Profiling Panel Research Articles

TWEAK and TNFɑ Pro-inflammatory Soluble Cytokines and their Specific Autoantibodies Secretion in Multiple Sclerosis Patients.

Multiple sclerosis (MS) is a complex, chronic inflammatory disease of the central nervous system, where immune dysregulation plays a critical role. We sought to explore the modulation of the pro-inflammatory cytokines tumor necrosis factor-alpha (TNFɑ) and TNF-like weak inducer of apoptosis (TWEAK), along with their respective autoantibodies, TNAb and TWAb, and to decipher potential associations between these and clinical characteristics which could assist personalized therapy in MS. We also assessed the complementarity to leading candidate biomarkers in MS patient monitoring, namely, glial fibrillary acidic protein (GFAP) and neurofilament light chain (NfL). Serum levels of these cytokines and their autoantibodies were measured in 150 MS patients and 186 healthy controls (HC) by ELISA. We found that sTWEAK levels were significantly higher, while sTNFɑ levels were lower in MS patients compared to HC. Additionally, we detected TWAb in 10% of MS patients, a prevalence significantly higher than in HC (4%) and TNAb in only one patient. TWAb-positive patients were significantly younger, had lower EDSS scores, a shorter disease duration, and predominantly presented with the relapsing-remitting form of MS. Together, these results provide new actors to be considered in the development of a biomarker profiling panel to be used in MS patient management. Further research is warranted to elucidate the clinical significance of these autoantibodies and their role in MS neuroinflammatory modulation.

Gene Expression Profiling of the Peritumoral Immune Cell Infiltrate of Penile Squamous Cell Carcinomas.

Penile carcinomas are rare tumors in Europe and need further investigations due to their inferior prognosis in late tumor stages. The presence of disparate immune cell infiltrates was observed in these tumors, which were subsequently demonstrated to give rise to divergent tumor prognoses. The objective was to further characterize this immune cell infiltrate with the use of immunohistochemistry and RNA expression. A total of twelve well-characterized cases of penile squamous cell carcinomas with known infection status by human papillomavirus (HPV) and p16 status were assessed. The cases were classified according to their morphological characteristics, including those exhibiting a pronounced peritumoral immune cell infiltrate and those with less peritumoral immune cell infiltration. The generation of RNA expression data was conducted using the nCounter® PanCancer Immune Profiling Panel. Computational models were employed to calculate the proportions of immune cells. To corroborate the findings, an immunohistochemical analysis was conducted using antibodies against CD20, CD3, CD4, CD8, MUM1, CD68, and CD117. Our cases were clustered according to the immune cell infiltrate detected via histology in a group with less immune cell infiltrate density and in a group with increased immune cell infiltrate density. Generally, all immune cells showed an increased amount in the group with pronounced immune cell infiltrate density. The clusters were found to relate to cell functions, the complement system, cytotoxicity, pathogen defense, regulation, and T-cell functions. In cases exhibiting a pronounced immune cell infiltrate, the top three genes that exhibited the greatest upregulation were GZMA, MICB, and GNLY. No relationship to HPV infection status was demonstrated. Immunohistochemistry validated the data gained via RNA expression and showed a correlation with EPIC and Cibersort. The clustering of cases based on immune cell infiltrate density revealed significant distinctions between groups with lower and higher immune cell infiltrate density. The group with increased immune cel infiltrate density showed a greater abundance of immune cells, aligning with key functions like cytotoxicity, pathogen defense, and T-cell regulation. Among these cases, the genes GZMA, MICB, and GNLY were significantly upregulated, suggesting their involvement in an increased immune response. The role of HPV infection status in our cases with regard to the peritumoral immune cell infiltrate remains inconclusive.

Widespread Adoption of Precision Anticancer Therapies After Implementation of Pathologist-Directed Comprehensive Genomic Profiling Across a Large US Health System.

