bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3121: Characterization of nab-paclitaxel resistant and gemcitabine resistant BxPC3 and MIA PaCa-2 pancreatic cancer cells
Authors: Arias Danielle M. , Collins John , Noel Pawan , Von Hoff Daniel D. , Han Haiyong
Published: July 2021

The gemcitabine (Gem) and nab-paclitaxel (Nab-p) combination is currently the FDA approved standard of care for patients with Stage IV metastatic pancreatic cancer. The response rate of the Gem and Nab-p regimen ranges from 23% to 46%, with many patients eventually becoming refractory. The specific aim of this research project is to determine which genes and pathways differ in Gem resistant (GemR) or Nab-p resistant (Nab-pR) MIA PaCa2 (MP2) and BxPC3 cells when compared with their parental cell lines. Cell proliferation assays were carried out to confirm resistance of the cell lines. The IC50 values of gemcitabine in the Gem resistant BxPC3 (BxPC3-GemR) and MP2 (MP2-GemR) are 26 and 64 fold higher than those in the corresponding parental cell lines, respectively. The IC50 values of nab-paclitaxel in the Nab-p resistant BxPC3 (BxPC3-Nab-pR) and MP2 (MP2-Nab-pR) are 13 and 140 fold higher than those in the corresponding parental cell lines, respectively. RNA sequencing (RNA-seq) was then performed to compare the gene expression profiles of the parental and resistant cell lines. Gene set enrichment analysis (GSEA) revealed that differentially expressed genes between Gem resistant and parental BxPC3 cell lines are often enriched in pathways associated with endoplasmic reticulum (ER) stress response (e.g. ATF-6 and XBP1) whereas in MP2 cells, The AMPK signaling pathway (e.g. PRKAB1, and PRKAG1) seemed to be activated in Gem resistant cells. NFkB signaling pathway (e.g. RIPK2 and NFKB2) was elevated in both Nab-p resistant BxPC3 and MP2 cell lines compared to their corresponding parental cell lines. Genes from these pathways were selected for further validation of their differential expression using RT-PCR. ATF6 which is a stress sensor and transducer in the ER is upregulated in BxPC3-GemR. PRKAB1 and PRKAG1 which regulate AMPK activity are upregulated in MP2-GemR. RIPK2 and NFKB1 are both up in the Nab-p resistant cell lines. Results from this study could provide insights on how to devise new therapeutic regimens to overcome resistance to gemcitabine and nab-paclitaxel.

Citation Format: Danielle M. Arias, John Collins, Pawan Noel, Daniel D. Von Hoff, Haiyong Han. Characterization of nab-paclitaxel resistant and gemcitabine resistant BxPC3 and MIA PaCa-2 pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3121.
Abstract 3159: Targeting the TGFΒ/TIMP-1/2 axes to re-educate pro-inflammatory/pro angiogenic NK cells in cancer patients
Authors: Gallazzi Matteo , Bruno Antonino , Noonan Douglas M. , Stetler-Stevenson William G. , Albini Adriana
Published: July 2021

Tissue inhibitors of metalloproteases (TIMPs) have been reported to exert dual roles in cancer, based on the different cancer type and interactions with the tumor microenvironments. As inhibitors of Matrix metalloproteases (MMP) they downregulate invasion, metastasis and angiogenesis. However, TIMP1 levels have been found increased in patients with different types of cancer. Natural Killer cells have been found to be phenotypically and functionally compromised in several solid and hematological cancers. We were the first in characterizing NK pro-angiogenic phenotype in the peripheral blood and tumor tissues of patients with Non-small Cell Lung cancer (NSCLC), colorectal cancers (CRC) and in pleural effusion (PE) of patients with metastatic cancers. Also, we found that pro-angiogenic NK cells in the peripheral blood of colorectal cancer patients release of TIMP-1 and TIMP-2, in a STAT3/STAT5-independent manner. Here, we investigated the effects of TIMP-1 and TIMP-2 recombinant proteins, using a TGFβ-mediated NK cell polarization model, in vitro. The effects of TGFβ, alone or in combination with TIMP-1 or TIMP-2 peptides, was determined by multicolor flow cytometry for surface antigens and cytokine productions, on cytolytic NK cells isolated from healthy subjects. Commercially available antibody membrane arrays was used to determine the modulation of the secretome, on FACS-sorted NK cells, from healthy subjects, following 72 hours of exposure to TGFβ, alone or in combination with TIMP-1 or TIMP-2 peptide. We found that NK cell from healthy donors, exposed to TGFβ, acquire the CD56brightCD9+CD49a+ decidual-like phenotype, together with decreased levels of the activation marker NKG2D. Administration of TIMP1 or TIMP2 peptides was effective in interfering with TGFβ-induced NK cell polarization towards the CD56brightCD9+CD49a+ decidual-like phenotype and in restoration of the levels of the NKG2D activation marker. We also observed that TGFβ-polarized NK cell, when co-treated with TIMP1 or TIMP2, exhibit a trend toward decreased production of VEGF and increased production of the anti-tumor cytokines IFNγ and TNFα Our results suggest a potential role of TIMPs in contrasting the angiogenic switch in NK cells and suggest a possible use of TIMPs peptides in the re-education of anergic/pro-angiogenic NK cells in cancer patients.

