The best sensitivity (77%) and specificity (60%) of this model was with 1 point per variable and cut-off ≥2 points. The sensitivity remained good in our 3 main ethnic subgroups (74–88%). We identified predictors of IVIg resistance and built a new score with good sensitivity and acceptable specificity in a non-Asian population.About 10–20% of patients with Kawasaki disease (KD) are unresponsive to intravenous immunoglobulin (IVIg) and are at increased risk of coronary artery abnormalities (CAAs). Early identification is critical to initiate aggressive therapies, but available scoring systems lack sensitivity in non-Japanese populations. We investigated the accuracy of 3 Japanese scoring systems and studied factors associated with IVIg unresponsiveness in a large multiethnic French population of children with KD to build a new scoring system. Children admitted for KD between 2011–2014 in 65 centers were enrolled. Factors associated with second line-treatment; i.e. unresponsiveness to initial IVIg treatment, were analyzed by multivariate regression analysis. The performance of our score and the Kobayashi, Egami and Sano scores were compared in our population and in ethnic subgroups. Overall, 465 children were reported by 84 physicians; 425 were classified with KD . Eighty patients (23%) needed second-line treatment. Japanese scores had poor performance in our whole population (sensitivity 14–61%). On multivariate regression analysis, predictors of secondary treatment after initial IVIG were hepatomegaly, ALT level ≥30 IU/L, lymphocyte count <2400/mm The level of coronary artery involvement mainly determines the prognosis of this systemic vasculitis affecting predominantly young children, although pericarditis, myocarditis and valvular dysfunction are not uncommon2. Occasionally, KD can be complicated during the acute phase by shock syndrome3, macrophage activation syndrome4, or myocardial infarction1. Although the mortality rate is relatively low during the acute phase, sudden death due to myocardial ischemia could occur many years later in children or adults with coronary artery sequelae1.Kawasaki disease (KD) is the leading cause of acquired heart disease in childhood in developed countries6 and has reduced the prevalence of coronary artery abnormalities (CAAs) from 26–30% to 2.5–5% at 1 month after disease onset7. However, 30% to 40% of KD patients develop coronary dilatations within the first days of the disease8. In addition, approximately 10–20% of KD patients is considered resistant to IVIg and has been shown at increased risk of CAAs9. Therefore, early identification is critical to initiate more aggressive therapy11.The efficacy of early treatment with intravenous immunoglobulin (IVIg) is well established14. However, they lack sensitivity in North American16, European19 and other Asian populations21. Attempts to develop a scoring system with better performance in American multiethnic and Israeli populations were unsuccessful22. Moreover, reliable tools to detect children at high risk of IVIg resistance are essential because this population seems to have increased recently, from 7% to 23% in Japan23 and 10–20% to 38% in San Diego, USA16.Several scoring systems have been developed in Japanese populations to predict resistance to IVIg therapy and show good sensitivity (77–86%) and specificity (67–86%)25. In this study, we analyzed a large multiethnic French population of children with KD. To remain the most in keeping with the current practice, we investigated the necessity of a second-line treatment including possible IVIg re treatment after initial standard of 2 g/kg of IVIg, then we tested the accuracy of the Japanese scoring systems to predict IVIg resistance. Finally we attempted to build a new scoring system predicting secondary treatment in our large group of non-Asian patients with KD.Data on children with KD resistant to IVIg in Europe are scarce. In addition the definition of IVIg resistance based on persistence or recrudescence of fever after completion of IVIG infusion is not homogenous with a period of observation varying from 24 h to 36 hData were extracted from the Kawanet database, a national clinical and biological repository aiming to define the characteristics of KD in France. Patients admitted for KD in 65 French pediatric centers between January 1, 2011 and March 31, 2014 were prospectively or retrospectively enrolled in Kawanet by use of an online electronic case report form (e-CRF) implemented in a Web-secured system (CLEAN Web). The e-CRF contains demographic, clinical, biological, and radiologic information associated with treatments and 3-month follow-up.The study population was limited to children (≤18 years) and was classified according to the American Heart Association (AHA) criteria in 3 groups: complete KD , incomplete KD , and others not meeting those criteria. Records for patients in the last group were reviewed during a consensus conference with a nominal group technique by 6 experts from 5 centers  to determine whether patients had probable or a doubtful KD. Participants were given complementary clinical, biological and radiological information on submitted cases. Consensus was obtained if 5/6 experts (83%) provided the same response. Some patients were unclassifiable due to missing data or lack of consensus. Patients with probable KD were taken into account in analyses but those unclassified and doubtful KD were excluded.We analyzed demographic, clinical, biological and radiologic factors associated with unresponsiveness to IVIg. To avoid possible biases and controversies regarding the definition of IVIG resistance, we evaluated current practice decisions and analyzed in our KD cohort, the need for a second course of IVIg or second-line treatment (after the first IVIg infusion) with corticosteroids or anti-tumor necrosis factor agent.26. Data on CAAs were reviewed on original echocardiographic reports requested to investigators. Other cardiac complications such as myocarditis, pericarditis, and heart failure were also reported.CAAs were defined by detection of a coronary artery dilatation or aneurysms on 2-D echography at diagnosis or early follow-up. Because Z-scores were not available in most cases, coronary lesions were considered internal lumen diameter >3 mm in children <5 years old or >4 mm in children ≥5 years old, internal diameter of a segment measuring ≥1.5 times that of the adjacent segment; or a clearly irregular coronary lumen according to the Japanese Ministry of Health criteriaEthnicity was defined by origin of parents and classified into 5 groups: European Caucasian, North African/Middle Eastern, African/Afro-Caribbean, and Asian (Far East), or mixed (children with parents from different areas). Follow-up was defined by the number of days from diagnosis to the last visit in the center.4 IU/L (1 point), C-reactive protein (CRP) level ≥8 mg/dl (1 point), and illness days ≤4 days (1 point), with cut-off ≥312. The Sano score (0 to 3) includes CRP level ≥7 mg/dl (1 point), total bilirubine ≥0.9 mg/dl (1 point) and aspartate aminotransferase (AST) level ≥200 IU/L with cut-off ≥213. The Kobayashi score (0 to 11 points) includes sodium level ≤133 mmol/L (2 points), illness days ≤4 days (2 points), aspartate aminotransferase (AST) level ≥100 IU/L (2 points), neutrophil count ≥80% (2 points), platelet count ≤30.104 IU/L (1 point), CRP level ≥10 mg/dL (1 point), and age ≤12 months (1 point). We investigated the accuracy of the Kobayashi score using a cut-off ≥4 described in the original study published in 2006 in patients treated with IgIV 1 g/kg for 2 consecutive days as well as a cut-off ≥5 as described in the external validation study published in 2011 comparing children treated with IgIV 1 g/kg for 2 consecutive days and 2 g/kg in a single infusion27.Kobayashi, Egami and Sano scores which predict resistance to treatment in Japanese population were studied in our population and in ethnic subgroups. The Egami score (0 to 6 points) includes age at diagnosis ≤6 months (1 point), alanine aminotransferase (ALT) level ≥80 IU/L (2 points), platelet count ≤30.10Descriptive statistics were used for demographic, clinical, biological and radiologic data. Continuous data are described with mean± SD and categorical data with number (%). Data were compared by chi-square test or Fisher’s exact test for categorical variables and Student’s test or Mann-Whitney test for continuous variables. Two quantitative variables  were transformed into clinically relevant classes: age, <1 year, 1–4 years and >5 years; and time to treatment (time between the first day of fever and IVIg treatment), <5 days, 5–10 days and >10 days. Quantitative variables (laboratory variables) were transformed into binary variables by using the receiver operating characteristic (ROC) curve. To obtain the optimized cut-off value, the distance between the point  and any point on the ROC curve was minimized. The clinical relevance of the threshold was discussed. We estimated odds ratios (ORs) and 95% confidence intervals (CIs). Multivariate analysis involved logistic regression models to identify factors associated with IVIg unresponsiveness. Variables significant at p ≤ 0.20 on univariate analyses were included in the stepwise selection. Potential interactions between variables were tested. The best model was determined by using Akaike Information Criteria. We built a scoring system based on our multivariate analysis for the IVIg resistance model. For each patient, variables retained in the model determined number of criteria. ROC curves were used to obtain the optimized cut-off value for the score maximizing sensitivity and specificity to distinguish unresponsive and responsive patients to the first IVIg infusion. Sensitivity, specificity for our score and the Kobayashi, Egami and Sano scores which predict unresponsiveness to treatment in Japanese populations were studied in our population and in ethnic subgroups.Statistical analyses involved use of SAS v9.3 . All tests were two-tailed, with p < 0.05 considered statistically significant.The study protocol followed ethics guidelines (CPP no.CO-10–002) and was approved by the Comité Consultatif sur le Traitement de l’Information en matière de Recherche dans le domaine de la Santé  and the Commission Nationale de l’Informatique et des Libertés . Informed consent was obtained from parents or legal guardians of children included in the study.Overall, 84 physicians reported 467 patients from 65 French centers. Two adult patients were not included in the analyses. A