This review article aims to present the epidemiology and associated risk factors of allergic rhinitis (AR) in Asia. AR-related literature published on Asia was systematically reviewed and the associated risk factors were investigated.The prevalence of AR in Asia varied considerably depending on the geographical location, study design and population involved. Several risk factors were observed to have strong association with disease presentation across multiple studies. Among these, family income, family size, daily personal computer usage time, personal and parental education attainment, and stress level have shown some level of biological gradient influence when multiple risk levels were analyzed. This suggests that AR manifestation and presentation possibly might be strongly affected by various personal and family factors. These findings are beneficial as they may provide insights into modifiable factors that may influence AR presentation. In addition, these results indicate that strategies to reduce personal and family-related risk factors have to be developed in order to alleviate the odds of AR expression.The online version of this article (10.1186/s40413-018-0198-z) contains supplementary material, which is available to authorized users. According to the Phase III International Study of Asthma and Allergies in Childhood (ISAAC), the prevalence of AR varied between 0.8 to 14.9% in 6-7 years old and 1.4 to 39.7% in 13-14 years old worldwide  to 32% iIt is a prevalent yet underappreciated atopic disorder which is commonly characterized by the presence of at least one of the following clinical symptoms: persistent nasal obstruction and mucous discharge, sneezing, and itching .Although AR is commonly regarded as a mild and seasonal nuisance, it can trigger persistent mucosal inflammation which may synergize with other infective inflammation, resulting in severe outcomes including hospitalization . As suchApart from the demographic factors, smoking and drinking habits, pet adoption, education attainment, and family history were the risk factors of AR, commonly studied in Asian countries –11. ConvWhile AR is influenced by genetic predisposition, the symptom presentation also depends on environmental exposures . In addiMoreover, patients can experience adverse effects on social life, productivity at work and performance in school, especially for those who suffer from a more severe form of AR . The useDiagnosis of the disease is usually based on medical history of the patient in addition to skin prick test or blood test. However, misinterpretation can occasionally occur and this delays the golden treatment period which can result in other unexpected consequences, such as paying unnecessary medical expenditure and missing work .This review article aims to study the epidemiology of AR in Asia and identify significant modifiable risk factors associated with disease presentation. Several criteria have been employed to establish association between triggering factors and disease manifestation.The epidemiology and potential factors associated with AR manifestation were obtained from the Web of Science using the search terms of ‘rhinitis’, ‘risk’ and Asian countries. The list of Asian countries and independent territories used in the search is listed in Additional file Using these search terms, 56 articles were first identified. The articles were carefully reviewed and those with unclear study design or disease definition or which were conducted in a narrow pool of individuals were excluded. Apart from these 14 articles, additional 6 cross-referencing articles were also included. These 20 articles, published between 1994 and 2017, were evaluated closely for their study design, disease prevalence, disease definition, and the AR risk factor analyzed.The factors investigated in the 20 articles were further classified either as a potential risk factor or a co-morbidity. The association between potential modifiable risk factors and AR manifestation were evaluated using several important criteria established in literature. These criteria include the strength of association, consistency of the observed association, specificity, biologic gradient, biologic plausibility, coherence, analogy, and temporality. In addition, meta-analysis was conducted using the software/program-Stata/SE 11.2 with random effects model to evaluate the influence of modifiable risk factors with replicative results reported in at least three independent AR publications.Based on the methodology described, different articles published in Asia were reviewed. The reviewed articles have variable study design, disease definition and adopt different analysis parameters as shown in Table Though similar parameters were used to study the epidemiology of AR, a larger population group will help to further establish the prevalence of the disease as it better represents the targeted population. In addition, apart from the country of study, the disease prevalence differs depending on the disease definition and the study population. In the study conducted by Min et al. , AR prevApart from the general demographic factors, many modifiable risk factors for allergic diseases, such as smoking and drinking habits were investigated as summarised in Table We further classified these factors into a potential risk factors or co-morbidities category based on the following definitions. A typical risk factor is a demographical, physical, sociological or environmental component which potentially increases the risk of presenting a disease or is protective against the expression of an illness. On the other hand, if AR manifestation is linked to another disease occurrence, it will be known as a co-morbid of AR. As listed in Table Multiple papers have suggested the importance of age, gender, race, and nationality in affecting AR presentation Table . The assIn Li et al. , the oddMoreover, being married, a large number of members in the household, and parity were indicated to be beneficial for protecting one against AR. However, their influences towards protection of AR are likely to be interrelated as married individuals are usually with children and are therefore likely to report an increased parity number and household members.Apart from the demographical factors that are usually non-changeable to an individual, one’s behaviours, attitude, and encounters might have direct and indirect influences to the disease presentation. These factors are highly varied from one person to another and are often affected by their background and the social group they interact with.As stated in Table Coincidentally, people with more computer usage, higher education, higher stress level and lesser sleeping time were presented with higher AR susceptibility. Though several pathways were speculated for such association, the effects of confounders and bias could not be ruled out and further study is required to establish the direct association link between these factors.Stress might be one of the critical risk factors for AR presentation. Studies have shown the association between the level of stress in individuals with more frequent drinking and smoking habits, having higher daily computer usage, and higher education levels but with less sleeping time –27. BeinIn contrast, people with parasitic or past respiratory infections were reported to have higher odds of AR presentation. The results are contradictory with biological plausibility discussed in other literature. Phathammavong et al. , proposeIn Table Conversely, inconsistent results are observed for the association of breastfeeding with AR presentation across multiple studies , 31. ThiOn the other hand, genetic factor is long established to play an influential role in AR presentation  and a faAs suggested in multiple studies investigated, environmental factors are highly important in triggering AR. For instance, Table Similarly, outdoor exposures to heavy traffic, air pollution, and fume exposure were also reported to be positively correlated with AR manifestation. These factors are especially crucial for those whose occupations expose them to the allergens . ConstanVarious risk factors have shown strong association with AR presentation. Results are consistent for several risk factors across studies with different experimental setups and countries.In addition to the ORs, criteria such as biological gradient, biological plausibility and temporality are important in evaluating the association between risk factors and AR. The biological gradient of the factor can be established especially when it is studied in a continuous manner or in multiple exposure levels. This was demonstrated in various demographical factors such as in family income, family size, personal factors like computer usage, education attainment, stress levels and even in parental education attainment. Moreover, the association between the risk factors and AR manifestation are further strengthened when factors with similar roles in AR presentation, such as the common allergens like house dust mites, fungi, and molds, display comparable results.Furthermore, the listed factors can only be considered as a potential risk if its exposure is reasonably affected or altered the risk of AR development. Its biologic plausibility must also be coherent to the study results found. However, with reference to Table Last but not least, with reference to Fig. The studies chosen for this review are limited to articles published in Asia. Thus, the result might not be relevant and applicable to other nations outside Asia. In addition, the analysis might still be biased though several criteria have been used in establishing the significance of the potential AR risk factor in triggering or protecting against AR presentation. The analysed data could be affected by personal viewpoints in addition to errors occurred when translating data from primary literature to the review summaries, such as misrepresentation and misinterpretation of the original data. Thus, it is highly recommended for readers to refer to the original articles before extracting any information from this article. Furthermore, as most of the studies used in this review are observational studies, confounding effects cannot be ruled out and the association of a particular risk factor with the disease presentation might not be as straightforward as what is illustrated here.From the articles reviewed, family income, family size, computer usage, personal and parental education attainment and stress level are identified as risk factors with the greatest potential to influence AR presentation, and when compared to other factors, they fulfill most of the criteria listed. In contrast, more considerations are required in interpreting the effects of breastfeeding, parasitic infections and past respiratory infections to AR presentation. These factors show incoherent biological plausibility and more in-depth investigation and analysis is thus required.The results obtained from this review article can be used to improve the diagnosis of AR in clinical settings by identifying patients with risk factors strongly associated with AR manifestation. In addition, as personal and family-related modifiable factors are found to be strong AR triggering factors, strategies to alleviate personal stress levels and increase the awareness of allergy risk in a hygienic environment have to be developed.Additional file 1:List of countries and dependent territories used in the literature review search. (PDF 322 kb)


The value of player decisions has typically been measured by changes in possession expectations, rather than relative to the value of a player’s alternative options. This study presents a mathematical approach to the measurement of passing decisions of Australian Rules footballers that considers the risk and reward of passing options. A new method for quantifying a player’s spatial influence is demonstrated through a process called commitment modeling, in which the bounds and density of a player’s motion model are fit on empirical commitment to contests, producing a continuous representation of a team’s spatial ownership. This process involves combining the probability density functions of contests that a player committed to, and those they did not. Spatiotemporal player tracking data was collected for AFL matches played at a single stadium in the 2017 and 2018 seasons. It was discovered that the probability of a player committing to a contest decreases as a function of their velocity and of the ball’s time-to-point. Furthermore, the peak density of player commitment probabilities is at a greater distance in front of a player the faster they are moving, while their ability to participate in contests requiring re-orientation diminishes at higher velocities. Analysis of passing decisions revealed that, for passes resulting in a mark, opposition pressure is bimodal, with peaks at spatial dominance equivalent to no pressure and to a one-on-one contest. Density of passing distance peaks at 17.3 m, marginally longer than the minimum distance of a legal mark (15 m). Conversely, the model presented in this study identifies long-range options as have higher associated decision-making values, however a lack of passes in these ranges may be indicative of differing tactical behavior or a difficulty in identifying long-range options. By measuring a player’s decision relative to their options, we can quantitatively attribute value to a player’s decision-making abilities, further decoupling components of a player’s performance.Team sport athletes are consistently presented with situations in which their decisions effect the immediate state of a game. These consist of overt on-ball decisions relating to passing or shooting, however also include off-ball actions such as occupation of a given space. Whilst previous works have quantified the impact of a decision on some measure of possession expectation  or on meendEPV – startEPV) across all possessions. In The expected possession value (EPV) metric considers spatiotemporal data, match phase and player behaviors to quantify possession outcomes in basketball , 2016. CCommon amongst these studies is the valuation of player decisions with respect to some change in possession expectation. Another approach would be to value decisions relative to alternative options, however, modeling this problem presents unique challenges. While quantifying a decision after the fact can be done by measuring the change in a given objective, each option available to a player has an accompanying probability of success. Multiple studies have