bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1263: Targeting of CDCA8 suppresses hepatocellular carcinoma growth by restoring ATF3 tumor suppressor and inactivating oncogenic Akt signaling
Authors: Park Keon Uk , Ko Min Ji , Hwang Ilseon , Ryoo Hun-Mo , Lee Yun-Han
Published: August 2019

Background: Hepatocellular carcinoma (HCC) is the third most lethal cancer worldwide which remains a major challenge due to poor prognosis and limited treatment options. Cell division cycle associated 8 (CDCA8) is known as a component of a chromosomal passenger complex required for stability of the bipolar mitotic spindle and it is commonly overexpressed in human HCC. However, the functional role of CDCA8 in HCC progression remains to be clarified. In this study, we hypothesized if targeting of CDCA8 with small interfering (si) RNA could alter the course of HCC progression. We also investigated the molecular mechanism that mediates HCC cell death caused by CDCA8 silencing.

Methods: Human HCC cell lines, Huh1 and Huh7, were transfected with CDCA8 siRNA and tested for growth inhibition and apoptotic induction using MTS, FACS and Western blotting. To obtain insights into the molecular changes in response to CDCA8 knockdown, global changes in gene expression were examined using RNA sequencing.

Results: siRNA silencing of CDCA8 inhibited HCC cell growth and long-term colony formation by blocking cell cycle progression and by inducing apoptotic cell death. RNA sequencing data showed that, representatively, the anti-proliferative effects were driven by a subset of molecular alterations including the upregulation of tumor suppressive ATF3 and GADD34 genes and the downregulation of BGLAP, a key regulator of cell growth and invasiveness. Subsequent Western blot analysis affirmed that CDCA8 silencing induces the increase in the levels of ATF3 and GADD34 and the decrease in phosphorylated Akt in both Huh1 and Huh7 cells. Also, the same condition decreased the levels of PARP-1 and pro-caspase 3, accelerating apoptosis. Of importance, silencing of CDCA8 expression effectively suppressed HCC tumor growth in a murine xenograft model.

Conclusion: These findings suggest that targeting CDCA8 could be an attractive option for molecular therapy of HCC.

Citation Format: Keon Uk Park, Min Ji Ko, Ilseon Hwang, Hun-Mo Ryoo, Yun-Han Lee. Targeting of CDCA8 suppresses hepatocellular carcinoma growth by restoring ATF3 tumor suppressor and inactivating oncogenic Akt signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1263.
Abstract 1274: Partial reprogramming of melanoma cells confers drug resistance and increased vulnerability to channel antagonists
Authors: Granados Karol , Hüser Laura , Federico Aniello , Sachindra Sachindra , Wolff Gretchen , Umansky Viktor , Utikal Jochen
Published: August 2019

During melanoma progression, tumor cells undergo phenotypic switching, increasing melanoma plasticity and resistance to mitogen-activated protein kinase inhibitors (MAPKi). We propose a partial reprogramming model to target melanoma therapy resistance. Here we show that partially reprogrammed cells are a less proliferative and more dedifferentiated cell population, expressing a gene signature for stemness and suppressing melanocyte-specific markers. To investigate the development of resistance to MAPKi, cells were exposed to BRAF and MEK inhibitors. Partially reprogrammed cells become less sensitive to MAPKi, showing increased cell viability and decreased apoptosis. Based on genome expression data, we evaluated potential targets to restore sensitivity to MAPKi in partially reprogrammed cells. One of our candidates showed promising results by increasing apoptosis and differentiation after sequential treatment with BRAF and MEK inhibitors, in partially reprogrammed cells and human BRAFi-resistant cells. In summary, we show that partial reprogramming of melanoma cells is a feasible in vitro model to study resistance to MAPKi. Moreover, we suggest our candidate as a target to restore sensitivity of resistant cells to MAPKi which has significant clinical implications in the development of resistance to current treatments against melanoma.

Citation Format: Karol Granados, Laura Hüser, Aniello Federico, Sachindra Sachindra, Gretchen Wolff, Viktor Umansky, Jochen Utikal. Partial reprogramming of melanoma cells confers drug resistance and increased vulnerability to channel antagonists [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1274.
Abstract 1276: Inhibition of lipid metabolism decreases proliferation and increases radiosensitvity in EGFR-TKI resistant NSCLC cells with bFGF secretion
Authors: Wang Wei , Peng Shunli , Wang Rong , Ma yueyun , Zhong Longhui
Published: August 2019

