bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2947: Towards advanced precision oncology assays - Selection of immunohistochemistry chromogen impacts microdissection and downstream molecular analysis of archival specimens
Authors: Laun Sarah , Roberge Adam , Weigman Robert , Lewis Aubrey , Greisman Valarie , Liu Meeiyueh , Stephens Owen , Peterson Erich , Shin Ikjae , Emmert-Buck Michael , Tangrea Michael , Johann Donald J.
Published: September 2020

Introduction: Precision medicine promises to improve patient treatment outcomes based on the application of molecular profiling methodologies to pathological specimens. This approach enables the development of rationally derived therapy assignments for each individual patient's tumor (versus categorical treatment assignments). Since tumors are composed of heterogeneous cells forming clusters of subtypes, dissimilar molecular profiles may emerge depending on which region is sampled and analyzed. Additionally, a manual slide scrape may result in a reduced or nearly absent molecular signal (i.e. key driver). Clearly, for cancer there is a need for an efficient, upfront enrichment method to specifically isolate specific tumor cell populations. Immuno-Laser Capture Microdissection (immuno-LCM) is a technology based on immunohistochemistry (IHC) that can isolate specific cell types from a heterogeneous cellular population. We sought to determine the impact of various IHC chromogens on the efficacy of microdissection and downstream molecular analysis from immuno-LCM samples.

Methods: Histological tissue specimens (clinical colorectal and lung cancer PDX) were processed using traditional IHC methods with a variety of chromogens, including: Vector Black, Impact Vector Red, 3-amino-9-ethylcarbazole (AEC), and 3,3'-diaminobenzidine (DAB). These chromogens were evaluated for staining quality, microdissection efficiency, biomolecular extraction, and evaluation of specific molecular targets.

Results: Interestingly, DAB, the most commonly used chromogen, demonstrated a negative impact on the retrieval of biomolecules from immuno-LCM samples. However, chromogens such as Vector Black and Impact Vector Red showed much improved biomolecule extraction compared to DAB even though the yield of microdissected material was comparable. While other chromogens, such as AEC, demonstrated a greater efficiency of biomolecule extraction but showed incomplete immuno-LCM of specific IHC-stained cells.

Conclusions: The data indicate that traditional IHC chromogens should be carefully considered when using immuno-LCM to analyze biomolecules.

Citation Format: Sarah Laun, Adam Roberge, Robert Weigman, Aubrey Lewis, Valarie Greisman, Meeiyueh Liu, Owen Stephens, Erich Peterson, Ikjae Shin, Michael Emmert-Buck, Michael Tangrea, Donald J. Johann. Towards advanced precision oncology assays - Selection of immunohistochemistry chromogen impacts microdissection and downstream molecular analysis of archival specimens [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 2947.
Abstract 299: TAK1 signaling regulates p53 through a mechanism involving ribosomal stress
Authors: Bakin Andrei V. , Zonneville Justin , Wong Vincent
Published: September 2020

Triple-negative breast cancer (TNBC) is among the most aggressive forms of breast cancer with limited therapeutic options. TAK1 is implicated in aggressive behavior of TNBC, while means are not fully understood. Here, we report that pharmacological blockade of TAK1 signaling hampered ribosome biogenesis (RBG) by reducing expression of RBG regulators such as RRS1, while not changing expression of ribosomal core proteins. Importantly, TAK1 blockade upregulated expression of p53 target genes in cell lines carrying wild type (wt) TP53 but not in p53-mutant cells. By examining involvement of the ribosomal stress response, we found that p53 activation by blockade of TAK1 was prevented by depletion of ribosomal protein RPL11. Further, siRNA-mediated depletion of TAK1 or RELA resulted in activation of p53 signaling and this response was dependent on RPL11. Knockdown of RRS1 disrupted nucleolar organization and resulted in activation of p53. Genomic TCGA data showed that TNBCs express high levels of ribosome biogenesis regulators, and elevated RRS1 levels correlate with unfavorable prognosis. Cytotoxicity data showed that TNBC cell lines are more sensitive to TAK1 inhibitor compared to luminal and HER2+ cell lines. Together, the data indicate that TAK1 regulates p53 activation by controlling ribosome biogenesis factors, and the TAK1-ribosome axis is a potential therapeutic target in TNBC.

Citation Format: Andrei V. Bakin, Justin Zonneville, Vincent Wong. TAK1 signaling regulates p53 through a mechanism involving ribosomal stress [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 299.
Abstract 3001: Drug resistant PDX models for new drug discovery
Authors: Zhang Feifei , Long Yuan , Wen Danyi
Published: September 2020

Drug resistant is one of the biggest challenge for cancer therapy. How to understand the mechanism and to overcome the drug resistant with new therapeutics represent the hot focus for oncology drug discovery. Drug resistant patient-derived xenograft (PDX) models derived from surgery samples, biopsy samples, hydrothorax or ascetic fluid provides unique tool for new therapeutics R&D. We have successfully established an array of drug resistant PDX models. Representative drug resistant PDX includes: clinically AZD9291-resistant EGFR triple mutant (Ex19del, T790M, C797S) PDX model. In vivo efficacy results show that this triple mutant NSCLC PDX model was resistant to Erlotinib and AZD9291 and sensitive to the combination of Cetuximab and Brigatinib. We also established CD74-ROS1 fusion and G2032R mutant NSCLC PDX model; EML4-ALK fusion and L1196M mutant NSCLC PDX model; Olaparib resistant Ovarian cancer PDX model; Heceptin resistant gastric PDX model; Rituximab and ABT-199 resistant DLBCL PDX model, and so on. In conclusion, these precious PDX models provide insight into resistance mechanisms as well as serve as models to develop new therapeutics drug candidates that overcome the drug resistant.

