bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 207: CML as a prototypical malignancy
Authors: Sonkin Dmitriy , Simon Richard
Published: August 2018

Chronic myelogenous leukemia (CML) was the first malignancy for which clinical outcome was drastically improved by kinase inhibitor therapy. Kinase inhibitors targeting other well-known oncogenes have been introduced into clinical practice, but none have shown the same magnitude of clinical benefit as ABL1 inhibition in CML. CML is sometimes described as an atypical malignancy or a one-hit malignancy. However careful examination of accumulation of additional oncogenic alterations in genes such as TP53, CDKN2A/B, IKZF1 and RUNX1 during CML progression from chronic phase to blast phase indicates that this is not the case. Instead CML provides a window into how many types of cancer may look and behave at an early stage, prior to diagnosis and the development of additional genomic alterations. The remarkable clinical benefits of ABL1 inhibition is likely due to early detection of CML at a stage in which the tumor is driven by single oncogenic alteration which can be successfully controlled by the inhibitor. The majority of CML patients in chronic phase of the disease treated with ABL1 inhibitor achieve a major molecular response (MMR) which corresponds to 1000 times decrease in BCR-ABL fusion levels. As a result, there is drastic reduction in number of cells which may accumulate additional alterations in genes like TP53, CDKN2A/B, IKZF1, RUNX1 leading to greatly decreased chance of progression to blast crisis. Thinking of CML as a prototype for effective systemic treatment based on early cancer detection may help to provide strategies for improving treatment for other types of cancer.

Citation Format: Dmitriy Sonkin, Richard Simon. CML as a prototypical malignancy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 207.
Abstract 2075: Whole genome sequence analysis informs precision medicine of pediatric cancers
Authors: Tanasa Bogdan , Lee Alex , Breese Marcus , Shah Avanthi , Leung Stan , Liu Heng-Yi , Spillinger Aviv , Hazard Kimberly , Rangaswami Arun , Spunt Sheri , Lacayo Norm , Cooney Tabitha , Sweet-Cordero Eric Alejandro
Published: August 2018

Several recent studies (including BASIC3, iCAT, INFORM, PEDS-MIONCOSEQ) have used whole exome sequencing (WES) and RNA-seq in order to identify targetable chromosomal alterations in a large variety of pediatric cancers. Few studies have attempted to evaluate whether a more comprehensive approach including WGS and RNA-seq could be used to identify novel events relevant to the pathogenesis of advanced pediatric cancer.

We analyzed a total of 59 patients (37 solid tumors, 11 brain tumors, and 10 leukemia/lymphomas) to determine the feasibility of using whole-genome sequencing (WGS) technology in conjunction with RNA-seq in order to identify actionable/druggable alterations in the pediatric cancer genomes. WGS analysis has been performed on 75 samples, that were collected from 45 patients, either at diagnosis or at relapse.

For WGS analysis of germline-tumor pairs, after performing the sequence alignment with BWA-MEM to gender-specific hg38 genomes, we have used and verified a set of computational methods: 1) MuTect2 for SNV calling, 2) cn.mops for CNV calling, 3) DELLY and LUMPY for SV calling.

Here we present our findings on the sets of SNV, CNV, SV, and gene fusions that we have identified by WGSA and RNA-seq, respectively, with a particular emphasis on the druggable alterations, and on the tumor response in the murine PDX models of the pediatric cancers. Consistent with previous observations, the mutational burden across pediatric cancers was low. While common mutations were identified, there was a long-tail of mutations that occurred at a low frequency. As anticipated, samples obtained post-chemotherapy had a higher mutational burden than treatment naive samples. TP53 was the most commonly mutated gene, but we also identified SNVs in other genes commonly mutated in cancer, such as ASXL1, NOTCH2, and RB1.

Other novel recurring variants were discovered, further analysis of which is ongoing. Our results indicate that integrated WGS and RNA-seq analysis is feasible in the clinical setting and can reliably identify variants reported on commercially available gene panel testing. However, this approach also resulted in additional clinically relevant findings and allows for novel discovery that will further advance our understanding of these rare and highly aggressive pediatric malignancies.

Citation Format: Bogdan Tanasa, Alex Lee, Marcus Breese, Avanthi Shah, Stan Leung, Heng-Yi Liu, Aviv Spillinger, Kimberly Hazard, Arun Rangaswami, Sheri Spunt, Norm Lacayo, Tabitha Cooney, Eric Alejandro Sweet-Cordero. Whole genome sequence analysis informs precision medicine of pediatric cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2075.
Abstract 2107: A novel real-time cell imaging assay to quantify macrophage efferocytosis
Authors: Bravo Daniel , Wang Jianyong , Shi Yongchang
Published: August 2018

