Datasets:

suolyer

/

pile_pubmed-central

like 1

The content published in Cureus is the result of clinical experience and/or research by independent individuals or organizations. Cureus is not responsible for the scientific accuracy or reliability of data or conclusions published herein. All content published within Cureus is intended only for educational, research and reference purposes. Additionally, articles published within Cureus should not be deemed a suitable substitute for the advice of a qualified health care professional. Do not disregard or avoid professional medical advice due to content published within Cureus. Introduction ============ Total knee arthroplasty (TKA) is a promising treatment for end-stage osteoarthritis (OA) of the knee for alleviating pain and restoring the function of the knee. Some of the cases with bilateral TKA are symptomatic, necessitating revision arthroplasty in both the knees. A bilateral revision TKA can be done either in two stage or simultaneously as a single stage procedure. However, the decision to perform simultaneous bilateral revision TKA is debatable because of possible higher complexity and complication rate. Very few cases have been reported in the literature on this issue. There are various advantages of doing simultaneous bilateral revision TKA compared with staged bilateral revision TKA. These include single operation and single anesthesia as well as better rehabilitation of both knees, apart from a significant reduction in the hospital stay and hospital costs. Case presentation ================= A 67-year-old hypothyroid and hypertensive female presented to us with unstable and painful knees 14 years after primary bilateral TKA for advanced OA. She began developing pain in both the knees for last six months, followed by instability in both knees (right \> left). She was managed symptomatically with painkillers, bracing, and physiotherapy but her pain and instability were not relieved. On clinical examination, the active and passive knee range of motion was painful. The flexion was 0° to 100°, anterior--posterior laxity of 5--10 mm, and a mild valgus laxity. The plain radiographs showed malalignment and loosening of the implants (Figures [1](#FIG1){ref-type="fig"}-[2](#FIG2){ref-type="fig"}). The leucocyte counts, C-reactive protein, and erythrocyte sedimentation rate (ESR) were within normal limits. A three-phase bone scan was also found to be negative for infection. {#FIG1} {#FIG2} Bilateral revision TKAs were performed using modified Insall's midline approach with lateral retraction of the patella (Figure [3](#FIG3){ref-type="fig"}) \[[@REF1]\]. A joint wound swab was taken and sent for gram stain, culture, and sensitivity. It was found to be negative for any microorganisms. The original cemented TKA implants were removed carefully, preserving as much bone as possible. Revision TKA was done on both sides sequentially, under the same anesthesia, using Scorpio® Total Stabilizer (Stryker®, Mahwah, NJ) constrained implants with long femoral and tibial stems. {#FIG3} The knees were protected in hinged braces postoperatively. The drains were removed 48 hours postoperatively; continuous passive motion (CPM) and active knee flexion exercises were started on postoperative day one and gradually increased to 0°--90° of flexion (Figure [4](#FIG4){ref-type="fig"}). {#FIG4} The postoperative radiographs showed satisfactory implant positions (Figures [5](#FIG5){ref-type="fig"}-[6](#FIG6){ref-type="fig"}). The patient had no complaints and was able to flex the knee to 80° easily. The range of motion and quadriceps strengthening exercises continued without forced flexion. She gradually resumed full weight-bearing with the help of the walker. Three months after surgery, the brace was removed, and active pain-free range of motion of 0°--115° was achieved with complete stability. At four months, the patient had returned to full activity without the brace or cane. At the final follow-up of four years, the knee was fully stable, and the patient was pain-free with no loosening or wear of the implants. {#FIG5} {#FIG6} Discussion ========== Symptomatic instability and pain following primary TKA requires revision surgery. In one retrospective study of 49 TKA patients with bilateral simultaneous revision, no postoperative cardiovascular complications, stroke, or death were noted \[[@REF2]\]. The minor reported complications included transient, self-limited confusion (in three cases); pulmonary embolism (in one patient), which was treated successfully with an inferior vena cava filter and extended anticoagulation; posterior compartment syndrome (in one case), which was treated by fasciotomy; and stiff knee in one patient (that was manipulated under anesthesia at three months). In a retrospective cohort study, Carter, et al. \[[@REF3]\] found that 33 of 141 morbidly obese patients (23.4%) who had revision TKA had a complication compared to 10 of 96 patients with a BMI 18.5 - 25 (10.4%) (p = 0.011). The most common complication was wound healing. Kevin, et al. reviewed 60,355 revision TKA procedures done in the USA and noted that the most common causes of revision TKA were an infection in 25.2%, implant loosening in 16.1%, and implant failure/breakage in 9.7% cases \[[@REF4]\]. They found that revision of all the components was the most common type of procedure done (35.2%). Singh, et al. found a high prevalence (46.5%) of overall moderate to severe activity limitation at two years and 50.5% at five years following revision TKA \[[@REF5]\]. Significantly higher odds of moderate to severe overall activity limitation was noted both at two and five-year follow-ups in patients with a BMI of 40 or higher, age greater than 80 years, higher Deyo-Charlson score, and in females. Kasmire, et al. studied predictors of functional outcome after revision TKA by using various parameters, such as short-form 36 (SF-36), Western Ontario and McMaster Osteoarthritis Index (WOMAC), and Knee Society Scores (KSS) \[[@REF6]\]. The data was collected preoperatively and at two years follow-up in their 175 revision TKAs done for aseptic failure. All of the above-mentioned parameters improved significantly after revision TKA (p \< 0.001). Lower preoperative pain and higher clinical KSS were found to be predictors of a better outcome. Sheth, et al. found that the complication rates were different for bilateral TKA done simultaneously and as staged procedures \[[@REF7]\]. These authors reported aseptic revision (1.17% vs. 0.9%), septic revision (0.8% vs. 0.7%), mortality (0.28% vs. 0.1%), and adverse events (2.49% vs. 1.97%). According to Bohm, et al., simultaneous bilateral primary TKA patients required more blood transfusions, a shorter hospital stay, more transfers to a rehabilitation facility, and less frequency of knee infections than staged bilateral TKA patients \[[@REF8]\]. However, these patients had a higher rate of cardiac complications and in-hospital mortality rate. The three-year revision, however, was same in both the groups. In a meta-analysis of 14 studies, Hu, et al. showed that the prevalence of mortality immediately postoperatively, mortality at 30 days postoperatively, and neurological complications were significantly higher in simultaneous TKA compared to staged TKA patients \[[@REF9]\]. The prevalence of thromboembolic disease, infection, and cardiac complications were not significantly different between simultaneous TKA compared to staged TKA patients. According to Hersekli, et al., the amount of blood loss, intensive care unit days and perioperative complications were same between single- and two-staged operations (p \> 0.05) \[[@REF10]\]. However, hospital stay and overall cost were significantly less in single-staged operations. We faced the challenge in decision-making regarding the staging of the procedures in this reported case, where revision of the components was necessary for both knees. We could not find proper guidelines regarding bilateral revision TKA as there are only a few documented reports of simultaneous bilateral revision TKA. There is limited evidence to support the one-stage practice of doing bilateral revision TKAs, as its safety remains controversial. We chose to do a single-staged bilateral revision TKA in this case, as a two-staged procedure would have required two anesthesias, longer hospital stay, more hospital bills, and surgery-related complications, which were overcome by a single-staged procedure in this case. With the use of constrained implants and long stems of the prosthetic components, we achieved good knee stability and satisfactory range of motion immediately postoperatively and at the four year follow-up. Conclusions =========== Two-staged bilateral revision total knee replacement (TKA) has many disadvantages, such as requiring anesthesia to be given twice, a longer hospital stay, more hospital bills, and higher surgery-related complications, which can be overcome by a single stage procedure. In carefully selected patients, single-staged bilateral revision TKAs should be considered over two-staged procedures. The authors have declared that no competing interests exist. Consent was obtained by all participants in this study

J Med Radiat Sci 65 (2018) 275--281 Introduction {#jmrs290-sec-0005} ============ There is a growing interest globally in making sure that graduates emerge from higher education with the capabilities and competencies that will equip them not only to be 'work ready' on graduation but also prepared for the development of technology, new models of service delivery and advances for practice in the future.[1](#jmrs290-bib-0001){ref-type="ref"}, [2](#jmrs290-bib-0002){ref-type="ref"}, [3](#jmrs290-bib-0003){ref-type="ref"} In a profession, such as medical imaging, the health workforce needs graduates who are ready to understand and apply emerging technology alongside meeting the demands of ever changing healthcare systems.[4](#jmrs290-bib-0004){ref-type="ref"} This paper reports on the outcomes of a survey undertaken as part of preparation for the review and redesign of clinical placements in a medical imaging programme in New Zealand. The project embraced the goal of defining work ready plus graduates for the medical imaging workforce. Identification of the capabilities required of a medical imaging technologist (MIT) in their graduate years was critical for the development of the clinical experience programme, as it is clinical placement and emersion in work that is most likely to develop capability and work readiness skills in graduates. It was envisaged that by defining, for our regional context, the capabilities and work skills employers seek in our graduates, we would have the data we needed to review and if necessary rewrite the graduate profile and utilise fully and effectively the real‐life clinical experiences that support the development of these capabilities. The results are also impacting positively on lecturers teaching methods as they consider how they can develop these capabilities in students through teaching, learning, and assessment methodologies. The theoretical underpinning for this study was Scott\'s fellowship work for the Australian Teaching and Learning Council and the professional and graduate capability framework published for the Australian tertiary environment.[1](#jmrs290-bib-0001){ref-type="ref"} The Professional Capability Framework as used by Western Sydney University was used as the foundation for the development of a survey tool as it was current and had been validated in a range of disciplines that included health professions. In addition, it looks beyond graduation and standards for practice (as required by the New Zealand Medical Radiation Technologist Registration Board and the Medical Radiation Practice Board of Australia towards the generic skills graduates need to flourish in a profession in the future.[1](#jmrs290-bib-0001){ref-type="ref"}, [5](#jmrs290-bib-0005){ref-type="ref"} Hence the term work ready plus. Using a validated and comprehensive professional and graduate capability framework ensured that all potentially relevant capability options had been considered. It was deemed generalisable to the New Zealand health care environment due to the similarities between both the health and education systems. Figure [1](#jmrs290-fig-0001){ref-type="fig"} summarises the key elements of the professional capability framework. The overlapping aspects of professional capability are identified -- personal, interpersonal and cognitive which have been validated in a range of investigations, mainly focused on professional leadership.[1](#jmrs290-bib-0001){ref-type="ref"}, [5](#jmrs290-bib-0005){ref-type="ref"} These domains are underpinned by relevant role‐specific and generic competencies (the skills and knowledge found to be essential to the specific role of an MIT). The key terms "competence" and "capability" are problematic and therefore often confused. We adopted the definition that competence is the possession of the skills and knowledge necessary to perform the duties set down for a specific role. The New Zealand Medical Radiation Technologist Registration Board (MRTB) reviewed and updated their competencies for New Zealand registration in March 2017, so a list of competencies was current and available. We have adopted a definition of capability that goes beyond the skills to practice as a safe and competent practitioner, to embrace the concept of being work ready plus. Being "work ready *plus"* requires capabilities for not just today, for current practice but for the future. Capabilities include the ability to work with others from a range of professions and backgrounds, manage the unexpected, adopt new technology, to be changed implementation savvy, inventive, sustainability responsive, to learn from experience and to operate with a clear understanding of one\'s ethical position.[1](#jmrs290-bib-0001){ref-type="ref"} {ref-type="ref"} Permission was obtained to reproduce this figure.](JMRS-65-275-g001){#jmrs290-fig-0001} These capabilities require a mixture of emotional and cognitive intelligence, including the ability to determine when and when not to deploy these competences.[1](#jmrs290-bib-0001){ref-type="ref"} We believed this concept was less developed for the medical imaging profession in New Zealand. The Professional Capability Framework developed through a scholarship awarded by the Australian Teaching and Learning Council formed the basis for the development of a survey that asked practicing MITs and MIT clinical managers at the three largest placements sites in New Zealand to rate the capabilities deemed critical in a graduate to ensure they are "work ready *plus"*.[1](#jmrs290-bib-0001){ref-type="ref"} The items used in the survey fall into three domains which align with the capability domains identified in Figure [1](#jmrs290-fig-0001){ref-type="fig"}. These domains are discussed in more detail in Scott, Coates and Anderson[5](#jmrs290-bib-0005){ref-type="ref"} and Fullan and Scott.[6](#jmrs290-bib-0006){ref-type="ref"} This paper shares the results from the survey and discusses the impact these are having on curriculum review and development. Method {#jmrs290-sec-0006} ====== This study was carried within all the public (three District Health Board, which includes 2 hospitals on the Northshore, 3 Inner City and 1 in South Auckland), Radiology Services, in the Auckland Region, where Unitec Institute of Technology\'s MIT students are placed for clinical experience during their 3‐year training programme. Data collection period, April and August 2017. A prospective survey was selected as the method of data collection tool as it allowed us to collect anonymous responses from stakeholders with minimal disruption to the work environment. The survey was distributed electronically. The SurveyMonkey online tool was used to develop a rating scale questionnaire, using the statements and domains from the Australian Capability Framework.[1](#jmrs290-bib-0001){ref-type="ref"} The survey was trialled by three clinicians and during this process one question was removed that was perceived repetitive. The final questionnaire had 39 capability statements that were clustered into three domains: personal, interpersonal and cognitive. The first question of the questionnaire requested participants consent before proceeding with the survey. An open survey link was sent to MIT clinical managers for internal circulation. A participant information sheet was attached to the email invitation email. Participants were assured of the anonymity of their responses and this was achieved by using the anonymity function on SurveyMonkey. Ethics approval was granted by the Unitec Research Ethics Committee (UREC) -- No 2017--1002. Analysis design {#jmrs290-sec-0007} --------------- For the demographic variables of the survey, the data were represented either in the form of tables or graphs. Owing to the subjective nature of the data related to capabilities that participants were requested to provide in ordinal form (ranking), the average ranking measure was considered most appropriate to statistically determine which answer choice was most preferred overall. The answer choice with the largest average ranking is the most preferred choice. The calculations were conducted using Microsoft Excel. The questionnaire was organised with a total of 39 statements which were grouped into the three domain categories: personal capabilities included 15 statements, interpersonal capabilities had 11 statements and cognitive capabilities had 13 statements. Thus, the ranking for personal capabilities was from 1 to 15, interpersonal from 1 to 11 and cognitive from 1 to 13. The average ranking was calculated as follows:$${{Average}\mspace{720mu}{Ranking}} = \frac{x_{1}w_{1} + x_{2}w_{2} + \ldots + x_{n}w_{n}}{Total},$$where *w* represented the weight of ranked position and *x* represented the response count for the answer choice. Weights are applied in reverse order. The respondent\'s most preferred choice, which is ranked 1, has the largest weight and their least preferred choice has a weight of 1. In our case, the personal capabilities had 15 statements. The highest ranked statement had a weight of 15, second highest had 14, third highest had 13 and so on with the last ranked statement having a weight of 1. Similar weights, depending on the number of statements, were applied to the interpersonal and cognitive capabilities. Results {#jmrs290-sec-0008} ======= A total of 52 responses were received from a maximum sample size of 265. This indicates a response rate of 19.6%. However, it is not possible to exactly predict the size of the actual sample pool, as the surveys were distributed via the clinical managers to their staff. From the responses, 90% (47) of the respondents were female and the remaining 10% (5) were males. In terms of the position/title of the respondents, the majority of the respondents (76%) were senior qualified MITs and 15% team leader/clinical specialist. 