While much progress has been made in relating cis-regulatory variants to expression levels, the timing of gene activation and repression may also be important to the evolution of cis-regulatory sequences. We investigated allele-specific expression (ASE) dynamics within and between Saccharomyces species during the diauxic shift and found appreciable cis-acting variation in gene expression dynamics. Within-species ASE is associated with intergenic variants, and ASE dynamics are more strongly associated with insertions and deletions than ASE levels. To refine these associations, we used a high-throughput reporter assay to test promoter regions and individual variants. Within the subset of regions that recapitulated endogenous expression, we identified and characterized cis-regulatory variants that affect expression dynamics. Between species, chimeric promoter regions generate novel patterns and indicate constraints on the evolution of gene expression dynamics. We conclude that changes in cis-regulatory sequences can tune gene expression dynamics and that the interplay between expression dynamics and other aspects of expression is relevant to the evolution of cis-regulatory sequences.Evolution of  In yeast, cis-acting variants most likely occur within the small (~500 bp) intergenic region upstream of a gene, but could also occur within the coding or 3′ region of a gene. For each hybrid, we tested whether the number of variants in these regions predicts significant ASE levels or ASE dynamics using logistic regression.ASE is caused by Both ASE levels and dynamics were associated with the number of SNP and InDel variants, but these associations varied by hybrid and the type of variant. Within intra-specific hybrids, upstream SNPs and InDels were associated with both ASE levels and dynamics, but InDels showed stronger associations as measured by the odds ratio and the significance . SNPs anInter-specific hybrids only showed significant associations between ASE dynamics and upstream SNPs and InDels. Association between ASE and divergence may be weak or absent if most substitutions between species do not affect gene expression or if there are many substitutions that affect expression but they have random effects that cancel each other out. The inter-specific hybrids have much higher rates of divergence compared to the intra-specific hybrids: an average of 15 and 107 upstream InDels and SNPs, respectively, compared to 1.2 and 6.0 InDels and SNPs within the intra-specific hybrids (Table S5 in cis-regulatory element (CRE-seq) reporter assay . There were a total of 337 variants, an average of 4.2 SNPs and 0.72 InDels per gene. For any CRE sequences with more than a single difference between the Oak and ChII alleles, we also generated CREs for each ChII variant in the Oak allele and vice versa . The totALD5, showed both short- and full-length reporter expression notably different from the endogenous RNA-seq expression. One explanation for this difference is that we used an annotated TSS 515 bp upstream of the ATG, whereas some studies indicate a site much closer (78–84 bp) to the ATG  and 17 genes had no region correlated. CRE-seq regions correlated with RNA-seq tended to lie further away from the TSS, and in some cases showed a gradual increase in correspondence . We also the ATG .To determine whether any of the CRE regions contained variants that affect expression, we examined the 281/334 regions upstream of the 69 genes with one or more differences between the Oak and ChII alleles. One of the regions showed significant differences in expression levels between the Oak and ChII alleles, and 31 showed differences in expression dynamics  was higher than that of intergenic regions across the genome . Out of the 22 genes with one or more CRE regions showing differences in expression dynamics between the Oak and ChII alleles, 8 had two or more significant variants, 12 had only a single variant, and 2 had no significant variants. While the number of InDels with significant effects on expression dynamics was small, the ratio of significant SNPs to InDels (6.0) was not different from that of intergenic regions across the genome .YPS6 promoter showed increased expression over time in the RNA-seq data but not in the CRE-seq data. Even so, the Oak allele exhibited higher expression levels than the ChII allele in both the RNA-seq and CRE-seq assays, a difference that can be attributed to one of the two variants in the region. The ICL2 promoter showed increased expression over time in both the RNA-seq and CRE-seq assays, but not all CRE-seq allele differences were consistent with the RNA-seq allele differences. Region 1 of the ICL2 promoter showed ASE differences consistent with RNA-seq and multiple variants with effects on expression dynamics. However, region 3, which had only a single variant, showed allele differences in expression dynamics inconsistent with RNA-seq, whereby the Oak allele responded more strongly than the ChII allele to the diauxic shift.Variants with significant effects on CRE-seq expression were not always consistent with patterns of RNA-seq ASE . Only 16Cis-regulatory variants that affect expression levels have been associated with conserved promoter regions and disruption of transcription factor binding sites  and 28 genes had no region correlated. Similar to intra-specific comparisons, more regions showed differences in expression dynamics (113) than expression levels (2) between the two species’ alleles  a weak or no association between expression divergence and sequence divergence between species  5' species ; and  and China II (SX6) (S. paradoxus (N17 × YJF153 = YJF1453) and S. uvarum (CBS7001 × YJF153 = YJF1484). Diploid hybrids were generated by mixing strains of opposite mating type and selecting for dominant drug resistance markers present at the HO locus.Three strains of II (SX6) . The int6 cells/mL. Approximately 3 × 108 cells were taken at each timepoint, centrifuged, supernatant removed, and flash frozen in liquid nitrogen. A total of 19 samples were collected during the switch from fermentation to respiration with the most intense sampling occurring every 15 min after glucose depletion , libraries were generated by GTAC, and paired-end (2 × 101 bp) reads were generated using an Illumina HiSeq 2500, resulting in 5.2–5.9 million paired reads per strain. Reads were mapped to the S288c reference genome (R64-1-1) using BWA v0.7.5 (http://broadinstitute.github.io/picard/). SNPs and InDels were called using GATK’s HaplotypeCaller v3.3-0 , followed by China II  and China I .The genomes of the three A v0.7.5 , and dupr v3.3-0  followinr v3.3-0  to the SS. cerevisiae (S288c), S. paradoxus (N17), and S. uvarum (CBS7001).Variants were used to generate reference genomes for mapping RNA-sequencing reads. Reference genomes were generated using the S288c genome as a template and GATK’s FastaAlternateReferenceMaker command to incorporate variants present in each strain. Genome annotations were generated using liftOver  to transhttp://hannonlab.cshl.edu/fastx_toolkit/) and then mapped to combined reference genomes using Bowtie2 v2.1.0  from the same linear model used above are used to calculate the test statistic:iw and ie are the weights (read counts) and residuals for sample i out of T timepoints. For both tests, we used FDR cutoffs of 0.01. For all analyses, FDR was calculated using the method of ASE counts were normalized using DESeq’s blind method . DiffereTen permutations of the data were used to validate the statistical cutoffs. Permuting the counts for the two alleles independently at each timepoint yielded an average of between 0.3 and 2.0 false positive across the five hybrids at an FDR cutoff of 0.01 for the test of ASE levels. Permuting the timepoints yielded an average of between 2.3 and 7.7 false positives across the five hybrids at an FDR cutoff of 0.01 for the test of ASE dynamics.Genes with low read counts were removed from the analysis: either those with less than an average of 20 reads per timepoint or more than seven timepoints with no reads. This filter eliminated an average of 692 genes per hybrid and left a total of 4703 genes with data in all five hybrids.k-means clustering was used to group genes by their combined allele expression profile and by their allele imbalance profile. For the combined gene expression profiles, 19,633 genes from all five hybrids with a significant (FDR < 0.01) autocorrelation in the combined  profile over time were normalized  and clustered into 12 groups. For the allele imbalance profiles, allele frequencies were centered but not scaled for genes with significant ASE dynamics (n = 6135) and clustered into 12 groups. For both, 336 genes without any reads at one or more timepoints were removed since k-means clustering does not handle missing data.The numbers of SNPs and InDels for intra-specific hybrids were obtained from variant calling, and the corresponding counts for inter-specific hybrids were obtained from multiple sequence alignments without correction for multiple hits . Upstreap indicates whether a gene shows significant ASE or not, b1 is the regression coefficient, x is the predictive variable, and e is the error. We used a Bonferroni cutoff for significance to correct for the 60 different regressions based on 6 predictive variables: number of SNPs and InDels within upstream, downstream, and coding regions, and 10 response variables: ASE dynamics and ASE levels in each of the five hybrids.For logistic regression, we predicted genes with differences in ASE levels and dynamics using cis-regulatory sequences are cloned en masse, YFP is inserted between the CRE and the barcode, and the reporter library is then integrated into the genome. The activity of each CRE is measured by the ratio of barcode sequencing reads from an RNA relative to a DNA library of the pooled transformants. As described below, we used this reporter assay to measure expression of 7268 and 7232 synthetic CRE sequences, representing intra-specific and inter-specific allele differences upstream of 69 and 98 genes, respectively.A high-throughput reporter assay (CRE-seq) was used to measure the activity of CREs by sequencing . In thiscis-regulatory element, RS1–5 are restriction sites, and BC is a barcode . The synthetic oligos include a forward primer, RS1, CRE, RS2, RS3, RS4, BC, RS5, reverse primer, where CRE is the  barcode . The bar barcode . For eac barcode  were fou barcode . Inter-seach CRE . The intTSA1 core promoter (CRE-seq libraries were amplified (eight separate reactions of 10-cycle amplification), digested, gel purified, and ligated into pIM202 . This dipromoter  was inseURA3 locus (S. uvarum strain (YJF1450) in YPD overnight at 30°C to allow mating. Double selection with nourseothricin and dsdA was used to select diploid cells.The reporter gene (YFP) with synthetic CREs and corresponding barcodes were integrated at the A3 locus . BstBI-dA3 locus . We collALD5, GND2, and PHO3, were chosen to compare short- and full-length promoter constructs. Full-length promoters from the Oak (YJF186) and ChII (SX6) strains were amplified from the TSS to the next upstream coding sequence. Four barcodes per construct and restriction sites were incorporated into the primers used for PCR. Each full-length promoter with barcodes was cloned into the pIM202 plasmid and YFP was inserted between the full-length promoter and the barcode. BstBI-digested plasmids were transformed into the Oak strain at the URA3 locus. Each of the transformed strains were mated with SX6 to form diploids.Three genes, 6 cells/mL. Samples of 3 × 108 cells were taken for DNA and RNA extraction between 6 hr and 15 hr, which spans the switch from fermentation to respiration. A total of 19 samples were taken for RNA measurements for the intra-specific library, and a total of 27 samples were taken for the inter-specific library. For both libraries, sampling corresponded to the RNA-sequencing timepoints but with more dense sampling for the inter-specific library. For each library, samples were taken for DNA measurements at the first and last timepoints. Cells were centrifuged for 30 s at 1000 g, and the pellets were immediately frozen with liquid nitrogen.Pooled libraries were shaken at 250 rpm in 125 mL YPD at 30°C with an initial density of 3 × 10The abundance of each CRE in the library was measured by sequencing the barcodes from DNA, and the expression of each CRE was measured by sequencing the barcodes from RNA extracts. DNA was extracted from the first and the last timepoints using YeaStar Genomic DNA Kit (Zymo Research). RNA was extracted by YeaStar RNA Kit (Zymo Research) and residual DNA digested with DNase (Promega). mRNA was purified by oligo-dT (Invitrogen), and cDNA was made using SuperScript II Reverse Transcriptase (Invitrogen). mRNA was removed after first-strand DNA synthesized with RNase H (New England BioLabs). Combinations of indexed Ion Torrent primers were used to amplify barcodes in the library for each timepoint  using PhAfter demultiplexing the samples using the indexed Ion Torrent primer pairs, perfect matches to the CRE barcodes were found for 401 and 97 million reads from the intra- and inter-specific libraries. For one sample, three technical replicate libraries was generated (PCR and sequencing). The technical replicates showed an average correlation of 0.96 for barcode counts and were subsequently combined. We removed CREs that had either zero reads in more than a third of the RNA timepoint samples or those with less than 100 reads on average in either the RNA or DNA samples. This filter left 6820 intra-specific and 5013 intra-specific CREs with an average of 16.4 and 3.2 million reads per timepoint and a median of 1278 and 357 reads per barcode across