bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2292: Reconstitution of myeloid derived suppressor cells after the induction of lymphopenia and TIL therapy
Authors: Kodumudi Krithika N. , Wiener Doris , Weber Amy , Royster Erica , Kelley Linda , Sarnaik Amod , Pilon-Thomas Shari
Published: July 2016

Factors such as T regulatory (Treg) cells and myeloid derived suppressor cells (MDSC) limit the effectiveness of current immunotherapy protocols. Several reports have shown that administration of chemotherapeutic agents or total body irradiation (TBI) before adoptive transfer of tumor-specific T cells reduces or eliminates the immunosuppressive populations such as Tregs and MDSC. A previous study in our lab has shown that MDSC that reconstitute after lymphodepletion are highly suppressive compared to the endogenous MDSC in a B16 melanoma model. Blockade of MDSC expansion in combination with adoptive T cell transfer delayed tumor growth and improved survival rate. However, there is very little known about the MDSC reconstitution after the adoptive transfer of tumor infiltrating lymphocytes (TIL) in melanoma patients. Melanoma patients were treated with non- myeloablative therapy that included cyclophosphamide and fludarabine. At the time of TIL transfer, no circulating lymphoid or myeloid cells were detected in the PBMC. One day after completion of chemotherapy, up to 100 billion TIL were transferred followed by high dose bolus IL-2. At weeks 1-4 after TIL transfer, PBMC were collected and we investigated the reconstitution and the suppressive function of MDSC. MDSC were phenotyped by flow cytometry as HLA-DR− Lin− CD14+CD33+ or CD11b+CD14−CD33+. While 3-15% of MDSC was observed in the PBMC of melanoma patients as their baseline, after lymphodepletion and one week post TIL infusion, the percent of MDSC increased to 10-30% and gradually dropped to baseline by week 4. To examine suppressive function, MDSC were isolated from peripheral blood at 1-2 weeks after TIL infusion and co-cultured with healthy donor T cells or patient TIL. MDSC were highly suppressive and abrogated T cell proliferation and IFN-gamma secretion. Furthermore, in patients that responded to TIL therapy, the ratio of peripheral MDSC to total CD8+ T cells was lower. These studies demonstrate that MDSC that reconstitute post- lymphodepletion are suppressive and may potentially interfere with robust T cell function. Further studies are warranted to clarify the role of MDSC in the efficacy of TIL therapy. Blocking MDSC in combination with TIL therapies represent an improved strategy for the treatment of melanoma patients.

Citation Format: Krithika N. Kodumudi, Doris Wiener, Amy Weber, Erica Royster, Linda Kelley, Amod Sarnaik, Shari Pilon-Thomas. Reconstitution of myeloid derived suppressor cells after the induction of lymphopenia and TIL 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 2292.
Abstract 230: The role of integrated stress response (ISR) transcription factor ATF4 in metabolic adaptation of tumors to c-Myc activation
Authors: Tameire Feven , Monroy Maria A. , Koumenis Constantinos
Published: July 2016

The ability of cancer cells to adapt to non-cell autonomous (extrinsic) and cell autonomous (intrinsic) stresses is critical for maintaining cell viability and growth. The Integrated Stress Response (ISR) pathway is essential in cancer cell adaptation to extrinsic stresses such as hypoxia and nutrient deprivation both in vitro and in vivo. The ISR kinases, PERK and GCN2, phosphorylate eIF2α, transiently inhibiting protein translation during times of stress. Phosphorylation of eIF2α promotes the preferential translation of the transcription factor ATF4, which activates a gene expression program that enhances uptake and synthesis of amino acids, antioxidant defense and chaperone expression to promote recovery from nutrient deprivation and ER stress. Here, we demonstrate the importance of ISR signaling in MYC oncogene induced intrinsic stress both in vitro and in vivo.

We previously reported that activation of PERK, during c-Myc induction is vital for activating cytoprotective autophagy and suppressing c-Myc induced apoptosis. Here, we demonstrate GCN2 is also robustly activated during c-Myc induction. GCN2 activation after c-Myc induction regulates ATF4 transcription and translation. ATF4 was necessary for survival and suppression of oxidative stress during c-Myc activation. We have identified ATF4 targets that regulate amino acid uptake and involved in amelioration of oxidative stress. Furthermore, siRNA mediated knockdown or genetic knockout of GCN2 was synthetic lethal with c-Myc activation. Interestingly, PERK is still able to phosphorylate eIF2α in the absence of GCN2. However, eIF2α phosphorylation was completely inhibited in the absence of both kinases suggesting that these kinases compensate for each other and both are required for optimal phosphorylation of eIF2α after c-Myc activation. Consistent with our in vitro data, we observe higher levels of p-eIF2α and ATF4 in lymphoma cells compared to WT B cells in vivo in a mouse model of Myc driven lymphoma. These findings highlight a novel mechanism of stress response pathway activation to support c-Myc driven tumorigenesis involving an amino acid sensor kinase GCN2.

