bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 147: Exome sequencing identifies frequent histone methyltransferase mutations in metastatic SCC
Authors: Toland Amanda Ewart , Allain Dawn , Gillespie Jessica , Nagarajan Priyaharsini , Peters Sara , Ozer Hatice Gulcin , Olencki Thomas , Yilmaz Ayse Selen , Teknos Theodoros
Published: July 2016

Although only a small proportion of cutaneous squamous cell carinomas (cSCC) metastasize, approximately 3900-8800 individuals in the United States die of metastatic cSCC each year. Little is known about the molecular events that lead approximately 2-6% of cSCCs to metastasize. Treatment efforts are hampered by a lack of ability to predict which lesions are more likely to recur or metastasize as these known correlates do not have high specificity for prediction. Furthermore, there is no FDA approved therapy that is specifically indicated for metastatic cSCC. Deep-sequencing efforts have identified targetable mutations and pathways for several cancers. Exome and targeted sequencing of primary cSCCs led to the discovery that inactivating mutations in NOTCH pathway genes occur in over 75% of tumors; however NOTCH mutations are not indicative of metastasis. Published studies identified frequent mutations in KMT2C and KMT2D in cSCCs with aggressive features. To identify mutations that are unique to or enriched in metastatic samples which may help to identify novel therapeutics for treating aggressive cSCCs, we performed exome sequencing of 37 samples consisting of matched normal DNAs, primary tumors and metastatic cSCCs. Many of the mutations found in the metastatic samples were also observed in the matched primary tumors. We found an enrichment of mutations in chromatin remodeling genes. Epigenetic regulatory genes mutated in the metastatic samples included KMT2D (64%), KMT2C (44%), SETD2 (45%), KAT6A (36%), EP300 (18%), and KDM6A/UTX (9%). We performed additional targeted deep-sequencing to evaluate whether candidate genes were frequently mutated in non-aggressive cSCCs. Initial analyses of targeted-sequencing of 10

non-aggressive primary cSCCs and 7 additional metastatic cSCCs as well as data from published exome studies on non-aggressive cSCCs suggest that chromatin remodeling genes are mutated at a much higher rate in metastatic cSCCs. These data provide potential pathways for therapeutic intervention for this deadly cancer.

Citation Format: Amanda Ewart Toland, Dawn Allain, Jessica Gillespie, Priyaharsini Nagarajan, Sara Peters, Hatice Gulcin Ozer, Thomas Olencki, Ayse Selen Yilmaz, Theodoros Teknos. Exome sequencing identifies frequent histone methyltransferase mutations in metastatic SCC. [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 147.
Abstract 1492: Development of a CD123xCD3 bispecific antibody to treat acute myeloid leukemia (AML)
Authors: Gaudet Francois , Nemeth Jennifer F. , McDaid Ronan , Li Yingzhe , Harman Benjamin , Millar Hillary , Teplyakov Alexey , Wheeler John , Luo Jinquan , Tam Susan , Wu Sheng-Jiun , Chen Emily , Babich Alexander , Elsayed Yusri , Attar Ricardo
Published: July 2016

AML is a heterogeneous disease characterized by uncontrolled clonal expansion of leukemic stem cells (LSCs). Current therapies for AML are not curative, in part due to their inability to eradicate LSCs from the bone marrow. T cell redirection has been shown to be effective in heme malignancies and represents a promising approach to treat AML by targeting markers differentially expressed on the cell surface of cancer cells. One marker, CD123 (α-chain of the interleukin-3 receptor) is often present on AML LSCs and blasts. We developed a human bispecific antibody (CD123xCD3; Ab-178) capable of binding to the extracellular domain of CD123 and the ϵ chain of CD3 on T cells to induce T cell–mediated tumor cell killing. This bispecific IgG4 antibody can recruit T cells to CD123+ AML cells (MOLM-13, KG-1 and OCI-AML5) and induce T cell activation as evidenced by CD69 and CD25 up-regulation on T cells. Ab-178 potently killed these CD123+ AML cell lines in vitro (EC50 = 0.51-0.91 nM) but not a CD123− cell line (JIM3). Ab-178 was also able to induce tumor inhibition (MOLM-13 cells) and regression (KG-1 cells) in murine xenograft CD123+ AML models in the presence of human PBMCs. Furthermore, this antibody was able to kill AML blasts in primary AML blood samples ex vivo in the absence of exogenous T cells (autologous setting; EC50 = 0.83 nM). Related bispecific antibodies directed against a viral epitope (nullxCD3 or CD123xnull) did not activate T cells or cause tumor cell killing in the various assays tested. Ab-178 had no impact on T cell activation when incubated with T cells alone. These results indicate that Ab-178 can potently and specifically activate T cells in the presence of CD123+ AML cells and induce their killing. Furthermore, because of the antibody format, this molecule is expected to have a longer half-life compared to smaller bispecific biologic scaffolds. Ab-178 is currently being evaluated pre-clinically for its potential to treat patients with AML.

