- Email: [email protected]
185 Treatment of patient-derived NSCLC xenograft preclinical models using image-guided small animal irradiation
60
Wednesday 19 November 2014
Poster Session – Preclinical Models
BRAFV600E mutation levels correlate with tumor burden and suggest a possible role as a prognostic indicator. Studies are currently underway to examine RAS mutations in patient blood as a possible marker for acquired resistance to BRAF inhibitors, and to monitor disease progression in patients with BRAF wildtype tumors. 181 POSTER Aberrant Wnt signaling activation in human cancers: In vitro and in vivo models to facilitate Wnt targeted drug development 1
1
1
1
1
1
1
G. Liu , C. Dong , R. Zhang , L. Zhang , S. Qian , J. Cai , J. Zhang , J. Ning1 . 1 Crown Bioscience Inc., Molecular and Cellular Biology, Taicang, China Activation of Wnt signaling has emerged as one of the major oncogenic aberrations in human cancers, which has been demonstrated to play critical roles in the maintenance of the undifferentiated cancer stem/progenitor cell phenotype, as well as to directly stimulate the malignant growth of tumors. Mutations in the downstream signaling components, including APC, AXIN, and b-catenin, have been well described in several cancer types, and recent studies have further extended the Wnt activation mechanisms beyond these downstream mutations to upstream signaling molecules in this pathway. By screening primary cancer cells established from the patient derived xenograft models, we identified multiple cancer types that secrete Wnt stimulating ligands. We also successfully established primary cancer cell line harboring recurrent R-spondin fusion mutations. Inhibition of the activated Wnt signaling in these cancer cells results in reduced cancer cell growth, indicating the critical dependence of cancer cells on the autocrine Wnt signaling. Thus, these in vitro and in vivo models provide a valuable resource for the high throughput screening of Wnt antagonists, efficacy assessment of candidate Wnt inhibitors, biomarker analysis, as well as the preclinical development of Wnt targeted therapeutics. 182 POSTER TRAP1 represents a key mediator of stemness and glycolytic metabolism in colorectal cancer cells G. Lettini1 , F. Maddalena1 , L. Sisinni1 , V. Condelli1 , L. Del Vecchio2 , M. Gemei2 , T. Notarangelo1 , M. Landriscina3 . 1 CROB − IRCCS, Laboratory of Pre-Clinical and Translational Research, Rionero in Vulture (PZ), Italy; 2 CEINGE, Biotecnologie Avanzate, Napoli, Italy; 3 University of Foggia, Clinical Oncology Unit Department of Medical and Surgical Sciences, Foggia, Italy Background: Tumor cells undergo a metabolic shift from mitochondrial oxidative phosphorylation to a preferential glycolytic metabolism, known as Warburg effect. Furthermore, cancer stem cells (CSCs), known for being responsible for tumor initiation and growth, are located in a hypoxic microenvironment, heavily relying on anaerobic glycolysis. Recently, tumor necrosis factor receptor-associated protein 1 (TRAP1), a HSP90 mitochondrial molecular chaperone up-regulated in colorectal, prostate, breast and lung carcinomas, has been identified as a key regulator of tumor cell metabolism, being responsible for suppression of mitochondrial oxidative phosphorylation and post-translational stabilization of HIF1a. This study was designed to address the hypothesis that TRAP1 is relevant in determining specific features of the CSC phenotype and, more specifically, the reprogramming of their metabolism. Methods: TRAP1 levels were investigated in CD166-positive CSC fractions of HCT116 and HT29 colorectal carcinoma cells (CRCs) and primary stem cells derived from dental pulp by flow cytometry, cell sorting and upon colony formation assay. Interfering strategies were used to evaluate TRAP1 role in regulating glucose uptake and glycolytic metabolism. Results: TRAP1-interfered CRC HCT116 cells showed lower clonogenic potential than scramble cells in soft agar assay. Furthermore, CD166positive CSCs derived from CRC cell lines exhibited higher TRAP1 levels compared to non-CSC subpopulations and, consistently, TRAP1 stable interference resulted in the down-regulation of CD166 expression in HCT116 cells. In parallel experiment, TRAP1 expression was downregulated upon differentiation of primary stem cells derived from dental pulp. Finally, TRAP1 interference resulted in increased O2 consumption and in reduced glucose uptake, and the parallel inhibition of the expression of glucose transporters 1 and 2 and lactate production in CRC cells. Conclusion: These preliminary data suggest a potential role of TRAP1 in the maintenance of the CSC phenotype, likely due to its capacity to enhance glucose uptake and glycolytic metabolism, thus favoring the adaptation to a hypoxic microenvironment.
