bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3855: Refolded albumin inhibits IL-17B-driven pancreatic cancer migration
Authors: Liang Chi-Ming , Liao Ching-Chun , Liang Shu-Mei
Published: July 2016

Folding and refolding a protein may change its physical, chemical and biological properties. Here, we used a novel protein refolding protocol to convert globular albumin into another form, namely f-albumin, with melting point 10oC higher than albumin (83oC vs 72oC). f-albumin, unlike albumin, inhibited the migration/invasion of a variety of cancer cells. Recently, anti-IL-17RB antibodies have been shown to block the IL-17B-IL17RB signaling-mediated migration/metastasis of pancreatic cancer cells via silencing multiple chemokines, notably CCL20, CXCL1, IL-8 and TFF1. We thus examined whether f-albumin is effective in blocking the stimulatory effects of IL-17B. We found that f-albumin inhibited the migration of pancreatic cancer cells (BxPC3, MIA-paca2 and Panc1) with IC50 around 0.2 μM. Although IL-17B (50 ng/ml) increased the migration of pancreatic cells, this effect of IL-17B was abolished by f-albumin (0.2 μM). The inhibitory effect of f-albumin, however, was not necessarily correlated with its inhibition of CCL20, CXCL1, IL-8 or TFF1. Additional study revealed that IL-17B increased the phosphorylation of PI3K, Akt and MAPK in pancreatic cancer cells. f-albumin modulated not only the IL-17B-mediated phosphorylation of Akt but also the IL-17B-mediated phosphorylation of MAPK in an integrin-dependent manner. These findings demonstrated that f-albumin downregulates Akt/MAPK signaling via integrin to play a braking role in the IL-17B-driven pancreatic cancer migration.

Citation Format: Chi-Ming Liang, Ching-Chun Liao, Shu-Mei Liang. Refolded albumin inhibits IL-17B-driven pancreatic cancer migration. [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 3855.
Abstract 3873: Single cell cisplatin measurements by ICP-MS
Authors: Amable Lauren , Smith Stan , Stephan Chady
Published: July 2016

Cisplatin, carboplatin, and oxaliplatin are the most widely used class of cancer chemotherapy drugs in the Western world. Many cancers are initially sensitive to platinum treatment but drug resistance frequently occurs. Cellular uptake of cisplatin is related to tumor burden, thus lower intracellular cisplatin levels are associated with a decreased tumor response. Traditional methods to assess cisplatin uptake in cells involves digesting a cell population and measuring the total platinum content. This approach does not reflect the distribution and individual cellular variation of cisplatin uptake. Here, we present a new method, single cell inductively coupled plasma-mass spectrometry (SC-ICP-MS) to quantitate the platinum concentration within individual cells. Experiments were performed using the A2780 cisplatin-sensitive and the corresponding cisplatin-resistant A2780-CP70 ovarian cancer cell lines. Time course experiments were performed to measure the change of cisplatin uptake over time. Serum starvation experiments were also performed to examine if differences in cisplatin uptake were dependent on the cell cycle. SC-ICP-MS was performed by injecting cell suspensions and analyzing the platinum 195 isotope. Individual cellular cisplatin levels were collected and a histogram representing the cell population was generated using the instrument software (Syngistix Nano Application). Other metals, such as zinc, iron, and copper, were analyzed to measure the variation between cell lines after treatment. We observed a heterogeneous distribution of cisplatin concentrations within the sample reflecting that cisplatin uptake differs from cell to cell. Serum starvation affected the uptake of cisplatin within cells, changing the cisplatin distribution curve. SC-ICP-MS was also effective in measuring other metals within individual cells and differences were observed between the A2780 and A2780-CP70 cell lines. In conclusion, single cell ICP-MS analysis allows for the quantitation of cisplatin within individual cells. The heterogeneous distribution of cisplatin uptake more closely reflects what occurs within tumor cells. SC-ICP-MS allows for the development of strategies to increase cisplatin uptake, translating to better clinical responses.

Citation Format: Lauren Amable, Stan Smith, Chady Stephan. Single cell cisplatin measurements by ICP-MS. [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 3873.
Abstract 3877: A new frontier for molecular profiling of neoplastic bone tissue
Authors: Mueller Claudius , Liotta Lance A. , Chiechi Antonella , Picci Piero , Alberghini Marco , Memeo Lorenzo , Canzonieri Vincenzo , Espina Virginia
Published: July 2016

Background: Tissue is alive and reacting to ex vivo stress during “cold ischemia time”. Tumor cells react to the absence of vascular perfusion, hypoxia, acidosis, and accumulation of cellular waste, which can critically mask cancer related signaling network data. Standard formalin fixation of calcified tissue requires decalcification with formic/nitric acid solution, which greatly interferes with downstream molecular analysis. We developed a non-formalin fixative (TheraLin) for preserving biomarker molecules and tissue histomorphology in one step, which greatly facilitates the analysis of phosphoprotein signaling pathways in calcified tumor samples.