Tumor-associated macrophages (TAMs) are found in the microenvironment of solid tumors, representing one of the most abundant immune cell types within tumor stroma. Studies have demonstrated that the presence of TAMs correlates with tumor progression and poor prognosis. The engulfment of apoptotic tumor cells by TAMs (a phagocytic process known as efferocytosis) results in clearance of neoplastic antigens, which prevents antigen presentation and activation of effector T cells. Additionally, it has been shown that TAMs produce cytokines that create an immunosuppressive environment that facilitates tumor evasion of immune surveillance and promotes tumor growth. Furthermore, TAMs have been reported to be key players in the promotion of tumor angiogenesis and metastasis. Since TAMs play such an important role in cancer progression, inhibition of their function in the tumor microenvironment becomes an attractive approach of cancer immunotherapy. Traditionally, quantifying efferocytosis in vitro has been a technically challenging task. Our lab has established a novel high-throughput assay that can be used to screen drug candidates that inhibit TAM efferocytosis. Our new assay uses the IncuCyte ZOOM real-time imaging platform to visualize macrophage efferocytosis of apoptotic cells that are labeled with a pH-sensitive probe (pHrodo) that only emits fluorescence in acidic enviroments. This technology enables us to quantify phagocytic events of apoptotic cells that have been processed into the phagolysosome. By using this method, we quantitatively characterized the efferocytosis activity of TAM-like macrophages that were differentiated from primary human monocytes. We tested several agents (including small-molecule compounds and antibodies) and quantified their activities in blocking efferocytosis of TAMs via IC50 concentrations and potency. This new high-throughput assay platform yields highly robust and reproducible data (Z'=0.69), is fully automated, noninvasive (no washing, fixing or lifting of cells), and works with low cell numbers. Our results show that our efferocytosis assay facilitates high-throughput functional screening and can be utilized to identify and characterize new cancer drug candidates, as well as enables research of efferocytosis mechanisms and functions of phagocytic cells.

Citation Format: Daniel Bravo, Jianyong Wang, Yongchang Shi. A novel real-time cell imaging assay to quantify macrophage efferocytosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2107.
Abstract 2118: Single-cell analysis reveals dynamic interaction between myeloma and bone marrow microenvironment
Authors: Min Jaewoong , Ryu Daeun , Lee Hae-Ock , Jo Areum , Park Woong-Yang , Kim Seok Jin , Kim Kihyun
Published: August 2018

Multiple myeloma (MM) is a monoclonal plasma cell (MPC) malignancy primarily propagating in the bone marrow. To understand molecular signatures of MM in association with tumor microenvironment, we performed single-cell RNA sequencing for bone marrow biopsies from seven newly diagnosed myeloma patients. Clinical parameters of 7 MM patients differed, which were recapitulated in the single-cell transcriptome analysis. In comparison to the normal bone marrow from healthy donors, patient-specific monoclonal immunoglobulin gene expression as well as high levels of plasma cell markers distinguished the MPC clusters in myeloma patients. For other cell type identification, reference transcriptome for bone marrow immune cells were utilized. Major cell populations were determined to be monocytes, T cells, B cells, and erythroid cells. T cells manifested a mature phenotype encompassing from naïve to cytotoxic effector gene expression characteristics. B cells and erythroid cell populations were in various developmental stages. Monocytes demonstrated relatively constant gene expression throughout healthy donors to myeloma patients. In more advanced tumor stages, the composition and gene expression characteristics of MPCs and bone marrow immune cells were altered. Taken together, single cell RNA sequencing reveals gene expression characteristics of both myeloma and bone marrow immune cells which provide potential therapeutic strategies targeting tumor or immune compartment.

Citation Format: Jaewoong Min, Daeun Ryu, Hae-Ock Lee, Areum Jo, Woong-Yang Park, Seok Jin Kim, Kihyun Kim. Single-cell analysis reveals dynamic interaction between myeloma and bone marrow microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2118.
Abstract 2127: RSK2 phosphorylates T-bet to attenuate colon cancer metastasis and growth
Authors: Yao Ke , Peng Cong , Zhang Yuwen , Zykova Tatyana A. , Lee Mee-Hyun , Chen Hanyong , Ryu Joohyun , Rao Enyu , Ma Wei-Ya , liu kangdong , Bode Ann M. , Li Bing , dong zigang
Published: August 2018

Metastasis is the major cause of death for patients with solid malignancies. Approximately 80 and 20% of colorectal cancer (CRC) patients develop liver metastasis and lung metastasis, respectively. We found that at different stages of colon cancer, the patients' interferon (IFN) γ secretion from peripheral blood mononuclear cells (PBMCs), was decreased compared with healthy people. The ribosomal S6 kinase (RSK) family of kinases has multiple cellular functions, and we examined their role in this observed IFNγ decrease. Flow cytometry analysis of wild-type (WT) and RSK2 knockout (KO) mice, revealed that the level of IFNγ was significantly lower in RSK2 KO mice as compared to WT mice. Since IFNγ is a component of immunity, which contributes to protection against metastatic carcinomas, a colon cancer liver metastasis experiment was conducted. We found that metastasis was significantly increased in RSK2 KO mice, compared to WT mice. Transcription factor T-bet can directly activate Ifnγ gene transcription. In vitro kinase assay results showed that RSK2 phosphorylated T-bet at serines 498 and 502. We show that phosphorylation of T-bet by RSK2 is required for IFNγ expression, because knocking down RSK2 expression or over-expressing T-bet S498A/S502A mutants reduces IFNγ mRNA expression. To verify the function of the phosphorylation sites, a

constitutively active mutant T-bet (S498E/S502E) was over-expressed in bone marrow. Mutant T-bet restored the IFNγ mRNA levels and dramatically reduced the metastasis rate in these mice. Overall, these results indicate that phosphorylation of T-bet is required for inhibiting colon cancer metastasis and growth through a positive regulation of RSK2/T-bet/IFNγ signaling.

