Per2 bioluminescent rhythm previously recorded in the mouse dorsal vagal complex reveals a characteristic phase relationship between three distinct circadian oscillators. These signals represent core clock gene expression in the area postrema (AP), the nucleus of the solitary tract (NTS) and the ependymal cells surrounding the 4th ventricle (4Vep). Initially, the data suggests a consistent phasing in which the AP peaks first, followed shortly by the NTS, with the 4Vep peaking 8–9 h later. Wavelet analysis reveals that this pattern is not consistently maintained throughout a recording, however, the phase dynamics strongly imply that oscillator interactions are present. A simple phase model of the three oscillators is developed and it suggests that realistic phase dynamics occur between three model oscillators with coupling close to a synchronisation transition. The coupling topology suggests that the AP bidirectionally communicates phase information to the NTS and the 4Vep to synchronise the three structures. A comparison of the model with previous experimental manipulations demonstrates its feasibility to explain DVC circadian phasing. Finally, we show that simulating steadily decaying coupling improves the model’s ability to capture experimental phase dynamics.Analysis of ex vivo  Core canonical circadian clock genes and proteins function as the gears and cogs of the circadian clock5 which operate in complex feedforward and feedback loops to regulate their own transcription to produce 24 h oscillations6. In mammals, most cells in the brain and body contain components of the molecular clock8 and the synchronization of these molecular oscillations to the external world optimally aligns physiological functions to rhythmic environmental demands. The most predictable change in the environment is the 24-h day-night cycle, and in mammals, many circadian processes are entrained to it by the master pacemaker within the suprachiasmatic nucleus (SCN) in the hypothalamus11. This entrainment can be observed in rhythmic locomotor activity levels, body temperature fluctuations12 and circulating hormone concentrations13, to name a few. However, rhythmic clock gene expression occurs in other brain structures, such as the olfactory bulb14, the subfornical organ15, the habenula17, and the hippocampus18. Such daily transcriptomic changes can drive rhythms in neuronal activity and function of these brain regions22. From this extended neural circadian system in which extra-SCN oscillators can be distal and widely distributed23, several questions arise including (1) how this network of oscillators interact, (2) are some oscillators pacemakers and others followers, and (3) how are these oscillators synchronized to the external world. Typically these oscillators are spatially separate and may lack physical connections, so determining how oscillators interact to shape each other’s circadian timekeeping is recondite.Circadian rhythms are recurring biological processes with an intrinsic period of approximately 24 h. They pervade all physiology across phyla, from bacteria to mammals24. It is composed of readily delineated neuroanatomical structures including the area postrema (AP), the nucleus of the solitary tract (NTS), and the dorsal motor nucleus of the vagus (DMX)25 as well as the cerebrospinal fluid-containing central canal/4th ventricle running along its medial axis. Rhythmic clock gene expression is reported in vivo in the AP and NTS29. Further, using a bioluminescent reporter  of the clock protein, PER2, a recent study showed that rhythms in PER2::LUC were sustained for several days in culture in mouse DVC explants. Specifically, robust rhythms in PER2::LUC were observed in the AP and NTS as well as the ependymal cells surrounding the 4th ventricle (4Vep)30. Intriguingly, daily PER2::LUC expression appeared to organize in a distinct spatiotemporal order, peaking first in the AP, then the NTS, and lastly several hours later in the 4Vep.One way to circumvent these challenges is to focus on a brain region in which several circadian oscillators are in close physical proximity. In this regard, the dorsal vagal complex (DVC) in the medulla of the brainstem presents an accessible and tractable setting for the investigation of the principles via which multiple circadian oscillators can interact. The DVC is a key relay hub for visceral and blood-borne information which are important for a range of cardiovascular and metabolic functions30. Subsequent measurements on day five show reduced coherence between cells and an altered phase difference between the AP and NTS, indicating that dynamic processes are at play. Understanding the evolution of the phase pattern may be useful for understanding the mechanisms that lead to its formation. For example, if the relationship between DVC oscillator phases arises due to rhythmic input from peripheral tissues31 or brain sites32, then in ex vivo cultures we would not expect phase relationships to be maintained. If interactions between the oscillators contribute to their phasing, then some persistence of this pattern should be observed in our recordings. There are neuronal connections between the AP and the NTS in the coronal plane34, which are both conserved in our culture preparations and exhibit a daily variation in the magnitude of their signalling30. Further, severing these connections significantly reduces the NTS oscillator period, whilst leaving the AP period relatively unaffected30, indicating a possible asymmetry in the phase communication between the nuclei.The phase ordering between these DVC oscillators suggests there are complex relations between them, and at present, it is unclear as to how this spatiotemporal clock gene pattern in the DVC emerges or how it is sustained. Our aim with this combined modelling and experimental study is to gain an elementary understanding of physiological processes potentially responsible for this pattern of PER2::LUC phasing. Currently, we have observed a consistent phase pattern between DVC oscillators during the second day of culture recordings, which is associated with large single-cell coherence within these structuresIn this study, we develop a deeper understanding of how PER2::LUC oscillations in distinct structures of the DVC change in time and we will link our previous experimental observations together into a mathematical framework capable of predicting how a DVC-like phase pattern emerges from three interacting oscillators. Our mathematical approach will be conceptual, focusing on explaining our dataset in the simplest possible terms and discovering the elementary physiological mechanisms that may be present.30, which is summarised in Fig. 30). To extend this description and gain insight into the dynamic stability of the DVC relationship, wavelet decomposition35 of nulcei-wide PER2::LUC signals was used to understand how the rhythmic properties change over time.The phase difference (PD) relationship we reported in our earlier exclusively experimental study30) because between 12 and 36 h, the mean phase difference of the oscillators in our original data set is calculated as 2 h for the AN-PD and 8.4 h for the AV-PD. Since the PD in the initial 24 h in culture is currently interpeted as representing the PD in vivo, this suggests that the AP does phase-lead the NTS and 4Vep, but the mechanism(s) underlying this phase arrangement is attenuated ex vivo.The (PD) dynamics of a combination of 11 previously and newly generated recordings of coronal DVC slice cultures are shown in Fig. While the PD relationship is generally not maintained, we also observed PD time series ranging from unstable , which has period The intrinsic periods of each oscillator were estimated directly from experimental data. For the AP, we calculate this period as the time-averaged period of PER2::LUC bioluminescent signals from surgically isolated and spatially separated AP explants  and AP-4Vep (orange) are plotted in Fig. entclass1pt{minimaentclass1pt{minimaentclass1pt{minimas such that i, j. Reducing s is analogous to reducing all of the coupling parameters by the same fraction, and the result of varying the overall coupling strength is seen in Fig. s (inserts (iii) and (iv)). Our experimentally obtained PD dynamics  time Fig. E. We obsDVC Fig. F. We pre30. The pattern formed by this triad of oscillators is a simple phase ordering where the AP phase leads the three oscillators, with the NTS following approximately 2 h behind and the 4Vep in almost antiphase to the others, reaching its zenith around 9 h after the AP  dynamics. Our results show that the phase pattern is neither completely stable nor unstable ex vivo. In some cases, the PDs that define the pattern are remarkably stable, and in other cases, they are unstable  were modelled using the same phase oscillator framework as in the present study, and intraSCN-wide interactions emerge from many cellular interactions. Taking this approach and simulating single-cell oscillations could yield similar outcomes to those we present here, but the additional complexity provided by a large array of oscillators could aid in resolving why a phase lag is required in the whole-structure description. This is a limitation of a simple phase oscillator description and more mechanistically detailed models capturing the dynamics of intracellular clock gene expression may be necessary66.We restricted our modelling to coarse-grained nuclei-wide rhythms, without treating the single-cell oscillations of the AP and NTS that we report in our earlier work30. This indicates that the AP maintains its rhythmicity by utilizing action potential independent mechanisms, such as gap-junctions or that communication among non-neuronal cells. It is notable that both of these mechanisms are used by the SCN circadian pacemaker69. Using theoretical models to discriminate the role of these and other forms of intercellular communications will be useful in understanding this cell-cell coupling in these structures.A feature that could be included in a range of future models is the existence of both neuronal and non-neuronal couplings. Tetrodotoxin (TTX) blocks action potential-dependent communication, and its application has differential effects on the AP and NTS. The cellular oscillations within the NTS show more pronounced desynchrony compared to the AP following TTX application51. This emphasises that there are unknown processes underlying AP-NTS circadian coupling which require further investigation. An interesting possibility is that the glial barrier separating the NTS from