Showing papers by "Jonathan A. Fletcher published in 2012"

The clinicopathologic features of YWHAE-FAM22 endometrial stromal sarcomas: a histologically high-grade and clinically aggressive tumor.

Abstract: Endometrial stromal sarcoma (ESS) is a genetically heterogenous group of uterine sarcomas, of which almost half are associated with JAZF1 rearrangement. We recently identified a novel genetic fusion between YWHAE and FAM22A/B in ESS harboring t(10;17)(q22;p13) and herein describe the clinicopathologic features of 13 YWHAE-FAM22 ESS cases (11 primary and 3 metastatic) and compare them with 20 ESS cases with JAZF1 rearrangement. Ten of 11 primary uterine tumors contained morphologically high-grade areas composed of round cells arranged in nests with a delicate stromal capillary network. The tumor cells showed large nuclei with irregular nuclear contours and significant mitotic activity (>10 mitoses/10 HPF) in addition to focal tumor necrosis, in contrast to JAZF1 ESS, which lacked a nested growth pattern, were composed of cells with small round/oval nuclei, and typically had <5 MF/10 HPF. In 7 of the 11 uterine tumors, there was an additional cytologically bland and mitotically weakly active spindle cell component with a fibrous/fibromyxoid stroma (ESS, fibromyxoid variant). Two metastatic tumors (pulmonary) also contained round cell and spindle cell components, whereas 1 metastasis (vaginal) was composed solely of the spindle cell component. In both primary and metastatic tumors, the spindle cells were diffusely positive for estrogen and progesterone receptors and CD10, in contrast to the round cell areas, which were negative. Clinically, 10 of 12 patients with YWHAE-FAM22 ESS presented with FIGO stages II to III disease, in contrast to only 4 of 16 patients with JAZF1 ESS presenting with stages II to III disease (P<0.05). Tumors with YWHAE-FAM22 rearrangements constitute a distinct group of ESS, which is associated with high-grade morphology and aggressive clinical behavior compared to JAZF1 ESS. Thus, their distinction from typical JAZF1 ESS is important for prognostic and therapeutic purposes.

14-3-3 fusion oncogenes in high-grade endometrial stromal sarcoma

Abstract: 14-3-3 proteins are ubiquitously expressed regulators of various cellular functions, including proliferation, metabolism, and differentiation, and altered 14-3-3 expression is associated with development andprogressionof cancer. Wereport a transforming14-3-3 oncoprotein, which we identified through conventional cytogenetics and whole-transcriptome sequencing analysis as a highly recurrent genetic mechanism in a clinically aggressive form of uterine sarcoma: high-grade endometrial stromal sarcoma (ESS). The 14-3-3 oncoproteinresultsfromat(10;17) genomicrearrangement,leadingtofusion between 14-3-3e (YWHAE) and either of two nearly identical FAM22 family members (FAM22A or FAM22B). Expression of YWHAE– FAM22 fusion oncoproteins was demonstrated by immunoblot in t (10;17)-bearing frozen tumor and cell line samples. YWHAE–FAM22 fusion gene knockdowns were performed with shRNAs and siRNAs targeting various FAM22A exons in an t(10;17)-bearing ESS cell line (ESS1): Fusion protein expression was inhibited, with corresponding reduction in cell growth and migration. YWHAE–FAM22 maintains a structurally and functionally intact 14-3-3e (YWHAE) protein-binding domain, which is directed to the nucleus by a FAM22 nuclear localization sequence. In contrast to classic ESS, harboring JAZF1 genetic fusions, YWHAE–FAM22 ESS display high-grade histologic features, a distinct gene-expression profile, and a more aggressive clinical course. Fluorescence in situ hybridization analysis demonstrated absolute specificity of YWHAE–FAM22A/B genetic rearrangement for high-grade ESS, with no fusions detected in other uterine and nonuterine mesenchymal tumors (55 tumor types, n =8 27). These discoveries reveal diagnostically and therapeutically relevant models for characterizing aberrant 14-3-3 oncogenic functions. cytogenetic aberration | translocation | uterine neoplasm | NUT | leiomyosarcoma

Antibody therapy targeting the CD47 protein is effective in a model of aggressive metastatic leiomyosarcoma

