Endobronchial ultrasound-guided trans-bronchial needle aspiration (EBUS-TBNA) is minimally invasive technique used for diagnosis and/or staging of benign and malignant pulmonary and non-pulmonary disease. Previous studies have established the utility of EBUS-TBNA in narrowly defined indications and populations. In this pragmatic ‘real world’ study we have analysed the use of EBUS-TBNA for a variety of clinical presentations and its clinical application in conjunction with other invasive investigations.All EBUS-TBNA procedures performed at Sir Charles Gardiner Hospital in 2012–2014 were reviewed retrospectively, using relevant hospital databases.A total of 327 patients underwent 337 EBUS-TBNA procedures. EBUS-TBNA procedures were used to diagnose a wide spectrum of benign and malignant conditions. The main application was in the diagnosis and staging of malignant conditions (70.6%), and in the diagnosis of benign conditions such as sarcoidosis 40 (12.2%), and silicoanthracosis 17 (5.2%). EBUS-TBNA was sufficient to diagnose and stage the disease as a single stand-alone invasive procedure in 191 (59.2%) patients. EBUS-TBNA was the final invasive procedure undertaken in 283 (87.6%) patients. Only 13.3% of non small cell lung cancer (NSCLC) patients who had EBUS-TBNA as a first investigation required multiple procedures compared to 51.1% of all NSCLC patients undergoing EBUS-TBNA. Overall sensitivity, specificity, NPV and diagnostic accuracy for EBUS-TBNA were 89.7, 100, 85.1 and 89.9%, respectively and three minor complications (0.9%) occurred as a result of the procedure.EBUS-TBNA was undertaken for a wide variety of clinical conditions. Good diagnostic accuracy and safety profiles were demonstrated for the procedure, supporting its application as a first line investigation in the diagnosis and/or staging of a range of malignant and benign conditions. Our study was unique in its documentation of the use of EBUS-TBNA in a real-world setting in conjunction with other invasive modalities. EBUS-TBNA was utilised as a stand alone invasive procedure in more than half of the patients. Importantly, in NSCLC, when EBUS-TBNA was performed as primary diagnostic and staging investigation, less patients underwent subsequent invasive procedures. Endobronchial ultrasound-guided (EBUS) procedures utilise ultrasound imaging for visualisation and sampling of lung tissue and lymph nodes through the wall of the trachea and bronchi. EBUS is a minimally invasive procedure with a very low complication rate, which has become standard practice in many institutions internationally , 2. The EBUS is available in two forms: EBUS guided trans-bronchial needle aspiration (TBNA) via the linear probe (EBUS-TBNA) and the EBUS radial probe via a guide sheath (EBUS-GS). Each form has specific benefits and services different patient disease presentations.EBUS-TBNA uses a dedicated bronchoscope and allows real-time TBNA of mediastinal and hilar lymph nodes and masses. A frequent application of EBUS-TBNA is in the accurate nodal staging of lung cancer , 3, howeComputed tomography guided transthoracic needle aspiration (CT-TTNA) can be used to gain peripheral tissue samples from the lung and provides good diagnostic accuracy, however carries a risk of pneumothorax  and expoA strong evidence base addressing the effectiveness, efficacy and safety of the EBUS-TBNA technique already stands, however most studies have analysed the diagnostic performance in narrowly defined populations , 16, 20.We retrospectively reviewed all EBUS-TBNA procedures performed at Sir Charles Gardiner Hospital (SCGH) between 1 January 2012 and 31 December 2014. Data regarding the clinical application, diagnostic performance and safety of EBUS was extracted using relevant hospital databases. Patient details such as age, gender, smoking history and Eastern Co-operative Oncology Group Performance Status (ECOG)  were recEthics approval was granted by the SCGH Human Research Ethics Committee (Ref. No 2013–233). Waiver of consent was granted for inclusion of patients who had EBUS in 2012, 2013 and January–February 2014, while patients included in the study after February 2014 provided consent for access to their medical records.Patients at SCGH were generally presented to the lung multidisciplinary meeting (MDM) after an initial CT scan of the thorax and upper abdomen and, in the majority of the cases results of a positrom emission tomography (PET) scan, which guided recommendations for an EBUS-TBNA investigation. Mediastinal lymph nodes were sampled if they were enlarged radiologically (> 1 cm) in the short axis and/or were PET positive. In general, if there was a pulmonary mass or nodule and evidence of mediastinal or hilar lymph node involvement, the lymph node was sampled first on the grounds that diagnosis and staging might be achieved with one procedure. Lymph node sampling was undertaken by first needling the lymph node that constituted the highest stage (N3) and progressing down to the lowest stage. Rapid on-site evaluation (ROSE) was undertaken and the adequacy of the sample was assessed by the presence of lymphocytes and evaluation by the pathologist. The site and number of lymph node stations sampled and the number of passes per lymph node were determined by the operator. At least three needle passes were made per lymph node unless the diagnostic material was reported adequate on ROSE. If the lymph node samples were non diagnostic then the pulmonary lesion would be targeted for diagnostic purposes by EBUS-GS if suitable, or otherwise by CT-TTNA. Some patients were referred to our service from other sources and may have had a diagnosis already established but required staging or sampling for molecular analysis.Hilar and mediastinal lymphadenopathy or mediastinal masses were directly sampled by EBUS-TBNA. EBUS-TBNA was performed under general anaesthesia.For the purpose of this study, cases referred for EBUS-TBNA were divided into two categories based on the intention of the procedure: 1) to confirm, stage or exclude malignancy, 2) to obtain tissue diagnosis for suspected benign conditions, based on clinical or radiological evidence. Cases were also classified into four sub-groups, based on radiological manifestations: those with a lung mass or nodule only, those with a lung mass or nodule and hilar or mediastinal lymphadenopathy, those with a hilar or mediastinal mass only, or those with hilar or mediastinal lymphadenopathy only.malignant when malignant cells were present, and benign when normal lung or lymphatic tissue with no malignant cells were seen. Inadequate samples were excluded from sensitivity and specificity analysis, but included in diagnostic accuracy. Standard definitions of sensitivity and specificity were used.Pathology results were reported as Cyto- and histo-pathological specimens for cases in category one were considered true positive if malignant cells were reported and true negative if an adequate sample showed no malignant cells and further clinical follow-up, pathological findings, or medical imaging confirmed the result. False negative specimens failed to detect malignancy in an adequate sample and malignancy was later clinically, radiologically or pathologically detected in the same lymph node, mass or region.Cyto- and histo-pathological specimens in category two were considered true positive if the sample was adequate for diagnosis of a benign condition, and false negative when the sample was normal and diagnosis was established by other means. Specimens were considered true negative if pathology was not identified in an adequate sample.All available clinicial documentation, radiological investigations, invasive investigations and pathological results relevant to the same provision of care were reviewed and recorded. All additional invasive investigations and their temporal relationship to EBUS were recorded if they related to the same provision of service. Four patients, referred from external sources, with incomplete records were excluded from the analysis of additional invasive procedures. The minimum follow up time for records after EBUS-TBNA procedure was 12 months, however patients without a clear final diagnosis or with non-diagnostic results of EBUS were followed up for two years.Data was analysed using the IBM SPSS Version 22 statistical software package. A descriptive analysis was performed where categorical variables were expressed as absolute and relative frequencies, and continuous variables were calculated as means.The demographic characteristics of all patients are recorded in Table A total of 327 patients underwent 337 EBUS-TBNA procedures. Of these, 317 patients had one and ten patients had two EBUS-TBNA procedures.EBUS-TBNA sample site locations , and 30 for patients with suspected benign conditions (category 2). Within category 1, 249 (81.1%) procedures were performed for diagnosis with or without staging, 53 (17.3%) were for cancer staging, and 5 (1.6%) for material for molecular tests.There were 297 (90.8%) category 1 patients where EBUS-TBNA was requested to confirm, stage or exclude malignancy. Of these patients 166 (55.9%) presented with a lung mass/or nodule and hilar/mediastinal lymphadenopathy, 105 (35.4%) with hilar/mediastinal lymphadenopathy, 26 (8.8%) with a hilar or mediastinal mass.There were 30 (9.2%) category 2 patients where EBUS-TBNA was requested to obtain tissue diagnosis for suspected benign conditions.Of the 323 patients with records of their invasive investigations, EBUS-TBNA was sufficient to diagnose and stage the disease as a single stand-alone invasive procedure in 191 (59.1%) patients. One hundred and eight (33.0%) patients underwent other invasive investigations in addition to EBUS-TBNA, and 24 (6.7%) had multiple EBUS procedures  required EBUS-TBNA alone  mutation, although in the initial stages of this study such testing was undertaken after discussion at the MDM since the test did not have Government remuneration. Kirsten rat sarcoma (KRAS) mutation was not performed if the EGFR was positive, since EGFR and KRAS are mutually exclusive in a patient, and since KRAS is not yet a targetable lesion. Anaplastic lymphoma kinase (ALK) gene rearrangement testing was performed in patients with adenocarcinoma who were EGFR negative; BRAF was performed in patients with melanoma, and C-MYC mutations in patients with lymphoma. Of 60 EBUS-TBNA samples sent for molecular testing, positive results were reported for 25 (41.7%) and 35 (58.3%) were negative. KRAS mutations, which were not routinely tested, were identified in 12 (20%) samples, EGFR mutations in five (8.3%), and ALK rearrangement was demonstrated in two samples (3.3%). A BRAF gene mutation was detected in five (8.3%) patients with metastatic melanoma and a C-MYC mutation was detected in one (1.7%) patient with non-Hodgkin lymphoma..Three complications (0.9%) occurred as a result of EBUS-TBNA. Two procedures were complicated by haemorrhage. One case was registered on a postprocedural X-ray and resolved without additional interventions, the second occured at the time of TBNA and while haemostasis was restored during the procedure, the presence of hypoxia required an overnight admission for observation. One patient developed wheeze, dyspnoea and cough following EBUS-TBNA and was admitted for treatment and observation. There were no obvious complications during the procedure and the post-procedural symptoms were thought to be due to gastro-oesophageal reflux and laryngospasmThis study showed that EBUS-TBNA was utilised for a wide variety of clinical conditions in our institution. The main application was