Pneumatic dilation versus laparoscopic Heller myotomy for the treatment of achalasia: variables related to a good response.

Achalasia is a motor disorder characterized by esophageal aperistalsis and failure of lower esophageal sphincter relaxation. The cardinal symptoms are dysphagia, food regurgitation and weight loss. The most effective treatments are pneumatic dilation (PD) of the cardia and Heller esophageal myotomy with partial fundoplication. There is still controversy regarding which treatments should be initially done. The aims of this study were to evaluate clinical response and the variables related to good results in both treatments. Ninety-two patients with achalasia diagnosed by esophageal manometry were randomized to receive either PD or laparoscopic Heller myotomy with partial fundoplication. After the procedure, patients were followed up clinically and submitted to esophageal manometry and pH monitoring. Three months after treatment, 73% of the patients from PD group and 84% of the surgery group had good results (P = 0.19). After 2 years of follow-up, 54% of the PD group and 60% of the surgery group (P = not significant) were symptom free. Variables related to a good response to PD were a 50% drop in lower esophageal sphincter pressure (LESP) or a LESP <10 mmHg after treatment. Patients over 40 years old with LESP ≤32 mmHg before treatment and a drop in LESP >50% after treatment significantly achieved better responses after surgical treatment when compared with PD. The reflux rate was significantly higher in the PD group (27.7%) compared with the surgery group (4.7%), P = 0.003. We concluded that surgical treatment and PD for achalasia are equally effective even after 2 years of follow-up. The choice of treatment for achalasia should be based on the following parameters: treatment availability, rate of good results, complication rates, variables related to good responses and also the patient's wish.

Downregulation of IGFBP2 is associated with resistance to IGF1R therapy in rhabdomyosarcoma.

Agents targeting the insulin-like growth factor-1 receptor (IGF1R) are in clinical development, but, despite some initial success of single agents in sarcoma, response rates are low with brief durations. Thus, it is important to identify markers predictive of response, to understand mechanisms of resistance, and to explore combination therapies. In this study, we found that, although associated with PAX3-FKHR translocation, increased IGF1R level is an independent prognostic marker for worse overall survival, particularly in patients with PAX3-FKHR-positive rhabdomyosarcoma (RMS). IGF1R antibody-resistant RMS cells were generated using an in vivo model. Expression analysis indicated that IGFBP2 is both the most affected gene in the insulin-like growth factor (IGF) signaling pathway and the most significantly downregulated gene in the resistant lines, indicating that there is a strong selection to repress IGFBP2 expression in tumor cells resistant to IGF1R antibody. IGFBP2 is inhibitory to IGF1R phosphorylation and its signaling. Similar to antibodies to IGF1/2 or IGF2, the addition of exogenous IGFBP2 potentiates the activity of IGF1R antibody against the RMS cells, and it reverses the resistance to IGF1R antibody. In contrast to IGF1R, lower expression of IGFBP2 is associated with poorer overall survival, consistent with its inhibitory activity found in this study. Finally, blocking downstream Protein kinase B (AKT) activation with Phosphatidylinositide 3-kinases (PI3K)- or mammalian target of rapamycin (mTOR)-specific inhibitors significantly sensitized the resistant cells to the IGF1R antibody. These findings show that constitutive IGFBP2 downregulation may represent a novel mechanism for acquired resistance to IGF1R therapeutic antibody in vivo and suggest various drug combinations to enhance antibody activity and to overcome resistance.

Loss-of-function screen in rhabdomyosarcoma identifies CRKL-YES as a critical signal for tumor growth.