Precision therapies and immunotherapies have revolutionized cancer care, with novel genomic biomarker-associated therapies being introduced into clinical practice rapidly, resulting in notable gains in patient survival. Despite this, there is significant variability in the utilization of tumor molecular profiling that spans the timing of test ordering, comprehensiveness of gene panels, and clinical decision support through therapy and trial recommendations. To standardize testing, we designed a pathologist-directed test ordering system at the time of diagnosis using a 523-gene DNA/RNA hybrid comprehensive genomic profiling (CGP) panel and extensive clinical decision support tools. To comprehensively characterize the clinical impact of this protocol, we developed a novel natural language processing (NLP)-based approach to extract clinical features from physician chart notes. We assessed test actionability rates, therapy choice, and outcomes across a set of 3,216 patients with advanced cancer. We observed 49% of patients had at least one actionable genomic biomarker-driven-approved and/or guideline-recommended targeted or immunotherapy (IO) and 53% of patients would have been eligible for a precision therapy clinical trial from three large basket trials. When assessing CGP versus an in silico 50-gene panel, 67% of tumors compared with 33% harbored actionable alterations including clinical trials. Among patients with 6 months or more of follow-up, over 52% received a targeted therapy (TT) or IO, versus 32% who received conventional chemotherapy alone. Furthermore, patients receiving TT had significantly improved overall survival compared with patients receiving chemotherapy alone (P < .001). Overall, these data represent a major shift in standard clinical practice toward molecularly guided treatments (targeted and immunotherapies) over conventional systemic chemotherapy. As guidelines continue to evolve and more precision therapeutics gain approval, we expect this gap to continue to widen.

In vitro pharmacologic profiling aids systemic toxicity assessment of chemicals

An adapted in vitro pharmacology profiling panel (APPP) was developed that included targets used in the pharmaceutical industry alongside additional targets whose cellular functions have been linked to systemic toxicities. This panel of 83 target assays was used to profile the activities of 129 cosmetic relevant chemicals with diverse chemical structures, physiochemical properties and cosmetic use types. Internal data consistency was proved robust, as evidenced by the reproducibility between single concentration and concentration-response data and showed good concordance with data reported in the ToxCast and drug excipient datasets. We discuss how the data can be analyzed and multiple potential contexts of use illustrated by case studies, alongside other new approach methodologies, to support cosmetic chemical risk assessments that do not require data from new animal studies.

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...

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.

Implementation of a comprehensive genomic profiling (CGP) panel for precision oncology at a cancer center in Brazil.

e23520 Background: Assessing molecular alterations in solid tumors is essential for providing the best care in precision oncology. CGP tests utilize next-generation sequencing (NGS) to detect clinically relevant alterations and molecular signatures that are increasingly utilized in cancer careand can be performed in-house, which decreases test turnaround time (TAT) and costs. A paucity of studies have documented the experience of in-house testing at academic centers in Brazil. Methods: The TruSight Oncology (TSO) 500 assay/TSO 500 HRD are research use only (RUO) CGP assays that were validated for the detection of small variants (SVs), copy number variants (CNVs), microsatellite instability (MSI), tumor mutation burden (TMB) and genomic instability score (GIS) in DNA and gene fusions/splice variants in RNA in the NextSeq 500 system. Secondary analysis was performed with Illumina Connected Analytics. Tertiary analysis was performed with Illumina Connected Insights (ICI) and Sophia DDM. TMB ≥ 10mut/Mb was defined as TMB-High and GIS ≥ 42 was homologous recombination deficient (HRD). Results: We performed CGP on 40 FFPE samples (16 with HRD) with variable quality and block age most of which had known alterations detected by other genomics/molecular tests: 17 TNBC (16 paired DNA and RNA, 1 DNA only), 18 lung cancer (13 paired DNA and RNA, 5 DNA only), 1 endometrium cancer (DNA only, POLE mutated, 4 replicates), 1 colorectal cancer (DNA only, MSI unstable) and 11 sarcomas (RNA only). 84%, 84%, 90% and 89% of samples with DNA integrity number (DIN) &gt; 2.5 were qualified for analysis of SVs/TMB, MSI, CNVs, and GIS, respectively, while for samples with DIN &lt; 2.5 these values felt to 47%, 74%, 68% and 43%, respectively. RNA isolated from blocks dated up to 5 y.o. were qualified in 90% of cases while from older blocks only 6%. The 4 replicate (single sample) analysis showed 82% and 83% overall variant concordance in the ICI and Sophia DDM software, respectively. All previously known oncogenic alterations (SVs ≥5% VAF, fusions/splice, MSI, TMB) were detected (qualified samples) and with similar performance in ICI and Sophia, respectively: concordance 0.91/p &lt; 0.0001 vs 0.94/p &lt; 0.0001; sensitivity 100% vs 100%; specificity 98% vs 100%. TMB had 100% concordance with previous TMB results. Of note, 73% (8/11) TNBC samples analyzed for GIS were HRD and 75% (6/8) explained by BRCA1 germline mutation (25%) and BRCA1or RAD51C somatic promoter methylation (75%). Conclusions: This study demonstrates the utility of CGP and HRD testing in identifying multiple molecular alterations in FFPE tissue samples with high sensitivity and specificity. Pre-analytical variables directly impact data generation. BRCA1/RAD51C promoter methylation showed to be a potential biomarker for selecting metastatic TNBC patients for PARP inhibitor therapies. Offering CGP can potentially bring precision oncology to more patients with cancer in Brazil.

