bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2972: Urolithin A decreases cell proliferation of three genetically diverse glioblastoma multiforme cell lines
Authors: Hearne Alexandra Lynn , Brockway Dakota , Wu Shiyong , Li Yunsheng
Published: September 2020

Glioblastoma multiforme is one of the most common and malignant tumors of the central nervous system, remaining incurable with a median survival time of only 15 months. Over the past 50 years there has been little change in this overall survival rate, due to the many challenges in treating glioblastoma, including molecular complexity, tumor location, and resistance towards standard therapies. Therefore, an improved method for treating glioblastoma is urgently needed. Urolithin A is a natural biometabolite from ellagitannins and ellagic acid, which can be found in pomegranates, berries, and nuts. Urolithin A exhibits anti-cancer activities in several cancer cell lines, including prostate, colon, and bladder cancer cells. However, the anti-cancer activities of urolithin A have yet to be characterized in glioblastoma multiforme. In this project, we investigated whether urolithin A could reduce human glioblastoma cell proliferation by conducting clonogenic assays and cell cycle analysis in three genetically diverse human glioblastoma cell lines: U87MG, T98G, and LN229. Clonogenic assay results showed that urolithin A significantly decreased cell proliferation in a dose-dependent manner in all three glioblastoma cell lines. Depending on the different cell lines, the cell proliferation was inhibited at concentrations of 10 to 60 µM urolithin A. In addition, cell cycle analysis via flow cytometry found that urolithin A was capable of arresting cell cycle progression in either the G2/M or S phase of the cell cycle in U87 and LN229 or T98G cells, respectively.

In conclusion, we have found that urolithin A can reduce cell proliferation and alter cell cycle progression of three genetically diverse glioblastoma cell lines. Importantly, urolithin A can pass the blood-brain barrier and has previously shown to be non-toxic in humans, a prerequisite for a drug to treat glioblastoma. Therefore, urolithin A has the potential to be used as a novel drug which can be combined with current standard methods to improve the efficacy of glioblastoma treatment. In the future, we plan to investigate specific signaling pathways involved in apoptosis, cellular proliferation, and cell cycle arrest. Once we elucidate the specific mechanisms we will investigate the efficacy of urolithin A in vivo, using an animal model. If successful, urolithin A will have the potential to be used alongside radiation and chemotherapy to further reduce glioblastoma cell proliferation.

Citation Format: Alexandra Lynn Hearne, Dakota Brockway, Shiyong Wu, Yunsheng Li. Urolithin A decreases cell proliferation of three genetically diverse glioblastoma multiforme cell lines [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2972.
Abstract 2978: Cyclic GMP-independent inhibition of colon cancer cell growth by phosphodiesterase inhibitors
Authors: Islam Bianca N. , Hou Yali , Mylarapu Namratha , Browning Kaylin A. , Vega Kenneth J. , Browning Darren D.
Published: September 2020

There is growing interest in the potential use of phosphodiesterase (PDE) inhibitors for colon cancer chemoprevention, but the tumor suppressive mechanism is not understood. The present study has tested the idea that PDE inhibitors can suppress growth and promote apoptosis of colon cancer cell lines by increasing cGMP and activating cGMP-dependent protein kinase (PKG). Treatment of LS174T, Caco2, and HCT116 colon cancer cells with membrane permeable 8Br-cGMP had no effect on cell proliferation or viability. Endogenous PKG was undetectable in any of these cells using phosphorylation of the PKG substrate vasodilator-stimulated phosphoprotein (VASP) as a readout. Ectopic expression of PKG2 conferred modest inhibition of proliferation by cGMP treatment (15-25% over 3 days), but did not affect cell viability. The lack of effect of cGMP on colon cancer proliferation is incongruous with many reported effects of PDE inhibitors. To understand the reason for the discrepant observations, colon cancer cells were treated with sildenafil, PF-0447943, and TAK-063 that are specific inhibitors of the cGMP-targeting PDE5, 9, 10a (respectively). At clinically unachievable concentrations exceeding 10μM, all of these drugs reduced proliferation to various degrees in the different cell lines. TAK-063 was the most effective, and the only one that reduced cell viability. Notably, growth-inhibition by the PDE inhibitors was independent of PKG expression, and surprisingly, none of them increased cGMP levels. The inability of the PDE inhibitors to increase cGMP was due to the lack of endogenous cGMP generation, because treatment with the guanylyl-cyclase C agonist linaclotide conferred a robust cGMP response to a physiological dose of sildenafil (1µM). The growth inhibitor effects of high, non-physiological levels of PDE inhibitors is likely due to off-target effects, but apart from a transient inhibition of the ERK pathway, no common mechanism was identified. Taken together, these results refute the idea that cGMP elevating agents prevent colon carcinogenesis by inhibiting cancer cell growth, and support the inhibition of tumor initiation as a more likely mechanism.

