Higher Proportion Of CD4 Research Articles

Lymphocyte Function in Tertiary Lymphoid Structures Predicts Hepatocellular Carcinoma Outcome

An increasing number of studies have revealed a correlation between tertiary lymphoid structures (TLSs) and the outcome of hepatocellular carcinoma (HCC). Nevertheless, the associations between the heterogeneity of cellular composition and the overall survival (OS) in HCC remain unexplored. Here, we evaluated the cancer tissues from 150 HCC individuals treated at the Tumor Hospital Affiliated with Nantong University using multiplex immunofluorescence to determine the presence and characteristics of TLS and to investigate the relationship between intra-TLS immunologic activity, TLS maturation, and intratumoral immune cell infiltration. Prognostic factors influencing the outcome were identified through both univariate and multivariate analyses. Additionally, the levels of cytotoxic T-lymphocyte antigen-4 (CTLA-4), programmed death 1, programmed death-ligand 1, and lymphocyte activation gene-3 were determined, as well as their relationship with TLS features were determined. TLS was detected in 71 (47.3%) of the 150 HCC cases and was related to higher intratumoral infiltration levels of lymphocytes. Additionally, intra-TLS lymphocyte proliferation correlated with that of intratumoral lymphocytes, and the presence of TLS and a high proportion of mature TLS demonstrated a significant correlation with better prognosis (P = .013 and P = .03, respectively). Among TLS-positive tumors, a high proportion of B cells expressing activation-induced cytidine deaminase and a high proportion of CD8+ T cells expressing CD45RO were significantly related to improved OS (P = .01 and P < .001, respectively). Comparatively, a high proportion of CD21+CD20+ B cells demonstrated a significant correlation with poorer OS (P < .002). A markedly reduced number of CTLA-4+ cells in the stromal regions in TLS-negative tumors was observed compared with TLS-positive tumors (P = .01). These findings reveal a correlation between TLS presence and improved OS in HCC patients. However, TLS exhibited significant variation in maturation state, T- and B-cell proliferation, and expression of markers related to B- and T-cell function. Notably, these characteristics were also found to possess prognostic significance, indicating that certain TLS might hinder tumor immunity by inhibiting immune cells, whereas others may foster antigen-driven immune responses, likely influenced by the composition and functional status of intra-TLS lymphocytes.

Stem cell memory EBV-specific T cells control EBV tumor growth and persist in vivo.

Adoptive T cell therapy (ACT), the therapeutic transfer of defined T cell immunity to patients, offers great potential in the fight against different human diseases including difficult-to-treat viral infections, but persistence and longevity of the cells are areas of concern. Very-early-differentiated stem cell memory T cells (TSCMs) have superior self-renewal, engraftment, persistence, and anticancer efficacy, but their potential for antiviral ACT remains unknown. Here, we developed a clinically scalable protocol for expanding Epstein-Barr virus (EBV)-specific TSCM-enriched T cells with high proportions of CD4+ T cells and broad EBV antigen coverage. These cells showed tumor control in a xenograft model of EBV-induced lymphoma and were superior to previous ACT protocols in terms of tumor infiltration, in vivo proliferation, persistence, proportion of functional CD4+ T cells, and diversity of EBV antigen specificity. Thus, our protocol may pave the way for the next generation of potent unmodified antigen-specific cell therapies for EBV-associated diseases, including tumors, and other indications.

Efficacy and biomarker analysis of second-line nab-paclitaxel plus sintilimab in patients with advanced biliary tract cancer.