Primary and acquired EGFR-TKIs resistance restricts the benefit of EGFR-TKIs to EGFR mutant NSLCLC patients. Recent studies found that metabolic reprogramming might contribute to develop metastasis and drug resistance of tumor cells. Therefore, illustrating the metabolism change is meaningful and targeting the peculiar metabolic pathways might be considered as potential therapeutic strategies for EGFR-TKIs resistant NSCLC. We separated single clones from gefitnib resistant NSCLC cells, by exposing EGFR-mutant EGFR-TKIs sensitive PC-9 cells into increasing concentrations of gefitnib. Among the numerous clones, clone 5 was resistant to gefitinib or AZD9291 and named as PC-9GR-FGF because of high level of bFGF secretion. We found that bFGF promote the transcription of SREBP-1 and its downstream genes, acetyl-CoA carboxylase alpha (ACC), ATP citrate lyase (ACLY) and fatty acid synthase (FASN) in PC-9 cells. Compared to PC-9 cells, higher ACC expression was detected in PC-9GR-FGF cells. Basing on TCGA database (lung Adenocarcinoma, provisional), patients with high ACC expression showed significant shorter PFS (progression-free survival) and OS (overall survival) compared to those with low ACC expression in the EGFR-mutant NSCLC (p<0.05, N=32). Down-regulation of ACC by siRNA or TOFA, a small molecular ACC inhibitor, mederately decreased the proliferation of PC-9 cells whereas strongly decreased proliferation of PC-9GR-FGF cells in vitro and in vivo. Moreover, Reactive oxygen species (ROS) and oxidative stress scavenger, NAC and ferrostatin-1, failed to reverse the effect of ACC inhibition in PC-9 and PC-9GR-FGF cells. Furthermore, after down-regulation or inhibition of ACC, stronger G2/M arrest and more accumulation of DNA damage marker, γH2AX, were found in PC-9GR-FGF cells compared with PC-9 cells. In addition, ACC expression were increased after irradiation of X-ray in PC-9GR-FGF cells, and inhibition of ACC increased accumulation of γH2AX induced by X-rays in PC-9GR-FGF cells. Our research gives a view sight into lipid metabolism changes in EGFR-mutant NSCLC cells before and after acquired EGFR-TKIs resistance, and explores the possibility of combination strategy with radiotherapy and anti-cancer lipid metabolism therapy.

Citation Format: Wei Wang, Shunli Peng, Rong Wang, yueyun Ma, Longhui Zhong. Inhibition of lipid metabolism decreases proliferation and increases radiosensitvity in EGFR-TKI resistant NSCLC cells with bFGF secretion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1276.
Abstract 1298: The anticancer effects of RX-5902 result from inhibition of phosphorylated p68-mediated β-catenin nuclear translocation
Authors: Lee Young B. , George Christina , Kim Deog J.
Published: August 2019

Phosphorylated p68 (p-p68 or phospho-DDX5) has been shown to be associated with cell transformation, epithelial mesenchymal transition (EMT) and cell migration, and furthermore play a vital role in cell proliferation and tumor/cancer progression by promoting the nuclear translocation of β-catenin. RX-5902 is an oral, small molecule inhibitor of β-catenin nuclear translocation mediated by p-p68. Previous studies have shown that RX-5902 inhibits the growth of cancer cells at low nanomolar IC50 (10 to 24nM) and disrupts the p-p68-β-catenin signaling pathway through an interaction with p-p68 on Tyr 593. By interfering with the p-p68-β-catenin signaling pathway cancer cells undergo apoptosis. In this study, we expanded our investigation to study the effect of RX-5902 on nuclear β-catenin in several types of cancer cell lines that include colon, gastric, hepatic, head and neck, melanoma, breast, pancreatic, ovarian, and prostate. Western blot data, in the cell lines tested, showed a decrease of nuclear β-catenin protein as well as p-p68 and c-myc in a concentration-dependent manner. Furthermore, an in vivo study of the MDA-MD-231 xenograft model demonstrated the ability of RX-5902 to inhibit the tumor growth by decreasing p-p68, β-catenin, cyclin D1 and c-myc proteins. Collectively our findings in both in vitro and in vivo studies support the potential therapeutic effect of RX-5902 in multiple cancer indications through the disruption of the phospho-p68/nuclear β-catenin interaction and blocking the nuclear translocation of β-catenin.

Citation Format: Young B. Lee, Christina George, Deog J. Kim. The anticancer effects of RX-5902 result from inhibition of phosphorylated p68-mediated β-catenin nuclear translocation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1298.
Abstract 1311: Inhibition of gankyrin-tumor suppressor protein interactions due to small molecule induced conformational change
Authors: Kabir Abbas , Muth Aaron
Published: August 2019

Introduction: Gankyrin is a highly conserved, 25 kDa protein containing 7 ankyrin repeats that binds to the S6 ATPase subunit of the 26S proteasome. It plays a crucial role in cellular growth, proliferation and metastasis. It is found to be largely overexpressed in a variety of malignancies and its abnormal overexpression makes it a biomarker in certain cancers. Gankyrin promotes cellular growth and proliferation through a variety of protein-protein interactions. Specifically, it binds to and promotes degradation of numerous tumor suppressor proteins (TSPs) such as p53 and retinoblastoma protein by promoting their proteasomal degradation. In 2016, a small molecule binder of gankyrin (cjoc42), demonstrated an ability to inhibit gankyrin activity in a dose dependent manner by inhibiting gankyrin’s interaction with certain TSPs as well as the proteasome.

Methods: In this research, we hypothesize that small molecule binding of gankyrin brings about a conformational change that inhibits its interaction with certain TSPs as well as the proteasome. In order to test this hypothesis, we have performed a series of fluorescence based experiments. We investigated the effect cjoc42 binding to gankyrin has on its intrinsic tryptophan fluorescence as well as its effect on fluorescently conjugated gankyrin. Follow up experiments consisted of both simple western blotting and co-immunoprecipitation assays to further investigate the ability of cjoc42 to induce a conformational change in gankyrin.

Discussion: We observed a diminished fluorescence intensity for both the intrinsic and labeled gankyrin experiments when in the presence of cjoc42. Western blot and Co-IP experiments are currently underway to further support our findings.

Conclusion: Cjoc42, a small molecule binder of gankyrin, appears to induce a conformational change. This conformational change appears to play a prominent role in regulating certain protein-protein interactions. Furthermore, this suggests that gankyrin's binding to specific TSPs and the proteasome are modulated by this mechanism.