Use of Immune Profiling Panel to assess the immune response of septic patients for prediction of worsening as a composite endpoint

Sepsis induces intense, dynamic and heterogeneous host response modulations. Despite improvement of patient management, the risk of mortality and healthcare-associated infections remains high. Treatments to counterbalance immune response are under evaluation, but effective biomarkers are still lacking to perform patient stratification. The design of the present study was defined to alleviate the limitations of existing literature: we selected patients who survived the initial hyperinflammatory response and are still hospitalized at day 5–7 after ICU admission. Using the Immune Profiling Panel (IPP), a fully automated RT-qPCR multiplex prototype, we optimized a machine learning model combining the IPP gene expression levels for the identification of patients at high risk of worsening, a composite endpoint defined as death or secondary infection, within one week after sampling. This was done on 332 sepsis patients selected from two retrospective studies. The IPP model identified a high-risk group comprising 30% of patients, with a significant increased proportion of worsening events at day 28 compared to the low-risk group (49% vs. 28%, respectively). These preliminary results underline the potential clinical application of IPP for sepsis patient stratification in a personalized medicine perspective, that will be confirmed in a larger prospective multicenter study.

Real-World Impact of Comprehensive Genomic Profiling on Biomarker Detection, Receipt of Therapy, and Clinical Outcomes in Advanced Non-Small Cell Lung Cancer.

Therapeutic decision making for patients with advanced non-small cell lung cancer (aNSCLC) includes a growing number of options for genomic, biomarker-guided, targeted therapies. We compared actionable biomarker detection, targeted therapy receipt, and real-world overall survival (rwOS) in patients with aNSCLC tested with comprehensive genomic profiling (CGP) versus small panel testing (SP) in real-world community health systems. Patients older than 18 years diagnosed with aNSCLC between January 1, 2015, and December 31, 2020, who received biomarker testing were followed until death or study end (September 30, 2021), and categorized by most comprehensive testing during follow-up: SP (≤52 genes) or CGP (>52 genes). Among 3,884 patients (median age, 68 years; 50% female; 73% non-Hispanic White), 20% received CGP and 80% SP. The proportion of patients with ≥one actionable biomarker (actionability) was significantly higher in CGP than in SP (32% v 14%; P < .001). Of patients with actionability, 43% (CGP) and 38% (SP) received matched therapies (P = .20). Among treated patients, CGP before first-line treatment was associated with higher likelihood of matched therapy in any line (odds ratio, 3.2 [95% CI, 1.84 to 5.53]). CGP testing (hazard ratio [HR], 0.80 [95% CI, 0.72 to 0.89]) and actionability (HR, 0.84 [95% CI, 0.77 to 0.91]) were associated with reduced risk of mortality. Among treated patients with actionability, matched therapy receipt showed improved median rwOS in months in CGP (34 [95% CI, 21 to 49] matched v 14 [95% CI, 10 to 18] unmatched) and SP (27 [95% CI, 21 to 43] matched v 10 [95% CI, 8 to 14] unmatched). Patients who received CGP had improved detection of actionable biomarkers and greater use of matched therapies, both of which were associated with significant increases in survival.

