T-cell Gene Signature Research Articles

Abstract B091: Spatial Transcriptomics Unveils Intraductal Papillary Mucinous Neoplasm Heterogeneity: From Novel Clusters to Immune Dynamics

Abstract Intraductal papillary mucinous neoplasms (IPMNs), a common precursor to pancreatic ductal adenocarcinoma, present as a spectrum of benign indolent behavior and progression to invasive carcinoma, which highlights the importance of identifying biomarkers of IPMN progression. This study utilized whole transcriptome spatial transcriptomics to define the molecular characteristics of Epithelial, Immune, and Fibroblast cell populations of the three distinct histological sub-types of IPMN to understand the dynamics between the cell types and identify biomarkers of neoplastic progression. Fifty-two surgically resected IPMN cores from 21 patients with resected IPMNs comprising 24 Gastric, 14 Intestinal, and 14 Pancreaticobiliary with a distribution of low-grade dysplasia (LGD), high-grade dysplasia (HGD), and Invasive carcinoma (IC) were analyzed. Epithelial, Immune, and Fibroblast components were analyzed using the GeoMx spatial profiler. Unsupervised hierarchical clustering of epithelial areas of interest (AOI) identified 3 distinct clusters demonstrating a spectrum of molecular phenotypes in IPMN that were correlated with the three histological subtypes. Comparing the gene expression profile based on the shared genes expressed on all three subtypes, the Intestinal type tends to have an overexpression of genes whereas the Pancreaticobiliary represents a repressed phenotype relative to the Gastric phenotype. Supervised clustering using Moffitt’s classifier to identify the Epithelial-Mesenchymal transition demonstrates the Pancreaticobiliary subtype to be more Basal-like versus the Gastric and Intestinal inclined towards the classical phenotype. Supervised clustering based on the degree of dysplasia within the epithelial compartments of each of these subtypes, demonstrated a spectrum of molecular heterogeneity and identified biomarkers specific to High-Grade dysplasia in the Gastric and Intestinal subtype. Immune AOI deconvolution analysis demonstrated that Tregs were associated with Pancreaticobiliary, Macrophage enrichment associated with the Intestinal phenotype, while there is enrichment of CD8 T-cell and CD4 T-cell gene signatures associated with the Gastric subtype. Fibroblast AOI analyses demonstrated heterogenous myCAF- iCAF signatures between the subtypes. In summary, Spatial whole transcriptome analysis of IPMN identifies molecular signatures in epithelial and immune compartments that differentiate between the three histological subtypes of IPMN and helps understand their progression from Low-grade dysplasia to High-grade dysplasia. Citation Format: Amaya Pankaj, Michael J Raabe, Yuhui Song, Bidish K Patel, Katherine Xu, Joshua R Kocher, Nicholas J Caldwell, Maria L Ganci, Linda T Nieman, Mari Mino-Kenudson, Vikram Deshpande, Nabeel Bardeesy, Carlos Fernandez-Del Castillo, Martin J Aryee, David T Ting, Yasmin G Hernandez-Barco. Spatial Transcriptomics Unveils Intraductal Papillary Mucinous Neoplasm Heterogeneity: From Novel Clusters to Immune Dynamics [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B091.

Impact of LAG-3/FGL1 pathway on immune evasive contexture and clinical outcomes in advanced urothelial carcinoma

BackgroundAnti-programmed death-1 (PD-1)/anti-PD-ligand-1 (PD-L1) pathway inhibition is a standard regimen for advanced urothelial carcinoma (UC); however, its limited efficacy has been reflected in reported medium response rates. This study explored...

ILT2 and ILT4 Drive Myeloid Suppression via Both Overlapping and Distinct Mechanisms.

Solid tumors are dense three-dimensional (3D) multicellular structures that enable efficient receptor-ligand trans interactions via close cell-cell contact. Immunoglobulin-like transcript (ILT)2 and ILT4 are related immune-suppressive receptors that play a role in the inhibition of myeloid cells within the tumor microenvironment. The relative contribution of ILT2 and ILT4 to immune inhibition in the context of solid tumor tissue has not been fully explored. We present evidence that both ILT2 and ILT4 contribute to myeloid inhibition. We found that although ILT2 inhibits myeloid cell activation in the context of trans-engagement by MHC-I, ILT4 efficiently inhibits myeloid cells in the presence of either cis- or trans-engagement. In a 3D spheroid tumor model, dual ILT2/ILT4 blockade was required for the optimal activation of myeloid cells, including the secretion of CXCL9 and CCL5, upregulation of CD86 on dendritic cells, and downregulation of CD163 on macrophages. Humanized mouse tumor models showed increased immune activation and cytolytic T-cell activity with combined ILT2 and ILT4 blockade, including evidence of the generation of immune niches, which have been shown to correlate with clinical response to immune-checkpoint blockade. In a human tumor explant histoculture system, dual ILT2/ILT4 blockade increased CXCL9 secretion, downregulated CD163 expression, and increased the expression of M1 macrophage, IFNγ, and cytolytic T-cell gene signatures. Thus, we have revealed distinct contributions of ILT2 and ILT4 to myeloid cell biology and provide proof-of-concept data supporting the combined blockade of ILT2 and ILT4 to therapeutically induce optimal myeloid cell reprogramming in the tumor microenvironment.

A Dysfunctional T-cell Gene Signature for Predicting Nonresponse to PD-1 Blockade in Non-small Cell Lung Cancer That Is Suitable for Routine Clinical Diagnostics.

