bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1543: Wogonin suppresses the production of breast cancer-derived osteolytic factors
Authors: Kim Hyungkeun , Park Kwang-Kyun , Chung Won-Yoon
Published: August 2015

Breast cancer is the most frequent cancer in women and the main cause of its mortality is induced by metastasizing to distant organs. Breast cancer primarily metastasizes to the bone, lung, liver and brain, and the bone is the most susceptible to metastasis. When breast cancer metastasizes to the bone, the dominant lesion is osteolytic. This osteolytic bone metastasis is highly associated with the complex interaction between cancer cells and the bone microenvironment. Interleukin-1 beta (IL-1β) and IL-17 act as osteolytic factors and promote osteolytic lesions in breast cancer pateints by increasing osteoclastogenesis and decreasing osteoblastogenesis. Especially, both of IL-1β and IL-17 increase in patients with breast cancer and decrease disease-free survival in cancer patients. Wogonin, one of the major flavonoids in the roots of Scutellaria baicalensis Georgi, has been recognized as a potent anti-cancer agent through increased apoptosis and decreased proliferation. We investigated whether wogonin could reduce the cancer cell-induced osteolysis by controlling interaction between cancer cells and bone-related cells. Wogonin suppressed cell viability, DNA synthesis and migration in MDA-MB 231 cells. Wogonin reduced the secretion of IL-1β and IL-17 in MDA-MB 231 cells. Wogonin at non-cytotoxic concentrations inhibited the receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclast formation in mouse bone marrow-derived macrophages. Furthermore, wogonin blocked an increase in RANKL/osteoprotegerin mRNA ratio in the osteoblastic hFOB1.19 cells exposed to MDA-MB 231 cells-derived conditioned medium. Finally, oral administration of wogonin significantly inhibited osteolytic lesions in MDA-MB 231 cells-injected mice. Taken together, these results indicate that wogonin is a promising agent for preventing and treating cancer-cell mediated bone loss.

Citation Format: Hyungkeun Kim, Kwang-Kyun Park, Won-Yoon Chung. Wogonin suppresses the production of breast cancer-derived osteolytic factors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1543. doi:10.1158/1538-7445.AM2015-1543
Abstract 1544: Association of tumor location with the breast and clinicopathological characteristics
Authors: Rummel Seth , Costantino Nick , Hueman Matthew T. , Shriver Craig D. , Ellsworth Rachel E.
Published: August 2015

Introduction Tumor location within the breast varies, with the highest frequency in the upper outer quadrant (UOQ) and lowest in the lower inner quadrant (LIQ). What is not well understood is whether tumor location is prognostic. To determine whether tumor location is prognostic, associations between tumor site and clinicopathological characteristics were evaluated.

Methods The Clinical Breast Care Project database was queried to identify all patients whose tumor site- UOQ, upper inner quadrant (UIQ), central, (LIQ), lower outer quadrant (LOQ) - was determined by a single, dedicated breast pathologist between 2001 and 2013. Patients with multicentric disease (n = 122) or tumors spanning multiple quadrants (n = 381) were excluded from further analysis. Clinicopathological characteristics evaluated included age at diagnosis, ethnicity, BMI, tumor stage, grade, and size, and hormone receptor, HER2 and lymph node status. Data was analyzed using chi-square and Mann-Whitney tests for univariate analysis with multivariate analysis performed using principal components analysis and multiple logistic regression. Significance was defined as P<0.05.

Results Of the 980 patients with defined tumor location, 30 had bilateral disease. Tumor location in the UOQ (51.5%) was higher than in the UIQ (15.6%), LOQ (14.2%), central (10.6%) or LIQ (8.1%). Tumors in the central quadrant were significantly more likely to have higher tumor stage (P = 0.003) and size (P<0.001), positive lymph node status (P<0.001), and mortality rates (P = 0.011) compared to other quadrants. Analysis within each tumor size, however, found no difference in lymph node status by tumor location. After multivariate analysis, only tumor size and lymph node status remained significantly associated with mortality.

Discussion Although tumors in the central region are associated with less favorable outcome, this association was driven by significantly larger tumor size, which has been associated with positive lymph node status. Tumors in the central region are known to be more difficult to detect mammographically and the majority of tumors in this region are palpable, thus, tumor location is not an independent prognostic factor, but rather less favorable prognosis, including larger tumor size, higher grade and more lymph node involvement in the central region can be attributed to delayed diagnosis.

Citation Format: Seth Rummel, Nick Costantino, Matthew T. Hueman, Craig D. Shriver, Rachel E. Ellsworth. Association of tumor location with the breast and clinicopathological characteristics. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1544. doi:10.1158/1538-7445.AM2015-1544
Abstract 1549: Development and validation of a biomarker for prospective selection of Notch1 activation in patients with certain advanced solid tumors in a first-in-human phase1 study of the cancer stem cell targeting antibody OMP-52M51 (anti-Notch1)
Authors: Cancilla Belinda , Tam Raymond , Zhang Chun , Anderson Steve , Lewicki John , Hoey Tim , McCune Bryan , Johnson Lori , Idusogie Esohe , Kapoun Ann M.
Published: August 2015

The Notch pathway plays a key role in embryonic development, the regulation of stem and progenitor cells, and is implicated centrally in many forms of human cancer. Notch1 is known to be frequently activated in certain solid tumor types. OMP-52M51 is a humanized IgG2 antibody that inhibits the signaling function of the Notch1 receptor. Mouse xenograft studies using minimally-passaged, patient-derived xenografts have shown that OMP-52M51 impedes tumor growth and selectively eliminates CSCs in a range of tumor types particularly in tumors with activated Notch1 signaling.

