Global DNA methylation profiling uncovers distinct methylation patterns of protocadherin alpha4 in metastatic and non-metastatic rhabdomyosarcoma.

BACKGROUND: Rhabdomyosarcoma (RMS), which can be classified as embryonal RMS (ERMS) and alveolar RMS (ARMS), represents the most frequent soft tissue sarcoma in the pediatric population; the latter shows greater aggressiveness and metastatic potential with respect to the former. Epigenetic alterations in cancer include DNA methylation changes and histone modifications that influence overall gene expression patterns. Different tumor subtypes are characterized by distinct methylation signatures that could facilitate early disease detection and greater prognostic accuracy. METHODS: A genome-wide approach was used to examine methylation patterns associated with different prognoses, and DNA methylome analysis was carried out using the Agilent Human DNA Methylation platform. The results were validated using bisulfite sequencing and 5-aza-2'deoxycytidine treatment in RMS cell lines. Some in vitro functional studies were also performed to explore the involvement of a target gene in RMS tumor cells. RESULTS: In accordance with the Intergroup Rhabdomyosarcoma Study (IRS) grouping, study results showed that distinct methylation patterns distinguish RMS subgroups and that a cluster of protocadherin genes are hypermethylated in metastatic RMS. Among these, PCDHA4, whose expression was decreased by DNA methylation, emerged as a down-regulated gene in the metastatic samples. As PCDHA4-silenced cells have a significantly higher cell proliferation rate paralleled by higher cell invasiveness, PCDHA4 seems to behave as a tumor suppressor in metastatic RMS. CONCLUSION: Study results demonstrated that DNA methylation patterns distinguish between metastatic and non-metastatic RMS and suggest that epigenetic regulation of specific genes could represent a novel therapeutic target that could enhance the efficiency of RMS treatments.

The Polycomb group (PcG) protein EZH2 supports the survival of PAX3-FOXO1 alveolar rhabdomyosarcoma by repressing FBXO32 (Atrogin1/MAFbx).

The Polycomb group (PcG) proteins regulate stem cell differentiation via the repression of gene transcription, and their deregulation has been widely implicated in cancer development. The PcG protein Enhancer of Zeste Homolog 2 (EZH2) works as a catalytic subunit of the Polycomb Repressive Complex 2 (PRC2) by methylating lysine 27 on histone H3 (H3K27me3), a hallmark of PRC2-mediated gene repression. In skeletal muscle progenitors, EZH2 prevents an unscheduled differentiation by repressing muscle-specific gene expression and is downregulated during the course of differentiation. In rhabdomyosarcoma (RMS), a pediatric soft-tissue sarcoma thought to arise from myogenic precursors, EZH2 is abnormally expressed and its downregulation in vitro leads to muscle-like differentiation of RMS cells of the embryonal variant. However, the role of EZH2 in the clinically aggressive subgroup of alveolar RMS, characterized by the expression of PAX3-FOXO1 oncoprotein, remains unknown. We show here that EZH2 depletion in these cells leads to programmed cell death. Transcriptional derepression of F-box protein 32 (FBXO32) (Atrogin1/MAFbx), a gene associated with muscle homeostasis, was evidenced in PAX3-FOXO1 RMS cells silenced for EZH2. This phenomenon was associated with reduced EZH2 occupancy and H3K27me3 levels at the FBXO32 promoter. Simultaneous knockdown of FBXO32 and EZH2 in PAX3-FOXO1 RMS cells impaired the pro-apoptotic response, whereas the overexpression of FBXO32 facilitated programmed cell death in EZH2-depleted cells. Pharmacological inhibition of EZH2 by either 3-Deazaneplanocin A or a catalytic EZH2 inhibitor mirrored the phenotypic and molecular effects of EZH2 knockdown in vitro and prevented tumor growth in vivo. Collectively, these results indicate that EZH2 is a key factor in the proliferation and survival of PAX3-FOXO1 alveolar RMS cells working, at least in part, by repressing FBXO32. They also suggest that the reducing activity of EZH2 could represent a novel adjuvant strategy to eradicate high-risk PAX3-FOXO1 alveolar RMS.

High ALK mRNA expression has a negative prognostic significance in rhabdomyosarcoma.

