Children diagnosed at age ≥ 18 months with metastatic MYCN-nonamplified neuroblastoma (NBL-NA) are at high risk for disease relapse, whereas those diagnosed at age < 18 months are nearly always ...cured. In this study, we investigated the hypothesis that expression of genes related to tumor-associated inflammatory cells correlates with the observed differences in survival by age at diagnosis and contributes to a prognostic signature.
Tumor-associated macrophages (TAMs) in localized and metastatic neuroblastomas (n = 71) were assessed by immunohistochemistry. Expression of 44 genes representing tumor and inflammatory cells was quantified in 133 metastatic NBL-NAs to assess age-dependent expression and to develop a logistic regression model to provide low- and high-risk scores for predicting progression-free survival (PFS). Tumors from high-risk patients enrolled onto two additional studies (n = 91) served as independent validation cohorts.
Metastatic neuroblastomas had higher infiltration of TAMs than locoregional tumors, and metastatic tumors diagnosed in patients at age ≥ 18 months had higher expression of inflammation-related genes than those in patients diagnosed at age < 18 months. Expression of genes representing TAMs (CD33/CD16/IL6R/IL10/FCGR3) contributed to 25% of the accuracy of a novel 14-gene tumor classification score. PFS at 5 years for children diagnosed at age ≥ 18 months with NBL-NA with a low- versus high-risk score was 47% versus 12%, 57% versus 8%, and 50% versus 20% in three independent clinical trials, respectively.
These data suggest that interactions between tumor and inflammatory cells may contribute to the clinical metastatic neuroblastoma phenotype, improve prognostication, and reveal novel therapeutic targets.
This study was conducted to investigate the prognostic significance and biologic relevance of trkA expression levels in peripheral neuroblastic tumors (pNTs) (i.e., neuroblastoma, ...ganglioneuroblastoma, and ganglioneuroma).
Levels of trkA expression from a total of 265 pNTs were determined by quantitative polymerase chain reaction analysis with Genescan software. The results were analyzed according to histopathology (favorable histology FH vs. unfavorable histology UH according to the International Neuroblastoma Pathology Classification) and MYCN tumor status (amplified vs. nonamplified) along with clinical stage and outcomes of the patients.
The levels of trkA expression differed significantly between the group of patients who were alive and well (n = 170 patients) and the group that had progressed or died (n = 95 patients) and between the group that was alive (n = 188 patients) and the group that died (n = 77 patients). However, the trkA expression levels were not independent predictors of clinical outcome when the proportional hazards model contained the known prognostic variables of clinical stage, histopathology, and MYCN status (all tests were done in 196 patients). In the neuroblastoma category (n = 173 tumors), tumors in the FH/nonamplified MYCN subset (n = 112 tumors) expressed higher levels of trkA and showed an age-dependent neuroblastic differentiation: They were classified into either a poorly differentiated subtype (n = 91 tumors; all patients age < 1.5 years at diagnosis) or a differentiating subtype (n = 21 tumors; 57% of patients ages 1.5-5.0 years). Tumors in the UH/amplified MYCN subset (n = 30 tumors) expressed significantly lower levels of trkA and showed very limited neuroblastic differentiation. Tumors in the FH/amplified MYCN subset were very rare (n = 3 tumors) and expressed higher levels of trkA. Tumors in the UH/nonamplified MYCN subset (n = 28 tumors) had trkA levels in a wide range and showed limited neuroblastic differentiation.
For patients with pNTs, levels of trkA expression did not add significant information to prognostic grouping, as defined by the combination of clinical stage, histopathology, and MYCN status. There was a biologically relevant correlation between molecular properties (trkA expression and MYCN status) and histopathologic features of the tumors in the neuroblastoma category.
Molecular genetic alterations with prognostic significance have been described in childhood acute myeloid leukemia (AML). The aim of this study was to establish cost-effective techniques to detect ...mutations of FMS-like tyrosine kinase 3 (FLT3), nucleophosmin 1 (NPM1), and a partial tandem duplication within the mixed-lineage leukemia (MLL-PTD) genes in childhood AML.
Ninety-nine children with newly diagnosed AML were included in this study. We developed a fluorescent dye SYTO-82 based high-resolution melting (HRM) curve analysis to detect FLT3 internal tandem duplication (FLT3-ITD), FLT3 tyrosine kinase domain (FLT3-TKD), and NPM1 mutations. MLL-PTD was screened by real-time quantitative PCR.