for diagnosis and staging of lung cancers, with 81% of these being non small cell lung cancer (NSCLC). EBUS-TBNA has previously been shown to have an essential role in the diagnosis and staging of lung cancers. It is recommended in current international and Australian guidelines as a first line investigation of lung malignancies with mediastinal lymph node involvement , 18, 22,In our study, five EBUS-TBNA procedures were indicated for the specific purpose of determining molecular characteristics of lung cancers, and molecular testing was performed in 17.8% of EBUS-TBNA procedures. With further development of targeted oncological therapies it is expected that the use of EBUS-TBNA in this area is likely to expand in the future .The literature reports typical diagnostic yields for EBUS-TBNA ranging between 80 and 100% –29. Our The relatively high diagnostic accuracy of EBUS-TBNA in our study could be due to the availability of ROSE, which allows for confirmation of true positive results, reducing the need for unnecessary additional passes, and confirmation of sample adequacy through the presence of lymphocytes . FurtherEBUS-TBNA is often used in the diagnosis of sarcoidosis with various levels of diagnostic accuracy reported. Two meta-analyses reported a pooled diagnostic accuracy of 79% (range of 54 to 93% and SD 24%) , 34, andThe highest observed sensitivity was for samples with malignant final diagnosis (91%), however the NPV of this group was much lower than that of cases with benign final diagnosis . The low NPV mandates further sampling if clinical suspicion of alternative disease is ongoing and a negative result is obtained. In our study the sensitivity and specificity of EBUS-TBNA for NSCLC was 90.8 and 100%, respectively, higher than the median sensitivity of 89% (range 46 to 97%) reported in a recent meta-analysis .This study was unique in its recognition of the clinician’s real-world use of EBUS-TBNA in combination with other invasive investigations. In the past decade, the refinement of EBUS procedures has allowed patients to experience a less invasive and expediated process in diagnosis and staging of cancer. Ideally, the initial invasive investigation for lung cancer should simultaneously provide tissue for histopathological diagnosis, allow for additional molecular testing and accurately stage the disease. Minimising procedures required to diagnose and stage malignancy spares patients additional risks associated with procedures and importantly, expediates decision making and time to treatment commencement.In suspected cancer cases included in our study, 56.7% of EBUS-TBNA procedures were adequate as a stand alone invasive procedure for diagnosis and staging. This figure is reassuring and suggests an appropriate use of EBUS-TBNA in line with clinical practice guidelines.Although ideally diagnosis and staging are performed simultaneously with EBUS-TBNA, our study included a wider variety of clinical presentations including patients with extrathoracic disease requiring additional staging with EBUS-TBNA or metastatic spread from an intrathoracic origin requiring other invasive modalities. Inadequate prior samples, which may represent poor modality choice (EBUS or other) was recorded for 13 patients, representing 9.8% of those who underwent tests in addition to EBUS-TBNA.The largest radiological group requiring additional invasive sampling techniques were those with lung lesion and lymphadenopathy, where 89 (53.3%) required tests in addition to EBUS-TBNA. This group included 58 (65.2%) patients with established diagnosis where tissue sample was required for further tests or staging. Forty eight of these patients had undergone initial sampling with CT FNA, bronchoscopy, or other thechniques and EBUS was the last invasive modality. For 34 NSCLC cases EBUS-TBNA was done for staging and if it was considered as the first invasive investigation potentially multiple procedures may have been avoided. Furthermore, our study supports previous findings that when EBUS-TBNA is performed as primary diagnostic and staging method in NSCLC, patients are less likely to require subsequent invasive sampling procedures . Only 13Our hospital was the only institution performing EBUS-TBNA in the state at the time of data collection and thus some patients in this group may have not had access to the procedure, accounting for the choice of alternative initial invasive investigations.The literature reports an excellent safety profile, with a complication rate of 0.15% for EBUS-TBNA .We have assessed a heterogenous group of patients referred for EBUS-TBNA for different indications to one of the largest tertiary hospitals in Western Australia, and the only hospital in the state offering EBUS at the time. We recognise that this study has limitations. This was a single-centre retrospective study and the data is limited to the information available from hospital records. Due to its pragmatic approach, the study depicts the application of EBUS-TBNA procedures at our institution and our findings may have limited generalisability particularly given the performance of EBUS procedures under general anaesthesia.The diagnostic power and excellent safety profile of EBUS-TBNA already documented in the literature was reflected at our institution supporting its use as a first line investigation in patients with mediastinal and hilar lymphadenopathy with or without suspicious pulmonary nodules or masses. The application of EBUS-TBNA at SCGH is largely in the diagnosis and staging of lung cancer, with promising results to support its use in the diagnosis of a variety of benign and malignant conditions. EBUS-TBNA was used in conjunction with other invasive investigations with the aims of maximising diagnostic ability, providing material for molecular testing and accurately staging malignancy, while minimising invasive procedures required. EBUS-TBNA was effectively utilised as a stand alone invasive procedure in more than half of patients at our institution. Importantly, in NSCLC, when EBUS-TBNA was performed as primary diagnostic and staging investigation, patients underwent less subsequent invasive procedures.


At 24 months, retention rates for community-based ART and standard of care were 73.1% and 44.0%, respectively (p < .01). Significant differences in levels of adherence were observed when comparing community-based ART against persons receiving standard of care (p < .05). No differences in adherence levels were observed between groups of people receiving various frequencies of home visits. Our pilot programme in Koidu City provides new evidence that community-based ART has the potential to improve retention and adherence outcomes for HIV-infected persons, regardless of the frequency of home visits. Overcoming the barriers to HIV care requires a comprehensive, patient-centred approach that may include clinic-based and community-based interventions.Evaluations of community-based antiretroviral therapy (ART) programmes have demonstrated positive outcomes, but little is known about the impact of tapering community-based ART. The objective of this study was to assess 24-month HIV retention outcomes of a community-based ART programme and its tapered visit frequency in Koidu City, Sierra Leone. This retrospective, quasi-experimental study compared outcomes of 52 HIV-infected persons initiated on community-based ART against 91 HIV-infected persons receiving the standard of care from November 2009 to February 2013. The community-based ART pilot programme was designed to strengthen the standard of care through a comprehensive, patient-centred case management strategy. The strategy included medical, educational, psychological, social, and economic support. Starting in October 2011, the frequency of home visits was tapered from twice daily every day per week to once daily three days per week. Outcomes were retention in care at 12 and 24 months and adherence to ART over a three-month time period. Participants who received community-based ART had significantly higher retention than those receiving standard of care. At 12 months, retention rates for community-based ART and standard of care were 61.5% and 31.9%, respectively ( Since the early 2000s, antiretroviral therapy (ART) has been made available throughout the world. As UNAIDS promotes a strategy to have 90% of HIV-infected persons virally suppressed UNAIDS, , structuWhile some clinic-based strategies such as early initiation of ART have improved retention in care  to community-based ART as a key component of national HIV programmes  and are expected to attend monthly visits where medication refills and routine clinical monitoring occurs.In 2009, HIV/AIDS was highly stigmatised in Koidu City, and ethnographic research carried out by organisational collaborators revealed many stories of patients who faced extreme marginalisation, ostracisation and isolation after being diagnosed – several even died at home. This research prompted us to develop a community-based ART pilot programme that would strengthen the standard of care through a comprehensive, patient-centred, case management strategy. This strategy included medical, educational, psychological, social, and economic support . A commuKono and Krio as well as able to read and write English . Final selection was based on results of the training. The training was conducted with the Partners in Health Accompagnateurs Training Guide  during home visits and occur twice daily for seven days every week. In October 2011, home visits were reduced; they continued to occur once daily three times every week. A typical CHW work schedule was to visit participants on Mondays, Wednesdays, and Fridays. At the first visit of the week, CHWs counted pills to assess adherence to ART. During the other two visits per week, the programme placed emphasis on providing non-medical support . Other cThe programme considered ‘real-world’ circumstances and allowed for flexibility in scheduling and activities of home visits based on the needs and interests of the participants. For example, the patient-centred approach to case management allowed for more social support if participants did not have any retention or adherence issues. Other situations arose such illness that required unscheduled CHW attention. By strengthening supervisory systems for CHWs and making the abovementioned and other changes to the programme, we intended to balance flexibility, independence, and structure with the goal that we would continue to achieve the same level of comprehensive care as the more intensive version of the programme.Our primary outcome was retention in care, defined as having attended a clinic visit in the last 150 days .Age, sex, and ART initiation were demographic data available from the ART registries. We used these variables to test for differences between groups in and out of care as well as between groups receiving standard of care with and without community-based ART. Fisher’s exact tests were conducted to determine if there were significant differences in retention outcomes at 12 and 24 months. Three months of adherence data were calculated for persons who received community-based ART (at different home visit frequencies) and for persons who received standard of care. Adherence was analysed as a continuous variable, and adherence determinations of >100% were truncated at 100. Mean and median adherence of the study population was calculated. Student We initiated our pilot programme for patient care, not for human subjects research. We obtained ethical approval by the Sierra Leone Ethics and Scientific Review Committee and the