Biliary tract cancer (BTC) is a highly aggressive malignancy with limited second-line therapy. We conducted this phase 2 trial to evaluate the efficacy and safety of second-line nab-paclitaxel plus sintilimab in advanced BTC. Histologically confirmed advanced BTC patients with documented disease progression after first-line chemotherapy were enrolled. Subjects received nab-paclitaxel 125 mg/m2 on days 1 and 8 plus sintilimab 200 mg on day 1, administered every 3 weeks. The primary end point was the objective response rate (ORR). The secondary end points were progression-free survival (PFS), overall survival (OS), and adverse reactions. Simultaneously, next-generation sequencing, programmed cell death ligand 1 immunohistochemistry and multiplex immunofluorescence of tumor-infiltrating lymphocytes were applied to explore potential biomarkers. Twenty-six subjects were consecutively enrolled. The ORR was 26.9% (7/26), including two complete responses and five partial responses, which met the primary end point. The disease control rate was 61.5% (16/26). The median PFS was 169 days (about 5.6 months, 95% confidence interval [CI] 60-278 days). The median OS was 442 days (about 14.7 months, 95% CI 298-586 days). Grade 3 treatment-related adverse events (TRAEs) were mainly anemia (27%), leukopenia (23%), neutropenia (19%), and peripheral sensory neuropathy (8%). No grade 4 or 5 TRAEs occurred. Biomarker analysis suggested that positive PD-L1 and high proportions of CD8+ T-cell infiltration were correlated with improved clinical outcome. Nab-paclitaxel plus sintilimab is a potentially effective and tolerable second-line regimen for advanced BTC that deserves to be studied in large-scale trials. PD-L1 status and CD8+ T cell infiltration might be promising biomarkers for efficacy prediction.

Differences in microenvironment of lung cancer and pleural effusions by single-cell RNA sequencing

BackgroundDirect comparison of tumor microenvironment of matched lung cancer biopsies and pleural effusions (PE) from the same patients is critical in understanding tumor biology but has not been performed. This is the first study to compare the lung cancer and PE microenvironment by single-cell RNA sequencing (scRNA-seq). MethodsMatched lung cancer biopsies and PE were obtained prospectively from ten patients. We isolated CD45+ cells and performed scRNA-seq to compare the biopsies and PE. ResultsPE had a higher proportion of CD4+ T cells but lower proportion of CD8+ T cells (False detection rate, FDR = 0.0003) compared to biopsies. There was a higher proportion of naïve CD4+ T cells (FDR = 0.04) and naïve CD8+ T cells (FDR = 0.0008) in PE vs. biopsies. On the other hand, there was a higher proportion of Tregs (FDR = 0.04), effector CD8+ (FDR = 0.006), and exhausted CD8+ T cells (FDR = 0.01) in biopsies. The expression of inflammatory genes in T cells was increased in biopsies vs. PE, including TNF, IFN-ɣ, IL-1R1, IL-1R2, IL-2, IL-12RB2, IL-18R1, and IL-18RAP (FDR = 0.009, 0.013, 0.029, 0.043, 0.009, 0.013, 0.004, and 0.003, respectively). The gene expression of exhaustion markers in T cells was also increased in tumor biopsies including PDCD1, CTLA4, LAG 3, HAVCR2, TIGIT, and CD160 (FDR = 0.008, 0.003, 0.002, 0.011, 0.006, and 0.049, respectively). ConclusionsThere is a higher proportion of naïve T cells and lower proportion of exhausted T cells and Tregs in PE compared to lung cancer biopsies, which can be leveraged for prognostic and therapeutic applications.

CD8+T-cell response to mutated HLA-B*35-restricted Gag HY9 and HA9 epitopes from HIV-1 variants from Medellin, Colombia

The HLA-B*35 alleles have been associated with a slow or rapid progression of HIV-1 infection. However, the mechanisms related to HIV-1 progression have yet to be entirely understood. Several reports indicate that the binding affinity between the HLA-I molecule and peptides could be associated with an increased CD8+ T-cell response. Novel HLA-B*35-restricted mutated variants have been described from HSNQVSQNY (HY9) and HPVHAGPIA (HA9) epitopes. Bioinformatic analysis has indicated that these mutated epitopes show low and high binding affinity towards HLA-B*35, respectively. However, the polyfunctionality of CD8+ T-cells stimulated with these mutated and wild-type epitopes has yet to be reported. The results suggest that the low-binding affinity H124 N/S125 N/N126S mutated peptide in the HY9 epitope induced a lower percentage of CD107a+CD8+ T-cells than the wild-type epitope. Instead, the high-binding affinity peptides I223V and I223A in the HA9 epitope induced a significantly higher frequency of polyfunctional CD8+ T-cells. Also, a higher proportion of CD8+ T-cells with two functions, with Granzyme B+ Perforin+ being the predominant profile, was observed after stimulation with mutated peptides associated with high binding affinity in the HA9 epitope. These results suggest that the high-affinity mutated peptides induced a more polyfunctional CD8+ T-cell response, which could be related to the control of viral replication.

