PanCancer Immune Profiling Research Articles

Genes deregulated in giant cell arteritis by Nanostring nCounter gene expression profiling in temporal artery biopsies

ObjectiveTo identify differentially expressed genes in temporal artery biopsies (TABs) from patients with giant cell arteritis (GCA) with different histological patterns of inflammation: transmural inflammation (TMI) and inflammation limited to...

Small duct and large duct type intrahepatic cholangiocarcinoma reveal distinct patterns of immune signatures

PurposeDedicated gene signatures in small (SD-iCCA) and large (LD-iCCA) duct type intrahepatic cholangiocarcinoma remain unknown. We performed immune profiling in SD- and LD-iCCA to identify novel biomarker candidates for personalized medicine.MethodsRetrospectively, 19 iCCA patients with either SD-iCCA (n = 10, median age, 63.1 years (45–86); men, 4) or LD-iCCA (n = 9, median age, 69.7 years (62–85); men, 5)) were included. All patients were diagnosed and histologically confirmed between 04/2009 and 01/2021. Tumor tissue samples were processed for differential expression profiling using NanoString nCounter® PanCancer Immune Profiling Panel.ResultsWith the exception of complement signatures, immune-related pathways were broadly downregulated in SD-iCCA vs. LD-iCCA. A total of 20 immune-related genes were strongly downregulated in SD-iCCA with DMBT1 (log2fc = -5.39, p = 0.01) and CEACAM6 (log2fc = -6.38, p = 0.01) showing the strongest downregulation. Among 7 strongly (log2fc > 2, p ≤ 0.02) upregulated genes, CRP (log2fc = 5.06, p = 0.02) ranked first, and four others were associated with complement (C5, C4BPA, C8A, C8B). Total tumor-infiltrating lymphocytes (TIL) signature was decreased in SD-iCCA with elevated ratios of exhausted-CD8/TILs, NK/TILs, and cytotoxic cells/TILs while having decreased ratios of B-cells/TILs, mast cells/TILs and dendritic cells/TILs. The immune profiling signatures in SD-iCCA revealed downregulation in chemokine signaling pathways inclulding JAK2/3 and ERK1/2 as well as nearly all cytokine-cytokine receptor interaction pathways with the exception of the CXCL1/CXCR1-axis.ConclusionImmune patterns differed in SD-iCCA versus LD-iCCA. We identified potential biomarker candidate genes, including CRP, CEACAM6, DMBT1, and various complement factors that could be explored for augmented diagnostics and treatment decision-making.

Assessing the implications of sentinel lymph node removal in cervical cancer: an immunogenetic perspective – a SENTICOL ancillary study

BackgroundCervical cancer’s lymphatic spread primarily begins from the sentinel lymph nodes (SLNs), underlining their pivotal role in disease metastasis. However, these nodes’ immune gene expression profiles and immunoregulation mechanisms have...

HUNK as a key regulator of tumor-associated macrophages in triple negative breast cancer

ABSTRACT Triple-negative breast cancer (TNBC) lacks the expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). TNBC tumors are not sensitive to endocrine therapy, and standardized TNBC treatment regimens are lacking. TNBC is a more immunogenic subtype of breast cancer, making it more responsive to immunotherapy intervention. Tumor-associated macrophages (TAMs) constitute one of the most abundant immune cell populations in TNBC tumors and contribute to cancer metastasis. This study examines the role of the protein kinase HUNK in tumor immunity. Gene expression analysis using NanoString’s nCounter PanCancer Immune Profiling panel identified that targeting HUNK is associated with changes in the IL-4/IL-4 R cytokine signaling pathway. Experimental analysis shows that HUNK kinase activity regulates IL-4 production in mammary tumor cells, and this regulation is dependent on STAT3. In addition, HUNK-dependent regulation of IL-4 secreted from tumor cells induces polarization of macrophages into an M2-like phenotype associated with TAMs. In return, IL-4 induces cancer metastasis and macrophages to produce epidermal growth factor. These findings delineate a paracrine signaling exchange between tumor cells and TAMs regulated by HUNK and dependent on IL-4/IL-4 R. This highlights the potential of HUNK as a target for reducing TNBC metastasis through modulation of the TAM population.

HMGB1 Expression Levels Correlate with Response to Immunotherapy in Non-Small Cell Lung Cancer.

High-mobility group box 1 protein (HMGB1) is subject to exportin 1 (XPO1)-dependent nuclear export, and it is involved in functions implicated in resistance to immunotherapy. We investigated whether HMGB1 mRNA expression was associated with response to immune checkpoint inhibitors (ICI) in non-small cell lung cancer (NSCLC). RNA was isolated from pretreatment biopsies of patients with advanced NSCLC treated with ICI. Gene expression analysis of several genes, including HMGB1, was conducted using the NanoString Counter analysis system (PanCancer Immune Profiling Panel). Western blotting analysis and cell viability assays in EGFR and KRAS mutant cell lines were carried out. Evaluation of the antitumoral effect of ICI in combination with XPO1 blocker (selinexor) and trametinib was determined in a murine Lewis lung carcinoma model. HMGB1 mRNA levels in NSCLC patients treated with ICI correlated with progression-free survival (PFS) (median PFS 9.0 versus 18.0 months, P=0.008, hazard ratio=0.30 in high versus low HMGB1). After TNF-α stimulation, HMGB1 accumulates in the cytoplasm of PC9 cells, but this accumulation can be prevented by using selinexor or antiretroviral drugs. Erlotinib or osimertinib with selinexor in EGFR-mutant cells and trametinib plus selinexor in KRAS mutant abolish tumor cell proliferation. Selinexor with a PD-1 inhibitor with or without trametinib abrogates the tumor growth in the murine Lewis lung cancer model. An in-depth exploration of the functions of HMGB1 mRNA and protein is expected to uncover new potential targets and provide a basis for treating metastatic NSCLC in combination with ICI.

