It is critical for determining the optimum therapeutic solutions for T1‐2 colorectal cancer (CRC) to accurately predict lymph node metastasis (LNM) status. The purpose of the present study is to establish and verify a nomogram to predict LNM status in T1‐2 CRCs.A total of 16 600 T1‐2 CRC patients were enrolled and classified into the training, internal validation, and external validation cohorts. The independent predictive parameters were determined by univariate and multivariate analyses to develop a nomogram to predict the probability of LNM status. The calibration curve, the area under the receiver operating characteristic curve (AUROC), and decision curve analysis (DCA) were used to evaluate the performance of the nomogram, and an external verification cohort was to verify the applicability of the nomogram.Seven independent predictors of LNM in T1‐2 CRC were identified in the multivariable analysis, including age, tumor site, tumor grade, perineural invasion, preoperative carcinoembryonic antigen, clinical assessment of LNM, and T stage. A nomogram incorporating the seven predictors was constructed. The nomogram yielded good discrimination and calibration, with AUROCs of 0.72 , 0.70 (95% CI: 0.67‐0.74), and 0.74 (95% CI: 0.71‐0.79) in the training, internal validation, and external validation cohorts, respectively. DCA showed that the predictive scoring system had high clinical application value.We proposed a novel predictive model for LNM in T1‐2 CRC patients to assist physicians in making treatment decisions. The nomogram is advantageous for tailoring therapy in T1‐2 CRC patients. ADadenocarcinomaAUCthe area under the curve; ROC, receiver operating characteristicCEAcarcinoembryonic antigenCIconfidence intervalcLNMclinical assessment of lymph node metastasisCRCcolorectal cancerCSScause‐specific survival; DFS, disease‐free survivalCTchemotherapy; AUROC, area under receiver operating characteristic curveDCAdecision curve analysisFUSCCFudan University Shanghai Cancer CenterHRhazard ratioIQRinterquartile rangeLNMlymph node metastasisMADmucinous adenocarcinomaOSoverall survivalSDstandard deviationSEERsurveillance, epidemiology, and end resultsSRCCsignet ring cell carcinomaTNMtumor‐node‐metastasis1Ranking as the third leading cause of death among all malignancies, the incidence of colorectal cancer (CRC) has continued to rise over the past three decades worldwide, which has made CRC a critical problem for public health.Cases confined to the muscularis propria (T1 and T2) without LNM or distant metastasis are generally considered early neoplastic lesions that can be potentially cured by complete resection of the tumor.The Surveillance, Epidemiology, and End Results (SEER) database functionally offers a profusion of integral clinical and pathological information for various cancers that cover ∼28% of the American population. In this study, information on CRC patients was gathered from the SEER database and Fudan University Shanghai Cancer Center (FUSCC) cohorts. First, we divided the cases in the SEER database into a training set and an internal validation set. Then, we developed an intuitive and comprehensive nomogram to predict the probability of LNM in T1‐2 CRC patients in the training set, evaluated the model's efficacy in the internal validation set, and further validated the model's predictive power in the FUSCC external validation set.22.1The Ethical Committee and Institutional Review Board of the Fudan University Shanghai Cancer Center reviewed and approved this study protocol. All patients provided written informed consent.2.2AJCC Cancer Staging Manual, 7th Edition.International Classification of Diseases for Oncology, 3rd Edition (ICD‐O‐3). According to ICD‐O‐3 oncology codes, three histological subtypes of CRCs were classified as follows: adenocarcinoma , mucinous adenocarcinoma , and signet ring cell adenocarcinoma (8490). (3) Pathological grade: Well‐differentiated tumor was classified as grade I; moderately differentiated tumor was divided into grade II; poorly differentiated and undifferentiated tumors were identified as grade III and grade IV, respectively. (4) Pre‐CEA level: In the SEER cohort, the pre‐CEA level refers to “CS Site‐Specific Factor 1,″ where “Code 010″ means “Positive,” “Code 020″ means “Negative,” and other codes mean “Other.” In the FUSCC cohort, pre‐CEA levels were determined by an electrochemiluminescence immunoassay using a Roche Cobas e601 immunoassay analyzer . The normal upper limit of pre‐CEA was adopted as 5.2 ng/mL. (5) cLNM: The clinical assessment of LNM status was based on information from the diagnostic workup, including physical examination, imaging, diagnostic lymph node biopsy, and exploratory surgery without resection. Patients’ survival information was quantified by cause‐specific survival (CSS), disease‐free survival (DFS), and overall survival (OS), and those with missing survival information were excluded. Finally, 15 537 patients with T1‐2 CRC from the SEER database were divided into training and internal validation sets for model building and evaluation. Eligible patients from the FUSCC cohort were used as the external validation cohort for model validation.In this study, a total of 15 537 eligible patients from the SEER dataset and 1063 patients from the FUSCC cohort with T1‐2 CRC were acquired. The detailed inclusion and exclusion criteria of patients are shown in Figure 2.3P < .05 in the univariate model were used in the multivariate logistic regression analysis. The measure of the effect of each variable on LNM was presented as an odds ratio (OR) to identify independent risk factors. Based on the multivariable logistic regression analysis results, a nomogram integrating clinicopathological parameters with cLNM was formulated. The overall points for each patient in the training, internal validation and external validation cohorts were calculated using the established nomogram, after which a logistic regression analysis of the entire cohort was carried out using the overall points as a parameter. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the nomogram were calculated.Univariable and multivariable logistic regression analyses were used to calculate and validate the effect of variables in the training, internal validation, and external validation cohorts. The variables with 2.4The calibration of the nomogram was evaluated by the Hosmer‐Lemeshow test and displayed in the form of the calibration curve. The accuracy of nomogram is displayed in the form of ROC curve, and the discriminative ability of the nomogram to predict LNM status in T1‐2 CRC is quantitatively expressed by the area under the receiver operating characteristic curve (AUROC).2.5Decision curve analysis (DCA) is a new method to evaluate the potential clinical value of a risk prediction model, which can directly reflect the potential benefits of the new model once applied in clinical practice2.6Survival curves for different groups  were plotted using the Kaplan‐Meier method and the differences among them were compared using the log‐rank test. CSS in the SEER dataset was defined as the time of the diagnosis of CRC to the time when CRC cause‐specific death occurred. Death from CRC was considered an event, and death from other causes or survival at the end of the follow‐up was censored.2.7http://www.r-project.org) was used for all statistical analyses. The R statistical packages “rms,” “barplot,” “survival,” “Hmisc,” “MASS,” and “pROC” were used to plot the distribution of risk scores and LNM, plot calibration, and logistic ROC curves, build a nomogram, and draw Kaplan‐Meier curves, while “rmda” was used to draw the DCA curves and “forestplot” was used to draw the forest plot. Continuous variables were presented as the mean ± SD, and the difference was analyzed using one‐way ANOVA or Student's t‐test. The differences in categorical variables were analyzed by Fisher's exact test or a two‐tailed χ2 test. All statistical tests were two‐sided, and P‐values < .05 were regarded as statistically significant.R software  and FUSCC (n = 1063) cohorts. The LNM rate was 14.2% (9.6% for T1 stage and 18.9% for T2 stage) and 20.1% (9.8% for T1 stage and 23.4% for T2 stage) for all T1‐2 CRC patients in the SEER and FUSCC cohorts, respectively. The patients’ clinicopathological characteristics according to LNM status and a comparison of the clinicopathological factors in the training, internal validation, and external validation cohorts are listed in Table P = .010, Supporting Information Figure 2A). Similar findings were confirmed in the FUSCC cohort, with a 5‐year DFS of 89.6% versus 78.2%  and a 5‐year OS of 95.1% versus 82.2%  for the LNM‐negative group and LNM‐positive group.In the SEER database, the median follow‐up period was 24 ± 12 months (ranging from 3 to 47 months). The estimated 3‐year CSS rate was 90.7% for all patients . In the FUSCC cohort, 80 patients experienced recurrence, while the other 983 patients were still free of disease at the last follow‐up, with a median follow‐up time of 55.2 months (ranging from 4 to 82 months). The 5‐year DFS rate was 87.1%, and the 5‐year OS rate was 92.4% for all patients . In the SEER cohort, the LNM‐negative group had a higher CSS of 91.0% for 3‐year CSS compared with 89.9% in the LNM‐positive group . Moreover, the 5‐year DFS rates of the LNM‐negative group, LNM‐positive with CT group, and LNM‐positive without CT group were 89.6%, 79.6%, and 75.7%, respectively, in the FUSCC cohort . Furthermore, the Kaplan‐Meier analysis revealed no statistical significance between the LNM‐positive with CT and LNM‐positive without CT groups, while both of them had worse 5‐year OS rates than the LNM‐negative group . The above results showed that accurate assessment of LNM status in T1‐2 CRC patients to formulate specific therapeutic regimens (surgery or surgery plus adjuvant CT) had a crucial impact on the prognosis of patients.In view of the adjuvant chemotherapy (CT) status for the LNM‐positive group, we divided the LNM‐positive group into subgroups of LNM‐positive with CT and LNM‐positive without CT. Kaplan‐Meier