Our findings suggest that this model may be useful in predicting acquired resistance to treatments containing IMiDs and/or PIs.Identifying biomarkers associated with disease progression and drug resistance are important for personalized care. We investigated the expression of 121 curated genes, related to immunomodulatory drugs (IMiDs) and proteasome inhibitors (PIs) responsiveness. We analyzed 28 human multiple myeloma (MM) cell lines with known drug sensitivities and 130 primary MM patient samples collected at different disease stages, including newly diagnosed (ND), on therapy (OT), and relapsed and refractory  timepoints. Our findings led to the identification of a subset of genes linked to clinical drug resistance, poor survival, and disease progression following combination treatment containing IMIDs and/or PIs. Finally, we built a seven‐gene model (MM‐IMiD and PI sensitivity‐7 genes [IP‐7]) using digital gene expression profiling data that significantly separates ND patients from IMiD‐ and PI‐refractory RR patients. Using this model, we retrospectively analyzed RNA sequcencing (RNAseq) data from the Mulltiple Myeloma Research Foundation (MMRF) CoMMpass ( While the majority of patients receiving combination chemotherapy including IMiD and/or PI initially respond, most of them eventually develop resistance. Understanding the underlying mechanisms of nonresponsiveness and identifying biomarkers associated with drug resistance and disease progression have become critical for personalized medicine and development of novel therapeutic strategies.IRF4 and MYC expression leading to inhibition of MM cell growth [IMiDs mediate anti‐MM effects by binding to the E3 ubiquitin ligase cereblon (CRBN) , 2, 3, wl growth . IMiD rel growth , 13, 14.l growth , 20, 21 l growth  .In clinical practice, IMiDs and PIs are usually given in combination with each other as well other classes of drugs (such as dexamethasone) as standard of care. Developing a method to monitor both IMiD and PI drug sensitivity and detect disease progression during treatment is important for precision medicine. In the present study, we sought to identify transcriptional changes associated with treatment nonresponse by studying the candidate genes previously associated with IMID and/or PI sensitivity to determine whether measuring expression levels of such genes could serve as a biomarker for disease progression.NanoString nCounter technology, a direct multiplexed measurement of gene expression based on digital color‐barcoding technology , is a fl22.1We investigated the transcriptional expression of 121 genes selected from published literature , 26–33 a2.2All HMCLs used in this study were provided by the Bergsagel laboratory and fingerprinted to confirm their identity . The celPrimary human MM cells were recovered from bone marrow aspirates collected from all Mayo Clinic sites. Informed consent was given in writing for collection and research use under Institutional Review Board approval  in accordance with the Declaration of Helsinki. After collection, CD138+ cells were isolated by immunomagnetic bead selection .2.3Total RNA from HMCLs and CD138‐selected plasma cells from MM patient bone marrows were isolated using RNeasy Mini kit and the AllPrep DNA/RNA Kit (Qiagen), respectively. After spectrophotometric quantification , RNA was stored at −80°C until use.2.4CRBN, genes with altered transcription in cell lines with low versus normal CRBN expression, genes encoding proteins associated with CRBN activity [IKZF1, IKZF3, IRF4, and MYC). Since CRBN isoforms, including the isoform lacking exon 10, have been associated with IMiD sensitivity [CRBN were included, with one (CRBN 3) designed to span the exon 10/11 junction. Twenty‐one genes were selected by analyzing baseline gene expression levels associated with drug response in a cohort of 44 refractory MM patients before initiation of pomalidomide and dexamethasone therapy on a phase 2 clinical trial [A unique list including 121 gene candidates was selected for NanoString profiling Figure . We firsactivity , or addisitivity , four pral trial , 36, 37 An additional 10 genes included in the exploratory panel were selected based on data from 59 MM patients that exhibited differential responses to a first line of treatment containing IMiDs in the CoMMpass data  and RR samples (n = 57) were selected using edgeR [Z = −32.1129 + 0.3773DIRAS1 + 2.2558CRBN_2 + 0.8810CD53 + 1.6225PSMD14 – 0.6304CEP55 + 0.9362SK2 ‐ 1.7078PSMA7Z, the probability of “responder” is calculated by sigmoid functionBased on the value Differentially expressed genes between ND . Using this model, we retrospectively analyzed existing RNA sequencing (RNAseq) data from the primary samples of the Multiple Myeloma Research Foundation (MMRF) coMMpass  and Mayo Clinic MM registry  datasets to correlate the MM‐IP‐7 results with other clinical data such as survival, disease stage, and number of treatment protocols. Briefly, we submitted RNAseq data to MM‐IP‐7 to calculate probabilities (by ranking scores) and then compared estimated results with other clinical data in each dataset. Since the RNAseq data are highly correlated but have different scales when compared to NanoString, the probability of estimate from this analysis is based on rank order rather than actual cut point criteria.We first evaluated the performance of seven‐gene model  by five‐fold cross‐validation as described  between 52 ND and 57 RR samples  as most significantly downregulated in RR samples; meanwhile another seven genes  were shown as most significantly upregulated  and RR patients (n = 57), we further evaluated the predictive value of each differentially expressed gene for correlation with drug resistance and disease progression. Using edgeR software, we identified 45/121 differentially expressed genes between ND and RR samples (p ≤ 0.01)  that contained seven‐associated predictors, CRBN, CEP55, DIRAS1, SKA2, CD53, PSMA7, and PSMD14  and Mayo Clinic MM patient registry (n = 487), found that model prediction correlated with OS (CoMMpass data, Figure Using the NanoString profiling data obtained from all ND (1 Figure . We iden4 Figure . The per4 Figure . Using M4CRBN. We not only identified downregulation of CRBN in samples harvested during or after treatment with an IMiD‐based therapy, but also observed that downregulation became increasingly prominent in the relapsed and end stages samples. These results are consistent with both our own and other's prior reports, which demonstrated that with progressive IMiDs exposure, CRBN copy number and expression was reduced in MM cell lines and patient samples [We investigated the expression of 121 selected candidate genes that were previously demonstrated to associate with IMID and/or PI response to determine whether such genes could be combined into a biomarker assay of disease progression following combination chemotherapy regimens. We identified a subset of genes with changes in transcriptional expression during treatment and disease progression. The top downregulated gene in refractory and late stage disease was  samples , 43. AltCRBN, in RR samples we also detected non‐CRBN‐related transcriptional changes identified in patient samples classified as nonresponders to pomalidomide (Supplemental Data IFITM1 and upregulation of PRR11, BIRC5, PBK, DEPDC1, RNFT2, and CEP55. IFITM1 is one of the interferon‐stimulated genes upregulated by IMiD treatment and suggested to be involved in IMID‐mediated antimyeloma activity [BIRC5, PRR11, PBK, DEPDC1, RNFT2, and CEP55 appeared subsequent to drug exposure in our study. BIRC5 and CEP55 are genes incorporated within the gene expression‐based proliferation indices [PRR11, PBK, and DEPDC1, like BIRC5, correlated with short survival and poor drug response (Figure ETV4. Upregulation of ETV4 has been demonstrated in MM patient samples at the time of acquire IMiD resistance [In addition to activity , 45. Upr indices , which ie Figure , implyine Figure . This resistance .CRBN [CD53 [PSMD4 [Based on the NanoString profiling data from ND and RR samples, we identified a subset of genes Figure  whose trCRBN , CD53 [3BN [CD53 , and PSM3 [PSMD4 . We alsoIn the current study, we also demonstrated that NanoString nCounter technology is a reliable method for accurately detecting gene expression in MM samples. As described in non‐Hodgkin's lymphoma –25, 49, In summary, we developed a NanoString gene expression profiling‐based prediction model, which may serve as a useful tool for clinical investigation and therapy selection. This is the first study to build a multivariate ordinal model to predict clinical drug resistance, poor survival, and disease progression following treatment with IMIDs and PI, by using a comprehensive gene list generated from multiple other publications and the robust NanoString technology, which is currently in clinical practice.AKS has a consulting role for Amgen, Bristol‐Myers Squibb, Glaxo Smith Klein, Janssen, Ono, Regeneron, Skyline Diagnostics, and Tempus Inc. and has received research funding from Amgen, Celgene, and Janssen. The remaining authors declare no competing financial interests.Supporting informationClick here for additional data file.Supporting informationClick here for additional data file.Supporting informationClick here for additional data file.