Abstract 1594: Pancreatic cancer and T-cell crosstalk-induced MUC4 expression attenuates T-cell-mediated response

Abstract Background: While T-cell-based immunotherapy emerged as a promising tool for cancer management, clinical trials and translational studies revealed that cancer cells develop resistance by modulating tumor microenvironment and cell-intrinsic mechanisms. Pancreatic cancer (PC), consisting of dense stroma, immunosuppressive environment, and aberrant mucin expression, has a dismal survival rate and responds poorly to immunotherapies. MUC4, a member of the mucin family, has been reported to block lymphokine-activated killer cells and induce apoptosis of cytotoxic T-cells, suggesting its possible role in immune modulation. However, how the T-cell secretome influences MUC4 expression in PC and its role in modulating the T-cell response is poorly investigated. Method: The scRNA-seq data analysis and primary T-cell conditioned media (CM) were utilized to investigate the MUC4 and T-cell crosstalk. T-cell CM-treated murine PC cell line (KCT-3266) was analyzed by RNA-seq analysis followed by MUC4 silencing studies to investigate its role in evading T-cell response. The subcutaneous murine model was utilized to investigate the influence of MUC4 KO on immune cells and cytotoxic cell infiltration. The RNA isolated from tumor tissues from KrasG12D/+, Trp53R127H/+, Pdx-1-Cre (KPC), and Muc4 knockout (KPMC) murine models were used for PanCancer immune profiling. Results: The scRNA-seq analysis suggests that intratumoral T-cells positively correlate with MUC4 expression in PC patients. The activated T-cell CM consisting of predominantly IL-2, IFN-γ, and TNF-α cytokines, significantly induce the expression of MUC4 transcriptionally and translationally in both human (SW1990 and COLO357) and murine (KCT-3248 and KCT3266) PC cell lines. RNA-seq analysis from T-cell CM-treated cancer cells revealed that T-cell secretome induced the pathways related to cancer cell death. The CRISPR knockout of MUC4 in PC cells showed increased expression of cleaved caspase-3 after T-cell CM treatment, indicating that MUC4 plays a protective role against the T-cell CM-mediated killing. Subcutaneous implantation of MUC4 proficient and deficient PC cells on the flanks of C57BL/6 immunocompetent mice demonstrated a significantly higher infiltration of CD3 positive and cytotoxic T-cell infiltration in the MUC4 deficient tumors, resulting in significantly lower tumor weight. The PanCancer immune profiling also showed that depletion of Muc4 increases the T-cells and cytotoxic T-cell signaling scores in the KPMC tumor tissues compared to KPC. Conclusion: T-cell secretome induces MUC4 expression in pancreatic cancer cells, and the increased MUC4 expression reduces T-cell infiltration and protects PC cells against T-cell-mediated killing. Citation Format: Xiaoqi Li, Imran Khan, Rachel Kehrberg, Zahraa Wajih Alsafwani, Rakesh Bhatia, Sushil Kumar, Surinder K. Batra. Pancreatic cancer and T-cell crosstalk-induced MUC4 expression attenuates T-cell-mediated response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1594.

Abstract 7527: Investigating biomarkers for immune-related adverse events following immune checkpoint inhibitor therapy in recurrent lung cancer patients

Abstract Background: The application of immune checkpoint inhibitors (ICIs) as adjuvant therapy post-lung cancer surgery is expected to confer significant benefits. However, the risk factors for severe immune-related adverse events (irAEs) remain incompletely understood, and managing irAEs may require long-term steroid use. The identification of biomarkers for irAEs in patients with surgically resected lung cancer is an urgent need. Methods: We analyzed 43 lung cancer patients who received ICI treatment for recurrence postoperatively. Macrodissection of lung cancer lesions was performed on FFPE specimens from resected lungs, from which total RNA was extracted. We conducted gene expression analysis of 770 immune-related genes using the PanCancer Immune Profiling Panel (NanoString Technologies, Inc., Seattle, WA, USA). The relationship between gene expression changes and irAEs or the efficacy of ICI treatment was examined. For validation, immunostaining of proteins strongly associated with irAEs was performed to determine their expression status in tissue specimens. Results: Among the 43 patients (30 males, 13 females, median age 68), 28 had adenocarcinoma, and 15 had squamous cell carcinoma. PD-L1 positivity was observed in 36 patients. The treatment regimens included pembrolizumab monotherapy for 18 patients and combination immunotherapy for 25 patients, with 25 patients responding to treatment and 7 developing irAEs. There was a marked difference in gene expression between adenocarcinoma and squamous cell carcinoma, with a notably higher number of tumor-infiltrating lymphocytes (TILs) in adenocarcinoma. An increase in TILs was observed in cases with irAEs across both cancer types. Additionally, an increased CD8/Treg ratio and CD8/exhausted T cell ratio were noted in adenocarcinomas with irAEs. A strong correlation between IRF5 gene expression and the occurrence of irAEs in adenocarcinoma was also confirmed by immunohistochemistry. Conclusion: We have identified tumor immune microenvironments correlated with the onset of irAEs following ICI treatment in patients with postoperative recurrence of lung cancer. This discovery could contribute to perioperative treatment strategies and improve our understanding of tumor immune mechanisms. Citation Format: Yujin Kudo, Jun Matsubayashi, Satoshi Takahashi, Masaru Hagiwara, Masatoshi Kakihana, Toshitaka Nagao, Tatsuo Ohira, Norihiko Ikeda. Investigating biomarkers for immune-related adverse events following immune checkpoint inhibitor therapy in recurrent lung cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7527.

