Research at Children's Mercy Month 2021

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A Comprehensive Investigation Into The Influence Of Gender, Ethnicity And Age On Immunoglobulin Concentrations

Santosh Khanal, Earl F. Glynn, Cas LeMaster, Rebecca McLennan, Mark A. Hoffman, and Todd Bradley

This study investigated concentrations of immunoglobulin isotypes IgA, IgE, IgG, IgG1, IgG2, IgG3, IgG4 and IgM with respect to ethnicity, age and gender across a large cohort utilizing the Health Facts data warehouse from Cerner. Immunoglobulin concentrations from 443,442 patients were used, ranging in age from 0 to over 90 years, with varying comorbidities to represent a diverse population. African American and Asian patients had similar immunoglobulin concentrations to each other, but both were significantly different to other ethnicities, most noticeably, Caucasian patients. Apart from IgG1, immunoglobulin concentrations were significantly different between male and female patients. Immunoglobulin concentrations were also dependent on age, with the most significant increase in concentrations occurring in the early years of life. Although the distribution of IgA, IgG and IgM concentrations broadened as age increased, most of the immunoglobulin concentrations analyzed remained stable throughout the middle and late years of life. Upon further examination, male and female patients displayed similar concentrations over time for IgA, IgE and IgM, but for IgG, IgG1, IgG2 and IgG3, female patients had higher concentrations early in life and lower later in life when compared to male patients. This comprehensive investigation clearly demonstrates that immunoglobulin concentrations differ according to age, gender and ethnicity, which is significant and must be considered when deciphering test results for patient diagnosis and treatment.
A Descriptive Examination Of Medication Adherence In Adolescent Patients With Polycystic Ovary Syndrome (PCOS)

Joy Cui, Tania S. Burgert, and Emily Paprocki

Background: The PCOS Clinic is led by a multidisciplinary team that provides specialized treatment to adolescents with PCOS, a condition characterized by menstrual irregularity and hyperandrogenism. Common medications prescribed are insulin sensitizer metformin and oral contraceptives to improve hyperandrogenism and regulate menses. Anecdotally, patients were not taking the prescribed treatment when evaluated for follow up. The goal of this preliminary study was to investigate medication non-adherence and evaluate demographic and health differences between prescription adherent and non-adherent patients.

Methods: A secondary data analysis (n=50) was conducted on patients diagnosed with PCOS ages 13-18 years between August 1, 2015-December 31, 2020. Six patients were omitted due to no information on medication adherence (n=44). All subjects presented with signs of biochemical/clinical hyperandrogenism and irregular menses. Patients were divided into adherent and non-adherent groups based on medical record documentation. Race, ethnicity, medical history (psychiatric disorders, ADHD, and gastrointestinal issues), and insurance coverage were factors compared.

Results: 18 of 44 girls (41%) failed to comply with their prescribed metformin and/or oral contraceptive regimens at first follow up. The majority of patients evaluated were Caucasian (35/44, 70%). Caucasians were more likely to be adherent (63%) compared to non-adherent (37%). Out of the Caucasians who were adherent, 68% were non-Hispanic and 32% were Hispanic. All of the non-Caucasian, non-adherent patients were African American. Of the entire non-adherent group, 39% had a history of anxiety, depression, or ADHD, in contrast to 31% of the adherent group. We did not see an effect of insurance type or history of gastrointestinal issues (GI) on adherence. The most common reasons for non-adherence were GI side effects (44%), prescriptions not refilled (17%), belief that medications did not help (17%), and parent hesitation about daughters being on medications (17%). All patients with GI side effects were taking metformin and none had baseline GI concerns. Of the patients with GI side effects, 63% had a diagnosis of anxiety/depression. None of the other non-adherent groups included patients with psychiatric disorders.

Conclusion: Medication adherence is a barrier to treatment of adolescents with PCOS. Lack of treatment can contribute to menstruation irregularities and metabolic consequences. This increases risk for comorbidities including endometrial cancer, metabolic syndrome, and diabetes. Interventions to improve patient adherence like increased medication counseling regarding management of side effects is one possible intervention. More data is needed to explore the relationship of race and adherence with tailored interventions to remove barriers to care.
A Family Obesity Intervention Combining Motivation Interviewing And Resource Mobilization

Helena Laroche, Amy M J O'Shea, Jessica Angino, Bery Engebretsen, Sarai Rice, Marvin Dejear, Kathy Janz, and Linda Snetselaar

Background: Parents and children influence each other’s behaviors. Obesity prevention is challenging especially among families with few resources.

Methods: This is a randomized controlled trial of a family obesity intervention combining motivation interviewing and connection of families to organizations to obtain needed resources (resource mobilization). Participants are low-income families where one parent has a BMI ≥ 30 and a child aged 6-12 years. The trial compares health coaching using motivational interviewing and enhanced resource mobilization vs written education and basic resource mobilization. Primary outcomes include: Adult BMI; Adult and Child moderate-vigorous physical activity (MVPA) and sedentary time (accelerometers); Family Nutrition and Physical Activity (FNPA) Scale. Secondary outcomes include: composite measure; child BMI Z-score; sugar-sweetened beverages. The intention to treat analysis used linear mixed models and multiple imputation.

Results: 208 families randomized. Racially and ethnically diverse. Adults: 95% female, household income $28,469, BMI 39.5. Children: age 8.6 years, BMI-Z 1.1. Over 12 months: No statistically significant difference between treatment groups. In both groups: Improved FNPA (Increase by 2.8 (1.0) control and 2.2 (0.9) intervention). Increase in sedentary time and decrease in MVPA in children consistent with increasing age. Decrease in sugar-sweetened beverage intake in adults and children (Adults decrease 1.3 servings control and 0.6 serving intervention; Children decrease in 0.7 servings control and 0.5 servings intervention). Improved adult and child composite score. Significant association between receipt of basic resources and improved adult MVPA (increase 20 min/day, p=0.008), and clinically relevant association with adult BMI (-1.2 kg/m2, p= 0.056) and fruit and vegetable servings (+0.36 servings, p=0.053).

Conclusion: Health behavior outcomes among low-income families improved with the receipt of basic resources but do not improve further with the addition of health coaching simultaneously.
A New, Validated Assessment Tool Demonstrates Positive Outcomes In Home Visiting Model For Families Affected By Maternal Substance Use

Danielle Chiang, Oneta Templeton, and Emily Siedlik

The Team for Infants Exposed to Substance abuse (TIES) Program is a longstanding home-based family support program in the Social Work Department that provides a multi-disciplinary, community-based model to address the complex needs of families with young children affected by maternal substance use. TIES developers have created and psychometrically validated a comprehensive tool, the Individualized Family Service Plan (IFSP) goal attainment scale, to assess outcomes in key domains: reduction in maternal substance use, positive parenting, child and maternal health, and income and housing stability.

A virtually interactive poster was presented at the National Research Center for Early Childhood (NRCEC) in December 2020. The poster covers a brief summary of the TIES Program, including its defining characteristics; development of the IFSP goal attainment scale and identification of its key domains; how the goal attainment scale is administered and scored; the exploratory and confirmatory factor analysis conducted to validate the scale for the service population; successful program outcomes as identified by the goal attainment scale; and ongoing research and evaluation efforts underway to demonstrate the effectiveness of the TIES Program.
APOBEC4, A Novel Regulator Of Cellular Aging, Cell Death, And Tumorigenesis

Atul Ranjan, Alejandro Parrales, Atsushi Kaida, Shrikant Anant, and Tomoo Iwakuma

The tumor suppressor p53 is a transcription factor that controls the mRNA level of downstream target genes involved in the regulation of the cell cycle (e.g., p21, Gadd45) and apoptosis (e.g., Puma, Bax), thus leading to tumor suppression. p53 activity is mainly regulated by its main E3 ubiquitin ligase, MDM2, and inhibition of MDM2 leads to p53 activation and tumor suppression.A small molecule MDM2 inhibitor, Nutlin-3a which binds to the p53-binding pocket in MDM2 to activate p53, is presently in cancer clinical trials. However, it is largely unknown which factors play crucial roles in p53-mediated tumor suppression. Toward this goal, we performed an unbiased screening by treating cells, that were infected with a human whole-genome lentiviral shRNA library, with Nutlin-3a. This screen identified several surviving colonies that contained a shRNA for Apobec4. Apobec4 is a member of the APOBEC family of RNA/DNA editing cytidine deaminases but lacks the cytidine deaminase and mutagenic activity. Hence, little is known about its biological function. Here, we demonstrated that cells downregulated for Apobec4 attenuated p53-mediated cell cycle arrest and apoptosis. As a mechanism, Apobec4 bound to p53 and enhanced p53’s binding to the p53-responsive elements in the promoter regions of the p53 target genes. Additionally, we found that Apobec4 mRNA expression was upregulated by p53. Our study, for the first time, reveals that Apobec4, a new binding partner of p53, is crucial for the proper function of p53 and in turn is a novel transcriptional target of p53, thus forming a positive feed-forward loop.
Association Between Added Sugars From Infant Formulas And Rapid Weight Gain In The U.S. Infants And Toddlers

Kai Ling Kong, Brenda Burgess, Katherine Morris, Tyler Re, Holly Hull, Debra Sullivan, and Rocco Paluch

Background: Formulas often contain high amounts of added sugars, though little research has studied their connection to obesity.

Objectives: This study assessed contributions of added sugars from formulas during complementary feeding on total added sugar intakes, and association between these sugars and upward weight-for-age percentile [WFA %ile] crossing (participants crossing a higher threshold percentile had an upward crossing).

Methods: Data from three 24-h dietary recalls for infants (n = 97, 9 – 12 mo) and toddlers (n = 44, 13 – 15 mo) were obtained in this cross-sectional analysis. Foods and beverages with added sugars were divided into 17 categories. Pearson’s correlations were used to test relations between added sugar intake and upward WFA %ile crossing, followed by multivariable regressions when significant. Analysis of variance compared intakes of all, milk-based and table foods between primarily formula-fed vs. breastfed participants. Multivariate regressions were used to test effects of added sugars and protein from all foods vs. added sugars and protein from milk-based sources on upward WFA %ile crossing.

