Prognostic Relevance of KITand PDGFRAMutations in Gastrointestinal Stromal Tumors

Submitted by Sandra Infurna ([email protected]) on 2018-12-08T19:52:19Z No. of bitstreams: 1 alines_moreira_etal_IOC_2010.pdf: 211669 bytes, checksum: 328237a1369059808b2fce6b45e30253 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2018-12-08T20:04:17Z (GMT) No. of bitstreams: 1 alines_moreira_etal_IOC_2010.pdf: 211669 bytes, checksum: 328237a1369059808b2fce6b45e30253 (MD5)Made available in DSpace on 2018-12-08T20:04:17Z (GMT). No. of bitstreams: 1 alines_moreira_etal_IOC_2010.pdf: 211669 bytes, checksum: 328237a1369059808b2fce6b45e30253 (MD5) Previous issue date: 2010Instituto Nacional de Câncer. Centro de Transplante de Medula Óssea. Rio de Janeiro, RJ, Brasil.Instituto Nacional de Câncer. Divisão de Pesquisa Clínica. Rio de Janeiro, RJ, Brasil.Instituto Nacional de Câncer. Divisão de Pesquisa Clínica. Rio de Janeiro, RJ, Brasil.Consultoria em Patologia. Botucatu, SP, Brasil.Instituto Nacional de Câncer. Serviço de Cirurgia Abdominal-pélvica. Rio de Janeiro, RJ, Brasil.Instituto Nacional de Câncer. Centro de Transplante de Medula Óssea. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Genômica Funcional de Bioinformática. Rio de Janeiro, RJ, Brasil.Instituto Nacional de Câncer. Serviço de Cirurgia Abdominal-pélvica. Rio de Janeiro, RJ, Brasil.Instituto Nacional de Câncer. Divisão de Patologia. Rio de Janeiro, RJ, Brasil.Consultoria em Patologia. Botucatu, SP, Brasil.Instituto Nacional de Câncer. Centro de Transplante de Medula Óssea. Rio de Janeiro, RJ, Brasil.Instituto Nacional de Câncer. Divisão de Pesquisa Clínica. Rio de Janeiro, RJ, Brasil.Instituto Nacional de Câncer. Centro de Transplante de Medula Óssea. Rio de Janeiro, RJ, Brasil.Background: Prediction of biological behavior is crucial for selection of new therapeutic modalities in GIST. Here, we aimed to assess whether KIT and PDGFRA mutations have survival impact in gastrointestinal stromal tumors (GIST). Patients and Methods: Fifty-five Brazilian patients with completely resected GIST were examined for KIT and PDGFRA mutations. The 5-year disease-free survival (DFS) was analyzed. Results: KIT and PDGFRA mutations were identified in 74.5% and 7.3% of patients, respectively. The 5-year DFS rate for all patients was 52.8%. The 5-year DFS rate was lower in patients with tumors having inframe deletions or concomitant in-frame deletions and insertions affecting codons 557-558 than in patients with tumors having other exon 11 KIT mutations (p=0.023). Conversely, when the patients with concomitant deletioninsertion mutations affecting codons 557-558 were excluded from the analysis, deletions involving codons 557-558 had no influence on 5-year DFS rates. Conclusion: Our findings indicate that a specific KIT mutation may be associated with unfavorable behavior in GIST. This finding may have implications on selecting patients for adjuvant therapy

HDAC8, A Potential Therapeutic Target for the Treatment of Malignant Peripheral Nerve Sheath Tumors (MPNST).

