bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4264: Liquid biopsy and FFPE tissue based somatic BRCA1/2 variant screening in breast and ovarian cancer
Authors: Boga Ibrahim , Sonmezler Ozge , Bisgin Atil
Published: September 2020

Next generation sequencing (NGS) has widened its use since it provides highly precise and accurate genetic testing with reasonable costs in tumor genetic profiling from somatic materials such as FFPE (formalin fixed paraffin embedded) tissue and liquid biopsy [circulating cell free DNA (ccfDNA)]. It has a high value in differentiating treatment strategies especially in BRCA1/2 genomic profiling in breast and ovarian cancer. However, somatic BRCA1/2 testing still remains unclear algorithm versus germ-line testing approved in human genetics for many decades. Thus, we sum up our testing results in relation with clinical diagnosis. FFPE samples from 89 and 74 liquid biopsy samples from breast and/or ovarian cancer patients were collected and sequenced via the GeneReader NGS system for BRCA1 and BRCA2 genes. QCI-Analyze and QCI-Interpret bioinformatics tools were used for bioinformatics analyses. We detected 56 actionable variants in 48 patients (29.5%) out of 163 patients. The 46.4% (nvariant=26) of the mutations were detected in BRCA1 gene while 53.6% (nvariant=30) of them were in BRCA2 gene. The allele frequencies vary from 1.43% to 100% with an average of 43% in 89 FFPE tissue samples with the positivity rates of 30% (nsample=27) while the rate is 28.4% (nsample=21) in 74 liquid biopsy samples. Interestingly, 8 patients had more than one actionable variant and 5 of them had mutations both in BRCA1 and BRCA2 genes. To sum up, genetic testing for targeted treatments has become first-line clinical testing in cancer and taken its place in international guidelines. Thus, somatic testing to profile BRCA genes should be considered in breast and ovarian cancer algorithm since 5% percent of the patients additionally carry somatic actionable mutation with regarding 15% positivity in germ-line BRCA1/2 studies.

Citation Format: Ibrahim Boga, Ozge Sonmezler, Atil Bisgin. Liquid biopsy and FFPE tissue based somatic BRCA1/2 variant screening in breast and ovarian cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4264.
Abstract 4271: Standardized approach for monitoring plasma cell disorders
Authors: Johansson Ulrika , Li Na , Saksena Suraj , Golts Natalie , Gaylord Brent
Published: September 2020

Multi-color flow cytometry using consensus surface markers (CD138, CD38, CD19, CD45, CD27, CD56, CD81, and CD117), intracellular kappa and lambda, as well as other auxiliary markers is routinely being used for the identification and characterization of plasma cells. Here, we describe the development of a modular 10-color plasma cell panel for immunophenotyping of the plasma cell compartment. The panel was developed as an 8-color dried backbone, which can be supplemented with 2 liquid (drop in) reagents.

Lyophilized or dried reagent cocktails in a unit test format are known to drive workflow efficiency in clinical settings by eliminating the need for manual cocktailing (of reagents), and frequent batch to batch verification procedures. Longer shelf life and room temperature storage are additional appealing features for lyophilized and dried products. We have developed a dried-down 8-color plasma cell panel, which is manufactured in a single test-per-tube, ready to use format, and can be supplemented with liquid (drop in) reagents. The flexibility of being able to add 2 liquid reagents to the dried backbone provides the option of using intracellular kappa and lambda to examine clonality or using CD38-multi-epitope and/or anti-p63 (VS38c) reagent(s). The single test-per-tube format is ideally suited for MRD applications, where sample size is limited and there is a need for acquiring a large number of events for data analysis. Also, the dried reagent panel is comprised of multiple high performing polymer dyes, which are brighter than conventional dyes, and afford high resolution of the relevant cell populations. Furthermore, the dried reagent tubes have a long shelf life (12 months) and significantly enhance the workflow efficiency.

Comparison of the liquid and dried versions of the 10-color plasma cell panel using peripheral blood from healthy donors (as well as control cell lines) showed similar staining patterns, comparable population statistics, and fluorochrome brightness. The dried plasma cell tube(s) were also tested on bone marrow samples from healthy and diseased subjects in conjugation with intracellular staining for kappa and lambda. Our results demonstrate that the 10-color plasma cell panel can be used for identification of the plasma cell compartment (based on CD38 & CD138 expression), assessment of aberrant marker expression (CD19, CD27, CD81, CD117, CD45, CD56) for the identification of normal and abnormal plasma cells, and examining clonality in multiple patient samples.

Citation Format: Ulrika Johansson, Na Li, Suraj Saksena, Natalie Golts, Brent Gaylord. Standardized approach for monitoring plasma cell disorders [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4271.
Abstract 4276: Target enrichment to enable RNA-sequencing on FFPE samples: Application to a patient selection biomarkers in colorectal cancer
Authors: Rogers-Grazado Maximilian , Theilhaber Joachim , Pollard Jack , Protopopov Alexei
Published: September 2020

In proof of concept clinical studies, selecting for patients that are the most likely to respond to a given therapy will increase the chance of demonstrating clinical benefit for a new investigational agent.

