bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3990: Circulating tumor DNA assessed through amplicon-based next-generation sequencing and its clinical application
Authors: Zembutsu Hitoshi , Osumi Hiroki , Shinozaki Eiji , Yamaguchi Kensei , Ozaka Masato , Sasaki Takashi , Muramatsu Marie , Sasahira Naoki
Published: August 2019

Circulating tumor DNA (ctDNA) studies focusing on only one or a few genes to monitor the disease progress or treatment response are unlikely to find its clinical significance. Hence, the development of cell-free DNA (cfDNA) panel covering hundreds of mutation hotspots is important for the establishment of clinically practical ctDNA detection system. We enrolled 101 patients with metastatic colorectal cancer (mCRC) who received chemotherapy. Amplicon-based genomic profiling of 14 genes, which are commonly mutated in CRC, in plasma by next-generation sequencing (NGS) was performed to evaluate the feasibility of this assay, and was compared with their clinical parameters and RAS status in matched tissue samples. Somatic mutations of the 14 genes in plasma cfDNA were detected in 88 patients (87.1%) with mCRC. Mutations in TP53, KRAS and APC genes were detected in 70 (69.3%), 39 (38.6%) and 24 (23.7%) patients, respectively. Mutant allele frequencies in cfDNA were significantly associated with metastasis (liver, P=0.00004, lymph node, P=0.008, number of metastatic organs, P=0.0006), tumor markers (CEA, P=0.000007, CA19-9, P=0.006, LDH, P=0.00001), and tumor diameter (maximum, P=0.00002, sum of diameter, P=0.00009). The overall concordance rate of RAS status between ctDNA and matched tissue was 77.2% (78/101). Furthermore, we enrolled 76 patients with stage lV pancreatic cancer, and their genomic profiling of 14 genes in plasma were performed to evaluate the feasibility of the cfDNA panel. We observed that patients with lower ctDNA level showed significantly longer overall survival (P=0.00000026). Our data confirmed that mutant allele in cfDNA can be sensitively detected by amplicon-based NGS system. These results suggest that ctDNA could be a novel diagnostic biomarker to monitor changes in mutational status and tumor burden in patients with mCRC, and a predictive marker for survival in patients with advanced pancreatic cancer.

Citation Format: Hitoshi Zembutsu, Hiroki Osumi, Eiji Shinozaki, Kensei Yamaguchi, Masato Ozaka, Takashi Sasaki, Marie Muramatsu, Naoki Sasahira. Circulating tumor DNA assessed through amplicon-based next-generation sequencing and its clinical application [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 3990.
Abstract 4011: Notch 1/2/3 mutation as a potential biomarker for immunotherapy in smokers with non-small cell lung cancer via increasing tumor mutational burden
Authors: Hong Xiaohua , Shen Aijun , Xu Yu , Li Chengcheng , Wang Guoqiang , Liu Li , Qi Chuang
Published: August 2019

Background: Smoking status have been discovered to be associated with the response of anti-PD-1/PD-L1 treatment in advanced non-small cell lung cancer (NSCLC), on account of the smoking-induced higher tumor mutational burden (TMB). However, merely partial smokers with NSCLC exhibit high TMB and respond to anti-PD-1/PD-L1 therapy. The underlying signalling pathway that involved in the accumulation of mutational burden and response to immunotherapy in smokers with NSCLC has not been clarified.

Methods: Data from Rizvi study (n = 240, nivolumab, tissue-sample NGS) and OAK study (n = 642, atezolizumab vs. docetaxel, blood-sample NGS) was retrieved and analysed as training cohorts, followed with validation in our independent NSCLC cohort (n = 44, anti-PD-1/PD-L1, blood-sample NGS). In survival analysis, Kaplan-Meier curves were compared by log-rank test, and the hazard ratio (HR) was determined through a multivariable Cox regression model.

Results: In Rizvi cohort, mutation of Notch 1/2/3 was associated with higher TMB in smokers with NSCLC (P < 0.001) not in non-smokers with NSCLC (P = 0.908). Notch 1/2/3mut was associated with prolonged progression-free survival (PFS) compared with Notch 1/2/3wt in smokers with NSCLC (P = 0.015; HR, 0.51, 95% CI, 0.30-0.88), while not in non-smokers with NSCLC (P = 0.73; HR, 0.83, 95% CI, 0.30-2.35). Consistent results were observed in OAK study that Notch 1/2/3mut was associated with higher TMB (P < 0.001) and longer PFS exclusively in current smokers with NSCLC treated

with immunotherapy (P = 0.018; HR, 0.28, 95% CI, 0.10-0.81). No association of Notch 1/2/3 status and PFS was observed in smokers with NSCLC treated with chemotherapy (P = 0.80; HR, 0.92, 95% CI, 0.47-1.78). We further validated these results in our independent cohort. Similar upregulation of TMB was observed in the Notch1/2/3-mutated smokers with NSCLC, and Notch1/2/3mut improved DCR (100% vs. 41.2%, P = 0.065) and significantly prolonged PFS (P = 0.046; adjusted HR, 0.12, 95% CI, 0.01-0.96).

Conclusions: Notch1/2/3 mutation may serve as a novel predictor of response to anti-PD-1/PD-L1 treatment in smokers with NSCLC via upregulating TMB.

Citation Format: Xiaohua Hong, Aijun Shen, Yu Xu, Chengcheng Li, Guoqiang Wang, Li Liu, Chuang Qi. Notch 1/2/3 mutation as a potential biomarker for immunotherapy in smokers with non-small cell lung cancer via increasing tumor mutational burden [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 4011.
Abstract 4021: A seven-lncRNA signature has a good value of prognosis in esophageal squamous cell carcinoma
Authors: Weng Nuoqing
Published: August 2019

Background: Long non-coding RNAs (lncRNAs) were used as prognostic biomarkers in many types of cancer. We aimed to recognize a signature that can forecast the prognosis in patients with esophageal squamous cell carcinoma (ESCC).

