PanCancer Immune Profiling Research Articles

Evaluation of immune-biomarker expression in high-grade soft-tissue sarcoma: HLA-DQA1 expression as a prognostic marker.

High-grade soft-tissue sarcoma (STS) is a highly malignant neoplasm with a poor overall prognosis. Numerous prognostic factors determine tumor progression and patient outcomes. Various immune-associated cells identified in the tumor microenvironment have important roles in various tumor types. The present study was performed to evaluate the expression of immune-associated genes and to elucidate the association between these genes and the prognosis in high-grade STS. A total of 12 formalin-fixed, paraffin-embedded tissue samples of high-grade STS were subjected to gene expression analysis using the NanoString nCounter® System and another 35 samples were used for immunohistochemistry. For comparative analysis, the patients were divided into two groups according to overall survival (OS). The expression levels of 770 genes were first analyzed using the nCounter® PanCancer Immune Profiling Panel. Immunohistochemistry was then performed for the most significantly altered genes. Subsequently, the association between gene expression and prognosis of high-grade STS was evaluated. Of the 770 immune-associated genes analyzed, several genes were identified as being differentially expressed between the two groups. Based on gene expression levels and fold change, 13 representative genes were identified; 7 of the 13 candidate genes (C3, CD36, DOCK9, FCER2, FOS, HLA-DRB4 and NCAM1) were significantly overexpressed in the poor prognosis group, while the other 6 immune-associated genes (BIRC5, DUSP4, FOXP3, HLA-DQA1, HLA-DQB1 and LAG3) were increased in the good prognosis group. By immunohistochemistry, the expression of the 13 immune-associated genes was confirmed to be significantly different between the two groups. Expression of HLA-DQA1, HLA-DQB1 and HLA-DRB4 was observed in 74.3, 34.3 and 48.6% of tumors, respectively. HLA-DQA1 and HLA-DQB1 were significantly decreased, whereas HLA-DRB4 was significantly increased in the poor prognosis group. Of note, expression of HLA-DQA1 was associated with a significantly longer OS (P=0.028). In conclusion, HLA-DQA1 expression was significantly associated with long-term survival and may therefore be an immune biomarker for good prognosis in high-grade STS.

Abstract 6397: Focal adhesion kinase (FAK) inhibition overcomes cisplatin resistance in head and neck squamous cell carcinoma (HNSCC)

Abstract Introduction: FAK is a non-receptor tyrosine kinase activated in response to interactions between transmembrane integrins and extracellular matrix involved in the activation of the PI3K–Akt pathway, required for cell proliferation and survival. Amplification of chromosome region 8q23-24, encoding FAK, is significantly associated with smoking, higher grade of dysplasia and progression from premalignant lesions to HNSCC. Multimodality treatment (surgery, radiation and platinum-based chemotherapy) is standard for locally advanced HNSCC. Local relapse/distant metastasis develop in 25-40% of patients with occurrence of platinum resistance. We hypothesize that FAK inhibition overcomes platinum resistance and immunosuppression in HNSCC. Methods: RNA was extracted from normal and cancer tissue of 12 retrospectively surgically collected formalin-fixed paraffin-embedded HNSCC samples and analyzed by NanoString PanCancer Pathway and Immune Profiling panels. 93VU and SCC1 cell lines were used and cell-derived spheroids were generated on attachment plates. Cell proliferation, wound healing and apoptosis were measured by IncuCyte S3 Live-Cell Analysis System (Essen BioScience). Cells were treated with the following commercially available FAK small molecule inhibitors: NVP-TAE-226 (Novartis); PF-573228, PF-562271, PF-431396 (Pfizer); GSK2256098 (GlaxoSmithKline); VS-6063 (Verastem). FAK expression was measured by western blotting and qPCR analysis. Results: In The Cancer Genome Atlas (TCGA) HNSCC dataset, FAK RNA expression was significantly higher in stage IV compared to stage I–III cancers (p<0.001). Among the genes significantly overexpressed (>2-fold change) in the 12 HNSCC samples analyzed by Nanostring pathway and immune profile panels, 17 were involved in PI3K-Akt signaling activation and 15 of these were also involved in the anchorage-dependent cell proliferation and FAK activation including the alpha form of integrins (ITGA2, ITGA6, ITGA3), the beta form of integrins (ITGB4, ITGB6), collagens (COL5A2, COL1A1, COL27A1, COL4A6, COL11A1), and laminins (LAMA3, LAMB3, LAMC2). We screened and identified that the 93VU and SCC1 cell lines were tolerant to cisplatin up to a dose of 10 uM. TAE-226 was the most effective FAK inhibitor in significantly blocking cell proliferation and wound healing in 93VU and SCC1 cell lines. TAE-226 treatment also effectively inhibited spheroid growth and proliferation in 3D culture. FAK inhibition decreased expression of chemokines and cytokines known to promote immunosuppression and metastasis such as CSF3, CXCL1, CXCL2, IL8, GPI, LIF, and CCL1. Conclusion: Our data suggests that FAK expression is associated with higher cancer stage. Its inhibition might have a role in overcoming cisplatin resistance and in affecting immunosuppressive signals in HNSCC. Immunocompetent syngeneic HNSCC animal models are currently ongoing. Citation Format: Atish Mohanty, Rebecca Pharaon, Arin Nam, Holly Yin, Sue Chang, Linlin Guo, Raju Pillai, Prakash Kulkarni, Ravi Salgia, Erminia Massarelli. Focal adhesion kinase (FAK) inhibition overcomes cisplatin resistance in head and neck squamous cell carcinoma (HNSCC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6397.