Methods: Fifty specimens of neoplastic bone were collected at the Istituto Ortopedico Rizzoli (Bologna, Italy). Matched 0.5 cm samples were fixed in TheraLin preservative; or fixed in 4% buffered formalin and decalcified with formic/nitric acid solution. Specimens were processed into paraffin blocks and 5 μm tissue sections cut onto glass slides. Enriched tumor cell populations were procured using Laser Capture Microdissection. Reverse Phase Protein Microarrays were constructed with the microdissected tumor cells and probed with >40 antibodies to unmodified or phosphorylated proteins. Immunohistochemistry (>200 stains in total) was performed on routine clinical protein targets and a subset of phosphorylated proteins.

Results: TheraLin fixation greatly facilitated molecular profiling of bony tissues by eliminating the need for decalcification (18.5 hour average reduction in processing time) while maintaining or improving histomorphology (>85% of cases) and immunohistology (>75% of cases). In addition, we were able to demonstrate full compatibility with laser capture microdissection, reverse phase protein microarrays and downstream analysis of cell signal protein activation. Protein extractability from microdissected material was significantly increased (>2.5 fold, p<0.01) compared to formalin fixed, decalcified bone tissue.

Conclusion: TheraLin obviates the need for decalcification of bone tissue while simultaneously preserving histomophology and immunohistochemistry. TheraLin enables reliable quantification of phosphorylated signal transduction proteins, the substrates of kinase drug targets, and greatly facilitates tumor cell enrichment compared to standard formalin fixation and decalcification.

Citation Format: Claudius Mueller, Lance A. Liotta, Antonella Chiechi, Piero Picci, Marco Alberghini, Lorenzo Memeo, Vincenzo Canzonieri, Virginia Espina. A new frontier for molecular profiling of neoplastic bone tissue. [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 3877.
Abstract 3879: Comprehensive delineation of stromal proteins implicated in the multistage carcinogenesis and identification of Tenascin-C as stromal marker for metastasis of colon carcinoma
Authors: Chen Yongheng , Li Maoyu
Published: July 2016

Colon carcinoma (CC) is one of the most common malignancies threatening the human health. Early diagnosis or effective biomarker for intervention is still a challenge in clinical application for CC. The stroma in human cancer provides the microenvironment for cancer initiation and development, and furthermore, the stroma has been regarded as a therapeutic target for cancer. However, little is known about the dynamics of proteins within the stroma during the carcinogenic process. To discover the involvement of stroma in carcinogenesis and identify the biomarker candidates, the stromal components at the various stages of colonic carcinogenesis were purified by laser capture microdissection (LCM) and iTRAQ-based quantitative proteomic analysis was used for identifying the differentially expressed stromal proteins at different stages. In total, 222 differentially expressed proteins were identified. The expression of two of the top ranked proteins, extracellular matrix protein TNC and S100 family protein S100A9, was further confirmed by immunohistochemical analysis. Further analysis revealed that TNC was highly expressed in the stroma of metastasis and also abundantly secreted by the high metastatic cells. These results suggested that TNC was potential stromal marker for metastasis of colon carcinoma. Our results will contribute to investigating the role of stroma in the colonic carcinogenesis and might provide clues for pathogenesis, early diagnosis, and clinical therapy of CC.

Citation Format: Yongheng Chen, Maoyu Li. Comprehensive delineation of stromal proteins implicated in the multistage carcinogenesis and identification of Tenascin-C as stromal marker for metastasis of colon 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 3879.
Abstract 3890: NCI's Clinical Proteomic Tumor Analysis Consortium
Authors: Mesri Mehdi , Boja Emily , Hiltke Tara , Kinsinger Chris , Lee Jerry , Rodriguez Henry
Published: July 2016

The NCI Clinical Proteomic Tumor Analysis Consortium (CPTAC) constitutes a network of Proteome Characterization Centers (PCCs), which coordinate and conduct research and data sharing activities to comprehensively interrogate genomically characterized cancer biospecimens. Such integrated proteogenomics approach brings key insights into genomic abnormalities manifested at the protein network and pathway level. Established in late 2011, CPTAC works closely with genomics initiatives such as The Cancer Genome Atlas (TCGA) to systematically identify proteins that derive from alterations in cancer genomes and related biological processes, and provide this data with accompanying assays and protocols to the public. CPTAC investigators conducted the first in-depth comprehensive proteome (global, phosphoproteome and N-glycoproteome) study on understanding the impact of cold ischemia that has resulted in the development of an ischemic signature database and best practice procedures for biospecimen (tissue) sample collection/processing. The PCCs have deeply characterized three cancer types (colorectal, ovarian and breast tumors) previously analyzed by TCGA and additional tissue collections, all of which are accompanied by genomic datasets. Scientific findings examining 95 TCGA colon and rectal tumors using deep proteomic analysis include: a) five proteomic subtypes were identified, with the TCGA MSI/CIMP transcriptomic subtype separated into distinct proteomic subtypes including one subtype (subtype C) that displayed protein network features characteristic of epithelial-mesenchymal transition (EMT); b) copy number alterations (CNAs) on mRNA abundance analysis showed strong cis- and trans-effects; but few extending to the protein level; and c) chromosome 20q amplicon was shown to contain the largest global changes in both mRNA and protein levels, allowing for prioritizing driver genes. In addition to the publicly available proteomic datasets (https://cptac-data-portal.georgetown.edu/cptacPublic), proteomic targeted assays developed by the consortium are available at CPTAC's Assay Portal (http://assays.cancer.gov). This is a public resource of mass spectrometry-based quantitative targeted proteomic assays for measuring proteins of interest from the program. Lastly, monoclonal antibodies targeting cancer specific proteins and peptides are made available at CPTAC's Antibody Portal (http://antibodies.cancer.gov).