Archived Formalin-Fixed, Paraffin-Embedded (FFPE) tissue taken at the time of first biopsy and cancer diagnosis are a ready source of material for such patient selection assays; however if the selection assay is RNA-based sequencing based assay, FFPE tumor tissues are typically refractory to standard methods. Here we describe Target Enrichment, an RNA-sequencing technique that enabled analysis of FFPE tumor tissue. The Target Enrichment technique provides the necessary, high quality gene expression data required for identifying the mesenchymal subtype of CRC. In this experiment, RNA was extracted from a set of 192 histologically characterized, archived colorectal cancer (CRC) FFPE tumor tissues with sufficient representation of all Consensus Molecular Subtypes of CRC.

A major issue with FFPE tissue is that nucleic acids are not well preserved in the fixation process. The RNA extraction method was carefully optimized in advance toward improving yield and integrity of the total RNA. In order to accommodate the low FFPE RNA integrity, we used Target Enrichment, a technique derived from whole-exome sequencing that isolates and amplifies library fragments corresponding to only the transcribed regions of interest. After testing several protocols, we established a Target Enrichment technique to remove low-quality library fragments that would otherwise interfere and impair sequencing quality.

This method allowed us to generate high-quality sequencing data for samples that proved challenging using traditional RNA-seq methods. Fresh-frozen tissue samples were used as a benchmark and our Target Enrichment technique showed comparable performance across multiple sequencing quality metrics including mapping rate, expression profiling efficiency, number of genes detected, also the pass rate. Furthermore, data generated using our Target Enrichment technique aligned with the known frequencies of the Consensus Molecular Subtypes of CRC.

Overall, our Target Enrichment technique provides a protocol for generating high quality RNA-seq libraries from highly degraded FFPE RNA.

Citation Format: Maximilian Rogers-Grazado, Joachim Theilhaber, Jack Pollard, Alexei Protopopov. Target enrichment to enable RNA-sequencing on FFPE samples: Application to a patient selection biomarkers in colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4276.
Abstract 43: Fully integrated single instrument imaging-based digital PCR platform
Authors: Lin Robert , Zayac Andrew , Gallagher Steve , Jiang Lingxia , Linn Felicia , Hung Paul
Published: September 2020

Digital PCR (dPCR) has gained popularity in recent years for cancer research such as rare mutation detection, minimal residual disease, treatment selection, and recurrence monitoring. However, currently available technologies suffer from several limitations such as tedious workflow, long time-to-result, poor multiplexity, and inconsistent reagent digitization that severely hindered its broad adoption. We have designed and manufactured a dPCR platform that is capable of consistent sample digitization, thermal cycling and simultaneous interrogation of 20,000 partitions with walk-away workflow. The instrument integrates a loading and digitization manifold, a thermal cycler and an imaging engine with 5 optical channels into a single benchtop box. The Microfluidic Array Partitioning (MAP) consumable is a micro-injection molded, automation-ready plate that contains 16 individual units that can be loaded simultaneously. Each unit partitions an individual sample into 20,000 fluidically isolated partitions while utilizing >90% of inputted sample. We utilized an oncology standard assay, confocal microscopy, and real-time imaging to validate the dynamic range, sample utilization, and digitization consistency of the platform, respectively. Using the European Breast Cancer Standards ERM-AD623 we quantified replicates that were serially diluted from 1 × 105cp/μL down to 10cp/μL. We demonstrated that the dPCR platform can consistently quantify targets over 5-orders-of-magnitude of concentration with high consistency in both quantification and partition numbers. With confocal microscopy, the reagent inside the partitions are visualized to characterize sample utilization. Using conventional qPCR calibration dye ROX and real-time imaging, we show that over 99% of the partitions can be consistently analyzed for high quantification confidence. With the combination of ease-of-use, fast time-to-result and consistent performance, we believe this novel dPCR platform provides unparalleled usability and capability and will allow dPCR to fulfill its promise to impact cancer patient care.

Citation Format: Robert Lin, Andrew Zayac, Steve Gallagher, Lingxia Jiang, Felicia Linn, Paul Hung. Fully integrated single instrument imaging-based digital PCR platform [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 43.
Abstract 4306: Retrospective analysis of docetaxel in combination with ramucirumab for previously treated non-small cell lung cancer patients
Authors: Kitadai Rui , Yoshimura Akihiro , Yamada Tadaaki , Okuma Yusuke , Takeda Takayuki , Kanematsu Takanori , Uchino Junji , Nishioka Yasuhiko , Takayama Koichi
Published: September 2020

BACKGROUND: Current clinical trials demonstrated that combination regimens comprising chemotherapy and immunotherapy lead to better patient outcomes compared to chemotherapy alone as the first line of treatment for non-small cell lung cancer (NSCLC). In addition, the combination therapy of docetaxel (Doc) and ramucirumab (Ram) was considered one of the standard treatments for advanced or relapsed NSCLC patients. However, little is known about the therapeutic responders of this combination therapy among previously treated NSCLC patients. In the present study, we aimed to identify predictive factors for therapeutic response, including programmed death-ligand 1 (PD-L1) expression in tumors, for Doc treatment in combination with Ram.

METHODS: We retrospectively analyzed a total of 135 advanced or relapsed NSCLC patients who were refractory to platinum-based chemotherapy at eleven institutions in Japan between July 2016 and November 2018.

