Is prenatal exposure to triptans associated with an increased risk of attention-deficit/hyperactivity disorder (ADHD) in offspring?This population-based cohort study of 10 167 children found no associations between prenatal exposure to triptans and ADHD diagnosis or ADHD symptoms among offspring.These findings suggest that there is no increased risk of ADHD in offspring associated with prenatal exposure to triptans. Triptans are commonly used in the treatment of migraine. Prenatal exposure to triptans may be associated with adverse fetal neurodevelopment; however, there is limited information about the long-term safety of triptan use during pregnancy.To examine the association between maternal use of triptans during pregnancy and diagnosis and symptoms of attention-deficit/hyperactivity disorder (ADHD) among offspring.This study used data from the Norwegian Mother, Father and Child Cohort Study (recruitment 1999-2008), linked to national health registries. Live-born singleton children born to women with migraine before or during pregnancy were included. Two analytic samples were defined: one to assess ADHD diagnosis and one to assess ADHD symptoms. Data were analyzed from May 1 to November 30, 2021.Maternal self-report of triptan use during pregnancy. Exposed children were compared with 2 groups of unexposed children whose mothers reported migraine (1) during pregnancy and (2) before pregnancy only.An ADHD diagnosis was defined as diagnosis of hyperkinetic disorder or receipt of dispensed ADHD medication. Symptoms of ADHD at 5 years were measured by the Conners’ Parent Rating Scale, where a higher score indicates more symptoms of ADHD. Cox proportional hazards regression models and generalized linear models with inverse probability weights were used to estimate weighted hazard ratios (HRs) and standardized mean differences, respectively, with 95% CIs.The ADHD diagnosis sample comprised 10 167 children , and the ADHD symptoms sample comprised 4367 children . Children were followed up for a mean (SD) of 10.6 (2.2) years. Children with prenatal triptan exposure had no increased risk of ADHD diagnosis compared with unexposed children whose mothers had migraine during pregnancy  and compared with unexposed children whose mothers had migraine only before pregnancy . There were no differences in ADHD symptom scores between exposed and unexposed children.The findings of this study suggest that there is no increased risk of ADHD among offspring associated with prenatal exposure to triptans. This cohort study uses data from the Norwegian Mother, Father and Child Cohort Study to examine the association between maternal use of triptans during pregnancy and diagnosis and symptoms of attention-deficit/hyperactivity disorder (ADHD) among offspring. Prevalence estimates of triptan use during pregnancy among women with migraine vary between 9% and 25%.5 Many women with migraine discontinue use of triptans during pregnancy or switch to acetaminophen,7 which is in line with treatment recommendations.8 However, a substantial proportion of these women report high pain intensity.6 Moreover, untreated maternal migraine has been associated with several pregnancy complications.9Triptans are the most commonly used prescription medications for acute treatment of migraine, which is a chronic neurologic disorder characterized by moderate to severe headache in combination with other symptoms.10 Some studies found increased neurodevelopmental problems in exposed offspring compared with migraine controls, mainly among younger children.12 These associations have not been observed for preschool-aged children.13 Previous studies have mainly used parent-reported outcomes, and there is a need for research on diagnoses of neurodevelopmental problems, such as attention-deficit/hyperactivity disorder (ADHD). Attention-deficit/hyperactivity disorder is one of the most common behavioral disorders in childhood, with a worldwide prevalence of 5%.14Studies of prenatal exposure to triptans during pregnancy have not found an association with increased risks of malformations.15The aim of this study was to investigate the association between prenatal exposure to triptans and ADHD among offspring. Specifically, we aimed to investigate both ADHD diagnosis and ADHD symptoms; the latter was investigated so that we could identify children with problems who do not meet the diagnostic criteria.16 All pregnant women who could speak Norwegian were invited to participate in the study from 1999 to 2008, and for 41% of the pregnancies the women provided written consent to participate.17 The cohort includes 114 500 children, 95 200 mothers, and 75 200 fathers. An overview of all questionnaires in MoBa can be found at the Norwegian Institute of Public Health webpage.18 The present study used information from questionnaire 1 , Q3 , Q4 (sent out 6 months post partum), and Q5y (sent out when the child was 5 years old); Q2 was a questionnaire about nutrition and was not included in the study. This study was based on data file version 9, released for research in November 2015 and approved by the Regional Committees for Medical and Health Research Ethics, region South-East. The establishment of MoBa and initial data collection were based on a license from the Norwegian Data Protection Agency and approval from the Regional Committees for Medical and Health Research Ethics. The MoBa cohort is currently regulated by the Norwegian Health Registry Act. The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline was followed.This study used data from the Norwegian Mother, Father and Child Cohort Study (MoBa), linked to the Medical Birth Registry of Norway, the Norwegian Patient Registry (NPR), and the Norwegian Prescription Database using the mother’s personal identification number. eFigure 1 in the 19 The NPR contains information about diagnoses and procedures in specialist health care for individual patients from 2008.20 Diagnoses are recorded according to the International Statistical Classification of Diseases and Related Health Problems, Tenth Revision (ICD-10).21 The Norwegian Prescription Database contains information about all prescriptions dispensed to individual patients from pharmacies in Norway starting from 2004.22 Dispensed medications are coded according to the Anatomical Therapeutic Chemical (ATC) classification system.23The Medical Birth Registry of Norway is a national health registry containing information about all births in Norway since 1967, including stillbirths and elective abortions after week 12 of pregnancy.A flowchart of the study sample selection is presented in the 23 Additional information about exposure is included in the eMethods in the Women could report use of medications during pregnancy under specific indications in 2 prenatal questionnaires (Q1 and Q3) and 1 postpartum questionnaire (Q4), including migraine (Q1), migraine or headache (Q3), and headache or other pains (Q4). Because triptans are used specifically for migraine, we included triptans reported under any indication in the questionnaires. Use of triptans was coded according to the ATC classification system (code N02CC).26 Age at time of ADHD was defined as the age at the first instance of either a recorded diagnosis of hyperkinetic disorder (ICD-10 code F90) in the NPR between 2008 and 2015 or a filled prescription of ADHD medication in the Norwegian Prescription Database  from 2004 to 2016. Children with no event were censored at the end of follow-up. For these children, we did not have the exact age at time of censoring, so their age was estimated by subtracting the birth year from the year that follow-up ended (2016). The term ADHD diagnosis used in the rest of the article refers to either a recorded diagnosis or a dispensed medication.We used both diagnosis and