Reading typically involves phonological mediation, especially for transparent orthographies with a regular letter to sound correspondence. In this study we ask whether phonological coding is a necessary part of the reading process by examining prelingually deaf individuals who are skilled readers of Spanish. We conducted two EEG experiments exploiting the pseudohomophone effect, in which nonwords that sound like words elicit phonological encoding during reading. The first, a semantic categorization task with masked priming, resulted in modulation of the N250 by pseudohomophone primes in hearing but not in deaf readers. The second, a lexical decision task, confirmed the pattern: hearing readers had increased errors and an attenuated N400 response for pseudohomophones compared to control pseudowords, whereas deaf readers did not treat pseudohomophones any differently from pseudowords, either behaviourally or in the ERP response. These results offer converging evidence that skilled deaf readers do not rely on phonological coding during visual word recognition. Furthermore, the finding demonstrates that reading can take place in the absence of phonological activation, and we speculate about the alternative mechanisms that allow these deaf individuals to read competently. Knowing how to read is closely tied to our knowledge of the language we are reading. The written form is a visual representation of auditory words, and learning to read an alphabetic script typically involves associating letters with sounds. During reading acquisition, phonological processes are the first to develop5 and lexical access involves phonological mediation8; reading deficits, such as dyslexia, are associated with poor phonological awareness9. The extent to which phonological representations play a role in reading depends on reading proficiency but also on the transparency of the writing system12. More proficient readers make greater use of strategies that bypass phonology by recognizing orthographic units or even whole words14. Transparent orthographies, in which there is a regular correspondence between graphemes and phonemes, favour phonological coding during word recognition15 and may impact the acquisition of syntactic rules16. In contrast, in opaque orthographies, readers can process printed words by using alternative strategies, such as relying on the visual-orthographic structure. In a language with a transparent orthography, like Spanish, phonological codes are automatically accessed during reading18. In this study, we ask whether phonological processing is required for word identification in a transparent orthography. To do this, we examine a population that reads without access to the phonological forms of words, namely, deaf individuals.Learning to read is a transformative experience for socio-economic mobility and for our cognitive development19 but in the case of deaf readers, their lack of audition severely limits their access to phonology. Consequently, the connection between the written word and the sound-based form that it represents has led various authors and educators to highlight the importance of phonological coding and awareness for reading skills in this population22. Many studies that look at the role of phonology in deaf readers do so through meta-phonological tasks and/or phonological awareness tasks that require explicit phonological judgments, such as rhyme detection29. Since our aim is to determine whether phonology is automatically activated in word reading, we wish to avoid task-driven effects that might activate phonological representations: we focus on implicit reading tasks that do not involve making decisions about the phonological form of the word.A variety of factors, including visuo-perceptual processing and reading experience, impact reading outcomes30. Reading ability in English of primary school children has been linked to language ability but not phonemic awareness31. A major study of over 200 secondary school deaf readers (aged between 11 and 16 years old) of four different languages (with transparent and opaque orthographies) found that syntactic rather than phonological knowledge characterized skilled readers from those less skilled16. In a meta-analysis of 57 studies looking at reading in deaf individuals, phonological awareness predicted only 11% of the variance in reading ability; in contrast, overall language ability (either spoken or signed) predicted up to 35%32. Further support comes from specific studies that have found no behavioural evidence of phonological encoding in deaf readers33. Skilled deaf readers of French activated visual, orthographic and semantic codes during reading, but not phonological codes34. In an eye tracking study of the phonological and orthographic preview benefit in the parafovea in readers of English, both hearing and deaf readers benefited from orthographic coding in parafoveal vision but only the hearing group benefited from phonological coding35.Despite the evidence of phonological encoding from studies with explicit tasks, phonology seems to be much less important for deaf readers36 has been conducted in languages with opaque orthographies, particularly English. Since orthographic depth can modulate the role of phonological coding during reading12, the absence of phonological coding in deaf readers of languages like English31 or Hebrew16 may be due to the opaque orthography. With a transparent orthography, the properties of the orthographic representation lead us to expect the engagement of phonological encoding: a study of deaf readers of Spanish found evidence of phonological coding37. Critically, the deaf readers in that study had a broad range of reading abilities. In this study we focus on the minority of deaf readers who have good reading skills. For those deaf individuals who read a transparent orthography well, what role does phonology play?Much of the previous work on deaf readers41. Our goal is to discover whether these proficient deaf readers activate phonological representations and to what extent they differ from hearing readers. More broadly, we inquire how central phonology is for proficient reading in a language with a transparent orthography.The present study investigates the involvement of phonological processes during visual word recognition in deaf skilled readers of a transparent language (Spanish) using tasks that do not explicitly require phonological analysis  while measuring behavioural responses and electrophysiological activity (EEG), and to compare the group with hearing readers. EEG provides sufficient temporal precision to capture the time course of sub-lexical processes . An important feature of this study is that deaf participants are skilled readers with reading levels comparable to hearing readers of the same age. We carried out two experiments, each looking at different aspects of the word recognition process: the first focuses on sub-lexical processing during word recognition to look for evidence of phonological activation before lexical access takes place; the second experiment examines lexical analysis and the possible role of phonological coding during lexical access. We exploit the pseudohomophone effect, based on pseudowords that sound like real words, one of the strongest indicators of phonological processing in visual word recognition42. Compared with control pseudoword primes, pseudohomophone primes modulate the N250 component43 and we expect this effect for the hearing readers. If deaf readers activate phonological codes, we expect a comparable modulation; conversely, if they do not activate phonological codes the response should be similar for both types of prime (pseudohomophones and control pseudowords).In Experiment 1 we tested the role of phonological processing with a go/no-go semantic categorization task  with masked priming. In the critical, no-go trials, primes were pseudowords and pseudohomophones of the target word. Masked priming has revealed early phonological effects in visual word recognition: the N250 component, with a broad scalp distribution, reflects sub-lexical processing that involves associating orthographic and phonological representations, and takes place before lexical access has occurredIn the semantic categorization task all participants showed high levels of accuracy, pressing the button response when the animal names were presented as target . When the animal names were presented as prime (for just 50 ms), the response rate was close to zero  indicating that the primes were successfully masked. There were only few false alarms in the experimental trials  suggesting that participants paid attention to the content of the stimuli.As planned, we analysed the EEG signal of no-go trials in an early time window to examine the effect of Group , PrimeType  and topographic factors (see Methods) on the N250 component. Additionally, visual inspection of the grand average waveform revealed a divergence between prime types later on see Fig. ; analysiF = 4.10, p = 0.049, ŋ2 = 0.097; see Fig. t(19) = 2.55, p = 0.039). The two conditions did not differ in the deaf group (t(19) = 0.79, p = 0.439). No other significant effect was found  = 0.14, p = 0.712, ŋ2 = 0.004; PrimeType: F = 0.89, p = 0.351, ŋ2 = 0.023).There was a significant interaction Group × PrimeType  = 10.05, p = 0.003; F2 = 30.37, p < 0.001, ŋ2 = 0.209; see Table F1 = 21.70, p < 0.001; F2 = 16.00, p < 0.001, ŋ2 = 0.363), indicating that participants made the highest number of errors with pseudohomophones as compared to the other two types of stimuli (words: t(117) = 4.46, p < 0.001; pseudowords: t(117) = 4.36, p < 0.001). The interaction Group × WordType  = 15.24, p < 0.001; F2 = 24.22, p < 0.001, ŋ2 = 0.286) showed that this difference was present in the hearing group (pseudohomophones vs. words: t(114) = 6.57, p < 0.001; pseudohomophones vs. pseudowords: t(114) = 6.50, p < 0.001), but not in the deaf group (pseudohomophones vs. words: t(114) = 0.62, p = 0.950; pseudohomophones vs. pseudowords: t(114) = 0.53, p = 0.950).The deaf group was more accurate than the hearing group  = 10.39, p = 0.003; F2 = 424.59, p < 0.001, ŋ2 = 0.215; see Table F1 = 93.22, p < 0.001; F2 = 90.53, p < 0.001, ŋ2 = 0.710) indicated that responses to the words were faster as compared to the two types of nonwords (pseudohomophones: t(117) = 4.08, p < 0.001; pseudowords: t(117) = 3.34, p = 0.002). Importantly, this effect interacted with Group  = 6.57, p = 0.014; F2 = 8.03, p = 0.023, ŋ2 = 0.147), suggesting that the difference between pseudohomophones and words was statistically more evident in the hearing group as compared to the deaf group (hearing: t(114) = 3.99, p < 0.001; deaf: t(114) = 2.42, p = 0.024), while the difference between pseudowords and words was similarly present in both groups (hearing: t(114) = 2.63, p = 0.018; deaf: t(114) = 2.62, p = 0.018). These behavioural results were confirmed when by-item and by-participant variability were accounted for by mixed linear models analyses  = 18.31, p < 0.001, ŋ2 = 0.325, see Fig. t(39) = 2.88, p = 0.007; pseudohomophones vs. words: t(39) = 3.44, p = 0.003; pseudowords vs. words: t(39) = 6.29, p < 0.001). The interactions between WordType and the topographical factors  = 9.89, p < 0.001, ŋ2 = 0.206; WordType × Laterality: F = 3.62, p = 0.010, ŋ2 = 0.087; WordType × Anteriority × Laterality: F = 2.10, p = 0.057, ŋ2 = 0.052) indicated that these differences were more evident over central right electrodes , which is compatible with the N400 distribution. In addition, the factor WordType interacted with Group  = 3.47, p = 0.036, ŋ2 = 0.084; Group × WordType × Anteriority: F = 3.10, p = 0.037, ŋ2 = 0.075), indicating different N400 effects for deaf and hearing participants. While in the hearing group only pseudowords elicited a greater N400 as compared to words and pseudohomophones (pseudowords vs. words: t(19) = 3.93, p = 0.003; pseudowords vs. pseudohomophones: t(19) = 3.96, p = 0.003; pseudohomophones vs. words: t(19) = 0.84, p = 0.413), in the deaf group both pseudowords and pseudohomophones elicited a greater N400 as compared to words (pseudowords vs. words: t(19) = 5.09, p < 