PanCancer Immune Profiling Panel Research Articles

Identification of molecular signatures associated with early relapse after complete resection of lung adenocarcinomas

The only potentially curative treatment for lung adenocarcinoma patients remains complete resection of early-stage tumors. However, many patients develop recurrence and die of their disease despite curative surgery. Underlying mechanisms leading to establishment of systemic disease after complete resection are mostly unknown. We therefore aimed at identifying molecular signatures of resected lung adenocarcinomas associated with the risk of an early relapse. The study comprised 89 patients with totally resected stage IA–IIIA lung adenocarcinomas. Patients suffering from an early relapse within two years after surgery were compared to patients without a relapse in two years. Patients were clinically and molecular pathologically characterized. Tumor tissues were immunohistochemically analyzed for the expression of Ki67, CD45, CD4, CD8, PD1, PD-L1, PD-L2 and CD34, by Nanostring nCounter PanCancer Immune Profiling Panel as well as a comprehensive methylome profiling using the Infinium MethylationEPIC BeadChip. We detected differential DNA methylation patterns as well as significantly differentially expressed genes associated with an early relapse after complete resection. Especially, CD1A was identified as a potential biomarker, whose reduced expression is associated with an early relapse. These findings might help to develop biomarkers improving risk assessment and patient selection for adjuvant therapy as well as establish novel targeted therapeutic strategies.

T and B Lymphocyte Transcriptional States Differentiate between Sensitized and Unsensitized Individuals in Alpha-Gal Syndrome.

The mechanisms of pathogenesis driving alpha-gal syndrome (AGS) are not fully understood. Differences in immune gene expression between AGS individuals and non-allergic controls may illuminate molecular pathways and targets critical for AGS development. We performed immune expression profiling with RNA from the peripheral blood mononuclear cells (PBMCs) of seven controls, 15 AGS participants, and two participants sensitized but not allergic to alpha-gal using the NanoString nCounter PanCancer immune profiling panel, which includes 770 genes from 14 different cell types. The top differentially expressed genes (DEG) between AGS subjects and controls included transcription factors regulating immune gene expression, such as the NFκB pathway (NFKBIA, NFKB2, REL), antigen presentation molecules, type 2/allergic immune responses, itch, and allergic dermatitis. The differential expression of genes linked to T and B cell function was also identified, including transcription factor BCL-6, markers of antigen experience (CD44) and memory (CD27), chemokine receptors (CXCR3, CXCR6), and regulators of B-cell proliferation, cell cycle entry and immunoglobulin production (CD70). The PBMCs from AGS subjects also had increased TNF and IFN-gamma mRNA expression compared to controls. AGS is associated with a distinct gene expression profile in circulating PBMCs. DEGs related to antigen presentation, antigen-experienced T-cells, and type 2 immune responses may promote the development of alpha-gal specific IgE and the maintenance of AGS.

Cross-platform comparison of immune-related gene expression to assess intratumor immune responses following cancer immunotherapy

Neoadjuvant immunotherapy can induce immune responses within the tumor microenvironment. Gene expression can be used to assess responses with limited amounts of conventionally-fixed patient-derived samples. We aim to assess the cross-platform concordance of immune-related gene expression data. We performed comparisons across three panels in two platforms: Nanostring nCounter® PanCancer Immune Profiling Panel (nS), HTG EdgeSeq Oncology Biomarker Panel (HTG OBP) and Precision Immuno-Oncology Panel (HTG PIP). All tissue samples of 14 neoadjuvant GM-CSF treated, 14 neoadjuvant Provenge treated, and 12 untreated prostate cancer patients were radical prostatectomy (RP) tissues, while 6 prostatitis patients and 6 non-prostatitis subjects were biopsies. For all 52 patients, more than 90% of the common genes were significantly correlated (p < 0.05) and more than 76% of the common genes were highly correlated (r > 0.5) between any two panels. Co-inertia analysis also demonstrated high overall dataset structure similarity (correlation>0.84). Although both dimensionality reduction visualization analysis and unsupervised hierarchical cluster analysis for highly correlated common genes (r > 0.9) suggested a high-level of consistency across the panels, there were subsets of genes that were differentially expressed across the panels. In addition, while the effect size of the differential testing for neoadjuvant treated vs. untreated localized prostate cancer patients across the panels were significantly correlated, some genes were only differentially expressed in the HTG panels. Finally, the HTG PIP panel had the best classification performance among the 3 panels. These differences detected may be a result of the different panels or platforms due to their technical setting and focus. Thus, researchers should be aware of those potential differences when deciding which platform and panel to use.

