Lupinus albus L.; WL), we have in silico annotated and in situ mapped the main classes of DNA repeats identified with RepeatExplorer. A highly diverse and an abundance of satellite DNAs were found representing more than 10 families, where three of them were highly associated with CENH3-immunoprecipitated chromatin. Applying a strategy of several re-hybridization steps with different combinations of satDNA, rDNA, and LTR-RTs probes, we were able to construct a repeat-based chromosome map for the identification of most chromosome pairs. Two families of LTR retrotransposons, Ty1/copia SIRE and Ty3/gypsy Tekay, were highly abundant at pericentromeric regions, while the centromeric retrotransposon of WL (CRWL) from the CRM clade showed strong centromere-specific localization in most chromosomes and was also highly enriched with CENH3-immunoprecipitated chromatin. FISH mapping of repeat DNA showed some incongruences with the reference genome, which can be further used for improving the current version of the genome. Our results demonstrate that despite the relatively small genome of WL, a high diversity of pericentromeric repeats was found, emphasizing the rapid evolution of repeat sequences in plant genomes.Plant genomes are known to be mainly composed of repetitive DNA sequences. Regardless of the non-genic function of these sequences, they are important for chromosome structure and stability during cell-cycle. Based on the recent available whole-genome assembly of white lupin ( Lupinus L., commonly known as lupins, is a genus of flowering plants in the legume family Leguminosae, comprising more than 300 species  is distinct within the vast and polymorphous genus Lupinus because of a small variation of morphological characters with its origin attributed to the Mediterranean region. It is recognized as traditional food and feed of interest due to its high seed protein content  have fully sequenced genomes (The genus  genomes . Despite genomes . This st genomes .The WL genome is highly repetitive and shows a great diversity of centromere-associated repeats. ChIPseq data showed that CENH3-containing chromatin is highly associated with the centromeric retrotransposon of WL (CRWL) and with at least four centromeric satellite DNA (cenDNA) repeats: CL2-5bp, CL10-78bp, CL21-38bp, and CL55-8bp. In contrast, the most abundant satellite repeat CL1-170bp did not show significant enrichment with the immunoprecipitated DNA. The CENH3-immunoprecipitated chromatin accounts for around 11% of the WL genome . Along wL. albus cv. AMIGA (2n = 50)  . We furtThe repeat abundance and characterization of the WL genome were previously reported . BrieflyTandem repeats comprised ∼15% of the WL genome, distributed in 14 tandem repeat clusters .As shown previously, CRWL FISH signals were observed as narrow-distributed signals at the centromeric regions of most WL chromosomes . Remarkain silico and in situ observations of the main centromeric repeat distribution for all WL chromosomes:Below we describe the LalbChr01: This chromosome is in silico characterized by harboring the single 5S rDNA array and having multiple CL53-24bp arrays. The putative centromere site sits on the flanking region of the first CL53-24bp array as depicted from the high accumulation of CRWL in this region , CL2-5bp, and CL77-36bp.LalbChr04: This chromosome is in silico characterized by having a large CL53-24bp array and additional CL2-5bp and CL77-36bp on opposite flanking sides. This chromosome was not identified by in situ hybridization.LalbChr05: This chromosome is both in silico and in situ characterized by having the presence of CL1-170bp and CL21-38bp. Additionally in situ signals were observed for CL2-5bp.LalbChr06: This chromosome is in silico characterized by having the presence of CL10-78bp, CL2-CL53-24bp, and CL77-36bp. However, in situ signals were observed only for CL55-8bp (present in three chromosomes) and a weak CL2-5bp.LalbChr07: This chromosome is in silico characterized by having the presence of CL55-8bp and CL77-36bp. However, in situ signals were observed only for CL55-8bp (present in three chromosomes) and a weak CL2-5bp.LalbChr08: This chromosome is both in silico and in situ characterized by having a large CL53-24bp satDNA block and additionally a minor CL2-5bp array.LalbChr09: This chromosome is in silico characterized by a minor CL2-5bp array and low enrichment with CRWL. This chromosome was not identified by in situ hybridization.LalbChr10: This chromosome is in silico characterized by minor CL2-5bp and CL10-78bp arrays, but highly enriched with CRWL in the flanking regions  of the WL reference genome is available in the genome browser website,Lupinus is a genus of considerable agronomic importance with a couple of species with full genome assemblies available . These differences were mainly due to missing satDNA sequences at the highly repetitive (peri) centromeric regions in the reference genome. Tandem repeat-rich regions create a technical challenge to genome assembly  family  family .As described in We found that only two of the 10 most abundant satellite repeats occurred in the genome exclusively as long tandem arrays typical of satDNA. Both occupied specific genome regions. CL1-170bp was associated with pericentromeric knobs in a few chromosomes, extending to very large regions  or even present as two distant in the pericentromeric regions of both chromosome arms but excluded from the centromeric regions . Sometimes CL1-170bp arrays were truncated by other satDNAs or retrotransposons. The other case was CL53-24bp, which occurred also as long arrays closer to centromere-associated repeats, either centromeric satDNA or CRWL.As we have shown, a narrow and concentrated distribution of CRWL was observed in most assembled centromeres. A high association of CRWL and centromeric satellite DNA peaks was also observed, suggesting a more specific distribution of these repeats compared to other TEs. Furthermore, CRWL insertions were more frequently observed within CENH3-enriched cenDNA repeat arrays, which is also corroborated by FISH results, suggesting that cenDNA repeats may act as preferential insertion sites for CRWL. Common across several plants, CRMs tend to preferentially be inserted into functional centromeres .Remarkably, some chromosome assemblies lacked a large region of repeats, for instance, the Lalb_Chr01, which lacks a large pericentromeric region containing the CL1-170bp and another region that is most likely the functional centromere containing both CL2-5bp and CL10-78bp. The final assembly showed only the repeat CL53-24bp in the pericentromeric region of this chromosome, which is highly enriched in this chromosome. In fact, a gap in the assembly is observed upstream from the largest array of the CL53-24bp satDNA . Based on our repeat mapping results and taking into account that the 64 unplaced contigs from the WL assembly are mostly enriched with repeats, it is likely that some of these contigs represent fragmented peri- and centromeric regions.Surprisingly, large arrays of CL2-5bp were rarely found on the reference genome of WL, contrasting to the bright signals observed for this repeat on most centromeric regions. This could be caused by a potential assembly failure and/or, less likely, by missing long-read sequencing coverage of this particular repeat. The reason the assembly/sequencing failed within arrays of this particular repeat is unknown, possibly the very short monomer size and high homogenization could have hampered its assembly/sequencing. For the CL55-8bp satDNA, which showed the highest enrichment in the CENH3-ChIPseq analysis, we observed signals in three chromosome pairs, but only two short arrays were found in the reference genome assembly, on Lalb_Chr07 and Lalb_Chr21.Lupin and the high number of species found in the genus, it will be of high interest to perform comparative cytogenomic analysis in the future. Furthermore, WL is a promising crop that could help increase plant protein as part of our diet and that will attract more and more interest from breeders in the near future. Focusing on the genomics of this ancient crop will certainly help meet this ambitious goal.With the advancement of long-read sequence technologies, assembly of high-quality chromosome-scale reference genomes has become a feasible task for many non-model organisms. Here, we show that even the highly repeat-complex genome of WL has most of its repeats well assembled and validated based on our dense repeat FISH mapping strategy. Furthermore, the combination of a high-quality reference genome with a detailed FISH analysis of repeats allowed us to build a comprehensive chromosome-specific repeat profile of the WL genome. We also show the power of cytogenetics associated with genomics to solve gaps in the assembly and how it can support further improvement of the next versions of the reference genome. The highly repeat-diverse small genome of WL provides yet another good example of the rapid diversification of plant genomes. Considering the economic importance of several species of L. albus cv. AMIGA were germinated on Petri dishes with humid paper. After germination, the root tips were collected. For meiotic tissue, young flower buds were collected from flowering L. albus cv. AMIGA plants growing under controlled conditions in the greenhouse of the SupAgro—Montpellier. Pre-treatment and fixation of tissues were performed as described below.Seeds of in situ hybridization analysis were conducted as described in Chromosome preparations for L. albus genome annotation (see text footnote 1).Identification and characterization of moderately to highly repeated genomic sequences were achieved by graph-based clustering of genomic Illumina reads using RepeatExplorer2 pipeline . A totalin situ hybridization (FISH) probes were obtained as 5′–Cy3 or 5′–FAM-labeled oligonucleotides (Eurofins MWG Operon),2 or were PCR-amplified as described below. All DNA probes, except oligonucleotides, were labeled with Cy3- or Alexa 488-dUTP (Jena Bioscience) by nick translation, as described by 3 was applied. Microscopic images were recorded using a Zeiss Axiovert 200M microscope equipped with a Zeiss AxioCam CCD. Images were analyzed using the ZEN software (Carl Zeiss GmbH).Fluorescence L. albus using the forward and reverse primers, as given in ® SV Gel and PCR Clean-Up System (Promega). Sanger sequencing confirmed the correct amplification of PCR fragments. After confirmation, the PCR products containing the same class of repeat were collected and used for probe labeling by nick translation as described above.Fragments for probe labeling were amplified using gDNA from CENH3-ChIPseq was previously generated, as in q > 30 were used. Read coverage BIGWIG files were generated from original BAM files using the bamCoverage tool of the deepTools suite (ChIPseq Illumina paired-end reads were mapped to WL genome assembly (cv. AMIGA)  using Bols suite  with the4 employing the LAST program  can be found in the article/AM, BH, and AS performed all experiments. AM and BP designed the research. AM, BH, and BP wrote 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Depression is common in the human immunodeficiency virus (HIV)-hepatitis C virus (HCV) co-infected population. Demographic, behavioural, and clinical data collected in research settings may be of help in identifying those at risk for clinical depression. We aimed to predict the presence of depressive symptoms indicative of a risk of depression and identify important classification predictors using supervised machine learning.We used data from the Canadian Co-infection Cohort, a multicentre prospective cohort, and its associated sub-study on Food Security (FS). The Center for