Citation Format: Feven Tameire, Maria A. Monroy, Constantinos Koumenis. The role of integrated stress response (ISR) transcription factor ATF4 in metabolic adaptation of tumors to c-Myc activation. [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 230.
Abstract 2309: T-cell development from T cell-derived induced pluripotent stem cell
Authors: van der Stegen Sjoukje J.C. , Themeli Maria , Eyquem Justin , Mansilla-Soto Jorge , Sadelain Michel
Published: July 2016

The ability to differentiate T cells of defined specificity and function from T-cell derived induced pluripotent stem cells (TiPSC) may be useful for the treatment of a range of pathologies, including cancer, infection and immune deficits. We recently reported that genetic engineering of TiPSC to express a Chimeric Antigen Receptor (CAR) is an effective strategy to combine the unlimited availability of TiPSC and facilitate reprogramming of the T cell specificity and functional potential. In vivo, CD19-retargeted human TiPS-derived CAR T cells (CARTiPSC-T) display therapeutic potency in a lymphoma model. Surprisingly, despite expression of the endogenous αβ TCR, the CARTiPSC-T cells possessed innate-like phenotype and function, most similar to γδ T cells. Although innate T cells have anti-tumor activity, they lack some vital features for therapeutic efficacy including long-term memory and in vivo persistence, which characterize mature CD8+ and CD4+ αβ TCR T cells.

Additional research into the mechanisms underlying in vitro T cell differentiation of TiPSC is required to improve the development of mature TCRαβ T cells and facilitate the employment of their therapeutic potential. T cell lineage determination depends in part on the balance between Notch and TCR signaling, we are investigating their respective roles, as well as that of the CAR, in determining lineage commitment. We hypothesize that the combined CAR and early CD3/TCRαβ expression disrupts the TCR/Notch signaling balance, prohibiting mature TCRαβ T cell development.

To study the effects of the different Notch ligands, TiPSC were differentiated on OP9 cells expressing one of four Notch ligands (DLL1, DLL4, Jagged-1 or Jagged-2). Preliminary data suggests that DLL4 is able to facilitate T cell development to CD4/CD8 double positive (DP) stage, however, this is hindered by CAR expression.

To determine the role of early TCRαβ expression, elimination of TCRαβ expression was facilitated by targeted disruption of the TCRα constant region using the CRISPR/Cas9 system. Elimination of early TCRαβ expression showed improved development towards DP stage of TiPSC on OP9-DLL1.

TiPSC are a valuable system for the study of human T cell differentiation. In addition, and most importantly they are further amenable to genetic engineering with TCRs or CARs, which may be useful for the generation of therapeutic “off-the-shelf”, antigen-specific T cells.

Citation Format: Sjoukje J.C. van der Stegen, Maria Themeli, Justin Eyquem, Jorge Mansilla-Soto, Michel Sadelain. T-cell development from T cell-derived induced pluripotent stem cell. [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 2309.
Abstract 2325: Immune checkpoint(s) expression in AML patients enrolled in a phase 1-2 study with guadecitabine
Authors: Fazio Carolina , Covre Alessia , Lofiego Maria , Taverna Pietro , Azab Mohammad , Lowder James N. , Coral Sandra , Maio Michele
Published: July 2016

Immune checkpoint(s) blockade is becoming the therapeutic mainstay in melanoma and lung cancer. Based on these important clinical achievements, immunotherapy with immunomodulating monoclonal antibodies (mAb) is being explored as a new therapeutic modality in most human malignancies, either alone or in combination with other immunomodulating agents. We have provided extensive evidence that the second generation DNA hypomethylating agent (DHA), guadecitabine (SGI-110), plays a promising role in potentiating the immunogenicity and the immune recognition of human malignancies through the up-regulation of the expression of different immune molecules on cancer cells. These findings led us to hypothesize that DHA could represent ideal partners for immune checkpoint(s) blocking agents to improve their therapeutic efficacy. Here, we studied the expression of programmed death 1 (PD-1), programmed death-ligand 1 (PD-L1), and 2 (PD-L2), and cytotoxic T lymphocyte-associated antigen 4 (CTLA4) in peripheral blood mononuclear cells of acute myeloid leukemia (AML) patients (N = 23), enrolled in a phase 1-2 study with guadecitabine. Patients included in this preliminary analysis were selected based on their responsiveness to therapy. Quantitative RT-PCR analyses showed a constitutive expression (gene/β-actin molecules >7.5E-05) of PD1, PD-L1, PD-L2 and CTLA-4 in 82.6%, 100%, 47.8% and 8.7% patients, respectively. Guadecitabine therapy up-regulated (≥2 fold) the expression of PD1, PD-L1, PD-L2 and CTLA-4 in 57.9%, 56.5%, 18.2% and 0% patients, respectively, at any of the time-points investigated during treatment. De novo expression of PD1, PD-L2 and CTLA-4 was observed in 100%, 41.6% and 38.1% of the immune checkpoint(s)-negative patients, respectively.

Though preliminary, these results further support the strong link between epigenetics/DNA methylation and immune response, in cancer patients, and strengthen the therapeutic potential of epigenetic immunomodulation, with “consolidated” and emerging immune checkpoint(s) blocking mAb.

