Differentiation in Neuroblastoma: Diffusion-Limited Hypoxia Induces Neuro-Endocrine Secretory Protein 55 and Other Markers of a Chromaffin Phenotype

Background: Neuroblastoma is a childhood malignancy of sympathetic embryonal origin. A high potential for differentiation is a hallmark of neuroblastoma cells. We have previously presented data to suggest that in situ differentiation in tumors frequently proceeds along the chromaffin lineage and that decreased oxygen ( hypoxia) plays a role in this. Here we explore the utility of Neuro-Endocrine Secretory Protein 55 ( NESP55), a novel member of the chromogranin family, as a marker for this process.Methodology/Principal Findings: Immunohistochemical analyses and in situ hybridizations were performed on human fetal tissues, mouse xenografts of human neuroblastoma cell lines, and on specimens of human neuroblastoma/ganglioneuroma. Effects of anaerobic exposure on gene expression by cultured neuroblastoma cells was analyzed with quantitative real-time PCR. Fetal sympathetic nervous system expression of NESP55 was shown to be specific for chromaffin cell types. In experimental and clinical neuroblastoma NESP55 immunoreactivity was specific for regions of chronic hypoxia. NESP55 expression also correlated strikingly with morphological evidence of differentiation and with other chromaffin-specific patterns of gene expression, including IGF2 and HIF2 alpha. Anaerobic culture of five neuroblastoma cell lines resulted in an 18.9-fold mean up-regulation of NESP55.Conclusions/Significance: The data confirms that chronic tumor hypoxia is a key microenvironmental factor for neuroblastoma cell differentiation, causing induction of chromaffin features and NESP55 provides a reliable marker for this neuronal to neuroendocrine transition. The hypoxia-induced phenotype is the predominant form of differentiation in stroma-poor tumors, while in stroma-rich tumors the chromaffin phenotype coexists with ganglion cell-like differentiation. The findings provide new insights into the biological diversity which is a striking feature of this group of tumors

Age dependence of tumor genetics in unfavorable neuroblastoma : arrayCGH profiles of 34 consecutive cases, using a Swedish 25-year neuroblastoma cohort for validation

Background: Aggressive neuroblastoma remains a significant cause of childhood cancer death despite current intensive multimodal treatment protocols. The purpose of the present work was to characterize the genetic and clinical diversity of such tumors by high resolution arrayCGH profiling. Methods: Based on a 32K BAC whole-genome tiling path array and using 50-250K Affymetrix SNP array platforms for verification, DNA copy number profiles were generated for 34 consecutive high-risk or lethal outcome neuroblastomas. In addition, age and MYCN amplification (MNA) status were retrieved for 112 unfavorable neuroblastomas of the Swedish Childhood Cancer Registry, representing a 25-year neuroblastoma cohort of Sweden, here used for validation of the findings. Statistical tests used were: Fisher's exact test, Bayes moderated t-test, independent samples t-test, and correlation analysis. Results: MNA or segmental 11q loss (11q-) was found in 28/34 tumors. With two exceptions, these aberrations were mutually exclusive. Children with MNA tumors were diagnosed at significantly younger ages than those with 11q-tumors (mean: 27.4 vs. 69.5 months; p=0.008; n=14/12), and MNA tumors had significantly fewer segmental chromosomal aberrations (mean: 5.5 vs. 12.0; p&lt;0.001). Furthermore, in the 11q-tumor group a positive correlation was seen between the number of segmental aberrations and the age at diagnosis (Pearson Correlation 0.606; p=0.037). Among nonMNA/non11q-tumors (n=6), one tumor displayed amplicons on 11q and 12q and three others bore evidence of progression from low-risk tumors due to retrospective evidence of disease six years before diagnosis, or due to tumor profiles with high proportions of numerical chromosomal aberrations. An early age at diagnosis of MNA neuroblastomas was verified by registry data, with an average of 29.2 months for 43 cases that were not included in the present study. Conclusion: MNA and segmental 11q loss define two major genetic variants of unfavorable neuroblastoma with apparent differences in their pace of tumor evolution and in genomic integrity. Other possible, but less common, routes in the development of aggressive tumors are progression of low-risk infant-type lesions, and gene amplifications other than MYCN. Knowledge on such nosological diversity of aggressive neuroblastoma might influence future strategies for therapy.De två sista författarna delar sistaförfattarskapet.</p