0.001; pseudohomophones vs. words: t(19) = 4.12, p = 0.003; pseudowords vs. pseudohomophones: t(19) = 1.09, p = 0.413). These differences were more evident over central and posterior sites . Similar ERP results were obtained using the same amount of items for each level of the factor WordType  or in the N400 response for pseudohomophones and control pseudowords. These deaf readers treated pseudohomophones as pseudowords; the fact that they sound like real words created no interference. We take this as evidence that these skilled deaf readers do not need to activate phonological coding during word reading. Even so, phonological coding might occur but not be apparent . Two pieces of evidence suggest that this is not the case. Firstly, in the semantic categorization task, pseudohomophones did not modulate deaf readers’ N250, suggesting that even at the sub-lexical level phonological coding is not activated. Secondly, in the lexical decision task, deaf readers responded significantly faster than hearing readers across all conditions. Previous studies have reported faster reaction times in word reading tasks for deaf compared to hearing readers19. This finding casts doubt on the assumption that deaf individuals need to access phonology in reading tasks to be competent readers21. Although some studies show a correlation between phonology and reading skills in deaf individuals56, our finding adds neuroimaging evidence to the body of studies that question the importance of phonological coding for proficient reading in the prelingually deaf59. Central to this issue is the type of deaf reader: this study has focused on highly skilled deaf readers, that is, individuals who have overcome the condition of deafness to acquire competent reading skills. Other deaf readers may have different processing mechanisms; a previous study with deaf readers of Spanish who were not highly skilled did find evidence of phonological encoding37.These results provide converging behavioural and electrophysiological evidence that these skilled deaf readers read words without activating phonology, as has already been shown behaviourally for other languages with transparent orthographies, such as German or Turkish14. The evidence for phonological coding in deaf children learning to read is mixed61 and more work is required to understand what phonology does and does not contribute to reading for different deaf individuals, but growing evidence suggests that the difference may lie at the sentence or discourse level rather than the lexical level19. This caveat limits the implications of this study for educational practice. Explicit training of phonological awareness may not help deaf individuals to achieve high reading proficiency31. Deaf readers do not need phonology during automatic stages of reading processing, suggesting that these readers avail of alternative strategies. One possibility is a visual, purely orthographic representation, supported by neuroimaging evidence that deaf readers have finely tuned orthographic representations but weak phonological sensitivity in the reading network62; another is mediation of the written word through the sub-lexical units of sign language63. Future studies should investigate how these processes support skilled deaf readers in acquiring a high level of literacy and how these mechanisms could be exploited and fostered to improve reading skills in deaf students.This leads to an important caveat for our finding in terms of what it tells us about deaf readers: our electrophysiological data support the claim that highly skilled deaf readers do not need to—and in fact do not—automatically activate phonological encoding. We have examined the end state of the process of acquiring reading but make no claims about the process of learning to read. For hearing learning readers, phonology seems to play an important role18, phonological mediation is not necessarily an automatic part of reading a language with a transparent orthography. Skilled deaf readers in the current study did not activate phonological codes, showing that phonological access is not necessary for competent reading in Spanish. Even though phonological knowledge may facilitate learning to read a transparent orthography, the present results demonstrate that this does not mean that phonology is required during skilled reading.To conclude, our findings tell us something important about reading in general. Contrary to previous claims64) and the two groups did not differ in non-verbal intelligence, Spanish reading comprehension, and Spanish vocabulary size (see Table Twenty adult severely (70–90 dB) to profoundly (> 90 dB) deaf adults  participated in the study. All participants were born deaf or lost their hearing before the age of 3 ; none had cochlear implants. They learned Spanish Sign Language before the age of 10 and used it as the main language for communication. Most of them learned to read at an early age, at school, except two individuals who learned to read after the age of 16. Twenty hearing Spanish readers  were included as a control group. All deaf participants and hearing controls were skilled readers in Spanish  pseudohomophones (40 items), which were created by replacing one letter of the target word by another letter that corresponded to the same phoneme , and (2) pseudowords (40 items), in which one letter of the target was replaced by another letter that corresponded to a different sound . Two lists were constructed such that each target word used to generate the nonwords was presented once in each list, either as a pseudohomophone or as a pseudoword.The experimental targets were 80 Spanish words  selected from the EsPal databaseIn addition, we selected an additional 20 Spanish words (4–6 letters long) corresponding to animal names , each of which was paired with a Spanish word with the same number of letters . Each word pair was not semantically related and was presented twice, once with the animal name as target and once with the animal name as prime. This was done to control for prime visibility.Participants were randomly assigned to one of the two lists. In total, each participant completed 120 trials: 80 experimental trials with nonword-prime/word-target pairs (40 pseudohomophone primes and 40 pseudoword primes), 20 trials with word-prime/animal-target pairs, and 20 trials with animal-prime/word-target pairs.The experiment was run in a silent and dimly lit room using Presentation software (version 0.70). Each trial began with a fixation cross for 500 ms, followed by a forward mask consisting of a row of hash marks (#####) for 500 ms (the number of hash marks matched the length of the prime). Then, a prime appeared in lowercase (25-pt. Courier New) and remained on the screen for 50 ms. The prime was followed immediately by the target stimulus, also in lowercase. Stimuli subtended a visual angle of 1.9 to 2.9 degrees depending on the number of letters. The target remained on the screen for 1500 ms or until the participant’s response see Fig. . ParticiF = 0.17, p = 0.68). The remaining clean epochs were averaged across conditions, with a baseline correction between – 200 and 0 ms.The electroencephalogram (EEG) was recorded with a 32-channel BrainAmp system (Brain Products GmbH) at a 500 Hz sampling rate. Scalp voltages were collected from 27 Ag/AgCl electrodes placed in an EasyCap recording cap . Two external electrodes were placed on the mastoids, with the right one as the online reference. An additional electrode at FCz served as ground, and four electrodes  recorded the electro-oculogram (EOG). Impedance was kept below 5KΩ for mastoids and scalp electrodes, and below 10KΩ for EOG electrodes. The EEG signal was re-referenced offline to the average activity of the two mastoids. A notch filter and a bandpass filter were applied . Ocular artifacts were corrected using Independent Component Analysis (ICA): the EEG of each subject was decomposed into independent components and those that explained the highest variance of EOG channels were identified and visually inspected before being removed from the original signal. Only trials corresponding to the experimental items were included in the analysis: all animal go trials and experimental trials with incorrect go-responses were excluded. For the remaining trials, EEG epochs of 1200 ms time-locked to the target presentation onset were obtained (including 200 ms pre-stimulus baseline). Residual artifacts exceeding ± 100 µV in amplitude were removed. Overall, 8.56% of trials were rejected, with no differences across experimental conditions  was applied to the N250 time window, which was defined based on visual inspection (230–270 ms). An additional late time window was added to the analysis after visually inspecting the ERP grand averages (500–700 ms). The average activity of three contiguous electrodes was calculated resulting in nine clusters: Left Anterior , Left Central , Left Posterior , Medial Anterior , Medial Central , Medial Posterior , Right Anterior , Right Central , Right Posterior . To probe the scalp distribution of any effects, these clusters were included in the statistical analyses as different levels of two topographical factors: Anteriority  and Laterality . Each ANOVA included the within-subject factor of PrimeType , the between-subject factor of Group , and the two topographical factors . The Greenhouse–Geisser procedure was applied when the sphericity assumption was violated. Effects of topographical factors will be reported only when they interact with the experimental factors. Two-tailed t-tests were conducted as follow-up analyses and corrected for multiple comparisons67.We analysed the data using RThe participants were the same as those in Experiment 1.65. For nonword trials, 80 Spanish base words between four and six letters long (mean number of letters 5.29) with a similar frequency to the first set were also selected . These words were then used to create two types of nonwords: (1) pseudohomophones by replacing one letter by another letter that corresponded to the same phoneme , and (2) pseudowords in which one letter was replaced by another letter that corresponded to a different sound . Two lists were constructed such that each base word used to generate the nonwords was presented once in each list, either as a pseudohomophone or as a pseudoword. Participants were randomly assigned to the two lists. In total, each participant completed 160 trials: 80 trials with words and 80 trials with nonwords (40 pseudohomophones and 40 pseudowords).For word trials, we selected 80 Spanish words between four and six letters long  from the EsPal databaseThe experiment was run in a silent and dimly lit room using Presentation software (version 0.70). Each trial began with a fixation cross at the centre of the screen for 500 ms, followed by a blank screen for 200 ms. Then, a stimulus (word or nonword) was presented in lowercase font  for 1500 ms or until the participant’s response . Trials with incorrect responses (5.94%) were excluded from RT analysis. RTs that were more than 2.5 SDs away from the mean of each condition and each participant were also excluded from the analysis of the response latencies (2.28%). A by-subject repeated-measures ANOVA was conducted on the error rates and the RTs including Group  as a between-subject factor, and WordType  as a within subject factor. Similar ANOVAs were conducted by item, including Group as a within-item factor and WordType as a between-item factor.We analysed the data using Rt-tests were conducted as follow-up analyses and corrected for multiple comparisons67.For the EEG data, a repeated-measures ANOVA was conducted on the average ERP amplitude within the N400 time window (300–500 ms). This analysis included Group  as a between-subject factor and WordType  as a within-subject factor. As in Experiment 1, two topographical factors were also added . The Greenhouse–Geisser procedure was applied when the sphericity assumption was violated. Effects of topographical factors will be reported only when they interact with the experimental factors. In both behavioural and EEG analyses, two-tailed Supplementary Information.