Results: Added sugars from formulas comprised 66% and 7% of added sugars consumed daily by infants and toddlers, respectively. A significant association was observed between upward WFA %ile crossing and added sugars from milk-based sources after controlling for gestational age, sex, age, introduction to solid foods, mean energy intakes, and maternal pre-pregnancy body mass index and education ( β = 0.003, 95% CI: 0.000, 0.007, p = 0.046 ). Primarily formula-fed participants consumed nearly twice the energy from added sugars ( p = 0.003 ) and gained weight faster (upward WFA %ile crossing = 1.1 ± 1.2 vs. 0.3 ± 0.6 , p < 0.001 ) than breastfed counterparts.

Conclusions: Added sugars in formulas predict rapid weight gain in infants and toddlers. Educating mothers on lower-sugar options may enhance childhood obesity prevention.
Characterizing Differential Antibody Responses To Covid-19 Vaccine And Pre-Existing Immunity To Covid-19

Elizabeth Fraley, Eric S. Geanes, Dithi Banerjee, Santosh Khanal, Daniel A. Louiselle, Nick Nolte, Rebecca L. Biswell, Bradley Belden, Angela Myers, Jennifer Schuster, Tomi Pastinen, Elin Grundberg, Rangaraj Selvarangan, and Todd Bradley

SARS-CoV-2 is a novel beta coronavirus that emerged in 2019 and is responsible for the on-going pandemic (COVID-19). The humoral immune response consists of antibodies of multiple isotypes that can block viral progression. We wanted to characterize antibody response to a vaccine (Pfizer, BNT162b2) in a group of Children’s Mercy employees. We compared responses of people with previous SARS-CoV-2 infection (COVID+) to those with no known previous infection (COVID-). We measured antibody isotypes IgG, IgM and IgA to SARS-CoV-2 spike protein subunit 1 (S1), spike protein subunit 2 (S2), receptor binding domain (RBD), and nucleocapsid (NP) at baseline (week 0), after the first dose (week 3) and after the second dose (week 7). At baseline, the COVID+ group had levels of IgG to S1, S2, RBD, NP that were significantly higher than the COVID- group. After the first dose, the COVID+ group continued to show significantly higher IgG levels to S1, S2, RBD, NP. The COVID- group had variable IgG levels, suggesting that protection after the first vaccine is not guaranteed. Due to the extremely high levels of IgG in COVID+ group after the first dose, these individuals may only require one vaccination. At the final time point, COVID- had similar IgG levels to S1, S2, and RBD as the COVID+ group; indicating that protection from the second vaccination was complete. Neutralizing antibody levels were also determined throughout the course of immunization. The COVID+ group had significantly higher neutralizing antibodies compared to the COVID- group at baseline and after the first dose. After the second dose, nearly all participants had high (<85%) neutralizing antibodies present. Finally, using the spike protein we identified regions of high IgG reactivity that may be excellent targets for future vaccine design.

Antibodies can target conserved sites on the virus and cross-react with multiple related coronaviruses. Children are often more recently infected with seasonal coronaviruses than adults and are more resistant to severe COVID-19 disease. It was previously reported that children have high levels of cross-reactive antibodies to SARS-CoV-2 despite never having infection. We wanted to confirm if children have higher levels of cross-reactive antibodies than adults, and further characterize these responses. We measured antibody isotypes in children never exposed to SARS-CoV-2 (cohort from 2017/2016). We found that a portion of children had pre-existing cross-reactive antibodies, and that these antibodies were also present in some adults with no prior history of SARS-CoV-2 infection.
Comparing Three Music Therapy Interventions For Anxiety And Relaxation In Youth With Amplified Pain

Ashley Scheufler, Dustin Wallace, and Emily Fox

Research in pediatric hospitals has shown that active music engagement, preferred music listening, and music-assisted relaxation can decrease anxiety and increase relaxation responses. However, there is little research on the use of music therapy with pediatric chronic pain conditions such as amplified pain syndromes. The purpose of the current study was to examine the effects of 3 specific music therapy interventions (active music engagement, live patient-selected music, and music-assisted relaxation) on anxiety and relaxation levels in youth (ages 10-18) participating in a 40 hr per week hospital-based intensive interdisciplinary pain treatment program. A sample of 48 patients participated in this study which utilized a 3-period, 3-treatment cross-over design with 3 interventions delivered in a quasi-randomized order determined by when the patients started the treatment program. State anxiety was measured via the state form of the State-Trait Inventory for Cognitive and Somatic Anxiety for Children and relaxation scores were assessed with a Visual Analog Scale. Statistically significant changes were found in anxiety and relaxation outcomes across all interventions provided. Results suggest that music therapy services (using active music engagement, live patient-selected music, and music-assisted relaxation) may be an effective modality to decrease anxiety and increase relaxation levels in pediatric patients with amplified pain syndromes.
Development Of A CRISPRi/a Whole-Genome Screen To Discover Novel Immunotherapy Targets In Pediatric Leukaemia

Jacqelyn Nemechek, Bradley C. Stockard, Kealan Schroeder, Fang Tao, Jennifer Pace, John C. Means, Scott T. Younger, and John M. Perry

Treatment of pediatric leukaemia often involves the use of high doses of anthracyclines, which results in severe side-effects including cardiotoxicity and the development of secondary cancers. Based on previous studies showing that anthracyclines such as doxorubicin (DXR) can target therapy-resistant leukemia stem cells (LSCs) at low, generally non-toxic doses, we repurposed DXR as a targeted inhibitor rather than a broadly cytotoxic chemotherapy. In contrast to typical clinical usage, low-dose DXR treatment reduces expression of multiple immune checkpoint (IC) inhibitors, which exposes LSCs to elimination by cytotoxic T lymphocytes. In the laboratory, anthracyclines can also stimulate immunogenic cell stress/death (ICD). Unlike other forms of cellular death, ICD can induce an anti-cancer immune response, effectively immunizing against cancer recurrence. Unfortunately, this effect is generally not observed in the clinic possibly due to the high-dose, combinatorial and thus immunosuppressive nature of current treatment regimens. Indeed, our data indicates this is due to fundamentally unique gene expression programs induced by high vs. low doses. Here, we will test the hypothesis, supported by preliminary data, that low to intermediate DXR exposure reduces IC expression and initiates stress-inducing ICD, respectively. Thus, discovering genes that inhibit or synergize with IC suppression and ICD induction will identify targets that can be activated or inhibited to enhance clinical efficacy of anthracyclines and other ICD-inducers at low concentrations that are not toxic to healthy tissues. Advances in CRISPR-Cas9 gene editing technology have made whole genome screens a practical reality. We are currently utilizing this technology to construct and validate a chemicalgenetic strategy for identifying IC suppressors/ICD inducers. We are optimizing combined genome-wide repression (using CRISPRi) and overexpression (using CRISPRa) screening systems on pediatric leukemia cells treated with low to high exposures of anthracyclines to discover genes that antagonize and synergize with IC suppression/ICD induction vs. drug resistance. Ultimately, this project will reveal novel therapeutic strategies for harnessing the immune system to enhance and potentiate the ICD response of low-dose DXR treatment, thus providing durable, less toxic cures for pediatric cancer.
Development Of Bispecific Antibodies Against Rituximab Resistant Large B Cell Lymphoma

Eric S. Geanes, Stephen Pierce, Santosh Khanal, Rebecca McLennan, and Todd Bradley

Lymphomas account for about 4% of all cancer diagnosis in the United States annually. The most common form of Non-Hodgkin’s lymphoma is known as diffuse large B-cell lymphoma (DLBCL). A frequently used treatment regimen against these lymphatic cancers involve a chemotherapy combination of cyclophosphamide, vincristine, doxorubicin, and prednisone, collectively known as CHOP. In addition to CHOP, the monoclonal antibody Rituximab has been shown to be an effective treatment against DLBCL, targeting the pan B-cell marker CD20. These therapies combined, known as R-CHOP, have been highly effective at treating DLBCL. However, 30-40% of DLBCL patients experience either relapse after R-CHOP treatment or have refractory disease. Mechanisms behind Rituximab resistance are not fully understood. Presumably, one cause for resistance is due to a decrease or loss of CD20 expression by lymphoma cells, in turn causing Rituximab to be less effective against these new cell populations.

We used single cell genomic analysis of B cells to identify alternative cell surface receptors that could be targets for broader immunotherapy. We identified two surface receptors, CD74 and IL-4 Receptor (IL-4R), that were more broadly expressed on all B cell populations as well as CD20 negative B cell populations. CD74 and IL4R were also found to be highly expressed in lymphoma tumor samples by gene expression analysis.

Two monoclonal antibodies are currently in clinical study against CD74 and IL4R, Milatuzumab and Dupilumab respectively. We developed bispecific antibodies to target both CD20 and CD74 together, and CD20 and IL-4R together. To determine the effectiveness of the antibodies, 3 lymphoma cell lines were used: Rituximab-sensitive SU-DHL-4, an intermediate Rituximab-resistant NU-DHL-1, and Rituximab-resistant SU-DHL-8. The individual CD74 and IL-4R antibodies showed no Antibody-dependent cellular cytotoxicity (ADCC) or Antibody-dependent cellular phagocytosis (ADCP), however the bispecific antibodies did have responses to the cell lines for both ADCC and ADCP. The ability of the antibodies to produce apoptosis was measured through Annexin V staining with an additional crosslinking antibody. The bispecific CD20/CD74 antibody and Rituximab had comparable apoptosis to Rituximab-sensitive SU-DHL-4 cells. Interestingly, the bispecific CD20/CD74 antibody exhibited increased apoptosis among Rituximab-resistant SU-DHL-8 cells compared to treatment with Rituximab. This finding provides evidence towards a possible additional therapeutic target of CD74 to help combat treatment resistance among lymphoma patients. These experimental discoveries open the door for the potential of using single cell genetic analysis to assist in identifying future therapeutic targets for not only lymphoma and cancer, but other diseases as well.
Eliminating MHC Restriction From The T Cell Receptor As A Strategy For Immunotherapy

