John Hicks

Background: Osteosarcoma (OS) is a fast-growing malignant bone tumor that occurs most often in children and teenagers. Development of pulmonary metastasis is the primary cause of treatment failure and mortality. Our previous studies demonstrated that cytoplasmic p27 interacts with PAK1, enhancing PAK1 phosphorylation and promoting OS pulmonary metastasis. However, the cellular functions of p27 and PAK1 are primarily regulated by phosphorylation, and the roles of specific phosphorylation residues in modulating OS metastatic potential remain unclear. Methods: To study tumor invasiveness and lung metastasis, we employed a CRISPR-based knock-in method to introduce specific mutations—p27-T157A, p27-T157D, PAK1-T423E, and PAK1-K299R—into the 143B OS cell line, followed by in vitro invasion and orthotopic xenograft mouse experiments. These residues were selected for their therapeutic potential, as T157 regulates p27 nuclear–cytoplasmic shuttling, while T423 and K299 modulate PAK1 kinase activity. Results: No significant differences in pulmonary metastasis were observed across p27 mutants compared to parental controls. However, the p27-T157D mutant exhibited increased cytoplasmic mislocalization, elevated PAK1-S144 phosphorylation, and enhanced in vitro invasiveness compared to the p27-T157A mutant and parental 143B cells. The PAK1-K299R mutant, designed to be kinase-dead, showed negligible S144 phosphorylation, consistent with loss of kinase activity. Unexpectedly, this mutant displayed increased T423 phosphorylation and in vitro invasiveness, and significantly enhanced pulmonary metastasis in vivo compared to the PAK1-T423E mutant and parental controls. Conclusions: These findings highlight the complexity of targeting specific p27 and PAK1 phosphorylation sites as an anti-metastatic strategy for OS. While p27-T157 phosphorylation influences cytoplasmic localization and invasiveness, it does not significantly alter metastatic outcomes. Conversely, PAK1-T423 phosphorylation is critical in driving OS metastatic potential, and the kinase-dead K299R mutant’s unexpected pro-metastatic effect suggests that kinase-independent mechanisms or compensatory pathways may contribute to metastasis. Our findings suggest the necessity for a more comprehensive understanding of the phosphorylation dynamics of p27 and PAK1 in metastatic OS. They also indicate that conventional kinase inhibition may be insufficient and underscore the potential benefits of alternative or combinatorial therapeutic strategies, such as targeting kinase-independent functions or other upstream kinases involved in these regulatory pathways.

Background: Metastasis continues to be a leading cause of mortality in osteosarcoma (OS) among pediatric and young adult populations, with few effective therapeutic options available. Despite immunotherapy advancements, its efficacy in OS is hindered by an incomplete understanding of the immunosuppressive tumor microenvironment (TME). Methods: We utilized multiplex imaging mass cytometry and phenoplexing to characterize immune and stromal cell populations within the TME of a tissue microarray comprising 51 primary OS tumors. The prognostic significance of TME cell abundance and spatial cell–cell distance was evaluated using Kaplan–Meier and Cox regression analyses. To investigate macrophage functionality in vivo, we employed orthotopic xenograft mouse models by co-injecting THP-1-derived M0 or M2 macrophages with 143B OS cells to assess their impact on tumor growth and pulmonary metastasis. Mechanisms of macrophage-mediated metastasis were explored using Luminex, ELISA, and transwell migration assays. Results: Our results showed that macrophages dominated the TME, with M0 and M2 subtypes significantly outnumbering M1 macrophages (M1) and other myeloid cells. T cells and myeloid-derived suppressor cells (MDSC) were the second and third most abundant immune populations, respectively. Among stromal cells, endothelial cells predominated over fibroblasts. While individual immunosuppressive cell populations (M2, MDSC, and Treg) showed no direct correlation with clinical outcomes, the collective abundance of M2 and MDSC was significantly associated with reduced metastasis-free survival (MFS, p = 0.0244) and recurrence-free survival (RFS, p = 0.0040). Notably, closer spatial proximity between M2 macrophages and immunosuppressive cells (p = 0.0248) or Ki-67⁺ cells (p = 0.0321) correlated with decreased MFS, suggesting the formation of an M2-centric immunosuppressive and pro-tumor hub. In vivo, co-injection of M2 macrophages with 143B cells significantly enhanced pulmonary metastasis (p = 0.0140). Luminex analysis identified M2-derived MIP-1α (CCL3) as a candidate chemokine driving OS cell metastatic potential. Conclusions: This study provides a high-resolution map of the OS TME, highlighting the prognostic significance of M2 and immunosuppressive cell interactions in driving metastasis, potentially through MIP-1α signaling. These findings establish a foundation for developing targeted immunotherapies to improve outcomes in metastatic OS.