Thymoma‐associated myasthenia gravis (TAMG) is one of the subtypes of myasthenia gravis with autoantibodies against the acetylcholine receptor (AChR‐Ab). We analyzed the clinical features of our cohort of TAMG patients and the changes in AChR‐Ab titer before and after thymectomy in order to identify factors predicting thymoma relapses.We retrospectively assessed: age of MG onset, MG clinical status according to MGFA (Myasthenia Gravis Foundation of America), epoch of thymectomy, post‐thymectomy status, oncological features and surgical approach. AChR‐Ab dosages were measured both before and after thymectomy. Linear regression models were applied to identify clinical determinants of AChR‐Ab titers and the Cox regression model was fitted to estimate the factors associated with the risk of thymoma recurrence.P < 0.001); the decrease was more pronounced in female patients (P = 0.05), in patients diagnosed with MG at an older age (P = 0.003), and in those who had lower MG stage before surgery (P = 0.02) or higher Masaoka‐Koga stage (P = 0.005). The risk of relapse was closely linked with the age of the patient, the Masaoka‐Koga stage and the surgical approach.The study sample included 239 MG patients, 27 of whom experienced one or more recurrences (median follow‐up time: 4.8 years). The AChR‐Ab titers decreased after first thymectomy  titer before and after thymectomy and the clinical features of thymoma‐associated myasthenia gravis (TAMG) patients in order to identify clinical and serological predictors of thymoma recurrence. Our study emphasizes the importance of a multidisciplinary approach among Neurologists, Oncologists and Thoracic Surgeons, and identifies subgroups of MG patients with high risk of thymoma recurrence who therefore need a more accurate follow‐up. Myasthenia gravis (MG) is the most frequent neurological disease associated with thymoma, constituting a real subtype of disease named TAMG (thymoma‐associated myasthenia gravis).Thymoma is a disease with malignant potential with a recurrence rate ranging from 5% up to 17% after complete resection.et al. analyzed AChR‐Ab titers in both thymomatous and nonthymomatous patients and they found a significant decrease in AChR‐Ab titer after thymectomy in nonthymomatous MG but not in patients with thymoma, suggesting that the pathogenic role of the thymus differs according to pathology.So far, to our knowledge, no other studies have been conducted to investigate the changes in AChR‐Ab titers before and after thymectomy in a large cohort of TAMG patients. Kim The aim of our study was to investigate the association of AChR‐Ab titers with the clinical characteristics of our cohort of TAMG patients and to evaluate their role in predicting thymoma recurrence.A retrospective analysis of a prospectively maintained database was conducted for all patients undergoing thymectomy for thymoma between January 1987 and December 2016 and regularly followed in the Clinic of Myasthenia Gravis and Thymus‐related disorders of the Department of Clinical and Experimental Medicine, Neurology Unit, and Division of Thoracic Surgery (Pisa). MG diagnosis was based on characteristic signs and symptoms of MG together with the anti‐AChR antibody positive test; patients with an anti‐AChR antibody negative test were excluded from the present study.Surgical approaches were sternotomy, robotic‐assisted VATS, cervicotomy and thoracotomy. Patients who did not undergo a radical tumor excision according to surgical reports were excluded.Patients were regularly followed up by the oncologist (A.C.) together with the neurologists, at intervals of three to four months for the first two years postoperatively, every six to eight months for the following three years and, then, annually or whenever it was required by the deterioration of the symptoms. A follow‐up CT scan was performed every year in the first five years after surgery and every two years thereafter to detect any recurrence of thymoma.Age of MG diagnosis, MG clinical status, age of the patient at the time of thymectomy, time between age at MG onset and thymectomy, surgical approach and oncological features were retrospectively assessed from the medical records of all the patients.MG clinical status was assessed before and one year following thymectomy according to MGFA classification.AChR‐Ab serum titers had been measured before and immediately after surgery by radio‐immune assay method (RIA).Each patient gave an informed written consent for inclusion in the study that was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of the Pisa University Hospital .The associations between the serological levels of AChR‐Ab and the clinical variables were assessed with linear regression models with the antibody concentrations before surgery  or the differences after versus before surgery as dependent variables. The estimated marginal means , only the former was included in the multiple regression models.Concerning surgery, 50 patients underwent thymectomy in other hospitals before our clinical evaluation. Due to the strong association between Masaoka‐Koga stage and the WHO classification of thymoma (P = 0.006); neither sex nor age at MG diagnosis nor Masaoka‐Koga stage were significantly associated with AChR‐Ab levels before surgery (not shown).The levels of AChR‐Ab before surgery were significantly higher in individuals with worse MGFA stage (P < 0.001). The reduction of the AChR‐Ab levels after surgery adjusted for before surgery levels are shown in Table P = 0.05), in cases diagnosed at older ages , in cases with lower MGFA stage (P = 0.02) and higher Masaoka‐Koga stage (P = 0.005) : the estimates were 0.96 (95% CI: 0.91–1.00), 0.94 (95% CI: 0.88–1.00) and 0.61 (95% CI: 0.45–0.82) at five years, and 0.96 (95% CI: 0.9–1.00), 0.83 (95% CI: 0.72–0.94) and 0.41 (95% CI: 0.25–0.67)) at 10 years for the Masaoka‐Koga stages I/IIa, IIb and III/IV, respectively , 27 relapsed. All first recurrences were pleural, except one paravertebral and one lung; moreover, four patients presented a recurrence both in pleural and diaphragmatic sites. Overall, the recurrence‐free survival probabilities at five and 10 years after thymectomy were 0.89, with 95% confidence interval (CI) 0.83–0.94, and 0.78 (95% CI: 0.71–0.86), respectively. The recurrence‐free survival probability was heterogeneous for Masaoka‐Koga stage (P = 0.02) and worse Masaoka‐Koga stage . Moreover, patients who underwent sternotomy or robotic thymectomy VATS experienced fewer recurrences than others . The relevant reduction of the antibody titer after surgery emphasizes the role of thymectomy as “disease modifying” treatment in MG with AChR‐Ab emphasizing that in patients with thymoma surgical radical treatment plays the same role as in patients with nonthymomatous MG.Our data highlight a significant decrease of AChR‐Ab levels after intervention .Presurgery levels of AChR‐Ab or their change after surgery were not associated with thymoma recurrence, although further studies with a larger sample size are warranted. So far, no clinical and serological biomarkers have been established to identify patients with a higher risk of tumor recurrence. Buckley P < 0.001 = 0.02). Recently, Tian et al. also found that older patients had a lower risk of recurrence (P = 0.009).In our cohort of patients, we found that the risk of thymoma recurrence was higher in patients with early‐onset MG (P = 0.006) in accordance with literature data that show a direct correlation between antibody titer and MG severity.P = 0.02). We hypothesized that this subclass of thymoma patients could benefit from thymectomy more than others even if larger studies are needed to confirm this speculation.Antibody titers before thymectomy were significantly correlated with the severity of MG  before thymectomy experienced less MG exacerbations than others, within one year after thymectomy. This could be attributed to the beneficial effect on myasthenia gravis of higher doses of steroids administered in these patients in preparation for surgery to control myasthenic symptomatology. Furthermore, the literature data show that presurgical steroid treatment enhances the effectiveness of thymectomyOur data suggest that the recurrence‐free survival probability at 10 years is around 0.78 overall and 0.96, 0.83 and 0.41 in Masaoka‐Koga stages I/IIa, IIb and III/IV, respectively.In our cohort of TAMG patients, 27 (11%) experienced one or more recurrences with a median follow‐up time after the first thymectomy of 4.8 years. These results are comparable with the study of the Japanese Association for Chest Surgery.P = 0.007) while WHO histological type was evaluated only in the univariate model (P = 0.04) because of its tight relationship with Masaoka classification.In accordance with the literature data,P = 0.002). Recent reports indicated that for early‐stage thymoma there is not a significant difference between minimally invasive surgery and traditional open operation for experienced surgeons.In our cohort, sternotomy and robotic thymectomy VATS were associated with a lower risk of recurrence than thoracotomy and cervicotomy  in patients with an aggressive histologic thymoma.The authors have no conflicts of interest to declare.


