bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5235: In vitro validation of drug-target interactions revealed in silico by Comprehensive Resource of Biomedical Relations with Network Representations and Deep Learning (CROssBAR) in HCC
Authors: Nalbat Esra , Rifaioglu Ahmet Sureyya , Dogan Tunca , Martin Maria Jesus , Cetin-Atalay Rengul , Atalay Volkan
Published: September 2020

Computational methods are more commonly employed for our understanding of cellular signaling mechanisms with the aim to develop cures for diseases including cancer. Identification of disease specific targeted drugs requires systems level analysis of cellular networks. Our research group developed a comprehensive resource, called CROssBAR, which connects various biological data sources in order to provide a well-connected resource with a focus on drug discovery and precision medicine. CROssBAR aims to predict and reveal novel drug-target interactions using deep learning algorithms. CROssBAR predictions were validated on hepatocellular carcinoma cells which is the 5th most prevalent and 3th deadliest cancer worldwide. We have selected 2 new small molecule kinase inhibitors Lenvatinib, Sunitinib along with Sorafenib and Regorafenib which are currently used in clinics. CROssBAR identifies novel and known gene targets for Lenvatinib, Sunitinib in comparison with Sorafenib and Regorafenib. Genes from SAPK/JNK and PI3K/Akt pathway that were not previously associated with hepatocellular carcinoma therapeutics were determined. All selected small molecules were bioactive on Huh7 and Mahlavu cells by SRB assay. Cell death was characterized by Annexin V which was due to G1/S cell cycle arrest. Further analysis of proteins involved in the SAPK/JNK, PI3K/Akt pathways and apoptotic markers such as PARP and Caspases were examined with immunoblotting upon treatment. As a result, cellular stress mechanisms activated PI3K/Akt and SAPK/JNK pathways which caused programmed cell death in HCC. With our deep learning approach, we were able to demonstrate a correlation between Lenvatinib and Sunitinib and their novel targets in HCC. Our validation results have illustrated that the comprehensive CROssBAR tool developed by our research group achieved in vitro significant predictions of new drugs and their targets against HCC that were not previously reported. Hence with this study we were able to demonstrate the use and validity of the CROssBAR tool along with new targets for primary liver cancer therapeutics.

Citation Format: Esra Nalbat, Ahmet Sureyya Rifaioglu, Tunca Dogan, Maria Jesus Martin, Rengul Cetin-Atalay, Volkan Atalay. In vitro validation of drug-target interactions revealed in silico by Comprehensive Resource of Biomedical Relations with Network Representations and Deep Learning (CROssBAR) in HCC [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 5235.
Abstract 5259: HSPA6 may be a new target by thyraquinone inhibiting migration and invasion of breast cancer cells
Authors: Shen Shiyi , Xiao Ting , Zhou Ju , Wei Chunli , Fu Junjiang
Published: September 2020

Background and Objective: Breast cancer is the malignant tumor with the highest incidence and the second highest mortality among women, and the incidence of breast cancer in China is increasing year by year. The treatment of breast cancer includes radiotherapy, chemotherapy, endocrine therapy, biological targeted therapy and traditional Chinese medicine adjuvant therapy; but the efficacy still needs to be further improved to benefit the patients. Thymoquinone (TQ) is a key component of black grass seed oil and has anti-cancer properties in a variety of tumors. Preliminary studies in our laboratory demonstrated that TQ has a significant inhibitory effect on the migration and invasion of triple negative breast cancer cells.

Methods: To find the new target genes for the inhibition of migration and invasion of breast cancer cells by TQ, RNA-sequencing (RNA-seq) was conducted with and without TQ in human breast cancer cell line BT-549 with high invasion and migration characteristics. Quantitative and semi-quantitative RT-PCR was used to further verify the genes regulated by TQ, and then Western Blotting was used to compare the changes of the genes regulated by TQ at the protein level. Finally, the molecular mechanism of TQ inhibiting migration and invasion of human breast cancer cells was investigated by increasing or decreasing the expression of these genes.

Results: Eighteen differentially expressed genes, which were closely related to the carcinomas in the experimental group and the control group, were obtained by RNA-seq. HSPA6 gene changed significantly, and showed an up-regulation after adding TQ, which was consistent with the results of RNA-seq. By RT-PCR and Western Blotting from different cell lines (BT549, MCF-7, ZR-75-30, SiHa, Caski), we found that the expression of HSPA6 in the experimental group was higher than that in the control group. The subsequent TQ treatment after knockdown / over-expression will be completed for HSPA6 protein functional effects by TQ.

Conclusion: HSPA6 may be a new target gene for TQ to inhibit the migration and invasion of breast cancer cells. The success of this study will provide a new candidate target for clinical treatment of breast cancer. Funding support: National Natural Science Foundation of China (Grant No. 81672887, 81172049)

Citation Format: Shiyi Shen, Ting Xiao, Ju Zhou, Chunli Wei, Junjiang Fu. HSPA6 may be a new target by thyraquinone inhibiting migration and invasion of breast cancer cells [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 5259.
Abstract 5267: Utilizing stimulated Raman scattering microscopy to study intracellular distribution of label-free ponatinib in live cells
Authors: Sepp Kristel , Lee Martin , Bluntzer Marie T. , Helgason G. Vignir , Hulme Alison N. , Brunton Valerie G.
Published: September 2020

Ponatinib is a clinically approved tyrosine kinase inhibitor used to treat chronic myeloid leukemia (CML). Drug resistance is a widespread problem in CML treatment, where ponatinib resistant patients have very limited treatment options. In this study stimulated Raman scattering (SRS) microscopy was used to allow label-free imaging of intracellular ponatinib with high sensitivity and specificity in live human CML cell lines, in the context of ponatinib resistance.