BACKGROUND: Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase aberrantly expressed in cancer, but its clinical and functional importance remain controversial. Mutation or amplification of ALK, as well as its expression levels assessed by conventional immunohistochemistry methods, has been linked to prognosis in cancer, although with potential bias because of the semi-quantitative approaches. Herein, we measured ALK mRNA expression in rhabdomyosarcoma (RMS) and determined its clinical impact on patients' stratification and outcome. METHODS: Specimens were obtained from RMS patients and cell lines, and ALK expression was analysed by quantitative RT-PCR, western blotting, IHC, and copy number analysis. RESULTS: High ALK mRNA expression was detected in the vast majority of PAX3/7-FOXO1-positive tumours, whereas PAX3/7-FOXO1-negative RMS displayed considerably lower amounts of both mRNA and protein. Notably, ALK mRNA distinguished unfavourable PAX3/7-FOXO1-positive tumours from PAX3/7-FOXO1-negative RMS (P<0.0001), and also correlated with larger tumour size (P<0.05) and advanced clinical stage (P<0.01), independently of fusion gene status. High ALK mRNA levels were of prognostic relevance by Cox univariate regression analysis and correlated with increased risk of relapse (P=0.001) and survival (P=0.01), whereas by multivariate analysis elevated ALK mRNA expression resulted a negative prognostic marker when clinical stage was not included. CONCLUSION: Quantitative assessment of ALK mRNA expression helps to improve risk stratification of RMS patients and identifies tumours with adverse biological characteristics and aggressive behaviour.

Use of minimal disseminated disease and immunity to NPM-ALK antigen to stratify ALK-positive ALCL patients with different prognosis.

We studied the prognostic value of minimal disseminated disease (MDD) and anti-ALK immune response in children with NPM-ALK-positive anaplastic-large cell lymphoma (ALCL) and evaluated their potential for risk stratification. NPM-ALK transcripts were analyzed by RT-PCR in bone marrow/peripheral blood of 128 ALCL patients at diagnosis, whereas ALK antibody titers in plasma were assessed using an immunocytochemical approach. MDD was positive in 59% of patients and 96% showed an anti-ALK response. Using MDD and antibody titer results, patients could be divided into three biological risk groups (bRG) with different prognosis: high risk (bHR): MDD-positive and antibody titer ≤ 1/750, 26/128 (20%); low risk (bLR): MDD negative and antibody titer >1/750, 40/128 (31%); intermediate risk (bIR): all remaining patients, 62/128 (48%). Progression-free survival was 28% (s.e., 9%), 68% (s.e., 6%) and 93% (s.e., 4%) for bHR, bIR and bLR, respectively (P<0.0001). Survival was 71% (s.e., 9%), 83% (s.e., 5%) and 98% (s.e., 2%) for bHR, bIR and bLR (P=0.02). Only bHR and histology other than common type were predictive of higher risk of failure (hazard ratio 4.9 and 2.7, respectively) in multivariate analysis. Stratification of ALCL patients based on MDD and anti-ALK titer should be considered in future ALCL trials to optimize treatment.

Consequences of heat shock protein 72 (Hsp72) expression and activity on stress-induced apoptosis in CD30+ NPM-ALK+ anaplastic large-cell lymphomas.

Understanding the mechanisms that control stress-induced apoptosis is critical to explain how tumours respond to treatment, as cancer cells frequently escape drug toxicity by regulating stress response through heat shock protein (HSP) expression. The overexpression of Hsp72, in particular, results in increased incidence of cell transformation, and correlates with poor prognosis in a wide range of cancers. We have shown that Hsp72 assists folding of oncogenic NPM-ALK kinase in anaplastic large-cell lymphomas (ALCLs), but its role in the maintenance of the malignant phenotype remains uncertain. Therefore, we assessed Hsp72 expression in ALCLs, investigating more in detail the mechanisms that regulate its status and activity. We found that Hsp72 is unique among the HSPs involved in tumourigenesis to be overexpressed in ALK(+) tumours and cell lines and to be induced by stress. Different from other HSPs, Hsp72 prevents cell injury, Bax activation and death by apoptosis in ALK(+) cells, acting both upstream and downstream of mitochondria. Conversely, Hsp72 is underexpressed in ALK(-) ALCL cells, and it is unable to protect cells from apoptosis under stress. Moreover, when Hsp72 expression is reduced following NPM-ALK inhibition, lymphoma cells undergo apoptosis, demonstrating the importance of Hsp72 in regulating ALCL stress response and drug sensitivity.