Citation Format: Carolina Fazio, Alessia Covre, Maria Lofiego, Pietro Taverna, Mohammad Azab, James N. Lowder, Sandra Coral, Michele Maio. Immune checkpoint(s) expression in AML patients enrolled in a phase 1-2 study with guadecitabine. [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 2325.
Abstract 2327: IFNgamma regulates PD-L1 expression through activating IRF1 transcription in tumor cells
Authors: Lu Chunwan , Paschall Amy V. , Redd Priscilla S. , Lebedyeva Iryna , Liu Kebin
Published: July 2016

The emerging clinical success of checkpoint blockade immunotherapy in human cancer patients has highlighted the critical importance of PD-L1, a ligand for the T cell inhibitory receptor PD-1, as a molecular target in cancer immunotherapy. PD-L1 is constitutively expressed in tumor cells and is also inducible by inflammatory cytokines such as IFNgamma. It has been proposed that tumor cells may sense the elevated IFNgamma from activated host T cells as a “threat” in the tumor microenvironment and adapt it by up-regulating PD-L1. The aim of this study is to elucidate the molecular mechanism underlying IFNgamma regulation of PD-L1 expression in tumor cells. We observed that PD-L1 is constitutively expressed, albeit at low level, in multiple types of cancer cells, including pancreatic, colon, breast, and sarcoma cells. IFNgamma treatment dramatically increased PD-L1 expression level and IFNgamma up-regulates PD-L1 expression through the Jak-STAT1 signaling pathway in vitro and in vivo. Chromatin immunoprecipitation assay did not identify IFNgamma-activated pSTAT1 binding to the pd-l1 promoter. Instead, we observed that IFNgamma activates IRF1 transcription and IRF1 is required for IFNgamma-induced PD-L1 expression. Chromatin immunoprecipitation analysis shows that pSTAT1 is associated with the irf1 but not the pd-l1 promoter. Analysis of the irf1 promoter DNA sequence revealed a pSTAT1-binding consensus sequence, and electrophoretic mobility shift assay indicates that pSTAT1 directly binds to this DNA element of the irf1 promoter. Furthermore, we demonstrated that IRF1 is associated with the pd-l1 promoter chromatin near the irf1 transcription initiation site. The pd-l1 promoter region contains a putative IRF1-binding consensus sequence and electrophoretic mobility shift assay shows that IRF1 binds to this DNA element of the pd-l1 promoter region. Taken together, our data indicate that IFNgamma activates pSTAT1 that binds to the irf1 promoter to activate irf1 transcription. IFNgamma-induced IRF1 then binds to the pd-l1 promoter to activate pd-l1 transcription in tumor cells.

Citation Format: Chunwan Lu, Amy V. Paschall, Priscilla S. Redd, Iryna Lebedyeva, Kebin Liu. IFNgamma regulates PD-L1 expression through activating IRF1 transcription in tumor 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 2327.
Abstract 2347: The amphiphatic β(2,2)-amino acid derivative, LTX-401 induces complete regression of experimental hepatocellular carcinomas
Authors: Mauseth Brynjar , Shi Ji-Hua , Camilio Ketil , Rekdal Øystein , Sveinbjørnsson Baldur , Line Pål-Dag
Published: July 2016

Hepatocellular carcinoma (HCC) is the sixth most common cancer in the world and third cause of cancer-related deaths. Because HCC is a relatively chemotherapy-resistant cancer, and many patients are restricted to surgical resection or liver transplantation, there is an urgent need to develop new and improved therapeutic regiments. In the present study, we have investigated the therapeutic effect of a novel oncolytic compound, LTX-401, designed for the local treatment of solid malignancies. In vitro analysis revealed that LTX-401 displayed potent anticancer activity against a broad variety of cancer cell lines. In our model cell line (JM1 HCC), LTX-401 was found to induce necrotic (lytic) cell death followed by the release of potent immune stimulants in the form of Danger-Associated Molecular Pattern molecules (DAMPs) such as HMGB1 and ATP. Ultrastructural analysis by transmission electron microscopy also revealed that a substantial portion of LTX-401-treated JM1 cells contained significantly affected mitochondria. Additionally, cytochrome c was detected in cell supernatants after treatment. The anticancer effect of LTX-401 was investigated in a rat hepatocellular carcinoma model by inoculating JM1 cells into the liver (intrahepatic) of syngeneic Fischer 344 rats. LTX-401 induced a complete regression of HCC tumors by intratumoral injection. Tumor-specific protective immune responses were obtained in previously cured animals, as re-challenge with live and equivalent tumor cells did not lead to tumor growth. Furthermore, intratumoral treatment with LTX-401 was able to eradicate diffuse distant untreated metastasis in the liver. Immuno-histological analysis showed T-cell infiltration into treated tumors. Taken together, intrahepatic treatment with LTX-401 can induce complete regression and long lasting protective tumor-specific immune responses.