measured the risk of passes in football. In In this study we value a player’s passing decisions through consideration of the risk and reward of their options. We measure the risk of a pass through modeling of individual and team spatial control, and reward via a measure of field equity detailed in There exist many methods for representing a player’s spatial occupancy. One common approach, particularly in football, is that of Voronoi tessellations which bound a player’s owned space as the space in which they could occupy before any other player. Simple applications of this approach do not consider player orientation, velocity, or individual physical capabilities e.g., . Taki anUnderlying these models is an assumption that spatial ownership is binary. That is, each location on the field is owned completely by a single player, determined by the time it would take them to reach said location, henceforth referred to as their time-to-point. Through observations of contests, we propose that ownership of space is continuous. For a given location, if the time-to-point of the ball is greater than the time-to-point of at least two players, then no single player owns the space completely. This distinction is important if we wish to quantify spatial occupancy (and its creation) relative to the ball, given its time-to-point, as we need to account for changes in field formations that could occur between possessions.Recent papers have addressed this. The density of playing groups was explored with Gaussian mixture models in commitment modeling, where we fit the distribution of player commitment to contests in four dimensions . The result is a realistic representation of player behaviors when presented with the opportunity to participate in a contest.Given its contested and dynamic nature, a continuous representation of space control is preferable e.g., . FurtherSpatiotemporal player tracking data was collected from the 2017 and 2018 AFL seasons. Data were collected by local positioning system (LPS) wearable Catapult Clearsky devices , situated in a pouch positioned between the players’ shoulder blades. Positional data in the form of Cartesian coordinates was recorded at a frequency of 10 Hz for all 44 players. To ensure consistent tracking and field dimensions, analyzed matches were limited to those played at Docklands Stadium, Melbourne, VIC, Australia. Play-by-play transactional data  were manually collected by Champion Data . These events are henceforth referred to as transactions. Consolidation of transaction and tracking data was used to infer ball position from possession, as ball tracking data is not available in Australian Rules football. Datasets were joined via unix timestamps present in both datasets. Transactions were recorded to the nearest second, hence it was assumed they occurred at the beginning of a second when matched to 10 Hz tracking data. If the location of one or more players was lost during a passage of play, said passage was omitted from the analysis. In total, data from 60 matches was used in this study. A total of 2236 passes across 60 matches were analyzed in this study.A player’s velocity and displacement direction were calculated from raw positional data. Displacement direction was extracted from consecutive tracking samples . A player’s change in displacement direction was considered as the angle between two vectors, In this study, only player decisions following a mark were included, given that a mark provides the player with time to make an informed decision. In Australian Rules football, a mark is a kick greater than 15 m that is received by a player on the full . To locate the destination of a player’s kick following their mark, the next transaction must also be a mark. If the next possession following a kick is not a mark, we are unable to reliably locate the intended target, given a reliance on transactions to infer ball position.pt and ct respectively) and field formation  were recorded. A player was considered as having committed to a contest if their Euclidean distance from the location of the contest was less than 2 m at ct. Using a player’s position at pt and their commitment , a model was developed that quantified the probability a player would commit to a contest across a continuous space within their vicinity.For analysis purposes, a contest was defined as a transaction following a pass in which at least one player from each team was involved and the ball location (for both the preceding kick and the receive) could be inferred from the consolidated datasets. In this study, the contest transaction types were spoils and contested marks. The former is an attempted pass that was physically prevented by the opposition and the latter is a mark in which multiple players attempted to receive the ball. For each contest, interest related to two moments – the pass that preceded the contest and the contest itself. For each moment, the time  used to estimate their probability density functions (PDFs). KDE is a form of data smoothing in which the PDF of a dataset is estimated, the form of which depends on the chosen kernel function and bandwidth inputs . KDE hasx is as follows:Individually, these distributions represent the density of the data-sets in four dimensions. If a player’s positional information and the ball location is known, the probability they will commit to a contest at location w is a weighting factor equal to the size of the commitment dataset divided by the total number of samples, and fc=1 and fc=0 are the PDFs for the datasets where commitment = 1 and commitment = 0 respectively. A player’s commitment probability (Pr(x)) considers their position relative to x, their velocity, and the ball’s time-to-point. Ball time-to-point to a location is equal to the distance between the ball and the location, divided by ball velocity. Ball velocity was estimated as 18.5 m/s after manually timing kicks from two quarters of a single AFL match and taking the average, however we note that this is a rough estimation as distances were estimated from manually recorded transactions. This represents a novel method for combining the distributions of two datasets of unequal sample size, where the resulting metric quantifies the probability that a new point belongs to each distribution. The combination of these distributions in a 2D space is illustrated in where A) or after (B) it bounces, and unsuccessful passes in which an opponent does the same (C and D respectively). For each option, we calculate the probability (p) and value (e) of each event (Eq. 3). As we consider players to be moving objects who exhibit spatial influence over locations not at their present position, the player with the ball could theoretically kick to any location within a radius equal to their maximum kicking distance. The typical maximum range of elite footballers has been found to be between 55 and 63 m :From the EO of a pass, we calculate the value of a decision  as the EO of the pass that was executed, divided by the maximum EO contained in a player’s kicking range  is the sum of the influence of its players:i is the commitment probability array for player i, from Eq. 2. An attacking team’s influence is a measure of the commitment of its players. From the influence of each team, we calculate the attacking team’s spatial dominance (DOM) as:where Pra(x)INF and o(x)INF are the influence of the attacking team and their opponent at x.where x is the proportion of space they own. Logically, greater spatial dominance translates to a higher chance of a successful pass. Given that dominance is a relative measure, it is possible for a team to have high dominance over a location where influence is low. In such a case, while the probability of a successful pass is high due to their dominance, the probability that their players will reach the location is low, hence such a location is likely a poor passing location. To account for this, we calculate the probability of a successful mark (Ap and Cp from Eq. 3) as a team’s dominance multiplied by their influence.The attacking team’s dominance at Ap as the risk of a pass, where higher values indicate a safer passing option.Given that a team’s desired outcome is a successful pass resulting in a mark, this probability (Eq. 7) is of particular importance when analyzing a pass. We refer to Bp and Dp from Eq. 3).If a pass does not result in a mark, the probability that either team would win the ball is simply equal to their dominance . Descriptive statistics (mean ± SD) were produced for all metrics. Spearman’s correlation coefficient (ρ) was used to measure the correlation of decision-making metrics with location. KDE was used to fit the distribution of analyzed variables, finding that the decision-making metrics are not normally distributed. The Mann–Whitney U-test was used to assess differences between the characteristics of decisions and alternatives  in both the 2017 and 2018 regular AFL playing seasons, and one team who finished in the bottom 10 in the same seasons (Team B). Participation in the play-off finals in AFL is between the top 8 teams, hence the choice of cut-off criteria ensured one team who participated in the finals, and one non-finalist team. Furthermore, the distribution of team samples is heavily skewed, hence importance was placed on selecting teams with adequate sample sizes. This skew in team samples is due to this study’s focus on matches played at a single stadium, hence teams who played more matches at this stadium appear more frequently in the dataset. Differences between team-level statistics were measured using the Mann–Whitney U-test. Within-team decision-making is analyzed for both teams. We fit the distribution of mean decision-making characteristics for each player on the team. All analyses were carried out in the Python programing language, using SciPy (We explore team level trends in decision-making by comparing two teams. Teams were selected by taking the teams with the highest samples who fit the following criteria – one team who finished in the top 8 (ng SciPy  and the ng SciPy  packagesw) of 0.14. Resultant motion models for four different player velocities for ball time-to-point of 2 s are visualized in t = 2 s), and between ball time-to-point (whole second integers from 1 to 4 s) and peak commitment probabilities (ρ = -1 for velocity = 4 m/s). At higher velocities, the probability that a player will commit to a contest decreases as the relative angle increases. For a velocity of 8 m/s or greater, player’s exhibit minimal influence on space in the negative y- axis . As velocity increases, we also note that the shape of a player’s commitment inverts.Motion models were produced from 46220 instances of player commitment. Within the dataset there were 6392 instances of player commitment (Commitment = 1), and 39828 instances of no commitment (Commitment = 0), producing a weighting coefficient . An example decision-making output is visualized in A very weak correlation was noted between vertical displacement from center and DV of decisions (ρ = 0.06). Horizontal displacement from the attacking team’s goal is positively correlated with DV (ρ = 0.56).The distributions of decision-making characteristics are presented in The distributions of passing characteristics for two teams are presented in This study demonstrates a method for measuring characteristics of player pass decision-making in invasion team sports. Previous studies of player decisions have measured decisions relative to some current measure of possession expectation e.g., , rather The methodology presented in this study has practical applications in performance analysis. Understanding the components of player behavior that contribute to their overall performance can be used by sporting teams to target coaching or recruitment practices. For example, understanding if a player has poor execution but good decision-making, or vice versa, provides meaningful insights into said players individual performance. This concept could be explored further in future work by analyzing player decisions in response to match events. It is likely that a player’s decision-making abilities vary based on external stimulus such as opposition pressure. Understanding these components of player performance allows for more specific recruitment of player types. For example, should teams tend toward a style of decision-making , the quantification of player decisions would allow for more informed recruitment.A major component of the decision-making modeling were player motion models, fit on the weighted distributions of player commitment to contests. While previous studies have developed probabilistic motion models with arbitrary bounds  or from A key finding in this study are the novel insights into the decision-making and passing tendencies of Australian Rules footballers. Previous studies have identified the importance of kicking in the AFL  but therIn contrast to player decisions, the optimal alternative passes that were identified by the model presented in this study were long distance kicks, less frequently to unmarked individuals. While the distribution of dominance was similarly bimodal for alternatives, the peak at absolute dominance (DOM = 1.0) was less intense than for decisions. The higher density for passes to areas of dominance between 0.5 (a 50/50 contest) and 1.0 suggests kicks to areas in which multiple teammates have an opportunity to receive the ball. This is reinforced by the distribution of influence for alternatives  where moTeam level analysis revealed that the less successful team in the 2017/2018 season had higher average DV than the more successful team. Furthermore, while within-team distribution of player averages were similar, the player variance of DV was more positively skewed for the less successful team. Of particular interest is the finding that the less successful team executed passes of higher value, potentially suggestive of a difference in playing styles. Future research into player and team-level decision-making, should consider contextual information such as match conditions, score deficits, and tactical styles.The tactical behavior of teams has been explored via network behavior, in which the connectivity between players is quantified via the frequency of passing between them in soccer .BS, KJ, and SR: conceptualization. BS and KJ: methodology. TB: data extraction. BS and TB: data cleaning. BS: data analysis and visualization. BS and SR: writing. BS, SR, KJ, and TB: drafting.KJ and TB were employed by Champion Data. 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.


