bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2610: A high performance blood test for multiple cancer early screening
Authors: Xu Linhao , Wang Jun , Ma Weifeng , Liu Xin , Yang Ting , Li Sihui , Hu Qian , Tao Jinsheng , Ye Zhujia , Chen Zhiwei , Fan Jian-Bing
Published: July 2021

Introduction: Early detection is critical to improving cancer patient survival and quality of life. Cell-free DNA (cfDNA) methylation profiling is a promising diagnostic approach for early detection of cancer, even before symptoms appear. We previously reported a non-invasive and cost-effective methylation test, Aurora, which is able to detect three major cancer types with high specificity and sensitivity especially at early stages (I/II), including lung cancer (LC), breast cancer (BC), colorectal cancer (CRC). We have improved this test to include two additional major cancer types, gastric cancer (GC) and esophageal cancer (EC). Taking together, these five cancer types represent 56% of new cancer cases and 60% of cancer-related deaths in China.

Methods: We developed a novel high-throughput targeted methylation profiling platform, Aurora 2.0 (an improved version of Aurora 1.0, as presented in AACR 2020), to efficiently enrich and capture cancer-specific DNA methylation markers in plasma, prior to analysis via next-generation sequencing on a MiSeqDx system. About 100 cases each for GC and EC patients (mostly at Stage I/II), together with ~200 healthy controls, have been analyzed by using Aurora 2.0 and used for model development and validation.

Results and Discussion: cfDNA methylation profiles have been obtained from 409 plasma samples, comprising 206 healthy controls, 98 GC and 105 EC patients. Logistic regression model has been developed with the Area Under Curve (AUC) of 94.0% and 93.5% for GC and EC, respectively. Overall, the performance (AUC) of Aurora 2.0 for LC/BC/CRC/GC/EC are 97.3%, 96.2%, 92%, 94% and 93.5%, respectively. An independent cohort (~2,000 plasma samples, five cancer types including lung, breast, colorectal, gastric and esophageal cancers with healthy controls) study is undergoing to further validate this test prospectively. The superior performance and cost-effectiveness (~$100) of the test could make early cancer screening of multiple major cancers in symptom-free and average-risk populations more achievable.

Citation Format: Linhao Xu, Jun Wang, Weifeng Ma, Xin Liu, Ting Yang, Sihui Li, Qian Hu, Jinsheng Tao, Zhujia Ye, Zhiwei Chen, Jian-Bing Fan. A high performance blood test for multiple cancer early screening [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 2610.
Abstract 2612: Personalized cancer risk and prevention: analysis of risk factors for specific cancers and integration into cancer risk assessment paradigms
Authors: Elespuru Rosalie K. , Fischer Catherine
Published: July 2021

Cancer risk assessment is performed generally in relation to assessment of the safety of new medical products or environmental exposures, especially when DNA modification is a factor. The risk assessment paradigms used for products subject to regulatory review are independent of specific cancers, multiple exposures, and individual risk factors. A default cancer risk assessment for regulatory review includes “uncertainty factors” to account for differential susceptibility and other variables involved in extrapolating from experimental systems to human risk. However, there is a growing amount of evidence that suggests differing risk for different types of cancer as well as the importance of combinations of risk factors not limited to genetic or environmental effects. In the era of personalized medicine and cancer genomics, can we begin to assess personalized cancer risk, instead of overall cancer risk? What models would assist in this paradigm shift? There are several cancers for which specific genetic, environmental, viral and other associations have been established and could be used in personalized cancer risk assessments, including colon, liver, and lung cancer. A search was conducted on PubMed for available literature on epidemiology, genetic susceptibility, and risk factors associated with the major types of cancer, including ~25 pathological types of cancer in 17 different organs. Risk factors associated with each included chemical exposures and tobacco, as well as genetic, epigenetic, microbial, viral, dietary, exercise, and other factors known for involvement in the development of cancer in humans. The diversity of cancers and evidence for specific risk factors is summarized. Common risk factors, including smoking and obesity, are supplemented with a substantial set of individual, often non-overlapping risk factors for diverse cancers. Surprisingly, little evidence was found that genetic-environmental interactions are significant risk factors for most cancers. From this assessment we can begin to ask whether and for what cancers a personalized risk approach seems feasible.

Disclaimer: The findings and conclusions in this presentation are those of the authors and should not be construed to represent any agency determination or policy.

Citation Format: Rosalie K. Elespuru, Catherine Fischer. Personalized cancer risk and prevention: analysis of risk factors for specific cancers and integration into cancer risk assessment paradigms [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 2612.
Abstract 2613: Cryptotanshinone and dihydrotanshinone epigenetically suppress human colorectal cancer cell growth
Authors: Su Zheng-Yuan , Chen Yu-Hsin Chen , Wu Tien-Yuan
Published: July 2021

Most cancer cases are closely related to many risk factors such as environment and diets. Carcinogenesis may also be controlled by epigenetic regulation. Salvia miltiorrhiza Bunge (Danshen) is a common Chinese herbal medicine, and it has been reported that it has anti-cancer, anti-inflammatory, anti-coagulant, and anti-thrombotic activities. In this study, we studied the effect and epigenetic mechanisms of cryptotanshinone (CTS) and dihydrotanshinone (DHTS), the major active ingredients in Danshen, against human colorectal cancer HCT116 cells. The results showed that CTS and DHTS suppressed the proliferation of HCT116 cells for 72-hour treatment and induced mRNA and protein expressions of p21 and DLEC1. Pro-apoptosis proteins such as PARP, caspase-3, and caspase-9 are cleaved and activated in HCT116 cells treating CTS and DHTS. Moreover, CTS and DHTS also increased the protein levels of epigenetics regulating enzymes, including histone deacetylases (HDACs) and DNA methyltransferases (DNMTs) in HCT116 cells. Therefore, it is suggested that CTS and DHTS may affect tumor suppressor genes, such as p21 and DLEC1, by regulating epigenetic mechanisms, which induce apoptosis of colorectal cancer HCT116 cells.

