G12D. Arid1a cooperates with Kras in the adult pancreas as postnatal silencing of Arid1a following sustained KrasG12D expression induces rapid and irreversible reprogramming of acinar cells into mucinous PDAC precursor lesions. In contrast, Arid1a silencing during embryogenesis, concurrent with KrasG12D activation, leads to retention of acinar cell fate. Together, our results demonstrate Arid1a as a critical modulator of Kras-dependent changes in acinar cell identity, and underscore an unanticipated influence of timing and genetic context on the effects of SWI/SNF complex alterations in epithelial tumorigenesis.Mutations in members of the SWI/SNF chromatin remodeling family are common events in cancer, but the mechanisms whereby disruption of SWI/SNF components alters tumorigenesis remain poorly understood. To model the effect of loss of function mutations in the SWI/SNF subunit Arid1a in pancreatic ductal adenocarcinoma (PDAC) initiation, we directed shRNA triggered, inducible and reversible suppression of Arid1a to the mouse pancreas in the setting of oncogenic Kras The pancreas produces many different hormones, as well as several substances important for digestion. To perform these roles, the pancreas contains different types of cells; for example, acinar cells make digestive enzymes that help to break down food. But, like other cells in the body, pancreatic cells can accumulate mutations in their DNA that cause them to divide, acquire an altered identity and form a cancerous tumor.The DNA of cells is packed into a structure called chromatin. While the DNA sequence is essentially the same across all normal cells of a given individual, chromatin can be more or less compacted in the different cell types that comprise our body tissues. A collection of proteins called the SWI/SNF complex can reorganize the chromatin to change how tightly the DNA is packed. This determines which genes in the DNA are accessible and can be activated, and which ones cannot.Around 25% of pancreatic cancers contain mutations in genes that produce proteins of the SWI/SNF complex. These mutations normally occur with an additional mutation that over-activates the gene that produces a potentially cancer-causing protein called Kras. Livshits et al. have now genetically engineered mice to investigate how one such SWI/SNF complex protein, called Arid1a, affects how pancreatic cancer develops using a genetic approach that made possible to temporarily halt the production of Arid1a in acinar cells by feeding these mice an antibiotic. The gene that produces Kras was also over-activated in the pancreases of the mice, making them more likely to develop cancer.Within just two weeks of stopping the production of Arid1a, the acinar cells stopped producing digestive enzymes and started making other proteins that are typically found in cancerous cells, indicating that Arid1a is involved in maintaining the normal identity and activity of these cells. Restoring the ability of altered acinar cells to produce normal levels of Arid1a (by removing the mice from the antibiotic diet) did not reverse these changes. Biochemical experiments showed that acinar cells with reduced levels of Arid1a have altered chromatin. In particular, the genes that produce digestive enzymes, which are normally active in healthy pancreases, were less accessible in mice who had over-active Kras and reduced levels of Arid1a.The results presented by Livshits et al. provide the first evidence of how alterations to Arid1a can lead to irreversible changes in the identity and activity of pancreatic acinar cells. These results will need to be carefully considered by researchers who are developing treatments for cancer patients with mutations in Arid1a and other SWI/SNF proteins. In particular, methods that attempt to restore the functions of absent SWI/SNF proteins to cancer cells are unlikely to treat the cancer successfully. Aberrant chromatin regulation is a hallmark of cancer . NumerouSWI/SNF complexes regulate chromatin accessibility and thus gene expression by re-positioning nucleosomes and interacting with available transcription factors (TFs) at enhancer regions . While tAs the SWI/SNF complex dictates chromatin accessibility by available TFs, such context dependencies might broadly relate to development or pathological cell state. Indeed, in drosophila and mammalian systems, SWI/SNF complex dependent regulation of gene expression controls cell identity and differentiation in developmental as well as regenerative contexts . How losARID1A or other SWI/SNF components occur in up to 25% of cancer patients , a setting in which mutations in patients . PDAC isoncogene , while aoncogene . PDAC cac stages .Ptf1a, Nr5a2 and Pdx1, when combined with oncogenic Kras, accelerates ADM and PanIN development  technology with embryonic stem (ES) cell genetic engineering (G12D (Col1a1 homing cassette that enables insertion of a single copy of a construct into the Col1a1 locus via recombinase-mediated cassette exchange (RMCE)  . The Ptfe (RMCE) . Thus ine (RMCE) .Col1A1 targeting cassette downstream of a tetracycline-response element (TRE) driven promoter; this configuration enables inducible and reversible expression of a downstream transgene in cells expressing the rtTA and supplied with the tetracycline analog doxycycline (dox)  . KC-RIK Kras (after completion of pancreas development (We placed KC-RIK-shArid1a mice on a doxycycline (dox) diet at five weeks of age. This enabled us to suppress Arid1a in the presence of existing oncogenic Kras  but afteelopment , mimickielopment . KC-RIK-elopment . Consistelopment .Surprisingly, a substantial portion of shArid1a mice became moribund within two weeks of enrollment on dox chow, while shRen mice remained healthy . Acute mKras mutation rapidly perturbs the identity and function of pancreatic acinar cells, enhancing their sensitivity to metaplastic and PanIN-inducing signals stemming from oncogenic Kras.The phenotypes described above were on target, as the same changes in histology and cell fate markers were observed in mice carrying two distinct Arid1a shRNAs . FurtherG12D allele from ES cell-derived KC-RIK-shArid1a and KC-RIK-shRen mice though one round of breeding. These C-RIK-shRNA mice were then placed on dox at five weeks of age, after the completion of pancreas development, as in the KC-RIK experiments. In contrast to settings involving oncogenic Kras, these mice did not become moribund following dox administration, and pancreata from both shArid1a and shRen mice appeared normal  or proliferation (Ki67) were noted  and a concomitant decrease in expression of markers of acinar differentiation (Cpa1) . ImportaG12D, CAGS-LSL-RIK, and TRE-shRNA alleles are activated upon Cre expression, leading to concomitant activation of oncogenic Kras and rtTA, allowing simultaneous silencing of Arid1a in the embryos of pregnant mice treated with dox  whereas, when occurring concomitant with KrasG12D activation, it paradoxically promotes retention of the acinar cell phenotype (E7 cohort). Taken together, these data reveal a temporal, context-dependent role for Arid1a in regulating acinar identity in response to oncogenic Kras.In both E7 and P5 cohorts, mKate2 positive pancreas tissue was readily observed in highly chimeric on dox mice at 7 weeks of age, indicating that Arid1a silencing during pancreas morphogenesis does not in and of itself impair pancreatic cell fitness . Howeverr tissue , a featur tissue , the retr tissue . Of noter tissue . Thus, AIn addition to uncoupling the timing of oncogene activation and tumor suppressor gene loss, our system is reversible, and as such enables assessment of whether ongoing gene suppression is needed to sustain pathological phenotypes. To test the stability of the acinar-to-mucinous reprogramming triggered by Arid1a depletion, we placed cohorts of KC-RIK-shRen and KC-RIK-shArid1a mice on dox at 5 weeks of age for two weeks to establish the acute Arid1a phenotype. Mice were then withdrawn from doxycycline to restore Arid1a expression .non-reversible reprogramming of cell fate that is dependent on cellular and genetic context.Remarkably, Arid1a restoration did not reverse the acinar reprogramming phenotype. As expected, pancreata from mice withdrawn from dox retained expression of the mKate2 lineage marker, but lost the shRNA-linked GFP fluorescence signal . Stainin+GFP+ epithelial cells sorted from 3 KC-RIK-shRen, or shArid1a and mice after 5 days of dox treatment. This timepoint occurs after maximal Arid1a suppression but before any observed histological changes, thus reducing the confounding effect of secondary changes in gene expression resulting from tissue inflammation (We next sought to understand the mechanism(s) whereby Arid1a regulates pancreas cell identity. Perturbation of the SWI/SNF chromatin-remodeling complex is expected to impact the expression of SWI/SNF target genes . In ordeammation . Principammation . Unsuperammation .Further analysis of the RNAseq data using an FDR threshold of 0.05 detected 711 downregulated and 505 upregulated genes . Among tIn parallel with mucin upregulation characteristic of PanIN development in KC-RIK-shArid1a mice, GSEA identified downregulation of genes associated with acinar function, such as those encoding the digestive enzymes Cel, Klk1 and Amy1 , which w+GFP+ cells sorted from KC-RIK-shRen and shArid1a mice after 5 days on dox, as in our transcriptional profiling experiments. ATAC-seq assesses chromatin accessibility genome-wide, and therefore provides a direct readout of the chromatin remodeling activity of SWI/SNF complexes. Importantly, ATAC-seq requires substantially fewer cells than current ChIP-seq protocols and, as such, is appropriate for analyzing chromatin states in the limited cell numbers that can be recovered from the preneoplastic murine pancreas.Building on recent observations , we hypoWe found that knockdown of Arid1a resulted in striking changes in chromatin accessibility, with shArid1a chromatin profiles that clearly separated from shRen by PCA . 