Citation Format: Mehdi Mesri, Emily Boja, Tara Hiltke, Chris Kinsinger, Jerry Lee, Henry Rodriguez. NCI's Clinical Proteomic Tumor Analysis Consortium. [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 3890.
Abstract 3894: A xenograft mouse model coupled with in-depth cell surface proteome analysis facilitates further elucidation of K-Ras driven tumorigenesis in lung carcinoma
Authors: Ye Xiaoying , Whiteley Gordon , Nissley Dwight , McCormick Frank , Blonder Josip
Published: July 2016

The purpose of this study was to develop a proteomic approach for analysis of cell surface proteins in tissue specimens and applied it on a K-Ras driven mouse model of metastatic lung carcinoma. Metastatic disease is the leading cause of lung cancer-related mortality in the United States. In spite of continuous efforts, effective treatments targeting oncogenic K-Ras in lung and other K-Ras driven malignancies are slow to develop. To expand the treatment options for lung cancer and facilitate better understanding of metastasis new targets need to be identified and characterized at the surface of cancer cells, preferably in their native tissue microenvironment. Towards this goal we developed a mass-spectrometry (MS)-based glyco-proteomic approach targeting specifically cell surface proteins in tissue specimens and applied it on a xenograft lung cancer mouse model. We used A549 cells expressing endogenous K-RasG12S to induce lung tumor xenografts in BALB/c mice via tail vein injection. A comparative surface glyco-proteomics of cultured A549 cells, dissected tumor tissue (TT), adjacent tumor (AT) tissue and normal mouse lung tissue (NT) obtained from saline-injected age-matched littermates yielded high enrichment (i.e., ≥80%) of surface proteins. It resulted in extensive catalogue/map of more than 400 glycoproteins identified on tumor cell surface. More than 50% of proteins identified on the surface of A549 cultured cells were unambiguously identified in mouse TT, featuring proteins and pathways implicated in metastasis, invadopodia formation and cancer cell migration. These markers also provide insight regarding species determination of tissue origin (i.e., human vs. mouse) differentiation of tumor parenchyma from stroma, and regulation of tumor immune response.

Citation Format: Xiaoying Ye, Gordon Whiteley, Dwight Nissley, Frank McCormick, Josip Blonder. A xenograft mouse model coupled with in-depth cell surface proteome analysis facilitates further elucidation of K-Ras driven tumorigenesis in lung 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 3894.
Abstract 3914: Repurposing disulfiram for use as an anticancer drug: a story about metabolism and metal binding
Authors: Wehbe Moe , Anantha Malathi , Backstrom Ian , Leung Ada , Chen Kent , Shi Minghan , Malhotra Armaan , Edwards Katarina , Sanche Léon , Bally Marcel B.
Published: July 2016

Disulfiram (DSF) is an FDA approved agent for the treatment of alcoholism with over 60 years of clinical use. It was identified in several high-throughput screen to be effective against glioma tumor initiating cells; this activity is amplified more than 100-fold in the presence of copper (Cu2+). DSF is metabolized to diethyldithiocarbamate (DDC), an established copper chelating agent. One mechanism attributed to the copper DDC (Cu(DDC)2) complex is proteosome inhibition. Cu(DDC)2 is very poorly soluble in aqueous solution; making it difficult to purse development for any cancer indication. A novel liposomal formulation of Cu(DDC)2 is described here; which represents the first iv injectable Cu(DDC)2. 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol (CHOL) (55:45 molar ratio) liposomes were prepared by extrusion methods in the presence of 300 mM unbuffered CuSO4 to ∼100 nm in size. DDC was added to these pre-formed liposomes at room temperature, unentrapped drug was removed using column chromatography. The therapeutic activity of this formulation was determined in vitro against a variety of cancer cell lines. The toxicity and pharmacokinetic (PK) behaviour of this formulation was determined in CD-1 mice. DDC was tested in the presence and absence of Cu2+ in vitro with U251, U87, MDA231-BR and A549 cell lines giving IC50 values greater than >10 μM in the absence and from 300- 900 nM in the presence of Cu2+. The liposomal Cu(DDC)2 formulation was as active as unencapsulated Cu(DDC)2 when tested in vitro. The maximum tolerated dose of liposomal Cu(DDC)2 was determined to be 8mg/kg given iv using a Q2D schedule for two weeks. The PK studies suggested that Cu(DDC)2 was rapidly released from circulating liposomes. Initial efficacy studies were completed using convection enhanced delivery of liposomal Cu(DDC)2 in the F98 rat glioma model. Treatment resulted in a statistically significant delay in tumour growth upon injection of 10 μL of 0.5 mg Cu(DDC)2 /mL solution. Further anti-tumor efficacy studies are being conducted in a subcutaneous leukemia xenograft model (MV-4-11). This work describes a novel formulation of Cu(DDC)2, the active agent generated when administering DSF and Cu to treat cancer.