the AP can limit communication between these structures. Experimentally, we showed PER2::LUC expression is rhythmic in this area and that the barrier is more permeable at night than during the day30. Therefore, this barrier is potentially a fourth DVC oscillator that interfaces with both the AP and NTS.Identification of the physiological mechanisms that lead to a phase lag in the AP-NTS coupling is also an aim for future studies. We have used this parameter to tune the AP-NTS phase difference because realistic phase differences are unobtainable without it see . We estiOur model fitting process indicates phase communication is negligible from the AP to the 4Vep and significantly stronger from the 4Vep to the AP, which is reflected by the parameters in Fig. 41. This could position the 4Vep as a receiver and integrator of circadian signals, relaying the rhythmic output of the rostrally positioned hypothalamic nuclei70 to the dorsal vagal complex.It is currently not known how the ependymal cells in the 4Vep communicate with the AP. Unlike neurons, ependymal cells do not have axonal projections and it is unknown whether they secrete factors locally within the DVC to influence the other circadian oscillators. Despite the uncertainty, it is interesting to speculate that the unidirectionality arises as a consequence of the 4Vep cells’ access to the cerebral spinal fluid, which contains oscillating concentrations of signalling factors secreted by other circadian oscillators73 which vary over the day-night cycle. Circadian oscillations within the DVC are altered in unhealthy metabolic or cardiovascular states29, and the relationship between oscillator phases may encode functionally relevant information. Here, we have provided a simple explanation for how phase difference patterns are maintained between the three DVC oscillators. With relatively simple network coupling, a phase model tuned to the DVC can reproduce the phase difference dynamics observed from wavelet analysis of rhythms in PER2::LUC expression. Further experimental and modelling work will be key to illuminating how intrinsic circadian oscillators interact with recurrent input from the periphery to shape the activity of the DVC across 24 h.The DVC serves as a site for integrating central (brain) and peripheral physiology. It is implicated in many processes including regulation of food intake and control of heart and lung activity30 supplemented with new experimental data using laboratory animals. Here, we used nine PERIOD2::LUCIFERASE (PER2::LUC) mice of both sexes, aged from 2 to 4 months. Mice were on a C57Bl6j background and were generated from breeding stock kindly supplied to HDP by Dr. Pat Nolan of MRC Harwell UK. These mice were originally created and generously gifted to the scientific community by the laboratory of Dr. J. S. Takahashi74. All mice were bred in the Animal Services Unit at the University of Bristol under standard 12:12 h light-dark conditions with ad libitum access to water and food. Experiments were performed in accordance with the UK Animal (Scientific Procedures) Act 1986 and were approved by the local AWERB committee. The study was conducted consistent with ARRIVE 2.0 guidelines75.This study is based on a reanalysis of previously published resultsd-glucose (Sigma). For the AP-disconnection study, the APs were totally cut off the DVC using the surgical blade and further placed on the same culture membrane near the remaining parts of the DVC.Nine adult PER2::LUC mice were killed by approved Schedule 1 UK Animal (Scientific Procedures) Act 1986 protocol. Briefly, animals were terminally anaesthetised with the intra-peritoneal injection of sodium pentobarbital (80 mg/kg) and decapitated. Brains were immediately removed and cut into 250 μm thick coronal brainstem slices containing the intermediate part of the DVC (at the level of the AP) in the ice-cold Hank’s Balances Salt Solution  supplemented with 1 mg/ml penicillin-streptomycin  and 0.01 M HEPES (Sigma) using a vibroslicer . Following, the DVC was manually dissected with the scalpel and placed on the 30 mm Millicell cell culture inserts  in glass coverslip sealed Fluorodish culture dishes  with sterile culture medium  supplemented with 0.1 mM luciferin , B27 , 1 mg/ml penicillin-streptomycin (Gibco Invitrogen Ltd), 10 mM HEPES (Sigma) and 3.5 g/L 30, we further binned the datapoints into 1 h epochs. Raw images were initially analysed in FIJI  using a polygon region of interest tool for the whole brain areas.Images were taken with the Olympus Luminoview LV200  fitted with a cooled Hamamatsu ImageEM C900-13 EM-CCD camera and a 76 .Raw PER2::LUC time series were detrended using a sinc filter (cutoff frequency of 48 h) and time-frequency spectrum’s were calculated by a continuous wavelet transform using the Python package pyBOATN bins. High values of y correspond to a prominent peak whereas low values indicate a more uniform distribution of phases. See Supplementary Fig. Phase difference traces were used to construct 15 min-binned histograms (PD measured in minutes). A histogram with a unimodal peak suggests that the PD remained at the value of the peak for a considerable portion of the recording. The prominence of the maximum peak in a histogram was assessed using the metric A state of constant PD between any two oscillators is identified if the rate of change of PD is less than 0.01 rad/h . Under the assumption of identical bilateral oscillations and symmetrical interactions between the AP and the two unilateral structures, we model the NTS as a single oscillator. In the absence of interactions, an oscillator will evolve at its intrinsic frequency, to estimate the coupling parameters in each of the sub-systems individually describing the AN-PD and the AV-PD, we focused on the top 5 most stable PD datasets that were unimodal in their PD distribution  method and 82.The systems of ODEs within this article were solved in Python using the SciPy function Supplementary Information.


Selective dorsal rhizotomy (SDR) can reduce the spasticity in patients with spastic cerebral palsy (SCP) and thus improve the motor function in these patients, but different levels of improvement in motor function were observed among patients after SDR. The aim of the present study was to subgroup patients and to predict the possible outcome of SDR based on the pre-operational parameters. A hundred and thirty-five pediatric patients diagnosed with SCP who underwent SDR from January 2015 to January 2021 were retrospectively reviewed. Spasticity of lower limbs, the number of target muscles, motor functions, and other clinical parameters were used as input variables for unsupervised machine learning to cluster all included patients. The postoperative motor function change is used to assess the clinical significance of clustering. After the SDR procedure, the spasticity of muscles in all patients was reduced significantly, and the motor function was promoted significantly at the follow-up duration. All patients were categorized into three subgroups by both hierarchical and K-means clustering methods. The three subgroups showed significantly different clinical characteristics except for the age at surgery, and the post-operational motor function change at the last follow-up in these three clusters was different. Three subgroups clustered by two methods could be identified as “best responders”, “good responders” and “moderate responders” based on the increasement of motor function after SDR. Clustering results achieved by hierarchical and K-means algorithms showed high consistency in subgrouping the whole group of patients. These results indicated that SDR could relieve the spasticity and promote the motor function of patients with SCP. Unsupervised machine learning methods can effectively and accurately cluster patients into different subgroups suffering from SCP based on pre-operative characteristics. Machine learning can be used for the determination of optimal responders for SDR surgery. Several studies reported that selective dorsal rhizotomy (SDR) is a safe procedure for reducing spasticity of lower extremities in pediatric SCP4. The motor function of these children is significantly improved after SDR when supplemented with rehabilitation therapy5. Patients who is about to accept SDR procedure should meet the surgical indications, which has been established for decades6. Nonetheless, it is reported that different levels of increased motor function were observed among patients after undergoing SDR procedure7. Predicting the possible prognosis of SDR basing on certain pre-operational parameters is of great clinical importance.Treatment approaches for children with spastic cerebral palsy (SCP) are aimed to promote the moving abilities of them8. These mathematical methods can be used for analysis of data in different fields. Machine learning methods have been used in clinical practice for diagnosis, classification and evaluation of heart failure patients with good results9. Therefore, machine learning can also be used for classification of SCP patients before undergoing SDR surgery. Machine learning comprises supervised or unsupervised method, depending on the algorithm used10. Unsupervised machine learning is used for sorting the entire dataset into several subsets based on the level of similarity11. The data in this case has no target attributes, and the computer should determine the inherent structure and patterns of the dataset. The differences between several sorted subsets are clarified by evaluating the characteristics of the different subsets.Machine learning is used for analysis of real-life data using mathematical modelsThe present study sought to classify children suffering from SCP using the unsupervised machine learning method to identify subjects eligible for SDR surgery and improve the outcomes of patients who undergo the procedure.7. In brief, the child was placed in a prone position with the head lowered, and the surgical incision was typically made at the L2–L3 interspace. After laminectomy, we made an incision of about 1.2 cm in the midline of the dura mater. With guidance from neurophysiological monitoring, we carefully test every nerve root/rootlet in the surgical area and to transect or protect nerve fibers according to the rhizotomy protocol7. The post-operative rehabilitation program was applied to these children 3 days after SDR , a score of 1 indicating MAS grade 1 , a score of 2 represented a MAS grade 1+ , a score of 3 represented MAS grade 2 (moderate increase in muscle tone), a score of 4 