Citation Format: Bianca N. Islam, Yali Hou, Namratha Mylarapu, Kaylin A. Browning, Kenneth J. Vega, Darren D. Browning. Cyclic GMP-independent inhibition of colon cancer cell growth by phosphodiesterase inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2978.
Abstract 2980: Effects of cetuximab and CD-3379 on cytokine/chemokine balance in the tumor micro-environment (TME) of squamous cell carcinoma of the head and neck (SCCHN)
Authors: Wang Dongsheng , Nannapaneni Sreenivas , Lesinski Gregory B. , Shin Dong M. , Saba Nabil F. , Chen Georgia Z.
Published: September 2020

Background: The Anti-EGFR agent cetuximab has shown promising outcomes and remains to be the only FDA-approved non-immunotherapeutic targeted agent for the treatment of squamous cell carcinoma of the head and neck (SCCHN). There is evidence that immune modulation represents a mechanism by which cetuximab elicits its clinical activity in SCCHN. As resistance inevitably occurs to cetuximab, there is a need to further elucidate the biological mechanisms by which cetuximab affects immune effectors in the tumor microenvironment (TME). We aimed to explore the effect of EGFR inhibition on the cytokine/chemokine balance in the TME.

Methods: Three SCCHN cancer cell lines UM-SCC47 (HPV positive), JHU022 (HPV negative), and SCC-1C (HPV negative and resistant to cetuximab) were treated with cetuximab, CD-3379 (a HER3 inhibitor), and their combination. After 48 and/or 72 hours, the supernatants of the treated and untreated cells were collected and subjected to multiplexed immunoassay [Meso Scale Discovery (MSD) U-PLEX Assays] to study the cytokine/chemokine alteration. Changes in immune suppressive chemokines were confirmed by ELISA. A potential effect of a chemokine neutralizing antibody on apoptosis was further evaluated using patient-derived organotypic cancer spheroids (PDOCSs).

Results: Our results identified 10 down regulated cytokines/chemokines, including IL6, IL-8, IL-2, IL-12, IL-27, IL-4, IL-5, TGF-β, GM-CSF, and IFN-γ in at least one SCCHN cell line. Three cytokines/chemokines were up regulated, including IL-15, MCP-3, and MCP-1. MCP-1 (CCL2) is a modulator of myeloid-derived suppressor cells (MDSCs). Enhancing MCP-1 by cetuximab and/or CD-3379 in the SCCHN cell lines was confirmed by ELISA. Treating multiple PDOCSs with the same combination along with MCP-1 neutralizing antibody enhanced apoptosis in comparison to cetuximab, CD-3379, or the cetuximab/CD-3379 combination.

Conclusions: Our findings suggest that cetuximab in combination with HER3 inhibition affects secretion of cytokines/chemokines which are both immune stimulatory and suppressive in the TME of SCCHN. Alteration of the selected cytokines/chemokines may enhance the clinical activity of EGFR targeting in SCCHN.

Citation Format: Dongsheng Wang, Sreenivas Nannapaneni, Gregory B. Lesinski, Dong M. Shin, Nabil F. Saba, Georgia Z. Chen. Effects of cetuximab and CD-3379 on cytokine/chemokine balance in the tumor micro-environment (TME) of squamous cell carcinoma of the head and neck (SCCHN) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2980.
Abstract 2983: A synthetic derivative of 1,2,3-triazole-pyrimidine hybrid reverses multidrug resistance mediated by MRP7
Authors: Wang Jing-Quan , Lei Zi-Ning , Teng Qiu-Xu , Wang Bo , Ma Li-Ying , Liu Hong-Min , Chen Zhe-Sheng
Published: September 2020

Multidrug resistance protein 7 (MRP7) is an important member of ABC transporter superfamily and has been revealed to transport a wide range of chemotherapeutic agents including taxanes, epothilone B, vinca alkaloids, daunorubicin and etoposide. In our previous study, a 1,2,3-triazole-pyrimidine hybrid 25 was synthesized and found significantly reversing multidrug resistance (MDR) mediated by ABCB1. In this study, we evaluated the efficacy of compound 25 in reversing MDR mediated by MRP7 in vitro. The results showed that 25 significantly sensitized cells overexpressing MRP7 to anticancer drugs that are MRP7 substrates at 3 μ;;M. Mechanism study showed that 25 reversed MRP7-mediated MDR by increasing the intracellular accumulation of anticancer drugs and decreasing drug efflux, without altering protein expression level or intracellular localization. Ligand-protein interactions were also modeled by docking with homology MRP7 model. Since limited researches on synthetic MRP7 modulators have been published, our findings provide a valuable prototype for structures which have the potential to be used in combination with conventional anticancer drugs to overcome MDR-mediated by MRP7.

Citation Format: Jing-Quan Wang, Zi-Ning Lei, Qiu-Xu Teng, Bo Wang, Li-Ying Ma, Hong-Min Liu, Zhe-Sheng Chen. A synthetic derivative of 1,2,3-triazole-pyrimidine hybrid reverses multidrug resistance mediated by MRP7 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2983.
Abstract 3006: Anticancer and multidrug resistance-reversing activities of novel antimicrobial peptides
Authors: Teng Qiu-Xu , Lei Zi-Ning , Luo Xiaofang , Wang Jing-Quan , Qin Zuodong , Wurpel John N. , Chen Zhe-Sheng
Published: September 2020