Institutional Review Board at Baylor College of Medicine to use programmatic and clinic records for research purposes.Between November 2009 and February 2011, 52 persons were enrolled in the programme and were initiated on community-based ART. During the same time frame, 91 persons who were ART-naive or ART-experienced were enrolled and only received standard of care. No significant differences in age, gender, or number of ART-experienced persons were observed among groups who were retained in care and LTFU as well as among groups who received standard of care with and without community-based ART.p < .01) than those who were only receiving standard of care. Comparative outcomes at 12 months are presented in Of the 52 HIV-infected persons who were receiving community-based ART with home visits twice daily, 32 persons (61.5%) were retained in care at 12 months. During the same time period, 29 (31.9%) of 91 persons only receiving standard of care were retained in care. Participants receiving standard of care plus community-based ART were significantly more likely to be retained in care (p < .01)  were retained in care, and 6 persons who had been lost to follow-up re-engaged in care. The 24-month retention rate (from initiation of community-based ART to 12-month censor after the taper) was 73.1% for participants in the community-based ART programme. In the standard of care group, 29 persons continued in care when the community-based ART programme was transitioned from twice daily to three times per week. After observing this group for another 12-month period, 28 persons (96.6%) were retained in care, and 12 persons who had been lost to follow-up re-engaged in care. The 24-month retention rate was 44.0% for participants in the standard of care group. At 24 months, the community-based ART programme continued to demonstrate significantly higher retention rates than the standard of care only (p < .01) .p = .02). Mean adherence for participants of the community-based ART programme who received home visits once daily three times per week was also significantly higher than mean adherence for participants who only received standard of care (p = .03). Differences in mean adherence among variations of community-based ART and the standard of care are presented in Thirty-two persons who received community-based ART with home visits twice daily had a mean and median observed adherence (SD) of 89.4% (6.9) and 91.1%, respectively. Thirty-one (96.9%) of these persons who received community-based ART with home visits three times per week had a mean and median adherence (SD) of 85.5% (8.4) and 86.8%, respectively. In the standard of care group, 58 persons had a mean and median adherence (SD) of 78.6% (18.2) and 81.8%, respectively. Mean observed adherence for persons who received community-based ART with home visits twice daily every day per week was significantly higher than persons receiving the standard of care only  are needed to assess the effect of tapering community-based ART over time in settings similar to ours in Sierra Leone.In summary, a pilot programme in Koidu City provides new evidence that community-based ART has the potential to improve retention and adherence outcomes for a group of HIV-infected persons, regardless of the frequency of home visits. Overcoming the barriers to HIV care requires a comprehensive, patient-centred approach that may include clinic-based and community-based strategies. In rural Sierra Leone, HIV patients routinely face a myriad of retention and adherence challenges, suggesting that a comprehensive but flexible patient-centred approach to community-based ART may be a viable intervention. Using these findings, the National HIV/AIDS Secretariat in Sierra Leone opted to use our community-based ART pilot programme as the model for national scale-up of community-based HIV care.


Electronic medical records (EMRs) contain a wealth of information that can support data-driven decision making in health care policy design and service planning. Although research using EMRs has become increasingly prevalent, challenges such as coding inconsistency, data validity, and lack of suitable measures in important domains still hinder the progress.The objective of this study was to design a structured way to process records in administrative EMR systems for health services research and assess validity in selected areas.On the basis of a local hospital EMR system in Singapore, we developed a structured framework for EMR data processing, including standardization and phenotyping of diagnosis codes, construction of cohort with multilevel views, and generation of variables and proxy measures to supplement primary data. Disease complexity was estimated by Charlson Comorbidity Index (CCI) and Polypharmacy Score (PPS), whereas socioeconomic status (SES) was estimated by housing type. Validity of modified diagnosis codes and derived measures were investigated.Visit-level  and patient-level records  were generated. The International Classification of Diseases, Tenth Revision, Australian Modification (ICD-10-AM) codes were standardized to the International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) with a mapping rate of 87.1%. In all, 97.4% of the ICD-9-CM codes were phenotyped successfully using Clinical Classification Software by Agency for Healthcare Research and Quality. Diagnosis codes that underwent modification (truncation or zero addition) in standardization and phenotyping procedures had the modification validated by physicians, with validity rates of more than 90%. Disease complexity measures (CCI and PPS) and SES were found to be valid and robust after a correlation analysis and a multivariate regression analysis. CCI and PPS were correlated with each other and positively correlated with health care utilization measures. Larger housing type was associated with lower government subsidies received, suggesting association with higher SES. Profile of constructed cohorts showed differences in disease prevalence, disease complexity, and health care utilization in those aged above 65 years and those aged 65 years or younger.The framework proposed in this study would be useful for other researchers working with EMR data for health services research. Further analyses would be needed to better understand differences observed in the cohorts. Secondary use of electronic medical records (EMRs) data by clinicians, researchers, data analysts, and computer scientists has led to promising findings in population health research such as patient-utilization stratification , treatmeDifferent research teams have described EMR data processing methods ,8-18. HoAlthough the standardization of diagnosis codes of different nosologies or different versions of the same nosology has been reported previously ,21, the Measures of patient complexity, disease severity, and socioeconomic status (SES) are not readily available in most datasets  but haveThis study has attempted to address some of these challenges common to the use of EMR data for health services research by detailing a structured framework for EMR data processing. Furthermore, the study proposed and validated methods for standardization of diagnosis codes and construction of disease phenotypes and also proposed and tested derived measures of disease complexity and SES, which could be applicable to other datasets with similar data fields.The National University Hospital (NUH) is a 1000-bed Academic Medical Center (AMC) in Singapore . Being 1The cascade architecture of PASS is patient → visit → record as shown in Patient ID is common in each table and Visit ID is available across (2) to (5). These IDs were used to link features across tables.The National University Hospital EMR system adopted International Classification of Diseases, Ninth Revision (ICD-9), Clinical Modification (CM) codes before 2010 and then migrated to the more updated the International Classification of Diseases, Tenth Revision (ICD-10), Australian Modification (AM) codes afterward. To standardize the ICD codes, we transformed ICD-10-AM codes to ICD-9-CM format using Australia Consortium for Classification Development (ACCD) backward mapping tables . ICD-10 0 digits to the ICD code). Mapping will be performed again thereafter. ICD-10-AM codes that were left unmapped after code modification were excluded from further analyses. A diagnosis will be classified as primary diagnosis (PD) if it is indicated as the hospital’s main diagnosis  or zero addition . OtherwAs the ACCD backward mapping table is well established and defined, we regard the mapping from original ICD-10-AM codes (no truncations or zero additions) to ICD-9-CM codes as valid . TherefoICD codes have good utility for clinical research where the researcher needs the granularity for identification and attribution of pathology at an individual level ,50. Howeessential hypertension and cancer of breast). For ICD codes that could not be classified directly using CCS, the approach outlined in To extend the utility of our dataset to support health services research, we sought ways to phenotype the more than 10,000 ICD-9-CM codes (both PD and SD) into meaningful groups. To that end, we grouped the ICD-9-CM codes (including those converted from ICD-10-AM codes) using CCS to 283 mutually exclusive disease categories  and then fully join the data for each visit by drawing on data across tables . Age and date exclusion criteria were applied to 3 of the tables (2-4) before the join. The diagnosis table was then also filtered using Visit ID from the other tables (2-4) to filter out diagnoses not related to visits within our cohort after applying the earlier exclusion criteria. The tables (2-5) were fully joined thereafter. The joined data were further linked to demographics through Patient ID. Patient-level records were then generated by aggregating visits by Patient ID. Patient-related exclusion criteria were then applied after obtaining patient-level records.R version 3.2.0 [R package multidplyr [Aggregation and analysis were undertaken using on 3.2.0 . R packaltidplyr  was usedThe following exclusion criteria were applied to streamline the data for subsequent analysis:Any inpatient visit with admission date before 2005 or discharge date after 2013 was dropped. This ensured that the entire period of each inpatient visit was captured.Visits when patients were aged less than 21 years were excluded in this study as subsequent analysis is focused on adult patients.Patients with no PD were excluded.Patients with birth years 1900 or earlier were excluded .Patients with invalid diagnoses  were removed.The final cohort analyzed was an adult cohort aged 21 years and above, with valid age and at least one PD record.The main source variables in the database had to be extracted and processed to generate secondary variables useful for cohort profiling and other health services research. In addition, we attempted to generate proxies for clinical and socioeconomic indicators unavailable in the dataset, namely, disease complexity and SES. Summary and details of all the extracted variables can be found in the On the basis of the source variables from the 5 PASS tables, we generated a series of secondary variables falling in categories of (1) demographics (including SES), (2) health care utilization, (3) disease indicators, and (4) disease complexity. For categorical source variables, we created dummy variables for visits, such as whether a visit is an emergency department (ED) visit or whether it has a specific CCS disease, and then we aggregated them to patient-level by adding new categories, summation, or logic operation. For