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Aims: Both acute placental inflammation and positive mid-gestational cervico-vaginal fetal fibronectin assays have been independently correlated with preterm delivery. We conducted this study to examine the relationship between positive mid-gestational fetal fibronectin (fFN) assays and histological evidence of acute placental inflammation at delivery among women presenting with symptomatic preterm labor. Methods: This retrospective chart review included women who underwent cervico-vaginal fFN testing for preterm contractions between 24-34 weeks gestation and also had placental histological analysis after delivery. Women with a multiple gestation, cerclage, preterm premature rupture of membranes, intercourse or vaginal bleeding within 24 h before the assay were excluded. The primary outcome was histological evidence of acute placental inflammation defined as acute chorioamnionitis, acute deciduitis, funisitis, or microabscess formation. Results: Of 82 women who met all study inclusion criteria, 45% were fFN positive. Women with positive assays were no more likely to have histological evidence of acute inflammation noted at birth than women with negative assays (45% vs. 26%, P=0.07). The assay had a sensitivity of 58.6%, specificity of 62.3%, positive predictive value of 46.0%, and negative predictive value of 73.3% for predicting acute inflammation at delivery. Conclusions: No association exists between positive fetal fibronectin assays and acute histologic placental inflammation at birth. [ABSTRACT FROM AUTHOR]

Oxidative stress, inflammation, and endoplasmic reticulum (ER) stress sequentially occur in bronchopulmonary dysplasia (BPD), and all result in DNA damage. When DNA damage becomes irreparable, tumor suppressors increase, followed by apoptosis or senescence. Although cellular senescence contributes to wound healing, its persistence inhibits growth. Therefore, we hypothesized that cellular senescence contributes to BPD progression. Human autopsy lungs were obtained. Sprague-Dawley rat pups exposed to 95% oxygen between Postnatal Day 1 (P1) and P10 were used as the BPD phenotype. N -acetyl-lysyltyrosylcysteine-amide (KYC), tauroursodeoxycholic acid (TUDCA), and Foxo4 dri were administered intraperitoneally to mitigate myeloperoxidase oxidant generation, ER stress, and cellular senescence, respectively. Lungs were examined by histology, transcriptomics, and immunoblotting. Cellular senescence increased in rat and human BPD lungs, as evidenced by increased oxidative DNA damage, tumor suppressors, GL-13 stain, and inflammatory cytokines with decreased cell proliferation and lamin B expression. Cellular senescence-related transcripts in BPD rat lungs were enriched at P10 and P21. Single-cell RNA sequencing showed increased cellular senescence in several cell types, including type 2 alveolar cells. In addition, Foxo4-p53 binding increased in BPD rat lungs. Daily TUDCA or KYC, administered intraperitoneally, effectively decreased cellular senescence, improved alveolar complexity, and partially maintained the numbers of type 2 alveolar cells. Foxo4 dri administered at P4, P6, P8, and P10 led to outcomes similar to TUDCA and KYC. Our data suggest that cellular senescence plays an essential role in BPD after initial inducement by hyperoxia. Reducing myeloperoxidase toxic oxidant production, ER stress, and attenuating cellular senescence are potential therapeutic strategies for halting BPD progression.

Background: Central nervous system (CNS) cancer is the 10th leading cause of cancer-associated deaths for adults, but the leading cause in pediatric patients and young adults. The variety and complexity of histologic subtypes can lead to diagnostic errors. DNA methylation is an epigenetic modification that provides a tumor type-specific signature that can be used for diagnosis., Methods: We performed a prospective study using DNA methylation analysis as a primary diagnostic method for 1921 brain tumors. All tumors received a pathology diagnosis and profiling by whole genome DNA methylation, followed by next-generation DNA and RNA sequencing. Results were stratified by concordance between DNA methylation and histopathology, establishing diagnostic utility., Results: Of the 1602 cases with a World Health Organization histologic diagnosis, DNA methylation identified a diagnostic mismatch in 225 cases (14%), 78 cases (5%) did not classify with any class, and in an additional 110 (7%) cases DNA methylation confirmed the diagnosis and provided prognostic information. Of 319 cases carrying 195 different descriptive histologic diagnoses, DNA methylation provided a definitive diagnosis in 273 (86%) cases, separated them into 55 methylation classes, and changed the grading in 58 (18%) cases., Conclusions: DNA methylation analysis is a robust method to diagnose primary CNS tumors, improving diagnostic accuracy, decreasing diagnostic errors and inconclusive diagnoses, and providing prognostic subclassification. This study provides a framework for inclusion of DNA methylation profiling as a primary molecular diagnostic test into professional guidelines for CNS tumors. The benefits include increased diagnostic accuracy, improved patient management, and refinements in clinical trial design., Competing Interests: M.S. is scientific advisor and shareholder of C2i Genomics, Heidelberg Epignostix and Halo Dx, and a scientific advisor of Arima Genomics, and received research funding from Lilly USA. Other authors declare no conflict of interest., (© The Author(s) 2023. Published by Oxford University Press, the Society for Neuro-Oncology and the European Association of Neuro-Oncology.)

Competing Interests: The authors declare no conflict of interest.

Next-Generation Sequencing (NGS) allows rapid analysis of multiple genes for the detection of clinically actionable variants. This study reports the analytical validation of a targeted pan cancer NGS panel CANSeq TM Kids for molecular profiling of childhood malignancies. Analytical validation included DNA and RNA extracted from de-identified clinical specimens including formalin fixed paraffin embedded (FFPE) tissue, bone marrow and whole blood as well as commercially available reference materials. The DNA component of the panel evaluates 130 genes for the detection of single nucleotide variants (SNVs), Insertion and Deletions (INDELs), and 91 genes for fusion variants associated with childhood malignancies. Conditions were optimized to use as low as 20% neoplastic content with 5 ng of nucleic acid input. Evaluation of the data determined greater than 99% accuracy, sensitivity, repeatability, and reproducibility. The limit of detection was established to be 5% allele fraction for SNVs and INDELs, 5 copies for gene amplifications and 1,100 reads for gene fusions. Assay efficiency was improved by automation of library preparation. In conclusion, the CANSeq TM Kids allows for the comprehensive molecular profiling of childhood malignancies from different specimen sources with high quality and fast turnaround time., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Schilter, Smith, Nie, Stoll, Felix, Jarzembowski and Reddi.)