Abstract PS4-37: A pro-tumorigenic mechanism of M2 tumor-associated macrophages (TAM) in triple-negative breast cancer

Abstract Introduction:Triple-Negative Breast Cancer (TNBC) comprises approximately 30% of all breast cancers in Indian women. Given their aggressive nature, TNBCs have high rates of systemic metastasis and mortality with only chemotherapy available for treatment. The success of immunotherapy in solid tumors has raised the hope for their utility in TNBCs as well. But only a subset of patients have a clinical response to check-point inhibitors. The cellular and molecular mechanisms that mediate the immunological response or tolerance are just beginning to be understood. Compared to ER/PR+ breast cancer, TNBC features a unique tumor microenvironment (TME) characterized by a large number of tumor-infiltrating lymphocytes (TILs) and tumor-associated macrophages (TAMs). The density of TILs in and of themselves do not accurately predict response to neoadjuvant chemotherapy or survival. M2 tumor-associated macrophages (M2-TAMs) have been reported to associate with solid tumors to facilitate epithelial to mesenchymal transition (EMT), tumor invasiveness, metastasis, and resistance to therapy. In this study, we have characterized presence of M2-TAM in the TNBC immune environment by examining expression of several biomarkers. Methods:Surgically excised tumor specimens from 88 Indian women with TNBC were accessed from the longitudinal observational series of SJNAHS tissue bank under IERB approved protocols and assayed on nCounter® PanCancer Immune Profiling Panel which comprised of 740 genes and characterises 14 different immune cell types. Transcriptome analysis using Non-negative Matrix factorization (NMF) based unsupervised clustering was done to arrive at stable subtypes characterized by different tumour microenvironments within TNBC. CIEBERSORT tool was used to analyse for distribution of cell types. Identification of macrophages was done by Immunohistochemistry (IHC) for CD68 and CD163 markers in TNBCs and a control group of ER+HER2-.Results:NMF analysis with an enriched immune gene signature of 111 genes, yielded 3 subtypes (ST) (37%, 27% and 36% with relative proportions of ST1, ST2 and ST3) within the TNBC. The three subtypes had distinct survival patterns with ST1 having the best prognosis with enriched TH1 gene signature and ST3 had poorest (log rank p=0.5). On application of this gene signature to TNBC groups in METABRIC and TCGA data, similar pattern emerged with a significant survival pattern of ST 3 having the poorest outcome (p=0.005). A closer examination of ST 3 revealed a signature enriched for M2 macrophages also known as TAM. Immunohistochemistry for Pan macrophage marker CD68 and an M2 specific marker CD163 between TNBC and ER/PR+ revealed difference in the two groups was significantly different (Mann Whitney p=0.03), with TNBC exhibiting 2.4 fold higher expression of CD163. CD 68, was enriched in ST2 and ST3. CD163 which is an M2 specific marker was highest in ST3 as compared to ST 1&amp; 2 (p=0.04). This group also correlated with signal molecules secreted by macrophages containing growth factors, cytokines and chemokines, such as TGF-β, VEGF, IL-10 and CXCL and interleukins like IL4 and IL6 suggestive of TAM recruitment and polarization. It is likely that the TAM enriched subgroup is likely to be unresponsive to PD1/PDL1 inhibitors but could be targeted by novel therapeutic strategies to directly target M2-TAM. In vitro and In vivo analysis to target M2-TAM to evaluate the response to these therapeutic compounds in cell lines as well as a mouse syngeneic model is underway.Conclusions and future directions: As a cell type within the tumor microenvironment, that promotes invasion, M2-TAMs makes an ideal therapeutic target in TNBC. In addition, this sub-type lends itself to easy identification by a simple IHC assay. Citation Format: Aruna Korlimarla, Jyothi Prabhu, Chantirika Ragulan, Gnanapriya Shivakumar, Krisha Desai, Maggie Cheang, Srinath BS, Ravi Diwakar, Sandhya Apachu, Rekha Kumar, TS Sridhar, Savitha RajaRajan, Rohini Kaluve, Annie Alexander, Anguraj Sadanandam. A pro-tumorigenic mechanism of M2 tumor-associated macrophages (TAM) in triple-negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS4-37.

