bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3834: Epigenetic modification of oncogenes or tumor suppressor genes by thymoquinone in triple negative breast cancer
Authors: Khan Md. Asaduzzaman , Zheng Meiling , Fu Junjiang
Published: August 2019

Background and Objective: Triple negative breast cancer (TNBC) is a molecular subtype, and most aggressive and deadly among breast cancers. Different molecular subtypes of breast cancer have distinct molecular mechanisms, and in case of TNBC, the mechanisms are complex, and still obscure. Development of therapeutic strategies are thus also difficult against TNBC. Thymoquinone (TQ) is a natural compound, which has been found effective against TNBC cells, but the mechanism of TQ action in TNBC is not clear. In this study, we have focused on TQ’s genome-wide epigenetic effect on TNBC cell line BT549. Methods: The BT549 cells were treated with 1-10 µM of TQ for cellular growth analysis, and isolated DNA for dot blot analysis with anti-5-methyl cytosine (anti-5MC) antibody to observe whole genome methylation status affected by TQ. Also the chromatin immunoprecipitation (ChIP) was performed with anti-methyl binding domain protein (anti-MBD1) to identify DNA sections, of which methylation status is changed by TQ.

Results: Cell viability assay showed that TQ dose-dependently reduced BT549 cell growth. DNA dot blot analysis results indicated no obvious changes in total methylation status change in genomic DNA. However, ChIP-seq confirmed that methylation level was affected (either increased or decreased) in certain genomic locations by TQ treatment. Some of the locations are proximal or close to some genes, indicating that this epigenetic modification might have influence on these genes’ expression pattern.

Conclusion: Thus, here in this preliminary research study, we indicate some genes, which might be epigenetically targeted by TQ in TNBC cells. Funding support: National Natural Science Foundation of China (Grant No. 81850410555, 81672887, 81172049)

Citation Format: Md. Asaduzzaman Khan, Meiling Zheng, Junjiang Fu. Epigenetic modification of oncogenes or tumor suppressor genes by thymoquinone in triple negative breast 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 3834.
Abstract 3842: Co-targeting BET and MEK as salvage therapy for MAPKi and checkpoint inhibitor resistant melanoma
Authors: Villanueva Jessie , Echevarria-Vargas Ileabett M. , Reyes-Uribe Patricia I. , Guterres Adam , Ronghe Amruta , Burgos Delaine M. Zayas-Bazan , Liu Qin , Xu Xiaowei , Speicher David W.
Published: August 2019

Despite novel therapies for melanoma, drug resistance remains a significant hurdle to achieving optimal responses. NRAS mutant melanoma is an archetype of therapeutic challenges in the field, which we used to test drug combinations to avert drug resistance. We show that the BET family member BRD4 is expressed at high levels in NRAS-mutant melanoma, and that high levels of BRD4 are associated with poor patient survival. Combining BET and MEK inhibitors synergistically curbed the growth of NRAS mutant melanoma and prolonged the survival of mice bearing tumors refractory to MAPK inhibitors and immunotherapy. Transcriptomic analysis revealed that combining BET and MEK inhibitors mitigated a MAPK- and checkpoint-inhibitor resistance transcriptional signature, downregulated the transcription factor TCF19, and induced apoptosis. Notably, TCF19 levels were downregulated in post-treatment biopsies from patients that responded to targeted or immunotherapies. In contrast, TCF19 levels were upregulated in tumor samples from non-responder patients. Furthermore, global proteomic analysis of PDX derived from patients resistant to immune checkpoint inhibitor indicate that the major downstream effects of concurrent BET and MEK inhibition is lipid metabolism. We are investigating the impact and mechanism whereby lipids modulate therapy response. Together our studies demonstrate that co-targeting MEK and BET can offset therapy resistance, offering a potential salvage strategy for melanomas with no other therapeutic options, and possibly other treatment-resistant tumor types.

Citation Format: Jessie Villanueva, Ileabett M. Echevarria-Vargas, Patricia I. Reyes-Uribe, Adam Guterres, Amruta Ronghe, Delaine M. Zayas-Bazan Burgos, Qin Liu, Xiaowei Xu, David W. Speicher. Co-targeting BET and MEK as salvage therapy for MAPKi and checkpoint inhibitor resistant melanoma [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 3842.
Abstract 3852: Development of1,2-bis(hydroxymethyl)benzo[g]pyrrolo[2,1-a]phthalazine hybrids as potent anticancer agents to small cell lung cancer
Authors: Chen Tai-Lin , Jain Vicky , Lin Yi-Wen , Su Tsann-Long , Lee Te-Chang
Published: August 2019