Streptococcal toxic shock syndrome (STSS) has a high mortality rate, and most patients die within a few days of onset. We report an elderly patient with STSS, necrotizing fasciitis and septic shock caused by group G streptococcus who was successfully treated with multidisciplinary therapy. The elderly patient suffered from streptococcal toxic shock syndrome and went into shock rapidly and progressively. However, prompt surgical treatment, antimicrobial agents, and blood purification therapy such as polymyxin B immobilized fiber column direct hemoperfusion and continuous hemodiafiltration could have saved the patient's life. The mortality rate is approximately 30%, with elderly populations showing remarkably worse prognosis.22: 97% (on room air). Electrocardiogram revealed atrial fibrillation. Multiple areas of erythema were found on both the right and left lumbar and lower abdominal areas, right proximal femoral areas, and left proximal inner femoral area. There were blisters, ruptured blisters, swelling, and aching pain on the right inner femoral area.A 71‐year‐old woman, 154 cm tall, weighing 46 kg, with a history of hypertension and chronic kidney disease, developed lower leg edema 1 month before hospitalization. She complained of right lower leg pain and a red flare on the day of hospitalization. She visited our emergency room due to the development of dysarthria and gait disturbances. When she arrived at the emergency room, her Glasgow Coma Scale (GCS) score was E4V5M6. However, although she was able to communicate, she had slurred speech and dysarthria. Her pupillary diameter was 3 mm bilaterally, indicating no anisocoria, and the light reflex was normal. Physical examination revealed the following: temperature: 36.9°C, blood pressure: 114/72 mmHg, heart rate: 116 beats/min, respiratory rate: 25 breaths/min, and SpO3, elevated C‐reactive protein of 16.7 mg/dl, and elevated procalcitonin level of 30.65 mg/dl, indicating leukopenia with evidence of inflammation and bacterial infection. Her blood urea nitrogen level was 68.9 mg/dl, and creatinine was 7.29 mg/dl, indicating impaired renal function, and prothrombin time (PT) was 19.3 s, PT‐INR was 1.63, activated partial thromboplastin time was 39.2 s, fibrin degradation products were 33.9 μg/ml, and D‐dimer level was 10.08 μg/ml, indicating prolonged coagulation and delayed activation of the fibrinolytic system. Blood gas analysis showed increased lactate levels of 4.59 mmol/L.Laboratory examination at admission revealed a low white blood cell (WBC) count of 1000 cells/mmBrain computed tomography (CT) examination performed to evaluate her neurological status showed an old brain infarction, with no new brain hemorrhage or space occupying lesions. Thoraco‐abdominal and pelvic CT examination displayed predominant right lower leg swelling, subcutaneous edema, and increased adipose tissue signals in the entire right femoral region Figure .Streptococcus dysgalactiae subsp. equisimilis (SDSE) was detected from culture of blood and genital wound samples, we de‐escalated the antibiotics to penicillin‐G on ICU Day 3. Since the patient had wound infection with Pseudomonas aeruginosa, ciprofloxacin was additionally administered. Thrombomodulin‐α was administered for the treatment of disseminated intravascular coagulation (DIC) from ICU Day 1 to Day 15. Furthermore, since her urine output was limited to 100 ml/day due to renal failure, continuous high‐flow CHDF was performed with a dialysate fluid rate of 2000 ml/h for 5 days, which was gradually tapered. Due to the substantial drainage from the wound, crystalloid fluid, fresh frozen plasma, and albumin preparations were administered.Her blood pressure gradually decreased after hospitalization, requiring vasopressors, and hence, continuous administration of noradrenaline at 0.05 μg/kg/min was initiated. The patient then developed disturbance of consciousness (GCS score was E4V3M4). Based on the clinical, laboratory, and imaging findings, we diagnosed necrotizing fasciitis from the right lumbar to lower abdominal regions along with septic shock. She was admitted to the intensive care unit (ICU) because his blood pressure could not be maintained, and she was also observed to have disturbance of consciousness. At her admission to the ICU, the patient's GCS score was E4V2M2, heart rate was 100 beats/min, blood pressure was 80/63 mmHg, and Sequential Organ Failure Assessment score was 11 points. Her systolic blood pressure subsequently decreased to approximately 60 mmHg, GCS score was E3V1M1, and respiration became unstable. Therefore, we performed endotracheal intubation and commenced mechanical ventilation. Fluid resuscitation was used for shock by administering 1000 ml of extracellular fluid and 500 ml of albumin in ICU. Since her hemodynamic parameters did not respond to fluid loading and noradrenaline administration, the noradrenaline dose was increased to 0.65 μg/kg/min. Figure Wound lavage was performed daily, along with blunt scraping of subcutaneous and inter‐fascial tissue and debridement Figure . Thereaf3Initial symptoms of STSS are fever, pain, and swelling of extremities, with rapid clinical deterioration leading to multiple organ failure, including acute respiratory distress syndrome, DIC, and renal disturbance, with extremely high mortality.When STSS is suspected, prompt diagnosis and early initiation of treatment with whole‐body management, antibiotic administration, and surgical intervention are critical.We used CHDF and PMX‐DHP in addition to hemodynamic management and infection control as a compassionate treatment. CHDF functions as an artificial kidney, removing various mediators of shock. In our case, we used an AN69ST membrane to absorb cytokines. The AN69ST membranes absorb high mobility group box protein 1 (HMGB‐1), which is a late mediator of septic shock.PMX‐DHP removes intrinsic cannabinoids, which are critical mediators in the early stages of septic shock and is effective in the treatment of severe sepsis.Since necrotic tissue has a poor blood supply, penetration of necrotic tissue by antibiotics is generally low. Therefore, early surgical resection of the necrotic tissue to prevent progression of tissue necrosis is prioritized in STSS with comorbid necrotic fasciitis.Here, we reported a case of STSS with necrotizing fasciitis and septic shock. Early multidisciplinary treatment resulted in a favorable outcome despite her advanced age. Many patients with STSS rapidly progress the symptoms which make difficult to rescue. The earliest diagnosis and treatment of STSS are critical. When we see a case with soft tissue infection which has atypical symptoms with rapid progression, STSS should be considered at high priority with proactive treatment.Akiko Kurachi: Conceptualization; writing – original draft. Yusuke Ishida: Conceptualization; writing – original draft; writing – review and editing. Koichi Nakazawa: Writing – review and editing. Toshio Okada: Writing – review and editing. Takumi Kishida: Conceptualization; writing – original draft. Hiroyuki Uchino: Writing – review and editing.The authors declare that they have no competing interests associated with this manuscript.Written informed consent was obtained from the patient to publish this report in accordance with the journal's patient consent policy.


Direct and indirect Coombs tests were both negative, suggesting a Coombs‐negative hemolytic anemia. A first blood smear did not show schistocytes and the coagulation profile was in order. Medical history was negative for oxidant drugs assumption, prosthetic heart valves, and liver and kidney diseases. Chest radiography was normal, and a complete abdominal echography did not show hepatomegaly or splenomegaly.An 83‐year‐old male was admitted to the emergency department for abrupt onset of jaundice and asthenia. Vital signs were in range, and physical examination was unremarkable except for jaundice and hyperchromic urine. Full blood count on admission showed his hemoglobin concentration to be 81 g/L with no alteration in white blood cell (WBC) or platelets count. Biochemical tests were significant for high bilirubin level 417.07 μmol/L (indirect bilirubin 369.53 μmol/L) and abnormal lactate dehydrogenase level 1520 μmol/L. The hypothesis of hemolytic anemia was confirmed by further laboratory tests, which showed undetectable haptoglobin and increased reticulocyte count 196 × 10tightness in the chest and dizziness and an electrocardiogram was compatible with a non‐ST‐segment elevation myocardial infarction. A new set of lab tests showed severe anemia with 66 g/L of hemoglobin, and the patient undergone blood transfusion. In the suspicion of a hemolytic crisis in the context of glucose‐6‐phosphate dehydrogenase (G6PD) deficiency, an accurate anamnesis was collected and the patient confirmed the assumption of fava beans 3 days earlier after a long time. G6PD levels could not be assessed overnight, but a second blood smear was significant for bite, blister, and ghost cells .Meanwhile, the patient clinical course was complicated by pain and s Figure  and B, wG6PD deficiency diagnosed in 83‐yer‐old patient with no previous similar event in an unfavorable setting (emergency department by night). It highlights the necessity of high clinical suspicion of G6PD deficiency in the context of Coombs‐negative hemolytic anemia, even in elderly patients. It is worth noting that different species of fava beans have a different capacity in inducing oxidative stress. Lastly, blood smear can still provide useful morphological data in supporting the diagnosis, regardless the immediate availability of G6PD test.This is a case of Fabio Giglio conceived the study; D. Sannipoli, D. T. Clerici, and Fabio Giglio provided all clinical and hematological data; A. Boletini and P. Ronchi performed and analyzed morphology; D. Sannipoli and Fabio Giglio wrote the paper. All authors approved the paper.The authors declare no conflict of interest.


The authors aimed to evaluate psychometric properties of the Concise Associated Symptom Tracking (CAST) Scale and validate the clinical utility of measuring irritability by updating and replicating a previously published outcome calculator from the Combining Medications to Enhance Depression Outcomes (CO‐MED) trial.Participants were 292 adults from the Establishing Moderators and Biosignatures of Antidepressant Response in Clinical Care (EMBARC) study who had completed the CAST scale at baseline. The scale's five‐domain  structure was evaluated with confirmatory factor analysis. Correlations with other clinical measures were used to confirm convergent and divergent validity. Logistic regression analyses from CO‐MED were used to estimate individual outcomes in EMBARC.s=0.50), Hamilton Rating Scale for Depression anxiety subscale (rs=0.24), Mood and Anxiety Symptoms Questionnaire anxious arousal scale (rs=0.44), Quick Inventory of Depressive Symptomatology Self‐Report insomnia items (rs=0.38), and Altman Self‐Rating Mania Scale (rs=0.39), respectively. Individual outcomes of remission (area under the curve [AUC]=0.805) and no meaningful benefit (AUC=0.779) were predicted with high accuracy among EMBARC participants using their baseline and week 4 scores for depression and irritability and model estimates from CO‐MED.Cronbach's alpha for the CAST scale was 0.78. Model fit for the five‐domain structure was adequate . Scores on irritability, anxiety, panic, insomnia, and mania were correlated with scores on the Anger Attack Questionnaire irritability item (rMeasuring irritability may help predict clinical course. The CAST scale is a valid measure of depression‐associated symptoms, including irritability. Symptoms of anxiety, irritability, panic, and mania are often associated with depression.The Concise Associated Symptom Tracking Scale is a valid measure of these depression‐associated symptoms.Measuring irritability along with overall depression severity may help predict clinical course. DSM‐5  study. To test the CAST scale's psychometric properties, we evaluated its five‐domain  structure with confirmatory factory analysis, measuring internal consistency with Cronbach's alpha coefficient, and demonstrating construct validity through correlation of the CAST domains with other clinical assessments at baseline. We then validated the CAST scale's clinical utility in measuring irritability as a symptom of major depression by updating the previously published CO‐MED calculator  and testhttps://clinicaltrials.gov/ct2/show/NCT01407094). Briefly, EMBARC participants were ages 18–65, met criteria for current episode of major depressive disorder on the Structured Clinical Interview for DSM‐IV Axis I Disorders, scored ≥14 on the 16‐item Quick Inventory of Depressive Symptomatology Self‐Report (QIDS‐SR) at both screening and randomization visits, did not meet criteria for a failed antidepressant trial during the current episode as measured by the Massachusetts General Hospital Antidepressant Treatment Response Questionnaire . At baseline, participants were randomly assigned to treatment with either escitalopram plus placebo, sustained‐release bupropion plus escitalopram, or extended‐release venlafaxine plus mirtazapine. Postrandomization visits were conducted at weeks 1, 2, 4, 6, 8, 10, and 12 for the acute phase and at weeks 16, 20, 24, and 28 for the continuation phase.For the present study, we used data from the CO‐MED trial participants to update the previously published logistic regression analyses of remission and no meaningful benefit as clinical outcomes . The CO‐Clinicians conducted the structured interview  for HAMDThe 16 items (each scored from 0 to 3) of the QIDS‐SR are based on the nine symptom domains of major depressive disorder . Total sThe 16 items of the CAST Self‐Report Scale assesses symptoms across five domains: anxiety , irritability , mania , insomnia , and panic . Each individual item is rated on a 5‐point Likert scale as 1, strongly disagree; 2, disagree; 3, neither agree nor disagree; 4, agree; or 5, strongly agree . In prevThe ASRM is a five‐item self‐reported scale designed to evaluate for the presence and severity of manic and hypomanic symptoms over the past 7 days. Each item consists of five possible responses, with scores ranging from 0 to 4. Item scores are added for a total score; 0 is the lowest possible score and 20 is the maximum possible .The 30‐item short‐form adaptation of the MASQ  was usedAt the baseline visit of the EMBARC study, participants completed the Massachusetts General Hospital Anger Attack Questionnaire (AAQ) . We usedIn the CO‐MED trial, separate logistic regression analyses were used to predict individual outcomes of remission  and no meaningful benefit (<30% reduction from baseline) at week 8 by using scores for depression (QIDS‐C) and irritability  at baseline and week 4 . Model βs) between the five domains and our clinical assessments for convergent and divergent validity.As previously mentioned, the modified intent‐to‐treat sample for the present study consisted of all EMBARC study participants who were randomly assigned to receive sertraline or placebo and who had completed the CAST scale at baseline of EMBARC N=292). Psychometric properties of the CAST scale were validated with EMBARC participants only. We used a confirmatory factor analysis implemented in PROC CALIS in SAS to validate the scale's five‐domain structure. We defined acceptable model fit a priori as a goodness‐of‐fit index ≥0.90, comparative fit index ≥0.90, and root mean square error of approximation ≤0.08 . We esti2. Psychobaseline depression from CO‐MED×(baseline depression in