Citation Format: Feifei Zhang, Yuan Long, Danyi Wen. Drug resistant PDX models for new drug discovery [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 3001.
Abstract 3029: Population pharmacokinetics and dose regimen evaluation of monalizumab, a monoclonal antibody targeting NKG2a, in patients with advanced solid tumors
Authors: Song Xuyang
Published: September 2020

Background: Monalizumab (IPH2201) is a humanized IgG4 monoclonal antibody that targets NKG2a, resulting in suppression of inhibitory signaling by tumors on NK cells. We aimed to develop a population pharmacokinetics (PK) model of monalizumab and to identify and quantitate the impact of patient/disease characteristics on PK variability, and to compare body weight-based and fixed dosing regimens of monalizumab.

Methods: Data were pooled from two clinical studies: NCT02671435 (Phase 1/2; monalizumab + durvalumab in all comers) and NCT02643550 (Phase 2; monalizumab + cetuximab in SCCHN). 95 patients provided monalizumab PK data following 0.4−10 mg/kg every 2 weeks (Q2W), 22.5 mg−750 mg Q2W, or 750 mg every 4 weeks (Q4W). Population PK modeling was performed using a non-linear mixed effects modeling approach in NONMEM software (v. 7.2). Impact of demographics, clinical indices, and biomarkers on PK was explored. The appropriateness of the final model was tested using diagnostics including visual predictive check (VPC).

Results: Monalizumab PK profiles were best described using a linear 2-compartment model. The clearances (CL) and central volume of distribution (V1) were 0.27 L/day and 3.6 L with a modest between-subject variability of 40% and 30% for CL and V1, respectively. Body weight on CL and volume of distribution resulted in ~30% impact; other evaluated covariates showed no impact on PK parameters, indicating no need for dose adjustment. VPC results demonstrated good predictability of the final population PK model. Simulations indicated that similar overall PK exposures are expected following either the fixed 750 mg Q2W or the body-weight based 10 mg/kg Q2W regimens. Both regimens are projected to result in maintenance of trough exposures >2 µg/mL in ≥95% of patients where nonlinear clearance is expected to be fully saturated.

Conclusions: A population PK model of monalizumab was developed and validated. No dose adjustments were needed based on patient or disease characteristics. Similar PK is expected following both body weight-based and fixed dosing regimens. These results support further evaluation of 750 mg Q2W or equivalent dose in future Phase 2/3 clinical studies.

Citation Format: Xuyang Song. Population pharmacokinetics and dose regimen evaluation of monalizumab, a monoclonal antibody targeting NKG2a, in patients with advanced solid tumors [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 3029.
Abstract 3093: Quantifying the confounders of panel-based tumor mutational burden (TMB) measurement
Authors: Budczies Jan , Kazdal Daniel , Allgäuer Michael , Christopoulos Petros , Rempel Eugen , Pfarr Nicole , Weichert Wilko , Fröhling Stefan , Thomas Michael , Peters Solange , Endris Volker , Schirmacher Peter , Stenzinger Albrecht
Published: September 2020

Retrospective data including subgroup analyses in clinical studies have sparked strong interest in developing tumor mutational burden (TMB) as a predictive biomarker for immune checkpoint blockade. While individual factors interfering with panel sequencing based measurement of TMB (psTMB) have been discussed in recent studies, an integrative study quantifying, comparing and combining all potential confounders is still missing. We separated different potential confounders of psTMB measurement including “panel size”, “germline mutation filtering”, “biological variance” and “technical variance” and developed a specific error model for each of these factors. Published experimental psTMB data were fitted to the error models to quantify the contribution of each of the confounders. The total psTMB variance was obtained as sum over the different variance contributions. Using a typical large panel (size 1 to 1.5 Mbp) total errors of 57%, 42%, 34% and 28% were observed for tumors with psTMB of 5, 10, 20 and 40 muts/Mbp. Even for large panels, the stochastic error connected to the panel size represented the largest of all contributions to the total psTMB variance, especially for tumors with TMB up to 20 muts/Mbp. Other sources of psTMB variability could be kept under control, but rigorous quality control, best practice laboratory workflows and optimized bioinformatics pipelines are essential. We developed an integrated framework for the analysis of psTMB variability and provide a set of recommendations for implementation of psTMB as a laboratory developed test. Our approach highlights the stochastic portion of psTMB variability. The stochastic error connected inherently with psTMB could in part explain the suboptimal performance of the biomarker in some of the recent lung cancer studies.