Association of B cells in tumor microenvironment (TME) with clinical benefit to programmed cell death protein-1 (PD-1) blockade therapy in esophageal squamous cell carcinoma (ESCC).

237 Background: Previous studies have indicated B cells as potential predictive markers for anti-PD-1 blockade therapy in several cancer types. The study explored whether B cells in TME and B cell related gene signatures are associated with clinical benefit (CB) for ESCC patients receiving anti-PD-1/PD-ligand 1 (PD-L1) therapy. Methods: Sixty-six ESCC patients treated with PD-1/PD-L1 blockade-based immunotherapy were enrolled. Tumor response was evaluated per RECIST 1.1, and CB was defined as complete response, partial response or stable disease at least 6 months. Transcriptome of formalin-fixed paraffin-embedded ESCC tissues were generated by NanoString nCounter platform with Human PanCancer Immune Profiling panel (n=25), Gene Set Enrichment Analysis (GSEA) was performed to identify the differential immune-related pathways, and CIBERSORT was applied to estimate the levels of infiltrating immune cells. The expression of CD20 was evaluated by immunohistochemistry (IHC) (n=66). Results: Of 66 enrolled patients (M: F= 65: 1, median age of 59), 44 and 22 were of recurrent and de novo metastatic ESCC. Forty and 26 received PD-1/PD-L1 blockade alone and PD-1/PD-L1-based combination immunotherapy, respectively. The response rate was 17%, and the CB rate was 24%. The median progression-free-survival (PFS) and overall survival are 1.8 and 5.5 months, respectively. B cell signature was significantly increased in patients with CB ( P &lt; 0.05) and associated with longer progression-free survival and overall survival (both P &lt; 0.05). The genes related to B cells, B cell functions, and T cell functions were up-regulated in patients with CB compared to that with non-CB (all P &lt; 0.05). Naïve B cells and plasma cells were significantly increased in patients with CB (both P &lt; 0.05). The expression levels of stromal CD20 by IHC trended to increase in patients with CB ( P = 0.08). Conclusions: B cells in tumor microenvironment may be associated with CB of anti-PD-1/PD-L1 therapy in patients with ESCC. (Funded by MOST 105-2314-B-002 -186 -MY3; MOHW107-TDU-B-211-114017; MOST 108-2314-B-002 -076 -MY3; MOST 107-2314-B-002-199 -).

Distribution pattern of tumor infiltrating lymphocytes and tumor microenvironment composition as prognostic indicators in anorectal malignant melanoma

Anorectal malignant melanoma (ARMM) is a rare disease with poor prognosis. Determining ARMM prognosis precisely is difficult due to the lack of proper assessment techniques. Immunotherapy has proven effective against cutaneous malignant melanoma and may show efficacy in ARMM. Herein, we assessed the immune profile of ARMM to identify possible prognostic biomarkers. Twenty-two ARMM formalin-fixed and paraffin-embedded samples were evaluated using an nCounter® PanCancer Immune Profiling Panel. Validation was performed through immunohistochemical staining for CD3, CD8, Foxp3, CD68, CD163, and PD-L1. RNA analysis revealed significantly decreased scores for pathways involved in cell regulation and function, as well as chemokines, in recurrent patients compared to nonrecurrent patients. In cell-type profiling, the recurrent cases displayed significantly low tumor infiltrating lymphocyte (TIL) scores. Recurrence/death prediction models were defined using logistic regression and showed significantly lower scores in recurrent and deceased patients (all, P < 0.001) compared to those in nonrecurrent and surviving patients. The high total TIL and tumor-associated macrophage (TAM) groups had significantly better overall survival outcomes compared to the low total TIL and TAM groups (P = 0.007 and P = 0.035, respectively). In addition, the presence of CD3 + TILs in the invasion front was an independent favorable prognostic indicator (P = 0.003, hazard ratio = 0.21, 95% confidential interval, 0.01–0.41). Patients with inflamed or brisk-infiltration type tumors also had a significantly better overall survival than that of patients with immune-desert/excluded and absent/non-brisk type tumors (P = 0.03 and P = 0.0023, respectively). In conclusion, TILs have a strong prognostic value in ARMM, and the quantification of TILs and an analysis of the TIL phenotype and infiltration pattern during pathological diagnosis are essential to guide treatment strategies and accurate prognosis in ARMM.