analysis revealed that LNM‐positive patients with CT in the SEER cohort had the best 3‐year CSS compared with the LNM‐negative group and LNM‐positive patients without CT . For all T1 CRC patients, the nomogram yielded an AUROC of 0.68 (95% CI: 0.66‐0.72), 0.66 (95% CI: 0.64‐0.70), and 0.71 (95% CI: 0.68‐0.75) in the training, internal validation, and external validation cohorts, respectively, indicating that the nomogram had favorable discrimination in T1 patients . In terms of all T2 patients, good discrimination ability was also observed among the training , internal validation , and external validation  cohorts . Similar findings were confirmed for the cLNM‐negative subgroup patients in the training, internal validation, and external validation cohorts, with AUROCs of 0.68 (95% CI: 0.65‐0.71), 0.66 (95% CI: 0.63‐0.70), and 0.72 (95% CI: 0.70‐0.75), respectively .3.4The DCA is a new strategy for evaluating alternative predictive treatment methods and has advantages over AUROC in clinical value evaluation. The DCA curves for the developed nomogram and cLNM in the training, internal validation, and external validation cohorts are shown in Figure 4The accurate assessment of LNM status is critical for tailored therapy in T1‐2 CRC. In this study, a nomogram incorporating clinical and pathological information in parametric quantities was built to evaluate LNM in T1‐2 CRC patients individually. The characteristics of the nomogram were confirmed regarding its identification and calibration, which contributed to a wide range of applications. The calibration curve and ROC curve showed that the possibility of LNM predicted by the nomogram is highly consistent with the actual LNM status. Moreover, the DCA curve indicated that the nomogram could obtain more potential clinical benefits than cLNM in clinical practice for evaluation of LNM in T1‐2 CRC.According to current guidelines, whether additional radical surgery plus regional lymphadenectomy should be performed after endoscopic resection of T1 CRC remains controversial. If endoscopic resection is not oncologically safe, which is dictated to a large degree by the probability of LNM, the patient might demand additional surgery to undergo lymphadenectomy.In fact, this study showed that patients with different LNM statuses and LNM‐positive patients with or without CT had significantly different survival rates. Randomized clinical trials have shown that stage III patients receiving postoperative adjuvant CT had a survival advantage over those receiving surgery alone.At present, the diagnosis of young CRC patients has increased. Relevant research has demonstrated that age was an independent predictive factor of LNM in T1‐2 CRC patients, with younger age associated with more promising outcomes.In this filed, much work on the prognostic factors and LNM status of CRC has been reported recently. A few researchers reported that their nomogram scoring systems had exceptional capabilities in predicting the LNM and survival of CRC patients.However, this research still has some limitations. First, therapy information except for surgery, such as specific radiotherapy and chemotherapy therapeutics, was not available in the SEER database to be incorporated into the analysis. Second, this was a retrospective study based on limited clinical records and hence was not free from potential selection bias. In addition, the model needs further prospective multicenter clinical research to prove its clinical effectiveness.5In conclusion, we developed and validated a nomogram for predicting the LNM probability of T1‐2 CRC patients. This novel nomogram had sufficient discrimination and calibration capabilities, in addition to exceptional clinical effectiveness, and could be a convenient‐to‐use tool for clinicians to select treatment strategies for patients with T1‐2 CRC.SBM, ZZ, and WXD had the idea for this study. WQX and LYH supervised the acquisition of the data. SBM, ZZ, and WXD undertook the statistical analysis. QGL, RJW, and LZ provided statistical advice. All authors contributed to interpretation of the results. SBM and ZZ wrote the article. QGL, SJC, and GXC revised the article and other authors contributed to the content. All authors approved the final version of the manuscript, including the authorship list.The authors declare that they have no competing interests.The Ethical Committee and Institutional Review Board of the Fudan University Shanghai Cancer Center reviewed and approved this study protocol. All patients signed written informed consent.Supplementary Figure 1Additional file 1: : Kaplan‐Meier survival analysis in all patients. (A) Cause‐specific survival of all patients in the SEER cohort. (B) Disease‐free survival of all patients in the FUSCC cohort. (C) Overall survival of all patients in the FUSCC cohort. Supplementary Figure 2: Kaplan‐Meier analysis of cause‐specific survival (CSS), disease‐free survival (DFS) and overall survival (OS) according to LNM and adjuvant chemotherapy (CT) status. (A) CSS between LNM‐negative and LNM‐positive subgroups in the SEER cohort. (B) CSS among LNM‐negative, LNM‐positive with CT, and LNM‐positive without CT subgroups in the SEER cohort. (C) DFS between LNM‐negative and LNM‐positive subgroups in the FUSCC cohort. (D) DFS among LNM‐negative, LNM‐positive with CT, and LNM‐positive without CT subgroups in the FUSCC cohort. (E) OS between LNM‐negative and LNM‐positive subgroups in the FUSCC cohort. (F) OS among LNM‐negative, LNM‐positive with CT, and LNM‐positive without CT subgroups in the FUSCC cohort. Supplementary Figure 3: Discriminative ability of the seven‐independent risk clinicopathologic characteristics to LNM status in the (A) training, (B) internal validation, and (C) external validation cohorts. Supplementary Figure 4: Subgroup analyses of the nomogram in different T stages. Performance of the nomogram to predict LNM in different T stages of the (A‐D) training, (E‐H) internal validation, and (I‐L) external validation cohorts. Supplementary Figure 5: Subgroup analyses of the nomogram in cLNM‐negative subgroup. Performance of the nomogram to predict LNM in cLNM‐negative subgroup of the (A and B) training, (C and D) internal validation, and (E and F) external validation cohorts. Supplementary Figure 6: Decision curve analysis in different T stages of the (A and B) training, (C and D) internal validation, and (E and F) external validation cohorts. The gray line and black line represent the assumption regarding all patients with and without LNM, respectively. The red line represents the nomogram, and the blue line represents the cLNM. Supplementary Figure 7: Decision curve analysis in cLNM‐negative subgroup of the (A) training, (B) internal validation, and (C) external validation cohorts. The gray line and black line represent the assumption regarding all patients with and without LNM, respectively. The red line represents the nomogram. Supplementary Figure 8: Decision curve analysis in different T stages in cLNM‐negative subgroup of the (A and B) training, (C and D) internal validation, and (E and F) external validation cohorts. The gray line and black line represent the assumption regarding all patients with and without LNM, respectively. The red line represents the nomogram.Click here for additional data file.Supplementary Table 1file 2: . Comparison of clinicopathological characteristics in the training, internal validation and external validation cohorts (N(%)). Supplementary Table 2. Point assignments and predictive scores for each variable in the nomogram model. Supplementary Table 3. Standardized net benefit using the nomogram for specific optimal thresholds.Additional Click here for additional data file.


Gene amplification chiefly manifests as homogeneously stained regions (HSRs) or double minutes (DMs) in cytogenetically and extrachromosomal DNA (ecDNA) in molecular genetics. Evidence suggests that gene amplification is becoming a hotspot for cancer research, which may be a new treatment strategy for cancer. DMs usually carry oncogenes or chemoresistant genes that are associated with cancer progression, occurrence and prognosis. Defining the molecular structure of DMs will facilitate understanding of the molecular mechanism of tumorigenesis. In this study, we re‐identified the origin and integral sequence of DMs in human colorectal adenocarcinoma cell line NCI‐H716 by genetic mapping and sequencing strategy, employing high‐resolution array‐based comparative genomic hybridization, high‐throughput sequencing, multiplex‐fluorescence in situ hybridization and chromosome walking techniques. We identified two distinct populations of DMs in NCI‐H716, confirming their heterogeneity in cancer cells, and managed to construct their molecular structure, which were not investigated before. Research evidence of amplicons distribution in two different populations of DMs suggested that a multi‐step evolutionary model could fit the module of DM genesis better in NCI‐H716 cell line. In conclusion, our data implicated that DMs play a very important role in cancer progression and further investigation is necessary to uncover the role of the DMs. Cytogenetically, gene amplification chiefly manifests as homogeneously stained regions (HSRs) or double minutes (DMs).Currently, it has been documented that extrachromosomal chromatins arose from circularization of co‐amplified DNA fragments from multiple chromosomal loci.Because of the diversity of extrachromosomal chromatins in size, amount, molecular composition and organization in single or multiple cells,The human colorectal adenocarcinoma cell line NCI‐H716 carries DMs, and the molecular structure of them has not been explicitly defined.22.12 at 37°C. Cells were authenticated by short tandem repeat profiling analysis .The human colorectal cancer cell line NCI‐H716 with spontaneous DMs was purchased from American Type Culture Collection . The cells were cultured in RPMI‐1640 medium  supplemented with 10% foetal bovine serum (Invitrogen) in a humidified atmosphere of 5% CO2.2Metaphase chromosomes of the NCI‐H716 cells were prepared as