General Features and Novel Gene Signatures That Identify Epstein-Barr Virus-Associated Epithelial Cancers

Epstein-Barr virus (EBV) is associated with various types of human malignancies, including nasopharyngeal carcinoma (NPC), EBV-associated gastric carcinoma (EBVaGC), and oral squamous cell carcinoma (OSCC). The present study aimed to identify gene signatures and common signaling pathways that can be used to predict the prognosis of EBV-associated epithelial cancers (EBVaCAs) by performing an integrated bioinformatics analysis of cell lines and tumor tissues. We identified 12 differentially expressed genes (DEGs) in the EBVaCA cell lines. Among them, only four DEGs, including BAMBI, SLC26A9, SGPP2, and TMC8, were significantly upregulated. However, SLC26A9 and TMC8, but not BAMBI and SGPP2, were significantly upregulated in EBV-positive tumor tissues compared to EBV-negative tumor tissues. Next, we identified IL6/JAK/STAT3 and TNF-α/NF-κB signaling pathways as common hallmarks of EBVaCAs. The expression of key genes related to the two hallmarks was upregulated in both EBV-infected cell lines and EBV-positive tumor tissues. These results suggest that SLC26A9 and TMC8 might be gene signatures that can effectively predict the prognosis of EBVaCAs and provide new insights into the molecular mechanisms of EBV-driven epithelial cancers.

Low-affinity CAR T cells exhibit reduced trogocytosis, preventing fratricide and antigen-negative tumor escape while preserving anti-tumor activity

ABSTRACTChimeric antigen receptor (CAR) T cells using the high-affinity CD19 binding domain FMC63 are an effective treatment for patients with relapsed and aggressive B cell lymphoma. However, antigen loss and poor CAR T cell persistence remain common causes for relapse in these patients. Using primary patient samples, we now show that FMC63-based CAR T cells confer rapid antigen loss in all major tumor types currently approved for treatment with CD19 CAR T cells via trogocytosis, the stripping of antigen from tumor cells by CAR T cells. We show that CAR T cell-mediated trogocytosis can be dramatically reduced across a wide range of B cell malignancies by replacing FMC63 with a low affinity CD19 antibody. This reduction in trogocytosis does not alter the direct anti-tumor activity of CD19 CAR T cells but prevents the emergence of antigen-negative tumor cells and significantly increases CAR T cell viability by reducing fratricide of CD19 CAR T cells following trogocytosis.TEASERA reduction in CAR affinity does not affect tumor killing but prolongs T cell persistence and prevents antigen-negative tumor escape.