Molecular Profiling of Exceptional Responders to Cancer Therapy

The vast majority of metastatic cancers cannot be cured. Palliative treatment may relieve disease symptoms by stopping or slowing cancer growth and may prolong patients' lives, but almost all patients will inevitably develop disease progression after initial response. However, for reasons that are not fully understood, a very few patients will have extraordinary durable responses to standard anticancer treatments. We analyzed exceptional responders treated at Fox Chase Cancer Center between September 2009 and November 2017. An exceptional response was defined as a complete response lasting more than 1 year or a partial response or stable disease for more than 2 years. Tumor samples were analyzed using an Ambry Genetics test kit with a 142-gene panel. Messenger RNA expression was evaluated using NanoString's nCounter PanCancer Pathways Panel and Immune Profiling Panel and compared with matched controls for gender, age, and cancer type. Twenty-six exceptional responders with metastatic bladder, kidney, breast, lung, ovarian, uterine, and colon cancers were enrolled. Mutations were identified in 45 genes. The most common mutation was an EPHA5 nonsynonymous mutation detected in 87.5% of patients. Mutations in DNA damage repair pathway genes were also frequent, suggesting increased genome instability. We also found varying expression of 73 genes in the Pathways panel and 85 genes in the Immune Profiling panel, many of them responsible for improvement in tumor recognition and antitumor immune response. The genomic instability detected in our exceptional responders, plus treatment with DNA damage compounds combined with favorable anticancer immunity, may have contributed to exceptional responses to standard anticancer therapies in the patients studied. With recent advances in the treatment of cancer, there is increased emphasis on the importance of identifying molecular markers to predict treatment outcomes, thereby allowing precision oncology. In this study, it was hypothesized that there is a "specific biologic signature" in the biology of the cancer in long-term survivors that allows sensitivity to systemic therapy and durability of response. Results showed that DNA damage repair pathway alterations, combined with favorable anticancer immunity, may have contributed to exceptional responses. It is very likely that an in-depth examination of outlier responses will become a standard component of drug development in the future.

Co-stimulatory and co-inhibitory Immune Markers in Solid Tumors With MET Alterations

The implication of MET alterations in solid tumors and the immune microenvironment remains elusive. Formalin-fixed, paraffin-embedded samples of 21 patients with solid tumors harboring MET alterations were used for immunohistochemical staining. Extracted RNA was analyzed with the NanoString nCounter human PanCancer immune profiling panel (NanoString Technologies, Inc., WA, USA). Patients were diagnosed with lung (n = 10), breast (n = 5), genitourinary (n = 3) or colorectal cancer (n = 3). Eleven had a MET missense mutation, four had an exon 14 splice site mutation and six had MET amplification. CD6, CCL19, CD40LG, XCR1, MAGEA1, ATM and CCL19 genes were significantly differentially expressed in MET-altered cancers. MET alterations may have a role in various solid tumors as potential therapeutic targets and combination therapy candidates with immune checkpoint inhibitors.