Invasive mammary carcinomas that spontaneously develop in female cats are associated with high mortality, and resemble the most aggressive human breast cancers, especially triple-negative breast cancer (TNBC). Transcriptome studies showed that TNBCs are a heterogeneous group that includes a potentially hormone-dependent subtype named luminal-AR. Some authors proposed an immunohistochemical definition of the luminal-AR subtype, which is not only positive for Androgen Receptor (AR), but also either positive for the transcription factor Forkhead box A1 (FOXA1), or negative for basal markers. The objectives of this study were to describe AR and FOXA1 expressions in feline mammary carcinomas (FMCs), their prognostic value, and if their coexpression could define a “luminal-AR” subtype of triple-negative mammary carcinomas in cats.In a previously described retrospective cohort of 180 female cats with FMCs, with a 2-year follow-up post-mastectomy, we assessed AR, FOXA1, ER, PR, Ki-67, HER2, and CK14 expressions by automated immunohistochemistry.n = 7) associated with a better cancer-specific survival by multivariate survival analysis  compared to AR+ FOXA1–CK14+ triple-negative FMCs (n = 46) (HR = 1.00), independently of the pathologic tumor size and pathologic nodal stage. The non-basal-like subtype of triple-negative FMCs that coexpresses AR and FOXA1 (the AR+ FOXA1+ CK14– subgroup) could represent the equivalent of the luminal-AR subgroup of human triple-negative breast cancer.Of the 180 FMCs, 57 (32%) were luminal; i.e., ER and/or PR positive, and 123 (68%) were triple-negative  FMCs. AR overexpression  and FOXA1 index ≥1%  were associated with a longer disease-free interval, overall survival, and cancer-specific survival in cats with FMC. Analysis of AR, FOXA1 and CK14 coexpression in triple-negative FMCs showed that AR+ triple-negative FMCs were heterogeneous: there existed an AR+ FOXA1+ CK14– subgroup . At least three factors justify that invasive mammary carcinomas that spontaneously arise in pet cats were proposed as relevant animal models for human breast cancer. The first is their high frequency: mammary tumors are reported to constitute 17% of neoplasms in female cats , 3, and FOXA1) gene [Transcriptome studies showed that triple-negative breast cancers (TNBCs) are a heterogeneous group. Among the major subtypes, the luminal-Androgen Receptor  subtype appears to be a stable subgroup, characterized by high expression of downstream Androgen Receptor (AR) targets and coactivators . Indeed,A1) gene . The traA1) gene . In the A1) gene , 26. In A1) gene .Since transcriptome studies are expensive, some authors proposed an immunohistochemical definition of the luminal-AR subtype of triple-negative breast cancer. Guiu et al. (2015) hypothesized that these two markers, AR and FOXA1, are needed to identify the luminal-AR TNBC subtype by immunohistochemistry . Other aAs most of feline invasive mammary carcinomas are triple-negative , 13, 19,The objectives of this study were thus 1) to investigate for the first time AR and FOXA1 expression in a large series of feline mammary carcinomas, 2) to assess their relationships with other clinical and pathological features of FMCs, 3) to investigate their prognostic significance in the feline patient, and 4) to put the first cornerstone for a better characterization of triple-negative FMCs, with identification of a luminal-AR subtype.The present cohort of 180 female cats has been previously described . BrieflyHematoxylin-Eosin-Saffron (HES)-stained sections of the FMCs were used to assess multicentricity, the histological type of feline mammary carcinoma, the pathologic tumor size (pT) in millimeters, presence/absence of lymphovascular invasion (LVI), central necrosis, squamous differentiation, and tumor-associated lymphohistiocytic inflammation, as previously described , 33. Thehttps://blast.ncbi.nlm.nih.gov/Blast.cgi), amino acid identity is 87% between human AR and feline AR proteins, and 89% between human FOXA1 and feline FOXA1 proteins. Specificity of the anti-AR clone SP107 in cats was validated on normal feline tissues: AR immunoreactivity was found in the testis , epididymis, uterus, ovary, hyperplastic mammary gland, cutaneous sebaceous glands, but absent in the gastrointestinal tract, liver, lung, heart, thyroid, parathyroid, hematopoietic and lymphoid tissues, skeletal muscle, adipose tissue, and nervous system, as expected in humans (https://www.proteinatlas.org). Similarly, the anti-FOXA1 clone SP88 cross-reacted with feline FOXA1, and was considered specific, as immunoreactivity was restricted to the hyperplastic mammary gland, cutaneous sweat glands, oviduct, bronchial epithelium, urinary bladder urothelium, and rare lymphocytes, as described in humans (https://www.proteinatlas.org).The immunohistochemical expression of Estrogen Receptor alpha (ERα), Progesterone Receptor (PR), Human Epidermal Growth Factor Receptor 2 (HER2), the proliferation marker Ki-67, Cytokeratin 14 (CK14), Androgen Receptor (AR), and Forkhead box A1 (FOXA1) were assessed using a Benchmark XT stainer  . Positive internal controls were the peritumoral mammary gland for ERα, PR, AR and FOXA1, cutaneous sebaceous glands for ERα and AR, the epidermis and hair follicles for Ki-67 and CK14, and sweat glands for FOXA1. For HER2 IHC, the pathway HER2 4-in-1 control slides (Roche Diagnostics) were used as external positive controls.A medical doctor specialist in breast cancer pathology (DL) and three certified veterinary pathologists , blinded to the clinical outcome or clinicopathologic data, evaluated the immunostochemical data. ERα and PR were considered positive at a 10% threshold, as previously reported for dogs –40 and cThe 180 invasive feline mammary carcinomas were classified as luminal  or triple-negative , including basal-like triple-negative carcinomas , as previously described , 18, 19.Nuclear AR expression was quantified as an index (percentage of positive neoplastic cells), and as an Allred score, whereas cytoplasmic AR immunoreactivity was not considered in scoring. The Allred score is the sum of a proportion score, reflecting the percentage of AR-positive cells by immunohistochemistry , and an intensity score , for a possible total score of 8 points. AR overexpression was defined by Allred scores of 7–8 points, and AR positivity was defined as AR index ≥25%. FOXA1 expression was quantified as the percentage of positive neoplastic cells  in at least 500 cancer cells (FOXA1 index in %). The prognostic cutoffs  were determined by receiver-operating-characteristic curve analyses calculated for 2-year cancer-specific survival.p-value of each covariate. The significance threshold was set at < 0.05.We have used the MedCalc® statistical software  for all of the statistical analyses. Statistical associations between the clinicopathologic characteristics were evaluated using Chi-2 tests for categorical variables, one-way analysis of variance between a continuous and a categorical variable, and linear regression analysis among continuous variables. Univariate survival analyses were performed using the Kaplan-Meier method and log-rank tests, while multivariate survival analyses relied on Cox proportional hazards models. The results are expressed as the Hazard Ratio (HR), its 95% confidence interval (95% CI), and the The main characteristics of the 180 female cats are reported in Table The pT  was measurable in 150 cases, and imprecise in the 30 remaining cases, due to positive tumor margins. A hundred and one patients (56%) had a positive pathologic nodal stage (pN+), and 8 (4%) had distant metastases (M1) at diagnosis. The 180 FMCs were diagnosed at stage I in 45 cats (25%), stage II in 23 cats (13%), stage III in 104 cats (58%) and stage IV in 8 cats (4%).Central necrosis was present in 160 cases (89%), squamous differentiation in 81 cases (45%), lymphovascular invasion (LVI) in 110 cases (61%), and moderate to severe tumor-associated inflammation in 103 cases (57%). According to the Elston and Ellis grading system, 10 cases (5%) were grade I, 82 (46%) were grade II and 88 (49%) were grade III FMCs.The mean ERα index was 10.0 ± 13.3% , with 88% of cases (158/180) showing at least one ER+ neoplastic cell, but only 49 FMCs (27%) were ER-positive. The mean PR index was 3.0 ± 11.0% , most FMCs  were totally devoid of PR expression, and only 13 (7%) were PR-positive. None of the carcinomas overexpressed HER2 . The mean Ki-67 index was 45 ± 14% . Fifty-seven FMCs (32%) were luminal , including 8 luminal-A (Ki-67 index < 20%) and 49 luminal-B (Ki-67 ≥ 20%) FMCs, and 123 (68%) were triple-negative .At 15% threshold for CK14 positivity, 132/180 (73%) FMCs were CK14+, including 39 (68%) of the 57 luminal FMCs and 93 (76%) of the 123 triple-negative FMCs.In the mammary gland surrounding FMCs, nuclear AR expression was usually intense but patchy, restricted to luminal cells in hyperplastic lobules and ducts; this level of expression corresponded to Allred scores ranging from 2  to 6 . In FMCs, positive immunohistochemical staining for AR was most commonly nuclear, and was observed in neoplastic cells as well as scarce stromal cells including endothelial cells and cancer-associated fibroblasts (data not shown). Cytoplasmic AR immunoreactivity was observed in 82 FMCs (46%), but was not taken into account for AR scoring. The mean percentage of AR-positive neoplastic cells (AR index) was 45 ± 25% . Most of the carcinomas  contained at least one AR-positive neoplastic cell, whereas only few FMCs  were completely devoid of AR expression. At 10% threshold for AR positivity, 92% of the cases (165/180) were positive for AR. When AR expression was quantified as an Allred Score, 33/180 (18%) FMCs overexpressed