74% (39) had over 6 years experience. Average ranking reported by domain {#jmrs290-sec-0009} ---------------------------------- Table [1](#jmrs290-tbl-0001){ref-type="table"} shows the average rankings for the domain personal capabilities with the top five clearly visible. ###### Average ranking scores for personal capabilities Statements -- personal capabilities Average ranking score ------------------------------------------------------------------------- ----------------------- Being willing to face and learn from errors 11.56 Wanting to do as good a job as possible 11.21 Understanding personal strengths and limitations 11.12 Remaining calm under pressure or when things take an unexpected turn 10.98 Having energy, passion and enthusiasm for the profession and role 10.94 Willingness to persevere when things are not working out as anticipated 8.35 Pitching in and undertaking menial tasks as required 7.44 Being true to one\'s personal values and ethics 7.27 Deferring judgment and not jumping in too quickly to resolve a problem 7.1 Maintaining a good work/life balance and keeping things in perspective 6.61 Being willing to take a hard decision 6.52 Bouncing back from adversity 6.37 Being confident to take calculated risks 5.71 Being willing to take responsibility for projects and how they turn out 5.62 Tolerating ambiguity and uncertainty 4.69 John Wiley & Sons, Ltd Table [2](#jmrs290-tbl-0002){ref-type="table"} shows the average rankings for the domain interpersonal capabilities. These rankings have a flatter profile with eight capabilities ranking higher than 5. ###### Average ranking scores for interpersonal capabilities Statements -- interpersonal capabilities Average ranking score ------------------------------------------------------------------------------------------------------------- ----------------------- Being transparent and honest in dealings with others 7.64 Empathising and working productively with people from a wide range of backgrounds 7.57 Listening to different points of view before coming to a decision 7.13 Understanding how the different groups that make up a work place operate and influence different situations 7.02 Giving and receiving constructive feedback to/from work colleagues and others 6.73 Being able to develop and contribute positively to team‐based programs 6.1 Being able to work with senior staff within and beyond the organisation without being intimidated 5.87 Motivating others to achieve positive outcomes 5.85 Being able to develop and use networks of colleagues to solve key workplace problems 4.77 Influencing people\'s behaviour and decisions in effective ways 3.96 Working constructively with people who are 'resistors' or are over‐enthusiastic 3.58 John Wiley & Sons, Ltd Table [3](#jmrs290-tbl-0003){ref-type="table"} shows the average rankings for the domain cognitive capabilities. All cognitive capabilities achieved an average ranking score of more than 5. ###### Average ranking scores for cognitive capabilities Statements -- cognitive capabilities Average ranking score ------------------------------------------------------------------------------------------------------------ ----------------------- Diagnosing the underlying causes of a problem and taking appropriate action to address it 9.2 Making sense of and learning from experience 9.19 Being able to identify the core issue from a mass of detail in any situation 8.04 Using previous experience to figure out what\'s going on when a current situation takes an unexpected turn 7.96 Having a clear, justified and achievable direction in area of responsibility 7.85 Thinking creatively and laterally 6.92 Seeing the best way to respond to a perplexing situation 6.72 Setting and justifying priorities for daily work 6.71 Adjusting a plan of action in response to problems that are identified during its implementation 6.67 Recognising patterns in a complex situation 5.94 Seeing and then acting on an opportunity for a new direction 5.88 Recognising how seemingly unconnected activities are linked 5.23 Tracing out and assessing the likely consequences of alternative courses of action 5.15 John Wiley & Sons, Ltd Discussion {#jmrs290-sec-0010} ========== Personal domain capabilities {#jmrs290-sec-0011} ---------------------------- The results from the personal domain show a clear top five capability rated highly by the respondents. Namely: remaining calm under pressure or when things take an unexpected turn; understanding personal strengths and limitations; being willing to face and learn from errors; wanting to do as good a job as possible; having energy, passion and enthusiasm for the profession and role. These five capabilities had strong face validity when presented to a meeting of national managers. They certainly provide a clear mandate as to which personal qualities should be incorporated into the graduate profile. The challenge in curriculum design will be to find ways to highlight, reinforce and role model these capabilities. The literature provides limited guidance, however Fraser & Greenhalgh[3](#jmrs290-bib-0003){ref-type="ref"} suggest that capability can be strengthened by the use of feedback, self‐reflection, and consolidation, with students following a nonlinear education model. Therefore, the incorporation of directed educator and supervisor feedback could assist in the recognition and development of this capability. To further consolidate these skills, it would be advantageous to encourage students to observe these capabilities in others and reflect as to how their own developing practice incorporates and builds this capability. Interpersonal domain capabilities {#jmrs290-sec-0012} --------------------------------- We have noted that in the interpersonal domain, the six top ranked qualities (all with a ranking above 6) have an alignment to the competencies identified for those working in interprofessional teams. Ponzer et al.[7](#jmrs290-bib-0007){ref-type="ref"} published the five core competencies that form the basis of many interprofessional education activities which have been modified for specific contexts and are frequently used to describe strong interprofessional teams.[8](#jmrs290-bib-0008){ref-type="ref"} Table [4](#jmrs290-tbl-0004){ref-type="table"} compares these. ###### Frequently used statements of attributes of effective interprofessional teams compared to the top six interprofessional capabilities in this study ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Interpersonal capabilities compared to interprofessional team attributes -------------------------------------------------------------------------------------------------------------- -------------------------------------------------------------- Understanding how the different groups that make up a work place operate and influence different situations\ Mutual understanding of roles and recognition of difference\ Empathising and working productively with people from a wide range of backgrounds\ Good patient‐ care/co‐operation\ Being able to develop and contribute positively to team‐based programs\ Mutual trust and respect\ Giving and receiving constructive feedback to/from work colleagues and others\ The importance of good communication for teamwork\ Listening to different points of view before coming to a decision\ Assertiveness needed for effective conflict management\ Being transparent and honest in dealings Be aware of ethical issues ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------- John Wiley & Sons, Ltd Following the World Health Organisation report in 2010[9](#jmrs290-bib-0009){ref-type="ref"} there is a growing international commitment to the promotion of collaborative practice in healthcare delivery supported by interprofessional education to ensure graduates have the capabilities required for collaborative roles on graduation. In 2012, the national boards and the Australian Health Ministers' Advisory Council, conducted an independent Review of the National Registration and Accreditation Scheme for health professionals.[10](#jmrs290-bib-0010){ref-type="ref"} As part of this review, the Health Practitioner Regulation Agency (AHPRA) reviewed the performance of each of these accreditation authorities against the domains of the Quality Framework for the Accreditation Function to inform the decisions on how to continue to implement the accreditation function under the National Law. Following this review process, all the current profession‐specific accreditation authorities were asked to consider opportunities to increase cross‐profession collaboration and innovation and support interprofessional learning.[10](#jmrs290-bib-0010){ref-type="ref"} There is a close working relationship between New Zealand and Australian registration and accrediting bodies and considerable influence in both directions. The growth of the Australian interprofessional agenda is likely to have a growing impact on New Zealand health professional registration and accreditation requirements. The capabilities for team work and collaborative practice recognised by practicing MITs in New Zealand appears to support the growing international agenda supporting collaborative practice models of care. There has been some substantial work in the interprofessional education and team working space around both learning and assessment methods that we can use to guide our curriculum planning.[11](#jmrs290-bib-0011){ref-type="ref"}, [12](#jmrs290-bib-0012){ref-type="ref"}, [13](#jmrs290-bib-0013){ref-type="ref"} Simulation with other professionals, interprofessional activities within the academic curriculum and opportunities to observe and engage with interprofessional teams while on placement (evidenced in a clinical portfolio) align to these capabilities. Cognitive domain capabilities {#jmrs290-sec-0013} ----------------------------- In the cognitive abilities domain diagnosing underlying causes of a problem, taking appropriate action and making sense of learning from experience are the most highly rated, followed by being able to identify the core issue from a mass of detail in any situation and using previous experience to figure out what\'s going on when a current situation takes an unexpected turn, are capabilities that aid problem solving. Overall the profile of preference in this domain is relatively flat. We note alignment to the concepts of clinical reasoning and critical thinking as it is described in the health professions. In the literature, the terms clinical reasoning, clinical judgment, problem‐solving, decision‐making and critical thinking are often used interchangeably. The term clinical reasoning is used to describe the process by which clinicians collect cues, process the information, come to an understanding of a patient problem or situation, plan and implement interventions, evaluate outcomes, and reflect on and learn from the process.[14](#jmrs290-bib-0014){ref-type="ref"}, [15](#jmrs290-bib-0015){ref-type="ref"}, [16](#jmrs290-bib-0016){ref-type="ref"} The clinical reasoning process is also described as dependent upon a critical thinking "disposition".[17](#jmrs290-bib-0017){ref-type="ref"} The American Philosophical Association defined critical thinking as purposeful, self‐regulatory judgment that uses cognitive tools such as interpretation, analysis, evaluation, inference, and explanation of the evidential, conceptual, methodological, criteriological or contextual considerations on which judgment is based.[18](#jmrs290-bib-0018){ref-type="ref"} We have noted that some students have a problem understanding how these capabilities are demonstrated in the work place. Responses in this domain are assisting us to define what capabilities are associated with critical thinking and clinical reasoning in the medical imaging profession and how they are evidenced in clinical practice. We are turning our attention to building processes to support the development of these capabilities within our class‐based learning, simulated learning, and clinical supervision. We will also incorporate post‐practicum experiences that will encourage students to appraise their experiences, seek clarification and comparisons and link their learning to the future, including securing employment.[19](#jmrs290-bib-0019){ref-type="ref"} The goal is to develop the student\'s ability to make judgements and decisions about their work experiences and learning that will position them as future critical thinkers, life longer enquirers and learners. Conclusion {#jmrs290-sec-0014} ========== Identification of the core capabilities that our stakeholder community rate highly has proved informative in assisting us to describe a "work ready *plus"* medical imaging graduate for the New Zealand context. The results have provided data to the curriculum development team allowing them to align the graduate profile to these expectations and raised awareness among academic staff of the need to include these capabilities in the curriculum. In addition, it has enabled a dialog with stakeholders about capability in the profession, refreshing and revising the involvement of the professional community in the academic programme. Scott reminds us that capability cannot be taught, people cannot be trained in it; but it can be learnt through exposure to educational experiences which entail coming to grips with real world dilemmas. Clinical placements provide this learning experience; it is here students learn what others do when the unexpected happens and develop the skills to make sense of what is unfolding to successfully resolve the situation. This naturally occurring curriculum of the workplace is often tacit and therefore not clearly visible to learners and students needs support.[19](#jmrs290-bib-0019){ref-type="ref"} These results provide a blue print for conceptualising the key opportunities a clinical placement offers beyond learning technical skills and competencies; highlighting the capabilities that can be learnt and developed on placement, bringing these learning opportunities to the attention of students and clinical supervisors alike and bringing a new clarity to the design of support for learning on placement. We now have descriptors of capability that will allow us to be more specific in our communication of the capabilities our graduates should aspire to (beyond but building on those established by the regulatory body) and we are incorporating these into the curriculum design process for both teaching and assessment purposes. They will inform clinical supervision and clinical learning, allowing clinical supervisors to focus on highlighting experiences that can develop these capabilities. This study is informing curriculum planning and energising discussions around the design of simulation, class room teaching activities and clinical placements designed to develop these capabilities. Conflict of Interest {#jmrs290-sec-0016} ==================== The authors declare no conflict of interest. We acknowledge the help and support of the Radiology Services and Research Office staff at Auckland and Waitemata District Health Boards, Auckland New Zealand. This project was kindly supported by the Unitec Strategic Fund, Unitec Research Office.

1. Introduction {#sec1-jcm-08-02039} =============== Venous thromboembolism (VTE) is a common cause of morbidity and mortality in hospitalized and non-hospitalized patients \[[@B1-jcm-08-02039]\]. The American Society of Hematology and American College of Chest Physicians guidelines recommend low-molecular-weight heparin (LMWH) as a first-line pharmacological option for most patients at risk of VTE \[[@B2-jcm-08-02039],[@B3-jcm-08-02039]\]. Several prophylactic doses and types of LMWH are used worldwide, which is reflected by differences in national summaries of product characteristics (SPCs) and dosing regimens of randomized controlled trials (RCTs). There is no high-quality evidence or guidance on the optimal prophylactic LMWH dose. Preceding systematic reviews on thrombosis prophylaxis have not specifically assessed benefits and harms associated with different LMWH doses \[[@B4-jcm-08-02039],[@B5-jcm-08-02039],[@B6-jcm-08-02039],[@B7-jcm-08-02039],[@B8-jcm-08-02039],[@B9-jcm-08-02039],[@B10-jcm-08-02039]\]. In addition, there have been very few direct comparisons of prophylactic LMWH dose regimens, and therefore indirect evidence could provide a 'second best' estimate of benefits and harms. There is no generally accepted definition of different prophylactic LMWH dose categories, which is why we previously categorized LMWH thrombosis prophylaxis regimens as either 'low-dose' or 'intermediate-dose', based on different registered doses in SPCs worldwide \[[@B11-jcm-08-02039]\]. Using this approach in a previous meta-analysis, we found that intermediate-dose LMWH, compared with placebo or no treatment, was associated with a significant decrease in symptomatic VTE, at the cost of an increase in major bleeding \[[@B11-jcm-08-02039]\]. The main objective of the current study was to perform a systematic review with meta-analysis and trial sequential analysis (TSA) comparing benefits and harms of low-dose LMWH versus placebo or no treatment for thrombosis prophylaxis in all types of patients at risk of VTE \[[@B12-jcm-08-02039]\]. 2. Materials and Methods {#sec2-jcm-08-02039} ======================== We conducted this systematic review according to a pre-published protocol on PROSPERO (<https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42019124722>) following the methodology suggested by Jakobsen et al, the Cochrane Handbook for Systematic Reviews of Interventions, the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) statement, and the GRADE (Grading of Recommendations Assessment, Development, and Evaluation) recommendations \[[@B12-jcm-08-02039],[@B13-jcm-08-02039],[@B14-jcm-08-02039],[@B15-jcm-08-02039]\]. 2.1. Study Selection {#sec2dot1-jcm-08-02039} -------------------- ### 2.1.1. Patients {#sec2dot1dot1-jcm-08-02039} Studies were considered for inclusion irrespective of language, blinding, publication status, or sample size. We included RCTs with adult patients allocated to receive thrombosis prophylaxis using either low-dose LMWH, placebo, or no treatment, regardless of their underlying disease or whether they were admitted to the hospital or visited the outpatient clinic. ### 2.1.2. Interventions {#sec2dot1dot2-jcm-08-02039} The experimental intervention was low-dose LMWH, irrespective of LMWH type or duration of treatment. We a priori defined 'low dose' in our protocol according to the SPCs as approved by the US Food and Drug Administration, the European Medicines Agency, and several national authorities ([Table 1](#jcm-08-02039-t001){ref-type="table"}). If different LMWHs or (weight-adjusted) doses were used in one trial, we classified the dose according to what was used most frequently. We included trials evaluating ultra-low-molecular-weight heparins and LMWHs not listed in [Table 1](#jcm-08-02039-t001){ref-type="table"} (e.g., LMWHs we were unable to classify into a specific dose) in a sensitivity analysis. The control intervention was placebo or no treatment. Co-interventions such as mechanical compression devices were allowed if they were applied in both treatment groups. ### 2.1.3. Outcomes {#sec2dot1dot3-jcm-08-02039} Predefined co-primary outcomes were all-cause mortality, symptomatic VTE, and major bleeding. Secondary outcomes were serious adverse events (SAE), clinically relevant non-major bleeding, and any VTE (including both symptomatic and asymptomatic events). All outcomes were assessed at maximum follow-up. VTE was defined as deep vein thrombosis or pulmonary embolism, and the diagnosis was accepted when objectified by an imaging technique or autopsy. We made no distinction between distal or proximal, or lower versus upper extremity thrombosis. Major bleeding and clinically relevant non-major bleeding were defined according to trial criteria. SAE were defined according to the International Conference on Harmonisation of Good Clinical Practice definitions (ICH-GCP) \[[@B16-jcm-08-02039]\]. 2.2. Data Sources and Searches {#sec2dot2-jcm-08-02039} ------------------------------ We searched the Cochrane Central Register of Controlled Trials (CENTRAL) in The Cochrane Library, PubMed/MEDLINE, EMBASE and Web of Science ([Table S1](#app1-jcm-08-02039){ref-type="app"}). References of identified studies were screened to identify further relevant trials. Finally, we searched the World Health Organization's International Clinical Trials Registry and ClinicalTrials.gov for ongoing trials ([Table S2](#app1-jcm-08-02039){ref-type="app"}). The search was last updated on 10 June 2019. 2.3. Data Extraction and Quality Assessment {#sec2dot3-jcm-08-02039} ------------------------------------------- Two authors (RJE, WB) independently identified trials for inclusion. Trials excluded on the basis of full text were listed with reasons for exclusion. We extracted information on characteristics (year of publication, country, numbers of sites and patients enrolled), participants (age, sex, eligibility criteria), interventions (type, dose, and duration of LMWH treatment), and outcomes. We resolved differences in opinion through discussion. Two authors (R.J.E., W.B.) independently assessed risks of bias of the included trials according to the revised Cochrane risk of bias tool version 2 \[[@B17-jcm-08-02039]\] in the following five domains: "Bias arising from the randomization process'', "Bias due to deviations from intended interventions'', "Bias due to missing outcome data'', "Bias in measurement of the outcome'', "Bias in selection of the reported result''. RCTs were classified as 'overall low risk of bias' when all bias domains were judged as 'low risk'. Conversely, trials were classified as 'overall high risk of bias' when 'some concerns' or 'high risk' was judged in one or more domains \[[@B18-jcm-08-02039]\]. Publication bias was assessed by inspecting funnel plots for signs of asymmetry when 10 or more trials were included in the analyses \[[@B12-jcm-08-02039],[@B14-jcm-08-02039]\]. 2.4. Data Synthesis and Analysis {#sec2dot4-jcm-08-02039} -------------------------------- We calculated relative risk (RR) with both conventional 95% confidence intervals (CIs) and TSA-adjusted CI if there were two or more trials for each outcome. ### 2.4.1. Assessment of Significance {#sec2dot4dot1-jcm-08-02039} We used adjusted thresholds for statistical significance to correct for multiplicity issues due to repeated testing. An alpha of 0.025 was used for the co-primary and secondary outcomes to keep the family-wise error rate at a maximum of 5% \[[@B14-jcm-08-02039]\]. In case of statistically significant RR, we calculated numbers needed to treat (NNT) or numbers needed to harm (NNH) with 97.5% CI. ### 2.4.2. Meta-Analysis {#sec2dot4dot2-jcm-08-02039} Data were pooled using both a fixed-effect and a random-effects model. In case of discrepancy between the models, we emphasized the most conservative estimate. Analyses were performed on an intention-to-treat basis whenever possible or otherwise using an 'available-case analysis'. ### 2.4.3. Trial Sequential Analysis {#sec2dot4dot3-jcm-08-02039} Conventional meta-analyses may result in type-I errors due to risks of random error when few data have been collected or due to repeated significance testing when a meta-analysis is updated with new trials \[[@B19-jcm-08-02039],[@B20-jcm-08-02039],[@B21-jcm-08-02039],[@B22-jcm-08-02039],[@B23-jcm-08-02039]\]. TSA is a sequential meta-analysis method that combines required information size estimation (i.e., the number of patients needed to detect an a priori specified relative risk reduction) with an adjusted threshold for statistical significance \[[@B21-jcm-08-02039],[@B22-jcm-08-02039]\]. This adjusted threshold is more conservative when data are sparse and becomes progressively more lenient as the accumulated sample size approaches the required information size. Accordingly, the TSA-adjusted CI is initially wider than the conventional 95% CI, but when the required information size has been reached, they become identical. The required information size is calculated on the basis of the unweighted event proportion in the control group, the assumption of a plausible relative risk reduction/increase (RRR/RRI), and the anticipated heterogeneity variance (D^2^) of the meta-analysis. We applied TSA to all outcomes, using the control event proportion from the actual meta-analyses; D^2^ as suggested by the meta-analysis; alpha of 2.5%; beta of 90%; and an anticipated RRR/RRI of 20%. ### 2.4.4. Assessment of Heterogeneity {#sec2dot4dot4-jcm-08-02039} Statistical heterogeneity I^2^ was explored by the chi-squared test with significance set at a *p*-value of 0.10. The quantity of heterogeneity was also measured by D^2^ \[[@B24-jcm-08-02039]\]. Clinical heterogeneity was explored by conducting explorative subgroup analyses. ### 2.4.5. Subgroup Analysis {#sec2dot4dot5-jcm-08-02039} We performed subgroup analyses according to overall risk of bias (low vs. high), type of patients, LMWH type, duration of the intervention (less vs. more than 30 days), and length of follow-up (less vs. more than 30 days). Statistically significant subgroup differences (test of interaction *p* \< 0.05) provided evidence of an intervention effect pending the subgroup. ### 2.4.6. Sensitivity Analysis {#sec2dot4dot6-jcm-08-02039} All analyses were re-conducted including trials that evaluated LMWH types not covered by [Table 1](#jcm-08-02039-t001){ref-type="table"}. In addition, sensitivity TSAs were conducted using an RRR as suggested by the overall low-risk-of-bias studies and using a D^2^ of 25% if the actual D^2^ was 0%. In case of rare events (\<2% in the control group), TSA was also performed using Peto's odds ratio. SAE are often inconsistently reported and, in addition to assessing SAE according to trial reporting, we estimated the number of patients with one or more SAE using two methods: (1) the highest proportion of either reported mortality, symptomatic VTE, or major bleeding in each trial and (2) all mortality, SAE, symptomatic VTE, and major bleeding events cumulated in each trial. The idea is that the 'true proportion' of SAE should lie between these two extremes. Finally, to assess the impact of attrition bias on the primary outcomes, we imputed missing outcome data in best-/worst-case and worst-/best-case scenarios \[[@B14-jcm-08-02039]\]. 