The Oncotype DX assay was recently reported to predict risk for distant recurrence among a clinical trial population of tamoxifen-treated patients with lymph node-negative, estrogen receptor (ER)-positive breast cancer. To confirm and extend these findings, we evaluated the performance of this 21-gene assay among node-negative patients from a community hospital setting.n = 220) were patients who died from breast cancer. Controls (n = 570) were breast cancer patients who were individually matched to cases with respect to age, race, adjuvant tamoxifen, medical facility and diagnosis year, and were alive at the date of death of their matched case. Using an RT-PCR assay, archived tumor tissues were analyzed for expression levels of 16 cancer-related and five reference genes, and a summary risk score (the Recurrence Score) was calculated for each patient. Conditional logistic regression methods were used to estimate the association between risk of breast cancer death and Recurrence Score.A case-control study was conducted among 4,964 Kaiser Permanente patients diagnosed with node-negative invasive breast cancer from 1985 to 1994 and not treated with adjuvant chemotherapy. Cases . At 10 years, the risks for breast cancer death in ER-positive, tamoxifen-treated patients were 2.8% , 10.7% (95% CI 6.3–14.9%), and 15.5% (95% CI 7.6–22.8%) for those in the low, intermediate and high risk Recurrence Score groups, respectively. They were 6.2% (95% CI 4.5–7.9%), 17.8% (95% CI 11.8–23.3%), and 19.9% (95% CI 14.2–25.2%) for ER-positive patients not treated with tamoxifen. In both the tamoxifen-treated and -untreated groups, approximately 50% of patients had low risk Recurrence Score values.After adjusting for tumor size and grade, the Recurrence Score was associated with risk of breast cancer death in ER-positive, tamoxifen-treated and -untreated patients (In this large, population-based study of lymph node-negative patients not treated with chemotherapy, the Recurrence Score was strongly associated with risk of breast cancer death among ER-positive, tamoxifen-treated and -untreated patients. Approximately 65% of women currently diagnosed with invasive breast cancer have lymph node-negative disease at diagnosis , and 85%Multigene assays may provide information on patient prognosis and response to therapy that is superior and/or complementary to that from standard histopathological and immunohistochemical techniques ,8. HowevUsing a multistep approach, a 21-gene assay (Oncotype DX) was recently developed for use in paraffin-embedded tumor tissue to predict risk for distant recurrence or death in lymph node-negative breast cancer patients . ApproxiTwo subsequent studies were conducted to evaluate the performance of the Recurrence Score in predicting distant recurrence-free survival in lymph node-negative breast cancer patients not treated with chemotherapy. Among 668 tamoxifen-treated patients in the National Surgical Adjuvant Breast and Bowel Project (NSABP) B-14 clinical trial , the RecTo evaluate the Oncotype DX assay in a third independent study population of lymph node-negative breast cancer patients not treated with chemotherapy, we conducted a case-control study among a large population of women who were diagnosed and treated at 14 hospitals and clinics within the Northern California Kaiser Permanente health plan. A pre-specified primary aim of the study was to assess the degree to which the Recurrence Score would predict the risk of breast cancer-specific mortality among women with ER-positive, node-negative breast cancer treated with tamoxifen , eitherWe conducted a case-control study nested within a cohort of breast cancer patients. The Northern California Kaiser Permanente tumor registry, a contributor to the Surveillance, Epidemiology, and End Results (SEER) program of cancer registries, was used to identify all female health plan members who were diagnosed with lymph node-negative invasive breast cancer from 1985 to 1994. Northern California Kaiser Permanente is a nonprofit, integrated health services delivery organization that provides care for over 3 million members at 14 Northern California Kaiser hospitals and 23 outpatient clinics. The Kaiser Permanente membership is racially and ethnically diverse and is demographically similar to the general population of northern California, although it tends to under-represent the extremes of the socioeconomic spectrum . The stuBreast cancer patients were eligible if their nodal status was documented to be negative by pathologic assessment, they were diagnosed before age 75 years, and their initial disease was not treated with chemotherapy. Patients were excluded for the following reasons: inflammatory or bilateral breast cancer or evidence of metastasis (including lymph nodes) at initial diagnosis; prior invasive cancer (breast or other) at diagnosis; or unknown/unconfirmed treatment with tamoxifen.Using the Kaiser Permanente tumor registry and electronic membership databases, patients were followed until death due to breast cancer, death from another cause, bilateral breast cancer, termination of membership, or December 2002, whichever came first.Cases were patients whose first event was death from breast cancer. At each case's death, up to three controls were randomly selected from patients alive and under follow up  . ControlThe medical records of cases and controls were reviewed to confirm the initial diagnosis, treatment and cause of death, and to obtain information on eligibility criteria. Pathologic tumor size was obtained from pathology reports. Information on ER and progesterone receptor (PgR) status of the index tumor  was also abstracted from the medical record.Cases and controls were selected and eligibility was determined before laboratory measurement of gene expression. Of the 402 cases identified as potentially eligible by the tumor registry, 269 were determined to be eligible by chart review. The eligibility of 27 cases could not be determined because of incomplete or unavailable medical records and were considered lost to the study. Similarly, 722 of the 989 controls initially matched to eligible cases were determined to be eligible by chart review and the eligibility of 29 could not be determined. Of those eligible by chart review, 31 cases and 91 controls were lost because of missing tumor blocks. Another four cases were lost because we were unable to find at least one matched control. This left 234 cases and 631 controls available for pathology studies.All pathology and laboratory procedures  were conducted blinded to the case-control status of patient specimens. In addition, all batches of pathology materials sent to Kaiser Permanente Regional Laboratory, NSABP Pathology, and Genomic Health, Inc. included a mixture of cases and controls.in situ) were excluded from the study (n = 18). Specimens with regions on the slide having prominent nontumor elements  where the nontumor elements were both sufficiently localized to be amenable to macro-dissection and sufficiently abundant  were sent to NSABP Pathology for macro-dissection (n = 59). Macro-dissection to obtain enriched tumor tissue was performed using a safety blade cleaned with RNaseZAP  by NSABP Pathology on six (10 μm) sections of the region enriched in tumor tissue. For all other specimens, three (10 μm) sections were prepared by Kaiser Permanente Regional Laboratory, placed into a microcentrifuge tube, and sent to Genomic Health, Inc.For eligible cases and controls, the hematoxylin and eosin stained slides from biopsies and/or surgeries performed at the time of the index diagnosis were reviewed using pre-specified criteria in order to identify the most representative block and to evaluate whether sufficient tumor tissue was present. Specimens with no tumor or very little tumor , tumor size was obtained from a pathology review of all hematoxylin and eosin stained slides from all surgeries at diagnosis. Tumor grade, based on review of an hematoxylin and eosin slide from the most representative block, was assessed independently by two board certified , assistant professors in departments of pathology, using the modified Bloom-Richardson grading criteria . ER stat® 7900 HT instruments .Gene expression analysis in fixed paraffin-embedded tumor tissue was performed by Genomic Health, Inc., as described previously ,12. BrieExpression of each gene was measured in triplicate wells, and then normalized relative to a set of five reference genes . Reference-normalized expression measurements range from 0 to 15, where a 1-unit increase reflects approximately a twofold increase in RNA.r = 0.86) was also observed between Recurrence Score results from core biopsies and resections among 49 patients with locally advanced breast cancer.The potential impact of tumor heterogeneity on Recurrence Score results was examined in two small studies (unpublished data). In a study of 20 patients and 60 blocks (two to five blocks/patient), analysis of variance was used to examine the variability of Recurrence Score between blocks from the same patient (tissue sections did not undergo macro-dissection). The standard deviation in Recurrence Scores  between blocks was 3.0 Recurrence Score units. For 16 of the 20 patients, the standard deviation between blocks was less than 2.5 Recurrence Score units. A similarly high concordance (Pearson's Recurrence Score cutoff points were pre-specified and classified patients into low risk (Recurrence Score <18), intermediate risk (Recurrence Score 18–30) and high risk (Recurrence Score ≥31) categories. All methods used for measurement of gene expression levels and for the calculation of Recurrence Score and gene group scores were identical to those used in the study conducted by Paik and coworkers .Statistical analyses were conducted entirely by Kaiser Permanente researchers following a pre-specified plan. As specified, results were generated for patient groups, based on ER status and tamoxifen treatment.The Recurrence Score was examined as a continuous variable in 50-unit increments, for consistency and comparison with previously published Recurrence Score findings. In additional analyses, women were categorized into presumptive low, intermediate, and high risk groups based on cutoff points , and when categorized according to Recurrence Score quartiles.When examining the relationships between risk of breast cancer death and the expression level of individual genes, the expression levels were treated as continuous variables. When examining gene groups, the proliferation gene group and the HER2 gene group scores were treated as continuous variables both with and without transformation based on a threshold value  and wheP values are for the addition of the given factor to a model including all other factors.Conditional logistic regression was used to calculate odds ratios as estimates of the relative risks for breast cancer death associated with the Recurrence Score, or a component of the Recurrence Score . In addition, we estimated relative risks adjusted for tumor size and tumor grade . Model parameter estimation was done by maximum likelihood, and 95% Wald's confidence limits were calculated. Statistical significance was assessed via the likelihood ratio test . In multn = 16) had matched controls who were also ER-negative. We therefore conducted conditional logistic regression analyses among those patients not treated with tamoxifen and generated relative risk estimates for ER-negative patients using terms for interaction with ER status. Finally, we conducted analyses of the full study population, using conditional logistic regression and interaction terms for ER status and tamoxifen therapy to obtain relative risk estimates for the different pre-specified patient groups characterized by these factors. However, because results were not materially different, we present only those results from analyses conducted within patient groups.For women with ER-positive tumors, analyses were performed separately for those treated and not treated with