Abstract 1576: Molecular profiling of the desmoplastic reaction within the colorectal tumor microenvironment using the nCounter® platform

Abstract Background & Objectives: Desmoplastic reaction (DR) refers to the presence of fibrosis at the tumor's invasive margin within the tumor microenvironment (TME). DR has been associated with cancer aggressiveness across various studies in colorectal cancer (CRC). Previously, we have suggested a histological classification of DR into 3 prognostic categories; mature, intermediate or immature, based on the presence of keloid-like collagen and myxoid stroma at the extramural desmoplastic front, and we have shown that survival outcome differs significantly among the DR categories. Although DR is significantly associated with patient prognosis, its molecular background remains unclear. NanoString nCounter® analysis platform enables the multiplex gene expression analysis with up to 800 genes from a single mRNA sample. The main focus of our study is the evaluation of the molecular profiles that may drive the development of the DR types. Methods: Four CRC patients of each DR category were included in this study (n = 12). Surgical specimens were split into two halves, one of which was frozen and the other was fixed in formalin. DR was assessed on slides of the formalin-fixed tissues whereas the fresh frozen tissues samples were used for the molecular profiling. Four µm thick sections from the formalin-fixed samples were stained with hematoxylin and eosin. The extramural tumor front within the slides was assessed for DR classification. Samples containing myxoid stroma with area greater than a microscopic field of a ×40 objective lens were classified as immature. Samples with absence of size-significant myxoid stroma and presence of keloid-like collagens were classified as intermediate. Samples were classified as mature if neither size-significant myxoid stroma nor keloid-like collagens were present. Ten µm thick sections from the fresh frozen samples were stained with hematoxylin. Two regions of interest per tissue sample were selected: a) fibrotic stromal regions and b) tumor areas adjacent to the fibrotic stroma. A total area of 5mm2 was microdissected from each region of interest. RNA was extracted from each sample and over 1400 gene expressions were measured for each sample using the nCounter® PanCancer Progression and Immune Profiling panels. Nanostring nSolver™ analysis software was used for the data analysis. Results: Genes associated with cell cycle and function, epithelial to mesenchymal transition and metastasis showed significantly different expression among the stromal and tumor regions of the 3 DR categories. The distribution of immune cells within the TME was also shown to be significantly different between the DR types. Although differential gene expression was observed among all the DR classes, the immature class had the most distinct pattern of gene expression compared to the other two classes. Conclusion: This is to our knowledge the first study reporting differences in gene expression between mature, intermediate and immature DR types. Citation Format: Ines P. Nearchou, Tadakazu Ao, Satsuki Mochizuki, Sarah Warren, Hirofumi Harashima, Peter D. Caie, Hideki Ueno. Molecular profiling of the desmoplastic reaction within the colorectal tumor microenvironment using the nCounter® platform [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1576.

Abstract 5672: Pharmacodynamics and potential predictive biomarkers of ABBV-428, a first-in-class mesothelin (MSLN)-CD40 bispecific, in patients with advanced solid tumors

Abstract Background: ABBV-428 is a first-in-class bispecific antibody against CD40 and MSLN designed to stimulate CD40 for tumor-specific immune activation with limited systemic toxicity (Cancer Immunol Res 2019;7:1864). In phase I findings, ABBV-428 was well tolerated up to 3.6mg/kg; 3/50 patients had long-term stable disease (SD; Luke et al. ESMO 2019). Here, pharmacodynamic and potential predictive biomarkers were assessed in the blood and tumor tissues in this phase I open-label, multicenter study (NCT02955251). Methods: Patients ≥18 y had advanced solid tumors and progression/intolerance to standard therapies. ABBV-428 (≤3.6 mg/kg) was administered biweekly. CD40 receptor occupancy, absolute B cell counts, immune costimulatory activation markers, and serum cytokines were assessed at baseline and after treatment. MSLN expression (H-score) by IHC was reported at baseline. CD40/MSLN colocalization was assessed with a fluorescent multiplex assay. Paired tumor biopsy samples were collected at baseline and after treatment (C1D1, C2D1) to measure CD8+ T cells and PD-L1+ cells (IHC) and gene expression (NanoString Pancancer immune profiling panel). Immunologic constant of rejection (ICR) gene signature scores (J Immunother Cancer 2018;6:50) and IFNγ signatures (J Clin Invest 2017;127:2930) were used to characterize the tumor immune inflammatory microenvironment. BAP1 gene was sequenced (Sanger) in mesothelioma samples in 3 patients with long-term SD. Results: In peripheral blood, there was >90% CD40 receptor occupancy starting at 0.8 mg/kg. There was a transient decrease in circulating B cells 2-24 hrs after administration of ABBV-428, followed by an increase in B cell costimulatory activation markers (CD80, CD86, CD69) across dose groups with no evidence of cytokine response (IFNγ, IL-10, IL-12p70, IL-6, IL-8, TNFα, IL-1β, IL-2). There was no consistent change in tumor-infiltrating CD8+ T cells or PD-L1+ cells after ABBV-428 treatment in paired tumor biopsy samples from 13 patients. Patients with SD had higher ICR scores and a higher IFNγ gene signature at baseline than those with progressive disease. There was no correlation between baseline MSLN expression level and clinical response. Wide intertumor variability of MSLN/CD40 colocalization was observed from 4 tumor biopsy samples. No well-established recurrent mutations were seen in BAP1, which are correlated with better prognosis in mesothelioma patients. Conclusions: These data show that ABBV-428 binds and activates CD40 in the blood leading to B cell activation with no evidence of systemic cytokine production or significant change in the tumor immune microenvironment. Evidence of inflamed immune tumor microenvironment was observed at baseline from long-term SD patients. These results may explain the clinical observations from the current ABBV-428 clinical trial. Citation Format: Hua Fang, Shiming Ye, Anita Reddy, Tolga Turan, Andrew Woolley, Debra T. Chao, Cyril Ramathal, William R. Henner, Kinjal Hew, Michael McDevitt, Joel Hayflick, Frances Fan, Kathryn Allaire, Lawrence Fong. Pharmacodynamics and potential predictive biomarkers of ABBV-428, a first-in-class mesothelin (MSLN)-CD40 bispecific, in patients with advanced solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5672.

