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Incomplete removal of paraffin and organic contaminants from tissues processed for diagnostic histology has been a profound barrier to the introduction of Raman spectroscopic techniques into clinical practice. We report a route to rapid and complete paraffin removal from a range of formalin-fixed paraffin embedded tissues using super mirror stainless steel slides. The method is equally effective on a range of human and animal tissues, performs equally well with archived and new samples and is compatible with standard pathology lab procedures. We describe a general enhancement of the Raman scatter and enhanced staining with antibodies used in immunohistochemistry for clinical diagnosis. We conclude that these novel slide substrates have the power to improve diagnosis through anatomical pathology by facilitating the simultaneous combination of improved, more sensitive immunohistochemical staining and simplified, more reliable Raman spectroscopic imaging, analysis and signal processing. [ABSTRACT FROM AUTHOR]

Pulmonary tuberculosis (TB) is caused by inhalation of Mycobacterium tuberculosis, which damages the bronchial epithelial barrier to establish local infection. Matrix metalloproteinase-1 plays a crucial role in the immunopathology of TB, causing breakdown of type I collagen and cavitation, but this collagenase is also potentially involved in bronchial epithelial repair. We hypothesized that the extracellular matrix (ECM) modulates M. tuberculosis-driven matrix metalloproteinase-1 expression by human bronchial epithelial cells (HBECs), regulating respiratory epithelial cell migration and repair. Medium from monocytes stimulated with M. tuberculosis induced collagenase activity in bronchial epithelial cells, which was reduced by ~87% when cells were cultured on a type I collagen matrix. Matrix metalloproteinase-1 had a focal localization, which is consistent with cell migration, and overall secretion decreased by 32% on type I collagen. There were no associated changes in the specific tissue inhibitors of metalloproteinases. Decreased matrix metalloproteinase-1 secretion was due to ligand-binding to the a2β1 integrin and was dependent on the actin cytoskeleton. In lung biopsies, samples from patients with pulmonary TB, integrin α2β1 is highly expressed on the bronchial epithelium. Areas of lung with disrupted collagen matrix showed an increase in matrix metalloproteinases-1 expression compared with areas where collagen was comparable to control lung. Type I collagen matrix increased respiratory epithelial cell migration in a wound-healing assay, and this too was matrix metalloproteinase-dependent, since it was blocked by the matrix metalloproteinase inhibitor GM6001. In summary, we report a novel mechanism by which a2ß1-mediated signals from the ECM modulate matrix metalloproteinase-1 secretion by HBECs, regulating their migration and epithelial repair in TB. [ABSTRACT FROM AUTHOR]

For several decades, a multitude of studies have documented the ability of Raman spectroscopy (RS) to differentiate between tissue types and identify pathological changes to tissues in a range of diseases. Furthermore, spectroscopists have illustrated that the technique is capable of detecting disease‐specific alterations to tissue before morphological changes become apparent to the pathologist. This study draws comparisons between the information that is obtainable using RS alongside immunohistochemistry (IHC), since histological examination is the current GOLD standard for diagnosing a wide range of diseases. Here, Raman spectral maps were generated using formalin‐fixed, paraffin‐embedded colonic tissue sections from healthy patients and spectral signatures from principal components analysis (PCA) were compared with several IHC markers to confirm the validity of their localizations. PCA loadings identified a number of signatures that could be assigned to muscle, DNA and mucin glycoproteins and their distributions were confirmed with antibodies raised against anti‐Desmin, anti‐Ki67 and anti‐MUC2, respectively. The comparison confirms that there is excellent correlation between RS and the IHC markers used, demonstrating that the technique is capable of detecting compositional changes in tissue in a label‐free manner, eliminating the need for antibodies. [ABSTRACT FROM AUTHOR]