dispensed medication to identify children with ADHD, as has been done in previous studies.28 in Q5y. The selected items are in the areas of inattention and hyperactivity or impulsivity 30 in Q1. Satisfaction with life was measured by the 5 items of the Satisfaction With Life Scale31 in Q1. Migraine is associated with disability and reduced quality of life,33 and a previous study reported reduced life satisfaction among patients with migraine compared with healthy controls.34Covariates were identified through use of directed acyclic graphs  and by weighting unexposed children by the probability of being unexposed divided by their conditional probability of being unexposed. Estimates derived from models including IPTWs are referred to as weighted estimates. We used 95% CIs to describe the precision of our estimates, corresponding to an α of .05 for a 2-sided test.Data were analyzed from May 1 to November 30, 2021. We estimated propensity scores and calculated stabilized inverse probability of treatment weights (IPTWs) to estimate the average treatment effect.For ADHD diagnosis, Cox proportional hazards regression models with robust SEs were used to estimate crude and weighted hazard ratios (HRs) with 95% CIs. Children were followed up from birth until the first instance of ADHD (diagnosis or dispensed medication) occurring after 3 years of age or censored at the end of follow-up. Year of birth was included in the regression models. Some children died (n = 28) or emigrated (n = 105) during the follow-up period, but we do not know the dates of death or emigration. These children were therefore censored at the time of their most recent appearance in any of the registries or at the end of 2016 if they did not appear in any of the registries. We assessed the proportional hazard assumption by visually examining the hazard curves.38 Generalized linear models with robust SEs were used to estimate crude and weighted mean differences in CPRS-R(S) z scores with 95% CIs.For ADHD symptoms, we estimated censoring weights in addition to treatment weights to account for loss to follow-up because only 43.0% of mothers (4367 of 10 167) returned Q5y and completed the CPRS-R(S). The probability of being uncensored was estimated using logistic regression. Only variables with complete information were included in the censoring model. Stabilized inverse probability of censoring weights (IPCWs) were estimated and multiplied with IPTWs, resulting in a combined weight in which participants in the 5-year sample were upweighted to represent participants who were lost to follow-up.39 with 30 imputations was used to impute missing values in covariates, under the assumption of missing at random. Additional information about the imputation procedure can be found in the eMethods in the 40 was used in all analyses.Multiple imputation by chained equations41 . We also compared results based on trimmed and untrimmed weights. To examine potential exposure misclassification, we applied probabilistic bias analysis with different scenarios for sensitivity and specificity  may have more symptoms of ADHD, we restricted the analysis of ADHD symptoms to children with no missing items. To assess the robustness of our models, we included different model specifications of the IPTWs, including models with prematurity and low birth weight , models without paternal and child factors, and models with maternal ADHD symptoms, measured by the Adult ADHD Self-Report ScaleMothers were classified as having migraine before or during pregnancy in 11.2% of the pregnancies (10 167 of 90 683). Of the women with migraine during pregnancy, 86.8% (4188 of 4823) also reported migraine before pregnancy. The ADHD diagnosis sample consisted of 10 167 children (5231 boys [51.5%]) born to 8412 mothers  with migraine before or during pregnancy. The ADHD symptoms sample consisted of 4367 children (2191 boys [50.2%]) born to 3855 mothers  with migraine before or during pregnancy. In the ADHD diagnosis sample, 832 children (8.2%) were exposed to triptans during pregnancy. In the ADHD symptoms sample, 401 children (9.2%) were exposed to triptans during pregnancy. Characteristics of the samples are presented in The mean (SD) age at time of diagnosis was 8.6 (2.1) years , and children were followed up for a mean (SD) of 10.6 (2.2) years . In total, 377 of 10 167 (3.7%) children received a diagnosis of ADHD. Prenatal exposure to triptans was not associated with an increased risk of ADHD compared with unexposed children whose mothers had migraine during pregnancy  and compared with unexposed children whose mothers had migraine only before pregnancy  . The hazz score 2 or more SDs from the mean, which is considered to indicate clinically relevant problems.43 Children with ADHD had a mean (SD) CPRS-R(S) score at 5 years of 2.0 (0.8)  or between triptan-exposed children and triptan-unexposed children whose mothers had migraines only before pregnancy  (The mean (SD) CPRS-R(S) score was 1.39 (0.4) , and 5.5% of the children (241 of 4367) had a to 0.07) .Probabilistic bias analysis of nondifferential exposure misclassification showed that specificity would have to be unrealistically low to change our conclusion. In realistic scenarios, bias due to nondifferential exposure misclassification could have biased our results by 2% toward the null .15 Moreover, we used modern methods to account for potential biases from loss to follow-up, confounding, and missing data.This study has some strengths, including the prospective design and long follow-up time in MoBa, with access to detailed information about potential confounders of the association between prenatal triptan exposure and childhood ADHD. In addition, we were able to assess both ADHD diagnosis and symptoms. Including the symptom perspective is important because an additional 5% of children beyond those with a diagnosis of ADHD did not meet the full diagnostic criteria but still had symptoms of ADHD according to a review of neurodevelopment outcome measures.47 Potential misclassification also applies to the outcomes. Attention-deficit/hyperactivity disorder symptoms were reported by parents and might be associated with maternal migraine and childhood ADHD. However, the mean symptom scores corresponded well with ADHD diagnosis, given that they were measured at the mean age of 5 years and 9 years, respectively, and we observed similar results. The validity of ADHD diagnosis in the NPR has not been established, but a study from Denmark found a positive predictive value of 0.87 for ADHD diagnoses in children registered in the Danish Psychiatric Central Research Registry.48This study also has some limitations. First, misclassification of exposure may have affected the results. We expect any misclassification to be nondifferential with bias toward the null, which could have led to not detecting an actual increased risk of ADHD. If a woman reported use of multiple migraine medications and indicated use both before and during pregnancy in Q1, we assumed that she was exposed to all the reported migraine medications before and during pregnancy, even though that might not have been the case. In Q4, triptan exposure was reported retrospectively 6 months after birth, and there was no specific migraine indication mentioned in this questionnaire. Reporting of late pregnancy exposure could therefore be subject to poor recall. However, the probabilistic bias analysis showed that the specificity would have had to be very low to change our conclusion. We did not have information about dosage, and dose-dependent effects cannot be ruled out. Another limitation is that migraine was self-reported. However, according to a previous study, self-reported migraine was confirmed in 82% of pregnant women.49 This finding affects the generalizability of our results