Citation Format: Brynjar Mauseth, Ji-Hua Shi, Ketil Camilio, Øystein Rekdal, Baldur Sveinbjørnsson, Pål-Dag Line. The amphiphatic β(2,2)-amino acid derivative, LTX-401 induces complete regression of experimental hepatocellular carcinomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2347.
Abstract 2360: GP350 containing exosomes as DNA vaccine carriers
Authors: Colluru Viswa Teja , McNeel Douglas G.
Published: July 2016

Background: Plasmid DNA vaccination is a safe and economical vaccine modality that has been demonstrated to elicit cellular and humoral immunity in preclinical models and human clinical trials. We have previously shown that enriched mature naive B cells, and not myeloid/DC lineages, are capable of expanding Ag specific CD8 T cells in vivo in mice, and in vitro with human cells. In this study, we sought a means to directly deliver plasmid DNA to B cells as a means to specifically activate this antigen-presenting cell type.

Methods: Exosomes were isolated from EBV+ lymphoblastic cell lines (LCL) and from HEK293T cells that had been transfected to express the EBV protein GP350. Plasmid DNA was labeled with a fluorescent peptide nucleic acid (PNA) probe for detection by imaging and conventional cytometry. Exosomes were transfected with fluorescently labeled plasmid DNA and incubated with either human CD21 transgenic mouse splenocytes or human PBMC and assayed for uptake and activation by FACS. Plasmid DNA transfected exosomes were coincubated with human PBMC and evaluated for their ability to expand antigen-specific CD8+ T cells by different methods.

Results: EBV LCL and HEK293T derived exosomes were successfully transfected with plasmid DNA as verified by image assisted flow cytometry. Transient transfection of HEK293T cells with plasmids encoding a transgene led to release of exosomes containing the transgene product. EBV LCL and HEK 293T-GP350 derived exosomes increased delivery of plasmid DNA to both human B lymphocytes and transgenic hCD21 mouse B lymphocytes, when compared to naked DNA alone, in an hCD21 dependent fashion. At 24h, plasmid DNA containing B lymphocytes exhibited increased activation and upregulation of CD80/86 costimulatory molecules. Further, human B lymphocytes treated with exosome-plasmid DNA complexes led to antigen mRNA production and expansion of Ag specific CD8 T cells without the need for APC subset enrichment. Studies comparing the immunogenicity of GP350-exosome mediated delivery versus naked DNA delivery are underway in different mouse models.

Conclusion: GP350 containing exosomes can successfully increase delivery of plasmid DNA to B lymphocytes even in the presence of other phagocytic cells like macrophages or dendritic cells. This led to detectable antigen production and expansion of cognate Ag specific CD8 T cell expansion without the need for B cell enrichment. Studies examining in vivo immunogenicity of exosomal plasmid DNA delivery are underway, but results to date suggest this approach may be a simple method to boost vaccine efficacy through increased delivery of DNA directly to relevant antigen-presenting B cells.

Citation Format: Viswa Teja Colluru, Douglas G. McNeel. GP350 containing exosomes as DNA vaccine carriers. [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 2360.
Abstract 2368: Inhibition of epidermal growth factor receptor pathway by epidermal growth factor antibodies in non-small cell lung cancer
Authors: Codony-Servat Jordi , Molina-Vila Miguel Angel , Bertran-Alamillo Jordi , Rosell Rafael , D’Hondt Erik
Published: July 2016

Introduction: The Epidermal Growth Factor Receptor (EGFR) signaling system is frequently unbalanced in non-small-cell lung cancer (NSCLC). Epidermal Growth Factor (EGF) could be an attractive target in these tumors. We used a “cancer vaccine” composed of human recombinant EGF conjugated to a carrier protein P64K. The vaccine is intended to induce antibodies against patients’ own EGF that would block EGF-EGFR interaction.

Objectives: In NSCLC cell lines we assessed whether anti-EGF antibodies can inhibit the pathway activated by EGF-EGFR binding and whether sera from immunized patients can block downstream key signal transduction proteins. NSCLC cell lines were selected to determine whethergenetic alterations in EGFR, KRAS, ALK, BRAF or MET can have an impact on inhibition of EGFR pathway by anti-EGF antibodies.

Experimental Procedures: Western blotting was used to evaluate the capacity of antibodies and sera from patients to inhibit EGFR activation pathway. MTT assays were performed to evaluate the effects on tumor cell proliferation.

Results: The anti-EGF antibodies showed a dose-dependent inhibitory effect on EGF-induced activation of EGFR and downstream pathway molecules such as Akt and ERK 1/2. This inhibition was observed in all cell lines tested, including KRAS, EGFR and BRAF mutated cells as well as cells with ALK translocations or MET amplification. Sera from 10 patients immunized with the P64K-EGF vaccine were then tested. All sera reversed EGF-induced activation of EGFR and ERK 1/2, but some sera were significantly more potent than others. By contrast, two control sera from non-vaccinated patients failed to inhibit EGF-induced phosphorylation of EGFR and ERK 1/2. Finally, anti-EGF antibodies were also able to block the stimulatory effects of EGF on the growth of some NSCLC cell lines.

Conclusions: Anti EGF antibodies and sera from patients immunized with an “EGF vaccine”, called otherwise EGF Pathway Targeted Immunization inhibits EGFR signaling in NSCLC cell line models. This effect is independent of cells’ genetic background.

