This study aimed to better characterize the clinicopathologic characteristics, outcomes, and prognostic factors of AITL in China.We retrospectively analyzed 312 patients with AITL enrolled between January 2011 and December 2020 from five institutions in China.The median age was 65 years, with 92.6% advanced stage, 59.7% elevated LDH, 46.1% anemia, and 44.0% hypergammaglobulinemia. The majority of patients (84.9%) received anthracycline‐based regimens with or without etoposide, and only 6.1% underwent autologous stem cell transplantation following first remission. The 5‐year OS and PFS estimates were 43.4% and 25.0% with no significant improvement of survival between patients treated during 2011–2015 and 2016–2020, respectively. Both the International Prognostic Index (IPI) and the prognostic index for PTCL, not otherwise specified (PIT), were predictive for OS. In multivariate analysis, age >70 years, elevated LDH, and albumin level <35 g/L were independent prognostic factors for OS. Combining these three factors, a novel prognostic model (the Chinese AITL score) was constructed, which stratified patients into low‐, intermediate‐, and high‐risk groups, with 5‐year OS rates of 69.0%, 41.5%, and 23.7%, respectively. This new model was successfully validated in an independent cohort.Patients with AITL were mainly treated with anthracycline‐based regimens, and the outcomes were still unsatisfactory in China. Our novel prognostic model may improve our ability to identify patients at different risks for alternative therapies. Patients with AITL in China are mainly treated with anthracycline‐based regimens with low rate of autologous stem cell transplantation and unsatisfactory outcomes. A novel prognostic model combining 3 factors  was proposed which stratified patients into the low, intermediate, and high‐risk subgroups with differing outcomes. In the training cohort, a retrospective analysis was first conducted to characterize the clinicopathologic characteristics and then identify prognostic factors for proposing a new prognostic model. A total of 312 patients diagnosed with AITL between January 2011 and December 2020 at five institutions in China were retrospectively evaluated. The inclusion criteria were as the following: (1) pathologically confirmed diagnosis of AITL according to the World Health Organization (WHO) classificationThis study was conducted in accordance with the Declaration of Helsinki and was approved by the institutional review board of each participating center and informed consent was waived because of the use of anonymized data.2.2At the time of enrollment, pathological specimens including slides stained with hematoxylin–eosin, immunohistochemistry, and in situ hybridization for Epstein–Barr virus encoded RNA (EBER‐ISH) were reviewed by expert hematopathologists from the pathology department of each of the eight participating centers to confirm the diagnosis. The diagnosis of AITL was based on the presence of partial or total effacement of the lymph node architecture, prominent vascularity with arborization of high endothelial venules, an extrafollicular follicular dendritic cell meshwork, an atypical population of CD4+ T cells with expression of at least two TFH markers , and the presence of large CD20+ B immunoblasts with or without the evidence of Epstein–Barr virus (EBV) infection. The positivity of Tfh markers was defined as the expression in at least 20% of the tumor cells. Patients with concurrent/secondary diffuse large B‐cell lymphoma and nodal peripheral T‐cell lymphoma with TFH phenotype (according to the 2016 revision of the WHO classification) were excluded from analysis.2.3Clinical data were collected by case‐report forms. The clinical data included age, sex, B symptoms, immune‐related symptoms, Eastern Cooperative Oncology Group (ECOG) performance status, Ann Arbor stage, involvement of extranodal sites, International Prognostic Index (IPI) score,Bone marrow involvement was diagnosed based on bone marrow biopsy. Splenic involvement was supported by diffuse FDG uptake on positron emission tomography (PET), the vertical length of spleen over 13 cm, and nodular lesions or mass on computed tomography (CT) or PET/CT in our study. The involvement of other extranodal sites was determined using diagnostic tools available at diagnosis, including CT, enhanced CT, and PET‐CT. Treatment responses were evaluated according to the 2007 Revised Response Criteria for Malignant Lymphoma2.4p < 0.05) were incorporated into the multivariate analyses. Factors identified as an independent prognostic factor on OS were used to create a novel prognostic score. The performance of the new prognostic model was compared to that of the IPI and PIT scoring systems using a measure of goodness of fit  and concordance index (Harrell C‐index), with low AIC value indicating better fit and high Harrell C‐index indicating better discrimination. Statistical analyses were performed using SPSS (version 20.0) software. Statistical significance was set at p < 0.05.Progression‐free survival (PFS) was defined as the time from diagnosis to the first progression, relapse, or any‐cause death. Overall survival (OS) was defined as the time from diagnosis to any‐cause death or the last follow‐up. Survival analyses of PFS and OS were performed using the Kaplan–Meier method. Log‐rank test was used to compare the survival rates between the two groups. To identify the prognostic factors, univariate and multivariate analyses were performed using Cox proportional hazards regression models. Significant covariates (with 33.1The median age at diagnosis was 65 years (range: 22–88 years), and the patients were predominantly male (58.3%). The patient characteristics and laboratory investigations are summarized in Table 3.2As summarized in Table 3.3The majority of the patients (84.9%) were treated with first‐line anthracycline‐based regimens, including 181/312 (58.0%) of the patients treated with cyclophosphamide, vincristine, doxorubicin, and prednisolone (CHOP); 62/312 (19.9%) with cyclophosphamide, vincristine, doxorubicin, etoposide, and prednisolone (CHOEP); and 19 /312 (7%) with other anthracycline‐containing regimens. The remaining 13.4% of the patients received other chemotherapy regimens without anthracycline or supportive care (2.6%). Chidamide, a histone deacetylase inhibitor (HDACi), was added to chemotherapy in 72/312 (23.1%) patients. Only 19/312 (6.1%) of the patients underwent autologous stem cell transplantation (ASCT) as consolidation following the first remission. In total, 51 patients (16.3%) received maintenance therapy following remission, including chidamide maintenance in 32 patients (10.3%), thalidomide/lenalidomide maintenance in 16 (5.1%), and chidamide combined with thalidomide/lenalidomide in 3 (1.0%).n = 155] vs. 33.3% in the CHOEP group [n = 54], p = 0.302) or ORR  between the CHOP and CHOEP groups. Similarly, adding chidamide to the CHOP/CHOEP regimen did not significantly improve the CR rate  or ORR. The major salvage therapies for the relapsed/refractory patients included regimens containing gemcitabine and platinum (n = 41), programmed cell death protein 1 (PD‐1) blockade (n = 14), and chidamide plus azacytidine (n = 8).Treatment response was documented in 241 patients treated with curative intent. Of the 241 patients, 91 (37.8%) achieved CR, and another 92 (38.2%) achieved PR, yielding an ORR of 75.9%. No significant differences were observed in the CR rate . The 1‐, 3‐, and 5‐year OS rates were 82.6%, 59.2%, and 43.4% for the entire group, with an apparent plateau at approximately 6 years Figure . The 1‐,By the end of the follow‐up time, 121 deaths had been recorded, with 43.8% of deaths occurring within the first year after diagnosis and 81.8% occurring within the third year after diagnosis. The most common cause of death was disease progression (91.3%), followed by infection (7.0%).p = 0.063; Figure p = 0.043; Figure To further analyze the effect of maintenance therapy, we selected patients who were treated with curative intent chemotherapy and achieved a CR or PR. A cohort of 183 patients fulfilled this criteria, of which 48 patients received chidamide, thalidomide, or lenalidomide maintenance therapy. Notably, patients who received chidamide, thalidomide, or lenalidomide maintenance therapy following the first remission tended to have better OS than patients who did not receive maintenance therapy, with 3‐year OS rates of 90.0% versus 64.3% , elevated LDH , and albumin level < 35 g/L .In univariate analysis, ECOG performance status ≥2, age >70 years, anemia, albumin level <35 g/L, platelet count <100 × 10p = 0.009) significantly influenced OS. However, in multivariate analysis, the Ki‐67 index was not retained as an independent prognostic factor for OS.We also evaluated pathological features as possible prognostic factors, including PD‐1 expression, background CD8, CD20, CD30 expression, EBER‐ISH, and Ki‐67 index. In univariate analysis, only a Ki‐67 index >60%  and the PIT scores , as shown in Figures The prognostic values of the PIT and IPI scoring systems were evaluated in this study. Both PIT and IPI were predictive of OS. The 5‐year OS rates were significantly different according to the IPI scores . Among the IPI, PIT, and the Chinese AITL scoring systems, the Chinese AITL scoring system demonstrated the greatest discriminant power, with the highest Harrell C‐index (0.6585 vs. 0.608 for IPI score and 0.610 for PIT score) and lowest AIC (1152 vs. 1159 for IPI score and 1157 for PIT score).Combining the three factors identified in the multivariate analysis, we constructed a new prognostic model, the Chinese AITL score. Patients were stratified into three risk groups: low‐risk group, 0 adverse factors; intermediate‐risk group, 1 factor; and high‐risk group, 2–3 adverse factors. The median OS was not reached in the low‐risk group, 50 months in the intermediate‐risk group, and 27 months in the high‐risk group Figure . The 5‐yp < 0.001) in this validation cohort , pleural effusion/ascites (14.1%), and a positive Coombs test (44.7%) appeared to be distinctive autoimmune manifestations of AITL. To our best knowledge, this analysis represents the largest AITL cohort reported to date, allowing for a real‐world assessment of clinicopathological features, treatment outcomes, survivals, and prognostic factors for patients treated in the contemporary era in China.Most patients (84.9%) were treated with CHOP‐like regimens, and a small proportion received etoposide (19.9%), which may partly reflect the older age of our cohort. In previous studies, adding etoposide to CHOP was reported to improve the ORR and event‐free survival, but not OS, and only for younger patients with AITL (age ≤ 60 years).Upfront ASCT is recommended to improve the prognosis of PTCLs. In the Nordic Lymphoma Group (NLG T‐01) study, the largest prospective trial evaluating ASCT consolidation, and the 5‐year PFS was 44% in patients who were consolidated with ASCT.Although the majority of patients with AITL are sensitive to chemotherapy, the response