Citation Format: Moe Wehbe, Malathi Anantha, Ian Backstrom, Ada Leung, Kent Chen, Minghan Shi, Armaan Malhotra, Katarina Edwards, Léon Sanche, Marcel B. Bally. Repurposing disulfiram for use as an anticancer drug: a story about metabolism and metal binding. [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 3914.
Abstract 3915: PBA2, a dual BCR-ABL and GSK3βkinases inhibitor, represses a wide array of Imatinib resistant cell lines in chronic myeloid leukemia
Authors: Yang Ke , Fu li-Wu
Published: July 2016

Despite the clinical efficacy of the BCR-ABL (Tyrosine Kinase Inhibitors) TKIs for the treatment of Chronic Myeloid Leukemia (CML), BCR-ABL-T315I mutation still confers higher-level resistance to imatinib and the second generation BCR-ABL TKIs. Ponatinib, as the third generation BCR-ABL TKIs, has the potential activity against CML with T315I mutant BCR-ABL whereas high frequency of serious adverse events limits its clinical use. Through a compound library screen, we identified a novel compound PBA2, a dual kinases inhibitor of GSK3β and BCR-ABL, as a promising agent for the treatment of imatinib-resistant CML. Not only can PBA2 inhibit cell growth but also induce terminal cell differentiation in a series of kinase mutant CML cell lines as well as T315I mutation. Remarkably, the mechanism of action of this compound involved the simultaneous inhibition effect of BCR-ABL and GSK3β, which could be a fruitful approach to suppress the β-catenin/CBP signal pathway that promoted the interaction of β-catenin/p300, leading to growth inhibition and subsequent terminal differentiation. More importantly, we found a novel molecular link between CBP and BCR-ABL. We showed that CBP could regulate BCR-ABL expression through activating CREB which binds to the BCR-ABL promoter in CML cells to start transcription, and the BCR-ABL in turns affect the expression level of CBP. This positive feedback pathway between CBP and BCR-ABL represents a novel way for the treatment of CML, and the novel compound PBA2 targeting at this loop deserves further investigation in clinical trial.

Citation Format: Ke Yang, li-Wu Fu. PBA2, a dual BCR-ABL and GSK3βkinases inhibitor, represses a wide array of Imatinib resistant cell lines in chronic myeloid leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3915.
Abstract 3933: CETSA as a diagnostic tool to guide personalized breast cancer therapy
Authors: Öberg Anette , Laursen Henriette , Packham Sylvia , Martinez Molina Daniel , Seki Takahiro , Cao Yihai , Hartman Johan , Bergh Jonas , Nordlund Pär , Lööf. Sara
Published: July 2016

The increased knowledge of fundamental cancer biology has translated into the development of a large number of targeted drugs. However, the potential for breakthroughs in cancer therapy is hampered by the lack of knowledge regarding which patients would benefit from a specific drug. Therefore, there is a need of diagnostic tools that can predict if the individual patient will respond or develop resistance to a specific treatment plan.

In the present project we aim to meet this challenge by developing stringent clinical diagnostic assays for assessing target engagement (TE; i.e. drug binding to its target protein in situ) of current and emerging drugs in breast cancer treatment. The key to develop these clinical TE assays is our novel technology Cellular Thermal Shift Assay (CETSA). This method allow for the first time direct monitoring of biophysical drug TE in cells, animals and patient samples. The main aim of the project is to correlate the results obtained by CETSA on breast cancer biopsies to the treatment outcome for the biopsied patient, with the goal of creating a diagnostic tool for guiding personalized breast cancer treatment. We also aim to identify new biomarkers of efficacy and resistance for specific drugs, by implementing proteome wide CETSA based on a quantitative mass spectrometry read out.

Our recent data demonstrates that CETSA can assess TE in intact cells of drugs commonly used in breast cancer treatment, such as microtubule inhibitors, anthracyclins and CDK4/6 inhibitors. Further we show that CETSA can be applied in vivo on human breast cancer Xenografts and breast cancer patient samples. These data further strengthens the potential of CETSA to become a valuable clinical tool and complement other diagnostic methods to improve efficacy and minimize adverse effects of cancer drugs.