all timepoints for the intra- and inter-specific libraries, respectively. The read count distribution of intra- and inter-specific libraries was normalized using DESeq2’s blind method . ExpressCRE-seq regions were tested for correlations (Pearson) with RNA-seq using the average expression of all barcodes for a given CRE region. For inter-specific CRE-seq, correlations were measured between the 19 timepoints closest to the RNA-seq timepoints. CREs with significant differences in expression levels and dynamics were tested using the weighted linear model and weighted Durbin–Watson test, respectively. When testing for differences in expression levels using the linear model, we used the average expression across all timepoints for each barcode rather than treating each timepoint as an independent measure of expression levels. We chose this more conservative test to avoid cases where one barcoded CRE had substantially higher or lower expression across all timepoints compared to the other replicate barcodes. Consequently, the power to detect differences in average CRE expression levels based on four barcode replicates was reduced. The test for expression dynamics was not affected by this problem since expression dynamics was measured by changes in the ratio of expression levels over time.S. cerevisiae and S. uvarum alleles. The left  and right  promoter regions were separately tested using the genotype of the left and right portions of the CRE, respectively  feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: i) Acceptance summary:The authors use RNAseq in yeast hybrids to study the effect of cis-variation on evolutionary divergence in gene expression and expression dynamics. What sets this study apart from previous work is that the authors use hybrids across different genetic distances, separate expression levels and dynamics by sampling across different time points during an environmental shift, and also investigate 3' sequences. The main conclusions confirm that SNPs and InDels both affect gene expression as well as dynamics, and that on average, InDels have larger effects compared to SNPs, especially on expression dynamics. Moreover, the results also reveal negative selection on expression levels, with the effect of some cis mutations compensated by other cis variation, which ultimately results in complex interactions between the different cis-acting polymorphisms. Together, the results further our understanding of how cis sequence variation supports divergence in gene expression levels and dynamics.Decision letter after peer review:Cis-regulatory variants affect gene expression dynamics in yeast" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Naama Barkai as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.In general, this is a solid and interesting study. None of the reviewers have suggested additional experiments, but each reviewer did suggest a few tweaks to the data analysis and discussion.1. Please consider to also take the effect size into account (i.e. by how much a certain genetic variation affects transcription). This could be done in several ways, the easiest one perhaps by repeating some analysis only for alleles that have a strong influence and comparing the results to the complete dataset.2. Reconsider the statistics as suggested by reviewer 3.3. Please expand a few sections to improve clarity, as suggested by the different reviewers, including a better discussion of the results in the light of what was already known from previous studies (eg work by Naama Barkai's team) or generally expected (namely that cis variation affects gene expression).The individual reviews are found below. They give more details and a few comments that, although not essential, you might also want to consider when revising your manuscript.Reviewer #1 (Recommendations for the authors):It would be useful to give the reader an idea of the average magnitude of divergence in expression levels and dynamics. What is the average difference in expression level  or dynamics . I wonder whether it is then possible to filter out mutations that likely have a very large effect on transcription levels or dynamics? As far as I can tell, all variations are lumped together, irrespective of the magnitude of their effect. What happens if you study the effect of variations that occur around genes that show the largest divergence in expression levels or dynamics? I understand that in the interspecific hybrids, there may be too many variants linked to one gene, but in the intraspecific hybrids, it may be possible to identify variants that are linked to genes that show very large expression divergenceI am not sure whether I get Figure 2. Is it really true that the odds ratio is largest for 3' sequences? If so, that merits more discussion I think, as most readers would likely expect that 5' differences (in the promoter rather than the terminator) would have larger differences.At first sight, the novel insight generated by this study might be quite limited? It would be useful to explicitly discuss which of their results confirm previous findings, and which are novel (such as perhaps the effect size of 5' vs 3' variation?)Reviewer #3 (Recommendations for the authors):I list my comments in rough order of importance:– I missed an analysis of the types of genes that were subject to dynamic ASE. Were these genes enriched in certain functional categories? Were they more or less likely to be essential? More or less conserved at the protein level?– Line 380: This section was not clear to me. What is the purpose of the "known" variants? That they determine thresholds for declaring new variants as true positives? The description of the "tranche filter" in line 386 did not clarify this. I suggest expanding or clarifying this section.– Line 428: Provide information on how FDR was calculated.– Line 535: Unless I completely missed it, I did not see information on how the CRE-Seq libraries were sequenced? Technology, machine, read length,…?– Line 134: 15 SNPs and 107 Indels must be the wrong way around. Probably there are more SNPs than indels? That would also better match the numbers presented in the Methods.– Abstract, line 13: The "but" connecting the two clauses seems off. The two statements aren't really in contrast, they're just two different statements.– Line 215: "gaps" sounds like a reference genome assembly gaps, why not call this "indels"?– In Table 1, I'd put the "Both" row at the bottom, not in between the two rows it combines. Reviewer #1 (Recommendations for the authors):It would be useful to give the reader an idea of the average magnitude of divergence in expression levels and dynamics. What is the average difference in expression level  or dynamics . I wonder whether it is then possible to filter out mutations that likely have a very large effect on transcription levels or dynamics? As far as I can tell, all variations are lumped together, irrespective of the magnitude of their effect. What happens if you study the effect of variations that occur around genes that show the largest divergence in expression levels or dynamics? I understand that in the interspecific hybrids, there may be too many variants linked to one gene, but in the intraspecific hybrids, it may be possible to identify variants that are linked to genes that show very large expression divergence.Figure S3 is intended to provide some indication of the effect size for expression levels (average ASE deviation from 0.5) and expression dynamics (ASE standard deviation over time). We used the standard deviation rather than the maximum to avoid outlier time-points. From this we see that most differences are small, <5%, but there is a long tail of larger effects.To examine how the magnitude of ASE influences the odds ratios we split the data into three equal groups of genes based on the sum of squared deviations from a 0.5 allele frequency. We used this criteria rather than two separate measures for ASE levels and dynamics since the latter would have resulted in comparison of odds ratios from a different set of genes for ASE levels and dynamics. The results from genes with small, medium and large ASE are similar except that InDels are better predictors of dynamics compared to levels for genes with small ASE differences, but InDels have similar predictive ability for genes with large ASE differences. We have now added the analysis of effect size along with a figure . We also point out in the discussion that genes with large ASE difference are more likely to exhibit both ASE levels and ASE dynamics, thereby explaining the similar associations.Within species there are some large effect genes with only a single upstream variant. However, because we couldn't exclude coding or 3' variants we didn't do any further analysis of these potentially large effect variants.In examining the odds ratios for different effect sizes, we noticed subtle differences between the values in Figure 2 and the supporting tables. The original supporting tables have the correct numbers and we have updated Figure 2 accordingly. These differences don't affect our interpretations or conclusions.I am not sure whether I get Figure 2. Is it really true that the odds ratio is largest for 3' sequences? If so, that merits more discussion I think, as most readers would likely expect that 5' differences (in the promoter rather than the terminator) would have larger differences.Correct, but comparing regions is problematic. The 3' region is small (80 bp) and may have a higher proportion of functional variants compared to the 5' region, which include all variants between the two genes. Thus, comparison between regions is problematic. We have removed such comparisons from the results and have added this point to the discussion: "the low odds ratios for 5' compared to 3' region SNPs could be a consequence of the size of the region. The small (80 bp) 3' regions may have a higher proportion of functional variants than the larger 5' regions, which likely contain a mixture of large effect promoter variants diluted by more numerous non-functional variants outside of the promoter region."At first sight, the novel insight generated by this study might be quite limited? It would be useful to explicitly discuss which of their results confirm previous findings, and which are novel (such as perhaps the effect size of 5' vs 3' variation?)cis-regulatory variation is somewhat limited. Nevertheless, we believe the results to be important for future studies of cis-regulatory variants and how they affect gene expression.Our work is novel in that we examine expression dynamics, not just expression levels. Our results show these two aspects of expression are largely similar to one another and so our insight into We have added explicit statements in the discussion regarding results that have been found before. "Previous studies have characterized cis-regulatory variants underlying gene expression levels. […] One notable difference between ASE levels and dynamics is that 5' InDels show a stronger association with ASE. Below, we discuss these results and some of the limitations of our study."Reviewer #3 (Recommendations for the authors):I list my comments in rough order of importance:– I missed an analysis of the types of genes that were subject to dynamic ASE. Were these genes enriched in certain functional categories? Were they more or less likely to be essential? More or less conserved at the protein level?Genes with ASE dynamics were enriched for ribosome biogenesis, carboxylic acid metabolic process and oxidation-reduction process, as might be expected for genes that change during the diauxic shift. We did not include a characterization of genes  as this was not an objective of our study. However, the list of significant genes for each hybrid is available on the OSF repository .– Line 380: This section was not clear to me. What is the purpose of the "known" variants? That they determine thresholds for declaring new variants as true positives? The description of the "tranche filter" in line 386 did not clarify this. I suggest expanding or clarifying this section.S. cerevisiae strains and S288c."We clarified that an independent set of variants was used to determine thresholds for calling new variants as true positives based on 99.9 sensitivity. "Single nucleotide polymorphisms (SNPs) and insertions/deletions (InDels) were called using GATK’s HaplotypeCaller v3.3-0  following InDel realignment, base recalibration and variant recalibration using an independently derived set of SNPs and InDels. […] Using GATK's tranche filter set to 99.9 (percent sensitivity to the independent set of variants), we identified 222,589 SNPs and 20,485 InDels within the three – Line 428: Provide information on how FDR was calculated.We now state: "FDR was calculated using the method of Benjamini and Hochberg (1995)."– Line 535: Unless I completely missed it, I did not see information on how the CRE-Seq libraries were sequenced? Technology, machine, read length,…?We now state: "Libraries were sequenced on an Ion Torrent Proton machine at St. Louis University's Genomics Core Facility." We did not included read length because length varies within a run on the Proton platform.– Line 134: 15 SNPs and 107 Indels must be the wrong way around. Probably there are more SNPs than indels? That would also better match the numbers presented in the Methods.Corrected.– Abstract, line 13: The "but" connecting the two clauses seems off. The two statements aren't really in contrast, they're just two different statements.Corrected.– Line 215: "gaps" sounds like a reference genome assembly gaps, why not call this "indels"?Corrected.– In Table 1, I'd put the "Both" row at the bottom, not in between the two rows it combines.Done.