tamoxifen. Because ER status was not a matching factor but was associated with our outcome, fewer than half of the ER-negative cases  had three controls each, 66 cases (30.0%) had two controls each, and 12 cases (5.5%) had one control each. The distribution of factors available from the tumor registry  among the evaluable cases and controls was generally similar to the distribution of these factors among the 239 potentially eligible cases and controls who were lost to the study. However, both lost cases and lost controls were slightly more likely than evaluable patients to be younger, to be not white, or to have smaller tumors.Breast cancer deaths occurred a median of 4.9 years after diagnosis. Among the cases and controls, the median tumor size was 1.5 cm (range 0.2–7.0 cm). Cases and controls were similar with respect to matching factors including age, race, diagnosis year, and treatment with tamoxifen (Table n = 19) who had ER-negative tumors according to RT-PCR assay and were not treated with tamoxifen . In addition, a small number of ER-negative patients were treated with tamoxifen (nine cases and three controls).For pre-specified analyses stratified by ER status and tamoxifen therapy, there were 55 cases and 150 matched controls who were tamoxifen treated and had ER-positive tumors according to RT-PCR assay. There were 110 cases and 251 matched controls who had ER-positive tumors according to RT-PCR assay and were not treated with tamoxifen. There were 16 cases with matched controls (The distributions of tumor size and/or tumor grade in patients categorized on the basis of the Recurrence Score as low risk (Recurrence Score <18), intermediate risk (Recurrence Score 18–30), or high risk (Recurrence Score ≥31) are shown in Table In ER-positive patients treated with tamoxifen, the risk of breast cancer death was positively and strongly associated with Recurrence Score when analyzed as a continuous variable, when categorized into quartiles, or when categorized based on pre-specified cutoff points Table .In ER-positive patients not treated with tamoxifen, the risk of breast cancer death was also positively associated with Recurrence Score Table . As expeLarger tumors and higher grade tumors were associated with an increased risk of breast cancer-specific mortality in both tamoxifen-treated and -untreated ER-positive patients Table . When th1, Ki-67, MYLBL2, STK15, and survivin). Positive associations were also observed for genes in the invasion group. The risk for death was negatively associated with expression of the ER-related genes . However, the associations between expression of the ER-related genes and risk were generally stronger for the tamoxifen-treated patients, especially with respect to the quantitative expression of the ER gene. Risk for death due to breast cancer was not statistically significantly associated with CD68, HER2, or GRB7 gene expression in patients treated or untreated with tamoxifen.The relationships between the expression of the individual genes that comprise the Recurrence Score and risk of breast cancer death were generally similar in tamoxifen-treated and -untreated ER-positive patients Figure . The risThe associations between expression of the gene group scores  and risk of breast cancer death were generally stronger than those for individual genes Figure .1, Ki-67, STK15, survivin). For a few genes, associations were in different directions for ER-negative and ER-positive patients . Note that estimates for ER-negative patients were often imprecise because of small numbers.After adjusting for tumor size and tumor grade, the risk of breast cancer death was positively associated with the Recurrence Score (continuous variable in 50-unit increments) among ER-negative patients . Risk for death was also strongly associated with the proliferation gene group . For some individual genes, associations appeared to be more strongly positive in the ER-negative than in the ER-positive patients , 10.7% (95% CI 6.3–14.9%), and 15.5% (95% CI 7.6–22.8%) for patients with Recurrence Score values in the low, intermediate, and high risk categories, respectively , 17.8% (95% CI 11.8–23.3%), and 19.9% (95% CI 14.2–25.2%) for patients in the low, intermediate, and high risk Recurrence Score groups, respectively. Again, when cross-classifying patients by Recurrence Score and tumor size and grade, an improvement in risk prediction was suggested primarily for those with Recurrence Score values categorized as low risk.In this large, population-based study of lymph node-negative patients not treated with chemotherapy, we found that the Recurrence Score was strongly associated with risk of breast cancer death among ER-positive patients treated with tamoxifen. We also found that the Recurrence Score was strongly associated with risk of breast cancer death among ER-positive patients not treated with tamoxifen and among ER-negative patients. In addition, we found that these associations remained after accounting for tumor size and grade, and that the Recurrence Score was able to identify a larger subset of patients with low risk of breast cancer death than was possible with either of these standard prognostic indicators.Several limitations should be considered when interpreting our results. We lacked ER status from the medical record for a substantial proportion of patients, and we therefore classified ER status based on gene expression. However, the estimates of relative risk were not materially changed when analyses were restricted to the 84% of patients with ER status from the charts (data not shown). Because of the diagnosis years of the study, only approximately 30% of patients were treated with tamoxifen. Although this is consistent with what has been reported for other patient populations during this period , it limiCurrently, adjuvant hormonal and/or cytotoxic chemotherapy are recommended for most women with early-stage invasive breast cancer. Treatment decisions are based on axillary node status, age, tumor size, histologic tumor type, tumor grade, hormone receptor status , and coexisting medical conditions . HormonaA growing number of studies suggest that multigene expression assays may be able to provide important prognostic and/or predictive information for breast cancer patients -42. A vaThis is the third study to evaluate the performance of the Recurrence Score among patients not treated with systemic chemotherapy ,13. The Our relative risk estimates for the Recurrence Score in ER-positive, tamoxifen-treated patients are generally quite similar to those observed in the NSABP B-14 study. Relative risks associated with expression of individual genes were also very similar. In the NSABP B-14 study, the 10-year risk of breast cancer death was 3.1% (95% CI 1.2–5.0%) in the low risk group, 12.2% (95% CI 6.7–17.6%) in the intermediate risk group, and 27.0% (95% CI 20.4–33.6%) in the high risk group. Although very similar to our findings for the low and intermediate risk groups, the 10-year risk for the NSABP B-14 high risk group was higher than ours. Patients in the two studies had a comparable age distribution, and although those in the NSABP B-14 study were more likely to have larger tumors, their tumors were also more likely to be well differentiated. Therefore, there is uncertainty regarding the extent to which differences in the distribution of prognostic factors in the two study populations may explain the difference observed in absolute risk estimates for the high risk group. In the Kaiser population, tumor size and tumor grade remained statistically significantly associated with risk of breast cancer death in most multivariate models that also included the Recurrence Score, whereas only tumor grade remained independently associated with risk in the NSABP B-14 study. Tumor size was determined by pathology in the Kaiser study and by clinical examination in the NSABP B-14 study. Clinical examination of tumor size is generally less accurate and could have resulted in attenuated relative risk estimates.The Recurrence Score has now been shown to be strongly associated with risk of breast cancer-specific mortality among lymph node-negative, ER-positive, tamoxifen-treated patients participating in a clinical trial and among similar patients from the community setting. In both study populations, the Recurrence Score was able to identify a large subset of patients (approximately 50% or more) who were at very low risk of breast cancer death at 10 years. In both studies it was also observed that although the Recurrence Score was correlated with tumor size and grade, there were a number of patients with large and/or moderately or poorly differentiated tumors with low risk Recurrence Score values. Results from our study, and to a lesser extent the NSABP B-14 study, suggest that combining Recurrence Score, tumor grade, and tumor size provides better risk classification than any one of these factors alone. Other studies, either retrospective or prospective, will be needed to confirm our findings among lymph node-negative, ER-positive patients not treated with tamoxifen and among ER-negative patients. Two studies have been performed ,44 and oCI = confidence interval; ER = estrogen receptor; NSABP = National Surgical Adjuvant Breast and Bowel Project; PgR = progesterone receptor; RT-PCR = reverse transcriptase polymerase chain reaction.The following authors received support for study-related activities from Genomic Health, Inc., but have no other competing financial or nonfinancial interests: LAH, MJK, AC, NTB, DG, CPQ, BL, and LF. The remaining co-authors  are employees or consultants for Genomic Health, Inc.LAH participated in the design of the study, directed the data analysis, and drafted the manuscript. SS participated in the design of the study, in interpreting results, and in writing the manuscript. MKJ and AC performed statistical analyses and participated in interpreting results. NTB and CA participated in the development of the study methods and coordinated data collection. MP participated in the development of the study methods and in data collection. JB participated in the development of the study methods, in interpreting results, and in writing the manuscript. MW, DW, and JH participated in the design of the study and in interpreting results. DG participated in data collection and in interpreting results. CPQ participated in the study design, co-directed the data analysis, and participated in interpreting results. BL provided methods for estimating cumulative risks and participated in interpreting results. LF participated in interpreting results. All authors read and approved the final manuscript.A Word document providing additional detail regarding the statistical analyses for estimating 10-year recurrence probabilities.Click here for file