Abstract 6513: 3,3'-diindolylmethane enhances tumor regression after radiation through protecting normal cells to modulate anti-tumor immunity

Abstract Preclinical and clinical data indicate that radiotherapy acts as an immune modifier, having both immunostimulatory and immunosuppressive effects on the tumor immune microenvironment (TIME). 3.3'-Diindolylmethane (DIM), the acid-catalyzed dimer of the bioactive indole found in cruciferous vegetables, has long been known for its anticancer properties and has been proposed for use as a cancer prevention agent. The current study characterizes how DIM influences TIME and tumor regression after fractionated radiation treatment using preclinical breast cancer models. We first compared tumor graft models using immune-competent and immune-deficient mouse strains to find out whether immune cells are required for DIM's effects. Tumor targeted radiation was done using narrow-beam radiation with shielding. Tumor regression was monitored after DIM and radiation combined treatment and radiation alone. Our results indicated that DIM enhanced tumor regression after radiation treatment in immune-competent but not immuno-deficient mice. Data from nCounter® PanCancer immune profiling panel showed DIM increased intratumoral immune cells including cytotoxic T cells, neutrophils and macrophages after radiation and enhanced immunological processes such as adhesion and antigen processing. Expression of several MHC Class II molecules and CD74, a gene encoding chaperone and transport cofactor that assists MHC II molecules folding and presenting, significantly increased in the combined treatment of radiation and DIM. The results showing that DIM treatment alone does not affect tumor growth suggested that DIM's radioprotective effect on non-tumoral cells, including stromal cells and immune cells, plays a critical role in promoting a microenvironment that favors anti-tumor immunity. Additional experiments showed that DIM protected normal cells from radiation-caused immediate injuries in vitro and in vivo. In summary, DIM's radioprotective effects on immune cells and other normal cells are important in promoting a TIME that favors anti-tumor immunity. As a result, DIM increases the efficacy of radiation treatment on tumor regression. This study supports the development of DIM as an adjunct agent for radiation treatment. Citation Format: Renxiang Chen, Lijun Li, Yun-Tien Lin, Arslon Humayun, Albert J. Fornace Jr., Heng-Hong Li. 3,3'-diindolylmethane enhances tumor regression after radiation through protecting normal cells to modulate anti-tumor immunity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6513.

Abstract 4561: Oncolytic vaccinia virus encoding IL-7 and IL-12 systemically sensitizes tumors to anti-PD-1 antibody in a syngeneic mouse model

Abstract Immune checkpoint blockade targeting PD-1, PD-L1 and CTLA-4 have demonstrated remarkable clinical responses in patients with advanced cancers; however, a durable benefit is obtained in only a subset of patients. To overcome this therapeutic limitation, a number of immunotherapies and their combinations have been investigated. Among these, in situ vaccination using tumor-selective oncolytic viruses has proved an attractive approach. Viruses not only directly kill cancer cells through their replication, but also activate antitumor immunity triggered by the release of tumor antigens from killed cells. We have explored this approach using tumor-selective oncolytic vaccinia viruses (VVs) carrying immunomodulatory genes. We previously demonstrated that intratumoral treatment with a VV carrying human IL-7 and murine IL-12 genes (hIL-7/mIL-12-VV) enhanced immune responses in treated tumors and induced complete tumor regression in syngeneic mouse models (AACR-NCI-EORTC 2019). Here, using a bilaterally tumor-bearing mouse model, we examined whether unilateral virus injection affected untreated distant tumors. All tumors that had been directly injected with hIL-7/mIL-12-VV completely regressed. At the same time, untreated tumors in three of six mice also disappeared. We further confirmed significant increases in tumor-infiltrating lymphocytes in both treated and untreated tumors after treatment with hIL-7/mIL-12-VV. Unlike the treated tumors, the untreated distant tumors showed no detectable viral DNA by qPCR. PD-L1 expression was also upregulated in this model following treatment with hIL-7/mIL-12-VV in both treated and untreated tumors. This led us to examine the effects of combining this virotherapy with anti-PD-1 antibody. While monotherapies with anti-PD-1 antibody did not show significant antitumor efficacy, hIL-7/mIL-12-VV prior to this agent elicited improved antitumor activity in the virus-untreated distant tumors, suggesting that hIL-7/mIL-12-VV systemically sensitized tumors to anti-PD-1 antibody. Evidence that intratumoral immune status was changed following hIL-7/mIL-12-VV was found in the upregulation of various immune pathways analyzed using the Nanostring Pancancer Immune Profiling panel, and the increased clonality of T-cell repertoire in tumors. These results support the clinical development of VV carrying IL-7 and IL-12 as both monotherapy and as a combination partner with anti-PD-1 antibody for patients with advanced tumors. Citation Format: Shinsuke Nakao, Midori Yamashita, Mamoru Tasaki, Tatsuya Kawase, Motomu Nakatake, Hajime Kurosaki, Masamichi Mori, Masahiro Takeuchi, Takafumi Nakamura. Oncolytic vaccinia virus encoding IL-7 and IL-12 systemically sensitizes tumors to anti-PD-1 antibody in a syngeneic mouse model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4561.

Prognostic value of novel immune-related genomic biomarkers identified in head and neck squamous cell carcinoma

BackgroundThe immune response within the tumor microenvironment plays a key role in tumorigenesis and determines the clinical outcomes of head and neck squamous cell carcinoma (HNSCC). However, to date, a...