EMBARC)+βbaseline irritability from CO‐MED×(baseline irritability in EMBARC)+β percent change in depression from CO‐MED×(percent change in depression in EMBARC)+βpercent change in irritability from CO‐MED×(percent change in irritability in EMBARC). We then compared these estimated probabilities with observed occurrence of these outcomes by using receiver operating characteristic (ROC) curves. We conducted all analyses with SAS, version 9.4; threshold of significance was set at p<0.05.To validate the scale's clinical utility, we updated previously described logistic regression analyses from the CO‐MED trial, which had remission and no meaningful benefit at week 8 as outcomes , by replAt baseline, mean±SD scores for the CAST scale's five domains  were 16.32±3.96, 8.84±2.66, 7.57±2.65, 6.64±2.06, and 4.54±2.00, respectively. Detailed frequency of responses to individual items of the scale is shown in Table In confirmatory factor analyses, goodness of fit, comparative fit index, and root mean square error of approximation were 0.93, 0.92, 0.06, respectively, for the CAST scale's five‐domain structure. Because three out of three a priori defined criteria were met, the model fit was deemed acceptable. The standardized factor loadings for the anxiety, irritability, mania, insomnia, and panic domains ranged from 0.36 to 0.85, 0.48 to 0.85, 0.45 to 0.78, 0.62 to 0.68, and 0.61 to 0.93, respectively , indicating that these items provided adequate discrimination. Table Cronbach's alpha for the 16‐item CAST scale was 0.78, whereas those for the irritability, anxiety, mania, insomnia, and panic domains were 0.77, 0.68, 0.71, 0.59, and 0.72, respectively.s=0.17–0.24) and HAMD‐17 (rs=0.11–0.34). The mania domain was not significantly correlated with these measures of depression severity (Table s=0.24) and the MASQ anxious arousal scale (rs=0.30). Items related to irritability on other scales, such as the AAQ and MASQ, were significantly correlated only with the CAST scale's irritability domain . The irritability domain was poorly correlated with scales for mania (rs=0.06 with ASRM) and insomnia (rs=0.09 with the sum of the insomnia items on the QIDS‐SR). The mania, insomnia, and panic domains were positively correlated with the ASRM (rs=0.39), the sum of insomnia items on the QIDS‐SR (rs=0.38), and the MASQ's anxious arousal subscale (rs=0.44), respectively.The irritability, anxiety, insomnia, and panic domains were positively, albeit modestly, correlated with measures of overall depression severity, namely the QIDS‐SR  in the CO‐MED trial (12), we found that individual‐level prediction of remission (area under the curve [AUC]=0.805) and no meaningful benefit (AUC=0.779) in the EMBARC study were similar to those of the CO‐MED trial  (Figure In this study of a large sample of adult outpatients with major depressive disorder, we found confirmatory evidence for the psychometric properties and five‐domain structure of the 16‐item CAST scale. Furthermore, we extended the clinical utility of CAST as a measure of irritability by updating and validating a previously reported individual‐level calculator for prediction of acute‐phase treatment outcomes of remission and no meaningful benefit.Our findings are consistent with previous studies that have found the CAST scale to have sound psychometric properties , 21. AddAlthough the multidimensional nature of anxiety among patients with major depressive disorder has been reported , similarThis study had several limitations. The findings were obtained through unplanned secondary analyses, and as such the analyses may not have been adequately powered. Validation of the psychometric properties of the CAST scale was not a primary goal of EMBARC. Hence, the EMBARC study design did not include separate measures of irritability, insomnia, and panic beyond the baseline visit. Furthermore, EMBARC's inclusion and exclusion criteria may make the findings less generalizable to all patients with major depressive disorder . When avIn this large sample of adult outpatients with major depressive disorder, we found the 16‐item CAST scale to have good psychometric properties. Furthermore, we replicated findings that irritability is an important symptom domain, which can help predict individual‐level outcomes of remission or no meaningful benefit when combined with measures of depression severity. Thus, this study informs research on irritability among adults with major depressive disorder and guides clinical practice by improving the accuracy of clinical course prognostication.Supplementary MaterialClick here for additional data file.


This post hoc analysis compared the efficacy and safety of suprachoroidally administered triamcinolone acetonide (CLS‐TA) to other commonly available treatments for non‐infectious uveitis.Results from the PEACHTREE study were compared between subjects randomised to CLS‐TA not requiring rescue therapy and those subjects randomised to control, who subsequently required rescue therapy. Endpoints included best corrected visual acuity (BCVA), central subfield thickness (CST), treatment emergent adverse events and intraocular pressure (IOP) related safety findings.p = 0.115). Unrescued CLS‐TA subjects showed a mean gain of 15.7 versus 10.9 letters in rescued control subjects (p = 0.080). A significantly greater mean reduction in CST was observed for unrescued CLS‐TA subjects versus rescued control subjects . Of unrescued CLS‐TA subjects, 4.9% experienced IOP elevations ≥30 mm Hg at any visit versus 10.9% of rescued control subjects. Further, use of IOP‐lowering medications appeared lower in unrescued CLS‐TA subjects versus rescued control subjects (7.2% vs. 13.0%). There were no IOP‐lowering surgical interventions in either group.In this analysis, there were 83 unrescued CLS‐TA subjects and 46 rescued control subjects. At Week 24, 51.9% of the unrescued CLS‐TA subjects gained ≥15 letters in BCVA, compared to 37.0% of the rescued control subjects (CLS‐TA subjects experienced significantly greater reduction in CST and tended towards greater improvement in BCVA, compared with rescued control subjects. Suprachoroidally administered CLS‐TA showed a lower incidence of IOP‐related safety findings. Compared to baseline, ME, as measured by central subfield thickness (CST), reduced by a mean of 153 µm versus 18 μm (p < 0.001) for the CLS‐TA and control arms, respectively. All subjects were allowed to receive rescue therapy based on a set of pre‐defined criteria or at the investigators' discretion. The control arm received rescue therapy more frequently (71.9%) compared to the CLS‐TA arm , with IVT and periocular corticosteroids being the most commonly prescribed rescue therapies. AEs of elevated intraocular pressure (IOP), not temporally related to the injection procedure, occurred in 11.5% of CLS‐TA arm and 15.6% of the control arm, likely reflecting the more frequent IVT and periocular corticosteroid rescue in the control arm. Cataract AE rates were comparable (7.3% and 6.3%) in the CLS‐TA arm versus the control arm.Suprachoroidal injection of CLS‐TA was assessed in the phase 3 PEACHTREE study (NCT02595398) in subjects with non‐infectious uveitis (NIU) complicated by ME.Suprachoroidal administration, with anatomically precise drug delivery, has the potential to yield durable safety and efficacy benefits over current therapies.22.1The PEACHTREE full study design and main findings have been reported elsewhere.Rescue therapy was permitted at any time during the study if any of the following criteria were met in the study eye: A decrease of ≥10 letters in ETDRS BCVA from baseline (day 0); an increase in CST of ≥100 µm or 20%, whichever is lower, from baseline (day 0) based on the CST measurement at the clinical site; a ≥ 1.5 step increase from baseline (day 0) in the level of inflammation  or an increase from 3+ to 4+; and/or the investigator judged the uveitic complications in the study eye had not improved and the condition required additional treatment. The choice of rescue therapy was left to the discretion of the investigator.2.2Efficacy endpoints evaluated in this post hoc analysis included: Proportion of subjects with ≥15‐letter improvement from baseline in BCVA, mean change from baseline in BCVA letter score, mean change from baseline in CST and proportion of subjects with retinal thickness < 300 µm, all at 24‐weeks. Safety endpoints evaluated included mean change from baseline in IOP at 24‐weeks and incidences of IOP elevations ≥30 mm Hg, proportions of subjects requiring ≥1 additional IOP‐lowering medications and proportions of subjects requiring surgical intervention for IOP. All IOP endpoint assessments omitted IOP values measured immediately following the study treatment injection procedures to exclude procedure related, volume driven events. Safety endpoints also included the incidences of serious AEs (SAEs) and treatment‐emergent AEs (TEAEs) reported during the entire 24‐week study period.2.3In this post hoc analysis, endpoints were evaluated in the subset of CLS‐TA subjects from the PEACHTREE intent‐to‐treat (ITT) population  who did not require rescue therapy and the subset of subjects in the control group that received at least one rescue therapy at any time during the study up to Week 24. Descriptive statistics were used to summarise continuous variables, while counts and percentages were used to summarise categorical variables. The number of subjects who received rescue was summarised. Demographic and disease characteristics were evaluated across subsets in order to assess for clinically important imbalances.p value were calculated based on an analysis of variance (ANOVA) model with fixed effects for treatment group and country.The number and percentage of subjects with an improvement from baseline of 15 letters or more in BCVA and a CST of less than 300 μm were summarised. Differences between treatments were tested using a Cochran–Mantel–Haenszel chi‐square test stratified by country. For mean changes from baseline in BCVA and CST the estimate of the between‐treatment difference, 95% confidence interval and p values are nominal and are presented for descriptive purposes only. For the purpose of informing future studies, a p value ≤0.050 was considered statistically significant. All analyses and summaries were produced using SAS® version 9.4  or higher for Windows.Endpoints based on the assessment of IOP were enumerated and included a clinically relevant IOP of ≥30 mm Hg. IOP lowering medications and IOP‐lowering surgeries were enumerated. The number and percentage of subjects experiencing TEAEs or SAEs during the study were summarised. All analyses were done on the ITT population as observed; no methods were used to impute values for missing data. No adjustments were made to the alpha level to account for multiple comparisons. Due to the post hoc nature of this analysis, and the fact that data from the two groups being compared do not reflect random samples, 33.1PEACHTREE randomised a total of 160 subjects .p = 0.414, ANOVA). In the unrescued CLS‐TA subjects, mean baseline CST was 480.6 μm and in the rescued control subjects mean baseline CST was 519.7 μm . In the unrescued CLS‐TA subjects, mean IOP at baseline was 13.5 mm Hg and in the rescued control subjects mean baseline IOP was 13.3 mm Hg.The two treatment groups were balanced with respect to age, race, gender, the mean duration of ME diagnosis in the study eye , the anatomical location of uveitis and the onset type, duration and course of uveitis .The percentage of subjects with a gain from baseline of ≥15 letters in BCVA was 51.9% (41/79) in the unrescued CLS‐TA group compared to 37.0% (17/46) in the rescued control group at Week 24 .At Week 24, unrescued CLS‐TA subjects who completed the study showed a mean improvement of 15.7 letters, compared to a 10.9 letter improvement in the rescued control subjects who completed the study .Analysis of CST showed a similar trend. Of the 77 unrescued CLS‐TA subjects with gradable images at Week 24, a total of 47 (61.0%) had a CST < 300 μm compared to 22/44 (50.0%) rescued control subjects .  trended toward better BCVA and CST outcomes than rescued control patients (n = 34).3.4At Week 24, unrescued CLS‐TA subjects who completed the study showed a mean IOP of 15.2 mm Hg, representing an increase from baseline in IOP of 1.6 mm Hg. Rescued control subjects showed a mean IOP of 15.4 mm Hg at Week 24 reflecting a mean increase of 2.0 mm Hg from baseline.Unrescued CLS‐TA subjects tended to experience fewer IOP‐related events with lower incidents of IOP ≥30 mm Hg at any visit compared to control subjects who received rescue therapies (4.9% vs. 10.9%). No subject experienced an IOP over 37 mm Hg. The use of IOP lowering medications in subjects who only received CLS‐TA appeared lower than control subjects who received rescue therapy (7.2% vs. 13.0%) . . In this post hoc analysis, the primary objective then was to compare safety and efficacy results from the unrescued CLS‐TA subjects in PEACHTREE to that of rescued control subjects (surrogate active comparator arm). Rescue therapy in the control arm consisted primarily of IVT and/or periocular corticosteroids.Results of this post hoc analysis are consistent with the results reported for PEACHTREE.There is limited data on the comparative effectiveness of therapies for uveitic ME. The PeriOcular and INTravitreal Corticosteroids for Uveitic Macular Edema Trial (POINT) study, an NIH‐funded prospective clinical trial, compared three commonly administered local corticosteroids for uveitic ME. At 6 months, IVT triamcinolone acetonide and IVT dexamethasone implant outperformed periocular corticosteroid, both achieving approximately nine letters of improvement compared to approximately four letters, respectively.Limitations of this study include its sample size and post hoc design, which cannot replace a prospective comparative effectiveness trial. One source of potential bias, likely related to the post hoc nature of this study, is that the two groups are not necessarily homogeneous, resulting in potential differences in disease characteristics. In addition, while there were pre‐defined criteria for rescue therapy administration, the choice of rescue therapy and route of administration were at the investigators' discretion. Thus, rescue medication use in the control group comprised a heterogenous mixture of medications/routes as compared to a single comparator agent/administration route, yet provided a real‐world snapshot of current practice. Although all rescued control subjects ultimately received corticosteroids, including IVT or periocular corticosteroids in 80.4% of rescued control subjects, CLS‐TA subjects had longer corticosteroid exposure compared to rescued control subjects, possibly biasing efficacy measures in favour of the unrescued CLS‐TA group and safety measures in favour of rescued control group. In a recently published paper, longitudinal modeling for CLS‐TA treated eyes showed that CST reached 90% of maximal improvement before the analogous 90% maximal response in BCVA was observed.Dr. Singer reports receiving consulting fees from Allergan, Aerie, Genentech, Regeneron, Novartis and Eyepoint as well as grant support from Optos, Appelis, Kodiak, Unity, Astellis, Ionis, Clearside, Allergan, Aerie, Genentech, Regeneron, Novartis and Eyepoint. Dr. Merrill reports receiving consulting fees from Santen, Gilead and Eyepoint as well as grant support from Clearside. Dr. Yeh reports receivning consulting fees from Clearside, Adverum and Santen. Dr. Hall, Dr. Ciulla and Mr. Kapik are employees of Clearside and hold stock in the company.