In vitro intervention strategies can change the status of ERS and induce the inhibition of ERS‐related pathway. Therefore, this study is designed to explore the role and molecular mechanism of cartilage stem cells (ACSCs) of ERS in chondrocytes after hip replacement.Endoplasmic reticulum stress (ERS) is present in chondrocytes of osteoarthritis, and the intensity of ERS is related to the degree of cartilage degeneration. Human cartilage cell lines C28/I2 were cultured as the control group. The ERS inducer was added into C28/I2 as ERS group. The third ERS + stem cells group was formed by adding cartilage stem cells into ERS group, and further transfection of si‐PERK was defined as si‐PERK + ERS + stem cells group. Cell cycle and apoptosis in the four groups were determined by flow cytometry. The protein expression of GRP78, PERK, ATF4, TMEM119, CDK4, Cyclin D, and BMP6 in chondrocytes in the four groups were investigated by western blot, and the distribution of PERK, TMEM119, and BMP6 in chondrocytes were observed by immunofluorescence assay. In addition, the transcriptional levels of Bcl2, Bax, and Caspase 3 were also determined by RT‐PCR.0/G1 and G2/M, while cartilage stem cells weakened the effects. The apoptosis rates in control group, ERS, ERS + stem cells, si‐PERK + ERS + stem cells were 0%, 21.3%, 18.9%, and 15.9%, respectively, and the difference of apoptosis rate between the latter three groups and control group was statistically significant (P < 0.01). Stem cells could weaken the ERS‐induced cell apoptosis, especially reducing the number of cells in the late stage of apoptosis from 5.4% to 1.1%. The protein level of GRP78, PERK, ATF4, TMEM119, and BMP6 in the group of ERS, ERS + stem cells, and si‐PERK + ERS + stem cells were all significantly higher than those in control group, and the group of ERS + stem cells was the highest, all of the differences were significant (P < 0.01). However, the protein level of CDK4 and Cyclin D presented an absolutely opposite trend and the difference was still significant (P < 0.05). The group of si‐PERK + ERS + stem cell was lower than those in the group of ERS + stem cell but higher than those in the group of ERS (P < 0.05). The level of Caspase 3 in the latter three groups was significantly higher than those in the control group, and the group of ERS was the highest (P < 0.01). Besides, the relative level of Bcl‐2/Bax in control group was 1, but the group of ERS was about 0.5, and there was significant difference (P < 0.01). The ratio of Bcl‐2/Bax in the group of ERS + stem cells was more than 2 and significantly higher than those of other groups.In cell cycle assay, ERS increased the accumulation of cells in GACSCs could reduce ERS‐induced chondrocyte apoptosis by PERK and Bax/Bcl‐2 signaling pathway. The normal articular surface is covered with hyaline cartilage. Once the hyaline cartilage is damaged, it will cause pain, instability, and stiffness of the joints, and accelerate the degeneration of the joints, leading to the occurrence of osteoarthritiset al. demonstrated that ERS of chondrocytes increased during the development of osteoarthritis (OA), resulting in increased apoptosis of chondrocytes and reduced protective responseEndoplasmic reticulum stress (ERS) is a kind of subcellular pathological state caused by various stimulations and subsequent accumulation of unfolded or misfolded proteins in the endoplasmic reticulum lumenArticular cartilage has also been proven to have a type of cartilage stem cells (ACSCs) with self‐proliferation and multi‐directional differentiation potentials. Such stem cells can spontaneously aggregate to the damaged site, which is conducive to the rapid establishment of the microenvironment for cartilage repair. ACSCs have been used in treatment for early joint injuries, inhibiting further develop of cartilage damage, as well as reducing the incidence of osteoarthritis alone or in combination with artificial replacementHowever, the research on ERS in the field of osteoarthritis is still quite limited, and the inhibitory effect and mechanism of ACSCs on ERS are unclear. Therefore, there are two main purposes of this study: (i) to explore the role of ACSCs in ERS in chondrocytes after hip replacement by observing the changes of related genes and proteins and (ii) to elucidate the molecular mechanism of ACSCs inhibitory effects on ERS. We hope that this research can provide new ideas for alleviating ERS in chondrocytes, prevent related complications after hip replacement, and help patients reduce pain to a greater extent.−5/UI penicillin, and 100 mg/L streptomycin. The cell mixture was cultured at 37°C in a humidified 5% CO2 incubator.Human cartilage cell lines C28/I2were obtained from Chinese Academy of Sciences . Cells were grown in DMEM medium supplemented with 10% fetal bovine serum (FBS), 1 × 102 andcultured for 72 h after transfection of 80 nmol/L PERK siRNAs. The transfection was performed by Lipofectamine 2000 Transfection Reagent.We bought human PERK siRNAs from Santa Cruz Biotechnology. Cells were put in plates containing Dulbecco's modified Eagle medium and 10% FBS. Then, plates were placed in incubator with 5% COin vitro to create hypoxia environment. Four groups were arranged in this study. A group contained normal human cartilage cells C28/I2 and worked as the control. Moderate endoplasmic reticulum stress was induced in human cartilage cells C28/I2 by exposure to tunicamycin, and these cells were further divided into three groups including B, C, and D group. Group B contained only C28/I2 cells that were subject to ERS. Group C contained the co‐culture system of cartilage stem cells and chondrocyte ERS model. The constructed si‐PERK interfering plasmid was transfected into chondrocyte ERS model to form D group together with cartilage stem cells.Hypoxia injury model was established by using hypoxia bag The cells, before and after transfection and at logarithmic growth stage, were digested into a single‐cell suspension and centrifuged. The supernatant was discarded and cells were stained using Annexin V/propyl iodide. Apoptosis was detected by flow cytometry according to the manufacturer's instructions.Cells were collected for protein expression determination. After digestion with 0.25% trypsin, the cells were washed with precooled PBS three times. Protein lysates were added to lyse the cells, and the total proteins in the cells were extracted. Equivalent protein samples were taken and subjected to SDS‐PAGE electrophoresis. Separated proteins on the gel were transferred to cellulose nitrate membrane. Reaction was conducted in 5% skim milk powder sealant for 2 h. Primary anti‐GRP78, PERK, ATF4, TMEM119, CDK4, CyclinD, and BMP6 were added and incubated at 4°C overnight. Secondary antibodies were added and incubated at room temperature for 2 h. The mixture was then washed with phosphate buffered solution (PBST) three times. ECL was used for chemiluminescence. Photos were taken and gray value of each strip was analyzed by the Image J Image analysis system.Total RNA was extracted from cells by Trizol method and cDNA was synthesized according to the instructions of reverse transcription kit. PCR amplification was performed as follows: 95°C 5 min, 95°C 20 s, 60°C 30 s, and 72°C 20 s for 40 cycles. U6 was taken as a control. The primers were listed in the following Table The cultured C28/I2 cells were washed three times with PBS and blockaded with 10% rabbit serum at 37°C for 30 min. Then, the cells were incubated with the primary antibodies for ERK, TMEM119, or BMP6 overnight at 4°C (1: 100). Afterwards, the cells were incubated with Alexa Fluor 488 Goat Anti‐Rabbit IgG (1: 100) for 30 min at 37°C and washed with PBS. Observation and photography were performed with an Olympus multifunction microscope .t‐tests. P < 0.05 was considered statistically significant.Quantitative data were analyzed using SPSS 20.0  and expressed as the mean ± SD. Statistical differences between groups were compared using ANOVA and two‐tailed 0/G1 and G2/M phases of chondrocytes in ERS group were lower than the corresponding phases in control group. The peak values of G0/G1 and G2/M phases in ERS + stem cell group were lower than those in control group, while higher than those phases in ERS group, suggesting that cartilage stem cells alleviated the effects on cell cycle caused by ERS. Besides, the peak values of G0/G1 and G2/M phases in si‐PERK + ERS + stem cell group were lower than those in ERS + stem cell group, while higher than ERS group. The silence of PERK pathway reduced the inhibitory effects of stem cells on endoplasmic reticulum stress. These results indicated that ERS could regulate the cell cycle of stem cells in chondrocytes.The cell cycle results in the control, ERS, ERS + stem cell and si‐PERK + ERS + stem cells groups were showed in Fig. P < 0.01).Phosphatidylserine (PS) is normally located on the inner side of the cell membrane, but, in the early stage of apoptosis, PS flips from the inner side of the membrane to the membrane surface. Annexin V can bind to PS with high affinity and cells marked with Annexin V are considered as apoptotic cells. PI can penetrate into cells with damaged membrane. Therefore, the total proportion of Annexin V/PI‐ quadrant and Annexin V/PI quadrant are considered as apoptotic cells. The apoptosis rates of chondrocytes in the control, ERS, ERS + stem cell and si‐PERK + ERS + stem cell groups were showed in Fig. Stem cells could weaken the ERS‐induced cell apoptosis, especially reducing the number of cells in the late stage of apoptosis from 5.4% to 1.1%. Besides, the apoptosis rate in si‐PERK + ERS + stem cells group was 15.9%. Elimination of the PERK gene could reduce the proportion of apoptotic cells in chondrocytes, indicating that the PERK pathway was the main factor inducing cell apoptosis during endoplasmic reticulum stress. These results demonstrated that the ERS‐induced cell apoptosis was decreased by cartilage stem cells.P < 0.01). The expression values in si‐PERK + ERS + stem cell group was higher than those in ERS group, but lower than those in ERS + stem cells group (P < 0.01). The protein expression values of CDK4 and Cyclin D in ERS, ERS + stem cell, and si‐PERK + ERS + stem cell groups were all markedly lower than those in control group, and the values in ERS group were lowest among them (P < 0.05). The values in si‐PERK + ERS + stem cell group were lower than those in ERS + stem cell group but higher than those in ERS group (P < 0.05). Based on these results mentioned above, we proposed that cartilage stem cells mediated the expression of various transcription factors.Western blot assay was used to determine the protein expression values of GRP78, PERK, ATF4, TMEM119, CDK4, Cyclin D, and BMP6 in chondrocytes in the four groups. As Fig. Immunofluorescence assay was applied to study the location of PERK, TMEM119, and BMP6 in chondrocytes. As shown in Fig. P < 0.01). The relative expression of Caspase 3 in si‐PERK + ERS + stem cells group was higher than that in ERS + stem cells group (P < 0.01). Besides, the relative expression level of Bcl‐2/Bax in control group was 1, the levels in ERS was about 