duration is often short, with relapse frequently that result in poor survival. In our study, responding patients (CR or PR response) who received chidamide maintenance therapy showed significantly superior OS and similar PFS than patients who did not receive maintenance therapy. The superiority of OS on the basis of similar PFS of the maintenance group indicates that chidamide maintenance may prolong the survival with tumor, slow the progression of the disease, and result in more opportunities for further treatment. In addition, chidamide is an oral tablet with the advantage of being convenient to use as long‐term maintenance. Previously, long‐term treatment with romidepsin and pralatrexate was only reported in case reports.Overall, the survival of patients with AITL treated with standard chemotherapy regimens was disappointing, with 5‐year OS and PFS estimates of 43.4% and 25.0%, respectively. These outcomes fall within the range of those reported in previous large population‐based studies (5‐year OS rates of 32%–44% and 5‐year PFS rates of 18%–33%).9/L were predictive of poor prognosis.To date, only a few studies have aimed to identify prognostic factors of AITL, yielding controversial results. However, no well‐defined clinical or pathological prognostic factors have been identified. A study from the International Peripheral T‐cell Lymphoma Project suggested that age >60 years, performance score ≥2, more than one site of extranodal involvement, presence of B symptoms, and platelet <150 × 10Few studies have attempted to identify pathologic prognostic factors of AITL, and none of them have proven clinical value. Our study specifically investigated the potential prognostic value of PD‐1 expression, background CD8, CD20, CD30 expression, and Ki‐67 index. In 2006, Went et al. evaluated the expression of 19 markers using a tissue microarray immunohistochemical analysis in a cohort of patients with AITL and PTCL‐NOS. They proposed a modified PIT model that incorporated a Ki‐67 index ≥80%.As shown in Table Although this study provided novel information regarding AITL, it also has some limitations. The most important limitations of our study was the lack of a centralized pathologic review. The antibodies utilized and the interpretations of immunohistochemical stains may vary in different participating centers. Second, the possibility of unrecognized biases could not be ruled out owing to the retrospective nature of the study. Third, genetic alterations and expression patterns were not included in this study. A better prognostic model may rely on the inclusion of genetic factors in addition to immunohistochemical features and clinical factors.In conclusion, patients with AITL are mainly treated with anthracycline‐based regimens, and the outcomes are still unsatisfactory. Age >70 years, elevated LDH level, and albumin level <35 g/L at the initial diagnosis are independent prognostic factors for OS. Our novel prognostic model combining the abovementioned three factors may improve the risk stratification for AITL.Wei Chong: Conceptualization ; data curation ; formal analysis ; investigation ; writing – original draft ; writing – review and editing . Wei Li: Data curation (supporting); investigation (supporting). Liping Tan: Data curation (supporting); investigation (supporting). Shan Liu: Data curation (supporting). Chang Xue: Data curation (supporting). Kexin Ren: Data curation (supporting). Zirong Zhang: Data curation (supporting). Caili Liu: Data curation (supporting). Fang Bao: Data curation (supporting). Hui Lai Zhang: Conceptualization (supporting); formal analysis (supporting); investigation (supporting). Hui Zhou: Conceptualization (supporting); formal analysis (supporting); methodology (supporting). Zhi‐Ming Li: Conceptualization (supporting); formal analysis (supporting); methodology (supporting). Huijing Wu: Conceptualization (supporting); formal analysis (supporting); methodology (supporting). Liqun Zou: Conceptualization (supporting); formal analysis (supporting); methodology (supporting). Lihong Liu: Conceptualization (supporting); formal analysis (supporting); methodology (supporting). Hongmei Jing: Conceptualization (supporting); formal analysis (supporting); methodology (supporting). Wei Zhang: Conceptualization ; data curation ; formal analysis ; investigation ; methodology ; writing – review and editing .National Natural Science Foundation of China (Nos. 81872902 and 82073917). Natural Science Foundation of Beijing Municipality (No. 7202154).The authors declare no potential conflicts of interest.The study was conducted in accordance with the Declaration of Helsinki and was approved by the institutional review board of each participating center. The requirement for informed consent was waived because of the use of anonymized data.Table S1Click here for additional data file.


Insulin‐like growth factor‐I and its binding proteins are involved in cancer development, progression, and metastasis. In urothelial carcinoma, the impact of this pathway is still poorly investigated. The present large cohort study aimed to evaluate the association of preoperative circulating levels of insulin‐like growth factor‐I, insulin‐like growth factor‐I binding protein‐2 and ‐3 on outcomes after radical cystectomy.A retrospective cohort study of the plasma specimens from 1036 consecutive urothelial carcinoma patients who were treated with radical cystectomy. The primary and secondary outcomes were adverse histopathological features and survival outcomes. Binominal logistic regression and multivariable Cox regression analyses were performed to assess the association of plasma levels of insulin‐like growth factor‐I, insulin‐like growth factor‐I binding protein‐2 and ‐3 with outcomes.P < 0.001). On multivariable Cox regression models, lower levels of both insulin‐like growth factor‐I binding protein‐2 and ‐3 plasma levels were associated with recurrence‐free survival, cancer‐specific survival, and overall survival. insulin‐like growth factor‐I binding protein‐2 and ‐3 levels and improved the discrimination of a standard reference model for the prediction of recurrence‐free survival, cancer‐specific survival, and overall survival .On multivariable analysis adjusting for the effects of preoperative variables, lower insulin‐like growth factor‐I binding protein‐2 levels were associated with an increased risk of lymph node metastasis and (any non‐organ confined disease) any non‐organ confined disease. Insulin‐like growth factor‐I binding protein‐3 levels were also inversely independently associated with lymph node metastasis. Receiver operating characteristic curve analysis showed that the addition of insulin‐like growth factor‐I binding proteins biomarkers to a reference model significantly improved the discriminating ability for the prediction of lymph node metastasis (+10.0%, Preoperative insulin‐like growth factor‐I binding protein‐2 and ‐3 are significantly associated with features of biologically and clinically aggressive urothelial carcinoma. These biomarkers improved prognostic urothelial carcinoma models. AUCarea under the curveCIconfidence intervalCISin situcarcinoma CSScancer‐specific survivalDCAdecision curve analysisHRhazard ratioIGFBPinsulin‐like growth factor‐I binding proteinIGF‐Iinsulin‐like growth factor‐IIGF‐IRinsulin‐like growth factor‐I receptorIQRinterquartile rangeLNMlymph node metastasisNOCDnon‐organ confined diseaseORodds ratioOSoverall survivalRCradical cystectomyRFSrecurrence‐free survivalROCreceiver operating characteristicSEstandard errorUCurothelial carcinomaRC with/without perioperative systemic chemotherapy is the standard treatment of muscle‐invasive non‐metastatic bladder UC; RC is also recommended for very high‐risk non‐muscle‐invasive bladder UC including Bacillus Calmette–Guérin unresponsive disease.IGF‐I is produced by liver cells,To definitively assess the relationship of preoperative plasma levels of IGF‐I, IGFBP‐2, and ‐3 with adverse histopathologic features and survival outcomes in bladder UC, we studied a large consecutive cohort of patients with non‐metastatic bladder UC treated with RC and pelvic lymphadenectomy.All procedures described in the present study were undertaken with the approval and oversight of the Institutional Review Board for the Protection of Human Subjects of two institutions (IRBs: 1011011386 and 069826900). This study is a retrospective analysis of prospectively collected plasma samples obtained from a consecutive cohort of patients who were treated with RC for non‐metastatic bladder UC at two medical institutions. The extent of lymphadenectomy and choice of urinary diversion were at the surgeon's discretion. Patients with any concomitant secondary malignancy, concomitant upper tract UC, or missing data were excluded. We included any patients who did not receive neoadjuvant chemotherapy. Adjuvant chemotherapy was administered to 167 patients (16.1%) at the clinicians' discretion based on tumor stage and overall health status. No patient received perioperative radiotherapy.g. The top layer corresponding to plasma was decanted using sterile transfer pipettes and immediately frozen and stored at −80°C in polypropylene cryopreservation vials . For quantitative measurements of IGF‐I, IGFBP‐2, and ‐3 levels, we used quantitative immunoassays . Every sample was run in duplicate, and the mean was used. Differences between the two measurements for IGF‐I, IGFBP‐2, and ‐3 were minimal .Sample collection was performed for our biomarker database between 2003 and 2015. Preoperative serum and plasma samples were collected typically on the morning of the day of surgery after an overnight fast. Specimen collection and measurement have been described in detail previously.All surgical specimens were processed according to standard pathological procedures as previously described.Clinical and radiological follow‐up was performed following institutional protocols and current guidelines. Routine follow‐up usually included physical examination, radiological imaging, and urine cytology every 3 months for 2 years. Between the second and the fifth years, follow‐up was performed semiannually. Afterward, in most cases, an annual follow‐up was performed. Tumor recurrence was defined as the occurrence of loco‐regional recurrence or distant metastasis on radiological imaging. Cause of death was abstracted from medical charts and/or from death certificates.U tests, Kruskal–Wallis tests, or calculation of Spearman's rank correlation coefficient (ρ) and subsequent significance testing, as appropriate. For stratification concerning Kaplan–Meier survival curves, 5‐year survival rates, and patient characteristics, group classification (low vs high) was performed using median plasma levels of IGF‐I, IGFBP‐2, and ‐3.Report of categorical variables included frequencies and proportions. Reporting of continuous coded variables focused on medians and IQR. Concerning preoperative plasma levels of IGF‐I, IGFBP‐2, and ‐3, which were routinely treated as continuous variables, group comparisons were performed using the