Cancer is a growing concern in public health problems worldwide. Although the development of advanced chemotherapies has already partly reduced the cancer death rate and improved patients' prognosis, the phenomenon of multidrug resistance (MDR), which impairs the efficacy of a serial structure-unrelated anticancer agent, casts a large shadow over the bright future of chemotherapies. MDR has various mechanisms to induce acquired drug resistance of cancer cells after a relatively long period of chemotherapy. One of the most common causes of MDR is the expression of ATP-binding cassette (ABC) transporters, which act as efflux pumps on cancer cell membranes and transport anticancer drugs out of cancer cells thereby reducing the intracellular drug concentration. The growing resistance of cancer cells to current anticancer drugs leads to the urgent need to develop anticancer drugs with a new mechanism of action. Over the past decade, antimicrobial peptides have entered the view of scientists as a new generation of anticancer drugs. In this study, the anticancer effects and the reversal activities against ABC transporter mediated MDR of four novel antimicrobial peptides were investigated. The results showed that, at non-toxic concentrations in vitro, peptides #7, #14, #22, and #24 significantly sensitized ABCB1- and ABCC1-overexpressing cell lines, including both drug-selected MDR cancer cell lines and ABCB1 or ABCC1 gene transfected HEK293 cell lines, to the substrate anticancer drugs paclitaxel and vincristine. Among the antimicrobial peptide compounds, #14 and #24 with the most potent reversal ability were selected for further investigation. These peptides increased the intracellular accumulation of [3H]-paclitaxel in ABCB1 overexpressing cells and that of [3H]-vincristine in ABCC1 overexpressing cells by suppressing the efflux function of ABCB1 and ABCC1 transporters, without causing alteration in the expression levels and localization patterns of ABCB1 and ABCC1 in overexpressing cells. Furthermore, the results of the ATPase assay showed that both two peptides stimulated ABCB1 ATPase activity. Taken together, these results demonstrated that peptide #14 and #24 reverse ABCB1- and ABCC1-mediated MDR through blocking the function of ABCB1 and ABCC1 without affecting the transporters' expression and cellular localization, which suggests that antimicrobial peptides may be used as a novel prospective cancer therapeutic strategy in the combination with traditional anticancer agents.

Citation Format: Qiu-Xu Teng, Zi-Ning Lei, Xiaofang Luo, Jing-Quan Wang, Zuodong Qin, John N. Wurpel, Zhe-Sheng Chen. Anticancer and multidrug resistance-reversing activities of novel antimicrobial peptides [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3006.
Abstract 3020: Tumor pharmacokinetics of the metastatic cancer inhibitor MBQ-167 in mice
Authors: Del Mar Maldonado Maria , Cruz-Collazo Ailed M. , Borrero-García Luis D. , Rosado-González Gabriela T. , Ruiz-Calderon Jean F. , Duconge Jorge , Dharmawardhane Suranganie
Published: September 2020

The Rho GTPases Rac and Cdc42 are potential targets against metastatic disease. Our group characterized the small molecule MBQ-167 as an effective dual Rac/Cdc42 inhibitor that reduces HER2 positive and triple negative tumor growth and metastasis in mice by ~90%. To continue validating this drug for FDA approval, we recently elucidated the plasma pharmacokinetics and the tissue distribution of MBQ-167 in mice (Maldonado et al., 2019). However, further studies are needed to characterize the tumor pharmacokinetics of MBQ-167 to determine drug exposure, time-to-peak, and the length of time that MBQ-167 remains in tumors after administration. The purpose of this study was to determine the tumor pharmacokinetics of MBQ-167 in a single dose input scheme (10 mg/kg BW) following intraperitoneal (IP) administration. Thirty-five female BALB/c mice (5 mice/group) were injected with 2.5 × 105 4T1 murine metastatic breast cancer cells at the mammary fat pad. After tumor establishment, a single dose of MBQ-167 (10 mg/kg BW) was administered via IP. Tumors were collected at 0.5, 1, 3, 6, 9,12, and 24 hours following drug administration. We used a validated bioanalytical method using supercritical fluid chromatography coupled with tandem mass spectrometry (SFC-MS/MS) to quantify MBQ-167 in tumors. Pharmacokinetic parameters were obtained by non-compartmental analysis using WinNolin® software. Pharmacokinetic analysis revealed that MBQ-167 has an elimination half-life (t1/2) of 8.64 hours in tumors, which is much longer than its half-life in plasma (2.17 hours), indicating preferential absorption and retention of this lipophilic small molecule in tumor tissues. The area under the curve (AUC0-t) was 4.39 %ID⋅hr/g with a maximum concentration (Cmax) of 1268.9 ng/g in tumors. Moreover, the time-to-peak concentration in tumors was 0.5 hours. Hence, this study supports the continued development of MBQ-167 as a potential anti-cancer therapeutic.

Citation Format: Maria Del Mar Maldonado, Ailed M. Cruz-Collazo, Luis D. Borrero-García, Gabriela T. Rosado-González, Jean F. Ruiz-Calderon, Jorge Duconge, Suranganie Dharmawardhane. Tumor pharmacokinetics of the metastatic cancer inhibitor MBQ-167 in mice [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3020.
Abstract 3101: Utility of a new targeted next generation sequencing test for liquid biopsy samples from patients with NSCLC
Authors: Reese Jordan , Maxwell Victoria , Jackson Leisa , Pestano Gary , Mellert Hestia S.
Published: September 2020