Seventeen cases of epithelioid osteoblastoma were reviewed. The tumors most commonly arose from the vertebrae (7 cases), followed by the mandible (3), sacrum (2), bones of the foot (2), and femur, rib, and scapula (1 each). Patients' ages ranged from 5 to 33 years. The tumors measured from 2.0 to 6.5 cm in the greatest diameter (mean = 4.1 cm) and most patients presented with low-grade pain at the affected site. Imaging studies showed expansile lytic lesions with cortical thickening and a mild rim of sclerosis. Histologically all tumors were characterized by active production of bone with a fibrovascular stroma containing microtrabecular aggregates of bone matrix. The osteoblastic proliferation was atypical and showed enlarged cells with prominent nucleoli and abundant cytoplasm imparting them with a striking epithelioid appearance. Immunohistochemical studies showed variable results that caused difficulties for interpretation; 4 of 12 cases showed strong nuclear positivity for FOS, 2 of 12 cases showed strong diffuse nuclear positivity for FOSB; the remaining cases showed variable, sometimes overlapping patterns, considered to be indeterminate. Ki-67 proliferation marker showed low nuclear positivity (∼2%) in 10 cases and a slight increase (<10%) in two cases. Clinical follow-up was available in 14 patients; one patient experienced a recurrence at six months that was treated with additional curetting; the remainder of the patients were all alive and well without evidence of recurrence from 1 to 22 years (median follow up = 3 years). Epithelioid osteoblastoma is an unusual variant of osteoblastoma that has the potential for simulating a malignancy and does not appear to be associated with a more aggressive behavior., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Objectives: Partnerships between low- to middle-income countries (LMICs) and high-income countries (HICs) is one strategy to mitigate observed health disparities. Cambodia's Angkor Hospital for Children (AHC), an LMIC institution, faces shortages in health care resources, including pathology services. A partnership was created with Children's Wisconsin (CW), an HIC hospital, including provision of pathology services. We describe our established pathology workflow, examine cases seen in AHC patients, and evaluate the impact of CW's interpretations., Methods: AHC provides clinical history and impression and ships samples to CW, which processes the samples, and pathologists provide interpretations, sending reports electronically to AHC. For analysis, final diagnoses were considered "concordant," "refined," or "discordant" based on agreement with the clinical impression. Cases were also classified as "did not change management" or "changed management" based on how CW interpretation affected clinical management., Results: We included 347 specimens (177 malignant, 146 benign, 24 insufficient for diagnosis). Of these cases, 31% were discordant and 44% of cases with clinical follow-up had a change in management with CW interpretation., Conclusions: Inclusion of pathology services in LMIC-HIC partnerships is crucial for resolving health disparities between the institutions involved. The described partnership and established pathology workflow can be adapted to the needs and resources of many institutions., (© The Author(s) 2022. Published by Oxford University Press on behalf of American Society for Clinical Pathology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Pediatric tumors are uncommon, yet are the leading cause of cancer-related death in childhood. Tumor types, molecular characteristics, and pathogenesis are unique, often originating from a single genetic driver event. The specific diagnostic challenges of childhood tumors led to the development of the first World Health Organization (WHO) Classification of Pediatric Tumors. The classification is rooted in a multilayered approach, incorporating morphology, IHC, and molecular characteristics. The volume is organized according to organ sites and provides a single, state-of-the-art compendium of pediatric tumor types. A special emphasis was placed on "blastomas," which variably recapitulate the morphologic maturation of organs from which they originate. SIGNIFICANCE: In this review, we briefly summarize the main features and updates of each chapter of the inaugural WHO Classification of Pediatric Tumors, including its rapid transition from a mostly microscopic into a molecularly driven classification systematically taking recent discoveries in pediatric tumor genomics into account., (©2021 The Authors; Published by the American Association for Cancer Research.)

Introduction: 131 I-meta-iodobenzylguanidine ( 131 I-MIBG) is effective in relapsed neuroblastoma. The Children's Oncology Group (COG) conducted a pilot study (NCT01175356) to assess tolerability and feasibility of induction chemotherapy followed by 131 I - MIBG therapy and myeloablative busulfan/melphalan (Bu/Mel) in patients with newly diagnosed high-risk neuroblastoma., Methods: Patients with MIBG-avid high-risk neuroblastoma were eligible. After the first two patients to receive protocol therapy developed severe sinusoidal obstruction syndrome (SOS), the trial was re-designed to include an 131 I-MIBG dose escalation (12, 15, and 18 mCi/kg), with a required 10-week gap before Bu/Mel administration. Patients who completed induction chemotherapy were evaluable for assessment of 131 I-MIBG feasibility; those who completed 131 I-MIBG therapy were evaluable for assessment of 131 I-MIBG + Bu/Mel feasibility., Results: Fifty-nine of 68 patients (86.8%) who completed induction chemotherapy received 131 I-MIBG. Thirty-seven of 45 patients (82.2%) evaluable for 131 I-MIBG + Bu/Mel received this combination. Among those who received 131 I-MIBG after revision of the study design, one patient per dose level developed severe SOS. Rates of moderate to severe SOS at 12, 15, and 18 mCi/kg were 33.3%, 23.5%, and 25.0%, respectively. There was one toxic death. The 131 I-MIBG and 131 I-MIBG+Bu/Mel feasibility rates at the 15 mCi/kg dose level designated for further study were 96.7% (95% CI: 83.3%-99.4%) and 81.0% (95% CI: 60.0%-92.3%)., Conclusion: This pilot trial demonstrated feasibility and tolerability of administering 131 I-MIBG followed by myeloablative therapy with Bu/Mel to newly diagnosed children with high-risk neuroblastoma in a cooperative group setting, laying the groundwork for a cooperative randomized trial (NCT03126916) testing the addition of 131 I-MIBG during induction therapy., (© 2021 Wiley Periodicals LLC.)

Background: Mitochondrial [Formula: see text]-oxidation of fatty acids is the primary energy source for the heart and carried out by Hydroxy Acyl-CoA Dehydrogenase (HADH) encoded trifunctional protein. Mutations in the genes encoding mitochondrial proteins result in functionally defective protein complexes that contribute to energy deficiencies, excessive reactive oxygen species (ROS) production, and accumulation of damaged mitochondria. We hypothesize that a dramatic alternation in redox state and associated mitochondrial dysfunction is the underlying cause of Fatty Acid Oxidation (FAO) deficiency mutant, resulting in heart failure. Mitochondrial co-enzymes, NADH and FAD, are autofluorescent metabolic indices of cells when imaged, yield a quantitative assessment of the cells' redox status and, in turn, that of the tissue and organ., Method: We utilized an optical cryo-imager to quantitively evaluate the three-dimensional distribution of mitochondrial redox state in newborn rats' hearts and kidneys. Redox ratio (RR) assessment shows that mitochondrial dysfunction is extreme and could contribute to severe heart problems and eventual heart failure in the mutants., Results: Three-dimensional redox ratio (NADH/FAD) rendering, and the volumetric mean value calculations confirmed significantly decreased cardiac RR in mutants by 31.90% and 12.32%, in renal mitochondrial RR compared to wild-type control. Further, histological assessment of newborn heart myocardial tissue indicated no significant difference in myocardial tissue architecture in both control and severe (HADHA e4-/- ) conditions., Conclusion: These results demonstrate that optical imaging can accurately estimate the redox state changes in newborn rat organs. It is also apparent that the FAO mutant's heart tissue with a low redox ratio is probably more vulnerable to cumulative damages than kidneys and fails prematurely, contributing to sudden death.