Mann–Whitney P‐values were two‐sided, and statistical significance was set at 0.05. All statistical analyses were performed using R Version 3.6.3 .The primary outcome was the adverse histopathological features including LNM, ≥pT3 disease, or any NOCD. Binominal logistic regression analysis was performed for evaluating the association of pretreatment plasma levels of IGF‐I, IGFBP‐2, and ‐3 with LNM, ≥pT3 disease, or any NOCD . The AUC of ROC curves was calculated to determine the predictive accuracy of multiple logistic regression models. DeLong's test was used to test for statistical significance between different AUCs. The secondary outcome was survival outcomes including RFS, CSS, and OS. Association between pretreatment IGF‐I, IGFBP‐2, and ‐3 with RFS, CSS, and OS were assessed in univariable and multivariable Cox regression models. Clinical and pathological tumor grade was excluded as a variable for all predictive models since virtually all RC patients had high‐grade bladder UC. Separate Cox regression models that featured either preoperative clinical variables or postoperative histopathological variables were created. The discriminative ability of these models after inclusion of IGF‐1, IGFBP‐2, and/or ‐3 was tested using Harrel's concordance index (C‐index). The additional clinical net benefit of all biomarkers was also evaluated using DCA. Separate reference models, that represented either the preoperative or postoperative setting, were created, to which IGF‐I, IGFBP‐2, and/or ‐3 were added to assess the additive predictive value of each biomarker. All reported ρ = −0.07, P = 0.03), IGF‐I and IGFBP‐3  and IGFBP‐2 and IGFBP‐3 . Median plasma levels of IGFBP‐2 and IGFBP‐3 were significantly lower among patients with adverse pathologic features such as lymphovascular invasion, LNM, or positive soft tissue surgical margins , pT3/4 disease  or any NOCD . Pretreatment levels of IGFBP‐2 and IGFBP‐3 were inversely associated with LNM , pT3/4 disease  or any NOCD .On univariable analyses, pretreatment levels of IGF‐I were not associated with either LNM . ROC curve analysis showed that the addition of IGFBP‐2 and ‐3 biomarkers to a preoperative reference model comprising age, sex, and clinical tumor stage improved the discriminatory ability for prediction of LNM , pT3/4 disease , and any NOCD . When the significant biomarkers from the multivariable models were added in DCA, there was an improvement in net benefit relative to the reference model for prediction of LNM . The 5‐year RFS, CSS, and OS estimates were 62.5% (95% CI 59.2–66.0), 66.6% (95% CI 63.3–70), and 59.6% (95% CI 53.6–60.5), respectively. Stratified by the median pretreatment levels, patients with high IGFBP‐2 and ‐3 had better RFS, CSS, and OS compared to those with IGFBP‐2 and ‐3 below the median . IGFBP‐2 was also independently associated with any NOCD  increased the accuracy for the prediction of LNM by a significant margin .P = 0.002). On multivariable Cox regression models, only elevated IGFBP‐3 plasma levels remained independently associated with favorable OS  in bladder UC. In agreement with our previous study, we failed to find an association between plasma IGF‐I levels and adverse pathologic features or survival outcomes. In contrast, we did find an association of lower pretreatment levels of IGFBP‐2 and ‐3 with adverse pathologic features after RC . We also found on multivariable analyses that patients with a lower preoperative IGFBPs level had a higher probability of harboring LNM and/or NOCD. In addition, survival analyses demonstrated that in both pretreatment and posttreatment settings, higher levels of both IGFBP‐2 and ‐3 were independently associated with improved RFS, CSS, and OS. While these promising results are consistent with those of previous studies that assessed prognostic/predictive levels of plasma IGFBPs.Our negative results for the IGF‐I levels might be explained by the fact that 99% of circulating IGF‐I are bound to IGFBPs.The most important histopathological prognostic variables after RC and LN dissection are tumor stage and LN status.To our knowledge, this study is the first with an acceptable sample size to assess the impact of the IGF‐I axis in bladder UC patients. The main limitations of our study are its retrospective and multicenter design as well as the short median follow‐up of 37 months. It has, however, previously been reported that more than 90% of all patients who experienced disease recurrence would do so within the first 24 months after RC.In the conclusion, we externally validated that elevated preoperative plasma levels of IGFBP‐2 and ‐3 are associated with RFS, CSS, and OS. In addition, both biomarkers had a significant impact on the prediction of adverse pathologic features such as LNM and advanced tumor stage. Thus, both biomarkers could help identify patients, who may benefit more from a neo‐adjuvant chemo/immune checkpoint therapy. Both biomarkers exhibit the potential effect to improve the discriminatory power of current prognostic models and refine decision‐making to enable individualized patient care.Reza Sari Motlagh: Conceptualization; data curation; formal analysis; methodology; writing – original draft. Victor M Schuettfort: Conceptualization; data curation; formal analysis; methodology. Keiichiro Mori: Data curation. Satoshi Katayama: Data curation; formal analysis. Pawel Rajwa: Data curation; supervision. Abdulmajeed Aydh: Data curation; supervision. Nico C Grossmann: Data curation; supervision. Ekaterina Laukhtina: Data curation; supervision. Benjamin Pradere: Data curation. Hadi Mostafaei: Data curation. Fahad Quhal: Data curation. Mohammad Abufaraj: Supervision. Richard Lee: Supervision; writing – review and editing. Pierre I Karakiewicz: Methodology; supervision. Yair Lotan: Supervision. Eva Comprate: Supervision. Marco Moschini: Supervision. Paolo Gontero: Supervision. Shahrokh F Shariat: Conceptualization; methodology; supervision; writing – review and editing.None declared.1011011386 and 069826900.N/A.N/A.N/A.Table S1. Association of the pretreatment IGF and its binding proteins with clinicopathologic characteristics in 1036 patients treated RC for UC of the bladder.Table S2. Postreatment multivariable cox regression analysis for prediction of RFS, CSS and OS in 1036 patients treated with RC for UC of the bladder.Table S3. Preoperative multivariable logistic regression analysis for prediction of lymph node involvement, pt3/4 disease and any NOCD in 498 stage t2 patients treated with RC for UC of the bladder.Table S4. Preoperative multivariable cox regression analysis for prediction of RFS, CSS and OS in 498 stage t2 patients treated with RC for UC of the bladder.Table S5. Preoperative multivariable logistic regression analysis for prediction of lymph node involvement, pt3/4 disease and any NOCD in 336 stage t1 patients treated with RC for UC of the bladder.Table S6. Preoperative multivariable cox regression analysis for prediction of RFS, CSS and OS in 336 stage t1 patients treated with RC for UC of the bladder.Click here for additional data file.


Conversion surgery (CS), which aims to cure after systematic therapy, is only scarcely reported in the field of hepatocellular carcinoma (HCC). However, advancements in systemic therapy for HCC are expected to increase the candidates eligible for CS because of the higher response rate. The aim of this study was to clarify the characteristics of patients who underwent CS after tyrosine kinase inhibitor (TKI) therapy.n = 292) and lenvatinib  from July 2009 to October 2020 were retrospectively enrolled. The endpoint of this analysis was overall survival (OS), and factors associated with CS are revealed.In all, 364 patients who were treated with first‐line sorafenib , and their etiology included viral hepatitis, especially hepatitis B virus (HBV) (P = 0.049). Improvements or maintenance of preoperative modified albumin–bilirubin grade from baseline were observed in five (83.3%) patients, and partial radiologic response was observed in four (66.7%) patients. The median OS and 3‐year survival rate of patients treated with CS were “not reached” and 80.0%, respectively.Six patients underwent CS after TKI therapy, and of these four (1.4%) and two (2.7%) patients received SOR and LEN, respectively. At baseline, patients who underwent CS were significantly younger  after tyrosine kinase inhibitor (TKI) therapy for hepatocellular carcinoma experienced long survival. The patients who underwent CS were relatively young, and exhibited radiological response to TKIs, and their liver function was either maintained or improved. Additional treatments other than curative resection showed fewer or marginal survival benefits.It has been reported that a higher response rate can lead to better outcomes after liver resection in patients treated with conversion chemotherapy for initially unresectable colorectal liver metastases.As described above, the number of patients who undergo CS after systematic therapy will increase in the future as the response rate increases as a result of advancements in systematic therapy for HCC. The aims of this study are to clarify the characteristics of patients who underwent CS after first‐line TKI (SOR and LEN) treatment and to propose this strategy for advanced HCC.In all, 364 patients treated with first‐line SOR or LEN at the Musashino Red Cross Hospital from July 2009 to October 2020 were retrospectively enrolled. The diagnosis of HCC was based on pathological or radiologic findings, such as typical arterial enhancement of the tumor followed by a washout pattern in the portal venous phase or the equilibrium phase on dynamic computed tomography (CT) or magnetic resonance imaging, in accordance with practice guidelines.Inclusion criteria for TKI treatment were as follows: metastatic or locally advanced HCC that was unresectable or refractory to TACE; primarily BCLC stage B or CChild–Pugh (C–P) classification as well as the albumin–bilirubin (ALBI) and modified ALBI (mALBI) grades were used to assess liver function. The ALBI score was calculated on the basis of serum albumin and total bilirubin values as previously reported.Written informed consent was obtained from all study participants. This study was approved by the ethics committee of the Musashino Red Cross Hospital. The investigation was conducted in accordance with the Declaration of Helsinki.SOR was orally administered at a dose of 800 mg/day. A reduced initial dose was allowed for patients with advanced age (≥80 years) and low body weight (<50 kg). LEN was orally administered at a dose of 8 mg/day to patients weighing <60 kg and 12 mg/day to those weighing ≥60 kg. SOR and LEN were reduced or interrupted if a patient developed any unacceptable grade 2 or any grade 3 drug‐related adverse events (AEs). If a patient developed AEs, dose reduction or temporary interruption was maintained until the symptoms resolved to less than grade 2. AEs were assessed according to the National Cancer Institute Common Terminology Criteria for Adverse Events, version 5.0.The endpoint of this analysis was