bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1310: Abrogation of KRas-addicted tumors by GSK3 suppression-mediated upregulation of β-catenin and c-myc
Authors: Kazi Aslamuzzaman , Xiang Shengyan , Yang Hua , Delitto Daniel , Trevino Jose , Jiang Rays H. , Ayaz Muhammad , Lawrence Harshani , Kennedy Perry , Sebti Said M.
Published: August 2019

The significant involvement of mutant KRas in human cancer development underscores the need to develop approaches that disable mutant KRas-driven tumors. Targeting KRas directly is challenging, and such identifying vulnerabilities specific for mutant KRas tumors is an important alternative approach. In this study, we discovered that glycogen synthase kinase 3 (GSK3) is required for the in vitro and in vivo growth and survival of human mutant KRas-dependent tumors but not for mutant KRas-independent tumors. Pharmacological inhibition with the GSK3 inhibitor SB as well as siRNA depletion of GSK3 lead to tumor suppression that is mediated at least in part by inhibition of the phosphorylation of the GSK3 substrates c-Myc on T58 and β-catenin on S33/S37/T41. CRISPR/Cas9 targeted knock out studies demonstrated that c-Myc and β-catenin upregulation mediate the antitumor activity of SB. Importantly, GSK3 blockade inhibits the in vivo growth of G12D, G12V, and G12C mutant KRas primary and metastatic patient-derived xenografts from pancreatic cancer patients who progressed on chemo- and radiation therapies. This discovery warrants advanced pre-clinical and clinical investigations of GSK3 inhibitors in mutant KRas-dependent cancers.

Note: This abstract was not presented at the meeting.

Citation Format: Aslamuzzaman Kazi, Shengyan Xiang, Hua Yang, Daniel Delitto, Jose Trevino, Rays H. Jiang, Muhammad Ayaz, Harshani Lawrence, Perry Kennedy, Said M. Sebti. Abrogation of KRas-addicted tumors by GSK3 suppression-mediated upregulation of β-catenin and c-myc [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 1310.
Abstract 1329: Preclinical evaluation of TAS6417 as a highly effective, pan-mutation-selective EGFR tyrosine kinase inhibitor
Authors: Udagawa Hibiki , Hasako Shinichi , Ohashi Akihiro , Abe Naomi , Haruma Tomonori , Komori Toshiharu , Terasaka Miki , Fujita Ryoto , Funabashi Kaoru , Yasuda Hiroyuki , Miyadera Kazutaka , Goto Koichi , Costa Daniel B. , Kobayashi Susumu S.
Published: August 2019

Objectives: Three generations of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have developed to treat advanced non-small cell lung cancer (NSCLC) with EGFR mutations. However, each generation of TKI has different spectrum of activity against the different mutations. Currently, no EGFR TKIs are approved for NSCLC with EGFR insertion 20 mutation. Previously, we have shown that TAS6417, a novel EGFR TKI, is effective for cells harboring EGFR insertion mutations in vitro and in vivo. In this study, we asked whether TAS6417 is effective for tumors harboring other EGFR mutations observed in clinic.

Methods: Genetically engineered Ba/F3 cells expressing EGFR mutants, lung cancer cell lines including newly established cell lines established from tumors harboring EGFR insertion 20 were used to evaluate efficacy of erlotinib, afatinib, osimertinib, poziotinib, and TAS6417 by cell viability analyses. The selectivity indexes (the wild type EGFR/mutant EGFR ratio) were calculated to predict a therapeutic window for each TKI. Effects of each EGFR TKI on EGFR signaling and apoptosis were evaluated by western blotting. Finally, murine xenograft models were used to evaluate efficacy of TAS6417 and all generation of EGFR TKIs for the treatment of NSCLC with EGFR mutations.

Results: Cell viability assays demonstrate that TAS6417 was as potent as poziotinib against common EGFR mutations (L868R and exon 19 deletions) and the most potent against cells harboring the T790M resistant mutations. In addition, TAS6417 was effective in cells harboring EGFR rare mutations (G719X L861Q, and S768I). Even though IC50s were higher than those of poziotinib in cells harboring EGFR insertion 20, the selectivity indexes (the wild type EGFR/mutant EGFR ratio) were higher in TAS6417, indicating a wider therapeutic window. TAS6417 was effective in mice injected with cells harboring EGFR mutations with no body weight loss.

Conclusions: Our preclinical results confirm that TAS6417 is a potent EGFR TKI with a broader spectrum of activity and a wider therapeutic window and support clinical trials in the near future.