Method: Using a 2520 probe microarray, we retrospectively analysed lncRNAs expression profiles in 141 samples of ESCC and 81 paired non-cancer specimens that were served as the training set from SUN YAT-SEN University Cancer Center (Guangzhou, China) to evaluate the association between the signature and clinical outcomes. We randomly selected 40 paired tumor tissues and adjacent normal tissues from the above samples for the quantitative RT-PCR to ensure the credibility of microarray data. Then we conducted the quantitative RT-PCR in another 103 samples of ESCC to identify the signature. We applied Kaplan-Meier method and log-rank tests to assess the prognostic power of signature with overall survival (OS) and disease-free survival (DFS).

Result: Being differently expression between esophageal squamous cell carcinoma tissues and no-cancer specimens, 338 lncRNAs were selected after filtration. A further analysis were conducted that identified a seven-lncRNA signature in the training set. We calculated the risk score according to the signature and classified ESCC patients as high-risk or low-risk group. Patients in high-risk group had shorter overall survival (HR = 3.555, 95%CI 2.195-5.757, p < 0.0001) and disease-free survival (HR 2.537, 95%CI 1.646-3.909, p < 0.0001) when compared with low-risk group in the training set, and we found the same conclusion in the independent set for OS (HR = 2.662, 95%CI 1.588-4.464, p < 0.0001) and DFS (HR 2.389, 95%CI 1.447-3.946, p < 0.0001). The multivariate Cox proportional hazards regression analysis indicated that the signature is an independent factor for predicting survival. Combing the signature and TNM stage was more powerful than TNM stage alone in prognosing outcomes both in the training set (AUC: 0.778 vs 0.681, p = 0.0127) and in the independent set ((AUC: 0.796 vs 0.713, p = 0.0539).

Conclusion: The seven-lncRNA signature might win a place as a valuable prognostic factor in patients survival with ESCC and guide a more individual treatment when cooperating with the TNM stage system.

Citation Format: Nuoqing Weng. A seven-lncRNA signature has a good value of prognosis in esophageal squamous cell carcinoma [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 4021.
Abstract 4025: STEAP4 ISH and IHC diagnostics for Tarloxotinib activation in EGFR/HER2 mutant cancers
Authors: Yu Hui , Silva Shevan , Estrada-Bernal Adriana , Rivard Chris , Hirsch Fred , Abbatista Maria , Smaill Jeff , Doebele Robert C. , Patterson Adam , Vellanki Avanish , Tirunagaru Vijaya G.
Published: August 2019

Background: Tarloxotinib is a clinical-stage prodrug that releases a potent, irreversible EGFR/HER2 inhibitor (Tarloxotinib-E) selectively in severely hypoxic regions of tumors. One-electron reduction leads to loss of the trigger moiety under oxygen-deficient conditions, releasing the neutral, diffusible ‘warhead’ Tarloxotinib-E. CRISPR/cas9-mediated disruption of the metalloreductase STEAP4 has demonstrated its role as the dominant plasma membrane reductase that facilitates one-electron reduction of Tarloxotinib.

Methods: Cancer cell lines, PDX and resected tumor tissues processed in Formalin Fixed Paraffin Embedded (FFPE) blocks were evaluated. RNAScope technology was used to develop a method to assess STEAP4 mRNA expression by in situ hybridization using specific probes for human STEAP4. mRNA ISH technology for STEAP4 was optimized on a Leica Bond RX autostainer for detection of STEAP4 in FFPE specimens. STEAP4 antibodies were evaluated for Immunohistochemistry (IHC) analysis. STEAP4 IHC was employed as cross-reference, using an anti-STEAP4 antibody validated against STEAP4 knockout and over-expressing cell line and tumor models. Staining data was compared to quantitative PCR results for mRNA ISH and Western blot analysis for IHC. STEAP4 mRNA expression was assessed in FFPE sections of various EGFR/HER2 mutant tumor xenografts and EGFR/HER2 mutant patient tumor specimens.

Results: The STEAP4 mRNA ISH assay was optimized and validated using FFPE sections from cell lines and STEAP4 overexpressing tumor blocks and compared to wild type tumors with minimal expression. High level of STEAP4 mRNA was observed in STEAP4 overexpressing tumors. STEAP4 mRNA ISH analysis revealed variable levels of expression in EGFR/HER2 mutant xenograft tumors. STEAP4 mRNA and protein expression level correlated with Tarloxotinib conversion to Tarloxotinib-E. STEAP4 mRNA expression in EGFR/HER2 mutant patient tumor specimens will be presented.

Conclusion: STEAP4 reduces Tarloxotinib and leads to the conversion to the active drug, Tarloxotinib-E, in hypoxic tumors. Assessment of STEAP4 expression in individual patients is important to guide patient stratification in Tarloxotinib clinical trials and represents an attractive biomarker to enroll tarloxotinib-responsive patients in clinical trials.