Ponatinib has an alkyne moiety in its structure that makes it inherently Raman active in the cellular silent region of the Raman spectrum. SRS microscopy represents a powerful imaging tool for visualizing label-free drug molecules within cells with high resolution, without the need for additional labels, or nanoparticle sensors as used in many other optical imaging technologies. It provides Raman imaging with minimal spectral distortion and a quantitative output, allowing the intracellular concentrations of drug molecules to be accurately determined.

Intracellular visualization of ponatinib was achieved at biologically relevant, nanomolar concentrations for the first time using SRS. It was determined that ponatinib is sequestered into the lysosomes, with a higher lysosomal concentration found in drug resistant cells. This was associated with increased lysosome biogenesis. Target engagement studies showed that treatment with chloroquine reduced ponatinib accumulation in lysosomes, but did not re-sensitise cells to ponatinib, confirming BCR-ABL independent resistance mechanism in this CML cell model.

In summary, we have visualized intracellular ponatinib localization for the first time using SRS, demonstrating that acquired drug resistance can influence drug uptake and localisation in CML, which in turn has an effect on target engagement.

Citation Format: Kristel Sepp, Martin Lee, Marie T. Bluntzer, G. Vignir Helgason, Alison N. Hulme, Valerie G. Brunton. Utilizing stimulated Raman scattering microscopy to study intracellular distribution of label-free ponatinib in live cells [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 5267.
Abstract 527: Isolation of large and diverse monoclonal antibody panels against the multipass membrane protein targets Kv1.3, P2X7, and Claudin 18.2
Authors: Stafford Lewis J. , Willis Sharon , Rucker Joseph , Doranz Benjamin , Chambers Ross
Published: September 2020

Monoclonal antibodies (MAbs) are a well-established treatment approach in oncology and other diseases. Nevertheless, multipass membrane proteins are largely inaccessible as MAb targets due to their poor expression, membrane-dependent structure, small extracellular regions, and high sequence conservation between humans and rodents. Integral Molecular's MPS Antibody Discovery platform specifically addresses each of these challenges. Using this platform, we have isolated large and diverse panels of functional (agonist/antagonist) MAbs against targets including Claudin 18.2 and the ion channels P2X7 and Kv1.3. A key feature that enables the success of the platform is the use of divergent species for immunization. Chickens are immunized with a combination of DNA and Lipoparticles (high-concentration membrane proteins) to obtain high-titer immune responses against the native membrane protein. Our optimized phage-display protocols allow the isolation of large panels of MAbs against diverse epitopes. The MPS Antibody Discovery platform offers the potential for discovering MAbs against difficult targets in cancer with a success rate over 95%.

Citation Format: Lewis J. Stafford, Sharon Willis, Joseph Rucker, Benjamin Doranz, Ross Chambers. Isolation of large and diverse monoclonal antibody panels against the multipass membrane protein targets Kv1.3, P2X7, and Claudin 18.2 [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 527.
Abstract 5272: Molecular analysis of Sunitinib resistant renal cell carcinoma after sequential application of Cabozantinib
Authors: Zaccagnino Angela , Vynnytska-Myronovska Bozhena , Stockle Michael , Junker Kerstin
Published: September 2020

Introduction: The tyrosine kinase inhibitors Sunitinib and Pazopanib have been established in firstline treatment of metastatic renal cell carcinoma (mRCC) patients especially in the good prognosis group. Despite the primary response many patients become resistant. Resistance mechanisms include activation of MET and AXL - downstream pathways have been reported. Cabozantinib (Cbz), a MET/AXL/VEGFR inhibitor, showed clinical benefits as second-line agent. However, why the therapy becomes ineffective and how the long-term administration with Sunitinib influences sensitivity of tumor cells towards Cbz remain to be answered.

Methods: We referred to previously generated Sunitinib -resistant cells (786-O/S) and their untreated counterpart (786-O/WT) to test effect of Cbz in vitro. Drug sensitivity was determined via WST-1 cell proliferation assay, whereas cell pathways analysis in response to Cbz was evaluated at different time points by means of immunoblotting.

Results: By challenging the two cell phenotypes with different concentrations of Cbz we found that the 786-O/WT cells were more sensitive to the drug in comparison to 786-O/S (IC50 value of 9.9 µM versus 12.2 µM, respectively). The 786-O/WT cells responded to short-term incubation with Cbz by decreasing the expression of the receptor MET and its downstream intracellular pathways: the activated Src (pSrc) and p38 MAPK (p-p38 MAPK). Besides, Erk protein showed a slight increased activity (pErk) after 8 and 24h drug exposure. In contrast, no significant regulation of MET, nor pSrc and p-p38 MAPK kinase expression was reported in the 786-O/S cells. Remarkably, pErk level was induced after Cbz incubation in a time-dependent manner. Finally, phosphorylated S6 (pS6) was used as a marker of PI3K/Akt/mTOR pathway. Cbz decreased the level of pS6 in 786-O/WT, while enhancing it in the 786-O/S cells.

Conclusions: Our work provides a preclinical model for sequential therapy in mRCC. The initial in vitro data confirmed Cbz as a potent tool to contrast the progress of RCC in the first-line setting. Still, switching to Cbz after Sunitinb resistance resulted in Erk and mTOR pathways activation and might be interpreted as overlapping survival mechanism. This ongoing work will provide a better knowledge of the protein expression patterns of possible cross-resistance in RCC by analyzing additional cell lines including Pazopanib-resistant ones.