We previously reported the frequency of Notch pathway activation across a large panel of human tumors (n>600) by an Immunohistochemistry (IHC) assay that detects the activated form of Notch1 using an antibody that specifically recognizes the Notch1 intracellular domain (ICD). Using this test and a rigorous H-score cut-off, we found elevated Notch1 ICD in 7-53% of the following cancers: chemo-resistant breast (29%), gastric (13%), cholangiocarcinoma (20%), esophageal (27%), hepatacellular carcinoma (HCC, 7%), small cell lung cancer (SCLC, 12%), pancreatic (12%) and colorectal cancer (53%).

Here we developed a specific CLIA-validated IHC assay capable of identifying patients with Notch1 activation. The assay has a rapid turn-around time and shows robust precision, reproducibility and accuracy. The sensitivity of the method was confirmed by comparing results obtained on xenograft tumor tissue samples with known levels of Notch1 expression and response to anti-Notch1 therapy. From these results, the cut-off of the assay was set. This predictive biomarker was specifically designed to screen patients for prospective selection in the first-in-human Phase1 study of OMP-52M51 (anti-Notch1) in certain advanced solid tumors (NCT01778439). Detailed assay validation data and its application to the analysis of clinical trial samples will be highlighted.

Citation Format: Belinda Cancilla, Raymond Tam, Chun Zhang, Steve Anderson, John Lewicki, Tim Hoey, Bryan McCune, Lori Johnson, Esohe Idusogie, Ann M. Kapoun. Development and validation of a biomarker for prospective selection of Notch1 activation in patients with certain advanced solid tumors in a first-in-human phase1 study of the cancer stem cell targeting antibody OMP-52M51 (anti-Notch1). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1549. doi:10.1158/1538-7445.AM2015-1549
Abstract 1555: An automated cage-based method of randomized group distribution in pre-clinical in vivo studies
Authors: Ibsen Eric M. , Kumer Jeffrey L.
Published: August 2015

To more closely emulate clinical research methodology, researchers are increasingly utilizing clinically-similar group randomization and distribution methods for oncology studies in animals. It has been common practice to re-house animals into new cages with new cagemates following randomized group distribution. In male animals in particular, this frequently results in high levels of stress, injury and sometimes death due to fighting to re-establish social dominance hierarchies. Since some animals suffer more and others less, based on their relative position within the newly-established hierarchy, the levels of stress vary across the study subject population. The full impact of such stress on anticancer agent efficacy is not known but is widely believed to significantly diminish study integrity. For this reason, many researchers do not re-house male animals, choosing instead to co-house animals from various groups. It is believed that the exposure of cagemates to test agents in excreta and saliva during grooming as well as significantly increasing the risk of accidental mis-dosing can likewise negatively impact study integrity. The commonly-used manual and spreadsheet-based randomization methods do not enable cage-based randomized distribution based on qualitative or quantitative parameters. Commercially available pre-clinical trial applications now offer a solution to the above-mentioned problems.Randomization or distribution of animals based on the mean tumor volume or other parameters within the same cage allow for distribution to groups based on cage means. The variety of cage-based randomization methods available include: pure randomization, stratified sampling randomization and block randomization across multiple numeric parameters. Also, deterministic and matched-pair distribution methods can be used as well.Subjects in cages may be excluded initially based on recorded qualitative observations, and then by acceptable value ranges for numeric parameters. ANOVA results of group means are displayed instantly to ensure similarity between groups. Using software to perform standardized cage-based randomization and distribution is more clinically relevant, mitigating the negative impacts associated with post-randomization re-housing of animals. This significantly decreases the negative impact on study integrity relative to spreadsheet-based methods.

Note: This abstract was not presented at the meeting.

Citation Format: Eric M. Ibsen, Jeffrey L. Kumer. An automated cage-based method of randomized group distribution in pre-clinical in vivo studies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1555. doi:10.1158/1538-7445.AM2015-1555
Abstract 156: LncRNA BC200 is induced by estradiol and regulates apoptosis in MCF-7 breast cancer cells
Authors: Singh Ramesh , Mo Yin-yuan
Published: August 2015