Citation Format: Francois Gaudet, Jennifer F. Nemeth, Ronan McDaid, Yingzhe Li, Benjamin Harman, Hillary Millar, Alexey Teplyakov, John Wheeler, Jinquan Luo, Susan Tam, Sheng-Jiun Wu, Emily Chen, Alexander Babich, Yusri Elsayed, Ricardo Attar. Development of a CD123xCD3 bispecific antibody to treat 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 1492.
Abstract 1518: Lung adenocarcinoma differential expression analysis using the Maverix RNA-Seq pipeline
Authors: Fitzsimons Michael S. , Lee Mei-Chong Wendy , Lee Byung-In , Subramanian Lenin
Published: July 2016

Background

RNA-Seq is a powerful means of identifying changes in gene expression in cancerous tissue. Widespread adoption of RNA-Seq is hampered by lack of familiarity with the appropriate analysis tools and excessive turn around time. Maverix Biomics, Inc offers a scalable, reliable, validated, and easily accessible differential expression pipeline for translational and clinical researchers to analyze and explore RNA-Seq data. Here we describe the pipeline, demonstrate the available tools and visualizations, and provide benchmarks for completion time from lung adenocarcinoma.

Results

Lung adenocarcinoma samples appear very different compared to their normal counterparts in terms of overall gene expression and exhibit many differentially expressed genes. For analysis completion time, parallelization is far superior than the theoretical curve without parallelization. It took 7.9 hours to analyze two samples, but took only 13.1 hours to analyze 25 samples. We found 2246 significant genes common to three different differential expression tools: DESeq, edgeR, and cuffdiff.

Conclusions

The Maverix RNA-Seq pipeline is demonstrated here to be highly scalable, easy to use, and includes a range of tools and visualizations for understanding differential expressions results. This pipeline is a great analytic option to accelerate translational and clinical research in the field of oncology.

Citation Format: Michael S. Fitzsimons, Mei-Chong Wendy Lee, Byung-In Lee, Lenin Subramanian. Lung adenocarcinoma differential expression analysis using the Maverix RNA-Seq pipeline. [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 1518.
Abstract 1521: Transcriptional networks of the normal human kidney
Authors: Lindgren David , Hansson Jennifer , Nilsson Helen , Krawczyk Krzysztof , Johansson Martin , Axelson Håkan
Published: July 2016

The kidney is a complex but highly structured organ composed of more than 20 different cell types. Disturbances among these cell types leads to a plethora of diseases of acute or chronic type, some of which are life threatening. In addition, kidney cancer represents about 3 percent of all malignancies. Considerable efforts have been made to profile the different cell types of the kidney. However, these are mainly based on studies in rodents. In the current study we present a method, called composition based cellular stratification that define gene signatures specific to different cell types and regions of the kidney. The method uses correlation-based analyses of gene expression microarrays of needle core biopsies obtained from histologically normal kidney tissues. For example, a HNF-driven gene expression signatures of proximal tubuli cells could be defined. This signature was validated in publically available data at the gene expression as well as at the protein level. We employ these gene expression signatures as a framework to study expressional changes observed in renal cell carcinoma, thus increasing the possibility to pin-point cell specific alterations associated with the transformation of renal epithelial cells.

Citation Format: David Lindgren, Jennifer Hansson, Helen Nilsson, Krzysztof Krawczyk, Martin Johansson, Håkan Axelson. Transcriptional networks of the normal human kidney. [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 1521.
Abstract 1523: Cross-disciplinary methods for personalizing screening modalities for early gastric cancer intervention
Authors: Walker Rachel , Mejia Jaime , Enderling Heiko , Pimiento Jose M. , Coppola Domenico
Published: July 2016

Background.

Up to 80% of patients in the early stages of gastric cancer are asymptomatic, making early diagnosis and hence effective treatment challenging. The poor prognosis of patients with late diagnoses clearly necessitates the identification of markers for early detection and the development of streamlined screening protocols. This is particularly crucial in high-risk populations, for example regions in which Helicobacter Pylori infection and associated chronic inflammation, responsible for over 70% of gastric cancers worldwide, is prevalent. At present, an efficient and personalized program for early detection does not exist.

Methods.

Specific biomarkers for the progression of gastric cancer have been identified in preliminary studies, including CD44, Lgr5 and CD133. In retrospective tissue samples incremental increases in expression of these markers was observed during the progression from normal gastric tissue to precancerous histologic lesions such as intestinal metaplasia and ultimately dysplasia and carcinoma. We developed a mechanistic mathematical model capable of simulating these marker-positive population dynamics, calibrated using the existing clinical data. The predictive capability of the model is validated by comparison of simulated to actual marker expression patterns in an independent test cohort of endoscopic gastric biopsies taken at sequential points during individual patients’ disease progression.

Results.

Based on clinically obtainable, patient-specific tissue conditions, the computational model can simulate the dynamics of marker-positive cells and, when calibrated and validated with clinical data from specific patient populations, may forecast disease progression and suggest optimal screening schedules for individual patients.

Conclusions.

The tools of mathematical oncology have the potential to directly inform clinical screening protocols. Verified prognostic factors for disease progression can be incorporated into mathematical models to improve diagnostic accuracy and allow clinically-actionable screening optimization. This can guide personalized screening schedules according to current marker expression on a case-by-case basis to achieve more efficient prognosis and reduced healthcare costs.