Because PD-1 blockade is only effective in a minority of patients with advanced-stage non-small cell lung cancer (NSCLC), biomarkers are needed to guide treatment decisions. Tumor infiltration by PD-1T tumor-infiltrating lymphocytes (TIL), a dysfunctional TIL pool with tumor-reactive capacity, can be detected by digital quantitative IHC and has been established as a novel predictive biomarker in NSCLC. To facilitate translation of this biomarker to the clinic, we aimed to develop a robust RNA signature reflecting a tumor's PD-1T TIL status. mRNA expression analysis using the NanoString nCounter platform was performed in baseline tumor samples from 41 patients with advanced-stage NSCLC treated with nivolumab that were selected on the basis of PD-1T TIL infiltration by IHC. Samples were included as a training cohort (n = 41) to develop a predictive gene signature. This signature was independently validated in a second cohort (n = 42). Primary outcome was disease control at 12 months (DC 12 m), and secondary outcome was progression-free and overall survival. Regularized regression analysis yielded a signature using 12 out of 56 differentially expressed genes between PD-1T IHC-high tumors from patients with DC 12 m and PD-1T IHC-low tumors from patients with progressive disease (PD). In the validation cohort, 6/6 (100%) patients with DC 12 m and 23/36 (64%) with PD were correctly classified with a negative predictive value (NPV) of 100% and a positive predictive value of 32%. The PD-1T mRNA signature showed a similar high sensitivity and high NPV as the digital IHC quantification of PD-1T TIL. This finding provides a straightforward approach allowing for easy implementation in a routine diagnostic clinical setting.

Tyrosine Kinase Inhibition Activates Intratumoral γδ T Cells in Gastrointestinal Stromal Tumor.

γδ T cells are a rare but potent subset of T cells with pleiotropic functions. They commonly reside within tumors but the response of γδ T cells to tyrosine kinase inhibition is unknown. To address this, we studied a genetically engineered mouse model of gastrointestinal stromal tumor (GIST) driven by oncogenic Kit signaling that responds to the Kit inhibitor imatinib. At baseline, γδ T cells were antitumoral, as blockade of either γδ T-cell receptor or IL17A increased tumor weight and decreased antitumor immunity. However, imatinib therapy further stimulated intratumoral γδ T cells, as determined by flow cytometry and single-cell RNA sequencing (scRNA-seq). Imatinib expanded a highly activated γδ T-cell subset with increased IL17A production and higher expression of immune checkpoints and cytolytic effector molecules. Consistent with the mouse model, γδ T cells produced IL17A in fresh human GIST specimens, and imatinib treatment increased γδ T-cell gene signatures, as measured by bulk tumor RNA-seq. Furthermore, tumor γδ T cells correlated with survival in patients with GIST. Our findings highlight the interplay between tumor cell oncogene signaling and antitumor immune responses and identify γδ T cells as targets for immunotherapy in GIST.

Regulatory T Cell Biomarkers Identify Patients at Risk of Developing Acute Cellular Rejection in the First Year Following Heart Transplantation.

Acute cellular rejection (ACR), an alloimmune response involving CD4+ and CD8+ T cells, occurs in up to 20% of patients within the first year following heart transplantation. The balance between a conventional versus regulatory CD4+ T cell alloimmune response is believed to contribute to developing ACR. Therefore, tracking these cells may elucidate whether changes in these cell populations could signal ACR risk. We used a CD4+ T cell gene signature (TGS) panel that tracks CD4+ conventional T cells (Tconv) and regulatory T cells (Treg) on longitudinal samples from 94 adult heart transplant recipients. We evaluated combined diagnostic performance of the TGS panel with a previously developed biomarker panel for ACR diagnosis, HEARTBiT, while also investigating TGS' prognostic utility. Compared with nonrejection samples, rejection samples showed decreased Treg- and increased Tconv-gene expression. The TGS panel was able to discriminate between ACR and nonrejection samples and, when combined with HEARTBiT, showed improved specificity compared with either model alone. Furthermore, the increased risk of ACR in the TGS model was associated with lower expression of Treg genes in patients who later developed ACR. Reduced Treg gene expression was positively associated with younger recipient age and higher intrapatient tacrolimus variability. We demonstrated that expression of genes associated with CD4+ Tconv and Treg could identify patients at risk of ACR. In our post hoc analysis, complementing HEARTBiT with TGS resulted in an improved classification of ACR. Our study suggests that HEARTBiT and TGS may serve as useful tools for further research and test development.

MTORC1 upregulates B7-H3/CD276 to inhibit antitumor T cells and drive tumor immune evasion

Identifying the mechanisms underlying the regulation of immune checkpoint molecules and the therapeutic impact of targeting them in cancer is critical. Here we show that high expression of the immune checkpoint B7-H3 (CD276) and high mTORC1 activity correlate with immunosuppressive phenotypes and worse clinical outcomes in 11,060 TCGA human tumors. We find that mTORC1 upregulates B7-H3 expression via direct phosphorylation of the transcription factor YY2 by p70 S6 kinase. Inhibition of B7-H3 suppresses mTORC1-hyperactive tumor growth via an immune-mediated mechanism involving increased T-cell activity and IFN-γ responses coupled with increased tumor cell expression of MHC-II. CITE-seq reveals strikingly increased cytotoxic CD38+CD39+CD4+ T cells in B7-H3-deficient tumors. In pan-human cancers, a high cytotoxic CD38+CD39+CD4+ T-cell gene signature correlates with better clinical prognosis. These results show that mTORC1-hyperactivity, present in many human tumors including tuberous sclerosis complex (TSC) and lymphangioleiomyomatosis (LAM), drives B7-H3 expression leading to suppression of cytotoxic CD4+ T cells.