Epidemiologic Studies Depression Scale-10 (CES-D-10) was administered in the FS sub-study; participants were classified as being at risk for clinical depression if scores ≥ 10. We developed two random forest algorithms using the training data (80%) and tenfold cross validation to predict the CES-D-10 classes—1. Full algorithm with all candidate predictors (137 predictors) and 2. Reduced algorithm using a subset of predictors based on expert opinion (46 predictors). We evaluated the algorithm performances in the testing data using area under the receiver operating characteristic curves (AUC) and generated predictor importance plots.We included 1,934 FS sub-study visits from 717 participants who were predominantly male (73%), white (76%), unemployed (73%), and high school educated (52%). At the first visit, median age was 49 years (IQR:43–54) and 53% reported presence of depressive symptoms with CES-D-10 scores ≥ 10. The full algorithm had an AUC of 0.82 (95% CI:0.78–0.86) and the reduced algorithm of 0.76 (95% CI:0.71–0.81). Employment, HIV clinical stage, revenue source, body mass index, and education were the five most important predictors.We developed a prediction algorithm that could be instrumental in identifying individuals at risk for depression in the HIV-HCV co-infected population in research settings. Development of such machine learning algorithms using research data with rich predictor information can be useful for retrospective analyses of unanswered questions regarding impact of depressive symptoms on clinical and patient-centred outcomes among vulnerable populations.The online version contains supplementary material available at 10.1186/s12874-022-01700-y. With shared modes of transmission, co-infection of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) is common, with approximately 2.3 million co-infected individuals worldwide , 2. DeprThe presence of significant depressive symptoms may have an impact on outcomes in patients, even in the absence of a clinical depression diagnosis; for example, the presence of depressive symptoms is associated with non-adherence to antiretroviral therapy among people living with HIV, which may lead to increased viral load and suppressed immune function , 8. ScreMachine learning includes robust techniques that enable accurate outcome predictions in medical research. In mental health research, machine learning has been used to predict current or future onset, disease course and treatment outcomes for psychiatric disorders including depression, anxiety, and schizophrenia , 16, 17.We leveraged a non-parametric supervised machine learning technique using cohort data to develop classification algorithms to predict the presence of depressive symptoms indicative of a risk for clinical depression and characteristics important for prediction of depressive symptoms in HIV-HCV co-infected individuals in Canada.We used data from the Canadian HIV-HCV Co-Infection Cohort (CCC), an open multicenter prospective cohort study, ongoing since 2003 and an associated sub-study, the Food Security and HIV-HCV co-infection study (FS sub-study) . The CCCThe FS sub-study is a mixed methods study conducted within the CCC between 2012 and 2015. All CCC participants were invited to participate and study visits were integrated into the biannual CCC visits. The FS sub-study recruited 725 participants and they were followed up for a maximum of 5 visits. The study collected data on food insecurity, general and mental health (including depression screening), treatment adherence and health care utilization using a self-administered questionnaire .Depression screening was performed only in the FS sub-study; thus, the analytic sample in this study only included FS sub-study participant visits. The FS sub-study visits were merged with corresponding CCC visit data. As the two study visits for CCC and FS sub-study were, on occasion, not on the same day, information from visits within 3 months of each other were considered ‘concurrent’. We used three exclusion criteria to create the final study sample—1) participant visits were excluded if no depression screening measure (see below) was available at that visit; 2) participant visits were excluded if there was no corresponding CCC visit (within the 3-month window), as the predictors used in this analysis were derived from the CCC; and 3) all visits for a participant were excluded if no data was available for a predictor in all of their study visits.Depression screening was conducted in the FS sub-study using the Center for Epidemiologic Studies Depression Scale-10 (CES-D-10), which is a shortened version of the CES-D-20 scale . The CESWe selected candidate predictors (x) from the CCC data based on the literature and subject matter expertise. We included predictors from five major categories: questions related to mental health, HR-QoL, sociodemographic, behavioural, and clinical characteristics; see Table We assessed proportion of missing data for each predictor. For predictors with < 5% missing data, we carried the value from the last visit forward. For predictors with ≥ 5% missing data and when data were missing for a predictor for all visits for a participant, we used an additional category of “no response” for categorical variables, which we hypothesized could be informative in the prediction algorithm (See Table SWe used the supervised machine learning technique of Random Forests (RF), an ensemble learning approach which uses bootstrap aggregation of multiple decision trees, combining predictions from these many trees ; see morWe conducted several analyses to provide additional details about the classification characteristics from the main analysis and assess robustness of the results: A) Using one visit per individual , to assess difference in performance compared to the use of multiple visits per individual; B) Algorithms using three different CES-D-10 thresholds—8, 13, and 15—based on suggested cut-offs in the literature , 30; C) ranger and caret; for performance evaluation, we used the performance assessment function in R by Wong et. al. (2019) [The final tuned algorithms were implemented in the testing data, which was not used in the development stage. The tuning parameters are shown in Table S. (2019) , 37–39.Of the 1973 FS sub-study visits in a total of 725 participants, 39 study visits were excluded based on the exclusion criteria described in the methods—16 visits (2 participants) with no CES-D-10 score, 18 visits (5 participants) with no con-current CCC visit and all 5 visits from 1 participant with no predictor data  in all visits. Thus, 717 participants with a total of 1934 visits contributed to the final study sample. The participant characteristics at the first visit included in the sample are described in Table The training data consisted of 1548 visits and testing data consisted of 386 visits. The algorithms in the primary analysis showed acceptable calibration, as seen in Figure SThe results for the additional analyses A-D are shown in Table We developed a random forest algorithms using patient data from a cohort study that reliably predicted the presence of depressive symptoms indicative of a risk of clinical depression in a vulnerable HIV-HCV co-infected population. The algorithms used a set of selected candidate predictors (x = 137) from the cohort. The full algorithm using all candidate predictors performed better, with an AUC of 0.82, which indicates a 82% chance of distinguishing between CES-D-10 classes compared 0.76 for the reduced algorithm which used a smaller subset (x = 46) . The preWe developed this tool to assess if patient data that is commonly collected in clinical charts and research studies could be useful in predicting presence of depressive symptoms, which is seldom directly measured routinely for all patients, nor measured repeatedly over time. This tool will be most useful for conducting longitudinal clinical and epidemiologic research rather than for clinical care. It may prove useful to help identify people at risk for depression, study how this risk changes over time and with various interventions.Most studies using machine learning have predicted the future onset of depressive symptoms , 40, 41 CCC collects extensive demographic, behavioral and clinical data. Using the full range and diversity of available predictors did show excellent discrimination in the full algorithm. However, for greater applicability, we chose to use a subset of 46 predictors that may be more readily available in other research settings, and despite using one third the number of predictors and less granular data, the overall discrimination was still acceptable. The additional analysis using only one visit per participant had a comparatively lower AUC, which may have been due to the smaller sample size and thus lower variability in the available data.The algorithm we developed was a purely prediction algorithm and hence estimation of the strength of the effect of individual predictors is not possible. Further analysis with different modeling strategies would be needed for this assessment. However, the algorithm does provide some insight into factors that may be important for classification. The five most important predictors are related two main themes—i. SES  and ii. overall health status . SES is a known strong determinant of depression. Receiving welfare and being from a low-income household has been associated with an elevated risk of food insecurity, and mental health issues , 45. In This study thus has many strengths. The CCC is generalizable to the HIV-HCV co-infected patients engaged in care in Canada, due to the recruitment from a variety of clinical settings . The sample used to develop these algorithms was generalizable to the parent CCC  compared to a cut-off of 10. However, the full algorithm using a cut-off of 15 appears to have a higher AUC (0.87) than that of using a cut-off 10 (0.82). It will be important to assess in future studies whether this higher threshold may be more applicable to the co-infected population. However, since the CES-D-10 cut-off of 10 has been validated in HIV populations in Canada , we deciWith a high proportion of participants with depressive symptoms in this population, it is important not to miss possible cases. Even if the algorithms we developed are considered to have acceptable discrimination (≥ 0.7) based on arbitrary thresholds, we would still misclassify a fair proportion of cases and thus this possible misclassification needs to be considered. Finally, this algorithm is applicable when the majority of the predictors are collected. However, in settings where such data is not available, especially completely clinical non-research setting implementing routine screening tools like the CES-D-10 should be considered, especially given the high prevalence of depressive symptoms we observed in this co-infected population.Depressive symptoms indicative of a risk for clinical depression were common in our population of people living with HIV-HCV co-infection. The random forest algorithms we developed shows promise in accurately predicting an elevated risk of clinical depression using data on patient characteristics collected in research settings. The algorithms identified important characteristics for depressive symptoms classification including employment, HIV clinical stage, revenue source, BMI, and education. Such machine learning algorithms can be used in research settings especially cohort studies where such data may be available to predict presence of depressive symptoms and use this information to understand the impact of depressive symptoms on clinical, health service and patient-reported outcomes in vulnerable populations.Additional file 1.