Citation Format: Jan Budczies, Daniel Kazdal, Michael Allgäuer, Petros Christopoulos, Eugen Rempel, Nicole Pfarr, Wilko Weichert, Stefan Fröhling, Michael Thomas, Solange Peters, Volker Endris, Peter Schirmacher, Albrecht Stenzinger. Quantifying the confounders of panel-based tumor mutational burden (TMB) measurement [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 3093.
Abstract 3094: Development of a prognostic and predictive 32 gene signature for gastric cancer
Authors: Park Sunho , Cheong Jae-Ho , Wang Sam C. , Hong ChangJin , Porembka Matthew , Hwang Tae Hyun
Published: September 2020

Clinical staging at initial cancer diagnosis guides the use and timing of chemotherapy and radiation. However, current pretreatment stratification modalities for gastric cancer (GC) are inadequate and improved tools are needed. In addition, while adjuvant chemotherapy after surgery improves survival of resectable gastric cancer, few predictive biomarkers exist to select patients who might benefit from the current standard chemotherapy.

To develop prognostic and/or predictive signatures that are clinically relevant for GC patients, we performed a pan-cancer analysis of somatic mutation of 6,681 patients with 19 different cancer types using the machine learning algorithm NTriPath and identified a 32-gene GC signature. To determine clinical utility of the 32-gene GC signature, we used expression of the 32-gene GC signature from 567 GC patients to discover genetic subtypes. We found that the 32-gene GC signature are prognostic and predictive of response to adjuvant 5-fluorouracil and platinum doublet and pembrolizumab. We further validated the preditive value of the 32-gene GC signature using an independent cohort with metastatic GC treated with Immune Checkpoint Blockades.

Finally, we built a genetic-subtype based risk-scoring model to predict overall survival that was validated in three independent cohorts (n = 976). The risk score accurately predicted survival as a continuous function and was significantly associated with survival independent of TNM stage, age, tumor location and Lauren type.

Taken together, the 32-gene driven genetic subtype and the genetic-subtype based risk score can guide the use of the current standard of care adjuvant chemotherapy regimen and/or immunotherapy for gastric cancer patients and should be confirmed in prospective clinical trials.

Citation Format: Sunho Park, Jae-Ho Cheong, Sam C. Wang, ChangJin Hong, Matthew Porembka, Tae Hyun Hwang. Development of a prognostic and predictive 32 gene signature for gastric 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 3094.
Abstract 3243: Allogeneic anti-PTK7 CAR-T cells for the treatment of solid tumors
Authors: Levitsky Katie , Li Zejun , Pham Minh Thu , Maeng Kelly , Hanley Luke , Sagert Jason , Terrett Jon , Dutta-Simmons Jui , Allen Melanie
Published: September 2020

Protein Tyrosine Kinase 7 (PTK7), also known as colon cancer carcinoma kinase 4 (CCK-4), is a highly conserved catalytically inactive tyrosine kinase with inherent signal transduction activity. PTK7 is a member of the Wnt signaling pathway and thought to function in cell proliferation, adhesion, migration and apoptosis. PTK7 has been shown to be highly expressed in certain cancer types including colon, breast, lung, pancreatic, renal and ovarian. Downregulation of PTK7 by shRNA has been shown to reduce tumor growth and metastases development in xenograft mouse models. Furthermore, high PTK7 expression in triple negative breast cancer and NSCLC patients has been linked to poor prognosis. PTK7 therefore appears to be an attractive target for solid cancer therapeutic intervention and has been targeted clinically with an Antibody Drug Conjugate (PF-06647020). However, the protein is also expressed to a lesser degree in some normal adult tissues, particularly in the stromal cells of the ovary, placenta, uterus and lung. We have developed a human/murine cross-reactive anti-PTK7 CAR construct and assessed the ability of allogeneic anti-PTK7 CAR-T cells bearing that construct to target cancer cells and elicit toxicity in mice. These anti-PTK7 CAR-T cells were shown to be efficacious in vitro and in immunocompromised mouse xenograft models of breast, lung, colon, pancreatic and ovarian cancer. However, consistent in all xenograft models studied, a drop in body weight was observed shortly after injection from which all mice rapidly recovered to above baseline. Latent toxicity that was more variable amongst the xenograft experiments was also observed. Understanding and developing appropriate mitigation strategies to address the observed toxicities may be required to develop further a safe and efficacious allogeneic anti-PTK7 CAR-T cell therapy for solid tumor cancers.

Citation Format: Katie Levitsky, Zejun Li, Minh Thu Pham, Kelly Maeng, Luke Hanley, Jason Sagert, Jon Terrett, Jui Dutta-Simmons, Melanie Allen. Allogeneic anti-PTK7 CAR-T cells for the treatment of solid tumors [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 3243.
Abstract 3256: TIGIT humanized BALB/c mouse: A novel animal model for preclinical studies of human TIGIT antibodies
Authors: Ju Cunxiang , Zhang Mingkun , Yang Xiaoliu , Li Song , Sun Hongyan , Li Shuai , Tang Jing , Zhao Jing , Gao Xiang
Published: September 2020

