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Spalt-Like Transcription Factor 4 (SALL4), a member of the SALL family, is a regulator of embryonic stem cell development that plays a crucial role in cell renewal and proliferation. SALL4 expression is normally repressed in most adult organs but is reactivated in a multitude of solid and blood cancers and upon treatment with hypomethylating agents (HMA). There is outstanding clinical need for the development and validation of a less invasive SALL4 expression assay via peripheral blood, which can be used for monitoring patients on HMA treatment and SALL4-targeting therapy such as molecular glue degraders.

Using the Simoa (Single Molecule Array) platform, a bead-based digital enzyme-linked immunosorbent assay, we cross-tested 5 commercial SALL4 antibodies and 13 antibody pair combinations, using the best antibody pair to test the matrix effects of human plasma, buffy coats, and bone marrow aspirates. Quantitative assay development further included creating recombinant protein standards, antibody pair screening and validation, and pre-analytical optimization. Upon completion of these steps, a retrospective cohort analysis via patient peripheral blood samples is currently being conducted to validate assay accuracy and precision. Additionally, we are aiming to conduct prospective studies to explore the role of the SALL4 protein biomarker in risk stratification and treatment monitoring.

Thus far, for quantitative assay development, we have successfully expressed and purified full-length SALL4 which will be used to calibrate the standard curve and determine the assay’s limit of quantification. We also optimized multiple pre-analytic steps, including our cell lysis protocol, isolation of buffy coats using density gradient separation, and sample collection method comparison. The best antibody pair can detect both SALL4A and SALL4B isoforms, with stronger signal detected with the SALL4A isoform. For the retrospective cohort validation, we have obtained samples for analysis for liver cancer, myelodysplastic syndrome (MDS), and acute myeloid leukemia (AML) with plans to expand to breast cancer and lung cancer.

In conclusion, we have developed an ultrasensitive SALL4 protein expression diagnostic assay for use in monitoring patient treatment response and for guiding targeted therapy by analyzing peripheral blood for both solid and liquid tumors. Future steps include validating the diagnostic assay for specificity and sensitivity in a retrospective cohort analysis and conducting prospective studies to explore the role of the SALL4 protein biomarker for risk stratification, monitoring on HMA treatment, and guiding targeted therapy.

2023 AMA Research Challenge

Ultrasensitive detection of SALL4 oncoproteins for the use in therapeutic monitoring

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Background
Links between cancer and microbes date back four millennia (Sepich-Poore et al. 2021. Science). Recently, we showed that microbial DNA is detectable in tumor tissues and patient blood from dozens of human cancer types (Poore et al. 2020. Nature; Narunsky-Haziza et al. 2022. Cell), suggesting that cell-free microbial DNA (cf-mbDNA) could augment human-centric liquid biopsies of cancer, which currently lack sensitivity at early-stage disease. Thus, we set out to build and evaluate the first multi-omic, multi-species (human+microbe) blood tests for early-stage lung cancer detection with two intended use populations: screening-eligible individuals without known disease (i.e., a “rule-in” test with high specificity), and lung nodule-bearing individuals (i.e., a “rule-out” test with high sensitivity).

Methods
We created a cancer-specific, microbial reference database using ¬de novo metagenome assembly on non-human DNA from 5187 whole-genome sequenced blood and tumor tissue samples. This microbial database was bioinformatically evaluated across 18,782 samples and ten international cohorts, with predictions made using machine learning. Diagnostic performance was measured with and without the addition of human analytes to test for synergy. Source tracking was performed among paired lung tumors, pre-operative plasma, and post-operative plasma to identify what proportion of cf-mbDNA was tumor derived. After tuning the tests using discovery cohort samples, we applied them to validation cohorts comprising (i) screening-eligible individuals without known disease and (ii) lung nodule-bearing individuals.