Despite advances in scaling up new vaccines in low- and middle-income countries, the global number of unvaccinated children has remained high over the past decade. We used 2000–2019 household survey data from 154 surveys representing 89 low- and middle-income countries to assess within-country, economic-related inequality in the prevalence of one-year-old children with zero doses of diphtheria–tetanus–pertussis (DTP) vaccine. Zero-dose DTP prevalence data were disaggregated by household wealth quintile. Difference, ratio, slope index of inequality, concentration index, and excess change measures were calculated to assess the latest situation and change over time, by country income grouping for 17 countries with high zero-dose DTP numbers and prevalence. Across 89 countries, the median prevalence of zero-dose DTP was 7.6%. Within-country inequalities mostly favored the richest quintile, with 19 of 89 countries reporting a rich–poor gap of ≥20.0 percentage points. Low-income countries had higher inequality than lower–middle-income countries and upper–middle-income countries . Zero-dose DTP prevalence among the poorest households of low-income countries declined between 2000 and 2009 and between 2010 and 2019, yet economic-related inequality remained high in many countries. Widespread economic-related inequalities in zero-dose DTP prevalence are particularly pronounced in low-income countries and have remained high over the previous decade. The Immunization Agenda 2030 (IA2030) envisions a world where everyone, everywhere, at every age, fully benefits from vaccines to improve health and well-being . There hThere are distinct measurement and programmatic considerations surrounding populations of zero-dose children versus those who are under-vaccinated . Whereas children who are under-vaccinated have had at least one contact with the health system, zero-dose children often lack access to basic health services, including immunization services. For instance, children who received fewer other health services and interventions, and whose mothers received fewer services , are at higher risk of being unvaccinated ,8. A stuMajor global immunization initiatives have adopted an intensified focus on reaching zero-dose children. IA2030 includes an objective of extending immunization services to regularly reach zero-dose children , and makes a call for better data to map and track at the subnational level children who are unvaccinated . Gavi 5.Few studies have characterized socioeconomic inequalities in zero-dose children. Across 92 low- and middle-income countries, an estimated 7.7% of one-year-old children did not receive any of four basic vaccines delivered in infancy, with higher zero-dose prevalence among the poorest 12.5%) than the richest (3.4%) wealth quintiles .5% than ; gender-To date, there have been no large multi-country studies that report the state of inequality for zero-dose children and that assess the latest status of inequality and change over time. In this study, we quantify the extent of economic-related inequality, including change in inequality over time, in zero-dose DTP prevalence, exploring variation by country income level grouping, and assessing patterns of inequality in countries with high prevalence or a high number of children with zero-dose DTP. Measuring and reporting socioeconomic inequalities in immunization can help inform strategies to equitably expand the reach of immunization programs and services.Disaggregated data were sourced from 88 Demographic Health Surveys (DHSs) and 66 Multiple Indicator Cluster Surveys (MICSs) in 89 low- and middle-income countries. DHS and MICS are large-scale, nationally representative household surveys that collect data through standardized face-to-face interviews with women aged 15–49 years, repeated approximately every three to five years. The data used in this analysis are the product of reanalysis of DHS and MICS microdata by the International Center for Equity in Health at the Federal University of Pelotas.Zero-dose DTP prevalence was defined as the percentage of children aged 12–23 months who did not receive any of the three DTP-containing vaccine doses recommended in infancy at the time of the DHS or MICS. In certain countries, the sample specified different age ranges to align with national vaccination schedules and ensure that our indicator is consistent with results from national survey reports and recent publications on this topic. Surveys with an age range of 18–29 months are: Bosnia and Herzegovina, 2006 and 2011; Egypt, 2014; Jamaica, 2011; Peru, 2009 and 2019; Ukraine, 2012; Albania, 2008; Guyana, 2006; North Macedonia, 2005; Serbia, 2005; Suriname, 2006; and Tajikistan, 2005. Surveys with a 15–26-month age range are: Turkey, 2013; Kazakhstan, 2006; and Republic of Moldova, 2005 and 2012.Household economic status was based on a household wealth index, which considered ownership of household assets and access to certain services. Principal component analysis was used to construct country-specific indices and generate wealth quintiles ranging from quintile 1 (poorest) to quintile 5 (richest). Information about wealth quintiles is available in the DHS or MICS datasets.Countries were considered for inclusion based on the availability of data for children with zero-dose DTP from a DHS or MICS conducted between 2010 and 2019. The analysis included countries categorized by the World Bank Group as low-, lower–middle-, or upper–middle-income countries in July 2021. Countries included in the change over time analysis had additional data available from a survey conducted between 2000 and 2009 . Ten country surveys with an insufficient sample size (less than 25 for any subgroup) were excluded from the analysis.A subset of 17 countries was identified for further analysis, based on (a) reporting more than 25% national zero-dose DTP prevalence according to the latest DHS or MICS between 2010 and 2019 or (b) being in the top 10 countries with the highest numbers of children with zero-dose DTP in 2019 [To assess the latest situation of inequality (using data from 2010–2019), we calculated summary measures of inequality using zero-dose DTP prevalence data disaggregated by wealth quintiles. Difference, a measure of absolute inequality, was calculated as prevalence in the poorest quintile minus prevalence in the richest quintile. Ratio, a measure of relative inequality, was calculated as prevalence in the poorest quintile divided by prevalence in the richest quintile.Absolute and relative complex summary measures of inequality , which take into account the situation across all wealth quintiles, were also calculated . The sloTo assess how inequalities have changed over time, annual absolute change was calculated using two data points, from 2010 to 2019 and from 2000 to 2009 (spanning about ten years). Annual absolute change was calculated as the difference in zero-dose DTP prevalence in the recent versus older surveys, divided by the number of years between the two surveys. It was calculated to quantify change in national average, and in the most and least advantaged subgroups.Annual absolute excess change compares the annual absolute change in the least and most advantaged subgroups. For example, to arrive at absolute excess change for economic status, the annual absolute change in quintile 5 is subtracted from the annual absolute change in quintile 1, yielding annual absolute excess change in percentage points. A positive excess change indicates change favoring the richest subgroup . A negative annual excess change indicates change favoring the poorest subgroup .A survey sampling design was taken into account when calculating point estimates of disaggregated data and corresponding 95% confidence intervals (95% CI). We ascertained statistical significance with 95% CIs.Analyses were conducted using Stata 16 and Stata 17 software and data visuals were developed using Tableau software (version 2021.4).The analysis of the latest situation of economic-related inequality included 89 countries, where the median national zero-dose DTP prevalence was 7.6%, ranging from 0% in Republic of Moldova to 72.7% in South Sudan (note: the South Sudan MICS 2010 occurred in the period leading up to South Sudan gaining independence in 2011) and 45.0% in Central African Republic . While on = 22), the difference between the median prevalence in the poorest quintile (20.7%) and richest quintile (6.3%) was 14.4 percentage points. Lower–middle-income countries (n = 41) and upper–middle-income countries (n = 26) had comparatively smaller gaps, at 8.9 percentage points and 2.7 percentage points, respectively.Across 89 countries, the median zero-dose DTP prevalence among the poorest quintile (9.9%) was higher than the median prevalence among the richest quintile (5.0%) . Across Slope index of inequality demonstrated similar patterns of inequality as difference across country income groupings, with the absolute extent of inequality tending to be slightly higher when taking all subgroups into account. Concentration index demonstrated similar patterns as ratio.The change over time analysis included 65 countries with available data . The medOverall, changes in inequality and national average over time demonstrated different patterns across countries . SeveralMany countries showed little or no absolute change in national average or economic-related inequality over the past ten years. Egypt and Mongolia sustained low zero-dose DTP prevalence , with absolute economic-related inequality remaining around 2 percentage points or less. In other countries, such as Benin, high zero-dose DTP prevalence remained unchanged (above 15%), with a sustained gap of at least 25 percentage points between the poorest and richest quintiles.In Congo, Côte d’Ivoire, and Haiti, the national zero-dose DTP prevalence remained largely unchanged between the two surveys (in the range of 14–20%), though the situation across wealth quintiles changed in divergent ways. In Haiti, the gap between the rich and poor widened due to increased prevalence among the poorest quintile (by more than 6 percentage points across ten years) and prevalence decreasing among the richest (by more than 7 percentage points across ten years). In Congo and Côte d’Ivoire, the zero-dose DTP prevalence increased among the richest quintile (by more than 9 percentage points across ten years) and decreased among the poorest quintile (by around 4–6 percentage points across ten years).n = 13) and/or highest numbers of children with zero-dose DTP (n = 9). This subset consists of seven low-income countries, nine lower–middle-income countries, and one upper–middle-income country. All 17 countries reported higher prevalence in the poorest quintile than the richest, with difference ranging from 2.7 percentage points in Mexico to 53.2 percentage points in South Sudan (median difference of 32.1 percentage points across 17 countries).A subset of 17 countries had zero-dose DTP prevalence of more than 25% according to the latest available survey since 2010  revealed divergent patterns, particularly with regards to how inequality changed over time. Notably, India and Indonesia achieved substantial reductions in economic-related inequality, while the situation in Guinea and Madagascar became worse, in terms of both inequality and national average. Country-specific studies are required to explore contextual factors that underlie existing socioeconomic inequalities and identify lessons learned in areas where the situation is improving. While cross-cutting strategies to improve equity in vaccination coverage have been identified, including improving the relationships between the health system and mothers who are socioeconomically disadvantaged, setting-specific approaches should be tailored to account for local considerations ,23.Since the inception of the Expanded Program on Immunization in the 1970s, all countries have included DTP3 in the national vaccination schedules, as per the recommendation of the WHO. There are complex reasons why children from socioeconomically disadvantaged subgroups lack access to vaccinations. Women’s low social status is a barrier that cuts across family, community, and health facility environments, and manifests through educational attainment, income, decision-making, and resource allocation ,24. DeliOthers have explored the reasons underlying socioeconomic inequalities in vaccine coverage. For instance, across 46 low- and middle-income countries, pro-rich inequality in the receipt of four basic vaccines in childhood was associated with higher antenatal care visit attendance among wealthier women . A studyDeriving estimates and assessing inequalities of zero-dose children, a population defined by its lack of contact with health systems, is subject to measurement limitations . Our useOur analysis used a lack of DTP vaccination as a measure of zero-dose children, following the operational definition used by IA2030, with reports by World Health Organization and UNICEF  which caAlongside indicators of vaccination coverage and drop-out, monitoring and reporting on zero-dose children is a stated priority of IA2030 and Gavi 5.0. The indicator provides valuable insights on children who lack access to routine immunizations and can help to inform actions to equitably expand the reach of immunization programs and basic healthcare services. In other words, actions should be equity-oriented to achieve national improvements alongside narrowing socioeconomic inequality (faster improvement among the poorest subgroups). To this end, our analysis of economic-related inequalities in zero-dose DTP prevalence serves as an important baseline assessment to benchmark future efforts to reduce the number of zero-dose children. Socioeconomic inequalities in zero-dose children, alongside other dimensions of inequality, should be monitored regularly as new data become available. Investments should expand and strengthen data collection and analysis, with a focus on disaggregated data. Further in-depth analysis of country-level inequalities is warranted, including exploration of the drivers of inequality and context-specific strategies to address them.