Citation Format: Hui Yu, Shevan Silva, Adriana Estrada-Bernal, Chris Rivard, Fred Hirsch, Maria Abbatista, Jeff Smaill, Robert C. Doebele, Adam Patterson, Avanish Vellanki, Vijaya G. Tirunagaru. STEAP4 ISH and IHC diagnostics for Tarloxotinib activation in EGFR/HER2 mutant cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4025.
Abstract 4036: The efficacy of immune checkpoint inhibitors in patients with EGFR mutated non small cell lung cancer in retrospective analysis
Authors: Yamada Tadaaki , Hirai Soichi , Yoshimura Akihiro , Watanabe Satoshi , Uchino Junji , Takayama Koichi
Published: August 2019

Background The treatment with EGFR-TKIs leads to initial response in most patients with EGFR mutated non-small cell lung cancer (NSCLC). In contrast, little is known the subpopulation of NSCLC patients with EGFR mutations who had clinical outcomes to treat for immune checkpoint inhibitors (ICIs). Therefore, to seek for the eligible cases to treat with ICIs, we retrospectively analyzed the correlation between clinical feature and the efficacy of ICIs in patients with EGFR mutations.

Methods/Materials and methods We analyzed a retrospective study of patients with advanced NSCLC harboring with EGFR mutations who were treated with ICIs after the resistance to EGFR-TKIs between February 2016 and April 2018 at six institutions in Japan. The association between clinical outcome and the efficacy or ICIs was investigated.

Results We enrolled 27 patients who have EGFR activating mutation. The objective response and disease control rates were higher in patients with uncommon EGFR mutations than in those with common EGFR mutations (71% versus 35.7%, and 57% versus 7%, p= 0.14 and p < 0.01, respectively). Patients with uncommon EGFR mutations or without T790M mutations was associated with a significantly longer median progression-free survival than those with common EGFR mutations or with T790M mutations (p = 0.003, 0.03, respectively).

Conclusions Patients with uncommon EGFR mutations and without T790M mutations are associated with best outcomes for the treatment with immunotherapy among EGFR mutated NSCLC in the retrospective analysis. Further research is needed to validate for the clinical biomarkers involved in ICI responders with EGFR mutation.

Citation Format: Tadaaki Yamada, Soichi Hirai, Akihiro Yoshimura, Satoshi Watanabe, Junji Uchino, Koichi Takayama. The efficacy of immune checkpoint inhibitors in patients with EGFR mutated non small cell lung cancer in retrospective analysis [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 4036.
Abstract 4049: Cellular phenotypes of spleen cells in cancer patients targeted for adoptive cellular therapy
Authors: Cole Kathryn , Ly Quan , Cox Jesse L. , Hollingsworth Michael A. , Padussis James C. , Foster Jason M. , Vargas Luciano M. , Talmadge James E.
Published: August 2019

The spleen is a rich resource of large numbers of lymphocytes, with potential for use in adoptive cellular therapy (ACT). This is of particular relevance to patients with a distal pancreatic ductal adenocarcinoma (PDAC) who frequently undergo a pancreatectomy that required a splenectomy to clear the nodal basin. In this study we compared the phenotypes of eight paired sets of peripheral blood (PB) and spleen cell populations to assess the frequency of myeloid derived suppressor cells (MDSC) and lymphocyte subpopulations. Because a positive clinical response to ACT requires a low frequency of T-regs and MDSC’s, both systemically and in the tumor microenvironment these cells were one of the foci for our studies. A high frequency of these cells negatively correlate with survival and positively with tumor burden. Indeed, in this study we observed a significant decrease in the frequency of granulocytic, monocytic and immature MDSC’s in the spleen vs PB (4.8±1.6 vs. 54.4±6.1; 0.2±0.1 vs. 0.9±0.4; and 0.6±0.1 vs. 2.3±0.8 percent respectively) but not T-regulatory (T-reg) cells (0.8+0.5 vs. 0.1+0.04 percent) supporting the potential superiority of spleen cells relative to PB as a cellular source for ACT. In addition to a significantly higher number of spleen cells as compared to the PB for expansion/infusion, spleens have a higher frequency of T-cells as compared to the PB including CD3+ (35+5 vs. 16+4%), CD4+ (19+2 vs. 10+2%) and CD8+ (10+1 vs. 5+2%) T-cells. The frequency of transitional memory (Ttm) T-cells, which have a high proliferative potential were also significantly increased in the spleen vs. PB (37.5±6 vs. 26±3.0%) as was the frequency of PD1+CD8+ T-cells (8+2 vs 1+0.5%), which have been shown to have tumor specific cytotoxicity in cancer patients. In summary the increased frequency of PD-1+CD8+ cells, a Ttm memory phenotype and low levels of an exhaustion phenotype (TIM3 and Lag3 expression) in spleen cells vs the PB is supportive of the potential utility of these cells for expansion and therapeutic utility for these cells.

Citation Format: Kathryn Cole, Quan Ly, Jesse L. Cox, Michael A. Hollingsworth, James C. Padussis, Jason M. Foster, Luciano M. Vargas, James E. Talmadge. Cellular phenotypes of spleen cells in cancer patients targeted for adoptive cellular therapy [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 4049.
Abstract 4072: A combinational therapy of oncolytic virus and nature killer cells for melanoma treatment
Authors: Chen Lin , Chen Xianju , Tan Xiankui , Li Meng , Hu Fang , Zhang Haiyan , Wang Xiaoyuan
Published: August 2019

Immunotherapy has obtained substantial impact on the clinical treatment of melanoma which is largely resistant to current therapies as chemotherapy and radiotherapy. Oncolytic virotherapy of melanoma is an appealing approach for inducing not only tumor cell death but also antitumor immunity. Furthermore, NK cells, a lineage of innate lymphocytes, not relying on peptides presented in an HLA class I context, have gained more and more attention as anti-melanoma effector cells. However, the combination of oncolytic adenovirus and NK cells has not yet been explored in melanoma. In this study, the combinational efficacies of oncolytic virus H101 and NK cells was tested in vitro. The combination of H101 and NK cells resulted more efficient killing of tumor cells (HT29, A549 and HepG2) compared to the monotherapies, and displayed no significant cytolytic effect of normal cells (HUVEC and MRC-5). In addition, we present a patient with right maxillary sinus malignant melanoma who was treated with the combination of H101 and adoptive NK cells. We observed intratumoral injections of H101 without dose-limiting toxicity. H101 injections stimulated self-immunity response with transient whole body fever. Furthermore, NK injections caused tumor temperature rise although contralateral cheek temperature was normal and body temperature was normal. Data are consistent with the hypothesis that virus-infected cancer cells attracted NK accumulation within tumor. The homing NK cells attacked targeted cancer cells, causing local temperature rise. Two courses of combination therapy in conjunction mediated the significant durable regression of melanoma, which had been ongoing for >22 months, and it represents a new immunotherapy approach for the treatment of these patients.