2.5. GRADE {#sec2dot5-jcm-08-02039} ---------- We used GRADE to assess the quality of the body of evidence associated with each outcome \[[@B15-jcm-08-02039]\]. 3. Results {#sec3-jcm-08-02039} ========== Our search strategy identified 10,374 records. After removal of duplicates and selections based on titles and abstracts, 312 records remained. A total of 271 reports were excluded on the basis of full text, and 41 records reporting 44 RCTs with a total of 22,579 patients were included ([Figure 1](#jcm-08-02039-f001){ref-type="fig"}). 3.1. Characteristics of the Included Studies {#sec3dot1-jcm-08-02039} -------------------------------------------- Detailed characteristics of the 44 included trials are presented in [Table S3](#app1-jcm-08-02039){ref-type="app"}. The year of publication ranged from 1988 to 2018. Forty trials were in English, two in German, one in French, and one in Chinese. Three trials were published as abstracts only, and the Chinese trial was assessed as abstract only due to lacking translation capacity. There were 24 single-center and 20 multicenter trials. Nine different types of LMWH preparations were used, and several types of patients were evaluated: orthopedic or immobilized patients (16 trials), surgical patients (13 trials), ambulatory cancer patients (8 trials), acutely ill medical patients (4 trials), and neurological patients (3 trials). 3.2. Bias Risk Assessment {#sec3dot2-jcm-08-02039} ------------------------- Six trials including 8172 patients were considered at overall low risk of bias ([Table S4](#app1-jcm-08-02039){ref-type="app"}). Thirty-eight trials were classified as overall high risk of bias. We did not suspect publication bias except for the outcome any VTE, in which asymmetry in the funnel plot was observed ([Figures S1--S4](#app1-jcm-08-02039){ref-type="app"}). Sensitivity analyses of imputed missing data suggested potential for attrition bias in all primary outcomes, since the imputed effect estimates in the best/worse and worse/best scenario's suggested benefit and harm, respectively ([Table S5](#app1-jcm-08-02039){ref-type="app"}). Results of a post-hoc sensitivity analysis excluding trials published before 2005 were comparable to those of the main analyses ([Table S6](#app1-jcm-08-02039){ref-type="app"}). 3.3. Co-Primary Outcomes {#sec3dot3-jcm-08-02039} ------------------------ ### 3.3.1. All-Cause Mortality {#sec3dot3dot1-jcm-08-02039} Twenty-three trials with 15,487 patients reported data on all-cause mortality, including five trials with 4960 patients at overall low risk of bias. Mortality proportions were 8.0% in the LMWH group and 6.2% in the control group ([Figure 2](#jcm-08-02039-f002){ref-type="fig"}). Meta-analysis of low-risk-of-bias trials showed no statistically significant effect on all-cause mortality (RR 1.03; 95%CI 0.92 to 1.16; *p* = 0.60; *I^2^* = 0%; TSA-adjusted CI 0.88 to 1.20; [Table 2](#jcm-08-02039-t002){ref-type="table"}). When assessing all trials, the conventional meta-analysis results remained similar, while TSA suggested futility, rejecting a 20% RRR or RRI in mortality. All sensitivity analyses were consistent with the primary analysis ([Table 2](#jcm-08-02039-t002){ref-type="table"}, [Table S7](#app1-jcm-08-02039){ref-type="app"}). Subgroup analyses showed no statistically significant tests of interaction ([Table S8](#app1-jcm-08-02039){ref-type="app"}). The overall level of certainty of the evidence was low ([Table 2](#jcm-08-02039-t002){ref-type="table"}). ### 3.3.2. Symptomatic Venous Thromboembolism {#sec3dot3dot2-jcm-08-02039} Twenty-five trials with 15,920 patients reported data on symptomatic VTE, including five trials with 4878 patients at overall low risk of bias. Symptomatic VTE proportions were 1.1% in the LMWH group and 1.8% in the control group ([Figure 3](#jcm-08-02039-f003){ref-type="fig"} and [Figure 4](#jcm-08-02039-f004){ref-type="fig"}). Meta-analysis of low-risk-of-bias trials showed a statistically significant beneficial effect on symptomatic VTE, which was not confirmed by TSA (RR 0.65; 95%CI 0.45 to 0.94; *p* = 0.02; *I^2^* = 0%; TSA-adjusted CI 0.15 to 3.05; [Table 2](#jcm-08-02039-t002){ref-type="table"}). When including all trials, both conventional meta-analysis and TSA showed a beneficial intervention effect (RR 0.62; 95%CI 0.48 to 0.81; *p* = 0.0006; *I^2^* = 0%; TSA-adjusted CI 0.44 to 0.89; NNT 137; 97.5%CI 87 to 330; [Table 2](#jcm-08-02039-t002){ref-type="table"}; [Figure 3](#jcm-08-02039-f003){ref-type="fig"} and [Figure 4](#jcm-08-02039-f004){ref-type="fig"}). The primary analysis results were confirmed by three out of four sensitivity analyses ([Table 2](#jcm-08-02039-t002){ref-type="table"}, [Table S7](#app1-jcm-08-02039){ref-type="app"}). The direction of the intervention effect consistently suggested benefit in all subgroups, and there were no statistically significant tests of interaction ([Table S8](#app1-jcm-08-02039){ref-type="app"}). The overall level of certainty of the evidence was moderate ([Table 2](#jcm-08-02039-t002){ref-type="table"}). ### 3.3.3. Major Bleeding {#sec3dot3dot3-jcm-08-02039} Thirty-three trials with 13,091 patients reported data on major bleeding, including five trials with 4960 patients at overall low risk of bias. Major bleeding proportions were 0.9% in the LMWH group and 0.8% in the control group ([Figure 5](#jcm-08-02039-f005){ref-type="fig"}). Meta-analysis of low-risk-of-bias trials showed a non-statistically significant increase in major bleeding (RR 1.70; 95%CI 0.77 to 3.74; *p* = 0.19; *I^2^* = 0%; [Table 2](#jcm-08-02039-t002){ref-type="table"}). TSA could not be conducted, since less than 5% of the required information size was accrued. When including all trials, both conventional meta-analysis and TSA showed no statistically significant effect (RR 1.07; 95%CI RR 0.72 to 1.59; *p* = 0.74; *I ^2^*= 0%; TSA-adjusted CI 0.18 to 5.73; [Table 2](#jcm-08-02039-t002){ref-type="table"}). Sensitivity analyses were consistent with the primary analyses ([Table 2](#jcm-08-02039-t002){ref-type="table"}, [Table S7](#app1-jcm-08-02039){ref-type="app"}). Subgroup analyses showed that low-dose LMWH for more than 30 days was associated with higher risk of major bleeding as compared to shorter treatments (RR 2.20; 95% CI 1.00 to 4.82 vs RR 0.84; 95%CI 0.53 to 1.32, *p* = 0.04 for test of interaction; [Table S8](#app1-jcm-08-02039){ref-type="app"}). The overall level of certainty of the evidence was low to moderate ([Table 2](#jcm-08-02039-t002){ref-type="table"}). 3.4. Secondary Outcomes {#sec3dot4-jcm-08-02039} ----------------------- ### 3.4.1. Serious Adverse Events {#sec3dot4dot1-jcm-08-02039} Eight trials with 5180 patients reported data on SAE, although events were generally not defined according to ICH-GCP. SAE proportions were 5.4% in the LMWH group and 3.8% in the control group ([Supplement Figure S5](#app1-jcm-08-02039){ref-type="app"}). The one trial at overall low risk of bias, including 1150 patients, showed no statistically significant intervention effect on SAE (RR 0.89; 95%CI 0.68 to 1.17; *p* = 0.42; TSA-adjusted CI 0.41 to 1.96; [Table 2](#jcm-08-02039-t002){ref-type="table"}). This result was confirmed in both conventional meta-analysis and TSA of all trials regardless of bias risk (RR 0.98; 95% CI 0.78 to 1.25; *p* = 0.89; *I^2^* = 0%; TSA-adjusted CI 0.37 to 2.58; [Table 2](#jcm-08-02039-t002){ref-type="table"}). As predefined sensitivity analysis, we categorized mortality, symptomatic VTE, and major bleeding events from 37 trials as SAE and used these data to estimate the proportion of patients with one or more SAEs: the results were consistent with those of the primary analysis ([Table S9](#app1-jcm-08-02039){ref-type="app"}). Subgroup analyses showed no statistically significant tests of interaction. The overall level of certainty of the evidence was very low to low ([Table 2](#jcm-08-02039-t002){ref-type="table"}). ### 3.4.2. Clinically Relevant Non-Major Bleeding {#sec3dot4dot2-jcm-08-02039} Five trials with 3372 patients reported data on clinically relevant non-major bleeding. Clinically relevant non-major bleeding proportions were 1.0% in the LMWH group and 0.7% in the control group ([Figure S6](#app1-jcm-08-02039){ref-type="app"}). No trials were at overall low risk of bias. Meta-analysis of all trials showed no statistically significant intervention effect on clinically relevant non-major bleeding (RR 1.50; 95%CI 0.72 to 3.12; *p* = 0.28; *I^2^* = 0%; [Table 2](#jcm-08-02039-t002){ref-type="table"}), and TSA could not be conducted, since less than 5% of the required information size was accrued. Sensitivity analyses were consistent with the primary analysis ([Table 2](#jcm-08-02039-t002){ref-type="table"}, [Table S7](#app1-jcm-08-02039){ref-type="app"}). Subgroup analyses showed no statistically significant tests of interaction. The overall level of certainty of the evidence was very low ([Table 2](#jcm-08-02039-t002){ref-type="table"}). ### 3.4.3. Any Venous Thromboembolism {#sec3dot4dot3-jcm-08-02039} Thirty trials with 5849 patients reported data on any VTE, including three trials with 1254 patients at overall low risk of bias. Proportions of any VTE were 10.7% in the LMWH group and 17.6% in the control group ([Figure S7](#app1-jcm-08-02039){ref-type="app"}). Meta-analysis of the low risk of bias trials showed a statistically significant beneficial effect on any VTE, which was not confirmed by TSA (RR 0.57; 95%CI 0.38 to 0.84; *p* = 0.005; *I^2^* = 0%; TSA-adjusted CI 0.11 to 2.82; [Table 2](#jcm-08-02039-t002){ref-type="table"}). When including all trials, both conventional meta-analysis and TSA showed a beneficial intervention effect (RR 0.61; 95%CI 0.50 to 0.75; *p* \< 0.00001; *I^2^* = 47%; TSA-adjusted CI 0.49 to 0.82; NNT 15; 97.5%CI 11 to 21; [Table 2](#jcm-08-02039-t002){ref-type="table"}). The primary analysis results were confirmed by all sensitivity analyses ([Table 2](#jcm-08-02039-t002){ref-type="table"}, [Table S7](#app1-jcm-08-02039){ref-type="app"}). Subgroup analyses showed no statistically significant tests of interaction. The overall level of certainty of the evidence was low to moderate ([Table 2](#jcm-08-02039-t002){ref-type="table"}). 4. Discussion {#sec4-jcm-08-02039} ============= In this systematic review on low-dose LMWH versus placebo or no treatment, LMWH was not associated with a statistically significant intervention effect on mortality, major bleeding, clinically relevant non-major bleeding, or SAE. Conversely, we found a large beneficial intervention effect on both symptomatic VTE and on any VTE which included asymptomatic events detected through screening. These effects were consistent among subgroup and sensitivity analyses, but the effect size varied per patient type, and the quality of the evidence was moderate. In the TSAs of mortality, symptomatic VTE, and any VTE, the adjusted monitoring boundaries were crossed (respectively, for futility and for benefit), indicating a low risk of random error. The intervention effects of low-dose LMWH on SAE and bleeding events remain inconclusive, as TSA monitoring boundaries were not crossed, and quality of evidence was low. There was a suggestion of publication bias in the reporting of any VTE, and attrition bias may have influenced the primary outcomes. 4.1. Considerations on the Optimal Prophylactic Dose {#sec4dot1-jcm-08-02039} ---------------------------------------------------- Previous systematic reviews did not observe a mortality benefit for patients receiving LMWH thrombosis prophylaxis compared to patients receiving placebo or no treatment, which is confirmed by our results including TSA. Although it was previously thought that LMWHs might improve survival in cancer patients, later systematic reviews found no survival benefit in cancer patients receiving different prophylactic doses of LMWH \[[@B6-jcm-08-02039],[@B9-jcm-08-02039]\]. Additionally, we detected no beneficial effect on mortality in any patient category in a previous meta-analysis on intermediate-dose LMWH \[[@B11-jcm-08-02039]\]. Nevertheless, we cannot exclude the possibility of a smaller intervention effect than 20% RRR/RRI on mortality; this would require many more randomized patients, as we used a 20% RRR for calculating the required information size in TSA. In line with previous literature, we found a consistent beneficial intervention effect on VTE in subgroup analyses according to patient type, although effect sizes varied among subgroups. The overall incidence of symptomatic VTE was low, resulting in an NNT of 137. Effect estimates were rather similar regardless of bias risk (low risk RCTs estimated an RRR of 35%, while all RCTs combined estimated an RRR of 41%), suggesting we may base our conclusions on the more accurate estimates derived from the meta-analyses of all trials. Previous systematic reviews on thrombosis prophylaxis have found larger relative risk reductions \[[@B6-jcm-08-02039],[@B7-jcm-08-02039],[@B10-jcm-08-02039],[@B25-jcm-08-02039]\]. This could indicate that low-dose LMWH may be slightly less effective for the prevention of VTE than more frequently used higher doses. However, this indirect comparison should be viewed with caution, as differences between reviews regarding study selection criteria could also explain the difference. A direct comparison in a homogeneous patient population is required for strong inferences about the efficacy of low-dose LMWH compared to higher doses. Finally, evidence on adverse events remains inconclusive. The point-estimate of the low-risk-of-bias trials suggested a 70% RRI in major bleeding which was not statistically significant, while the estimate including all trials was neutral. This difference may relate to bias risk but could also be explained by trial characteristics: cancer and treatment duration are risk factors for major bleeding, and three out of five low-risk-of-bias trials included oncological patients who were generally treated for a longer duration \[[@B26-jcm-08-02039]\]. The increased risk of major bleeding in the subgroup of oncological patients was comparable to that reported in previous systematic reviews for this patient category \[[@B6-jcm-08-02039],[@B7-jcm-08-02039]\]. Conversely, the risk of major bleeding for other patient types was low compared to that indicated in other systematic reviews \[[@B10-jcm-08-02039],[@B11-jcm-08-02039],[@B25-jcm-08-02039]\]. This may be explained by the low LMWH dose but also by differences in included patients or co-interventions. Data on clinically relevant non-major bleeding were reported by only a few trials, and analyses were inconclusive. There was no apparent effect on SAE, confirmed by sensitivity analyses in which we incorporated data from nearly all available trials. Assessment of these two outcomes was hampered by wide variations in definitions and reporting between trials, resulting in low- to very low quality evidence and limiting inferences on the harms of low-dose LMWH. 4.2. Implications for Clinical Practice {#sec4dot2-jcm-08-02039} --------------------------------------- In general, clinicians will not prescribe thrombosis prophylaxis without considering both effectiveness and harms. This balance may differ depending on patients' characteristics such as disease type, severity of illness, or surgery. In prespecified subgroup analyses according to patient type, we found that, in surgical patients, low-dose LMWH reduced both symptomatic and any VTE, without evidence for increased major bleeding. In orthopedic patients, there was a statistically significant reduction in any, but not in symptomatic, VTE, with no evidence for increased major bleeding events. Although not statistically significant, there was a 39% RRR in symptomatic VTE, and the discrepancy may be explained by low power. In oncological patients, a beneficial effect on symptomatic VTE, but not on any VTE, was found. Additionally, the direction of the intervention effect suggested an increase in major bleeding. There were no statistically significant beneficial or harmful effects in acutely ill medical patients, suggesting either that there was a very small intervention effect with concurrent high numbers needed to treat or that a low LMWH dose is insufficient for this type of patient. Recent guidelines have recommended an individualized approach towards thrombosis prophylaxis in acutely ill medical patients \[[@B2-jcm-08-02039]\]. On the basis of our results, one could hypothesize that medical patients deemed at high risk of VTE will mainly benefit from higher doses of thrombosis prophylaxis. Finally, only very few neurological patients were included, limiting inferences for this subgroup. This systematic review provides a general overview of the effects of low-dose LMWH: although there are differences between patient subgroups, there also are many similarities in the direction of effects. Overall, we found that low-dose LMWH was most effective in surgical, orthopedic, and oncological patients, while the estimated RRI for bleeding events was low in most prespecified patient subgroups, except for oncological patients. These results should be viewed in the perspective of the limited quality of the evidence and the inherent limited power of subgroup analyses. In cases where physicians are in doubt whether a patient should receive thrombosis prophylaxis or no prophylaxis at all or when a higher prophylactic dose is deemed inappropriate with respect to bleeding risk, clinicians may consider a low-dose LMWH for thrombosis prophylaxis, especially in surgical and orthopedic patients. 4.3. Strengths and Limitations {#sec4dot3-jcm-08-02039} ------------------------------ Strengths of this review include its systematic and transparent methodology according to recommendations by the Cochrane Handbook, the PRISMA statement, and the GRADE working group. We used a prespecified protocol, a comprehensive search strategy without language restrictions, although we did assess one Chinese article as abstract only, independent data extraction and bias assessment by two authors, and incorporation of bias risk assessment in the results and conclusions. Finally, we applied TSA to all outcomes to assess the risks of random error and to estimate the required information size. Nevertheless, several important limitations apply. Our main goal was to make general inferences on the efficacy and safety of low-dose LMWH, using all available evidence. Consequently, there was a high amount of clinical heterogeneity between trials. The balance between thrombosis and bleeding may vary depending on patient subgroup characteristics: relying on overall effect estimates could obscure more subtle associations or lead to wrong inferences about a subpopulation. However, the distinction between different patient populations is somewhat arbitrary in any systematic review, and we attempted to account for clinical heterogeneity by conducting several preplanned subgroup analyses. This approach offers the benefit of increased power of the meta-analysis, and we found the direction of the intervention effects was equal in most subgroups. A second limitation concerns the inclusion of trials comparing low-dose LMWH to an inactive comparator, which led to the selection of mainly older trials or trials assessing LMWH in specific patient types or countries, limiting the generalizability of our results. In a post-hoc sensitivity analysis excluding trials published before 2005, the results remained comparable, although no inferences could be made for subgroups due to the very limited sample size. Third, to estimate the effect of low-dose LMWH on SAEs we conducted a sensitivity analysis to estimate the proportion of patients having one or more SAEs. For this purpose, we categorized mortality, symptomatic VTE, SAE, and major bleeding events from 37 trials as SAE. In reality, not all symptomatic VTE and major bleeding events are SAEs by definition (i.e., a distal leg thrombosis may be classified as adverse event, while pulmonary embolism can be a serious adverse event), but making this distinction was impossible on the basis of insufficiently detailed trial reports. Last, the best-/worst- and worst-/best-case analyses we performed to explore the influence of missing outcome data were probably overpowered to detect potential attrition bias, since the incidence of lost to follow-up was higher than the incidence of the primary outcomes. 