Citation Format: Jordi Codony-Servat, Miguel Angel Molina-Vila, Jordi Bertran-Alamillo, Rafael Rosell, Erik D’Hondt. Inhibition of epidermal growth factor receptor pathway by epidermal growth factor antibodies in non-small cell lung 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 2368.
Abstract 2370: Therapeutic nano-DC vaccine For HER2 positive breast cancer
Authors: Shen Haifa , Xia Xiaojun
Published: July 2016

Harnessing a patient's own immune capacity to eradicate malignant cells is a promising approach for cancer treatment, and therapeutic vaccine represents an important arm of cancer immunotherapy. Our research aims to develop a potent therapeutic cancer vaccine for breast cancer which is traditionally considered as poorly immunogenic. We have recently shown that porous silicon microparticles (PSMs) can stimulate type I interferon (IFN) expression in dendritic cells (DCs), and thus serve as a potent adjuvant for a therapeutic cancer vaccine (Xia et al: Cell Reports 2015, 11:957-966). This action is mediated by the TRIF/MAVS pathways, and is independent of the cell surface or endosomal Toll-like receptors. In addition, the nanometer-size pores in PSMs can serve as a reservoir for sustained antigen release. We have assembled a Nano-DC vaccine comprising of bone marrow-derived DCs internalized with HER2 antigen peptide-loaded PSMs. Treatment of murine models of HER2 positive breast cancer with this Nano-DC vaccine not only activates and expands antigen-specific CD8+ T cells, but also promotes a Th2-to-Th1 transition in the tumor microenvironment to favor anti-tumor activity. Thus, our result supports the development of a therapeutic Nano-DC vaccine for breast cancer.

Citation Format: Haifa Shen, Xiaojun Xia. Therapeutic nano-DC vaccine For HER2 positive 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 2370.
Abstract 2371: Multi-cell phylogenetic inference of copy number evolution in breast cancer
Authors: Davis Alexander J. , Gao Ruli , Sei Emi , Tsai Pei-Ching , Casasent Anna , Zhang Amy , Shi Xiuqing , Wang Yong , Waters Jill , Meric-Bernstam Funda , Edgerton Mary , Navin Nicholas
Published: July 2016

Aneuploidy is a hallmark of breast cancer, but little is known about how these complex genomic rearrangements evolve during tumor growth. To investigate this question, we developed a Highly-Multiplexed Single Nucleus Sequencing (HM-SMS) method to profile genome-wide copy number in individual tumor cells and compared multiple cells to infer evolutionary lineages. We applied this method to analyze thousands of single cells from 12 triple-negative breast cancer patients and 10 ductal-carcinoma-in-situ (DCIS) patients. Integer copy number profiles were calculated from a combination of sequencing data and ploidy estimation using flow cytometry. A multi-cell segmentation algorithm was used to identify common CNA events that are shared between tumor cells in each patient. Trinary event matrices were calculated for each patient to determine the presence or absence of individual copy number aberrations (CNAs) in individual cells. Using the maximum parsimony criterion, we inferred a phylogenetic tree for each tumor and estimated the temporal order of the CNAs.. Our data suggest that most tumors consisted of 1-4 major clonal subpopulations that shared a common evolutionary lineage, suggesting that they evolved from a single normal cell in the breast tissue. Furthermore our data support a punctuated model of copy number evolution, which which the majority of CNAs were acquired in a short evolutionary burst, followed by one or more stable clonal expansions to form the tumor mass.

Citation Format: Alexander J. Davis, Ruli Gao, Emi Sei, Pei-Ching Tsai, Anna Casasent, Amy Zhang, Xiuqing Shi, Yong Wang, Jill Waters, Funda Meric-Bernstam, Mary Edgerton, Nicholas Navin. Multi-cell phylogenetic inference of copy number evolution in 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 2371.
Abstract 2382: Novel integration of genomic and morphological information reveals tumor tissue heterogeneity
Authors: Lee Chen-Chung , Predki Paul , Raub Christopher , Shibata Darryl , Taylor Clive , Kartalov Emil , Anderes Kenna
Published: July 2016

Cancer treatment decisions today are predominantly based upon decades-old histological methods, which more recently have been combined with simple phenotypic or molecular tests. To advance this paradigm, we have developed a digital molecular morphology platform that integrates histological images with next-generation sequencing (NGS) or PCR data. This allows us to visualize DNA mutations and RNA expression levels across a histological image, and to correlate mutational or expression status to cell morphological features. Thus, providing a novel approach to mapping tumor heterogeneity. The basic approach is to overlay a microstructure on top of H&E-stained FFPE slides to create a large number of tissue-bottomed micro-wells, which can be as small as a single cell or contain clusters of cells. Reagents are added to the micro-wells and biochemical reactions are carried out and analyzed in place (PCR) or partially offline (NGS). In the case of NGS, barcodes are used to reference the resulting sequence back to the tissue image. Software is then used to analyze and visualize the resulting genetic information in the context of the original histological image. Our initial work has focused on FFPE cancer biopsies using targeted cancer genes and gene panels. Here we demonstrate the use of this approach to measure and visualize KRAS G12V mutational status in colorectal cancer tissues, using both PCR and NGS detection modalities. We show that with this method we are able to efficiently extract DNA from the tissue while maintaining a leak-proof physical integrity from one micro-well to another. Further, we are able to PCR-amplify DNA in the micro-wells for direct fluorescence detection, or to create libraries for sequencing. Detection of KRAS G12V correlates well with malignant morphological features in the H&E stained tissue, and appears to present a more sensitive way to detect mutations than PCR or NGS from bulk FFPE tissue alone. We are currently applying this technology to a variety of research and clinical applications, ranging from assessment of tumor heterogeneity and evolution, to prediction of patient outcome, to post-operative NGS-based characterization of surgical margins. This integration of cellular and molecular data promises to more fully enable precision medicine to guide the course of treatment and improve individual patient outcomes.

