Andrew J. Murphy

SIX2 and CITED1, markers of nephronic progenitor self-renewal, remain active in primitive elements of Wilms' tumor

PURPOSEnSIX2 and CITED1 are transcriptional regulators that specify self-renewing nephronic progenitor cells of the embryonic kidney. We hypothesized that SIX2, which promotes and maintains this stem cell population, and CITED1 remain active in Wilms tumor (WT).nnnMETHODSnTo evaluate expression domains and the pathogenic significance of SIX2 and CITED1 across WT, the Childrens Oncology Group provided 40 WT specimens of stages I to IV (n = 10 per stage), which were enriched for unfavorable histology (n = 20) and treatment failure (relapse or death, n = 20). SIX2 and CITED1 protein expression was evaluated qualitatively (immunohistochemistry) and quantitatively (Western blot, or WB). Gene transcription was estimated using quantitative real-time polymerase chain reaction (qRT-PCR).nnnRESULTSnSIX2 was visualized by immunohistochemistry in 36 (94.7%) of 38 specimens. Protein and messenger RNA expression of SIX2 were quantitatively similar across all stages of disease (P = .48 WB; P = 0.38 qPCR), in favorable or unfavorable histology (P = 0.51 WB; P = 0.58 qPCR), and in treatment failure or success (P = 0.86 WB; P = 0.49 qPCR). Although CITED1 expression paralleled SIX2 qualitatively, no quantitative correlation between SIX2 and CITED1 expression was observed (Spearman correlation coefficient, 0.28; P = 0.08). As in the fetal kidney, overlapping, but also distinct, WT cellular expression domains were observed between SIX2 and CITED1.nnnCONCLUSIONnSIX2 and CITED1 remain active across all disease characteristics of WT. Activity of these genes in WT potentially identifies a population of self-renewing cancer cells that exhibit an embryonic, stemlike phenotype. Taken together, these transcriptional regulators may be fundamental to WT cellular self-renewal and may represent targets for novel therapies that promote terminal differentiation.

Race Disparities in Wilms Tumor Incidence and Biology

BACKGROUNDnWilms tumor (WT) is thought to arise in children of Black African ancestry with greater frequency than in Whites. To clarify the biological basis for race disparities in WT, we first verified that Black children residing in Tennessee have an increased incidence of WT, and second, established molecular profiles in WT that are specific to race.nnnMATERIALS AND METHODSnTo assess race disparities in WT epidemiology, the Tennessee Cancer Registry (TCR) was queried for all in-state patients less than 20 y of age and registered between 1999 and 2008. To explore race disparities in WT biology, six Black and four White WT specimens acquired in Tennessee were analyzed using imaging mass spectrometry (IMS).nnnRESULTSnTCR data show that Black children are over-represented among WT patients (29%) relative to all other childhood cancers (18.5%; P = 0.01). WT ranked the fifth most common cancer diagnosis among Blacks, but ninth among Whites. The diagnosis of WT occurred 79% more frequently among Blacks (n = 28) than Whites (n = 69; P = 0.01), and proportionally more Blacks tended to present with distant disease. Although overall survival from WT was not statistically different between Blacks (92.9%) and Whites (94.0%), Black males showed the lowest survival (85%; P = 0.21). IMS analysis identified peptide spectra from both WT blastema and stroma that independently classify specimens according to race with greater than 80% accuracy.nnnCONCLUSIONSnIn Tennessee, Black children appear more susceptible than Whites to develop WT. Race-specific molecular profiles can be determined that may help to clarify pathways of Wilms tumorigenesis and the biological basis for race disparities in WT incidence and biology.

Molecular characterization of Wilms' tumor from a resource-constrained region of sub-Saharan Africa

Sub‐Saharan African children have an increased incidence of Wilms tumor (WT) and experience alarmingly poor outcomes. Although these outcomes are largely due to inadequate therapy, we hypothesized that WT from this region exhibits features of biological aggressiveness that may warrant broader implementation of high‐risk therapeutic protocols. We evaluated 15 Kenyan WT (KWT) for features of aggressive disease (blastemal predominance and Ki67/cellular proliferation) and treatment resistance (anaplasia and p53 immunopositivity). To explore the additional biological features of KWT, we determined the mutational status of the CTNNB1/β‐catenin and WT1 genes and performed immunostaining for markers of Wnt pathway activation (β‐catenin) and nephronic progenitor cell self‐renewal (WT1, CITED1 and SIX2). We characterized the proteome of KWT using imaging mass spectrometry (IMS). The results were compared to histology‐ and age‐matched North American WT (NAWT) controls. For patients with KWT, blastemal predominance was noted in 53.3% and anaplasia in 13%. We detected increased loss to follow‐up (p = 0.028), disease relapse (p = 0.044), mortality (p = 0.001) and nuclear unrest (p = 0.001) in patients with KWT compared to controls. KWT and NAWT showed similar Ki67/cellular proliferation. We detected an increased proportion of epithelial nuclear β‐catenin in KWT (p = 0.013). All 15 KWT specimens were found to harbor wild‐type CTNNB1/β‐catenin, and one contained a WT1 nonsense mutation. WT1 was detected by immunostaining in 100% of KWT, CITED1 in 80% and SIX2 in 80%. IMS revealed a molecular signature unique to KWT that was distinct from NAWT. The African WT specimens appear to express markers of adverse clinical behavior and treatment resistance and may require alternative therapies or implementation of high‐risk treatment protocols.