Results
Microbial DNA discriminated blood samples from 24 cancer types simultaneously with an average pairwise area under the receiver operating characteristic (AUROC) curve of 95.5%. A median of 81.4% of cf-mbDNA was directly attributable to intratumoral microbial DNA via source tracking, indicating its predominant tumor origin. The addition of human cell-free tumor DNA (ctDNA) fragments and two plasma-derived proteins (CEA, osteopontin (OPN)) to cf-mbDNA significantly and synergistically improved diagnostic performance (Delong’s test: p<0.001). Application of the joint host+microbial test to screening-eligible, stage I lung cancers versus healthy smokers showed a sensitivity of 68.2% at 96.2% specificity. Then, collectively across two independent validation cohorts comprising lung nodule-positive individuals, the test significantly outperformed PET-CT and clinical risk scores, with an overall specificity of 49.5% at 95.7% sensitivity.

Conclusion
A multi-omic, multi-species test diagnoses early-stage lung cancer better than existing methods. In January 2023, the FDA provided Breakthrough Designation status for this approach to determine malignancy likelihood of lung nodules for patients who refuse or lack access to PET-CT testing.

Early-stage detection of lung cancer using a multi-omic, multi-species test

Background
Digital microscopy is becoming increasingly popular in the fields of pathological education, tissue-based research, and clinical diagnosis, offering many advantages over light microscopy. With technological advancements and the ease of proliferation of reads across digital methods, pathologists now can view slides across different centers in real time, which allows for a plethora of new modalities of treatment that were not available before due to the limitations of handling slides, such as teleconsultation, quality assurance, and collaborative efforts. The aim of this study was to compare digital versus light microscopy in kidney transplant biopsies at independent sites in the context of a large, multi-center clinical trial [Clinical Trials in Organ Transplantation (CTOT-08) NCT01289717]. 
Methods
The primary objective of this pilot study is to compare digital versus light microscopy in diagnostic kidney transplant biopsies across multiple centers to validate its use in multi-center clinical trials. To achieve this objective, we aim to quantify the level of intra-rater variability by comparing the same reads (cases) across various diagnoses from pathologists at multiple sites between microscopy platforms (digital vs. light). A secondary aim is to evaluate the extent of inter-rater variability, regardless of the platform. For 32 randomly chosen transplant kidney biopsies, glass slides and corresponding digital images were assessed by 3 pathologists in a blinded and randomized fashion. The glass slides were scanned as high-resolution images and viewed with computer software. 
Results
Pathology reads from digital microscopy were comparable to light microscopy with respect to overall diagnosis and individually quantified Banff 2007 criteria. Our analysis revealed no significant differences within raters (intra-rater variability) between the two platforms for the primary diagnoses of acute cellular rejection (ACR) (p=0.646 light vs. digital), interstitial fibrosis and tubular atrophy (i.e., “chronic Injury”) (p=0.317) or borderline ACR (p=0.805) In contrast, the pairwise kappa statistics among raters revealed lower levels of agreement for borderline rejections and chronic injury when compared to acute cellular rejection.
Conclusion
Digital microscopy of kidney allograft biopsies is reliable and comparable to traditional light microscopy. The use of digital microscopy enables improved collaboration and allows for central or duplicate reading of kidney transplant biopsies in the context of a large, multi-center clinical trial. This pilot study confirmed the reliability of digital microscopy when compared to standard light microscopy but showed a low level of agreement among pathologists for some diagnoses, stressing the importance of refined criteria and alternative methods to enhance diagnostic accuracy.




Digital versus light microscopy in kidney transplant biopsies: A multicenter evaluation comparison pilot study

Introduction

Adverse outcomes and complications relating to SARS-CoV-2 (COVID-19) infection may be influenced by a number of factors, including pre-existing comorbidities and past medical history. Despite traumatic brain injury (TBI) being a public health crisis worldwide, the relationship between the prevalence of a recent TBI and adverse outcomes following COVID-19 infection remains poorly understood. This study aims to identify whether patients with a recent TBI face increased risk of mortality and adverse outcomes associated with COVID-19, as compared to patients without a recent TBI.