The prognostic value of two antibodies targeting citrullinated antigens, anti-Sa and anti-cyclic citrullinated peptide (CCP), present at inclusion, was evaluated prospectively in a cohort of 165 consecutive patients with recent-onset or early polyarthritis (EPA) followed for up to 30 months. Patients were treated according to current Good Clinical Practice standards. Predefined outcomes were severe arthritis and persistent arthritis. At inclusion, a median of 3 months after disease onset, 133 (81%) patients fulfilled at least four American College of Rheumatology criteria for rheumatoid arthritis and 30 (18%) had erosive changes on radiographs of hands and feet. Disease-modifying anti-rheumatic drugs were used in close to 80% of the patients at 30 months. Joint damage increased linearly over time, whereas disease activity declined markedly and remained low at each follow-up. Autoantibodies were identified in 76 (46%) patients: rheumatoid factor (RF) in 68 (41%), anti-CCP in 53 (33%), and anti-Sa in 46 (28%). All three antibodies were correlated, but anti-Sa antibodies best predicted severity at 18 and 30 months. RF and anti-CCP performed less well. For both outcomes, anti-Sa alone performed better than any combination of antibodies. The presence of any autoantibody identified about 50 to 60% of the patients with poor outcomes. In multivariate analysis, anti-Sa (odds ratio (OR) 8.83), the presence of erosions at inclusion (OR 3.47) and increasing age (OR 1.06/year) were significantly associated with severity, whereas RF and anti-CCP were not significant predictors. Persistent arthritis was present in up to 84% of patients; autoantibodies were specific but poorly sensitive predictors of this outcome. We conclude that assays for antibodies against citrullinated antigens differ in their ability to predict poorer outcomes in patients with EPA. In our EPA cohort treated in accordance with current standards, detection of anti-Sa but not of RF or anti-CCP antibodies, in combination with clinical and radiological variables present at the first encounter, allowed the identification of a subgroup of EPA patients suffering more rapid and more severe joint damage over 30 months. Rheumatoid arthritis (RA) is a chronic inflammatory arthritis that frequently starts at the peak of productive life and is a major cause of invalidity, morbidity, and premature mortality . RA is cSufficiently specific and sensitive prognostic markers that could be used with confidence in the individual patient with early or recent-onset polyarthritis (EPA) and recent-onset RA are still lacking -11. For in vivo citrullinated proteins, such as anti-keratin antibodies, antiperinuclear factor, anti-citrullinated (pro)filaggrin and anti-Sa/citrullinated vimentin [Recently, antibodies targeting determinants resulting from the deimination of peptidylarginine to peptidylcitrulline residues have been described in the serum of patients with RA . These ivimentin ,22. The vimentin ,24. The vimentin . Assays vimentin -27, althvimentin -31.The purpose of our study was to assess the prognostic role of anti-Sa and of anti-CCP antibodies, relative to RF, in patients with polyarthritis evaluated and treated early after the onset of disease, irrespective of the fulfillment or not of diagnostic criteria for a specific disease entity. We now present an analysis of our cohort at 30 months of follow-up.Consecutive adult patients with synovitis affecting at least three joints for at least 1 month and less than 12 months and evaluated at the Centre hospitalier universitaire de Sherbrooke (CHUS) were asked to participate to the study. The CHUS is the single regional rheumatology referral center for about 400,000 people, and is the site of practice of six of the seven rheumatologists in the area. Patients with bacterial or crystal-induced arthritis, patients with a defined connective tissue disease , patienA rheumatologist completed a structured physical examination, including 68 tender and 66 swollen-joint count assessments and identification of extra-articular manifestations. A trained nurse coordinator performed a structured interview at the inclusion visit and at each of the follow-up visits scheduled at 18, 30, 42 and 60 months after the onset of inflammatory arthritis. The time of onset of arthritis was assumed to be the month during which the patient indicated that joint symptoms/signs had appeared or, in patients with previous musculoskeletal complaints (such as osteoarthritis), at the time that the signs or symptoms of inflammatory arthritis appeared, additive to the previous signs and symptoms. Scheduling the follow-up evaluations relative to the onset of symptoms rather than to the establishment of diagnosis sought to ensure that the cohort was more homogeneous in disease duration on follow-up. [Variables assessed included demographics, tender and swollen joint counts, duration of morning stiffness, use of DMARDs and corticosteroids at and between each visit, modified Health Assessment Questionnaire (M-HAQ) , erythro . A DAS28 ,42, wherPersistent arthritis was defined as the presence of at least one joint with synovitis and/or the current use of DMARDs or at least 10 mg of prednisone equivalent per day . FulfillGenomic DNA was extracted from leukocytes present in 2.5 ml of EDTA-treated whole blood using Wizard DNA extraction kit , then stored at 4°C. Genomic typing was performed by using PCR with sequence-specific primers specific for HLA class II molecules ,44. Primin vitro citrullinated bovine myelin basic protein (MBP). MBP and vimentin have similarly high proportions of arginine. Most positive results in MBP-ELISA correlated with positive anti-Sa results on the original WB method using human placenta.The Sa antigen was first detected in human spleen/placenta extracts by western blotting (WB)  and subs2 in IMDM  enriched with 10% fetal bovine serum containing 3.024 g/L sodium bicarbonate (pH 7.2). At confluence, cells were trypsinized, washed, incubated for 5 min on ice in 1 ml of buffer containing 40 mM Tris-HCl pH 7.5, 1 mM EDTA pH 8.0, 0.150 NaCl, and mechanically detached. The resultant cell pellet was submitted to three freeze-thaw cycles, and the supernatant was clarified by centrifugation. The concentration of protein was measured by the Bradford assay (Bio-Rad), and the extracts were stored at -80°C. In preparation for WB, the extracts (2.5 μg per tested serum) were auto-citrullinated for 3 hours at 37°C in 50 mM citrullination buffer . The reaction was stopped with EDTA . An equal volume of 2× WB loading buffer was added to the citrullinated extract for long-term storage in aliquots at -20°C. WB was performed as described [ECV 304 cells were cultured at 37°C under 5% COescribed , with ECin vitro by incubation for 3 hours at 37°C in citrullination buffer containing 0.2 U rabbit PADI 2 (Sigma) per μg of MBP. Microtiter plates  were coated overnight at 4°C with native or citrullinated bovine MBP at 1 μg per well. The plates were blocked for 1 hour at room temperature (20 to 25°C) with PBS containing 1% (w/v) BSA, then washed three times with PBS containing 0.05% Tween 20 (PBST). The plates were incubated for 1 hour at room temperature with 100 μl per well of human serum diluted 1:300 in PBS containing 1% (w/v) BSA (PBS-BSA), and then washed three times with PBST. Bound human IgG was detected with a goat anti-human IgG, alkaline phosphatase-labeled conjugate  diluted 1:1000 in PBS-BSA. The reaction was detected with 1 mg/ml p-nitrophenyl phosphate (Sigma) in substrate buffer  for 20 min at room temperature. The absorbance, A, was read at 405 nm. Each sample was run in duplicate and in parallel on native MBP (N), on citrullinated MBP (C), and on BSA (B). Results were given by the equation A405 = AC – AN. A positive test was defined as A405 > 0.2. A known positive control serum was used on each plate for reagent quality control. The BSA control was used to detect spurious intrinsic individual serum stickiness for which some RA sera are notorious. When a serum gave high A405 when used on BSA alone, a positive anti-Sa ELISA result had to be confirmed on WB to be reported as positive. All the anti-Sa assays described here were developed and performed on coded sera at the McGill University Autoimmune Research Laboratory.Bovine MBP (Sigma) was citrullinated The principal independent variables were anti-Sa (MBP-ELISA), anti-CCP (positive if more than 20 U/ml), and RF (positive if at least 40 IU/ml) antibodies. The degree of association between anti-Sa and anti-CCP assays was measured by using the phi coefficient. Sensitivity, specificity and positive likelihood ratio (LR) were calculated for each of the three outcome measures at 18 and 30 months into disease and for each of the 13 following putative prognostic markers present at the inclusion visit: anti-Sa, anti-CCP, RF, sex, age (in years), duration of disease (in months), duration of morning stiffness (positive when at least 1 hour), symmetry of joint swelling, number of joints with synovitis, M-HAQ score (positive when at least 1.0), RA-associated HLA-DRB1 alleles ('shared epitope'), CRP levels (positive when higher than 8.0 mg/L), and an erosion component of the SvH score of at least 5. The predictive ability of anti-Sa, anti-CCP, and RF in the early identification of patients with each outcome was also studied by using the area under a receiver operating characteristics curve. Confidence intervals were calculated for these estimates. After these estimations, a logistic regression approach was used to estimate the independent contribution of RF, anti-Sa, and anti-CCP present at inclusion to predict the development of outcomes at 30 months. Each autoantibody was evaluated separately and in combination, after inclusion in the multivariate statistical model of prognostic factors listed above. A final statistical model was constructed with the stepwise logistic regression approach. By using the variables included in the final model, with and without the inclusion of anti-Sa and anti-CCP antibodies, the odds ratio (OR) was calculated to estimate the contribution of each marker adjusted for the other markers in the prediction of the severity outcome.Up to May 2004, 260 patients agreed to participate and were included. Eleven additional patients declined to participate or had very incomplete inclusion data and were excluded. At this time, 165 patients had reached the 30-month evaluation mark. Of these patients,5 had died and 11 failed to come to their scheduled appointment. We therefore report on the 149 patients (retention rate 90.3%) with a complete 30-month assessment.The baseline characteristics at entry of the 165 patients are summarized in Table The prevalence of autoantibodies was low. RF was found in 68 (41.2%) patients, anti-CCP in 53 (33.1%), and anti-Sa in 46 (28.0%). As reported previously ,29, the Upon follow-up, the clinical status of the patients improved markedly Table . The DASa priori assumption about the rate of damage progression in our patients, as this should be highly dependent on the characteristics of the patients included and (possibly) the treatments used. The threshold for the upper third of SvH score was calculated as a total score of more than 10 at 18 months and more than 15 at 30 months, that is, about 2% and 3.5%, respectively, of the maximal SvH score. Not surprisingly, functional impairment was the criterion most determinant for selection in the group with severe disease at inclusion, whereas radiological damage became the major reason for selection during follow-up.Severe arthritis was present in 56 (38%) patients at 30 months. Our inclusive definition of severity (upper third of SvH score and/or M-HAQ ≥ 1.0) did not make any Only 28 (18.79%) patients still fulfilled at least four ACR criteria for RA at 30 months. At inclusion or on follow-up, 20 patients fulfilled criteria for inflammatory rheumatic diseases other than RA: 13 had arthritis associated with skin psoriasis (present at inclusion in 12), 3 had benign sarcoid arthritis, 3 had spondylarthropathy, and 1 had scleroderma. Autoantibodies were present at low frequency in these patients: RF in four, anti-Sa in two, and anti-CCP in one. At each of the follow-up visits, more than two-thirds of the patients  did not fulfill criteria for a specific diagnosis and might thus be classified as undifferentiated arthritis.a priori definition , persisIn all three evaluated outcomes, RF had a higher sensitivity than anti-Sa, whereas anti-Sa had a higher specificity than RF (except for the fulfillment of RA criteria). Anti-CCP also had a slightly better specificity than RF for predefined outcomes. However, despite their high specificity for established RA, anti-CCP antibodies performed poorly. The same differences between anti-CCP and anti-Sa antibodies, although present, were less marked at the 18-month evaluation (data not shown). As noted above, anti-Sa alone performed better than any combination of antibodies in predicting severity. The conclusions were not significantly different when the analysis was restricted to patients fulfilling RA criteria at inclusion (data not shown). As noted in Table As illustrated in the multiple regression model for severity Table , model AHowever, anti-Sa