Citation Format: Anette Öberg, Henriette Laursen, Sylvia Packham, Daniel Martinez Molina, Takahiro Seki, Yihai Cao, Johan Hartman, Jonas Bergh, Pär Nordlund, Sara Lööf. CETSA as a diagnostic tool to guide personalized breast cancer therapy. [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 3933.
Abstract 3936: Identification and validation of the potential biomarker insulin-like growth factor binding protein acid-labile subunit for breast cancer in African American women
Authors: Tadi Uppala Padma P. , Garberoglio Carlos , Lum Sharon , Davis Willie , Leung Hon-Chiu Eastwood , Liebman Michael , Oda Keiji , Patel Utkarsh P.
Published: July 2016

Breast cancer is the most frequently diagnosed cancer in women, with an estimated 40, 730 breast cancer deaths in the US in 2015. African American (AA) women have a lower breast cancer incidence rate, but a higher breast cancer death rate, than non-Hispanic White women. Research indicates that breast tumor biology in AA women is different from that in Caucasian women. AA women are more likely to be diagnosed with breast cancer at an earlier age and with more aggressive form of the disease, characterized by higher grade and negative estrogen and progesterone receptor status. Because of the aggressive nature of these tumors and current lack of targeted therapies, identification of novel relevant protein markers is of great importance. The purpose of this study was to validate serum proteins that were previously identified by serum proteomic profiling in 22 serum samples by 2D-DIGE/MS analysis and a subset of samples by shotgun LC/MS technology. Methods and new data: The current study included serum samples from 15 African American breast cancer patients and 12 healthy controls. Patients were grouped into triple negative (TN), HER2 and Luminal A and B subtypes. Proteins of biological significance were validated using western blot analysis. For ceruloplasmin, and insulin-like growth factor binding protein acid-labile subunit (IGFBP-ALS), one-way ANOVA was used to compare mean density among the three groups. For Vitamin D Binding protein (VDB), a two-sample t-test was used to compare the density between the groups. Due to the small sample size, we have also conducted nonparametric tests. IGFBP-ALS was significantly lower in triple negative breast cancer patients (p = 0.016) and in HER2 (p = 0.025) subtypes. There was no significant difference in VDB protein in the luminal A and B subtypes (p = 0.98). Future efforts will focus on validating the identified panel of biomarkers to gain insight into their role(s) in the etiology of aggressive breast tumors. Funded by Susan G Komen for the Cure and SEED grant SPH.

Citation Format: Padma P. Tadi Uppala, Carlos Garberoglio, Sharon Lum, Willie Davis, Hon-Chiu Eastwood Leung, Michael Liebman, Keiji Oda, Utkarsh P. Patel. Identification and validation of the potential biomarker insulin-like growth factor binding protein acid-labile subunit for breast cancer in African American women. [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 3936.
Abstract 3941: Novel biomarkers and multiplexed NGS to stratify FFPE NSCLC by tumor infiltrating lymphocytes and histopathology phenotypes
Authors: Mongan Ann , Rozenzhak Sophie , Bien Geoffrey , Chi David , Nishikawa Hiroyoshi , Hyland Fiona , Godsey Jim
Published: July 2016

There is increasing evidence supporting the association of tumor infiltrating lymphocytes (TIL) and drug sensitivity of solid tumors. In particular, primary and meta-analyses have reported a positive correlation between TIL level and outcome in advanced non-small cell lung cancer (NSCLC) treated with checkpoint inhibitors such as PD-1 and PD-L1. Recent trials with immunotherapies have started including TIL assessment in the study protocol in recognition of this metric as a predictive and prognostic biomarker. TIL levels are typically quantified by visual assessment of H&E-stained tumor sections. While this approach is generally accepted as the standard, there is an increased recognition that visual assessment of H&E-stained slides lacks precision and is relatively subjective (Salgado R, 2015; Schalper KA, 2015). Furthermore, as investigators are often also interested in measurements of additional biomarkers such as IFNg as well as the drug targets, a gene panel approach offers a convenient solution to objectively quantify expression levels of these informative markers. Here we report the discovery and verification of a unique gene expression signature that is capable of stratifying FFPE samples of NSCLC tumors by TIL levels and histopathology phenotypes (adenocarcinoma vs. squamous cell carcinoma). Gene expression was measured by an RNA Ion AmpliSeq Gene Expression research panel* containing 200 assays. Each research sample was measured with replicates at library generation step and sequencing step. Technical replicates were found to have >0.99 correlation among each other. Assays on the panels were also found to be robust with respect to low input amount (1-10 ng RNA). *For Research Use Only. Not for use in diagnostic procedures.

Citation Format: Ann Mongan, Sophie Rozenzhak, Geoffrey Bien, David Chi, Hiroyoshi Nishikawa, Fiona Hyland, Jim Godsey. Novel biomarkers and multiplexed NGS to stratify FFPE NSCLC by tumor infiltrating lymphocytes and histopathology phenotypes. [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 3941.
Abstract 3945: Antigen-specific immunity and regulation as predictive biomarkers for anti-tumor vaccination
Authors: Johnson Laura E. , Olson Brian M. , McNeel Douglas G.
Published: July 2016