The prevalence of fatty liver disease is potentially increasing in adolescents and young adults (AYAs) due to the obesity and alcohol pandemics. The aim of this study was to assess the prevalence of alcohol-associated fatty liver disease (ALD) and nonalcoholic fatty liver disease (NAFLD) in a representative U.S. cohort utilizing transient elastography to directly measure hepatic steatosis and suspected fibrosis.® measurements in the National Health and Nutrition Examination Survey (NHANES) database (2017–2018) were included in the analyses. Those with viral hepatitis, pregnancy, or ALT/AST > 500 U/L were excluded. The population was divided into those with excessive alcohol consumption (ALQ130) and those without. Controlled attenuation parameter (CAP) score ≥ 248 dB/m was used to identify suspected ALD and NAFLD. In those with evidence of ALD, the following cutoffs of liver stiffness measurement (LSM) were used for suspected fibrosis: F ≥ F2 at LSM ≥ 7.5 kPa and F ≥ F3 at ≥ 9.5 kPa, respectively. In those with suspected NAFLD, the following LSM cutoffs were used: F ≥ F2 at 6.1 and F ≥ F3 at ≥ 7.1, respectively. Cutoffs were chosen based on published literature to maximize sensitivity.AYAs (age 15–39 years) with valid FibroScanComparing to those without, subjects with excessive alcohol consumption tended to be older (29.8 vs 28.5 years), have a higher BMI (29.3 vs 28.9 kg/m2), and be from a White ethnicity (65.3% vs. 55.4%). In subjects with excessive alcohol consumption, suspected ALD was present in 56.59% (95% CI 41.57–70.49). In those with suspected ALD, suspected significant fibrosis (F ≥ F2) was present in 12.3% (95% CI 4.74–28.34) and advanced fibrosis (F ≥ F3) was present in 6.31% (95% CI 0.69–39.55). Similarly, in subjects without excessive alcohol consumption, suspected NAFLD was present in 40.04% (36.64–43.54). In those with suspected NAFLD, suspected significant fibrosis (F ≥ F2) was present in 31.07% (27.25–35.16) and suspected advanced fibrosis (F ≥ F3) was present in 20.15% (16.05–24.99).A significant percentage of AYAs are at risk for ALD and NAFLD and a subset of these subjects is at risk for significant fibrosis. Efforts should focus on increasing awareness of the prevalence of ALD and NAFLD in this population and to mitigate modifiable risk factors.The online version contains supplementary material available at 10.1186/s12876-022-02430-7. Non-alcoholic fatty liver disease (NAFLD) and alcoholic liver disease (ALD) are the two most common causes of chronic liver diseases and are characterized by the accumulation of excessive amounts of fat within the hepatocytes. NAFLD is currently considered as the most common cause of chronic liver disease worldwide and is the second leading cause of liver transplantation in the United States . The preSimilarly, ALD counts for one of the very relevant, most prevalent and fastest growing liver diseases in the United States with an overall prevalence of 2%. Moreover, ALD is one of commonest causes of end-stage liver disease, with 50% of the overall mortality in cirrhosis patients being attributed to alcohol, and 50% of alcohol-related mortality being attributed to liver disease –5. The iWorrisomely, the demographic pattern of alcohol consumption has further shifted to the younger age groups resulting in a more substantial loss of productive life years and an increase in the alcohol-attributable mortality in the adolescents and young adults (AYAs), which is the age group most responsible for disability-adjusted life years (DALY) , 9.The relationship between ALD and NAFLD is a two-sided and a rather complex one. For instance, numerous previous studies showed a possible protective effect of mild to moderate alcohol consumption on the development and progression of NAFLD –12, howeDespite the significant prevalence and burden, the overall awareness of these two entities remains limited in the general population , 18. OurThe National Health and Nutrition Examination Survey (NHANES) database is a major project of the National Center for Health Statistics (NCHS) , which iThis survey-based program annually examines a nationally representative sample of ~ 5000 individuals located in counties across the United States. The NHANES interview includes demographic, socioeconomic, dietary, and health-related questions. The examination includes –but is not limited to- medical, physiological and laboratory measurements performed by highly trained medical personnel at a central laboratory, in addition to interview questionnaires and standardized physical examination.® and was utilized for this analysis.Data from the survey is used in epidemiological and health-related studies and helps designing further health programs and services. The survey was approved by the Institutional Review Board at the Center for Disease Control and Prevention, and informed consent was obtained from all participants. Data from NHANES 2017–2018 is the most recent survey cycle that provided transient elastography information as determined by FibroScan® were included in the analysis. Those with chronic viral hepatitis B or C, missing alcohol consumption data, pregnancy, Alanine Aminotransferase {ALT}, Aspartate Aminotransferase {AST} values > 500 U/L, or missing or incomplete elastography data were excluded. The inclusion criteria and participants stratification algorithm is shown in Fig. Participants between 15 and 39 years with demographic , alcohol intake data and a valid FibroScanAlcohol consumption = )/(365 days) using The Alcohol Use Questionnaire (ALQ) 130 {In the past 12 months, on those days that {you/SP} drank alcoholic beverages, on the average, how many drinks did {you/he/she} have?} and ALQ 121 {During the past 12 months, about how often did {you/SP} drink any type of alcoholic beverage? PROBE: How many days per week, per month, or per year did {you/SP} drink?} in the NHANES database. Excessive alcohol consumption was defined as male > 2 drinks/day and female > 1 drink/day, and the population was stratified into those with excessive alcohol consumption and those without. A “current smoker” was defined as any adult who has smoked 100 cigarettes in his/her lifetime and who currently smokes cigarettes. A “former smoker” was defined as any adult who has smoked at least 100 cigarettes in his/her lifetime but who had quit smoking at the time of interview. A “never smoker” was defined as any adult who has never smoked, or who has smoked less than 100 cigarettes in his/her lifetime. Controlled attenuation parameter (CAP) score ≥ 248 dB/m was used to define steatosis and to further stratify the population into suspected ALD and suspected NAFLD [Daily alcohol consumption was calculated as: ed NAFLD .In addition to CAP score and liver stiffness measurement (LSM), the following socio-demographic information and laboratory data were collected for analysis: age, sex, race/ethnicity, alcohol intake status, smoking status , Body Mass Index {BMI}, ALT, AST, total bilirubin, albumen, Mean Corpuscular Volume {MCV}, platelet count, and Haemoglobin A1C {HBA1C}.Stata Statistical Software: Release 17. College Station, TX: StataCorp LLC.).In those with evidence of ALD, the cut-offs of LSM used for suspected fibrosis were F ≥ F2 at LSM ≥ 7.5 kPa and F ≥ F3 at ≥ 9.5 kPa, respectively. In those with suspected NAFLD, LSM cut-offs were F ≥ F2 at 6.1 and F ≥ F3 at ≥ 7.1, respectively. These cut-offs were chosen based on the published literature to maximize sensitivity –22. We cAmong the total number of noninstitutionalized civilian population screened in the database N = 9254), our final sample consisted of 1319 AYAs after excluding those with viral hepatitis, pregnancy, ALT or AST > 500 U/L, missing data on alcohol consumption, and/or missing VCTE data. We reported the baseline characteristics of the study population (N = 1319), along with those with excessive alcohol consumption (N = 100) compared to those without (N = 1219) in Table 54, our fIn the excessive alcohol consumption group, the characteristics of those with ALD were compared to those without in Table Finally, the prevalence and stage of suspected fibrosis for ALD and NAFLD were separately collected and compared in both groups.3/uL p < 0.001) and HbA1C .The prevalence of excessive alcohol intake (N = 100) was 8.49% (95% CI 6.43–11.13) in the general population. In comparison to those without excessive alcohol consumption (N = 1219), subjects with excessive alcohol consumption (N = 100) were older , more likely to be male , of Caucasians descendants , current smokers , had higher AST , ALT  and MCV , and had lower platelet counts  had ALD compared to 43.41% (95% CI 29.51–58.43) who had no evidence of liver involvement based on CAP < 248 dB/m. The prevalence of suspected fibrosis in the ALD group was as follows , Caucasians , current smokers , had higher AST  and ALT , comparable albumin  and MCV  levels, with higher platelet count  and HbA1C .Moreover, compared to those with no evidence of ALD based on low CAP score, subjects with ALD (N = 52) tended to be older , more males , with higher BMI , the prevalence of NAFLD was 40.04% (95% CI 36.64–43.54) compared to 59.96 (95% CI 56.46–63.36) who had no liver involvement. The prevalence of suspected fibrosis in the NAFLD group was as follows , Hispanics , former smokers , had higher AST  and ALT , lower albumin , higher platelet counts  and higher HbA1C .Also comparably, those with NAFLD (N = 506) tended to be older , more males  than females, had significantly higher BMI (34.1 vs 25.5 kg/mThe main findings of this study are the following: (1) a significant percentage of AYAs consume excessive amounts of alcohol placing them at risk of ALD; (2) in those with excessive alcohol consumption, 57% have evidence of ALD based on CAP and 12% are at risk of having significant suspected fibrosis based on LSM; (3) in those without excessive alcohol consumption, 40% have evidence of NAFLD with 31% at risk of significant fibrosis; (4) male gender, obesity, smoking, and certain ethnicities were associated with higher burden of liver disease.Unhealthy alcohol use and obesity are the two major drivers of the burden of fatty liver disease (FLD) across the globe , 24. Alc2) with slight male predilection more .Compared to the most recently reported US prevalence of both diseases from the same database on 2008 cycle (age cut off: 18 or above), our results show a significantly higher prevalence for NAFLD (40.04% vs. 11.01%) and ALD (56% vs. 3.94%), confirming previously predicted exponential growth of the trends and burden of these two diseases [Ethnic factors play a role, as evidenced by epidemiological studies comparing different ethnic groups observing the rates of alcoholic cirrhosis. Ethnicity factors are postulated to play a role in liver metabolism via several genetic polymorphisms . In our Although alcohol consumption is increasing in younger people more rapidly than ever, our study demonstrated that excessive alcohol intake is slightly higher in older individuals in the AYAs group. We believe that the addictive and cumulative nature of alcohol consumption is responsible for such findings.Hart et al. did an exciting analysis showing how high BMI and alcohol consumption have a supra-additive interaction to cause liver disease . AnotherObesity is one of the most important modifiable risk factors which determines the risk of cirrhosis in heavy drinkers .3/uL p < 0.001) and HbA1C . Hence, no unique pattern of liver enzymes or other markers has been seen.Current study also exhibits interesting comparative findings blood chemistry in AYAs. AYA with excessive alcohol consumption had higher AST , ALT  and MCV , and had lower platelet