This study aimed to compare longitudinal change of the psychological distress of a group with psychological first aid (PFA) experience and a group without PFA experience among physicians and other healthcare professionals from before the novel coronavirus disease (COVID-19) pandemic to during the pandemic. The baseline survey was conducted in January 2020 (T1). The respondents in T1 were invited to participate in March (T2) and November 2020 (T3). Psychological distress was assessed by the Kessler 6 Scale. Participants were divided into two categories: a group with and a group without PFA experience. Participants were further divided between physicians and healthcare professionals other than physicians, because physicians are more likely to experience morally injurious events. A mixed-model repeated-measures ANOVA was conducted as an indicator of the group differences. In T1, 398 healthcare professionals participated. The longitudinal analysis of healthcare professionals other than physicians showed that psychological distress was significantly greater in the group without PFA experience than in the group with PFA experience (T1 vs. T3). This study showed psychological distress among healthcare professionals other than physicians was significantly greater in the group without PFA experience than in the group with PFA experience during the COVID-19 pandemic, but the results were not consistent among physicians.  Since early 2020, the novel coronavirus disease COVID-19) has spread throughout the world. Mental health problems have been reported among healthcare professionals responding to COVID-19 9 has spr,4. A sysA number of strategies have been recommended to support the mental health of healthcare professionals during COVID-19 outbreaks . Some syHowever, some systematic reviews concluded there was a lack of evidence that interventions, such as PFA, during or after disease epidemics and pandemics were beneficial to the resilience and mental health of healthcare professionals ,14. A prIn addition, previous studies have not revealed the target populations for which PFA training is effective ,16, but This study aimed to compare the longitudinal change in the psychological distress of a group with PFA training experience and a group without PFA training experience, comparing physicians with other healthcare professionals from before the pandemic (January 2020) to during the pandemic (November 2020) in Japan.The Disaster Psychiatric Assistance Team (DPAT) and Disaster Medical Assistance Team (DMAT) are trained healthcare professionals who have the mobility to work in an acute phase of disasters. They are among the major disaster medical relief teams in Japan who respond at the onset of a disaster for a couple of days. DPAT and DMAT members  usually work at their own hospital. The national or prefectural government requests a deployment to their hospitals at a time of need. The selected members provide rescue activities to the affected areas of the disaster or accident sites for several days, and return to their normal working hospital after the rescue activity. The recruited participants included DPAT and DMAT members who met the following inclusion criteria in this study: (1) native Japanese speaker or nonnative speaker with Japanese conversational abilities; (2) aged 18 years or older; (3) able to receive an e-mail of the written guide for this study from the DPAT office or the DMAT office; (4) physically and psychologically capable of understanding and providing consent for study participation.Healthcare professionals belonging to DPAT or DMAT were recruited for this internet-based cohort survey. The baseline survey of this study was conducted from 29 January to 21 February 2020 (T1). The respondents in T1 were invited to participate from 11 March to 2 April 2020 (T2) [We decided on the timing of conducting the surveys for the following reasons. We conducted a survey (T1) on the participants of our study before the outbreak of COVID-19 in Japan from January to February 2020. After the start of the survey (T1), the COVID-19 outbreak occurred, and the infection of COVID-19 spread in Japan. We conducted a survey (T2) on the same participants from March to April 2020 to investigate their mental health during the COVID-19 outbreak. Survey (T3) was conducted from October to November 2020 as a survey six months after the survey (T2).This study was ethically approved by the research ethics committee of the National Hospital Organization Disaster Medical Center (No. 2019-19), and the research ethics committee of the Graduate School of Medicine and Faculty of Medicine at the University of Tokyo (No. 2019164NI-(1)(2)(3)). This study was also ethically approved to use the information of DPAT and DMAT by the Ministry of Health, Labor and Welfare of Japan. Informed consent was obtained by the participant reading an ethical document, and completing a consent form on the web page. This study was conducted in accordance with the Strengthening the Reporting of Observational Studies in Epidemiology statement .Psychological distress was assessed by the Kessler 6 Scale (K6). It is a self-report questionnaire designed to evaluate psychological distress, rating one’s condition for the prior month . The K6 Items about PFA training experience were originally developed through discussion among healthcare professionals and researchers  who were engaged in mental health among healthcare professionals or PFA in Japan. The question about the experience of taking PFA training was asked at T1: “Have you ever taken psychological first aid (PFA) training?”, and was answered by a binary (yes/no). Resilience was assessed by the Tachikawa Resilience Scale (TRS). TRS was developed on the basis of data obtained from unstructured interviews with Japanese motor vehicle accident survivors, and as a concise scale of resilience for Japanese populations . TRS conDemographic variables, such as sex, age, an affiliation of DMAT or DPAT, hospital affiliation, e-mail address, occupation, years of occupational experience, and years of DMAT or DPAT experience, were retrieved at T1 survey. Contact with a patient of COVID-19 at their own workplace was measured at T2 and T3. We did not survey about contact with a patient of COVID-19 at their own workplace at T1 because there were very few cases of COVID-19 in Japan at T1.t-test to evaluate the difference in continuous variables.We analyzed the dataset of participants who completed all questions of the self-report questionnaire. Participants were divided into two categories: a group with PFA training experience, and a group without PFA training experience. Participants were further divided into two groups according to their occupation: physicians, and healthcare professionals other than physicians. We used a chi-squared test to evaluate the difference in categorical variables in the two groups between physicians and healthcare professionals other than physicians, and used a Student’s p < 0.05. All analyses were conducted using SPSS version 26.0 J for Windows . An adjustment for multiple comparisons was not made because all the statistical tests were conducted simultaneously in a regression model.A mixed model repeated measures ANOVA with an unstructured covariance matrix was conducted using a group (group with PFA training experience vs. group without PFA training experience) × time  interaction as an indicator of the group differences, adjusted for sex, years of DMAT or DPAT, and Tachikawa resilience scale  ,27,28. Wt-test between physicians and healthcare professionals other than physicians, there were significant differences in sex, age, years of occupational experience, and years of DMAT or DPAT experience. There were not significant differences in PFA training experience between the two groups.At T1, of 9733 DMAT and DPAT members, 592 (response rate: 6.1%) agreed to participate in this study, and 476 (4.9%) completed all questions. After excluding 78 participants who were clerical workers and not healthcare professionals, the final sample comprised 398 (4.1%) healthcare professionals. Among the 398 participants in T1, 90 participants (follow up rate: 22.6%) answered at T2, and 80 participants (20.1%) answered at T3. The characteristics of participants are shown in p = 0.03). Among healthcare professionals other than physicians (mainly nurses), being in the group with PFA training experience was associated with a significant decrease in psychological distress from T1 to T3 compared to being in the group without PFA training experience .The results of a mixed-model ANOVA are shown in p = 0.02). The estimated mean of psychological distress among physicians increased in both groups from T1 to T2, and increased in the group with PFA training experience and decreased in in the group without PFA training experience from T2 to T3. Among healthcare professionals other than physicians (mainly nurses), the estimated mean of psychological distress increased in both groups from T1 to T2, and decreased in the group with PFA training experience and increased in the group without PFA training experience from T2 to T3.The results of a mixed model ANOVA, adjusted for sex, years of DMAT or DPAT, and the TRS, are shown in This longitudinal study investigated the relationship between the experience of PFA training and psychological distress among healthcare professionals during the COVID-19 pandemic. The result of this longitudinal analysis among healthcare professionals other than physicians (mainly nurses) showed that psychological distress was significantly greater among those without PFA training experience than those with PFA training experience (T1 vs. T3). Among physicians, psychological distress was not significantly different between the group with PFA training experience and the group without PFA training experience in the adjusted model .The result of this longitudinal analysis showed that psychological distress among healthcare professionals other than physicians (mainly nurses) increased from January (T1) to April (T2) 2020, but decreased in the group with PFA training experience and increased in the group without PFA training experience from April (T2) to November T3), and psychological distress was significantly greater in the group without PFA training experience than in the group with PFA training experience (T1 vs. T3). In the period from January to April 2020, the first domestic case of COVID-19 was confirmed on 16 January; from there, the number of COVID-19 cases rapidly increased, and the first state of emergency was declared on 7 April in Japan . The fin, and psyAmong physicians, psychological distress increased in both groups from January (T1) to April (T2) 2020, and increased in the group with PFA training experience and decreased in the group without PFA training experience from April (T2) to November (T3). Psychological distress was not significantly different between the group with PFA training experience and the group without PFA training experience in the adjusted model . The psychological distress among physicians in both groups may have increased due to their anxiety associated with the COVID-19 outbreak and the increased workload in the workplace in the wake of the COVID-19 pandemic from January to April 2020, as well as in healthcare professionals other than physicians (mainly nurses). From April to November, psychological distress