Analysis Of Immune Phenotypes Research Articles

Altered immune surveillance of B and T cells in patients with persistent residual lung abnormalities 12 months after severe COVID-19

BackgroundPost-COVID-19 respiratory sequelae often involve lung damage, which is called residual lung abnormalities, and potentially lead to chronic respiratory issues. The adaptive immune response, involving T-cells and B-cells, plays a critical role in pathogen control, inflammation, and tissue repair. However, the link between immune dysregulation and the development of residual lung abnormalities remains unclear.Methods109 patients discharged with residual lung abnormalities after a critical COVID-19 were followed for 12 months and divided as full recovery patients (FRG, n = 88) and persistent lung abnormalities (PLAG, n = 21). Cell profiling analysis was done using flow cytometry at 24 h of not antigen-specific in vitro stimulation. Plasma or supernatant levels of IFN-g, IL-4, IL-10, IgM, and IgG were assessed, and 10 patients (5 FRG, 5 PLAG) were randomly selected for detailed immune cell phenotyping and functional analysis of peripheral blood mononuclear cells using flow cytometry.ResultsCompared to the FRG group, PLAG exhibited an increase of unswitched (p = 0.0159) and decreased double-negative activated B-cells (p = 0.0317), systemic IL-10 levels were lower, displayed reduced frequency of total B-cells, and impaired spontaneous IgM (p = 0.0357) and IgG (p = 0.0079) release in culture. Regarding T-cells, PLAG patients showed a reduction in effector memory CD4 + cells (p = 0.0159) and an increase in CD4 + TEMRA cells (p = 0.0079) following in vitro stimulation. Notably, the PLAG group also exhibited higher frequencies of central memory CD4 + Th2 (GATA3+) T-cells in response to activation than the FRG group (p = 0.0079).ConclusionsPatients with residual lung abnormalities 12 months post-critical COVID-19 exhibit impaired B-cell function, increased unswitched B-cells, and higher frequencies of CD4 + TEMRA T-cells following in vitro activation. These immune imbalances may contribute to ongoing lung dysfunction and warrant further investigation as a potential mechanism in residual lung abnormalities. Larger studies are necessary to confirm these findings.

Combined Molecular Subclass and Immune Phenotype Correlate to Atezolizumab Plus Radiation Therapy Response in Invasive Bladder Cancer: BPT-ART Phase 2 Study.

Bladder preservation therapy in combination with atezolizumab and radiation therapy trial, which was a multicenter, open-label, single-arm phase 2 study, showed a promisingly high interim clinical complete response (cCR) rate of 84.4% (38/45). In the present study, we aimed to identify potential tissue biomarkers for achieving cCR using bladder preservation therapy in combination with atezolizumab and radiation therapy. We used tumor tissue samples of the bladder and blood samples collected from patients at baseline to analyze the tumor immune microenvironment at baseline using an integrated approach of immunophenotyping, genomic, and tumor-infiltrating lymphocyte (TIL) profiling. Immune phenotype analysis revealed that cCR rates of patients with the desert phenotype were as similarly high as patients with excluded/inflamed phenotypes (73.3% [11/15] vs 93.3% [14/15], P = .33) despite lower programmed death-ligand 1 expression levels in the desert phenotype. Genomic and TIL profiling then revealed that increased CD8+ and CD204+ TIL infiltration, high CD8:forkhead box protein P3 ratios in the stroma of the excluded/inflamed phenotypes, and gene alterations, such as CDK12, GNAS, NOTCH2, and AR1D1A, were associated with a high cCR rate (93.3%). Furthermore, the characteristics of these dual TILs, CD8-forkhead box protein P3 ratios, and gene alterations (especially FGFR3) bifurcated the desert phenotype into 2 subgroups with different cCR rates (100% [11/11] and 0% [0/4]). These potential subgroups, defined by combined molecular subclass and immune phenotype, could lead to the identification of good responders to atezolizumab plus radiation therapy for invasive bladder cancer. However, given the small cohort size and limited number of tumor samples, these findings should be viewed as hypothesis-generating and require further validation in larger studies.

EP.11B.02 Correlation Analysis of Peripheral Blood Cell Immune Phenotype with Prognosis of Immunotherapy in Advanced Non-Small Cell Lung Cancer

EP.11B.02 Correlation Analysis of Peripheral Blood Cell Immune Phenotype with Prognosis of Immunotherapy in Advanced Non-Small Cell Lung Cancer

Abstract PR-08: Reprogramming and selective recruitment of distinct neutrophil subpopulations restrain cancer metastasis

