bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 6305: Monitoring the activities of ectonucleotidases CD39 and CD73 and their specific modulation by antibodies and small molecules using homogenous HTS formatted platform
Authors: Goueli Said A. , Hsiao Kevin , Zegzouti Hicham
Published: September 2020

The success of immune oncology for treating a wide variety of cancers ushered a new paradigm in cancer therapy. However, due to some limitations in some patients, it is imperative to identify other pathways that are exploited by cancer cells to circumvent immune surveillance, and possibly synergize immune checkpoint treatment in those cases. Recent recognition of significant increase in adenosine levels in the tumor microenvironment and that adenosine/adenosine receptors play a powerful role as immunosuppressant and attenuating several effector T cell functions have spurred interest in targeting ectonucleotidases that generate adenosine. The two main enzymes responsible for generating adenosine in the microenvironment are the ectonucleotidases CD39 and CD73, the former utilizes both ATP and ADP and producing AMP while the latter utilizes AMP and generates adenosine. Thus, these two enzymes combined are the major source for the bulk of adenosine produced in the microenvironment. They were shown to be validated targets in oncology leading to several clinical trials that include small molecules as well as antibodies, showing positive and encouraging results in the preclinical arena. To facilitate the development of novel drugs to target these enzymes, we have developed a platform that can be utilized to monitor the activities of both enzymes in vitro (biochemical) as well as in cells (cell based) assays. It is very sensitive, and the assays are homogenous and enabled us to monitor the activity of both purified enzymes and enzymes bound to the cellular membranes. Most significant is the ability to discern the selectivity of known small molecule inhibitors as well as selective monoclonal antibodies. Our recent expansion of testing of many small molecules and several antibodies have confirmed the utility of this approach for discovering novel therapeutics that might synergize other regiments such as chemotherapies, and in the design of targeting inhibitors. This should speed up screening for novel inhibitors leading to more effective cancer therapy

Citation Format: Said A. Goueli, Kevin Hsiao, Hicham Zegzouti. Monitoring the activities of ectonucleotidases CD39 and CD73 and their specific modulation by antibodies and small molecules using homogenous HTS formatted platform [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6305.
Abstract 6313: A transcription block survival assay for the discovery of functional inhibitors of transcription factors
Authors: Madden Sarah K. , Mason Jody M.
Published: September 2020

Transcription factor dysregulation is often key to oncogenesis and cancer progression. Direct inhibition of transcription factors provides a logical therapeutic strategy but the broad and shallow interaction interfaces of their protein-protein and protein-DNA interactions are challenging to target using traditional small-molecule approaches. The larger size of peptides provides an opportunity to span such interfaces with the exquisite affinity and selectivity required, and a number of peptide-based inhibitors are starting to emerge.

In this study we focused on inhibiting basic leucine zipper transcription factors (bZIPs) as proof of principle. A key therapeutically relevant protein-protein interaction of bZIPs is the dimerization interface. Developing efficient peptide inhibitors of bZIP transcription factors has generally focused on either rational design or screen-based assays to identify peptides best able to inhibit dimerization. However, a major step in which bZIPs induce cancer initiation and progression is through DNA binding and the most effective inhibitors of bZIP dimerization do not necessarily translate into those best able to block the bZIP-DNA interaction. An assay that is able to screen for peptides that inhibit dimerization of the target transcription factor and – importantly – also selects according to the ability to inhibit protein-DNA interactions, holds significant promise in the development of more potent inhibitors.

To achieve this aim we have developed a Transcription Block Survival assay. Here key binding sites are placed into a gene essential for E.coli survival. The target transcription factor then blocks transcription of this essential gene by binding to these sites, leading to cell death. When a peptide inhibitor from a peptide library is introduced into the system that is able to effectively inhibit DNA binding of the transcription factor, the gene is transcribed, restoring growth. Libraries can have up to two million peptides and allow the assay to select for peptides that not only bind to the target transcription factor, but that are also able to differentiate peptides on their ability to inhibit the transcription factor from binding DNA.

The assay has been fully optimized for one bZIP target, and is currently undergoing the final stages of optimization for two other bZIP targets. Future work will focus on screening peptide libraries against these systems. This assay provides a route towards more efficient selection of potent inhibitors of transcription factors for the treatment of cancer.

Citation Format: Sarah K. Madden, Jody M. Mason. A transcription block survival assay for the discovery of functional inhibitors of transcription factors [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 6313.
Abstract 6316: High throughput small molecule screening with synthetic 3D lung-mimetic hydrogels in the rare lung cancer lymphangioleiomyomatosis
Authors: Pietrobon Adam , Yockell-Lelievre Julien , Doré Carole , Tam Roger Y. , Delaney Sean P. , Shoichet Molly , Stanford William L.
Published: September 2020