Citation Format: Rachel Walker, Jaime Mejia, Heiko Enderling, Jose M. Pimiento, Domenico Coppola. Cross-disciplinary methods for personalizing screening modalities for early gastric cancer intervention. [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 1523.
Abstract 153: Patterns of genetic progression in high grade prostatic intraepithelial neoplasia
Authors: Jung Seung-Hyun , Shin Sun , Lee Sug Hyung , Chung Yeun-Jun
Published: July 2016

High grade prostatic intraepithelial neoplasia (HGPIN) is considered a neoplastic lesion that precedes prostate cancer (PCA). Genetically, HGPIN is known to have some driver mutations such as TMPRSS2-ERG fusion and 8p loss. However, we still do not know how the whole picture of somatic mutations in HGPIN is and what are added during the progression to PCA. To identify the genomic differences between HGPIN and PCA, we analyzed 20 regions of paired HGPIN and PCA from 6 patients by whole-exome sequencing and array-comparative genomic hybridization. As expected, the number of total mutations and CNAs of HGPINs were significantly fewer than those of PCAs. Mutations in FOXA1 and CNAs (1q and 8q gains) were detected in both HGPIN and PCA (common), suggesting their roles in early PCA development. Mutations in SPOP, KDM6A, and KMT2D were PCA-specific, suggesting their roles in HGPIN progression to PCA. The 8p loss was either common or PCA-specific. HRAS and FOXA1 mutations were PIN-specific. Our data show that PCAs are direct descendants of HGPINs in most cases that require more genomic alterations to progress to PCA. The nature of heterogeneous HGPIN population that might attenuate genomic signals should further be studied. Our results provide a clue to explain the long latency from HGPIN to PCA and provide useful information for clinical decision making in the genetic diagnosis of HGPIN and PCA.

Citation Format: Seung-Hyun Jung, Sun Shin, Sug Hyung Lee, Yeun-Jun Chung. Patterns of genetic progression in high grade prostatic intraepithelial neoplasia. [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 153.
Abstract 1538: Vimentin expression as a potential immunomarker of predicting aggressive disease in clear cell renal cell carcinoma
Authors: Mohamed Mahmoud , Hu Lulin , Liu Haiyan , Lin Fan , Williams Heinric
Published: July 2016

Vimentin is a type III intermediate filament protein that is expressed in mesenchymal cells. Overexpression of vimentin has been detected in prostate cancer, lung cancer, gastrointestinal cancer, and melanoma. Although vimentin has been used for diagnosing and classifying renal neoplasms, little is known regarding its prognostic value. This study examines the prognostic significance of vimentin expression in clear cell renal cell carcinoma (ccRCC), chromophobe renal cell carcinoma (chRCC) and oncocytoma. Our patient group (n = 88) consisted of 63 men and 25 women with a mean age of 59 years at the time of diagnosis. Paraffin embedded specimens from these patients’ renal tumors were selected for this study which included ccRCC (n = 66), chRCC (n = 11) and oncocytoma (n = 11). Vimentin expression was determined by a tissue microarray technique (TMA) performed by a single pathologist (HL). Tumor staining results were classified as negative (<5%), weak positive (5-25%), intermediate positive (25-50%), moderate positive (50-75%), or strong positive (>75%). Clinicopathologic data was also included tumor histology, tumor size, T stage, grade, presence of sarcomatoid differentiation, presence of metastasis, and survival outcome following diagnosis were determined for each patient. Chi square statistical test analysis was performed in order to test the association of clinical parameters with vimentin immunoreactivity. Our results show that positive (>5%) vimentin expression was present in 100% of ccRCC cases, 9% of oncocytoma, and 0% of chRCC cases. In addition, vimentin expression in ccRCC was associated with metastasis (p<0.047) but was not significantly associated with pathologic T stage (P<0.185), Fuhrman grade (P<0.636), or the presence of sarcomatoid differentiation (P<0.855). With a median follow up of 7.2 years, there were 26 deaths in the ccRCC and none in the chRCC or oncocytoma groups. Positive vimentin staining was not associated with overall survival in the ccRCC group. Our results suggest that vimentin may be valuable predictor of metastatic disease in ccRCC patients.

Citation Format: Mahmoud Mohamed, Lulin Hu, Haiyan Liu, Fan Lin, Heinric Williams. Vimentin expression as a potential immunomarker of predicting aggressive disease in clear cell renal cell 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 1538.
Abstract 1559: CCR5-CCRL2 regulates CCL5-induced organ specific metastasis: the in vitro and in vivo studies
Authors: Hsieh Chia-Ling , Liu Che-Ming , Sung Shian-Ying
Published: July 2016

