Gain-of-function mutations in human androgen receptor (AR) are among the major causes of drug resistance in prostate cancer (PCa). Identifying mutations that cause resistant phenotype is of critical importance for guiding treatment protocols, as well as for designing drugs that do not elicit adverse responses. However, experimental characterization of these mutations is time consuming and costly; thus, predictive models are needed to anticipate resistant mutations and to guide the drug discovery process. In this work, we leverage experimental data collected on 68 AR mutants, either observed in the clinic or described in the literature, to train a deep neural network (DNN) that predicts the response of these mutants to currently used and experimental anti-androgens and testosterone. We demonstrate that the use of this DNN, with general 2D descriptors, provides a more accurate prediction of the biological outcome  in AR mutant-drug pairs compared to other machine learning approaches. Finally, the developed approach was used to make predictions of AR mutant response to the latest AR inhibitor darolutamide, which were then validated by in-vitro experiments. Resistance to drug treatments is a common occurrence across many diseases, but it is especially prevalent and lethal in cancers, where it represents a major obstacle for long-term therapies. The acquired drug resistance can be caused by a number of different mechanisms by which cancer cells can escape the treatment . One of The main drug target in PCa is the androgen receptor (AR), a nuclear hormone receptor whose increased activation is one of the principal drivers of PCa. Multiple decades of research on the AR has led to a number of targeted drug treatments that have significantly improved patient survival and well-being. However, despite the major gains in AR targeted treatments, resistance invariably develops to all current drugs ,4. One iPrevious research had identified a number of new AR mutants from circulating cell-free DNA of PCa patients using next generation sequencing technology . The inhNaman et al. , in 2016Recent advances in machine learning theory have led to significant progress and qualitative changes in QSAR and chemoinformatics practice. Interpretable linear QSAR models have become increasingly replaced by support vector machines (SVM), random forest (RF), artificial neural networks (ANN), and other non-linear techniques that demonstrate robust performance on large biological datasets ,14. MoreIn this work, we utilized a DNN, along with general proteochemometric descriptors, to predict more differentiated AR mutant-drug responses on a significantly extended experimental dataset, compared to our previous study. Furthermore, we implemented a structure-independent protocol where, by using protein sequence-based descriptors and 2D drug fingerprints, we avoided the drug docking step in feature construction, saving time and making the model more generalizable. Notably, such an approximation allows the consideration of mutations that occur outside of the ligand-binding domain (LBD) and DNA binding domain (DBD) of the AR, where no structural information is available. As mentioned above, the resulting DNN model distinguishes four AR mutant response phenotypes and provides rather accurate discrimination between them.AR mutant data for training was collected as described by Lallous et al.  in whichiLearn python package [Using the fasta-formatted amino acid sequences of the AR variants (trimmed to only include the range in which the current substitutions occur), mutant sequences were generated for each single or multiple mutant in the dataset. These sequences were then used to generate z-scale descriptors using the  package . Z-scale package ,26. Drug package ,28. Z-scGiven that certain drug response phenotypes are less frequent than others (resistant phenotype is relatively rare), there is a high imbalance between classes that can negatively affect the accuracy of the model (the model can get high accuracy by just predicting the majority class). To address this imbalance, we over-sample the minority classes according to the borderline synthetic minority over-sampling technique (SMOTE) method, which outperformed the regular SMOTE method and random oversampling in cross validation ,30.To justify the use of the more complex DNN method for this task, we benchmarked the corresponding results against outcomes from SVM and RF models. In particular, we obtained 5-fold cross validation predictions by SVM and RF and utilized area under the receiver operating characteristic curve (AUC) statistics. Hyperparameter tuning was performed using a random search 5-fold cross-validation on a dictionary of a range of parameters. The parameters that gave the best validation accuracy were chosen. SVM used an radial basis function kernel with The general overview of our approach can be seen in Interestingly, SVM and random forest have fairly high AUC on the training set, despite the large dimension of the input, and the DNN performs only slightly better than SVM and the same as random forest . HoweverThe shallow models had difficulty discriminating between the antagonist and mixed phenotypes and thus do not generalize well to test data. This can further be seen in We next used the trained DNN model to predict the response of 44 common mutants to darolutamide, a leading AR antagonist which has not yet shown a resistant, mixed-response phenotype. These 44 mutants had been tested with bicalutamide and enzalutamide but not with darolutamide, therefore making them an ideal validation set for our model. To make these predictions, we compute Z-scale descriptors and Morgan circular fingerprints according to the same protocol as above and then use our trained DNN model to predict the response phenotype based on these descriptors. Our model predicts that, of these 44 mutants, 12 are predicted to be non-responsive, 31 are predicted to be antagonized by the drug, and a single mutant, E666D, is predicted to have the resistant, mixed-response phenotype. Prediction results for the two baseline methods are also included; however, both methods under-perform compared to the DNN as they tend to over predict for the mixed-response phenotype.These mutants were then tested experimentally in PC3 cell lines transfected with the corresponding AR mutants with increasing concentration of darolutamide, as described in the experimental methods section and according to a similar protocol used to produce training data. Interestingly, we see that the experimental responses of these mutants match almost exactly to the predictions made by the model, as can be seen in A possible explanation for the lack of resistant phenotype in response to darolutamide may be found by looking at the chemical structures of current anti-androgens . BicalutIn this work, we demonstrated the potential of deep learning in combination with flexible and informative proteochemometric descriptors to predict adverse drug responses of a wide range of acquired AR mutations observed in clinical samples and reported in the literature over the years. Indeed, the predictions made by the model for darolutamide are nearly perfectly confirmed in experimental validation and show that darolutamide appears to be less prone to resistance. However, future work will look to predict and validate many more mutants and double mutants that may be activated by darolutamide. As more data becomes available for this problem, the superior accuracy and ease of use of our DNN approach would be expected to grow. We recognize that the applicability domain of this particular model is limited to mutants of the AR and to compounds similar to the current anti-androgens. However, this modeling approach could also easily be extended to other protein targets and used to predict for other drug compounds, either experimental or in clinical use. The developed DNN model would also be of high practical utility, either in the clinical context by warning of resistance causing mutations to current therapies or in the drug development context to screen lead compounds for their likelihood to elicit resistant phenotypes.PC3 cells lacking the AR were maintained in RPMI 1640 media  and 5% FBS  at 37 https://github.com/osnow/AR_mutant_response. The DNN model was implemented in Keras [All scripts were written in python and are available along with input datasets at in Keras  and the in Keras . Input fin Keras  and the in Keras .Raw AR drug response data were converted to the 4 classes as follows: if the average of response values across all drug concentrations was less than 5% activation, the sample was classified as non-responsive. If the response at 50 Optimal cut-off values for RF and SVM can be seen in