Citation Format: Abbas Kabir, Aaron Muth. Inhibition of gankyrin-tumor suppressor protein interactions due to small molecule induced conformational change [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1311.
Abstract 1319: Repotrectinib, a new generation ROS1 inhibitor, is highly potent against fusion ROS1s and emerging resistance mutations
Authors: Deng Wei , Zhai Dayong , Zhang Xin , Lee Dong , Rogers Evan , Whitten Jeffrey , Cui J. Jean
Published: August 2019

Chromosome rearrangements of ROS1 have been identified as oncogenic drivers in many malignancies, especially non-small cell lung cancer (NSCLC). Crizotinib is the only approved treatment for ROS1+ NSCLC. The efficacy of crizotinib varies among different types of ROS1 fusion partners in patients with ROS1-rearranged NSCLC, and the most dominant fusion CD74-ROS1 was associated with a higher rate of brain metastases and shorter overall survival. Unfortunately, the emergence of drug resistance to crizotinib and other ROS1-targeted therapies represents a major obstacle. The most common resistance mutations to crizotinib in ROS1+ NSCLC are the solvent front mutation (SFM) ROS1 G2032R and gatekeeper mutation ROS1 L2026M. Repotrectinib was rationally designed to overcome resistance mutations. The activity of repotrectinib against multiple fusion ROS1s and corresponding resistance mutations is outlined in the Table. Repotrectinib potently inhibited both wildtype and mutated fusion ROS1s including SFMs and gatekeeper mutations. In cell growth assays using engineered Ba/F3 cells expressing ROS1 fusions with several different partners, such as SDC4, CD74, TPM3 and EZR, repotrectinib demonstrated superior potency in comparison to other ROS1 inhibitors against multiple ROS1 mutations, especially the solvent front and gatekeeper mutations. In xenograft tumor model studies, repotrectinib resulted in tumor regression in the tumors carrying WT or SFM ROS1 fusion genes. Overall, repotrectinib demonstrated a strong inhibition profile against WT and various mutated ROS1s across different fusion partners when compared to many other ROS1 TKIs. A Phase 1/2 clinical trial of repotrectinib is currently enrolling ROS1+ NSCLC patients (NCT03093116).

Ba/F3 Cell Proliferation IC50 (nM)No Kinase Domain MutationROS1 G2032RROS1 L2026MInhibitorCD74-ROS1SDC4-ROS1EZR-ROS1TPM3-ROS1CD74-ROS1SDC4-ROS1EZR-ROS1TPM3-ROS1EZR-ROS1TPM3-ROS1Repotrectinib<0.20.2<0.1<0.13.33516.3<0.2<0.1Crizotinib14.619.619.431.1266.24661660500.695.6236.2Lorlatinib0.20.30.20.3160.7352.9190.5434.91.61.9Entrectinib10.5ND1.59.41813ND2947109313.340.7Ceritinib42.859.833.110513911883885.8543.712.666.5Brigatinib2138.725.86111721473360.6300024.441.3Cabozantinib0.530.44.511.3169.439.560.73.412.6Ensartinib39.5ND118.6433.1371.8ND17574814543.31463

Citation Format: Wei Deng, Dayong Zhai, Xin Zhang, Dong Lee, Evan Rogers, Jeffrey Whitten, J. Jean Cui. Repotrectinib, a new generation ROS1 inhibitor, is highly potent against fusion ROS1s and emerging resistance mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1319.
Abstract 132: Direct interactions of oral bacteria and oral squamous cell carcinoma cells
Authors: Kovac Stefan , Napier Tiara , Scoffield Jessica , Amm Hope
Published: August 2019

Head and neck cancers (HNC) are the sixth most common cancer worldwide with more than 90% being squamous cell carcinoma. HNC can occur in the oral cavity, oropharynx, larynx or hypopharynx, or nasal cavities. Oral squamous cell carcinoma (OSCC) is estimated to be diagnosed in 50,000 patients this year with an overall 5-year survival rate of 64%. The tumor microenvironment has been shown to play a vital role in the progression of many human cancers, including OSCC. Given the complexity of the oral microbiome, we hypothesize that OSCC and oral bacteria interact to affect the tumorigenic properties of OSCC.

Methods: OSCC cells were cultured in the presence of oral pathogenic (Streptococcus mutans, Enterococcus faecalis, Fusobacterium nucleatum, Aggregatibacter actinomycetemcomitans) and non-pathogenic (Streptococcus parasanguinis) bacteria. OSCC cells (A253, Cal27, FaDu, SCC-4, SCC- 9, SCC-25, SCC-152) were assessed for changes in cell proliferation, gene expression, and capacity to invade.

Results: Pathogenic oral bacteria increased proliferation in A253 OSCC cells. Enterococcus faecalis increased proliferation of SCC-4, SCC-25 and SCC-152 cells. No increase in proliferation was seen in FaDu and Cal27 cells. When examining changes in gene expression, oral bacteria increased MMP2 expression in A253 cells.Enterococcus faecalis increased JAG1, markers of hedgehog signaling (GLI1, GLI2), cell proliferation (CCDN1, CDKN1A), and cell invasion (MMP2) in SCC-4. In SCC-25 cells, Enterococcus faecalis increased JAG1, GLI1, and GLI2. Despite having no effect in total proliferation, Enterococcus faecalis increased HES1 (a marker of Notch activity), GLI1, and GLI2 in Cal27 cells. In SCC-9 cells, GLI1 expression was increased with all treatments. Streptococcus mutans increased MMP9 and HES1. Aggregatibacter actinomycetemcomitans increased MMP2 and MMP9. Enterococcus faecalis and Fusobacterium nucleatum also increased HES1.