Citation Format: Lin Chen, Xianju Chen, Xiankui Tan, Meng Li, Fang Hu, Haiyan Zhang, Xiaoyuan Wang. A combinational therapy of oncolytic virus and nature killer cells for melanoma treatment [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 4072.
Abstract 4085: DDR2 inhibition enhances response to anti-PD-1 immunotherapy
Authors: Tu Megan M. , Lee Francis Y. , Jones Robert T. , Kimball Abigail K. , Saravia Elizabeth , Graziano Robert F. , Coleman Brianne , Menard Krista , Yan Jun , Michaud Erin , Chang Han , Abdel-Hafiz Hany A. , Rozhok Andrii I. , Duex Jason E. , Agarwal Neeraj , Chauca-Diaz Ana , Johnson Linda K. , Ng Terry L. , Cambier John C. , Clambey Eric T. , Costello James C. , Korman Alan J. , Theodorescu Dan
Published: August 2019

Therapies targeting PD-1 are used in multiple cancer types. While a fraction of patients show durable therapeutic responses, most remain unresponsive, highlighting an urgent need to better understand and improve these therapies. Using an in vivo screening approach with a customized shRNA pooled library, we identified DDR2 as a leading target for the enhancement of response to anti-PD-1 immunotherapy. Using isogenic in vivo murine models across five different tumor histologies, bladder, breast, colon, sarcoma and melanoma, we show that DDR2 depletion increases sensitivity to anti-PD-1 treatment compared to monotherapy. Combination treatment of tumor-bearing mice with anti-PD-1 and dasatinib, a tyrosine kinase inhibitor of DDR2, also led to tumor load reduction and in some cases, complete clearance. RNAseq and CyTOF analysis revealed higher CD8+ T cell populations in tumors with DDR2 depletion and those treated with dasatinib when either was combined with anti-PD-1 treatment. Our work provides strong scientific rationale for targeting DDR2 in combination with PD-1 inhibitors.

Citation Format: Megan M. Tu, Francis Y. Lee, Robert T. Jones, Abigail K. Kimball, Elizabeth Saravia, Robert F. Graziano, Brianne Coleman, Krista Menard, Jun Yan, Erin Michaud, Han Chang, Hany A. Abdel-Hafiz, Andrii I. Rozhok, Jason E. Duex, Neeraj Agarwal, Ana Chauca-Diaz, Linda K. Johnson, Terry L. Ng, John C. Cambier, Eric T. Clambey, James C. Costello, Alan J. Korman, Dan Theodorescu. DDR2 inhibition enhances response to anti-PD-1 immunotherapy [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 4085.
Abstract 4093: Synergy of CTL tumor cytotoxicity with myxoma oncolytic virotherapy
Authors: Chen Meixuan , Matos Ana L. , Yuvaraj Padhmavathy , Belmont Laura , McFadden Grant , Anderson Karen S.
Published: August 2019

Background: Checkpoint blockade (CKB) are in clinical trials in breast cancer, but responses may be limited by the low tumor micro-immune profile and somatic mutation burden. Oncolytic viruses may function by activating the immune microenvironment, and improve clinical responses when combined with CKB against cancers like melanoma. In this study, we evaluate the synergy of cytotoxic T lymphocytes (CTLs) combined with oncolytic myxoma virus in an in vitro model of human breast cancer.

Method: BMLF1-CTLs were generated from human HLA-A*02 PBMCs, which were stimulated with antigen presenting cells pulsed with the EBV BMLF1 HLA-A*02 peptide (GLCTLVAML). MCF-7 was engineered to express the full-length BMLF1 gene from EBV. To assess the impact of myxoma virus infection on CTL cytotoxicity, target MCF-7 cells (+/- BMLF1) were co-cultured with BMLF1-CTLs (E:T ratio=5:1) and myxoma virus vMyx-M135KO-GFP (vMyx135KO), at multiplicity of infection (MOI) ranges from 0.1 to 10 ffu/cell, in vitro for 48 hours. Cytotoxicity was measured by flow cytometry with propidium iodide (PI). Cell surface expression of MHC class I and class II were measured by flow cytometry. Statistical comparisons were performed using an unpaired t-test and variation among and between groups was calculated using ANOVA. Statistical significance was defined as p<0.05.

Results: At the highest MOI (10 ffu/cell), cell surface MHC class I expression on MDA-MB-231 was decreased 38-fold (p<0.01), compared to untreated control. MCF-7 was more sensitive to MHC class I downregulation (MOI 1 ffu/cell, 37.3-fold reduction, p<0.002). No significant change of cell surface expression of MHC class II was detected in either breast cancer cell lines at any MOI. In MCF-7-BMLF1- cells, baseline cytotoxicity remained low in both CTL and vMyx135KO (MOI=0.1 ffu/cell) only treatments (14% vs. 10%). With antigen expression (MCF-7-BMLF1+), CTL cytotoxicity increase to 21% (p=0.02). At a MOI of 0.1 ffu/cell, vMyx135KO viral infection further sensitized cells to CTL cytotoxicity (21% vs. 36%, p<0.001)

Conclusions: Oncolytic myxoma virus infection can decrease the MHC class I expression in human breast cancer cell lines, but only at high multiplicities of infection. At low level of virus, myxoma virus infection can enhance CTL cytotoxcity of breast cancer cell line.