Abstract 5261: Immunologic signatures of peripheral blood cells in patients with advanced pancreatic cancer under lenvatinib-pembrolizumab maintenance therapy

Abstract Background: The treatment of pancreatic cancer is limited by chemotherapy toxicities which impacts quality of life. This is a phase 2 clinical trial of lenvatinib and pembrolizumab as maintenance therapy in patients with advanced pancreatic cancer. We observed preclinical activity of this combination in pancreatic mouse models. We hypothesize that this combination may address unmet needs to minimize toxicities associated with standard treatments, stimulate anti-tumor T cell immunity, and improve the progression free and overall survival. Exploratory studies aim to characterize immune-related systemic changes in peripheral blood over the course of therapy and to identify global changes in expression of tumor-related genes in needle biopsy samples of liver metastatic lesions. Methods: Multiplex gene expression analysis of unsorted peripheral blood mononuclear cells (PBMC) was performed using PanCancer Immune Profiling Panel on NanoString nCounter platform. PBMC samples from pre- through week 32 of treatment were assessed for differential expression of 730 immune profiling genes defining T cell functions and associated immune categories. Results: PBMC immune profiling from 4/5 patients show a distinctive pattern of increased activation of T cells during the first 9 weeks of treatment followed by a sharp inhibition starting at weeks 9-12 after 3-4 cycles of treatment (CD3E, CD247, LCK, TBX1, TCF7, STAT4, IL2RG). Subsequent upregulation of macrophage-derived scavenger, inflammatory, and immunosuppressive gene expression patterns continued for the remainder of treatment (CD14, MSR1, MARCO, CD68, TGFB1, IL10RA). The median PFS of first 10 patients was 21.5 (9-32) weeks. Conclusions: Immunotherapy resistance may be related to rapidly developing terminal differentiation and unresponsiveness of T cells in parallel with upregulation of macrophage-derived inflammatory and immunosuppressive processes. Studies are on-going to prevent inhibition of the T-cells. Citation Format: Vincent Chung, Ferdynand Kos, Edwin Manual, Shannon Dempsey, Don J. Diamond. Immunologic signatures of peripheral blood cells in patients with advanced pancreatic cancer under lenvatinib-pembrolizumab maintenance therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5261.

Abstract 1526: The distinct tumor microenvironment of advanced hepatocellular carcinoma with high PVR or high PD-L1 expression

Abstract Background: The combination of anti-PD-L1 and anti-VEGF has been the standard-of-care for advanced hepatocellular carcinoma (HCC), while additionally targeting TIGIT is under active investigation. We aimed to explore the expression and clinical significance of PVR (CD155), the ligand of TIGIT, within the tumor microenvironment (TME) of advanced HCC. Methods: All HCC tumor specimens were obtained at the advanced stage of HCC. Immunohistochemistry (IHC) was employed to evaluate membranous staining of PVR and PD-L1 of tumor specimens, and semiquantitative scores with IHC 0, 1, 2, or 3 were given if < 1%, ≥1% but < 5%, ≥5% but < 10%, or ≥10% of tumor cells (TCs) and tumor-infiltrating immune cells (ICs) were PVR or PD-L1 positive. Selected tumor specimens underwent immune gene expression profiling using the nCounter Analysis System (NanoString Technologies, Inc.) with the PanCancer Immune Profiling Panel. Results: The study enrolled 67 advanced HCC patients, with median age of 59.2 years (male:female = 57:10). Forty-eight (71.6%) of the patients were hepatitis B virus (HBV) surface antigen positive, and 23 (34.3%) had received sorafenib before the study. Thirteen (19.4%) and 18 (26.9%) tumor tissues showed positive PVR expressions (IHC scores ≥1) in TCs and ICs, respectively; Seventeen (25.4%) and 36 (53.7%) tumor tissues showed positive PD-L1 expressions in TCs and ICs, respectively. The expressions of PVR in TCs or ICs showed no association with clinical factors, such as sex, age, HBV infection, sorafenib treatment, or overall survival. Interestingly, in both TCs and ICs, the intensities of PVR and PD-L1 expression were inversely correlated for those with at least IHC scores ≥1 in either PVR or PD-L1 (TCs, n = 26, r = -0.6966, p < 0.0001; ICs, n = 46, r = -0.4548, p = 0.0015). Immune profiling of the selected tumor specimens showed that those with high PVR but low PD-L1 expressions had significantly lower CD8 T cells scores, lower cytotoxic cells scores, and lower CD8/Treg ratio than those with high PD-L1 but low PVR expressions. Conclusions: There was an inverse correlation observed between expressions of PVR and PD-L1 in the advanced HCC tissues. The TME of those with high PVR expression was distinct from those with high PD-L1 expression. These findings may have important clinical implications for developing combination immunotherapy in advanced HCC. (This work is supported by grants from the Ministry of Science and Technology, Taiwan, MOST 110-2314-B-002-204-MY3). Citation Format: Li-Chun Lu, Yi-Hsuan Lee, Tsung-hao Liu, Yu-Yun Shao, Ann-Lii Cheng, Chih-Hung Hsu. The distinct tumor microenvironment of advanced hepatocellular carcinoma with high PVR or high PD-L1 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1526.