Abstract PD10-08: PD10-08 Immune cell infiltration associated with poor anti-proliferative response to aromatase inhibitors in postmenopausal women with primary ER-positive HER2-negative breast cancer

Abstract Background: Aromatase inhibitors (AIs) are one of the main treatment strategies for the clinical management of estrogen receptor-positive (ER+) breast cancer (BC). Despite prolonged time to recurrence and initial clinical responses, >20% of patients eventually relapse, and previous studies have shown an association of poor anti-proliferative response to AIs and worse outcome. High immune activity in ER+ tumors may be associated with worse outcome, in contrast to ER-negative BC where immune infiltration is a feature associated with better outcome. Our work focused on understanding the correlations between immune cell infiltration and response to AI. Methods: All patients with ER+ HER2- tumors within the bottom 15% of Ki67 anti-proliferative responders to AIs (poor responders [PRs]; n=177) were selected from the PeriOperative Endocrine Therapy for Individualizing Care (POETIC) trial and matched on baseline Ki67 levels to good responders (GRs) within the 50% showing the best response (n=190). Response to AI was measured by the Ki67 percentage change after 2 weeks of treatment. PRs were further divided into groups expressing high ESR1 (PRs ESR1HIGH; n=119) and low ESR1 (PRs ESR1LOW; n=58) levels to represent PR subgroups that showed partial or no response to AIs. The percentage of stromal tumor-infiltrating lymphocytes (TILs) was assessed. Multiple immunofluorescence was performed for ER, CD3, CD20, CD68, FOXP3, and CD3/FOXP3 in 15 baseline samples from each of the GR, PR ESR1HIGH, and PR ESR1LOW populations and immune cell density in stromal or tumor compartments was estimated. Spearman correlations of TILs with Consensus tumor microenvironment (TME) deconvolution and Molecular Signatures Database hallmark gene sets were conducted. The relationship between the immune markers’ density and genes, hallmark gene sets and Consensus TME was assessed. Results: The percentage of TILs was significantly higher in the PR ESR1HIGH and PR ESR1LOW compared to the GRs (adjusted p< 0.05). As expected, TILs were highly correlated with T cells (particularly T-regulatory cells) and immune hallmark gene sets. There was a tendency for higher density of each of the immune markers in PRs compared to GRs, with significant differences being observed in stromal B-cell marker CD20 density (p< 0.05). Analysis showed a significant correlation between TILs and stromal FOXP3 marker density (FDR< 0.05), and stromal biomarker density was highly correlated to the gene expression of the encoding genes of the same tumors (CD3/CD3D, FOXP3/FOXP3, and CD20/MS4A1) (FDRs< 0.05). There was also a strong and significant correlation between the stromal expression of CD20, CD3, FOXP3, and CD3/FOXP3 with the immune hallmark gene sets (FDRs< 0.05). Finally, the immune phenotyping showed the expected correlations with TME deconvolution, with particularly strong correlations of CD20 and CD3 with B- and T-cell gene signatures, respectively (FDRs< 0.05). Conclusions: Different immune features indicated a broad involvement of several immune cell types in PRs to AIs, suggesting that the immune system might be associated with resistance of ER+ breast tumors to AI treatment. Spatial gene expression profiling is ongoing to characterize these tumors further and investigate potential mechanisms of AI resistance. Citation Format: Anastasia Alataki, Gene Schuster, Lila Zabaglo, Perry Maxwell, Elena López-Knowles, Elizabeth Folkerd, David Evans, Kally Sidhu, Holly Tovey, Nicholas Turner, Stephen Johnston, Maggie Chon U Cheang, John Robertson, Manuel Salto-Tellez, Ian Smith, Judith Bliss, Mitch Dowsett. PD10-08 Immune cell infiltration associated with poor anti-proliferative response to aromatase inhibitors in postmenopausal women with primary ER-positive HER2-negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD10-08.

Immune signatures in cutaneous melanoma correlate with survival independently of immunotherapy treatment.

Immune checkpoint inhibitors (ICIs) have fundamentally improved survival from advanced cutaneous melanoma. Significant efforts have been made to understand the ICI response to identify ways to further improve outcomes. One such approach has been to investigate gene expression associated with response to ICI, which has identified various immune-related mRNA signatures, including a 6-gene IFN-γ signature (IFN-γ6 ), an expanded immune signature (IFN-γ18 ), an effector T-cell gene signature (Teff ), and a Teff -associated and IFN-γ-associated gene signature (Teff + IFN-γ). Given that these signatures appear to reflect expression from T-cells and the level of tumour-infiltrating immune cells has been associated with survival, we hypothesised that the prognostic value of the signatures is not limited to ICI treatment and investigated if they were associated with survival also in patients who never received ICI. The signatures were not present in melanoma cell lines when compared with tumour samples, confirming that the signatures were likely derived from the samples' non-tumour (immune) components. We acquired expression and survival data from five melanoma cohorts with a wide range of disease stages, treatments, and metrics for survival, and correlated the expression signatures with survival. All four signatures were significantly associated (P<0.05) with survival in 4/5 cohorts, with hazard ratios ranging from 0.69 to 0.92. We conclude that these immune signatures' association with survival is not specific to ICI-treated patients, but present in a number of settings.

A novel immune signature predicts immunotherapy responsiveness and reveals the landscape of the tumor immune microenvironment in head and neck squamous cell carcinoma.