LncRNA BC200 is a small functional RNA which has been implicated in the regulation of protein synthesis in neurons. It is dysregulated in invasive carcinomas of the breast. In this study, we reported that BC200 is upregulated in breast cancer, as detected by RT-PCR analysis of breast cancer tissue scan, suggesting its possible role as an oncogene. We then demonstrated that BC200 is transcriptionally induced by estradiol (E2) treatment in ER positive breast cancer cell lines through multiple functional estrogen response elements (EREs), including the full ERE sequence ∼800bp from transcriptional start site. Luciferase reporter assay combined with mutation analysis indicated that the full ERE region is critical to the estradiol-induced promoter activity. Furthermore, BC200 is crucial for cell growth and viability; its knockdown induces apoptosis in MCF-7 cells. This may explain in part why estrogen can promote cell proliferation in ER positive breast cancer cells. To better study its role in breast cancer development and progression, we generated BC200 knockout MCF-7 cell cells using CRISPR/Cas9 technique. BC200 knockout leads to the release of cytochrome c from mitochondria and promotes the cleavage of PARP-1, both of which are an indicator of cell apoptosis. This was further confirmed by TUNEL assay. It has been reported that hnRNP A2/B1 alters the bcl-X pre-mRNA splicing in favor of anti-apoptotic bcl-XL. Of great interest, BC200 knockout enhances expression of pro-apoptotic bcl-XS form at both mRNA and protein level. RNA pulldown assays using biotin-labeled BC200 probe suggested that BC200 can bind to hnRNP Q and hnRNP A2/B1. On the other hand, RNA immunoprecipitation using hnRNP A2/B1 antibody in BC200 knockout MCF-7 cells showed a significant reduction in enrichment of bcl-X mRNA as compared to control cells. We propose that the regulation of bcl-X alternative splicing depends upon endogenous expression of BC200 that would help hnRNP A2/B1 to position itself on bcl-X mRNA and modulate splicing in favor of bcl-XL. Thus, loss of BC200 leads to generation of bcl-XS protein synthesis and to apoptosis. Together, these results suggest that BC200 functions as a negative regulator of apoptosis in breast cancer and thus, it may serve as a potential target for cancer therapies.

Citation Format: Ramesh Singh, Yin-yuan Mo. LncRNA BC200 is induced by estradiol and regulates apoptosis in MCF-7 breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 156. doi:10.1158/1538-7445.AM2015-156
Abstract 1560: FGFR4 is expressed in the tumor and stromal compartments of human gliomas of all grades and histologies
Authors: Gjerde Christiane H. , Mutlu Ercan , Leiss Lina , Kristensen Bjarne Winther , Rahman Mohummad Aminur , Enger Per Øyvind
Published: August 2015

BACKGROUND: Fibroblast growth factor receptor (FGFR) 4 is reportedly expressed in several cancer types, and is a high-affinity transmembrane receptor for FGF2 which is expressed human astrocytomas. However, the expression of FGFR4 across gliomas of different grades and histologies has not been extensively investigated.

METHODS: We performed immunohistochemistry (IHC) of 178 grade II-IV human gliomas from our tumor bank, including 117 GBMs and 61 lower grade gliomas of astrocytic and oligodendroglial lineage. The samples were scored (0-2) separately for staining intensity and fraction of area that was stained and whether staining was predominantly cytoplasmic or nuclear. This scoring was performed separately for tissue from tumor center and periphery, as well as in tumor areas around pseudopalisading (PP) and large necroses. We also performed western blot analysis (n = 22) and real-time qPCR (n = 37). On one tumor sample we performed FISH for EGFR (red)/Centromere chromosome 7 (green), combined with immunofluorescence (IF) for FGFR4.

RESULTS: I) FGFR4 is uniformly expressed in human gliomas, although at a varying degree, as confirmed by IHC, western blotting and RT-qPCR. II) FGFR4 expression was detected both in the nuclei and cytoplasm. III) Both a homogenous and a granular staining pattern was observed the cytoplasm, suggesting involvement of different cell types, whereas FISH demonstrated expression in the tumor and stromal cell compartments. IV) Tumors of grade II and III show a statistically significant higher expression than tumors of grade IV as shown by RT-qPCR. V) There is no difference in expression by IHC between tumors of grade II, III and IV.

Citation Format: Christiane H. Gjerde, Ercan Mutlu, Lina Leiss, Bjarne Winther Kristensen, Mohummad Aminur Rahman, Per Øyvind Enger. FGFR4 is expressed in the tumor and stromal compartments of human gliomas of all grades and histologies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1560. doi:10.1158/1538-7445.AM2015-1560
Abstract 1564: Evaluation and characterization of anti-RalA autoantibody as a potential biomarker in human prostate cancer
Authors: li jitian , Dai Liping , Casiano Carlos A. , Zhang Jianying
Published: August 2015

Autoantibodies against intracellular tumor associated antigens (TAAs) are commonly found in various types of human cancers. Their utility in providing insights into tumor biology and as potential cancer biomarkers is well documented. In this study, serum autoantibodies to the Ras family small GTP binding protein (RalA) were detected by immunofluorescence assay, enzyme-linked immunosorbent assay (ELISA), and immunoblotting in 353 sera from patients with prostate cancer (PCa), benign prostatic hyperplasia (BPH), and normal controls. Expression of RalA in prostate tumor tissues was evaluated by immunohistochemistry. The cumulative positive rate of autoantibodies against RalA in PCa reached 18.4%, higher than that in sera with BPH (14.7%) and normal human sera (NHS) (2.25%, P<0.01). Twenty seven of 32 (84.4%) sera with positive OD values were consistently positive in immunoblots. The sensitivity and specificity of anti-RalA antibody in PCa detection was 18.4% and 97.8%, respectively. Representative PCa sera showed a cytoplasmic immunofluorescence staining pattern, with more intense staining in the perinuclear regions or partial staining in intracellular membrane. This pattern was similar to that produced by monoclonal RalA antibody. Strong expression of RalA was observed in 76.5% of prostate tumor tissues. Anti-RalA and PSA simultaneously used as diagnostic markers improved the sensitivity of PCa detection to 57.4%. In addition, the appearance of autoantibodies to RalA was assessed over time in the serially collected samples after surgery in cases. In all cancer patients with anti-RalA positive, autoantibodies were detected in 6 of 54 serial samples taken after surgery with a median lead time of 121 days (Interquartile Range, IQR 0-360). In conclusion, autoantibody to RalA might be a potential serological biomarker for the immunodiagnosis of early stage PCa. We are currently identifying additional novel TAAs targeted by serum autoantibodies in PCa by using two-dimensional gel electrophoresis (2DE) and liquid chromatography-tandem-mass spectrometry (LC-MS/MS). The combination of RalA with PSA, and with other antigens in PCa-specific TAA arrays has the potential for significantly improving the sensitivity of early PCa detection and management in diverse populations.