Abstract 6843: Comprehensive multi-omics approaches for identification of genetic alterations in head and neck squamous cell carcinoma

Abstract Background: Head and neck squamous cell carcinoma (HNSCC) is one of the most difficult and challenging malignancies, with a high level of heterogeneity and a wide range of treatment responses, regardless of the clinical stage. Oral squamous cell carcinoma (OSCC) is the most common head and neck cancer and accounts for over 90% of cancers that develop in the mucosal epithelium of the oral cavity. Additional carcinogenic risk factors, including tobacco, alcohol, and HPV infection are also associated with the pathogenesis of oral carcinogenesis. Unfortunately, today the failure of standard treatment modalities such as surgery, radiotherapy, and chemotherapy underscore the need for comprehensive genomic profiling to provide a better picture of the genetic alterations and gain an improved understanding of the complex signaling networks involved in the development of potential treatment resistance as well as the identification of novel biomarkers that discriminate between smokers and non-smokers. Methods: A retrospective analysis was performed on biopsied Formalin-Fixed Paraffin-Embedded (FFPE) samples from OSCC patients with and without a history of smoking and treated +/- chemotherapy. Patient samples representing matched normal tissue to serve as controls were compared to tumor squamous cell carcinoma samples and profiled using a single assay panel that includes 517 genes by both DNA and RNA NGS, microsatellite instability (MSI), and tumor mutational burden (TMB). Further analysis was also performed using the NanoString nCounter® PanCancer Immune Profiling Panel (PCIP), 770-comprehensive gene expression panel Results: The Neo ComprehensiveTM Solid Tumor pan cancer assay enabled simultaneously analysis of both DNA and RNA in one integrated workflow with an accompanying DragenTM bioinformatics analysis platform and provided comprehensive genomic profiling of sequence and structural variants, as well as genomic signatures such as TMB and MSI in OSCC patients. Utility of the NanoString PCIP highlighted immune-relevant gene expression changes within the different sample cohorts. The detailed molecular information highlighted by our study emphasizes the need for improved patient classification based on smoking status in the management of OSCC and also in establishing potential therapeutic options based on the many altered cell signaling pathways that we identified. By combining a multi-omics approach with expression data for immune-related genes and associated overall survival data, we provide an effective strategy and workflow for the identification of prognostic biomarkers in OSCC. Citation Format: Kirsteen Maclean, Qinqin Zha, Lakshmi Chandramohan, Anna Juncker-Jensen. Comprehensive multi-omics approaches for identification of genetic alterations in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6843.

Abstract B025: IL6 signaling in pre-malignant obese endometrium and endometrial cancer

Abstract Objective: Obesity associated chronic inflammatory status plays a role in the increased endometrial cancer incidences, IL-6 is one of those inflammatory molecules. To explore the potential of targeting pro-inflammation factors for endometrial cancer prevention, using multi-platform and multi-model analysis, we investigated IL6 and its associated signaling in pre-malignant endometrium and low-grade endometrial cancer. Methods: Illumina microarray and Nanostring nCounter® Pancancer Immune profiling panel gene expression profiling was performed on pre-malignant obese endometrium from diet induced obese Wistar rats, and endometrium from obese patients, lean endometrium was used as control. Clariom D assay was performed on RNA extracted from the gland and stroma of human obese endometrial carcinoma (OEC) and obese normal endometrium (ONE). IPA, nSolver, Transcriptome Analysis Console (TAC), and CIBERSORTx were used for data analysis. Ishikawa cells were 2D and 3D cultured, or 3D co-cultured with endometrial stroma cell line SHT290. Western blot was used for testing cell signal activity. Results: Pronounced IL-6 signaling (Z-score:2.3333, p=2.48E-02) was shown in pre-malignant obese endometrium of diet induced obese rats compared with lean littermates: NF-kB signaling (Z-score:2.714, p=3.47E-02), STAT3 signaling (Z-score:2.53, p=6.56E-04), and tumor microenvironment pathway (Z-score: 3.441, p=6.81E-05) were enhanced; PTEN signaling (Z-score:-1.997, p=2.76E-03) was attenuated. IL6R (FC=1.64, p=0.017), MMP2 (FC=1.938, p=0.026), IL1R2 (FC=1.75, p<0.0001), and CCL2 (FC=2.251, p=0.002) expression were increased. IL6 induced stronger STAT3 signaling, and weaker activation of MAPK signaling in Ishikawa cells 3D co-cultured with stroma cells, compared with 3D cultured Ishikawa cells without stroma cells. No IL-6 induced activation of MAPK signaling was observed with activation of Stat3 signaling in 2D-cultured Ishikawa cells. In pre-malignant obese endometrium from patients with high serum IL-6 levels, attenuated pro-inflammation signaling was seen compared with that from patients with low serum IL6 level: expression of IL1A (FC=-2.21, p=0.001), IRF8 (FC=-1.83, p=0.012), GAGE1 (FC=-3.49, p=0.028), and LBP (FC=-2.03, p=0.038) were decreased. Compared with pre-malignant obese endometrium, more regulatory T cell (Tregs, 0.1276 vs 0.077, p=0.04), and less eosinophils (0.003 vs 0.022, p=0.028) in the stroma, and more neutrophils (0.0145 vs. 0, p=0.02) in the tumor glands were observed in obese EC, implicating immuno-suppression in OEC. Conclusion: Due to the elevated systemic pro-inflammation status, pre-malignant obese endometrium demonstrated activated IL-6 signaling. IL-6 induced cell signaling varied by cellular microenvironment. In pre-malignant endometrium from obese women with high systemic IL-6 level, attenuated inflammation signaling was observed compared to those with low serum IL6 levels. Immuno-suppression was implicated in obese EC. Citation Format: Qian Zhang, Brenda Melendez, Xiaoping Su, Mikayla Bowen, Joseph Celestino, Melinda Sue Yates, Karen Lu. IL6 signaling in pre-malignant obese endometrium and endometrial cancer [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr B025.