indicating a MAS grade 3 (significant increase in muscle tone) and a score of 5 denoting a MAS grade 4 (affected part rigid in flexion or extension)13. Muscles assessed in this study included bilateral hip adductors, hamstrings, gastrocnemius, and soleus. Muscles evaluated as MAS score 3 or higher before the SDR procedure were referred as target muscles.Muscle tone of muscles in bilateral lower extremities in all patients was pre-operatively and post-operatively assessed by one physiotherapist using the modified Ashworth Scale (MAS)15. GMFM-66 is an observational clinical tool for evaluation of motor function changes in cerebral palsy16. GMFM-66 scoring system is a four-point-scale comprising 66 items grouped into five dimensions of gross motor function. A 5-year-old child without motor disabilities exhibits the maximum score (a score of 100). GMFM-66 score is highly correlated with the GMFCS grade, but the score is more accurate as a tool for motor function evaluation compared with use of the classification system.The motor function of all patients was examined by one single physiotherapist who conducted muscle tone assessment. The gross motor function classification system (GMFCS) and gross motor function measure-66 (GMFM-66) were utilized to assess the motion ability of the participants. GMFCS is a five-grade classification system for determination of motor function of patients presenting with level I  to level V (dependent on humans and equipment) SCPhttp://www.gmfmer.ca)17. The expected natural evolution could serve as a historical control to measure and compare GMFM-66 score evolution in children with different GMFCS level, and thus normalizing the improvement achieved by SDR and the post-SDR rehabilitation program in the cohort. It has to be mentioned that the monthly expected natural evolution was calculated up to the age of 96 months, and the expected natural evolution of those older than 96 months were taken as zero in this study.Considering that the motor function of children with spastic cerebral palsy would be improved owing to the natural growth, we utilized the equation to calculate the expected natural evolution score over a definite period of time, which is accessible online . After clustering, the post-operative GMFM-66 score change was taken as outcome measure to compare the effect of SDR among these three subgroups.Variables including age, pre-operational GMFCS level, GMFM-66 score, number of target muscles, and MAS scores of bilateral hip adductors, hamstrings, gastrocnemius, and soleus, were used as input data for unsupervised machine learning calculations. Correlation analyses were conducted between the input variables before machine learning analysis. GMFCS level was eliminated from the input data as the correlation coefficient between GMFCS score and GMFM-66 was more than 0.8 . The input variables were scaled before clustering. Subsequently, hierarchical clustering and K-means clustering were performed for the eleven variablest test for statistical comparison, and for data that do not follow normal distribution, we employed Wilcoxon signed-rank test for comparison. For comparisons between subgroups, we use ANOVA with Tukey's test or Kruskal–Wallis test with Dunn's multiple comparison test as appropriate. A value of p < 0.05 was considered statistically significant. Data were analyzed by SPSS software version 24.0 for Windows .Continuous variables with normal distribution were presented as mean ± SD, whereas variables that with skewed distribution were reported as median . For data that follow normal distribution, we used paired This study was conducted in accordance with the relevant guidelines and the Declaration of Helsinki. It is a retrospective study of clinical data and it has been approved by the Ethics Review Committee, Children’s Hospital of Shanghai, Shanghai Jiao Tong University . Because of the retrospective nature of the study, the informed consent for inclusion was waived by the ethics committee of Children’s Hospital of Shanghai.A total of 135 cases (99 boys and 36 girls) were included in the current study. The mean age of the participants was 6.0 ± 1.9 years old, with age of subjects at SDR ranging from 3 to 12 years. All cases were diagnosed with SCP, including 12 hemiplegias, 58 diplegias, and 65 quadriplegias, respectively. The median GMFCS level of the subjects before the operation was level 3, whereas the average GMFM-66 score was 55.8 ± 14.1. The pre-operative muscle tone of lower extremities of the patients was atypical. The median MAS score of bilateral adductors and hamstrings was 3.0, and the MAS score of distal muscles was markedly high with a score of 4.0 for bilateral gastrocnemius and bilateral soleus. Altogether, 6.5 ± 1.8 muscle groups were evaluated ≥ MAS score 3 during the physical assessment and were marked as target muscles.All the subjects underwent SDR. A mean of 66.4 ± 7.5 roots (rootlets) was tested during the surgery using the intraoperative neurophysiological monitoring system, and 11.1 ± 5.6 sensory rootlets were partially incised based on the rhizotomy protocol. The mean follow-up duration for the participants was 568 days, and the anticipated expected natural evolution according to the algorithm increased 1.03 in median value when compared with pre-operational status. During the post-operational assessment, the MAS score of muscles in lower extremities reduced significantly, which in detail, from 3 to 1 in right adductors, 3 to 1 in left adductors, 3 to 2 in right hamstrings, 3 to 2 in left hamstrings, 4 to 2 in right Gastrocnemii, 4 to 2 in left Gastrocnemii, 4 to 1 in right Soleus and 4 to 1 in left Soleus . Principal component analysis was performed to reduce the data dimensionality to two principal components PC1 and PC2) for visualization of the clustering results  and exhibit different degrees of mobility restriction caused by elevated muscle tone. SDR is an effective neurosurgical procedure for alleviation of spasticity in lower limbs and is used for treatment of SCP patients7. In this current study, it is observed that the spasticity of muscles in lower extremities reduced significantly after the surgery, confirming the surgical effect in relieving muscle tone.Cerebral palsy is a group of disorders that affects the ability to move, maintain posture and balance4. The findings of the current study showed that the patient’s motor function significantly improved after SDR procedure with supplementation of the regular rehabilitation, which is consistent with results from previous studies. Differences were observed in post-operative improvement of patients during the follow-up, though all patients met the indications for the surgical procedure. The purpose of this study is to estimate the short-term motor function change by pre-operational physical assessment and other parameters. This might help to select best candidates for SDR. Machine learning is a recently emerging data analysis approach widely used for analysis of data collected in various fields, which was used in the current study for classification of the patients into different subgroups to identify optimal responders to SDR26. Owing to the fact that there’s no standards defining “good” or “bad” surgical outcome, the patients could not be labeled, thus the supervised machine learning could not be used. Patients can only be categorized into various groups based on the similarity of pre-operational clinical characteristics by unsupervised machine learning, and then the surgeons might be able to predict the outcome of the subjects. Postoperative assessment was conducted to evaluate the effectiveness of the clustering method. This method differs from a previous data analysis approach as it compares patients with different outcomes, which is critical in clinical set-ups.Pre-operational assessment of patients is essential, and it is applied by neurosurgeons to determine patients eligible for SDR surgery22. The visualization by principal component analysis showed clear cluster boundaries among different clusters, demonstrating the validity of the two clustering methods. After the clustering, the change of GMFM-66 after SDR showed that different surgical outcome existed among three subgroups clustered by both clustering algorithms. As the motor function promotion at post-operational follow up is much higher than the expected natural evolution of motor function, it is confirmed that SDR is helpful to all the patients with SCP who met surgical indications in this study. The different levels of improvement in three subgroups suggested that all the patients could be categorized into three subgroups, best SDR candidates, good SDR candidates and moderate SDR candidates, in an order basing on the level of post-operative motor function promotion. Analysis of the clinical characteristics of the participants showed that the pre-operational GMFM-66 scores of patients defined as best SDR candidates were the highest relative to that of the other two clusters. They also had the lowest number of target muscle groups and relatively mild degree of spasticity, which in detail, the number of target muscle groups less than 4 and the MAS score of target muscles less than 4. On the contrary, patients defined as moderate SDR candidates exhibited the most severe clinical manifestations, namely, the number of target muscle groups at 8 and the MAS score of target muscles mostly greater than 4.Pre-operational assessment scores of the cases were used as the parameters for the machine learning process in this study. The results showed that unsupervised machine learning through hierarchical and K-means clustering methods effectively and accurately categorized all patients into three subgroups. Principal component analysis dimensionality reduction was conducted to visualize the data and data were compared among the three clusters to validate the accuracy of the classification methods. Principal component analysis is mainly used to downscale data from high-dimensional space to low-dimensional space for utilization in subsequent calculations27. The principle of the algorithm used in hierarchical method is relatively simple. Data are presented as clusters, and the two closest clusters are merged into one cluster until all clusters are combined into a single cluster. Hierarchical clustering results are presented as a dendrogram. In addition, K-means clustering method was used for grouping of subjects in this study. In the K-means clustering approach, a number K is determined by elbow method, and then the computer divides the dataset