Key words: anti-RalA autoantibody; tumor-associated antigens (TAAs); cancer immunodiagnosis; prostate cancer

Citation Format: jitian li, Liping Dai, Carlos A. Casiano, Jianying Zhang. Evaluation and characterization of anti-RalA autoantibody as a potential biomarker in human prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1564. doi:10.1158/1538-7445.AM2015-1564
Abstract 1566: Association of a serum HPV16 IgG signature and risk of oropharyngeal cancer
Authors: Anderson Karen S. , Langseth Hilde , Wallstrom Garrick , Posner Marshall , Cheng Julia N. , Alam Rizwan , Chowell Diego , Mork Jon
Published: August 2015

Introduction: Human papillomavirus type 16 (HPV16) infection is associated with an increasing fraction of oropharyngeal cancer. The aim of our study was to determine the association of HPV16 antibodies (Abs) and oropharyngeal cancer (OPC) risk in sera obtained prior to clinical diagnosis.

Methods: We identified 92 participants with incident oropharyngeal cancers and 460 age-, gender-, region- and year- matched controls from the Janus Serumbank in Norway. Serum samples were collected from cases, on average, 9.2 years before diagnosis (range, -0.6-14.9 years). 11 cases had serum samples from multiple time points. IgG Abs to the HPV16 antigens E1, E4, E5, E6, E7, L1, L2, and the N-terminal and C-terminal fragments of E2 (NE2, CE2) were quantified in blinded fashion using a custom RAPID ELISA assay. Full-length genes were expressed as GST fusion proteins with a mammalian protein expression system, incubated with sera, and bound IgG was measured. The ratio of relative luminescence units (RLU) values for each antigen to control GST protein was determined. Cutoff values were pre-established as 3 SD>median of 247 healthy controls. A logistic regression classifier of a 7-Ab biomarker panel (E1, NE2, CE2, E4, E5, E6, E7) for the predictive diagnosis of OPC was trained on an independent sample set, and applied to these samples. P-values were calculated by Fisher's exact test. Odds ratios (ORs) of cancer and 95% CIs were calculated.

Results: HPV16 early antigen seropositivity was present in 13% of patients with OPC and 1% of controls (OR, 17.0; 95% CI, 5.0-74.0, p<0.0001). The probability of seropositivity was inversely associated with the time from serum collection and diagnosis (0-2 years, 84.9; >10 years, 5.4). Abs to CE2 were strongly associated with cases 0-2 years pre-diagnosis (OR, 112.0; 95% CI, 15.0-971.2, p<0.0001).

Conclusion: HPV16 Abs to multiple early viral antigens are detectable years prior to diagnosis of oropharyngeal cancer, and the probability of seropositivity increases closer to diagnosis. A model for screening would help identify individuals at risk for whom interventions and surveillance may be evaluated.

Citation Format: Karen S. Anderson, Hilde Langseth, Garrick Wallstrom, Marshall Posner, Julia N. Cheng, Rizwan Alam, Diego Chowell, Jon Mork. Association of a serum HPV16 IgG signature and risk of oropharyngeal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1566. doi:10.1158/1538-7445.AM2015-1566
Abstract 1569: Development of a noninvasive and sensitive urine screening test for liver cancer targeting circulation-derived cancer DNA biomarkers
Authors: Jain Surbhi , Chen Sitong , Lin Selena Y. , Clemens Adam , Hann Hei-won L. , Chang Ting-Tsung , Hu Chi-Tan , Chen Shun-Hua , Song Wei , Su Ying-Hsiu
Published: August 2015