Characterization of Sentinel Lymph Node Immune Signatures and Implications for Risk Stratification for Adjuvant Therapy in Melanoma

Although sentinel lymph node (SLN) biopsy is a standard procedure used to identify patients at risk for melanoma recurrence, it fails to risk-stratify certain patients accurately. Because processes in SLNs regulate anti-tumor immune responses, the authors hypothesized that SLN gene expression may be used for risk stratification. The Nanostring nCounter PanCancer Immune Profiling Panel was used to quantify expression of 730 immune-related genes in 60 SLN specimens (31 positive [pSLNs], 29 negative [nSLNs]) from a retrospective melanoma cohort. A multivariate prediction model for recurrence-free survival (RFS) was created by applying stepwise variable selection to Cox regression models. Risk scores calculated on the basis of the model were used to stratify patients into low- and high-risk groups. The predictive power of the model was assessed using the Kaplan-Meier and log-rank tests. During a median follow-up period of 6.3years, 20 patients (33.3%) experienced recurrence (pSLN, 45.2% [14/31] vs nSLN, 20.7% [6/29]; p = 0.0445). A fitted Cox regression model incorporating 12 genes accurately predicted RFS (C-index, 0.9919). Improved RFS was associated with increased expression of TIGIT (p = 0.0326), an immune checkpoint, and decreased expression of CXCL16 (p = 0.0273), a cytokine important in promoting dendritic and T cell interactions. Independent of SLN status, the model in this study was able to stratify patients into cohorts at high and low risk for recurrence (p < 0.001, log-rank). Expression profiles of the SLN gene are associated with melanoma recurrence and may be able to identify patients as high or low risk regardless of SLN status, potentially enhancing patient selection for adjuvant therapy.

An Immune Senescence and Exhaustion-Related RNA Profile Predicts Clinical Outcomes in Acute Myeloid Leukemia

Background Chemotherapy refractoriness and disease relapse continue to be significant obstacles to therapeutic success in AML. We have recently identified bone marrow (BM) transcriptomic profiles that stratify patients with AML into an immune-infiltrated and an immune-depleted subtype and that refine the accuracy of survival prediction in response to conventional chemotherapy beyond that afforded by cytogenetic and molecular prognosticators (Vadakekolathu J, et al. Sci. Transl. Med. 2020; 12: eaaz0463). Importantly, AML CD8+ T cells exhibit features of immune exhaustion and senescence (IES), including the upregulation of natural killer (NK) cell-associated transcripts, which persist only in chemotherapy non-responders (Knaus HA, et al. JCI Insight 2018; 3: e120974). The aim of the current study was to determine whether IES correlate with immune infiltration and with clinical outcomes in AML. Methods We used prior knowledge and gene set enrichment analysis (GSEA) to derive a 68-gene IES signature score (computed as the cohort-wide average of gene expression) from publicly available RNA-sequencing datasets (TCGA and Beat-AML Master Trial; 162 and 281 AML patients, respectively). The wet-lab AML cohorts used in this study included a total of 382 BM samples from children and adults with AML treated with curative intent. BMs were collected at time of diagnosis, complete remission (CR) and relapse (PMCC, SAL and CHOP series). BM RNAs were profiled on the nCounter platform using the PanCancer Immune Profiling Panel (NanoString Technologies, Seattle, WA). Results In the AML discovery cohort, the IES score significantly correlated with BM lymphoid infiltration (R=0.958) and with CD8+ T cell (R=0.88), cytotoxic T cell (R=0.90) and tumor inflammation signature scores (R=0.82, P&amp;lt;0.0001 for all; Fig. 1A). The novel IES gene signature showed minimal overlap with previously discovered interferon (IFN)-related gene sets with prognostic relevance in AML (Fig. 1B) and was higher in TP53-mutated (P&amp;lt;0.0001) and RUNX1-mutated (P=0.0071) compared with WT AML. Overall survival (OS) was significantly shorter in patients with higher than median IES scores (Fig. 1C). IES signature scores were also associated with worse clinical outcomes in Beat-AML cases (median OS of 10.6 and 21.1 months, respectively, in patients with higher and lower than median IES scores; Fig. 1D) and were higher in primary induction failures (PIF) compared with chemotherapy responders (Fig. 1E). The top IES genes associated with survival prediction both in TCGA and Beat-AML, although not overlapping with published IFN-related gene sets, were still significantly enriched in type I/II IFN signaling pathways and in molecular functions indicative of heightened ion-gated channel activity, as shown by protein-protein interaction network analyses. The above findings were validated in independent wet-lab cohorts comprising adults (PMCC series; n=290; SAL series; n=46) and children (CHOP series; n=46) with AML. In the ELN adverse risk subgroup (PMCC cohort), both relapse-free survival (RFS) and OS were significantly shorter in patients with higher than median compared with lower than median IES scores (median RFS time of 6.93 versus not reached [log-rank P=0.0053], and median OS of 10.5 months versus 18 months [log-rank P=0.0011], respectively). In contrast, the IES signature score failed to stratify survival in patients with ELN favorable and intermediate risk. Finally, a pairwise comparison of matched diagnostic, CR and relapse BM samples (SAL series; n=22 patients and CHOP series; n=46 patients) showed significantly higher IES signature scores at time of CR, congruent with chemotherapy-induced acceleration of IES, and at time of post-chemotherapy relapse compared with disease onset (Fig. 1F). Notably, CD8A, TBX21, a Th1 transcription factor, and markers of NK cells and cytotoxic T lymphocytes, including KLRK1, KLRD1, KLRC2, GNLY, and granzymes, were among the top ranked immune genes associated with AML relapse (Fig. 1G). Conclusions Patients with immune-infiltrated AML exhibit features of IES, which may correlate with adverse-risk molecular features, chemotherapy refractoriness and shorter survival. Molecular circuits reflective of IES might also underpin AML relapse after conventional induction chemotherapy. IES T cells could be functionally rejuvenated by novel immunotherapies being investigated in AML. Figure 1 Disclosures Church: NanoString Technologies, Inc.: Current Employment. Davidson-Moncada:Macrogenics: Current Employment. Tasian:Incyte Corporation: Research Funding; Aleta Biotherapeutics: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Research Funding. Gojo:Amphivena: Research Funding; Merck: Research Funding; Genentech: Research Funding; Amgen: Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees. Luznik:WindMil Therapeutics: Patents &amp; Royalties: Patent holder; AbbVie: Consultancy; Merck: Research Funding, Speakers Bureau; Genentech: Research Funding. Rutella:Kura Oncology: Research Funding; MacroGenics, Inc.: Research Funding; NanoString Technologies, Inc.: Research Funding.