Conclusions: Both the chemical and spatial interactions between OSCC and oral bacterium may play a role in tumor progression and migration by regulating proliferation of cancer cells and cellular signaling. A direct infection model, as opposed to interactions with bacterial spent media, showed more potential in terms of affecting cell viability and likely gene regulation as well. Future experiments will further explore the interactions, including the complexity of the oral biofilm in cancer patients and the presence of multiple bacteria.

Citation Format: Stefan Kovac, Tiara Napier, Jessica Scoffield, Hope Amm. Direct interactions of oral bacteria and oral squamous cell carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 132.
Abstract 1327: UM-9107: A selective wild-type and T315I Bcr-Abl inhibitor with in vivo activity against chronic myelogenous leukemia
Authors: Phadke Sameer , Lopez-Barcons Lluis , Johnson Taylor K. , Lachacz Eric J. , Merajver Sofia D. , Soellner Matthew B.
Published: August 2019

Purpose: Long-term treatment of chronic myelogenous leukemia (CML) with the Bcr-Abl inhibitor imatinib, is initially successful but gives rise to several drug-resistant mutations. The most prevalent of these is the T315I ‘gatekeeper’ mutation that is sensitive to the second-line therapy, ponatinib. However, ponatinib causes serious vascular adverse events, including fatalities, in over 25% of patients, in part due to its target promiscuity. We have designed a novel Bcr-Abl inhibitor (UM-9107) that shows significant in vitro and in vivo anti-CML activity against wild-type and drug-resistant mutants, including the gatekeeper mutant. Furthermore, UM-9107 has excellent selectivity across the kinome and in vitro surrogate toxicity assessment models demonstrate a low likelihood of vascular adverse events as compared to ponatinib.

Experimental Design: We designed a series of compounds to specifically interact with a specific kinked conformation of the phosphate-binding loop of Abl. Since only a small proportion of kinases display this kinked conformation, we hypothesized that our compounds would be selective for Abl. We have analyzed the compound series biochemically and in vitro. The most potent compound, UM-9107, has been comprehensively characterized including: biochemical inhibition, cellular target engagement, kinome-wide profiling, in vitro cell proliferation, pharmacokinetic studies, and in vivo activity in xenograft models of CML.

Results: We demonstrate that UM-9107 potently inhibits all clinical mutants of Bcr-Abl, including the T315I gatekeeper mutant. UM-9107 shows robust activity against CML cell lines, comparable to ponatinib, with significantly fewer off-targets, as determined by kinome-wide profiling. Phenotypic profiling of UM-9107 in human primary cells shows low risk of vascular adverse events as indicated by specific biomarker readouts. UM-9107 demonstrates good oral bioavailability and in vivo activity in xenograft models of CML, with low toxicity.

Conclusions: The success of imatinib has been revolutionary and has led to an increase in the number of patients on long-term therapy. This inevitably gives rise to drug resistance through point mutations in the Bcr-Abl protein. Our results suggest that UM-9107 is a safe and efficacious preclinical candidate for the treatment of imatinib-resistant CML.

Citation Format: Sameer Phadke, Lluis Lopez-Barcons, Taylor K. Johnson, Eric J. Lachacz, Sofia D. Merajver, Matthew B. Soellner. UM-9107: A selective wild-type and T315I Bcr-Abl inhibitor with in vivo activity against chronic myelogenous leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1327.
Abstract 1333: Isolation, characterization and expansion of circulating tumor cells in solid cancers
Authors: Kulasinghe Arutha , Kapeleris Joanna , Kenny Liz , Warkiani Majid , Vela Ian , Thiery Jean-Paul , O'Byrne Ken , Punyadeera Chamindie
Published: August 2019

Metastasis in cancer patients is reflected by measurable levels of circulating tumor cells (CTCs) in the blood of cancer patients. CTCs represent cancer cells from the primary and metastatic sites, thereby providing a comprehensive representation of the tumor burden of an individual patient. Recent advancements have shown that PD-1/PD-L1 immune checkpoint therapies have durable responses in a number of solid tumor types.

Our study was designed to use multiple CTC enrichment platforms for the capture of CTCs and novel culture formulations for the ex vivo expansion of CTCs. Head and Neck cancer (n=300) and lung cancer (n=80) patients were recruited to investigate the prognostic role of CTCs.

We evaluated multiple CTC isolation technologies (CellSearch, filtration, CD45 depletion, inertial microfluidics) using matched patient samples which showed that epitope-independent CTC isolation captured a greater proportion of CTCs. Molecular alterations present in the primary tissue were confirmed in the CTCs by 3D-DNA FISH (EGFR-amplification, ALK-translocations). In HNC, the presence of CTC clusters associated with the development of distant metastatic disease (P=0.0313). HNC CTC-positive patients had shorter progression free survival (PFS) (Hazard ratio [HR]: 4.946; 95% confidence internal [CI]:1.571-15.57; P=0.0063) and PD-L1-positive CTCs were found to be significantly associated with worse outcome ([HR]:5.159; 95% [CI]:1.011-26.33; P=0.0485).

