bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3083: Novel inhibitor GZ17-06.12 suppresses pancreatic cancer tumorigenesis
Authors: Paul Santanu , Stehno-Bittel Lisa , Dhar Animesh
Published: July 2016

Purpose: Pancreatic ductal adenocarcinoma (PDAC) or pancreatic cancer is one of the most lethal malignancies in humans. The survival rate for two years is very limited in most of cases (10%) after diagnosis of this disease. This is because current therapeutic regimens have limited effectiveness. Novel therapeutic targets are needed to treat this disease. GZ17-06.12 contains a nutraceutical for its anti-cancer properties, in several cancers. Crude extracts of the plant Arum palaestinum Boiss used in traditional Israeli and Palestinian medicine, have demonstrated cytostatic and cytotoxic activities in colon, prostrate and lung carcinoma. Therefore, we hypothesize that GZ17-06.12 will inhibit tumor progression and metastasis in PDAC.

Experimental Procedure: In this study, we have determined cell proliferation, pancosphere formation and apoptosis following treatment of different doses oGZ17-06.12 for 24-72 hours using MiaPaCa-2 and S2-007 human pancreatic cancer cells. Cell cycle distribution and apoptosis were measured using flow cytometic analysis. Orthotopic pancreatic cancer model in athymic mice was developed and GZ17-06.12 was given orally for 20 days to those mice. Proliferative markers, pEGFR/pAkt and apoptotic markers, Bax/Bcl-2, were monitored following treatment with GZ17-06.12 in both in vivo and in vitro models. Metastatic markers, MMP-2 and MMP-9 were measured in metastatic tissues in orthotopic models.

Results: GZ17-06.12 inhibited proliferation of pancreatic cancer cell lines in a dose- and time-dependent manner. Cell cycle analysis suggested that cells were arrested in S and G2 phase following treatment. In addition, GZ17-06.12 induced apoptosis in both in vitro and in vivo pancreatic cancer. Moreover, the compound significantly inhibited epidermal growth factor receptor and Akt phosphorylation. Furthermore, GZ17-06.12 decreased the number and size of the pancospheres in S2-007 cells with concomitant inhibition of pancreatic cancer stem cell markers, DCLK1, Lgr5 and EpCam. The effect of GZ17-06.12 suppressed tumor growth and metastatic potential as indicative of MMP-2 and MMP-9 activity in primary and metastatic tumors.

Conclusions: GZ17-06.12 significantly reduces tumorigenesis and metastasis in both in vitro and in vivo pancreatic cancer models, inhibiting proliferation with concomitant increase of apoptosis.

Citation Format: Santanu Paul, Lisa Stehno-Bittel, Animesh Dhar. Novel inhibitor GZ17-06.12 suppresses pancreatic cancer tumorigenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3083.
Abstract 3087: Combination of ONC201 and bevacizumab significantly impacts colorectal cancer growth and metastasis in vivo
Authors: Wagner Jessica , El-Deiry Wafik S.
Published: July 2016

ONC201 is a first-in-class small molecule anti-tumor agent (Allen et al., 2013; Wagner et al., 2014; Prabhu et al., 2015; Kline et al., 2015) that upregulates endogenous TRAIL and activates the integrated stress response leading to increased DR5 expression, among other anti-tumor effects. ONC201 recently completed a phase I clinical trial in advanced solid tumors and is currently in multiple phase I/II clinical trials in select advanced cancers (NCT02250781, NCT02324621, NCT02420795, NCT02392572, NCT02609230, NCT02525692, NCT02038699). Colorectal cancer (CRC) remains one of the leading causes of death from cancer worldwide; predominantly due to its metastatic nature. Increasing clinical evidence including randomized clinical trials has demonstrated that there is an increased efficacy from certain cytotoxic chemotherapeutic agents when placed in combination with molecular agents that target the vascular endothelial growth factor (VEGF) pathway. Bevacizumab (Avastin), a monoclonal antibody that targets VEGF, has become an important therapeutic option for patients with advanced metastatic CRC. The single agent activity of ONC201 in preclinical models of colorectal cancer have been well documented, however a combinatorial regimen for this investigational agent has not been prioritized for this indication. We investigated the efficacy of ONC201 in combination with bevacizumab in preclinical models of colorectal cancer based on the previously reported cooperative efficacy with this combination in preclinical GBM models. While each agent showed efficacy as a single agent, the combination of ONC201 with bevacizumab appeared synergistic in the suppression of CRC xenograft growth including a significant impact on tumor regression and occasional complete tumor regressions. The ONC201 combination with bevacizumab also suppressed metastasis from xenografted human colorectal cancer cells. Mechanistic studies and angiogenesis imaging studies are ongoing to demonstrate the importance of this combination on both tumor growth, migration, and invasion. Our results demonstrate that combining anti-angiogenic bevacizumab with ONC201 enhances anti-tumor efficacy in preclinical models of colorectal cancer and suggest that further testing of the combination is warranted in the clinic.