To identify novel signaling pathways necessary for rhabdomyosarcoma (RMS) survival, we performed a loss-of-function screen using an inducible small hairpin RNA (shRNA) library in an alveolar and an embryonal RMS cell line. This screen identified CRKL expression as necessary for growth of alveolar RMS and embryonal RMS both in vitro and in vivo. We also found that CRKL was uniformly highly expressed in both RMS cell lines and tumor tissue. As CRKL is a member of the CRK adapter protein family that contains an SH2 and two SH3 domains and is involved in signal transduction from multiple tyrosine kinase receptors, we evaluated CRKL interaction with multiple tyrosine kinase receptor signaling pathways in RMS cells. While we saw no interaction of CRKL with IGFIR, MET or PI3KAKT/mTOR pathways, we determined that CRKL signaling was associated with SRC family kinase (SFK) signaling, specifically with YES kinase. Inhibition of SFK signaling with dasatinib or another SFK inhibitor, sarcatinib, suppressed RMS cell growth in vitro and in vivo. These data identify CRKL as a novel critical component of RMS growth. This study also demonstrates the use of functional screening to identify a potentially novel therapeutic target and treatment approach for these highly aggressive pediatric cancers.

Global methylation analysis identifies PITX2 as an upstream regulator of the androgen receptor and IGF-I receptor genes in prostate cancer.

The insulin-like growth factor-I (IGF-IR) and androgen (AR) receptors are important players in prostate cancer. Functional interactions between the IGF-I and androgen signaling pathways have crucial roles in the progression of prostate cancer from early to advanced stages. DNA methylation is a major epigenetic alteration affecting gene expression. Hypermethylation of tumor suppressor promoters is a frequent event in human cancer, leading to inactivation and repression of specific genes. The aim of the present study was to identify the entire set of methylated genes ("methylome") in a cellular model that replicates prostate cancer progression. The methylation profiles of the P69 (early stage, benign) and M12 (advanced stage, metastatic) prostate cancer cell lines were established by treating cells with the demethylating agent 5-aza-2'-deoxycytidine (5-Aza) followed by DNA microarray analysis. Comparative genome-wide methylation analyses of 5-Aza-treated versus untreated cells identified 297 genes overexpressed in P69 and 191 genes overexpressed in M12 cells. 102 genes were upregulated in both benign and metastatic cell lines. In addition, our analyses identified the PITX2 gene as a master regulator upstream of the AR and IGF-IR genes. The PITX2 promoter was semi-methylated in P69 cells but fully methylated (i. e., silenced) in M12 cells. Epigenetic regulation of PITX2 during the course of the disease may lead to orchestrated control of the AR and IGF signaling pathways. In summary, our results provide new insights into the epigenetic changes associated with progression of prostate cancer from an organ confined, androgen-sensitive disorder to an aggressive, androgen-insensitive disease.

Insulin-like growth factor 1 receptor antibody induces rhabdomyosarcoma cell death via a process involving AKT and Bcl-x(L).

Insulin-like growth factors (IGFs) and their receptor, IGF-1 receptor (IGF1R), have important roles in growth, development, stress response, aging and cancer. There are many agents that inhibit IGF1R in oncology clinical development, and in some cases, they have been associated with rapid tumor regression. However, it is not clear by which process these targeted agents induce cancer cell death and how to predict such tumor responses. Here, we showed that IGF1R antibody led to rapid cell death and tumor regression in some rhabdomyosarcoma (RMS) cells. Mechanistic analysis revealed a rapid onset of mitochondrial-dependent apoptosis, including mitochondrial depolarization, cytochrome C release and the activation of specific caspases. The antibody sensitive cells had greater dependence on AKT for maintaining downstream signaling and the expression of a constitutively active AKT, which restored AKT-signaling in these cells, inhibited anti-IGF1R induced cell death. Further analysis showed IGF1R antibody-induced hypophosphorylation of BAD and activation of downstream BAX. Interestingly, the examination of RMS cell lines and tumors revealed an inverse correlation between elevated IGF1R and Bcl-2 level (P=0.033), with the sensitive cells lacking Bcl-2 expression. The overexpression of BAD specific target, Bcl-x(L), conferred resistance, whereas Bcl-x(L) knockdown sensitized cells lacking Bcl-2 to anti-IGF1R-induced cell death. We propose that RMS pathogenesis involves increased IGF1R expression that enhances AKT and Bcl-x(L)-mediated cell survival, and the blockage of IGF1R results in inhibition of survival signal from Bcl-x(L) and cell death in the sensitive Bcl-2 negative cells.