Blood-based methods for profiling tumor-sourced nucleic acids have become increasingly important in the diagnostic workup for cancer patients, particularly in non-small cell lung cancer (NSCLC). Blood-based assays address several limitations of tissue-based testing approaches such as inaccessible lesions, limited tissue, extended time to results, and invasive specimen collection procedures. Broad-profiling next-generation sequencing (NGS) methods are used to detect rare variants in blood samples. Here we describe the utility of a newly developed 52 gene NGS test, which uses the GeneStudio S5 Prime platform. We report on performance verification of the assay using contrived specimens as well as studies which evaluated clinical specimens at diagnosis and during monitoring. Thirty-eight cell-free nucleic acid (cfNA) specimens were evaluated from donors previously diagnosed with NSCLC. Specimens were collected in blood collection tubes containing preservatives (Streck cfDNA BCT) and shipped at ambient temperature to our central testing Laboratory. cfNA was isolated from plasma and reverse transcribed. The sequencing metrics for these studies included an average of 15 million mapped reads, 94.4% of on target reads, and a mean sequencing depth of 52,000. Additionally, the average molecular coverage was 2400 reads per amplicon with 99.9% uniformity at 500x coverage. Detected variants and their allelic frequencies were compared with reference results from a validated ddPCR method. Eleven variants (6 EGFR and 5 KRAS) were detected in the cohort with allele frequencies ranging from 0.90 - 96.16%. Results were highly concordant between the two methods (R2=0.992) with 90.3% sensitivity and 100% specificity. In addition, we present a case study to further demonstrate clinical utility of NGS for longitudinal monitoring over 540 days for clearance followed by re-emergence of EGFR positive cfNA harboring the EGFR C797S resistance mutation. These studies add to the growing body of knowledge supporting the utility of molecular detection for actionable and emerging biomarkers in liquid biopsy.

Citation Format: Jordan Reese, Victoria Maxwell, Leisa Jackson, Gary Pestano, Hestia S. Mellert. Utility of a new targeted next generation sequencing test for liquid biopsy samples from patients with NSCLC [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3101.
Abstract 3106: Accurate estimation of circulating tumor DNA fraction from ultra low-pass whole-genome sequencing
Authors: Finkle Justin , Lo Christine , Boulos Hala , Driessen Terri , Zhu Wei , Tell Robert
Published: September 2020

Early detection of recurrence is critical to improve the survival of cancer patients. Blood-based “liquid biopsies” provide a minimally invasive method to detect variants, and ultra low-pass whole-genome sequencing (LPWGS) can accurately and sensitively detect low fractions of circulating tumor DNA in blood plasma. We previously conducted LPWGS on approximately 400 samples (primarily from lung, breast, prostate, and pancreatic cancers) residual to samples previously analyzed with our xF liquid biopsy assay. In this analysis, we demonstrate that circulating tumor fraction estimates from LPWGS correlate with the maximum detectable pathogenic variant allele fraction, and where there are discrepancies the LPWGS estimates are more reasonable. We also show differences in tumor fractions and ploidy between cancer cohorts and stages. Our results suggest LPWGS provides an accurate measure of the fraction of circulating DNA derived from tumors across cancer cohorts and stages.

Citation Format: Justin Finkle, Christine Lo, Hala Boulos, Terri Driessen, Wei Zhu, Robert Tell. Accurate estimation of circulating tumor DNA fraction from ultra low-pass whole-genome sequencing [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3106.
Abstract 310A: Transcription factors FOXP3 regulate ATF3 expression
Authors: Yang Wei-Hsiung , Wang Chiung-Min , Yang William
Published: September 2020

Activating transcription factor 3 (ATF3), a stress sensor, is rapidly induced by a variety of physiological and pathological signals, including those from cancer cells, and is essential in the complex processes of stress response. Several lines of evidence have demonstrated that ATF3 is involved in host defense and cancer development. Several studies also provide the relationship between ATF3 and p53 indicating that ATF3 binds to and stabilizes p53 while ATF3 is a negative regulator of TP53 gene expression. FOXP3, a well-known breast and prostate tumor suppressor, is a novel transcriptional repressor for several oncogenes including SKP2. However, whether ATF3 is the target protein of FOXP3 remains unknown. Herein, we demonstrate that ATF3 expression is regulated by FOXP3. First, we observed that overexpression of FOXP3 reduced ATF3 protein level in several cancer cell lines (H1299, HepG2, and Ishikawa cells). Secondly, FOXP3 dose-dependently reduced ATF3 promoter activity in gene reporter assay in several cell lines (MCF7, H1299, MDAMB231, and Saos2 cells). Since FOXP3 is regulated by post-translational modifications (PTMs), we next investigated whether PTMs affect FOXP3-mediated ATF3 expression. Interestingly, we observed that phosphorylation mutation on FOXP3 (Y342F) abolished FOXP3-mediated ATF3 expression. However, other PTM mutations on FOXP3, including S418 phosphorylation, K263 acetylation and ubiquitination, and K268 acetylation and ubiquitination, did not alter FOXP3-mediated ATF3 expression. Finally, FOXP3 binding site was found on ATF3 promoter region, around -870 bp region. Taken together, our results suggest that FOXP3 functions as a novel regulator of ATF3 and this novel event may be involved in tumor development and progression.

Citation Format: Wei-Hsiung Yang, Chiung-Min Wang, William Yang. Transcription factors FOXP3 regulate ATF3 expression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 310A.
Abstract 3110: In depth comparison of cell free DNA blood collection tubes
Authors: Desharnais Joel , Vasquez Jacob M. , Reshatoff Katya J. , Bui Quyen , Chen Han-Mei , Tsvetanova Billyana , Lu Jerry , Murray Amber N. , Costa Gina L.
Published: September 2020

Liquid biopsy continues to gain traction as a minimally invasive method to monitor biomarkers associated with malignancy and metastasis such as cell-free DNA (cfDNA). However, cell-free DNAs are present in low quantities and are prone to rapid degradation and contamination, which presents challenges for the detection of rare cfDNA targets. Higher molecular weight genomic DNA from poorly stabilized leukocytes can also serve to contaminate plasma, compromising liquid biopsy assay results by increasing the total amount of extracted DNA while diluting rare cfDNA targets; leading to sample rejection or false negative results. Efficient and robust preservation of cfDNA and prevention of leukocyte lysis is thus essential for the accuracy of downstream tests, and several blood collection tubes (BCTs) designed to stabilize cfDNA are now commercially available.

