordance for pulmonary tumours according to WHO diagnostic criteria. METHODS: Fifty-two unselected lung and bronchial biopsies were diagnosed by a thoracic pathologist based on a broad spectrum of immunohistochemical (IHC) stainings, molecular data and clinical/radiological information. Slides stained with H&E, thyroid transcription factor-1 (TTF-1) clone SPT24 and p40 were scanned and provided digitally to 20 pathologists unaware of reference diagnoses. The pathologists independently diagnosed the cases and stated if further diagnostic markers were deemed necessary. RESULTS: In 31 (60%) of the cases, ≥80% of the pathologists agreed with each other and with the reference diagnosis. Lower agreement was seen in non-small cell neuroendocrine tumours and in squamous cell carcinoma with diffuse TTF-1 positivity. Agreement with the reference diagnosis ranged from 26 to 45 (50%-87%) for the individual pathologists. The pathologists requested additional IHC staining in 15-44 (29%-85%) of the 52 cases. In nearly half (17 of 36) of the malignant cases, one or more pathologist advocated for a different final diagnosis than the reference without need of additional IHC markers, potentially leading to different clinical treatment. CONCLUSIONS: Interpathologist diagnostic agreement is moderate for small unselected bronchial and lung biopsies based on a minimal panel of markers. Neuroendocrine morphology is sometimes missed and TTF-1 clone SPT24 should be interpreted with caution. Our results suggest an intensified education need for thoracic pathologists and a more generous use of diagnostic IHC markers.

Versican but not decorin accumulation is related to malignancy in mammographically detected high density and malignant-appearing microcalcifications in non-palpable breast carcinomas.

BACKGROUND: Mammographic density (MD) and malignant-appearing microcalcifications (MAMCs) represent the earliest mammographic findings of non-palpable breast carcinomas. Matrix proteoglycans versican and decorin are frequently over-expressed in various malignancies and are differently involved in the progression of cancer. In the present study, we have evaluated the expression of versican and decorin in non-palpable breast carcinomas and their association with high risk mammographic findings and tumor characteristics. METHODS: Three hundred and ten patients with non-palpable suspicious breast lesions, detected during screening mammography, were studied. Histological examination was carried out and the expression of decorin, versican, estrogen receptor α (ERα), progesterone receptor (PR) and c-erbB2 (HER-2/neu) was assessed by immunohistochemistry. RESULTS: Histological examination showed 83 out of 310 (26.8%) carcinomas of various subtypes. Immunohistochemistry was carried out in 62/83 carcinomas. Decorin was accumulated in breast tissues with MD and MAMCs independently of the presence of malignancy. In contrast, versican was significantly increased only in carcinomas with MAMCs (median ± SE: 42.0 ± 9.1) and MD (22.5 ± 10.1) as compared to normal breast tissue with MAMCs (14.0 ± 5.8), MD (11.0 ± 4.4) and normal breast tissue without mammographic findings (10.0 ± 2.0). Elevated levels of versican were correlated with higher tumor grade and invasiveness in carcinomas with MD and MAMCs, whereas increased amounts of decorin were associated with in situ carcinomas in MAMCs. Stromal deposition of both proteoglycans was related to higher expression of ERα and PR in tumor cells only in MAMCs. CONCLUSIONS: The specific accumulation of versican in breast tissue with high MD and MAMCs only in the presence of malignant transformation and its association with the aggressiveness of the tumor suggests its possible use as molecular marker in non-palpable breast carcinomas.

Cytoskeletal Organization Correlates to Motility and Invasiveness of Malignant Mesothelioma Cells.

Malignant mesothelioma (MM) is a rare but highly aggressive cancer that primarily originates from the pleura, peritoneum or pericardium. There is a well-established link between asbestos exposure and progression of MM. Direct invasion of the surrounding tissues is the main feature of MM, which is dependent on dysregulated communication between the mesothelium and the microenvironment. This communication is dependent on the dynamic organization of the cytoskeleton. We have analyzed the organization and function of key cytoskeletal components in MM cell lines of increasing malignancies measured as migratory and invasive properties, and we show that highly malignant and invasive MM cells have an organization of the actin filament and vimentin systems that is distinct from the less malignant MM cell lines. In addition, the Hippo tumor suppressor pathway was inactivated in the invasive MM cells, which was seen as increased YAP nuclear localization.

Effusion cytology of malignant mesothelioma enables earlier diagnosis and recognizes patients with better prognosis.

A conclusive diagnosis of malignant mesothelioma (MM) can be based on effusion cytology using the guidelines for the cytopathologic diagnosis of epithelioid and mixed-type MM. Briefly, the diagnosis is obtained when the mesothelial phenotype of malignant cells is established by ancillary techniques. This study is based on the comparison of the overall survival rates of patients with MM when diagnosed by effusion cytology, histopathology, or a combination of both. A total of 144 patients were diagnosed with epithelioid and mixed-type pleural MM at Karolinska University Hospital between 2004 and 2013. The diagnosis was obtained by histopathology in 74 cases and by cytological examination of pleural effusion in 70 cases. In 29 of the latter cases, a diagnostic biopsy was obtained simultaneously. A total of 104 patients received chemotherapy. All diagnoses were supported by clinical findings, including computer tomography scans. The median time between first symptoms and diagnosis was similar for cytology and histopathology. However, a delay of more than 6 months after first symptoms was seen in many patients in the histopathology group, resulting in late onset of treatment. The overall survival and proportion of long-term survival were significantly better for cases diagnosed by cytology. Similarly, a better survival, following a cytological diagnosis, was also seen in patients who were only provided the best supportive care. Accurate cytological diagnosis enables conclusive diagnosis of MM. Our finding enables the initiation of treatment as soon as the cytological diagnosis is established, avoiding further delay and deterioration of patient survival and possibilities for treatment.