Background: Jaundice within the first 1–2 weeks of a neonate’s life will generally self-resolve; however, if it lasts longer than this time frame it warrants further work up. Direct or conjugated hyperbilirubinemia can suggest neonatal cholestasis, which in turn reflects marked reduction in bile secretion and flow. The differential diagnosis for neonatal cholestasis is broad. Neonatal choledocholithiasis is a rare cause of neonatal cholestasis, but should be considered on the differential diagnosis for patients presenting with elevated conjugated bilirubin. Case presentation: We describe an infant who presented with neonatal cholestasis. He subsequently underwent work up for biliary atresia, as this is one of the more time-sensitive diagnoses that must be made in neonates with conjugated hyperbilirubinemia. He was ultimately found to have choledocholithiasis on magnetic resonance cholangiopancreatography. He was managed conservatively with optimizing nutrition and ursodeoxycholic acid therapy. Conclusions: We found that conservative management, specifically optimizing nutrition and treating with ursodeoxycholic acid, can be a sufficient approach to facilitating resolution of the choledocholithiasis and conjugated hyperbilirubinemia.

Background: In this study, we investigate the impact of epigallocatechin gallate (EGCG), the most abundant and potent catechin in green tea, on a mouse model of inflammatory bowel disease (IBD) and the underlying mechanisms of action. Methods: C57BL/6J mice were subjected to dextran sulfate sodium (DSS)-induced IBD-like disease and then randomly divided into three groups: Model group (MD), low-dose EGCG group (LE, 20 mg/kg/d), and high-dose EGCG group (HE, 50 mg/kg/d). DSS-induced clinical and macroscopic changes were monitored daily. Intestinal permeability was assessed by FITC-Dextran assay. Results: Both high- and low-dose EGCG treatment alleviated clinical manifestations including body weight loss and disease activity index (DAI) of DSS-induced colitis. The DAI score was significantly improved after two days of EGCG treatment. At the end of the study, the macroscopic severity score (MSS) of HE and LE treatment groups were 2.4 ± 1.2, and 2.2 ± 1.0, respectively, significantly lower than that of the controls (5.0 ± 2.1). EGCG treatment also prevented colon shortening, and improved intestinal permeability and histopathological changes. In addition, EGCG treatment attenuated colon inflammation by suppressing colonic levels of pro-inflammatory cytokines IL-6, MCP-1, and TNF-alpha, and inhibited CD3+ T cell and CD68+ macrophage infiltration. Conclusion: EGCG is effective in inflammatory colitis because it reduces cellular and molecular inflammation, and reduces intestinal permeability.

Hemolytic uremic syndrome (HUS) can occur as a primary process due to mutations in complement genes or secondary to another underlying disease. HUS sometimes occurs in the setting of glomerular diseases, and it has been described in association with Denys-Drash syndrome (DDS), which is characterized by the triad of abnormal genitourinary development; a pathognomonic glomerulopathy, diffuse mesangial sclerosis; and the development of Wilms tumor. We report the case of a 46, XX female infant who presented with HUS and biopsy-proven thrombotic microangiopathy. Next generation sequencing of genes with known mutations causative of atypical HUS found that she was homozygous for the Complement Factor H H3 haplotype and heterozygous for a variant of unknown significance in the DGKE gene. Whole exome sequencing identified a de novo heterozygous WT1 c.1384C > T; p.R394W mutation, which is classically associated with Denys-Drash syndrome (DDS). At the time of bilateral nephrectomy five months after her initial biopsy, she had diffuse mesangial sclerosis, typical of Denys-Drash syndrome, without evidence of thrombotic microangiopathy. This unique case highlights HUS as a rare but important manifestation of WT1 mutation and provides new insight into the genetics underlying this association.

Background: Osteosarcoma (OS) is the most common pediatric bone cancer. Despite advances in treatment regimens, the survival rate remains 60–70%. There is an urgent need to identify prognostic biomarkers, so that targeted therapies can be developed to improve the outcome.Procedure: Our laboratory has previously identified that circulating serum amyloid A (SAA) and CXC chemokine ligand 4 (CXCL4) are upregulated in patients with OS. In this study, we tested if they could be used as prognostic biomarkers. We used enzyme‐linked immunosorbent assays to measure their concentrations in serum samples (n = 233) and immunohistochemistry to examine their expressions in primary tumors (n = 37). Prognostic significance of the serum concentrations and tumor expressions of the biomarkers was then evaluated.Results: Patients with “high SAA” and “low CXCL4” circulating levels at diagnosis significantly correlated with a worse outcome (HR = 1.68, P = 0.014), which was independent of the metastatic status. These patients also exhibited a significantly higher rate of poor histologic response to chemotherapy. Furthermore, low tumor expression of CXCL4 correlated with poor survival (HR = 3.57, P = 0.005).Conclusions: Our results demonstrate that circulating SAA and CXCL4 may serve as prognostic biomarkers in OS. Targeting CXCL4 has been reported, suggesting that it may be exploited as a therapeutic target in OS.

