Sian Gibson

Clusterin, a haploinsufficient tumor suppressor gene in neuroblastomas.

BACKGROUND Clusterin expression in various types of human cancers may be higher or lower than in normal tissue, and clusterin may promote or inhibit apoptosis, cell motility, and inflammation. We investigated the role of clusterin in tumor development in mouse models of neuroblastoma. METHODS We assessed expression of microRNAs in the miR-17-92 cluster by real-time reverse transcription-polymerase chain reaction in MYCN-transfected SH-SY5Y and SH-EP cells and inhibited expression by transfection with microRNA antisense oligonucleotides. Tumor development was studied in mice (n = 66) that were heterozygous or homozygous for the MYCN transgene and/or for the clusterin gene; these mice were from a cross between MYCN-transgenic mice, which develop neuroblastoma, and clusterin-knockout mice. Tumor growth and metastasis were studied in immunodeficient mice that were injected with human neuroblastoma cells that had enhanced (by clusterin transfection, four mice per group) or reduced (by clusterin short hairpin RNA [shRNA] transfection, eight mice per group) clusterin expression. All statistical tests were two-sided. RESULTS Clusterin expression increased when expression of MYCN-induced miR-17-92 microRNA cluster in SH-SY5Y neuroblastoma cells was inhibited by transfection with antisense oligonucleotides compared with scrambled oligonucleotides. Statistically significantly more neuroblastoma-bearing MYCN-transgenic mice were found in groups with zero or one clusterin allele than in those with two clusterin alleles (eg, 12 tumor-bearing mice in the zero-allele group vs three in the two-allele group, n = 22 mice per group; relative risk for neuroblastoma development = 4.85, 95% confidence interval [CI] = 1.69 to 14.00; P = .005). Five weeks after injection, fewer clusterin-overexpressing LA-N-5 human neuroblastoma cells than control cells were found in mouse liver or bone marrow, but statistically significantly more clusterin shRNA-transfected HTLA230 cells (3.27%, with decreased clusterin expression) than control-transfected cells (1.53%) were found in the bone marrow (difference = 1.74%, 95% CI = 0.24% to 3.24%, P = .026). CONCLUSIONS We report, to our knowledge, the first genetic evidence that clusterin is a tumor and metastasis suppressor gene.

Coordinated oncogenic transformation and inhibition of host immune responses by the PAX3-FKHR fusion oncoprotein

Tumors have evolved elaborate mechanisms for evading immune detection, such as production of immunoinhibitory cytokines and down-regulation of major histocompatibility complex (MHC) expression. We have studied PAX3-FKHR as an example of an oncogenic fusion protein associated with an aggressive metastatic cancer. We show that PAX3-FKHR alters expression of genes that are normally regulated by Janus kinase/signal transducer and activator of transcription (STAT) signaling pathways. This occurs as a result of a specific interaction between PAX3-FKHR and the STAT3 transcription factor, which results in a dramatic reduction in tumor MHC expression, and an alteration in local cytokine concentrations to inhibit surrounding inflammatory cells and immune detection. Collectively, these data show that an oncogenic transcription factor can promote tumor growth and tissue invasion while inhibiting local inflammatory and immune responses. This is the first time that an immunomodulatory role has been described for an oncogenic fusion protein.

Clinical and pathological features of paediatric malignant rhabdoid tumours

Malignant rhabdoid tumours (MRT) and their central nervous system (CNS) counterparts atypical teratoid/rhabdoid tumours (ATRT) are rare, highly aggressive malignant neoplasms of childhood. Although there are isolated reports of long‐term survival with intensive, multimodal therapy, outcomes are generally poor.

Beta-catenin expression in pediatric fibroblastic and myofibroblastic lesions: A study of 100 cases

Nuclear immunoreactivity for β-catenin is a useful adjunct for diagnosis of adult desmoid-type fibromatoses, many of which exhibit mutations within the APC/β-catenin (Wnt) pathway. Pediatric fibromatoses represent a heterogeneous group of lesions that are diagnostically challenging, especially on biopsy. We studied β-catenin expression in a variety of pediatric fibroblastic and myofibroblastic lesions. Immunohistochemical nuclear expression of β-catenin was assessed in 100 tumors. High-level expression of β-catenin was found in 42% of usual-type or deep fibromatoses (21 of 50). Such expression was not seen in any of the other lesions, including fibrous hamartoma of infancy (0 of 18), juvenile hyaline fibromatosis (0 of 7), infantile digital fibromatosis (0 of 6), myofibromatosis (0 of 5), lipofibromatosis (0 of 4), calcifying aponeurotic fibroma (0 of 3), palmar-plantar fibromatosis (0 of 2), fibromatosis colli (0 of 1), or torticollis (0 of 1). High-level β-catenin staining is seen in deep “adult-type” fibromatoses occurring in children, although to a lesser frequency than in adult fibromatoses. This indicates that a subset of deep fibromatoses in childhood shares similar mechanisms of tumorigenesis with those in adults. β-catenin is not expressed in other common pediatric fibroblastic and myofibroblastic lesions, and the Wnt pathway does not appear to play a role in their pathogenesis.