into K clusters. The algorithm achieves clustering by randomly assigning a number (1 to K) to each data. Subsequently, the cluster centroid for each cluster is computed and each dataset is assigned to the cluster with the closest centroid21. These two clustering methods were utilized in classification of patients as they are relatively easy to understand and are widely used in scientific fields. The results indicated the feasibility of the two strategies. However, differences between the two methods were observed in some patients (7/135). Nonetheless, the two methods were effective in sorting the patients enrolled in this study as the consistency of these two clustering methods was high.Various machine learning methods use different algorithms and principles. Hierarchical clustering method was used for classification of patients in this study. Classification by hierarchical method is achieved by continuous merging of clusters from the bottom to the top or sorting out clusters from top to bottom28. The DBSCAN was also used to classify the dataset in this study. However, the clustering results were not satisfying which were not fully discussed in this paper (Supplementary Fig. Other data clustering methods, such as DBSCAN have been reportedThis study was a single center retrospective study. A relatively small sample size, short follow-up duration and the retrospective nature of the study limit application of the study findings. Moreover, post-operative rehabilitation variables were not included as input variables when classifying the patients due to the challenges in quantification of these variables. Exclusion of post-operative rehabilitation variables in classification may affect the accuracy of the classification results. However, all cases included in this study received standard post-operative rehabilitation therapy; therefore, exclusion of the variables may not have significantly affected the results. What’s more, owing to the nature of unsupervised machine learning, we didn’t split the whole dataset into a training and testing set. Instead, we used the change of GMFM-66 score after SDR to evaluate whether the clustering have clinical significance. Further research should be conducted with a larger population and longer follow-up duration with participants recruited from several centers to verify the validity of the study findings.SDR could relieve the spasticity and promote motor function of patients with SCP. Unsupervised machine learning is a feasible and effective method for clustering SCP patients based on pre-operative characteristics. K-means and hierarchical methods effectively and accurately grouped the patients included in this study. Patients clustered by machine learning approach had different pre-operational clinical features and varying levels of improvement in post-SDR motor function, indicating that the clustering approach can be accurately used for prognosis of SCP patients. The results indicate that machine learning is an effective method for determining eligibility of SCP patients before undergoing SDR surgery.Supplementary Figures.


Compressive postoperative seromas in the cervical spine are a rare but significant complication following cervical laminectomy and instrumented fusion. There is a paucity of cases reported in the literature, with a majority of the reported cases attributing seroma formation to the use of recombinant human bone morphogenetic protein-2 (rhBMP-2). In this article, we report four cases of compressive postoperative seroma in the absence of rhBMP-2 use and highlight similarities in their clinical presentations. We postulate that seroma formation is a significant complication of the dead space that results following posterior instrumentation in the cervical spine, with or without the use of rhBMP-2. The typical presentation is one of the gradual delayed neurological deterioration several days following the index surgery and after drain removal. Neurological deterioration can be reversed rapidly with early recognition and drainage of the seroma. Postoperative seromas causing spinal cord compression in the cervical spine are a rare but significant complication following cervical laminectomy and instrumented fusion. While seromas are not uncommon following posterior spinal surgery, the accumulation of sufficient amounts of fluid to cause mass effect on the spinal cord and neurological deficits and/or myelopathy are much rarer. A large majority of information in the literature on this condition is from case reports that focused on the role of recombinant human bone morphogenetic protein-2 (rhBMP-2) in seroma formation -4, rhBMPCase 1A 67-year-old gentleman with a significant past medical history of severe cardiomyopathy on dual antiplatelet therapy, diabetes mellitus, and chronic kidney disease presented with a one-year history of predominantly left-sided upper and lower limb dysfunction requiring a wheelchair for ambulation. On examination, he had significant spasticity and weakness over his left upper and lower limbs (3-4/5), with positive Hoffman’s sign bilaterally. Magnetic resonance imaging (MRI) of the brain revealed no stroke disease to account for his predominantly unilateral symptoms, while MRI of the cervical spine demonstrated multilevel degenerative changes with severe cord compression over C3-C6 and myelomalacia at C4-C5 as well as a mild kyphotic deformity over the involved segments  putty mixed with morselized autograft from the laminectomies in the lateral gutters following decortication. Hemostasis was secured using an oxidized regenerated cellulose (ORC) hemostatic agent, and vancomycin powder was applied to the implants. The wound was closed in layers over a closed suction drain placed deep into the fascia. Surgery was uneventful with stable neuromonitoring throughout.The patient awoke from surgery with no new neurological deficits, and the drain was removed on postoperative day three after the drain output turned serous and drainage was 60 mL over 24 hrs. Initial rehabilitation in the first week progressed uneventfully. On postoperative day seven, the patient complained of mild weakness of his left-hand grip strength (4/5), which had previously been intact. This subsequently progressed to involve the previously uninvolved right upper limb with loss of motor power in elbow flexion (4/5) and extension (3/5). An MRI was performed in view of his progressive upper limb neurological deterioration, which revealed a large posterior fluid collection that was homogeneously T2 hyperintense compressing the spinal cord  after a Valsalva maneuver. All intraoperative cultures were negative. The wounds were closed in layers over two suction drains placed beneath the fascia. A postoperative MRI scan was performed to confirm the re-expansion of the thecal sac  accompanied by an upgoing plantar reflex in the right lower limb as well as significant ataxia with an inability to stand without support. Motor power was full in the upper limbs. Imaging revealed multilevel degenerative changes with segmental ossification of the posterior longitudinal ligament (OPLL) and severe cord compression over C3-C6 with cord signal changes at C5-C6  following laminectomy, while the left upper limb MEP, bilateral lower limb MEP, and bilateral somatosensory evoked potentials (SSEP) all remained stable.The patient woke up from surgery with no new neurological deficits. The drain was removed on day four after the drain output decreased to 30 mL over 24 hours. On postoperative day six, the patient was noted to have new-onset decreased motor power of his left shoulder abduction, elbow flexion and extension, and wrist extension (3/5). An urgent MRI was performed, which revealed a large T2 hyperintense fluid collection that was compressing the cord  following decompression of the seroma and subsequently recovered back to his preoperative baseline one month later. His ambulatory status improved following a period of rehabilitation. By four months postoperatively, he was able to regain independent ambulatory status.Case 3A 73-year-old gentleman with a known history of hypertension, hyperlipidemia, and cervical myelopathy, for which he had previously declined surgical intervention, presented to the emergency department with an incomplete cervical spinal cord injury  following a fall onto his face on the same day. On admission, he was noted to have significant weakness in his bilateral upper and lower limbs 2-3/5) with a neurological level of injury at C5. Urgent MRI revealed multilevel degenerative changes with severe cord compression over C3-T1, cord signal changes at C3-C4 and C6-C7, and mild cervical kyphosis at C3-C5 . Imaging revealed a cervical kyphosis, which was correctable to <10 degrees on neck extension, as well as multilevel degenerative changes with severe cord compression over C3-C6 and myelomalacia at C3-C4  and grip strength (4/5) compared to his examination on postoperative day one, and an urgent MRI was performed. This revealed a large T2 hyperintense fluid collection that was compressing the cord Figure , Panel BUnlike in the preceding cases, an image-guided aspiration of the collection was performed, which yielded 75 mL of dark hemoserous fluid. No drain was placed. A post-aspiration MRI confirmed adequate drainage of the seroma Figure , Panel CA summary of the four cases is presented in Table Seroma formation is a known complication of spinal surgery, which in the literature has been most commonly attributed to the use of rhBMP-2 ,8. WhileOur four cases presented, which occurred over the course of a short two-year period, highlight that this complication may not be as rare as portrayed in the literature and does not solely occur with the use of rhBMP-2. The clinical course is typically one gradual and delayed neurological deterioration that begins several days postoperatively and after the removal of surgical drains, which would have prevented further fluid build-up, given the serous nature of the fluid. Other cases reported in the literature have described neurological deterioration occurring as early as three days and up to six weeks postoperatively -6,9. NeuThis typical sequence of events is in keeping with the pathophysiology of seroma formation, which authors have attributed to an inflammatory cytokine-mediated response resulting in soft tissue swelling and the production of fluid in the surgical field ,7,8. ThiThe majority of papers on this subject have proposed rhBMP-2 as the main risk factor responsible for seroma formation. Instead, we postulate that in addition to rhBMP-2 