Citation Format: Ke Yao, Cong Peng, Yuwen Zhang, Tatyana A. Zykova, Mee-Hyun Lee, Hanyong Chen, Joohyun Ryu, Enyu Rao, Wei-Ya Ma, kangdong liu, Ann M. Bode, Bing Li, zigang dong. RSK2 phosphorylates T-bet to attenuate colon cancer metastasis and growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2127.
Abstract 2131: Paracrine cytokine pathways mediate metastasis of breast cancer to lymphatics
Authors: Ji Kyungmin , Zhao Zhiguo , Moin Kamiar , Xu Yong , Gillies Robert J. , Mattingly Raymond R. , Sloane Bonnie F.
Published: August 2018

Lymphatics, rather than blood vessels, are the primary route for breast cancer metastasis. The presence of breast cancer cells in regional lymphatics, i.e., lymphatic metastasis, is an important prognostic factor for patients. Delineating molecular mechanisms by which the breast cancer cells migrate toward and infiltrate into lymphatics is crucial to designing new therapies to prevent metastatic dissemination. To study tumor:lymphatic interactions, we are using a three-dimensional (3D) heterotypic co-culture model of human breast cancer cells (hBCCs) grown with human microvascular lymphatic endothelial cells (hLECs) in novel chambers that we designed and fabricated. These chambers support growth of the 3D co-cultures, live-cell confocal imaging in real time and noninvasive collection of conditioned media for secretomic analyses. We use live-cell assays developed in our laboratory for quantitative analysis of temporal and dynamic changes in BCC:LEC interactions in correspondence with changes in their malignant and proteolytic phenotypes. We cultured hLECs in the presence and absence of human MDA-MB-231 (231) triple-negative BCCs in 3D cultures for 4 days. In mono-cultures, the 231 cells grow in clusters that exhibit a stellate morphology and hLECs form branching networks with central nodes. We observed that the volumes of 3D structures formed by LECs and 231 cells were significantly greater in co-cultures than in mono-cultures of either cell type. In addition, 231 cells infiltrate into the LEC networks with the infiltration increasing over the 4-day period as assessed by the degree of overlap between 231 cells and hLECs in 3D reconstructions of the co-cultures. Moreover, soluble factors from LECs increase invasive outgrowths of 231 structures. This was demonstrated in 231 cells grown in media conditioned by LECs and in parallel co-cultures of 231 cells and LECs. The induction of invasiveness by LEC conditioned media is reduced by boiling and repeated freeze/thawing, suggesting that the active factor(s) is a protein. Our preliminary results suggest that LECs secrete soluble factors that may be therapeutic targets for reducing invasion of BCCs into lymphatic networks.

Citation Format: Kyungmin Ji, Zhiguo Zhao, Kamiar Moin, Yong Xu, Robert J. Gillies, Raymond R. Mattingly, Bonnie F. Sloane. Paracrine cytokine pathways mediate metastasis of breast cancer to lymphatics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2131.
Abstract 2132: Anti-vascular inflammatory nanoparticles inhibit tumor progression and metastasis through vascular normalization
Authors: Choi Young Sun , Kang Soo Hyun , Bae Jong-Sup , Kim In-San , Lee You Mie
Published: August 2018

Vascular networks in cancer are structurally and functionally abnormal. Abnormal blood vessel development in cancer plays an important role in cancer growth and metastasis, and blocks delivery of chemotherapeutic drugs. Recent studies have revealed that the normalization of abnormal tumor vasculature is one of the promising approaches to treat cancer. Inflammatory microenvironment is one of the newly focused hallmarks of cancer, and tumor vasculature has similar features to that of inflammatory vessels. Thus, vascular inflammation is emerged as an attractive target for cancer treatment. We hypothesized that amelioration of vascular inflammation in tumor may induce vascular normalization leading to the inhibition of tumor progression and metastasis. Here, we investigated the function of ant-vascular nanoparticles in tumor growth and metastasis in LLC allograft tumor models and MMTV-PyMT, spontaneous breast tumor models using TFG and TFMG which are engineered a protein-cage nano-particle (ferritin) with both EPCR-targeting peptides (γ-carboxyglutamic acid (Gla) domain of protein C) and PAR-1-activating peptides (TRAP) of thrombin on its surface. As a result, tumor growth was significantly inhibited in the nanoparticle-injected group, and the lymph node metastasis was also decreased in both animal models. It has been found that the survival rate of the nanoparticle-injected group was prolonged. TFG and TFMG normalized abnormal tumor vasculature, that is, pericytes coverage in vasculatures was significantly increased but the hypoxic regions was decreased in the nanoparticle injected group. Furthermore, when the experiment was conducted by administering cisplatin and nanoparticles together in the LLC allograft tumor model, the tumor size was synergistically increased in the case of the nanoparticle and cisplatin combination administration compared to the cisplatin alone group. Taken together, anti-vascular inflammation nanoparticles with anti-vascular inflammatory properties normalized the abnormal blood vessels and inhibited tumor formation and metastasis. It is suggested that inducing normalization of cancer blood vessels by improving the inflammatory state of blood vessels may be a promising therapeutic method for effective cancer treatment.