previously described.2.3https://earray.chem.agilent.com/) for interrogating the genome of DMs with high‐resolution and accuracy. Data were quality controlled and extracted using Feature Extraction , and subsequently analysed by Agilent Genomic Workbench .DNA processing, microarray handling and data analysis were performed according to the protocol by manufacturer  with minor modifications. Oligonucleotide‐based Human Genome Microarrays (Agilent Technologies) containing 60 K, 180 K, 400 K and 1 M features were used for hybridization, among which 2 × 400 K format was used to examine genome‐wide copy number alteration Figure . The 4 ×2.4Genomic coordinates of DMs were provided by high‐resolution array‐CGH analysis. Customized NimbleGen 2.1M sequence capture arrays were fabricated using SeqCap v2 software. The captured library preparation and NimbleGen Sequence Capture Arrays were performed according to the manufacturer's protocol, which was adapted from the company's application notes . The captured DNAs were generated by ligating PacBio SMRTbell™ adapters to both ends of linear DNA fragments. These DNA fragments were sequenced on the PacBio RS as a continuous circle. Then, clear data were mapped onto reference human genome (GRCh37/hg19) using Burrows‐Wheeler Aligner (BWA) to filter out the reads without SVs. Subsequently, all the identified fusion sequences were also verified with the BLAST‐Like Alignment Tool (BLAT) and Basic Local Alignment Search Tool (BLAST).2.5Genomic DNA of the NCI‐H716 cells was extracted, quantified and purified with HiSeq X Ten protocol . DNA fragments were ligated with adaptor oligonucleotides to form paired‐end DNA libraries with an insert nucleotide of 500 bp. This library was amplified by PCR with adaptor‐specific primers and sequenced by Illumina HiSeq X Ten instrument to obtain up to ∼150 million reads . Reads that aligned to genomic regions were collected for mutation identification and subsequent analysis. Samtools mpileup and bcftools were used to do variant calling and identify single nucleotide polymorphisms (SNPs), insertions and deletions (INDELs). Control‐FREEC was utilized to do copy number variations (CNVs) detection. And BreakDancer was applied to detect SV information.2.6Sequences of Junction VII and Junction VIII were acquired using GenomeWalker universal kit and Advantage 2 PCR Kit  according to the manufacturer's instructions. Normal human DNA was used as control. All the junction sequences were validated by Sanger sequencing.33.16. However, a dramatically higher amplification level was observed in sub‐amplicons , twice of the major amplicons  on chromosome 8q24.12‐21 and Amplicon H4 on 10q26.13 Figure . Based o3.2Since DMs in the cells contained sequences from different chromosome fragments, we speculate that the way in which DMs arranged might be crucial to cell evolution. To this end, we chose six available BACs based on amplification level within these amplicons, as representative FISH probes to localize the sub‐amplicons in the cells. The amplified regions on DMs were verified by M‐FISH analyses, and co‐localization of the four amplified DNA fragments was shown in Figure Furthermore, we observed duplicated signals of H1a, H2d and H3c located within one individual DMs from Population one, indicating a more complex molecular structure of DMs similar to a ‘diploid’ structure  and identified a total number of 3,120,201 single nucleotide polymorphisms (SNPs), which including 1,689,436 heterozygote SNPs and 1,430,765 homozygote SNPs. We selected a total of 34 heterozygote SNPs in amplified regions from chromosome 8 for the study Table  and foun3.3Because of heterogeneity of DMs, we hypothesized that break‐rejoin model might be a possible mechanism to induce the occurrence of DMs. To elucidate the mechanism, we designed a NimbleGen capture array to detect the sequence of DMs. First, DMs DNA was enriched with a NimbleGen capture array and sequenced with PacBio RS system with average read length of 1100 bp on the basis of circular consensus sequencing. The sequencing reads were then aligned to the human genome to obtain the breakpoint sequences. On the basis of the CGH microarray results, 15 breakpoints within the above four major amplicons were identified and named J1‐J15 in sequential order Figure , and theTo acquire the entire sequence map of DMs, junction sequences must be fully ascertained. Therefore, we cloned the rearrangement breakpoints J7, J8, J10, and J12 of DMs by chromosome walking technique starting from the boundary sequence obtained through CGH microarray and high‐throughput sequencing. Then, the rejoining patterns were manifested by alignment of the cloned sequence to the human reference genome Figure . As show3.42 ratios ≈ 7) than those in the other amplification regions .The regions of each amplicon in DMs were validated through high‐density array CGH. The amplified regions on DMs were verified by M‐FISH analyses, and the results confirmed two populations of DMs in NCI‐H716 cells. Precise boundaries and junction sequences of each amplicon were identified by high‐throughput sequencing and chromosome walking analysis. Based on our research data, we reconstructed the molecular structure of DMs Figure . In deta4Previous studies have reported the existence of endogenous DMs carrying fragments from chromosome 8 and 10 in NCI‐H716 cell line,Our study employed the high‐throughput sequencing technique, providing an unprecedented platform to detect DMs in cancer cells.In this study, we aimed to reveal the mechanisms of DMs formation by deciphering the junction events. As a result, we found three ‘junctions’ Figure  in DM PoTill now, several different models have been proposed for the formation of DMs.In NCI‐H716 cells, DM Population one might have participated in the formation of DM Population two. It can be speculated from the following evidence. First, DM Population one included three integral amplicons ; in contrast, DM Population two only contained partial sequences of these amplicons . Second, more powerful evidence was from the formation of Junction IV (green bar), which does not exist on chromosomes, shared by both populations Figure . These SIn summary, we re‐identified two populations of DMs in human colorectal adenocarcinoma cell line NCI‐H716, confirming their heterogeneity in cancer cells and managed to construct their molecular structure, which were not investigated before. Based on our analysis, we propose that both NHEJ/MMEJ and FoSTeS/MMBIR pathways may mediate the rearrangements in DMs, and the complex structure of DMs in NCI‐H716 cell line may be generated by multi‐step evolutionary process involving various mechanisms. Further anatomy on DMs will enhance our understanding the biological significance of extrachromosomal chromatins in cancer.The authors declare that they have no competing interests.Xueyuan Jia: Conceptualization ; Methodology ; Software (lead); Writing – original draft (lead); Writing – review and editing (lead). Rongwei Guan: Data curation (lead); Software . Xiaobo Cui: Data curation ; Software ; Writing – review and editing . Jing Zhu: Methodology ; Software ; Validation . Peng Liu: Methodology ; Validation . Ling Zhang: Visualization (lead). Dong Wang: Methodology ; Software . Yang Zhang: Conceptualization ; Methodology . Kexian Dong: Methodology ; Software . Jie Wu: Validation ; Visualization . Wei Ji: Methodology ; Validation . Guohua Ji: Resources . Jing Bai: Project administration ; Supervision . Jingcui Yu: Resources ; Supervision . Yang Yu: Conceptualization ; Supervision . Wenjing Sun: Data curation ; Resources (lead); Validation (lead); Writing – review and editing . Feng Zhang: Conceptualization (lead); Funding acquisition ; Methodology (lead); Supervision . Songbin Fu: Funding acquisition (lead); Project administration (lead); Supervision (lead); Writing – review and editing .Supplementary MaterialClick here for additional data file.


Endobronchial ultrasound‐guided transbronchial lung biopsy (EBUS‐TBLB) and fluorodeoxyglucose positron emission tomography (FDG‐PET) have been widely used in the diagnosis of peripheral pulmonary lesions (PPLs). This study was conducted to determine the diagnostic value of EBUS‐TBLB combined with FDG‐PET in the assessment of PPLs.The clinical data of 76 patients with PPLs who received both FDG‐PET and EBUS‐TBLB from January 2016 to February 2018 were retrospectively evaluated. Further subgroup analysis was performed according to lesion diameter (≤20 mm or >20 mm). Related diagnostic indices were calculated and compared between groups.P < 0.01 and P < 0.05, respectively), and its Youden's index was also at a higher level. When stratified by lesion diameter, the combined approach showed a significantly higher diagnostic accuracy rate (P < 0.05) and a higher Youden's index for PPLs >20 mm than PPLs ≤20 mm. In addition, we found that positive bronchus sign and probe within the probe were two important factors conducing to enhancing the diagnostic accuracy rate for EBUS‐TBLB.When combining EBUS‐TBLB with FDG‐PET, the diagnostic accuracy rate, sensitivity, specificity, Youden's index, positive predictive value, and negative predictive value for PPLs were 86.8%, 90.2%, 73.3%, 63.5%, 93.2%, and 64.7%, respectively. In addition, the diagnostic accuracy rate of the combined approach was significantly higher than the single EBUS‐TBLB and FDG‐PET (An integrated approach combining EBUS‐TBLB with FDG‐PET is particularly useful for diagnosing PPLs, and the improved diagnostic yields were especially evident for PPLs >20 mm. Lung cancer is one of the most frequently diagnosed malignancies on a global scale. max) is a critical index for evaluating FDG uptake, and SUVmax >2.5 is generally regarded as a cutoff value for diagnosing malignant lesions.max of 2.5 was adopted for distinguishing malignant from benign PPLs. According to the findings of Mizugaki et al. FDG‐PET has a diagnostic accuracy rate of 78.5% for PPLs.A variety of medical techniques have been adopted to diagnose PPLs. Among these techniques, fluorodeoxyglucose positron emission