Lymphangioleiomyomatosis (LAM) is a rare lung cancer characterized by immature smooth muscle-like cell invasion of the lung parenchyma, leading to cystonodular destruction and respiratory decline. Interestingly, LAM is a monogetic disease caused by loss of TSC2, conferring constitutive mTORC1 activation. Currently, there are no approved therapeutics which exhibit LAM cell-specific cytotoxicity. Here, we describe a novel 3D drug screening platform which couples a rationally designed, synthetic, lung-mimetic hydrogel to high content imaging. We have designed and synthesized a hyaluronic acid-based hydrogel which mimics endogenous lung tissue. Human LAM cell models maintain mTORC1 activation in the hydrogel compared to matched controls, reflective of human LAM lesions. We observe LAM cells to exhibit both protease-dependent and independent modes of invasion: the former imparted by gel crosslinking with an MMP-cleavable peptide, the latter enabled by methylcellulose conjugation. Proliferation is inhibited as LAM cells actively invade through the gel. We coupled our culture system to high content confocal microscopy, permitting measurements of cell viability and invasion depth in response to therapeutic screening. We subsequently tested an 800-drug library of Health Canada-approved small molecule cancer therapeutics on LAM cells and matched controls. Surprisingly, LAM cells exhibited pan-therapeutic resistance as measured by both invasion and viability metrics. Enrichment analysis revealed categories of therapeutics which demonstrated LAM-selective cytotoxicity and/or anti-invasiveness. We performed a refinement screen on select therapeutics and identified AURA inhibition as an effective LAM cell-specific therapeutic avenue. Work is ongoing to establish the most efficacious small molecule inhibitor. In conclusion, we describe a novel drug screening platform which enables high content measurements of viability and invasion in response to drug screening in a lung-mimetic system. We have identified AURA inhibition as a potential therapeutic avenue for LAM patients using this screening platform. Future work will involve target validation, elucidation of the mechanism of action, and testing in a pre-clinical mouse model. As these small molecules are Health Canada-approved, we anticipate rapid clinical translation of our preferred candidate.

Citation Format: Adam Pietrobon, Julien Yockell-Lelievre, Carole Doré, Roger Y. Tam, Sean P. Delaney, Molly Shoichet, William L. Stanford. High throughput small molecule screening with synthetic 3D lung-mimetic hydrogels in the rare lung cancer lymphangioleiomyomatosis [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 6316.
Abstract 6331: Cloaking VEGF in small extracellular vesicles as a cause of adaptive resistance to anti-VEGF therapy
Authors: Ma Shaolin , Mangala Lingegowda S. , Hu Wen , Bayaktar Emine , Hu Wei , Jennings Nicholas B. , Coleman Robert L. , Lopez-Berestein Gabriel , Sood Anil K.
Published: September 2020

Despite the wide use of anti-vascular endothelial growth factor (VEGF) therapy (AVT) for many solid cancers, most of them become resistant to such therapy, resulting in progressive disease. Therefore, new biomarkers and strategies for blocking adaptive resistance of cancer to AVT are needed. Small extracellular vesicles (small EVs, with a size of <200 nm) have been extensively studied as crucial intercellular messengers by delivering DNA, RNA, and proteins to recipient cells. We hypothesized that the packaging of angiogenic factors, especially VEGF, into small EVs could cause adaptive resistance to AVT by evading recognition by therapeutic antiboides. In this study, we carried out a series of in vitro (immunoblot analysis, flow cytometry, tube formation, and immunofluorescence staining) and in vivo (subcutaneous mouse models and Matrigel plug angiogenesis assay) experiments to determine the roles of small EVs in AVT resistance. We found that cancer-derived small EVs increasingly package VEGF and other factors in response to AVT. Further studies showed that bevacizumab cannot recognize the VEGF packaged inside small EVs (eVEGF) and eVEGF was protected from being neutralized by bevacizumab. Notably, eVEGF was transferred to endothelial cells, triggered intracrine signaling, and subsequently increased the number of tubes formed in an in vitro angiogenesis assay. In addition, VEGF was present at higher levels in serum small EVs in mouse models of adaptive resistance of ovarian cancer to bevacizumab than in models of sensitivity to it. Ovarian cancer cell-derived eVEGF increased tumor growth despite treatment with bevacizumab. Collectively, our data provide a new mechanism whereby eVEGF evades recognition by therapeutic antibodies and promotes tumor angiogenesis and progression. These findings have implications for biomarkers and new therapeutic strategies for ovarian cancer therapy.

Citation Format: Shaolin Ma, Lingegowda S. Mangala, Wen Hu, Emine Bayaktar, Wei Hu, Nicholas B. Jennings, Robert L. Coleman, Gabriel Lopez-Berestein, Anil K. Sood. Cloaking VEGF in small extracellular vesicles as a cause of adaptive resistance to anti-VEGF 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 6331.
Abstract 6338: High-throughput screening program to target ovarian cancer leader cells
Authors: Karimnia Nazanin , Bilandzic Maree , Plebanski Magdalena , Stephens Andrew N.
Published: September 2020

