Outcomes of non-anaplastic stage III and 'inoperable' Wilms tumour treated in the UKW3 trial.

BACKGROUND AND PURPOSE: To describe the outcome of patients with stage III Wilms tumours (WT) treated in the UKW3 trial. MATERIAL AND METHODS: Patients with a pathologically confirmed stage III non-anaplastic WT at nephrectomy (Group A) or with an 'inoperable' tumour at diagnosis managed by biopsy and pre-operative chemotherapy (Actinomycin D-Vincristine-Doxorubicin) but stage I or II at subsequent nephrectomy (Group B) were included. RESULTS: The 4-year overall (OS)/event free survival (EFS) for Group A (n = 117) patients was 90%(95%CI:83-94)/81%(CI:73-87) and for Group B (n = 32) 94%(CI:77-98)/88%(CI:70-95). The 4-year OS/EFS of patients with pathological stage III WT according to whether they received flank/abdominal radiotherapy (95 patients) or not (37 patients, 22 from UKW3 pooled with 17 patients from UKW2) were 91%(CI:83-95)/82%(CI:73-89), and 84%(CI:67-92)/78%(CI:61-89), respectively. The 4-year OS/EFS for patients having one reason to be stage III versus two or three was 92%(CI:84-96)/83%(CI:73-90) and 85%(CI:70-93)/78%(CI:61-88), respectively. CONCLUSION: Our findings question the inclusion of biopsy or pre-operative chemotherapy as sole criterion for assigning a tumour stage III. Selected patients with pathological stage III WT can survive without radiotherapy. Whilst cautious interpretation is needed due to the post hoc nature of these analyses, further biological studies may better characterise those who could benefit from reduced therapy.

Olive ingestion causing a false suspicion of relapsed neuroblastoma: A case of "oliveblastoma?"

Measurement of the urine catecholamine metabolites homovanillic acid (HVA) and vanillylmandelic acid (VMA) are the standard method for detecting disease recurrence in neuroblastoma. We present a case of abnormal concentrations of catecholamine metabolites that prompted investigations for relapsed neuroblastoma. However, further study revealed that the abnormal biochemistry was likely due to ingestion of olives. Olive ingestion should be considered when interpreting urine HVA and VMA results, and excluded if concentrations are unexpectedly abnormal.

How to use lymph node biopsy in paediatrics.

Lymphadenopathy is a common finding in children. It often causes anxiety among parents and healthcare professionals because it can be a sign of cancer. There is limited high-quality evidence to guide clinicians as to which children should be referred for lymph node biopsy. The gold standard method for evaluating lymphadenopathy of unknown cause is an excision biopsy. In this Interpretation, we discuss the use of lymph node biopsy in children.

Surgical complications after immediate nephrectomy versus preoperative chemotherapy in non-metastatic Wilms' tumour: findings from the 1991-2001 United Kingdom Children's Cancer Study Group UKW3 Trial.

PURPOSE: To compare surgical complication rates after immediate nephrectomy versus delayed nephrectomy following preoperative chemotherapy in children with non-metastatic Wilms' tumour enrolled in UKW3, both in randomised patients and in those for whom the treatment approach was defined by parental or physician choice. METHODS: Records for all patients enrolled into UKW3 were reviewed. Any record of tumour rupture or surgical complication was extracted and comparisons made between the two treatment strategies in both populations of randomised and non-randomised patients. RESULTS: Of 525 children enrolled, 205 patients were randomised to either immediate nephrectomy (n=103) or pre-operative chemotherapy followed by delayed nephrectomy (n=102). Of the 320 children not randomised, data were available on 189 cases treated with immediate nephrectomy and 103 treated with pre-operative chemotherapy. There were significantly fewer surgical complications in randomised children given pre-operative chemotherapy before surgery compared to children undergoing immediate nephrectomy (1% vs. 20.4%, P<0.001); this difference was most marked for tumour rupture (0% vs. 14.6%, P<0.001). CONCLUSIONS: Delayed nephrectomy for Wilms' tumour, preceded by pre-operative chemotherapy was associated with fewer surgical complications compared with immediate nephrectomy.

Subtype-specific FBXW7 mutation and MYCN copy number gain in Wilms' tumor.