HDAC isoform-specific inhibitors may improve the therapeutic window while limiting toxicities. Developing inhibitors against class I isoforms poses difficulties as they share high homology among their catalytic sites; however, HDAC8 is structurally unique compared to other class I isoforms. HDAC8 inhibitors are novel compounds and have affinity for class I HDAC isoforms demonstrating anti-cancer effects; little is known about their activity in malignant peripheral nerve sheath tumors (MPNST). Recently, we demonstrated anti-MPNST efficacy of HDAC8i in human and murine-derived MPNST pre-clinical models; we now seek to consider the potential therapeutic inhibition of HDAC8 in MPNST.Four Human MPNST cell lines, a murine-derived MPNST cell line, and two HDAC8 inhibitors (PCI-34051, PCI-48012; Pharmacyclics, Inc. Sunnyvale, CA) were studied. Proliferation was determined using MTS and clonogenic assays. Effects on cell cycle were determined via PI FACS analysis; effects on apoptosis were determined using Annexin V-PI FACS analysis and cleaved caspase 3 expression. In vivo growth effects of HDAC8i were evaluated using MPNST xenograft models. 2D gel electrophoresis and mass spectrometry were used to identify potential HDAC8 deacetylation substrates.HDAC8i induced cell growth inhibition and marked S-phase cell cycle arrest in human and murine-derived MPNST cells. Relative to control, HDAC8i induced apoptosis in both human and murine-derived MPNST cells. HDAC8i exhibited significant effects on MPNST xenograft growth (p=0.001) and tumor weight (p=0.02). Four potential HDAC8 substrate targets were identified using a proteomic approach: PARK7, HMGB1, PGAM1, PRDX6.MPNST is an aggressive sarcoma that is notoriously therapy-resistant, hence the urgent need for improved anti-MPNST therapies. HDAC8 inhibition may be useful for MPNST by improving efficacy while limiting toxicities as compared to pan-HDACis

Summary of 2D comparison and MS analysis of S462 treated with DMSO and PCI3.

<p>¹ Corresponding gel location/spot in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133302#pone.0133302.g004" target="_blank">Fig 4</a></p><p>² PCI3 vs DMSO difference. Differences are calculated from spot percentages.</p><p>Summary of 2D comparison and MS analysis of S462 treated with DMSO and PCI3.</p

Summary of 2D comparison and MS analysis of S462 treated with DMSO and PCI3.

<p>¹ Corresponding gel location/spot in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133302#pone.0133302.g004" target="_blank">Fig 4</a></p><p>² PCI3 vs DMSO difference. Differences are calculated from spot percentages.</p><p>Summary of 2D comparison and MS analysis of S462 treated with DMSO and PCI3.</p

Potential HDAC8 acetylation targets.

<p>Proteomics analysis of potential HDAC8 acetylation substrates. Differences in spots (pan-acetyl lysine) in S462 cells treated with DMSO or PCI3 (5μM/24h) were analyzed. Several spots were then analyzed via MS yielding potential HDAC8 acetylation targets: PRDX6, PGAM1, HMGB1, PARK7/DJ-1.</p

Involvement of signaling molecules in the prediction of response to imatinib treatment in metastatic GIST patients

Imatinib therapy has undoubtedly contributed to the treatment of metastatic gastrointestinal stromal (GIST) tumors that were previously untreatable. However, disease progression during treatment with tyrosine kinase inhibitors remains an issue in clinical practice not fully explained by KIT and PDGFRA mutation status. We investigated the role of three important signaling molecules (insulin-like growth factor 1 receptor [IGF1R], protein kinase C-? [PKC?], and Raf kinase inhibitor protein [RKIP]) that have been implicated in GIST pathogenesis as potential biomarkers for prediction of response to imatinib treatment. We retrospectively reviewed 76 patients with metastatic GIST submitted to imatinib treatment between 2002 and 2007, and analyzed 63 of them. Insulin-like growth factor 1, total PKC?, phosphorylated PKC?, and RKIP immunohistochemical expression were correlated with objective response to imatinib treatment and progression-free and overall survival. Median follow-up was 31.2 mo (95% confidence interval, 26.3-36.1 mo). There was a statistically significant association between IGF1R expression and type of response to imatinib treatment (P = 0.05)-that is, higher IGF1R expression was related to lower objective response. However, IGF1R higher expression did not affect progression-free and overall survival. Insulin-like growth factor 1, but not PKC? and RKIP, emerges as a potential biomarker for prediction of response to imatinib treatment in metastatic GISTs. Validation studies are warranted.This work was supported by an unrestricted grant from Novartis Brazil. Financial support was also provided by Fundacao do Cancer and Instituto Nacional de Cancer (INCA), Rio de Janeiro-Brazil

Effects of HDAC8 inhibition on MPNST cell cycle and apoptosis.