was not significantly changed among physicians in the group with PFA training experience, but decreased in the group without PFA training experience in the adjusted model, which was different from the results of healthcare professionals other than physicians (mainly nurses). Potential risks from delivering PFA training were reported, such as confusion about role boundaries, inadequate skills development, and inconsistent use of PFA guidance, which could potentially harm both a person in distress and PFA providers ,33. PFA In addition, psychological distress among physicians was not significantly different between the group with PFA training experience and the group without PFA training experience in the adjusted model, adjusted for sex, year of DMAT or DPAT, and Tachikawa resilience scale . The results suggest that gender, years of occupational experience, and resilience may have been associated with psychological distress among participants of this study, as shown in previous studies ,27,28. AThe result of this longitudinal analysis for healthcare professionals other than physicians (mainly nurses) showed that psychological distress was significantly greater among those without PFA training experience than in the group with PFA training experience (T1 vs. T3). However, psychological distress among physicians was not significantly different between the group with PFA training experience and the group without PFA training experience in the adjusted model . The reason why the results differed by occupation may be related to the nature of the work. Since healthcare professionals other than physicians were mainly responsible for non-critical decision-making tasks, such as patient care during the COVID-19 pandemic, they were able to apply the content of the PFA training to their daily work. On the other hand, physicians have to make important decisions about patients and be extra vigilant when examining and reviewing patients during the COVID-19 pandemic. Previous studies showed that the nature of their work worsened the mental health of physicians more than it did for other healthcare professionals during a severe acute respiratory syndrome outbreak and the COVID-19 pandemic ,19. SincThis study has several limitations. Firstly, the participants were not asked when they took the PFA training, so we do not know how long before January 2020 they might have taken it. Participants with PFA training experience gained different knowledge and skills depending on when they attended PFA training, which may have different effects on participants’ psychological distress during the COVID-19 pandemic. Second, the response rate was low, which may limit the external validity of this study. Non-responders could have been too stressed to respond or not at all stressed, and, therefore, not interested in this survey. Additionally, the low response and follow up rates may have caused biases in the analysis. Due to this limitation of this study, in order to show the association between experience of PFA training and psychological distress during the COVID-19 pandemic, it is necessary to investigate in the study with higher response and follow up rates. Third, motivated healthcare professionals tend to register as DPAT and DMAT members in general; thus, DPAT and DMAT members are not representative of healthcare professionals in Japan. Finally, this study was an observational study; in the future, interventional studies, such as randomized controlled trials, would be necessary to show reliable results.This longitudinal study investigated the relationship between the experience of PFA training and psychological distress among healthcare professionals during the COVID-19 pandemic. The result of this longitudinal analysis showed that psychological distress among healthcare professionals other than physicians (mainly nurses) increased from January (T1) to April (T2) 2020, and decreased in the group with PFA training experience and increased in the group without PFA training experience from April (T2) to November (T3), and psychological distress was significantly greater among those without PFA training experience than in the group with PFA training experience (T1 vs. T3). Among physicians, psychological distress increased in both groups from T1 to T2, and increased in the group with PFA training experience and decreased in the group without PFA training experience from T2 toT3, and psychological distress was not significantly different between the group with PFA training experience and the group without PFA training experience in the adjusted model . In the future, further research, especially an interventional study, is needed to show reliable results of the effectiveness of PFA training on mental health for different occupations of healthcare professionals.


A large number of patients with head and neck squamous cell carcinoma (HNSCC) have an advanced-stage disease (stages III to IVB) that do not respond to therapy despite aggressive, site-specific multimodality therapy and most of them will develop disease recurrence. This is the description of a phase IIb randomized multicenter trial that involves the enrolment of 96 patients. The aim of the study is to verify whether electrochemotherapy performed with bleomycin of head and neck squamous cell carcinoma (HNSCC) relapses of the oral cavity and oropharynx is able to lead to an increase in the objective response rate in comparison with the systemic treatment with cetuximab + platinum-based therapy + 5-fluorouracil. The primary objective is to verify the objective response rate of patients in the control arm compared to the treatment arm.Background: A significant proportion of patients with head and neck squamous cell carcinoma (HNSCC) have advanced-stage disease (stages III to IVB) that do not respond to therapy despite aggressive, site-specific multimodality therapy. A great number of them will develop disease recurrence, with up to 60% risk of local failure and up to 30% risk of distant failure. Therapy can be very demanding for the patient especially when important anatomical structures are involved. For these reasons, therapies that preserve organ functionality in combination with effective local tumor control, like electrochemotherapy (ECT), are of great interest. Until few months ago, systemic cetuximab + platinum-based therapy + 5-fluorouracil represented the standard treatment for HNSCC relapses with a median overall survival of 10.1 months and an objective response rate of 36%. Recently the results of KEYNOTE-048 study were published and a new combination of monoclonal antibody named pembrolizumab and chemotherapy emerged as standard first line therapy of recurrent or metastatic tumor that overexpress tissue PDL-1 (Programmed Death 1 ligand). Nevertheless, a variable percentage from 10 to 15% of patients with recurrent/metastatic disease have a tumor that does not overexpress tissue PDL-1, and therefore, according to the results of the KEYNOTE-048 study, does not benefit from replacement of cetuximab with pembrolizumab. These patients will be treated with the “gold standard”: cetuximab, cisplatin/carboplatin and 5-fluorouracil. Aim: To verify whether electrochemotherapy performed with bleomycin of HNSCC relapses of the oral cavity and oropharynx (single relapse on T) is able to lead to an increase in the objective response rate in comparison with the systemic treatment with cetuximab + platinum-based therapy + 5-fluorouracil in patients with PDL-1 negative tumors. Methods: The phase IIb study involves the enrolment of 96 patients who meet the inclusion criteria (48 in the control arm and 48 in the treatment arm). The control arm involves the treatment of HNSCC with systemic treatment (cetuximab + platinum-based therapy + 5-fluorouracil). The treatment arm involves the ECT with bleomycin. The primary objective is to verify the objective response rate of patients in the control arm compared to the treatment arm. Electroporation (EP) is a technique in which a short and intense electrical field is applied to cells in order to increase the permeability of the cell membrane. As a consequence of the electroporation, molecules that usually cannot enter the cell membrane can reach the cytoplasm through either diffusion or active transport. The combination of cytotoxic drug and EP is called electrochemotherapy (ECT). This study will use the CLINIPORATOR™  which induces electroporation by applying electric pulses, thus allowing the transmembrane and intracellular transfer of molecules or compounds for which cell membranes are normally resistant or minimally permeable.The first electrochemotherapy clinical trials took place at the Institute Gustave Roussy  ,2,3,4. TA significant proportion of patients with head and neck squamous cell carcinoma (HNSCC) have an advanced-stage disease (stages III to IVB)  that do p < 0.0086). On the other hand, patients whose tumor does not express tissue PDL-1, should be yet treated with the combination of cetuximab-platinum and 5-fluorouracil [Since many patients with relapsed or metastatic disease are unresponsive to therapy, morbidity is high with reduced survival. Palliative systemic therapy with doublet, platinum-based therapy, more effective compared with single agents determine an improvement of response rate but not of survival. A combination of monoclonal antibody (cetuximab or pembrolizumab) and chemotherapy emerged as standard first line therapy of recurrent or metastatic tumors . More inrouracil , as wellRecurrent extended neoplasms of the head and neck especially if they involve important anatomical structures can be challenging for the surgeon and very demanding for the patient. Surgical treatment of these lesions could be disfiguring and followed by functional impairment. Moreover, chemo-radiotherapy can be associated with anatomical and functional complications, and importantly, radiotherapy has often already been performed up-front, thus limiting the possibility of re-doing it.For these reasons, therapies like ECT, that preserve organ functionality in combination with effective local tumor control, are of great interest ,18,19,20ECT has proven to be effective in the treatment of tumors of the skin, subcutaneous and mucosal tissue with several advantages including high success rate in local tumor control  with limited damage to healthy tissue with good cosmetic and functional results. In fact, surgery and radiotherapy could be associated with not only a scar, but also volume loss, retraction and other deformities, even for small tumors. ECT, by leaving the treated tissue in site for resorption by the body, without stroma and vascular destruction, allows progressive healing with limited compromise of aesthetics or loss of function, with limited side effects and advantageous cost/benefit ratio. In fact, the technology involved and the drug used are not too expansive and the treatment may not require hospitalization since it can be performed under local or general anesthesia. Usually only one session of treatment is required.The aim of the study is to evaluate efficacy of bleomycin electrotherapy in treatment of recurrent tumors located in the oral cavity and oropharynx in a two arms prospective, randomized, controlled, phase IIb clinical trial.In this study 96 patients will be enrolled, 48 each arm, which meet the inclusion criteria . All patients have primary tumors of the