Expression and role of TYRO3 and AXL as potential therapeutical targets in leiomyosarcoma.

BACKGROUND: Leiomyosarcoma (LMS) are 15% of adult sarcomas and remain seldom curable in metastatic phase. The TAM receptors and their ligands are overexpressed or activated in multiple malignancies, including LMS. METHODS: The TAM receptor and ligand expression was evaluated in LMS cell lines and 358 sarcoma samples by either gene expression or immunohistochemistry. TYRO3 and AXL were knocked down. Crizotinib and foretinib were investigated in vitro. RESULTS: High expression of TYRO3 and AXL was detected in LMS cell lines. TYRO3 or AXL gene knockdown reduced cell proliferation/colony formation. Crizotinib and foretinib decreased TYRO3 and AXL phosphorylation, apoptosis, G2/arrest and reduced colony formation. Immunohistochemistry performed in 107 sarcomas showed higher expression of TYRO3 and GAS6 in LMS vs other sarcomas and nuclear TYRO3 only in LMS. Microarray gene expression performed in 251 sarcomas revealed significantly higher expression of TYRO3 and GAS6 in LMS than other sarcomas. Leiomyosarcoma patients with high expression of GAS6 or PROS1 present a significantly worse PFS. CONCLUSIONS: Leiomyosarcoma patients, especially those whom develop metastasis, express higher levels of TYRO3 and GAS6. Crizotinib and foretinib showed effective antitumour activity in LMS through TYRO3 and AXL deactivation indicating that clinical trials using TYRO3 and AXL inhibitors are warranted in advanced LMS.

Inhibition of the NOTCH pathway using γ-secretase inhibitor RO4929097 has limited antitumor activity in established glial tumors.

Notch signaling is altered in many cancers. Our previous findings in primary pediatric ependymoma support a role for NOTCH in glial oncogenesis. The present study evaluates the γ-secretase inhibitor RO4929097 in glial tumor models. The expression of Notch pathway genes was evaluated using real-time RT-PCR in 21 ependymoma and glioma models. NOTCH1 mutations were analyzed by DNA sequencing. RO4929097 activity was evaluated in vitro and in vivo, as a single agent and in combination, in glioma and ependymoma models. Notch pathway genes are overexpressed in ependymomas and gliomas along with FBXW7 downregulation. NOTCH1 mutations in the TAD domain were observed in 20% (2/10) of ependymoma primary cultures. Blocking the Notch pathway with the γ-secretase inhibitor RO4929097 reduced cell density and viability in ependymoma short-term cultures. When combined with chemotherapeutic agents, RO4929097 enhanced temozolomide effects in ependymoma short-term cultures and potentiated the cytotoxicity of etoposide, cisplatinum, and temozolomide in glioma cells. RO4929097, in combined treatment with mTOR inhibition, potentiated cytotoxicity in vitro, but did not enhance antitumor effects in vivo. In contrast, RO4929097 enhanced irradiation effects in glioma and ependymoma xenografts and showed tumor growth inhibition in advanced-stage IGRG121 glioblastoma xenografts. RO4929097-mediated effects were independent of NOTCH1 mutation status or expression levels, but associated with low IL-6 levels. In established glial tumor models, NOTCH inhibition had limited effects as a single agent, but enhanced efficacy when combined with DNA-interfering agents. These preclinical data need to be considered for further clinical development of NOTCH inhibitors in glial tumors.

Antibody phage display libraries: contributions to oncology.

Since the advent of phage display technology, dating back to 1985, antibody libraries displayed on filamentous phage surfaces have been used to identify specific binders for many different purposes, including the recognition of tumors. Phage display represents a high-throughput technique for screening billions of random fusion antibodies against virtually any target on the surface or inside cancer cells, or even soluble markers found in patient serum. Many phage display derived binders targeting important tumor markers have been identified. Selection directed to tumoral cells' surfaces lead to the identification of unknown tumoral markers. Also the improvement of methods that require smaller amounts of cells has opened the possibility to use this approach on patient samples. Robust techniques combining an antibody library displayed on the phage surface and protein microarray allowed the identification of auto antibodies recognized by patient sera. Many Ab molecules directly or indirectly targeting angiogenesis have been identified, and one of them, ramucirumab, has been tested in 27 phase I-III clinical trials in a broad array of cancers. Examples of such antibodies will be discussed here with emphasis on those used as probes for molecular imaging and other clinical trials.

Astrocytes reverted to a neural progenitor-like state with transforming growth factor alpha are sensitized to cancerous transformation.

