Determining tumor necrosis factor-alpha inhibitors (anti-TNF-α) failure is still a challenge in the management of moderate-to-severe psoriasis. Thus, our comprehensive systematic literature review aimed to gather information on the criteria used to define anti-TNF-α failure. We also aimed to discover the main reasons for anti-TNF-α failure and define subsequently administered treatments.We conducted a systematic review following review and reporting guidelines (Cochrane and PRISMA). International (Medline/PubMed and Cochrane Library) and Spanish databases , and gray literature were consulted to identify publications issued until April 2021 in English or Spanish.Our search yielded 58 publications. Of these, 37 (63.8%) described the criteria used to define anti-TNF-α primary or secondary failure. Criteria varied across studies, although around 60% considered Psoriasis Area and Severity Index (PASI)-50 criteria. Nineteen (32.8%) reported the reasons for treatment failure, including the lack or loss of efficacy and safety-related problems, mainly infections. Finally, 29 (50%) publications outlined the treatments administered after anti-TNF-α: 62.5% reported a switch to another anti-TNF-α and 37.5% to interleukin (IL)-inhibitors.In the treatment of psoriasis, the primary and secondary anti-TNF-α failure criteria differ widely in the scientific literature.The strictest efficacy criteria for defining anti-TNF-α failure, or those recommended by guidelines such as PASI75, were underused both in clinical trials and observational studies.Most studies failed to consider patient-reported outcomes in assessing psoriasis treatment efficacy, which contrasts with recent recommendations on the inclusion of patient-reported HRQoL as a supporting criterion when considering clinical outcomes.Our findings suggest a need to standardize the management of anti-TNF-α failure and reflect the incorporation of new targets, such as IL-inhibitors, in the treatment sequence. Therapeutic options for moderate-to-severe psoriasis have dramatically increased in recent years due to the incorporation of new biologic agents. These include tumor necrosis factor-alpha inhibitors (anti-TNF-α)  and the interleukins (IL)-inhibitors  . ChoosinNotwithstanding, observations show that around 20% of patients discontinue the first course of anti-TNF-α and treatment discontinuation rates increase with time. The main reason for patients’ discontinuation seems to be a failure to respond or a loss of response to anti-TNF-α therapy . PredictTherefore, there is a clear need to standardize the management of anti-TNF-α failure in patients with moderate-to-severe psoriasis. With this overarching objective in mind, we conducted the present systematic literature review, primarily to gather information on the criteria used to define treatment failure. Additionally, we investigated the main reasons for anti-TNF-α treatment discontinuation, the therapeutic options chosen after anti-TNF-α failure, the current recommendations on treatment choice after anti-TNF-α failure, and the main factors to consider when choosing a new therapy after anti-TNF-α failure in moderate-to-severe psoriasis patients. Taken together this information might provide us useful insights into current practices with a view to establishing the foundations for future standardization.Supplementary Tables 1 and 2 for completed PRISMA checklists). This systematic approach minimized the risks of publication bias, ensuring the robustness of our results. To this end, we consulted international (Medline/PubMed and Cochrane Library) and Spanish national databases . In addition, we undertook a broad search in the gray literature. The search targeted a wide range of publications, including original articles, systematic and narrative reviews, or clinical practice guidelines, published up until April 2021 in English or Spanish, focusing mainly on the criteria used to define anti-TNF-α failure in moderate-to-severe psoriasis patients. We also searched for the reasons why anti-TNF-α was discontinued in addition to the therapeutic options used after anti-TNF-α failure and the treatment recommendations for patients failing to respond to anti-TNF-α. We conducted a systematic literature review following the Cochrane methodology  and repoSupplementary Tables 3 and 4 for further details). Furthermore, we manually searched the reference lists of relevant articles obtained. Besides the database search, a broad gray literature search was also performed. Supplementary Table 5 describes the sources and search strategies applied for the gray literature.To consult international and national databases, we used either free-text or Medical Subject Heading (MeSH) terms related to psoriasis, anti-TNF-α treatment, and treatment failure combined with Boolean operators (AND/OR) , publication screening was conducted in two phases. In the first phase, articles were screened by title and abstract according to the inclusion and exclusion criteria. In the second phase, articles selected for full-text reading in the previous phase were revised, considering the objectives of the review.Two researchers independently assessed the compliance of the original studies included to the reporting guidelines and the level of evidence of the publications. Any discrepancies were resolved by consensus and, if necessary, by consulting a third reviewer. To evaluate the compliance with the reporting guidelines, we applied the 30-point Consolidated Standards of Reporting Trials (CONSORT) statement for randomized clinical trials  and the Supplementary Table 6 for further details).The variables recorded for the included articles comprised: the first author; the year of publication; the period of the study; the type of publication; the characteristics of the treatment and population; the outcomes evaluated; and their compliance with the reporting guidelines and level of evidence  and Spanish databases (IBECS [n = 326] and MEDES [n = 157]) in addition to six publications from gray literature sources. After the removal of duplicates, a total of 1153 remained for title and abstract screening. Of these, 1037 were excluded in the first phase as they did not meet the eligibility criteria. We finally assessed 116 full-text articles, considering the review objectives, and identified 54 publications. Additionally, four further publications were recovered through the manual search of the reference lists of relevant articles (n = 16), we reviewed the CONSORT criteria in eight reports (50%) that had a randomized controlled design, of which six (75.0%) were compliant with the CONSORT reported items. Among the observational studies (n = 38), 23 (60.5%) had a retrospective design, whereas the rest  followed a prospective design. We applied the STROBE criteria in 37 publications based on cohort observational designs and excluded a case-control study. Among the observational studies analyzed, 16 (43.2%) had good compliance with STROBE guidelines. Supplementary Table 7 provides a summary of all the studies included.We retrieved 1507 publications in international  publications—twenty-two observational studies (59.5%), 14 clinical trials (37.8%), and one clinical practice guideline (2.7%)—dealing with the current criteria used to define anti-TNF-α failure. Of these, 35 (94.6%) covered criteria for primary failure or lack of response, whereas 18 (48.6%) discussed criteria for secondary failure.n = 33; 94.3%) defined it using clinical criteria such as Psoriasis Area and Severity Index (PASI) or Physician’s Global Assessment (PGA). Nonetheless, the standards of these criteria varied from study to study, reporting most of them  a less stringent PASI, inability to achieve a 50% reduction in PASI or PASI50. Supplementary Table 8 shows the details of the criteria reported in each study. Three of the studies (8.6%) included patient-reported health-related quality of life (HRQoL), measured by the Dermatology Life Quality Index (DLQI), in combination with clinical criteria to assess primary therapeutic failure. One of them defined treatment failure as not improving DLQI by 50% or below the value of 10 and/or failure to achieve PASI50 [Regarding primary failure, most studies  criteria also varied from study to study. Loss of efficacy after reaching PASI50 was the most frequent criteria used . Additionally, two publications (11.1%) did not specify the criteria used [The standards of secondary failure  publications, specifically 17 (89.5%) observational studies and two (10.5%) clinical trials, which dealt with the main reasons for anti-TNF-α treatment discontinuation. Of these, sixteen also provided us detailed information to estimate the distribution of patients among the different causes for discontinuation. Accordingly, most anti-TNF-α discontinuations were due to efficacy-related reasons, mainly a lack or loss of efficacy or due to disease remission. The following reasons for discontinuation were safety-related and, more specifically, adverse events occurrence, with infections cited as the most commonly reported adverse event causing treatment withdrawal. Finally, other reasons for discontinuation that were not related to the treatment itself were patients’ preferences , lack of adherence to treatment, comorbidities unrelated to psoriasis, or a lack of patients’ follow-up. n = 35; 62.5%), while the others involved changes to an IL inhibitor  mainly to an IL-12/IL-23 inhibitor , followed by an IL-17 inhibitor  or an IL-23 inhibitor . When we considered the three anti-TNF-α evaluated—etanercept, infliximab, and adalimumab—separately, more studies showed a change of adalimumab to other therapeutic targets rather than other agents: for adalimumab, 50% (n/N = 9/18) of changes were to an IL inhibitor, compared with 31.6% (n/N = 6/19) for etanercept and 37.5% (n/N = 6/16) for infliximab. See Supplementary Figure 1 reflects the treatment changes after anti-TNF-α failure reported over time, showing the other therapeutic targets to be adopted in the last ten years, such as IL-12/IL-23 inhibitors.Twenty-nine publications (50%), including 17 (58.6%) observational studies and 12 clinical trials (41.4%), met the last objective: namely, they described the therapeutic options used after anti-TNF-α failure in moderate-to-severe psoriasis patients. Among these, we identified a total of 56 treatment changes after anti-TNF-α failure: most of these consisted in a shift to another anti-TNF-α  were clinical practice guidelines from European settings ,21,22 anAnti-TNFs have become the predominant biological option to treat moderate-to-severe psoriasis patients due to the availability of biosimilars. However, determining anti-TNF-α failure to respond to these treatments and deciding the best alternative is one of the most challenging aspects of psoriasis management. Within this scenario, our systematic literature review aimed to cover key questions in the management of anti-TNF-α failure and, primarily, to know about the criteria used to define treatment failure and whether these criteria are uniform among studies.The primary and secondary failure criteria differed widely among the studies appraised; however, failure to achieve PASI50 was the most commonly cited criteria. The predominance of PASI50 in the studies implies that stricter criteria such as the PASI75, recommended by the European consensus in 2011  as the sAnother important question raised in our review relates to the most common reasons for anti-TNF-α discontinuation. As expected, efficacy-related reasons were the most frequent. The most-reported included inefficacy in terms of both lack and loss of efficacy. However, some studies include positive causes, such as remission of psoriasis symptoms, which accounted for up to 24% of the reasons for discontinuation . The secWith respect to the biologic agents chosen after anti-TNF-α failure, our findings reflect a change in treatment sequence in the last ten years, incorporating new targets, mainly IL-inhibitors. Among these, the IL-12/IL-23 inhibitor was predominantly chosen, followed by IL-17 inhibitors. These changes in the chosen treatment are not surprising as they reflect the chronological treatment approval sequence  and are We wish to describe certain limitations and strengths related to the design of the current review. With respect to the limitations, we acknowledge that we appraised a wide range of publications, including those with low reporting quality or a low level of evidence, such as narrative reviews. We recognize that the latter might introduce the author’s bias, which might likewise affect the results and conclusions of our study. Additionally, the inclusion criteria were limited to English and Spanish publications, possibly resulting in the omission of critical publications in other languages. Also, we are aware that treatment options for psoriasis continue to increase rapidly and, therefore, the validity of our results could be limited in time. For example, the IL-17 inhibitor, bimekizumab, was approved at the time of the review, so we could not include it in the search. On the other hand, the articles reviewed correspond to studies addressed to evaluate original molecules and no biosimilars were included. This is because for the approval of biosimilars, extrapolation of data is allowed, so that in some cases biosimilars are approved for the treatment of psoriasis without having been directly studied in comparative trials for this pathology but for others . In factOur findings suggest the need to standardize the current management of anti-TNF-α failure in psoriasis patients. The appraised evidence illustrates significant heterogeneity in the criteria used to define anti-TNF-α failure. Among the various criteria, PASI50 was chosen in a high proportion of studies and was applied indistinctly of the study period, even though the stricter PASI75 has only been recommended in recent years and new biologic treatments allow patients to achieve PASI75 and even PASI90 or PGA 0 or 1. Another finding is that most studies failed to consider patient-reported outcomes in assessing psoriasis treatment, which contrasts with recent recommendations on the inclusion of patient-reported HRQoL as a supporting criterion when considering clinical outcomes. The reasons for anti-TNF-α discontinuation also varied widely among studies, with efficacy or safety-related issues being the most common. However, our results also suggest that other factors, such as patients’ treatment preferences, should be considered. Finally, although the recommendations on which treatment to choose after anti-TNF-α failure are scarce, our findings reflect a pattern in the last ten years, with greater incorporation of new targets, mainly IL-inhibitors such as IL-12/IL-23, IL-17, and IL-23 inhibitors in the treatment sequence for moderate-to-severe psoriasis patients.Click here for additional data file.


Aberrant epigenetic remodeling events contribute to progression and metastasis of breast cancer (Bca). The specific mechanims that epigenetic factors rely on to mediate tumor aggressiveness remain unclear. We aimed to elucidate the roles of epigenetic protein PHF6 in breast tumorigenesis.Published datasets and tissue samples with PHF6 staining were used to investigate the clinical relevance of PHF6 in Bca. CCK-8, clony formation assays were used to assess cell growth capacity. Cell migration and invasion abilities were measured by Transwell assay. The gene mRNA and protein levels were measured by quantitative real-time PCR and western blot. Chromatin immunoprecipitation (ChIP)-qPCR assays were used to investigate transcriptional relationships among genes. The Co-immunoprecipitation (Co-IP) assay was used to validate interactions between proteins. The CRISPR/Cas9 editing technology was used to construct double HIF knockout (HIF-DKO) cells. The subcutaneous xenograft model and orthotopic implantation tumor model were used to asess in vivo tumor growth.In this study, we utilized MTT assay to screen that PHF6 is required for Bca growth. PHF6 promotes Bca proliferation and migration. By analyzing The Cancer Genome Atlas breast cancer (TCGA-Bca) cohort, we found that PHF6 was significantly higher in tumor versus normal tissues. Mechanistically, PHF6 physically interacts with HIF-1α and HIF-2α to potentiate HIF-driven transcriptional events to initiate breast tumorigenesis. HIF-DKO abolished PHF6-mediated breast tumor growth, and PHF6 deficiency in turn impaired HIF transcriptional effects. Besides, hypoxia could also rely on YAP activation, but not HIF, to sustain PHF6 expressions in Bca cells. In addition, PHF6 recuits BPTF to mediate epigenetic remodeling to augment HIF transcriptional activity. Targeting PHF6 or BPTF inhibitor (AU1) is effective in mice models. Lastly, PHF6 correlated with HIF target gene expression in human breast tumors, which is an independent prognostic regulator.Collectively, this study identified PHF6 as a prognostic epigenetic regulator for Bca, functioning as a HIF coactivator. The fundamental mechanisms underlying YAP/PHF6/HIF axis in breast tumors endowed novel epigenegtic targets for Bca treatment.The online version contains supplementary material available at 10.1186/s12967-023-04031-8. Currently, breast cancer (Bca) has become the second most common malignancy worldwide after lung cancer, leading to an estimated annual death of 41,760 cases in women . AlthougMost breast cancer cells express estrogen receptor (ERα), and drugs targeting the ERα pathway have become the mainstay of breast cancer treatment . Since t2-dependent HIFα isoform, the HIF-1β subunit, and other essential cofactors. HIF mainly binds to the hypoxia-responsive elements (HREs) in the promoters of downstream targets, thereby inducing transcription of VEGFA, LOX, ANGPTL4, and others [As is well documented, hypoxic environment in Bca is significantly related to the invasive phenotype, distant metastasis, and treatment resistance of cancer cells . Active d others . Under td others . Althougd others . Loss ofd others . AlthougIn this study, we identified that PHF6 is an oncogenic regulator in Bca that correlates with poor prognosis of patients. Mechanistically, we uncovered the potential associations between PHF6 and HIF activation. PHF6 protein could recruit BPTF to drive the expressions of HIF downstream targets. We further elucidated the upstream mechanisms of high PHF6 in Bca, highlighting epigenetic vulnerabilities in Bca treatment.2 at 37 °C. The lipofectamine 3000  was utilized to conduct the transfection under the manufacturer’s instructions. The specific siRNA targeting PHF family members and negative controls were purchased from GenePharma . ShRNA for stable knockdown of YAP or the negative control construct using a lentiviral system, and YAP or PHF6 overexpression plasmid was constructed and validated by GenePharma . The specific shRNAs sequences targeting YAP were as the following:shYAP-1:5ʹ-GACAUCUUCUGGUCAGAGATT-3ʹ;shYAP-2:5ʹ-GGUGAUACUAUCAACCAAATT-3ʹ. shBPTF-1:5ʹ-CGCCACTAACAGAGAAGGATT-3ʹ;shBPTF-2:5ʹ-GCGGCAGCTAATGAAGAAATT-3ʹ. shPHF6-1:5ʹ-GCACCATAAGTGCATGCTCTT-3ʹ;shPHF6-2: 5ʹ-CCATTATAAGTGCATGTTGTT-3ʹ.Human breast cancer cell lines , breast epithelial cell line MCF-10A, and human embryonic kidney 293 T cells were purchased from America Type Culture Collection . All cells were cultured with Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum  and 100 U/ml penicillin/streptomycin . Cells were maintained in a humidified incubator with 5% COThe 50 paired breast cancer specimens and adjacent normal control tissues were collected from the department of General Surgery, Zhongshan Hospital, Fudan University, Shanghai from 2008 to 2020. We finally selected 35 pairs with complete clinical information. All patients have signed the informed consent and the study was approved by the Ethics Review Committee of Ethics Committee of ZhongShan Hospital, Fudan University, Shanghai.In order to generate HIF-1α and HIF-2α knockouts, Bca cells  were transfected with CRISPR/Cas9 using PX459, a gift from Feng Zhang  . The sgRTotal RNA from tissue specimens or cultured cells was purified using TRIzol reagent  and RNA concentration was detected. An equal amount of RNA (2 μg) was reverse transcribed into complementary DNA (cDNA) using Superscript