bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3024: Targeting the RNA binding protein LIN28B in Group 3 medulloblastoma decreases proliferation and promotes apoptosis
Authors: Shahab Shubin W. , Rokita Jo Lynne , Juraschka Kyle , Kumar Sachin , Taylor Michael , Schnepp Robert W. , MacDonald Tobey J. , Kenney Anna M.
Published: July 2021

Medulloblastoma (MB) is the most common pediatric malignant brain tumor and is currently divided into WNT, SHH, Group 3 and Group 4 subtypes. Even with multimodal chemotherapy, radiotherapy and surgery, many children with Group 3 MBs do not survive. While the molecular aberrations underlying WNT- and SHH-driven MBs are relatively well understood, the oncogenic drivers that lead to Group 3/4 MBs are poorly defined, limiting therapeutic progress. In addition to genetic mutations and alterations, cancers display dysregulated transcription and translation. RNA-binding proteins (RBPs) play key roles in both transcription and translation, and a subset of RBPs are differentially expressed in many different cancers. Indeed, we have previously demonstrated an oncogenic role for the RBP LIN28B in neuroblastoma and it is known to be upregulated in Wilms tumor, hepatoblastoma, germ cell tumors, leukemia among others. LIN28B is a key regulator of let-7 family miRNAs, which in turn inhibit LIN28B and other oncogenes. We hypothesize that LIN28B plays an important role in Group 3 MB and that a better understanding of LIN28B and LIN28B-driven networks will reveal novel therapeutic vulnerabilities. In support of our hypothesis we find that among the four subtypes, LIN28B levels are highest in Group 3 MB, and that overexpression is associated with significantly worse survival. Down-regulation of LIN28B results in significant reduction in cell proliferation by CellTiter-Glo and increased apoptosis by Caspase-Glo (as well as induction of cleaved PARP on immunoblots). In contrast overexpression of LIN28B increases Group 3 cell proliferation and tumor sphere formation. In addition we find that PDZ-binding kinase (PBK) a downstream target of LIN28B is downregulated when LIN28B is depleted. PBK knock down also leads to decreased proliferation of Group 3 MB cells. Finally, in order to robustly define the signaling networks downstream from LIN28B that are involved in Group 3 MB metastasis, we have performed who transcriptome RNA-seq profiling of two group 3 cell lines following LIN28B depletion and plan to interrogate a subset of these based on expression change and functional relevance to LIN28B-mediated Group 3 MB metastasis. This work will help define the role for LIN28B in Group 3 MB aggressiveness and pave the way for similar studies in other cancers.

Citation Format: Shubin W. Shahab, Jo Lynne Rokita, Kyle Juraschka, Sachin Kumar, Michael Taylor, Robert W. Schnepp, Tobey J. MacDonald, Anna M. Kenney. Targeting the RNA binding protein LIN28B in Group 3 medulloblastoma decreases proliferation and promotes apoptosis [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 3024.
Abstract 3071: A method for infratentorial structures segmentation for tumor treating fields treatment planning
Authors: Glozman Yana , Shamir Reuben R. , Bomzon Zeev
Published: July 2021

Introduction: Tumor treating fields (TTFields) is an FDA approved treatment for the management of glioblastoma multiform (GBM) and mesothelioma (MPM) and is associated with a significant extension to patients' survival. We have developed software to optimize the location of the TTFields transducer arrays (TAs) on GBM patients' head to maximize the field in the tumor region. To that end, we first segment the patients' head MRIs into five normal and three abnormal tissues. However, the current method for head segmentation was found to be less accurate at the infratentorial regions, mark some sinuses with cerebrospinal fluid (CSF), and does not identify the cerebellum and brain-stem. Therefore, it is assuming the infratentorial structures have the same electrical properties as in the cerebrum.

Methods: We have developed a new method for segmentation of the head MRI that overcomes these limitations. As in the previous method, we have incorporated an atlas that is composed of an MRI image and tissue probability maps (TPM). The TPM assign each voxel in the atlas MRI with a value in the range of 0-1. This value reflects the probability that the specific tissue resides in that voxel. We have generated new customized TPMs for the cerebellum, brain stem and sinuses. Moreover, we have carefully revised the other TPMs to ensure best results. Last, we have incorporated these TPMs in a new atlas-based segmentation method. We have validated the method on 10 GBM patients T1w +gad head MRIs.

Results: The average Dice coefficient between gold-standard and algorithms' segmentation was 68.4% (SD=11.2%) that is similar to the value observed with the current method implemented in the treatment-planning software (67.8%; SD=10.6%). However, the new method presented here extends the current method with the cerebellum (average Dice = 84%) and brainstem (average Dice = 67%). Moreover, the sinuses are marked with air, and erroneous CSF segments in the sinuses were removed.

Conclusions: We have presented a novel method for the segmentation of the head that facilitates accurate infratentorial and supratentorial structures' segmentation. Moreover, the method eliminates erroneous CSF segmentation in the sinuses. These improvements pave the way for TTFields planning for infratentorial tumors.