Abstract Neutrophils are integral to the metastatic tumor microenvironment (TME), and discrete subsets of neutrophils have been associated with pro- and anti-tumor responses. However, the composition of neutrophils in the metastatic TME and strategies to harness neutrophils for the treatment of metastatic disease remain poorly understood. Here, we studied the role of neutrophils in metastatic disease using mouse models of metastatic pancreatic ductal adenocarcinoma (PDAC). By utilizing mouse PDAC cells driven by mutant KRAS and p53 with controllable expression of ovalbumin, an immunogenic antigen (“TumorAg ON” or “TumorAg OFF”), we found that TumorAg ON PDAC engendered more than 10-fold higher recruitment of neutrophils into liver metastatic lesions compared to TumorAg OFF PDAC. RNA sequencing analysis of micro-dissected metastatic lesions showed that TumorAg ON PDAC elicited transcription of genes associated with neutrophil chemotaxis, including Cxcr2 and its ligands. Multimodal immune phenotyping and imaging mass cytometry (IMC) analysis further revealed that neutrophils within metastatic lesions consisted of CXCR2high and CXCR2low subpopulations and were spatially associated with distinct subsets of tumor cells, T cells, and myeloid cells. In both mouse models of liver and lung metastasis, small molecule inhibition of CXCR2 using SX-682 led to enrichment of CXCR2low neutrophils and reduced metastatic burden in TumorAg ON PDAC. In contrast, CXCR2 inhibition had no impact on metastatic burden in TumorAg OFF PDAC. To define the subpopulations of neutrophils in further depth, we also performed single-cell spatial transcriptomics using Xenium. Notably, CXCR2 inhibition in TumorAg ON PDAC, but not in TumorAg OFF, led to enrichment of neutrophil subsets that expressed higher levels of Mpo, Nos2, and Ly6g and lower levels of Cxcr2 and Siglecf into metastatic lesions. In this setting, depletion of neutrophils using anti-Ly6G antibodies worsened metastatic burden, highlighting anti-tumor properties of these neutrophil subsets in the presence of antigenic recognition. Indeed, in a highly immune-resistant model of PDAC, 6419c5, the use of tumor-targeting listeria vaccine CRS-207 along with CXCR2 inhibition significantly reduced metastatic burden, whereas CXCR2 inhibitor or CRS-207 alone had no impact. To extend the clinical relevance of these observations, we analyzed serial tissue biopsies collected from patients with metastatic PDAC in the liver treated with immunotherapy regimen incorporating CRS-207 (NCT03190265). Consistent with our mouse model results, IMC analysis revealed increased proportion of MPOhigh NOS2high neutrophils upon treatment, suggesting that induction of tumor antigen response results in an effective remodeling of the neutrophil compartment . Taken together, our results show that neutrophils may acquire anti-tumor properties under appropriate inflammatory conditions and that distinct subpopulations of neutrophils may be leveraged for the treatment of metastatic disease. Citation Format: Jae W Lee, Diana Caroline Vargas Carvajal, Yeonju Cho, Sarah M Shin, Erin M Coyne, Alexei Hernandez, Courtney D Cannon, Xuan Yuan, Zhehao Zhang, Nicole E Gross, Soren Charmsaz, Daniel H Shu, Katherine M Bever, Dung T Le, Luciane T Kagohara, Elizabeth M Jaffee, Won Jin Ho. Reprogramming and selective recruitment of distinct neutrophil subpopulations restrain cancer metastasis [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 PR-08.

Artificial Intelligence-Powered Spatial Analysis of Tumor-Infiltrating Lymphocytes as a Potential Biomarker for Immune Checkpoint Inhibitors in Patients with Biliary Tract Cancer.

Recently, anti-programmed cell death-1/anti-programmed cell death ligand-1 (anti-PD1/L1) immunotherapy has been demonstrated for its efficacy when combined with cytotoxic chemotherapy in randomized phase 3 trials for advanced biliary tract cancer (BTC). However, no biomarker predictive of benefit has been established for anti-PD1/L1 in BTC. Here, we evaluated tumor-infiltrating lymphocytes (TIL) using artificial intelligence-powered immune phenotype (AI-IP) analysis in advanced BTC treated with anti-PD1. Pretreatment hematoxylin and eosin (H&E)-stained whole-slide images from 339 patients with advanced BTC who received anti-PD1 as second-line treatment or beyond, were employed for AI-IP analysis and correlative analysis between AI-IP and efficacy outcomes with anti-PD1. Next, data and images of the BTC cohort from The Cancer Genome Atlas (TCGA) were additionally analyzed to evaluate the transcriptomic and mutational characteristics of various AI-IP in BTC. Overall, AI-IP were classified as inflamed [high intratumoral TIL (iTIL)] in 40 patients (11.8%), immune-excluded (low iTIL and high stromal TIL) in 167 patients (49.3%), and immune-desert (low TIL overall) in 132 patients (38.9%). The inflamed IP group showed a substantially higher overall response rate compared with the noninflamed IP groups (27.5% vs. 7.7%, P < 0.001). Median overall survival and progression-free survival were significantly longer in the inflamed IP group than in the noninflamed IP group (OS, 12.6 vs. 5.1 months; P = 0.002; PFS, 4.5 vs. 1.9 months; P < 0.001). In the TCGA cohort analysis, the inflamed IP showed increased cytolytic activity scores and IFNγ signature compared with the noninflamed IP. AI-IP based on spatial TIL analysis was effective in predicting the efficacy outcomes in patients with BTC treated with anti-PD1 therapy. Further validation is necessary in the context of anti-PD1/L1 plus gemcitabine-cisplatin.

Immune Phenotype-Genotype Associations in Primary Clear Cell Renal Cell Carcinoma and Matched Metastatic Tissue

Adjuvant immunotherapy has been recently recommended for patients with metastatic clear cell renal cell carcinoma (ccRCC), but there are no tissue biomarkers to predict treatment response in ccRCC. Potential predictive biomarkers are mainly assessed in primary tumor tissue, whereas metastases (METs) remain understudied. To explore potential differences between genomic alterations and immune phenotypes in primary tumors and their matched METs, we analyzed primary tumors (PTs) of 47 ccRCC patients and their matched distant METs by comprehensive targeted parallel sequencing, whole-genome copy number variation analysis, determination of microsatellite instability, and tumor mutational burden. We quantified the spatial distribution of tumor-infiltrating CD8+ T cells and coexpression of the T-cell-exhaustion marker thymocyte selection-associated high mobility group box (TOX) by digital immunoprofiling and quantified tertiary lymphoid structures. Most METs were pathologically "cold." Inflamed, pathologically "hot" PTs were associated with decreased disease-free survival, worst for patients with high levels of CD8+TOX+ T cells. Interestingly, inflamed METs showed a relative increase in exhausted CD8+TOX+ T cells and increased accumulative size of tertiary lymphoid structures compared with PTs. Integrative analysis of molecular and immune phenotypes revealed BAP1 and CDKN2A/B deficiency to be associated with an inflamed immune phenotype. Our results highlight the distinct spatial distribution and differentiation of CD8+ T cells at metastatic sites, and the association of an inflamed microenvironment with specific genomic alterations.