Methods

We queried the TriNetX United States Collaborative Network and identified patients with a recent TBI within 1 year of a hospital encounter for COVID-19. A propensity-match analysis was performed using baseline demographics and past medical history to compare patients with a recent TBI and COVID-19 to a cohort of patients with COVID-19 without recent TBI. We collected outcome measures within 1 month of COVID-19 diagnosis relating to overall mortality, intensive care unit (ICU) admission, and in-hospital complications. A Kaplan-Meier survival (KMS) analysis was performed to determine overall survival differences.

Results
A total of 13,970 patients were identified in each cohort. The mean age was 59 years old, and over 51% of patients were male. Compared to patients without a recent TBI, patients with a recent TBI had increased risk of hospitalization (OR 3.7 [95%-CI 3.5-4.0]; p<0.001), intubation (OR 2.4 [95%-CI 1.9-3.0]; p<0.001), ICU admission (OR 3.7 [95%-CI 3.3-4.0]; p<0.001), respiratory failure (OR 1.5 [95%-CI 1.4-1.6]; p<0.001), hepatic injury (OR 1.7 [95%-CI 1.3-2.3]; p=0.001), and overall mortality (OR 2.5 [95%-CI 2.2-2.8]; p<0.001) within 1 month of COVID-19 diagnosis. Recent TBI patients had reduced risk of cardiac injury (OR 0.8 [95%-CI 0.7-0.9]; p<0.001). KMS analysis demonstrates that patients with prior TBI had increased mortality within 1 month of COVID-19 diagnosis (HR 2.5 [95%-CI 2.2-2.9]; p<0.001).

Conclusion 

Patients with recent TBI up to 12 months prior to a separate hospital encounter for COVID-19 diagnosis have increased risk of hospitalization, intubation, ICU admission, respiratory failure, hepatic injury, and overall mortality within 1 month of COVID-19 diagnosis. We hope that these results improve medical decision-making and management of patients with a recent TBI and COVID-19.

The Impact of a Traumatic Brain Injury Within 1 Year of COVID-19 Infection: A Propensity-Match Analysis

Glioblastoma Multiforme (GBM) is one of the most common and aggressive high-grade primary brain tumors that arises from the glial cells of the central nervous system and occurs most often in the frontal and temporal lobes. Despite current treatment options, long-term prognosis of GBM remains low with no significant improvement rates. Chimeric antigen receptor (CAR)-expressing T cells target the B cell marker CD19 and have shown increased efficacy in B cell lymphomas, and lymphoblastic leukemias suggesting benefits in GBM. This study aims to quantify the persistence of CAR-T cells in the context of GBM tumor volume following neurosurgical debulking procedure. A radiology software known as the mint Lesion was utilized to provide Tumor Response Assessment by Criteria (TRAC) reports that consist of volumetric measurements of the tumor lesions (target enhancing lesions, non-target enhancing lesions, and non-target non-enhancing lesions). Fluorescence-activated cell sorting (FACS) analysis takes a sample mixture of cells and sorts them into different populations based on specific biomarkers and characteristics. B7-H3 CAR-T cell persistence was increased in patients with a larger post-surgical tumor volume (3.04% Subject 001 with 2438 mm² volume and 2.38% Subject 003 with 224 mm² tumor volume). However, the number of CAR-T cells per 1 mm² was significantly reduced in patients with a larger post-surgical tumor volume (0.0012 cells/mm² Subject 001 and 0.010 cells/mm² Subject 003). This study shows that although there is an overall increased CAR-T cell persistence for a larger tumor volume, the number of CAR-T cells per 1 mm² is significantly smaller for larger tumor volumes, while illustrating that a larger post-surgical tumor bulk volume is correlated with an increased CAR-T cell persistence within 1-week post-infusion. These findings provide novel information and complement other correlative studies as part of the Phase 1 interventional clinical trial for glioblastoma patients.

Glioblastoma Multiforme Tumor Volume and Persistence of Chimeric Antigen Receptor T Cells Following Neurosurgical Debulking

Background
Healthcare disparity is the United States has been a long-standing issue. A disease of interest is diabetic retinopathy, one of the leading causes of blindness in the United States, as the minorities are disproportionately affected. Treatment regimens often consist of panretinal photocoagulation (PRP) and intravitreal injections (IVI) with anti-vascular endothelial growth factor (anti-VEGF). However, the efficacy of the treatment regimens as demonstrated in clinical trials have been limited in real-world settings as these necessitate frequent follow-ups, a challenge given that 25 to 51% of patients with diabetic retinopathy are lost to follow-ups. Furthermore, the effect of LTFU in itself on final outcomes have not been studied to date. Therefore, we examined factors associated with LTFU, the effect of LTFU on proliferative diabetic retinopathy, and its interaction with different treatments.