We questioned whether the introduction of a subjective evaluation of patient-specific goals, could be used as a valid method to assess the effectiveness of inpatient rehabilitation.In this prospective cohort study, all admissions to the UHN hemodialysis rehabilitation service between April 2013 and August 2016 were included. We introduced a system of subjective assessment, performed by the team at the time of admission and discharge. We evaluated Functional Independence Measure (FIM®) score and KDQoL for objective measures of physical function and patient-reported quality of life.Success, Partial success, and Not Successful were 28 [interquartile range (IQR) 20–34], 24 [IQR18–31], 16 [IQR 11–34] respectively. The median PCS at discharge for those deemed to have Success was 37.4 , and for those with Partial success & Not Successful 28.8 . There was no correlation with MCS scores .A total of 201 patients were included. The median FIM score at discharge correlated well with the subjective staff evaluation. FIM score changes for those with evaluations for These results suggest the subjective staff evaluation is a brief but valid assessment of patient outcome for dialysis patients undergoing inpatient rehabilitation. The goal of rehabilitation is to optimize an individual’s functioning despite disease, injury or other health conditions. Specific activity goals are determined in the context of the individual’s lifestyle and resources, as well as with regard to how they interact with their physical, attitudinal and social environment . Thus whThe objective of this study was to evaluate if the subjective staff evaluation was validated by objective measures of rehabilitation success  score ) and by Data were obtained from an ongoing prospective cohort study of patients undergoing rehabilitation and hemodialysis (HD) at the Toronto Rehabilitation Institute, University Health Network, Toronto. All admissions to the hemodialysis rehabilitation service between April 2013 and August 2016 were included. Patient characteristics were determined from the prospectively-maintained clinical database and included age, sex, time on renal replacement therapy, cause of renal failure, reason for rehabilitation, comorbidity condition, hemoglobin, albumin, serum creatinine, and depressive symptoms. The study was approved by the University Health Network research ethics board. All patients received a thorough explanation of the study and only patients able and willing to provide written consent were included.not successful’); 30–69% ; ≥70% . To ensure team consensus, a minimum of three different health care disciplines had to be involved in the discharge evaluation .At the time of admission, realistic goals were set by the rehabilitation clinicians and the patient collectively, and documented in the chart . At the The objective change in physical function, attributable to rehabilitation, was quantified using the change in FIM®  score ovA subset of patients also completed the KDQoL-36  and 58.4 [IQR 50.1–63.1] respectively.The subjective staff evaluation corresponded well with discharge FIM® scores  and MCS scores . A similar trend was not seen with change in baseline MCS change .The subjective staff evaluation correlated only weakly with absolute change in FIM® scores (8 IQR 20–4, PartiaThe data presented in this study suggest that the routine use of a staff subjective evaluation is a valid quick assessment tool that corresponds with objective measures of physical function and patient-reported quality of life. The assessment is rapid and easily applied and has the added advantage of being specific to an individual’s needs and goals. We therefore suggest it may be an appropriate method to assess the success of our rehabilitation program although acknowledge that further work is required to assess if it can be used to estimate prognosis and long term outcomes. Similar methods have been proposed in other areas of rehabilitation such as paediatric rehabilitation, neurorehabilitation and in those using prosthetics for example , 5. HoweWe chose to study a small group of hemodialysis patients undergoing rehabilitation as part of a project to assess the value of subjective team evaluation. Although dialysis patients have a unique and often complex pattern of comorbidity, the nature of their impairment and disability is similar to the general rehabilitation population. Consistent with this expectation, recent studies show the magnitude of improvement with rehabilitation, in patients recently started onto dialysis, to be similar to that of non-dialysis patients –8. The sThe data are limited by several factors. We did not include a validation cohort or long-term follow up data. A total of 12 patients did not have FIM data at baseline or follow up for unclear reasons. Another 37 did not have discharge FIM scores, mostly because these patients became acutely unwell and were transferred emergently to another acute care institution. Thus it is possible that our results are biased towards the inclusion only of those doing well with rehabilitation. Similarly a substantial number of patients did not complete both admission and discharge quality of life questionnaires limiting generalizability. Our study is also of relatively small size, and may not be generalizable across units with smaller teams or fewer health disciplines.We propose that the use of a simple staff subjective evaluation is a valid clinical tool that can be easily used to assess improvement in functional and physical aspects after rehabilitation. As little to no change was seen in the mental health scores on the KDQoL, it remains unclear whether the evaluation tool is able to capture changes in emotional well-being, were they to have occurred. Further studies are needed to determine whether this subjective evaluation can be extended to other patient subgroups.


We have previously demonstrated that checkpoint kinase 2 (CHK2) is a critical negative regulator of androgen receptor (AR) transcriptional activity, prostate cancer (PCa) cell growth, and androgen sensitivity. We have now uncovered that the AR directly interacts with CHK2 and ionizing radiation (IR) increases this interaction. This IR-induced increase in AR–CHK2 interactions requires AR phosphorylation and CHK2 kinase activity. PCa associated CHK2 mutants with impaired kinase activity reduced IR-induced AR–CHK2 interactions. The destabilization of AR – CHK2 interactions induced by CHK2 variants impairs CHK2 negative regulation of cell growth. CHK2 depletion increases transcription of DNAPK and RAD54, increases clonogenic survival, and increases resolution of DNA double strand breaks. The data support a model where CHK2 sequesters the AR through direct binding decreasing AR transcription and suppressing PCa cell growth. CHK2 mutation or loss of expression thereby leads to increased AR transcriptional activity and survival in response to DNA damage. Recently, the DDR has been a focus of PCa research since the androgen receptor (AR), a major driver of PCa, modulates the transcription of DDR genes and DNA repair  is activated to maintain cell viability and genome stability . ProstatA repair . We haveA repair .CHK2 is a serine/threonine protein kinase that plays a crucial role in sensing DNA damage and initiating the DDR, which is comprised of cell cycle arrest, DNA repair, and apoptosis . CHK2 coCHK2 association with PLK1 leads to its localization at centrosomes where it regulates mitotic entry . CHK2 auHere, we uncovered novel molecular interactions between CHK2 and AR that provide mechanistic insight into our observation that CHK2 negatively regulates prostate cancer growth. We demonstrate that AR directly binds CHK2, and that this interaction increases with ionizing radiation (IR). The IR-induced increase in AR – CHK2 interaction requires AR phosphorylation on both S81 and S308 and CHK2 kinase activity, which is impaired in PCa associated CHK2 mutations. Furthermore, these CHK2 mutants exhibit diminished effects on prostate cancer cell growth. CHK2 depletion increases transcription of DNAPK and RAD54, clonogenic survival, and resolution of DNA double strand breaks (DSBs) following IR, while altering the kinetics of IR-induced γH2AX foci.We previously showed that AR co-immunoprecipitated with CHK2 immune complexes in several prostate cancer cell lines . To deteWe observed the reciprocal binding of AR and CHK2 by Far western. Expression, purification, and Far western blotting were performed as described above using FLAG-wtCHK2 and FLAG-CHK2 mutants K373E or T387N as targets and HA-wtAR as a probe. This also showed that AR and CHK2 directly bind . In thesSince IR is a standard of care for patients with localized advanced PCa, and CHK2 is a mediator of the DDR, we wanted to assess the impact of IR on AR – CHK2 interactions. Moreover, we wanted to test the influence of AR phosphorylation and CHK2 mutation on any effect of IR on the AR – CHK2 interaction. LNCaP cells were transduced with lentiviral particles containing wtAR or AR mutants S81A or S308A . The assThe requirement for AR phosphorylation for the IR-induced increase in AR – CHK2 association led us to test if AR phosphorylation on S81 and S308 was increased in response to IR. AR was immunoprecipitated from irradiated cells, and phospho-S81 and phospho-S308 were measured by western blotting using phospho-specific antibodies to