John Szarejko, Thomas M. Yankee, and Douglas Myers

A key hallmark of cancer cells is their ability to evade the immune system through a number of mechanisms including the down regulation of antigen presenting major histocompatibility complexes (MHC). Chimeric antigen receptor (CAR) technology addresses this problem by allowing for a patients’ T cells to be redirected toward different tumor antigens in an MHC independent manor. Although CAR therapy has been successful in the treatment of pediatric B cell malignancies, clinical outcomes have been disappointing for the treatment of most pediatric cancers. Some of these clinical limitations may be attributed to the unregulated surface expression of CARs, which leads to tonic signaling and T cell exhaustion. Moreover, CARs currently used clinically often induce cytokine release syndrome and can damage healthy tissues through on-target/off-tumor effects. While first and second generation (Gen1 and Gen2) CARs contain signaling elements from the T cell receptor (TCR) and costimulatory receptors, they lack key regulatory components necessary for proper T cell activation. To address these limitations, we designed a CAR which incorporates a single chain antibody (scFv) into the endogenous TCR complex. We hypothesize that incorporation of our CAR into the TCR complex will mimic endogenous T cell activation and regulation by including contributions from all CD3 members (CD3δ, CD3ε, CD3γ, and CD3ζ). Furthermore, we propose that endogenous regulation of CAR signaling will prevent the tonic signaling observed with Gen1 and Gen2 CAR constructs and allow for the formation of memory T cells. In contrast to the unregulated expression of Gen1 and Gen2 CARs, surface expression of our novel CAR was limited by the availability of the endogenous CD3 proteins. TCR-based CARs also induced the production of more physiologic levels of cytokines than Gen1 and Gen2 CARs, indicating that the risk of cytokine release syndrome may be lower with TCR-based CARs. Finally, using a xenograft model of B cell lymphoma, we have demonstrated the ability of TCR-based CARs to target and control tumor burden in vivo. Currently, we are examining the effector potential of our TCR-based CAR against pediatric solid tumors. Additionally, we are developing TCR-based CARs capable of signaling not only through the TCR complex but also through endogenous co-stimulatory receptors. In summary, we present a novel platform for CAR therapy that is likely to be less toxic and more broadly applicable than other CARs.
Enhanced Recovery After Surgery (ERAS) Vs Traditional Care In Pediatric Cerebral Palsy Patients Undergoing Bilateral Multilevel Lower Extremity Orthopedic Surgery: A Pilot Study

Nichole M. Doyle, Kathryn Keeler, Todd A. Glenski, Ezra Goodrich, and Marisha Madhira

Introduction: Children with cerebral palsy (CP) often undergo musculoskeletal surgeries throughout their developmental years. Historically, standard of treatment for these patients utilizes an epidural catheter post-operatively along with valium and narcotic as needed for analgesia. Epidural catheter placement in this patient population may not always possible or safe, and having an epidural catheter may decrease mobility and prevent the initiation of physical therapy. In a collaborative effort to improve the patient experience, an enhanced recovery after surgery (ERAS) protocol was developed utilizing peripheral nerve blocks instead of epidural catheters. We hypothesize that by eliminating the use of epidural catheters, patients will be able to be mobilized earlier, pass physical therapy sooner and as a result have a decreased length of hospital stay.

Methods: This IRB approved cohort study has a primary objective of assessing the effectiveness of our ERAS protocol versus non-ERAS in CP patients undergoing bilateral multilevel lower extremity surgeries. 10 consecutive CP patients from June to Sept 2021 undergoing surgery were compared with controls from 2018 to 2020 matched by Gross Motor Function Classification System (GMFCS) score, procedure performed and age. Data collected included type regional/neuraxial anesthesia performed, post-operative need for patient-controlled anesthesia (PCA), narcotic use, valium use, and length of stay.

Results: There were 10 patients in both the control group and the ERAS group. The average GMFCS in the control and ERAS group was 4.20. Post-operative narcotic use in morphine equivalents in the ERAS group was 0.26 mg/kg compared to 0.54 mg/kg in the control group. Post-operative valium use was 0.64 mg/kg in the ERAS group versus 0.60 mg/kg in the control group. Post-operative need for a PCA was zero in the ERAS group compared to 4 in the control group. Post-operative discharge from physical therapy was 1.70 days in the ERAS group versus 2.44 days in the control group. Length of stay was on average 2.20 days in the ERAS group compared to 3.00 days in the control group. (Table 1)

Conclusion: The primary difference for a patient in the ERAS pathway is receiving peripheral nerve blocks instead of an epidural catheter. In the ERAS group there was a decrease in the post-operative narcotic consumption and the average length of stay. By replacing an indwelling epidural catheter with regional anesthesia peripheral nerve blocks at the beginning of the surgery, this ERAS protocol has the potential to improve patient outcomes and improve the patient/family experience.
Experience Using A Combination Of Variant Prioritization Tools In A Large Rare Disease Cohort

Ana S A Cohen, Isabelle Thiffault, Emily Farrow, Warren A. Cheung, Jeffrey J. Johnston, Tricia N. Zion, Lauren E. Bartik, Margaret Gibson, Adam Walter, Laura M B Puckett, Nyshele L. Posey, Brittany D. McDonald, Mary M. Elfrink, Suzanne Herd, Neil Miller, and Tomi Pastinen

We are embarking on the systematic assessment of all exome negative rare disease families consenting to our large-scale genomic program “Genomic Answers for Kids” (GA4K). GA4K aims to collect genomic data and health information from 30,000 children and their families. As it has become clear in recent years, the true challenge lies in the interpretation of such data. With this in mind, GA4K is employing an advanced set of genomic tools to uncover a greater number of candidate variants in these patients. These tools include novel sequencing technologies to test beyond the exome as well as machine-learning analysis methods. We combined two publicly available tools to aid with variant prioritization: Exomiser (Smedley et al. 2015; PMID: 26562621) and AMELIE (Birgmeier et al. 2020; PMID: 32434849). Both tools (E/A) rely on structured phenotyping (with HPO terms) but apply algorithms that explore different features of the variants/genes. Therefore, we hypothesized that combining them would improve speed and accuracy of our analysis of genomic data.

A review of the combined top 50 ranked E/A candidate variants for each proband was carried out for the first 1090 cases. Bioinformatically selected variants were then manually prioritized based on multiple criteria (zygosity, segregation, population frequency, gene function, etc.). In ~40% of cases, the top variants selected from the combined E/A files were consistent with those identified by lengthy expert review. In addition, no strong E/A candidates were identified in ~6% of cases which were positive for a variant that would not have been annotated by these tools (such as copy number variants, deep intronic variants, structural variants, repeat expansions, etc). Moreover, ~34% of cases were deemed negative by both expert analysis and combined E/A ranking, giving us an overall consistency of 80%. Finally, in ~8% of cases, these tools pointed us towards new candidates that may not have otherwise been considered. Thus, combined analytical approaches using Exomiser and AMELIE are effective for variant and disease gene prioritization and automated review of diagnostic interpretation. Furthermore, this ranking method also helped prioritize among genes of unknown significance that should be pursued for further investigations (e.g. through collaborations established on GeneMatcher, other testing technologies, or other modelling), and also provides a means to communicate larger sets of ranked candidate variants. Negative cases are further investigated using PacBio long-read sequencing, along with whole genome bisulphite sequencing and single cell analysis (scATAC/scRNA sequencing).
Exploration Of Genetic Variation Beyond Leukocyte-Derived Germline DNA In A Pediatric Rare Disease Cohort

Tricia N. Zion, Daniel A. Louiselle, Laura M B Puckett, Nyshele L. Posey, Shelby H. Neal, Mary M. Elfrink, Brittany D. McDonald, Alexandra Greathouse, Bradley Belden, Suzanne Herd, Adam Walter, Margaret Gibson, Warren A. Cheung, Jeffrey J. Johnston, Ana S A Cohen, Isabelle Thiffault, Emily Farrow, Neil Miller, Tomi Pastinen, and Elin Grundberg

Current standard of practice for most children with suspected genetic disease is to undergo clinical molecular sequencing on germline-derived DNA taken from a blood draw. While there are exceptions for known somatic genetic diseases (ex. Sturge-Weber syndrome, McCune-Albright syndrome), somatic sources of DNA are not routinely clinically tested due to the invasive nature of a skin punch biopsy and lack of existing structures in some hospital systems for obtaining surgical tissue that might otherwise be discarded^1,2. Germline whole-exome sequencing (WES) currently has a diagnostic yield of between 20-30% in pediatric cohorts, with germline whole-genome sequencing (WGS) increasing this yield by 10-20%^3,4– at least a small percentage of non-diagnostic cases in these cohorts can be attributed to unidentified somatic variation/mosaicism ^5,6.

There are several uses for DNA and RNA derived from tissue as opposed to germline specimens including:
1. Identification of causative somatic genetic variants
2. Identification of somatic mosaicism in selected tissues
3. Functional validation of variants of interest
4. Improved variant annotation
5. Delineation of germline vs. acquired genetic variation in individuals with hematological cancers
Our study seeks to develop a systematic method of routine tissue collection for genetic studies as well as to provide broader evidence for the utility of these specimens in clarifying genetic diagnosis.
Genetically Engineered Mouse Models (GEMM) Core At CMRI

Laramie Pence and Jay L. Vivian

The Genetically Engineered Mouse Models (GEMM) core is a new CMRI resource created to support investigators in the design, generation, and analysis of novel genetically modified mouse strains. The development of new animal models will provide CMRI researchers an unprecedented tool to investigate the etiology and treatment of pediatric disease. The GEMM core staff have comprehensive expertise in both molecular biology and methods for genetic manipulation of the mouse genome, and the facility is outfitted with the necessary equipment to employ state-of-the-art technologies. Recently the core staff have developed a genotyping approach that uses Next Generation Sequencing to identify the full gamut of alleles present in CRISPR-edited founder animals. This procedure streamlines what is sometimes a challenging genotyping task and provides a reliable means to select useful founders. The GEMM core will continue to employ cutting edge methods like these to create and characterize custom mouse models to address the specific research needs of CMRI researchers. In addition to novel model generation the GEMM core offers a host of other services: cryopreservation of sperm and embryos to protect and archive important strains and storage of these samples in the Children’s Research Institute Biorepository (CRIB), colony maintenance and support for difficult breeding colonies, importation of mice from outside facilities via rederivation, and design and implementation of an array of genotyping methods to maintain the correct allele representation within colonies. These supporting services round out the core's ability to meet the needs of CMRI researchers and to achieve the goal of creating animal models that will advance research in pediatric medicine.
High-Resolution Epigenome Analysis In Nasal Samples Derived From Children With Respiratory Viral Infections Reveals Striking Changes Upon Sars-Cov-2 Infection