Despite its relatively low incidence ovarian cancer is one of the most lethal gynecological malignancies, characterized by high recurrence rate following primary therapy and almost universal acquisition of chemoresistance. Recently, we identified a subpopulation of “Leader Cells” (LCs) that control invasion, progression and acquired chemoresistance. Therapies targeting leader cells are likely to be pivotal to improving prognosis, particularly in patients who are refractory to standard-of-care. To investigate the role of LCs in chemoresistance, we used CRISPR to integrate an in-frame T2A-GFP expression cassette downstream of the native LC marker gene, Keratin 14 (KRT14). Genomic integration drives the LC specific expression of GFP, facilitating visualization and quantitative tracking of the LC subset. The status of GFP as a surrogate marker of LCs in vitro was confirmed by immunostaining and expression analyses. Chemotherapeutic intervention studies and co-staining with the apoptosis marker cleaved caspase 3, demonstrated a significant enrichment of LCs that represent a non-apoptotic subset of cells resistant to standard chemotherapy. High-throughput drug screening, combining large functional drug libraries with high-throughput viability and invasion assays, was used to identify drugs that could inhibit LC function in vitro. Ovarian cancer cell lines (SKOV3 and COV362.4 expressing KRT14-T2A-GFP) were screened against 1450 FDA-approved drugs, and 218 hits identified that inhibited LC viability, invasive capacity or both. Amongst the hits that we identified, the neural signaling pathway emerged as a potential novel mediator for LC function. Further validation experiments confirmed that inhibition of the neural signaling pathway did not affect viability but synergized with chemotherapy to reduce cisplatin IC50 by 10-20-fold. Our data demonstrate that the high-throughput screen developed is a viable approach to identify opportunities for drug repurposing. Moreover, the neural signaling pathway appears to be important in LC function and inhibiting this pathway results in increased sensitivity to chemotherapy in vitro. Ongoing work will evaluate the applicability of this new tool for clinical screening programs, and further, validate existing drugs for potential repurposing.

Citation Format: Nazanin Karimnia, Maree Bilandzic, Magdalena Plebanski, Andrew N. Stephens. High-throughput screening program to target ovarian cancer leader 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 6338.
Abstract 6360: Development of a new substituted 1,3-dioxoisoindoline series that are RPS6K1 protein kinase inhibitors as potential cancer therapeutics
Authors: Sridhar Jayalakshmi , Komati Rajesh , Bratton Melyssa
Published: September 2020

The 40S ribosomal S6 kinase 1 (S6K1), a conserved serine/threonine protein kinase, is the principal kinase effector downstream of the PI3K/mTOR regulatory signaling pathway. Over-expression of RPS6K1 has been associated with cell transformation and elevated proliferation rates in tumors, poor prognosis and an increased risk of local recurrence. RPS6K1 has been found to play an important role in the progression of ER-positive breast cancer, HER2 positive breast cancer and node-negative premenopausal breast cancer. Overexpression of RPS6K1 is closely correlated with aggressive progression and poor prognosis in prostate cancer patients. Inhibition of this kinase can prove to be beneficial for the treatment of several types of cancer including breast cancer, prostate cancer, non-small cell lung cancer, etc. Our laboratory has identified 7-amino-5-amido-1,3-dioxoisoindolines as S6K1 inhibitors with low micromolar IC50 values (2-5 μM). These compounds also inhibited the growth of prostate cancer cells DU-145 and breast cancer cells MCF-7 (ER-positive) and SKBR3 (HER2-positive). The IC50 values of growth inhibition of SKBR3 breast cancer cell lines by the compounds RJ19, RJ22 and RJ28 are 3.3, 1.9 and 4.7 μM, respectively. The core structure 5,7-diamino-1,3-dioxoisoindoline was found to selectively inhibit RPS6K1 over 99 other oncologic protein kinases. This series of compounds have the potential to become viable therapeutics for many types of cancer. The results from the synthesis of 7-amino-5-amido-1,3-dioxoisoindoline derivatives, the structure activity studies, and their efficacy studies will be presented.

Citation Format: Jayalakshmi Sridhar, Rajesh Komati, Melyssa Bratton. Development of a new substituted 1,3-dioxoisoindoline series that are RPS6K1 protein kinase inhibitors as potential cancer therapeutics [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 6360.
Abstract 6363: Small molecule PG3-Oc partially restores the p53 pathway transcriptome and proteome in tumors with mutant p53 through ATF4 transcriptional modulation by ERK1/2 and CDK9
Authors: Tian Xiaobing , Ahsan Nagib , El-Deiry Wafik S.
Published: September 2020

The tumor-suppressor p53 prevents cancer development via initiating cell-cycle arrest, cell death, repair, or anti-angiogenesis processes. Over 50% of human cancers harbor cancer-causing mutations in p53. p53 mutations not only abrogate its tumor-suppressor function, but also endow mutant p53 with a gain of function (GOF), creating a proto-oncogene that contributes to tumorigenesis, tumor progression, and chemo- or radiotherapy resistance. Thus, targeting mutant p53 and/or restoring a wild-type p53 signaling pathway provides an attractive strategy for cancer therapy. We previously reported that prodigiosin, a natural product, has potent anti-cancer activity against tumors with mutated p53. The novel synthetic analogue of prodigiosin, PG3-Oc, is potent in reducing both short-term and long-term proliferation in a broad panel of mutant p53 cell lines. PG3-Oc treatment induces genome-wide restoration of p53 target genes, which are mediated by activation of ATF4. We demonstrate that ERK1/2 and CDK9 kinase functions are required for ATF4 transcriptional activity in mutant p53-expressing cancer cells. We have characterized a mechanism of PG3-Oc that engages the apoptosis pathway by upregulation of the pro-apoptotic p53 target gene PUMA in a time-and dose-dependent manner through the ATF4/PUMA axis in colorectal cancer cell lines. We further report the impact of PG3-Oc on the p53 transcriptome and proteome and the role of ATF4. Our results demonstrate feasibility of functional cellular reprogramming to restore p53 pathway signaling and provide novel insights into mechanisms of signaling leading to cell death and tumor suppression with potential clinical relevance in cancer therapy.