AR (Allred scores 7–8 points), including 12/57 (21%) luminal FMCs and 20/123 (16%) triple-negative FMCs.p < 0.001): only 33% (11/33) of FMCs with AR overexpression were pN+ compared to 61% (90/147) of AR-negative FMCs; (2) lymphovascular invasion : only 37% (12/33) of FMCs with AR overexpression were LVI+ compared to 67% (99/147) of AR-negative FMCs, and (3) clinical stage at diagnosis : only 41% (13/33) of FMCs with AR overexpression were diagnosed at stage III or IV compared to 67% (99/147) of AR-negative FMCs. In addition, the AR index was negatively associated with (4) the pathologic tumor size (R2 = 0.026 and p = 0.048), (5) the Elston and Ellis histological grade (p = 0.002): grade I and II FMCs had an AR index of 51 ± 24% compared to 39 ± 24% for grade III carcinomas, and (6) tumor-associated inflammation (p = 0.016): FMCs with moderate to severe tumor-associated inflammation had an AR index of 41 ± 25%, compared to 50 ± 25% in FMCs with absent to mild tumor-associated inflammation.AR overexpression (Allred score ≥ 7) was negatively correlated with (1) pathologic nodal stage : only 20% (4/20) of AR-overexpressing triple-negative FMCs were grade III compared to 50% (51/103) of AR-negative triple-negative FMCs, and (2) lymphovascular invasion : only 40% (8/20) of AR-overexpressing triple-negative FMCs were LVI+ compared to 67% (69/103) of AR-negative triple-negative FMCs.In triple-negative FMCs, AR overexpression was negatively correlated with (1) the Elston and Ellis histological grade . In the 180 FMCs, the mean FOXA1 index was 3.8 ± 9.9% . Seventy-eight (43%) of the carcinomas contained at least one FOXA1-positive neoplastic cell whereas the other 102 (57%) were completely devoid of FOXA1 expression. At a FOXA1 index ≥1%, 64/180 (36%) FMCs were FOXA1+, including 28/57 (49%) luminal FMCs and 36/123 (29%) triple-negative FMCs.p = 0.020): only 44% (28/64) of FOXA1-positive FMCs were pN+ compared to 63% (73/116) of FOXA1-negative FMCs, (2) Elston and Ellis histological grade : 89% (57/64) of FOXA1-positive FMCs were grade II or III compared to 98% (114/116) of FOXA1-negative FMCs, (3) clinical stage at diagnosis : 47% (30/64) of FOXA1-positive FMCs were stage III or IV compared to 71% (82/116) of FOXA1-negative FMCs, (4) lymphovascular invasion : only 44% (28/64) of FOXA1-positive FMCs were LVI+ compared to 70% (81/116) of FOXA1 negative FMCs, and (5) the Ki-67 proliferation index : 50% (32/64) of FOXA1-positive FMCs had a high proliferation index (≥ 42%) compared to 66% (77/116) of FOXA1-negative FMCs. FOXA1 index ≥1% was positively correlated to (6) PR positivity : 42% (27/64) of FOXA1-positive FMCs were PR+ compared to only 9% (10/116) of FOXA1-negative FMCs, (7) AR (p = 0.032): FOXA1-positive FMCs had an AR index of 50 ± 25% compared to 42 ± 25% in FOXA1-negative FMCs, and finally FOXA1 was positively correlated with (8) the luminal phenotype : 44% (28/64) of FOXA1 positive FMCs were luminal compared to 25% (29/116) of FOXA1 negative FMCs.FOXA1 index ≥1% was negatively correlated with the (1) pathologic nodal stage : only 47% (17/36) of FOXA1-positive triple-negative FMCs were LVI+, compared to 69% (60/87) of FOXA1-negative triple-negative FMCs. The FOXA1 index of was also negatively correlated to (2) clinical stage (p = 0.026): stage I–II triple-negative FMCs had a FOXA1 index of 2.7 ± 6.2% compared to only 0.9 ± 2.4% for stage III–IV triple-negative carcinomas, (3) pathologic nodal stage (p = 0.049): the 70 pN+ triple-negative FMCs had a FOXA1 index of only 0.9 ± 2.6%, compared to 2.5 ± 5.7% in the 53 pN0-pNX triple-negative carcinomas, (4) Elston and Ellis histological grade (p = 0.046): grade I–II triple-negative FMCs had a FOXA1 index of 2.3 ± 5.3%, compared to only 0.7 ± 2.0% for grade III triple-negative carcinomas, and finally FOXA1 expression was also positively correlated with (5) the AR index (R2 = 0.044 and p = 0.019).In triple-negative FMCs, FOXA1 index ≥1% was negatively correlated with (1) lymphovascular invasion , pathologic nodal stage , distant metastasis , lymphovascular invasion , and PR positivity . By multivariate survival analysis, AR overexpression was associated with longer disease-free interval, independently of the pathologic tumor size and distant metastasis (Table AR overexpression (Allred score ≥ 7) was associated with longer disease-free interval  by univariate survival analysis, and the other clinicopathologic parameters associated with overall survival were patient age at diagnosis , the pathologic tumor size , pathologic nodal stage , distant metastasis , lymphovascular invasion , histological grade , cutaneous ulceration , tumor-associated inflammation , and PR positivity . By multivariate survival analysis, AR overexpression was associated with longer overall survival, independently of the pathologic tumor size and distant metastasis (Table AR overexpression (Allred score ≥ 7) was also associated with longer overall survival , pathologic nodal stage , distant metastasis , lymphovascular invasion , tumor-associated inflammation , and PR positivity . The favorable prognostic value of AR overexpression in terms of cancer-specific survival was confirmed by multivariate analysis independently of the pathologic tumor size, pathologic nodal stage and distant metastasis  was associated with longer cancer-specific survival  and overall survival . In triple-negative FMCs also, AR overexpression was associated with longer disease-free interval  and cancer-specific survival .In luminal FMCs as well, AR overexpression was associated with favorable outcome in terms of cancer-specific survival , and longer cancer-specific survival  and cancer-specific survival . However, FOXA1 index ≥1% was not significantly associated with outcome in triple-negative carcinomas.In the 180 FMCs studied, FOXA1 index ≥1% was associated with longer disease-free interval  characterized by double positivity to AR and FOXA1 (AR+ FOXA1+ CK14–), defined by AR positivity (AR index ≥25%), FOXA1 positivity (index ≥1%) and CK14 negativity (CK14 <  15%), and a basal-like subgroup  with loss of FOXA1 expression (AR+ FOXA1– CK14+). Both subgroups did not significantly differ by tumor stage, nodal status, stage at diagnosis, or histological grade (data not shown). However, the non basal-like AR+ FOXA1+ CK14– subgroup was associated with longer disease-free interval  were AR-positive (AR index ≥25%). Among AR-positive triple-negative FMCs, we identified two subgroups, a non-basal-like subgroup  compared to the basal-like AR+ FOXA1– CK14+ subgroup (HR = 1.00) independently of the pathologic tumor size  and pathologic nodal stage   contained AR-positive neoplastic cells. At 10% threshold for AR positivity, 92% of the cases would be considered positive for AR. In women, the frequency of AR expression in invasive ductal mammary carcinomas varies considerably depending on the studies and the thresholds used –51. At tIn our cohort, 43% of the carcinomas contained FOXA1-positive neoplastic cells. At FOXA1 index ≥1, 36% of the FMCs were considered positive for FOXA1. The prognostic cutoff for FOXA1 used in breast cancer varies considerably –55, reacThe second objective of this study was to assess the relationships between AR, FOXA1, and the other clinical and pathological features of feline invasive mammary carcinomas. In the present study, there was a negative correlation between AR overexpression and pathologic tumor size, in agreement with results reported in human breast cancer , 51. ForFOXA1 positivity in FMCs was negatively associated with pathologic nodal stage, including in triple-negative FMCs, as reported in human breast cancer , 69, 70.p = 0.06) with the risk of disease progression being higher for FOXA1-negative TNBCs than for FOXA1+ TNBCs [The third objective of this study was to investigate the prognostic significance of AR and FOXA1 in FMCs, in order to evaluate their significance in the feline patient. AR overexpression was associated with longer disease-free interval, overall survival, and cancer-specific survival by univariate and multivariate survival analyses, independently of clinical stage at diagnosis . AR overexpression was associated with favorable outcomes in both luminal and triple-negative FMCs. In human breast cancer also, AR overexpression is associated with better outcomes in patients with luminal , 77 and 1+ TNBCs .The fourth and last objective of our study was to better characterize AR-positive triple-negative FMCs in order to define a “luminal-AR” subtype. Indeed, gene expression studies have shown that TNBCs in women are a heterogeneous group, comprising a stable luminal-AR subtype, which has been reported by Lehmann et al. , 21, BurIn conclusion, we have shown that AR and FOXA1 were mainly associated with favorable features such as lower stage and lower histological grade, but also correlated together, as reported in human breast cancer, despite the fact that AR was more commonly expressed in FMCs than in breast cancers, and conversely FOXA1 was less frequently expressed in cats than in women with invasive mammary carcinomas. Both AR and FOXA1 were strong and favorable prognostic factors, independently of tumor stage at diagnosis. Within AR-positive triple-negative FMCs, we identified a non-basal AR+ FOXA1+ CK14– subgroup with AR and FOXA1 coexpression, which appears similar to the luminal-AR TNBC subgroup described in humans, and is associated with a favorable outcome. Female cats with invasive mammary carcinomas may thus be interesting spontaneous animal models to investigate new cancer therapy strategies such as anti-androgen or anti-androgen receptor molecules, especially in the aggressive and potentially non-hormone-responsive triple-negative basal-like AR+ FOXA1–CK14+ subgroup.