OBX-115, an interleukin 2 (IL2)-sparing engineered tumor-infiltrating lymphocyte (TIL) cell therapy, in patients (pts) with immune checkpoint inhibitor (ICI)-resistant unresectable or metastatic melanoma.

9515 Background: Investigational unengineered TIL cell therapy has shown promising activity in ICI-resistant metastatic melanoma but requires systemic high-dose IL2, which has well-described high-grade toxicity frequently requiring specialized management and limiting pt eligibility. OBX-115 autologous engineered TIL cell therapy does not require co-administration of IL2 due to its regulatable expression of membrane-bound IL15 (mbIL15) using the FDA-approved small-molecule drug acetazolamide (ACZ) to provide cytokine support for TIL expansion and persistence. Methods: This Phase 1 study (NCT05470283) assesses the OBX-115 regimen in pts with ICI-resistant unresectable/metastatic melanoma. OBX-115 is manufactured from the pt’s tumor tissue (surgical excision or core needle biopsy [CNB]). After lymphodepletion (cyclophosphamide, fludarabine), pts receive OBX-115 followed by ACZ once daily for up to 7 days; ACZ redosing (up to 7 days) is permitted at Wk 6 for non-responders. No systemic IL2 is administered. Peripheral blood and tumor tissue is collected for longitudinal immune profiling. Results: As of 02 Jan 2024, 9 pts (median age, 50 y) with ICI-resistant metastatic melanoma were infused with OBX-115 (median study follow up, 17 wks; range, 2–58 wks). Median lines of prior therapy was 3 (range, 1–6). OBX-115 was successfully manufactured for all pts, including from CNB tumor tissue (n = 5). The infusion product had a high proportion of CD8+ cells (≥96%) and stem-like cells (CD8+CD39-CD69-; median 76%) with very low PD-1 expression in the CD8+ population ( &lt; 1%). Post-infusion safety included no DLTs, 3 Gr 3 nonhematologic TEAEs in 2 pts (abdominal pain, ALT elevation, syncope), and no Gr 4 nonhematologic TEAEs. ACZ was redosed and well-tolerated in 4 of 5 eligible pts. Among patients with minimum 12-wk follow-up (n = 6), ORR per RECIST v1.1 was 50% (2 CR, 1 PR, 3 SD); all responses occurred between Wk 6–18 and were ongoing, with longest response sustained &gt; 12 mo. One pt developed new metastatic disease (liver) and progressed at Wk 24, despite continued target lesion reduction; no pt developed brain metastasis. Post-infusion ctDNA was not detectable in any of the responders at Day 14 or 42. Though OBX-115 had minimal NK cells ( &lt; 1%), post-infusion peripheral blood and tumor biopsies showed NK cell expansion, consistent with trans-presentation of mbIL15 to circulating NK cells. Updated efficacy data on all infused pts will be presented. Conclusions: OBX-115regulatableengineeredTIL cell therapy was well-tolerated and produced consistently deepening and durable responses, indicating that OBX-115 may mediate CRs and durable clinical benefit in ICI-resistant metastatic melanoma without high-dose IL2. OBX-115 investigation continues in this and an ongoing Phase 1/2 multicenter study (NCT06060613). Clinical trial information: NCT05470283 .

Final survival outcomes and exploratory biomarker analysis from the randomized, phase 2 neoSCORE trial: Two versus three cycles of neoadjuvant sintilimab plus chemotherapy for resectable non-small cell lung cancer.