Citation Format: Angela Zaccagnino, Bozhena Vynnytska-Myronovska, Michael Stockle, Kerstin Junker. Molecular analysis of Sunitinib resistant renal cell carcinoma after sequential application of Cabozantinib [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 5272.
Abstract 5280: Kinase fusions drive endocrine resistance in estrogen receptor-positive breast cancer
Authors: Liu Bo , Ross Dara S. , Schram Alison M. , Razavi Pedram , Lagana Stephen M. , Zhang Yanming , Scaltriti Maurizio , Bromberg Jacqueline F. , Ladanyi Marc , Hyman David M. , Drilon Alexander , Zahir Ahmet , Benayed Ryma , Hechtman Jaclyn F. , Chandarlapaty Sarat
Published: September 2020

Oncogenic kinase activation by gene fusions can promote cancer development and tumor progression, however, kinase fusions have not been characterized extensively in breast cancer. In this study, we characterized kinase fusions in a large cohort of 4854 breast cancer patients using clinical DNA and/or RNA next generation sequencing platforms. Twenty-seven cases harboring kinase fusions were identified, including 11 FGFR (5 FGFR2, 3 FGFR3, 3 FGFR1), 5 BRAF, 4 NTRK1, 2 RET, 2 ROS1, 1 ALK, 1 ERBB2, and 1 MET. Eight patients with a history of endocrine therapy had available pre-treatment samples, of which six were negative for kinase fusion, and ESR1 hotspot mutations were not observed in any of these kinase fusion-positive samples. These findings suggest a potential role for kinase fusions in endocrine therapy resistance and prompted us to model the kinase fusions in human breast cancer cell lines. Ectopic expression of LMNA-NTRK1 fusion kinase activated growth factor signaling cascades, including PI3K-AKT and MAPK-ERK pathways, and promoted hormone-independent growth in MCF7 and T47D cells. Enforced expression of the LMNA-NTRK1 fusion conferred resistance to the ER antagonist fulvestrant and combined treatment of fulvestrant and the Trk inhibitor larotrectinib completely blocked the growth of LMNA-NTRK+ breast cancer cells. Similarly, expression of the EML4-ALK fusion also activated growth factor signaling pathways and caused resistance to estrogen depletion and induced sensitivity to the ALK inhibitor, Ceritinib. Treatment of xenografted LMNA-NTRK1 expressing tumors confirmed the efficacy of the combined treatment of antiestrogen and NTRK1 inhibition in vivo. Two patients with acquired LMNA-NTRK1 fusions and metastatic disease received larotrectinib and demonstrated clinical benefit. Overall, our findings demonstrate that kinase fusions promote endocrine resistance in ER-positive breast cancer, and suggest that fusion screening in advanced breast cancer, particularly those with ER-positive breast cancer at progression on hormone therapy can identify rare tumors harboring targetable kinase fusions.

Citation Format: Bo Liu, Dara S. Ross, Alison M. Schram, Pedram Razavi, Stephen M. Lagana, Yanming Zhang, Maurizio Scaltriti, Jacqueline F. Bromberg, Marc Ladanyi, David M. Hyman, Alexander Drilon, Ahmet Zahir, Ryma Benayed, Jaclyn F. Hechtman, Sarat Chandarlapaty. Kinase fusions drive endocrine resistance in estrogen receptor-positive breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5280.
Abstract 5303: GSTP1 positive prostate cancer
Authors: Vidal Igor D. , Glavaris Stephanie , Jones Tracy , Hicks Jessica , Trock Bruce J. , Yegnasubramanian Srinivasan , Nelson William , De Marzo Angelo M.
Published: September 2020

Introduction: GSTP1 is a member of the Glutathione-S-transferase family, known to be silenced by CpG island DNA methylation in approximately 90% of prostatic adenocarcinomas. However, the cases that are not densely methylated have not been extensively characterized in terms GSTP1 expression or clinico-pathological features.

Description of experimental procedures: We analytically validated an automated IHC assay against human GSTP1 using well-known cell line controls and human prostate cancer tissues. We examined 33 TMAs from 1296 prostatectomy patients using the index tumor with 4-fold redundancy from patients from 1993 to 2015. TMA slides stained for GSTP1 were scanned and uploaded to a web based image scoring platform (TMAJ). The images were reviewed by two different pathologists with expertise in prostate cancer. The scoring system followed a two-tiered approach, noted as positive (carcinomas with any nuclear and/or cytoplasmic staining), and as negative (carcinoma cells with no nuclear or cytoplasmic stain) in the presence of benign stromal or inflammatory cells that served as internal controls. The GSTP1 positive staining was correlated with Gleason grade groups, pathological stage, and age.

Results: After exclusion for inadequate tissue staining, GSTP1 was evaluated in 4326 TMA cores with carcinoma. GSTP1 IHC staining was positive in 190 cores (4.3%), corresponding to 75 patients (5.9% of patients had GSTP1 positive cancer). Of these 190 positive cores, 109 showed homogeneous (100% positive) tumor cell staining while 81 showed heterogeneous staining, with mixed positive and negative regions. In 43 patients (3.3%), all TMA cores containing carcinoma stained positively. In a number of standard slides from prostatectomies, GSTP1 positive IHC staining cases were also found to be positive for GSTP1 mRNA by in situ hybridization. There was no correlation between GSTP1 positive staining, Gleason grade group, pathological stage, margin status or patient age. Preliminary analysis in a subset of cases showed no correlation with TMPRSS2-ERG status or PTEN loss.