Citation Format: Jessica Wagner, Wafik S. El-Deiry. Combination of ONC201 and bevacizumab significantly impacts colorectal cancer growth and metastasis in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3087.
Abstract 3092: Physiological mimicry to characterize the average topology of the MYC and VEGF promoter G-quadruplexes
Authors: Morgan Rhianna K. , Brooks Tracy A.
Published: July 2016

DNA G-quadruplexes (G4s) have become increasingly present in modern drug discovery efforts. The main focus centers on cancer therapeutic development, as G4s are more concentrated in oncogenic promoters than the rest of the genome. Two of the most well described G4s to date are within the MYC and the VEGF promoters; stabilization of either structure fosters transcriptional silencing. Downregulation of MYC decreases cell proliferation and alters metabolism; it is promising for a number of cancers, such lymphomas, leukemia, colorectal cancer and more. VEGF downregulation would modulate angiogenesis, which is of therapeutic benefit for almost all solid tumors. The physiological structure of both of these promoter G4s has been elucidated using plasmid DMS footprinting, which incorporates DNA supercoiling in addition to stabilizing cations or molecules. The current study examines a variety of anhydrous or crowding co-solvents for their ability to recapitulate the physiological structure in single-stranded ex vivo conditions. The co-solvents tested included glucose, acetonitrile, polyethylene glycol, dextran sulfate, sucrose, ficoll, and extracted nucleoplasm, ranging from 0-40%. Significant changes in structure, including loop directionality and number of competing isoforms, were examined by electronic circular dichroism. Electromobility shift assays differentiated inter- and intra-molecular structures, further distinguishing the distribution of isoforms. DMS footprinting with select molecular crowding and dehydrating agents was performed and compared to plasmid footprints, to validate physiological mimicry ex vivo. Ongoing studies examining the physiological conditions regulating G4 stability and function include moving from plasmid to in cell DMS footprinting in order to most completely identify the physiologic isoform, and to using optimized co-solvents to examine the effects of small molecules ex vivo and better predict active hits for in vitro evaluation. Overall the VEGF and MYC promoter G4 structures are promising therapeutic targets for anti-angiogenic and anti-proliferative therapy, respectively. This comprehensive understanding of physiological principles governing the above G4 formation will best inform future drug discovery efforts for these and other oncogenic promoters.

Citation Format: Rhianna K. Morgan, Tracy A. Brooks. Physiological mimicry to characterize the average topology of the MYC and VEGF promoter G-quadruplexes. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3092.
Abstract 3097: Structural and biophysical characterization of anti-apoptotic protein Bcl-2 and GTPase Rac1 interaction
Authors: Ajumal Shani , Kang Jia , Joseph Thomas , Pervaiz Shazib , Swaminathan Kunchithapadam
Published: July 2016

We have recently reported a novel mechanism linking the pro-oxidant activity of the anti-apoptotic protein Bcl-2 to its interaction with the small GTPase Rac1. In this current study, we investigate the nature of this interaction using activated (GTP loaded) Rac1 and Bcl-2 peptides (spanning the BH3 domain and its adjacent loop region) using coimmunoprecipitation, isothermal titration calorimetry (ITC), X-ray crystallography, docking/modeling, fluorescence spectroscopy, molecular dynamics and mutational studies. Our results will develop to translational relevance in the treatment of human cancers that are refractory to chemotherapy due to overexpression of Bcl-2.

Citation Format: Shani Ajumal, Jia Kang, Thomas Joseph, Shazib Pervaiz, Kunchithapadam Swaminathan. Structural and biophysical characterization of anti-apoptotic protein Bcl-2 and GTPase Rac1 interaction. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3097.
Abstract 310: Mechanism of sorafenib resistance in acute myeloid leukemia
Authors: Lindblad Oscar , Cordero Eugenia , Puissant Alexandre , Macaulay Lucy , Kabir Nuzhat N. , Sun Jianmin , Haraldsson Karin , Borg Åke , Levander Fredrik , Stegmaier Kimberly , Pietras Kristian , Rönnstrand Lars , Kazi Julhash U.
Published: July 2016

Activating mutations in FLT3 occur in up to 35% of patients with AML and correlate with poor prognosis. Therapy directed against FLT3 has been shown to induce response in patients with AML, but these responses are almost always transient. Dual PI3K/mTOR inhibitors have displayed promising results in the treatment of solid tumors, and of hematological cancers. In this report we describe that a dual PI3K/mTOR inhibitor is effective against sorafenib-responsive, and -resistant, AML cell lines both in vitro and in vivo. We generated two cell lines by sustained treatment with sorafenib. Parental cell lines carry the FLT3-ITD mutation and are highly responsive to FLT3 inhibitors, while sorafenib-resistant cell lines display resistance to multiple FLT3 inhibitors. Next generation sequencing did not show any significant difference in the mutational burden in between responsive and resistant cell lines. While next generation sequencing identified FLT3-D835Y with an allele-depth of 67:37 in a resistant cell line, Sanger sequencing and protein mass-spectroscopy did not identify any acquired mutations in the kinase domain of FLT3 in the resistant cells. Moreover, sorafenib treatment effectively blocked FLT3 activation in resistant cells, while it was unable to block colony formation or cell survival, suggesting that the resistant cells are no longer dependent on FLT3. Gene expression analysis of sorafenib-sensitive and -resistant cell lines, as well as of blasts from patients with sorafenib-resistant AML, suggested an enrichment of the PI3K/mTOR pathway that correlated with the resistant phenotype, which was further supported by phospho-specific-antibody array analysis. The selective PI3K/mTOR inhibitor, gedatolisib, efficiently blocked proliferation, colony and tumor formation of resistant cell lines as well as induces apoptosis. Taken together, our data suggest that aberrant activation of the PI3K/mTOR pathway results in FLT3-inhibitors-resistance and a dual specific PI3K/mTOR inhibitor is an effective treatment in both tyrosine kinase inhibitor sensitive and resistant AML.