Gonadal dysgenesis (GD) is associated with increased risk of gonadal malignancy. Determining a patient’s risk and appropriate timing of gonadectomy is challenging, but immunohistochemical markers (IHM) may help establish the diagnosis of malignant germ cell tumors (GCT). Our objective was to identify the prevalence of specific IHM expression in patients with GD and determine if the patterns of expression can help identify malignancy versus pre-malignancy state. We evaluated the published literature using the Grading of Recommendation, Assessment, Development, and Evaluation (GRADE) system to provide recommendations on the predictive role of IHM in the detection of germ cell malignancy. The data for this retrospective study included karyotype, gonadal location, external masculinization score, age at time of gonadectomy or biopsy, microscopic description and diagnosis of gonadal tissue, and immunohistochemical staining, including octamer binding transcription factor (OCT) 3/4, placental-like alkaline phosphatase (PLAP), β-catenin, alpha-fetoprotein (AFP), and stem cell factor receptor CD117 (c-KIT). Patients with complete or partial GD who had undergone gonadectomy or gonadal tissue biopsy were included. The study included 26 patients with GD, 3 of whom had evidence of GCT (11.5 %, gonadoblastoma, dysgerminoma): 2 had Swyer syndrome, 1 had 46,XY partial GD. One patient with XY partial GD had gonadoblastoma-like tissue. All 4 patients (15 %) had strong expressions of 4 tumor markers (OCT 3/4, PLAP, β-catenin, CD117), as did 5 other patients (19 %, ages 2–14 months) without GCT: 4 had XY GD, 1 had 46,XX GD. β-catenin was expressed in 96 % of patients in a cytoplasmic pattern, CD117 in 78 %, OCT 3/4 in 55 %, PLAP in 37 %, and AFP in 1 patient (4 %). Tumor marker expression was not specific for ruling out malignancy in patients <1 year. In patients older than 1 year, expression of all three markers (OCT 3/4, PLAP, CD117) may be instrumental in the decision-making process for gonadectomy, even in the absence of overt germ cell malignancy. Our literature review suggests that OCT 3/4 expression is most helpful in predicting risk of malignancy. Additional criteria are needed to stratify risk in patients younger than 1 year of age, as these markers are not reliable in that age group.

Gonadal dysgenesis, a condition in which gonadal development is interrupted leading to gonadal dysfunction, is a unique subset of disorders of sexual development (DSD) that encompasses a wide spectrum of phenotypes ranging from normally virilized males to slightly undervirilized males, ambiguous phenotype, and normal phenotypic females. It presents specific challenges in diagnostic work-up and management. In XY gonadal dysgenesis, the presence of a Y chromosome or Y-chromosome material renders the patient at increased risk for developing gonadal malignancy. No universally accepted guidelines exist for identifying the risk of developing a malignancy or for determining either the timing or necessity of performing a gonadectomy in patients with XY gonadal dysgenesis. Our goal was to evaluate the literature and develop evidence-based medicine guidelines with respect to the diagnostic work-up and management of patients with XY gonadal dysgenesis. We reviewed the published literature and used the Grading of Recommendation, Assessment, Development, and Evaluation (GRADE) system when appropriate to grade the evidence and to provide recommendations for the diagnostic work-up, malignancy risk stratification, timing or necessity of gonadectomy, role of gonadal biopsy, and ethical considerations for performing a gonadectomy. Individualized health care is needed for patients with XY gonadal dysgenesis, and the decisions regarding gonadectomy should be tailored to each patient based on the underlying diagnosis and risk of malignancy. Our recommendations, based on the evidence available, add an important component to the diagnostic and management armament of physicians who treat patients with these conditions.

The detection of microscopic hematuria in a child’s urine prompts evaluation for renal and urinary bladder causes. Microscopic hematuria identified during a routine physical examination by the pediatrician is much more common than macroscopic hematuria. Persistent microscopic hematuria is particularly worrisome and may require a percutaneous needle core kidney biopsy to determine whether the etiology is secondary to glomerular disease, tubulointerstitial disease, urinary tract infection, urinary tract structural abnormalities, medications, or toxins. This paper reviews the epidemiology, pathologic features, pathogenesis, treatment, and outcome of familial hematuria (Alport syndrome [hereditary nephritis]), thin basement membrane nephropathy), IgA nephropathy, Henoch-Schönlein purpura, and acute postinfectious glomerulonephritis.