counts  leads to an increase in stiffness, which causes irreversible scarring and fibrosis, thereby causing significant liver illnesses. Hepatic fibrosis has been established as a dynamic process with the potential for considerable resolution. Unlike an alcoholic liver disease, there is no unique elevation pattern of liver enzymes for diagnosing NAFLD.We used transient electrography to quantify hepatic steatosis and suspected fibrosis. Due to considerable inter-observer variability, traditional USS imaging is not a very sensitive diagnostic tool to assess mild liver steatosis compared to liver biopsy . The FDABeyond these demographic results, our study extends the literature in a number of ways. First, we extricated the CAP score by Fibroscan and determined the suspected fibrosis occurrences in NAFLD and ALD individuals in our two cohorts of excessive alcohol and non-excessive alcohol consumption. While alcohol consumption is a well-thought-out cause of hepatic steatosis in the ALD process, NAFLD spans the spectrum of hepatic steatosis in the absence of alcohol consumption.An epidemiological study by Abeysekera et al. had coherent conclusions to our findings. The participants with steatosis and alcohol use disorder were reported to have four times higher risk of developing fibrosis . In the In subjects without excessive alcohol consumption (N = 1219), suspected NAFLD was present in 40.04% (95% CI 36.64–43.54) of individuals.In the fraction of individuals with suspected ALD, we found that for stage F0-F1 prevalence of suspected fibrosis was 87.7% (95% CI 71.66–95.26), for subsequent significant fibrosis stage (F ≥ F2) prevalence was 12.3% (95% CI 4.74–28.34), and advanced fibrosis stage F ≥ F3 6.31% (95% CI 0.69–39.55). For individuals with suspected NAFLD (N = 506), the prevalence of suspected fibrosis was stratified. For the stage F0-F1 prevalence was 68.93% (95% CI 64.84–72.75), for subsequent stage of suspected significant fibrosis F ≥ F2 prevalence was 31.07 (95% CI 27.25–35.16) and for suspected advanced fibrosis stage F ≥ F3 prevalence was 20.15% (95% CI 16.05–24.99).These results were coherent with the findings observed in a community-based ABCD  study of 890 individuals by Petta et al., which showed a stark high 60% prevalence of NAFLD in the general population and amongst NAFLD patients, there was a high prevalence of advanced fibrosis in specific variants  . NAFLD wOur study has several limitations. Firstly, despite using strict exclusion criteria, responses entail a degree of recall error considering the nature of the questionnaire for alcohol consumption history. This also leads to the generalizability of our findings to other patient populations requires clarification. For diagnosing NAFLD, we used the Fibroscan, which has limited sensitivity and validity to detect fibrosis equivalent to stage F2 or less , 45. SinOur study draws attention to the growing prevalence of FLD in AYA. Since there is limited evidence regarding understanding factors contributing to FLD in AYA, we need to focus more on mitigating these risk factors. Large randomized control trials are a curial necessity to have early and accurate detection of diffuse liver diseases in AYAs. Our study showed that a significant percentage of AYAs are at risk for ALD and NAFLD, and a fraction of these subjects are at risk of developing liver fibrosis. Efforts should focus on increasing awareness of the burden of ALD and NAFLD in this population and mitigate the modifiable risk factors. Educational programs, lifestyle interventions, and awareness are essential to acknowledge the prevalence of these two clinical entities, which are often intertwined in AYAs.Additional file 1: Numbers of excluded individuals among study participants with corresponding criteria.


Stimulator of interferon (IFN) genes (STING) is a protein that promotes type I IFN production essential for T‐cell activation. In this study, we aim to characterize STING expression comprehensively using The Cancer Genome Atlas (TCGA) database, cell lines, and patient tumor samples stained with immunohistochemistry.n = 499 patients from the TCGA database was analyzed. Methylation was evaluated on STING and cGAS in five STING‐negative NSCLC cell lines.Two cohorts were evaluated comprising 721 non–small cell lung cancer (NSCLC) patients and 55 NSCLC cell lines for STING and cyclic GMP‐AMP synthase (cGAS) expression using immunohistochemistry. Moreover, an independent cohort of p = 0.033). STING protein expression is significantly higher in adenocarcinoma (AC) and is lost with increasing stages of AC. STING‐positivity is significantly higher in mutant EGFR and KRAS tumors. STING‐positive NSCLC patients identified with immunohistochemistry (H‐score > 50) have increased survival . Treatment of STING‐negative cell lines with a demethylating agent restores STING expression.STING RNA expression positively correlates with T cell function and development genes, negatively correlates with cell proliferation and associated with increased survival (5‐year‐overall survival [OS] 47.3% vs. 38.8%, STING is ubiquitously expressed in NSCLC and associated with T cell function genes, AC histology, EGFR, and KRAS mutations and improved overall survival. The treatment for patients with non–small cell lung cancer (NSCLC) has been changed by introducing anti‐programmed death‐1 (PD‐1) immunotherapy. Long‐term responses in advanced‐stage disease that were previously not anticipated now have been accomplished, and 5‐year overall survival (OS) increased to 20% in unselected and up to 40% in PD‐L1STING  is a protein responsible for controlling anticancer immune responses to leaked self‐ or non‐self DNA.Recent studies in hepatocellular,The cytoplasmic DNA sensor cGAS Our study aims to analyze the presence and the expression landscape of STING and cGAS protein according to key clinicopathological parameters, including stage, sex, histological type, mutational status, and survival. Furthermore, to validate our results on the human tissues, we analyzed the methylation of STING and cGAS in NSCLC cell lines using demethylating agents.22.1N = 419 AC and N = 302 SCC, Table Tumor tissue samples from a total of 721 patients were included in our study  using the Ventana Benchmark XT autostainer, like previously described.2.3Mycoplasma‐free by the Cell Technologies Shared Resource (CTSR) at the University of Colorado Cancer Center. For methylation studies, all cell lines were grown to 70% confluency in duplicate T25 flasks with Roswell Park Memorial Institute 1640 media containing 10% fetal bovine serum and Pen/Strep. Cell media was then replaced with media alone or media containing 10 µM 5‐Aza‐2ʹ‐deoxycytidine demethylating agent (Sigma‐Aldrich) for 72 h. Cell lines were then lyzed with Cell Lysis Buffer , followed by sonication and centrifugation. Cell lysate protein concentration was determined using the BCA assay .Cell lines used in studies were obtained from ATCC and fingerprinted for authenticity and certified as Western blot analysis was performed using 4%–20% precast Criterion gels . Specimens were loaded with 40 µg total protein per lane and run at 70 V for 3 h, followed by protein transfer to polyvinylidene difluoride membrane  for 500 mAmp*h. Primary antibody was diluted in milk/T‐Tris‐buffered saline (T‐TBS)  and added to membranes, and incubated overnight at 4°C on a rocker, followed by horseradish peroxidase‐secondary antibody after washing with T‐TBS. Membranes were revealed using the Clarity ECL reagent  and recorded on radiographic film.2.4www.cbioportal.org). Heat maps of gene sets were generated using UCSC Xena (http://xena.ucsc.edu) browser to probe RNAseq (PolyA + IlluminaHiSeq Percentile) data.Provisional LUAD and LUSC data sets from The Cancer Genome Atlas (TCGA) were analyzed for messenger RNA (mRNA) expression of STING using the proprietary program BioGAP. TCGA Methylation data were generated using Illumina 450k Methylation data obtained from cBioPortal (2.5χ2 test. Receiver operating characteristic (ROC) curves were used to define optimal survival cutoffs for STING mRNA expressions, and survival analysis was performed using the Kaplan–Meier method and the log‐rank test. OS was calculated from the date of diagnosis to the date of death or last available follow‐up. Hazard ratios and confidence interval (CI) were calculated using Cox's proportional hazards method. A two‐sided p value of 0.05 was statistically significant. We used SPSS, version 24.0.0.0 (IBM Corporation).Associations between clinicopathologic characteristics and STING or cGAS expression were analyzed using the 33.1n = 576) and SCC (n = 553). Johnston et al. identified a gene set of lung cancer‐associated T cell signature genes that indicate T cell function in lung tumors.Figure n = 249, n = 199 Stage I–II and n = 46 Stage III–IV), and bottom 50% , with a cutoff value of 27.5 FKPM  was significantly higher compared with the bottom 75%  in terms of STING expression  and below the 50th percentile  according to STING RNA  and bottom 75%  STING‐expressor patients with SCC    Figure . STING e) Figure . Express) Figure .3.3p = 0.003) . STING H‐score in this cohort was much higher in early‐stage cases and decreased with more advanced stage  (Tables p = 0.04) and in KRAS mutant versus wild type   Figure , and in ) Tables . Both AC) Figure .n = 421)   showed significantly increased survival in the “annotated” cohort  Figure . When pa3.4n = 422) and SCC . The cGAS promoter shows slight methylation in AC  but a large increase in SCC . Here we investigated methylation of STING and cGAS in five STING‐negative NSCLC cell lines confirmed by IHC. Cell lines were treated with demethylating agent 5ʹAZADC followed by western blot analysis of STING and cGAS protein expression. STING expression increased from baseline in each of the 5ʹAZADC‐treated cell lines, while cGAS expression increased in 4/5 of the cell lines.Since methylation of STING and cGAS promoters has been implicated in the loss of STING or cGAS protein expression, we analyzed TCGA NSCLC methylation data and sought to restore STING or cGAS expression in cell lines using the demethylating agent 5ʹAZADC Figure . TCGA an4n = 721 individual cases. This is complemented with TCGA analysis and cell line functional data to validate our results. We found that the STING is extensively expressed in NSCLC, and the proportion of STING‐positive cases was more significant in AC than SCC in both individual cohorts. In addition, we showed that STING expression is reduced with a higher tumor stage, irrespective of histology. These data may have future relevance with the emergence of STING agonist therapy and or in a potential combination with anti‐PD immunotherapy.T cell responses and the immune microenvironment are critical factors for therapeutic efficacy. The reported response rates for anti‐PD immunotherapy in NSCLC are still about 20%.The STING pathway might be defective in lung tumors that can alter responses, as reported in other cancers. Others showed that STING/cGAS expression was lost in tumor tissues; however many of these studies involve only a small number of cases.Activating STING to transform immunologically refractive cold tumors to a hot phenotype is a potential new therapeutic approach.p = 0.02) in a pooled NSCLC cohort, and a nonsignificant improve in overall survival when stratified according to histologies . We found stage, age. and PD‐L1 significant in the multivariate analysis, but STING‐high expression in NSCLC remained prognostic only in the univariate analysis, possibly due to lack of STING specific treatment. Analysis of TCGA RNA expression data of n = 499 AC patients confirmed our finding that