Previous studies of soluble factors in the tumor associated stromal cells demonstrated the elevation of CCL5 expression in prostate cancer patient sera that correlated wit malignant cancer progression. To evaluate the expression level of chemokine receptors in response to chemokines released by reactive stroma, quantitative PCR to evaluate the mRNA level of chemokine receptors was performed. We noticed the significant increased of CCRL2 (86-fold) and CXCR4 (45-fold) in androgen independent prostate cancer (AIPC) cells. This increased of CCRL2 expression also confirmed in malignant lung, breast and rental cells. Knockdown of CCRL2 declined 75% of migration activities induced by CCL5, suggests CCRL2 involve in CCL5 induce cancer migratory activities (t-test, p<0.001). IHC analysis of CCRL2 in paired prostate cancer patients was performed and revealed increasing of CCRL2 expression in malignant prostate cancer locus, whereas no CCRL2 can be detected in the benign region of same patient. Proximity ligation assays (PLAs) of benign, high grade tumor and tissues collected from patients less than 5-year survive demonstrated the PLA signals only in the lethal progression on patients. In vivo xenograft mice model studies demonstrated the organ specific cancer metastasis can be enhanced after overexpressed CCRL2 in cancer cells. Molecular analysis of CCRL2 demonstrated that CCRL2 regulated EZH2 expression in prostate cancer cells. In summary, out studies indicated the increased expression of CCRL2 play a central role in cancer cell migration induced by chemokine CCL5. The released of CCL5, secreted by bone stromal cells after interaction with dormancy cancer cell, may induce directional migration and invasion of circulating cancer cells. Clinical analyses demonstrated increased of CCRL2 in malignant prostate cancer. Inhibition of these homing mechanisms might significantly decreased cancer cell metastatic activities of AIPC patients.

Citation Format: Chia-Ling Hsieh, Che-Ming Liu, Shian-Ying Sung. CCR5-CCRL2 regulates CCL5-induced organ specific metastasis: the in vitro and in vivo studies. [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 1559.
Abstract 1575: Intratumoral injection of interleukin-17 inhibits distant metastasis of radiation-induced recurrent tumor
Authors: Li Yi-Nan , Chen Fang-Hsin , Hong Ji-Hong , Chiang Chi-Shiun
Published: July 2016

Interleukin-17 (IL-17) is a pro-inflammatory cytokine that leads to the production of GM-CSF, G-CSF, IL-1, IL-6, TNF-α. It can be secreted by various cells, including T cells, NKT cells, fibroblasts, and even tumor cells. In addition, reports have shown that IL-17 could also promote tumor growth by recruiting and expanding granulocytic myeloid suppressor cells (G-MDSCs). In our preliminary study, we found that Tramp-C1, a murine prostate cancer cell line, had remarkable IL-17 level when it grew in pre-irradiated (PreIR) stroma. In contrast, B16F0, a murine melanoma cell line, had much less IL-17 and stronger tumor bed effect, in terms of tumor growth delay, but lung metastasis were formed gradually as B16F0 developed. When the recombinant IL-17 was directly injected into the B16F0 tumors growing in PreIR stroma, we found that the probability of lung metastasis was decreased despite a slight increase of tumor growth at primary site. This indicates that IL-17 may have different effects on irradiated tumor bed and distant metastatic site. The influence of IL-17 on tumor microenvironments of distant metastatic sites and irradiated tumor bed is currently under investigation. In conclusion, we propose a new therapeutic approach of reducing distant metastasis following radiation therapy.. This work was kindly supported by MOST-104-2314-B-182 -024 grant to Fang-Hsin Chen.

Citation Format: Yi-Nan Li, Fang-Hsin Chen, Ji-Hong Hong, Chi-Shiun Chiang. Intratumoral injection of interleukin-17 inhibits distant metastasis of radiation-induced recurrent tumor. [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 1575.
Abstract 1593: Stat3 signaling in erbB3-mediated epithelial-mesenchymal transition in erbB2-positive breast cancer cells
Authors: Lyu Hui , Wu Ying , Zhou Yan , Liu Bolin
Published: July 2016

Introduction: Understanding the mechanism of metastatic program is essential to reducing the mortality of cancer patients. It is well known that epithelial-mesenchymal transition (EMT) contributes to cancer metastasis. Recently, numerous studies have documented the role of erbB3-mediated oncogenic signaling in breast cancer progression. In this study, we have focused on investigating the effect of Stat3 activation on EMT-induced by erbB3 receptor in erbB2-positive (erbB2+) breast cancer cells.

Methods: Immunofluorescence analyses were performed to study the localization and expression of EMT markers in erbB2+ breast cancer cells. Lentiviral vector containing shRNA was used to specifically knockdown erbB3. Western blot analyses were performed to assess the expression and activation of proteins. Cell growth (MTS) assays were used to determine cell viability. Wound healing and transwell assays were used to examine the motility and migration of cells.

Results: We discovered that ectopic expression of erbB3 facilitated EMT, as evidenced by change of cell morphology, induction of mesenchymal markers Vimentin, Snail, Slug, ZEB1 and repression of the epithelial marker E-cadherin in erbB2+ breast cancer cells. Wound healing and transwell assays indicated that the capability of cell motility and migration was enhanced. In contrast, specific knockdown of erbB3 abrogated the observations described above. Moreover, elevated expression of erbB3 potently stimulated activation of Stat3, which has been validated to act as a promoter of tumor invasion and metastasis in many type cancers. Suppression of Stat3 signaling via its inhibitor, S3I-201, significantly alleviated EMT and cell motility/migration that were triggered by activation of erbB3. To further assess the clinical relevance of erbB3 signaling in cancer metastasis, we took advantage of a publicly accessible portal that allows analysis of the effect of genes on survival using 4,142 breast cancer samples (http://kmplot.com/analysis/). Kaplan-Meier survival curves showed that the erbB2+ breast cancer patients with erbB3-high expression tended to have a shorter Distance Metastasis-free Survival (DMFS).