Gliomas, the most frequent primitive central nervous system tumors, have been suggested to originate from astrocytes or from neural progenitors/stem cells. However, the precise identity of the cells at the origin of gliomas remains a matter of debate because no pre-neoplastic state has been yet identified. Transforming growth factor (TGF)-alpha, an epidermal growth factor family member, is frequently overexpressed in the early stages of glioma progression. We previously demonstrated that prolonged exposure of astrocytes to TGF-alpha is sufficient to trigger their reversion to a neural progenitor-like state. To determine whether TGF-alpha dedifferentiating effects are associated with cancerous transforming effects, we grafted intracerebrally dedifferentiated astrocytes. We show that these cells had the same cytogenomic profile as astrocytes, survived in vivo, and did not give birth to tumors. When astrocytes dedifferentiated with TGF-alpha were submitted to oncogenic stress using gamma irradiation, they acquired cancerous properties: they were immortalized, showed cytogenomic abnormalities, and formed high-grade glioma-like tumors after brain grafting. In contrast, irradiation did not modify the lifespan of astrocytes cultivated in serum-free medium. Addition of TGF-alpha after irradiation did not promote their transformation but decreased their lifespan. These results demonstrate that reversion of mature astrocytes to an embryonic state without genomic manipulation is sufficient to sensitize them to oncogenic stress.

Isolation of osteosarcoma-associated human antibodies from a combinatorial Fab phage display library.

Osteosarcoma, a highly malignant disease, is the most common primary bone tumor and is frequently found in children and adolescents. In order to isolate antibodies against osteosarcoma antigens, a combinatorial osteosarcoma Fab library displayed on the surface of phages was used. After three rounds of selection on the surface of tumor cells, several osteosarcoma-reactive Fabs were detected. From these Fabs, five were better characterized, and despite having differences in their VH (heavy chain variable domain) and Vkappa (kappa chain variable domain) regions, they all bound to a protein with the same molecular mass. Further analysis by cell ELISA and immunocytochemistry suggested that the Fabs recognize a membrane-associated tumor antigen expressed in higher amounts in neoplasic cells than in normal tissue. These results suggest that the human Fabs selected in this work are a valuable tool for the study of this neoplasia.

High yield bacterial expression and purification of active recombinant PA28alphabeta complex.

The PA28 complexes (also termed REG or 11S complexes) are described as activators of the 20S proteasome, a major intracellular protease in eukaryotic cells. They bind to the ends of the barrel-shaped 20S proteasome, and activate its peptidase activities. The interferon gamma inducible PA28alphabeta, made of the two related subunits PA28alpha and beta, is under sustained investigation as it plays important roles in the production by the proteasome of class I antigen peptides. However, in vitro studies of this complex have been impaired by the difficulty of producing large amount of this protein, mainly due to the poor solubility of its beta subunit when expressed in Escherichia coli. Here we describe the construction of a bicistronic vector, allowing simultaneous production of functional human PA28alpha and beta subunits in E. coli. Co-expression of the two proteins allows efficient formation of active PA28alphabeta complexes, that remain soluble and can be easily purified by regular chromatographic procedures.

Construction of a human Fab phage display library from antibody repertoires of osteosarcoma patients.

Osteosarcoma is the commonest type of primary malignant bone tumor, frequently found in adolescents at sites of rapid bone growth. Despite current management protocols, up to half of the patients succumb to this disease. Moreover, there is no well-characterized molecular marker for diagnosis and prognosis. Since phage display methodology allows the selection of human antibody fragments with potential use in clinical applications, we applied this procedure to construct a recombinant Fab (antigen binding fragment) library from patients with osteosarcoma. We used peripheral blood lymphocyte total RNA from 11 osteosarcoma patients and cloned recombinant Fab representing the micro, gamma and kappa chain antibody repertoires of these individuals. The resulting library was cloned in the pComb3X vector and attained 1.45 x 10(8) different functional forms. BstO I fingerprinting and DNA sequencing analysis of randomly selected clones revealed the diversity of the library, demonstrating that Fab harbors Vkappa chains from subgroups I to V, biased towards the A27 fragment, as normally reported for the human repertoire. Analysis of the VH repertoire revealed that our library has a slight bias towards the VH4 family, instead of the usually reported VH3. This is the first description of a phage display library from osteosarcoma patients. We believe these human Fab fragments will provide a valuable tool for the study of this neoplasia and could also contribute to improvements in the diagnosis of this disease.

Unusual early-onset Huntingtons disease.

Huntington's disease is an autosomal dominant progressive neurodegenerative disorder characterized by involuntary movements, cognitive decline, and behavioral disorders leading to functional disability. In contrast to patients with adult onset, in which chorea is the major motor abnormality, children often present with spasticity, rigidity, and significant intellectual decline associated with a more rapidly progressive course. An unusual early-onset Huntington's disease case of an 11-year-old boy with severe hypokinetic/rigid syndrome appearing at the age of 2.5 years is presented. Clinical diagnosis was confirmed by polymerase chain reaction study of the expanded IT-15 allele with a compatible size of 102 cytosine-adenosine-guanosine repeats L-Dopa mildly ameliorated rigidity, bradykinesia, and dystonia. We conclude that Huntington's disease should be included in the differential diagnoses of regressive syndromes of early childhood.

Neuronal differentiation distinguishes supratentorial and infratentorial childhood ependymomas.