Citation Format: Matteo Gallazzi, Antonino Bruno, Douglas M. Noonan, William G. Stetler-Stevenson, Adriana Albini. Targeting the TGFΒ/TIMP-1/2 axes to re-educate pro-inflammatory/pro angiogenic NK cells in cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3159.
Abstract 3164: Modeling the metastatic niche interactions between patient tumor and mesenchymal cells to identify drivers of chemotherapy drug resistance
Authors: Deems Liam , Shahar Amit , Pepicelli Amy , Murphy Cheryl , Solomon Daniel , Deems David , Shvartsman Dmitry
Published: July 2021

Cancer research relies on a plethora of disease-relevant tumor models, but patient specific models are still being developed. In this study, we show the utility of patient-specific pancreatic, ovarian and breast cancer models in the context of the metastatic niche, composed of co-cultured mesenchymal stem cells (MSC) in three-dimensional tumor spheroids. Additionally, different ratios of MSC's were seeded with the cancer spheroids to interrogate the effect of MSC's on chemotherapeutic drug response in defined media conditions. The patient-specific and treatment-naive cancer models represent a diverse range of disease type, progression grade, and genetic profiles. These models were co-cultured with MSC's sourced from both bone marrow and adipose tissue. These co-culture models were tested with a panel of common, broad-range chemotherapeutic agents as well as several disease-specific drugs. The resulting drug responses show that the presence of MSC's within the context of the metastatic niche causes attenuation towards a higher chemotherapeutic drug resistance. The tumor models exhibit a varied chemotherapeutic response when MSC's are seeded in 50:1, 5:1, or 1:1 ratio with cancer cells, suggesting that even a small presence of MSC's may have a significant effect on the drug resistance of metastatic tumor spheroids. While this MSC-induced drug response attenuation is observed across the broad spectrum of patient profiles and disease types, the strength of the effect is not homogenous. The observed variability in the strength of the effect was impacted by the disease type of the cancer cells, the source of the MSC's, and the specific drug used in the metastatic tumor model. We propose that using these reproducible and easily scaled models of metastatic tumors to find effective chemotherapeutic drugs could increase our understanding of the tumor microenvironment, the onset of metastasis, and the process of cancer grafting into other tissues. Discovering effective and targeted therapies utilizing a more advanced model of the metastatic tumor niche could increase the success rate of an eventual clinical trial.

Citation Format: Liam Deems, Amit Shahar, Amy Pepicelli, Cheryl Murphy, Daniel Solomon, David Deems, Dmitry Shvartsman. Modeling the metastatic niche interactions between patient tumor and mesenchymal cells to identify drivers of chemotherapy drug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3164.
Abstract 3169: The therapeutically inflamed tumor microenvironment drives melanoma progression
Authors: Bradshaw Andrew M. , Kuo Erica , Grahovac Jelena , Sylakowski Kyle , Sander Cindy , Edington Howard , Kirkwood John M. , Wells Alan
Published: July 2021

Even the most therapy responsive melanomas eventually evade immune checkpoint inhibition. Interestingly, initial responses seem to benefit from localized inflammation, but this can become prohibitive when treating advanced disease. Subsequently, there is an urgent need to elucidate how inflammation impacts the tumor microenvironment (TME). Here, inhibition of EGFR and Ras/Raf/MEK is shown to drive the release of a pro-inflammatory secretome that activates the TME to promote progression. This secretome was enriched with immune recruiting cytokines and members of the transforming growth factor beta (TGFβ) superfamily. Both healthy and cancer associated fibroblasts (CAFs) were hyperactivated in response to the inflamed secretome with induction of the inflammatory marker, alpha smooth muscle actin (αSMA). Pharmacologic inhibition of TGFβ receptor type I (TGFβRI) using SB431542 or clinically relevant TEW-7197, diminished CAF αSMA phenotype, disrupted TGFβ and phospo-SMAD2/3 signaling, and blunted CAF expression of tumor promoting extracellular matrix and adhesion molecules. Invasion of melanoma spheroids was enhanced ~2.5-fold in CAF coculture transwells. However, treatment with MEK inhibitor AZD6244 and TEW-7197 only limited invasion of mixed melanoma and CAF multi-cell spheroids in vitro. Prompted by this, the extracellular matrix of patient-derived and melanoma xenograft tumors was analyzed for inflammatory protein expression. Micrometastatic foci were flanked by peri-tumor αSMA+ CAFs and enriched for inflammatory Tenascin-C (TNC) at the invasive front. The analysis of human melanoma tissue microarrays corroborated the experimentally observed expression of αSMA CAFs and TNC which statistically increase with malignant tumor grade. Conversely, the anti-invasive Decorin (DCN), decreased in malignant melanoma while collagen-1 increased during late stage tumor fibrosis. These findings prompted the use of ex vivo human skin organ cultures (SOCs) to define how cell matrix impacted melanoma. Addition of DCN to the SOC eliminated melanoma vertical invasion whereas pathological TNC promoted progression. Thus, a BRAFV600E resistant patient-derived metastatic cell line was used to establish intrasplenic xenografts that were treated after one week using AZD6244 and TEW-7197. Both monotherapy and combination therapy were well tolerated and resulted in reduced splenic tumor burden, αSMA staining, and halted metastasis to the liver.

