Agrobacterium-mediated transformation in Chinese cabbage (Brassica rapa). For the transformation, two plant expression vectors containing genes encoding for the EpCAM recombinant protein along with the fragment crystallizable (Fc) region of immunoglobulin M (IgM) and Joining (J)-chain tagged with the KDEL endoplasmic reticulum retention motif (J-chain K) were constructed. The vectors were successfully transformed and expressed in the Chinese cabbage individually using Agrobacterium. The transgenic Chinese cabbages were screened using genomic polymerase chain reaction (PCR) in T0 transgenic plant lines generated from both transformants. Similarly, the immunoblot analysis revealed the expression of recombinant proteins in the transformants. Further, the T1 transgenic plants were generated by selfing the transgenic plants (T0) carrying EpCAM–IgM Fc and J-chain K proteins, respectively. Subsequently, the T1 plants generated from EpCAM–IgM Fc and J-chain K transformants were crossed to generate F1 plants carrying both transgenes. The presence of both transgenes was validated using PCR in the F1 plants. In addition, the expression of Chinese cabbage-derived EpCAM–IgM Fc × J-chain K was evaluated using immunoblot and ELISA analyses in the F1 plants. The outcomes of the present study can be utilized for the development of a potential anti-cancer vaccine candidate using Chinese cabbage.The epithelial cell adhesion molecule (EpCAM) is a tumor-associated antigen and a potential target for tumor vaccine. The EpCAM is a cell-surface glycoprotein highly expressed in colorectal carcinomas. The objective of the present study is to develop an edible vaccine system through  Plant expression systems, such as those in tomatoes ,2, carroBeta vulgaris subsp. Vulgaris), Nicotiana tabaccum, and N. Benthamiana [1q complement-dependent cytotoxicity [Among the valuable recombinant proteins expressed in plants, an epithelial cell adhesion molecule (EpCAM) is a tumor-associated antigen (TAA) that has been shown to be highly expressed on cancer cells over the last several decades and expressed using diverse heterologous expression including plants such as Swiss chard  analysis of the genomic DNA was conducted to confirm the presence of transgenes in transgenic plants (EpCAM–IgM Fc) and (J-chain K) randomly selected Chinese cabbage regenerants obtained by transforming each vector A. All teExpression of the EpCAM–IgM Fc protein in leaf tissues of transgenic plants was analyzed using Western blotting . In the 0 transgenic line #2, with the highest expression of EpCAM–IgM Fc, was self-crossed to generate a T1 line #2 (1 plants (1 plants obtained from the transformation using the plant expression vector carrying the gene encoding EpCAM–IgM Fc had the expected amplified bands at 837 bp (EpCAM), 1053 bp (IgM–Fc), and 1023bp (HTP) (1. Transgenic plants in T1 carrying the J-chain K transgene were randomly selected for PCR testing (1 plants obtained from selfing had the expected amplified band at 543 bp (J-chain K) (The T line #2 . PCR was1 plants . All tesbp (HTP) A. In the testing B. All ofchain K) B.1 transgenic lines, expression of the EpCAM–IgM Fc protein was confirmed by Western blotting , IgM Fc (1053 bp), and J-chain K (543 bp), except for M209-8, in which only the J-chain K gene was detected without the EpCAM and IgM Fc genes  showed the 75 kDa size protein band. Furthermore, in the EpCAM detection immunoblot, all tested F1 transgenic lines (#1–9) showed the 75 kDa size protein band , whereas EpCAM–IgG FcT had almost no absorbance signal (0.02) , whereas EpCAM–IgG FcT showed an absorbance signal (0.165) (Enzyme-linked immunosorbent assay (ELISA) was conducted for the recognition of Chinese cabbage-derived EpCAM–IgM Fcantibody . In the l (0.02) . However (0.165) . In the  (0.165) .0 and T1 Chinese cabbage, including F1 plants from a crossing between EpCAM–IgM Fc and J-chain K T1 transgenic plants. Transgenic Chinese cabbage expressing EpCAM–IgM Fc and J-chain K were obtained from Agrobacterium-mediated transformation. PCR analysis revealed that all tested T0 transgenic plants carrying EpCAM–IgM Fc and J-chain K transgenes had PCR bands of both transgenes, indicating that these genes were properly embedded in the plant genome. Western blotting showed variable expression of the EpCAM–IgM Fc transgene in the T0 transgenic plants. In EpCAM–IgM Fc, among the tested T0 transgenic plants, transgenic line #2 had the highest level of EpCAM–IgM Fc. Thus, EpCAM–IgM Fc transgenic line #2 was selected for self-crossing to generate T1 plants. Regarding the J-chain K, according to Western blot analysis, its expression was not observed in the T0 transgenic plants.This study demonstrated the successful expression of EpCAM–IgM Fc as anti-colorectal cancer IgM Fc fusion antigenic proteins, creating a candidate for a cancer vaccine in transgenic T1 transgenic plants, the existence of EpCAM–IgM Fc and J-chain K transgenes was confirmed by PCR, respectively. However, Western blotting revealed that only the EpCAM–IgM Fc protein expression was detected in the T1 plants, and the J-chain K protein was not detected. It appeared that J-chain protein expression is difficult to detect. Currently, there are no reports demonstrating J-chain expression in transgenic plants. However, the function of the J-chain is active when it is co-expressed with IgM or immunoglobulin A (IgA) antibodies [In both EpCAM–IgM Fc and J-chain K Ttibodies . Despitetibodies .1 transgenic line highly expressing EpCAM–IgM Fc proteins was selected to cross with the T1 transgenic line carrying the J-chain K transgene to generate F1 plants carrying two transgenes. Since transgenic Chinese cabbage plants have the ability of self-fertilization and cross-fertilization, this crop has been efficiently bred to build useful characteristics in terms of growth physiology, taste, and disease resistance [1 plants having two different transgenes encoding EpCAM–IgM Fc and J-chain K. The immunoglobulin Fc-fusion protein is the protein linked to the immunoglobulin Fc fragment. The Fc-fused proteins obtain beneficial biological and pharmacological effects from the Fc fragment [Agrobacterium-mediated transformation. However, in this study, each transgene was individually transferred to each Chinese cabbage plant, and these plants were successfully cross-fertilized to obtain both transgenes. To confirm whether an anti-human IgM Fc µ-chain antibody can recognize Chinese cabbage-derived EpCAM–IgM Fc × J-chain K, an ELISA analysis was conducted. The binding reaction between EpCAM–IgM Fc × J-chain K and the anti-human IgM Fc µ-chain antibody showed an absorbance signal indicating that the EpCAM–IgM Fc × J-chain K was structurally formed to be recognized by the anti-IgM Fc µ-chain antibody. In previous studies, EpCAM alone and fused with IgG Fc have been expressed in diverse plants [Brasscia species has glycosylation apparatus for protein glycosylation [The Tsistance ,40,41. Ifragment ,43. The fragment ,45. Furtfragment . The Fc-fragment . In genefragment . The IgMfragment . The J-cfragment . In IgA,fragment . In IgM,fragment . The penfragment . Therefoe plants ,27,28, hsylation ,50. In t1 transgenic seedlings carrying both transgenes. These results revealed that both transgenes were stably inserted, and the EpCAM–IgM Fc protein gene was expressed in F1 transgenic plants reproduced from crossing between EpCAM–IgM Fc and J-chain K T1 transgenic plants. Furthermore, these results suggested that two genes can co-exist through cross-breeding in Chinese cabbage T1 transgenic plants. In conclusion, the transgenic Chinese cabbage expressing EpCAM–IgM Fc can be applied to express anti-colorectal cancer IgM Fc fusion recombinant vaccine candidate proteins.All considered, we confirmed that each EpCAM–IgM Fc and J-chain K transgene expression cassette was