Beta4 integrin promotes osteosarcoma metastasis and interacts with ezrin.

The development of pulmonary metastasis is the major cause of death in osteosarcoma, and its molecular basis is poorly understood. In this study, we show that beta4 integrin is highly expressed in human osteosarcoma cell lines and tumor samples. Furthermore, highly metastatic MNNG-HOS cells have increased levels of beta4 integrin. Suppression of beta4 integrin expression by shRNA and disruption of beta4 integrin function by transfection of dominant-negative beta4 integrin was sufficient to revert this highly metastatic phenotype in the MNNG-HOS model without significantly affecting primary tumor growth. These findings suggest a role for beta4 integrin expression in the metastatic phenotype in human osteosarcoma cells. In addition, we identified a previously uncharacterized interaction between beta4 integrin and ezrin, a membrane-cytoskeletal linker protein that is implicated in the metastatic behavior of osteosarcoma. The beta4 integrin-ezrin interaction appears to be critical for maintenance of beta4 integrin expression. These data begin to integrate ezrin and beta4 integrin expression into a model of action for the mechanism of osteosarcoma metastases.

Rapamycin induces feedback activation of Akt signaling through an IGF-1R-dependent mechanism.

Rapamycin and several analogs, such as CCI-779 and RAD001, are currently undergoing clinical evaluation as anticancer agents. In this study, we show that inhibition of mammalian target of rapamycin (mTOR) signaling by rapamycin leads to an increase of Akt phosphorylation in Rh30 and RD human rhabdomyosarcoma cell lines and xenografts, and insulin-like growth factor (IGF)-II-treated C2C12 mouse myoblasts and IGF-II-overexpressing Chinese hamster ovary cells. RNA interference-mediated knockdown of S6K1 also results in an increase of Akt phosphorylation. These data suggest that mTOR/S6K1 inhibition either by rapamycin or small interfering RNA (siRNA) triggers a negative feedback loop, resulting in the activation of Akt signaling. We next sought to investigate the mechanism of this negative feedback regulation from mTOR to Akt. Suppression of insulin receptor substrate (IRS)-1 and tuberous sclerosis complex-1 by siRNAs failed to abrogate rapamycin-induced upregulation of Akt phosphorylation in both Rh30 and RD cells. However, pretreatment with h7C10 antibody directed against insulin-like growth factor-1 receptor (IGF-1R) led to a blockade of rapamycin-induced Akt activation. Combined mTOR and IGF-1R inhibition with rapamycin and h7C10 antibody, respectively, resulted in additive inhibition of cell growth and survival. These data suggest that rapamycin mediates Akt activation through an IGF-1R-dependent mechanism. Thus, combining an mTOR inhibitor and an IGF-1R antibody/inhibitor may be an appropriate strategy to enhance mTOR-targeted anticancer therapy.

Insulin-like growth factor type 1 (IGF-1) and IGF binding protein-3 in patients with Ewing sarcoma family of tumors.

BACKGROUND: Ewing sarcoma family of tumors (ESFTs) are the second most common bone tumor, that most often affects persons ages 3-40 years. The ESFTs rely on signaling through the insulin-like growth factor-1 receptor (IGF-1R) for growth and transformation. The current studies were performed to determine the levels of IGF-1 and IGF binding protein-3 (IGFBP-3) in patients with ESFT. The authors then performed an exploratory analysis to evaluate whether IGF parameters could differentiate event free or overall survival in ESFT patients. METHODS: The authors measured serum levels of IGF-1 and IGFBP-3 by using a radioimmunoassay from 111 patients with ESFT with a median follow-up of 13 years from diagnosis. RESULTS: The IGF-1 levels were lower among patients with metastatic disease to the bones or the bone marrow compared with patients without metastasis to these sites (p2 = 0.021 and 0.0038, respectively). IGFBP-3 is known to sequester IGF-1; the ratios of IGFBP-3 to IGF-1 were evaluated. Patients with metastatic disease to any site had higher IGFBP-3 to IGF-1 ratios than patients with localized disease (p2 = 0.0067). There was a trend toward increased survival in patients with localized disease who had high IGFBP-3 to IGF-1 levels. Metastatic patients showed a similar trend. CONCLUSIONS: Levels of IGF-1 and IGFBP-3 in ESFT patients can identify patients with the most widespread disease. The IGFBP-3 to IGF-1 ratio in patients with either localized or metastatic disease identified patients with a trend toward increased survival. Further prospective evaluation with higher patient numbers might show a prognostic role for the IGFBP-3 to IGF-1 ratio in patients with ESFT.