tomography (FDG‐PET) is an important approach to discriminate between malignant and benign PPLs. As for FDG‐PET, maximum standardized uptake value , this has been widely used in the diagnosis of PPLs.To our knowledge, there are few studies regarding an integrated approach which combines EBUS‐TBLB with FDG‐PET for diagnosing PPLs. This study was conducted to determine the role of EBUS‐TBLB combined with FDG‐PET in the assessment of PPLs.From 1 January 2016 to 28 February 2018, 76 consecutive patients with PPLs who received EBUS‐TBLB at the First Affiliated Hospital of Zhejiang University and FDG‐PET at tertiary care centers were enrolled in this retrospective study. PPLs were generally defined as lesions encircled by normal lung parenchyma without showing any evidence of endobronchial abnormalities. EBUS‐TBLB was performed within four weeks after FDG‐PET was examined. Whether PPLs were malignant or not was ultimately confirmed by the histological diagnosis based on the invasive procedures , or imaging and clinical follow‐up examinations. All PPLs were divided into two groups on the basis of lesion diameter: ≤20 mm and >20 mm. This study was approved by the ethics committee at the First Affiliated Hospital of Zhejiang University. All patients provided their written informed consent.Bronchoscopy was carried out with a flexible bronchoscope  under local anesthesia. For patients receiving EBUS‐TBLB, ultrasound analysis was carried out employing an EBUS system , equipped with a 20 MHz mechanical radial miniprobe . A thorough examination of the target bronchus with the bronchoscope was performed. Once the target PPL was localized, we measured the distance between it and the outer orifice of the bronchoscope working channel for the purpose of performing TBLB at the same PPL site. Subsequently, the miniprobe was withdrawn, and biopsy forceps were inserted into the bronchoscope working channel to acquire five or more biopsy specimens in the same location and depth indicated by the minprobe.18F‐FDG. Regions of interest (ROIs) were drawn over the lesions with the highest uptake of 18F‐FDG, and then the SUVmax was calculated automatically by the computer. In this study, SUVmax >2.5 was regarded as a cutoff value for the diagnosis of malignant lesions, which was consistent with previous studies.FDG‐PET scan was performed using an integrated FDG‐PET scanner . All patients were informed to fast over six hours (blood glucose <8 mmol/L) prior to the examination and were subsequently intravenously injected with FDG at a level of 5.5–7.4 MBq/kg bodyweight. After resting for an hour, patients received a whole‐body FDG‐PET scan. Tumor/background (T/B) ratio was utilized to determine the tumor uptake of max of different subgroups. All statistical analyses were performed using R version 3.5.1 software. A two‐tailed P‐value < 0.05 was regarded as statistically significant.Categorical variables were reported as median . Diagnostic indices including diagnostic accuracy rate, sensitivity, specificity, Youden's index, positive predictive value and negative predictive value were calculated, respectively. In addition, we also calculated the diagnostic accuracy rate of different clinical characteristics, such as age, bronchus sign on CT, and probe position. These characteristics are influencing factors, either for EBUS‐TBLB or PET.Baseline information of the eligible patients is described in Table P < 0.05) and a higher Youden's index.All lesions were detected and punctured by EBUS‐TBLB. The value of EBUS‐TBLB in the diagnosis of PPLs was presented with a diagnostic accuracy rate of 69.7%, sensitivity of 62.3%, specificity of 100%, Youden's index of 62.3%, positive predictive value of 100%, and negative predictive value of 39.5%  for benign PPLs, 7.8 (2.8–10.3) for malignant PPLs, 3.6 (1.7–8.6) for PPLs ≤20 mm, and 7.3 (2.4–10.3) for PPLs >20 mm  (Table P < 0.05) and a higher Youden's index.The median SUVnd 7.3 2.–10.3 fornd 7.3 2.–10.3 forP < 0.01 and P < 0.05, respectively). Furthermore, whether for PPLs >20 mm or ≤20 mm, the combined approach showed a significantly higher diagnostic accuracy rate than EBUS‐TBLB , whereas no significant difference was found in comparison with FDG‐PET (Table P < 0.05) and a higher Youden's index for PPLs >20 mm than PPLs ≤20 mm. In total, 24 patients were identified by FDG‐PET as benign PPLs. Despite the negative FDG‐PET results, 13 patients were eventually confirmed to have malignant PPLs. Of these, seven (54%) could be identified by EBUS‐TBLB.When combining EBUS‐TBLB with FDG‐PET, the diagnostic accuracy rate, sensitivity, specificity, Youden' index, positive predictive value, and negative predictive value for PPLs were 86.8%, 90.2%, 73.3%, 63.5%, 93.2%, and 64.7%, respectively (Table P < 0.001) (Table P = 0.002). We also observed that the diagnostic accuracy rate of PET for patients aged over 65 years was significantly higher than that for patients aged ≤65 years (P = 0.015).The diagnostic accuracy rate of EBUS‐TBLB for negative bronchus sign was 33.3% 14.6%–57.0%), which was significantly lower than that , for positive bronchus sign  were observed. To be exact, the diagnostic accuracy rate, negative predictive value, and Youden's index were 86.8%, 64.7% and 63.5%, respectively. In general, 86.8% of diagnostic accuracy rate indicates that the probability of misdiagnosis made by the combined approach is 13.2%, and 64.7% of negative predictive value means a benign PPL diagnosed by the combined approach has 64.7% probability to be a true‐negative result. In addition, we also observed an increased Youden's index (sensitivity+specificity‐1), which meant the ability of identifying the true‐positive and true‐negative patients has been improved.In this study, the diagnostic accuracy rate, sensitivity and specificity of EBUS‐TBLB were 69.7%, 62.3%, 100%, respectively, which is consistent with previous studies.max of 2.5, and PPLs with SUVmax >2.5 were considered positive.max than larger nodules, having been demonstrated by our findings where PPLs >20 mm displayed a significantly higher level of SUVmax than PPLs ≤20 mm. Additionally, our results are consistent with those of Khalaf et al. who found FDG‐PET had a significantly lower diagnostic value in the assessment of small‐size pulmonary nodules.In the present study, we adopted the traditional cutoff SUVet al.Whether for EBUS‐TBLB or FDG‐PET, we observed a significant higher diagnostic accuracy rate for PPLs >20 mm than PPLs ≤20 mm. Allowing for this, it is understandable why the combined approach is more beneficial for the diagnosis of PPLs >20 mm, which is in agreement with the findings from Mizugaki Several limitations of this study should be noted. First, due to the single‐center, small sample size and retrospective nature of this study, the statistical power in our study may be lowered to some extent. Thus, a prospective, multicenter study with a larger sample size is encouraged to further confirm our findings. Second, since the accurate diagnosis of EBUS‐TBLB for PPLs is influenced by the operators' proficiency on this technique, and this study was conducted in the tertiary care center, the findings of this one‐center study may not be generalizable to the primary medical centers. Finally, as EBUS‐TBLB was performed using UM‐S20‐20R in the present study, our findings may not be applicable to EBUS‐TBLB using UM‐S20‐17R. In future, a new study combining UM‐S20‐17R with UM‐S20‐20R may be conducive to enhancing the diagnostic yields.In conclusion, an integrated approach combining EBUS‐TBLB with FDG‐PET is particularly useful for diagnosing PPLs, with a diagnostic accuracy rate of 86.8%, sensitivity of 90.2%, specificity of 73.3%, Youden's index of 63.5%, positive predictive value of 93.2%, and negative predictive value of 64.7%. Meanwhile, the improved diagnostic yields were especially evident for PPLs >20 mm. Based on the results of EBUS‐TBLB and FDG‐PET, clinicians can make a preliminary judgment on the malignant or benign nature of PPLs, and decide on further surgical procedures or long‐term follow‐up.No authors report any conflict of interest.


Lumbar disc herniation (LDH) is mainly caused by annular fiber disruption with a discrete leakage of nucleus pulposus pressing on a nerve, resulting in back pain and radiating pain. Most patients with LDH can be treated conservatively, but there are many different conservative treatments. Furthermore, most previous studies did not evaluate the long-term efficacy of these treatments and the prognosis. Therefore, an effective and safe therapeutic strategy is lacking for patients with LDH. In this study, we evaluated Xiao Sui Hua He decoction (XSHHD) in the treatment of LDH.This was a rigorous prospective observational 3-year follow-up study. We recruited 69 participants with ruptured lumbar disc herniation (RLDH) between February 2014 and February 2016. Patients took XSHHD orally twice a day for 6 months. The primary outcome measurements were visual analogue scale (VAS) pain score, Oswestry disability index (ODI) and straight leg raising test (SLRT). The secondary outcome measurements was nucleus pulposus protrusion volume on magnetic resonance imaging (MRI). Clinical outcomes were measured at baseline (Visit 1), and at 3, 6, 12, and 36 months ..P < .001). There were no statistically significant differences at 6 months vs 36 months for SLRT and ODI. VAS scores  after 3 years of treatment were statistically significantly different compared with baseline . The baseline protrusion volume was 2018.61 ± 601.16 mm3, and the volume decreased significantly to 996.51 ± 387.42 mm3 at 36 months . The volume of protrusion resorption rate (VPRR) at 36 months was 47.24 ± 23.99%, with significant resorption in 23 cases, partial resorption in 23 cases, no resorption in 15 cases, and increased volume in 2 cases.Sixty-three patients were followed-up for 3 years after treatment. SLRT and ODI after non-surgical treatment improved significantly compared with baseline (This study showed that non-surgical treatment with XSHHD was effective, and the study clarified the natural outcomes in