183 POSTER Spatio-temporal characterization of tumor growth and invasion A.M. Jimenez1 , O. Yogurtcu2 , M. Horn-Lee1 , P. Rao1 , S.X. Sun2 , D. Wirtz1 . 1 Johns Hopkins University, Chemical and Biomolecular Eng, Baltimore MD, USA; 2 Johns Hopkins University, Mechanical Engineering, Baltimore MD, USA Background: Tumors are a complex arrangement of tissues made up of several components, including dense masses of cancer cells and extracellular matrix (ECM). Recent studies have revealed the crucial role extracellular matrix components have on single cancer cell behavior, but how the interaction of ECM components affect the growth dynamics of an entire tumor is not well understood. Materials and Methods: We designed a novel three-dimensional (3D) in vitro system and used human derived fibrosarcoma cell (HT1080) aggregates embedded in 3D collagen matrices in combination with live cell imaging, cryo-stat sectioning, and immunostaining to characterize tumor growth and invasion. Results: Multicellular aggregates grow linearly with a rate that decreases with increasing collagen concentration. The spatio-temporal cell density distribution of the aggregates differs with differing collagen concentrations. Both cell migration and proliferation are highly dependent on collagen concentration and local cell density. Conclusion: We identified cell migration as a key contributor to multicellular aggregate size and demonstrate that different cell phenotypes correlate with different invasion patterns of the cell aggregates at various collagen concentrations. 184 Molecular profiling of heterogeneous tumor cells
POSTER
A. Chenchik1 , D. Deng1 , K. Bonneau1 , M. Makhanov1 , M. Coram2 , G. Dolganov2 , S.S. Jeffrey2 . 1 Cellecta Inc., Mountain View CA, USA; 2 Stanford University, Palo Alto CA, USA Molecular profiling of heterogeneous circulating tumor cells (CTCs) and tumor biopsy samples at the single-cell level is critical for identifying different cancer cell subpopulations and understanding their value in predicting metastatic cancer progression and responses to treatment. Unfortunately, the isolation and comprehensive characterization of hundreds to thousands of individual cancer cells present formidable analytical and technical challenges. To this end, we developed a high-throughput (HT) HiCellex technology to obtain the genome-wide expression and mutation profiles of hundreds to thousands of epithelial cancer cells at the single cell level. Importantly, HiCellex allows selective molecular profiling of a complete set of viable cells at the single-cell level in the presence of contaminating leukocytes without physical separation of individual cells. The genetic profiling technology uses unique cell-specific molecular barcodes to label mRNAs in individual viable cancer cells by a spliceosome-mediated trans-splicing mechanism. Barcoded trans-splicing constructs, necessary for the labeling of the entire population of pre-mRNAs, are delivered and expressed in a pool of target cancer cells with lentiviral vectors. Digital expression data are generated by HT sequencing of barcoded amplified cDNAs and easily cluster to each cell in silico using cell-specific barcodes. This presentation will provide results of development and validation of genome-wide and multiplex RT-PCR profiling assay of the 500 most informative subtyping and metastatic signature genes (MGS) in model cancer cell lines, CTC and tumor samples. The developed HiCellex 500 MGS profiling assay is a cost-effective approach to effectively characterize, subtype and identify prognostic and diagnostic biomarkers in heterogeneous cancer cells at the single-cell level. 185 POSTER Treatment of patient-derived NSCLC xenograft preclinical models using image-guided small animal irradiation N. Papadopoulou1 , A. McKenzie1 , J. King1 , M. Page1 , R. Kumari1 . 1 Precos Ltd, Crown Bioscience, Loughborough Leicestershire, United Kingdom Background: Radiotherapy is a primary, adjuvant or neoadjuvant treatment for a number of different cancers such as glioblastoma, breast, lung and prostate. Image-guided micro-irradiation (IGMI) is widely used to treat cancer patients providing more accurate treatment plans and reduced side effects. However in the preclinical setting the use of IGMI is less common with traditional irradiation studies utilising whole body irradiation with lead shielding to focus the radiation to a specific area on the animal or simple single beam techniques. The development of the image-guided small animal radiation research platform (SARRP) allows the treatment of animal models of cancer more accurately and with planned protocols