Differences in immune-related gene expressions and tumor-infiltrating lymphocytes according to chemotherapeutic response in ovarian high-grade serous carcinoma

BackgroundHigh-grade serous carcinoma (HGSC) of the ovary is the most common subtype of epithelial ovarian cancer (EOC) and has an overall poor prognosis. There is increasing awareness of the importance of immune cell populations and tumor-infiltrating lymphocytes (TILs) in various immune pathways in the tumor microenvironment. The present study evaluated immune-related gene expressions and TIL levels, as well as associated chemotherapeutic responses, to elucidate the correlation between gene expression and TIL levels in HGSC.Materials and methodsFresh tissue samples from 12 HGSC patients were included in this study. Depending on their response to adjuvant chemotherapy, the patients were divided into two groups: chemosensitive (CS) or chemoresistant (CR). The expression levels of 770 genes were analyzed using the nCounter® PanCancer Immune Profiling Panel of the NanoString nCounter® Analysis System. Quantitative real-time polymerase chain reaction (qPCR) was performed to validate the NanoString data obtained. The TIL levels in representative sections were examined via hematoxylin and eosin staining. Gene and TIL levels were subsequently correlated with the chemotherapeutic response.ResultsSeveral genes were differentially expressed in the two study groups. Eleven representative genes were selected for further evaluation. Of those, 9 genes (IRF1, CXCL9, LTB, CCL5, IL-8, GZMA, PSMB9, CD38, and VCAM1) were significantly overexpressed in the CS group; whereas expressions of 2 genes (CD24 and CD164) were increased in the CR group. Results of qPCR were consistent with those of the NanoString nCounter® analysis. Stromal TIL levels were significantly associated with adjuvant chemotherapeutic response (p = 0.001).ConclusionsSignificant differences between the CS and CR groups were observed in the expression levels of immune-related genes. Immune-related gene expressions were significantly higher in the CS group, which also had higher levels of TILs. We, therefore, suggest that, in patients with HGSC, immune-related gene expressions and TIL levels may be associated with chemotherapeutic sensitivity.

Abstract B062: Kaiso influences immune signaling of breast cancer exosomes

Abstract Introduction: Exosomes are communication vesicles act as mediator of intracellular transfer of genetic information, act an important role in intercommunication between tumor cells and immune cells. However, the mechanism underlining this cell-cell communication is not well understanding, particularly in African American breast cancer patients. Recently, our lab has demonstrated that Kaiso, a novel bi-modal transcription factor is highly expressed in African American breast cancer and notably, high Kaiso expression correlates with breast cancer aggressiveness and the disparity in survival outcomes of breast cancer patients of African American compared to European American patients. However, the differential expression and biological consequences of Kaiso in immune signaling of breast cancer exosomes has not been studied yet. Herein we demonstrate the biological role of Kaiso in immune signaling in breast cancer exosomes. Methods: In this study we utilized Nanostring immune profiling technology along with multiple in vitro and in vivo assays were used to study the role of Kaiso in breast cancer immune escape. Results: Nanostring pan cancer immune profiling demonstrated that European American breast cancer exosomes exhibited higher expression of TILs markers, T cell activation markers and CD8+T Cells markers compared to African American, while we observed an increase in the expression of the anti-phagocytic molecule CD47 in breast cancer patient exosomes of African American compared to European American patients. In addition to that CD47 and SIRP-α (Signal Regulatory Protein) are highly expressed in Kaiso-scrambled MDA-MB-231 cells (sh-Scr) and exosomes, whereas THBS1, which is a regulator of CD47 expression and is regarded as angiogenesis inhibitor is significantly increased in sh-Kaiso MDA-231 cells and exosomes. Additionally, we observed that Kaiso directly binds methylated sequences in the promoter region of CD47 and THBS1 by ChIP assay. Furthermore, in vivo sh-Kaiso cells injected into athymic mice exhibited delayed tumor formation after four weeks with smaller tumor size as compared to sh-SCR cells, and we observed higher expression of THBS1 with lower expression of CD47 and SIRP-α molecules by IHC and exosomes isolated from invivo tumors, indicating that Kaiso is associated with macrophage mediated immune escape. Conclusion: These findings demonstrate the important role of kaiso in immune signaling through exosomes which may be related with more aggressive cancer phenotype in breast cancer specially in African Americans. Citation Format: Shakir U Ahmed, Brittany Davis, Benjamin Adu Addai, Balasubramanyanam Karanam, Melissa Davis, William Grizzle, Honghe Wang, Clayton C Yates. Kaiso influences immune signaling of breast cancer exosomes [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr B062.

Sa1466 EUS HAS THE POTENTIAL TO EVALUATE MEMBERS OF THE TUMOR NECROSIS FACTOR (TNF) SUPERFAMILY AND IDENTIFY DRUG TARGETS ON AN INDIVIDUAL PDAC PATIENT BASIS TO GUIDE PRECISION IMMUNE-ONCOLOGY ELIGIBILITY

The FDA has approved use of the programmed cell death-1 (PD-1) inhibitor, pembrolizumab, in MSI-High solid tumors. This is a rare event among pancreas ductal adenocarcinoma (PDAC) patients, occurring at a frequency of 0.8%. In order to decipher if other cancer immunotherapy targets are potential therapies for PDAC patients, the Tumor Necrosis Factor (TNF) Receptor Superfamily has gained prominence, due to its ability to activate antitumor T-cell immunity. Members include CD70, TNFSF4 (OX-40L) and TNFRSF9 (41BB). It is important to develop strategies to aid patient selection criteria to guide therapeutic decision-making for clinical trials. We sought to determine if EUS fine needle biopsy (FNB) treatment naïve PDAC specimens are suitable to evaluate the TNF Superfamily, and in turn identify potential targets on an individual patient basis. The cohort of 13 EUS FNB patient specimens, 7 of whom with ≥ stage III disease, a CA19-9 level of 227.5 (IQR 58-605) U/ml and overall mortality of 77% at 22.2 (IQR 13.4-36.1) months was evaluated by the Nanostring nCounter® PanCancer Immune Profiling Panel. This is a highly multiplexed mRNA gene expression panel designed to quantitate the expression of 770 genes. A ≥ 2-fold change in expression when compared to pancreas control tissue was deemed to be reflective of altered expression. The digital mRNA profiling panel revealed that 44.8% and 10% of evaluated genes had either increased or reduced expression, when compared to normal pancreas controls. Of the 30 TNF Superfamily gene members evaluated, 17 (56.7%) and 1 (3.3%) were up and downregulated in PDAC and 4 (13.3%) were each up and down regulated in surgical resection chronic pancreatitis specimens (n=3), respectively. (Figure 1) TNFRSF10C (alias TRAILR3) and TNFRSF11B (alias Osteoprotegerin) expressions were elevated in 12 (92.3%) PDAC specimens x12.2-fold (range x1.8 – x36.2 fold) and x6.5-fold (range x1.8 – x40.9 fold), respectively. CD70, an immune checkpoint, had a tumor-restricted expression pattern and was upregulated in 10 (77%) PDAC patients (x3.2-fold) but downregulated in chronic pancreatitis (x-4.23-fold). (Figure 2) EUS has a central role in guiding precision immune-oncology eligibility for clinical care and clinical trials. Using digital mRNA profiling, CD70 had a tumor-restricted expression pattern and was identified in 77% of PDAC patient specimens. Therefore, this suggests that such patients may be sensitive to monoclonal antibody therapy that targets and neutralizes CD70.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