Abstract B16: The immune profile of sentinel lymph nodes in melanoma

Abstract Background: Immune cell composition and immune-mediated processes occurring in the sentinel lymph node (SLN) of melanoma patients regulate generation of tumor-specific effector T-cell responses and ultimately tumor control. We hypothesized that comprehensive analysis of these immune-associated processes in the SLN biopsy may identify patients at high risk of melanoma recurrence. Methods: We analyzed 38 formalin-fixed, paraffin-embedded sentinel lymph nodes from melanoma patients undergoing SLN biopsy from 2009-2012 using the NanoString nCounter® PanCancer Immune Profiling panel. The panel included 770 immune related genes. Results: Of 38 patients, 19 were SLN negative and 19 were SLN positive. Among SLN-negative patients (n=19), the median Breslow depth was 1.25 mm, and 21% (n=4) had developed recurrence at median follow-up of 6.2 years. Among SLN-positive patients, the median Breslow depth was 3.35 mm and 37% (n=7) had developed recurrence at median follow-up of 6.5 years. There were 31 genes significantly differentially expressed (P&amp;lt;0.01) in SLN positive compared to SLN negative, including higher relative expression of B-cell signaling markers (CD19, CD22, Pax5, CXCR5, CD79B). Among all 38 patients, there were 13 genes differentially expressed (P&amp;lt;0.05) between patients with and without recurrence, including higher relative expression of IL6 in patients with recurrence compared to no recurrence. Further analysis is ongoing. Conclusion: In this retrospective series, we found the immunologic gene expression profile of melanoma SLNs was significantly different in SLN negative versus SLN positive. There were also significant differences in the profile between patients who developed melanoma recurrence versus no recurrence regardless of SLN status. We aim to build on these pilot data. The goal is to use this profile to predict patients at high risk of recurrence after SLNB who may benefit from adjuvant therapy. Citation Format: Georgia M. Beasley, Eda K. Holl, Norma E. Farrow, Margaret G. Leddy, April K. Salama, Brent A. Hanks, Smita K. Nair. The immune profile of sentinel lymph nodes in melanoma [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr B16.