Poster Session – Preclinical Models similar to those utilised in the clinic. We have established a panel of Caucasian NSCLC patient-derived xenograft (PDX), which are sensitive or have acquired resistance to standard of care treatments. Methods: NSCLC samples obtained from untreated patients undergoing surgery were collected with ethical consent, disaggregated and implanted subcutaneously in MF-1 nude mice (Harlan, UK) admixed with a human stromal cell component to generate patient-derived xenograft (PDX) models. Resistant models were generated in vivo through repeated cycling of treatment for up to 10 weeks with standard of care or targeted agents. Patient-derived xenografts were maintained in vivo and monitored by calliper measurements three times weekly and mice recruited to the study when mean tumour volume was approximately 100–200 mm3 . Irradiation protocols and dose calculation were designed to deliver appropriate irradiation dose (2−3 Gy/day/mouse) to the tumours either alone or in combination with a targeted agent/chemotherapeutic, whilst sparing the surrounding normal tissue. Response to treatment was evaluated by tumour growth inhibition and clinical condition of mice monitored daily. Results: Mice treated with IGMI using the SARRP tolerated irradiation doses either alone or in combinations. Response and resistance to irradiation across a panel of Caucasian NSCLC PDX and resistant models will be reported along with any outcome of the combination treatments to re-sensitize resistant cancer to radiotherapy. Conclusions: The SARRP platform allows the evaluation of irradiation alone or in combination with anti-cancer agents in small animals with reduced side effects and improved safety outcome, allowing these novel preclinical models to be used effectively for drug discovery programmes and to derive irradiation schedules and regimens suitable for testing subsequently in clinical trials. 186 POSTER The use of Quantitative Textural Analysis imaging biomarkers to predict response to temsirolimus treatment in advanced HCC subjects R. Korn1 , R. Osarogiagbon2 , R. Newbold3 , D. Burkett4 , J. Sachdev5 . 1 Scottsdale Healthcare Research Institute TGEN, Imaging Endpoints, Scottsdale, USA; 2 Baptist Cancer Center, Oncology, Memphis, USA; 3 Scottsdale Medical Imaging, Radiology, Scottsdale, USA; 4 Imaging Endpoints Core Lab, Radiology, Scottsdale, USA; 5 Scottsdale Healthcare Research Institute-TGEN, Virginia G Piper Cancer Center, Scottsdale, USA Background: The treatment of advanced HCC can be challenging. Being able to predict who will respond to therapy would represent a major step forward in disease control. We have developed an exploratory CT imaging biomarker that may predict treatment response to temsirolimus (T) after sorafenib (S) failure using quantitative textural analysis (QTA) of hepatic tissue and tumors on CT scans. Material and Methods: Pre and Post venous phase contrast CT scans through the abdomen were obtained in 10 subjects enrolled in a IIT from a single institution prior to treatment with T after S failure. QTA was performed on the axial slice of the liver containing both uninvolved hepatic tissue and tumor in the same slice. QTA parameters were generated at a fine filter level (SSF 3) and displayed as histogram derived features of mean, SD, mean positive pixel (MPP), entropy, skewness and kurtosis. Statistical Correlation between QTA parameters and subsequent CT tumor responses by mRECIST criteria, Barcelona score, tumor markers, OS, PFS were performed using spearman correlations, regression analysis and ROC analysis. P values <0.05 were considered significant. Results: A single arm Simon two-stage phase II trial was conducted to test the activity of T in previously S treated HCC patients. Of the 26 subjects enrolled 10 subjects qualified for QTA analysis as having both pre and post contrast venous phase CT scans available for evaluation. Of the 10 subjects, 7 had PR and 3 had SD as best response by mRECIST. The mean change in tumor size was −26.7% (median −34.4%, range −52.5% to 11.3%) for the 10 subjects. Using QTA there was a significant correlation between baseline tissue-tumor texture and best mRECIST response as measured by MPP (rho = 0.7551, p = 0.0011). ROC analysis showed that a MPP cutoff value of 7.95 had the best separation of predicting PRs with a 85% sensitivity and 100% specificity. No significant correlation was noted between QTA and PFS, OS, serum tumor markers, Barcelona scores. Conclusion: This limited analysis of responding HCC patients indicates that textural analysis from CT scans may provide an imaging biomarker for predicting response to T therapy following S failure. Although preliminary, these findings merit further investigation.