Angiogenesis is a hallmark of cancer development, especially in lung cancer and colorectal cancer. Unfortunately, the rapid development of resistance to anti-angiogenesis agents has limited the clinical benefits for the treatment. To improve the efficacy of anti-angiogenesis agents, we designed and synthesized a series of new hybrid molecules that are composed of benzophthalazine (anti-angiogenesis moiety) and bis(hydroxymethyl)pyrrole (DNA interstrand crosslinking moiety). We demonstrate that these compounds are the dual function inducing DNA crosslinking and anti-angiogenesis. 1,2-Bis(hydroxymethyl)benzopyrrolo[2,1-a]phthalazine derivatives displayed potent cytotoxicity to leukemia and a batch of solid tumor cell lines. Among them, small cell lung cancer (SCLC) cells were the most susceptible cells with the IC50 values ranged from dozens of nanomolar to submicromolar. Significantly, we observed total tumor remission by treatment of SCLC H526 (TP53 WT) xenografts with formulated compound 8 at the dose of 20mg/kg, 5 times/week for 2 weeks. In addition, compound 8 was also more potent than cisplatin in suppression of growth of SCLC H211 (TP53 mutant) xenografts. Combination treatment of H211 cells with compound 8 and cisplatin displayed the synergistically cell killing. Our preliminary results further showed that co-treatment of compound 8 and low dose cisplatin significantly suppressed tumor growth and prolonged the survival rate of mice bearing H211 xenografts. In contract to cisplatin, treatment of mice with compound 8 did not cause body weight lost, indicating low systematic toxicity of compound 8. In summary, our results indicate that bis(hydroxymethyl)benzopyrrolo[2,1-a]phthalazine hybrids, either alone or in combination with other therapeutic drugs, are potent anticancer agents against SCLC and deserve for future development.

Note: This abstract was not presented at the meeting.

Citation Format: Tai-Lin Chen, Vicky Jain, Yi-Wen Lin, Tsann-Long Su, Te-Chang Lee. Development of1,2-bis(hydroxymethyl)benzo[g]pyrrolo[2,1-a]phthalazine hybrids as potent anticancer agents to small cell lung 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 3852.
Abstract 3866: Decursin inhibits tumor growth, migration and invasion through the regulation of CXCR7 expression in gastric cancer
Authors: Kim Solbi , Kim Nayoung , Joo Mina , Jeon HueungJin , Ryu Hyewon , Lee Hyo Jin
Published: August 2019

Background: CXCR7 is overexpressed in a variety of cancer and is known to promote cell migration, invasion and metastasis. Therefore, inhibition of CXCR7 can prevent tumor progression. In this study, we investigated the role of decursin on the CXCR7 expression and its antitumor effects in gastric cancer.

Methods: CXCR7-overexpressing gastric cell lines were established, and the expression of membrane CXCR7 was confirmed by flow cytometry and immunocytochemistry. Cell viability and anoikis assay were performed using CCK-8 reagent. The effect of decursin on cell migration and invasion was tested using transwell system, and the cleavage of PARP and caspase-3 was performed in western blotting and was confirmed by flow cytometry using apoptosis detection kit. The inhibition of tumor growth by decursin was also demonstrated in vivo by measuring tumor growth using subcutaneous injection mouse model.

Results: CXCR7 overexpression significantly increased the invasion and migration, and tumor growth in gastric cancer cells. Administration of decursin decreased the membrane expression of CXCR7 in a concentration dependent manner and significantly inhibited tumor progression by suppressing cell migration, invasion and proliferation. In addition, decursin treatment impeded in vivo tumor growth in BALB/c nude mice. CXCR7-induced STAT3 and c-Myc activation were inhibited by decursin, whereas pro-apoptotic pathway was activated.

Conclusion: Decursin inhibited the cell growth, migration and invasion via downregulating the membrane expression of CXCR7 in gastric cancer in vitro and suppressed tumor growth in vivo suggesting that decursin might be used as a therapeutic agent alone or in combination with other anticancer agents in gastric cancer.

Citation Format: Solbi Kim, Nayoung Kim, Mina Joo, HueungJin Jeon, Hyewon Ryu, Hyo Jin Lee. Decursin inhibits tumor growth, migration and invasion through the regulation of CXCR7 expression in gastric 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 3866.
Abstract 3879: Inhibition of CXCR4 driven colorectal cancer progression by Nef-M1 peptide
Authors: Katkoori Venkat R. , Anderson Zeynoire , Manne Upender , Bumpers Harvey
Published: August 2019

Introduction: The Nef-M1 peptide (Nef-M1) competes effectively with the natural ligand of CXCR4, SDF-1α, and exhibits anti-tumorigenic activity in human colorectal cancer (CRC). However, the molecular mechanisms of action of Nef-M1 on CRC remain unclear. In this study, we evaluated the inhibition of CXCR4 driven molecular signaling mechanisms that is involved in CRC progression.

Experimental Design: Cell line derived xenografts (CDX), patient derived explant (PDE) and in vitro cell based models were used to examine the mechanisms of action of Nef-M1. The severe combined immunodeficient (SCID) mice with tumor were treated intraperitoneally with either Nef-M1 or scrambled amino acids sequence Nef-M1 (sNef-M1) that is the control. Preparation of CRC tissue, explant complete media, and cultures and treatment for 48 hours were performed for the PDE study. Sections from tumors were evaluated by immunostaining (IHC) for signaling proteins thus, implicating CRC progression. Western blot (WB) analyses were also performed on lysates of both cell lines and tumors to assess the effect of Nef-M1 on the signaling pathways that promote CRC progression.