RESULTS: Our observations showed that PD-L1 expression in tumors is not associated with the efficacy of combined therapy of Doc and Ram in previously treated NSCLC patients. Analysis of the patient clinical profiles indicated that prior treatment with immune checkpoint inhibitors (ICIs) is a reliable predictor for the good progression-free survival (PFS) to this combination therapy (P=0.041).

CONCLUSIONS: Our retrospective study indicated that combination regimens comprising chemotherapy and ICIs followed by Doc and Ram could be an optimal therapeutic option for NSCLC patients regardless of the PD-L1 status of tumors. Further investigations are required to strengthen clinical evidence demonstrating the effectiveness of the combination therapy of Doc plus Ram in previously treated NSCLC patients.

Citation Format: Rui Kitadai, Akihiro Yoshimura, Tadaaki Yamada, Yusuke Okuma, Takayuki Takeda, Takanori Kanematsu, Junji Uchino, Yasuhiko Nishioka, Koichi Takayama. Retrospective analysis of docetaxel in combination with ramucirumab for previously treated non-small cell lung cancer patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4306.
Abstract 4311: PPIB expression associates with tumor progression and unfavorable disease-free survival of non-small cell lung cancer
Authors: Kim Jeongwon , Chung Joon-Yong , Hewitt Stephen M. , Hwang Ilseon , Song Joon Seon , Choi Chang-Min , Cho Eunho , Song Kwon-Ho , Kim Tae Woo , Ylaya Kris
Published: September 2020

Background: PPIB (Peptidylprolyl isomerase B) is involved in cellular transcriptional regulation, immune response, chemotaxis, and proliferation. Recent studies have shown that PPIB is linked to tumor progression and chemoresistance. The role of PPIB in non-small cell lung cancer (NSCLC) has not been explored. We aimed to investigate the significance of PPIB in NSCLC and its mechanism of action.

Methods: The role of PPIB was assessed in 5 human NSCLC cell lines. PPIB expression was examined using RNA in situ hybridization (RNA-ISH) on tissue microarrays consisting of 544 NSCLC cases and Ki-67 expression was evaluated by immunohistochemical assays (IHC). RNA-ISH and IHC scores were evaluated by digital image analysis and used to compare patients' prognostic outcomes.

Results: PPIB overexpression promoted proliferation, colony formation and migration of NSCLC tumor cells. We also confirmed the oncogenic properties of PPIB expression in PPIB transfected human bronchogenic epithelial cells. In NSCLC tumor samples, high PPIB expression correlated with lymph node metastasis and recurrence (p = 0.018 and 0.026, respectively), while Ki-67 overexpression correlated with advanced tumor stage (p < 0.001). Patients with high PPIB expression assessed by RNA-ISH (PPIBhigh), exhibited significantly shorter disease-free survival (DFS) time than those who were PPIBlow [HR (95% CI) = 1.53 (1.11-2.10), p = 0.008]. Multivariate analysis revealed that combined Ki-67high/PPIBhigh expression was an independent predictor of shorter DFS [HR (95% CI) = 1.71 (1.26-2.31), p < 0.001].

Conclusions: These findings suggest that PPIB plays a role in NSCLC tumor growth and is associated with unfavorable disease-free survival. PPIB may have value as a prognostic marker and a potential target molecule for NSCLC treatment.

Citation Format: Jeongwon Kim, Joon-Yong Chung, Stephen M. Hewitt, Ilseon Hwang, Joon Seon Song, Chang-Min Choi, Eunho Cho, Kwon-Ho Song, Tae Woo Kim, Kris Ylaya. PPIB expression associates with tumor progression and unfavorable disease-free survival of non-small cell lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4311.
Abstract 4316: Frequent microsatellite instability in Chinese solid tumor patients
Authors: Wang Huilin , Zhu Jiang , Liang Guanbiao , Wang Yongyong , Guo Jianji , Zheng Min , Fu Qin , Shi Weiwei
Published: September 2020

Background Microsatellite instability (MSI) has important clinical implications related to therapeutic options and prognostic assessment. Few studies have been done on the frequency of MSI in cancers and the correlation between microsatellite status and clinicopathological features in Chinese solid tumor patients.

Methods A total of 10149 Chinese solid tumor patients were collected for genome testing using a targeted next-generation sequencing (NGS) panel (OrigiMed, Shanghai). Microsatellite status and other clinicopathological features were examined. Fisher's exact test was used for statistical analysis.

Results MSI-high (MSI-H) was observed in 188 solid tumors (1.8%). Cancer subtypes with significantly higher MSI frequencies included colorectal adenocarcinoma (8.4%), gastric adenocarcinoma (6.9%) and intrahepatic cholangiocarcinoma (2.7%). No statistical significant difference was found in the proportion of MSI in women and men (1.9 % vs 1.7%, respectively). The proportion MSI tumors was higher in older patients (1.3% at 60-69 years, 2.8% at 70-79 years,P<0.0001) and younger patients (1.4% at 50-59 years, 3.0% at 40-49 years, P<0.0001). Compared with microsatellite stable (MSS) carcinomas, MSI was significantly correlated with the oldest and youngest age ranges that were tested, tumors found in the stomach and intestine, and in colorectal adenocarcinoma and gastric adenocarcinoma subtypes. In addition, 123 of the 188 samples (65.4%) harbored at least one MMR gene mutations predicted as loss of functions, including nonsense mutations or truncations in MSH2, MSH6 or MLH1. 2.3% (217/9354) MMR gene mutation was detected in MSS samples (p<0.0001).

