ObjectivesPleural mesothelioma is a cancer arising in the cells that line the lungs and chest wall with poor survival and poor response to first-line therapy. Organoid models of cancer can faithfully recapitulate the genetic and histopathological characteristics of individualized tumors and have potential to be used for precision medicine, however methods of establishing patient-derived mesothelioma organoids have not been well established in the published literature.Materials and methodsLong-term mesothelioma patient-derived organoids were established from ten malignant pleural effusion fluids. Mesothelioma patient-derived organoids were compared to the corresponding biopsy tissue specimens using immunohistochemistry labelling for select diagnostic markers and the TruSight Oncology-500 sequencing assay. Cell viability in response to the chemotherapeutic drug cisplatin was assessed.ResultsWe established five mesothelioma patient-derived organoid cultures from ten malignant pleural effusion fluids collected from nine individuals with pleural mesothelioma. Mesothelioma patient-derived organoids typically reflected the histopathological and genomic features of patients’ matched biopsy specimens and displayed cytotoxic sensitivity to cisplatin in vitro.ConclusionThis is the first study of its kind to establish long-term mesothelioma organoid cultures from malignant pleural effusions and report on their utility to test individuals’ chemotherapeutic sensitivities ex vivo.

Supplementary Figures 1-7. Supplementary Figure 1: Assembly of the PWD genome and alignment to the C57BL/6 genome. Supplementary Figure 2: RNA-seq read assignment. Supplementary Figure 3: MeDIP-seq quality controls. Supplementary Figure 4: Histology and distribution of tumors in B6 x PWD consomic mice. Supplementary Figure 5: Transcriptome analyses identify modifier candidate genes on other chromosomes than chromosome 5. Supplementary Figure 6: Strain-specific DNA methylation on chromosome 5 is correlated to CpG availability. Supplementary Figure 7: Assessment of cell types in the intestinal crypt.

An investigator-initiated, Australia-wide multi-centre retrospective observational study was undertaken to investigate the real-world prevalence of programmed death ligand-1 (PD-L1) expression in non-small cell lung carcinoma (NSCLC). Multiple centres around Australia performing PD-L1 immunohistochemistry (IHC) were invited to participate. Histologically confirmed NSCLC of any stage with a PD-L1 IHC test performed for persons aged ≥18 years between 1 January 2018 and 1 January 2020, and eligible for review, were identified at each centre, followed by data extraction and de-identification, after which data were submitted to a central site for collation and analysis. In total data from 6690 eligible PD-L1 IHC tests from histologically (75%) or cytologically (24%) confirmed NSCLC of any stage were reviewed from persons with a median age of 70 years, 43% of which were female. The majority (81%) of tests were performed using the PD-L1 IHC SP263 antibody with the Ventana BenchMark Ultra platform and 19% were performed using Dako PD-L1 IHC 22C3 pharmDx assay. Reported PD-L1 tumour proportion score (TPS) was ≥50% for 30% of all tests, with 62% and 38% scoring PD-L1 ≥1% and <1%, respectively. Relative prevalence of clinicopathological features with PD-L1 scores dichotomised to <50% and ≥50%, or to <1% and ≥1%, were examined. Females scored ≥1% slightly more often than males (64% vs 61%, respectively, p=0.013). However, there was no difference between sexes or age groups (<70 or ≥70 years) where PD-L1 scored ≥50%. Specimens from patients with higher stage (III/IV) scored ≥1% or ≥50% marginally more often compared to specimens from patients with lower stage (I/II) (p≤0.002). Proportions of primary and metastatic specimens did not differ where PD-L1 TPS was ≥1%, however more metastatic samples scored TPS ≥50% than primary samples (metastatic vs primary; 34% vs 27%, p<0.001). Cytology and biopsy specimens were equally reported, at 63% of specimens, to score TPS ≥1%, whereas cytology samples scored TPS ≥50% slightly more often than biopsy samples (34% vs 30%, respectively, p=0.004). Resection specimens (16% of samples tested) were reported to score TPS ≥50% or ≥1% less often than either biopsy or cytology samples (p<0.001). There was no difference in the proportion of tests with TPS ≥1% between PD-L1 IHC assays used, however the proportion of tests scored at TPS ≥50% was marginally higher for 22C3 compared to SP263 (34% vs 29%, respectively, p<0.001). These real-world Australian data are comparable to some previously published global real-world data, with some differences noted.