Citation Format: Chen-Chung Lee, Paul Predki, Christopher Raub, Darryl Shibata, Clive Taylor, Emil Kartalov, Kenna Anderes. Novel integration of genomic and morphological information reveals tumor tissue heterogeneity. [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 2382.
Abstract 2385: Understanding the dynamic interplay between genetically different cancer cell clones in glioblastoma
Authors: Guo Min , Heller Susanne , Thorslund Jessie , Orre Lukas , Lehtiö Janne , Nistér Monica , Hägerstrand Daniel
Published: July 2016

Glioblastoma is the most frequent and aggressive brain tumor in adults. Based on genomic data glioblastomas can be classified into at least four subclasses: proneural, neural, classical and mesenchymal. However, recent publications have shown that individual glioblastoma can be heterogeneous and contain a mixture of these four subclasses. Moreover, it has been shown in other studies that intratumoral communication between genetically distinct cancer cell subclones in glioblastomas can affect the overall tumor growth via secreted proteins. In this project we aim to investigate the difference between protein secretomes from different tumor cell clones from within genetically heterogeneous glioblastomas.

For this we have used the U343 cell culture system, consisting of U343MG, U343MGa, U343MGa-31L and U343MGa-Cl2:6. These cultures were derived from a single glioblastoma, and can be divided into at least two categories based on their mutually exclusive expression patterns of FN1 and GFAP. Here we show that these different cultures display different characteristics with regard to matrigel invasion capacity, neurosphere formation capacity, and gene and protein expression. Combinatorial co-culture and conditioned media based experiments showed that the U343MG culture elicit anti-proliferative effects on U343MGa-31L via secreted factors. To identify proteins that are secreted by U343MG we have used Secretome Protein Enrichment with Click Sugars (SPECS) followed by mass spectrometry analysis. We detected 150 proteins by more than 2 peptides in U343MG conditioned media. Several of these proteins were growth factors and matrix proteins, including FN1. We are now performing a combined secretome and gene expression analysis of all U343 cultures to identify candidate secreted proteins that most likely are involved in the observed signaling effects between U343MG and U343MGa-31L. Finally, a functional genomic approach will be taken to experimentally pinpoint mediators of these inter-clonal effects.

This study shows that subclones from a heterogeneous glioblastoma can display different phenotypic characters and affect each other via secreted factors. Further knowledge about cell-to-cell communication through secreted proteins in glioblastoma may provide novel therapeutic targets.

Citation Format: Min Guo, Susanne Heller, Jessie Thorslund, Lukas Orre, Janne Lehtiö, Monica Nistér, Daniel Hägerstrand. Understanding the dynamic interplay between genetically different cancer cell clones in 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 2385.
Abstract 2389: Microarray analysis of polysome-associated mRNAs from different regions of the same human glioblastoma reveals intratumoral heterogeneity
Authors: Hajj Glaucia N. , Lupinacci Fernanda S. , Roffé Martín , Kuasne Hellen , Santos Tiago G. , Andrade Victor P. , Sanematsu Paulo , Martins Vilma R. , Rogatto Silvia R.
Published: July 2016

Glioblastoma is among the most aggressive tumor type and less responsive to chemotherapeutic agents, thus a better understanding of the behavior of these tumors may help to develop new treatments for this disease. Currently, many genome-wide projects attempt to define general patterns of gene expression based on deep sequencing or microarray data from total mRNA populations. However, this approach provides little information about the molecular mediators of tumor biology, because the expression levels of mRNAs do not necessarily reflect the levels of proteins. The identification of mRNAs target of translational alterations in tumors can show gene expression profiles that better reflect the population of proteins. In this work we were able to identify mRNAs differentially translated in a human glioblastoma that presented histologically different parts. The sample was divided in 8 pieces that were classified as high or low grade based on histological characteristics. Actively translating ribosomes and their associated mRNAs were isolated biochemically through a polysomal profile. Total and polysomal mRNA was then extracted and submitted to a microarray analysis. By comparing high vs low grade tumor pieces, we were able to identify more differentially translated genes (138 genes) than differentially expressed genes (43 genes). Among the differentially translated mRNAs, there are many related to proliferation, development and cancer. By Integrated Pathway Analysis, we identified the TGF-β pathway among the the most relevant for grade progression. Thus, the technique of isolating mRNAs engaged in translation can be used to identify biomarkers of tumor progression, leading to new therapeutic approaches.