Inflammatory bowel disease (IBD) is a heterogenous disease in which the microbiome has been shown to play an important role. However, the precise homeostatic or pathological functions played by bacteria remain unclear. Most published studies report taxa-disease associations based on single-technology analysis of a single cohort, potentially biasing results to one clinical protocol, cohort, and molecular analysis technology. To begin to address this key question, precise identification of the bacteria implicated in IBD across cohorts is necessary.We sought to take advantage of the numerous and diverse studies characterizing the microbiome in IBD to develop a multi-technology meta-analysis (MTMA) as a platform for aggregation of independently generated datasets, irrespective of DNA-profiling technique, in order to uncover the consistent microbial modulators of disease. We report the largest strain-level survey of IBD, integrating microbiome profiles from 3,407 samples from 21 datasets spanning 15 cohorts, three of which are presented for the first time in the current study, characterized using three DNA-profiling technologies, mapping all nucleotide data against known, culturable strain reference data.Lachnospiraceae strains consistently decreased in both the gut luminal and mucosal contents of patients with IBD, and demonstrate that these strains are correlated with inflammation-related pathways that are known mechanisms targeted for treatment. Furthermore, comparative MTMA at the species versus strain level reveals that not all significant strain associations resulted in a corresponding species-level significance and conversely significant species associations are not always re-captured at the strain level.We identify several novel IBD associations with culturable strains that have so far remained elusive, including two genome-sequenced but uncharacterized We propose MTMA for uncovering experimentally testable strain-disease associations that, as demonstrated here, are beneficial in discovering mechanisms underpinning microbiome impact on disease or novel targets for therapeutic interventions. Ruminococcus gnavus strains in patients but not in control subjects, and these strains were found to harbor genes that conferred them an adaptive advantage in disease . A diagnosis of UC was confirmed by a qualified gastroenterologist at the Foothills Medical Center, University of Calgary. Informed consent for use of biopsy samples was obtained by the University of Calgary Intestinal Inflammation Tissue Bank and the study was approved by the University of Calgary, Conjoint Health Research Ethics Board (ID: 18142 and 14-2429).Mucosal biopsies were collected at the University of Calgary during initial diagnosis or follow-up endoscopy from September 2007 to April 2013 from control and UC subjects  following instructions provided by the manufacturer.The 16S rRNA V4 region was PCR-amplified using fusion primers designed against surrounding conserved regions and tailed to incorporate Illumina adaptors and indexing barcodes as described previously . AmplicoV1 through V9 16S rRNA gene analysis was performed on the G3 PhyloChip  using laBiopsies were completely defrosted in RNA-later before performing RNA purification with the AllPrep RNA Mini kit (Qiagen). Defrosted biopsies were transferred into a tube containing 350 μl RLT buffer with β-mercaptoethanol , three 3.5 mm glass beads, and 0.25 ml of 0.1 mm glass beads . Disruption and homogenization were carried out in a MagNA Lyser  two times for 15 s at 3,500 or 6,500 rpm. RNA purification was performed according to the kit manufacturer’s instructions. DNA contaminations in RNA samples were removed by Turbo DNA-free kit following the manufacturer’s instructions . RNA concentrations were measured using a Nano-Drop 2000 Spectrophotometer . RNA integrity was checked on 1% agarose gel electrophoresis and 2100 Bioanalyzer system . In addition, RNA quality was considered acceptable if the RNA integrity number was ≥ 6 and the rRNA ratio was ≥ 1.5. Host transcriptome RNAseq was carried out by Macrogen  using TruSeq Stranded mRNA Sample Prep Kit (Illumina) with Illumina HiSeq 4000 2 × 150 reads following the manufacturer’s protocol.via manual curation. Clinical metadata was stored in this database as a series of label:value pairs attached to the biospecimen from which the data files were generated.For the integrated meta-analysis, we obtained 13 additional cohorts where UC and/or CD subjects were profiled with WGS.NGS and/or 16S.NGS techniques against control subjects using either mucosal biopsies or stool biospecimens . 16S.NGS1 is a reference database of archaeal and bacterial genomic identifiers organized by strain. StrainSelect assigns a consistently formatted identifier for known strains that have been isolated and have had their genome sequenced and/or their 16S rRNA gene sequenced and shared publicly.StrainSelectChristensenella minuta YIT 12065. Two independent BRCs, DSM, and JCM, also propagated sub-cultures of this strain with their own unique catalog numbers, DSM 22607 and JCM 16072. The University of California at Davis, Beijing Genome Institute, Washington University, and South China University of Technology each procured the strain from one of the BRCs then separately sequenced the extracted DNA and submitted their optimal assembly to public databases which are now downloadable under 4 different assembly identifiers: GCF_001571425, GCF_001652705, GCF_001678855, and GCF_003628755. A novice user of these public databases can misinterpret these four assemblies as four different genomes from four different strains, but they would be incorrect. In building the StrainSelect database, we sought to overcome confusion by tracing through the synonymous identifiers for sub-cultures and genomic data records and assigning a consistently formatted identifier for the strain, which in this example is StrainSelectID: t__520.A single strain encapsulates all the descendants of single isolation in pure culture and is usually disseminated by a succession of cultures ultimately derived from a single colony . The iniTaxonomic placement and nomenclature were adapted from GTDB . In casevia DADA2 applying default settings for filtering, learning errors, dereplication, amplicon sequence variant (ASV) inference, and chimera removal  and aligned to StrainSelect , version 2019 (SS19) using USEARCH  .Empirical OTUs (eOTUs) generated using image-scoring procedures were annotated against SS19 to obtain strain-level annotations as described previously .via Kraken  strain-level differential abundances when integrating five or more datasets in a meta-analysis.via Welch’s t-tests, and adjusted p-values were determined with the Benjamini-Hochberg correction. Fold change and variance were calculated using the metafor package in R using the escalc function with measure = MD  and performed prevalence filtering at 5% (zero_cut = 0.95). Following this a pseudo-count of 1 was added to all samples and bins, and data were normalized via a clr-transformation (as implemented in clr function in the compositions package in R). Significant differences for bins were calculated via t-tests, and adjusted p-values were determined with the Benjamini-Hochberg correction. The clr-transformation followed by the t-test for DA in NGS data results in high concordance across related cohorts  were acquired from the same subject, only the earliest time point was retained. If the clinical metadata did not resolve the relative timepoints, then the biospecimen that yielded the greatest number of reads post-filtering was retained. Fold change and variance were calculated using the ure = MD . Standarrma.mv function in the metafor package in R  as implemented in the age in R . A multip-values, log-2 fold changes, and 95% confidence intervals were obtained for comparisons.From a table of strain-level observations of log-2 fold changes and associated standard errors in each isolated dataset, random draws were taken to simulate a collection of observations from 1 to 3 DNA-profiling technologies, 1 to 2 biospecimen-types, 1 to 2 disease-subtype contrasts, and 2 to 21 datasets. A total of 7,500 of the random draws were made simulating 2 or 3 single-technology meta-analyses (STMAs) within an MTMA allowing comparisons between STMA and MTMA. STMA and MTMA were then run on these simulated observations as described in the MTMA section above and For strains of interest, protein sequences were predicted by Prodigal . KEGG ormygene package in R  from the pathway enrichment analysis for each strain were compared to identified pathways unique to the strains determined as significantly decreased in IBD. Note that the top 20 pathways were compared only for pathways with adjusted p < 0.1.Mucosal biopsies from patients where both microbiome (PhyloChip) and host-expression profile were available in SG-Cohort-1, were examined for gene-expression patterns correlating with the abundance of each strain of interest  in individual studies could be confirmed across cohorts, we identified microbiome studies where both clinical metadata and microbiome-sequencing data were publicly available or were generated from three of our recent studies . We intenologies . Patientnologies . We devenologies . These cnologies . Thus wenologies . Furthernologies , we hypop < 0.01) in STMA or MTMA was determined by integrating data pertaining to each DNA-profiling technology only or all simulated data agnostic of technology  may fail to eliminate; given that they are restricted to trends observed in one DNA-profiling technology only. To demonstrate this added value of a comprehensive view of the microbiome in disease facilitated by MTMA compared to STMA, permutations were run to simulate two or more observations of taxa from two or more DNA-profiling technologies. For each simulated taxon, significance  and intestinal-mucosa from UC and CD subjects  that were significantly enriched and one strain (D. invisus t__28462) that was decreased in controls compared to case subjects. The association of these strains with the disease was supported by > 12 cohorts and also supported in both stool and mucosa  in SG-Cohort-1 had both microbiome (PhyloChip) and host-gene expression profiles (RNAseq) of their mucosal samples available for analysis. Gene-expression patterns indicated significant enrichment of immune-related and signal-transduction pathways associated with these four strains . For thes in IBD . To detes in IBD . This anLachnospiraceae strains; It is apparent from our re-analysis of published datasets that there exist significant variations across cohorts, even when datasets are analyzed through a standardized pipeline . MTMA enLachnospiraceae strains that are decreased in IBD stools and mucosa, besides their genome assemblies. Just as NIH projects such as “Most Wanted” identified abundant 16S rRNA amplicons that inspired a search for their genomes of origin  can be found below: JR contributed to the conception and design of work, acquisition, analysis and interpretation of data, and drafted and substantively revised the manuscript. ER contributed to the acquisition of data. C-EC, GK, PB, and MC contributed to the analysis and interpretation of data, and revision of the manuscript. NN contributed to the analysis of data. AH contributed to the design of work, acquisition, analysis and interpretation of data, and substantively revised the manuscript. SI and TD contributed to the conception and design of work, acquisition, analysis and interpretation of data, and substantively revised the manuscript. KD contributed to the conception and design of work, interpretation of data, and substantively revised the manuscript. All authors contributed to the article and approved the submitted version.


