Carlos S. Alvarado

Expression of Inhibitor-of-Apoptosis Protein (IAP) Livin by Neuroblastoma Cells: Correlation with Prognostic Factors and Outcome

Livin is a recently identified member of the Inhibitor-of-Apoptosis protein (IAP) family of antiapoptosis proteins, and expression has been reported in melanoma and some types of carcinoma. We evaluated livin expression in paraffin-embedded tumor tissue from 68 patients with neuroblastoma (NB) and 7 NB cell lines by immunoperoxidase using an anti-livin monoclonal antibody. Eighteen (26.5%) of the 68 NB tumor tissues showed high livin expression, 36 (53%) showed low-intermediate expression, and 14 (20.5%) were negative. Similarly, 4 NB cell lines showed high livin expression, and 3 showed intermediate expression. In primary NB tissue, livin was observed mainly in tumor neuropil, an extension of tumor cell cytoplasm, and the cytoplasm itself. By reverse transcriptase–polymerase chain reaction, livin expression was confirmed in all 7 NB lines and in frozen tissue from 1 of 3 primary tumors examined to date, in agreement with immunohistochemical data; both livin α and β isoforms were detected. In the NB cases, we further analyzed the correlation between livin expression and clinical and biological features with established prognostic significance (i.e., age at diagnosis, stage, histology, and MYCN oncogene status), and patients’ outcome. Livin expression alone did not appear to have an effect on survival; however, patients with high livin expression and amplified MYCN had significantly decreased survival compared with patients lacking both markers or with either of these markers alone. These results suggest that (a) livin is expressed in primary and cultured neuroblastoma cells and (b) high livin expression may identify a subset of neuroblastoma patients with a particularly poor prognosis among those with MYCN amplified tumors.

Bexarotene Is Active Against Subcutaneous Panniculitis-Like T-Cell Lymphoma in Adult and Pediatric Populations

INTRODUCTION Subcutaneous panniculitis-like T-cell lymphoma (SPTL-AB) and cutaneous gamma/delta T-cell lymphoma (CGD-TCL) are rare T-cell lymphomas with varying clinical courses. There is no standard treatment, although chemotherapy and hematopoietic stem cell transplantation are commonly used. We describe results using bexarotene for children and adults with these disorders. METHODS We identified 15 patients (12 adults, 3 children) who were treated with bexarotene between 2000 and 2010 from the Memorial Sloan-Kettering Cancer Center lymphoma database, the Stanford Cancer Center Registry, and the National Cancer Institute (NCI) pediatric lymphoma database. There were 8 females and 7 males, with a median age of 45 years (range, 3 years to 85 years). All patients had stage IV disease. Two of 15 and 4 of 15 patients had documented CGD-TCL and SPTL-AB, respectively; others were presumed to have SPTL-AB. Bexarotene was administered at flat doses corresponding to 91 to 339 mg/m(2)/d. Two of 15 patients received concurrent denileukin diftitox. Two children received bexarotene as maintenance therapy and were not evaluable for response. RESULTS Among those treated with bexarotene alone, the overall response rate (ORR) was 82% (6/11 complete response [CR], 3/11 partial response [PR]). One of the 2 patients treated with concomitant denileukin diftitox responded for an ORR of 10/13 (77%), including 54% CR and 23% PR. Median progression-free survival was 38.4 months; median duration of response was 26.3 months. Six patients developed hypothyroidism and 9 developed hyperlipidemia; one patient developed dose-limiting hypertriglyceridemia. One pediatric patient developed insulin-dependent diabetes mellitus. CONCLUSIONS In this retrospective series, bexarotene showed a high response rate in SPTL-AB and CGD-TCL. It was generally well-tolerated with durable responses; therefore, bexarotene represents a promising therapy for children and adults with these disorders.

Adjuvant chemotherapy for advanced nasopharyngeal carcinoma in childhood.