Background: Immune-checkpoint blockade (ICB) has been routinely implemented to treat head and neck squamous cell carcinoma (HNSCC) patients. However, only a few patients benefit from immune checkpoint inhibitor (ICI) therapies. Methods: In this study, we used a combined cohort (including the GSE41613, GSE65858, TCGA, and CELL cohorts) to identify hub genes significantly associated with ICB and activated CD8+ T-cell gene signatures. We performed single-sample gene set enrichment analysis (ssGSEA) to quantify the expression of hub genes; we then constructed a novel immune signature named "the IMS" that can predict immunotherapy responsiveness, prognosis, immune infiltration, and clinical characteristics. Data from the GSE102349 external cohort and the pembrolizumab cohort obtained from a clinical trial were used to validate the efficiency of the IMS. In addition, we revealed potential mechanisms of the antitumor response by analyzing the HNSCC single-cell database. Finally, we used the LASSO algorithm to build an IMS-related risk model. Results: The high IMS group was associated with significant immune activation, better prognosis, and increased immunotherapy responsiveness; thus, the IMS potentially represents a candidate biomarker for ICB. Moreover, a tumor microenvironment with a higher IMS underwent remarkable metabolic reprogramming characterized by enrichment in the glycolysis/gluconeogenesis, oxidative phosphorylation, and citrate cycle (TCA cycle) pathways. We also revealed key information on cellular crosstalk between the IMS and other immune lineages, which may mechanistically explain immune escape. In addition, we constructed and validated a risk prediction model (CD2, TBC1D10C, and CD3E) that could stratify HNSCC patients based on survival and response to ICB treatment. Conclusion: IMS is a signature closely correlated with the tumor immune microenvironment. The findings of this study contribute to the understanding of the immune landscape in HNSCC patients. IMS may aid in the clinical management of HNSCC patients through the identification of effective immunotherapies for specific patients.

Targeting CD96 overcomes PD-1 blockade resistance by enhancing CD8+ TIL function in cervical cancer

BackgroundNovel therapies are needed to treat recurrent and advanced cervical cancer (CC), as their prognosis remains very poor. Although therapies targeting the programmed cell death protein 1 (PD-1) pathway have...

The Identification of a Tumor Infiltration CD8+ T-Cell Gene Signature That Can Potentially Improve the Prognosis and Prediction of Immunization Responses in Papillary Renal Cell Carcinoma.

BackgroundCD8+ T cells, vital effectors pertaining to adaptive immunity, display close relationships to the immunization responses to kill tumor cells. Understanding the effect exerted by tumor infiltration CD8+ T cells in papillary renal cell carcinoma (papRCC) is critical for assessing the prognosis process and responses to immunization therapy in cases with this disease.Materials and ApproachesThe single-cell transcriptome data of papRCC were used for screening CD8+ T-cell-correlated differentially expressed genes to achieve the following investigations. On that basis, a prognosis gene signature associated with tumor infiltration CD8+ T cell was built and verified with The Cancer Genome Atlas data set. Risk scores were determined for papRCC cases and categorized as high- or low-risk groups. The prognosis significance for risk scores was assessed with multiple-variate Cox investigation and Kaplan–Meier survival curves. In addition, the possible capability exhibited by the genetic profiles of cases to assess the response to immunization therapy was further explored.ResultsSix hundred twenty-one cell death-inhibiting RNA genes were screened using single-cell RNA sequencing. A gene signature consisting of seven genes (LYAR, YBX1, PNRC1, TCF25, MYL12B, MINOS1, and LINC01420) was then identified, and this collective was considered to be an independent prognosis indicator that could strongly assess overall survival in papRCC. In addition, the data allowed papRCC cases to fall to cohorts at high and low risks, exhibiting a wide range of clinically related features as well as different CD8+ T-cell immunization infiltration and immunization therapy responses.ConclusionsOur work provides a possible explanation for the limited response of current immunization checkpoint-inhibiting elements for combating papRCC. Furthermore, the researchers built a novel genetic signature that was able to assess the prognosis and immunotherapeutic response of cases. This may also be considered as a promising therapeutic target for the disease.

Immune and Epigenetic Landscape of TP53-mutated Acute Myeloid Leukemia (AML) and Higher-Risk Myelodysplastic Syndromes (HR-MDS)

Immune and Epigenetic Landscape of TP53-mutated Acute Myeloid Leukemia (AML) and Higher-Risk Myelodysplastic Syndromes (HR-MDS)

Abstract 1792: Engineered exosome- mediated STAT6 knockdown in tumor associated macrophages (TAMs) results in potent single agent activity in a hepatocellular carcinoma (HCC) model