Citation Format: Oscar Lindblad, Eugenia Cordero, Alexandre Puissant, Lucy Macaulay, Nuzhat N. Kabir, Jianmin Sun, Karin Haraldsson, Åke Borg, Fredrik Levander, Kimberly Stegmaier, Kristian Pietras, Lars Rönnstrand, Julhash U. Kazi. Mechanism of sorafenib resistance in acute myeloid leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 310.
Abstract 3105A: Titanium(IV) regulation by serum transferrin and citrate sheds new insight into the use of chemical transferrin mimetics for Ti(IV) anticancer drug development
Authors: Tinoco Arthur D. , Loza-Rosas Sergio A. , Vazquez Alexandra , Rivero Kennett I. , Negron Lenny M. , Saxena Manoj , Sharma Shweta , Delgado Yamixa , Sanchez Annelis , Zambrana Nicole , Parks Timothy B.
Published: July 2016

A recently resolved structure of Ti(IV) bound human serum transferrin (sTf) and cell viability studies reveal that the small molecule citrate and the iron(III) transport protein sTf work together to maintain Ti(IV) in a nontoxic speciation in the body. Identification of this biomolecular interaction elucidates important factors that must be considered when developing Ti(IV)-based anticancer agents. Chemical transferrin mimetic (cTfm) ligands are being employed in a Ti(IV) anticancer drug design strategy to take advantage of the structural features of the sTf metal binding site favorable for Ti(IV) coordination to facilitate Ti(IV) cytotoxicity. The very strong Fe(III) binding property of these ligands are exploited to create stable Ti(IV) complexes that are able to deliver Ti(IV) into cells but exhibit lability in the presence of Fe(III). These complexes are intended to operate by the dual function of iron depletion and Ti(IV) attack at intracellular sites. Work with the cTfm ligands N,N’-di(o-hydroxybenzyl)ethylenediamine-N,N’-diacetic acid (HBED) and deferasirox reveal the structural features of the cTfm ligands that maximize the stability of the Ti(IV) complex outside the cell but enable sufficient lability to trigger cytotoxicity within the cell. The features are those that minimize induced dissociation due to the presence of citrate and sTf. Preliminary results suggest that while Fe(III) depletion may play an important role in the activity of the Ti(IV) cTfm complexes, the two lead complexes may operate via different mechanisms of action.

Citation Format: Arthur D. Tinoco, Sergio A. Loza-Rosas, Alexandra Vazquez, Kennett I. Rivero, Lenny M. Negron, Manoj Saxena, Shweta Sharma, Yamixa Delgado, Annelis Sanchez, Nicole Zambrana, Timothy B. Parks. Titanium(IV) regulation by serum transferrin and citrate sheds new insight into the use of chemical transferrin mimetics for Ti(IV) anticancer drug development. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3105A.
Abstract 3109: The TERT variant rs2736100_AC is associated with reduced risk of upper track urothelial carcinomas
Authors: Yuan Xiaotian , Li Ping , Björkholm Magnus , Xu Dawei
Published: July 2016

Upper tract urothelial carcinomas (UTUCs) are originated from urothelium, and consist predominantly of renal pelvic carcinomas (RPCs) and ureter carcinomas (UTs). The UTUC incidence has increased over the past two decades, and most UTUCs have become invasive when diagnosed. Therefore, it is urgently needed to identify high-risk populations for the preventive intervention and monitoring. Recent genome-wide association study has provided strong evidence that common single nucleotide polymorphisms (SNPs) are associated with susceptibility of various malignancies. However, little is known about the susceptibility loci to UTUC. Here we genotyped the telomerase reverse transcriptase (TERT) SNPs rs2736100 and rs2736098, two SNPs associated with a risk of other malignancies, in Han Chinese patients with UTUC (n = 217) and evaluated the relationship between the rs2736100 polymorphism and UTUC risk by comparing with that in 289 healthy controls. Genotyping was performed using a TaqMan® SNP Genotyping Assays kit. Neither AA nor CC genotypes differed significantly between cases and control subjects, while the AC carriers were associated with the reduced risk of UTUC (OR = 0.6043; 95% CI: 0.4039 - 0.9042; P = 0.0185). When RPCs (n = 100) and UTs (n = 117) were analyzed separately, such protective effect remained significant in UTs (OR = 0.5728; 95% CI: 0.3540 - 0.9269; P = 0.031). The AC genotype was lower in RPC patients than in controls, but the difference was not statistically significant (OR = 0.6484; 95% CI: 0.3811 - 1.103; P = 0.142). In conclusion, we show for the first time that the rs2736100 AC genotype is protective against UTUC development.

Citation Format: Xiaotian Yuan, Ping Li, Magnus Björkholm, Dawei Xu. The TERT variant rs2736100_AC is associated with reduced risk of upper track urothelial carcinomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3109.
Abstract 3111: Elevated expression of hyaluronan synthase 2 associates with poor prognosis in diffusely infiltrating astrocytomas
Authors: Poukka Mari J. , Haapasalo Hannu , Rilla Kirsi J. , Tyynelä-Korhonen Kristiina , Soini Ylermi , Pasonen-Seppänen Sanna M.
Published: July 2016

Diffusely infiltrating astrocytomas are central nervous system tumors originating from astrocytic glial cells or their precursor cells. In this retrospective study we investigated the content of hyaluronan and the expression of hyaluronan metabolizing enzymes and its cell surface receptor, CD44, in these aggressive tumors. Hyaluronan is a large extracellular matrix molecule synthesized by three hyaluronan synthases (HAS1-3) on the plasma membrane and degraded by two hyaluronidases (HYAL1-2). In many tumors, aberrant hyaluronan metabolism implicates aggressive disease progression and metastatic potential.