Reverse Transcriptase . Quantitative real-time PCR (qRT-PCR) was performed on the ABI 7500 real-time PCR system  using the PowerUP SYBR Green Master Mix .The cultured cells were lysed using 1 × Cell lysis buffer  containing protein inhibitor cocktail  and Protein concentration was measured using a BCA protein quantification kit . An equal amount of protein (20 μg) was separated by 10% SDS-PAGE and transferred onto a nitrocellulose membrane . The membranes were blocked with 5% non-fat milk at room temperature for one hour and then incubated with primary antibodies overnight at 4 °C. After washing with 1 × TBST, the membranes were further incubated with Horseradish Peroxidase (HRP)-conjugated secondary antibodies at room temperature for 1 h. GAPDH was used as an internal loading control. Primary antibodies used in the study were listed as the following: PHF6 ; HIF1α ; HIF2α ; BPTF ; SNF2L ; RBBP4 ; β-actin .The effect of PHF family members inhibition (siRNAs) on cell viability was measured by MTT assay (Sigma-Aldrich). Briefly, MCF-7 cells (2000 cells/well) were cultured in 96-well plates for 24 h or 48 h. After incubation with 20 μL of MTT solution (5 mg/ml in PBS) for an additional 4 h at 37 °C, the supernatant solution was removed, 150 μL of DMSO was added to dissolve the formazan crystal, and the absorbance of each well was measured with a microtiter reader  at a wavelength of 490 nm. The absorbance values for control cells were set as 1 for normalization. For the CCK-8 assays, Bca cells were seeded into a 96-well plate at 2000 cells/well with 100 µl of 10% FBS DMEM. According to the protocol of CCK-8 solution , 10 µl of CCK-8 solution diluted in 100 µl of complete culture medium replaced the original medium of each group at indicated points. After the cells were incubated in the dark at 37 °C for an additional 2 h, we detected viable cells by using absorbance at a 450-nm wavelength. For colony formation assays, we planted Bca cells into the six-well plate at a density of 1000 cells/well and then cultured them in complete culture medium at 37 °C for 14 d. After the cells were gently washed in PBS twice, they were fixed with 4% paraformaldehyde for 30 min. Then 0.1% (w/v) crystal violet was applied for staining the fixed cells for 30 min. ImageJ software  was used to count the numbers of colonies. Three independent assays were conducted to analyze cell proliferation abilities.5 cells per well were seeded in the upper chamber in 100 μL of serum-free medium, and 600 μL of complete medium was added to the lower chamber as a chemoattractant at the same time. After incubated for 24 h at 37 °C, the cells remaining at the upper surface of the membrane were removed with cotton swabs, and the cells on the lower surface of the membrane are the migrated cells. After fixed with 4% paraformaldehyde and stained with 0.1% crystal violet solution, the cells that passed through the filter were photographed by microscope. The transwell invasion assay was carried out as described above, except that 100 μL of 1:8 DMEM-diluted Matrigel  was added to each well at 37 °C for 6 h before the cells were seeded onto the membrane, followed by incubating for 48 h.Transwell plates ) were used for the transwell assay. The 1 × 10® 2000 transfection reagent  along with 200 ng of YAP/YAP-mutant construct and 10 ng of Renilla luciferase pRL-TK plasmid . After 48 h, the dual luciferase assay was performed using the Dual-Luciferase® Reporter Assay System , under the manufacturer’s protocol. Luciferase activity was detected as the ratio of firefly luciferase signal to Renilla luciferase signal. All measurements were normalized to the control group alone. Each experiment was performed in triplicate.A total of 100 ng of pGL3-Basic plasmid  with inserts of the PHF6 promoter sequence (TSS:− 2000 ~  + 50) were co-transfected into MDA-MB-231 and MCF7 cells using Lipofectamine2 for 24 h, crosslinked with 1% formaldehyde.for 10 min at room temperature, and quenched in 0.125 M glycine. Cells were lyzed in buffer , sonicated, and subjected to immunoprecipitation overnight in the presence of salmon sperm DNA/protein A beads with antibodies against PHF6, HIF-1, RNA BPTF, or IgG at 4 °C. Precipitated chromatin DNA was extensively washed, eluted with freshly prepared elution buffer , decrosslinked at 65 °C for 4 h followed by treatment with proteinase K at 45 °C for 45 min, purified with phenol/chloroform/isoamyl alcohol , and quantified by real-time qPCR assay.Cells were exposed to 20% or 1% OFor immunoprecipitation, protein was extracted by HEPES lysis buffer  with protease-inhibitor cocktail (Roche). Briefly, the lysates were incubated with 20 protein A/G-conjugated agarose beads (Roche) and 5 mg antibody or anti-flag beads (Sigma) at 4 ℃ overnight. After washed 3 times by PBST, beads were heated with SDS-loading and analyzed by western blotting.https://tcga-data.nci.nih.gov/tcga/). The expression levels of PHF6 and its correlation with the clinical characteristics of breast cancer patients were analyzed. The expression of PHF6 mRNAs was normalized to Log2 counts and then analyzed. Kaplan–Meier survival analysis and log-rank tests were performed using the OS and RFS data. The Meta-validation Bca cohort containing 2032 samples was derived from the Kaplan–Meier Plotter dataset .The TCGA-Bca cohort were obtained from The Cancer Genome Atlas  were injected into the second left mammary fat pad of female NSG or NOD/SCID mice (6–8 weeks old). Mice that were implanted with MCF-7 cells were administrated subcutaneously with a slow-release 17-estradiol pellet (0.72 mg/60-day release/pellet) one day before cell implantation. Tumor size was measured every week and tumor volume was calculated (length × width2 × 1/2). Primary tumors were harvested, photographed, and weighed. In vivo animal experiments were reviewed and approved by the Experimental Animal Ethics Committee of ZhongShan Hospital, Fudan University, Shanghai.Female BABL/c nude mice (6-week old) were obtained from the Shanghai SLAC Animal Center  and randomly assigned into indicated groups. 2 × 10P < 0.05 was considered to be statistically significant. All statistical analysis was conducted by GraphPad Prism .All experimental results were presented as Mean ± Standard deviation. The differences between groups were analyzed using Student’s t-test or one-way ANOVA. Kruskal–Wallis (K-W) test was used to assess associations between PHF6 and clinical features. The OS and RFS were analyzed using the Kaplan–Meier curves and compared by the log-rank test. The To elucidate the roles of PHF family proteins in human breast cancer progression, we conducted the MTT screen assays using siRNAs targeting specific members in MCF-7 cells. Relative to other proteins, PHF6 was the most potent hit Fig. : Fig S1A2 for 12 h, the whole cell lysates were subjected to the Co-Immunoprecipitation (Co-IP) assay with anti-PHF6 antibody or control antibody IgG. The Co-IP assay demonstrated the physical interactions between endogenous HIF1α/2α and PHF6 in hypoxic breast cancer cells  of HIF-1α, HIF-2α, or both via the CRISPR/Cas9 technology were stably generated and the western bot was used to detect HIF-1/2α proteins under the hypoxia condition  bioinformatic results indicated that hypoxia signaling was significantly enhanced in breast cancer patients with upregulated PHF6 expression Fig. A. Given 2 for 24 h. Apparently, PHF6 occupied the chromatin HRE regions of VEGFA, ANGPTL4, and LOX, relative to control IgG  could notably elevate PHF6 expressions . Accordingly, we conducted the immunohistochemistry (IHC) assay in the collected samples and confirmed the higher protein levels of PHF6 in tumor versus normal tissue sections  in Fig. P = 0.0043, Fig. http://kmplot.com/analysis/index.php?p=service&cancer=breast). We conducted Kaplan–Meier analysis in the Meta-validation cohort and observed that patients with high PHF6 had shorter relapse-free survival (RFS) time than those with low PHF6, in line with the notion that PHF6 correlates with tumor recurrence  is recently regarged as an essential cause that influences epigenetic reprogramming . The redgression , 31. Congression , 33. Forgression . Besidesgression . As regagression . Apart fgression . Haiquangression . BesidesHippo signaling pathway participates in multiple biological processes of tumors. When the Hippo pathway is activated, MST1/2 phosphorylates and activates LATS1/2, which in turn phosphorylates YAP/TAZ and inhibits the activity of YAP/TAZ . InhibitThe nucleosome remodeling factor complex (NURF) containing the SNF2L subunit catalyzes the sliding of ATP-dependent nucleosomes on the DNA template to construct the active regions of the promoter to activate target genes . PreviouWe also raised several prolems in the current study that need further considerations. First of all, owing to financial limits, we failed to conduct the Chromatin Immunoprecipitation Sequencing (ChIP-Seq) and Assay for Targeting Accessible-Chromatin with high-throughout Sequencing (ATAC-Seq) to confirm the regulations between PHF6/BPTF regulators and HIF transcriptions based on the genome scale. Besides, more pre-clinical models were warranted to confirm the inhibitory efficacy of AU1 on Bca cells. Last of all, we intended to collect more samples in our hospital to validate the prognostic significance of YAP/PHF6/BPTF axis, thereby constructing HIF-related signature models for prediction.YAP activation promotes elevated PHF6 in Bca tissues, predicting poor prognosis of patients. PHF6 recruits chromatin remodeling regulator BPTF to maintain HIF transcriptional activity. Under hypoxia, PHF6 interacts with HIF1α/HIF-2α to promote their recruitment to the HRE, thereby driving breast cancer progression. Targeting PHF6/BPTF is effective in breast cancer mouse models. As a result, the YAP/PHF6/HIF axis has clinical significance and targeting this crosstalk highlights an essential strategy for Bca treatment.Additional file 1: Figure S1. Quantification of expressions of each PHF family member in control or siRNA group.Additional file 2: Table S1. Summary of siRNA sequences that target each PHF family member.