Abstract Background: Tumor-associated macrophages (TAMs) promote tumor progression and resistance to immune checkpoint inhibitors and are thus attractive targets for cancer immunotherapy. The STAT6 transcriptional network is an important driver of the immune-suppressive M2 macrophage program in the tumor microenvironment (TME). Previous attempts to therapeutically target these transcriptional networks have not been successful. Exosomes serve as an efficient, natural, intercellular communication system that can deliver nucleic acids and other macromolecules. Leveraging the potential of exosomes, we have developed a novel, engineered exosome therapeutic candidate loaded with antisense oligonucleotides (ASO) targeting STAT6 (exoASO-STAT6), that effectively silences STAT6 expression in TAMs. Results: In vitro and in vivo studies demonstrate an enhanced delivery of ASO to M2 macrophages. exoASO showed a 2x improvement in uptake vs free ASO in human M2 macrophages in vitro. Following IV administration, exoASO demonstrated up to 11x increase in uptake in monocytes and MDSCs in the blood, Kupffer cells in liver and TAMs and MDSCs in the tumor. In vitro treatment with exoASO-STAT6 resulted in 90% target gene KD in human, mouse and cynomolgus monkey M2 macrophages, which was persistent for up to 10 days. Additionally, exoASO-STAT6 demonstrated greater potency than free ASO. STAT6 KD resulted in a 7x decrease in M2 marker CD163 and a 25x increase in pro-inflammatory cytokines such as IL-12 or TNFα, demonstrating effective macrophage reprogramming. In vivo efficacy studies in CT26 showed potent dose-dependent single agent activity of exoASO-STAT6, with a cumulative dose of 36 μg of ASO resulting in 94% TGI and 80% complete responses. CD8 T-cell depletion abrogated anti-tumor activity and the complete responders were resistant to tumor cell re-challenge, demonstrating a CD8-T cell mediated immunological memory response. In an orthotopic model of HCC that is resistant to anti-PD-1 or anti-CSF1R therapy, IV administration of exoASO-STAT6 significantly attenuated tumor growth, as observed by a 61% reduction in tumor mass and complete elimination of tumor lesions in 50% of treated mice. exoASO-STAT6 therapy resulted in a decrease in M2 markers such as Tgfb1 and Ccl17 and an increase in M1 markers such as IL1b. A significant increase in interferon and cytotoxic T-cell gene signatures was also observed, demonstrating effective reprogramming of the TME. Conclusion: exoASO-STAT6 is a novel therapeutic that selectively targets STAT6, a key transcription factor in TAMs. This therapy results in effective macrophage reprogramming to a pro-inflammatory M1 phenotype and potent single agent anti-tumor activity in multiple checkpoint refractory tumor models. In sum, exoASO-STAT6 represents a first-in-class strategy to target TAMs in a highly selective manner. Citation Format: Sushrut Kamerkar, Charan Leng, Olga Burenkova, Su Chul Jang, Christine McCoy, Kelvin Zhang, William Dahlberg, Marie Leblanc, Hugo Quillery, Sylvie Maubant, Olivier Duchamp, Kyriakos Economides, Timothy Soos, Dalia Burzyn, Sriram Sathyanarayanan. Engineered exosome- mediated STAT6 knockdown in tumor associated macrophages (TAMs) results in potent single agent activity in a hepatocellular carcinoma (HCC) model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1792.

DNMT3A Mutations Identify a Prognostic Subgroup in Peripheral T-Cell Lymphoma

Background Mutational profiling of angioimmunoblastic T-cell lymphoma (AITL) and peripheral T-cell lymphoma not otherwise specified (PTCL-NOS) has revealed recurrent mutations in DNMT3A, a de novo methyltransferase. DNMT3A catalyzes the conversion of cytosine to 5-methylcytosine (5-mC) while interacting with histones and transcription factors to influence gene expression. While the DNMT3A mutational profile in PTCL entities indicates loss-of-function, hotspot change-of-function mutations (e.g., DNMT3AR882H/C) have been observed with their frequency differing between PTCL entities. Despite the high occurrence of DNMT3A mutations in PTCLs (~30% of cases), their functional consequences have not been extensively studied. Herein, we examined DNMT3A mutations in AITL and the novel molecular subgroups of PTCL-NOS (i.e., PTCL-TBX21 and PTCL-GATA3) and observed distinct biological and prognostic significance associated with DNMT3A mutations in the PTCL-TBX21 subgroup. Methods PTCL-NOS cases (n = 141) were utilized following PTCL-TFH exclusion. Using previously described molecular classification methods, cases were classified as PTCL-TBX21 (n = 80) or PTCL-GATA3 (n = 61). A separate cohort of AITL cases (n = 176) were included for comparative purposes. Clinical outcome data were assessed with the Kaplan-Meier method. Mutation data were generated from DNA-sequencing (n = 224) or RNA-sequencing methods (n = 46). Gene expression comparisons were conducted using BRB-ArrayTools. Immune-cell signatures were generated from the CIBERSORT and/or xCell computational tools. 5-mC DNA immunoprecipitation sequencing (MeDIP-Seq) was performed on available PTCL-TBX21 cases (n = 7) or healthy tonsil controls (n = 2). Four of these cases carried DNMT3A mutations (n = 3 DNMT3AR882, n = 1 DNMT3AQ886) while the remaining cases (n = 3) were wild type for DNMT3A. In vitro analyses of ectopic expression of the DNMT3AR882H mutant or DNMT3A knockdown were conducted using healthy-donor CD3+ T-cells or the CD8+ T8ML1 PTCL cell line. Following corrections for false discoveries, p-values &amp;lt; 0.05 were considered significant. Results DNMT3A-mutated PTCL-TBX21 cases had an inferior overall survival, with DNMT3A mutated residues skewed toward the methyltransferase domain. In contrast to the DNMT3A mutation profile seen in AITL, PTCL-TBX21 featured DNMT3AR882H/C mutations at a frequency (30%) similar to other hematological malignancies. Gene expression profiling revealed that DNMT3A-mutant PTCL-TBX21 cases were enriched for activated CD8+ T-cell gene signatures and showed association with the previously described TH1/αβ cytotoxic T-cell lymphoma subgroup. Following MeDIP-Seq, assessment of differentially methylated regions comparing DNMT3AR882/Q886 PTCL-TBX21 cases to wild type found hypomethylation in pathways associated with T-cell activation, TCR signaling, and TH1 responses. In vitro analyses demonstrated that ectopic expression of the DNMT3AR882H mutant or DNMT3A knockdown lead to enhanced proliferation and NF-κB signaling in T8ML1 cells in comparison to control vectors. In primary CD3+ T-cell cultures, ectopic expression of the DNMT3AR882H mutant protein resulted in the preferential outgrowth of CD8+ T-cells. Conclusions Taken together, our findings establish mutations in DNMT3A as a novel prognostic marker in PTCL-TBX21. The integrated expression, methylation, and in vitro findings suggest that disruption of DNMT3A leads toward an activated and cytotoxic phenotype and could potentially drive oncogenic TCR signaling. Clinically, as these cases were associated with the TH1/αβ cytotoxic T-cell lymphoma subgrouping, these findings should be taken into consideration for future treatment strategies regarding PTCL-NOS patients as current standard-of-care treatments may be particularly inadequate in the treatment of PTCLs with cytotoxic phenotype. Disclosures No relevant conflicts of interest to declare.