780 ALTA-002, a SIRPα-directed TLR9 agonist antibody conjugate activates myeloid cells and promotes anti-tumor immunity

BackgroundNovel therapies engaging both innate and adaptive immune responses may engender durable anti-tumor immunity. Activation of toll-like receptor 9 (TLR9) by unmethylated CpG oligonucleotides promotes innate inflammatory responses and induces adaptive immunity. Immune cells expressing TLR9 encompass B cells and myeloid cells (including dendritic cells and plasmacytoid dendritic cells). Recently, several TLR9 agonists have demonstrated clinical benefit in patients with melanoma when administered intra-tumorally.1 Specifically designed for systemic administration, we developed a novel Toll-like Receptor Agonist Antibody Conjugate (TRAAC) molecule comprised of a differentiated TLR-9 agonist (T-CpG) conjugated to an antibody against SIRPα (ALTA-002). Signal regulatory protein α (SIRPα) is a myeloid inhibitory receptor that suppresses immune activation following binding of its ligand CD47. Blockade of CD47-SIRPα myeloid checkpoint pathway has been shown to promote myeloid-mediated anti-tumor functions leading to the induction of adaptive immunity.2 Additionally, SIRPα is highly expressed in various tumor types including renal cell carcinoma and melanoma.3 Here we present preclinical data demonstrating that ALTA-002 delivers T-CpG to SIRPα expressing myeloid cells, triggering TLR9 signaling, cell activation and immune modulation resulting in robust anti-tumor efficacy.MethodsIn vitro activity of ALTA-002 was evaluated using human PBMCs co-cultured in the presence of SIRPα positive and negative tumor cells. Anti-tumor efficacy of mouse ALTA-002 surrogate was evaluated in multiple syngeneic tumor models with varying immunogenicity profiles.ResultsIn vitro co-culture of human PBMC and SIRPα positive or negative tumor cells with ALTA-002 stimulates myeloid cells, leading to increased IRF7 induction, expression of co-stimulatory molecules, and cytokine secretion. In vitro treatment with ALTA-002 led to enhanced phagocytic engulfment by human monocyte-derived macrophages across multiple SIRPα positive and negative tumor cell lines. Following systemic delivery of a mouse ALTA-002 surrogate, durable anti-tumor activity was observed in both SIRPα positive and negative expressing tumors. ALTA-002 treated mice with eradicated tumors suppressed tumor growth upon tumor re-challenge, indicating tumor-specific immune memory.ConclusionsThese results demonstrate the unique properties of systemically administered ALTA-002, which integrates TLR9 activation and blockade of CD47-SIRPα interaction on myeloid cells to engender both innate and adaptive anti-tumor immune responses. These data support the development of ALTA-002 as an anti-cancer therapeutic for a variety of tumor malignancies.ReferencesHamid O, Ismail R, Puzanov I. Intratumoral Immunotherapy-Update 2019. Oncologist 2020;25(3):e423-e4382.Kuo T, Chen A, Harrabi O. Targeting the myeloid checkpoint receptor SIRPα potentiates innate and adaptive immune responses to promote anti-tumor activity. J Hematol Oncol 2020;13:160–1783.Yanagita T, Murata Y, Tanaka D. Anti-SIRPα antibodies as a potential new tool for cancer immunotherapy. JCI Insight 2017;2(1):e89140.Ethics ApprovalIn vivo studies were approved by the Institutional Animal Care and Use Committee of Tallac Therapeutics.

Evolution of HER2-low expression from primary to recurrent breast cancer

About a half of HER2-negative breast cancer (BC) show HER2-low expression that can be targeted by new antibody-drug conjugates. The main aim of this study is to describe the evolution of HER2 expression from primary BC to relapse by including HER2-low category in both primary and recurrent BC samples. Patients with matched primary and relapse BC samples were included. HER2 was evaluated according to ASCO/CAP recommendations in place at the time of diagnosis. A cutoff of >10% cells staining for HER2-positivity was applied. HER2-negative cases were sub-classified as HER2-low (IHC = 1 + /2+ and ISH not amplified), or HER2-0 (IHC-0). 547 patients were included. The proportion of HER2-low cases was 34.2% on the primary tumor and 37.3% on the relapse samples. Among HER2-negative cases, HER2-low status was more frequent in HR-positive vs triple-negative tumors (47.3% vs 35.4% on primary tumor samples, 53.8% vs 36.2% on relapse samples). The overall rate of HER2 discordance was 38.0%, mostly represented by HER2-0 switching to HER2-low (15%) and HER2-low switching to HER2-0 (14%). Among patients with a primary HER2-negative tumor, the rate of HER2 discordance was higher in HR-positive/HER2-negative vs triple-negative cases (45.5% vs 36.7% p = 0.170). This difference was mostly driven by cases switching from HER2-0 to HER2-low. HER2-low expression is highly unstable during disease evolution. Relapse biopsy in case of a primary HER2-0 tumor may open new therapeutic opportunities in a relevant proportion of patients.