Expression of ETV6-NTRK in classical, cellular and mixed subtypes of congenital mesoblastic nephroma

Aim:  Congenital mesoblastic nephroma (CMN) is the commonest renal tumour of infancy, with classical, cellular and mixed histological subtypes described. A specific ETV6‐NTRK3 fusion‐gene product is reported in association with the cellular variant. The aim was to investigate the relationship between the presence of this product and morphological phenotype using paraffin‐embedded archival material.

PAX5 Expression in Nonhematopoietic Tissues: Reappraisal of Previous Studies

The Pax gene family encodes transcription factors with similar structures but distinctive roles in development and with limited expression in adult tissues. Reexpression of PAX proteins is frequently observed in human cancers, reflecting recapitulation of embryologic or developmental function. Defining expression of PAX family members is important in the immunohistochemical differential diagnosis of cancer, understanding oncogenesis, and defining targets for therapy. Immunostaining for PAX5 has become a commonly used technique in differential diagnosis of B-lineage hematologic malignancies. In seeking to define the range and degree of expression of PAX5 in nonhematologic pediatric cancers by immunohistochemical analysis with the anti-PAX5 monoclonal antibody routinely used in research and diagnosis, we observed strong immunostaining in a number of malignant tissues, including Wilms tumor. The pattern of expression of PAX5 in Wilms tumor was found to be identical to that of PAX2, raising the possibility of antibody cross-reactivity. This was subsequently confirmed by Western blotting and immunostaining of transfected cells and quantitative reverse transcriptase-polymerase chain reaction. Since the same PAX5 monoclonal antibody has been used consistently in the literature, these findings indicate a need for reappraisal of published PAX5 immunostaining results.

Ultrasound-guided core needle biopsy for the diagnosis of rhabdomyosarcoma in childhood

Most commonly a tissue diagnosis of rhabdomyosarcoma (RMS) in children is made by biopsy as opposed to primary resection. Open surgical procedures are often recommended to obtain sufficient material for accurate and complete diagnostic work up. Our institution has routinely used image‐guided needle biopsies for soft tissue tumour diagnosis. We therefore sought to assess diagnostic accuracy and completeness, and procedure safety of consecutive patients diagnosed by needle biopsies in a single institution.

Platelet‐derived growth factor receptors and ligands are up‐regulated in paediatric fibromatoses

Aims:  Platelet‐derived growth factors (PDGF) and their receptors (PDGFR) play an essential role in pathways involved in the regulation of cell proliferation, growth and function. Overexpression of PDGF/R is reported in a wide range of solid tumours. The aim was to determine levels of PDGF/R expression in paediatric fibromatoses and myofibromatosis.