Citation Format: Hibiki Udagawa, Shinichi Hasako, Akihiro Ohashi, Naomi Abe, Tomonori Haruma, Toshiharu Komori, Miki Terasaka, Ryoto Fujita, Kaoru Funabashi, Hiroyuki Yasuda, Kazutaka Miyadera, Koichi Goto, Daniel B. Costa, Susumu S. Kobayashi. Preclinical evaluation of TAS6417 as a highly effective, pan-mutation-selective EGFR tyrosine kinase inhibitor [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 1329.
Abstract 1347: Peripheral blood TCRB chain convergence in chronic viral infection and cancer: Emerging trends from a novel immune repertoire biomarker
Authors: Looney Timothy J.
Published: August 2019

Background T cell receptor (TCR) convergence refers to the phenomenon whereby antigen-driven selection enriches for TCRs having shared antigen specificity but different nucleotide sequences. Previous work has demonstrated the potential utility of TCR convergence as a predictive biomarker for response to checkpoint blockade and dendritic cell based immunotherapy for cancer. The extent to which convergence arises owing to chronic viral infection is not yet established. Here we sought to identify features of chronic cytomegalovirus (CMV) infection using TCRB profiling of peripheral blood (PBL) total RNA.

Methods Total RNA from PBL was obtained from 35 blood donors of known CMV status, then used for TCRB sequencing via the Oncomine TCRB-LR assay (amplicon spanning CDR 1, 2 and 3) and the Ion Torrent S5. In parallel, we prepared libraries via the Oncomine TCRB-SR assay (CDR3 only). Data were used to identify TCRB repertoire features correlated with CMV status and compare repertoire features across the two assays. For context, we compare CMV-related convergence to previous reports detailing convergent T cell responses in individuals with cancer.

Results T cell clone evenness was reduced in CMV positive individuals irrespective of age, predictive of CMV status (AUC=.86, p=2E-4, Wilcoxon), and strongly correlated between LR and SR assays (Spearman cor=.96). TCR convergence was elevated in CMV positive individuals and uncorrelated with evenness (Spearman cor = -.03) such that the combination of convergence and evenness improved the performance of a logistic regression classifier (AUC= .93).

Conclusions We identify reduced T cell evenness and elevated TCR convergence as features of chronic CMV infection. CMV infection appears to significantly alter the T cell repertoire, suggesting that CMV status may be required for proper interpretation of T cell expansion in the context of immunotherapy for cancer. TCR convergence may detect T cell responses to a broad range of viral and tumor neoantigens and thus may serve as a particularly useful biomarker for the identification of immunogenic tumors having few genetic alterations.

Citation Format: Timothy J. Looney. Peripheral blood TCRB chain convergence in chronic viral infection and cancer: Emerging trends from a novel immune repertoire biomarker [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 1347.
Abstract 1360: MMP-degraded vimentin is elevated in serum from patients with non-small cell lung cancer - development and biological validation of a new serum biomarker
Authors: Nissen Neel I. , Karsdal Morten , Willumsen Nicholas
Published: August 2019

Background: Non-small cell lung cancer (NSCLS) accounts for 85% of lung cancers. The five-year survival rate decreases from 47% in stage I to 1% in stage IV, emphasizing the need for early detection. In lung cancer, the intermediate filament protein vimentin has been shown to be applicable for clinical pathology and associate with epithelial-to-mesenchymal transition. Vimentin has been found extracellularly and may be a target for proteolytic cleavage by matrix metalloproteases (MMPs). In fact, a MMP-degraded and citrullinated vimentin fragment (VICM) has shown biomarker potential in serum from patients with NSCLC. The aim of this study was to determine whether (un-citrullinated) MMP-degraded vimentin (VIM) can serve as a biomarker in patients with NSCLC.

Methods: A monoclonal antibody was raised against a MMP-generated vimentin fragment identified by mass spectrometry. A competitive enzyme-linked immunosorbent assay (ELISA) was developed and technically validated. Levels of VIM were measured in serum from patients with NSCLC (n=40) and healthy controls (n=20) and compared to the equivalent citrullinated vimentin fragment (VICM).

Results: The VIM ELISA assay was highly specific to MMP-degraded vimentin and with no cross-reactivity to VICM and the full-length vimentin. VIM was significantly elevated in serum from patients with NSCLC (p=0.0003) compared to controls whereas VICM was not (p=0.0741). In support, the diagnostic accuracy (AUROC) for VIM was 87.1% (p=<0.0001) vs 64% (p=0.0685) for VICM.

Conclusion: The newly developed VIM assay detected significantly elevated levels of MMP-degraded vimentin in serum from patients with NSCLC compared to controls. Furthermore, this non-citrullinated vimentin fragment (VIM) shows stronger accuracy in separating NSCLC patients from healthy controls than the citrullinated equivalent (VICM), suggesting its biomarker potential and biological relevance in NSCLC. Future studies are needed to validate and expand on these findings.