Purpose: With the emergence of the coronavirus disease-2019 (COVID-19) pandemic, institutions were tasked with developing individualized pre-procedural testing strategies that allowed for re-initiation of elective procedures within national and state guidelines. This report describes the experience of a single US children's hospital (Children's Wisconsin, CW) in developing a universal pre-procedural COVID-19 testing protocol and reports early outcomes., Methods: The CW pre-procedural COVID-19 response began with the creation of a multi-disciplinary taskforce that sought to develop a strategy for universal pre-procedural COVID-19 testing which (1) maximized patient safety, (2) prevented in-hospital viral transmission, (3) conserved resources, and (4) allowed for resumption of procedural care within institutional capacity., Results: Of 11,209 general anesthetics performed at CW from March 16, 2020 to October 31, 2020, 11,150 patients (99.5%) underwent pre-procedural COVID-19 testing. Overall, 1.4% of pre-procedural patients tested positive for COVID-19. By June 2020, CW was operating at near-normal procedural volume and there were no documented cases of in-hospital viral transmission. Only 0.5% of procedures were performed under augmented COVID-19 precautions (negative pressure environment and highest-level personal protective equipment)., Conclusion: CW successfully developed a multi-disciplinary pre-procedural COVID-19 testing protocol that enabled resumption of near-normal procedural volume within three months while limiting in-hospital viral transmission and resource use.

Consolidation using high-dose chemotherapy with autologous stem cell transplantation (ASCT) is an important component of frontline therapy for children with high-risk neuroblastoma. The optimal preparative regimen is uncertain, although recent data support a role for busulfan/melphalan (BuMel). The Children's Oncology Group (COG) conducted a trial (ANBL12P1) to assess the tolerability and feasibility of BuMel ASCT following a COG induction. Patients with newly diagnosed high-risk neuroblastoma who did not progress during induction therapy and met organ function requirements received i.v. busulfan (every 24 hours for 4 doses based on age and weight) and melphalan (140 mg/m 2 for 1 dose), followed by ASCT. Busulfan doses were adjusted to achieve to an average daily area under the curve (AUC) <5500 µM × minute. The primary endpoint was the occurrence of severe sinusoidal obstruction syndrome (SOS) or grade ≥4 pulmonary complications within the first 28 days after completion of consolidation therapy. A total of 146 eligible patients were enrolled, of whom 101 underwent BuMel ASCT. The overall incidence of protocol-defined unacceptable toxicity during consolidation was 6.9% (7 of 101). Six patients (5.9%) developed SOS, with 4 (4%) meeting the criteria for severe SOS. An additional 3 patients (3%) experienced grade ≥4 pulmonary complications during consolidation. The median busulfan AUC was 4558 µM × min (range, 3462 to 5189 µM × minute) for patients with SOS and 3512 µM × min (2360 to 5455 µM × minute) (P = .0142). No patients died during consolidation. From the time of study enrollment, the mean 3-year event-free survival for all 146 eligible patients was 55.6 ± 4.2%, and the mean 3-year overall survival was 74.5 ± 3.7%. The BuMel myeloablative regimen following COG induction was well tolerated, with acceptable pulmonary and hepatic toxicity., (Copyright © 2021. Published by Elsevier Inc.)

Pediatric adrenal tumors are unique entities with specific diagnostic, prognostic, and therapeutic challenges. The adrenal medulla gives rise to peripheral neuroblastic tumors (pNTs), pathologically defined by their architecture, stromal content, degree of differentiation, and mitotic-karyorrhectic index. Successful risk stratification of pNTs uses patient age, stage, tumor histology, and molecular/genetic aberrations. The adrenal cortex gives rise to adrenocortical tumors (ACTs), which present diagnostic and prognostic challenges. Histologic features that signify poor prognosis in adults can be meaningless in children, who have superior outcomes. The key clinical, pathologic, and molecular findings of pediatric ACTs have yet to be completely identified., Competing Interests: Disclosure None., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Lesions of bone featuring osteoclast-like giant cells comprise a diverse group of entities, including giant cell tumor (GCT) of bone, chondroblastoma, and aneurysmal bone cyst, among others. The receptor activator of nuclear factor-κB ligand (RANKL) has been implicated in the pathogenesis of GCT of bone and may play a role in the pathogenesis of other giant cell-rich lesions as well. In addition, RANKL inhibitors (denosumab) have also been shown to have some efficacy in treating some giant cell-rich lesions. Herein, we examine RANKL expression by RNA in situ hybridization in a total of 84 osseous lesions with a focus on chondroblastoma, GCT, fibrous dysplasia, and aneurysmal bone cyst. The lesions were tested for RANKL expression using a chromogenic RNA in situ hybridization assay. RANKL expression was identified in 24/25 (96%) GCT, 24/26 (92%) chondroblastomas, 6/7 (86%) aneurysmal bone cysts, and 3/16 (19%) patients with fibrous dysplasia. RANKL expression was statistically lower in chondroblastoma and aneurysmal bone cyst compared with GCT. RANKL reactivity in fibrous dysplasia was exclusively seen in the 3 cases with osteoclast-type giant cells. Our results indicate a high proportion of chondroblastomas, GCTs, and aneurysmal bone cysts express RANKL while reactivity in fibrous dysplasia is dependent on the presence of osteoclast-type giant cells. On the basis of the success of denosumab therapy for GCTs, our results indicate that it may be a potential therapeutic option in other primary osseous tumors.