Citation Format: Zheng-Yuan Su, Yu-Hsin Chen Chen, Tien-Yuan Wu. Cryptotanshinone and dihydrotanshinone epigenetically suppress human colorectal cancer cell growth [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 2613.
Abstract 2647: HOXB7, a key transcriptional regulator in triple negative breast cancer progression
Authors: Jin Kideok
Published: July 2021

Despite major advances in breast cancer treatment and management one-third of women with ER-positive, and nearly 30% of women with ER-negative breast cancer will have recurrent disease within fifteen years. Our laboratory and others have accumulated a large body of evidence for the involvement of HOXB7 in various aspects of breast cancer progression: tamoxifen resistance, DNA repair, epithelial-mesenchymal transition, promotion of metastasis in a transgenic mouse model and human cells as well as angiogenesis suggesting a role for HOXB7 not only in the epithelium but also strongly implicating its control on the microenvironment of the tumor. In this project, we focused on understanding factors driven by HOXB7 that lead to triple-negative breast cancer (TNBC). In the previous studies, we found that HOXB7 expression results in the recruitment of stromal components and enhances the formation of lung metastasis in transgenic crosses of HOXB7xHER2 mice. Interestingly, HOXB7 expression was increased in MDA-MB-231 Bone and Brain metastatic cells. Therefore, we hypothesize that HOXB7, through its ability to mediate EMT and recruit pro-angiogenic molecules, is capable of promoting invasion and metastasis. In this report, we established HOXB7 overexpressing MDA-MD-231 cells. We screened secretomes from MB231-HOXB7 cells using Human Cytokine Array Q440 to quantitatively detect 440 human inflammatory factors, growth factors, chemokines, receptors, and cytokines simultaneously. We identified upregulation of IL-8, IL-6, VEGF, ARG2, CXCL1, CF XIV, Procalcitonin, Syndecan-3, AMICA, IL-11, CXCL14, ROBO3, Follistatin, SOST, and ALCAM in the secretomes from MB231-HOXB7 cells. We ranked the expression level of each factor by real-time qRT-PCR and determined that IL-8 and CXCL1 were the top candidates. We confirmed by ELISA that IL-8 and CXCL1 secreted from tumor conditioned medium (TCM) was upregulated compared to the control cells. Our data showed that the HUVEC tube formation was increased by MDA-MD-231-HOXB7 conditioned media. Furthermore, fibroblast and M2 type macrophage migration were promoted by TCM of MDA-MD-231-HOXB7 cells. Significantly, Depletion of IL-8 and CXCL1 using specific shRNAs in MDA-MD-231-HOXB7 cells decreased the HUVEC tube formation and the cell migration of fibroblast and macrophage. In addition, CXCL1 and IL-8 signaling pathway by Reparixin, an inhibitor of the CXCR1/2 abrogated the tube formation and migration induced by MDA-MD-231-HOXB7 cells. These findings implicate IL-8 and CXCL1 signaling as a critical event in HOXB7 overexpression TNBC growth and metastasis via crosstalk between cancer cells and stromal components.

Citation Format: Kideok Jin. HOXB7, a key transcriptional regulator in triple negative breast cancer 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 2647.
Abstract 2651: Combination of an anti-angiogenic antibody with PD1/PDL1 blockade agents produces synergistic anti-tumor efficacy in preclinical models
Authors: Xue Liting , Zhang Haoyu , Wang Qian , Li Feng , Li Xinxin , Yu Xiaohong , Lu Zhihong , Huang Yue , Yang Wenqing
Published: July 2021

Vascular endothelial growth factor (VEGF) and its receptor (VEGFR) play a fundamental role in tumor growth and angiogenesis. Signalling through VEGF/VEGFR not only induces cancer angiogenesis but also affects immune cells, making VEGF/VEGFR a pair of well-documented cancer therapeutic targets. Recently more and more studies have focused on combination of anti-VEGF/VEGFR agents and immune checkpoint inhibitors in preclinical and clinical settings. BD0801 is a humanized rabbit anti-VEGF monoclonal antibody currently in clinical development stage. In this study, we have revealed that BD0801 presents more potent activity than Bevacizumab in blockade of VEGF/VEGFR2 binding (IC50=275 ng/ml for BD0801; IC50=1451 ng/ml for Bevacizumab) and VEGFR2 activation in HUVEC assays. BD0801 also showed enhanced inhibitory effects on the proliferation (IC50=87 ng/ml for BD0801; IC50=476 ng/ml for Bevacizumab) and migration of the HUVECs, compared to Bevacizumab. We found that BD0801 exhibits dose-dependent tumor growth inhibitory activities in both lung cancer PC9 xenograft and lung cancer 3LL murine syngeneic tumor models. In addition, we obtained PK parameters of BD0801 using lung cancer PC9 tumor bearing mice and successfully made correlations between the in vitro function, PK profile and in vivo efficacy of BD0801. Notably, combination of BD0801 with either anti-PD-1, or anti-PD-L1 antibodies showed synergistic anti-tumor efficacy in both lung and colorectal cancer mouse models. Mechanistic studies were conducted to reveal possible mechanism of actions (MOA) underlying the modification in tumor microenvironment. Our data demonstrated that the MOA of the anti-tumor synergy may involve enhanced T-cell mediated immunity, including increased tumor infiltration of CD8+ and CD4+ T cells and reduced double positive CD8+PD-1+ T cells, as well as improved vasculature normalization. Taken together, these data provide a foundation for combining BD0801 with immunotherapy for cancer treatment and support further clinical development plans for BD0801 toward this direction.

