Alberto Donfrancesco

Antagomir-17-5p Abolishes the Growth of Therapy- Resistant Neuroblastoma through p21 and BIM

We identified a key oncogenic pathway underlying neuroblastoma progression: specifically, MYCN, expressed at elevated level, transactivates the miRNA 17-5p-92 cluster, which inhibits p21 and BIM translation by interaction with their mRNA 3′ UTRs. Overexpression of miRNA 17-5p-92 cluster in MYCN-not-amplified neuroblastoma cells strongly augments their in vitro and in vivo tumorigenesis. In vitro or in vivo treatment with antagomir-17-5p abolishes the growth of MYCN-amplified and therapy-resistant neuroblastoma through p21 and BIM upmodulation, leading to cell cycling blockade and activation of apoptosis, respectively. In primary neuroblastoma, the majority of cases show a rise of miR-17-5p level leading to p21 downmodulation, which is particularly severe in patients with MYCN amplification and poor prognosis. Altogether, our studies demonstrate for the first time that antagomir treatment can abolish tumor growth in vivo, specifically in therapy-resistant neuroblastoma.

Deregulated expression of miR-26a and Ezh2 in rhabdomyosarcoma.

172 Cell Cycle 2009; Vol. 8 Issue 1 Rhabdomyosarcoma (RMS) is a highly malignant pediatric tumor that is thought to derive from mesenchymal cells already committed to become skeletal muscle cells.1 The majority of RMS tumors continues to express high levels of early inducers of muscle differentiation, such as MyoD and myogenin, indicating that aberrant differentiation pathways may participate in rhabdomyosarcoma tumorigenesis.2 Pediatric RMS is classified into two major types, embryonal RMS (ERMS) and the more aggressive alveolar RMS (ARMS). The embryonal and alveolar types of RMS differ in histology, genetic markers, current treatment protocols and prognosis. In particular, most alveolar rhabdomyosarcomas contain either the common t(2;13)(q35;q14) or the rare t(1;13)(p36;q14) translocations that form respectively PAX3-FKHR and PAX7-FKHR fusion proteins.1 Since RMS is a highly invasive malignant neoplasm, its complete surgical resection is often difficult. Therefore most of young patients require a multimodal therapy that is associated with significant acute toxicity and long-term side effects. These clinical drawbacks raise the necessity of alternative therapies tailored towards new molecular targets. Recently, the strong relationship between microRNA (miRNA) deregulated expression and cancer became evident also in human sarcomas.3 miRNAs are short non-coding RNA molecules able to regulate gene expression, affecting the stability and/or translation of target mRNAs.4 They have been established to have an important role in development and lineage differentiation, including that of the skeletal muscle system.4 A growing number of reports confirmed that clustering of the samples on the basis of their miRNA expression might be helpful for determining the developmental origin of tissues and even the cancer diagnosis.5 Numerous data suggest that each tumor type is characterized by a specific miRNA expression profile, and sometimes a given miRNA can be upregulated in some tumors and downregulated in others.6 Moreover, differences in miRNA profiles also exist among various histopathological variants of the same tumor type.7 To investigate whether miRNAs are differentially expressed in human rhabdomyosarcoma we analyzed the levels of a selected group of 20 miRNAs among those already shown to be involved in skeletal muscle differentiation and potentially in cancerogenesis (Table 1). Using RealTime polymerase chain reaction technology, we measured the expression of mature miRNA molecules in two human rhabdomyosarcoma cell lines (Table 1), embryonal RD and alveolar Rh-30, the latter expressing the PAX3-FKHR fusion protein. As rhabdomyosarcoma is believed to derive from skeletal muscle progenitor cells, we compared miRNA expression in RD and Rh-30 with that of normal human proliferating myoblasts cultured in growth medium (Skeletal Muscle Cells, Promo Cell, Germany) SkMC GM (Table 1). In RD cells eight miRNAs (40.0%) were upregulated and two (10.0%) were downregulated, compared to SkMC GM with more than a 2-fold change. No significant difference was observed for the remaining 10 miRNAs (50.0%) analyzed. Conversely, in Rh-30 cells, only one miRNA (5.0%) was upregulated whereas twelve (60.0%) were downregulated at different degrees, and seven (35.0%) remained unchanged (Table 1). These results show a pronounced downregulation of the expression of several miRNAs in the Rh-30 cell line compared to SkMC GM control cells. This is in accordance with the aggressive character of Rh-30 cells, and with the observation that a lower expression of miRNAs has been reported in tumors than in their normal tissue counterparts.4,5,8 Consistently, an overall lower expression of miRNAs has been clinically related to a more differentiated and aggressive phenotype in hepatocellular carcinomas, where mature miRNA expression level has been found to be the lowest, and patient survival the poorer.9 A substantial difference in microRNA expression between the two cell lines is also evident. The expression of 19 out of 20 miRNAs (95.0%) in RD is significantly higher than in Rh-30 (RD/Rh-30 column in Table 1), supporting the assumption that RD cells are characterized by a more differentiated phenotype. This has been already supported by the poor expression of Pax3, a known inhibitor of muscle differentiation that, conversely, exerts transcriptional activity in Rh-30 under the form of the PAX3-FKHR fusion protein.10 To evaluate the modulation of miRNAs in a human skeletal muscle differentiation model, we treated the SkMC myoblasts for 10 days with a differentiating medium (PromoCell).11 Under these conditions more than 70% SkMC converted into multinucleate myotubes (SkMC DM). Results on miRNA expression revealed a general upregulation of the miRNAs analyzed in differentiating SkMC DM compared to SkMC GM (Table 1). Particularly, miR-1 and miR-26a, two major inducers of myogenesis, are strongly expressed among those involved in muscle differentiation (Table 1). Interestingly, they appeared to be the most upregulated of this group of miRNAs also in RD cells, strengthening the assumption that RD cells could be arrested at a later phase of differentiation compared to Rh-30 cells.10 Among miRNAs previously involved in other types of cancer, miR17-5p, miR-19a, miR-19b miR-20 and miR-106a showed a coordinated expression in each cell line, as expected, since they belong to the two homologous clusters, miR-17-92 and miR-106a.12 They were not consistently modulated, being modestly upregulated in RD and SkMC DM and downregulated in Rh-30 (Table 1). Recently, beyond a wellknown oncogenic role in several human cancers,7 the miR-17-92 cluster has been reported to have tumor-suppressive features, representing a context-dependent miRNA.13 miR-221 and miR-222, generally well expressed in other human tumors compared to normal tissue,7 were downregulated in both tumor cell lines (Table 1). In a recent report on primary rhabdomyosarcomas these miRNAs have been shown to be not upregulated.4 To spotlight the clinical relevance of the miRNAs measured in RD and Rh-30 cell lines, we performed a Real Time PCR analysis of selected miRNAs (bolded in Table 1) on a group of five rhab*Correspondence to: Rossella Rota; Laboratory of Angiogenesis; Ospedale Pediatrico Bambino Gesù; Research Institute; Piazza S. Onofrio 4; Roma 00165 Italy; Tel.: 39.06.6859.2648; Fax: 39.06.6859.2904; Email: rota@opbg.net/Antonio Giordano; Sbarro Institute for Cancer Research and Molecular Medicine; Center for Biotechnology; College of Science and Technology; Temple University; BioLife Science Bldg. Ste. 333; 1900 North 12th Street; Philadelphia; PA 19122 USA; Tel.: 2152049520; Fax 2152049522; email: giordano@temple.edu

c-Kit is preferentially expressed in MYCN-amplified neuroblastoma and its effect on cell proliferation is inhibited in vitro by STI-571.