This retrospective study demonstrates the long-term outcomes of treating prostate cancer using intensity modulated (IMRT) with incorporation of MRI-directed boost.From February 2009 to February 2013, 78 men received image-guided IMRT delivering 77.4 Gy in 44 fractions with simultaneously integrated boost to 81–83 Gy to an MRI-identified lesion. Patients with intermediate-risk or high-risk prostate cancer were recommended to receive 6 and 24–36 months of adjuvant hormonal therapy, respectively.Median follow-up was 113 months (11–147). There were 18 low-risk, 43 intermediate-risk, and 17 high-risk patients per NCCN risk stratification included in this study. Adjuvant hormonal therapy was utilized in 32 patients (41%). The 10-year biochemical control rate for all patients was 77%. The 10-year biochemical control rates for low-risk, intermediate-risk, and high-risk diseases were 94%, 81%, and 88%, respectively (p = 0.35). The 10-year rates of local control, distant control, and survival were 99%, 88%, and 66%, respectively. Of 25 patients who died, only four (5%) died of prostate cancer. On univariate analysis, T-category and pretreatment PSA level were associated with distant failure rate (p = 0.02). There was no grade =3 genitourinary and gastrointestinal toxicities that persisted at the last follow-up.This study demonstrated the long-term efficacy of using MRI to define an intra-prostatic lesion for SIB to 81–83Gy while treating the entire prostate gland to 77.4 Gy with IMRT. Our study confirms that modern MRI can be used to locally intensify dose to prostate tumors providing high long-term disease control while maintaining favorable long-term toxicity. Dose escalation has been a critical topic of investigation in the management of prostate cancer with radiotherapy –8. The eStudies evaluating patterns of failure following conventionally fractionated radiation demonstrated that 90% of local recurrences typically occur in the intraprostatic dominant nodule . The intMultiparametric MRI uses various T1 and T2 sequences, dynamic contrast enhancement, and diffusion-weighted imaging to identify prostate cancer versus normal prostatic tissue –17. UsinHerein, we report the long-term results of patients receiving a simultaneous integrated boost up to 83 Gy to intraprostatic lesions identified by MRI. This treatment strategy was devised in order to increase the therapeutic ratio by delivering radiation therapy focally to the region of prostate cancer. While there are many studies looking at the safety and short-term outcomes of simultaneous integrated boost to intraprostatic lesions using MRI, this is one of the few studies to present data with substantially longer follow-up.Institutional review board approval was obtained prior to performing this retrospective analysis. Patients treated between February 2009 and February 2013 at our institution with MRI-guided boost for prostate cancer were analyzed in this study.A multiparametric MRI was used to identify the intra-prostatic lesion (IPL) for treatment planning. T2-weighted, diffusion-weighted, and dynamic contrast-enhanced (DCE) images were obtained to define the IPL. A genitourinary diagnostic radiologist identified prostate lesions. Following the MRI, four fiducial markers were placed in the prostate. Patients were then simulated with computed tomography (CT) in the treatment position. The IPL was designated on MRI within the planning system.Volume and organs at risk (OAR) definition and treatment planning were performed in Eclipse . The rectum and bladder were contoured as solid organs. The prostate was contoured to generate a low-dose target volume. The IPL was contoured to create the high-dose target volume. In patients with multiple IPLs, each IPL was included in the high-dose target volume. If an IPL was not identified on MRI, the region of biopsy positivity was contoured to create the high-dose target volume. The prostate volume was expanded by 3 mm to create the PTV for the lower dose with no expansion used for the SIB high-dose volume. The PTV low-dose volume (prostate plus 3 mm) received 77.4 Gy in 1.8 fractions with a simultaneous integrated boost of 83 Gy delivered to the PTV high-dose volume. Patients without an IPL on MRI had 81 Gy delivered to the PTV high-dose volume. Seminal vesicles received 75–77.4 Gy if involved and only 54 Gy if not involved. The adjacent dose-limiting normal structures including bladder, rectum, and femoral heads were contoured as organs at risk. This was our standard technique at that time based in large part on our experience performing a prospective trial utilizing IMRT and ProstaScint-based SIB .3 of the rectum or bladder could receive ≥81 Gy.Normal tissue dose constraints were as follows: ≤30% of the rectum or bladder could receive ≥70 Gy; ≤10% of the rectum or bladder could receive ≥75 Gy; and ≤1.8 cmRadiotherapy was delivered using intensity-modulated radiotherapy techniques (IMRT). IMRT plans were generated using seven-field IMRT, nine-field IMRT, or volumetric modulated arc therapy  using 6- or 18-MV x-rays. Image guidance using kV matching of the four implanted fiducial markers was performed daily to localize the prostate.Androgen deprivation therapy (ADT) was administered at the discretion of the treating physician and was recommended for intermediate- and high-risk diseases. Patients in the intermediate-risk group were advised to receive 6 months of ADT (leuprolide), and patients in the high-risk group were advised to receive 24–36 months of ADT. After radiotherapy, patients were evaluated at 3–12-month intervals with serum PSA measurement, physical examination, and toxicity assessments.Treatment outcomes were defined in terms of biochemical control (BC), overall survival (OS), local control (LC), and distant control (DC) rates. Length of follow-up and overall survival was determined as the time from completion of all treatment to date of last follow-up or death. Biochemical failure was defined as the time from end of radiation treatment to biochemical relapse defined per the American Society for Radiation Oncology-Phoenix definition . Local fSeventy-eight patients with clinical T1–3, N0 M0 prostate cancers were treated with conventional fractionated radiotherapy to the prostate gland. The 10-year treatment outcomes are listed in Here, we report our long-term outcomes using standard treatment of 77.4 Gy to localized prostate cancer with focal boost to intraprostatic lesion, identified on multiparametric MRI, to 81–83 Gy. Previously, we reported the feasibility of this approach with 3-year biochemical control, local control, distant control, and overall survival rates of 92%, 98, 95%, and 95% respectively . HoweverMultiple randomized trials have shown that dose-escalated RT leads to improved biochemical control of prostate cancer Table 3 There have been several retrospective and phase II studies which demonstrated the feasibility of simultaneous integrated boost for prostate cancer Table 4 The benefits of a boost with brachytherapy has been well documented , 33. In While there have been retrospective studies that assess the feasibility and safety of simultaneous integrated external beam radiation boost to intraprostatic lesions, the FLAME trial was the first phase III clinical trial to assess the addition of a focal boost on rates of biochemical relapse and other endpoints . The FLARecently, there has been a shift in the radiation treatment paradigm for prostate cancer toward hypofractionation Table 3.The results of the FLAME and ProstaScint trials support the continued efforts to use advanced technologies to deliver focal boosts to prostate tumors. The development of prostate-specific membrane antigen (PSMA)-targeted imaging provides new opportunities to safely boost prostate tumors. Although multiparametric MRI is consistently used to identify prostate cancer given its high sensitivity, specificity, and predictive value, it is possibly advantageous to use the newest imaging when designing radiation boost techniques , 39. MulThis treatment approach was initiated to increase the therapeutic ratio of radiation treatment to prostate cancer but does have limitations. It is limited by its retrospective nature and by the fact that most patients had low-risk and intermediate-risk diseases (80%). The future applicability of this regimen is limited given the overall shift in practice