Dear Editor, We read with interest the systematic review by Sharma et al.[1] that highlighted the value of rare disease registries as important clinical and research resources to attain better patient outcomes. As the authors report, there are few rare eye disease registries: they identify a total of 26 registries, of which almost half are devoted to hereditary diseases. Notably, most registries are nationally based and have been established in high-income countries or regions spanning western Europe, the United Kingdom, and North America. We agree there remains an unmet need for registries that have international reach for a broad spectrum of rare eye conditions. To this end, we recently established the International Vitreoretinal B-Cell Lymphoma Registry.[2] This multinational initiative, which was not included in the author’s list of rare eye diseases, aims to provide the medical and general communities with real-world information about outcomes of standard diagnostic and therapeutic practices.[2] Vitreoretinal lymphoma affects an estimated one in two million persons annually, typically older adults.[3] Its etiology is unknown, and it may occur as an isolated—or primary—condition, or in association with primary central nervous system lymphoma. Its five-year survival rate is low (approximately 30%)[3,4]; yet, this cancer often “masquerades” as uveitis, delaying a diagnosis and the initiation of appropriate treatment. The rarity of vitreoretinal lymphoma, along with historical and current limitations related to inconsistent reporting in the literature and classification systems, have challenged efforts to improve outcomes.[3,5] Ophthalmologists across the globe contribute to the International Vitreoretinal B-Cell Lymphoma Registry by lodging retrospectively collected, management-focused data without patient identifiers via a secure online platform. We echo the call by Sharma et al.[1] to increase the number and scope of registries for rare eye diseases and applaud the efforts of the Women Ophthalmologists Society to establish the first rare eye disease registry in India. Financial support and sponsorship This work was supported by the Australian Department of Industry, Science, Energy and Resources through an Innovation Connections Grant, in partnership with South Bank Day Hospital (Grant number ICG001790). Conflicts of interest There are no conflicts of interest.

Supplementary tables 1-7. Supplementary Table 1: GSEA signatures and references (Wnt target genes are taken from the HALLMARK Signature). Supplementary Table 2: 58 differentially expressed genes on chromosome 5, B6 vs C5 APC min normal intestine. Supplementary Table 3: 116 differentially expressed genes not located on chromosome 5, B6 vs C5 APC min normal intestine. Supplementary table 4: Differentially expressed genes on chromosome 5 regulated in cis, trans, or both. Supplementary table 5: Candidate modifier genes differentially expressed between B6 or PWD chromosome 5. Supplementary Table 6: Polymorphisms in human orthologs of mouse genes located on chromosome 5, polymorphic between B6 and PWD, and expressed in the intestine. Supplementary Table 7: PWD polymorphisms on chr5 in genes that are expressed in the intestine and/or in adenoma.

Introduction Vitreoretinal lymphoma is a rare ocular cancer with high morbidity and mortality despite treatment. Diagnosis by cytopathology is often delayed, and various molecular and image-based investigations have been developed. Diverse treatments are used, but there is a limited medical evidence to differentiate their effectiveness. We designed an international registry that would collect diagnostic, treatment and outcomes data, to establish new evidence for the management of this cancer.Methods and analysis The International Vitreoretinal B-Cell Lymphoma Registry will accrue data retrospectively for individuals aged 18 years or older, diagnosed with new or recurrent vitreoretinal B-cell lymphoma on or after 1 January 2020. A steering committee of subspecialised ophthalmologists identified 20 key clinical data items that describe patient demographics, tissue involvements, diagnostic testing, ocular and systemic treatments and treatment complications, and visual acuity and survival outcomes. Customised software was designed to permit collection of these data across a single baseline and multiple follow-up forms. The platform collects data without identifiers and at 3 month reporting intervals. Outcomes of the project will include: (1) descriptions of clinical presentations, and diagnostic and therapeutic preferences; (2) associations between clinical presentations, and diagnostics and treatments, and between diagnostics and treatments (assessed by ORs with 95% CIs); and (3) estimations of rates of vision loss, and progression-free and overall survival (assessed by Kaplan-Meier estimates).Ethics and dissemination The registry has received Australia-wide approval by a national human research ethics committee. Sites located outside Australia are required to seek local human research ethics review. Results generated through the registry will be disseminated primarily by peer-reviewed publications that are expected to inform clinical practice, as well as educational materials.