Citation Format: Neel I. Nissen, Morten Karsdal, Nicholas Willumsen. MMP-degraded vimentin is elevated in serum from patients with non-small cell lung cancer - development and biological validation of a new serum biomarker [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 1360.
Abstract 1364: A Non-Small Cell Lung Cancer Reference Material
Authors: Tomson Farol L. , Huang Catherine , Clement Omo , Garlick Russell , Anekella Bharathi
Published: August 2019

Lung cancer is the leading cause of cancer death in the United States as well as globally. The major diagnosis (~85%) of lung cancer patients is non-small cell lung carcinoma (NSCLC). About 10-50% of NSCLCs have several activating EGFR driver mutations which have been clinically validated as therapeutic biomarkers for anti-EGFR drugs. Identifying these driver mutations through liquid biopsies is a promising alternative to traditional biopsies; however, the precision of mutation analysis for some of these targets, e.g., T790M, especially in advanced-stage metastatic NSCLC patients remains a challenge. To address this, we developed a platform of single and low complexity, highly patient-like circulating tumor DNA (ctDNA) EGFR reference standards designed for PCR assays that are used for EGFR-based patient testing. We mixed genomic DNA from the highly characterized human cell line (GM24385) with four synthetic EGFR variant-containing DNA sequences: T790M, L858R, G719S, and an ex19 deletion, in both individual and combined format. Using digital PCR we were able to precisely blend the material at the defined variant allele frequencies of 1% and 0.1%. In addition, wild-type reference material without any synthetic EGFR variants was also created as a comparable negative control. The materials were size-selected (160-170bp) after fragmentation for more patient-like ctDNA size characteristics. After size-selection the material is further refined to improve conversion efficiency. In order to make the material comparable to native ctDNA, such as that isolated from cancer patients, we encapsulated the DNA fragments into a synthetic plasma matrix. The process of DNA size selection followed by encapsulation and formulation into a synthetic plasma matrix enhances the commutability of these reference standards and improves the long-term stability of the samples to at least two years. The allele frequencies of the reference materials were confirmed by both dPCR and NGS. The Archer Reveal ctDNA 28 Kit was used for NGS library preparation while an Illumina MiSeq, using v2 (2x150 bp) PE chemistry reagents, was used for sequencing. Additional evaluation of these materials was conducted by laboratories that provide EGFR RT-PCR tests for patient anti-EGFR therapy stratification. The data from the dPCR, NGS and RT-PCR assays were comparable and highlight the broad compatibility of these EGFR ctDNA reference standards on a variety of assay testing platforms for NSCLC.

Citation Format: Farol L. Tomson, Catherine Huang, Omo Clement, Russell Garlick, Bharathi Anekella. A Non-Small Cell Lung Cancer Reference Material [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 1364.
Abstract 1379: Insights into analytical factors impacting variant concordance of plasma-tumor NGS testing
Authors: Stetson Daniel
Published: August 2019

Next generation sequencing (NGS) of circulating free DNA (cfDNA) is transforming the treatment paradigm in oncology. Sensitive non-invasive cfDNA assays could revolutionize clinical trial design with improved patient safety. However, there are substantial challenges to working with cfDNA, particularly in an early-stage setting. It has been shown that solid tumors from early stage disease shed less DNA into the bloodstream making the detection of somatic variants difficult. Because of this, the current landscape of commercial cfDNA assays are focused on advanced stage patients and may lack the sensitivity and specificity to interrogate early stage samples. To understand the limitations of the currently available cfDNA assays on early-stage oncology samples, we have designed several studies to examine the factors that impact the concordance of plasma-tumor testing and hinder the ability to detect low frequency somatic variants. Three comparison studies have been completed examining both the inter- and intra- variability of commercially available plasma-based NGS assays. Two inter-vendor studies utilized matched sets of 24 tumor-plasma samples purchased from a commercial bio-banking company. The plasmas were aliquoted into 2 ml volumes and sent to the commercial vendor for sequencing with the currently available cfDNA assay, while the tumor-normal tissues were sequenced by an external tumor sequencing vendor. The unique study design allowed for confident determination of true positive, false positive and importantly, false negative variants from the reported plasma variants. The third comparison study also used commercially purchased early-stage patient and normal samples to examine the intra-vendor assay reproducibility. Summarizing the results from all three studies, we found that the majority of discordant variants were from technical rather than biological factors. The technical factors identified include mutational bias, bioinformatic filtering, and variant annotation, while the biological factors found were clonal hematopoiesis and tumor heterogeneity. Through a detailed examination of the discordant variants from the first inter-vendor comparison, we believe that less than 15% of discordance was from biological sources. Overall these findings differ from the current literature which suggests that the sub-clonal nature of a tumor is the cause of most discordance in plasma testing. We have begun to establish guidelines to identify these technical factors in our current and past datasets, with the hope of liquid biopsy testing improvement for early-stage cancer patients.

Citation Format: Daniel Stetson. Insights into analytical factors impacting variant concordance of plasma-tumor NGS testing [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 1379.
Abstract 1380: Clinical relevance of cell-free DNA analysis for bile juice assessed through amplicon-based deep sequencing in bile duct and pancreatic cancer patients with obstructive jaundice
Authors: Muramatsu Marie , Muramatsu Marie , Ito Hiromichi , Zembutsu Hitoshi
Published: August 2019