Abstract: Antibodies against CD47, which block tumor cell CD47 interactions with macrophage signal regulatory protein-α, have been shown to decrease tumor size in hematological and epithelial tumor models by interfering with the protection from phagocytosis by macrophages that intact CD47 bestows upon tumor cells. Leiomyosarcoma (LMS) is a tumor of smooth muscle that can express varying levels of colony-stimulating factor-1 (CSF1), the expression of which correlates with the numbers of tumor-associated macrophages (TAMs) that are found in these tumors. We have previously shown that the presence of TAMs in LMS is associated with poor clinical outcome and the overall effect of TAMs in LMS therefore appears to be protumorigenic. However, the use of inhibitory antibodies against CD47 offers an opportunity to turn TAMs against LMS cells by allowing the phagocytic behavior of resident macrophages to predominate. Here we show that interference with CD47 increases phagocytosis of two human LMS cell lines, LMS04 and LMS05, in vitro. In addition, treatment of mice bearing subcutaneous LMS04 and LMS05 tumors with a novel, humanized anti-CD47 antibody resulted in significant reductions in tumor size. Mice bearing LMS04 tumors develop large numbers of lymph node and lung metastases. In a unique model for neoadjuvant treatment, mice were treated with anti-CD47 antibody starting 1 wk before resection of established primary tumors and subsequently showed a striking decrease in the size and number of metastases. These data suggest that treatment with anti-CD47 antibodies not only reduces primary tumor size but can also be used to inhibit the development of, or to eliminate, metastatic disease.

Efficacy and Safety of Regorafenib in Patients With Metastatic and/or Unresectable GI Stromal Tumor After Failure of Imatinib and Sunitinib: A Multicenter Phase II Trial

Abstract: Purpose Metastatic GI stromal tumor (GIST) is a life-threatening disease with no therapy of proven efficacy after failure of imatinib and sunitinib. Regorafenib is a structurally unique inhibitor of multiple cancer-associated kinases, including KIT and platelet-derived growth factor receptor (PDGFR), with broad-spectrum anticancer activity in preclinical and early-phase trials. Because KIT and PDGFR-α remain drivers of GIST after resistance to imatinib and sunitinib, we performed a multicenter single-stage phase II trial of regorafenib in patients with advanced GIST after failure of at least imatinib and sunitinib. Patients and Methods Patients received regorafenib orally, 160 mg daily, on days 1 to 21 of a 28-day cycle. Disease assessment was performed every two cycles per RECIST 1.1. Primary end point was clinical benefit rate (CBR), defined as objective responses (ie, complete or partial response [PR] as well as stable disease [SD] ≥ 16 weeks). Serial tumor biopsies were obtained from consenting patien...

Cyclin D1 as a diagnostic immunomarker for endometrial stromal sarcoma with YWHAE-FAM22 rearrangement

Abstract: Endometrial stromal sarcoma (ESS) characterized by YWHAE-FAM22 genetic fusion is histologically higher grade and clinically more aggressive than ESS with JAZF1-SUZ12 or equivalent genetic rearrangements, hence it is clinically important to recognize this subset of ESS. To identify diagnostic immunomarkers for this biologically defined ESS subset, we compared gene expression profiles between YWHAE-FAM22 ESS and JAZF1-rearranged ESS. These studies showed consistent upregulation of cyclin D1 in YWHAE-FAM22 ESS compared with JAZF1-SUZ12 ESS. Immunohistochemically, the high-grade round cell component of all 12 YWHAE-FAM22 ESS demonstrated diffuse (≥70%) moderate to strong nuclear cyclin D1 staining, and this diffuse positivity was not seen in 34 ESSs with JAZF1 and equivalent genetic rearrangements or in 21 low-grade ESS with no demonstrable genetic rearrangements. In a series of 243 non-ESS pure uterine mesenchymal and mixed epithelial-mesenchymal tumors, only 2 of 8 undifferentiated endometrial sarcomas with nuclear uniformity and 1 of 80 uterine leiomyosarcomas demonstrate diffuse cyclin D1 immunoreactivity. Both cyclin D1-positive undifferentiated endometrial sarcomas showed diffuse strong CD10 staining, which is consistently absent in the high-grade round cell component of YWHAE-FAM22 ESS. The low-grade spindle cell component of YWHAE-FAM22 ESS showed a spatially heterogenous cyclin D1 staining pattern that was weaker and less diffuse overall. Our findings indicate that cyclin D1 is a sensitive and specific diagnostic immunomarker for YWHAE-FAM22 ESS. When evaluating high-grade uterine sarcomas, cyclin D1 can be included in the immunohistochemical panel as an indicator of YWHAE-FAM22 ESS.

