Assessing vascular effects of adding bevacizumab to neoadjuvant chemotherapy in osteosarcoma using DCE-MRI.

BACKGROUND: The purpose of this study was to assess the impact of bevacizumab alone and in combination with cytotoxic therapy on tumour vasculature in osteosarcoma (OS) using DCE-MRI. METHODS: Six DCE-MRI and three (18)F-FDG PET examinations were scheduled in 42 subjects with newly diagnosed OS to monitor the response to antiangiogenic therapy alone and in combination with cytotoxic therapy before definitive surgery (week 10). Serial DCE-MRI parameters (K(trans), v(p), and v(e)) were examined for correlation with FDG-PET (SUV(max)) and association with drug exposure, and evaluated with clinical outcome. RESULTS: K(trans) (P=0.041) and v(p) (P=0.001) significantly dropped from baseline at 24 h after the first dose of bevacizumab alone, but returned to baseline by 72 h. Greater exposure to bevacizumab was correlated with larger decreases in v(p) at day 5 (P=0.04) and week 10 (P=0.02). A lower K(trans) at week 10 was associated with greater percent necrosis (P=0.024) and longer event-free survival (P=0.034). CONCLUSIONS: This is the first study to demonstrate significant changes of the plasma volume fraction and vascular leakage in OS with bevacizumab alone. The combination of demonstrated associations between drug exposure and imaging metrics, and imaging metrics and patient survival during neoadjuvant therapy, provides a compelling rationale for larger studies using DCE-MRI to assess vascular effects of therapy in OS.

Prognostic factors for local and distant control in Ewing sarcoma family of tumors.

BACKGROUND: Advances in the treatment of Ewing sarcoma family of tumors (ESFT) are the result of improvements in systemic and local therapies. The individual contributions of each treatment component cannot be analyzed separately; improvements in local and systemic control can influence each other. PATIENTS AND METHODS: We reviewed the records of 220 patients treated on institutional protocols from 1979 to 2004. Factors predictive of local and distant recurrence were analyzed. RESULTS: The median age at diagnosis was 13.7 years. Ninety-five patients relapsed at a median of 1.6 years. The 5-year overall survival estimate was 63.5% +/- 3.5%. The estimated 5-year cumulative incidence (CI) of local failure was 25.1% +/- 3.0%. Local failure was associated with treatment era (P < 0.001), tumor size (P = 0.037) and type of local control (P = 0.021). Systemic treatment intensification improved local control. The estimated 5-year CI of distant recurrence was 22.5% +/- 2.9%. Patients with localized disease (P < 0.001), smaller tumors (P = 0.018) and those who received surgery +/- radiation for local control (P = 0.023) had lower CI of distant failure. CONCLUSIONS: Successful treatment of ESFT requires optimal systemic and local therapy. Both treatment modalities are intertwined and the control of both local and distant disease is the result of the combined approach.

STAT3 is activated in a subset of the Ewing sarcoma family of tumours.

STAT3 is an oncogene that regulates critical cellular processes and whose constitutive activation has been demonstrated to correlate with biological and clinical features in many types of human malignancy. In this study, STAT3 activation was assessed in the Ewing sarcoma family of tumours (ESFT), which is characterized by fusion of the EWS gene with one of several Ets transcription factors, most commonly EWS-FLI1. STAT3 activation was assessed by immunohistochemistry using a monoclonal antibody specific for tyrosine(705)-phosphorylated STAT3 (pSTAT3(tyr705)) and a tissue microarray containing 49 paraffin-embedded ESFT tumours with known EWS translocations. Twenty-five (51%) tumours were pSTAT3(tyr705)-positive, as defined by more than 10% tumour cell immunostaining. STAT3 activation correlated with tumour site at presentation, with pSTAT3(tyr705)-negative ESFT involving axial sites predominantly (p = 0.008). Notably, among 31 patients who presented with localized disease, high-level STAT3 activation correlated with better overall survival (p = 0.02). STAT3 activation was not directly related to EWS-FLI1 expression, since EWS-FLI1 transfection did not result in STAT3 activation. Furthermore, detailed molecular analysis indicated that STAT3 activation may be seen with EWS-FLI1 or EWS-ERG and appears to be independent of EWS-FLI1 fusion type. In conclusion, STAT3 activation is present in approximately half of ESFT and correlates with clinical features. The role of STAT3 activation in ESFT pathogenesis seems to be independent of the type of EWS/Ets translocation.

Immunomagnetic purging of Ewing's sarcoma from blood and bone marrow: quantitation by real-time polymerase chain reaction.

PURPOSE: A propensity for hematogenous spread with resulting contamination of autologous cell products complicates cellular therapies for Ewing's sarcoma. We used a new approach to purge artificially contaminated cellular specimens of Ewing's sarcoma and show the capacity for real-time polymerase chain reaction (PCR) to quantify the contamination level of Ewing's sarcoma in such specimens. PATIENTS AND METHODS: Binding of monoclonal antibody (MoAb) 8H9 to Ewing's sarcoma cell lines and normal hematopoietic cells was studied using flow cytometry. Using real-time PCR--based amplification of t(11;22), levels of Ewing's contamination of experimental and clinical cellular products were monitored. Purging was accomplished using immunomagnetic-based depletion. Monitoring of the function of residual hematopoietic progenitors and T cells was performed using functional assays. RESULTS: MoAb 8H9 shows binding to Ewing's sarcoma but spares normal hematopoietic tissues. Nested real-time PCR is capable of detecting contaminating Ewing's sarcoma cells with a sensitivity of one cell in 10(6) normal cells. After 8H9-based purging, a 2- to 3-log reduction in contaminating Ewing's sarcoma was shown by real-time PCR, with purging to PCR negativity at levels of contamination of 1:10(6). Levels of contamination in clinical samples ranged from 1:10(5) to 10(6). Therefore, 8H9-based purging of clinical samples is predicted to reduce tumor cell contamination to a level below the limit of detection of PCR. CONCLUSION: These results demonstrate a new approach for purging contaminated cellular products of Ewing's sarcoma and demonstrate the capacity of real-time PCR to provide accurate quantitative estimates of circulating tumor burden in this disease.