Citation Format: Liting Xue, Haoyu Zhang, Qian Wang, Feng Li, Xinxin Li, Xiaohong Yu, Zhihong Lu, Yue Huang, Wenqing Yang. Combination of an anti-angiogenic antibody with PD1/PDL1 blockade agents produces synergistic anti-tumor efficacy in preclinical models [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 2651.
Abstract 2654: Differential effect of MGMT methylation between moderate or high vascular profile in IDH wild-type glioblastoma
Authors: del Mar Álvarez-Torres Maria , Fuster-García Elies , Juan-Albarracín Javier , Puig Josep , Balaña Carmen , Capellades Jaume , Emblem Kyrre Eeg , García-Gómez Juan Miguel
Published: July 2021

A significant association between MGMT methylation and prolonged overall survival (OS) was observed for patients with glioblastoma (GB) that present moderately relative cerebral blood volume (rCBV) 1. However, that study should be updated to the current glioma WHO classification. In the current study we assess the combined effect of rCBV and MGMT methylation combined on OS in patients with IDH wild-type GB.

An independent cohort of 95 IDH wild-type GB patients with OS>30 days and with tumor resection was included for this study. The rCBV values of the highest angiogenic tumor regions were automatically calculated using ONCOhabitats analysis services2. Finally, the whole cohort was divided into moderately and highly vascularized GB by the median of the previously calculated rCBVs (rCBVmedian=9.56).

We observed a significant association between MGMT methylation and prolonged OS for the whole cohort (p = 0.003, HR = 0.49), as well as for patients with moderate rCBV (p = 0.002, AUC = 0.30). The difference in OS between patients with MGMT methylated and patients with MGMT unmethylated was 4 times higher in the group with moderate rCBV than for the group with high rCBV (205 vs. 42 days). Finally, no association was found between MGMT methylation and OS in patients with high rCBV (p > 0.05) (Table below).

Our results highlight the combined prognostic effect of MGMT methylation and moderate vascularity in IDH wild-type GB patients. As demonstrated, non-invasive MRI perfusion markers may identify those patients who will mostly benefit in terms of prolonged OS owing to their MGMT methylation status.

1: doi.org/10.1007/s00330-020-07297-4

2: doi.org/10.1016/j.ijmedinf.2019.05.002

Number of patientsMedian OS (days)ResultsStatistical resultsTotalMeth MGMTUnmethMGMTMeth MGMTUnmethMGMTΔOSHR [95% CI]pAUCAll9551445894501390.49 [0.30-0.78]0.0028*0.61Moderate rCBV4221216534482050.30 [0.14-0.64]0.0020*0.72High rCBV533023493451420.68 [0.37-1.26]0.21800.52

Citation Format: Maria del Mar Álvarez-Torres, Elies Fuster-García, Javier Juan-Albarracín, Josep Puig, Carmen Balaña, Jaume Capellades, Kyrre Eeg Emblem, Juan Miguel García-Gómez. Differential effect of MGMT methylation between moderate or high vascular profile in IDH wild-type glioblastoma [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 2654.
Abstract 2661: Role of NKX2-1 in thyroid neoplasms induced by iodine-deficiency and radiation
Authors: Shirai Yo-Taro , Takizawa Yoshinori , Iwadate Manabu , Paiz Jorge , Yokoyama Shigetoshi , Miyakoshi Masaaki , Ward Jerrold M. , Kimura Shioko
Published: July 2021