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Abstract Genetic predisposition affects the penetrance of tumor-initiating mutations, such as APC mutations that stabilize β-catenin and cause intestinal tumors in mice and humans. However, the mechanisms involved in genetically predisposed penetrance are not well understood. Here, we analyzed tumor multiplicity and gene expression in tumor-prone ApcMin/+ mice on highly variant C57BL/6J (B6) and PWD/Ph (PWD) genetic backgrounds. (B6 × PWD) F1 APCMin offspring mice were largely free of intestinal adenoma, and several chromosome substitution (consomic) strains carrying single PWD chromosomes on the B6 genetic background displayed reduced adenoma numbers. Multiple dosage-dependent modifier loci on PWD chromosome 5 each contributed to tumor suppression. Activation of β-catenin–driven and stem cell–specific gene expression in the presence of ApcMin or following APC loss remained moderate in intestines carrying PWD chromosome 5, suggesting that PWD variants restrict adenoma initiation by controlling stem cell homeostasis. Gene expression of modifier candidates and DNA methylation on chromosome 5 were predominantly cis controlled and largely reflected parental patterns, providing a genetic basis for inheritance of tumor susceptibility. Human SNP variants of several modifier candidates were depleted in colorectal cancer genomes, suggesting that similar mechanisms may also affect the penetrance of cancer driver mutations in humans. Overall, our analysis highlights the strong impact that multiple genetic variants acting in networks can exert on tumor development. Significance: These findings in mice show that, in addition to accidental mutations, cancer risk is determined by networks of individual gene variants.

Primary central nervous system lymphoma (PCNSL) is a rare brain cancer that remains challenging to study. Epidemiology of PCNSL in the Australian population, which is racially and ethnically diverse, has not been examined previously. Using ICD-O-3.1 Morphology and Topography Codes to identify cases, we analyzed complete datasets from the comprehensive Australian Cancer Database (1982–2014, adults aged ≥20 years) to establish incidence rates and trends of PCNSL, and to define survival outcomes of individuals diagnosed with PCNSL, including the predominant diffuse large B-cell lymphoma (DLBCL) type. Age-standardized incidence of PCNSL increased by an average annual 6.8% percent over the study period, with current incidence of 0.43 (95% confidence interval, 0.41–0.46) per 100,000 person-years, in comparison to 21.89 (21.41–22.38) per 100,000 person-years for non-CNS lymphoma. Increase in incidence was characterized by an acute rise between 1996 and 1999, was more pronounced with increasing age, and was driven by increasing incidence of DLBCL. Overall survival for persons diagnosed with PCNSL improved significantly across the study period, with 5-year survival probability increasing from 0.21 (95% confidence interval, 0.16–0.26) to 0.33 (0.30–0.36), and median survival increasing from 318 to 600 days, between 1982–1999 and 2000–2014. Increase in survival was significantly higher for persons with DLBCL versus non-DLBCL PCNSL, but substantially lower than that for persons with non-CNS lymphoma, who had a 5-year survival probability of 0.62 (0.62–0.62) and a median survival of 3388 days in 2000–2014. This study links increasing incidence of PCNSL in Australia to increasing incidence of DLCBL, including in younger adults, and highlights the improving, but low, survival outcome of this cancer.