and could affect the association between prenatal exposures and childhood ADHD, according to a previous study.50 Even though we used IPCW to address loss to follow-up as recommended, there could be additional factors associated with dropout not included in the censoring model, such as migraine and ADHD. In addition, we were not able to include educational level as a factor when the IPCWs were combined with IPTWs estimated from imputed data, which is a limitation, because educational level has been found to be an important factor associated with participation in MoBa.50Second, there could be bias from self-selection and loss to follow-up in MoBa. The participation rate was 41%, and participants were less likely to be young mothers and more likely to be married or cohabiting, have a high educational level, and have a healthier lifestyle during pregnancy compared with the general population.52 A recent study using quantitative genetic modeling concluded that the association between migraines and ADHD was almost completely explained by genetic factors.53 We included 2 comparison groups with expected different migraine severity, finding no systematic differences between the groups. Moreover, we tried to account for migraine severity by using satisfaction with life as a proxy. However, this measure is not specific to migraines and may not reflect differences in migraine severity, although the unexposed groups had slightly higher mean scores than the exposed group.Third, although we took several measures to address bias from confounding by indication, this could have affected our results. Migraine severity is likely to be an important confounder; women using triptans during pregnancy are likely to have more severe migraines than women not using triptans, and migraines are heritable and associated with ADHD in children.Fourth, we had too few exposed children to be able to investigate triptan exposure by trimester or to examine specific triptans. For the same reason, we cannot rule out small effects.This cohort study found no association between prenatal triptan exposure and ADHD diagnosis or ADHD symptoms at 5 years of age. This study adds to the growing literature on the safety of triptan use during pregnancy and expands it to an important neurobehavioral outcome.


Fgf 4, 8, 9, and 17 are all expressed in the AER. An exception to this paradigm is the salamander (axolotl) developing and regenerating limb, where key Fgf ligands are expressed in the mesenchyme. The mesenchymal expression of Amex.Fgf8 in axolotl has been suggested to be critical for regeneration. To date, there is little knowledge regarding what controls Amex.Fgf8 expression in the axolotl limb mesenchyme. A large body of mouse and chick studies have defined a set of transcription factors and canonical Wnt signaling as the main regulators of epidermal Fgf8 expression in these organisms. In this study, we address the hypothesis that alterations to one or more of these components during evolution has resulted in mesenchymal Amex.Fgf8 expression in the axolotl. To sensitively quantify gene expression with spatial precision, we combined optical clearing of whole-mount axolotl limb tissue with single molecule fluorescent in situ hybridization and a semiautomated quantification pipeline. Several candidate upstream components were found expressed in the axolotl ectoderm, indicating that they are not direct regulators of Amex.Fgf8 expression. We found that Amex.Wnt3a is expressed in axolotl limb epidermis, similar to chicken and mouse. However, unlike in amniotes, Wnt target genes are activated preferentially in limb mesenchyme rather than in epidermis. Inhibition and activation of Wnt signaling results in downregulation and upregulation of mesenchymal Amex.Fgf8 expression, respectively. These results implicate a shift in tissue responsiveness to canonical Wnt signaling from epidermis to mesenchyme as one step contributing to the unique mesenchymal Amex.Fgf8 expression seen in the axolotl.The expression of fibroblast growth factors (Fgf) ligands in a specialized epithelial compartment, the Apical Ectodermal Ridge (AER), is a conserved feature of limb development across vertebrate species. In vertebrates,  Ambystoma mexicanum (axolotl), a primary model for regeneration research (Mus musculus), Fgf signaling is similarly required for limb development , a thickened epidermal structure at the distal dorsoventral boundary of the limb bud. Fgf signals from the AER act on the underlying mesenchyme to sustain its growth and patterning. Of the AER Fgfs, Mmu.Fgf8 is the only one that is individually required for normal limb development in mice  family is necessary for normal limb development and for limb regeneration in the salamander research . In lesselopment . In mamm in mice .Fgfs 8, 9, and 17 are expressed in the mesenchymal compartment, unlike in other vertebrates, while Amex.Fgf4 transcription is negligible or absent  and mouse. In mice, AER expression of Mmu.Fgf8 has been shown to be dependent on mesenchymal Mmu.Fgf10 acting through the epidermis-specific receptor isoform Mmu.Fgfr2b  and Wnt3 (mouse), together with BMPR1a-dependent signaling, activate canonical Wnt signaling within AER cells to induce Fgf8  and limb amputations, suggesting its role may be restricted to wound response  in situ hybridization  datasets for the axolotl limb bud  and blasdization  with Ce3dization  and lighFgf8 mesenchymal expression.We identify conserved and divergent features of the axolotl limb signaling network and implicate enhanced canonical Wnt signaling in the axolotl limb mesenchyme as one of the regulators of Amex.Fgf8 in the mammalian AER , an inhibitor of the canonical Wnt pathway that acts by stabilizing Axin2 and consequently increasing the degradation of β-catenin. IWR treatment induced a similar decrease in Amex.Axin2 and Amex.Fgf8 transcripts when compared to Dimethyl sulfoxide (DMSO) treated controls  or in DMSO control for 3, 6 and, in a separate experiment, 12 hr. As expected, Amex.Axin2 expression was upregulated in all CHIR treatment conditions compared to controls .Prior to amputation, axolotls were anesthetized in 0.03% benzocaine (Sigma) solution until they no longer responded to physical stimuli. For expression profiling axolotl limb buds were harvested at stage 45–46. For expression profiling of blastema samples, forelimbs of axolotl snout-to-tail 3–4 cm were amputated through the middle of the zeugopods and allowed to regenerate for 7 days before harvesting. Samples were fixed in 4% Paraformaldehyde PFA at 4°C overnight (14–17 hr) and then dehydrated progressively in a series of methanol–phosphate-buffered saline (PBS): 25–75%, 50–50%, 75–25%, and 100% methanol, each step on ice for at least 30 min. Samples were stored until usage at −20°C in 100% methanol. Prior to staining, limbs were rehydrated in progressively in 25–75%, 50–50%, 75–25%, and 100–0% PBS–methanol.Axolotls 3–4 cm from snout-to-tail were amputated as described above. Drug treatments were performed for different durations always starting at 5-day postamputation.12-hr IWR treatment, 4 axolotls were treated for 12 hr with 25 µM IWR-1-endo , in a 30 ml water bath, and 4 axolotls with the corresponding amount of DMSO as a control. All samples from this experiment were stained and imaged together on the same day.For the 12-hr CHIR treatment, 4 axolotls were treated for 12 hr with 50 µM CHIR-99021 , in a 30-ml water bath, and 4 axolotls with the corresponding amount of DMSO as a control. All samples from this experiment were stained and imaged together on the same day.For the 3- and 6-hr CHIR treatment, 6 axolotls were treated with 50 µM CHIR or a DMSO control for either 3 or 6 hr, in a 15-ml water bath. Limbs were stained and imaged in two rounds.For the Fgfr2b for which