PURPOSE: Wilms' tumor (WT), the most common pediatric renal malignancy, is associated with mutations in several well-characterized genes, most notably WT1, CTNNB1, WTX, and TP53. However, the majority of cases do not harbor mutations in these genes. We hypothesized that additional drivers of tumor behavior would be contained within areas of consistent genomic copy number change, especially those associated with the WT risk groups defined by the International Society of Paediatric Oncology (SIOP). EXPERIMENTAL DESIGN: We analyzed high-resolution (Affymetrix 250K single nucleotide polymorphism array) genomic copy number profiles of over 100 tumors from selected risk groups treated under the SIOP protocols, further characterizing genes of interest by sequencing, Multiplex Ligation-dependent Probe Amplification, or fluorescence in situ hybridization. RESULTS: We identified FBXW7, an E3 ubiquitin ligase component, as a novel Wilms' tumor gene, mutated or deleted in approximately 4% of tumors examined. Strikingly, 3 of 14 (21%) of tumors with epithelial type histology after neoadjuvant chemotherapy had FBXW7 aberrations, whereas a fourth WT patient had germline mutations in both FBXW7 and WT1. We also showed that MYCN copy number gain, detected in 9 of 104 (8.7%) of cases, is relatively common in WT and significantly more so in tumors of the high risk diffuse anaplastic subtype (6 of 19, 32%). CONCLUSIONS: Because MYCN is itself a target of FBXW7-mediated ubiquitination and degradation, these results suggest that a common pathway is dysregulated by different mechanisms in various WT subtypes. Emerging therapies that target MYCN, which is amplified in several other pediatric cancers, may therefore be of value in high risk Wilms' tumor.

Expression of hepatocyte growth factor and its receptor met in Wilms' tumors and nephrogenic rests reflects their roles in kidney development.

PURPOSE: Hepatocyte growth factor (HGF) and its receptor Met are known to play diverse roles in both organogenesis and cancer. Wilms' tumor (WT) is a prototype for the link between abrogated development and neoplasia, with dysregulation of growth factor/receptor pathways playing key roles. Despite this, an understanding of the HGF/Met axis in the process is lacking. EXPERIMENTAL DESIGN: Observing copy number alterations at the loci for these genes in WTs and their precursor lesions nephrogenic rests, we examined protein expression by immunohistochemistry and investigated the effects of HGF on an in vitro model of kidney development. RESULTS: HGF was preferentially expressed in the blastemal cells of nephrogenic rests but not WTs. Met expression was infrequent and restricted to well-differentiated epithelial cells and stroma in both lesions. In an independent cohort of favorable histology WTs on a tissue microarray, HGF was expressed in 15 of 193 (8%) cases and correlated with a predominance of epithelial cells, whereas Met expression was observed in 25 of 179 (14%) cases and was associated with stromal subtypes. In a mouse mesonephric cell line model, we observed Met expression in culture conditions reflecting both mesenchymal and epithelial differentiation, whereas HGF was up-regulated in association with acquisition of a more epithelial-like phenotype. This could be mimicked by exogenous exposure of mesenchymal-like cells to recombinant HGF. CONCLUSIONS: These data show that the relatively infrequent expression of HGF and Met in WT tumorigenesis reflects their roles in nephrogenesis, particularly the mesenchymal-to-epithelial transition, rather than a dependence on oncogenic signaling pathways.

Allele loss at 16q defines poorer prognosis Wilms tumour irrespective of treatment approach in the UKW1-3 clinical trials: a Children's Cancer and Leukaemia Group (CCLG) Study.

Survival from Wilms tumour is excellent. Hence, better markers are required to restrict treatments causing late sequelae to those at highest risk of relapse. We investigated the prognostic significance of loss of heterozygosity (LOH) on 1p and 16q in 426 favourable histology Wilms tumours treated with either immediate nephrectomy (63%) or preoperative chemotherapy (37%). Four years RFS and OS were 84.6% and 92.0%, respectively. 10.3% tumours had LOH 1p, 14.6% LOH 16q, with 2.6% at both loci. In multivariate analysis, LOH 16q was associated with an increased risk of relapse (hazard ratio (HR) 2.69, 95%CI: 1.47-4.92) and death (HR 2.67, 95%CI: 1.17-6.06). LOH 1p showed no significant associations. These results were not influenced by treatment approach. LOH 16q is an adverse risk factor in favourable histology Wilms tumour, regardless of initial approach to therapy. Its relationship with histological risk groups defined after neo-adjuvant chemotherapy requires analysis in a larger series, and is the subject of the current SIOP WT 2001 trial.

Genomic imbalances in rhabdomyosarcoma cell lines affect expression of genes frequently altered in primary tumors: an approach to identify candidate genes involved in tumor development.