total of 355 patients (76%) fulfilled criteria for complete or incomplete KD. Among the 110 other patients, records for 13 were not usable because of missing data. The 97 remaining patients were classified as probable KD, 70 (72%), and doubtful KD, 27 (28%): meaning that in 27/452 (6%) cases, the diagnosis of KD was challenged by experts , and although mean delay to treatment was almost the same for the 3 subgroups, patients with incomplete KD were more frequently treated after 10 days of fever (22% vs. 11% for complete KD and 7% for probable KD) (P = 0.02). Only 3/425 had initial combined therapy with IVIG plus steroids.For the 425 patients classified with KD (AHA + experts confirmation), 242 were boys. The mean age at disease onset was 2.8 ± 2.4 years range 0.1 to 14.4) and mean delay to IVIg treatment 6.6 ± 3.6 days  were European Caucasian descendants, 50 (12%) North African/Middle Eastern, 43 (10%) African/Afro-Caribbean, 11 (3%) Asian and 47 (11%) mixed (Table Information on response to the first IVIG treatment was available for 415/425 patients (98%) with KD; 92 (22%) needed second-line treatment. On univariate analysis, IVIg unresponsiveness was strongly associated with clinical or radiological hepatic or cardiac involvement Table . BiologiWe could estimate the Egami, Sano and Kobayashi scores (with cut-off ≥4 and ≥5) for 328, 211 and 334 patients, respectively . For patients without AHA criteria, expert agreement with the referring physicians for KD diagnosis was high, 72%, reflecting a high level of knowledge of KD among French pediatricians, with a possibility of 6% over diagnosis. As a whole, the clinical characteristics of French KD patients agreed with those previously known in terms of sex ratio, age of onset, and distribution of clinical symptoms31. Patients of African/Afro-Caribbean ethnicities tended to be younger than European Caucasians and North African/Middle Eastern patients and had significantly less adenitis and more erythema and desquamation of the bottom. Unfortunately, our Asian population (n = 11) was too small to allow any comparison.Kawanet aimed to analyze the degree of awareness of pediatricians and to have a thorough picture of what is called KD in France, how the disease is managed and the response to standard treatment (IVIg) but it is not a tool dedicated to calculating KD incidence 7 is due to our definition of cardiac complications — presence of any coronary dilatation on any echocardiography ever, even if the dilatation disappeared — whereas previous rates were estimated at 1 month of disease evolution33. Because of the little published information, we could not confirm a possible reduced or increased risk of coronary dilatation in African/Afro-Caribbean patients35. Unfortunately, absence of Z-scores in Kawanet limited a more accurate analysis of cardiac complications1. Therefore, we focused on unresponsiveness to treatment. A relatively high proportion of our patients (22%) required secondary treatment (including IVIg re treatment) after standard treatment with 2 g/kg IVIg. Overall, the Egami, Sano and Kobayashi predicting IVIg resistance did not provide enough performance in our multi ethnic KD population. However, and to our knowledge, for the first time, we observed a better performance of two of these scores in African/Afro-Caribbean patients compared to other ethnic groups, with the exclusion of Asians. Prediction of IVIg resistance is crucial to intensify initial treatment combined with IVIg to prevent cardiac complications37, but none of the currently published scores have good sensitivity in Caucasian populations38. In our multiethnic population, we identified predictive factors of IVIg resistance based on real-life practice, and built a scoring system and obtained for the first time, good sensitivity (77%) and acceptable specificity (60%) in our non-Asian population. The sensitivity remained good in our 3 main ethnic subgroups (74 to 88%). Besides our new score, other predictors of IVIg resistance recently identified include proportion of neutrophils, hemoglobin level, CRP level, procalcitonin level, erythrocyte sedimentation rate, albumin level, creatinine level, sodium level, N-terminal pro-brain natriuretic peptide, interleukin-6 and −10 levels40 and baseline Z-score ≥2 or echocardiogram alteration, all found to predict high risk KD patients41.Although our KD cohort was early diagnosed and treated with an accurate regimen of IVIg, we observed a high rate of cardiac complications, especially coronary dilatations, 49%, that did not differ among our 3 ethnic populations. This discrepancy with the 26–30% coronary complications without IVIg treatment described in the literature19, we maximized sensitivity of the score despite an acceptable slight decrease in specificity.The strengths of the Kawanet study are the wide data collection, which was predominantly prospective, the multiethnic distribution of patients, and the involvement of independent experts for case adjudication. In addition, the definition of IVIG unresponsiveness was based on current practice data rather than on any potentially controversial definition. However, the limitations include selection biases: adult patients were not included, potentially most severe patients in intensive care units were not recruited, the recruitment was voluntary and finally, our study could not compare Asian and non-Asian patients. In order to minimize risk of aneurysms associated with unresponsiveness to IVIgWe identified predictors of IVIg unresponsiveness in our cohort of KD patients and built a new score with good sensitivity and acceptable specificity in the non-Asian population. Our score, which should be tested in other multiethnic KD cohorts, is sensitive enough to be clinically useful in making decisions about initial and alternative treatments in non-Asian populations.


Talin1 was significantly associated with advanced TNM stage (p = 0.034) as well as gender (p = 0.029) in mRNA levels. Similarly, IHC results indicated that a low level of cytoplasmic expression of Talin1 was significantly associated with advanced TNM stage (p = 0.028) as well as gender (p = 0.009) in CRC patients. Moreover, decreased expression of cytoplasmic Talin1 protein was found to be a significant predictor of worse disease-specific survival (DSS) (p = 0.011) in the univariate analysis. In addition, a significant difference was achieved (p = 0.039) in 5-year survival rates of DSS: 65% for low, 72% for moderate, and 88% for high Talin1 protein expression. Observations showed that lower expression of Talin1 at both the gene and protein level may drive the disparity of CRC patients’ outcomes via worse DSS and provide new insights into the development of progression indicators because of its correlation with increased tumor aggressiveness.To explore the proper prognostic markers for the likelihood of metastasis in CRC patients. Seventy-seven fresh CRC samples were collected to evaluate the mRNA level of the selected marker using Real-time PCR. Moreover, 648 formalin-fixed paraffin-embedded CRC tissues were gathered to evaluate protein expression by immunohistochemistry (IHC) on tissue microarrays. The results of Real-Time PCR showed that low expression of  It is ranked as third in incidence but second in mortality in different countries, and most of deaths are caused by metastasis; hence, the prevention of subsequent metastasis is the main focus of clinical research4. Robust determinants of CRC prognosis include local involvement, lymphovascular invasion, positive surgical margin, regional lymph node metastasis, pre-operative elevation of CEA, and high tumor grade6. Moreover, traditional biomarkers, colono/sigmoidoscopy, imaging techniques (magnetic resonance imaging (MRI) and computed tomography (CT)) play specific roles as the gold standard in colorectal screening; however, they are not satisfactory enough7. Unfortunately, the early spread of tumor cells is usually not detectable, and access to more detailed information related to the identification of metastasis-specific molecular markers is urgently warranted.Colorectal cancer (CRC) is one of the frequent forms of solid tumor diagnosed in both sexes worldwide8. The cancer patient's blood contains diverse tumor-derived materials, including circulating tumor cells (CTCs), and exosomes enable the minimally invasive monitoring of tumor evolution over time in the clinic10. It seems that achieving the molecular signature based on CTCs and exosomes, which is completely representative of the tumor, plays a significant role in precise theranostics and can be utilized in combination with clinicopathological staging. From a prognostic perspective, the identification of tumor metastasis-specific molecular markers which cover cancer heterogeneity and increase longitudinal surveillance of patients is essential for precision medicine11.Histopathological analysis as a prevalent standard test is applied to assess novel molecular biomarkers through simultaneous analysis of hundreds of tissue samples in the clinical setting13. To the best of our knowledge, just a couple of studies have investigated the direct correlation between molecular metastasis cascades in CTCs and exosomes. Hence in the present study, databases were screened in order to analyze the common expression profile of these biomarkers as an essential step in understanding the mechanism of metastasis and in discovering the prognostic markers for CRC14. This study was performed using integrated analysis of bioinformatics data related to the molecular markers involved in CRC metastasis in both CTCs and exosomes. Using the GEO, Exocarta, and Vesiclepedia databases, similarities in the expressed genes among the above-mentioned biomarkers were identified. Both gene ontology (GO) analysis and KEGG pathway15 were examined to identify key candidate genes, and it was found that Talin1 could provide significant prognostic value in CRC metastasis.Although large‐scale studies have been carried out to identify novel biomarkers with great prognostic value, investigations of metastasis-related molecular markers should be increased to actualize the exact prediction of the time of metastasis. Advances in high throughput technology in omics data, especially transcriptomic approaches such as microarrays and RNA sequencing, would increase our knowledge of the molecular markers involved in CRC metastasis, resulting in the identification of potential therapeutic targets for CRC patientsTalin1 in fresh colorectal tumor samples and their adjacent normal