Conclusion: Activation of Stat3 signaling by erbB3 promotes EMT and shifts the cellular phenotype toward a mesenchymal state that is advantageous for cancer metastasis. Our data shed a new sight on erbB3's role in erbB2+ breast cancer metastasis and may facilitate the development of novel strategy for treatment of tumor progression.

Keywords: ErbB3, Stat3, EMT, Metastasis, Breast Cancer

Citation Format: Hui Lyu, Ying Wu, Yan Zhou, Bolin Liu. Stat3 signaling in erbB3-mediated epithelial-mesenchymal transition in erbB2-positive breast 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 1593.
Abstract 160: Identification of the carcinoma and immune cells in the breast cancer by single-cell RNA sequencing
Authors: Chung Woosung , Eum Hye Hyeon , Kim Kyu-Tae , Lee Kyung-Min , Jo Arum , Han WonShik , Lee Hae-Ock , Park Woong-Yang
Published: July 2016

Summary: Breast cancer is a heterogeneous type of cancer accompanying extensive immune cell infiltration. Although its mortality rate has been decreasing with early diagnosis and introduction of molecular targeted therapies, it is still one of the world leading cause of cancer death in women. In breast cancer, molecular subtyping is used for the patient stratification and treatment decisions, emphasizing the importance of inter-patient tumoral heterogeneity in the tumor cell behavior. Tumor cells within a patient may also manifest intra-tumoral heterogeneity, which may cause treatment resistance. Single cell gene expression profiling would reveal both inter- and intra-tumoral heterogeneity in breast cancer encompassing tumor cells and associated stromal and immune cells.

Experimental Process: From 4 different subtype primary breast cancer and 2 lymph node metastases, single cells were captured and amplified through C1™Single-cell Auto Prep System (Fluidigm) with the SMARTer Ultra Low RNA Kit. Sequencing libraries were constructed with the Nextera XT DNA sample Prep Kit and sequenced through a Hiseq2500 (Illumina) as 100-bp paired end mode of the TruSeq Rapid PE Cluster kit and Truseq Rapid SBS kit.

Computational Process: RNA sequencing reads were aligned to the modified human genome reference using 2-pass mode of STAR_2.4.0b, and transcript per million (TPM) values were obtained as the relative gene expression levels of single cells through RSEM v1.2.17.

Results: As most breast cancers originate from the mammary epithelium, we distinguished carcinoma cells from associated non-tumor cells by epithelial cell adhesion molecule (EPCAM) signature genes. We validated the distinction with chromosomal gene expression patterns and gene set enrichment analyses using tumor, stromal, or immune genesets. With or without removal of non-tumor cells, TNBC tumor cells showed the highest gene expression heterogeneity, recapitulating the known inter-patient heterogeneity. TNBC tumor cells from a single patient were categorized into 6 different TNBC-subtypes, further demonstrating intra-tumoral heterogeneity. In addition, immune cells identified in the TNBC tumor were naïve B cells and regulatory T cells, suggesting non-activated status of the immune system.

Conclusions: The high resolution gene expression profiling revealed features of breast cancer transcriptome which may provide clues for molecular directed therapies targeting the tumor or the immune compartment.

Citation Format: Woosung Chung, Hye Hyeon Eum, Kyu-Tae Kim, Kyung-Min Lee, Arum Jo, WonShik Han, Hae-Ock Lee, Woong-Yang Park. Identification of the carcinoma and immune cells in the breast cancer by single-cell RNA sequencing. [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 160.
Abstract 1603: Highly expressed integrin-α8 in multiple myeloma early relapse induces epithelial to mesenchymal transition-like features
Authors: Ryu Jiyeon , Koh Youngil , Lee Hyun Jung , Chung Hyun Sub , Yoon Sung-Soo
Published: July 2016

Multiple myeloma (MM) is characterized by clonal proliferation of malignant plasma cells in the bone marrow niches. Despite the remarkable advances of treatment for MM,still many patients of MM struggle early relapses and its mechanism is not well understood. Patients with early relapse after autologous bone marrow transplantation need to be biologically characterized to better understand and reach complete remission. To define altered gene expression in MM relapse, we performed microarray with whole bone marrow aspirates from 16 relapsed MM patients who underwent auto-BMT. Gene expression profile was compared (P < 0.01) and divided into two groups on the basis of their progression-free survival (PFS) at the point of 12 months. To discern the significance of the result, only four patients with shortest and longest duration of PFS was also nexamined, and interestingly both of the analysis showed common up-regulated genes including PDGFB, ITGA7, and ITGA8. Their associated pathways were focal adhesion, ECM-receptor interaction, and regulation of actin cytoskeleton (P < 0.05). By performing qRT-PCR, we validated three genes and only ITGA8 was corresponded with microarray data that early relapsed patients showed higher expression of ITGA8. Several integrin family members are well documented to involve in MM progression that it mediates MM cell adhesion, migration and homing through BM microenvironment. However, integrin-α8 is largely unknown in MM. Due to ITGA8 was highly expressed in MM relapse, we functionally overexpressed integrin-α8 in MM cell lines. Surprisingly, it activated genes showing stem-like features including HIF1a, VEGF, OCT4, and Nanog and EMT-related genes, such as N-cadherin, Slug, Snail and CXCR4. These, consequently enhanced invasion and migration abilities and reduced adhesion activity, indicating the mechanism of MM progression, metastasis as well as relapse. Our speculated specific gene, integrin-α8, up-regulated in MM early relapse, regulates EMT feature in MM that suggesting a potential marker for relapse and attractive clinical target to achieve complete remission.