Citation Format: Meixuan Chen, Ana L. Matos, Padhmavathy Yuvaraj, Laura Belmont, Grant McFadden, Karen S. Anderson. Synergy of CTL tumor cytotoxicity with myxoma oncolytic virotherapy [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 4093.
Abstract 4104: Expression of the immune checkpoint receptor TIGIT in seminoma
Authors: Lennartz Maximillian , Blessin Niclas C. , Hinsch Andrea , Simon Ronald , Kluth Martina , Fischer Kristine , Hube-Magg Claudia , Li Wenchao , Mandelkow Tim , Debatin Nicolaus F. , Höflmayer Doris , Sauter Guido , Izbicki Jakob R. , Minner Sarah , Büscheck Franziska , Uhlig Ria , Dum David , Krech Till , Luebke Andreas M. , Wittmer Corinna , Jacbosen Frank , Burandt Eike , Steurer Stefan , Wilczak Waldemar
Published: August 2019

A characteristic feature of testicular seminoma is the abundance of immune cells in the tumor microenvironment, raising the possibility that immune checkpoint inhibitors could be a therapeutic option in these tumors. TIGIT (T cell immunoreceptor with Ig and ITIM domains) is an inhibitory immune checkpoint receptor in analogy to PD-1 and drugs targeting TIGIT are currently tested in clinical trials. Little is known about the expression of these proteins in testicular seminomas. Here, we employed immunohistochemistry to determine the relative abundance of TIGIT and PD-1 in relation to the total CD3+immune cell infiltration in a tissue microarray (TMA) constructed from 78 seminoma patients. The fraction of TIGIT+and PD-1+lymphocytes was highly variable in individual cancers and ranged from 2.3% to 69.4% (mean:32.2±14.7%) for TIGIT and from 0.8% to 56.5% (mean:21.6±13.2%) for PD-1. The same high degree of variability was also found for the ratio of PD-1 to TIGITpositive cells that varied from a dominance of TIGIT (PD-1:TIGIT ratio=0.02) in 74% of patients to a predominance of PD-1 (PD-1:TIGIT ratio=12.5) in 23% of patients. In summary, the immune checkpoint receptors TIGIT and PD-1 are abundantly expressed in human seminomas. Once available, anti-TIGIT antibodies, possibly in combination with anti-PD-1 drugs, may be a promising therapeutic option for clinical studies in this cancer type.

Citation Format: Maximillian Lennartz, Niclas C. Blessin, Andrea Hinsch, Ronald Simon, Martina Kluth, Kristine Fischer, Claudia Hube-Magg, Wenchao Li, Tim Mandelkow, Nicolaus F. Debatin, Doris Höflmayer, Guido Sauter, Jakob R. Izbicki, Sarah Minner, Franziska Büscheck, Ria Uhlig, David Dum, Till Krech, Andreas M. Luebke, Corinna Wittmer, Frank Jacbosen, Eike Burandt, Stefan Steurer, Waldemar Wilczak. Expression of the immune checkpoint receptor TIGIT in seminoma [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 4104.
Abstract 4108: Preclinical evaluation of half-life extended Affimer® biotherapeutics targeting the PD-L1 pathway
Authors: Basran Amrik , Jenkins Emma , Adam Estelle , Laurent Floriane , Writer Michele , Oumie Assa , Sivula Jyrki , West Maureen , Stanley Emma , Hillman Jennifer , Robles-munoz Viviana
Published: August 2019

Programmed Cell Death-Ligand 1 (PD-L1) binds to its receptor, Programmed Cell Death protein (PD-1) and is expressed on a range of activated immune cells and is upregulated by tumor cells and by immune cells in the tumor microenvironment. PD-L1 immunotherapies have emerged as effective treatments both as monotherapies and in combinations, providing long term responses in a subset of patients.

Phage display selections have identified a range of potent Affimer biotherapeutic antagonists to both human and mouse programmed death-ligand 1 (PD-L1). We have demonstrated when formatted as bivalent molecules they have improved sub nM affinity, half-life extension in a mouse PK study, blocking activity in vitro and in vivoefficacy in syngeneic or xenograph mouse models.

Affimer biotherapeutics are a 14kDa monomeric scaffold protein based on the human protease inhibitor Stefin A, lacking post-translational modifications such as disulfide bonds and glycosylation. Large diverse phage display libraries have been created by engineering in two nine amino acid peptide loops into the scaffold backbone. We have identified a range of affinity competitive binders to mouse or human PD-L1 confirmed by performing competition ELISA and SPR using Fc formatted antigen. Half-life extension of lead Affimer proteins was achieved by fusing to human IgG1Fc or as an in-line fusion (ILF) fused to serum albumin binding Affimers. We demonstrated avidity for binding to target antigen, binding to cells in vitro and blockade of the PD-1L/PD-1 interaction using Promega cell based assay. The PK properties of the formatted Affimers in mouse showed half-life extension from 40-120 hours depending on the clone and half-life extension technology used.

We have demonstrated that the Affimer scaffold has the necessary properties to be developed as an immunotherapy. The anti-PD-L1 Affimer scaffold proteins were formatted as Fc fusion and in-line fusion formats which showed in vivo efficacy. These molecules will be developed further for both bispecific and drug conjugates to generate new novel biotheraputics that will give greater efficacy in the clinic.