0.5, indicating ERS‐induced apoptosis (P < 0.01). Bcl‐2/Bax expression ratio in ERS + stem cells group was more than 2, and the increased value suggested that apoptosis caused by ERS was weakened. Transfection of si‐PERK reduced the effect of stem cells, but the Bcl‐2/Bax expression ratio was still higher than that in ERS group. These results showed that cartilage stem cells could influence the expression of Bcl2, Bax, and Caspase 3 after treated with ERS.The relative expression levels of Bcl2, Bax, and Caspase 3 in the control, ERS, ERS + stem cell, and si‐PERK + ERS + stem cell groups were showed in Fig. Endoplasmic reticulum (ER) is a fine membrane system in cells, which plays an important role in cell growth, survival, and functionChondrocytes are the only cellular components found in normal articular cartilage. Chondrocytes are in a special microenvironment without vascular, nerve and lymphatic distribution, and various physical and chemical factors are likely to stimulate ERSCartilage stem cells (ACSCs) can be grown into mature chondrocytes. ERS regulates cell apoptosis by activating the unfoldable protein response (UPR). UPR includes three signaling pathways, namely PERK, ATF6, and IRE1. PERK is endoplasmic reticulum type I transmembrane protein. It is activated by autodimerization and phosphorylation after dissociation. Activated PERK phosphorylates translation initiation factor 2α (EIF2α), which selectively facilitates the translation of ATF‐4, thus the ERS stress response was activated and the apoptosis process was initiated. In our study, the apoptosis rate in ERS group was significantly higher than the control group. Addition of ACSCs decreased the cell apoptosis induced by ERS, while further transfection of si‐PERK weakened the inhibitory role of ACSCs on ERS‐induced cell apoptosis. Moreover, the expressions of PERK and ATF‐4 were found significantly up‐regulated by ERS.2+‐binding, and controlling the activation of trans‐membrane ERS sensorsCo‐culture of ACSCs with ERS cells promoted the protein expression of PERK and ATF‐4, while further transfection of si‐PERK abolished the promoting role. GRP78, also referred to as BIP, is a central regulator of endoplasmic reticulum function due to its roles in protein folding and assembly, targeting misfolded protein for degradation, ER CaOur study showed ERS and ACSCs up‐regulated the expression of GRP78, PERK, ATF4, TMEM119, and BMP6, which knockdown of PERK weakens the up‐regulation role. ERS decreased the level of CDK4 and CyclinD, while the addition of ACSCs increased the expression of these genes. Further knockdown of PERK weakened the increased role of ACSCs. Bcl‐2 family is an important regulatory signal molecule of apoptosis signal transduction and Caspase is the terminal molecule of apoptosis. Our study showed that ERS induction could increase the expression levels of Bax/Bcl‐2 and Caspase‐3. Adding of ACSCs alleviated the apoptosis levels induced by ERS, while further transfection of si‐PERK abolished the decreased role of ACSCs. From above results, we speculated ACSCs can reduce ERS‐induced chondrocyte apoptosis by PERK and Bax/Bcl‐2 signaling pathway.There are still limitations in this study. We know that the gene transcription and protein expression are not necessarily the same, so it is better to test the Bcl‐2, Bax, and Caspase 3 protein expressions by using western‐blot assay. Besides, we found that stem cells could up‐regulate PERK protein expression and inhibit cell apoptosis. However, activation of PERK pathway could trigger apoptosis by ERS induction. The complex relationship among stem cell, ERS, and PERK needs more investigation. Factors like the concentration of stem cells and degree of stress may have pivotal effects on the regulation of ERS and underlying mechanism of stem cells in chondrocytes. Moreover, the study is only conducted at cell level; animal and human models are necessary for further verification.


BRCA1/2 significantly contribute to hereditary breast and/or ovarian cancer. Here, we report a novel BRCA2 duplication of exons 22–24 in a female patient with bilateral breast cancer at age 35 and 44. The duplicated region was initially detected by gene panel sequencing and multiplex ligation‐dependent probe amplification. However, the location and orientation of the duplicated region was unknown. Therefore, it was initially classified as a variant of unknown significance.Germline mutations in BRCA2 duplication was achieved by targeted enrichment of the whole‐genomic BRCA2 locus including exons and introns, and subsequent high‐throughput sequencing. Subsequently, bioinformatics tools and a breakpoint‐spanning PCR were used for identification of location and orientation of the duplication.The spatial directional characterization of the The duplicated region was arranged in tandem and direct orientation (Chr13(GRCh37):g.32951579_32960394dup; NM_000059.3 c.8754 + 651_9256+6112dup p.(Ala3088Phefs*3)). It is predicted to result in a frameshift and a premature stop codon likely triggering nonsense‐mediated mRNA decay. Consequently, it is regarded as pathogenic.This case study demonstrates that a comprehensive characterization of a structural variant by breakpoint assessment is crucial for its correct classification. Therefore, sequencing strategies including non‐coding regions might be necessary to identify cancer predispositions in affected families. BRCA2 duplication of exons 22–24 in a female patient with bilateral breast cancer. The duplicated region was initially detected by gene panel sequencing and multiplex ligation‐dependent probe amplification. Since the breakpoints are located in introns, the duplication could only be further characterized by high‐throughput sequencing including noncoding regions.In our manuscript we report a novel  Germline pathogenic variants (PVs) in one of the genes significantly increase the lifetime risk to develop hereditary breast and/or ovarian cancer . About 25% of HBOC can be explained by pathogenic BRCA1/2 variants  , which has been first introduced in 2003. MLPA is a semi‐quantitative PCR‐based technique that can determine the copy numbers of different target sequences, simultaneously  Figure  was firsWithin the patient's family, multiple incidences of different cancer types appeared, including one other incidence of breast cancer Figure . Her matBRCA1/2 via high‐resolution melt (HRM) analysis and denaturing high‐performance liquid chromatography (DHPLC). Additionally, she was tested for CNVs in BRCA1 via MLPA . No PV was identified.The proband's young age at the initial breast cancer diagnosis and the development of bilateral breast cancer before the age of 50, suggested a genetic predisposition for the disease. In 2010, after the second incidence of breast cancer, she was first examined in our clinic. Genomic DNA was isolated from EDTA blood samples using the QIAamp DNA blood Midi kit (Qiagen) and was tested for single‐nucleotide variants in TP53 can be identified . On this occasion, MLPA testing for BRCA2  was also performed, which was implemented as a routine screening in our clinic in 2012. Both techniques detected a duplication of exons 22–24 of BRCA2, but its location and orientation within the genome of the duplicated region remained unknown. Due to the large introns surrounding exon 22 and exon 24, determining the duplication's orientation and breakpoints using PCR‐based sequencing techniques would have been labor‐intensive and inefficient. Therefore, the duplication has initially been classified as a variant of unknown significance (VUS), according to the ENIGMA classification criteria for BRCA1/2 variants (Version 2.5.1 29 June 2017).Since other types of cancer occurred in the family, including sarcoma, the patient was tested for Li‐Fraumeni syndrome (MIM #151623). In approximately 70% of families fulfilling the criteria for classical Li‐Fraumeni syndrome, a PV in BRCA2 locus of the patient was amplified via a custom‐designed targeted enrichment strategy (Agilent SureSelectQXT) and sequenced on a NextSeq 500 (Illumina) according to the manufacturer's instructions. Briefly, 50 ng of the patient's genomic DNA was enzymatically fragmented and adaptor‐tagged. The DNA fragments of interest hybridized to biotin‐labeled cRNA baits targeting the genomic loci of BRCA1 (Chr17(GRCh37):g.40600254_41756008) and BRCA2 (Chr13(GRCh37):g.32414335_33482189), including exonic, intronic, and surrounding intergenic regions. The cRNA‐DNA hybrids were captured using streptavidin‐coated magnetic beads. After washing and amplification, the enriched DNA fragments were submitted to paired‐end sequencing using a NextSeq 500 Mid Output V2 kit for 2 × 150 bp paired‐end reads (Illumina). A mean coverage of 422 reads with 99.61% of bases covered by at least 20 reads was achieved.To further characterize the duplication, the complete https://github.com/imgag/ngs-bits) confirmed the duplication covering exons 22–24 of BRCA2  sequenced with the same targeted enrichment method. By analyzing the reads, we were able to determine the breakpoint and its direct tandem orientation. Additionally, a breakpoint‐spanning PCR was performed, using the following primers for validation, 5′‐CTCTAGAGTACTGTGAGTGG‐3′ (Chr13(GRCh37):g.32960177–32960196) and 5′‐GAGACGGAGTTTTGCTCTTG‐3′ (Chr13(GRCh37):g.32951708–32951727). The breakpoints of the duplication were located within the homologous region TAAAAATACAAAA: Chr13(GRCh37):g.32951579_32960394dup (Figure The in silico copy number variation detection tool CnvHunter (p Figure . TherefoBRCA2 duplication. Unfortunately, additional family members including the proband's parents did not consent to genetic testing for the duplication. Therefore, it is unknown whether this duplication emerged de novo in the patient. The patient was advised to undergo a risk‐reducing salpingo‐oophorectomy. An earlier discovery of the BRCA2 duplication might have ultimately resulted in the same therapy, but a bilateral mastectomy would have been offered.Considering this classification, the patient's sisters (Figure Here, we demonstrate the rapid and accurate identification of a large genomic rearrangement via a targeted high‐throughput sequencing approach. With this method, we were able to reclassify a variant of unknown significance to a pathogenic variant. This highlights the importance of thorough characterization of LGRs, such as duplications, by the identification of their location and orientation to determine their pathogenicity. This might also require targeting intronic regions, which are mostly excluded in high‐throughput sequencing panels. In this context, targeted enrichment is a powerful and cost‐effective tool for complete coverage of target regions and the characterization of large genomic rearrangements.The authors declare that they have no competing interests.J.L.v.L., J.B., and S.S. performed the high‐throughput sequencing, analyzed the data, wrote, and revised the manuscript; G.S. analyzed the data and provided scientific advice; W.H. performed the bioinformatics analysis; S.M. contacted the patients; R.B. analyzed the MLPA results; B.A., B.S., and D.S. revised the manuscript; D.S. supervised the work.