numerical source variables, such as inpatient length-of-stay (LOS), hospital charges, and the components, a simple summation over all visits led to the features at patient-level. Hospital charges (full cost of care before subsidy) was adjusted using Monetary Authority of Singapore Web-based inflation calculator  for healAs clinical indicators and investigation results  that proR package icd [In our dataset, the Charlson comorbidities were identified using ICD-9-CM codes , and botkage icd  was usedkage icd . PPS quakage icd . PPS at Validity of CCI and PPS were assessed to ensure that these measures were consistent with theoretical understanding and literature. To assess convergent validity, Spearman rank correlation between PPS and CCI was computed. This measures the degree to which PPS and CCI, that should be measuring disease complexity, are in fact related. To assess criterion validity, Spearman rank correlations between health care utilization  and PPS and CCI were computed. This measures the extent to which higher CCI and PPS is associated with higher health care utilization, under the assumption that clinically complex patients require more health care utilization . The 95%To estimate the SES of PASS patients, we used housing type as a proxy, given the lack of a direct indicator of SES in the dataset. We then validated the use of housing type as a proxy for SES as part of the study.Each residential block and house in Singapore has a postal code assigned. Using the postal code data of each patient, we were able to determine the block and, consequently, housing type for each patient. The latest postal codes captured in PASS EMR were used, as patients’ past addresses were not available. For all Housing Development Board (HDB) blocks (public housing), we obtained information of flat types by postal codes collected using OneMap Singapore  from the2) and Kruskal-Wallis rank-sum test [Criterion validity of housing type as a proxy for SES was assessed through studying the relationship between housing type and 2 measures: (1) subsidy status and (2) relative subsidy received (RSR). Subsidy status indicates whether a patient received government subsidized care or nonsubsidized  care where nonsubsidized care is costlier and involves higher out-of-pocket payments. Typically, the lower the income level of an individual, the more likely one is to opt for subsidized care given the lower cost . RSR indsum test ,70, respAmong 10,795,573 visits during the study period, 7,778,761 satisfied our inclusion criteria and constitute our visit-level data. The visit-level data comprised 7,367,495 outpatient visits and 411,266 inpatient visits. An increasing trend was observed in the number of visits from 2005 to 2013 . The visThere was a total of 4,842,705 diagnoses belonging to our patient cohort after visit-level aggregation, of which 19.2% was coded in ICD-10-AM, with the remainder in ICD-9-CM. The ICD-10-AM codes in our cohort were standardized to ICD-9-CM codes with a mapping rate of 90.3% for PD codes, 78.2% for SD codes, and 81.4% overall using ACCD backward mapping tables. This resulted in 4,670,111 ICD-9-CM codes in the cohort, with 16.2% converted directly from ICD-10-AM by the ACCD backward mapping table. As mentioned in the Methods section, the ACCD backward mapping table has been validated previously; hence, the team regarded these 16.2% of codes that were mapped directly through ACCD as valid. Detailed statistics for code mapping rates are presented in In addition, there were 172,594 codes that could not be mapped through ACCD. Of these, 23,800 (13.79%) ICD-10-AM codes were converted after truncation and 29,005 (16.80%) converted after zero addition . These 5In total 810,459 (87.1%) of the total ICD-10-AM codes were successfully converted to ICD-9-CM codes (97.2% of PD and 83.5% of SD). These converted codes and the original ICD-9-CM codes form a pool of 4,722,916  ICD-9-CM codes in our cohort. The unmapped codes, which consisted of 119,789 (12.9%) of the total ICD-10-AM codes, or 471 unique codes were excluded.The ICD-9-CM codes were then phenotyped to CCS codes, which resulted in 282 mutually exclusive groups. Out of the 4,722,916 ICD-9-CM codes, 4,178,862  were converted to CCS codes directly through the CCS. These 4,178,862 (88.48%) are regarded to be valid conversions, given the previous validation done on the CCS . DetaileIn addition, 27,240  ICD-9-CM codes were converted after truncation and 392,386  converted after zero addition  through In total, 4,598,488 (97.4%) of the ICD-9-CM codes in our cohort were successfully converted to CCS codes , number of SOC visits , and number of ED visits ; PPS was found to have an even stronger correlation with health care utilization measures, with exception being number of ED visits: number of inpatient visits , number of SOC visits , and number of ED visits . CCI and PPS were also found to be positively correlated . On the basis of multivariate regression analysis, which adjusted for gender, race, age, and observed period, health care utilization was expected to increase when there was a unit increase in CCI (P<.001), 1.32 (P<.001), and 1.23 (P<.001), respectively. Health care utilization was also expected to increase when there was a unit increase in PPS; the number of inpatient visits, SOC visits, and ED visits were expected to change by a factor of 1.10 (P<.001), 1.08 (P<.001), and 1.03 (P<.001), respectively.CCI was found to be positively correlated with health care utilization measures, including number of inpatient visits , further confirming the observation. The median and mean RSR of patients by housing type were plotted in 24=245232, P<.001). The mixed group is a composite group and, hence, it was difficult to interpret the results for this group. On the basis of multivariate regression analysis, which adjusted for nationality, the odds of receiving subsidized care only rather than nonsubsidized care only were higher in patients who lived in smaller housing types when compared with patients who lived in private housing (P<.001), and those who stayed in 5-room flats or executive housing had the lowest OR of 2.97 (P<.001). A relatively smaller effect size, but the same trend, was observed when mixed group was compared with nonsubsidized group. Patients who stayed in 2-room flats or smaller and 5-room flats or executive housing had the highest and lowest ORs of receiving both subsidized and nonsubsidized (mixed) care rather than only nonsubsidized care, respectively. The ORs were 3.29 (P<.001) and 1.65 (P<.001), respectively. RSR was also expected to be higher for patients who stayed in smaller housing after adjusting for nationality (P<.001) more relative subsidy than those who stayed in private housing, and patients who stayed in 5-room flats or executive housing were expected to receive 9.8% (P<.001) more relative subsidy than those who stayed in private housing.For all the patients with valid housing type data, the proportions by subsidy status categories within each housing type are presented in  housing . Patientionality . PatientThe detailed demographic, medical, and utilization characteristics of the cohort are shown in The patients older than 65 years had a greater prevalence of chronic diseases and disease complexity scores as compared with those younger than or at 65 years. They also had almost 7 times the median hospital charges, median LOS that was 3 days longer, and 3 times the median SOC visits during the study period compared with those younger than or at 65 years.Conversion to ICD-10 codes from other codes such as ICD-9 or International Classification of Primary Care is commonly applied in medical or health care studies to increase granularity for identification and attribution of pathology at an individual level. However, given that our dataset is prepped for future health services studies, our primary objective in code standardization was to balance code sparsity with granularity. In this regard, backward mapping from ICD-10 to ICD-9 codes was a more suitable method for this system, and the phenotyping of these standardized codes using broader CCS codes provided different levels of granularity in line with our objectives. Our study showed that standardization of diagnosis codes to ICD-9-CM codes from ICD-10-AM and phenotyping to broader CCS groups through open source-mapping tables could achieve high mapping rates of more than 81% and 88%, respectively. The mapping rates could be further improved through code modification to rates in excess of 97% for ICD PD. Code modification through truncation or zero addition as applied in our study was a robust way of improving the mapping rates as shown by high validity when assessed by independent physicians. Overall, we also showed that bias resulting from code modification was small in our dataset, given that modified codes only constituted less than 2% of total ICD codes and 9% of total CCS codes and that high validity was observed even with these modifications. Given the frequent shifts in ICD codes, these results assure health services researchers that the use of open source-mapping tables together with code modification can rapidly standardize diagnosis coding with low biases and high validity to facilitate retrospective longitudinal analyses. However, we advise caution to researchers who wish to use the ICD-9-CM codes  directly, without collapsing to CCS codes for their studies as there were ICD-9-CM codes that were unmapped to. Further details on this can be found in CCI and PPS were introduced as proxy measures of disease complexity. CCI and PPS demonstrated positive correlation with health care utilization measures in keeping with theoretical understanding that patients with more complex disease consume more health care . CCI andSES is a key determinant of health outcomes and health care utilization . NeitherOur finding on the suitability of housing type as a proxy for SES is useful, given that most clinical and administrative databases collect addresses but not direct SES information or other proxies. Our method of estimating SES would serve well in countries where methods of estimating SES such as area-based estimates ,35, insuUnlike in countries where zip codes are area-based, the postal codes in Singapore are assigned to each individual building; hence, they serve almost like an address. Housing in Singapore can be divided into 3 main classes: private housing, public housing, and public rental housing. The private housing caters mainly to the upper-middle to upper income groups, whereas the public housing caters to the middle-class population, with 80% of the permanent population living in public housing as owner-occupiers . EligibiFinally, our cohort was found to be similar in profile with the Singapore national population. Comparison with National Census data in 2010  found a With increasing digitization of medical records, use of wearables and Internet-of-Things–connected devices in health care, the amount of data generated by health care systems is growing at a tremendous rate ,90. Bein