Citation Format: Amrik Basran, Emma Jenkins, Estelle Adam, Floriane Laurent, Michele Writer, Assa Oumie, Jyrki Sivula, Maureen West, Emma Stanley, Jennifer Hillman, Viviana Robles-munoz. Preclinical evaluation of half-life extended Affimer® biotherapeutics targeting the PD-L1 pathway [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 4108.
Abstract 411: Rapid and sensitive detection of EGFR C797S mutations using a blood-based droplet digital PCR assay
Authors: Edwards Victoria , Jackson Leisa , Reese Jordan , Mellert Hestia S. , Pestano Gary
Published: August 2019

The adoption of third-generation tyrosine kinase inhibitors (TKIs) to treat non-small cell lung cancer (NSCLC) for EGFR positive patient cases has led to the emergence of acquired resistance pathways. Approximately 40% of EGFRT790M-positive NSCLC cases display an acquired mutation of amino acid 797 (C797S) that mediates this resistance, which can develop after a median of 10 months following treatment. The effectiveness of treatment for patients with EGFRC797S may depend on whether the mutation is present in cis or trans with EGFRT790M, the type of sensitizing mutation (EGFRdel19 or EGFRL858R), and previous treatment(s). Patients with EGFRdel19 or EGFRL858R in conjunction with EGFRT790M and EGFRC797S remain resistant to all current targeted therapies. Several fourth-generation inhibitors are in development but have yet to be validated in clinical trials. We have developed a blood-based test that can detect two of the most common C797S nucleotide mutations (T>A and G>C) in cis or trans with EGFRT790M using the droplet digital PCR (ddPCR) technology and assays. Analytic sensitivity and specificity were assessed using synthetic DNA designed to mimic the EGFRC797S variants and their locus detected in the assay. Normal healthy donors as well as reference ddPCR positive and negative NSCLC donor samples were assessed for clinical specificity and sensitivity. Finally, the precision of the assay was evaluated with both clinical and analytical samples. Specifically, we evaluated the assay at high, medium, and low mutation frequencies over three consecutive days, including repeat runs on one day, and with multiple operators. This assay is capable of accurately and precisely detecting multiple EGFRC797S variants and their locus from blood specimens in the clinical laboratory. Consistent with other ddPCR blood-based EGFR variant assays we have developed, the limit of detection was 0.02%for C797S variants detected in both cis and trans with the T790M mutation. Once validated, the EGFR C797S assay described here may be of utility in diagnosing patients who have developed resistance to osimertinib.

Citation Format: Victoria Edwards, Leisa Jackson, Jordan Reese, Hestia S. Mellert, Gary Pestano. Rapid and sensitive detection of EGFR C797S mutations using a blood-based droplet digital PCR assay [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 411.
Abstract 4124: Expressions of co-inhibitory / co-stimulatory molecules may impact immune checkpoint therapies
Authors: Daftarian Pirouz M. , George Marybeth , Kleiman Eden , Ouerkaxi Wushouer , Yamamura Amy , Li Zhongliang , Zang Mingfa , Yu Derron , Hwang Eunmi , An Annie X. , Lin Ann E. , Li Henry
Published: August 2019

Screening patient peripheral blood using our proposed non-invasive method can boost the accuracy in selecting proper candidates for immune checkpoint therapy in the clinical setting, thereby leading to higher success in treatment. The immune system is regulated by a sophisticated network of modulatory molecules. In chronic infections and cancers, T cell exhaustion can arise when clearance of antigens is incomplete due to sustained expression of co-inhibitory molecules. T cell exhaustion has been exploited by some cancers, and also described in chronic infections with latency. Such exhausted T cells may be reversed with the right immune checkpoint blockade, thereby restoring effector T cell function. We have optimized a T cell Exhaustion Recall Antigen Assay, demonstrating T cell exhaustion may be reversed by immune checkpoint blockades in certain hosts. We hypothesize that understanding of the capacity and expressions of co-inhibitory molecules assist in predicting responders for specific immune checkpoint therapies. We have used a flow cytometry-based approach to examine changes in an array of immune checkpoint molecules on activated T cells and antigen presenting cells. We first tested the effect of anti-PD1 on tumor antigen-specific activated T cells using splenocytes from PMEL T cell receptor (TCR) transgenic mouse, which expresses mouse homologue of human premelanosome protein, or gp100. Activated CD3+CD8+ gp100-specific T cell population and IFNγ production were measured by flow cytometry. Secondly, we evaluated the expression levels of various immune checkpoint molecules post anti-CD3 stimulation of human peripheral blood mononuclear cells (PBMC) from characterized healthy hosts. Surprisingly, we found high expressions of co-inhibitory and co-stimulatory molecules including CTLA-4, PD1, PDL-1/PDL2, TIGIT, LAG3, TIM3, GITR, are associated with poor response to immune checkpoint blockades. Levels of activated T cells and cytokine production in donors with high checkpoint receptors showed lower T cell activations and cytokine production. Screening patient peripheral blood using our proposed non-invasive method can boost the accuracy in selecting proper candidates for immune checkpoint therapy in the clinical setting, thereby leading to higher success in treatment.