Personalized treatment for patients with pancreatic or bile duct cancer is limited because cancer cells could not be detected in some patient. Cell-free DNA (cfDNA) sequencing is expected to enable a precision oncology approach for these patients. The development of gene panel for cfDNA covering hundreds of mutation hotspots is important for the establishment of clinically practical cfDNA detection system. We enrolled 40 patients with bile duct or pancreatic cancer which caused obstructive jaundice. Amplicon-based genomic profiling of 14 genes, which are commonly mutated in digestive cancer, in preoperative bile juice by next-generation sequencing (NGS) was carried out to evaluate the feasibility of this assay, and was compared with their clinical parameters. One or more somatic mutation(s) of the 14 genes in bile cfDNA were detected in 25 patients (62.5%) with bile duct or pancreatic cancer. Mutations in TP53, KRAS and APC genes were detected in 19 (47.5%), 11 (27.5%) and 4 (10.0%) patients, respectively. Mutant allele frequencies in plasma were not associated with CEA nor CA19-9. Of the 40 patients with bile duct or pancreatic cancer, the cancer cells could be detected by cytology in 18 patients (45.0%). On the other hand, mutant alleles from cancer cells could be detected in 14 patients who showed negative cytology. Hence, 32 patients (80.0%) could be diagnosed as cancer before surgical operation by using the information from liquid biopsy (cfDNA analysis using bile juice) in addition to that from cytology. Our data confirmed that mutant allele in bile cfDNA could be sensitively detected by amplicon-based NGS system. These results suggest that the detection of cfDNA in bile juice using high-sensitive deep sequencing system could be a novel diagnostic tool, and these technologies could increase the ratio of definitive diagnosis of refractory cancer such as bile duct or pancreatic cancer.

Citation Format: Marie Muramatsu, Marie Muramatsu, Hiromichi Ito, Hitoshi Zembutsu. Clinical relevance of cell-free DNA analysis for bile juice assessed through amplicon-based deep sequencing in bile duct and pancreatic cancer patients with obstructive jaundice [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 1380.
Abstract 1393: Automated adeno/squamous-cell NSCLC classification from diagnostic slide images: A deep-learning framework utilizing cell-density maps
Authors: Jaber Mustafa I. , Beziaeva Liudmila , Szeto Christopher W. , Elshimali John , Rabizadeh Shahrooz , Song Bing
Published: August 2019

Background: The most common form of lung cancer, non-small cell lung cancer (NSCLC), is further categorized into two major histopathological subtypes: ~40% Adenocarcinoma (LUAD), and ~30% squamous cell carcinoma (LUSC). Classifying patients accurately is important for prognosis and therapy decisions, but requires costly pathologist review. Here we present an automated algorithm to differentiate LUAD and LUSC diagnostic whole slide images (WSIs).

Methods: 488 subtyped NSCLC high-resolution diagnostic WSIs were obtained from TCGA sources. Adjacent normal regions were identified and excluded from analysis. Cancer cell-density maps were created based on cell counts within discrete patches. These maps were then binned into ten ranges of cell counts (20-30 cells per patch, 30-40, etc. up to >110 cells per patch). 2D color patches were transformed into 1D descriptive vectors using the inception v3 deep learning framework. Samples were randomly split into 70% training and 30% testing sets. Ten LUAD/LUSC linear SVM classifiers (one for each cell-density bin) were trained on such transformed data. Subtype prediction in unseen testing samples was achieved by averaging subtype predictions from the 10 subsequent models.

Results: 338 TCGA diagnostic WSIs (164 LUAD and 174 LUSC) were used to train, and 150 (71 LUAD and 79 LUSC) were used to test. The proposed system achieved an AUC of 0.9068 in test samples, corresponding to a classification accuracy of 83.33%. The (heretofore excluded) adjacent normal regions were classified correctly almost as accurately as tumor regions (74.7%).

Conclusions: This fully-automated histopathology subtyping method generates maps of regions-of-interest within WSIs, providing novel spatial information on tumoral organization. For example, our results on test data show tumor patches of size 100 square microns with 60 to 100 cells distinguish LUAD from LUSC better than other cell-density ranges. Moreover, adjacent normal tissue may provide additional insights into tumorigenesis mechanisms.

Citation Format: Mustafa I. Jaber, Liudmila Beziaeva, Christopher W. Szeto, John Elshimali, Shahrooz Rabizadeh, Bing Song. Automated adeno/squamous-cell NSCLC classification from diagnostic slide images: A deep-learning framework utilizing cell-density maps [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 1393.
Abstract 1395: Residual convolutional neural network for predicting response of transarterial chemoembolizationin hepatocellular carcinomafrom CT imaging
Authors: Peng Jie
Published: August 2019

Purpose: We aimed to develop and validate a model of deep learning for the preoperative prediction of response of patients with intermediate stage hepatocellular carcinoma undergoing transarterial chemoembolization.

Methods: The training and two independent validation CT images were acquired for 540 patients from the Nan Fang Hospital (NFH), 84 patients from Zhu Hai Hospital Affiliated with Jinan University (ZHHAJU) and 136 patients from the Sun Yat-sen University Cancer Center (SYUCC). We built a predictive model from the outputs using transfer learning techniques of a residual convolutional neural network (ResNet50). Then, we revaluated the prediction accuracy for each patch in two independent validation cohorts.

