Glenn M. Marshall

Expression of the Gene for Multidrug-Resistance–Associated Protein and Outcome in Patients with Neuroblastoma

BACKGROUND Overexpression of the gene for the multidrug-resistance-associated protein (MRP) has been linked with resistance to chemotherapeutic agents (multidrug resistance) in vitro. The expression of MRP by neuroblastoma cells correlates with N-myc oncogene amplification, a well-established prognostic indicator in patients with neuroblastoma. METHODS To relate MRP gene expression to established prognostic markers and the clinical outcome of neuroblastoma, we analyzed MRP expression in specimens of primary tumors from 60 patients with neuroblastoma. RESULTS Levels of MRP gene expression were significantly higher in tumors with N-myc amplification than in tumors without such amplification (P < 0.001). High levels of MRP expression were strongly associated with reductions in both survival and event-free survival (P < 0.001) in the overall study population and in subgroups of patients without N-myc amplification and patients with localized disease. For the overall study population, the five-year cumulative survival rates in the groups with high and low levels of MRP expression were 57 percent (95 percent confidence interval, 37 to 78 percent) and 94 percent (95 percent confidence interval, 86 to 100 percent), respectively. In contrast, expression of the MDR1 multi-drug-resistance gene was not predictive of survival or event-free survival. After adjustment by multivariate analysis for the effects of N-myc amplification and other prognostic indicators, high levels of MRP expression retained significant prognostic value for poor survival (relative hazard, 14.9; P = 0.01) and poor event-free survival (relative hazard, 9.7; P = 0.004), whereas N-myc amplification had no prognostic value. CONCLUSIONS High levels of MRP gene expression in patients with neuroblastoma correlate strongly with poor outcome. The findings suggest that expression of this multidrug-resistance gene accounts for the association between N-myc amplification and reduced survival.

The Critical Role of the Class III Histone Deacetylase SIRT1 in Cancer

Gene expression and deacetylase activity of the class III histone deacetylase SIRT1 are up-regulated in cancer cells due to oncogene overexpression or loss of function of tumor suppressor genes. SIRT1 induces histone deacetylation and methylation, promoter CpG island methylation, transcriptional repression, and deacetylation of tumor suppressor proteins. SIRT1 may play a critical role in tumor initiation, progression, and drug resistance by blocking senescence and apoptosis, and promoting cell growth and angiogenesis. SIRT1 inhibitors have shown promising anticancer effects in animal models of cancer. Further screening for more potent SIRT1 inhibitors may lead to compounds suitable for clinical trials in patients.

Prognostic Significance of MYCN Oncogene Expression in Childhood Neuroblastoma

PURPOSE To assess the significance of MYCN gene expression as a prognostic factor in patients with neuroblastoma of various ages, and to determine whether it can predict for outcome independently of MYCN gene amplification. PATIENTS AND METHODS The level of MYCN gene expression in 60 specimens of primary untreated neuroblastoma was determined by reverse-transcriptase polymerase chain reaction (RT-PCR) analysis. RESULTS High levels of MYCN gene expression were associated with advanced tumor stage (P=.0005), with the presence of MYCN gene amplification (P < .0001), but not with older age at diagnosis. Among patients who lacked MYCN gene amplification, the levels of MYCN gene expression were significantly greater in the tumors of infants compared with those of older children (P < .0005). High MYCN expression was strongly associated with reduced survival and event-free survival in the overall study population (P < .005), and also in the subset of patients aged older than 1 year at diagnosis (P < .001). In contrast, MYCN expression did not appear to be predictive of outcome in infants. After adjustment for the effect of MYCN amplification, high levels of MYCN expression retained significant prognostic value for poor survival (relative hazards, 30.3; P=.003) in children aged older than 12 months at diagnosis. CONCLUSION High MYCN gene expression is strongly predictive of poor outcome in older children with neuroblastoma, but not in infants. The findings help explain the controversy in the literature about the prognostic value of MYCN gene expression and highlight the different biology of neuroblastoma that presents in infants and older children.

ODC1 Is a Critical Determinant of MYCN Oncogenesis and a Therapeutic Target in Neuroblastoma