antibodies are not widely available yet, and their presence in the model might have wiped out significant associations of RF and/or anti-CCP with severe arthritis. After the removal of anti-Sa from the analysis, neither RF nor anti-CCP was significantly predictive of severe arthritis Table , model BFinally, we tested the interaction of erosion SvH score and anti-Sa, the two major initial predictors of severe arthritis on the development of joint damage Fig. . Each ofIn the multiple regression model for persistence at 30 months (data not shown), disease duration for at least 4 months at inclusion was the single significant prognostic marker. No antibody was statistically associated with the outcome of persistence at that time.The main conclusion from our EPA study is that assays targeting different citrullinated antigens have distinct prognostic values for poor outcomes, in addition to their differences in sensitivity and specificity . When prAt first sight, the lower prevalence of anti-Sa (28% versus 41% for RF) seemed a significant disadvantage. However, the sensitivity of anti-Sa for severe arthritis increased over time in our cohort, as 29%, 39%, and 45% of all severe patients had anti-Sa at inclusion, 18 months, and 30 months, respectively. This observation held true even though anti-Sa positive patients received as aggressive DMARD treatments as the other patients with persistent disease. The lack of statistically significant association of antibodies with persistence was probably due in part to the dual definition of persistence that we used, namely the presence of synovitis and/or being treated. In the absence of a more specific diagnostic test for persistence, we consider this definition to be the best currently available in treated recent-onset arthritis. The major limitation with our definition of persistence was the unexpectedly high (80%) frequency of ongoing use of DMARDs at 30 months. We expect that most patients in protracted remission will slowly taper their DMARDs over the planned follow-up of up to 5 years. The confounding influence of prolonged treatment on our definition of persistence should thus progressively attenuate over these additional 30 months, and the prognostic value of antibodies for persistence might then become assessable.The second important conclusion is that autoantibodies were present early in only 50 to 60% of the patients who developed any of the predefined poor outcomes at 30 months. Autoantibodies present early into disease therefore characterize a large, but limited, subset of EPA patients with poor outcomes. Detection of autoantibodies in EPA patients must thus be used in combination with additional variables present at inclusion (for example elevated erosion SvH scores and increasing age) to best predict, and possibly prevent, the development of severe disease at 30 months (and beyond).The third conclusion is that our results should be generally applicable to EPA patients evaluated by rheumatologists. Several reasons support that opinion. First, selection biases were minimized by the strict exclusion of patients with diseases known to have a good prognosis  and by a retention rate of 90% at 30 months, without individual missing data. Second, the patients were thoroughly evaluated with widely available tools (except for anti-Sa), early in their disease (median 3 months), and at consistent intervals relative to disease onset. Third, the predefined severity outcome included both functional disability and radiographic damage. Functional impairment and invalidity are the best indicators of severity, but they occur too late into disease to be useful prognostic indicators. Radiographic changes, such as those quantified with the SvH score, are therefore used as surrogates of severity in early disease. Because disability and radiographic damage are poorly correlated in early arthritis ,48, bothOur observations regarding the prognostic usefulness of anti-CCP in EPA patients are somewhat at variance with other reports. The reasons for differences in prognostic value between anti-CCP and anti-Sa assays therefore merit further discussion. A first possible explanation relates to deficiencies of the specific commercial anti-CCP assay used in this paper. That explanation is unlikely. We observed no significant differences in sensitivities or specificities between commercial anti-CCP assays . Such differences would be unlikely, because all commercially available anti-CCP assays use the same antigen strips  and diffA second and more likely possibility for the poor performance of anti-CCP might reside in the design of the assay. The objectives of current anti-CCP assays are to attain the maximal sensitivity for patients with established RA or RA-like disease, while maintaining a reasonable specificity. This approach favors a low threshold to report positive results. Because EPA and RA are very heterogeneous diseases, a more appropriate design would be to identify subsets of patients with worse outcomes. In our cohort, most anti-CCP positive sera had high titers  but, significantly, 11 patients had low or moderate titers (60 U/ml and below). The presence of low or moderate titers of anti-CCP at inclusion did not seem to be associated with severe arthritis at 30 months. Indeed, using a cut-off of 61 U/ml afforded the best OR estimate for the development of severe arthritis, although the predictive value of this level of anti-CCP was still not statistically significant Table , model Cin vivo by modified arginine-rich proteins. At present it remains to be confirmed whether citrullinated proteins in general might perform better than CCP in ELISA at identifying severe subsets of EPA patients.A third explanation for the prognostic discrepancy between anti-CCP and anti-Sa implies hypothetical qualitative differences between the anti-Sa and the anti-CCP assays, as suggested by the moderate degree of association between anti-Sa and anti-CCP results remaining constant across different cut-offs for positive anti-CCP . These qualitative differences between assays would translate into genuine consequent associations with outcomes. Antibodies present in sera from RA patients recognize citrullinated peptidic residues in the context of adjoining amino acid residues and of peptide conformation ,20. The A fourth contributing factor to explain the poor performance of anti-CCP in our cohort is the influence of aggressive treatment on EPA prognosis. An earlier time of introduction, a higher intensity of use, and a more prolonged maintenance of DMARDs, with the objective of controlling disease, distinguish our current cohort from previous EPA cohorts established during the early 1990s. This more aggressive approach is illustrated by the high prevalence of DMARDs still used at 30 months, as well as by the marked decrease in disease activity over time, as assessed by the DAS28-3, the swollen joint counts, and the M-HAQ. Possibly because of this relatively intensive use of DMARDs, more than half of the patients were in clinical remission at 30 months. According to this hypothesis, the presence of anti-CCP antibodies would indeed identify patients with a poor natural course, as suggested in previous EPA or early RA cohorts -27,50. IFinally, our data stress the importance of intrinsic aggressiveness of arthritis in causing progressive joint damage. At inclusion, 18% of the patients already demonstrated clinically significant erosive changes. That represents a very early progression to the pannus phase, and an already missed opportunity to intervene during the optimal 'therapeutic window' in these patients. Severe outcomes at 30 months happened mostly in those patients who already had significant erosive joint damage at first evaluation. Early erosive changes are therefore an excellent surrogate marker for an aggressive arthritis. Whether the use of more sensitive imaging techniques such as ultrasound or magnetic resonance imaging  would inThis study reports the first direct comparison of RF, anti-CCP and anti-Sa antibodies as prognostic markers in a cohort of patients with recent-onset inflammatory polyarthritis treated according to current standards. In this cohort, anti-Sa antibodies were present at inclusion in 45% of the patients who subsequently developed severe outcomes at 30 months. Anti-Sa antibodies were the single serological marker independently predictive of poor outcomes and, together with the presence of joint erosions at inclusion and with increasing age, characterized a subgroup of patients with more rapid and more severe joint damage. In contrast, anti-CCP antibodies were not independently associated with severe outcomes in our patients. Using higher thresholds for positive results slightly improved the performance of the anti-CCP assay, but the predictive value of anti-CCP antibodies remained inferior to that of RF. Although anti-Sa and anti-CCP assays both use citrullinated antigens, our data suggest that the two assays differ in their ability to predict poor outcomes in patients treated aggressively early after the onset of inflammatory polyarthritis.ACR = American College of Rheumatology; BSA = bovine serum albumin; CCP = cyclic citrullinated peptide; CHUS = Centre hospitalier universitaire de Sherbrooke; CRP = C-reactive protein; DAS28-3 = Disease Activity Score using 28 joints and 3 variables (tender and swollen joint counts and CRP); DMARD = disease-modifying anti-rheumatic drug; ELISA = enzyme-linked immunosorbent assay; EPA = early (or recent-onset) polyarthritis; LR = likelihood ratio; MBP = myelin basic protein; M-HAQ = modified Health Assessment Questionnaire; OR = odds ratio; PADI = peptidylarginine deiminase; PBS = phosphate-buffered saline; RA = rheumatoid arthritis; RF = rheumatoid factor; SvH = Sharp–van der Heijde; WB = western blotting.The author(s) declare that they have no competing interests.GB designed the research protocol, played a leading role in the design and coordination of the study, and drafted the manuscript. NC performed the statistical analysis and helped to draft the manuscript. PC helped in the design of the research protocol, longitudinally contributed to the coordination of the study, and helped in drafting the manuscript. AJF participated in the performance and the coordination of the study, and helped to draft the manuscript. PL participated in the performance and the coordination of the study, and helped to draft the manuscript. TN participated in the design of the protocol, contributed to the coordination of the study, supervised the statistical analysis, and reviewed the draft manuscript. ZJZ developed the anti-Sa assays and performed a large portion of the assays for the study. CD participated in the design of the protocol and performed the immunogenetic tests. HAM designed the research protocol, contributed to the performance of the study, supervised the development and performance of anti-Sa assays, and helped to draft the manuscript. All authors read and approved the final manuscript.


Dear Editor,An Iranian study was recently published in your prestigious journal, in which the aim was to determine the association between health-promoting lifestyles and quality of life (QoL) among a sample of 389 adult beta-thalassemia major (TM) patients . The finWe conducted a retrospective cohort study enrolling 704 TM patients in 2016 in Shiraz, southern Iran. Our study aimed to identify changes in the survival rate of TM patients over the past two decades. Two different approaches, including a cohort analysis  and a peA downward trend was found in both the 20-year (p< 0.001), and 30-year (p< 0.001) survival rates of TM patients from 1995 to 2007, but an upward trend was found from 2007 through 2016 . The perWith these results in mind, the poor QoL reported among Iranian TM patients -3 could In conclusion, it is suggested that factors affecting QoL should be addressed in future studies to obtain important information for policymakers to improve the QoL in this population, thereby improving survival.