50,000–To define the TFs that might be responsible for these changes, we searched for transcription factor motifs enriched in regions genomic regions showing reduced accessibility following Arid1a suppression using the HOMER suite of tools for motif discovery . SeveralCela2a). Increased chromatin closing observed upon Arid1a knockdown at these TF target sites was associated with reduced gene expression at adjacent genes. As shown in To further connect these chromatin changes with the transcriptional repression of the acinar identity genes identified above , we perfKras mutations accelerates acinar to ductal reprogramming and specification of mucinous, pancreatic cancer precursor lesions that cannot be reversed by restoring endogenous Arid1a expression. Thus, our data suggest that Arid1a can provide a potent barrier to Kras driven acinar to ductal metaplasia and, consequently, early stage pancreatic neoplasia. Once disrupted by Arid1a mutation, these altered cells are reprogrammed in such a manner that Arid1a is no longer needed to sustain the aberrant cell fate.Mutations in chromatin remodelers are prevalent across the human cancer spectrum, making identification of tissue specific relationships between chromatin regulation and tumorigenesis an important goal in understanding neoplastic progression. In this study, we used a rapid and robust mouse-modeling platform to study the consequences of inducible and reversible suppression of the Arid1a SWI/SNF subunit in a mouse model of pancreatic cancer. Suppression of Arid1a in adult acinar cells harboring oncogenic Kras mutation is characterized by reduced chromatin accessibility accompanied by loss of acinar morphology, reduced expression of acinar genes, and upregulation of a signature characteristic of mucinous lesions. These features imply that genomic areas rendered accessible by Arid1a harbor elements involved in preventing acinar to duct like trans-differentiation in the context of mutant Kras, a critical rate-limiting step in Kras driven pancreatic neoplasia. Indeed, our transcriptional and chromatin profiling data imply that Arid1a disruption, in the presence of oncogenic Kras, reduces the chromatin accessibility of key TFs to the enhancers of genes needed to sustain acinar cell fate. At least one such factor, Nr5a2, can physically interact with the SWI/SNF complex via the Baf60 subunit  that co-occur with KRAS and ARID1A can then contribute to further PanIN progression. While studies to explore the role of ARID1A in later stage PDAC progression are in progress, the irreversibility of the Kras-induced PanIN phenotype indicates that impaired SWI/SNF function may not be required for tumor maintenance. If confirmed, the identification of genotype-specific dependencies produced by SWI/SNF dysfunction rather than restoration of its normal activity may be a more effective way to exploit SWI/SNF mutations for new therapeutic approaches. While some proposed vulnerabilities, such as ARID1B, extend across multiple tissues and mutational landscapes  was changed twice weekly. Chimeric mouse cohorts were generated through the Mouse Genetics core and Center for Pancreatic Cancer Research (CPCR) at MSKCC. For caerulein experiments, mice were given eight hourly intraperitoneal injections of PBS or 50 μg/kg caerulein one day apart. shRNA and genotyping primer sequences shRNAs targeting the following sequences were cloned into mir30-based targeting constructs as described previously . shRen: Col1a1 primer paired with a transgene specific primer:Genotyping of chimeric mice and their progeny was performed as previously described using a common Common: TTCAGACAGTGACTCTTCTGC shRen.713: GTATAGATAAGCATTATAATTCCTA shArid1a.6421: TATACTATACAACTTCTCCAGGCG shArid1a.1803: TATGAATCTTGAGAAAGCTCCTGTAB_300798 and Cell Signaling Technology Cat# 2956S, RRID:AB_1196615), mKate2 , Arid1a , Ki67 (BD BD55609), Cpa1 (R and D AF2765), Clusterin , Sox9 , CK19 Troma III , Cleaved-Caspase 3 , CK8 , E-cadherin (BD BD610181). For immunofluorescence, the following secondary antibodies were used: donkey-anti-chicken CF488 , goat-anti-chicken AF488 and AF647 , donkey anti-rabbit AF594 , goat anti-rabbit AF594 (Life Technologies A11037). Slides were counterstained with DAPI and mounted in ProLong Gold (Life Technologies). For Arid1a, pERK and Mesothelin immunohistochemistry, Vector ImmPress HRP kits were used for secondary detection. Tissues were counterstained with haematoxylin or Alcian blue, dehydrated and mounted with Permount (Fisher). Images were acquired on a Zeiss AxioImager microscope using Axiovision software. CD45 Immunohistochemistry was performed on a Bond Rx autostainer (Leica Biosystems) with heat mediated antigen retrieval using citrate buffer. Antibodies used were rat monoclonal CD45 primary antibody (Abcam 25386). Rabbit anti rat  was used as secondary and Bond Polymer Refine Detection (Leica Biosystems) was used according to manufacturer’s protocol. Sections were then counterstained with hematoxycilin, dehydrated and film coverslipped using a TissueTek-Prisma and Coverslipper (Sakura). Whole slide scanning (40x) was performed on an Aperio AT2 (Leica Biosystems). Quantification of GFP, Cpa1, Ki67 and c-Casp3 was performed using FIJI on sections from >3 animals per condition. For histological analysis of mucinous lesions, PanINs were classified and graded by a veterinary pathologist blinded to genotype, using established criteria , Arid1a , Cpa1 (R and D AF2765), Amylase (Sigma A8273) and Actin-HRP (Sigma A3854). Blots were developed with SuperSignal West Femto substrate (ThermoFisher).2+ and Ca2+ supplemented with 0.1 mg/ml DNase I  and 0.1 mg/ml Soybean Trypsin Inhibitor (Sigma T9003-250mg) for 30 min while rotating at 37°C. After washing with PBS, samples were further digested with 0.05% Trypsin-EDTA at 37°C. Trypsin digestion was neutralized with FACS buffer (10 mM EGTA and 2% FBS in Ca2+ Mg2+in PBS) containing STI. After an additional wash, samples were subsequently digested with 2 U/ml dispase (Roche) in PBS containing DNase I and STI for 20 min at 37°C. Samples were then washed in FACS buffer containing DNase I and STI, filtered through a 40 μm strainer and subjected to FACS. Samples were collected in FACS buffer with an additional 10% FBS for ATAC-seq or sorted directly into TRIzolLS for RNAseq. Each sample was used for either RNAseq or ATAC-seq.Pancreata from KC-RIK-shRNA mice were chopped with scissors, finely minced with a blade and dissociated with digestion buffer: 1 mg/ml Collagenase V dissolved in HBSS with Mghttp://software.broadinstitute.org/gsea/msigdb) and published expression signatures in organoid and mouse models . Sequencing and library preparation was performed at the Integrated Genomics Operation (IGO) at MSKCC. Ribosomal RNA was depleted using RiboZero (Illumina) and stranded KAPA RNA libraries were prepared from 1 ug RNA. Approximately 40 million paired-end 50 bp reads were sequenced per replicate on a HiSeq 2000. Resulting RNA-Seq data was analyzed by removing adaptor sequences using Trimmomatic . Reads we models .Total RNA was isolated using the TRIZOL (Thermo Fisher Scientific), and cDNA was obtained from 500 ng of RNA using the Transcriptor First Strand cDNA Synthesis Kit (Roche) after treatment with DNAse I (Invitrogen) and according to manufacturer’s instructions. The following gene-specific primer sets for mouse sequences were used: Arid1a_F TCCCAGCAAACTGCCTATTC, Arid1a_R CATATCTTCTTGCCCTCCCTTAC, Agr2_F ACAACTGACAAGCACCTTTCTC, Agr2_R GTTTGAGTATCGTCCAGTGATGT, Klk1_F GCCCTGGCAAGTGGCTGTGT, Klk1_R AAGCCGGTGTTGGGCAGAGG, Msl1_F CCCATCGAAGTGGTCAGTCTC, Msln_R GGTGTATGACGGTCAGCTTAGA, Cel_F AAGTTGCCCGTGAAAAAGCAG, Cel_R ATGGTAGCAAATAGGTGGCCG, Amy1_F TCACACGGGTGATGTCAAGTT, Amy1_R GTCTGGGTTAATGCTCACTTCTT, Hprt_F TCAGTCAACGGGGGACATAAA and Hprt_R GGGGCTGTACTGCTTAACCAG. qRT-PCR was carried out in triplicate (5 cDNA ng/reaction) using SYBR Green PCR Master Mix (Applied Biosystems) on the ViiA 7 Real-Time PCR System (Life technologies). Hprt served as endogenous normalization control.+ cells isolated by FACS were lysed in 50 ul lysis buffer (http://broadinstitute.github.io/picard/). Density profiles for genome browser tracks were created using the BEDTools suite (http://bedtools.readthedocs.io). Enriched regions were discovered using MACS2 and scored against input sequence . Dynamic regions between two conditions were discovered using a similar method, with the second ATAC library replacing input. Peaks were then filtered against genomic ‘blacklisted’ regions (http://mitra.stanford.edu/kundaje/akundaje/release/blacklists/mm9-mouse/mm9-blacklist.bed.gz) and those within 500 bp were merged. All genome browser tracks and read density tables were normalized to a sequencing depth of ten million mapped reads. K-means clustering was performed on all dynamic peaks, with the optimal k determined by silhouette analysis. Dynamic peaks were annotated using linear genomic distance and motif signatures were obtained using the ‘de novo’ approach with Homer v4.5 (http://homer.ucsd.edu/). For analyses of TF density, transcription factor ChIP-seq enriched regions were discovered by comparison with matched input using MACS2, as with the ATAC-seq data. TF peaks overlapping sites decreasing in accessibility were classified using 'bedtools intersect' with an overlap of 1 bp. Gene density of these peaks was calculated using RefSeq annotated gene boundaries ± 30 kb of gene start or end, and these gene names were then matched with either differentially expressed genes, or genes with expression not changing by more than 10%. ATACseq data have been deposited to GEO under accession GSE114567.50,000 mKate2s buffer  and subj In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Fiona Watt as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Arid1a restrains Kras-dependent changes in acinar cell identity" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:In their manuscript, Livshits et al. investigate the role of the chromatin remodeling protein Arid1a in pancreatic carcinogenesis and acinar cell identity. To study the consequences of Arid1a-deficieny on acinar fate in the presence and absence of oncogenic Kras activation the authors utilize a very elegant ES cell RNAi based mouse model that facilitates inducible and reversible targeting of Arid1a independent on the Cre-mediated activation of oncogenic Kras during embryogenesis. In addition to phenotypic analyses using the described mouse model system the manuscript provides first mechanistic insights into pancreatic carcinogenesis in the context of Arid1a deficiency using RNA-Seq and ATAC-Seq analysis. Together, the authors demonstrate that Arid1a can function as a barrier to Kras-driven pancreatic cancer initiation, albeit the impact of Arid1a-deficiency underlies a significant context-dependency. The manuscript significantly extends our understanding of the involvement of chromatin remodeling processes in general and the function of Arid1A in particular in pancreatic cancer development. All together the manuscript is perfectly written and follows a logical order and the conclusions drawn from the data are largely justified.1) Please provide more information about the lesions in the mice. The authors state that the ductal lesions found in the Arid1a KD mice can be described as PanIN or IPMN-lesions. In Figure 2D only PanINs have been quantified. How have the lesions been distinguished from IPMNs so that only PanINs were counted? Do the Arid1a KD mice give rise to both, PanINs and IPMN? As BRG1-deficiency in the context of oncogenic Kras activation in the pancreas results to development of IMPN lesions , formation of IPMN might be a likely phenotype in Arid1A kd mice. As the severity of PanINs can be more important than their total numbers, it is important to illustrate the PanIN quantification in Figure 2D for the different PanIN stages separately. Please state how many mice have been observed until an age of app. 1 year ? Was there a significant number of mice to justify the authors´ statement that Arid1a kd animals do not progress to PDAC? Can the authors show that Arid1a is still absent after one year of Dox treatment? How exactly have those lesions been quantified? Counting the lesions per field might lead to misleading numbers, if the Arid1 kd pancreata suffer from pancreatic atrophy. How is the number of ADMs normalized to pancreatic size?Figure 3J: Arid1a kd and control mice are challenged with caerulein to induce pancreatitis. The authors state that they observe comparable ADM formation and pancreatic inflammation in both genotypes. Could the authors please quantify the inflammatory response  to objective the statement of comparable inflammation? In the same line: is the caerulein-induced damage comparable in both models? If blood samples from the mice were available, lipase and amylase serum levels could be assessed. Comparable damage in both models is important to justify the statement of equal regenerative potential in both models.2) Please provide more information about the transcriptional analysis. The unsupervised hierarchical clustering depicted in Figure 6B shows 3 samples for each Arid1a shRNA. In contrast, the PCA in Figure 6A only shows two dots for A1A.1803. Is the dot hidden behind another one or have the authors excluded one sample? If so, what has been the rationale for excluding the sample? In the same line: in ATAC-Seq, there are four replicates for shRen, but 3 in shArid1A. Why? Has a shArid1A sample been excluded? The transcriptional analysis in cell extracts from KC-Ren and KC-shArid1a cells