Citation Format: Young Sun Choi, Soo Hyun Kang, Jong-Sup Bae, In-San Kim, You Mie Lee. Anti-vascular inflammatory nanoparticles inhibit tumor progression and metastasis through vascular normalization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2132.
Abstract 2139: Evaluation of a novel hypoxia-activated prodrug strategy in colorectal cancer cells
Authors: Hussain Omar , Johansson Henrik , Allison Simon J. , Pedersen Daniel S. , Phillips Roger M.
Published: August 2018

Introduction: Current chemotherapeutic drugs are often ineffective against cancer cells that reside within the tumor microenvironment due to contributing factors such as hypoxia (low O2 tension) and acidic extracellular pH (pHe). Hypoxia is known to drive the aggressive cancer phenotype in many solid tumors, making it a key component of drug development. The use of hypoxia-activated prodrugs (HAPs) has been extensively studied over 40 years but to date no HAP has been approved for use in humans, largely because of poor activity and toxicity profiles. The design of prodrugs that are activated by both hypoxia and acidic pHe may provide a greater degree of selectivity and activity than HAPs alone, and this study was designed to evaluate a series of novel bromosugar-based HAPs under hypoxic and acidic pHe conditions.

Method: A library of 13 HAPs was evaluated against HCT116 colorectal cancer cells and ARPE-19 human retinal epithelial noncancerous cell line. Cytotoxicity was determined under (i) aerobic condition (21% oxygen) at pHe 7.4 or mild acidic conditions (pHe 6.5) and (ii) hypoxic condition (0.1% oxygen) at pHe 7.4 or mild acidic conditions (pHe 6.5). ARPE-19 cells were only tested under aerobic conditions and pHe 7.4 to reflect normal physiologic conditions. Compounds were tested at different concentrations ranging from 0.1-100µM from which an IC50 value was determined. Cell survival was measured after a 96-hour continuous drug exposure using the MTT assay.

Results: Among the compounds screened, DS10 demonstrated the most enhanced potency against HCT116 cells under hypoxic conditions with an IC50 value of 28.18 ± 7.01 µM as compared to an IC50 value of 66.39 ± 8.10 µM under normoxic conditions at pHe 7.4. Potency was further increased when DS10 was evaluated under a combination of hypoxic and acidic pHe conditions with an IC50 value of 16.30 µM ± 4.11. ARPE-19 cells showed an IC50 value of 42.95 µM ± 5.03 under aerobic conditions.

Discussion: The results of this study demonstrated that bromosugar derivatives may have the potential to selectively target hypoxic cancer cells in an acidic extracellular environment. Specifically, DS10 exhibited potential HAP properties with the ability to target cells that reside within the hypoxic and acidic microenvironment of tumors. Selectivity to tumor cells as opposed to noncancer cells was observed under these conditions, suggesting that DS10 is a promising compound to take forward for further evaluation.

Citation Format: Omar Hussain, Henrik Johansson, Simon J. Allison, Daniel S. Pedersen, Roger M. Phillips. Evaluation of a novel hypoxia-activated prodrug strategy in colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2139.
Abstract 2142: High variability of TIGIT expression in Hodgkin's lymphoma
Authors: Simon Ronald , Blessin Niclas C. , Kluth Martina , Fischer Kristine , Hube-Magg Claudia , Li Wenchao , Makrypidi-Fraune Georgia , Wellge Björn , Mandelkow Tim , Debatin Nicolaus F. , Sauter Guido , Wilczak Waldemar , Hinsch Andrea
Published: August 2018

Hodgkin's lymphoma (HL) is characterized by a high background of inflammatory cells which play an important role for the pathogenesis of the disease. T cell immunoreceptor with Ig and ITIM domains (TIGIT) is an inhibitory immune checkpoint receptor and a putative target for novel immune therapies. To study patterns of TIGIT expression in the T cell background surrounding malignant cells including Hodgkin cells, Reed-Sternberg cells and histiocytic cells, a microenvironment (ME) tissue microarray (TMA) was constructed from tissue punches measuring 2 mm in diameter obtained from formalin-fixed tissue samples of Hodgkin lymphoma lymph nodes (n=40) and normal human tonsil (n=2) as a reference. The ME-TMA was stained with brightfield and multiplex fluorescence immunohistochemistry (IHC) in order to evaluate expression levels of TIGIT and PD-1 as well as standard lymphocyte markers (CD8, CD4, FOXP3) in the lymphocytic background. TIGIT and PD-1 expression was found in all (100%) analyzed HL samples. In general, TIGIT localized to the same cells as PD-1. IHC based identification of T cell subtypes revealed TIGIT and PD-1 expression on CD8+ cytotoxic T cells, CD4+ helper T cells and FOXP3+ regulatory T cells. Strikingly, expression levels of TIGIT and PD-1 were highly variable among the analyzed samples irrespective of the histological subtype of HL. Interestingly, highest levels of both proteins were found in one sample of nodular lymphocytic-predominant HL (NLPHL). The high variability of TIGIT and PD-1 expression was also independent from the T cell subtype. HL with high TIGIT/PD-1 expression on CD8 positive cells sometimes showed low TIGIT/PD-1 expression on CD4 or FOXP3 positive T cells and vice versa. However, the T cell subtypes differed with respect to the TIGIT:PD-1 ratio. In the majority of analyzed HL, FOXP3+ regulatory T cells expressed higher levels of TIGIT than of PD-1. This was different for CD8+ and CD4+ cells, where similar fractions of HL showed either more TIGIT as PD-1, or more PD-1 as TIGIT, or comparable levels of both receptors. In conclusion, TIGIT (and PD-1) expression is highly variable between patients with Hodgkin's lymphoma. TIGIT may play a particularly important role in FOXP3 regulatory T cells in the lymphocytic background.