LDH. Lumbar disc herniation (LDH) is mainly caused by annular fibers disruption with a discrete leakage of nucleus pulposus pressing on a nerve, resulting in back pain, radiating pain in the lower extremities, and paresthesia as the main manifestations of the disease. Many clinical guidelines, including the Prognosis Research Trial and meta-analyses, acknowledge the effectiveness of conservative treatment for patients with LD H–4.Herbal medicine is one of the most important parts of traditional Chinese medicine (TCM), with a history of thousands of years of use. Currently, herbal medicine is widely used in the treatment of LDH as an alternative and complementary therapy to Western medicine in China.According to whether the posterior longitudinal ligament is ruptured, LDH can be divided into ruptured and non-ruptured types. Ruptured lumbar disc herniation (RLDH) is one of the most commonly accepted surgical indications due to the large-sized and dissociative protrusions and patients’ severe clinical symptoms. However, if RLDH can be controlled by drugs in the acute stage, resorption of the protrusions may occur after a period of time, and the clinical symptoms will also be alleviated. Even large nucleus pulposus protrusions can be absorbed spontaneously.Some previous studies have evaluated non-surgical treatment  of RLDH,–7 but moThis study was a rigorous prospective observational 3-year follow-up study.We enrolled 66 patients with single-segment RLDH from the outpatient clinic of Suzhou TCM Hospital affiliated to Nanjing University of Traditional Chinese Medicine. The recruitment period was 24 months and extended between February 2014 and February 2016. The inclusion criteria were: (1) age 18–60 years; (2) symptom duration: ≤ 6 months; (3) visual analogue scale (VAS) score ≥ 4/10; (4) MRI data consistent with the symptoms of single-segment RLDH, with an interrupted posterior margin of the “black line” [Astragalus mongholicus , Stephania tetrandra , Chinese angelica , Semen brassicae , Ligusticum wallichii , radix Clematidis , fructus Chaenomeles lagenariae , white Atractylodes rhizome , Pberetima , and Aulastomum gulo .Patients were instructed to take 120 mL Xiao Sui Hua He decoction (XSHHD) orally twice a day for 6 months. XSHHD was developed by Professor Jiang Hong, and was modified according to the Fang Ji Hang Qi decoction and Bu Yang Huan Wu decoction, which were the ancient Chinese prescriptions for thousands of years. XSHHD is widely used in the treatment of LDH in Suzhou TCM Hospital, and we performed a series of clinical and experimental studies to show that XSHHD is effective for LD H, 9. XSHHIn the acute phase (within 2 weeks), patients were prescribed bed rest, and were permitted to use celecoxib temporarily, if necessary . Rehabilitation training was increased during the remission stage, because this training can enhance the strength of the lumbar muscles, and prevent waist injury.At baseline (Visit 1), all patients underwent MRI, and begin to take XSHHD. At 3, 6, and 12 months, patients visited the hospital again  and underwent clinical examinations. At 36 months, the final visit (Visit 5) was performed as an outpatient consultation, and each patient underwent repeat MRI. During the follow-up, each patient visited the hospital within 3 days of the arranged time point. We made appointments for the patient at the outpatient clinic for the follow-up examinations, and recorded follow-up information and scores in a follow-up registration form. When serious adverse effects occurred, we provided immediate appropriate treatment, recorded the adverse effect, and stopped the medicine.At Visits 1 and 5, back pain and sciatica were evaluated using VAS ranging from 0 to 1 0.At Visits 1–5, the symptoms of nerve root compression were measured by the straight leg raising test (SLRT), which waAt Visits 1–5, spinal dysfunction was measured using the Oswestry disability index (ODI ). The ODI·Significant Resorption: VPRR ≥60%·Partial Resorption: VPRR ≥30% to < 60%·None Resorption: VPRR ≥ − 20% to < 30%·Increased: VPRR < − 20%.The secondary outcome measurement was nucleus pulposus protrusion volume on MRI. The final visit of volume of protrusion resorption rate (VPRR) was calculated as follows: (Visit 1 protrusion volume − Visit 5 protrusion volume) / Visit 1 protrusion volume *100%. Disc herniation resorption was classified as follows:3) was calculated by the equation:Protrusion volume measurement was performed using a Siemens 1.5 T superconducting MRI unit . The image data were processed using picture archiving and communication systems (PACS). The volume of the protrusion was calculated by the method described by Auti o. The T2Wi represents the area of the protrusions of layer i, and the total layer number is n.Where IS represents interlayer spacing, LT represents layer thickness, APP < .05 was considered statistically significant.Statistical analyses were performed using SPSS Statistics software (version 23.0). Continuous data are presented as mean ± standard deviation (SD), and categorical data are presented as frequencies. The comparison of SLRT and ODI was performed by the Friedman M test of the non-parametric test. Comparisons of protrusions volumes were performed by the paired t-test. VAS  comparisons were performed by Wilcoxon’s nonparametric test, and Spearman’s correlation analysis was used to compare OIR and VPRR. This study was conducted in accordance with the Declaration of Helsinki (WMA) and the International Ethical Guidelines for Biomedical Research involving human subjects (CIOMS). This was study was reviewed and approved by the Ethics Committee of Suzhou Hospital of Traditional Chinese Medicine, and the approval number was 2017-LYP-013.Of 1031 patients screened, 69 participants were recruited, and 66 concluded 6 months’ treatment and follow-up. Three patients received surgical treatment, among whom 2 patients had progressive pain seriously affecting work and life during the treatment, and 1 patient developed cauda equina syndrome during the treatment. The 66 patients constituted 46 males and 20 females, aged 18 to 58 years, with an average of 36.27 ± 9.97 years. Lesions were located at L3/4 in 4 cases, L4/5 in 27 cases, and L5/S1 in 35 cases. Patients underwent bed rest from 1 to 3.5 weeks, with an average of 2.19 ± 0.54 weeks. Patients took XSHHD from 8 weeks to 6 months, with an average duration of 16.86 ± 5.55 weeks. The study flow diagram is presented in Fig. P < .001). VAS  scores 3 years after treatment were statistically significanly different compared with baseline values  , improved significantly after treatment compared with baseline, and the differences were statistically significant P < .00. VAS (leP = .517). However, a statistically significant difference was found in pair-wise comparisons at the other visit points (P < .001),indicating that non-surgical treatment with XSHHD was effective. The curative effect continued to improve from 3 months to 12 months, and thereafter, the curative effect gradually decreased. However, the overall clinical efficacy at 36 months was equivalent to that at 6 months.There was no statistically significant difference between Visit 5 and 3 for ODI . However, a statistically significant difference was found in pair-wise comparisons at the other visit points (P < .01), indicating that clinical function steadily improved from 3 months to 6 months, with the best function at 12 months. With longer follow-up function decreased, but was still significantly improved compared with baseline, which was consistent with the clinical efficacy.There was no statistically significant difference between Visit 5 and 3 for SLRT . The VPRR at 36 months was 47.24 ± 23.99%, with significant resorption in 23 cases, partial resorption in 23 cases, no resorption in 15 cases, and increased volume in 2 cases (Table Comparisons of the MRI findings in the 63 patients between Visit 5 and baseline are shown in Table P < .001), showing that the higher the improvement rate of ODI in patients was, the better the protrusion resorption rate (RR).After 36 months’ treatment, the Spearman rank correlation analysis was performed on the 63 patients with VPRR and OIR. It was found that there was a positive correlation between the two , and the findings are considered the natural outcome of RLDH. Long-term, there is a certain recurrence rate with RLDH. In this study, after 1 year of treatment, patients’ symptoms were slightly worse than before, but their overall condition was satisfactory, with no major adverse effects.“Resorption” refers to the protrusion reduction or even disappearance without surgical intervention in LDH patients. Since Guinto et al. first discovered protrusion resorption on CT in 1984, this pheCurrently, RLDH is generally included in the clinical indications for surgery. However, non-surgical treatment is a definite option for patients without cauda equina syndrome whose life and work are not seriously affected. In a 2-year prospective randomized controlled trial conducted by Atlas et al., 924 patiMany studies have shown that TCM can achieve good results in the treatment of LD H, 29. In Most of the patients in this study were satisfied with the treatment results, and no adverse effects or complications occurred during the study period. Our results showed that XSHHD is safe, and that non-surgical treatment with XSHHD is a feasible treatments for RLDH.As a rigorous prospective observational 3-year follow-up study, all patients in this study were followed–up for clinical efficacy  and functional recovery (SLRT). Additionally, MRI examinations were performed at baseline and at the last follow-up. Our study proved that non-surgical treatment with XSHHD is effective, and we clarified the natural outcome of the RLDH. Owing to the small sample size and lack of a randomized control group in this study, it is difficult to compare the current treatment with other treatments. This study has limitations, and large-sample prospective randomized case–control studies are needed to further evaluate the efficacy of treatment with XSHHD.