Sequential intensified chemotherapy with stem cell rescue for children and adolescents with desmoplastic small round-cell tumor.

The RMS4.99 study was designed to explore the role of early sequential intensified chemotherapy (SICT) with PBSC rescue in patients with soft tissue sarcoma with a poor prognosis. Fourteen patients with desmoplastic small round-cell tumor (DSRCT) were included in this study. Initial chemotherapy was followed by a course of CY and etoposide with subsequent PBSC harvest, then three consecutive intensified chemotherapy combinations followed by PBSC rescue and G-CSF administration: first cycle thiotepa (150 mg/m(2) x 2 on day 1) and melphalan (60 mg/m(2) on day 2), second cycle CY (2 g/m(2) on days 1 and 2) and thiotepa (150 mg/m(2) x 2 on day 3), third cycle melphalan (80 mg/m(2) on day 1). The interval between cycles had to be kept as short as possible. Then patients underwent surgery or radiotherapy or both, after which six courses of vincristine, actinomycin D, CY were administered. Ten patients received SICT, which was well tolerated. With a median follow-up of 27 months only three patients are alive without evidence of disease. The 3-year event-free and overall survival rates were 15.5 and 38.9%, respectively. The prognosis for pediatric patients with DSRCT did not improve after administering intensified chemotherapy early in their treatment, so different strategies are needed.

Treatment of pediatric non-Hodgkin lymphomas in a country with limited resources: results of the first national protocol in Nicaragua.

We report the results of a protocol for the diagnosis and treatment of pediatric non-Hodgkin lymphomas (NHL) conducted in Nicaragua in the context of an international collaborative program. Fifty-three children with NHL treated between 1996 and 2003 were retrospectively evaluated. Therapy was designed based on local drug availability and affordability with dose and schedule adaptations for Burkitt and lymphoblastic lymphomas. With a median follow-up of 3 years, the projected 9-year overall survival was 63% and event-free survival 53%. The treatment was efficacious, feasible, and well tolerated in spite of the local socio-economical conditions.

Anaplastic large cell lymphomas: a study of 75 pediatric patients.

In this article, we describe the morphologic and immunophenotypic features of 75 cases of pediatric anaplastic large cell lymphoma (ALCL). According to the World Health Organization classification, 49 cases were common subtype ALCL, and respectively, 3, 6, and 17 cases were small cell, lymphohistiocytic, or mixed histologic variants. Anaplastic lymphoma kinase positivity was detected in 90.7% of the tumors and, using a panel of 9 T-cell surface markers, 88% could be assigned to the T-cell lineage. A molecular analysis for the T-cell receptor gamma (TCR- gamma) and the heavy chain of the immunoglobulin H rearrangements was performed on 6/9 ALCLs with a null immunophenotype, and a TCR clonal pattern was detected in 5/6 cases. In addition, 94.1% were immunoreactive for 1 or more cytotoxic proteins (Tia1, granzyme B, or perforin), and 15% expressed CD56. Clusterin, CD83, and Pax5, respectively, expressed in 91.3%, 1.7%, and 0% of the ALCLs, were useful biomarkers for the differential diagnosis with Hodgkin's lymphomas.

Prevalence and clinical implications of bone marrow involvement in pediatric anaplastic large cell lymphoma.

Anaplastic large cell lymphoma (ALCL) harbors the reciprocal chromosomal translocation t(2;5)(p23;q35) in approximately 80% of the cases. The genes involved are nucleophosmin (NPM) and anaplastic lymphoma kinase (ALK) and the resulting chimeric NPM-ALK protein is thought to play a key role in the pathogenesis of t(2;5) positive ALCL. Few data on bone marrow (BM) involvement in ALCL have been published and they mostly rely on morphological examination of BM smears. We studied 52 ALCL for NPM-ALK expression by RT-PCR: 47/52 biopsies were positive. In 41 of the 47 cases we obtained the BM at diagnosis and investigated the prevalence of minimal BM infiltration by RT-PCR and real-time PCR. Minimal disseminated disease was positive in 25/41 patients (61%), of whom six had morphologically infiltrated BM. Survival analysis demonstrated a 5-year progression-free survival of 41 +/- 11% for patients with molecularly positive BM vs 100% for patients with negative BM (P = 0.001). These results suggest that minimal BM involvement at diagnosis is a common event in pediatric ALCL and that minimal BM disease monitoring could identify patients at risk of relapse.