Citation Format: Yana Glozman, Reuben R. Shamir, Zeev Bomzon. A method for infratentorial structures segmentation for tumor treating fields treatment planning [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 3071.
Abstract 3072: Clinically relevant MAPK pathway inhibition reverses stem cell fate defects and sensitize BRAF mutant glioma to immune modulatory therapies
Authors: Petritsch Claudia K. , Grossauer Stefan , Wang Wei , Barrette Anne Marie , Park Jongwhi
Published: July 2021

Pediatric low-grade gliomas (pLGG) are the most common central nervous system (CNS) tumor in children. Although the majority of pLGG has unregulated MAPK pathway activity, and clinical MAPK pathway inhibitors are available, pLGG treatment remains challenging. Tumor progression, rebound, and therapy resistance limit the success of MAPK pathway inhibitors, especially in high risk subgroups, such as BRAF V600E mutant CDKN2A deleted gliomas.To define mechanisms for progression and overcome resistance, we generated BRAF V600E mutant, CDKN2A deleted murine glioma models of different grades and drug sensitivities. Here, we use genetically engineered mice to target expression of BRAF V600E and deletion of CDKN2A to various germinal and non-germinal areas, assessing neuro-anatomical aspects of oncogene sensitivity. Using neural stem cell (NSC)- and astrocyte-, and oligodendrocyte progenitor cell (OPC)-selective driver line, we investigate the likely origin for pLGG and therapy resistant glioma stem cells, developing models that faithfully recapitulate human tumor development. Our numerous immunocompetent, orthotopic BRAF V600E mutant glioma model derived from endogenous tumors exhibit a range of sensitivities to MAPK inhibitor therapies, allowing us to define mechanisms for therapy resistance. In syngeneic, orthotopic BRAF V600E mutant CDKN2A deleted gliomas, we define MAPK pathway inhibitor-induced-changes in the tumor microenvironment, including the immune infiltrate, and investigate immune modulatory therapy as novel treatment strategy for children with BRAF mutant gliomas. Our numerous models for BRAF mutant glioma will facilitate research on tumor development, progression and the tumor-immune interactions. Collectively, our results describe genetic alteration- and microenvironment-based mechanisms for BRAF mutant tumor initiation and progression, and suggest therapeutic combination therapies for glioma.

Citation Format: Claudia K. Petritsch, Stefan Grossauer, Wei Wang, Anne Marie Barrette, Jongwhi Park. Clinically relevant MAPK pathway inhibition reverses stem cell fate defects and sensitize BRAF mutant glioma to immune modulatory therapies [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 3072.
Abstract 3083: Role of S100A10 in the metastatic colonization of breast cancer stem cells
Authors: Shimono Yohei , Yanagi Hisano , Watanabe Takashi , Nishimura Tatsunori , Hayashi Takanori , Okada Seiji , Suzuki Motoshi , Kawada Kenji , Minami Hironobu , Gotoh Noriko
Published: July 2021

Breast cancer is now the most frequently diagnosed cancer and the leading global cause of cancer death in women. Despite advances in the diagnosis and treatment of human breast cancer, advanced stage, metastatic breast cancers are difficult to cure. Among the common target organs for breast cancer metastasis, post recurrence survival is worst when multiple metastasis was observed in the liver. Cancer stem cells (CSCs) are a sub-population of the cells that retain high tumorigenic capacity and, at the same time, are able to sustain the formation of tumors that recreate the cellular diversity of the parent lesions from which they have been originally isolated. We and others have shown that in epithelial malignancies such as breast and colorectal cancers, self-renewal ability of cancer cells with CSC properties is epigenetically regulated by their expression of microRNAs, such as with miR-200c, miR-142 and miR-221. Because of their highly tumorigenic properties, CSCs with are associated with metastatic progression, especially at the initial steps of metastases. We have previously established the breast cancer patient-derived tumor xenograft (PDX) mouse model in which CSC marker CD44+ cancer cells formed spontaneous microscopic metastases in the liver. Comparison of gene expression profiles between the CD44+ human breast cancer cells metastasized to the liver (‘metastasized CSCs') and those in the primary site (‘primary CSCs') revealed that S100A10 was upregulated in metastasized CSCs than in primary CSCs. Knockdown of S100A10 in breast cancer cells reduced the matrix metalloproteinase activity and suppressed their invasion abilities in transwell cell invasion assays in which upper surface of transwell inserts were precoated with Matrigel. Knockdown of S100A10 suppressed, and overexpression of S100A10 enhanced the 3D organoid formation capacities of breast cancer PDX cells in vitro. Finally, constitutive knockdown of S100A10 significantly reduced their metastatic ability to the liver in vivo. Upregulation of S100 family protein expression is observed in many human cancers, including breast, lung, bladder and gastric cancers. These findings suggest that S100A10 functions as a metastasis promoter of breast CSCs by conferring both invasion ability and CSC properties in breast cancers.