Methods
A retrospective cohort study at a large safety net hospital examined 271 eyes of 271 patients with PDR that received PRP or IVI over a 2-year period. LTFU was defined as failure to return to care for at least 6 months. Baseline patient characteristics were collected. Ocular data was gathered at the visit before being LTFU, the return visit after being LTFU, and the final clinic visit. Univariate analyses and multivariate regression with adjustment for multiple comparisons to control confounders were conducted. 

Results
A history of LTFU was associated with subsequent neovascularization elsewhere (NVE) and tractional retinal detachment (TRD) but not with final visual acuity (VA). Among those LTFU, those who received IVI were not at higher risk of TRD than those receiving PRP.

Conclusion
Undergoing PRP is associated with LTFU. LTFU is independently associated with subsequent development of NVE and TRD, but not with the final visual acuity. One rationale in opting for PRP over IVI for managing PDR was the risk of the patient being LTFU, but our study did not demonstrate a difference between those who received PRP prior to LTFU and those who received IVI prior to LTFU in terms of the rate of subsequent TRD formation. Further studies should expand upon those findings to design better interventions strategies. 


Out of Sight, Not Out of Mind: The Effect of Loss to Follow-Up on on Proliferative Diabetic Retinopathy

Introduction: 
Current AAOS guidelines recommend that children younger than thirty-six months with a femur fracture be evaluated for non-accidental trauma. However, the likelihood of abuse in walking-age children presenting with these fractures has been found to be less than 10%. These guidelines could subject up to 63% of cases to unnecessary evaluation by Child Protective Services. The purpose of this study was to identify how patient age and femur fracture morphology impact the likelihood that providers assess a fracture as non-accidental and pursue further investigation of possible child abuse. 

Methods:
This study evaluated a questionnaire completed by 75 clinicians from multiple specialties. There were 5 subgroup populations (6, 12, 18, 24, 35 months old) combined with diaphyseal and metaphyseal femur fracture types. For each age group, the same radiograph was presented and described as an oblique diaphyseal fracture in one scenario and spiral diaphyseal fracture in another. Similarly, some cases were described as a corner fracture in one scenario and classic metaphyseal lesion in another. This design assessed if fracture nomenclature, in addition to patient age, impacted clinician suspicion of abuse. Participants assessed case presentations and reported their perceived likelihood the case represented non-accidental trauma utilizing a Likert scale from 1 to 5 corresponding to a percent likelihood. They also reported the percent likelihood they would pursue further investigation. 

Results:
75 questionnaires were analyzed. Mean likelihood of abuse rating in all fracture types for patients 18-35 months was 3.8, corresponding to 61-80% perceived likelihood of abuse. Mean likelihood for further investigation of child abuse in patients 18-35 months of age was 3.7, corresponding to 61-80% clinician likelihood to further investigate. Fracture nomenclature did not significantly impact clinician ratings. In the linear mixed model, the strongest predictors of perceived high likelihood of abuse were respondent specialty, with emergency medicine (p<0.001) and advanced practice providers (p=0.006) having the highest likelihood ratings. 

Conclusions: 
Clinicians are trained to have a high index of suspicion for child abuse in pediatric patients. However, this study demonstrates a high assessment of chance for abuse and low threshold for pursuing further investigation for femoral shaft fractures in walking-age children which contradicts literature that reports actual rates of non-accidental trauma to be lower than 10%. This bias may exhaust hospital and community resources in evaluating toddler femur fractures for abuse and cause unnecessary stress for patients and families.