patients in the upper quartile (STING expression) exhibit significantly better 5‐year‐OS, including early‐ and late‐stage diseases. In contrast, STING RNA expression was not proved to be a positive prognosticator in SCC in none of the comparisons according to the same analysis. Furthermore, TCGA RNA expression data showed that STING was associated with T‐cell promotion and development genes underpinning its rule as an immune activator. Since STING is a consequence of detecting non‐self DNA in the tumor microenvironment, STING expression could be increased in those tumors with common mutations, such as TP53.Positive STING protein expression (H‐score > 50) was associated with significantly improved overall survival (58 vs. 35 months, The underlying mechanism for STING and cGAS loss has been attributed to hypermethylation of the promoter regions for both genes. Given that epigenetic modifications are common in NSCLC, it is reasonable to expect methylation to play a role in STING and cGAS expression. TCGA analysis of methylation for both STING and cGAS genes showed much higher methylation in SCC than in AC. The differences in methylation between AC and SCC might explain why STING expression is more remarkable in AC than in SCC. Recent studies combining PD‐L1 inhibitors with low‐dose demethylating agents such as azacytadine have been shown to improve outcomes.Moreover, a recent study showed that cisplatin treatment increases the activation of the STING/cGAS pathway and is associated with higher PD‐L1 expression in multiple NSCLC preclinical models in both AC and SCC.Of note, our study has possible clinical and therapeutic implications. Others showed in a preclinical ovarian cancer study that survival of mice treated with a combination of STING, carboplatin, agonist, and anti‐PD‐1 antibody was the longest.Limitations of this study include that we have no comprehensive data collected on specific treatments in subgroups. Moreover, our "annotated cohort" is from overwhelmingly early‐stage NSCLC patients, and we have no data on the potential clinical behavior without STING targeted specific treatments administered in advanced‐stage patients.5Our sizable clinical patient cohort shows that STING is extensively expressed in NSCLC. High STING tumor expression correlates with improved survival, early‐stage disease, and EGFR and KRAS mutations. Our data are further supported by the coincidence of STING and T‐cell activation genes, cell line demethylation, and TCGA data. Furthermore, our study provides multiple aspects of STING expression's translational relevance. Along with other studies on immunotherapy and STING associations, our data serve as a reasonable basis for further exploring the exact clinical role of STING in NSCLC.The authors declare no conflict of interest.Conceptualization: David Dora, Christopher J. Rivard, Fred R. Hirsch, and Zoltan Lohinai. Methodology: Charles Caldwell jr, Christopher J. Rivard, Kenichi Suda, and Hui Yu. Validation: Charles Caldwell jr, Christopher J. Rivard, Gareth Rivalland, and Kim Ellison. Formal analysis: Hui Yu, Gareth Rivalland, Leslie Rozeboom, Rafal Dziadziuszko, and Paul Mitchell. Investigation: Charles Caldwell jr, Christopher J. Rivard, Zoltan Lohinai, and David Dora. Resources: Christopher J. Rivard and Fred R. Hirsch. Data curation: Hui Yu, Gareth Rivalland, Paul Mitchell, Thomas John, Inigo San Millan, and Shengxiang Ren. Visualization: Charles Caldwell jr, Christopher J. Rivard, and David Dora. Supervision: Zoltan Lohinai, Christopher J. Rivard, David Dora, and Fred R. Hirsch. Project administration: David Dora, Zoltan Lohinai, Christopher J. Rivard, Leslie Rozeboom, Rafal Dziadziuszko, Paul Mitchell, Thomas John, Inigo San Millan, and Shengxiang Ren. Writing‐original draft: Charles Caldwell jr, David Dora, Zoltan Lohinai, and Christopher J. Rivard. Writing‐review and editing: David Dora, Zoltan Lohinai, and Christopher J. Rivard.This study evaluates RNA and tissue protein expression of the immune‐checkpoint STING in a sizable cohort of NSCLC patients. We find that increased STING expression is associated with increased expression of T‐cell activation genes, early stage, EGFR, and KRAS mutations and improved survival.Supporting information.Click here for additional data file.


Longitudinal data regarding the association of normal systolic blood pressure maintenance  and ≥elevated SBPmaintain (follow‐up SBP ≥ 120 mm Hg). Annualized PVC was defined as PVC divided by the interscan period.We analyzed 95 adults  without baseline CV disease who underwent serial coronary computed tomographic angiography with mean 3.5 years of follow‐up. All participants were divided into two groups of normal SBPmaintain, those with ≥elevated SBPmaintain had higher annualized total PVC (mm3/year) . Baseline total plaque volume (β = .10) and the levels of SBPmaintain (β = .23) and follow‐up high‐density lipoprotein cholesterol (β = −0.28) were associated with annualized total PVC . The optimal cutoff of SBPmaintain for predicting plaque progression was 118.5 mm Hg . SBPmaintain ≥ 118.5 mm Hg  and baseline total plaque volume  independently influenced coronary plaque progression .Compared to participants with normal SBPmaintain is substantial to attenuate coronary atherosclerosis progression in conditions without established CV disease.Normal SBP Finally, 95 participants without established CV disease at index CCTA were included in the present study. All participants were divided into two groups of normal SBPmaintain (follow‐up SBP < 120 mm Hg) (n = 40) and more than elevated SBPmaintain (follow‐up SBP ≥ 120 mm Hg) (n = 55) based on the follow‐up SBP levels, as per the 2017 ACC/AHA guidelines. Laboratory tests were conducted within 1 month of all CCTA examinations. All blood samples were collected after a minimum of 8 h fasting period. SBP and diastolic blood pressure (DBP) were measured on the right arm using an automatic manometer with an appropriate cuff size after the participants rested for ≥5 min.The study design and protocol of the Progression of AtheRosclerotic PlAque DetermIned by Computed TomoGraphic Angiography Imaging (PARADIGM) registry has been reported previously.2.23) was defined as the difference of plaque volume between the index and follow‐up CCTA at the per‐patient level. Annualized PVC (mm3/year) was defined as PVC divided by interscan period. Plaque progression was defined as the difference in plaque volume between follow‐up and index CCTA was more than zero. All methods were performed following the relevant guidelines and regulations. The appropriate institutional review board committees of Severance Cardiovascular Hospital approved the protocol of the present study.Data acquisition and post‐image processing were in accordance with SCCT guidelines.2.3t‐test or Mann–Whitney U test, as appropriate. Categorical variables were compared using the χ2‐test or Fisher's exact test, as appropriate. Linear regression models were used to identify the association between clinical variables and annualized total PVC. Logistic regression models were used to identify the independent predictors of coronary plaque progression. Variables with p < .05 in the univariate analyses were considered confounding variables and entered into multivariate regression analyses, respectively. Except for the nonmodifiable variables of age, gender, and baseline plaque volume, other independent variables achieved at follow‐up CCTA were included in the regression models. In the receiver operating characteristic analysis, the optimal cutoffs of follow‐up SBPmaintain for predicting coronary plaque progression was determined using the Youden index. All statistical analyses were performed using the Statistical Package for the Social Sciences version 19 and SAS . A p value of <.05 was considered significant for all analyses.Continuous variables are expressed as mean ± SD or medians [interquartile range], as appropriate. Categorical variables are presented as absolute values and proportions. Continuous variables between two groups were compared using the independent 33.1maintain, those with ≥elevated SBPmaintain had significantly higher levels of SBP, DBP, and BMI at both CCTAs. No significant difference in statin use after index CCTA between participants with normal SBPmaintain and those with ≥elevated SBPmaintain was observed.The mean age of participants was 56.7 ± 8.5 years and the proportion of men was 40.0%. The mean interscan period was 3.5 ± 1.4 years. Table 3.2maintain, the presence of plaque  and total plaque volume (mm3)  were higher in those with ≥elevated SBPmaintain at baseline (Table 3/year) was lower in participantes with normal SBPmaintain than in those with ≥elevated SBPmaintain    Table .3.33 increase) , SBPmaintain (per‐1 mm Hg increase)  and follow‐up high‐density lipoprotein cholesterol (HDL‐C) (per 1‐mg/dL increase)  were associated with the annualized total PVC. The multivariate linear regression analysis showed that baseline total plaque volume , SBPmaintain , and follow‐up HDL‐C  were independently associated with the annualized total PVC.Table 3.4maintain cut‐off for predicting coronary plaque progression was found to be 118.5 mm Hg, with 78.2% sensitivity and 62.5% specificity  , baseline plaque volume (per 1‐mm3 increase) , and SBPmaintain ≥ 118.5 mm Hg  were associated with plaque progression. The multivariate logistic regression analysis showed that baseline plaque volume  and SBPmaintain ≥ 118.5 mm Hg  independently influenced on plaque progression  Figure . In the 4maintain, together with baseline total plaque volume, was independently associated with the progression of coronary atherosclerosis in subjects without established CV disease at baseline and (2) to maintain normal SBP levels was an important factor in attenuating coronary plaque progression in this population. Despite the characteristics of participants in the current study being completely different from those of the SPLINT study, these results showed the significance of normal SBPmaintain even in conditions of low CV risk burden. In addition, these findings could be substantial evidence to support the reinforced ACC/AHA guideline for hypertension.Data on the history of coronary atherosclerosis in subjects without established CV disease has been scarce because the performance of CCTA in this population is not yet justified despite the outstanding advances in CCTA technique. It was possible for the current study to evaluate this issue because PARADIGM is, to the best of our knowledge, the largest serial CCTA registry to date. The major findings of the present study were (1) SBPAlthough the prevalence of hypertension definitely increases in clinical practice under the application of the 2017 ACC/AHA guideline,maintain in the present study. This study identified that the optimal cutoff of SBPmaintain for predicting coronary plaque progression was 118.5 mm Hg; this cutoff value might be influenced by the small number of newly developed cases of Stage 1 and Stage 2 hypertension during follow‐up periods. However, this result supports the rationality of reinforced ACC/AHA guideline for hypertension and reveals the significance of strict SBP control even in low CV risk status. Further investigation to assess the cost–benefit or cost‐effectiveness problems of strict SBP control as a primary preventive strategy should be necessary in clinical practice.Recent studies have focused on the independent predictors for subclinical atherosclerosis in the absence of CVRFs.maintain than in those with ≥elevated SBPmaintain. After adjusting for baseline volume of densely calcified plaque and statin use at follow‐up, normal SBPmaintain was related to