In a proof of principle study, we were able to demonstrate for the first time, short-term patient derived CTC cultures outside the patient’s body from 7/18 HNC samples (4/7 HPV-positive). Recently, we have preliminary data that suggests that PD-L1 is frequently expressed on CTCs in HNC and lung cancer and an immunoscore may be able to identify patients likely to benefit from immunotherapy-a current unmet clinical need. Expanding CTCs outside the patient’s body allows for the recapitulation of the molecular diversity present within the tumor, understanding of the disease progression and testing of therapies.

Citation Format: Arutha Kulasinghe, Joanna Kapeleris, Liz Kenny, Majid Warkiani, Ian Vela, Jean-Paul Thiery, Ken O'Byrne, Chamindie Punyadeera. Isolation, characterization and expansion of circulating tumor cells in solid cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1333.
Abstract 1352: Serum neurofilament light (NfL), glial fibrillary acidic protein (GFAp) and tau protein are possible serum biomarkers for activity of brain metastases and gliomas
Authors: Porter Jason , Hepner Adriana , Pandey Manjari , Prouet Philippe , Hare Felicia , Nasir Syed S. , Boles Madison , Zetterberg Henrik , Blennow Kaj , Martin Michael
Published: August 2019

Introduction: While the morbidity and mortality attributable to gliomas and brain metastases is significant, there are currently no approved biomarkers for detecting and monitoring intracranial disease and imaging remains the primary modality. Blood levels of the neurofilament light (NfL) and tau, as well as glial fibrillary acidic protein (GFAp) show promise as biomarkers for brain injury. We therefore hypothesized that these biomarkers would correlate with CNS activity of gliomas and brain metastases.

Methods: Serum concentrtaions of NfL, tau and GFAp were measured using the ultrasensitive single molecule array (Simoa) technology (Quanterix, Lexington, MA). Levels of the three proteins were then correlated with status of gliomas and brain metastases in the patients. Statistical analysis was performed using GraphPad Prism7 and all p-values were 2-tailed with an accepted 2-sided alpha level of 0.05 as statistically significant.

Results: 36 patients were enrolled with varying malignancies. Both serum NfL and GFAp levels were significantly associated with the state of intracranial disease (ANOVA psNfL= 0.0307; ANOVA pGFAp=0.0348). In contrast, serum tau levels were discordant with brain involvement with cancer (ANOVA pTau= 0.1453).

Conclusion: Serum NfL and GFAp both apparently vary closely with presence and activity of gliomas and brain metastases. Our studies are among the first to identify possible candidate serum biomarkers for screening and monitoring cancer patients for CNS involvement with malignancy. Further studies in larger populations are needed to expand these findings.

Citation Format: Jason Porter, Adriana Hepner, Manjari Pandey, Philippe Prouet, Felicia Hare, Syed S. Nasir, Madison Boles, Henrik Zetterberg, Kaj Blennow, Michael Martin. Serum neurofilament light (NfL), glial fibrillary acidic protein (GFAp) and tau protein are possible serum biomarkers for activity of brain metastases and gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1352.
Abstract 1359: Detection of PD-L1 positive circulating tumor cells from patient with bladder cancer
Authors: Suh Yongjoon , Kim Seong-Kun , Lee Hye Seon , Jeong Yun-Gyu , Moon Jeong-Mi , Kim Myoung Shin , Jeon Byung Hee , Ha U-Syn
Published: August 2019

Immunotherapy with PD-L1 inhibitors greatly enhanced the clinical outcomes of patients with late stage metastatic bladder cancer. However, a concern has been raised in the front-line clinical use of PD-L1 inhibitors (permbrolizumab and atezolizumab) as a single treatment to PD-L1-low patients, due to a poorer overall survival compared with patients treated with plantinum-based chemotherapy. In accordance, reliable evaluation of PD-L1 expression is required for selecting patients relevant for treatment with PD-L1 inhibitors. However, due to the regional heterogeneity in primary tumor, evaluation of PD-L1 levels in tissue biopsy might be less reliable than the levels in circulating tumor cells (CTCs). Here, we developed the evaluating system of PD-L1 levels in CTCs isolated from the blood of patients with bladder cancer. Initially, CTCs were enriched by Smart Biopsy Cell Isolator (CytoGen), which utilizes the size-based gravity filtration for CTC isolation. The isolated CTCs were then validated with the expression of EpCAM, Vimentin and CK as well as a negative selection with CD45, and were analyzed for PD-L1 expression status by immunocytochemical staining. Using the evaluating system, we show that PD-L1 (+) cancer cell was efficiently recovered from the healthy human blood samples in a spike and recovery experiment. Furthermore, we evaluated PD-L1 expression status in CTCs isolated from ~25 patients with bladder cancer. Collectively, this study implicates the possibility that PD-L1 expression status could be analyzed at the levels of CTCs instead of the matching primary tumor tissue biopsy, probably with a better clinical diagnostic option.