those sites . There wTo further determine if CHK2 kinase activity was necessary for the AR – CHK2 association and what impact IR has on CHK2 mutant association with AR, we tested if CHK2 mutants that are found in PCa and have impaired kinase activity could interact with the AR by co-IP. We expressed FLAG-wtCHK2, FLAG-K373E, or FLAG-T387N in combination with HA-wtAR in LNCaP cells. FLAG-CHK2 immunoprecipitations were performed and HA-AR association was evaluated . In respSince CHK2 regulates the cell cycle and PCa cell growth , we inveReports in the literature suggest that the AR is a critical regulator of genes in the DDR  ThereforWe next examined the impact of CHK2 interactions on cell survival and sensitivity to IR . LNCaP cTo assess the effect of IR-induced DNA damage in the presence or absence of CHK2 knockdown, we performed comet assays to measure DNA breaks , and immSince supraphysiological doses of androgen have been associated with transcription dependent double strand breaks , we alsopcf.org). While androgen deprivation therapy (ADT) is effective initially, most patients will relapse and develop incurable CRPC. Recently, there has been an emphasis on understanding the link between the DDR and AR, since radiation is a standard of care for locally advanced PCa where the AR is a major driver, and PARP inhibitors may be efficacious in CRPC patients with mutations in DDR genes  and C4-2 (RRID:CVCL_4782) cells (a gift from Dr. L. W. K. Chung) were grown in DMEM:F12 (Invitrogen) with 5% non-heat inactivated serum (Gemini) and 1% Insulin-Transferrin-Selenium-Ethanolamine (ITS) (ThermoFisher). CWR22Rv1 (Rv1) (RRID:CVCL_1045)  and 293T (RRID:CVCL_0063) cells (gift from Dr. Tim Bender) were grown in DMEM (Invitrogen) with 10% heat-inactivated serum. For growth experiments, phenol-red free DMEM:F12 media with 5% Charcoal-Stripped Serum (CSS) (Sigma) was used. Commercial DNA fingerprinting kits  verified cell lines. The following STR markers were tested: CSF1PO, TPOX, TH01, Amelogenin, vWA, D16S539, D7S820, D13S317 and D5S818. Allelic score data revealed a pattern related to the scores reported by the ATCC, and consistent with their presumptive identity. Cell lines are tested monthly for mycoplasm or three passages following reconstitution from frozen stocks.LNCaP (RRID:Transfection: Fugene 6 (Promega); TransIT-2020 (Mirus Bio). Inhibitors: Enzalutamide , BML-277 (Santa Cruz Biotech). Antibodies: CHK2 (2G1D5), pCHK2 T68, ERK1/2 (137F5), Actin, FLAG-Tag, V5-Tag, HA-Tag, γH2AX ; AR, pAR S308 (in-house); pAR S81 (Millipore); Cy3-labeled donkey anti-rabbit (Jackson ImmunoResearch). Western blotting performed as previously described .To measure direct protein interactions, the protocol was adapted from CHK2 or AR protein was immunoprecipitated from 1 mg cell lysate from LNCaP cells transiently transfected with FLAG-wtAR/FLAG-S81A/FLAG-S308A or FLAG-wtCHK2/Flag-K373E/Flag-T387N plus HA-wtAR for 48 hr; treated with radiation. Immunoprecipitations (IP) were performed with either agarose or magnetic beads, proteins were separated by 7.5% SDS-PAGE; and immunoblotted with AR, pAR S81, pAR S308, CHK2, pCHK2 T68, HA, or ERK1/2 antibodies. Non-cropped versions of the Far western and IP western blots are shown in Assay was performed as previously described . BrieflyATGAGTACAAGCCCTGAG); DNAPKc RV (ATATCAGAGCGTGAGAGC) (Tm = 60 deg). RAD54B FW (ATAACAGAGATAATTGCAGTGG); RAD54B RV (GATCTAATGTTGCCAGTGTAG) (Tm = 60 deg). PSMB6 FW (CAAACTGCACGGCCATGATA); PSMB6 RV (GAGGCATTCACTCCAGACTGG) (Tm = 60 deg). The relative standard curve method was used to determine transcriptional fold changes as we have done previously  were performed as previously described . RNA coneviously . RNA staLNCaP cells were transduced with lentiviral particles expressing vector or CHK2 shRNAs and treated with 0-6Gy of radiation 72 hr after transduction. Cells were trypsinized, counted, and appropriate numbers were plated in triplicate with the appropriate growth media for colony formation assays . After 10–14 days, colonies consisting of 50–70 cells were counted using crystal violet. All counts were made by a single observer in a blinded manner using a colony counter pen to maximize consistency and minimize bias. Plotted is the surviving fraction (number of colonies counted/(number of cells seeded x PE) where PE = plating efficiency=number of colonies counted/number of cells seeded) following radiation.All Comet Assay steps were performed in the dark. Cells were washed twice with PBS, scraped and suspended in PBS. Cells were combined with molten LMAgarose  and placed into comet suitable slides. Samples were left at 4°C for 15 min in order to create a flat surface. Slides were immersed in lysis solution  for 40 min at 4°C and then in alkaline solution for 30 min at room temperature. Slides were electrophoresed in 200 mM NaOH, 1 mM EDTA in water; pH >13 at 21Volts (300mA) for 30 min at 4°C. After the electrophoresis, slides were gently drained, washed twice in dH2O for 10 min and immersed in 70% ethanol for 5 min. Samples were left to dry overnight at RT. Samples were stained with SYBR Green at 4°C for 5 min and left to dry completely overnight. For quantification, images were acquired using a fluorescence microscope  equipped with a 20×, 0.5 NA objective and a camera (DP70). Images were acquired with DPController software. Images were analyzed by ImageJ software and graphs generated using Prism (GraphPad Software). All imaging was performed at ∼24°C.LNCaP cells were transduced with lentivirus expressing vector or CHK2 shRNAs on 1 μg/ml fibronectin-coated coverslips and treated with radiation 48 hr after transduction. Cells were allowed to recover from IR exposure up to 24 hr. Coverslips were washed 3X with PBS, permeabilized with 0.2% Triton-X for 5mins, washed 3x quick and 3 × 5 min with PBS-Tween 20 at 0.05% (PBS-T), blocked with 2% FBS/BSA/donkey serum in PBS-T for 2 hr at room temperature, and incubated with γH2AX or 53BP1 antibodies overnight at 4°C. Coverslips were then washed by rocking for 3x quick and 3 × 10 min in PBS-T and incubated with donkey anti-rabbit or anti-mouse secondary antibody (Invitrogen) for 1 hr at room temperature. Coverslips were then washed by rocking for 3x quick and 3 × 10 min in PBS-T and mounted with Vectashield containing DAPI (ThermoFisher). Images were acquired with a LSM 880 confocal microscope (Carl Zeiss) and signals were measured using ImageJ software.Each experiment was performed independently a minimum of three times and each experiment had technical replicates for measuring the endpoint. An independent experiment is defined as an experiment performed on a different day with a different cell line passage number. The number of independent experiments is reported in each figure legend. All data are shown, no outliers were removed. Statistical analysis was performed using GraphPad Prism 8.2.1 and the test used is reported in each figure legend. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:The manuscript presents novel and compelling data on the interaction between CHK2 and Androgen Receptor in prostate cancer cells, by which CHK2 suppresses prostate cancer cell growth. These data explain how CDK2 mutations in prostate cancer lead to increased androgen receptor activity and tumor cell survival in response to DNA damage induction.Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, including Wilbert Zwart as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Jonathan Cooper as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Kavita Shah (Reviewer #3).Thank you for submitting your article "Checkpoint kinase 2 regulates prostate cancer cell growth through physical interactions with the androgen receptor" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.In this study, Gioeli and colleagues build on their 2015 Cancer Research paper showing that AR transcriptionally represses CHK2 and its negative effects on PCa growth and androgen sensitivity, by investigating mechanisms underlying this CHK2/AR cross-talk in the specific context of radiation-induced signaling and DNA damage response. They propose that their findings indicate that wild-type CHK2 can sequester AR and prevent its transcriptional enhancement of DNA repair genes, which in turn confers PCa radioresistance and survival under DNA damage.Overall this is an interesting and conceptually novel study. The authors convincingly show that AR and CHK2 interact physically, that IR transiently stabilizes this interaction, and that AR activity is required for this interaction. However, a number of issues need to be addressed in a revised manuscript:Essential revisions:lower %tail DNA/tail moments relative to the pLKO controls, indicative of radioresistance. Although the AR/CHK2 interaction peaks at 1 hour, this is not the optimal time frame in which to evaluate the levels of unrepaired DNA breaks which would compromise PCa growth and survival by triggering tumor-inhibitory responses to radiation. γH2AX typically appear as punctate foci 6-12 hours following DNA damaging stresses. The diffuse staining in