use, a variety of other contributing factors may also increase the risk of compressive seroma formation. First, the use of instrumentation using an open technique, in which a wide subperiosteal elevation of the paraspinal muscles is performed, leaves a large dead space for seromas to accumulate. This has been previously raised by other authors as a likely risk factor , and we Second, the use of not only rhBMP-2 but, in fact, any biologically active foreign material, such as allograft , hemostaFinally, the fact that in all four cases, neurological deterioration only occurred after postoperative day five suggests that fluid accumulation in the surgical field likely only peaks beyond this time frame. Premature removal of surgical drains prior to this peak may increase the risk of compressive seroma formation. We recommend keeping surgical drains until daily fluid output is on a consistent downward trend and below 30 mL over 24 hours. In some cases, this may necessitate drains being kept longer than routinely performed.Management of this complication lies first and foremost in its early detection, which begins with being aware of the typical sequence of events and having a high index of suspicion in a patient who may only have mild or non-specific but progressive symptoms in the postoperative course. Early drainage of the compressive seroma generally results in immediate symptomatic improvement. While we elected to re-open the wound for exploration and drainage in Cases 1 to 3, an image-guided percutaneous aspiration was performed in case 4, which yielded equally satisfying results without the morbidity of repeat surgery. This technique was described in a recent study , and we We acknowledge the limitations of our study. Having only small case numbers without a control group, we are unable to draw any definitive conclusions regarding the causation of this complication. However, we believe that the similarities in the cases presented brought forth several hypotheses regarding the risk factors potentially predisposing to postoperative seromas as discussed earlier, which can then be investigated further in future larger-scale studies.These four cases add to the currently limited body of literature describing this complication and highlight the typical presentation of compressive postoperative seromas in the cervical spine. Our cases refute the prevailing understanding that rhBMP-2 is the main culprit for postoperative seroma formation, and we postulate that compressive seroma formation may not be as rare a complication as the literature seems to suggest, with multiple contributing risk factors. We urge clinicians to have a high index of suspicion in the event of gradual neurological deterioration in the days following drain removal, and advocate early MRI scan to confirm the diagnosis. Early drainage of the fluid collection allows good relief of compressive symptoms, and radiologically-guided aspiration may be a feasible alternative to open drainage in selected cases.


Effective treatments for advanced/recurrent head and neck squamous-cell carcinoma are limited. For cases not curable by conventional local therapies, the immune checkpoint inhibitor pembrolizumab shows modest response rates. Quad-shot, a hypofractionated palliative radiotherapy regimen (14.8 Gy in four twice-daily fractions), can provide symptomatic relief, contributes to local control and may potentiate the effects of immune checkpoint inhibitors. In this study, 15 patients with advanced/recurrent head and neck squamous-cell carcinoma will be treated with pembrolizumab combined with up to three administrations of quad-shot before cycles four, eight and 13. Outcomes include disease response, survival and treatment toxicity. Correlative multiomics analysis of blood and saliva will identify molecular biomarkers of response to immune checkpoint inhibitor and the immune-related impact of quad-shot.Clinical trial registration: This study (WFBCCC 60320) is registered on NCT04454489  Advanced and recurrent head and neck cancers are difficult to treat. Most patients receive systemic therapies, such as chemotherapy or immunotherapy, with modest rates of cancer control. We aim to test the effectiveness of an immunotherapy drug called pembrolizumab in combination with a type of low-dose radiation therapy called quad-shot. Patients will receive pembrolizumab every 3 weeks and will be treated with one to three low-dose radiation therapy courses targeted at their cancer in the head and neck approximately every 12 weeks. We plan to measure how well the cancer responds to treatment, how long this response lasts, how long patients survive and treatment side effects. In a pilot study, 15 advanced/recurrent head and neck squamous-cell carcinoma patients will be treated with pembrolizumab plus quad-shot hypofractionated palliative radiotherapy. Response, survival and toxicity will be measured. Head and neck squamous-cell carcinoma (HNSCC) is a significant cause of morbidity and mortality worldwide. Approximately 10% of patients with HNSCC have metastatic disease at presentation, 20–30% will develop metastatic disease after definitive treatment and 40–60% will progress within 3 years after curative therapy . ImmunotThis study protocol aims to address this unmet clinical need by evaluating possible increased ICI effectiveness with the addition of a prospective immunosensitizing strategy and generating molecular and cellular immune-responsive models to define biomarkers predictive of treatment response and improve patient selection for ICIs in A/R/M HNSCC.The currently preferred first-line treatment for recurrent, unresectable or metastatic HNSCC is pembrolizumab as monotherapy or in combination with platinum and 5-fluorouracil chemotherapy, per the results of KEYNOTE-048 , in whicCombining pembrolizumab with chemotherapy may not be the ideal strategy for increasing pembrolizumab response. Although the KEYNOTE-048 study did not aim to directly compare the two arms, there are a few observations worth considering. First, the addition of chemotherapy as utilized in KEYNOTE-048 likely contributed to the observed increases in toxicity compared with pembrolizumab alone. Second, the median duration-of-response for patients responding to pembrolizumab monotherapy was high compared with the control cetuximab chemotherapy arm . Finally, the median duration-of-response for patients who responded to pembrolizumab combined with chemotherapy was much lower , suggesting the absence of a significant synergistic interaction or immunosensitizing effect of the chemotherapy regimen. These findings highlight the limitations of current standard of care immunotherapy-based regimens for first-line treatment of A/R/M HNSCC and the need to investigate combinations of immunotherapy-based systemic regimens with other potentially immunosensitizing therapies. A review of actively recruiting clinical studies investigating ICIs in combination with different immunosensitizing therapies is summarized in Palliative radiotherapy (RT) is a mainstay in the standard of care treatment of recurrent or metastatic HNSCC . PalliatIn prior studies, approximately 64% of patients were eligible to receive more than one QS cycle . The equThe mechanism of conventionally fractionated (1.8–2 Gy per fraction) RT is to induce clonogenic cell death in target cells by generating irreparable DNA damage. This is achieved indirectly through the generation of radical species such as reactive oxygen species and directly by inducing DNA damage . BecauseImmune checkpoint inhibition has been studied in combination with RT in preclinical models with the goal of increasing ICI efficacy through RT-mediated escalation of tumor immunogenicity . The comThe combination of ICI and RT was safe for patients with intermediate- or high-risk HNSCC in preliminary studies ,26. EarlNo clear clinical evidence exists to support the timing of ICI combined with RT. In a mouse model, a synergistic effect was described when RT was given in the days prior to ICI or on the same day . Other pClinicalTrials.gov (NCT04454489).We report the design and methods of a single-center study to investigate the efficacy and tolerability of a short course (or courses) of QS RT combined with the PD-1 inhibitor pembrolizumab. These standard-of-care treatments have the potential to act synergistically to improve response, and this study will investigate the combination of these two treatments in a novel sequence. The clinical hypothesis is that the delivery of one or more courses of QS RT in the proposed sequence with pembrolizumab is feasible and improves both response rates and duration of tumor response. This study will be conducted at the Wake Forest Baptist Comprehensive Cancer Center and is registered on This is a single-arm, nonrandomized study to evaluate the feasibility, efficacy and tolerability of the combination of QS RT and pembrolizumab immunotherapy for patients with A/R/M HNSCC. Fifteen eligible patients will be enrolled and treated with standard of care pembrolizumab at a dose of 200 mg every 3 weeks until progression or treatment intolerance . QS RT cWhenever tumor tissue is available, PD-L1 level will be tested using the Dako 22C3 assay , and resPatients will be removed from the treatment portion of the protocol if one of the following criteria applies: disease progression , unacceptable adverse events, intercurrent illness that prevents further administration of treatment, patient decision to withdraw from the study or changes in patient condition rendering the patient ineligible for further treatment in the judgment of the treating oncologist. New patients will be enrolled to replace those who do not complete at least one QS treatment, those who do not undergo initial imaging response assessment and those who cannot provide scheduled correlative blood and saliva samples.Saliva and blood will be collected for correlative studies. A comprehensive translational analysis will interrogate the molecular and cellular immune response to ICI alone and in combination with RT in order to generate a proposed immune-sensitive profile and to describe the mechanisms of immune sensitization with hypofractionated RT in immune unresponsive patients.Eligibility and exclusion criteria are summarized in The primary objective is to determine the overall response rate for pembrolizumab given with QS RT. Secondary objectives are to determine the following: response rate at the target and nontarget lesions, durability of response at the target lesion, PFS, OS and tolerability of the combination