Unrelated bone marrow transplantation for high-risk anaplastic large cell lymphoma in pediatric patients: a single center case series.

OBJECTIVES: The use of allogeneic stem cell transplantation in NHL patients is not yet clearly defined, especially in children and adolescents, but this option offers the advantages of a tumor-free graft and the possible induction of a graft-vs.-tumor effect. PATIENTS AND METHODS: We report the results of four consecutive pediatric patients affected by anaplastic large cell lymphoma (ALCL) and treated with allogeneic stem cell transplantation from an unrelated donor. The conditioning regimen was based on total body irradiation given in association with etoposide in three patients, and with thiotepa and cyclophoshamide in one patient. Graft-vs.-host disease (GVHD) prophylaxis consisted of cyclosporin, a short course of methotrexate and rabbit antithymocyte globulin. RESULTS: All patients had rapid engraftment within 3-4 wk for neutrophils and platelets, and achieved a stable full donor chimerism that has been maintained to the last follow-up visit. One patient later developed a restrictive pneumonopathy. This patient had been heavily pretreated during the course of the disease having suffered four relapses and had received a cumulative dose of bleomycin of 160 mg/m(2). After a follow-up of 11-42 months, all patients are alive in complete hematological and molecular remission; and three of them without any chronic GVHD. CONCLUSIONS: The increasing number of volunteer bone marrow donors and the reduced toxicity of unrelated stem cell transplantation, especially in children, make this therapeutic option worth more extensive investigation in the treatment of high-risk failure ALCL, although more data is needed to evaluate the long-term benefits. In this regard, the presence of factors predictive of worst outcome such as an early relapse (within 12 months from diagnosis), a refractory or relapsing ALCL and the persistent detection on blood or bone marrow of nucleophosmin-anaplastic lymphoma kinase protein (NPM-ALK) transcript may help select the patients eligible to allogeneic related or unrelated stem cell transplantation.

Prospective analysis of minimal bone marrow infiltration in pediatric Burkitt's lymphomas by long-distance polymerase chain reaction for t(8;14)(q24;q32).

The chromosomal translocation t(8;14)(q24;q32) represents a characteristic marker for Burkitt's lymphoma (BL). This translocation involves the MYC oncogene on chromosome 8 and the immunoglobulin heavy-chain (IgH) locus on chromosome 14. Since the translocation does not produce a fusion gene, we established a long-distance polymerase chain reaction (LD-PCR) assay that can detect the t(8;14) at the genomic level. The sensitivity of the LD-PCR was 10(-4). We used the LD-PCR assay to prospectively study 78 BL patients and found a specific PCR product in 52 of them. Among the 52 positive patients, we could test both the tumor and the bone marrow (BM) at diagnosis in 33 and determined the prevalence of minimal disseminated disease (MDD) at diagnosis. In 12/33 patients, BM was positive by LD-PCR and in 10 of them we conducted a study of minimal residual disease (MRD). Eight out of 10 children showed a clearance of MRD after one cycle of chemotherapy. The only two patients who did not achieve a negative MRD status died of disease progression. The comparative analysis of sensitivity of BM aspirate, BM biopsy and LD-PCR in t(8;14)-positive patients demonstrated a superiority of the molecular method in the assessment of MDD. The LD-PCR for t(8;14) is an important tool to study minimal BM infiltration at diagnosis and to determine its response kinetics in BL.

Cell division cycle control in embryonal and alveolar rhabdomyosarcomas.