Results: The present study revealed that PDE recapitulate multiple biological features of the disease and these were found to be very similar to the corresponding original CRC. CXCR4 overexpressing CRC cells displayed activation of CXCR4/CREB signaling as demonstrated by an increased activation of CREB, and expression of B-Myb and APOBEC3B (A3B). IHC analysis for Ub-H2B, a stem cell signaling protein that correlates with advanced disease and metastasis indicated that Nef-M1 treated CDX or PDE tumors had low expression of Ub-H2B. However, sNef-M1 treated tumors had high expression of Ub-H2B. WB analyses indicated that Nef-M1 not only suppressed the expression of Ub-H2B, but also significantly suppressed the expression of A3B. Cells expressing CXCR4 became susceptible to Nef-M1-induced inhibition of Akt, mTOR activation and Ub-H2B expression.

Conclusions: These results indicate that Nef-M1 suppresses CXCR4 driven activation of CXCR4/CREB signaling and the expression of Ub-H2B. Therefore, Nef-M1 inhibition of these signaling pathways may be a promising therapeutic strategy for CRC. This work was supported by NIH/NCI Workforce Diversity Grant R21-CA171251.

Citation Format: Venkat R. Katkoori, Zeynoire Anderson, Upender Manne, Harvey Bumpers. Inhibition of CXCR4 driven colorectal cancer progression by Nef-M1 peptide [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 3879.
Abstract 3891: A phase 1 study to evaluate the pharmacokinetics (PK) and safety of niraparib in Chinese patients with epithelial ovarian cancer (OC)
Authors: Zhang Jian , Gao Yu Nong , Lou Ge , Yin Ru Tie , Hou Jian Mei , Yan James , Li Jing , Zhang Zhi Yi , Wu Xiao Hua
Published: August 2019

Purposes: To characterize the PK and safety of niraparib as a maintenance therapy in Chinese OC patients.

Methods: Eligible patients were randomized 1:1:1 to 100, 200, or 300 mg once daily cohort. Plasma samples were collected following single and multiple dosing and analyzed using a validated LC/MS/MS method. PK parameters were analyzed by standard non-compartmental approach with WinNonlin. A population PK (Pop PK) model was derived from pooled PK data of current study and two previous PK studies in Caucasian: the dose escalation and expansion study (PN001), and the ENGOT-OV16/NOVA sub-study. Non-linear mixed effect modeling was done with NONEM®.

Results: 36 patients were randomized and included in the PK and safety analysis set. Niraparib was rapidly absorbed after dosing with tmax~3h. The exposure of niraparib was dose proportional, while other PK parameters such as t1/2, accumulation ratio were dose independent (Table 1). The POP-PK analysis model was established using PK data from 144 Caucasians and 39 Chinese. Chinese patients had a slightly higher Cmax than Caucasian patients but similar total exposure (AUC0-∞) after a single dose. Simulation by final Pop PK model indicated comparable total exposure (AUC0-∞) and Cmax between Chinese and Caucasian patients at the steady-state following multiple daily doses. Treatment emergent AEs (TEAEs) occurred in 97.2% of all patients treated with niraparib. The most frequent (>20%) TEAEs were consistent with the known safety profile of PARP inhibitors. TEAEs of Grade 3/4 reported in ≥ 5% patients included decreased platelet count (13.9%), decreased neutrophil count (11.1%), anemia (8.3%) and increased gamma-glutamyl transferase (5.6%).

Conclusions: The PK profile of niraparib in Chinese was consistent with its known profile in Caucasian. Niraparib in Chinese OC patients has a manageable safety profile, similar to that observed in global studies. (NCT03551171)

Citation Format: Jian Zhang, Yu Nong Gao, Ge Lou, Ru Tie Yin, Jian Mei Hou, James Yan, Jing Li, Zhi Yi Zhang, Xiao Hua Wu. A phase 1 study to evaluate the pharmacokinetics (PK) and safety of niraparib in Chinese patients with epithelial ovarian cancer (OC) [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 3891.
Abstract 3907: Synergistic effect of targeting both CDK4/6 and mTORin sarcoma cell lines
Authors: Wang Xiaochun , Goldstein David , Crowe Philip , Yang Jia-Lin
Published: August 2019

Background: The phosphatidylinositide-3-kinase/Protein kinase B (AKT)/ mammalian target of rapamycin (mTOR) signaling is central for cancer growth, survival and motility. One of the key restraints on cell growth downstream is retinoblastoma (Rb) tumour suppressor, which is negatively regulated by the cyclin-dependent protein kinases (CDK) 4/6 protein. We hypothesize that blocking both the CDK4/6 and the mTOR allows the double brakes to tumour growth.

Aim: Our aim is to investigate whether combination therapy using mTOR and CDK4/6 inhibitors (palbociclib and ridaforolimus) would have a synergistic growth inhibitory effect in sarcoma.

Methods: The effect and mechanism of palbociclib and ridaforolimus was investigated in a panel of twelve sarcoma cell lines by Crystal-violet colorimetric and Western blot assays. HT1080 fibrosarcoma metastatic mouse model was investigated for in vivo therapeutic study.