oral cavity, rhino or oropharynx with histological confirmation of local recurrence, with no indication for surgery or irradiation. The patients must have a PDL-1 negative HSNCC or evident contraindications to the use of pembrolizumab.Multicenter, two arms, prospective, randomized, controlled, phase IIb clinical trial of intravenous administration of bleomycin combined with electroporation in patients with recurrent squamous cell carcinoma of oral cavity and oropharynx PDL-1 negative or with evident contraindications to the use of pembrolizumab. Patients randomized to the treatment arm that either have their disease progress, locally or systemically, at any point during follow-up will be crossed over to the control arm.A total of 96 patients (48 patients each arm) will be enrolled.To verify if the treatment with ECT and bleomycin is superior in terms of objective response to treatment with cetuximab + therapy based on platinum + 5-fluorouracil. The clinical response will be evaluated by RECIST 1.1 criteria on CT and/or MR images at 2 months from baseline.Overall survival;Evaluation of progression free survival time;Evaluation of the duration of the response ;Evaluation of disease control, defined as complete response, partial response and stable disease between the two groups;The quantification of the impact of treatment on the patient quality of life, in terms of pain reduction and bleeding.Before enrolment investigators will assess the eligibility of the patients. When a patient is eligible for participation, detailed study information will be provided. The patients who decide to participate in the study will sign informed consent witnessed by a member of the research group. Inclusion and exclusion criteria are summarized in In the control group, patients whose tumor does not express tissue PDL-1 will be treated with the combination of cetuximab, cisplatin/carboplatin and 5-fluorouracil. In addition, patients with evident contraindications to the use of pembrolizumab, in the opinion of the investigator, can be enrolled in this Protocol.2) and cetuximab (at a dose of 400 mg/m2), as a 2 h intravenous infusion (loading dose), then 250 mg/m2, as an infusion intravenously 1 h per week per day 1 and 5-FU (at a dose of 1000 mg/m2 per day for 4 days) every 3 weeks followed by maintenance of cetuximab with 250 mg/m2 every week or 500 mg/m2 every 2 weeks.Carboplatin (AUC 5 IV day 1) or cisplatin (at a dose of 100 mg/mEach patient will be subjected to electrochemotherapy with bleomycin. ECT is a combination treatment including the injection of an anticancer drug (bleomycin) followed by the delivery of electric pulses using appropriate electrodes and electric pulses. Bleomycin will be injected intravenously to ensure that the whole tumor field will be infiltrated by the drug and that the concentration of bleomycin in the interstitial tissue will be sufficient to kill all the dividing tumor cells while completely sparing the normal non-dividing cells.Bleomycin is active on any type of tumor because electroporation affects all the cell types and, once cells are electro-permeabilized, it can enter all the cells and interact with the cell DNA in the same way whatever the cell type and whatever the genetic expression of the cell.2.Bleomycin will be prepared in the pharmacy of each Institution using the standard procedures for this drug. It will be injected in an intravenous bolus over a short time, up to 5 min at a dose of 15,000 IU/mAppropriate electrodes will be inserted in and around the lesions 8 min after the end of the bolus injection of bleomycin. The electric pulses are generated by a Cliniporator™  and will be delivered by means of appropriate electrodes. Both the electrodes and pulse generator have CE certification. ECT treatment will be performed according to ESOPE protocol ,6.Cliniporator™  will be used to deliver electric voltage with the following parameters: 8–96 pulses of 140–1000 V, of 100 μs duration, at 5000 Hz repetition frequency. The electrodes will be chosen between linear, hexagonal, finger and expandable deployable endoscopic ones depending on the size and tumor localization. Generally, linear needle electrodes will be used to treat cutaneous, lymph node and subcutaneous lesions smaller than 3 cm while use of the hexagonal will be limited in wider tumors. Finger and expandable deployable endoscopic electrodes will be used to reach less accessible lesions, such as those localized in the oro-pharynx (tonsil and base of tongue). The surgeon will perform multiple procedures of the electrode in the tumor tissue covering the complete area of the lesion to be treated and possibly a margin area of free tissue growths of 3–5mm (overtreatment) around the lesion itself. The procedure can require one or more electrode insertion and pulse applications and must be completed within 40 min from the end of bleomycin injection.If the procedure will be performed under local anesthesia, the last will be induced between bleomycin injection and the beginning of the electrode’s insertion; while if the procedure will be performed under general anesthesia, bleomycin injection will follow the induction of anesthesia itself.The patients will be randomized 1:1 to ECT or standard treatment.In To verify if the treatment with ECT and bleomycin is superior in terms of objective response to treatment with cetuximab + therapy based on platinum + 5-fluorouracil. Tumor response will be evaluated by means of clinical and radiological examination. Biopsy with histological examination is performed on indication .In case of progression disease : revaluation of the lesion stage will be done and crossover to control arm would be considered. The appearance of one or more new lesions is also considered progression.In case of stable disease or partial response: revaluation of the lesion stage and the possibility of a second treatment with ECT will be considered.Objective evaluation of tumor response will be performed in accordance with RECIST criteria (version 1.1) .During the 12-months follow-up period, complete regression duration, appearance of recurrence, causes of death will be recorded. Overall and progression-free survival will be evaluated with Kaplan–Meyer estimation analysis. Overall survival (OS) will be calculated from the date of recruitment to the date of death from any cause or last follow-up. Local Disease Free Survival (LDFS) will be calculated from the date of recruitment to the date of first evidence of local recurrence or last follow-up. Disease recurrence rates and percentages will be compared between two groups. Time of failure of the treatment will be calculated from the date of recruitment to the date of local disease progression. Median and minimum–maximum value will be considered.QOL, pain and satisfaction questionnaires scores will be computed.Quality of life will be measured by means of the EORTC QLQ-C30, EORTC QLQ-H&N35, EQ-5D-5L questionnaires at the time of inclusion, 4 and 8 weeks after treatment and at every follow-up visit ,24,25.There is no specific toxicity to be expected, with no side effects with the exception of pain and muscular contraction just during the treatment and pain thereafter.However, specific follow-up will be made during the first 2 post-procedure days in order to register any serious adverse event. The occurrence of an adverse event refers to any clinically significant event that results in death or is life-threatening or that requires the patient’s hospitalization or prolongation of hospitalization.Power of adverse events not listed in this classification will be evaluated according to the NCI-CTC classification (version 3.0): Mild (grade 1), does not affect the patient’s usual daily activities; Moderate (grade 2), perturbs the patient’s usual daily activities; Severe (grade 3), prevents the patient carrying out his usual daily activities; Very severe (grade 4), necessitates intensive care or is life-threatening; Death (grade 5).Patients in the treatment group who show disease progression, local or distant, will be assigned to the control arm (Cross-Over). For patients with stable or partial disease a second ECT session may be considered.The events that eventually occur in the course of the treatment period and/or the follow up, attributable or not to the research, will be considered an adverse event (AE) and will be registered on the applicable case report form. Intercurrent events are any clinical or biological abnormalities not present at the start of the trial. The events will be identified either through patients’ spontaneous statements or following questioning by the researcher and during the examinations.The National Cancer Institute’s Common Toxicity Criteria for Adverse Events, version 4.0 (CTCAEv4) will be used to estimate the toxicity .The study will last 48 months and the patients will be followed up for a minimum of 12 months.The patients will be visited 1, 2, 4, 6, 8, 10, and 12 months after the treatment. CT/MRI or PET-CT will be performed at 2, 6 and 12 months from treatment. The cut-off point for tumor response evaluation has been fixed at 2 months. The visits scheme is showed in Patients must sign individual informed consent form before they undergo any study procedures. Patients who fulfil all inclusion criteria and do not meet any exclusion criteria will be enrolled.At Visit 1, patients will be interviewed and their study eligibility determined according to the inclusion and exclusion criteria and then will be randomized 1:1 to ECT or standard treatment. The investigator will collect and document the patient’s medical history with special emphasis on oncology history, previous chemotherapy, and response to prior therapy. Patients will be asked about concomitant treatments, medications, and events at each patient contact and each visit. Treatment information will include nature of treatment, start date, end date, and reason for treatment. Drug information gathered and documented will include dose, route of administration, frequency, start date, end date, and indication, as applicable. Continuation and discontinuation of treatments and drugs will be verified with the patient at each visit.The patient will undergo a comprehensive physical examination that includes weight, height, and vital signs. Patients observed with an active infection of any kind will be excluded from study participation.The ECOG (Eastern Cooperative Oncology Group) Performance Status  will be CBC and coagulation profiles will be measured at the enrolment visit (Visit 1). Patients with an absolute neutrophil count below 1000/mL, platelet count below 70,000/mL, and/or INR above 1.5 will be excluded from study participation.Pain will be assessed at all visits using a 100 mm anchored visual analogy scale with 0 being “No Pain” and 100 mm being “Pain as bad as it could possibly be.”EORTC QLQ-C30, EORTC QLQ-H&N35, EQ5d questionnaires, will be completed at each follow up visit to measure the patient’s quality of life.All patients will undergo pre-anesthesia assessment in accordance with the usual and customary anesthesia assessment procedures at the clinical site. Patients found not to be acceptable for either general anesthesia or monitored anesthesia care (conscious sedation) will be excluded from study participation.After confirmation of continued study eligibility, the day of the treatment the patients will be undergo ECT treatment (treatment group) or directly to cetuximab + platinum + 5-fluorouracil therapy (control group).Patients randomized to treatment group will undergo ECT treatment in accordance with established clinical and institutional practices.Pain will be assessed immediately before discharge from the treatment facility. Patients will be encouraged to report symptoms to investigators at any time but will be specifically queried and evaluated for toxicity and adverse events at each visit.The use of general anesthesia or monitored anesthesia care  will be determined by the anesthesiologist based on usual and customary practices for invasive procedures at the respective clinical site.In the treatment arm, after induction of anesthesia or sedation, the patient will be prepared and draped for the electroporation procedure according to usual and customary procedures for invasive surgical procedures.Once the lesion has been treated, the usual and customary wound dressing will be applied and the patient will be transferred to post-anesthesia care for further monitoring and pain management. Toxicity and adverse events due to bleomycin will be evaluated after recovery from anesthesia/conscious sedation.Post-procedure analgesia is prescribed by the investigator in accordance with usual and customary practices at the respective clinical site. Recording of the drugs used for pain control will be done.The investigator will establish the need for post-procedure hospitalization on the base of the patient’s response to the electrochemotherapy and anesthesia/monitored anesthesia (conscious sedation) care, as well as usual and customary clinical practice at the study site. Routine post procedure hospitalization, therefore, is not considered an adverse event.Patients will undergo to the same procedures as previously described: clinical evaluation; photographic documentation; EORTC QLQ-C30, EORTC QLQ-H&N35, EQ-5D-5L questionnaires, pain evaluation with VAS score and blood samples as per normal clinical practice and CD8 and CD16 dosages.The patients will undergo clinical evaluation; photographic documentation; evaluation treatment response according to RECIST criteria (version 1.1); CT, MRI or PET-CT ; photographic documentation; EORTC QLQ-C30, EORTC QLQ-H&N35, EQ-5D-5L questionnaires, pain evaluation with VAS score, blood samples as for normal clinical practice and CD8 and CD16 dosages.Biopsy will be performed only in the case of clinical evidence or radiologic suspect.The patients will undergo the same procedures as previously described for visit 3.The patients will undergo the same procedures as previously described for visit 4Sample size estimation was done by using the superiority hypothesis in two independent parallel sample proportions ,32,33,34We assume that ECT can provide an increase of about 20% of the OR .The test statistic used is the one-sided T-test. The significance level of the test was targeted at 0.15. Group sample sizes of 48 in group one and 48 in group two achieve 81% power to detect a difference between the group proportions of 0.2000. The proportion in group one (the treatment group) is assumed to be 0.36 under the null hypothesis and 0.56 under the alternative hypothesis. The proportion in group two (the control group) is 0.36. The significance level actually achieved by this design is 0.16.Continuous variables will be presented as mean ± standard deviation if normally distributed; as median and interquartile range (IQR) if not normally distributed. The difference between follow-up values and baseline values will be computed and the null hypothesis  will be tested by two-sided Student’s t-test  or by Mann–Whitney test . ANOVA test  or Kruskall–Wallis test  will be used to test the null hypothesis by adjusting for proper covariates. For categorical variables, the chi square test for multiple comparisons will be used.The objective response will be reported as a categorical variable and marked as a response rate.Survival analysis will be performed with the Kaplan–Meier test, and the difference between the curves with the log-rank test.Quality of life and pain assessment will be evaluated as continuous variables. The difference between the follow-up values and baseline values will be calculated and the null hypothesis (no effect) will be tested with two-tailed Student’s t-test  or with the Mann–Whitney test . The ANOVA test will be used to verify the null hypothesis by adjusting for appropriate covariates  or with the Kruskal–Wallis test.p < 0.05.Differences will be considered significant at In HNSCC with advanced-stage disease, the drug of choice is platinum combined with fluorouracil (FU) or a taxane; these regimens generally resulted in a 30% response rate, median progression-free survival (PFS) of 3–4 months, and median overall survival (OS) of 6–8 months ,35,36,37Long-term survival is possible in a few numbers of patients with locally recurrent, non-metastatic HNSCC that is candidate to salvage surgery and/or reirradiation ,42,43,44Recently the results of KEYNOTE-04, a randomized, multicenter phase 3 study of patients with untreated locally incurable HNSCC tumors showed that a new combination of monoclonal antibody and chemotherapy emerged as standard first line therapy of recurrent or metastatic tumors . After sECT was investigated on 36 patients with recurrent and mucosal head and neck tumors. This prospective multicenter trial  showed aElectrochemotherapy ECT has been shown to be more effective than other therapeutic options in locally advanced SCC treatment. Di Monta et al.  in theirThe ECT proved to be an interesting antitumoral therapy in 93 patients with advanced chemo and radio-refractory HN neoplasms . Five peIn a large number of patients with recurrent/metastatic HNSCC, survival outcomes remain poor and a need for new therapeutic options that can prolong the survival and improve quality of life of these patients is urgent.This study is the first two arms prospective controlled, randomized, phase IIb clinical trial that will compare efficacy of bleomycin electrotherapy in treatment of recurrent tumors located in the oral cavity and oropharynx with a standard therapy.


T cells infected with human T-cell leukemia virus type 1 (HTLV-1) acquire various abnormalities during a long latent period and transform into highly malignant adult T-cell leukemia-lymphoma (ATL) cells. This can be described as “clonal evolution”, in which a single clone evolves into ATL cells after overcoming various selective pressures in the body of the infected individuals. Many studies have shown that the genome and epigenome contain a variety of abnormalities, which are reflected in gene expression patterns and define the characteristics of the disease. The latest research findings suggest that epigenomic disorders are thought to begin forming early in infection and evolve into ATL through further changes and accentuation as they progress. Genomic abnormalities profoundly affect clonal dominance and tumor cell characteristics in later events. ATL harbors both genomic and epigenomic abnormalities, and an accurate understanding of these can be expected to provide therapeutic opportunities. Genetic abnormality is the main feature of cancer and greatly alters cell fate. The proliferative cycle is essential in the early stages of cancer development since DNA replication errors and abnormal repair functions associated with cell division are the main requirements for development. Through the analogical process of “natural selection” advocated by Charles R. Darwin , cells tOn the other hand, epigenetic abnormalities result from a series of processes regulated by multiple complexes and often do not require excessive cell division as a condition for development . Almost Human T-cell leukemia virus type 1 (HTLV-1) mainly infects CD4+ T cells and disrupts host signaling pathways and gene expression patterns with viral gene products such as Tax and HBZ ,8. As a ATL is a neoplastic disease with a very poor prognosis in which one of the numerous infected cells evolves clonally over 30 to 50 years. Recent comprehensive genome, epigenome, and gene expression analyses have provided macroscopic views of aggressive type ATL . HoweverA series of intensive studies shows that HTLV-1 causes the immortalization of infected cells by viral gene products after infection, resulting in abnormal proliferation. Individual studies of oncogenic viruses are essential in pathophysiology and drug discovery. Furthermore, the study of viral tumorigenesis is an excellent model for understanding the nature of cancer initiation, diversity, clonal selection, and evolution. These viral infections can be regarded as the first hit of a multistep carcinogenic process. Tracing the precancerous cell population formed by the infection along the timeline may help unravel the multistep process.In this review article, we pick up some of the latest studies on the mechanism of HTLV-1 induced tumorigenesis from the viewpoint of host epigenomic abnormalities, especially modifications that significantly influence gene expression patterns, and clonal evolution with genomic abnormalities, and discuss the future aspects.The essential nature of epigenetics has been detailed in excellent reviews ,5,6,12. One of the most remarkable properties of the epigenome in contrast to the genome is its flexibility. This means that it is susceptible to change, and conversely, it is theoretically possible to restore the epigenome to its original state . This poAnother property of the epigenome is its heritability. This is somewhat surprisingly similar to the nature of DNA. Although the inheritance of the epigenome across generations of individuals is controversial, the inheritance of the epigenome from cell to cell is evident; epigenetic changes, such as DNA methylation and histone modifications, once written or erased, can be passed on to the next generation of cells to form populations with the same characteristics ,13. ThisSeveral studies have suggested the existence of HTLV-1-triggered epigenetic abnormalities. Their findings show how (1) transcriptome analysis of infected cells purified with specific surface markers and monoclonal tumor cells showed a common expression pattern across cases ; (2) miRA genome-wide ChIP assay revealed epigenetic changes in infected and ATL cells and demonstrated large-scale changes in trimethylation of the 27th lysine residue of histone H3 molecules (H3K27me3) . In ATL An important finding needs to be pointed out here. The enzyme that catalyzes H3K27me3, an enhancer of zeste homolog 2 (EZH2), physically interacts with HTLV-1 Tax. This interaction is thought to disrupt the scope of target genes originally regulated by EZH2, resulting in a genome-wide change in the pattern of H3K27me3. By inactivating the function of EZH2 with an inhibitor during the process of immortalization by Tax, it is possible to stop the growth of infected cells by Tax . This isIn addition to EZH2, the interaction of Tax with other histone-modifying enzymes such as SUV39H1 and HDAC1 has been reported ,19. SimiIn addition, the insertion of the HTLV-1 genome has been implicated as a possible effect on the host epigenome. CTCF-binding sequences on the provirus suggest a new relationship between the provirus and host epigenome . Some stA polycomb family is a group of factors that regulate chromatin structure using H3K27 methylation. Polycomb