Common Misperceptions of Child Abuse in Toddler Femur Fractures

Introduction: Hand-foot-mouth disease (HFMD) is a common childhood infectious disease. Atypical skin findings of HFMD, often associated with coxsackievirus A6 (CVA6), were first reported in 2008, with increasing reports worldwide since. Atypical lesions of HFMD often involve sites beyond the palms and soles and tend to have unusual, polymorphic morphology. 
Methods: A systematic review was conducted on clinical features and outcomes of pediatric HFMD with atypical cutaneous manifestations.
Results: Eighty-five studies were included, representing 1,359 cases with mean age 2.4 years and a male predominance of 61%. The most reported morphologies were vesicles (53%), papules (49%), and bullae (36%). Other morphologies included eczema herpeticum-like (19%), purpuric/petechial (7%), and Gianotti Crosti-like (4%). Common atypical sites included the arms and/or legs (47%), face (45%), and trunk (27%). CVA6 was identified in 63% of cases. Symptoms resolved in a mean of 10 days. Overall, 16% of cases received treatment, most commonly with acyclovir, intravenous antibiotics, or topical steroids. The most common complications were nail changes (21%) and desquamation (4%) which occurred a mean of three and two weeks after symptoms, respectively. 
Conclusion: Due to unusual morphologies resembling other conditions, HFMD with atypical cutaneous findings may be misdiagnosed, leading to inappropriate and unnecessary investigations, hospitalization, and treatment. Greater awareness of atypical presentations of HFMD is warranted to improve patient care and counseling on infection control precautions.

Atypical Cutaneous Findings of Hand-Foot-Mouth Disease in Children: A Systematic Review

Background: Life adversities, such as neighborhood disadvantage and lower household incomes, are associated with well-established impairments on language development, which predict academic difficulties throughout school. These language delays are observable by age 2, but it remains unclear if adversities experienced in utero affect neurological underpinnings of language development before delays emerge. Of interest are 3 white matter tracts implicated in early language skills: the corpus callosum (CC), inferior fronto-occipital fasciculus (IFOF), and uncinate (Unc).

Methods: A longitudinal cohort of 160 term-born (>36 weeks gestation) neonates were recruited from the eLABE study at Washington University, which oversampled St. Louis families exposed to poverty. Structural equation modeling was used to create 2 latent constructs of prenatal adversity. The first, “social disadvantage”, aggregates neighborhood disadvantage, family income, insurance status, maternal education, and diet. The second, “maternal psychosocial stress”, models maternal depression, stress, life adversities, and perceived discrimination. At birth, diffusion MRI data were collected, and parameters for white matter tract development were created using probabilistic tractography in FSL. In-person follow-up at age 2 evaluated child language skills. To assess the independent and interactive effects of prenatal adversities and neonatal white matter microstructure on language skills, stepwise linear regression models were conducted after adjustment for child sex, gestational age, and postmenstrual age at scan. 


Results: Prenatal social disadvantage (p=.00) and maternal psychosocial stress (p=.00) predicted significantly lower language scores by age 2. However, in a joint model, only the effects due to social disadvantage remained significant. Lower CC fractional anisotropy predicted significantly lower language scores (p=.02) independently of social disadvantage (p=.00). IFOF and Unc were not predictive of language ( p > .05). To examine the relationship between CC maturation and social disadvantage, a moderation analysis was conducted after dichotomizing families’ income into above and below 200% the federal poverty line (FPL), as most federal assistance programs use this value as a threshold for benefit eligibility. The interaction term was not significant (p= .11), but upon plotting results by income group, the slope for infants above 200% FPL was somewhat stronger. 

Conclusion: This study suggests that prenatal adversity, particularly social disadvantage, and immature CC myelination are strong predictors of language delays by age 2. These data can be used as empirical evidence to advocate for increased funding of federal assistance programs that benefit pregnant people (ex., WIC) and downstream fetal brain development. By continuing to explore the relationship between prenatal adversities on fetal brain development and emerging language skills, future studies will continue to identify specific neurological markers of early language development to allow clinicians to intervene before delays emerge.