Neuroblastoma is a frequently lethal childhood tumor in which MYC gene deregulation, commonly as MYCN amplification, portends poor outcome. Identifying the requisite biopathways downstream of MYC may provide therapeutic opportunities. We used transcriptome analyses to show that MYCN-amplified neuroblastomas have coordinately deregulated myriad polyamine enzymes (including ODC1, SRM, SMS, AMD1, OAZ2, and SMOX) to enhance polyamine biosynthesis. High-risk tumors without MYCN amplification also overexpress ODC1, the rate-limiting enzyme in polyamine biosynthesis, when compared with lower-risk tumors, suggesting that this pathway may be pivotal. Indeed, elevated ODC1 (independent of MYCN amplification) was associated with reduced survival in a large independent neuroblastoma cohort. As polyamines are essential for cell survival and linked to cancer progression, we studied polyamine antagonism to test for metabolic dependence on this pathway in neuroblastoma. The Odc inhibitor alpha-difluoromethylornithine (DFMO) inhibited neuroblast proliferation in vitro and suppressed oncogenesis in vivo. DFMO treatment of neuroblastoma-prone genetically engineered mice (TH-MYCN) extended tumor latency and survival in homozygous mice and prevented oncogenesis in hemizygous mice. In the latter, transient Odc ablation permanently prevented tumor onset consistent with a time-limited window for embryonal tumor initiation. Importantly, we show that DFMO augments antitumor efficacy of conventional cytotoxics in vivo. This work implicates polyamine biosynthesis as an arbiter of MYCN oncogenesis and shows initial efficacy for polyamine depletion strategies in neuroblastoma, a strategy that may have utility for this and other MYC-driven embryonal tumors.

Association of High-Level MRP1 Expression With Poor Clinical Outcome in a Large Prospective Study of Primary Neuroblastoma

PURPOSE We have previously shown in a retrospective study that expression of the multidrug transporter gene MRP1 (ABCC1) is associated with outcome in neuroblastoma. We have now undertaken a prospective analysis to examine the independent prognostic significance of MRP1 expression in a large cohort of primary untreated neuroblastomas. PATIENTS AND METHODS Two hundred nine diagnostic neuroblastoma samples from patients prospectively enrolled onto the Pediatric Oncology Group biology protocol 9047 were analyzed for expression of the MRP1, MDR1, MYCN, and TRKA genes using real-time polymerase chain reaction. Expression levels were correlated with established prognostic indicators and disease outcome. RESULTS MRP1 expression was detected in all tumors analyzed, and levels were significantly higher in tumors with versus without MYCN amplification (P < .0001). High levels of MRP1 were highly predictive of both event-free survival (EFS; P < .001) and overall survival (OS; P < .001). High-level MYCN and low-level TRKA were also predictive of poor outcome. MDR1 expression demonstrated no prognostic significance. After adjustment for the effect of statistically significant prognostic indicators in multivariate models, MRP1 expression retained significant prognostic value for both EFS (hazard ratio = 3.0; P = .0011) and OS (hazard ratio = 2.5; P = .0095), whereas MYCN amplification did not have prognostic significance. CONCLUSION The results of this prospective study confirm our earlier findings and support a clinically relevant role for MRP1 gene expression in neuroblastoma. These findings have implications for the biology, prognosis, and treatment of this disease and provide evidence that MRP1 is a bone fide molecular target for reversing chemotherapy resistance in aggressive drug-refractory neuroblastoma.

Expression of multidrug transporter MRP4/ABCC4 is a marker of poor prognosis in neuroblastoma and confers resistance to irinotecan in vitro

Members of the multidrug resistance–associated protein (MRP) family of transporters are believed to contribute to cytotoxic drug resistance and chemotherapy failure. We observed frequent MRP4 overexpression in aggressive primary neuroblastoma, a disease for which we have previously shown MRP1 to be a prognostic indicator. High MRP4 expression correlated with MYCN oncogene amplification and was significantly associated with poor clinical outcome. Although MRP4 is known to transport some nucleoside analogues, it has not previously been associated with resistance to drugs used to treat solid tumors. We now show that it mediates substantial resistance in vitro to the topoisomerase I poison irinotecan/CPT-11 and its active metabolite SN-38. These results suggest that MRP4 will be a useful prognostic marker for neuroblastoma and that clinical trials of irinotecan as a neuroblastoma treatment should monitor MRP4 expression. The same may be true for other tumor types expressing high levels of the transporter.

Epigenetic silencing of BIM in glucocorticoid poor-responsive pediatric acute lymphoblastic leukemia, and its reversal by histone deacetylase inhibition.

Glucocorticoids play a critical role in the therapy of lymphoid malignancies, including pediatric acute lymphoblastic leukemia (ALL), although the mechanisms underlying cellular resistance remain unclear. We report glucocorticoid resistance attributable to epigenetic silencing of the BIM gene in pediatric ALL biopsies and xenografts established in immune-deficient mice from direct patient explants as well as a therapeutic approach to reverse resistance in vivo. Glucocorticoid resistance in ALL xenografts was consistently associated with failure to up-regulate BIM expression after dexamethasone exposure despite confirmation of a functional glucocorticoid receptor. Although a comprehensive assessment of BIM CpG island methylation revealed no consistent changes, glucocorticoid resistance in xenografts and patient biopsies significantly correlated with decreased histone H3 acetylation. Moreover, the histone deacetylase inhibitor vorinostat relieved BIM repression and exerted synergistic antileukemic efficacy with dexamethasone in vitro and in vivo. These findings provide a novel therapeutic strategy to reverse glucocorticoid resistance and improve outcome for high-risk pediatric ALL.