TIGIT is an immune-checkpoint receptor mainly expressed on activated and memory T cells, regulatory T cells (Tregs), and NK cells. Engaged with CD155 and CD112, TIGIT can transmit an inhibitory signal that negatively regulate the cell functionality. TIGIT blockade antibodies that release the inhibitory signals and direct potent tumor killing are currently under active development. However, animal models appropriate for preclinical evaluation of human TIGIT antibodies are still largely unmet. To solve this problem, we established a TIGIT humanized mouse model (hTIGIT) in which the extracellular domain of mTIGIT was replaced with human counterparts while the trans-membrane and cytoplasmic domain kept intact. These hTIGIT mice express similar level of chimeric TIGIT as compared to mTIGIT in wildtype mice. Surprisingly, the growth of subcutaneously engrafted CT26 tumor in BALB/c-hTIGIT mice, but not MC38 tumor in B6-hTIGIT mice, was strongly inhibited when treated with human TIGIT antibody (anti-TIGIT). TILs analysis on CT26 engrafted BALB/c-hTIGIT revealed significantly higher levels of intra-tumoral NK and Treg cells, and more interesting, Treg was remarkably deleted after anti-TIGIT intervention. Furthermore, Fc mutation that blocks ADCC effect completely abolished its therapeutic activity. These data suggested that tumor infiltrated Treg clearance by ADCC may contribute to the tumor inhibition observed in BALB/c-hTIGIT model. Taken together, BALB/c-hTIGIT mouse is a novel non-human animal model that can recapitulate the tumor environment and appropriate for direct preclinical efficacy study of human TIGIT antibodies.

Citation Format: Cunxiang Ju, Mingkun Zhang, Xiaoliu Yang, Song Li, Hongyan Sun, Shuai Li, Jing Tang, Jing Zhao, Xiang Gao. TIGIT humanized BALB/c mouse: A novel animal model for preclinical studies of human TIGIT antibodies [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 3256.
Abstract 3260: The preclinical characterization of CS1001, an anti-PD-L1 IgG4 monoclonal antibody and its activity beyond T cell regulation
Authors: Zhang Juan , Li Zhenhu , Tang Liang , Liu Linlin , Ren Zhaoxiang , Ma Jingshu , Shi Qingmei , Bao Yuanwu , Lu Liang , Zheng Yong , Yang Baotian , Li Jing , Wang Xinzhong Jon
Published: September 2020

CS1001 is a high-affinity, fully human programmed death ligand-1 (PD-L1) blocking IgG4 monoclonal antibody. So far three therapeutic anti-PD-L1 antibodies (atezolizumab, avelumab, durvalumab) have been approved by US FDA in different cancer indications. CS1001 selectively bound to PD-L1 and blocked PD-L1/PD-1 ligation. The crystal structure of CS1001 complexed with human PD-L1 solved at 2.3Å revealed that CS1001 bound perpendicularly to PD-L1 and the binding epitope region well overlapped with the hPD1-binding region. In the Mixed Lymphocyte Reaction (MLR) assay, CS1001 effectively induced the proliferation of CD4+ T lymphocytes and the production of interferon-γ (IFN-γ) and interleukin-2 (IL-2). CS1001 employs IgG4 isotype and lacks antibody-dependent cellular mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). Furthermore, the in vivo efficacy of CS1001 in inhibiting tumor growth was demonstrated in the subcutaneous MC38-hPD-L1 murine colon carcinoma (murine MC38 cells expressing human PD-L1) in human PD-1 knock-in mouse (Biocytogen) model. In another dual-humanized animal model, i.e. PD-1/PD-L1 dual knock-in mice implanted with MC38-hPD-L1 tumor (Biocytogen) where human PD-L1 expressed in both tumor and host system, it is interestingly noticed that CS1001 not only enhanced the cytotoxic T cell/regulatory T cell ratio, but also significantly upregulated M1 macrophage & down-regulated MDSC population, suggesting that anti-PD-L1 antibody may play the pro-anti-tumor immune response through modulating myeloid cells that also express PD-L1, thus a non-ADCC/CDC antibody design could be beneficial. CS1001 is currently explored clinically in 5 registration trials as monotherapy or in combination with standard of care therapies. CS1001 monotherapy or combo with chemotherapy or targeted therapy were safe and well tolerated. CS1001 demonstrated promising efficacy in multiple tumor types and support full development of CS1001 as mono/combo therapy for multiple indications in ongoing and planned clinical trials including cHL, ENKTL, GC, EC, and NSCLC.

Citation Format: Juan Zhang, Zhenhu Li, Liang Tang, Linlin Liu, Zhaoxiang Ren, Jingshu Ma, Qingmei Shi, Yuanwu Bao, Liang Lu, Yong Zheng, Baotian Yang, Jing Li, Xinzhong Jon Wang. The preclinical characterization of CS1001, an anti-PD-L1 IgG4 monoclonal antibody and its activity beyond T cell regulation [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 3260.
Abstract 3264: CRISPR-Cas9 library screen in primary T cells to identify cancer immunotherapeutic targets
Authors: Yue Yangbo , Li Qian
Published: September 2020

Background: Immunotherapy, especially checkpoint blockers targeting programmed cell death protein 1 (PD-1) pathways, has transformed cancer treatment. Current checkpoint blockers are limited by low response rate, side effect and treatment relapse. An efficient and high-throughput screen platform in primary immune cells will accelerate the identification of key regulators in the immune response to tumor. New targets identified from the screen will be validated in in vitro and in vivo models that recapitulate key components of tumor-immune interaction.