Citation Format: Andrew M. Bradshaw, Erica Kuo, Jelena Grahovac, Kyle Sylakowski, Cindy Sander, Howard Edington, John M. Kirkwood, Alan Wells. The therapeutically inflamed tumor microenvironment drives melanoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3169.
Abstract 3175: FAT1 and the immunosuppressive milieu in glioblastoma tumors
Authors: Irshad Khushboo , Srivastava Chitrangda , Malik Nargis , Gupta Yakhlesh , Suri Vaishali , Mahajan Swati , Gupta Deepak , Suri Ashish , Chattopadhyay Parthaprasad , Sinha Subrata , Chosdol Kunzang
Published: July 2021

INTRODUCTION: Glioblastoma tumors harbor an immunosuppressive microenvironment which is sustained by key players such as TGF-β1/2 and IL-10 cytokines; and mediators of the PD-1/PD-L1/2 pathway. Previously, our lab has shown the role of a new gene, FAT1, in promoting gliomagenesis by supporting the production of pro-inflammatory cytokines (IL-1β, IL-6, etc.) in glioma cells. Here, we show a potent role of FAT1 gene in regulation of anti-inflammatory genes in human glioma tumors, cell lines and primary cultures. Our findings indicate an additional way in which FAT1 might contribute to gliomagenesis. METHODS: q-PCR was used to analyse transcript levels of FAT1 and genes of the TGF-β1/2, IL-10 and PD-L1/2 pathways in fresh-frozen glioblastoma samples relative to normal brain RNA. Gene expression correlation was analysed using Spearman's test and semi-supervised clustering. TCGA expression data was extracted to analyse the correlation of FAT1 with anti-inflammatory genes. Primary tumor cultures and cell lines were transfected with FAT1-specific siRNA to check the effect on expression of anti-inflammatory cytokines, as well as effect on migratory properties of monocytes towards FAT1-knockdown tumor cells (supernatants). RESULTS: Significant positive correlation was found between upregulation of FAT1 and that of anti-inflammatory genes in fresh-frozen glioblastoma samples as well as TCGA datasets. Upon siRNA-mediated knockdown of FAT1 in in-vitro glioma cell cultures, we observed decreased anti-inflammatory genes' expression. We also found increased transmigration of monocytes towards the supernatants from siFAT1-transfected cells. CONCLUSION: FAT1 favors the immunosuppressive milieu in glioblastoma by supporting expression of anti-inflammatory genes. This is an emerging role of the new oncogene FAT1 and crucial to our understanding of the contributions of FAT1 to hallmarks of cancer.

FUNDING: SERB Start-Up Grant (DST, India); DHR Young Scientist Scheme (ICMR, India)

Citation Format: Khushboo Irshad, Chitrangda Srivastava, Nargis Malik, Yakhlesh Gupta, Vaishali Suri, Swati Mahajan, Deepak Gupta, Ashish Suri, Parthaprasad Chattopadhyay, Subrata Sinha, Kunzang Chosdol. FAT1 and the immunosuppressive milieu in glioblastoma tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3175.
Abstract 3179: Deconvolving bulk gene expression to chart the immune activity and crosstalk in the tumor microenvironment
Authors: Wang Kun , Patkar Sushant , Lee JooSang , Gertz E. Michael , Robinson Welles , Schischlik Fiorella , Schäffer Alejandro , Ruppin Eytan
Published: July 2021

The tumor microenvironment is a complex mixture of cell-types that interact with each other to affect tumor growth and clinical outcomes. To accelerate the discovery of such interactions, we develop CODEFACS (Confident Deconvolution For All Cell Subsets), a deconvolution tool that infers cell-type-specific gene expression in each sample from bulk expression measurements. We additionally developed LIRICS (Ligand Receptor Interactions between Cell Subsets), a pipeline that analyzes the deconvolved gene expression to further identify clinically relevant ligand-receptor interactions between cell-types. Using 15 benchmark datasets, we first demonstrate that CODEFACS substantially improves our ability to reconstruct cell-type-specific transcriptomes from individual bulk samples compared to the state-of-the-art method, CIBERSORTx. We next analyze the TCGA using CODEFACS and LIRICS to uncover tumor-immunological cell-type-specific interactions that are differentially activate in mismatch repair deficient tumors. This analysis shows that T-cell co-stimulating interactions are associated with higher survival rates in these tumors independent of their mutation burden levels. Finally, using machine learning, we identify key cell-type-specific ligand-receptor interactions that predict patient response to immune checkpoint blockade therapy in melanoma. Taken together, we present and validate a computational framework for analyzing large bulk gene expression datasets at a cell-type specific resolution that has many potential future applications, complementing single cell transcriptomics in a cost-effective manner.

Citation Format: Kun Wang, Sushant Patkar, JooSang Lee, E. Michael Gertz, Welles Robinson, Fiorella Schischlik, Alejandro Schäffer, Eytan Ruppin. Deconvolving bulk gene expression to chart the immune activity and crosstalk in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3179.
Abstract 3185: Integrin signaling is critical for myeloid-mediated support of T-ALL
Authors: Lyu Aram , Nam Seo Hee , Hu Zicheng , Arasappan Dhivya , Horton Terzah M. , Ehrlich Lauren I.
Published: July 2021