Placenta-mediated pregnancy complications are a major challenge in the management of maternal-fetal health. Maternal thrombophilia is a suspected risk factor, but the role of thrombotic processes in these complications has remained unclear. Endothelial protein C receptor (EPCR) is an anticoagulant protein highly expressed in the placenta. EPCR autoantibodies and gene variants are associated with poor pregnancy outcomes. In mice, fetal EPCR deficiency results in placental failure and in utero death. We show that inhibition of molecules involved in thrombin generation or in the activation of maternal platelets allows placental development and embryonic survival. Nonetheless, placentae exhibit venous thrombosis in uteroplacental circulation associated with neonatal death. In contrast, maternal EPCR deficiency results in clinical and histological features of placental abruption and is ameliorated with concomitant Par4 deficiency. Our findings unveil a causal link between maternal thrombophilia, uterine hemorrhage, and placental abruption and identify Par4 as a potential target of therapeutic intervention., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Since the original description of pathogenic germline DICER1 variation underlying pleuropulmonary blastoma (PPB), the spectrum of extrapulmonary neoplasms known to be associated with DICER1 has continued to expand and now includes tumors of the ovary, thyroid, kidney, eye, and brain among other sites. This report documents our experience with another manifestation: a primitive sarcoma that resembles PPB and DICER1-associated sarcoma of the kidney. These tumors are distinguished by their unusual location in the peritoneal cavity, associated with visceral and/or parietal mesothelium. A total of seven cases were identified through pathology review in children presenting at a median age of 13 years (range 3-14 years). Primary sites of origin included the fallopian tube (four cases), serosal surface of the colon (one case), and pelvic sidewall (two cases). One case had pathologic features of type I PPB, another type Ir (regressed) PPB, and the remaining five had features of type II or III PPB with a mixed primitive sarcomatous pattern with or without cystic elements. All had a pathogenic DICER1 variation identified in germline and/or tumor DNA. PPB-like peritoneal tumors represent a newly described manifestation of DICER1 pathogenic variation whose pathologic features are also recapitulated in DICER1-related renal sarcoma, cervical embryonal rhabdomyosarcoma, and some Sertoli-Leydig cell tumors with heterologous elements. Tumors arising from the fallopian tube or elsewhere in the abdomen/pelvis, especially those with heterogeneous rhabdomyosarcomatous and/or cartilaginous differentiation, should prompt consideration of germline and tumor DICER1 testing.

Nontuberculous mycobacteria (NTM) infect children with increasing frequency worldwide. Using blood and lymph node tissue from children with NTM lymphadenitis, and uninfected lymph node tissue from community controls, we evaluated helper T (TH) cells in functional assays of TH1/TH17 differentiation and measured the concentration of their associated cytokines at the site of infection. Circulating TH cells from infected children were attenuated in their TH1/TH17 differentiation capacity and expressed less interferon γ and interleukin 17 after polyclonal stimulation. Similar differences were observed at the site of infection, where most cytokine concentrations were unchanged relative to controls. Our data are consistent with a model wherein TH1/TH17 differentiation is attenuated in NTM-infected children., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Core biopsy (CB) is increasingly popular for assessing solid lesions in children. To date, pediatric literature is limited regarding factors contributing to diagnostically inadequate or inaccurate CB. Therefore, we retrospectively examined radiologic/pathologic factors associated with adequacy/accuracy of CB in pediatric patients. A search of the surgical pathology database for CB between January 2007 and December 2014 yielded 134 CB from 99 patients. Age, sex, anatomic site of lesion, CB diagnosis, and final diagnosis were acquired from the electronic medical record. Image guidance modality, lesion size, and CB sampling device were obtained from radiology records. CB hematoxylin and eosin slides were reviewed for fragmentation, percentage of fibrosis, and percentage of necrosis. Overall, CB length was measured using cellSens software and a DP71 camera. Groups were compared using 2-sided homoscedastic Student's t tests; 87.3% (117/134) CB were diagnostic; final diagnosis was available for 105 cases, with a concordance rate of 80.0% (84/105). Image guidance modality, lesion site (extremity vs nonextremity), and CB needle gauge did not significantly differ between diagnostic versus nondiagnostic CB or concordant versus discordant CB. Diagnostic CB had less necrosis and fibrosis than did nondiagnostic CBs (6.8% vs 29.7%, P = .0002 and 10.3% vs 29.1%, P = .0006). Nondiagnostic and discordant CB were more likely to be from bony lesions than soft tissue ( P = .01 and P = .0248). CB is valuable for diagnosing solid lesions in children, with good overall diagnostic rates regardless of lesion size, location, or imaging modality used for biopsy. Nondiagnostic and discordant CB were more often obtained from bony lesions; sampling via open biopsy may be more useful in that setting. Nondiagnostic and discordant CB have more necrosis and fibrosis, suggesting that on-site evaluation of CB tissue viability-for example, by touch imprint or fine needle aspiration-may be useful in further enhancing CB utility.

Introduction: The utility of cytologic evaluation of effusion fluids in adults is well established but has been less well documented in the pediatric population. We examined the clinicopathologic characteristics of children with malignant effusions to establish the value of cytologic examination of these specimens., Materials and Methods: Pleural, pericardial, peritoneal, and intraoperative washing specimens obtained between January 2000 and October 2015 were identified via surgical pathology database search. Relevant clinical and pathologic data was recorded. Statistical analysis of patient groups was performed via two-sided heteroscedastic Student t tests, and Kaplan-Meier analysis was used to compare overall survival between patient groups., Results: 474 effusion/washing specimens obtained from 342 patients were identified: 179 pleural effusions, 220 peritoneal fluids, 28 pericardial effusions, and 47 intraoperative washing specimens. Of these, 13.7% (56 of 474) effusion specimens were positive for malignancy. Among cancer patients with effusions, malignant effusions were seen in 54.5% of patients with rhabdomyosarcoma and 28.8% of patients with hematolymphoid malignancies. Regarding patient outcomes, malignant effusions were associated with statistically significantly shorter overall survival (P = 0.019). When compared with stage IV patients with benign effusions, those with malignant effusions had a shorter 5-year overall survival (P = 0.042); multiple malignant effusions were associated with a dramatically shorter survival than a single malignant effusion., Conclusions: The overall rate of malignant effusions in our patient population was 13.7%. Malignant effusions in children, particularly multiple, portend a poor prognosis. Our observations emphasize the importance of accurate cytologic diagnosis of effusion fluids, and reinforce the value of their cytologic evaluation., (Copyright © 2016 American Society of Cytopathology. Published by Elsevier Inc. All rights reserved.)