Figure 8B indicates the timepoints of detection are prior to formation of DDR complexes at DSB sites or potentially technical issues . Either issue calls the accuracy of foci quantitation into question.1) Concerns were raised on the experimental design and interpretation of data in Figure 8 and Supplementary Figure 2. Assuming the model in Figure 9 is correct, then under IR treatment, CHK2 depletion should significantly decrease unrepaired DNA breaks by increasing the frequency/efficacy of DNA repair through AR activity. This should manifest, over time, in fewer unresolved γH2AX foci and 2) The alkaline comet assay detects not just DNA double strand breaks but also DNA nicks and abasic sites (through initiation of base excision repair) which will abound following IR treatment. To assess whether these have been repaired properly or not, the assay should be repeated at later timepoints (12-18 hours) when the changes in %tail DNA and tail moments will better represent efficiency and extent of repair. The authors may find a far more striking difference between their control and CHK2-depleted cells, if they repeat the γH2Ax experiment at 12-18 hours following IR when the punctate foci (indicative of repair efficiency or lack thereof) will become more apparent. The difference in comet tail DNA metrics will also most clearly reflect unrepaired genotoxic DNA intermediates at a longer time point. The data, as they stand, do not support their model. Similarly, the relatively small changes in DNA PKc and RAD54 transcripts in Figure 7 do not convince of their assertion that CHK2 depletion increases PCa survival through AR-mediated transcription of DNA repair genes. Such transcript-level changes alone in these factors cannot be extrapolated to their functional effects on DNA repair. The increase in clonogenic survival 14 days post-IR in CHK2-depleted cells is fairly modest and does not necessarily support that CHK2 inhibits radioresistance . As the authors show in Figure 6, LNCaP cells are generally more growth inhibited by wt CHK2 so the differences in Figure 8A may not reflect radioresistance but just a general growth advantage upon CHK2 loss.3) It is unclear what PCa cell growth actually refers to in this study – is the reduction in cell numbers due to a proliferation arrest or cell death? The known CHK2 interactomes could affect either (or both) pathway. Some textual and/or experimental elaboration of this point is warranted, considering this is a central premise of the study.4) The findings from this study taken in context with the previous 2015 study would suggest that the CHK2/AR interaction is mutually inhibitory, as the earlier study suggested that CHK2 is transcriptionally repressed by AR. However, this mutual repression and the notion of negative feedback regulation from CHK2 to AR is not discussed here, for instance in the model in Figure 9. This aspect should be addressed, at least textually, to provide a complete picture of this crosstalk in PCa.blocked IR-induced CHK2-AR interactions". This is a strong statement considering the blot in Figure 3B doesn't appear representative of the quantitation shown – it seems the variants reduce binding but certainly don't seem to block it completely.5) "Kinase-impaired CHK2 variants, including the K373E variant associated with 4.2% of PCa, 6) It seems IR increases the CHK2/AR interaction by increasing CHK2 phosphorylation and therefore its activity, which is an interesting finding. However, both the experimental design and Figure 4 (which describes this effect) are hard to understand. For example, the blots on the left vs. the respective quantitation on the right are confusing. The graphs look different (- or + radiation of the V5-CHK2 cells) but the blots look very similar. It would help if Figure 4 included a schematic of the experimental design.7) Statistical analyses and quantifications should be provided for all figure and supplementary panels, when appropriate .8) Did the authors check whether CHK2 directly phosphorylates AR, and whether this phosphorylation is required for their binding?9) Although the authors show that CHK2 binding with AR peaks at 1 hour, it would be ideal if the others would have tested additional early time points  to exactly determine the peaking of signal. Are these data available?10) As ARS81 is phosphorylated by CDK1, does CDK1 inhibition  impact IR induced association of CHK2 and AR?11) Please check whether the ERK1/2 control blots in Figure 6B (C42) vs. 6C (22Rv1) have been accidentally duplicated. Essential revisions:1) Concerns were raised on the experimental design and interpretation of data in Figure 8 and Supplementary Figure 2. Assuming the model in Figure 9 is correct, then under IR treatment, CHK2 depletion should significantly decrease unrepaired DNA breaks by increasing the frequency/efficacy of DNA repair through AR activity. This should manifest, over time, in fewer unresolved γH2AX foci and lower %tail DNA/tail moments relative to the pLKO controls, indicative of radioresistance. Although the AR/CHK2 interaction peaks at 1 hour, this is not the optimal time frame in which to evaluate the levels of unrepaired DNA breaks which would compromise PCa growth and survival by triggering tumor-inhibitory responses to radiation. γH2AX typically appear as punctate foci 6-12 hours following DNA damaging stresses. The diffuse staining in Figure 8B indicates the timepoints of detection are prior to formation of DDR complexes at DSB sites or potentially technical issues . Either issue calls the accuracy of foci quantitation into question.We thank the reviewers for suggesting that we look at later times for DNA damage and DNA repair activity. When we examine DNA damage using the comet assay we do in fact observe statistically significantly less DNA breaks with CHK2 knockdown at 24hrs. The comet assay shows less % DNA in tail, shorter DNA tail length, and lower tail moment with CHK2 knockdown. Interestingly, we observe more γH2AX foci early and quicker resolution of the foci with CHK2 depletion, suggesting a more rapid resolution of the DNA damage when CHK2 is knocked down. These data are now included in the resubmitted manuscript.With regards to the kinetics of DNA repair following irradiation, data in the literature demonstrate that DSB repair has short half-life of 10 to 60 minutes depending on the type of DNA repair utilized . DSBs are also repaired faster in cancer cells (PMID: 3182330), DSB repair is fast when NHEJ is utilized (PMIDs: 21530420), and the CWR22Rv1 cells have a mutation in BRCA2 and the LNCaP lineage displays “BRCAness” , which results in reliance on the more rapid NHEJ. Additionally, cells in G1 cannot use HR and will use NHEJ to repair DSBs and the majority of the PCa cells are in G1. Furthermore, NHEJ is utilized in G2 (PMID: 18155970). Thus, given the system that we are using, the data in the literature supports the importance of looking at early time points for DSBs and DNA repair foci.In response to IR, γH2AX foci can be cleared in a little as 3 hours , although as noted by the reviewers, foci can remain for much longer and importantly, the remaining low number of foci may reflect unresolved DSB repair that could compromise PCa cell growth/survival. This is reflected in our observation of a statistically significant reduction in DSBs in the CHK2 knockdown. We have also extended our time course to 24hrs and have now included data for both γH2AX and 53BP1 foci in the resubmitted manuscript.We have rigorously optimized foci staining (no primary/no secondary controls) and do our antibody incubations in detergent containing buffer. We have added additional details to our Materials and methods for clarity. We have also developed an unbiased method for foci quantitation as described in Figure 6—figure supplement 1. We strongly believe the reported data are an accurate reflection of the γH2AX and 53BP1 foci in PCa cells in response to IR.2) The alkaline comet assay detects not just DNA double strand breaks but also DNA nicks and abasic sites (through initiation of base excision repair) which will abound following IR treatment. To assess whether these have been repaired properly or not, the assay should be repeated at later timepoints (12-18 hours) when the changes in %tail DNA and tail moments will better represent efficiency and extent of repair. The authors may find a far more striking difference between their control and CHK2-depleted cells, if they repeat the γH2Ax experiment at 12-18 hours following IR when the punctate foci (indicative of repair efficiency or lack thereof) will become more apparent. The difference in comet tail DNA metrics will also most clearly reflect unrepaired genotoxic DNA intermediates at a longer time point. The data, as they stand, do not support their model. Similarly, the relatively small changes in DNA PKc and RAD54 transcripts in Figure 7 do not convince of their assertion that CHK2 depletion increases PCa survival through AR-mediated transcription of DNA repair genes. Such transcript-level changes alone in these factors cannot