OS, which was the time from TKI treatment initiation until death by any cause or the last follow‐up. Baseline factors between patients who received TKI with or without CS were compared. The factors analyzed for significance included age, sex, chronic liver disease etiology, serum data , C–P score, ALBI score, HCC condition , first‐line TKI (SOR or LEN), and TKI therapy duration.t‐test or Mann–Whitney U‐test was used to analyze the distribution of continuous variables. Cumulative survival rates of patients were determined using the Kaplan–Meier method. A P‐value <0.05 was considered statistically significant. GraphPad Prism software  was used to analyze statistical significance.Chi‐squared and Fisher's exact tests were used to compare categorical data. Student's A 48‐year‐old female was referred to the Musashino Red Cross Hospital for back pain, and a liver tumor was discovered by ultrasonography and CT scan. She had no significant medical history. Abdominal dynamic CT scan revealed a tumor 13.4 cm in size in the left lobe of the liver with invasion of the left PV (Vp3) Fig. . No extrWith respect to background liver disease, she was positive for hepatitis B surface antigen (HBsAg) and HBV DNA, with titers of 1.92 IU/mL and 4.6 log IU/mL, respectively. Her alanine aminotransferase (ALT) level was 20 U/L. She was diagnosed with chronic hepatitis B  and immediately treated with tenofovir alafenamide fumarate (TAF). Her liver function showed a C–P score of A (5 points) and an ALBI score of −2.594. LEN was administrated at 8 mg/day. No AEs occurred, and therefore she could be treated without LEN reduction. Two months after the initial treatment, dynamic CT revealed no change in tumor size but it did reveal decreased intratumor vascularity, which was determined to be stable according to RECIST 1.1 and partial response by mRECIST Fig. . HoweverAfter 8 months of LEN treatment, both the AFP and PIVKA‐II levels had significantly declined to 4.5 ng/mL and 1072.1 mAU/mL, respectively, and dynamic CT revealed PR according to RECIST 1.1 and mRECIST Fig. . Based oShe then continued LEN treatment, experienced no recurrence at 5 months after LEN administration, and maintained CR status.Between July 2009 and October 2020, 364 patients began TKI therapy, and of these, 292 and 72 patients received SOR and LEN, respectively, as first‐line TKI therapy. The patient characteristics are presented in Table n = 3, 50.0%) and HCV . The median ALBI score was −2.478 . The patients who underwent CS were significantly younger (P = 0.019) and their etiology included viral hepatitis, especially HBV (P = 0.049), compared with patients who did not undergo CS. The median ALBI score tended to be lower in CS patients . No significant differences were observed in sex, C–P Class, BCLC stage, EM, the extent of PVTT, median AFP concentration, median TKI therapy duration, or first‐line TKI regimen.Six patients underwent CS after TKI therapy, and of these, four  and two  patients received SOR and LEN, respectively, as first‐line TKI therapy (Table n = 6) or without CS (n = 358), are shown in Figure Survival curves, according to whether patients underwent CS  mm, the median number of tumors was 5 , and five cases (83.3%) were out of the “up‐to‐seven” criteria. The median period of TKI administration was 278.5  days. Improvements in and maintenance of the preoperative mALBI grade were observed in two and three cases, respectively, compared with those at the time of introduction of TKI therapy. Though the ORR of patients received TKI without CS was 24.0%, radiologic partial response was observed in four (66.7%) cases in patients who underwent CS. One case was BCLC stage A, which was unrefractory to TACE and SOR therapy, and therefore CS was performed. According to the pathological findings, three cases were moderately differentiated and three were poorly differentiated. HCC recurrence was observed in five cases (83.3%) at a median of 5.6  months after surgery.Based on these results, the patients who underwent CS were relatively young and responded to TKI treatment, and their liver function was either maintained or improved.This study provides valuable evidence of the benefits of CS after first‐line TKI (SOR and LEN) therapy in terms of a better prognosis in patients with advanced HCC. Based on a comparison of characteristics of patients who underwent CS and those who did not, patients who underwent CS were relatively young and responded well to TKIs, and their liver function was either maintained or improved. Six patients underwent CS after TKI therapy, and of these, four  and two  received SOR and LEN, respectively, as a first‐line TKI therapy. Although no significant differences were observed, the proportion of patients who underwent CS was numerically higher in patients who received LEN than SOR. This might reflect differences in the ORR.In our presented case Figs , 2, the In this retrospective study, the median OS of patients who received CS was long Fig. , and theThis study had several limitations. First, this was a case report and retrospective study in which the exclusion of unidentified biases was impossible. The small number of patients in this study precluded the performance of more solid comparison analyses. It would have been more persuasive if we had compared patients who had CS after TKIs with those who had surgery from the beginning. Second, regarding CS, we were unable to determine the best method or timing of the surgery. Recommended treatments were discussed and decided by the tumor board; however, the preference of each patient strongly influenced the final decision. A follow‐up study of a larger cohort is needed for further analysis, including the prognostic factors after CS. A multicenter, single‐arm observational study of LEN followed by surgical resection for initially unresectable HCC is in progress and results are expected. Third, systemic treatment options to be administered to advanced HCC during the observation period have changed significantly over the last decade. Not only can systematic chemotherapy now be used with SOR and LEN, but combination therapy of atezolizumab and bevacizumab (ATZ + BEV) was approved because of its ability to significantly prolong the progression‐free survival and OS of patients with unresectable HCC compared with SOR alone when used as a first‐line therapy.In conclusion, CS after TKI treatment is a useful strategy for advanced HCC. When patients start systematic therapy, they should be considered for CS if an antitumor effect and good background liver function are observed. The application of CS may lead to better prognosis in terms of longer survival in patients with advanced HCC.


To determine whether there is a correlation between the type of ossification and radiological parameters, modified thoracic JOA scores, and complications in patients with thoracic ossification of ligamentum flavum treated by posterior thoracic surgery.This retrospective cohort study included 48 patients with thoracic myelopathy caused by single-level thoracic ossification of ligamentum flavum who underwent thoracic posterior approach surgery in our Hospital o between December 2013 to December 2018. Patients were divided into unilateral, bilateral, and bridged groups in axial position, and beak and round groups in sagittal position. The differences were analyzed according to the ossification morphology.P = 0.884). In sagittal view, there was no significant difference in preoperative JOA score between the two groups (P = 0.710), while the postop JOA score and the recovery rate in the beak group were significantly lower than that of the round group . Two-way ANOVA showed that sagittal morphology had a significant effect on postop JOA score (P = 0.028), but axial morphology don’t (P = 0.431); there was no interaction between them (P = 0.444). For the recovery rate, sagittal morphology also had a significant effect (P = 0.043), but axial ossification don’t (P = 0.998); there was no interaction between them (P = 0.479).In axial myelopathy, there was no significant difference in preop and postop JOA scores and RR among the three groups in axial position (Sagittal morphology had a significant adverse effect on postop JOA score and surgical outcome, while axial morphology had no effect on surgical outcome, and there was no interaction between sagittal morphology and axial morphology. Thoracic ossification of ligamentum flavum (TOLF) is one of the ectopic ossification diseases of the spinal ligament. It can lead to thoracic spinal canal stenosis and is the most common cause of thoracic myelopathy . LigamenPrevious studies have shown that the incidence of ossification of ligamentum flavum (OLF) is very low , which m. [In the past, OLF was often thought to be mainly related to genetic and dietary factors. However, following the reports of worldwide OLF cases, biomechanical factors came to be seen as the main cause of OLF . Kuh et .  classifiThe aim of this study was to conduct a retrospective cohort analysis to determine whether there is a correlation between the type of ossification and radiological parameters, modified thoracic JOA scores, and complications in patients with thoracic myelopathy due to single-level TOLF treated by posterior thoracic surgery.This retrospective clinical study included 54 patients with thoracic myelopathy due to single-level TOLF who underwent thoracic posterior approach surgery at our Hospital between December 2013 and December 2018. Six patients were lost to follow-up, and 48 patients (88.89%) were included in the cohort. All patients were followed up for at least 2 years postoperatively. Criteria for the diagnosis of thoracic myelopathy due to OLF were based on clinical, radiological, and pathological assessments. The age, sex, preoperative underlying diseases, and duration of symptoms in patients were recorded. Computerized tomography (CT) and Magnetic resonance imaging (MRI) examinations were performed for all patients, and the ossified segments, canal occupation rate, intramedullary signal change, dural ossification, and OLF ossification type were recorded. In addition, according to the results of radiology examination and previous studies, patients were divided into unilateral, bilateral, and bridged groups in the axial position and beak and round groups in the sagittal position  intraoperative x-rays were used to locate the OLF segment and expose the posterior spine through a midline incision at the posterior part of the ossified segment; (2) the spinous process was excised, and the lamina and articular process were excised by rongeurs and high-speed drill; (3) the epidural fat and dura were dissected under the ossified mass; (4) the ossified mass was carefully removed with rongeurs. For increase stability, posterior internal fixation was performed by pedicle screws. (5) Sutures were made layer by layer, and the subfascial drain was placed for posterior wounds.