Coexpression for c‐Kit receptor and its ligand stem cell factor (SCF) has been described in neuroblastoma (NB) cell lines and tumors, suggesting the existence of an autocrine loop modulating tumor growth. We evaluated c‐Kit and SCF expression by immunohistochemistry in a series of 75 primary newly diagnosed neuroblastic tumors. Immunostaining for c‐Kit was found in 10/75 and for SCF in 17/75, with 5/10 c‐Kit–positive tumors also expressing SCF. For both, c‐Kit and SCF staining were predominantly found in the most aggressive subset of tumors, i.e., those amplified for MYCN: c‐Kit was detected in 8/14 amplified vs. 2/61 single copy (p<0.001), and SCF in 9/14 amplified vs. 8/61 single copy tumors (p<0.001). Furthermore, the association of c‐Kit expression with advanced stage (3 or 4) (p=0.001) and of SCF expression with adrenal primary (p=0.03) was substantiated. The in vitro activity of the tyrosine kinase inhibitor STI‐571 (imatinib mesylate, Gleevec, Glivec) on NB cell lines positive or negative for c‐Kit was also assessed. When cells were grown in 10% fetal calf serum, the 4 c‐Kit‐positive cell lines tested were sensitive to STI‐571 growth inhibition to a different extent (ranging from 30 to 80%); also the c‐Kit‐negative cell line GI‐CA‐N was slightly affected, suggesting that other STI‐571 targets operate in regulating NB proliferation. In addition, c‐Kit‐positive cell lines SK‐N‐BE2(c) and HTLA230, grown in SCF only, remained sensitive (40 and 70% of growth inhibition, respectively), while, in the same conditions, proliferation of the c‐Kit‐negative cell line GI‐CA‐N was not affected. Immunoprecipitation of c‐Kit from cell lysates of SK‐N‐BE2(c) and HTLA230 cells grown in SCF and subsequent western blot analysis of the immunoprecipitates revealed a sharp decrease of c‐Kit phosphorylation after STI‐571 treatment. These data demonstrate that both c‐Kit and SCF are preferentially expressed in vivo in the most aggressive neuroblastic tumors and that their signaling is active in promoting in vitro NB cell proliferation that can be selectively inhibited by treatment with STI‐571.

High-dose carboplatin in combination with etoposide (JET regimen) for childhood brain tumors.

Fourteen patients aged 1 to 15 years with medulloblastoma (six patients), low-grade astrocytoma (four patients), and high-grade astrocytoma (four patients) were treated with carboplatin and etoposide (JET regimen). Six patients had been treated previously, two of them with cisplatin at conventional doses. Carboplatin was administered at 500 mg/m2/day over 5 h on days 1 and 2, in association with pulsed etoposide at 100 mg/m2/day on days 1, 2, and 3. Courses were repeated at 3-week intervals. The disease-specific response rates were as follows: five of six with three complete responses and two partial responses for medulloblastoma; zero of four for low-grade astrocytoma; and two of four with two partial responses for high-grade astrocytoma. Myelosuppression was the main side effect: anemia (hemoglobin less than 8.0 g/dl), thrombocytopenia (less than 25,000/microliter) and leukopenia (less than 1,000 white blood cells/microliters) were noted in 19 of 54, 10 of 54, and 7 of 54 courses, respectively. Gastrointestinal toxicity was very mild, and nephro- and neurotoxicity were not observed. No audiometric abnormalities were demonstrated in seven of seven patients who had not previously received cisplatin, and preexisting audiometric abnormalities were not worsened by the administration of carboplatin in one cisplatin-pretreated patient. The combination of carboplatin and etoposide administered in this study appears to be effective and well tolerated in children with brain tumors. Further studies on a larger number of patients are needed to ascertain its real activity in childhood brain tumors.

Deferoxamine, cyclophosphamide, etoposide, carboplatin, and thiotepa (D-CECaT): a new cytoreductive chelation-chemotherapy regimen in patients with advanced neuroblastoma.

Thirteen patients with Stage III (3 patients) or Stage IV (10 patients) neuroblastoma were treated with a new iron chelation-cytotoxic therapy regimen. Deferoxamine given for five consecutive days followed by 3 days of cyclophosphamide, etoposide, carboplatin, and thiotepa (D-CECaT) caused moderate to severe myelotoxicity. In 39 courses there were four episodes of sepsis; platelet and packed red blood cell transfusions were required in 72% and 82% of courses, respectively. Mild nausea and vomiting occurred in 52% of courses. Objective responses after two courses were observed in 12 of 13 patients. Three of four partial responses were achieved in previously treated relapsed patients, and seven of eight complete responses (four of which were surgically documented) were achieved in previously untreated patients. This cytoreduction regimen appears to be an improvement over other initial induction regimens and may be worth testing in larger populations.