Citation Format: Yohei Shimono, Hisano Yanagi, Takashi Watanabe, Tatsunori Nishimura, Takanori Hayashi, Seiji Okada, Motoshi Suzuki, Kenji Kawada, Hironobu Minami, Noriko Gotoh. Role of S100A10 in the metastatic colonization of breast cancer stem 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 3083.
Abstract 3093: Nix mediated mitophagy maintains ovarian cancer cell stemness
Authors: Khaket Tejinder Pal , Wang Qi-En
Published: July 2021

Ovarian cancer is the most frequent cause of death among gynecologic malignancies. Despite the availability of effective therapeutics against cancer, the overall 5-year survival rate of ovarian cancer is still ~30-40%. Most of the lethality are due to cancer relapse and drug resistance, where cancer stem cells (CSCs) are supposed to be the key regulators. CSCs also known as tumor-initiating cells, are a small subgroup of tumor cells that can develop tumors through their capability of self-renewal and differentiation. CSCs can canonically nurture therapeutic resistance, tumor invasion, metastasis, and recurrence, which tremendously attract the scientific community for the unique CSC-based cancer therapeutics. While changing phenotypical features of CSCs complicates their identification and thereby metabolism-based alternate strategy seems to be worthy anti CSC therapy. Though autophagic degradation of mitochondria (mitophagy) based mitochondrial dynamics endowed cancer cells with stemness, but wide informational gaps and missing mechanistic knowledge further necessitates the study on mitophagy dependent CSC regulation. In this study, we investigated the potential involvement of mitophagy in maintenance of cancer cell stemness. In our experimental conditions, FACS sorted CD117/44+/+, ALDHbr and ovarian cancer stem cell like cells (CSCLCs) from OVACR4 and OVCAR3 cells showed higher mitochondrial translocation of cytosolic LC3 that directly indicates the progression of mitophagy in these subpopulations. Reduced mitocontent in CSCLCs as compare to bulk cells also supports the mitochondrial degradation. To analyze the involvement of mitophagy in stemness, mitophagy has been targeted with mDIVI a well-known mitophagy inhibitor, which downregulate the sphere forming ability and protein levels of stemness marker such as SOX2 in ovarian cancer cells. Furthermore, the mechanistic detail of mitophagy involvement in cancer stemness were evaluated. Among different mitophagy proteins, BNIP3L/NIX is highly expressed in ALDHbr and CD117/44+/+ sorted cells as compare to ALDHdim and CD117/44-/- cells, respectively. The functional implication of NIX in ovarian CSCs was verified by gene silencing approach. NIX silencing diminished the sphere forming ability and stem cell marker OCT4 that supported its crucial involvement in ovarian cancer cells stemness. In summary, our findings provide a mechanistic detail of mitophagy underlying maintenance of the CSC population in ovarian cancer and suggest that targeting of NIX based mitophagy could be exploited for the eradication of CSC and thereby tumor recurrence and drug resistance in ovarian cancer.

Citation Format: Tejinder Pal Khaket, Qi-En Wang. Nix mediated mitophagy maintains ovarian cancer cell stemness [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 3093.
Abstract 3096: A novel ZIP4-NOTCH3-HDAC4 axis in ovarian cancer stem cells
Authors: Xu Yan , Fan Qipeng , Emerson Robert
Published: July 2021

High grade serous ovarian cancer (HGSOC) is one of the most deadly and heterogenic cancers. We have recently shown that ZIP4 (gene name SLC39A4), a zinc transporter, is a novel cancer stem cell (CSC) marker in HGSOC. 100-200 ZIP4+, but not ZIP4-, cells from both PE04 and PEA2 cells formed larger tumors than those from 100-200 ALDH+ cells in mice. Mechanistically, we found that ZIP4 was an upstream regulator of another CSC-marker, NOTCH3, in HGSOC cells. NOTCH3 was functionally involved in spheroid formation in vitro and tumorigenesis in vivo in HGSOC. Drug-resistance is one of the main characteristics of CSCs, While ZIP4 converts drug-resistance to cisplatin (CDDP) and doxorubicin (DOX) as we reported previously, we unexpectedly found that ZIP4 induced a sensitization of HGSOC cells to histone deacetylase inhibitors (HDACis). In particular, only those HDACis against the Class IIa HDACs showed ZIP4-dependent sensitization. ZIP4 selectively up-regulated HDAC IIa HDACs, including HDAC4 and 5, with little or no effects to HDACs in other classes. ZIP4 knockout (KO) and HDAC4 knockdown (KD) increased cell resistance to LMK-235, a selective HDAC4/5 inhibitor. LMK-235 and HDAC4 knockdown (KD) inhibited spheroid formation in vitro and tumor development in vivo. Collectively, we revealed a novel ZIP4-NOTCH3-HDAC4 axis, which is functionally involved and important in CSC-related activities in vitro and tumorigenesis in vivo, and provide an innovative targeting strategy to CSC.

Citation Format: Yan Xu, Qipeng Fan, Robert Emerson. A novel ZIP4-NOTCH3-HDAC4 axis in ovarian cancer stem 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 3096.
Abstract 312: Targeted NanoSpheres (TNS)
Authors: Kang HyungGyoo , Nagy Jon , Upton Bryon , Upton Racheal , Triche Timothy , Triche Timothy
Published: July 2021

