Quantitative phase imaging has gained popularity in bioimaging because it can avoid the need for cell staining, which, in some cases, is difficult or impossible. However, as a result, quantitative phase imaging does not provide the labelling of various specific intracellular structures. Here we show a novel computational segmentation method based on statistical inference that makes it possible for quantitative phase imaging techniques to identify the cell nucleus. We demonstrate the approach with refractive index tomograms of stain-free cells reconstructed using tomographic phase microscopy in the flow cytometry mode. In particular, by means of numerical simulations and two cancer cell lines, we demonstrate that the nucleus can be accurately distinguished within the stain-free tomograms. We show that our experimental results are consistent with confocal fluorescence microscopy data and microfluidic cyto-fluorimeter outputs. This is a remarkable step towards directly extracting specific three-dimensional intracellular structures from the phase contrast data in a typical flow cytometry configuration.

HIF1A (Hypoxia-Inducible-Factor 1A) expression in solid tumors is relevant to establish resistance to therapeutic approaches. The use of compounds direct against hypoxia signaling and HIF1A does not show clinical efficiency because of changeable oxygen concentrations in solid tumor areas. The identification of HIF1A targets expressed in both normoxia and hypoxia and of HIF1A/hypoxia signatures might meliorate the prognostic stratification and therapeutic successes in patients with high-risk solid tumors. In this study, we conducted a combined analysis of RNA expression and DNA methylation of neuroblastoma cells silenced or unsilenced for HIF1A expression, grown in normoxia and hypoxia conditions. The analysis of pathways highlights HIF-1 (heterodimeric transcription factor 1) activity in normoxia in metabolic process and HIF-1 activity in hypoxia in neuronal differentiation process. HIF1A driven transcriptional response in hypoxia depends on epigenetic control at DNA methylation status of gene regulatory regions. Furthermore, low oxygen levels generate HIF1A-dependent or HIF1A-independent signatures, able to stratify patients according to risk categories. These findings may help to understand the molecular mechanisms by which low oxygen levels reshape gene signatures and provide new direction for hypoxia targeting in solid tumor.

Neuroblastoma (NB) and malignant cutaneous melanoma (CMM) are neural crest cells (NCC)-derived tumors and may have a shared genetic basis, but this has not been investigated systematically by genome-wide association studies (GWAS). We took a three-staged approach to conduct cross-disease meta-analysis of GWAS for NB and CMM (2101 NB cases and 4202 controls; 12 874 CMM cases and 23 203 controls) to identify shared loci. Findings were replicated in 1403 NB cases and 1403 controls of European ancestry and in 636 NB, 508 CMM cases and 2066 controls of Italian origin. We found a cross-association at locus 1p13.2 (rs2153977, odds ratio = 0.91, P = 5.36 × 10⁻⁸). We also detected a suggestive (P < 10⁻⁷) NB-CMM cross-association at 2q37.1 with opposite effect on cancer risk. Pathway analysis of 110 NB-CMM risk loci with P < 10⁻⁴ demonstrated enrichment of biological processes such as cell migration, cell cycle, metabolism and immune response, which are essential of human NCC development, underlying both tumors. In vitro and in silico analyses indicated that the rs2153977-T protective allele, located in an NB and CMM enhancer, decreased expression of SLC16A1 via long-range loop formation and altered a T-box protein binding site. Upon depletion of SLC16A1, we observed a decrease of cellular proliferation and invasion in both NB and CMM cell lines, suggesting its role as oncogene. This is the largest study to date examining pleiotropy across two NC cell-derived tumors identifying 1p13.2 as common susceptibility locus for NB and CMM risk. We demonstrate that combining genome-wide association studies results across cancers with same origins can identify new loci common to neuroblastoma and melanoma arising from tissues which originate from neural crest cells. Our results also show 1p13.2 confer risk to neuroblastoma and melanoma by regulating SLC16A1.