The HRM methodology correlated well with gold standard Sanger sequencing with less cost. Among the 99 patients studied, the FLT3-ITD mutation was associated with significantly worse event-free survival (EFS). Patients with the NPM1 mutation had significantly better EFS and overall survival. However, HRM was not sensitive enough for minimal residual disease monitoring.
High-resolution melting was a rapid and efficient method for screening of FLT3 and NPM1 gene mutations. It was both affordable and accurate, especially in resource underprivileged regions. Our results indicated that HRM could be a useful clinical tool for rapid and cost-effective screening of the FLT3 and NPM1 mutations in AML patients.
Background: ASNase is a critical treatment component in childhood acute lymphoblastic leukemia (ALL). As ASNase is a bacterial protein, production of anti-ASNase Ab, often associated with clinical ...allergy, poses a frequent problem during ASNase therapy. Sixty percent of higher risk ALL patients, who received native ASNase in induction, were Ab(+) by 5 months into therapy (Panosyan et al., 2004). Moreover, 22%–29% of these patients had no clinical allergy (silent hypersensitivity) and were at increased risk for relapse.
Patients and Methods: Physicians from 13 institutions submitted samples for 48 children in first remission, aged 14 to 252 months, on various SR or HR ALL protocols with suspected ASNase allergy. The male/female ratio was 1.4. The serum samples were assessed for ASNase activity and anti-ASNase Ab as reported earlier (Avramis, et al., Blood 2002). Ab titer is expressed as the numeric ratio relative to an Ab negative control.
Results: Allergic symptoms ranged from localized skin rash and swelling to respiratory difficulties and/or anaphylaxis. Neutralizing anti-PEG-ASNase Ab's were seen in 41 patients (85%). The results were examined with respect to age, gender, ASNase formulation received, time of post-ASNase administration, and the physician's clinical observations. Higher Ab ratios > 1.1 correlated with low or no ASNase activity post-PEG-ASNase (46/48 patients) or post-Native ASNase dosing (2 patients). Fourteen patients had cutaneous manifestations (rash, hives, urticaria). All had neutralizing Ab and no enzymatic activity. Nine had higher Ab ratios and 5 had lower Ab ratios. Eight additional patients had localized or generalized swelling; all had higher Ab ratios. Similarly, over the entire CCG-1961 study, 526/1100 patients had a clinical reaction; 476/526 had higher Ab ratios (90%).
Conclusions: We found neutralizing Ab in 85% of patients with apparent clinical ASNase allergy. Neutralizing Ab was correlated with lower or absent ASNase activity. Lower Ab ratios may be associated with earlier time points in the anamnestic response. In the remaining 15% of patients with an apparent clinical reaction, we found no Ab and substantial ASNase activity. Monitoring ASNase activity and Ab may be useful for guiding ASNase therapy. Patients with no Ab and substantial activity, might be rechallenged with the same product despite an apparent allergic reaction.
Known clinical and genetic markers have limitations in predicting disease course and outcome in juvenile myelomonocytic leukemia (JMML). DNA methylation patterns in JMML have correlated with outcome ...across multiple studies, suggesting it as a biomarker to improve patient stratification. However, standardized approaches to classify JMML on the basis of DNA methylation patterns are lacking. We, therefore, sought to define an international consensus for DNA methylation subgroups in JMML and develop classification methods for clinical implementation.
Published DNA methylation data from 255 patients with JMML were used to develop and internally validate a classifier model. Accuracy across platforms (EPIC-arrays and MethylSeq) was tested using a technical validation cohort (32 patients). The suitability of both methods for single-patient classification was demonstrated using an independent cohort (47 patients).
Analysis of pooled, published data established three DNA methylation subgroups as a
standard. Unfavorable prognostic parameters (
mutation, elevated fetal hemoglobin, and older age) were significantly enriched in the high methylation (HM) subgroup. A classifier was then developed that predicted subgroups with 98% accuracy across different technological platforms. Applying the classifier to an independent validation cohort confirmed an association of HM with secondary mutations, high relapse incidence, and inferior overall survival (OS), while the low methylation subgroup was associated with a favorable disease course. Multivariable analysis established DNA methylation subgroups as the only significant factor predicting OS.
This study provides an international consensus definition for DNA methylation subgroups in JMML. We developed and validated methods which will facilitate the design of risk-stratified clinical trials in JMML.