Intramedullary Ewing sarcoma of the spinal cord: consequences of molecular diagnostics. Case report.

Molecular biological techniques have begun to transform modern medicine. These techniques have shown promise in the pathological diagnosis of difficult or uncommon tumors. Accurate molecular diagnosis of the small round-cell tumors, for example, is especially important because divergent therapies may be required to eradicate such disparate lesions as neuroblastoma, lymphoma, rhabdomyosarcoma, central primitive neuroectodermal tumors/medulloblastoma, or Ewing sarcoma (ES). The authors present an unusual case of a primary, extraosseous ES arising from the intramedullary spinal cord, in which molecular studies were required for specific diagnosis and therapeutic guidance.

Insulin and IGF-1 induce different patterns of gene expression in mouse fibroblast NIH-3T3 cells: identification by cDNA microarray analysis.

The IGF-1 receptor and the related insulin receptor are similar in structure and activate many of the same postreceptor signaling pathways, yet they mediate distinct biological functions. It is still not understood how the specificity of insulin vs. IGF-1 signaling is controlled. In this study, we have used cDNA microarrays to monitor the gene expression patterns that are regulated by insulin and IGF-1. Mouse fibroblast NIH-3T3 cells expressing either the wild-type human IGF receptor or the insulin receptor were stimulated with either IGF-1 or insulin, respectively. Thirty genes, 27 of which were not previously known to be IGF-1 responsive, were up-regulated by IGF-1 but not by insulin. Nine genes, none of which was previously known to be insulin responsive, were up-regulated by insulin but not by IGF-1. The IGF- and insulin-induced regulation of 10 of these genes was confirmed by Northern blot analysis. Interestingly, more than half of the genes up-regulated by IGF-1 are associated with mitogenesis and differentiation, whereas none of the genes specifically up-regulated by insulin are associated with these processes. Our results indicate that under the conditions used in this study, IGF-1 is a more potent activator of the mitogenic pathway than insulin in mouse fibroblast NIH-3T3 cells.

A phase I/II dose escalation and activity study of intravenous injections of OCaP1 for subjects with refractory osteosarcoma metastatic to lung.