Ifosfamide/carboplatin/etoposide (ICE) as front-line, topotecan/ cyclophosphamide as second-line and oral temozolomide as third-line treatment for advanced neuroblastoma over one year of age

Children affected by advanced neuroblastoma have a discouraging prognosis, but intensive induction chemotherapy may increase the complete response rate. The combination of ifosfamide, carboplatin and etoposide (ICE) was used for the first time as front‐line regimen in patients with stage 4 neuroblastoma over the age of 1 y. Similarly, second‐line treatment for children with relapsed neuroblastoma, particularly after high‐dose chemotherapy, has been unsatisfactory. The combination of topotecan and cyclophosphamide was studied in resistant or relapsed solid tumors. Furthermore, there is a need for effective palliative treatment in patients failing therapy. Temozolomide, a new dacarbazine analog with optimal oral bioavailability, is being used in an ongoing phase II study as an alternative to oral etoposide. Seventeen patients with stage 4 neuroblastoma have entered the ICE study; 15/16 (94%) major responses after induction were observed and 6/16 (37%) evaluable patients are disease free after a median of 51 mo. Twenty‐one patients with relapsed/refractory disease (of whom 13 neuroblastomas) entered the topotecan/cyclophosphamide study: 7/21 (33%) patients responded. Forty‐one patients entered the temozolomide study (of whom 16 had neuroblastomas): stable disease and symptom relief were obtained in 15/30 (50%) evaluable patients. Intensive induction with ICE resulted in a faster response with high response rate; a larger study with longer follow‐up is needed to confirm a survival advantage. Second‐line treatment was effective in obtaining remissions, some of them long lasting. Third‐line treatment did not elicit measurable responses in neuroblastoma, but achieved prolonged freedom from disease progression and excellent palliation in several patients.

Spindle cell (Kaposiform) hemangioendothelioma with Kasabach‐Merritt syndrome in an infant: Successful treatment with α‐2A interferon

A two-month-old infant developed a vascular tumor of the right flank which upon biopsy proved to be a spindle cell hemangioendothelioma. The increased capillary bed characterizing the neoplasm caused a severe thrombocytopenia together with a consumption coagulopathy (Kasabach-Merritt syndrome). The patient, who was dependent on platelet transfusions, improved quickly after interferon alpha-2a was given at the dosage of 3,000,000 U/m2, with resolution of the Kasabach-Merritt syndrome after three weeks and a 75% decrease of the tumor volume within three months of treatment.

Desmoplastic small round cell tumour in children and adolescents

BACKGROUND Desmoplastic small round cell tumour (DSRCT) is a rare highly aggressive neoplasm, and clinical studies are scarce. PROCEDURE We report six cases of children and adolescents (median age 14 years, range 6.9-17.5) with DSRCT (5 abdominal, 1 paratesticular) registered by the Italian Cooperative Group (ICG) for soft tissue sarcoma over a 9-year period. Patients received a multidisciplinary treatment, including aggressive initial or delayed surgery and radiotherapy. Chemotherapy regimen was based on the use of ifosfamide, vincristine, dactinomycin, and a few doses of antharacyclines (doxorubicin or epirubicin). RESULTS Complete surgical resection was possible only for the paratesticular tumour. Among the patients with abdominal lesions, macroscopically radical excision was possible in only one case. All patients received multidrug chemotherapy, and tumour reduction was obtained in the 4 evaluable patients. No relapses were evident in the irradiated fields in the 4 patients who received radiotherapy. Two patients remained progression-free 22 and 63 months after diagnosis, one is in third complete remission, whereas three died 10-25 months after diagnosis. CONCLUSIONS DSRCT is a chemosensitive tumour, but survival rates remain disappointing despite aggressive multimodality therapy. Our results support surgical tumour removal and radiotherapy to achieve local control. Our experience and a review of the literature suggest that patients with localised abdominal tumours or a paratesticular primary may have a better prognosis.

Prognostic factors in pleuro‐pulmonary blastoma

To evaluate the prognostic factors in a series of children affected by pleuropulmonary blastoma (PPB).

Temozolomide in resistant or relapsed pediatric solid tumors

We report the off‐label study aimed at investigating the use of temozolomide (TMZ) as single agent in relapsed or resistant pediatric solid tumors. The drug was administered at the dose of 215 mg/m2/day × 5 days or 180 mg/m2/day × 5 days in patients with prior craniospinal irradiation (CSI) or autologous bone marrow transplantation (ABMT).