The tumor-targeted, nanoparticle delivery technology has shown the possibility of increasing the delivered dose of virtually any small therapeutics to tumor cells while limiting the drug uptake by normal tissues, thereby greatly increasing the effective MTD. By refining the early formulations and after extensive testing, we developed a novel targeted liposomal nanoparticle, Targeted NanoSpheres (TNS). We showed this novel nano-formulated platform can overcome many deficiencies of conventional nanoparticles and therefore has enhanced potential as an effective delivery vehicle for virtually any type of cancer for which a suitable tumor cell surface antigen is present. We have developedNV103, a first-in-class anti-CD99 TNS (CD99-TNS/Irinotecan), for the treatment of Ewing Sarcoma (EWS), a highly lethal pediatric and young adult bone and soft tissue tumor driven by a fusion protein, typically EWS/FLI1 in over 85% of cases. Cancer cells have the ability to develop resistance to the chemotherapy. Our present salvage/rescue study with NV103 showed that NV103 can overcome irinotecan resistance of Ewing tumor with simple change of dose schedule (dose intensification), not increasing the dose. The results from our salvage/rescue studies with NV103 suggested that successful dose intensification schedules with NV103 could be used to treat relapsed or resistant patients in future clinical trials. Furthermore, Targeted Nanosphere (TNS) can be used to overcome drug resistance of various cancers.This platform is also being investigated for other types of cancer that either express CD99, or other tumor antigens like GD2 and B7-H3.

Citation Format: HyungGyoo Kang, Jon Nagy, Bryon Upton, Racheal Upton, Timothy Triche, Timothy Triche. Targeted NanoSpheres (TNS) [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 312.
Abstract 3125: Analysis of intratumoral protein and gene expression heterogeneity in gastric cancer with a clustering analysis
Authors: Tsujio Gen , Yashiro Masakazu , Yamamoto Yurie , Sera Tomohiro , Sugimoto Atsushi , Kushiyama Shuhei , Nishimura Sadaaki , Yoshi Mami , Tamura Tasuro , Toyokawa Takahiro , Tanaka Hiroaki , Lee Shigeru , Muguruma Kazuya , Ohira Masaichi
Published: July 2021

Background/Aim: Intratumoral heterogeneity has been reported in various cancer types and may cause cancer metastasis and chemotherapy treatment resistance, including resistance to molecular targeted therapy. In gastric cancer, the intratumoral heterogeneity of cancer-associated molecules, such as HER2, has been reported and relates to the acquisition of chemotherapy resistance. To reveal whether intratumoral heterogeneity is related to cancer etiology, such as the mechanism of metastasis, and the development of new treatment strategies, such as combination therapy, we investigated intratumoral heterogeneity of gastric cancer with operative specimen and Cancer Genome Atlas (TGCA) data.

Methods: The study included 503 patients who underwent gastrectomy and immunohistochemical staining for gastric cancer between 2001 and 2006 and TGCA data. We conducted clustering analysis based on protein and gene expression data for FGFR2IIIb (FGFR2), HER2(ERBB2), CXCR2(CXCR2), CXCR4(CXCR4), cMet(MET), TGFβ1(TGFB1), IGF1R(IGF1R), and p-smad2(SMAD2), and we analyzed the relation of FGFR2IIIb (FGFR2) and HER2(ERBB2) expression with the expression of other proteins and genes. Additionally, we investigated clinicopathological feature correlations.

Result/Discussion: We divided gastric cancer specimens into nine clusters by immunohistochemical staining and 11 clusters using TCGA data. According to immunohistochemical staining, the high HER2 and FGFR2IIIb expression groups formed a cluster with high expression of various proteins. Analysis of stage 3-4 samples revealed more high protein expression clusters than the analysis of stage1-2, and this suggests that cancer acquires heterogeneity with cancer progression. In the TGCA data of stage 3-4, high FGFR2 and MET and high ERBB2 and SMAD2 or IGF1R expression formed clusters, which is consistent with the clustering analysis that was performed with immunohistochemical staining results.

Conclusion: Our findings suggest that the existence of a combination of intratumoral heterogeneity and clusters may change with cancer progression.

Citation Format: Gen Tsujio, Masakazu Yashiro, Yurie Yamamoto, Tomohiro Sera, Atsushi Sugimoto, Shuhei Kushiyama, Sadaaki Nishimura, Mami Yoshi, Tasuro Tamura, Takahiro Toyokawa, Hiroaki Tanaka, Shigeru Lee, Kazuya Muguruma, Masaichi Ohira. Analysis of intratumoral protein and gene expression heterogeneity in gastric cancer with a clustering analysis [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 3125.
Abstract 313: Synergistic interaction between gingerol, shogaol and paradol with platinum-based chemotherapeutic drugs against naïve and resistant breast cancer cells
Authors: Bawadood Aziza S. , Al-Abbasi Fahad A. , El-Halawany Ali M. , Al-Abd Ahmed M.
Published: July 2021

Breast cancer is one of the most common female cancers and contributes 25.4% of the total diagnosed malignancies in women in 2018. Shogaol, paradol, and gingerol are obtained from many plants belonging to the family Zingiberaceae such as dried ginger and grain of paradise and shows different pharmacological activities. The current study evaluated the anticancer potential of these compounds in combination with chemotherapeutic platinum drugs against breast cancer cells. Cytotoxicity assay, apoptosis assay, immunofluorescent staining cell surface marker analysis, and wound healing assay were used for evaluating the cytotoxic potential of these compounds alone and in combination with platinum chemotherapeutics agents against naïve and resistant breast cancer cell lines (MDA-MB-231 and MCF-7adr, respectively). Platinum drugs showed synergism in combination with shogaol and gingerol against the resistant breast cancer cells (MCF-7adr) with a Combination Index (CI) values of 0.39 and 0.54, respectively. A significant increase was observed in the percentages of early and late apoptotic cells for MDA-MB-231 cells treated with 6-shogaol for 24h. Significant decrease in CD44+/CD24- subpopulation was recorded in both cell lines using flowcytometric analysis. In the Wound healing assay, cells treated with the test compounds migrated significantly slower than control and needed a longer time to heal the scratch. In conclusion, gingerol, shogaol, and paradol compounds showed synergistic cytotoxicity with platinum-based drug and augmented their migration inhibitory activity, apoptotic effects, and tumor-associated stem cell suppressive the potential within naïve and resistant breast cancer cells.