Pleiotrophin , WNT1‐inducible‐signaling pathway protein 2 , chordin‐like protein 1 , transgelin , and R‐spondin‐1, were the proteins most significantly associated with age. Weighted gene co‐expression network analysis identified two of nine modules (clusters of highly correlated proteins) to be significantly associated with chronological age and demonstrated that the biology of aging overlapped with complex age‐associated diseases and other age‐related traits. The correlation between proteomic age prediction based on elastic net regression and chronological age was 0.8 (p < 2.2E−16). Pathway analysis showed that inflammatory response, organismal injury and abnormalities, cell and organismal survival, and death pathways were associated with aging. The present study made novel associations between a number of proteins and aging, constructed a proteomic age model that predicted mortality, and suggested possible proteomic signatures possessed by a cohort enriched for familial exceptional longevity.Aging is a complex trait characterized by a diverse spectrum of endophenotypes. By utilizing the SomaScan Age‐associated proteomic changes studied using SomaScan assay showed a plethora of novel proteins and pathways to be associated with aging in older adults. Predicted age using elastic net regression captured mortality better than actual chronological age. Clusters of highly correlated proteins associated with chronological age were also associated with diverse phenotypes and traits giving clues for shared biology. Demographic and clinical baseline characteristics are summarized in Table 2.2p < 1 × 10−5). Of these, the majority, 427 (56.6%) proteins, were positively associated with aging while the remaining 327 were negatively associated. The top proteins that were significantly positively correlated with age included pleiotrophin (PTN), WNT1‐inducible‐signaling pathway protein 2 (WISP‐2), chordin‐like protein 1 (CRDL1), R‐spondin‐1 (RSPO1), transgelin (TAGL), EGF‐containing fibulin‐like extracellular matrix protein 1 (FBLN3), and growth/differentiation factor 15  transport and regulation” pathway was most strongly associated with age, followed by pathways involved in extracellular matrix remodeling, post‐translational modification and clotting Table . Analysi2.3Next, we performed an unbiased weighted gene co‐expression network analysis (WGCNA) in order to investigate the association between protein networks and age. In this analysis, 4,265 proteins were clustered into nine modules based on co‐expression analysis done in our subjects, with each module characterized by module eigengene (ME). The nine modules include black (230 proteins), blue , brown (486 proteins), green (420 proteins), magenta (31 proteins), pink (98 proteins), red (390 proteins), turquoise , and yellow (458 proteins) Figures .p = 1.0 × 10−60). Gene significance (GS) for age and module membership in green module showed significant correlation , diabetes , hypertension , frailty , and mortality . This module was also negatively associated with gait velocity  and overall cognition  and positively associated with male gender   and nominally with less frailty, death, and stroke , 0.80 (p < 2.2E−16), and 0.78 (p < 2.2E−16), respectively  and cumulative frailty index  better than chronological age  and the frailty index  in the unified model.We compared the predictive validity of chronological age, constructed proteomic age, and the frailty index for all‐cause mortality. The median follow‐up time was 4.79 ± 2.88, and 51 deaths were reported in 525 participants in the validation set. The proteomic age predicted all‐cause mortality better than chronological age , also known as heparin binding growth factor, was the protein most strongly associated with chronological age in our study. PTN is secreted as a cell signaling cytokine, and as its name suggests, it is involved in a plethora of functions such as cell growth, migration, and survival in diverse tissues, including brain and bone. In the central nervous system, PTN acts as an important neuromodulator that plays a role in neurogenesis, learning, and long‐term memory , may have pro‐survival effects by contributing to Wnt3a mediated vascular smooth muscle survival , a bone morphogenetic protein‐4 antagonist, that also has been identified in other studies employing the SomaScan platform  and NT‐pro‐BNP. The relationship between GDF‐15 and age has been noted in a recent SomaScan study, as has been its association with diabetes, cardiovascular disease, and mortality  transport and uptake by insulin‐like growth factor‐binding proteins (IGFBPs) (R‐HSA‐381426) to be the top pathway related to age. IGF‐1 is an endocrine and autocrine/paracrine growth factor that has diverse effects on development, cell growth, differentiation, and tissue repair , post‐translational protein phosphorylation (R‐HSA‐8957275), platelet degranulation (R‐HSA‐114608), and formation of fibrin clot  may also have important implications for functional aging, as they have been shown to be involved in processes such as maintenance of skeletal muscle integrity, Alzheimer's disease, platelet function, and extracellular matrix degradation  or offspring of parents with usual survival . The primary goal of this longitudinal study was to identify genotypes that confer longevity and successful aging. Participants were recruited systematically using public records such as voter registration lists or through contacts at community organizations, synagogues, and advertisements in Jewish newspapers in the New York City area. Potential participants were contacted by telephone to assess interest and eligibility. Exclusion criteria include the following: a score >2 on the AD8  were considered significant. Gender and cohort stratified analyses were performed to understand the possible differential effect of gender and cohort status on age regulated proteomic profile.Baseline characteristics of participants were compared using descriptive statistics. Relative fluorescence unit (RFU) values observed after data normalization procedure for each SOMAmer reagent were natural log‐transformed. Outliers were removed using median absolute deviation method. The preliminary objective of this study was to identify the association of SOMAmer reagents with chronological age using linear regression analysis. Analyses were adjusted for gender and cohort status (OPUS or OPEL). Beta estimate is defined as increase or decrease in specific log (SOMAmer reagent) concentrations with each 1 unit (1 year) of increase in age. Initial normalization procedures carried out by SomaLogic adjusted for changes associated with the experimental setup like inter‐sample differences within a plate and variance across assay runs, individual sample variance on the basis of signaling differences between microarrays or Agilent scanners. The Bonferroni corrected 4.4® Pathway Analysis ) according to the fit of the protein set. We also investigated top diseases and bio‐functions associated with aging. Top networks were checked for concordance with pathway analysis using Reactome  is used to express network interconnectedness. Hierarchical clustering of proteins were based on topology overlap dissimilarity (1‐TOM), and modules were defined from branches of cluster trees using dynamic tree cut method  and other age‐associated phenotypes including frailty index, death, stroke, diabetes, hypertension, myocardial infarction, lipid levels, physical measures , and cognitive phenotypes . We have selected all prominent age‐associated phenotypes whose data were available in our cohort.First principle component of a module is defined as Module eigengene Each module is characterized by a highly connected gene called a hub gene. A hub gene was defined based on highest module membership (MM). MM is measured as correlation of individual protein expression profile with the module eigengene of a given module. Hub genes were analyzed for associated module with age.4.6We constructed a proteomic chronological age predictor using penalized regression model with the glmnet R package  with 95% confidence intervals (CIs) to predict incident all‐cause mortality based on chronological age, proteomic age (predicted), and frailty index. We constructed cumulative frailty index in our cohort as discussed in the supplementary methods. All models were adjusted for gender and cohort status. Time scale was follow‐up time in years to date of death or final contact. Proportional hazard assumptions of all models were tested graphically and analytically and were adequately met. All survival analyses were carried out using coxph function in R.None declared.Nir Barzilai, Joe Verghese, Sofiya Milman, and Sanish Sathyan contributed to the design of the study and interpretation of the data. Sanish Sathyan, Erica F. Weiss, Sofiya Milman, Joe Verghese, and Nir Barzilai contributed to the acquisition of data and writing of the manuscript. Sanish Sathyan, Emmeline Ayers, Tina Gao, and Nir Barzilai contributed to the analysis of the data. Sanish Sathyan, Emmeline Ayers, Tina Gao, Erica F. Weiss, Sofiya Milman, Joe Verghese, and Nir Barzilai contributed to the critical revisions of the manuscript. All the authors approved the final version of the manuscript and agreed to be accountable for all aspects of the work.Supplementary MaterialClick here for additional data file.Tables S9–S18Click here for additional data file.


Smoking rates are high in adults with anxiety disorders (ADs), yet little is known about the safety and efficacy of smoking‐cessation pharmacotherapies in this group.n = 192; generalized anxiety disorder [GAD], n = 243; panic disorder [PD], n = 277) and in a nonpsychiatric cohort . Participants were randomly assigned to varenicline, bupropion, nicotine‐replacement therapy (NRT), or placebo plus weekly smoking‐cessation counseling for 12 weeks, with 12 weeks follow‐up. General linear models were used to test the effects of treatment group, cohort, and their interaction on neuropsychiatric adverse events (NPSAEs), and continuous abstinence weeks 9–12 (treatment) and 9–24 (follow‐up).Post hoc analyses in 712 smokers with AD , GAD (5.4%), and PD (6.2%) was higher versus NPC (2.1%), regardless of treatment. Across all treatments, smokers with PTSD (odds ratio [OR] = 0.58), GAD (OR = 0.72), and PD (OR = 0.53) had lower continuous abstinence rates weeks 9–12 (CAR9–12) versus NPC. Varenicline demonstrated superior efficacy to placebo in smokers with GAD and PD, respectively ; NRT was superior to placebo in smokers with PD . While there was no statistically significant effect of any treatment on CAR9–12 for smokers with PTSD, varenicline improved 7‐day point prevalence abstinence at end of treatment in this subcohort.Individuals with ADs were more likely than those without psychiatric illness to experience moderate to severe NPSAEs during smoking‐cessation attempts, regardless of treatment. While the study was not powered to evaluate abstinence outcomes with these subgroups of smokers with ADs, varenicline provided significant benefit for cessation in those with GAD and PD, while NRT provided significant benefit for those with PD. In this planned secondary analysis we compare the safety and efficacy of varenicline, bupropion, NRT, and placebo across AD subcohorts , and compare clinical characteristics and rates of clinically significant neuropsychiatric adverse events (NPSAEs) and cessation in smokers with AD versus a cohort of smokers without psychiatric disorders.The multinational EAGLES  conducted at the request of the FDA and European Medicines Agency. It was an active treatment‐  and placebo‐controlled trial of varenicline and bupropion for 12 weeks, with a 12‐week nontreatment follow‐up assessment. Institutional review boards or ethics committees at participating institutions approved the study procedures. The study adhered to the Declaration of Helsinki and