successfully transferred to the Chinese cabbage, and cross-fertilization between both transgenic plants carrying one of the transgenes generated Fhpt) gene for Chinese cabbage (B. rapa L. ssp. pekinensis) transformation [Nicotiana plumbaginifolia was fused to N-terminus of the cleavage site (Thr17-Ala23). Chinese cabbage was transformed with the plant expression vector pRCV2 EpCAM–IgM Fc (Agrobacterium tumefaciens strain LBA 4404 carrying pRCV2 EpCAM–IgM Fc was applied for plant transformation. Agrobacteria were grown in a yeast extract peptone (YEP) medium.The gene encoding the human EpCAM protein  was fused to the gene encoding human IgM Fc polypeptides  to construct the EpCAM–IgM Fc fusion protein, and this was cloned under the CaMV promoter in the plant expression vector pRCV2 to generate pRCV2 EpCAM–IgM Fc carrying the hygromycin phosphotransferase (M–IgM Fc A. This vAgrobacterium-mediated transformation to express EpCAM–IgM Fc and the J-chain tagged with the KDEL endoplasmic reticulum retention motif (J-chain K)  was used for chain K) B. The seAgrobacterium cell stock was cultured in 50 mL of a YEP medium containing kanamycin (50 mg/L) and acetosyringone (5 mg/L) until an optical density (OD) 600 value of 1.0 was obtained. The Agrobacterium cells were pelleted and washed. Then, they were resuspended in 50 mL of the YEP medium. Agrobacterium cells were applied to infect hypocotyl explants by immersing them in the bacterial inoculum for 10 min (3 (4 mg/L), acetosyringone (5 mg/L), cefotaxime (200 mg/L), hygromycin (10 mg/L), and 0.8% plant agar (pH 5.6). After cultivation for 3 and 8 weeks, the number of calli and shoots that formed on the explants were determined, respectively  and adjusted to 20 ng/μL, and the DNA was used for PCR amplification. The status of transgenic plants was confirmed by PCR using HTP (hygromycine) and target gene  specific primers. The reaction solution for PCR analysis contained 20 ng of gDNA, 5.0 μL of Takara buffer mixture buffer , 1.0 μL each of 10 pmol/μL of primer set, and autoclaved distilled water, to reach a total volume of 20 μL. The primer set for EpCAM -GCA GGC TAT GGC TAC TCA ACG AAG G/reverse primer (R)-CTC GAG TTT TAG ACC CTG CAT TGA G), IgM , J-chain K , and hygromycine  genes were used for DNA amplification to confirm the presence of the transgene in the plants. The PCR reaction was performed as follows: denaturation at 95 ℃ for 10 min in the beginning, a repeated cycling step of denaturation at 95 ℃ for 1 min, annealing at 56 ℃ for 40 s, and extension at 72 ℃ for 1 min (35 times). This was followed by a final denaturation at 72 ℃ for 10 min. A non-transgenic Chinese cabbage plant was used as a negative control, whereas plant expression vectors containing the EpCAM–IgM Fc and J-chain K genes were used as positive controls.To confirm the existence of transgene encoding EpCAM–IgM Fc, genomic DNA was prepared from the fresh leaves of transgenic and non-transgenic Chinese cabbage plants according to the DNA extraction kit  using the mini-prep method and protocol. DNA concentration was measured using a Nanovuev/v) Tween 20). The blot was incubated for 1 h 30 min at room temperature (RT) with either mouse anti-human EpCAM antibody # MAB960-500  diluted in blocking buffer at 1:500 or goat anti-human IgM Fc µ-chain conjugated to horseradish peroxidase (HRP)  diluted in blocking buffer at 1:5000 and then incubated for 1 h and 30 min at RT with a secondary antibody goat anti-mouse IgG 2a heavy chain conjugated to horseradish peroxidase  diluted in blocking buffer at 1:5000. The goat anti-human IgM Fc µ-chain antibody recognized the IgM Fc portion of EpCAM–IgM Fc, whereas the anti-human EpCAM antibody detected EpCAM. Protein bands were visualized by exposing the membrane to X-ray film  using a chemiluminescence substrate (Pierce). Non-transgenic plants and tobacco-derived human EpCAM–IgM Fc (50 ng) [To confirm the expression of EpCAM–IgM Fc in transgenic Chinese cabbage, 100 mg of leaf tissue was harvested from in vitro tissue culture and homogenized to generate leaf extracts in 1× PBS. A volume of 20 μL of leaf extract samples (100 mg of leaf/300 μL) mixed with 5 μL of protein loading buffer  was loaded on 10% SDS-PAGE and transferred to a nitrocellulose membrane . The membrane was incubated in blocking buffer  in Tris-Buffered Saline (TBS) plus 0.5% ( (50 ng)  were use0 transgenic plant validation by PCR and Western blot analysis, the transgenic plantlets (T0) with well-developed roots were transplanted into 10 cm pots containing soil and transferred to a glasshouse at the National Institute of Horticultural and Herbal Science (NIHHS), Wanju, Korea from 2015 to 2017 (0 plants were grown in 2015 and selfed to produce seeds (T1). In 2016, the T1 seeds were sown and the plants were grown for 4–6 weeks with vernalization treatment (1 plants containing the EpCAM–IgM Fc genes were crossed with the T1 plants with J-chain K during the blooming stage for the generation of the F1 transgenic lines in 2017. The crossing resulted in the generation of the F1 transgenic line (After T to 2017 B. The plreatment C. The T1nic line D.T) [T) × J-Chain KT [C) × J-Chain KC proteins diluted in 0.05 M carbonate/bicarbonate buffer . After overnight incubation, the plates were washed with 1X PBS plus 0.5% (v/v) Tween 20 (1X PBS-T) four times and blocked with 5% skim milk  in 1X PBS-T for 1 h at RT. Then, the plates were treated with rabbit anti-human IgG Fcγ antibody conjugated to HRP  or goat anti-human IgM Fc µ-chain antibody conjugated to HRP  diluted 1:5000 in 1X PBS-T or 1X PBS and incubated for 2 h at RT. After washing four times with 1X PBS-T, the plates were detected using 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate and TMB stop solution . The plates were read using an Epoch Microplate Spectrophotometer  at 450 nm.The Maxisorp 96-well immuno plates  were coated and incubated overnight at 4 ℃ with 10 ng per well of purified tobacco plant-derived EpCAM–IgG Fc (EpCAM–IgG FcT) , tobaccoChain KT , and Chit-test to determine the differences in absorbance of each group  using Microsoft Excel software . The difference between each group  was compared for statistical significance at 0.05 and 0.01 probabilities .Statistical analysis consisted of using Student’s


The Clinical Research Experience Internship Program (CREST) provides participants with foundations in scientific research appropriate for high school students interested in clinical careers in health-related disciplines . The overall program goal is to provide research and career experiences to individuals from disadvantaged backgrounds, racial/ethnic minorities, and others who are underrepresented in these fields. The focus of this presentation is that part of the CREST program that has been ongoing since 2013. One program mentor has provided an internship experience to 22 high school students training in and work experience with community-based programs designed to improve health for older adults (identified as ‘community gerontology’). This poster presentation describes some of these experiences . Students have worked with older adults in senior housing facilities, supported housing complexes for veterans, and congregate meal sites for older adults. Because of the program experience, the CREST program helps dispel common stereotypes about older adults and encourages students exploring possible clinical career options to consider focusing on older adults as client populations. Program components are described which can allow conference participants to decide on the applicability of this type of programming for their own communities. Qualitative data are presented that provide insights into these experiences as these influence choice of college major and projected career paths and attitudes about working in community-based healthcare with older people.