Conclusions This study revealed MSI-H in approximately 1.8% of Chinese solid tumor patients and a significant association between MSI-H and MMR gene mutations. These results suggest that there are specific tumor types and clinicopathological features associated with a higher frequency of MSI, indicating a bias during tumorigenesis. Also, these results may potentially contribute to more effective treatment options and a better prognostic assessment for solid tumor patients.

Citation Format: Huilin Wang, Jiang Zhu, Guanbiao Liang, Yongyong Wang, Jianji Guo, Min Zheng, Qin Fu, Weiwei Shi. Frequent microsatellite instability in Chinese solid tumor patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4316.
Abstract 4346: Training Minority Community Leaders on Prostate Cancer. Effect of an Educational Strategy in Los Angeles, CA & Tallahassee, FL
Authors: Aristizabal Carolina
Published: September 2020

Background: Certain groups are disproportionately impacted by prostate cancer (PCa) compared to other racial/ethnic groups such as Blacks. While the burden of PCa in Hispanics is low, there is limited understanding of the disparities in subpopulations of Hispanics. We developed and culturally adapted, and updated all the scientific data, translated, implemented and evaluated a Prostate Cancer Advocacy Training (PCAT) for Hispanic populations.

Methods: Culturally and language specific content on PCa cause, risk factors, epidemiology, detection, diagnosis and treatment was delivered through a workshop and simultaneously broadcasted in Spanish in Los Angeles County (n=29) and in English in Tallahassee, Florida (n=9). Pre- and post-test surveys assessed impact.

Results: The following pre vs. post differences were statistically significant (p < 0.05), in knowledge (5.0±1.6 vs 6.3±1.1) and advocacy intentions (3.9±0.9 vs 4.3±0.8). On correctly identifying warning signs for PCa (50% vs 87%), intent to inform and educate patients, family, and friends about PCa within the next three months, (69% vs 95%), to ensure that high quality research is sensitive to the priorities of patients (63% vs 84%), to help increase patient recruitment, compliance and retention for clinical trials within the next month (62% vs 84%), intent to engage in PCa patient education within the next three months, (67% vs 92%); and in engaging in PCa community outreach within the next three months, (67% vs 94%) There were no significant differences due to race/ethnicity.

Conclusion: The PCAT led to increased knowledge, awareness, and intention to engage patients and community health workers in advocacy and engagement regarding PCa in the next three months. Results suggest that delivering culturally and language specific educational information increases engagement of Hispanic and African American patient/community advocates. This has implications for reducing cancer health disparities.

Citation Format: Carolina Aristizabal. Training Minority Community Leaders on Prostate Cancer. Effect of an Educational Strategy in Los Angeles, CA & Tallahassee, FL [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4346.
Abstract 4353: The Fibrolamellar Registry: A patient-based medical registry can address medical care
Authors: Newcomb Julie Latone , Lett Siobhan , Migler Rachael D. , Simon Elana P. , Simon Sanford M.
Published: September 2020

Advances in genomics and proteomics have enabled more precise characterizations of tumors with the consequence that many cancers are being segregated into smaller categories. With this larger number of categories, more cancers are being categorized as rare. The downside to such categorizations is that the reduced numbers of patients in each category makes it difficult to gather enough information about each cancer. We are a group of patients and caregivers who have joined together to form a repository for patient-shared data and reports in an IRB-approved, non-profit medical registry for the rare and usually lethal childhood liver cancer fibrolamellar hepatocellular carcinoma (FLC). Since the Fibrolamellar Registry is patient-run and patient-owned, we have the trust of the patient community that the records will not be sold for profit. This has enabled us, in our first 3 years, to gather detailed medical records, scans and tests from over 170 patients. With input from scientists and clinicians who study FLC, we have written 600 questions of specific interest to this disease. Most of our patients have also opted to allow these medical records to be shared with a tissue FLC repository that already has samples from 110 patients. Many of patients with FLC get hyperammonenic encephalopathy. These patients do not respond to lactulose and, unfortunately, often enter a coma and die from the condition. From evaluation of our medical records the results indicate that patients do not have hyperammonenic encephalopathy as a result of liver failure. Instead, it is the result of an alteration of the expression of ornithine transcarbamylase, as a consequence of the expression of oncokinase that drives fibrolamellar. These alterations of ornithine transcarbamylase shift the urea acid cycle, resulting in the buildup of ammonia. This suggests that hyperammonemic encephalopathy should be treated with agents that modulate the urea acid cycle rather than lactulose. These results are an example of the power of a patient-run medical registry.