Abstract PD1-05: Atezolizumab in combination with trastuzumab emtansine or with trastuzumab and pertuzumab in patients with HER2-positive breast cancer and atezolizumab with doxorubicin and cyclophosphamide in HER2-negative breast cancer: Safety and biomarker outcomes from a multi-cohort Phase Ib study

Abstract Background: Chemotherapy and HER2-targeted antibodies are standard-of-care (SOC) treatments (tx) for HER2+ breast cancer (BC) in several settings. Atezolizumab (atezo) + chemotherapy improves progression-free survival (PFS) and overall survival (OS) in PD-L1+ advanced triple-negative BC. Due to the potential for effective tumor cell death and anti-tumor immunity, this study investigated the safety and clinical activity of novel atezo-based combination therapy in HER2+ and HER2-negative BC. In addition, the study evaluated changes in the tumor microenvironment with the combination of immune checkpoint inhibition (atezo) with antibody-dependent cellular cytotoxicity (ADCC) agents (trastuzumab [T] + pertuzumab [P]) or with antibody drug conjugates (ADCs; trastuzumab emtansine [T-DM1]). Methods: GO29831 (NCT02605915) is a multi-cohort Phase Ib study evaluating the safety of atezo-containing tx combinations in patients (pts) with BC. Evaluation of clinical activity and biomarkers were exploratory objectives. PD-L1 status by VENTANA SP142 IHC assay, CD8 IHC and RNA-based gene expression signatures were assessed in pre-tx and on-tx biopsies. See table for patient populations and interventions. Results: At clinical data cutoff (Dec 17, 2018), 76 pts were safety evaluable across 8 tx cohorts. Patient demographics in this completely enrolled study were heterogeneous between cohorts (e.g., hormone receptor status, no. of prior therapies in metastatic setting). No new safety signals were observed beyond the established safety profiles of the individual drugs. Two up-front cycles of atezo combination therapy with anti-HER2 therapy had no detrimental effect on the pathologic complete response (pCR) rates expected with SOC regimens. Increases in PD-L1-expressing tumor-infiltrating immune cells were observed in both HER2+ early BC (eBC) and metastatic BC (mBC) with the combination of atezo + T-DM1 or atezo + T + P. Consistent increases in CD8+ T cells in the tumor area were observed only in eBC. Increases in RNA gene signatures associated with antigen presentation, cytolytic activity and immune checkpoints were observed with both atezo + T-DM1 and atezo + T + P in eBC, whereas statistically significant increases in B-cell (P = 0.0322) and T-cell gene signatures (P = 0.0049) were observed only with atezo + T + P. Conclusions: These tx combinations were tolerable, and safety signals were in line with the known safety profiles of the individual drugs. Biomarker analyses showed that combining atezo with ADCC or ADC agents promoted the activation of the adaptive immune system in the tumor microenvironment. TableCohortPatient PopulationInterventionNo. of Pts1APts with HER2+ mBC or LABCAtezo 1200 mg, T 6 mg/kg, P 420 mg q3w61BPts with HER2+ mBC or LABCAtezo 1200 mg, T-DM1 3.6 mg/kg q3w61EPts with HER2-negative mBC or LABCAtezo 840 mg, doxorubicin 60 mg/m2, cyclophosphamide 600 mg/m2 and pegfilgrastim or G-CSF q2w for 4 cycles, followed by atezo 1200 mg q3w31FPts with HER2+ mBCAtezo 1200 mg, T 8-mg/kg loading dose (maintenance: 6 mg/kg), P 840-mg loading dose (maintenance: 420 mg) and docetaxel 75 mg/m2 q3w62APts with HER2+ operable LABC or inflammatory eBC (tumor &amp;gt; 2 cm)Neoadjuvant atezo 1200 mg + T 8 mg/kg (c2: 6 mg/kg) + P 840 mg (c2: 420 mg) for 2 cycles, followed by 6 cycles of docetaxel 75 mg/m2, carboplatin AUC 6 mg/mL x min, T 6 mg/kg, P 420 mg q3w prior to breast surgery202BPts with HER2+ operable LABC or inflammatory eBC (tumor &amp;gt; 2 cm)Neoadjuvant atezo 1200 mg + T-DM1 3.6 mg/kg q3w for 2 cycles, followed by 6 cycles of docetaxel 75 mg/m2, carboplatin AUC 6 mg/mL x min, T 6 mg/kg, P 420 mg q3w prior to breast surgery202CPts with HER2+ mBC or LABC with PD on mBC tx or ≤ 6 mo of adjuvant txAtezo 1200 mg, T-DM1 3.6 mg/kg q3w142DPts with HER2+ mBC or LABC with PD on T- and P-containing regimen ≤ 12 wkAtezo 1200 mg, T 8-mg/kg loading dose (maintenance: 6 mg/kg), P 840-mg loading dose (maintenance: 420 mg) q3w1AUC, area under the curve; c2, cycle 2; G-CSF, granulocyte colony-stimulating factor; LABC, locally advanced breast cancer; PD, progressive disease; pt, patient; q2w, every 2 weeks; q3w, every 3 weeks. NCT02605915 Citation Format: Erika Paige Hamilton, Virginia Kaklamani, Carla Falkson, Gregory A Vidal, Patrick J Ward, Monika Patre, Stephen Y Chui, Jacob Rotmensch, Kushagra Gupta, Luciana Molinero, Yijin Li, Leisha A Emens. Atezolizumab in combination with trastuzumab emtansine or with trastuzumab and pertuzumab in patients with HER2-positive breast cancer and atezolizumab with doxorubicin and cyclophosphamide in HER2-negative breast cancer: Safety and biomarker outcomes from a multi-cohort Phase Ib study [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD1-05.