Uveitis is a broad term that refers to a large group of eye disorders categorized by intraocular inflammation, a leading cause of visual impairment. Historically, treatment of noninfectious uveitis has depended on corticosteroid drugs. Owing to the myriad of side effects caused by corticosteroids, immunomodulatory therapy has become the preferred treatment for chronic noninfectious intraocular inflammation. Recently, biological response modifiers have established a new era in uveitis therapy, with the range of targets continuing to expand. In this review, we aimed to convey up-to-date information on the treatment of noninfectious uveitis to the general ophthalmologist.

Introduction This is a phase 1, open-label, single-centre, uncontrolled, dose-escalation study to evaluate the feasibility, tolerability and pharmacokinetic profiles of a single dose of liposomal curcumin, administered via an existing tunnelled indwelling pleural catheter (TIPC) directly to the tumour site in individuals with diagnoses of malignant pleural effusion. Primarily, we aim to determine a maximum tolerated dose of liposomal curcumin administered via this method. Methods and analysis We will use a 3+3 expanded cohort for predefined dose-escalation levels or until a predefined number of dose-limiting toxicities are reached. Participants will be administered a single dose of liposomal curcumin (LipoCurc, SignPath Pharma) via their existing TIPC as a sequential enrolling case series with the following dose cohorts: 100, 200 and 300 mg/m2. Primary endpoints are determination of the maximum tolerated dose within the predetermined dose range, and determination of the feasibility of intrapleural administration of liposomal curcumin via an existing TIPC. Secondary endpoints include determination of the safety and tolerability of intrapleural administration of liposomal curcumin, median overall survival, effects on quality of life and on feelings of breathlessness, and the pharmacokinetics and concentrations of curcumin from the plasma and the pleural fluid. Important inclusion criteria include age ≥18 years, an existing TIPC, a pleural biopsy or pleural fluid cytology-proven diagnosis of malignant pleural effusion and for whom no antitumour therapy of proven benefit is available or has been previously declined, eastern cooperative group performance status <2. Ethics and dissemination The study protocol has been approved by the Southern Adelaide Local Health Network Human Research Ethics Committee (HREC) (approval number: HREC/20/SAC/11). Study results will be published in peer-reviewed journals, and presented at conferences, in field of medical oncology and respiratory medicine. Trial registration number ACTRN12620001216909. Protocol version number V.1.0.

Primary central nervous system lymphoma (PCNSL) may manifest initially in the eye (termed vitreoretinal lymphoma or VRL) or in non-ocular CNS compartments, or in both. The nature of the onset of PCNSL implies two clinical specialists — ophthalmologists and neuro-oncologists — independently may assess the primary presentation of this rare malignancy. Clinically relevant perspectives on expectations of PCNSL manifestation in both ocular and non-ocular CNS compartments would help inform management practices in each specialty, which should impact clinical outcomes. A recent increase in the number of published PCNSL cohort studies provides new opportunity to review the current prevalence rates of ocular involvement, and the timing of this involvement over the course of disease. In PCNSL cohorts defined by non-ocular CNS compartment involvement, with or without ocular involvement (termed "PCNSL ± ocular involvement" cohorts), mean rates of concomitant VRL at diagnosis, or at any time during the course, are 10% and 16%, respectively. Only a few individuals within this cohort group present with exclusive eye disease (<5%), and the rate of secondary ocular involvement is only 5–9%. In PCNSL cohorts defined by the involvement of the ocular compartment, with or without non-ocular CNS involvement (termed "VRL ± non-ocular CNS involvement" cohorts), 58% of persons have a primary ocular diagnosis, which carries a 50% risk of secondary involvement in the CNS beyond the eye. Rates of non-ocular CNS involvement with VRL at diagnosis or over the course of disease are 41% and 69%, respectively.