tissues as the control were evaluated using Real-Time PCR. Then, to explore the protein localization and expression level of Talin1 in CRC samples as well as its correlation with clinicopathological features and survival outcome in CRC patients, the findings from the analysis of gene expression data were validated and confirmed on a large series of CRC tissue microarrays (TMAs) using the immunohistochemistry (IHC) technique.The evaluation and validation of biological fluids markers as debatable matter should be assessed on tissue samples because of the lack of a consensus over isolation and characterization. Therefore, this step was taken in the current study to optimize the novel findings in the broad range of samples. The mRNA expression of TLN1, TMEM51, GNPNAT1, RPS4X, MARCKSL1, FARP1, RHOB, HSP90AB1, LFNG, AHNAK, RPSA, CNPY2, PRDX1, SLC25A5, and TGFBI, as shown in Fig. p value < 0.05 as the cut-off criteria are summarized in Supplementary Table To find the valuable molecular marker between CTCs and tumor-derived exosomes that was affected in metastasis after online analysis of the total similarity of their expressed genes, the following 15 genes were found: 17, even as a regulators of cancer stemness, metastasis, and drug resistance18. Talin because of structural and functional significance with integrin cytoplasmic tails resulting in extracellular integrin compartments rearrangements and integrin activation19. Specifically, Talin1, which functionally contributes to the extravasation and metastasis of colon cancer CTCs20, was selected for the current evaluation. Talin1 protein expression was significantly localized in the cytoplasm that mediates integrin interactions with the extracellular matrix (ECM). From another side, for cell spreading and migration, the link between ECM- bound integrins and intracellular F actin is vital21. Talin1 mediates both inside-out and outside-in signaling, which leads to subsequent cascades23 and also has a direct effect on the regulation of insulin signaling, actin dynamic, and granule trafficking24. Talin serves as tension-sensing anchoring as well as a unique mechanotransductive point to link the cells to the ECM. It has been proven using cryoelectron microscopy (cryo-EM) full-length structure examination that the function of Talin could be regulated as a two-way mode of auto-inhibition, and the fast switching between its inactive conformations and active could regulate Focal adhesion kinase (FAK) turnover25. In our evaluation, focal adhesion among cellular components, cadherin, and RNA binding between molecular function were spotlighted. Also, cytoskeletal anchoring at plasma membrane, platelet aggregation, cell–cell adhesion, and junction throughout the biological process were considered significantly highlighted. Additionally, the KEGG pathway  of which came from males and 25 (32.6%) from females . The median age of patients was 60 years ; 37 (48%) patients were aged ≤ 60 years and 40 (52%) were aged > 60. Eight (10.4%) cases were diagnosed as stage I, 25 (32.6%) stage II, 27 (35%) and 17 (22%) stage III and IV respectively. Thirty-two (41.6%) cases were low-grade adenocarcinomas, and 14 (18.2%) and 31 (40.2%) were moderated and poorly differentiated adenocarcinomas, respectively.Talin1 is ubiquitously expressed. Websites like Uniprot27 and Genecards28 showed it as localized within the cytosol, extracellular, and in the cytoskeleton. The expression profiles of Talin1 were decreased in patients with CRC compared with the healthy group (p < 0.043) according to Real-Time PCR  as well as gender (p = 0.029), indicating an association between the low level expression of Talin1 and an increased TNM stage  and stages II and III (p = 0.028). Further, we did not observe any statistically significant differences in the median level of Talin1 mRNA expression between other stages  and 27.2 (SD = 3.4) months, respectively.From the 77 CRC samples, 70 (91%) patients had no history of recurrence, while 7 (9%) patients were positive. In 42 (54.5%) metastasis occurred, while 35 (45.5%) patients were negative for it. Thirty (39.0%) patients had no history of metastasis, recurrence, or cancer-related death, while 47 (61.0%) patients for these parameters were positive. During the follow-up period, cancer-related death in 26 (33.8%) patients was reported. The mean duration of follow-up time was 20 months , and the range was from 1 to 33 months. The mean DSS time for patients with high and low expression of Talin1 mRNA expression had shorter DSS compared to patients with high expression of Talin1 but without a statistically significant association   Fig. A.Figure Talin1 mRNA were 24.9 (SD = 0.89) and 21.7 (SD = 3.2) months, respectively. The Kaplan–Meier curve showed that low level Talin1 expression is not significantly related to PFS (p = 0.698)  technology has covered the evaluation of clinical values of huge number of candidate markers, 760 cases were included in this research, and for final scoring, 648 CRCs tissues were left. The FFPE population samples consisted of 303 (46.8%) male and 262 (40.4%) female patients, with a male/female ratio of 1.15. The median age of patients was calculated as 60 years . To clarify, 298 (46.0%) were ≤ 60 and 267 (41.2%) were > 60. Tumor size ranged from 1 to 20 cm, and tumors were classified into two groups. Three hundred and eighty-two (59.0%) of cases had tumors that were smaller in size than the mean (size ≤ 4.5) and 181 (27.9%) had tumors larger than the mean (size > 4.5). In this study, 90 (13.9%) patients had stage I, 254 (39.2%) stage II, 198 (30.6%) stage III, and 22 (3.4%) stage IV tumors. Moreover, 212 (32.7%) cases were grade I, 316 (48.8%) were grade II, and 34 (5.2%) were grade III; VI was found in 87 (13.4%) cases, and LNI and NI were observed in 212 (32.7%) cases. Tumor recurrence was seen in 96 (14.8%) patients, and 90 (13.9%) patients showed distant metastasis during the follow-up period.Talin1 protein was evaluated using IHC on TMA sections by three scoring methods. H-score consist of staining intensity and percentage of positive tumor cells. In the CRC samples, Talin1 was expressed at varying intensities in the cell cytoplasm and ECM. Cytoplasmic and ECM expressions of Talin1 were detected in 643 (99.2%) and 628 (96.9%) CRC cases, respectively. The mean expression of Talin1 was 149 in cytoplasm and 138 in ECM. Talin1 expression based on the H-score demonstrated that low, moderate, and high levels of cytoplasmic expression of Talin1 were observed in 205 (31.6%), 349 (53.9%), and 94 (14.5%) CRC samples, respectively. Moreover, low, moderate, and high ECM expression of Talin1 was found in 235 (36.3%), 379 (58.5%), and 34 (5.2%) CRC patients, respectively  as well as gender (p = 0.009)  (p = 0.004). The results of the Mann–Whitney U test revealed a statistically significant difference in the median level of Talin1 protein expression between stages I and II (p = 0.013) and stages II and III (p = 0.001). We did not find any statistically significant differences in the median level of Talin1 protein expression between other stages of CRC cases  Table . The non p = 0.02 as well Talin1 protein expression and advanced TNM stages (p = 0.038). No association was found between cytoplasmic Talin1 protein expression and the other clinicopathological characteristics  patients had no history of metastasis, recurrence, or cancer-related death, while 111 (31.7%) patients for these parameters were positive. In 91 (26.0%) patients metastasis and in 96 (27.4%) ones recurrence occurred. In 97 (27.7%), patients cancer-related death was documented during the mean duration of 45.0 months (SD = 27.6) as follow-up time with a median of 41.0 months ; the range was from 1 to 117 months.Talin1 and DSS  , 86 (SD = 3.7), and 47 (SD = 4.0) months, respectively. Furthermore, the 5-year survival rates for DSS in patients whose samples expressed three groups of cytoplasmic Talin1 expression was 88%, 72%, and 65%, respectively (p = 0.039). Besides, there was no significant differences between PFS and patients with cytoplasmic Talin1 expression levels (p = 0.079) , 81 (SD = 3.9), and 74 (SD = 4.3) months, respectively.Kaplan–Meier curve results indicated significant differences between the patients with three groups of cytoplasmic expression rates of 48) Fig. A. The meTalin1 expression was an independent prognostic factor of DSS or PFS and to assess the clinical significance of various parameters that might influence survival outcomes in CRC patients29. Among all of the clinicopathological parameters, only cytoplasmic Talin1 expression (p = 0.050), LNI (p = 0.010), NI (p = 0.008), distant metastasis (p < 0.001), tumor recurrence (p < 0.001), and age (p = 0.009) affecting DSS in univariate analysis. In addition, in multivariate analysis, only age (p = 0.015) was significantly related to DSS in these cases , respectively. Moreover, the results of univariate and multivariate analyses indicated that the listed clinicopathologic variables were not affecting the DSS or PFS of CRC patients.Kaplan–Meier analysis showed that there were no differences between DSS or PFS and the patients with three groups of ECM expression of 30, the identification and characterization of both molecular markers as powerful tools in non-invasive cancer monitoring are significant steps to improving our understanding of molecular and cellular mechanisms of cancer metastasis31. Furthermore, the identification of new prognostic markers for the likelihood of metastasis that can stratify patients into various classification is clearly warranted. Therefore, considering the heterogeneity and complexity of CRC, this study was conducted to predict prognoses of CRC patient based on the evaluation of their gene expression network and to the clinical significance of their common molecular marker by bioinformatics analysis in multiple fashions. We take advantage of the limited available data related to CTC and exosome collection assays throughout online web tools for tracking CRC metastasis. “Wet-lab” experiments using patient specimens for identifying cancer biomarkers through bioinformatics analysis were considered vital. The best application of that can be develop a practically manageable gene based on applying p values and enrichment pathway analysis32.Because cross-talking between cancer cells and exosomes plays a vital role in the CRC dynamic network, especially in metastasis7. Since the limitation of these various methods are considerable33 shifting to CTCs and TDEs as a liquid biopsy approach35 