The multi-step carcinogenesis model, including the adenoma-carcinoma sequence, has been proposed in many cancers including those of colon, pancreas, and thyroid. BRAF activation is among the reported gene mutations important for thyroid carcinogenesis. However, the role of NKX2-1, a master regulator of thyroid development and function, in thyroid carcinogenesis remains elusive. In this study, we investigated the role of NKX2-1 in thyroid neoplasm formation using thyroid-specific Nkx2-1 conditional KO mice, Nkx2-1(fl/fl);TPO-Cre (cKO mice). Nkx2-1(fl/fl) (WT) and cKO mice were each allotted to four groups: 1. Control Diet, 2. Control Diet + Radiation, 3. Low Iodine Diet (LID), 4. LID + Radiation. Whole body gamma-irradiation (2 Gy, 3 times every other day) was carried out at 4-8 weeks of age, followed by switching the diet to either Control Diet or LID. Thyroid histology was examined at 1.1-1.3 years after the start of the diet. In both WT and cKO mice, iodine-deficiency, regardless of radiation, induced diffuse hyperplasia (goiter) in more than 50% of the mice, and increased the incidence of focal hyperplasia considered as a preneoplastic lesion. Adenoma formation was only observed in LID-treated mice regardless of radiation, suggesting that iodine-deficiency, but not radiation, is a key factor for mouse thyroid adenoma formation. The incidence of adenoma formation was significantly higher in cKO mice than WT mice in LID + Radiation group (14% for WT vs 52% for cKO, in progress). In addition, carcinoma formation was observed only in LID + Radiation-treated mice, suggesting that radiation is necessary to proceed from adenoma to cancer in thyroid carcinogenesis. Further, there is a tendency for higher incidence of carcinoma in LID + Radiation-treated cKO mice (4% for WT vs 11% for cKO, in progress). These results suggest that the loss of Nkx2-1 can promote adenoma formation induced by iodine-deficiency, and possibly facilitate carcinogenesis induced by LID + Radiation. In summary, our results suggest that NKX2-1 may function as a tumor suppressor in thyroid carcinogenesis. We are now trying to investigate the molecular mechanisms by which loss of NKX2-1 affects thyroid proliferation and facilitates neoplasm formation by examining the molecular profiles of mouse thyroids after LID and/or radiation treatment.

Citation Format: Yo-Taro Shirai, Yoshinori Takizawa, Manabu Iwadate, Jorge Paiz, Shigetoshi Yokoyama, Masaaki Miyakoshi, Jerrold M. Ward, Shioko Kimura. Role of NKX2-1 in thyroid neoplasms induced by iodine-deficiency and radiation [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 2661.
Abstract 2673: The cytoplasmic adaptor FRS2beta fashions a cytokine-rich inflammatory microenvironment that promotes breast cancer carcinogenesis
Authors: Gotoh Noriko , Takeuchi Yasuto , Kimura Natsuko , Murayama Takehiko , Machida Yukino , Iejima Daisuke , Nishimura Tatsunori , Yamamoto Mizuki , Inoue Jun-ichito , Akashi Kouichi , Saya Hideyuki , Kuroda Masahiko , Kitabayashi Issay , Tojo Arinobu
Published: July 2021

It is widely held that pro-inflammatory changes often precede the onset of breast cancer. However, the underlying molecular mechanisms are largely unknown. Here, we demonstrate that FRS2β, a membrane-linked adaptor protein expressed in a small subset of luminal epithelial cells, triggers the inflammatory changes in precancerous mammary tissues and is responsible for the onset of the disease. FRS2β-deficiency in a mouse mammary tumor virus (MMTV)-ErbB2 genetic background markedly attenuated mammary tumorigenesis. Importantly, tumor cells derived from MMTV-ErbB2 mice failed to generate tumors when grafted in the FRS2β-deficient precancerous mammary tissue niche. we observed that the co-localization of FRS2β and the NEMO subunit of the IκB kinase (IKK) complex on early endosomes led to activation of nuclear factor-κB (NFκB). Moreover, the expression of numerous cytokines, including CXC chemokine ligand (CXCL)12, was elevated owing to the activation of NFκB. CXCL12 may in turn activate NFκB in an autocrine and paracrine loop, leading to the inflammatory changes that promote tumorigenesis. The elucidation of the FRS2β-dependent NFκB-activation pathway uncovers a hitherto unknown molecular link between the tissue inflammation and the onset of breast cancer.

Citation Format: Noriko Gotoh, Yasuto Takeuchi, Natsuko Kimura, Takehiko Murayama, Yukino Machida, Daisuke Iejima, Tatsunori Nishimura, Mizuki Yamamoto, Jun-ichito Inoue, Kouichi Akashi, Hideyuki Saya, Masahiko Kuroda, Issay Kitabayashi, Arinobu Tojo. The cytoplasmic adaptor FRS2beta fashions a cytokine-rich inflammatory microenvironment that promotes breast cancer carcinogenesis [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 2673.
Abstract 2684: Single cell transcriptomics reveals the effect of PD-L1 and TGF-B blockade on the tumor microenvironment
Authors: Stone Erica L. , Lim Yoong Wearn , Coles Garry L. , Sandhu Savreet K. , Johnson David S. , Adler Adam S.
Published: July 2021

Background: The anti-tumor activity of anti-PD-1/PD-L1 therapies correlates with T cell infiltration in tumors. Thus, a major goal in oncology is to find strategies that enhance T cell infiltration and efficacy of anti-PD-1/PD-L1 therapy. TGF-β has been shown to contribute to T cell exclusion and anti-TGF-β improves anti-PD-L1 efficacy in vivo. However, TGF-β inhibition has frequently been shown to induce toxicity in the clinic, and the clinical efficacy of combination PD-L1 and TGF-β blockade has not yet been proven. To identify strategies to overcome resistance to PD-L1 blockade, the transcriptional programs associated with PD-L1 and/or TGF-β blockade in the tumor microenvironment should be further elucidated.

Results: We used single-cell RNA sequencing to characterize the transcriptomic effects of PD-L1 and/or TGF-β blockade on cells in the tumor and surrounding microenvironment. Combination treatment led to upregulation of immune response genes, including multiple chemokine genes such as CCL5, in CD45+ cells, and down-regulation of extracellular matrix genes in CD45- cells. Analysis of publicly available tumor transcriptome profiles showed that the chemokine CCL5 was strongly associated with immune cell infiltration in various human cancers. Further investigation with in vivo models showed that intratumorally administered CCL5 enhanced cytotoxic lymphocytes and the anti-tumor activity of anti-PD-L1.