Impact of Fixation of Camel Lymph Node Cells on Marker Expression Stability in Flow Cytometry

Single cell immunophenotyping by flow cytometry has proven a useful and high sensitive method for the analysis of immune cell composition and phenotype in different lymphatic and non-lymphatic tissues. Fixation of stained cells is usually recommended when the cells need to be preserved for later analysis by flow cytometry to avoid changes in cell morphology and expression of the level of cellular antigens. In the present study, a stain-fix approach was used in combination with flow cytometry to investigate the impact of fixation of camel lymph node cell suspension (n = 5 camels) after labeling with monoclonal antibodies to some leukocyte antigens on their cellular composition and expression density of immune cell markers. The obtained results indicated that camel lymph node cell suspension stained with fluorochrome-conjugated mAbs to leukocyte antigens and fixed with paraformaldehyde (PFA) will keep stable values for their immune cell composition for at least six days when analyzed by flow cytometry. However, if cell subsets were to be identified, fixation may result in different values that were obtained when analyzing fresh stained unfixed cells. Especially the instability in the fluorescence intensity of CD14, CD172a, and MHCII will lead to significant changes in the frequency of monocyte subsets (classical versus intermediate or non-classical) and the identification of macrophage functional subtype (M1 versus M2). Similarly, the instability in CD44 expression may affect the identified phenotype of T cells with significantly lower frequency of activated T cells. In conclusion, flow cytometric data collected from stained and PFA-fixed cell suspension prepared from camel lymph nodes should be interpreted with care if the functional subtype of cells is to be identified based on surface molecule expression.

Abstract CT160: A phase I trial to evaluate allogeneic NKG2DL-targeting chimeric antigen receptor-grafted γδ T cells in subjects with advanced solid tumors or hematological malignancies (the ANGELICA Trial)

Abstract Background: Adoptive cellular therapy (ACT) has been transformative in the treatment of hematological malignancies. Limitations of ACTs include difficulty identifying suitable tumor antigens for solid tumors, the ability to target only one antigen per construct which commonly results in secondary resistance due to antigen escape, and largely autologous approaches which are challenging in heavily pre-treated patients with poor marrow function and those with rapidly progressive disease who may not receive their personalised cellular products in time. This is a phase I study evaluating a novel off-the-shelf allogenic mRNA-electroporated NKG2D ligand-targeting CAR-grafted γδ T cell therapy. NKG2DLs are a group of 8 types of stress-induced cancer antigens which are preferentially and widely expressed on tumor cells from diverse tissue origins but are not typically present on normal tissue. NKG2DL-targeting γδ T cells will be manufactured from peripheral blood mononuclear cells isolated from healthy donors enrolled on the donor protocol of the ANGELICA trial. ANGELICA is a phase I study evaluating the safety, tolerability and recommended phase 2 dose (RP2D) of NKG2DL-targeting CAR-grafted γδ T cells in patients with treatment refractory tumors. Methods: Patients will be enrolled in a 3+3 design. Dose escalation will be performed at 3 dose levels: 1x108, 3x108 and 1x109 per infusion (adjusted for body weight). Lymphodepletion with fludarabine 25mg/m2/day and cyclophosphamide 250mg/m2/day will given for 3 days and completed at least 2 days prior to the first cycle of treatment. Patients will receive 4 doses of weekly infusions for the first cycle, with up to 5 subsequent infusions every 2 months as maintenance. Patients will receive intravenous zoledronic acid 1mg prior to each cellular infusion and subcutaneous IL-2 1x106 IU/m2 within 2 hours of each cellular infusion as pre-clinical data demonstrated cancer cell sensitisation and prolonged γδ T cell survival with these adjuncts. Dose-limiting toxicities will be assessed over the first 8 weeks. Adverse events, response rates (RECIST v1.1), survival outcomes and immunomonitoring (immune cell phenotyping and serum cytokine analysis) will be assessed. The trial is currently enrolling healthy donors; enrolment of patients at the first dose level will begin in February 2024. NCT05302037 TABLE 1: NAND Dose Level and Schedule Dose Level Dose per infusion Infusion schedule Number of patients 1 Weight 65kg and above: 1x108.Weight less than 65kg: 1.5x106. Cycle 1: weekly x 4 infusions. Maintenance phase: 2-monthly x 5 infusions. 3-6 2 Weight 65kg and above: 3x108.Weight less than 65kg: 4.6x106. 3-6 3 Weight 65kg and above: 1x109.Weight less than 65kg: 1.5x107. 3-6 Citation Format: Joan Choo, Wee Kiat Tan, Lucas Luk, Jieming Zeng, Teck Guan Soh, Sou Yen Soon, Jedidah Lieow, Calista Wong, Mei Yan Pang, Sudipto Bari, Michelle Poon, Liang Piu Koh, Wee Joo Chng, Anand Jeyasekharan, Lip Kun Tan, Esther Chan, Raghav Sundar. A phase I trial to evaluate allogeneic NKG2DL-targeting chimeric antigen receptor-grafted γδ T cells in subjects with advanced solid tumors or hematological malignancies (the ANGELICA Trial) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT160.