OP0044 WHOLE KIDNEY TRANSCRIPTOMIC ANALYSIS OF FORMALIN-FIXED PARAFFIN-EMBEDDED LUPUS NEPHRITIS KIDNEY BIOPSY TISSUE USING THE NANOSTRING NCOUNTER PLATFORM

Background:There is an ongoing effort to elucidate the molecular pathways that are key to kidney injury in lupus nephritis (LN). One approach is to study the transcriptome utilising kidney tissue obtained during diagnostic renal biopsy [1]. In clinical practice the most common tissue that is surplus to diagnostic requirements is formalin-fixed paraffin-embedded (FFPE) tissue. However, due to RNA degradation, transcriptomic analysis has been sub-optimal and challenging using standard procedures. The NanoString technology platform has the advantage that reliable detection of transcripts can be achieved even with degraded RNA. In this study we explored the utility of NanoString technology in identifying transcripts in RNA isolated from archival FFPE kidney biopsy sections in a cohort of patients with LN.Objectives:To explore the utility of the NanoString platform in elucidating a renal transcriptomic signature in formalin-fixed paraffin-embedded Lupus Nephritis kidney biopsy tissue.Methods:We utilised well defined Class III (n=11); Class IV (n=22) and Class V (n=24) LN FFPE kidney biopsies from female patients attending the Imperial College Healthcare NHS Trust. We excluded biopsies with mixed lesions or chronic lesions (i.e. significant glomerular scarring). Kidney biopsies from patients with Thin Basement Membrane (TBM; n=14) disease were used as controls. Six 10 micron thick sections were obtained from each biopsy and RNA isolated using the Qiagen RNeasy FFPE Kit. 100 micrograms of RNA was used for the detection of transcripts. We used the NanoString PanCancer immune profiling panel (770 transcript probes) and an additional 30 custom designed probes, enabling us to detect 800 transcripts, including 40 reference genes. Transcript analysis was performed according to manufacturer’s instructions using the NanoString nSolver software. When analysing differential gene expression (DGE) we used Benjamini-Hochberg adjustment to account for multiple testing. The threshold for statistical significance was an adjusted P value of 0.05 (5% false discovery rate).Results:Transcriptomic data passing NanoString nSolver quality control metrics was obtained from all sections. Notably sections included biopsies up to 16 years old (range: 1-16 years). Our transcript panel contained several Type I interferon (IFN) responsive genes, including sialic acid-binding Ig-like lectin 1 (Siglec-1). We detected a type I IFN signature using 8 IFN-responsive transcripts in 39 (68.4%) of the LN biopsies but in none of the TBM biopsies. This signature was present in Class III (n=9, 81.8%), Class IV (n=16, 72.7%) and Class V (n=14, 58.3%) LN samples. When we performed DGE analysis using TBM as the baseline we detected significantly increased expression across the classes (Class III = 202; Class IV = 357 and Class V = 237 differentially expressed transcripts, Figure 1).Conclusion:We have successfully identified transcriptomic signatures in RNA samples derived from a relatively large cohort of FFPE LN samples. Consistent with published reports we could detect a type I IFN signature in the LN kidney tissue [1]. Consistent with a recent study [1], we detected increased expression of OPN (osteopontin) and FN1 (fibronectin-1) in proliferative (Class III and IV) but not Class V LN. We are now performing clinical correlations to determine if the differentially expressed transcripts are clinically informative.

Dynamics of Cancer- and Immune-Pathway Gene Expression During Colitis and Recovery from Gut Injury in Mice Fed the Total Western Diet

Abstract Objectives Consumption of a Western type diet is a known risk factor for colorectal cancer. Our group previously developed the total Western diet (TWD) for rodents with energy and nutrient profiles that emulate a typical Western diet. In prior studies, consumption of the TWD in C57BL6/J mice markedly enhanced gut inflammation and colon tumorigenesis. In this study, our objective was to assess the dynamics of immune and cancer pathway gene expression during the onset and resolution of DSS-induced colitis. Methods Male C57BL/6 J mice were fed the standard AIN93G diet or the TWD diet for 45 days; on day 21, mice were provided 1% (w/V) DSS in the drinking water for 10 days. On days 33 and 45, the colitis disease activity index was determined. Also, days 21, 33, and 45, colon mucosa was collected (n = 8 per group) and total mRNA was isolated for gene expression analysis using the NanoString nCounter Mouse PanCancer Immune Profiling Panel, which targets 750 cancer- and immune-related genes. Results Results of these targeted gene expression analyses point to striking up-regulation of hundreds of genes associated with interferon response, inflammation, innate immunity, adaptive immunity, and chemokines and receptor pathways in mice fed TWD as compared to the standard AIN diet during active colitis. In a pattern that mirrored the persistent elevation in inflammation and mucosal injury observed in our prior longitudinal studies with TWD and DSS exposure, dysregulation of many of these genes persisted through recovery from gut injury, in addition to the stimulation of other pathways, such as B-cell activation and antigen processing. Conclusions This study is the first to assess the dynamics of immune and cancer pathway gene expression during the onset and resolution of DSS-induced colitis and the first to employ highly multiplexed, direct digital detection NanoString technology for analysis of a colitis transcriptome in mouse colon mucosa. Our observations indicate that consumption of the TWD markedly enhanced colitis, delayed recovery from gut injury, and enhanced colon tumorigenesis likely via extensive changes in expression of immune-related genes in the colon mucosa. Funding Sources Supported by the Hatch Capacity Project from the USDA National Institute of Food and Agriculture and the Dr. Robert and Carol Deibel Family Endowment.