An Unusual Location for a Nonurachal Bladder Adenocarcinoma

Malignant bladder neoplasms represent a significant disease burden not only for urologists but also the broader medical community. While the majority of bladder tumors are urothelial in origin, up to two percent are found to be adenocarcinomas. Among bladder adenocarcinomas, roughly one-tenth are urachal and are frequently located at the dome of the bladder where urachal remnants can often be found. We describe a case of bladder adenocarcinoma that presented at the dome of the bladder but ultimately exhibited a nonurachal histology. A 65-year-old male with a history of myocardial infarction and cerebrovascular accident with residual right-sided hemiparesis and aphasia was referred to our clinic for evaluation of a bladder mass discovered in the setting of painless gross hematuria. Diagnostic cystoscopy demonstrated a large mass at the dome of the bladder, and subsequent transurethral resection revealed stage T1 mucinous adenocarcinoma arising in a villous adenomatous lesion without the presence of muscle in the specimen. The patient underwent a robotic-assisted laparoscopic partial cystectomy with extended bilateral pelvic lymph node dissection. Postoperatively, the patient experienced short-lived paralytic ileus and was discharged on postoperative day 5. Follow-up surveillance imaging at 6 months with CT chest, abdomen, and pelvis, repeat office cystoscopy, and negative tumor markers postoperatively indicated no evidence of disease recurrence. Characterization of bladder adenocarcinomas into urachal and nonurachal subtypes is critical in differentiating the operative management and oncologic outcomes of the respective neoplasms. However, given the paucity of literature describing treatment approaches to bladder adenocarcinoma in general, existing methods have largely mirrored genetically similar neoplasms, including ovarian and colon adenocarcinomas. Although there is still much to be understood regarding the potential mechanisms of carcinogenesis of nonurachal adenocarcinomas, further investigation may pave the way for a more standardized treatment paradigm and provide insight into the potential utility of modern immunotherapies.

Systematic tissue collection during clinical breast biopsy is feasible, safe and enables high-content translational analyses

Systematic collection of fresh tissues for research at the time of diagnostic image-guided breast biopsy has the potential to fuel a wide variety of innovative studies. Here we report the initial experience, including safety, feasibility, and laboratory proof-of-principle, with the collection and analysis of research specimens obtained via breast core needle biopsy immediately following routine clinical biopsy at a single institution over a 14-month period. Patients underwent one or two additional core biopsies following collection of all necessary clinical specimens. In total, 395 patients were approached and 270 consented to the research study, yielding a 68.4% consent rate. Among consenting patients, 238 lesions were biopsied for research, resulting in 446 research specimens collected. No immediate complications were observed. Representative research core specimens showed high diagnostic concordance with clinical core biopsies. Flow cytometry demonstrated consistent recovery of hundreds to thousands of viable cells per research core. Among a group of HER2 + tumor research specimens, HER2 assessment by flow cytometry correlated highly with immunohistochemistry (IHC) staining, and in addition revealed extensive inter- and intra-tumoral variation in HER2 levels of potential clinical relevance. Suitability for single-cell transcriptomic analysis was demonstrated for a triple-negative tumor core biopsy, revealing substantial cellular diversity in the tumor immune microenvironment, including a prognostically relevant T cell subpopulation. Thus, collection of fresh tissues for research purposes at the time of diagnostic breast biopsy is safe, feasible and efficient, and may provide a high-yield mechanism to generate a rich tissue repository for a wide variety of cross-disciplinary research.

