RESUMEN
BACKGROUND: Cell-free DNA (cfDNA) profiles of 5-hydroxymethylcytosine (5-hmC), an epigenetic marker of open chromatin and active gene expression, are correlated with metastatic disease burden in patients with neuroblastoma. Neuroblastoma tumors are comprised of adrenergic (ADRN) and mesenchymal (MES) cells, and the relative abundance of each in tumor biopsies has prognostic implications. We hypothesized that ADRN and MES-specific signatures could be quantified in cfDNA 5-hmC profiles and would augment the detection of metastatic burden in patients with neuroblastoma. METHODS: We previously performed an integrative analysis to identify ADRN and MES-specific genes (n = 373 and n = 159, respectively). Purified DNA from cell lines was serial diluted with healthy donor cfDNA. Using Gene Set Variation Analysis (GSVA), ADRN and MES signatures were optimized. We then quantified signature scores, and our prior neuroblastoma signature, in cfDNA from 84 samples from 46 high-risk patients including 21 patients with serial samples. RESULTS: Samples from patients with higher metastatic burden had increased GSVA scores for both ADRN and MES gene signatures (p < .001). While ADRN and MES signature scores tracked together in serially collected samples, we identified instances of patients with increases in either MES or ADRN score at relapse. CONCLUSIONS: While it is feasible to identify ADRN and MES signatures using 5-hmC profiles of cfDNA from neuroblastoma patients and correlate these signatures to metastatic burden, additional data are needed to determine the optimal strategies for clinical implementation. Prospective evaluation in larger cohorts is ongoing.
Asunto(s)
Ácidos Nucleicos Libres de Células , Neoplasias Primarias Secundarias , Neuroblastoma , Humanos , Niño , Ácidos Nucleicos Libres de Células/genética , Recurrencia Local de Neoplasia , Neuroblastoma/patología , PronósticoRESUMEN
PURPOSE: Neuroblastoma is the most common extracranial solid tumor in childhood. We previously showed that circulating cell-free DNA (cfDNA) and tumor biopsy derived 5-hydroxymethylcytosime (5-hmC) profiles identified patients with neuroblastoma who experienced subsequent relapse. Here, we hypothesized that 5-hmC modifications selectively enriched in cfDNA compared with tumor biopsy samples would identify epigenetic changes associated with aggressive tumor behavior and identify novel biomarkers of outcome in patients with high-risk neuroblastoma. METHODS: 5-hmC profiles from cfDNA (n = 64) and tumor biopsies (n = 48) were compared. Two neuroblastoma cell lines underwent chromatin immunoprecipitation followed by sequencing (ChIP-Seq) for H3K27me3, H3K4me3, and H3K27ac; kethoxal-associated single-stranded DNA sequencing; hmC-Seal for 5-hmC; and RNA-sequencing (RNA-Seq). Genes enriched for both H3K27me3 and H3K4me3 in the included cell lines were defined as bivalent. Using bivalent genes defined in vitro, a bivalent signature was established in three publicly available cohorts of patients with neuroblastoma through gene set variation analysis. Differences between tumors with high or low bivalent signatures were assessed by the Kaplan-Meier method and Cox proportional hazards models. RESULTS: In cfDNA compared with tumor biopsy derived 5-hmC profiles, we found increased 5-hmC deposition on Polycomb Repressive Complex 2 target genes, a finding previously described in the context of bivalent genes. We identified 313 genes that bore bivalent chromatin marks, were enriched for mediators of neuronal differentiation, and were transcriptionally repressed across a panel of heterogeneous neuroblastoma cell lines. In three distinct clinical cohorts, low bivalent signature was significantly and independently associated with worse clinical outcome in patients with high-risk neuroblastoma. CONCLUSION: Low expression of bivalent genes is a biomarker of worse outcome in patients with high-risk neuroblastoma.
Asunto(s)
5-Metilcitosina/análogos & derivados , Ácidos Nucleicos Libres de Células , Neuroblastoma , Humanos , Histonas/genética , Histonas/metabolismo , Pronóstico , Neuroblastoma/genéticaRESUMEN
A deep learning model using attention-based multiple instance learning (aMIL) and self-supervised learning (SSL) was developed to perform pathologic classification of neuroblastic tumors and assess MYCN-amplification status using H&E-stained whole slide digital images. The model demonstrated strong performance in identifying diagnostic category, grade, mitosis-karyorrhexis index (MKI), and MYCN-amplification on an external test dataset. This AI-based approach establishes a valuable tool for automating diagnosis and precise classification of neuroblastoma tumors.
RESUMEN
Background: Cell free DNA (cfDNA) profiles of 5-hydroxymethylcytosine (5-hmC), an epigenetic marker of open chromatin and active gene expression, are correlated with metastatic disease burden in patients with neuroblastoma. Neuroblastoma tumors are comprised of adrenergic (ADRN) and mesenchymal (MES) cells, and the relative abundance of each in tumor biopsies has prognostic implications. We hypothesized that ADRN and MES specific signatures could be quantified in cfDNA 5-hmC profiles and would augment the detection of metastatic burden in patients with neuroblastoma. Methods: We previously performed an integrative analysis to identify ADRN and MES specific genes (n=373 and n=159, respectively). Purified DNA from cell lines was serial diluted with healthy donor cfDNA. Using Gene Set Variation Analysis (GSVA), ADRN and MES signatures were optimized. We then quantified signature scores, and our prior neuroblastoma signature, in cfDNA from 84 samples from 46 high-risk patients including 21 patients with serial samples. Results: Samples from patients with higher metastatic burden had increased GSVA scores for both ADRN and MES gene signatures (p < 0.001). While ADRN and MES signature scores tracked together in serially collected samples, we identified instances of patients with increases in either MES or ADRN score at relapse. Conclusions: While it is feasible to identify ADRN and MES signatures using 5-hmC profiles of cfDNA from neuroblastoma patients and correlate these signatures to metastatic burden, additional data are needed to determine the optimal strategies for clinical implementation. Prospective evaluation in larger cohorts is ongoing.
RESUMEN
Neuroblastoma is the most common extra-cranial solid tumor in childhood and epigenetic dysregulation is a key driver of this embryonal disease. In cell-free DNA from neuroblastoma patients with high-risk disease, we found increased 5-hydroxymethylcytosine (5-hmC) deposition on Polycomb Repressive Complex 2 (PRC2) target genes, a finding previously described in the context of bivalent genes. As bivalent genes, defined as genes bearing both activating (H3K4me3) and repressive (H3K27me3) chromatin modifications, have been shown to play an important role in development and cancer, we investigated the potential role of bivalent genes in maintaining a de-differentiated state in neuroblastoma and their potential use as a biomarker. We identified 313 genes that bore bivalent chromatin marks, were enriched for mediators of neuronal differentiation, and were transcriptionally repressed across a panel of heterogenous neuroblastoma cell lines. Through gene set variance analysis, we developed a clinically implementable bivalent signature. In three distinct clinical cohorts, low bivalent signature was significantly and independently associated with worse clinical outcome in high-risk neuroblastoma patients. Thus, low expression of bivalent genes is a biomarker of ultra-high-risk disease and may represent a therapeutic opportunity in neuroblastoma.