PAX5 Expression in Rhabdomyosarcoma

To the Editor: PAX5, a member of the PAX family of transcription factors, is a possible target for cellular immunotherapy as it has a limited expression pattern in adult tissues and is known to be expressed in a variety of cancers, most notably in B-cell malignancies. We have been undertaking an assessment of the expression of PAX5 in a range of pediatric solid tumors and were interested in the recent article by Sullivan et al on PAX5 expression in rhabdomyosarcoma (RMS). They reported PAX5 expression in 34 of 51 (67%) cases of alveolar RMS (ARMS), but no expression in any of 55 embryonal RMS (ERMS). On the basis of the data from a subset of 7 ARMS, in which genetic information was available, the authors suggest a correlation between PAX5 expression and the presence of translocations involving t(2;13)(q35:q14) or t(1;13)(p36;q14) resulting in PAX3/ FKHR or PAX7/FKHR fusion genes, respectively. We have undertaken an analysis to examine this hypothesis in a larger number of RMS cases with established translocation status. The histopathology files at Great Ormond Street Hospital were searched for cases of RMS diagnosed between 2000 and 2008 in which PAX gene fusion status had been determined in our diagnostic laboratory by molecular analysis (typically by reverse transcription polymerase chain reaction, or, in a minority of cases, by karyotype analysis or fluorescence in situ hybridization). In all cases employing reverse transcription polymerase chain reaction, replicate assays were performed and positive controls showed a single clear band of appropriate size and negative controls showed no DNA product. A total of 42 cases were identified and archived paraffin-embedded tissue was available in 25 cases (9 ERMS, 15 ARMS, and 1 sclerosing RMS). Immunohistochemistry for PAX5 was performed on 4-mm-thick sections. These were deparaffinized with xylene and rehydrated in graded alcohol solutions, before blocking of endogenous peroxidase activity with 3% hydrogen peroxide in phosphate buffered-saline. Antigen retrieval was performed in citrate buffer, pH 6.2, using a pressure-cooking device, before incubation with monoclonal antibody to PAX5 (BD Biosciences, clone 24) at a dilution of 1:100. Visualization of antibody staining was with the Dako Envision system and slides were counterstained with hematoxylin. An internal positive control with a section of tonsil was included and primary antibody was omitted in negative controls. The stained slides were reviewed independently by 2 examiners who were blind to the PAX3/PAX7 gene fusion status. Only nuclear staining was considered positive and scoring excluded infiltrating or tumor-associated PAX5-positive lymphocytes. We used an identical scoring system to Sullivan et al to permit a comparison of data, as follows: 0, no reactivity; 1+, rare positive tumor cells; 2+, 1% to 10% positive cells; 3+, 10% to 50% of positive cells; and 4+, >50% of positive cells. Histologic subtype (ERMS/ARMS) had been determined on the basis of morphology and immunohistochemistry in all cases by formal pathologic diagnosis at Great Ormond Street Hospital, a department with extensive experience in the diagnosis of pediatric small round cell tumors. In agreement with Sullivan et al, we saw no PAX5 positivity in any of the 9 ERMS cases, all of which were also negative for PAX3 or PAX7 fusion genes. A single case of sclerosingtype RMS was also negative for PAX5 immunostaining and PAX3/ PAX7 translocations. Of the ARMS cases, translocations involving PAX3 or PAX7 were confirmed in 7 of 15 (47%) and 9 ARMS cases (60%) showed some degree of nuclear staining for PAX5 (Table 1 and Fig. 1). Interestingly, in contrast to the data presented by Sullivan et al we did not see a consistent correlation between PAX5 staining in ARMS and PAX3/PAX7 translocation status. Although the majority (6 of 7, 86%) of translocation positive ARMS samples showed some degree of PAX5 immunostaining, translocation negative ARMS samples were not consistently PAX5 negative. Indeed, 3 of 8 (38%) of these translocation negative ARMS showed some degree of PAX5 staining, and translocation negative ARMS samples made up the majority of the samples with the greatest degree (3+ or 4+) of PAX5 staining (Table 2). Thus, our data suggest that PAX5 immunoreactivity in RMS, although correlating with histologic subtype, is not specific for translocationpositive tumors. Our data indicate that ARMS with either PAX3 or PAX7 translocations, or with no translocation, may express PAX5 and that although PAX5 immunoreactivity seems more common in translocation positive tumors, PAX5 staining cannot be used as a surrogate marker for translocation status in place of molecular genetic investigations. Sequence homology within the PAX gene family makes cross-reactivity of the PAX5 antibody with other PAX family proteins a possibility. Nevertheless, the lack of widespread PAX5 immunostaining in a subset of PAX3/PAX7 translocation-positive tumors makes crossreactivity of the PAX5 antibody with PAX3 or PAX7 very unlikely. The biologic relevance of PAX5 expression in a subset of ARMS remains unclear. Although some degree of immunostaining is seen in approximately 60% of the cases in this study (and in that of Sullivan et al), this includes a significant number of TABLE 1. Alveolar Rhabdomyosarcoma PAX5 Staining

Rapid and accurate determination of MYCN copy number and 1p deletion in neuroblastoma by quantitative PCR.

MYCN amplification and 1p36 deletion are adverse prognostic factors in neuroblastoma, and rapid accurate determination of MYCN amplification is essential for risk stratification. MYCN copy number and 1p36 deletion status were determined by fluorescence in situ hybridization (FISH) and real time PCR in a diagnostic pathology laboratory setting on 35 consecutive patients with neuroblastoma. The PCR technique was technically successful in all cases and results were generally available within 24 hr of biospy. There was no discordance between FISH and PCR results. Real time PCR is a reliable, accurate, and simple technique that can be applied to small neuroblastoma biopsies allowing rapid diagnosis.