Conclusion: GSTP1 protein is present in approximately 5.9% of clinically localized prostate cancers and in approximately 50% of these positive cases, all TMA cores with carcinoma stained positively.

Citation Format: Igor D. Vidal, Stephanie Glavaris, Tracy Jones, Jessica Hicks, Bruce J. Trock, Srinivasan Yegnasubramanian, William Nelson, Angelo M. De Marzo. GSTP1 positive prostate 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 5303.
Abstract 5307: The impact of image analysis and multiplex hybridization on PD-L1 diagnostic case triage
Authors: Humphries Matthew P. , Bingham Victoria , Sidi Fatima Abdullahi , Craig Stephanie G. , McQuaid Stephen , James Jacquline , Salto-Tellez Manuel
Published: September 2020

Introduction: Immune checkpoint blockade therapy is a new paradigm in cancer treatment with durable tumor regression and prolonged stabilization of disease in patients with advanced cancers, including non-small cell lung cancer (NSCLC). However, clinical assessment using immunohistochemistry (IHC) remains complex and ambiguous.

Methods: PD-L1 IHC staining was performed as a routine clinical reflex test on 802 NSCLC cases over an 18 month period using an automated staining system with PD-L1 clone SP263. PD-L1 expression was assessed using image analysis (IA) by 2 individuals and concordance was evaluated between the recorded manual clinical score and the digitally generated score. Additionally, a subset of 330 samples, representing cases with either varying PD-L1 expression or difficulty in assessment, were stained by multiplex immunofluorescence. Sensitivity and specificity was compared between manual assessments of both chromogenic and multiplex IHC.

Results: PD-L1 expression by IA showed significant concordance (R²=0.8248) to manual assessment. Sensitivity and specificity was 86.8% and 91.4%, respectively. Discordant cases were mainly due to spurious or poor staining quality, difficult classification by IA and overabundant macrophages and a low IA detection threshold. Evaluation of IA scores 10-49% and >70% revealed 96.8% concordance to manual assessment. Multiplex assessment enabled PD-L1+/CD68+ macrophages to be readily identified within PD-L1+/CK+ or PD-L1-/CK+ tumor nests. Manual assessment of multiplex vs. chromogenic IHC was highly concordant, with a sensitivity and specificity respectively of 97.8% and 91.8%.

Conclusions: Deployment of IA in routine PD-L1 IHC assessment of NSCLC represents a beneficial time saving process and could aid case triage saving >50% of pathologist review time. However cases close to diagnostic thresholds still require a more comprehensive pathological evaluation. Multiplex IHC provides a higher level of confidence in PD-L1 assessment and could be offered for challenging cases by centers with access to appropriate expertise and specialist equipment.

Citation Format: Matthew P. Humphries, Victoria Bingham, Fatima Abdullahi Sidi, Stephanie G. Craig, Stephen McQuaid, Jacquline James, Manuel Salto-Tellez. The impact of image analysis and multiplex hybridization on PD-L1 diagnostic case triage [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 5307.
Abstract 5331: Targeted protein degradation for the treatment of TRK fusion-driven cancers
Authors: Han Xiaoran , Chen Liqun , Zhang Chengwei , Jiao Bingyang , Chen Yanke , Liu Jing , Plewe Michael , Wang Jialiang
Published: September 2020

Small molecule-mediated targeted protein degradation offers a systemic approach to deplete disease-causing proteins. The tropomyosin receptor kinase (TRK) family kinases are receptors for neurotrophic factors and primarily function in the central nervous system. As results of chromosomal rearrangement events, TRK kinases are re-expressed as fusion proteins and implicated in a wide diversity of human malignancies. These fusion proteins universally retain the kinase domain and function as key oncodrivers. Two TRK kinase inhibitors, entrectinib and larotrectinib, have been approved to treat TRK fusion-expressing cancers with compelling clinical evidence, highlighting the significance of TRK fusion kinases as oncology targets. In the current study, we report the development of selective TRK degraders. Lead compounds induced rapid degradation of different TRK fusion variants identified in patients at subnanomolar concentrations. Degradation of the fusion kinases significantly compromised TRK-dependent cancer cell growth with low nanomolar IC50. The lead compound exhibited approximately 16% oral bioavailability in mouse and effectively controlled TRK-driven xenograft tumor growth. Pharmacodynamic analysis demonstrated sustained degradation of the TRK fusion in xenograft tumors. Collectively, these findings provide the framework to develop a novel approach that modulates oncogenic TRK fusion kinases for cancer treatment.

Citation Format: Xiaoran Han, Liqun Chen, Chengwei Zhang, Bingyang Jiao, Yanke Chen, Jing Liu, Michael Plewe, Jialiang Wang. Targeted protein degradation for the treatment of TRK fusion-driven cancers [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 5331.
Abstract 5340: Novel small-molecule antagonists of the PD1/PD-L1 axis
Authors: Magnez Romain , Leleu-Chavain Natascha , Tardy Morgane , El Bouazzati Hassiba , Klupsch Fréderique , Bailly Christian , Millet Régis , Quesnel Bruno , Thuru Xavier
Published: September 2020