Rhabdomyosarcomas (RMS) are the most common pediatric soft tissue sarcomas. They resemble developing skeletal muscle and are histologically divided into two main subtypes; alveolar and embryonal RMS. Characteristic genomic aberrations, including the PAX3- and PAX7-FOXO1 fusion genes in alveolar cases, have led to increased understanding of their molecular biology. Here, we determined the effect of genomic copy number on gene expression levels through array comparative genomic hybridization (CGH) analysis of 13 RMS cell lines, confirmed by multiplex ligation-dependent probe amplification copy number analyses, combined with their corresponding expression profiles. Genes altered at the transcriptional level by genomic imbalances were identified and the effect on expression was proportional to the level of genomic imbalance. Extrapolating to a public expression profiling dataset for 132 primary RMS identified features common to the cell lines and primary samples and associations with subtypes and fusion gene status. Genes identified such as CDK4 and MYCN are known to be amplified, overexpressed, and involved in RMS tumorigenesis. Of the many genes identified, those with likely functional relevance included CENPF, DTL, MYC, EYA2, and FGFR1. Copy number and expression of FGFR1 was validated in additional primary material and found amplified in 6 out of 196 cases and overexpressed relative to skeletal muscle and myoblasts, with significantly higher expression levels in the embryonal compared with alveolar subtypes. This illustrates the ability to identify genes of potential significance in tumor development through combining genomic and transcriptomic profiles from representative cell lines with publicly available expression profiling data from primary tumors.

Efficacy and tolerability of high-dose methotrexate in central nervous system positive or relapsed lymphoproliferative disease following liver transplant in children.

Childhood post-transplant lymphoproliferative disease (PTLD) is a heterogeneous condition in which treatment varies, from the reduction of immunosuppression to moderately intensive chemotherapy. While low-dose chemotherapy/rituximab has been found to be effective, moderately intensive chemotherapy is required for patients who relapse, have classic non-Hodgkin lymphoma or have fulminant PTLD. Methotrexate (Mtx) is highly effective in lymphomas and crosses the blood-brain barrier. However, there are no data in the literature regarding its safety in post-liver transplant patients. We describe four cases of high-grade lymphomas (three diffuse large B cell and one T-cell lymphoblastic), post-liver transplant, for which chemotherapy including high-dose Mtx (HDMTX) was the treatment of choice. In total, 20 doses of HDMTX (1-5 g/m(2)) were given. The treatment was well tolerated and all four patients had a good response. One case of central nervous system (CNS) diffuse large B-cell lymphoma was treated with HDMTX alone. We conclude that, in the absence of significant organ damage, HDMTX can safely be given to liver transplant patients, but should only be administered in specialist oncology units. Proof of effectiveness as a single agent in CNS lymphoma needs further studies.

c-KIT overexpression, without gene amplification and mutation, in paediatric renal tumours.

AIMS: To investigate the presence and prognostic relevance of KIT expression in paediatric renal tumours, and to determine whether receptor overexpression is associated with gene amplification and/or mutation. METHODS: Immunohistochemistry without antigen retrieval for CD117 was carried out on tissue microarrays consisting of 274 Wilms' tumours, 13 clear cell sarcomas of the kidney (CCSK), 10 mesoblastic nephromas (MN), and 7 rhabdoid tumours of the kidney (RTK). In addition, gene copy number was investigated by chromogenic in situ hybridisation (CISH), and overexpressing tumours were sequenced for KIT mutations in exons 9, 11, 13 and 17. RESULTS: Only 8/200 (4.0%) Wilms' tumours exhibited any degree of moderate-strong KIT staining in any of their assessable cell types. This small group of KIT-positive tumours had a shorter time to relapse (p = 0.0044, log-rank test). There were no positive MNs or RTKs; however 3/11 (27.3%) CCSKs were strongly positive, with an additional two cases weakly reactive. No cases exhibited gene amplification or mutation. CONCLUSIONS: KIT overexpression in rare in Wilms' tumours, although does appear to confer a worse prognosis, in particular for patients primarily treated with preoperative chemotherapy. CCSKs are associated with an increased expression of KIT, however, in the absence of gene amplification and/or activating mutation. The potential of anti-KIT therapeutic strategies in the treatment of paediatric renal tumours appears to be limited.

Multifaceted dysregulation of the epidermal growth factor receptor pathway in clear cell sarcoma of the kidney.