Citation Format: Aziza S. Bawadood, Fahad A. Al-Abbasi, Ali M. El-Halawany, Ahmed M. Al-Abd. Synergistic interaction between gingerol, shogaol and paradol with platinum-based chemotherapeutic drugs against naïve and resistant breast 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 313.
Abstract 3133: Clonal facilitation drives estrogen receptor positive (ER+) breast cancer growth and survival
Authors: Emond Rena , Grolmusz Vince K. , Griffiths Jason , Chen Jinfeng , Adler Frederick R. , Bild Andrea
Published: July 2021

Competition between different lineages of cells within a tumor has been included in thinking about cancer evolution and progression, but positive interactions like cooperation have received far less study. We have developed isogenic estrogen positive breast cancer cell lineages sensitive and resistant to CDK4/6 inhibition in order to investigate the mechanisms cell interactions under selective pressure. When these sensitive and resistant lineages are grown in co-culture under ribociclib treatment, sensitive survive and proliferate better than in mono-culture despite the effects of competition. Conditioned media taken from resistant cells grown in monoculture conditions reproduces this advantage, suggesting that secretion of some factor from resistant cells underlies the effect. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays were used to identify factors secreted by cell lineages. While the less active estrone metabolite was detected in all lineages, the highly active estradiol was only detectable in mono- or coculture with resistant cells. As sensitive cells are more dependent on estrogen signaling than resistant cells, they benefit more from the increase in local estradiol, leading to facilitative growth when cocultured. Cooperative signaling between isogenic cancer cells is enhanced through addition of estradiol and blocked by small molecule inhibitors targeting estrogen signaling pathways. We have developed mathematical models that quantify the strength of competition and facilitation between cell lines and evaluated the effects of treatments that modify these interactions. These analyses support the conclusion that these heterogeneous cell lineages cooperate to survive during therapy through the increased production and sharing of local estradiol. This multidisciplinary approach reveals interactions between ER+ cancer lineages during treatment and provides insight into cell cooperation and competition during treatment, and how these interactions promote cancer cell growth and survival during treatment.

Citation Format: Rena Emond, Vince K. Grolmusz, Jason Griffiths, Jinfeng Chen, Frederick R. Adler, Andrea Bild. Clonal facilitation drives estrogen receptor positive (ER+) breast cancer growth and survival [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 3133.
Abstract 3146: Heterogeneity of the antigen-expression pattern in primary and recurrent glioblastoma patients
Authors: Dufner Vera , Meyer-Hofmann Moritz , Feldheim Jonas , Maurus Katja , Monoranu Camelia , Nerreter Thomas , Hudecek Michael , Hagemann Carsten , Ernestus Ralf-Ingo , Löhr Mario
Published: July 2021

Introduction: Immunotherapy leads to promising results in the treatment of glioblastoma (GBM) patients. Recent clinical trials revealed a survival benefit of patients treated with monospecific CAR-T-cells. However, antigen escape mechanisms due to heterogeneous surface expression of target antigens still constitute a critical hurdle in the immunotherapeutic treatment of GBM and are a major reason for non-response or relapse. Therefore, we aim to characterize GBM's heterogeneity, the intratumoral distribution and changes of surface antigens in relapse.

Methods: We performed immunohistochemical staining of the ten in vitro and in vivo most targeted antigens in CAR-T cell therapy (HER2, EGFRvIII, CD70, B7H3, Il13Rα2, NY-ESO1, GD2, CD133, CSPG4 and EphA2). We analyzed 35 patients, who underwent surgery for primary (PT) as well as recurrent GBM (RL). To address intrapersonal heterogeneity, we examined five fields of view per slide. As controls, we stained cerebral, cerebellar and brain stem slides of three healthy specimens. We quantified the percentage of low, medium and high antigen expressing cells on each slide by applying a specialized macro in ImageJ. We further weighted this expression score by multiplication with 1 (low), 2 (medium) or 3 (high expression). Finally, we calculated the median expression values of all tumors combined for each antigen.

Summary: Median values of EGFRvIII (PT: 0.14; RL: 0.73), HER2 (PT: 0.67; RL: 4.00) and NY-ESO1 (PT: 0.00; RL: 0.16) expression were very low. Antigen expression of CSPG4 (PT: 4.67; RL: 2.78), Il13Rα2 (PT: 3.84; RL: 2.78), GD2 (PT: 7.50; RL: 27.03) and B7H3 (PT: 8.81; RL: 16.62) was mild. CD133 (PT: 52.16; RL: 56.13), CD70 (PT: 19.18; RL: 25.02) and EphA2 (PT: 60.65; RL: 55.46) showed moderate antigen expression. None of the evaluated antigens displayed high expression. Median values of HER2 (p=0.005, Cohen's r=0.48), EGFRvIII (p=0.042, Cohen's r=0.344), and GD2 (p=0.013, Cohen's r=0.42) were significantly increased in RL compared to PT. We found significant intrapersonal expression differences for each antigen. However, none of the healthy brain tissue specimens expressed any of the ten antigens.