Citation Format: Ronald Simon, Niclas C. Blessin, Martina Kluth, Kristine Fischer, Claudia Hube-Magg, Wenchao Li, Georgia Makrypidi-Fraune, Björn Wellge, Tim Mandelkow, Nicolaus F. Debatin, Guido Sauter, Waldemar Wilczak, Andrea Hinsch. High variability of TIGIT expression in Hodgkin's lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2142.
Abstract 2179: Population remodeling in the acquisition of endocrine resistance in breast cancer
Authors: Hu Rong , Sengupta Surojeet , Sevigny Catherine , Zhang Zhen , Wang Yue , Clarke Robert
Published: August 2018

The cell-cell interactions that occur within the breast tumor microenvironment are critical determinants of cancer cell fate. In the face of treatment, the theory of clonal evolution dominates current thinking. Thus, individual cells acquire a mutation(s) that provides a selection advantage where Darwinian selection acts at the single-cell level. We studied the effect of antiestrogen treatment on the population remodeling of admixtures of sensitive LCC1 (S) cells and resistant LCC9 (R) human breast cancer cells. R cells were derived from S cells by selection against antiestrogens. We labeled S cells with GFP and R cells with mCherry, mixed the cells in different ratios, and treated with antiestrogen or vehicle. Unlike other resistance phenotypes (e.g., MDR1-driven cytotoxic drug models), R cells do not outcompete S cells, nor do S cells restrain R cell growth. Rather, R cells protect S cells from treatment. Full communication of the resistance phenotype occurs at a 1:1 mixture of S:R; ~75% of the R phenotype transfer is evident at 10:1. These results reflect improved fitness conferred by population interactions, not clonal evolution. SILAC analysis of S and R cells (pure populations) implicate differential regulation of estrogen (E2) response-early; E2 response-late; glycolysis; fatty acid metabolism; mTORC1 signaling; peroxisome; unfolded protein response (UPR); oxidative phosphorylation in S vs. R cells. Clustering of iTRAQ-TAT data from pure and admixed S:R populations shows that the 1:1 and 5:1 treated, and the 1:1 untreated admixtures, cluster with pure R cells. Untreated 5:1 admixed cells cluster with untreated S cells. Thus, R and S cells alter each other's molecular signatures. Studies using scratch labeling and small-molecule inhibitors show that R cells communicate with S cells via both juxtacrine and paracrine interactions mediated by gap junctional intracellular communication and secreted microvesicles, respectively. The molecular features modified reflect our unifying hypothesis of signaling and cell function control in endocrine resistance.

Citation Format: Rong Hu, Surojeet Sengupta, Catherine Sevigny, Zhen Zhang, Yue Wang, Robert Clarke. Population remodeling in the acquisition of endocrine resistance in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2179.
Abstract 2194: Subclonal variation and evolutionary dynamics of BRAF mutations in cancer
Authors: Gopal Priyanka , Sarihan Elif I. , Abazeed Mohamed E.
Published: August 2018

Tumors have genetically distinct subclones that compete for space and resources and differentially resist efforts to make them extinct. We studied the intratumoral heterogeneity of BRAF mutations across several cancer types. We identified BRAF driver mutations as predominately clonal in some cancer types (e.g. melanoma) and subclonal in others (e.g. lung adenocarcinoma). Clonality corresponded to the amplification of BRAF and prevalence of V600 mutations in each cancer type. We mathematically and experimentally modeled the propagation and selection of tumors containing BRAF mutations and determined that the speed of clonal sweeps were associated with the extent of activation of MAPK signaling pathway and BRAF copy number. Consistent with these findings, tumors with "hard" sweeps were more likely to respond to BRAF and/or MEK inhibitors. Furthermore, some PDX models treated with cytotoxic therapy underwent BRAF mutant subclone expansion over time and this effect is mitigated by inhibitors of BRAF and/or MEK. Treatment strategies based on subclone composition resulted in significantly improved tumor control. Our study uncovers patterns of distinct BRAF clonal evolutionary trajectories and advances therapeutic strategies on the basis of BRAF mutation identity and subclone composition that merit testing in patients.

Citation Format: Priyanka Gopal, Elif I. Sarihan, Mohamed E. Abazeed. Subclonal variation and evolutionary dynamics of BRAF mutations in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2194.
Abstract 2200: Intratumoral heterogeneity of copy number variation in lung cancer harboring L858R via immunohistochemical heterogeneous staining
Authors: Hashimoto Takafumi
Published: August 2018

Background: Acquired resistance to EGFR-TKI is an inevitable event for advanced lung cancer patients harboring the EGFR-activating mutation. Although intratumoral heterogeneity is commonly observed in several cancers, such as renal cell carcinoma, few studies have shown its presence in EGFR-mutated lung cancer. We analyzed the intratumoral heterogeneity in EGFR-mutated (L858R) lung cancer through immunohistochemistry (IHC) for the primary site and targeted sequencing for specific cases in order to clarify the mechanism of acquired resistance.