Wednesday 19 November 2014
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187 POSTER Targeted genomic profiling of penile squamous cell carcinoma using the Oncomine cancer research panel A.S. McDaniel1 , D. Hovelson1 , A. Cani1 , C.J. Liu1 , Y. Zhang1 , S. Sadis2 , S. Bandla2 , P. Williams2 , D. Rhodes2 , S.A. Tomlins1 . 1 University of Michigan, Pathology, Ann Arbor MI, USA; 2 ThermoFisher Scientific, Ann Arbor MI, USA Background: Penile squamous cell carcinoma (PeSCC) is a rare cancer notable for significant morbidity and mortality as well as an incomplete understanding of the underlying molecular alterations and lack of effective non-surgical therapeutic approaches. Material and Methods: A retrospective cohort of 60 formalin fixed, paraffin embedded (FFPE) tumor samples from 44 PeSCC cases (including 14 matched primary/metastasis pairs) was subjected to targeted next generation sequencing (NGS) using the Oncomine Cancer Research Panel encompassing actionable recurrent somatic alterations in ~125 oncogenes and tumor suppressors identified by analysis across multiple cancer types. Sequencing of multiplexed templates was performed using the IonTorrent Proton system. Sequence analysis was performed in Torrent Suite 4.0, with sequence alignment by TMAP, and variant calling using the Torrent Variant Caller plugin. HPV infection status for each sample was assessed using additional genomic DNA for PCR with the GP5/6 and My09/11 consensus primer sets for viral detection and typing. Results: Highly multiplexed targeted NGS yielded an average of 1,136,032 mapped reads per sample with high coverage (mean >450×) over targeted bases using 20 ng of input genomic DNA. High risk HPV was detected in five cases (Four with HPV 16, one with HPV 33). All classes of genomic alterations were evaluated, including single nucleotide variants (SNVs), insertions, deletions, stopgains, and copy number variants (CNVs). Frequently altered genes included CDKN2A (20 of 44), TP53 (19 of 44), NOTCH1 (9 of 44), FBXW7 (8 of 44), PIK3CA (7 of 44), NFE2L2 (6 of 44), and HRAS (6 of 44). Notably, tumor specimens from four cases harbored amplifications of EGFR and one case demonstrated CDK4 amplification; genes for which approved and investigational targeted therapies are available. Importantly, cases with multiple tumor samples profiled showed significant differences in 4 of 14 paired samples, indicating heterogeneity for actionable mutations such as EGFR amplification between primary tumors and metastases. Conclusions: We evaluated a cohort of PeSCC FFPE specimens using an NGS panel of recurrently altered cancer-associated somatic variants, providing detailed molecular analysis of this disease for the first time. The scope of mutations identified was similar to squamous cell carcinomas from other locations such as the lung and head and neck region. We identified a subset of cases harboring mutations with immediate therapeutic potential, including EGFR amplifications. This research suggests that NGS profiling of PeSCC may have utility as part of a precision medicine approach to aid clinical decision making. 188 POSTER RANBP2 knock-down is synthetic lethal with BRAF V600E in colon cancer L. Vecchione1 , V. Gambino1 , G. d’Ario2 , S. Tian3 , A. Schlicker1 , S. Mainardi1 , B. Diosdado1 , I. Simon3 , M. Delorenzi2 , C. Lieftink1 , R. Beijersbergen1 , S. Tejpar4 , R. Bernards1 . 1 NKI/AVL, Division of Molecular Carcinogenesis, Amsterdam, Netherlands; 2 Swiss Institute for Bioinformatics, Bioinformatics Core Facility, Lausanne, Switzerland; 3 Agendia BV, Agendia BV, Amsterdam, Netherlands; 4 KULeuven, Laboratory of Digestive Oncology, Leuven, Belgium Background: Approximately 8−15% of colon (CC) patients carry an activating mutation in BRAF. This CC subtype is associated with poor outcome and with resistance to chemotherapeutic treatments. We recently showed that BRAF (V600E) colon cancers have a characteristic gene expression signature, which is found also in subsets of KRAS mutant and KRAS-BRAF wild type (WT2) tumors. Tumors having this gene signature, referred to as ‘BRAF-like’, have a similar poor prognosis irrespective of the presence of the BRAF (V600E) mutation. By using a shRNA-based genetic screen in BRAF mutant CC cell lines we aimed to identify genes necessary for survival and growth of BRAF mutant CC in order to reveal novel targets for therapy. Method: We selected 363 genes that are specifically overexpressed in 89 BRAF mutant tumors as compared to 608 WT2 type tumors, based on gene expression profiles generated in two independent datasets The 363 genes list was used to generate a shRNA library consisting of 1815 hairpins targeting these genes (BRAF library) selected from the TRC human genome-wide shRNA collection (TRC-Hs1.0). Vaco432 and WIDR (BRAFV600E) CC cell lines and Lim1215 (WT2) CC cell line were infected with the BRAF library and screened for shRNAs that are selectively synthetic lethal with BRAFV600E mutation. Cells stably expressing the shRNA