isoform-specific probes were designed. Unique transcript sequences were identified by BLAST alignment of the sequence against the axolotl transcriptome Amex.T_v47 , frozen on dry ice, and sectioned using a cryostat to a thickness of 12 µm. HCR staining was performed according to the All image analysis was performed in Fiji . First, Axolotl limb buds and blastemas contain blood cells that are highly auto fluorescent and can compromise the accuracy of signal quantifications. To address this issue, we acquired an image of the autofluorescence in an empty channel (488-nm illumination). We then segmented the gene expression domains, as well as the autofluorescent signal and subtracted autofluorescent regions from the gene expression domains.x = 6.0, y = 6.0, z = 1.74 pixels, corresponding to 3.84 µm in all directions. We then thresholded manually the resulting image, isolating the expression domains in the 647- and 546-nm channel or autofluorescent structures in the 488-nm channel. Different thresholds were used for each channel, experiment, and imaging session, to account for fluctuations in laser power or staining efficacy but the same threshold was applied to all samples of the same experiment. The resulting segmentation masks were applied to the cropped raw data, after background subtraction, for 3D average intensity measurement using the 3D Roi Manager in Fiji  with a sigma of  in Fiji .Axin2 expression in the epidermis, the mask of Amex.Fgf8 expression domain was subtracted from the Axin2 mask to isolate the epidermal Amex.Axin2 expression domain  in Fgf8 expression for the two different staining rounds resulted not statistically different (Wilcoxon rank sum), so samples were combined for further analysis. For the 12-hr CHIR treatment, pharmacological treatment was performed once, and limbs were stained and imaged together. There are eight data points for Amex.Fgf8 and four for Amex.Axin2, because four of the Amex.Fgf8 stained samples were costained with a different marker. For the 12-hr IWR treatment, pharmacological treatment was performed once, and limbs were stained and imaged together.For all pharmacological treatments sibling, animals were randomly allocated to the treatment or control group. For the 3- and 6-hr CHIR treatment, pharmacological treatment was performed once, and limbs were stained and imaged in two rounds (4 limbs per condition per round). Data were normalized only to the DMSO samples imaged in the same round. After normalization, the effect on Amex.Axin2 and Amex.Fgf8 expression, the mean HCR intensity fluorescence in the segmented gene expression domains for a single blastema was used as a data point. Samples were grouped according to the treatment condition, and intensity data for each data point were normalized to the average of the DMSO control samples for the corresponding staining and treatment condition. Statistical comparison between sample groups and the corresponding DMSO controls was performed using the nonparametric Wilcoxon rank sum test.After rotation and cropping, image analysis was performed automatically and with identical parameters for treatment and corresponding control groups. For the evaluation of the effect of pharmacological Wnt signaling perturbation on Amex.Axin2 HCR signal intensities in epidermis and mesenchyme  and epidermis  using the FeaturePlot function. For Single-cell data were reanalyzed to match the latest axolotl transcriptome annotation . The datAll images were processed in Fiji and adjusted in brightness and contrast only for clarity (without gamma adjusting). Figure data plots were generated using R software. Figures were assembled in Adobe Illustrator. The authors investigate the upstream regulators of axolotl Fgf8 expression in limb development and regeneration with the aim of identifying the reasons for its unique mesenchymal localization. The results suggest some refractoriness of the axolotl epidermis to activate Wnt signaling although Fgf8 expression in the anterior mesenchyme is still under Wnt control. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Review Commons.][Editors' note: this paper was reviewed by  Reviewer #1 ):I have three comments on the data interpretation from the figures as I see them.1. Fgf10 expression.Page 6 says 'Surprisingly, we additionally found Amex.Fgf10 transcripts in a small and distinct cell population located in the basal layer of the distal epidermis. To exclude that this population is of mesenchymal origin we performed HCR in situ hybridization and lineage tracing using Prrx1-Cre transgenic axolotls .And on page 9 it says 'In our study we also report that Amex.Fgf10 expression is present not only in the axolotl limb mesenchyme but also in a small cell population localized in the basal layer of the limb bud and blastema epidermis.The images in Supplementary figure 1 do not show the purple dots in the basal epidermis, they are on the outer layers of the epidermis .There is a whole paragraph on the hypothesis that epidermal Fgf10 is playing an important role based on something I can't see in the figures.We confirm that we detect Amex.Fgf10 in a portion of the basal epidermis of the axolotl blastema and limb bud. Basal epidermis expression is visible, albeit weak, in Figure 4B and 4B’ of our updated manuscript. We have added arrows to indicate the basal epidermis expression in this figure to facilitate the reader’s interpretation. To make our imaging data easier to evaluate, we magnified all the panels in Figure 4.We also confirm that in Supplementary Figure 4 of the current version of the manuscript Amex.Fgf10 is detected in the basal epidermis. To facilitate the interpretation of the figure we enhanced the DAPI channel contrast, making the outer epidermal layers more visible. We also labeled the box outlined in supplementary Figure 4-figure supplement 1A4A, indicating that it corresponds to the region magnified in Figure 4-figure supplement 1B and 1C.2. Fgfr2 expression.Page 6 says 'In the axolotl, using HCR, we detected Amex.Fgfr2b transcripts through the entire basal epidermis of the blastema  and limb bud , consistent with Amex.Fgf10 signaling to the epidermis as in other vertebrates.The image in Figure 4B shows Fgfr2b (purple) in the apical layers of the epidermis, not the basal.Therefore It is unlikely that Fgfr2b is playing a signaling role between the epidermis and mesenchyme via fgfg10 as it is 'so far away' from the mesenchyme.We thank the reviewer for pointing out this additional lack of clarity in one of our figures. In the previous version of our figures, the right magenta structures in the blastema outer epidermis corresponded to autofluorescence and did not represent specific HCR signal. We acquired new data for Fgfr2b expression and updated the figure panel  with a clearer image. We also updated the limb bud Fgfr2b image .3. Wnt3a expression. If Wnt 3a is expressed in the mesenchyme why don't you see it in the single cell data in Figure 3B, B'? I don't see any Wnt3a expression in the mesenchyme in Figure 4, perhaps some dots in the limb bud (B') but certainly not in the blastema mesenchyme (B).Whether these genes are expressed in particular domains or not is important for constructing hypotheses so it should be clear to the reader.We thank the reviewer for raising this important point. We confirm that we detect Wnt3a transcripts mainly in the basal epidermis of the axolotl limb bud and blastema. We acquired new data for Wnt3a expression and updated the corresponding figure panels . We agree with the reviewer that the images show expression in the basal epidermis, consistent with the single cell analysis. We therefore removed the sentence regarding expression of Wnt3a in the mesenchyme. We think that basal epidermis is the main site of Wnt3a expression in the axolotl limb, as visualized