Citation Format: Julie Latone Newcomb, Siobhan Lett, Rachael D. Migler, Elana P. Simon, Sanford M. Simon. The Fibrolamellar Registry: A patient-based medical registry can address medical care [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4353.
Abstract 4426: Redox regulation of tumor cell heterogeneity in the PyMT mammary tumor model revealed by single cell RNA sequencing
Authors: Ren Zuen , Dharmaratne Malindrie , Kulkarni Ameya , Fard Atefeh Taherian , Mar Jessica , Galbo Phillip M. , Liu Yang , Zheng Deyou , Liang Huizhi , Benard Outhiriaradjou , Suyama Kimita , Prystowsky Michael B. , Norton Larry , Hazan Rachel B.
Published: September 2020

Tumor heterogeneity is known to drive breast cancer progression and metastasis. Current therapies target mostly inter-tumor heterogeneity (different patients) without taking into consideration intratumor heterogeneity (individual tumor). Hypoxia is a hallmark of aggressive tumors that is known to select for intratumor cells that are prone for metastasis. Here we show that knockdown of a ROS scavenging enzyme, Glutathione Peroxidase 2, in the PyMT mammary tumor model results in highly hypoxic and metastatic tumors. Single cell RNA-seq on both GPx2 knockdown and control tumors revealed a high degree of intratumor heterogeneity resulting in several clusters that were either of luminal/epithelial or basal-like/mesenchymal type shared by both tumors. Interestingly, GPx2 loss caused increases in a mesenchymal-like/quiescent and a highly proliferative luminal population, implying that GPx2 loss might drive a cancer stem-like population that is conducive of metastasis. Metascape pathway analysis of differentially expressed genes identified pathways that were significantly overrepresented in GPx2 knockdown tumors involving mitochondrial oxidative phosphorylation and response to hypoxia, consistent with the high levels of reactive oxygen species in these tumors. Finally, differential gene expression analysis on mesenchymal and luminal subpopulations predicts novel candidate gene signatures stimulated by GPx2 loss, that might be used to identify or target metastatic cells in tumors. In sum, our data imply that oxidative stress conveys a metastatic phenotype via effects on tumor cell heterogeneity.

Citation Format: Zuen Ren, Malindrie Dharmaratne, Ameya Kulkarni, Atefeh Taherian Fard, Jessica Mar, Phillip M. Galbo, Yang Liu, Deyou Zheng, Huizhi Liang, Outhiriaradjou Benard, Kimita Suyama, Michael B. Prystowsky, Larry Norton, Rachel B. Hazan. Redox regulation of tumor cell heterogeneity in the PyMT mammary tumor model revealed by single cell RNA sequencing [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4426.
Abstract 4468: Cross-species analysis and immunophenotyping using of a focused panel of immune-responsive genes
Authors: De Velasco Marco A. , Kura Yurie , Sakai Kazuko , Nakagaki Hideki , Nishio Kazuto , Uemura Hirotsugu
Published: September 2020

Immunotherapy has transformed the landscape of cancer therapy, unfortunately, not all patients can reap the benefits and identifying those patients that may benefit remains challenging. It is the consensus that patients with inflamed tumors are more likely to benefit from immunotherapies targeting immune checkpoints, and therapies aiming to stimulate or boost T cell anti-tumor immunity and modulate tumor inflammation are being investigated. Determining which type of immune therapy to administer depends largely on the patients immunophenotype. Various methods aimed at determining a tumor's immune profile are emerging or are available, of these, immune profiling by gene expression analysis is common. Several platforms are available, however, methods to analyze results vary. We previously used a transgenic mouse model of prostate cancer to identify target genes and developed a focused panel of immune-related genes. In addition, we designed a scoring system to profile the tumor's immune phenotype and assess tumor immune responses. Our aim is to extend the applicability of this analysis model to any platform capable of measuring these gene targets. As proof of concept for our analytical approach, we extended our assessment to human cancer by defining cancer specific immune profiles and assessing their clinical relevance. Our focused panel consisted of 115 genes (six reference/normalization genes and 109 immune-related genes) and our scoring system was based on an enrichment algorithm using various immune cell-specific signatures and immune related processes. We evaluated the robustness of this immunophenotyping approach using mRNA data from a cohort consisting of 8477 patients spanning 28 cancer types from The Cancer Genome Atlas (TCGA) Research Network. Our computational pipeline utilized unsupervised and machine learning approaches to identify and evaluate phenotype clusters from gene expression or immune signatures. In this study, Louvain clustering outperformed traditional hierarchical clustering. Using Louvain clustering, we identified distinct clusters from which immunophenotypes could be defined and compared these to various molecular and clinical features. Here, we summarize our findings and show that a minimal gene set effectively identified cancer immunophenotypes. This cross-species analysis corroborates our previous observation and paves the way for further development of our analysis model.

Citation Format: Marco A. De Velasco, Yurie Kura, Kazuko Sakai, Hideki Nakagaki, Kazuto Nishio, Hirotsugu Uemura. Cross-species analysis and immunophenotyping using of a focused panel of immune-responsive genes [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4468.
Abstract 4499: Cancer targets early B-cell precursors to generate metastasis-promoting Bregs by promoting their premature emigration from the bone marrow and expansion in the circulation
Authors: Ragonnaud Emeline , Moritoh Kanako , Bodogai Monica , Garaud Soizic , Chen Chen , Wang Xin , Willard-Gallo Karen , Biragyn Arya
Published: September 2020