8048 Background: In the neoSCORE trial (NCT04459611), three cycles of neoadjuvant sintilimab plus chemotherapy achieved a numerically higher major pathological response (MPR) rate compared to two cycles. However, the relationship between MPR and survival in resectable non-small cell lung cancer (NSCLC) patients receiving neoadjuvant immunotherapy is not currently fully elucidated. Furthermore, reliable predictive biomarkers are still lacking. Here, we report the survival and biomarker results of the study. Methods: Eligible patients with stage IB-IIIA resectable NSCLC were randomized 1:1 to receive either two or three cycles of neoadjuvant treatment with sintilimab (200mg) plus platinum-doublet chemotherapy (Q3W). After surgery, patients received totally four doses of perioperative immuno-chemotherapy. The primary endpoint was the MPR rate. Secondary endpoints included the complete pathological response (pCR) rate, objective response rate (ORR), 2-year disease-free survival (DFS) rate, 2-year overall survival (OS) rate, and safety. Tumor samples (n = 19) were obtained for immune biomarker analysis via mass cytometry time of flight (CyTOF). Multiplexed immunofluorescence (mIF) was performed on FFPE tumor samples (n = 42). Results: At the data cutoff (15/1/2024), 55 patients received neoadjuvant immuno-chemotherapy and underwent surgical resection, with a median follow-up of 36.1 months. DFS (HR = 1.25, P= 0.595) and OS (HR = 0.88, P= 0.830) were similar between the two-cycle and three-cycle groups. The median DFS and OS were not reached in both groups. The 2-year DFS rate in the two-cycle and three-cycle groups were 76.9% and 65.5%, and the 2-year OS rates were 84.6% and 86.2%, respectively. In a multivariable Cox analysis including clinical characteristics, therapy, and pathological response, only MPR was significantly associated with longer DFS (HR = 0.32, P= 0.044). The AUC for the percentage of residual viable tumor in predicting DFS was 0.649 ( P= 0.061). CyTOF analysis demonstrated that increased infiltration of CD8+CD38+CD103- T cells after neoadjuvant immuno-chemotherapy was significantly associated with MPR ( P= 0.035). The mIF confirmed that patients achieving MPR showed higher frequencies of CD8+CD38+CD103- T cells than those without MPR ( P= 0.005). Additionally, patients with higher proportions of CD8+CD38+CD103- T cells had a trend towards improved DFS (HR = 0.39, P= 0.061) and OS (HR = 0.45, P= 0.259). Conclusions: Neoadjuvant sintilimab plus chemotherapy was feasible and demonstrated a robust and persistent survival benefit in resectable NSCLC. MPR was associated with improved DFS. Increased CD8+CD38+CD103- T cells were associated with MPR, with a trend towards a better survival benefit, suggesting that it could be a promising predictive biomarker. Clinical trial information: NCT04459611 .

Immunogenicity Analysis and Identification of Potential T-Cell Epitopes in C129R Protein of African Swine Fever Virus.

The highly conserved C129R protein of AFSV was utilized in the development of an ASFV recombinant adenovirus vaccine, demonstrating strong immunogenicity. In this study, we immunized 6-week-old female C57BL/6J mice via subcutaneous injection with 10 μg of purified C129R protein. Humoral and cellular immune effects were assessed using ELISA, flow cytometry, and ELISpot assays. Additionally, 19 peptides of the C129R protein were synthesized and screened for the use of bioinformatics. Positive T-cell epitopes were screened using ELISpot. The results indicated a higher proportion of CD4+ and CD8+ T lymphocytes in immunized mice compared to control mice. ELISA analysis revealed a serum titer of approximately 1:1, 638, 400 in the experimental group of mice. Additionally, peptides C11(53-61aa), C14(81-89aa), C16(97-105aa), and C18(116-124aa) from the C129R protein were able to activate mice spleen lymphocytes to produce IFN-γ. These findings suggest that the C129R protein significantly enhances both humoral and cellular immunity in immunized mice. Moreover, peptides C11, C14, C16, and C18 may serve as potential T-cell epitopes for the C129R protein. These results lay the groundwork for the further exploration of ASFV C129R protein and the identification of novel ASF vaccine antigens.