by both the single-cell analysis and in situ hybridization. This is relevant in consideration of the fact that Wnt3a expression is sufficient to induce ectopic Fgf8 expression in the chick limb epidermis, and Wnt3 expression is necessary for Fgf8 expression in the mouse limb epidermis. The axolotl epidermis lacks Fgf8 expression despite the presence of this putatively upstream ligand. Downstream Wnt signaling activation appears in the axolotl biased toward the mesenchyme, as reflected by the Axin2 co-stain in Figure 4D, 4D’.Reviewer #1 (Significance (Required)):Valuable data on the relationship between signaling genes during axolotl limb development and regeneration. All our current concepts on this subject are based on the chick and the mouse developing limbs, but these can't regenerate. Do regenerating limbs have different gene relationships during development which could explain why the axolotl can regenerate and other limbs can't.Reviewer #2 ):This is a very interesting study investigating the upstream regulators of axolotl Fgf8 expression in limb development and regeneration with the aim of identifying the reasons for its unique mesenchymal localization. Using scRNAseq and HCR, the authors identify both conserved and divergent gene expression features. Overall, the results suggest some refractoriness of the axolotl epidermis to activate Wnt signaling although Fgf8 expression in the anterior mesenchyme is still under Wnt control.I will mention a few points for consideration by the authors.- One important point is the stage at which the study has been performed (particularly in limb development), regarding the activation and/or maintenance of Fgf8 expression. It is known that Wnt signaling is continuously required for Fgf8-AER expression both in mouse and chick, but in principle the inductor could be present only for the initial activation. The authors may consider discussing the difference between activation and maintenance. Also, I think it would be helpful to show a drawing/picture of a St 52 axolotl hindlimb indicating the corresponding mouse/chick stages.We thank the reviewer for giving us the opportunity to clarify this point. Our expression and pharmacological perturbation data is acquired for both the limb bud and blastema at stages subsequent to the initiation of Fgf8 expression, and therefore addresses the maintenance aspect of Fgf8 expression. We have clarified this in the Discussion section of the manuscript:Fgf8 expression was present after 3h of treatment but disappeared when the perturbation was performed for longer durations, possibly due to indirect effects or negative feedbacks triggered by the ubiquitously high Wnt signaling environment. The positive upstream regulatory role of Wnt signaling is similar to that seen in chicken and mammals yet occurring in a different tissue compartment . Our experiments have been performed at stages of limb development and regeneration subsequent to the initiation of Amex.Fgf8 expression and therefore indicate a function of canonical Wnt signaling at least in maintaining Amex.Fgf8 expression. In mouse and chick, canonical Wnt signaling is required for both the initiation and maintenance of endogenous Fgf8 expression and, when activated ectopically, can induce ectopic epidermal Fgf8 expression . Interestingly, in the axolotl, canonical Wnt activation was not sufficient to induce ectopic Amex.Fgf8 expression in the ectoderm.”“A positive effect of CHIR treatment on Amex.We have also mentioned explicitly in the main text (as quoted below) that HCR in situ hybridizations were performed on stage 44-45 axolotl forelimbs. We think forelimbs at this stage are analogous to the stage 52 hindlimb buds that were used for the generation of the single cell dataset analyzed. We decided to perform our experiments in the forelimb to be consistent with the blastema single cell dataset analyzed and with the large corpus of available limb literature that focuses on the forelimb.“To this end, we first analyzed published scRNA seq datasets for the axolotl stage 52 axolotl hindlimb bud  and 7 days-post-amputation axolotl forelimb blastema . Both single cell datasets were reanalyzed to match the latest axolotl transcriptome annotation  and re-clustered, using known markers for cluster assignment . We then used HCR in situ hybridization in tissue sections and whole-mount cleared limb preparations of forelimb buds and blastemas of comparable stages  to validate the expression domains and acquire spatial information.”Although we are confident that the stages analyzed represent the maintenance phase of Fgf8 expression, we are unable to directly compare stages with that of mouse and chick, especially given that the genes we examine show different expression patterns.- Is the number of Sp6 expressing cells in the mesenchyme cluster negligible? Is Sp6 expression in the mesenchyme detected by HCR?We thank the reviewer for raising this question. We assessed Sp6 expression in the limb bud and blastema using HCR and included the images in Figure 2 and in a new Figure 2-figure supplement 1. We refer to this new data in our manuscript as follows:Sp6 is expressed in the epidermis of the axolotl limb bud and regenerating blastema. Interestingly, we detected Amex.Sp6 also in the mesenchyme of the axolotl limb bud, in a domain proximal to Amex.Fg8 expression . This proximal mesenchymal expression of Amex.Sp6 was absent in the blastemas that we analyzed and is unlikely to regulate the more distal Amex.Fg8 expression in the axolotl limb bud.”“We performed whole mount HCR staining in regenerating axolotl limb blastemas and limb buds  and confirmed that Amex.Would it be possible to have an idea of the nameless cluster continuation of the epidermal cluster that contains many Sp8 positive cells.The additional cluster that contains Sp8 positive cells corresponds to epidermal small secretory cells. We now provide two new supplementary figures , where we name the different clusters for the single cell datasets and show UMAPs for some of the markers used to assign the clusters to specific cell populations.Is the small population of Fgf10 expressing cells in the epithelial layer only found in the blastema or also in limb buds? Can this impact the use of Fgf10 as marker to identify the mesenchymal cluster?The small population of Fgf10-expressing epithelial cells is also present in limb buds. We indicated epidermal Fgf10 expression in figure 4B and 4B’ of the updated manuscript with an arrow to facilitate the interpretation from the reader. We think that indeed Fgf10 is not a reliable mesenchymal marker. We identified the mesenchymal cluster in our analysis using different markers (Prrx1) as is now visible in the new supplementary figures Figure 1-figure supplements 1 and 2.Prrx1 is considered a mesenchymal marker however its expression is preferentially seen in some regions of the mesenchyme . Does Prrx1 expression show dV or other bias?While we detect Prrx1 transcripts in all the blastema mesenchyme, we agree with the reviewer’s observation that expression of Prrx1 is spatially biased. The bias is present along the anterior-posterior axis in the images from our study. Expression of Prrx1 in present in the entire blastema mesenchyme, but stronger anteriorly.The results with CHIR are very clear after 3h treatment but fading afterward disregarding Wnt signalling remaining upregulated according to axin. Have the authors considered a possible explanation?We agree with the reviewer that the effect of CIHIR treatment on Fgf8 expression changes after the 3h treatment. We think that this could be due to indirect effects or negative feedback triggered by prolonged exposure to an induced high Wnt signaling environment. We reported this possibility in the manuscript discussion as follows:Fgf8 expression was present after 3h of treatment but disappeared when the perturbation was performed for longer durations, possibly due to indirect effects or negative feedbacks triggered by the ubiquitously high Wnt signaling environment.”