T-cell acute lymphoblastic leukemia (T-ALL) is a malignant transformation of immature T cells. Although T-ALL harbors multiple pro-leukemic genetic alterations, T-ALL blasts require exogenous signals to survive. We recently reported that tumor-associated myeloid cells provide critical support for T-ALL in mouse models and in pediatric patient samples, at least partially through activation of IGF1R signaling. However, IGF1 alone is not sufficient to support T-ALL survival, implicating additional mechanisms in myeloid-mediated T-ALL support. We previously found that T-ALL cells make prolonged and frequent contacts with tumor-associated myeloid cells in live tissue sections; thus, we hypothesized that cell-cell interactions are essential for myeloid-mediated support of T-ALL. Here, we confirm that cell contact is required for myeloid cells to support T-ALL cell survival in vitro. Analysis of transcriptional profiling data revealed enrichment of gene signatures associated with integrin pathways in T-ALL cells relative to healthy T cells, implicating integrin-mediated cell adhesion as a potential mechanism underlying contact-dependent T-ALL support. Blockade of integrin αL ± integrin β1 or their ligands ICAM-1 ± VCAM-1 via antibodies or pharmacologic inhibitors significantly decreased T-ALL survival and reduced activation of IGF1R in T-ALL cells co-cultured with tumor-associated myeloid cells. Consistent with these findings, inhibition of integrins or their ligands in vivo via antibody blockade or genetic manipulation resulted in a significant reduction of T-ALL burden at multiple leukemic sites and prolonged survival. Consistent with mouse models, blockade of integrin-mediated cell adhesion significantly reduced survival of primary pediatric T-ALL cells co-cultured with myeloid cells. Furthermore, analyses of published transcriptomic datasets from pediatric T-ALL patients showed that enrichment of integrin pathways correlates with elevated myeloid gene signatures and poor patient prognosis. Together, our studies demonstrate that tumor-associated myeloid cells promote survival of T-ALL cells by activating integrin signaling, implicating the associated integrins and downstream signals as targets for therapeutic intervention.

Citation Format: Aram Lyu, Seo Hee Nam, Zicheng Hu, Dhivya Arasappan, Terzah M. Horton, Lauren I. Ehrlich. Integrin signaling is critical for myeloid-mediated support of T-ALL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3185.
Abstract 3186: Differential expression of PD-L1 between primary and metastatic Middle-Eastern epithelial ovarian carcinoma and its clinico-pathological correlation
Authors: Parvathareddy Sandeep K. , Siraj Abdul K. , Annaiyappanaidu Padmanaban , De Vera Felisa , Kong Yan , Qadri Zeeshan , Ahmed Saeeda O. , AlDossari Hassan , Al-Kuraya Khawla S.
Published: July 2021

Ovarian cancer (OC) is one of the most common gynecologic cancer, which has the worst prognosis and highest mortality rate. The lack of curative treatment and the high relapse rate, especially in advanced OC, continues to present a clinical challenge, highlighting the need for new therapeutic strategies. This study was conducted to compare the expression of PD-L1 in Middle-Eastern primary epithelial ovarian carcinoma to that in peritoneal metastases and its correlation with clinico-pathological parameters. In total, 194 treatment naïve paired EOC and peritoneal metastases were analyzed by immunohistochemistry for PD-L1 expression. Clinico-pathological information was available for all patients. Significant differences in PD-L1 expression were found between primary EOC and peritoneal metastasis (p < 0.0001). We found discordant tumor cell PD-L1 expression between primary tumors and corresponding peritoneal metastasis in 34% (66/194) of cases. Furthermore, PD-L1 expression in peritoneal metastasis samples was significantly associated with adverse prognostic factors, such as high proliferative index (Ki67) (p = 0.0039) and high histologic grade (p = 0.0330). In conclusion, the heterogeneity of PD-L1 expression in EOC suggests its assessment as a potential biomarker for PD-L1 blockade may require analysis of metastatic lesions.

Citation Format: Sandeep K. Parvathareddy, Abdul K. Siraj, Padmanaban Annaiyappanaidu, Felisa De Vera, Yan Kong, Zeeshan Qadri, Saeeda O. Ahmed, Hassan AlDossari, Khawla S. Al-Kuraya. Differential expression of PD-L1 between primary and metastatic Middle-Eastern epithelial ovarian carcinoma and its clinico-pathological correlation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3186.
Abstract 3198: In vitro exhausted T cell assays
Authors: Janss Thibaut J. , Lamy Juliette , Arnold Johan , Pattijn Sofie
Published: July 2021

The need to better understand the tumor microenvironment (TME) dictates the characterization of the cell types involved, the roles they play and how they respond to treatment. To develop better cancer immunotherapies, in vitro primary immune cell bioassays offer an early assessment of their effects on the various players of the TME. Exhausted T cells were first discovered in LCMV virus infections where chronic stimulation due to remaining virus antigens leads to T cells that stop responding to stimulation and lose their functionality (cytokine secretion and cytotoxic activity). The same phenotype was afterwards found in the tumor microenvironment where a lot of T cells were found to be “anergic”. Reversing their loss of functionality as a therapeutic strategy has made the characterization of these exhausted T cells a priority. Their phenotype is currently defined by an increase in the expression of a specific set of cell surface markers such as PD-1, TIM-3 and LAG-3. As exhaustion builds, the expression of these markers increases while T cell functionality decreases. The loss of functionality is defined by a reduction in cytokine secretion, proliferation, and cytotoxic activity in the presence of an antigen. To evaluate this phenotype in more depth, transcription factors can also be monitored. For example, BLIMP-1 and BATF, which play a role in PD-1 expression, are shown to increase following exhaustion. New therapeutics that can reverse exhausted phenotype lead to a new population of T cells called reinvigorated T cells which possesses high effector activity such as cytotoxicity towards cancer cells. Using primary immune cells, in vitro bioassays were developed to better screen the potential effect of new therapeutics on exhausted T cells. Their ability to reverse the exhausted phenotype and facilitate the anti-tumour immune response can as a result be assessed early in the drug development process.