Osteosarcomas are malignant tumors arising from skeletal tissue and occur most frequently during childhood and adolescence. Osteosarcoma was once fatal in more than 80% of patients who presented with apparently localized disease. Chemotherapy, better surgical techniques, and improved staging methods now allow most patients to be treated with limb-sparing surgery and to be cured of their disease. However, many patients still die of metastatic disease and new approaches are still needed. The lung is the most frequent metastatic site and is treated with chemotherapy and surgical resections. Multiple resections for repeated recurrences that are limited to the lung are not uncommon but are limited by the amount of lung tissue that can be removed and become futile as recurrences become more frequent. Although a main component of initial therapy, chemotherapy has not been shown to be of benefit for recurrent disease. Direct introduction of therapeutic genes into malignant cells in vivo may provide effective treatment of solid tumors. The proposed study will use the adenoviral vector Ad-OC-E1a (OCaP1), which contains a murine osteocalcin (OC) promoter to regulate the production of the adenoviral E1a protein to allow for restricted viral replication and subsequent lysis of tumor cells. The OC promoter is developmentally regulated, with peak expression in the neonate. It functions primarily in osteoblasts found in growing bone and is highly expressed in osteogenic sarcomas. Because adenovirus is quickly cleared by normal tissues, especially the liver, systemic administration has been problematic. Although bioavailability would be decreased following exposure to the liver, the OcaP1 construct should not be hepatotoxic due to OC-restricted tissue expression of the Ela protein. Metastatic disease to the lung is a major problem and often is the cause of death for patients with osteogenic sarcoma. Treatment of pulmonary metastases could potentially be accomplished using intravenously administered OcaP1 since the material would pass through the lung prior to reaching the systemic circulation. In animal models using OC expressing tumors, OCaP1 has been effective at reducing lung metastases following intravenous injection. This protocol is a phase I/II investigational study of bolus intravenous injections of Ad-OC-E1a for the treatment of chemotherapy-refractory osteogenic sarcoma that has metastasized to the lungs. Initially patients will receive one injection of 1 x 10(10), 1 X 10(11), 1 X 10(12), or 5 x 10(12) viral particles of Ad-OC-E1a using a standard Phase I dose escalation design that studies 3 to 6 patients per dose group. After safety has been established in the first part of the trial, we will evaluate the anti-tumor activity of OCaP1. Because the matrix associated with osteogenic sarcoma may not change despite tumor necrosis, radiographic evaluation alone has not been considered sufficient to evaluate response in this disease. Histologic criteria that assess the amount of necrosis have been shown to have prognostic significance and are a key component of the anti-tumor response assessment. Therefore, the anti-tumor assessments will be carried out in patients for whom resection of their pulmonary metastases is clinically indicated. These patients will receive one injection of OCaP1 and 28 to 42 days later undergo their planned pulmonary resection. Responses will be graded using radiographic and histologic objective response criteria that are considered standard for osteogenic sarcoma. A total of 14 to 25 patients, depending upon whether objective anti-tumor responses occur, will be studied in this part of the protocol.

Antigenicity of fusion proteins from sarcoma-associated chromosomal translocations.

Synovial sarcoma (SS), clear cell sarcoma (CCS), and desmoplastic small round cell tumor (DSRCT) are soft-tissue malignancies occurring primarily in adolescents and young adults. These tumors contain specific chromosomal translocations that fuse the 5' region of one gene with the 3' region of another, resulting in the formation of characteristic fusion proteins. These translocations are unique to tumor cells and may be required for persistence, thereby serving as targets for immunotherapy. It was hypothesized that the fusion breakpoint sequences associated with SS, CCS, and DSRCT can serve as tumor-specific neoantigens. To test this, peptides corresponding to the fusion breakpoints were designed and assessed for ability to bind to various class I HLA molecules. Two peptides derived from the SS breakpoint specifically bind the HLA-B7 antigen, and a 10-amino acid minimal epitope was identified for this interaction. Specific binding of a SS peptide and a CCS peptide to HLA-B27 molecule was also observed. Finally, a peptide designed from the DSRCT breakpoint specifically binds the HLA-A3 molecule, and a 9-amino acid optimal epitope was identified for this interaction. The physiological/immunological relevance of these peptide/MHC interactions was demonstrated by the induction of SS-specific CTLs from normal donor lymphocytes using in vitro stimulation with autologous, peptide-pulsed dendritic cells and by the ability of these CTLs to lyse human SS tumor cells endogenously expressing the full-length fusion protein. These results suggest that sequences in the fusion region of sarcoma-associated chimeras can bind class I HLA molecules and serve as neoantigens. These may be useful for the development of novel immunotherapies for sarcoma patients with appropriate HLA molecules and tumors bearing these translocations.

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.

Insulin-like growth factor 1 (IGF-1)-induced twist expression is involved in the anti-apoptotic effects of the IGF-1 receptor.