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.

A comprehensive analytical validation framework for immunofluorescence assay design using CellScape precise spatial multiplexing

Abstract Multiplex immunofluorescence (mIF) is a powerful immunology research tool that enables deep phenotyping of cells within the tissue microenvironment. Careful optimization of multiplex antibody panels for specificity, sensitivity, and reproducibility, is crucial to achieving robust data and is a requirement for assay repeatability and standardization. Here, we describe our validation procedure for multiplex assay panels. Experiments were performed using the CellScape™ platform for precise spatial multiplexing. To design a ready-to-use panel for immune cell phenotyping, individual antibodies were first screened for specificity and then optimized for labeling sensitivity. Signal-to-noise, inter-assay reproducibility, and intra-assay precision were evaluated in a multi-stage testing matrix to validate each antibody in isolation and assembled into a panel. Measurements of both signal and quantitative immune phenotyping across technical replicates showed a high degree of intra-assay precision and inter-assay reproducibility. Pairing the CellScape platform with comprehensively validated antibody panels streamlined deep immune profiling. The 15-plex, ready-to-use VistaPlex™ immune profiling panel for human FFPE tissues was validated on human tonsil, lung, breast, and colon tissues demonstrating the ability to positively identify epithelial cells and 22 cellular immune phenotypes most critical to understanding tumor immune responses.

Abstract 1539: A comprehensive analytical validation framework for antibodies used in the CellScape multiplex immunofluorescence assay

Abstract Background: Multiplex immunofluorescence (mIF) is a powerful tool that enables deep phenotyping of cells within the tumor microenvironment (TME). Careful optimization of multiplex antibody panels for specificity, sensitivity and reproducibility is crucial to achieving robust data and is a precondition for assay repeatability and standardization. We describe our validation procedure for VistaPlex™ multiplex assay panels. Methods: Experiments were performed using the CellScape™ platform for precise spatial multiplexing. The platform enables automated cyclic mIF for readily available fluorophore-conjugated antibodies compatible with fresh frozen or FFPE tissues. The use of high dynamic range imaging further facilitates quantitative phenotyping, while excellent imaging resolution provides single-cell discrimination with high accuracy. To design a ready-to-use panel for immune cell phenotyping, individual antibodies were first screened for specificity and then optimized for labeling sensitivity. Signal-to-noise, inter-assay reproducibility, and intra-assay precision were evaluated across serial sections in a multi-stage testing matrix to validate each antibody in isolation and assembled into a panel. Resulting from this effort, we introduce a 15-plex ready-to-use VistaPlex immune profiling panel for human FFPE tissues. Results: Image analysis of signal-to-noise ratios demonstrated robust performance of multiplexed antibodies across multiple tissue sections and tissue types. Measurements of both signal and quantitative immune phenotyping across technical replicates showed a high degree of intra-assay precision and inter-assay reproducibility. The 15-plex panel was validated on human tonsil, lung, breast, and colon tissues. Conclusions: The CellScape platform enables simultaneous detection of multiple protein biomarkers on a single tissue section for deep immune cell profiling in the TME. Rigorously validated VistaPlex panels further streamline this spatial biology workflow and will aid in the generation of consistent and reliable results for end users. Citation Format: Charles E. Jackson, Arne Christians, Jannik Boog, Matthew H. Ingalls, Xenia Meshik, Adam Northcutt, Kayla E. Cashion, J Spencer Schwarz, Oliver Braubach. A comprehensive analytical validation framework for antibodies used in the CellScape multiplex immunofluorescence assay [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 1539.