Citation Format: Glaucia N. Hajj, Fernanda S. Lupinacci, Martín Roffé, Hellen Kuasne, Tiago G. Santos, Victor P. Andrade, Paulo Sanematsu, Vilma R. Martins, Silvia R. Rogatto. Microarray analysis of polysome-associated mRNAs from different regions of the same human glioblastoma reveals intratumoral heterogeneity. [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 2389.
Abstract 2399: System analysis of adapted and quiescent/dormancy(QD) models of drug resistance in melanoma cell lines
Authors: Liu Feng , Panchal Parvita Paresh , Wang Zi , Farhat Ahmed , Garcia Angela , Fagundes Tcharles , Filipp Fabian , Meyskens Frank L.
Published: July 2016

The biological basis for inherent and acquired drug resistance in melanoma is a prominent issue, highlighted by the recent discovery of various pathways leading to acquired resistance to the mutant BRAF inhibitor Vemurafenib (Plx4032). In order to dissect patterns of drug resistance, we treated 3 melanoma cell lines (A375, 1205Lu and SK-Mel28) under 2 diverse conditions: (1) Increasing drug concentrations over time(4 months)- Melanoma cells treated with increasing concentrations of Plx4032 (initial and increasing concentrations of 0.1, 0.5, 1 and 5 μM PLx4032) exhibited an adaptation response. After an initial pause in proliferation, these cells proliferated in the continuing presence of the drug. Cells able to grow in the presence of 10 or 20 μM of Plx4032 were cultured as pools. RNA-Seq profiling and system biology analysis comparing the gene expression in the parental SK-Mel28 cells and adapted A2-1 revealed three major altered pathways: the PDGF-MAPK-DUSP-HIF2α, TGF-β-SMAD9-FGF1 and EDNRB-GNAI2-PLCB4-PKA-MITF pathway. Our qRT-PCR and western blot experiments have confirmed an up-regulation of PDGF which by-passed the BRAF gene and activated ERK1/2 kinases. These changes were accompanied by increase of NRAS accumulation in the adapted cells. (2) Using high initial drug concentrations of PLx4032 (>10 μM), > 99.9 of cells were killed, but some single cells (Q/D) survived for at least 4 weeks in the continuous presence of drug. After the drug was removed cellular proliferation reoccured 2-3 weeks later (designated post-dormant cells 1, PD1 cells). These cells did not show increased IC50s to Plx4032 after 72 hour of retreatment. However, when treated with high dose of drug again, the PD1 cells produced more dormant cells (PD2 cells), suggesting inheritance of the DQ trait. RNA-Seq profiling studies of the DQ cells are underway. The drug-induced dormancy/quiescence and adaptive response represent 2 different pathways of how cells survive treatment and develop resistance and provide a beginning to understanding the biological basis of the well established clinical phenomenon of long term clinical dormancy of melanoma and potentially other cancers.

Citation Format: Feng Liu, Parvita Paresh Panchal, Zi Wang, Ahmed Farhat, Angela Garcia, Tcharles Fagundes, Fabian Filipp, Frank L. Meyskens. System analysis of adapted and quiescent/dormancy(QD) models of drug resistance in melanoma cell lines. [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 2399.
Abstract 2403: Molecular mechanisms orchestrating intratumoral heterogeneity in Kras-driven carcinomas
Authors: Azimi Roxana
Published: July 2016

The considerable variability within tissue microenvironments as well as the multiclonality of cancers leads to intratumoral heterogeneity. This increases the probablility of cellular states that promote resistance to therapy and eventually lead to reconstitution of the tumor by treatment-resistant cancer cells, which in some cases have properties of normal tissue stem cells. Wnt signals are important in the maintenance of stem cells in various epithelial tissues, including in lung development and regeneration. We hypothesized that Wnt signals contribute to tumor heterogeneity in genetically engineered KrasG12D; Tp53Δ/Δ (“KP”) mouse lung adenocarcinomas (LUAD). We observed that a subpopulation of LUAD cells exhibited high Wnt reporter activity and had increased tumor forming ability, which could be suppressed by silencing of Wnt signaling pathway components or by small molecule Wnt inhibitors in vitro and in vivo. KP LUAD cells show hierarchical features with two distinct populations, one with increased Wnt reporter activity and another forming a niche that provides the Wnt signal. Lineage-tracing experiments in the autochthonous KP tumors demonstrated that Wnt responder cells have increased tumor propagation ability in vivo. Strikingly, selective ablation of the Wnt responder cells resulted in tumor stasis. CRISPR-based targeting or small molecules targeting Wnt signaling reduced tumor growth and prolonged survival in the autochthonous KP mouse lung cancer model. Interestingly, we found that a similar Wnt-driven mechanism operates to establish an intratumoral hierarchy also in other Kras-driven carcinomas. These results indicate that maintenance of heterogeneity within tumors may be advantageous for the tumor cell population collectively, which may be utilized as a therapeutic target to homogenize cancer cell phenotypes within tumors.