Abstract 914: Combination analysis of tumor-associated collagen frameworks and tumor immune phenotype of lung carcinomas using virtual staining

Abstract An emerging predictive parameter of immunotherapy response is the patient’s tumor immune status, generally classified as “inflamed”, "immune excluded” and “desert”. Historically, classification was performed semi-quantitatively with somewhat subjective parameters as evidenced by the recent Delphi Workshop consensus. There are a variety of approaches to classifying immune status, most based upon analysis of H&amp;E images (counting tumor infiltrating lymphocytes, TILs), or counting subpopulations of immune cells using immunologic staining. However, use of multiplexed or multiple stains has recently been explored to better quantify the counts and types of immune cells in individual tissue compartments. While the immune microenvironment represents an important parameter in predicting patient response, other regions such as the extracellular matrix (ECM) may yield complementary information, as collagen-rich ECMs may present a barrier to drug diffusion and the orientation of fibers may direct the migration of malignant cells. Here we present a proof of concept virtual staining enhanced image analysis pipeline, which converts autofluorescence signals from a single tissue section into virtual H&amp;E and Masson’s Trichrome along with virtual detection of pan cytokeratin (AE1/AE3/PCK26) and CD45 (LCA) positive cells. We apply image analysis to the panel of virtual stains to study the spatial and case-by-case heterogeneity of tumor collagen frameworks and immune phenotype. Using a standard slide scanner (Axio Scan Z1, Zeiss), multiple autofluorescence images were captured from unstained sections (4-6um thick) of lung tissue. The virtual staining was performed by four deep neural networks trained in a supervised learning fashion. Select chemical stains were performed on previously scanned tissues and reviewed by pathologists side-by-side with virtual stains to ensure consistency and quality control. Four perfectly registered WSI virtual stain images were generated from each tissue section. The multi-stain results were rendered from a variety of lung cancers, including tissue microarray slides. Image analysis was performed using HALO (Indica Labs) and custom python scripts. We identified unique areas of tumor and adjacent stroma with heterogeneous immune phenotype and collagen characteristics, suggesting that an interplay might exist that could be utilized to improve patient selection or prognosis when deploying advanced AI tools. Future work includes applying this virtual stain technique retrospectively to cases with known immunotherapy treatment responses to determine the prognostic significance of the combined immune/ECM phenotype. Citation Format: Serge Alexanian, Yair Rivenson, Ning Xuan, Brian Cone, Zihang Fang, Sean Meyering, Luis A. Carvalho, David Palacios, Raymond Kozikowski. Combination analysis of tumor-associated collagen frameworks and tumor immune phenotype of lung carcinomas using virtual staining [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 914.

Integrated analysis revealing novel associations between dietary patterns and the immune system in older adults.

With the expanding ageing population, there is a growing interest in the maintenance of immune health to support healthy ageing. Enthusiasm exists for unravelling the impact of diet on the immune system and its therapeutic potential. However, a key challenge is the lack of studies investigating the effect of dietary patterns and nutrients on immune responses. Thus, we have used an integrative analysis approach to improve our understanding of diet-immune system interactions in older adults. To do so, dietary data were collected in parallel with performing immunophenotyping and functional assays from healthy older (n = 40) participants. Food Frequency Questionnaire (FFQ) was utilised to derive food group intake and multi-colour flow cytometry was performed for immune phenotypic and functional analysis. Spearman correlation revealed the strength of association between all combinations of dietary components, micronutrients, and hallmarks of immunesenescence. In this study, we propose for the first time that higher adherence to the Mediterranean diet is associated with a positive immune-ageing trajectory (Lower IMM-AGE score) in older adults due to the immune protective effects of high dietary fibre and PUFA intake in combating accumulation or pro-inflammatory senescent T cells. Furthermore, a diet rich in Vit A, Vit B6 and Vit B12 is associated with fewer features of immunesenescence [such as accumulation of terminally differentiated memory CD8 T cells] in older adults. Based on our findings we propose a future nutrition-based intervention study evaluating the efficacy of adherence to the MED dietalongside a multi-nutrient supplementation on immune ageing in older adults to set reliable dietary recommendations with policymakers that can be given to geriatricians and older adults. Insight box: There is a growing interest in the maintenance of immune health to boost healthy ageing. However, a key challenge is the lack of studies investigating the effect of dietary patterns and nutrients on immune responses. Thus, to do so we collected dietary data in parallel with performing immunophenotyping and functional assays on healthy older (n = 40) participants, followed by an integrative analysis approach to improve our understanding of diet-immune system interactions in older adults. We strongly believe that these new findings are appropriate for IB and will be of considerable interest to its broad audience.

Abstract C079: Comprehensive immunophenotypic profiling sheds light on the dynamic interplay between immune cells in the 4T1 breast cancer model upon anti-PD1 and anti-CTLA4 immunotherapy