The work presented here evaluates LBgard® Blood Tubes (Biomatrica), the Streck Cell-Free DNA BCTs® (Streck), and PAXgene Blood ccfDNA Tubes (PreAnalytix) for analysis of cfDNA. Healthy donor blood was collected into collection tubes, and the samples were either stored for several days at a range of temperatures or subjected to various shipping conditions. Several metrics were evaluated, including hemolysis, plasma recovery, cfDNA preservation, contamination with genomic DNA (gDNA), and quantitation of cfDNA targets. Differences were observed between the various tubes evaluated, especially when tested under real-world stress shipping conditions. Data reports performance metrics and evaluations across the multiple formulations of BCTs and highlights the criticality of collection tube selection in biomarker investigation and study.

Citation Format: Joel Desharnais, Jacob M. Vasquez, Katya J. Reshatoff, Quyen Bui, Han-Mei Chen, Billyana Tsvetanova, Jerry Lu, Amber N. Murray, Gina L. Costa. In depth comparison of cell free DNA blood collection tubes [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3110.
Abstract 3148: FOXC1 expression is associated with a triple-negative basal-like phenotype in breast cancer
Authors: Cho William C. , Ma Victor W. , Cheuk Wah , So Grace Y. , Chin Rebecca Y.
Published: September 2020

Introduction: Triple-negative breast carcinoma (TNBC) is a group of aggressive tumors that are resistant to hormonal therapy. Forkhead box C1 (FOXC1) has been shown to play a crucial role in cancer pathogenesis by cancer stem cell regulation, epithelial to mesenchymal transition and tumor progression in basal-like breast cancer (BLBC). This study examined the expression of FOXC1 in basal-like TNBC, which may potential be exploited as a treatment target for this group of tumors.

Materials and Methods: Immunohistochemical Peroxidase-DAB system and Opal immunofluorescence staining system were employed to detect FOXC1 and cytokeratin 14 (CK14) expressions in 44 formalin-fixed paraffin-embedded (FFPE) invasive ductal breast carcinoma (IDC) and 150 IDC samples on tissue microarray. CK14 was used to identify the BLBC subtype. The staining was scored semi-quantitatively according to the intensity with a mean score from 0-4.

Results: CK14 identified 15 (34%) BLBC in the FFPE tissues and 24 (16%) BLBC in the samples on tissue microarray. Of the 44 FFPE tissues, FOXC1 expression was observed in 100% of the BLBC and 83% of TNBC, while it was only found in 15% of non-TNBC. The mean staining intensity of FOXC1 was lower in non-TNBC (0.30) compared to TNBC (2.16) and basal-like TNBC (2.60). These findings were confirmed in the tissue microarray, which also revealed a strong positive correlation of FOXC1 with basal-like TNBC. In addition, our results showed that FOXC1 was more frequently expressed in ER-negative (22%), PR-negative (21%) and HER2-negative (19%) breast cancers than in ER-positive (3%), PR-positive (3%) and HER2-positive (5%) breast cancers. Moreover, FOXC1 expression was significantly associated with high tumor grade (p = 0.000041) and high tumor-infiltrating lymphocytes (TILs) (p = 0.000001).

Conclusion: FOXC1 is highly expressed in TNBC, particularly among the basal-like subgroup, as opposed to other breast cancers. It accurately classifies the BLBC subtype which may be used as a surrogate marker for this molecular subtype. We find that FOXC1 has an inverse correlation with ER, PR and HER2, as well as a positive correlation with tumor grade and TILs. Our finding suggests that FOXC1 may serve as a potential treatment target for basal-like TNBC.

Citation Format: William C. Cho, Victor W. Ma, Wah Cheuk, Grace Y. So, Rebecca Y. Chin. FOXC1 expression is associated with a triple-negative basal-like phenotype in breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3148.
Abstract 3149: An assay quantifying pro-peptides of type XI collagen (PRO-C11) in serum provides novel insight into the pathology of non-small cell lung cancer (NSCLC)
Authors: Willumsen Nicholas , Kehlet Stephanie N. , Jensen Tina M. , Nissen Neel I. , Karsdal Morten A.
Published: September 2020

Background: The tumor microenvironment (TME) is characterized by an extensive number and activity of cancer-associated fibroblasts (CAFs). CAFs are the most abundant cell type in the TME of stroma-rich tumors and dictate patient outcome. One of the most specific CAF genes is COL11A1 which encodes the α1-chain of type XI collagen. Type XI collagen is synthesized as a pro-collagen and proteolytically cleaved by bone-morphogenetic protein 1 (BMP-1) to yield mature type XI collagen. Cleaved pro-peptides released to the circulation could serve as biomarkers of type XI collagen formation and provide novel insight into patients with cancer. We developed and evaluated an assay targeting the N-terminal pro-peptide of type XI collagen for quantifying this specific fragment in the circulation (serum) of patients with non-small cell lung cancer (NSCLC).