Citation Format: Xiaobing Tian, Nagib Ahsan, Wafik S. El-Deiry. Small molecule PG3-Oc partially restores the p53 pathway transcriptome and proteome in tumors with mutant p53 through ATF4 transcriptional modulation by ERK1/2 and CDK9 [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 6363.
Abstract 6371: Gaseous nitric oxide at high concentration is a powerful anti-tumor agent both in-vitro and in-vivo
Authors: Confino Hila , Lisi Steve , Kalaora Rinat , Goldshtein Matan , Avniel Amir
Published: September 2020

Nitric Oxide (NO) acts as a signaling molecule in the body in different pathologic conditions, including cancer. Beyond Air is developing an innovative gaseous Nitric Oxide (gNO) based tumor ablation method. The highly diffusible nature of NO may ease its spreading inside the tumor, while its short half-life may limit its effect to the tumor site and not affect the surrounding area.

According to our preliminary data, exposure of gNO at 10,000-50,000ppm for a very short time (in some cases one minute or less) efficiently eradicates breast, colon, lung and prostate cancers and shows a potent mouse tumor's destruction in-vivo.

Methods: Mouse colon and breast cancer cell lines were exposed to gNO at 150-50,000 ppm for 10 seconds - 180 seconds. Cell viability was examined 24hrs later by XTT assay.

Mouse colon tumors were treated by gNO at 10,000ppm - 50,000ppm intratumorally by hypodermic needles or local exposure of the tumor using a plastic box or tube.

Results: To test if gNO at 150-50,000ppm can decrease cancer cell viability, cells were exposed (I) directly to NO in a 96 well plate or (II) in an acrylic glass box. Cell viability was tested 24hrs later by XTT. According to our findings, less than 3% of 4T1 cells remain viable when exposed to 10,000ppm NO for 3 minutes or 50,000ppm NO for 1 minute in a 96-well plate. ~1% of CT26 cells remain viable after exposure to 50,000ppm NO for 10 seconds in a 96 well plate; while less than 10% remain viable after 1 minute of exposure of 50,000ppm NO in a box.

To test if gNO at 10,000-50,000ppm can destroy mouse colon cancer tumors in-vivo, tumors were exposed to gNO locally by hypodermic needles or a plastic cover. Air treated tumor bearing mice served as the control group.

A complete tumor destruction was observed when the skin was removed from the tumor and the entire tumor was exposed to 10,000ppm gNO for 10 minutes.

Conclusion: In the current work, gNO at high concentration showed a substantial cytotoxic effect on cancer cells in-vitro and in-vivo. The proposed project represents development of a novel treatment for cancer. Our innovative gNO based treatment may serve to treat solid tumors locally or be used as an adjunctive therapy for surgery and can be synergized with common anticancer therapies, such as chemotherapy and radiotherapy, to further enhance the effect.

Citation Format: Hila Confino, Steve Lisi, Rinat Kalaora, Matan Goldshtein, Amir Avniel. Gaseous nitric oxide at high concentration is a powerful anti-tumor agent both in-vitro and in-vivo [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 6371.
Abstract 6409: Targeted degradation of endogenously tagged proteins for phenotypic studies using HaloPROTAC3 and HaloTag technologies
Authors: Caine Elizabeth A. , Mahan Sarah , Riching Kristin , Murphy Nancy , McDougall Mark , Corona Cesear , Heid Chris , Daniels Danette L. , Urh Marjeta
Published: September 2020

To understand a protein's function inside the cell, studies are often done to remove it using CRISPR-mediated knockout or RNAi knockdown methods. These approaches have challenges in either obtaining efficient or complete loss, are not possible if the protein target is essential for cell growth, and are genetic methods, not directly protein loss. To overcome these hurdles, we have employed a highly precise and temporally controlled target protein degradation strategy utilizing HaloPROTAC, a HaloTag Proteolysis-targeting chimera small molecule which will specifically degrade HaloTag fusion proteins in live cells. HaloPROTAC3, developed by Prof. Craig Crews at Yale University, recruits HaloTag fusion proteins to VHL E3 ligase complexes, resulting in ubiquitination and degradation of the HaloTag fusion via the ubiquitin-proteasomal pathway. For phenotypic studies, removal of the endogenous proteins is often required, therefore we have developed efficient protocols for the introduction of HaloTag via CRISPR/Cas9 gene editing into either the endogenous N- or C-terminal loci of any target protein. We have additionally appended the 11 amino acid HiBiT to the HaloTag-target protein, allowing for highly quantitative and kinetic monitoring of degradation to be tracked in live cells without the use of antibodies. Shown here are several key therapeutic proteins, BRD4 and B-catenin, which have been endogenously tagged with HiBiT-HaloTag via CRISPR/Cas9 and show rapid and sustained degradation (80-90% loss) after treatment with HaloPROTAC3. For BRD4, this loss was similar to what was observed using a BET-family specific PROTAC. For B-catenin, further phenotypic studies were done to show a muted response to Wnt3a stimulation and TCF/LEF promoter gene activation after degradation of endogenous B-catenin. These examples demonstrate that the technologies can be used readily to elicit robust degradation of target proteins, with control over the protein level and the time frame for which the protein will be lost, in order to more fully understand protein function. Together, these provide new opportunities for phenotypic studies based directly on control of protein levels and loss, with the ability to study the roles of essential proteins not amenable to genetic knockout or knockdown approaches.