IntroductionThe National Resident Matching Program (NRMP) requires all Match participants to adhere to a strict code of conduct known as the Match Participation Agreement, yet Match violations continue to occur. We sought to determine how interview experiences, including Match violations, impact applicants’ perceptions and rankings of residency programs.MethodsAn electronic survey was sent to all accredited medical school Deans of Student Affairs and Association of American Medical Colleges Student Representatives for distribution to fourth-year medical students. Questions assessed pressures that residency programs placed on applicants during interview season and their impact on applicants. Both quantitative and qualitative data were collected.ResultsOf the 433 included respondents, 31.2% (n = 135) reported breaches of the NRMP Match Participation Agreement. Of those, 63% (n = 85) had a negative perception of the violating programs, and 37.8% (n = 51) were less likely to rank those programs highly. Violations included asking applicants about the locations of their other interviews , pressuring applicants to reveal their ranking , explicitly requesting applicants to reveal their ranking , asking applicants to provide a commitment before Match day , and other behavior that was felt to ignore the spirit of the Match . Implying that applicants would match into a program if they ranked it highly  was received positively by 65.2% (n = 105) of applicants experiencing this breach, with 42.2% (n = 68) ranking the program more highly. Three major themes impacting applicants’ impressions of residency programs emerged from the qualitative data: interview experience, professionalism, and post-interview communication (PIC). Respondents overwhelmingly agreed that PIC should either be eliminated or that programs should set clear expectations for PIC.ConclusionsMatch violations continue to occur, despite the NRMP Match Participation Agreement. With the notable exception of communication implying that applicants would match into a program, applicants overwhelmingly view programs that commit these violations negatively and often rank these programs lower as a result. One of the most important and daunting tasks facing medical students is obtaining a residency position at their preferred program. Likewise, residency programs have a stake in securing highly desirable candidates. It is therefore unsurprising that the application process has historically been fraught with attempts by both programs and applicants to influence each other’s decisions. These tensions culminated in the late 1940s, with hospitals offering residency positions to students prior to their fourth year and giving them as little as 12 hours to make vital career decisions  impression of a program” and 28.4% (n = 123) provided “comments regarding the Match interview process that positively impacted [their] impression of a program.” Twenty-seven percent (n = 117) gave suggestions to improve the interview process. All of the comments comprised three overarching themes: interview experience , professionalism , and PIC.Regarding interview experience, applicants preferred to interview at programs where the residents were enthusiastic and involved during the interview process. Applicants also preferred interviewers who are well prepared and familiar with their application, as demonstrated by the following quote:“The thing that had the greatest negative impact on my impression of a program was how unprepared interviewers were for my interview. Some had obviously never seen my application, CV, or essay…”Regarding professionalism, applicants disliked programs that made negative comments about other programs, asked personal questions , or probed them to divulge the other places they applied, interviewed, or ranked. Several applicants reported that certain questions caused them to rank programs lower. One applicant said:“… Any sort of questioning that was not friendly or just combative, that program was ranked much lower.”Another reported:“… Unethical questions about me being a woman with children going into surgery placed a program at the bottom of my rank list despite enjoying my time with the residents and program director.”Yet another said:“… what I did have a problem with was being asked point blank if my home program, where I rotated, or where I completed a research year, offered me an interview (which happened frequently).”Regarding PIC, applicants overwhelmingly agreed that PIC should either be eliminated or that programs should set clear expectations for PIC.Representative quotes include:“… Some of my [PIC] from a program was toeing the line of breaking the Match rules on their part . Overall, I think virtually all [PIC] undermines the spirit of the Match.”“Even without explicitly asking for how you ranked them, I disagree with the use of ‘ranked to match’ or ‘highly ranked’ communications from [program directors] … I think there should be no [PIC].”“… faculty demanded letters of intent proving I was ranking them first in order to be ranked competitively … I mean, I was interested in their programs and would have happily gone there. But if they’re ranking applicants based on how likely they think that applicant is to come to their program…”Many applicants reported appreciating programs that did not place pressure on applicants to engage in PIC:“Programs that openly endorsed the Match process, saying they believe it works and puts people where they belong was a huge positive to me. I really appreciated those who went out of their way to say [PIC] was discouraged and did not influence their decisions. I felt those program directors were more trustworthy and honestly and truly wanted the best for their program.”Applicants’ suggestions included:“Every program should have a written post-interview contact policy clarifying if they do or do not want thank you letters, letters of interest, etc. and what the program’s policy is on sending [PIC]”“No [PIC] … follow NRMP guidelines and make sure all interview committee members are knowledgeable of said guidelines.”Table Our results show that Match violations still commonly occur and can negatively affect applicants’ impression and impact the ranking of programs. We expand upon earlier literature by assessing both overt match violations and those actions that ignore the spirit of the Match without explicitly violating the Match Participation Agreement (MPA) ,6,8-10. Applicants in this cohort were frequently asked where else they interviewed , which is similar to another study of a large single academic institution showing that 72% of applicants were asked at least once about their other interviews . Other pNotably, a majority (65.2%) of applicants were positively to very positively impacted by programs implying they would match at said program if it was ranked highly, and 42.2% were more likely to much more likely to rank the program highly. It may be tempting for programs to utilize this tactic to increase their likelihood of matching desired applicants. However, we feel that this is ethically inappropriate, and programs should remain cognizant of the fact that this is still a Match violation and can trigger disciplinary action from the NRMP. The program director was the most common perpetrator of Match violations, closely followed by other faculty, residents, and program coordinators. As the leader of the resident selection process, it is likely that the attitudes of the program director permeate throughout the program. Our findings suggest that positive changes would be most impactful if encouraged and spread by the program director.Our collection and examination of qualitative data expand upon the aforementioned insights gained from our quantitative data. This study is unique in its emphasis on qualitative data from students entering a broad range of specialties. To our knowledge, there are only two other studies to date that examined student comments related to unethical match behavior, both of which were specialty-specific ,12. In tStudents described negative perceptions of programs that appeared disorganized and ill-prepared, asked violating questions about personal life or questions that appeared generally disrespectful, and were not familiar with their applications. The underlying implication is that students felt they put a great deal of effort into attending and preparing for each interview and took care to treat others respectfully, but these courtesies were not reciprocated. These findings suggest that better preparation prior to interviews on behalf of the interviewer, as well as maintenance of a professional and respectful interview environment, would improve applicants’ perceptions and ranking of a given program. These are easily made improvements if one is cognizant of them, and they can likely improve a program’s reputation and advance their goal of recruiting desirable applicants.PIC was also identified as an area of concern. Students felt that it ignores the spirit of the Match, creates confusion, and adds stress to the application process. Students also negatively perceived being asked to commit to a program after the interview. Even positive PIC from programs, such as telling an applicant that they are highly ranked, was frequently viewed as confusing. The underlying implication was that students felt these messages could be disingenuous and unfairly pressured them to respond in kind. Indeed, several specialty-specific studies found that PIC impacted applicant and program rankings for a noteworthy minority of individuals (approximately 20%-30%) -16, whicLimitations of our study include the potential for recall bias as well as negativity bias; applicants with more negative match experiences may have been more likely to respond to the survey than those with positive experiences. Another limitation is a relatively small sample size. We were unable to send our survey directly to applicants’ personal emails and had to rely on third parties. Thus, we are unsure of exactly how many applicants received the survey, as some intermediaries may have chosen not to forward it. For this reason, we were unable to calculate an exact response rate. Despite this limitation, we believe our results are valid as they both are consistent with and expand upon prior studies ,9,10. ThMatch violations continue to occur. The most frequently reported Match violations include asking about other interviews, explicitly pressuring applicants to reveal the locations of other interviews, and asking applicants to provide a commitment prior to Match Day. Additionally, applicants described several program behaviors which, while not explicitly forbidden, were interpreted to violate the spirit of the Match. These included unethical behavior, pressure to attend a second-look visit, inappropriate PIC, or other undue pressure.With the notable exception of communication implying that applicants would match into a program, applicants overwhelmingly view programs that commit Match violations negatively and often rank these programs lower as a result. PIC in particular was consistently identified as unwelcome and confusing. Many applicants favored either establishing explicit PIC expectations or eliminating it entirely. The majority of Match violations were committed by Program Directors, and these leaders are uniquely positioned to lead the reform of the residency interview process. Based on these results, elimination of Match violations would likely be beneficial to both applicants and programs.