Results: We developed a CRISPR-Cas9 genome targeting system that can achieve efficient gene disruption in primary CD8+T cells isolated from mouse (~60%) or human (~70%). We have applied this method to a pooled CRISPR library screen for key modulators of T cell-induced cytotoxicity to cancer cells in vitro and in vivo. This library contains sgRNAs targeting nearly all membrane proteins expressed in both murine and human T cells. In our in vitro model, cancer cells expressing chicken ovalbumin (OVA) were co-cultured with OVA-specific CD8+T cells isolated from OT-I transgenic mice. The proliferation and function of CD8+ T cell were damped by tumor cells in an antigen dependent way. In our in vivo model, OVA-specific CD8+T cells were transplanted into C57BL/6 mice implanted with OVA-expressing tumor. The accumulation, proliferation and function dynamics of transplanted CD8+T cells were monitored in tumors. They infiltrated into tumor in 3 days, substantially decreased in number in the following 4 days, and then continuously increased. Importantly, most CD8+T cells (~70%) in tumor were CD107a negative, which means they were non-functional.

Summary: We have established a high-throughput platform for identification of membrane proteins in primary CD8+ T cells to regulate T cell proliferation and function in tumor microenvironment.

Citation Format: Yangbo Yue, Qian Li. CRISPR-Cas9 library screen in primary T cells to identify cancer immunotherapeutic targets [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 3264.
Abstract 3269: Ablation of Cbl-b gene function leads to increased activation and resistance to immunosuppression in human primary T lymphocytes
Authors: Parmantier Eric , Kalouche Georges , Soulard Clara , Lanneau Christophe , Boisrobert-Blais Sophie , Nicolazzi Céline , Orsini Cécile
Published: September 2020

Purpose: The role of Cbl-b as an essential modulator of T cell activation has been widely documented, particularly in mouse studies. To substantiate the effects of Cbl-b ablation in human T cells, gene editing conditions were optimized, and gene KO efficiency was evaluated along with T cell response to stimulatory antibodies.

Experimental design: Non-preactivated human primary T cells obtained from several donors were electroporated with the CRISPR/Cas9-gRNA ribonucleoprotein complex targeting CBLB gene or controls. Cells were then activated in presence of anti-CD3 and anti-CD28 antibodies with or without the immunosuppressive factors PGE2 or TGFβ. Gene editing efficiency was measured using targeted NGS technology and cytokine release (IL2, IFNγ, TNFα), membrane activation markers (CD25, CD69) and cell proliferation were evaluated at various timepoints.

Results: Initial studies showed moderate gene editing efficiencies with on average 45% and 25% of out-of-frame mutations using 2 gRNAs targeting different regions of CBLB gene and were correlated with increased IL-2 release. By optimizing gene editing experimental conditions, out-of-frame mutations in CBLB gene reached more than 70% and were associated with higher levels of secreted IL2, IFNγ and TNFα early after T cell stimulation and increased CD25 membrane expression at later timepoints, compared to controls. Furthermore, CBLB gene-edited T cells were less susceptible to PGE2- and TGFβ-mediated inhibition of cell proliferation.

Conclusion: The efficient gene editing in human non-preactivated T cells, through the delivery of the CRISPR/Cas9-gRNA ribonucleoprotein complex, confirms that Cbl-b acts as a negative regulator of T cell activation and further validates its role in transducing immunosuppressive environmental cues.

Citation Format: Eric Parmantier, Georges Kalouche, Clara Soulard, Christophe Lanneau, Sophie Boisrobert-Blais, Céline Nicolazzi, Cécile Orsini. Ablation of Cbl-b gene function leads to increased activation and resistance to immunosuppression in human primary T lymphocytes [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 3269.
Abstract 3300: Association of molecular characteristics with survival in advanced non-small cell lung cancer patients treated with checkpoint inhibitors
Authors: Zhao Dan , Mambetsariev Isa , Li Haiqing , Chen Chen , Fricke Jeremy , Fann Patricia , Kulkarni Prakash , Xing Yan , Lee Peter , Bild Andrea , Massarelli Erminia , Koczywas Marianna , Reckamp Karen , Salgia Ravi
Published: September 2020

Objectives: Immune checkpoint inhibitors (ICIs) have changed the landscape of lung cancer therapy. However significant proportions of patients have primary or acquired resistance to ICIs. Molecular characterization is critical for patient selection and overcoming resistance to checkpoint inhibitors. The purpose of this study is to investigate the molecular characteristics associated with ICIs outcomes in advanced non-small cell lung cancer (NSCLC) patients.

Materials and methods: All advanced stage NSCLC patients at City of Hope who received ICIs (pembrolizumab, nivolumab, atezolizumab, and durvalumab) were identified retrospectively. Overall survival (OS, from the start of the ICIs), Pathology and information on genomic alterations (GAs) including next-generation sequencing (NGS) data, tumor mutation burden (TMB), and Programmed death-ligand 1 (PD-L1) levels were collected. Chi-square and Fisher's exact test, Log-rank test were used for comparison of demographics, and survival curves respectively. Univariate and multivariate COX proportional hazards model was used for survival analysis.