Citation Format: Elizabeth A. Caine, Sarah Mahan, Kristin Riching, Nancy Murphy, Mark McDougall, Cesear Corona, Chris Heid, Danette L. Daniels, Marjeta Urh. Targeted degradation of endogenously tagged proteins for phenotypic studies using HaloPROTAC3 and HaloTag technologies [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 6409.
Abstract 6436: A potential treatment for malignant glioblastoma: Silver nanoparticles and low-level laser combination
Authors: Atluri Rohini , Korir Daniel , Tyagi Nishant , Kim Eunyoung , Choi Tae-Youl , Simmons Denise P.
Published: September 2020

Glioblastomamultiforme (GBM) is a dreadful primary brain tumor characterized with extremely poor prognosis and survival rate, despite available treatments. The failure of current therapies to eliminate specific subpopulations within heterogeneous GBM has been considered a key factor contributing to the inevitable recurrence after treatments. To address this concern, it is necessary to develop novel treatments, which can kill all the GBM cells or at least convert the heterogeneous GBM population to homogeneous populations, which can be easily targeted. In this study, we investigated the effects of Ag-PMMA-PAA nanoparticles (Silver np's) on human malignant glioma(U251) cells and Silver np's role in combinational use with low-level He-Ne laser (632.8nm) against U251 cells.The Silver np's were synthesized using a green chemistry approach and were then characterized using spectroscopic methods. Dose and time dependent studies revealed that the maximum killing (81%) of U251 cells occurred at 225μM Silver np's over 6 hours. Moreover, combined HeNe laser exposure resulted in overall cell death of 92%. Western analysis suggests caspase family members, other than caspase 9, are involved in killing Silver np treated U251 cells or the combination treatment. Thus, our Silver np and low-level laser combination treatment could have a potential application in enhancing chemotherapy for malignant glioma.

Citation Format: Rohini Atluri, Daniel Korir, Nishant Tyagi, Eunyoung Kim, Tae-Youl Choi, Denise P. Simmons. A potential treatment for malignant glioblastoma: Silver nanoparticles and low-level laser combination [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 6436.
Abstract 6442: Investigation of custom biomarkers on circulating tumor cells from clinical samples using RarePlex® Developer Kits
Authors: Lo Edward , Campton Daniel , Ramirez Arturo , Costandy Lillian , Gardner Brady , Houston Ryan , Itamoto Heather , Werbin Jeffery L. , Gadi VK , Mojica Tanisha , Clein Alisa , Higano Celestia , Sabath Daniel E. , Kaldjian Eric P. , George Tad
Published: September 2020

Enumeration and phenotypic profiling of circulating tumor cells (CTCs) can give important information about tumor progression, presence of therapeutic targets, and metastatic potential. New and informative cancer-specific biomarkers are being discovered at a rapid pace, so there is a strong need for tools that enable investigator driven assays to best study and utilize these biomarkers. Through the RareCyte platform, we provide sensitive and specific assays that are optimized and validated for CTC enumeration and biomarker expression. RarePlex Developer Kits enable the addition of user-selected antibodies against biomarkers of interest to a CTC detection assay. Here we demonstrate the application of RarePlex Developer Kits to study the presence of a variety of cancer related biomarkers. Using the Developer strategy, we present results for several biomarkers, including HER2, ER, PR, EGFR, Ki67, AR, ARv7, PDL1, and PSMA. We also characterized clinical samples from prostate (AR and ARv7) and breast (HER2 and ER) cancer patients. The biomarkers demonstrated proper localization on or within model CTC control cells when using default antigen retrieval and fixation conditions. For each biomarker, fluorescence intensity cut-offs that segregated negative and positive cell lines were statistically defined to maximize classification accuracy. For clinical samples, breast and prostate cancer sample staining showed expected localization based on available clinical information. In conclusion, RarePlex Developer Kits provide a flexible tool for custom CTC assay development that enables researchers to develop assays in their own lab for characterization of phenotypic heterogeneity.

Citation Format: Edward Lo, Daniel Campton, Arturo Ramirez, Lillian Costandy, Brady Gardner, Ryan Houston, Heather Itamoto, Jeffery L. Werbin, VK Gadi, Tanisha Mojica, Alisa Clein, Celestia Higano, Daniel E. Sabath, Eric P. Kaldjian, Tad George. Investigation of custom biomarkers on circulating tumor cells from clinical samples using RarePlex® Developer Kits [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 6442.
Abstract 6450: Circulating hepsin as a novel serum biomarker in prostate cancer patients
Authors: Beard John , Eddington Sam , Bowman Noah , Cole Adam , O'Brien Timothy , Johnson Blake P.
Published: September 2020