Results: In the training (NFH) set, the deep learning model had a high accuracy of 84.3% and an areas under the receiver operating characteristic curves (AUROC) of 0.97, 0.96, 0.95 and 0.96 for complete response (CR), partial response (PR), stable disease (SD) and progressive disease (PD), respectively. In the validation (ZHHAJU) set, the deep learning model had an accuracy of 85.1% and an AUROC of 0.98, 0.96, 0.95 and 0.94 for CR, PR, SD and PD, respectively. In the other validation (SYUCC) set, the deep learning model had an accuracy of 82.8% and an AUROC of 0.97, 0.96, 0.95 and 0.95 for CR, PR, SD and PD, respectively.

Conclusion: The proposed ResNet model showed excellent performance for the individualized and non-invasive estimation of TACE therapy, which may help clinicians to better identify patients with HCC who can benefit from the interventional treatment.

Citation Format: Jie Peng. Residual convolutional neural network for predicting response of transarterial chemoembolizationin hepatocellular carcinomafrom CT imaging [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 1395.
Abstract 1399: Ex-vivo targeting of urothelial carcinomas by fluorescent pHLIP imaging agents
Authors: Golijanin Borivoj , Moshnikova Anna , Engelman Donald M. , Andreev Oleg A. , Reshetnyak Yana K. , Amin Ali , Golijanin Dragan
Published: August 2019

Introduction: The detection of urothelial carcinomas of the bladder and upper tract is not satisfactory. Incomplete findings by contemporary imaging modalities provide a challenge in patient care, so there is a definitive need to improve diagnosis and treatment methods for urothelial carcinomas.

Experimental procedures: ICG (indocyanine green) near infrared fluorescent (NIRF) dyes were conjugated with a pH-Low Insertion Peptide (pHLIP®), which senses and targets the acidity at the surfaces of cancer cells. In a few cases IR800 pHLIP® was used. Urinary neoplasm (kidney, ureter and bladder) specimens obtained through radical surgery were irrigated and incubated ex-vivo with ICG pHLIP® followed by fluorescence imaging. Pathology findings were correlated with the ICG pHLIP® NIRF imaging.

Results: In the upper urothelial tract, nine of ten patients with nephroureterectomy had urothelial carcinomas (UTUC). Eighteen (100%) malignant lesions were found in NIRF imaging. Only thirteen (72%) of malignant lesions were found under white light macroscopic examination. ICG pHLIP® NIRF imaging gave a 28% increase in diagnosis of UTUC in ex-vivo specimens. There was no non-specific uptake in the non-tumoral tissue. The ICG pHLIP® imaging agent identified malignant upper tract urothelial lesions with 100% specificity and sensitivity.

In the lower urinary tract, thirty-eight radical cystectomy specimens were incubated with pHLIP® conjugated with one of the NIRF dyes (ICG or IR800). In the thirty-eight bladders, eighty-one lesions were identified using fluorescent pHLIP®(specificity (97%) and sensitivity (100%)). White light cystoscopy did not detect 20.5% of lesions identified under NIRF cystoscopy (p<.001). Normal tissue did not show any uptake by the construct; however, entrapment of the dye in the necrotic tumor bed was noted. All regions showing the NIRF signal of pHLIP® were confirmed by standard pathological analysis.

Conclusions: The ICG pHLIP® NIRF revealed additional lesions not seen with white light ex-vivo cystoscopy or preoperative ureteroscopy. pHLIP® based agents promise to improve diagnostic accuracy for urothelial carcinomas and may enable targeted treatment.

Citation Format: Borivoj Golijanin, Anna Moshnikova, Donald M. Engelman, Oleg A. Andreev, Yana K. Reshetnyak, Ali Amin, Dragan Golijanin. Ex-vivo targeting of urothelial carcinomas by fluorescent pHLIP imaging agents [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 1399.
Abstract 14: A new orally available drug for the treatment of metastatic cancers
Authors: Zhou Kun , Cheong Jae Eun , Zaffagni Michaela , Xu Yingjie , Sun Lijun , Zetter Bruce
Published: August 2019

Metastatic cancers account for the great majority of cancer deaths. We conducted a screen to indetify known drugs that selectively inhibit the viability of metastatic tumor cells. Results of the screen identified members of the FDA-approved benzimidazole methylcarbamate family (e.g. mebendazole (MBZ) and albendazole (ALB)) as potential therapies for metastatic disease. Earlier work also supports a role for this chemical family in the potential treatment of activity in multiple cancers, but progress has been stalled by their poor water solubility and poor bioavailability for systemic delivery to disseminated tumors. We therefore synthesized a novel compound (OBD9) containing the scaffold of MBZ coupled to an oxetane group to enhance aqueous solubility to 361 μM. OBD9 demonstrates significant cytotoxicity toward a variety of cancer cell types including colon, lung, and prostate cancers (IC50: 0.9-2 μM). In a mouse xenograft model of human A549 lung cancer cell line OBD9 dramatically inhibited the growth of established tumors at 30 or 90 μM without noticeable toxicity. In a mouse xenograft model using highly aggressive PCMLN3 prostate cancer cells, oral administration of OBD9 at 30 mg/kg also significantly repressed growth of established tumors with no visible toxicity.