Conclusions: Taken together, our data could be leveraged translationally to improve anti-PD-L1 plus anti-TGF-β combination therapy, for example through companion biomarkers, and/or to identify novel targets that could be modulated to overcome resistance.

Citation Format: Erica L. Stone, Yoong Wearn Lim, Garry L. Coles, Savreet K. Sandhu, David S. Johnson, Adam S. Adler. Single cell transcriptomics reveals the effect of PD-L1 and TGF-B blockade on 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 2684.
Abstract 2692: CXCR4 as a therapeutic target in angiosarcoma
Authors: Ghosh Anindita , Brown Robert E. , Bull Joan M. , Elzamly Shaimaa M. , Buryanek Jamie J.
Published: July 2021

Angiosarcomas account about 2-3% of adult soft tissue sarcomas. This is a rare form of malignancy with remarkable clinical heterogeneity in term of presentation and behavior and can occur in any location of the body. Although radiation and chemotherapy are the usual form of therapy, frequent recurrence and metastasis is observed. The prognosis of angiosarcoma is relatively poor, and the overall 5-year survival rate is approximately 35%. Previous studies have shown the SDF-1 (stromal derived factor-1)/CXCR4 pathway is involved in the progression and the metastasis of angiosarcoma. CXCR4 is a chemokine receptor involved in metastatic spread of malignant tumors and is upregulated by many factors, including hypoxia and the histone methyltransferase EZH2. CXCR4 in turn activates the JAK/STAT3 pathway, which regulates the immune environment by inducing an infiltrate of tumor associated macrophages (TAMs) of the M2 (immunosuppressive) phenotype. Sarcomas in general have increased TAMs of the M2 phenotype and accumulating evidence indicates that a high number of TAMs are associated with an unfavorable prognosis in a variety of malignancies including angiosarcoma. The monocyte chemokine CCL2 has already been documented previously in canine hemangiosarcoma as one of the primary chemoattractants for M2 macrophages. The SDF-1/CXCR4 pathway has also been documented in other malignancies as a regulator of M2 macrophage infiltration and the immunosuppressive environment. It has also been reported that blockade of CXCR4 selectively reduces M2-polarized macrophages. We studied the expression of EZH2, p-STAT3, CXCR4, and CD163 (M2 macrophage marker), in four known cases of angiosarcoma. Two cases involved right lower leg soft tissue and two cases involved tissues from aortic wall. In all of our four cases the tumor cells of angiosarcoma stained positive for EZH2, p-STAT3, CXCR4, and CD163. This supports our hypothesis that indeed in angiosarcoma the M2 macrophages has an association with CXCR4 and pSTAT3. Clinical implications of this are that plerixafor or other CXCR4 antagonists could be used to treat angiosarcoma. By blocking CXCR4, the progression and metastasis of angiosarcoma could be abrogated and the immunosuppressive environment can be normalized. Our study indicates that CXCR4 could be considered a therapeutic target in angiosarcoma and the CXCR4 antagonist plerixafor should be further investigated as a potential agent to improve patient outcomes.

Citation Format: Anindita Ghosh, Robert E. Brown, Joan M. Bull, Shaimaa M. Elzamly, Jamie J. Buryanek. CXCR4 as a therapeutic target in angiosarcoma [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 2692.
Abstract 2701: Molecular profiling to assess the immune response to neoadjuvant SABR in early breast cancer
Authors: Spears Melanie , Talebian Vida , Liao Linda , Hopkins Megan , Lynn Kalan , Lock Michael , Kornecki Anat , Bartlett John M. , Brackstone Muriel
Published: July 2021

Radiation therapy is used successfully in the treatment of breast cancer. Stereotactic radiation therapy (SBRT) is well established in the treatment of lung cancer, brain metastases and various other metastatic sites. Localized radiotherapy can promote dendritic cells maturations and activation and enhances phagocytosis of cells by antigen-presenting cells (APCs) increased presentation of tumor-associated antigenic peptides, and T cells being primed from naïve towards memory phenotypes In this study we evaluated the ability to implement a three fraction SBRT regimen for low-risk primary carcinoma of the breast prior to lumpectomy and profiled the immune microenvironment using NanoString's GeoMx Digital Spatial Profiling (DSP) platform and we performed NanoString gene expression profiling using the Human v.1.1 PanCancer immune profiling panel.

NanoString's DSP platform was used to analyze 25 patient samples from the SIGNAL 2.0 clinical trial. For analysis, region of interest were selected comparing the tumor microenvironment (TME, CD45+ve) and tumor (pan cytokeratin) in pre and post treated FFPE slides. A panel of 60-antibodies were analyzed in each region. For gene expression profiling total RNA was extracted from the frozen tissue and the human V.1.1 PanCancer Immune Profiling Panel was used. We observed notable differences in the immune microenvironment gene expression patterns in samples pre- and post-treatment. We identified a total of 175 differentially expressed genes (DEGs) using a 5% adjusted p-value across the entire patient population regardless of which radiation arm. Pathway analysis identified genes associated with activation of immune response, signaling by interleukins and adaptive immune response were significantly changed in the post treatment samples. Deconvolution of the immune cell mRNA gene expression data demonstrated that there were significant changes in the cellular composition after radiotherapy. There were significant increase in expression levels of macrophages, dendritic cells, neutrophils and CD8 T cells post radiation treatment. In conclusion we have demonstrated through proteomic and transcriptomic profiling, SBRT elicits an immune response with increases in the innate response.