5. Conclusions {#sec5-jcm-08-02039} ============== In a wide variety of patients at risk of VTE, there was very low to moderate-quality evidence that low-dose LMWH for thrombosis prophylaxis did not decrease all-cause mortality but reduced the incidence of symptomatic and asymptomatic VTE, while results on the intervention effects on major bleeding, clinically relevant non-major bleeding, and SAE remain inconclusive. We would like to thank S. van der Werf, medical information specialist, for her assistance with the development and execution of the search strategy. ###### Click here for additional data file. The following are available online at <https://www.mdpi.com/2077-0383/8/12/2039/s1>, Figure S1: Forest plot of SAE, stratified for patient type; Figure S2: Forest plot of clinically relevant non-major bleeding, stratified for patient type; Figure S3: Forest plot of any VTE, stratified for patient type; Figure S4: Funnel plot of all-cause mortality; Figure S5: Funnel plot of symptomatic VTE; Figure S6: Funnel plot of major bleeding; Figure S7: Funnel plot of any VTE; Table S1: Search strategy; Table S2: Characteristics of included randomized trials, stratified by patient type; Table S3: Ongoing trials; Table S4: Risk of bias assessment; Table S5: Sensitivity analysis: best-worse and worst-best case scenario's; Table S6: Sensitivity analysis: trials with publication year ≥ 2005; Table S7: Sensitivity analysis: including LMWH types not a priori defined; Table S8: Subgroup analysis: co-primary outcomes; Table S9: Sensitivity analysis: proportion of SAE and cumulative SAE. F.K. developed the original idea for this study. R.J.E., W.B., J.W., R.O.B.G., K.M., I.C.C.v.d.H., and F.K. contributed to the design of the study including the development of the protocol. R.J.E. and W.B. acquired the data. R.J.E. and F.K. performed the statistical analysis. R.J.E., W.B., J.W., R.O.B.G., K.M., I.C.C.v.d.H., and F.K contributed to interpretation of the data. R.J.E. drafted the first version of the manuscript, and all authors contributed critically to subsequent versions. R.J.E., W.B., J.W., R.O.B.G., K.M., I.C.C.v.d.H., and F.K approved the final manuscript. The corresponding author attests that all listed authors meet authorship criteria and that no others meeting the criteria have been omitted. RJE is supported by a personal grant from the 'Groninger AGIKO programme', funded by the University of Groningen. This program supports young clinicians who combine medical specialist training with obtaining a PhD degree. The university had no role in the design or conduct of the study, analysis or interpretation of the data, review or approval of the manuscript, or the decision to submit the manuscript for publication. KM reports grants from Bayer, Sanquin, and Pfizer; speaker fees from Bayer, Sanquin, Boehringer Ingelheim, BMS, and Aspen; travel support from Bayer, and consulting fees from Uniqure outside the submitted work; JW is a member of the task force at the Copenhagen Trial Unit to develop theory and software of Trial Sequential Analysis; other authors have disclosed no potential conflicts of interest. {#jcm-08-02039-f001} {#jcm-08-02039-f002} {#jcm-08-02039-f003} {#jcm-08-02039-f004} {#jcm-08-02039-f005} jcm-08-02039-t001_Table 1 ###### LMWH dose definitions. LMWH Type A Priori Defined as Low-Dose LMWH Dose Used in Included Trials ------------ ----------------------------------- ------------------------------ Bemiparin \<3500 IU 2500 IU Certoparin \<5000 IU 3000 IU Dalteparin \<5000 IU 2500 IU ^a^ Enoxaparin \<40 mg 20 mg Nadroparin \<5700 IU 2850--3800 IU ^b,c^ Parnaparin \<4250 IU 3200 IU Reviparin \<3436 IU 1750 IU Tinzaparin \<4500 IU 3500 IU ^d^ IU: International Units; LMWH: low-molecular-weight heparin; mg: milligrams; ^a^ Sandset et al. used weight-dependent doses of 3000--5500 IU; ^b^ Fraisse et al. used weight-dependent doses of 3800--5700 IU; ^b^ Yoo et al. used weight-dependent doses of 2850--5700 IU; ^c^ Xiao-Li et al. used weight-dependent doses of 41--62 IU/kg; ^d^ Sorensen et al. and Lassen et al. used weight-dependent doses: 50 IU/kg. jcm-08-02039-t002_Table 2 ###### Conventional meta-analysis and trial sequential analysis outcomes. ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Outcome Included Trials Trials (Patients) Conventional Meta-Analysis ^a^ Primary TSA ^a^\ Sensitivity TSA ^a^\ Sensitivity TSA ^a^\ Certainty of Evidence α 2.5%; ß 90%; RRR 20%;\ α 2.5%; ß 90%; RRR Based on Low Risk Trials; D^2^ Model Variance Based α 2.5%; ß 90%; RRR 20%; D^2^ 25% D^2^ Model Variance Based ---------------------------------------- ----------------- ------------------------ --------------------------------- --------------------------------- ------------------------------------------------------------------------ ---------------------------------- -------------------------- Mortality Low bias risk 5 (4.960) RR 1.03 (0.92 to 1.16) RR 1.03 (0.88 to 1.20) Insufficient data (\<5% of DIS) RR 1.03 (0.86 to 1.23) Low ^d,\ e,\ f^ All 23 (15.487) RR 0.99 (0.85 to 1.14) RR 1.02 (0.89 to 1.16) ^b^ Insufficient data (\<5% of DIS) RR 1.02 (0.90 to 1.15) Low ^d,\ g^ Symptomatic VTE Low bias risk 5 (4.878) RR 0.65 (0.45 to 0.94) RR 0.67 (0.15 to 3.05) RR 0.67 (0.32 to 1.38) 0.67 (0.15 to 3.05) Moderate ^e^ All 25 (15.920) RR 0.62 (0.48 to 0.81) RR 0.62 (0.44 to 0.89) ^c^ RR 0.62 (0.42 to 0.92) RR 0.62 (0.20 to 1.95) Moderate ^g^ Major bleeding Low bias risk 5 (4.960) RR 1.70 (0.77 to 3.74) Insufficient data (\<5% of DIS) Insufficient data (\<5% of DIS) Insufficient data (\<5% of DIS) Moderate ^f^ All 33 (13.091) RR 1.07 (0.72 to 1.59) RR 1.01 (0.18 to 5.73) ^c^ RR 1.01 (0.52 to 1.93) RR 1.09 (0.75 to 1.60) Low ^e,\ f,\ g^ SAE Low bias risk 1 (1.150) RR 0.89 (0.68 to 1.17) RR 0.89 (0.41 to 1.96) RR 0.89 (0.27 to 2.96) RR 0.89 (0.36 to 2.23) Low ^e,\ f,\ h^ All 8 (5.180) RR 0.98 (0.78 to 1.25) RR 0.98 (0.37 to 2.58) Insufficient data (\<5% of DIS) RR 0.98 (0.77 to 1.24) Very low ^d,\ e,\ f,\ g^ Clinically relevant non-major bleeding Low bias risk 0 (0) \- \- \- \- \- All 5 (3.372) RR 1.50 (0.72 to 3.12) Insufficient data (\<5% of DIS) Insufficient data (\<5% of DIS) Insufficient data (\<5% of DIS) Very low ^d,\ e,\ f,\ g^ Any VTE Low bias risk 3 (1.254) RR 0.57 (0.38 to 0.84) RR 0.57 (0.11 to 2.82) RR 0.57 (0.32 to 1.01) Not performed (D^2^ \>25%) Moderate ^e,\ i,\ k^ All 30 (5.849) RR 0.61 (0.50 to 0.75) RR 0.63 (0.49 to 0.82) RR 0.63 (0.50 to 0.80) Not performed (D^2^ \>25%) Low ^e,\ i,\ j,\ k^ ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ^a^ Small discrepancies of the intervention effect estimates between traditional RevMan meta-analyses and the TSA-adjusted results may occur due to different pooling methods (for example the inclusion of zero-event trials in TSA analyses); ^b^ TSA monitoring boundary for futility crossed; ^c^ sensitivity analysis using Peto's odds ratio showed similar results; ^d^ downgraded for inconsistency, since point estimates varied widely; ^e^ downgraded for imprecision, since TSA-adjusted confidence interval crossed '1'; ^f^ downgraded for imprecision, since conventional confidence interval crossed '1'; ^g^ downgraded for risk of bias, since (some) included trials were at high risk of bias; ^h^ downgraded for indirectness, since only one trial was included under assessment; ^i^ downgraded for risk of publication bias, since there was important asymmetry in the funnel plot; ^j^ downgraded for inconsistency, since point estimates varied widely and there was moderate statistical heterogeneity; ^k^ upgraded, since there was a strong association. α: two-sided significance level, ß: power; D^2^: diversity; DIS: diversity-adjusted information size; OR: odds ratio; RR: relative risk; RRR: relative risk reduction; SAE: serious adverse events; TSA: trial sequential analysis; VTE: venous thromboembolism.

INTRODUCTION {#sec1-1} ============ Bonding of composite resin to dentin has always been a challenge for the clinicians. Although there is an improvement in handling and bonding characteristics of the newer dental materials,\[[@ref1][@ref2]\] the collagen structure and stability of the dentin bond strength are still a challenge. Studies have proved that collagen in the hybrid layer is affected by enzymatic degradation, leading to bond failure over time.\[[@ref3][@ref4]\] Collagen fibrils in tissue are stabilized by lysyl oxidase-mediated covalent intermolecular cross-linking.\[[@ref5]\] The biomechanical properties of type I collagen can be improved by introducing more cross-links within and/or between the fibrils with the treatment of specific chemical agents. Grape seed extract, which is a naturally occurring cross-linker, mainly composed of proanthocyanidins (PAs), could be a good candidate to fulfill such role. It is a natural plant metabolite and showed to possess low toxicity and ability to induce exogenous cross-links.\[[@ref6]\] PA-based compounds can improve dentin collagen physical properties.\[[@ref7][@ref8]\] Another such important agent is ascorbic acid. It is an essentially required component in the synthesis of hydroxyproline and hydroxylysine in collagen. Hydroxyproline serves to stabilize the collagen triple helix. Hydroxylysine is necessary for the formation of the intermolecular crosslinks in collagen. It is believed that ascorbate modulates collagen production through its effect on prolyl hydroxylation.\[[@ref9]\] Hence, we can employ it as an effective chairside procedure to overcome the disadvantage of reduced bond strength of composite resin to dentin. The present study was carried out to evaluate if collagen cross-linking agents such as PA and sodium ascorbate can affect the shear bond strength of composite resin bonded to the dentinal surfaces of the teeth. The null hypothesis was that "there was no difference in shear bond strength of resin composite with dentin with or without treated with collagen cross-linking agent PA and sodium ascorbate." SUBJECTS AND METHODS {#sec1-2} ==================== One hundred freshly extracted human permanent molars were used in this study with the following exclusion/inclusion criteria. Inclusion criteria {#sec2-1} ------------------ Permanent molars with intact crownAll teeth should be free of caries, any visible discoloration, or crack and without any restoration. Exclusion criteria {#sec2-2} ------------------ Teeth which did not meet our inclusion criteria were excluded from the study. The roots of the specimens were mounted in self-cure acrylic resin, with occlusal surface parallel to the floor and extending 4 mm above the surface. Dentin surface was prepared with the help of slow-speed sectioning diamond disc under copious water supply and remove complete enamel portion and exposing the dentinal surface, then the surface of dentin was finished with wet 600 grit silicon carbide paper under running water to produce a standardized smear layer. After that, the dentinal surfaces of the specimens were acid etched with 35% phosphoric acid (SwissTec, COLTENE, Switzerland) as per manufacturers\' recommendation. The specimens were randomly divided into three groups based on the surface treatment of dentin as follows: Group I (*n* = 20) -- No dentin pretreatment was doneGroup II (*n* = 40) -- 10% sodium ascorbate pretreatment. This group was further divided into two subgroups based on the pretreatment time as follows:Subgroup IIa (*n* = 20) -- The etched dentin surface was treated with 10% sodium ascorbate solution for 5 min and rinsed with water, after which they were blot dried leaving a moist dentinal surface for bondingSubgroup IIb (*n* = 20) -- The etched dentin surface was treated with 10% sodium ascorbate solution for 10 min and rinsed with water, after which they were blot dried leaving a moist dentinal surface for bonding.Group III (*n* = 40) -- 6.5% PA pretreatment. This group was further subdivided into subgroups based on pretreatment time as follows:Subgroup IIIa (*n* = 20) -- The etched dentin surface was treated with 6.5% PA solution for 5 min and rinsed with water, after which they were blot dried leaving a moist dentinal surface for bondingSubgroup IIIb (*n* = 20) -- The etched dentin surface was treated with 6.5% PA solution for 10 min and rinsed with water, after which they were blot dried leaving a moist dentinal surface for bonding. Bonding for composite resin build-up {#sec2-3} ------------------------------------ Before application of bonding agent, a transparent plastic tube (3 mm in diameter and 5 mm height) was placed on the prepared occlusal surface, and the outer diameter was delineated with lead pencil as a reference mark for the application of the bonding agent. Then, plastic tube was removed, and bonding agent (One Coat Bond SL, SwissTec, COLTENE, Switzerland) was applied according to the manufacturer\'s instructions. Then, it was cured with light-emitting diode light (Woodpecker, Guilin Woodpecker Medical Instruments Co., Ltd., China) for 30 s (according to manufacturer\'s instruction). Composite resin buildup {#sec2-4} ----------------------- Transparent plastic tube was then fixed manually on the prepared surface of each tooth. A microhybrid composite resin (SwisTec, COLTENE, Switzerland) was applied in five 1 mm layers, and each layer was photopolymerized for 40 s, reaching 5 mm in total height, during which the light was moved around the tube to assure curing of the entire composite resin cylinder. The plastic tube was then removed, and excess composite resin was removed using a polishing disc (Sof-Lex™; 3M Espe, St. Paul, MN, USA). The samples were then stored in distilled water at 37°C for 48 h. Thermocycling and shear bond strength testing {#sec2-5} --------------------------------------------- After 48 h, samples were transferred from distilled water to normal water at 37°C for 24 h and then thermocycled for 500 cycles between 5°C and 55°C with a dwell time of 30 s each and transfer time between two baths was 5 and 10 s. After thermocycling, shear bond strength tests were performed on a universal testing machine (Unitek, 9450 PC, FIE, India) at a cross-head speed of 1 mm/min until the composite cylinder was dislodged from the tooth. Shear bond strength was calculated as the ratio of fracture load and bonding area, expressed in megapascals (MPa). The results were tabulated and subjected to statistical analysis. RESULTS {#sec1-3} ======= Obtained data analyzed and expressed in mean ± standard deviation (SD). Mean was compared by one-way analysis of variance and Tukey *post hoc* test *P* = 0.05 was taken as statistically significant. The results obtained through statistical analysis depicted that dentin pretreatment significantly increases the shear bond strength of the composite resin with dentin; duration of application of pretreatment materials on dentin also plays a significant role \[Tables [1](#T1){ref-type="table"}-[3](#T3){ref-type="table"}\]. ###### Shear bond strength in different groups irrespective of time of treatment  ###### Shear bond strength in different groups taking time of treatment into consideration  ###### Shear bond strength comparison between group/subgroup (Tukey\'s HSD test)  Mean difference was found to be maximum between Groups I and IIb and minimum between Groups IIIa and IIIb. All the between-group comparisons except between Groups IIIa and IIIb were significant statistically \[[Table 3](#T3){ref-type="table"}\]. On the basis of above evaluation, the following order of shear bond strength was observed in different groups/subgroups: IIb \> IIa \> IIIb \~ IIIa \> I" with "IIb \> IIa \> IIIb \> IIIa \> I". It was observed that shear bond strength values in Group I was of lower order, whereas shear bond strength values in Group II were of higher order. Shear bond strength values in Group III were of middle order. In Group I, shear bond strength ranged from 13.14 to 17.00 MPa with a mean value of 15.36 and a SD of 1.16 MPa. In Group IIa, shear bond strength ranged from 16.31 to 23.07 MPa with a mean value of 20.09 and a SD of 1.87 MPa. In Group IIb, shear bond strength ranged from 18.74 to 29.99 MPa, with a mean value of 22.55 and a SD of 2.51 MPa. In Group IIIa, shear bond strength ranged from 12.36 to 20.26 MPa, with a mean value of 17.01 and a SD of 2.10 MPa. In Group IIIb, shear bond strength ranged from 17.11 to 20.66 MPa, with a mean value of 18.46 and a SD of 0.97 MPa \[[Table 2](#T2){ref-type="table"}\]. DISCUSSION {#sec1-4} ========== The results obtained through statistical analysis depicted that sodium ascorbate and PA application before bonding significantly improve the bond strength of composite with dentin \[[Table 1](#T1){ref-type="table"}\]. The increase in bond strength in the present study may be attributed to improve dentin collagen stability, due to the higher number of collagen cross-links. Matrix metalloproteinases (MMPs) are such class of enzymes that are known to degrade collagen. These proteins are found in the dentin but more abundant on enamel--dentin junction and in the predentin.\[[@ref10]\] The MMPs converted from Pro-MMP which are trapped or bound in the dentin during its formation by lowering the pH to 4.5 or below.\[[@ref11][@ref12]\] Application of self-etching adhesives (acidic resins) to dentin increases 14-fold in MMP enzyme activity.\[[@ref13]\] Thus, adjunctive collagen pretreatment strategies have been proposed to improve dentin adhesion, through the use of agents that maintain the stability of collagen toward enzymatic degradation.\[[@ref14]\] These agents include the use of substances that are considered to be inhibitors of MMPs and cysteine cathepsins. Thus, pretreatment with collagen cross-linking agents can promise improvement in the bonding mechanism preserving the integrity of collagen hybrid layer. Various natural as well as synthetic cross-linking agents such as glutaraldehyde, tannic acid, PA, genipin, and cocoa seed extract have been used to strengthen the hybrid layer and have shown positive results in improving the bond strength to significant levels.\[[@ref15][@ref16][@ref17]\] Results of this study show that sodium ascorbate application significantly improves bond strength compared with control and PA application \[[Table 3](#T3){ref-type="table"}\]. In Group IIa (10% sodium ascorbate for 5 min), shear bond strength ranged from 16.31 to 23.07 MPa, with a mean value of 20.09 Mpa and a SD of 1.87 MPa. In Group IIb (10% sodium ascorbate for 10 min), shear bond strength ranged from 18.74 to 29.99 MPa, with a mean value of 22.55 MPa and a SD of 2.51 MPa. These results for the sodium ascorbate in the present study were found to be time dependent (*P* \< 0.001), whereas for PA difference was found to be insignificant (*P* = 0.096) \[Table [1](#T1){ref-type="table"} and [2](#T2){ref-type="table"}\]. The major action of the sodium ascorbate is in the stabilization of the collagen, as a cofactor of hydroxylation of proline and lysine.\[[@ref18]\] A study based on energy dispersive spectroscopy analysis has shown that calcium ion concentration after demineralization dropped from the 28.62 to 12.77% and increased to 15.99% after sodium ascorbate treatment for 10 min, With this may due to chemical interaction of sodium ascorbate with collagen fibres in dentine.\[[@ref19]\] However, shear bond strength of composite to deep dentin after treatment with 6.5% PA and 10% sodium ascorbate found the statistically significant better results with PA than the sodium ascorbate.\[[@ref20]\] PAs, which form a complex subgroup of the flavonoid compounds, have been found in a wide variety of fruits, vegetables, flowers, nuts, seeds, and bark. PA from grape seed extract has been shown to safely and effectively cross-link collagen in both *in vitro* and *in vivo* models and also inhibit MMP activity.\[[@ref21][@ref22]\] Considering its wide spectrum of benefits and high biocompatibility, we have selected this agent for this study. Studies done by Han *et al*. (2003) and Bedran-Russo *et al*. and found the improvement in ultimate tensile strength and shear bond strength, respectively, after pretreatment with PA.\[[@ref7][@ref8]\] These findings also corroborate with the results of these studies. A statistically significant improvement was seen after pretreatment with PA (*P* \< 0.001) \[[Table 1](#T1){ref-type="table"}\]. The proposed mechanisms for interaction between PA and proteins include covalent, ionic, hydrogen bonding, and hydrophobic interactions.\[[@ref7][@ref23]\] In the present study, molars were selected since they are most commonly restored, due to high incidence of caries. Therefore, validation and extension of our results await further investigations. The results of the *ex vivo* assays may not be directly comparable with the *in vivo* conditions, where all other parameters are to be considered. *In vivo* research is must to assess the clinical outcome and analysis of these agents so as to make out most of the benefits to the clinical adhesive dentistry. CONCLUSIONS {#sec1-5} =========== We can conclude that the treatment of dentinal surfaces with collagen cross-linking agent increases the shear bond strengths. Results for sodium ascorbate were found to be time dependent, whereas for PA, differences were nonsignificant. Financial support and sponsorship {#sec2-6} --------------------------------- Nil. Conflicts of interest {#sec2-7} --------------------- There are no conflicts of interest.