of QS RT and pembrolizumab. The exploratory objectives are to evaluate the effect of QS on pembrolizumab-mediated immune activation and to determine possible mechanisms of any observed immunosensitizing effects of the proposed ICI plus QS RT treatment regimen.The primary outcome measure is the overall treatment response rate according to Response Evaluation Criteria in Solid Tumors 1.1. Follow-up imaging with CT, MRI or PET/CT will be obtained every five to six immunotherapy treatments. For the purposes of tumor measurements, follow-up imaging will be performed using the same modality (CT or MRI) used for baseline imaging measurements. PET/CT may be utilized for follow-up imaging at the clinician's discretion. Response in the target lesion(s) and in other, nontarget head and neck lesions will be assessed and documented during the course of the study by a fellowship-trained neuroradiologist with subspecialty expertise in head and neck imaging. The maximum response documented in the target lesion(s) will address the primary objective. Duration-of-response at the target lesion(s) will be defined as the duration of time from when the measurement criteria are met for complete or partial response (whichever is recorded first) to the first date of documented recurrent or progressive disease. PFS will be defined as the duration of time from registration to the time of progression, death or date of last contact; patients lost to follow-up will be censored. OS will be defined as the duration of time from registration to the date of death or date of last contact; patients lost to follow-up will be censored. The study treatment ends for all patients with the last administration of immunotherapy. Patients will be followed after the conclusion of treatment for PFS and OS outcomes. Subsequent visits can be accomplished in person or over the phone, and clinical and imaging data will be collected from medical records as available. Treatment toxicities will be monitored and documented during the treatment and for 30 days from the end of immunotherapy. Toxicities will be graded according to Common Terminology Criteria for Adverse Events (CTCAE) version 5.0. Patient-reported outcomes will be obtained at each time point using a targeted checklist of head and neck cancer-specific Patient-Reported Outcome–CTCAE version 1.0 items.Saliva and blood will be collected before each ICI treatment for the first five cycles and before RT on the first day of each QS treatment. For patients treated with subsequent QS cycles , samples will be collected before the ICI cycles that are administered before and after each additional QS. Levels of immunoregulatory miRNAs will be quantified in plasma using PCR-based techniques. Immune miRNA analysis will be utilized to create an immune response curve in order to select the best-response sample for in-depth immune response investigation, employing targeted analysis with single-cell RNA sequencing and complementary mass spectrometry-based omics  on peripheral blood mononuclear cells and plasma. We will utilize an integrated data processing pipeline to align and interpret multiomics profiles to identify molecular drivers of response to ICI combined with QS RT. Expression of the top five candidate biomarkers identified, as determined by fold change and statistical significance, will be secondarily validated by western blot or mass spectrometry-based targeted analysis. Oral microbiota profiling will be performed using 16S rRNA gene sequencing to characterize the effect of ICI and the combination with QS RT on bacterial diversity and composition in saliva.The gross tumor volume will be defined as the primary and/or nodal disease to be targeted. The clinical target volume will be defined as areas at risk of subclinical disease around the gross tumor volume that may be delineated at the discretion of the treating radiation oncologist. The planning target volume will be a 0.3- to 1-cm isotropic expansion from the gross tumor volume or clinical target volume (if present). Each cycle of QS RT will comprise 14.8 Gy in four fractions (3.7 Gy per fraction) delivered twice daily (at least 6 h apart) over two consecutive days before the administration of pembrolizumab. All patients will receive at least one cycle of QS RT; up to three QS cycles may be administered per protocol if deemed appropriate by the treating radiation oncologist. The first administration of QS RT will occur before cycle four (between cycles two and three or three and four) of pembrolizumab. If deemed appropriate by the treating radiation oncologist, a second QS may occur prior to pembrolizumab cycle eight (between cycles six and seven or seven and eight) and a third QS may occur prior to pembrolizumab cycle 13 (between cycles 11 and 12 or 12 and 13). Therefore, the total RT dose over the course of the study will depend on the number of QS RT treatments delivered: 14.8 Gy in four fractions for those who complete one QS RT cycle, 29.6 Gy in eight fractions for those who complete two QS RT cycles or 44.4 Gy in 12 fractions for those who complete three QS RT cycles. Upon planning the initial cycle of QS RT, a composite plan will be generated to assess the total dose to the target and organs at risk. Repeat CT simulation may be performed in order to plan subsequent cycles. QS RT will not be given on the same day as pembrolizumab infusion, and it is recommended to occur no more than 7 days prior to the next pembrolizumab infusion.For this single-arm pilot study, the planned sample size is 15 patients. With a sample size of 15 patients, the maximum width of a Clopper–Pearson exact 95% CI is 0.52. Since the interval may not be symmetric, this corresponds to approximately ± 0.26, if the response rate is as high as seven of 15. If the response rate is lower than this, the width of the CI will be less. With this sample size, we will be able to determine whether the response rate in this treatment is sufficient to warrant future examination. Based on the preliminary data presented in According to internal metrics, our institution saw an average of 2.6 patients meeting these inclusion criteria per month over the 3 years prior to study design, and 3.5 arrivals per month were expected over the following year. Considering ineligibility and patient declination, we expect to accrue 15 patients over the course of 2 years.The primary analytic objective is to measure the overall response rate according to Response Evaluation Criteria in Solid Tumors 1.1. We also aim to determine the percentage of patients with either a complete or partial response as well as the corresponding Clopper–Pearson exact 95% CI around this measure. These results will provide useful information concerning the potential efficacy of this treatment for planning future studies.There are six secondary outcomes of interest for this pilot study: 1) response rate for the target lesion(s), 2) response rate for the nontarget lesion(s), 3) duration-of-response for the target lesion(s), 4) PFS, 5) OS and 6) tolerability assessed using Patient-Reported Outcome–CTCAE. The analytical approach for each measure is as follows: 1) for the response rate measures, we will estimate the percentage of responders  and corresponding Clopper–Pearson exact 95% CI; 2) for the duration-of-response among responders, we will estimate the mean and median response duration, with the corresponding 95% CI for the mean and interquartile range for the median; 3) for time-to-event measures (PFS and OS), we will estimate Kaplan–Meier survival curves and median time-to-event times as well as percent PFS and OS at 6 months and 1 year post-treatment; 4) for tolerability, we will estimate the percentage of patients with different adverse events assessed using the Patient-Reported Outcome–CTCAE and corresponding Clopper–Pearson exact 95% CI.Additional exploratory subgroup analyses are limited by the small sample size but will be considered if adequate sample size is achieved in each subgroup. A main consideration for exploratory analysis will be to compare response rates and other oncological outcomes  between patients with HPV-associated oropharyngeal squamous-cell carcinoma and those with non-HPV-associated oropharyngeal cancer/nonoropharyngeal HNSCC.In order to improve therapeutic outcomes for patients with A/R/M HNSCC, we aim to assess response to, survival associated with and toxicity of a novel combination of pembrolizumab and QS RT. Extensive laboratory correlative analyses are planned to elucidate the immune-related impact of this RT regimen combined with ICI. Additionally, the data provided by this clinical trial may provide evidence supporting future confirmatory clinical trials investigating this paradigm.Of patients with head and neck squamous-cell carcinoma, up to 20–30% develop metastatic disease and up to 40–60% experience locoregionally recurrent disease.Systemic therapy alone is associated with limited response rate and survival.Quad-shot (QS) radiotherapy (RT) is a low-dose palliative regimen that provides symptomatic relief and local control.The tumor microenvironment is characterized by changes that shield cancer cells from immune recognition and targeting.Palliative hypofractionated RT induces changes in the immunoregulatory microenvironment, such as increased tumor immunogenicity, induction of MHCs, alteration of cytokine expression and changes in T-cell and antigen-presenting cell function.Hypofractionated RT  is considered to optimally synergize with immune checkpoint inhibition.In this single-center study, 15 patients will be treated with pembrolizumab at a dose of 200 mg every 3 weeks.QS RT (14.8 Gy in four twice-daily fractions) will be delivered at least once prior to cycle four of pembrolizumab and up to three times (before cycles eight and 13).Overall response, target and nontarget lesion response, durability of response, progression-free survival, overall survival and tolerability will be measured.Integrative multiomics, including proteomics, metabolomics, immunoregulatory miRNA expression, oral microbiome analysis and single-cell RNA sequencing, will be performed on blood and saliva samples collected before and during treatment to evaluate the effect of QS RT on the immune response to pembrolizumab.This study of a novel combination of QS RT and pembrolizumab is the first of its kind.Preliminary results will inform future clinical trials, and planned correlative analyses will improve understanding of the mechanisms of pembrolizumab combined with RT in advanced/recurrent head and neck squamous-cell carcinoma.