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 &amp;lt; 1%, ≥1% but &amp;lt; 5%, ≥5% but &amp;lt; 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 &amp;lt; 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 2291: Bravo automation of Agilent Avida targeted enrichment for high-throughput detection of genomic alteration and DNA methylation

Abstract Background: Current genomic and epigenomic profiling of cancer tissue DNA or cfDNA (cell-free DNA) in liquid biopsy relies upon separate, time- and sample-consuming technologies for somatic variant detection or methylation analysis. Here we describe workflow and performance of the Agilent Bravo automated liquid handling platform with the Agilent Avida targeted enrichment solution for next generation sequencing of somatic variants and methylation profiling. This solution can effectively analyze low-input tumor DNA or cfDNA samples. The Avida Duo workflow enables highly sensitive detection of single nucleotide variant (SNV), insertions and deletions (INDEL), copy number variation (CNV) and DNA methylation profiles from a single sample, without any sample splitting. Methods and Results: Panels, reagents, and automated workflows for Avida DNA, Avida Methyl, and Avida Duo Methyl (combined DNA &amp; methylation) kits were developed to accommodate up to 96 samples on the Bravo NGS workstation. The automated solution supports independent single-day workflows for somatic variants or methylation sample preparation. The Avida Duo analysis combines both workflows without sample splitting, streamlining the process and reducing sample consumption. Leveraging a focused cancer hotspot panel, we demonstrate excellent reproducibility and low allele frequency (SNV at sub 1%) variant detection in cfDNA samples and reference standards with as little as 10ng DNA input. We exhibit similar performance with a larger ~300 kb target region tumor profiling panel. Finally, we demonstrate DNA methylation detection in the standalone or combined (“Duo”) workflows across differentially methylated regions (DMRs) with a panel of ~3400 targets. Conclusions: Automation of the Avida targeted enrichment solution with the Bravo NGS workstation enables a sensitive, high-throughput, end-to-end analysis for detection of genomic alterations and DNA methylation changes from a single low-input cfDNA or tumor DNA sample. Citation Format: Ashraf Wahba, Tony Ho, Sarah Johns, Aswati Aravind, Heng Wang, Neelima V. Mehendale, Gilbert P. Amparo, Khine Win, Manuel Gomez, Grace Zhao, Douglas N. Roberts. Bravo automation of Agilent Avida targeted enrichment for high-throughput detection of genomic alteration and DNA methylation [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 2291.

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 3904: Multiplexed flow cytometry based immunophenotyping paired with functional ex vivo (MFLEX) drug profiling informs potential efficacy of therapeutic agents in acute myeloid leukemia