Konner Winkley, Boryana Koseva, Dithi Banerjee, Warren A. Cheung, Rebecca L. Biswell, Daniel A. Louiselle, Nyshele L. Posey, Margaret Gibson, Ferdaus Hassan, Rangaraj Selvarangan, Tomi Pastinen, and Elin Grundberg

DNA methylation patterns of the human genome can be modified by environmental stimuli and provide dense information on gene regulatory circuitries. We studied genome-wide DNA methylation in nasal samples from infants (<6 >months) applying whole-genome bisulfite sequencing (WGBS) to characterize epigenome response to 10 different respiratory viral infections including SARS-CoV-2. We identified virus-specific differentially methylated regions (vDMR) with human metapneumovirus (hMPV) and SARS-CoV-2 followed by Influenza B (Flu B) causing the weakest vs. strongest epigenome response with 496 vs. 78541 and 14361 vDMR, respectively. We found a strong replication rate of FluB (52%) and SARS-CoV-2 (42%) vDMR in independent samples indicating robust epigenome perturbation upon infection. Among the FluB and SARS-CoV-2 vDMRs, around 70% were hypomethylated and significantly enriched among epithelial cell-specific regulatory elements whereas the hypermethylated vDMRs for these viruses mapped more frequently to immune cell regulatory elements, especially those of the myeloid lineage. The hypermethylated vDMRs were also enriched among genes and genetic loci in monocyte activation pathways and monocyte count from large GWAS. Finally, we perform single-cell RNA-sequencing characterization of nasal mucosa from children positive for FluB and SARS-CoV-2 infections to functionally analyze the epigenome perturbations. Using these data, we support the trends identified in methylation data and highlights the important role for monocytes. All together, we find evidence indicating genetic predisposition to innate immune response upon a respiratory viral infection. Our genome-wide monitoring of infant viral response provides first catalogue of associated host regulatory elements. Assessing epigenetic variation in individual patients may reveal evidence for viral triggers of childhood disease.
Low Dose Doxorubicin Inhibits Immune Checkpoint Upregulation In Acute Leukemias

Bradley C. Stockard, Jacqelyn Nemechek, Kealan Schroeder, Jennifer Pace, and John M. Perry

Background: Chemoresistant leukemia stem cells (LSCs) can cause relapse in leukemia. Doxorubicin (DXR) has been identified as an inhibitor of an immune checkpoint (IC) mechanism of resistance. The objective of this study was to determine the DXR doses that inhibit IC expression upregulation in vitro.

Methods: Kasumi and Jurkat cell lines were treated with low and high doses of DXR determined by previous kill curve experiments. Cells were collected at 12h timepoints over the course of 48h and analyzed for expression of CTLA-4, PD-1, PD-L1, TIGIT, and TIM-3 via flow cytometry. Statistical analysis was done using Tukey’s multiple comparisons test.

Results: 10 and 25 nM DXR treated Kasumi cells showed significantly lower CTLA-4, TIGIT, and TIM-3 expression at 36h (p < 0.0001) and 48h (p < 0.001) compared to 100 nM treated cells. PD-L1 showed lower expression at 48h (p < 0.0001). 10 and 25 nM DXR treated Jurkat cells showed significantly lower CTLA-4, TIGIT, and TIM-3 expression at 36h (p<0.01) and 48h (p<0.0001) compared to 100 nM treated cells. TIGIT and TIM-3 also showed lower expression at 24h (p<0.0001). PD-L1 showed lower expression at 48h (p < 0.0001).

Conclusions: Our results show that in mixed cell populations, low dose DXR prevents the upregulation of multiple ICs within the first two days of treatment. Overall, this presents a promising strategy for preventing a mechanism of resistance using an established chemotherapeutic agent.
Many Clinical Laboratories Performing Next-Generation Sequencing Have No Future Plans To Migrate To GRCH38

Lisa A. Lansdon, Maxime Cadieux-Dion, Byunggil Yoo, Neil Miller, Ana S A Cohen, Lee Zellmer, Lei Zhang, Emily Farrow, Isabelle Thiffault, Elena Repnikova, Linda D. Cooley, Joseph Alaimo, Binu Porath, John Herriges, Carol Saunders, and Midhat Farooqi

Introduction: Analysis of clinical next-generation sequencing (NGS) data requires the Human Reference Genome (HRG) for alignment. Build GRCh38 was released in December 2013 and resolved ~1,000 issues from GRCh37, including erroneous calls within clinically-relevant genes such as F5, ADAMTSL2, RECQL4, NCF1, and RPS17, among others. As most clinical laboratories are using build GRCh37, we were interested to learn their plans for migration to GRCh38, including their proposed timelines and related concerns; therefore, we conducted a survey to define the current landscape of genome alignment in clinical NGS.

Methods: 71 clinical laboratories performing constitutional NGS testing were invited to participate in an unvalidated online survey to understand general laboratory characteristics as well as motivation for migrating or not migrating to GRCh38.

Results: 33 of 71 laboratories responded (46%), 28 of which met criteria for downstream analyses. The overwhelming majority of laboratories (26; 93%) reported using build GRCh37, whereas only 2 laboratories (7%) said that they had already migrated to GRCh38. Surprisingly, 15 of the 26 laboratories who had not yet migrated indicated that they had no future plans to migrate to GRCh38. The most common factors affecting migration for these 26 laboratories were that the benefit of GRCh38 did not outweigh the time and monetary cost to migrate (14; 54%) and, also, that there were insufficient staff available to facilitate the migration (12; 46%).

Conclusion: Clinical laboratories are divided about migrating to build GRCh38 due to limited resources. This is expected to change within the next 1 to 2 years as a variety of large-scale databases (i.e. gnomAD, Genomics England, ClinVar, HGMD, etc.) have already transitioned. We conclude that increased awareness of clinically-relevant variation that may be missed by NGS pipelines using build GRCh37 is needed, and orthogonal bioinformatics approaches to reduce the likelihood of missing such variants should be considered.
Mice With Human-Like Bile Acid Composition Develop PFIC-2- Like Cholestasis

Anthony Nickel, Jay L. Vivian, Chengpeng Bi, Yazen Alnouti, James F. Daniel, and Iván L. Csanaky

Bile acids (BAs) are essential for lipid absorption, bile formation, and regulation of intermediary metabolism. Primary BAs are synthesized in the liver and conjugated with glycine or taurine (T) before biliary excretion. Humans produce two primary BAs, cholic acid (CA) and chenodeoxycholic acid (CDCA). BA synthesis is regulated by the farnesoid X receptor (FXR), which represses Cyp7a1, the rate-limiting enzyme of BA synthesis. Although mice are commonly used models, mice efficiently hydroxylate CDCA by Cyp2c70 at the 6β-position; thus, their dominant BAs are the hydrophilic muricholic acids (MCAs), in contrast to the hydrophobic human BA-pool. These species differences complicate the translational research of BAs. For example, because of the hydrophilic BA composition, the PFIC-2 model, bile salt export pump (Bsep)-null mouse shows moderate changes in phenotype compared to human PFIC-2 patients. To better understand the role of human-like BA metabolism, we engineered Cyp2c70-null mice.

BA concentrations in liver, bile, and plasma, and hepatic gene expression, were quantified in male and female wild-type and Cyp2c70-null mice. Hepatic and plasma concentrations of total (Σ)BAs increased in male mice (liver: +73%, plasma: +5-fold), and were markedly higher in female null mice (liver: +1.79-fold, plasma: +14-fold) compared to WT mice. MCA formation was practically abolished, and CDCA and its metabolites increased markedly, creating a human-like, hydrophobic BA composition in Cyp2c70-null mice. Contrary to significant increase in concentrations of CDCA and LCA in livers of Cyp2c70-null mice, hepatic FXR-SHP and intestinal FXR-Fgf15 pathways were minimally activated. Biliary excretion of ΣBAs decreases in male (-18%) and more in female (-25%) Cyp2c70-null mice, contrary to increased biliary excretion of CDCA (~16-fold) and LCA (~32-fold). Decreased ΣBA excretion was due to reduced biliary excretion of TCA in male mice (-19%), more markedly in female mice (-46%). The ratio of TCDCA/TCA in WT liver is 0.12, whereas it is 1.6 in male and 2.9 in female Cyp2c70-null mice. Murine Bsep has a higher affinity for TCDCA than TCA; therefore, TCDCA can inhibit biliary transport of TCA, especially in female (-46%) Cyp2c70-null mice. Since (T)CA is the natural ligand for murine FXR, due to lower excretion of TCA, the intestinal FXR-Fgf15 pathway (responsible for Cyp7a1 suppression) is not activated. In contrast, the hepatic FXR-pathway is activated, causing 12-α-hydroxylase Cyp8b1 suppression, especially in female Cyp2c70-null mice. In summary, Cyp2c70-null mice have a human-like hydrophobic BA-pool and exhibit PFIC-like cholestatic features, which will help clarify the pathomechanisms of various pediatric liver diseases.
Mutant P53 Degradation By Potential HSP40/J-Domain Protein Inhibitors Derived From A Natural Compound

Mohamed A.A. Alalem, Atul Ranjan, Satomi Yamamoto, Atsushi Kaida, Alejandro Parrales, Sana Farooki, Shrikant Anant, and Tomoo Iwakuma