the decreased risk of progression in dense calcified plaque . Considering the recent data showing that (1) the usefulness of coronary artery calcium score (CACS) to determine therapeutic targets in various clinical conditionsA recent experimental study has suggested that high blood pressure per se exacerbates atherogenesis of coronary arteries in the absence of proatherogenic humoral factors.Previous studies have evaluated the effect of specific antihypertensive agents on CV events and the progression of coronary atherosclerosis in patients with established coronary artery disease (CAD). The Comparison of Amlodipine vs Enalapril to Limit Occurrences of Thrombosis (CAMELOT) trial reported that the administration of amlodipine to patients with CAD and normal blood pressure resulted in reduced adverse CV events and attenuated atherosclerosis progression in 1991 patients with angiographically documented CAD and DBP < 100 mm Hg during a follow‐up of 24 months.The present study has some limitations. First, consecutive changes of clinical variables during follow‐up periods were not available. Second, this study only included an extreme selection of participants from the PARADIGM registry who had no established CV disease. Therefore, the characteristics of our participants could not represent the overall participant characteristics of the PARADIGM registry. Third, the major proportion of the overall PARADIGM registry was East Asians. In this PARADIGM substudy, all participants were East Asians; hence, this might limit the generalizability of the findings. Fourth, the target blood pressure might be somewhat different in an elderly population.maintain, annualized total PVC was significantly higher in subjects with ≥elevated SBPmaintain in the absence of baseline CV disease. Both baseline total plaque volume and SBPmaintain had an independent association with the risk of coronary plaque progression. The present study suggests that the endeavor to maintain normal SBP is important to attenuate coronary atherosclerosis progression even in conditions without established CV disease. Further prospective studies with larger sample sizes and longer follow‐up durations should be necessary.In conclusion, compared to subjects with normal SBPKi‐Bum Won and Hyuk‐Jae Chang contributed to the conception and design of the work. Ki‐Bum Won, Hyung‐Bok Park, Ran Heo, Byoung Kwon Lee, Fay Y. Lin, Martin Hadamitzky, Yong‐Jin Kim., Ji Min Sung, Edoardo Conte, Daniele Andreini, Gianluca Pontone, Matthew J. Budoff, Ilan Gottlieb, Eun Ju Chun, Filippo Cademartiri, Erica Maffei, Hugo Marques, Pedro de Araújo Gonçalves, Jonathon A. Leipsic, Sang‐Eun Lee, Sanghoon Shin, Jung Hyun Choi, Renu Virmani, Habib Samady, Kavitha Chinnaiyan, Daniel S. Berman, Jagat Narula, Jeroen J. Bax, James K. Min, and Hyuk‐Jae Chang contributed to the acquisition, analysis, and interpretation of data. Ki‐Bum Won drafted the manuscript. Hyuk‐Jae Chang critically revised the manuscript. All authors gave final approval and agree to be accountable for all aspects of work ensuring integrity and accuracy.Dr. James K. Min receives funding from the Dalio Foundation, National Institutes of Health, and GE Healthcare. Dr. Min serves on the scientific advisory board of Arineta and GE Healthcare, and has an equity interest in Cleerly. Dr. Habib Samaday has equity interest in Covanos. Dr. Leipsic serves as a consultant and has stock options in HeartFlow and Circle Cardiovascular Imaging; and receives grant support from GE Healthcare and speaking fees from Philips. All other authors declared no conflicts of interest.Supplementary Material.Click here for additional data file.Supplementary figure 1. Receiver operating characteristic curve of optimal SBPmaintain for predicting coronary plaque progression. SBPmaintain, systolic blood pressure maintenance.Click here for additional data file.Supplementary table 1. Changes of coronary plaque subtypes according to SBPmaintain.Click here for additional data file.


To test whether this mild phenotype is recurrent in other insect species, we analysed resilin in the spotted‐wing fruit fly Drosophila suzukii. As quantified by DT autofluorescence by microscopy, DT intensities in the trochanter and the wing hinge are higher in D. suzukii than in D. melanogaster, while in the proboscis the DT signal is stronger in D. melanogaster compared to D. suzukii. To study the function of Pro‐Resilin in D. suzukii, we generated a mutation in the proresilin gene applying the Crispr/Cas9 technique. D. suzukii pro‐resilin mutant flies are flight‐less and show a hdw phenotype resembling respective D. melanogaster mutants. DT signal intensity at the wing hinge is reduced but not eliminated in D. suzukii hdw flies. Either residual Pro‐Resilin accounts for the remaining DT signal or, as proposed for the hdw phenotype in D. melanogaster, other DT forming proteins might be present in Resilin matrices. Interestingly, DT signal intensity reduction rates in D. suzukii and D. melanogaster are somehow different. Taken together, in general, the function of Pro‐Resilin seems to be conserved in the Drosophila genus; small differences in DT quantity, however, allow us to hypothesise that Resilin matrices might be modulated during evolution probably to accommodate the species‐specific lifestyle.Resilin is a protein matrix in movable regions of the cuticle conferring resistance to fatigue. The main component of Resilin is Pro‐Rresilin that polymerises via covalent di‐ and tri‐tyrosine bounds (DT). Loss of Pro‐Resilin is nonlethal and causes a held‐down wing phenotype ( Drosophila suzukii. In proresilin mutant D. suzukii flies the di‐tyrosine signal is reduced indicating reduction or elimination of proresilin. Reduction or elimination of dityrosine and proresilin in D. suzukii causes wing posture defects preventing flight.Di‐tyrosine represents proresilin in several patches in the cuticle of the wing hinge region in  Drosophila suzukii and Drosophila melanogaster adults is similar.Resilin/di‐tyrosine distribution in D. suzukii and D. melanogaster.Reduction or elimination of Resilin/di‐tyrosine causes an identical phenotype in Besides proresilin, resilin matrices in Drosophila species probably contain also other proteins.A general positive correlation between body size and Resilin/di‐tyrosine amounts is not apparent in Drosophila species. After mass crossing of the flies generated by injected eggs, we continuously checked for the occurrence of the held‐down wing phenotype in our CCproresilin  stock. Flies with this phenotype were isolated and kept together to generate a stock for further analyses. For determination of the mutation, genomic DNA was prepared from these and control flies by standard methods to amplify and sequence the respective region using the primers ATTCCGATCAGCAGCAGTCC and GTCCGCCATTACCATTACCG. Sequences, determined by Microgen Europe B.V., were analysed by the BLAST software at the NCBI and the SignalP 6.0 software on the DTU Health Tech site.To mutagenize the 2.3For body size measurements, flies were submerged in halocarbon oil (Sigma‐Aldrich) on a glass slide and photographed on a Nikon AZ100 microscope at a magnification of 10x using the in‐built NIS software that allows length determination.For morphological analyses, flies were observed on an inverse Axio Observer Z1 (Zeiss) equipped with an Axiocam Mono camera at the systems biology group of C. Dahmann at the TU Dresden. The filters were: AF405 (DT specific filter): Ex: 357/44 nm Brightline HC, splitter HC BS 389 nm, Em: 420/40 nm ET Bandpass; DAPI: Ex: 359/48 nm, splitter: 395 nm, Em: 445/50 nm; and GFP: Ex: 470/40 nm, splitter: 495 nm, Em: 525/50 nm. These objectives were used: EC Plan‐Neofluar 5x/0.67 M27 DRY, EC Plan‐Neofluar 10x/0.3 M27 DRY, and EC Plan‐Neofluar 20x/0.5 M27 DRY. The light source was a halogen lamp. Flies were immobilised by freezing at −20°C for 4 min, then dissected and mounted in ROTI®mount Aqua. Images were taken with the Zeiss inbuilt software. They were prepared using ImageJ and Adobe Illustrator CS6.D. melanogaster wild‐type wing hinge signal extracted by the profile‐program comparing the intensities obtained by the DT and DAPI filters (DT > DAPI). The threshold of 7500 counts was used to produce the numbers of DT dots in the wing hinges of all used lines. These area counts where than compared and calculated with the total individual numbers (n) per line.In this study, we aimed at identifying Resilin matrices indirectly by DT autofluorescence. For this purpose, we used a specific filter system adjusted to the published emission range . All statistical analyses were performed using XLstat 2020 (AddinSoft®). All computed p‐values are listed in Supporting Information: Data Data were analysed using Student's δs:dt = (mean body size species 1/mean intensity species 1) – (mean body size species 2/mean intensity species 2). If there is a body size‐proportional correlation , the coefficient approximates 0. If the coefficient is different from 0, we suppose a nonbody size dependent difference of intensity. Indeed, for most cases tested in this study, we obtain a coefficient of approximately 0  that is needed for the secretion of Pro‐Resilin to the extracellular space. According to the SignalP 6.0 software, the deletion of M11A12 would shift the signal peptide cleavage site to the position between residues 17 and 18 concomitantly reducing the probability of signal peptide cleavage (0.810029). The new cleavage site, if functional, would cause the deletion of four N‐terminal amino acids of the secreted protein. We assume that disruption of the signal peptide prevents the efficient secretion of Pro‐Resilin into the differentiating cuticle, thereby causing the observed phenotype. In addition, N‐terminal truncation of the protein might destabilise it and/or reduce its activity. Together, despite the deletion, low amounts of aberrant Pro‐Resilin may be secreted correctly and display residual function, thereby explaining the relatively mild nonlethal phenotype. Alternatively, redundant DT‐forming proteins may act in parallel to Pro‐Resilin thereby alleviating the defect. Indeed, the genomes of many insect species harbour more than one gene coding for Pro‐Resilin‐like proteins , 13% (trochanter), 16% (wing hinge), and 25% (cibarium)   and conserved in other Drosophila species (orthology ID 2370). As Cpr56F is, probably like in D. melanogaster, not expressed in the wing hinge, at least a third DT‐forming protein may be postulated to be incorporated into Resilin matrices in at least some body parts in Drosophilae. Overall, based on our data, we conclude that the Resilin matrix composition and probably function are largely conserved between D. melanogaster and D. suzukii. Despite this, we detected minor differences in DT signal intensity and distribution  suggesting that resilin matrices may be optimized in related species during evolution to accommodate slightly different lifestyles. The biomechanics consequences of these differences, once investigated, may serve to advance in biomimetics approaches.To evaluate the effects of the r Figure  and S3. ) Figure . The DT ) Figure . In otheSteven Lerch performed most histological experiments, analysed the data and contributed to the writing of the first draft of the manuscript. Yang Yang assisted Steven Lerch in microscopy in some experiments. Justin Flaven‐Pouchon performed the statistical analyses of the data. Nicole Gehring generated the CRISPR/Cas9 flies. Bernard Moussian conceived and planned all experiments. He analysed the data and wrote the manuscript.The authors declare no conflicts of interest.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.