repressive complex 2 (PRC2), which contains either H3K27 methyltransferases EZH1 or EZH2, serves as the basic unit for inducing H3K27me3 .Interestingly, when referring to the expression patterns of various cell types, the expression of EZH1 and EZH2 shows an inverse correlation. For example, undifferentiated hematopoietic stem cells have high H3K27me3 that is induced by high EZH2 expression to maintain their multipotency. On the other hand, expression of EZH1 is high, and that of EZH2 is low in mature lymphocytes. This indicates that either EZH1 or EZH2 functions primarily in the formation and maintenance of the necessary H3K27me3 pattern. When EZH2 is overexpressed in mature T cells with high EZH1 expression, the coexistence and function of EZH1-PRC2 and EZH2-PRC2 are phenomenally consistent with the overall increase in H3K27me3 in tumor cells . This fiNotably, preclinical studies have shown the concept that targeting both EZH1 and EZH2 can normalize the accumulation of H3K27me3 not only in high-grade tumor cells but also in cells in a precancerous state infected with HTLV-1 present in the peripheral blood of infected individuals  Figure C. RecentHow does a characteristic infected cell population, formed by viral genes and a series of epigenetic changes, subsequently evolve into ATL? This is revealed by the comprehensive genetic analyses of the ultimately evolved ATL cells.Genomic studies of ATL have a long history, and early results of chromosome analysis showed chromosomal abnormalities in 96% of cases . SubsequPLCG1 (36%), PRKCB (33%), CARD11 (24%), VAV1 (18%), IRF4 (14%), and FYN (4%) mutations. Other mutations include signaling factors such as STAT3 (21%) and NOTCH1 (15%), transcription factors such as IKZF2 (35%), TP53 (18%), GATA3 (15%), and IRF4 (14%), epigenetic factors such as TET2 (8%) and EP300 (6%), chemokine receptors such as CCR4 (29%) and CCR7 (11%), and structural variants in the CD274 (encoding PD-L1) (27%), which is important for immune evasion, were also identified [A comprehensive study by Kataoka et al. has provided an overview of genetic mutations and copy number variations (CNV) in ATL cells . The mosentified .Although somewhat different in frequency and pattern, Shah et al. reported similar data . The freThe most obvious feature of the genome of ATL is that it is characterized by many abnormalities in the TCR pathway. However, the genomic abnormalities in each case are highly diverse, and it is at the same time clear that there is no single mechanism for the development of ATL. After HTLV-1 infection, a polyclonal population of infected cells is formed by viral gene products and epigenomic disorders. Over the next several decades, a single infected clone is thought to gain dominance and proliferate through various genomic aberrations. So then, what mechanisms are involved in clonal selection during this long latent period? A possible approach is to consider genomic mutations as patterns and address them quantitatively by setting up axes such as time scale, mutant clone size, and disease history.One important implication is that many of the genetic abnormalities can be detected before the disease onset. Rowan et al. used deep sequencing to go back in time and detect most of the genetic abnormalities during the carrier phase and showed that they clonally expanded as the disease progressed . MarçaisThis disease has three features that provide analytical advantages in tracing and understanding this evolutionary process. The first is that the “first hit” in clonal evolution is defined as “HTLV-1 infection”. In general, cancer cells of origin are formed by various internal factors  and external factors . However, it is not easy to detect them early in clinical specimens and study their characteristics and mechanisms of cancer development. On the other hand, ATL always has a background of direct effects of HTLV-1 infection, and its interrelationship with subsequent clonal evolution can be studied more deeply.Second, by analyzing the viral genome inserted into the host genome (provirus), it is possible to distinguish individual polyclonally infected cells. It has been shown that each infected cell clone has a randomly inserted virus somewhere in the host genome of approximately 6 billion base pairs, and by using this insertion site information as an ID, each clone can be distinguished and traced with extreme accuracy. Furthermore, the size of each clone can be easily estimated by quantifying the chimeric reads between the end of the provirus and the host genome, or the paired-end reads spanning the two, and other analysis techniques have also been established ,39,40. IThird, a highly accurate analytical method using specific surface antigens of HTLV-1-infected cells has been established; surface antigens such as CD4+, CD25+, CCR4+ , and CADDiversity is the essence of cancer. In the case of solid tumors, methods to estimate diversity and evolution are often used, mainly by sequencing analysis of a large number of pathological regions. On the other hand, in the case of circulating hematological tumors, it is difficult to read out the exact clonal composition based only on genomic information from a single time point in peripheral blood. We recently analyzed a time series of clinical specimens from the same infected individuals over a period of about ten years. We succeeded in depicting the process of competition among individual infected clones, followed by the acquisition of genomic abnormalities by some infected clones and the increase in clone size . High-deAccurately capturing the properties of each clone from a heterogeneous population is difficult using conventional bulk methods. Single-cell RNA-seq (scRNA-seq) is a methodology that can overcome these technical challenges. Rather than estimating clonal structure by extrapolating data from bulk samples, it physically distinguishes cells and sequences each cell individually. We have constructed a new pipeline to identify infected cell populations by extracting viral genes (HBZs) that are constantly expressed in infected cells from sequencing reads and mapping them to clustering. In addition, an analysis method that can identify clones with genetic mutations by extracting mutant RNA reads was also incorporated into the pipeline. Using this method, it is possible to distinguish each mutated subclone by clustering each genetic mutation . A new iThe tumor cells in the peripheral blood of ATL patients were not homogeneous but were a heterogeneous population consisting of multiple infected clones with different characteristics. The characteristics  of each infected clone were consistent with the patterns of genetic characteristics, suggesting that genomic abnormalities define the fundamental clonal structure. Analysis of these cases at different times showed that the clone prevalence ratio was altered, with one clone increasing relative to the other. This can be described as a process of competition and selection between multiple infected clones.Gene expression patterns suggested that the clones that had actually increased relative abundance were more proliferative than those that were initially dominant. The use of different signaling pathways was also revealed. These data represent a moment of clonal selection in the infected cell population and the fact that the characteristics of each clone correlate well with the pattern of genetic abnormalities.Another important finding is the gradual acquisition of genetic abnormalities. A multistep evolutionary process with genetic abnormalities was detected by following the same patient along the time axis ,36,37 FB. IntegrThe critical insight here is that the clones before acute transformation have already acquired some genetic abnormalities and have increased clone size compared to other infected cell populations. This premalignant clone showed a distinctly abnormal gene expression pattern compared to normal cells. This indicates that, although this infected clone has become dominant in the population, it has not progressed to the final stage and appears to be in the process of natural selection. This situation is probably common in carriers with increasing numbers of infected cells and patients with indolent types of ATL. The difference between clones that progress to the final stage and those that stop before the final stage cannot be accurately predicted at present. If we can identify the characteristics of malignant clones based on patterns of gene abnormalities, gene expression, and epigenomic abnormalities, it will be beneficial for prognosis prediction and therapeutic drug development.CCR4 and CADM1. It has also been reported that some of these are induced by HBZ [How are polyclonal cell populations evolving using genomic and epigenomic abnormalities? An important suggestion is that the epigenome is a common feature across cases. In the single-cell analysis, epigenomic abnormalities were commonly detected in comparative analysis of competing cell populations and cells before and after progression . Gene sud by HBZ ,8,46 andd by HBZ . The eviThe genomic abnormalities detected in ATL cases are very complex. Although they share common features such as the TCR signaling pathway, they are more complex than other typical leukemias and some solid tumors and appear to involve clonal individuality rather than common properties. It is not clear what combination of genetic abnormalities is responsible for the underlying hallmarks of tumor cells, such as abnormal proliferation and evasion from the apoptotic form of regulated cell death (RCD). The fact that the pattern of genetic abnormalities varies among cases may indicate redundancy among genetic abnormalities. Considering the direct effects of viral genes and the commonality among clones, epigenomic disorders are thought to begin forming early after infection and evolve into ATL through further changes and accentuation as they progress C. To theIt should be noted that the genomic variations referred to here are only within the range that can be considered with the current sensitivity of the analysis. It cannot be determined from which point of time they are introduced. Genomic instability caused by HTLV-1 has been pointed out, and there may be genomic variations that do not lead to clonal expansion beyond a distinguishable level. Many passenger mutations and subclonal structures caused by neutral evolution have been detected in ATL cases. The function of mutations in noncoding regions is also largely unknown.PD-L1 [It is also necessary to consider the role of selective pressure. Environmental factors such as oxidative stress, nutrient conditions, and therapeutic agents may contribute to the evolution of polyclonal populations resulting from HTLV-1 infection. In addition, evidence of immunological selective pressure is certainly documented at the genomic level, as exemplified by structural variation in PD-L1 . BecausePD-L1 ,49.The evolutionary path of T cells infected with HTLV-1 should have been inscribed in the ATL cells that appear as the disease. Characterizing the heterogeneous ATL cell population may help us identify opportunities for durable therapeutic intervention. A multifaceted understanding of the path from infection to ATL and HAM from the elements of virology, genomics, epigenomics, and host factors would lead to a significant advance in managing HTLV-1 infectious diseases.