Methods: Twenty resected primary lung cancers known to harbor EGFR L858R were analyzed. IHC was performed using an L858R mutant-specific rabbit monoclonal antibody (clone43B2; Japan) and the samples were scored by staining intensity (0-3) and proportion. For cases with heterogeneous L858R protein expression, the nucleic acids were extracted from each differently stained lesion, and targeted sequencing (Oncomine Cancer Research Panel) was performed. Single-nucleotide variations (SNVs) and copy number variations (CNVs) from each site were then analyzed. The cell proliferation and apoptosis were also evaluated by the ki-67 labeling index and TUNEL staining.

Results: Among 20 cases, 3 (15%) showed heterogeneous staining, defined as having both an L858R IHC-positive part and an IHC-negative one. Genetic analyses for cases with heterogeneous staining revealed an increase in the copy number of EGFR in the IHC-positive part (CNV 19.54) compared to the negative part (CNV 3.03), and an increase in the copy number of CCNE1 was observed in the IHC-positive part (CNV 18.59) compared to the negative part (CNV 2.59) in one case (case 1). In another case (case 2), an increase in the copy number of EGFR was observed in the IHC-positive part (CNV 8.21) compared to the negative part (CNV 2.90), and an increase in the copy number of MDM2 was observed in the IHC-positive part (CNV 6.65) compared to the negative part (CNV 1.37). Case 3 did not show any CNV. In three cases, no SNV changes were observed. An increase in the ki-67 labeling index in the L858R-positive part in case 1 and increased apoptosis in the L858R-positive part in case 2 were observed, suggesting the functional significance of CNV changes.

Conclusion: These cases exhibiting L858R IHC intratumoral heterogeneity suggest a heterogeneous effect on the cell activity due to CNV heterogeneity.

Citation Format: Takafumi Hashimoto. Intratumoral heterogeneity of copy number variation in lung cancer harboring L858R via immunohistochemical heterogeneous staining [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2200.
Abstract 2209: Lung cancer risk prediction using DNA methylation markers
Authors: Guida Florence , Nøst Therese H. , Relton Caroline , Brennan Paul , Sandanger Torkjel M. , Chadeau-Hyam Marc , Johansson Mattias
Published: August 2018

There is an urgent need to improve lung cancer risk assessment as current screening criteria miss a large proportion of cases and result in a high rate of false positives on low-dose CT screening. A fixed effect meta-analyses of 4 epigenome-wide association studies of lung cancer revealed differential DNA methylation at 16 CpG sites (FDR<0.05). The current study aimed to evaluate the extent to which such methylation markers can improve upon smoking-based risk-discrimination among ever smokers. We used data on 662 ever smoking lung cancer case-control pairs from 4 individual prospective cohorts that measured DNA methylation using the Illumina Infinium HumanMethylation450 BeadChip in peripheral blood samples before diagnosis: The Italian part of the European Prospective Investigation into Cancer (EPIC) cohort, the Melbourne Collaborative Cohort (MCCS), the Norwegian Women and Cancer cohort (NOWAC) and the Northern Sweden Health and Disease Study (NSHDS). We adopted a training-testing design where the training was performed on MCCS and NSHDS (N=511 case-control pairs), and the testing on EPIC-Italy and NOWAC (N=151 case-control pairs). Logistic regressions with lasso penalties were performed in the training set to select the best set of CpGs jointly predicting lung cancer. A methylation score was trained by fitting a logistic regression model including the selected CpGs in the training set. A baseline score based on self-reported smoking information (duration, cigarettes/day for current smokers and time since smoking cessation for former smokers) was also developed. The discriminative performances of both scores, as well as integrated model where both smoking and methylation info was incorporated, were assessed by the AUC under the ROC curves in the validation set. The methylation score based on the 9 selected CpG sites yielded an AUC of 0.78 [0.73-0.84] compared to 0.73 [0.68-0.79] for the baseline-smoking score. The model integrating both scores yielded an AUC of 0.79 [0.73-0.84], a notable 0.06-increase in AUC from using the smoking score alone (P=0.008 for difference in AUC). In conclusion, specific methylation biomarkers have a strong potential to improve lung cancer risk assessment and current USPSTF criteria for CT-screening. During the conference, we will present absolute risk estimates based on the integrated risk prediction model.

Citation Format: Florence Guida, Therese H. Nøst, Caroline Relton, Paul Brennan, Torkjel M. Sandanger, Marc Chadeau-Hyam, Mattias Johansson. Lung cancer risk prediction using DNA methylation markers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2209.
Abstract 2245: Deconvolution of diverse immune cell populations within tumors using ACE Transcriptome
Authors: Levy Eric , Boyle Sean M. , Milani Pamela , Luo Shujun , West John , Chen Richard
Published: August 2018

Comprehensive tumor immuno-genomic characterization is becoming an important tool for identifying new biomarkers correlated with patient response to immunotherapy. Both the abundance and composition of tumor-infiltrating lymphocytes have been associated with tumor progression and patient outcome. Prior studies have shown that an enrichment of cytotoxic immune cells, and depletion of immunosuppressive cells, can indicate better clinical outcome.