Our material consisted of 165 diffusely infiltrating astrocytomas (WHO grades II-IV). Tumor samples were processed into tissue microarray (TMA) blocks. The TMA sections were stained for hyaluronan, CD44, HAS1, HAS2, HAS3 and HYAL2. The staining intensity of astrocytomas was evaluated with a four-level scoring (0-3; no color, weak, moderate and strong) by two independent observers. The immunostaining results were compared with χ2 -test or with Kruskal-Wallis test for correlation with clinicopathological parameters and survival analyses were done with Kaplan-Meier log rank test and Cox regression.

Hyaluronan and CD44 were strongly expressed in astrocytic gliomas but their expression did not correlate with WHO grade, cell proliferation activity by Ki-67, p53 status, epidermal growth factor receptor (EGFR) amplification or IDH1 mutation. Whereas HAS2 staining intensity showed a significant correlation with IDH1 mutation. Tumors with high HAS2 expression were IDH1 negative (p = 0.003). In addition, in non-parametric tests increased HAS2 staining intensity showed association with increased cell proliferation (p = 0.013) and in log rank test with decreased overall survival of patients (p = 0.001). Variables included in the multivariable Cox regression analysis were HAS2 staining intensity, p53 status, EGFR amplification, IDH1 mutation and WHO tumor grade; in this analysis HAS2 expression turned out to be a significant independent prognostic factor (p = 0.008). This study indicates that elevated expression of HAS2 is associated with glioma progression and suggests that HAS2 has a prognostic significance in diffusely infiltrating astrocytomas.

Citation Format: Mari J. Poukka, Hannu Haapasalo, Kirsi J. Rilla, Kristiina Tyynelä-Korhonen, Ylermi Soini, Sanna M. Pasonen-Seppänen. Elevated expression of hyaluronan synthase 2 associates with poor prognosis in diffusely infiltrating astrocytomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3111.
Abstract 3120: B-Catenin gene expression and its mutations: promising and key component in the pathogenesis of acute myeloid leukemia (AML)
Authors: Maqbool Mohsin , Iqbal Sobuhi , Sharma Atul , Bakhshi Sameer , Vishnubhatla Sreenivas , Upadhyay Ashish Datt , Kumar Lalit
Published: July 2016

INTRODUCTION AND AIM: Wnt signaling pathway plays a critical role in stem cell self-renewal and proliferation of leukemic stem cells and its association with the pathogenesis of acute myeloid leukemia (AML) has also been described. Beta-Catenin, a key regulator of Wnt signaling is highly expressed in cancers. Its role has been recently documented in AML however its prognostic significance in AML is not clear. The present study was carried to determine the potential prognostic implications of B-catenin expression and mutations in de novo AML patients.

METHODS: We performed the gene expression of B-Catenin and its mutations analysis in exon 3. We investigated the expression of B-Catenin gene in bone marrow and/or blood samples by quantitative PCR using SYBR-Green chemistry and analysis was done by ΔΔCT Method. 18S RNA and B2M genes were used as reference genes. B-Catenin mutations analysis was by done direct DNA Sanger sequencing.

RESULTS: Total 117 de novo non M3 AML patients and 40 healthy controls were included for the gene expression and analyzed for their clinicopathologic and prognostic significance. Median age for entire cohort (n = 117) was 17.0 years (Range 1-60 years) with male:female ratio of (1.8:1). FLT3-ITD mutation was positive in 11 (9.5%) patients. Only 4% of AML patients showed genetic alterations in exon 3 of B-catenin and no significant correlation with base line characteristics. B-catenin showed a significantly increased mRNA expression with a 5 fold rise as compared to healthy controls (p<0.05). B-catenin over expression was significantly associated with poor survival at median followup of 17 months and having less CR rates (p = 0.03). There was no significant correlation of B-catenin over expression vs baseline clinical characteristics like Hb, TLC, Platelets, Cyto group (p = 0.11), blast percentage (p = 0.28), Flt3-itd mutations (p = 0.42).

CONCLUSION: These results provide the evidence for WNT signaling activation existence in AML patients and suggest that B-Catenin could be the key component in the pathogenesis and can be prognostically important marker in AML. However B-catenin expression in larger cohort of samples and with longer follow-up is required to confirm our findings.

Citation Format: Mohsin Maqbool, Sobuhi Iqbal, Atul Sharma, Sameer Bakhshi, Sreenivas Vishnubhatla, Ashish Datt Upadhyay, Lalit Kumar. B-Catenin gene expression and its mutations: promising and key component in the pathogenesis of acute myeloid leukemia (AML). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3120.
Abstract 313: Type II interleukin 1 receptor-modulated SETBP-1/SET/PP2A/p-ERK signaling reduces the therapeutic activity of regorafenib against human colorectal cancer cells
Authors: Mar Ai-Chung , Chu Chun-Ho , Yang Shung-Haur , Jiang Jeng-Kai , Shiau Chung-Wai , Lee Te-Chang
Published: July 2016