be extrapolated to their functional effects on DNA repair. The increase in clonogenic survival 14 days post-IR in CHK2-depleted cells is fairly modest and does not necessarily support that CHK2 inhibits radioresistance . As the authors show in Figure 6, LNCaP cells are generally more growth inhibited by wt CHK2 so the differences in Figure 8A may not reflect radioresistance but just a general growth advantage upon CHK2 loss.We are very appreciative of the suggestion to look at later time points for the comet assay. As mentioned above in our response to the first comment, when we look at 24hrs the comet assay shows less % DNA in tail, shorter DNA tail length, and lower tail moment with CHK2 knockdown. This data is now included in the manuscript.The reviewers’ point that the changes in DNA PKc and RAD54 transcripts when CHK2 is knocked down may be insufficient to drive our observations of increased DNA repair and survival with CHK2 knockdown in PCa is valued. This has been a point of discussion in my laboratory since we first examined the level of DDR transcripts reported in PMIDs: 24027197 and 24027196 and only observed differences in DNA PKc and RAD54. Another study (PMID: 30305041) showed that a different set of DDR genes is regulated by AR. The discrepancy in the DDR gene subsets regulated by the AR in these studies may be system dependent, however, it also indicates that the dogma that AR regulates DDR gene expression needs further examination. We have added text in the Results and Discussion of the manuscript that we feel more completely addresses this issue.3) It is unclear what PCa cell growth actually refers to in this study – is the reduction in cell numbers due to a proliferation arrest or cell death? The known CHK2 interactomes could affect either (or both) pathway. Some textual and/or experimental elaboration of this point is warranted, considering this is a central premise of the study.For our earlier study when we first described that CHK2 functioned as a tumor suppressor gene and that CHK2 knockdown increased cell growth, we performed preliminary studies of the cell cycle distribution based on DNA content and apoptosis by western blotting of cleaved caspase and PARP. Normal growing PCa cells have low to not detectable levels of cleaved caspase and PARP, making it difficult to determine if there was a reduction in apoptosis with CHK2 knockdown. This would favor a model whereby CHK2 knockdown leads to increased proliferation. Preliminary cell cycle analysis by flow cytometry in LNCaP cells +/- CHK2 KD indicated an increase in S and G2/M, however, this would have to be explored further to be conclusive. We have not examined proliferation/death in the context of IR. Since DSBs induce apoptosis we expect the increased clonagenic survival with CHK2 knockdown is due to the increase in DSB repair  and corresponding reduction in DSB-induced apoptosis. We have added text to the manuscript addressing this point.4) The findings from this study taken in context with the previous 2015 study would suggest that the CHK2/AR interaction is mutually inhibitory, as the earlier study suggested that CHK2 is transcriptionally repressed by AR. However, this mutual repression and the notion of negative feedback regulation from CHK2 to AR is not discussed here, for instance in the model in Figure 9. This aspect should be addressed, at least textually, to provide a complete picture of this crosstalk in PCa.We have added text discussing the negative feedback model. In brief, we suggest that AR represses CHK2 to turn off the DDR, while CHK2 negatively regulates AR to dampen growth while damaged DNA is being repaired.5) "Kinase-impaired CHK2 variants, including the K373E variant associated with 4.2% of PCa, blocked IR-induced CHK2-AR interactions". This is a strong statement considering the blot in Figure 3B doesn't appear representative of the quantitation shown – it seems the variants reduce binding but certainly don't seem to block it completely.We agree that the CHK2 variants association with the AR is slightly enhanced with IR and not completely blocked. We have changed the wording to more accurately reflect the reduced binding in the presence of IR.6) It seems IR increases the CHK2/AR interaction by increasing CHK2 phosphorylation and therefore its activity, which is an interesting finding. However, both the experimental design and Figure 4 (which describes this effect) are hard to understand. For example, the blots on the left vs. the respective quantitation on the right are confusing. The graphs look different (- or + radiation of the V5-CHK2 cells) but the blots look very similar. It would help if Figure 4 included a schematic of the experimental design.We have completely revised the Far Western data reported in the paper, which is now shown in Figure 1. We now include a schematic of the technique, in Figure 1—figure supplement 1, and thank the reviewer for the suggestion. The data initially reported in Figures 4 and 5 have been removed for clarity and robustness.7) Statistical analyses and quantifications should be provided for all figure and supplementary panels, when appropriate .We have now included a description of the statistical analysis for each figure in the legend.8) Did the authors check whether CHK2 directly phosphorylates AR, and whether this phosphorylation is required for their binding?We have examined whether CHK2 phosphorylates the AR on select sites, as AR phosphorylation has long been an interest of my laboratory. We have spent considerable effort in the intervening time since first submitting our manuscript (and in prior years) in an effort to answer this question. However, our experiments have not yet yielded robust reportable data.9) Although the authors show that CHK2 binding with AR peaks at 1 hour, it would be ideal if the others would have tested additional early time points  to exactly determine the peaking of signal. Are these data available?The data demonstrating CHK2 and AR peak binding at 1 hour was with endogenous proteins in LNCaP cells. To address the question raised by the reviewers we performed co-IP experiments at earlier time points following IR in LNCaP. In the process of conducting these experiments, we learned that the endogenous co-IP does not always work for reasons that are unclear, despite considerable effort on our part to determine reliable experimental conditions for a robust co-IP of endogenous CHK2 and AR. In contrast, the co-IP of exogenous proteins is robust. Therefore, we attempted to use the co-IP of exogenous CHK2 and AR to refine the kinetics of the interaction. However, exogenous expression of CHK2 leads to basal CHK2 T68 phosphorylation making it more difficult to define the kinetics of CHK2 – AR co-IP since the interaction is dependent on CHK2 T68 phosphorylation that is induced by IR. We could show data summarizing multiple replicates of the endogenous co-IP of CHK2 and AR, however, we feel this would be disingenuous without having defined the conditions to reliably perform the endogenous protein co-IP.10) As ARS81 is phosphorylated by CDK1, does CDK1 inhibition  impact IR induced association of CHK2 and AR?We thank the reviewers for raising this interesting question. We have one replicate showing that inhibition of CDK1 with RO3306 reduces CHK2-AR interaction by co-IP. One challenge with this type of experiment is that RO3306 treatment results in complete cell cycle arrest at the G2/M transition. This is an inherent issue with all experiments that pharmacologically inhibit cell cycle regulators – it is difficult to separate the observations due to inhibition of the protein of interest from the fundamental alteration in the cell cycle that could indirectly effect the readout. For that reason, we did not pursue this approach further. The other challenge is that while CDK1 does phosphorylate the AR on S81, our data demonstrates that CDK9 is the predominate S81 kinase (PMID: 20980437). This raises the larger issue that the context of CDK1 phosphorylation of S81 may be functionally different then the CDK9 phosphorylation of S81 that happens during transcription of AR target genes; this question cannot be adequately addressed within the scope of this manuscript.When considering that CHK2 may not be phosphorylating the AR (see response to concern # 8), we asked the fundamental question of whether CHK2 facilitated CDK1 – AR association, with subsequent plans to examine AR pS81. We used CHK2 knockdown to determine the impact on the co-IP of CDK1 and AR. However, when cells were transduced with CHK2 shRNAs, the co-IP experiments of endogenous CDK1 and AR were not consistent, similar to the lack of consistency of endogenous CHK2 – AR co-IPs (see response to concern #9). These technical challenges have limited our ability to further explore this interesting questions at this time.11) Please check whether the ERK1/2 control blots in Figure 6B (C42) vs. 6C (22Rv1) have been accidentally duplicated.Thank you for bringing this to attention. We have ensured that the correct corresponding blots are now shown.