Characterization of the chromosomal translocation t(10;17)(q22;p13) in clear cell sarcoma of kidney

Abstract: Clear cell sarcoma of kidney (CCSK) is classified as a tumour of unfavourable histology by the National Wilms' Tumor Study Group. It has worse clinical outcomes than Wilms' tumour. Virtually nothing is known about CCSK biology, as there have been very few genetic aberrations identified to act as pointers in this cancer. Three cases of CCSK bearing a chromosomal translocation, t(10;17)(q22;p13), have been individually reported but not further investigated to date. The aim of this research was to characterize t(10;17)(q22;p13) in CCSK to identify the genes involved in the translocation breakpoints. Using fluorescently labelled bacterial artificial chromosomes (BACs) and a chromosome-walking strategy on an index case of CCSK with t(10;17)(q22;p13) by karyotype, we identified the chromosomal breakpoints on 17p13.3 and 10q22.3. The translocation results in rearrangement of YWHAE on chromosome 17 and FAM22 on chromosome 10, producing an in-frame fusion transcript of ∼3 kb, incorporating exons 1-5 of YWHAE and exons 2-7 of FAM22, as determined by RT-PCR using YWHAE- and FAM22-specific primers. The YWHAE-FAM22 transcript was detected in six of 50 further CCSKs tested, therefore showing an overall incidence of 12% in our cohort. No transcript-positive cases presented with stage I disease, despite this being the stage for 31% of our cohort. Tumour cellularity was significantly higher in the cases that were transcript-positive. Based on the chromosome 10 breakpoint identified by FISH and the sequences of the full-length transcripts obtained, the FAM22 members involved in the translocation in these CCSK cases include FAM22B and FAM22E. Elucidation of the role of YWHAE-FAM22 in CCSK will assist development of more efficient and targeted therapies for this childhood cancer, which currently has poor outcomes.

ROR2 is a novel prognostic biomarker and a potential therapeutic target in leiomyosarcoma and gastrointestinal stromal tumour

Abstract: Soft-tissue sarcomas are a group of malignant tumours whose clinical management is complicated by morphological heterogeneity, inadequate molecular markers and limited therapeutic options. Receptor tyrosine kinases (RTKs) have been shown to play important roles in cancer, both as therapeutic targets and as prognostic biomarkers. An initial screen of gene expression data for 48 RTKs in 148 sarcomas showed that ROR2 was expressed in a subset of leiomyosarcoma (LMS), gastrointestinal stromal tumour (GIST) and desmoid-type fibromatosis (DTF). This was further confirmed by immunohistochemistry (IHC) on 573 tissue samples from 59 sarcoma tumour types. Here we provide evidence that ROR2 expression plays a role in the invasive abilities of LMS and GIST cells in vitro. We also show that knockdown of ROR2 significantly reduces tumour mass in vivo using a xenotransplantation model of LMS. Lastly, we show that ROR2 expression, as measured by IHC, predicts poor clinical outcome in patients with LMS and GIST, although it was not independent of other clinico-pathological features in a multivariate analysis, and that ROR2 expression is maintained between primary tumours and their metastases. Together, these results show that ROR2 is a useful prognostic indicator in the clinical management of these soft-tissue sarcomas and may represent a novel therapeutic target.