In many industrialized countries routine vaccination with the 23-valent pneumococcal polysaccharide vaccine (PPSV-23) is recommended to prevent pneumococcal disease in the elderly. However, vaccine-induced immunity wanes after a few years, and there are controversies around revaccination with PPSV-23. Here, we systematically assessed the effectiveness and safety of PPSV-23 revaccination.We conducted a systematic literature review in MEDLINE, EMBASE, and Cochrane Central Register of Controlled Trials from inception to June 2015. We included all study types that compared effectiveness, immunogenicity and/or safety of PPSV-23 as a primary vs. a revaccination dose in persons aged 50 years and older. With respect to immunogenicity, we calculated the ratio of geometric mean antibody concentrations and opsonophagocytic indexes at identical time-points after primary and revaccination. Additionally, we compared rates and severity of adverse events (AEs) after primary and revaccination.n = 5) or (ii) two groups consisting of participants receiving PPSV-23 who were either vaccine-naïve or had received a first PPSV-23 dose 3 to 13 years earlier (n = 5). Three studies used electronic data bases to compare AEs after primary vs. revaccination doses of PPSV-23 after 1 to 10 years and one study had a cross-sectional design. Number of participants in the non-register-based and register-based studies ranged from 29 to 1414 and 360 to 316,000, respectively. 11 out of 14 included studies were at high risk of bias, three studies had an unclear risk of bias. None of the studies reported data on clinical effectiveness. Immunogenicity studies revealed that during the first two months antibody levels tended to be lower after revaccination as compared to primary vaccination. Thereafter, no obvious differences in antibody levels were observed. Compared to primary vaccination, revaccination was associated with an increased risk of local and systemic AEs, which, however, were usually mild and self-limiting. The risk and severity of AEs appeared to decrease with longer intervals between primary and revaccination.We included 14 observational studies. 10 studies had a prospective design and analysed data on (i) the same individuals after a first and a second dose of PPSV-23 given 1 to 10 years later  contains supplementary material, which is available to authorized users. Streptococcus pneumoniae (PPSV-23) and a 13-valent conjugate vaccine (PCV-13).Pneumococcal disease is a major cause of morbidity and mortality in the elderly population worldwide –3. TherePPSV-23 has been available since 1983. There is evidence that its protective effect declines already 3 to 5 years after vaccination , 8. At tAfter licensure of PCV-13 had been extended for adults in 2011, many NITAGs continue to recommend PPSV-23 for elderly , 15, 16.The purpose of this review was therefore to systematically assess differences in the effectiveness, immunogenicity and safety of revaccination as compared to primary vaccination with PPSV-23 in the elderly population.The systematic review was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) statement  and was We evaluated all original studies that reported effectiveness, immunogenicity or safety of revaccination with PPSV-23 as compared to a primary PPSV-23 dose in persons aged 50 years or older, irrespective of underlying comorbidities. No restrictions were made regarding publication language and publication status (published/unpublished studies). We excluded studies in which other vaccine types were used  or if a revaccination dose was administered within 1 year of the first PPSV-23 dose.Regarding vaccine effectiveness, we considered all clinical outcomes including invasive pneumococcal disease, pneumonia , as well as non-specific outcomes such as all-cause mortality or hospitalization. Immunogenicity data were considered, if geometric mean antibody concentrations (GMCs) or opsonophagocytic indexes (OPIs) were measured at the same interval after primary vaccination and revaccination dose , respectively. Since seroconversion is difficult to define in the absence of a validated protective threshold, we evaluated absolute antibody levels (GMC or OPA titers) rather than seroconversion rates. Regarding safety, we considered all types of local and systemic AE that were reported in the included studies.Electronic databases searched were MEDLINE, EMBASE, and Cochrane Central Register of Controlled Trials (date of last search 26.06.2015). The search strategy included both keywords and MESH terms related to effectiveness, immunogenicity or safety of PPSV-23. For complete search strategy, see Additional file Two reviewers  independently screened titles, abstracts and full text articles. From eligible studies, two reviewers (CR and GF) extracted study data and assessed methodological quality, using standardized extraction forms. The extraction forms were pilot tested with the first two identified studies. After calibration of the extraction process, CR and GF finally extracted the following information: author, publication year, country, study design, study population, age of participants, study size, number of PPSV-23 doses administered, time span between first and revaccination doses, outcome measures, laboratory methods, serotypes measured, confounders considered in the statistical analysis and potential conflicts of interests of the authors. In case of disagreements regarding the screening process, data extraction, and quality assessment a final decision was made by consensus or resolved by a third reviewer (GF [literature screening] or TH [data extraction process]).http://www.casp-uk.net/checklists). According to the suggestions of the Cochrane Collaboration, we gave particular attention to the domains risk of selection bias, detection bias and attrition bias in observational studies. For each study, risk of bias appraisal was expressed as considered judgment as either “low”, “high” or “unclear”.We used the Cochrane risk of bias tool to assess risk of bias for RCTs  and the Abstracted data were aggregated in tables. Risk ratios (RR), odds ratios (OR) and corresponding 95% confidence intervals (95% CIs) were either calculated or directly extracted from the publications, if available. To assess differences in serotype-specific GMCs after the primary and revaccination dose, we graphically displayed numbers with 95% CI for the most commonly analyzed serotypes 4, 6B, 14 and 23 F. We also calculated GMC (and OPI) ratios  after revaccination/GMC (OPI) after primary vaccination) with corresponding 95% CIs at each available time point. A ratio of > 1 indicates a higher antibody level after revaccination than after primary vaccination. A ratio of < 1 indicates a lower antibody level after revaccination than after primary vaccination. Ratios with 95% CIs were calculated by back transforming the mean difference GMCs between the vaccination groups on the logarithmic scale .Ratios are displayed separately for each study and for the most commonly analyzed serotypes. Due to marked differences within studies (immunogenicity differences between serotypes) and between studies , we did not perform meta-analyses. Calculations were performed using STATA 12 .The literature search yielded a total of 1162 titles and two additional studies were identified through other sources   or retrospective (n = 3) cohort studies, and one study had a cross-sectional design (for study characteristics see Table n = 10), the remaining included only patients with chronic obstructive pulmonary disease [Of the 14 studies, two studies were randomized controlled trials (RCTs) , 31. How disease , 29, pat disease  or renal disease , 29, 31.In 6 studies two different study groups were compared: one group consisted of participants who had received a primary PPSV-23 dose, and the second group comprised those who had received one , 23, 25 None of the studies reported effectiveness of PPSV-23 against clinical outcomes. Three studies  reportedIn 11 (78%) studies risk of bias was high , 31, 32.Nine studies used enzyme-linked immunosorbent assays (ELISAs) to measure serotype-specific anti-pneumococcal immunoglobulin G (IgG) levels, but only three studies , 29, 31 Within studies, serotype-specific GMCs differed widely between serotypes vaccination, GMC ratios (GMC after second dose divided by GMC after first dose) were mostly below 1, indicating higher short-term immunogenicity of the first PPSV23 dose as compared to the second dose, although these results were statistically not significant  pre-vaccination antibody levels was assessed, three studies found an association –13 and oIn studies that used ICD-9 codes to assess differences in vaccine-related unplanned medical visits, differences were smaller and ranged from 0.3 to 1.3% after primary and from 0.7 to 1.9% after revaccination doses , 32, 34.or fourth-dose recipients [or second dose [Of four studies in which safety of a third dose was analyzed, one study found more local and systemic AEs among third-dose recipients than among second-dose recipients , whereascipients . In the ond dose , 32.presumptive medically attended injection site reaction than those who received a primary or a third dose [Four studies provided adjusted safety analyses , 27, 30.ird dose .Overall, local AEs and systemic AEs after revaccination were reported more frequently in studies in which patient diaries were used Table . Two of Comparative data on the effectiveness of primary vs. revaccination with PPSV-23 are still lacking. Since we did not identify studies with clinical endpoints, evidence had to be derived from immunogenicity studies only. These studies indicated that antibody responses are reduced during the first months after revaccination as compared to primary vaccination with PPSV-23. However, differences were observed only for some serotypes and were restricted to early time points (i.e. one or two months after revaccination). Thereafter immunogenicity of primary vaccination and revaccination was comparable. After a period of more than two years antibody levels against some serotypes were even higher after revaccination than after primary vaccination. Regarding safety, our systematic review indicates that revaccination with PPSV-23 is associated with a higher frequency of local and systemic, self-limiting adverse events, but not with severe sequelae.. showed that serotype-specific protection varies widely [Measuring the efficacy/effectiveness against clinical outcomes has been advocated as the best way to assess the protective effect of immunizations , 36. Hows widely , serotyps widely , 40, we In addition to serotype-specific IgG, measurement of functional antibodies as assessed by OPA is considered as a valid surrogate parameter for vaccine protection from pneumonia or bacteremia , 41, 42.It remains unclear why the rise in antibody levels after revaccination with pneumococcal polysaccharide vaccine is lower than after primary vaccination and if this might affect clinical effectiveness . SeveralData on the safety of a revaccination vs. primary PPSV-23 dose were more consistent and indicate that revaccination with PPSV-23 is associated with a higher number of non-severe AEs as compared to the primary dose. However, since in the majority of studies participants of the revaccination groups were older and/or had more comorbidities than the comparison group, interpretation of the unadjusted rates of AEs has to be taken with caution. The fact that revaccination increases the risk of AEs is consistent with Arthus-type hypersensitivity reactions in which antigen-antibody complexes cause local symptoms after (re-)vaccination , 51. SinThis study has several strengths. It is based on a comprehensive systematic review of the currently available literature, comprising immunogenicity as well as safety data. By calculating the ratios of GMTs, we provide a measure of relative immunogenicity of revaccination, which is independent of differences of laboratory assays used in different studies. The usefulness of this approach was limited by differences in the choice and numbers of serotypes analyzed in different studies.Interpretation of the immunogenicity data was complicated by differences in the composition of study populations  and differences regarding the follow-up period and time points at which blood sampling was performed. In addition, risk of bias was high in the majority of studies, limiting the internal validity of the results. Since no study reported adjusted immunological data, it remains unclear to what extend confounders such as comorbidities might have influenced the results. Furthermore, in some studies it remained unclear how selection of participants into the cohorts was performed. For these and further reasons related to heterogeneity of the study designs, it was not possible to calculate meta-analytical estimates.In conclusion, comparative data on the effectiveness of primary vs. revaccination with PPSV-23 are still missing, mainly due to the lack of randomized controlled trials using clinical endpoints. The available evidence from observational studies indicates that revaccination with PPSV-23 is likely to induce long-term antibody levels that are comparable to those after primary vaccination. However, due to differences in study design, study populations, different intervals between doses and high or unclear risk of bias in the included studies, these results have to be taken with caution and it remains unclear how immunogenicity results translate into clinical protection. Since primary vaccination with PPSV-23 has been shown to protect from pneumococcal disease, it seems likely that – based on similar antibody levels - also revaccination may confer. In view of the waning immunity and increasing risk of severe pneumococcal disease with age, the benefits of revaccination with PPSV-23 probably outweigh the increased risk of non-severe local and systemic AE. Revaccination could therefore be considered in the elderly after informing about benefits and possible harms of revaccination. In order to obtain strong evidence for the effectiveness of PPSV-23 in the elderly, RCTs or carefully designed observational studies using clinical relevant endpoints are urgently needed.