Immuno-therapy has become a leading strategy to fight cancer. Over the past few years, immuno-therapies using checkpoint inhibitor monoclonal antibodies (mAbs) against programmed death receptor 1 (PD-1) and programmed death ligand 1 (PD-L1) have demonstrated improved survival compared with chemotherapy. We describe the identification and characterization of an innovative series of synthetic compounds (named PyrDLones, patented) endowed with nanomolar activity against PD-L1. PyrDLones properties were characterized using several biophysical techniques including microscale thermophoresis (MST) and fluorescence resonance energy transfer (FRET) measurements. In vitro, selected small molecules demonstrate a high affinity for human PD-L1, potently disrupt the PD-L1:PD-1 interaction (<2nM), and inhibit Src homology region 2 domain-containing phosphatase (SHP2) recruitment to PD-1 (<4nM). As a result, upon binding to and inhibiting PD-L1, these molecules reactivate proliferation of CTL-L2 cells expressing PD-1 (EC50=44nM). More than 150 molecules have been synthesized and a dozen of highly potent “PD-L1 silencing compounds” have been identified, based on in vitro measurements. Structure-activity relationships have been defined and an in silico drug-target model supporting the mechanism of action has been built. Experiments are on-going to delineate further the molecular mechanism of action of the PyrDLones (drug-induced PD-L1 dimer formation), the cellular consequences of the PD-L1 silencing (reactivation of cytotoxic T cells in the tumor microenvironment) and to characterize their activity in vivo. In summary, we have discovered a novel series of potent, small molecule PD-L1 antagonists, amenable to the design of orally active drugs for immuno-therapy of cancers.

Citation Format: Romain Magnez, Natascha Leleu-Chavain, Morgane Tardy, Hassiba El Bouazzati, Fréderique Klupsch, Christian Bailly, Régis Millet, Bruno Quesnel, Xavier Thuru. Novel small-molecule antagonists of the PD1/PD-L1 axis [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 5340.
Abstract 5343: Experimental targeted alpha-particle therapy against liver metastasis of HER2-positive gastric cancer in mice
Authors: Hasegawa Sumitaka , Li Huizi Keiko
Published: September 2020

Gastric cancer (GC) is one of the common cancer type and a leading cause of cancer-related death worldwide. Liver metastasis of gastric cancer (LMGC) is often found in GC patients and a fatal condition that is hard to be cured. HER2 overexpression is detected in a subset of about 10-20% of GC. HER2-targeting antibody trastuzumab has been used clinically for the treatment against advanced GC positive for HER2. Recently, targeted alpha-particle therapy has emerged as a new therapeutic option of cancer. Radionuclide astatine-211 is produced by cyclotron and emits alpha-particle at its decay with about 7 hours-half-life. Alpha-particle beam induces irreparable DNA damages to kill the cells. Here we investigated the therapeutic efficacy and toxicities of At-211-armed trastuzumab in a mouse model of LMGC positive for HER2. Trastuzumab was conjugated with At-211 to produce alpha-emitting antibodies targeting HER2 ([At-211]-trastuzumab). To generate a mouse LMGC model, we injected luciferase-labelled HER2-positive human metastatic NCI-N87 GC cells into splenic vein of severe combined immunodeficiency mice. Bio-distribution data showed that the maximum uptake of [At-211]trastuzumab in the liver metastatic GC tumors was approximately 12% of injected dose per tissue gram at 24 hours after injection. [At-211]trastuzumab was excreted mostly in the urine. For experimental therapeutics, a single dose of systemic injection of [At-211]trastuzumab (1 MBq) significantly reduced a tumor burden in the liver to extend the survival of model mice significantly compared to controls. Transient leukocytopenia was observed in mice received 1 MBq of [At-211]trastuzumab at 5-7 days after injection. No body weight loss was so far found in the mice treated with [At-211]trastuzumab. No Biochemical abnormalities for Liver and kidney function were found. These data suggest that targeted alpha-particle therapy using [At-211]trastuzumab is effective for LMGC.

Citation Format: Sumitaka Hasegawa, Huizi Keiko Li. Experimental targeted alpha-particle therapy against liver metastasis of HER2-positive gastric cancer in mice [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5343.
Abstract 5350: RBE, in vivo, for a 212Pb-conjugated anti-HER2/neu antibody
Authors: Liatsou Ioanna , Josefsson Anders , Cortez Angel , Hobbs Robert F. , Brayton Cory , Torgue Julien , Sgouros George
Published: September 2020

The relative biological effectiveness is defined as the absorbed dose ratio (Dref(x)/Dtest(x)) of a reference radiation, Dref, to a test radiation, Dtest, that leads to a particular biological end-point, x. The majority of reported RBE values are obtained from in vitro irradiation of cells in monolayer culture wherein clonogenic survival is the end-point. As alpha-particle emitter radiopharmaceutical therapy (RPT) becomes a viable cancer therapy modality, RBE determinations, in vivo, are essential to assessing the potential efficacy and toxicity of alpha-emitter RPT. The aim of the study was to obtain the relative biological effectiveness, RBE, for alpha-particle emissions delivered by Bi-212, following the decay of Pb-212, conjugated onto an anti-HER2/neu antibody. Photon irradiation (200 kVp, 13 mA, 4 Gy/min, 10 × 10 mm2 collimator), delivered by a small animal radiation research platform (SARRP) was used as the reference radiation to irradiate the femurs of 6-8 weeks old female, neu/N transgenic mice (MMTV-neu). The biological endpoint was the reduction in hematopoietic cells in the region of the femur used for the RBE calculation. Alpha-particle emissions were delivered to this region by administering 325 kBq (8.8 μCi) of 212Pb-TCMC-7.16.4 intravenously (tail vein). Mice were sacrificed (n=3) at 1-7 days post-212Pb-TCMC-7.16.4 antibody administration. The long bones (femurs) were harvested, fixed with formalin and counted for radioactivity. The samples were also examined for histopathologic changes to assess the marrow cellularity. Clinical chemistry and hematological analysis were also performed on blood collected by cardiac puncture. The nadir in blood counts was observed 5 days after radiolabeled antibody injection. An absorbed dose to the marrow from the 212Bi alpha-particle emissions of 1.6 Gy yielded a marrow cellularity reduction of 50 %. The absorbed dose from XRT to achieve the same reduction in marrow cellularity was 5.7 Gy. This gives an RBE of 3.6 for a 212Pb-anti-HER2/neu antibody. Since hematologic toxicity is dose-limiting in almost all antibody-based RPT, in vivo measurements of RBE are critical to avoiding it. 2 Gy to the red marrow of low LET radiation is a threshold between low platelet toxicity grades (1 and 2) vs high (3 and 4) grade toxicity. These results suggest that the toxicity threshold for a 212Pb-antibody would be less than 1 Gy.