We previously reported that breast cancer (BC) generates metastasis-promoting regulatory B cells (CD25+ tumor-evoked Bregs). Because phenotypically these cells resembled bone marrow (BM) Rag+ B-cell precursors, such as large Pre-B cells, we hypothesized whether cancer uses them to generate Bregs. To investigate this idea, we used mouse and human BM aspirates, human PBMCs from BC patients, and mice with different orthotopic cancers, such as highly metastatic 4T1 breast cancer cells and EMT6. We report that breast cancer indeed uses BM B-cell precursors to generate Bregs. However, to do this, cancer expresses thymic stromal lymphopoietin (TSLP). Mechanistically, cancer-produced TSLP downregulates surface expression of CXCR4 and α4β1 (VLA-4) of B-cell precursors, thereby impairing their BM retention and increasing their exit into the circulation. TSLP also enables survival and proliferation of these BM emigrants, while other factors such as 5-lipoxygenase metabolites convert them into Bregs. The loss of TSLP expression in cancer cells alone or TSLPR deficiency in B cells not only reduce pre-B cells in the circulation, but also retard lung metastasis. Overall, our data reveal a previously unknown function of cancer-produced TSLP as inducers of premature B-cell precursor emigration from BM to support Breg generation. The results also suggest that the TSLP-pre-B cell axis can be an attractive therapeutic target.

Citation Format: Emeline Ragonnaud, Kanako Moritoh, Monica Bodogai, Soizic Garaud, Chen Chen, Xin Wang, Karen Willard-Gallo, Arya Biragyn. Cancer targets early B-cell precursors to generate metastasis-promoting Bregs by promoting their premature emigration from the bone marrow and expansion in the circulation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4499.
Abstract 4501: RNA seq analysis reveals altered immune specific gene signature in African American bladder cancer
Authors: reddy Karthik reddy kami , Karanam Balasubramanyam , Coarfa Cristian , Lotan Yair , Bollag Roni J. , Terris Martha K. , Putluri Nagireddy
Published: September 2020

Understanding specific functional gene signature among bladder cancer (BLCA) patients of diverse race/ethnicities remain largely unexplored. Even though the incidence rate of BLCA in African Americans (AA) is nearly half as that of European Americans (EA), but AA have the worst survival. This survival difference can be attributed to unequal access to health care, and social economic status, which in turn delays the treatment in AA patients. Here, we analyzed RNA-seq data from AA and EA patient cohort compare with adjacent benign tissues and identified a set of signature genes which are significantly altered in AA patients. Gene Set Enrichment Analysis (GSEA) reveals that these genes are significantly enriched in immune pathways. Several known cytokines, cell differentiated markers and transcription factors including, growth differentiation marker 2 (GDF2), Carcinoembryonic Antigen Related Cell Adhesion Molecule 1 (CEACAM1), Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA4) were downregulated and fibroblast growth factor 1 (FGF1), Caudal type homeobox 1 (CDX1) were upregulated in AA compare to EA. To interrogate the function of specific genes in tumor, we have used an integrated repository portal for tumor-immune system interactions (TISIDB). Using this disease association analysis, we identified a common relationship of these genes with immune response, cytokine signaling and regulation of lymphocyte activation. Our findings indicate that an altered tumor-immune interplay could be a factor for AA BLCA progression and provide the rationale to examine immune specific checkpoint inhibitors to target BLCA on subset of patients from the AA community.

Key words: Racial disparity, Immune system, Bladder cancer.

Citation Format: Karthik reddy kami reddy, Balasubramanyam Karanam, Cristian Coarfa, Yair Lotan, Roni J. Bollag, Martha K. Terris, Nagireddy Putluri. RNA seq analysis reveals altered immune specific gene signature in African American bladder cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4501.
Abstract 4512: Mouse model for in vivo evaluation of efficacy and potential cytokine release syndrome of chimeric antigen receptor (CAR) T cell therapy
Authors: Jiao Jing , Ye Chunting , Cai Danying , Cheng Mingshan , Keck James
Published: September 2020

Chimeric antigen receptor (CAR) T-cell therapy holds great promise as cancer immunotherapy. While impressive responses have been achieved in patients with hematological malignancies there are challenges for CAR T therapy such as toxicity complications due to cytokine release syndrome (CRS) and limitations due to CAR T cell exhaustion. As increasing number of novel CAR T products are in the discovery and preclinical phase of development, preclinical models to assess efficacy as well as potential toxicity of CAR T in vivo are needed. Here, we describe a new NSG™ mice model to examine CAR T cells for efficacy and potential CRS.

Immunodeficient NSG™ mice are commonly used to evaluate CAR T functions. However, human T cells can cause acute xeno-GVHD in NSG™ limiting the use of the model system. A system to study human T cell function in the absence of xeno-GVHD is required. A new NSG™ derivative NSG-MHC Class I/II knockout (KO) strain has MHC class I and MHC class II deficiency in addition to B, T and NK cell deficiency. NSG-MHC Class I/II KO mice are known to have a delayed onset of GVHD and can provide a useful background to study human immunity in the absence of GVHD [M. A. Brehm et al., FASEB J. 33, 3137–3151 (2019)]. Human B-cell lymphoma Raji tumor cells express high levels of CD19 and CD22 which are targets for CAR T therapy. Using NSG-MHC Class I/II KO mice and luciferase-tagged Raji-Luc tumor model we found that compared to controls CD22 CAR T and CD19 CAR T showed significant tumor growth inhibition by Xenogen imaging analysis. No body weight loss was observed with the CAR T treatments. As high tumor burdens are usually associated with the potential of causing CRS we examined CD19 CAR T cells on low and high tumor burdens and the data will be presented in context of efficacy. We have established a new mouse model using NSG-MHCI/II KO mice to enable evaluation of CAR T efficacy and propensity to induce cytokines simultaneously in vivo. Furthermore PBMC-humanized NSG-MHCI/II KO mice can also be established and used to examine CAR T cells prepared from the same PBMC donor to mimic autologous CAR T cell therapy.