“A positive effect of CHIR treatment on Amex.Please, indicate the AP or DV axis in the limb bud HCR images.We have not indicated the AP or DV axis in the limb buds, as without the ulna and radius as a reference, it is difficult to reliably distinguish between the axes when specific markers are not stained.Please, indicate the number of samples analysed for each gene expression.We indicated the number of samples analyzed in the figure legends.Reviewer #2 (Significance (Required)):I think this study addresses a topic of interest to a broad audience, is clearly presented and opens new avenues of research towards deciphering the uniqueness of the mesenchymal Fgf8 expression in the axolotl.Reviewer #3 ):Summary:The manuscript by Glotzer et al. tries to address if Wnt signaling is responsible for the mesenchymal expression of FGF-8 in axolotl limbs buds and limb blastemas. Axolotls are different than mice and chicks in that FGF-8 is not expressed in epidermal cells at all. The authors used already available scRNAseq dataset to look at the expression of genes known to play a role in mediating/controlling the expression of FGF-8 in the AER of mice and chicks. The conclusion is that some unidentified gene is likely responsible for the fact that FGF-8 is restricted to mesenchymal cells in axolotls and or the epidermal cells in mice and chicks.Major comments:The key conclusions are that most of the genes expressed in mice and chick developing limbs can be observed during axolotl limb development and regeneration. Another conclusion is that most genes involved in FGF-8 regulation are also expressed in axolotls similar to what is observed in mice and chicks even though FGF-8 is restricted to mesenchymal cells.The data and the methods are presented in such a way that they can be reproduced and references are provided in an appropriate manner.The experiments are adequately replicated and statistical analyses are adequate.Minor comments:Specific experimental issues that are easily addressable.In Figure 2 B and C the stages of the blastema does not seem to be the same. Panel C looks as if it might be a late bud blastema and B an early bud? Instead of a diagram in A and A' an actual picture of a representative blastema and developing limb might be better.We agree with the reviewer that the Sp8 images in Figure 2 looked different to the other panels, likely because the images in panel B were from tissue sections while the images in the other panels were single planes from whole mount data. We have generated new whole mount data for Sp8 expression and updated the corresponding panels.We would prefer to keep the diagrams in A and A’, as they allow us to schematically depict the epidermal and mesenchymal compartments.Figure 4 D' the expression of Rspo2 is not 100% convincing. It would be good to include in the supplementary data the other n for the expression of this gene in axolotl developing limbs.We have generated additional whole mount data for Rspo2 expression and updated the panels in figure 4 for both the blastema and limb bud . We also included another limb bud image in the supplementary data, displaying that a DV bias in Rspo2 expression is present also in the developing limb.Supp Figure 1: the expression of FGF-10 does not seem to correspond to cells. In panels B and C it looks like blotches that are not specific.We have enhanced the DAPI contrast in this image. We think that now it is more clearly visible that the HCR signal is in close proximity to the nuclei. HCR signal corresponds to quasi-single molecules of RNA, and preferentially localizes to the cytoplasm and not to nuclei, as expected.On page 8 the last sentence of the section just before Discussion, it should be Figure 5E and not 3E.We thank the reviewer for pointing out this mistake that we corrected it in the current version of the manuscript.Reviewer #3 (Significance (Required)):Describe the nature and significance of the advance  for the field. Axolotls are unique amongst tetrapod vertebrates in many ways: they can regenerate their limbs and they don't have an AER during limb development or limb regeneration. Interestingly, almost all the genes that are expressed in animals with AER during limb development are also expressed during axolotl limb development. FGF-8 is an important AER marker in mice and chicks. In axolotls FGF-8 is also expressed during both limb development and regeneration but it is never observed in the epidermis. Axo.FGF-8 is exclusively in mesenchymal cells. The authors tried to understand how can this be and what drives the expression of FGF-8 in mesenchymal cells in axolotls as oppose to epidermal cells.The authors demonstrated that multiple genes known to drive the expression of FGF-8 in mice and chicks are also present in axolotls. Some of these genes are in the epidermis (SP6 and SP8) in axolotls although FGF-8 is in the mesenchyme. The true mechanism driving FGF-8 expression in the mesenchyme has not been elucidated in this manuscript but significant new data is presented that helps understand better the similarities and differences between axolotls and other tetrapods.Place the work in the context of the existing literature . This is one of the first paper that tries to unravel the molecular mechanism behind the expression of mesenchymal FGF-8 expression in axolotls. It has been known for almost 20 years that axolotl FGF-8 is in the mesenchyme. Understanding why it is in the mesenchyme could be important for evolutionary reasons. It could also shed light on other aspects unique to salamanders such as limb regeneration. It is possible that the expression of FGF-8 in the mesenchyme is a crucial aspect of limb regeneration in addition to positional reasons?State what audience might be interested in and influenced by the reported findings.Every scientist with an interest in limb development/regeneration, evolutionary biology of patterning and limb development will be interested in this manuscript.- Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.Limb regeneration, cellular signaling, pattern formation.


This prospective study validates a new PRO measure to monitor the presence of MD in the IMF among women receiving breast RT.There has been an increasing interest in patient-reported outcome (PRO) measures in both the clinical and research settings to improve the quality of life among patients and to identify when clinical intervention may be needed. The primary purpose of this prospective study was to validate an acute breast skin toxicity PRO measure across a broad sample of patient body types undergoing radiation therapy. Between August 2018 and September 2019, 134 women undergoing adjuvant breast radiotherapy (RT) consented to completing serial PRO measures both during and post-RT treatment and to having their skin assessed by trained trial radiation therapists. There was high patient compliance, with 124 patients (92.5%) returning to the clinic post-RT for at least one staff skin assessment. Rates of moist desquamation (MD) in the infra-mammary fold (IMF) by PRO were compared with skin assessments completed by trial radiation therapists. There was high sensitivity (86.5%) and good specificity (79.4%) between PRO and staff-reported presence of MD in the IMF, and there was a moderate correlation between the peak severity of the MD reported by PRO and assessed by staff (rho = 0.61,  The prospective outcomes and support initiative (POSI) is a program designed to develop, collect, and use patient-reported outcomes (PROs) to guide clinical care, drive quality improvement, and empower