This systematic review and meta-analysis examines data from studies of patients with first-episode psychosis to estimate the prevalence of clinically relevant neuroradiological abnormalities detected using magnetic resonance imaging. How common are neuroradiological abnormalities in first-episode psychosis?In this systematic review and meta-analysis, we found approximately 6% of patients with first-episode psychosis had an abnormality that required a change in clinical management. The number of patients needed to scan to detect 1 clinically relevant abnormality was estimated to be 18.These findings support the routine use of magnetic resonance imaging as part of the initial assessment in patients presenting with first-episode psychosis. Individuals presenting with first-episode psychosis (FEP) may have a secondary (“organic”) etiology to their symptoms that can be identified using neuroimaging. Because failure to detect such cases at an early stage can have serious clinical consequences, it has been suggested that brain magnetic resonance imaging (MRI) should be mandatory for all patients presenting with FEP. However, this remains a controversial issue, partly because the prevalence of clinically relevant MRI abnormalities in this group is unclear.To derive a meta-analytic estimate of the prevalence of clinically relevant neuroradiological abnormalities in FEP.Electronic databases Ovid, MEDLINE, PubMed, Embase, PsychINFO, and Global Health were searched up to July 2021. References and citations of included articles and review articles were also searched.Magnetic resonance imaging studies of patients with FEP were included if they reported the frequency of intracranial radiological abnormalities.2I index. The robustness of results was evaluated using sensitivity analyses. Publication bias was assessed using funnel plots and Egger tests.Independent extraction was undertaken by 3 researchers and a random-effects meta-analysis of pooled proportions was calculated. Moderators were tested using subgroup and meta-regression analyses. Heterogeneity was evaluated using the Proportion of patients with a clinically relevant radiological abnormality ; number of patients needed to scan to detect 1 such abnormality (number needed to assess [NNA]).Twelve independent studies (13 samples) comprising 1613 patients with FEP were included. Of these patients, 26.4%  had an intracranial radiological abnormality, and 5.9%  had a clinically relevant abnormality, yielding an NNA of 18. There were high degrees of heterogeneity among the studies for these outcomes, 95% to 73%, respectively. The most common type of clinically relevant finding was white matter abnormalities, with a prevalence of 0.9% , followed by cysts, with a prevalence of 0.5% .This systematic review and meta-analysis found that 5.9% of patients presenting with a first episode of psychosis had a clinically relevant finding on MRI. Because the consequences of not detecting these abnormalities can be serious, these findings support the use of MRI as part of the initial clinical assessment of all patients with FEP. Since its introduction more than 40 years ago, structural MRI has become increasingly available, and the costs of scanning have progressively reduced.4 However, there is no consensus as to whether MRI scanning should be a mandatory part of the clinical assessment of patients presenting with a first episode of psychosis (FEP). Some guidelines recommend scanning all patients with FEP,5 while others recommend that MRI be restricted to cases in which a secondary cause is suspected.6The early diagnosis of secondary psychosis, where a psychotic disorder is caused by another medical condition, is an indispensable but complex diagnostic task in psychiatry. Because several causes of secondary psychosis result in structural brain abnormalities,7 to more than 10%.8 Beyond guidelines for individual clinical management, there is also the issue of population health. In otherwise healthy individuals, white matter hyperintensities reliably predict later cognitive decline, greater cerebrovascular risk, and increased mortality in epidemiological studies,10 suggesting that the presence of neuroradiological abnormalities may be an indicator of neurological health.Most radiological abnormalities in patients with FEP are incidental and do not require any clinical action. However, a minority of abnormalities lead to a change to a patient’s clinical care. A barrier to resolving the debate about the routine use of MRI in the assessment of FEP has been uncertainty about the prevalence of clinically relevant abnormalities, with estimates ranging from 0%We sought to clarify the prevalence of intracranial abnormalities in FEP by undertaking the first meta-analysis of such studies to our knowledge. We also sought to establish the prevalence of clinically relevant abnormalities that led to a change in diagnosis or management. In addition, we examined the influence of study, patient, and imaging characteristics on outcome.MOOSE)11 and Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines,12 and the study was prospectively registered on PROSPERO (CRD42020140917). See the eMethods in A systematic review and meta-analysis was conducted in accordance with Meta-analysis of Observational Studies in Epidemiology  or diagnosis. Abnormalities were also grouped into the following neuroanatomical categories: white matter, vascular (excluding white matter), ventricular, cyst, pituitary, tumor, cerebral atrophy, and other  by a psychiatrist (G.B.) and a neuroradiologist (A.M.), with the latter blind to diagnosis  was calculated, based on the reciprocal of the prevalence estimate,18 which is comparable with the numbers-needed-to-treat19 statistic: NNA = 1 / .For each study, the proportion of patients with FEP and a radiological abnormality was calculated, along with the 95% CI based on the Score (Wilson) method.We also estimated the prevalence of the neuroanatomical subtypes of abnormalities  and field strength (3 T vs <3 T) using subgroup analysis based on the Cochran Q statistic. For continuous variables, we explored the effect of sample age, psychosis duration, and year of publication using meta-regression provided at least 6 studies could be included.24Heterogeneity was assessed using the Cochran 25 was performed using the dmetar package.26 This was performed to identify any study with an excessive influence on the pooled effect size and/or that contributed substantially to between-study heterogeneity.Sensitivity analyses were performed to determine the effect of studies (1) with a mean patient age older than 35 years, (2) where assessment was performed by a nonradiologist, and (3) based on a research sample. Influential study analysis using the leave-1-out paradigm8; however, it was possible to estimate the proportion of abnormalities in the FEP subgroup group based on published details and consultation with the study authors . . Antipsychotic status at the time of neuroimaging was reported in 6 samples (n = 714). Among these, the proportion of patients receiving antipsychotic medication was 65%. Duration of psychosis was reported in 6 samples (n = 665) and ranged between 4 and 52 weeks with the exception of 1 study, which had a mean duration of 90 weeks.35The FEP sample size ranged from 20Scanner field strength was reported in 10 samples, with 1.5 T (n = 6) being the most common. MRI scans were interpreted by a neuroradiologist in 9 samples. In the other 3 samples, MRI scans were reported by a general radiologist (n = 1) or a psychiatrist (n = 1), or the clinician was unspecified (n = 1). In 6 samples, raters were blind to clinical status (unreported in 8 samples).The quality assessment score ranged from 4 to 8 of 10 , with a corresponding NNA of 4  . In the other samples, clinically relevant abnormalities were grouped with non–clinically relevant abnormalities.33 The pooled prevalence was 5.9% , with a corresponding NNA of 18 . The 2I statistic was 73%, indicating moderate heterogeneity.The proportion of patients with any abnormality was calculable for 12 samples (11 studies) because 1 study only reported the presence or absence of white matter abnormalities.CI, 3-7) . The I2 As part of the secondary analysis, we calculated the prevalence of specific neuroanatomical abnormalities among patients with FEP . OverallWe also calculated the pooled risk ratio of neuroanatomical abnormalities in patients with FEP vs healthy controls. Patients with FEP had a relative risk of 2.8  for any radiological abnormality compared with heathy controls  or sample age . There were insufficient samples (k = 3) to explore the effect of psychosis duration. Subgroup analysis found no association for the effect of sample type  or field strength .Meta-regression found no association between the prevalence of clinically relevant abnormalities and publication year . For the meta-analysis of relative risk for clinically relevant abnormalities, the study by Sommer et al8 was identified as influential, and removal adjusted the pooled relative risk to 2.1 .We repeated the analysis excluding samples (1) with a mean patient age older than 35 years, (2) where assessment was performed by a nonradiologist, and (3) that recruited patients