(1) D1 and D2 were the maximum distances measured from the bilateral ossification mass to the inner edge of the lamina, where the larger one was Dmax. D is the perpendicular distance from the intersection of the canal occupation rate (APD) and the posterior vertebral wall to X1 or X2. Canal occupation rate (COR) was calculated using the following formula : COR = .Fig. 3Sc(2) The intramedullary hypersignal was observed at the sagittal position of MRIT2-weighted image before surgery.(3) Preoperative and postoperative neurological status were assessed by using modified Japanese Orthopaedic Association (JOA) Scores . RecoverP value < 0.05 was considered to be statistically significant  was used for the analysis. The measurement data between the two groups were compared using the independent sample T-test or Mann–Whitney test, according to the normal distribution and homogeneity of variance. ANOVA test or Kruskal–Wallis test was used to compare the measurement data among the three groups according to whether they were in accordance with normal distribution and homogeneity of variance. According to the expected value, counting data were compared between the two groups using Pearson test or continuous corrected Chi-square test. Counting data were compared between the three groups using Pearson's test or Fisher's exact probability method according to the expected value. nt Table .Table 1Rn = 14), Bilateral (n = 18), and Bridged (n = 16), and descriptive features of patients grouped by sagittal location as Beak (n = 19) and Round (n = 29). It can be seen that there was no significant difference in the number of patients with the three ossification types in the axial view (P > 0.05), while in the sagittal view, there were more patients with round type than beak type (P < 0.05). Statistical comparisons showed that the descriptive characteristics of patients with various ossification types were similar (P > 0.05). In terms of comorbidities, there were no significant differences among patients with different ossification types (P > 0.05).A total of 48 patients, 29 males and 19 females, with a mean age of 58.35 years (range 31–77 years) with thoracic myelopathy due to single-level TOLF were included in the study. Table P < 0.001). For umCOR, there was no difference as 45.36% ± 11.09% in the unilateral group, 47.41% ± 13.60% in the bilateral group and 51.23% ± 14.57% in the bridge group (P = 0.470).Table P < 0.001) and bridged group (P < 0.001), while there was no significant difference between bilateral group and bridged group (P = 0.278), or in umCOR among the three groups. In the unilateral group, 1 case was located in the upper thoracic vertebrae (T1–T4), 2 in the middle thoracic vertebrae (T5–T9), and 11 in the lower thoracic vertebrae (T10–T12). In the bilateral group and the bridged group, the data were 3, 1, 14, and 4, 4, 8, respectively. There were no significant differences in ossification levels among the three groups (P = 0.323). There were also no significant differences (P > 0.05) between the three ossification types in the presence of a high signal on MRIT2-weighted image and the presence of dural ossification on CT.In order to clarify intra-group differences, pairwise comparisons were made between the three groups in terms of COR, umCOR, and surgical methods Table , revealin = 8, 57.14%), while the bridged group had the highest proportion of laminectomy and internal fixations . Although there was no difference between the three groups (P = 0.88), there was a significant difference between the unilateral group and the bridge group (P = 0.029)  (P = 0.685), and no significant difference in the ossification segment, which was the same as in the axial position (P = 0.906). Also, ossification mostly occurred in the lower thoracic vertebrae. There was no significant difference between the two groups on imaging with high signal and dural ossification. In terms of selecting surgical methods, most patients from the two groups chose laminectomy and internal fixation; however, no significant difference was found (P > 0.05).On the sagittal plane Table , there wP < 0.001); a bilateral group from 6.92 ± 1.22 to 8.83 ± 1.06 (P < 0.001); the bridge group improved from 6.63 ± 1.77 to 8.50 ± 1.75 (P < 0.001), but there was no difference in the improvement rate among them (P = 0.884). In the sagittal position (Table P < 0.001), while in a round group it changed significantly from 6.95 ± 1.59 to 8.93 ± 1.43 (P < 0.001). There was no significant difference in preoperative JOA score between the two groups (P = 0.710), while a significant difference was observed in postoperative JOA score and recovery rate between the two groups (P = 0.017) (P = 0.034).In terms of the axial position Table , there wP = 0.098, P = 0.476, respectively), and there was no interaction between them (P = 0.383) (Table P = 0.028) (Table P = 0.043), while axial ossification had no effect on the recovery rate (P = 0.998), and there was no interaction between the two groups (P = 0.479)  this was a single-center retrospective study, and due to the low incidence of single-level TOLF, the sample size was small, which should be addressed by further multi-center prospective large sample size studies; (2) the maximum duration of neurological function recovery after TOLF was unclear, and follow-up time of 2 years may be insufficient.In this study, COR was more effective than umCOR in assessing spinal cord compression, even though some ossified ligamentum flavum compressed only one side of the spinal cord in the axial position. Surgery can be an effective way to restore spinal cord function regardless of the type of ossification. In sagittal classification, beak-type ossification had a significant adverse effect on surgical prognosis, while axial classification had no effect, and there was no interaction between sagittal and axial ossification morphology.


The prevalence and clinical importance of cardiac abnormalities in dogs with acute pancreatitis (AP) is unknown.Twelve dogs with AP and 60 archived serum samples from dogs with suspected AP.Two‐phase study. Phase I: Analysis of archived serum samples from dogs with clinical signs of AP and high Spec cPL concentrations. High sensitivity troponin I (TnIH) and N‐terminal pro‐B‐type natriuretic peptide (NT‐proBNP) concentrations were measured in achieved serum samples. Phase II: Prospective observational study. Dogs with AP underwent echocardiography and Holter monitoring. Serum cardiac troponin I (cTnI) and plasma NT‐proBNP concentrations were measured. Previously described disease severity indices were calculated for each dog.Phase I: 41 of 60 dogs suspected of having AP had abnormally high TnIH concentrations and 13 of 60 had abnormally high serum NT‐proBNP concentrations. Higher TnIH concentrations were observed in dogs with Spec cPL concentration >2000 μg/L as compared to those with concentrations of 1000‐2000 μg/L. Phase II: 11 of 12 dogs diagnosed with pancreatitis had abnormal cTnI concentrations  and 7 of 12 dogs had plasma NT‐proBNP concentrations above the reference interval . Supraventricular and ventricular ectopic beats occurred in 3 dogs. Echocardiographic abnormalities were detected in 5 dogs. Cardiovascular variables were not associated with indices of disease severity.Myocardial injury is common in dogs with AP, but clinical consequences appeared to be uncommon in our small cohort. Cardiac biomarkers should be interpreted with caution in dogs with AP. AoaorticAPacute pancreatitisfullAPPLEacute patient physiologic and laboratory evaluation scorebpmbeats per minuteCAPScanine acute pancreatitis severity scoreCRPC‐reactive proteincTnIADVIA Centaur Troponin I Ultra‐assayDCMdilated cardiomyopathyECGelectrocardiogramEFejection fractionFSfractional shorteningIVSIiterventricular septal thicknessLAleft atrialLVleft ventricleLVAleft ventricular areaLVEDVleft ventricular end‐diastolic volumeLVESVleft ventricular end‐systolic volumeLVIDleft ventricular internal diameterLVPWleft ventricular posterior wall thicknessM‐CAImodified canine activity indexNT‐proBNPN‐terminal pro‐B‐type natriuretic peptideRIreference IntervalSMODSimpson's method of discsSpec cPLspecific canine pancreatic lipase immunoreactivitySVstroke volumeTnIHSiemens Atellica IM high sensitivity troponin I assay1Pancreatitis is the most common disorder of the exocrine pancreas in dogs.In contrast to humans with AP, available data regarding cardiovascular abnormalities in dogs with AP is limited and consists of retrospective studies, case reports, and anecdotal information. Over 70% of dogs with high DGGR lipase activities in 1 study had abnormally high cardiac troponin I concentrations, but the reason why the clinician requested concurrent cardiac biomarker assessment in each case was not reported.22.1Two‐phase study. In phase I of the study, the database of the Texas A&M Gastrointestinal Laboratory was searched from 1/4/22 to 2/23/22 to identify serum specimens from dogs with suspected AP. Cases were selected if the medical records revealed that clinical signs were consistent with AP and the measured cPLI concentration  was ≥400 μg/L. A total of 60 samples were selected, with 20 samples encompassing each of the following cPLI ranges: 400‐999, 1000‐2000, and >2000 μg/L. These groupings were selected to allow for evaluation of cardiac biomarker concentrations over a range of abnormal cPLI concentrations.Dogs presenting to Michigan State University College of Veterinary Medicine with suspected AP were eligible for prospective enrollment in phase II of the study. The study protocol was approved by the Institutional Animal Care and Use Committee at Michigan State University, and informed consent was obtained from all owners. The diagnosis of AP was based on an integration of clinical signs, serum cPLI concentrations, and abdominal ultrasound findings.2.2In phase I of the study, all dogs had serum concentrations of high sensitivity troponin‐I (TnIH)  and serum N‐terminal pro‐B‐type natriuretic peptide  measured via commercial assays. Serum and plasma NT‐proBNP quantification have been analytically validated for canine samples.In phase II of the study, all dogs meeting enrollment criteria had serum samples submitted for measurement of C‐reactive protein (CRP) and cardiac troponin I (cTnI) concentration and a plasma sample submitted for the measurement of NT‐proBNP concentration. The assays used for measurements of CRP , cTnI , and NT‐proBNP  are validated for use in dogs.2.3In phase II, dogs underwent continuous Holter monitoring with a 3‐lead 3‐channel system for 48 hours . Holter's were removed early only if the animal died or if specifically requested by the owner. The Holter tracings were analyzed by an automated system  and reports were verified by a board‐certified veterinary cardiologist (RAS). When greater than 24 hours of data was available, each 24‐hour period was analyzed separately and was only included into the analysis if there was >21 hours of usable data. Data obtained included: normal heart rate parameters (min/mean/max heart rate and number of pauses greater than 2 seconds), frequency of isolated ventricular premature complexes, ventricular couplets, ventricular triplets, and episodes of