reveals genes and signatures associated with carcinogenesis and acinar cell fate. Could the authors please validate the distinct expression of some of these genes in their isolated cells and in the tissue of the mice? Have RNA-Seq and ATAC-Seq been performed using cells from identical animals?3) Please solidify the analysis of transcriptional programs and chromatin accessibility by demonstrating clearer relationships between the two. For example, the text states "gene sets comprising the targets of these transcription factors were enriched among the downregulated genes in GSEA analysis of transcriptional profiles derived from shArid1a animals ." However, Figure 7H only shows that genes whose chromatin accessibility decreased showed reduced expression. The authors should analyze the data to correlate expression and accessibility at target sites of key acinar transcription factor binding sites, and separately determine the accessibility at genes whose expression changes in shArid1a. […] 1) Please provide more information about the lesions in the mice. The authors state that the ductal lesions found in the Arid1a KD mice can be described as PanIN or IPMN-lesions. In Figure 2D only PanINs have been quantified. How have the lesions been distinguished from IPMNs so that only PanINs were counted? Do the Arid1a KD mice give rise to both, PanINs and IPMN? As BRG1-deficiency in the context of oncogenic Kras activation in the pancreas results to development of IMPN lesions , formation of IPMN might be a likely phenotype in Arid1A kd mice. As the severity of PanINs can be more important than their total numbers, it is important to illustrate the PanIN quantification in Figure 2D for the different PanIN stages separately.G12D”, second paragraph). Regarding PanIN grades, histological analysis 2 weeks after shArid1a inhibition in this adult cohort reveals the majority of lesions are consistent with PanIN-1, though a small number of PanIN-2 lesions were present as well. No PanIN-3 lesions were observed. Similarly, few IPMNs were identified. These data have been included in new Figure 2D. The differences between Arid1a KD and Brg1-deficiency mouse models are discussed in Results subsection “Temporal context dictates the outcome of Arid1a depletion” (first paragraph), and in the fourth and fifth paragraphs of the Discussion.We now quantify and classify ductal lesions as ADM, PanIN , or IPMN, confirming that Arid1a suppression in adult pancreatic cells following expression of mutant Kras during embryonic development predominantly drives development of low grade PanIN lesions ? Was there a significant number of mice to justify the authors´ statement that Arid1a kd animals do not progress to PDAC? Can the authors show that Arid1a is still absent after one year of Dox treatment?G12D”, last paragraph). This supports a role for Arid1a suppression constraining Kras driven preneoplastic development rather than malignant progression as addressed in the original and revised manuscript.A substantial portion of the 66 KC-shArid1a mice placed on dox as adults became morbid and required euthanasia prior to 1 year of age in the absence of PDAC, likely due to the exocrine dysfunction as described in the text. 11 shArid1a mice were analyzed at >1 year of age; of these, one had progressed to cancer, while the rest retained mucinous lesions as pictured. We have confirmed the stability of Arid1a knockdown in mucinous PanIN lesions in this aged cohort .This frequency of PDAC development after a year is consistent with historically reported rates in mouse models where mutant Kras is expressed in the developing pancreas in the absence of genetic modification of other tumor suppressors (subsection “Acute Arid1a knockdown induces rapid changes in adult acinar cell fate in the setting of KrasHow exactly have those lesions been quantified?Quantification was performed by a veterinary pathologist experienced in murine pancreas histology (J. Wilkinson) blinded to genotype. To clarify our methods to the reader, we added a supplementary figure  showing our scoring criteria and examples of the quantified lesions , and extended our Materials and method’s description.Counting the lesions per field might lead to misleading numbers, if the Arid1 kd pancreata suffer from pancreatic atrophy. How is the number of ADMs normalized to pancreatic size?increased pancreas:body weight ratio compared to controls at the time when mice were analyzed, likely due to the stromal expansion that accompanies preneoplastic initiation  . As suggested, we also updated the graphs that reported “lesion per field” to express lesions relative to mm2 of tissue area analyzed .While we agree that counting lesions per field can be misleading in the setting of pancreatic atrophy, shArid1a mice displayed a slightly Figure 3J: Arid1a kd and control mice are challenged with caerulein to induce pancreatitis. The authors state that they observe comparable ADM formation and pancreatic inflammation in both genotypes. Could the authors please quantify the inflammatory response  to objective the statement of comparable inflammation? In the same line: is the caerulein-induced damage comparable in both models? If blood samples from the mice were available, lipase and amylase serum levels could be assessed. Comparable damage in both models is important to justify the statement of equal regenerative potential in both models.To exclude the possibility that genotype specific differences in acute pancreatic damage in response to caerulein prevents accurate evaluation of the regenerative capacity of shArid1a expressing pancreatic cells, we have more fully characterized the time course of pancreatic inflammation, exocrine stress, and exocrine regeneration in shArid1a and shRen expressing mice. Scoring inflammation based on histological analysis and CD45 immunohistochemistry reveals similar levels of inflammation at D2 post caerulein in both shArid1a and shRen expressing mice, as well as comparable resolution at D9. .While no blood samples were available to measure serum pancreatic enzyme levels as proxy for pancreatic damage, we compared molecular markers of acinar stress and acinar differentiation after caerulein treatment. As shown in new Figure 3—figure supplement 2C, caerulein treatment induced a comparable increase in clusterin, a well-established anti-apoptotic gene that is transiently expressed in damaged acini following pancreatic damage and downregulated upon regeneration , in both genotypes at D2 that is markedly reduced upon regeneration (D9). Furthermore, we now show an inverse expression pattern of the differentiated acinar marker Cpa1 in both shRen and shArid1a acinar cells undergoing injury-induced regeneration . Importantly, as shown in Figure 3—figure supplement 3B, Arid1a levels remained suppressed at all stages of response and regeneration following acute pancreatitis, thus confirming the absence of counter selection against Arid1a suppressed cells and expansion of Arid1a expressing cells. These results provide strong evidence that Arid1a suppression does not alter the damage:regeneration capacity of adult acinar cells and that Arid1a is dispensable for restoring acinar fate after injury. These issues are now detailed fully in the results .2) Please provide more information about the transcriptional analysis. The unsupervised hierarchical clustering depicted in Figure 6B shows 3 samples for each Arid1a shRNA. In contrast, the PCA in Figure 6A only shows two dots for A1A.1803. Is the dot hidden behind another one or have the authors excluded one sample? If so, what has been the rationale for excluding the sample?There are 3 dots for A1A.1803 in the PCA plot in Figure 6A, with one inadvertently cropped during composition of the figure. We updated Figure 6A to correct this error. Importantly, the results are not affected by this correction.In the same line: in ATAC-Seq, there are four replicates for shRen, but 3 in shArid1A. Why? Has a shArid1A sample been excluded?No samples were excluded – we were only able to generate ATAC libraries of sufficient quality from 3 shArid1a and 4 control animals and all available results are presented.The transcriptional analysis in cell extracts from KC-Ren and KC-shArid1a cells reveals genes and signatures associated with carcinogenesis and acinar cell fate. Could the authors please validate the distinct expression of some of these genes in their isolated cells and in the tissue of the mice?We now include validation of the RNAseq data by qRT-PCR performed in RNA extracted from FACS-sorted shRNA expressing epithelial cells 5 days after dox administration in adult mice with histologically validated Arid1a knockdown . qRT-PCR analyses confirmed significant deregulation of the expression of both carcinogenesis-associated genes  and acinar identity genes  upon Arid1a knockdown . Moreover, histological validation of select up- and down-regulated genes at 2 weeks post dox treatment shows that early transcriptional changes induced by Arid1a suppression  are maintained in the resultant metaplastic pancreas .Have RNA-Seq and ATAC-Seq been performed using cells from identical animals?Technical challenges involved in dissociating epithelial cells from fibrotic pancreata undergoing Kras driven metaplasia precluded our ability to collect sufficient cells to perform both ATAC and RNA SEQ from the same animals. This is clarified in the revised Materials and methods section.3) Please solidify the analysis of transcriptional programs and chromatin accessibility by demonstrating clearer relationships between the two. For example, the text states "gene sets comprising the targets of these transcription factors were enriched among the downregulated genes in GSEA analysis of transcriptional profiles derived from shArid1a animals ." However, Figure 7H only shows that genes whose chromatin accessibility decreased showed reduced expression. The authors should analyze the data to correlate expression and accessibility at target sites of key acinar transcription factor binding sites, and separately determine the accessibility at genes whose expression changes in shArid1a.We performed a comprehensive analysis of gene expression and chromatin accessibility at targets of pancreatic TFs necessary for maintenance of acinar differentiation  and acinar regeneration (Pdx1) that previously demonstrated to constrain Kras driven preneoplastic development . We compared ATAC-SEQ profiles following Arid1a suppression to high confidence, experimentally-defined datasets of TF binding sites defined by ChIP-seq . This analysis showed a significant enrichment of TF binding sites at ATAC-seq peaks that lose accessibility upon Arid1a KD compared with sites not changing in accessibility . Composite plots of average ATAC-seq signal at these TF binding sites also demonstrated decreased accessibility in Arid1a KD compared with control . Importantly, increased chromatin closing observed upon Arid1a KD at these TF target sites is associated with reduced gene expression at target genes, now documented in two ways. First, by associating these TF-ATAC sites with genes by linear genomic distance and then using GSEA, we were able to find significant enrichment of these peaks in genes that become downregulated upon Arid1a KD as compared with control . Second, by computing the density of these TF-ATAC sites per gene, we found increased density in genes downregulated compared with genes whose expression is upregulated or unchanged . While the TF density was calculated per gene, we note that this is not simply an effect of underlying gene size discrepancies in the different expression categories . Lastly, we note that various intersections of ATAC-seq dynamics and TF binding sites failed to yield a correlation with genes that become upregulated upon Arid1a KD.


