Orthotopic Wilms tumor xenografts derived from cell lines reflect limited aspects of tumor morphology and clinical characteristics.

BACKGROUND: Wilms tumor (WT) is a pediatric tumor of the kidney, the treatment of which includes heavy chemotherapy. Affected children would likely benefit from more targeted therapies with limited side effects. Establishment of relevant orthotopic WT xenografts is important to better understand mechanisms of WT growth and for preclinical drug testing. PROCEDURE: Here we established and characterized orthotopic xenografts from WT cell lines WiT49, CCG-99-11, and WT-CLS1 to ascertain in what aspects each of them recapitulated WT histology, immunophenotype, invasion, and metastatic spread. RESULTS: WiT49 xenografts recapitulated near triphasic WTs with clear WT1 staining and anaplastic features, but with tumor restricted to the kidney. On the contrary both CCG-99-11 and WT-CLS1 xenografts conveyed metastatic disease. CCG-99-11 showed a blastemal phenotype whereas WT-CLS1 xenografts did not properly reflect any specific WT subtype. CONCLUSIONS: From the three tested cell lines, orthotopic WiT49 xenografts best reflect the triphasic pattern of classical WT.

Absence of Epstein-Barr and cytomegalovirus infection in neuroblastoma cells by standard detection methodologies.

Indications exist in the scientific literature that infection with human herpes family viruses may contribute to the pathogenesis of neuroblastoma (NB). However, systematic investigations regarding viral presence in NB cells have been scarcely reported. Here, the presence of DNA from Epstein-Barr virus (EBV) and human cytomegalovirus (HCMV) was assessed by PCR in 12 NBs, supplemented with RNA in situ hybridization, immunohistochemical detection, and high-throughput DNA sequencing. These standard methods did not detect infection by EBV or HCMV in NB cells in any tumor, while occasional immune cells were positive for EBV RNA or HCMV protein in four cases.

Generation of trisomies in cancer cells by multipolar mitosis and incomplete cytokinesis.

One extra chromosome copy (i.e., trisomy) is the most common type of chromosome aberration in cancer cells. The mechanisms behind the generation of trisomies in tumor cells are largely unknown, although it has been suggested that dysfunction of the spindle assembly checkpoint (SAC) leads to an accumulation of trisomies through failure to correctly segregate sister chromatids in successive cell divisions. By using Wilms tumor as a model for cancers with trisomies, we now show that trisomic cells can form even in the presence of a functional SAC through tripolar cell divisions in which sister chromatid separation proceeds in a regular fashion, but cytokinesis failure nevertheless leads to an asymmetrical segregation of chromosomes into two daughter cells. A model for the generation of trisomies by such asymmetrical cell division accurately predicted several features of clones having extra chromosomes in vivo, including the ratio between trisomies and tetrasomies and the observation that different trisomies found in the same tumor occupy identical proportions of cells and colocalize in tumor tissue. Our findings provide an experimentally validated model explaining how multiple trisomies can occur in tumor cells that still maintain accurate sister chromatid separation at metaphase-anaphase transition and thereby physiologically satisfy the SAC.

SIX1 protein expression selectively identifies blastemal elements in Wilms tumor.

BACKGROUND: Wilms tumor (WT) is the most common renal neoplasm in children. Histologically, most WTs consist of three tissue elements: blastema, epithelium, and stroma. Some cases also show diffuse or focal anaplastic features. Previous studies have shown that a predominance of blastemal cells in post-chemotherapy WT specimens is associated with a poor clinical course. However, there is currently no molecular marker for blastemal cells, and risk stratification for post-nephrectomy treatment is therefore often based on clinico-histological parameters alone. PROCEDURE: In the present study, three public gene expression microarray datasets, including 82 WTs and 8 normal fetal kidneys, were used to establish a consensus gene expression profile of WT. By bioinformatic analyses, 17 genes overexpressed in WT compared to fetal kidney were then selected for evaluation of their protein expression in WT cell lines and in the different histological components in paraffin-embedded WT tissue sections by immunofluorescence. RESULTS: Most of the evaluated proteins were expressed in all three common histological components. A prominent exception was SIX1, being expressed predominantly in blastemal elements in 24/25 pediatric cases containing blastema. Anaplastic elements exhibited highly variable SIX1-positivity. The SIX2 protein, known to be co-expressed with SIX1 during nephrogenesis, only exhibited blastemal-predominant expression in half of the SIX2 evaluated cases. CONCLUSIONS: Genes highly expressed in WT compared to fetal kidney are generally overexpressed in all of the three common WT tissue elements. An exception is the predominant expression of SIX1 in blastemal cells, hereby identifying this protein as a candidate marker for blastema.