This cohort study investigates the association between participants’ capacity to complete push-ups at baseline and cardiovascular disease events during 10 years of follow-up. Is there an office-based objective measurement that clinicians can use to assess the association between fitness and cardiovascular disease risk?This longitudinal cohort study of 1104 occupationally active adult men found a significant negative association between baseline push-up capacity and incident cardiovascular disease risk across 10 years of follow-up. Participants able to complete more than 40 push-ups were associated with a significant reduction in incident cardiovascular disease event risk compared with those completing fewer than 10 push-ups.Push-up capacity is a no-cost, fast, and simple measure that may be a useful and objective clinical assessment tool for evaluating functional capacity and cardiovascular disease risk. Cardiovascular disease (CVD) remains the leading cause of mortality worldwide. Robust evidence indicates an association of increased physical fitness with a lower risk of CVD events and improved longevity; however, few have studied simple, low-cost measures of functional status.To evaluate the association between push-up capacity and subsequent CVD event incidence in a cohort of active adult men.Retrospective longitudinal cohort study conducted between January 1, 2000, and December 31, 2010, in 1 outpatient clinics in Indiana of male firefighters aged 18 years or older. Baseline and periodic physical examinations, including tests of push-up capacity and exercise tolerance, were performed between February 2, 2000, and November 12, 2007. Participants were stratified into 5 groups based on number of push-ups completed and were followed up for 10 years. Final statistical analyses were completed on August 11, 2018.Cardiovascular disease–related outcomes through 2010 included incident diagnoses of coronary artery disease and other major CVD events. Incidence rate ratios (IRRs) were computed, and logistic regression models were used to model the time to each outcome from baseline, adjusting for age and body mass index (BMI) . Kaplan-Meier estimates for cumulative risk were computed for the push-up categories.A total of 1562 participants underwent baseline examination, and 1104 with available push-up data were included in the final analyses. Mean (SD) age of the cohort at baseline was 39.6 (9.2) years, and mean (SD) BMI was 28.7 (4.3). During the 10-year follow up, 37 CVD-related outcomes (8601 person-years) were reported in participants with available push-up data. Significant negative associations were found between increasing push-up capacity and CVD events. Participants able to complete more than 40 push-ups were associated with a significantly lower risk of incident CVD event risk compared with those completing fewer than 10 push-ups .The findings suggest that higher baseline push-up capacity is associated with a lower incidence of CVD events. Although larger studies in more diverse cohorts are needed, push-up capacity may be a simple, no-cost measure to estimate functional status. However, unlike anthropometric measurements and serum biomarkers, physical activity and CRF assessments have largely been neglected by clinicians. The most commonly used physical activity assessments are the patient’s self-reported history and health and lifestyle questionnaires.2 However, objectively measured CRF levels are often significantly lower than expected based on self-reported physical activity.20 Although good performance on accurate and objective CRF assessment tools such as exercise tolerance tests has been inversely associated with future CVD,22 these examinations are expensive, time-consuming, and often require professional facilities and trained personnel to administer. The use of these tools remains limited to particular occupations and targeted patient populations. To our knowledge, no study has examined the association of push-up capacity, a simple, no-cost, surrogate measure of functional status, with future cardiovascular events. In this study, we examined baseline performance on commonly performed physical fitness assessments  and its association with subsequent incident CVD events in a cohort of occupationally active men. We hypothesized that higher fitness levels would be associated with lower rates of incident CVD.In multiple recent scientific and policy statements, the American Heart Association has promoted assessment of physical activity in clinical settings and workplaces.STROBE) reporting guideline for cohort studies.23 A retrospective cohort was assembled from records of active career firefighters from 10 Indiana fire departments who underwent periodic medical surveillance between January 1, 2000, and December 31, 2010, at 1 local medical clinic under contract with the fire departments. Male firefighters aged 18 years or older who had no job restrictions at the time of their initial examination were included in the study. Each underwent baseline and periodic physical examinations, including tests of push-up capacity and maximal or submaximal exercise tolerance tests between February 2, 2000, and November 12, 2007. The study protocol was reviewed by the institutional review board of the Harvard T.H. Chan School of Public Health, which also determined that the retrospective review of the firefighters’ deidentified records was exempt from further review and informed consent.This study followed the Strengthening the Reporting of Observational Studies in Epidemiology . Numbers of push-ups were arbitrarily divided into 5 categories in increments of 10 push-ups for each category. Exercise tolerance tests were performed on a treadmill using a modified Bruce protocol until participants reached at least 85% of their maximal predicted heart rates, requested early termination, or experienced a clinical indication for early termination according to the American College of Sports Medicine Guidelines (maximum oxygen consumption [V̇o2max]).26Data collected during baseline and follow-up visits included complete physical examinations, anthropometric measures, and laboratory results, as well as clinical and occupational outcomes. All examinations were voluntary, and participants were allowed to bypass any part of the examination. The clinical examination was conducted by trained nurse practitioners and consisted of standardized measurements of height, weight, blood pressure, and resting heart rate.27Occupational activity status was provided by the fire departments to the medical clinic, and the vital status of firefighters who were retired was verified through a registry of Indiana firefighter deaths constructed during an earlier mortality study.The main outcome that we examined in this study was incident CVD-related events among participants. Cardiovascular disease–related events were defined as incident diagnosis of coronary artery disease or other major CVD event . Participants were followed up from the date of enrollment  until an outcome event or December 31, 2010, whichever came first. Cardiovascular disease events were verified clinically at return-to-work evaluations, fitness-for-duty examinations, or subsequent periodic examinations, all performed by the same clinic.2 test of independence for categorical data and the t test or the analysis of variance test, as appropriate, for continuous data. The primary analyses were restricted to firefighters who completed a test of baseline push-up capacity. Incidence rate ratios (IRRs) were computed using the Poisson regression model with log-link function. Cox proportional hazards regression models were used to model the association of V̇o2max categories and push-up groups with the time to each outcome of interest from baseline, adjusting for age and body mass index (BMI) ; the resulting hazard ratios together with the corresponding 95% CIs and P values were reported. Kaplan-Meier estimates for the cumulative risk of CVD event were computed in the different push-up categories, compared using the log-rank test, and presented as a P < .05 indicating statistical significance. Final statistical analyses were completed on August 11, 2018.Continuous characteristics were presented as mean (SD), whereas categorical variables were shown as frequency or percentage. Covariates were compared among groups using the χP < .001; BMI, P < .001; systolic blood pressure, P < .001; diastolic blood pressure, P < .001; total cholesterol level, P = .02; low-density lipoprotein cholesterol level, P = .04; triglycerides, P < .001; glucose level, P < .001; and smoking status, P < .001), although it was significantly positively associated with the estimated V̇o2max (P < .001). The baseline characteristics of firefighters without push-up data were similar and not statistically different from those who completed the push-up capacity evaluation on all markers of CVD risk.A total of 1562 eligible firefighters underwent baseline examination between February 2, 2000, and November 12, 2007, and were included in the initial retrospective cohort. Mean (SD) age at baseline for all participants was 39.6 (9.2) years and the mean (SD) BMI was 28.7 (4.3). o2max baseline.During the 10-year follow-up period, 37 CVD-related outcomes  occurred among firefighters with recorded push-up capacity data at baseline and were included in our analyses . We obseo2max groups with CVD outcomes. Even after adjusting for age and BMI, we observed an independent association of push-up capacity with CVD outcomes. Increased capacity was associated with a lower risk for CVD outcomes, with the comparison of the 21- to 30-push-ups group vs the 0- to 10-push-up group being statistically significant , although the other group comparisons did not reach statistical significance.The This study found that push-up capacity was inversely associated with 10-year risk of CVD events among men aged 21 to 66 years. Thus, push-up capacity, a simple, no-cost measure, may provide a surrogate estimate of functional status among middle-aged men. Participants able to perform 11 or more push-ups at baseline had significantly reduced risk of subsequent CVD events. To our knowledge, this is the first study to report the inverse relationship between push-up capacity at baseline and subsequent CVD-related outcomes in an occupationally active male cohort.29 In those studies, the authors found that a higher level of muscular strength was associated with lower cardiometabolic risk independent of cardiorespiratory fitness in the cohorts observed. Muscular strength has been shown to have an independent protective effect for all-cause mortality and hypertension in healthy males and is inversely associated with metabolic syndrome incidence and prevalence.30 However, most of those studies were either cross-sectional32 or conducted with adolescent participants.29 Our retrospective cohort study provides further insights into the association of greater fitness, specifically muscular strength, with CVD-related outcomes in an occupationally active cohort across 10 years of follow-up. Statistical adjustment for age and BMI suggested that some of the risk reduction seen with higher push-up categories was accounted for by these characteristics. There was also evidence that differences in established CVD risk factors  may explain much of the residual differences in outcome. Nonetheless, the results of this study support an inverse association between push-ups and CVD events among middle-aged men. In the present study, push-up capacity was more strongly associated with future CVD risk than was V̇o2max as estimated by submaximal stress tests. Previous studies have documented that submaximal stress tests can overestimate or underestimate CRF owing to the assumptions involved with extrapolating from submaximal to maximal results.34Previous cross-sectional studies have incorporated push-ups in the assessment of muscular fitness and its correlation with cardiometabolic risk markers.37 However, push-up capacity is a functional test. Many fire and police departments have neglected to provide periodic medical examinations or functional tests such as stress tests owing to cost