Protein Kinase C Inhibitor AEB071 Targets Ocular Melanoma Harboring GNAQ Mutations via Effects on the PKC/Erk1/2 and PKC/NF-κB Pathways

Abstract: Somatic GNAQ mutations at codon 209 have been identified in approximately 50% of uveal melanomas (UM) and have been reported to be oncogenic through activating PLCβ-PKC-Erk1/2 pathways We hypothesized that PKC may provide new opportunities for therapeutic targeting of UM carrying GNAQ mutations To test this hypothesis, UM cells harboring wild type or mutant GNAQ were treated with the PKC inhibitor AEB071 (sotrastaurin) or infected with lentivirus expressing shRNAs targeting PKC isoforms Notably, AEB071 at low micromolar concentrations significantly inhibited the growth of UM cells harboring GNAQ mutations through induction of G1 arrest and apoptosis However, AEB071 had little effect on UM cells carrying wild type GNAQ AEB071-mediated cell inhibition in the GNAQ mutated UM was accompanied by inhibition of Erk1/2 phosphorylation, NF-κB, decreased expression of cyclin D1, survivin, Bcl-xL and XIAP, and increased expression of cyclin-dependent kinase inhibitor p27Kip1 AEB071 suppressed the expression of PKC α, β, δ, e and θ in GNAQ mutated UM cells Our findings from shRNA-mediated knockdown studies revealed that these PKC isoforms are functionally important for UM cells harboring GNAQ mutations Furthermore, inhibitors of Erk1/2 and NF-κB pathways reduced viability of UM cells Together, our findings demonstrate that AEB071 exerts antitumor action on UM cells carrying GNAQ mutations via targeting PKC/Erk1/2 and PKC/NF-κB pathways Targeted PKC inhibition with drugs such as AEB071 offers novel therapeutic potential for UM harboring GNAQ mutations

Sorafenib Inhibits Many Kinase Mutations Associated with Drug-Resistant Gastrointestinal Stromal Tumors

Abstract: Sorafenib has substantial clinical activity as third- or fourth-line treatment of imatinib- and sunitinib-resistant gastrointestinal stromal tumors (GIST). Because sorafenib targets both angiogenesis-related kinases (VEGFR) and the pathogenetic kinases found in GIST (KIT or PDGFRA), the molecular basis for sorafenib efficacy in this setting remains unknown. We sought to determine the spectrum of activity of sorafenib against different mutant kinases associated with drug-sensitive and drug-resistant GIST. We compared the activity of imatinib and sorafenib against transiently expressed mutant forms of KIT and PDGFRA, including various secondary mutations that have been identified in imatinib-resistant or sunitinib-resistant GISTs. We also examined these drugs against four GIST cell lines, three of which are imatinib resistant. In our in vitro studies, we determined that sorafenib inhibited imatinib-resistant mutations in exons encoding the ATP/drug-binding pocket and in exons encoding the activation loop, with the exception of substitutions at KIT codon D816 and PDGFRA codon 842. Notably our data indicate that sorafenib is more effective than imatinib or sunitinib for inhibiting the kinase activity of drug-resistant KIT mutants (as assessed by biochemical IC50). We hypothesize that a major determinant of the efficacy of sorafenib for treatment of advanced GIST is the activity of this agent against KIT or PDGFRA-mutant kinases. These results have implications for the further development of treatments for drug-resistant GIST. Mol Cancer Ther; 11(8); 1770–80. ©2012 AACR .

The protein kinase C inhibitor enzastaurin exhibits antitumor activity against uveal melanoma.

Abstract: GNAQ mutations at codon 209 have been recently identified in approximately 50% of uveal melanomas (UM) and are reported to be oncogenic through activating the MAPK/Erk1/2 pathway. Protein kinase C (PKC) is a component of signaling from GNAQ to Erk1/2. Inhibition of PKC might regulate GNAQ mutation-induced Erk1/2 activation, resulting in growth inhibition of UM cells carrying GNAQ mutations. UM cells carrying wild type or mutant GNAQ were treated with the PKC inhibitor enzastaurin. Effects on proliferation, apoptosis, and signaling events were evaluated. Enzastaurin downregulated the expression of several PKC isoforms including PKCβII PKCθ, PKCe and/or their phosphorylation in GNAQ mutated cells. Downregulation of these PKC isoforms in GNAQ mutated cells by shRNA resulted in reduced viability. Enzastaurin exhibited greater antiproliferative effect on GNAQ mutant cells than wild type cells through induction of G1 arrest and apoptosis. Enzastaurin-induced G1 arrest was associated with inhibition of Erk1/2 phosphorylation, downregulation of cyclin D1, and accumulation of cyclin dependent kinase inhibitor p27Kip1. Furthermore, enzastaurin reduced the expression of antiapoptotic Bcl-2 and survivin in GNAQ mutant cells. Inhibition of Erk1/2 phosphorylation with a MEK specific inhibitor enhanced the sensitivity of GNAQ wild type cells to enzastaurin, accompanied by p27Kip1 accumulation and/or inhibition of enzastaurin-induced survivin and Bcl-2 upregulation. PKC inhibitors such as enzastaurin have activity against UM cells carrying GNAQ mutations through inhibition of the PKC/Erk1/2 pathway and induction of G1 arrest and apoptosis. Inhibition of the PKC pathway provides a basis for clinical investigation in patients with UM.