Seven children with advanced nasopharyngeal carcinoma younger than 20 years of age diagnosed between 1975 and 1986 (inclusive) were treated with a uniform adjuvant chemotherapy regimen, which consisted of vincristine (1.5 mg/m2; day 1), doxorubicin (45 mg/m2; day 1), 5‐fluorouracil (8 mg/kg; days 1 through 5), and cyclophosphamide (7 mg/kg; days 1 through 5). This combination chemotherapy was given for 12 to 24 months after completion of radiation therapy. The radiation doses to the primary sites ranged from 6000 cGy to a maximum of 6800 cGy. The radiation doses for neck prophylaxis ranged from 4500 cGy to a total of 5000 cGy. Involved sites were irradiated to at least an additional boost of 1000 cGy. One patient had an external dose 6000 cGy to the primary site boosted with brachytherapy of 3000 cGy at the surface of an ovoid. After chemotherapy myelosuppression occurred in all patients and was tolerable. All seven patients are surviving, six disease‐free, for 22 months to 12 years (median, 4 years). This study suggests that the combination of radiation therapy and chemotherapy as used here has acceptable toxicity and is effective and further suggests that children with nasopharyngeal carcinoma, even in its advanced stage at diagnosis, may be curable.

PHA665752, a small-molecule inhibitor of c-Met, inhibits hepatocyte growth factor-stimulated migration and proliferation of c-Met-positive neuroblastoma cells

Backgroundc-Met is a tyrosine kinase receptor for hepatocyte growth factor/scatter factor (HGF/SF), and both c-Met and its ligand are expressed in a variety of tissues. C-Met/HGF/SF signaling is essential for normal embryogenesis, organogenesis, and tissue regeneration. Abnormal c-Met/HGF/SF signaling has been demonstrated in different tumors and linked to aggressive and metastatic tumor phenotypes. In vitro and in vivo studies have demonstrated inhibition of c-Met/HGF/SF signaling by the small-molecule inhibitor PHA665752. This study investigated c-Met and HGF expression in two neuroblastoma (NBL) cell lines and tumor tissue from patients with NBL, as well as the effects of PHA665752 on growth and motility of NBL cell lines. The effect of the tumor suppressor protein PTEN on migration and proliferation of tumor cells treated with PHA665752 was also evaluated.MethodsExpression of c-Met and HGF in NBL cell lines SH-EP and SH-SY5Y and primary tumor tissue was assessed by immunohistochemistry and quantitative RT-PCR. The effect of PHA665752 on c-Met/HGF signaling involved in NBL cell proliferation and migration was evaluated in c-Met-positive cells and c-Met-transfected cells. The transwell chemotaxis assay and the MTT assay were used to measure migration and proliferation/cell-survival of tumor cells, respectively. The PPAR-γ agonist rosiglitazone was used to assess the effect of PTEN on PHA665752-induced inhibition of NBL cell proliferation/cell-survival and migrationResultsHigh c-Met expression was detected in SH-EP cells and primary tumors from patients with advanced-stage disease. C-Met/HGF signaling induced both migration and proliferation of SH-EP cells. Migration and proliferation/cell-survival were inhibited by PHA665752 in a dose-dependent manner. We also found that induced overexpression of PTEN following treatment with rosiglitazone significantly enhanced the inhibitory effect of PHA665752 on NBL-cell migration and proliferation.Conclusionc-Met is highly expressed in most tumors from patients with advanced-stage, metastatic NBL. Furthermore, using the NBL cell line SH-EP as a model, PHA665752 was shown to inhibit cMet/HGF/SF signaling in vitro, suggesting c-Met inhibitors may have efficacy for blocking local progression and/or metastatic spread of c-Met-positive NBL in vivo. These are novel findings for this disease and suggest that further studies of agents targeting the c-Met/HGF axis in NBL are warranted

Effective adjuvant chemotherapy for advanced nasopharyngeal carcinoma in children : A final update of a long-term prospective study in a single institution

Purpose: The purpose of this study was to determine the efficacy and toxicity of a doxorubicin/eyclophosphamide-based chemotherapy and local radiation therapy in children with locally advanced or metaslatic nasopharyngeal carcinoma (NPC). Patients and Methods: Twelve patients aged 6 to 20 years old were treated with a chemotherapy regimen comprised of vincristine (1.5 mg/m2) and doxorubicin (45 mg/m2) on day 1 and cyclophosphamide (210 mg/m2) and 5-fluorouracil (240 mg/m2) on days 1 to 5. Chemotherapy was administered every 3 weeks for 1 to 2 years. Radiotherapy to the primary site (59 to 68 Gy) and to the neck (59 to 66 Gy) was given before or after 2 to 4 courses of chemotherapy. Results: All patients achieved a complete response 4 to 16 months from the start of therapy (median 7 months). Nine patients have remained tumor free from 2 to 21 years (median 11 years) from diagnosis. One child was lost to follow-up and one died of tuberculosis; both were disease-free. One child developed a secondary osteosarcoma in the left mandible. Chemotherapy caused grade 4 neutropenia and thrombocytopenia in four patients. There were no therapy-related deaths and the most common late effect of therapy was neck fibrosis, which was observed in all patients. We conclude that the chemotherapy and radiotherapy regimen used in this study is highly effective for children and adolescents with locally advanced NPC and is associated with tolerable toxicity.