Mutant p53 (mutp53) functions as an oncogene by promoting metastasis and drug resistance, which is referred to as gain of function (GOF). Accumulation of mutp53 in cancer cells is crucial for their GOF activity while depletion of mutp53 significantly inhibits cancer progression. Hence, targeting mutp53 for degradation is a promising approach for cancer therapy. We recently demonstrated that DNAJA1 binds to and protects unfolded type of mutp53 from degradation. Targeting DNAJA1 could, therefore, inhibit cancer progression by inducing degradation of unfolded mutp53. However, no inhibitors of DNAJA1 or JDPs/HSP40 are clinically available. To identify potential DNAJA1 inhibitors, we performed in-silico docking using a natural compound library with the J-domain of DNAJA1 as a probe. After validation, we identified a plumbagin derivative that decreased the protein levels of unfolded mutp53. CETSA studies confirmed intracellular binding of this compound with DNAJA1. We furthermore synthesized several analogs of this compound and focused on a compound, namely PLTFBH, as it efficiently reduced the protein levels of both DNAJA1 and unfolded mutp53. PLTFBH showed nonspecific cytotoxicity in a manner independent of DNAJA1 or mutp53. However, it showed specific inhibition of cancer cell migration dependent on DNAJA1 and mutp53, since filopodia formation and migration of cells lacking DNAJA1 or mutp53 were minimally affected by PLTFBH. These findings could pave the way toward discovery of a promising targeted therapy for various cancer conditions.
Neighborhood Walkability And Obesity Among Children’s Mercy Primary Care Patients

Jordan A. Carlson, Robin Shook, Ann M. Davis, Amy Papa, Chelsea Steel, Carolina M. Bejarano, Janelle R. Noel-Macdonnell, Shelly Summar, Kelsey Dean, and Sarah Hampl

Background and Purpose. Despite evidence of the importance of neighborhood walkability features in relation to physical activity and obesity, research has been limited in informing localized practice due to small sample sizes and limited geographic coverage. This demonstration study integrated data from the Children’s Mercy pediatric health system with nationally available neighborhood walkability data to inform local decision making around neighborhood environments and childhood obesity.

Methods. Height/weight from clinic visits available in the Electronic Health Record (EHR) and home neighborhood walkability measures from the U.S. Environmental Protections Agency (EPA) were obtained for 15,989 6-17 year olds. Multilevel models accounted for the nested data structure and were adjusted for neighborhood income and child sociodemographics. Findings were mapped across the metropolitan area to inform where stronger neighborhood walkability improvement efforts are likely needed to combat childhood obesity.

Results. In 6-8 year olds, no neighborhood walkability measures were associated with BMIz or obesity. In 9-17 year olds, greater street connectivity and overall walkability were associated with a 0.01-0.04 lower BMIz (Ps = .009-.017), and greater residential density, street connectivity, and overall walkability were associated 5-7% lower odds of being overweight/obese (Ps = .004-.044) per standard deviation increase in environment variable. 45.9% of children in the lowest walkability tertile were overweight or obese, whereas 43.1% of children in the highest walkability tertile were overweight or obese. Maps revealed areas with low walkability and a high income-adjusted percent of children overweight/obese.

Conclusions. Integrating electronic health records with neighborhood environment data is a replicable process that can inform local practice by highlighting the importance of neighborhood features locally and pointing to areas most in need of health promotion efforts. In the Kansas City area, present findings showed that fewer children were overweight/obese in more walkable neighborhoods. The findings point to recommendations for primary care provider and local decision makers (e.g., community leaders, city officials). Children in low-walkable neighborhoods need to be supported to gain access to other/more physical activity opportunities and through advocacy efforts that aim to improve neighborhood-based opportunities for physical activity. Opportunities exist for improving neighborhood walkability through the use of smart growth and pedestrian-oriented development.
Novel HSP40/J-Domain Protein Inhibitors To Deplete Misfolded Mutant P53

Shigeto Nishikawa, Atsushi Kaida, Atul Ranjan, Alejandro Parrales, Mohamed A.A. Alalem, David Johnson, and Tomoo Iwakuma

Background:

Accumulation of oncogenic mutant p53 (mutp53) greatly contributes to cancer progression. Heat shock protein 40 (HSP40), also known as J-domain protein (JDP), has been implicated in stabilization of misfolded mutp53. Recently, we demonstrate that knockdown of DNAJA1 results in degradation of mainly misfolded mutp53 and inhibition of tumor growth. Since no HSP40/JDP inhibitors are currently available in clinics, these findings have prompted us to identify potential HSP40/JDP inhibitors to induce mutp53 degradation.

Methods/Design:

To identify compounds that potentially bind to DNAJA1, we performed an in-silico docking study for the J-domain of DNAJA1. Identified compounds and their analogs were validated for their abilities to deplete misfolded p53 and/or DNAJA1 in multiple cancer cell lines.

Results:

The top 33 compounds were tested for their abilities to reduce the levels of misfolded mutp53 and DNAJA1, allowing us to identify the best candidate, namely #7-3. Eighteen commercially available analogs of #7-3 were further examined. Of these, a compound, namely B#2, had the strongest activity to deplete both DNAJA1 and misfolded mutp53. B#2 reduced misfolded mutp53 in a concentration-dependent and a time-dependent manner without affecting mRNA levels of mutp53. Importantly, B#2 showed minimal effects on the levels of wild-type p53 and DNA contact mutp53.

Conclusions:

Our study, for the first time, has identified a small compound that may inhibit DNAJA1, leading to depletion of misfolded mutp53. This compound and its analogs could be used to inhibit tumor progression as potential novel anti-cancer agents.
Parent Decision Making With Fetal Surgery

Kara Hansen, Kelly Trowbridge, Marni Scott, Kathryn Simpson, and Allie Wayne

Background: Parents who find out that their unborn baby is diagnosed with Spina Bifida have the option of fetal surgery during their pregnancy, a surgery that was studied in the Management of Myelomeningocele (MOMS) Trial and was published in 2011. Parents face a short timeframe and decision making that includes weighing risks and benefits of surgery for both mother and baby.

Study Aims: To learn more about how parents make the decision to pursue fetal surgery for Spina Bifida. To determine the use of the Shared Decision Making framework to describe parent interaction with the healthcare team.

Methods: We conducted a qualitative study using semi-structured interviews and purposive sampling with 10 parents who chose fetal surgery. Interviews were recorded and transcribed verbatim. We used an inductive approach to analyze data from the interviews and conducted thematic analysis.

Preliminary Findings: Parents’ decision making focused primarily on their baby and the potential benefits their child could gain from fetal surgery. Decision-making was swift, early and conclusive. Parents reported gathering information through online groups and being heavily guided by the experience of other parents in these groups who underwent fetal surgery. Parents described the influence of the healthcare team often in interpersonal terms, valuing relationships with those that were supportive, compassionate and normalizing.

Preliminary Conclusions: Through this initial qualitative study, we concluded that parents find comfort and meaning in fetal surgery as a mechanism for caring for their unborn child, and they arrive at that decision primarily through their values, other parents’ shared experiences and compassion received from the healthcare team. We hope to use this information to shape parents’ care in the Fetal Health Center and lay the groundwork for future studies on the psychosocial impact of fetal surgery.
Pediatric Bariatric Patients’ Perception Of Health Status During Covid-19 Pandemic

Bingjie Li, Brooke Sweeney, Denise Skinner, Bryce Miller, and Amy Papa

Objective: To determine how pediatric patients in the process of preparing for bariatric surgery in the Weight Management Clinics at Children’s Mercy Hospital believe their weight, sleep, food, and exercise quantities impact their health during the COVID-19 pandemic, as well as their overall opinions on the severity of COVID-19.

Methods: Participants (N=52, 67% female) received three surveys over the course of six months (Baseline, 3 months, 6 months). They were asked to evaluate how strongly they believed the above factors would affect the severity of their illness if they were to contract COVID-19. They rated on a 5-point Likart scale, and their responses reflected whether these beliefs changed during the course of the pandemic.

Results: 30/52 completed baseline assessments, 27 completed the 3 month survey, and 13 completed the 6 month survey. Of these, 10 completed all three surveys. Preliminary results showed that participants’ beliefs toward the severity of COVID-19 were decreased across most parameters, with the largest decrease occurring when asked their agreement with the statement, “I am more worried about my health now, than before COVID-19,” dropping from an average of 3.1 to 2.4. Participants reported the highest agreement when asked “I believe COVID-19 is a serious disease” with an average of 4.7 at baseline, 4.6 at 3 months, and 4.5 at 6 months. Participants reported the lowest agreement when asked “If I get COVID-19, I think the foods I am eating now will affect how sick I get” with an average of 2.3, 2.6, and 2.3 at baseline, 3 months, and 6 months, respectively.

Conclusion: We know weight gain has been common in children and adults during the pandemic, but participants in our study, while understanding the severity of COVID, are not attributing it to their own health risk. We hypothesized that over time, participants would have improved health awareness and express a stronger correlation between their weight, sleep, food, and exercise and the severity of their illness were they to contract COVID-19. Preliminary results show that participants became less worried over time across multiple parameters, except for their awareness of COVID-19. Strengths of this study include initiation of data collection at the beginning of the pandemic from a high-risk population, while limitations include the reduced number of participants who completed all surveys, which makes extrapolation of data over time difficult. Further research is needed to understand how to motivate people to improve their health during COVID-19.
Pediatric Proximal Phalanx Base Fractures In Fingers: Identifying The Need For Surgical Management

John Schutz, Nicole Look, Andy Lalka, Hannah Korrell, Gabriela Cleary, Johanna Hold, Jennifer Nance, Sarah E. Sibbel, and Micah K. Sinclair

Introduction: Fractures of the base of the proximal phalanx are among the most common finger fractures in children. Immobilization with or without a closed reduction of the digit for 3-4 weeks can lead to good results. The purpose of this retrospective study is to evaluate the change in angular deformity with and without reduction of proximal phalanx base fractures at final follow up.

Methods: A retrospective review of skeletally immature patients treated for a proximal phalanx base fracture between years 2002-2019. The variables collected included: demographics, initial and final angulation and displacement on the lateral and anteroposterior views, treatment group, malunions, Salter Harris classification, and time union. Patients with less than 3-weeks follow up, adequate medical record details, or missing radiographs were excluded.