Citation Format: Jiyeon Ryu, Youngil Koh, Hyun Jung Lee, Hyun Sub Chung, Sung-Soo Yoon. Highly expressed integrin-α8 in multiple myeloma early relapse induces epithelial to mesenchymal transition-like 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 1603.
Abstract 161: Single-cell RNA sequencing presents explicit expression signatures in primary breast cancer
Authors: Eum Hye Hyeon , Lee Hae-Ock , Chung Woosung , Kim Kyu-Tae , Lee Kyung-Min , Jo Arum , Han WonShik , Park Woong-Yang
Published: July 2016

Intratumoral heterogeneity has emerged as a problem to be resolved for precise diagnosis and effective treatment of cancer. Breast cancer is one of the most heterogeneous cancer in its molecular subtypes and its intratumoral genomic diversity has been demonstrated as well. Single-cell sequencing approach would reveal heterogeneity in gene expression signatures and the common gene expression characteristics across tumor cells within a tumor. In this study, we isolated live individual cells and amplified cDNAs using C1™Single-cell Auto Prep System (Fluidigm) then performed RNA sequencing using HiSeq2500 System (Illumina). Single-cell transcriptomes were acquired from four primary tumor (ER+/HER2-, ER-/HER2+, ER+/HER2+ and triple negative breast cancer) and two matched lymph node metastases (ER+/HER2+ and triple negative breast cancer). Because the tumor tissue isolates may include stromal and immune cells in the tumor microenvironment, we defined ‘epithelial cell adhesion molecule signature’ to filter out non-tumor populations (Poster# presented by Woosung Chung et. al) After the removal of non-tumor cells, collected tumor single-cell transcriptomes largely recapitulated bulk tumor-expression profiles. The ER+/HER2- tumor cells showed highly activated estrogen receptor-associated pathways. Although both ER-/HER2+ and ER+/HER2+ tumors had the HER2 gene amplification and overexpression, HER2/HER3 pathway is activated only in ER-/HER2+ tumor cells. The association of gene expression for cancer stem cells and chemoresistance, such as IL8 and CXCL1, with HER2/HER3 signaling pathway activation was prominent. By contrast, the ER+/HER2+ tumor cells had highly activated estrogen receptor signaling, presenting them as the luminal type. The triple negative breast cancer cells highly expressed adhesion molecules and proteases suggesting their invasive nature, yet demonstrated extensive gene expression heterogeneity. Collectively these data revealed the power of single cell gene expression profiling in the precise characterization of tumor cell properties. The explicit gene expression signatures for patient specific tumors may be defined as subtype-specific signatures and helpful to unmask targets for molecular directed therapies.

Citation Format: Hye Hyeon Eum, Hae-Ock Lee, Woosung Chung, Kyu-Tae Kim, Kyung-Min Lee, Arum Jo, WonShik Han, Woong-Yang Park. Single-cell RNA sequencing presents explicit expression signatures in primary breast cancer. [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 161.
Abstract 1630: Dysregulation of Hippo pathway in metastatic prostate cancer
Authors: Huang Jen-Ming , Chu Gina C-Y , Kim Hyung L. , Zhau Haiyen E. , Chung. Leland W.K.
Published: July 2016

Background: Prostate cancer (PCa) is prevalently diagnosed and more than 80% of late stage PCa patients develop bone metastases. We hypothesized that the selective lodging of the PCa cells in the bone can be contributed by increased RANK-mediated cell signaling network. Cancer cell-derived RANKL orchestrates a complex bone-remodeling program facilitating PCa bone colonization through the activation of specific transcription factors (TFs) promoting PCa-bone microenvironment interaction through increased epithelial to mesenchymal transition (EMT), cell growth, and osteomimicry. Overexpression of RNAKL in indolent LNCaP cells drives cancer bone and soft tissue metastases.

Methods and Results: Microarray gene expression was performed to compare RANKL- and Neo-vector- transfected LNCaP cells, LNCaPRANKL and LNCaPneo, respectively. qRT-PCR and western blot were performed to confirm the expression of RANKL-targeted genes in these cells. We found the new extracellular diffusible signal mediated by RANKL, regulating Hippo pathway in the aggressive PCa cells. Recombinant RANKL-treated indolent PCa cells, LNCaP and LAPC4, were shown to gain increased aggressiveness. The RANKL-treated indolent PCa cells exhibited down-regulation of Hippo pathway core kinases (Mst1/2 and Lats1/2) but up-regulation of YAP/TAZ expressions. By deleting downstream effectors of Hippo pathway, YAP/TAZ, interrupted the aggressive PCa cells to migrate, proliferate and invade and reversed their EMT phenotype. Inhibitions of YAP/TAZ in aggressive PCa cells via genetic knockdown approach resulted in E-cadherin increase and Vimentin decrease. With the identification of crosstalk between Hippo and TGF-β, or Wnt pathways, this opened up a new network and targeting opportunity for PCa bone metastasis. The metastatic PCa cells with YAP/TAZ deletions were illustrated for sensitization to other therapeutic agents such as TGF-β inhibitors. These results prompted us to study the mechanism, validate the functional significance, and devise potential targeting strategies of Hippo pathway in PCa bone metastasis.