We have studied the working mechanisms behind OBD9 and found that the Wnt signaling pathway is suppressed by OBD9 treatment. Both qPCR and Western blot data suggest that the beta-catenin/TCF complex in the Wnt signaling pathway are inhibited by OBD9. Among the downstream targets of Wnt signaling pathway, the oncogene cMyc is also significantly decreased in different cancers including colon, lung and prostate cancer. Overall, our in vitro and in vivo data suggest that OBD9 potentially represents a novel therapeutic option for multiple cancers especially metastatic cancer through targeting Wnt signaling pathway and the downstream oncogene cMyc.

Citation Format: Kun Zhou, Jae Eun Cheong, Michaela Zaffagni, Yingjie Xu, Lijun Sun, Bruce Zetter. A new orally available drug for the treatment of metastatic 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 14.
Abstract 140: Comprehensive analysis of immune evasion in breast cancer by single-cell RNA-seq
Authors: Yin Jianhua , Zhou Hanlin , Yan Chen , lv Ruqian , Wang Ting , He Chenyang , Huang Chen , Hou Yong , Zhu Shida , Wang Ling , Dean Michael , Wu Kui , Hu Hong , Li Guibo
Published: August 2019

The tumor microenvironment is composed of numerous cell types, including tumor, immune and stromal cells. Cancer cells interact with the tumor microenvironment to suppress anticancer immunity. In this study, we molecularly dissected the tumor microenvironment of breast cancer by single-cell RNA-seq. The breast tumor microenvironment was profiled by analyzing the single-cell transcriptomes of 52,163 cells from the tumor tissues of 15 breast cancer patients. The tumor cells and immune cells from individual patients were analyzed simultaneously at the single-cell level. Among more than 40,000 tumor cells, we identified distinct cell clusters with different molecular subtypes and gene signatures in the same tumor, which revealed intra-tumor heterogeneity in breast cancer. We comprehensively profiled the functional state of T cells, macrophages, B cells, CAFs in breast cancer tumor microenvironment, which highlight the immune cell and CAF diversity in the breast cancer TME. Moreover, we analyzed intercellular communication in the breast cancer TME using a dataset of human ligand receptor pairs, which showed distinct cell interaction models across tumors. The evolution of the tumor microenvironment during clinical treatment was dissected by comparing tumor microenvironment of breast cancer pre- and post-chemotherapy. This study investigated the diversity of the cell types in the tumor microenvironment and provided information on the mechanisms of escape from clearance by immune cells in breast cancer, which could be a beneficial resource for immunotherapy development.

Citation Format: Jianhua Yin, Hanlin Zhou, Chen Yan, Ruqian lv, Ting Wang, Chenyang He, Chen Huang, Yong Hou, Shida Zhu, Ling Wang, Michael Dean, Kui Wu, Hong Hu, Guibo Li. Comprehensive analysis of immune evasion in breast cancer by single-cell RNA-seq [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 140.
Abstract 1408: Diffuse optical spectroscopic imaging reveals regimen-dependent changes in breast tumors throughout neoadjuvant chemotherapy
Authors: Tank Anup , Peterson Hannah , Pera Vivian , Tabassum Syeda , Ko Naomi , Leproux Anais , Tromberg Bruce , Roblyer Darren
Published: August 2019

Diffuse Optical Spectroscopic Imaging (DOSI) is a non-invasive near-infrared imaging modality than can provide label-free functional and metabolic information about breast tumors, probing depths up to several centimeters. Previous work has shown that DOSI has prognostic value for pathological response at key timepoints during neoadjuvant (presurgical) chemotherapy (NAC), including baseline, 24 hours after the first infusion, week 1, and at the midpoint of therapy. To date there have been almost no reports on how specific NAC regimens affect the prognostic capability of DOSI. Here we present a large multi-center DOSI dataset consisting of 54 breast tumors and 313 unique measurements taken across three timepoints during NAC: Baseline, week 1, and midpoint of treatment. We will show for the first time that the manifestation of DOSI response parameters (oxyhemoglobin, de-oxyhemoglobin, water, and lipid) is highly dependent on the specific NAC regimen used. Subjects were treated with either a Maximum Tolerated Dose (MTD) regimen, in which a higher dose is administered less frequently, or a Metronomic (MET) regimen, in which a smaller dose is given more frequently. MTD regimen of cytotoxic agents has traditionally been the most effective method to treat breast cancer as it aims to kill the maximum amount of tumor cells before regrowth. MET administration of cytotoxic therapies has shown antiangiogenic properties aiming to limit toxic side effects and drug resistance, while potentially inducing tumor dormancy and immunomodulation. Subjects treated with MTD regimen and also responding to this treatment exhibited an oxyhemoglobin flare in the first day of NAC. In contrast, subjects treated with MET presented only small fluctuations in oxyhemoglobin throughout the first week of NAC. The oxyhemoglobin flare has been theorized to be induced by an inflammatory effect significant for cell death, which could be in response to specifically the MTD regimen and its higher dosing. This study indicates that the specific regimen must be considered when using DOSI for NAC monitoring. It also suggests DOSI may provide biological insight into the regimen-specific mechanisms of action.