Higher concordance of PD-L1 expression between biopsies and effusions in epithelioid than in nonepithelioid pleural mesothelioma.

BACKGROUND: Malignant mesothelioma (MM) is a therapy-resistant tumor, often causing an effusion. Drugs targeting the programmed cell death 1 (PD-1)/programmed cell death ligand 1 (PD-L1) pathway have shown promising results, but assessment of PD-L1 expression to select patients for therapy has mainly been performed on histologic tissue samples. In a previous study, we showed that MM effusions are suitable for PD-L1 assessment with results comparable to those reported in histologic studies, but no studies have compared PD-L1 expression in histologic and cytologic samples. METHODS: PD-L1 expression was determined immunohistochemically (clone 28-8) in 61 paired samples of effusions and biopsies from patients with pleural MM, obtained at the time of diagnosis. Only cases with >100 tumor cells were included. Membranous staining in tumor cells was considered positive at ≥1%, >5%, >10%, and >50% cutoff levels. RESULTS: Of 61 histologic samples, PD-L1 expression was found in 28 and 7 samples at ≥1% and >50% cutoffs, respectively; the corresponding figures for cytology were 21 and 5, respectively. The overall percentage agreement between histology and cytology was 69% and 84%, with a kappa (κ) of 0.36 and 0.08 at ≥1% and >50% cutoffs, respectively. The concordance between cytology and histology tended to be higher for epithelioid MM versus nonepithelioid MM at a ≥1% cutoff. PD-L1 positivity in biopsies, but not in effusions, correlated with the histologic subtype at a ≥1% cutoff. CONCLUSIONS: A moderate concordance of PD-L1 expression between biopsies and effusions from pleural MM, especially for the epithelioid subtype, indicates biological differences between the 2 types of specimens. Cytology and histology may be complementary.

Nuclear Syndecan-1 Regulates Epithelial-Mesenchymal Plasticity in Tumor Cells.

Tumor cells undergoing epithelial-mesenchymal transition (EMT) lose cell surface adhesion molecules and gain invasive and metastatic properties. EMT is a plastic process and tumor cells may shift between different epithelial-mesenchymal states during metastasis. However, how this is regulated is not fully understood. Syndecan-1 (SDC1) is the major cell surface proteoglycan in epithelial cells and has been shown to regulate carcinoma progression and EMT. Recently, it was discovered that SDC1 translocates into the cell nucleus in certain tumor cells. Nuclear SDC1 inhibits cell proliferation, but whether nuclear SDC1 contributes to the regulation of EMT is not clear. Here, we report that loss of nuclear SDC1 is associated with cellular elongation and an E-cadherin-to-N-cadherin switch during TGF-ß1-induced EMT in human A549 lung adenocarcinoma cells. Further studies showed that nuclear translocation of SDC1 contributed to the repression of mesenchymal and invasive properties of human B6FS fibrosarcoma cells. The results demonstrate that nuclear translocation contributes to the capacity of SDC1 to regulate epithelial-mesenchymal plasticity in human tumor cells and opens up to mechanistic studies to elucidate the mechanisms involved.

Diagnostic and Prognostic Utility of the Extracellular Vesicles Subpopulations Present in Pleural Effusion.

Extracellular vesicles (EVs), comprising exosomes, microvesicles, and apoptotic bodies, are released by all cells into the extracellular matrix and body fluids, where they play important roles in intercellular communication and matrix remodeling in various pathological conditions. Malignant pleural mesothelioma (MPM) is a primary tumor of mesothelial origin, predominantly related to asbestos exposure. The detection of MPM at an early stage and distinguishing it from benign conditions and metastatic adenocarcinomas (AD) is sometimes challenging. Pleural effusion is often the first available biological material and an ideal source for characterizing diagnostic and prognostic factors. Specific proteins have previously been identified as diagnostic markers in effusion, but it is not currently known whether these are associated with vesicles or released in soluble form. Here, we study and characterize tumor heterogeneity and extracellular vesicle diversity in pleural effusion as diagnostic or prognostic markers for MPM. We analyzed extracellular vesicles and soluble proteins from 27 pleural effusions, which were collected and processed at the department of pathology and cytology at Karolinska University Hospital, representing three different patient groups, MPM (n = 9), benign (n = 6), and AD (n = 12). The vesicles were fractionated into apoptotic bodies, microvesicles, and exosomes by differential centrifugation and characterized by nanoparticle tracking analysis and Western blotting. Multiplex bead-based flow cytometry analysis showed that exosomal markers were expressed differently on EVs present in different fractions. Further characterization of exosomes by a multiplex immunoassay (Luminex) showed that all soluble proteins studied were also present in exosomes, though the ratio of protein concentration present in supernatant versus exosomes varied. The proportion of Angiopoietin-1 present in exosomes was generally higher in benign compared to malignant samples. The corresponding ratios of Mesothelin, Galectin-1, Osteopontin, and VEGF were higher in MPM effusions compared to those in the benign group. These findings demonstrate that relevant diagnostic markers can be recovered from exosomes.