Abstract Immuno-oncology has revolutionized cancer treatment by harnessing the immune system to target and eliminate tumor cells. In this study, we employed a combination of fluorescence-activated cell sorting (FACS) and Cytek full spectrum flow cytometry to investigate the impact of anti-PD1 (programmed cell death protein 1) and anti-CTLA4 (cytotoxic T-lymphocyte-associated protein 4) treatments on the 4T1 breast cancer model. We comprehensively analyzed 25 surface markers on immune cells to gain insights into the dynamic changes induced by immunotherapy. The 4T1 murine breast cancer model is known for its aggressive and metastatic nature, resembling triple-negative breast cancer in humans. We established a syngeneic orthotopic tumor graft of 4T1 cells in immunocompetent mice and treated them individually with anti-PD1 or anti-CTLA4 antibodies and compared the results to the respective isotype controls. Using FACS, we characterized immune cell populations within the tumor microenvironment and spleenocytes at multiple time points (before treatment, day 5, and day 17) during the treatment course. The Cytek full spectrum flow cytometry platform allowed simultaneous measurement of 25 surface markers, enabling a detailed analysis of immune cell phenotypes and functional states in a single experiment. Our findings revealed significant alterations in the immune landscape following anti-PD1 or anti-CTLA4 treatments. We observed an increase in tumor-infiltrating lymphocytes (TILs) and specifically CD4+ as well as CD8+ T cell infiltration within the tumor, accompanied by upregulation of T cell activation markers, such as CD69, CD38, CD44, PD1 and CD25. The immune cell composition within 4T1 tumors included CD4+ and CD8+ TILs, as well as increased populations of monocytes and macrophages. Additionally, a decrease in regulatory T cells (TRegs) was observed, indicating a potential restoration of immune balance. Moreover, F4/80+ CD206+ tumor-associated macrophages (TAMs) were identified in the 4T1 tumors vs. the spleen. This was associated with increased granzyme B production by CD8+ T cells and a shift towards an effector memory phenotype, suggesting improved cytotoxic function. To gain insights into spatial infiltration patterns of immune cells, we performed Immunohistochemistry (IHC) stainings of various immune cell markers in the 4T1 tumors. In line with the FACS results, we observed increased immune cell infiltration in tumors treated with anti-PD1 and anti-CTLA4 antibodies. Our comprehensive immunophenotypic analysis, utilizing Cytek full spectrum flow cytometry and IHC sheds light on the dynamic interplay between immune cells and the 4T1 breast cancer model during anti-PD1 and anti-CTLA4 immunotherapy. These findings provide valuable insights into the mechanisms underlying the therapeutic efficacy of immune checkpoint blockade in this aggressive cancer model. Citation Format: Christos Nikolaou, Simon Heller, Stefan Kaulfuss, Carlo Stresemann, Alexandra Eichten, Oliver von Ahsen, Martin Lange. Comprehensive immunophenotypic profiling sheds light on the dynamic interplay between immune cells in the 4T1 breast cancer model upon anti-PD1 and anti-CTLA4 immunotherapy [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C079.

Measurable Residual Disease As Assessed By Flow Cytometry As Predictor of Survival in Pediatric AML Patients

Introduction Pediatric acute myeloid leukemia (pedAML) is a heterogeneous group of hematological malignancies classified according to morphology, immunophenotyping, and genomics. The optimal adjustment of therapy based on level of response is not well defined. Aim: Herein, we present the results of a retrospective study aiming to analyze the prognostic value of measurable residual disease (MRD) as determined by flow cytometry (FC-MRD) in pediatric AML. Patients and Methods All pediatric and adolescent patients (aged 0-18 years old) with AML treated in our unit between 1/2013-6/2023 were included in this study. French American British (FAB) morphological classification, immune-phenotype analysis, and molecular characterization were performed on diagnosis, and all patients were treated according to AML-BFM protocols. Final risk classification was based on evaluation of response and on genetic abnormalities at diagnosis. MRD was assessed by FC-MRD after Induction 1 and 2, and, either after course 5 or before hematopoietic stem cell transplantation (HSCT). Complete Remission (CR) was defined as regenerative or normocellular bone marrow with &amp;lt;5% blasts; in peripheral blood ≥1.0x10 9/L neutrophil granulocytes and ≥80x10 9/L thrombocytes or Remission with partial regeneration ( CRp: regenerative or normocellular bone marrow with &amp;lt;5% blasts; in the blood: ≥1.0x10 9/L leukocytes, ≥0.5x10 9/L, neutrophil granulocytes and ≥50x10 9/L, self-sustained platelets), lasting at least 4weeks; no extramedullary disease. Overall survival (OS) was defined as the time interval from the treatment initiation date to the date of death due to any cause. Progressive free survival (PFS) was defined as the time interval from the response date to the date of disease relapse or death due to any cause. The probabilities of OS and PFS were estimated with the Kaplan-Meier method. Results Overall, there were 52 patients (median age at diagnosis:7 years, 37males/15 females) diagnosed with AML, of whom: 40 patients with de novo AML, 5 patients with therapy- or myelodysplasia-related AML and 7 patients with mixed-lineage leukemia. Cytogenetics were considered favorable for 16 (31.0%), intermediate for 4 (7.6%), and adverse for 32 (61.4%) patients. Standard-risk patients (n=16) received only chemotherapy, whereas all high-risk patients, and intermediate-risk patients with available matched donors, underwent HSCT should they have reached remission. Forty-four patients (84.6%) achieved complete response (CR). Among these patients, 43 patients (97.7%) achieved CR after 1 course of induction, and 1 patient (2.3%) required at least 2 courses. Twenty-four patients (46.1%) underwent HSCT during either their first (20/24) or second (4/24) CR. Overall, 12 patients (23.07%) died, 4 patients due to resistant disease, 3 patients due to relapse, 2 patients due to serious complications upon diagnosis, 1 patient due to infection and 2 patients due to transplant-related complications. With a median follow-up time of 3.7 years (range 0.1-11.1years), the five-year OS and PFS of the entire cohort was 74.0% and 70.0%, respectively. The level of FC-MRD was a significant prognostic factor for OS and PFS. More specifically, achievement of FC-MRD &amp;lt; 0.1% at all time points was associated with a significant improvement in PFS and OS. (figure 1). FC-MRD ≥ 0.1% after course 5 or before HSCT was the only independent adverse prognostic factor that was statistically significant for both progression-free (P=0.001) and overall survival (P&amp;lt;0.001) (figure1). Conclusions We investigated the association between FC-MRD and survival in Greek pediatric patients with AML. In our cohort, achieving an MRD status &amp;lt;0.1% according to FC is associated with significant improvement in the outcome of pediatric patients with AML.

Ablative and Immunostimulatory Effects of Histotripsy Ablation in a Murine Osteosarcoma Model.