Residential opioid rehabilitation aims to improve the mental health and quality of life of opioid users through abstinence and residential program participation. This study aimed to determine the depression, anxiety, stress and quality of life amongst maintenance to abstinence (MTA) program residents. Secondary study aims were to assess the personal characteristics of MTA clients, addiction and risk taking behaviours, factors associated with program completion, as well as to assess the reliable change in participants’ mental health and quality of life on exit.Retrospective analysis of routinely collected data (2013–2017) from surveys completed by 100 clients. Outcome measures were: Depression, Anxiety, Stress Score (DASS-42), World Health Organisation Quality of Life 8 questions (WHOQOL-8) and Kessler Psychological Distress Scale (K10). Other variables included demographics, drug use, other addictions, aggression, self-harm, suicidal ideation/attempts, and risk taking behaviours. Statistical methods included Chi-square, Fisher’s exact, t-tests, repeated measures analysis of variance and the Reliable Change Index.All mean DASS-42, WHOQOL-8 and K10 scores improved significantly in all participants from entry to exit (p < 0.001). The majority of participants demonstrated reliable improvement across all psychometric measures. Completion rates for the MTA program were 51%. Depression (p = 0.023), anxiety (p = 0.010) and stress (p = 0.015) DASS-42 scores decreased significantly more in completers compared to non-completers. The rate of improvement in mean WHOQOL-8 scores and psychological distress scores (K10) was not statistically significantly different between completers and non-completers over time. There was no significant difference between completers and non-completers on socio-demographics, self-reported drug addiction or risk taking behaviour on program entry, except for suicidal thoughts while intoxicated (p = 0.033). Completers were more satisfied with their relationships (p = 0.044) and living place (p = 0.040) on program entry.Overall, completers and non-completers demonstrated improved mental health and quality of life from entry to exit, regardless of program completion. Depression, anxiety and stress reduced more markedly in program completers. Policy makers and programmers could use these findings to further validate their own programs to improve mental health and quality of life of opioid users. Residential opioid rehabilitation aims to improve the mental health and quality of life of opioid users through participation in a residential program which includes psychological support. A maintenance to abstinence (MTA) program has the specific goal of achieving abstinence from maintenance therapies, also known as opioid agonist medications, such as Methadone or Buprenorphine. Although the standard of care for treatment of opioid use disorder is with maintenance therapies , this prOpioid use impacts the individual’s mental health, physical health, social wellbeing and has an impact on Australian society. The negative effects on the individual’s mental health are profound  with mucThere is an abundance of literature evaluating the utility of different treatment modalities on the mental health and quality of life of opioid users, however there is a gap in the literature for evaluating programs such as the MTA program with a goal of achieving abstinence from maintenance therapies, particularly in Australia.The UK National Treatment Outcome Research study, a prospective cohort study, studied individuals undergoing treatment for drug addictions in four main treatment modalities  . They foLength of stay has been shown to be a predictor of reliable change in the psychological recovery and well-being of individuals after completion of a residential substance use treatment  includinThe aforementioned programs, similar to the MTA program, have shown improvements in the quality of life and mental health of participants. The high burden of disease and poor psychometric outcomes for opioid users, as well as the social and economic costs to society highlight that there is great need for programs to address opioid addiction. Moreover, the availability of residential rehabilitation in those with opioid use disorder is of particular importance as this population is more likely to suffer homelessness and social exclusion limiting their access to outpatient services . This stThis study aimed to primarily determine the depression, anxiety, stress and quality of life levels amongst MTA program residents at program entry and exit. Further aims of this study were to assess the personal characteristics of individuals participating in the Maintenance to Abstinence (MTA) programs and to identify which factors on entry were associated with MTA program completion. The study also aimed to assess the changes in psychometric parameters over time, and evaluate the reliability of the change in these measures between entry and exit of the program.This study was a retrospective analysis of routinely collected data from all individuals aged 18 years and over who attended the MTA program between 2013 and 2017 and consented to participating in research evaluation. All participants of the MTA program who consented for this data to be used for research purposes were included in this study.The maintenance to abstinence program (MTA) is a residential rehabilitation program for individuals with opioid dependence and misuse who aim to achieve abstinence. Entry to the program requires an opioid use history of over 2 years and a personal desire to achieve abstinence, not driven by a condition of bail or other contributing legal pressures.Individuals attend the program voluntarily and are assisted in reducing, and then achieving abstinence from their maintenance therapy (either Buprenorphine or Methadone) during their stay. Their maintenance medication is then reduced as per the NSW clinical guidelines for the treatment of opioid dependence , howeverThe program also incorporates psychological support such as counselling and stress management exercises, as well as the opportunity for social engagement through becoming involved in activities, education and low intensity work on site. The aims of these practices are to build self-esteem and reduce mental illness in the residents throughout their rehabilitation. The program also supports the physical health of the residents through regular healthy meal times and encouragement to exercise. The program includes guidance and counselling to teach strategies to cope and remain abstinent after leaving the program . These iParticipants may leave voluntarily at any point throughout the program, as well as at completion of the program. Upon achieving abstinence, participants may also be given the choice to progress into a therapeutic community program in the same facility. Participants engage with a group and are responsible for domestic tasks, and participate in leisure activities with support from therapists and psychologists. Participants may be asked to leave the program if they are unable to follow the restrictions of substance use in the program, as well as general social and safety requirements of the facility. These participants are considered to have left the program “involuntarily” .Data was routinely collected by staff and through computer based questionnaires. Data was collected on entry to the program, at the halfway point (6 weeks) and on exit of the program . Participants provided written consent for this data to be used for research purposes. De-identified data was provided to the research team.Participants were considered to have completed the program if they completed the 91 days in the MTA program, or had achieved abstinence early and progressed to the Therapeutic Community program at the same location.Ethics approval was given by the University of Western Sydney Human Research Ethics Committee (EC00314), approval number H11353.The primary outcomes measures were quality of life and mental health status of the participants. This involved the use of the psychometric tools the Depression, Anxiety, Stress Scale (DASS) , the WorThe DASS-42 scale measures levels of depression, anxiety and stress as separate subscales Each subscale has a minimum score of zero, maximum score of 42. A depression score of 0–9 indicates normal levels of depressive symptoms, a score of 10–13 mild, 14–20 moderate, 21–27 severe and over 28 extremely severe depression. An anxiety score of 0–7 indicates normal anxiety levels, 8–9 mild, 10–14 moderate, 15–19 severe and over 20 extremely severe anxiety. A stress score of 0–14 indicates normal stress levels, 15–18 mild, 19–25 moderate, 26–33 severe and over 34 extremely severe stress .WHOQOL-8 is validated for use in Australia . It is aThe Kessler Psychological Distress Score (K10) measures levels of psychological distress. A high K10 score correlates to high levels of psychological distress, and may indicate an increased likelihood of depression or an anxiety disorder. A score under 20 indicates low levels of psychological distress, a score from 20–24 mild levels, 25–29 moderate levels, and over 30 severe levels of psychological distress [The Modified Monash Model (MMM)  is a tooFurther variables that were collected were the participants’ age, gender, primary substance of use, age at first use, other addictions, aggression, self-harm and suicidal ideation/attempts, risk taking behaviour and gambling problems.p value of less than 0.05 was considered to be significant. Repeated measures analysis of variance (RMAV), using SPSS version 24 [Descriptive statistics were used to determine the demographics of the cohort, as well as the means of the various outcome measures. Chi-square, Fisher’s exact, and t-tests were used to determine the characteristics associated with program completers versus non-completers. A rsion 24 , was useThe Reliable Change Index was calculated for the total K10, WHOQOL-8 and DASS-42 scores in line with Turner and Deane  and was A change score for an individual was considered significant if it was outside the two standard deviations. Participants within the two standard deviations were classified as ‘reliably not improved’. Participants on either side of the normal curve of two standard deviations were classified as either ‘reliably improved’ or ‘reliably declined’. Each category of reliable change was compared with length of stay in the program. Given the small numbers and the non-normal distribution of length of stay, it was not possible to compare the mean length of stay with each reliable change category. Thus, length of stay was divided into three categories: less than 46 days (1st quartile), 47–82 days (second quartile) and more than 83 days. Following this, a Fisher’s