Major lower-limb amputation (LLA) predisposes patients post-operatively to a significant decline in daily-life functioning. The physical condition of amputee patients prior to surgery is significantly deteriorated due to chronic peripheral vascular disease (PVD) and diabetes, which accounts for the majority of LLAs in the adult population. A common strategy called pre-rehabilitation has been used in multiple patient populations to prepare the patient for undergoing a surgical event and to improve post-operative patient outcomes. Pre-rehabilitation might enhance the outcome of dysvascular LLA patients and reduce the high post-operative mortality rates. However, experience of experts with pre-rehabilitation and feasibility of a pre-rehabilitation program in this group remains unknown.To investigate the experiences of medical professionals and researchers in the field of LLA with the use of pre-rehabilitation in general and in particular PVD patients. Additionally, the study examines their opinions regarding need for and feasibility of a pre-rehabilitation program for dysvascular patients at risk for an LLA.Two explorative focus group discussions were organized with in total 16 experts in the field of treatment and research of LLA. Transcribed data were coded using the Atlas.ti software package. Thematic analysis with inductive approach was opted to arrange and interpret codes.The experiences of the experts with pre-rehabilitation in dysvascular patients were scarce. The experts described dysvascular patients at risk for an LLA as a difficult group for pre-rehabilitation due to short time window prior to surgery, older age, multiple co-morbidities and lack of motivation for behavioral change. The experts concluded that a pre-rehabilitation program should focus on patients who have sufficient time in advance before the amputation for pre-rehabilitation and who are motivated to participate.Although in general the effects of pre-rehabilitation are promising, pre-operative rehabilitation in dysvascular patients at risk for an LLA seems not feasible. Future research could focus on a better monitoring of dysvascular patients and the development of pre-rehabilitation in subgroups of younger dysvascular LLA patients. Major lower-limb amputation (LLA) is a drastic life-changing event predisposing the patient to a significant decline in daily-life functional performance, participation and autonomy post-operatively. PeripherAll these demographics give an indication of the extent to which poor physical conditioning and low pre-operative activity levels, likely in this patient group, can impact one’s life. The general physical condition of these patients is already significantly deteriorated prior to surgery due to the illness  preceding and leading to the amputation, which results in an even lower level of physical fitness after the amputation.,10 The pThe relationship between pre- and post-operative physical and psychosocial well-being described above implies that taking strategic measures in terms of pre-operative care might have the potential to enhance the functional outcomes of dysvascular LLA patients and reduce the high post-operative mortality rates in this patient group. Therefore, it seems plausible to aim at optimizing physical and psychosocial functioning of the patients before stressful surgery, which is the case with an LLA.The process of enhancing and optimizing the functional capacity of the individual to enable him or her to withstand a stressful surgical event associated with inactivity has been termed pre-operative rehabilitation . It involThese findings retrieved from the current literature show that pre-rehabilitation might be beneficial for multiple patient groups which are listed for elective surgery. Making productive use of the pre-operative waiting periods by trying to optimize the physical and psychosocial functioning of patients can be an important determinant of post-operative outcomes. In their studies Taylor et al. (2005) and Pinzur et al. (1992) indicate that the pre-operative level of functioning of dysvascular LLA patients, including functional and ambulation status, and medical comorbidities, among others, can be used to predict post-operative outcomes, such as, for instance, prosthetic usage, maintenance of ambulation, and survival.,35 HowevThis focus group study attempts to provide answers to these questions by investigating the experiences of medical professionals and researchers in the field of LLA with the use of pre-rehabilitation in general and in PVD and/or diabetes patients at risk for LLA in particular. Their opinions and ideas regarding the need for and feasibility of development and implementation of a pre-rehabilitation program in the current rehabilitation care pathway for dysvascular patients who run the risk of undergoing a major LLA are also being explored. In general, a focus group with experts provides an open discussion on an unexplored topic. Regarding pre-rehabilitation in LLA patients a focus group can provide individual and shared ideas about content, feasibility, and barriers for the development and implementation of a pre-rehabilitation program.The Medical Ethics Committee of the University Medical Center Groningen declared that the study does not fall within the Dutch law on ‘Medical Research involving Human Subjects’ (the WMO) and that therefore, a formal approval was not required from the committee for the conduction of this focus group study involving medical professionals and researchers (METc 2016/040).A qualitative research consisting of two focus group discussions with medical professionals and researchers in the field of LLA, based on the thematic analysis approach ,37, is aThis study included experts . Each participant received a personal coding number to ensure their anonymity and preserve their privacy. The analysis of the transcriptions consisted of three phases. A detailed flowchart of the thematic analysis is presented in A total of 16 experts participated in this focus group study. Of these 16 participants, 5 were female (31%) and 11 were male (69%). Additional information on of the participating experts is presented in The data and information presented in the following sections are the opinions and statements of the experts. The quotes represent the literal translation of what the experts said during the focus groups.The experiences of experts with pre-rehabilitation were inventoried at the start of each focus group and divided into two categories–namely, experiences with the use of pre-rehabilitation in general and in PVD and/or diabetes patients at risk for LLA in particular. The experiences reported among the experts were diverse and involved a variety of patient groups. Two experts (a physiotherapist and rehabilitation psychologist) had some experience with pre-rehabilitation in dysvascular patients at risk of or undergoing a LLA. The physiotherapist reported the use of physiotherapy involving treadmill training for patients with intermittent claudication. The rehabilitation psychologist described her experience with meeting the patient pre-operatively, if sufficient amount of time is available, to evaluate his/her expectations for the surgery and the post-operative period, as well as to offer support and help with decision making. The specific details regarding the described experiences and the characteristics of the reported pre-rehabilitation interventions are shown in Three experts who were actively involved in research on pre-rehabilitation and had direct experience with the effect of the applied pre-rehabilitation interventions in other patient groups shared their experiences with the effect of the interventions they have observed. In relation to his research into the effect of pre-operative arm exercising for esophageal cancer patients on the prevention of post-operative pulmonary complications expert 6 stated:‘[…] It proved effective. And in addition to the fact that they progressed physically in terms of arm muscle strength and clearing of sputum from the airways, it was also very obvious to notice that it (pre-rehabilitation) gave them a lot of self-confidence. And the exact working mechanism–well, we haven’t discovered it fully […] So whether it is purely exercising or actually the contact and the self-confidence as well as the mutual contact between patients–this stayed a bit unclear.’ (Expert 6)Expert 3, who was part of a research group investigating pre-rehabilitation effect  in an orthopedic patient population , said:[…] it certainly had an effect at orthopedic level, for instance, in relation to muscle memory. In fact, you will train someone who has pain so you shouldn’t be under the illusion that you help someone improve in a pain zone. But you can improve muscle memory so that patients recover quicker.’ (Expert 3)‘The abovementioned positive effects of pre-rehabilitation were supported by a third expert who shared his experience and observations from a research project with patients undergoing thoracic surgery:‘They (the patients) are first seen by the cardiologist and before they enter the surgical care pathway they go to the rehabilitation center to be clinically admitted and to start training there. Before they start training an exertion test is performed. Based on the results of the test an exercise program is determined. Afterwards they get called for the surgery and enter the rehabilitation care pathway. And the statement ‘Better in, better out’–we endorse it. We see really positive results both at psychosocial and physical level.’ (Expert 16)No negative experiences with pre-rehabilitation have been shared by the experts. Further, there was significant scarcity of experience with pre-rehabilitation in the dysvascular patient group.All experts unanimously agreed that a better pre-operative physical and mental condition could significantly improve the post-operative recovery of our target group of patients. One expert stated:‘I think that if you can and the more you improve the nutritional state and general condition of everyone who undergoes a surgery, the better he recovers…better in, better out!And also improving the psychological condition of the patient. So in an ideal world…I absolutely agree it is needed.’ (Expert 1)‘I don’t know if it is really needed…need is, of course, a strong word but it would certainly be super desirable, yes!’ (Expert 2). Another expert supported this statement by saying that a need for a pre-rehabilitation program ‘suggests that you keep someone for 6 weeks on antibiotics at any costs so that he/she still can do physiotherapy’ (Expert 1). As stated by the majority of experts, the problem with deciding whether the program is needed or not for the dysvascular patient group was that such a program has to be feasible in the first place. In an ideal world, as implied by the experts, proper physical and mental preparation before a surgery would be feasible and would undoubtedly give effective results. Despite the fact that this patient group seems be in need of a better pre-operative conditioning and preparation, realistically speaking this might not be feasible. According to another expert ‘there are actually not so many possibilities to start a pre-rehabilitation training program. Training is, in my opinion, always good, in all aspects of muscle strength, endurance, etc. That’s good! The better the condition of the patient before the surgery, and that holds not only for the leg which will be amputated, but also for the rest of the extremities for which you take care to maintain muscle strength, the better the outcomes. You also give advice to the patient how to make transfers, etc. But what I find most difficult is the moment of the amputation, which often comes very quickly so that you actually don’t have any time to prepare for it (the amputation).’ (Expert 11) In addition to this, the patient is also ‘not fit enough or not motivated enough to start following a program. In a few cases, in case there are such, I think there is certainly a need for it (pre-rehabilitation)’ (Expert 10).However, doubts were expressed about the need for pre-rehabilitation in dysvascular LLA patients. As one expert said, The central topic of the focus group discussions was the feasibility of developing and implementing a pre-rehabilitation program for dysvascular LLA patients in the current rehabilitation care pathway. It was brought up in both focus group discussions from the very beginning since feasibility takes a central place in the decision-making process of whether to ultimately work towards developing and implementing a pre-rehabilitation program.Two major themes for feasibility of a pre-rehabilitation program emerged from the analysis of the focus group transcripts. The experts mentioned both factors that would facilitate the development and implementation of pre-rehabilitation program for dysvascular patients at risk for a LLA, as well as factors which would represent a significant barrier. The latter aspect turned out to be significantly more predominating during both focus group discussions, which is reflected in the text, mentioned below.Considering the timeframe, in the dysvascular patient group a major barrier to implementing a pre-rehabilitation program is the short time window between taking the decision to amputate and the amputation itself due to the acute nature of the event. This was also the most commonly reported barrier by the experts in both focus groups. A reason which was mentioned for the occurrence of this issue was the unpredictability of the moment of amputation and the fact that patients may delay for months their visit to the doctor until their condition becomes significantly aggravated. Not until critical limb ischemia develops and progresses to the point of severe pain, skin ulcers, sores, or gangrene do people seek medical care and become hospitalized. They try to delay the surgery as long as possible and do not come to the outpatient clinic from fear of losing their limb despite their inability to walk for months. There is rarely a time frame of 6–12 weeks in this patient group which also makes it very difficult, in some cases even impossible, to initiate a pre-rehabilitation program well in advance. One of the experts said that ‘the time frame is a day or two, because usually they (the patients) are admitted just before the surgery’ (Expert 5) According to another one, the ‘time window is often very short, because people try to delay the surgery. And if they come then it is usually one or two days before they get operated’ (Expert 1).Patient-related factors, in relation to the feasibility of a pre-rehabilitation program were also mentioned by the experts, focusing on lack of motivation, age and fragility. The lack of motivation for behavior and lifestyle change was described by the experts as the greatest problem in the dysvascular patient group. Expert 1, a vascular surgeon, stated:‘[…] this category patients are just difficult to motivate for lifestyle advice, exercise and other things…because they are old, and…maybe…they often come from a particular socio-economic class.’. (Expert 1)The overall opinion of the experts in both focus groups was that these patients have low motivation to improve their physical and mental condition before amputation. One of the rehabilitation physicians stated that ‘the ideal amputee patient who is very motivated is already exercising in the gym. He has already stopped smoking and eats healthy so he has taken the necessary initiatives. So pre-rehabilitation shouldn’t be focused on this group of patients, because they already initiate it themselves. It’s about the group which is less motivated’ (Expert 2). Another factor which was pointed out as a burden to the motivation of patients to initiate a pre-operative rehabilitation is pain. Expert 3, a physiotherapist, stated that ‘patients have pain before the amputation and are not motivated to exercise. Therefore, a mental support and physical exercise would not be sufficient to help the patient. Pain management is also important in such cases.’Related to Age and fragility, co-morbidity and complications were other important factors mentioned as a burden to initiating a pre-operative rehabilitation in dysvascular patients since the majority of them are elderly above the age of 65 years.The goal of this study was to investigate the experiences of medical professionals and researchers in the field of LLA with the use of pre-operative rehabilitation in general and dysvascular patients in particular. The study also explored the ideas and opinions of the experts regarding the need for and feasibility of development and implementation of a pre-rehabilitation program for dysvascular patients at risk of undergoing a major LLA. The shared experience by the participating experts in the two focus groups showed that pre-rehabilitation is an intervention which is currently used in a number of patient populations with positive results and has the potential to improve the post-operative patient outcomes. However, our focus group study showed that pre-rehabilitation might not be a feasible option for elderly dysvascular patients, due to the short time window before amputation, lack of motivation for behavior and lifestyle change in this patient group, and multiple comorbidities and medical complications typically present in these patients.The results of our study addressing the potential benefits of implementing a pre-rehabilitation program are, in that respect, comparable with the results presented in the study of Landry et al.  In this Although the number of publications on pre-rehabilitation is not extensive, there is mounting evidence suggesting that the provision of rehabilitation before surgery in general, not specifically in LLA’s, is beneficial. For instance, Marcinkowski et al.  reportedAs stated before, the incidence of LLA has not decreased in the past decade compared to the years 1992–1993. This refA possible suggestion for the initiation of a timely pre-operative intervention in elderly dysvascular patients could be to monitor the dysvascular patients with or without diabetes from the moment they enter the healthcare system with a vascular-related healthcare request. It is advisable to raise awareness on the realistic risks of amputation in these patients in good time, as early as they start experiencing signs of vascular problems which have the potential to lead to serious aggravation and eventually to a major LLA; to explore with great attention through conversations with the patient the thoughts and believes that drive his/her behaviour and that define the health-related choices he/she makes on a daily basis. By doing so, the medical professional will get a better understanding of where the patient's motivation for behavioral change lies, and thus will be in a much better position to influence the patient and the future development of the disease he/she is suffering from, also potentially preventing life-changing and impairing events such as an LLA.The fact that two focus groups were included in this study, in which representatives from various disciplines within the medical and research field took part, can be regarded as a strength. By having mixed focus groups all experts participated in the discussions on all topics, which made it possible for us to gather a great diversity of opinions and viewpoints. Although we did not set a point of saturation in advance, it is highly likely that data saturation was reached during the second focus group meeting because the same issues were identified and discussed within the group, eventually not resulting in new insights. A limitation of the study is the lack of member check implying that respondent validation of the transcripts of the focus group discussions was not requested from the participants due to time restrictions. However, at the end of both focus group discussions a summary of the participants’ answers and the discussion findings was made by the moderator and the participants were asked whether they agreed with the moderator’s summary of their answers.The present study provides valuable insights into experts’ experiences and opinions on pre-rehabilitation and the need for and feasibility of developing and implementing a pre-rehabilitation program for dysvascular LLA patients. Experts seem to see a beneficial role for a pre-rehabilitation program for dysvascular lower-limb amputees and have positive opinions about its importance and effectivity, however they express strong doubts about the feasibility of implementing such a program for the dysvascular LLA patient population in the current rehabilitation care pathway. The uncertainty and unpredictability of the amputation moment combined with the suboptimal physical and mental condition of the elderly dysvascular LLA patients represents a burden to the implementation of a pre-rehabilitation program in this patient population. The initiation of a timely pre-operative intervention in elderly dysvascular patients could be to at least monitor the dysvascular patients with or without diabetes from the moment they enter the healthcare system with a vascular-related healthcare request. Furthermore, focusing on another LLA patient group, such as for instance younger dysvascular patients, could offer an opportunity for complementary development and implementation of a pre-rehabilitation program.S1 Appendix(PDF)Click here for additional data file.S2 Appendix(PDF)Click here for additional data file.S1 File(DOC)Click here for additional data file.