Multi-omics Analysis Reveals Immune Features Associated with Immunotherapy Benefit in Patients with Squamous Cell Lung Cancer from Phase III Lung-MAP S1400I Trial.

Identifying molecular and immune features to guide immune checkpoint inhibitor (ICI)-based regimens remains an unmet clinical need. Tissue and longitudinal blood specimens from phase III trial S1400I in patients with metastatic squamous non-small cell carcinoma (SqNSCLC) treated with nivolumab monotherapy (nivo) or nivolumab plus ipilimumab (nivo+ipi) were subjected to multi-omics analyses including multiplex immunofluorescence (mIF), nCounter PanCancer Immune Profiling Panel, whole-exome sequencing, and Olink. Higher immune scores from immune gene expression profiling or immune cell infiltration by mIF were associated with response to ICIs and improved survival, except regulatory T cells, which were associated with worse overall survival (OS) for patients receiving nivo+ipi. Immune cell density and closer proximity of CD8+GZB+ T cells to malignant cells were associated with superior progression-free survival and OS. The cold immune landscape of NSCLC was associated with a higher level of chromosomal copy-number variation (CNV) burden. Patients with LRP1B-mutant tumors had a shorter survival than patients with LRP1B-wild-type tumors. Olink assays revealed soluble proteins such as LAMP3 increased in responders while IL6 and CXCL13 increased in nonresponders. Upregulation of serum CXCL13, MMP12, CSF-1, and IL8 were associated with worse survival before radiologic progression. The frequency, distribution, and clustering of immune cells relative to malignant ones can impact ICI efficacy in patients with SqNSCLC. High CNV burden may contribute to the cold immune microenvironment. Soluble inflammation/immune-related proteins in the blood have the potential to monitor therapeutic benefit from ICI treatment in patients with SqNSCLC.

Deciphering Transcriptomic and (Epi)Genetic Signatures of Central Nervous System Infiltration in Patients Diagnosed with Diffuse Large B-Cell Lymphoma

Introduction Central Nervous System (CNS) involvement in Diffuse Large B Cell Lymphoma (DLBCL) is a rare complication with bad prognosis. The CNS Prognostic Index is employed to categorize patients at a high risk of CNS relapse; however, its effectiveness is not accurate. The discovery of precise biomarkers for predicting CNS involvement in DLBCL patients remains unmet. Our objective was to characterize the immune, transcriptomic, genomic and epigenomic hallmarks of systemic tumors at diagnosis of patients who later experienced relapse in the CNS. By exploring these biological profiles, we sought to gain valuable insights into the underlying mechanisms and potential biomarkers associated with CNS relapse in DLBCL. Methods We conducted a retrospective study including 38 patients diagnosed with nodal DLBCL who experienced relapse within the first two years after diagnosis. Among these patients, 12 individuals relapsed specifically in the CNS, while 26 relapsed in systemic sites. As a control group, we included 21 patients who did not relapse. DNA and RNA were extracted from FFPE tumor samples collected at diagnosis. To evaluate immune cell infiltration, we utilized the nCounter PanCancer Immune Profiling Panel (Nanostring); cell-of-origin (COO) was determined using the Lymph2Cx assay. Transcriptomic profiles were generated by bulk RNA sequencing and mutational analysis was performed using a custom targeted NGS panel, which included the most frequently mutated genes observed in lymphoid neoplasms. Methylation profiles were obtained using the Illumina Infinium MethylationEPIC Array 850K. Results Immune profiling analysis showed cell composition was not significantly different between groups; however, we observed distinct immune gene expression patterns at diagnosis in patients who experienced CNS relapse. Multiple genes were overexpressed in the CNS-relapsed group compared to non-relapsed group, which encompassed various functional categories including chemokine signaling such as IL7R (FC=2; P=0.05) and CCR6 (FC=3.4; P=0.01); adhesion like VCAM1 (FC=1.6; P=0.03) and ITGA4 (FC=2.2; P=0.006); and immune regulation such as CD1A (FC=4.8; P=0.001), LTB (FC=3.3; P=0.009) and BTLA (FC=2.3; P=0.02) (Fig.1A). The ITGA4 gene also presented higher expression levels in the CNS-relapsed group when compared to both systemic-relapsed and non-relapsed groups combined (FC=1.7; P=0.03). No significant differences were observed in the COO classification between groups. Whole-genome transcriptomic data revealed enrichment in gene sets associated with proliferation and survival in the CNS-relapsed group (Fig.1B). Analysis of genetic alterations indicated that patients with CNS relapse exhibited an increased mutation load and frequency in PIM1, IGLL5, and KMT2D genes. Finally, analysis of the CNS relapse-related epigenome showed 343 differentially hypomethylated CpG sites compared to both systemic and non-relapsed groups together, affecting a total of 211 genes. Pathway enrichment analysis of these genes revealed a link between CNS infiltration and cell migration and locomotion. Conclusions Tumors from DLBCL patients that will relapse in the CNS present a higher chemokine, adhesion and proliferation signatures at diagnosis. Of notice, BTLA, CCR6 and IL7R proteins have been previously reported to be implicated in B cell entry into the CNS in B-cell malignancies. Interestingly, patients with CNS relapse demonstrated significant overexpression of the ITGA4 gene, which encodes the integrin α4 subunit (CD49d) of the Very Late Antigen-4. Engagement between CD49d on B cells and its endothelial ligand VCAM-1 is required for migration of these cells across the blood-brain barrier. In fact, Natalizumab (anti-CD49d mAb) is administered in multiple sclerosis to impede entrance and accumulation of B cells in the brain. Initial experiments in mice have shown increased expression of CD49d in lymphoma B cells that have infiltrated the brain, compared to those in the spleen. Herein, we have demonstrated that DLBCL cells at diagnosis already own specific transcriptomic and (epi)genetic hallmarks that could confer a higher capacity to infiltrate the CNS. Validation of these findings in larger cohorts of patients could improve selection of patients with an increased risk of CNS involvement in need of prophylactic CNS-directed therapies at diagnosis.