and standard-of-care treatment toward hypofractionation. While our study demonstrates durable biochemical control and overall survival with low toxicity using a simultaneous integrated boost in the setting of conventional fractionation, more recent trials have altered the standard of care to hypofractionated courses of radiotherapy. Therefore, studies which focus on dose-escalated SIB in the setting of hypofractionation are needed. Currently, the HEIGHT trial is an ongoing interventional clinical trial investigating the delivery of hypofractionated boost to the dominant tumor lesion identified by multiparametric MRI. The lesion will be treated with an absolute dose of 89.3–91.2 Gy, while the prostate receives 76 Gy over 38 fractions. Questions regarding the feasibility, safety, and efficacy of MRI guided boost to tumors with hypofractionation will be answered.In summary, our experience demonstrated that an MRI-guided focal boost to intraprostatic lesions produces durable long-term biochemical and local control without severe (grade 3 or greater) toxicity. MRI-guided boost to prostate lesions is an effective strategy to enhance biochemical control in patients. Our study provides validation that MRI-guided boost can be safely employed without increased risk of increased long-term toxicity. This was likely true because we left the normal tissue dose limitations in place when administering the boost doses. Given the evolution of prostate cancer treatment with shorter treatment courses, further studies evaluating dose-escalated boost in the setting of hypofractionation and stereotactic body radiotherapy are needed. The incorporated use of MP-MRI and PSMA PET-directed boost techniques with hypofractionation or stereotactic body radiotherapy should be investigated in future trials to potentially improve both disease control and treatment-related toxicities.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Mayo Clinic Institutional Review Board. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.SS conceptualized the study and performed the formal statistical analysis. BL wrote this study. All authors were involved with review, editing, and final approval of this manuscript.SS is a writer and editor for UptoDate.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


We present the early results of a novel partial bulky-tumor irradiation using carbon ions and protons. This approach explores the radiation-induced bystander and abscopal effects in patients with recurrent unresectable bulky tumors who failed previous state-of-the-art treatments including radio-chemotherapy. Early tumor response, symptom relief and minimal toxicity suggest that the partial bulky-tumor irradiation was an effective, safe and well tolerated treatment. Abscopal effects elucidate an immunogenic pathway contribution.Background: We present the early results of a novel partial bulky-tumor irradiation using particles for patients with recurrent unresectable bulky tumors who failed previous state-of-the-art treatments. Methods: First, eleven consecutive patients were treated from March 2020 until December 2021. The targeted Bystander Tumor Volume (BTV) was created by subtracting 1 cm from Gross Tumor Volume (GTV) surface. It reflected approximately 30% of the central GTV volume and was irradiated with 30–45 Gy RBE  in three consecutive fractions. The Peritumoral Immune Microenvironment (PIM) surrounding the GTV, containing nearby tissues, blood-lymphatic vessels and lymph nodes, was considered an organ at risk (OAR) and protected by highly conservative constraints. Results: With the median follow up of 6.3 months, overall survival was 64% with a median survival of 6.7 months; 46% of patients were progression-free. The average tumor volume regression was 61% from the initial size. The symptom control rate was 91%, with an average increase of the Karnofsky Index of 20%. The abscopal effect has been observed in 60% of patients. Conclusions: Partial bulky-tumor irradiation is an effective, safe and well tolerated treatment for patients with unresectable recurrent bulky disease. Abscopal effects elucidate an immunogenic pathway contribution. Extensive tumor shrinkage in some patients might permit definitive treatment—otherwise previously impossible. The last decades have witnessed remarkable technological, physical and biological developments in the field of radiation oncology resulting in improved treatment effectiveness and reduced complication rates. High-precision tumor identification and localization, as well as dose delivery, permits safe dose escalation, in combination with smart drugs and immunotherapies. However, the resulting improvements in local control and survival do not apply to all oncologic patients. A large patient population is still lacking the benefit of those modern radio-oncology therapies remaining hopeless and predestined to palliative or best supportive care (BSC). These are patients affected by unresectable bulky tumors, whose clinical status is often further complicated by the presence of a large hypoxic tumor component that is resistant to all available non-surgical approaches, (oligo) metastases and poor general conditions, the presence of which only eases the recommendation of the tumor boards for BSC. This is understandable given the large volume of these tumors and their close relationship with neighboring critical organs, which do not allow the ablative radiation dose to be delivered through conventional radiotherapy. Furthermore, the inside of the tumors, especially those large and solid, is represented by a very specific microenvironment that differs a lot from the outer tumor portion in terms of the vasculature, oxygenation level and metabolic profile. The larger the tumor is, the more pronounced these differences are, leading to chronic tumor hypoxia and subsequently resulting in biologically more aggressive tumor behavior, the formation of an immunosuppressive peri-tumoral environment, radio- and chemo-resistance, treatment failure, disease progression and poor prognosis ,2. ConveIn 2016, a novel, unconventional radiotherapy approach for high-dose PArtial Tumor irradiation targeting HYpoxic segment (PATHY) was developed to improve the therapeutic ratio of these large unresectable tumors by exploiting the bystander and abscopal effects . The bysIn 2020, carbon ions and protons were integrated into the PATHY concept (Particle-PATHY). Their specific physical and biological features make them ideally suited to achieve the goals required more efficiently, in terms of immunomodulatory properties, such as the neutralization of tumor hypoxia, known as a strong immunosuppressor, as well as sparing the outer circumferences of tumor and normal tissue, i.e., the PIM-volume . Compared to photons, particles can significantly reduce the “integral immune dose” sparing more efficiently PIM. Furthermore, carbon ions induce significantly stronger immunogenic cell death than photons or protons in both normoxic and hypoxic conditions ,18,19. IWe conducted a retrospective review approved by the local ethic committee to assess the impact of Particle-PATHY on Quality of Life (QoL), tumor downsizing (neoadjuvant effect), tumor control and survival among patients affected by recurrent unresectable bulky tumors unsuitable for conventional radio-chemotherapy. This series includes the first eleven consecutive patients treated at our institution from March 2020 until December 2021 that were previously subjected to BSC and who were referred for Particle Therapy but considered unsuitable for conventional Particle Therapy by our New Patient Review Board .The hypothesis is that for effective immunomodulation manifested by a clinically relevant bystander and abscopal effects, the entire tumor volume may not need to be irradiated but only a partial volume to initiate the immune cycle in radiation-spared PIM.Simulation and immobilization: CT and MRI scans were performed with 2 mm slice thickness as per institutional practice and were subsequently co-registered at the level of the bulky tumor. Depending on the tumor site, patients were positioned prone or supine and immobilized using a thermoplastic mask and customized vacuum cushion. Simulation CT was performed without whilst the MRI with contrast media. The MRI protocol consisted of T1ce, T2w (including FLAIR and MutliVane techniques) and DWI sequences.Volumes