AR splicing variants or nonsense mutations of AR. Post-translational modifications of ARs can also contribute to ligand-independent transactivation of the AR. Other mechanisms for AR axis activation are mediated by pathways that bypass the AR. Recent studies revealed that the glucocorticoid receptor can upregulate a similar transcription program to that of the AR, thus bypassing the AR. ARAT agents are essentially ineffective for CRPC driven by these AR-independent mechanisms. This review article describes recent efforts to overcome these refractory machineries for the development of next-generation AR axis blockade in CRPC.Second-generation androgen receptor axis-targeted (ARAT) agents, namely abiraterone and enzalutamide, enable stronger blockade of the androgen receptor (AR) axis and longer survival of men with castration-resistant prostate cancer (CRPC). However, the extent of the improved survival remains insufficient and the majority of patients eventually develop resistance to these novel agents. Some patients develop resistance against ARAT treatment through mechanisms termed “complete AR independence” or “AR indifference”, and no longer require activation of the AR axis. However, a considerable proportion of CRPC patients remain persistently dependent on AR or its downstream signaling pathways. Ligand-independent activation of the AR, an AR axis-dependent mechanism, is mediated by truncated forms of ARs that lack the ligand-binding domain (LBD), arising as products of  In the United States, prostate cancer (PC) is the most commonly diagnosed malignancy, and the second highest cause of cancer-related deaths among men. It is estimated that 164,690 new cases of PC will be diagnosed and 29,430 patients will die of PC in 2018 [Abiraterone acetate is an oral drug that selectively and irreversibly inhibits the CYP17A1 microsomal enzyme, leading to inhibition of testosterone biosynthesis in the testes, adrenal glands, and PC cells. Enzalutamide is an oral AR antagonist with eight-fold higher affinity for its target than bicalutamide , and alsDespite the significant survival benefits for treatment groups compared with placebo groups, some individuals fail to respond to these drugs from the beginning of treatment  response) because of intrinsic resistance. Although the remaining patients initially respond to treatment, they eventually develop acquired resistance, typically within a few months.Treatment of ARAT-refractory CRPC is a clinical challenge, because effective treatment in this setting is quite limited. Docetaxel, cabazitaxel, sipuleucel-T, and Ra-223 are current candidates for patients with ARAT-refractory CRPC, but their survival benefits are very limited or remain unproven in a restricted sense. Moreover, treatment with ARAT agents can significantly alter the biological characteristics of CRPC, and consequently require specific attention in the management of ARAT-refractory CRPC.This article reviews the current understanding of the molecular bases underlying intrinsic and acquired resistances to second-generation ARAT agents, focusing on their relationships with the AR signaling axis, and summarizes novel treatment strategies based on these mechanisms.Several reported mechanisms for resistance to second-generation ARAT agents can be classified into three groups by their dependency on AR and its downstream signals ,11,12,13In this category, despite castration levels of serum testosterone, some mechanisms still activate AR to induce expression of downstream target genes. Activation of the altered androgen biosynthesis pathway in a tumor, amplification and overexpression of the AR gene, genetic mutation of the AR, constitutively active AR splice variants, and phosphorylation of the AR by the Src and Akt pathways have been reported as possible mechanisms. These mechanisms can be further classified by the presence or absence of ligand dependency.Although abiraterone inhibits CYP17A1 and suppresses androgen synthesis, studies involving animal models have shown that CYP17A1 is overexpressed in tumors and that key genes involved in the androgen synthesis pathway, such as CYP11A1, AKR1C3, and HSD17B3, are upregulated during treatment ,15. SincA gain-of-function N367T mutation in 3-beta-hydroxysteroid dehydrogenase type 1 (3βHSD1) generated during treatment with abiraterone can inhibit degradation of 3βHSD1 and promote DHT synthesis from DHEA via 5α-androstenedione as an alternative pathway . LikewisIn 1995, Taplin and colleagues reported that AR expression was elevated in more than 80% of CRPC cases . A similIn CRPC, AR mutations are found in 5–30% of tumors, circulating tumor cells (CTCs), and ctDNA ,24,27. TAR F877L (previously reported in the literature as F876L), a phenylalanine-to-leucine substitution at amino acid 877, was shown to convert enzalutamide from a potent antagonist to a partial agonist in PC cell lines ,29,30. SAR T878A (previously reported as T877A) is a missense mutation that leads to AR activation by progesterone and first-generation antiandrogens such as flutamide. Since inhibition of CYP17A1 by abiraterone promotes elevated expression of intracellular progesterone in tumor tissues, while DHEA and testosterone are suppressed, it is suggested that the T878A mutation creates malignant clones to overcome abiraterone inhibition. Chen et al.  reportedL702H is a mutation that leads to glucocorticoid-mediated activation of ARs. Carreira et al.  revealedTo overcome resistance by inhibiting these mechanisms of ligand-dependent AR transactivation, several clinical trials on approaches such as combined administration of enzalutamide and abiraterone, dose escalation of abiraterone, novel AR antagonists, and progesterone receptor inhibitors are underway .In CRPC, the splicing mechanism for AR mRNA produces splice variants that lack the LBD, which plays an important role in cancer proliferation under androgen ablation ,35. AmonRegarding AR-V7 expression, one mechanism is that ADT-induced AR gene transcription and the recruitment of splicing factors like U2AF65 and alternative splicing factor/pre-mRNA-splicing factor SF2 (ASF/SF2) to AR pre-mRNA contribute to enhanced AR-V7 levels in PC cells . AnotherMany studies have focused on the functional significance of AR-V7. AR-V7 retains the nuclear localization signal and promotes the expression of target genes by translocation in a ligand-independent manner . TherefoAdditionally, in-depth studies have also been conducted on other AR variants, including AR-V1, AR-V3, AR-V9, and ARv567es ,48,49. SHan et al.  identifiIn the presence of low androgen levels, the AR interacts with Src kinase and the p85α regulatory subunit of phosphoinositide 3 kinase (PI3K) to activate mitogen-activated protein kinase (MAPK) and Akt pathways and enhance cell proliferation and survival in a non-genomic manner. Activated MAPK and Akt by non-genomic signaling also enhance genomic AR signals by phosphorylating the AR or transcriptional coactivators ,55. ThomTo establish novel treatments that can suppress non-genomic AR signaling, several clinical trials of combination therapy with second-generation ARAT agents and molecular targeted drugs such as tyrosine kinase inhibitors are currently being conducted.This category means that AR downstream genes are expressed by activation of not the AR, but other receptors or pathways. Mechanisms involving the glucocorticoid receptor (GR) and the progesterone receptor (PR) have been reported as described below.Resistance to enzalutamide or abiraterone can occur through increased expression of the GR because it shares response elements with the AR in multiple gene targets. Arora et al.  reportedThese findings suggest that combined inhibition of GRs and ARs could be a promising treatment strategy. Kach et al.  reportedPR expression is increased in CRPC, and two isoforms (A and B) are expressed in interstitial fibroblasts and smooth muscle cells in the prostate to regulate cell proliferation. Sustained production of progesterone by enzalutamide or abiraterone may be involved in acquired resistance .In this category, tumor cells proliferate under ADT without activation of the AR or expression of AR target genes.TMPRSS2-ERG genomic translocation in AR-negative small-cell carcinoma also existed in AR-positive adenocarcinoma [RB1 and TP53 can contribute to its pathogenesis [Neuroendocrine prostate cancer (NEPC) is a clinically aggressive subtype of PC with pathological features of neuroendocrine differentiation that are enriched in the advanced disease setting after ADT . Althougarcinoma ,66,67. Iarcinoma . Moreoveogenesis ,70. Thesogenesis  showed togenesis .In recent years, development of novel drugs has been underway for efficient targeting of the above-described ligand-dependent and ligand-independent AR activation processes. These drugs include agents that antagonize the AR more strongly, degrade the AR, target the N-terminal and DNA-binding domain of the AR, or inhibit AR dimer formation ,74,75,76Apalutamide (ARN-509) is a non-steroidal anti-androgen agent that binds directly to the LBD of the AR and prevents AR translocation, DNA binding, and AR-mediated transcription, similar to enzalutamide. Recently, the phase three SPARTAN trial showed that metastasis-free survival and time to symptomatic progression in men with non-metastatic CRPC were significantly prolonged by apalutamide compared with placebo . It was Darolutamide (ODM-201) is an oral, investigational, high-affinity AR antagonist with activity against known AR mutants like F877L, W741L, and T878A that confer resistance to other second-generation anti-androgens, has minimal brain barrier penetration, and does not significantly increase serum testosterone [TRC253 is a novel high-affinity competitive inhibitor of the AR. TRC253 is also a pan-inhibitor of multiple AR mutants, including F877L, and is under development for treatment of men with PC in a phase one/two clinical study (NCT02987829).Seviteronel (VT-464) is a non-steroidal anti-androgen that specifically acts as an androgen synthesis inhibitor through inhibition of the enzyme CYP17A1. It has approximately 10-fold selectivity for the inhibition of 17,20-lyase over 17α-hydroxylase, resulting in less interference with corticosteroid production relative to abiraterone acetate, and could thus be administrable without a concomitant exogenous glucocorticoid. In addition, seviteronel appears to exert greater efficacy as an anti-androgen than abiraterone in vitro . SeveralGaleterone (TOK-001) is a selective multi-targeted agent that inhibits CYP17, antagonizes the AR, and reduces AR expression in PC cells by increasing AR degradation . A phaseNiclosamide, an FDA-approved anti-helminthic drug, was identified as a potent AR-V7 inhibitor in PC cells in a screening of known compounds . NiclosaEPI-506 is a pro-drug for one of the four stereoisomers of its predecessor, EPI-001. EPI analogs covalently bind to the AR N-terminal domain to block the transcriptional activity of AR and its splice variants and reduce the growth of CRPC xenografts ,82. A phAs described above, AR amplification drives PC toward castration-resistant progression. However, it has been difficult to target AR amplification because the molecular mechanisms for AR amplification have been unknown. In this regard, Takeda et al.  recentlyAs the mechanism of the disease has continued to become more apparent, the number of new therapeutic agents have increased. However, this has brought about new clinical challenges. First, it is important for doctors to predict which drugs can be effective, but research on predictive factors including biomarkers has not yet progressed. With the increasing importance of companion diagnosis, liquid biopsy has attracted much attention in PC, because tissue sampling from metastatic lesions is often difficult. Annala et al.  reportedGiven the fact that no medicine can be expected to provide a cure for CRPC, novel therapeutic agents multiply questions for clinical use, such as whether to use the agents in combination or in sequence, how to set goals for treatment with individual medications, how to judge their effectiveness, and how to decide whether to change treatment. Further studies are needed to resolve these questions, and clinical trials based on multi-arm multi-stage platform protocols such as the STAMPEDE trial  may becoDespite the increasing number of treatments proven to prolong overall survival, none of them have shown promising results in terms of a cure. One of the main reasons for this appears to lie in the associated heterogeneity. There are multi-dimensional classes for tumor heterogeneity including four types of diversity . Tumor cThe pivotal mechanisms associated with treatment resistance of metastatic CRPC are clearly inter-tumoral and metachronous heterogeneities. It seems very difficult, at least to date, to treat such cases once the disease has acquired these high extents of heterogeneity and plasticity, although recent reports have shown promising results for reversing neuroendocrine differentiation of CRPC ,70. In tUnfortunately, most of these studies targeted only high-risk cases or failed to prove the superiority of multiple therapies in low-risk cases. It remains unknown whether a subset of low-risk cases may be cured by multiple treatments more frequently compared with ADT alone. A substantial proportion of patients with low-risk PC achieve durable responses with almost complete remission by ADT alone, which lowers the benefit of combined treatment. Instead of the current prognostic risk stratification, precise prediction of treatment responses based on better understanding of potentially heterogeneous mechanisms will be essential for this strategy. Ideally, treatment drugs should be selected in a personalized manner, incorporating multiple factors of the disease and the patient . Companion diagnostics using genetic tests, liquid biopsy, and other technologies will be increasingly available for clinical decision-making for multiple treatments in the first-line early intervention setting. Otherwise, the number needed to treat and time needed for follow-up will be much larger to examine the benefit of combined treatment in this population.Clearly, management of AEs accompanying multiple treatments is very important. Increased cost of the treatments is another inevitable issue. In summary, further studies are warranted to understand and evaluate tumor heterogeneity in CRPC. When we aim to cure patients with advanced PC, multiple personalized treatments based on the simultaneous use of potent weapons to the right patient at the right time in the right way with good control of associated AEs and using not only medicine but also surgery and radiotherapy are desirable.The widespread use of second-generation ARAT agents has multiplied novel biological and clinical questions, including increased tumor heterogeneity. More effort is needed to clarify unmet clinical needs and underlying biological mechanisms, and to find a solution that can be applied to patients by frequently going back and forth between the benchside and the bedside.