The contribution of coding mutations to oncogenesis has been largely clarified, whereas little is known about somatic mutations in noncoding DNA and their role in driving tumors remains controversial. Here, we used an alternative approach to interpret the functional significance of noncoding somatic mutations in promoting tumorigenesis. Noncoding somatic mutations of 151 neuroblastomas were integrated with ENCODE data to locate somatic mutations in regulatory elements specifically active in neuroblastoma cells, nonspecifically active in neuroblastoma cells, and nonactive. Within these types of elements, transcription factors (TF) were identified whose binding sites were enriched or depleted in mutations. For these TFs, a gene expression signature was built to assess their implication in neuroblastoma. DNA- and RNA-sequencing data were integrated to assess the effects of those mutations on mRNA levels. The pathogenicity of mutations was significantly higher in transcription factor binding site (TFBS) of regulatory elements specifically active in neuroblastoma cells, as compared with the others. Within these elements, there were 18 over-represented TFs involved mainly in cell-cycle phase transitions and 15 under-represented TFs primarily regulating cell differentiation. A gene expression signature based on over-represented TFs correlated with poor survival and unfavorable prognostic markers. Moreover, recurrent mutations in TFBS of over-represented TFs such as EZH2 affected MCF2L and ADP-ribosylhydrolase like 1 expression, among the others. We propose a novel approach to study the involvement of regulatory variants in neuroblastoma that could be extended to other cancers and provide further evidence that alterations of gene expression may have relevant effects in neuroblastoma development. Significance: These findings propose a novel approach to study regulatory variants in neuroblastoma and suggest that noncoding somatic mutations have relevant implications in neuroblastoma development.

The genetic aetiology and the molecular mechanisms that characterize high‐risk neuroblastoma are still little understood. The majority of high‐risk neuroblastoma patients do not take advantage of current induction therapy. So far, one of the main reasons liable for cancer therapeutic failure is the acquisition of resistance to cytotoxic anticancer drugs, because of the DNA repair system of tumour cells. PARP1 is one of the main DNA damage sensors involved in the DNA repair system and genomic stability. We observed that high PARP1 mRNA level is associated with unfavourable prognosis in 3 public gene expression NB patients’ datasets and in 20 neuroblastomas analysed by qRT‐PCR. Among 4983 SNPs in PARP1 , we selected two potential functional SNPs. We investigated the association of rs907187, in PARP1 promoter, and rs2048426 in non‐coding region with response chemotherapy in 121 Italian patients with high‐risk NB. Results showed that minor G allele of rs907187 associated with induction response of patients ( P = .02) and with decrease PARP1 mRNA levels in NB cell line ( P = .003). Furthermore, rs907187 was predicted to alter the binding site of E2F1 transcription factor. Specifically, allele G had low binding affinity with E2F1 whose expression positively correlates with PARP1 expression and associated with poor prognosis of patients with NB. By contrast, we did not find genetic association for the SNP rs2048426. These data reveal rs907187 as a novel potential risk variant associated with the failure of induction therapy for high‐risk NB.

Noncoding cis-regulatory variants have gained interest as cancer drivers, yet progress in understanding their significance is hindered by the numerous challenges and limitations of variant prioritization. To overcome these limitations, we focused on active cis-regulatory elements (aCRE) to design a customized panel for the deep sequencing of 56 neuroblastoma tumor and normal DNA sample pairs. To search for driver mutations, aCREs were defined by reanalysis of H3K27ac chromatin immunoprecipitation sequencing peaks in 25 neuroblastoma cell lines. These regulatory genomic regions were tested for an excess of somatic mutations and assessed for statistical significance using a global approach that accounted for chromatin accessibility and replication timing. Additional validation was provided by whole genome sequence analysis of 151 neuroblastomas. Analysis of HiC data determined the presence of candidate target genes interacting with mutated regions. An excess of somatic mutations in aCREs of diverse genes were identified, including IPO7, HAND2, and ARID3A. CRISPR-Cas9 editing was utilized to assess the functional consequences of mutations in the IPO7-aCRE. Patients with noncoding mutations in aCREs showed inferior overall and event-free survival independent of age at diagnosis, stage, risk stratification, and MYCN status. Expression of aCRE-interacting genes correlated strongly with negative prognostic markers and low survival rates. Moreover, a convergence between the biological functions of aCRE target genes and transcription factors with mutated binding motifs was associated with embryonic development and immune system response. Overall, this strategy enabled the identification of somatic mutations in regulatory elements that collectively promote neuroblastoma tumorigenesis. Significance: Assessment of noncoding cis-regulatory variants and long-range interaction data highlight the combined effect of somatic mutations in regulatory elements in driving neuroblastoma.