attracted much attention and help to discover markers which are important in diagnostic, prognostic and cancer staging37, estimation of relapse risk, therapeutic targets identification39, and proper monitoring of patients40. Regardless of the metastatic site, CTC enumeration are enough as a proper cancer-monitoring index whenever CEA and other marker levels are not measurable42.Traditional biomarkers , as well as colon/sigmoidoscopy, play an unsatisfactory specificity role in CRC screening43. EMT plays an important role in the invasion and metastasis of CRC, which is mediated by FAK. However, whether FAK participates in EMT, through the EGF/EGFR signaling pathway, remains unknown44. Talin1 can activate integrin's as core regulators45 and play an important role as a focal adhesion proteins as well as be mediators of adhesion to the ECM46. Therefore, it must be considered extensively to determine its role clearly. FAK-Talin binding is required for adhesion turnover and cell motility and in our view has a significant point in common with molecules between circulating tumor cells (CTCs) and tumor-derived exosomes (TDEs).Although several mechanisms are involved in cancer progression, recent evidence points to EMT and CSCs as having significant roles in these processesTwist 49, E-cadherin, vimentin, N-cadherin, Snail, Slug, integrin's, cytokeratin's, fibronectin, b-catenin, ZEB1, ZEB2, and TGFb51. The correlation analysis was performed between two markers  and the results showed that there is no significant correlation between cytoplasmic expression of them in these series of CRC patients .In malignant tumors, EMT is crucial for acquisition of a mesenchymal phenotype with invasive and metastatic properties leading to tumor progression. The important markers related to this process include Talin1 was selected due to its most important biological roles in migrant cells. Assembly and disassembly of FAK including Talin are essential for cell migration; meanwhile, molecular mechanisms details remained poorly understood and must be elucidated52. Up and down regulation of FAK protein expression might have a profound effect on signal transduction; for example, FAK expression levels were increased in primary CRC compared with normal mucosa and decreased in liver metastases53. In addition, our findings point towards Talin1 targeting as a promise for the future, and we supposed that EMT and stemness have been extensively investigated in the field of CTC-related research54. With regard to metastasis, the identification of molecular markers that robustly define a subset of CTCs which are enriched in CSCs are vital55. Thus, an improved understanding of the EMT/CTC/CSC connections may uncover the novel therapeutic targets and underline tumorigenicity mechanisms56.Additionally, Most of the fifteen genes that were obtained from our bioinformatics analysis, have been previously implicated in cancer development, but Talin1 expression in CRC in various areas such as by western blot analysis, HPLC-Chip/MS analysis, and enzyme-linked immunosorbent assay (ELISA). Nevertheless, no study was found that addressed the expression of Talin1 either in mRNA or protein levels in CRC tissue. Hence, due to the existence of information gaps regarding the expression of Talin1 in both mRNA and protein levels, the present study was designed to examine that in a well-characterized series of 77 fresh and 648 FFEP CRC tissue samples. This is also the first report of localization of Talin1 expression in cytoplasm and ECM in a large number of tumor samples with evaluation of its impact on CRC prognosis. The current results showed that low level mRNA expression of Talin1 is associated with increases in TNM stages. Furthermore, the results of TMA revealed an adverse association between the cytoplasmic expression levels of Talin1 and increasing TNM stage and gender, while no association between the ECM expression of Talin1 and the clinicopathological parameters was found. It was determined that the localization of Talin expression depends completely upon its function related to integrins. Moreover, a statistically significant difference was observed in the median level of Talin1 mRNA and protein expression between different stages. To clarify, the comparison of low stages (I–II) and high stages (III) showed that Talin1 mRNA and protein cytoplasmic expression levels are associated with CRC aggressiveness. It is noteworthy that the continuous refinement of the TNM system, T (tumor), N (node), and M (metastasis) staging define the prognosis and patient outcome and play a pivotal role in decision-making for treatment58. Accumulating data underlines the importance of structuring the management of CRC patient's survival by stages at diagnosis and prognostic stratification60. In addition, LNI, NI, distant metastasis, tumor recurrence, and age were prognostic variables in univariate analysis that depicted the associations between these parameters and more aggressive tumor behavior. In cytoplasmic Talin1 expression, only age was a prognosis factor affecting the DSS in multivariate analysis. It was previously shown that age and comorbidities worsened the survival rate of CRC patients61. In this study, the results exhibited that low level cytoplasmic expression of Talin1 compared with ECM expression is related to the degree of malignancy and progression in CRC cases. Talin1 cytoplasmic expression can be considered more significant in comparison with ECM ones63.Several studies have previously investigated levels of Talin1 expression, analysis, and localization in various tissues. The first study related to Talin expression showed that it increased64, but eight years later the reduced expression of Talin in liver metastases compared with matched primary human CRC was reported65. Barbazan et al. introduced one molecular pattern in CRC for CTC detection67. Their research was continued by Insua et al., who included Talin1 as a gene involved in CTC extravasation during metastatic dissemination in their gene expression panel, which was composed of GAPDH, VIL1, CLU, TIMP1, TLN1, LOXL3, and ZEB2. This panel was checked as a prognostic and predictive tool in metastatic CRC blood samples. Patients with higher gene panel expression compared with low ones had reduced PFS and OS rates (p = 0.003 and p = 0.001), respectively65. According to the current results, this prognostic value seems to be related to Talin1 expression accompanied by the other markers, which were important in the panel53. One recent study revealed that Talin1 expression was upregulated in CRC, and the proliferation, migration, and invasive ability of the CRC cell line was significantly reduced by its knockdown compared with the control. In the current study, however, it was downregulated in advanced tumor progression68. Analysis of Talin1 using ELISA showed no significant correlation between its preoperative levels in the serum of patients and age and gender; however, a significant correlation between Talin1 levels and tumor grade, TNM stage, and lymph node metastasis was found69.Previous articles have shown that there are many challengeable reports regarding Talin1 protein tend to have a worse prognosis for DSS. Moreover, CRC patients who expressed a lower level of cytoplasmic Talin1 had a worse 5-year survival rate for DSS compared with those with high expression. Thus, low expression level of Talin1 can be considered a worse prognostic factor of DSS in CRC patients. Talin1 levels could not be used as an independent prognostic factor of DSS. More prolonged follow-up time may increase the prognostic value of Talin1. In mRNA expression, the current finding showed that patients with low Talin1 mRNA expression had shorter DSS compared to patients with high expression of Talin1, but the difference was not statistically significant. This might be due to the limited sample size and shorter follow-up period in comparison with FFEP samples, because all fresh tissue samples were collected on 2017.The results of the Kaplan–Meier curve on protein determined that tumors with low-level expression of the Talin1 in some malignancies in its importance in cancer development, invasion, migration, and metastasis, and even introduces it as a cancer stem cell (CSCs) marker, because in the current study, Talin1 was a common marker between CTCs and exosomes72. Therefore, it must be mentioned that Talin1 can be an extremely helpful factor for a worse prognosis of DSS in CRC patients. Moreover, the use of IHC can verify the Real-Time PCR results and establish Talin1 status in routine clinical practice.The current study was similar to other studies conducted on Talin1 mRNA expression is associated with more aggressive tumor behavior in CRC patients. Furthermore, we found that decreased cytoplasmic expression of Talin1 rather than its ECM expression has a clinical significance in CRC cases and is associated with increased invasiveness and poor prognosis risk for DSS in univariate analysis. To sum up, investigation of the expression pattern of Talin1 in the cytoplasm of tumor biopsies as a predictor and prognostic indicator of cancer progression in CRC patients’ tumor biopsies is useful. Extending the follow-up time might increase the prognostic value of Talin1 as an independent prognostic factor, but that must be confirmed in further studies.The current study is the first to exhibit that low level https://www.ncbi.nlm.nih.gov/geo) based on the GPL13497 (Agilent-026652 Whole Human Genome Microarray 4 × 44 K v2) platforms, containing the CTC in CRC patients (n = 6) and healthy subjects (n = 3). The GEO2R online analysis tool to detect the DEGs was applied, using a p value < 0.05 and |logFC|≥ 2 as cut-off criteria.To find the common molecular marker between CTCs and exosomes, first, GSE31023 gene expression and CTC profiling by array were obtained from the NCBI GEO database  determines whether a prior defined set of genes shows statistical significance76. Thus, the initially extracted markers in this article were subjected to STRING (https://string-db.org/) and EnrichR (amp.pharm.mssm.edu/Enrichr/) to obtain a better comprehensive of the significantly related pathway, which is described in Supplementary Table Molecular markers are valuable tools in the development of cancer screening and monitoring strategies. GO and GSEA are the common useful methods employed in this study to suggest new markers among the CTCs and exosomes in CRC samples. GO is useful for annotating genes and identifying characteristic biological attributes for high-throughput