In this study, we investigated the mRNA level of several genes involved in cell cycle regulation in alveolar (ARMS) and embryonal rhabdomyosarcomas (ERMS). p21(Cip1), Cyclin D1, Cyclin D2, Cyclin D3, CDK2, and CDK4 were evaluated by RT-PCR. All (13 out of 13) ERMS expressed the p21(Cip1) gene compared with only 40% (4 out of 10) of the ARMS. Moreover, the amount of p21(Cip1) mRNA was noticeably higher in the ERMS samples than in the positive ARMS specimens. p27(Kip1) protein were analysed by immunohistochemical and immunoblotting. A noticeable difference was observed, in that ERMS had higher amounts of the cell cycle inhibitor compared with the ARMS. Finally, treatment of two rhabdomyosarcoma cell lines, RH-30 and RD, with butyrate, resulted in complete growth inhibition and in the upregulation of the p21(Cip1) and p27(Kip1) levels. Our results demonstrate that ERMS have a much higher level of p27(Kip1) and p21(Cip1) than the alveolar types, explaining, at least in part, the distinct features and outcomes (i.e. a poor prognosis of the alveolar type) of the two forms of this childhood solid cancer. Moreover, the data on butyrate-treated cell lines suggest that the two genes are potential novel therapeutic targets for the treatment of rhabdomyosarcomas.

MAGE, BAGE and GAGE gene expression in human rhabdomyosarcomas.

MAGE, BAGE and GAGE genes encode tumor-associated antigens that are presented by HLA class I molecules and recognized by CD8(+) cytolytic T lymphocytes. These antigens are currently regarded as promising targets for active, specific tumor immunotherapy because MAGE, BAGE and GAGE genes are expressed in many human cancers of different histotype and are silent in normal tissues, with the exception of spermatogonia and placental cells. MAGE, BAGE and GAGE gene expression has been extensively studied in different tumors of adults but is largely unknown in many forms of pediatric solid cancer. Using RT-PCR, we analyzed MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-6, BAGE, GAGE-1,-2 or -8 and GAGE-3,-4,-5,-6 or -7b gene expression in 31 samples of pediatric rhabdomyosarcoma, the most frequent form of malignant soft tissue tumor in children. MAGE genes were expressed in a substantial proportion of patients (MAGE-1, 38%; MAGE-2, 51%; MAGE-3, 35%; MAGE-4, 22%; MAGE-6, 35%), while expression of BAGE (6%); GAGE-1, GAGE-2 and GAGE-8 (9%); and GAGE-3, GAGE-4, GAGE-5, GAGE-6 and GAGE-7B (16%) was less frequent. Overall, 58% of tumors expressed at least 1 gene, and 35% expressed 3 or more genes simultaneously. Our data suggest that a subset of rhabdomyosarcoma patients could be eligible for active, specific immunotherapy directed against MAGE, BAGE and GAGE antigens.

Neonatal leukemoid reaction and early development of bronchopulmonary dysplasia in a very low-birth-weight infant.

The factors controlling the recruitment of inflammatory cells and the activation of the cytokine cascade in low-birth-weight premature infants have been implicated in the sequence of multiorgan inflammatory diseases, including the chronic lung disease of prematurity, bronchopulmonary dysplasia. This article describes a 982-gram, 25 (+2 days) weeks' gestation male infant, who had a leukemoid reaction throughout the first week of life, followed by early development of bronchopulmonary dysplasia.

Geldanamycin abrogates ErbB2 association with proteasome-resistant beta-catenin in melanoma cells, increases beta-catenin-E-cadherin association, and decreases beta-catenin-sensitive transcription.

Beta-catenin undergoes both serine and tyrosine phosphorylation. Serine phosphorylation in the amino terminus targets beta-catenin for proteasome degradation, whereas tyrosine phosphorylation in the COOH terminus influences interaction with E-cadherin. We examined the tyrosine phosphorylation status of beta-catenin in melanoma cells expressing proteasome-resistant beta-catenin, as well as the effects that perturbation of beta-catenin tyrosine phosphorylation had on its association with E-cadherin and on its transcriptional activity. Beta-catenin is tyrosine phosphorylated in three melanoma cell lines and associates with both the ErbB2 receptor tyrosine kinase and the LAR receptor tyrosine phosphatase. Geldanamycin, a drug which destabilizes ErbB2, caused rapid cellular depletion of the kinase and loss of its association with beta-catenin without perturbing either LAR or beta-catenin levels or LAR/beta-catenin association. Geldanamycin also stimulated tyrosine dephosphorylation of beta-catenin and increased beta-catenin/E-cadherin association, resulting in substantially decreased cell motility. Geldanamycin also decreased the nuclear beta-catenin level and inhibited beta-catenin-driven transcription, as assessed using two different beta-catenin-sensitive reporters and the endogenous cyclin D1 gene. These findings were confirmed by transient transfection of two beta-catenin point mutants, Tyr-654Phe and Tyr-654Glu, which, respectively, mimic the dephosphorylated and phosphorylated states of Tyr-654, a tyrosine residue contained within the beta-catenin-ErbB2-binding domain. These data demonstrate that the functional activity of proteasome-resistant beta-catenin is regulated further by geldanamycin-sensitive tyrosine phosphorylation in melanoma cells.