Autologous haematopoietic stem cell transplantation (AHSCT) is increasingly used to treat people with multiple sclerosis (MS). Supported by an evolving evidence base, AHSCT can suppress active inflammation in the central nervous system and induce long-term changes in immune cell populations, thereby stabilizing, and, in some cases, reversing disability in carefully selected MS patients. However, AHSCT is an intensive chemotherapy-based procedure associated with intrinsic risks, including profound cytopenia, infection, and organ toxicity, accompanied by an on-going degree of immuno-compromise and general deconditioning, which can be associated with a transient increase in functional impairment in the early stages after transplantation. Although international guidelines and recommendations have been published for clinical and technical aspects of AHSCT in MS, there has been no detailed appraisal of the rehabilitation needed following treatment nor any specific guidelines as to how this is best delivered by hospital and community-based therapists and wider multidisciplinary teams in order to maximize functional recovery and quality of life. These expert consensus guidelines aim to address this unmet need by summarizing the evidence-base for AHSCT in MS and providing recommendations for current rehabilitation practice along with identifying areas for future research and development. Multiple Sclerosis (MS) is an acquired chronic immune-mediated inflammatory disease of the central nervous system (CNS) and is the commonest cause of non-traumatic disability in adults of working age , 2.Approximately 85% of people with MS present with a relapsing remitting course (RRMS) characterized by distinct episodes of new or worsening neurological dysfunction (relapses) followed by complete or partial recovery (remission) . In the Clinical presentation varies dependent on the site of lesions within the CNS. Clinical features may include abnormalities of muscle strength, sensation, balance, coordination, vision, cognition, speech, swallowing, bladder, bowel, and sexual function as well as tremor, pain, fatigue, heat sensitivity, and changes in mood and personality . MS can Currently, there is no known cure for MS. Medical treatment aims to modify the course of the disease process and control symptoms as they develop . In RRMSDMTs have various levels of efficacy but many patients respond poorly and continue to accumulate disability . There iIn recent years an increasing evidence base and published professional guidelines have supported more widespread use of AHSCT as a treatment option in patients with highly active RRMS. MS is now the fastest growing indication for AHSCT across Europe .AHSCT provides a “one-off” treatment as opposed to DMTs, which generally require on-going administration , 16. AHSAlthough AHSCT is not thought to be directly associated with repair or regeneration of damaged myelin or nerve fibers, previous studies have demonstrated reversal of disability in some patients following treatment which could be explained by the prolonged suppression of inflammation and physiological CNS repair . SecondaOutcomes for individuals with MS could be maximized through rehabilitation; both by optimizing a patient's physical fitness and by guiding and stimulating neuroplasticity. In MS, evidence suggests that neuroplasticity can play a role in limiting the clinical impact of damage . Task orWith AHSCT, there is a rapid and prolonged suppression of CNS inflammation associated with a sustained interval free of immunosuppressive treatments; therefore, the early administration of a neuro-rehabilitation program represents a unique opportunity to maximize its effect, possibly in association with axonal repair.Similar to the medical management for MS, rehabilitation intervention is determined by the symptoms and clinical presentation for each patient and therefore a full clinical assessment is essential with the rehabilitation being goal directed and person centered , 25. ThiCurrently, there is no established rehabilitation pathway for patients with MS either before or after AHSCT. Although pre-habilitation and rehabilitation have been explored in malignant hematological diseases where AHSCT is routinely used , there iThe following guidelines and recommendations have been established by consensus to support therapists with the rehabilitation of patients with MS undergoing AHSCT with the goal of optimizing management and overall outcome.Following approval by the processes of the EBMT Autoimmune Diseases Working Party (ADWP), an authorship group was convened from clinicians from relevant professional groups active in or associated with the ADWP and Nurses Group (NG) of the European Society for Blood and Marrow Transplantation (EBMT) with experience in AHSCT for neurological ADs as well as patient consumer representation via the EBMT Patient Advocacy Committee. As such, this group included therapists, hematologists, neurologists, specialist nurses, and patient representation, with access to patients with MS who had undergone AHSCT. An important point is that several areas of specialism have collaborated to share expertise in respective areas.In the absence of specific studies or guidelines for rehabilitation before or after HSCT for MS, based on knowledge within the authorship group and literature searching, the aim was to produce an expert consensus as a starting point. A systematic review was considered to be premature and therefore was not undertaken. Whilst the structure of the guideline and recommendations broadly considered the principles of the AGREE process , this waThe target readership primarily includes specialist therapists directly involved in planning rehabilitation for patients with MS undergoing AHSCT as well as clinical teams and their members involved in the planning and delivery of AHSCT. As such the guidelines are written in a technical style for the target readership of health professionals, and, although they are not intended as a primary resource for patient information, they should be made accessible to them via their health professionals. These guidelines cover adults, young people, and children. They are intended to be holistic aiming to address physical rehabilitation as well as cognitive, communication, and psychosocial factors. Specific evidence in the area of rehabilitation in AHSCT for MS is very limited, and therefore all recommendations are consensus and based on agreed best practice within the group, whilst the need for future systematic research in specific areas is recognized.The aim of these guidelines and recommendations is to suggest a pathway for rehabilitation, recognizing the challenges that patients face at each stage. A further aim was to generate questions for future research in this field. We have divided the rehabilitation process into four phases:Phase 1: Assessment and pre-habilitation 4 weeks before starting treatment,– Phase 2: Acute rehabilitation,– Phase 3: Sub-acute rehabilitation, and– Phase 4: Community rehabilitation including vocational rehabilitation.– Each phase of the pathway has a different emphasis but each treatment plan must still be tailored to the individual and will change depending on the stage of a patient's rehabilitation. These guidelines therefore outline the assessment process and propose outcome measures and standardized assessment tools as well as guidance on therapeutic interventions.Patients with MS being considered or offered AHSCT should meet the criteria suggested in current EBMT guidelines and recommendations and further updates, and treatment should be approved by an appropriately constituted multi-disciplinary team (MDT) .In line with EBMT recommendations, care should be provided with a co-ordinated multidisciplinary approach by a team responsible for initial assessment of suitability for AHSCT, and an extended team, who facilitate referral, assessment, and delivery of rehabilitation throughout the four phases of the pathway.The MDT membership should include:Neurologist(s) with an interest in MSHematologist(s) with an interest in HSCTSpecialist MS nurse(s)Specialist HSCT nurse(s)Rehabilitation specialist(s)Specialist therapists  including physiotherapists, occupational therapists, speech and language therapistsNeuropsychologists and counselorsDieticiansPain management specialistsInfectious disease specialistsOther specialist nurses including continence and tissue viability nurses, late effects nursesGPs and district nursing teams.For detailed general and MS specific literature on AHSCT, please refer to EBMT guidelines for AHSCT in MS and neurological diseases , 28, 29 The process is summarized in Pre-transplant workup, including wash out of DMTs Patients should be reviewed by both MS neurologists and transplant Hematologists who are experienced in using AHSCT in this context. Patients are initially assessed and provided with extensive counseling to ensure that they are familiar with the benefits and risks of AHSCT ] which results in ablation of haematopoietic and immune cells throughout the body  requiring blood product transfusions, and a range of other organ toxicities . Worsening of existing neurological symptoms can occur often in the context of fever, drug reactions, and infection . Neurolofunction . Thus, afunction .Secondary complications include deconditioning, reduced caloric intake due to nausea and mucositis and limited activity in the immediate post-transplant period . ProlongLong-term effects include risks of infertility , autoimmUtilizing a biopsychosocial model, rehabilitation aims to optimize a patients' health, functional independence and well-being, comprising a comprehensive programme covering the physical, cognitive, psychological, and social aspects of their care. Previous recommendations have advocated the need for physical therapy before and after HSCT to promote recovery of functional capacity and improve quality of life . These wThe paucity of evidence in the field of rehabilitation means that it is not possible to make firm recommendations. However, a summary of the basis for AHSCT alongside consensus guidance is presented to support therapists and clinicians caring for these patients to optimize collaborative care and identify areas for future development.beginning of acute treatment and would include physical and psychological assessments that establish a baseline functional level, identifies impairments, and provides targeted interventions that improve a patient's health to reduce the incidence and the severity of current and future impairments”  should be recorded at key points throughout the patient's journey to monitor any change in key domains within the biopsychosocial model of care as outlined by the World Health Organization's International Classification of Functioning, Disability and Health .Patients should undergo a detailed assessment , 35 at tThere are no tools or OMs specifically available for use for patients with MS receiving AHSCT and future research into this area is needed. Whilst these guidelines suggest a spectrum of tools and OMs, these are not exhaustive and must be used based on a patient's presentation and their phase of rehabilitation. For example, a patient may not be able to mobilize acutely in the initial days following their transplant, and a 10 m walk test will only be appropriate when they progress and start to mobilize.To assess someone's cognitive function, there are a number of standardized assessment tools. For example, the Minimal Assessment of Cognitive Function in MS (MACFIMS), and the Montreal Cognitive Assessment (MoCA) have been shown to be a valuable screening tool for MS patients , 39. TheFor some patients, an assessment of their cognition through function may be more beneficial. For speech therapy, there are assessments for dysarthria for example the Frenchay Dysarthria Assessment (FDA-2)  and for Much like the assessment tools, a series of reliable and validated outcome measures (OMs) should be recorded at key points throughout the transplantation treatment to monitor any change in key domains within the biopsychosocial model of care as outlined by the World Health Organization's International Classification of Functioning, Disability and Health . In accoThe National Multiple Sclerosis Society suggested a number of measures  but Cohe– 10 m walk test– Berg balance– Modified fatigue impact scale– Rivermead Mobility index– MS QOL 54 measure– Dynamic gait index– Activities of daily living (ADL)– Nine hole peg test.Prior to the transplant, it is recommended that baseline level of impairment function and participation is ascertained through a formal MDT assessment process. This should include a full medical and physical assessment, including a medication and social history A physical assessment should include joint range of movement, muscle strength, length and tone, balance, sensation, cerebellar signs, proprioception, movement analysis, and endurance including respiratory function. The patient's cognition, swallowing, communication, nutritional status, functional independence, and endurance should also be assessed including determining how they transfer, mobilize, and complete personal activities of daily living including washing, dressing, feeding as well as their ability to do domestic tasks. A range of standardized outcome measures normally form part of the full assessment (see above).It is important that any risk factors for potential deterioration are identified. These may include fatigue, existing muscle weakness or spasticity, reduced respiratory function, poor cough, contractures, pressure sores, postural changes, urinary tract infections and other infections, constipation, deconditioning, compromised nutrition, and pain . As wellIn Phase 1, the key focus is optimizing the patients' physical, social, and emotional functioning and well-being prior to them undergoing AHSCT. Patients should be given individualized advice and education to help them self-manage their rehabilitation and prevent risks occurring. Advice leaflets provide guidance on exercise, nutrition, and any necessary lifestyle changes supporting a self-care and self-management paradigm. If appropriate, carers and family members should also be provided with advice and education to support the individual. Specialist consultation may be necessary/beneficial in some cases.During phase 1, “Pre-habilitation,” can be provided as part of an intensive community rehabilitation package or in an outpatient setting. Residential rehabilitation may be required based on a patient's presentation. Pre-habilitation during this phase will be patient centered based on a full assessment.The aims of this stage are not only to enhance neuromuscular systems and respiratory function but also to reduce the risk of secondary complications. The following should be considered, as appropriate:– Breathing exercises and the use of respiratory adjuncts to optimize respiratory function .