Citation Format: Pirouz M. Daftarian, Marybeth George, Eden Kleiman, Wushouer Ouerkaxi, Amy Yamamura, Zhongliang Li, Mingfa Zang, Derron Yu, Eunmi Hwang, Annie X. An, Ann E. Lin, Henry Li. Expressions of co-inhibitory / co-stimulatory molecules may impact immune checkpoint therapies [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 4124.
Abstract 4125: Precise and reproducible measurement of tumor mutational burden using panel sequencing: the role of panel size and of mutation types
Authors: Budczies Jan , Rempel Eugen , Allgäuer Michael , Kazdal Daniel , Endris Volker , Schirmacher Peter , Stenzinger Albrecht
Published: August 2019

Tumor mutational burden (TMB) is an emerging biomarker for patient stratification for immune checkpoint inhibition. Implementing TMB measurement by panel sequencing, experimental setup and bioinformatics pipelines need to be optimized to minimize sources of error and variance. To gain insight into contributors to TMB variance, combinatorial calculations were performed based on the hypothesis that each base in the coding sequence was mutated with the same probability. Simulations of sequencing panels in the TCGA data were performed to check for additional contributors to the variance. A narrow definition of TMB including only missense mutations was compared to a broader definition additionally including other mutation types. The quantity under investigation was TMB measured in relatively to the size of the panel (per Mbp). The combinatorial contribution to the coefficient of variation (CV) of TMB turned out to be inversely proportional to the square root of the panel size and inversely proportional to the square root of the TMB. For a tumor with TMB of 10 muts/Mbp, the combinatorial CV increased drastically for panels sizes smaller than 1.5 Mbp. The combinatorial variance represents a persistent contribution to the error of TMB calculation that cannot be avoided. Simulations in WES data confirmed the combinatorial variance as main contributor to the imprecision of TMB estimation. In many instances, the fold change between the number of all mutations in the coding sequence and the number of missense mutations was similar for tumors of the same cancer type. However, the fold changes were different in specific cancer types for example significantly higher in melanoma (1.66) compared lung adenocarcinoma (1.48). In particular, the former tumors were observed to include a higher proportion of synonymous mutations than the latter. The combinatorial source of error implies that a panel of least 1.5 Mbp is needed to precisely determine TMB for tumors ranging around typical currently used diagnostic thresholds (10 muts/Mbp). Additional sources of error might even favor larger panel sizes such as 3 Mbp. The combinatorial error can be reduced by including all mutations types instead of only missense mutations and simultaneously adjusting TMB with a cancer type specific factor.

Citation Format: Jan Budczies, Eugen Rempel, Michael Allgäuer, Daniel Kazdal, Volker Endris, Peter Schirmacher, Albrecht Stenzinger. Precise and reproducible measurement of tumor mutational burden using panel sequencing: the role of panel size and of mutation types [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 4125.
Abstract 4133: The in vitro melanoma tumor microenvironment conditions macrophages to an immunosuppressive M2-like phenotype, which is reversible by oncolytic virus ORCA-010 with immune modulators
Authors: Milenova Ioanna E. , Ven Rieneke van de , Dong Wenliang , Beusechem Victor W. van , Gruijl Tanja D. de
Published: August 2019

The melanoma tumor environment suppresses the immune reaction, favoring tumor escape from the immune system. This suppressive environment is in part sustained by tumor associated M2-like macrophages. In order to activate the immune system, one approach is macrophage-targeted therapies, that repolarize M2-like macrophages to M1-like macrophages. ORCA-010 is a potency enhanced, replication selective oncolytic adenovirus, with strong anti-tumor activity.

As a model of macrophage polarization in the tumor microenvironment, human melanoma cell lines were co-cultured in vitro with CD14+ monocytes. The effects to induce M1-like macrophages by addition of lipopolysaccharide (LPS) and interferon (IFN)-gamma, JAK2 or p38 MAPK inhibitors combined with ORCA-010, was assessed in the co-cultures. Cell phenotypes were characterized by flow cytometry.

Our data demonstrate that ORCA-010 significantly reduces M2-like macrophage marker CD163 on CD45+ cells in co-cultures, and increases co-stimulatory M1-like macrophage markers CD80 and CD86 expression on macrophages. CD80 is significantly up-regulated by ORCA-010 with LPS and IFN-gamma. Next, the macrophage polarizing effects of combining ORCA-010 with p38 MAPK and JAK2 inhibitors, known for their favorable myeloid M1 and dendritic cell skewing properties, were assessed in the melanoma co-cultures. The inhibitors JAK2 (AG490) and p38 MAPK (LY2228820) both significantly reduced CD163 levels, however did not induce increased CD80 and CD86 by themselves. Only when combining these with ORCA-010 did we observe a CD86 up-regulation.

In a B16.OVA immunocompetent mouse model, ORCA-010 appeared to enhance recruitment of CD8+ T cells to the tumor. Interestingly, combining ORCA-010 with anti-PD-1 resulted in a significant increase of recruitment of CD8+ T cells at the tumor, which could be further enhanced by co-treatment with a p38 inhibitor.

Our data suggest that ORCA-010 induces a repolarization of the M2-like macrophages to a favorable M1-like phenotype. This can be further enhanced by the combined administration of ORCA-010 with a TLR4 agonist and IFNγ, or with a JAK2 inhibitor.

This work is supported by the European Union Horizon2020 ITN-EID grant 643130 “VIRION”.

Citation Format: Ioanna E. Milenova, Rieneke van de Ven, Wenliang Dong, Victor W. van Beusechem, Tanja D. de Gruijl. The in vitro melanoma tumor microenvironment conditions macrophages to an immunosuppressive M2-like phenotype, which is reversible by oncolytic virus ORCA-010 with immune modulators [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 4133.
Abstract 4141: The induction of iNKT cells and CIK cells toward anti-tumor phenotypes
Authors: Wongkajornsilp Adisak , Htwe Khin Su Su , Sawatpiboon Nathawadee , Duangsa-ard Sunisa , Kasetsinsombat Kanda
Published: August 2019

Natural killer T (NKT) cells and cytokine-induced killer (CIK) cells contain features of both T cells and NK cells that occasionally brought confusion to several studies. These cells have been reported to possess either anti-tumor or immunosuppressive properties. NKT cells could be directly isolated from the peripheral blood, while CIK cells require ex vivo culture of peripheral blood lymphocytes for 2-3 weeks with IFN-γ, IL2 and anti-CD3. NKT type I cells carry invariant TCRαβ recognizing α-Galcer loaded CD1d on antigen-presenting cells. We investigated whether NKT type I cells, as well as CIK cells, could be induced toward Th1 phenotype that should elicit anti-tumor activity. The inducers included ODN, checkpoint inhibitors and CDKi. We observed the tipping toward Th1 phenotype in both cell types, especially on CIK cells. It is concluded that both NKT type I cells and CIK cells could be induced toward the anti-tumor Th1 phenotype.