<p><b>A.</b> PI staining/FACS analyses showing the effect of HDACis (PCI2 0.5μM/48h, PCI3/4 5μM/48h) on human and murine-derived MPNST cell cycle progression. PCI2 reduced S-phase with a modest increase in G1 and G2 in S462 cells. STS26T exhibited a PCI2-induced G2 arrest and a decrease in S-phase. The effect of PCI2 on murine-derived MPNST cell cycle was modest. All 3 MPNST cell lines exhibited S-phase arrest when treated with either HDAC8i. Additionally, an increase in sub G1 population is observed (depicted in the respective histograms), suggesting cell death. <b>B.</b> HDAC8i (PCI3/4 5μM/96h) induced human MPNST cell apoptosis (Annexin V/PI staining FACS analyses). The response of these cell lines to HDAC8i-induced apoptosis was recapitulated and further confirmed via WB for cleaved caspase 3 (CC3). Due to the higher sensitivity toward HDAC8i compared to human MPNST cell lines, murine MPNST cells were treated for 48h. PCI2 (0.5μM/48h) and both HDAC8i (PCI3/4 5μM/48h) induced marked apoptosis in murine-derived MPNST cells (Annexin V/PI staining FACS analyses, CC3 WB). * p<0.05.</p

Effect of HDAC8 inhibition <i>in vivo</i>.

<p>SCID mice bearing MPNST6IEPVI xenografts were treated with PCI4 (20 mg/kg BID) or vehicle (10 mice/group). Tumor growth and weight curves are depicted showing that PCI4 abrogated the growth of MPNST6IEPVI tumors (p = 0.001 and 0.02 for tumor size and weight, respectively).</p

Mocetinostat combined with gemcitabine for the treatment of leiomyosarcoma: Preclinical correlates

<div><p>Leiomyosarcoma (LMS) is a malignant soft tissue sarcoma (STS) with a dismal prognosis following metastatic disease. Chemotherapeutic intervention has demonstrated to have modest clinical efficacy with no curative potential in LMS patients. Previously, we demonstrated pan-HDAC inhibition to have a superior effect in various complex karyotypic sarcomas. In this study, our goal is to evaluate the therapeutic efficacy of mocetinostat alone and in combination with gemcitabine in LMS. Human leiomyosarcoma (LMS) cell lines were used for <i>in vitro</i> and <i>in vivo</i> studies. Compounds tested included the class I HDAC inhibitor, mocetinostat, and nucleoside analog, gemcitabine. MTS and clonogenic assays were used to evaluate the effect of mocetinostat on LMS cell growth. Cleaved caspase 3/7 analysis was used to determine the effects of mocetinostat on apoptosis. Compusyn software was used to determine <i>in vitro</i> synergy studies for the combination of mocetinostat plus gemcitabine. A LMS xenograft model in SCID mice was used to test the impact of mocetinostat alone, gemcitabine alone and the combination of mocetinostat plus gemcitabine. Mocetinostat abrogated LMS cell growth and clonogenic potential, and enhanced apoptosis in LMS cell lines. The combination of mocetinostat plus gemcitabine exhibited a synergistic effect in LMS cells <i>in vitro</i>. Similarly, mocetinostat combined with gemcitabine resulted in superior anti-LMS effects <i>in vivo</i>. Mocetinostat reduced the expression of gemcitabine-resistance markers RRM1, RRM2, and increased the expression of gemcitabine-sensitivity marker, hENT1, in LMS cells. LMS are aggressive, metastatic tumors with poor prognosis where effective therapeutic interventions are wanting. Our studies demonstrate the potential utility of mocetinostat combined with gemcitabine for the treatment of LMS.</p></div

Mocetinostat regulates gemcitabine-resistant markers expression.

<p>Mocetinostat reduces RRM1 and RRM2, and increases hENT1 expression in LMS cell lines.</p