Aberrant activation, nuclear localization, and phosphorylation of yes‐associated protein‐1 in the embryonic kidney and Wilms tumor

The Yes‐associated‐protein‐1 (YAP1) is a novel, direct regulator of stem cell genes both in development and cancer. FAT4 is an upstream regulator that induces YAP1 cytosolic sequestering by phosphorylation (p‐Ser 127) and therefore inhibits YAP1‐dependent cellular proliferation. We hypothesized that loss of FAT4 signaling would result in expansion of the nephron progenitor population in kidney development and that YAP1 subcellular localization would be dysregulated in Wilms tumor (WT), an embryonal malignancy that retains gene expression profiles and histologic features reminiscent of the embryonic kidney.

Concordance of imaging modalities and cost minimization in the diagnosis of pediatric choledochal cysts

PurposeGiven evolving imaging technologies, we noted significant variation in the diagnostic evaluation of pediatric choledochal cysts (CDC). To streamline the diagnostic approach to CDC, and minimize associated expenses, we compared typing accuracy and costs of ultrasound (US), intraoperative cholangiography (IOC), and magnetic resonance cholangiopancreatography (MRCP).MethodsRecords of 30 consecutive pediatric CDC patients were reviewed. Blinded to all clinical data, two pediatric radiologists reviewed all US, MRCPs, and IOCs to type CDCs according to the Todani classification. When compared with pathologic findings, the concordance between and accuracy of each diagnostic test were determined. Inflation-adjusted procedure charges and collections for imaging modalities were analyzed.ResultsMean typing accuracy overlapped for US, IOC, and MRCP. Inter-rater reliability was 87xa0% for US (κxa0=xa00.77), 80xa0% for IOC (κxa0=xa00.62), and 60xa0% for MRCP (κxa0=xa00.37). MRCP procedure charges (

SIX2 Effects on Wilms Tumor Biology

1204.69) and collections (

Associations between pediatric choledochal cysts, biliary atresia, and congenital cardiac anomalies

420.85) exceeded IOC and US combined (

Chronic biloma after right hepatectomy for stage IV hepatoblastoma managed with Roux-en-Y biliary cystenterostomy ☆

264.80 charges, pxa0=xa00.0002;

Peptide spectra in Wilms tumor that associate with adverse outcomes

93.40 collections, pxa0=xa00.0021).ConclusionOur data support the use of US alone in the diagnosis of pediatric CDC when no intrahepatic biliary ductal dilatation is visualized. However, when dilated intrahepatic ducts are encountered on US, MRCP should be utilized to distinguish a type I from a type IV CDC, which may alter the operative approach.

Volume-outcome effects for children undergoing resection of renal malignancies

Wilms tumor (WT) blastema retains gene expression profiles characteristic of the multipotent nephron progenitor pool, or cap mesenchyme (CM), in the developing kidney. As a result, WT blastema and the CM are believed to represent contextual analogues of one another. Sine oculis homeobox 2 (SIX2) is a transcription factor expressed specifically in the CM, provides a critical mechanism for CM self-renewal, and remains persistently active in WT blastema, although its purpose in this childhood malignancy remains unclear. We hypothesized that SIX2, analogous to its function in development, confers a survival pathway to blastema, the putative WT stem cell. To test its functional significance in WT biology, wild-type SIX2 was overexpressed in the human WT cell line, WiT49. After validating this model, SIX2 effects on anchorage-independent growth, proliferation, invasiveness, canonical WNT pathway signaling, and gene expression of specific WNT pathway participants were evaluated. Relative to controls, WiT49 cells overexpressing SIX2 showed significantly enhanced anchorage-independent growth and early-passage proliferation representing surrogates of cell survival. Interestingly, overexpression of SIX2 generally repressed TCF/LEF-dependent canonical WNT signaling, which activates and coordinates both differentiation and stem pathways, but significantly heightened canonical WNT signaling through the survivin promoter, a mechanism that exclusively maintains the stem state. In summary, when overexpressed in a human WT cell line, SIX2 enhances cell survival and appears to shift the balance in WNT/β-catenin signaling away from a differentiation path and toward a stem cell survival path.