Germline mutation of the tumour suppressor PTEN in Proteus syndrome

Proteus syndrome (PS, OMIM 176920) is a hamartomatous disorder characterised by overgrowth of multiple tissues, connective tissue and epidermal naevi, and vascular malformations.1 These presentations are usually apparent at birth or soon after and continue to develop as the patient ages. It is named after the Greek god Proteus who, legend has it, could change his shape at will to avoid capture. It is probably the disease suffered by the Elephant Man.2 Tumours, mostly benign but some malignant, have also been reported in PS, generally presenting by the age of 20 years and including papillary adenocarcinoma of the testis, meningioma, and cystadenoma of the ovaries.3 Given the predominantly sporadic nature of this syndrome and the mosaic distribution of lesions, it has been suggested that PS may be caused by somatic mosaicism for a genetic change that is lethal in the non-mosaic state.4 Clinical overlap, in the form of tissue overgrowth, macrocephaly, and the presence of lipomas, exists between PS and another hamartoma syndrome, Bannayan-Riley-Ruvalcaba syndrome (BRR, OMIM 153480),5 in which up to 60% of affected subjects are known to carry a germline mutation of the tumour suppressor gene PTEN .6 BRR also shows partial clinical overlap with Cowden syndrome (CS, OMIM 158350), in which affected subjects are at risk of developing hamartomas in multiple organs including the breast, thyroid, central nervous system, skin, and gastrointestinal tract, as well as malignant tumours of the breast, thyroid, and endometrium. PTEN is mutated in the germline in up to 80% of patients with CS.6 Two recent reports have shown germline, and probably germline mosaic, PTEN mutations in a subset of patients with PS or a PS-like disorder,7,8 although other studies of patients with PS have been unable to confirm these findings.9,10 PTEN …

MYCN Expression Is Not Prognostic of Adverse Outcome in Advanced-Stage Neuroblastoma With Nonamplified MYCN

PURPOSE The clinical significance of MYCN expression in children with neuroblastoma (NB) remains controversial. To determine the prognostic significance of MYCN expression in the absence of MYCN amplification, we analyzed MYCN mRNA and protein expression in tumors from 69 patients. PATIENTS AND METHODS Sixty-nine NB tumor samples with nonamplified MYCN from patients with stage C or D disease were obtained from the Pediatric Oncology Group Neuroblastoma Tumor Bank. MYCN mRNA was analyzed using a real-time reverse transcriptase polymerase chain reaction assay, and MYCN protein was examined by Western blot analyses. RESULTS The estimated 5-year event-free survival (EFS) and survival (S) rates plus SE for the cohort were 57% +/- 17% and 60% +/- 16%, respectively. Infants younger than 1 year had significantly higher rates of EFS and S than children >/= 1 year of age (P =.003 and P <.001, respectively); patients with stage C disease had better outcome than those with stage D NB (P <.001); and patients with hyperdiploid tumors had better outcome than those with diploid NB (P <.001). Surprisingly, outcome was slightly better for patients with high versus low levels of MYCN mRNA expression (4-year S, 70% +/- 13% v 50% +/- 16%; P =.290), and for patients with tumors that expressed MYCN protein (4-year S, 73% +/- 19% v 53% +/- 15%, respectively; P =.171). CONCLUSION High levels of MYCN expression are not prognostic of adverse outcome in patients with advanced-stage NB with nonamplified MYCN. A trend associating high levels of MYCN expression with improved outcome was observed.

Divergent mechanisms of glucocorticoid resistance in experimental models of pediatric acute lymphoblastic leukemia.

Cell line models of glucocorticoid resistance in childhood acute lymphoblastic leukemia (ALL) almost invariably exhibit altered glucocorticoid receptor (GR) function. However, these findings are incongruous with those using specimens derived directly from leukemia patients, in which GR alterations are rarely found. Consequently, mechanisms of glucocorticoid resistance in the clinical setting remain largely unresolved. We present a novel paradigm of glucocorticoid resistance in childhood ALL, in which patient biopsies have been directly established as continuous xenografts in immune-deficient mice, without prior in vitro culture. We show that the GRs from six highly dexamethasone-resistant xenografts (in vitro IC(50) >10 micromol/L) exhibit no defects in ligand-induced nuclear translocation and binding to a consensus glucocorticoid response element (GRE). This finding contrasts with five commonly used leukemia cell lines, all of which exhibited defective GRE binding. Moreover, whereas the GRs of dexamethasone-resistant xenografts were transcriptionally active, as assessed by the ability to induce the glucocorticoid-induced leucine zipper (GILZ) gene, resistance was associated with failure to induce the bim gene, which encodes a proapoptotic BH3-only protein. Furthermore, the receptor tyrosine kinase inhibitor, SU11657, completely reversed dexamethasone resistance in a xenograft expressing functional GR, indicating that pharmacologic reversal of glucocorticoid resistance in childhood ALL is achievable.