Aberrant CpG methylation is a universal epigenetic trait of cancer cell genomes. However, human cancer samples or cell lines preclude the investigation of epigenetic changes occurring early during tumour development. Here, we have used MeDIP-seq to analyse the DNA methylome of APCMin adenoma as a model for intestinal cancer initiation, and we present a list of more than 13,000 recurring differentially methylated regions (DMRs) characterizing intestinal adenoma of the mouse. We show that Polycomb Repressive Complex (PRC) targets are strongly enriched among hypermethylated DMRs, and several PRC2 components and DNA methyltransferases were up-regulated in adenoma. We further demonstrate by bisulfite pyrosequencing of purified cell populations that the DMR signature arises de novo in adenoma cells rather than by expansion of a pre-existing pattern in intestinal stem cells or undifferentiated crypt cells. We found that epigenetic silencing of tumour suppressors, which occurs frequently in colon cancer, was rare in adenoma. Quite strikingly, we identified a core set of DMRs, which is conserved between mouse adenoma and human colon cancer, thus possibly revealing a global panel of epigenetically modified genes for intestinal tumours. Our data allow a distinction between early conserved epigenetic alterations occurring in intestinal adenoma and late stochastic events promoting colon cancer progression, and may facilitate the selection of more specific clinical epigenetic biomarkers.

Cellular hierarchies and signals that govern stemness and differentiation of intestinal adenoma cells are not well defined. In this study, we used organotypic culture to investigate the impact of β‐catenin and BMP signals in cells that form intestinal adenoma in the mouse. We found that activation of β‐catenin signaling by loss of APC or transgenic induction of oncogenic mutant β‐catenin (Ctnnb1mut) initiates the conversion of untransformed intestinal cells to tumor cells. These tumor cells display cancer stem cell (CSC) traits such as increased expression of the CSC markers Cd133 and Cd44, a high capacity for self‐renewal and unlimited proliferative potential. Subsequent inactivation of transgenic Ctnnb1mut results in the reversion of tumor cells to normal intestinal stem cells, which immediately reinstall the cellular hierarchy of the normal intestinal epithelium. Our data demonstrate that oncogenic activation of β‐catenin signaling initiates the early steps of intestinal cellular transformation in the absence of irreversible genetic or epigenetic changes. Interestingly, we found that tumor cells in culture and in adenoma produce BMP4, which counteracts CSC‐like traits by initiating irreversible cellular differentiation and loss of self‐renewal capacity. We conclude that the opposition of stemness‐maintaining oncogenic β‐catenin signals and autocrine differentiating BMP signals within the adenoma cell provides a rationale for the formation of cellular hierarchies in intestinal adenoma and may serve to limit adenoma growth.

The short isoform of single-minded 2 (SIM2s), a basic helix–loop–helix/PAS (bHLH/PAS) transcription factor, is upregulated in pancreatic and prostate tumours; however, a mechanistic role for SIM2s in these cancers is unknown. Microarray studies in prostate DU145 cells identified the pro-cell death gene, BNIP3 (Bcl-2/adenovirus E1B 19 kDa interacting protein 3), as a novel putative target of SIM2s repression. Further validation showed BNIP3 repression in several prostate and pancreatic carcinoma-derived cell lines with ectopic expression of human SIM2s. BNIP3 levels are enhanced in prostate carcinoma cells upon short interfering (si)RNA-mediated knockdown of endogenous SIM2s. Chromatin immunoprecipitation and promoter studies show that SIM2s represses BNIP3 through its activities at the proximal promoter hypoxia response element (HRE), the site through which the bHLH/PAS family member, hypoxia-inducible factor 1α (HIF1α), induces BNIP3. SIM2s attenuates BNIP3 hypoxic induction via the HRE, and increased hypoxic induction of BNIP3 occurs with siRNA knockdown of endogenous SIM2s in prostate PC3AR+ cells. BNIP3 is implicated in hypoxia-induced cell death processes. Prolonged treatment of PC3AR+ cells with hypoxia mimetics, DP and DMOG, confers hypoxia-induced autophagy, measured by enhanced LC3-II levels and SQSTM1/p62 turnover. We show that PC3AR+ cells expressing ectopic SIM2s have enhanced survival in these conditions. Induction of LC3-II and turnover of SQSTM1/p62 are attenuated in PC3AR+/SIM2s DMOG and hypoxia-treated cells, suggesting that SIM2s may attenuate autophagic cell death processes, perhaps through BNIP3 repression. These data show, for the first time, SIM2s cross-talk on an endogenous HRE. SIM2s' functional interference with HIF1α activities on BNIP3 may indicate a novel role for SIM2s in promoting tumourigenesis.

