Peripheral Blood Mononuclear Cells Samples Research Articles

Abstract PO4-09-09: Deep Immunoprofiling Demonstrates Racial Differences in the Peripheral Immune System in Women at Risk for Breast Cancer

Abstract Background: Disparities in breast cancer outcomes between Non-Hispanic Black (NHB) and Non-Hispanic White (NHW) women have been attributed to NHB women presenting with more aggressive subtypes and later stage disease, differences in treatment receipt and adherence, access to care and tumor biology. However, previous work has shown that these known factors do not fully account for the observed disparities in outcomes. Systemic immune fitness is a factor not previously studied that may play a role in dictating disease progression and therapeutic responses. Numerous factors, including age and race, impact immune system function and understanding these changes is critical to address disparities in breast cancer outcomes. Here, we developed a pipeline for deep immunoprofiling of peripheral blood mononuclear cells (PBMC) at single cell resolution by full-spectrum flow cytometry. Using this pipeline, we sought to investigate whether there are differences in immune fitness between NHB and NHW women at high risk for developing breast cancer. Methods: PBMC were collected and processed from age-matched NHB and NHW women (n=20/cohort) identified as being at high risk for developing breast cancer through our institution’s B-PREP (Breast Cancer Personalized Risk Assessment Education and Prevention) clinic. Two custom antibody panels and protocols for comprehensive immunoprofiling of T and B cell subsets (T/B Panel), as well as monocytic, NK, and dendritic cell subsets (M/N/D Panel), along with functional and exhaustion markers were designed, optimized, and implemented. Traditional gating strategies were used to quantify proportions of canonical cell populations, and unbiased high dimensionality reduction analysis was performed using the OMIQ analysis platform to assess phenotypes and functional status of various immune cell subsets. Results: Significant differences between cohorts in abundance as well as phenotype of various cell populations were observed. Specifically, CD4+ follicular helper T cells, TEMRA CD8, and CD45RA Terminal Effector CD8+ cells were enriched in NHB women while PD-1+/CD3+ cells and PD-1+/CD8+ cells were enriched in the NHW women (Table). Additionally, several immunosuppressive and systemic inflammatory immune cell types were enriched in NHB women (Table). Conclusions: Deep immunoprofiling using single-cell spectral flow cytometry revealed differences in peripheral immune cells between NHB and NHW women at high risk for breast cancer. The significant enrichment of immunosuppressive monocytes and several activated NK cell populations in PBMC samples from NHB women relative to those from NHW women indicate an altered immune environment that may influence breast cancer development. Additional analysis, including a larger sample size and patients with invasive cancer, are ongoing to further understand if differences in immune fitness between NHB and NHW women are associated with disparities in breast cancer outcomes. Table: PBMC populations (% of CD45+) that are significantly enriched in NHB relative to NHW patients Citation Format: Esther Ogayo, Milos Spasic, Tasnim Rahman, Olga Kantor, Tari King, Peter van Galen, Sandra McAllister, Elizabeth Mittendorf. Deep Immunoprofiling Demonstrates Racial Differences in the Peripheral Immune System in Women at Risk for Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-09-09.

Abstract PO5-02-05: Assessing Vaccine-Mediated Cellular Immune Responses in Patients Receiving Combined Vaccine and Chemotherapy Treatment

Abstract Background: We showed recently that immunizing breast cancer patients with the P10s-PADRE vaccine in combination with chemotherapy activates both humoral and cellular immune responses. Induction of specific antibody response was easily determined by measuring anti-P10s antibodies, however, we observed increase in serum interferon-gamma (IFNg) and tumor necrosis factor-alpha (TNFα) as well as activation of Natural Killer (NK) and T-cells, accurate assessing of which proved complicated due to the non-specific impact of chemotherapy. This study was undertaken to further distinguish the vaccine-induced cellular immune responses from the immune effects induced by chemotherapy, and to evaluate their potential contributions to anti-tumor activity. Methods: Peripheral blood mononuclear cells (PBMCs) from twenty-five subjects who received a combination of P10s-PADRE and standard-of-care chemotherapy were utilized for gene expression analysis and various immune assays. PBMC samples collected before and after treatment were subjected to flow cytometry and ELISpot assays, as well as bulk and single-cell RNA sequencing analyses. For functional assays PBMCs were stimulated with P10s, P10s-PADRE, anti-CD3/CD28, and tumor cell lysates. Differential gene expression analysis, gene set analysis, Hiearchical Clustering, and principal component analysis were performed. Results: Non-specific (anti-CD3/CD28) and specific (peptide, cancer cells) stimulation of PBMCs significantly activated immune cells in post-immune samples compared to pre-immune samples as measured by cytokine secretion, with IFNg being the dominant cytokine secreted. Flow cytometry showed activation of both natural killer and CD8+ cells measured by a significant increase in CD69 expression. These data plus detection of Ab response suggests induction of both humoral and cellular immune responses. However, bulk RNA-seq performed on 24 patients indicate a dramatic reduction of B, T, and NK cells in the post-immune samples. Deconvolution reanalysis of the bulk sequencing data together with complementary scRNA-seq analysis of PBMCs of select subjects supports vaccine-mediated activation of both CD8+ and NK cells and suggests NK cells as a source of detected serum increase in IFNg and TNFα. Conclusions: The P10s-PADRE vaccine elicits substantial and quantifiable cellular and humoral immune responses, despite the deleterious effects of chemotherapy on effector immune cells involved. The evidence indicates that the activation of both CD8+ and NK cells is facilitated by the vaccine. It is probable that the NK cells are the primary contributors of IFNγ and TNFα. These findings carry considerable implications for the structuring of advanced phase trials and the potential application of the vaccine in treating other forms of malignancies. Citation Format: Donald Johann, Fariba Jousheghany, Emily Blitz, Bernice Nounamo, Sam Makhoul, Eric Siegel, Thomas Kieber-Emmons, Behjatolah Monzavi-Karbassi. Assessing Vaccine-Mediated Cellular Immune Responses in Patients Receiving Combined Vaccine and Chemotherapy Treatment [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-02-05.