Heart failure with preserved ejection fraction (HFpEF) is characterized by a complex, heterogeneous spectrum of pathologic features combined with average left ventricular volume and diastolic dysfunction. HFpEF is a significant public health problem associated with high morbidity and mortality rates. Currently, effective treatments for HFpEF represent the greatest unmet need in cardiovascular medicine. A lack of an efficient preclinical model has hampered the development of new devices and medications for HFpEF. Because large animal models have similar physiologic traits as humans and appropriate organ sizes, they are the best option for limiting practical constraints. HFpEF is a highly integrated, multiorgan, systemic disorder requiring a multipronged investigative approach. Here, we review the large animal models of HFpEF reported to date and describe the methods that have been used to create HFpEF, including surgery-induced pressure overloading, medicine-induced pressure overloading, and diet-induced metabolic syndrome. In addition, for the first time to our knowledge, we use two established clinical HFpEF algorithms (HFA-PEFF and H2FPEF scores) to evaluate the currently available large animal models. We also discuss new technologies, such as continuous remote pressure monitors and inflatable aortic cuffs, as well as how the models could be improved. Based on current progress and our own experience, we believe an efficient large animal model of HFpEF should simultaneously encompass multiple pathophysiologic factors, along with multiorgan dysfunction. This could be fully evaluated through available methods . Although many models have been studied, only a few studies completely meet clinical score standards. Therefore, it is critical to address the deficiencies of each model and incorporate novel techniques to establish a more reliable model, which will help facilitate the understanding of HFpEF mechanisms and the development of a treatment. Heart f2.Accurately understanding the pathophysiology of a disease is essential for developing an animal model. To date, the precise mechanism for the pathophysiology of HFpEF is incompletely understood. Unlike HFrEF, which largely results from ischemic heart disease or structural heart disease and is therefore easily translated into an animal model, HFpEF is a systemic condition with both cardiac and extra-cardiac features . ClinicaIn addition, recent data have shown an important role for the peripheral vascular system in HFpEF. Patients with HFpEF have abnormalities in the systemic vasculature, endothelium, adipocytes, and skeletal muscle , 17. Ano3.in vivo testing and are considered the last preclinical step before testing in humans. Therefore, large animal models of HFpEF are needed that can simulate HFpEF in humans. Currently, several types of large animal models of HFpEF are available that we will discuss here. To apply objective standards for evaluating the modeling of this complex clinical syndrome in large animals, we used clinical scores, as previously done in mice  one 4.2.LVEF should be maintained above 50%, which can be measured using transthoracic echocardiography, along with LV dimension and volume. However, because animals cannot verbalize, detecting symptoms in animals is not straightforward. As an alternative, we can use surrogate measurements that provide evidence of HF, such as blood natriuretic peptide level  and results of a stress or exercise test. Reduced exercise reserve caused by weakness or fatigue is a typical feature of HFpEF. If a model does not include any of these, it should not be regarded as a complete preclinical model.4.3.The HFA-PEFF score includes the following echocardiography measurements for LV function and morphology: LV wall thickness, LV end diastolic diameters, left atrial volume index, LV mass index, mitral inflow (E wave), tissue Doppler septal and lateral wall mitral velocity (e'), peak tricuspid regurgitation velocity (if applicable), and global longitudinal strain. In large animal studies, these parameters can be recorded by using transthoracic echocardiography and should be included for each animal. Therefore, an experienced echocardiographer is required.4.4.Similar to the HFA-PEFF score, the H2FPEF score includes LV diastolic function analysis, as well as the following extracardiac factors.Obesity. Body weight can be easily measured for a large animal every time the animal is sedated for an echocardiography procedure.Hypertension. In large animals, monitoring blood pressure without invasive procedures can be difficult. Recently, with the development of telemetry technology, some implantable devices have become available for use in large animals. For example, the implantable easyTel +  device  device  can be uAge. Most large animals are enrolled in studies at a young age. For example, a pig will be considered “aging” when it reaches 13 to 15 years , and tissue Doppler mitral velocity (e'), which should be acquired by using transthoracic echocardiography. For recording atrial fibrillation, a telemetry device for pigs is recommended that can be implanted into the pig's body and provide continuous electrocardiogram (EKG) monitoring  . For cow4.5.A major cardiac pathophysiologic change in patients with HFpEF is diastolic dysfunction. Therefore, hemodynamic assessment is the gold standard and may be an advantage for the verification of a model if included in a study. In large animals, hemodynamic measurements, including LV PV-loop, dp/dt, or tau index , can be 5.5.1.Aortic banding mimics high blood pressure by constricting the aorta, which in turn induces hypertension and further causes chronic LV pressure overload and hypertrophy. This model is primarily used in pigs. Constrictors can be placed in the aortic root, ascending aorta, or descending aorta. In these studies, LV fibrosis is consistently documented as the major pathologic change. The two types of constrictors are the fixed-size or pressure constrictor –49, or tCurrently, aortic banding is the most widespread, well-known method for inducing HFpEF. However, in the 12 studies we reviewed, only 4 studies –46, 50 w5.2.An aortic stent has effects similar to aortic banding but is less invasive because it requires a percutaneous procedure instead of open-chest surgery. The stent is ingrown into the aortic wall and does not allow normal aortic growth due to the stent's constant size, which causes partial antegrade obstruction and stenosis in the aorta . To date5.3.Renal wrap has been used primarily in dogs. Briefly, the procedure includes performing a midline abdominal incision, followed by wrapping a single kidney or both kidneys through silk or sterilized cellophane without constricting the renal vessels. This method produces persistent renal hypertension due to perinephritis. The hypertension causes LV pressure overload and induces LV hypertrophy. Because dogs can reach “old” age more easily than other animal models, these studies may be useful for examining age as a factor. For example, Munagla et al.  wrapped 5.4.For the renal clamping procedure, an adjustable clamp made of pure silver or stainless steel is used, and each clamp is attached to the renal artery at around 1 cm distal from its origin to apply an arbitrary extent of ligation. Many studies have described the use of clamps to induce hypertension in dogs, but none focused on HFpEF. Recently, renal embolization has been used as a new technique to induce hypertension by injecting microspheres through the renal artery to embolize the whole kidney. To date, one study has described renal embolization as one of multiple ways to induce HFpEF. Therefore, this study is discussed in the section “combination models.”5.5.Deoxycorticosterone acetate (DOCA) is a mineralocorticoid receptor agonist that acts as a precursor to aldosterone. The medication is administered subcutaneously (100 mg/kg) in pellets and is released over 90 days, with the aim of promoting sodium and water retention. One study  demonstrIn most cases, DOCA was combined with other methods to introduce HFpEF, which is discussed in the section “combination models.”5.6.Diet can be used as a complementary method for inducing metabolic syndrome in heart failure models. Diets can vary according to the aim of the research, but the most common diet is the Western diet , 57, whi5.7.Because HFpEF in humans is a highly heterogeneous syndrome, more researchers are looking to develop models that introduce multiple risk factors, such as a high-fat diet, hypertension, and diabetes. These models may accurately mimic multi-system dysfunction that closely represents human pathophysiologic change .Sorop et al.  introducIn another combination model in which multiple methods were used to induce risk factors, renal wrap and DOCA were performed in old dogs . At the The combination of diet and hypertension—the most common multiple comorbidities of HFpEF—has also been applied as a model. In a study of mini-pigs fed a diet high in fat, fructose, and salt combined with DOCA, the pigs developed obesity, hypertension, and diastolic dysfunction with pulmonary hypertension . This moIn another study, Schwarzl et al.  induced 6.Using the two most common clinical score systems, we evaluated large animal models of HFpEF that are currently used in the research field. When we combined all of the single-factor studies or used the most popular single-factor study of aortic banding to compare with the combination models, we found among the large animal studies that HFA-PEFF criteria were easier to meet than those of the H2FPEF score, whether the study was a single-factor or multiple-factor design . For theFor the models that received a lower score under the H2FPEF, we believe there are several reasons. First, most models failed to produce obesity, even with a high-fat diet, which is an interesting topic. In our own experience, we realize not every animal has the same appetite for a high-fat diet. Animals may lose weight if given a food that they do not want to eat. Also, some other factors introduced into the model, such as renal dysfunction, may further affect the animals' appetite. Second, many models lacked methods for monitoring. For example, atrial fibrillation is an important factor to consider, and most studies made no mention of EKG. Currently, as telemetry becomes more and more popular, we believe the tool can be used to monitor EKG for a long time in animals. For example, in our own studies, we implant an EKG recorder into the pig's body to provide a continuous EKG monitor. We hope that this allows us to capture the EKG changes during the whole period and make the model more reliable.Echocardiography plays an important role in both of the score systems studied. Therefore, an experienced echocardiographer is essential for the whole study. Unfortunately, many studies did not provide good echocardiographic evaluations, especially for diastolic function. Although hemodynamic measurement is the gold standard for evaluating diastolic function, it is invasive, complicated, expensive, and lacks repeatability and is thus rarely used in clinical evaluations. Therefore, a method that is consistent with clinical application should be applied to validate the model. For example, echocardiography can be used to monitor the animal weekly by noninvasive means and show a clear trend of diastolic function change. In contrast, hemodynamic measurement can be scheduled only 2 or 3 times during the study period because of its invasiveness. Some authors reported that echocardiography was not good because of the animals’ anatomical variation. In our own experience, at least with pigs, good images can be easily obtained through the subcostal view, with clear mitral valve flow and tissue Doppler signals. Therefore, we strongly suggest that diastolic function data be evaluated by using echocardiography and that hemodynamic measurement be reserved for additional verification.It is important to note that the clinical scores evaluated in this study do not cover all facets of HFpEF. For example, the scores do not account for pathology, change of peripheral vascular function, or stress/exercise tests, which are valuable for understanding the pathological changes in the disease. Just as in the combination model  discusse7.HFpEF is a complex disease with multiple contributing comorbidities that make it difficult to develop effective preclinical models. Progress in the field requires agreement on key features of animal models of HFpEF. Two new clinical scores have recently been developed to define these features. With this review, we are the first to our knowledge to propose using these scores to evaluate large animal models of HFpEF and create a checklist for optimizing translational research. Although most animal models do not meet all criteria, some multifactorial models resemble human HFpEF and may be the future of research. Our proposed approach aims to fill major gaps in HFpEF pathophysiology and facilitate the development of new therapeutics.