While common lab approaches are used to profile tumor samples for the presence and enumeration of immune cell types, these approaches can be limited by the number of markers and throughput. Thus, there is interest in using an accurate computational method leveraging NGS data to more comprehensively profile the distribution of immune cells in the tumor microenvironment. Through the use of marker genes that are expressed in a cell type specific manner, it is possible to computationally predict the fractions of multiple cell types in a mixed sample. This approach relies on an input of reference gene expression signatures, which are used to estimate the proportions of the cell types represented in the reference. The generation and validation of these reference signatures are critical for ensuring the accuracy of the results on a given platform.

We have used the ACE Cancer Transcriptome, from the ACE ImmunoID platform, to produce high-quality gene expression profiles of purified immune cells representing many lineages, including B cells, monocytes, NK cells, and T cells. These profiles were used to create reference signatures of immune cell type specific genes, enabling quantification of their cellular abundances. In addition, we test the accuracy of the deconvolution approach on mixtures of immune cells. Finally, we validate the approach on tumor samples with immune cells quantified by widely accepted orthogonal methods. Through these results, we show that deconvolution of TILs through our ACE Cancer Transcriptome can be a robust platform for elucidating the composition of immune cells in a tumor sample.

Citation Format: Eric Levy, Sean M. Boyle, Pamela Milani, Shujun Luo, John West, Richard Chen. Deconvolution of diverse immune cell populations within tumors using ACE Transcriptome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2245.
Abstract 2259: Prostate cancer molecular subtype impacts interpretation of clinical parameters
Authors: Liu Deli
Published: August 2018

Background: Molecular characterization of prostate cancer has revealed distinct subtypes with underlying genomic alterations. One of these core subtypes (SPOP mutant prostate cancer) has previously only been identifiable through DNA sequencing, making the impact on prognosis and routinely utilized risk stratification parameters unclear. We aimed to identify prostate cancer molecular subtypes from transcriptional data alone and determine associations with clinicopathologic outcomes and long-term prognosis.

Methods: We developed a novel gene expression signature, classifier (SCaPT), and decision tree to predict SPOP mutant subclass from RNA gene expression data and classify common prostate cancer molecular subtypes. We then validated and further interrogated the association of prostate cancer molecular subtypes with pathologic and clinical outcomes in retrospective and prospective cohorts of 8,158 patients. Associations with clinicopathologic variables, metastasis-free and prostate cancer-specific survival were the main outcomes.

Findings: The SCaPT model prediction showed high sensitivity and specificity in multiple cohorts across both RNA-seq and microarray gene expression platforms. We predicted 8~9% of cases to be SPOP mutant from both retrospective and prospective cohorts. Surprisingly, we found routine clinical risk factors were disconjugate when molecular subtype was considered. The SPOP mutant subclass was associated with lower frequency of positive margins, extraprostatic extension, and seminal vesicle invasion at prostatectomy. However, SPOP mutant cancers were associated with higher pretreatment serum prostate-specific antigen (PSA). The association between SPOP mutant status and higher PSA level was validated in three independent cohorts. Despite high pre-treatment PSA, the SPOP mutant subtype was associated with a favorable prognosis with improved metastasis-free survival, particularly in patients with “high-risk” preoperative PSA levels.

Interpretation: Using a novel gene expression signature to assign the SPOP mutant subclass and a decision tree algorithm to define prostate cancer molecular subclasses, we found the SPOP mutant subclass associated with higher preoperative PSA, less adverse pathological features, and favorable prognosis. These findings suggest a paradigm where interpretation of common risk stratification parameters, particularly PSA, must be modified to account for the molecular subtype of prostate cancer.

Citation Format: Deli Liu. Prostate cancer molecular subtype impacts interpretation of clinical parameters [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2259.
Abstract 2263: The dysregulation of circadian genes across cancers
Authors: Liu Zexian , Lin Huan , Liu Zekun , Yu Kai , Zhao Qi , Zhang Xiaolong , Wang Yan , Zuo Zhixiang
Published: August 2018

As the daily oscillation in physiological processes, circadian is critical for health while disruption of the circadian rhythm has been associated with cancer in humans. Generally, circadian rhythm was proposed to play important roles in tumor suppression, while there were also evidences that disruption of canonical circadian pathway have anti-cancer effects. In this study, we systematically analyzed the aberrances including mutation, somatic copy number alternation, differential DNA methylation and differential expression of circadian genes across cancers based on TCGA data. It is observed that core circadian network genes (CCNGs) such as PER1/2/3, CLOCK, CRY1/2, ARNTL were highly mutated in a number of cancers such as uterine corpus endometrial carcinoma, skin cutaneous melanoma, stomach adenocarcinoma and colon adenocarcinoma. Generally, the expression for the majority of CCNGs was down regulated in a variety of the cancers such as bladder urothelial carcinoma, head and neck squamous cell carcinoma, breast invasive carcinoma, cervical and endocervical cancers, prostate adenocarcinoma, glioblastoma multiforme and stomach adenocarcinoma, while the expression of a number of CCNGs were related with overall survival in various cancers. The alternations of somatic copy number and DNA methylation were observed to be consistent with the differential expression of these CCNGs. Furthermore, the PCA based circadian gene expression signature was found to be related with the overall survivals and immunophenotypes among cancers, which indicated that circadian rhythm might play critical roles in regulation of immune signaling state in tumor environment. Taken together, our study provided comprehensive analyses of the aberrances and functional roles of circadian genes in cancer, while the results should be helpful for further investigation of the molecular mechanisms and therapy development for cancer.