To establish a prognostic model to estimate the cancer‐specific survival (CSS) for urothelial carcinoma of bladder (UCB) patients after radical cystectomy (RC).n = 4323) and validation cohort (n = 4327). We performed Cox regression analysis to identify prognostic factors and Kaplan‐Meier analysis to assess survival outcome. A nomogram predicting CSS was constructed. Its performance was validated by calibration curves, the receiver operating characteristic (ROC) curves, concordance index (C‐index), decision curve analysis (DCA), the net reclassification improvement (NRI), and the integrated discrimination improvement (IDI).A total of 8650 candidates (2004–2011) obtained from the Surveillance, Epidemiology, and End Results (SEER) database were randomly split into development cohort (The nomogram incorporated marital status, T stage, N stage, tumor size, and chemotherapy. In validation cohort, C‐index of the nomogram was 0.707. AUC of the nomogram and AJCC stage were 0.767 versus 0.674. Calibration plots for 3‐ and 5‐year CSS displayed good concordance. DCA curves of the nomogram exhibited larger benefits than the AJCC stage. The NRI and IDI indicated the nomogram outperformed AJCC stage.We have established a prognostic nomogram with improved discriminative ability and clinical benefits for UCB patients after RC. The nomogram alongside an easy access web tool may assist clinicians in optimizing the postoperative management. This population‐based study has established a nomogram and its derived webserver to predict cancer‐specific survival probabilities for patients with urothelial carcinoma of the bladder after radical cystectomy. The model’s discriminative abilities, accuracy and clinical benefits were validated and performed better than traditional AJCC stage. The prognostic roles of various factors and predictive models for UCB have been studied for years. Traditionally, clinicians rely on the American Joint Committee of Cancer (AJCC) stage guidelines which comprise primary tumor (T) status, regional lymph node (N), and distant metastasis (M).Nomograms are now one of the most widely used prediction tools that provide tailored individual prognostic information by incorporating significant demographic, clinical, pathological or treatment features, and presenting simple visualized results of statistical analysis.In this study, we aim to establish a novel prognostic nomogram to assess relevant prognostic factors and estimate the cancer‐specific survival for patients after RC. Our study is based on a large population derived from the Surveillance, Epidemiology, and End Results (SEER) database. In addition, we demonstrated the nomogram's discriminative ability and clinical practicality by comparing it with the AJCC stage. We then developed a novel web tool for easy access of our model and improved counseling of patients after RC.22.1Patient records were retrieved from the SEER database by SEER*Stat version 8.3.6. SEER contains cancer incidence data collected by 18 population‐based cancer registries which cover nearly 35% U.S. population. The inclusion criteria were as follows: (a) bladder cancer cases diagnosed from 2004 to 2011; (b) patients who received RC and lymph node dissection; (c) age not under 18 years old; (d) with clear classification of races; (e) with complete information of AJCC stages and tumor‐node‐metastasis (TNM) stages; (f) with clear marital status; (g) with clear racial information; (h) with definitive survival duration or follow‐up time. Patients with distant metastasis (M1) or missing information of any of above conditions were excluded from this study. Since SEER is a publicly available database and all records have been de‐identified, no additional ethical approval or informed consent was required after the SEER Research Data Agreement was signed for accessing data.2.2This study collected information of variables including age at diagnosis, gender, race, marital status, year of diagnosis, histologic subtype, histologic grade, AJCC stage, T stage, N stage, radiotherapy, chemotherapy, primary tumor size, SEER cause‐specific death classification, vital status, and survival time (months). In SEER database, records between 2004 and 2011 were coded with the sixth edition of AJCC stages. Age was a numeric factor and for application simplicity, it was converted into categorical form according to real‐world experience and previous studies.2.3The total records were randomly split into development and validation cohorts in a ratio of 1:1. The univariate Cox proportional hazards regression model was applied in the development cohort for estimating the hazard ratio (HR) and corresponding 95% confidence interval (CI) to identify potential significant prognostic factors. The factors were then incorporated into multivariate analytic model to determine their independent association with CSS in the same cohort. Survival analyses were performed by the Kaplan–Meier method and log‐rank tests were used to estimate the differences of CSS stratified by each factor. Based on final screened variables, the nomogram was constructed for visualized prediction of 3‐ and 5‐year survival probability of development cohort. In addition, a web tool was created using the shiny package of R 3.6.1 for easy access and convenient application of this model.Z test was used to assess the differences.The internal validation of the nomogram was conducted in development cohort and the external validation was performed using validation cohort. The discriminative ability of the nomogram was assessed by the concordance index (C‐index) and the receiver operating characteristic (ROC) curves with the calculated area under the curve (AUC). Calibration plots were employed for comparing nomogram‐predicted and actual outcomes of 3‐ and 5‐year survival time. Both discrimination analyses and calibration plots used bootstrapping with 500 resamples. In addition, decision curve analysis (DCA) was applied to estimate the clinical usefulness and benefits of the nomogram by comparing the threshold probabilities range of the model to that of the AJCC staging system.http://www.R‐project.org, The R Foundation for Statistical Computing) via RStudio software version 1.2.5033 and EmpowerStats . A p < 0.05 was considered statistically significant.All statistical analyses were conducted by R version 3.6.1 (33.1n = 4323) and validation cohort (n = 4327) in a ratio of 1:1 . Differences between the two cohorts in each variable are statistically insignificant (p > 0.05).A total of 8650 M0 patients who underwent RC and lymph node dissection were retrieved and screened for further analyses according to the inclusion criteria. They were randomly assigned into development cohort (3.2p = 0.017), never‐married status , higher T stage , lymph node metastasis , and larger primary tumor size  were associated with worse CSS. While the administration of chemotherapy appeared to be a protective factor . Kaplan–Meier analyses demonstrate different survival outcomes stratified by each variable , indicating that factor stratifications were appropriate and acceptable.The univariate Cox proportional hazards regression analysis identified nine factors which were associated with CSS in the development cohort. Sequential multivariate Cox regression analysis incorporating these variables was performed to finally determine five independent prognostic factors as following: marital status, T stage, N stage, chemotherapy, and primary tumor size Table . To namee Figure . Log‐ran3.3The prognostic nomogram predicting 3‐ and 5‐year bladder CSS probability was established based on the screened factors using 4323 patients from the development cohort , which was 0.695 and 0.683, respectively. Discriminative ability of the nomogram was also examined by ROC curves  and 1.4% (p < 0.001), respectively, in the validation cohort and 1.7% (p < 0.001) and 1.6% (p < 0.001), respectively, in the development cohort. These results suggest that the nomogram has greater potential to accurately predict prognosis compared with traditional AJCC staging system.Accuracy analysis showed that the NRI for 3‐ and 5‐year survival time were 0.202 (95% CI: 0.143–0.294) and 0.180 (95% CI: 0.128–0.269), respectively, in the validation cohort and 0.187 (95% CI: 0.139–0.285) and 0.165 (95% CI: 0.088–0.257), respectively, in the development cohort. Furthermore, the IDI for 3‐ and 5‐year survival were 1.5% (3.5https://prediction‐calculator.shinyapps.io/DynNomapp/. To utilize this tool, researchers and clinicians can simply input clinical and demographic features. Then, the output figures and tables generated by the tool can be read to directly learn the predicted survival probability across different time.An online web tool based on our nomogram can be accessed at 4UCB is one of the major health issues across the globe, with both high morbidity and mortality. Although various treatment modalities have been developed, RC remains the standard therapy of muscle‐invasive UCB and high‐risk non‐muscle‐invasive UCB.Our study also assessed the clinical value of constructed nomogram by comparing it with traditional AJCC staging system. The nomogram outperforms AJCC stage both in the development and validation cohorts. In detail, our prognostic model shows better discriminative ability and accuracy for predicting 3‐ and 5‐year survival probabilities with higher C‐indices and AUCs, positive NRI and IDI. Besides, DCA curves demonstrate that our nomogram possesses better potential of clinical utility than the AJCC stage.The nomogram for CSS probability prediction incorporated five factors including marital status, T stage, N stage, primary tumor size, and chemotherapy. Notably, to reduce selection bias and maintain the completeness of data, patients with unknown tumor size were reserved in our analysis. Each finally included variable was independently associated with survival outcomes of UCB patients, which was consistent with prior studies. Sammon et al. found that being married was an indicator of lower all‐cause mortality for both men and women after RC, compared with their SDW or never‐married counterparts.The need for prognostic prediction after RC for UCB patients has induced the development of various postoperative models.Our study has certain limitations to note. First, as the study is based on the SEER database, there is a lack of potential important factors, such as preoperative laboratory results, lymphovascular invasion, surgical margin status, comorbid conditions, and socioeconomic status. Second, we excluded patients with unknown histologic grade, AJCC stage, T stage, or N stage, which might introduce selection bias despite the small fraction. However, the large number of patients and the population‐based design could strengthen our model and reduce potential confounding impact. Finally, the results should be taken with caution due to the study's retrospective nature. Therefore, large prospective clinical trials for external validation are needed. Once it is further validated, our model could provide a foundation for future improved predictive tool incorporating potential multi‐omics profiles.5This large population‐based study revealed several demographic, clinicopathologic factors, and therapeutic features that were significantly associated with survival outcome of UCB patients after RC. We established and validated a prognostic nomogram to better predict 3‐ and 5‐year CSS probabilities than the AJCC stage for these patients. In addition, we developed a web tool for easy access to and improved utility of our model. This novel instrument may help clinicians in patient counseling, follow‐up scheduling, and potential clinical trial design for UCB patients who underwent RC. Still, large prospective clinical studies for external validation are needed.The authors declare that they have no conflict of interest.PH and ZQY conceptualized and supervised the conduct of the study; YJB and MYL conducted data collection and statistical analysis; ZQY and XH prepared the manuscript for submission; all authors reviewed and critiqued the manuscript for content.