Canine lymphoma is a common cancer of one of the white blood cells that is commonly treated with chemotherapy. Many papers have been published about this disease and treatment, but few comprehensive reviews of the outcomes reported after chemotherapy, and the prognostic factors that influence the outcomes, have been published. Knowledge of the potential outcomes for this disease is important for veterinarians as they guide dog owners in the decision making about treatment options. This review evaluated the papers published on this topic and found that there is a wide variation in outcomes reported, as well as the factors that influence the outlook. Additionally, while individual papers have shown a much better survival, over the last 40 years, the overall improvement in outcome has been modest at best. The use of uniform reporting in the studies is needed, and novel approaches are likely needed to significantly improve the outlook for dogs with this disease.While canine lymphoma is a relatively common and important disease seen by veterinarians, there are limited comprehensive reviews of the literature regarding the remission and survival times following chemotherapy, and the associated prognostic factors. This comprehensive thematic review covers the available veterinary literature covering treatment outcomes and identified prognostic factors. A lack of standardised approaches to evaluate and report the outcomes was identified, including factors that would alter the duration of responses by weeks, or occasionally months. After publication of the suggested reporting criteria, this has improved but is still not uniformly applied. The prognostic factors included for evaluation varied from as few as three to seventeen, with over 50 studies using only univariate analysis. Individual papers reported much longer outcomes than others, but assessing the outcomes overall, there has been minimal change over the last 40 years. This supports the belief that novel approaches for lymphoma therapy will be required to substantively improve outcomes.  Canine lymphoma is common in clinical practice with an estimated frequency of about 20/100,000 dogs ,4. The mWhile over 1000 papers have been published on canine lymphoma, there is variation, however, in the patient and lymphoma details provided, as well as the inclusion/exclusion criteria used, and inconsistency in the inclusion of potential prognostic factors. Many summaries have been published in textbooks and review articles, but few provide a critical review of the studies , insteadFull classification of the lymphoma types in veterinary studies is often lacking, including in many recent studies that do not classify the disease beyond immunophenotype. This contrasts with studies of lymphoma in people where histological and genomic classifications are widely used as inclusion criteria in treatment studies . People The authors’ aim in this review was to evaluate the veterinary literature and report on the variability in the current information that is available, to identify the gaps that are revealed, and to highlight the need for best practice in the reporting of lymphoma studies in the veterinary literature. This information will highlight the deficiencies in how canine lymphoma treatment studies have been reported.A search was performed in the Web of Sciences  using the terms (dog or canine) and (lymphoma or lymphosarcoma) for all years until the end of 2021. All papers were accessed where available either electronically or hardcopy. Most papers in languages other than English were not available for review. The papers were reviewed for outcomes after the use of chemotherapy, if additional therapies were used, the inclusion/exclusion criteria for the study, and the details of the statistical analysis. The data extracted included if the case recruitment was prospective or retrospective; if cases were naïve (no prior chemotherapy), relapsed, a mixture, or not specifically stated; if the immunophenotype of the cases was known; if histological classification was included; if specific anatomic locations were included/excluded; the chemotherapy protocol(s) used; and if cases with early relapse/death were excluded. When the preceding information was not available, this was recorded. Chemotherapy protocols were divided into CHOP-like, COP-like , MOPP-like , other combinations, and single agents. The CHOP-like protocol included cyclophosphamide or chlorambucil, a vinca alkaloid, an antitumour antibiotic , and usually prednisone/prednisolone, with or without L-asparaginase. COP-like protocols included cyclophosphamide or chlorambucil, a vinca alkaloid, and prednisone/prednisolone with or without L-asparaginase. MOPP-like protocols included mustargens/CCNU/bleomycin, vincristine/vinblastine, procarbazine, and usually prednisone/prednisolone. Other combinations were combinations of two or more drugs that did not fit the above criteria. There were many single agents used, most often doxorubicin. The use of prednisolone/prednisone was variable across these protocols and was not always clearly indicated if used as a standard dose or duration, leading to its use not altering the group allocation. Some studies included a variety of treatments and reported only the overall outcome, which were included in a treatment group named as mixed. Response to treatment definitions were extracted when defined in the studies. The rates of complete remission (CR), partial remission (PR), progression free or remission times, survival times, and one-, two-, and three-year survival percentages were recorded where available. When both a CR and PR rate, or the CR rate was 100%, an overall response rate (ORR) was calculated if not reported. When outcomes for dogs achieving a CR or PR were reported separately, this was documented. In some papers that compared different protocols, the outcome information was identified for each protocol, in addition to the overall. When the protocols were similar, varying only in duration, or had drugs substitutions but remained within the definitions previously given, they were included as a single protocol if there was no significant difference in outcome. When there was a significant outcome difference between the treatment protocols, they were individually included. Specific anatomical localisations of lymphoma were separated from multicentric or nodal lymphoma. In studies without designation of anatomical location, the cases were designated as mixed, with the majority likely to be multicentric or nodal. Studies reporting relapsed or resistant lymphoma were considered separately to naïve cases. Prognostic factors included in the study were evaluated. Each paper was reviewed to identify which factors were included in the analysis, and the details recorded included if univariate or multivariate analysis was performed. Substage was recorded as indicated by the attending veterinarian when included in the record or based on the history and examination findings. Some variables reported in this review were combined. Prednisolone and prednisone were considered equivalent if prior use or inclusion in the protocol was evaluated in the study. Treatment as a prognostic variable referred to differing protocols, either drugs used or duration/dosage of the drugs. Coagulation included clotting parameters or thromboelastography changes. For neutrophils and monocytes, both cytophilia and ratios were combined. The genetic/molecular markers group includes genotype analysis, specific molecular markers, and karyotype evaluation. If the The statistical methods used in the papers varied. When assessing prognostic factors, only studies assessed with multivariant analysis were included in this paper. The criteria for a factor to be included in the multivariant analysis was recorded. When only univariate analysis was included in the paper, the information was tabulated in the All results were tabulated in Excel spreadsheets and statistical analysis was limited to descriptive statistics . To simplify the discussion in this paper, remission was used to designate all variations of remission and progression free survival, while survival was used to designate the various definitions of overall survival including lymphoma specific survival. Correlation evaluations were performed in IBM SPSS version 26 using the Pearson’s coefficient. Each journal article was designated as a paper; within each paper, each individually assessable population of cases was designated as a group. The dogs included in each group were designated as cases.The initial database search found 5882 papers including duplicates. These were reviewed for those describing clinical disease in dogs, refining this to 1179 individual papers. Evaluation of these papers identified 156 from which treated lymphoma outcome information was available, involving 211 groups and a total of 10,567 cases. There were 112 papers identified in which prognostic variables for outcome were reported. Treatment with vaccine or other immunotherapy was reported in seven papers with outcomes for a control group treated only with chemotherapy, with or without a placebo, reported with the non-immunotherapy groups included ,13,14,15Treatment response was defined in the The timing of the assessment of the dogs and the minimum duration of response required to be classified as such varied between papers. For many papers, neither the timing nor the duration of response were defined, and the strongest response was recorded and used, or the response at a specific day in the protocol was used (day 22) , after oThere was inconsistency in the literature for the starting point of remission duration. This varied from the date of diagnosis, the date treatment commenced, the date when the greatest response was achieved, the end of induction, the end of the first cycle, the end of the chemotherapy protocol, or it was not clearly defined. In addition, standardised terminology was not used to describe the duration of the first remission with multiple terms used for similar measures. A list of the terms and the number of studies utilising them is in Relapse and progression were not defined consistently. Many papers did not offer a definition, others used the identification of progressive disease as meaning relapse. A small number of papers required a return to the nodal size at the time of diagnosis and/or new nodal enlargement for a patient to be considered to have relapsed.Survival time definitions were variable with many not specifying the start date. When defined, it varied between the date of diagnosis, the date of starting chemotherapy, and the