Dr. Louis "Pepper" Dehner is an internationally renowned surgical pathologist, especially in the subspecialty of pediatric pathology. Although his clinical and academic expertise are broad, with over 400 published articles, some of his most intriguing contributions have been in the area of pediatric renal and genitourinary pathology. This review focuses on the entities in these following organ systems where he has focused his efforts: malignant rhabdoid tumor, renal medullary carcinoma, Ewing sarcoma/peripheral neuroectodermal tumor, and the DICER1-related lesions cystic nephroma, embryonal rhabdomyosarcoma of the uterine cervix, and Sertoli-Leydig cell tumor., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Bone marrow (BM) nvolvement is common in stage 4/M neuroblastoma patients and profoundly impacts clinical decision-making and predicts outcomes, but to our knowledge no standard exists for immunohistochemical evaluation of staging BMs. We examined the use of three immuno-stains-synaptophysin, tyrosine hydroxylase (TH), and PGP9.5-in detecting metastatic neuroblastoma in BM. We retrospectively selected 174 BM core biopsies from 41 neuroblastoma patients. Immunohistochemistry for synaptophysin, TH, and PGP9.5 was performed. These slides and the hematoxylin and eosin (H&E)-stained slide from each BM were randomized and independently scored by three pathologists as positive, negative, or indeterminate. Cohen's κ coefficients (interobserver agreement), McNemar's test (for frequencies of positive/indeterminate interpretations), and sensitivities for each stain/combination were calculated. Interobserver agreement was higher for all immunostains (synaptophysin, 78%-90%, κ  =  0.548-0.787; TH, 77%-92%, κ  =  0.481-0.788; and PGP9.5, 83%-90%, κ  =  0.601-0.740) than for H&Es (77%-84%, κ  =  0.434-0.572). Indeterminate interpretations were more frequent with H&Es (8.9%) and synaptophysin (6.0%) than with PGP9.5 (3.5%) or TH (3.3%). TH (76%) and PGP9.5 (70%) were the immunostains most likely to correctly resolve indeterminate H&E interpretation. Mean sensitivity among all three pathologists for detection of metastasis compared to the consensus diagnosis was 42.5% for H&E alone, 70.7% to 78.8% for H&E plus one immunostain, and 81.6% to 85% for H&E plus two immunostains. Immunohistochemistry enhanced sensitivity for tumor detection particularly dramatically in cases of prior chemotherapy. PGP9.5 and TH showed good interobserver agreement, fewer indeterminate interpretations, and resolved indeterminate H&E diagnoses at the highest frequencies. Therefore, we recommend H&E and two immunostains, specifically PGP9.5 and TH, for optimal detection of metastatic neuroblastoma in BM.

Chronic inflammation plays a key tumor-promoting role in lung cancer. Our previous studies in mice demonstrated that neutrophils are critical mediators of tumor promotion in methylcholanthrene (MCA)-initiated, butylated hydroxytoluene (BHT)-promoted lung carcinogenesis. In the present study we investigated the role of neutrophil myeloperoxidase (MPO) activity in this inflammation promoted model. Increased levels of MPO protein and activity were present in the lungs of mice administered BHT. Treatment of mice with N-acetyl lysyltyrosylcysteine amide (KYC), a novel tripeptide inhibitor of MPO, during the inflammatory stage reduced tumor burden. In a separate tumor model, KYC treatment of a Lewis Lung Carcinoma (LLC) tumor graft in mice had no effect on tumor growth, however, mice genetically deficient in MPO had significantly reduced LLC tumor growth. Our observations suggest that MPO catalytic activity is critical during the early stages of tumor development. However, during the later stages of tumor progression, MPO expression independent of catalytic activity appears to be required. Our studies advocate for the use of MPO inhibitors in a lung cancer prevention setting.

Background: Of 4,706 peripheral neuroblastic tumors (pNTs) registered on the Children's Cancer Group and Children's Oncology Group Neuroblastoma Study between 1989 and 2010, 51 cases (1.1%) had genotype-phenotype discordance characterized by MYCN amplification (indicating poor prognosis) and Favorable Histology (indicating better prognosis)., Procedure: To distinguish prognostic subgroups in the genotype-phenotype discordant pNTs, two subgroups, "conventional" and "bull's eye," were identified based on the nuclear morphology. The "conventional" tumors (35 cases) included: Neuroblastoma, poorly differentiated subtype (NB-PD, 26 cases) with "salt-and-pepper" nuclei; neuroblastoma, differentiating subtype (4 cases); ganglioneuroblastoma, intermixed (3 cases); and ganglioneuroma, maturing subtype (2 cases). The "bull's eye" tumors included NB-PD with prominent nucleoli (16 cases). Clinicopathologic characteristics of these two subgroups were analyzed. N-myc protein expression was tested immunohistochemically on available tumors., Results: No significant difference was found between these two subgroups in the distribution of prognostic factors such as age at diagnosis, clinical stage, histopathology category/subtype, mitosis-karyorrhexis index, ploidy, 1p LOH, and unbalanced 11q LOH. However, prognosis of the patients with "conventional" tumors (5-year EFS 85.7 ± 12.2%; OS 89.3 ± 10.3%) was significantly better than those with "bull's eye" tumors (EFS 31.3 ± 13.0%; OS 42.9 ± 16.2%; P = 0.0010 and 0.0008, respectively). Immunohistochemically all (11/11) tested "conventional" tumors were negative, and 10/11 tested "bull's eye" tumors were positive for N-myc protein expression., Conclusions: Based on the presence or absence of prominent nucleoli (the putative site of RNA synthesis/accumulation leading to N-myc protein expression), two prognostic subgroups, "conventional" with a better prognosis and "bull's eye" with a poor prognosis, were distinguished among the genotype-phenotype discordant pNTs., (Copyright © 2012 Wiley Periodicals, Inc.)

Embryonal rhabdomyosarcoma (ERMS) is the most common childhood sarcoma and is a component of the familial pleuropulmonary blastoma (PPB)-predisposition syndrome. Using the PPB model, we hypothesized that DICER1 mutations would be found in familial and sporadic forms of ERMS. Blood samples from four children with familial PPB and ERMS, and 52 sporadic ERMS tumors were tested for DICER1 mutations. Germline DICER1 mutations were found in all four patients with familial PPB and 2 of 52 (3.8%) sporadic ERMS had somatic mutations. Our findings confirm the pathogenetic relationship between ERMS and PPB suggesting that ERMS may result from abnormal miRNA regulation., (Copyright © 2011 Wiley Periodicals, Inc.)