We have reported the results of two phase I trials using a DNA vaccine encoding prostatic acid phosphatase (PAP) in patients with biochemically recurrent prostate cancer. In both trials, PAP-specific proliferating and IFNγ-producing T cells developed in some patients, and persistent Th1-type immunity was associated with an increase in PSA doubling time. In the current study, we sought to determine if one or more measures of antigen-specific or antigen non-specific immunity present prior to treatment was associated with subsequent immune response as a possible predictive biomarker. Specifically, patients who had developed PAP-specific, IFNγ-secreting immune responses, detectable at least twice over one year after immunization, were defined as immune “responders”. The frequency of peripheral T cell and B cell lymphocytes, natural killer cells, monocytes, dendritic cells, myeloid derived suppressor cells, and regulatory T cells did not differ between the immune responder and non-responder groups. PAP-specific immune responses, detected as cytokine secretion following antigen stimulation of peripheral blood mononuclear cells in vitro, were detected in many patients pre-immunization. PAP-specific expression of granzyme B, IFNγ, and IL-6 was not significantly different between immune responding (n = 7) and non-responding patients (n = 23), however non-responder patients tended to have higher PAP-specific IL-10 production (p = 0.09). Using a trans vivo delayed-type hypersensitivity (DTH) assay to assess for antigen-specific immune regulation, responder patients tended to have preexisting PAP-specific bystander regulatory responses that suppressed DTH to a recall antigen, a response previously demonstrated to be IL-35-mediated (p = 0.049). While the number of patients analyzed was low, these results suggest that different measures of antigen-specific regulation might help predict outcome from vaccination. These will be prospectively evaluated in an ongoing randomized, placebo-controlled, phase II trial evaluating this vaccine (NCT01341652).

Citation Format: Laura E. Johnson, Brian M. Olson, Douglas G. McNeel. Antigen-specific immunity and regulation as predictive biomarkers for anti-tumor vaccination. [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 3945.
Abstract 3948: Lysosomal acid phosphatase 2 is an unfavorable prognostic factor associated with better survival in colorectal cancer patients receiving chemotherapy
Authors: Chen. Chi-Long
Published: July 2016

Colorectal cancer (CRC) is one of leading malignancies worldwide. The therapeutic modality for advanced CRC including stage III and IV, is operation following by chemotherapy. For stage II CRC, surgery is the main therapeutic modality but the usage of adjuvant chemotherapy is still controversial and there is no reliable indicator for their purpose. In this study, we found that the expression of lysosomal acid phosphatase 2 (ACP2) in the tumor tissue of CRC was elevated and was correlated with poor outcome in stage II CRC patients, especially. In addition, in CRC patients with high ACP2 expression tended to have more sensitive for 5-FU treatment in our cohort. In CRC of stage II patients, multivariate Cox proportion hazard model analysis revealed that ACP2 expression and T stage are independent prognostic factor for overall survival (HR: 3.274, CI: 1.413-7.586, p = 0.006; HR: 3.742, CI: 1.058-13.233, p = 0.041). Our data indicated that high expression of ACP2 was a negative prognosticator and could predict 5-FU chemotherapy response in stage II CRC. Moreover, we used HCT116 cells transfected with ACP2 lentivirus to create stable shACP2-HCT116 clone. Our results showed knockdown ACP2 could induce chemoresistance and more mesenchymal-like phenotype of CRC cell line. Therefor, targeting ACP2 might be a novel chemotherapy factor and might a new strategy in stage II CRC patients.

Citation Format: Chi-Long Chen. Lysosomal acid phosphatase 2 is an unfavorable prognostic factor associated with better survival in colorectal cancer patients receiving chemotherapy. [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 3948.
Abstract 3953: Highly accurate genetic profiling of circulating tumor cells using a label-free inertial microfluidic approach coupled with droplet PCR-based next-generation sequencing
Authors: Lee Yi Fang , Consugar Mark , Helzer Kimberly , Tan Mei Hui , Emrick Lori , Bhagat Ali Asgar
Published: July 2016

Background: Circulating tumor cells (CTCs) could provide significant insights into cancer metastatic events and potentially even inform clinical decisions. However, the scarcity of CTCs, compounded by the abundance of circulating white blood cells, poses a major technical challenge in the isolation, yield and downstream molecular analysis. Moreover, next-generation sequencing (NGS) of CTCs has been challenged by low CTC purity and nucleic acid yields commonly derived from existing technologies. Here, we present a label-free microfluidic approach that utilizes curvilinear microchannel geometry for high purity CTC enrichment from 7.5 ml whole blood.

Methods: We showed high purity isolation of CTCs using a label-free inertial focusing microfluidics platform, ClearCell® FX. The lung cancer cell line, NCI-H1975, with known EGFR, CDKN2A and TP53 mutations, were spiked into 7.5ml healthy donor peripheral blood samples at varying concentrations (50, 100, 200 cells). DNA was extracted using the QIAamp Micro kit. The Thunderbolts Cancer Panel droplet digital PCR NGS library preparation method was used to enrich 230 loci in 50 known cancer genes from the isolated CTC gDNA. The libraries were sequenced on a MiSeq system.

Results: The CTCs were sufficiently enriched with an average recovery of 45.8% and purities ranging from 1% - 70%. All libraries were successfully generated and sequenced with ≥1400× mean coverage, even with as little as 2 ng of starting material. Known EGFR, CDKN2A and TP53 mutations in H1975 were successfully detected in all the samples (n = 9), with a significant variant frequency that correlates with CTC purity levels.