Results: 346 NSCLC patients were identified. Univariate and multivariate analysis found the association of OS with PD-L1 level ≥50% (Hazard ratio [HR], 0.19; 95% confidence interval [CI], 0.06-0.59; P<0.01), EGFR (HR 7.38; 95% CI, 1.15-47.42; P<0.05), and TET2 (HR 0.15; 95% CI, 0.03-0.90; P<0.05). The median OS was not reached [NR] for the 12 patients who had genomic alterations (GAs) in TET2 (12/108, 11%) versus (vs) 11.5 months in TET2 negative patients (98/108, 89%). Interestingly, GAs in TET2 and FANCA were mutually exclusive and patients who had GAs in FANCA gene (6%) had shorter OS (5.5 months vs 14.5 months, Log-rank test, P<0.05).

Conclusions: We described the clinical and molecular features of NSCLC patients treated with ICIs. The association of GAs in TET2 with longer OS and its mutual exclusivity with FANCA GAs were insightful for developing novel therapeutic strategies to improve ICIs outcomes in NSCLC.

Keywords: Lung cancer, molecular, next-generation sequencing (NGS), immune checkpoint inhibitors, TET2, FANCA, PD-L1, TMB, immunotherapy

Citation Format: Dan Zhao, Isa Mambetsariev, Haiqing Li, Chen Chen, Jeremy Fricke, Patricia Fann, Prakash Kulkarni, Yan Xing, Peter Lee, Andrea Bild, Erminia Massarelli, Marianna Koczywas, Karen Reckamp, Ravi Salgia. Association of molecular characteristics with survival in advanced non-small cell lung cancer patients treated with checkpoint inhibitors [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 3300.
Abstract 3311: B cell like neutrophil found in peripheral blood of melanoma patients
Authors: Meyer Melissa A. , Dinh Huy Q. , Zhu Yanfang , Liang Shu , Seumois Gregory , Vijayanand Pandurangan , Catz Sergio D. , Hedrick Catherine C.
Published: September 2020

An expansion of neutrophils often marks cancer. To date, neutrophils are assumed to be a relatively homogenous population, but recent studies describe both pro- and anti-tumor functions in neutrophils. This suggests there is more heterogeneity in neutrophils than previously appreciated. To better understand and target neutrophil functions in cancer, we must dissect neutrophil heterogeneity and how it changes from health to disease. To this end, we employed single-cell transcriptome technology to profile immature neutrophils from bone marrow of healthy individuals. Intriguingly, we identified a new neutrophil subset that expresses several B cell markers, including CD79a/b, components of the B cell receptors and PAX5, a lineage-specific transcriptional factor. We termed this population the BNeut. Using flow cytometry, we found that BNeuts are confined to the bone marrow of healthy individuals but expand into the blood of melanoma patients at an early stage, suggesting BNeuts might be a biomarker for disease. Additionally, we find BNeuts in the mouse models of different cancer types, including breast and lung carcinoma. The BNeut appears and functions more similar to a neutrophil than a B cell. BNeuts possess a neutrophil-like transcriptional profile, large cytoplasm with segmented nucleus, and produce ROS and NETs. Using imaging, we show that the BNeuts are single cells co-expressing both CD66b and CD79b, and lack the B cell surface marker CD19. To understand why neutrophils might use a B cell program, we explored different B cell functions including antigen presentation, a function that is usually absent from neutrophils. We show BNeuts are excellent at phagocytosis, including uptake of tumor cells. Further, BNeuts have increased expression of antigen presentation-related genes compared to other neutrophils and can modulate CD4+ T cell responses. Together, we have identified a novel neutrophil subset expressing B cell markers that is preferentially expanded in the blood of melanoma patients and poised to control T cell responses during disease.

Citation Format: Melissa A. Meyer, Huy Q. Dinh, Yanfang Zhu, Shu Liang, Gregory Seumois, Pandurangan Vijayanand, Sergio D. Catz, Catherine C. Hedrick. B cell like neutrophil found in peripheral blood of melanoma 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 3311.
Abstract 3323: In situ multiplex analysis of resident microglia and infiltrating macrophages in glioblastoma
Authors: Goulange Nicolas , Finan-Marchi Amanda , Tliba Maroua , Motte Manon , Estermann Fanny , Vernay Alexandre , Coton Jean-Philippe , Burrer Renaud
Published: September 2020

Glioblastoma mutliforme (GBM) is the most malignant primary brain tumor. Resident microglia and peripheral infiltrating macrophages account for up to half of the non-neoplastic cells in a GBM. These tumor-associated macrophages have been implicated in proliferation, angiogenesis, and immunosuppression in GBM and can influence the efficacy of chemo-, radio-, and immunotherapies. However, certain cancer specific interactions have been associated with either microglia or macrophages, necessitating an approach that can delineate the two populations. Multiplex immunofluorescence offers a technical advantage that allows for the profound phenotyping of cells in the tumor microenvironment as well as their spatial organization. We have designed a multiplex immunofluorescence protocol to differentiate microglia and infiltrating M1/M2 macrophages in GBM in situ. Human GBM tissues were investigated with a multiplex panel consisting of GFAP, TMEM119, CD68, c-maf, and CD163. After multi-spectral acquisition, the microglia and macrophage populations could be easily phenotyped and localized in all of the tissues examined. Microglia and peripherally recruited M1 and M2 macrophages were quantified. The spatial distribution of the populations within the tumor core, edge, or margin was evaluated. The approach presented here demonstrates the power of multiplex immunohistochemistry in the phenotyping and spatial analysis of resident and recruited immune cell populations on a single tissue section and the potential application of this method in clinical studies.