Inadequate health human resources is a key challenge to advancing child survival in Ghana. Nurses are an essential human resource to target because they represent the largest portion of the health workforce. Building on lessons learned from our pilot pediatric nurse training project and World Health Organization guidelines for transforming and scaling up health professional education, this project aimed to; train 500 pediatric nurse specialists through a one-year training program; develop and integrate a critical mass of pediatric nursing faculty and establish a national standardized pediatric nursing curriculum. This study aimed to evaluate the effectiveness of a national pediatric nurse training program in Ghana at the end of 4 years, including eight cohorts with 330 graduates.This was a mixed-method evaluation with surveys, focus groups and a pre-test/post-test design. Before and after surveys were used to measure knowledge and confidence at baseline and graduation. Objective Structured Clinical Examinations (OSCE) were used to measure clinical skills at baseline, graduation, and 14 months follow-up. At the end of every module, surveys were used to measure students’ satisfaction. Focus groups at graduation qualitatively measured program outcomes. Repeat focus groups and surveys at 14 months after graduation captured the graduates’ career progress, experiences reintegrating into the health system and long-term program outcomes.Overall, the graduates completed the program with significantly increased knowledge, confidence, and clinical skills. They also had increased job satisfaction and were able to apply what they learned to their jobs, including leadership skills and gender-sensitive care. Data from 14-month follow-up OSCEs showed that all graduates remained competent in communication, physical assessment, and emergency care, although some obtained a lower mark compared to their performance at graduation. This finding is linked with the observation that the amount of mentorship, support from leadership and equipment that the graduates accessed from their respective facilities varied.Mixed-methods evaluations demonstrated significant increases in knowledge confidence and skills by completing the program and maintenance of skills more than 1 year after graduation. Findings have implications for those working on the design, implementation, and evaluation of nursing education interventions in low- and middle-income countries.The online version contains supplementary material available at 10.1186/s12912-021-00550-1. Globally, health professional education has not kept pace with health care challenges  which haUnderinvestment in nursing education has resulted in capacity constraints and challenges including a shortage of nursing faculty, outdated curricula that are mainly didactic “talk and chalk” rather than practice-based curricula, insufficient training in patient- and family-centred care, and shortage of teaching equipment , 4. TraiSickKids-Ghana Pediatric Nursing Education Partnership (PNEP) was established as an international effort to reduce child mortality in Ghana by scaling up pediatric nursing education across the country to further increase the concentration of highly skilled pediatric nurses who are critical to improving health outcomes for children [Ghana has been investing in the training of pediatric nurses since 2010 in an effort to address health system shortcomings and reduce child mortality from preventable causes. A pilot pediatric nurse training program was established in 2010 and a 2013 evaluation of the pilot  indicatechildren , 9. PNEPGood Health and Wellbeing and its target of achieving universal health coverage), #4 , and #5 .Through PNEP, a one-year post-basic, practice-focused pediatric nursing education program was established at three training sites in Ghana with a target of training 500 pediatric nurses by September 2020. PNEP partners worked to ensure equitable recruitment from underserved areas into the program, increased recognition and remuneration for nurses upon graduation, and effective deployment and retention in the Ghanaian health care system. With an additional focus on positioning nurses as leaders who can not only improve patient outcomes but also advocate for gender and health equality and closing the global gap in health leadership positions held by women , PNEP’s A rigorous monitoring and evaluation plan was established at the onset of PNEP to support continuous learning and improvement and to ensure quality education standards were met. This manuscript presents the evaluation of PNEP’s one-year pediatric nursing education program in Ghana and focuses on program outcomes  at graduation and 14 months after graduation; reintegration to the Ghanaian health system 14 months after graduation; and overall satisfaction with the program.PNEP’s one-year pediatric nursing education program, hereafter referred to as ‘the program”, is delivered by the Ghana College of Nurses and Midwives, hereafter referred to as ‘the College’. Nurse educators from SickKids and Ghanaian nursing faculty collaborated to develop a national, accredited, competency-based curriculum that would be taught at all program sites. Program curriculum focuses on foundational pediatric nursing principles such as family centred care, strengths-based nursing, and gender equality, the provision of primary care and community care, managing acute and changing conditions in hospital and emergency settings, and nursing leadership development.To be admitted into the program, students must already be registered nurses of good standing with the Nursing and Midwifery Council of Ghana, hold a minimum of a diploma certificate in nursing, and have at least 3 years of work experience. Delivery of the program includes interactive lectures, case-based learning, simulation, and experiential learning through an intensive clinical practicum to ensure integration of theory with practice. Students spend 30% of their time in class and the skills lab and 70% of their time in selected facilities at training centres for the clinical practicum.To support country-wide recruitment into the program, the country was divided into three zones  with Accra , Kumasi , and Tamale  serving as the regional training centres. Classrooms and Skills Labs were established at each training centre. PNEP employed a step-wise approach to recruitment of cohorts of students at each training centre over 5 years with the cumulative total of 501 students  at graduation and 14 months after graduation;Assess graduates’ reintegration to the Ghanaian health system 14 months after graduation; andExamine if graduates were satisfied with the program and uncover areas for improvement.The Monitoring & Evaluation (M&E) Plan for PNEP was a mixed methods, qualitative/quantitative design with concurrent data collection and analysis that was developed in consultation with key stakeholders and received approval through the SickKids Quality Improvement Committee in May 2016 and from the Ghana Health Service Ethics Review Committee in August 2016. PNEP’s M&E plan is built on the notion that evaluation of nursing education programs is a process that starts during education design and continues through delivery of the program, graduation of students and when graduates have gone back to work within the health system –14. SpecThis study focuses on the sections of the larger evaluation plan related to cohort data and the graduates’ perception of the program’s effectiveness. Appendix A 21-question multiple-choice exam, covering the key content of the program curriculum, was used to measure nurses’ knowledge before they started the program, and again at graduation , 15.A seven-item questionnaire was used to measure nurses’ confidence in seven key areas related to clinical practice before they started the program, and again at graduation , 15. A fObjective Structured Clinical Examinations (OSCEs), considered the gold standard for assessment of clinical competence , 17, werPhysical AssessmentCommunicationEmergency: Assessing and Responding to a Deteriorating PatientClinical competence was measured at graduation and 14 months after graduation, using three OSCE stations to evaluate students in key areas required for every patient encounter:Fourteen months after graduation was chosen (instead of a longer lag time) due to the time limitations of the PNEP project period. Similarly, a baseline OSCE was conducted with only one cohort (Kumasi Cohort 2), due to time and resource constraints within PNEP. Prior to the baseline assessment, students were given an introduction to the structure of an OSCE since many students had not encountered this type of exam in their previous training. The baseline OSCE covered Physical Assessment and Communication stations. Competencies in Emergency Management were considered more advanced and not assessed at baseline.All OSCEs were marked using a five-point scale adopted from Bondy’s validated scale . The sca1 = Not competent.2 = Towards competent.3 = Competent.4 = Towards proficient.5 = Proficient.Detailed scale definitions are presented in Appendix The region where the nurses worked before entering the program;The type of institution where they worked ; andThe number of years of experience as a nurse.Seven to eight individuals from seven respective cohorts were invited to participate in in-person facilitated focus groups when they finished the program . Informed written consent was obtained from all participants for all focus groups and facilitators were hired to run the focus groups with standardized questionnaires that were developed specifically for these focus groups. The graduates were asked to comment on the quality of the curriculum content, the teaching format, and ideas for improving the program. Maximum variation sampling was used  using thFocus group facilitators were external to the College. Focus group data were recorded, transcribed, and coded by two researchers. Codes were further analyzed using a thematic analysis approach . RefreshFourteen months after graduation, three additional respective focus groups