Embryonal rhabdomyosarcoma of the uterine cervix is an uncommon presentation of the most common soft-tissue sarcoma in the first decades of life. Unlike embryonal rhabdomyosarcoma in other anatomic sites, in which 70-80% of cases present before 9 years of age, the average age in our series of 14 cervical cases was 12.4 years (median, 13 years), with an age range of 9 months to 32 years at diagnosis. Of the 14 cases, 12 presented as a polyp at the cervical os; two patients had an infiltrative mass in the cervix without a botryoid polyp. The polyps measured 1.5-5 cm and all had the histopathological pattern of the sarcoma botryoides variant of embryonal rhabdomyosarcoma, with condensations of primitive and differentiated rhabdomyoblasts beneath the surface epithelium and around endocervical glands. Nodules of benign-appearing cartilage were present in the stroma of six cases (43%). One of the embyronal rhabdomyosarcomas from the youngest patient, 9 months old, also had a distinctive microscopic focus of immature tubular profiles in a primitive stroma; these tubules expressed epithelial and neuroendocrine markers. Two patients had a pleuropulmonary blastoma, one diagnosed 9 years before the embryonal rhabdomyosarcoma of the cervix and the other recognized synchronously. This latter 9-year old had a DICER1 germline mutation. One patient presented with hirsutism and had a Sertoli-Leydig cell tumor, an incidentally detected cervical embryonal rhabdomyosarcoma, and nodular hyperplasia of the thyroid. Although a pleuropulmonary blastoma was not documented in the latter patient, ovarian sex-cord stromal tumors and nodular hyperplasia of the thyroid are manifestations of the pleuropulmonary blastoma family tumor and dysplasia syndrome (OMIM 601200). Embryonal rhabdomyosarcoma of the cervix must be distinguished from other rare entities, including adenosarcoma, malignant mixed Mullerian tumor and low-grade stromal sarcoma, as the former has a better prognosis; 12 of our 14 patients remain disease-free following conservative surgery and chemotherapy. Our study suggests that cervical embryonal rhabdomyosarcoma may be another pathological manifestation in the spectrum of extrapulmonary pathology in the setting of pleuropulmonary blastoma., Competing Interests: Disclosure/conflict of interest The authors declare no conflict of interest.

Ku70 was first characterized as a nuclear factor that binds DNA double-strand breaks in nonhomolog end-joining DNA repair. However, recent studies have shown that Ku70 is also found in the cytoplasm and binds Bax, preventing Bax-induced cell death. We have shown that, in neuroblastoma cells, the binding between Ku70 and Bax depends on the acetylation status of Ku70, such that, when Ku70 is acetylated, Bax is released from Ku70, triggering cell death. Thus, to survive, in neuroblastoma cells, cytoplasmic Ku70 acetylation status is carefully regulated such that Ku70 is maintained in a deacetylated state, keeping Bax complexed with Ku70. We have shown that overexpression of CREB-binding protein (CBP), a known acetyltransferase that acetylates Ku70, releases Bax from Ku70, triggering apoptosis. Although we have shown that blocking deacetylase activity using non-type-specific inhibitors also triggers Ku70 acetylation and Bax-dependent cell death, the targets of these deacetylase inhibitors in neuroblastoma cells remain unknown. Here, we demonstrate that, in neuroblastoma cells, histone deacetylase 6 (HDAC6) binds Ku70 and Bax in the cytoplasm and that knocking down HDAC6 or using an HDAC6-specific inhibitor triggers Bax-dependent cell death. Our results show that HDAC6 regulates the interaction between Ku70 and Bax in neuroblastoma cells and may be a therapeutic target in this pediatric solid tumor.

Clusterin is a ubiquitously expressed glycoprotein with multiple binding partners including IL-6, Ku70, and Bax. Clusterin blocks apoptosis by binding to activated Bax and sequestering it in the cytoplasm, thereby preventing Bax from entering mitochondria, releasing cytochrome c, and triggering apoptosis. Because increased clusterin expression correlates with aggressive behavior in tumors, clusterin inhibition might be beneficial in cancer treatment. Our recent findings indicated that, in neuroblastoma cells, cytoplasmic Bax also binds to Ku70; when Ku70 is acetylated, Bax is released and can initiate cell death. Therefore, increasing Ku70 acetylation, such as by using histone deacetylase inhibitors, may be therapeutically useful in promoting cell death in neuroblastoma tumors. Since clusterin, Bax, and Ku70 form a complex, it seemed likely that clusterin would mediate its anti-apoptotic effects by inhibiting Ku70 acetylation and blocking Bax release. Our results, however, demonstrate that while clusterin level does indeed determine the sensitivity of neuroblastoma cells to histone deacetylase inhibitor-induced cell death, it does so without affecting histone deacetylase-inhibitor-induced Ku70 acetylation. Our results suggest that in neuroblastoma, clusterin exerts its anti-apoptotic effects downstream of Ku70 acetylation, likely by directly blocking Bax activation.

Background: Previously, we have shown that endothelial microparticles (EMPs) injected into mice induce acute lung injury (ALI) [1]. In this study, we hypothesize that EMPs induce ALI by initiating cytokine release in the lung, leading to recruitment and activation of neutrophils., Materials and Methods: C57BL/6J male mice (8-10 wk old) were intravenously injected with EMPs (200,000/mL), LPS (2 mg/kg), or both. Bronchoalveolar lavage (BAL) and serum levels of IL-1β and TNF-α were analyzed by enzyme-linked immunoassay (ELISA). Morphometric analysis was performed on H and E stained lung sections. Myeloperoxidase (MPO) levels were determined via an enzymatic assay and immunofluorescence of stained sections., Results: EMPs led to significantly increased pulmonary and systemic IL-1β and TNF-α levels, which correlated with increased neutrophil recruitment to the lung. MPO levels in the lungs were increased significantly following injection of EMPs or LPS, compared to PBS. In mice treated with EMPs and LPS either simultaneously or successively, the cytokine and MPO levels were significantly increased over that of either treatment alone., Conclusion: EMPs contribute to lung injury through the initiation of a cytokine cascade that increases recruitment of neutrophils and subsequent release of MPO. Furthermore, treatment of mice with both EMPs and LPS induced greater lung injury than either treatment alone, suggesting that EMPs prime the lung for increased injury by other pathogens. Therapies aimed at reducing or blocking EMPs may be a useful strategy for attenuating lung injury., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Pleuropulmonary blastoma (PPB) is a rare pediatric lung tumor that is often part of an inherited cancer syndrome. PPBs consist of mesenchymal cells that are susceptible to malignant transformation and cysts lined by epithelial cells. In a subset of patients, overgrowth of the cysts by mesenchymal cells leads to sarcoma formation. Here, we show that 11 multiplex PPB families harbor heterozygous germline mutations in DICER1, a gene encoding an endoribonuclease critical to the generation of small noncoding regulatory RNAs. Expression of DICER1 protein was undetectable in the epithelial component of PPB tumors but was retained in the malignant mesenchyme (sarcoma). We hypothesize that loss of DICER1 in the epithelium of the developing lung alters the regulation of diffusible factors that promote mesenchymal proliferation.