Ependymomas are glial neoplasms occurring in any location throughout the central nervous system and supposedly are derived from radial glia cells. Recent data suggest that these tumors may have different biological and clinical behaviors according to their location. Pediatric supratentorial and infratentorial ependymoma (SE and IE) were compared with respect to clinical and radiological parameters and immunohistochemistry (IHC). Neuronal markers were specifically assessed by IHC and quantitative PCR (qPCR). No single morphological or radiological characteristic was associated with location or any neuronal marker. However, there was a significant overexpression of neuronal markers in SE compared with IE: neurofilament light polypeptide 70 (NEFL)-positive tumor cells were found in 23 of 34 SE and in only 4 of 32 IE (P < .001). Among SE, 10 of 34 exhibited high expression of NEFL, defined as more than 5% positive cells. qPCR confirmed the upregulation of neuronal markers (NEFL, LHX2, FOXG1, TLX1, and NPTXR) in SE compared with IE. In addition, strong NEFL expression in SE was correlated with better progression-free survival (P = .007). Our results support the distinction of SE and IE. SEs are characterized by neuronal differentiation, which seems to be associated with better prognosis.

Portrait of ependymoma recurrence in children: biomarkers of tumor progression identified by dual-color microarray-based gene expression analysis.

BACKGROUND: Children with ependymoma may experience a relapse in up to 50% of cases depending on the extent of resection. Key biological events associated with recurrence are unknown. METHODOLOGY/PRINCIPAL FINDINGS: To discover the biology behind the recurrence of ependymomas, we performed CGHarray and a dual-color gene expression microarray analysis of 17 tumors at diagnosis co-hybridized with the corresponding 27 first or subsequent relapses from the same patient. As treatment and location had only limited influence on specific gene expression changes at relapse, we established a common signature for relapse. Eighty-seven genes showed an absolute fold change ≥2 in at least 50% of relapses and were defined as the gene expression signature of ependymoma recurrence. The most frequently upregulated genes are involved in the kinetochore (ASPM, KIF11) or in neural development (CD133, Wnt and Notch pathways). Metallothionein (MT) genes were downregulated in up to 80% of the recurrences. Quantitative PCR for ASPM, KIF11 and MT3 plus immunohistochemistry for ASPM and MT3 confirmed the microarray results. Immunohistochemistry on an independent series of 24 tumor pairs at diagnosis and at relapse confirmed the decrease of MT3 expression at recurrence in 17/24 tumor pairs (p = 0.002). Conversely, ASPM expression was more frequently positive at relapse (87.5% vs 37.5%, p = 0.03). Loss or deletion of the MT genes cluster was never observed at relapse. Promoter sequencing after bisulfite treatment of DNA from primary tumors and recurrences as well as treatment of short-term ependymoma cells cultures with a demethylating agent showed that methylation was not involved in MT3 downregulation. However, in vitro treatment with a histone deacetylase inhibitor or zinc restored MT3 expression. CONCLUSIONS/SIGNIFICANCE: The most frequent molecular events associated with ependymoma recurrence were over-expression of kinetochore proteins and down-regulation of metallothioneins. Metallothionein-3 expression is epigenetically controlled and can be restored in vitro by histone deacetylase inhibitors.

Candidate genes on chromosome 9q33-34 involved in the progression of childhood ependymomas.

PURPOSE: The molecular pathogenesis of pediatric ependymoma remains unclear. Our study was designed to identify genetic changes implicated in ependymoma progression. PATIENTS AND METHODS: We characterized 59 ependymoma samples (33 at diagnosis and 26 at relapse) using array-comparative genomic hybridization (aCGH). Specific chromosomal imbalances were confirmed by fluorescent in situ hybridization, and candidate genes were assessed by real-time quantitative polymerase chain reaction (qPCR), immunohistochemistry, sequencing, and in vitro functional studies. RESULTS: aCGH analysis revealed a significant increase in genomic imbalances on relapse compared with diagnosis, such as gain of 9qter and 1q (54% v 21% and 12% v 0%, respectively) and loss of 6q (27% v 6%). Supervised tumor classification showed that gain of 9qter was associated with tumor recurrence, age older than 3 years, and posterior fossa location. Using a candidate-gene strategy, we found an overexpression of two potential oncogenes at the locus 9qter: Tenascin-C and Notch1. Moreover, Notch pathway analysis (qPCR) revealed overexpression of Notch ligands, receptors, and target genes (Hes-1, Hey2, and c-Myc), and downregulation of Notch repressor Fbxw7. We confirmed by immunohistochemistry the overexpression of Tenascin-C and Hes-1. We detected Notch1 missense mutations in 8.3% of the tumors (only in the posterior fossa location and in case of 9q33-34 gain). Furthermore, inhibition of Notch pathway with a gamma-secretase inhibitor impaired the growth of ependymoma stem cell cultures. CONCLUSION: The activation of the Notch pathway and Tenascin-C seem to be important events in ependymoma progression and may represent future targets for therapy. We report, to our knowledge for the first time, recurrent oncogenic mutations in pediatric posterior fossa ependymomas.