Results: Five hundred and eighty-four patients met the inclusion criteria and were categorized into non-operative, closed reduction, and operative groups. The average age at time of fracture was 10.8 years. Salter Harris II fractures were the most common injury (86.3%, P=0.001). The largest mechanism of injury being sports 51.6% followed by fall 32.4%. The small 53.9% and ring 15.4% fingers were injured most frequently (P=0.015). There were four malrotations, three in the non-operative and one in the closed reduction group for an overall (0.68%) malrotation rate. Non-operative, closed reduction, and operative groups initial and final median coronal deformity: (2 vs 16 vs 13.5, P=0.0001) and (2 vs 4 vs 1.5, P=0.0001) respectively, differed significantly between and within groups. Similarly initial and final median sagittal angular deformity (2 vs 8 vs 11, P=0.0001) and (2 vs 3 vs 3, P=0.0022) respectively, differed significantly between and within groups. Initial median AP displacement (0 vs 0.85 vs 1.6, P=0.0001) was significantly different between and within groups.

Conclusion: A limited number of proximal phalanx base fractures require surgical management. The great majority can be treated with closed reduction in the emergency room or clinical setting without sedation, resulting in equivalent outcomes of minimal angular deformity. Current treatment methods have led to good results with correction of angular deformity to 2° in both the sagittal and coronal planes at final follow-up.
Plan To Prevent Delegation Of Authority Log Errors

Danielle Wolfe and Hugo Escobar

Introduction:

Accuracy with Delegation of Authority Logs (DOAs) is essential to ensure correct assignment of tasks for research staff. An error can lead to reportable deviations and impact participant safety. Delegation templates are not universal and use different terminology. Lack of uniformity can lead to errors when assigning delegated tasks. Tasks can also overlap among the study members, furthering confusion. As a study evolves, or new staff are added, delegation of responsibilities can be missed. The fluidity of DOAs can lead to reportable errors. We designed a quality improvement project to promptly identify and prevent DOA errors.

Methods:

Bi-weekly meetings including a Principle Investigator (PI) and Research Coordinator (RC) were held to develop a process to examine accuracy of DOAs for each study. Considering DOAs fluctuate based on amendments, and change of study members, a plan was developed for continual monitoring throughout the life of the study. Activity included a three-pronged approach: 1) create universal tool with standardized definitions and assignments for a guide in absence of a DOA template; 2) develop process verifying DOAs for accuracy at study start-up by having a second RC review DOA; and 3) develop process to ensure accuracy of DOAs throughout the study with twice yearly DOA reviews for active studies. A template DOA form was drafted and accepted by the team which provides consistency among defined roles. Next, all RCs were asked to review DOAs of active studies and report errors to the QI team. Finally, a goal was established to decrease the amount of errors found by 50% each year.

Results:

The initial review of DOAs showed 7 (44%) of the 16 studies examined had errors. Examples included missing end dates for staff or lack of updated tasks based on recent certifications. Interestingly, there were also errors related to tasks assigned at study initiation. After a Plan-Do-Study-Act (PDSA) cycle to review the initial data, it was determined DOAs should be verified at the time of study start-up by backup RC. This added step was presented and accepted by the research team. At the next bi-annual review, the error rate was noted to be 18%. The team commitment to this project helped achieve a decrease of 26% in error rate after just one review.

Conclusion

DOAs are fluid documents requiring updates throughout the life of the study. Lack of continuous monitoring and confusing tasks can lead to untimely updates and errors. With standardization of definitions and research roles, combined with the implementation of a periodic review system of active DOAs, we are able to decrease reported errors.
Practices And Procedures In Clinical Pediatric Exercise Laboratories In North America

Kelli M. Teson, Jessica S. Watson, Sandy Knecht, Wayne Mays, Tracy Curran, David D. Williams, Paul Rebovich, and David A. White

Inter-institutional differences in clinical pediatric exercise laboratory (CPEL) practices may affect consistency of patient care and efficacy of multi-center research. Purpose: To 1) describe current practices and procedures in CPELs; and 2) explore differences in CPELs that employ an exercise physiologist (ExP) to those that do not. Methods: A survey (min 37, max 68 items) was distributed to CPELs in the U.S and Canada focusing on three domains: 1) staffing (min 6, max 12 items); 2) volumes, reporting, and interpretation (min 12, max 22 items); and 3) procedures/protocols (min 19, max 34 items). Results: Of the n=73 responses, n=18 were excluded for being ineligible, not completing the survey, or were duplicated responses, producing a final sample of n=55. Most responses were from the U.S. (92.7%), represented a children’s hospital with university affiliation (83.6%), and reported to be cardiology specific (58.2%). ExPs are employed in 56.4% of CPELs (88.2% ExPs with master’s degree or higher). Physicians, cardiovascular techs, respiratory therapists, or nurses were responsible for conducting clinical exercise stress tests (ESTs) in CPELs without an ExP. Emergency life-support, professional, and clinical certifications were required in 92.3%, 27%, and 21.2% of CPELs, respectively. 9.6% of CPELS had no certification requirements. The median volume was 201-400 ESTs/yr. with 20% of the sample performing >800 ESTs/yr. Treadmill and cycle were the primary modalities (80% and 10% of ESTs, respectively). Institution specific exercise protocols were used in 20% of CPELs. 72% of CPELs provide services in addition to ESTs such as cardiac/pulmonary rehab. Non-parametric testing found that those CPELS with an ExP perform a higher volume of ESTs (p < 0.001), are more likely to perform metabolic ESTs (p=0.028), participate in more research (p < 0.001), and provide services in addition to ESTs (p=0.001). Conclusions: Inter-institutional differences in CPELs staffing and operation may warrant efforts for standardization.
Quantitative Assessment Of Glioblastoma Cell Phenotypes Establishes Cell Migration As A Robust Readout Of CRK AND CRKL Activity

Taeju Park, Neka Large, and Tom Curran

Background: The expression levels of CT10 regulator of kinase (Crk) and Crk-like (CrkL) are elevated in many human cancers, including glioblastoma (GBM), and are believed to contribute to poor prognosis. Although Crk and CrkL have been proposed as therapeutic targets in these tumors, the lack of a reliable, quantitative assay to measure Crk and CrkL activity has hindered development of inhibitors.

Methods: Here, we knocked down Crk, CrkL, or both using small interfering RNAs (siRNAs) in a human GBM cell line, U-118MG, to determine the respective, quantitative contributions of Crk and CrkL to cellular phenotypes.

Results: The combined use of specific and potent Crk and CrkL siRNAs induced effective knockdown of CrkII, CrkI, and CrkL. Whereas Crk knockdown did not affect cell morphology, proliferation, adhesion, or invasion, CrkL knockdown caused shrinkage of cells and inhibition of cell proliferation, adhesion, and invasion. Crk/CrkL double knockdown resulted in more pronounced morphological alterations and more robust inhibition of proliferation, adhesion, and invasion. Furthermore, Crk/CrkL double knockdown completely blocked cell migration, and this effect was rescued by transient overexpression of CrkL but not of Crk. Quantification of protein levels indicated that CrkL is expressed more abundantly than CrkII and CrkI in U-118MG cells.

Conclusions/Significances: These results demonstrate both the predominant role of CrkL and the essential overlapping functions of Crk and CrkL in U-118MG cells. Furthermore, our study indicates that migration of U-118MG cells depends entirely on Crk and CrkL. Thus, impedance-based, real-time measurement of tumor cell migration represents a robust assay for monitoring Crk and CrkL activities.

Support: This work was supported by Tom Keaveny Endowed Fund for Pediatric Cancer Research (to TP) and by a MCA Partners Advisory Board grant from Children’s Mercy Hospital (CMH) and the University of Kansas Cancer Center (KUCC) (to TP).
Recurrent Neonatal Herpes Simplex Virus Infection Associated With IRF7 And UNC93B1 Variants

Megan Tucker, Heather Menden, Sheng Xia, Nikita Raje, and Venkatesh Sampath

BACKGROUND: Neonatal herpes simplex virus (HSV) is a devastating disease with high mortality. In adults and children, genetic variants in the Toll-like receptor 3 (TLR3) pathway increase susceptibility to herpes simplex encephalitis (HSE), but the genetic basis of susceptibility to neonatal HSV is unknown. We hypothesized that deleterious variants in the TLR3 pathway increased vulnerability to HSE in neonates. We investigated immunogenetic studies in an infant with neonatal skin, eye, mouth (SEM) HSV followed by HSE.

OBJECTIVES: To combine exome sequencing with in vivo and in vitro immunological functional analysis to discover the immunogenetic basis of HSV vulnerability in proband.

DESIGN/METHODS: The proband developed SEM HSV1 on day 7 of life and recovered fully with acyclovir. At 1 year of age he presented with seizures and was diagnosed with HSV1 HSE. Exome sequencing was performed to identify pathogenic genetic variants. An immune work up including peripheral blood monocyte (PBMC) functional TLR assay was done. Wild type and mutated alleles were transfected into THP1 monocyte cell line stably expressing an interferon regulatory factor 3 (IRF3) promoter-driven luciferase reporter. Poly(I:C) (1ug/mL), a TLR3 ligand, was used to stimulate THP1 for 24hr prior to luciferase assay and qRT-PCR for interferon (IFN) α and β gene expression.

RESULTS: We identified rare missense mutations in IRF7 (Arg100Pro) and UNC93B1 (Pro404Ser) genes. Immune work up was normal except for a total loss of PBMC cytokine response to TLR3 stimulation. Luciferase assays in THP1 showed dramatically reduced TLR3-driven IRF3 promoter activity in response to poly(I:C) with IRF7 and UNC93B1 variants. Similarly, IFNα and IFNβ expression induced by poly(I:C) was enhanced by wild type IRF7 and UNC93B1 alleles, but strongly suppressed by mutant IRF7 and UNC93B1 alleles. Combining the 2 mutant alleles compounded disruption of TLR3 signaling.