Neuroblastoma (NB) is the most common extracranial solid neoplasm of infancy and childhood. Whereas most low-risk patients do well, children with high-risk tumors often fail intensive treatment. Identification of novel biomarkers is critical to improve prognostication, tailor therapy, and develop new therapeutic targets. Differential RNA-level expression between tumor cells with neuroblastic (N-type) and Schwannian stromal (S-type) phenotypes was used to identify genes of potential interest based on tumor cell type-specific regulation. Gene expression microarray analysis revealed marked differences between N-type and S-type cells in their levels of BCL6 messenger RNA, a transcriptional regulator overexpressed in a variety of hematopoietic malignancies. S-type cells express higher levels of Bcl6 RNA and protein than N-type, and protein levels are significantly limited by proteasome function. An NB tumor tissue microarray linked to clinicopathologic data was immunohistochemically stained to measure Bcl6 protein levels. Bcl6 was detected in both the neuroblastic and Schwannian stromal regions, as distinguished histologically, and correlated with outcome. We found that expression in neuroblastic regions differentiates outcomes, in that Bcl6 expression in neuroblastic regions is associated with increased time to relapse and increased overall survival compared with absent expression in neuroblastic regions, regardless of Schwannian stromal expression. Thus, our findings suggest that Bcl6 may be useful as a prognostic marker and might represent a potential therapeutic target for high-risk NB.

Pleuropulmonary blastoma (PPB) is a malignant neoplasm of the lung that presents in early childhood. The early form of the disease, cystic type I PPB, can be clinically and pathologically deceptive because of its resemblance to some developmental lung cysts. This study reviews 51 cases of type I PPB and 6 lung cysts from relatives of children with PPB. Type I PPB is a delicate multilocular cyst with variable numbers of primitive mesenchymal cells beneath a benign epithelial surface. Rhabdomyoblasts and cartilage nodules are seen in 49% and 40% of cases, respectively. Tumors in the youngest subset of patients, from birth to 2 months of age, are more uniform in composition and cellularity compared with those in older groups. Early tumors have a subtle transition between normal developing lung and tumor, showing bland interstitial mesenchymal cells uniformly expanding the alveolar septa. Presumed regressive changes including cyst wall necrosis are common. This phenomenon may explain the variable and sometimes sparse tumor cellularity seen in some type I PPBs. On a biologic level, this process supports the concept that not all type I PPBs are fated to progress to a type II or III PPB. Factors that control the balance between progression and regression may be important in predicting tumor behavior and determining which patients will benefit from adjuvant chemotherapy. In the meantime, recognition of this lesion as a neoplasm with malignant potential rather than a developmental cystic malformation is vital so the child can receive complete excision and appropriate follow-up care.

The cytotoxic mechanism of the histone deacetylase inhibitor (HDACI) Trichostatin A (TSA) was explored in a neuroblastoma (NB) model. TSA induces cell death in neuroblastic-type NB cells by increasing the acetylation of Ku70, a Bax-binding protein. Ku70 acetylation causes Bax release and activation, triggering cell death. This response to TSA depends on the CREB-binding protein (CBP) acetylating Ku70. TSA-induced cell death response correlates with CBP expression. In stromaltype NB cell lines with low levels of CBP and relative resistance to TSA, increasing CBP expression disrupts Bax-Ku70 binding and sensitizes them to TSA. Reducing CBP expression in neuroblastic cell types causes resistance. Cytotoxic response to TSA is Bax-dependent. Interestingly, depleting NB cells of Ku70 also triggers Bax-dependent cell death, suggesting that conditions that leave Bax unbound to Ku70 result in cell death. We also show that CBP, Ku70, and Bax are expressed in human NB tumors and that CBP expression varies across cell types comprising these tumors, with the highest expression observed in neuroblastic elements. Together, these results demonstrate that CBP, Bax, and Ku70 contribute to a therapeutic response to TSA against NB and identify the possibility of using these proteins to predict clinical responsiveness to HDACI treatment.

Purpose: The discovery and validation of new prognostic factors and further refinement of risk group stratification are needed to improve clinical interpretation of neuroblastoma. Our laboratory isolated and characterized a developmentally regulated gene named TUBEDOWN against which we have raised a monoclonal antibody (OE5). Tubedown becomes down-regulated postnatally yet remains strongly expressed in some neuroblastomas. The purpose of this study is to define the utility of Tubedown expression as a new measure of the differentiation status and aggressiveness of neuroblastic tumors., Experimental Design: Tubedown protein expression was quantitatively assessed in neuroblastic tumors (neuroblastomas, ganglioneuroblastomas, and ganglioneuromas) and normal adrenal tissues using Western blot and OE5 immunohistochemistry. Regulation of Tubedown expression during retinoic acid-induced neuronal differentiation in neuroblastoma cell lines was assessed by Western blotting., Results: High levels of Tubedown expression are observed in tumors with significant neuroblastic component, unfavorable histopathology, advanced stage, high-risk group, and poor outcome. In contrast, more differentiated subsets of neuroblastic tumors, ganglioneuroblastomas with favorable histopathology and ganglioneuromas, express low levels of Tubedown. In vitro, marked retinoic acid-induced neuronal differentiation responses of neuroblastoma cells are associated with a significant decrease in Tubedown expression, whereas limited neuronal differentiation responses to retinoic acid were associated with little or no decrease in Tubedown expression., Conclusions: Our results indicate that the levels of Tubedown expression are linked to the differentiation status and aggressiveness of neuroblastic tumors and represent an independent prognostic factor for neuroblastoma. Tubedown expression may be useful to more accurately define different neuroblastic tumor subsets and ultimately provide more adequate assessment and treatment for neuroblastoma patients.

A 7-year-old boy presented with a 1-year history of back pain radiating to his left scapula and arm, with tenderness to palpation over the area. Laboratory studies were unremarkable. Computed tomographic scan showed a mixed lytic and sclerotic process at the T5-T6 level of the vertebral column in continuity with an adjacent paravertebral soft tissue mass. Nuclear medicine scan demonstrated increased uptake in the T5 vertebral body. Histology revealed invasive squamous cell carcinoma infiltrating mature, gliotic neural tissue, with areas of necrosis and prominent perivascular space involvement. Associated vertebral fragments showed bone destruction, reactive bone formation, and fibrosis. By immunohistochemistry, the carcinoma cells were positive for cytokeratin AE1/AE3, cytokeratin 5/6, EMA, and MIC-2 (membranous staining). The mature neural tissue was positive for GFAP; immature neural elements were not identified. Based on morphologic, immunohistochemical, and clinical features, this lesion was diagnosed as invasive squamous cell carcinoma arising within an intravertebral and paravertebral teratoma.