were conducted with a total of 38 graduates. Graduates were asked to describe their experiences of going back to the workforce and to discuss their challenges and achievements. Facilitators of these focus groups were staff at the College with little or no prior contact with the graduates. The same sampling strategy as above was used.A follow-up survey was sent to all graduates 14 months after they finished the program in order to track retention within the Ghanaian health system and understand their career trajectory. Of particular interest was whether the graduates remained in their respective health facilities and whether they were promoted to a higher nursing rank with increased salary .Overall Satisfaction, measured using a five-point scale for both in-class modules and clinical practicums.Graduates’ satisfaction with the program and areas for improvement were measured using course evaluation surveys throughout the year. For this study, the focus will be on All statistical analysis was performed using IBM SPSS Statistics, version 26. Descriptive statistics were conducted to summarize the demographic characteristics and the 14 months follow-up survey data and to calculate the level of confidence, knowledge, and clinical skills (OSCE station mean scores). Wilcoxon Signed Ranks for paired samples was used to measure changes in both knowledge  and confidence  before and after the program. Focus group data were recorded, transcribed, and coded by two researchers. Codes were further analyzed using a thematic analysis approach .The analysis is presented as aggregate data. Cohort-specific analysis, however, has been conducted throughout, to ensure that quality of education was consistent across training sites and over time.This study focuses on data for the eight cohorts that have so far graduated from the program. Appendix N = 330) across the Accra, Tamale and Kumasi training centres and there were no missing responses. On average, students had 6 years of work experience and were in their early 30s. The range was large for both age and years of experience; this shows that the program attracted both novice and experienced nurses. More than half entered the program with a diploma certificate, as this is the minimum entry requirement for the program at the College. The majority were from district hospitals. Students were recruited from all 16 regions of Ghana, with half being from regions identified as underserved.Table n = 293, P = 0.000). Variations existed within and between cohorts in terms of baseline knowledge ; however, across all cohorts, graduates had increased knowledge compared to baseline and the change was statistically significant.The Wilcoxon signed-rank test showed that the mean knowledge test score increased from 52% ±11.2 at baseline to 71% ±9.2 at graduation, a 37% increase, which is statistically significant . Results in Table Tables Table A thematic analysis was conducted Table  of sevenOf the 125 students eligible to take part in a 14 months follow-up survey, 124 participated (one graduate could not be reached). Of the 124 participants, all had remained in the Ghanaian health system with 119 being in same facility as at baseline, and five having moved to a different hospital. Of the 124 participants, 120 were promoted to Nursing Officer rank, an indicator of recognition and increased remuneration. Three were not promoted as they were already Nursing Officers at baseline, and one participant could not be promoted due to protocols in the military hospital where the graduate worked.As shown in Table n = 330). Overall satisfaction ranged between 4.0 and 4.6 among cohorts. For clinical practicum, satisfaction ranged from 2.9–4.9. Due to the varied nature of the facilities in which the students completed their practicum, an overall mean satisfaction score is not reported.For the in-class portion of the program, the mean Overall Satisfaction score for all cohorts on a five-point scale was 4.35 ± 0.18 , job satisfaction and by graduates advocating for a clearly defined pediatric nurse role in their health care setting. Despite areas for improvement, and system-level challenges  outside the control of the program, the mixed methods evaluation demonstrates that the program was successful in creating a critical mass of highly skilled, knowledgeable, competent and confident pediatric nurse leaders.In the program, graduates had statistically significant increases in knowledge and confidence and focus group discussions confirmed these quantitative findings. The largest change in confidence was related to physical assessment and history taking, which was notable because all focus group participants identified these two as areas they wanted to improve on.At graduation, focus group participants reported a more positive attitude towards their patients and talked about having learned valuable leadership skills and competencies to provide gender-sensitive care. Subsequently, at 14 months follow-up, they gave concrete examples of how they applied those skills in their practice, including teaching their colleagues new skills and advocating for their patients to improve patient outcomes. All graduates reported high levels of job satisfaction at 14 months follow-up due to their newly acquired competencies and self-perception as being leaders who can make positive changes happen.The significance of these results becomes more pronounced within the current context of health care provision in African countries. Disrespectful care and maltreatment of mothers (the primary caregivers) and patients as well as stigmatization of adolescents seeking health care services by health workers has been a known but largely ignored problem in many African countries, including Ghana –23. FemaResearch has shown that transition of nurses from ‘competent’ to ‘proficient’ depends on a range of interconnected factors, including having the opportunity to practice the learned skills, receiving ongoing mentorship from their colleagues, a supportive work environment and an organizational culture that is open to change , 24–28. A series of clinical observations and stakeholder interviews at clinical facilities where graduates are practicing are being planned for a future date to better understand how the results of this educational program influence organizational improvements in hospitals across Ghana and what strategies could be developed to support the graduates so that they can contribute to decreasing under-5 mortality in Ghana. One area where institutions like the College could exert direct influence in supporting graduates is through ongoing professional development opportunities via short courses and networking opportunities. In the case of PNEP, 14 months follow-up OSCEs were structured to not only complete the evaluation but also facilitate professional development sessions and debriefing with the faculty and graduates. The College is now in the process of expanding the number of professional development courses available.Students’ overall satisfaction with the program was high, evident both from the overall satisfaction score for all eight cohorts and from focus group data. While the satisfaction rate for the in-class component of the program was consistently high among all cohorts, satisfaction with the clinical practicum varied slightly, likely due to variations in size and scope of clinical sites as well as number of preceptors available to mentor the students. The program team closely monitored satisfaction rates at each clinical site and made changes to the list of sites chosen as practicum sites as needed. One area for improvement revealed by focus group participants was the ratio of content to the time allocated to each module. Students consistently felt that the intensity and rigour of the program was more consistent with a first-degree program than a post-basic program. This feedback, along with other stakeholder consultations, informed continuous revisions to the program in each academic year.Study strengths include evaluating the longer-term outcomes of the program (at 14 months) which will offer stakeholders and perhaps other groups doing similar work, a broader perspective on the extent to which this type of program may strengthen the nursing workforce. Other strengths include the triangulation of designs and data types  for more robust conclusions and sharing the lessons learned in real-time for continuous quality improvement.There are some limitations associated with this evaluation. Evidence shows that OSCEs with a larger number of stations tend to have higher reliability . In the Another limitation is that at the time of writing, results for eight of 11 cohorts had been included due to the timing of PNEP’s project period which ended in September 2020. Three cohorts graduated in September 2020 and a 14-months follow-up (November 2021) would only be possible well after PNEP ended. Given the patterns to date with the first eight cohorts it can be inferred that graduates will demonstrate similar results however further analysis after all 11 cohorts are completed would be necessary to validate this.PNEP’s project period ended in September 2020, however the program will continue forward under the operational leadership of the College. Although operating the program independently of PNEP presents new challenges for the College, the program has been setup to be sustainable in a manner that can continue to build the knowledge, confidence and skills of pediatric nurses. Continuing investment in education at the post-graduate level is required to develop nursing leaders in clinical, education and policy roles who can affect systems-level changes that ultimately benefit patients and in PNEP’s case - children. This evaluation has generated evidence regarding the benefits that a high-quality nursing education program can offer to nurses, the children they treat, and national health systems. Through high quality education, the program will continue to contribute to the ultimate outcome of reducing under-5 mortality and advancing multiple Sustainable Development Goals.Additional file 1.Additional file 2.