Wednesday 19 November 2014
Poster Session – Preclinical Models
BRAFV600E mutation levels correlate with tumor burden and suggest a possible role as a prognostic indicator. Studies are currently underway to examine RAS mutations in patient blood as a possible marker for acquired resistance to BRAF inhibitors, and to monitor disease progression in patients with BRAF wildtype tumors. 181 POSTER Aberrant Wnt signaling activation in human cancers: In vitro and in vivo models to facilitate Wnt targeted drug development 1
1
1
1
1
1
1
G. Liu , C. Dong , R. Zhang , L. Zhang , S. Qian , J. Cai , J. Zhang , J. Ning1 . 1 Crown Bioscience Inc., Molecular and Cellular Biology, Taicang, China Activation of Wnt signaling has emerged as one of the major oncogenic aberrations in human cancers, which has been demonstrated to play critical roles in the maintenance of the undifferentiated cancer stem/progenitor cell phenotype, as well as to directly stimulate the malignant growth of tumors. Mutations in the downstream signaling components, including APC, AXIN, and b-catenin, have been well described in several cancer types, and recent studies have further extended the Wnt activation mechanisms beyond these downstream mutations to upstream signaling molecules in this pathway. By screening primary cancer cells established from the patient derived xenograft models, we identified multiple cancer types that secrete Wnt stimulating ligands. We also successfully established primary cancer cell line harboring recurrent R-spondin fusion mutations. Inhibition of the activated Wnt signaling in these cancer cells results in reduced cancer cell growth, indicating the critical dependence of cancer cells on the autocrine Wnt signaling. Thus, these in vitro and in vivo models provide a valuable resource for the high throughput screening of Wnt antagonists, efficacy assessment of candidate Wnt inhibitors, biomarker analysis, as well as the preclinical development of Wnt targeted therapeutics. 182 POSTER TRAP1 represents a key mediator of stemness and glycolytic metabolism in colorectal cancer cells G. Lettini1 , F. Maddalena1 , L. Sisinni1 , V. Condelli1 , L. Del Vecchio2 , M. Gemei2 , T. Notarangelo1 , M. Landriscina3 . 1 CROB − IRCCS, Laboratory of Pre-Clinical and Translational Research, Rionero in Vulture (PZ), Italy; 2 CEINGE, Biotecnologie Avanzate, Napoli, Italy; 3 University of Foggia, Clinical Oncology Unit Department of Medical and Surgical Sciences, Foggia, Italy Background: Tumor cells undergo a metabolic shift from mitochondrial oxidative phosphorylation to a preferential glycolytic metabolism, known as Warburg effect. Furthermore, cancer stem cells (CSCs), known for being responsible for tumor initiation and growth, are located in a hypoxic microenvironment, heavily relying on anaerobic glycolysis. Recently, tumor necrosis factor receptor-associated protein 1 (TRAP1), a HSP90 mitochondrial molecular chaperone up-regulated in colorectal, prostate, breast and lung carcinomas, has been identified as a key regulator of tumor cell metabolism, being responsible for suppression of mitochondrial oxidative phosphorylation and post-translational stabilization of HIF1a. This study was designed to address the hypothesis that TRAP1 is relevant in determining specific features of the CSC phenotype and, more specifically, the reprogramming of their metabolism. Methods: TRAP1 levels were investigated in CD166-positive CSC fractions of HCT116 and HT29 colorectal carcinoma cells (CRCs) and primary stem cells derived from dental pulp by flow cytometry, cell sorting and upon colony formation assay. Interfering strategies were used to evaluate TRAP1 role in regulating glucose uptake and glycolytic metabolism. Results: TRAP1-interfered CRC HCT116 cells showed lower clonogenic potential than scramble cells in soft agar assay. Furthermore, CD166positive CSCs derived from CRC cell lines exhibited higher TRAP1 levels compared to non-CSC subpopulations and, consistently, TRAP1 stable interference resulted in the down-regulation of CD166 expression in HCT116 cells. In parallel experiment, TRAP1 expression was downregulated upon differentiation of primary stem cells derived from dental pulp. Finally, TRAP1 interference resulted in increased O2 consumption and in reduced glucose uptake, and the parallel inhibition of the expression of glucose transporters 1 and 2 and lactate production in CRC cells. Conclusion: These preliminary data suggest a potential role of TRAP1 in the maintenance of the CSC phenotype, likely due to its capacity to enhance glucose uptake and glycolytic metabolism, thus favoring the adaptation to a hypoxic microenvironment.