Methods: A monoclonal antibody was raised against the C-terminal neo-epitope on the N-terminal pro-peptide of type XI collagen and employed in the development of a competitive ELISA (PRO-C11). PRO-C11 was measured in serum from 40 stage 3+4 NSCLC patients and compared to 32 age-matched healthy controls. PRO-C11 was correlated with demographic and clinical characteristics (age, BMI, smoking, metastasis, histological subtype).

Results: The PRO-C11 assay is technically robust with specificity only for the BMP-1 generated type XI collagen pro-peptide neo-epitope. PRO-C11 was significantly elevated in NSCLC compared to age-matched healthy controls (p<0.0001). No correlation (Pearson, p>0.05) was seen between PRO-C11 and age, BMI, smoking status, metastasis (y/n) and histological subtype (squamous cell carcinoma vs. adenocarcinoma).

Conclusion: The present study validates a new serological assay quantifying the pro-peptide of type XI collagen. The biomarker has promising potential for NSCLC and warrants further investigations into stroma-rich cancers. Further studies are needed to evaluate the diagnostic/prognostic use of PRO-C11 in NSCLC.

Citation Format: Nicholas Willumsen, Stephanie N. Kehlet, Tina M. Jensen, Neel I. Nissen, Morten A. Karsdal. An assay quantifying pro-peptides of type XI collagen (PRO-C11) in serum provides novel insight into the pathology of non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3149.
Abstract 3155: Interferon-gamma-induced melanoma plasticity and response to PD-1 blockade therapy
Authors: Kim Yeon Joo , Sheu Katherine M. , Tsoi Jennifer , Abril-Rodriguez Gabriel , Grasso Catherine , Hoffmann Alexander , Smale Stephen T. , Graeber Thomas G. , Puig-Saus Cristina , Ribas Antoni
Published: September 2020

Melanoma is a cancer of melanocytes, which develop from neural crest cells during embryogenesis. One of the ways melanoma can resist targeted therapy or adoptive T cell transfer therapy (ACT) is by dedifferentiation, whereby the tumor becomes less melanocytic and more neural crest-like. ACT-induced dedifferentiation and therapy resistance was a result of tumor necrosis factor (TNF) from antitumor T cells (Landsberg, Nature 2012), which can serve as a positive control for inflammatory cytokine-induced melanoma plasticity.

To study whether immune checkpoint blockade induces melanoma dedifferentiation, we analyzed RNA-seq data from paired biopsies of 68 patients before and during anti-PD-1 therapy. Surprisingly, the tumors of patients with complete or partial response dedifferentiate following therapy (paired p= 0.00015), but those of stable or progressive disease do not (paired p= 0.31 and 0.32, respectively). As the presence of interferon-gamma signatures in biopsies is best correlated with response to anti-PD-1 therapy, we hypothesized that interferon-gamma mediates dedifferentiation.

We dedifferentiated four human melanoma cell lines by in vitro exposure to long-term (three to five weeks) interferon-gamma and compared it to three days of TNF as positive control. Although TNF and long-term interferon-gamma induced similar phenotypic changes based on flow cytometry, ATAC-seq showed that they each drive different global chromatin remodeling. Motif enrichment analyses revealed STAT6, TFAP2C, and SOX proteins are key transcription factors involved in interferon-gamma-induced dedifferentiation. Inferred protein activity analysis of the matching RNA-seq data showed that these regulators, as well as IRF3 and HMGA1, alter in activity selectively with dedifferentiation.

In conclusion, dedifferentiation may be a marker of positive response to anti-PD-1 therapy, mediated by chronic exposure to T cells producing interferon-gamma.

Citation Format: Yeon Joo Kim, Katherine M. Sheu, Jennifer Tsoi, Gabriel Abril-Rodriguez, Catherine Grasso, Alexander Hoffmann, Stephen T. Smale, Thomas G. Graeber, Cristina Puig-Saus, Antoni Ribas. Interferon-gamma-induced melanoma plasticity and response to PD-1 blockade therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3155.
Abstract 3162: Early change in peripheral blood CD4+T cells associated with the clinical outcome in gastrointestinal cancer patients receiving immune checkpoint inhibitors
Authors: Lu Zhi-Hao , Liu Chang , Wang Yan-ni , Li Shuang , Jiao Xi , Zou Jian-ling , Wang Zheng-hang , Qi Chang-song , Zhang Xiao-tian , Li Jian , Shen Lin
Published: September 2020

Purpose: Predictive biomarkers for immune checkpoint inhibitors (ICIs) are urgently needed in advanced gastrointestinal (GI) carcinoma.

Methods: Peripheral blood lymphocyte, lymphocyte subset and early dynamic changes of them were retrospectively analyzed in patients with GI cancer treated with ICIs. Cox regression and Kaplan-Meier analyses were conducted to analyze survival.