Age dependence of tumor genetics in unfavorable neuroblastoma : arrayCGH profiles of 34 consecutive cases, using a Swedish 25-year neuroblastoma cohort for validation

Background: Aggressive neuroblastoma remains a significant cause of childhood cancer death despite current intensive multimodal treatment protocols. The purpose of the present work was to characterize the genetic and clinical diversity of such tumors by high resolution arrayCGH profiling. Methods: Based on a 32K BAC whole-genome tiling path array and using 50-250K Affymetrix SNP array platforms for verification, DNA copy number profiles were generated for 34 consecutive high-risk or lethal outcome neuroblastomas. In addition, age and MYCN amplification (MNA) status were retrieved for 112 unfavorable neuroblastomas of the Swedish Childhood Cancer Registry, representing a 25-year neuroblastoma cohort of Sweden, here used for validation of the findings. Statistical tests used were: Fisher's exact test, Bayes moderated t-test, independent samples t-test, and correlation analysis. Results: MNA or segmental 11q loss (11q-) was found in 28/34 tumors. With two exceptions, these aberrations were mutually exclusive. Children with MNA tumors were diagnosed at significantly younger ages than those with 11q-tumors (mean: 27.4 vs. 69.5 months; p=0.008; n=14/12), and MNA tumors had significantly fewer segmental chromosomal aberrations (mean: 5.5 vs. 12.0; p&lt;0.001). Furthermore, in the 11q-tumor group a positive correlation was seen between the number of segmental aberrations and the age at diagnosis (Pearson Correlation 0.606; p=0.037). Among nonMNA/non11q-tumors (n=6), one tumor displayed amplicons on 11q and 12q and three others bore evidence of progression from low-risk tumors due to retrospective evidence of disease six years before diagnosis, or due to tumor profiles with high proportions of numerical chromosomal aberrations. An early age at diagnosis of MNA neuroblastomas was verified by registry data, with an average of 29.2 months for 43 cases that were not included in the present study. Conclusion: MNA and segmental 11q loss define two major genetic variants of unfavorable neuroblastoma with apparent differences in their pace of tumor evolution and in genomic integrity. Other possible, but less common, routes in the development of aggressive tumors are progression of low-risk infant-type lesions, and gene amplifications other than MYCN. Knowledge on such nosological diversity of aggressive neuroblastoma might influence future strategies for therapy.De två sista författarna delar sistaförfattarskapet.</p

Histological evidence of hypoxia-dependence of expression of NESP55 and of other genes in neuroblastoma xenografts.

<p>Nuclear HIF2α immunoreactivity was used as operational definition of hypoxia. Serial sections were used to compare the different results.</p><p><i>Abbreviations and symbols</i>: IHC: immunohistochemistry; ISH: <i>in situ</i> hybridization; CgA: chromogranin A.</p><p><b>⇑</b>(sp): positivity specific for regions of chronic hypoxia.</p><p><b>⇑</b>(gr/w): weak positive gradient of expression in regions of chronic hypoxia but expression not specific to these regions.</p><p>⇑*: few positive cells, but exclusively in regions of chronic hypoxia.</p><p>ND: not analyzed.</p><p><b>⇑</b>(gr/str): strong positive gradient of expression in regions of chronic hypoxia but expression not specific to these regions.</p><p><b>⇓</b>(partial): partial down-regulation in regions of chronic hypoxia.</p><p><b>⇓</b>(complete): complete down-regulation in regions of chronic hypoxia.</p><p><b> = </b>: no discernible chronic hypoxia-dependence of expression intensity.</p><p><b>⇑</b>(ref): reference method for identification of regions of chronic hypoxia.</p

Chromogranin A immunoreactivity of the human fetal sympathetic nervous system.

