Explore the vast range of titles available.

Optimisation and validation of a multiplex immunofluorescence (mIF) workflow, from staining to digital image analysis (DIA), ensure assay robustness. Chromogenic immunohistochemistry (IHC) and fluorescent singleplexes are fundamental in this process, particularly when biomarkers are co‐expressed. We describe our experience developing two mIF panels and the various parameters of staining, scanning and DIA to consider when standardising a digital pathology workflow. Multiplex immunofluorescence is a powerful tool for the simultaneous detection of tissue‐based biomarkers, revolutionising traditional immunohistochemistry. The Opal methodology allows up to eight biomarkers to be measured concomitantly without cross‐reactivity, permitting identification of different cell populations within the tumour microenvironment. In this study, we aimed to validate a multiplex immunofluorescence workflow in two complementary multiplex panels and evaluate the tumour immune microenvironment in colorectal cancer (CRC) formalin‐fixed paraffin‐embedded tissue. We stained CRC and tonsil samples using Opal multiplex immunofluorescence on a Leica BOND RX immunostainer. We then acquired images on an Akoya Vectra Polaris and performed multispectral unmixing using inform. Antibody panels were validated on tissue microarray sections containing cores from six normal tissue types, using qupath for image analysis. Comparisons between chromogenic immunohistochemistry and multiplex immunofluorescence on consecutive sections from the same tissue microarray showed significant correlation (rs > 0.9, P‐value < 0.0001), validating both panels. We identified many factors that influenced the quality of the acquired fluorescent images, including biomarker co‐expression, staining order, Opal‐antibody pairing, sample thickness, multispectral unmixing and biomarker detection order during image analysis. Overall, we report the optimisation and validation of a multiplex immunofluorescence process, from staining to image analysis, ensuring assay robustness. Our multiplex immunofluorescence protocols permit the accurate detection of multiple immune markers in various tissue types, using a workflow that enables rapid processing of samples, above and beyond previous workflows.

H3K27-altered Diffuse Midline Glioma (DMG) is a universally fatal paediatric brainstem tumour. The prevalent driver mutation H3K27M creates a unique epigenetic landscape that may also establish therapeutic vulnerabilities to epigenetic inhibitors. However, while HDAC, EZH2 and BET inhibitors have proven somewhat effective in pre-clinical models, none have translated into clinical benefit due to either poor blood–brain barrier penetration, lack of efficacy or toxicity. Thus, there remains an urgent need for new DMG treatments. Here, we performed wider screening of an epigenetic inhibitor library and identified inhibitors of protein arginine methyltransferases (PRMTs) among the top hits reducing DMG cell viability. Two of the most effective inhibitors, LLY-283 and GSK591, were targeted against PRMT5 using distinct binding mechanisms and reduced the viability of a subset of DMG cells expressing wild-type TP53 and mutant ACVR1 . RNA-sequencing and phenotypic analyses revealed that LLY-283 could reduce the viability, clonogenicity and invasion of DMG cells in vitro, representing three clinically important phenotypes, but failed to prolong survival in an orthotopic xenograft model. Together, these data show the challenges of DMG treatment and highlight PRMT5 inhibitors for consideration in future studies of combination treatments.

Insulin-like growth factor-1 (IGF-1) is associated with prostate cancer (PCa) development and lethality and exhibits immunosuppressive properties in other models. We investigated IGF-1’s tumor-intrinsic immune effects in PCa to understand mechanisms underlying its poor immunotherapy response. Transcriptional profiling of human (DU145, 22Rv1) and murine (Myc-CaP) PCa cells revealed that IGF-1 suppresses cytokine signalling, antigen processing and presentation, and additional immune regulatory pathways. We further examined the expression of components involved in cancer cell recognition and immune evasion: the antigen processing machinery and PD-L1 checkpoint. IGF-1 downregulated key elements such as transporters associated with antigen processing (TAPs), endoplasmic reticulum aminopeptidase-1 (ERAP-1), and Class I β2-microglobulin, without significantly altering Class I allele expression. These changes were associated with reduced surface presentation of Class I complexes on Myc-CaP cells, suggesting disrupted peptide transport, processing, and/or presentation. In contrast, IGF-1 upregulated the immune checkpoint CD274 (PD-L1) via IGF receptor/AKT/ERK-dependent signalling. Analysis of TCGA Firehose Legacy PCa data showed higher CD274 expression in tumors with elevated IGF1 and IGFBP5. Multiplex immunofluorescence in primary PCa confirmed increased PD-L1 in patients with high serum IGF-1, supporting its role in immune evasion. Overall, these findings reveal a novel IGF-1-driven immunosuppressive mechanism that may underlie PCa’s resistance to immunotherapy.