Alternative lengthening of telomeres--an enhanced chromosomal instability in aggressive non-MYCN amplified and telomere elongated neuroblastomas.

Telomere length alterations are known to cause genomic instability and influence clinical course in several tumor types, but have been little investigated in neuroblastoma (NB), one of the most common childhood tumors. In the present study, telomere-dependent chromosomal instability and telomere length were determined in six NB cell lines and fifty tumor biopsies. The alternative lengthening of telomeres (ALT) pathway was assayed by scoring ALT-associated promyelocytic leukemia (PML) bodies (APBs). We found a reduced probability of overall survival for tumors with increased telomere length compared to cases with reduced or unchanged telomere length. In non-MYCN amplified tumors, a reduced or unchanged telomere length was associated with 100% overall survival. Tumor cells with increased telomere length had an elevated frequency of APBs, consistent with activation of the ALT pathway. The vast majority of tumor biopsies and cell lines exhibited an elevated rate of anaphase bridges, suggesting telomere-dependent chromosomal instability. This was more pronounced in tumors with increased telomere length. In cell lines, there was a close correlation between lack of telomere-protective TTAGGG-repeats, anaphase bridging, and remodeling of oncogene sequences. Thus, telomere-dependent chromosomal instability is highly prevalent in NB, and may contribute to the complexity of genomic alterations as well as therapy resistance in the absence of MYCN amplification and in this tumor type.

Evaluation of CITED1, SIX1, and CD56 protein expression for identification of blastemal elements in Wilms tumor.

OBJECTIVES: Successful further treatment of Wilms tumors (WTs) after preoperative chemotherapy and surgery depends on correct histopathologic risk stratification, including quantification of remaining blastemal elements. In the present study, we assessed the usefulness of protein markers for the detection of WT blastema. METHODS: Expression of the candidate blastemal protein markers CITED1, SIX1, and CD56 was evaluated by immunofluorescence regarding sensitivity and specificity for staining blastema in a tissue microarray containing cores from 30 WTs, a small number of rarer pediatric renal neoplasms, and normal postnatal kidney. RESULTS: CITED1, SIX1, and CD56 were expressed in blastema in 100%, 89%, and 74%, respectively, of the WTs with this component present. However, they were also expressed in 64%, 25%, and 79%, respectively, of epithelial WT elements and 48%, 52%, and 62%, respectively, of stromal WT elements. CONCLUSIONS: SIX1 showed the highest specificity, CITED1 the highest sensitivity, and CD56 low specificity and sensitivity for detection of postchemotherapy WT blastema. Cytokeratin staining proved to be a useful way to determine rudimentary tubular elements not readily recognized by routine staining.

Intratumour diversity of chromosome copy numbers in neuroblastoma mediated by on-going chromosome loss from a polyploid state.

Neuroblastomas (NBs) are tumours of the sympathetic nervous system accounting for 8-10% of paediatric cancers. NBs exhibit extensive intertumour genetic heterogeneity, but their extent of intratumour genetic diversity has remained unexplored. We aimed to assess intratumour genetic variation in NBs with a focus on whole chromosome changes and their underlying mechanism. Allelic ratios obtained by SNP-array data from 30 aneuploid primary NBs and NB cell lines were used to quantify the size of clones harbouring specific genomic imbalances. In 13 cases, this was supplemented by fluorescence in situ hybridisation to assess copy number diversity in detail. Computer simulations of different mitotic segregation errors, single cell cloning, analysis of mitotic figures, and time lapse imaging of dividing NB cells were used to infer the most likely mechanism behind intratumour variation in chromosome number. Combined SNP array and FISH analyses showed that all cases exhibited higher inter-cellular copy number variation than non-neoplastic control tissue, with up to 75% of tumour cells showing non-modal chromosome copy numbers. Comparisons of copy number profiles, resulting from simulations of different segregation errors to genomic profiles of 120 NBs indicated that loss of chromosomes from a tetraploid state was more likely than other mechanisms to explain numerical aberrations in NB. This was supported by a high frequency of lagging chromosomes at anaphase and polyploidisation events in growing NB cells. The dynamic nature of numerical aberrations was corroborated further by detecting substantial copy number diversity in cell populations grown from single NB cells. We conclude that aneuploid NBs typically show extensive intratumour chromosome copy number diversity, and that this phenomenon is most likely explained by continuous loss of chromosomes from a polyploid state.