Hepsin, a type II transmembrane serine protease, is commonly overexpressed in a variety of epithelial cancers, where its overexpression and concurrent proteolytic activity correlate with tumor progression. Recent studies indicate that Hepsin undergoes activation via autocatalysis and subsequent ectodomain shedding, thus highlighting its potential as a serum-based biomarker. Using transgenic mouse models of prostate adenocarcinoma, Hepsin overexpression has previously been implicated in disease progression and, notably, metastases that are neuroendocrine in nature. Collectively, these findings led us to explore the potential diagnostic utility of Hepsin expression in prostate cancer progression, inclusive of metastatic variants. Here, we reanalyzed pre-existing, publicly available gene expression datasets (2 datasets, 326 patient samples), which revealed a significant upregulation of Hepsin gene expression in tumor tissue versus benign tissue and further pronounced upregulation in androgen-dependent and -independent metastatic lesions. To further examine the potential correlation of Hepsin overexpression to androgen-independence, we reanalyzed in vitro data comparing LNCaP parental versus androgen-independent LNCaP subclones. This analysis revealed a stepwise increase in Hepsin expression over the course of androgen deprivation, which was accompanied by a concomitant decrease in PSA expression. Lastly, to evaluate circulating hepsin protein levels as they occur in patient serum, a total of 424 subjects suspected of prostate cancer were analyzed for circulating Hepsin levels from 2015 to 2018 at the time of biopsy and followed for recurrence or survival until the end of the study. Serum Hepsin levels, as determined by enzyme-linked immunosorbent assay, rendered a specificity of 89% (Gleason < 6 (3+3); Hepsin positive, >100 ng/mL). Within this cohort, 18 patients presented with biochemical recurrence, of which 44% were Hepsin positive and exhibited a 22-month accelerated clinical path to recurrence versus Hepsin negative patients. Collectively, these results highlight the longitudinal diagnostic potential of circulating Hepsin levels in the prostate cancer setting.

Citation Format: John Beard, Sam Eddington, Noah Bowman, Adam Cole, Timothy O'Brien, Blake P. Johnson. Circulating hepsin as a novel serum biomarker in prostate cancer patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6450.
Abstract 6468: Prognostic genomic alterations associated with recurrence after primary therapy for patients with luminal B breast cancer
Authors: Liu Chun-Yu , Chen Ji-Lin , Yang Yi-Ting , Tsai Yi-Fang , Chao Ta-Chung , Tan Kien Thiam , Wang Wan-Lun , Lien Pei-Ju , Chen Shu-Jen , Tseng Ling-Ming
Published: September 2020

Purpose: Luminal type breast cancers account for the most common subtypes of breast cancers, among which luminal B subtype is relatively faster tumor growing and confers substantial risk of recurrence after primary treatment despite adjuvant endocrine therapy. Understanding the molecular alterations underlying the poorer survival outcome for luminal B tumors represents an unmet medical need.

Experimental design: The archival samples of 16 recurrences within 5 years, 3 recurrences between 5-10 years and 24 non-recurrence tumor tissues from luminal B breast cancer patients were retrieved. Genomic alterations of these 43 breast cancer samples were detected using deep next-generation sequencing of a 440-gene panel. The mRNA levels of candidate genes in breast cancer patients were analyzed from The Cancer Genome Atlas (TCGA) database.

Results: Overall, genomic alterations were found in 340 genes, including single nucleotide variants and copy number variation. Comparing to the non-recurrence tumors, NUP98 (31.3%), MAPK4 (18.8%), PTEN (18.8%), PER1 (18.8%) and TRIP11 (18.8%) showed higher population frequency in the recurrence tumors, and IGF2R (50.0%), PIM1 (45.8%) and ROS1 (37.5%) were dominate in the non-recurrence tumors. After a validation with the public clinical dataset, we identified three potential prognostic biomarkers, including NUP98, PER1, and TRIP11. We observed missense mutation or copy number deletion of NUP98, PER1 and TRIP11 in early recurrence tumors. Data from TCGA database exhibited patients with breast cancer harbored lower NUP98, PER1 and TRIP11 transcripts levels. Notably, lower NUP98, PER1 and TRIP11 expression levels correlated with worse recurrence-free survival in luminal B breast cancer. Interestingly, based on algorithm for visualization of protein interaction network, we found these 3 genes were all involved in androgen receptor signaling network.

Conclusion: Our study identified NUP98, PER1, and TRIP11 which potentially served as biomarkers to predict recurrence in luminal B breast cancer. Further research is required to understand the association between genomic alterations and androgen receptor signaling as well as its clinical impact.

Citation Format: Chun-Yu Liu, Ji-Lin Chen, Yi-Ting Yang, Yi-Fang Tsai, Ta-Chung Chao, Kien Thiam Tan, Wan-Lun Wang, Pei-Ju Lien, Shu-Jen Chen, Ling-Ming Tseng. Prognostic genomic alterations associated with recurrence after primary therapy for patients with luminal B 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 6468.
Abstract 6473: Outcomes of advanced renal cell carcinoma with sarcomatoid features post-cytoreductive nephrectomy in clear cell and non-clear cell disease
Authors: Adashek Jacob J. , Bilotta Alyssa , Chahoud Jad , Li Jiannong , Spiess Philippe E.
Published: September 2020

Background: Cytoreductive nephrectomy in the setting of treatment of advanced renal cell carcinoma with sarcomatoid features (sRCC) has been highly contested in terms of added benefit for survival. Patients with sRCC are known to have a poor prognosis and more aggressive tumor biology.