Citation Format: Ioanna Liatsou, Anders Josefsson, Angel Cortez, Robert F. Hobbs, Cory Brayton, Julien Torgue, George Sgouros. RBE, in vivo, for a 212Pb-conjugated anti-HER2/neu antibody [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 5350.
Abstract 5352: Acute total body ionizing radiation induces long-term cardiac effects and immediate changes in oxidative carbonylation of cardiac proteins in the rat
Authors: Rosen Elliot T. , Kryndushkin Dmitry , Aryal Baikuntha , Gonzalez Yanira , Chehab Leena , Dickey Jennifer , Rao V. Ashutosh
Published: September 2020

Radiation-induced heart disease represents a significant health risk in the event of an accidental radiation exposure as well as to cancer patients who receive acute doses of irradiation as part of radiation therapy. We utilized the Wistar-Kyoto and spontaneously hypertensive rat models, previously shown to demonstrate drug-induced cardiomyopathy, to evaluate the acute and long-term effects of sub-lethal total body irradiation at two, four, and fifty-two weeks. We examined irreversible oxidative protein carbonylation in the heart immediately following irradiation. Both males and females sustained reduced growth and anemic conditions over a one-year period as reflected by reduced body weight and low red blood cell count. Increased inflammation was detected via elevated IL-6 serum levels selectively in males at four weeks. Serum cardiac troponin T and I analyses revealed signs of cardiomyopathy at earlier timepoints, but high variability was observed, especially at one year. Echocardiography at two weeks following 5.0Gy treatment revealed inflammation and a decrease in cardiac output in the short term, but significant differences were not observed at the one-year timepoint. Cardiac output was decreased in both males and females after two weeks, and statistically significant in females. Following 10.0 Gy irradiation, the heart tissue showed an increase in total protein oxidative carbonylation accompanied by DNA damage indicated γ-H2AX. Using proteomic analyses, several novel proteins showed a marked carbonylation profile including those associated with mitochondrial homeostasis and cardiac contractility. Overall, we present findings of acute oxidative protein, DNA damage, and long-term cardiomyopathy in the irradiated animals, along with oxidative proteomic markers of acute damage.

Citation Format: Elliot T. Rosen, Dmitry Kryndushkin, Baikuntha Aryal, Yanira Gonzalez, Leena Chehab, Jennifer Dickey, V. Ashutosh Rao. Acute total body ionizing radiation induces long-term cardiac effects and immediate changes in oxidative carbonylation of cardiac proteins in the rat [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 5352.
Abstract 5368: A multiparameter assay for HER2 protein detection on circulating tumor cells in non-small cell lung cancer
Authors: Lo Edward , Campton Daniel , Costandy Lillian , Itamoto Heather , Huston Ryan , Gardner Braden , Reese Steve , Ramirez Arturo , Kaldjian Eric P. , George Tad , Wang I-Ming , Pirie-Shepherd Steven
Published: September 2020

Lung cancer biopsy can be difficult to perform successfully; it has a 30% adverse event risk and often provides insufficient material to test. Therefore, a liquid biopsy method to analyze the tumor is advantageous. Analysis of circulating tumor cells (CTCs) by multiparameter immunofluorescence (IF) microscopy allows non-invasive characterization of cancer cell biomarker expression. HER2 is a well-known therapeutic target in breast cancer and studies have shown that HER2 status on CTCs may provide prognostic information about response to anti-HER2 therapies. Less is understood about its frequency and clinical importance in non-small cell lung cancer (NSCLC). The RareCyte platform is uniquely suited for CTC identification and phenotypic characterization with a sensitive, accurate, simple and repeatable workflow from blood collection to CTC characterization and single-cell isolation. We developed an assay for detecting HER2 expression on lung CTCs using the RareCyte platform and validated the assay on model CTC spike-in samples with high (BT-474), medium (MDA-MB-453), low (H1650 and OVCAR-3) and absent (MDA-MB-468) HER2 protein levels. Blood was processed to slides using the AccuCyte Sample Preparation System and stained with anti-HER2 together with the RarePlex Kit for CTC identification (nuclear dye, anti-CD45 to exclude WBC, and anti-cytokeratin/EpCAM for CTCs). The spike-in samples showed the expected trend in per-cell HER2 mean fluorescence intensity values. The percentage of HER2-positive cells was > 90% for the HER2-high, medium and low cell lines, and < 5% for the HER2-negative cell line. The assay was applied to 10 advanced stage (III or IV) post-treatment NSCLC patient samples. At least 1 CTC was found in 6 samples (range 0-2043) and HER2 expression was confirmed in 5 out of the 6 samples, indicating this assay is useful for characterizing HER2 expression in CTCs in NSCLC.