the International Conference on Harmonization Good Clinical Practice Guidelines. All patients signed informed consent. The main outcome paper provides details of the study design  for primary PD , PTSD (n = 192), or GAD (n = 243), confirmed by the Structured Clinical Interview for DSM‐IV‐TR Axis I Disorders  and social phobia disorder (n = 48) were also enrolled in the parent trial but not included in the present analysis because of the small sample size . Participants could have a comorbid psychiatric diagnosis, provided it did not prevent the participant from complying with study requirements. If the comorbid diagnosis was a substance‐use disorder, the participant was required to be in sustained full remission to be eligible. AD participants must have been considered psychiatrically stable as evidenced by no exacerbations of their psychiatric condition in the prior 6 months and if on pharmacotherapy, to have been on a stable dose for ≥3 months. They could not be considered at high risk of self‐injurious or suicidal behavior. Nonpsychiatric cohort (NPC) participants were confirmed via SCID to have no major psychiatric disorders .Eligible participants were adults aged 18–75 years who smoked an average of ≥10 cigarettes/day with an exhaled carbon monoxide (CO) >10 parts per million (ppm) at screening and wanted to stop smoking. AD subcohort participants met diagnostic criteria based on the 2.3In EAGLES, participants were stratified into one of four psychiatric subcohorts that included those individuals with AD, or the NPC, while also stratifying into one of four geographic regions. Subsequently, participants were randomized to study treatment in a 1:1:1:1 ratio using a computer‐generated schedule. They were not randomized based on their specific AD diagnosis but stratified by whether they had any AD. Participants received either varenicline 1 mg twice daily, bupropion 150 mg twice daily, transdermal nicotine patch (NRT) 21 mg/day with taper, or placebo for 12 weeks. Treatment was administered in a double‐blind, triple‐dummy fashion , and all assessments were blinded throughout the study. All participants received brief weekly smoking cessation counseling  that lasted <10 min and was based on US Agency for Healthcare Research and Quality Guidelines  and secondary efficacy was CARs weeks 9–24 (CAR9–24). Continuous abstinence was defined by self‐report of no smoking (even a puff), with an expired CO concentration of ≤10 ppm  was used to model the safety and efficacy endpoints of interest. Design terms for treatment, diagnostic subcohort, their interaction, and region (US or non‐US), had forced inclusion (efficacy included cohort by region interaction as well). Several baseline characteristics, both numerical and categorical, formed the set of candidate covariate terms. Safety analyses included all participants who took ≥1 dose of randomized treatment. Efficacy analyses of CARs included all randomized participants. Consistent with the Russell standard , with smokers in all three AD subcohorts  having an increased incidence of moderate‐to‐severe NPSAEs versus NPC smokers (2.1%). Across the AD subcohorts, the rates of treatment discontinuation due to NPSAEs were similar, ranging from 1.3% (GAD) to 2.1% (PTSD).Table Figure 3.3Table 3.4Figure The secondary efficacy endpoint, weekly PPA at weeks 12 and 24, are presented in Figure Figure 3Observed 7‐day point prevalence of abstinence during treatment and follow‐up and odds ratios at week 12 and week 24 by cohort. The following variables were included in the odds‐ratio model: treatment group, anxiety disorder subcohort, treatment‐by‐subcohort interaction, and region (US or non‐US) and region‐by‐cohort interaction. CI, confidence interval; GAD, generalized anxiety disorder; NPC, nonpsychiatric cohort; NRT, nicotine replacement therapy ; OR, odds ratio; PD, panic disorder (with/without agoraphobia); PPA, point prevalence of abstinence; PTSD, posttraumatic stress disorderObserved CAR9–24 for varenicline, bupropion, NRT, and placebo ranged from 7 to 13% (PTSD), 6–22% (GAD), 5–21% (PD), and 11–26% (NPC), with placebo consistently the lowest . Within AD subcohorts, estimated ORs versus placebo for CAR9–24 were >2 for all active treatments, with the exception of bupropion and varenicline in smokers with PTSD. However, the 95% CIs for all comparisons included one, with the exception of NRT in smokers with PD, which continued to demonstrate longer‐term efficacy in that subcohort.4Our results from the largest, prospective, placebo‐controlled RCT of smoking cessation medications in smokers with AD provide evidence that varenicline, bupropion, and NRT are generally well tolerated in smokers with PTSD, GAD, and PD, and that varenicline and NRT aid quitting in these diagnostic subgroups, albeit at rates lower than in smokers without mental health conditions.Like smokers with other mental health conditions  for the CAR9–12 data and the significant effect  for varenicline versus placebo for 7‐day PPA at week 12 Figure . Our finAmong smokers with GAD, we observed a pattern of results similar to that found in smokers with heterogeneous mental health conditions that we described in our initial report  to 47% (PD) less likely to quit than smokers without mental health conditions. The incidence of moderate to severe NPSAEs was similar across the three AD subcohorts and not related to any specific smoking cessation treatment. Despite limited power in some diagnostic subcohorts, compared with placebo and counselling, varenicline boosted odds of quitting threefold in smokers with PTSD (95% CI = 0.67–15.67), 4.5‐fold in smokers with GAD (95% CI = 1.20–17.10), and 8.5‐fold in PD smokers (95% CI = 1.57–45.78). Additionally, among PD smokers, NRT improved quit rates approximately 7.5‐fold versus placebo (95% CI = 1.37–40.35) while also tripling the quit rate in smokers with PTSD (95% CI = 0.64–14.47). Our results demonstrate that the first‐line  smoking cessation treatments are effective at least in the short‐term for smokers with PTSD, GAD, and PD. Smokers with AD, particularly those with PTSD, are not all alike with respect to their baseline smoking and psychiatric characteristics, AE patterns, and treatment response. Differences between distinct anxiety and trauma‐related disorders should be further explored. Regardless of whether there are larger clinical trials in the future, smoking cessation efforts utilizing these first‐line treatments should be considered for smokers with ADs.C. R. A. and J. L. H. report no conflicts of interest. A. E. E. reports research grants to her institution from Forum Pharmaceuticals and Pfizer and personal fees for consultation or advisory board services from Charles River Analytics, Karuna Pharmaceuticals, Alkermes, Pfizer, and Reckitt Benck iser. A. E. E.'s writing of the manuscript was supported by a National Institute on Drug Abuse Career Award in Patient‐Oriented Research, K24 DA030443. R. M. A. reports his university receiving grants from Alkermes and Pfizer and providing consulting and/or advisory board services to Arena Pharmaceuticals, Cerecor, Pfizer, and US WorldMeds. R. M. A.'s writing of this manuscript was supported, in part, by National Institute on Alcohol Abuse and Alcoholism Grant #s U10 AA008401 and 1R44AA024643, and NIDA Grant # UO1 DA041731. C. R., D. L., and T. M. are employees and stockholders of Pfizer. The opinions expressed in this article are those of the authors and do not necessarily reflect the views of their employers.All authors, C. R. A., J. L. H., C. R., D. L., T. M., A. E. E., and R. M. A., were involved in the analyses and/or interpretation of data; D. L. performed the statistical analyses; C. R. A. drafted the initial manuscript; and all authors have critically revised the manuscript for content and have approved the final version and the decision to submit for publication.Supporting informationClick here for additional data file.


Advanced lung disease in adult cystic fibrosis (CF) drives most clinical care requirements. The aim was to evaluate outcome (time to death while in the study) in a cohort of adult CF patients with severe lung disease, and to determine the association among baseline patient characteristics and outcome.1) < 30% of predicted. Outcomes of all patients, including their date of death or transplantation, were determined till January 1st, 2016. Clinical data were recorded at the entry date.A retrospective cohort study was performed and clinical records between 2000 and 2015 were reviewed. Severe lung disease was defined as forced expiratory volume in the first second (FEV2)  and use of tobramycin inhalation therapy . Median survival was 37 (95% CI = 16.4–57.6) months. The best cut-off point for BMI was 18.5 kg/m2. Median survival in patients with BMI < 18.5 kg/m2 was 36 months (95% CI = 18.7–53.3).Among 39 subjects included in the study, 20 (51.3%) died, 16 (41.0%) underwent bilateral lung transplantation, and 3 were alive at the end of the study period. Two variables were independently associated with death: body mass index .Median survival of CF subjects with FEV Cystic fibrosis (CF) causes premature death . The pro1) is the main parameter used to assess the severity of lung disease in subjects with CF [Lung disease is the strongest predictor of death in CF, and forced expiratory volume in 1 second , indicates timing of referral in CF. Other indicators for a pretransplant assessment are malnutrition, diabetes, frequent exacerbations and/or an increasing need for intravenous antibiotic therapy, recurrent or massive haemoptysis, relapsing or complicated pneumothorax, or the need for intensive care unit admission [Given the shortage of organs, the resulting waiting times, and the unpredictable evolution of end-stage CF, CF patients eligible for lung transplant should be referred to a transplant centre at an appropriate time . An FEV1dmission , 9.1 < 30% of predicted) refuse or do not meet the criteria for lung transplant. On the other hand, the demand of lung transplantation continues to exceed the supply of suitable donor organs and the median waiting time for lung transplantation has increased [A significant number of CF patients with severe lung disease  of a cohort of adult CF patients with severe lung disease, seen at the Adult CF Centre of Hospital de Clínicas de Porto Alegre (HCPA), and to determine the association among baseline patient characteristics and outcome.1 < 30% of predicted was analysed.This was a retrospective cohort study in which chart review was conducted to identify patients with CF diagnosed with severe lung disease, evaluated, or monitored at the Adult CF Centre of HCPA . Clinical records of Adult CF Program between 2000 and 2015 were reviewed. A cohort of those individuals with severe lung disease defined as FEVHospital de Clínicas de Porto Alegre (HCPA) is a tertiary care teaching hospital located in Porto Alegre, the capital of the state of Rio Grande do Sul, in southern Brazil. The Adult CF Centre of HCPA is one of the largest adult CF centres in Brazil.The diagnosis of CF should be based on clinical features and a positive sweat chloride (> 60 mmol/L) or, in cases with a borderline sweat test result, the presence of a known disease-causing mutation on each cystic fibrosis transmembrane conductance regulator (CFTR) gene .1 < 30% of predicted, we selected patients who had an FEV1 < 30% predicted for the first time. It had to be persistently in this range  was defined as their “entry date” to create a life table for the group.For this survey, we reviewed our CF data base and identified all patients who were, between January 1st, 2000 to December 31st, 2015, 18 years or older (adult CF clinic receive patients 16 years or older). To more specifically look at those patients with FEVThe outcomes of all patients, including their date of death or transplantation, were followed till January 1st, 2016. This date