Citation Format: Yongjoon Suh, Seong-Kun Kim, Hye Seon Lee, Yun-Gyu Jeong, Jeong-Mi Moon, Myoung Shin Kim, Byung Hee Jeon, U-Syn Ha. Detection of PD-L1 positive circulating tumor cells from patient with bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1359.
Abstract 1372: Detection of early stage pancreatic cancer using 5–hydroxymethylcytosine signatures in circulating cell free DNA
Authors: Collin Francois , Ning Yuhong , Guler Gulfem D. , Phillips Tierney , McCarthy Erin , Scott Aaron , Ellison Chris , Ku Chin-Jen , Chau Kim , Ashworth Alan , Quake Stephen R. , Levy Samuel
Published: August 2019

Pancreatic cancers are typically diagnosed at late stage where disease prognosis is poor as exemplified by a 5-year survival rate of 8.2%. Earlier diagnosis would be beneficial by enabling surgical resection or earlier application of therapeutic regimens. We investigated the detection of pancreatic ductal adenocarcinoma (PDAC) in a non-invasive manner by interrogating changes in 5-hydroxymethylated cytosines (5hmC) in circulating cell free DNA in the plasma of a PDAC cohort (n=51) in comparison with a non-cancer cohort (n=41). 5hmC profiles from PDAC and non-cancer samples were generated using a previously published modified hMe-Seal protocol that utilizes chemical labeling of 5hmC by β-glucosyltransferase and allows detection of cell free 5hmC from small amounts of cfDNA (1). We found that 5hmC sites are enriched in a disease and stage specific manner in exons, 3’UTRs and transcription termination sites. Our data show that 5hmC density is reduced in promoters and histone H3K4me3 associated sites with progressive disease suggesting increased transcriptional activity. 5hmC density is differentially represented in thousands of genes, and a stringently filtered set of the most significant genes points to biology related to pancreas (GATA4, GATA6, PROX1, ONECUT1) and/or cancer development (YAP1, TEAD1, PROX1, ONECUT1, ONECUT2, IGF1 and IGF2). Regularized regression models were built using 5hmC densities in a comprehensive set of genes with the most variable 5hmC counts and performed with an AUC = 0.94 - 0.96 on training data. We tested the ability to classify PDAC and non-cancer samples with the Elastic net and Lasso models on three independent pancreatic cancer 5hmC data sets (n = 26, 23 and 7) compared with corresponding independent non-cancer cohorts (n =103, 53 and 10), and found validation performance to be AUC = 0.74 - 0.97. The findings suggest that 5hmC changes enable classification of PDAC patients with high fidelity and are worthy of further investigation on larger cohorts of patient samples. Reference: 1. Song, C. - X. et al. 5 - Hydroxymethylcytosine signatures in cell-free DNA provide information about tumor types and stages. Cell Res 27, 1231 (2017).

Citation Format: Francois Collin, Yuhong Ning, Gulfem D. Guler, Tierney Phillips, Erin McCarthy, Aaron Scott, Chris Ellison, Chin-Jen Ku, Kim Chau, Alan Ashworth, Stephen R. Quake, Samuel Levy. Detection of early stage pancreatic cancer using 5–hydroxymethylcytosine signatures in circulating cell free DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1372.
Abstract 1382: Megakaryocyte potentiating factor as a predictive biomarker for therapies against malignant mesothelioma
Authors: Cao Liang , Yu Yunkai , Thomas Anish. , Zhang Jingli , Onda Masanori , Meltzer Paul , Hassan Raffit , Pastan Ira
Published: August 2019

Objectives: Effective biomarkers for malignant mesothelioma (MM) are needed for clinical management and the development of cell surface mesothelin-targeted therapies. We evaluated serum megakaryocyte potentiating factor (MPF) as a biomarker predictive of clinical outcome in MM patients, for patient monitoring, and for developing mesothelin-targeted therapies.

Patients and Methods: Serial serum samples from MM patients in two clinical trials of an anti-mesothelin immunotoxin were tested for serum MPF. For the purpose, we developed a new MPF assay and conducted both analytical and clinical validation. Correlative studies were performed to determine the test effectiveness in treatment monitoring and outcome prediction. MPF was further evaluated for an association with response to an anti-mesothelin therapy and for disease monitoring.

Results: Our data showed that in MM patients, elevated serum MPF is a predictor of poor survival with a hazard ratio (HR) = 2.46 (Log Rank P = 0.003; n = 95). In refractory MM patients, elevated MPF is a strong predictor of poor outcome with a HR = 6.12 (Log Rank P = 0.0007; n = 57). There was a significant reduction of serum MPF in patients with elevated baseline and radiological response, with an average change from -52% to -78% after 1 to 6 cycles. Using a -50% change as the cutoff, MM patients with a positive MPF-response had significantly improved progression-free survival (PFS) (P < 0.0001), with median extended from 1.9 to 11.3 months. These MPF-response patients further exhibited improved overall survival (OS) (P = 0.004), with median extended from 8.8 to 22.3 months. In refractory MM patients, there was an association between elevated pre-treatment serum MPF and radiological response to an experimental anti-mesothelin therapy (P = 0.033). Furthermore, in these response patients, serum MPF was monitored between 32.2 and 63.8 months and found to reflect the response and disease progression.

Conclusion: In MM patients, elevated MPF was observed in 2/3rd of the patients and is a predictor for poor survival, throughout the course of the disease. Following systemic therapies, a > -50% reduction in MPF was associated with improved clinical outcome, both PFS and OS. An elevated baseline serum MPF may be a predictor of response to an experimental anti-mesothelin therapy in refractory MM patients, however, the finding needs to be confirmed in a larger study.