– Cardiovascular exercise. The evidence suggests that adults with MS should engage in at least 30 min of moderate intensity aerobic activity twice a week as well as strength training of major muscle groups biweekly to optimize fitness . High im– Strengthening and stretching programmes – Spasticity management – The use of neuro-technologies – Cognitive rehabilitation , 57, 58,– Nutrition ,– Relaxation ,– Fatigue management ,– Pain management – Providing strategies to accomplish transfers and ADL  and– The provision of equipment including aids and orthotics as appropriate.All activities should be underpinned by a self-management approach, where possible.Rehabilitation during this phase will be goal directed and patient centered helping prevent secondary problems through gentle mobilization. As such it aims to prevent cachexia and optimize respiratory function. At this stage, the provision of appropriate levels of exercise will be influenced by platelet count and fatigue. In most cases platelet count will have recovered by day +15 post-transplant although secondary immune thrombocytopenia may occur in 5–7% of patients later on. Mental health and well-being should be monitored throughout this phase.The main consideration for rehabilitation at phase 2 is the individual's immunity. The immune system begins to recover around 2 weeks after infusion of the blood stem cells, during which patients are likely to remain in hospital for close monitoring and continuation of supportive treatment such as antibiotics. Even with neutrophil recovery, the immune system remains suppressed for several months. Any infections may adversely affect patients physically and cognitively . InfectiFollowing AHSCT, patients invariably have cytopenias increasing the risk of infection and bleeding. At this time, physical function does need to be monitored so it is essential that skilled therapists are involved in guiding rehabilitation.Thrombocytopenia is sometimes considered a contraindication to exercise and therefore platelet count should be monitored and exercise prescribed as appropriate. A previous consensus publication  has prop9/L contraindication to exercise– <20 × 109/L—gentle non-resistant exercises– 20–30 × 109/L—minimal resistance (0.5–1 kg exercises)– 30–50 × 109/L—progressive resisted exercises– 50–150 × 109/L—no restrictions.– >150 × 10Following the transplant and when medically appropriate, the individual is reassessed in line with the WHO ICF  by an apFollowing the assessment, carers including nursing staff and family should be provided with manual handling advice to facilitate safe transfers and mobility whilst encouraging independence, provided there is no negative impact. They should also be provided with any positioning or seating guidance to optimize respiratory function and posture and to aid spasticity management if this continues to be a problem. These patients will also need advice on minimizing the risk of infections, which may include breathing exercises and be on an appropriate diet.The following should be assessed by an MDT as appropriate:Posture—in lying, sitting, standing, walkingSeatingProvision of any specialist equipment requiredRespiratory statusTransfers and mobilityEnduranceSwallowCommunicationCognitionPersonal activities of daily living and participation.For specific symptom management, please see Multiple Sclerosis in Adults: Management . The fol– Cardiovascular exercise—many post-AHSCT patients have reduced aerobic capacity and decreased physical activity. Physical activity can restore exercise tolerance and improve function in patients after AHSCT .– Progressive strengthening training can improve walking and balance but should be carefully prescribed for individual patients – Stretching programme as appropriate – Positioning and seating– Spasticity management—commenced or continued with specific exercises, medication, and splinting as appropriate – Handling, facilitation, and specific exercise techniques for postural realignment– Mobility progression – Balance exercises – Functional task practice– Swallow guidance – Communication strategies – Cognitive rehabilitation , 57, 58– Fatigue management—please note that graded physical therapy has been shown to be effective in minimizing post-HSCT fatigue – Pain management .During this phase, the patients need to be seen every day by a physiotherapist to ensure that their respiratory function is monitored and optimized and their physical function is assessed as well as receiving rehabilitation specific to their needs. Each patient will respond and react differently to the transplant process so each rehabilitation package is bespoke. Regular occupational therapy input is also required to ensure that independent function is optimized and speech and language therapy is provided as required.Following the transplant and when medically stable, the individual should receive a period of intense inpatient or outpatient rehabilitation to optimize physical fitness, independence, and the outcome of the transplant. The timing of this will depend on the patient. This will likely be 8 weeks following the transplant, as prior to this, patients will be too weak to fully participate and are at the highest risk of EBV/CMV reactivation. Even with neutrophil recovery, the immune system remains suppressed for several months and patients may need readmission for infections and other complications. Therapists and patients also need to be aware that there is a risk of “late effects” associated with AHSCT. These may be the result of the transplant regimen and altered post-transplant immune reconstitution, but may also be driven by pre-treatment of the underlying neurological disease .The rehabilitation in this phase should be aimed at treating both the neurological presentation and addressing the neuromuscular weakness and other disabilities now that the active inflammation has subsided.Rehabilitation should include a full assessment and a treatment programme as above and should aim to further progress the patient. Therapy should consider cardiovascular workout equipment as well as the use of neuro-technologies where possible to optimize the effects of the AHSCT. Neuro-technology enables repetitive task practice. More evidence is emerging with regards to their benefits in neuro-rehabilitation . Neuro-tOverall rehabilitation should follow a biopsychosocial, multi-disciplinary model with the patient having an allocated key worker, accessing the health care professionals required with a structured rehabilitation approach including access to groups and rehabilitation assistance sessions where appropriate. Utilizing a patient centered approach, patients and their carers should be involved with their goal setting. There should also be family meetings if required and opportunities for education to fully engage the patient in their rehabilitation. Following a self-management and self-care approach will ensure motivation, self-efficacy and the sustainability of behavior change once the patient has been discharged from formal rehabilitation.The rehabilitation programme provided in this phase should be intensive. The intensity will vary depending on the patient and their pre-morbid and post-transplant presentation. For active individuals who tolerated the transplant procedure well, up to 4 h of therapy intervention could be received in a typical working day with input from physiotherapy, occupational therapy, speech and language therapy, and psychology as appropriate. Therapy groups can be considered providing immune status is taken into account.Manual handling advice as well as seating and positioning programmes should be reviewed and any changes communicated as appropriate. As well as their therapy sessions, patients should be provided with exercise programmes to complete in their own time as part of their self-management programme. Patients should also be provided with advice regarding rest periods and fatigue management. The discharge planning and links into the community are made at this time. This includes exploring any on-going community support and return to work programmes.This phase of rehabilitation occurs when the individual has been discharged home. Therapists and individuals need to be aware of late-effects of the AHSCT. A fever episode might cause deterioration in function but patients should be made aware that such conditions are usually fully reversible. The focus of rehabilitation during this phase is a continuum of the inpatient goals within their normal home environment, integrating the patient back into their home life and promoting independence. It is important that patients continue with their individualized rehabilitation programmes and progress their mobility and independence as able.Returning to work in a timely manner is important for this patient group as many are in employment and may have dependants. Patients are generally not encouraged to return to work until 3 months post-transplant due to infection risks and in some cases delayed up until 6 months post-transplant depending on immune recovery. When and as appropriate, patients should be referred to appropriate vocational rehabilitation services for support. Specialist occupational therapists are crucial to carry out pre-employment physical and functional testing and liaising with the patient and their employer about returning to work . A speciIt is important that on-going support and guidance is available after the patient has returned home. This may require referrals to appropriate community-based teams including therapy teams, specialists  or social services. If appropriate, carers and family members should also be provided with advice and education to assist the individual going home. This may include advice on manual handling including the use of equipment, positioning, seating, and nutritional or feeding advice.Regaining neurological function can continue in the first couple of years following AHSCT and it is important to optimize this period of time with a tailored, comprehensive rehabilitation programme. Patients who have undergone AHSCT should have a comprehensive review of all aspects of their care at least once a year, which needs to be carried out by healthcare professionals with expertise in MS and its complications. The review process and follow up treatments must be individually tailored to need. It may result in onward referral for further specialist support e.g., spasticity management, orthotics or specialist equipment available as required. Hydrotherapy can be considered providing immune status is taken into account by transplant physician and specialist hydrotherapist.The consensus recommendations are summarized in FR, HH, JS, BS, and HJ conceived the proposal and developed and wrote the manuscript with the other co-authors after approval by the EBMT Autoimmune Diseases Working Party . FR, HH, CB, and SM are co-opted into the ADWP as physiotherapists with established experience and interaction with EBMT transplant centers and their patients. BS, JB, GM, PM, and AI are neurologists specialized in MS, including experience in AHSCT. JS, RS, MK, RG, KK, BW, and MP are hematologists experienced in AHSCT for MS. HJ is the EBMT Nurses Group representative on the ADWP. BV is the Chair of the EBMT Patient Advocacy Group, and thereby provided patient representation. FR and AI created the figures. All authors reviewed the final manuscript.FR and HH work for Hobbs Rehabilitation, JS declares honoraria for speaking at educational meetings from Sanofi, Jazz, Janssen, Gilead, and Mallinckrodt, BV declares honoraria for speaking at educational meetings and consultancy from Janssen, Takeda, and Amgen. PM reports travel support and speaker honoraria from unrestricted educational activities organized by Novartis, Bayer HealthCare, Bayer Pharma, Biogen Idec, Merck-Serono, and Sanofi Aventis. He also discloses consulting to Magenta Therapeutics and Jasper Therapeutics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer EP declared a shared affiliation, with no collaboration, with two of the authors RS and CB to the handling Editor. The Handling Editor declared a past co-authorship with one of the authors GM.