ECOA-4. Hypoxia alters the DNA methylation profile of glioblastoma tumor cells

Glioblastoma (GBM) is the deadliest and most vascularized brain tumor in adults; however, blood circulation is highly inefficient in these tumors, contributing to areas of cell death (necrosis) within the tumor, which is likely due to oxygen deprivation (hypoxia). Hypoxia plays a major role in tumor growth, invasion, and resistance to therapy. Hypoxic stress has been linked to several changes that are fundamental to the malignant progression of GBM and other tumor types. Pimonidazole (PIMO) is an exogenous marker of hypoxia that is used to delineate hypoxic regions in several tumor types. To date, a clear hypoxia gene signature has not been specifically described for GBM. We hypothesize that specific cellular pathways are differentially regulated in hypoxic tumor niches and can serve as novel actionable targets for treatment-resistant tumor cells in GBM. Over the past 3 years, we have administered PIMO to 35 patients with primary IDH1/2 wild-type GBM and isolated PIMO-positive and PIMO-negative tumor cells by laser capture microdissection using a PIMO-specific antibody on frozen tumor specimens. Total genomic DNA was isolated and subjected to DNA methylation profiling using the Illumina Infinium Methylation EPIC array. Our preliminary results suggest that PIMO-positive (hypoxic) tumor cells display a distinct DNA methylation profile that corresponds to changes in expression of a set of genes associated with immune regulation, angiogenesis, and proliferation. Furthermore, multiple CpG sites within the promoter of some genes are differentially methylated in hypoxic cells, suggesting these genes may be epigenetically regulated under hypoxia. In conclusion, our results indicate that hypoxia is associated with distinct epigenetic alterations in tumor cells which may alter how these cells respond to low oxygen levels and can further be utilized to uncover the epigenomic vulnerabilities of hypoxic tumor cells in GBM.

Hsp70 Interacts with the TREM-1 Receptor Expressed on Monocytes and Thereby Stimulates Generation of Cytotoxic Lymphocytes Active against MHC-Negative Tumor Cells

The search for and analysis of new ligands for innate immunity receptors are of special significance for understanding the regulatory mechanisms of immune response. Here we show that the major heat shock protein 70 (Hsp70) can bind to and activate TREM-1, the innate immunity receptor expressed on monocytes. The Hsp70–TREM-1 interaction activates expression of TNFα and IFNγ mRNAs in monocytes and stimulates IL-2 secretion by РВМСs. Moreover, incubation of РВМСs with Hsp70 leads to an appearance of cytotoxic lymphocyte subpopulations active against the MHC-negative tumor cells. In addition, both the CD4+ Т-lymphocytes and CD14+ monocytes are necessary for the Hsp70 signal transduction and a consequent activation of the cytotoxic lymphocytes. We believe that data presented in this study will broaden the views on the involvement of Hsp70 in the antitumor immunity.

Efficacy of KD033, an anti-human-PD-L1-IL-15 bispecific protein, in human-PD-L1 positive and negative murine tumor models.

e14556 Background: KD033 is a clinical-stage bispecific fusion molecule consisting of a high-affinity anti-human-PD-L1 antibody and human IL-15. Preclinical studies have demonstrated that targeting IL-15 with anti-PD-L1 antibody resulted in increased efficacy, safety and maximal tolerated dose of the fusion protein compared to administration of free IL-15, as well as reduction of tumor growth in both PD-L1 positive and negative tumor models (1). The goal of the current study is to directly compare KD033 efficacy when PD-L1 is present or absent on the surface of the same tumor. Methods: KD033 was administered in the human-PD-L1/PD-1 transgenic C57/Bl6 mice subcutaneously transplanted with human-PD-L1 positive and negative MC38 colon carcinoma cells. This animal model allowed the evaluation of the impact of the presence or absence of human-PD-L1 expression on the tumor cell surface without altering human-PD-L1 expression on immune cells. Results: KD033 treatment resulted in significant tumor growth reduction in both human-PD-L1 positive and negative MC38 tumors. Analysis of peripheral immune cell populations showed similar increases of CD8 T and NK cells between human-PD-L1 positive and negative MC38- bearing mice after KD033 administration. Immunohistochemistry demonstrated a significant increase in CD8 T-cell infiltration into the human-PD-L1 positive MC38 tumors, whereas NK cells infiltration was more pronounced in the human-PD-L1 negative MC38 tumors. Analysis of tumor gene transcription after KD033 treatment highlighted differences in gene signatures between human-PD-L1 positive and negative MC38 tumors following KD033 treatment. Conclusions: These results showed that the efficacy of anti-PD-L1-IL-15 fusion protein is not limited to PD-L1 tumor expression as KD033 was efficacious in both PD-L1 positive and negative tumors. Mol Cancer Ther February 1 2021 (20) (2) 347-356; DOI: 10.1158/1535-7163.MCT-20-0457