Introduction ============ Sickle cell disease (SCD) is a genetic disorder in which polymerization of deoxygenated sickle hemoglobin (HbS) leads to decreased deformability of the normally flexible erythrocytes. These rigid sickle-shaped red blood cells (RBC) can occlude the microvasculature leading to the sudden onset of painful vaso-occlusive episodes (VOC).^[@b1-1050083],[@b2-1050083]^ After HbS deoxygenates in the capillaries, it takes some time (seconds) for HbS polymerization and the subsequent flexible-to-rigid transformation. If the transit time of RBC through the microvasculature is longer than the polymerization time, sickled RBC will lodge in the microvasculature.^[@b3-1050083]^ Any trigger that decreases microvascular blood flow will prolong the transit time, promoting the entrapment of sickled RBC, resulting in vaso-occlusion. This physiology of SCD, described decades ago,^[@b4-1050083],[@b5-1050083]^ is fundamental to understanding the triggering of VOC. Patients report that stress, cold, and pain itself can trigger the onset of VOC^[@b6-1050083]^ but the frequency of VOC is highly variable. To date, the mechanism of how such events might trigger regional vaso-occlusion has not been fully elucidated. Psychological stress is an exacerbating factor in many chronic illnesses, such as SCD,^[@b7-1050083]--[@b10-1050083]^ coronary artery disease and myocardial ischemia.^[@b11-1050083],[@b12-1050083]^ Stress is significantly associated with increased pain intensity, reductions in social and physical activities and greater health care utilization.^[@b8-1050083],[@b13-1050083],[@b14-1050083]^ Day-to-day stressors have been associated with onset of pain and the course of VOC in SCD.^[@b9-1050083],[@b10-1050083]^ Stress is well-known to modulate autonomic nervous system (ANS) activity which in turn plays a major role in the regulation of regional blood flow.^[@b15-1050083]^ Interestingly, SCD children with greater mental-stress-induced autonomic reactivity had more severe clinical disease.^[@b16-1050083],[@b17-1050083]^ SCD subjects also have augmented ANS-mediated vasoconstriction in response to sighing, hypoxia, and pain.^[@b18-1050083]--[@b20-1050083]^ Therefore, autonomic dysregulation in SCD represents a plausible physiological link between mental stress and sickle RBC retention in the microvasculature.^[@b16-1050083],[@b18-1050083]--[@b21-1050083]^ Further understanding of this proposed mechanism of VOC triggering would not only help to predict disease manifestations, but would also open up opportunities for therapeutic intervention in disorders such as SCD in which preservation of microvascular blood flow is important.^[@b22-1050083]^ To address the role of mental stress in the physiology of SCD, we objectively quantified microvascular blood flow, measured by photoplethysmography, in response to standardized mental stress tasks in subjects with SCD and in controls. We also assessed cardiac ANS balance by analysis of heart rate variability in response to mental stress. We correlated photoplethysmogram-derived physiological indices with subjective indices of perceived stress assessed from standardized anxiety questionnaires. The aim of this study was to determine the relationship of peripheral and cardiac ANS responses with mental stress in SCD. Methods ======= The study was conducted under an institutional review board-approved protocol at the Children's Hospital Los Angeles with approved consent/assent. Twenty SCD subjects with Hb SS, S-β^0^, S-β^+^ or SC genotype and 16 age- and race-matched controls from the patients' family and friends were recruited. Experimental setup and study protocol ------------------------------------- All studies were performed in an ANS laboratory under strictly controlled settings.^[@b18-1050083]^ Neuropsychological stress was assessed at baseline using the State-Trait Anxiety Inventory (STAI) questionnaire.^[@b23-1050083]^ The STAI Y-1 and Y-2 evaluate "anxiety at this moment, aka *state anxiety*" and "how people generally feel, aka *trait anxiety*", respectively. Following 5 minutes of baseline recording, the stress induction protocol was presented through psychological software (E-prime 2.0, Psychology Software Tools, USA). The protocol consisted of a memory task (N-back)^[@b24-1050083]^ and a conflict test (Stroop),^[@b25-1050083],[@b26-1050083]^ presented in a randomized order, followed by a pain anticipation (PA) test ([Figure 1](#f1-1050083){ref-type="fig"}). During the N-back task, the subjects were asked to respond when the current letter matched the letter from n steps (n=zero, one, two, or three back) earlier in the sequence. During the Stroop task, the participants were asked to identify the font color of a word, not the written name of the word. We measured state anxiety between tasks. During the PA task, subjects read the following sentence on their computer screen: "You will receive a maximum pain stimulus in one minute. When you cannot tolerate the pain any longer, say STOP and the device will cool down to normal level immediately". However, no pain stimulus was actually applied. {#f1-1050083} Physiological measurements and analysis parameters -------------------------------------------------- All the physiological monitoring sensors were attached to the subjects' left arm. Microvascular blood flow was measured using photoplethysmography (Nonin Medical Inc., USA) and laser Doppler flowmetry (Perimed, Sweden). Respiration (thoracic and abdominal bands, zRip DuraBelt, Philips), the electrocardiogram and continuous blood pressure (Nexfin, Amsterdam) were recorded. Recorded data from all devices were exported for processing and analysis in MATLAB. The photoplethysmogram amplitude was normalized to its own 95^th^ percentile value during the full study. The average microvascular blood flow was calculated over the 5 min baseline period, the N-back, Stroop and PA tasks. The percent decrease in the amplitude of the photoplethysmogram or microvascular perfusion waveforms ([Figure 2](#f2-1050083){ref-type="fig"}; 2^nd^ and 3^rd^ signals, respectively) from the baseline mean was interpreted as a vasoconstriction response.^[@b18-1050083],[@b27-1050083]^ {#f2-1050083} Cardiac autonomic balance was assessed by analysis of the R-to-R interval and heart rate variability^[@b19-1050083],[@b28-1050083],[@b29-1050083]^ during baseline and mental stress tasks. The following power spectral indices were calculated: low frequency power, reflecting a combination of cardiac sympathetic and parasympathetic activity; high frequency power, reflecting parasympathetic activity;^[@b29-1050083],[@b30-1050083]^ and the ratio of low frequency power to high frequency power, reflecting sympathovagal balance.^[@b30-1050083]^ Percent changes in mean microvascular blood flow and mean spectral indices from baseline to tasks were calculated. The Student *t*-test (or Wilcoxon sign rank) or χ^2^ test was used to test baseline group differences and task differences. Robust regression was used to correlate vasoconstriction response and state anxiety during the PA task. Repeated measures analysis of variance was used to test differences in N-back and Stroop sublevels and accuracy scores. All statistical analyses were performed using STATA/IC 14.1 (StataCorp LP, TX, USA) with nominal significance set at *P*≤0.05. The methods are described in detail in the *Online Supplementary Methods S1*. Results ======= Data from a total of 20 SCD patients and 16 controls were analyzed. Transfused and non-transfused subjects with SCD were grouped together and healthy and sickle cell trait subjects (controls) were combined after it had been demonstrated that these factors were not statistically significant in the analyses. The percentage of HbS (HbS%) was considered to be zero in patients with sickle cell trait as the cellular distribution of HbS differs in sickle cell trait and does not contribute to sickling under the conditions of the experiments in this study, making the HbS% in sickle cell trait not comparable to that in transfused or non-trait sickle phenotypes. The subjects' characteristics are summarized in [Table 1](#t1-1050083){ref-type="table"}. Nine (45%) SCD subjects were on chronic transfusion, nine (45%) were being treated with hydroxyurea and two (10%) were not receiving either treatment. The characteristics of both groups were balanced except for hemoglobin concentration on the study day. Sixty-one percent of subjects had a level of education equivalent to high school or superior. Seventy-two percent reported that they had a high level of competitiveness on the visit screening questionnaire. ###### Population characteristics.  Vasoconstriction due to mental stress ------------------------------------- As determined from the photoplethysmogram, there was a significant drop in microvascular blood flow during both cognitive tasks (N-back and Stroop, *P*\<0.0001) and the PA task (*P*\<0.0001) ([Figure 3](#f3-1050083){ref-type="fig"}). [Figure 2](#f2-1050083){ref-type="fig"} (signal 2) shows a typical response of vasoconstriction in one subject. The drop in microvascular blood flow from baseline was greater during the PA task than during the cognitive tasks. A similar vasoconstriction response was observed when the microvascular blood flow was assessed by laser-Doppler flowmetry. Subjects had higher anxiety scores immediately after completing the tasks than at baseline (STAI Y-1, mean difference=6; *P*=0.0007). Eighty-five percent of patients with SCD and 75% of controls showed vasoconstriction compared to baseline during at least one cognitive task. Eighty-five percent of SCD patients and 87.5% of controls had decreases in mean blood flow during the PA task. There was no difference in the magnitude of responses between individuals with SCD and controls. Demographic variables such as age, gender, race, number of days from last menstruation, and laboratory values were not associated with the magnitude of the vasoconstriction response. {#f3-1050083} The Stroop test caused greater vasoconstriction than the N-back task, irrespective of the order in which the tests were presented (*P*=0.019) ([Figure 3](#f3-1050083){ref-type="fig"}). Subjects who were randomized to perform the Stroop task first had greater anxiety responses than did the subjects who performed the N-back task first (mean difference=10; *P*=0.03). Overall the accuracy score was significantly lower for the Stroop task than for the N-back task in all subjects (mean score difference=25, *P*\<0.001). The accuracy score for the Stroop and N-back tasks decreased as the difficulty increased from zero-back to three-back in the N-back task and from level one to level three in the Stroop task (*P*\<0.0001) ([Figure 4](#f4-1050083){ref-type="fig"}) but there was no further change in blood flow with increasing difficulty. Once the subjects manifested vasoconstriction, in comparison with baseline vascular tone, the vasoconstriction remained throughout the whole task regardless of the difficulty of the tasks. {#f4-1050083} Vasoconstriction response to perceived anxiety during pain anticipation ----------------------------------------------------------------------- On robust regression, the effect of state anxiety on blood flow response was greater in SCD patients than in controls (*P*=0.03 for the interaction), suggesting that higher anxiety at baseline (STAI Y-1) in SCD subjects is associated with less change in blood flow (coefficient = −1.85, *P*=0.002) in response to pain anticipation ([Figure 5](#f5-1050083){ref-type="fig"}). State anxiety had no effect on change in blood flow in control subjects. To understand why SCD subjects would have less response with high anxiety, we looked at the baseline blood flow. We found that highly anxious subjects tended to have a lower mean baseline blood flow (*Online Supplementary Figure S1*), meaning they were already vasoconstricted at baseline, limiting them from further vasoconstriction. This trend was not seen among controls. (*Online Supplementary Figure S2A*, *B*: high-anxiety SCD responder and low-anxiety SCD responder). {#f5-1050083} Cardiac autonomic response -------------------------- Since the ANS regulates blood flow and SCD subjects have dysautonomia,^[@b15-1050083],[@b28-1050083],[@b31-1050083],[@b32-1050083]^ we explored the effect of mental stress responses on cardiac autonomic balance. In com parison to the value at baseline, there was a significant decrease in R-to-R interval, signifying an increase in heart rate, during all tasks (*P*\<0.0001) ([Figure 6A](#f6-1050083){ref-type="fig"}). As for the microvascular blood flow response, the R-to-R interval was less during the Stroop task than during the N-back task (*P*=0.002). ![Autonomic nervous system responses to mental stress. (A) R-to-R interval (sec) and (B) high frequency power (sec^[@b2-1050083]^/Hz, shown on a log scale) in response to the N-Back and Stroop tasks in all subjects. There is a significant decrease in R-to-R interval and parasympathetic withdrawal during mental stress tasks compared to baseline. SE: standard error of mean; HFP: high frequency power.](10583.fig6){#f6-1050083} There was significant parasympathetic withdrawal during the N-back and Stroop tasks as reflected by the drop in high frequency power (*P*=0.002 and *P*\<0.0001, respectively) ([Figure 6B](#f6-1050083){ref-type="fig"}) The Stroop task caused stronger parasympathetic withdrawal than the N-back task (*P*\<0.0001). There was more sympathetic activation during the Stroop test (low-to-high power ratio: *P*=0.03), but not during the N-back task. We did not analyze autonomic reactivity during the PA task because the 1-minute test period was not long enough to derive spectral indices.^[@b29-1050083]^ Discussion ========== VOC is a significant complication of SCD and a major cause of morbidity and mortality.^[@b33-1050083]^ The frequency of VOC is related in part to hemoglobin-F content, white blood cell count, inflammatory status and other factors.^[@b34-1050083]--[@b36-1050083]^ However, there is still significant variability in crisis frequency among SCD subjects with otherwise similar hematologic status. Pain crises can be promoted by preceding dehydration, infection, injury, exposure to cold or emotional stress.^[@b37-1050083],[@b38-1050083]^ Much of the research in past decades has focused on adhesion and processes attributed to occlusion in the post-capillary venule, and to decreased flow due to nitric oxide depletion.^[@b39-1050083]^ While stress and cold are often mentioned, very little attention has been paid to decreased flow due to neurally induced vasoconstriction.^[@b32-1050083],[@b40-1050083]^ SCD patients undergo a tremendous amount of stress not only due to environmental challenges but also the illness-related stress of painful episodes, repeated medical procedures and life-threatening complications. Stress causes ANS hyperreactivity by enhancing the sympathetic nervous system and dampening the parasympathetic system in SCD subjects compared to non-SCD individuals.^[@b16-1050083],[@b17-1050083]^ Sympathetic and parasympathetic responses have been related to clinical vaso-occlusion in SCD, through ANS modulation of regional blood flow.^[@b15-1050083],[@b17-1050083]^ SCD is probably the best example of a disorder in which decreased microvascular perfusion can be directly related to the pathology of the disorder, because the increase in transit time from decreased blood flow promotes entrapment of rigid red cells in small vessels.^[@b3-1050083],[@b5-1050083]^ To our knowledge, this is the first study to quantify regional blood flow modulated by ANS reactivity under mental stress in SCD. Our data show that experimental mental stress caused a decrease in regional blood flow in all participants. While we thought that SCD subjects would exhibit stronger vasoconstriction because of their hyperresponsiveness to sympathetically induced stimuli, such as sighing,^[@b28-1050083]^ we did not detect a difference in stress-induced vasoconstriction between SCD patients and controls. We did find a significantly higher anxiety response score (*P*=0.03) in subjects who were exposed to the more difficult mental stress test first (Stroop). We also found that the degree of vasoconstriction was proportional to the magnitude of the stress. Subjects reported that overall the Stroop task was more stressful: accuracy scores were lower and there was also a greater decrease in blood flow with this cognitive stressor task. However, different sublevels of difficulty within a task type did not correlate with levels of vasoconstriction. This finding suggests that consecutive stressful events could make SCD patients more vulnerable to vaso-occlusion. We think that variability in the vasoconstriction response to stress may account in part for differences in clinical severity among SCD patients who have the same hemoglobin phenotype. The frequency of VOC and intensity of pain are higher among patients found to have high anxiety and stress scores on standard psychological assessments.^[@b8-1050083],[@b41-1050083],[@b42-1050083]^ We tried to correlate the vasoconstrictive response with clinical severity. As our SCD patients were either on chronic transfusion or hydroxyurea, the number of VOC was too low to detect differences in this current relatively small sample. Along with a strong vasoconstriction response, significant autonomic reactivity was seen in all subjects. The Stroop test was consistently more stressful, and induced greater vasoconstriction as well as greater autonomic reactivity. There was both sympathetic activation as well as parasympathetic withdrawal during this cognitive task. Mental stressors are known to influence autonomic function by sympathetic or parasympathetic tone alterations. Higher anxiety induces atherosclerosis via enhanced sympathetic modulation, increasing the risk of cardiovascular disease.^[@b43-1050083]^ In addition, mental stress and anxiety have been linked to impaired endothelial function via autonomic dysfunction.^[@b43-1050083]--[@b45-1050083]^ Endothelial function, quantified by flow mediated dilation, decreases as a result of mental stress tasks.^[@b46-1050083]^ Similarly, in SCD, a synergistic interaction between impaired local vascular function and the exaggerated neurally mediated vasoconstrictive response could further reduce peripheral blood flow, setting the stage for VOC. Consistent with the findings of our previous study,^[@b18-1050083]^ anticipation of pain caused significant vasoconstriction and this response was quantitatively greater than that of the calibrated experimental stress tasks ([Figure 3](#f3-1050083){ref-type="fig"}). We do not have strong evidence to conclude that the presence of SCD alone influences mental stress-induced vasoconstriction but anxiety seems to be a modifying factor. Interestingly unlike control subjects, SCD subjects who were highly anxious had less vasoconstriction during the PA task and *vice versa*. We think that this pattern of response occurred because highly anxious subjects were already vasoconstricted at baseline and this limited the magnitude of further vasoconstriction. So the fact that SCD subjects have less change in the vasoconstriction response to the stressors than controls actually reflects their chronically vasoconstricted state. Although not statistically significant, the trend of lower baseline blood flow with high anxiety in SCD can be seen in *Online Supplementary Figure S1*, which also shows the significant variability in baseline measures. Photoplethysmogram and microvascular perfusion signals from Perimed do not have absolute units, so measurements made as percent changes from baseline are more reliable, basically correcting for baseline variability and allowing detection of the differences seen in [Figure 5](#f5-1050083){ref-type="fig"}. These findings may be related to pain catastrophization and increased psychophysical pain sensitivity due to frequent pain episodes.^[@b7-1050083],[@b47-1050083],[@b48-1050083]^ Over the years, pain catastrophization may increase the frequency of pain and severity of pain crises.