Citation Format: Zexian Liu, Huan Lin, Zekun Liu, Kai Yu, Qi Zhao, Xiaolong Zhang, Yan Wang, Zhixiang Zuo. The dysregulation of circadian genes across cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2263.
Abstract 2278: Scalability and reliability improvements to the Iso-Seq analysis pipeline enables higher throughput sequencing of full-length cancer transcripts
Authors: Tseng Elizabeth , Clark Tyson , Ashby Meredith H.
Published: August 2018

The characterization of gene expression profiles via transcriptome sequencing has proven to be an important tool for characterizing how genomic rearrangements in cancer affect the biological pathways involved in cancer progression and treatment response. More recently, better resolution of transcript isoforms has shown that this additional level of information can be useful in stratifying patients into cancer subtypes with different outcomes and responses to treatment. The Iso-Seq protocol developed at PacBio is uniquely able to deliver full-length, single molecule cDNA sequences, allowing for the unambiguous, direct determination of splice variants. This has led to the identification of novel cancer biomarker candidates and has yielded new insights into gene fusion events that contribute to cancer progression.1,2,3

Recent improvements to the Iso-Seq bioinformatics pipeline increases the speed and scalability of data analysis while boosting the reliability of isoform detection and cross-platform usability. Here we report evaluation of Iso-Seq runs of human UHRR samples with spiked-in synthetic RNA controls on the Sequel System and show that the new pipeline recovers both more human and synthetic isoforms while reducing the number of false positives. We also share the results of sequencing the well-characterized HCC-1954 breast cancer and matched normal cell lines, which will be made publicly available. Combined with the recent simplification of the Iso-Seq sample preparation protocol, the new analysis pipeline completes a streamlined workflow for revealing the most comprehensive picture of transcriptomes at the throughput needed to characterize cancer samples.

1. Kohli M, et. al. (2017) Androgen Receptor Variant AR-V9 Is Coexpressed with AR-V7 in Prostate Cancer Metastases and Predicts Abiraterone Resistance. Clin Cancer Res. ePub ahead of print. doi: 10.1158/1078-0432.CCR-17-0017

2. Komor MA, et. al. (2017) Identification of differentially expressed splice variants by the proteogenomic pipeline Splicify. Mol Cell Prot. ePub ahead of print. doi: 10.1074/mcp.TIR117.000056

3. Weirather JL, et. al. (2015) Characterization of fusion genes and the significantly expressed fusion isoforms in breast cancer by hybrid sequencing. Nucleic Acids Res, 43 (18), e116.

Citation Format: Elizabeth Tseng, Tyson Clark, Meredith H. Ashby. Scalability and reliability improvements to the Iso-Seq analysis pipeline enables higher throughput sequencing of full-length cancer transcripts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2278.
Abstract 2287: Precise common and rare germline CNV calling with GATK
Authors: Babadi Mehrtash , Lee Samuel K. , Smirnov Andrey , Lichtenstein Lee , Gauthier Laura D. , Howrigan Daniel P. , Poterba Timothy
Published: August 2018

We propose, implement, and evaluate a novel method (GATK gCNV) for accurate discovery of rare and common copy-number variations (CNVs) from read-depth data obtained from whole genome sequencing (WGS), whole exome sequencing (WES), or custom gene panels. GATK gCNV utilizes a sophisticated Bayesian model to learn bias factors arising from sequencing and library preparation. This model accounts for ploidy of sex chromosomes and autosomal aneuploidies, treats GC bias probabilistically, and automatically determines the necessary level of model complexity in a data-driven manner. Unlike most existing read-depth methods, GATK gCNV maintains a high level of sensitivity in common CNV regions, due to a hierarchical hidden Markov model used for accurate genotyping of multi-allelic loci. Furthermore, GATK gCNV performs bias modeling and CNV discovery simultaneously and self-consistently, resulting in significantly improved sensitivity and precision. Our implementation utilizes the PyMC3/Theano framework for performing automatic differentiation variational inference (ADVI). In addition, GATK gCNV automatically scatters large tasks across multiple machines using the Cromwell/WDL framework, enabling the scalable processing of large cohorts.

We use GATK gCNV to compute copy-number transmission and de novo rates in a cohort of WES trios and observe consistency with observed population metrics. Furthermore, we benchmark GATK gCNV for sensitivity, precision, and reproducibility of both rare and common CNV calls. Using a cohort of WES blood normal samples from The Cancer Genome Atlas (TCGA), we show that GATK gCNV calls are in remarkable concordance with Genome STRiP calls on matched WGS data and gCNV substantially outperforms XHMM and CODEX. We also validate GATK gCNV calls on WGS data against calls made using PacBio long reads.

Citation Format: Mehrtash Babadi, Samuel K. Lee, Andrey Smirnov, Lee Lichtenstein, Laura D. Gauthier, Daniel P. Howrigan, Timothy Poterba. Precise common and rare germline CNV calling with GATK [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2287.