Citation Format: Thibaut J. Janss, Juliette Lamy, Johan Arnold, Sofie Pattijn. In vitro exhausted T cell assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3198.
Abstract 328: Selective fluorogenic probe for rapid detection of cathepsin L activity
Authors: Schleyer Kelton A. , Fetrow Ben , Fatland Peter , Liu Jun , Chaaban Maya , Ma Biwu , Cui Lina
Published: July 2021

Cathepsin L (CTL) is a cysteine protease that demonstrates upregulated activity and altered trafficking in various cancers. Its upregulation in many cancers correlates positively with tumor grade while its excessive secretion makes elevated serum levels a possible biomarker for aggressive cancer types. The overlapping substrate specificity of cathepsin family members makes selective detection of activity from a single cathepsin difficult, and CTL activity is particularly difficult to parse from its close homologue CTV and the ubiquitous CTB. This complicates clinical sample assays for CTL activity, which generally rely on simple fluorescent probes lacking specificity for CTL over other cathepsins. We have developed a fluorogenic probe, CTLAP, that is rapidly activated by CTL and displays good selectivity over CTB and CTV, the closest competing analytes for CTL. The novel chemical structure of CTLAP facilitates markedly higher signal generation and improved selectivity for CTL when compared to Z-FR-AMC, the commercial probe commonly used to detect CTL activity in mixed samples. Optimum selectivity for CTL is achieved within 10 min of incubation, suggesting that CTLAP is amenable for rapid detection of CTL, even in the presence of competing cathepsins.

Citation Format: Kelton A. Schleyer, Ben Fetrow, Peter Fatland, Jun Liu, Maya Chaaban, Biwu Ma, Lina Cui. Selective fluorogenic probe for rapid detection of cathepsin L activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 328.
Abstract 337: p16INK4A-deficiency predicts for response to combined HER2 and CDK4/6 inhibition in HER2+ breast cancer brain metastases
Authors: Ni Jing , Kabraji Sheheryar , Xie Shaozhen , Wang Yanzhi , Pan Peichen , He Xiaofang , Liu Zongming , Long Henry , Brown Myles , Winer Eric P. , Dillon Deborah , Lin Nancy , Zhao Jean
Published: July 2021

Approximately 50% of patients with metastatic HER2-positive (HER2+) breast cancer develop brain metastases (BCBMs). To identify predictors of response and resistance to brain-penetrant, small molecule targeted therapies, we performed whole exome sequencing, RNAseq and quantitative immunohistochemistry of HER2+ BCBM tissues from 21 patients. We found that the tumor suppressor p16INK4A (p16) was deficient in the majority of HER2+ BCBMs. Orthotopic HER2+ BCBM patient-derived xenograft (PDX) models with p16 deficiency were resistant to the brain-penetrant HER2 inhibitor, tucatinib or the brain penetrant CDK4/6 inhibitor, abemaciclib, when used as single agents. However, p16-deficiency predicted response to combined tucatinib and abemaciclib in orthotopic HER2+ BCBM PDX models. These data establish the rationale for a biomarker-driven clinical trial of combined CDK4/6- and HER2-targeted agents for patients with HER2+ BCBM.

Citation Format: Jing Ni, Sheheryar Kabraji, Shaozhen Xie, Yanzhi Wang, Peichen Pan, Xiaofang He, Zongming Liu, Henry Long, Myles Brown, Eric P. Winer, Deborah Dillon, Nancy Lin, Jean Zhao. p16INK4A-deficiency predicts for response to combined HER2 and CDK4/6 inhibition in HER2+ breast cancer brain metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 337.
Abstract 348: Development of an RNA based diagnostic panel to the tumor microenvironment to match cancer therapies for colorectal cancer
Authors: Strand-Tibbitts Kristen , Culm-Merdek Kerry , Santps Valerie Chamberlain , Benjamin Laura , Carter Julia , Douglass Larry , Luštrik Roman , Cvitkovič Robert , Ausec Luka , Rosengarten Rafael
Published: July 2021

The most utilized targeted therapies in colorectal cancer (CRC) are focused on EGFR inhibition and anti-angiogenesis. In the ~5% of patients with microsatellite instability (MSI-H) or high tumor mutational burden (TMB), checkpoint inhibitors (CPIs) have been approved. Oncxerna has developed an RNA expression-based approach to characterize the ‘dominant' biology of a patient's tumor microenvironment with the diagnostic hypothesis to prospectively pair those patients with therapies and known mechanism of action that directly target these biologies. We developed an RNA-based gene expression panel (TME Panel-1) and machine learning (ML) algorithms to prospectively predict a patient's response to anti-angiogenesis or immune modulators, such as CPIs. In this study, we explore the potential of the TME Panel-1 to identify dominant biologies present in colorectal cancer specimens procured from the Wood-Hudson Cancer Research Lab. Total RNA expression counts from FFPE slides were analyzed with the ML algorithms and used to assign each sample into one of four subgroups. The respective prevalence of the subgroups are similar to those observed in gastric cancer and ovarian cancer samples, suggesting that the TME-Panel 1 has potential to be used to develop pan-tumor diagnostics. We will present these results, correlations with clinical outcomes and other relevant biomarkers for CRC. In summary, we conclude that RNA-based descriptors of biology may be a useful approach to enrich for better response to targeted therapies whose mechanism of action is to modify the TME biology.