Hepatocellular carcinoma (HCC), or liver cancer, is an aggressive disease and is regarded as one of the fastest growing cancers in the United States in incidence. The 5-year survival rate of HCC patients drops dramatically from a poor 26%, in early-stage cancer, to a mere 2% in late stages of this disease. The only available early detection biomarker is serum alpha-fetoprotein, which can detect only 40-60% of cases. Our goal is to develop a screening test for early detection of liver cancer, in order to meet this urgent need for new and better biomarkers for HCC. Cancer is a disease of the genome and epigenome, and if we can detect these underlying genetic mutations and epigenetic modifications in the periphery, we can effectively detect cancer early. Hence, we chose a panel of both HCC-associated genetic and DNA methylation biomarkers. We have previously shown that urine contains fragmented cell-free DNA derived from the circulation and that we can detect cancer-related DNA, both mutated and methylated DNA, in the urine of such patients. We have also demonstrated that the concentration of tumor-derived DNA in plasma and in urine is similar in patients with tumors. In order to demonstrate the feasibility of detecting HCC DNA biomarkers in the circulation derived urine DNA, we have developed short amplicon (∼50 bp) PCR-based assays targeting HCC-specific mutations in TP53 and CTNNB1 genes and HCC-specific methylation in GSTP1, RASSF1A and CDKN2A genes. Urine DNA isolated from samples of hepatitis, cirrhosis, and HCC patients were tested in each of the assays and analyzed for individual marker and panel performance. The performance of the markers and our approach towards the development of a targeted next generation urine DNA based HCC screening test is discussed.

Citation Format: Surbhi Jain, Sitong Chen, Selena Y. Lin, Adam Clemens, Hei-won L. Hann, Ting-Tsung Chang, Chi-Tan Hu, Shun-Hua Chen, Wei Song, Ying-Hsiu Su. Development of a noninvasive and sensitive urine screening test for liver cancer targeting circulation-derived cancer DNA biomarkers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1569. doi:10.1158/1538-7445.AM2015-1569
Abstract 1575: Serum circulating miR–Test application: Standardized protocol for miR–Test clinical application
Authors: Montani Francesca , Marzi Matteo , Dezi Fabio , Dama Elisa , Carletti Rose Mary , Veronesi Giulia , Nicassio Francesco , Di Fiore Pier Paolo , Bianchi Fabrizio
Published: August 2015

Lung cancer is leading cause of cancer death worldwide. Being lung cancer asymptomatic in its early stages, the majority of patients are diagnosed with advanced disease. Therefore, it is vital that screening programs and novel diagnostic tools are developed to increase lung cancer early detection. The development of a minimally invasive blood-based diagnostic tool would be ideal as a first-line screening procedure. An increasing number of studies are demonstrating that fluctuations of circulating miRNAs are associated to lung cancer. Recently, we described a serum circulating miRNA signature (miR-Test) diagnostic for asymptomatic, early stage, lung cancer, that was validated in a large cohort of individuals (N = 1115) enrolled in the lung cancer screening program COSMOS (Continuous Observation of SMOking Subjects). However, the transfer to the clinic of a blood test based on circ-miRNAs requires the establishment of standardized operating procedures (SOPs), working instructions and guidelines for all pre-analytical and analytical procedures.

We identified possible sources of variability affecting circulating miRNAs, analyzed their impact on the miR-Test performance, and defined a standardized protocol to optimize miR-Test application. Analysis of all possible technical and biological variation affecting circ-miRNAs level, revealed two main sources of variability: one related to analytical procedures for miRNAs extraction and quantification, and the other due to pre-analytical conditions, on how samples are prepared. The extraction causes the main source of analytical imprecision.

In conclusion, we identified an optimal protocol for the application of miR-Test for lung cancer early diagnosis.

Citation Format: Francesca Montani, Matteo Marzi, Fabio Dezi, Elisa Dama, Rose Mary Carletti, Giulia Veronesi, Francesco Nicassio, Pier Paolo Di Fiore, Fabrizio Bianchi. Serum circulating miR–Test application: Standardized protocol for miR–Test clinical application. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1575. doi:10.1158/1538-7445.AM2015-1575
Abstract 1581: A study of pulmonary and peripheral vein blood as sources of circulating tumor cells in early lung cancer
Authors: Murlidhar Vasudha , Reddy Rishindra M. , Azizi Ebrahim , Zhao Lili , Grabauskiene Svetlana , Zhang Zhuo , Ramnath Nithya , Lin Jules , Chang Andrew C. , Carrott Philip W. , Lynch William R. , Orringer Mark B. , Wicha Max S. , Palanisamy Nallasivam , Beer David G. , Nagrath Sunitha
Published: August 2015

Lung cancer is one of the most prevalent cancers world-wide. It is usually diagnosed at an advanced stage with low survival rates. Early detection and intervention can have a profound impact in improving survival. Circulating tumor cells (CTCs), or cells shed by the tumor into the blood circulation, offer a less invasive method of tumor biopsy. Surgical lobectomy for early stage lung cancers provides access to the pulmonary vein (PV) draining the affected lobe, and is presumably an enriched source of CTCs shed by the primary lung tumor. Analyzing peripheral (Pe) and pulmonary vein (PV) blood combined could potentially shed light onto optimal markers and disease progression. We hypothesize that blood from the pulmonary vein has a higher abundance of CTCs than peripheral blood, thereby providing a larger yield of CTCs from early lung cancer patients for downstream analysis. A cohort of greater than 30 lung cancer patients was enrolled in a pilot study. PV and Pe blood specimens from these patients were obtained at different time points in the perioperative time period, and were processed through a high-throughput microfluidic device, the OncoBean Chip that isolates CTCs by immuno-affinity based capture. A higher number of PV CTCs were detected than from Pe. In addition, rare CTC clusters were observed in the PV blood in some cases, which were further characterized for protein expression. Gene expression analysis was performed for comparison of CTCs obtained from these two different sources. Evaluating different CTC sources along the venous drainage of a tumor could potentially offer an insight into CTC biology and spread of the disease.