Results: Palbociclib showed anti-proliferation in all cell lines (IC50s: 0.1-1.1µM), while ridaforolimus had growth inhibition in 10/12 cell lines (IC50s: 0.4-26nM). The palbociclib-ridaforolimus combination achieved synergistic effect (CIs: 0.1-0.9) in 9/12 cell lines, including applying the drugs in different sequence (together or pre-treatment with one drug for 24-48 hours) and ratio (1:1, 1:2, 1:4, 2:1 or 4:1). The Western blot demonstrated that the 2-drug combination further blocked both CDK/Rb/E2F and AKT/mTOR pathways, via promoting apoptosis. In the experimental lung metastatic HT1080 mouse model, using palbocilib (10 and 30mg/kg, every other day) and ridaforolimus (0.1 and 0.3mg/kg, daily for 5 days per week) for 4 weeks synergistically (CI = 0.7) inhibited the development of lung metastases (mean number of colonies: 132 in low combination dose and 38 for high combination dose) compared to monotherapy [243 (palbociclib) and 255 (ridaforolimus) in low dose, 113 and 97 for high dose) and vehicle control (286). In the orthotopic fibrosarcoma HT1080 mouse model, the palbociclib-ridaforolimus combination therapies achieved significant (p<0.001) and synergistic (CI=0.00375) growth inhibitory effect compared to monotherapies and control groups.

Conclusions: This study demonstrated that palbociclib-ridaforolimus combination is active in a variety of sarcoma subtypes and worthy of further development towards a clinical trial.

Citation Format: Xiaochun Wang, David Goldstein, Philip Crowe, Jia-Lin Yang. Synergistic effect of targeting both CDK4/6 and mTORin sarcoma cell lines [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 3907.
Abstract 3914: mTOR pathway components dictate cell response to Src inhibitors in prostate cancer cells
Authors: Dai Yao , Siemann Dietmar W.
Published: August 2019

Src inhibitors have been actively evaluated in the clinic for the treatment of advanced prostate cancer however the effects are modest. One interpretation is that oncogenic signaling pathways are redundant and blocking one pathway is not sufficient to yield considerable efficacies. mTOR pathway is commonly hyperactivated in prostate cancer. However, the functional role of mTOR in Src inhibitor-mediated response remains largely unknown. In the current study, we combined mTOR inhibitor rapamycin and Src inhibitor dasatinib to detect functional behaviors in prostate cancer cells. Cell proliferation and clonogenic survival were detected by MTT and colony formation assay, respectively. Wound-healing” scratch assay was conducted to monitor cell migration, and Matrigel-based transwell assay was applied to assess cell invasion. Protein expression was detected by Western blotting. Gene silencing was detected by siRNA transfection. We found that two agents combined together showed greater inhibition on proliferation and clonogenic survival than either agent alone. Such combination effects were additive or synergistic depending on the cell types, as reflected by combination index calculation. In addition, cell migration and invasion were inhibited in a greater extent by rapamycin plus dasatinib, compared to either treatment alone. At the molecular level, co-targeting mTOR and Src lead to clear inhibition of Src and its downstream pathway focal adhesion kinase (FAK), Akt and mTOR downstream components S6RP and 4E-BP1. Interestingly, knockdown 4EBP1, but not S6RP, attenuated dasatinib-mediated functional inhibition. These data indicate that adding mTOR inhibitors to Src-based intervention may achieve better treatment outcomes. Such combination could be evaluated as a novel treatment strategy for prostate cancer patients who are less responsive to Src inhibitors. In addition, 4EBP1 may be used as a candidate predictive biomarker for monitoring clinical effectiveness of Src inhibitors in prostate cancer.

Citation Format: Yao Dai, Dietmar W. Siemann. mTOR pathway components dictate cell response to Src inhibitors in prostate cancer 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 3914.
Abstract 392: LKB-inactivated lung cancer cells exhibit dependency on INSL4 signaling
Authors: Yang Rongqiang , Li Steven W. , Chen Zirong , Zhou Xin , Ni Wei , Fu Dongtao A. , Lu Jianrong , Kaye Frederic J. , Wu Lizi
Published: August 2019

Lung cancer is the leading cause of cancer deaths worldwide. The LKB1 (STK11) tumor suppressor gene is commonly inactivated in non-small cell lung carcinomas (NSCLC), a major form of lung cancer. Currently, no effective treatments are available for LKB1-inactivated lung cancer. Elucidation of critical signaling downstream of LKB1-inactivation has the potential to uncover rational therapeutic targets for this unique and prevalent molecular subset of lung cancers. In this study, we identified INSL4 (insulin-like 4), a member of the insulin/IGF/relaxin superfamily, as a novel downstream target of LKB1-deficiency through gene expression profiling analysis. We observed that INSL4 expression was highly induced through aberrant CRTC-CREB activation subsequent to LKB1 inactivation. INSL4 was highly expressed in human LKB1-deficient NSCLC cells and primary tumors while undetectable in all normal tissues except in the placenta. High INSL4 expression was associated with more advanced lung cancer and worse patient survival. INSL4 depletion inhibited the growth and viability of LKB1-null NSCLC cells in vitro and blocked the growth of lung cancer xenografts. Moreover, expression profiling identified an INSL4-regulated gene program that is associated with the regulation of cell cycle, growth, and survival. In conclusion, LKB1 deficiency induces an autocrine INSL4 signaling that critically supports the growth and survival of lung cancer cells. Therefore, aberrant INSL4 signaling is a promising therapeutic target for LKB1-deficient lung cancers.