Mechanism of association between BMI and dysfunction of the gastro-oesophageal barrier in patients with normal endoscopy

Abstract: Introduction The association between body mass index (BMI) and gastro-oesophageal pressure gradient (GOPG) is incompletely understood. We examined the association between BMI and gastro-oesophageal (GO) barrier function and the effect of mechanically increasing intra-abdominal pressure on GO physiology. Methods (A) 103 dyspeptic patients with normal endoscopy underwent 24 h pH-metry and upper gastrointestinal manometry. Relationships between BMI and acid reflux, intragastric pressure (IGP), GOPG and lower oesophageal sphincter (LOS) pressure were calculated using bivariate correlations. (B) In 18 healthy volunteers, the effects of increasing IGP by abdominal belt on GO manometry were studied. Results (A) There was a linear correlation between BMI and oesophageal acid exposure in erect (R=0.35, p Conclusion The association between reflux and BMI may be largely explained by effects of increased intra-abdominal pressure. However, the reduced LOS pressure associated with BMI may be mediated by another mechanism or effects of chronic rather than acute elevation of intra-abdominal pressure.

The HSP90 Inhibitor, AT13387, Is Effective against Imatinib-Sensitive and -Resistant Gastrointestinal Stromal Tumor Models

Abstract: The majority of gastrointestinal stromal tumors (GIST) are characterized by activating mutations of KIT, an HSP90 client protein. Further secondary resistance mutations within KIT limit clinical responses to tyrosine kinase inhibitors, such as imatinib. The dependence of KIT and its mutated forms on HSP90 suggests that HSP90 inhibition might be a valuable treatment option for GIST, which would be equally effective on imatinib-sensitive and -resistant clones. We investigated the activity of AT13387, a potent HSP90 inhibitor currently being evaluated in clinical trials, in both in vitro and in vivo GIST models. AT13387 inhibited the proliferation of imatinib-sensitive (GIST882, GIST-T1) and -resistant (GIST430, GIST48) cell lines, including those resistant to the geldanamycin analogue HSP90 inhibitor, 17-AAG. Treatment with AT13387 resulted in depletion of HSP90 client proteins, KIT and AKT, along with their phospho-forms in imatinib-sensitive and -resistant cell lines, irrespective of KIT mutation. KIT signaling was ablated, whereas HSP70, a marker of HSP90 inhibition, was induced. In vivo, antitumor activity of AT13387 was showed in both the imatinib-sensitive, GIST-PSW, xenograft model and a newly characterized imatinib-resistant, GIST430, xenograft model. Induction of HSP70, depletion of phospho-KIT and inhibition of KIT signaling were seen in tumors from both models after treatment with AT13387. A combination of imatinib and AT13387 treatment in the imatinib-resistant GIST430 model significantly enhanced tumor growth inhibition over either of the monotherapies. Importantly, the combination of AT13387 and imatinib was well tolerated. These results suggest AT13387 is an excellent candidate for clinical testing in GIST in combination with imatinib.