data analysisTalin1, 760 FFPE tissues of CRC taken from 2010 to 2019 were collected from university-based hospitals  in Tehran, Iran. Hematoxylin and eosin (H&E) stained slides and archived medical records were retrieved to obtain patients’ clinicopathological characteristics, including age, gender, tumor size (maximum tumor diameter), tumor differentiation, TNM stage, venous invasion (VI), lymph node invasion (LNI), neural invasion (NI), distant metastasis, and tumor recurrence. In addition, 60 adjacent normal tissue samples were used to compare expression levels of the Talin1 markers in a range of tissue samples. From the date of surgery to the date of death was measured as Disease-specific survival (DSS). The interval between the primary surgery and the last follow-up visit was considered as Progression-free survival (PFS) if the patient showed no evidence of disease, metastasis, recurrence, CRC or disease-related death.In the current study, 77 CRC fresh tumor tissues and their adjacent normal tissue samples from 2018 to 2019 were collected from university-based hospitals (Firoozgar and Bahman) in Tehran, Iran. Patients who had undergone surgery without receiving any relevant chemotherapy or radiotherapy were included in this study. The specimens were embedded in RNA later solution, transferred to the laboratory, and frozen in liquid nitrogen. Patient information including gender, age, TNM stage, tumor differentiation, and distant metastasis was also recorded. To evaluate the protein expression of Talin1 expression level was detected in forty pairs of CRC and adjacent normal tissue samples. The primer sequences were as follows: GAPDH: 5′-AACTTTGGCATTGTGGAAGG-3′ F and 5′-CACATTGGGGGTAGGAACAC-3′ R. Talin1: 5′-.TTGGAGATGCCAGCAAGCGACT-3′ F and 5′-CCAGTTCTGTGGCTGCCTGATT-3′. The expression levels of Talin1 mRNA were normalized against GAPDH levels based on the 2−ΔΔCt approach77.Briefly, tissue specimens were lysed and homogenized in the presence of a highly denaturing guanidine-thiocyanate-containing buffer that inactivates RNases. Trizol reagent  added to provide appropriate binding conditions were grounded. Then, the total RNA was isolated based on the RNAeasy Mini Kit  protocol. One μg of RNA was reverse transcripted with PrimeScript 1st Strand complementary DNA Synthesis kit (Takara). Real-time PCR assay was performed on the Qiagen Rotor Gen Q system using the SYBR green Premix Dimer Eraser kit (TaKaRa cat number: RR820Q). The cycling conditions were an initial 30 s denaturation at 95 °C and 40 cycles . The GAPDH gene was set as internal control; 78. Briefly, our pathologist (Z.S.) examined the H&E slides to select the representative tumor area in each block. Then, using a precision arraying instrument , selected spots were punched into recipient blocks, and TMA blocks were constructed in three copies. In each TMA block, adjacent normal tissue samples were also included to compare the expression pattern of Talin1 with tumor tissue specimens80. Due to technical problems and an insufficient amount of tissue for scoring, some cases were lost during the staining and excluded from the study. Tumor heterogeneity is a major concern during the TMA procedure, and thus, at least two or three cores were evaluated from each sample to achieve better results82.Colorectal tissue TMAs were prepared as described previouslyTalin1 was evaluated using our IHC laboratory protocol y83. Sequential TMA sections at 60 °C for 30 min were dewaxed, in xylenes rehydrated, and then under ethanol treatment graded. 3% H2O2 (endogenous peroxidase) were applied as blocker for 20 min at room temperature. Antigen was retrieved by autoclaving tissue sections for 10 min in sodium citrate buffer (pH 6.0). Then, all of the slides were blocked  for 20 min. After serial dilution, 1:1000 for anti-Talin1 antibody was prepared as an optimal dilution for subsequent use. The tissue sections were incubated overnight at 4 °C with the following antibody dilutions: anti Talin1 antibody . The next day, after three washes in Tris-buffered saline (TBS), sections were incubated with anti-rabbit/anti-mouse envision IgG–HRPO  as the secondary antibody for 1 h. 3,3′-diaminobenzidine (DAB) (Dako) substrate as a chromogen for treating the TMA slides were used in 3 min at room temperature. Sections were counterstained with hematoxylin (Dako), in alcohol dehydrated, with xylenes cleared and mounted. TBS was replaced for negative controls, and only the secondary antibody was used. Meanwhile, human kidney tissue was used as a positive control for Talin1 staining.The protein expression of Our pathologist (Z.S.) using a semi-quantitative scoring examined the immunostained tissue arrays system in a coded manner. Consensus was achieved by the second investigator (Z.M.) in difficult cases for confirming the scoring.Talin1 marker was expressed at various intensities in the cell cytoplasm and ECM, each of which was analyzed separately. The intensities of Talin1 staining were scored on a 4-point scale, ranging from negative to strong . The percentages of positive tumor cells varied between zero and 100. Histochemical score (H-score) as an overall score was achieved by multiplying the intensity of staining by the percentage of positive cells; then, a final score of 0 to 300 was given to each core. In the current study, the mean H-scores were classified into three groups: 1. low expression (0–100), 2. moderate expression (101–200), and 3. high expression (201–300).In the CRC samples, the Talin1 expression and clinicopathological parameters. Kruskal–Wallis and Mann–Whitney U tests were applied for pairwise comparisons between groups. DSS and PFS curves were drawn using the Kaplan–Meier method, and the log-rank test was used to compare the estimated curves between groups with 95% confidence intervals (CI). To clarify that which variables affected DSS or PFS, the Cox proportional hazards regression model was applied. In all parts, a p-value of < 0.05 was considered statistically significant. As noted, in the first step, all quantified data was replicated an average of three times.SPSS software version 22.0  was utilized to analyze the data. Categorical data was reported by N (percentage) and quantitative data as mean (SD) and median . Pearson’s χ2 and Spearman’s correlation tests were used to analyze the significance of associations and correlations between The Research Ethics Committee of Iran University of Medical Sciences issued IR.IUMS.REC 1395.9221513203 for this study. All procedures, including obtaining informed consent from each human participant before surgery, were in accordance with the above-mentioned ethical standards.Supplementary Information 1.Supplementary Information 2.


More than 50% of the participants were found to have moderate or high 5-year CVD risk. Observed prevalence of MS among HIV patients was higher than other studies done in India. Considering a sizeable number of participants to be having moderate to high CVD risk, culturally appropriate lifestyle interventions need to be planned.Longer survival due to use of antiretroviral therapy (ART) has made human immunodeficiency virus- (HIV-) infected individuals prone to chronic diseases such as diabetes, hypertension, and cardiovascular diseases (CVD). Metabolic syndrome (MS), a constellation of risk factors which increase chances of the cardiovascular disease and diabetes, can increase the morbidity and mortality among this population. Hence, the present study was conducted with the objectives of estimating the prevalence and determinants of MS among ART naïve and ART-treated patients and assess their 5-year CVD risk using the reduced version of Data Collection on Adverse Effects of Anti-HIV Drugs (D : A : D) risk prediction model (D : A : D(R)). This hospital-based cross-sectional study included 182 adults aged ≥ 18 years. MS was defined using the National Cholesterol Education Program-Adult Treatment Panel-3 (NCEP ATP-3) criteria. Univariate and multivariate logistic regressions were done to identify the factors associated with MS. Prevalence of MS was 40.1%  = 33.0%–47.2%). About 24.7% of the participants had at least a single criterion for MS. Age >45 years (adjusted odds ratio (AOR) = 2.3; 95% CI = 1.1–4.9,  Human immunodeficiency virus (HIV) continues to be a major public health issue with 36.7 million people living with HIV globally and 53% of them receiving ART . With thA quarter of deaths due to noncommunicable diseases were attributed to CVDs in India in the year 2014 . MetabolA cross-sectional study was conducted among HIV-positive ART-naïve individuals and those receiving ART at a tertiary care teaching hospital in southern India during March–June 2017. The study included all the HIV-positive ART-treated and ART-naïve adult males and nonpregnant females aged ≥18 years, who visited the hospital during the study period.https://www.chip.dk/Tools-Standards/Clinical-risk-scores). This tool is based on a reduced model and estimates the risk of an individual developing cardiovascular disease (CVD) within the next 5 years. The D : A : D (R) does not include ART as a parameter, and it requires the following information: gender, age, smoking status, diabetes (diagnosis or on antidiabetic treatment), family CVD history, systolic blood pressure (SBP), total cholesterol, high-density lipoprotein (HDL), and cluster of differentiation 4 (CD4) count. The composite CVD outcome includes myocardial infarction, stroke, invasive coronary artery procedure (including coronary artery by-pass or angioplasty and carotid artery endarterectomy), or death from coronary heart disease. This D : A : D (R) model is valid for HIV-infected individuals aged 18–75 years. The 5-year risk of CVD was classified as low (< 1%), moderate (1 to 5%), high (5 to 10%), or very high (>10%) [A clearance was obtained from the Institutional Ethics Committee of Kasturba Medical College and Kasturba Hospital, Manipal, before the commencement of the study (IEC 26/2017). A written informed consent was obtained from the participants. Details including sociodemographic characteristics and behavioral factors such as diet, tobacco, and alcohol consumption were collected using a structured questionnaire (Supplementary Appendix 1). An enquiry was made regarding frequency of consumption of high salt items , high sugar items , high fat items , fish, and fruits and vegetables. The frequency of consumption of various food items was classified as daily, 2-3 times per week, at least once a week, and rarely. Current user of tobacco or alcohol was