Conclusion: We have demonstrated a highly accurate and sensitive workflow utilizing a combination of inertial focusing and digital droplet microfluidics to detect low numbers of rare CTCs and interrogate their molecular profiles from as little as 10 cells/mL frequency. This workflow has the potential for further development and adoption as a liquid biopsy approach for non-invasive genomic profiling of metastatic cancers.

Citation Format: Yi Fang Lee, Mark Consugar, Kimberly Helzer, Mei Hui Tan, Lori Emrick, Ali Asgar Bhagat. Highly accurate genetic profiling of circulating tumor cells using a label-free inertial microfluidic approach coupled with droplet PCR-based next-generation 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 3953.
Abstract 398: Real-life example of biobanking: results of the PATH Biobank
Authors: Anzeneder Tobias , Ohlms Ulla , Bodenmüller Heinz
Published: July 2016

Introduction: Since 2002, PATH Foundation has kept a biobank collecting high-quality fresh frozen breast cancer specimens adhering to uniform SOPs at seven certified breast cancer centres in Germany. Research groups from academia and industry can obtain samples after application and review. PATH Biobank is a not-for-profit organisation and asks for a cost recovery fee in exchange for sample allocation to sustainably finance the expenses it incurs.

Material and methods: The PATH Biobank consists of a centralized database and a decentralized bio repository. The samples are collected and stored at seven institutes for pathology at certified German breast cancer centres. Tumour tissue, along with normal (benign) adjacent tissue and blood serum aliquots are processed, labelled and stored according to uniform SOPs. Informed consent to biobanking and the use of the samples and data for research is obtained from the donors individually during pre-operation discussions.

Results: Since 2004, more than 8,600 breast cancer patients have given their informed consent to the storage and analysis of their tissue and blood serum for research purposes. Breast cancer tissue samples from 59% of donors could be stored due to size of the surgically removed tissue specimen. In addition, normal adjacent tissue is available from 62% of donors and blood serum aliquots can be derived from 92% of patients.

Using the annotating data, 96% of donors can be classified into the intrinsic molecular breast cancer subtypes in accordance with the St. Gallen Criteria. In 2008, PATH Biobank started to support research groups by providing breast cancer samples and data as well as informing the public about the projects on-line. In 2015 three co operations resulted in scientific publications.

One landmark collaborations started in 2012 and has been studying the frequency and background of defined mutations in breast cancer. A number of 701 tissue samples have been used for this study. All samples were subject to quality testing, only 11% failed due to less than 5% tumour content. Tumour content depended on clinical conditions and staging (ranging from 34% failure in samples derived from neo adjuvantly treated patients to 2% in cases with staging UICC IV). Additional results concerning the quality of samples from PATH Biobank will be presented.

Conclusions: For breast cancer research and biomarker studies, PATH Biobank can be a valuable resource.

Citation Format: Tobias Anzeneder, Ulla Ohlms, Heinz Bodenmüller. Real-life example of biobanking: results of the PATH Biobank. [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 398.
Abstract 3984: Novel t(X;21)(q26;q22) detected in a case of acute unclassifiable leukemia by application of anchored multiplex PCR-based next-generation sequencing
Authors: Johnson Laura , Wang Helen , Trifilo Katelyn , Kudlow Brian , Pappenhausen Peter R. , Hussaini Mohammad
Published: July 2016

In this study, we demonstrate real-time clinical application of novel anchored multiplex PCR (AMP) technology in hematologic disease. A 78-year-old man with a history of hairy cell leukemia treated with Cladribine (2013) and bladder cancer (2014) developed dyspnea in August 2015. Work up revealed bicytopenia and leukocytosis. Outside bone marrow biopsy was read as B lymphoblastic leukemia/lymphoma. Rebiopsy was performed at our NCI-designated cancer center with extensive work up resulted in reclassification as acute unclassifiable leukemia (rare) with deferral to molecular diagnostics for guidance regarding classification. MLL, BCR-ABL, and MDS FISH panel were negative. Next generation sequencing (NGS) interrogating more than 400 genes showed FLT3-ITD, truncation of RUNX1 exon 6, ASXL1 p.G646Wfs*12, MLL p.V297L, and EZH2 p.V674M and p.C443Y mutations. The molecular profile favored therapy-related AML. Interestingly, cytogenetics revealed 46,Y,t(X;21)(q26;q22)[20]. FISH confirmed RUNX1 rearrangement but the partner was unknown. The t(X;21)(q26;q22) appears to be novel. However, the rare t(3;21)(q26.2;q22) has been cited to segregate with therapy-related MDS/AML. Subsequent aberrant activation of RUNX1 and MECOM is postulated to be pivotal in pathogenesis. Therefore, it is of great interest to determine the translocation partner associated with RUNX1 in our case. This type of analysis is not easily amenable to either Sanger or conventional NGS approaches. Fortuitously, with the recent advent of AMP technology (Archer FusionPlex) novel fusion detection has become possible. AMP was key in the characterization of this sample and promotes discovery of novel fusions by targeting one (known) of the two genes involved in a translocation event. Application of this cutting edge technology lead to the discovery of the novel fusion RUNX1-G6PD in this case. The identity of the novel fusion partners was supported by the breakpoints originally observed by the karyotype analysis. G6PD has been previously reported to be upregulated in acute leukemia. This discovery raises the possibility convergent pathogenic pathways and carries potential biological, diagnostic, and therapeutic implications.