Citation Format: Adisak Wongkajornsilp, Khin Su Su Htwe, Nathawadee Sawatpiboon, Sunisa Duangsa-ard, Kanda Kasetsinsombat. The induction of iNKT cells and CIK cells toward anti-tumor phenotypes [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 4141.
Abstract 4151: Siglec-15 is a novel immunomodulatory effector of adaptive immune evasion in acute lymphoblastic leukemia
Authors: Pillsbury Claire , Fonseca Jairo A. , Dougan Jodi , Gardner Lori , Rabe Jennifer , Gearheart Christy , Porter Chris C.
Published: August 2019

Despite advances in therapy for acute lymphoblastic leukemia (ALL), it remains a leading cause of illness-related death in children and has poor long term disease-free survival and prognosis in recurrence in adults. Recent developments in immune-based therapies, such as CAR T cells, have shown great promise, but immune evasion mechanisms of leukemia remain poorly understood, and alternative strategies to engage the immune system are needed.

Our group has characterized a role for calcineurin (CN) as a driver of a potent immune escape mechanism in an aggressive murine model of BCR-ABL+ ALL. We performed RNA-seq analysis of control and CN-deficient ALL cells to identify downstream mediators of this effect. Siglec-15 emerged as one of the most differentially expressed genes, being preferentially downregulated in the more immunogenic CN-deficient cells. The role of this Siglec-family immunomodulator is yet unstudied in the context of leukemia and could represent a novel mechanism for immune evasion.

We first validated lower expression of Siglec-15 in CN-deficient leukemia cells by RT-qPCR. We next mined publicly available databases, which revealed higher expression of Siglec-15 in primary B-ALL and AML leukemias as compared to normal PBMCs, as well as in post-chemotherapy MRD samples as compared to at-diagnosis samples. Western blot analysis of human leukemia cell lines demonstrated upregulation of Siglec-15 expression in B-ALL, some AML, as well as DLBCL, as compared to normal PBMCs.

To determine if Siglec-15 mediates immune evasion in vivo, we knocked down Siglec-15 in the murine model of BCR-ABL+ ALL, which resulted in rapid suppression of the leukemia following engraftment in immunocompetent mice, an effect lost in immunocompromised counterparts. Preliminary studies with cells in which Siglec-15 was knocked-out using CRISPR/Cas9 revealed similar results. Importantly, Siglec-15-deficient cells do not have any changes in proliferation or susceptibility to DNA damage-induced apoptosis.

To address the capacity for therapeutic targeting of Siglec-15, mice with parental leukemia were treated with a monoclonal blocking antibody against Siglec-15, yielding an overall reduction in leukemic burden and prolonged survival. Taken together, these results suggest that Siglec-15 is a novel immunomodulatory effector and has a therapeutically exploitable role in immune evasion of ALL.

Citation Format: Claire Pillsbury, Jairo A. Fonseca, Jodi Dougan, Lori Gardner, Jennifer Rabe, Christy Gearheart, Chris C. Porter. Siglec-15 is a novel immunomodulatory effector of adaptive immune evasion in acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4151.
Abstract 4152: Irreversible electroporation induces immunogenic cell death and mediates durable response in orthotopic PDAC model in combination with anti-PD-1
Authors: Zhao Jun , Wen Xiaofei , Li Tingting , Melancon Marites , Gupta Sanjay , Peng Weiyi , Li Chun
Published: August 2019

Immunotherapy has only limited efficacy against pancreatic ductal adenocarcinoma (PDAC) because of the presence of an immunosuppressive tumor-associated stroma. Here, we showed that combined IRE and anti-PD-1 immune checkpoint blockade significantly suppressed tumor growth and prolonged the lives of immunocompetent mice bearing well-established orthotopic PDAC. Remarkably, more than 35% of mice had a durable response, and were found to be free of residual tumor upon necropsy. All mice that survived for 60 days after IRE and anti-PD1 treatments rejected tumor cell re-challenge. Analyses of splenocytes confirmed that these long-term survivors developed an anti-tumor memory T cell response. Further mechanistic studies unveiled that the efficacy of IRE + anti-PD-1 could be attributed to multiple factors, including rapid release of danger associated molecular patterns, activation of DCs, and alleviation of immunosuppressive tumor microenvironment. Neutralization of CD8+ T cells by anti-CD8 antibody nullified the antitumor effect of combined IRE and anti-PD-1, suggesting that tumor-infiltrating CD8+ T cells played a key role in mediating the anti-tumor efficacy of IRE + anti-PD-1.Given that both IRE and anti-PD-1 antibodies are already used in the clinic, our results support the translation of this combination as a promising approach for treating patients with PDAC.

Citation Format: Jun Zhao, Xiaofei Wen, Tingting Li, Marites Melancon, Sanjay Gupta, Weiyi Peng, Chun Li. Irreversible electroporation induces immunogenic cell death and mediates durable response in orthotopic PDAC model in combination with anti-PD-1 [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 4152.