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.

BACKGROUND: 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. PROCEDURE: We conducted a retrospective review of all patients diagnosed with MRT/ATRT at Great Ormond Street Hospital over the 20 years from 1989 to 2009. All cases were subjected to expert pathological review including INI-1 immunostaining. RESULTS: In a final cohort of 34 cases, overall survival was 17.4%, with median survival 10.1 months. Outcome in patients aged <3 years was significantly worse (median survival 6.2 months vs. 19.2 months). Data demonstrated a statistically significant benefit of radiotherapy (median survival 14.9 months vs. 6.6 months), although this analysis is confounded by the impact of patient age. There were four long-term survivors (>30 months), all of whom received chemotherapy with or without surgical resection or radiotherapy. In the present study, immunohistochemistry revealed no significant staining for either c-Erb or c-Met in any case, suggesting that targeting these molecules is unlikely to be of benefit in treating MRT/ATRT. CONCLUSIONS: In view of poor outcomes, there is a clear need for new treatment strategies and the identification of novel molecular targets for MRT/ATRT.

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

BACKGROUND: 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. METHODS: A retrospective review of consecutive biopsies of patients who were diagnosed with RMS or undifferentiated sarcoma in a single institution over a 9-year period. RESULTS: There were 24 children diagnosed with RMS or undifferentiated sarcoma who underwent 37 procedures (30 primary site and 7 draining lymph nodes). In the primary site diagnostic procedures, definitive diagnosis was made in all cases. In the majority of cases there was sufficient material for molecular analysis, cytogenetics and freezing. There were no complications of biopsy. CONCLUSIONS: In the hands of experienced operators, image-guided needle biopsies of RMSs allow for accurate diagnosis, allow sufficient material to be obtained for supplementary studies and research, and are associated with minimal morbidity.

Immunohistochemical findings in embryonal small round cell tumors with molecular diagnostic confirmation.

The diagnosis of pediatric tumors relies heavily on immunohistochemical staining of small tissue biopsies, since many entities share a "small blue cell" phenotype. More recently, molecular genetic analysis for detection of specific gene fusion products has become available. With the increased use of such molecular techniques, the authors have noted that tumors with proven molecular diagnoses can exhibit unusual patterns of immunohistochemical staining. This study examines pediatric tumors with a "small blue cell" phenotype in which molecular diagnoses were available where applicable. A panel of immunohistochemical stains was performed (S100, CD56, NB84, CD99 [MIC2], Bcl-2, CD117, CD34, desmin, MNF116, and WT1). In the 370 sections from 37 cases, all primitive neuroectodermal tumors, with and without the presence of t(11;22), demonstrated uniform membranous membrane staining with CD99 (MIC2) and focal staining with CD56, NB84, MNF116, and WT1. All rhabdomyosarcomas, both alveolar and embryonal, demonstrated uniform desmin, CD56, and cytoplasmic WT1 immunostaining. Desmoplastic small round cell tumors showed positive cytokeratin staining, with half having "dot-like" cytoplasmic desmin and WT1 positivity; some showed focal positivity for NB84, CD99, and Bcl-2. The "undifferentiated" sarcomas showed the widest range of staining, with no marker staining all cases. Neuroblastomas exhibited uniform strong staining for CD56 and NB84 and marked cytoplasmic Bcl-2 positivity, and some cases showed cytoplasmic WT1 expression. Blastematous Wilms' tumors showed uniform strong membranous staining for CD56, uniform cytoplasmic staining for Bcl-2, and nuclear expression of WT1. Embryonal pediatric malignancies can demonstrate apparently nonspecific expression patterns for several antigens, which may reflect developmental immaturity rather than specific differentiation pathways.

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.

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

Nuclear immunoreactivity for beta-catenin is a useful adjunct for diagnosis of adult desmoid-type fibromatoses, many of which exhibit mutations within the APC/beta-catenin (Wnt) pathway. Pediatric fibromatoses represent a heterogeneous group of lesions that are diagnostically challenging, especially on biopsy. We studied beta-catenin expression in a variety of pediatric fibroblastic and myofibroblastic lesions. Immunohistochemical nuclear expression of beta-catenin was assessed in 100 tumors. High-level expression of beta-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 beta-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. beta-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.

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.

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 biopsy. 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.

Assessing the total costs of blood delivery to hospital oncology and haematology patients.

OBJECTIVE: To determine direct costs associated with a blood transfusion session in two hospital settings. RESEARCH DESIGN AND METHODS: The study was conducted in two United Kingdom hospital sites during April 2004. Transfusion sessions for patients receiving units of red blood cells within either haematology or oncology departments were followed using time and motion techniques to measure the direct costs. Other data were collected from the centres to calculate the cost of disposables, blood wastage and blood bank machines. RESULTS: Total mean staff cost per transfusion of 2 units was 37.24 pounds sterling (9.68 pounds sterling for blood bank and 27.56 pounds sterling for ward procedures). The mean cost of disposables was 13.25 pounds sterling and the mean cost for blood products was 287.56 pounds sterling. The mean cost of wastage was 11.86 pounds sterling per transfusion. After including other derived costs, such as hospital stay, the mean cost for a transfusion of 2 units of blood was estimated to be 546.12 pounds sterling. CONCLUSION: This study estimates the cost of an average blood transfusion of 2 units to be 546.12 pounds sterling. It should be noted that significant indirect costs, such as those incurred by patients, their carers and societal costs, have not been considered. Against the background of finite blood resources and other factors such as patient quality of life, blood transfusion may not represent the best choice for patient care. Alternative treatments should be considered.