Regorafenib, a newly approved multi-kinase inhibitor by US FDA, has recently demonstrated overall survival benefits in late-stage colorectal cancer (CRC) patients. We have preliminarily observed that the expression type II interleukin 1 receptor (IL1R2), an IL-1 decoy receptor, was closely associated with regorafenib resistance in human CRC cells. We also found that IL1R2 expression was associated with poor prognosis and 5-year survival of CRC patients. In this study, we conducted experiments to elucidate the mechanisms underlying which IL1R2 is involved in regorafenib resistance. We first demonstrated that silencing of IL1R2 in HT29 cells overcame its resistance to regorafenib, whereas ectopic expression of IL1R2 in HCT116 cells reduced its sensitivity to regorafenib in either in vitro or in vivo assay systems. In addition, we established regorafenib-resistant DLD-1 cells (DLD-1-R) by long-term culturing DLD-1 cells in the presence of regorafenib, and we observed that protein expression of IL1R2 was increased in DLD-1-R cells compared to the parental DLD-1 cells. Furthermore, our results showed that IL1R2 expression was positively associated with p-ERK levels, which are crucial for survival of cancer cells. Pretreatment of HT29, IL1R2-overexpressing HCT116, and DLD-1-R cells with MEK/ERK inhibitor U0126 significantly reversed their regorafenib resistance in in vitro and in vivo systems. Mechanistically, we revealed that increased p-ERK levels in IL1R2-overexpressing cells were due to the decrease of PP2A activity instead of its expression levels. Since PP2A is a phosphatase of ERK, we further demonstrated that IL1R2 mediated through transcriptionally enhancing the expression of SETBP-1. The formation of SETBP-1/SET/PP2A complex decreased the activity of PP2A. Taken together, our present study identified IL1R2 was a potential prognostic and staging biomarker of CRC patients. Significantly, we also demonstrated a new molecular mechanism involved in regorafenib resistance and the combination of regorafenib and MEK/ERK inhibitor is a rationale regime to overcome regorafenib resistance in CRC patients.

Citation Format: Ai-Chung Mar, Chun-Ho Chu, Shung-Haur Yang, Jeng-Kai Jiang, Chung-Wai Shiau, Te-Chang Lee. Type II interleukin 1 receptor-modulated SETBP-1/SET/PP2A/p-ERK signaling reduces the therapeutic activity of regorafenib against human colorectal cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 313.
Abstract 3147: Identification of developmental endothelial locus-1 on circulating extracellular vesicles as a novel biomarker for early breast cancer detection
Authors: Moon Pyong-Gon P. , Lee Jeong-Eun , Cho Young-Eun , Lee Soo Jung , Jung Jin Hyang , Chae Yee Soo , Bae Han-Ik , Kim Young-Bum , Kim In-San , Park Hoyong , Baek Moon-Chang
Published: July 2016

Purpose: Currently, there are no molecular biomarkers for the early detection of breast cancer (BC). This study focused on identifying surface proteins found on circulating extracellular vesicles (EVs) for detecting early-stage BC.

Experimental Design: Circulating EVs, isolated from the plasma of 10 patients with BC (stages I and II), and 5 healthy controls, were analyzed using LC-MS/MS. Developmental endothelial locus-1 protein (Del-1) was selected as a candidate biomarker. Two different enzyme-linked immunosorbent assays (ELISAs) were used to measure Del-1 in plasma samples from healthy controls (n = 81), patients with BC (n = 269), BC patients after surgical resection (n = 50), patients with benign breast tumors (n = 64), and patients with non-cancerous diseases (n = 98), in two cohorts.

Results: Plasma Del-1 levels were significantly higher (P < 0.0001) in patients with BC than in all controls and returned to almost normal after tumor removal. The diagnostic accuracy of Del-1 was area under the curve (AUC), 0.961 [95% CI, 0.924-0.983], sensitivity of 94.70% and specificity of 86.36% in test cohort, and 0.968 [0.933-0.988], 92.31% and 86.62% in validation cohort for early-stage BC by one type of ELISA. Furthermore, Del-1 maintained diagnostic accuracy for patients with early-stage BC using the other type of ELISA (0.946 [0.905-0.972], 90.90%, and 77.14% in the test cohort; 0.943 [0.900-0.971], 89.23%, and 80.99% in the validation cohort).

Conclusions: Del-1 on circulating EVs is a promising marker to improve identification of patients with early-stage BC and distinguish BC from benign breast tumors and non-cancerous diseases.

Citation Format: Pyong-Gon P. Moon, Jeong-Eun Lee, Young-Eun Cho, Soo Jung Lee, Jin Hyang Jung, Yee Soo Chae, Han-Ik Bae, Young-Bum Kim, In-San Kim, Hoyong Park, Moon-Chang Baek. Identification of developmental endothelial locus-1 on circulating extracellular vesicles as a novel biomarker for early breast cancer detection. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3147.
Abstract 3148: Clinical validation of a next-generation sequencing assay specifically for blood-drop liquid biopsy
Authors: Yeh Chen-Hsiung , Spurgin Jonathan , Ford Andrew , Athanasuleas John , Manne Upender
Published: July 2016

Next-generation sequencing (NGS) technology enables rapid analysis and turnaround of multiple genes for clinically actionable somatic variants. Genetic sequencing on tumor biopsy tissue to inform treatment decisions is the hallmark of precision medicine. However, a one-time, single-site tissue sample may not be a true representation of tumor dynamic profile. A liquid biopsy, based on circulating cell-free DNA (cfDNA), can capture the entire heterogeneity of the disease, and offer what tissue biopsies can’t, the opportunity to take serial samples in order to monitor tumor genomic evolution in real time. We have recently developed a proprietary cfDNA enrichment process that requires only droplet volumes of blood. Here, we presented clinical validation of the blood-drop liquid biopsy NGS assay interrogating 2855 mutation hotspots in 50 cancer-associated genes using the AmpliSeq cancer panel and Ion Torrent Proton sequencer.