Citation Format: Vasudha Murlidhar, Rishindra M. Reddy, Ebrahim Azizi, Lili Zhao, Svetlana Grabauskiene, Zhuo Zhang, Nithya Ramnath, Jules Lin, Andrew C. Chang, Philip W. Carrott, William R. Lynch, Mark B. Orringer, Max S. Wicha, Nallasivam Palanisamy, David G. Beer, Sunitha Nagrath. A study of pulmonary and peripheral vein blood as sources of circulating tumor cells in early lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1581. doi:10.1158/1538-7445.AM2015-1581
Abstract 1589: Investigation of circulating tumor cells from head and neck cancer patients undergoing radiation therapy: A pilot study
Authors: Eblan Michael J. , Myung Ja Hye , Caster Joseph M. , Chera Bhishamjit S. , Hong Seungpyo , Wang Andrew Z.
Published: August 2015

Background: Circulating tumor cell (CTC) enumeration provides prognostic information in patients with metastatic breast, prostate, colorectal and lung cancer. However, the effect of radiation therapy (RT) on CTCs in patients with primary head and neck malignancies has not been explored. The purpose of this exploratory study is to examine how CTCs, as measured by a novel cell capture technique, respond to RT in patients with head and neck cancer. Methods: A total of a 16 patients undergoing definitive radiation for primary, non-metastatic head and neck cancer were enrolled in this pilot study. Peripheral blood was collected at 5 time points, including at baseline prior to starting RT, at the first week, mid-point and final week during treatment, and at least 4 weeks after completion of RT. Quantification of CTCs was performed with a novel device that used a biomimetic combination of three cancer cell-specific antibodies (aEpCAM, aHer-2, and aEGFR) immobilized through poly(amidoamine) dendrimers and E-selectin, which respectively induce concurrent stationary binding and dynamic cell rolling of the tumor cells. Results: CTCs were successfully detected in all patients before the start of radiation (100%), including patients with HPV/p16 positive or negative tumors. The novel CTC device yielded up to a 4-fold enhancement in capture efficiency relative to the aEpCAM-functionalized surface. The average CTC count in patients before RT was 386 CTCs per mL (range, 18 to 1134 CTCs per mL), significantly higher than the average CTC count of 90 CTCs per mL after the completion of radiation (range, 5 to 393 CTCs per mL). All but one patient demonstrated a decrease in measured CTCs at sequential time points during the course of RT. The patient with an elevation in CTCs during RT had residual metastatic carcinoma detected after left level II selective cervical neck dissection, which was performed 12 weeks post-RT due to persistent radiographic lymph nodal abnormality. At a median follow-up of 1.5 months, no patient has had a local or distant failure. Conclusions: We have demonstrated that our novel technology can affectively capture CTCs in head and neck cancer patients. Importantly, our pilot data shows CTCs decrease over the course of RT, suggesting CTCs can be a predicative biomarker for radiotherapy response. Further study will investigate associations between CTC kinetics and clinical outcomes.

Citation Format: Michael J. Eblan, Ja Hye Myung, Joseph M. Caster, Bhishamjit S. Chera, Seungpyo Hong, Andrew Z. Wang. Investigation of circulating tumor cells from head and neck cancer patients undergoing radiation therapy: A pilot study. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1589. doi:10.1158/1538-7445.AM2015-1589
Abstract 159: LncRNA RoR enhances the c-Myc mRNA stability in colon cancer
Authors: Huang Jianguo
Published: August 2015

Background: Emerging evidence has implicated long non-coding RNAs (lncRNAs) as master gene regulators; and they are often aberrantly expressed in a variety of human diseases including cancer. One of the mechanisms of lncRNA-mediated gene expression involves modulation of translation or mRNA stability by interacting with RNA binding proteins. We have previously demonstrated that lncRNA-RoR is a strong negative regulator of p53 in response to DNA damage. The present study was to determine whether and how lncRNA-RoR regulates c-Myc in colon cancer.

Methods: MTT assays were used to determine cell growth. Colon cancer tissue array was used to determine the level of lncRNA-RoR in colon cancer tissues by qRT-PCR. Interaction between lncRNA-RoR and RNA binding proteins was confirmed by RNA co-immunoprecipitation and RNA precipitation. LncRNA-RoR knockout in colon cancer cells was through CRISPR/Cas9 system. Dual fluorescent in situ hybridization (FISH) was used to determine the relative levels of lncRNA-RoR and c-Myc mRNA, and their co-localizations in colon cancer specimens. The effect of lncRNA-RoR on tumor growth was determined by a xenograft mouse model.

Results: Ectopic expression of lncRNA-RoR upregulated, whereas RoR-siRNA inhibited c-Myc mRNA and protein level, which was further confirmed by lncRNA-RoR knockout. Mechanistically, we showed that lncRNA-RoR directly interacted with RNA binding proteins, hnRNP U and hnRNP D, both of which are implicated in stability of c-Myc mRNA. This interaction was further confirmed by RNA immunoprecipitation. In consistent with these findings, ectopic expression of lncRNA-RoR stimulated cell proliferation but knockout of lncRNA-RoR significantly decreased cell proliferation. Moreover, ectopic expression of lncRNA-RoR in HCT-116 cells significantly promoted tumor growth in a xenograft mouse model. Finally, qPCR array showed the significant upregulation of lncRNA-RoR in colon cancer tissues. Similarly dual FISH revealed a strong correlation between lncRNA-RoR and c-Myc mRNA in colon cancer tissues.