Conclusions: Inter- and intrapersonal heterogeneity constitutes a major obstacle for the implementation of GBM immune therapies which avoid antigen escape mechanisms.

Citation Format: Vera Dufner, Moritz Meyer-Hofmann, Jonas Feldheim, Katja Maurus, Camelia Monoranu, Thomas Nerreter, Michael Hudecek, Carsten Hagemann, Ralf-Ingo Ernestus, Mario Löhr. Heterogeneity of the antigen-expression pattern in primary and recurrent glioblastoma 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 3146.
Abstract 3177: Intrinsic and acquired resistance to RIG-I induced anti-tumor responses
Authors: Fraile-Bethencourt Eugenia , Khou Sokchea , Anand Sudarshan
Published: July 2021

The immune microenvironment plays a critical role in cancer orchestrating tumor progression and response to therapeutics. Strategies to manipulate the tumor immune microenvironment are invaluable to complement current standard of care treatments. Recently, approaches to target Pattern Recognition Receptors (PRRs) that recognize intracellular and extracellular pathogens have emerged as a promising strategy to enhance immune recognition of tumors. Among the PRRs, intracellular receptors that recognize DNA and RNA in the cells are unique in their ability to respond to both pathogenic and ‘self' nucleic acids that are generated as a result of aberrant cell division/transcription. We previously showed that targeting a cytosolic DNA sensor three prime exonuclease 1 (TREX1) enhanced immune responses in mouse tumor models including colorectal carcinomas (CRCs). Here we show that a cytosolic RNA sensing pathway, Retinoic acid Induced Gene 1 (RIG-I) acts as a robust immune activator across cancer types by enhancing the cellular and molecular correlates of anti-tumor immune responses. Using a stem loop hairpin RNA structure derived from a H1N1 sequence we elicited a potent RIG-1 response in CRC and breast cancers. Interestingly, activation of RIG also increased expression of TREX1 revealing potential cross-talk between the two pathways. We show that activation of tumor cell RIG-I alone is sufficient to decrease tumor burden with a concomitant broad cellular and molecular anti-tumor response. We observed a group of negative regulators of immune responses, including the NKG2a ligand - HLA-E, mono PARPs and the inhibitory checkpoint protein LAG3, are upregulated in RIG high tumors in cell culture, mouse and human tumors indicating how RIG-I high tumors may develop resistance to immune killing. Combination of RIG-I agonists synergized with inhibition of these resistance mechanisms in a tumor and context dependent manner. To understand intrinsic resistance to RIG signaling, we performed a focused RNAi screen and identified a few critical positive and negative modulators of RIG-I function in tumor cells. Taken together, our findings illustrate how tumor cell RIG-I is a potent inducer of cellular and molecular immune responses as well as highlight both intrinsic and acquired resistance to RIG-I induced anti-tumor responses.

Citation Format: Eugenia Fraile-Bethencourt, Sokchea Khou, Sudarshan Anand. Intrinsic and acquired resistance to RIG-I induced anti-tumor responses [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 3177.
Abstract 3189: Integrated analysis for tumor-immune interaction and its application to ENKTCL immunotyping
Authors: Liu Yanbin , Gao Yan , Shao Yun , Meng Fang , Huang Huiqiang , Qin Frank Xiaofeng
Published: July 2021

Immunotherapy represents a major breakthrough for multiple cancer types, yet it remains necessary to expand disease indications and improve treatment efficiency based on better understanding of tumor-immune interaction. Three currently validated biomarkers (PD-L1, MSI/dMMR and TMB) are not sufficient to predict the efficacy and patient selection for immune checkpoint therapy. We hereby propose a comprehensive tumor-immune interaction typing strategy by integrating WES, RNA-seq and TCR-seq omic technologies for the detection of gene mutations, copy number variation, fusion genes, clone variation, HLA typing, tumor antigen prediction, tumor microenvironment immune repertoire analysis and tumor lymphocyte infiltration composition under the condition of one-time sampling. Thus enabling the systematic analysis of the tumor microenvironment characteristics. Applied to a cohort of ENKTCL patients, we studied the interactions between the tumor cells and the immune system tumor microenvironment. We discovered changes of tumor immunogenicity caused by DNA variation, e.g. abnormalities in immune regulatory region 6q21, DNA repair region, cases of fusion, CNV and LOH events. These mutational events summarizes the tumor intrinsic factors in NKTL. To study the TME and “extrinsic” factors, we investigated mRNA signature enrichment patterns in patient samples and found upregulation of various immune signatures such as macrophages, T helper cells in NKT tissue samples. As examples, CD274 and PDCD1 are significantly upregulated in NKTL tissue. We also found lower TCR diversity in NKTL patients. These discoveries suggest the association of tumor microenvironment with tumor intrinsic factors to elucidate the NKTL disease etiology. Taking together, our multi-omics datasets may also provide useful resources for further interrogating with treatment and clinical outcomes.