Immune-Related Gene Expression Signatures Identify a Patient's Subset Achieving Durable Complete Response to Brentuximab Vedotin in Relapsed/Refractory Hodgkin Lymphoma

Background: In classical Hodgkin lymphoma (cHL), 20%-30% of patients experience relapse or progressive disease after standard chemotherapy, requiring salvage treatment strategies including targeted immunotherapy with brentuximab vedotin (BV). Molecular mechanisms underlying treatment resistance to BV in cHL are still poorly understood. The unique characteristics of cHL microenvironment, which represents >90% of the cellular infiltrate, make targeted gene expression profiling (T-GEP) a suitable technique to investigate immune-related determinants of BV sensitivity. Methods: 31 consecutive patients (pts) affected by relapsed-refractory (R/R) cHL treated at 2 italian Institutions from 2013 to 2018 with available formalin-fixed paraffin embedded (FFPE) tissue collected right before the start of BV therapy, were included in this retrospective study. FFPE tissue was profiled by T-GEP on the Nanostring platform, using the PanCancer Immune Profiling panel, encompassing 730 genes regulating the most relevant immunologic pathways. Disease response after BV treatment was evaluated by FDG-PET after 4, 8, 12 and 16 cycles. Results: Median age of the whole cohort was 38 years (y) (range 18-76). 18 pts (58%) were male. Median number of BV cycles was 8 (range 2-16). After a median follow-up of 3.5 y from the start of Brentuximab treatment, the overall survival (OS) and progression-free survival (PFS) rates were 87% and 32% respectively. Pts were classified as BV-responders in case of complete metabolic response (CMR) lasting more than 6 months (m); pts achieving less than CMR or short lasting (< 6 m) CMR were classified as non-responders (NR). By applying these criteria, 12 pts (39%) were classified as responders and 19 as non-responders. Differential expression (DE) analyses were performed and differentially expressed (DE) genes were selected by applying two statistical filters: (log2FC > 1 and p-value < 0.05). 61 genes were differentially expressed between responders and non-responders. The 61-gene signature included genes involved in JAK-STAT signaling, IL-17 signaling and cytokine-cytokine receptor interaction. Unsupervised clustering analyses identified two different pts clusters with clearly different BV-sensitivity profiles: cluster1 (22 pts) and cluster2 (9 pts). Among the 22 pts of cluster 1, only 4 pts were responders while 18 were non-responders. As opposite, 8 of 9 patients of Cluster2 were responders, while only 1 was non-responder (p=0.0005). In line with this, 4-y PFS rate of the 22 pts included in cluster1 was 19%, while the 4-y PFS rate of the 9 pts included in cluster2 was 66% (p=0.007). Median PFS of Cluster1 was only 5.7 months. The composition of the two clusters was not significantly different in terms of number of prior therapies and disease stage at the time of BV treatment. After achieving a CMR 6 of 9 pts were consolidated with autologous stem cell transplantation (ASCT) in cluster2. In cluster1, 1 patient was consolidated with ASCT after CMR, and 2 pts received allogeneic stem cell transplantation (allo-SCT) after BV-failure. Conclusions: With the limitation of the small sample size, these data suggest that the composition of cHL microenvironment could influence the activity of single agent BV. Pre-treatment microenvironment profiling with T-GEP could be a useful tool for selecting patients who may derive maximal benefit from BV in cHL.

Tumor cell cytoplasmic metallothionein expression associates with differential tumor immunogenicity and prognostic outcome in high-grade serous ovarian carcinoma.

The underlying mechanism of high T-cell presence as a favorable prognostic factor in high-grade serous ovarian carcinoma (HGSOC) is not yet understood. In addition to immune cells, various cofactors are essential for immune processes. One of those are metallothioneins (MTs), metal-binding proteins comprising various isoforms. MTs play a role in tumor development and drug resistance. Moreover, MTs influence inflammatory processes by regulating zinc homeostasis. In particular, T-cell function and polarization are particularly susceptible to changes in zinc status. The aim of the present study was to investigate a possible role of MT-mediated immune response and its association with prognostic outcome in ovarian cancer. A retrospective study was conducted on a clinically well-characterized cohort of 24 patients with HGSOC treated at the University Hospital of Essen. Gene expression patterns for anti-cancer immunogenicity-related targets were performed using the NanoString nCounter platform for digital gene expression analysis with the appurtenant PanCancer Immune Profiling panel, consisting of 770 targets and 30 reference genes. Tumor-associated immunohistochemical MT protein expression was evaluated using a semi-quantitative four-tier Immunohistochemistry (IHC) scoring. MT immunoexpression was detected in 43% (10/23) of all HGSOC samples. MT immunoexpression levels showed a significant association to survival, leading to prolonged progression-free and overall survival in positively stained tumors. Furthermore, T-cell receptor signaling gene signature showed a strong activation in MT-positive tumors. Activated downstream signaling cascades resulting in elevated interferon-gamma expression with a shift in the balance between T helper cells (TH1 and TH2) could be observed in the MT-positive subgroup. In addition, a higher expression pattern of perforin and several granzymes could be detected, overall suggestive of acute, targeted anti-cancer immune response in MT-positive samples. This is the first study combining broad, digital mRNA screening of anti-tumor immune response-associated genes and their relation to MT-I/II in ovarian cancer. MT overexpression is associated with molecular characteristics of an anti-cancer immune response and is a strong prognostic marker in ovarian HGSOC. The observed immune cell activation associated with tumor MT expression comprises but is not limited to T cells and natural killer cells.

