Boo Messahel

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.

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.

Relapsed intracranial ependymoma in children in the UK: patterns of relapse, survival and therapeutic outcome.

Relapsed ependymoma in children poses difficult dilemmas in management. Clinico-pathological and treatment data of 108 children with relapsed ependymoma in the United Kingdom (UK) treated between 1985 and 2002 were reviewed to identify prognostic factors affecting survival. The primary site was the most common site of relapse (84%). Overall 25% had metastatic relapse. Surgery at relapse was attempted in only 55%. Radiotherapy was delivered at relapse in 66% infants and 50% of older children were re-irradiated. Overall 5-year survival was 24% and 27% for children less than 3 years of age at initial diagnosis and older children, respectively. Multivariate analysis showed that, for infants, surgery (p=0.01) and radiotherapy (p=0.001) at relapse were independent predictors of survival. For older children regardless of the previous radiotherapy, repeat irradiation was associated with better outcome (p=0.05). Relapse was associated with poor outcome in both age groups. A survival advantage conferred by both radiotherapy and surgery at relapse is independently significant.

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/m2) 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.

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.

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.

Complex patterns of chromosome 9 alterations including the p16INK4a locus in Wilms tumours

Background: Previous data implicating genetic and epigenetic events on chromosome 9, including the CDKN2A/2B locus, as molecular predictors of Wilms tumour relapse, have been conflicting. Aims: To clarify this using genome-wide and focused molecular genetic analysis. Methods: Microarray-based comparative genomic hybridisation (aCGH) using genome-wide coverage was applied to 76 favourable histology Wilms tumours. Additional investigation of the 9p21 locus was carried out using loss of heterozygosity (LOH) and fluorescence in situ hybridisation (FISH), as well as immunohistochemistry for CDKN2A/p16INK4a on a paediatric renal tumour tissue microarray. Results: Approximately half of the tumours were found to show chromosome 9 copy number changes. Those cases which harboured alterations comprised at least four distinct patterns: gain of the entire chromosome, loss of 9p, gain of 9q34, or a more complex combination of gains/losses. None of these tumour groups showed any statistically significant correlation with clinicopathological variables. Deletion mapping of 9p by LOH revealed several regions of overlap, including the CDKN2A/2B locus in 4/34 (11.8%) tumours, which was confirmed to represent hemizygous deletions by FISH. CDKN2A/p16INK4a protein expression was predominantly negative in Wilms tumours as assessed by immunohistochemistry on a tissue array, reflecting the expression pattern in normal kidney. However, 38/236 (16.1%) non-anaplastic Wilms tumours, 4/9 (44.4%) anaplastic Wilms tumours, 5/7 (71.4%) rhabdoid tumours of the kidney, and 4/10 (40%) clear cell sarcomas of the kidney showed nuclear CDKN2A/p16INK4a immunoreactivity. Conclusions: These data reveal the complex nature of genetic alterations on chromosome 9 in Wilms tumours, but do not provide evidence for their involvement in or association with treatment failure.