Methods: Pts with clear cell RCC and non-clear cell RCC underwent cytoreductive nephrectomy in efforts to improve overall survival. Patients were stratified by presence or absence of histologic presence of sarcomatoid features within the tumor samples.

Results: Of 167 patients who underwent cytoreductive nephrectomy, 127 had clear cell, of which 14 had sarcomatoid features and 40 had non-clear cell RCC of which 13 had sarcomatoid features. Median age in the cohort was 62 (range, 56.5-69). The cohort included 119 male (71.3%) and 48 (28.7%) female patients. There were 121 patients with >T3 disease, 24 (14.4%) with T2a/b disease, and 22 (13.2%) with T1a/b disease. In all patients with advanced RCC having sRCC had a significant impact on survival post-cytoreductive nephrectomy (30 vs. 8 mos; HR 2.88; p <0.0001). Additionally, good-risk patients had significantly longer OS compared to intermediate- or poor-risk patients (56 vs 30 vs 10 mos; HR 0.21; p = 0.00016). For patients with clear cell RCC, having sRCC present conferred a poorer significant survival (30 vs 9 mos; HR 2.82; p = 0.0035). Patients with non-clear cell sRCC also had significantly worse outcomes compared to those without sarcomatoid features (30 vs 6.5 mos; HR 3; p = 0.009). When sRCC patients were stratified by whether there was >10% or ≤10% present within the sample, there was no significant difference in overall survival (8 vs 8.5; p = 0.32).

Conclusions: Sarcomatoid features within tumor histology confers significantly poor prognosis. Patients with sRCC regardless of clear cell vs non-clear cell histology have significantly shorter overall survival. Even among patients with 10% or less sarcomatoid features there was no overall survival benefit to cytoreductive nephrectomy. Based on our findings, there appears to be a limited to no role of cytoreductive nephrectomy, if sRCC is identified on pre-treatment biopsy.

Citation Format: Jacob J. Adashek, Alyssa Bilotta, Jad Chahoud, Jiannong Li, Philippe E. Spiess. Outcomes of advanced renal cell carcinoma with sarcomatoid features post-cytoreductive nephrectomy in clear cell and non-clear cell disease [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 6473.
Abstract 6483: Performance characteristics of the first FDA-cleared droplet digital PCR (ddPCR) IVD assay for monitoring chronic myelogenous leukemia
Authors: Shelton Dawne N. , Bhagavatula Prasanthi , Sepulveda Nathan , Beppu Lan , Radich Jerald
Published: September 2020

Introduction: Chronic myelogenous leukemia (CML) is a cancer of myeloid cells that accounts for 15-20% of all cases of leukemia. Most CML cases are caused by a translocation between chromosomes 9 and 22 which creates an abnormal fusion gene, BCR-ABL1. The BCR-ABL1 gene expresses an abnormal tyrosine kinase protein that causes unregulated growth and survival of granulocytes. CML is treated with TKIs (tyrosine kinase inhibitors). The concentration of BCR-ABL1 transcript RNA is a marker of disease progression and the effectiveness of TKI treatment.Bio-Rad's QXDx BCR-ABL %IS Kit is an in vitro diagnostic test for the quantification of BCR-ABL transcripts in total RNA from whole blood from individuals diagnosed with Chronic Myeloid Leukemia expressing BCR-ABL1 Fusion transcript types e13a2 and/or e14a2.

Materials and Methods: The QXDx BCR-ABL %IS Kit is a newly launched FDA-approved ddPCR assay from Bio-Rad Laboratories. Reproducibility of results across multiple sites, days, instruments, and users was evaluated using contrived samples consisting of BCR-ABL positive patient samples mixed with negative patient samples. Clinical performance of the kit was evaluated on patient samples, and compared to an already existing FDA-approved test. Clinical performance was further tested in the College of American Pathologists challenge.

Results: The reproducibility study noted negligible contributions to variance from site, instrument, day, and user for samples spanning from MR 0.7-4.2. The mean between the QXDx™ BCR-ABL %IS Kit and the comparator test using a Bland-Altman analysis was 0.16 MR units. The limits of agreement between the two methods indicate that the difference between the two methods should be between 0.47 and -0.14 MR on 95% of tested samples. The QXDx™ BCR-ABL %IS assay demonstrated excellent correlation with the comparator test using a Weighted Deming regression with a Pearson R correlation coefficient of 0.99, slope 1.037 and intercept of 0.1084. College of American Pathologists(CAP) challenges (n=6) were conducted from 2016-2019, QXDx™ BCR-ABL %IS system results were regressed vs the mean values published by CAP yielded a slope of 1 and R2=0.99.

Conclusions: The QXDx™ BCR-ABL %IS Kit on the QXDx™ AutoDG™ Droplet Digital PCR System can be used safely and effectively in the laboratory on t(9;22) positive CML patients for monitoring of treatment with Tyrosine Kinase Inhibitors (TKIs).