Citation Format: Liang Cao, Yunkai Yu, Anish. Thomas, Jingli Zhang, Masanori Onda, Paul Meltzer, Raffit Hassan, Ira Pastan. Megakaryocyte potentiating factor as a predictive biomarker for therapies against malignant mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1382.
Abstract 1389: Single-cell sequencing to identify genetic variant accelerating shedding of CTCs into peripheral blood in gastric cancer patients
Authors: Li Yilin , Zhang Xiaotian , Gong Jifang , Wang Daisy Dandan , Qi Mingxia , Li Yanyan , Liu Dan , Wang Chunxiang , Lin Peter Ping , Shen Lin
Published: August 2019

Circulating tumor cells (CTCs) are shed from primary or metastatic tumors into blood circulation, which may carry specific genetic characteristics related to hematogenous metastasis. Drawbacks of conventional EpCAM or cell size-dependent CTC detection and relevant single-cell sequencing technologies have limited discovery of specific genetic variants in CTCs. In the current study, a novel integrated SE-iFISH strategy, despite cell size or epithelial marker status, was applied to detect CTCs in advanced gastric cancer (GC) patients. Obtained CTCs were classified into diverse subtypes upon different chromosome ploidy. Fifty-three aneuploid CTCs identified by iFISH in 6 of naïve GC patients were isolated by a novel non-laser microscopic single cell manipulator (NMSCM), and subjected to modified Multiple Displacement Amplification (MDA) and subsequent single-cell sequencing. Compared with the conventional PCR, MDA generates 2 to 100-kb products with a lower error rate and achieves high fidelity whole genome amplification. Sequencing targeting 50 tumor genes of single CTC as well as paired primary tumor specimens was performed. Moreover, leukocytes genomic DNA as germline controls were included. Obtained results showed that high frequent SNV in genes such as TP53 were detected in both CTCs and the paired primary tumor mass. Furthermore, CTCs had considerable

genetic heterogeneity compared to the paired primary tumor lesions. In particular, the CDKN2AA68V variant is frequently detected in CTCs but the paired primary GC tumor. We propose that the specific CDKN2AA68V variant carried by GC CTCs with high frequency, but low in the examined whole population of cancer cells in paired primary tumor lesions, may serve as the driver to promote CTCs shed into peripheral blood and to further facilitate hematogenous metastasis. Systematic in vitro and ex vivo studies are in progress to investigate roles of CDKN2A mutation in hematogenous dissemination in GC.

Citation Format: Yilin Li, Xiaotian Zhang, Jifang Gong, Daisy Dandan Wang, Mingxia Qi, Yanyan Li, Dan Liu, Chunxiang Wang, Peter Ping Lin, Lin Shen. Single-cell sequencing to identify genetic variant accelerating shedding of CTCs into peripheral blood in gastric cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1389.
Abstract 1391: Development of in vitro multiplex colorectal cancer associated mutation detection assay powered with xenonucleic acid
Authors: Sha Michael Y. , Lee Anna J. , Powell Michael J. , Zhang Aiguo
Published: August 2019

Precise detection of cancer associated mutations have many advantages including diagnosis and monitoring of the disease and monitoring development of drug resistance. However, unlike germline mutations, somatic mutations in cancer are found at a much lower frequency. For this reason it remains a challenge especially when trying to detect mutations from limited amount of patient tissue biopsy or liquid samples. Currently, detection and genotyping of these mutations is heavily dependent on deep NGS methods. These procedures, however, are costly and time consuming. Here, we developed an alternative high-throughput method that combines DiaCarta’s proprietary xenonucleic acid (XNA) technology that allows target enrichment, and Luminex MAGPIX platform technology that enables simultaneous detection of up to 50 mutation targets. Briefly, multiplex PCR amplification of cancer associated genes is conducted in the presence of XNA. In this reaction, XNA specifically suppresses the wild-type DNA from being amplified and thus, only generates amplicons from the genes that carry mutations. The amplicons are then hybridized in a single tube that contains a set of magnetic beads. Each bead is uniquely color-coded for identification, and each corresponds to a single mutation of interest. The beads are analyzed on Luminex platform for genotyping of the mutations. Using this method, we compared conditions with or without XNA for target amplification. Our preliminary data show that when we tested the cancer oncogene KRAS exon 2 mutation variations, we find that 100% correct allele call was made with XNA for all 7 reference mutations genes. However, in conditions without XNA, the PCR product was composed of mostly wild-type sequences, and thus, only two correct allele calls (28.6%) were made. Consequently we have showed a clear advantage of XNA technology for efficient mutant target enrichment, followed by Luminex MAGPIX platform technology for simultaneous detection of multiple genetic targets. Multiplex capacity of both PCR and Luminex MAGPIX platform allows a significant reduction in the total amount of patient samples needed for precise mutation detection from each assay, especially for the mutations with low allelic frequency. Further development on this assay will provide a more reliable, sensitive and quick method for simultaneously detecting mutations associated with cancer in a single reaction.

Citation Format: Michael Y. Sha, Anna J. Lee, Michael J. Powell, Aiguo Zhang. Development of in vitro multiplex colorectal cancer associated mutation detection assay powered with xenonucleic acid [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1391.
Abstract 1396: Detection and local histological staging of prostate cancer foci in H&E whole slide images using convolutional neural networks
Authors: Stürenberg Carolin , Khan Umair , Sandeman Kevin , Gencoglu Oguzhan , Malen Adrian , Erickson Andrew , Heikkinen Timo , Rannikko Antti , Mirtti Tuomas
Published: August 2019