However, the effect of CD34+ cell therapy on health care expenditures before and after treatment is unknown. We determined the effect of CD34+ cell therapy on cardiac‐related hospital visits and costs during the 12 months following stem cell injection compared with the 12 months prior to injection. Cardiac‐related hospital admissions and procedures were retrospectively tabulated for patients enrolled at one site in one of three double‐blinded, placebo‐controlled CD34+ trials in the 12 months before and after intramyocardial injections of CD34+ cells vs placebo. Fifty‐six patients were randomized to CD34+ cell therapy (n = 37) vs placebo (n = 19). Patients randomized to cell therapy experienced 1.57 ± 1.39 cardiac‐related hospital visits 12 months before injection, compared with 0.78 ± 1.90 hospital visits 12 months after injection, which was associated with a 62% cost reduction translating to an average savings of $5500 per cell therapy patient. Patients in the placebo group also demonstrated a reduction in cardiac‐related hospital events and costs, although to a lesser degree than the CD34+ group. Through 1 January 2019, 24% of CD34+ subjects died at an average of 6.5 ± 2.4 years after enrollment, whereas 47% of placebo patients died at an average of 3.7 ± 1.9 years after enrollment. In conclusion, CD34+ cell therapy for subjects with refractory angina is associated with improved mortality and a reduction in hospital visits and expenditures for cardiac procedures in the year following treatment.Patients with refractory angina who are suboptimal candidates for further revascularization have improved exercise time, decreased angina frequency, and reduced major adverse cardiac events with intramyocardial delivery of CD34 + progenitor cell injections compared to the 12 months prior to injections (P = .002).The total cardiovascular hospitalizations, procedures and emergency room visits are significantly reduced in patients with refractory angina in the 12 months following intramyocardial CD34 This report demonstrates for the first time that patients who received these cells experienced reduced health care utilization and costs in the year following therapy compared with the year before their administration. Furthermore, patients who received CD34+ cell therapy showed reduced mortality compared with patients who received placebo.Intramyocardial delivery of CD342As mortality from coronary artery disease declines, a growing number of patients experience refractory angina each year.+ have demonstrated heightened efficacy in promoting neovascularization of myocardium.+ cells with similar designs.There has been ongoing interest in therapeutic angiogenesis as a method of improving cardiac perfusion.+ cell therapy on cardiac‐related events, hospitalizations, and costs is unknown. Although these patients are believed to have high health care costs, investigation into their health care expenditures is limited.+ cells compared with placebo. We then conducted a cost analysis to determine whether CD34+ cell therapy was associated with a reduction in hospital expenditures within the first year after treatment.Despite these promising results, the effect of CD343+ stem cells between 2004 and 2013: a phase I/IIa (NCT00081913), phase II ACT‐34 , and phase III RENEW (NCT 01508910). All patients had provided written, informed consent. Enrollment criteria required that patients have Canadian Cardiovascular Society (CCS) class 3‐4 chronic refractory angina despite optimal medical management and be deemed ineligible for further revascularization by percutaneous coronary intervention (PCI) involving stenting or bypass surgery with a minimum of seven anginal episodes per week with limited exercise capacity. All enrolled patients underwent bone marrow cell mobilization with granulocyte colony‐stimulating factor  followed by apheresis on day 5 of mononuclear cells enriched for CD34+ cells using a commercially available device.+ cells  or placebo delivered by intramyocardial injections with the NOGA Myostar catheter following electromechanical endocardial mapping using the NOGA system  to identify ischemic regions of myocardium prior to injection of CD34+ cells. A standard of care arm was implemented in the phase III trial only and was not included in this retrospective review.A retrospective chart review was performed for patients at The Minneapolis Heart Institute Foundation at Abbott Northwestern Hospital  who enrolled in one of three clinical trials evaluating CD34+ cells and for 12 months following injection. Hospital visits for cardiac rhythm abnormalities  were excluded, as were admissions for deep vein thrombosis, enhanced external counterpulsation therapy, and other research studies. Tabulation of hospital visits resulting in coronary procedures included coronary artery bypass grafting, PCI, percutaneous transluminal coronary angioplasty without stenting, coronary angiography, and right heart catheterization. Median total variable costs within the Allina Healthcare system were used to estimate costs of these procedures and any admissions for congestive heart failure or chest pain with and without intervention. Mortality data were obtained from the electronic medical record or from death certificates.Cardiac‐related hospital admissions and cardiovascular procedures were tabulated from electronic medical records for 12 months preceding injection of autologous CD34+ cells or placebo, as well as associated hospital costs. A paired t test was used to compare number of events and health care expenditures before and after cell therapy. A P value of <.05 was considered statistically significant. Because this was an exploratory retrospective analysis with no a priori power calculations performed, the findings of statistical significance should be interpreted with caution.Descriptive statistics include percentage reduction of cardiac‐related emergency department (ED) visits and hospital admissions before and after injection of CD344+ cells were included in this study. The mean age was 57.9 ± 7.5 years, and 13.5% of patients were female. Baseline characteristics are shown in Table + cell therapy experienced an average of 1.57 ± 1.39 cardiac‐related hospital visits 12 months before injection compared with 0.78 ± 1.90 cardiac‐related hospital visits 12 months after injection  12 months before injection compared with 0.32 ± 0.75 events (n = 6) 12 months after injection (P < .0001). Overall, the cell therapy group experienced a 73% reduction in coronary procedures in the 12 months following their injection. The total median variable costs of all cardiac hospital visits and procedures significantly decreased by 62% in the cell therapy group following injection , compared with eight (47%) in the placebo group. In the CD34+ group, five deaths were due to cardiovascular disease, two deaths were due to cancer, and cause of death was unavailable for two subjects. The average time between treatment with CD34+ cells and death was 6.5 ± 2.4 years. In contrast, placebo patients died an average of 3.7 ± 1.9 years after enrollment, resulting in a significantly shorter timespan compared with the CD34+ group (P = .02).Mortality in the CD34+ treatment group  also experienced fewer hospitalizations and procedures in the year following injection, from 1.26 ± 0.87 hospital visits and procedures before injection to 0.63 ± 1.42 hospital visits after injection, although this decline was not significant because of the smaller sample size. However, postinjection placebo subjects did exhibit a significant 70% reduction in their health care expenditures, that translated to a significant cost savings of $3700 per subject, although less than the total cost savings per patient in the CD345+ stem cells, significant reductions in cardiac‐related ED visits and hospital admissions, interventional coronary procedures, and associated hospital expenditures were observed compared with the 12 months before injection.In the 12 months following intramyocardial injection of CD34+ therapy is consistent with results from a recent analysis of a European cell therapy trialThe reduction in hospital visits following CD34+ cell product and is the only cost study that included a placebo group. We observed that mortality was halved for those patients that received CD34+ compared with patients who received placebo. Furthermore, among patients who eventually died, those that received CD34+ cells lived twice as long as those who received placebo, supporting a true therapeutic benefit for this therapy.Importantly, ours is the first study to document a reduction in mortality in addition to the decrease in health care costs and adverse events with the CD345 CD34+ cells per kilogram body weight) exhibited a significant reduction in angina frequency and a significant improvement in exercise tolerance time at 12 months compared with placebo patients, which was also confirmed in the meta‐analysis.The decrease in hospital visits among placebo patients after enrollment may reflect a placebo effect or a real benefit of G‐CSF. Alternatively, the process of intramyocardial injection itself may exert an antianginal effect or stimulate neovascularization. However, Hossne et al+ cells suggests that cell therapy warrants further investigation as a cost‐effective treatment option for patients with refractory angina. It is also possible that the cost benefit of this therapy demonstrated over the first year will continue to grow over time. Because cell therapy is associated with significant costs of procedures and cell processing, it is hoped that the reductions in expenditures demonstrated with this therapy will justify the eventual insurance coverage of this therapy once Food and Drug Administration approval is obtained.Given that the long‐term mortality rates among this patient population are relatively low,+ group. Our findings of no signs of risk or early adverse cardiovascular events or increase in hospitalizations support the contention that the procedures associated with CD34+ cell mobilization, apheresis, and intramyocardial injections are safe without an accumulation of risk in the postprocedure period. However, in spite of these benefits, the placebo group demonstrated greater mortality than the CD34+ cell therapy group extending over the long‐term follow‐up period.Our work also supports the observation that placebo‐treated patients accrue substantial benefit from participation in clinical trials, as patients in the placebo group also demonstrated a reduction in cardiac‐related hospital events and costs, although to a lesser degree than the CD34T.J.P. declared advisory role with Caladrius Biosciences, Blue Rock Therapeutics, and University of Washington; honoraria with Caladrius Biosciences, Blue Rock Therapeutics, and NovoNordisk; and research funding from CSL Behring, Intracellular Therapeutics, Eli Lilly, Merck, and Janssen Pharmaceuticals. D.W.L. declared leadership position and ownership interest with Caladrius Biosciences. L.I.S. declared leadership position with Minneapolis Heart Institute Foundation, Comcast, and Critical Value Consulting & Analytics; research funding from NIH; and ownership interest with Comcast. J.H.T. declared research funding from National Heart, Lung, and Blood Institute. The other authors indicated no conflicts of interest.G.L.J., J.H.T.: collection/assembly of data, manuscript writing, final approval of manuscript; T.D.H., T.J.P., D.W.L.: conception and design, data analysis and interpretation, final approval of manuscript; R.F.G., L.I.S., C.E.S.: data analysis and interpretation, final approval of manuscript.