Citation Format: Melanie Spears, Vida Talebian, Linda Liao, Megan Hopkins, Kalan Lynn, Michael Lock, Anat Kornecki, John M. Bartlett, Muriel Brackstone. Molecular profiling to assess the immune response to neoadjuvant SABR in early breast 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 2701.
Abstract 2702: Prostate stromal transcriptome as biomarker of aggressive prostate cancer
Authors: Zhou Yinglu , Ma Chaoran , Fanelli Giuseppe Nicoló , Stefanizzi Lavinia , Gazeeva Elizaveta , Mucci Lorelei A. , Loda Massimo , Tyekucheva Svitlana , Penney Kathryn L.
Published: July 2021

Background: Prostate cancer has a heterogeneous prognosis. Discovery of molecular biomarkers that improve identification of potentially lethal prostate cancer could enhance the ability to determine which patients would benefit most from immediate treatment. Most prognostic biomarkers have focused on those in prostate tumors. However, it is increasingly recognized that the tumor microenvironment contributes to cancer aggressiveness and progression. We therefore examined whole transcriptome expression of the prostate stroma and associations with clinically significant, high-grade prostate cancer.

Methods: We performed RNA-sequencing (Illumina TruSeq RNA Exome library preparation) of macrodissected stromal samples (adjacent to and distant from tumor) from 363 radical prostatectomy specimens from Health Professionals Follow-up Study and Physicians' Health Study participants. We performed a differential expression analysis comparing the gene expression and pathways of stroma adjacent to and distant from tumor. We also performed a cluster analysis and computed a single sample gene set enrichment analysis (ssGSEA) score to assess the association of our previously reported 25 stromal signature genes (Tyekucheva et al. 2017) to determine the association with Gleason score.

Results: Eighty-eight genes as well as eight Gene Ontology pathways and fourteen Canonical pathways were differentially expressed (FDR<0.05) between tumor adjacent to and distant from tumor. Pathways included cell-substrate adhesion, muscle development, and cardiomyopathy. 30% of men had high-grade (Gleason score 8-10) tumors. We observed higher expression of the individual signature genes in the cluster enriched for high Gleason score tumors, and a higher stromal signature ssGSEA score was associated with high Gleason score (p=0.04).

Conclusions: Pathways differentially expressed between stroma adjacent to and distant from tumor suggest known differences in cell types by location or a potential field effect. We observed that the direction of the association of the stromal signature with Gleason score agreed with our previous report. We are currently analyzing the full expression dataset to identify additional stromal genes associated with Gleason score and lethal prostate cancer, and performing multi-plex immunohistochemistry to assess stromal cell-type heterogeneity in regions of tumor and normal prostate across tissue microarrays.

Citation Format: Yinglu Zhou, Chaoran Ma, Giuseppe Nicoló Fanelli, Lavinia Stefanizzi, Elizaveta Gazeeva, Lorelei A. Mucci, Massimo Loda, Svitlana Tyekucheva, Kathryn L. Penney. Prostate stromal transcriptome as biomarker of aggressive 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 2702.
Abstract 2706: Utilizing RNAscope to assess STK11-LoF-dependent transcriptional phenotypes in human NSCLC biopsies and evaluate its potential to predict anti-PD1 therapy response
Authors: Sarausky Hailey M. , Lenahan Sean M. , Seward David Joseph
Published: July 2021

Our work demonstrates RNAscope allows simultaneous quantitative assessment of multiple transcripts in situ using formalin fixed paraffin embedded (FFPE) human non-small cell lung cancer (NSCLC) biopsies and suggests this methodology could be used as a rapid surrogate biomarker for genotype/transcriptome profiling stemming for Serine Threonine Kinase 11 (STK11) loss of function (LoF). Recent clinical studies have correlated anti-PD1 therapy resistance in NSCLC with somatic disruption of STK11. Historically, rapid assessment of STK11 status using immunohistochemistry (IHC) has been a challenge. Our work in human NSCLC cell lines suggests that STK11 loss correlates with changes in tumor intrinsic cytokine expression. Several recent studies have suggested such changes may drive immune evasion. We therefore wondered whether cytokine expression profiles from primary human NSCLC biopsies might be used as a reliable biomarker to predict anti-PD1 therapy response. Here we present our findings using multiplexed RNAscope probes on KRAS-driven primary human NSCLC biopsies with and without STK11 loss. This initial work focuses on cytokines identified using our cell culture models as a proof of principle approach. Using CRISPR-Cas9 we engineered paired STK11 KO cell lines derived from 3 independent KRAS-driven human lung adenocarcinomas (NCI-H441, NCI-H1792, NCI-H2009). RNAseq and differential gene expression analysis revealed STK11-loss-dependent increases in IL-6, IL-8 and IL-32 in all three lines. We then created FFPE cell blocks using these cell lines and validated the RNAseq expression changes with multiplexed RNAscope probes in situ. Next, we informatically searched the molecular pathology archives at the UVM medical center and identified primary KRAS-driven human NSCLC biopsies with and without predicted STK11 loss. We then performed multiplexed RNAscope analysis on these samples to quantify IL-6, IL-8 and IL-32 gene expression as a function of tumor genotype in vivo. Our data suggest RNAscope represents a valid methodology to exploit quantitative differential gene expression as a reliable and reproducible surrogate biomarker of somatic STK11 disruption in NSCLC. Our future studies aim to establish whether genotype-dependent cytokine profiles predict anti-PD1 therapy response in KRAS-driven lung adenocarcinomas.