Background: Osteosarcoma (OS) is the most frequently occurring malignant bone tumor in humans, primarily affecting children and adolescents. Significant advancements in treatment options for OS have not occurred in the last several decades, and the prognosis remains grim with only a 70% rate of 5-year survival. The objective of this study was to investigate the focused ultrasound technique of histotripsy as a novel, noninvasive treatment option for OS. Methods: We utilized a heterotopic OS murine model to establish the feasibility of ablating OS tumors with histotripsy in a preclinical setting. We investigated the local immune response within the tumor microenvironment (TME) via immune cell phenotyping and gene expression analysis. Findings: We established the feasibility of ablating heterotopic OS tumors with ablation characterized microscopically by loss of cellular architecture in targeted regions of tumors. We observed greater populations of macrophages and dendritic cells within treated tumors and the upregulation of immune activating genes 72 h after histotripsy ablation. Interpretation: This study was the first to investigate histotripsy ablation for OS in a preclinical murine model, with results suggesting local immunomodulation within the TME. Our results support the continued investigation of histotripsy as a novel noninvasive treatment option for OS patients to improve clinical outcomes and patient prognosis.

T-cell senescence induced by peripheral phospholipids.

We present an integrated analysis of the clinical measurements, immune cells, and plasma lipidomics of 2000 individuals representing different age stages. In the study, we explore the interplay of systemic lipids metabolism and circulating immune cells through in-depth analysis of immune cell phenotype and function in peripheral dynamic lipids environment. The population makeup of circulation lymphocytes and lipid metabolites changes dynamically with age. We identified a major shift between young group and middle age group, at which point elevated, immune response is accompanied by the elevation of specific classes of peripheral phospholipids. We tested the effects in mouse model and found that 10-month-dietary added phospholipids induced T-cell senescence. However, the chronic malignant disease, the crosstalk between systemic metabolism and immunity, is completely changed. In cancer patients, the unusual plasma cholesteryl esters emerged, and free fatty acids decreased. The study reveals how immune cell classes and peripheral metabolism coordinate during age acceleration and suggests immune senescence is not isolated, and thus, system effect is the critical point for cell- and function-specific immune-metabolic targeting. • The study identifies a major shift of immune phenotype between young group and middle age group, and the immune response is accompanied by the elevation of specific classes of peripheral phospholipids; • The study suggests potential implications for translational studies such as using metabolic drug to regulate immune activity.

Artificial intelligence (AI) analysis of tumor-infiltrating lymphocytes (TILs) in hematoxylin and eosin (H&amp;E) slides to explore immune phenotypes in papillary thyroid cancer.

6100 Background: Different biomarkers have been discovered to predict response to immunotherapy but the variability of response exists. TILs are a potential biomarker as infiltration of CD8+ T cells in the tumor microenvironment(TME) was related to treatment response to PD-1 blockade. Advanced well-differentiated papillary thyroid cancer(PTC) has limited standard-of-care treatment options and can exhibit a poor prognosis when exhausted. Our study aimed to classify the TME of PTC according to TILs. Methods: Using the TCGA PTC data, samples were classified into three immune phenotypes (immune-’desert’, immune-’excluded’, and ‘inflamed’) based on peri-tumor lymphocyte density from analysis of H&amp;E slides using an AI model(Lunit SCOPE). Immune and stromal cell infiltration was inferred using xCell. Cytolytic scores(CYT) and thyroid differentiation scores(TDS) were calculated using mRNA expression. Results: Total of 383 samples were included. Immune phenotype analysis showed 183(47.7%) ‘desert’, 133(34.7%) ‘excluded’, and 67(17.4%) ‘inflamed’. Median age at diagnosis was the lowest in ‘inflamed’(inflamed 41.5 vs excluded 47 vs desert 50, p = 002). TNM stages were as follows in the order of stages 1 to 4: inflamed 49/4/8/3, excluded 64/13/32/16, and desert 86/28/40/19. BRAF V600E mutation was most prevalent in ‘excluded’(97/133, 72%), followed by ‘inflamed’(32/67, 47.7%) and ‘desert’(51/183, 27.8%). CYT was the highest in ‘inflamed’(median: inflamed 186 vs excluded 53 vs desert 39, p &lt; 0.001). TDS was the highest in ‘desert’(median: inflamed -0.3 vs excluded -0.69 vs desert 0.73, p &lt; 0.001). The 6 cell types with the most statistically significant difference in infiltration were activated dendritic cells(aDCs), DCs, B cells, epithelial cells(EpCs), CD8+ central memory T cells, and CD4+ memory T cells. The ‘inflamed’ showed the highest median value for all of these cells except EpCs. The cells with the highest median value in ‘excluded’ included EpCs, sebocytes, and immature DCs. The cells with the highest median value in ‘desert’ were lymphatic endothelial cells(ECs), ECs, and osteoblasts. Three immune phenotypes were associated with differential outcomes in disease-free survival(log-rank for trend, p-value = 0.01). There was no difference in overall survival. Conclusions: Our results suggest that 15% of PTC can have immune features which were related to a higher rate of response to immunotherapy across different tumor types. A previous study showed that PTC with a low TDS or BRAF V600E mutation is related to increased immune response indices. In our study, TDS or BRAF V600E mutation status could not distinguish the ‘inflamed’ from other phenotypes. This suggests that immune phenotype classification through TILs can provide distinct information about the TME of PTC.