Citation Format: Anup Tank, Hannah Peterson, Vivian Pera, Syeda Tabassum, Naomi Ko, Anais Leproux, Bruce Tromberg, Darren Roblyer. Diffuse optical spectroscopic imaging reveals regimen-dependent changes in breast tumors throughout neoadjuvant chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1408.
Abstract 1411: Semi-quantitative mapping of oncological therapies with mass spectrometry imaging
Authors: Baluya Dodge , Shrestha Bindesh , Cressman Erik N.
Published: August 2019

Hepatocellular carcinoma (HCC) is one of the challenging cancers to treat with occurrence steadily rising in the United States. While surgical resection is considered curative, it is not available to patients who have underlying liver problems, such as chronic liver disease. Liver transplantation may be considered in these cases but recurrence is a possibility, and transplantation occurs for <5% of all HCC cases. Doxorubicin (DXR), a drug derived from a natural product extracted from several wild strains of Streptomyces, is used to treat different types of cancer, particularly liver cancer such as HCC. While DXR use for treatment shows positive efficacy, the side effects such as cardiomyopathy and typhlitis, are easily triggered when the systemic concentration dose reaches a particular threshold. Also, there is no confirmation that the drug reaches the target area with a viable therapeutic concentration. In this work, we use mass spectrometry imaging (MSI) platform on Waters SYNAPT G2-Si MALDI to gather spatial information and quantity of the DXR to determine the delivery efficacy. To generate a calibration curve that is suitable for MSI, a phantom tissue template was created. Livers were extracted from pig and homogenized using a Qiagen Tissuelyzer with a 30% w/v in deionized water, and created DXR tissue homogenate solutions (20, 10, 5, 2, 1µM) using serial dilution. The resulting solutions were pipetted into a gelatin mold before it was frozen using liquid nitrogen and stored at -80 °C until needed. For the ‘unknown’ samples, the liver was explanted from a pig that was treated with doxorubicin, then flash-frozen using liquid nitrogen and kept at -80 °C until needed. The tissue was sectioned using a Leica cryotome and mounted on glass microscope slides. For each analysis, two adjacent sections were selected, one was submitted for MSI analysis and the other for IHC staining. A good correlation (R>0.99) of the DXR signal from the tissue homogenates showed that the calibration curve could be used to comparably measure the DXR amount in the similar tissue type. MSI of the explanted liver showed the distribution of DXR towards a particular section. While this experiment showed only the response for DXR, this approach can be applied to other drug therapies to show the localization and concentration, thereby helping the effectivity assessment of the therapy complementary with the other evaluation tools for a successful treatment.

Citation Format: Dodge Baluya, Bindesh Shrestha, Erik N. Cressman. Semi-quantitative mapping of oncological therapies with mass spectrometry imaging [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 1411.
Abstract 1425: Chimeric antigen receptors based on T cell receptor-like antibodies
Authors: Wu Ling , Brzostek Joanna , Sankaran Shvetha , Tan Triscilla , Chan Conrad , Yap Jiawei , Lai Junyun , MacAry Paul , Gascoigne Nicholas
Published: August 2019

T cells are essential in adaptive immunity and play an indispensable role to eliminate abnormal or virus infected cells. By merging antibody technology and cell engineering, T cells equipped with a chimeric antigen receptor (CAR) can be redirected to the target in a non-MHC restricted fashion. Within the past few years, clinical trials using CAR T cells engineered to recognize B cell cancers have shown high rates of response (70%-90%) and durability of response that are unprecedented in acute and chronic leukaemia. However, severe toxicity has been observed due to massive, and to some extent nonspecific, T cell activation. Although CARs have been improved and investigated heavily, the extent to which a CAR is similar to its parental TCR extra- or intracellularly, and whether critical elements involved in normal TCR signalling are kept or rewired in CAR signalling, are unclear. A better understanding of these questions would greatly facilitate improvements in CAR technology and its usefulness in clinical practice. A CAR targeting a peptide-MHC complex (using an Fv from an antibody that recognizes MHC-peptide: in other words, a TCR-like Ab) mimics the process of TCR recognition and takes advantage of CAR technologies developed so far. It is thus an excellent study object to compare TCR and CAR signalling. Well-built research methods on TCR can be transferred immediately to TCR-like CAR, while the knowledge and findings generated through the studies of TCR-like CAR can in turn renew our perceptions on TCR.

TCR-like antibodies targeting the EBV epitopes LMP1125-133, LMP2A426-434 or EBNA1562-570, were engineered as CARs. We find that conformation change, if any, upon ligand binding is not enough to activate downstream TCR signalling, but oligomerization is a crucial factor. Although CAR with a TCR-like specificity can recruit CD8 co-receptor to the immunological synapse, it is dispensable for activation of the T cell. Furthermore, the activation kinetics of CAR and TCR are distinctly different. TCR shows a pulse-like activation, whereas CAR shows an activation that gradually plateaus and remains steady. These unique properties of CAR identified in our study demonstrate that CAR signalling properties may be amenable to modifications leading to better specificity and activity in vivo. Our long-term goal is that the TCR-like CAR can be used to combat EBV induced Nasopharyngeal Carcinoma.