Citation Format: Rongqiang Yang, Steven W. Li, Zirong Chen, Xin Zhou, Wei Ni, Dongtao A. Fu, Jianrong Lu, Frederic J. Kaye, Lizi Wu. LKB-inactivated lung cancer cells exhibit dependency on INSL4 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 392.
Abstract 3946: Tumor checkpoint inhibitor profiling for an optimal clinical response
Authors: Maniyar Rachana R. , Chakraborty Sanjukta , Rahoma Ghada Ben , Degliuomini John J. , Wallack Marc , Geliebter Jan , Tiwari Raj K.
Published: August 2019

The last three decades have seen a steady rise in melanoma, the most aggressive form of skin cancer. The discovery of checkpoint inhibitors has revolutionized the treatment landscape for melanoma; anti-CTLA4 and anti-PD1 showing great successes in clinic. While checkpoint molecules and co-stimulatory molecules are traditionally present on cells involved in immune activation, mounting evidence suggests that tumor cells also express these molecules. Our laboratory has characterized five patient-derived melanoma cell lines, MEL-2, MEL-V, 3MM, KFM, and GLM-2 and screened them for the expression of a comprehensive list of 29 co-stimulatory and co-inhibitory molecules compared to normal adult melanocytes. We see a differential mRNA expression of many of these immune-regulatory molecules, including BTLA, HVEM, CD160, CD226 and TIM1. Western blots and immunofluorescence confirmed the presence of these molecules at the protein level. A flow cytometry analysis demonstrated that BTLA, HVEM, CD160, TIM1 and CD226 are present on the membrane of these patient derived melanoma cells; implying that they are capable of engaging their respective ligands and exerting a functional role in immunomodulation. These findings were additionally validated in melanoma tissues, obtained from patients, by immunohistochemistry. Interestingly, treatment of MEL-2, MEL-V, KFM and GLM-2, our BRAFV600E containing patient derived cells, with BRAFV600E inhibitor PLX4032 led to the upregulation of these molecules. This upregulation was coupled with an increase in transcription factor MITF, a binding site for which is present in the promoter region of all the upregulated molecules. Co-culture experiments with immune cells, demonstrate a modulatory role played by the HVEM/BTLA/CD160 axis molecules that are present on tumor cells. The presence of these additional immune-regulatory molecules signify the existence of a robust tolerance mechanism induced by these checkpoint inhibitors. Our studies underscore the importance of characterizing tumor profiles to enable the selection of an optimal treatment regimen combining targeted small molecule inhibitors, and checkpoint inhibitors to maximize T cell killing, and tumor clearance.

Citation Format: Rachana R. Maniyar, Sanjukta Chakraborty, Ghada Ben Rahoma, John J. Degliuomini, Marc Wallack, Jan Geliebter, Raj K. Tiwari. Tumor checkpoint inhibitor profiling for an optimal clinical response [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 3946.
Abstract 3962: Chemotherapy induces alteration of programmed death ligand-1 expression in gastric cancer
Authors: Yamashita Kohei , Iwatsuki Masaaki , Iwagami Shiro , Eto Kojiro , Koga Yuki , Kiyozumi Yuki , Baba Yoshifumi , Yoshida Naoya , Baba Hideo
Published: August 2019

Background Cancer immunotherapy targeting the programmed cell death 1 (PD-1) / programmed death ligand 1 (PD-L1) pathway have been demonstrated favorable therapeutic effect in gastric cancer (GC). However, it remains unclear that PD-L1 expression in GC tissue can be a biomarker to predict the therapeutic effect of immune checkpoint inhibitors. We hypothesize that prior therapy, including chemotherapy and radiation therapy can lead to the alteration of PD-L1 status, resulting in imprecise evaluation of PD-L1 expression. The aim of this study is to clarify the effect of cytotoxic agents on PD-L1 expression in GC cells in time and dose depending manner. Methods PD-L1 expression of GC cell lines were confirmed by quantitative PCR and western blotting. Two gastric cancer cell lines, which have low PD-L1 expression, are selected and used in subsequent in vitro experiment. We evaluated the expression of PD-L1 in these cell lines by quantitative PCR and western blotting after exposure to cytotoxic agents at various concentrations and exposure times. Furthermore, we examined whether the expression of PD-L1 induced by cytotoxic agents was maintained after ceasing the 5-FU exposure. The gene pathway regulating PD-L1 induction was also examined. Results The PD-L1 expression of the selected GC cell lines were upregulated, depending on the concentration and exposure time of cytotoxic agents. Furthermore, the PD-L1 expression induced by cytotoxic agents was maintained even after cessation of exposure. We clarify that the upregulation of PD-L1 expression was induced via p38 MAPK activation. Conclusion We demonstrated that cytotoxic agent can alter the expression of PD-L1 in GC cells through the activation of p38 MAPK pathway in dose and time depending manner. Our findings suggested that the modified administration of immune checkpoint inhibitors can improve the antitumor response, leading to promising new treatment strategy against GC.