date of strongest remission. The majority included all causes of death, while lymphoma-specific survival was used in a smaller number. Most papers reported both a remission and survival time (88 papers); 43 reported only a remission time and 25 only survival times.The outcome for chemotherapy treatment was reported in 173 papers including 197 groups and 10,301 cases. Of these, 136 papers with 151 groups and 9191 cases reported a combination protocol with the remainder having a single agent protocol. There were two groups from two papers treated only with prednisone or prednisolone ,59. Of tThere was variability in the exclusion criteria regarding treatment prior to the use of chemotherapy. Only patients that had not received prior chemotherapy were included in 85 papers with 107 groups involving 5269 cases ,135,136;Specification of the anatomical location as an exclusion criterion varied across the literature. There were papers that specifically excluded cases with bone marrow ,102, cutThe outcome for multicentric or generalised nodal lymphoma treated with a CHOP-like protocol was reported in 43 papers with 54 groups and 4382 cases ,174,175.There were an additional 26 papers including 33 groups and 1606 cases ,161,171 There were 21 papers with 27 groups that included patients treated with a COP-like protocol ,165,167.Eleven studies included 14 groups and 481 cases with multicentric or nodal B-cell lymphoma treated with a CHOP-like protocol ,124,161.T-cell multicentric or nodal lymphoma treated with chemotherapy was reported in 8 papers with 9 groups and 476 cases. Of these, 3 papers with 3 groups included 107 cases treated with a CHOP-like protocol ,123,126.Single agent chemotherapy was reported in 35 papers with 39 groups and 1230 cases of dogs with generalised lymphoma. Of these, 13 papers with 16 groups and 466 cases described the treatment of naïve lymphoma ,131,177,The complete remission rate for doxorubicin-treated dogs was reported for 14 groups and ranged from 22 to 82%, with a median of 65.5% and mean of 61%. Partial remission rates were reported in eight groups and ranged from 0 to 56%, with a median of 12.5% and mean of 17%. The overall response rate ranged from 33 to 94% with a median of 79.5% and mean of 73%. The median remission time was reported for 15 groups, ranging from 14 to 246 days, with a median of 147 and mean of 140 days. The reported mean remission time in four groups ranged from 156 to 203 days, with a median of 180 and mean of 180 days. Median survival times were reported for 13 groups, ranging from 100 to 423 days, with a median of 230 and mean of 237 days. Mean survival times were reported for five groups and ranged from 198 to 351 days. One group from one paper had relapsed lymphoma cases ; the remThere were 25 papers with 27 groups and 1116 cases of relapsed generalised lymphoma where outcomes for chemotherapy treatment were reported ,149,150.Eleven papers reported on lymphoma that was described as neither multicentric nor nodal and treated with chemotherapy, including six reporting outcomes for cutaneous lymphoma ,68,179, In the six papers reporting on cutaneous lymphoma, two used CCNU, one masitinib, one an experimental drug VDC-1101, one the combination VELCAP-EL based on a CHOP-like protocol with additional alkylating agents or CCNU, and one had a mix of treatment protocols. Remission rates were reported in all papers. The CR rate ranged from 0 to 64% with a median of 17% and mean of 22%. The PR rate ranged from 27 to 100% with a median of 50% and mean of 51%. The ORR ranged from 44 to 100% with a median of 78% and mean of 73%. The MRTs reported in five papers ranged from 38 to 106 days, with a median of 85 and mean of 72 days. Survival times ranged from 130 to 281 in the two papers where this was reported, with a median of 207 and mean of 205 days.In the four studies reporting on gastrointestinal lymphoma, one was limited to colorectal disease, one was intestinal and the final two had predominately gastrointestinal involvement. In one the treatment was VELCAP-SC, one used single agent L-asparaginase, and the other two involved several different protocols. Remission rates were reported in two studies with one study having a CR rate of 50% and a PR rate of 6% and the other a CR rate of 47% and a PR rate of 9%. The MRT ranged from 50 to 1318 days, and the MST ranged from 77 to 1845 days. The studies with colorectal lymphoma had the longest remission and survival outcomes ; removing this paper, the ranges were 50 to 86 days and 77 to 147 days for remission and survival, respectively. As the papers available for review covered nearly five decades, from 1973 to 2021, the authors looked at the change over time in reported remission and survival durations. Generalised lymphoma was selected as this represents the most frequent presentation of lymphoma. A similar evaluation for B-cell lymphoma was undertaken as the number of groups of T-cell lymphoma were small. The results are shown graphically in p = 0.070), and a steady median overall survival time  when all generalised lymphoma types are included. When the results are restricted to papers reporting B-cell lymphoma outcomes, both the median first remission duration  and median overall survival  showed a slight decreasing trend over time. For both data sets, there was significant correlation between remission and survival . A median first remission duration was available from 99 papers and a median overall survival from 109 papers for generalised lymphoma. When restricted to B-cell lymphoma, a median first remission duration was available for 19 papers and a median overall survival for 18 papers. The trend lines show a slight increase in the median first remission duration over time  compared to others, and some papers including selected stages. Evaluation was undertaken for both remission and survival in 34 papers, remission only in seven, and survival only in six. A significant influence on both outcomes was found in three papers, for remission in three papers, and survival in eight papers. Clinical stage was prognostic for remission in 14.6% of papers and for survival in 27.5% of papers where included. The anatomical location was evaluated as a prognostic variable in 13 papers involving 1139 cases. The most common anatomical locations where involvement was reported were the anterior mediastinum or bone marrow. Eight papers included both remission and survival, remission alone was evaluated in three, and survival in two. It was found to be prognostic for both outcomes in three papers and for survival in one. This represents 27.3% of papers when included for remission and 40% for survival. The prognostic effects of the immunophenotype of the lymphoma was included in 28 papers with 3926 cases. Both remission and survival outcomes were evaluated in 21 papers, remission alone in four, and survival in four. Immunophenotype was prognostic for both outcomes in seven papers, remission in six, and survival in three papers. It was prognostic for remission in 52% and survival in 41.7% of papers that included these outcomes.Anaemia, hypercalcaemia, and thrombocytopenia were the only parameters evaluated in more than 10 papers. The remain parameters are included in the Anaemia was included as a variable in 22 papers involving 1874 cases. Anaemia was most often reported as categorical data, present or not. It was evaluated as a prognostic variable for both remission and survival in 14 papers, for only remission in three, and survival in five. Anaemia was prognostic for remission in two papers and for survival in six, being 11.8% and 31.6%, respectively, of papers where evaluated. The presence of hypercalcaemia was evaluated in 28 papers with 2838 cases. The effects on both remission and survival were included in 22 papers, remission only in four, and survival in two. It was found to be significantly prognostic for both outcomes in three papers, for remission in one, and for survival in two papers. This equates to being prognostic for remission in 11.8% and survival in 31.6% of papers where included. Thrombocytopenia was included as a prognostic variable in 13 papers with 1323 cases. Both remission and survival were included in nine papers, remission alone in three, and survival in one. It was found to be prognostic for remission in two and survival in three papers. Thrombocytopenia was identified as a positive prognostic factor for survival in one paper . When inThe response to treatment, most often indicated as the patient achieving complete remission, was included in 10 papers involving 1211 cases. Both remission and survival were included in eight papers, remission alone in one, and survival alone in one paper. It was identified as prognostic for both outcomes in five papers and for survival in three. It was prognostic in 62.5% of papers for remission and 88.9% for survival when included. This review shows there is a large degree of variability in the literature for outcomes for lymphoma and prognostic variables. Variability in the results of the papers have arisen from the variable inclusion/exclusion criteria, the variety of definitions used, the analyses performed, and potentially the number of cases included. Despite there being many papers in the literature, it is difficult to acquire a clear view of the topic or evidence to inform best practice. A basic measure in dogs with lymphoma treated with chemotherapy is response. We described the literature in which the criteria used to assess this fundamental feature is inconsistent, including when it is assessed, and the duration required to be considered a response. Prior to 2010, a veterinary scheme for describing responses in nodal lymphoma was lacking, which was addressed in the VCOG review. However, since the VCOG paper  was publA key feature in the decision making in lymphoma is first remission duration rather than overall survival. Survival is affected by other factors such as the owners’ willingness to pursue repeated chemotherapy, for financial or logistical reasons, and the availability of euthanasia for veterinary patients as an option. There is varied terminology and methodology used to report remission duration. The initial date used ranged from the date of diagnosis through to completion of a fixed portion of a protocol. This could be a difference of 60 days when studies have a mean and median reported MRT of about 200 days. This would contribute to some of the variability in outcomes, but with a wide range of median remission times, from 73 to 622 days for nodal lymphoma treated with CHOP-like chemotherapy, there are likely other factors that