Citation Format: Kristen Strand-Tibbitts, Kerry Culm-Merdek, Valerie Chamberlain Santps, Laura Benjamin, Julia Carter, Larry Douglass, Roman Luštrik, Robert Cvitkovič, Luka Ausec, Rafael Rosengarten. Development of an RNA based diagnostic panel to the tumor microenvironment to match cancer therapies for colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 348.
Abstract 360: The dilemma of the current diagnostic tests for MSI in prostate cancer
Authors: Dikoglu Esra , Naizhen Xu , O'Connor Luke P. , Pinto Peter , Merino Maria J.
Published: July 2021

Background:

Understanding the relationship between tumor genomics and the immune response in cancer has gotten more attention with the advance of immunotherapy. Microsatellite instability (MSI) is a molecular marker that provides prognostic and predictive information in many types of tumors including prostate cancer (PC). In PC, MSI-H and dMMR have been reported anywhere from 1% in primary tumors to up to 12% in metastasis. Reliable testing strategies for MSI/MMR status are critical for clinical management of patients with PC. MSI detection methods include PCR based molecular tests, NGS-based MSI detection or immunohistochemical staining (IHC). We observe technical difficulties in our daily practice with current molecular diagnostic tests.

Design:

We examined MMR protein expression (MSH2, MSH6, MLH1, PMS2) and PD-L1 by IHC in 30 PC.

Results:

Among 30 PC tested, 1 tumor (3%) was completely negative for MLH1 and PMS2 and 1 tumor (3%) revealed loss of PMS2 by IHC even though gene panel did not reveal any mutation in PMS2. The PD-L1 IHC was 44% positive, but the single MMR negative biopsy was PD-L1 negative. PD-L1 expression in PC samples did not show correlation with defective MMR expression.

Conclusion:

In our study, controversial results were obtained. Based on our experience; even though many exons of MMR genes are covered with these panels, there are some exons do not get enough coverage to be analyzed. This low coverage problem creates false negative results. There are also pseudogene pairs of these genes, especially PMS2. For some specific regions, even though there is enough coverage it is impossible to know if the pathogenic variant is on the PMS2 or the pseudogene without additional test. This result suffers from false positive results without a confirmatory test. It is also known that 5% to 11% of MSI-H cases demonstrate intact MMR staining and localization (proficient MMR, pMMR) due to retained antigenicity and nonfunctional protein. So far, in regular practice we use MMR analyzing strategies which set up for colon cancer where the tumor is uniform. PC is more complex; most of the time more than one clone is involving.We believe MSI detection for PC requires improvement the technics of detection, robust set up of testing strategy with high sensitivity and specificity, analyzing strategy and training of pathologists. Due to technical difficulties of the detection, we believe that the prevalence of MSI-high/dMMR PC might be higher than reported in the literature so far.

Citation Format: Esra Dikoglu, Xu Naizhen, Luke P. O'Connor, Peter Pinto, Maria J. Merino. The dilemma of the current diagnostic tests for MSI in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 360.
Abstract 386: Evaluation of CPa9-HNE - a peripheral biomarker based on neutrophil extracellular traps (NETs) - for predicting outcome in patients with metastatic melanoma treated with anti-PD-1 therapy
Authors: Jensen Christina , Kverneland Anders , Donia Marco , Mortensen Joachim H. , Karsdal Morten A. , Svane Inge Marie , Willumsen Nicholas
Published: July 2021

Background:

Peripheral biomarkers associated with response and resistance to anti-PD-1 therapy in metastatic melanoma patients represent an unmet medical need. High neutrophil activity is associated with immune checkpoint inhibitor failure, amongst others due to release of neutrophil extracellular traps (NETs) that have been shown to protect tumor cells against cytotoxic attacks. Human neutrophil elastase (HNE) and calprotectin are the major NETs constituents. We hypothesized that HNE mediated degradation of calprotectin (CPa9-HNE), when measured in serum, has peripheral biomarker potential in metastatic melanoma patients treated with anti-PD-1 therapy.

Methods:

A monoclonal antibody was raised against a specific HNE mediated degradation fragment of calprotectin (CPa9-HNE) and used to develop an ELISA for quantifying CPa9-HNE in serum. CPa9-HNE was then measured in pre-treatment serum from metastatic melanoma patients treated with anti-PD-1 therapy (pembrolizumab) (n=35). The biomarker levels were associated with progression-free survival (PFS) and overall survival (OS) by univariate and multivariate Cox regression analyses adjusting for PDL1 expression (≥1%), lactate dehydrogenase (LDH), BRAF mutational status, and c-reactive protein (CRP).

Results:

High pre-treatment CPa9-HNE levels predicted worse PFS (HR=3.32, 95%CI=1.25-8.82, p=0.016) and OS (HR=11.31, 95%CI=2.27-56.33, p=0.003) when compared to low CPa9-HNE levels by univariate Cox regression. By multivariate Cox regression, high CPa9-HNE was found to be independently predictive of poor PFS (HR=8.22, 95%CI=1.30-52.14, p=0.025) and OS (HR=76.87, 95%CI=4.73-1248.57, p=0.002) when adjusted for PDL1 expression, LDH, BRAF mutations, and CRP.

Conclusions:

A non-invasive biomarker of HNE mediated degradation of calprotectin (CPa9-HNE) was associated with poor PFS and OS in metastatic melanoma patients treated with anti-PD-1 therapy. These findings support a link between neutrophil activity/NETs and response to anti-PD-1 and indicate promising non-invasive biomarker potential to stratify melanoma patients for anti-PD-1 therapy in future clinical studies.