population-based research in British Columbia, Canada . To dateInterest in the use of PRO measures to improve clinical care has been increasing. Basch et al.  have shoBreast cancer patients receiving radiotherapy (RT) are educated to expect skin toxicity and are directed to contact the nursing line if they develop symptoms that require medical attention. Data on breast skin toxicity are not systematically collected as part of routine practice, and PRO instruments have not yet been made widely available for breast RT patients. As a result, there are only modest data on the rates of patients experiencing severe skin toxicity during or immediately after RT treatment and even less on how this impacts their quality of life. It is important to have a baseline understanding of toxicity and then to be able to track if and how the introduction of new technology and advances in breast RT techniques impact the patient experience over time. Up to 40% of women receiving adjuvant RT are expected to develop some moist desquamation (MD), a painful and worrisome radiation skin reaction observed when radiation dose exceeds the survival tolerance of cells in the basal layer ,9,10. A High-grade skin toxicity, such as MD, generally peaks 10 to 14 days after RT completion for conventional fractionation schemes. In order to ultimately bring PROs into routine clinical practice to support patients in the post-RT acute reaction phase, a PRO tool would ideally be used one- and two weeks post-RT completion. This poses a challenge for assessment and intervention as patients are generally not in clinic at that time. Remote monitoring of symptoms using electronic PRO instruments is still in the development and pilot stage . We inveThe primary purpose of this prospective study was to validate an acute breast skin toxicity PRO questionnaire for use in routine clinical care and research across a broad sample of patient body types undergoing RT.Specific objectives included: (1) to establish correlations between patient-reported acute skin toxicity and staff assessments of skin toxicity, with a particular focus on MD in the IMF; (2) to establish estimates of current rates of MD across our breast RT population according to known risk factors including body mass index (BMI) greater than 25, IMFs (more than 1 cm deep), and cup size D and larger.All patients older than 18 years of age receiving adjuvant whole breast RT and post-mastectomy RT for breast cancer were eligible to participate. Patients requiring a boost following or prior to tangential irradiation were eligible for the study, as well as those requiring locoregional RT.Patients were ineligible if they were not expected to be able to complete the POSI study questionnaire at the designated time points post-RT. Patients who could complete the questionnaire but did not attend the skin assessments post-RT were still eligible. The questionnaire was only available in English, but patients were eligible if a translator or family member fluent in English was available for translation.Breast RT patients were offered participation in this study over a one-year period between August 2018 and September 2019 at three RT treatment centers. Trial radiation therapists obtained consent at the time of the computed tomography (CT) simulation appointment. Patients consenting to this trial were asked to come 10 min early or stay after treatment to complete the POSI study questionnaire by electronic tablet and meet with the trial radiation therapist for skin assessment at baseline and during the penultimate week and last week of RT. As an optional part of the study, patients were asked if they were willing and reasonably confident that they would be able to return to the clinic for skin assessments one- and two weeks post-RT. Patients were also asked again at the time of their last RT treatment appointment. Those showing interest had appointments scheduled with the trial radiation therapist. POSI data from all patients contributed to the study of whether or not the patient returned for skin assessments post-RT.The following baseline data were collected on all patients at CT appointment and during the treatment planning process: age, smoking status, breast cup and bra band size, BMI, and measured IMF depth (in treatment position). IMF depth was measured using a ruler placed in the fold at the deepest point with the patient in the treatment position at CT simulation. The treatment technique and planned dose and fractionation were captured during treatment planning. Previous or current use of chemotherapy and hormonal therapy was also recorded.Treatment prescriptions included 42.5 Gy in 16 fractions and 50 Gy or 50.4 Gy in 25 or 28 fractions. Opposed tangential fields using field-in-field forward planning and step and shoot intensity modulated radiation therapy (IMRT) were used, with single or combined beam energies of 6 MV, 10 MV, and 15 MV. The majority of treatments used 6 MV or 6 MV and 10 MV combined.The 12-question POSI questionnaire (available in The POSI questionnaire was completed at baseline and up to four subsequent time points: (1) penultimate week of treatment, (2) last week of treatment, (3) one week after treatment, and (4) two weeks after treatment. POSI questionnaire data were collected from patients during clinic appointments by means of electronic tablets. All patients were offered access to an electronic or paper version of the questionnaire at one- and two weeks post-RT, in addition to being offered the opportunity to complete this by electronic tablet if they returned to the clinic for skin assessments at these two post-RT time points. As some patients received an additional boost to the tumor bed, the timing of one- and two weeks post-treatment was defined as post-tangential field treatment for this study.Patients were categorized into five groups as follows: (1) BMI below 25 and IMF less than 1 cm, cup size from A to C; (2) BMI 25 or greater and IMF 1 cm or greater, cup size D or greater; (3) BMI below 25 and IMF 1 cm or greater, cup size D or greater; (4) BMI 25 or greater and IMF less than 1 cm, any cup size; (5) post-mastectomy. Group 1 was considered to be at the lowest risk for MD, while group 2 was considered to be at the highest risk for MD ,10. PartThe expectation was to obtain skin assessments one- and two weeks post-RT in addition to POSI questionnaire data on 10–20% of all breast RT patients seen in the clinic during the course of the study. The study was planned to run for one year or until at least 15 patients in each of groups 1 and 2 had been clinically assessed one- and two weeks post-RT in addition to completing POSI. By including a minimum of 15 patients in both the highest risk group (group 2) and lowest risk group (group 1), we aimed to detect a difference between the expected rate of MD of 40% in the highest risk group compared to 0% in the lowest risk group, using a two-tailed alpha of 5%, and a power of 80% for a Fisher’s exact test.Trial radiation therapists scored the degree of skin change using established criteria, National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) V4.0, at each clinic visit while on RT for all patients and at one- and two weeks post-tangential RT for those patients who chose to return. The NCI CTCAE criteria are not specifically designed to distinguish the presence or absence of MD since different degrees of MD are included in both grade 2 (patchy MD primarily in skin folds) and grade 3 (MD outside skin folds) on this scale. Thus, a modified scale expanding on the NCI CTCAE criteria was used to include MD occurring in skin folds and to quantify the size of the area affected, adapted from Wright et al. . The modPatients were advised to apply a water-based moisturizer daily starting at the beginning of RT treatment. At the