Donation after circulatory death (DCD) liver grafts have a poor prognosis after transplantation. We investigated whether the outcome of DCD donor organs can be improved by heme oxygenase 1 (HO-1)-modified bone marrow-derived mesenchymal stem cells (BMMSCs) combined with normothermic machine perfusion (NMP), and explored its underlying mechanisms.BMMSCs were isolated, cultured, and transduced with the HO-1 gene. An NMP system was established. DCD rat livers were obtained, preserved by different methods, and the recipients were divided into 5 groups: sham operation, static cold storage (SCS), NMP, BMMSCs combined with NMP, and HO-1/BMMSCs combined with NMP (HBP) groups. Rats were sacrificed at 1, 7, and 14 days after surgery; their blood and liver tissue samples were collected; and liver enzyme and cytokine levels, liver histology, high-mobility group box 1 (HMGB1) levels in monocytes and liver tissues, and expression of Toll-like receptor 4 (TLR4) pathway-related molecules were evaluated.After liver transplantation, the SCS group showed significantly increased transaminase levels, liver tissue damage, and shorter survival time. The HBP group showed lower transaminase levels, intact liver morphology, prolonged survival time, and decreased serum and liver proinflammatory cytokine levels. In the NMP and SCS groups, HMGB1 expression in the serum, monocytes, and liver tissues and TLR4 pathway-related molecule expression were significantly decreased.HO-1/BMMSCs combined with NMP exerted protective effects on DCD donor liver and significantly improved recipient prognosis. The effect of HO-1/BMMSCs was greater than that of BMMSCs and was mediated via HMGB1 expression and TLR4 pathway inhibition. Liver transplantation is the most effective treatment for various end-stage liver diseases . Donor gCurrently, liver graft preservation methods mainly include SCS, hypothermic machine perfusion (HMP), subnormothermic machine perfusion (SNMP), and normothermic machine perfusion (NMP). SCS has failed to provide optimal DCD liver preservation, resulting in many complications. HMP may cause sinusoidal endothelial damage because it increases vascular resistance and shear stress . MoreoveIn order to enhance the protective effects of NMP on organs, NMP combined with cellular therapy may be a novel strategy. Previously, some researchers used NMP to perfuse bone marrow mesenchymal stem cells (BMMSCs) into the kidney and showed that the BMMSCs could survive in the NMP system and colonize the organ . AlthougDue to their immunomodulatory functions, BMMSCs are commonly used for cellular therapy and play an important role in the study of organ damage repair and transplantation immune regulation . Howevern = 25) were used as a source of BMMSCs. Male SD rats aged 7–8 weeks (220–240 g) were used to establish an orthotopic liver transplantation (OLT) model. Recipients were randomly divided into 5 groups: sham operation (Sham), SCS, NMP, BMMSCs combined with NMP (BP), and HO-1/BMMSCs combined with NMP (HBP). Thirty (6 in each group) recipients were used for survival rate analysis, and the other 120 recipients were used for sample collection following OLT . All animals were housed in standard animal facilities for at least 3 days before use. Animals received humane care and were maintained in accordance with the guidelines established by the Committee on Laboratory Resources, National Institutes of Health. All experiments were approved by the Ethics Committee of Tianjin First Central Hospital.All animals were obtained from China National Institutes for Food and Drug Control. Male Sprague-Dawley (SD) rats aged 3–4 weeks  was injected. After 10 min of heparinization, the diaphragm was cut open and the thoracic aorta was clamped with an artery clamp; the heart was compressed to induce cardiac arrest; and the abdominal cavity was covered with 37 °C warm saline for 30 min. During this period, a temperature-sensing probe is placed in the abdominal cavity of the rat to detect and maintain the body temperature between 35 and 37 °C.2O. All DCD livers were subjected to in situ flushing with 10 mL UW solution and SCS livers were stored at 4 °C for 4 h. Similarly, the NMP protocol also lasted for 4 h. The NMP livers were simply mechanically perfused. In BP group, the livers were perfused with 1.5–3 × 107 BMMSCs through the portal vein. In the HBP group, the livers were perfused with HO-1/BMMSCs 1.5–3 × 107. All BMMSCs were perfused into the liver through a 100-μm microthrombotic filter and stored in vitro for 4 h.The NMP system mainly consists of an organ chamber , a membrane oxygenator, a peristaltic pump, a temperature and pressure sensor, a filter, and a heat exchanger   were perfused into the liver using NMP, and liver fluorescence was measured using in an vivo imaging system on postoperative day (POD) 1.The levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), glutamyl transpeptidase (GGT), total bilirubin (TBil), and serum albumin were detected using an automatic biochemical analyzer .The liver tissue was fixed in 10% neutral formalin solution, and the samples were dehydrated, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). The tissue blocks were then embedded in optimal cutting temperature compound and snap-frozen in liquid nitrogen for frozen sections.The serum was collected from the peripheral blood, and the serum levels of interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and high mobility group box 1 (HMGB1) were measured using an ELISA kit  according to the manufacturer’s instructions.Samples were dehydrated, embedded in paraffin, sectioned, and subjected to HO-1 and cytokeratin 19 (CK19) staining . HO-1 staining assessments were performed using light microscopy while CK19 staining was assessed using a fluorescence microscope.low and CD43high monocytes based on the fluorescence intensity of CD43 [low monocytes and CD43high mononuclear cells were isolated and collected by cell sorting .Monocytes were identified in the whole blood after red blood cell lysis. The peripheral blood was incubated with anti-CD172a, anti-CD43, and anti-HMGB1 antibodies . Flow cytometry was used to detect monocytes with high CD172a expression. Monocytes were divided into two groups of CD43 of CD43 . The meaProteins extracted from the liver tissue were analyzed by western blotting as previously described . The expTotal RNA from the liver tissue was isolated using TRIzol reagent , and cDNA was synthesized using a cDNA reverse transcription kit  in accordance with the manufacturer’s instructions. β-actin was used as an internal control. The primer sequences are listed in Table r) between groups. P < 0.05 was considered statistically significant.SPSS 13.0  and GraphPad 8.0  were used for statistical analysis. Data are expressed as mean ± standard deviation. Significance was tested using one-way analysis of variance. The count data were expressed as percentage (%), and significance was tested using the chi-squared test. Survival analysis using Kaplan–Meier survival curves and log-rank tests (Mantel-Cox) were performed. The Pearson correlation method was used to determine the correlation coefficient . The survival time in the BP group (37.5 days) was higher than that in the NMP group (16 days), but significantly lower than that in the HBP group (P < 0.05). Hence, HO-1/BMMSCs combined with NMP can significantly prolong the survival time of recipients after OLT and are more effective than BMMSCs combined with NMP  and lowest in the SCS group (2.5 days), which was significantly lower than that in the other groups (BP group 7.5 days P < 0.05), but significantly lower than that in the HBP group (P < 0.05), indicating that HO-1 transduction could improve BMMSC survival and HO-1 expression in liver grafts.Immunohistochemistry showed that there were greater numbers of HO-1-positive cells in the livers of the HBP group compared to those in other groups Fig. A. WesterP < 0.05). In addition, these levels were significantly lower in the BP and HBP groups than those in the NMP group at POD 7 and 14 (P < 0.05); however, on POD 14, the levels in the HBP group were significantly lower than those in the BP group (P < 0.05). These results suggested that HO-1/BMMSCs combined with NMP can improve hepatobiliary function after OLT more significantly than BMMSCs combined with NMP , 1, 7, and 14. In the SCS group, on POD 7 and 14, normal lobular structure was partially destroyed and infiltration of inflammatory cells into the liver grafts was observed. The hepatic tissue structure in the BP and HBP groups was significantly improved at POD 7 and 14, and hepatic injury in the HBP group was lower than that in the BP group. These results indicated that HO-1/BMMSCs combined with NMP can improve liver graft histopathology more significantly than BMMSCs combined with NMP Fig. B. The SuP < 0.05), but significantly lower than that in the HBP group (P < 0.05), indicating that HO-1/BMMSCs combined with NMP can reduce bile duct injury more significantly than BMMSCs combined with NMP.Transplantation of DCD livers is associated with a high rate of biliary