defined as daily or occasional consumption during the current year, and past user was defined as one who has quit for more than a year. A person who used tobacco or alcohol either currently or in the past was considered to have “ever used” any substance. Physical activity was estimated using the International Physical Activity Questionnaire (IPAQ), and the participants were classified as inactive, minimally active, and health-enhancing physical activity (HEPA) . The 5-yh (>10%) .Anthropometric measures such as weight (kg), height (cm), and waist circumference (cm) were measured. Body mass index (BMI) was computed and classified based on Indian classification . Two blot-test were used to find out association between categorical and continuous variables, respectively. The Mann–Whitney test was used to compare mean DAD scores. Univariate and multivariate logistic regressions were done to identify the factors associated with MS and unadjusted and adjusted odds ratio (OR), respectively, with 95% confidence intervals (CI) have been reported. The variables which were significant in univariate analysis were included in the multivariate analysis. A p value of <0.05 was considered to be statistically significant.The data were entered and analyzed using the Statistical Package for Social Sciences (SPSS) version 15. Continuous variables were summarized using mean (standard deviation (SD)) or median (interquartile range (IQR)), and categorical variables have been expressed as percentages. The chi-square test and independent-sample The study included a total of 182 (123 ART-treated and 59 ART-naïve) participants who visited the outpatient facility of the department of medicine or hospitalized as an inpatient during the months of March to June 2017. The recruitment of study participants is shown in As shown in p=0.23). There was no difference in the prevalence of MS across the two genders .A quarter of the participants (24.7%) had at least one criterion for MS, and 3.8% of the participants had all the five criteria for MS. The overall prevalence of MS among participants was observed to be 40.1% (95% CI = 33.0%–47.2%) with a higher prevalence among individuals on ART as compared to ART-naïve individuals (43.1% vs 33.9%). However, this difference was not statistically significant , whilst the least common was BP ≥130/85 or being on antihypertensives (28.6%).Distribution of the overall morbidities among the participants was diabetes (15.9%), hypertension (20.3%), and hypothyroidism (5.5%). As shown in p=0.004) and low HDL  as compared to males. Males displayed a higher frequency of elevated TG , BP , and fasting blood sugar  against females.Females had higher prevalence of abdominal obesity . The median D : A : D score was significantly higher among males as compared to females . A significant difference was also noted between median D : A : D scores of the two groups, i.e. ART-treated vs ART-naïve individuals .The prevalence of MS in the present study was 40.1%. This is higher when compared to studies done elsewhere in India in which prevalence ranged from 19.8 to 26.6% . StudiesThe current study did not observe any statistically significant difference in MS prevalence between ART-treated and ART-naïve individuals. However, many studies have reported prevalence of MS to be higher among patients on ART as compared to their ART-naïve counterparts , 11, 29.The current study found a quarter of the participants to be having at least one risk factor for MS. The proportion of participants with at least one abnormal MS component ranged from one-third to almost two-thirds of the study population among various other studies , 32. Thep < 0.001), which is akin to findings of the present study [The overall median 5-year CVD risk observed in the present study was higher than the study findings from Cameroon (0.6% (0.3–1.3)) in which 80% of participants were females . As 63% nt study . While int study . Anothernt study . ParticiThe present study found that age > 45 years and high BMI to be positively associated with MS. Association between these traditional CVD risk factors and MS have been demonstrated in several studies among HIV-positive individuals , 28, 35.Studies by Pongthananikorn et al. from India  and TiozOur study systematically dissected all components of metabolic syndrome in HIV-infected individuals in India. We further estimated their 5-year cardiovascular risk. Our study has highlighted the importance of higher BMI, a modifiable risk factor for MS, which could guide future preventive interventions. However, our study has certain limitations. As this study was carried out in a tertiary referral center, MS prevalence in the study setting may not be generalizable to the general HIV+ population. The information collected on tobacco, alcohol consumption, and dietary factors was minimal, and this might have affected the results. Larger prospective studies are necessary to provide better understanding of the factors associated with MS.Prevalence of MS is high among the HIV-infected study participants. Predictive factors for MS in this study were age and BMI, both being traditional risk factors. None of the HIV-related factors such as duration of infection, exposure to ART, and CD4 count were associated with presence of MS in this population. More than half of the study participants had moderate or high risk of development of CVD in the next five years. It is of utmost importance to develop specific interventions to prevent, diagnose, and treat MS among HIV-infected patients to reduce their long-term morbidity and mortality. Early identification and instituting appropriate management will help to reduce future complications and mortality due to CVD.


To introduce a modified rotational atherectomy (RA) procedure and investigate the early and midterm outcomes of the RA-facilitating diversified percutaneous coronary intervention (PCI) in a large group of aged patients with higher cardiovascular risk.  Previous studies about the outcomes of RA were limited with small sample size and low-risk population.  Between January 2013 and November 2015, 1169 consecutive patients treated with modified RA-facilitated PCI were retrospectively enrolled, including de novo calcified lesions and in-stent restenosis. Patients were regularly followed up for at least 1 year. Major adverse cardiac events (MACE) were analyzed for all participants by different strategies. Cox regression analysis was performed to identify risk factors for the events.  The median age of patients was 75 years, with 11.7% of patients on maintenance hemodialysis. Most lesions (99.9%) were complex (American Heart Association type B2/C), and 68.3% were treated with RA + drug-eluting-stent (DES). Successful angiography was achieved in 97.8% cases, with 1.7% (20/1169) experiencing coronary perforation (including guidewire perforation). The incidence of MACE was 20.5% and 26.8% at 1-year and 2-year follow-up and were mainly driven by target lesion revascularization (TLR) . The strategy of RA + DES had the lowest 2-year MACE, compared with the RA + drug-coated balloon and RA + plain old balloon angioplasty .  The modified RA technique is a safe and effective tool in the contemporary PCI era, even in high-risk patients. The TLR rate was relatively high but acceptable in such complex lesions. In contemporary PCI practice, rotational atherectomy (RA) is considered as an adjunctive tool for the management of fibrotic or heavily calcified coronary lesions by “differential cutting” and “orthogonal displacement of friction” –3. RA haHowever, the volume of RA was reported to vary widely in these two decades and between individual centers, ranging from 0.6%–20% , 11. In On the other hand, utilization of RA never stopped in daily practice. In particular, with advancement in PCI devices and perioperative preparation with new drugs, the contemporary RA technique has also evolved in the past two decades yielding improved PCI performance , 14. StuIn the past 5 years, the Sapporo Cardio Vascular Clinic (SCVC) has modified the RA technique and achieved favorable results for complex PCI . With thBetween January 2013 and November 2015, data of a consecutive series of patients treated with RA-facilitated PCI were retrospectively collected from SCVC. The indications for RA included (1) moderate-to-severe superficial calcification lesions observed on intravascular ultrasound (IVUS)/optical coherence tomography (OCT) images or linear calcium density images on both sides of the target lesion visible under fluoroscopy; (2) calcified lesions making the passage of imaging probes difficult, where inadequate stent delivery or expansion can be expected; (3) calcified ostial and true bifurcating lesions; (4) chronic total occlusion (CTO) lesions, in which the guidewire has been correctly positioned but low-profile balloons cannot be advanced; and (5) selected cases of diffuse in-stent restenosis. The decision to perform RA and PCI was at an experienced and high-volume operator's discretion. Cases with stand-alone RA, bailout use of suboptimally expanded stents, rotablation at the same PCI stage, and incomplete follow-up data were excluded. Clinical follow-up was conducted regularly at least once within 2-years in outpatient clinics by operators. Coronary computed tomography angiogram was routinely performed within 1 year, and a coronary angiogram was suggested when clinically driven . Informed consent was obtained from each patient. Data were collected using a standardized case report form to record demographic and clinical characteristics and procedural and follow-up data.Immediately before the intervention, patients received intra-arterial or intravenous heparin (70–100 IU/kg) to maintain the activated clotting time over 250 sec. Dual antiplatelet therapy with aspirin and thienopyridine  was used at least one year after PCI and continued as long as possible with aspirin or another type of antiplatelet drug. RA was performed using a Rotablator™ . In accordance with the Japanese insurance policy, a maximum of two burrs were used when required. The choice of stenting (DES/BMS), DCB, or POBA alone was dependent on the operator's judgment.Concordant with the general principle of RA performance , 14, theQCA was performed by standard methods and definitions using CAAS software version 5.9.1.  . AngiogrEarly major adverse cardiac events (MACE) were the combination of all-cause death and the first occurrence of target lesion revascularization (TLR) during hospitalization and 30 days after PCI. Midterm MACE included 12-month and 24-month rates of all-cause death, cardiac death, the first occurrence of hospitalization due to heart failure, definite stent thrombosis, and TLR. TLR was defined as repeat revascularization within 5 mm proximal or distal to the target lesion or coronary artery bypass graft surgery (CABG) of the lesion in the same epicardial vessel.P < 0.10 in univariate Cox models were entered into a multivariable Cox model. All P values were two-sided and not adjusted for multiple testing. P values <0.05 were regarded as statistically significant.All analyses were performed using SPSS software version 24.0 . Continuous variables with abnormal distribution were presented as median . Categorical variables were presented as proportions (%). Clinical event rates were calculated using the Kaplan–Meier analysis, and the differences between groups were assessed with a log-rank test. Hazard ratios (HR) and 95% confidence intervals (CI) were calculated using the Cox proportional hazards model, with assumptions of proportional hazards based on the Schoenfeld residuals. Collinearity diagnosis was performed using the Spearman correlation and Belsley's criterion. For clinically similar variables, we selected the variable that we considered to be more clinically relevant. Basing on the literature , 18 and During the study period, among 1374 patients treated with RA, excluding 205 lost to follow-up, 1169 patients with 1453 lesions were included. The median age was 75 (IQR: 69–81) years, and 32.1% of patients were aged >80 years . The preThe success rate of angiography was 97.8%. QCA data were available for 1056 cases (89.7%). Pre-QCA demonstrated that the reference vessel diameter was 2.72 (2.37–3.20) mm, lesion length 30.67 (22.78–39.91) mm, and the proportion of lesions with angulation >45° (moderate/excessive) was 52.9%. The other cases were unmeasurable because of severe stenotic lesions without clear visualization of contrast filling. As shown in Supplementary The median follow-up time was 498 days (IQR 324–727 days), and the follow-up rate was 100% and 54.8% at 12 and 24 months, respectively. Six deaths occurred during hospitalization  and 23 in the first 30 days . No defiAmong different RA-facilitated PCI strategies, the survival curve of RA + DES started to separate at six months after PCI from that of the other two strategies. RA + DES had the lowest 2-year MACE of 14.5%, compared with the RA + DCB and RA + POBA  1.41, 95% CI 1.08–1.85), hemodialysis , LVEF level , RA + DCB versus RA + DES , and RA + POBA versus RA + DES  were significantly related to total MACE . MeanwhiThis retrospective study presents the “real-world” utility of modified RA in the contemporary coronary angioplasty era with regular follow-up. We demonstrated that (1) the modified RA procedures were safe both in severe calcification and in ISR lesions with about 98% success rate and no definite acute occlusion occurring within the first month even with RA + POBA alone; (2) RA + DES had the best midterm outcome compared with DCB or RA + POBA; (3) the rate of MACE was mainly driven by a high but acceptable TLR rate, considering the severity of patients; and (4) diabetes, hemodialysis, low LVEF, and stent-less RA were significant predictors for MACE and TLR in patients receiving RA.It has been >30 years since Jerome Ritchie, David Auth, and colleagues introduced RA as a technique for endovascular treatment of obstructive atherosclerotic disease . AlthougRA was shown to be safe for complex PCI in different trials with complication rates about 3–9% , 8, mainThe multivariable analysis revealed hemodialysis as a significant risk factor for MACE and TLR, which is consistent with previous studies , 26, 27.Among the three RA-facilitated PCI strategies, RA + DES proved to be the most effective to reduce MACE, with the metallic support of stent struts and antiproliferative drugs. Theoretically, RA and DES could act synergistically in complex lesions as RA can avert stent coating damage and DES can effectively suppress neointimal proliferation. In contrast, the ROTAXUS trial demonstrated that rotablation does not increase the efficacy of DES in calcified lesions compared with balloon dilation , due to To our knowledge, this is the largest retrospective study analyzing the safety and efficacy of “real-world” RA in the contemporary coronary angioplasty era, especially with advanced-age patients. The follow-up rate was more than 85%, and patients were strictly evaluated , ensuring high quality of the data. However, some limitations should be acknowledged. First, the choice of RA was based on the operators' discretion, which was more aggressive as the initial strategy than recommended. Secondly, there was no comparison between the modified RA and the standard method. Interventional coronary angiography driven by clinical symptoms would also numerically decrease MACE prevalence from the misdiagnosis of silent ischemia.The present study demonstrated the modified RA technique as a safe and effective tool for high-risk patients in the contemporary PCI era, while RA + DES emerged as the most favorable interventional strategy with the lowest MACE rate. Diabetes, hemodialysis, low LVEF level, and stent-less RA strategy were independent risk factors of MACE in patients who underwent the RA procedure.


Reproducibility of FFQs measures the consistency of the same subject at different time points. We performed a meta-analysis to explore the reproducibility of FFQs and factors related to reproducibility of FFQs.A systematic literature review was performed before July 2020 using PubMed and Web of Science databases. Pooled intraclass and Spearman correlation coefficients  were calculated to assess the reproducibility of FFQs. Subgroup analyses based on characteristics of study populations, FFQs, or study design were performed to investigate factors related to the reproducibility of FFQs. A total of 123 studies comprising 20,542 participants were eligible for the meta-analysis. The pooled crude intraclass correlation coefficients ranged from 0.499 to 0.803 and 0.499 to 0.723 for macronutrients and micronutrients, respectively. Energy-adjusted intraclass correlation coefficients ranged from 0.420 to 0.803 and 0.507 to 0.712 for macronutrients and micronutrients, respectively. The pooled crude and energy-adjusted Spearman correlation coefficients ranged from 0.548 to 0.851 and 0.441 to 0.793, respectively, for macronutrients; and from 0.573 to 0.828 and 0.510 to 0.744, respectively, for micronutrients. FFQs with more food items, 12 months as dietary recall interval (compared to less than 12 months), and a shorter time period between repeated FFQs resulted in superior FFQ reproducibility.In conclusion, FFQs with correlation coefficients greater than 0.5 for most nutrients may be considered a reliable tool to measure dietary intake. To develop FFQs with higher reproducibility, the number of food items and dietary recall interval should be taken into consideration.The online version contains supplementary material available at 10.1186/s12966-020-01078-4. The FFQ is the most commonly used tool to assess individual usual dietary intake in nutritional epidemiological studies, especially for investigating the relationship between dietary and health outcomes , 2. FFQsReproducibility reflects reliability and refers to the similarity of the same method at different timepoints . ReproduNumerous studies have been devoted to assess the reproducibility of FFQs before applying FFQ to different populations. The Spearman and intraclass correlation coefficients (ICCs) to assess the reproducibility of 134-item FFQs with approximately 6 months apart ranged from 0.46 to 0.79 and from 0.34 to 0.71, respectively, for 25 nutrients in the Shanghai Diet and Health Study . The repFurthermore, the characteristics of FFQs may affect their reproducibility. A previous study reported that the ICCs of an FFQ comprising 255 items ranged from 0.69 (fat) to 0.84 (vitamin A) in Moroccan adults . A shortAlthough the reproducibility of FFQs has been evaluated in various studies, there has yet to be a comprehensive meta-analysis of the reproducibility studies and definition of reference ranges for reproducibility coefficients. Moreover, no study has systematically explored the factors related to the reproducibility of FFQs. Therefore, we conducted a meta-analysis to systematically assess the reproducibility of FFQs and to explore the factors related to the reproducibility of FFQs.A systematic review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guideline; the relevant checklist is provided in PRISMA Checklist.We conducted a comprehensive literature search for published studies from PubMed and Web of Science databases before July 2020. The literature search was conducted by two independent researchers. The search strategy used employed the terms “FFQ OR food frequency questionnaire” AND “reproducibility OR repeatability OR reliability”.The potentially relevant articles were evaluated by two independent reviewers based on the inclusion. The original studies were obtained from the database. After removing duplicates, we screened the studies according to title and abstract. After reading the full texts, the eligible articles were obtained by exclusion criteria.Articles were included if they met the following criteria: (1) FFQs were used to measure nutrient intake; (2) the age range of target healthy populations was between 8 and 86 years; (3) the study assessed the reproducibility of FFQs; (4) the study was published in English; and (5) the reproducibility of FFQs was measured with the intraclass correlation coefficient (ICC) and Pearson correlation coefficient or SCC.The exclusion criteria were: (1) food intake was assessed using FFQs; (2) FFQs were used to assess a specific nutrient; (3) the target population was unhealthy people or specific populations, such as individuals who were overweight or malnourished; (4) the participants were less than 8 years old; (5) the article investigated diet-disease relationships; and (6) the full text was unavailable through web searches.Data were extracted from each study by independent reviewers. The extracted contents included the following, excluding authors and published years: (1) characteristics of participants including sample size, age, gender distribution, and region; (2) characteristics of FFQs including food items and dietary recall interval; (3) characteristics of study design including administration method and interval between two FFQs; and (4) statistics