weekly assessment with the physician, further recommendations to manage acute radiation dermatitis  were made as needed.Descriptive statistics were used to describe the patient population. For each POSI question with a 0–3 severity score, the peak score (highest score across the five time points) was tested for correlation with the peak staff-reported MD score and the peak POSI open skin under the breast score using the Spearman correlation coefficient across the aggregate patient sample. A Spearman’s rho correlation coefficient of 0.6 or greater was considered to signify a good relationship. Sensitivity and specificity of the patient response of “Yes” to “open skin under the breast” from the POSI questionnaire versus the staff-assessed presence of MD in the skin fold was tested using a 2 × 2 table.Univariate and multivariate analyses of POSI outcomes of pain, pain medication, fatigue, anxiety, activity, and sleep interruption versus peak staff-assessed MD or peak POSI open skin score, smoking, chemo, hormone, and radiation dose were performed using logistic regression. All outcome measures were dichotomized for the clinical significance of the problem .A total of approximately 1000 breast RT patients were treated during the one-year study period. Participant recruitment and study completion status are shown in For remote POSI questionnaire completion, 50% of the time, a phone call prompt was conducted because the questionnaire had not been completed within two days of the planned date. Recruitment targets of a minimum of 15 patients in each category were met, except only four patients were recruited in group 3. Baseline patient and treatment characteristics are described in Treatment dose as a function of body type is shown in As shown in Overall, 52 patients (38.8%) reported MD (open skin) in the IMF for at least one of the POSI questionnaire time points. Patients estimated the peak area of MD to be under 1 cm in 9/134 patients (6.7%), between 1 and 2.5 cm in 12 patients (9.0%), and greater than 2.5 cm in 31 patients (23.1%).Trial radiation therapists reported MD in 37 patients (27.6%) for at least one of the skin assessment visits. The peak area of MD was measured by staff to be under 1 cm in 16 patients (11.9%), between 1 and 2.5 cm in 4 patients (3.7%), and greater than 2.5 cm in 16 patients (11.9%).The staff-assessed peak MD area observed by trial radiation therapists and POSI open skin score versus body type is shown in p < 0.001). The sensitivity and specificity for POSI open skin to indicate any staff-assessed MD were found to be 86.5% and 79.4%, respectively, as shown in p < 0.001).The peak POSI open skin score had a moderate Spearman correlation with the peak staff skin score that was statistically significant  and total dose of 48 Gy or greater were statistically significantly associated with MD as assessed by staff or by POSI. Whole breast dose of 48 Gy or greater was also correlated with clinically significant pain, fatigue, sleep interruption, and antibiotic use, while group 2 also correlated with antibiotic use. Group 3 had only four patients, so data are not included in this analysis. Details of these results are shown in p < 0.001).This prospective study validates the POSI questionnaire to monitor the presence of MD in the IMF among women receiving breast RT. There is high sensitivity (86.5%) and good specificity (79.4%) for open skin in the IMF as assessed by POSI to indicate the presence of MD as assessed by staff. A moderate correlation overall was found between the peak staff skin score and peak POSI open skin score . When the body types with a higher risk for MD were grouped together , the incidence of MD among those patients was 41.7%. Overall rates of MD in a given setting will depend heavily on how the patient population is distributed with respect to various body types. This may explain some of the variation in reported MD rates from study to study ,13,22.However, body type alone is not the only factor that determines the incidence of developing MD. We found in this study that the risk of developing MD is strongly dependent on the prescribed dose. On multivariable analysis, patients receiving 48 Gy or greater were more than three times more likely to develop MD compared to patients receiving 45 Gy or less. This is consistent with other published results on the effect of RT fractionation with acute toxicity or body image at the end of treatment ,23. As wBoth patients and staff reported some difficulty with measuring the dimensions of MD areas which may have impacted the overall correlation between peak staff skin score and peak POSI open skin score. Similar issues were commented on in the study by Berthelet et al., who found that while the interobserver reliability for MD was high, there was poor agreement among observers documenting the area of skin reaction  . A less Many patients in this study reported clinically significant (“quite a bit” or “very much”) pain, fatigue, interference of sleep or activity, and even the use of pain medication at some point during their RT treatment or shortly afterward. Total whole breast dose was a strong predictor of pain, fatigue, sleep interruption, and antibiotic use, and peak POSI open skin score was correlated with all symptoms to a lesser degree. Other factors could have contributed to decreased quality of life for patients. Fractionation and race were factors that could play a role in patient-reported breast pain, breast symptoms, or fatigue . Lapen eThe effect of other treatment factors on quality of life, such as the use and timing of chemotherapy and adjuvant hormonal therapy, as well as the use of boost or locoregional RT, will need to be further studied. Future analysis of peak POSI and staff-reported skin scores in any area of the breast  may show an improved correlation to these other qualities of life questions. This study reports on the experience of patients receiving external beam breast RT and may not apply to patients receiving brachytherapy. Some studies suggest that patients receiving interstitial partial breast brachytherapy report improved cosmetic outcomes with similar long-term quality of life outcomes compared to external beam RT ,31.This prospective study describes the validation of a PRO tool with clinical reported outcomes by trained radiation therapists for evaluating MD. This PRO measure may significantly reduce the burden on trial staff in studies of interventions aimed at reducing acute treatment toxicity, and the problem of inter-observer variability for repeated skin assessments may also be overcome. The shortcoming of the NCI CTCAE categorization of radiation dermatitis grade 2 to include both bright erythema and patchy MD will also be alleviated using this PRO measure. In routine clinical use, this tool could help identify patients experiencing severe acute skin toxicity at home and likely requiring assistance to manage their skin reaction after completion of RT.However, this study should be interpreted in the context of its strengths and limitations. The distribution of participants by body type is expected to approximately reflect the distribution across the general RT breast population. This prospective study validates the use of a PRO tool for enabling patients to self-report the presence of MD in a mixed population of body types and breast habitus and correlates well with clinical evaluation for MD by trial radiation therapists. Compliance with this PRO tool is high both during and post-RT treatment. This is a highly accessible means of enabling patients to report acute skin toxicity from breast RT. Future research will investigate the timing and impact of treatment factors on PRO scores, refinement of this POSI questionnaire, and comparison of this questionnaire to objective assessments.