^[@b47-1050083],[@b49-1050083]^ From a standpoint of neural physiology, repeated acute pain creates a central neural pathological pain connectome^[@b50-1050083]^ that leads to baseline chronic pain and chronic vasoconstriction. Although baseline blood flow was not statistically significantly lower, probably due to insufficient study power, we suspect that the above-described phenomenon is the explanation for our findings and warrants further study. We showed that neurally mediated vasoconstriction is a biophysical marker of mental stress in SCD patients and controls. Mental stress has been identified as a trigger for pain crises in SCD and its connection with a decrease in microvascular perfusion seems to make a causal link to VOC. The probability of vaso-occlusion is predicted to be related to the relation between time to polymerization of deoxy HbS and microvascular flow.^[@b3-1050083],[@b5-1050083]^ Obviously, HbS is the major pathology in SCD. However, neurally mediated changes in microvascular flow certainly play a significant and unappreciated role. Individual variation in patterns of vasoconstriction with different ANS reactivity may offer a possible biological explanation for the variability in the frequency of VOC in SCD patients with similar hemoglobin phenotype. Identifying the high-risk individuals who show a phenotype of chronic vasoconstriction and repeated pain crises, and targeting them with neuro-modulatory cognitive-based therapies may improve vascular and neural physiology in SCD. In the primary stage of a crisis, implementing these learned cognitive-based therapies or distraction and relaxation techniques will help to improve the prognosis during acute pain. Microvascular flow in response to stress may also serve as an important surrogate endpoint for therapy in SCD and other diseases in which small vessel blood flow and reactivity are important. Some limitations of this study should be acknowledged. One limitation was that the small sample size did not allow us to detect a difference in the magnitude of vasoconstriction between groups and correlate it with a clinical outcome such as VOC. Since the concept that mental stress causes vasoconstriction has not been studied in SCD, prior effect size was not known to permit sample size calculation. Another reason for lack of difference between groups is that over 90% of our patients are on hydroxyurea or chronic transfusion and thus clinical crises are relatively uncommon. Any real magnitude differences would be more likely to emerge in studies with larger samples and untreated patients. However, the primary aim of this study was to understand the changes in peripheral and cardiac responses to mental stress. The fundamental study design presented here was able to detect changes in physiological signals with millisecond accuracy and clearly showed vasoconstriction responses and ANS reactivity due to mental stress in all subjects. We think that the consequences of these findings are mechanistically related to the pathophysiology of sickle cell vaso-occlusion. This work was supported by grants from the National Institutes of Health National Heart, Lung, and Blood Institute (U01 HL117718). The authors thank Justin Abbott for his contribution to the data collection. Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: [www.haematologica.org/content/105/1/83](http://www.haematologica.org/content/105/1/83)

INTRODUCTION {#s1} ============ Upper track urothelial carcinoma (UTUC) is less common than bladder urothelial carcinoma and accounts for 5--10% of all urothelial carcinoma.^[@b1]^ The incidence of ureteral urothelial carcinoma (UUC) is approximately half that of pyelocaliceal urothelial carcinoma.^[@b2]^ UUC has a worse prognosis than pyelocaliceal urothelial carcinoma.^[@b3],[@b4]^ Owing to different anatomical considerations and oncological outcomes in UTUC, these different malignant entities must be evaluated independently.^[@b5]^ The gold standard treatment for UTUC is radical nephroureterectomy with excision of the bladder cuff, regardless of the tumour location.^[@b6]^ Recently, conservative surgery such as endoscopic ablation or segmental ureteral resection, which allows preservation of the upper urinary renal unit, has also been applied.^[@b7]^ However, preoperative histological evaluation through biopsy of the upper urinary tract is difficult, because ureteroscopy is an invasive procedure and usually requires general anaesthesia. Furthermore, the accuracy of ureteroscopic biopsy in predicting tumour stage and grade is limited, and the limitations of endoscopic biopsy must be balanced against the possible advantage of avoiding radical surgery.^[@b8]^ Thus, accurate preoperative prediction of tumour grade could be helpful in selecting more appropriate therapeutic options. CT urography (CTU) is an imaging modality with high diagnostic accuracy in the detection of UTUC and has replaced intravenous excretory urography and ultrasonography as the first-line imaging test for investigating high-risk patients.^[@b9]^ Even though several studies have investigated diffusion-weighted MRI (DW-MRI) as an imaging assessment for predicting tumour grade of UTUC,^[@b10]--[@b11]^ characteristic CTU findings that can predict tumour grade of UUC have not been identified, to the best of our knowledge. In this study, we aimed to evaluate the correlation between CTU imaging variables, including tumour size and imaging features, and histological grade of UUC, and to identify CTU imaging features that allow prediction of high-grade UUC, which should be treated by radical surgery. METHODS AND MATERIALS {#s2} ===================== Patients -------- This retrospective single-centre study was approved by the institutional review board at and written informed consent was not required. We searched institutional patient information systems to identify all consecutive patients with UUC who had undergone nephroureterectomy between January 2005 and July 2016. A total of 79 consecutive patients who underwent surgery with removal of a surgical specimen for histological analysis were registered. The inclusion criteria for this study were as follows: (i) tumours only located in the ureter, (ii) patients had undergone CTU scan prior to surgery and (iii) histologicalal confirmation of UUC with clear statement of histological grade according to the WHO 2004 classification system. Four patients were excluded because histological grade was not available in the pathological reports, and two patients did not undergo a CTU scan. Ultimately, 73 patients (52 males and 21 females; mean age, 68.92 ± 9.08 years) with 81 UUCs were included in our study. All pathological data were reviewed by a board-certificated pathologist, and all tumours were classified into low-grade and high-grade groups according to the WHO 2004 classification system and pathologic T stage of the tumours was assessed according to the TNM staging system. CTU technique ------------- All CTU examinations were performed using various CT scanners from 16-channel to 128-channel MDCT scanners (Somatom Sensation 16, Siemens Healthcare, Brilliance 64, Philips Medical Systems, Best, Netherlands or Somatom Definition Flash 128, Siemens Healthcare Forchheim, Germany). Scanning parameters of the most frequently used CT scanner (Brilliance 64, Philips Medical Systems, Best, Netherlands) were as follows: tube voltage, 120 kVp; effective tube current, 300 mAs; section thickness, 5 mm; pitch and speed, 0.891:1; rotation time, 0.75 s and collimation, 64 × 0.625 mm for 64-channel MDCT. Before acquisition of contrast-enhanced scans, simple unenhanced scans were obtained, after which 2 ml kg^--1^ non-ionic contrast material containing 300--350 mg ml^−1^ of iodine \[iomeprol (Iomeron 300, Bracco Altana Pharma, Konstanz, Germany), iopamidol (Pamiray 300, Dongkook Pharmaceutical, Seoul, Republic of Korea) or iobitridol (Xenetix 300, Guerbet, Villepinte, France)\] was intravenously administered at a rate of 3.0 ml s^−1^ using a standard power injector. For CTU, in addition to the unenhanced scan, two-phase studies were performed with combinations of corticomedullary and excretory phases at our institution. The corticomedullary phase began 30--40 s after contrast administration, and excretory phases began 300 s after contrast administration, respectively. Image analysis -------------- Two radiologists (DJS and STH with 17 and 3 years of experience, respectively, in interpreting genitourinary images) independently reviewed all CTU images on a picture archiving and communication system workstation (INFINITT PACS, INFINITT Healthcare, Seoul, Republic of Korea). The readers knew all patients had been diagnosed with UUC, but were informed of neither the histological grade nor the findings listed in the initial radiological report. They evaluated the following CTU imaging features: tumour location, tumour size, tumour enhancement value, multiplicity, periureteral infiltration, enlarged retroperitoneal lymph nodes with a short axis of more than 1 cm, and hydronephrosis grade. Tumour location was categorized into three groups (proximal, middle, and distal) according to anatomic ureteral segmentation. Tumour size was determined as the maximal length or diameter of the whole tumour presenting as ureteral soft-tissue mass or enhancing wall thickening on the axial, sagittal, or coronal CTU images. In patients with multiple lesions, the largest one was selected for size measurement. Tumour enhancement value was calculated as the difference between attenuation values in the corticomedullary phase and unenhanced phase. On corticomedullary phase images, the readers drew a circular ROI that included the enhancing solid portion of the tumour, avoiding adjacent mesenteric fat. The ROI was as large as possible to minimize noise. A ROI of the same size was placed in the corresponding location on the unenhanced scan image. The readers also reported hydronephrosis grade according to the modified version of the Society for Foetal Urology Hydronephrosis Grading System ([Table 1](#t1){ref-type="table"}). ###### Modified version of the Society for Fetal Urology Hydronephrosis Grading System Grade 0 1 2 3 4 --------------------------------- --------------- ------------------------------ -------------------------------------- -------------------------------------------------------------- -------------------------------------------------- Ureter and pelvocalyceal system No dilatation Local dilation of the ureter Ureteral and renal pelvis dilatation Ureteral and renal pelvis dilatation plus calices dilatation Further dilatation of ureter, pelvis and calices Renal parenchymal thickness Normal Normal Normal Normal Thin Statistical analysis -------------------- Descriptive statistics of means, standard deviations and frequencies were used to describe patient characteristics. Univariate logistic regression modelling, Mann--Whitney *U* tests, and *Χ*^2^ tests were used to assess the correlation between CTU imaging variables and histological tumour grade. Multiple logistic regression analysis using a backward selection method was performed to identify significantly independent CTU imaging variables that could predict high-grade tumours. Spearman correlation analysis was used to assess the correlation between tumour size and hydronephrosis grade. *Χ*^2^ test and linear-by-linear association were used to investigate the correlation of hydronephrosis grade and peritumoural intfiltration with pathologic T stage. A receiver operating characteristic (ROC) curve was constructed to identify the cut-off value of effective factors that provided the best diagnostic accuracy. Interobserver agreement was calculated using kappa statistics for nominal values, including hydronephrosis grade, peritumoural infiltration, multiplicity and presence of enlarged retroperitoneal lymph nodes. Intraclass correlation was calculated for continuous values including tumour size and contrast enhancement value. The scores were used to define agreement as follows: 0.41--0.60 denoted moderate agreement; 0.61--0.80, good agreement and greater than 0.81, excellent agreement. Statistical analysis was done using IBM SPSS Statistics version 22.0 for Windows (IBM Corp., Armonk, NY). A *p* value of less than 0.05 was considered statistically significant. RESULTS {#s3} ======= Images of the 73 patients with 81 UUCs were reviewed. The lesions were unilateral in all cases. 15 patients (20.5%) had low-grade UUCs ([Figure 1](#f1){ref-type="fig"}) and 58 patients (79.5%) had high-grade UUCs ([Figure 2](#f2){ref-type="fig"}). 22 (27.1%) lesions were located in the proximal ureter, 14 (17.2%) in the middle ureter, and 45 (55.5%) in the distal ureter. Eight (5.8%) patients had multiple lesions in the ipsilateral ureter. Clinicopathological characteristics of the patients are summarized in [Table 2](#t2){ref-type="table"}. {#f1} {#f2} ###### Clinicopathological characteristics of enrolled patients Characteristic Data -------------------------------------------------------- ----------------- Age (years)[*^a^*](#tb2fn1){ref-type="fn"} 68 ± 9 (43--86) Sex[*^b^*](#tb2fn2){ref-type="fn"}  Male 52 (71.2)  Female 21 (28.8) Hitologic grade of UTUC[*^b^*](#tb2fn2){ref-type="fn"}  High grade 58 (79.5)  Low grade 15 (20.5) Data are presented as mean (range) values. Data are presented as number (percentage) of patients. CTU imaging variables (tumour size, multiplicity, peritumoural infiltration, hydronephrosis grade, contrast enhancement value, presence of enlarged retroperitoneal lymph nodes) with respect to histological grade of UUCs are summarized in [Table 3](#t3){ref-type="table"}. The readers had excellent agreement for the other CT variables (*к* = 0.862 for hydronephrosis grade, intraclass correlation = 0.829 for tumour size, intraclass correlation = 0.892 for contrast enhancement value). In addition, there were good or moderate interobserver agreements for the other subjective assessments (*к* = 0.748 for multiplicity, *к* = 0.546 for periureteral infiltration). Tumour size was significantly larger in the high-grade group than in the low-grade group according to reader 1 (*p* = 0.028). Hydronephrosis grade was significantly higher in the high-grade group than in the low-grade group (*p* \< 0.001 for both readers). There was no significant difference in multiplicity, peritumoural infiltration, contrast enhancement value, or presence of enlarged retroperitoneal lymph nodes between the two groups. ###### Clinical characteristics of the enrolled patients according to histological grade Grade -------------------------------------- ----------------------- ---------------------- ---- ---------------------------------------- **Reader 1** Tumour size (mm) 39.7 (10--140) 23.3 (1--41) 0.028^[*b*](#tb3fn2){ref-type="fn"}^ Hydronephrosis grade \<0.001^[*c*](#tb3fn3){ref-type="fn"}^ 4 22 (37.9) 1 (6.7) 23 3 27 (46.6) 2 (13.3) 29 2 6 (10.3) 5 (33.3) 11 1 2 (3.4) 2 (13.3) 4 0 1 (1.7) 5 (33.3) 6 Enhancement value 56.4 (2--120) 51.2 (6--92) 0.508 Peritumoural infiltration 0.07^[*c*](#tb3fn3){ref-type="fn"}^ Present 17 (29.3) 1 (6.7) 18 Absent 41 (70.7) 14 (93.3) 55 Multiplicity 0.55^[*c*](#tb3fn3){ref-type="fn"}^ Present 7 (12.1) 1 (6.7) 8 Absent 51 (87.9) 14 (93.3) 65 Enlarged retroperitoneal lymph nodes 0.611^[*c*](#tb3fn3){ref-type="fn"}^ Present 11 (19.0) 2 (13.3) 13 Absent 47 (81.0) 13 (86.7) 57 Reader 2 Tumour size(mm) 43.10 (11--140) 34.50 30.14 (15--58) 27.50 0.234^[*b*](#tb3fn2){ref-type="fn"}^ Hydronephrosis grade \<0.001^[*c*](#tb3fn3){ref-type="fn"}^ 4 22 (37.9) 1 (6.7) 23 3 27 (46.6) 4 (26.7) 31 2 4 (7.0) 2 (13.3) 6 1 4 (7.0) 2 (13.3) 6 0 1 (1.7) 6 (40.0) 7 Enhancement value 55.5 (2--121) 58.9 (19--101) 0.793 Peritumoural infiltration 0.127^[*c*](#tb3fn3){ref-type="fn"}^ Present 8 (13.8) 0 (0.0) 8 Absent 50 (86.2) 15 (100.0) 65 Multiplicity 0.239^[*c*](#tb3fn3){ref-type="fn"}^ Present 5 (8.6) 0 (0.0) 5 Absent 53 (91.4) 15 (100.0) 68 Enlarged retroperitoneal lymph nodes 0.611^[*c*](#tb3fn3){ref-type="fn"}^ Present 11 (19.0) 2 (13.3) 13 Absent 47 (81.0) 13 (86.7) 57 Pathologic T stage \<0.001^[*c*](#tb3fn3){ref-type="fn"}^ Ta 4 (6.9) 6 (40.0) 10 T1 14 (24.1) 8 (53.3) 22 T2 11 (19.0) 0 (0.0) 11 T3 29 (50.0) 1 (6.7) 30 Data are presented as number (percentage) of patients. Mann--Whitney *U* test. Pearson's *Χ*^2^ test. Univariate logistic regression analysis revealed that hydronephrosis of Grade 3 or higher was significantly associated with high-grade tumour for both readers, and tumour size was significantly associated with high-grade tumour for reader 1. Multivariate logistic regression analysis using a backward selection method demonstrated that only hydronephrosis of Grade 3 or higher was a significant independent predictor of high-grade tumour for both readers ([Table 4](#t4){ref-type="table"}). Other CTU imaging variables, including tumour size, were omitted as independent variables in multivariate logistic regression analysis. In addition, there was no significant correlation between tumour size and hydronephrosis grade according to Spearman correlation analysis. ###### Results of the multivariate logistic regression analysis with backward selection of independent variables predictive of high-grade tumours Univariate logistic Multivariate logistic with variable selection -------------------------------------- ---------------------- ----------------------------------------------- -------------------- ------- **Reader 1** Tumour size (mm) 1.050 (1.006--1.096) 0.025 Grade of hydronephrosis 4 110 (5.83--2074.45) 0.002 72 (3.67--1411.89) 0.005 3 67.50 (5.09--893.63) 0.001 48 (3.48--661.60) 0.004 2 6 (0.51--69.75) 0.152 6 (0.47--75.34) 0.165 1 5 (0.27--91.51) 0.278 8 (0.31--206.37) 0.21 0 0.16 0.097 Enhancement value 1.01 (0.98--1.03) 0.536 Peritumoural infiltration 5.81 (0.71--47.69) 0.102 Multiplicity 1.92 (0.22--16.95) 0.557 Enlarged retroperitoneal lymph nodes 1.47 (0.29--7.53) 0.646 **Reader 2** Tumour size (mm) 1.027 (0.99--1.06) 0.146 Grade of hydronephrosis 4 126 (6.82--2328.09) 0.001 68 (3.46--1336.27) 0.005 3 40.5 (3.81--430.28) 0.002 24 (2.11--273.59) 0.009 2 12 (0.80--180.97) 0.073 16 (0.72--354.80) 0.08 1 12 (0.780--180.97) 0.073 16 (0.72--354.80) 0.08 0 0.01 Enhancement value 0.99 (0.97--1.02) 0.718 Peritumoural infiltration 5.22 (0.24--113.30) 0.293 Multiplicity 3.25 (0.12--81.44) 0.4737 Enlarged retroperitoneal lymph nodes 1.47 (0.29--7.53) 0.646 Values in parentheses are 95% confidence intervals. Pathologic T stage did not significantly correlate with peritumoral infiltration and hydronephrosis grade, respectively ([Table 5](#t5){ref-type="table"}). ###### Pathologic T stage correlation with periureteral infiltration and hydronephrosis grade -------------------- ------------------------------ ---------------------------------------- ----------- ------------------ ----------- ------------ ---------------------------------------------------------------------------- **Reader 1** **Peritumoral infiltration** **Hydronephrosis grade (*****n*****)** **Total** ***p*****value** Pathologic T stage Present Absent 0 1, 2 3, 4 0.194^[*a*](#tb5fn1){ref-type="fn"}^, 0.308^[*b*](#tb5fn2){ref-type="fn"}^ Ta-T1 5 (15.6) 27 (84.4) 4 (12.5) 9 (28.1) 19 (59.4) 32 (100.0) T2 4 (36.3) 7 (63.7) 0 (0.0) 4 (36.4) 7 (63.7) 11 (100.0) T3-4 9 (30.0) 21 (70.0) 2 (6.7) 2 (6.7) 26 (86.7) 30 (100.0) Total 18 (24.7) 55 (75.3) 6 (8.2) 15 (20.6) 52 (71.2) 73 (100.0) **Reader 2** **Peritumoral infiltration** **Hydronephrosisgrade (*n*)** **Total** ***p*value** Pathologic T Stage Present Absent 0 1, 2 3, 4 0.403^[*a*](#tb5fn1){ref-type="fn"}^, 0.173^[*b*](#tb5fn2){ref-type="fn"}^ Ta-T1 1 (3.1) 31 (96.9) 5 (15.6) 7 (21.9) 20 (62.5) 32 (100.0) T2 1 (9.0) 10 (91.0) 0 (0.0) 3 (27.3) 8 (72.7) 11 (100.0) T3-4 6 (20.0) 24 (80.0) 2 (6.7) 2 (6.7) 26 (86.7) 30 (100.0) Total 8 (11.0) 65 (89.0) 7 (9.7) 12 (16.6) 54 (73.7) 73 (100.0) -------------------- ------------------------------ ---------------------------------------- ----------- ------------------ ----------- ------------ ---------------------------------------------------------------------------- Data in parentheses are percentages. *p* value from the *Χ* ^2^ test for correlation of peritumoural intfiltration with pathologic T stage. *p* value from the *Χ* ^2^ test for correlation of hydronephrosis grade with pathologic T stage. ROC curve analysis showed that the best cut-off point of hydronephrosis grade was 2.5 for the prediction of high-grade tumour. The area under the curve (AUC) using the final model was 0.856 for reader 1 and 0.813 for reader 2 ([Figure 3](#f3){ref-type="fig"}). For clinical application in practice, the optimal cut-off grade of hydronephrosis was set at Grade 3, which corresponded to a prediction of high-grade UUC with an AUC of 0.830 and sensitivity and specificity of 88 and 79%, respectively, for reader 1, and AUC of 0.763 and sensitivity and specificity of 86 and 80%, respectively, for reader 2 ([Figure 4](#f4){ref-type="fig"}). {#f3} {#f4} DISCUSSION {#s4} ========== As with most other malignancies, the most accurate independent predictors of prognostic outcome in UUC are tumour stage and grade.