Citation Format: Roxana Azimi. Molecular mechanisms orchestrating intratumoral heterogeneity in Kras-driven carcinomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2403.
Abstract 2418: Microenvironment-on-chip: Development of a microfluidics-based tumor ecosystem
Authors: Torga Gonzalo , Lin Ke-Chih , Austin Robert H. , Pienta Kenneth J.
Published: July 2016

Despite its limited clinical predictive capacity, conventional cell culture remains the most frequently used preclinical model in biomedical research. For many years, microfluidics-based organ-on-chips have been proposed to overcome conventional cell culture limitations but given their inability to provide reliable and reproducible data, their usage remains anecdotal.

Immune, stromal and cancer cells constitute a complex adaptive system in each tumor microenvironment. To study the causes and consequences of tumor heterogeneity in these complex adaptive systems, we have developed a platform to model and investigate tumor microenvironments at primary and metastatic sites in which multiple nuclear-labelled cell types can be grown in a multi-welled, communicating habitat with controlled gradients for temperature, pH, nutrients, and oxygen tension. In conjunction with labelled-nuclei, genetically-engineered cell lines with fluorescence-based biosensors can be generated to detect specific genetic expression changes in response to cell to cell interactions as well as varying environmental conditions over time.

One of the major caveats for microfluidics prototypes is reproducibility. The proposed design is easily adaptable to most of the commercially available incubation microscopy systems, allowing the experiments to be replicated by independent groups in an effort to provide reliable and reproducible data.

The ability to establish dynamic gradients within individual habitats longitudinally over time and space provides the capacity to study the effect of chemokines and drugs on the tumor ecosystem in order to discover key interactions between host cells and cancer cells and to develop improved therapeutic strategies.

In contrast to previous organ-on-chip models, our technology is not limited to a mere observational platform for automated cell counting and tracking but also provides flexibility for downstream capacities such as immunofluorescence, in situ hybridization or single-cell sequencing, as well as retrieval of single cells or specific subpopulations based on the fluorescent reporters and labelled nuclei.

The presented engineered microenvironment allows continued in vitro quantitative studies of the interactions of multiple cell types as well as varying environmental conditions. Here we propose a cancer-on-chip platform that is able to recapitulate key components and interactions to mimic different tumor microenvironments in a comprehensive manner, yet simple enough to provide reliable and reproducible data.

Citation Format: Gonzalo Torga, Ke-Chih Lin, Robert H. Austin, Kenneth J. Pienta. Microenvironment-on-chip: Development of a microfluidics-based tumor ecosystem. [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 2418.
Abstract 2419: Anastomotic recurrences are clonally related to primary tumors in sporadic colorectal carcinoma
Authors: Vakiani Efsevia , Shah Ronak H. , Iacobuzio-Donahue Christine , Solit David B. , Weiser Martin R.
Published: July 2016

Background:

Anastomotic recurrences occur in 2-10% of colorectal carcinoma (CRC) cases after surgical resection of the primary tumor. To date there are no molecular data investigating their genetic profile and multiple theories exist as to their pathogenesis including re-growth of tumor cells present within local lymphatics after initial surgery, seeding of anastomosis by metastatic tumor cells and a second primary tumor. The aim of our study was to compare the genomic profile of anastomotic recurrences to that of matched primary tumors and, where available, to that of distant metastases.

Design:

Thirty-six tumors from 14 patients were genotyped using a hybridization capture-based next-generation sequencing assay for targeted deep sequencing of all exons and selected introns of 341 key cancer genes. All patients had resection of their primary tumor with clear margins and recurred either at the anastomotic line or in the peri-anastomotic area 1.1 to 7.0 years following resection of their primary tumor. In 3 patients 2 consecutive anastomotic recurrences were sequenced, while in 6 patients a distant metastasis that occurred 1.2 to 3 years prior to the anastomotic recurrence was also analyzed. All tumors were microsatellite stable except in one patient with genetically confirmed Lynch syndrome.

Results:

A total of 254 somatic mutations were detected including 140 mutations in the microsatellite stable cases. The most commonly mutated genes (mutated in > 3 patients) were APC, KRAS, TP53, PIK3CA, ATM and PIK3R1. In 13/14 patients the anastomotic recurrence(s) and primary tumor shared between 50-100% of mutations, including mutations in key driver genes such as APC, KRAS and TP53, consistent with these tumors being clonally related. In the patient with the Lynch syndrome the 2 tumors showed distinct somatic mutations suggestive of independent primaries. We identified eleven genetic events present in a distant metastasis and not in a primary tumor and none of these events was detected in the anastomotic recurrence arguing against re-seeding of the anastomotic site by a metastatic clone. All five patients with isolated anastomotic recurrence were free of disease 0.9 to 6.1 years following resection of their recurrent CRC.

Conclusions:

Anastomotic recurrences appear to be clonally related to the primary tumor in sporadic CRC cases. Our data also provide molecular data in support of the treatment of anastomotic recurrences as localized disease.

Citation Format: Efsevia Vakiani, Ronak H. Shah, Christine Iacobuzio-Donahue, David B. Solit, Martin R. Weiser. Anastomotic recurrences are clonally related to primary tumors in sporadic colorectal 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 2419.