To establish a survival prognostic model for pseudomyxoma peritonei (PMP) treated with cytoreductive surgery (CRS) plus hyperthermic intraperitoneal chemotherapy (HIPEC) based on Bayesian network (BN).453 PMP patients were included from the database at our center. The dataset was divided into a training set to establish BN model and a testing set to perform internal validation at a ratio of 8:2. From the training set, univariate and multivariate analyses were performed to identify independent prognostic factors for BN model construction. The confusion matrix, receiver operating characteristic (ROC) curve and the area under curve (AUC) were used to evaluate the performance of the BN model.p < 0.05). Based on independent factors, the BN model of training set was established. After internal validation, the accuracy and AUC of the BN model were 70.3% and 73.5%, respectively.The univariate and multivariate analyses identified 7 independent prognostic factors: gender, previous operation history, histological grading, lymphatic metastasis, peritoneal cancer index, completeness of cytoreduction and splenectomy  survival prognostic model for PMP which has a resonable level of predictive performation. The BN model can help oncologists to make clinical decisions. Tumor cells and mucus enter the abdominal and pelvic cavity through the perforated appendix wall, accumulate and redistribute in the abdomen and pelvis, leading to mucinous ascites, peritoneal implantation, omentum cake, and organ involvement particularly to the ovary and the spleen.There are many factors affecting the prognosis of PMP, such as age, peritoneal cancer index (PCI), completeness of cytoreduction (CC), histological grading, lymphatic metastasis, vascular invasion, stripped peritoneum area, number of anastomosis.In recent years, machine learning method has been widely used in medical field.22.1Our institute is a medical center specialized in treating peritoneal metastases from gastrointestinal and gynecological malignancies, mainly using the CRS + HIPEC and postoperative integrated treatment approaches. Each patient treated at our center has been entered into a prospectively established database, which contained detailed clinicopathological information on 1980+ patients. From this database, we screened 453 PMP patients underwent CRS + HIPEC for the first time from December 2004 to July 2021. All patients met the following inclusion and exclusion criteria.9/L and platelet ≥80 × 109/L; (4) serum bilirubin, aspartic aminotransferase and alanine aminotransferase <2 × the upper limit of normal (ULN); (5) serum creatinine <1.2 × ULN; and (6) cardiac and pulmonary functions can stand major operation.Major inclusion criteria were: (1) pathological diagnosis of PMP; (2) Karnofsky performance status (KPS) score > 60; (3) white blood cells ≥3.5 × 10Major exclusion criteria were: (1) lung, brain, bone or liver metastases; (2) serum bilirubin, aspartic aminotransferase and alanine aminotransferase ≥2 × ULN; (3) serum creatinine ≥1.2 × ULN; (4) severe mesenteric contracture; and (5) major organ functions cannot stand major operation.2.2After general anesthesia, a midline xiphoid‐pubic incision was performed to enter the abdomen. Once the abdominal wall was opened, characteristics and volume of ascites were recorded and evaluation of PCI was conducted, according to Sugarbaker's principle.CC score was evaluated after CRS according to Sugarbaker's criteria.After CRS, open HIPEC was performed. The chemotherapy drugs were docetaxel 120 mg + cisplatin 120 mg or cisplatin 120 mg + mitomycin C 30 mg, each dissolved in 3000 ml of heated saline at 43°C for 60 min.Then, digestive tract and urinary tract reconstructions were performed after HIPEC. Intestinal stoma was conducted if necessary. Drainage tubes were placed and the incision was sutured with reduced tension. After operation, patient was delivered to the intensive care unit for recovery and transferred to the surgical oncology ward when the condition stabilized.2.3The follow‐up consisted of physical examination, tumor response evaluation and survival information. The frequency of follow‐up was once every 3 months within 2 years after CRS + HIPEC, once every 6 months for the third year after CRS + HIPEC and once every year thereafter.2.4Overall survival (OS): OS was defined as the time interval from the date of clinical diagnosis to the date of death or the last follow‐up.2.5t‐test or rank sum test. Categorical variables were presented as number (percentage), analyzed by x2 test and Fisher's exact method. Kaplan–Meier method was used to estimated OS and log‐rank test was used for comparison between groups. p value <0.05 was considered significant. Univariate and multivariate COX regression analyses were conducted to identify the independent risk factors on OS. R software  was used for BN model development and evaluation.SPSS 26.0  were used for data collection and analysis. Continuous variables were reported as median (range) and compared with 2.6Bnlearn” package (version 4.7) was used for BN structure learning, parameter learning and inference. To evaluate the BN model performance, all PMP patients were randomly split in training set and testing set with a ratio of 8:2. The training set was used to establish the BN model and the testing set performed internal validation. From the training set, univariate and multivariate analyses were performed to screen for independent prognostic factors for BN model construction. We selected OS as the target variable and 36 months as the target cut‐off point time. As the “Bnlearn” package can only deal with discrete variables, discretization of the data was completed prior to the construction of the model. After establishment of the dataset and discretization of variables into discrete variables, a BN model was established.The “2.7ROCR” package (version 1.0–11), the receiver operating characteristic curve (ROC) and the area under curve (AUC) were calculated to evaluate the overall performance of the BN model.The confusion matrix is a cross table containing the observed and predicted classes with relevant statistics, which can be obtained by internal validation. The accuracy of the BN model is defined by the following equation: Accuracy = [true positive (TP) + true negative (TN)]/[TP + false positive (FP) + TN + false negative (FN)]. Using the “33.1A total of 453 PMP patients undergoing CRS + HIPEC between 2004 and 2021 were included. There were 207 (45.7%) males and 246 (54.3%) females for the whole cohort. Patients ranged in age from 24 to 81 years (median 55). In terms of histological grading, there were 248 (54.7%) cases with low grade, 158 (34.9%) cases with high grade, and 47 (10.4%) cases with high grade with signet ring cells. The median duration of CRS + HIPEC was 630 min (range: 95–1080 min). The median number of resected organs and peritoneum were 3 (0–10) and 5 (0–9), respectively. The median PCI was 30 (range:1–39). There were 227 (50.1%) cases with CC0‐1 and 226 (49.9%) cases with CC2‐3.p > 0.05) (Table There were 362 (80.0%) patients in the training set and 91 (20.0%) patients in the testing set. The baseline characteristics of the training set and testing set were balanced, and there were no statistically significant differences in clinicopathological characteristics of two sets    Table . Factors5) Table . Kaplan–5) Table .3.3The confusion matrix of internal validation is listed in Table 4The development and utilization of cancer survival prediction models are of great significance for physicians to make clinical decisions. In this study, we constructed a BN model to predict survival of PMP patients based on the 7 independent prognostic factors. After internal validation, the BN model showed a reasonable level of predictive performance with the accuracy being 70.3% and the AUC being 73.5%.The univariate and multivariate analyses of training set showed that gender, previous operation history, histological grading, lymphatic metastasis, PCI, CC and splenectomy were the independent prognostic factors. Chua et al.Among 7 independent prognostic factors selected by multivariate analysis for our study, there were two factors associated with CRS + HIPEC, which were splenectomy and CC score. Our study showed that splenectomy provided a significantly better survival comparing with non‐splenectomy for PMP patients. The reason may be that splenectomy enhances the likelihood of complete cytoreduction. However, a studyIn 2001, Sugarbaker systematically studied CRS + HIPEC+ early postoperative intraperitoneal chemotherapy (EPIC) for PMP, demonstrating that this therapy was the optimal treatment strategy for PMP patients. This treatment embodies the advantages of comprehensive treatment based on surgery, integrating the synergistic effects of surgical resection, regional chemotherapy, hyperthermia and large volume liquid lavage. CRS can remove all visible tumor tissues and HIPEC can eliminate micro‐metastases and free tumor cells. Current studiesCurrently, HIPEC regimens vary in different treatment centers. Oxaliplatin and mitomycin C are the most commonly basic chemotherapy drugs for HIPEC. There is no international consensus on the best drug and dose for HIPEC. Therefore, international peritoneal cancer centers need to strengthen cooperation and conduct multi‐center, large sample randomized controlled clinical trials to explore HIPEC protocol with high efficacy and less toxicity.The nomogram is a graphical representation that has been used to predict cancer survival in recent years. Two studiesIn recent years, BN has been widely used in artificial intelligence, systematic biology, disease diagnosis and prognosis, scientific decision‐making and other fields. The application value of BN in medical field is also prominent. The BN survival prediction model has the following advantages: (1) The model is presented in the form of tree graph, which is simple and intuitive; (2) The correlation between variables can be found and the conditional probability of each variable can be calculated and predicted; (3) The inference function of BN can guide treatment decision‐making.There were three major deficiencies in this study: (1) The survival prognosis model established in this study was based on single‐center data, and only conducted internal validation without external validation; (2) The time span of the cases included in this study was long, which resulted in heterogeneity of the cases; (3) Preoperative tumor markers, Ki‐67, P53 and other pathological indicators were not included in this study.For the results of this study, the prediction accuracy of the BN model remains to be further improved. In future study, we will expand the sample size, include more variables and conduct external validation to improve the prediction accuracy of the survival prognostic model of PMP.5To conclude, this study established a BN‐based survival prediction model for PMP from 7 independent prognostic factors, which could help clinical treatment decision making and outcome prediction.Yan Li contributed to study concept. Yan Li and Xin Zhao contributed to study design. Xin Zhao contributed to literature research. Xin Zhao, Xin‐Bao Li, Yu‐Lin Lin, Ru Ma, Da‐Zhao Xu and Ying Zhang contributed to data acquisition. Xin Zhao and Yan Li contributed to data analysis/interpretation. Xin Zhao contributed to statistical analysis. Xin Zhao contributed to manuscript definition of intellectual content. Xin Zhao and Yan Li contributed to manuscript editing. Yan Li contributed to manuscript revision/review. Yan Li contributed to manuscript approval.This work was supported by Natural Science Foundation of China (82073376).None.