Citation Format: Dawne N. Shelton, Prasanthi Bhagavatula, Nathan Sepulveda, Lan Beppu, Jerald Radich. Performance characteristics of the first FDA-cleared droplet digital PCR (ddPCR) IVD assay for monitoring chronic myelogenous leukemia [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 6483.
Abstract 6491: Prognostic potential of carcinoembryonic antigen expression patterns in lung adenocarcinoma
Authors: Zheng Ming-Yi , Kuo Yen-Shou , Chou Yu-Ting
Published: September 2020

Carcinoembryonic antigen (CEA) is one of the most commonly used tumor markers. Although the suitability of CEA as an effective marker for lung cancer remains debatable, characteristics such as noninvasiveness and lack of other reliable markers make CEA an attractive option in lung cancer. Divergent CEA expression patterns before and after EGFR-tyrosine kinase inhibitor (TKI) treatment have been reported. However, whether these patterns are associated with tumor progression remain elusive. We hypothesized that divergent patterns of serum carcinoembryonic antigen at initial diagnosis (CEAIn) and disease progression (CEAPd) may reflect the diversity of lung cancer cells selected by TKI, conferring distinct progression profiles in patients. Our objective was to verify the prognostic potential of divergent CEA patterns before and after TKI treatment in lung cancer patients with wild-type or mutant EGFR. Of the 1736 lung cancer patients identified from the cancer registry group between 2011 to 2016, we selected 517 patients with advanced stage adenocarcinoma, data on EGFR mutation status and CEAIn, among whom were 288 patients with data on CEAPd, eligible for inclusion in the correlation analysis of clinical characteristics and survival. EGFR wild-type lung adenocarcinoma patients displayed lower CEAIn level than those with EGFR mutations. Multivariable analysis revealed that CEAIn expression was associated with poor progression-free survival in patients harboring mutant EGFR but not in those with wild-type EGFR. Moreover, CEAIn and CEAPd were associated with the good and poor post-progression survival, respectively, only in the EGFR-mutant and not in the wild-type group. In vitro cell line experiments revealed that CEA expression, cancer dissemination, and chemoresistant properties can be affected by EGFR-TKI selection. EGFR-mutant patients, exhibiting high CEAIn (≥ 5 ng/mL) and low CEAPd (< 5 ng/mL), showed a potential toward displaying new metastasis. Our findings support the notion that EGFR mutation status is a critical factor in determining prognostic potential of CEAIn and CEAPd in patients under EGFR-TKI treatment, and divergent CEAIn and CEAPd patterns are associated with distinct cancer progression profiles.

Citation Format: Ming-Yi Zheng, Yen-Shou Kuo, Yu-Ting Chou. Prognostic potential of carcinoembryonic antigen expression patterns in lung adenocarcinoma [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 6491.
Abstract 6499: Small slide, big world: Fast detection of altered proteins and RNA levels in clinical samples using Nanostring nCounter® GeoMxTM Digital Spatial Profiler
Authors: Gong Jingjing , Farren Matthew R. , Shaib Walid , Lesinski Gregory , Warren Sarah , Liang Yan
Published: September 2020

Although many new drugs have been approved and more clinical trials are being conducted for various disease therapies in recent decades, efficient identification of reliable therapeutic targets is still a big challenge during drug development. Therefore, tremendous efforts have been put into advancing the technology to quickly determine the altered biomarkers in clinical samples. Nanostring nCounter GeoMxTM Digital Spatial Profiling (GeoMx DSP) has been used in multiple projects and exhibited its outstanding performance on collecting analyzable digital counts for up-to 96 proteins and 20,000 RNAs in a specific area on a single FFPE slide. Tissue regions of interests are identified by fluorescent staining of morphology markers, and either proteins or RNAs in the specific defined regions are collected as the digital nCounter count format. By using this state-of-art technology, spatial distributions of immune- and cancer- relevant proteins were identified in 71 tumor cell rich-, 70 immune cell rich-, and 69 stromal cell rich- area respectively in three Pancreatic cancer patient groups including surgery alone, neoadjuvant FOLFIRINOX (a combination of fluorouracil, folinic acid, irinotecan and oxaliplatin) alone or combined with stereotactic body radiation therapy (F+SBRT). Our results showed that differential regulations among immune checkpoint markers, pan-immune markers and tumor markers in these treatment groups. In tumor-rich regions, CD44 is present at higher levels in FOLFIRINOX treated patients than surgery-alone group. VISTA was significantly down-regulated in F+SBRT group. Also, several key factors from pStat3, PTEN and Bcl2 pathways are also regulated by treatment methods. In immune-rich regions, both T cells markers (CD3 and CD4) and B cell markers (CD19 and CD20) were regulated differently by treatment methods. GeoMx DSP platform provides a novel and more precise view of the spatial regulation of micro-world in the tissue, which enable us to explore more about internal regulation within sub-population of the tissue cells.

GeoMxTM DSP platform is only for RESEARCH USE ONLY. Not for use in diagnostic procedures.

Citation Format: Jingjing Gong, Matthew R. Farren, Walid Shaib, Gregory Lesinski, Sarah Warren, Yan Liang. Small slide, big world: Fast detection of altered proteins and RNA levels in clinical samples using Nanostring nCounter® GeoMxTM Digital Spatial Profiler [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 6499.