Triggered release and targeted drug delivery of potent anti-cancer agents using hyperthermia-mediated focused-ultrasound (FUS) is gaining momentum in the clinical setting. In early phase studies, tissue biopsy samples may be harvested to assess drug delivery efficacy and demonstrate lack of instantaneous cell death due to FUS exposure. We present an optimised tissue cell recovery method and a cell viability assay, compatible with intra-cellular doxorubicin. Flow cytometry was used to determine levels of cell death with suspensions comprised of: (i) HT29 cell line exposed to hyperthermia (30 min at 47 °C) and/or doxorubicin, or ex-vivo bovine liver tissue exposed to (ii) hyperthermia (up to 2 h at 45 °C), or (iii) ablative high intensity FUS (HIFU). Flow cytometric analysis revealed maximal cell death in HT29 receiving both heat and doxorubicin insults and increases in both cell granularity (p < 0.01) and cell death (p < 0.01) in cells recovered from ex-vivo liver tissue exposed to hyperthermia and high pressures of HIFU (8.2 MPa peak-to-peak free-field at 1 MHz) relative to controls. Ex-vivo results were validated with microscopy using pan-cytokeratin stain. This rapid, sensitive and highly quantitative cell-viability method is applicable to the small masses of liver tissue typically recovered from a standard core biopsy (5–20 mg) and may be applied to tissues of other histological origins including immunostaining.

Background This Phase 2a dose expansion study was performed to assess the safety, tolerability and preliminary efficacy of the maximum tolerated dose of the oral histone de-acetylase (HDAC) inhibitor CXD101 in patients with relapsed / refractory lymphoma or advanced solid organ cancers and to assess HR23B protein expression by immunohistochemistry as a biomarker of HDAC inhibitor sensitivity. Methods Patients with advanced solid-organ cancers with high HR23B expression or lymphomas received CXD101 at the recommended phase 2 dose (RP2D). Key exclusions: corrected QT > 450 ms, neutrophils < 1.5 × 10 9 /L, platelets < 75 × 10 9 /L, ECOG > 1. Baseline HR23B expression was assessed by immunohistochemistry. Results Fifty-one patients enrolled between March 2014 and September 2019, 47 received CXD101 (19 solid-organ cancer, 28 lymphoma). Thirty-four patients received ≥80% RP2D. Baseline characteristics: median age 57.4 years, median prior lines 3, male sex 57%. The most common grade 3–4 adverse events were neutropenia (32%), thrombocytopenia (17%), anaemia (13%), and fatigue (9%) with no deaths on CXD101. No responses were seen in solid-organ cancers, with disease stabilisation in 36% or patients; the overall response rate in lymphoma was 17% with disease stabilisation in 52% of patients. Median progression-free survival was 1.2 months (95% confidence interval (CI) 1.2–5.4) in solid-organ cancers and 2.6 months (95%CI 1.2–5.6) in lymphomas. HR23B status did not predict response. Conclusions CXD101 showed acceptable tolerability with efficacy seen in Hodgkin lymphoma, T-cell lymphoma and follicular lymphoma. Further studies assessing combination approaches are warranted. Trial registration ClinicalTrials.gov identifier NCT01977638 . Registered 07 November 2013.