<p><b>A</b>: Sympathetic paraganglion and adjacent sympathetic ganglion of a term fetus. Same region as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012825#pone-0012825-g001" target="_blank">Figure 1</a> panel E, analyzed in a consecutive section. <b>B</b>: High-power view of a sympathetic ganglion from the same section. <i>Symbols</i>: black arrowhead: sympathetic paraganglion; arrow: sympathetic ganglion, red arrowhead: SIF cell. Black double arrowheads indicate axonal stainings and red double arrowheads indicate perinuclear stainings of ganglion cells.</p

NESP55-independent evidence of a chromaffin hypoxic phenotype in neuroblastoma xenografts.

<p><b>A</b>: Columns show <i>HIF2</i>α, <i>IGF2</i> and <i>GAP 43 in situ</i> hybridization results, as indicated. The specificities of these expressions within the early sympathetic nervous system are shown in the upper row, representing consecutive sections of a 12 week fetal specimen containing a sympathetic ganglion and a sympathetic paraganglion, as indicated. The rows below represent consecutive sections from three different neuroblastoma xenograft tumors, as indicated. Silver grains appear in black in panels showing <i>IGF2</i> expression (brightfield illumination). In all other panels silver grains appear in white (darkfield illumination). <i>IGF2</i> ISH is used here as a combined marker for a chromaffin phenotype and for tumor hypoxia. <i>Symbols</i>: arrows: sympathetic ganglion; arrowheads: sympathetic paraganglion; asterisks: vascular structures. <b>B</b>: Chromogranin A expression in a SK-N-FI neuroblastoma xenograft. Left and middle panels depict chromogranin A immunoreactivity at two different magnifications. Right panel shows a <i>chromogranin A in situ</i> hybridization result in darkfield view from a consecutive section. Middle and right panels represent the same tumor region as depicted in the bottom row of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012825#pone-0012825-g005" target="_blank">Fig. 5: A</a>.</p

NESP55 immunoreactivity and histological evidence of hypoxia in neuroblastoma xenografts.

<p>Columns represent NESP55/HIF2α immunoreactivities and <i>IGF2</i>/<i>VEGFA in situ</i> hybridization (ISH) results, as shown. Rows represent different cell lines, as described. In each row the same tumor region is shown for the different analyses. NESP55/HIF2α results are produced from consecutive sections. <i>IGF2</i>/<i>VEGFA</i> results are from consecutive sections that are adjacent, but not consecutive, to the former sections. HIF2α immunoreactivity and <i>IGF2</i>/<i>VEGFA</i> expressions were chosen as markers for cellular hypoxia. <i>IGF2</i> ISH results and <i>VEGFA</i> ISH results for Kelly are shown in brightfield view (silver grains appear in black). <i>VEGFA</i> expression in SK-N-BE(2) is represented by an x-ray film autoradiography and <i>VEGFA</i> ISH results for SH-SY5Y and SK-N-FI xenografts are shown in darkfield view (silver grains appear in white). <i>Symbol</i>: n: areas of necrosis.</p

Schematic summary of the studied effects of diffusion-limited hypoxia in neuroblastoma.

<p>x-axis symbolizes the distance from tumor capillary and y-axis symbolizes the relative levels of marker gene expression and of oxygen tension (pO₂). Photo images exemplify the microvascular-dependence of NESP55 immunoreactivity and of changes in cell morphology. Photo images are from the same tumor region of an infant neuroblastoma, taken from consecutive sections. Black triangle symbolizes the region of increasing chromaffin metaplasia parallel to decreasing tissue oxygen tension.</p