Effect of Kaiso on immune signaling of breast cancer exosomes.

3088 Background: Exosomes are communication vesicles that act as mediators of intracellular transfer of genetic information, an important role in intercommunication between tumor cells and immune cells. However, the mechanism underlining this cell-cell communication is not well understanding, particularly in African American breast cancer patients. Recently, our lab has demonstrated that Kaiso, a novel bi-modal transcription factor is highly expressed in African American breast cancer and notably, high Kaiso expression correlates with breast cancer aggressiveness and the disparity in survival outcomes of breast cancer patients of African American compared to European American patients. However, the differential expression and biological consequences of Kaiso in immune signaling of breast cancer exosomes has not been studied yet. Herein we demonstrate the biological role of Kaiso in immune signaling in breast cancer exosomes. Methods: In this study we utilized Nanostring immune profiling technology along with multiple in vitro and in vivo assays were used to study the role of Kaiso in breast cancer immune escape. Results: Nanostring pan cancer immune profiling demonstrated that European American breast cancer exosomes exhibited higher expression of TILs markers, T cell activation markers and CD8+T Cells markers compared to African American (p < 0.05, FDR), while we observed an increase in the expression of the anti-phagocytic molecule CD47 in breast cancer patient exosomes of African American compared to European American patients. In addition to that CD47 and SIRP-α (Signal Regulatory Protein) are highly expressed in Kaiso-scrambled MDA-MB-231 cells (sh-Scr) and exosomes, whereas THBS1, which is a regulator of CD47 expression and is regarded as angiogenesis inhibitor is significantly increased in sh-Kaiso MDA-231 cells and exosomes. Additionally, we observed that Kaiso directly binds methylated sequences in the promoter region of CD47 and THBS1 by ChIP assay. Furthermore, in vivo sh-Kaiso cells injected into athymic mice exhibited delayed tumor formation after four weeks with smaller tumor size as compared to sh-SCR cells (p < 0.05), and we observed higher expression of THBS1 with lower expression of CD47 and SIRP-α molecules by IHC and exosomes isolated from in vivo tumors (p < 0.05), indicating that Kaiso is associated with macrophage mediated immune escape. Conclusions: These findings demonstrate the important role of kaiso in immune signaling through exosomes which may be related with more aggressive cancer phenotype in breast cancer, especially in African Americans.

Immune profiling and clinical outcomes in patients treated with ramucirumab and pembrolizumab in phase I study JVDF.

3089 Background: In Study JVDF (NCT02443324), we combined ramucirumab (VEGFR2 antagonist) and pembrolizumab (PD-1 antagonist) to simultaneously target the tumor microenvironment and immune checkpoints in patients with advanced non-small cell lung cancer (NSCLC), gastric or gastroesophageal junction adenocarcinoma (G/GEJ), urothelial carcinoma (UC) or biliary tract cancer (BTC). We reported that this combination was associated with increased antitumor activity in patients with PD-L1 positive tumors by immunohistochemistry (IHC) compared with PD-L1 negative tumors.1) Here we explore the association between baseline gene expression profiles and clinical outcomes. Methods: JVDF was a nonrandomized phase 1a/b trial that treated patients with intravenous ramucirumab at 8 mg/kg on days 1 and 8 (G/GEJ, BTC) or 10 mg/kg on day 1 (G/GEJ, NSCLC, UC) plus pembrolizumab (200 mg day 1) every 3 weeks.1 Baseline tumor samples from 53 patients across 7 study cohorts were analyzed with the NanoString PanCancer Immune Profiling Panel for RNA expression and the DAKO PD-L1 IHC 22C3 pharmDx assay for PD-L1 protein expression. Clinical outcomes included progression-free survival (PFS), overall survival (OS), and objective response rate (ORR). Results: Across cohorts, PD-L1 gene expression was correlated with increased IFNγ gene expression and immune-related gene signatures (T effector, T cell-inflamed (TIS)), and trended with PD-L1 protein expression. Expression of immune checkpoint-related genes and myeloid-derived suppressor cell /regulatory T cell markers was increased in the NSCLC TPS≥50% PD-L1 IHC subgroup (N=8), while no clear pattern of expression was observed in other cohorts. Higher T effector and TIS scores appeared associated with better survival and response in NSCLC cohorts (mean TIS: 1.21±0.80 in responders (N=7) vs -0.13±0.57 in non-responders (N=7); p=0.004), and a trend was observed in G/GEJ cohorts (mean TIS: -0.18±0.30 (N=5) vs -0.39±0.21 (N=13); p=0.207). Of note, partial responses and stable disease were also observed in NSCLC and G/GEJ patients with a low baseline inflammatory signature score. Additional analyses are ongoing and will be presented. Conclusions: Baseline PD-L1 gene and protein expression tends to correlate with immune-related gene expression, and an inflamed tumor microenvironment may be associated with better clinical outcomes with ramucirumab and pembrolizumab. However, interpretation is limited by lack of control arm and sample size.1) Herbst et al. Lancet Oncol. 2019 Aug; 20 (8):1109-1123. Clinical trial information: NCT02443324 .