Exploring Psoriasis Inflammatory Microenvironment by NanoString Technologies.

Psoriasis is a chronic inflammatory skin disease whose molecular mechanisms and microenvironment are poorly understood. We performed gene expression analysis through the nCounter® PanCancer Immune Profiling Panel (NanoString Technologies, Seattle, WA, USA) on 22 FFPE punch biopsies from 19 psoriasis-affected patients. A subset of five cases was analyzed before (T0) and after 6 months (T6) of treatment with dimethyl fumarate (DMF) to address immune microenvironment changes. Molecular comparisons according to biopsy site and age of onset showed a different distribution of innate immune cells (mast cells, macrophages, NK cells, and DC cells) and pathways (complement regulation and transporter functions). The analysis according to PASI (Psoriasis Area and Severity Index) led to non-significant results, suggesting no link between molecular expression profile and clinical amount of skin disease. In DMF-treated patients, we observed a strong immunomodulatory effect after treatment: A subversion of exhausted CD8 T cells, NK CD56dim cells, Tregs, neutrophils, CD45+ cells, T cells, B cells, and macrophages was reported between the two analyzed time-points, as well as the reduction in pro-inflammatory pathways and molecules, including cytotoxicity, pathogen defense, antigen processing, adhesion, cell cycle, chemokines, cytokines, and interleukins. The inflammatory psoriatic microenvironment can be modulated using DMF with encouraging results, achieving an immune-tolerant and non-inflammatory condition through the regulation of both innate and adaptive immunity.

Immunologic constant of rejection as a predictive biomarker of immune checkpoint inhibitors efficacy in non-small cell lung cancer

BackgroundAnti-PD1/PDL1 immune checkpoint inhibitors (ICI) transformed the prognosis of patients with advanced non-small cell lung cancer (NSCLC). However, the response rate remains disappointing and toxicity may be life-threatening, making urgent identification of biomarkers predictive for efficacy. Immunologic Constant of Rejection signature (ICR) is a 20-gene expression signature of cytotoxic immune response with prognostic value in some solid cancers. Our objective was to assess its predictive value for benefit from anti-PD1/PDL1 in patients with advanced NSCLC.MethodsWe retrospectively profiled 44 primary tumors derived from NSCLC patients treated with ICI as single-agent in at least the second-line metastatic setting. Transcriptomic analysis was performed using the nCounter® analysis system and the PanCancer Immune Profiling Panel. We then pooled our data with clinico-biological data from four public gene expression data sets, leading to a total of 162 NSCLC patients treated with single-agent anti-PD1/PDL1. ICR was applied to all samples and correlation was searched between ICR classes and the Durable Clinical Benefit (DCB), defined as stable disease or objective response according to RECIST 1.1 for a minimum of 6 months after the start of ICI.ResultsThe DCB rate was 29%; 22% of samples were classified as ICR1, 30% ICR2, 22% ICR3, and 26% ICR4. These classes were not associated with the clinico-pathological variables, but showed enrichment from ICR1 to ICR4 in quantitative/qualitative markers of immune response. ICR2-4 class was associated with a 5.65-fold DCB rate when compared with ICR1 class. In multivariate analysis, ICR classification remained associated with DCB, independently from PDL1 expression and other predictive immune signatures. By contrast, it was not associated with disease-free survival in 556 NSCLC TCGA patients untreated with ICI.ConclusionThe 20-gene ICR signature was independently associated with benefit from anti-PD1/PDL1 ICI in patients with advanced NSCLC. Validation in larger retrospective and prospective series is warranted.

A Microenvironment-Related Nine-Gene Signature May Predict Survival in Mycosis Fungoides Patients at Diagnosis.

Mycosis fungoides (MF) is the most common cutaneous lymphoma characterized by an indolent course. Prognosis is stage-based but this approach does not reflect the different outcomes within stages. Considering that tumor microenvironment is known to be involved in MF pathogenesis and progression, we decided to investigate 99 MF cases by using the PanCancer Immune Profiling Panel. We identified and validated a signature of 9 genes able to predict MF survival and distinguish a high-risk group with a worse outcome from a low-risk group of cases with a better outcome. At the molecular level, low-risk vs. high-risk cases reported a global upregulation of immune genes, enriched in cytokines, and a higher density of dendritic cells and mast cells, possibly associated with a more favorable clinical course.

17th International Conference on Malignant Lymphoma, Palazzo dei Congressi, Lugano, Switzerland, 13 - 17 June, 2023.