CONCLUSION: We identified 2 variants (IRF7, UNC93B1) that disrupted TLR3-responsiveness to HSV in vitro and in vivo in an infant with recurrent HSV disease complicated by HSE. This is the first report of human HSV disease associated with IRF7 mutations. Neonatal HSV may be a phenotype for immunodeficiency due to variants in TLR3 pathway genes. Infants with severe neonatal HSV may warrant genetic screening to identify variants that increase risk of recurrence, and prolonged acyclovir prophylaxis should be considered.
Resting Energy Expenditure Equations Have Lower Validity For Overweight And Obese Versus Healthy Weight Adolescents

Paige Posson, Paul R. Hibbing, and Robin Shook

Objective: The pediatric obesity epidemic has created an urgent need for improved treatment and prevention plans. Clinicians often rely on metabolic equations to predict resting energy expenditure (REE), as other direct measures of REE are often impractical for use in a clinical setting. However, few equations have been validated for use in overweight and obese adolescents. The purpose of this research was to assess whether REE equations have comparable validity for overweight and obese versus healthy weight adolescents.

Methods: Ten equations were used to predict REE for 109 adolescents (n = 77 males; 36.7% overweight or obese). Nine equations were age specific, and the tenth, non-age specific equation was included due to its widespread use on adolescent populations. 95% equivalence testing was used to assess how well each equation agreed with the criterion measure of indirect calorimetry.

Results: For healthy weight adolescents, all ten equations were significantly equivalent to the criterion measure within ±8.4% (p < 0.05), whereas for overweight or obese participants only three equations were equivalent within the same range (p < 0.05).

Summary: Prediction equations tend to be biased toward higher accuracy in healthy weight versus overweight/obese adolescents, unless the original sample specifically included overweight/obese participants. The findings of this research underscore the importance of sample diversity in original development procedures for prediction equations. Clinicians should carefully consider the characteristics of the individual or group being assessed when choosing a prediction equation as commonly utilized equations may not be valid for adolescents across all weight status groups.
Role Of Selective Intubation To Inform Decision Making For Surgical Treatment Of Acquired Lobar Overinflation (ALO) In The Setting Of Pulmonary Hypertension (PH): A Case Report

Lilah Melzer, Brian Birnbaum, and Alvin Singh

Introduction: Bronchopulmonary dysplasia (BPD) remains one of the most common complications of preterm birth. Acquired lobar overinflation (ALO) is a rare, but serious complication of advanced BPD. Characterized by hyperinflation of the affected lobe, it can cause mediastinal shift with collapse of the contralateral, healthy lung resulting in decreased pulmonary reserve. Pathogenesis remains a mystery, but current hypotheses include a combination of intraluminal obstruction and bronchomalacia in the setting of barotrauma from prolonged intubation and lung immaturity¹. We describe one case of acquired lobar overinflation in a patient with severe BPD and pulmonary hypertension (PH).

Case Description: The patient is a 4-month-old infant with history of prematurity (born at 26 weeks gestational age), BPD and hypoxemia requiring 0.5 L/min of supplemental oxygen who was admitted to the intensive care unit for respiratory distress secondary to adenovirus. He required Venovenous Extracorporeal Membrane Oxygenation (VV ECMO), and subsequently developed severe pulmonary hypertension requiring Sildenafil, Bosentan, and IV Treprostinil along with eventual tracheostomy. Serial imaging demonstrated hyperaeration of the left lingula with subsequent mediastinal shift and compression atelectasis of the right lung. Bronchoscopy showed significant malacia and obstruction at the orifice of the left lingula on exhalation. Perfusion scan showed patchy perfusion consistent with pulmonary vascular disease. Due to lack of clinical improvement with aggressive PH management and mechanical ventilation, the right mainstem was selectively intubated. Imaging demonstrated increased aeration of the right lung with improvement of oxyhemoglobin saturations and pCO2. A left lingular lobectomy was performed, and post-operatively over time, the residual left lung re-expanded and pulmonary pressures, oxygenation and ventilation status started improving. After 18 months, he was discharged home on oral Sidenafil, Bosentan and subcutaneous Treprostinil and is currently undergoing evaluation for decannulation.

Conclusion:

Achieving optimal oxygenation and ventilation in patients with severe lobar overinflation and pulmonary hypertension can be challenging. Selective intubation may help to further delineate between healthy versus diseased lung. In this case, right mainstem intubation was both therapeutic and diagnostic; it improved ventilation and facilitated expansion of the previously compressed right lung. More importantly it demonstrated adequate function of the right lung, which is crucial information when considering surgical resection and determining overall prognosis.
Role Of Therapeutic Apheresis In Treatment Of Intrahepatic Cholestasis In Pediatric Sickle Cell Disease

Amy Johnson MD, MBA; Thomas Cochran; Nazia Iqbal; Amanda Graul-Conroy; Lejla Music Aplenc; and Gerald Woods

Current standard of practice for most children with suspected genetic disease is to undergo clinical molecular sequencing on germline-derived DNA taken from a blood draw. While there are exceptions for known somatic genetic diseases (ex. Sturge-Weber syndrome, McCune-Albright syndrome), somatic sources of DNA are not routinely clinically tested due to the invasive nature of a skin punch biopsy and lack of existing structures in some hospital systems for obtaining surgical tissue that might otherwise be discarded^1,2. Germline whole-exome sequencing (WES) currently has a diagnostic yield of between 20-30% in pediatric cohorts, with germline whole-genome sequencing (WGS) increasing this yield by 10-20%^3,4– at least a small percentage of non-diagnostic cases in these cohorts can be attributed to unidentified somatic variation/mosaicism ^5,6.

There are several uses for DNA and RNA derived from tissue as opposed to germline specimens including:
1. Identification of causative somatic genetic variants
2. Identification of somatic mosaicism in selected tissues
3. Functional validation of variants of interest
4. Improved variant annotation
5. Delineation of germline vs. acquired genetic variation in individuals with hematological cancers
Our study seeks to develop a systematic method of routine tissue collection for genetic studies as well as to provide broader evidence for the utility of these specimens in clarifying genetic diagnosis.
Targeting Vulnerabilities Of Cancer Cells Lacking Wild-Type P53

Alejandro Parrales, Shigeto Nishikawa, Atul Ranjan, Peter McDonald, Anuradha Roy, Mitchell Braun, Frank Schoenen, Jenna Wang, Steve Rogers, Melinda Broward, Tyce Bruns, Shrikant Anant, Dan Dixon, Douglas Thamm, Scott Weir, Kathleen Neville, Joy M. Fulbright, and Tomoo Iwakuma

Identification of cancer-specific molecular targets is crucial for development of safe and efficient chemotherapy drugs. Given that mutations and deletions in the p53 gene are frequent and mostly occurs only in cancer cells, identification of compounds that selectively target cancer cells lacking wild-type p53 (wtp53) activity would significantly accelerate development of safe anti-cancer therapies. Here, we performed a high-throughput screen of a chemical library consisting of ~15,000 compounds using several p53-null or -mutated canine and human osteosarcoma cell lines as well as osteoblast cell lines carrying wtp53 as negative controls. This screening identified a new compound, namely KU0171032, as an inducer of cell death in multiple osteosarcoma cells, with minimal effects on non-transformed cells with wtp53. KU0171032 also induced apoptosis much more effectively in p53-null or -mutated cell lines as compared with those with wtp53. Depletion of wtp53 in several cancer cell lines significantly enhanced sensitivity to KU0171032 with increased DNA damage, G2/M cell cycle arrest, and caspase-3 cleavage. Moreover, KU0171032 efficiently inhibited in vivo tumor growth of cancer cells carrying mutant p53 or those knocked down for wtp53. Mechanistically, KU0171032 caused prolonged DNA damage with reduced ATM signaling and impaired DNA double-strand break repair in p53-null/mutated cells, while it caused minimal DNA damage without activation of p53 in wtp53-expressing cells unlike other chemotherapy drugs. Thus, KU0171032 targets a vulnerability of cancer cells lacking wtp53 and could be used for development of novel anti-cancer agents with minimal side effects.
The 2020 Kansas City Regional Report Card On Physical Activity For Children And Youth: A Regional Physical Activity Profile

Elizabeth Wilson, Jordan A. Carlson, Emily Meissen-Sebelius, Matt Kleinmann, Shelly Summar, Russ Pate, Harold Kohl III, and Robin Shook

Background: The Physical Activity Guidelines for Americans recommend that children and youth 6-17 years old engage in at least 60 minutes of moderate-to-vigorous physical activity each day. Research has demonstrated that CM patients who engage in less than the recommended amount of activity face increased risk of obesity. In addition, The Physical Activity Alliance has made calls for communities to develop physical activity plans specific to their regions and emphasized the need for surveillance and reporting of physical activity to enhance efforts to increase rates of physical activity. Given this background, this report card characterizes current physical activity in children and youth in the Kansas City region, establishing a baseline for ongoing surveillance and providing a tool for community advocacy.

Methods: Data were obtained through publicly available sources. Much of the data was drawn from the CM community health needs assessment. Two types of grades, an overall indicator grade and a data quality grade, were assigned in nine physical activity indicators: 1) Active Play; 2) Active Transportation; 3) Community and Built Environment; 4) Family and Peers; 5) Organized Sports Participation; 6)Physical Activity; 7) Physical Fitness; 8) School; and, 9) Sedentary Behavior. Overall indicator grades were derived from the percentage of children or youth meeting indicator criteria, subject matter expert input, and discussion with the regional Kansas City Physical Activity Plan core workgroup. The data quality grade was based on how closely sources aligned with indicator criteria and five standards of data quality: data available at the local level (i.e., Kansas City metro region or county-by-county; required criterion), collected at multiple time points (to track change over time), stratified samples, and publicly available (i.e., free and easily accessible via public websites).

Results: Indicator grades ranged from B- in Organized Sport Participation to F in Active Transportation. Sufficient data were not available in Active Play, Physical Fitness, Family and Peers, or School indicators. Data quality grades ranged from A in Overall Physical Activity to F in Schools.