Citation Format: Ling Wu, Joanna Brzostek, Shvetha Sankaran, Triscilla Tan, Conrad Chan, Jiawei Yap, Junyun Lai, Paul MacAry, Nicholas Gascoigne. Chimeric antigen receptors based on T cell receptor-like antibodies [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 1425.
Abstract 1426: Expansion of CD8+PD-1+ T cells restored TCR repertoire as a prognostic marker to adoptive T cell immunotherapy of advanced pancreatic cancer
Authors: Qiao Guoliang
Published: August 2019

Purpose: We have previously reported the clinical benefits of autologous adoptive T cell immunotherapy in advanced pancreatic cancer (APC). We therefore aimed to further identify the core functional population of CD8+ T cells which exerted on the aberrant TCR repertoire and subsequently contribute to favorable prognosis after adoptive T cell therapy(ACT).

Experimental Design: PBMC from APC patients (n=25) treated on a protocol of S-1 plus adoptive T cell immunotherapy were expanded ex vivo with a cytokine cocktail. The different fractions of CD8+PD-1+, CD8+LAG-3+, CD8+TIM-3+ and CD8+4-1BB+ T cells from time dependent segment were quantitated and expression of co-inhibitory molecules and biological functions on tumor killing and comparative analysis TCR repertoire from each patient were measured. The multivariate Cox hazards regression analysis was performed to identify the key T cell subset that affected the TCR repertoire recovery and subsequently associated with clinical outcome.

Results: The CD3+, CD3+CD4+, and CD3+CD8+ T cells within the expanded cell product reached peak levels by day 15. CD8+ T cells exhibited enhanced expression of PD-1, LAG-3, and TIM-3, but not 4-1BB, after ex vivo expansion. Survival analysis showed that patients with a ratio of post/pre CD8+PD-1+ T cells > 2 had significantly favorable progressive-free survival (PFS) (median PFS time 180 days vs 85 days, P=0.002) and overall survival (OS) (median OS time 238 days versus 142 days, P=0.024). The sorted CD8+PD-1+ T cells displayed enhanced anti-tumor activity, and increased IFN-γ secretion after co-culture with autologous tumor cell lines. TCR repertoire diversity simultaneously rose during the ex vivo expansion and was associated with an increase in CD8+ T cells and decline in CD8+/CD28- T cells. Moreover, an increased TCR diversity was associated with significantly favorable PFS (median PFS time 166 days vs 79 days, P=0.043) and OS (median OS time 216 days versus 112 days, P=0.031) and was associated with the extent of expansion of the CD8+PD-1+ T cells (r2=0.464, P=0.001). Cox proportional hazards analysis showed that post/pre CD8+PD-1+ T cells after expansion ex vivo was an independent prognostic marker associated with both OS(P=0.009) and PFS(P=0.012).

Conclusion: CD8+PD-1+ T cells, after ex vivo expansion over 15 days, are tumor-reactive. The extent of CD8+PD-1+ T cell expansion could restore TCR repertoire and was an independent prognostic factor associated with clinical outcomes in APC patients treated with ACT.

Citation Format: Guoliang Qiao. Expansion of CD8+PD-1+ T cells restored TCR repertoire as a prognostic marker to adoptive T cell immunotherapy of advanced pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1426.
Abstract 143: Immune landscape in lung adenocarcinoma drawn by single-cell RNA sequencing
Authors: KU Bo Mi , Kim Nayoung , Lee Kyung Young , Sun Jong-Mu , Lee Se-hoon , Ahn Jin Seok , Park Keunchil , Kim Hong Kwan , Lee Hae-Ock , Ahn Myung-Ju
Published: August 2019

Immune cells in the tumor microenvironment from an ecosystem that modulates cancer progression. However, the exact nature and dynamics of tumor microenvironment in lung cancer remain largely unknown. To depict the baseline landscape of the composition of tumor microenvironment at different stages, we conduceted single-cell RNA sequencing of 58 samples from 43 lung adenocarcinoma patients. Here, we analyzed 11 early-stage lung tumor and normal pairs that had been resected before systemic therapy, 11 late-stage lung tumor biopsy, and 5 metastatic pleural-effusion or 10 metastatic brain tumor samples and revealed differential immune profiles encompassing lymphoid and myeloid compartment. In normal lung, large numbers of macrophages and T cells consistently comprised the immune cell repertoire across patients. In paired cancer tissues, decrease in macrophage but increase in B cell proportions were evident indicating the activation of humoral immunity. In addition, diverse effector T cell populations mark the cancer tissues both at the primary and metastatic sites, corroborating the activation of cellular adaptive immunity. These data provide valuable insight of tumor-driven immune changes in lung cancer.

Citation Format: Bo Mi KU, Nayoung Kim, Kyung Young Lee, Jong-Mu Sun, Se-hoon Lee, Jin Seok Ahn, Keunchil Park, Hong Kwan Kim, Hae-Ock Lee, Myung-Ju Ahn. Immune landscape in lung adenocarcinoma drawn by single-cell RNA sequencing [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 143.