Ichthyosaurs are extinct marine reptiles that display a notable external similarity to modern toothed whales. Here we show that this resemblance is more than skin deep. We apply a multidisciplinary ...experimental approach to characterize the cellular and molecular composition of integumental tissues in an exceptionally preserved specimen of the Early Jurassic ichthyosaur Stenopterygius. Our analyses recovered still-flexible remnants of the original scaleless skin, which comprises morphologically distinct epidermal and dermal layers. These are underlain by insulating blubber that would have augmented streamlining, buoyancy and homeothermy. Additionally, we identify endogenous proteinaceous and lipid constituents, together with keratinocytes and branched melanophores that contain eumelanin pigment. Distributional variation of melanophores across the body suggests countershading, possibly enhanced by physiological adjustments of colour to enable photoprotection, concealment and/or thermoregulation. Convergence of ichthyosaurs with extant marine amniotes thus extends to the ultrastructural and molecular levels, reflecting the omnipresent constraints of their shared adaptation to pelagic life.
Residual melanins have been detected in multimillion-year-old animal body fossils; however, confident identification and characterization of these natural pigments remain challenging due to loss of ...chemical signatures during diagenesis. Here, we simulate this post-burial process through artificial maturation experiments using three synthetic and one natural eumelanin exposed to mild (100 °C/100 bar) and harsh (250 °C/200 bar) environmental conditions, followed by chemical analysis employing alkaline hydrogen peroxide oxidation (AHPO) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Our results show that AHPO is sensitive to changes in the melanin molecular structure already during mild heat and pressure treatment (resulting, e.g., in increased C-C cross-linking), whereas harsh maturation leads to extensive loss of eumelanin-specific chemical markers. In contrast, negative-ion ToF-SIMS spectra are considerably less affected by mild maturation conditions, and eumelanin-specific features remain even after harsh treatment. Detailed analysis of ToF-SIMS spectra acquired prior to experimental treatment revealed significant differences between the investigated eumelanins. However, systematic spectral changes upon maturation reduced these dissimilarities, indicating that intense heat and pressure treatment leads to the formation of a common, partially degraded, eumelanin molecular structure. Our findings elucidate the complementary nature of AHPO and ToF-SIMS during chemical characterization of eumelanin traces in fossilized organismal remains.
Abstract
Melanin pigments play a critical role in physiological processes and shaping animal behaviour. Fossil melanin is a unique resource for understanding the functional evolution of melanin but ...the impact of fossilisation on molecular signatures for eumelanin and, especially, phaeomelanin is not fully understood. Here we present a model for the chemical taphonomy of fossil eumelanin and phaeomelanin based on thermal maturation experiments using feathers from extant birds. Our results reveal which molecular signatures are authentic signals for thermally matured eumelanin and phaeomelanin, which signatures are artefacts derived from the maturation of non-melanin molecules, and how these chemical data are impacted by sample preparation. Our model correctly predicts the molecular composition of eumelanins in diverse vertebrate fossils from the Miocene and Cretaceous and, critically, identifies direct molecular evidence for phaeomelanin in these fossils. This taphonomic framework adds to the geochemical toolbox that underpins reconstructions of melanin evolution and of melanin-based coloration in fossil vertebrates.
Fossilized eyes permit inferences of the visual capacity of extinct arthropods
. However, structural and/or chemical modifications as a result of taphonomic and diagenetic processes can alter the ...original features, thereby necessitating comparisons with modern species. Here we report the detailed molecular composition and microanatomy of the eyes of 54-million-year-old crane-flies, which together provide a proxy for the interpretation of optical systems in some other ancient arthropods. These well-preserved visual organs comprise calcified corneal lenses that are separated by intervening spaces containing eumelanin pigment. We also show that eumelanin is present in the facet walls of living crane-flies, in which it forms the outermost ommatidial pigment shield in compound eyes incorporating a chitinous cornea. To our knowledge, this is the first record of melanic screening pigments in arthropods, and reveals a fossilization mode in insect eyes that involves a decay-resistant biochrome coupled with early diagenetic mineralization of the ommatidial lenses. The demonstrable secondary calcification of lens cuticle that was initially chitinous has implications for the proposed calcitic corneas of trilobites, which we posit are artefacts of preservation rather than a product of in vivo biomineralization
. Although trilobite eyes might have been partly mineralized for mechanical strength, a (more likely) organic composition would have enhanced function via gradient-index optics and increased control of lens shape.
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