complications, especially ischemic cholangiopathy. Since the expression of CK19 is high in normal bile duct epithelial cells, but low in injured bile duct epithelial cells, the expression levels can be used to evaluate biliary tract injury. Immunofluorescence analyses showed greater numbers of CK19-positive cells in the livers of the HBP group compared to those in the other groups Fig. A. CK19 eP < 0.05). IL-1β and TNF-α levels in the HBP and BP groups were lower than those in the NMP group on POD 7 and 14. The proinflammatory cytokine levels in the HBP group were lower than those in the BP group on POD 14 (P < 0.05). The mRNA levels of IL-1β, IL-6, and TNF-α in the liver tissue were similar to the serum levels. On POD 7 and 14, the mRNA liver tissue levels of IL-1β, IL-6, and TNF-α in the HBP and BP groups were significantly lower than those in the SCS and NMP groups; however, the levels in the HBP group was lower than those in the BP group on POD 14 (P < 0.05). These results suggested that HO-1/BMMSCs combined with NMP can reduce proinflammatory cytokine expression more effectively than BMMSCs combined with NMP . On POD 7 and 14, serum HMGB1 levels were lower in the HBP and BP groups than those in the NMP group, and on POD 14, the levels were lower in the HBP group than those in the BP group . On POD 7 and 14, the MFI of HMGB1 on monocytes in the SCS group continued to increase and was higher than that in the NMP group (P < 0.05); however, on POD 14, the MFI of HMGB1 in the HBP group increased slightly but was lower than that in the BP group   Fig. D.Fig. 8P < 0.05).Western blotting was used to detect HMGB1 expression in the two monocyte groups Fig. E. HMGB1 P < 0.05; Fig. P < 0.05). These results indicated that HO-1/BMMSCs combined with NMP can reduce serum HMGB1 level and inhibit HMGB1 expression in monocytes and liver grafts more effectively than BMMSCs combined with NMP.HMGB expression in the liver tissues of BP and HBP groups was significantly lower than that in the liver tissues of SCS and NMP groups on POD 7 , and it is necessary to establish a double-circulation method to perfuse the human donor liver . MoreoveCurrently, NMP technology has gained much attention. In combination with other treatments, it can be used for resuscitation of marginal donor liver in vitro. In this study, HO-1/BMMSCs combined with NMP was used to preserve the DCD donor liver, thus overcoming the short survival time of BMMSCs, and improving donor liver quality and postoperative survival rate. We hope that NMP can be applied in clinical conditions, providing a new direction for the preservation and resuscitation of DCD donor livers in the future.In summary, we established a stable and reliable NMP system to enhance BMMSC colonization in the liver, thus avoiding the possible occurrence of pulmonary embolism or other complications caused by systemic application of BMMSCs. Additionally, our results demonstrated that HO-1/BMMSCs combined with NMP exerted protective effects on DCD donor livers and significantly improved recipient prognosis. The effect of HO-1/BMMSCs was greater than that of BMMSCs and was mediated via TLR4 pathway inhibition. These data are of great significance for resuscitation of marginal donor liver in vitro and expansion of donor liver pool.Additional file 1. A schematic diagram of the NMP system. A stable NMP system was established. It mainly consisted of an organ chamber , a membrane oxygenator, a peristaltic pump, a temperature and pressure sensor, a filter, and a heat exchanger. Individual portal perfusion, bile duct intubation, and continuous bile production during perfusion were observed.


In multivariate analysis, LVI was associated with worse OS in N0 [HR 1.37 ] and ≥26 [HR 1.58 ], but not RS 0–10. No interaction between LVI and chemotherapy benefit was seen. Black race was associated with worse OS in N0  and N+  disease. LVI adds prognostic information in ER+, HER2−, N0 BCA with RS 11–100, but does not predict chemotherapy benefit. Black race is associated with worse OS, but not LVI.Lymphovascular invasion (LVI) and Black race are associated with poorer prognosis in early breast cancer (EBC). We evaluated the association between LVI and race, and whether LVI adds prognostic benefit to the 21-gene recurrence score (RS) in EBC. Women with ER+ HER2− EBC measuring up to 5 cm, with 0–3 involved axillary nodes, diagnosed between 1 January 2010 and 1 January 2014, who underwent surgery as first treatment and had available RS, were identified in the NCDB database. Bivariate associations between two categorical variables were examined using chi-square test. Multivariate Cox proportional hazards model were used to assess the association of LVI, race, and other covariates with overall survival (OS). 77,425 women, 65,018 node-negative (N0), and 12,407 with 1–3 positive (N+) nodes, were included. LVI was present in 12.7%, and associated with poor grade, RS 26–100, and N+ (all  Multiparameter gene expression assays also provide prognostic information for distant recurrence in hormone receptor-positive (HR+), HER2 negative breast (HER2−) cancer9. The 21-gene recurrence score (RS) assay is both prognostic for recurrence and predictive of chemotherapy benefit in EBC13, including women with up to three positive axillary nodes15. The extent to which LVI adds prognostic information or predictive information for chemotherapy benefit to the 21-gene RS or other assays has not been adequately evaluated16.Lymphovascular invasion (LVI), the presence of tumor cells within the lumen of lymphatic or vascular system in the tumor primary site, is associated with a higher risk of regional lymph node metastasis at diagnosis and subsequent distant recurrence in early breast cancer (EBC)18, survival gaps are also seen for Black women receiving contemporary adjuvant therapy on NCI-sponsored clinical trials22, suggesting that factors other than care disparities contribute to their poorer prognosis. For example, in the TAILORx trial, Black race was associated with a 1.4-fold higher risk of recurrence after adjustment for clinicopathologic characteristics, despite similar chemotherapy and endocrine therapy use and similar RS distribution23. Thus, biological factors that are not captured by the 21-gene RS, including racial differences in the tumor microenvironment25, may contribute to the worse outcomes for Black women with EBC.Black race has also been associated with worse prognosis in EBC. Although disparities in access to care play a role in Black patients’ poorer outcomes28. Studies evaluating racial differences in BCA tumor microenvironment have described the differential expression of genes associated with inflammation and angiogenesis31, as well as increased microvessel density31 and increased numbers of tumor-associated macrophages32, which correlate with increased lymphangiogenesis and LVI35, in tumors belonging to Black women. Although no studies have directly compared racial differences in LVI in BCA, a multinational study evaluating clinicopathologic characteristics of urothelial tumors in white and Asian patients showed a significant racial difference in the rate of LVI36. No prior studies have evaluated the impact of LVI, RS, and race in the same population. We, therefore, sought to assess whether LVI adds prognostic or predictive information to the 21-gene RS, and whether there is an association between LVI and race that may contribute to racial disparities in EBC.The presence of LVI represents a manifestation of an interaction between the tumor and its microenvironment. The molecular mechanisms underlying LVI are complex and not fully defined, but pathways involving cytokine-receptor interactions have been implicatedAs shown in Fig. As shown in Table p = 0.0365).Also shown in Table p < 0.0001 for all). Demographic factors associated with poorer OS in univariate analysis were Black race, older age, lower median educational level, and lower median income   in univariate analysis were axillary nodal involvement, larger tumor size, higher grade, increasing RS, presence of LVI, and increased number of comorbidities p < 0.000 for all.p < 0.0001) and Black race . LVI was prognostic in the 65,018 patients with N0  but not in the 12,407 patients with N+  disease, whereas Black race was prognostic in both N0  and N+  disease. Other factors associated with poorer OS included N+ disease (p < 0.0001), larger tumor size (p < 0.0001), grade 3 histology (p < 0.0001), higher RS , older age (p < 0.0001), lower median income (p = 0.0001), and greater number of comorbidities (p < 0.0001).As shown in Table p = 0.35) ] and 26–100 [HR 1.58 ], but not in patients with RS 0–10 [HR 1.1 ], although there was no statistical interaction between LVI and RS (interaction p = 0.003) and in N+ patients (p = 0.001), but not in N0 patients (p = 0.134). There was no interaction between LVI and chemotherapy benefit in the 39,402 patients with N0 disease and an RS 11–25 (interaction p = 0.46) , and was inversely associated with median educational and income levels (p < 0.0001). Both presence of LVI and Black race were