183 POSTER Spatio-temporal characterization of tumor growth and invasion A.M. Jimenez1 , O. Yogurtcu2 , M. Horn-Lee1 , P. Rao1 , S.X. Sun2 , D. Wirtz1 . 1 Johns Hopkins University, Chemical and Biomolecular Eng, Baltimore MD, USA; 2 Johns Hopkins University, Mechanical Engineering, Baltimore MD, USA Background: Tumors are a complex arrangement of tissues made up of several components, including dense masses of cancer cells and extracellular matrix (ECM). Recent studies have revealed the crucial role extracellular matrix components have on single cancer cell behavior, but how the interaction of ECM components affect the growth dynamics of an entire tumor is not well understood. Materials and Methods: We designed a novel three-dimensional (3D) in vitro system and used human derived fibrosarcoma cell (HT1080) aggregates embedded in 3D collagen matrices in combination with live cell imaging, cryo-stat sectioning, and immunostaining to characterize tumor growth and invasion. Results: Multicellular aggregates grow linearly with a rate that decreases with increasing collagen concentration. The spatio-temporal cell density distribution of the aggregates differs with differing collagen concentrations. Both cell migration and proliferation are highly dependent on collagen concentration and local cell density. Conclusion: We identified cell migration as a key contributor to multicellular aggregate size and demonstrate that different cell phenotypes correlate with different invasion patterns of the cell aggregates at various collagen concentrations. 184 Molecular profiling of heterogeneous tumor cells
POSTER
A. Chenchik1 , D. Deng1 , K. Bonneau1 , M. Makhanov1 , M. Coram2 , G. Dolganov2 , S.S. Jeffrey2 . 1 Cellecta Inc., Mountain View CA, USA; 2 Stanford University, Palo Alto CA, USA Molecular profiling of heterogeneous circulating tumor cells (CTCs) and tumor biopsy samples at the single-cell level is critical for identifying different cancer cell subpopulations and understanding their value in predicting metastatic cancer progression and responses to treatment. Unfortunately, the isolation and comprehensive characterization of hundreds to thousands of individual cancer cells present formidable analytical and technical challenges. To this end, we developed a high-throughput (HT) HiCellex technology to obtain the genome-wide expression and mutation profiles of hundreds to thousands of epithelial cancer cells at the single cell level. Importantly, HiCellex allows selective molecular profiling of a complete set of viable cells at the single-cell level in the presence of contaminating leukocytes without physical separation of individual cells. The genetic profiling technology uses unique cell-specific molecular barcodes to label mRNAs in individual viable cancer cells by a spliceosome-mediated trans-splicing mechanism. Barcoded trans-splicing constructs, necessary for the labeling of the entire population of pre-mRNAs, are delivered and expressed in a pool of target cancer cells with lentiviral vectors. Digital expression data are generated by HT sequencing of barcoded amplified cDNAs and easily cluster to each cell in silico using cell-specific barcodes. This presentation will provide results of development and validation of genome-wide and multiplex RT-PCR profiling assay of the 500 most informative subtyping and metastatic signature genes (MGS) in model cancer cell lines, CTC and tumor samples. The developed HiCellex 500 MGS profiling assay is a cost-effective approach to effectively characterize, subtype and identify prognostic and diagnostic biomarkers in heterogeneous cancer cells at the single-cell level. 185 POSTER Treatment of patient-derived NSCLC xenograft preclinical models using image-guided small animal irradiation N. Papadopoulou1 , A. McKenzie1 , J. King1 , M. Page1 , R. Kumari1 . 1 Precos Ltd, Crown Bioscience, Loughborough Leicestershire, United Kingdom Background: Radiotherapy is a primary, adjuvant or neoadjuvant treatment for a number of different cancers such as glioblastoma, breast, lung and prostate. Image-guided micro-irradiation (IGMI) is widely used to treat cancer patients providing more accurate treatment plans and reduced side effects. However in the preclinical setting the use of IGMI is less common with traditional irradiation studies utilising whole body irradiation with lead shielding to focus the radiation to a specific area on the animal or simple single beam techniques. The development of the image-guided small animal radiation research platform (SARRP) allows the treatment of animal models of cancer more accurately and with planned protocols