employed to assess reproducibility between repeated FFQs including ICC and Pearson correlation coefficient or SCC in relation to energy, macronutrients, and micronutrients . Macronutrients included protein, fat, plant fat, animal fat, MUFA, PUFA, n-3 PUFA, n-6 PUFA, SFA, linoleic acid, linolenic acid, EPA, DHA, trans-fat, cholesterol, lipid, carbohydrate, sucrose, sugar, starch, fiber, soluble fiber, insoluble fiber, and alcohol. Minerals included selenium (Se), magnesium (Mg), calcium (Ca), iron (Fe), iodine (I); zinc (Zn), copper (Cu), potassium (K), phosphorus (P), sodium (Na), and manganese (Mn). Vitamins included vitamin A, retinol, carotene, β-carotene, vitamin C, vitamin D, vitamin E, vitamin K, thiamin, riboflavin, niacin, vitamin B6, folate and vitamin B12.2). I2 greater than 50% indicated the presence of heterogeneity. z-values were converted using inverse Fisher’s transformation to obtain correlation coefficients and 95% CIs to account for results. Sensitivity analysis was performed to explore the when to further explore the source of heterogeneity.The pooled correlation coefficients were calculated based on the ICC and SCC values obtained from each article. We converted Pearson correlation coefficients into SCCs if the latter were lacking. Fisher’s transformation was used to convert each correlation coefficient to an approximately normally distributed z-value. The standard error of z was calculated. After appropriate conversion, random effects meta-analyses were used to combine data. The heterogeneity of the z-values among studies was determined by calculating the inconsistency index  population characteristics including age , gender, and region; (2) characteristics of the reproducibility studies including sample size  and time interval between repeated FFQs ; and (3) characteristics of FFQ design including FFQ items , dietary recall interval (≥12 months and < 12 months), administration mode (interviewer-administered or self-administered). All statistical analyses were performed using Stata Software . A The flow chart of the study selection is shown in Fig. n = 63) or more than 6 months (n = 55).An overview of the retrieved studies assessing the reproducibility of FFQs is presented in Table 2 > 50%).As shown in Table 2 > 75%).Table To assess the impact of age on the degree of reproducibility of two FFQ measures, we performed subgroup analysis according to age Fig. . As showBased on subgroup analysis according to sex Fig. , pooled Subgroup analysis according to region revealed that the ICCs of reproducibility ranged from 0.369 (retinol) to 0.829 (thiamin), 0.560 (Zn) to 0.830 , 0.400 (vitamin K) to 0.839 (β-carotene), 0.310 (starch) to 0.859 (lipid), and 0.563 (vitamin D) to 0.861  in the regions of Africa, Oceania, Asia, Europe, and America, respectively  and from 0.485 to 0.788 (median = 0.643), respectively . In terms of energy and 24 macronutrients, all ICC and SCC values exceeded 0.5, except for I, soluble fiber, trans-fat, n-3 PUFA, and n-6 PUFA. Moreover, we identified that FFQs with more food items, 12 months as dietary recall interval, and shorter time periods between repeated FFQs resulted in superior FFQ reproducibility.To evaluate the ability of FFQs to accurately evaluate long-term dietary intake in different age groups, we conducted subgroup analysis according to age which revealed that the correlations of the reproducibility of FFQs exceeded 0.5 for most nutrients in the elderly, adolescents, and adults, indicating that the reliability of FFQs was relatively consistent across age groups. However, the reproducibility of FFQs for adults was higher than that for the elderly and adolescents for most nutrients. A potential reason for the lower correlation in adolescents was that older individuals may have more established dietary habits than younger individuals . FurtherGender differences in the reproducibility of FFQs were observed in this study. The degree of reproducibility was generally higher in women than in men for most nutrients, suggesting that women have more stable long-term dietary intake than that of men . GeneralIn addition, we observed that the reproducibility of FFQ was low when the sample size was large. This low correlation was not the true reproducibility coefficients between FFQs, but might be caused by irrelevant factors in the operation process. As the large sample sizes may facilitate the management of more participants; consume time, resources, and effort; induce loss to follow-up and put a burden on researchers. However, a small sample size may limit representativeness, which induces large differences in within-person nutrient intakes, leading to less reliable correlation coefficients and ICCs . TherefoFFQs with more items presented better reproducibility for most nutrients, indicating that long FFQs collated more reliable information  and enabFFQs were used to assess regular dietary habits over extended periods. The correlation coefficients of the study assessing the reproducibility of FFQs over more than a 1-year period were higher than those over short periods for most nutrients. Relatively high correlations for 1 year indicated that FFQs can provide an accurate estimation of long-term dietary habits. The reasons for lower correlations of FFQs over less than 1 year may be related to the seasonal availability of food . In addiThe combined correlation coefficients were higher when FFQs were administered over a short period (≤6 months) compared with those over a long time interval (> 6 months), suggesting that shorter intervals between repeated FFQ administrations were a key factor contributing to high reproducibility of FFQs, in accordance with a previous review . A possiThe main strength of this study is that it is the first meta-analysis to comprehensively analyze FFQ reproducibility. Current research is based on a large number of different populations with a wide age range which revealed good reproducibility of nutrient intake, making FFQs suitable for analyzing dietary intake among different subgroups of age, sample size, gender, and region. We comprehensively evaluated the reproducibility of FFQs by analyzing the intake of 50 nutrients, which strengthened the conclusions of this study.This study has some limitations. First, our screening criteria excluded articles that assessed the effectiveness of specific nutrients, which may have affected our results in different ways. Second, learning ability and lifestyles, such as education level and body mass index, may have influenced FFQ reliability. However, the relevant data were not available in the included articles. Third, we did not evaluate the quality of included studies because there are currently no tools to assess the quality of reproducibility studies for FFQs. Further studies are needed to establish such tools to evaluate the quality of reproducibility studies for FFQ.In conclusion, FFQs with correlation coefficients greater than 0.5 for most nutrients may be considered a reliable tool to measure dietary intake. In addition, factors related to FFQ design may be associated with the reproducibility of FFQs, such as FFQ items and dietary recall intervals. To increase the reproducibility of FFQs, the following points should be considered before developing FFQs. First, pilot studies are warranted to explore the appropriate number of FFQ items based on the characteristics of the study population. Second, 12 months is suggested as the dietary recall interval. Third, when performing reproducibility studies for FFQs, a sample size with sufficient statistical power, but no larger, is recommended.Additional file 1. PRISMA Checklist.Additional file 2 Supplemental Table 1. The overview of the retrieved studies assessing the reproducibility of FFQs.Additional file 3 Supplemental Table 2. Pooled intraclass correlation coefficients for energy and nutrients stratified by age.Additional file 4 Supplemental Table 3. Pooled spearman correlation coefficients for energy and nutrients stratified by age.Additional file 5 Supplemental Table 4. Pooled intraclass correlation coefficients for energy and nutrients stratified by sex.Additional file 6 Supplemental Table 5. Pooled spearman correlation coefficients for energy and nutrients stratified by sex.Additional file 7 Supplemental Table 6. Pooled crude intraclass correlation coefficients for energy and nutrients stratified by regions.Additional file 8 Supplemental Table 7. Pooled energy-adjusted intraclass correlation coefficients for energy and nutrients stratified by regions.Additional file 9 Supplemental Table 8. Pooled crude spearman correlation coefficients for energy and nutrients stratified by regions.Additional file 10 Supplemental Table 9. Pooled energy-adjusted spearman correlation coefficients for energy and nutrients stratified by regions.Additional file 11 Supplemental Table 10. Pooled intraclass correlation coefficients for energy and nutrients stratified by sample size.Additional file 12 Supplemental Table 11. Pooled spearman correlation coefficients for energy and nutrients stratified by sample size.Additional file 13 Supplemental Table 12. Pooled intraclass correlation coefficient for energy and nutrients stratified by time interval (6 months as cut-point).Additional file 14 Supplemental Table 13. Pooled spearman correlation coefficient for energy and nutrients stratified by time interval (6 months as cut-point).Additional file 15 Supplemental Table 14. Pooled intraclass correlation coefficient for energy and nutrients stratified by time interval (12 months as cut-point).Additional file 16 Supplemental Table 15. Pooled spearman correlation coefficient for energy and nutrients stratified by time interval (12 months as cut-point).Additional file 17 Supplemental Table 16. Pooled intraclass correlation coefficient for energy and nutrients stratified by items of FFQ.Additional file 18 Supplemental Table 17. Pooled spearman correlation coefficient for energy and nutrients stratified by items of FFQ.Additional file 19 Supplemental Table 18. Pooled intraclass correlation coefficient for energy and nutrients stratified by dietary recall interval.Additional file 20 Supplemental Table 19. Pooled spearman correlation coefficient for energy and nutrients stratified by dietary recall interval.Additional file 21 Supplemental Table 20. Pooled intraclass correlation coefficient for energy and nutrients stratified by administration mode.Additional file 22 Supplemental Table 21. Pooled spearman correlation coefficient for energy and nutrients stratified by administration mode.