ENPP1 gene and AR in kidney transplant patients. We analyzed 449 subjects without AR and 98 with AR from a retrospective cohort of kidney transplant patients from Southern Brazil. K121Q polymorphism was genotyped using allelic discrimination-real-time PCR. Cox regression analysis was used to evaluate freedom of AR in kidney transplant patients according to genotypes. Q allele frequency was 17.6% in recipients without AR and 21.9% in those with AR (P = 0.209). Genotype frequencies of the K121Q polymorphism were in Hardy-Weinberg equilibrium in non-AR patients (P = 0.70). The Q/Q genotype (recessive model) was associated with AR  after adjusting for confounders factors. Our findings suggest a novel association between the ENPP1 121Q/Q genotype and AR in kidney transplant recipients.The identification of risk factors for acute rejection (AR) may lead to strategies to improve success of kidney transplantation. Ectonucleotidases are ectoenzymes that hydrolyze extracellular nucleotides into nucleosides, modulating the purinergic signaling. Some members of the Ectonucleotidase family have been linked to transplant rejection processes. However, the association of Ectonucleotide Pyrophosphatase / Phosphodiesterase 1 (ENPP1) with AR has not yet been evaluated. The aim of this study was to evaluate the association between the K121Q polymorphism of  Kidney transplantation has become the treatment of choice for a substantial proportion of patients with end-stage renal disease. Transplantation has been shown to provide better quality of life and longer life expectancy than dialysis treatment , 2. HoweAR is a potentially destructive allograft immune response that may occur at any time during the life span of a grafted organ. A number of factors are known to be associated with increased risk of AR, including recipient’s age, ethnicity, HLA sensitization and occurrence of delayed graft function (DGF). However, these factors alone may not fully account for all rejection episodes , 7. CurrEctonucleotidases are ectoenzymes that hydrolyze extracellular nucleotides into nucleosides. They consist of four families, namely, ectonucleoside triphosphate diphosphohydrolases (NTPDases), ectonucleotide pyrophosphatase/phosphodiesterases (NPPs), ecto-5'-nucleotidases and alkaline phosphatases, and they are involved in the modulation of purinergic signaling . DiffereENPP1 gene is a risk factor for diabetes mellitus (DM) [Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) belongs to the NPP family. This enzyme was first reported as a surface marker of B lymphocytes, being previously called plasma-cell differentiation antigen-1 (PC-1). ENPP1 is a cell membrane protein with an extracellular active site catalyzing the release of nucleoside 5-monophosphatase from nucleotides and their derivatives . This entus (DM) , 19 and tus (DM) , 21, butENPP1 gene might play a role in the AR process and, if so, could be used as predictor biomarkers of AR. Therefore, the aim of the present study was to evaluate the association between the ENPP1 K121Q polymorphism and AR in a cohort of kidney transplant recipients.Considering the potential involvement of different ectonucleotidases in transplant rejection, and a possible role of ENPP1 in immune modulation, we hypothesized that polymorphisms in the ENPP1 K121Q polymorphism differs between ethnic groups [This nested case-control study was undertaken within a cohort of kidney transplant recipients from a tertiary teaching hospital in Southern Brazil. It was designed in accordance with STROBE and STREGA guidelines for reporting of genetic association studies , 23. Sixc groups , 25, we Rejections that occurred within the first post-transplant year were diagnosed by an experienced transplant pathologist according to the Banff classification currently available –29. DataENPP1 K121Q polymorphism. DNA was extracted using a standardized salting-out procedure. Genotyping of the K121Q (A/C) polymorphism (rs1044498) in exon 4 of the ENPP1 gene was performed using primers and probes contained in the Human Custom TaqMan Genotyping Assay 20x . Primer and probe sequences used for genotyping were: 5-AGCCTCTGTGCCTGTTCAG-3’ (forward primer), 5’-ACACACAGAACTGTAGTTGATGCA-3’ (reverse primer), 5’-AGTCGCCCTTGTCCTT-3’ (VIC probe), and 5’-TCGCCCTGGTCCTT-3’ (FAM probe). All reactions were conducted in 96-well plates, in a total of 5 μl volume using 2 ng of genomic DNA, TaqMan Genotyping Master Mix 1x (Thermo Fisher Scientific Inc.) and Custom TaqMan Genotyping Assay 1x, and ran on the 7500 Fast Real-Time PCR System (Thermo Fisher Scientific Inc.).Peripheral blood samples were collected from all patients for DNA extraction and genotyping of the 2 test. Allele and genotype frequencies were compared between groups of patients using χ2 tests. Clinical and laboratory characteristics were compared between groups by using unpaired Student’s t-test or χ2, as appropriate. Variables are shown as mean ± SD or absolute numbers (percentages). The magnitude of the associations with AR were estimated using odds ratios (ORs) with 95% confidence intervals (95% CI). Hazard ratio (HR) and 95% CI obtained from a Cox regression model was used to evaluate freedom of AR episodes in patients according to the presence of the 121Q/Q genotype (recessive model). The selection criteria for variables included in the Cox analysis were a well-established association with AR or P values < 0.10 obtained in the univariate Cox regression. In addition, logistic regression analysis was performed to test the independent association of the ENPP1 K121Q polymorphism with AR, adjusting for the same covariates used in the Cox analysis. Results for which P values were less than 0.05 were considered statistically significant. Statistical analyses were performed using SPSS version 18.0 .Allelic frequencies were determined by gene counting, and departures from the Hardy–Weinberg equilibrium (HWE) were verified using χAmong the 449 renal transplant recipients included in this study, 98 presented biopsy proven acute cellular rejection and three hundred and fifty one patients did not have AR episodes (non-AR group). The median time for biopsy-proven AR was 12.5 days . The main underlying diseases for CKD were: hypertension (22.8%), diabetic nephropathy (11.4%), primary glomerulonephritis (13.9%), and secondary glomerulonephritis (2%). Twenty one percent of the patients had an unknown underlying disease. Patient’s demographic and clinical characteristics are shown in Maintenance immunosuppression was achieved with prednisone, tacrolimus and mycophenolate for 269 patients (50.9%), 97 patients (18.4%) used prednisone, mycophenolate and cyclosporine, and the remaining patients (30.7%) received other combined therapies.vs. Q/Q; OR = 3.064; 95% CI 1.267–7.408; P = 0.020) or recessive  inheritance models .Genotype frequencies were in HWE in controls (P = 0.70). The presence of Q/Q genotype was associated with AR assuming either additive  and ecto-5'-nucleotidases (CD73), as major nucleotide metabolizing enzymes, are known to regulate immunity and inflammation and, possibly, to protect against hypoxic and ischemic tissue injuries . Accordiet al , studyinENPP1 Q/Q genotype has an indirect negative effect on CD39 e CD73 activities, since mutated ENPP1 will generate less substrate for the other ectonucleotidases in the cascade. This might lead to increased activity of T effector lymphocytes and thus predispose to AR , is known to be an inhibitory mediator of T effector lymphocytes in various immune diseases –40. CD39se to AR . To confENPP1 K121Q polymorphism remained highly associated with AR after adjustment for HLA-DR mismatches, pregnancies, blood transfusions, recipient age, DGF, and induction therapy.It is established that HLA sensitizing events, such as pregnancies, blood transfusions, and prior transplants, might increase the risk of AR . LikewisENPP1 K121Q polymorphism is differentially distributed across ethnicities [nicities , 20. In nicities  and othenicities –52. BaseThe present study has limitations to be acknowledged, including its retrospective design, and being a single center study without a confirmation cohort. However, we believe that the study of the K121Q polymorphism and its relationship with AR is worth evaluation in other populations in order to confirm or deny the present findings.ENPP1 K121Q polymorphism and AR in kidney transplant patients. The precise mechanisms behind this finding are uncertain and need to be further elucidated. Screening of this polymorphism may be useful to predict those patients (carriers of Q/Q genotype) more prone to experience rejection and, therefore, may need a more intense vigilance and perhaps more intense immunosuppressive therapy.In conclusion, our findings support an association between the S1 FigFlowchart showing the strategy used to select patients for inclusion in the study.(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(XLS)Click here for additional data file.