One nanogram of cfDNA prepared from 20 uL of plasma/serum was used as template to amplify 207 mutation hotspot amplicons. Deep sequencing of total 95 clinical specimens (≥ 1000× average coverage across the capture regions) achieved high sensitivity for detecting somatic variants at low allele fraction (AF). The variants at AF of ≥ 10% in most samples (90/95), except 5 cases, were confirmed by another laboratory. Repeated sequencing of 15 samples, and progressively diluted samples from two reference standard DNAs with known mutations and AF demonstrated reliable detection sensitivity at 1% for most single nucleotide variants (SNV) with high within-run and between-run reproducibility. Validation also revealed sensitivity and specificity for detecting variants at 5% AF was 100% (95% CI = 95.7-100.0) using Tru-Q 4 NGS reference standard DNA, and NA19240 or NA12878 HapMap individual, respectively.

Overall, our new-generation liquid biopsy NGS assay, with the capability to multiplex and simultaneously sequence multiple patient samples using finger-stick blood, is a robust and sensitive method for mutation analysis of clinical significance, further expedite treatment decision-making and identify targeted therapies for eligible patients in a time- and cost-efficient manner.

Citation Format: Chen-Hsiung Yeh, Jonathan Spurgin, Andrew Ford, John Athanasuleas, Upender Manne. Clinical validation of a next-generation sequencing assay specifically for blood-drop liquid biopsy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3148.
Abstract 3150: Analysis of the extracellular vesicles content present in the plasma of patients with head and neck squamous cell carcinoma for identification of molecular markers for treatment response
Authors: Rodrigues-Junior Dorival Mendes , Tan Soon Sim , Lim Sai Kiang , Iyer N Gopalakrishna , Luiz Vettore Andre
Published: July 2016

Previous data showed that induction chemotherapy (IC) with cisplatin and paclitaxel followed by chemoradiotherapy (CRT) based on cisplatin is safe, well tolerated and improves overall survival for patients with locally advanced head and neck squamous cell carcinoma (HNSCC) (stage III -IV). However, approximately 30% of these patients have incomplete response to IC and CRT and there is no clinical marker able to segregate these patients from those that respond to the treatment. The recent discovery of extracellular vesicles (EVs) in different body fluids carrying proteins and nucleic acids may indicate an alternative route for new specific markers present in the plasma of patients with HNSCC. In this study, Cholera toxin B chain (CTB) and Annexin V (AV) which respectively binds GM1 ganglioside and phosphatidylserine, were used to isolate EVs from plasma of HNSCC patients that respond and not respond to IC and CRT. The proteins in the vesicles were identified using antibody array through a pool of 6 responders’ patients (R) in comparison to 6 patients with incomplete response (IR). A total of 327 proteins were identified into CTB-EVs, from which 54 and 20 proteins were exclusive to R and IR patients, respectively. Beside this, 271 proteins were identified into AV-EVs, from which 20 proteins were present in R patients and other 20 proteins were present only in IR ones. A preliminary analysis of this data revealed that HNSCC resistant patients have elevated levels of TGFβ3 in CTB-EVs while patients with incomplete response have elevated levels of HSP90AB1 in AV-EVs. This study demonstrated the successful use of CTB and AV to isolate circulating plasma EVs for biomarker discovery in HNSCC and represents a novel technology that can be applied to segregate patients that may respond or not to IC or CRT.

Citation Format: Dorival Mendes Rodrigues-Junior, Soon Sim Tan, Sai Kiang Lim, N Gopalakrishna Iyer, Andre Luiz Vettore. Analysis of the extracellular vesicles content present in the plasma of patients with head and neck squamous cell carcinoma for identification of molecular markers for treatment response. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3150.
Abstract 316: FTY720 enhances the efficacy of paclitaxel and of carboplatin in drug-resistant ovarian cancer cells
Authors: Kreitzburg Kelly M. , Alvarez Ronald D. , Landen Charles N. , Yoon Karina J.
Published: July 2016

Objectives: Our long-term goal is to develop effective therapies for ovarian cancer by targeting molecules that contribute to drug resistance in this tumor type. Preliminary RNA-seq data were generated using a patient-derived xenograft model. These data identified the sphingosine-1-phosphate (S1P) signaling pathway as one of the pathways most affected by carboplatin and paclitaxel, the current standards of care for ovarian cancer. We then examined whether a synthetic analog of sphingosine, FTY720, enhanced the cytotoxicity of platinum and taxane resistant ovarian cancer cell lines.

Methods: To assess the efficacy of FTY720 + carboplatin and of FTY720 + paclitaxel, we exposed three pairs of parental and drug resistant human ovarian cancer cell lines to each combination. The cell lines used were: SKOV3-ip1 and SKOV3-TR (taxane resistant), A2780 and A2780-CP20 (platinum resistant), and HeyA8 and HeyA8-MDR (taxane-platinum resistant). We used alamarBlue cell proliferation assays to determine the efficacy of FTY720 as a single agent or in combination with paclitaxel or carboplatin, and immunoblots to assess the expression of proteins involved in the S1P pathway.