Objective: The aim of this study was to explore the potential effects of diet-induced weight loss on molecular biomarkers of colorectal cancer risk in serum and colorectal tissue.Methods: This single-arm exploratory study included 20 adults with BMI ≥ 30 kg/m2 completing an 8-week, complete, low-energy liquid diet. Pre- and postintervention anthropometric measurements, fasting blood draws, and endoscopic examinations to procure colorectal biopsies were performed. Fasting insulin, glucose, insulinlike growth factor 1 (IGF-1), C-reactive protein (CRP), and blood lipids were measured in serum, and tissue markers of apoptosis (M30), colonocyte proliferation (Ki-67), and insulin signaling (phospho-mTOR) were assessed using immunohistochemical staining.Results: Participants achieved substantial weight loss (mean = 13.56%). Mean concentrations of insulin, glucose, and cholesterol were significantly reduced (P < 0.05), but IGF-1 and CRP were not. Colorectal tissue expression of Ki-67 was significantly reduced (preintervention mean score = 7, postintervention mean score = 3.9, mean % change -43.8; P = 0.027). There were no significant changes in M30 or phospho-mTOR.Conclusions: Weight loss in individuals with obesity was associated with improvements in insulin sensitivity and blood lipid profiles and a significant reduction in tissue Ki-67 expression. This is one of the first studies to demonstrate potential cancer-relevant changes in colorectal tissue following weight loss achieved through diet. [ABSTRACT FROM AUTHOR]

The Amur tiger (Panthera tigris altaica) is an endangered tiger subspecies. An adult zoo-bred female was found collapsed, and died despite supportive treatment. Hematology and biochemistry showed pancytopenia and hyperglobulinemia, and serum protein electrophoresis revealed a monoclonal band in the ß-globulin region. Necropsy demonstrated hemoabdomen, multifocal lytic bone marrow lesions, splenomegaly, and hemorrhagic hepatic nodules, with left medial lobe rupture. There were mutifocal hemorrhages in the subcutis, lung, epicardium, and intestinal mucosa. Histopathology demonstrated plasmacytoid cells infiltrating the bone marrow, liver and spleen, and circulating within blood vessels. On immunohistochemistry, cell infiltrates of the three tissues were positive for λ light chains, bone marrow infiltrates were positive for MUM-1 and bone marrow and spleen infiltrates were positive for CD20. These findings indicate that this animal died of hemoabdomen subsequent to multiple myeloma. This is the first time this disease has been reported in a tiger. [ABSTRACT FROM AUTHOR]

The impact of anti-tumor necrosis factor (TNF) therapies on inducible TNF-dependent activity in humans has never been evaluated in vivo. We aimed to test the hypothesis that patients responding to anti-TNF treatments exhibit attenuated TNF-dependent immune responses at the site of an immune challenge. We developed and validated four context-specific TNF-inducible transcriptional signatures to quantify TNF bioactivity in transcriptomic data. In anti-TNF treated rheumatoid arthritis (RA) patients, we measured the expression of these biosignatures in blood, and in skin biopsies from the site of tuberculin skin tests (TSTs) as a human experimental model of multivariate cell-mediated immune responses. In blood, anti-TNF therapies attenuated TNF bioactivity following ex vivo stimulation. However, at the site of the TST, TNF-inducible gene expression and genome-wide transcriptional changes associated with cell-mediated immune responses were comparable to that of RA patients receiving methotrexate only. These data demonstrate that anti-TNF agents in RA patients do not inhibit inducible TNF activity at the site of an acute inflammatory challenge in vivo, as modeled by the TST. We hypothesize instead that their therapeutic effects are limited to regulating TNF activity in chronic inflammation or by alternative non-canonical pathways. [ABSTRACT FROM AUTHOR]

Numerous gene signatures, or modules have been described to evaluate the immune cell composition in transcriptomes of multicellular tissue samples. However, significant diversity in module gene content for specific cell types is associated with heterogeneity in their performance. In order to rank modules that best reflect their purported association, we have generated the modular discrimination index (MDI) score that assesses expression of each module in the target cell type relative to other cells. We demonstrate that MDI scores predict modules that best reflect independently validated differences in cellular composition, and correlate with the covariance between cell numbers and module expression in human blood and tissue samples. Our analyses demonstrate that MDI scores provide an ordinal summary statistic that reliably ranks the accuracy of gene expression modules for deconvolution of cell type abundance in transcriptional data. [ABSTRACT FROM AUTHOR]