Dental caries inequalities still severely burden individuals’ and society’s health, even in countries where fluoride toothpastes are widely used and the incidence of dental caries has been decreasing. School-based fluoride mouth-rinse (S-FMR) programs, a population strategy for dental caries prevention, might decrease dental caries inequalities. This study investigated the association between S-FMR and decreasing dental caries prevalence and caries-related inequalities in 12-year-olds by Japanese prefecture.We conducted an ecological study using multi-year prefecture-level aggregated data of children born between 1994 and 2000 in all 47 Japanese prefectures. Using two-level linear regression analyses (birth year nested within prefecture), the association between S-FMR utilization in each prefecture and 12-year-olds’ decayed, missing, or filled permanent teeth (DMFT), which indicates dental caries experience in their permanent teeth, were examined. Variables that could explain DMFT inequalities between prefectures, such as dental caries experience at age 3 years, dentist density, and prefectural socioeconomic circumstances, were also considered.High S-FMR utilization was significantly associated with low DMFT at age 12 . S-FMR utilization explained 25.2% of the DMFT variance between prefectures after considering other variables. Interaction between S-FMR and dental caries experience at age 3 years showed that S-FMR was significantly more effective in prefectures where the 3-year-olds had high levels of dental caries experience.S-FMR, administered to children of all socioeconomic statuses, was associated with lower DMFT. Utilization of S-FMR reduced dental caries inequalities via proportionate universalism. In fact, interventions that depend on individual motivation often increase health inequalities.13,14 In contrast, interventions aimed at changing social environment are beneficial regardless of individual socioeconomic circumstances and can reduce health inequalities. They are sometimes more beneficial for people with poor socioeconomic circumstances.3,15,16 Such interventions are known as population strategies.17 Water fluoridation is an example of a population strategy16,17 that reduces dental health inequalities.15,18 Unfortunately, water fluoridation has not been established in Japan except on United States Army bases, even though it is recommended by Japanese Society for Oral Health19 and Ministry of Health, Labour and Welfare stated for technical support to fluoridation.20 Japanese government and advocate activities are conducted in several municipalities.Health intervention dissemination is often affected by socioeconomic circumstances.21 The Japanese government has published guidelines for fluoride (FMR) mouth-rinse.22 However, the proportion of schoolchildren receiving S-FMR differs among prefectures23 because some prefectures have not included S-FMR as part of the local public health policy, and the final decision to introduce S-FMR is made by each school’s administrators. S-FMR works as a “geographical targeting population approach,” a population approach for specific areas.24,25 Thus, it might partially contribute to children’s dental health improvement and decrease inequalities between prefectures in Japan.Some schools in Japan have adopted another population strategy for dental caries prevention, namely a school-based fluoride mouth-rinse (S-FMR) programs. S-FMR offers the possibility of decreasing dental caries inequalities among schoolchildren.21,26 In contrast, using fluoride toothpaste at home depends on individual household efforts and may thus be affected by social determinants.27 A recent Cochrane systematic review showed that simultaneously receiving FMR and using fluoride toothpaste is more effective than the use of fluoride toothpaste alone, but not to a significant degree.28 However, another review showed that the use of a topical fluoride application, including mouth-rinse, significantly reduced dental caries.29 Thus, the effectiveness of S-FMR is questionable in countries like Japan, where fluoride toothpaste is widely used.S-FMR changes dental caries prevention strategies at the school level. It improves schoolchildren’s dental health regardless of their socioeconomic status, and is more effective for schoolchildren with poor dental status.Here, we examined the effectiveness S-FMR on children’s dental caries experience and inequalities at the population level between prefectures in Japan, accounting for prefectural differences in socioeconomic circumstances, fluoride toothpaste utilization, and the recent decline in dental caries.30 Ecological study is appropriate when the prevention or intervention implications are at a population level.31 This study design is sometimes criticized for weak causal inference, since associations observed on a population level are not necessarily applicable on an individual level. However, this “ecological fallacy” can be avoided when causation has been shown in previous studies.32 Causal relationships between the use of fluoride mouth-rinse and caries prevention have been established in randomized controlled trials.29 Thus, we believe that the ecological study design has greater benefits than disadvantages for our research.To examine S-FMR effectiveness in decreasing children’s dental caries experience at a population level and dental caries inequalities between prefectures, we chose an ecological study design using each prefecture as one unit. Ecological studies are frequently used to investigate the spatial patterns of diseases and interventions.In addition, to consider recent declines in dental caries and high utilization of fluoride toothpaste in Japan, we used data from multi-year birth cohorts, aggregated data to the prefecture level, and employed multilevel analysis. We obtained the prefecture-level aggregated variables of all 47 prefectures in Japan relating to children born between 1994 and 2000 from open data from previous surveys conducted when the children were aged 3, 7, and 12 years. The variables were grouped according to the age of each birth cohort . Thus, t33 To consider dental caries experience before receiving S-FMR, mean numbers of decayed, missing, or filled primary teeth (dmft) of 3-year-old children were obtained from dental health checkups at local health centers, which are also required by law, conducted between 1997 and 2003.34 Dental caries experience of primary teeth could represent dental caries risk for permanent teeth in each prefecture because past caries experience is the best predictor of future dental caries onset in children.35Our outcome variable was the mean number of dental caries experienced by 12-year-old children, indicated by decayed, missing, or filled permanent teeth (DMFT), because it reflects the population benefit of S-FMR. These data were obtained from records of school dental health checkups, which are required by law, conducted between 2006 and 2012.36 Children aged 6–7 years are in the first grade of elementary school in Japan. Longer periods of S-FMR exposure are more beneficial to prevent dental caries.21 Because the proportion of S-FMR varied annually, we used the data between 2001 and 2007 for each birth cohort, which indicated the S-FMR proportion in each prefecture when children were aged 7 years. This survey was conducted biennially. Mean values of the results of 1 year prior or later were used for the years in which the survey was not conducted.The S-FMR variable was the proportion of children in the prefecture who received S-FMR, obtained from the National Survey on School-based Fluoride Mouth-rinse Programs in Japan.37 and average number of times any toothpaste was bought per capita in a year38; 2) dentist density (per 100 000 people)39; 3) average sugar consumption per capita in each prefecture38; and 4) mean annual income of each prefecture.40 The survey of dentist density was conducted biennially. Mean values of the results of one year prior or later were used for the years the survey was not conducted. We could not consider water fluoridation because it was not conducted in Japan. Since only aggregated open data were used, ethical approval was not needed.To consider other factors that possibly contribute to dental caries and caries-related inequalities between prefectures, we obtained the following variables from surveys conducted when children were 7 years old (between 2001 and 2007): 1) average consumption of fluoride toothpaste per capita in each prefecture, calculated by multiplying domestic utilization of fluoride toothpasteWe applied multivariable multilevel linear regression models to examine S-FMR’s contribution to decreasing dental caries experience and caries-related inequalities. For the current data set, every prefecture had seven birth cohorts. Therefore, the year was treated as level 1, nested within the prefecture as level 2. To determine the contribution of a 1% increment in the S-FMR coverage on dental caries and caries-related inequalities, S-FMR was treated as a continuous variable. Other variables except dentist density were also treated as continuous variables. Continuous variables were centered on the grand mean values. Dentist density was divided into quartile categories, as it was skewed.41 Then, the associations between S-FMR and DMFT at age 12 years in each situation were estimated. Model fitting was evaluated by Akaike’s information criterion (AIC) and the likelihood ratio test.We constructed five models, sequentially adding independent variables: a model to check whether there is a significant DMFT difference at 12 years old between prefectures with no explanation variable (model 1); a model to examine contributions of fluoride toothpaste dissemination, mean annual income, sugar consumption, and dentist density to the DMFT difference at 12 years old (model 2); a model to examine contribution of dental caries experience at 3 years old (model 3); a model to examine the contribution of S-FMR (model 4); and a model to examine whether S-FMR contributes to decreasing DMFT inequalities at 12 years old between prefectures, adding interaction terms of dental caries experience at 3 years old and S-FMR utilization (model 5). If model 5 shows that S-FMR is more effective in lowering DMFT for 12-year-olds in the prefectures with high dental caries experience at age 3, it indicates that S-FMR contributes to improving dental caries inequalities between prefectures. In addition, to estimate the modified S-FMR effects by dental caries experience at age 3, we assumed three situations: dental caries experience at age 3 was low (mean −1 standard deviation [SD]); middle (mean); or high (mean +1 SD).P-values