exact test was used to determine whether there was an association between reliable change categories and length of stay.The inclusion criteria was met by 100 individuals, 14 individuals did not consent to their data being used for research purposes and were therefore excluded from this study.Table The sample population is comprised of 56% males and 44% females. The mean age is 38.9 (SD = 7.7) years, with 80% of participants aged between 30 and 49 years. The majority of participants (62.4%) were from a major city . There wTwelve percent of participants on entry reported non-drug addictions , one third reported a problem with aggression and about half reported problems with self-harm or suicidality and 59% reported risk taking behaviour more than once a month. The only statistically different behaviour between completers and non-completers reported on entry was that a significantly higher number of completers experienced suicidal thoughts while intoxicated (p = 0.033). Most variables had very small numbers Table .Table 2STable WHOQOL-8 showed only 21% of participants reporting a “good” quality of life on entry to the program whilst 48% reported “poor” or “very poor” quality of life (Table Table The mean reported depression score at entry is “moderate depression” for completers (20.5) and non-completers (20.2) when scored on the DASS-42 scoring system, the mean on exit is “normal” (7.8) for completers and “mild depression” for non-completers (12.8). Anxiety on entry was “severe” for completers (16.5) and non-completers (16.9), which dropped to “normal” for completers (6.4) and “moderate” for non-completers (12.3) on exit. Stress levels on entry were “moderate” for both completers (20.8) and non-completers (21.5), dropping to “normal” for completers (10.2) and “mild” for non-completers (16.2) on exit. Depression (p = 0.023), anxiety (p = 0.010) and stress (0.015) scores decreased significantly more for those that completed the MTA program overtime compared to those who did not. Only anxiety (p = 0.047) and stress (p = 0.018) had a significant group (completion) effect indicating that these scores averaged over time were significantly greater in the non-completion group. There was no statistically significant difference on depression, anxiety and stress on entry between completers and non-completers (results not shown).Completers had higher mean scores for all WHOQOL-8 scores when compared to non-completers on entry and exit. All mean WHOQOL-8 scores improved significantly over time from entry to exit, all with a p value of < 0.001 but the rate of improvements were not statistically significantly different between completers and non-completers over time. Only the total quality of life score (p = 0.010) quality of life (p = 0.017), health (p = 0.023), daily living (p = 0.018) and relationships (0.050) had a significant group (completion) effect indicating that these domains averaged over time were significantly higher in the completion group. There was no statistically significant difference on WHOQOL-8 scores on entry between completers and non-completers, with the exception of daily living (p = 0.042) (results not shown).Mean K10 scores on entry correlated with “moderate levels of psychological distress” (28.1) for completers and “severe” for non-completers (31.0), which dropped to “none” for completers (19.3) and “mild” for non-completers (23.7) on exit. The K10 score improved significantly over time from entry to exit (p < 0.001) but the rate of improvements were not statistically significantly different between completers and non-completers over time (p = 0.525). K10 scores had a significant group (completion) effect (p = 0.017) indicating that these domains averaged over time were significantly greater in the non-completion group.There was no statistically significant difference for psychological distress on entry between completers and non-completers.Table The MTA program appears to improve the mental health and quality of life of those with opioid addiction through involvement in the program, regardless of whether or not they complete the program. Only depression, anxiety and stress reduced more markedly in program completers. Quality of life and psychological distress improved for both completers and non-completers but this improvement was not significantly higher among completers. Reliable improvement was shown across all parameters for the majority of participants, with 74% showing reliable improvement in K10 score, 76.3% showing reliable improvement in WHOQOL-8 score and 72.8% showing reliable improvement in overall DASS-42 score. This data strengthens the notion that an MTA program is effective in improving mental health and quality of life in opioid users. Factors measured on entry that were associated with completion were suicidal thoughts while intoxicated and the participant’s satisfaction with their relationships and living place. This information could be used to develop strategies around improving these measures, or monitoring those who scored lower in these measures which are associated with non-completion.Improvements in mental health and quality of life should be expected when an individual is supported to remain abstinent from opioid drugs, as the relationship between the use of these substances and poor mental health and quality of life measurements is clearly documented , 5, 9. Ton entry to the program when comparing the level of satisfaction. Living place and relationships have been described as “fundamental human needs” since psychologist Maslow first coined the phrase in 1943 [An interesting finding of the study was the association between completion, and less dissatisfaction with living place and relationships according to the WHOQOL-8 score  in 1943 . Living  in 1943 , 30. ThiAnother statistically significant association with completers was higher incidence of suicidal thoughts while intoxicated in the 4 weeks before entry to the program. No other self-harm, suicidality while sober, or other risk taking behaviour was associated with completion. It may be of interest to look at an association with levels of alcohol consumption between the two groups to see if this result may be explained by levels of alcohol intoxication. This finding may be due to chance considering that there were no other associations found.Quality of life and mental health significantly improved over time (entry to exit) for both completers and non-completers Table . This deReliable improvement was seen across all outcome measures for the majority of participants, strengthening the conclusions of this study. Individuals who remained in the program for more than 83 days had a higher proportion of individuals who showed reliable improvement across all measures. The majority of people who stayed less than 46 days also showed reliable improvements. Increased length of stay was not found to be a statistically significant indicator of improved mental health and quality of life outcomes for participants. Those with the longest length of stay (more than 83 days) having the highest proportion of reliable improvement across all measures may give some support to the current literature which indicates that increased length of stay, particularly of 90 days or more, relates to higher rates of reliable improvement , howeverStudy limitations were the small sample size, self-reported data and relatively high missing data for some variables, especially the K10 data. The MTA program data was only available over a four year period, which led to the small sample size for this study and may therefore have been underpowered to detect differences between completers and non-completers. Another limitation is that this study does not draw comparison to other treatment modalities such as individuals remaining on maintenance therapies, nor those in a therapeutic community program. Furthermore, the study took place in one particular setting so may not be generalisable to other settings.The findings of this study support the body of literature which discusses the utility of residential rehabilitation for opioid users on improving their psychosocial wellbeing , 15. TheA study was conducted on the Therapeutic Community program at the same location . CompletFuture research for both the Buttery’s MTA program, as well as other residential opioid rehabilitation programs, would be to conduct long term follow up studies. It would be of interest to examine the same outcome measures of mental health and quality of life at intervals after exit from the program. A survey detailing relapse of opioid or other drug use would be of interest to determine whether abstinence is maintained long term in those who completed the program. Further studies of MTA programs could aim to compare the characteristics of individuals with different dose reduction regimens, as well as withdrawal effect profiles.This study adds to the evidence supporting the use of MTA programs such as The Buttery’s. It validates residential MTA rehabilitation as a means of intervention for those affected by opioid use, even if they do not complete the full program. Another potential impact from this study may be that MTA programs continue to improve the mental health and quality of life outcomes for those suffering from opioid use. This may subsequently reduce the impact that these individuals may have on other health services later.This study may assist rehabilitation program providers to further improve their understanding of the needs of individuals throughout their recovery. In particular, it points to the important and basic needs of individuals to have satisfactory relationships and living arrangements in order to best support their recovery. This may give them a greater chance of program completion. This finding could be used to help identify individuals who are at risk of program drop out, and potentially lower their risk by focussing on improving their satisfaction with both their living place and relationships.The MTA program in particular are incorporating the findings of this study into practice. Specifically they are reviewing the need for flexibility in length of stay, as well as developing a profile of the significant associations with completion, in order to reflect on how better to meet the needs of the client group who exit the program early.In conclusion, the use of the MTA program shows reliable improvements in opioid users’ mental health and quality of life, irrespective of program completion. Depression, anxiety and stress show increased rates of improvements amongst those that complete the program. These findings may help guide decisions about service requirements for people who are recovering from drug misuse and support the continued use of residential opioid rehabilitation programs.