Despite a growing number of descriptive studies that show Single-minded 2 (Sim2) is not only essential for murine survival, but also upregulated in colon, prostate and pancreatic tumours, there is a lack of direct target genes identified for this basic helix-loop-helix/PAS transcription factor. We have performed a set of microarray experiments aimed at identifying genes that are differentially regulated by SIM2, and successfully verified that the Myomesin2 (Myom2) gene is SIM2-responsive. Although SIM2 has been reported to be a transcription repressor, we find that SIM2 induces transcription of Myom2 and activates the Myom2 promoter sequence when co-expressed with the heterodimeric partner protein, ARNT1, in human embryonic kidney cells. Truncation and mutation of the Myom2 promoter sequence, combined with chromatin immunoprecipitation studies in cells, has lead to the delineation of a non-canonical E-box sequence 5'-AACGTG-3' that is bound by SIM2/ARNT1 heterodimers. Interestingly, in immortalized human myoblasts knock down of Sim2 results in increased levels of Myom2 RNA, suggesting that SIM2 is acting as a repressor in these cells and so its activity is likely to be highly context dependent. This is the first report of a direct SIM2/ARNT1 target gene with accompanying analysis of a functional response element.

Aim: Antibody fragments, appropriately formulated, can penetrate through the ocular surface and thus have potential as therapeutic agents. The aim was to investigate the influence of protein fragment format on the kinetics and extent of ocular penetration in vitro. Methods: Immunoglobulin single chain variable domain fragments of a murine monoclonal antibody with specificity for rat CD4 were engineered with a 20 or 11 amino acid linker by assembly polymerase chain reaction, expressed in Escherichia coli and purified by chromatography. Fab fragments of the parental antibody were prepared by papain digestion. Antibody fragments were formulated with a penetration and a viscosity enhancer and were applied to the surface of perfused pig corneas for up to 10 hours in vitro. Penetration was quantified by flow cytometry on rat thymocytes. Results: 20-mer antibody fragments formed natural monomers and dimers following purification that could be separately isolated, while 11-mer fragments were dimeric. All formats of fragment (20-mer monomers and dimers, 11-mer dimers, Fab) showed penetration through the pig cornea after 6 hours of intermittent topical administration. Conclusion: Antibody fragments of different shapes and sizes can penetrate the cornea after topical administration, thereby increasing the potential of this class of proteins for topical ophthalmic use.

Aims Antibody fragments have been shown to penetrate into the anterior chamber when applied to the cornea. The aim of this study was to investigate whether such fragments could penetrate into the vitreous cavity after topical administration to the ocular surface of rabbits. Methods An engineered single-chain variable-domain antibody fragment with specificity for an irrelevant rat determinant was applied as a 50 μ l eye drop to the eyes of live rabbits at 20-min intervals over 12 h. Eye drops contained 0.8–1.1 mg/ml protein in a buffered salt solution supplemented with penetration and viscosity enhancers. Samples were collected by paracentesis from the vitreous cavity immediately postmortem. Antibody fragments in these samples were quantified by measuring the binding activity to specific antigen, using flow cytometry. Results Topically applied antibody fragments were detectable in the vitreous of rabbit eyes after 4–12 h but had cleared at 12 h following the final eye drop. Concentrations of the antibody fragment in the vitreous samples were estimated to be 50–150 ng/ml at 12 h. Penetration of the parental whole antibody into the vitreous was not observed. Conclusion Antibody fragments penetrate into the vitreous chamber of the rabbit eye after topical administration to the ocular surface. Such fragments may have therapeutic potential for diseases affecting the posterior segment.