Comparison of skin biopsy sample processing and storage methods on high dimensional immune gene expression using the Nanostring nCounter system

BackgroundDigital multiplex gene expression profiling is overcoming the limitations of many tissue-processing and RNA extraction techniques for the reproducible and quantitative molecular classification of disease. We assessed the effect of different skin biopsy collection/storage conditions on mRNA quality and quantity and the NanoString nCounter™ System’s ability to reproducibly quantify the expression of 730 immune genes from skin biopsies.MethodsHealthy human skin punch biopsies (n = 6) obtained from skin sections from four patients undergoing routine abdominoplasty were subject to one of several collection/storage protocols, including: i) snap freezing in liquid nitrogen and transportation on dry ice; ii) RNAlater (ThermoFisher) for 24 h at room temperature then stored at − 80 °C; iii) formalin fixation with further processing for FFPE blocks; iv) DNA/RNA shield (Zymo) stored and shipped at room temperature; v) placed in TRIzol then stored at − 80 °C; vi) saline without RNAse for 24 h at room temperature then stored at − 80 °C. RNA yield and integrity was assessed following extraction via NanoDrop, QuantiFluor with RNA specific dye and a Bioanalyser (LabChip24, PerkinElmer). Immune gene expression was analysed using the NanoString Pancancer Immune Profiling Panel containing 730 genes.ResultsExcept for saline, all protocols yielded total RNA in quantities/qualities that could be analysed by NanoString nCounter technology, although the quality of the extracted RNA varied widely. Mean RNA integrity was highest from samples that were placed in RNALater (RQS 8.2 ± 1.15), with integrity lowest from the saline stored sample (RQS < 2). There was a high degree of reproducibility in the expression of immune genes between all samples with the exception of saline, with the number of detected genes at counts < 100, between 100 and 1000 and > 10,000 similar across extraction protocols.ConclusionsA variety of processing methods can be used for digital immune gene expression profiling in mRNA extracted from skin that are comparable to snap frozen skin specimens, providing skin cancer clinicians greater opportunity to supply skin specimens to tissue banks. NanoString nCounter technology can determine gene expression in skin biopsy specimens with a high degree of sensitivity despite lower RNA yields and processing methods that may generate poorer quality RNA. The increased sensitivity of digital gene expression profiling continues to expand molecular pathology profiling of disease.

Characterization of the immune profile of oral tongue squamous cell carcinomas with advancing disease.

We investigated whether a unique immune response was instigated with the development of oral tongue squamous cell carcinomas (OTSCC), with/without nodal involvement, with/without recurrent metastatic disease, or within tumor involved nodes. One hundred and ten formalin‐fixed paraffin‐embedded samples were collected from a retrospective cohort of 67 OTSCC patients and 10 non‐cancerous tongue samples. Targets including CD4, CD8, FOXP3, PD‐L1, and PD‐1 were analyzed by immunohistochemistry. The Nanostring PanCancer Immune Profiling Panel was used for gene expression profiling. Data were externally validated in the The Cancer Genome Atlas (TCGA) head and neck (HNSCC), melanoma and lung squamous cell carcinoma (LSCC) cohorts. A 24‐immune gene signature was identified that discriminated more aggressive OTSCC cases, and although not prognostic in HNSCC was associated with survival in other TCGA cohorts (improved survival for melanoma, P < .001 and worse survival for LSCC, P = .038). OTSCC exhibited concordant gene and immunohistochemical (IHC) features characterized by a TH‐2 biased, proinflammatory profile with upregulated B cell and neutrophil gene activity and increased CD4, FOXP3, and PD‐L1 expression (P < .001 for all by IHC). Compared to less advanced disease, nodal involvement and recurrent OTSCC did not induce a different immune response although recurrent disease was characterized by significantly higher PD‐L1 expression (P = .004 by SP263, P = .013 by 22C3, P = .004 for gene expression). Identification of a gene signature associated with different prognostic effects in other cancers highlights common pathways of immune dysregulation that are impacted by the tumor origin. The significant immunosuppressive signaling in OTSCC indicates primary failure of immune system to control carcinogenesis emphasizing the need for early, combination therapeutic approaches.

Prognostic role of PD-L1 and immune-related gene expression profiles in giant cell tumors of bone.

Giant cell tumor of bone (GCTB) is a locally aggressive and rarely metastatic tumor, with a relatively unpredictable clinical course. A retrospective series of 46 GCTB and a control group of 24 aneurysmal bone cysts (ABC) were selected with the aim of investigating the PD-L1 expression levels and immune-related gene expression profile, in correlation with clinicopathological features. PD-L1 and Ki67 were immunohistochemically tested in each case. Furthermore, comprehensive molecular analyses were carried out using NanoString technology and nCounter PanCancer Immune Profiling Panel, and the gene expression results were correlated with clinicopathological characteristics. PD-L1 expression was observed in 13/46 (28.3%) GCTB (and in 1/24, 4.2%, control ABC, only) and associated with a shorter disease free interval according to univariate analysis. Moreover, in PD-L1-positive lesions, three genes (CD27, CD6 and IL10) were significantly upregulated (p < 0.01), while two were downregulated (LCK and TLR8, showing borderline significance, p = 0.06). Interestingly, these genes can be related to maturation and immune tolerance of bone tissue microenvironment, suggesting a more immature/anergic phenotype of giant cell tumors. Our findings suggest that PD-L1 immunoreactivity may help to select GCTB patients with a higher risk of recurrence who could potentially benefit from immune checkpoint blockade.