Purpose Despite recent advances, approximately 50% of patient with metastatic melanoma eventually succumb to the disease. Patients with melanomas harboring a BRAF mutation (BRAF Mut ) have a worse prognosis than those with wildtype (BRAF WT ) tumors. Unexpectedly, interim AVAST-M Phase III trial data reported benefit from adjuvant anti-VEGF bevacizumab only in the BRAF Mut group. We sought to find mechanisms underpinning this sensitivity. Methods We investigated this finding in vitro and in vivo using melanoma cell lines and clones generated by BRAF V600E knock-in on a BRAF WT background. Results Compared with BRAF WT cells, isogenic BRAF V600E clones secreted more VEGF and exhibited accelerated growth rates as spheroids and xenografts, which were more vascular and proliferative. Recapitulating AVAST-M findings, bevacizumab affected only BRAF V600E xenografts, inducing significant tumor growth delay, reduced vascularity and increased necrosis. We identified 814 differentially expressed genes in isogenic BRAF V600E /BRAF WT clones. Of 61 genes concordantly deregulated in clinical melanomas ROR2 was one of the most upregulated by BRAF V600E . ROR2 was shown to be RAF-MEK regulated in BRAF V600E cells and its depletion suppressed VEGF secretion down to BRAF WT levels. The ROR2 ligand WNT5A was also overexpressed in BRAF Mut melanomas, and in ROR2-overexpressing BRAF V600E cells MEK inhibition downregulated WNT5A and VEGF secretion. Conclusions These data implicate WNT5A-ROR2 in VEGF secretion, vascularity, adverse outcomes and bevacizumab sensitivity of BRAF Mut melanomas, suggesting that this axis has potential therapeutic relevance.

Key Clinical Message Gliosarcoma, a rare cerebral neoplasm, has not been linked to hippocampal changes in cats. We report a case of complex partial seizures with orofacial involvement, revealing gliosarcoma concurrent with bilateral hippocampal sclerosis. A 16‐year‐old neutered female domestic shorthair cat presented with acute inappetence, ataxia, disorientation, and vacant staring. Brain MRI revealed an ill‐defined, round, intra‐axial mass in the right piriform lobe, showing hyperintensity on T2W, T2‐FLAIR, and T2*W, and hypointensity on T1W images. The lesion exhibited mass effect and contrast enhancement in its center. Bilateral hyperintensity on T2‐FLAIR images and contrast enhancement were observed in the hippocampus. Brain histologic and immunohistochemical analysis revealed cerebral gliosarcoma with concurrent hippocampal sclerosis. Feline LGI1‐antibody testing on the serum and/or CSF was not performed due to insufficient biomaterial. Although retrospective testing on brain tissue was considered, it ultimately proved unfeasible, preventing us from ruling out antibody‐associated limbic encephalitis. In conclusion, cerebral gliosarcoma should be included in feline intracranial tumor differentials, warranting brain MRI and feline LGI1‐antibody testing in cats showing complex partial seizures with orofacial involvement. In our case, the prognosis remained poor due to the presence of a high‐grade glioma.

Multiplex immunofluorescence (mIF) imaging can provide comprehensive quantitative and spatial information for multiple immune markers for tumour immunoprofiling. However, application at scale to clinical trial samples sourced from multiple institutions is challenging due to pre‐analytical heterogeneity. This study reports an analytical approach to the largest multi‐parameter immunoprofiling study of clinical trial samples to date. We analysed 12,592 tissue microarray (TMA) spots from 3,545 colorectal cancers sourced from more than 240 institutions in two clinical trials (QUASAR 2 and SCOT) stained for CD4, CD8, CD20, CD68, FoxP3, pan‐cytokeratin, and DAPI by mIF. TMA slides were multi‐spectrally imaged and analysed by cell‐based and pixel‐based marker analysis. We developed an adaptive thresholding method to account for inter‐ and intra‐slide intensity variation in TMA analysis. Applying this method effectively ameliorated inter‐ and intra‐slide intensity variation improving the image analysis results compared with methods using a single global threshold. Correlation of CD8 data derived by our mIF analysis approach with single‐plex chromogenic immunohistochemistry CD8 data derived from subsequent sections indicates the validity of our method (Spearman's rank correlation coefficients ρ between 0.63 and 0.66, p ≪ 0.01) as compared with the current gold standard analysis approach. Evaluation of correlation between cell‐based and pixel‐based analysis results confirms equivalency (ρ > 0.8, p ≪ 0.01, except for CD20 in the epithelial region) of both analytical approaches. These data suggest that our adaptive thresholding approach can enable analysis of mIF‐stained clinical trial TMA datasets by digital pathology at scale for precision immunoprofiling.