Sequential testicular biopsies in childhood acute lymphocytic leukemia.

Between August 1978 and November 1981, 33 boys with acute lymphocytic leukemia (ALL) (26 non‐T, 7 T‐cell) younger than 16 years underwent bilateral wedge testicular biopsies at the time of initial diagnosis. Seven (4 non‐T, 3 T‐cell) demonstrated focal leukemic infiltrates. Rebiopsy after successful induction therapy without testicular irradiation showed eradication of leukemic infiltrates in five, persistent focal infiltrates in one, and a diffuse infiltrate in one. The two patients who had persistent leukemic involvement had a T‐cell phenotype, and in one of them overt testicular disease developed 2 years later. All 33 patients were followed prospectively for a minimum of 3 years. Fifteen (14 non‐T, 1 T‐cell) remained in remission for 3 years and underwent another testicular biopsy before the cessation of therapy. Two patients, both non‐T and both of whom were free of testicular involvement at diagnosis, showed testicular infiltrates at that time. Of the seven boys with positive specimens at diagnosis, only two remained disease‐free for 3 years and showed no testicular involvement upon the completion of chemotherapy. In this study, microscopic testicular involvement by lymphoblasts occurred in 21% of newly diagnosed boys with ALL; this occurred only if the leukocyte count exceeded 25,000/μ1. These patients in general had a poor prognosis, probably reflecting the overall heavy tumor burden. However, it was not possible to predict accurately those patients who would have leukemic testicular infiltrates at the cessation of chemotherapy by performing biopsy of the testes at the time of initial diagnosis or after induction therapy.

“Hamartoma” of the Spleen (Splenoma) in Children

Hamartomas of the spleen or splenomas, are uncommon benign tumorous growths in this organ which have not been well characterized in children. We report four patients, 4 to 11 years old, who had splenomegaly and splenic “hamartomas” associated with different hematologic conditions (refractory microcytic anemia, sickle cell anemia, hereditary spherocytosis, and dyserythropoietic hemolytic anemia). All patients had total splenectomy as a primary therapeutic approach or to lessen their transfusion requirements. In only one patient was a focal splenic mass identified preoperatively with contrasted computed tomography (CT) scans and magnetic resonance imaging (MRI). None of the patients showed a mass by ultrasonography. Gross examination showed enlarged spleens (315–724 g) which on cut surface revealed a single nodule in one and multiple bulging nodules in three specimens. The nodules varied from 1.3 to 7 cm and were indistinct from the surrounding nonlymphoid splenic (i.e., red pulp) parenchyma. Histology of the nodules showed red splenic pulp with variable histiocytic proliferation, focal extramedullary hematopoiesis, lympho-plasmacytosis, fibrosis, and siderotic-calcific deposits. Intranodular small T- and B-cell lymphoid aggregates but no organized secondary follicles or periarteriolar sheaths were seen. Proliferation antigen Ki-67 (Mib-1) immunostains showed a low (< 5%) proliferation index in the nodules and surrounding tissue. Reticulin stains did not show a capsule or border between the normal spleen and the nodules. The critical histologic differential diagnosis for these lesions is with benign vascular tumors. These can be identified by their more disorderly pattern, by immunohistochemistry and by their higher proliferation index. It is our contention that these splenic nodules are not true hamartomas, as they seem to result from remote ischemic or infectious/inflammatory insults, leading to the fibro-inflammatory reaction and deposition of calcium and hemosiderin that is better designated with the descriptive term of splenoma. Review of the literature and our own experience indicates that most children with splenic hamartomas or splenomas as we prefer to call them, have an underlying hematologic disorder likely made worse by a state of hypersplenism that explains the consistent improvement in the blood values after splenectomy.