Citation Format: Kohei Yamashita, Masaaki Iwatsuki, Shiro Iwagami, Kojiro Eto, Yuki Koga, Yuki Kiyozumi, Yoshifumi Baba, Naoya Yoshida, Hideo Baba. Chemotherapy induces alteration of programmed death ligand-1 expression in gastric 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 3962.
Abstract 3973: Nivolumab-based ELISA revealed increased serum PD-1 levels in advanced RCC patients
Authors: Mizutani Kosuke , Horie Kengo , Kato Taku , Nakane Keita , Kawakami Kyojiro , Fujita Yasunori , Ito Masafumi
Published: August 2019

Introduction: Antibody therapy has been successfully used for several caners, but the efficacy is still limited. To select patients that respond well to the therapy, development of companion diagnostics is essential. Although PD-L1 expression determined by immunohistochemistry has been shown to be a marker to select patients with non-small-cell lung cancer who will benefit from antibody therapy, its potential for Nivolumab treatment in renal cell carcinoma (RCC) patients is unclear. Detection of the soluble form of PD-1 in serum has been reported, but there are few studies demonstrating its clinical role in RCC. Antibody-based assays may rely on characteristics and quality of antibody used. We hypothesized that the use of Nivolumab as a capture antibody in ELISA would give more clinically relevant results to evaluate the serum PD-1 levels in RCC patients.

Materials and Methods: After approval of the study by the Medical Review Board of Gifu University, a total of 71 participants were recruited (22; healthy volunteers, 29; localized RCC and 20; RCC patients with metastasis or recurrence). Serum PD-1 levels were determined using PD-1 Human ELISA Kit (Thermo Fisher Scientific Inc., MA, USA). For Nivolumab-based ELISA, a capture antibody included in the kit was changed to Nivolumab. Nivolumab was coated to 96-well microplates using a commercially available kit.

Results: Medians of the serum PD-1 levels measured by conventional ELISA were 31.8, 29.8 and 34.3 pg/ml in healthy volunteers, localized RCC patients and those with metastasis or recurrence, respectively, which showed no statistically significant difference. When the same set of patients were analyzed by Nivolumab-based ELISA, medians were 24.6, 23.7 and 46.4 pg/ml and the serum PD-1 levels were significantly elevated in patients with metastasis or recurrence (P = 0.013) compared to controls and localized patients. Of the 29 localized patients, 3 patients with relatively higher serum PD-1 levels at initial diagnosis relapsed later. Among 6 patients with metastasis who were treated with Nivolumab, well responders showed relatively lower serum PD-1 levels before treatment.

Conclusion: The serum PD-1 levels determined using Nivolumab were increased in advanced RCC patients. Our results suggest that the detection of target molecules using a therapeutic antibody may yield more clinically relevant information, thus providing a novel approach to develop companion diagnostics.

Citation Format: Kosuke Mizutani, Kengo Horie, Taku Kato, Keita Nakane, Kyojiro Kawakami, Yasunori Fujita, Masafumi Ito. Nivolumab-based ELISA revealed increased serum PD-1 levels in advanced RCC 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 3973.
Abstract 3981: Exosomal microRNA profiles in peritoneal fluids as a therapeutic biomarker for peritoneal metastasis of gastric cancer
Authors: Ohzawa Hideyuki , Saito Akira , Kumagai Yuko , Yamaguchi Hironori , Hosoya Yoshinori , Sata Naohiro , Kitayama Joji
Published: August 2019

Objectives: Peritoneal metastasis (PM) frequently occurs in patients with gastric cancer and has poor prognosis even with systemic chemotherapy. Intraperitoneal chemotherapy (IPC) using paclitaxel (PTX) combined with systemic chemotherapy has demonstrated a remarkable clinical efficacy for gastric cancer with PM. However, there is no reliable biomarker to reflect the response of peritoneal lesions as well as to predict the outcome of the patients receiving IPC. Although peritoneal fluids are considered to contain many factors derived from disseminated tumor cells in peritoneal cavity, little information is available so far. In this study, we examined exosomal miRNA profiles derived from peritoneal fluids in patients with gastric cancer and explored possible biomarkers useful for IPC of the patients with PM.

Methods: Peritoneal fluids were collected from 2 groups; with or without PM. Peritoneal fluids were obtained either laparoscopically, during open surgery or from subcutaneous infusion port connected to an intraperitoneal catheter. Exosomal fractions were isolated using ultracentrifuge method (150,000 xg, 70 min, 4°C) and the presence of exosome was confirmed with nanotracking analysis. Total RNA including small RNA was extracted from exosomal fractions and expression analyses of miRNAs were performed using RT-PCR.

Results: At first, we comprehensively analyzed the expression of miRNAs between 11 samples with PM and 14 samples without PM and identified 11 miRNAs which showed particularly different expression pattern between the 2 groups. Among them, 4 miRNAs (miR-21-5p,miR-223-3p, miR-342-3p and miR-92a-3p) were selected and further analysed using 51 samples derived from the peritoneal fluids of gastric cancer. Expression of these 4 miRNAs were significantly up-regulated in samples with PM which was consistent with formeranalysis. Moreover, expression of miR-21-5p was significantly upregulated in in samples fromT4 tumor (invaded serosa or adjacent structure) as compared with those from T1 to T3 tumor.

Conclusion: We identified several dysregulated exsosomal miRNAs derived from peritoneal fluids of the patients with PM from gastric cancer, which were supposed to reflect the tumor burden on peritoneum. Exosomal miRNA profiles might be useful biomarkers to determine the presence of PM as well as the response to IPC.