concerns.39 The push-up examination requires no special equipment, is low cost or no cost, can easily be performed in almost any setting within 2 minutes, and provides an objective estimate of functional status. It is a quantitative measurement that is easily understood by both the clinician and the patient. The use of push-up capacity could assist clinicians in presenting objective information on CVD risk and in formulating physical activity and weight reduction prescriptions for patients. Fonarow et al40 suggested that the workplace is an important setting for promoting cardiovascular health; however, current workplace wellness programs often lack the appropriate physical activity assessment tools and the resources for exercise tolerance tests. Therefore, push-up capacity could potentially be an easily implemented tool and used as part of a comprehensive workplace wellness program.The use of age and BMI in occupational settings as determinants of fitness for duty has been avoided owing to concerns related to the Americans with Disabilities Act.41 As a result, it is recommended that individuals with low CRF  receive cardiovascular risk reduction management.21 Again, a significant limitation of this recommendation is the requirement to perform costly and time-consuming maximal exercise stress tests. Most fire departments have been unable to adopt this recommendation for career firefighters, let alone for the volunteer firefighters who compose 70% of the fire service nationally.42 The use of push-up capacity has the potential to improve the feasibility and implementation of physical fitness assessment in workplace settings. Based on the current study, CVD risk factor reduction should be recommended, including lifestyle measures for those with low push-up capacity, especially those capable of 10 or fewer push-ups.Previous cross-sectional research in firefighters established that those with low CRF were significantly more likely to have abnormal results on maximal stress tests, worse CVD risk profiles, and a higher prevalence of metabolic syndrome, suggesting a higher future risk of CVD events.17 Thus, our study avoided participant recall bias and other well-known limitations of self-reported data. Our sample size and follow-up period of 10 years also provided sufficient data on CVD-related outcomes and exit events to observe significant associations. Another strength is the generalizability of the study cohort to other US firefighters. The cohort of Indiana career firefighters is similar with respect to age, race/ethnicity, and diet to other multistate cohorts.44 Our within-group analyses and comparisons and use of a statewide database of death certificates mitigated concerns for bias owing to a healthy worker effect or loss to follow-up.The major strength of this study is the use of an objective surrogate measure of functional status, push-up capacity, from baseline physical examinations. Although push-up capacity may be influenced by technique and practice, the use of a standardized protocol for push-up performance by clinic staff would minimize the effect of technique on capacity. Previous studies have used self-reported physical activity questionnaires to assess duration, intensity, and frequency of physical activity; most of these categorized physical activity by total activity time.The current study has several limitations. First, the study assessed the association between push-ups and CVD events. The results do not support push-up capacity as an independent predictor of CVD risk. Second, because the study cohort consisted of middle-aged, occupationally active men, the study results may not be generalizable to women, older or nonactive persons, other occupational groups, or unemployed persons. Further studies are needed to examine the association between push-up capacity and CVD incidence in a more general population of middle-aged men as well as among older individuals and women. The fitness evaluation was voluntary for the firefighter population used for this study; thus, the baseline measurements were missing information for approximately one-fourth of participants. However, the characteristics of participants with missing push-up information were similar to those with available push-up information. Nonetheless, this decreased the statistical power. In addition, given that the tests performed were not adjusted for multiple comparisons, some of the significant findings could be due to chance.In this 10-year longitudinal study, participants able to complete more than 40 push-ups were associated with a significant reduction in incident CVD event risk compared with those completing fewer than 10 push-ups, which may be explained by significant differences in recognized CVD risk factors at baseline among the groups. The findings suggest that being able to perform a greater number of push-ups at baseline is associated with a lower incidence of CVD events among active adult men. Thus, results from this study suggest that it is reasonable for clinicians to assess functional status during clinical evaluations by using basic questions regarding activity. Further research is warranted to determine the association of push-up capacity with CVD risk in the general population and the potential use of push-ups as a clinical assessment tool.


A partial response to first-line treatment was observed in 7% of patients treated with anti-programmed cell death protein (PD)-1 monotherapy and in 21% with combined anti-cytotoxic T lymphocyte antigen (CTLA)-4 plus anti-PD-1 therapy. Median progression-free survival was 2.5 months for patients treated in the pre-ICI era compared to 3.5 months in the post-ICI era (hazard ratio (HR) 0.43; 95% confidence interval (CI) 0.28–0.67; p < 0.001). The estimated one-year overall survival rate increased from 25.0% to 41.9% and the median overall survival improved from 7.8 months to 10.0 months, respectively . Thus, the introduction of ICI as first-line treatment appears to have significantly improved the real-world survival of patients with metastatic UM, despite relatively low response rates compared to cutaneous melanoma. With the lack of therapies proven effective in randomized trials, these data support the current treatment with ICI in patients with metastatic UM.Uveal melanoma (UM) is the most common intraocular malignancy in adults and shows a high rate of metastatic spread. As randomized clinical trials with immune checkpoint inhibitors (ICI) have not been performed in patients with metastatic UM, we analyzed the real-world outcomes in a nationwide population-based study. Clinical data of patients with UM were extracted from the Danish Metastatic Melanoma database, a nationwide database containing unselected records of patients diagnosed with metastatic melanoma in Denmark. Survival before (pre-ICI,  Uveal melanoma (UM) is the most common intraocular malignancy in adults and it arises in the melanocytes in the uveal tract of the eye, mainly in the choroid. With an incidence of 2–8 per million persons per year, it remains a rare disease and only constitutes ~3% of patients with metastatic melanoma ,2. UM isAt present, no systemic treatment has proven to result in survival benefit in patients with metastatic UM . With thIn the last decade, the success of ICI has revolutionized the treatment of patients with metastatic melanoma and numerous other tumor types. In 2011, ipilimumab was the first approved ICI for melanoma, showing an objective response rate (ORR) of 11–15% in patients with metastatic CM . HoweverOverall, substantially lower response rates on ICI are reported in metastatic UM compared to CM. Still, data is limited, and randomized clinical trials have not been performed. Most data originate from small, retrospective cohorts and largely consist of highly selected patients with no comparison to other treatments. Therefore, we analyzed the real-world outcomes of treatment with ICI and chemotherapy in unselected patients with metastatic UM in a nationwide, population-based study.n = 2) or orbital melanoma (n = 1) were excluded from the analysis. The Danish Metastatic Melanoma Database (2011-41-6802) and the Copenhagen Epidemiological Uveal Melanoma Study database (2016-41-4897) were approved by the Danish Data Protection Agency in 2011 and 2016, respectively.We performed a nationwide, retrospective population-based study of all registered patients with metastatic UM in Denmark. All patients with metastatic melanoma that are not amenable for local treatment or progressed after local treatment of metastases are referred to the department of clinical oncology in one of the three reference centers . Patients with an initial oncological evaluation between 1 January 2011 and 31 December 2018 were included. All records were retrieved from the Danish Metastatic Melanoma database (DAMMED), which has an estimated coverage of >95% of metastatic melanoma patients in Denmark. The database was locked on May 6, 2019. As the records were categorized as ocular melanoma in the Danish Metastatic Melanoma database, diagnosis of primary UM was confirmed via the Copenhagen Epidemiological Uveal Melanoma Study database (COEUS) . This daPatients were treated according to best practice which consisted of chemotherapy (temozolomide), immunotherapy  or best supportive care. Few patients were included in clinical trials. Patients were analyzed according to actual received treatment or in time periods depending on date of drug approval for first-line treatment in melanoma in Denmark: pre-ICI era (2011–2013) versus post-ICI era (2014–2018).Tumor response was assessed according to the Response Evaluation Criteria in Solid Tumor (RECIST) guidelines . DurableProgression-free survival (PFS) was defined as the time from initiation of systemic treatment to the date of documented disease progression or last follow-up. Overall survival (OS) was defined as the time from initiation of first-line systemic treatment to death or last follow-up. In patients who did not receive any systemic treatment, OS was calculated from the date of initial oncological evaluation.p-values were two-sided and p-values < 0.05 were considered statistically significant. SPSS Statistics version 25.0 software  was used for statistical analysis.Statistical significance of baseline characteristics was evaluated using chi-square tests. Survival was estimated using the Kaplan-Meier method and compared using the log-rank test. Follow-up duration was estimated using the Kaplan-Meier method with the time from initiation of first-line treatment, or date of initial oncological evaluation in patients who did not receive systemic treatment, to date of last follow-up and censored for death. Hazard ratios (HR) and corresponding 95% confidence intervals (CI) were calculated with the Cox proportional hazards model. All A total of 126 patients with metastatic UM, referred for systemic treatment of distant metastases between 2011 and 2018, were retrieved from the Danish Metastatic Melanoma database. At the time of data analysis, median follow-up was 23 months and 19% of patients were still alive with a minimum follow-up time of 5 months. Thirty-two patients (25%) were treated before approval of the first ICI as first-line treatment, pre-ICI era (2011–2013), and 94 patients (75%) were treated after first-line treatment with ICI became available, post-ICI era (2014–2018). The baseline characteristics of all metastatic UM patients are summarized in In the pre-ICI era, first-line treatment consisted of chemotherapy (temozolomide) in the majority of patients 84%: . Four paIn the post-ICI era, only five patients (5%) received