Investigating the platelet contribution to diet induced PVAT leukocyte infiltration

Background: Perivascular adipose tissue (PVAT) is an adipocyte and leukocyte depot that encircles most peripheral arteries and aids in the regulation of vascular tone. Under normal physiological conditions PVAT mediates vascular relaxation, but under obesogenic conditions, such as those caused by poor nutrition, PVAT loses its anti-contractile properties, thus contributing to increased vascular tone. This change in PVAT phenotype is marked by the replacement of native anti-inflammatory leukocytes with pro-inflammatory counterparts which facilitate an inflamed, hypertensive phenotype. As platelets have been shown to aid in the infiltration of leukocytes into infected tissues from the circulation, we investigated whether platelets are significant contributors to the leukocyte recruitment that marks the PVAT phenotypic/functional shift resulting from poor nutrition. Methods: To determine the impact of platelets on the diet induced phenotypic shift in PVAT leukocyte composition, male C57BL6/J mice were administered a platelet depleting antibody or vehicle control for 2 weeks prior to the isolation of the thoracic and abdominal PVAT depots whereupon leukocyte composition was determined via flow cytometry. Dietary interventions included either a control (10% fat, 7% sucrose, 0.3% salt Research Diets Inc. D12450J) or Western (40% fat, 30% sucrose, 8% salt Research Diets Inc. D06111701) diet to either maintain PVAT or facilitate disruption. Two cohorts of mice were utilized with one fed diet for 8 weeks beginning at the time of weaning (4 weeks of age) and another for 2 weeks beginning at 10 weeks of age to elucidate whether platelets are an early or ongoing facilitator of PVAT perturbation. Results: Compared to control diet, Western diet shifted the lymphocyte content of the thoracic PVAT depot such that there was a higher proportion of CD4+ T cells; however, this was not observed in abdominal PVAT. With regards to the myeloid cell population, Western diet facilitated a reduction in thoracic PVAT nonclassical monocytes and macrophages while additionally increasing the proportion of classical monocytes in abdominal PVAT. Furthermore, a Western diet nominally reduced the proportion of proliferative macrophages in both thoracic and abdominal PVAT. Conclusions: A Western diet influences the composition of both the thoracic and abdominal PVAT depots by altering monocyte polarization, as well as influencing macrophage and T cell distribution within the tissue. This shift in leukocyte populations is congruent with an overall phenotypic switch towards a more pro-inflammatory and thus vasoconstrictive environment as previously noted to be driven by obesogenic dietary interventions. Funding: ACRI/ABI AWD-037176. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Advanced in immunological monitoring of HIV infection: profile of immune cells and cytokines in people living with HIV-1 in Benin

BackgroundImmune cells and cytokines have been linked to viremia dynamic and immune status during HIV infection. They may serve as useful biomarkers in the monitoring of people living with HIV-1 (PLHIV-1). The present work was aimed to assess whether cytokines and immune cell profiles may help in the therapeutic follow-up of PLHIV-1.MethodsForty PLHIV-1 in treatment success (PLHIV-1s) and fifty PLHIV-1 in treatment failure (PLHIV-1f) followed at the University Hospital of Abomey-Calavi/Sô-Ava in Benin were enrolled. Twenty healthy persons were also recruited as control group. Circulating cytokines and immune cells were quantified respectively by ELISA and flow cytometry.ResultsPLHIV-1 exhibited low proportions of CD4 + T cells, NK, NKT, granulocytes, classical and non-classical monocytes, and high proportions of CD8 + T cells, particularly in the PLHIV-1f group, compared to control subjects. Eosinophils, neutrophils and B cell frequencies did not change between the study groups. Circulating IFN-γ decreased whereas IL-4 significantly increased in PLHIV-1s compared to PLHIV-1f and control subjects even though the HIV infection in PLHIV-1s downregulated the high Th1 phenotype observed in control subjects. However, Th1/Th2 ratio remained biased to a Th1 phenotype in PLHIV-1f, suggesting that high viral load may have maintained a potential pro-inflammatory status in these patients. Data on inflammatory cytokines showed that IL-6 and TNF-α concentrations were significantly higher in PLHIV-1s and PLHIV-1f groups than in control subjects. Significant high levels of IL-5 and IL-7 were observed in PLHIV-1f compared to controls whereas PLHIV-1s presented only a high level of IL-5. No change was observed in IL-13 levels between the study groups.ConclusionOur study shows that, in addition to CD4/CD8 T cell ratio, NK and NKT cells along with IL-6, TNF-α, IL-5 and IL-7 cytokines could serve as valuable immunological biomarkers in the therapeutic monitoring of PLHIV-1 although a larger number of patients would be necessary to confirm these results.