Conclusion: It is well known that c-Myc mRNA has a very short half-life and regulation of c-Myc mRNA stability is complex. Our results suggest a novel mechanism of regulation of c-Myc mRNA stability involving lncRNA-RoR, hnRNP U and hnRNP D.

Citation Format: Jianguo Huang. LncRNA RoR enhances the c-Myc mRNA stability in colon cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 159. doi:10.1158/1538-7445.AM2015-159
Abstract 1590: CD133-expressing circulating tumor cells might predict poor prognosis in patients with head and neck cancer
Authors: Hsieh Jason Chia-Hsun , Shiao Harber Hung-Bo , Chao Bill Yuan-Jhih , Lin Ming-Jung , Chang John Wen-Cheng , Wang Hung-Ming , Wu Tyler Min-Hsien
Published: August 2015

PURPOSE: The prognostic value of circulating tumor cells (CTCs) number in patients with head and neck carcinoma (HNSCC) has been reported to be positive. The hypothesis of the additional markers on CTCs might predict clinical outcomes have been proposed. This study aimed to elucidate the prognostic/predictive value of CD133-expressing CTCs in HNSCC patients.

PATIENTS AND METHODS: In a prospective single-center study, CTCs were enumerated in the peripheral blood of 24 subjects, including 14 patients with HNSCC and 10 healthy donors, at baseline and 2 weeks after starting first dose of chemotherapy. CTCs were measured using an immunomagnetic separation technique by combined negative and positive selection strategies. Immunofluorescence staining methods were applied for surface marker identification. Tumor response at 12 weeks after chemotherapy of the whole cohort was performed by computed tomograpgy or magnetic resonance imaging scans following RECIST criteria 1.1.

RESULTS: In the first step of efficacy evaluation phase, an average recovery rate of 85.5% and a R2 value of 0.9932 were found when compared between spiked and recovered blood samples. A CTCs detection rate of 100.0% (14/14) was found by this protocol and the number could successfully differ HNSCC patients from healthy donors (P = 0.001). The mean and median (range) of baseline CTC number were 105.0 and 87.2 (34.5-358.1) cells/mL of blood. The higher baseline CTCs number was associated with higher tumor stage (AJCC 7th edition, P = 0.003). The higher absolute number (>5cells/mL of blood) of baseline CD133-expressing CTCs could predict poor survivals (Kaplan-Meier survival curves, P = 0.013). In all assessable patients, a high correlation rate of 92.9% was found between changes of CTCs (decline or elevation) and CT responses (progressive disease [PD] and partial response [PR]/Stable disease [SD]) with a P value of 0.005 (Fisher exact test).

CONCLUSION: The number of CD133-expressing CTCs might be a useful prognostic for survivals and predictors for imaging response evaluation at 12 weeks in patients with HNSCC. Further investigation in large-scale prospective trials is warranted on the basis of the findings in this study.

Citation Format: Jason Chia-Hsun Hsieh, Harber Hung-Bo Shiao, Bill Yuan-Jhih Chao, Ming-Jung Lin, John Wen-Cheng Chang, Hung-Ming Wang, Tyler Min-Hsien Wu. CD133-expressing circulating tumor cells might predict poor prognosis in patients with head and neck cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1590. doi:10.1158/1538-7445.AM2015-1590
Abstract 1596: Detection of PD-L1 in cell surface vimentin positive circulating tumor cells is associated with poor survival in cancer patients
Authors: Satelli Arun , Brownlee Zachary , Noh Hyangsoon , Meng Qing H. , Kopetz Scott , Overman Michael , Li Shulin
Published: August 2015

Circulating tumor cells (CTCs) are the cells that detach from the primary tumor of patients and enter in to the blood stream and these can be represented as the seeds of metastasis. Increasing evidence has shown the detection of circulating tumor cells (CTCs) from blood of cancer patients parallels with tumor burden and is associated with poor prognosis. Although CTC counts change indicates a good modality to detect therapeutic response to drug treatments, there is an increasing need of a reliable marker that can be used to predict survival in cancer patients. One of the most prevalent markers detected in cancer patients is the cell surface glycoprotein called PD-L1 (also called B7-H1 and CD274). Aberrant expression of PD-L1 has been reported in several cancer types. Here in this study we tested the hypothesis that detection of PD-L1 in CTCs is associated with poor prognosis. To validate the hypothesis, we isolated cell-surface vimentin (CSV) positive CTCs from colorectal cancer patients using 84-1 method and analyzed for PD-L1 expression. Our results indicated that PD-L1 detection in CTCs was associated with poor overall survival (HR, 2.43; 95% CI, 1.11 to 5.35; p, 0.0264) in colorectal cancer patients undergoing treatment. These findings thus suggest that PD-L1 detection in CSV CTC could serve as a new prognostic tool. We further extend our observations in other types of cancers including breast, prostate and sarcoma.