MAX) gene have been associated with familial pheochromocytomas and paragangliomas with an autosomal dominant pattern of inheritance, a median age at onset of 33 years and an overall frequency estimated at 1.9%. We describe a deleterious MAX variant associated with hereditary pheochromocytoma in a family with four affected individuals.Pheochromocytomas are rare catecholamine-producing neuroendocrine tumours arising from chromaffin cells of the adrenal medulla or extra-adrenal sympathetic paraganglia. Recent studies have indicated that up to 40% of pheochromocytomas could be attributable to an inherited germline variant in an increasing list of susceptibility genes. Germline variants of the MYC-associated factor , in the latter individual, his two siblings and their father were tested and the same variant was identified in all of them. Both siblings were subsequently diagnosed with pheochromocytoma  and choose to remain on active surveillance before they were submitted to adrenalectomy. All the tumours secreted predominantly norepinephrine, accordingly to the typical biochemical phenotype ascribed to variants in the MAX gene.The first patient presented with bilateral pheochromocytoma in 1995; genetic testing was proposed to his oldest son, when he was diagnosed with a bilateral pheochromocytoma with a synchronous neuroblastoma. Upon the identification of the MAX variant in the same family. It is also the first case with a synchronous pheochromocytoma and neuroblastoma in carriers of a MAX deleterious variant. This report draws attention to some ill-defined features of pheochromocytoma and other malignancies associated with a MAX variant and highlights the importance of understanding the genotype-phenotype correlation in hereditary pheochromocytoma and the impact of oriented genetic testing to detect, survey and treat patients and kindreds at risk.This case series is, to our knowledge, the one with the largest number of individuals with hereditary pheochromocytoma with a deleterious  Pheochromocytomas are rare catecholamine-producing neuroendocrine tumours arising from chromaffin cells of the adrenal medulla (80% to 85% of the cases) or extra-adrenal sympathetic paraganglia (15% to 20% of the cases), the latter also referred as extra-adrenal pheochromocytomas or paragangliomas , kinase signalling group  and Wnt signalling group (CSDE1 and MAML3)  is a gene associated with regulation of cell proliferation, differentiation, and death   , 11. The8 years) . As a ki8 years) .MAX deleterious variant in four affected relatives.We report a family with hereditary pheochromocytoma carrying a The patients gave their written consent to sample collection, genetic testing, and the use of genetic test data for the purposes of research. Written informed consent for publication of their clinical details and/or clinical images was obtained from the patients and relative of the patient 2.In November 2010, a 27-year old male, the oldest of the three children of patient 2, was admitted to our hospital for evaluation of an inguinal pain. He had no medical or medication history and was a competitive swimmer. He reported a 3 month-history of inguinal pain, resting tachycardia and a weight loss of 10 kg in the last year that he ascribed to intense exercise and diet. At his first observation on the internal medicine ward, his blood pressure (BP) was 200/100mmHg with a resting heart rate of 140bpm (sinus tachycardia with signs of left ventricular volume overload on EKG) but presented an otherwise normal physical examination. Whole body (WB)-CT scan revealed a posterior mediastinal mass (59x80 mm) paravertebral to T9-T11 vertebral bodies, bilateral hypervascular adrenal lesions (46x39 mm and 15.5 mm on the left and 80x110mm on the right) and a retroperitoneal periaortic and peri-common iliac artery lesion (135x185mm) with an extensive encasement of this vessel with a marked compression of the venous structures Figure 1123I-metaiodobenzylguanidine (123I-MIBG) showed left adrenal and para-aortic and common iliac trace uptake and on echocardiogram there were signs of catecholaminergic cardiomyopathy with severe left ventricular dysfunction (ejection fraction ~20%).A biopsy of the retroperitoneal mass was performed and was suggestive of a paraganglioma (tumour cells reactive with chromogranin and synaptophysin); for this reason, Endocrinology observation was required. The hormonal workup revealed catecholamine hypersecretion Table 1,After multidisciplinary review of the case, a metastatic pheochromocytoma and paraganglioma were presumed and surgery was proposed. He needed hospitalization for pre-operative blood pressure and chronotropic control .He underwent bilateral adrenalectomy with incomplete resection of tumoral mass from the left iliac vessels in February 2011 and pathology was consistent with medullar hyperplasia on the right adrenal gland, and pheochromocytoma with evidence of angioinvasion on the left adrenal gland with tumour cells reactive with chromogranin, synaptophysin and S100. The extra-adrenal tumour was suggestive of well differentiated neuroblastoma  with a Ki-67 index of 10%.123I-MIBG performed on April 2011 showed an intense uptake on his left iliac artery.On the immediate post-operative period, his blood pressure and his left ventricular ejection fraction normalised and there was no evidence of catecholamine hypersecretion. MAX germline truncating variant c.97C>T, p.(Arg33Ter) (NM_002382.3) in a blood sample. Subsequently, given the unexpected association of neuroblastoma with a variant of the MAX gene, the presence of loss of heterozygosity (LOH) was evaluated in a formalin–fixed and paraffin–embedded sample of the neuroblastoma tumour (from a delimited area with >50% tumour cells), using the same NGS panel, and the variant allele frequency (VAF) was 71.2%, which is compatible with LOH. During the follow-up at this centre he presented with recurrence of catecholamine hypersecretion and hypertension. After multidisciplinary team review, it was decided to start palliative chemotherapy for neuroblastoma; despite chemotherapy cycles with cyclophosphamide, doxorubicin, vincristine and topotecan from 2011 to July´2013 there was a disease progression with multiple abdominal and pelvic lymphadenopathies . The posterior mediastinal mass’ size remained stable and was deemed unresectable; it was assumed as a probable pheochromocytoma metastasis but with no histological evidence.Due to significant residual tumoral lesions, the patient was referred to the Portuguese Oncology Institute of Porto . Next generation sequencing (NGS), using the TruSight Hereditary Cancer Panel (Illumina) identified the 123I-MIBG on April 2014 documented multiple abdominal and pelvic focus of intense uptake, mainly on the left side. 131I- MIBG therapy was performed on June’2014 with no apparent response on post-treatment MIBG scan.On March 2014 he was submitted to exploratory laparotomy with debulking; pathology was consistent with poorly differentiated neuroblastoma with no lymph node parenchyma identified. He was afterwards submitted to right nephrostomy and later, on October 2014, to a double-J stent placement, as a preparation for a new surgical debulking. On the 2nd November 2014 he was admitted to the emergency department with an urosepsis with an acute on chronic kidney disease and severe hyperkalaemia. Despite initiation of broad-spectrum antibiotics, vasopressor support and emergent continuous veno-venous hemofiltration, he died 24 hours later, at the age of 31 years old.2), his BP was 156/96mmHg with a resting heart rate of 80bpm and an otherwise normal physical examination. He had no signs of Cushing’s syndrome, such as rounded face, thin skin, easy bruising, or purple striae.In March 1995, a 32-year old male was referred to our Endocrinology outpatient clinic with complains of daily paroxysms of headaches, diaphoresis, and facial pallor for the last 6 months. A year before he was diagnosed with hypertension, but he had refused anti-hypertensive treatment. At observation he presented a class I obesity  were normal.Our investigation revealed an increase in 24-hour urinary normetanephrines and metanephrines Table 2;In May 1995, a right adrenalectomy and subtotal left adrenalectomy was performed. Pathology revealed adrenal hyperplasia and the genetic testing for RET mutation was negative.123I-MIBG showed no abnormal uptake. After discussing the treatment options with the patient, he opted to resume alpha and beta-block therapy and maintain close clinical, analytical, and imaging surveillance. In June 2000, after clinical worsening with increased frequency of paroxysms associated with retrosternal chest pain, he was submitted to totalization of left adrenalectomy; this time pathology reported a tumoral lesion of 25x15 mm consistent with a pheochromocytoma of low mitotic index. Post-operatively he was started on glucocorticoid and mineralocorticoid hormone replacement and his anti-hypertensive drug was stopped. He has been asymptomatic and with no evidence of catecholamine hypersecretion ever since. After the identification of a deleterious MAX variant in the index case , he was found to carry the same variant.At his immediate post-operative follow-up visit he presented normal blood pressure, but 3 months after surgery he relapsed with recurrence of hypertension and paroxysms associated with elevated urinary metanephrines. Abdominal-CT revealed a residual lesion on the left adrenal gland but MAX germline variant in his father (patient 2) and older brother (patient 1). He was overweight (BMI 29.4kg/m2) but his physical examination was otherwise normal (blood pressure was 124/74 mmHg and heart rate 90bpm). Biochemical testing revealed a small rise in urinary normetanephrines  and orthostatic tachycardia requiring treatment with a calcium channel blocker, initiated by his primary care physician. Over the years there was a progressive increase in the levels of plasma and urinary normetanephrines .Post-operatively he was started on glucocorticoid and mineralocorticoid replacement and his anti-hypertensive drug was stopped. Until the last appointment there was no biochemical evidence of catecholamine hypersecretion or relapse of hypertension.MAX germline mutation in his father and his older brother. He was 14 years old, had a class I obesity (BMI 31.9kg/m2) with no other medical or medication history nor endocrine hypersecretion features and at examination his blood pressure was 132/67mmHg and heart rate of 85bpm with an otherwise normal physical exam. Genetic testing confirmed that he was a carrier of the same MAX variant previously identified in the family. From the age of 15 years old, he underwent annual screening with 24-hour urinary metanephrines and abdominal-CT every two years with both remaining normal until 2017  family of proteins; this MYC/MAX/MXD1 network regulates cell proliferation, differentiation and apoptosis  can be held accountable for this varying accumulation.In addition to the frequently bilateral PPGL and the early onset, our patients’ tumours secreted predominantly norepinephrine in a greater proportion than epinephrine. This biochemical phenotype is intermediate between the established epinephrine producing tumours due to for PNMT . An inteMAX gene heterozygous variant, c.299G>C . Our case is, to the best of our knowledge, the first with a simultaneous PPGL and neuroblastoma presenting as two distinct entities in association with a MAX pathogenic variant. LOH observed in the neuroblastoma (VAF=71.2%) supports a causal relation between the MAX germline pathogenic variant and the origin of this tumour The embryological common origin and the importance of MYC/MAC/MXD1 network in the development of this neural crest cells tumours are identified as the basis of this association  and biennial imaging (with MRI) surveillance of first-degree relatives  for the publication of any potentially identifiable images or data included in this article.DB, LF and CC co-wrote the manuscript. MA was the pathologist responsible for the diagnosis of patient 2. JL, CS and MT carried out the genetic analysis that first demonstrated the presence of the MAX gene deleterious variant for patient 2 (in 2011) and the remaining patients (in 2012). RC is the main physician of the patients; APS was the main physician of patient 1 after his referral IPOFG. LF, APS, MT and MC contributed to the revision of the manuscript. All authors contributed to the article and approved the submitted version.The 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.