Citation Format: Yanbin Liu, Yan Gao, Yun Shao, Fang Meng, Huiqiang Huang, Frank Xiaofeng Qin. Integrated analysis for tumor-immune interaction and its application to ENKTCL immunotyping [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 3189.
Abstract 341: Predicting clinical endocrine response in advanced breast cancers using a reproducible low-dimensional biomarker
Authors: Nath Aritro , Cohen Adam , Chang Jeffrey , Bild Andrea
Published: July 2021

In the absence of reliable and effective prognostic biomarkers, endocrine therapy remains the standard of care for all advanced and metastatic estrogen receptor-positive (ER+) breast cancers. Attempts to develop biomarkers using the baseline tumor transcriptome or genome of advanced ER+ breast cancers have so far been unsuccessful due to predictive models with poor reproducibility in independent studies. Here we present an approach to develop a low-dimensional biomarker that estimates the risk of adverse events on endocrine therapy using the baseline tumor transcriptome of patients. Using a framework for supervised dimensionality reduction of the gene expression feature space, we constructed an endocrine response signature (ENDORSE) modeled on the survival outcomes of ER+ breast cancers from METABRIC. ENDORSE outperformed transcriptome-wide and knowledge-based signature models while significantly improving upon routine histopathological and genomic classifiers in cross-validation analyses. The ENDORSE risk estimate accurately predicted the outcomes for endocrine therapy in three independent clinical trials for ER+ breast cancers. Further, analysis of the phenotypes enriched in high-risk categories show endocrine resistance was not associated with rates of proliferation, but instead with a potential loss of DNA damage repair and cell-matrix interaction pathways.

Citation Format: Aritro Nath, Adam Cohen, Jeffrey Chang, Andrea Bild. Predicting clinical endocrine response in advanced breast cancers using a reproducible low-dimensional biomarker [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 341.
Abstract 365: Detection of homologous recombination deficiency across cancer types by a real-time activity-based functional biomarker
Authors: Lee Chih-Ying , Lei Kai-Hang , Huang Shih-Han , Ko Min-Yu , Chang Ko-Yu , Lin Po-Han , Chen Yu-Li , Cheng Wen-Fang , Chi Peter
Published: July 2021

Homologous recombination (HR) is an error-free repair pathway to eliminate DNA double-strand breaks and crucial for maintaining genome stability. Pathogenic mutations in genes involved in HR, such as BRCA1 and BRCA2, lead to homologous recombination deficiency (HRD) and sensitive cells to poly(ADP-ribose) polymerase inhibitors (PARPi). As such, the selection of HRD cancer patients becomes an important clinical need. In addition to sequencing HR-related genes, recent studies have developed several approaches to detect HRD including genomic scar score, mutational signatures analysis, and RAD51 foci formation assay. However, these assays are not on a real-time basis and provide an indirect estimation of HR status. To overcome these limitations, we have successfully developed a virus-based functional assay to directly quantify HR activity in cells. Our method detects HR status in a real-time and tumor-only manner. By using this activity-based functional assay, we reveal a universal activity threshold for identifying HRD across cancer types. Here, we present a promising method to accurately detect primary ovarian cancer cells with HRD. Clinical samples that be quantified as HRD show a significant response to PARPi. Therefore, our method can serve as a functional biomarker and companion diagnostic for PARPi.

Citation Format: Chih-Ying Lee, Kai-Hang Lei, Shih-Han Huang, Min-Yu Ko, Ko-Yu Chang, Po-Han Lin, Yu-Li Chen, Wen-Fang Cheng, Peter Chi. Detection of homologous recombination deficiency across cancer types by a real-time activity-based functional biomarker [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 365.
Abstract 397: ECM2 mutation can serve as a potential efficacious predictor of immunotherapy in melanoma patients
Authors: Yu Qitao , Zeng Aiping , Zhao Yun , Huang Xiaochun , Xie Wenzhuan , Huang Mengli , Bei Ting
Published: July 2021

Background: Epithelial-mesenchymal transition (EMT) has been reported to play a pivotal role in tumor immunosuppression and immune evasion. In multiple solid tumors, PD-L1 expression was much higher in “mesenchymal” compared with that in “epithelial”, indicating patients with mesenchymal phenotypes are more likely to benefit from PD-1/PD-L1 immunotherapy. ECM2 encodes a putative extracellular matrix protein, which involves in cell-cell and/or cell ECM recognition processes, thereby may affecting EMT. However, the predictive value of ECM2 in cancer immunotherapy has not been revealed.

Method: Seven independent public cohorts of NSCLC, melanoma, and pan-cancer, including Rizvi, Van Allen, MSKCC, OAK/POPLAR, Miao, and Samstein cohorts were used to investigate the correlation between ECM2 mutation and clinical events, such as objective response rate (ORR), overall survival (OS), and progression free survival (PFS).