Citation Format: Jing Jiao, Chunting Ye, Danying Cai, Mingshan Cheng, James Keck. Mouse model for in vivo evaluation of efficacy and potential cytokine release syndrome of chimeric antigen receptor (CAR) T cell therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4512.
Abstract 4562: Next generation Oncos oncolytic adenovirus with novel anti-cancer double-transgenes shows synergistic anticancer effect in melanoma mouse model
Authors: Kuryk Lukasz , Moller Anne-Sophie
Published: September 2020

Oncolytic virotherapy is a promising anti-cancer strategy utilizing genetically engineered viruses to preferentially infect, replicate in and lyse cancer cells. To date, Talimogene laherparepvec is the only FDA approved oncolytic virus (OV), however, many other types of OVs are showing promising results in clinical studies. One example is ONCOS-102, an adenovirus serotype 5/3 chimera expressing GM-CSF as a transgene, which recently reported 33% ORR in advanced anti-PD1 refractory melanoma.

Genetically engineered OVs can be armed with different co-stimulatory molecules in order to boost the anti-tumour immune responses. Building on the ONCOS-102 backbone, we have engineered two next generation ONCOS viruses, ONCOS-210 and ONCOS-212, expressing novel double transgenes designed to inhibit tumor growth and metabolism. Here, we describe the characterization and pre-clinical in vitro and in vivo testing of ONCOS-210 and ONCOS-212.

The oncolytic properties of ONCOS-210 and ONCOS-212 were confirmed in 4 melanoma cell lines in vitro, demonstrating robust cell lysis and induction of immunogenic cell death. Anti-cancer effects of the viruses were also assessed in i) immunodeficient xenograft and ii) humanized xenograft mouse models to further understand the anti-cancer and immune stimulatory potency of the constructs. Intra-tumoral ONCOS-210 and ONCOS-212 resulted in robust anti-tumor activity in both immune-deficient and immune-competent mice. The double transgenes were shown to act synergistically in vivo, in line with the design hypotheses and proposed mode of action to induce cell lysis and prevent tumor growth. Moreover, the vectors were able to increase tumor infiltration of various immune subsets including CD4+, CD8+, CD8+ expressing PD1+ or CD8+ expressing Granzyme B T cells and reduction of regulatory T-cells and MDSC in the tumour microenvironment, suggesting an ability to prime immune-suppressive tumors to better respond to immunotherapy.

These encouraging preclinical findings will be further investigated to elucidate the mode of action and perform toxicological studies to bring ONCOS-210 and ONCOS-212 towards clinical testing.

Citation Format: Lukasz Kuryk, Anne-Sophie Moller. Next generation Oncos oncolytic adenovirus with novel anti-cancer double-transgenes shows synergistic anticancer effect in melanoma mouse model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4562.
Abstract 4585: LAMP1 targeting of the large T antigen of Merkel cell polyomavirus elicits potent CD4+ T cell responses and prevents tumor growth
Authors: Rosean Claire Buchta , Sinha Pratima , Koelle David M. , Nghiem Paul , Heiland Teri
Published: September 2020

The majority of Merkel cell carcinomas (MCC), a rare and highly-aggressive type of neuroendocrine skin cancer, are associated with Merkel cell polyomavirus (MCPyV) infection. MCPyV integrates into the host genome, resulting in expression of a truncated form of the viral large T antigen (LT) in infected cells and making LT an attractive target for therapeutic cancer vaccines. While induction of tumor-reactive CD8+ T cells is a major goal of cancer therapy, CD4+ T cells provide essential support to CD8+ T cells by promoting their expression of cytotoxic effector molecules and increasing their migratory capacity. Cytokines secreted by CD4+ T cells, such as IFNγ, can also exert desirable effects on the tumor microenvironment. Therefore, we set out to design a cancer vaccine that promotes potent, antigen-specific CD4+ T cell responses to MCPyV-LT. To activate antigen-specific CD4+ T cells in vivo, we utilized our nucleic acid platform, UNITE (UNiversal Intracellular Targeted Expression), which fuses a tumor-associated antigen with lysosomal-associated membrane protein 1 (LAMP1). This lysosomal targeting technology results in enhanced antigen presentation and a balanced T cell response. LTS220A, encoding a mutated form of MCPyV-LT that abrogates its pro-oncogenic properties, was introduced into the UNITE platform. Intradermal vaccination with LTS220A-UNITE promoted a potent, antigen-specific CD4+ T cell response to MCPyV-LT. Additionally, prophylactic vaccination with LTS220A-UNITE prevented growth of B16F10 tumors expressing LTS220A in 100% of mice. Therefore, we find that DNA vaccination using the UNITE platform enhances CD4+ T cell responses to MCPyV-LT and prevents tumor growth when given prophylactically. Future studies will test the efficacy of LTS220A-UNITE for use as a therapeutic cancer vaccine.