for trends were calculated. Interaction terms were not examined in the sensitivity analysis.We also conducted sensitivity analysis because the S-FMR distribution was skewed. In the sensitivity analysis, we divided the S-FMR variable into deciles and treated it as a categorical variable. To evaluate the dose-response relationship of S-FMR on DMFT at age 12 years, All analyses were conducted using the Stata 13.1 software package .Table Table P < 0.001 for trend).Sensitivity analysis using S-FMR deciles showed that over the 40th percentile of S-FMR utilization showed significantly lower DMFT at age 12 years compared to that at under the 10th percentile of the S-FMR utilization group , the calculated number of DMFT decline was 14 458  in this hypothetical situation. Thus, S-FMR would have large benefits for population dental health. However, this estimated reduction in the number of caries needs careful interpretation because this is an ecological study and the effect of S-FMR differs by children’s risk factors and other characteristics, such as caries risk and fluoride utilization. S-FMR contributed to prefecture-level DMFT variance even in the present situation where fluoride toothpaste is widely used. This result was robust in our sensitivity analyses. The association between S-FMR and DMFT for 12-year-olds was modified by dental caries experience at age 3 years (dmft at 3 years old) in the prefectures. S-FMR had more beneficial associations with DMFT for 12-year-olds when mean numbers of dental caries experienced at age 3 years in the prefecture were high. Thus, S-FMR seemed to decrease inequalities in dental caries between prefectures.This study showed that each 1% increase in S-FMR utilization was significantly associated with 0.011 lower DMFT for 12-year-olds. In Japan, there were 1 314 403 children aged 12 years in 2000.43 However, the external validity of randomized trials may be limited in real-world settings. Participants who are included and followed in randomized intervention studies might differ from the general population.32,43,44 Additionally, according to the “inverse care law” concept first discussed by Hart JT in 1971, interventions that depend on individual behavior are more effective among low-risk than high-risk populations.13,14,45 Although randomized controlled trials showed little additional effects of fluoride mouth-rinse beyond fluoride toothpaste alone on dental caries prevention,29 our study showed a beneficial effect of S-FMR in a society where fluoride toothpaste is widely used. One possible reason for this finding is that the utilization of fluoride toothpaste among children is affected by their socioeconomic status.46 Utilization of fluoride toothpaste is likely low in children of low socioeconomic status.27 Unfortunately, such children tend to be at high risk for dental caries.18 In contrast, S-FMR is an intervention that changes school environment as a means of improving schoolchildren’s dental health. Therefore, its advantages are easily conferred to children of low socioeconomic status. This may partly account for the results of our study.Our study examined the effect of FMR under actual social conditions, rather than in an experimental or randomized interventional context. Randomization has many advantages for estimating intervention effects.13,47 Upstream interventions  change health determinants at the social, political, and life circumstance levels and decrease health inequalities.3,18,25,48,49 Downstream interventions  alter health determinants at the individual level and increase health inequalities because such interventions are often more effective for low-risk than high-risk individuals.13,43 The use of fluoride toothpaste at home is a downstream intervention, as it depends on individual health behavior and socioeconomic status. In some countries, toothpaste is considered cosmetic, and many families are unable to obtain it because of its high cost.3 In addition, an intervention that provided free fluoride toothpaste for children in England did not decrease dental caries inequalities related to deprivation.27 On the other hand, some upstream interventions have been shown to reduce dental health inequalities. Several reviews have indicated that caries inequalities across social classes have decreased in countries where water fluoridation has been implemented.18,48,50 Although water fluoridation is one of the most cost-effective public health strategies,3,15 it is unfortunately not conducted in Japan. Results of the present study showed geographical and socioeconomic inequalities in dental caries. Therefore, upstream interventions are also needed in Japan. S-FMR, an intervention that changes school environments to improve schoolchildren’s health, is a relatively upstream public health intervention conducted in Japan.Health interventions can be divided into two types: upstream interventions and downstream interventions.28 In contrast, usage of fluoride in prefectures with higher prevalence of caries is not prevalent; therefore, additional effects of S-FMR are greater and inequalities are reduced. Figure 21 Therefore, more widespread S-FMR implementation is recommended. The importance of “proportionate universalism” was proposed to reduce health inequalities. Proportionate universalism refers to interventions that improve the health of high risk groups more than that of low risk groups.51 S-FMR seems to work via proportionate universalism to reduce health inequalities.This study suggests that S-FMR as a population strategy could be effective for reducing dental caries and caries-related geographical inequalities, even in areas/countries where fluoride toothpaste is widely used and dental caries prevalence has been declining. Theoretically, it is possible that S-FMR is not randomly distributed because prefectures or schools with high dmft for 3-year-olds might be more likely to implement S-FMR. In such situations, S-FMR would work as a geographical targeting approach and contribute to reducing inequality in dental caries among prefectures. In this study, S-FMR was not significantly associated with dmft for 3-year-olds; prefectures with higher caries levels did not tend to conduct S-FMR. However, S-FMR reduced inequalities in DMFT for 12-year-olds; thus, it worked as a geographical targeting approach. The possible reason for reduction of inequalities by S-FMR is that children in prefectures with lower caries prevalence already tended to use fluoride; therefore, the effects of S-FMR on caries rates and inequalities are attenuated.31 However, we intended to investigate the population effect of S-FMR on dental health and its contribution to inequalities in dental caries between prefectures. Ecological studies are appropriate for implications of prevention or intervention implications at a population level.31 In fact, ecological studies are frequently used in geographical epidemiology.30 Additionally, the ecological fallacy can be avoided when individual-level causation has been shown in previous studies.32 Thus, ecological fallacy was less of a concern in the present study because the effect of fluoride on dental caries prevention has been demonstrated in many previous studies.50,52 Second, some schoolchildren might have received fissure sealant on their permanent teeth, but we did not have information on fissure sealant. However, we believe that this bias did not seriously affect the result because no fissure sealant programs were conducted at the prefecture level. Basically, Japanese children who receive fissure sealant in dental clinics do so under on individual basis, not at school. A significant association between S-FMR and fissure sealant coverage is therefore unlikely. Thus, it is improbable that the absence of data on fissure sealant resulted in overestimation of the effect of S-FMR in this study. Third, the S-FMR data in the present study included FMR utilization in preschool, kindergarten, elementary school, junior high school, and schools for special-needs education. In Japan, children in junior high school and some children in schools for special-needs education are 13 years or older. This could cause some bias because S-FMR for them would not have had causal effects on the outcome of the present study . However, S-FMR utilization excluding that in junior high schools and schools for special-needs education in each prefecture, which was available only for 2010,23 and S-FMR utilization in the prefecture were highly correlated . Therefore, the results of this study were reliable. Fourth, we obtained information on dental caries from mandatory dental health check-up surveys, in which many dentists in Japan examine children’s dental status. Inter-validity between the dentists in this survey has not been evaluated, and might have produced a degree of bias. However, the dentists employed universal caries diagnosis criteria. Nonetheless, some dentists may underestimate children’s dental caries, whereas others may overestimate it. Such “non-differential misclassification” bias would lead to underestimation of the results. We observed significant associations even with this potential bias, and therefore consider that this bias did not seriously affect our results. Finally, we followed birth cohorts at the prefecture level. Some individuals might have moved to other prefectures during the follow-up, which might have caused some bias. However, this bias would also be “non-differential misclassification” because moving to other prefectures would not be associated with S-FMR utilization or dental caries. Therefore, we believe the associations we observed in the present study are robust.The strength of this study was that we investigated the impact of S-FMR on population dental health and caries-related geographical inequalities using multilevel analyses of longitudinal data. We also considered other possible factors that could explain the geographical inequalities in dental caries. The present approach enabled us to examine the impact of S-FMR under actual social conditions. On the other hand, this study has several limitations. First, this was a prefecture-based ecological study. In general, ecological study designs do not allow causal inferences because an association observed between variables on an aggregated level does not necessarily represent the association at an individual level .In conclusion, this study showed that S-FMR was associated with low dental caries experience and decreasing caries-related inequalities between prefectures, even in a country where fluoride toothpaste is widely used and dental caries prevalence has been decreasing.eFigure 1. eTable 1. eTable 2. Abstract in Japanese.