Citation Format: Hideyuki Ohzawa, Akira Saito, Yuko Kumagai, Hironori Yamaguchi, Yoshinori Hosoya, Naohiro Sata, Joji Kitayama. Exosomal microRNA profiles in peritoneal fluids as a therapeutic biomarker for peritoneal metastasis of gastric 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 3981.
Abstract 399: ctDNA a promising predictive marker for treatment with PD-1 inhibitors in KRAS mutated NSCLC after platinum based chemotherapy
Authors: Hiltermann T.Jeroen.N. , Hijmering-Kappelle Lucie B.M. , Azpurua Maria L. Aguirre , Wekken Anthonie.J. vd , Hiddinga Birgitta.I. , Timens Wim , Groen Harry.J.M. , Schuuring Ed
Published: August 2019

Introduction: Immunotherapy is a breakthrough in non-small cell lung cancer (NSCLC), showing long lasting responses in metastasized disease. However, only a limited number of patients responds to treatment. Selection of patients by known biomarkers (eg PD-L1, TMB) and combining PD-(L)1 inhibitors with other treatments (eg. chemotherapy or CTLA4 inhibitors) enriches the population for responders, however still around 60% do not respond with a durable response. We hypothesized that in KRAS mutant positive advanced NSCLC the detection of changes of ctDNA levels will be a relevant predictive marker in immunotherapy ( and stronger than eg PD-L1 expression in tumor tissue)..

Purpose: To test ctDNA as an early predictive marker for durable response in KRAS mutated lung cancer, in patients treated with PD-1 inhibitors after progression on platinum containing chemotherapy.

Methods: Patients with advanced NSCLC treated with nivolumab with a known KRAS or BRAF mutation from primary biopsy, with disease progression after platinum based chemotherapy are eligible. ctDNA obtained from EDTA plasma at baseline, 1, 2, 4, 6 weeks and thereafter every 12 weeks compared to durable response as primary outcome (ie. benefit from treatment> 6month). Secondary outcomes were tumor response according to RECISTv1.1, progression-free and overall survival. ctDNA was determined and analyzed using a digital droplet PCR assay (Biorad) that was confirmed by a second technique (Idylla). ctDNA was categorized according to its response pattern in time: increasing, decreasing and undetectable KRAS mutant allele frequencies over time.

Results: In this ongoing study 31 patients with a known KRAS and 2 patients with a BRAF mutation participated. 24 (72%) had detectable levels of KRAS (n=22) or BRAF (n=2) in plasma. Concordance between Idylla and ddPCR was good and correlation was high (R2 =0,52). Of first 27 patients ctDNA increasing n=11, decreasing n=10, no change n=6. Data on all included patients will be presented.

Conclusion: Early detection of decreasing KRAS/BRAF mutant alleles in ctDNA predicted durable tumor responses, and increasing levels were associated with early disease progression.

Citation Format: T.Jeroen.N. Hiltermann, Lucie B.M. Hijmering-Kappelle, Maria L. Aguirre Azpurua, Anthonie.J. vd Wekken, Birgitta.I. Hiddinga, Wim Timens, Harry.J.M. Groen, Ed Schuuring. ctDNA a promising predictive marker for treatment with PD-1 inhibitors in KRAS mutated NSCLC after platinum based chemotherapy [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 399.
Abstract 4013: CoA Synthase (COASY): A novel predictive marker and functional protein that mediates radiation resistance via PI3K signaling in rectal cancer
Authors: Ferrandon Sylvain , DeVecchio Jennifer , Duares Leonardo , Chouhan Hanuman , Davenport Jacqueline , Karagkounis Georgios , Orloff Matthew , Liska David , Kalady Matthew F.
Published: August 2019

Purpose: Rectal cancer response to neoadjuvant chemoradiation is prognostic for disease-free and overall survival, with better responders enjoying better outcomes. Understanding the biology underlying better responses to therapy could lead to more personalized treatment approaches. The purpose of this study was to evaluate a newly identified biomarker and explore its mechanism of action.

Experimental Design: Pretreatment human rectal cancer samples underwent DNA microarray analysis and profiles were compared to post-chemoradiation histologic regression scores. A candidate gene, COASY (which encodes for Coenzyme A Synthase), was stably knocked down by shRNA in colorectal cell lines, and the effects on response to radiation were tested in a xenograft model. The biological function of COASY was first assessed by Gene Set Enrichment Assay (GSEA). Its effects on cell death, cell survival, and DNA repair capacity in colorectal cell lines was analyzed. Immunoprecipitation of COASY followed by LC-MS/MS analysis was done to identify COASY partners.

Results: COASY gene expression was overexpressed in rectal cancer compared to normal rectum and was significantly correlated to the tumor regression score. Tumors from cell lines knocked down for COASY had a strong delayed tumor growth, less proliferation, and more apoptosis after irradiation than the control cell line using in vivo xenograft models. In addition, a higher cell death rate after irradiation was observed in vitro in two colorectal cancer cell lines knocked

down for COASY. Mechanistically, a physical interaction between COASY and the PI3K regulatory subunit PI3K-P85α was identified; which led to a modulation of AKT and mTOR phosphorylation after irradiation. Furthermore, there were more residual double DNA strand breaks after irradiation in cell lines knocked down for COASY accompanied by a decrease in DNA-PK and MRE11 protein levels.

Conclusion: COASY serves as a predictive biomarker for rectal cancer response to neoadjuvant chemoradiation and its increased levels correlate with poor response. In addition to its role as a predictive biomarker, COASY has biological relevance leading to increased radiation resistance via the PI3K pathway-dependent increase in DNA repair capacity.