Background and Objective: Endobronchial ultrasound (EBUS) is a minimally invasive endobronchial technique, which uses ultrasound along with a bronchoscope to visualize the airway wall and structures that are adjacent to it. Indications for endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) are samplings of mediastinal, hilar lymph nodes, and tumors adjacent to airway walls. EBUS-TBNA has been used in our clinic since 2009. The aim of the study is to evaluate the sensitivity, specificity, positive and negative predictive value, and diagnostic accuracy of cytological and histological specimens, and the safety of EBUS-TBNA in an unselected patient population that has been referred to our hospital. Materials and Methods: We have retrospectively analyzed the medical documentation of 215 patients who had EBUS-TBNA performed in our clinic from April 2009 to February 2014. Results: There were 215 patients who underwent EBUS-TBNA. A total of 296 lymph nodes were sampled. EBUS-TBNA was diagnostic in 176 (81.9%) cases of cytological, 147 (68.4%) cases of histological, and 191 (88.9%) cases of the combined evaluation. In the lung cancer patients, EBUS-TBNA cytology had a sensitivity of 72.9% and histology of 72.9%, and in the sarcoidosis group, it had a cytology of 55.8% and histology of 64.5%. As all positive cytology and histology specimens were assumed to be true positive, specificity and positive predictive value (PPV) were 100%. The sensitivity and diagnostic accuracy was significantly higher when cytology and histology specimens were combined, compared with cytology or histology results evaluated separately (p < 0.05) (for lung cancer 84.1% and for sarcoidosis 78.8%). The sensitivity and diagnostic accuracy of EBUS-TBNA procedures increased significantly over time, with increased experience. There were no complications with EBUS-TBNA in our clinical practice. Conclusions: EBUS-TBNA had a high diagnostic yield and was safe in the diagnosis of lung cancer and sarcoidosis. It was most informative when cytology and histology were combined. The informative value of EBUS-TBNA histology increased with our experience. Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is a minimally-invasive, endobronchial technique that uses ultrasound along with bronchoscopy to visualize the airway wall and structures adjacent to it, and allows real-time guidance in the sampling of mediastinal and hilar lymph nodes, and tumors ,2.Mediastinoscopy was the gold standard for invasive mediastinal staging, providing definite tissue diagnosis at 100% specificity and almost 80% sensitivity . Other oThe majority of the initial clinical trials with EBUS-TBNA were well-designed prospective trials, in reference centers with high expertise, which used highly preselected patient populations, and were enriched by selecting for particular diseases—which probably provided the highest diagnostic yield ,12. NowaIn Lithuania, EBUS was introduced for the first time in the Department of Pulmonology and Immunology of the tertiary Hospital of the Lithuanian University of Health Sciences, in 2009.The aim of this study is to evaluate the sensitivity, specificity, positive and negative predictive value, diagnostic accuracy of cytological and histological specimens, and the safety of EBUS-TBNA in an unselected patient population, that was referred to our hospital.Patients with mediastinal or hilar lymphadenopathy, who were referred to EBUS-TBNA between April 2009 and February 2014, were enrolled in this retrospective study. A chest computed tomography was routinely performed on all patients, and those with enlarged hilar or mediastinal lymph nodes (nodes with a short-axis diameter greater than 10 mm) were selected for EBUS procedures.The procedures were performed under conscious sedation, using midazolam and phentanyl, by three different pulmonologists, each with equal training and expertise. A conventional flexible bronchoscopy was performed before all EBUS procedures, to investigate the tracheobronchial tree for intraluminal pathology, using a flexible bronchoscope. Local anesthesia was achieved with a 10% lidocaine solution, sprayed in the pharyngeal area, combined with a bolus dose of 2% lidocaine, applied to the vocal cords and tracheobronchial tree. EBUS-TBNA was performed using a linear curved-array ultrasonic bronchoscope BF TYPE UC180F . This bronchoscope had an outer diameter of 6.9 mm, a 2.0 mm instrument channel, and 30 degree oblique forward-viewing optics. An electronic convex-array ultrasound transducer was attached at the distal tip of this instrument and covered by a balloon, which was inflatable with water. Scanning allowed for a penetration of 5 cm and was performed at a frequency of 7.5 MHz. Image processing was performed by Aloka ProSound α5sx . TBNA was performed by passing a dedicated 22- or 21-G needle . A central stylet was passed through the working channel of the bronchoscope, and then advanced through the airway wall, and into the lymph nodes under real-time ultrasound control. An integrated color–power Doppler ultrasound  was used to exclude vessels prior to needle puncture. After stylet removal, suction was applied using a syringe, while manipulating the needle back and forth approximately 10–15 times within the lesion. A minimum of three needle passes per lymph node were performed. After the sampling, suction was released slowly and the needle was retracted. Lymph node aspirates were smeared onto glass slides and air-dried for cytological examination, and put into the separated container and filled with formalin for histological examination of cell blocks. No rapid onsite cytology was performed.EBUS-TBNA was considered as diagnostic if a clear, definite cytological or histological diagnosis of lung cancer, sarcoidosis, or another disease was obtained (positive specimens), or if lymphocytes and normal lymphoid tissue were found (negative specimens) . It was Complications with EBUS-TBNA were classified as major  and minor  .The study was approved by the Kaunas Regional Biomedical Research Ethics Committee .2) test. For comparisons of data in more than two groups, we used an analysis of variance (ANOVA), and, in two groups, the Student’s t-test. Diagnostic sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy (yield) were calculated using the following standard definitions: sensitivity—the probability of getting a positive test result in a subject with the disease; specificity—the probability of getting a negative test result in a subject without the disease; positive predictive value—the probability of having the disease of interest in a subject with a positive test result; negative predictive value—the probability of not having a disease in a subject with a negative test result; diagnostic accuracy—a proportion of correctly classified subjects among all subjects. The unit of analysis was patients. Values of p < 0.05 were considered to indicate statistical significance.Data were analyzed using the SPSS 16.0 (version for Windows)  statistical software package. Data were presented as frequencies or means ± standard deviation. Categorical data were analyzed using the chi-square  were women and 144 (66.9%) were men. The mean age of patients was 58.6 ± 14.9 years (range 21–83). The main reasons for performing EBUS-TBNA were the following: suspected lung cancer, where primary diagnosis was needed (some together with staging) ; staging of known lung cancer ; suspected sarcoidosis ; and mediastinal lymphadenopathy of unknown origin . Out of the total296 lymph nodes that were sampled, subcarinal and right paratracheal were most frequently biopsied .p < 0.0001).EBUS-TBNA was diagnostic in 176 (81.9%) cases of cytological, 147 (68.4%) cases of histological, and 191 (88.9%) of the combined evaluation. The positive findings were found slightly more often in histologic (cell block) specimens compared with cytology smears, but cytologic specimens had less non-diagnostic results. The highest rate of positive results and lowest rate of non-diagnostic results were found when cytology and histology specimens were combined . Among pp < 0.05). Additionally, the sensitivity and diagnostic accuracy of EBUS-TBNA procedures increased significantly over time . Significantly higher NPV was detected for the histological and combined evaluation in the sarcoidosis group, compared with lung cancer (p < 0.05). No significant differences in diagnostic accuracy between these two groups were detected. No significant differences in the sensitivity, NPV, and diagnostic accuracy was found when comparing younger and older (>70 years of age) patients .The overall diagnostic yield, grouped by final diagnosis, is presented in p < 0.05 for combined results compared to cytology or histology separately). As all positive cytology and histology specimens were assumed to be true positive, specificity and PPV were 100%.While analyzing the most frequently biopsied lymph nodes, the following results were found: In subcarinal lymph nodes, sensitivity, NPV, and diagnostic accuracy were 67.6%, 50.0%, and 75.5%, respectively, for cytology specimens; 67.3%, 49.3%, and 75.2%, respectively, for histology specimens; and 80.6%, 62.5% and 85.3%, respectively, when both specimens were combined. In comparison, sensitivity, NPV, and diagnostic accuracy of the samples from the right paratracheal lymph node were 63.9%, 37.1% and 70.3%, respectively, for cytology specimens; 60.7%, 35.1% and 67.6%, respectively, for histology specimens; and 78.7%, 50.0% and 82.4%, respectively, when both specimens were combined . One of the early studies on EBUS-TBNA, performed by Yasufuku et al. , on 70 pMany factors could have influenced these discrepancies. For example, the majority of these previously reported trials were prospective, well-conducted clinical studies; the patients that were investigated in these trials were usually preselected, with a high prevalence of lung cancer, and referred to large bronchoscopy centers; and EBUS-TBNA was carried out by experienced investigators. These reasons probably led to better results and might not have reflected the real clinical situation. Further studies showed that the value of EBUS-TBNA in daily clinical practice differed from the value in clinical trials. The sensitivity could have depended on the design of the trials, with retrospective studies that indicated a lower diagnostic yield. Agarwal et al. showed that the diagnostic yield for diagnosing sarcoidosis in retrospective studies was only 74.3%, compared with 83.9% in prospective studies .The efficacy of EBUS-TBNA also seemed to depend on the investigated pathology. The highest sensitivity of EBUS-TBNA was found in the patients with known lung cancer . For exaIn diagnosis of sarcoidosis, the value of EBUS-TBNA seemed to be lower than that of lung cancer, with reported diagnostic yields ranging from 54 to 93%, with pooled diagnostic accuracy being 79% [p < 0.05) with increased experience . In our center, patients were selected for EBUS-TBNA after a computed tomography or positron emission tomography–computed tomography scanning.Another factor that might have influenced the sensitivity of EBUS-TBNA, was the method in which a patient was selected for the procedure. It was reported that sensitivity was higher in the studies that enrolled patients on the basis of a positive computed tomography or positron emission tomography result   comparedEBUS-TBNA obtained cytology samples do not always provide sufficient information. Sometimes additional tissue is required, which can be achieved by processing sampled material to cell blocks. This enables histological diagnosis and immunohistochemical staining. With the development of new treatments for lung cancer, which have different degrees of efficacy and toxicity, an accurate pathologic classification has become essential. This can be enabled by cell blocks, which are recommended for routine use during lung cancer diagnosis ,27. In oPatients with isolated mediastinal lymphadenopathy (IML) were frequently presented to physicians . In thisEBUS-TBNA allowed for the ability to obtain both cytological and histological samples via either a 22-G needle or the more recent 21-G needle. In our study, we used a 22-G needle. Some studies, which compared the samples that were obtained with both needles, did not demonstrate a difference in diagnostic yield within the groups . HoweverEBUS-TBNA had high diagnostic yield and was safe in the diagnosis of lung cancer and sarcoidosis. The informative value of EBUS-TBNA histology increased with our experience. It is the main diagnostic standard in our daily clinical practice, and surgical methods are only used as an alternative.