Result: Across all the analyzed cancer types, ECM2 mutation has a strong connection with better outcomes to immune checkpoint inhibitors only in melanoma cancer. ECM2-mutated group was significantly associated with higher ORR (75.0% vs 13.9%, P = 0.013), longer OS (35.9 months vs 8.5 months, 95% CI, 0.02-1.09, P = 0.032) than ECM2-wide-type group. PFS between these two groups was comparable (8.15 months vs 2.8 months, 95% CI, P = 0.25). Furthermore, melanoma patients harboring ECM2 mutation exhibited significantly higher TMB compared with ECM2-wide-type patients in Van Allen cohort (1279.5 mut/Mb vs 125.5 mut/Mb, P = 0.0056).

Conclusion: This study demonstrated that ECM2 mutation can serve as a potential predictor for a favorable response to ICIs in melanoma patients.

Citation Format: Qitao Yu, Aiping Zeng, Yun Zhao, Xiaochun Huang, Wenzhuan Xie, Mengli Huang, Ting Bei. ECM2 mutation can serve as a potential efficacious predictor of immunotherapy in melanoma 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 397.
Abstract 398: Tumor mutational burden estimated by whole-exome sequencing across multiple cancer types
Authors: Xu Fei , Wei Jinwang , Zhang Xiaochen , Liu Jun , Dai Chun , Wang Guan , Xu Qiang
Published: July 2021

Purpose: Tumor mutational burden (TMB) is emerging as a promising biomarker in immune checkpoint inhibitor therapy. Although whole-exome sequencing (WES) is the gold standard to quantify TMB, most TMB measurements in routine diagnostic applications were done by gene panels due to their lower cost and technical accessibility. Therefore, data is lacking for WES derived TMB in several major tumors. However, panel derived TMB can be misleading because research found its correlation with WES derived TMB was compromised when a subset of samples with high TMB values were excluded from the dataset. In this study, we aim to establish the landscape of WES derived TMB in six tumor types in Chinese patients, test the association between WES-TMB and patient survival in immunotherapy, and compare the performance between WES-TMB and panel-TMB.

Methods: We recruited 357 patients who had received immune checkpoint inhibition (ICI) therapy, representing six types of cancers (see Results for the list). We analyzed the tumors and patient-matched blood samples using WES and called somatic mutations of the patients. We defined our WES-TMB as the total number of somatic nonsynonymous mutations in the tumor exome. In each of the tumor types tested, we assigned the patients with the upper tertile TMB values as the high-TMB group and the remaining two thirds of patients as the low-TMB group.

Results: The distribution of WES-TMB medians are highly variable among different tumors, ranging from 2.71 (cholangiocarcinoma), 2.97 (nervous system tumor), 3.69 (gastric cancer), 4.31 (hepatocellular carcinoma), 4.64 (colorectal cancer), to 5.67 (lung cancer) mutations/Mb. This trend is almost the same as other TMB studies done with panel sequencing, despite the values derived from WES tend to be lower. Additionally, TMB was significantly associated with overall survival in colorectal patients receiving ICI. The high-TMB group also showed better overall survival in hepatocellular carcinoma and gastric cancer.

Conclusion: TMBs derived from panel are overestimated compared to that derived from WES although that doesn't affect the rank of TMBs in different cancer types. It is rational to define TMB subgroups by percentile within different cancer types. These results support TMB as a biomarker to predict clinical benefit to ICI therapy in colorectal cancer.

Citation Format: Fei Xu, Jinwang Wei, Xiaochen Zhang, Jun Liu, Chun Dai, Guan Wang, Qiang Xu. Tumor mutational burden estimated by whole-exome sequencing across multiple cancer types [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 398.
Abstract 403: The landscape of PD-L1 expression in lung cancer harboring EGFR mutations
Authors: Zhang Linlin , Gong Fuyu , Meng Fanlu , Wang Xin , Zhong DianSheng
Published: July 2021

Background: Results from previous studies indicated that lung cancer patients harboring EGFR sensitizing mutations may receive unfavorable outcomes from immunotherapy. The present study aims to investigate the landscape of PD-L1 expression in lung cancer harboring EGFR mutations.

Methods: Tissue samples were subjected to NGS in a College of American Pathologists-certified and Clinical Laboratory Improvement Amendments-accredited lab for driver oncogene mutations and PD-L1 expression.

Results: A total of 22143 lung cancer patients were selected, including 12278 (55.4%) male and 9865 (44.6%) female with a median age of 63. There are 9560 patients has the EGFR mutation, and the frequency of EGFR mutation is 43.2%. Among the EGFR mutation patients, 1602 patients have been collected tumor tissue, and the PD-L1 expression have been tested by FDA-approved SP263 and 22C3 antibodies. Among all the EGFR mutation cases assessed PD-L1 expression (n=1602), low PD-L1 expression levels (<1%) were more than half proportion with 56.5% (n=905), PD-L1 expression level of 1%-49% accounted for 31.2% (n=905), PD-L1 expression level of 50%-79% accounted for 9.1% (n=146), PD-L1 expression level of 80%-89% accounted for 1.8% (n=29), and PD-L1 expression level higher than 90% accounted for 1.4% (n=22).

Conclusion: Our study supported that NSCLC patients harboring sensitizing oncogenic mutations may potentially benefit from anti-PD-1/L1 especially for those with positive indicators of immunotherapy with strong positive PD-L1 expression.

Citation Format: Linlin Zhang, Fuyu Gong, Fanlu Meng, Xin Wang, DianSheng Zhong. The landscape of PD-L1 expression in lung cancer harboring EGFR mutations [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 403.