Differential Expression of LMNA/C and Insulin Receptor Transcript Variants in Peripheral Blood Mononuclear Cells of Leukemia Patients

Background: Recent research has identified alternative transcript variants of LMNA/C (LMNA, LMNC, LMNAΔ10, and LMNAΔ50) and insulin receptors (INSRs) as potential biomarkers for various types of cancer. The objective of this study was to assess the expression of LMNA/C and INSR transcript variants in peripheral blood mononuclear cells (PBMCs) of leukemia patients to investigate their potential as diagnostic biomarkers. Methods: Quantitative TaqMan reverse transcriptase polymerase chain reaction (RT-qPCR) was utilized to quantify the mRNA levels of LMNA/C (LMNA, LMNC, LMNAΔ10, and LMNAΔ50) as well as INSR (IR-A and IR-B) variants in PBMCs obtained from healthy individuals (n = 32) and patients diagnosed with primary leukemias (acute myeloid leukemia (AML): n = 17; acute lymphoblastic leukemia (ALL): n = 8; chronic myeloid leukemia (CML): n = 5; and chronic lymphocytic leukemia (CLL): n = 15). Results: Only LMNA and LMNC transcripts were notably present in PBMCs. Both exhibited significantly decreased expression levels in leukemia patients compared to the healthy control group. Particularly, the LMNC:LMNA ratio was notably higher in AML patients. Interestingly, IR-B expression was not detectable in any of the PBMC samples, precluding the calculation of the IR-A:IR-B ratio as a diagnostic marker. Despite reduced expression across all types of leukemia, IR-A levels remained detectable, indicating its potential involvement in disease progression. Conclusions: This study highlights the distinct expression patterns of LMNA/C and INSR transcript variants in PBMCs of leukemia patients. The LMNC:LMNA ratio shows promise as a potential diagnostic indicator for AML, while further research is necessary to understand the role of IR-A in leukemia pathogenesis and its potential as a therapeutic target.

Monocyte response to SARS-CoV-2 protein ORF8 is associated with severe COVID-19 infection in patients with chronic lymphocytic leukemia.

The open reading frame 8 (ORF8) protein, encoded by the SARS-CoV-2 virus after infection, stimulates monocytes/macrophages to produce pro-inflammatory cytokines. We hypothesized that a positive ex vivo monocyte response to ORF8 protein pre-COVID-19 would be associated with subsequent severe COVID-19. We tested ORF8 ex vivo on peripheral blood mononuclear cells (PBMCs) from 26 anonymous healthy blood donors and measured intracellular cytokine/chemokine levels in monocytes by flow cytometry. The % monocytes staining positive in the sample and change in mean fluorescence intensity (ΔMFI) after ORF8 were used to calculate the adjusted MFI for each cytokine. We then tested pre-COVID-19 PBMC samples from 60 CLL patients who subsequently developed COVID-19 infection. Severe COVID-19 was defined as hospitalization due to COVID-19. In the 26 normal donor samples, the adjusted MFI for interleukin (IL)-1β, IL-6, IL-8, and CCL-2 were significantly different with ORF8 stimulation vs controls. We next analyzed monocytes from pre-COVID-19 PBMC samples from 60 CLL patients. The adjusted MFI to ORF8 stimulation of monocyte intracellular IL-1β was associated with severe COVID-19 and a reactive ORF8 monocyte response was defined as an IL- 1β adjusted MFI ≥ 0.18 (sensitivity 67%, specificity 75%). The median time to hospitalization after infection in CLL patients with a reactive ORF8 response was 12 days versus not reached for patients with a non-reactive ORF8 response with a hazard ratio of 7.7 (95% CI: 2.4-132, p=0.005). These results provide new insight on the monocyte inflammatory response to virus with implications in a broad range of disorders involving monocytes.

Molecular Analysis and Ex Vivo Infectivity of Seronegative Occult Hepatitis C Virus: A Study in Single Haemodialysis Centre.