contribute. The VCOG paper [The variability in the survival times reported is due to factors that approximate those mentioned regarding remission such as the definition of the duration’s starting point. While survival times were longer than remission times by approximately 50%, a different starting point could account for about 10% of the result. As mentioned above, survival was affected by the owner’s decisions on further treatment or euthanasia. When clearly defined specific subtypes or anatomical locations are considered, which would better match the information from people, the number of papers and included cases decreases. The most common form of lymphoma in dogs is nodal or multicentric DLBCL. There are only 11 papers that included groups confined to B-cell lymphoma, of which only five were restricted to DLBCL. As shown in The change in outcome over time as measured by either remission duration or survival has not changed in a clinically significant degree as indicated in There is a reasonable body of literature evaluating variables as prognostic indicators for outcome for lymphoma in dogs. However, we found variability in the study design and how the factors were evaluated. There were more than 45 factors that were included in at least one paper, as we grouped some related ones together, but the most evaluated in any single paper was 17, with some including as few as three. This is confounded by the differing criteria used as such for the inclusion of variables in the multivariate analysis. The limited inclusion of prognostic factors in many of the studies has the potential to bias the results of the analysis if a confounding variable is not included. We did not detail the results of univariate analysis in the manuscript as these did not remain significant when multivariate analysis was performed with a reasonable frequency, and lack of the latter makes interpretation difficult. What we found interesting was that the majority of the commonly cited prognostic factors were prognostic in less than 50% of the papers in which they were evaluated, many in less than 20%. If we exclude treatment type and molecular/genetic markers as diverse groups, and factors evaluated in fewer than 10 papers, only immunophenotype and response to treatment were prognostic in greater than 50% of the papers evaluated, and immunophenotype only for remission, and 41.7% for survival. Hypercalcaemia and substage are commonly cited as strong predictors of outcome for dogs with lymphoma, but these were identified as prognostic in 38.5% and 11.8% of papers, respectively, for remission and 35.1% and 31.6% of papers, respectively, for survival. We must question whether this has arisen from reliance on a small number of studies, or a weakness in the methods of the studies that did not show these to be prognostic indicators. There is variability in the assessment by veterinarians on substage , which cThe way prognostic indicators were evaluated was not consistent. The clinical stage of lymphoma was prognostic for remission in 14.6% of papers and for survival in 27.5% of papers. Some papers included stage I disease, and as would be expected, these dogs did better, so overall stage was reported as prognostic. Other excluded high-grade disease, stage V, and did not find stage to be prognostic, possibly missing the fact that stage V might have a worse outlook. There is no information on the influence of stage V lymphoma on outcomes when differences in how this was diagnosed are considered. Bone marrow involvement might differ from the involvement of a non-lymphoid organ, or the degree of bone marrow involvement might influence outcome.Disappointingly, there were potentially prognostic variables that were evaluated in few studies. Some, such as dogs experiencing toxicity or dose modification, are often cited as being prognostic, but these were included in only five and six papers, respectively. The histological classification was only evaluated in eight papers but was prognostic for survival in 42.9% of papers in which it was included. Histology is being performed less often with the common use of cytology, immunocytochemistry, and flow cytometry to diagnose and classify patients. The failure to include a reasonable range of factors when assessing prognosis might miss important information that could influence treatment choices and decisions when they are omitted, or potentially bias statistical analysis.The authors propose the use of the VCOG  recommenFurther work is required to develop a consensus on the prognostic indicators that should be included in lymphoma studies and if the current staging system needs refinement for use in outcome studies. Having a consensus on substage classification for dogs with lymphoma is required as misclassification included in the statistical analysis has an unknown effect. When evaluating prognostic factors, future studies should endeavour to include a reasonable number of factors in the analysis and not exclude potentially confounding factors.


We introduce the problem of variable-length (VL) source resolvability, in which a given target probability distribution is approximated by encoding a VL uniform random number, and the asymptotically minimum average length rate of the uniform random number, called the VL resolvability, is investigated. We first analyze the VL resolvability with the variational distance as an approximation measure. Next, we investigate the case under the divergence as an approximation measure. When the asymptotically exact approximation is required, it is shown that the resolvability under two kinds of approximation measures coincides. We then extend the analysis to the case of channel resolvability, where the target distribution is the output distribution via a general channel due to a fixed general source as an input. The obtained characterization of channel resolvability is fully general in the sense that, when the channel is just an identity mapping, it reduces to general formulas for source resolvability. We also analyze the second-order VL resolvability. Thus, wCorollary 3.For any finite alphabet target source Remark 8.As in the case of FL resolvability and FL source coding problems, e X, Han  has showand, hence, from Corollary 3, it is concluded thatIn addition, if a general sourcesatisfies the uniform integrability and the strong converse property (cf. Han ), then ewhere Remark 9.Han and Verdú  have disE[Ln] in  replacedCorollary 4.For any general target source So far, we have considered the problem of VL resolvability, in which the approximation level is measured by the variational distance between In this subsection, we address the following problem.Definition 4.A resolution rate where The infimum of all is called the VL-resolvability or simply the  See Here, we establish another main theorem, which characterizes Theorem 4.For any general target source Remark 11.It should be noticed that the approximation measure considered here is not the normalized divergencewhich has been used in the problem of FL δ-resolvability . The ach form as , if the As we have seen in Lemma 1, we generally have Corollary 5.For any general target source Corollary 5 indicates that the .h.s. of . By 88)vD(0)-resProof of Theorem 4.(1)Converse Part:R be Let tisfying  and yi yi107) y we used  and 92)(108)H[δ+(2)Direct Part:R is We modify the achievability scheme in the proof of the direct part of Theorem 3. Although the proof of this part is quite similar to that of the direct part of Theorem 3, we give here the full proof in order to avoid subtle possible confusions. We may assume that Let We also defineLetting, for We set (a)Construction of Mapping Index the elements in (cf. –63)). F). F63)).If(b)Evaluation of Average Length:The average length i(m) and . For xi∈ain from  and the ain from (132)PX˜It should be noticed that  also imp(m))and  into (13(m))and , we obtaThus, we obtain from 137)limows from . Since wlimows fr(c)Evaluation of Divergence:The divergence R is In view of , we obtaDTheorem 6.For any input process In particular,(with divergence)Remark 13.It can be easily verified that the variational distance satisfiesand, therefore, we have formulas  and (169for any given channel X˜n||Xn) , and forregardless of channel Remark 14.It is obvious that Theorems 5 and 6 reduce to Theorems 3 and 4, respectively, when the channel where where Proof of Theorems 5 and 6.(1)Converse Part:Because of the general relationship , to provR be mean Let tisfying  and(179ain from (181)H[δSince  implies .The converse part of Theorem 6 can be proven in an analogous way with due modifications.(2)Direct Part:Because of the general relationship , to provLet Then, by the same argument to derive  and (82)R is Thus, we obtainThe direct part of Theorem 6 can be proven in the same way as Theorem 4 with due modifications. Fixing n obtain . A key sThe first term on the r.h.s. can be bounded asnγas in , where tnγas in , the secave used . Here, D□So far, we have analyzed the first-order VL resolvabilities and established various first-order resolvability theorems. One of the next important steps is the second-order analysis, and so, in this section, we generalize VL resolvabilities in the second-order setting.second-order resolution rates [We now turn to considering the on rates ,27,29. FDefinition 9.A second-order resolution rate where When Theorem 8.For any input process In particular, in the case where (with divergence)Remark 16.As discussed in eplacing  and Converse Part:We will show the converse part of Theorem 7. The converse part of Theorem 8 can be proved in an analogous way.L be VL Let tisfying  and yi yi212) yave used  and (191(2)Direct Part:We will show the direct part (achievability) of Theorem 7 by modifying the argument of Theorems 3 and 5, whereas the direct part of Theorem 8 can be proved in a similar manner by modifying that of Theorem 4 instead of Theorem 3.L is VL LettingWe use the same achievability scheme as in the proof of Theorem 3 with slightly different parameter settings. For We here defineTherefore, we obtainL is VL Since □VL source/channel resolvability, in which a given target probability distribution is approximated by transforming VL uniform random numbers. We have investigated the problem of In this paper, we have first analyzed the fundamental limits on the VL source resolvability is equal to the minimum achievable rate of VL source codes with an error probability of less than or equal to FL source resolvability and the minimum achievable rate of FL source codes [channel resolvability is closely related.Then, we have addressed the problem of found in . Indeed,VL intrinsic randomness [normalized divergence as an approximation measure results in the same general formula as with the variational distance and the unnormalized divergence, which does not necessarily hold in the case of mean/VL resolvability (cf. Remark 11). It is also noteworthy that whereas only the case of When ndomness ,36, in wWhen As noted in Remark 10, the order of arguments in divergence iscussed . Investi