Abstract OT1-01-01: SOLTI-1503 PROMETEO: Combination of talimogene laherparepvec (T-VEC) with atezolizumab in patients with residual breast cancer after standard neoadjuvant multi-agent chemotherapy

Abstract Background: New strategies as immune blockade inhibitors and oncolytic viruses are opening new possibilities in cancer therapy. Preliminary results in melanoma showed that the combination of Talimogene Laherparevec (T-VEC) with an anti PD-L1 or anti CTLA4 has greater efficacy than either therapy alone, without additional safety concerns beyond those expected for each individual agent. The presence of residual cancer after neoadjuvant chemotherapy (NAC) in early breast cancer (BC) patients is an unmet medical need. We hypothesize that combining T-VEC with Atezolizumab may offer clinical benefit in the preoperative setting for early BC patients with intermediate to high risk of recurrence who present residual disease (RD) after standard NAC. Methods: PROMETEO is an open-label, multicenter trial of T-VEC in combination with Atezolizumab in patients with residual disease after completing standard NAC. 30 patients with triple negative breast cancer (TNBC) or Luminal B-like/Her2- (ER/PR&amp;gt;10% and Ki-67 ≥30% or ER/PR 1-10% and Ki-67 ≥20%) will be included. RD must be confirmed by core-biopsy and have a diameter ≥ 15mm measured by magnetic resonance imaging. Adequate organ function and ECOG PS 0-1 are required. T-VEC is administered intratumorally in week 1 (106 plaque-forming units/mL [pfu/mL]), then in week 4 and every 2 weeks thereafter (108 pfu/mL) for four injections. Atezolizumab (840 mg) is administered intravenously every 2 weeks for four infusions, beginning in week 4. BC surgery will be carried out 1 to 3 weeks after completion of study treatment. Primary objective is to evaluate the effect of T-VEC + Atezolizumab by comparing the expression of a gene signature tracking activated CD8 T-cells between RD at surgery and RD after NAC. We expect a relative increase of ≥20% in the expression of this CD8 T-cell gene signature in 20% of patients or more, defined as the mean expression of PRF1, CD8A, GZMM, CD8B and FLT3LG, between post and pre-treatment samples. Secondary endpoints include pathologic complete response rate and residual cancer burden, objective response rate, and safety. Two safety data reviews - after 4 and 10 patients complete treatment - are planned. A total of 4 sites in Spain are participating in the trial. To date, 4 patients have been included and it is expected to complete the first safety data review in September 2019. The study recruitment is planned to be completed by October 2020. Clinical trial identification: NCT03802604 Citation Format: Tomás Pascual, Patricia Villagrasa, María J Vidal, Sergi Ganau, Begoña Bermejo, Ana Julve, Esther Zamora, Ignacio Miranda, Estela Vega, Cristina Marquez, Mafalda Oliveira, Juan Miguel Cejalvo, Luis de la Cruz, Manel Juan, Jordi Canes, Xavier Gonzalez, Aleix Prat. SOLTI-1503 PROMETEO: Combination of talimogene laherparepvec (T-VEC) with atezolizumab in patients with residual breast cancer after standard neoadjuvant multi-agent chemotherapy [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr OT1-01-01.

Abstract 1176: Immunogenomic profiling identifies a subgroup of squamous cell lung cancers with immunosuppressed tumor microenvironment and correlates TGF-beta and Wnt/beta-catenin signaling as predictive of low PD-L1 expression