Autocrine Transforming Growth Factor-beta Growth Pathway in Murine Osteosarcoma Cell Lines Associated with Inability to Affect Phosphorylation of Retinoblastoma Protein.

Purpose. Production of active transforming growth factor-beta (TGF-beta ) by human osteosarcoma may contribute to malignant progression through mechanisms that include induction of angiogenesis, immune suppression and autocrine growth stimulation of tumor cell growth.To study events associated with induction of cell proliferation by TGF-beta , we have evaluated the TGF-beta pathway in two murine osteosarcoma cell lines, K7 and K12.Results. Northern and immunohistochemical analyses show that each cell line expressesTGF-beta1 and TGF-beta3 mRNA and protein. Both cell lines secrete activeTGF-beta 1 and display a 30-50% reduction in growth when cultured in the presence of a TGF-beta blocking antibody. Expression of TGF-beta receptors TbetaRI, TbetaRII and TbetaRIII is demonstrated by affinity labeling with (125) -TGF-beta 1, and the intermediates, Smads 2, 3 and 4, are uniformly expressed. Smads 2 and 3 are phosphorylated in response toTGF-beta , while pRb phosphorylation in each osteosarcoma cell line is not affected by either exogenousTGF-beta or TGF-beta antibody.Conclusions. The data implicate events downstream of Smad activation, including impaired regulation of pRb, in the lack of a growth inhibitory response toTGF-beta , and indicate that this murine model of osteosarcoma is valid for investigating the roles of autocrineTGF-beta in vivo.

Acute lymphoblastic leukemia with the (8;14)(q24;q32) translocation and FAB L3 morphology associated with a B-precursor immunophenotype: the Pediatric Oncology Group experience.

Five pediatric patients are described with acute lymphoblastic leukemia (ALL) who at presentation had clinical findings suggestive of B cell ALL and lymphoblasts with FAB L3 morphology and the characteristic t(8;14)(q24;q32). However, the leukemia cells of all five patients failed to express surface immunoglobulin (sIg) and kappa or lambda light chains. Based on initial immunophenotyping results consistent with B-precursor ALL, four of these cases were initially treated with conventional ALL chemotherapy. These four patients were switched to B cell ALL treatment protocols once cytogenetic results became available revealing the 8;14 translocation. The fifth case was treated with B cell ALL therapy from the outset. Four of the five patients are in complete remission at 64, 36, 29 and 13 months from diagnosis. One patient relapsed and died 6 months after initial presentation. These five unusual cases with clinical B cell ALL, the t(8;14), and FAB L3 morphology, but negative sIg, demonstrate the importance of careful and multidisciplinary evaluation of leukemic cells with morphology, cytochemistry, immunophenotyping and cytogenetic analysis. Future identification of patients with this profile will allow us to expand our knowledge regarding prognostic significance and optimal treatment for this rare subgroup of patients.

AP-2 may contribute to IGF-II overexpression in rhabdomyosarcoma.

The human insulin-like growth factor II gene is regulated in a development-dependent manner and is not expressed in most adult tissues. However, high levels of insulin-like growth factor II mRNA are detected in many human tumors including rhabdomyosarcoma, an embryonal tumor of skeletal muscle origin. In this study, we demonstrate that the developmentally regulated transcription factor AP-2 is expressed at higher levels in human fetal skeletal muscle and rhabdomyosarcoma cells compared to human adult skeletal muscle. Endogenous insulin-like growth factor II mRNA derived from the P3 as well as transfected P3 promoter activity were modestly and consistently increased to the same extent following treatment of the rhabdomyosarcoma cell line RD with forskolin, a compound implicated in AP-2 transactivation. This effect of AP-2 on increased transcriptional activity was confirmed by nuclear run-on assays. Expression of AP-2B, a dominant-negative inhibitor of AP-2, suppressed the P3 promoter activity in AP-2 expressing RD cells. Furthermore, five AP-2 protected regions corresponding to six AP-2 specific binding sites were detected in the insulin-like growth factor II P3 promoter. These data together suggest that AP-2 may contribute to the high expression of IGF-II in rhabdomyosarcoma cells.

Albumin, IgG, retinol-binding protein, and alpha1-microglobulin excretion in childhood.

Urine concentrations of two high molecular weight proteins, albumin and IgG, and two low molecular weight proteins, alpha1-microglobulin (A1M) and retinol-binding protein (RBP), were measured in 657 healthy children from birth to 18 years of age. The urinary levels of RBP and A1M suggest an age-dependent decline, whereas the levels of albumin and IgG show an uneven distribution. Reference values for albumin, IgG, A1M, and RBP for each group are reported.