The increasing burden of heart failure with preserved ejection fraction (HFpEF) has become a global health problem. HFpEF is characterized by systematic inflammation, cardiac metabolic remodeling, and fibrosis. Eosinophils act as an essential but generally overlooked subgroup of white blood cells, which participate in cardiac fibrosis, as reported in several recent studies. Herein, we explored the role of eosinophils in a “two‐hit” preclinical HFpEF model. The peripheral eosinophil counts were comparable between the normal chow and HFpEF mice. Deficiency of eosinophils failed to alter the phenotype of HFpEF. Conclusively, the development of HFpEF is independent of eosinophils in terms of the functional, biochemical, and histological results. This is the first report to evaluate the role of eosinophils in heart failure with preserved ejection fraction (HFpEF), where a novel preclinical model of HFpEF and eosinophil‐deficient mice was utilized. Results indicated that the development of HFpEF is independent of eosinophils, providing new insights into the roles of eosinophils in cardiovascular diseases. Herein, a novel preclinical model of HFpEF and eosinophil‐deficient mice was utilized to examine the association of eosinophils with HFpEF. However, we found no elevation of peripheral blood eosinophils in HFpEF mice. Mice with eosinophil deficiency showed no alternation of cardiac function and histological performance.22.1All animal experiments complied with the ARRIVE guidelines and were carried out in accordance with the UK Animals (Scientific Procedures) Act, 1986, and associated guidelines. All animal studies were approved by the Animal Care and Use Committee of Fuwai Hospital, Peking Union Medical College of Medicine (No. FW‐2022‐0041). A total of 50 male wild type  and 50 ΔdblGATA1 knock‐out  C57BL/6N mice, which have complete ablation of the eosinophil lineage,N[w]‐nitro‐l‐arginine methyl ester  for 12 weeks, respectively for establishment of HFpEF, as reported previously.Mice were randomly assigned into two groups with cage as the experimental unit, which were exposed to normal diet (chow) or high‐fat diet  plus 2.2A total of 150 μL peripheral blood was obtained from the eye socket. The blood samples were diluted to three times  by adding normal saline, and then evaluated by blood cell counter 2120i (Siemens).2.3Both systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured noninvasively in conscious mice using the tail‐cuff instrument . Animals were placed in individual holders on a temperature‐controlled platform (37°C), and recordings were performed under steady‐state conditions after sufficient acclimatization. The averaged values of three measurements of each mouse were included in the analysis.2.4Echocardiography was undertaken by employing the Visual Sonics Vevo 2100 imaging system (FUJIFILM). The mice were anesthetized with 3% isoflurane and maintained with 1%–2% isoflurane in 95% oxygen. Anesthesia was adjusted to obtain a target heart rate of 470 ± 50 beats per minute (bpm). Left ventricular (LV) ejection fraction (EF) was measured from the short‐axis M‐mode at the mid‐ventricular level. Apical four‐chamber view was used to obtain tissue Doppler imaging (TDI) mode and Pulse‐wave Doppler (PWD) mode for analysis of myocardial velocity  and blood flow velocity , respectively. All parameters were measured three times, and means were used for analysis. At the end of the procedures, all mice recovered from anesthesia without difficulties.2.5Mice ran uphill (10°) on the FT‐200 treadmill (Techman) starting at a warm‐up speed of 4 m/min for 4 min after which speed was increased to 9 m/min for 2 min. The pace was then increased by 2 m/min per 2 min till mice were exhausted, which was defined as the inability of the mouse to return to running within 10 s of direct contact with an electric‐stimulus grid. Record running time and calculate running distance.2.6After anaesthetization, the hearts and bilateral lungs were harvested from the mice and weighed. The lungs were dried under 65°C for 3 days and weighed again. Length of tibias was measured by a vernier caliper. Then hearts were fixed in 4% PFA for 60 ± 6 h and imbedded in paraffin. Slides (5‐μm thick) were acquired, baked at 68°C for 45 min, and stained with hematoxylin and eosin (H&E) or Sirius Red solution by routine methods.Wheat germ agglutinin  staining was used to examine cross cross‐sectional area of cardiomyocytes, an indicator of cardiac hypertrophy. For immunofluorescence staining, the paraffin sections were incubated with the antibodies at 4°C overnight, followed by goat antimouse  or goat antirat  conjugated with Alex Fluro 488 or 594 secondary antibodies for 1 h at room temperature. After washing, the nuclei were stained with 4′,6‐diamidino‐2‐phenylindole. The sections were then observed under a laser scanning confocal microscope (Leica) at ×400 magnification and five randomly chosen high‐power fields (HPF) were measured per animal. Primary antibodies in the study included rat anti‐mouse Siglec‐F antibody  and mouse anti‐mouse cardiac Troponin T . For each mouse, five HPF images were obtained, and the averaged eosinophil count or cross‐sectional area was utilized for quantitative analysis.2.7−ΔΔCt methods. The levels of serum transforming growth factor β (TGFβ) was measure using mouse TGFβ ELISA kit  according to the manufacturer's instructions.Total RNA was extracted from heart tissue with Trizol reagent (Life Technologies) according to the manufacturer's instructions. Reverse transcription was performed using a Prime‐ScriptTMRT Reagent Kit with gDNA Eraser (Takara). The quantitative real‐time PCR process of Gapdh and Nppb (encoding B‐type natriuretic peptide [BNP]) was conducted with PowerUp™ SYBR™ Green Master Mix (Applied Biosystem) on a QuantStudio 3 Real‐Time PCR system (Applied Biosystem). The data were calculated via comparative 22.8N for each study were recorded in the figure legends. Statistical data are presented as the mean ±  standard deviation (SD) and were analyzed using GraphPad 8.0 (GraphPad Software). Outliers, which means higher than mean +2 SD or lower than mean −2 SD, would be excluded during analysis. Comparisons between two groups were analyzed by unpaired student t‐test. Comparisons among four groups were evaluated by two‐way analysis of variance. Statistical significance was set at p < .05 for all comparisons.Accurate 33.1l‐NAME recapitulated a bulk of clinical features of patients of HFpEF, including obesity , both of which were considered to be normal. Eosinophil‐deficient mice did not show significantly altered diastolic function, as demonstrated by the comparable ratios of E to e' in WT‐HFpEF and Gata‐HFpEF groups . This preclinical model is favored because it recapitulates the numerous systemic and cardiovascular features of HFpEF people, such as diastolic dysfunction, preserved LVEF, exercise intolerance, elevated LV filling pressures, microvascular dysfunction, modest cardiomyocyte hypertrophy, capillary rarefaction, predisposition to AF, and modest myocardial fibrosis.In this study, we assessed the effects of eosinophils in HFpEF. A novel, well‐recognized “two‐hit” model was applied, characterized by metabolic inflammation (induced by obesity) and hypertensive stress , an essential cytokine regulating the migration and infiltration of monocytes/macrophages, was significantly activated in response to pressure overload, and the inhibition of MCP‐1 alleviated diastolic dysfunction.Despite several advances recently, the role of eosinophils in myocardium remains to be elucidated due to inconsistent observations. For instance, higher eosinophil count was considered as a risk factor or predictor of several cardiovascular diseases. Cardiac syndrome X (CSX), characterized by exertional angina pectoris and normal coronary angiography results, may involve microvascular spasm and endothelial dysfunction. It's reported that patients with CSX had higher circulating eosinophil counts compared with control persons.There are some limitations to be noted. First, molecular mechanisms underlying the marginally insignificant upregulation of BNP in Gata1‐chow mice compared with WT‐chow mice were not explored. Additionally, we observed a trend of mild spontaneous cardiac hypertrophy and fibrosis in Gata1 KO mice in terms of E/e', HW/TL and Sirius Red staining. However, none of the indicators changed sharply, and most importantly, the running distance is comparable between the two groups, indicating the clinical significance of these changes is limited. Besides, it's infeasible to upregulate the eosinophils by IL‐5 to further verify the conclusion because administrating cytokines for a long time could be difficult due to high cost, and the adverse effects of IL‐5 such as pro‐anaphylaxis, body loss and global immune alternations result in failure of modeling and unreliable interpretation of results. Lastly, the roles of eosinophils in other HFpEF phenogroups (such as age‐related phenogroup and hypertension‐related phenogroup) were not discussed, although the “two‐hit” model is generally considered the best preclinical model of HFpEF to imitate clinical conditions of the majority of patients.Conclusively, the present study revealed that eosinophils did not drive the development of cardiometabolic HFpEF. However, more investigations are required to explore the mechanisms of mild spontaneous cardiac hypertrophy and fibrosis caused by eosinophil deficiency.Qi Pan: Conceptualization; formal analysis; investigation; methodology; software; validation; and writing—original draft. Cheng Chen: Conceptualization; formal analysis; methodology; software; and validation. Zhaoting Gong: Data curation and resources. Guihao Chen: Funding acquisition; project administration; supervision; visualization; and writing—review & editing. Yuejin Yang: Data curation; funding acquisition; project administration; resources; supervision; and writing—review & editing.The authors declare no conflict of interest.All animal studies were approved by the Animal Care and Use Committee of Fuwai Hospital, Peking Union Medical College of Medicine (No. FW‐2022‐0041).Supporting information.Click here for additional data file.