In this study we investigated the molecular mechanisms whereby insulin-like growth factor 1 (IGF-1) induced Twist gene expression and the role of Twist in the anti-apoptotic actions of the IGF-1 receptor. In NIH-3T3 fibroblasts overexpressing the human IGF-1 receptor (NWTb3), treatment with IGF-1 (10(-8) m) for 1 and 4 h increased the level of Twist mRNA as well as protein by 3-fold. In contrast, insulin at physiological concentrations did not stimulate Twist expression in NIH-3T3 fibroblasts overexpressing the human insulin receptor. The IGF-1 effect was specific for the IGF-1 receptor since, in cells overexpressing a dominant negative IGF-1 receptor, IGF-1 failed to increase Twist expression. Pre-incubation with the ERK1/2 inhibitor U0126 or expression of a dominant negative MEK-1 abolished the effect of IGF-1 on Twist mRNA expression in NWTb3 cells, suggesting that Twist induction by IGF-1 occurs via the mitogen-activated protein kinase signaling pathway. In vivo, IGF-1 injection increased the mRNA level of Twist in mouse skeletal muscle, the major site of Twist expression. Finally, using an antisense strategy, we demonstrated that a reduction of 40% in Twist expression decreased significantly the ability of IGF-1 to rescue NWTb3 cells from etoposide-induced apoptosis. Taken together, these results define Twist as an important factor involved in the anti-apoptotic actions of the IGF-1 receptor.

Metastasis-associated differences in gene expression in a murine model of osteosarcoma.

Despite advances in the management of osteosarcoma (OSA) and other solid tumors, the development of metastasis continues to be the most significant problem and cause of death for cancer patients. To define genetic determinants of pulmonary metastasis, we have applied cDNA microarrays to a recently described murine model of OSA that is characterized by orthotopic tumor growth, a period of minimal residual disease, spontaneous pulmonary metastasis, and cell line variants that differ in metastatic potential. Microarray analysis defined 53 genes (of 3166 unique cDNAs) that were differentially expressed between the primary tumors of the more aggressive (K7M2) and less aggressive (K12) OSA models. By review of the literature, these differentially expressed genes were assigned to six nonmutually exclusive metastasis-associated categories (proliferation and apoptosis, motility and cytoskeleton, invasion, immune surveillance, adherence, and angiogenesis). Functional studies to evaluate K7M2 and K12 for differences in each of these metastasis-associated processes revealed enhanced motility, adherence, and angiogenesis in the more aggressive K7M2 model. For this reason, 10 of the 53 differentially expressed genes that were assigned to the motility and cytoskeleton, adherence, and angiogenesis categories were considered as most likely to define differences in the metastatic behavior of the two models. Ezrin, a gene not described previously in OSA, with functions in motility, invasion, and adherence, was 3-fold overexpressed in K7M2 compared with K12 by microarray. Differential expression for RNA was confirmed by Northern analysis and for protein by immunostaining. Alterations in ezrin protein levels and concomitant cytoskeletal changes in our model confirmed predictions from the arrays. The potential relevance of ezrin in OSA was suggested by its expression in five of five human OSA cell lines. This work represents a rationale approach to the evaluation of microarray data and will be useful to identify genes that may be causally associated with metastasis.

Resistance training and gait function in patients with Parkinson's disease.

OBJECTIVE: To determine whether patients with Parkinson's disease who are enrolled in a resistance training program can gain strength similar to that of normal control subjects and whether these gains in strength would improve their gait function. DESIGN: Subjects included 14 patients with mild-to-moderate Parkinson's disease of either gender and six normal control subjects of similar age. The training consisted of an 8-wk course of resistance training twice per week, focused primarily on the lower limbs. The primary outcome measures consisted of exercise performance monitoring and quantitative gait analysis before and after the training course. RESULTS: Both the patients with Parkinson's disease and normal control subjects significantly increased their performance with resistance training. Subjects with Parkinson's disease had gains in strength similar to those of normal elderly adults. Patients with Parkinson's disease also had significant gains in stride length, walking velocity, and postural angles compared with pretreatment values. CONCLUSIONS: Patients with mild-to-moderate Parkinson's disease can obtain increases in performance or strength similar to that of normal adults of the same age in a resistance training program. Resistance training can produce functional improvements in gait and may, therefore, be useful as part of a physical rehabilitation and/or health maintenance program for patients with Parkinson's disease.