In occult hepatitis C virus infection (OCI), hepatitis C virus ribonucleic acid (HCV RNA) is detectable in peripheral blood mononuclear cells (PBMCs) but is not evident in serum or plasma. Understanding of OCI in patients with seronegative anti-HCV antibodies is limited. In this study, six HCV isolates from haemodialysis (HD) patients with seronegative OCI were identified by molecular assays and phylogenetic analysis. The virus infectivity was assessed ex vivo using a primary naïve PBMC culture system. HCV isolates obtained from the PBMCs of 10 patients with chronic HCV infection (CCI) were characterised concurrently and used as positive controls in the cell culture. Sequence analysis of the 5' untranslated region (UTR) and non-structural 5B (NS5B) region revealed that HCV genotype 3 was the most prevalent virus type in both the OCI and CCI groups. One of the occult HCV isolates was identified as a mixed type. The mean viral load (log10 RNA copies/106 cells) in the PBMC samples of the OCI group (M = 3.4, SD = 0.7) was lower than that of the CCI group (M = 4.6, SD = 1.7). Upon culture, de novo OCI-HCV replicates were detected in five out of six naïve PBMC cultures. Analysis of the replicates showed a single guanine addition in the domain III of 5'-UTR but the overall molecular structure was retained. Seronegative OCI is an active form of infection that replicates at a low level in PBMCs. Seronegative OCI may share the same route of transmission as CCI. The retained viral competency may have an implication for its persistence.

Combinational therapy of CAR T-cell and HDT/ASCT demonstrates impressive clinical efficacy and improved CAR T-cell behavior in relapsed/refractory large B-cell lymphoma

BackgroundApproximately two-thirds of patients with relapsed or refractory large B-cell lymphoma (R/R LBCL) do not respond to or relapse after anti-CD19 chimeric antigen receptor T (CAR T)-cell therapy, leading to...

Single-cell 5′ RNA sequencing of camelid peripheral B cells provides insights into cellular basis of heavy-chain antibody production

Camelids produce both conventional tetrameric antibodies (Abs) and dimeric heavy-chain antibodies (HCAbs). Although B cells that generate these two types of Abs exhibit distinct B cell receptors (BCRs), whether these two B cell populations differ in their phenotypes and developmental processes remains unclear. Here, we performed single-cell 5′ RNA profiling of peripheral blood mononuclear cell samples from Bactrian camels before and after immunization. We characterized the functional subtypes and differentiation trajectories of circulating B cells in camels, and reconstructed single-cell BCR sequences. We found that in contrast to humans, the proportion of T-bet+ B cells was high among camelid peripheral B cells. Several marker genes of human B cell subtypes, including CD27 and IGHD, were expressed at low levels in the corresponding camel B cell subtypes. Camelid B cells expressing variable genes of HACbs (VHH) were widely present in various functional subtypes and showed highly overlapping differentiation trajectories with B cells expressing variable genes of conventional Abs (VH). After immunization, the transcriptional changes in VHH+ and VH+ B cells were largely consistent. Through structure modeling, we identified a variety of scaffold types among the reconstructed VHH sequences. Our study provides insights into the cellular context of HCAb production in camels and lays the foundation for developing single-B cell-based camelid single-domain Ab screening.

Combination of IFN-gamma with STING agonist and PD-1 immune checkpoint blockade: a potential immunotherapy for gastric cancer.

Suppression of the cGAS-STING pathway is an immune escape mechanism in cancer cells. The critical role of this pathway in gastric cancer (GC) is not fully understood. Herein, we evaluated the effect of the interferon-gamma (IFN-gamma), STING agonist, PD-1 immune checkpoint blockade, and their combination on the cGAS-STING pathway in GC. Expression of cGAS and STING in tumor tissue samples and adjacent normal tissue (ANT) biopsies of fifty new GC patients was evaluated by quantitative real-time PCR (qRT-PCR). Moreover, cGAS and STING expression levels were examined in Peripheral Blood Mononuclear Cells (PBMC) samples of forty GC patients and twenty-five healthy subjects. The apoptosis rate of cancer cells was analyzed by Annexin V-FITC/PI. Cell proliferation was measured by the BrdU assay. Also, IFN-β levels were evaluated in the supernatants of the treated groups. The cGAS expression was decreased in patients with distant metastasis. Co-cultures treated with IFN-gamma showed an elevated level of cGAS and STING expressions in PBMC and cancer cells. The rate of apoptosis increased in all the treatment groups. In addition, the rate of proliferation in PBMCs increased in different treated groups. The main role of PBMCs in cytotoxicity was determined by a comparative analysis of the viability of cells treated with all treatments, both with and without PBMCs. The production of IFN-β was elevated in all treated groups. The current study suggests that a combination therapy using IFN-gamma, STING agonist, and anti-PD-1 antibody can provide a promising approach to the treatment of GC.

Abstract LB135: Effects of acute psychological stress on DNA damage levels in peripheral blood mononuclear cells: An experimental study with healthy human volunteers