Citation Format: Arun Satelli, Zachary Brownlee, Hyangsoon Noh, Qing H. Meng, Scott Kopetz, Michael Overman, Shulin Li. Detection of PD-L1 in cell surface vimentin positive circulating tumor cells is associated with poor survival in cancer patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1596. doi:10.1158/1538-7445.AM2015-1596
Abstract 1598: Isolation and characterization of circulating melanoma cells by size filtration and fluorescent in situ hybridization
Authors: Yanagita Masahiko , Luke Jason J. , Paweletz Cloud P. , Ivanova Elena , Kirschmeier Paul T. , Hodi Stephen F. , Jänne Pasi A.
Published: August 2015

Somatic mutations in BRAF are predictive of the clinical efficacy of BRAF and/or MEK inhibitors for advanced melanoma. Resistance to treatment is observed for most patients despite a high response rate to kinase inhibitors. While repeated tumor biopsy is the gold standard for evaluating genetic changes following treatment, this is not generally feasible. The analysis of circulating melanoma cells (CMCs) has the potential to be a non-invasive means of interrogating the biology underlying response and resistance.

Here we report on the isolation, detection, and characterization of CMCs from patients using size filtration followed by immunoflourescence and/or fluorescent in situ hybridization (FISH) analyses. Blood from a cohort of 20 patients with advanced melanoma was collected in an initial proof of concept experiment. CMCs were identified in 14 out of 20 melanoma blood samples (70%) with a range of 2.5-99 CMCs per 3mL of blood by immunofluorescence (IF) using a cocktail of 4 melanoma specific antibodies (HMB45, MART-1, Tyrosinase and MiTF). Cells that appeared tumor like by microscopy, but that did not stain for CD45 or the cocktail of melanoma specific antibodies were also detected. To further characterize these cells, FISH was performed with a set of 4 melanoma-specific probes (RREB1, MYB, Centromere 6, CCND1). In a cohort of 13 patients with melanoma specimens that had paired blood specimens, CMCs were detected in all 13 specimens ranging from 7.2-76 CMCs per 3mL of blood with more identification by FISH in 10 out 13 samples. Additionally, cells that were double negative by IF were observed to be positive by FISH.

CMCs are detectable in the majority of patients with advanced melanoma. Use of IF and FISH can help detect and characterize CMCs. These tools will be useful in characterizing treatment related changes in CMCs.

Citation Format: Masahiko Yanagita, Jason J. Luke, Cloud P. Paweletz, Elena Ivanova, Paul T. Kirschmeier, Stephen F. Hodi, Pasi A. Jänne. Isolation and characterization of circulating melanoma cells by size filtration and fluorescent in situ hybridization. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1598. doi:10.1158/1538-7445.AM2015-1598
Abstract 1607: Identification of low number circulating tumor cells (CTCs) for cancer treatment monitoring
Authors: George Thomas J. , Sheng Weian , Varillas Jose I. , Liu Chen , Fan Z. Hugh
Published: August 2015

For most solid tumors, the clinical use of CTCs is limited by current instrument restrictions on detection of infrequent CTCs. Based on the FDA-approved criteria used in current CTC detection instruments, we developed a microfluidic device capable of capturing, enumerating and releasing low numbers of CTCs in the peripheral blood of patients with solid tumors. The device is the size of a microscope slide, consisting of an inlet, an outlet, and eight parallel channels connecting them. Micromixers inside the channels enhance interactions between CTCs and capture agents on the channel surfaces. Epithelial cell-adhesion molecule (EpCAM) antibodies are immobilized onto the surfaces via avidin-biotin chemistry. A blood sample from a cancer patient is introduced through the inlet. Cells captured by anti-EpCAM studded channels are verified as CTCs using triple-color IHC definitions (EpCAM+, cytokeratin+, CD45-, and DAPI+). Using pancreatic cancer as a model for a low frequency CTC condition, device capture efficiency and cell purity was tested under various flow rates (shear stresses) using L3.6pl cells spiked into blood. Capture efficiency was defined as the% of target cells isolated relative to target cells present in a sample, representing device sensitivity. Cell purity was defined as the% of target cells isolated relative to all cells captured (both target and non-target cells), representing device specificity. Capture efficiency (90%) and cell purity (84%) were further optimized by changing the flow rate. A higher flow rate resulted in higher cell purity as non-target bound cells are dislodged by shear forces. This capability of parameter adjustment and performance control is enabled by the microfluidics platform used as opposed to CTC isolation using other commercially approved technologies. The device was then clinically tested in detecting CTCs and monitoring the response to anti-cancer drug treatment in patients with advanced pancreatic cancer. We identified CTCs in 113 out of 115 clinical blood samples (98%). CTC enumeration (CTC number/mL of blood) both correlated with and predated changes in the radiographic tumor size over the treatment period, ranging from 10 to 35 cycles (2 weeks per cycle) of therapy. Microfluidic devices possess the capacity to enumerate infrequent CTCs which also appear to correlate with treatment response.

Note: This abstract was not presented at the meeting.

Citation Format: Thomas J. George, Weian Sheng, Jose I. Varillas, Chen Liu, Z. Hugh Fan. Identification of low number circulating tumor cells (CTCs) for cancer treatment monitoring. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1607. doi:10.1158/1538-7445.AM2015-1607