Citation Format: Nicolas Goulange, Amanda Finan-Marchi, Maroua Tliba, Manon Motte, Fanny Estermann, Alexandre Vernay, Jean-Philippe Coton, Renaud Burrer. In situ multiplex analysis of resident microglia and infiltrating macrophages in glioblastoma [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 3323.
Abstract 3331: Role of innate immune cells in the development of vessel co-opting CRC liver metastases
Authors: Mayer Thomas Z. , Kim Diane H. , Rada Miran , Petrillo Stephanie , Lazaris Anthoula , Metrakos Peter
Published: September 2020

Approximatively 50 % of patients who undergo resection for primary colorectal cancer will develop liver metastases. Liver metastases present as two major histopathological growth patterns (HGP) the desmoplastic and the replacement HGP. In the desmoplastic HGP, the cancer cells are surrounded by a desmoplastic stromal ring preventing their contact with the normal liver. In the replacement HGP, the cancer cells infiltrate the normal liver and replace hepatocytes near the tumour periphery. We have found that the desmoplastic HGP tumors derive their blood supply through angiogenesis while the replacement HGP tumors are non-angiogenic and co-opt the blood vessel of the liver they invade. Furthermore, we have published that patients under chemotherapy presenting with a vessel co-opting phenotype have a worse pathological response and five-year overall survival compared to patients presenting with angiogenic liver metastases. Their prognosis is worse when treated with bevacizumab an anti-VEGF antibody used alongside chemotherapy. CRCLM are considered immunologically cold lesions, with low numbers of infiltrating T-cells, and thought to be unlikely to benefit from immunotherapies. We have recently found that neutrophils and macrophages but not lymphocytes infiltrate vessel co-opting lesions to a greater extent than angiogenic lesions. Using flow cytometry and the Nanostring GeoMx spatial protein profiling platform capable of detecting up to 96 proteins simultaneously on FFPE tissue slices, we established the immunoprofile of immune cells recruited to angiogenic or vessel co-opting CRCLM lesions in chemonaive and treated patients. Many studies have demonstrated that neutrophils and macrophages play an important role in tumor progression, modification of the ECM, angiogenesis, and immunosuppression. We hypothesize that innate immune cells can facilitate blood vessel co-option of CRCLM lesions and contribute to an immunosupressive environment favorable to tumor growth. Fully understanding the role of the innate immune system in the developmend of vessel co-opting CRCLM tumors could allow for the development of immunotherapies capable of converting vessel co-opting lesions into angiogenic lesions that are more successfully treated with chemotherapy and anti-angiogenic therapies.

Citation Format: Thomas Z. Mayer, Diane H. Kim, Miran Rada, Stephanie Petrillo, Anthoula Lazaris, Peter Metrakos. Role of innate immune cells in the development of vessel co-opting CRC liver metastases [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 3331.
Abstract 3374: Development of novel and improved monoclonal antiMUC16 antibodies for the targeted therapy of ovarian cancer
Authors: Laster Ian , Yeku Oladapo , Spriggs David
Published: September 2020

There is a need for the development of better targeted therapies in the treatment of ovarian cancer. Monoclonal antibodies directed towards MUC16, a diagnostic and prognostic marker for ovarian cancer, with improved selectivity and specificity would be an instrumental component in the construction of novel methods of drug delivery and immunotherapies. C57BL/6 mice were immunized with various MUC16 peptides and truncated MUC16 constructs to extend the scope of MUC16 epitopes within the proximal COOH portion of the retained elements. Three rounds of hybridomas were made from harvested murine splenocytes, and subsequent rounds of peptide-based ELISA and cell-based flow cytometry were performed to select the most promising subclones from each of the fusions. Over 50 hybridomas producing antibodies against MUC16 have been identified and screened for reactivity with MUC16 peptide sequences and native, full-length MUC16 protein presented on the surface of ovarian cancer cell line cells. The antibodies were isotyped, and 10 have been selected for initial characterization. Non-overlapping epitopes were mapped via sequential and tandem peptide-based ELISA. With improved selectivity and specificity, these monoclonal antibodies will be used in the construction of antibody drug conjugates (ADCs) as potential targeted therapies in the treatment of ovarian cancer. Efficacy testing of the candidate antibodies in Matrigel invasion and direct growth inhibition assays is underway. Comparative effectiveness of the binding, internalization, and killing by ADCs in ovarian cancer cell lines will be presented. Antibodies against key MUC16 epitopes may have value in the development of antigen directed therapy.

Citation Format: Ian Laster, Oladapo Yeku, David Spriggs. Development of novel and improved monoclonal antiMUC16 antibodies for the targeted therapy of ovarian cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3374.
Abstract 3384: Characterization of the tumor microenvironment and liquid biopsy in head and neck and non-small cell lung cancer
Authors: Kulasinghe Arutha , Kapeleris Joanna , Kenny Liz , Hughes Brett , Warkiani Majid , Vela Ian , Thiery Jean-Paul , O'Byrne Ken , Punyadeera Chamindie
Published: September 2020

Metastasis in cancer patients is reflected by measurable levels of circulating tumor cells (CTCs) in the blood of cancer patients. CTCs represent cancer cells from the primary and metastatic sites, thereby providing a comprehensive representation of the tumor burden of an individual patient. Recent advancements have shown that PD-1/PD-L1 immune checkpoint therapies have durable responses in a number of solid tumor types.