Abstract Introduction: Acute Myeloid Leukemia (AML) is a heterogenous hematological malignancy with a complex clonal architecture that requires in-depth immunophenotyping for disease characterization. Conventional AML ex vivo models for preclinical drug screening lack functional readouts that provide single cell level data to identify drug activity in defined AML subsets with unique immune signatures. Here, we present a novel platform that links Multiparameter Flow cytometry with high-throughput EX vivo drug screening (MFLEX) in AML patient-derived cell models to simultaneously detect phenotypic changes and predict cell-subset specific drug responses in AML. Method: We developed a 21-color high-dimensional immune profiling panel that captures AML heterogeneity by delineating primitive and mature AML blasts along with normal hematopoietic lineages. The panel also includes markers for functional measurement of apoptosis, proliferation, differentiation, and expression of BCL2 family proteins in lymphoid and myeloid cell subsets. Ex vivo culture conditions were optimized using physiologically relevant stroma-free cytokine-rich media that supports primary AML cells with minimal phenotypic shift, allowing for high throughput functional drug sensitivity testing. Timepoints and dosing conditions for AML standard of care (SoC) agents were optimized to support combination screens. Multi-parameter datasets were analysed for functional differences and visualized using dimension reduction methods. Results: We tested MFLEX on clinically annotated AML samples to understand sensitivity profiles to venetoclax and combination partners: 5-azacytidine, cytarabine, fludarabine and gilteritinib. We identified distinct drug sensitivity profiles for venetoclax in AML patients with varying leukemic differentiation states where M0/M1 subtypes were more sensitive to therapy than M4/M5. We also observed cell lineage specific sensitivities, with myeloid blast being more sensitive to venetoclax compared to autologous CD4+ and CD8+ T-cells and this observed difference was correlated with higher expression of BCL2. Our platform demonstrated that patients who previously failed venetoclax therapy could benefit from a combination with 5-azacytidine or fludarabine and this was patient-specific. We further evaluated MFLEX’s predictive value for patient clinical response by systematically comparing venetoclax sensitivity in patient-derived xenograft models in vivo vs ex vivo and found a high correlation between drug responses. Conclusion: We have established a unique, immune drug screening platform, MFLEX, that has high translational value and can be implemented in clinical trials to identify drug response patterns, novel combinations, and potential biomarkers of response for patient stratification in AML. Citation Format: Reecha Shah, Heba Wander, Michelle Hsueh, Muskan Floren, Wei Liu, Patricia Cheung, Courtney Andersen, Michael White, Nathan Kingston, Pablo Morentin Gutierrez, Lisa Drew, Giulia Fabbri, Elena Bibikova, Daniel Auclair. Multiplexed flow cytometry based immunophenotyping paired with functional ex vivo (MFLEX) drug profiling informs potential efficacy of therapeutic agents in acute myeloid leukemia [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 3904.

Abstract 5187: VistaPlex immunofluorescence assay panels provide reliable deep spatial phenotyping for diverse immuno-oncology applications

Abstract Multiplex immunofluorescence (mIF) is a powerful tool that enables deep phenotyping of immune and cancer cells across a wide variety of tissue types. The initial effort and cost required to develop high-plex mIF panels is significant and poses a considerable hurdle for adoption of spatial biology. To streamline high-plex assay development, we developed a process for creating and validating multiplex antibody panels for use in immuno-oncology applications that leverage CellScape™, an automated end-to-end platform for Precise Spatial Multiplexing. Our multiplex assay development was a rigorous, multi-stage, iterative process to evaluate antibodies for suitability, specificity, and reproducibility. These assay panels are formulated as ready-to-use VistaPlex™ assay kits and are intended to enable rapid, reliable spatial phenotyping of key immune populations. To confirm the utility of VistaPlex, we deployed the Human FFPE Spatial Immune Profiling Panel to stain and image various FFPE tissue samples on the CellScape platform. With CellScape, tissues are mounted in enclosed fluidic chambers while on-instrument staining, imaging and signal erasure are conducted in iterative rounds. The panel presented here contains antibodies against 16 common biomarkers used in immune-oncology applications. Among these are CD3, CD4, CD8, CD20, and CD45 to detect cell lineages, PD-1 and PD-L1, to interrogate immune checkpoints, and markers that inform about cell state and activation. Spatial immune profiling data were obtained from human tonsil, colon, breast, and lung tissue samples. Deep immune phenotyping was performed using unsupervised clustering analysis and AI-assisted cluster labelling, and biomarker distributions were studied in the context of cellular neighborhoods and other proximity analyses. Overall, we identified more than 19 cell phenotypes with varying frequencies and spatial arrangements across tissue samples. Our data are robust and demonstrate that VistaPlex Assay kits for spatial profiling present an efficient and cost-effective solution for users that want to break into spatial biology. Citation Format: Arne Christians, Charles E. Jackson, Jannik Boog, Matthew H. Ingalls, Adam Northcutt, Kayla E. Cashion, Jonah Rauer, Karen Kwarta, J Spencer Schwarz, Oliver Braubach. VistaPlex immunofluorescence assay panels provide reliable deep spatial phenotyping for diverse immuno-oncology applications [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 5187.