Citation Format: Edward Lo, Daniel Campton, Lillian Costandy, Heather Itamoto, Ryan Huston, Braden Gardner, Steve Reese, Arturo Ramirez, Eric P. Kaldjian, Tad George, I-Ming Wang, Steven Pirie-Shepherd. A multiparameter assay for HER2 protein detection on circulating tumor cells in 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 5368.
Abstract 5400: Pharmacogenomics in the primary and metastatic osteosarcoma: Gene expression profile associated with outcome
Authors: Trujillo-Paolillo Alini , Tesser-Gamba Francine , Petrilli Antonio Sergio , Alves Maria Teresa , Garcia-Filho Reynaldo Jesus , Oliveira Renato , Toledo Silvia Regina
Published: September 2020

Purpose: Osteosarcoma (OS) is the most common malignant bone tumor in children and adolescents. Drug resistance and unfavorable outcome are problems that still affect an important percentage of OS patients. Thus, the aim of the present study was to analyze the expression of genes related to pharmacogenomics in OS.

Procedure: The expression of 32 target genes in 80 paired specimens (pre-chemotherapy primary tumors, post-chemotherapy primary tumors and pulmonary metastases), from 33 patients diagnosed with OS, were analyzed by the real-time PCR methodology. Five normal bone specimens were used as calibrators (control). The target genes are related to drug transport, drug detoxification, doxorubicin and folate pathway, DNA repair, cell cycle and apoptosis. Results: The higher ABCC3 gene expression in pre-chemotherapy primary tumors was associated with worse event-free survival (EFS) (p=0.048, HR=3.41). The higher TOP2A gene expression in post-chemotherapy primary tumors was associated with worse overall survival (OAS) and EFS (p=0.015, HR=5,37; p=0.006, HR=6.36; respectively). The lower MTHFR gene expression in pulmonary metastases was associated with worse OAS and EFS (p=0.027, HR=3.27; p=0.024, HR=3.1; respectively), as well as the lower BCL2L1 gene expression (p=0.018, HR=3.53; p=0.019, HR=3.29). Furthermore, lower expression of the RALBP1, ABCC2 and SOD1 genes in the pulmonary metastasis specimens were associated with worse EFS (p=0.022, HR=3.26; p=0.048, HR=3.16; p=0.027, HR=3.14; respectively). Moreover, the pulmonary metastases presented higher expression of the ABCC1 and ABCC4 genes (p=0.049; p =0.039; respectively) and lower expression of the ABCC10, ERCC2, MSH2, SLC22A1, SOD1 and TOP2A genes (p=0.049; p=0.043; p=0.043; p=0.017; p=0.048; p=0.005; respectively) than pre-chemotherapy primary tumors.

Conclusion: Therefore, the pharmacogenomics profile may be useful as prognostic factors as well as therapeutic targets. Thus, our findings may, in the future, contribute to clinical management in the primary and metastatic OS.

Citation Format: Alini Trujillo-Paolillo, Francine Tesser-Gamba, Antonio Sergio Petrilli, Maria Teresa Alves, Reynaldo Jesus Garcia-Filho, Renato Oliveira, Silvia Regina Toledo. Pharmacogenomics in the primary and metastatic osteosarcoma: Gene expression profile associated with outcome [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 5400.
Abstract 541: CLL-1 specific chimeric antigen receptor T cells (CART) as a novel AML therapy
Authors: Freitas Antonio , Gautron Anne-Sophie , Sugita Mayumi , Goswami Srinjoy , Galetto Roman , Garton Andrew , Smith Julianne , Gouble Agnes , Becker Michael W. , Roboz Gail J. , Guzman Monica L.
Published: September 2020

Acute myeloid leukemia (AML) is a disease with high incidence of relapse that is originated and maintained from leukemia stem cells (LSCs). Hematopoietic stem cells (HSCs) can be distinguished from LSCs by an array of markers including CD123, CLL-1, amongst others. Thus, these are thought to be unique candidates to eliminate LSCs using a variety of targeted approaches, including chimeric antigen receptor (CAR) T cells. Here, we evaluated the potential of CLL-1 as a therapeutic target for LSCs using allogeneic gene-edited CAR T cells (UCARTCLL1). UCARTCLL1 cells are TCRαβneg human T cells derived from healthy donors, and expressing an anti-CLL-1 CAR under the control of the endogenous TRAC promoter. This is achieved following a 2-in-1 TRAC knock-out CAR knock-in strategy using TALEN® gene-editing technology. UCARTCLL1 cells are also knocked-out for the beta 2 microglobulin gene. We first assessed the expression of CLL-1 across samples from 41 AML patients and 4 healthy donors. The AML samples included 32 de novo and 9 relapse samples. Using multi-parameter flow cytometry, we evaluated CLL-1 expression on blasts and phenotypically defined leukemic stem cells (CD34pos CD38neg). We found that the majority of the blasts were positive for CLL-1. Furthermore, we found that 36 out of 41 primary AML samples had phenotypically defined stem cells (CD34pos CD38neg) from which 35 showed more than 0.1% CLL-1pos cells. Previous work has shown that individuals with more than 0.1% putative LSCs will have worse outcomes (Van Renhen et al). Thus, this data suggests that there is a subset of putative CLL-1 positive LSCs that if not eliminated by current therapies could lead to disease relapse. Therefore, we sought to determine the therapeutic potential of UCART cells targeting CLL-1. First, we evaluated cytotoxic efficacy of UCARTCLL1 cells at different effector to target ratios in 7 primary AML samples with different levels of CLL-1 expression. We observed a significant cytotoxic effect specific to the CLL1pos subsets. Notably, CLL-1pos cells in the AML samples treated with 5:1 and 1:1 ratios were completely eliminated. Currently, we are evaluating the in vivo activity of UCARTCLL1 cells in AML-PDX mouse models to determine their efficacy at eliminating AML blasts, progenitors and LSCs.