Symptomatic intracranial hemorrhage (sICH) after intravenous thrombolysis with recombinant tissue-plasminogen activator (rt-PA) for acute ischemic stroke is associated with a poor functional outcome. We aimed to develop a score assessing risk of sICH including novel putative predictors—namely, pretreatment with statins and severe renal impairment.We analyzed our local cohort (Berlin) of patients receiving rt-PA for acute ischemic stroke between 2006 and 2016. Outcome was sICH according to ECASS-III criteria. A multiple regression model identified variables associated with sICH and receiver operating characteristics were calculated for the best discriminatory model for sICH. The model was validated in an independent thrombolysis cohort (Basel).sICH occurred in 53 (4.0%) of 1,336 patients in the derivation cohort. Age, baseline National Institutes of Health Stroke Scale, systolic blood pressure on admission, blood glucose on admission, and prior medication with medium- or high-dose statins were associated with sICH and included into the risk of intracranial hemorrhage score. The validation cohort included 983 patients of whom 33 (3.4%) had a sICH. c-Statistics for sICH was 0.72 (95% CI 0.66–0.79) in the derivation cohort and 0.69 (95% CI 0.60–0.77) in the independent validation cohort. Inclusion of severe renal impairment did not improve the score.We developed a simple score with fair discriminating capability to predict rt-PA-related sICH by adding prior statin use to known prognostic factors of sICH. This score may help clinicians to identify patients with higher risk of sICH requiring intensive monitoring. Symptomatic intracranial hemorrhage (sICH) occurs in 2–4% of patients receiving intravenous recombinant tissue-plasminogen activator (rt-PA) for acute ischemic stroke –3. sICH Therefore, the accurate identification of patients with higher risk of rt-PA-related hemorrhage is of high relevance. Several predictive instruments for estimated risk of sICH after thrombolytic therapy have been developed in recent years  –10. HoweFrom January 2006 to March 2016, all consecutive patients with acute ischemic stroke admitted to our tertiary care center  and undergoing intravenous thrombolysis were included in a prospective database. Our prospective database was approved by the ethics committee of the Charité—Universitätsmedizin Berlin . An ethics committee approval was not required for the current analysis, in accordance with laws and regulations in the Federal State of Berlin. Stroke severity on admission was assessed using the National Institutes of Health Stroke Scale (NIHSS) by a trained neurologist. Demographics, vascular risk factors (applying criteria used in prior research), admission blood pressure, glucose levels, creatinine, estimated glomerular filtration rate (eGFR), time from onset of stroke symptoms until treatment, previous medication including statin, and anti-platelet use were recorded. Simvastatin 40 mg and simvastatin 80 mg  were defined as medium- and high-dose statin use, respectively , 15, 16.For categorical variables, proportions were calculated by dividing the number of events by the total number of patients, excluding missing or unknown cases. For continuous variables, median and interquartile ranges (IQR) were calculated.2 test was used for comparison of categorical variables. Comparisons of continuous variables were performed using the Mann–Whitney U-test. All factors with a P-value ≤0.10 were included in a multiple backward stepwise logistic regression analysis in order to identify characteristics that where associated with sICH. Based on the β-coefficients of the final multiple binary logistic regression model, we calculated the area under ROC (c-statistics) and 95% CI of the score as a measure of discrimination of sICH. The resulting risk of sICH score (RICH score) was validated in an independent cohort of ischemic stroke patients treated with intravenous thrombolysis from the University Hospital Basel, Switzerland. Comparison with the previously published scores MSS  stroke unit with rt-PA and included in our prospective database. Patients with a diagnosis of a stroke mimic at discharge , additional intraarterial thrombolysis and/or thrombectomy , and patients with unknown stroke onset and onset-to-treatment time >270 min  were not included in our analysis. A total of 1,336 patients were eligible for analysis and score development . Table Symptomatic ICH according to ECASS-III-criteria occurred in 53 patients (4.0%). In multiple logistic regression analysis, older age, higher stroke severity measured by NIHSS upon admission, higher blood glucose upon admission, and treatment with medium or high-dose statins before the acute event were associated with sICH Table . SystoliThe score was then validated and compared with other scores in the thrombolysis registry of the University Hospital Basel, which included 983 patients of whom 33 (3.4%) had a sICH. Table For ease of use of the RICH score, we developed a risk calculator giving the probability of sICH after thrombolysis based on individual patient data . In addition, we developed a final ordinal score based on the results of the multiple logistic regression analysis to further facilitate the use of the score in the clinical setting. For this, we dichotomized continuous variables and assigned one point for each risk factor Table . Figure The RICH score is an easy and quick computable score for the risk of sICH in patients with an acute stroke treated with intravenous rt-PA. Its simplicity and fair discriminatory ability may help clinicians to identify patients with higher or low risk of sICH after rt-PA. All items of the score are easy and quick to obtain and do not require interpretation. While incorporating well-known risk factors of sICH such as age, stroke severity, and admission levels of blood glucose, our score differs from other published scores in considering pretreatment with medium or high-dose statins as additional risk factor. Furthermore, in contrast to many other scores, the RICH score does not include early signs of ischemic infarction on CT, which may facilitate its use.Several scores for the risk of sICH after rt-PA have been published in recent years –10. Mostvia different mechanisms. More specifically, a higher dose-dependent risk of cerebral hemorrhage after thrombolytic therapy may be attributable to antithrombotic and anticoagulative properties of statins  of the University Hospital Basel, the University Basel, the Swiss Heart Foundation, and the Swiss National Science Foundation. AR has received honoraria for lectures from Bayer and Ever Pharma.


Our aim was to evaluate clinically relevant long-term outcomes of transvaginal mesh or native tissue repair in women with recurrent pelvic organ prolapse (POP).We performed a 7-year follow-up of a randomized controlled trial on trocar-guided mesh placement or native tissue repair in women with recurrent POP. Primary outcome was composite success, defined as absence of POP beyond the hymen, absence of bulge symptoms, and absence of retreatment for POP. Secondary outcomes were adverse events, pain, and dyspareunia. Multiple imputation was used for missing data of composite success and pain; estimates are presented with 95% confidence intervals (CI).Between August 2006 and July 2008, 194 women were randomized; 190 underwent surgery. At 7 years, 142 (75%) were available for analysis, of whom, the primary outcome could be calculated in 127. Composite success was 53%  for mesh and 54%  for native tissue. Repeat surgery for POP was 25% for mesh and 16% for native tissue  and occurred in untreated compartments in the mesh group and treated compartments in the native tissue group. Mesh exposure rate was 42%; pain with mesh 39% and native tissue 50% ; dyspareunia with mesh 20% and native tissue 17% .Seven-year composite success rates appeared similar for mesh and native tissue. Mesh did not reduce long-term repeat surgery rates due to de novo POP in nonmesh-treated vaginal compartments. Mesh exposure rates were high, though significant differences in pain and dyspareunia were not detected.Clinical trial registration. ClinicalTrials.gov, NCT00372190. The 10-year rate of repeat surgery for pelvic organ prolapse (POP) and urinary incontinence (UI) is as high as 17% –4. The cA limited number of randomized clinical trials (RCT) compared short-term safety and efficacy of nonabsorbable mesh with native tissue repair . Long-teThe study reported focused on the long-term follow-up of that RCT, with the primary aim being comparison of long-term composite success  7 years after surgery. The secondary aim was to describe long-term adverse events, with particular focus on pain and dyspareunia.www.clinicaltrials.gov, NCT00372190). Patients with recurrent POP were 1:1 randomized between a first-generation nonabsorbable TVM   and conventional vaginal native tissue repair. Repairs comprised anterior or posterior colporrhaphies; a Manchester Fothergill procedure; or vaginal hysterectomy with high uterosacral ligament suspension, sacrospinous ligament suspension, or a combination. Details regarding design, randomization, sample size, surgical interventions, and 1-year outcomes have previously been published [clinicalTrials.gov, NCT00372190. Informed consent was obtained prior to inclusion in the extended follow-up.The original trial was performed in 13 centers in The Netherlands between August 2006 and July 2008 , a subspecialist in urogynecology, was blinded to the index procedures and performed all interviews and physical examinations at the original study sites. Participants completed the same validated urogynecologic questionnaires as were used at baseline and 1 year in the index study: Global Impression of Improvement questionnaire (PGI-I), visual analog scale (VAS), EuroQol-5D (EQ-5D), Urogenital Distress Inventory (UDI), Defecatory Distress Inventory (DDI), Incontinence Impact Questionnaire (IIQ), and the short form of the Pelvic Organ Prolapse Urinary Incontinence Sexual Questionnaire PISQ-12) –12. The  –12. TheSubjective prolapse symptoms were considered present if a patient answered affirmatively to the question: Do you see or do you feel a vaginal bulge? Pain was considered present if a patient answered affirmatively to the question: Do you experience pain in the lower abdomen or genital region? Dyspareunia was considered present if a patient answered affirmatively to the question: Do you experience pain during intercourse? The degree of bother caused by dyspareunia was registered using a 4-point Likert scale . SUI was considered present if a patient answered affirmatively to the question: Do you experience involuntary urine loss during physical exercise, coughing, or sneezing? De novo SUI was considered present if the participant had no SUI according the baseline questionnaire but answered affirmatively at 7 years or had received treatment for SUI after the trial’s index surgery within the 7-year follow-up.Pain was assessed with a 10-point VAS, where 0 denoted no pain and 10 the worst imaginable. Prior to physical examination, women were requested to undergo two VAS for pain: one at rest and one during physical activity.Physical examination consisted of POP staging using the Pelvic Organ Prolapse Quantification (POP-Q) system . A thoroIn contrast with the primary outcome at 1 year, defined as an anatomic failure in treated vaginal compartments, the primary outcome of the study reported here was composite success, defined as a combination of absence of POP beyond the hymen in a treated or nontreated compartment, absence of bulge symptoms, and absence of retreatment for POP in a treated or nontreated compartment . If no PSecondary outcomes were anatomic recurrences overall and failures per treated vaginal compartment at 7 years, defined as POP stage ≥ II, POP > hymen, de novo SUI, surgery-related complications for mesh, pain perception, and dyspareunia. Descriptive statistics with numbers, percentages, and risk differences with 95% CI were used to summarize baseline characteristics and primary and secondary outcomes. The 95% CI for differences between median values was estimated with the Hodges–Lehmann estimator .To account for missing data of women who were alive but did not attend the 7-year follow-up visit, a multiple imputation approach was used for composite success and UDI domain of pain. Analyses were performed using the combination of 50 data sets generated with the fully conditional method (chained equations) for imputation of missing values. Values imputed for pain <0 or >100 were truncated to 0 or 100, and composite success was imputed as a binary variable. Missing data of women who died during the 7-year follow-up were not imputed in the main analysis, but were imputed as failures in a sensitivity analysis of composite success. After multiple imputation, composite success was analyzed using a binomial distribution, an identity link, and covariate treatment group. UDI domain pain was analyzed using a linear model, with covariate treatment group and pain at baseline.Analysis was according the intention-to-treat principle. Statistical analyses were performed using SPSS Statistics for Windows, version 22.0 and analyses of pain and composite success with the STAT package .One hundred and ninety-four women were randomized in the index trial between August 2006 and July 2008, of whom 190 underwent surgical treatment. In this 7-year follow-up, 142 (75%) women were available for analysis  , systematic long-term follow-up, physical exam by one independent observer (blinded to the index procedures), and the high response rate. Furthermore, the extensive assessment of pain and dyspareunia with data at both baseline and 7 years after index surgery is unique. The 2006 American College of Obstetricians and Gynecologists Committee Opinion on Ethical Guidelines in Innovative Practice state that: “without adequate data on risks and benefits of new treatments, patients are unable to provide truly informed consent” . Our lonFindings in this study strengthen the necessity of long-term observations on safety and efficacy when surgical devices are used in POP surgery. In every woman with recurrent POP, a native tissue repair can be reconsidered, preferably based on a prediction model . Only if