Conclusions: Our results showed that dysregulated Hippo pathway in the aggressive PCa cells. With deletions of YAP/TAZ, PCa metastatic abilities were inhibited. Our findings might explain why PCa cells prefer bone as primary metastatic sites. In addition, targeting YAP/TAZ could be a potential therapy for PCa bone metastasis.

Citation Format: Jen-Ming Huang, Gina C-Y Chu, Hyung L. Kim, Haiyen E. Zhau, Leland W.K. Chung. Dysregulation of Hippo pathway in metastatic prostate cancer. [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 1630.
Abstract 1641: Complex behavior of ALDH1A1 and IGFBP1 in liver metastasis of colorectal cancer
Authors: Kim Jin Cheon , Ha Ye Jin , Tak Ka Hee , Roh Seon Ae , Kim Chan Wook , Kim Tae Won , Cho Dong-Hyung , Kim Seon-Kyu , Kim Seon-Young , Kim Yong Sung
Published: July 2016

The present study aimed to identify upregulated genes associated with colorectal cancer (CRC) liver metastasis (CLM) using our dataset (GSE50760) previously established by RNA sequencing and to verify their biological behavior. Candidate genes were assessed for their role in tumor proliferation, invasion, and the expression of epithelial-mesenchymal transition/CRC stem cell (EMT/CSC)-related molecules. The role of the identified genes in CLM was verified in mice after intrasplenic transplantation of CRC cells. Of nine candidate genes, ALDH1A1 and IGFBP1 were selected and showed few post-translational changes. The upregulation of ALDH1A1 and IGFBP1 significantly and time-dependently decreased cell proliferation (p ≤ 0.001-0.003) and suppressed invasiveness by ≥3 fold over control cells (p < 0.001) in the SW480 cell line, whereas it had a mild effect on reducing SW620 cell proliferation. EMT/CSC-related molecules showed different patterns of expression in CLM tissues and treated CRC cells. The cadherin switch, namely, N-cadherin upregulation with E-cadherin downregulation, was not observed in CLM tissues and treated CRC cells. Persistent overexpression of β-catenin, vimentin, and ZO-1 in IGFBP1-overexpressing SW480 cells may have contributed to CLM development in mice implanted with IGFBP1-overexpressing cells (CLM occurrences: SW480/IGFBP1 transfected mice vs. SW480/vector and /ALDH1A1 transfected mice, 4/8 vs. 0/10, p = 0.023). ALDH1A1 and IGFBP1 were differentially overexpressed in CLM and may play a dual role, functioning as tumor suppressors and metastasis promoting genes in CRC. Unidentified behaviors of these genes may facilitate the reassessment of their potential value as CLM modulators and as therapeutic targets.

Citation Format: Jin Cheon Kim, Ye Jin Ha, Ka Hee Tak, Seon Ae Roh, Chan Wook Kim, Tae Won Kim, Dong-Hyung Cho, Seon-Kyu Kim, Seon-Young Kim, Yong Sung Kim. Complex behavior of ALDH1A1 and IGFBP1 in liver metastasis of colorectal cancer. [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 1641.
Abstract 1646: A glioblastoma methylation assay (GaMA) developedfrom genomic analysis of glioma spheroid cultures predicts response toradiation therapy in patients with glioblastoma
Authors: Wang Qianghu , Ezhilarasan Ravesanker , Eskilsson Eskil , Gumin Joy , Yang Jie , Jaffari Mona , Tang Ming , Aldape Kenneth D. , Lang Frederick F. , Verhaak Roel G.W. , Sulman Erik P.
Published: July 2016

Radiation therapy (RT) remains one of the most effective treatments for patients with GBM and has been repeatedly demonstrated to improve survival; yet response to RT is variable. We explored the relationship between methylation status and radiation response to develop a predictor of RT response using the epigenetic data of glioma sphere-forming cells (GSCs). The DNA methylomes of 42 GSCs were profiled using Illumina Infinium 450K methylation bead arrays. 15 GSCs were irradiated with 2-, 4-, and 6-Gy RT and response determined using clonogenic assays. We discovered 168 CpG probes capable of distinguishing sensitive from resistant GSCs. To validate, we analyzed 362 TCGA GBM samples, 272 that received standard 60Gy RT and 90 treated with low or no RT. Using the glioblastoma methylation assay (GaMA) signature, we classified the samples as either RT sensitive or resistant. Survival was significantly different between the predicted sensitive vs resistant patients for those treated with standard RT (median 21.0m vs 14.7m, p<0.005). GaMA did not predict a survival difference among patients receiving no/low-dose RT, suggesting a predictive, but not prognostic, role for the signature. Using the ENCODE ChIP-Seq Significance Tool, we observed that the transcription factor EZH2 was significantly associated with the radiation resistant promoters in the GaMA signature. Among the hypermethylated genes with EZH2 binding sites, the NR2F2 promoter had the greatest number of hypermethylated CpG sites correlated to RT resistance. NR2F2 has previously been identified as negatively associated with activation of the wnt/β-catenin, a pathway associated with RT resistance of mammary progenitor cells. Expression of WNT1 in TCGA GBM cohort was negatively associated with NR2F2 expression. Our GSC RT response-based methylome analysis corroborates this association and provides a rationale for the methylation signature as a predictive biomarker of radiation response.