Chordoma is a rare malignant bone tumour with a poor prognosis and limited therapeutic options. We undertook a focused compound screen (FCS) against 1097 compounds on three well-characterized chordoma cell lines; 154 compounds were selected from the single concentration screen (1 µ m), based on their growth-inhibitory effect. Their half-maximal effective concentration (EC50) values were determined in chordoma cells and normal fibroblasts. Twenty-seven of these compounds displayed chordoma selective cell kill and 21/27 (78%) were found to be EGFR/ERBB family inhibitors. EGFR inhibitors in clinical development were then studied on an extended cell line panel of seven chordoma cell lines, four of which were sensitive to EGFR inhibition. Sapitinib (AstraZeneca) emerged as the lead compound, followed by gefitinib (AstraZeneca) and erlotinib (Roche/Genentech). The compounds were shown to induce apoptosis in the sensitive cell lines and suppressed phospho-EGFR and its downstream pathways in a dose-dependent manner. Analysis of substituent patterns suggested that EGFR-inhibitors with small aniline substituents in the 4-position of the quinazoline ring were more effective than inhibitors with large substituents in that position. Sapitinib showed significantly reduced tumour growth in two xenograft mouse models (U-CH1 xenograft and a patient-derived xenograft, SF8894). One of the resistant cell lines (U-CH2) was shown to express high levels of phospho-MET, a known bypass signalling pathway to EGFR. Neither amplifications ( EGFR, ERBB2, MET) nor mutations in EGFR, ERBB2, ERBB4, PIK3CA, BRAF, NRAS, KRAS, PTEN, MET or other cancer gene hotspots were detected in the cell lines. Our findings are consistent with the reported (p-)EGFR expression in the majority of clinical samples, and provide evidence for exploring the efficacy of EGFR inhibitors in the treatment of patients with chordoma and studying possible resistance mechanisms to these compounds in vitro and in vivo. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. [ABSTRACT FROM AUTHOR]

In the twenty-first century, diabetic patients are likely to be one of the major beneficiaries from the advancement of regenerative medicine through cellular therapies. Though the existence of a specific self-renewing stem cell within the pancreas is still far from clear, a surprising variety of cells within the pancreas can differentiate towards a β-cell phenotype: ductular cells, periductular mesenchymal cells and β-cells themselves can all give rise to new β-cells. Extra-pancreatic adult somatic stem cells, in particular, those originating from bone marrow may also be capable of differentiating to β-cells, though equally well the beneficial effects of bone marrow cells may reside in their contribution to the damaged islet vasculature. Forced expression of the β-cell-specific transcription factor Pdx1 in hepatocytes also holds promise as a therapeutic strategy to increase insulin levels in diabetic individuals. Embryonic stem (ES) cells are clearly another possible source for generating β-cells, but ES cells are beyond the scope of this review, which focuses on adult stem and progenitor cells capable of producing β-cells. Despite considerable endeavour, we still have much to learn in the field of pancreatic regeneration prior to any clinically applicable therapy based upon adult stem cells. Copyright © 2006 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Simple Summary: Ovarian clear cell carcinoma (OCCC) is a rare subtype of epithelial ovarian cancer that has a poor response to chemotherapy. Here, we assessed the immunological features of a series of 33 OCCCs and identified an immune-related gene expression signature that correlated with a patient's risk of recurrence. Additionally, using multiplex immunofluorescence, we assessed the spatial distribution and abundance of immune cell populations at the protein level and identified that tumour-associated macrophages (TAM) and regulatory T cells are excluded from the vicinity of tumour cells in low-risk patients, suggesting that high-risk patients have a more immunosuppressive microenvironment. We also found that TAMs and cytotoxic T cells were also excluded from the vicinity of tumour cells in ARID1A mutated OCCCs, suggesting that the exclusion of these immune effectors could determine the host response in ARID1A mutant OCCCs. Ovarian clear cell carcinoma (OCCC) is a rare subtype of epithelial ovarian cancer characterised by a high frequency of loss-of-function ARID1A mutations and a poor response to chemotherapy. Despite their generally low mutational burden, an intratumoural T cell response has been reported in a subset of OCCC, with ARID1A purported to be a biomarker for the response to the immune checkpoint blockade independent of micro-satellite instability (MSI). However, assessment of the different immune cell types and spatial distribution specifically within OCCC patients has not been described to date. Here, we characterised the immune landscape of OCCC by profiling a cohort of 33 microsatellite stable OCCCs at the genomic, gene expression and histological level using targeted sequencing, gene expression profiling using the NanoString targeted immune panel, and multiplex immunofluorescence to assess the spatial distribution and abundance of immune cell populations at the protein level. Analysis of these tumours and subsequent independent validation identified an immune-related gene expression signature associated with risk of recurrence of OCCC. Whilst histological quantification of tumour-infiltrating lymphocytes (TIL, Salgado scoring) showed no association with the risk of recurrence or ARID1A mutational status, the characterisation of TILs via multiplexed immunofluorescence identified spatial differences in immunosuppressive cell populations in OCCC. Tumour-associated macrophages (TAM) and regulatory T cells were excluded from the vicinity of tumour cells in low-risk patients, suggesting that high-risk patients have a more immunosuppressive microenvironment. We also found that TAMs and cytotoxic T cells were also excluded from the vicinity of tumour cells in ARID1A-mutated OCCCs compared to ARID1A wild-type tumours, suggesting that the exclusion of these immune effectors could determine the host response of ARID1A-mutant OCCCs to therapy. Overall, our study has provided new insights into the immune landscape and prognostic associations in OCCC and suggest that tailored immunotherapeutic approaches may be warranted for different subgroups of OCCC patients. [ABSTRACT FROM AUTHOR]

Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) functions as a critical stress sentinel that coordinates cell survival, inflammation, and immunogenic cell death (ICD). Although the catalytic function of RIPK1 is required to trigger cell death, its non-catalytic scaffold function mediates strong pro-survival signaling. Accordingly, cancer cells can hijack RIPK1 to block necroptosis and evade immune detection. We generated a small-molecule proteolysis-targeting chimera (PROTAC) that selectively degraded human and murine RIPK1. PROTAC-mediated depletion of RIPK1 deregulated TNFR1 and TLR3/4 signaling hubs, accentuating the output of NF-κB, MAPK, and IFN signaling. Additionally, RIPK1 degradation simultaneously promoted RIPK3 activation and necroptosis induction. We further demonstrated that RIPK1 degradation enhanced the immunostimulatory effects of radio- and immunotherapy by sensitizing cancer cells to treatment-induced TNF and interferons. This promoted ICD, antitumor immunity, and durable treatment responses. Consequently, targeting RIPK1 by PROTACs emerges as a promising approach to overcome radio- or immunotherapy resistance and enhance anticancer therapies. [Display omitted] • RIPK1's scaffolding function protects cancer cells from immune detection and death • RIPK1-specific PROTAC degraders enhance innate immune signaling and necroptosis • RIPK1 PROTACs boost the immunostimulatory and antitumor effects of RT and ICB • PROTAC-mediated RIPK1 depletion suppresses skin inflammation Tumor cells frequently hijack RIPK1's scaffolding function to resist cell death and avoid immune detection. Mannion et al. describe the development of a RIPK1-selective PROTAC degrader that boosts the immunostimulatory and antitumor activity of radiotherapy (RT) and immune checkpoint blockade (ICB), heating up tumors and driving long-lasting antitumor immunity. [ABSTRACT FROM AUTHOR]

Highlights • BAP1 expression may correlate with response to vinorelbine in mesothelioma. • BAP1 expression does not correlate with response to combination mitomycin, vinblastine and cisplatin. • BAP1 expression should be assessed as a biomarker in ongoing prospective trials of vinorelbine in mesothelioma. Abstract Introduction BRCA1 associated protein-1 (BAP1) is a key tumor driver in mesothelioma and a potential biomarker predicting response to several targeted therapies in clinical testing. Whether it also modulates response to cytotoxic chemotherapy is undetermined. This study used retrospective response analysis of BAP1 expression in archival tumor biopsies taken from patients in the MS01 trial (NCT00075699). We aimed to determine if BAP1 expression correlated with overall survival within the three treatment arms in this trial, namely active symptom control (ASC); ASC plus mitomycin, vinblastine and cisplatin (MVP); and ASC plus vinorelbine. Materials and methods We used immunohistochemical analysis of tumor samples from the MS01 trial to identify subgroups with and without nuclear BAP1 expression. We performed correlative analysis of clinical characteristics (age at diagnosis, sex and histological subtype) and overall survival within treatment arms with nuclear BAP1 expression. Results 89 tumor samples from the 409 patients originally in the trial were available for analysis. Of these, 60 samples harbored a positive internal control, in the form of positive staining of inflammatory cells for BAP1, and were carried forward for analysis. Correlative analysis suggested no significant association between loss of nuclear BAP1 expression and age at diagnosis, sex and histological subtype. Kaplan Meier survival analysis revealed a small, though non-significant, overall survival disadvantage associated with BAP1 expression in tumors from patients treated with vinorelbine. Discussion This exploratory analysis suggests BAP1 expression may modify response to vinorelbine in MPM, possibly due to prevention of mitotic microtubule formation. We suggest ongoing and planned clinical studies of vinorelbine in MPM assess BAP1 expression as a predictive biomarker of response. [ABSTRACT FROM AUTHOR]