ARID1A truncating mutations were identified as the by far most common alterations of SWI/SNF complexes in urothelial bladder cancer. As current ARID1A protein expression data in bladder cancer are inconsistent and incomplete we examined if the frequency of truncating ARID1A mutations translates into a similar frequency of cases showing ARID1A protein loss. We applied a validated ARID1A antibody conducting a comprehensive immunohistochemistry-based expression analysis in urothelial bladder cancer (n = 362) including carcinoma in situ (CIS) cases. While observing increased median ARID1A protein levels in all carcinoma subgroups compared to normal urothelial controls (n = 21), the percentage of cases showing ARID1A protein loss was positively correlated with increasing stage and grade culminating in a rate of 14.1% in muscle-invasive disease. ARID1A-depletion did neither increase EZH2 protein or trimethylated H3K27 levels in vitro nor did ARID1A expression correlate with EZH2 or H3K27me3 amounts in human bladder carcinomas. Importantly, ARID1A-deficiency was neither associated with enhanced sensitivity towards inhibition of EZH2 enzymatic activity nor depletion of EZH2 protein. In summary, ARID1A truncating mutations, potentially translating into ARID1A protein loss in a subset of high-grade bladder cancers, are the most common SWI/SNF genetic alterations in bladder cancer. Our data do not support ARID1A-deficiency as predictive biomarker for EZH2-inhibitor treatment response in bladder cancer underlining the need for future bladder cancer-specific, drug screens for successfull discovery of ARID1A-deficiency-based targeted drugs.Bladder cancer therapy relies on aggressive treatments highlighting the need for new, targeted therapies with reduced side effects. SWI/SNF complexes are mutated in ~20% across human cancers and dependency of SWI/SNF-deficient tumors on EZH2 has been uncovered recently. To systematically dissect the frequency of genetic alterations in SWI/SNF complexes potentially contributing to their inactivation, mutations and copy number variations in 25 SWI/SNF subunit genes were analyzed making use of publicly available sequencing data for 408 muscle-invasive bladder carcinoma samples.  More thiagnosed ,5 highliMYC, CCNE1 and CDKN1A [Genes encoding subunits of the SWI/SNF (Switch/Sucrose Non-Fermentable) nucleosome remodeling complexes have been reported to be mutated in approximately 20% of all human cancers and functional/mechanistic studies support their role as tumor suppressors ,7. SWI/Sd CDKN1A –10. Mored CDKN1A ,12. The d CDKN1A . Two majd CDKN1A . ARID1A,d CDKN1A , is the d CDKN1A  and a poet al. demonstrated that reintroduction of the core SWI/SNF subunit SNF5 into a SNF5-deficient tumor cell line results in eviction of PRC1 and PRC2 from the P16INK4A locus leading to activation of p16 expression [Numerous studies have uncovered a genetic antagonism between SWI/SNF and Polycomb repressive complex (PRC) genes –19. Polypression . Wilson pression . Moreovepression ,22 but tARID1A truncating mutations as the most common inactivating events in SWI/SNF complexes. As current ARID1A protein expression data for bladder cancer are inconsistent and incomplete, a comprehensive immunohistochemistry (IHC)-based ARID1A protein expression analysis including carcinoma in situ (CIS) cases was conducted for the first time. Moreover, direct evidence of ARID1A’s tumor suppressor capability in urothelial bladder cancer cells is scarce and we thus analyzed the effect of ARID1A protein loss on growth of urothelial cells as well as potential underlying mechanisms. Finally, we investigated the putative dependence of ARID1A-depleted and ARID1A-mutated urothelial cells on EZH2 using in vitro models for bladder cancer. So far, our data do neither support a genetic antagonism between ARID1A and EZH2 nor ARID1A-deficiency as predictive biomarker for EZH2-inhibitor treatment response in bladder cancer, underscoring the need for future bladder cancer-specific, high-throughput drug screens for successfull discovery of ARID1A-deficiency-based targeted drugs.In order to systematically analyze the potential applicability of treatments based on SWI/SNF-deficiency—including dependency on EZH2—to bladder cancer, we comprehensively dissected the frequency of inactivating alterations in known SWI/SNF subunit genes in bladder carcinomas revealing http://www.cbioportal.org/) [ARID1A and TP53/RB1 was performed in cBioPortal using data sets of four independent studies [Genetic alterations (mutations and copy number variations (CNVs)) of 25 SWI/SNF subunit genes were analyzed in two TCGA cohorts,one comprising 408  and the  studies ,24,26,27https://varbank.ccg.uni-koeln.de/). Illumina Realtime Analysis (RTA) software v.1.8 was applied to filter primary data according to signal purity. Sequencing reads were subsequently aligned against the human genome reference build hg19 using the BWA alignment algorithm [To detect alterations in SWI/SNF, PRC1/PRC2 complexes and Ras pathway genes in the bladder cancer cell lines J82 and RT112 as well as bladder cancer tissues, whole exome sequencing was performed. Libraries were constructed from 2 μg non-degraded gDNA (0.7% agarose gel) with SureSelect Human All Exon V6 . Sequencing was carried out on a HiSeq2000 (Ilumina) at the Cologne Center for Genomics. Data analysis was performed using the VARBANK pipeline v.2.3 and the corresponding filter interface . Described ARID1A truncating mutations in the bladder cancer cell lines HT1376, JMSU-1 and VM-CUB-1 [Absence of genetic alterations in SWI/SNF and PRC2 subunit genes in the cell line SCaBER was verified using sequencing data provided by the COSMIC database  was compared to OS of patients exhibiting putative passenger mutations, gene amplification/gain and wildtype ARID1A gene sequence using publicly available sequencing data of two TCGA cohorts comprising 127 [ARID1A’s mutational status of the above mentioned studies can be accessed and downloaded via the cBioPortal  [Overall survival (OS) of bladder cancer patients showing sing 127  and 408 sing 127  muscle-ial.org/) .Formalin-fixed, paraffin-embedded (FFPE) tumorous and normal urothelial tissue samples used to construct a tissue microarray (TMA) were obtained from the diagnostic archive of the Institute of Pathology, Medical Faculty of the RWTH Aachen. This retrospective, anonymized study was conducted in accordance with local Institutional Review Board (IRB)-approved protocols  of the Medical Faculty at RWTH Aachen University and the principles expressed in the Declaration of Helsinki. The TMA comprised 362 tumor and 21 normal urothelial samples from 284 patients. Clinico-pathologic variables of urothelial bladder cancer cases included in the tissue microarray are summarized in TM Flex peroxidase blocking reagent (Dako) for 5 min to block endogenous peroxidase activity. Immunostaining was performed with antibodies specific for ARID1A , EZH2  and H3K27me3 . Subsequently, tissue sections were incubated with a HRP-conjugated secondary reagent (Dako) for 15 min. The peroxidase reaction was visualized with the DAB+ Substrate Chromogen System (Dako). The sections were then counterstained using Mayer`s haematoxylin. Nuclear expression of ARID1A, EZH2 and H3K27me3 was assessed by two experienced pathologists (RK and NTG) according to an adapted immunoreactive score (IRS) developed by Remmele and Stegner (1987) [For pre-treatment, TMA sections (2 μm) were incubated in antigen retrieval solution with pH 6 or 9  at 95°C for deparaffinization, rehydration and epitope retrieval. FFPE slides were then treated with EnVisionr (1987) .To analyze the subcellular ARID1A protein expression in J82 cell clones, cell pellets were fixed in 4% formalin solution for 30 min at room temperature. Subsequently, fixed cells were embedded in 3% agarose followed by a dehydratization step. Dehydrated agarose-embedded cells were finally covered with paraffin. After preparation of FFPE sections on microscopic slides, ARID1A protein detection was accomplished following the protocol for immunohistochemistry described above.1×NuPAGE LDS Sample Buffer (Invitrogen) supplemented with 50mM dithiothreitol (Life Technologies). Heat denatured samples were loaded on 4–12% gradient gels  and then transferred onto 0.2μm PVDF membranes (Whatman)  for immunodetection. Blots were blocked in TRIS-buffered saline (TBS) containing 0.1% Tween-20 (TBS-T) and either 5% non-fat dry milk (Merck) or BSA (Roth) for 1h at room temperature. Blocked blots were then incubated with the primary antibody overnight at 4°C, diluted in blocking solution either containing 5% non-fat dry milk or BSA. The following primary antibodies were used: ACTB , ARID1A , CCND1 , EZH2 , H3K27me3 , MYC , p21  , p16 , p53 . After washing three times (TBS + 0.1% Tween-20), blots were incubated with secondary peroxidase-conjugated antibodies (DAKO) diluted in blocking solution containing 5% non-fat dry milk for 1h at room temperature. After washing three times (TBS + 0.1% Tween-20), antibody detection was accomplished with Pierce ECL Western blotting Substrate (Thermo Scientific).Total cell protein lysates were obtained by sonification of cells in an appropriate volume of TRIzol® (Invitrogen) RNA extraction. Extracted RNA was quantified using the NanoDrop ND1000 spectrophotometer (Thermo Scientific). The A260 nm/A280 nm ratio was generally between 1.9 and 2.0. Subsequently, cDNA was synthesized using 1μg of total RNA and the reverse transcription system (Promega) as described previously [Total RNA from human cell lines was isolated using the standard procedure for eviously . After cSYBR Green PCR mix (Bio-Rad Laboratories) and the iCycler IQ5 (Bio-Rad Laboratories) as described previously [Primer3web software (version 4.0.0) (http://primer3.ut.ee/). All reactions were performed in triplicates including negative controls without cDNA. Specificity of amplicons was confirmed by size estimation on agarose gels and melt curve analysis. Obtained data were analyzed using the comparative Ct (threshold cycle) method. Complete reaction conditions, primer sequences and lengths of amplicons are listed in cDNAs were amplified by semi-quantitative real-time PCR using eviously . Gene-spVenor® GeM Mycoplasma Detection Kit (Minerva Biolabs).The human urothelial bladder cancer cell lines , the squamous cell carcinoma bladder cell line SCaBER and the SV40 large T-antigen-immortalized normal urothelial cell line UROtsa were authenticated using Multiplex Cell Authentication by Multiplexion (Heidelberg) as described recently . J82, JM5/well) were seeded in six-well plates and cultured overnight. After 24h, culture medium was replaced and cells transfected for 72h using HiPerFect transfection reagent (Qiagen), ARID1A-specific siRNAs (20nM) (Qiagen) and EZH2-specific siRNAs (40nM) (Qiagen), respectively. Hs_ARID1A_6: 5‘-CTCGGTATCACCGTTGATGAA-3‘; Hs_ARID1A_4: 5‘-CAGAGTTTACTCTGTACGAAT-3‘. Hs_EZH2_6: 5’-CAGGATGGTACTTTCATTGAA-3’; Hs_EZH2_7: 5’-AACCATGTTTACAACTATCAA-3’. Equal amounts of a scrambled siRNA served as a negative control . To generate stably transfected ARID1A knockdown single-cell clones, J82 cells were transfected using FuGene HD Transfection Reagent (Promega) following the manufacturer's instructions and the vector pGeneClip Neomycin either encoding an ARID1A-specific shRNA  or a scrambled shRNA sequence (5‘-GGAATCTCATTCGATGCATAC-3‘) as negative control . Selection of stable single-cell clones was achieved by culturing transfectants in complete growth medium containing 700μg/ml G418 (Life Technologies) for at least two weeks.To transiently knockdown ARID1A and EZH2 expression, urothelial cells (1-2x104 cells/well) and cultivated for 96 h . Thereafter, the cell number was determined using the CASY® Cell Counter and Analyzer (OLS OMNI Life Science). Colony growth was analyzed by conducting a 2D colony formation assay. Briefly, UROtsa or J82 cells were seeded in six-well plates (1000 cells/well) containing growth medium. Medium was replaced every two days and cells were cultivated for two weeks . Afterwards, cell colonies were fixed and stained using a 0.5% crystal violet staining solution . Densitometrical evaluation of photographs was accomplished by using ImageJ Software . Experiments were performed in triplicate.Increase in cell number was recorded for UROtsa cells over a period of 96 h. Briefly, cells were seeded in 12-well culture plates (2x102O). Densitometrical evaluation of photographs was accomplished by using ImageJ Software . Experiments were performed in triplicate. Growth IC50 values were determined by plotting lines of best fit using a non-linear regression model. All experiments were performed in triplicate.For short-term drug exposure, 1000–3000 cells/per well were seeded in 96-well plates. 