Direct overlay of US images onto the according anatomy is possible with augmented reality (AR) technologies. Our purpose was to explore the performance of US-guided needle placement with and without AR in situ US. The punctures were performed in two different phantoms, a leg phantom with soft tissue lesions and a vessel phantom. Time to puncture and number of needle passes were recorded for each puncture. Data are reported as median [range] according to their non-normal distribution.Three untrained operators and two experienced radiologists performed 200 US-guided punctures: 100 with and 100 without AR in situ US resulted in reduced time  and number of needle passes  compared to the conventional technique. The initial gap in performance of untrained versus experienced operators with the conventional US  was reduced to 12.5 s [3–101] versus 13 s [3–100] and 1 [1–4] versus 1 [1–4] when using AR in situ US, respectively.AR in situ US could be a potential breakthrough in US applications by simplifying operator’s spatial orientation and reducing experience-based differences in performance of US-guided interventions. Further studies are needed to confirm these preliminary phantom results.AR  In sAugmented reality (AR) is increasingly gaining impact in medicine, particularly for guidance purposes since its introduction in the 1980s –12. Somein situ US view with the US image as hologram, displayed at the exact anatomic location in reality, creating a direct sonographic view for the operator. We hypothesised that AR in situ US would decrease inter-operator variability in performance, by simplifying the challenge of spatial orientation.We investigated if US with AR technology would achieve an AR No institutional review board was needed for this prospective phantom study.in situ US system is composed of a conventional US system , custom developed software, industry-grade head-mounted AR displays  and physical extensions of the US transducer handles .Three untrained operators (orthopaedic surgeons) without any experience in US and two experienced radiologists (with 9 and 11 years of training) performed 200 US-guided punctures with a linear transducer using a conventional US  and with the AR in situ US, and > 4 weeks later, each operator repeated the punctures using the conventional US, in order to avoid a possible training effect after the first puncture session. The location of the needle tip was documented after each puncture by an experienced radiologist in US in the leg phantom and verified by fluid aspiration in the vessel phantom. Time to puncture and number of needle passes as well as location (correct or incorrect) of needle tips were documented for each puncture using the Redcap software  [The punctures were performed using a 20-gauge yellow needle (7-cm long) in a leg phantom  including 20 soft tissue lesions of varying lesions ranging from 4 to 11 mm in diameter. The leg phantom size was 81 × 20 × 20 cm; the weight 11.5 kg. Ten of those lesions were marked and numbered, ensuring that each operator punctured the same lesions in the same order Figs. a. Furthele, USA) .Fig. 2TDescriptive statistic was performed using the software PRISM , USA). Medians and ranges were used to report the non-parametric data. By purpose, being this a proof-of mechanism phantom study for which sample size was not preliminarily estimated, and based on consultation with the statistician, we did not perform statistical testing for significance.in situ US  compared to the conventional US technique  compared to conventional US ; conventional US, 94% (94/6)).The time to puncture was overall reduced using AR in situ US, the difference between experienced and untrained operators was smaller  compared to the untrained operators  Fig. . When usin situ US view with the US image as hologram, displayed at the exact anatomic location in reality, would reduce inter-operator variability in performance, by simplifying the challenge of spatial orientation. As expected, we observed a relevant difference in performance of untrained versus experienced operators using conventional US-guided punctures. This difference decreased when enforcing untrained operators with AR in situ US technology. The operator dependency is taught to be caused by differences in ability for spatial orientation. We assume that AR in situ US simplifies spatial orientation for US-guided interventions, since the US image is directly superposed on the anatomy in a 1:1 ratio and therefore the operator can directly aim the tip of the needle into the US image.Ultrasound-guided punctures might be challenging and require training and expertise to be reliable and reproducible. We investigated if the combination of US with AR technology achieving an AR in situ US saved time and needle passes thorough all operators. We think this can simply be explained as the time turning the head to the monitor’s image can be spared. Therefore, it is plausible that untrained operators needing more corrections also needed to turn their head more often to the monitor and thus needing more time in total compared to the radiologists. The differences between untrained operators versus radiologists were more pronounced in the leg phantom, as the punctured targets were more difficult to puncture—the targets to puncture were in different depth within the soft tissue and deeper below the surface compared to the vessel phantom, where the blood vessel was a few centimetres below the surface and was for all punctures at the same height.Not only the operator dependent differences were diminished, but also intraindividual differences, such as AR in situ technology; however, only needle placement accuracy were compared between the two methods (AR US versus standard US). Maas et al. [There are only few comparable US studies published using a similar technique with tracking the US probe and overlying the US image to its anatomical position , 20. Thes et al. . The mans et al. , 21, 22.in situ US, which could be important in case of patient motion, particularly considering other potential application such as breast mass biopsies, liver biopsies, and also injections for magnetic resonance arthrography or therapeutic joint injections. One other advantage of the use of AR in situ US using a head-mounted device is the better mobility and easier handling of the entire system. Further, even if we investigated feasibility with a linear probe, other probes (independent of the manufacturer) could be easily adaptable to the here presented technology. Also, the cost of head-mounted devices has decreased over the last decades rapidly, making a potential cost-effectiveness of such system realistic in future.Other advantages here not investigated could be the direct view to the patient at all times with AR in situ US; on a separate day (> 4 weeks later), the same punctures were repeated using the conventional US. With this, we tried to reduce the difference of operator-dependent performance as a result of a potential learning curve.Since this is the first study describing this potential breakthrough in US imaging, comparable study methodologies were difficult to identify. Two different phantoms, namely a leg phantom with soft tissue lesions and a phantom with vessels, were used to simulate two common clinical applications. Certainly, this is one of the main limitations of the study. External validity for clinical situations is not claimed and is subject of current investigations at our institution. We identified a further limitation that was difficult to address in designing this phantom study: repetitive interventions allow a learning curve and could have biased the measured outcomes. Therefore, first, the initial punctures were all performed using AR in situ US technology could be a potential breakthrough for US-guided interventions by simplifying the operator’s spatial orientation, with a potential for reducing the inter- and intra-operator variability.In conclusion, this proof-of mechanism phantom study showed that AR