^[@b14]^ However, preoperative tumour staging is difficult in UUC because the accuracy of imaging and endoscopic biopsy for T categorization remains unsatisfactory. It is not possible to differentiate a T1 lesion from T2 UUC on CTU, and it is also difficult to obtain representative muscularis tissue with ureteroscopic biopsy. Even though T3 lesions can be characterized by periureteral infiltration, current imaging modalities cannot reliably identify microscopic invasion. Periureteral infiltration, which represents the invasiveness of UUC on CT, can cause overstaging due to additional inflammatory changes, while understaging can occur due to microscopic invasion.^[@b15]^ In our study, there was no significant correlation between periureteral infiltration on CT and tumour grade. In addition, periureteral infiltration on CT did not significantly correlate with pathologic T stage. In clinical practice, tumour grade is a crucial factor in determining whether radical surgery or endoscopic conservative treatment is optimal for UUC, because accurate tumour staging is only available postoperatively based on the pathological evaluation of radical nephroureterectomy specimens. Ureteroscopic evaluation and biopsy definitively set up the diagnosis of UUC and provide fundamental information for risk stratification and clinical management. Several studies have reported that biopsy tumour grade accurately predicts surgical tumour grade in 78--91.6% of patients.^[@b16]--[@b18]^Contrary to these reports, it has been shown that ureteroscopic biopsy performance is inadequate in predicting final pathological grade.^[@b19],[@b20]^ Tumour grade is misinterpreted in more than one third of patients with conservatively managed UTUC,^[@b19]^ and 15% of high-grade tumours are underestimated as low-grade urothelial carcinoma.^[@b20]^ DW-MRI has shown potential as an biomarker in oncological imaging practice, and apparent diffusion coefficient (ADC) values obtained from DW-MRI may help predict tumour invasiveness and metastatic potential of UTUC.^[@b21]^ Some researchers report that high-grade UTUCs have significantly lower ADC values than low-grade tumours.^[@b10],[@b11]^ More recently, however, others have found no significant correlation between ADC value and histological grade of UTUC.^[@b12],[@b13]^ Furthermore, different imaging sequences, parameters, and MRI scanners can cause inconsistency in ADC measurement. Thus, our study aimed to determine whether CTU imaging features reproducible in routine practice could preoperatively predict the histological grade of UUC. There have been a number of studies demonstrating an association between hydronephrosis and advanced clinicopathological features and poor oncologic outcomes in UTUC.^[@b22]--[@b27]^Pyelocaliceal urothelial carcinomas usually do not result in urinary tract obstruction except in tumours involving the ureteropelvic junction. In contrast, UUCs are more likely to have hydronephrosis compared to pyelocaliceal urothelial carcinomas.^[@b4]^ A few studies that focused on UUC alone also reported a predictive role of hydronephrosis in advanced pathological features.^[@b5],[@b28]^ Cho et al found that 86% of patients with hydronephrosis of Grade 3 or 4 had an invasive tumour of T2 stage or greater.^[@b28]^ However, their research was based on various imaging assessments using CT, excretory urography, and renal ultrasonography. In our study, there was no significant correlation between hydronephrosis grade and pathologic T stage. Luo et al reported that hydronephrosis of Grade 2 or higher was associated with non-organ-confined disease,^[@b5]^ although their imaging review was not performed either in consensus or independently, and the specificity was limited to 37.3%. Chung et al assumed that hydronephrosis may cause outward expansion and longitudinal thinning of the already narrow ureter or renal pelvis wall, which may facilitate the seeding of cancer cells to regional or distant organs.^[@b22]^ Even so, the mechanism of the development of hydronephrosis and its relationship with tumour invasiveness is not fully understood.^[@b5]^ To the best of our knowledge, however, our study is the first evaluation of the association between hydronephrosis grade and tumour grade in pure UUC, and adequate diagnostic performance (sensitivity and specificity over 79%) was obtained at a cut-off point of hydronephrosis Grade 3 in the prediction of high-grade tumours. Cho et al reported that the tumour diameter of UUC correlated with pathological T stage and 80% of patients with a tumour diameter of 1.5 cm or greater had invasive UUC.^[@b28]^ In their study, however, tumour diameter was measured on axial CT images and was classified as less than 1.5 cm, greater than or equal to 1.5 cm but less than 2.5 cm, and 2.5 cm or greater. In our study, in which the largest tumour size was measured on multireconstructed images, tumour size did not independently predict tumour grade. In addition, our study showed no significant association between tumour size and hydronephrosis grade. At the time of diagnosis, patients with UTUC and a contralateral normal kidney can be classified as having low-risk UTUC or high-risk UTUC.^[@b29]^ Preoperative clinical factors associated with low-risk UTUC include low-grade ureteroscopic biopsy, low-grade cytology, tumour size \<1 cm, no invasive features on cross-sectional imaging, unifocal disease, and the availability of feasible close follow-up.^[@b29]^ According to the current European guidelines on UTUC,^[@b7]^ diagnostic ureteroscopy with biopsy should be performed in the preoperative assessment of UTUC. On the other hand, the routine use of ureteroscopy is not advocated for the confirmation of UTUC.^[@b30]^ Based on the results of our study, the need for ureteroscopy and biopsy may be obviated in patients with UUC causing hydronephrosis of Grade 3 or higher. The current study has limitations. First, the study population was relatively small due to the rarity of UUC, and because the study was conducted retrospectively at a single institution, the possibility of selection bias should be considered. Prospective multicentre studies with larger sample size are needed to validate our results. Second, the direct imaging-pathological correlation was not obtained in tumour size assessed on CTU. Consequently, tumour size could have been overestimated if there was concomitant inflammation. In conclusion, high-grade hydronephrosis on preoperative CTU was significantly associated with high-grade UUC. The results of the current study may help develop algorithms for risk stratification of patients with pure UUC. Radical surgical treatment should be considered in patients with UUC causing hydronephrosis of Grade 3 or higher regardless of tumour size and absence of peritumoural infiltration on CTU.

Introduction {#S1} ============ Despite significant improvements in outcome,([@R1]--[@R3]) relapse remains the leading cause of treatment failure for children with acute lymphoblastic leukemia (ALL) and occurred in 11 to 36% of those with high-risk B-precursor ALL.([@R4]--[@R10]) Mechanisms by which genomic variation influence relapse risk could involve somatically acquired mutations or inherited genetic variations, which could affect intrinsic resistance to chemotherapy([@R11]--[@R13]) or host pharmacokinetics of anti-leukemic agents.([@R14]--[@R16]) Some studies report that black and Hispanic children with ALL have inferior outcomes to non-Hispanic white children.([@R17]--[@R21]) Reasons for these differences are likely multifactorial, including differences in treatment adherence and access to therapy,([@R22]--[@R24]) in the incidence of favorable and unfavorable presenting features and cytogenetics,([@R25]--[@R27]) and in the frequency of genetic variants affecting pharmacokinetics and pharmacodynamics of antileukemic agents which segregate with ancestry.([@R28]) It remains uncertain whether racial disparities persist with modern intensive ALL regimens. We performed a genome wide association study (GWAS) in a large cohort of children with high-risk B-ALL to identify inherited genetic variations associated with relapse. We performed an analysis adjusting for both treatment and ancestry to identify single nucleotide polymorphisms (SNPs) which increased risk across ancestries (ancestry-agnostic SNPs). Because racial disparities in relapse persisted in this trial, we also performed analyses within each of the three largest ancestral groups (white, black, Hispanic) to identify ancestry-specific variations associated with relapse. We also interrogated relapse SNPs for associations with risk of central nervous system (CNS) relapse, relapse among patients randomized to receive either escalating-dose methotrexate and asparaginase (i.e., Capizzi regimen) or high-dose methotrexate during the first interim maintenance (IM1), and for associations with the pharmacokinetics of antileukemic agents or the intrinsic sensitivity of leukemia cells to chemotherapy. Finally, to assess robustness of relapse SNPs across different therapies, we tested for replication in an independent cohort. Methods {#S2} ======= Patients and treatment {#S3} ---------------------- For the discovery cohort, germline DNA was obtained at remission in children and young adults with newly diagnosed B-precursor ALL enrolled on COG AALL0232 (NCT00075725, <https://clinicaltrials.gov/ct2/show/NCT00075725>).([@R8]) This protocol involved a 2×2 factorial randomization for induction steroid (prednisone ×28 days vs. dexamethasone ×14 days) and interim maintenance 1 regimen (Capizzi escalating-dose methotrexate with pegylated-asparaginase vs. high-dose methotrexate). Exclusion criteria are described in [Figure 1](#F1){ref-type="fig"} and the [Supplementary Methods](#SD1){ref-type="supplementary-material"}. The replication cohort comprised children treated on prior generation protocols who would have met the eligibility criteria of AALL0232 ([Supplementary Methods and Supplementary Table 1](#SD1){ref-type="supplementary-material"}). All studies were approved by the institutional review boards of participating institutions, and all patients and/or guardians provided age appropriate consent/assent in accordance with the Declaration of Helsinki. Genotyping and genetic ancestry {#S4} ------------------------------- Genotyping and genetic imputation was performed as described in the [Supplementary Methods](#SD1){ref-type="supplementary-material"}. Genetic ancestry was defined using STRUCTURE v2.2.3.([@R29]) For categorization of patients into discrete ancestral groups, individuals were classified based on inferred genetic ancestry as white \[Northern European (CEU) \>90%\], black \[West African (YRI) \>70%\], Hispanic \[Native American([@R30]) \>10% and Native American greater than West African\], or Other, including Asian \[East Asian (CHB/JPT) \>90%\]. Quality control steps for both patients and SNPs are detailed in the [Supplementary Methods](#SD1){ref-type="supplementary-material"}. Identification of relapse associated SNPs {#S5} ----------------------------------------- The approaches to perform GWASs for relapse are detailed in the [Supplementary Methods](#SD1){ref-type="supplementary-material"}. GWASs were performed to identify SNPs using an ancestry-agnostic ([Supplementary Table 2 and Supplementary Figure 1](#SD1){ref-type="supplementary-material"}) and an ancestry-specific approach ([Supplementary Figures 2a--c](#SD1){ref-type="supplementary-material"}). Treatment arm and site specific annotation of relapse SNPs {#S6} ---------------------------------------------------------- SNPs associated with relapse were further characterized in subsets of patients based on their IM1 randomization (the Capizzi arm with escalating-dose methotrexate plus pegylated-asparaginase vs. the high-dose methotrexate arm) while adjusting for induction randomization, rapid early response, and ancestry as categorical variables. Additionally, SNPs were tested for their association with CNS relapse (isolated or combined with other sites), with isolated hematologic or other extramedullary relapse treated as competing risks. Significant association thresholds for all analyses were determined by profile information criteria (Ip),([@R31]) which balances false positives and negatives while addressing the effects of multiple testing. Association with orthogonal pharmacologic data {#S7} ---------------------------------------------- SNPs associated with relapse (ancestry-specific or ancestry-agnostic) were evaluated for association with drug resistance in HapMap cells lines (prednisone, asparaginase, mercaptopurine, methotrexate polyglutamate accumulation), primary ALL cells from newly diagnosed patients (prednisone, vincristine, mercaptopurine, asparaginase, *in vivo* leukocyte count decrease following methotrexate), or for association with increased drug clearance (asparaginase allergy, methotrexate clearance, dexamethasone clearance), as described in the [Supplementary Methods](#SD1){ref-type="supplementary-material"}. SNPs were considered supported by orthogonal data if the risk allele for relapse was associated (at P\<0.05) with *in vitro* drug resistance, decreased methotrexate polyglutamate accumulation, smaller leukocyte decrease after methotrexate, more rapid drug clearance, or greater incidence of asparaginase allergy. Evaluation of relapse-associated SNPs in replication cohort {#S8} ----------------------------------------------------------- Relapse-associated SNPs were evaluated in an independent replication cohort (n=719) for their association with relapse using a Cox proportional hazard regression, with patients censored at the time of competing events (i.e. remission death, second malignancy) or last follow-up and adjusting for treatment categorized into 6 groups ([Supplementary Table 2](#SD1){ref-type="supplementary-material"}).([@R5], [@R9], [@R32]) AALL0232 ancestry-agnostic SNPs were evaluated in all patients while adjusting for treatment and ancestry. AALL0232 ancestry-specific SNPs were evaluated in the same ancestry subset of the replication cohort while adjusting for treatment and, in blacks and Hispanics, percent ancestry. The replication cohort SNPs were evaluable if they passed quality control steps as described for the discovery cohort ([Supplementary Methods](#SD1){ref-type="supplementary-material"}). Differences in genotyping platforms between the discovery and replication cohorts, as well as the smaller size of the replication cohort, resulted in 595 of the 1,017 relapse SNPs from the discovery cohort being evaluable in the replication cohort. Validated SNPs were those associated with relapse at P\<0.05 and with identical risk alleles. Quantitative contribution of SNPs to ancestral differences in relapse {#S9} --------------------------------------------------------------------- To identify SNPs which most contributed to ancestry-associated differences in relapse risk, a classification and regression tree analysis was performed separately in blacks and Hispanics considering treatment arm and validated ancestry-agnostic and ancestry-specific SNPs as potential branches. Branches were limited to two levels with each new branch needing to contain at least 20% of the initial ancestral patient group (representing \~1% or at least 22 patients from the discovery cohort for the smallest group, those with black ancestry). The impact of these SNPs on the risk of relapse associated with black or Hispanic ancestry was then evaluated in a competing risk regression model of relapse including the SNPs, treatment, and ancestry. Statistical analysis {#S10} -------------------- Statistical and bioinformatics analyses were performed using R versions 3.2.2, including the "survival", "cmprsk", "rpart", and "forestplot" packages. Association studies of orthogonal phenotypes were performed either in R or PLINK version 1.07. Results {#S11} ======= Patient Characteristics {#S12} ----------------------- Of 3,084 children and young adults enrolled on AALL0232, germline genotype and relapse data were available for 2,652, and 2,225 were included in the GWAS for relapse ([Figure 1](#F1){ref-type="fig"}). To identify covariates to include in the GWAS, we examined the importance of treatment group and ancestry on relapse risk. Consistent with findings in the entire randomized cohort,([@R8]) patients treated with Capizzi-methotrexate had a higher relapse risk than those treated with high-dose methotrexate ([Supplementary Table 3](#SD1){ref-type="supplementary-material"}). Because patients with slow early response did not differ by their induction steroid assignment but did differ by IM1 randomization, patients with slow early response were combined for multivariable and GWAS analyses ([Supplementary Table 2](#SD1){ref-type="supplementary-material"}). Blacks \[P=2.66×10^−4^, hazard ratio (HR)=2.31\] and Hispanics (P=2.17×10^−5^, HR=1.77) had an increased relapse risk compared to whites ([Supplementary Table 3](#SD1){ref-type="supplementary-material"}). The effects of ancestry and treatment groups remained significant in multivariate analyses ([Supplementary Table 3](#SD1){ref-type="supplementary-material"}, [Figure 2](#F2){ref-type="fig"}). Blacks and Hispanics also had a higher risk of any CNS relapse than whites (P=0.016, HR=2.54 for blacks; P=0.0018, HR=2.08 for Hispanics). Association of SNPs with relapse {#S13} -------------------------------- Following quality control steps, 11,180,806 SNPs were evaluated for their association with relapse. A total of 302 SNPs representing 175 unique genetic loci (LD blocks) were associated with relapse in an analysis adjusting for both treatment and percent genetic ancestry (i.e. their association with relapse was "agnostic" to ancestry; [Supplementary Table 4, Supplementary Figure 4](#SD1){ref-type="supplementary-material"}). An additional 715 SNPs representing 424 unique genetic loci were associated with relapse in ancestry-specific analyses, with 280 SNPs (179 loci) associated with relapse in Hispanics, 258 SNPs (167 loci) in blacks, 173 SNPs (72 loci) in whites, 2 SNPs (2 loci) in both blacks and whites, and 2 SNPs (1 locus) in both blacks and Hispanics ([Supplementary Tables 5--7, Supplementary Figures 3, 5--7](#SD1){ref-type="supplementary-material"}). Of the 1,017 relapse SNPs, 192 were associated with relapse in patients treated on the Capizzi arm, 186 in patients treated on the high-dose methotrexate arm, and 18 in both treatment groups; 621 SNPs were not associated with relapse in either group alone but were associated with relapse in the combined cohort ([Supplementary Tables 4--7, Supplementary Figures 4--7](#SD1){ref-type="supplementary-material"}). Of the 302 ancestry-agnostic SNPs, 54 were also associated with an increased risk of CNS relapse ([Supplementary Table 4, Supplementary Figure 4](#SD1){ref-type="supplementary-material"}). Of these, 25 were associated with increased CNS relapse in patients treated on the Capizzi arm, 14 in patients on the high-dose methotrexate arm, and 4 in patients on both arms. Because of the association between ancestry and CNS relapse risk, we evaluated ancestry-specific SNPs for their association with CNS relapse and identified 18 SNPs associated with increased CNS relapse risk in whites, 38 SNPs in blacks, and 52 SNPs in Hispanics ([Supplementary Tables 5--7, Supplementary Figures 5--7](#SD1){ref-type="supplementary-material"}). Because of the importance of minimal residual disease (MRD) in defining high-risk patients,([@R33]) we also evaluated relapse SNPs for their adverse impact in the 1,931 patients with end of induction (day 29) MRD less than 0.1%. 