24h after cell seeding, GSK126 (Selleckchem) or DMSO as negative control were added to the cells and these were continuously exposed for 72h. Cell viability was finally estimated using CellTiter-Glo® luminescent cell viabilty assay (Promega). For long-term clonogenic assays, cells were seeded (1000 cells/well) in six-well plates and 24h after cell seeding continuously exposed to GSK126 or DMSO control for 10 days. Medium containing fresh GSK126 was replaced every 2–3 days. Cell colonies were fixed and stained using a 0.5% crystal violet staining solution  and the repeated measures ANOVA (Tukey’s multiple comparison test) test were used to compare more than two groups.For statistical analyses SPSS 19.0  and GraphPad Prism 5.0  were applied. Differences were considered significant if the two-sided P values were < 0.05. To determine the potential prognostic value of ARID1A was identified as the by far most commonly altered subunit of SWI/SNF complexes in urothelial carcinomas (106/408 = 26%) with inactivating truncating mutations (frameshift and splice mutations resulting in premature stop codons as well as nonsense mutations) being the predominant mutational type  followed by alterations in the genes BRD9 (12%) and SMARCA2 (10%)  (35/133 = 26%) and tumors of non-papillary origin developing from carcinoma in situ (67/269 = 25%)  in 25 known SWI/SNF subunit genes using a publicly available bladder cancer cohort comprising 412 muscle-invasive bladder cancer (MIBC) cases . For 408A2 (10%) . Interes9 = 25%) . Similar9 = 25%) .ARID1A truncating mutations are correlated with reduced ARID1A mRNA expression as expected from prelimary work [ARID1A mRNA levels in cases exhibiting truncating mutations compared to patient samples without these alterations  (ARID1A gene locus (not shown).Subsequently, we analyzed if ary work . Indeed,equence) . This obARID1A gene alterations potentially resulting in loss of functional ARID1A protein (truncating mutations and deep deletions) are associated with worse patient prognosis. Comparing overall survival (OS) of patients harboring truncating mutations and deep deletions (median OS: 28.2 months) to those patients showing ARID1A gene amplification/gain and wildtype gene sequence (median OS: 44.9 months) revealed no significant association of potentially deleterious ARID1A genetic alterations with overall survival , were applied to knockdown endogenously expressed ARID1A mRNA and protein in J82 cells. As a control, J82 wildtype cells were transfected using the same vector backbone containing a scrambled shRNA sequence  and normal urothelial (NU) controls (n = 21) for ARID1A protein expression loss as a potential surrogate for in vitro models. First, J82 high-grade urothelial bladder cancer cells, neither showing inactivating alterations in subunit genes of the SWI/SNF complex nor genetic alterations in the PRC2/PRC1 complex or Ras pathway genes that might disguise the dependence [in vitro model we transiently knocked down ARID1A expression in SV40 large T-antigen-immortalized normal urothelial UROtsa cells  [in vitro model to analyze potential effects of ARID1A loss on the expression of key cell cycle-related genes  previously associated with ARID1A gene function [CDKN1A transcript (FC: 1.5) and p21 protein (range FC: 1.3–1.5) levels  function [Subsequently, we performed RT-NHUC)  showing function . Interes) levels . MYC hasfunction . We obsein vitro growth of TERT-NHUC, we applied a second normal urothelial cell in vitro model (UROtsa), that has been immortalized by SV40 large T-antigen, to study the effect of ARID1A on cellular growth [CDKN1A transcript (FC: 3.2) and p21 protein levels (range FC: 1.5–1.6)  .ARID1A being the most commonly altered subunit gene [ARID1A has been identified as one of the top-altered genes with mutational frequencies ranging from 13 to 38% between studies [Current bladder cancer therapy mainly relies on non-specific treatments, i.e. chemotherapy or BCG-based immunotherapy in addition to surgery depending on stage and grade of the disease ,5 highlinit gene . In urot studies ,37,43–47 studies , resulti studies ,22. The ARID1A that might contribute to inactivation of SWI/SNF complexes in urothelial bladder carcinomas. We analyzed mutations and CNVs in 25 SWI/SNF subunit genes  making use of publicly available sequencing data for 408 [ARID1A truncating mutations as the by far most common alterations in SWI/SNF complexes. Although a previously performed meta-analysis considering SWI/SNF subunit gene mutations in several human carcinomas including bladder cancer came to similar results, the conclusions drawn for bladder carcinomas have been limited as sequencing data for only 9 cases have been included [SWI/SNF complexes are assemblies of more than 10 subunits that are encoded by 29 known subunit genes, resulting in a great diversity in SWI/SNF complex composition . Here, w for 408  as well  for 408  and idenincluded .ARID1A mutations also translates into a similar frequency of cases showing ARID1A protein loss [in situ, has not been considered so far in any of the previously conducted studies. For these reasons we started our analysis with a profound specificity validation of the ARID1A antibody applied and performed a thorough, comprehensive IHC-based ARID1A expression analysis in a large cohort of urothelial bladder carcinomas including carcinoma in situ (CIS) cases. We observed increased ARID1A protein levels in all urothelial carcinoma subgroups with strongest expression in early tumor stages compared to NU controls. As the SWI/SNF complex is involved in DNA repair [ARID1A truncating mutations result in ARID1A protein expression loss [ARID1A gene expression reduction/loss and should be addressed in future studies.Thus, we subsequently focused on ARID1A analyzing if the frequency of truncating ein loss . Currentein loss ,39 assocein loss . Recentlein loss . ImportaA repair ,12, thisA repair . ImportaA repair ,39. In sion loss –37, we hion loss , might aARID1A and TP53 mutations are mutually exclusive and that ARID1A acts in concert with p53 to regulate target gene expression including CDKN1A in gynecological cancers [ARID1A and TP53/RB1 genetic alterations in bladder carcinoma samples using sequencing data of independent studies [ARID1A might not be sufficient to initiate tumor development, but that additional TP53 inactivation might be necessary to drive oncogenic transformation of urothelial cells. In concordance with this notion, ARID1A knockout alone did not result in tumor formation in the murine urinary bladder [Previous studies suggested a functional redundancy between SWI/SNF complexes and the RB1 pathway in cell cycle control ,50. In c cancers . Althoug studies ,24,26,27 studies . In cont studies . Even thARID1A truncating mutations did not sensitize urothelial cells to treatment with the small molecule EZH2-inhibitor GSK126, contrasting recent work using several tumor cell lines of different origin  was based on the absence of inactivating alterations in subunit genes of the SWI/SNF complex as well alterations in PRC2/PRC1 complex and Ras pathway genes [ARID1A-knockdown. Of note, even though further investigation is needed, we observed resistance of the normal urothelial model UROtsa but response of all bladder cancer cells analyzed towards GSK126 treatment (range: 6.5–13.4μM), potentially relevant for clinical translation. The focus of our study was to test the potential of ARID1A-deficiency to predict response to already available EZH2 small-molecule inhibitors such as GSK126 affecting its enzymatic activity [ARID1A knockdown or ARID1A wildtype expression. Importantly, EZH2 knockdown did neither inhibit cellular growth of J82 cells deficient for ARID1A nor J82 cells showing wildytpe ARID1A expression. In contrast to current data in bladder cancer [ARID1A-mutated and SWI/SNF wildtype bladder cancer cells. Taken together, we assume that ARID1A-deficiency (ARID1A truncating mutations and/or expression loss) is not a suitable predictive biomarker for EZH2 inhibitor treatment response in urothelial bladder cancer.Having demonstrated ARID1A protein loss in a significant portion (10%) of high-grade urothelial bladder carcinomas of different stages including CIS, we analyzed for the first time if a functionally antagonistic relationship between ARID1A and EZH2 exists in urothelial cells, and secondly if inhibition of EZH2 methyltransferase activity could be a potential therapeutic option for ARID1A-deficient bladder carcinomas. In contrast to the initial observation that SWI/SNF complex inactivation (by SMARCB1 depletion) results in EZH2 up-regulation and enhanced H3K27-trimethylation in murine embryonic fibroblasts and CD8+ T cells , we did ma cells ,22) but activity . Notablyr cancer ,53, we eARID1A alterations in urothelial bladder and a demonstrated loss of ARID1A protein expression, predominantly in a subset of high-grade bladder carcinomas including CIS, it seems worthwile to promote research on the identification of specific vulnerabilities conferred by ARID1A-deficiency. Considering molecular variability between different tumor entities and consequent difficulties in the identification of globally effective cancer treatment strategies, we suggest that bladder cancer-specific high-throughput drug discovery screens should be performed in near future to promote successful discovery of novel targeted drugs.Regarding the high frequency of genetic S1 Fig(PPTX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S3 Fig(DOCX)Click here for additional data file.S4 Fig(DOCX)Click here for additional data file.S5 Fig(DOCX)Click here for additional data file.S6 Fig(DOCX)Click here for additional data file.S7 Fig(DOCX)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.