chemotherapy as first-line treatment. Ipilimumab, pembrolizumab and combined ipilimumab/nivolumab were given as first-line treatment in 21%, 46% and 20% of patients, respectively. No systemic treatment was administered to one patient in the pre-ICI era (3%) and to seven patients in the post-ICI era (7%). All but one of these patients had an elevated LDH and/or ECOG performance status of 2 or 3.p < 0.001; The median PFS of first-line treatment in the pre-ICI era was 2.5 months versus 3.5 months in the post-ICI era .The median OS was 7.8 months in the pre-ICI era versus 10.0 months in the post-ICI era , of which one is ongoing for more than 3.5 years.n = 86) of which 67% of patients were treated with anti-PD-1 antibodies in first line, showing an ORR of 4.7% and a median PFS between 2.8 and 3.1 months [Randomized clinical trials with ICI have not been performed in metastatic UM patients. Still, numerous metastatic UM patients are currently treated with ICI outside of clinical trials. In this nationwide population-based study, we showed that objective responses were observed in 7% and 21% of patients treated with first-line pembrolizumab or combined ipilimumab/nivolumab, respectively. Furthermore, with the introduction of ICI as first-line treatment the median OS increased from 7.8 to 10.0 months and a rise in one-year OS rate from 25.0% to 41.9% was observed. Compared to clinical trials, real-world data generally show lower response rates and poorer survival as the unselected patient population usually has inferior baseline characteristics than trial participants . In our 1 months . Althoug1 months . Howeverp = 0.48) [No clinical trials have been performed to compare ICI with chemotherapy in metastatic UM. Mignard et al. retrospectively analyzed patients with metastatic UM treated with chemotherapy or ICI. No objective responses were observed with ICI and survival did not significantly differ between ICI and chemotherapy  . The lac = 0.48) . HoweverOur study has some limitations inherent to its retrospective design and rarity of the disease. For example, ECOG performance score and LDH were not documented in about half of the patients in the pre-ICI era. Although, known baseline characteristics are well balanced between the pre-ICI and post-ICI era, the missing data might conceal some differences. One disbalance between the groups is present as the liver resections were all performed on patients in the post-ICI era. However, only three patients obtained clinical benefit from the surgery and are unlikely to have biased the results as two showed progressive disease on first-line treatment and only one patient showed stable disease for approximately 10 months on pembrolizumab. Furthermore, the treatments in the post-ICI era are diverse and subgroups per treatment remain too small to draw firm conclusions on response rates, despite representing one of the biggest cohorts of metastatic UM patients treated with ICI.p < 0.001). In addition, after physicians became familiar with pembrolizumab, with its favorable toxicity profile, a higher proportion of patients were treated; in the last two years, all patients with a poor ECOG performance score received systemic treatment. Still, comparing the pre-ICI and post-ICI era seems the fairest comparison possible, compared to per treatment, as choice of treatment might be dependent on patient characteristics, e.g., less fit patients might not get treated with combined ipilimumab/nivolumab but with pembrolizumab monotherapy to reduce the risk of toxicity. As no major changes were made in the treatment of primary UM in the last decade and no effective adjuvant treatment is available, the bias in time (2011–2013 versus 2014–2018) is considered to be absent or minimal. This is substantiated by literature as no association between survival and time was present over the past few decades [Nevertheless, the analysis of our unselected patient population is likely to underestimate the benefit of ICI treatment in metastatic UM. Survival differences between the treatments might be undervalued as 50% of patients in the pre-ICI era received ipilimumab either as first-line treatment within a clinical trial or as second-line treatment. If only patients treated with temozolomide in the pre-ICI era and patients treated with ICI in the post-ICI era are analyzed, the difference in OS increases  and the full results of combination as first-line treatment (NCT02626962) might give more insight in the response rate to combined ipilimumab/nivolumab. However, no randomization takes place in these trials so comparison with other treatments will be difficult and it is unlikely that a randomized clinical trial with ICI will be performed in metastatic UM. Besides clinical trials, further investigation to determine predictive biomarkers for response to ICI is warranted to select patients for ICI treatment, e.g., specific germline mutations , tumor mThe introduction of ICI as first-line treatment appears to have significantly improved the real-world survival of patients with metastatic UM. Despite relatively low response rates, the response rate in patients treated with combined ipilimumab/nivolumab is still promising. Thus, with the lack of therapies proven effective in randomized trials, these data support the current treatment with combination ICI in patients with metastatic UM if participation in a clinical trial is not possible.


The effects of thyroid hormone disrupting chemicals (THDCs) on eye development of zebrafish were investigated. We expected THDC exposure to cause transcriptional changes of vision-related genes, which find their phenotypic anchoring in eye malformations and dysfunction, as observed in our previous studies. Zebrafish were exposed from 0 to 5 days post fertilization (dpf) to either propylthiouracil (PTU), a thyroid hormone synthesis inhibitor, or tetrabromobisphenol-A (TBBPA), which interacts with thyroid hormone receptors. Full genome microarray analyses of RNA isolated from eye tissue revealed that the number of affected transcripts was substantially higher in PTU- than in TBBPA-treated larvae. However, multiple components of phototransduction  were responsive to both THDC exposures. Yet, the response pattern for the gene ontology (GO)-class “sensory perception” differed between treatments, with over 90% down-regulation in PTU-exposed fish, compared to over 80% up-regulation in TBBPA-exposed fish. Additionally, the reversibility of effects after recovery in clean water for three days was investigated. Transcriptional patterns in the eyes were still altered and partly overlapped between 5 and 8 dpf, showing that no full recovery occurred within the time period investigated. However, pathways involved in repair mechanisms were significantly upregulated, which indicates activation of regeneration processes. During the last years, research on the effects of endocrine disrupting chemicals (EDCs) on fish has mainly focused on the disruption of the sex steroid hormone system, which can result in reproductive impairment of exposed organisms ,2. While−1 dry weight in sediment samples and up to 1.2 μg kg−1 wet weight in fish [−1 (water), 330 to 3800 pg g−1 dry weight (sediment), and <0.29 to 1.7 ng g−1 lipid weight (fish) [In recently published data from our own research, we exposed zebrafish embryos to THDCs with different modes of action: propylthiouracil (PTU), a pharmaceutical that inhibits TH synthesis , and tet in fish . Measuret (fish) . Even tht (fish) , it has t (fish) .In our previous study, we showed that exposure of zebrafish embryos to these two different THDCs resulted in congruent eye pathologies, such as smaller eye size, altered cellular structure and reduced pigmentation of the retina . The obsUntil now, investigations on specific molecular interactions of the eyes and TH system have been limited to the fundamental studies of Bagci  and HoubAdditionally, we investigated the reversibility of transcriptional changes after a recovery period of 3 days in clean water until 8 dpf. This scenario is relevant in an environmental context, where short-term peak exposure is likely to occur, which is especially critical during early development. The results from this study provide new insights into the molecular pathways associated with adverse effects of TH disruption in developing fish, and, at the same time, advance our knowledge of the role of THs in the regulation of fish eye development.Both treatments, as well as the subsequent recovery phase, had significant impact on transcript levels in the eyes of exposed larvae. From a total of 43,803 probes on the microarray, Generally, the number of differentially expressed transcripts was much higher in PTU-treated fish than in TBBPA-treated ones  after 3 days of recovery when the percentage of up-regulated transcripts was higher than the percentage of down-regulated transcripts. In contrast, in TBBPA-treated fish, the proportions of up- or down-regulated transcripts did not change  between 5 and 8 dpf.The proportion of either up- or down-regulated transcripts in the four different transcript lists (two compounds with two time points each) was calculated , resultiThe three most enriched  gene ontology (GO) classes for each of the eight transcript lists were identified. Each list had a distinct response pattern with different GO classes involved. The top three enriched GO classes for each list are shown in the pie charts in The list of all differentially expressed transcripts was used to perform a cluster analysis based on their expression pattern across treatments, resulting in five clusters see  that werIn TBBPA-treated fish, the number of differentially expressed transcripts was much lower than for PTU-treated fish, even though the phenotypic effects in our previous study  were simThe most obvious differences between the two compounds were, e.g., the response pattern for the GO class sensory perception, where at 5 dpf we found over 90% down-regulation in PTU-treated fish compared to over 80% up-regulation in TBBPA-treated fish. Another difference was found for the GO-class proteolysis, where the PTU treatment caused almost 70% up-regulation, whereas for TBBPA it was only 10%.When comparing 5 and 8 dpf for each compound, it becomes obvious that the recovery period caused a change of the response pattern. In the PTU-treated group, the percentage of up-regulated transcripts increased for nearly all GO-classes, e.g., from almost 70% to over 90% in the GO-class proteolysis or from under 10% to over 40% in the GO-class sensory perception. In TBBPA-treated fish, the most obvious change was the general decrease of differentially expressed transcripts from 5 to 8 dpf, which makes the response pattern difficult to compare.Additionally, selected GO classes were visualized as examples in heatmaps showing the different response patterns in the four contrast groups see  after thIn combination with the observed impairment of visual capacities in PTU- and TBBPA exposed zebrafish in our previous study , this leBased on our previous work that revealed adverse changes in the development of eye structure and function in PTU- and TBBPA-exposed zebrafish larvae , the preThe first basic finding of our analyses was that PTU had a much stronger impact on transcriptional changes in the eyes of exposed fish than TBBPA, even though similar biological processes were affected. The number of differentially expressed transcripts was over 10-times higher for PTU, and the difference was even greater after the recovery period of 3 days. This was surprising, as morphological and physiological changes in the