Spatially Resolved Multi-Omics Single-Cell Analyses Inform Mechanisms of Immune Dysfunction in Pancreatic Cancer

As pancreatic ductal adenocarcinoma (PDAC) continues to be recalcitrant to therapeutic interventions including poor response to immunotherapy, albeit effective in other solid malignancies, a more nuanced understanding of the immune microenvironment in PDAC is urgently needed. We aimed to unveil a detailed view of the immune micromilieu in PDAC using a spatially-resolved multimodal single cell approach. We applied single cell RNA sequencing, spatial transcriptomics, multiplex immunohistochemistry and mass cytometry to profile the immune compartment in treatment naive PDAC tumors and matched adjacent normal pancreatic tissue, as well as in the systemic circulation. We determined prognostic associations of immune signatures, and performed a meta-analysis of the immune microenvironment in PDAC and lung adenocarcinoma (LUAD) on single-cell level. We provide a spatially-resolved fine map of the immune landscape in PDAC. We substantiate the exhausted phenotype of CD8 T cells and immunosuppressive features of myeloid cells, and highlight immune subsets with potentially underappreciated roles in PDAC, that diverge from immune populations within adjacent normal areas, particularly CD4 T cell subsets and NKT cells that are terminally exhausted and acquire a regulatory phenotype. Differential analysis of immune phenotypes in PDAC and LUAD revealed the presence of extraordinarily immunosuppressive subtypes in PDAC, along with a distinctive immune checkpoint composition. Our study sheds light on the multilayered immune dysfunction in PDAC and presents a holistic view of the immune landscape in PDAC and LUAD, providing a comprehensive resource for functional studies and the exploration of therapeutically actionable targets in PDAC.

Changes in the molecular profiles of large-vessel vasculitis treated with biological disease-modifying anti-rheumatic drugs and Janus kinase inhibitors.

Giant cell arteritis and Takayasu arteritis are two types of primary large-vessel vasculitis (LVV). Although glucocorticoids (GC) are the standard treatment for LVV, the disease relapse rates are high. Recent clinical trials on biological disease-modifying anti-rheumatic drugs (bDMARDs) and Janus kinase (JAK) inhibitors have demonstrated their efficacy in reducing LVV relapse rates and GC dosages. However, the control of residual inflammation and degenerative alterations in the vessel wall remains an outstanding requirement in the clinical management of LVV. The analysis of immune cell phenotypes in patients with LVV may predict their response to treatment with bDMARDs and JAK inhibitors and guide their optimal use. In this mini-review, we focused on molecular markers, including the immune cell proportions and gene expression, in patients with LVV and in mouse models of LVV treated with bDMARDs and JAK inhibitors.

Abstract 4512: My Pediatric and Adult Rare Tumor Network (MyPART): integrating longitudinal, clinical, molecular, and patient-reported outcomes for rare tumors

Abstract Background: Rare tumors are defined as &amp;lt;15 cases/100,000 people/year and present an unmet need due to lack of effective medical treatments, paucity of biospecimens and research models, and difficulty accruing to clinical trials. MyPART (My Pediatric and Adult Rare Tumor Network) was established to: 1) engage patients/advocates as partners in rare tumor research, 2) provide expertise and personalized health care to children and young adults with rare tumors, and 3) build databases and tools to advance research on new treatments for rare tumors. We have developed a natural history and biospecimen acquisition study (NCT03739827), enrolling patients with rare solid tumors, their family members, and participants with germline mutations at increased rare tumor risk. Method: Participants in our natural history study enroll remotely or come to the National Institutes of Health Clinical Center. Participants complete medical and family history forms, patient reported outcomes (PROs), and provide tumor samples and blood and/or saliva for DNA/RNA analysis. For patients coming to the Clinical Center, blood samples are collected for immune phenotyping and cytokine analysis. Clinical staff extract medical records for downstream analyses. Tumors are analyzed using a 500+ gene panel and genomic analysis. Participants are followed yearly to collect data on recurrence, progression, PROs, development of additional tumors, and response to treatments such as standard of care and any treatment trials they may join. Results: We report a clinical and molecular summary of the first 200 participants (66% female, 35% male), with neuroendocrine neoplasms (NEN), adrenocortical cancer (ACC), SDH-deficient gastrointestinal stromal tumor (sdGIST), chordoma, medullary thyroid cancer (MTC), and other less commonly enrolled tumors totaling 35 different tumor types. Participants come from 46 US states and 9 countries. Early results show tumor-specific differences in mutations, treatment/management, PROs, and self-reported non-tumor health issues. The most common pathogenic germline mutations are SDHA/SDHB/SDHC in sdGIST and RET in MTC and other tumors. The most common pathogenic tumor mutations are TP53 and CTNNB1 in ACC, SDHA/B and TP53 in sdGIST, MEN1 in NEN, RET and HRAS in MTC, and SMARCB1 in chordoma. Clinically significant levels of anxiety and pain are more frequent in ACC, NEN, and sdGIST. Conclusions: By developing methods to provide value to rare tumor patients, using a large catchment area and opportunity for remote participation, and collecting data in a tumor-agnostic way, we can learn about multiple rare tumors and share findings more quickly than if each rare tumor was studied alone. We describe the MyPART cohort and challenges to overcome for successful longitudinal rare tumor natural history studies. Citation Format: Shadin Ahmed, Margarita Raygada, Robin Lockridge, Mary Frances Wedekind Malone, Jaydira Del Rivero, John W. Glod, Barbara J. Thomas, Donna Bernstein, Markku M. Miettinen, Liqiang Xi, Jung Kim, Manoj Tygagi, Mark Raffeld, Kenneth Aldape, Ashkan A. Malayeri, Abby B. Sandler, Brigitte C. Widemann, Karlyne M. Reilly. My Pediatric and Adult Rare Tumor Network (MyPART): integrating longitudinal, clinical, molecular, and patient-reported outcomes for rare tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4512.