Abstract Increased levels of DNA damage in human peripheral blood mononuclear cells (PBMC), as indicated by classic alkaline comet and γ-H2AX assays, have been widely used in toxicology and epidemiology studies of potential environmental carcinogens. To date, however, little research has explored the possibility that exposure to acute psychological stresses in daily life may acutely increase levels of DNA damage and thus, much like repeated tobacco use, may cumulatively contribute to lifetime cancer risk. The purpose of the present experimental study was to provide a first critical test of the hypothesis that exposure of healthy humans to a brief psychological stress results in increased levels of DNA damage. We also tested preventative effects of a beta-adrenergic receptor blocker (propranolol), a mechanistic pathway for psychological stress effects on DNA supported by preclinical research. Healthy volunteers were recruited by multiple strategies including targeted outreach (telephone/email), with oversampling for age, sex, and minority status. Eligibility was confirmed by self-report, clinical testing (blood & urine), and MD review. On the experimental day, participants were randomized to receive either a single dose of propranolol (80mg) or a placebo. After a 60 min rest period with video distraction, all participants (n=123; 48.8% ≥ 40yr; 52% women; 52% minority) underwent a well-established, 15-minute stress challenge, a speech task and mental arithmetic (Trier Social Stress Test, TSST), with psychological stress confirmed by visual analog scales. Blood samples were collected (via IV line) immediately before and after the TSST. As the primary study outcome, DNA damage in PBMC was assessed (blind) with Comet IV software (% tail intensity) following single cell gel electrophoresis under alkaline conditions with and without in vitro exposure to 25uM H2O2 (comet assay). For a subset of participants with sufficient frozen PBMC samples available (n=43), levels of pan-nuclear γ-H2AX were determined with a validated, sensitive, immunoblot method that detects H2AX phosphorylation at Ser139, and expresses γ-H2AX as the ratio of p-Ser139-H2AX to total H2AX. Exploratory nonparametric analyses revealed that undergoing the TSST significantly increased levels of DNA damage in PBMC (H2O2 comet assay [p<.001] and γ-H2AX biomarker [p=.03], Wilcoxon tests) in the group of participants that was randomized to pretreatment with placebo. In the group pretreated with propranolol before the TSST, a significant increase in γ-H2AX was not found. Conclusion. Supporting the study hypothesis that acute stress can increase levels of DNA damage, the results of the present study highlight the potential importance of additional research to address the possibility that repeated exposures to acute life stresses may cumulatively contribute to increased lifetime risk of cancer. Citation Format: Dana H. Bovbjerg, John Schmitz, Kurt D. Ackerman, Scott R. Beach, Albert D. Donnenberg, Jessica Manculich, Matthew F. Muldoon, Patricia L. Opresko, Patrick M. Tarwater, Hong Wang, Frank J. Jenkins. Effects of acute psychological stress on DNA damage levels in peripheral blood mononuclear cells: An experimental study with healthy human volunteers [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 LB135.

Rapid Isolation and Detection of Breast Cancer Circulating Tumor Cells Using Microfluidic Sequential Trapping Array

AbstractCirculating tumor cells (CTCs) have garnered special attention as promising cancer biomarkers. Phenotypic changes of CTCs reveal invaluable information for oncologists in disease prognosis and adjusting their treatment options. Microfluidic technology has emerged as a promising tool for CTC isolation; however, two major hurdles remain to be solved in employing them in CTC analysis. First, a rapid CTC isolation scheme is needed to allow immediate use of patient samples for point‐of‐care treatment monitoring. Second, multiplexed and streamlined CTC imaging is needed to facilitate CTC detection. Here, a microfluidic CTC sequential trapping array (STA) is proposed which addresses these hurdles enabling pipette‐based CTC isolation and simultaneous profiling of multiple CTC protein expressions. The STA device isolates CTCs based on their size difference from blood cells and increases sample processing throughput through its parallel design configuration. It successfully isolates CTC from a depleted peripheral blood mononuclear cells sample of breast cancer patients with a high recovery rate of 80% and discriminates the number and types of CTCs in breast cancer based on their disease stage. These findings will open a new avenue in clinical translation of CTC profiling technologies. It will be an example for future translational developments in CTC‐based cancer management.

Senescent fibroblasts and innate immune cell activation might play a role in the pathogenesis of elderly atopic dermatitis

BackgroundElderly atopic dermatitis (AD) is a subtype of AD defined by age (≥60 years). The molecular characteristics of elderly AD remain to be clarified. ObjectiveWe sought to characterize the molecular features of skin lesions and peripheral blood mononuclear cells (PBMCs) in patients with AD across different age, focusing on elderly AD. MethodsSkin and PBMCs samples were used for RNA sequencing. Analysis of differentially expressed genes and gene set variation analysis were performed. Immunofluorescence staining, quantitative real-time PCR (qRT-PCR), flow cytometry and transwell assay were used for validation. ResultsCompared with healthy controls, the skin transcriptome of AD patients showed common signatures of AD, like barrier dysfunction and enhanced Th1/Th2/Th17 immune pathways. In PBMCs, the expression of Th1/Th2 response genes was more remarkable in adult AD, while expression of Th17-related genes was significantly higher in childhood AD. The gene modules associated with natural killer (NK) cells were downregulated in elderly AD. In skin lesions, elderly AD exhibited enrichment of macrophages, fibroblasts and senescence-associated secretory phenotype (SASP) related genes. The correlation among fibroblasts, SASP and innate immune cells were revealed by the co-localization of fibroblasts, macrophages and NK cells in the lesions across different age groups. Fibroblasts under inflammation or senescence could induce stronger chemotaxis of macrophages and NK cells. ConclusionWe identified the molecular phenotypes of skin lesions and PBMCs in elderly AD individuals. Fibroblasts, innate immune cells, and SASP might play important roles in the pathogenesis of elderly AD.