Citation Format: Antonio Freitas, Anne-Sophie Gautron, Mayumi Sugita, Srinjoy Goswami, Roman Galetto, Andrew Garton, Julianne Smith, Agnes Gouble, Michael W. Becker, Gail J. Roboz, Monica L. Guzman. CLL-1 specific chimeric antigen receptor T cells (CART) as a novel AML 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 541.
Abstract 5436: Purification and mRNA profiling of extracellular vesicles for early detection of hepatocellular carcinoma
Authors: Lee Yi-Te , Sun Na , Zhang Ryan Y. , Kao Rueihung , Chen Pin-Jung , Teng Pai-Chi , Wang Jasmine J. , Yang Yingying , Kim Minhyung , Posadas Edwin M. , You Sungyong , Yang Ju Dong , Agopian Vatche G. , Tseng Hsian-Rong , Zhu Yazhen
Published: September 2020

Hepatocellular carcinoma (HCC) is the 4th most common cause of cancer-related deaths worldwide. The poor prognosis of HCC is due to the fact that diagnosis is often made at an advanced stage in disease development. It is crucial to develop a non-invasive liquid biopsy-based diagnostic solution for early detection of HCC. In this study, we developed a covalent chemistry-based nanostructured silicon substrate (“EV Click Chip”) for the isolation of HCC extracellular vesicles (EVs). The EV Click Chip leverages specific click chemistry reactions and sensitive multi-marker cocktail antibody identification of the HCC EVs. The EV Click Chip also allows for the subsequent release of the captured HCC EVs and is optimal for the downstream molecular analysis. A well-validated 10 liver-specific genes were quantified using reverse transcription droplet digital PCR (RT-ddPCR) in HCC EVs purified by the optimized EV Click Chips for the detection of HCC. Our EV Click Chip-based HCC-EV Assay is able to differentiate HCC from non-HCC controls (chronic liver diseases, healthy donors and other cancers) and outperformed clinical AFP test for distinguishing early-stage HCC from at-risk cirrhotic patients.

Citation Format: Yi-Te Lee, Na Sun, Ryan Y. Zhang, Rueihung Kao, Pin-Jung Chen, Pai-Chi Teng, Jasmine J. Wang, Yingying Yang, Minhyung Kim, Edwin M. Posadas, Sungyong You, Ju Dong Yang, Vatche G. Agopian, Hsian-Rong Tseng, Yazhen Zhu. Purification and mRNA profiling of extracellular vesicles for early detection of hepatocellular carcinoma [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 5436.
Abstract 5442: The Collaborative Ocular Oncology Group study number 2: A prospective multi-center evaluation of uveal melanoma prognostic biomarkers
Authors: Decatur Christina L. , Durante Michael A. , Correa Zelia M. , Aaberg Thomas M. , Mruthyunjaya Prithvi , Schefler Amy C. , Harbour J. William
Published: September 2020

Introduction: Uveal melanoma (UM) is a highly aggressive eye cancer that leads to metastatic death in up to half of patients. UM has a propensity to undergo early micrometastasis prior to treatment of the primary tumor, with later emergence of overt metastatic disease. UM can be stratified into Class 1 (low metastatic risk) and Class 2 (high metastatic risk) using a prospectively validated 12-gene expression profile (GEP) molecular prognostication classifier. The Collaborative Ocular Oncology Group Study 2 (COOG2) is being conducted to prospectively evaluate new prognostic biomarkers in UM, including expression of PRAME, and prognostic driver mutations in BAP1, SF3B1, and EIF1AX.

Methods: The COOG2 is a 24 center, prospective study of UM patients undergoing prognostic testing using the CLIA-certified DecisionDx-UM test (Castle Biosciences, Inc) between July 2017 and December 2019. Molecular analysis is performed on residual clinical samples. Data collected include clinical annotations, patient outcomes, GEP, PRAME status, and mutations in BAP1, SF3B1, and EIF1AX.

Results: To date, there are 1300 participants enrolled. The baseline characteristics are as follows: 52% male, and 48% female; 87% non-Hispanic, 3% Hispanic, and 10% unknown. GEP classification is 66% class 1 and 34% class 2. PRAME status is 68% PRAME (-) and 32% PRAME (+). Targeted sequencing has been performed on 650 samples and is being analyzed.

Conclusions: The COOG2 will establish a new standard of prognostic testing accuracy from a single fine needle biopsy in UM. The resulting integrated prognostic testing platform will be used as a stratification tool for entry into clinical trials of adjuvant therapy in high-risk patients.

Citation Format: Christina L. Decatur, Michael A. Durante, Zelia M. Correa, Thomas M. Aaberg, Prithvi Mruthyunjaya, Amy C. Schefler, J. William Harbour. The Collaborative Ocular Oncology Group study number 2: A prospective multi-center evaluation of uveal melanoma prognostic biomarkers [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 5442.