Results: Eighty patients were enrolled. According to whether tumor progression occurred by 6 months after the initial ICI, patients were divided into two cohorts, 6m-progressive disease (PD) (N=40) and 6m-non-PD (nPD) (N=40).BaselineCD4+/CD8+ T cell (CD4/CD8) ratio was higher in the 6m-nPD cohort than in the 6m-PD cohort. Lymphocyte subsets were obtained both at baseline and before the second dose of ICIs in 61 patients. In total, 54.1% (33/61) of patients experienced tumor progression within 6 months of the initial ICI treatment, and these patients showed significant declines in CD4+ T cell counts (CD4-C1-decline) (p=0.0002) and the CD8+ T cell counts (CD8-C1-decline) (p=0.045) after the first dose of ICIs. In multivariate analyses, CD4-C1-decline (HR=12.66; 95% CI: 2.17-73.91; p=0.005) was an independent prognostic factor for OS. Furthermore, Stratified analyses indicated that CD4-C1-decline was a poor prognostic factor for tumor progression in patients with a deficient mismatch repair (dMMR)/microsatellite instability-high (MSI-H).

Conclusion: Early change in CD4+ T cell counts in peripheral blood samples may help as prognostic biomarkers for GI cancer patients treated with ICIs. Further prospective studies are needed to validate this conclusion.

Citation Format: Zhi-Hao Lu, Chang Liu, Yan-ni Wang, Shuang Li, Xi Jiao, Jian-ling Zou, Zheng-hang Wang, Chang-song Qi, Xiao-tian Zhang, Jian Li, Lin Shen. Early change in peripheral blood CD4+T cells associated with the clinical outcome in gastrointestinal cancer patients receiving immune checkpoint inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3162.
Abstract 3167: Immune checkpoint inhibition re-challenge after a planned treatment interruption in patients with pancreatic adenocarcinoma with mismatch repair deficient tumors
Authors: Florou Vaia , Nevala-Plagemann Chris , Barber Kristin , Mastroianni Jenna , Cavaliery Courtney Christine , Garrido-Laguna Ignacio
Published: September 2020

Background: Immune checkpoint inhibitors (CPI) are used in numerous solid cancers as well as hematologic malignancies. The optimal duration of immunotherapy to maintain durable responses has not been defined, neither is the efficacy of CPI when the disease progresses during a planned treatment interruption. Pembrolizumab has been approved for all mismatch repair deficient tumors regardless of histology, based on cumulative data of five clinical trials including multiple solid tumors. Only eight patients with pancreatic cancer were included in those trials used for CPI approval in this setting. Outside of this, there is currently no role for standard of care CPI in pancreatic cancer.

Methods: We screened all patients with pancreatic adenocarcinoma treated at our institution and identified two patients who had mismatch repair deficient tumors and received immune checkpoint inhibitors.

Results: Two patients with metastatic pancreatic cancer were retreated with an anti- PD-1 antibody after a planned treatment interruption which, did not occur in the context of disease progression or development of immune-related adverse events. Both patients were initially treated for more than one year (17 and 22 months each) with partial response of their disease, before therapy was discontinued after at least six months of stable radiographic findings were achieved. One patient had disease progression after ten months off therapy and the other 16 months after treatment discontinuation. Both were treated with the same checkpoint inhibitor upon progression and had radiographic regression of their disease within two months after treatment initiation. Both remain on CPI with continuing radiographic and clinical response.

Conclusions: CPI re-challenge may be an efficient therapy when discontinued after maximal response has been achieved. Only twelve cases have been described in the literature where patients were retreated with CPI upon progression of disease while off therapy. Our case series contributes to that body of literature and is the first to describe patients with pancreatic cancer re-challenged with CPI. Mismatch repair deficient pancreatic cancers are rare, but CPI have similar efficacy compared to other tumors with similar molecular features.

Citation Format: Vaia Florou, Chris Nevala-Plagemann, Kristin Barber, Jenna Mastroianni, Courtney Christine Cavaliery, Ignacio Garrido-Laguna. Immune checkpoint inhibition re-challenge after a planned treatment interruption in patients with pancreatic adenocarcinoma with mismatch repair deficient tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3167.
Abstract 317: Organoid and TIL co-culture system models the human tumor immune microenvironment and drug sensitivity
Authors: Tokumaru Yoshihisa , Oshi Masanori , Katsuta Eriko , Matsuhashi Nobuhisa , Futamura Manabu , Yoshida Kazuhiro , Takabe Kazuaki
Published: September 2020

BACKGROUNDS: Host tumor immune microenvironment has been shown to play a critical role on the outcome of the breast cancer patient. Among the immune cells, tumor infiltrating lymphocytes (TILs) were shown to be a predictive biomarker for the efficacy of chemotherapy by clinical trials. Triple negative breast cancer (TNBC) is known to have high infiltration of TILs compared with other subtypes. TILs were reported to be associated with the response to doxorubicin and cyclophosphamide, and with better survival in breast cancer patients. Recently, there are accumulating evidences that organoids generated from patient tumors are useful as models for drug sensitivity pre-clinical trials. Here, we hypothesized that co-culture of TILs with organoids will mimic human tumor immune microenvironment and enhance the drug sensitivity.

MATERIAL AND METHODS: GSE22226 that contains both clinical data and gene expression profile of untreated tumors was used as the breast cancer patient cohort that underwent neoadjuvant chemotherapy (NAC). Relative fractions of 22 types of infiltrating immune cells were estimated from tumor gene expression with the CIBERSORT method. Organoids of the same tumors grown as mouse xenografts were generated using standard methods. Organoids (1000/well of 96-w plate) were treated with doxorubicin (0.01-100 µM) for 2 days before addition of TILs of same patient (1-16 x103 per well). Cytotoxicity was assessed after another two days with the CellTiter-Glo 3D cell viability assay.