ventricular tachycardia. The frequencies of ventricular bigeminy and ventricular trigeminy were noted. The definitions of conduction disturbances used by the analysis software are included in Table 2.4In phase II, dogs underwent an echocardiogram  including standard 2D M‐mode, color Doppler, and spectral doppler imaging performed by 1 of 2 board certified veterinary cardiologists. All stored images and cine loops were subsequently reviewed by 1 board certified veterinary cardiologist (RAS). All echocardiograms were performed in right and left lateral recumbency in a quiet, dark room on an echocardiogram table. No sedation was utilized. Measurements included 2D aortic diameter (Ao) and left atrial diameter (LA) from the right parasternal short axis image of the heart base, as described and left ventricular area (LVA) in diastole and systole from the right parasternal short axis view.2.5full), as utilized in previous studies.Multiple severity indices have been proposed for use in dogs with AP and no studies have directly compared the utility of multiple severity indices in an independent cohort. In our study, disease severity was determined by the following indices: canine acute pancreatitis severity score (CAPS), modified canine activity index (M‐CAI), and the acute patient physiologic and laboratory evaluation score . For phase II of the study, Spearman's rank correlation coefficients were calculated to evaluate potential associations of cardiac variables  with disease severity indices. In addition, cardiac variables were compared between those dogs with a CAPS ≥11 and those dogs with a CAPS <11. The cut‐point of ≥11 was selected because this value has been suggested to be 89% sensitive and 90% specific for predicting short‐term case fatality in dogs with AP.33.1In phase I of the study, the median age of dogs enrolled was 11 years (range: 1‐19 years). Thirty‐three dogs were spayed females, 22 were neutered males, and 4 were intact males. The sex of 1 dog was not recorded. The median weight of the enrolled dogs was 13.3 kg (range: 3.2‐34.0 kg). Twenty‐three dogs were mixed breed dogs, 4 were Yorkshire terriers, 3 were Boxers, and 3 were Dachshunds. Two each of the following breeds were represented: Cavalier King Charles spaniel, Cocker spaniel, Labrador retriever, Maltese, Soft‐coated Wheaten terrier, and Havanese terrier. One each of the following breeds were also represented Beagle, Boston terrier, Bulldog, Cardigan Welsh corgi, Catahoula leopard dog, Golden retriever, Jack Russel terrier, Miniature pinscher, Miniature poodle, Pointer, Shiba Inu, Shih tzu, and Standard poodle. The breed for 2 dogs were unavailable.In phase II of the study, 19 dogs met initial enrollment criteria, but 7 dogs were subsequently removed because serum cPLI concentrations were either normal or in the diagnostic gray‐zone (200‐399 μg/dL). The median age of the 12 dogs undergoing analysis was 9.5 years (range: 8‐15 years). Seven were spayed females and 5 were neutered males. The median weight of enrolled dogs was 15.6 kg (range: 5.5‐51.0 kg). Six were mixed breed dogs and 1 each of the following breeds were represented: Brittany spaniel, Fox terrier, Golden Retriever, Labrador retriever, Shih tzu, and Yorkshire terrier. Eleven of 12 dogs survived. The median serum cPLI concentration was 1316 μg/dL (range: 491‐>2000 μg/dL).3.2In phase I of the study the median serum TnIH was 111 ng/L . Forty‐one of 60 dogs had increased serum TnIH concentrations. Twenty‐four of 60 dogs had serum TnIH concentrations above the laboratory provided cut‐off for myocardial injury (>135 pg/mL). A diagnostic gray zone has been recommended for concentrations between 50 and 135 pg/mL. The median serum NT‐proBNP concentration was 326 pmol/L . Thirteen of the 60 dogs had increased serum NT‐proBNP concentrations. Serum TnIH and serum NT‐proBNP concentrations in relation to cPLI concentrations are shown in Table In phase II of the study, the median serum cTnI concentration was 0.384 ng/mL . Eleven of the 12 dogs (91.7%) had increased serum cTnI concentrations. The serum cTnI concentration of the dog that died was 1.027 ng/mL. The median plasma NT‐proBNP concentration was 971 pmol/L (range: 250‐2215 pmol/L). Seven of the 12 dogs (58%) had increased plasma NT‐proBNP concentrations. The plasma NT‐proBNP concentration of the dog that died was 1096 pmol/L.3.3All 12 dogs were evaluated using a Holter monitor. The median supraventricular burden was 0.197% (range: 0.00%‐3.59%). The median ventricular burden was 0.022% (range: 0.00%‐4.64%). The top 3 conduction abnormalities noted included: dropped beats, premature ventricular ectopic beats, and ventricular ectopic beats. Further details on dog‐specific conduction disturbances can be found in Supplementary material SRoutine evaluation of stored echocardiographic images was performed by RAS. Seven of 12 dogs had no major structural abnormalities. However, mild structural abnormalities were noted in some dogs, including mild degenerative valve disease (3 dogs), mild reductions in indices of systolic function (2 dogs), pulmonary arterial hypertension (1 dog), and mild degenerative valve disease with mild pericardial effusion (1 dog).3.4Rs = 0.618, P = .032), but not M‐CAI , or APPLEfull . Serum cardiac troponin I concentrations were not different between those dogs with CAPS ≥11 and those with CAPS <11 with a P = .625. No dogs in our study had an APPLEfull score ≥40, preventing evaluation as a dichotomous variable. Plasma NT‐proBNP concentrations were not correlated with CAPS , M‐CAI , or APPLEfull . N‐terminal pro‐B‐type natriuretic peptide concentrations in plasma were not significantly different between those dogs with CAPS ≥11 and those with CAPS <11 with a P = .812. No dogs in our study had an APPLEfull score ≥40, preventing evaluation as a dichotomous variable.In phase II of the study, serum cardiac troponin I concentrations (cTnI) were correlated with CAPS , M‐CAI , or APPLEfull . Supraventricular burden was not different between those dogs with CAPS ≥11 and those with CAPS <11 with a P = .625. No dogs in our study had an APPLEfull score ≥40, preventing evaluation as a dichotomous variable. Ventricular burden was not correlated with CAPS , M‐CAI , or APPLEfull . Ventricular burden was not correlated with dichotomous CAPS values (≥11 vs <11) with a P = .876. No dogs in our study had an APPLEfull score ≥40, preventing evaluation as a dichotomous variable. No other comparisons to disease severity indices were significant.Supraventricular burden was not correlated with CAPS . Most (41/60) archived serum samples had increased (>50 pg/mL) serum TnIH concentrations and 24 of 60 dogs had serum TnIH concentrations above the laboratory provided cut‐off value for myocardial injury (>135 pg/mL). A smaller proportion of dogs (13/60) also had increased serum NT‐proBNP concentrations. The diagnosis of AP in phase II of the study was based on a complete clinical diagnosis based on a consistent history/physical examination, diagnostic imaging findings, and an elevated serum cPLI concentration (≥400 μg/dL).Cardiac troponins are quantitative biomarkers that are considered the gold standard for detection of myocardial injury.N‐terminal pro‐B‐type natriuretic peptide is a biomarker of myocardial stretch that has been validated for use in dogs.Although this study emphasizes cautious interpretation of cardiac biomarkers in dogs with concurrent AP, it should be noted that various other noncardiac disorders have been associated with elevated cardiac biomarker concentrations, including systemic inflammatory response syndrome, gastric dilatation and volvulus, and infectious diseases.In our study we utilized a Holter monitor to investigate the prevalence of conduction abnormalities in dogs with AP. This method was selected over routine brief ECG assessments (1‐5 minutes), to avoid confounding factors such as restraint, and to allow for the detection of intermittent arrythmias. Holter monitoring has been shown to be more sensitive than routine brief ECG assessments for the detection of arrythmias in other studies.Although not an aim of the current study, CAPS was not a useful prognostic indicator in this study cohort. A CAPS score ≥11 is reported to have a sensitivity of 89% and a specificity of 90% for predicting short term mortality (within 30 days of admission) in dogs with AP.A limitation of the present study was the small sample size in phase II. This could lead to a type II error and might mean that uncommon abnormalities were missed. However, it is the largest prospective evaluation of cardiovascular abnormalities of AP in dogs to date and offers a comprehensive structural, conduction, and biomarker assessment of such dogs. Additionally, phase I of the study thoroughly investigated the prevalence of cardiac biomarker abnormalities in a cohort of 60 dogs. Although only dogs with no evidence of preexisting cardiovascular disease were included in the study, given the lack of longitudinal follow up, it is possible that dogs may have gone on to develop primary cardiac disease. A further limitation of our study was the inability to evaluate the effect of cardiovascular abnormalities on mortality, given the low case fatality rate in our study cohort. The low case fatality rate may be related to either dog or treatment specific factors, or both. Two cardiac troponin assays were utilized in this study, limiting direct comparison between phase I and phase II of the study. This was unfortunately out of the control of the study investigators as 1 assay was discontinued by the manufacturer. The TnIH assay was internally analytically validated, but no external peer‐reviewed and published analytical validation study currently exists. The diagnosis of pancreatitis is notoriously challenging. In phase I of the study, we utilized dogs with clinical signs and an elevated serum cPLI concentration, without imaging data. Serum cPLI concentrations ≥400 μg/dL have a high sensitivity (81.0%‐90.9%) and specificity (74.1%‐81.1%) for the diagnosis of pancreatitis.In conclusion, increased serum and plasma cardiac biomarkers were common in dogs with suspected or diagnosed pancreatitis, but this did not translate into clinically important cardiac complications. Thus, cardiac biomarkers should be interpreted with caution in dogs with pancreatitis.Dr. Steiner serves as the director of the Gastrointestinal Laboratory at Texas A&M University, which offers measurement of serum canine pancreatic lipase immunoreactivity concentration , and measurement of cardiac troponin I (via TnIH) on a fee‐for‐service basis. Dr. Steiner also acts as a paid consultant for IDEXX Laboratories, which is the manufacturer of the Spec cPL assay and also offers cPLI testing using the Spec cPL on a fee‐for‐service basis. No other authors have a conflict of interest.Authors declare no off‐label use of antimicrobials.Approval was granted by the Michigan State University IACUC.Authors declare human ethics approval was not needed for this study.Appendix S1. Supporting InformationClick here for additional data file.