Citation Format: Claire Buchta Rosean, Pratima Sinha, David M. Koelle, Paul Nghiem, Teri Heiland. LAMP1 targeting of the large T antigen of Merkel cell polyomavirus elicits potent CD4+ T cell responses and prevents tumor growth [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4585.
Abstract 4609: TP53, PIK3CA, and BRAF somatic mutations in airway epithelial field of injury associated with lung cancer risk
Authors: Craig Daniel J. , Morrison Thomas , Khuder Sadik , Crawford Erin L. , Wu Leihong , Xu Joshua , Blomquist Thomas M. , Willey James C.
Published: September 2020

Background: Lung cancer is the leading cause of cancer-related death in men and women, and cigarette smoking is the most significant preventable risk factor. Early detection through annual low dose CT screening reduces mortality but has low positive predictive value and specificity, in part due to low lung cancer incidence (<10%) among those who currently meet screening criteria. Thus, there is a need for biomarkers that reliably detect those at highest lung cancer risk, thereby enabling more effectivescreening. The purpose of this study was to test the hypothesis that high risk for lung cancer is characterized by high prevalence of low variant allele frequency (VAF) somatic mutations among known lung cancer driver genes in normal airway epithelial cells (AEC).

Methods: Synthetic DNA internal standards (IS) were prepared for each of 11 lung cancer driver genes and mixed with each AEC genomic (g)DNA specimen prior to competitive multiplex PCR amplicon NGS library preparation. A custom Perl script was developed to separate IS reads and respective specimen gDNA reads from each target into separate files for parallel variant frequency analysis. This approach enabled reliable detection of mutations with VAF as low as 5 x 10-4 (0.05%). This method was then applied in a retrospective case-control study. Specifically, AEC specimens were collected by bronchoscopic brush biopsy from the normal airways of 19 subjects, including eleven lung cancer cases and eight non-cancer controls, and the association of lung cancer risk with AEC driver gene mutations was tested.

Results: TP53 mutations with 0.05-1.0% VAF were more prevalent (p<0.005) and significantly more enriched for tobacco smoke and age-associated mutation signatures in AEC from lung cancer cases compared to non-cancer controls matched for smoking and age. Further, PIK3CA and BRAF mutations in this VAF range were identified in AEC from cases but not controls.

Conclusions: Measurement of very low frequency mutations in the 0.05-1.0% VAF range enabled identification of an AEC somatic mutation field of injury associated with lung cancer risk. A biomarker comprising TP53, PIK3CA, and BRAF somatic mutations may better stratify individuals for optimal lung cancer screening and prevention outcomes.

Citation Format: Daniel J. Craig, Thomas Morrison, Sadik Khuder, Erin L. Crawford, Leihong Wu, Joshua Xu, Thomas M. Blomquist, James C. Willey. TP53, PIK3CA, and BRAF somatic mutations in airway epithelial field of injury associated with lung cancer risk [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4609.
Abstract 464: Allicin, a potent new ornithine decarboxylase inhibitor in neuroblastoma
Authors: Schultz Chad R. , Gruhlke Martin C. , Slusarenko Alan J. , Bachmann Andre S.
Published: September 2020

The purpose of this study was to test if allicin inhibits ornithine decarboxylase (ODC) in pediatric neuroblastoma (NB). The natural product allicin is a reactive sulfur species (RSS) from garlic (Allium sativum L.). NB is an early childhood cancer arising from the developing peripheral nervous system. In up to 25% of cases MYCN gene amplification is correlated to high tumor stage and poor patient prognosis. High stage NB relapses frequently despite multimodal therapy and relapsed tumors are virtually untreatable. Therefore, there is a clear need for specific, novel therapeutics. MYCN transactivates the E-box gene ODC1, and the gene product ODC is a rate-limiting enzyme in polyamine biosynthesis. The increase of polyamines (putrescine, spermidine, spermine) triggers cell hyperproliferation in NB and other MYC-driven cancers through the activation of Rb-regulated cell cycle progression. ODC is a validated drug target and α-difluoromethylornithine (DFMO) is an ODC inhibitor under investigation in phase II NB trials. Although a safe drug with clinical promise, DFMO has various challenges including the need for exceptionally high treatment doses and rapid renal clearance in the urine. In an effort to identify pharmacologically superior ODC inhibitors, we identified allicin as a potent ODC inhibitor using a specific radioactive in vitro activity assay with purified ODC. Allicin also inhibited ODC activity in actively growing NB cells, reduced polyamine levels, and induced apoptosis in cell cultures. ODC is a homodimer with 12 cysteines per monomer and allicin S-thioallylates cysteines. Allicin reacts with, and is titrated-out by, low molecular weight thiols like dithiothreitol (DTT). Removal of DTT from the ODC activity assay reaction resulted in significantly higher allicin potency (IC50 at 11 nM). Although allicin has multiple cellular targets, our data reveal that one mode of action is ODC inhibition, which suppresses polyamine accumulation and induces apoptosis. The natural product allicin could be used in conjunction with other anticancer treatments, the latter at a lower than usual dosage, to achieve drug synergism with good prognosis and less side-effects.

Citation Format: Chad R. Schultz, Martin C. Gruhlke, Alan J. Slusarenko, Andre S. Bachmann. Allicin, a potent new ornithine decarboxylase inhibitor in neuroblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 464.