Citation Format: Christina Jensen, Anders Kverneland, Marco Donia, Joachim H. Mortensen, Morten A. Karsdal, Inge Marie Svane, Nicholas Willumsen. Evaluation of CPa9-HNE - a peripheral biomarker based on neutrophil extracellular traps (NETs) - for predicting outcome in patients with metastatic melanoma treated with anti-PD-1 therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 386.
Abstract 395: Prediction of genomic based drug sensitivity signatures to enable optimal drug positioning
Authors: Gilvary Coryandar , Elemento Olivier , Madhukar Neel
Published: July 2021

Cancer by nature, is a heterogeneous disease, which can lead to highly variable patient responses for targeted therapies - even within the same clinically defined cancer type or subtype. Identifying drug sensitivity biomarkers, patient specific traits that are highly correlated with positive response, has proven to be an effective strategy to identify positioning opportunities and selecting patients. Here, we describe our biomarker identification workflow which identifies drug specific genomic sensitivity signatures, a collection of genomic aberrations that are found to be predictive of patient response, which can be used for patient selection as well as understanding a compound's mechanism of action. Using available drug efficacy data (in vitro or in vivo results) with paired baseline genomics data, we build diverse models to predict sample sensitivity to a drug. For every model, we train and measure the performance and how each genomic aberration contributes to model performance, those that do not sufficiently increase predictability are removed. This process is repeated, with each new model training on a smaller subset of genomic aberrations, until we have the minimum number of genomic aberrations needed to reach peak predictive performance. The resulting set of genomic aberrations make up a genomic sensitivity signature. Instead of using one type of algorithm to build these models, we train three distinct model types: linear, decision tree and custom kernel based. The multiple model architecture allows us to model the unique underlying biological mechanism of a compound's efficacy, such as synthetic lethal relationships or resistant pathway deactivation, ultimately giving us a collection of robust biomarker signatures. In addition to mechanistic understanding, clinical feasibility was a top priority. Therefore, our workflow architecture has been tested and validated to incorporate mutation, copy number alteration and/or gene expression based biomarkers either in combination or alone. This flexibility lowers the data required to use our workflow, making it more widely available to investigational drugs. Our identification of genomic sensitivity signatures can then be used for clinical patient selection or positioning a compound for a clinically defined cancer type, based on the prevalence of the predicted signature. Overall, this workflow is a powerful approach identifying diverse, mechanism based drug sensitivity signatures that can enable identifying the right patient for a therapy of interest.

Citation Format: Coryandar Gilvary, Olivier Elemento, Neel Madhukar. Prediction of genomic based drug sensitivity signatures to enable optimal drug positioning [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 395.
Abstract 40: Targeting SOX10-deficient cells to reduce resistance to targeted therapy in melanoma
Authors: Capparelli Claudia , Purwin Timothy J. , Tiago Manoela , Wilski Nicole , Pomante Danielle , Glasheen McKenna , Rosenbaum Sheera , Nguyen Mai Q. , Cai Weijia , Zheng Richard , Kumar Gaurav , Chervoneva Inna , Shimada Ayako , Snook Adam E. , Fortina Paolo , Xu Xiaowei , Hookim Kim , Cukierman Edna , Davies Michael A. , Herlyn Meenhard , Aplin Andrew E.
Published: July 2021

Intratumoral heterogeneity and cellular plasticity enable tumors to alter phenotypes and adapt to foreign microenvironments and resist targeted inhibitors. While the ability to switch between phenotypic states has been broadly characterized, the key mechanisms that underlie tumor plasticity remain poorly understood. We studied the neural crest lineage transcription factor, SOX10, in the context of cutaneous melanoma and resistance to targeted therapies. SOX10 is heterogeneously expressed in melanoma samples. Using bio-informatics as well as in vivo and 3D in vitro melanoma models, SOX10 loss was sufficient to induce an invasive but slow proliferating phenotype in vitro and in vivo that was associated with expression of a mesenchymal gene set. Interestingly, while SOX10 knockout initially induced a targeted inhibitor tolerant state, longer exposure of co-mixed populations of SOX10 proficient and SOX10 deficient to targeted therapy drives the clonal selection of SOX10 knockout cells. Furthermore, cell lines generated from xenograft tumors that have acquired resistance to either vemurafenib, paradox-breaking BRAFi or the combination of BRAFi + MEKi showed dramatically reduced SOX10 expression compared to their parental counterparts. Altogether these data suggest that acquired resistant clones may arise from drug tolerant persister cells. As a strategy to selectively target this invasive, drug-tolerant SOX10-deficient sub-population, we screened a drug compound library and identified a class of cIAP1/2 inhibitors to be synthetically lethal for SOX10-deficient cells. Our preliminary data suggest that birinapant can delay or prevent resistance to BRAFi/MEKi in vivo. Together, these data suggest that SOX10 mediates phenotypic switching in cutaneous melanoma and enables tumor adaptation to altered microenvironments and drug treatments which could be targeted using cIAP1/2 inhibitors.