In addition, the SEC6 signature was associated with lower overall survival, progression‐free interval, and disease‐specific survival in stage II and III patients. SEC6‐based risk stratification indicated that 5‐FU treatment was beneficial for low‐risk patients, whereas only aggressive treatments (FOLFOX and FOLFIRI) provided benefits to high‐risk patients in stages II and III. In summary, this novel risk‐score demonstrates the value of the secretome compartment as a reliable source for the retrieval of biomarkers with high prognostic and chemotherapy‐predictive capacity, providing a potential new tool for tailoring decision‐making in patient care.The necessity to accurately predict recurrence and clinical outcome in early stage colorectal cancer (CRC) is critical to identify those patients who may benefit from adjuvant chemotherapy. Here, we developed and validated a gene‐based risk‐score algorithm for patient stratification and personalised treatment in early stage disease based on alterations in the secretion of metastasis‐related proteins. A quantitative label‐free proteomic analysis of the secretome of highly and poorly metastatic CRC cell lines with different genetic backgrounds revealed 153 differentially secreted proteins (fold‐change >5). These changes in the secretome were validated at the transcriptomic level. Starting from 119 up‐regulated proteins, a six‐gene/protein‐based prognostic signature composed of IGFBP3, CD109, LTBP1, PSAP, BMP1, and NPC2 was identified after sequential discovery, training, and validation in four different cohorts. This signature was used to develop a risk‐score algorithm, named SEC6, for patient stratification. SEC6 risk‐score components showed higher expression in the poor prognosis CRC subtypes: consensus molecular subtype 4 (CMS4), CRIS‐B, and stem‐like. High expression of the signature was also associated with patients showing dMMR, CIMP TP53, KRAS, and BRAF mutations (among others) that lead to different pathogenesis and drug sensitivity [Colorectal cancer (CRC) is considered a heterogeneous disease with different outcomes according to the molecular subtypes , 2. Thissitivity , 2, 4, 5sitivity , 7.We hypothesised that protein expression profiles associated with invasive and metastatic capacity might be useful for building more accurate risk predictors. The conditioned medium or cellular secretome, including exosomes, has been demonstrated to be a rich source of metastatic effectors and biomarkers of metastasis in different tumours . Cancer For secretome characterisation, cell lines present multiple advantages over whole tumour tissues as they facilitate a more exhaustive and complete recovery of secreted factors. Cell lines constitute a useful resource to recover functional molecular data and design mechanistic studies overcoming the high heterogeneity of human CRC. Multiple studies have confirmed the capacity of cell lines to faithfully represent the molecular subtypes of CRC , 11. ConTo explore the secretome compartment, we performed a combined multi‐omic approach: first, we used a label‐free quantitative proteomic analysis of cell line supernatants, which was validated using a global gene expression analysis of the three cell lines. Then, by using iterative training of hazard ratios (HRs) and survival log‐rank tests in four different datasets from primary tumours containing all the CRC stages, we identified and validated a panel of six genes/proteins with strong prognostic power for stage II/III disease. A risk‐score algorithm was developed and tested with the currently existing CRC molecular classifications. Finally, the predictive value in response to chemotherapy was investigated.2 humidified atmosphere. Cells were regularly tested for mycoplasma contamination and authenticated by short tandem repeat determination.KM12SM and KM12L4 cells were obtained from Dr Fidler . SW620 cells were obtained from the ATCC . Cells were grown in Dulbeccos Modified Eagle Medium containing 10% fetal bovine serum  and antibiotics at 37 °C in a 5% COin silico analysis. Raw data files have been deposited to the Proteome Xchange Consortium via the PRIDE repository with accession number PXD032899.See See The proteins identified and quantified in the proteomic studies were analysed by systems biology in order to obtain the prediction of the enriched functions. Gene ontology (GO) analysis was performed using g:Profiler web site . Venn diet al [z‐score value. CRIS classification of the GSE14333, GSE39582, and TCGA COADREAD datasets was obtained from Isella et al [Different public gene expression datasets were used for prognosis analysis including datasets from the Gene Expression Omnibus . Then, patients were divided into two groups (i.e. high or low risk) by the optimal cut‐off method using X‐tile software [P value. Gene prognostic signature and risk‐score classification were validated in the GSE39582, TCGA COADREAD, and GSE17538 datasets.Gene selection for the prognostic signature was sequentially performed using the GSE39582, TCGA, COADREAD and, then, GSE14333 cohorts. Genes with a HR > 1 in GSE39582 were investigated for a significant log‐rank software . The optt tests for each protein or gene. ANOVA tests were performed in order to detect significant differences in risk‐score between three or more groups. F statistic and P value are shown.Univariate and multivariate Cox regression analysis were performed using ‘survival’ and ‘survminer’ R packages. Forest plot representation was obtained using the ‘ggplot2’ R package. Kaplan–Meier analysis was performed using OriginPro Version 2020 . The significance of gene expression differences between groups was obtained by using two‐sample 2 values of the quantified proteins in each triplicate showed a similar normal distribution pattern, confirming the robustness of the proteomic analysis   with the publicly available GSE59857 dataset, which contains the gene expression analysis for 155 CRC cell lines . Qualityn = 566) containing stage and relapse information. Sixty of 119 genes were found to have a HR > 1, according to the Cox model estimator  resulted in the selection of 8 genes with a log‐rank P value <0.05  resulted in the selection of six genes  showing a HR > 1 with P value <0.05 , P value: 6.34E−6, respectively  using the GSE14333 dataset Figure , as descy Figure .p < 0.001 and HR: 3.527, 95% CI (1.74–12.44), P value: 4.56E−04, respectively. In a similar way, tumour grade, American Joint Committee on Cancer (AJCC) stage, and SEC6 risk classification were independent risk factors for overall survival (OS) in the GSE17538 cohort in the multivariate analyses , CIMP+ status , or the presence of BRAF mutations  were significantly associated with higher expression of the SEC6 genes  representing the sum of GSE17358, GSE39582, and TCGA COADREAD , P value: 0.00687  and HR: 1.94, 95% CI (0.96–3.57), P value: 0.05 (stage III)  and HR: 4.25, 95% CI (2.06–8.76), P value: 2.21E−5 (stage III)  Figure . Finally) Figure . The res) Figure . These sP value: 6.45E−4. In contrast, high‐risk stage II and III patients did not significantly benefit from the use of FUFOL , whereas the number of patients who received more aggressive treatments (FOLFIRI or FOLFOX) was much lower. When high and low‐risk patients were examined together, stage III (but not stage II) patients showed a significant improvement in OS after chemotherapy Figure . HoweverL Figure , suggestL Figure . In 5‐FUThe necessity to predict recurrence and clinical outcome in early stage CRC is critical to identify those patients who may benefit from adjuvant chemotherapy and for the implementation of therapeutic guidelines for histologically similar tumours. An adequate stratification of stage II and III patients should facilitate a continuous follow‐up and adequate chemotherapy administration regardless of surgical approaches. Here, we have developed a prognostic six gene‐based signature (SEC6) following a combination of proteomic and transcriptomic analyses using a three‐step approach. First, 119 secreted proteins were identified as up‐regulated in metastatic versus non‐metastatic cells after the proteomic analysis of soluble factors. Second, differential expression of secreted proteins was validated at the transcriptional level using global gene expression analyses. Third, an iterative analysis of HRs and log‐rank tests for the 119 genes through 4 different datasets that included a total of 1,855 patients revealed a signature of six genes: IGFBP3, CD109, LTBP1, PSAP, BMP1, and NPC2 with robust prognostic power. Their overexpression was confirmed in metastatic cell lines and tissues. Our studies indicate that SEC6‐positive tumours were associated with lower OS and higher recurrence rates than SEC6‐negative tumours across four independent datasets. Prognostic value was independent of sex, age, or location of the tumours.+ status as well as BRAF mutations, which are commonly associated with worse prognosis in CRC [BRAF mutations in CRC [BRAF mutations have shown worse prognosis in metastatic CRC [Given the heterogeneity of CRC, different CRC classifications have been developed that associate with different outcomes and responses to chemo‐ and biological therapies . Accordis in CRC , 32. It s in CRC . Althougatic CRC , 31.Microarray‐derived gene expression signatures have shown great potential for patient stratification. However, the large number of genes usually involved in these signatures complicates the translation of these findings to clinically useful tests. Current tests are based on a limited number of genes, e.g. the seven‐gene Oncotype DX recurrence score . TherefoRegarding chemotherapy response, our results indicate that SEC6‐positive, high‐risk, stage II/III patients require aggressive therapies such as FOLFOX or FOLFIRI and they do not benefit from first‐line 5‐FU therapy in contrast to low‐risk patients. Although our SEC6 predictor needs to be further validated in larger cohorts of chemotherapy‐receiving patients and prospective analyses, SEC6 recapitulates the prognostic information to identify high‐risk CRC subtypes and facilitate the development of novel clinical tools for a correct patient stratification. The low number of biomarkers involved may simplify the development of clinical tools for predicting patient survival and personalising therapies according to the molecular characteristics of the tumours. In summary, these results confirm that the secretome analysis of aggressive metastatic cells constitutes a rich mine of information for the discovery of new prognostic and predictive biomarkers.JIC and JR designed the study and wrote the manuscript. JR, LPB, IB, BE, VR, RAB, MJ and AMR performed the experiments. JIC, JR, VR, MJFA and JII analysed the data. JIC supervised the whole study. JIC and JII participated in the acquisition of funding.Supplementary materials and methodsClick here for additional data file.Figure S1. Quality control and gene ontology analysis of the proteomic assayFigure S2. Quality control of the gene expression analysisFigure S3. Validation of the six‐gene risk‐score in GSE17538Figure S4. Association of risk score‐based classification with current colorectal cancer classificationsFigure S5. Analysis of differentially expressed genes between high‐ and low‐risk patients in GSE39582Click here for additional data file.Table S1. List of deregulated proteins identified and quantified in the secretome of KM12SM, KM12L4, and SW620Click here for additional data file.Table S2. Up‐ and down‐regulated proteins in KM12L4 and/or KM12SM compared with SW620Click here for additional data file.Table S3. List of gene alterations between KM12SM and SW620 cellsClick here for additional data file.Table S4. Determination of HRs and long rank P values for the up‐regulated secreted proteins in four CRC datasetsClick here for additional data file.Table S5. HRs of the six selected genes (SEC6) in different datasetsClick here for additional data file.Table S6. List of genes associated to cell migration, adhesion, EC organisation, and cell cycle in high‐risk CRC patientsClick here for additional data file.Table S7. HRs (95% CI) of high‐ and low‐risk patients classified by stages in survival analysesClick here for additional data file.