Proteomic and cellular characterization of Omicron breakthrough infections and a third homologous or heterologous boosting vaccination in a longitudinal cohort

BackgroundThe understanding of dynamic plasma proteome features in hybrid immunity and breakthrough infection is limited. A deeper understanding of the immune differences between heterologous and homologous immunization could assist in the future establishment of vaccination strategies. MethodsIn this study, 40 participants who received a third dose of either a homologous BBIBP-CorV or a heterologous ZF2001 protein subunit vaccine following two doses of inactivated coronavirus disease 2019 vaccines and 12 patients with BA.2.2 breakthrough infections were enrolled. Serum samples were collected at Days 0, 28, and 180 following the boosting vaccination and breakthrough and then analyzed using neutralizing antibody tests and mass spectrometer-based proteomics. Mass cytometry of peripheral blood mononuclear cell samples was also performed in this cohort. ResultsThe chemokine signaling pathway and humoral response markers (IgG2 and IgG3) associated with infection were found to be upregulated in breakthrough infections compared to vaccination-induced immunity. Elevated expression of IGKV, IGHV, IL-17 signaling, and the phagocytosis pathway, along with lower expression of FGL2, were correlated with higher antibody levels in the boosting vaccination groups. The MAPK signaling pathway and Fc gamma R-mediated phagocytosis were more enriched in the heterologous immunization groups than in the homologous immunization groups. ConclusionBreakthrough infections can trigger more intensive inflammatory chemokine responses than vaccination. T-cell and innate immune activation have been shown to be closely related to enhanced antibody levels after vaccination and therefore might be potential targets for vaccine adjuvant design.

Comparative single-cell RNA sequencing analysis of immune response to inactivated vaccine and natural SARS-CoV-2 infection.

Uncovering the immune response to an inactivated SARS-CoV-2 vaccine (In-Vac) and natural infection is crucial for comprehending COVID-19 immunology. Here we conducted an integrated analysis of single-cell RNA sequencing (scRNA-seq) data from serial peripheral blood mononuclear cell (PBMC) samples derived from 12 individuals receiving In-Vac compared with those from COVID-19 patients. Our study reveals that In-Vac induces subtle immunological changes in PBMC, including cell proportions and transcriptomes, compared with profound changes for natural infection. In-Vac modestly upregulates IFN-α but downregulates NF-κB pathways, while natural infection triggers hyperactive IFN-α and NF-κB pathways. Both In-Vac and natural infection alter T/B cell receptor repertoires, but COVID-19 has more significant change in preferential VJ gene, indicating a vigorous immune response. Our study reveals distinct patterns of cellular communications, including a selective activation of IL-15RA/IL-15 receptor pathway after In-Vac boost, suggesting its potential role in enhancing In-Vac-induced immunity. Collectively, our study illuminates multifaceted immune responses to In-Vac and natural infection, providing insights for optimizing SARS-CoV-2 vaccine efficacy.

Abstract 3858: Peripheral CD38+ effector memory (EM) T cells are dynamic biomarkers of response to neoadjuvant immune checkpoint blockade (NeoICB) in patients (pts) with resectable dedifferentiated liposarcoma (DDLPS) and undifferentiated pleomorphic sarcoma (UPS)

Abstract Introduction: NeoICB is a promising approach for pts with DDLPS and UPS. However, biomarkers to identify which pts benefit from this treatment and understanding the mechanisms of response and resistance to ICB remains a challenge. Methods: We conducted a randomized phase 2 trial of neoICB (anti-PD1 +/- CTLA4) in pts with resectable DDLPS (n=17) and UPS (n=10). Pts with UPS received concurrent neoICB and radiation. The primary endpoint of the trial was pathological response; 3 DDLPS and 9 UPS pts were responders (R) and 14 DDLPS and 1 UPS pts were non-responders (NR). Peripheral blood mononuclear cells (PBMCs) were collected at baseline, prior to cycle 2 of neoICB (C2D1), and at the time of surgery (SURG). We performed mass cytometry (CyTOF) to identify immune cells associated with response and their dynamic changes with neoICB. We designed a custom 41-parameter panel using CD45 live cell barcoding with 31 markers for immune profiling, of which 23 were selected to detect key cell lineage and 8 to detect signaling markers. Batch correction and high dimensional data analyses were performed using CyCombine and CyTOF workflow R packages. Longitudinal comparisons between immune cell populations were conducted using Mann-Whitney U Test. Results: Of 79 available PBMC samples from 27 pts, 63 passed quality control after thawing (≥30% viability) and data clean-up (at least 10,000 events). Data was down-sampled to 5000 events for analysis. Of 19 immune cell types identified, monocytes were the most common followed by CD4+ and CD8+ T cells. There was an early increase in CD38+ effector/effector memory (EM) T cells at C2D1 in pts with DDLPS (CD8+ p=0.005, CD4+ p<0.001) and a similar increase observed in pts with UPS (CD8+ C2D1 p=NS, SURG p=0.034; CD4+ C2D1 p=0.024, SURG p=0.06) and these cells had higher Ki67 expression than CD38- T cells (p<0.005). At baseline, R pts with DDLPS had higher fractions of CD38+ EM CD8+ T cells than NR pts (p=0.044). Analysis in the UPS cohort was limited by the low number of NR pts. Higher expression of TIGIT was observed on CD38+ EM CD8+ T cells compared to CD38- counterparts at all time points. Conclusion: Peripheral CD38+ EM CD8+ T cells are associated with response to neoICB in pts with DDLPS. Longitudinal peripheral immune profiling reveals an increase in CD38+ EM CD8+ and CD4+ T cells with neoICB in pts with DDLPS and UPS. These cells are proliferative and express higher levels of TIGIT, which could be a future target for therapeutic exploration. Importantly, changes in the peripheral immune populations may precede changes in the tumor microenvironment (TME) as preliminary analyses of the TME did not identify early (C2D1) changes. TME imaging mass cytometry analyses are currently underway to associate peripheral and intratumor changes in immune populations. Citation Format: Elise F. Nassif, Hsinyi Lu, Michael Stanford, Karen Millerchip, Diana Romero, Jared Malke, Brenna Matejka, Jeffrey E. Gershenwald, Susmita Kumari, Duncan H. Mak, Angelique J. Lin, Jared K. Burks, Wei-Lien Wang, Alexander J. Lazar, Neeta Somaiah, Cara Haymaker, Christina L. Roland, Emily Z. Keung. Peripheral CD38+ effector memory (EM) T cells are dynamic biomarkers of response to neoadjuvant immune checkpoint blockade (NeoICB) in patients (pts) with resectable dedifferentiated liposarcoma (DDLPS) and undifferentiated pleomorphic sarcoma (UPS) [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 3858.