Abstract Introduction: immune checkpoint inhibitors have revolutionized the treatment of non-small cell lung cancer (NSCLC), but only ~15% of patients will achieve durable clinical benefit. Our group and others have focused on characterizing the interaction between tumor genomic characteristics and immune microenvironment. Genes such as PTEN, CTNNB1, STK11 and KEAP1 have been associated with intra-tumor immune depletion, but a broader understanding of other key driver genes of NSCLC and their interplay with the immune microenvironment is currently lacking. To prospectively delineate the genomic and immune landscape of NSCLC, we launched the ICON Project (Immunogenomic Profiling of NSCLC). Methods: we prospectively collected tissue specimens from patients with stage I-IIIA NSCLC undergoing surgical resection. Samples were subjected to extensive immune-genomic profiling, including whole-exome and RNA sequencing, multiplex immunofluorescence (mIF), and flow cytometry. Results: from 2016-2018, 150 patients were accrued, with 78 samples having undergone genomic profiling. Most patients had stage I-II disease (74%) and non-squamous histology (70%). Most commonly mutated genes were TP53, EGFR, KRAS, CDKN2A, STK11, and mean TMB was 7.6 mut/Mb, which is comparable to TCGA (6.9mut/Mb, p=ns). Unsupervised clustering according to expression of CD8+ T cell related genes segregated tumors into immune-enriched (clusters 1 and 3) and immune-depleted (clusters 2 and 4) subgroups. We validated these findings through flow cytometry (CD8+GzB+ T cells) and mIF (CD8), with both methods showing CD8+ T cell infiltration to be enriched in clusters 1 and 3. Cluster 1 included squamous and non-squamous histology, tumors with epithelial and mesenchymal signatures, and high TMB. Cluster 2 included exclusively squamous histology with predominant mesenchymal tumor signature, high TMB, and was enriched for TP53 mutations. Cluster 3 had predominantly non-squamous histology, epithelial signature, and low TMB. Cluster 4 had exclusively non-squamous histology, predominant epithelial signature, low TMB, and was enriched for KEAP1 mutations. In addition, PD-L1 expression (by mIF) was negatively correlated with TGF-beta and WNT/Beta-catenin signaling pathway, as well as high EGFR and FGFR2 expression on malignant cells. Conclusion: CD8+ T-cell gene signature identifies immunosuppressed subgroups of lung cancer, especially among squamous cell carcinomas with a mesenchymal signature, that may be associated with resistance to immune-based therapies. TGF-beta and Wnt/beta-catenin pathways are associated with an immunosuppressed tumor microenvironment, and may be potential novel targets to restore anti-tumor immunity in lung cancers with low PD-L1 expression. Citation Format: Marcelo V. Negrao, Tatiana Karpinets, Jun Li, Alexandre Reuben, Cara Haymaker, Kyle G. Mitchell, Junya Fujimoto, Chi-Wan Chow, Edwin R. Parra, Lorenzo Federico, Jianhua Zhang, Ara A. Vaporciyan, Chantale Bernatchez, Tina Cascone, Boris Sepesi, Ignacio I. Wistuba, John V. Heymach, Jianjun Zhang, Don L. Gibbons, ICON Team. Immunogenomic profiling identifies a subgroup of squamous cell lung cancers with immunosuppressed tumor microenvironment and correlates TGF-beta and Wnt/beta-catenin signaling as predictive of low PD-L1 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1176.

Targeted expression profiling by RNA-Seq improves detection of cellular dynamics during pregnancy and identifies a role for T cells in term parturition

Development of maternal blood transcriptomic markers to monitor placental function and risk of obstetrical complications throughout pregnancy requires accurate quantification of gene expression. Herein, we benchmark three state-of-the-art expression profiling techniques to assess in maternal circulation the expression of cell type-specific gene sets previously discovered by single-cell genomics studies of the placenta. We compared Affymetrix Human Transcriptome Arrays, Illumina RNA-Seq, and sequencing-based targeted expression profiling (DriverMapTM) to assess transcriptomic changes with gestational age and labor status at term, and tested 86 candidate genes by qRT-PCR. DriverMap identified twice as many significant genes (q < 0.1) than RNA-Seq and five times more than microarrays. The gap in the number of significant genes remained when testing only protein-coding genes detected by all platforms. qRT-PCR validation statistics (PPV and AUC) were high and similar among platforms, yet dynamic ranges were higher for sequencing based platforms than microarrays. DriverMap provided the strongest evidence for the association of B-cell and T-cell gene signatures with gestational age, while the T-cell expression was increased with spontaneous labor at term according to all three platforms. We concluded that sequencing-based techniques are more suitable to quantify whole-blood gene expression compared to microarrays, as they have an expanded dynamic range and identify more true positives. Targeted expression profiling achieved higher coverage of protein-coding genes with fewer total sequenced reads, and it is especially suited to track cell type-specific signatures discovered in the placenta. The T-cell gene expression signature was increased in women who underwent spontaneous labor at term, mimicking immunological processes at the maternal-fetal interface and placenta.

Mutation load and an effector T-cell gene signature may distinguish immunologically distinct and clinically relevant lymphoma subsets

AbstractIdentifying follicular lymphoma (FL) patients with preexisting antitumor immunity will inform precision medicine strategies for novel cancer immunotherapies. Using clinical and genomic data from 249 FL patients, we determined the clinical impact of mutation load and an effector T-cell (Teff) gene signature as proxies for the likelihood of a functional immune response. The FL mutation load estimate varied between 0 and 33 mutations per Mb (median, 6.6), and 92% of FL patients with a high mutation load had high Teff gene expression (P = .001). The mutation load was associated with a benefit from rituximab maintenance: FL patients with low mutation loads experienced a profound benefit from rituximab maintenance (hazard ratio [HR], 0.29; 95% confidence interval [CI], 0.15-0.54; P ≮ .001). The Teff gene signature was prognostic as a continuous predictor (P = .008), and was used to separate FL patients into 2 groups, an “inflamed” subset (Teff-high; n = 74) and an “uninflamed” subset (Teff-low; n = 75), with longer progression-free survival (PFS) in the inflamed FL subset (PFS HR, 0.39; 95% CI, 0.21-0.70; P = .002). Furthermore, the subset of inflamed FL tumors demonstrated high expression of other T-cell signatures and counterregulatory genes, which also correlate with PFS. Mutation load and Teff gene expression may help identify immunologically distinct lymphoma subsets relevant for modern immunotherapies.