Conclusions: Less than half of children and youth meet the primary indicator criterion for Overall Physical Activity. Compared to the U.S. Physical Activity report card, the Kansas City region performs worse in Active Transportation and Community and Built Environment indicators and better in Overall Physical Activity, Sedentary Behaviors, and Organized Sports Participation indicators. Systematic surveillance of physical activity should be established in the region to monitor and improve efforts to increase regional physical activity.
The Cellular Genomics Of Human Breast Milk

Cas LeMaster, Stephen Pierce, Santosh Khanal, Staci Elliott, Allison Scott, Daniel A. Louiselle, William E. Truog, Devika Maulik, Tamorah Lewis, Tomi Pastinen, and Todd Bradley

Human breast milk (BM) has been nutritionally and immunologically established as a complex and beneficiary fluid to infants. While some components of BM, such as proteins and antibodies have been extensively studied, the cellular profile of BM has yet to be thoroughly characterized. BM contains a heterogeneous population of maternal cells, including leukocytes, epithelial cells, and mammary gland stem cells. These maternal cells can colonize and penetrate the infant gastrointestinal tract; however, the impact of different cell types and their physiological role is not well understood and necessitates further resolution. In this study, we aimed to define and characterize population cell types using single cell RNA sequencing. We sequenced freshly isolated BM cells from 8 new mothers within the first week of birth and again at two weeks of age, covering several gestational periods that range from extremely preterm (<28 >weeks), to preterm (28-36 weeks), and term (37-41 weeks) deliveries. Milk cells were pelleted and washed, and the soluble portion of the milk was collected for future tangential analysis. Library preparation was performed according to instruction in the 10X Chromium 3’ single-cell kit. Processing was performed using the 10X Genomics workflow. The Cell Ranger Software Suite was used for demultiplexing, barcoding, and UMI quantification. Samples were pre-processed in aggregate. Cell ranger yielded over 93,000 cells from 11 samples. Processing, clustering and marker identification were handled in R using the Seurat and SingleR packages. Cell types were assigned using CellMarker and Celldex reference datasets. We collected cell type data on over 54,000 cells across 27 clusters. The most abundant cells in BM were epithelial cells, macrophages, and neutrophils. Surprisingly, several neural cell types were found in BM. Future analysis will explore the differences in cell types based on gestational periods and infant health, as well as the cellular composition over time.
The Impact Of Antibody Fc Region Genetic Variance On Humoral Immunity

Stephen Pierce, Santosh Khanal, and Todd Bradley

Individuals have variable response to vaccination and immunotherapy that may be attributable to host genetics. A critical yet often overlooked mechanism driving vaccine and immunotherapy efficacy are immunoglobulin (Ig) constant region fragment crystallizable (Fc) effector functions that are critical for antibody stability and effector immune cell function. Our group will thus incorporate genomic, molecular biology and biochemical methods to investigate how Fc variance may impact Fc receptor (FcR) binding kinetics. We have established a next-generation sequencing analysis pipeline to identify and characterize genetic variance in the Ig constant region in various human populations. We will then engineer the observed variants into therapeutic monoclonal antibodies to determine the Fc:FcR binding kinetics of these natural and artificial Ig allotypes using Surface Plasmon Resonance – a highly sensitive biochemical application used to evaluate Receptor/Ligand binding kinetics in an in vitro environment. Finally, we will utilize cell-based functionality assays to determine the extent to which difference in binding kinetics impacts Fc:FcR effector function. Our experimental plan will thus provide us with conclusive data concerning Fc genetic variance, if specific SNP’s can impact Fc:FcR binding kinetics, and if this ultimately would impact Fc:FcR driven effector functions.
The Role Of Stem Cell-Like Memory T Cells In Anti-Cancer Immunosurveillance Against Therapy-Resistant Pediatric Cancer

Fang Tao, Sara McElroy, John Szarejko, Jacqelyn Nemechek, Jennifer Pace, Kealan Schroeder, Santosh Khanal, Todd Bradley, Douglas Myers, and John M. Perry

Leukemia is the most commonly diagnosed pediatric cancer. Acute lymphoblastic leukemia (ALL) has an 85% 5-year-survival rate; yet 20% of patients experience relapse driven by therapy-resistant cells and do not survive long-term. The Wnt/β-catenin and PI3K/Akt signalling pathways interact to confer resistance to cancer therapies. We previously found low dose doxorubicin (DXR) specifically target therapy-resistant leukemic stem cells (LSCs) by inhibiting immune check point genes on LSCs in a T-cell acute lymphoblastic leukemia (T-ALL) mouse model. However, the cellular and molecular mechanisms of how low DXR functions to achieve this anti-cancer immunity are not well understood. Wnt signaling was shown to arrest effector cell differentiation and generate memory stem cells, which preserves anti-tumor potential. As DXR inhibits the cooperative interaction of Akt and β-catenin, we hypothesized that low DXR facilitates immune reactivation against resistant LSCs by regulating T memory stem cells. In this study, we investigated the role of stem-cell like memory T cells in T-ALL. We found that the bone marrow and spleen of T-ALL mice showed exhausted naïve CD8 T cells. Intriguingly, these cells expressed higher levels of surface proteins with stem-like features. Low dose doxorubicin restored the naïve T cell pool, but paradoxically reduced the stem cell-like memory T cells. Further functional analysis of the reactive T memory stem cells by in vitro culture and allogeneic transplantation is ongoing. Furthermore, our single-cell sequencing analysis of flow cytometry sorted LSCs and blast cells revealed significant transcriptome changes after low DXR treatment, further illuminating the nature of T memory stem cells. Meanwhile, we are characterizing the human counterpart of T memory stem cells. These cells may ultimately be pharmacologically stimulated by low DXR while genetically engineered to express chimeric antigen receptors, allowing application of adoptive immunotherapy based on the tumor-specific stem memory T cells and preventing relapse in patients treated with immunotherapy.
Ulnar Epiphysiodesis: Success Of The Index Procedure

Tanner Campbell, Wade Faulk, Kristen Vossler, Allison Goodrich, Sarah E. Sibbel, and Micah K. Sinclair

Introduction: Pre-mature radial physeal closure is a relatively rare occurrence in children. When isolated growth arrest of the radius with continued ulnar growth occurs, the resulting ulnar positive deformity leads to altered wrist mechanics and pain. Timely epiphysiodesis of the distal ulna with and without ulnar-shortening osteotomy can address these issues, but continued ulnar overgrowth is a possible complication. We seek to evaluate the success rate of the primary epiphysiodesis of the ulna and associated clinical outcomes.

Materials & Methods: A chart review was conducted at two children’s hospitals from 2008 to 2019. Patients between the ages of 6 and 18 years old, with pre-mature distal radius physeal closure, with or without positive ulnar variance, and greater than two months follow up were included. We evaluated the following characteristics for each patient: demographics, initial cause of premature radial physeal closure, ulnar variance, additional procedures performed during epiphysiodesis, pre- and post-operative pain, range of motion, instability. Summary statistics were conducted and expressed as proportions, medians and means. A paired t-test evaluated change in ulnar variance for those who had an ulnar shortening osteotomy performed.

Results: Thirty-one patients were identified, and the median age at the time of surgery was 12.2 years (IQR: 3.4). Ulnar shortening osteotomies were performed in 53.1% of cases and distal radius osteotomy in 15.6%. Bone graft was utilized in 25.81% of the epiphysiodesis procedures. There were two failures of primary epiphysiodesis indicating an index success rate of 93.7%. The average combined ulnar variance correction was 4.30 mm (95%CI: 3.17, 5.43). The mean physeal time to closure was 134 days. Pre-operative symptoms were resolved for 90.6% cases at final follow up.

Conclusion: Ulnar epiphysiodesis successfully terminates ulnar physeal growth in 93.7% of cases. Pre-operative symptoms were completely resolved with a median physeal closure of just over 4 months. Ulnar variance was corrected on average by 4.30 millimeters when a radial or ulnar shortening osteotomy was performed at the time of epiphysiodesis.
Vancomycin Auc Monitoring In Individuals With Cystic Fibrosis

Claire Elson, Ellen Meier, Stephanie Duehlmeyer, and Christopher M. Oermann

Purpose: Antibiotic therapy is essential for the treatment of cystic fibrosis (CF) lung infections. Methicillin resistant Staphylococcus aureus (MRSA) infects 20-25% of people with CF (PCF) and is associated with increased morbidity. Treatment of pulmonary exacerbations (PE) often requires hospitalization including increased respiratory treatments and IV antimicrobials. IV vancomycin (IV VANC), which is commonly used for MRSA infections, requires serum concentration monitoring to ensure efficacy and minimize toxicity. Previous monitoring guidelines used trough concentrations to predict efficacy and toxicity. Recent guidelines changed to recommend area under the curve (AUC) modeling

Methods: Children’s Mercy Kansas City (CMKC), changed IV VANC monitoring from trough to AUC measurement on 01 May 2020. A retrospective chart review collected trough monitoring data for all PCF that received IV VANC at CMKC from 01 January 2019 to 31 December 2019. Data for all PCF treated with IV VANC after the AUC monitoring change was collected through 28 February 2021. Patient demographics, details of IV VANC therapy (dose, frequency, total exposure, nephrotoxicity), and monitoring data (serum concentrations and AUC modeling) were collected. Descriptive statistics were used to assess pre- and post-implementation data.

Results: Pre-AUC, 25 patients received 42 courses of IV VANC; 14 were female (56%), and the median age was 14.02 years (4.25-20.25). Median treatment duration was 9.62 days (1.79-26.54), and median daily vancomycin exposure was 71.43 mg/kg/day (49.58-99.29). Target vancomycin trough concentration (>15 mcg/mL) was reached during 18 courses (43%). The median time to therapeutic trough was 83.58 hours (11.55-273.55) and required a median of three phlebotomies (1-9). Post-AUC there have been 15 courses of IV VANC in 8 PCF; 5 were female (63%), and the median age was 17.96 years (7.60-20.10). Median treatment duration was 9.52 days (5.68-14.63) and median daily vancomycin exposure was 75 mg/kg/day (48.63-92.80). All treatment courses reached target vancomycin AUC (400-600 mcg/mL*hr); median time of 20.13 hours (11.6-106.12) and median three phlebotomies (2-8).

Conclusions: Changing to AUC monitoring for IV VANC among PCF, was not associated with a significant change in vancomycin daily exposure or duration. There was a 60% increase in individuals achieving therapeutic targets with AUC monitoring (n=15, 100%) compared to trough monitoring (n=18,40%). AUC monitoring was associated with a decreased time to therapeutic target by 63.45 hours. A difference in nephrotoxicity was not seen. Limitations include short post-implementation period (ten months) and small sample size. Ongoing data collection is planned.

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