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 3628: Combined mek and jak inhibition in vitro is an effective strategy in overcoming jak inhibitor resistance in primary myelofibrosis (PMF)

Abstract Introduction Primary myelofibrosis (PMF) is a myeloproliferative neoplasm arising from various mutations in myeloid-committed hematopoietic progenitor cells. Treatment of PMF is challenging as Jak inhibitor resistance is common, making the expansion of molecular characterization and novel therapeutic target identification in PMF essential. The main goals of this study are to determine both the presence of Ras activity in PMF and the impact on Ras/MAP Kinase pathway targeting in the disease. Methods Integrated genomic-transcriptomic sequencing analyses were performed on peripheral blood mononuclear cells (PBMCs) purified from 27 patients with PMF at a single institution; protein was then obtained from whole cell lysates. Ras activity was then assessed by applying Ras binding domain (RBD) to sample in an ELISA and was compared between patients with PMF, essential thrombocythemia (ET), polycythemia vera (PV) and healthy donors. Statistical analyses were conducted using two-way unpaired t-tests. The next set of in vitro studies aimed to evaluate the effects of MEK inhibition in PMF. Cell viability studies were first performed on human erythroleukemia (HEL) cells to identify IC50 values. Colony forming assays in methylcellulose-enriched media formulated for the growth of CD34+ cells were then performed in 4 conditions: Ruxolitinib and Cobimetinib monotherapy, combined therapies and untreated sample. Results As previously reported in our initial study, Ras activity in 21 patients with MF was 1.74 fold higher than healthy donors (13 volunteers, p=0.0304) and 1.64 fold higher than in PV/ET (12 patients, p=0.0462). We also reported previously on Ras activity from differential enrichment of myeloid and lymphoid cells from PBMC samples, where a key observation was that myeloid contribution to Ras activity in PMF patients with RAS/MAP Kinase pathway mutations was 3.8 times higher in Ras/MAP Kinase WT patients (p=0.0012), potentially suggesting that RAS pathway mutations have a predominance in myeloid cells. In HEL cell viability assays, cobimetinib and ruxolitinib, when utilized in tandem, resulted in more cell growth inhibition than either agent individually. This same finding was recapitulated in colony forming assays in 4 different patients with varying levels of sensitivity to Jak inhibitors, in all of whom ruxolitinib and cobimetinib lead to a greater reduction in colony formation than ruxolitinib monotherapy. Discussion Data from colony forming assays of bone marrow from patients with PMF identifies at least an additive effect of combined Jak and Ras/MAP Kinase (specifically Mek) inhibition on CD34+ colony formation. The specific observation that Ras pathway inhibition may be utilized as a means of overcoming Jak inhibitor resistance in PMF is a novel finding in this field and may be a valuable avenue for future early-phase clinical trials. Citation Format: Samuel Benjamin Reynolds, Sho Matono, Malathi Kandarpa, Kristen Pettit, Mark Ribick, Moshe Talpaz, Qing Li. Combined mek and jak inhibition in vitro is an effective strategy in overcoming jak inhibitor resistance in primary myelofibrosis (PMF) [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 3628.