An immune gene expression signature distinguishes central nervous system metastases from primary tumours in non–small-cell lung cancer

BackgroundDissemination of non–small-cell lung cancer (NSCLC) in the central nervous system is a frequent and challenging clinical problem. Systemic or local therapies rarely prolong survival and have modest activity regarding local control. Alterations in gene expression in brain metastasis versus primary tumour may increase aggressiveness and impair therapeutic efforts. MethodsWe identified 25 patients with surgically removed NSCLC brain metastases in two different patient cohorts. For 13 of these patients, primary tumour samples were available. Gene expression analysis using the nCounter® PanCancer Immune Profiling gene expression panel (nanoString technologies Inc.) was performed in brain metastases and primary tumour samples. Identification of differentially expressed genes was conducted on normalized data using the nSolver analysis software. ResultsWe compared gene expression patterns in brain metastases with primary tumours. Brain metastasis samples displayed a distinct clustering pattern compared to primary tumour samples with a statistically significant downregulation of genes related to immune response and immune cell activation. Results from KEGG term analysis on differentially expressed genes revealed a concomitant enrichment of multiple KEGG terms associated with the immune system. We identified a 12-gene immune signature that clearly separated brain metastases from primary tumours. ConclusionsWe identified a unique gene downregulation pattern in brain metastases compared with primary tumours. This finding may explain the lower intracranial efficacy of systemic therapy, especially immunotherapy, in brain metastasis of patients with NSCLC.

Abstract B63: Immune gene expression profiling of acute myeloid leukemia identifies predictors of survival and actionable targets for treatment

Abstract Background: Acute myeloid leukemia (AML) is characterized by clonal expansion of poorly differentiated myeloid precursors, resulting in impaired hematopoiesis and often bone marrow (BM) failure. The general therapeutic approach in patients with AML has not changed substantially in more than 30 years. Investigation of new strategies, including immunotherapy, remains a priority. Tumor phenotypes are dictated not only by major oncogene drivers, but also by the tumor immunologic microenvironment (TIME) which is inherently immunosuppressive, is equipped to hamper effector T-cell function and includes immune and inflammatory cells, soluble mediators such as interferon (IFN)-gamma and extracellular matrix components. Herein, we profiled the TIME of AML to identify gene signatures that are reflective of general immune status and predictive of antileukemia immune potential. Methods: We used the hybridization-based nCounter® system (NanoString Technologies®, Seattle, WA) and the RNA Pan-Cancer Immune Profiling PanelTM to analyze BM aspirates from 290 adults and 40 children with newly diagnosed, nonpromyelocytic AML. Data were normalized to a set of reference genes and log2-transformed for bioinformatics analysis. The clinical relevance of the association between immune gene signatures and patient outcome was further validated in silico using publicly available cancer transcriptomic datasets (The Cancer Genome Atlas; TCGA). Results: We identified distinct sets of co-expressed genes corresponding to innate immunity, adaptive immunity and IFN-gamma signaling. BM samples with IFN-gamma-dominant gene expression profiles showed up-regulation of actionable immune checkpoints, such as B7-H3, PD-L1 and CTLA-4. A set of 4 differentially expressed immune genes between children and adults with AML (FDR&amp;lt;0.005) was associated with adverse cytogenetic features, leukemia relapse and shorter patient survival, and also stratified survival in the TCGA cohort of adult patients with AML. Finally, all four genes were amplified, deleted or mutated in 7% of 51,175 TCGA solid tumors, with the highest frequency of mutations being detected in primary CNS lymphoma (40%) and diffuse large B-cell lymphoma (25%). Conclusions: Our study identified distinct immune gene programs in the TIME of patients with newly diagnosed AML. From a translational standpoint, immune-enriched and IFN-gamma-dominant AMLs may benefit from immune checkpoint blockade and from new immunotherapy approaches, including dual-affinity T-cell redirecting antibodies targeting CD123. Grant support: John and Lucille van Geest Foundation, UK; Roger Counter Foundation, UK; Qatar National Research Fund (#NPRP8-2297-3-494). Citation Format: Sergio Rutella, Jayakumar Vadakekolathu, Tressa Hood, Stephen Reeder, Sarah E. Warren, Patrick Danaher, Jan Davidson-Moncada, Alessandra Cesano, Joseph M. Beechem, Sarah K. Tasian, Mark D. Minden. Immune gene expression profiling of acute myeloid leukemia identifies predictors of survival and actionable targets for treatment [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B63.

NanoString Gene Expression Profiling in Chronic Lung Allograft Dysfunction (CLAD)

CLAD, mainly manifest as bronchiolitis obliterans syndrome (BOS), is the leading cause of late death in lung recipients. BOS develops despite routine immunosuppression and its pathogenesis is poorly understood. The histological correlate of BOS is bronchiolitis obliterans (BO), or airways fibrosis. We quantified over 700 immune-related genes in BO affected airways to better understand immune responses active in end-stage BOS. Formalin fixed paraffin embedded lung tissue was procured at retransplantation for BOS (n=6). Unused donor tissue was used as normal control (n=5). Histologies of interest were isolated using laser capture microdissection (LCMD). Within each BOS lung, airways histologically affected by BO pathology, defined as intraluminal fibrous expansion, were isolated. Additionally, airways unaffected by evident fibrosis were isolated. Within each donor lung, histologically normal airways were isolated. Cellular RNA was extracted and multiplex gene expression analysis was performed using the NanoString PanCancer Immune Profiling Panel. Linear regression was used to compare gene expression among groups. The primary comparison was differential expression in BO affected vs. normal donor airways. We also compared expression in BO affected vs. unaffected airways within BOS explant lungs. P-values were corrected for multiple comparisons using Benjamini-Hochberg. Sixty-seven genes were significantly different between BO affected vs. normal donor airways, of which 52 had a fold change (FC) <0.5 or >2. Along with genes in adaptive immunity, several genes related to innate and humoral activation were significantly increased in BO affected airways. Among the innate immune genes with increased expression in BO were toll-like receptor 1 (FC 2.31) and its adaptor molecule CD14 (FC 5.26) in addition to the complement subcomponent C1s (FC 3.06), while those in humoral immunity included bone marrow stromal antigen 2 (FC 3.41), important to the growth and development of B-cells. No genes were significantly differentially expressed between BO affected vs. unaffected airways within BOS explant lungs. Our results suggest innate and humoral immune responses are active in airways affected by BO pathology, and also in airways without evident fibrosis. Ongoing analyses of BO lesions through LCMD will provide new insights into CLAD pathogenesis.