PURPOSE: Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase overexpressed in a variety of human malignancies, against which targeted therapies have shown efficacy in lung and brain tumors. Clinical responses to EGFR inhibitors have been found to be highly dependent on the presence of activating mutations, whereas gene amplification, downstream activation of Akt, and abnormalities in PTEN are also reported predictive factors. We sought to evaluate these variables in pediatric renal tumors. EXPERIMENTAL DESIGN: We screened a series of 307 pediatric renal tumors for EGFR expression by immunohistochemistry and gene amplification by chromogenic in situ hybridization. In identifying a striking predilection for certain tumor types, we further analyzed the clear cell sarcomas of the kidney (CCSK) for mutations in EGFR and PTEN. RESULTS: Although only 23 of 177 (13.0%) nonanaplastic Wilms' tumors were EGFR positive, 4 of 11 (36.4%) anaplastic tumors showed receptor overexpression. In addition, 5 of 9 (55.6%) mesoblastic nephromas and 12 of 12 (100%) CCSKs were strongly immunoreactive for EGFR. In studying the CCSKs in more detail, we identified gene amplification in 1 of 12 (8.3%) cases and a somatic T790M EGFR mutation in a further case. These two samples additionally harbored mutations in PTEN. Downstream pathway activation, as assayed by phosphorylated Akt expression, was observed in 8 of 12 (66.7%) cases. CONCLUSIONS: Together, these data show dysregulation of the EGFR pathway at multiple levels in CCSKs. Identification of factors predictive of poor response to targeted therapy, including the drug resistance T790M mutation, may provide a rationale for upfront trials with irreversible inhibitors of EGFR in children with these tumors.

Single agent efficacy of rituximab in childhood immunosuppression related lymphoproliferative disease: a United Kingdom Children's Cancer Study Group (UKCCSG) retrospective review.

Survival in childhood lymphoproliferative disease (LPD) remains poor, particularly in non-transplant patients. The anti-CD20 antibody rituximab shows promise but data in children is scant. A retrospective study of 22 (aged 11 months to 18 years) children treated with rituximab is presented. Two had primary immunodeficiency, two had prolonged immunosuppression and 18 had post-transplant LPD (eight bone marrow, five liver, four heart, one kidney). Nine patients had multi-organ involvement and 13 single site disease. Seventeen out of 22 had rituximab alone. In 16, a dose of 375 mg/m2 i.v. weekly was used (less in one patient due to renal dysfunction). Twelve patients received four courses and ten patients received one to three courses. Fever was the main side-effect in four. Eight (47%) had single agent response; four complete and four partial. All had other treatment prior to rituximab. Median follow-up was 35 months (range 22 - 47 months). In childhood LPD unresponsive to standard treatment, rituximab showed single agent response and requires further evaluation.

Amplification and overexpression of CACNA1E correlates with relapse in favorable histology Wilms' tumors.

PURPOSE: The most well established molecular markers of poor outcome in Wilms' tumor are loss of heterozygosity at chromosomes 1p and/or 16q, although to date no specific genes at these loci have been identified. We have previously shown a link between genomic gain of chromosome 1q and tumor relapse and sought to further elucidate the role of genes on 1q in treatment failure. EXPERIMENTAL DESIGN: Microarray-based comparative genomic hybridization identified a microamplification harboring a single gene (CACNA1E) at 1q25.3 in 6 of 76 (7.9%) Wilms' tumors, correlating with a shorter relapse-free survival (P = 0.0044, log-rank test). Further characterization of this gene was carried out by measuring mRNA and protein expression as well as stable transfection of HEK293 cells. RESULTS: Overexpression of the CACNA1E transcript was associated with DNA copy number (P = 0.0204, ANOVA) and tumor relapse (P = 0.0851, log-rank test). Immunohistochemistry against the protein product Ca(V)2.3 revealed expression localized to the apical membrane in the distal tubules of normal kidney but not to the metanephric blastemal cells of fetal kidney from which Wilms' tumors arise. Nuclear localization in 99 of 160 (61.9%) Wilms' tumor cases correlated with a reduced relapse-free survival, particularly in cases treated with preoperative chemotherapy (P = 0.009, log-rank test). Expression profiling of stably transfected HEK293 cells revealed specific up-regulation of the immediate early response genes EGR1/EGR2/EGR3 and FOS/FOSB, mediated by activation of the MEK/ERK5/Nur77 pathway. CONCLUSIONS: These data identify a unique genetic aberration with direct clinical relevance in Wilms' tumor relapse and provide evidence for a potential novel mechanism of treatment resistance in these tumors.

Blastemal expression of type I insulin-like growth factor receptor in Wilms' tumors is driven by increased copy number and correlates with relapse.