A pathologist’s histological diagnosis is the gold standard of prostate cancer diagnostic measures. In addition to cancer grade, the stage of cancer determines the follow-up and possible adjuvant therapies after surgery. Not only glandular pattern recognition but also the assessment of three-dimensional extent and size of cancer, in relation to the whole organ, are parts of the subjective diagnosis, which may vary among pathologists. Furthermore, evaluation of lymph nodes for any possible metastases is time-consuming and missing cancer in lymph nodes can lead to undertreatment of the patient. For the abovementioned reasons, our team is developing an algorithm that calculates the amount of cancer tissue objectively, assisting the pathologist in the diagnostic procedure. In addition, we are creating an assistant for cancer detection in lymph nodes, which are removed for histological evaluation. All in all, we aim to create a tool that will help pathologists to a better, faster, more secure and more accurate diagnosis. Our material consists of full sets of scanned whole slide images from 302 prostates that were retrieved by radical prostatectomy. After supervised learning procedure, convolutional neural networks were employed for the classification of cancerous and non-cancerous regions in the images. A tiling based approach was used in which a slide was divided into square shaped small tiles. Millions of cancerous and non-cancerous tiles were sampled from the dataset for training and validation. During the convolutional neural network training, several different tile sizes were used, i.e., 256x256, 512x512, 1024x1024 pixels. Four different types of architectures were fine-tuned and trained for the task of tile-wise binary classification, namely InceptionV3, Xception, ResNet50 and a custom convolutional neural network architecture. In our preliminary assays for cancer detection, in both pixel-wise and tile-wise evaluation, InceptionV3 performed outstandingly well with an AUC score of 0.97 and 0.951, respectively. In conclusion, our algorithm has developed very well thus far with an accuracy in cancer detection of 97%. It is not only a versatile assisting tool, aiding pathologists to a more objective, standardized and accurate diagnosis, but also serves as a second opinion in difficult and challenging diagnostic cases.

Citation Format: Carolin Stürenberg, Umair Khan, Kevin Sandeman, Oguzhan Gencoglu, Adrian Malen, Andrew Erickson, Timo Heikkinen, Antti Rannikko, Tuomas Mirtti. Detection and local histological staging of prostate cancer foci in H&E whole slide images using convolutional neural networks [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1396.
Abstract 1400: C1GALT1 regulates malignant phenotypes of cholangiocarcinoma cells
Authors: Chen Po-Da , Chang Ai-An , Huang Min-Chuan , Wu Yao-Ming
Published: August 2019

Identifying molecular targets for cholangiocarcinoma is an urgent need to overcome the treatment failure and death from cancer cell invasion and metastasis. Core 1 β1,3-galactosyltransferase (C1GALT1) has been documented as the key enzyme in morphogenesis and cellular adhesion, for it adds galactose to form Gal-GalNAc-Ser/Thr structure (T antigen); whereas abnormal C1GALT1 expression and aberrant O-glycans are commonly associated with tumor progression. However, functional roles of C1GALT1 in cholangiocarcinoma remains unclear. With clinical correlation, the immunohistochemistry confirmed that higher expression of C1GALT1 in cancer tissue is associated with larger tumor size, advanced cell grade and overall staging. Consistently, stable knockdown of C1GALT1 regarding cholangiocarcinoma cell lines inhibits cell viability, migration, and invasion; whereas the overexpressed C1GALT1 promotes cell viability, migration, and invasion of cholangiocarcinoma cell lines. Additionally, control of C1GALT1 regulates the colony formation, as well as sphere formation of cholangiocarcinoma cells. Moreover, our data suggest that down-regulated C1GALT1 modifies AAL, VVA, PNA, LEL, and MALII lectin, in correlation with the cell malignant behaviors. Given that, C1GALT1 is sufficient to regulate malignant behaviors of cholangiocarcinoma cells with a significant correlation between gene expression and clinical prognosis, implying that C1GALT1 plays a crucial role in cancer progress of cholangiocarcinoma.

Citation Format: Po-Da Chen, Ai-An Chang, Min-Chuan Huang, Yao-Ming Wu. C1GALT1 regulates malignant phenotypes of cholangiocarcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1400.
Abstract 1403: Combining oligonucleotide library-based RNA fluorescence in situ hybridization and fluorescent immunohistochemistry on the same tissue section
Authors: Ach Robert A. , Tom-Moy May , Tsang Peter , Kroeger Kelly
Published: August 2019

The ability to visualize both RNA and protein expression in a single formalin-fixed paraffin-embedded (FFPE) tissue section is a powerful technique for studying biological problems such as the regulation of protein expression and RNA/protein interactions, as well as for antibody characterization and validation. We have leveraged our ability to chemically synthesize oligonucleotides in massively parallel reactions to produce DNA libraries that can be used to generate probes for the detection of DNA or RNA by Fluorescence in situ Hybridization (FISH). The sequences of the oligonucleotides in these libraries are selected in silico using empirically determined criteria so as to avoid repetitive elements or regions homologous to other non-targeted nucleic acids. We have used these oligonucleotide library-derived FISH probes in combination with a novel signal amplification technique to detect the localization of a variety of both coding and non-coding RNAs in fixed tissue culture cells and FFPE tissue sections, using both conventional fluorescence and structured illumination microscopy. We report here combining our RNA FISH methodology with antibody-based fluorescent immunohistochemistry (IHC) detection of proteins on the same FFPE slide. We show that our combined RNA FISH/IHC protocol works on a variety of RNA probe/antibody combinations for both nuclear and cytoplasmic proteins. We also show that our fluorescent assay can be converted to a colorimetric one for both the RNA and protein detection.

Citation Format: Robert A. Ach, May Tom-Moy, Peter Tsang, Kelly Kroeger. Combining oligonucleotide library-based RNA fluorescence in situ hybridization and fluorescent immunohistochemistry on the same tissue section [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1403.