Results: FTY720 as a single agent decreased cell viability in a dose-dependent manner in all three pairs of cell lines, with IC50 values ranging from 5 to 8 μM. When cells were exposed to the IC50 of FTY720 + a range of concentrations of carboplatin or paclitaxel, FTY720 had little effect in any of the three parental cell lines and in SKOV3-TR cells. Interestingly, however, FTY720 decreased the IC50 of carboplatin 15- to 16-fold and the IC50 of paclitaxel 20- to 90- fold in A2780-CP20 and HeyA8-MDR cells, respectively. We also observed that FTY720 altered the expression of sphingosine kinase2, S1P lyase, and acid ceramidase, each of which contributes to the ceramide-sphingosine-S1P rheostat that tightly regulates expression of S1P pathway proteins.

Conclusions: Our data show that FTY720 sensitized drug resistant ovarian cancer cell lines A2780-CP20 and HeyA8-MDR to carboplatin and to paclitaxel 15- to 90-fold. The data suggest that the S1P pathway may contribute to carboplatin and paclitaxel resistance in this cell type. We propose that proteins of the S1P pathway merit further study as effective molecular targets in drug resistant ovarian cancer cells.

Citation Format: Kelly M. Kreitzburg, Ronald D. Alvarez, Charles N. Landen, Karina J. Yoon. FTY720 enhances the efficacy of paclitaxel and of carboplatin in drug-resistant ovarian cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 316.
Abstract 3181: Expression changes of the chromatin modifier PBRM1 in human renal cell carcinomas in relation to histopathological features
Authors: Krause Hans , Högner Anica , Jandrig Burkhard , Kilic Ergin , Kempkensteffen Carsten
Published: July 2016

Recent next-generation sequencing studies of clear cell renal cell carcinoma (ccRCC) have identified point mutations in chromatin-modifying genes, among them PBRM1 which is now the second most frequently mutated gene in RCCs (in up to 41% ccRCCs cases) and belongs to the m1 TCGA (The Cancer Genome Atlas) subset. The occasional presence of PBRM1 mutations in the absence of VHL mutations indicates, that PBRM1 might be another driver tumor suppressor gene in ccRCCs. We were interested in examining PBRM1 expression at mRNA and protein levels and correlated these findings to histopathological features of RCC patients.

Relative gene expression (RGE) data were obtained by Q-PCR from matched tumor and adjacent normal fresh frozen ccRCC tissues from 57 patients who underwent radical nephrectomy at our clinic. Papillary (pRCC) as well as chromophobic cases (chRCC) were added to analyze the expression of splice variants of PBRM1. Comparative Western blot analyses as well as semi-quantitative immuno-histochemical staining (IHC) for PBRM1 and VHL were performed on all ccRCC cases.

In 78.9% of cases (45/57) PBRM1 mRNA was downregulated at least 1.5-fold, 7% (4/57) showed PBRM1 upregulation, and 14% (8/57) displayed no obvious expression changes between tumor and corresponding tumor tissue. Interestingly, 21 of 45 ccRCCs tumor tissues expressed splice variant 4 more abundantly when compared to normal tissue, whereas the normal tissue preferentially expressed splice variant 1 that includes an additional exon. Remarkably, this differential expression picture is completely reversed in pRCC and chRCC cases.

The majority of 57 ccRCCs displayed weak nuclear PBRM1 staining (52.6%), whereas 31.6% showed moderate and 15.8% strong staining. However, we were not able to demonstrate a significant correlation of IHC expression levels, neither to tumor staging nor to Fuhrman grading.

The observed high frequency of decreased expression of the chromatin-remodeling gene PBRM1 on mRNA (78.9%) and protein levels (52.6%), respectively, as wells as a high mutation rate (about 15% in our data set) indicate a substantial role of PBRM1 in the tumorigenesis of ccRCCs. Preferences in the expression of different PBRM1 splice variants warrant further investigation with regard to renal cell carcinoma development.

Citation Format: Hans Krause, Anica Högner, Burkhard Jandrig, Ergin Kilic, Carsten Kempkensteffen. Expression changes of the chromatin modifier PBRM1 in human renal cell carcinomas in relation to histopathological features. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3181.
Abstract 3183: Delivery of point-of-care management to patients with gynecologic malignancies using comprehensive genomic profiling
Authors: Rodriguez-Rodriguez Lorna , Hirshfield Kim M. , Rojas Veronica , Ali Siraj , DiPaola Robert S. , Gibbon Darlene , Isani Sara , Leiser Aliza , Riedlinger Gregory M. , Ganesan Shridar
Published: July 2016

INTRODUCTION: Gynecologic cancers were estimated to occur in 98,280 women in 2015 in the United States and result in 30,440 deaths (Siegel 2015). The most common forms of gynecologic cancers occur in the ovary and uterine corpus (Siegel, 2015). Various reasons for poor outcome in these cancers include the late stage of the disease at the time of diagnosis and therapy resistance. We sought to elucidate alternate treatment avenues based on the genomic profiles of recalcitrant or rare gynecologic tumors. In the process, we aim to determine both the clinical utility and the feasibility of using comprehensive genomic profiling (CGP) during clinical care to identify clinically relevant tumor genomic alterations for these patients while enabling point-of-care management.

METHODS: As part of our ongoing prospective clinical trial, we report on the initial 69 patients with gynecologic cancers that were refractory to standard therapy. CGP was performed by Foundation Medicine, Inc. Genomic mutations and alterations were reviewed by members of an expert, multidisciplinary institutional molecular tumor board (MTB). Consensus recommendations by the MTB were provided to the treating physician on genomically targeted, clinically relevant, FDA-approved, on- and off-label therapies and clinical trials. The outcomes were assessed.