for research. Removing studies under any 1 of these conditions did not result in pooled estimates becoming nonsignificant. Leave-1-out sensitivity analysis did not identify any influential samples for the meta-analysis of prevalence  and those reporting clinically relevant abnormalities (P = .70).Inspection of funnel plots suggested no clear evidence of publication bias  reported a higher prevalence of clinically relevant abnormalities (10.3%) in their first episode subsample compared with our meta-analytic estimate but essentially found no difference from healthy controls, who had a similarly high prevalence of clinically relevant abnormalities (11.8%).8 This study is notable for being the only one in our meta-analysis that reported the prevalence of clinically relevant abnormalities to be lower in patients with psychosis than in controls and was identified as an outlier in the leave-1-out sensitivity analysis. Studies exploring radiological abnormalities in patients with psychosis using computed tomography (CT) have yielded substantially lower estimates than MRI.39 This likely reflects the relative insensitivity of CT to detect intracranial abnormalities in patients with psychosis.Although the prevalence of neuroradiological abnormalities in FEP has previously been explored in systematic reviews,40 to 2.7%.18 In our study, we were able to derive the first meta-analytic estimate of the relative risk of clinically relevant brain abnormalities in FEP compared with asymptomatic healthy individuals. Our findings suggest a 2-fold increased risk, once adjusted for outliers. Research MRI studies have identified widespread differences in gray and white matter density in FEP compared with controls. However, these studies typically use voxel-based morphometry and involve alterations that are too small to be detected by the naked eye. Although most radiological abnormalities in FEP do not necessitate a change in management, it is worth noting that these apparently benign findings may be associated with relatively poor outcomes across the life span41 and a marker of neurovascular health.42 This suggests that they could reflect the macroscopic sequelae of suboptimal brain development and as such may represent determinants of a poor outcome, even if they do not lead to a diagnosis of secondary psychosis.In otherwise healthy individuals, the prevalence of incidental clinically relevant brain abnormalities found on MRI is estimated to be 1.4%45 White matter lesions are closely associated with neuroinflammatory processes in psychosis,46 as well as immune-mediated neurological disorders such as multiple sclerosis,47 supporting an etiological role of the immune system in psychosis.The most common neuroradiological abnormality was white matter abnormalities, predominantly small hyperintensities. They were also the most common clinically relevant abnormality reported. This finding is consistent with independent neuroimaging evidence that psychosis is associated with widely distributed anatomical and functional dysconnectivity.48Interestingly, we found the prevalence of brain tumors in FEP was very low (with the estimated NNA to detect 1 tumor of around 1000) despite this being one of the main concerns of psychiatrists. However, because all the studies in this meta-analysis excluded patients with clinical evidence suggestive of a secondary medical (“organic”) cause, our results are likely to underestimate the true prevalence of tumors in patients with FEP more broadly, as such cases are more likely to present with neurologic features, such as apraxia, visual field deficits, and anomia.The heterogeneity between studies in the proportion of patients with any type of abnormality was large. In contrast, heterogeneity for clinically relevant abnormalities was moderate. Between-study differences in design, eligibility criteria, neuroimaging methods, and radiological assessment may have contributed to this statistical heterogeneity. We explored its basis using subgroup analysis and meta-regression. The former found no difference between studies based on sample type, rater, or field strength, and the latter found that the effects of patient age and publication year were not significant. We were not able to explore the effect of psychosis duration because of insufficient data.49 was identified as an outlier in the meta-analysis of risk for clinical abnormalities, and its removal resulted in the risk ratio becoming significant. Furthermore, the results remained robust to several sensitivity analyses. Our group-level estimates assumed that each patient had a maximum of 1 type of abnormality, and findings did not change substantially at a group level when we excluded studies in which this assumption could not be confirmed.We assessed the robustness of the findings using sensitivity analyses. One study3 Another approach is to consider the economic implications. The financial costs of a brain MRI vary considerably, and therefore the economic case for routine screening is also likely to vary. In Europe, the average cost is around $264 (€250), including evaluation by a radiologist. Based on the estimated NNA, the cost to detect 1 clinically relevant abnormality is approximately $4752 (€4500). In comparison, the financial cost is substantially higher in the United States. However, the potential costs associated with failing to identify a clinically relevant abnormality  are also likely to be higher. While further analysis is indicated to explore the net economic benefits, provisional evaluation based on clinical grounds would favor offering MRI to all patients with FEP.Should MRI be routinely performed in patients with FEP? One approach to resolving this debate is to consider the net clinical benefit. We were able to ascertain that 1 in 18 patients had a change in management after an MRI, and therefore it could be argued they received some clinical benefit. In contrast, clinical risks associated with MRI scanning are minimal, and most patients find the procedure acceptable.This meta-analysis provides the most precise estimate of the prevalence of neuroradiological abnormalities in FEP in the literature to date. Subgroup and meta-regression permitted the exploration of moderating factors and causes of heterogeneity, such as study characteristics and imaging parameters. Furthermore, by comparing neuroradiological abnormalities in FEP with healthy controls, we were able to determine the specificity of these abnormalities. Importantly, in most studies, FEP samples were matched with healthy controls. Other strengths included a rigorous approach to study identification and data extraction. Furthermore, because the meta-analysis focused on patients with FEP, the findings are unlikely to have been confounded by the influence of chronic illness or its treatment.This study also had limitations. First, the studies we examined may not have included patients who were particularly unwell and/or lacked capacity. Second, around half of the studies involved patients who had undergone MRI as part of research rather than routine clinical care, and all the studies had excluded patients in whom there was clinical evidence of a potential secondary cause (based on examination and/or psychiatric assessment). These factors are likely to have resulted in an underestimate of the prevalence of clinically relevant radiological abnormalities in FEP, suggesting the true figure may be higher. Third, we assumed each patient had only 1 type of radiological abnormality. However, in a few studies, this could not be confirmed, which may have inflated the overall estimate . Fourth, because we used aggregate data, we were unable to explore the influence of potentially relevant patient-level characteristics. Fifth, information on duration of illness and antipsychotic exposure was unavailable in several studies. Finally, included studies mostly consisted of relatively small samples, which reduces statistical precision.52 and delusions of misidentification.53 Systematic assessment of these risk factors could complement the use of MRI to help clinicians identify patients with a secondary etiology. Further research is also indicated to explore the optimal MRI parameters for detecting radiological abnormalities.Follow-up data would help determine the proportion of clinically relevant radiological abnormalities that are treatable. Similarly, it would be useful to clarify whether the presence of radiological abnormalities are associated with adverse long-term clinical outcomes. If this was the case, this may suggest a role for MRI in providing prognostic information in addition to its diagnostic role. Secondary causes of psychosis are associated with particular clinical variables, such as visual hallucinationsThis systematic review and meta-analysis found that around 6% of patients presenting with psychosis have a clinically relevant radiological abnormality on MRI, with a corresponding NNA of 18. These findings provide a rationale for the use of MRI in the clinical assessment of all patients presenting with psychosis. As the availability of MRI increases and its costs decrease, it is becoming increasingly difficult to justify not making MRI a mandatory part of the clinical assessment of FEP.