definition: The targeted internal partial tumor volume is termed “Bystander Tumor Volume” (BTV). Its delineation was adopted from previously published studies on photon-based SBRT-PATHY [RT-PATHY ,8. SinceOnce the BTV was defined, the PIM was created A,B. The The OARs at the level of the treated area were delineated on MRT images and subsequently checked and refined on the co-registered simulation CT images. Those included the structures involved by or in close proximity to the tumor, such as parenchymatous and hollow organs, vessels and nerves.Treatment planning: Treatments were prescribed using either protons, carbon ions, or mixed proton-carbon treatment. Comparative plans (carbon ions vs protons) were performed for each patient. The choice of the particle-type was subordinated to the BTV-coverage: PIM-dose ratio. The goal was to maximize target coverage while minimizing dose to PIM. Radiotherapy plans were calculated on the native simulation CT scans using Ray Station 8 and 11 with Pencil Beam (for carbon ions) and Monte Carlo (for protons) algorithm.Dose prescription: The task of prescribing the PATHY dose addressed two separate dosimetric issues: concerning immunogenicity aiming to increase the likelihood of generating immune-mediated bystander and abscopal effects:(1)To deliver sufficiently high and possibly heterogeneously distributed radiation dose at the level of the targeted BTV. The intended dose was in the order of 10–15 Gy per fraction. In order to address heterogeneity in radiation dose delivery, dose-gradients were created within the BTV, by creating an additional BTV (BTV2) within the former BTV (BTV1) by subtracting 5 mm from the BTV1 surface in all directions whenever tumor volume would permit it B.(2)To ascertain simultaneously effective radiation sparing of the PIM in order to preserve the loco-regional immune system cells necessary to mediate the non-targeted radiation effects.Achieving the BTV prescription goal was subordinated to the possibility of creating a sufficiently sharp dose fall off between the BTV surface and the GTV surface surrounded by the PIM and was proportional to the thickness of peripheral tumor tissue in between the BTV and GTV surfaces. Treatment plans were calculated to deliver 30–36 Gy in three consecutive fractions (10–12 Gy per fraction) to the BTV1, and 36–45 Gy (12–15 Gy per fraction) to the BTV2 so that at least 95% of the BTVs received as minimum 95% of the prescription dose. The choice of prescribing the fractional doses was driven by PIM-sparing: 12 Gy to the BTV1 with 15 Gy to the BTV2 were preferred if allowing planned PIM-sparing which was easier to achieve in particularly large tumors. Otherwise, the fractional dose was reduced to 10 Gy to the BTV1 with 12 Gy to the BTV2 to lower the dose received by PIM. In those patients in whom neither of the two goals was possible, only one BTV (BTV1) was created and treated either with 12 Gy × 3 or 10 Gy × 3 A. ReportFollow-up: The RECIST-based [ST-based  assessmeST-based .All procedures performed in the present study were in accordance with the ethical standards. All patients signed informed consent.Patient and disease characteristics are summarized in Treatment characteristics are summarized in Clinical outcomes are summarized in The neoadjuvant effect was not induced within the short follow-up time by those typically late- and scarce responding chordomas and chondrosarcomas, as expected. The average tumor-volume regression was 890.4 cc corresponding to 61% of the mean GTV volume. LC had an average duration of 5 months (range 2–16).Overall survival was 64% with a median survival of 6.7 months (range 3–16). 46% of patients were progression-free with a median progression-free survival (PFS) of 5.2 months (range 2–16).Symptom relief was achieved in 10/11 patients (symptom control rate 91%). KPS was significantly improved in 10 (91%) patients by an average of 20% . Three patients (27%) experienced Grade 1 fatigue. No other side effects were observed. In ten patients (91%) treatment resulted in a reduction of analgesics and abrogation of strong opioids.The abscopal effect has been observed in three (60%) out of five patients that had assessable distant tumor disease either N+ or M+ . These wThis analysis presents the early results of a novel Particle-PATHY concept using high-dose, hypofractionated carbon ions and protons for patients with recurrent unresectable bulky tumors who failed previous state-of-the-art treatments including radio-chemotherapy. This analysis focuses on the initial response and early outcomes for patients with progressive disease and without any realistic standard therapeutic option, thus for whom even a short follow-up period may allow decent conclusions to be drawn. By delivering this short-course treatment, we were able to improve KPS and QoL, prolong survival, induce substantial tumor downsizing, effectively reduce severe symptoms and suspend or reduce the use of opioids . TreatmeThe majority of tumors progressed rapidly locally prior to initiation of PATHY-therapy, i.e., with a radiographic increase in size. This demonstrated the need for a fast and effective therapeutic response. In such large tumors with fast growth dynamics, any anti-tumor effect of conventional palliative radiotherapy would probably be poor requiring several weeks before reaching its maximum—thus frequently exceeding the estimated overall survival time of the patient. In our series, 64% of patients are still alive reaching 6.7 months (range 3–16) of median survival and 5.2 months (range 2–16) of median PFS. Additionally, LC was maintained on average for 5.4 months. This 3-days short-course treatment shows a favorable cost-effectiveness profile by minimizing requirements and efficient use of resources, i.e., equipment, treatment rooms and staff.The scope of this study was not to assess the underlying physiologic mechanisms resulting in favorable clinical outcomes. However, previous clinical and biomolecular data provided evidence of interaction with the immune system . Those pParticle-PATHY was delivered in this patient group with strictly palliative intent. In tumors exhibiting good response, i.e., extensive tumor shrinkage, the issue arises, if downsizing might permit more definitive and potentially even curative treatment. This would be in analogy to the use of pre-operative irradiation to convert borderline resectable or locally advanced unresectable bulky tumors to a resectable state. Radiotherapy has been used for decades as a neoadjuvant treatment aiming to downsize large tumors allowing for radical resection ,36,37. WParticle-PATHY delivered extraordinarily low radiation dose to the surrounding OARs–best reflected by the low PIM-dose . Since sThis study represents a first and early analysis. Hence, it has inherent limitations. The presently small patient numbers and short follow-up period preclude a more comprehensive statistical analysis. Additional follow-up is presently accumulating. We considered these retrospective data sufficiently promising to initiate a prospective, IRB-approved Study.The Particle-PATHY approach resulted in an effective, safe and well tolerated treatment.Our early data indicate that as a minimum, all patients gained improvement in their symptoms and quality of life without associated treatment related toxicity. The local tumor regrew in some patients within the follow-up period. However, in a significant amount of patients Particle-PATHY resulted in varying degrees of tumor downsizing. This holds the true prospect that in some patients, for whom no standard-of-care definitive treatment existed, Particle-PATHY can result in a shrinkage of tumor size that will permit the application of definitive treatment thereafter. This prospect will be explored in the future. The fact that abscopal effects in un-irradiated distant metastatic sites were observed in 3/5 patients points towards the involvement of immune-stimulating mechanisms.Optimum patient selection and definition of most suitable disease characteristics are currently explored in an ongoing, prospective study. The added benefit of particle therapy compared to photon RT will ultimately require randomized, multi-institutional studies.Tubin Slavisa M.D. reported an international patent application PCT/EP2019/052164 published as WO 2019/162050. The authors reported no other conflict of interest.