A 12-year-old female with developmental delay/mental retardation and a family history of gynecologic cancers presented with nonspecific abdominal complaints and was found to have a 4.5-kg, 25- x 23- x 15-cm pelvic mass with solid and cystic components and associated retroperitoneal and mesenteric lymphadenopathy. Laboratory studies revealed increased serum levels of CA-125 and inhibin B. Histologically, the tumor exhibited several different morphologic appearances including adult granulosa cell tumor, juvenile granulosa cell tumor (with areas of marked atypia), and Sertoli cell tumor. Immunohistochemically, the tumor was positive for calretinin, MIC-2 (CD99), S100 protein, PGP 9.5, and neuron-specific enolase. Electron microscopy of the Sertoli cell tumor-like areas showed Charcot-Bottcher filaments, a distinguishing feature of Sertoli cells. Together, these findings supported a diagnosis of mixed sex cord-stromal tumor including granulosa cell tumor of adult and juvenile types and intermediate- to high-grade Sertoli cell tumor, with large areas of markedly atypical sex cord-stromal tumor.

Eosinophils produce granulocyte macrophage colony-stimulating factor (GM-CSF), which enhances their survival and function. In T cells and fibroblasts, GM-CSF production is controlled predominantly by variable messenger RNA (mRNA) stability involving 3' untranslated region (3' UTR) adenosine-uridine-rich elements (AREs) and sequence-specific mRNA binding proteins. However, the mode of regulation of this critical cytokine remains unknown in eosinophils. Therefore, we measured GM-CSF mRNA decay in an eosinophil-like cell line (AML14.3D10) and, with a radiolabeled GM-CSF RNA probe, asked whether ARE-specific, mRNA binding proteins were present in cytoplasmic lysates of these cells. Human GM-CSF mRNA transfected into unstimulated AML14.3D10 cells decayed with a half-life of 6 min, which increased to 14 min after 1 h, and to 22 min after 2 h, of ionophore-mediated activation. GM-CSF RNA mobility shift assays using cytoplasmic extracts from resting or ionophore-stimulated AML14.3D10 cells revealed multiple RNA-protein complexes of 55, 60, 85, 100, and 125 kD. A 47-kD complex was also detected with an 80-base RNA probe containing four consecutive AUUUA motifs. On the basis of competition studies, all of the observed binding protein activities interacted with the 3' UTR AREs. In addition, binding activity increased 2.5-fold in cytoplasmic lysates from cells stimulated with calcium ionophore for 2 h, contemporaneous with GM-CSF mRNA stabilization. These data provide direct evidence that ionophore stabilizes GM-CSF mRNA in AML14.3D10 cells and simultaneously increases the activity of a series of AUUUA-specific mRNA binding proteins. We conclude that the interaction of AU-specific binding proteins may stabilize GM-CSF mRNA in activated eosinophil-like cell lines.

Granulocyte-macrophage colony stimulating factor (GM-CSF) mRNA levels are controlled post-transcriptionally by the 3'-untranslated region (UTR) adenosine-uridine-rich element (ARE). In untransformed, resting cells, the ARE targets GM-CSF mRNA for rapid degradation, thereby significantly suppressing protein expression. We used a rabbit reticulocyte lysate (RRL) cell-free system to examine translational regulation of GM-CSF expression. We uncoupled decay rates from rates of translation by programming the RRL with an excess of mRNAs. Capped, full-length, polyadenyl-ated human GM-CSF mRNA (full-length 5'-UTR AUUUA+A90) and an ARE-modified version (full-length 5'-UTR AUGUA+A90) produced identical amounts of protein. When the 5'-UTR was replaced with an irrelevant synthetic leader sequence (syn 5'-UTR), translation of syn 5'-UTR AUUUA+A90 mRNA was suppressed by >20-fold. Mutation of the ARE or removal of the poly(A) tail relieved this inhibition. Thus, in the absence of a native 5'-UTR, the ARE and poly(A) tail act in concert to block GM-CSF mRNA translation. Substitutions of different regions of the native 5'-UTR revealed that the entire sequence was essential in maintaining the highest rates of translation. However, shorter 10-12 nt contiguous 5'-UTR regions supported 50-60% of maximum translation. The 5'-UTR is highly conserved, suggesting similar regulation in multiple species and in these studies was the dominant element regulating GM-CSF mRNA translation, overriding the inhibitory effects of the ARE and the poly(A) tail.

Human eosinophils activated by calcium ionophore produce granulocyte-macrophage colony-stimulating factor (GM-CSF). In T lymphocytes GM-CSF messenger RNA (mRNA) stability is regulated by 3' untranslated region (UTR) adenosine-uridine-rich elements (AREs). We show endogenous GM-CSF mRNA is rapidly induced in an eosinophil cell-line (AML14.3D10) after activation with ionomycin. To calculate the decay rate of GM-CSF mRNA in activated cells, eosinophils were transfected with wild-type, full-length GM-CSF mRNA or a mutant version lacking the AUUUA motifs. In unstimulated cells, wild-type GM-CSF mRNA decayed with a half-life time (t1/2) of 6+/-2 min while the mutant decayed with a t1/2 of 20+/-4 min, demonstrating the dominant, destabilizing effect of multiple AUUUA motifs. Within 1 hr of activation by ionomycin, the half-life of transfected wild-type mRNA increased by 2.5-fold, which increased up to 4-fold after 2 hr of activation. The half-life of the mutant GM-CSF was unaffected by ionomycin, demonstrating that ionophore-mediated stabilization requires intact AUUUA motifs. Actinomycin D (ActD) stabilized wild-type GM-CSF mRNA as well, causing poly(A) tail elongation and translation inhibition. These data show that in eosinophil-like cell lines, GM-CSF mRNA is exquisitely unstable but can be markedly stabilized by calcium ionophore. Both effects require intact 3' UTR AREs.

We have previously shown that heterogeneous nuclear ribonucleoprotein C (hnRNP C) and nucleolin bound specifically to a 29 nt sequence in the 3'-untranslated region of amyloid precursor protein (APP) mRNA. Upon activation of peripheral blood mononuclear cells, hnRNP C and nucleolin acquired APP mRNA binding activity, concurrent with APP mRNA stabilization. These data suggested that the regulated interaction of hnRNP C and nucleolin with APP mRNA controlled its stability. Here we have directly examined the role of the cis element and trans factors in the turnover and translation of APP mRNA in vitro . In a rabbit reticulocyte lysate (RRL) translation system, a mutant APP mRNA lacking the 29 nt element was 3-4-fold more stable and synthesized 2-4-fold more APP as wild-type APP mRNA. Therefore, the 29 nt element functioned as an APP mRNA destabilizer. RNA gel mobility shift assays with the RRL suggested the presence of endogenous nucleolin, but failed to show hnRNP C binding activity. However, wild-type APP mRNA was stabilized and coded for 6-fold more APP when translated in an RRL system supplemented with exogenous active hnRNP C. Control mRNAs lacking the 29 nt element were unaffected by hnRNP C supplementation. Therefore, occupancy of the 29 nt element by hnRNP C stabilized APP mRNA and enhanced its translation.