Molecular confirmation of Ewing sarcoma.

OBJECTIVE: To analyze retrospectively results of reverse transcription polymerase chain reaction (RT-PCR) testing and demographic information in patients with known or suspected Ewing sarcoma/primitive neuroectodermal tumor family of tumors referred to the National Cancer Institute and to describe factors influencing the determination of molecular marker status. PATIENTS AND METHODS: Tumor samples from 76 patients from February 1997 to December 1999 were analyzed. In all cases, the diagnosis of this family of tumors was confirmed by histopathologic review. RESULTS: In 58 patients, the presence of a translocation associated with this family of tumors was confirmed using RT-PCR. Specifically, there were 45 Ewing sarcoma (EWS)-FLI type 1 translocations, four EWS-FLI type 2 translocations, five EWS-ERG translocations, and four less common EWS-FLI variants. Of patients with a confirmed translocation, four were confirmed only after nested RT-PCR techniques were used. In five patients who initially underwent needle biopsy, the diagnosis was confirmed only after open biopsy or repeat needle biopsy was undertaken. Samples from 18 patients were translocation-negative. Of these, seven samples were deemed inadequate for RT-PCR testing as a result of inappropriate tissue handling or the presence of necrotic material. Five patients were found to have a different diagnosis after complete histopathologic and molecular characterization. Six samples remained, in which adequate tissue was obtained with no evidence of a characteristic translocation. CONCLUSIONS: In apparently translocation-negative samples, close attention should be given to the possibility of an alternative diagnosis, the potential need for nested RT-PCR, and the possibility of an inadequate sample. Strong consideration should be given to the use of open biopsy as opposed to needle biopsy to avoid the need for repeat biopsies and the potential for inaccurate assessment of molecular marker status.

Targeting pediatric malignancies for T cell-mediated immune responses.

Successful immune targeting of malignancies hinges upon the ability to activate specific T-cell populations to recognize and attack tumor but spare normal vital tissues. Investigators in the field of tumor immunology are currently utilizing at least three distinct approaches toward this goal. In the first approach, molecular targets of cytolytic T cells which spontaneously develop in tumor-bearing patients have been identified and are subsequently used as immunogens in immunotherapy trials. Whereas this approach originally focused upon the identification of tumor antigens in the immune-responsive tumors malignant melanoma and renal cell carcinoma, it surprisingly led to the identification of a variety of molecules that are now known to be expressed in other common pediatric and adult tumors. In the second approach, tumor-specific molecules (eg, mutant p53 and chromosomal translocations) that have been identified in individual tumors during the study of neoplastic transformation are used as immunogens. Because chromosomal translocations are common in pediatric tumors, such targets may be of particular interest in pediatric oncology. In the third approach, immunization with whole tumor cell components is undertaken with the assumption that the most immunogenic molecules within the tumor will dominate the immune response induced. The benefits and limitations for each approach, particularly as it pertains to the development of immunotherapy for pediatric tumors, are discussed in this article.

XAGE-1, a new gene that is frequently expressed in Ewing's sarcoma.

Our previous expressed sequence tag database analysis indicates that XAGE-1 is frequently found in Ewing's sarcoma and alveolar rhabdomyosarcoma (U. Brinkmann et al., Cancer Res., 59: 1445-1448, 1999). Using Northern blots and RNA dot blots, we have now found that XAGE-1 is highly expressed in normal testis, in seven of eight Ewing's cell lines, in four of nine Ewing's sarcoma patient samples, and in one of one alveolar rhabdomyosarcoma patient sample. The gene is located on the X chromosome. The full-length cDNA contains 611 bp and predicts a protein of Mr 16,300 with a potential transmembrane domain at the NH2 terminus. XAGE-1 shares homology with GAGE/PAGE proteins in the COOH-terminal end. These findings could be valuable for cancer diagnosis and cancer immunotherapy.