IntroductionThe British Association of Endocrine and Thyroid Surgeons (BAETS) published guidance in 2012 advising against day-case thyroid lobectomy due to the unpredictable risk of post-operative haematoma. Due to the increasing pressures the National Health Service (NHS) faces, a position statement was released by BAETS in 2020 highlighting their support of day-case thyroid lobectomy in specific low-risk cases with the introduction of appropriate local protocols.ObjectiveWe aimed to assess post-operative complications of thyroid lobectomy at University Hospitals Bristol and Weston NHS Trust (UHBW), a tertiary centre for Otolaryngology, to consider the feasibility and safety of day-case surgery in our unit.MethodsWe conducted a retrospective audit of 140 elective thyroid lobectomy cases identified in the period of January 2017 to December 2018 at UHBW. Inclusion criteria included no previous thyroid surgery or major head and neck surgery, elective thyroid lobectomy procedure only, and surgery performed at UHBW by otolaryngology or maxillofacial teams. Exclusion criteria included emergency hemithyroidectomy; hemithyroidectomy as part of another procedure; and completion thyroidectomy.ResultsA total of 140 elective thyroid lobectomy procedures were performed between January 2017 and December 2018. Of them, 125 cases met inclusion criteria, and 15 cases were excluded: two emergency hemithyroidectomy, three incorrect coding, four hemithyroidectomy as part of another head and neck procedure, and six completion thyroidectomy. Mean age was 51.9 years (range: 20-88 years), with 30 (24.0%) male cases and 95 (76.0%) female cases. Mean in-patient stay was 1.4 days (range: 0-19 days), and mean hours in hospital were 34 hours. Two (1.6%) cases were discharged on the same day of their operation. Post-operative complications included five (4%) cases of post-operative haematoma, four (3.2%) cases of unilateral vocal cord palsy, one (0.8%) case of pneumonia, and three (2.4%) cases of wound infections. From those cases that developed post-operative haematoma, 80% (n=4/5) developed less than 24 hours after the procedure. One (20%) case of post-operative haematoma developed on day 6 after their procedure and required readmission for re-operation.ConclusionThe unpredictable nature of post-operative haematoma poses a significant risk to patients and medicolegal implications. Our findings suggest that there were no predictable risk factors amongst our patient cohort and therefore day-case thyroid lobectomy should be avoided. Further research is required to identify low-risk patients who could benefit from day-case surgery. Thyroid lobectomy, also known as hemithyroidectomy, is an operative procedure to remove one lobe of the thyroid gland. This can be performed for several reasons, including thyroid cancer, thyrotoxicosis, thyroid goitre, and thyroid nodule. When considering thyroid lobectomy as day-case surgery, the principal consideration is risk to patient safety. The development of a post-operative haematoma is a rare but unpredictable complication of thyroid surgery and has an associated risk of life-threatening acute airway compression . Other cThe Association of Anaesthetists and the British Association of Day Case Surgery guidelines clearly define day-case surgery as "the patient is admitted and discharged on the same day" . In 1990This retrospective audit aimed to assess the incidence of post-operative haematoma development as a post-operative complication of thyroid lobectomy procedures. This was to assess the safety and feasibility of performing this operation as a day-case surgery given the potential for cost-savings at Bristol Royal Infirmary, the tertiary head and neck surgery centre within University Hospitals Bristol and Weston NHS Trust (UHBW).Elective unilateral thyroid lobectomy cases performed between January 2017 and December 2018 at UHBW were identified through coding and retrospectively analysed. Inclusion criteria were no previous thyroid surgery or major head and neck surgery, elective thyroid lobectomy procedure only, and surgery performed at UHBW by otolaryngology or maxillofacial teams. Exclusion criteria included emergency hemithyroidectomy, hemithyroidectomy as part of another procedure, and completion thyroidectomy.Pre-operative data analysed included age, sex, smoking status, reason for hemithyroidectomy, comorbidities, antiplatelet and anticoagulant use, American Society of Anaesthesiologists (ASA) grade, and body mass index (BMI). Operative data collected included operation start time, surgeon seniority, and drain insertion. Post-operative data analysed included post-operative complications, date and time of discharge, readmission within 30 days, and mortality. Pathological data collected included specimen weight and histology.Data were collected and analysed by the authors using the trust electronic note systems in April 2019. This audit was registered with the Local Trust Audit Department, and ethical approval was not required.A total of 140 cases were identified through coding in the period of January 2017 to December 2018, with 125 meeting inclusion criteria; 15 exclusions were made: three cases of incorrect coding, six cases of completion thyroidectomy, four cases of as part of another head and neck procedure, and two cases of emergency hemithyroidectomy.Baseline characteristics are presented in Table Post-operative complications are listed in Table Mean in-patient stay for this cohort was 1.4 days, and mean hours in hospital from theatre start time to discharge were 34 hours. Two  cases were discharged on the same day within 9 hours post-operatively, with no reasons provided for their early discharge. No post-operative complications were recorded in these two cases. One  case was readmitted within 30 days, and this was one of the cases that had developed post-operative haematoma and required return to theatre.This single-centre retrospective audit showed an overall complication rate of 15.2% (n=19/125). There were four (3.2%) recorded cases of post-operative haematoma that developed within the immediate post-operative period prior to discharge. All of these cases required return to theatre for evacuation of haematoma within 24 hours of initial surgery. These cases highlight the potential implications and risk associated with day-case surgery for thyroid lobectomy. One (0.8%) case developed a haematoma on day 6 post-operatively and was readmitted for surgery; this was the only case to develop a haematoma after 24 hours following initial surgery in our cohort.A recent meta-analysis identified statistically significant risk factors for the development of post-operative haematoma in thyroid surgery as male gender, older age, Graves’ disease, hypertension, antithrombotic use, and low-volume hospitals . SimilarThe BAETS position statement on day-case thyroid lobectomy does include risk factors that increase the risk of haemorrhage , such asA proposed risk factor from our cohort is ASA grade. All post-operative haematoma cases were ASA 2 or above which means they have a “moderate but definite systemic disturbance” identified from their pre-operative screening . These pStatistical analyses are beyond the scope of this audit, and it is therefore not possible to comment definitively whether cases of post-operative haematoma shared risk factors as identified in the aforementioned meta-analysis and review ,10. HoweLimitationsThis was a single-centre audit and therefore had a small sample size. This could be improved through collaboration with other centres.This audit concludes that given the unpredictable nature of post-operative haematoma, the potential risk to patients and medicolegal implications are too great to perform day-case thyroid lobectomy at our centre. It therefore remains best practice to follow current UK guidance and European consensus and continue to perform this procedure as an elective inpatient admission. Further research into the risk factors for developing significant post-operative complications would be beneficial and would help to identify low-risk patient populations who could safely undergo day-case thyroid lobectomy.