Abstract 5376: Quantitative profiling of HLA class I and class II antigens and neoantigens in tissue biopsy and PBMC samples using an optimized mass spectrometry-based workflow

Abstract Major histocompatibility complex (MHC) molecules play a central role in orchestrating immune responses by presenting antigenic peptides derived from both self and foreign proteins. In the context of cancer, understanding the repertoire of tumor-associated antigens (TAAs) presented by MHC molecules (or HLA molecules in human) is crucial for deciphering how the immune system recognizes and responds to malignant cells. The identification of neoantigens, unique to individual tumors due to somatic mutations, has become a focal point in immunopeptidomic studies. One significant hurdle in systematic immunopeptidomics analysis is the high input material requirement. Here, we present a semi-automated workflow to robustly identify and quantify immunopeptides from reduced amounts of clinical tissue biopsy and peripheral blood mononuclear cell samples. At the core of immunopeptidomics is the enrichment of HLA-associated peptides, followed by identification using mass spectrometry and bioinformatics tools. We optimized the native lysis and a sequential immunoprecipitation workflow for both class I and class II immunopeptides while ensuring scalability and reproducibility. Leveraging the magnetic properties of the beads, 1,000 samples can be processed within a week by a single operator. For both tissue and PBMC samples, we performed a systematic ramping experiment starting with as little as 2.5mg tissue or 5 million PBMCs. In all experiments, The established sample preparation offers high reproducibility and identifications of good quality: 1) Class-I immunopeptides: >60% of the peptides identified are 9-mers, >80% predicted strong binders, and the expected amino acids are enriched at the anchor positions; 2) Class-II immunopeptides, >50% of the peptides identified are 14-to-16-mers, and >50% are predicted strong binders. Furthermore, the pipeline is highly sensitive as we could still identify over 2,800 class-I immunopeptides when processing 2.5 mg fresh frozen tissue and 2,000 - 3,000 class-I immunopeptides when starting from 5 million PBMCs. Furthermore, the developed immunopeptidomics workflow was deployed to profile a cohort of 12 cancerous and matched healthy lung tissue samples. Their Class-I immunopeptidomes clearly displayed a pattern where matched tissues from each subject cluster together, further underlining the fact that the intricate immune-tumor interface is highly personalized. Overall, we established a robust pipeline for for class-I and II immunopeptidome profiling from clinically relevant sample types. Taking advantage of the nature of mass spectrometry-based methods, customized targeted assays can be developed without the need for affinity reagent, allowing specific and absolute quantification of any immunopeptide of interest. Citation Format: Ilja E. Shapiro, Marco Tognetti, Tikira Temu, Oliver M. Bernhardt, Daniel Redfern, Yuehan Feng, Roland Bruderer, Lukas Reiter. Quantitative profiling of HLA class I and class II antigens and neoantigens in tissue biopsy and PBMC samples using an optimized mass spectrometry-based workflow [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 5376.

Abstract 7045: RNA sequencing approaches enable tissue specific B and T cell gene expression and immune repertoire profiling

Abstract The adaptive immune system defends the human body from pathogens through the recognition ability of antigen receptors from B and T cells. Different organs play various immune function roles, and therefore present varied B and T cell gene expression and immune repertoire profiles. Abnormality of these profiles may reflect immunological disorder, presence of neoplastic tissue, or response to infectious agent. High-throughput sequencing technology has facilitated the understanding of gene expression and related cell functions, but in-depth understanding of B and T cell phenotype and clonotype profiling is an emerging application that has yet to take full advantage of sequencing technology for diagnosis and prognosis of disease. Here, we share results of RNA sequencing of immune system tissues; including B cell and T cell specific gene expression and full-length immune repertoire profiling. Total RNA was extracted from various tissue samples including healthy donor peripheral blood mononuclear cells (PBMC), lymph nodes, bone marrow, spleen, thyroid, thymus and colon, as well as diseased PBMC and colon samples. RNA-seq and Immune-seq libraries were made from these samples and sequenced on Illumina NextSeq2000. Gene expression analysis and immune repertoire profiling from RNA-seq data were performed using edgeR and TRUST4. Immune repertoire sequencing data was processed using pRESTO and IgBlast. We correlated RNA-seq with Immune-seq results for B-cell receptor and T-cell receptor clonotypes. Differential expression and clonotype profiling comparisons were also performed between normal and diseased RNA samples. The effect of reference on immune repertoire detection sensitivity and accuracy was further investigated by comparing IMGT reference and AIRR-C Human IG Reference Sets. This study has identified tissue specific B/T cell phenotype and clonality patterns that relate to tissue functions and disease progression. RNA-seq enables highly multiplex immunophenotyping which is traditionally performed by flow cytometry. RNA-seq can detect high-abundant clones that correlate with Immune-seq, with the latter targeted approach providing more in depth clonality signatures. References comparison results show the importance of continued community effort to develop more representative and accurate immunorepertoire germ line references for clonotype annotation in diverse populations. Integrating both RNA sequencing and immune repertoire sequencing approaches allows accurate phenotype and clonotype determination for immune landscape in various tissues. Citation Format: Chen Song, Evan Janzen, Tessa Devoe, Ariel Erijman, Gautam Naishadham, Li Song, Bradley W. Langhorst. RNA sequencing approaches enable tissue specific B and T cell gene expression and immune repertoire profiling [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 7045.