Citation Format: Hailey M. Sarausky, Sean M. Lenahan, David Joseph Seward. Utilizing RNAscope to assess STK11-LoF-dependent transcriptional phenotypes in human NSCLC biopsies and evaluate its potential to predict anti-PD1 therapy response [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 2706.
Abstract 2716: P-selectin axis plays a key role in microglia immunophenotype and glioblastoma progression
Authors: Yeini Eilam , Ofek Paula , Pozzi Sabina , Albeck Nitzan , Ben-Shushan Dikla , Tiram Galia , Golan Sapir , Kleiner Ron , Sheinin Ron , Reich-Zeliger Shlomit , Grossman Rachel , Ram Zvi , Brem Henry , Hyde Thomas , Magod Prerna , Friedmann-Morvinski Dinorah , Madi Asaf , Satchi-Fainaro Ronit
Published: July 2021

Glioblastoma (GB) is an aggressive type of brain cancer with high mortality rate. It is a highly angiogenic tumor exhibiting an extremely invasive nature. As such, its brain microenvironment plays a crucial role in its progression. Microglia are the brain resident immune cells which have been shown to facilitate GB cell invasion and immune suppression. The mechanism by which GB cells alter microglia behavior is yet to be fully understood. One proposed mechanism involves adhesion molecules such as the Selectins family of proteins which are expressed on the surface of endothelial and immune cells and are involved in immune modulation and cancer immunity. We have previously shown that P-Selectin (SELP) is expressed by GB cells. Here, we investigated the factional role of SELP in GB-microglia interactions. First, we found that microglia cells facilitate the expression and secretion of SELP by GB cells, and that GB cells facilitate the expression of P-Selectin ligand by microglia. We then showed that SELP mediates microglia-enhanced GB invasion and proliferation in 2D and 3D in vitro models and has a role in microglia activation state. These findings were validated in vivo, showing that inhibition or downregulation of SELP leads to reduced tumor growth, increased overall survival and improved immune response. Single-Cells RNA-seq analysis of the tumors revealed an increase in pro-inflammatory microglia signature, reduction in cancer cell tumorigenesis potential and improved T cell activation. Our results indicated that SELP has an important role in GB progression and microenvironment activation. This work can improve our understanding of tumor-associated microglia function and the mechanisms by which GB cells suppress the immune system and invade the brain tissue.

Citation Format: Eilam Yeini, Paula Ofek, Sabina Pozzi, Nitzan Albeck, Dikla Ben-Shushan, Galia Tiram, Sapir Golan, Ron Kleiner, Ron Sheinin, Shlomit Reich-Zeliger, Rachel Grossman, Zvi Ram, Henry Brem, Thomas Hyde, Prerna Magod, Dinorah Friedmann-Morvinski, Asaf Madi, Ronit Satchi-Fainaro. P-selectin axis plays a key role in microglia immunophenotype and glioblastoma 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 2716.
Abstract 2726: Characterization of immune microenvironment and heterogeneity in breast cancer subtypes
Authors: Jain Drashti , Liao Linda , Hopkins Megan , Quintayo Mary Anne , Talebian Vida , Bayani Jane , Cheung Alison , Yaffe Martin , Bartlett John , Spears Melanie
Published: July 2021

Despite ongoing advancements in the clinical management of breast cancer (BC), the variability in the response to treatment requires continued understanding of the molecular biology of this disease. Receptor status for estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) along with pathological features dictate the course of therapy. Tumor cells in BC are phenotypically diverse between and within patients due to underlying differences in their cellular biology. This heterogeneity in the expression of key molecular targets renders some tumor cells resistant to therapy. While the etiology of intra-tumor heterogeneity is under investigation, it is equally important to characterize the heterogeneous nature of the tumor microenvironment as both of these compartments are engaged in the molecular crosstalk; with the latter known to contribute to tumor progression. Traditionally considered to be immunologically cold, recent advances have demonstrated that some BC subtypes elicit immunological response. However, further research is warranted to broadly illustrate the immune cell contexture in BC in a spatial context. In this study, the immune microenvironment was characterized through proteomic analysis using NanoString's GeoMX Digital Spatial Profiling (DSP) platform, in a cohort of early BC patients. This cohort consists of Luminal A, Luminal B, Basal, and HER2 BC subtypes. Correlation of proteomic and genomic data will reveal the role of genomic alterations in differential immune response. Protein markers were analyzed in both the tumor microenvironment (TME) and tumor compartments with beta-2-microglobulin, CD40, and CD11 significantly expressed in the TME compartment while PanCK, ERalpha, and HER2 were enriched in the tumor. Interestingly, CD127 was also expressed in the tumor compartment, indicating infiltration by memory T cells. Through this study we spatially characterize the immune microenvironment in BC subtypes, providing further evidence against the “immunologically cold” view of this disease. In addition, we demonstrate the potential clinical use of this novel platform in diagnosing and better stratifying BC patients based on spatial heterogeneity in tumor and TME.