Citation Format: Qianghu Wang, Ravesanker Ezhilarasan, Eskil Eskilsson, Joy Gumin, Jie Yang, Mona Jaffari, Ming Tang, Kenneth D. Aldape, Frederick F. Lang, Roel G.W. Verhaak, Erik P. Sulman. A glioblastoma methylation assay (GaMA) developedfrom genomic analysis of glioma spheroid cultures predicts response toradiation therapy in patients with glioblastoma. [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 1646.
Abstract 1651: Overexpression of hepatoma upregulated protein (HURP) promotes radioresistance in prostate cancer cells
Authors: Hassan Mohamed , El Khattouti Abdelouahid , Ma Tangeng , Espinoza Ingrid , Day W. Andrew , Vijayakumar Srinivasan , Gomez Christian R.
Published: July 2016

Despite progression in diagnosis and treatment, prostate cancer (PCa) still represents major cause of cancer-related mortality and morbidity in men. Although radiation therapy offers clinical benefit over other therapeutic modalities, the success of this therapeutic modality is commonly hampered by significant resistance in advanced tumor. So far, the mechanisms governing the acquired resistance to radiotherapy have not been discussed in detail. Hepatoma Up-Regulated Protein (HURP) is a cell cycle-regulated and microtubule-associated protein. It functions as a Ran GTPase effector and is involved in the stabilization of the mitotic kinetochore fibers. The expression of this protein is elevated in different tumor types and associated with tumor progression and aggressiveness. We recently reported on the value of HURP as an independent prognostic factor for high-risk PCa. The aim of this study was to address the role of HURP in the modulation of PCa resistance to γ- irradiation. Cell lines (LNCaP, PC3, and DU145) with regulated expression of HURP protein using a Lent viral Tet-On 3G Inducible Expression System were irradiated using a Cs-137 γ-source. Overexpression of HURP suppressed γ- irradiation- induced apoptosis. This suppression was associated with the expression of E2F1, p53, p21 together with the phosphorylation of apoptosis signal-regulating kinase1 (ASK1), c-jun-N-terminal kinase (JNK) and Ataxia-telangiectasia mutated (ATM) and histone family member X (H2AX). Also, inhibition of γ- irradiation- induced- cytochrome c release, cleavage of caspase-9, caspase-3, PARP, and reactive oxygen species (ROS) was noted. The observed resistance was consequence of HURP's ability to trigger the ubiquitination of p53 and ATM that, in turn, resulted in the inhibition of downstream pathways, which are essential for the modulation of γ- irradiation-induced apoptosis. Our data provide evidence for the involvement of HURP in the modulation of PCa cell resistance to γ-irradiation via a mechanism mediated by ubiquitination of ATM and p53. Understanding of the biological mechanisms underlying HURP-mediated resistance of PCa to available therapies may help to improve the treatment strategy and emphasize HURP as therapeutic target for PCa treatment.

Funding sources: Department of Defense Grant PC094680 and Prostate Cancer Foundation Creativity Award.

Citation Format: Mohamed Hassan, Abdelouahid El Khattouti, Tangeng Ma, Ingrid Espinoza, W. Andrew Day, Srinivasan Vijayakumar, Christian R. Gomez. Overexpression of hepatoma upregulated protein (HURP) promotes radioresistance in prostate 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 1651.
Abstract 166: Optimizing the detection of subclonal somatic variants with VarDict
Authors: Lai Zhongwu , Chapman Brad , Ahdesmäki Miika , Hofmann Oliver , Johnson Justin , Dry Jonathan
Published: July 2016

Detection of somatic variants in tumor tissues is far from a solved problem. Heterogenous tumor materials complicate variant assessment in low frequency subclonal populations, which can play an important role in the development of drug resistance. Loss of heterozygosity, copy number variation and other ploidy changes can further change the expected distribution of variant metrics.

We demonstrate improvements in the specificity and sensitivity of variant calling algorithms by optimising linear combinations of widely used filtering criteria such as allelic frequency, depth and p-value distributions. We specifically improve SNP and indels from the freely available VarDict caller (https://github.com/AstraZeneca-NGS/VarDictJava) and show that these optimisations can be generalised across a wide range of allelic frequencies. We validate our findings against synthetic and in vitro standards such as the ICGC-TCGA DREAM challenge truth set. Our proposed filtering strategy is widely applicable to other variant callers, and the updated filters for VarDict dramatically improve sensitivity and precision on low frequency variants which are crucial for our ability to reconstruct likely tumor populations from short read sequencing data and to study tumor evolution.

Citation Format: Zhongwu Lai, Brad Chapman, Miika Ahdesmäki, Oliver Hofmann, Justin Johnson, Jonathan Dry. Optimizing the detection of subclonal somatic variants with VarDict. [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 166.