was defined as “the end of the study period”.Pseudomonas aeruginosa, chronic infection with Burkholderia cepacia, chronic infection with methicillin resistant Staphylococcus aureus (MRSA), chronic infection with nontuberculous mycobacteria (NTM), pulmonary artery systolic pressure (PASP) estimated by Doppler echocardiography, use of inhaled dornase alfa, use of inhaled colistin, use of inhaled tobramycin, use of oral azithromycin, pulmonary exacerbations in the last year, lung transplantation status .Data on the following variables were recorded at “the entry date”: entry date, date of birth, sex, ethnicity, educational level, income level monthly, presence of the F508del mutation (homozygous or heterozygous), body mass index (BMI), pancreatic status, CF related diabetes (CFRD), history of pneumothorax, history of haemoptysis, history of previous diagnosis of allergic bronchopulmonary aspergillosis (ABPA), distal intestinal obstruction syndrome (DIOS), CF liver disease, liver transplant, chronic infection with P. aeruginosa, B. cepacia or MRSA was defined as patients having three or more positive isolates during the previous 12 months. CFRD was identified as use of insulin.In this study, pancreatic insufficiency was defined as the use of enzymes and pancreatic sufficiency as no use of enzymes. Chronic infection by The study was approved by the Ethics Committee of Hospital de Clínicas de Porto Alegre and Plataforma Brasil (protocol number 18–0055). Because this was a retrospective study, the Ethics Committee waived the need for informed consent. The authors signed a data use agreement, protecting the confidentiality of patient information. The clinical research complied with international and national standards for clinical study in human .Data analysis was performed using the SPSS 22.0 . Descriptive statistics  were calculated for characteristics of the study sample.Actuarial survival was determined by the Life Table method. Kaplan-Meier graphs was used to demonstrate survival over time. Patients who underwent lung transplantation were censored at the time of their operation, and patients who were alive were censored on the “the end of the study period”.p values were calculated. The likelihood ratio method was used to determine hazard ratios (HR), and the HR was used to approximate the relative risk (RR). Covariates with p < 0.1 entered into a forward stepwise multivariate Cox regression analysis. A p value > 0.10 was the criterion to remove covariates from the model. The non-collinear covariables that reached significance (p < 0.1) in the univariate analysis were included in a multivariate Cox regression analysis with enter method. The most significant variable of this multivariate analysis was correlated with the outcome (death) and submitted to the receiver operating curve (ROC) to determine the cut-off point.Cox proportional hazards regression methods was used to identify risk factors for death while on the study and to determine the association among baseline patient characteristics and outcomes. Time to death while in the study was the primary outcome. Univariate proportional hazards analyses were performed, and Wald chi-square We calculated sample size based on the study of Vizza et al.  In this 1 was 24.4% ± 3.9 of predicted  male subjects and 37 (94.9%) were Caucasian. The mean age at the entry date was 25.3 ± 8.8 years. The mean FVC was 40.5 ± 10.3% of predicted and the mean FEVTwenty subjects (51.3%) died (death outcome), 16 subjects (41.0%) underwent bilateral lung transplantation (censored at the date of transplantation), and 3 subjects were alive and still waiting for lung transplantation at the end of the study period . Among those 20 subjects who died, 14 did not fulfil lung transplantation criteria, 2 refused the lung transplantation referral and 4 died while waiting on waiting list. The time spent on waiting list for those 4 subjects who died while waiting for lung transplantation was 37.5 ± 10.6 months. The time spent on waiting list for those 16 subjects who underwent lung transplantation was 35.3 ± 24.9 months.Additional descriptive characteristics are provided in Tables 2  and use of tobramycin inhalation therapy . In multivariate Cox regression analysis with enter method both variables were associated with case status: BMI kg/m2  and use of tobramycin inhalation therapy .The results of univariate Cox proportional hazards regression methods for death while on the study and the association of case status (death) are shown in Table 2.In ROC curve analysis for BMI, the area under the curve was 0.694 and the best cut-off point selected was 18.5 kg/m1 < 30%. Median survival was 37 (95% CI = 16.4–57.6) months and mean survival was 50.5 (95% CI = 39.3–61.6) months. Figure 2 and BMI ≥ 18.5 kg/m2. Mean survival in patients with BMI < 18.5 kg/m2 was 39.9 (95% CI = 25.8–54.0) months  and in patients with BMI ≥ 18.5 kg/m2 mean survival was 65.5 (95% CI = 49.2–81.8) months , Log-rank test P = 0.028.Figure 1 < 30% of predicted was 37 months. The main factors associated with time to death while in the study were BMI and tobramycin inhalation therapy. In ROC curve analysis for BMI, the best cut-off point selected was 18.5 kg/m2. Mean survival in patients with BMI < 18.5 kg/m2 was significantly lower (39.9 months) than in patients with BMI ≥ 18.5 kg/m2 (65.5 months). At the end of the study period, 20 (51.3%) died, 16 subjects (41.0%) underwent bilateral lung transplantation, and 3 subjects were alive and still waiting for lung transplantation. There was no difference between patients with entry date in the study after 2007 from those with entry date in 2007 or before.In this retrospective cohort study, we demonstrated that the median survival of CF subjects with FEV1 < 30% of predicted was associated with a 2-year mortality rate of approximately 50% (38% in men and 56% in women). Milla et al. [1 < 30% predicted. The median survival was 3.9 years and only the rate of decline in percent of predicted FEV1 was a significant predictor of the risk of death. Vizza et al. [A measurement of lung function over time to assess disease progression has been the most useful predictor . In 1992a et al.  analyseda et al.  studied 1 less than 30% predicted. Out of them, 9 patients were transplanted. CF patients with a FEV1 < 30% and who did not receive a lung transplant had survived longer than CF patients who did receive a lung transplant . Two factors  were found to influence the mortality rate.Augarten et al.  evaluate1 was first observed to be less than 30% predicted. Median survival improved from 1.2 years in the 1990 group to 5.3 years in the 2002 group, with a marked improvement in survival from 1994. The use of nebulised recombinant human DNase was significantly associated with a reduced risk of death . Significantly increased risks were associated with a BMI under 19 , long term oxygen therapy , and nebulised antibiotics .George et al.  evaluate1 < 30% prior to lung transplant. Median transplant-free survival was 6.6 years. There was substantial heterogeneity in survival, with some patients with CF dying soon after reaching this lung function threshold and others living for many years.Ramos et al.  conducte1 < 30% of predicted and elevated PaCO2 > 50 mmHg [1 percentage predicted. Female sex, diabetes mellitus, B. cepacia infection, and the number of exacerbations negatively affected the survival of patients with CF, whereas FEV1 percentage predicted alone was not a sufficient predictor of early mortality [Another study reported a relationship between early mortality and FEV 50 mmHg . In a 5-ortality .The importance of heterogeneity of the disease and inter-population variations in determining the survival in adult CF patients in different populations was highlighted by a recent report . In cont2.Prior studies evaluating survival in patients with CF have also documented increased risk of death among patients with lower BMI , 14, 19.2 and 11.29 million inhabitants. There are three CF centers in Rio Grande do Sul, all of them located in the capital Porto Alegre. Almost all patients attending the Adult CF Centre of HCPA were from were living in the state of Rio Grande do Sul.In Brazil, the public health system provides ambulatory and hospital health carefree of charge. Also, CF medications are available through the public health system known as SUS (Sistema Único de Saúde) entirely free of any cost. Some CF medications are part of a protocol of medications that have been approved by Brazilian Health Ministry. The Health Secretary of each state is responsible for the delivery of the medications through public health care units or through public pharmacies. Usually, the CF care (CF Centers) is performed in each state in public hospital or university hospital. During the period of the study, the following medications were available: alfa dornase, colistin, tobramycin solution for inhalation, pancreatic enzymes, fat-soluble vitamins , ursodeoxicolic acid, azithromycin, dietary supplements, oral antibiotics for pulmonary infectious exacerbation. During the period of the study, new therapies such ivacaftor and ivacaftor/lumacaftor had not yet been approved in Brazil. State of Rio Grande do Sul, in southern Brazil, has a total area of around 281,748 kmIn Brazil there are six lung transplantation centers, two of them located in Porto Alegre. In Brazil, lung transplantation candidates are listed according to the chronological criterion. When there is organ supply, active patients compete according to waiting list time, provided there is ABO blood group identification, ribcage size compatibility, updated immunological reactivity test and, eventually, ABO compatibility (in the absence of receptors with the same ABO blood group as the donor). Patients who receive an organ that does not function within the first 48 h are an exception to the criteria above. In these situations, these receptors can be listed to receive another organ with urgency, being automatically placed at the top of the list . The meaP. aeruginosa [Pseudomonas aeruginosa. In Brazil, inhaled colistin therapy is less expensive then inhaled tobramycin [P. aeruginosa was colistin. We reserved the use of inhaled tobramycin for patients with advanced lung disease. Consequently, patients who receive inhaled tobramycin in our service are more likely to have worse disease severity. The association of tobramycin inhalation with time to death in the present study could interpreted as confounding by severity [Guidelines recommend tobramycin solution for inhalation in patients over 6 years with chronic ruginosa . Colistibramycin . So, durseverity .The present study has some potential limitations. First, the investigation was done in a single centre. Second, the sample size was too small and would be associated with low statistical power. Mainly, because too many variables entered Cox regression models. Third, the retrospective study design did not allow us to assess some potential risk factors for mortality (parameters that were not collected).1 < 30% of predicted. Also, the association with a long time in lung transplant waiting list, should suggest that early referral for consideration of lung transplantation is highly desirable.The clinical relevance of the present study is to alert Brazilian adult CF physicians to the lower survival rate of adult CF subjects with FEV1 < 30% of predicted was 37 months. The main factors associated with time to death while in the study were BMI and tobramycin inhalation therapy. The best cut-off point selected for BMI was 18.5 kg/m2. Mean survival in patients with BMI < 18.5 kg/m2 was significantly lower (39.9 months) than in patients with BMI ≥ 18.5 kg/m2 (65.5 months). It is hoped that targeting these individuals for more intense nutritional optimisation will improve the survival.In conclusion, we demonstrated that the median survival of CF subjects with FEV