Concomitant amplification and expression of PAX7-FKHR and MYCN in a human rhabdomyosarcoma cell line carrying a cryptic t(1;13)(p36;q14).

Alveolar rhabdomyosarcoma (ARMS) is associated with the specific chromosomal translocation (2;13)(q35;q14) or its rarer variant t(1;13)(p36;q14), which produces the fusion gene PAX7-FKHR. Here we describe the human cell line RC2, derived from an ARMS, which harbors a cryptic t(1;13)(p36;q14) and concomitantly shows amplification of the PAX7-FKHR fusion gene and of the MYCN oncogene. The t(1;13) and MYCN oncogene were studied by standard cytogenetic analysis and molecular techniques. The reverse transcriptase polymerase chain reaction demonstrated the expression of PAX7-FKHR mRNA in RC2 cells, although karyotype analysis failed to demonstrate a t(1;13)(p36;q14) chromosomal translocation or a derivative 13 chromosome. Double minute chromosomes were detected in all the metaphases studied. Fluorescence in situ hybridization analysis revealed multiple copies of the PAX7-FKHR fusion gene localized exclusively on a subset of double minutes, whereas multiple copies of MYCN were identified on other double minute chromosomes. Southern-blot analysis demonstrated that RC2 cells contain approximately 20 copies of the MYCN oncogene. So far no continuous RMS cell line carrying the t(1;13)(p36;q14) has been described, and PAX7-FKHR and MYCN amplifications have always been reported to occur separately in rhabdomyosarcoma (RMS). The availability of an ARMS cell line that harbors the t(1;13)(p36;q14) constitutes a useful tool for further understanding the role of the PAX7-FKHR fusion gene in RMS oncogenesis and may improve knowledge of the possible relation between PAX7-FKHR and MYCN amplification.

All-trans retinoic acid and interferon-alpha in the treatment of a patient with resistant metastatic osteosarcoma.

BACKGROUND: A boy age 14 years who was in complete remission from Stage IIB small cell osteosarcoma, which was misdiagnosed as Ewing sarcoma and consequently was treated, developed inoperable lung metastases when he was off therapy. He received second-line treatment for recurrent Ewing sarcoma, including chemotherapy and radiotherapy, and obtained only a temporary response. A compassionate treatment with all-trans retinoic acid (ATRA) and interferon-alpha (IFNalpha) was then undertaken. METHODS: The patient initially was treated according to the national SE91 protocol for nonmetastatic Ewing sarcoma. After a bilateral pulmonary recurrence, he received second-line chemotherapy and irradiation of the largest metastasis, with a temporary partial response. The patient was then treated with a combination of oral ATRA (90 mg/m(2) for 3 days per week) and subcutaneous IFNalpha (3 x 10(6) U/m(2) 5 days per week) for 4 months. The same therapy also was administered for the control of residual disease after surgery for a total duration of 1 year of ATRA/IFN treatment. During the first 3 weeks of therapy, ATRA pharmacokinetics were studied. RESULTS: After progression of the patient's disease, despite the administration of first-line and second-line chemotherapy, combined treatment with ATRA/IFNalpha yielded a partial remission, which allowed surgical resection of the largest metastasis. The same therapy was effective in preventing tumor recurrence after incomplete removal of the remaining metastases. Treatment was well tolerated, and the patient is in stable complete remission 14 months after the end of therapy. The pharmacokinetics results confirmed the indication of an intermittent schedule for oral ATRA therapy. CONCLUSIONS: ATRA/IFNalpha treatment may be considered as an alternative approach in the treatment of patients with metastatic osteosarcoma who have disease that is resistant to conventional chemotherapy and in the treatment of patients with minimal tumor residue.