Our study was designed to use multiple CTC enrichment platforms for the capture of CTCs and novel culture formulations for the ex vivo expansion of CTCs. Head and Neck cancer (n=350) and lung cancer (n=150) patients were recruited to investigate the prognostic role of CTCs. In parallel, a subset of HNC tumors were profiled using the NanoString GeoMx Digital Spatial Profiling (DSP) technology using a 44-plex antibody cocktail. We evaluated multiple CTC isolation technologies (CellSearch, filtration, CD45 depletion, Spiral, Straight and novel microfluidic chip technology) using matched patient samples which showed that epitope-independent CTC isolation captured a greater proportion of CTCs. Molecular alterations present in the primary tissue were confirmed in the CTCs by 3D-DNA FISH (EGFR-amplification, ALK-translocations). In HNC, the presence of CTC clusters associated with the development of distant metastatic disease (P=0.0313). HNC CTC-positive patients had shorter progression free survival (Hazard ratio [HR]: 4.946; 95% [CI]:1.571-15.57; P=0.0063) and PD-L1-positive CTCs were found to be significantly associated with worse outcome ([HR]:5.159; 95% [CI]:1.011-26.33; P=0.0485). In a proof of principle study, we were able to demonstrate for the first time, short-term patient derived CTC cultures outside the patient's body and exome sequencing of CTCs cultures confirmed the presence of mutational signatures consistent with The Cancer Genome Atlas (TCGA). Spatial characterization of the tumor microenvironment revealed immune subsets predictive of outcome to immunotherapy. Comprehensive characterization of the tumor microenvironment and liquid biopsy allows for the recapitulation of the molecular diversity present within the tumor, understanding of the disease progression and testing of therapies.

Citation Format: Arutha Kulasinghe, Joanna Kapeleris, Liz Kenny, Brett Hughes, Majid Warkiani, Ian Vela, Jean-Paul Thiery, Ken O'Byrne, Chamindie Punyadeera. Characterization of the tumor microenvironment and liquid biopsy in head and neck and 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 3384.
Abstract 3449: A novel rapid zebrafish model for validation of potential therapies for fatal pediatric brain tumors
Authors: Kaur Harpreet , Guo Huizi , Emmerich Kevin , White David , Green Peter , Hector Alara Michelle , Akhtarkhavari Sepehr , Bhargava Anukriti , Martin Allison , Shah Smit , Eberhart Charles G. , Mumm Jeffrey , Raabe Eric H.
Published: September 2020

Diffuse intrinsic pontine glioma (DIPG) and atypical teratoid/rhabdoid tumors (AT/RT) are deadly pediatric brain tumors. There is an urgent need of better therapeutics for improving survival and quality of life for these patients. Additionally, we need improved animal models to accelerate identification of novel therapeutics and improve through-put. In line with our long-term interests of validating therapies and developing model systems for pediatric brain tumors, we tested a repurposed drug quinacrine as a potential therapy using a new and faster model system in zebrafish. Quinacrine is a safe and widely used treatment for pediatric malaria and parasitic infections. We hypothesized that quinacrine will increase tumor cell death and decrease tumorigenicity of DIPG and ATRT. We used quinacrine in six patient-derived cell lines: three AT/RT (BT37, CHLA-05, CHLA-266) and three DIPG (JHHDIPG1, SUDIPGXIII, SF7761). In all tumor cell lines, quinacrine caused a dose-dependent reduction in proliferation (BrdU) and increase in apoptosis (cleaved caspase-3 and cleaved PARP) compared to vehicle-treated cells (P<0.01). Quinacrine had no effect on growth of normal hindbrain neural stem and progenitor cells. Using quinacrine fluorescence as a surrogate, we achieved micromolar concentration of quinacrine in the mouse and zebrafish brain without overt toxicity. Treatment of mice bearing ATRT flank tumors with quinacrine resulted in decreased tumor volume compared to vehicle-treated mice (P<0.05). To validate quinacrine orthotopically in DIPG, we developed a novel model in zebrafish wherein we injected fluorescent DIPG cells into the developing zebrafish blastula. Two days post injection, DIPG cells had homed to the zebrafish brain. Treatment of xenografted zebrafish with quinacrine for 72 hours decreased DIPG growth by 40% as measured by fluorescence, suggesting that minor groove binding drugs like quinacrine are a viable treatment strategy for these tumors. Using this new system, we were able to validate a potential therapeutic in 7 days versus 7 months in mice. Future studies are aimed at investigating the mechanism of quinacrine in these tumors and optimizing our zebrafish model for high-throughput screening of potential drugs.

Citation Format: Harpreet Kaur, Huizi Guo, Kevin Emmerich, David White, Peter Green, Alara Michelle Hector, Sepehr Akhtarkhavari, Anukriti Bhargava, Allison Martin, Smit Shah, Charles G. Eberhart, Jeffrey Mumm, Eric H. Raabe. A novel rapid zebrafish model for validation of potential therapies for fatal pediatric brain tumors [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 3449.