Citation Format: Drashti Jain, Linda Liao, Megan Hopkins, Mary Anne Quintayo, Vida Talebian, Jane Bayani, Alison Cheung, Martin Yaffe, John Bartlett, Melanie Spears. Characterization of immune microenvironment and heterogeneity in breast cancer subtypes [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 2726.
Abstract 275: Lack of electron acceptors contributes to redox stress and growth arrest in asparagine-starved sarcoma cells
Authors: Bauer Christoph , Quante Meret , Regina Carla , Schneider Michaela , Andrieux Geoffroy , Gorka Oliver , Groß Olaf , Boerries Melanie , Kammerer Bernd , Hettmer Simone
Published: July 2021

Background. Amino acids are integral components of cancer metabolism. Adequate availability of the non-essential amino acid asparagine is necessary to support growth and survival of various cancer cell types, including sarcoma cells.

Methods. Different mass spectrometry approaches were employed to determine changes in the metabolome of asparagine-deprived mouse and human sarcoma cells, which may be responsible for arrested growth and apoptosis due to asparagine depletion.

Results. Lower aspartate levels, higher aspartate/glutamine ratios and lower levels of tricarboxylic acid (TCA) cycle metabolites in asparagine-deprived sarcoma cells indicated a redirection of TCA cycle flux and were accompanied by reduced NAD+/NADH ratios, consistent with relative deficiency of electron acceptors in asparagine-starved cells. Elevated lactate/pyruvate ratios may be due to compensatory regeneration of NAD+ through increased pyruvate to lactate conversion by lactate dehydrogenase in asparagine-deprived cells. Supplementation with exogenous pyruvate, which was previously shown to serve as electron acceptor in cells challenged by reductive stress, restored aspartate levels, NAD+/NADH ratios, lactate/pyruvate ratios and cell growth in asparagine-deprived cells. Treatment with chemicals disrupting regeneration of NAD+ in the electron transport chain (e.g. metformin) further enhanced the anti-proliferative and pro-apoptotic effects of asparagine depletion.

Conclusions. We conclude that asparagine deprivation, through metabolic reprogramming, causes reductive stress, which, in turn, results in lower aspartate levels in asparagine-starved cells. These changes are major contributors to the growth arrest observed in asparagine-starved cells. Future studies are needed to further elucidate the cellular processes that contribute to lack of electron acceptors in asparagine-starved tumor cells.

Citation Format: Christoph Bauer, Meret Quante, Carla Regina, Michaela Schneider, Geoffroy Andrieux, Oliver Gorka, Olaf Groß, Melanie Boerries, Bernd Kammerer, Simone Hettmer. Lack of electron acceptors contributes to redox stress and growth arrest in asparagine-starved sarcoma 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 275.
Abstract 2783: Fast and accurate spatial phenotyping and immuno-profiling using Ultivue UltiMapper kits and Zeiss Axio Scan.Z1 slide scanner
Authors: Chatterjee Gourab , Murphy Michael , Neumann Markus , Doering Volker , Manesse Mael
Published: July 2021

Understanding the complexities of the tumor micro-environment in detail can vastly improve the accuracy of immuno-oncology research and accelerate the discovery of potential immunotherapy targets. Multiplexed immunofluorescence (mIF) assays have emerged as a critical approach for identifying complex cellular phenotypes in the tumor micro-environment, powered by the value of the spatial correlation of such phenotypes in a tissue specimen. Although many methods for multiplexed IF exist, they are often costly and require time-consuming assay development and long imaging times. The Ultivue UltiMapper kits enable rapid, pre-optimized staining of multiple targets simultaneously in a single FFPE tissue section. This technology is ready-to-use with conventional automated staining workflows and commercially available automated imaging systems. Here, we demonstrate the potential of accurate cellular phenotyping using the InSituPlex technology with fast whole-slide scanning using ZEISS Axioscan.Z1 an automated slide scanning system for fluorescence and transmitted light applications with modern cameras, a sophisticated focusing method and a powerful imaging software. The compounded effect of integrating these technologies can significantly improve the throughput of immuno-oncology research. A 4-target immunostaining assay was performed on multiple FFPE tumor sections using UltiMapper reagents. Briefly, slides were stained with a cocktail of primary antibodies using a Leica Biosystems BOND RX autostainer. Post-staining, the slides were coverslipped and imaged on ZEISS Axioscan.Z1. The resulting images were analyzed using IndicaLabs HALO analysis software. In this poster, we report on an efficient and streamlined workflow for mIF staining, imaging, and analysis of tumor samples. After nuclear segmentation of the resulting images, T-cell and macrophage populations were subtyped and identified through spatial identification of relevant biomarkers. The combined workflow presented here allows a user to get 4-plex, whole slide immunofluorescence images of FFPE tumor tissues within 6 hours. The automated whole slide scanning capability of ZEISS Axioscan.Z1 also allows a user to image up to 100 slides with minimal external input. The combination of optimized, fast staining UltiMapper reagents with ZEISS Axioscan.Z1 automated whole-slide fluorescence scanner enables rapid, deep immunoprofiling within the spatial context of the tumor, empowering translational and immuno-oncology research.

Citation Format: Gourab Chatterjee, Michael Murphy, Markus Neumann, Volker Doering, Mael Manesse. Fast and accurate spatial phenotyping and immuno-profiling using Ultivue UltiMapper kits and Zeiss Axio Scan.Z1 slide scanner [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 2783.