Most Wilms' tumors are of low stage, favorable histology, and have a high likelihood of cure with current multimodal therapy. Despite this, there remains a group of patients whose tumors recur for whom intensive salvage regimens result in survival of only 50%. Fitting a Cox proportional hazards model to microarray-based comparative genomic hybridization (aCGH) data on 68 Wilms' tumor samples, we identified a significant correlation between increased copy number at chromosome 15q26.3 insulin-like growth factor I receptor (IGFIR) and tumor relapse (adjusted P = 0.014). Wilms' tumors (13%) exhibited a low-level gain corresponding to three to four copies of the gene by aCGH analysis, 9 of 10 of which exhibited high IGFIR mRNA levels. Although IGFIR protein expression was restricted to the epithelial cells of fetal kidney and Wilms' tumors in most cases, 12% of tumors were also found to express IGFIR in the blastemal compartment. Blastemal IGFIR protein expression was associated with an increased copy number and a shorter relapse-free survival time (P = 0.027, log-rank test). In addition to the membrane localization, IGFIR was localized to the perinuclear region of the blastemal cells in 6% of Wilms' tumors. These data provide evidence that an increase in IGFIR gene copy number results in aberrant expression in the blastemal compartment of some Wilms' tumors and is associated with an adverse outcome in these patients. These findings suggest the possibility of use of targeted agents in the therapy of these children.

Immediate nephrectomy versus preoperative chemotherapy in the management of non-metastatic Wilms' tumour: results of a randomised trial (UKW3) by the UK Children's Cancer Study Group.

PURPOSE: To determine if patients receiving preoperative chemotherapy with vincristine and actinomycin D for non-metastatic Wilms' tumour have a more advantageous stage distribution and so need less treatment compared to patients who have immediate nephrectomy, without adversely affecting outcome. METHODS: Between 1991 and 2001, a total of 205 patients with newly diagnosed non-metastatic renal tumours, of which 186 had Wilms' histologies, were randomly assigned either to immediate surgery or to 6 weeks preoperative chemotherapy and then delayed surgery. Both groups of children received postoperative chemotherapy according to tumour stage and histology determined at the time of nephrectomy. RESULTS: There was a significant improvement in the stage distribution for patients with Wilms' histologies receiving delayed surgery compared to those having immediate nephrectomy (stage I: 65.2% versus 54.3%; stage II: 23.9% versus 14.9%; stage III: 9.8% versus 29.8%, chi2 test for trend=7.02, p=0.008). This improvement resulted in 20% fewer children receiving radiotherapy or doxorubicin yet event-free and overall survivals at 5 years of 79.6% and 89.0%, respectively, were similar in the two groups. CONCLUSION: Six weeks of preoperative chemotherapy with vincristine and actinomycin D results in a significant shift towards a more advantageous stage distribution and hence reduction in therapy, while maintaining excellent event free and overall survival in children with non-metastatic Wilms' tumour. Around 20% of survivors were therefore spared the late-effects of doxorubicin or radiotherapy. Our results suggest that all children with non-metastatic Wilms' tumour should receive chemotherapy prior to tumour resection.

Comparative genomic hybridization and BUB1B mutation analyses in childhood cancers associated with mosaic variegated aneuploidy syndrome.

We previously demonstrated that constitutional BUB1B mutations cause mosaic variegated aneuploidy, a condition characterized by constitutional aneuploidies and childhood cancer predisposition. To further investigate the role of BUB1B in cancer predisposition we performed comparative genomic hybridization analysis in an embryonal rhabdomyosarcoma from an MVA case with biallelic BUB1B mutations, revealing aneuploidies typical of sporadic E-RMS, with gain of chromosomes 3, 8, 13 and loss of chromosomes 9, 14, X. To investigate whether somatic BUB1B mutations occur in sporadic childhood cancers we screened 30 Wilms tumours, 10 acute lymphoblastic leukemias, nine rhabdomyosarcomas and 11 rhabdomyosarcoma cell lines for BUB1B mutations. We identified seven exonic and six intronic variants. Six of the exonic variants were synonymous and one resulted in a non-synonymous conservative missense alteration that was also present in a control. These data suggest that the genetic progression in rhabdomyosarcoma from MVA and non-MVA cases may be similar, but that somatic BUB1B mutations are unlikely to be common in sporadic childhood cancers known to be associated with MVA.

Clinical features of molecular pathology of solid tumours in childhood.

The outlook for children with cancer has improved substantially over the past 20 years, with over three-quarters of children now surviving in the long term. Better use of existing cytotoxic drugs and supportive care have made large contributions, but some of the improvement in survival is due to a greater knowledge of childhood cancer at the cellular and molecular levels. As in leukaemias, several childhood solid tumours carry balanced chromosomal translocations, resulting in fusion genes that encode chimeric proteins with new oncogenic properties. Many of these fusion genes, and other genetic aberrations are tumour specific and are related to outcome. Tumour biology now plays an important part in identifying appropriate treatment through more accurate diagnoses and new risk stratifications based on molecular markers.