Citation Format: Laura Johnson, Helen Wang, Katelyn Trifilo, Brian Kudlow, Peter R. Pappenhausen, Mohammad Hussaini. Novel t(X;21)(q26;q22) detected in a case of acute unclassifiable leukemia by application of anchored multiplex PCR-based next-generation 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 3984.
Abstract 3988: Detection of novel t(12;17)(p12;p13) in treatment-refractory/relapsed acute myeloid leukemia by anchored multiplex PCR(AMP)-based next-generation sequencing
Authors: Johnson Laura , Trifilo Katelyn , Wang Helen , Kudlow Brian , Padron Eric , Pappenhausen Peter R. , Hussaini Mohammad
Published: July 2016

Gene fusions are an important class of mutations and have been shown to drive many genetic diseases. While several technologies can be used to detect fusions, anchored multiplex PCR (AMP) next generation sequencing (NGS)-based detection offers the advantages of novel fusion detection and the ability to multiplex multitudinous genes. Here we report application of this technology in the evaluation of a 56-year-old man diagnosed with AML (2013). He was treated initially with Vidaza and later with CLAG-M induction (2014) with complete response and consolidation CLA (2015). He relapsed (4/2015) and was reinduced with 7+3 (Ida). He relapsed again and was reinduced with FCT but bone marrow biopsy showed persistent disease (75% blasts). Karyotype showed 46, XY, t(12;17)(p12;p13)[20]. It was postulated that ETV6 oncogene was involved based on 12p13 locus. The partner gene was unknown in this patient with multiply relapsed/refractory disease. AMP technology (Archer FusionPlex) confirmed involvement of the expected ETV6. However, more importantly, employing this technology lead to the discovery of a novel fusion partner, HIC1. The identity of the novel fusion partners was supported by the breakpoints originally observed by karyotype analysis. AMP promotes discovery of novel fusions by targeting one (known) of two genes involved in the fusion event. HIC1 regulates cellular growth and serves as a tumor suppressor gene. Inactivation of HIC1 has been associated with aggressive disease and poor survival in other cancer types. Interestingly, in vitro restoration of gene function by 5-aza-dC resulted in decreased cell proliferation and tumor aggressiveness in pancreatic cancer and head and neck squamous cell carcinoma suggesting the prospect of a therapeutic target. This discovery provides new insight into a possible leukemogenic pathway in AML and potential for targeted therapy.

Citation Format: Laura Johnson, Katelyn Trifilo, Helen Wang, Brian Kudlow, Eric Padron, Peter R. Pappenhausen, Mohammad Hussaini. Detection of novel t(12;17)(p12;p13) in treatment-refractory/relapsed acute myeloid leukemia by anchored multiplex PCR(AMP)-based next-generation 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 3988.
Abstract 3997: The impact of PD-L1 expression in patients with metastatic GEP-NETs
Authors: Kim Seung T. , Lee Won
Published: July 2016

Programmed death-ligand 1 (PD-L1), which is expressed on many cancer cells, interacts with PD1 expressed on the surface of T cells, inhibiting the T cells and blocking the antitumor immune response. Expression of PD-L1 in gastroenteropancreatic neuroendocrine tumors (GEP-NETs) has not been studied. We investigated the impact of PD-L1 expression in 32 patients with metastatic GEP-NET.

The expression of PD-L1 was evaluated using an anti-PD-L1 immunohistochemistry (IHC) antibody optimized for staining of formalin-fixed paraffin-embedded (FFPE) tissue samples. The correlation between PD-L1 and clinicopathological data including survival and response to systemic treatments was analyzed.

Primary sites were 24 foregut-derived GEP-NETs, including stomach (n = 1), duodenum (n = 2), biliary tract (n = 7), and pancreas (n = 14), and 8 hindgut-derived GEP-NETs of the distal colon and rectum. Among the 32 patients with metastatic GEP-NET analyzed in this study, 7 (21.9%) had expression of PD-L1 in tumor tissues. Expression of PD-L1 was significantly associated with high-grade WHO classification (grade 3) (p = 0.008) but not with gender, primary site, and number of metastatic sites (p>0.05). The status of PD-L1 expression was statistically associated with progression-free survival (PFS) for first-line systemic treatment (p = 0.047). Moreover, the status of PD-L1 expression could significantly predict overall survival (p = 0.037).

The expression of PD-L1 was associated with higher WHO tumor grade (grade 3) in metastatic GEP-NETs. PD-L1 expression had both predictive and prognostic value for survival of patients with metastatic GEP-NETs.

Citation Format: Seung T. Kim, Won Lee. The impact of PD-L1 expression in patients with metastatic GEP-NETs. [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 3997.