Poster Session – Preclinical Models similar to those utilised in the clinic. We have established a panel of Caucasian NSCLC patient-derived xenograft (PDX), which are sensitive or have acquired resistance to standard of care treatments. Methods: NSCLC samples obtained from untreated patients undergoing surgery were collected with ethical consent, disaggregated and implanted subcutaneously in MF-1 nude mice (Harlan, UK) admixed with a human stromal cell component to generate patient-derived xenograft (PDX) models. Resistant models were generated in vivo through repeated cycling of treatment for up to 10 weeks with standard of care or targeted agents. Patient-derived xenografts were maintained in vivo and monitored by calliper measurements three times weekly and mice recruited to the study when mean tumour volume was approximately 100–200 mm3 . Irradiation protocols and dose calculation were designed to deliver appropriate irradiation dose (2−3 Gy/day/mouse) to the tumours either alone or in combination with a targeted agent/chemotherapeutic, whilst sparing the surrounding normal tissue. Response to treatment was evaluated by tumour growth inhibition and clinical condition of mice monitored daily. Results: Mice treated with IGMI using the SARRP tolerated irradiation doses either alone or in combinations. Response and resistance to irradiation across a panel of Caucasian NSCLC PDX and resistant models will be reported along with any outcome of the combination treatments to re-sensitize resistant cancer to radiotherapy. Conclusions: The SARRP platform allows the evaluation of irradiation alone or in combination with anti-cancer agents in small animals with reduced side effects and improved safety outcome, allowing these novel preclinical models to be used effectively for drug discovery programmes and to derive irradiation schedules and regimens suitable for testing subsequently in clinical trials. 186 POSTER The use of Quantitative Textural Analysis imaging biomarkers to predict response to temsirolimus treatment in advanced HCC subjects R. Korn1 , R. Osarogiagbon2 , R. Newbold3 , D. Burkett4 , J. Sachdev5 . 1 Scottsdale Healthcare Research Institute TGEN, Imaging Endpoints, Scottsdale, USA; 2 Baptist Cancer Center, Oncology, Memphis, USA; 3 Scottsdale Medical Imaging, Radiology, Scottsdale, USA; 4 Imaging Endpoints Core Lab, Radiology, Scottsdale, USA; 5 Scottsdale Healthcare Research Institute-TGEN, Virginia G Piper Cancer Center, Scottsdale, USA Background: The treatment of advanced HCC can be challenging. Being able to predict who will respond to therapy would represent a major step forward in disease control. We have developed an exploratory CT imaging biomarker that may predict treatment response to temsirolimus (T) after sorafenib (S) failure using quantitative textural analysis (QTA) of hepatic tissue and tumors on CT scans. Material and Methods: Pre and Post venous phase contrast CT scans through the abdomen were obtained in 10 subjects enrolled in a IIT from a single institution prior to treatment with T after S failure. QTA was performed on the axial slice of the liver containing both uninvolved hepatic tissue and tumor in the same slice. QTA parameters were generated at a fine filter level (SSF 3) and displayed as histogram derived features of mean, SD, mean positive pixel (MPP), entropy, skewness and kurtosis. Statistical Correlation between QTA parameters and subsequent CT tumor responses by mRECIST criteria, Barcelona score, tumor markers, OS, PFS were performed using spearman correlations, regression analysis and ROC analysis. P values <0.05 were considered significant. Results: A single arm Simon two-stage phase II trial was conducted to test the activity of T in previously S treated HCC patients. Of the 26 subjects enrolled 10 subjects qualified for QTA analysis as having both pre and post contrast venous phase CT scans available for evaluation. Of the 10 subjects, 7 had PR and 3 had SD as best response by mRECIST. The mean change in tumor size was −26.7% (median −34.4%, range −52.5% to 11.3%) for the 10 subjects. Using QTA there was a significant correlation between baseline tissue-tumor texture and best mRECIST response as measured by MPP (rho = 0.7551, p = 0.0011). ROC analysis showed that a MPP cutoff value of 7.95 had the best separation of predicting PRs with a 85% sensitivity and 100% specificity. No significant correlation was noted between QTA and PFS, OS, serum tumor markers, Barcelona scores. Conclusion: This limited analysis of responding HCC patients indicates that textural analysis from CT scans may provide an imaging biomarker for predicting response to T therapy following S failure. Although preliminary, these findings merit further investigation.