Abstract B28: A novel intestinal microbiome-derived peptide modulates immune cell activity and the tumor microenvironment

Abstract The composition of the gut microbiota affects cancer development, progression, and response to therapy. A number of commensal bacteria, including Bifidobacterium, have been associated with increased response to immune checkpoint inhibitors in mouse tumor models, as well as in melanoma patients. We hypothesized that secreted peptides or proteins contribute to the effects mediated by Bifidobacterium strains in vivo. We used our unique bioinformatics-driven discovery platform to nominate putatively secreted Bifidobacterium-derived peptides for evaluation in immune cell effector assays. We have previously described bacterial peptides that induce secretion of proinflammatory cytokines (e.g., IL-6, TNF) by in vitro-generated mouse and human dendritic cells, as well as effector cytokine secretion (e.g., IFNγ, IL-2) by mouse splenic T lymphocytes in vitro. To investigate the function of Bifidobacterium-derived peptides in the context of the tumor microenvironment, we injected a candidate peptide, termed SG-A, directly into the tumors of tumor-bearing mice. We then performed immune phenotyping via the Nanostring PanCancer Immune Profiling panel, as well as by flow cytometry. Both Nanostring and flow cytometry analysis demonstrated an increase in CD45+ lymphocytes within the tumors of mice treated with SG-A. Peptide SG-A also induced upregulation of dendritic cell function genes (CD40, CD83, and CD86) and multiple effector cytokines and chemokines. Restimulation of tumor-draining lymph node cells with a tumor-derived peptide antigen also increased induction of IFNγ in SG-A treated animals (vs. vehicle-treated controls). Collectively, our results demonstrate the utility of the Second Genome discovery platform for leveraging microbiome science to identify novel immunomodulatory factors. This platform offers the potential to identify agents that may complement or enhance efficacy of existing approaches to immunotherapy for melanoma and other cancers. Citation Format: Helena Kiefel, Dhwani Haria, Yuliya Katlinskaya, Divya Ravichandar, Sunit Jain, Thomas Weinmaier, Shoko Iwai, Todd DeSantis, Toshi Takeuchi, Karim Dabbagh, Kareem Graham. A novel intestinal microbiome-derived peptide modulates immune cell activity and the tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr B28.

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.

The Oncocytic Variant of Poorly Differentiated Thyroid Carcinoma Shows a Specific Immune-Related Gene Expression Profile

Poorly differentiated thyroid cancer (PDTC) is a rare, follicular cell-derived neoplasm with an unfavorable prognosis. The oncocytic variant of PDTC may be associated with even more adverse outcome than classical PDTC cases, but its specific molecular features are largely unknown. Our aim was to explore the immune-related gene expression profile of oncocytic and classical PDTC, in correlation with clinical and pathological characteristics (including programmed death ligand 1 [PD-L1] expression) and outcome, and in comparison with a control group of well-differentiated follicular carcinomas (WDFCs), including conventional follicular carcinomas (FTCs) and Hürthle cell carcinomas (HCCs). A retrospective series of 48 PDTCs and 24 WDFCs was analyzed by means of NanoString technology employing the nCounter PanCancer Immune Profiling panel. Gene expression data were validated using quantitative real-time polymerase chain reaction. Oncocytic PDTCs showed a specific immune-related gene expression profile, with higher expression of LAIR2, CD274, DEFB1, IRAK1, CAMP, LCN2, LY96, and APOE, and lower expression of NOD1, as compared to conventional PDTCs. This molecular signature was associated with increased intratumoral lymphocytic infiltration, PD-L1 expression, and adverse outcome. Three of these genes, CD274, DEFB1, and IRAK1, as well as PD-L1 expression, were also the hallmarks of HCCs as compared to FTCs. By contrast, the panel of genes differentially regulated in PDTCs as compared to WDFCs was unrelated to the oncocytic phenotype. Our results revealed a distinctive immune-related gene expression profile of oncocytic PDTC and confirmed a more aggressive outcome in this cancer subtype. These findings may provide guidance when exploring novel immunotherapeutic options for oncocytic PDTC patients.

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 &amp;gt;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.

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)