RESULTS: Study outcomes were available for 64 patients. On average, patients had 4.97 genomic alterations per tumor (range 1-46), while the time from the testing laboratory report to the formulation of recommendations by the MTB was approximately 3-4 weeks. Targeted

treatment options were recommended by the MTB for 93% of patients; 39% of these patients had the MTB-based recommendations implemented by the treating physician. A response or clinical benefit was seen in 56% of the patients receiving MTB-based targeted therapy.

CONCLUSION: These data suggest that alternatives to standard therapies for recalcitrant gynecologic cancers can be found though CGP and subsequent analysis from members of an expert, multidisciplinary institutional MTB. The formulation of clinical recommendations by an MTB based on tumor genomic profiling results is a feasible option. Further research is needed to understand how this approach shapes clinical decision making.

Citation Format: Lorna Rodriguez-Rodriguez, Kim M. Hirshfield, Veronica Rojas, Siraj Ali, Robert S. DiPaola, Darlene Gibbon, Sara Isani, Aliza Leiser, Gregory M. Riedlinger, Shridar Ganesan. Delivery of point-of-care management to patients with gynecologic malignancies using comprehensive genomic profiling. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3183.
Abstract 3184: Genomic analyses reveal two distinct biologic entities of endometrial endometrioid carcinoma
Authors: Sugiyama Yuko , Fukui Nobuyuki , Gotoh Osamu , Hasumi Katsuhiko , Takazawa Yutaka , Noda Tetsuo , Mori Seiichi
Published: July 2016

Endometrial carcinoma (EC) is classified into two types: type 1 (endometrioid type) and type 2 (non-endometrioid type). Type 1 endometrioid adenocarcinoma (EA) has been generally considered a single disease entity and was developed from endometrial hyperplasia as a precursor lesion. Recently we identified two distinct subgroups in type 1 EA; groups 1 and 2 as tumors with and without endometrial hyperplasia, respectively. The purpose of this study is to identify genes involved those differentially expressed by groups 1 and 2 EA. Genomic analyses including transcriptome and exome sequencing and immunohistochemistry were performed. For the validation, these data were projected to the TCGA (The Cancer Genome Atlas Research Network) data. In the results of transcriptome, group 1 and 2 EAs identified differential regulation of multiple genes and pathways. As an example, estrogen receptor (ER) related pathways were up-regulated in group 1 EA. Immunohistochemical analyses further confirmed overexpression of ER and PgR in the protein level. In the result of exome sequencing, mutation was observed more frequently in group 2 and sixteen driver genes were more frequently mutated in group 2.

Citation Format: Yuko Sugiyama, Nobuyuki Fukui, Osamu Gotoh, Katsuhiko Hasumi, Yutaka Takazawa, Tetsuo Noda, Seiichi Mori. Genomic analyses reveal two distinct biologic entities of endometrial endometrioid carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3184.
Abstract 3188: Targeted semiconductor sequencing of 409 cancer-related genes for somatic mutations and copy number variations in multiple myeloma
Authors: Ikeda Hiroshi , Sasaki Yasushi , Ishiguro Kazuya , Ishida Tadao , Aoki Yuka , Tokino Takashi
Published: July 2016

Background:

2016's NCCN guideline recommended that induction therapy used conventional chemotherapy such as proteasome inhibitors and Imids in MM. Overall survival is improving by these Novel agents. However, we don't know how to choose these new agents. In the future, we can use next generation sequencer as a tool of drug choosing system. So we reviewed newly diagnosed 11 patients with MM who had received novel agents in our institution.The comprehensive analysis of genetic alterations in tumor by next generation sequencing can allow for the prediction of drug resistance and facilitate improvements in the treatment of MM patients.

Method:

DNA was extracted from magnetic bead-enriched bone marrow CD138-positive malignant plasma cells from 11 cases of MM, and CD138-negative cells were used as matched non-tumor cells. Forty nanograms of DNA were used for multiplex PCR amplification with an Ion Ampliseq Comprehensive Cancer Panel that offers targeted coverage of all exons in 409 tumor suppressor genes and oncogenes frequently cited and frequently mutated in human cancers. (covered regions: 95.4% of total). We sequenced 15,992 regions which obtained more than 1.5 megabases of target sequence.

Result:

Each sample underwent on average 8.3 million sequencing reads after quality filtering. The mean read depths were 539x, and >95% of targeted bases were represented by at least 20 reads. The average number of non-synonymous mutations detected per patient was 5.8 (range 0-11). We also detected copy number variations in which segments of the genome can be duplicated or deleted from sequencing data. We found several genetic alterations that may have been associated with the poor prognosis and poor response to chemotherapy of MM patients. Pathway assessment has shown that somatic aberrations within MM genomes are mainly involved in several important pathways, including cell cycle regulation, RTK-MAPK-PI3K and NF-kB.

Conclusion:

We performed targeted next-generation sequencing for rapid (2 days), standardized, and cost-effective gene analysis of malignant plasma cells from patients with MM. We can use targeted next-generation sequencing as tool of drug choosing system.

Citation Format: Hiroshi Ikeda, Yasushi Sasaki, Kazuya Ishiguro, Tadao Ishida, Yuka Aoki, Takashi Tokino. Targeted semiconductor sequencing of 409 cancer-related genes for somatic mutations and copy number variations in multiple myeloma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3188.