Use of VP‐16–213 in the treatment of familial erythrophagocytic lymphohistiocytosis

Five patients with familial erythrophagocytic lymphohistiocytosis (FEL), aged 6 weeks to 3 years, were treated with VP‐16–213. The drug dosage ranged from 100 to 200 mg/m2 administered biweekly until remission was achieved, and then at 1‐to 3‐week intervals as maintenance therapy. Intrathecal methotrexate was given to two patients with central nervous system involvement. All patients attained remission. Systemic relapses often ensued in all patients when VP‐16–213 was delayed because of myelosuppression, or after attempts to lengthen the treatment interval, but they initially responded again to a more intensive chemotherapy schedule. To date, four of the patients died from disseminated disease and terminal infections 15 to 20 months from the time of diagnosis. One child is alive and well 20 months from diagnosis. Three of the dead children had become refractory to the drug. Our observations show that VP‐16–213 induces remissions and prolongs survival in FEL. However, since the patients eventually become refractory to the drug and die of the disease, additional forms of therapy are required to improve the outlook of affected children.

Tissue factor/FVIIa activates Bcl-2 and prevents doxorubicin-induced apoptosis in neuroblastoma cells

BackgroundTissue factor (TF) is a transmembrane protein that acts as a receptor for activated coagulation factor VII (FVIIa), initiating the coagulation cascade. Recent studies demonstrate that expression of tumor-derived TF also mediates intracellular signaling relevant to tumor growth and apoptosis. Our present study investigates the possible mechanism by which the interaction between TF and FVIIa regulates chemotherapy resistance in neuroblastoma cell lines.MethodsGene and siRNA transfection was used to enforce TF expression in a TF-negative neuroblastoma cell line and to silence endogenous TF expression in a TF-overexpressing neuroblastoma line, respectively. The expression of TF, Bcl-2, STAT5, and Akt as well as the phosphorylation of STAT5 and Akt in gene transfected cells or cells treated with JAK inhibitor and LY294002 were determined by Western blot assay. Tumor cell growth was determined by a clonogenic assay. Cytotoxic and apoptotic effect of doxorubicin on neuroblastoma cell lines was analyzed by WST assay and annexin-V staining (by flow cytometry) respectively.ResultsEnforced expression of TF in a TF-negative neuroblastoma cell line in the presence of FVIIa induced upregulation of Bcl-2, leading to resistance to doxorubicin. Conversely, inhibition of endogenous TF expression in a TF-overexpressing neuroblastoma cell line using siRNA resulted in down-regulation of Bcl-2 and sensitization to doxorubicin-induced apoptosis. Additionally, neuroblastoma cells expressing high levels of either endogenous or transfected TF treated with FVIIa readily phosphorylated STAT5 and Akt. Using selective pharmacologic inhibitors, we demonstrated that JAK inhibitor I, but not the PI3K inhibitor LY294002, blocked the TF/FVIIa-induced upregulation of Bcl-2.ConclusionThis study shows that in neuroblastoma cell lines overexpressed TF ligated with FVIIa produced upregulation of Bcl-2 expression through the JAK/STAT5 signaling pathway, resulting in resistance to apoptosis. We surmise that this TF-FVIIa pathway may contribute, at least in part, to chemotherapy resistance in neuroblastoma.

Expression and functional role of inhibitor-of-apoptosis protein livin (BIRC7) in neuroblastoma

We evaluated the expression of the inhibitor-of-apoptosis protein (IAP)livin (BIRC7)in 59 cases ofneuroblastoma (NBL) by quantitative RT-PCR. We also examined the role of livin in protecting tumor cells from chemotherapy drugs. Livin expression varied significantly amongtumors. High levels of expression were observed in 17 of 39 patients with advanced stages (stages 3 and 4) and 6 of 20 patients with localized stages (stages 1 and 2). Livin-transfected, MYCN-amplified NBL cells showed increased resistance to doxorubicin and etoposide. Conversely, livin knockdown with siRNA enhanced spontaneous and drug-induced apoptosis in NBL cells. Multivariate analysis of prognostic factors showed that high livin expression worsened prognosis for patients with MYCN-amplified tumors. Our data suggest that (i) livin is frequently expressed in NBL and protects tumor cells with amplified MYCN oncogene from genotoxic agents; (ii) the antiapoptotic effect of livin in NBL is blocked by siRNA; (iii) in the sample studied, high livin expression enhanced the adverse prognostic impact of MYCN amplification. These findings suggest that livin may contribute to drug resistance in NBL.