The [We longitudinally investigated PD‐related brain pattern expression changes in 20 subjects with isolated rapid eye movement sleep behavior disorder to investigate whether this may be a suitable technique to study prodromal PD progression in these patients and to identify potential phenoconverters.18F]fluorodeoxyglucose‐PET brain scans ~3.7 years apart, along with baseline and repeated motor, cognitive, and olfactory testing within roughly the same time frame.Subjects underwent two [z scores above the receiver operating characteristic–determined threshold). At follow‐up, six additional subjects exhibited significant PD‐related brain pattern expression . PD‐related brain pattern expression increased in all subjects (P = 0.00008). Four subjects (20%), all with significant baseline PD‐related brain pattern expression, phenoconverted to clinical PD.At baseline, 8 of 20 (40%) subjects significantly expressed the PD‐related brain pattern (with Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder SocietySuprathreshold PD‐related brain pattern expression and greater score rate of change may signify greater shorter‐term risk for phenoconversion. Our results support the use of serial PD‐related brain pattern expression measurements as a prodromal PD progression biomarker in patients with isolated rapid eye movement sleep behavior disorder. © 2020 The Authors.  Isolated or idiopathic rapid eye movement sleep behavior disorder (iRBD) is known to be prodromal for alpha‐synucleinopathies, such as Parkinson's disease (PD), dementia with Lewy bodies (DLB), or more rarely, multiple system atrophy (MSA) in >80% of cases.not phenoconvert.However, this necessitates the availability of reliable biomarkers for the tracking and prediction of disease progression and phenoconversion to manifest alpha‐synucleinopathies. Equally important, these biomarkers must be able to identify those who will 18F]fluorodeoxyglucose positron emission tomography ([18F]FDG‐PET)‐based abnormal cerebral glucose metabolism known as the “PD‐related pattern” (PDRP) in subjects with iRBD. The PDRP is defined by the Scaled Subprofile Model Principal Component Analysis (SSM/PCA) method,Accordingly, this study focuses on the longitudinal change in expression of a characteristic pattern of 1Ffluorode18F]FDG‐PET‐based disease‐related metabolic brain pattern expression in patients with iRBD have not been performed before. We therefore studied 20 subjects with iRBD with glucose metabolic brain imaging, as well as motor, cognitive, and olfactory testing two times approximately 4 years apart. Our primary purpose was to investigate whether serial PDRP expression measurements may be a suitable technique to study prodromal PD progression in patients with iRBD.Repeated longitudinal measurements of FDG‐PET imaging, as well as motor, cognitive, and olfactory testing (see Table Twenty subjects with iRBD (3 Dutch and 17 German) were evaluated with baseline and follow‐up [18F]FDG‐PET imaging to z‐transform the iRBD PDRP scores.In addition, 16 age‐ and gender‐matched healthy control subjects (HCs)  underwent baseline FDG‐PET imaging an average of 3.7 ± 0.6 years apart. Sixteen HCs underwent baseline [18F]FDG‐PET imaging as well. All baseline and follow‐up scans were performed on a Siemens Biograph mCT64 or mCT40 PET/CT camera  at the University Medical Center of Groningen. Images were reconstructed with OSEM3D , time‐of‐flight, point‐spread‐function, Gaussian 8‐mm full‐width‐at‐half‐maximum spatial filter, and matrix size 256 (corresponding to a voxel size of 2 mm × 3.18 mm × 3.18 mm).Twenty RBD Screening Questionnaire–screened and video polysomnographically confirmed iRBD patients underwent baseline and follow‐up [Central nervous system depressants and any iRBD‐related medications  were discontinued in all HCs and subjects with iRBD for at least 24 hours before baseline and follow‐up imaging.18F]FDG‐PET template in Montreal Neurological Institute brain spaceAll iRBD and HC images were spatially normalized to an FDG‐PET imaging, as well as motor, cognitive, and olfactory testing . PDRP expression increased among all 20 (100%) subjects between baseline and follow‐up . The average absolute z score increase was by 2.1 ± 1.1 points, with an average z score increase of 0.6 ± 0.3 point per year.At baseline, 8 of 20 (40%) subjects with iRBD expressed a PDRP P < 0.33, 0.48, and 0.26, respectively). However, olfactory scores correlated significantly with PDRP z scores at baseline and follow‐up . In addition, UPDRS‐III score change per year did correlate significantly with PDRP z score change per year , which was from a phenoconverted subject with the highest PDRP z score of the entire iRBD cohort (9.11).UPDRS‐III, MoCA, and olfactory scores did not change significantly between baseline and follow‐up testing  or guessing (in the case of the olfaction test).One of the strengths of this study includes its longitudinal nature. Some of the limitations of this study include small sample size and lack of repeat measurements of the HC cohort for comparison. Investigation of greater numbers of subjects with iRBD will be necessary to confirm or modify the conclusions of this study. In addition, a separate investigation into expressions of the DLB‐ or MSA‐related patterns in this cohort was beyond the scope of this report.18F]FDG‐PET and other biomarkers, such as dopamine transporter imaging with [123I]N‐ω‐fluoropropyl‐2β‐carbomethoxy‐3β‐(4‐iodophenyl)nortropane single‐photon emission computed tomography ([123I]FP‐CIT‐SPECT).18F]FDG‐PET imaging may have an advantage, because [18F]FDG‐PET allows for quantitative analysis of changes in glucose uptake in all areas of the brain at once, whereas [123I]FP‐CIT‐SPECT is predominately used to visualize the dopaminergic nigrostriatal tract. [18F]FDG‐PET also likely has greater potential to identify which specific parkinsonian disorder will develop in a patient with iRBD.123I]FP‐CIT‐SPECT scans.Further longitudinal studies should also examine the relationship between FDG‐PET imaging. Rosalie V. Kogan organized all logistics and imaging appointments for patients in Groningen. Annette Janzen performed all of the neuroclinical assessments with German patients in Marburg, while Rosalie V. Kogan, Vita Gurvits, and Klaus L. Leenders performed the neuroclinical assessments with Dutch patients in Groningen. Rosalie V. Kogan performed all of the statistical analyses with help from Remco J. Renken and put together all of the figures and table with the support of Annette Janzen, with the exception of Supporting Information Klaus L. Leenders, Wolfgang H. Oertel, Geert Mayer, Jan Booij, Kathrin Reetz, Sebastiaan Overeem, Angelique Pijpers, and Rosalie V. Kogan assisted in the study protocol and design. Klaus L. Leenders and Wolfgang H. Oertel secured the funding for this project. Annette Janzen, Felix Bernhard, Elisabeth Sittig, Sebastiaan Overeem, and Angelique Pijpers recruited patients. Sanne K. Meles, Laura K. Teune, Fransje E. Reesink, and Jelmer G. Kok recruited HCs. Elisabeth Sittig organized and accompanied German patients on visits to Groningen for 8FFDG‐PETThis study was funded by Dutch “Stichting ParkinsonFonds” and the German “ParkinsonFonds Deutschland. The funders of this study did not have any role in designing this study, collecting, analyzing, or interpreting the data, or in writing this article. The corresponding author had access to and responsibility for all of the relevant data that were submitted for publication. The authors report no conflicts of interest. W.H.O. is a Hertie Senior Research Professor .REMPET Working Group list of authors: Jan Booij, MD, PhD, Department of Radiology and Nuclear Medicine, Amsterdam University Medical Centers, location Academic Medical Center, Amsterdam, the Netherlands; Kathrin Reetz, MD, Department of Neurology and JARA‐BRAIN Institute Molecular Neuroscience and Neuroimaging, RWTH Aachen University, Aachen, Germany; Sebastiaan Overeem, MD, PhD, Kempenhaeghe Sleep Medicine Center, Heeze, the Netherlands; Angelique Pijpers, MD, PhD, Kempenhaeghe Sleep Medicine Center, Heeze, the Netherlands; Felix Bernhard, MD, Department of Neurology, Philipps‐Universität Marburg, Marburg, Germany; David Vállez García, PhD, Department of Nuclear Medicine and Molecular Imaging, University of Groningen, University Medical Center Groningen, the Netherlands; Débora E. Peretti, Department of Nuclear Medicine and Molecular Imaging, University of Groningen, University Medical Center Groningen, the Netherlands; Laura K. Teune, MD, PhD, Department of Neurology, University of Groningen, University Medical Center Groningen, the Netherlands, and Department of Neurology, Wilhemina Hospital Assen, the Netherlands; Fransje E. Reesink, MD, PhD, Department of Neurology, University of Groningen, University Medical Center Groningen, the Netherlands; and Jelmer G. Kok, MD, PhD, Department of Neurology, University of Groningen, University Medical Center Groningen, the Netherlands.Appendix S1. Supporting InformationClick here for additional data file.