Wednesday 19 November 2014
61
187 POSTER Targeted genomic profiling of penile squamous cell carcinoma using the Oncomine cancer research panel A.S. McDaniel1 , D. Hovelson1 , A. Cani1 , C.J. Liu1 , Y. Zhang1 , S. Sadis2 , S. Bandla2 , P. Williams2 , D. Rhodes2 , S.A. Tomlins1 . 1 University of Michigan, Pathology, Ann Arbor MI, USA; 2 ThermoFisher Scientific, Ann Arbor MI, USA Background: Penile squamous cell carcinoma (PeSCC) is a rare cancer notable for significant morbidity and mortality as well as an incomplete understanding of the underlying molecular alterations and lack of effective non-surgical therapeutic approaches. Material and Methods: A retrospective cohort of 60 formalin fixed, paraffin embedded (FFPE) tumor samples from 44 PeSCC cases (including 14 matched primary/metastasis pairs) was subjected to targeted next generation sequencing (NGS) using the Oncomine Cancer Research Panel encompassing actionable recurrent somatic alterations in ~125 oncogenes and tumor suppressors identified by analysis across multiple cancer types. Sequencing of multiplexed templates was performed using the IonTorrent Proton system. Sequence analysis was performed in Torrent Suite 4.0, with sequence alignment by TMAP, and variant calling using the Torrent Variant Caller plugin. HPV infection status for each sample was assessed using additional genomic DNA for PCR with the GP5/6 and My09/11 consensus primer sets for viral detection and typing. Results: Highly multiplexed targeted NGS yielded an average of 1,136,032 mapped reads per sample with high coverage (mean >450×) over targeted bases using 20 ng of input genomic DNA. High risk HPV was detected in five cases (Four with HPV 16, one with HPV 33). All classes of genomic alterations were evaluated, including single nucleotide variants (SNVs), insertions, deletions, stopgains, and copy number variants (CNVs). Frequently altered genes included CDKN2A (20 of 44), TP53 (19 of 44), NOTCH1 (9 of 44), FBXW7 (8 of 44), PIK3CA (7 of 44), NFE2L2 (6 of 44), and HRAS (6 of 44). Notably, tumor specimens from four cases harbored amplifications of EGFR and one case demonstrated CDK4 amplification; genes for which approved and investigational targeted therapies are available. Importantly, cases with multiple tumor samples profiled showed significant differences in 4 of 14 paired samples, indicating heterogeneity for actionable mutations such as EGFR amplification between primary tumors and metastases. Conclusions: We evaluated a cohort of PeSCC FFPE specimens using an NGS panel of recurrently altered cancer-associated somatic variants, providing detailed molecular analysis of this disease for the first time. The scope of mutations identified was similar to squamous cell carcinomas from other locations such as the lung and head and neck region. We identified a subset of cases harboring mutations with immediate therapeutic potential, including EGFR amplifications. This research suggests that NGS profiling of PeSCC may have utility as part of a precision medicine approach to aid clinical decision making. 188 POSTER RANBP2 knock-down is synthetic lethal with BRAF V600E in colon cancer L. Vecchione1 , V. Gambino1 , G. d’Ario2 , S. Tian3 , A. Schlicker1 , S. Mainardi1 , B. Diosdado1 , I. Simon3 , M. Delorenzi2 , C. Lieftink1 , R. Beijersbergen1 , S. Tejpar4 , R. Bernards1 . 1 NKI/AVL, Division of Molecular Carcinogenesis, Amsterdam, Netherlands; 2 Swiss Institute for Bioinformatics, Bioinformatics Core Facility, Lausanne, Switzerland; 3 Agendia BV, Agendia BV, Amsterdam, Netherlands; 4 KULeuven, Laboratory of Digestive Oncology, Leuven, Belgium Background: Approximately 8−15% of colon (CC) patients carry an activating mutation in BRAF. This CC subtype is associated with poor outcome and with resistance to chemotherapeutic treatments. We recently showed that BRAF (V600E) colon cancers have a characteristic gene expression signature, which is found also in subsets of KRAS mutant and KRAS-BRAF wild type (WT2) tumors. Tumors having this gene signature, referred to as ‘BRAF-like’, have a similar poor prognosis irrespective of the presence of the BRAF (V600E) mutation. By using a shRNA-based genetic screen in BRAF mutant CC cell lines we aimed to identify genes necessary for survival and growth of BRAF mutant CC in order to reveal novel targets for therapy. Method: We selected 363 genes that are specifically overexpressed in 89 BRAF mutant tumors as compared to 608 WT2 type tumors, based on gene expression profiles generated in two independent datasets The 363 genes list was used to generate a shRNA library consisting of 1815 hairpins targeting these genes (BRAF library) selected from the TRC human genome-wide shRNA collection (TRC-Hs1.0). Vaco432 and WIDR (BRAFV600E) CC cell lines and Lim1215 (WT2) CC cell line were infected with the BRAF library and screened for shRNAs that are selectively synthetic lethal with BRAFV600E mutation. Cells stably expressing the shRNA