617 SNPs remained significant at the previously defined significance threshold, including 209 ancestry-agnostic SNPs ([Supplementary Tables 4--7, Supplementary Figures 4--7](#SD1){ref-type="supplementary-material"}). Association of relapse SNPs with orthogonal pharmacologic data {#S14} -------------------------------------------------------------- To explore possible mechanisms underlying the 1,017 SNPs associated with relapse, we tested for their association with orthogonal phenotypes including *in vitro* resistance to chemotherapy, decreased response to methotrexate *in vivo*, increased chemotherapeutic drug clearance *in vivo*, and asparaginase allergy *in vivo.* Of the 302 ancestry-agnostic SNPs, 54 were associated with one resistance/clearance phenotype and 10 were associated with more than one such phenotype ([Supplementary Table 4](#SD1){ref-type="supplementary-material"}). Of the 715 ancestry-specific SNPs, 128 were associated with one resistance/clearance phenotype and 32 with more than one phenotype ([Supplementary Tables 5--7](#SD1){ref-type="supplementary-material"}). 36 of the 162 relapse SNPs associated with CNS relapse were associated with at least one resistance/clearance phenotype. Of the 54 relapse SNPs associated with intrinsic leukemic asparaginase resistance (N=24 SNPs) or asparaginase allergy (N=30 SNPs), 20 were associated with relapse in the Capizzi arms, which included additional doses of asparaginase, compared to only eight associated with relapse in the high-dose methotrexate arms (Fisher's P=0.015). In contrast, relapse SNPs associated with decreased intracellular methotrexate polyglutamates (N=15 SNPs), rapid methotrexate clearance (N=19 SNPs), or decreased *in vivo* response to methotrexate (N=42 SNPs) were balanced equally in their association across IM randomization arm (19 of 76 SNPs significant in the Capizzi arm, 13 of 76 significant in the high-dose arm; Fisher's P=0.32). Relapse SNPs were associated with both pharmacokinetic and pharmacodynamic phenotypes. For example, the relapse SNP rs10496350 was associated with asparaginase allergy (which results in decreased exposure to asparaginase), and patients carrying at least one copy of the C risk allele had a higher (P adjusted for treatment and ancestry =2.94×10^−5^) five-year cumulative incidence of relapse (CIR, 37.5%) than did patients with GG genotype (five-year CIR 13.3%) as well as double the risk (P=0.006) of allergy (23.3% for CC or CG genotype vs. 10.8% for GG genotype, [Figure 3](#F3){ref-type="fig"}). Relapse SNPs were also associated with resistance to chemotherapeutic agents: for example, rs743535 (intronic within *CYP2E1*) was associated with both vincristine resistance (median lethal concentration for 50% of cells 0.27 μM for GG genotype vs. 2 μM for GA/AA genotypes, P=0.016) and increased five-year CIR (12% for the GG genotype vs. 20.6% for the GA or AA genotypes, P=2.42×10^−4^, [Figure 4](#F4){ref-type="fig"}). Replication cohort {#S15} ------------------ Of the 1,017 relapse SNPs, 595 were evaluable in the independent replication cohort of 719 patients and 32 replicated (representing 19 loci). Of 138 evaluable ancestry-agnostic SNPs, seven were associated with increased relapse in the replication cohort. 25 of the 457 evaluable ancestry-specific SNPs were also associated with increased relapse in the replication cohort in the same ancestry as was identified in the AALL0232 cohort, including three which increased relapse risk in blacks, 18 in Hispanics, and four in whites ([Table 1](#T1){ref-type="table"}). Of the 32 replicated SNPs, four were associated with an increased relapse in patients treated with high-dose methotrexate, two in patients treated with Capizzi-methotrexate, and two in both cohorts. Of the seven replicated ancestry-agnostic SNPs, four were associated with at least one unfavorable pharmacological phenotype: rs41530849 in *PTPN14* with both rapid methotrexate clearance and *in vitro* asparaginase resistance, rs743535 in *CYP2E1* with *in vitro* vincristine resistance, intergenic SNP rs2463380 with rapid methotrexate clearance, and the missense SNP rs16843643 in *FARP2* with a diminished *in vivo* response to high-dose methotrexate. Additionally, four ancestry-agnostic SNPs and 12 Hispanic-specific SNPs that were associated with CNS relapse in the discovery cohort were replicated in the independent replication cohort, and 23 SNPs were significant among MRD negative patients ([Table 1](#T1){ref-type="table"}). SNP contribution to excess relapse risk in black and Hispanic patients {#S16} ---------------------------------------------------------------------- Using classification and regression trees, we identified two SNPs in blacks (rs4710143 and rs16843643), and in Hispanics (rs9325870 and rs743535) most contributing to their excess relapse risk. In a multivariate model, these four SNPs attenuated the adverse risk associated with black (P=0.79) and Hispanic (P=0.065) ancestry group status ([Figure 5a](#F5){ref-type="fig"}). Additionally, ancestry did not improve the ability to predict relapse if SNPs and treatment group were already known (ANOVA P=0.19 comparing a model with treatment and SNPs as covariates to a model with treatment, SNPs, and ancestry). Patients carrying at least one risk allele for any of the four SNPs had higher relapse than did patients without any risk alleles, regardless of their ancestry ([Figure 5b](#F5){ref-type="fig"}). These variants were less prevalent in whites, with the average white patient carrying 0.21 risk alleles (of a possible eight, range in whites 0--2) compared to a mean of 1.28 in black patients (range 0--5), 0.79 in Hispanics (range 0--4), and 0.63 in patients of other ancestry (range 0--4; Mann-Whitney P\<1×10^−15^). Discussion {#S17} ========== Relapse in high-risk B-ALL remains a significant problem, and most patients who relapse do not survive. Although evaluation of early treatment response and MRD identifies many patients at high risk for relapse, many patients who relapse do not carry these adverse features.([@R33], [@R34]) Further identification of adverse biologic features is needed to allow further refinements in therapy. In this study, we focused on three primary implications of this genetic analysis: whether host genetic variation explained ancestry-related differences in relapse, whether the importance of genetic variation differed by major treatment arms, and how genetic variations were replicated for orthogonal pharmacologic phenotypes and in an independent ALL cohort. In this GWAS, we identified 1,017 SNPs associated with increased relapse risk in children with high-risk B-ALL. We identified both SNPs associated with relapse risk regardless of patient ancestry (ancestry-agnostic) as well as SNPs associated with relapse in an ancestry-specific fashion. Of these relapse SNPs, 7 ancestry-agnostic and 25 ancestry-specific SNPs were also associated with an increased relapse risk in an independent replication cohort ([Table 1](#T1){ref-type="table"}). Importantly, we identified genetic variants associated with increased relapse risk in an ancestry-specific manner across two generations of B-ALL protocols. The identified SNPs contribute to the higher risk of relapse in blacks and Hispanics but also identify patients in each ancestral group at high risk of relapse. Using only four SNPs (rs4710143, rs16843643, rs9325870, and rs743535), we identified 73% of blacks and 57% of Hispanics at high-risk of relapse ([Figure 5b](#F5){ref-type="fig"}). These SNPs were also associated with relapse risk in whites and patients of other ancestry. However, more than 50% of blacks and Hispanics carry at least one risk allele in these SNPs compared to 20% of whites, suggesting the increased relapse risk attributable to these SNPs is disproportionately distributed to blacks and Hispanics, simply on the basis of racial differences in allele frequency. The addition of ancestry group to a model including these SNPs and treatment group failed to improve the model (ANOVA P=0.19) suggesting these SNPs attenuate the adverse impact of ancestry on relapse. These data mirror findings in other malignant([@R35], [@R36]) and non-malignant diseases([@R37]--[@R42]) in which variants strongly associated with ancestry may be the cause of discrepant disease outcomes in different ancestral populations. Such variants offer the opportunity for therapy modification and risk stratification when their effects are stable across multiple settings, as are the replicated ancestry-specific variants identified in this study ([Table 1](#T1){ref-type="table"}). One of the principles of discovery research in pharmacogenomics is that the variants identified in any study will be influenced by the therapy that has been given. Because the randomly assigned methotrexate treatment arm had a significant effect on treatment outcome in AALL0232, we had a unique opportunity to test whether some genomic variants associated with relapse were more important in those receiving one treatment arm (high-dose methotrexate) versus the other (Capizzi methotrexate plus asparaginase). Interestingly, the SNPs directly associated with methotrexate pharmacology did not differentially distribute between the two treatment arms (Fisher's P=0.32), but relapse SNPs associated with asparaginase resistance or asparaginase allergy did cluster in the Capizzi arm (Fisher's P=0.015). Those in the Capizzi arm received more asparaginase but less methotrexate than those in the high-dose methotrexate arm. The association with asparaginase resistance/allergy in the Capizzi arm suggests that asparaginase exposure was more critical to preventing relapse among the patients whose methotrexate exposure was low (Capizzi treatment), and that treatment with high-dose methotrexate diminishes the importance of maximizing asparaginase. Therapeutic and patient differences may also explain differences in the SNPs associated with relapse in this cohort compared to prior analyses. In prior GWAS of ALL relapse risk and MRD,([@R43], [@R44]) the majority of patients were NCI standard-risk, in contrast to the high-risk population studied here. Moreover, all patients in the discovery cohort of this study also received delayed intensification and MRD-directed therapy intensification, whereas many patients in the prior GWASs([@R43], [@R44]) did not. In a review of the SNPs previously associated with relapse or MRD,([@R43], [@R44]) we identified five (rs35229355, rs7517671, rs10883699, rs7350429, and rs6773449) that associated with relapse (P\<0.05) after adjusting for both treatment and ancestry in the current discovery cohort. However, these SNPs did not reach the Ip selected P value threshold, nor were they replicated at least 20 times during iterative resampling. This finding highlights the importance of population and therapeutic differences on the association of pharmacogenomic variants and outcome. It is encouraging that many of the SNPs identified in the current GWAS were associated with relapse among patients treated on both the high-dose methotrexate and the Capizzi escalating-methotrexate/asparaginase arms, suggesting that some of these variants may be prognostic across therapies. The analysis of relapse SNPs' association with orthogonal pharmacologic phenotypes suggests mechanisms through which some relapse SNPs may be exerting their effects on relapse risk. Relapse SNPs were associated with both pharmacokinetic and pharmacodynamic phenotypes. For example, rs6786341 (an intronic variant in lactoferrin) was associated with more rapid methotrexate clearance, a phenotype which has previously been associated with decreased methotrexate polyglutamate accumulation and increased relapse risk.([@R45], [@R46]) The rs743535 variant in *CYP2E1* was associated with resistance to vincristine ([Figure 4](#F4){ref-type="fig"}). Variants in this gene have previously been implicated in inferior survival in non-Hodgkin's lymphoma([@R47]) and non-small cell lung cancer,([@R48]) potentially due to resistance to chemotherapeutic agents used in those diseases. Variants near *LZTS1*, which include promoter and enhancer marks in neural tissues,([@R49]) were associated with CNS relapse in Hispanics. Suppression of this gene has previously been implicated in metastatic potential in multiple solid tumors,([@R50]--[@R52]) suggesting these variants may alter leukemic trafficing into the CNS, thereby altering CNS relapse risk. Other identified CNS relapse SNPs likely contribute to CNS relapse through alterations in leukemic drug resistance or rapid drug clearance, as 36 of 162 CNS relapse SNPs were also associated with pharmakokinetic or drug resistance phenotypes. We identified several novel inherited risk variants for relapse in a large population of children with high-risk B-precursor ALL. Several of these are associated with the increased relapse risk specific to black and Hispanic ancestry and may contribute to the adverse outcomes attributed to "race." Many of these variants are associated with "inherited" leukemic resistance or rapid clearance of chemotherapy. These findings may allow personalized therapy to further improve outcomes for children with high-risk B-ALL. Supplementary Material {#S18} ====================== **Funding/Support** The work was supported by the National Institutes of Health \[grant numbers GM 92666, GM 115279, CA142665, CA 21765, CA 36401, CA98543 (COG Chair's grant), CA98413 (COG Statistical Center), CA114766 (COG Specimen Banking), U01-HG04603, RC2- GM092618, R01-LM010685, 5T32-GM007569\]; Leukemia Lymphoma Society (grant number 6168); and by the American Lebanese Syrian Associated Charities. **Role of funding source** The funders had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication. **Original Data Statement** Drs. Mary Relling and Seth Karol had full access to all data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. **Authors' contributions** MVR and JJY contributed to the conception and design of the study. EL, LBR, CAF, JRM, SWP, RJA, ELL, BD, SJ, C-HP, EAR, NJW, WLC, SPH, MLL, MD, WEE, JJY, and MVR contributed to the provision of study materials, patient recruitment, or acquisition of data. SEK, CC, XC, and MVR contributed to data analysis and interpretation. All authors contributed to the drafting and reviewing of the manuscript and gave their final approval to submit for publication. **Conflicts of interest** The authors declare no competing financial interests. **Data Availability:** Detailed information on the primary clinical trial (COG AALL0232) for the discovery cohort is available from: <https://clinicaltrials.gov/ct2/show/results/NCT00075725?term=0232&rank=3> {#F1} ![Association of non-white genetic ancestry with increased relapse risk\ Non-whites had an increased risk of relapse in the discovery cohort. The five-year cumulative incidence of relapse was higher in blacks \[23.7%, 95% confidence interval (CI) 14.7--32.7%, P=2.27×10^−4^, HR=2.32\] and Hispanics (19.3%, 95% CI 15.7--22.9%, P=8.23×10^−5^, HR=1.7) than whites (10.3%, 95% CI 8.9--12.8%). P values are adjusted for treatment.\ White: \>90% CEU; black: \>70% YRI; Hispanic: \>10% Native American and Native American \>YRI](nihms842426f2){#F2} {#F3} {#F4} ###### Relapse SNPs attenuate the adverse impact of black and Hispanic ancestry a: **Forest plot of relapse risk comparing multivariable models with and without four relapse SNPs** b: **Presence of a risk allele in any of the four SNPs confers high relapse risk regardless of ancestry** Risk alleles in any of four SNPs (rs4710143, rs16843643, rs9325870, and rs743535) confer increased relapse risk regardless of ancestry. (A) In multivariate models, these SNPs largely attenuate the adverse effect of black or Hispanic ancestry, while leaving unchanged the association between treatment arm and relapse. Treatment arms are described in [Supplementary Table 2](#SD1){ref-type="supplementary-material"}: For the rapid early response patients, Dex/Capizzi, Pred/Capizzi, Dex/HD, Pred/HD refer to the induction steroid (dex = dexamethasone, pred = prednisone) and the interim maintenance (Capizzi=escalating dose methotrexate plus asparaginase, HD = high-dose methotrexate). For the slow early response patients (SER), induction steroid groups were combined. (Hazard ratio (HR) from model without SNPs shown in blue, models with SNPs shown in red). \(B\) Whites, blacks, and Hispanics carrying risk alleles for any of these SNPs (dashed lines) have higher five-year relapse risks than do those without any risk alleles (solid lines) \[15.3% vs. 9.7% (P=0.025) for whites, 32.3% vs. 0% (P=1.28×10^−4^) for blacks, and 25.5% vs. 10.7% (P=3.72×10^−6^) for Hispanics\]. P values are adjusted for treatment.   ###### SNPs associated with relapse in discovery (n=2,225) and replication (n=719) cohorts ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- rsID Gene Risk allele RAF P: discovery cohort Hazard Ratio (95% CI): discovery cohort P: replication cohort Ancestry Additional phenotypes -------------------- ----------- ------------- ------- --------------------- ----------------------------------------- ----------------------- ---------- ------------------------------------ rs41530849 *PTPN14* T 0.006 4.26E-06 3.87\ 0.019 agnostic HD arm, MTX clearance, PPL ASP (2.17--6.89) **rs10205940** A 0.224 6.85E-06 1.42\ 0.044 agnostic HD arm, Capizzi arm, CNS (1.22--1.66) **chr23: 9863426** *SHROOM2* T 0.008 1.04E-05 2.45\ 0.049 agnostic HD arm, Capizzi arm, CNS (1.64--3.64) rs2463380 G 0.22 3.98E-05 1.52\ 0.045 agnostic HD arm, CNS, MTX clearance (1.24--1.86) **rs2710418** *NELL2* T 0.031 4.99E-05 1.98\ 0.021 agnostic HD arm (1.42--2.76) rs743535 *CYP2E1* A 0.124 5.00E-05 1.54\ 0.045 agnostic PPL Vinc (1.25--1.9) **rs16843643** *FARP2* C 0.012 0.000226 2.95\ 0.031 agnostic Capizzi arm, CNS, MTX WBC response (1.66--5.25) **rs775491** *BEST3* A 0.304 0.000265 1.61\ 0.0086 white (1.24--2.07) rs156008 *PCSK1* A 0.158 0.000297 1.65\ 0.024 white (1.26--2.16) **rs4710143** *RNASET2* G 0.074 0.000579 4.92\ 0.014 black Capizzi arm (1.98--12.2) **rs202408** C 0.144 0.000789 3.56\ 0.021 black (1.7--7.49) **rs7860525** T 0.134 0.00175 2.79\ 0.016 black (1.47--5.31) **rs9325870** *LZTS1* C 0.205 1.84E-05 2\ 0.036 Hispanic CNS (1.46--2.75) rs16999479 *DSCAM* G 0.016 0.000219 4.02\ 0.046 Hispanic CNS (1.92--8.42) **rs141707566** *GRIN2A* C 0.014 0.000289 2.76\ 0.037 Hispanic HD arm (1.59--4.77) **rs12535024** *DDC* C 0.181 0.00103 1.76\ 0.0499 Hispanic (1.26--2.47) **rs6786341** *LTF* T 0.012 0.00173 4.14\ 0.038 Hispanic MTX clearance (1.7--10.1) rs16945138 *DNAH9* T 0.007 0.00186 7.5\ 0.014 Hispanic (2.11--26.7) rs6651255 *GSDMC* C 0.425 0.00222 1.59\ 0.0029 Hispanic (1.18--2.13) ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- RAF: Risk allele frequency; CI: confidence interval Characteristics of validated SNPs are shown for the discovery cohort, with one SNP for each locus shown (SNPs removed through LD pruning are shown in [Supplementary Tables 4--7](#SD1){ref-type="supplementary-material"}). Bolded SNPs were significant at the Ip determined significance threshold when evaluated among patients who were end-induction minimal residual disease negative. SNPs are ordered by ancestry of discovery, with SNPs associated with relapse while adjusting for both treatment and ancestry (i.e. "ancestry agnostic") labeled as agnostic and ancestry-specific SNPs labeled with their associated ancestry group. Additional phenotypes include: association with relapse among patients treated on either first interim maintenance arm \[Capizzi arm, HD (high-dose methotrexate) arm\], association with CNS relapse (CNS), as well as association with *in vitro* resistance among primary patient lymphoblasts to asparaginase (PPL ASP) or vincristine (PPL Vinc), more rapid methotrexate clearance (MTX clearance), or diminished white blood cell decrease after *in vivo* methotrexate treatment (MTX WBC response).

"Introduction {#S0001}\n============\n\nThe rotator cuff tear is the most common tendinopathy in hum(...TRUNCATED)

"1. Introduction {#sec1-membranes-08-00069}\n===============\n\nPolymer electrolytes are regarded as(...TRUNCATED)

"1. Introduction {#sec1}\n===============\n\nAcute respiratory distress syndrome (ARDS) incorporates(...TRUNCATED)