Abstract 5266: Deciphering the systemic immune effects of abemaciclib in recurrent metastatic breast cancer: A multicenter, prospective study

Abstract Background: Abemaciclib, a CDK4/6 inhibitor, stands as a cornerstone for managing hormone receptor-positive, HER2-negative advanced or metastatic recurrent breast cancer. It is known to modulate the tumor immune microenvironment, enhancing anti-tumor responses. Yet, its systemic immunological impact remains to be elucidated. This investigation aims to delineate the influence of abemaciclib on both the gut microbiota and the systemic immune landscape in patients with breast cancer. Methods: A multicenter, prospective clinical trial was conducted involving 40 patients with hormone receptor-positive, HER2-negative advanced or metastatic recurrent breast cancer, scheduled for abemaciclib therapy. The trial's primary objectives were to evaluate the duration of treatment efficacy and the severity of gastrointestinal toxicity. Secondary objectives included assessing the interplay between circulating immune profiles, gut microbiota, and clinical outcomes. Patient samples, including peripheral blood mononuclear cells (PBMCs) for single-cell RNA sequencing and stool for 16S rRNA gene analysis, were collected at baseline and after 90 days of treatment initiation. Results: This interim report presents findings from 39 patients. We examined 75 PBMC samples and 78 stool samples. Preliminary analysis indicated that abemaciclib precipitated an upregulation of cytokines pivotal for anti-tumor immunity, notably IL-7 and IP-10, alongside an activation of circulating innate immune cells such as CD4-positive T cells and dendritic cells. Concurrently, gut microbiota analysis revealed a post-treatment reduction in alpha diversity, with notable shifts in microbial populations implicated in innate immune activation. Discussion: This study presents novel insights into the systemic immunomodulatory effects of abemaciclib. Our results suggest that abemaciclib's therapeutic efficacy may partially derive from its ability to alter the circulating immune profile and gut microbiota. These findings could pave the way for innovative immunotherapeutic approaches, integrating gut microbiome considerations into the treatment of hormone receptor-positive, HER2-negative breast cancer. Citation Format: Yukiko Fukui, Kosuke Kawaguchi, Yurina Maeshima, Yuri Fujimoto, Hiroshi Ishiguro, Kazuhiko Yamagami, Sachiko Takahara, Hirofumi Suwa, Masae Torii, Shigenori Nagai, Yasuaki Sagara, Wakako Tsuji, Hiroyasu Yamashiro, Takeshi Kotake, Shinji Fukuda, Kuniaki Saito, Yasuko Yamamoto, Masako Kataoka, Yuki Himoto, Atsushi Yonezawa, Yuki Nakamura, Wei Li, Sunao Tanaka, Satoshi Morita, Masahiro Takada, Masakazu Toi. Deciphering the systemic immune effects of abemaciclib in recurrent metastatic breast cancer: A multicenter, prospective study [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 5266.

Abstract 6428: Transcriptome analysis of tissues and peripheral blood revealed potential biomarkers for chemo/radio-immunotherapy in non-small cell lung cancer

Abstract Background: Combination of anti-PD-(L)1 antibody with chemotherapy has shown promising efficacy in advanced/metastatic non-small cell lung cancer (NSCLC). Dynamics of peripheral blood mononuclear cells (PBMC) might have impact on repertore of immune cells further resulting different immune infiltration in tumor microenvironment. Thus, monitoring changes in PBMC during therapy or measuring tumor microenvironment provides the possibility to predict the efficacy of tumor immunotherapy. Methods: Patients diagnosed with NSCLC (Stage III-IV)were enrolled in this study. Anti-PD-L1 and chemo/radio combination therapy was performed as adjuvant therapy. Tumor samples were obtained prior to adjuvant therapy PBMC samples were obtaind at multiple time points, including prior adjuvant therapy, 3 months after adjuvant therapy, and 3 months after adjuvant therapy. Tumor and PBMC mRNA sequencing were performed by Amoy Diagnostics Co Ltd. (Xiamen, China). Patients who achieved CR/PR/SD and PFS > 6 months were defined as well responders. Pathway enrichment and GSEA analyses were performed by clusterProfiler (version 4.0.5) on GO, KEGG, and from six different gene sets. Correlations between immune cellular signature ES scores and gene expression were estimated using Pearson correlation. Results: 28 patients were enrolled; mean age was 60.38±7.86 years, 27 (96.4%) patients were male. 21 (75.0%) patients were identified as responder and 7 (25.0%) were non-responder. In the baseline tumor samples, immune infiltration was more extensive in responders compared to non-responders. Cytotoxic lymphocytes, activated CD8+ T cell, and NK cells were significantly enriched in responders. High expression of CCL17 and down regulation of CTSG/PLK1/PRTN3, a signaling axis that inhibits histone H3 hydrolysis and promotes monocyte to macrophage differentiation, was significant in responders. Dynamic monitoring of PBMC before and after treatment showed significant upregulation of immune infiltration-related pathways in responders, including effector cells, NK cells and CD8+ T cells. In the non-responder group, the up-regulated pathways after treatment were mainly enriched in tumor growth rate, EMT, and matrix remodeling. Multi-time point analysis showed that CEACAM5/MKI67 level and tumor proliferation rate in non-responders were higher than those in responders at each time point after treatment. Conclusion: Baseline tumor transcripton signatures and dynamic changes in PBMC could be applied to develop novel biomarkers for immune-related therapy. Patients with stronger immune infiltration signals in baseline tumor samples and PBMC after treatment had better immune response. The upregulation of proliferation-related signals in PBMC predicts poor immune response. Citation Format: Jing Hu, Jianguo Zhang, Peng Cheng, Yanhua Chen, Changbin Zhu, Feilin Yi, Conghua Xie. Transcriptome analysis of tissues and peripheral blood revealed potential biomarkers for chemo/radio-immunotherapy in non-small cell lung cancer [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 6428.