p53 Modulation as a Therapeutic Strategy in Gastrointestinal Stromal Tumors

Abstract: The KIT-inhibitor imatinib mesylate (IM) has greatly improved the treatment of metastatic gastrointestinal stromal tumors (GIST) IM exhibits strong antiproliferative effects but fails to induce sufficient levels of apoptosis resulting in low pathologic complete remission rates and a high rate of secondary progression in the metastatic setting Upregulation of p53 by MDM2 inhibitors has been shown to induce apoptosis in p53 wildtype tumors Analyzing a series of 62 mostly untreated, localized and metastatic GIST we detected a low rate (3%) of inactivating p53 mutations, thus providing a rationale for further exploration of p53-directed therapeutic strategies To this end, we studied nutlin-3, an inhibitor of the p53 antagonist MDM2, and RITA, a putative p53 activator, in GIST cell lines Nutlin-3 effectively induced p53 at therapeutically relevant levels, which resulted in moderate antiproliferative effects and cell cycle arrest in p53 wildtype GIST cell lines GIST430, GIST48 and GIST48B P53 reactivation substantially improved the apoptotic response after effective KIT inhibition with sunitinib and 17-AAG in IM-resistant cell lines The commonly used imatinib-sensitive cell lines GIST882 and GIST-T1 were shown to harbor defective p53 and therefore failed to respond to nutlin-3 treatment RITA induced p53 in GIST48B, followed by antiproliferative effects and a strong induction of apoptosis Surprisingly, GIST-T1 was also highly sensitive to RITA despite lacking functional p53 This suggested a more complex, p53-independent mechanism of action for the latter compound No antagonistic effects from p53-activating drugs were seen with any drug combination Our data provide first evidence that modulation of the MDM2/p53 pathway may be therapeutically useful to improve the apoptotic response of KIT-inhibitory drugs in the treatment of naive GIST, with p53 mutation status being a predictive factor of response

Integrating Bioinformatics and Clinicopathological Research of Gastrointestinal Stromal Tumors: Identification of Aurora Kinase A as a Poor Risk Marker

Abstract: For completely resected primary gastrointestinal stromal tumors (GISTs), mitotic rate, tumor size, and tumor location are important risk factors for recurrence. However, molecular markers for recurrence are still lacking. We reanalyzed GIST gene expression profile GSE8167 available from the Gene Expression Omnibus (GEO) and confirmed the prognostic influence of one selected gene, aurora kinase A (AURKA), in another cohort of 142 patients using immunohistochemistry (IHC). Thirty-two cases in GSE8167 were classified into two risk groups with distinct recurrence-free survival (RFS) and expression profiles using modified criteria of Miettinen et al. from the Armed Forces Institute of Pathology (AFIP-Miettinen). AURKA was among the 19 genes common to the top 50 features of the high-risk phenotype and a 67-gene signature called the complexity index in sarcomas. AURKA was significantly overexpressed in the high-risk group, and patients with high AURKA expression had significantly worse RFS than those with low expression. In the IHC-validated cohort, AURKA expression was significantly higher in nongastric tumors than in gastric tumors and was significantly correlated with AFIP-Miettinen risk group. Univariate analysis showed that RFS was significantly influenced by tumor size, mitotic count, AFIP-Miettinen risk group classification, and AURKA expression. However, only tumor size (P = 0.017), mitotic count (P = 0.007), and AURKA expression (P = 0.039) were identified as independent unfavorable prognostic factors for RFS in multivariate analysis. By integrating bioinformatics and clinicopathological studies, AURKA was identified as a marker for high-risk GIST.

Comparative Gene Expression Profiling of Benign and Malignant Lesions Reveals Candidate Therapeutic Compounds for Leiomyosarcoma

Abstract: Leiomyosarcoma (LMS) is a malignant, soft-tissue tumor for which few effective therapies exist. Previously, we showed that there are three molecular subtypes of LMS. Here, we analyzed genes differentially expressed in each of the three LMS subtypes as compared to benign leiomyomas and then used the Connectivity Map (cmap) to calculate enrichment scores for the 1309 cmap drugs in order to identify candidate molecules with the potential to induce a benign, leiomyoma-like phenotype in LMS cells. 11 drugs were selected and tested for their ability to inhibit the growth of three human LMS cell lines. We identified two drugs with in vitro efficacy against LMS, one of which had a strongly negative enrichment score (Cantharidin) and the other of which had a strongly positive enrichment score (MG-132). Given MG-132's strong inhibitory effect on LMS cell viability, we hypothesized that LMS cells may be sensitive to treatment with other proteasome inhibitors and demonstrated that bortezomib, a clinically-approved proteasome inhibitor not included in the original cmap screen, potently inhibited the viability of the LMS cell lines. These findings suggest that systematically linking LMS subtype-specific expression signatures with drug-associated expression profiles represents a promising approach for the identification of new drugs for LMS.

Methods and compositions for diagnosing and treating cancer

Abstract: The invention provides assays, methods, systems, compositions, and kits for diagnosing and treating cancer