eyes were comparable in our previous study with the same exposure concentrations . HoweverAdditionally, many general metabolic processes were affected by the THDC treatment in the eyes of exposed zebrafish larvae, which is not surprising, as the primary function of THs is to regulate metabolism , which cDespite the difference in the amounts of affected transcripts, our analyses of the biological functions revealed that similar processes in the eyes of exposed larvae were affected by the two THDC treatments, especially with regard to transcripts involved in neurological system, visual and sensory perception. However, the most obvious differences between the two compounds were, e.g., the response pattern for the GO class sensory perception, where at 5 dpf we found over 90% down-regulation in PTU-treated fish compared to over 80% up-regulation in TBBPA-treated fish. Independent of the type of approach for our data analyses, important transcriptional changes of neuro-, sensory-, and vision-related genes were always detected for both compounds and time points, as well as the different intersects. This effect was more prominent in PTU-exposed fish, but also considerable in TBBPA-treated fish. These results provide strong evidence that the treatment with both THDCs did induce molecular changes that affect the eye development and function of exposed zebrafish larvae, as already demonstrated in our previous study at higher levels of biological organization, i.e., cellular changes in the retina, reduced eye size, and impaired visual performance of PTU- and TBBPA-exposed larvae . To undeOur analyses extend our previous findings and existing literature on TH regulation of eye development in vertebrates that can be disturbed by THDC exposure. It has been reported that in zebrafish, mice, and humans, THs regulate expression of opsins in the retina through the Thrb2 receptor . Suzuki A general neurotoxic mechanism of TBBPA might also play a role, as shown, e.g., by Chen , who expConsequently, even though the molecular initiating events in TH signaling may differ, the findings of the present study indicate that the toxicity pathways merge at a certain level to translate in uniform changes of eye development. Such observations are particularly relevant in the context of the current discussion about the adverse outcome pathway concept. Moreover, data from our own research ,15,23,26Interestingly, the response pattern of vision-related transcripts between 5 and 8 dpf clearly changed for both substances. While for TBBPA, only one up-regulated transcript (opsin) was still detectable at 8 dpf, the number of differentially expressed vision-related transcripts for PTU was still high, and the pattern changed from mainly down-regulated to more up-regulated at 8 dpf. Transcripts that were not affected at 5 dpf but became up-regulated after the depuration period were opsins (light-sensitive protein) and phosphodiesterase. Most of the transcripts that were down-regulated at 5 dpf, were still down-regulated but less apparent at 8 dpf. This change of response pattern for both substances provides evidence for activation of a recovery effect after the depuration period of 3 days, especially because transcripts from the GO class regeneration were strongly upregulated, which might be a compensatory response. Similar observations were made by Houbrechts , who obsGenerally, we identified different GO classes that can be regarded as a basic response to regeneration/recovery in the eyes of exposed larvae. Genes involved in tissue development, immune system, cell redox homeostasis, and positive regulation of cellular component organization were differentially expressed for both compounds and time points, which is in line with the cellular and morphological changes in the eyes observed in our previous study . These cThe activation of repair mechanisms in the eyes of exposed larvae at 8 dpf might not only be a response to cellular changes in the eyes but could be enforced by different other factors. There are multiple studies that describe the involvement of THs in regeneration processes of fish, for example, Bouzaffour  showed tAnother aspect that has to be considered regarding the activation of repair mechanisms is the interplay of the thyroid and immune system. Concerning immune-related genes, PTU and TBBPA exposure resulted in very different response patterns see . TBBPA hOur results show that within the 3-day-recovery period (after 5 days of THDC exposure), zebrafish larvae activated substantial repair and regeneration processes, associated with a general metabolic activation. At the same time, especially for PTU, many of the specific transcriptional responses that were detected after 5 days of exposure, were still present after the 3-day recovery phase  and the Commission Implementing Decision 2012/707/EU state that fish are non-protected animals until they are free feeding, i.e., 120 h post fertilization (hpf) for zebrafish . All exp2, 0.33 mM MgSO4, pH 7.6). PTU was directly dissolved in it.Tetrabromobisphenol-A  and propylthiouracil PTU, CAS 51-52-5) were purchased from Sigma Aldrich  with >98% purity. Exposure concentrations were 200 µg/L for TBBPA (0.37 µM) and 350 mg/L for PTU (2.04 mM). These sublethal concentrations were chosen based on our previously published study on TH disruption and eye development in zebrafish , as they-52-5 werDanio rerio) from an in-house laboratory colony at the University of Antwerp were used for egg production. They were kept in reconstituted freshwater with adjusted pH  and conductivity , a temperature of 28 ± 0.2 °C and a day/night cycle of 14/10 h. Water quality was monitored twice weekly, using Tetratest kits . Values for ammonium, nitrite, and nitrate were below 0.25, 0.3, and 12.5 mg/L, respectively. Adult zebrafish were fed four times per day, twice with granulated food  and twice with frozen Chironomidae larvae, Artemia sp. nauplii, Chaoboridae larvae and Daphnia sp.  alternately. Four breeding pairs  were placed in breeding tanks with a perforated bottom for egg production. Eggs were collected early in the morning after the lights were switched on. Eggs were collected, rinsed, and transferred to clean reconstituted water 30 min after fertilization. All eggs were checked using a stereomicroscope  and only healthy, fertilized eggs were further used. Eggs from the different breeding pairs were mixed and randomly distributed into pre-incubated 24-well plates  containing the exposure solutions latest 2 h after fertilization . Plates were incubated at 28.5 °C under a day/night cycle of 14/10 h. Exposure solutions were renewed daily with freshly prepared stock solutions to ensure constant chemical concentration levels and good water quality. Each exposure group was run in 6 biological replicates with 24 eggs per biological replicate/plate. After 5 days of exposure, 3 biological replicates of each treatment were sampled, resulting in 3 treatment groups which were used for the subsequent microarray analyses: control 5 dpf, PTU 5 dpf, and TBBPA 5 dpf. Larvae from the 3 remaining biological replicates of each treatment were carefully placed into new plates with clean water and raised until 8 dpf with daily water exchange. These larvae represent the 3 recovery groups for the microarray analyses: control 8 dpf, PTU 8 dpf, and TBBPA 8 dpf. The exposure set-up is summarized in Non-exposed adult wildtype zebrafish  hybridization oven (Agilent Technologies). After hybridization, the slides were rinsed in Agilent wash buffers, acetonitrile and in stabilization/drying solution (Agilent Technologies) to wash and protect against ozone-induced dye-degradation. Microarray slides were scanned with a Genepix 4100A confocal scanner  at a resolution of 5 μm and excitation wavelengths of 635 nm and 532 nm in an ozone-free location . Images were analyzed for spot identification and for quantification of fluorescent signal intensities with the Genepix Pro software 6.1 (Axon Instruments).Total RNA was linearly amplified and labelled with the Low Input Quick Amplification Labelling Kit (Agilent Technologies) according to the manufacturer’s instructions. In summary, 100 ng RNA was reverse transcribed into cDNA using oligo dT primers. Afterwards, cDNA was transformed into cRNA and amplified. The cRNA of each sample was labelled once with Cy3-CTP and once with Cy5-CTP. Subsequently, the labelled cRNA samples were purified with the RNeasy mini spin column kit . cRNA yield, quality, and dye incorporation efficiency were verified with the Nanodrop spectrophotometer. For final transcriptome analysis, we used Agilent’s Zebrafish Gene Expression Microarray V3 (AMADID 026437) in a 4 × 44k format, which is a full genome microarray containing 43,803 l design  where n p < 0.05 where p is the adjusted p value after false discovery rate (FDR) control according to the Benjamini-Hochberg procedure [Quality control of the microarray data was performed with the R software package “arrayQualityMetrics” , and norocedure . A relathttp://www.ncbi.nlm.nih.gov/geo) and are accessible through the GEO series accession number GSE121338 .Raw and analyzed microarray data, including the hybridization design, have been deposited in NCBI’s Gene Expression Omnibus . Transcripts were further analyzed in GOrilla (http://cbl-gorilla.cs.technion.ac.il), which is a tool for identifying enriched (significantly overrepresented) gene ontology (GO) terms in lists of genes compared to a relevant background gene list [Clustering analysis of transcript changes was performed with the MultiExperiment Viewer software 4.8.1  enriched GO classes of each list were selected to describe the most important affected biological processes in each treatment (see (a) In a first descriptive analysis, for each compound and time point, a separate GO enrichment analysis was carried out for significantly upregulated transcripts on the one hand and significantly down-regulated transcripts on the other hand, using all differentially expressed transcripts as the background list. This allowed us to determine which GO classes were characteristic of each specific treatment relative to the full set of differentially expressed genes in any of the treatments. We chose not to use all transcripts on the array as the background list since this would imply that the selection of the genes represented on the array, made by the manufacturer, would affect our analysis. This resulted in 7 different lists of enriched GO classes (TBBPA 8 dpf up-regulated gave no result). The top 3 (highest ment see .(b) In a second comparative analysis, all differentially expressed transcripts were used to perform a cluster analysis in MEV (k-means—Pearson correlation), resulting in 5 clusters see  containiIn conclusion, our study clearly demonstrates (i) how specific eye-/vision-related transcripts were differentially expressed after the THDC exposure, (ii) that the transcript profiles differed at least partly according to the mode of action of the TCDs, (iii) and that the recovery time of 3 days was not sufficient to fully reverse those effects, even though regeneration processes were clearly activated. These results confirm existing literature that shows that photoreceptors, optic primordia, optic nerve development, and opsin expression in fish are under the influence of THs and can be disrupted by THDC treatment. To the best of our knowledge, full-genome transcriptomic analyses of isolated eye tissue of THDC-exposed fish have not been performed so far and aids in understanding the underlying molecular mechanisms that translate into impairment of visual capacities of exposed fish.