Introduction: The Nordic Lymphoma Group (NLG) performed a dose-finding/expansion trial evaluating pixantrone, etoposide, bendamustine and, in CD20+ lymphomas, rituximab (P[R]EBEN) in patients (pts) with relapsed diffuse large B-cell (DLBCL) or peripheral T-cell (PTCL) lymphomas. The regimen was out-patient based, and applicable in frail, heavily pre-treated pts. Here we present a long-term follow-up of the trial and a per-protocol correlative biological analysis of pre-therapeutic tumour biopsies looking for gene expression profiles associated with long-term response. Methods: We enrolled 60 pts (37 DLBCL and 23 PTCL; age range: 39–84 yrs, median: 71 yrs). Time to event parameters were analyzed by Kaplan-Meier estimates, log-rank test and Cox regression models. Pre-therapeutic biopsies from 42 pts (25 DLBCL, 17 PTCL) were analyzed for gene expression by NanoString PanCancer Pathways and Immune profiling panels (altogether 1348 genes). Results: Of the original 60 patients, 22 were alive at the time of the present analysis. The median follow-up of surviving pts was 41 mo (range 26–76 mo). The 38 deaths were due to: lymphoma (24; 63%), infections (3; 8%) and other causes (11; 29%: 1 lung carcinoma, 5 acute myeloid leukemia-AML, 1 myelodysplasia-MDS, 1 lung embolism, 1 allotransplant related and 2 unknown). Of the 6 pts with AML/MDS, 5 were PTCL and 1 DLBCL. Of 58 evaluable pts, 38 (65%) had a complete (CR) and 1 (3%) a partial response (B: 51%; T: 70%). The median duration of response (DoR) of the 38 CR pts was 17 mo (range 0.5–55 mo; B: 18 mo; T: 17 mo). Four pts were bridged to allogeneic transplant. The overall 5-yr OS and PFS were 33% and 19%, respectively. The median OS for DLBCL was 16.0 mo [IQR 7.0>] and for PTCL 18.0 mo [IQR 8.0>]. The median PFS for DLBCL was 10.0 mo [IQR 4.0–32.0] and for PTCL 10.0 mo [IQR 5.0–26.0]. To better understand molecular alterations underlying the delay of relapse in this heavily pre-treated elderly population, we determined differentially expressed genes between short (<12 mo) and long term (>12 mo) responders. In DLBCL, 31 genes showed significant differential expression between short- and long-term responders (P < 0.05). The latter had higher expression of genes related to transcription factor activity. The gene most significantly associated with long-term response was Neutrophil Cytosolic Factor 4 (NCF4). In PTCL, 23 genes were differentially expressed between short- and long-term responders, the latter showing enrichment in Ras signaling pathway genes (e.g., Rac Family Small GTPase 3, RAC3, and phospholipase A2 group IIA, PLA2G2A). The research was funded by: Servier Laboratoires Keywords: Aggressive B-cell non-Hodgkin lymphoma, Aggressive T-cell non-Hodgkin lymphoma, Combination Therapies No conflicts of interests pertinent to the abstract.

Immunologic constant of rejection as a predictive biomarker of immune checkpoint inhibitors efficacy in non-small cell lung cancer.

e21162 Background: Immune checkpoint inhibitors (ICI) transformed non-small cell lung cancer (NSCLC) prognosis but response rate remains disappointing. Besides PDL1 expression, tumor mutational burden, quantification of tumor infiltrating lymphocytes or methylation profile failed to confirm their predictive role. The ICR (Immunologic Constant of Rejection) signature is defined as an immune phenotype quantifying the expression of 20 genes all involved in cancer immunity. Our first objective was the assessment of ICR signature predictive value and our secondary objectives were to compare ICR signature with other published immune signatures and to assess the prognostic value of ICR signature. Methods: Our series included 77 paraffin-embedded (FFPE) tumors derived from NSCLC patients treated with ICI as a single agent in at least the second line setting. We also collected clinical and biological data from 4 public data sets to expand and confront our local data. Transcriptomic analysis was performed using nCountertechnology Dx analysis by Nanostring based on microscopic imaging counting relative abundance of hundreds of transcripts using the “nCounter PanCancer Immune Profiling Panel” including the 20 ICR genes. Results: We applied the ICR classification to a series of 44 local tumor samples enhanced with 118 samples from public datasets. Hierarchical clustering on both local cohort and public datasets allowed us to distinguish the four ICR classes, from ICR 1 (“cold” tumors) to ICR 4 (“hot” tumors). Among the 5 datasets, we did not observe any significant difference between ICR groups for age, sex and smoking status. We observed a relative homogeneity in Durable Clinical Benefit (DCB) rate among ICR groups 2 to 4 (median DCB above 20%) unlike ICR group 1 (median DCB under 10%) leading us to consider ICR as a binary signature (ICR 1 vs ICR 2-4). ICR signature indeed showed a significant association with DCB among the 5 pooled datasets (OR = 4.40 [95% Confidence Interval 1.44-13.45] p = 0.950) with uniform distribution between them. Then, we conducted a logistic regression univariate analysis of DCB among various previously published immune signatures and ICR signature was the only one to show a significant association with DCB in this analysis ( p = 0.007). Conclusions: We identified a feasible transcriptomic signature with a promising signal of ICI efficacy prediction in the so far disappointing immune biomarkers field. In a near future, predict ICI efficacy will be a major challenge with three pivotal questions. When, balanced with the new data on ICI use in early-stage lung cancer or widely pretreated patients. How, assessing various techniques to develop composite signatures to better consider every driver of immune response. What, considering the amount of potential new therapeutic targets, starting by the 20 genes of ICR signature.