RESULTS: We first assessed the clinical relevance of TILs in response outcome of NAC by utilizing the previously reported cohort, in which Adriamycin, Cyclophosphamide and Taxane were used. We found that the number of TILs were significantly higher in the patients that achieved pathological complete response (pCR) when compared with the ones that did not. We established 18 PDX models all of which are from TNBC and we successfully expanded TILS 7 of these patients. We derived 8 organoids from the patients. We performed the experiment of adding TILs to the organoids. TILs effectively killed organoids when TILs were cultured at 1:8 or 1:16 ratio. Furthermore, the killing of organoids by doxorubicin was enhanced by the addition of TILs.

CONCLUSION: Amount of TILs associated with better outcome in NAC cohort. In pre-clinical setting, addition of TIL intensified the sensitivity of organoids to doxorubicin.

Citation Format: Yoshihisa Tokumaru, Masanori Oshi, Eriko Katsuta, Nobuhisa Matsuhashi, Manabu Futamura, Kazuhiro Yoshida, Kazuaki Takabe. Organoid and TIL co-culture system models the human tumor immune microenvironment and drug sensitivity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 317.
Abstract 3205: classifieRc: An interactive web interface for the molecular classification of colorectal cancer from RNA-sequencing data
Authors: Quinn Gerard , Sessler Tamas , Allen Wendy , Maguire Sarah , Dunne Philip , McArt Darragh , VanSteenhouse Harper , Gallagher Peter , Lees Andrea , Longley Dan , Seligmann Bruce , Wappett Mark , McDade Simon
Published: September 2020

Background: Transcriptional predictors are increasingly important for sub-typing cancer patients and understanding disease aetiology, predicting patient outcomes and response to treatment. Colorectal cancers (CRC) can be classified by transcriptionally inferred consensus (CMS) and intrinsic (CRIS) sub-types, annotating transcriptional profiles derived from colorectal cancer samples with these sub-types and other transcriptionally inferred predictors requires an experienced bioinformatician and is time-consuming.

Methods: Publically available R packages CMSclassifier, CRISclassifier, MCP-counter and DoRothEA have been integrated into a web interface using the R/Shiny framework which allows users to upload gene expression data which can be additionally normalized with DESeq2. Visualization of this data can be interacted with through using plotly.

Results: To address this bottleneck, we developed the classifieRC Shiny app, which enables rapid analysis, annotation of colorectal cancer transcriptional profiles with state-of-the-art transcriptional CRC sub-typing (CMS and CRIS) as well as estimation of cellular composition (MCP-Counter) and transcription factor activity predictions (DoRothEA). classifieRC can be accessed through a web-based interface, a locally deployable R-script or executable software, with capability of publishing datasets and their resulting analysis to Shiny.IO.

Conclusions: classifieRc enables researchers to rapidly annotate colorectal transcriptomic datasets with molecular sub-types and of functional predictions without the need for a dedicated bioinformatician, expediting insights related patient cohort analyses and novel discoveries. classifieRc provides an easy to use flexible framework for functional annotation transcriptomic datasets and a platform for development of other disease specific apps.

Citation Format: Gerard Quinn, Tamas Sessler, Wendy Allen, Sarah Maguire, Philip Dunne, Darragh McArt, Harper VanSteenhouse, Peter Gallagher, Andrea Lees, Dan Longley, Bruce Seligmann, Mark Wappett, Simon McDade. classifieRc: An interactive web interface for the molecular classification of colorectal cancer from RNA-sequencing data [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3205.
Abstract 3214: BART: An integrative bioinformatics toolkit and web server for functional transcription factor prediction
Authors: Wang Zhenjia , Ma Wenjing , Zhang Yifan , Magee Neal E. , Chen Yang , Zang Chongzhi
Published: September 2020

The goal of this bioinformatics tool is to predict functional transcription factors (TFs) from various queries of target genomic features. TFs play a key role in regulating gene expression in a dynamic chromatin structure. From a target gene set or a genomic region set, it is challenging to identify functional TFs that regulate these genes or bind in these regions, because of complexity of enhancer distributions and insufficient specificity of DNA sequence motifs. Here, we present BART (Binding Analysis for Regulation of Transcription), an interactive bioinformatics toolkit and web server to predict functional TFs that regulate a set of co-regulated genes or associate with a genomic profile. From a gene set, BART first uses a machine learning approach to generate a genome-wide cis-regulatory profile by leveraging hundreds of publicly available ChIP-seq profiles for active enhancer histone mark H3K27ac and DNase-seq profiles for genome-wide cis-regulatory elements. Then it identifies a list of TFs whose binding profiles most correlate with the cis-regulatory profile by comparing with tens of thousands of ChIP-seq datasets. BART can also identify TFs associated with differential chromatin interaction from a pair of three-dimensional (3D) genomic datasets, such as Hi-C. BART integrates over 12,000 ChIP-seq datasets for over 900 TFs for human and over 500 TFs for mouse to make accurate predictions. We demonstrate that BART outperforms existing methods in identifying the true TF from target gene sets. BART can be useful for any researcher in studying cancer gene regulation and cancer epigenetics, and demonstrates the power of utilizing public data for computational biology research. BART web server is freely available at: http://bartweb.org.

Citation Format: Zhenjia Wang, Wenjing Ma, Yifan Zhang, Neal E. Magee, Yang Chen, Chongzhi Zang. BART: An integrative bioinformatics toolkit and web server for functional transcription factor prediction [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3214.