Punica granatum L.) flowers (PF), leaves (PL), peel (PP) juice (PJ) and seeds oil (PSO).Platelet aggregation and advanced glycation end products (AGEs) and oxidative stress are known as key factors for the development of cardiovascular diseases and diabetic complications. In this context, fruit and vegetable consumption, good sources of antioxidant compounds have been largely reported as an effective way of preventing human against these diseases. The current study focuses on the evaluation of antioxidant, antiplatelet and anti-glycation activities of pomegranate  against adenosine diphosphate (ADP), Collagen and arachidonic acid (AA).50 values of 0.7 mg/mL and 0.63 mg/mL respectively. For anti-glycation activity, PP, PF and PL inhibited moderately the pentosidine-like AGEs formation compared to positive controls with AGE-IC50 value of 0.4 mg/mL. PJ and PSO haven’t any anti-AGE effect. All the extracts selectively inhibited platelet aggregation caused by one, two or three inducers in dose dependent manner. PF was the most potent inhibitor caused by all three inducers, with inhibitory effects ranging from 35.6 to 66.6%. PP and PJ exhibited antiplatelet effect against both ADP and collagen and PL and PSO only against AA.PF extract displayed the highest antioxidant activity against ABTS and lipid peroxidation with ICThese results suggest that some pomegranate extracts exert potential in vitro anti-glycative and antiplatelet activities. Punica granatum L.), has been widely known as a very potent antioxidant fruit [Pomegranate  which have been reported to be correlated with the progression of diabetes and aging , 13. In Tounsi pomegranate trees in October 2021 from Mahdia region, Tunisia. Variety authenticity was confirmed by taxonomist Dr. Faten Zaouay from the Department of Horticulture, Higher Agronomic Institute, Chott-Meriem  and a voucher specimen was deposited in our national collection maintained in duplicate at Gabes and Chott-Mariem (Sousse), with the code ‘TNl, TN2, TN3, TN5, TN5”.Leaves and fruits were harvested from Pomegranate extracts were prepared as described by our previous study . Fruits 2S2O8) solution (2.7 mM). For the evaluation of antioxidant capacity, the ABTS• + solution was diluted with phosphate buffer  to obtain the absorbance of 0.700 ± 0.020 at 660 nm. Then, ABTS• + solution was mixed with pomegranate extracts prepared at different concentrations. After incubation, the absorbance was measured at 734 nm. Ascorbic acid was used as the positive control. The percentage of inhibition of ABTS• + radical was calculated with the following formula:The antioxidant capacity of pomegranate extracts by the ABTS  assay was measured using a previous method . Briefly50) was determined.Acontrol refers to the solution containing pure MeOH instead of the sample, and Asample refers to the absorbance of pomegranate extract containing solutions. The effective concentration of sample necessary to decrease the absorbance ABTS• + by 50% . Solutions were incubated in 96-well microtiter plates at 37 °C for 24 h in a closed system before AGE fluorescence measurement. Fluorescence resulting from the incubation, under the same BSA (10 mg/mL) and tested extract conditions, was subtracted for each measurement. Pentosidine-like  AGEs fluorescence was measured using a microplate spectrofluorometer. The percentage of AGEs formation was calculated as follows for each extract concentration and the EC50 values were determined:Inhibition of pentosidine-like AGEs formation and ECications . BrieflyFresh blood was obtained from healthy volunteers with negative history of consumption of drug, beverages or foods that may affect aggregation for at least 10 days and preferably should have fasted overnight because the presence of chylomicron may also disturb the aggregation patterns. The study was approved by the local ethics committee of the University Hospital Hedi Chaker of Sfax, Tunisia.Venous blood was collected in a plastic tube containing trisodium citrate 109 mM. PRP was obtained by centrifuging at room temperature for 12 min at 200×g. PRP was removed carefully avoiding contamination with red cells or buffy coat, and stored at room temperature until tested. All the tests should be completed within 3 hours of preparing the PRP. The remaining blood was than centrifuged at 2000×g for 20 min to obtain platelet-poor plasma (PPP). We used a screening panel of aggregating agents: adenosine 5′-diphosphate , collagen (5 μg/mL) and arachidonic acid (2 mM).PRP and PPP were used to set, respectively, 0 and 100% light transmission in the aggregometer. Platelet aggregation was monitored for at least 5 minutes after adding an agonist.For pomegranate leaves (PL), flowers (PF), juice (PJ) and peel (PP) extracts, different concentrations were prepared previously for each extract dissolved in DMSO . For PSO, different concentrations were dissolved in 70% Polyethylene glycol (PEG) which is a widely used solvent in an in vivo to dissolve water-insoluble compounds. Ten microliters of each extract were added to 260 μL of control PRP, and then the mixture was incubated for at least 5 minutes (until 30 min) at 37 °C before adding agonists. Then collagen (5 μg/mL), AA (2 mM) or ADP (20 μmol/L) was added and platelet shape change and aggregation were monitored for 5 min. DMSO (0.5% v/v) was used as negative control and aspirin was used as positive control.The extent of platelet aggregation was calculated by the following formula:D = platelet aggregation in the presence of test compoundsS= platelet aggregation in the presence of solvent.50), were obtained from at least three determinations.The platelet aggregation inhibitory activity was expressed as percent inhibition by comparison with that measured for the vehicle (DMSO or PEG) alone. Each sample was measured in triplicate and the data are presented as mean ± SD. The values of effective concentrations required for 50% inhibition of platelet aggregation (ECP < 0.05).Results were expressed as the mean of at least three independent measurements, unless standard deviations have been reported (means ± SD) and analyzed using SPSS ver. 21.0, professional edition. For antioxidant activities, Duncan’s test was used to estimate the significance among the main effects at the 5% probability level , slightly larger than the standard ascorbic acid (0.52 mg/mL), however this difference was not statistically significant (p < 0.05). PP extract followed by PL and PJ extracts are able to effectively reduce the free radical ABTS. The same order was found in lipid peroxidation tests. However, PSO demonstrated the weakest antioxidant activity in both in vitro assays. It is reported that there is an established relationship between the phenolic content and the antioxidant capacity [The antioxidant capacities of pomegranate extracts were measured by ABTS and lipid peroxidation assays. The results were summarized in Table capacity . In our capacity , we stud50 value of 0.4 mg/ml  and weak compared to Rutoside trihydrate . However, results show that PJ and PSO haven’t any anti-AGE effect . In double blind study, Sohrab (2015) concluded that pomegranate (Punica granatum) juice decreases lipid peroxidation, but has no effect on plasma advanced glycated end-products in adults with type 2 diabetes [The anti-glycation capacities of pomegranate extracts evaluated by their inhibition of the formation of global fluorescent AGEs in the BSA/ribose system are depicted in Fig. ml Table . This andiabetes . Our resdiabetes . The ant50 values of pomegranate extracts or compounds with the mean values of three measurements. All the extracts selectively inhibited platelet aggregation caused by one, two or three inducers in dose dependent manner.Pomegranate parts were evaluated for their ability to inhibit platelet aggregation of human PRP induced by ADP, Collagen and AA as potent aggregation inducers. Table 50 value of 2.8 mg/mL, then against AA-induced platelet aggregation with an EC50 value of 3.85 mg/mL and with 4.86 mg/mL when aggregation was stimulated by ADP. Compared to Aspirin as positive control, PF, PP and PJ have inhibitory effect against aggregation induced by collagen. However, Aspirin inhibited aggregation induced by AA and ADP with as EC50 of 0.42 and 0.66 mg/ml respectively but no effect was found against collagen. In this study and in our previous study [Flowers extract was found to be the most potent inhibitor of platelet aggregation caused by all three inducers, with inhibitory effects ranging from 35.6 to 66.6% at 3.5 mg/mL. In fact, it was active against collagen-induced platelet aggregation with an ECus study , PF are us study . Hydrolyus study . On the us study , 28. So,50 values revealed that PP decreased ADP and collagen-induced platelet aggregation more efficiently than PJ.PP and PJ exhibited inhibitor effect against both ADP and collagen-induced platelet aggregation. However, no effect was shown for both extracts when AA was used as agonist. Our results do not confirm with that found by Mattiello et al., 2009 who show that both extracts inhibit platelet response to AA . The comThe difference in inhibitory effect between both extracts can be explained by the difference in antioxidant capacity. PP was more potent antioxidant than PJ against ABTS radical and lipid peroxidation in this study and also against DPPH radical and reducing power . This exIn conclusion, pomegranate flowers, leaves and peel have in vitro inhibitory effects on protein glycation and platelet aggregation. These effects were attributed to the antioxidant properties of several pomegranate active compounds. However, further research is necessary to confirm these results and to obtain a deeper understanding of its mechanism of action, before being proposed as a natural AGE inhibitor. The antioxidant property? Active compounds in pomegranate that potentially/ contribute to these properties.