associated with lower receipt of RS . Use of RS was associated with lower likelihood of receiving chemotherapy  ; however, the difference in frequency of LVI between white and Black women, whereas statistically significant, was numerically small in magnitude. Multivariable analysis of OS in women who did not undergo RS assay showed that LVI was associated with poorer OS in the entire cohort , and in both N0  and N+ patients . Black race was associated with poorer OS in the entire cohort  and in N+  women only  had numeric RS available. We compared characteristics of this group with the 119,321 patients who did not undergo RS analysis. Receipt of RS correlated with N0 disease, grade 2 histology, and absence of comorbidities  after adjustment for RS.Three previous studies have evaluated the correlation between LVI and the 21-gene RS in ER+ N0 EBC. Mutai et al.13, the optimal management for patients with N0 ER+ BCA and intermediate RS was unclear, and recommendations for adjuvant chemotherapy in this group of patients were often based on clinicopathologic criteria. Chen et al.39 showed that the presence of LVI was significantly associated with receipt of chemotherapy in patients with intermediate RS diagnosed before 2013. Our study, although not randomized, did not detect a chemotherapy benefit in patients with intermediate RS and LVI who received adjuvant chemotherapy. In contrast, a retrospective evaluation of patients enrolled on two International Breast Cancer Study Group trials of chemoendocrine therapy in N0 EBC found that the benefit of ovarian suppression with goserelin in premenopausal patients with ER+ disease was exclusively confined to patients with LVI40. In addition, analysis of the BIG 1–98 trial showed that patients with LVI had greater than average benefit from letrozole compared with tamoxifen41. These findings are consistent with results of the SOFT/TEXT trials, indicating greater benefit from ovarian function suppression plus an aromatase inhibitor compared with tamoxifen in those at higher recurrence risk42.Prior to the publication of the randomized TAILORx data18, later stage at diagnosis44, higher incidence of triple-negative disease45, and decreased adherence to chemotherapy and endocrine therapy regimens47. However, four clinical trials, which collectively included 26,138 patients, 2,370 (9%) of whom were characterized as Black  have shown poorer outcomes for Black women with HR+ EBC23, despite similar antineoplastic therapy, indicating that differences in tumor biology in HR+ EBC also play a role. This disparity is hypothesized to be at least partially due to racial differences in the tumor microenvironment25. Although Black women have higher rates of obesity, which has been associated with higher recurrence rates in HR+ HER2− disease49, evidence suggests that this racial disparity is not explained by obesity alone50. Our analysis, using real-world data from NCDB, indicates that the racial disparity in outcome of ER+ EBC is not explained by a greater incidence of LVI, or by substantially and clinically relevant differences in the 21-gene RS.Multiple factors contribute to poorer prognoses for Black breast cancer patients, including disparities in access to care43. An additional important limitation of our study is that the NCDB relies on local assessment of LVI, and does not distinguish between focal and extensive LVI. Prior reports indicate that up to 26% of local pathologists’ assessments of LVI are altered after pathologic review by an experienced breast pathologist51, and that extent of LVI correlates with poorer outcomes6. In addition, 10.67% of our cohort had unknown LVI status.Our study has several strengths and limitations, which are inherent in the use of NCDB data. Strengths include the large sample size, utilizing real-world data from CoC-accredited institutions, and representing the largest analysis evaluating the prognostic information provided by LVI, with a sufficient sample size to determine an association between LVI and Black race. Limitations include lack of information regarding cancer recurrence, both distant and locoregional, relatively short duration of follow-up, and relative under-representation of Black women in the EBC NCDB dataset53, and in patients felt to be able to tolerate chemotherapy53. Lower receipt of RS in patients with LVI may reflect clinician bias in favor of administering chemotherapy to patients with LVI, a known poor prognostic factor. Black women were also somewhat under-represented in our cohort, compared with patients who did not receive RS. Previous studies evaluating racial bias in receipt of genomic testing have shown conflicting results. One study utilizing NCDB data showed that Black women were less likely than white women to undergo RS assay, and more likely to have testing ordered in guideline-discordant scenarios54. However, a more recent study, utilizing data from the Georgia Cancer Registry, found no racial differences in receipt of RS, but found a 1 year lag in uptake of testing in Black women55.Our study is also limited by real-world biases in the ordering of RS. Comparison between our cohort and patients in the NCDB database who did not undergo RS assay show that patients in whom RS was ordered were more likely to have intermediate-grade tumors, N0 disease, and no comorbidities, confirming previously reported clinician tendencies to order RS in a state of clinical equipoiseIn summary, our data demonstrated that LVI adds prognostic information for OS in N0 ER+ HER2-BCA, with RS 11–100, but found no association between LVI and Black race in women undergoing RS assay. Despite the poorer prognosis associated with LVI, no chemotherapy benefit was seen in patient with RS 11–25 and LVI. Therefore the presence of LVI should not influence chemotherapy decision-making in patients with intermediate RS.56, and 80% of newly diagnosed breast cancers in the United States57. The NCDB Participant User File (PUF) is a HIPAA-compliant data file, which is made available to investigators from CoC-accredited cancer programs who complete an application process. The data used in the study are derived from a de-identified NCDB file. The American College of Surgeons and the Commission on Cancer have not verified and are not responsible for the analytic or statistical methodology employed, or the conclusions drawn from these data by the investigator. The NCDB PUF contains de-identified patient data, including demographic information, tumor site, and pathology data, first course of treatment, and mortality. The NCDB PUF does not contain information on recurrence. The NCDB began collecting information on both LVI and on results of gene expression assays in 2010.We utilized a data set derived from the 2005–2016 National Cancer Database (NCDB). NCDB is a nationwide, facility-based database jointly sponsored by the American Cancer Society and the American College of Surgeons Commission on Cancer (CoC). The NCDB contains data collected on over 34,000,000 cancer cases from over 1500 CoC-accredited hospitals, representing over 70% of newly diagnosed cancersThe study population included women age 75 and under-diagnosed between 1 January 2010 and 1 January 2014 with estrogen receptor-positive (ER+) HER2− BCA, measuring up to 5 cm, with 0–3 pathologically involved axillary nodes, treated with definitive surgery as first treatment, and with numeric RS available. Demographic information obtained included age, race, estimated annual household income and educational attainment. Race was classified as white, Black, other  or unknown. Clinical characteristics included tumor size in mm, histologic grade, axillary node involvement, LVI, and numeric RS. Micrometastatic nodal involvement (pN1mi) was classified as node positive. LVI was characterized as present, absent, or unknown. RS was characterized as low, intermediate, or high using TAILORx cutpoints, where 0–10 was defined as low, 11–25 as intermediate, and 26–100 as high.Bivariate associations between categorical variables were examined using the chi-square test. Multivariate Cox proportional hazards model were used to assess the association of LVI, chemotherapy, and RS on OS, while adjusting age, race, tumor size, grade, LN status, Charlson–Deyo comorbidity score, median income, and education level. The estimated HR for each variable in the model, along with its 95% CI, was reported. All tests are two-sided with a significance level ≤5%. Proportionality assumption was examined, and no violation was detected. All analyses were conducted using SAS 9.4 .NCDB PUF data are de-identified, and compliant with HIPAA. Hospitals, health-care providers, and patients are not identified. Patient-informed consent is not obtained prior to institutional data submission to NCDB. As this study utilizes de-identified patient data, with no attempt made to contact or re-identify the subjects, it is deemed exempt from oversight by the Institutional Review Board of Albert Einstein College of Medicine.Further information on research design is available in the Supplemental TablesReporting Summary Checklist