The Effects of Prenatal Adversity and Neonatal White Matter Microstructure on Child Language Skills at Age 2

Background
Breast cancer related lymphedema effects one in five breast cancer survivors. This debilitating complication is due to transection and obstruction of lymph flow during surgery. Although lymphedema has been met with new surgical and clinical treatments, little is known about the anatomy of the lymphatic system. Diagnostic dyes have become a vital tool in surgery and lymphatic mapping. Microsurgeons utilize florescent Indocyanine green (ICG) distally in extremities to identify afferent lymphatic pathways. In immediate lymphatic reconstruction (ILR) following mastectomies, ICG helps microsurgeons locate transected channels for anastomosis to a neighboring vein, restoring lymph flow. Isosulfane blue (ISB) is an easily visible dye favored by breast surgeons. In axillary reverse mapping, surgeons inject ISB into the proximal upper extremity, allowing them to locate and preserve lymphatic channels wherever possible. The benefit of utilizing both ICG and ISB during axillary dissection and ILR is reliable visualization of lymphatics intraoperatively. Using dual-dye technique, we aim to differentiate afferent lymphatic drainage from the distal and proximal upper extremity into the axilla.

Methods
To identify the distal and proximal lymphatic zones in breast cancer patients receiving ILR following axillary dissection, ICG and ISB were injected in the ipsilateral arm. ICG was injected intradermally at the first and third webspace and volar wrist, while ISB was injected at the same depth into the upper medial arm. Operative reports were reviewed to determine how often each dye was visualized during axillary dissection. Details of the anastomoses were also documented.

Results
Twenty-six breast cancer patients undergoing ILR following axillary lymph node dissection were reviewed. Twenty-one patients met the criteria of dual-dye technique. ICG was seen in 100% of axillary dissections. ISB was seen with ICG in 61.9% of procedures, indicating a shared lymphatic pathway. ISB was never seen alone. The average lumen size of all lymphatic channels was 1.12mm. An average of 2.46 channels were bypassed to an average of 1.65 veins per procedure. Venous lumen size ranged from 1 to 3 mm. 

Conclusion
Dual-dye technique allows for visualization of axillary lymphatic drainage by the breast and plastic surgeons intraoperatively. Utilizing dyes with different visual properties enabled us to determine if lymphatic flow originated from the distal or proximal upper extremity. This technique could provide useful anatomic data in correlating the development of lymphedema to regional lymphatic drainage within the arm, allowing potential anastomotic connections and clinical treatments. 


Differentiating Afferent Lymphatic Pathways Using Dual-Dye Technique During Immediate Lymphatic Reconstruction

Background: Clinical Practice Guidelines (CPGs) aim to offer guidance to physcians to improve patient outcomes. The Reporting Items for Practice Guidelines in Healthcare (RIGHT) Working Group created a set of rules for CPGs to aid guideline presentation and communication. CPGs in Plastic and Reconstructive surgery have not yet been evaluated by CPG metrics, such as RIGHT. This investigation aims to assess and recognize areas for improvement in currently available guidelines. 
Methods: Three reviewers extracted and reviewed all 5 guidelines listed on the American Society of Plastic Surgeons (ASPS) website in March 2023 and scored them according to the RIGHT checklist. RIGHT criteria in the form of 22 distinct statements were broken down into 35 items, and guidelines were assigned a “1” if they met the criteria and a “0” otherwise. Interrater agreeability through Fleiss’s kappa scores calculated for each guideline. 
Results: Among the 5 PRS guidelines checked against the RIGHT criteria, Fleiss’s kappa interrater agreeability ranged from 0.762 to 0.962, indicating strong agreement among independent raters. However, 4 (22.9%) RIGHT items were not met by any ASPS guidelines. There was a mean adherence of 21 RIGHT items. Guidelines were most lacking in categories concerning reporting of quality and review processes. 
Conclusion: Overall, many RIGHT framework recommendations were met fully by ASPS guidelines, but there is room for improvement in disclosure of the guideline development process. By evaluating fidelity to the RIGHT checklist, specific and actionable opportunities for improvement to guideline language and reporting can be implemented after identification.

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Ultrasensitive detection of SALL4 oncoproteins for the use in therapeutic monitoring

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