Carbonic anhydrases (CA) influence intra- and extracellular pH and ion transport in varied biological processes. We recently identified CA9 and CA12 as hypoxia-inducible genes. In this study we examined the expression of these tumor-associated CAs by immunohistochemistry in relation to necrosis and early breast tumor progression in 68 cases of ductal carcinoma in situ (DCIS) (39 pure DCIS and 29 DCIS associated with invasive carcinoma). CA IX expression was rare in normal epithelium and benign lesions, but was present focally in DCIS (50% of cases) and in associated invasive carcinomas (29%). In comparison, CA XII was frequently expressed in normal breast tissues (89%), in DCIS (84%), and in invasive breast lesions (71%). In DCIS, CA IX was associated with necrosis ( P= 0.0053) and high grade ( P = 0.012). In contrast, CA XII was associated with the absence of necrosis ( P = 0.036) and low grade ( P = 0.012). Despite this, augmented CA XII expression was occasionally observed adjacent to necrosis within high-grade lesions. Neither CA IX nor CA XII expression was associated with regional or overall proliferation as determined by MIB1 staining. Assessment of mammographic calcification showed that CA XII expression was associated with the absence of calcification ( n = 43, P = 0.0083). Our results demonstrate that induction of CA IX and CA XII occurs in regions adjacent to necrosis in DCIS. Furthermore, these data suggest that proliferation status does not influence expression of either CA in breast tissues, that hypoxia may be a dominant factor in the regulation of CA IX, and that factors related to differentiation, as determined by tumor grade, dominate the regulation of CA XII. The existence of differential regulation and associations with an aggressive phenotype may be important in the development of selective inhibitors of CAs, because the latter have recently been shown to prevent tumor invasion.

Previous preclinical research has shown that extracorporeal devices can be used to enhance the delivery and distribution of systemically administered anticancer drugs, resulting in increased intratumoural concentrations. We aimed to assess the safety and feasibility of targeted release and enhanced delivery of doxorubicin to solid tumours from thermosensitive liposomes triggered by mild hyperthermia, induced non-invasively by focused ultrasound. We did an open-label, single-centre, phase 1 trial in a single UK hospital. Adult patients (aged ≥18 years) with unresectable and non-ablatable primary or secondary liver tumours of any histological subtype were considered for the study. Patients received a single intravenous infusion (50 mg/m2) of lyso-thermosensitive liposomal doxorubicin (LTLD), followed by extracorporeal focused ultrasound exposure of a single target liver tumour. The trial had two parts: in part I, patients had a real-time thermometry device implanted intratumourally, whereas patients in part II proceeded without thermometry and we used a patient-specific model to predict optimal exposure parameters. We assessed tumour biopsies obtained before and after focused ultrasound exposure for doxorubicin concentration and distribution. The primary endpoint was at least a doubling of total intratumoural doxorubicin concentration in at least half of the patients treated, on an intention-to-treat basis. This study is registered with ClinicalTrials.gov, number NCT02181075, and is now closed to recruitment. Between March 13, 2015, and March 27, 2017, ten patients were enrolled in the study (six patients in part I and four in part II), and received a dose of LTLD followed by focused ultrasound exposure. The treatment resulted in an average increase of 3·7 times in intratumoural biopsy doxorubicin concentrations, from an estimate of 2·34 μg/g (SD 0·93) immediately after drug infusion to 8·56 μg/g (5·69) after focused ultrasound. Increases of two to ten times were observed in seven (70%) of ten patients, satisfying the primary endpoint. Serious adverse events registered were expected grade 4 transient neutropenia in five patients and prolonged hospital stay due to unexpected grade 1 confusion in one patient. Grade 3–4 adverse events recorded were neutropenia (grade 3 in one patient and grade 4 in five patients), and grade 3 anaemia in one patient. No treatment-related deaths occurred. The combined treatment of LTLD and non-invasive focused ultrasound hyperthermia in this study seemed to be clinically feasible, safe, and able to enhance intratumoural drug delivery, providing targeted chemo-ablative response in human liver tumours that were refractory to standard chemotherapy. Oxford Biomedical Research Centre, National Institute for Health Research.