Citation Format: Sylvain Ferrandon, Jennifer DeVecchio, Leonardo Duares, Hanuman Chouhan, Jacqueline Davenport, Georgios Karagkounis, Matthew Orloff, David Liska, Matthew F. Kalady. CoA Synthase (COASY): A novel predictive marker and functional protein that mediates radiation resistance via PI3K signaling in rectal 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 4013.
Abstract 4016: PIK3IP1 regulates prostate cancer cell migration and proliferation and is a noninvasive biomarker of the disease
Authors: Jedinak Andrej , Kaplan Katherine , Loughlin Kevin , Moses Marsha
Published: August 2019

Prostate cancer (PCa) is the second most common epithelial cancer in men after lung cancer. PCa shares similar clinical symptoms with benign prostate hyperplasia (BPH) making it very challenging for physicians to make an accurate diagnosis. Prostate specific antigen (PSA) has been used for several decades to facilitate clinical decision making but unfortunately lacks sensitivity and specificity and cannot effectively distinguish between these two prostate diseases. Therefore, there is a need for new biomarkers that can discriminate between these two diseases with high accuracy. Using a global proteomics approach supported by extensive validation, we have found significantly (P<0.002) elevated levels of Phosphoinositide-3-Kinase Interacting Protein1 (PIK3IP1) in the urine of PCa patients with early disease compared to patients with BPH. These data motivated us to examine the potential role of this protein in prostate cancer development and progression. We began by transiently transfecting multiple prostate cancer cell lines including DU145, PC-3 and LNCaP with a PIK3IP1 plasmid DNA and used these cells to conduct proliferation and migration studies in vitro. We found that there was a significant inhibition of PCa cell proliferation (P<0.05) and migration (P<0.007) in all cell lines tested. We validated these finding with DU145 stable clones established by lentiviral transduction with a PIK3IP1 vector. Stable clones overexpressing PIK3IP1 significantly suppressed proliferation and especially migration and invasion of DU145 cells. We next transiently transfected DU145, PC-3 and LNCaP cells with PIK3IP1 plasmid DNA and performed phospho-protein array experiments to identify signaling molecules that might be modulated by PIK3IP1 to mediate these activities. These data revealed altered levels of molecules involved in several pathways including MAPK. We are currently conducting further in vitro and in vivo studies to assess the potential effects of PIK3IP1 on the modulation of prostate cancer progression and metastasis. In summary, we have identified and validated the urinary detection of PIK3IP1 as a novel liquid biopsy biomarker for localized PCa and have demonstrated that it can modulate both the migration and proliferation of several PCa cells including DU145, PC-3 and LNCaP. (Supported by The Ellison Foundation and the Advanced Medical Research Foundation)

Note: This abstract was not presented at the meeting.

Citation Format: Andrej Jedinak, Katherine Kaplan, Kevin Loughlin, Marsha Moses. PIK3IP1 regulates prostate cancer cell migration and proliferation and is a noninvasive biomarker of the disease [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 4016.
Abstract 4017: Clinical-genomic models of node-positive breast cancer: training, testing, and validation
Authors: Huang Tzu-Ting , Lu Tzu-Pin , Lei Lei , Cheng Skye H.
Published: August 2019

Purpose: We developed and validated clinical-genomic models for stratifying node-positive breast cancer patients into low- and high-risk groups.

Experimental design: The four datasets were (1) training group (n = 112); (2) testing group (n = 46); (3) validation group (n = 388); and (4) external validation group (n = 655). Patients who had undergone mastectomy or breast-conserving surgery and mRNA microarray analysis of their primary tumor tissue with a pathological stage of N0-N2 were enrolled. The validation groups included node-positive patients. Using pre-set cutoffs obtained from the training group, the models were tested and validated in the three other independent groups.

Results: Two clinical-genomic models were developed to predict any recurrence (recurrence index [RI]-local recurrence [LR]) and distant recurrence (RI-DR). In the validation dataset, the RI-LR distinguished between low- and high-risk groups according to 10-year LR-free interval (LRFI; 100% vs. 93.3%, P = 0.014), relapse-free interval (RFI; 88.4% vs. 79.0%, P = 0.017), and relapse-free survival (RFS; 82.2% vs. 73.3%, P = 0.029). The RI-DR distinguished the low- from the high-risk group according to 10-year RFI (90.2% vs. 79.3%, P = 0.007) and RFS (81.0% vs. 74.2%, P = 0.031). RI-DR was associated with regional node irradiation in N1-N2 patients (hazard ratio: 3.7, 95% confidence interval 1.2-12.0) by multivariate analysis. The RI-DR- and RI-LR-gene panels performed similarly in the external validation dataset with hazard ratios of 1.27 (0.93-1.74, P= 0.127) and 1.54 (1.11-2.13, P= 0.0094), respectively, in high-risk patients.

Discussion: The RI-DR shows better performance in partitioning N1-N2 patients into low- and high-risk groups than the RI-LR for any recurrence, although the latter is superior for predicting LRR.

Citation Format: Tzu-Ting Huang, Tzu-Pin Lu, Lei Lei, Skye H. Cheng. Clinical-genomic models of node-positive breast cancer: training, testing, and validation [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 4017.