Citation Format: Claudia Capparelli, Timothy J. Purwin, Manoela Tiago, Nicole Wilski, Danielle Pomante, McKenna Glasheen, Sheera Rosenbaum, Mai Q. Nguyen, Weijia Cai, Richard Zheng, Gaurav Kumar, Inna Chervoneva, Ayako Shimada, Adam E. Snook, Paolo Fortina, Xiaowei Xu, Kim Hookim, Edna Cukierman, Michael A. Davies, Meenhard Herlyn, Andrew E. Aplin. Targeting SOX10-deficient cells to reduce resistance to targeted therapy in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 40.
Abstract 434: Characterizing viral mRNA and immuno-protein expression in head and neck squamous cell carcinoma using a novel automated RNAscope™/Polaris™ integrated assay
Authors: Pollan Sara , Hanifi Arezoo , Nagy Mate , Stavrou Nicholas , Parnell Erinn , Gozo Maricel , Attanasio Nickolas , William Josette , Au Qingyan
Published: July 2021

Background: Chronic viral infection can generate inflammatory microenvironments leading to neoplastic growth and cancer development. Cancer patients with chronic viral infection have been shown to exhibit worse outcomes compared to non-infected individuals. One such cohort, head and neck squamous cell carcinoma (HNSCC) patients infected with chronic human cytomegalovirus (CMV), have increased risk of death when receiving radiotherapy or radiochemotherapy. This is clinically significant as HNSCC already carries a high mortality rate and low response to surgery and chemotherapeutic treatment. The ability to detect and localize viral infection and immune response in the same patient tissues has been historically under-developed and may assist in stratifying patients for therapeutic intervention.

Methods: Immune infiltrate to tumor in CMV infected and non-infected samples was assessed in HNSCC patient tissue using RNAscope in-situ hybridization (ISH) probe to detect CMV mRNA and Vectra® Polaris™ automated multiplex protein detection of CD8, PD-L1, CD68 and panCK with OPAL dyes.

Results: We developed a novel automated RNAscope™/Vectra® Polaris™ integrated multiplex immunofluorescence (IF) assay with OPAL detection and quantification of signal using Indica HALO® algorithms. The results include quantitative and reproducible RNAscope fluorescent ISH counting, cell-by-cell expression profiles, multiplex protein quantification and whole-slide image analysis.

Conclusion: NeoGenomics Laboratories RNAscope™/Polaris™ integrated assay detects and quantifies CMV viral infection and protein expression of PD-L1 positive and negative cytotoxic T cells and macrophages within and adjacent to HNSCC tumor regions.

Citation Format: Sara Pollan, Arezoo Hanifi, Mate Nagy, Nicholas Stavrou, Erinn Parnell, Maricel Gozo, Nickolas Attanasio, Josette William, Qingyan Au. Characterizing viral mRNA and immuno-protein expression in head and neck squamous cell carcinoma using a novel automated RNAscope™/Polaris™ integrated assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 434.
Abstract 442: Pemetrexed and platinum plus pembrolizumab in patients with metastatic non-squamous non-small cell lung cancer by tumor burden at baseline: A post-hoc efficacy analysis of KEYNOTE-189
Authors: Gadgeel Shirish , Grey Jhanelle , Rizzo Maria Teresa , Peterson Patrick , Kim Jongseok , Rodríguez-Abreu Delvys
Published: July 2021

Background: Pemetrexed (pem) and platinum (plat) plus pembrolizumab (pembro) showed clinical improvement in metastatic nonsquamous (NSQ) non-small-cell lung cancer (NSCLC). The aim of this analysis was to assess the clinical benefit of pem+plat+pembro in patients by tumor burden at baseline.

Methods: The intention-to-treat population was analysed. Patients were randomized pem+plat+pembro (n=410) or pem+plat+placebo (n=206). The data cut-off date was May 20, 2019. OS, PFS, PFS-2, and ORR were evaluated by Kaplan-Meier and univariate Cox methods within subgroups identified by extent of distant metastasis (M1a vs. M1b), median number (≤3 vs. >3) of organ systems with lesions, and severity score of patient-reported symptoms (below median vs. above median) at baseline. These subgroup analyses were also replicated within PD-L1 TPS subgroups (<1%, 1-49%, ≥50%).

Results: A consistent benefit in OS, PFS, PFS-2 and ORR was observed in the pem+plat+pembro treatment arm, compared to the pem+plat+placebo arm, irrespective of staging (M1a vs. M1b), number (≤ 3 vs. 3) of organ systems with lesions and severity of patient-reported symptoms (Table 1). Results by PD-L1 expression in the subgroups were generally consistent regardless of the level of expression.

Conclusions: Pem+plat+pembro demonstrated consistent clinical benefit across all subgroups analysed regardless of baseline tumor burden. These results continue to support pem+plat+pembro as a standard of care in metastatic NSQ-NSCLC.

TABLE 1.OS, PFS, PFS-2, and ORR in subgroups by tumor burden at baselineTreatment armsNaOSPFSPFS-2ORRHRHRHROdds ratioM1aPembro1230.540.480.572.66Placebo53M1bPembro2850.580.510.504.21Placebo152≤ 3b organ systems with lesionsPembro2350.490.410.436.00Placebo118> 3b organ systems with lesionsPembro1750.670.590.601.80Placebo88≤ medianc severity score of symptomsPembro1700.510.490.432.72Placebo82> medianc severity score of symptomsPembro1870.600.480.554.04Placebo97a.There were 3 patients with missing data for M1a and M1bb.Median number of organ systems with lesions was 3. Lesions include the primary tumorc.Median score = 122.2, based on six questions from the patient-reported LC-13 instrument

Citation Format: Shirish Gadgeel, Jhanelle Grey, Maria Teresa Rizzo, Patrick Peterson, Jongseok Kim, Delvys Rodríguez-Abreu. Pemetrexed and platinum plus pembrolizumab in patients with metastatic non-squamous non-small cell lung cancer by tumor burden at baseline: A post-hoc efficacy analysis of KEYNOTE-189 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 442.