Abstract 2369: Spatial organization of the tumour immune microenvironment (TIME) in primary and metastatic melanoma is associated with patient outcome

Abstract Introduction The complex spatial cellular interactions of immune cell populations within the melanoma TIME are associated with patient prognosis and immunotherapy response. Novel multiplex immunofluorescence (mIHC) and spatial analysis techniques enable comprehensive investigation of these interactions. This study aimed to develop an automated spatial analysis workflow for large patient cohorts and to determine immunospatial patterns associated with outcome in patients (pts) with primary melanoma as well as pts with stage III melanoma treated with adjuvant immunotherapy. Methods Two cohorts with available clinical &amp; mIHC data were studied: one of primary melanoma pts (primary cohort, n=46), and another of baseline resected stage III melanoma pts who received adjuvant anti-PD-1 (stage III cohort, n=119). Melanoma tissue was stained for CD8+ T cells and the markers CD103, PD-1 and CD39 in both cohorts and the primary cohort was additionally stained for NK cells, B cells, and Langerhans cells (LCs). mIHC data was investigated using the SPIAT spatial analysis R package. Immune-melanoma and immune-immune spatial relationships were quantified throughout the entire tissue and analysed using neighborhood analysis and cell colocalization metrics including entropy and mixing interaction scores. Results In the primary melanoma cohort, 11 neighborhood metaclusters (NMCs) were identified from 1617 neighborhoods. NMCs with high B cells, CD39+CD103+ CD8+ T cells, or LCs were associated with improved outcomes in this cohort. Neighborhood composition varied significantly based on tissue localisation, with B cells, LCs and NK cells significantly increased in marginal and stromal neighborhoods (p&amp;lt;0.0001). Over 100 B cell aggregate (BCA) neighborhoods were identified predominantly at the tumor margins and adjacent stroma. These BCAs were likely early TLSs based on their localisation and immune composition. BCA composition was further compared based on patient outcomes, with those harboring increased CD103-PD-1+ CD8+ T cells associated with poor outcome. In-depth pairwise analysis of immune phenotypes with melanoma cells found that increased B cell-melanoma and CD103+PD-1- CD8+ T cell-melanoma interaction was associated with improved outcomes in the primary cohort. In the stage III cohort, increased intratumoral immune diversity was associated with reduced melanoma recurrence (p=0.0004). Conclusions In developing a cohort-wide TIME spatial analytic approach and workflow, we identified multiple immunospatial associations with patient outcome in primary and stage III metastatic melanoma. Intratumoral immune diversity and immune cell neighborhoods indicate beneficial anti-tumor immune functions contributing towards improved patient outcomes. Citation Format: Grace Heloise Attrill, Eva R. Shteinman, Xinyu Bai, Felix Marsh-Wakefield, Camelia Quek, Umaimainthan Palendira, Ismael A. Vergara, Georgina V. Long, Richard A. Scolyer, James S. Wilmott. Spatial organization of the tumour immune microenvironment (TIME) in primary and metastatic melanoma is associated with patient outcome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2369.

Artificial intelligence (AI)-powered immune phenotyping to predict outcomes of immuno-oncology (IO)-based regimens in hepatocellular carcinoma (HCC).

601 Background: Anti-PD-(L)1 monotherapy or combination with bevacizumab or ipilimumab are approved treatment options in HCC. Nevertheless, not all patients benefit from IO treatment, and discovery of predictive biomarkers for IO outcome has not been fully investigated. Immune phenotype analysis by AI-powered spatial tumor-infiltrating lymphocyte (TIL) analyzer in H&amp;E whole-slide images (WSI), Lunit SCOPE IO, has shown predictive effect across multiple cancer types. Here, we investigated the correlation between immune phenotype (IP) and the real-world outcomes in HCC. Methods: Real-world dataset of 177 patients with HCC were retrospectively collected from CHA Bundang Medical Center. Inflamed score was defined by the proportion of area with high intra-tumoral TIL density per 0.5 mm2-sized grid. IP status was sub-analyzed by PD-L1 combined positive score (CPS) and prognostic factors including Child-Pugh class and Barcelona Clinic Liver Cancer (BCLC) stage. We evaluated the correlation between IP and IO outcome by regimen (atezolizumab+bevacizumab [Atez+Bev], n=100; nivolumab+/-ipilimumab [Niv+/-Ipi], n=77). Results: The majority of patients (75.3%) were Child-Pugh class A (75.3%) and BCLC stage C (86.5%), respectively. Atez+Bev was mostly received as first-line therapy (92.0%), whereas Niv+/-Ipi as second-line or beyond (98.7%). The inflamed score was significantly higher in patients who had lymph node metastasis ( p=0.008), but it was not correlated with other metastatic sites, or any other prognostic factors. Of 177 patients, 105 (59.3%) patients were classified as having a high inflamed score (≥10%) and 72 (40.7%) patients as having lower than 10%. PD-L1 CPS was significantly higher in the sample with high score compared to those with low inflamed score ( p=0.01387). PFS of Niv+/-Ipi were significantly favorable in the patients with high inflamed score (3-m PFS rate; 44.0% vs 3.61%, 12-m PFS rate; 17.8% vs 0%, Hazard ratio [HR] 0.37, 95% confidence interval [CI] 0.22-0.63, p&lt;0.001), while PFS of Atez+Bev were not different according to inflamed score (3-m PFS rate; 67.5% vs 57.3%, 12-m PFS rate; 36.6% vs 31.0%, HR 0.80, 95% CI 0.48-1.32, p=0.400). Conclusions: Inflamed score, the proportion of area with high intra-tumoral TIL infiltration, can be a clinically significant predictor of PFS of Niv+/-Ipi, but it was not relevant for Atez+Bev treatment outcomes.