Abstract LB345: Correlating the blood immune repertoire and clinical features of del17p CLL patients

Abstract Introduction: Deletion in the short arm of chromosome 17 (del17p) is a strong, negative prognostic biomarker found in about eight percent of patients with untreated chronic lymphocytic leukemia (CLL). CLL patients with del17p exhibit resistance to therapy, accelerated disease progression, and global immunodeficiency. It is unclear the extent to which global immunodeficiency impacts disease course and therapy outcomes. The goal of this study was to document the blood immune repertoire of patients with untreated CLL who have del17p and associate with clinical features, time to first treatment (TFT), and overall survival (OS). Methods: Biobanked blood samples from treatment-naïve del17p CLL patients (n = 58) and age-matched controls (n = 5) were obtained. B cells and T cells were analyzed with comprehensive, cell type-specific flow cytometry panels and visualized with unbiased dimensionality reduction. Lab values were log-transformed and compared using t-tests and one-way ANOVAs. TFT was calculated from date of sample collection to date of first CLL-directed therapy or last known untreated date; OS was calculated from date of sample collection to death or last known alive status. Cox proportional hazards regression analyses (results reported as hazard ratio [HR] and 95% confidence interval [CI]) were employed to identify associations of lab values with OS and TFT. Results: Patients with del17p CLL had increased proportions of T regulatory cells (Tregs) among total CD4+ T cells compared to controls (CLL 11.1%, control 5.7%, p = 0.05). A higher proportion of CD8+ T cells was associated with longer TFT (HR 0.57, 95%CI 0.36-0.89); in contrast, higher proportions of TIGIT+ (HR 1.83, 95%CI 1.03-3.28) and TIM3+ conventional (non-Treg) CD4+ T cells were associated with shorter TFT (HR 1.70, 95%CI 1.02-2.84). A higher proportion of PD1+ CD8+ T cells was associated with shorter OS (HR 1.79, 95%CI 1.05-3.06), in addition to PD1+ conventional CD4+ T cell proportion (HR 2.21, 95%CI 1.06-4.60). CLL B cells expressed PD-1 and inducible T cell costimulator (ICOS) at significantly lower median fluorescence intensities (MFIs) than control B cells (p &amp;lt; 0.0001 for both). The CXCR5+ MFI (HR 1.0002, 95%CI 1.0001-1.003) and CD23+ MFI (HR 1.88, 95%CI 1.01-3.50) of CLL B cells were associated with shorter TFT. Discussion: In this cohort of treatment-naïve del17p CLL patients, we determined that patients with higher proportions of exhausted CD4+ and CD8+ T cell phenotypes had significantly shorter OS. Cytotoxic T cell proportion positively associated with TFT, whereas proportions of CD4+ T cell exhaustion phenotypes negatively associated with TFT. Higher CXCR5+ and CD23+ MFIs of CLL B cells also associated with shorter TFT. The preliminary findings from our study suggest that the blood immune repertoire has strong associations with clinically relevant outcomes such as time to first treatment and overall survival in patients with untreated del17p CLL. Citation Format: Ethan J. Katcher, Kim A. Gwin, Sameer A. Parikh, Kari G. Rabe, Petra K. Lothert, Preeti Trisal, Esteban Braggio, Susan L. Slager, Nicholas Strong, Daniel L. Van Dyke, Zhiquan Wang, C. Chris Huang, Anita K. Gandhi, Kay L. Medina, Neil E. Kay. Correlating the blood immune repertoire and clinical features of del17p CLL patients [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 LB345.

Investigating peripheral blood monocyte and T-cell subsets as non-invasive biomarkers for asymptomatic hepatic steatosis: results from the Multi-Ethnic Study of Atherosclerosis.

Circulating immune cells have gained interest as biomarkers of hepatic steatosis. Data on the relationships between immune cell subsets and early-stage steatosis in population-based cohorts are limited. This study included 1,944 asymptomatic participants of the Multi-Ethnic Study of Atherosclerosis (MESA) with immune cell phenotyping and computed tomography measures of liver fat. Participants with heavy alcohol use were excluded. A liver-to-spleen ratio Hounsfield units (HU) <1.0 and liver attenuation <40 HU were used to diagnose liver fat presence and >30% liver fat content, respectively. Logistic regression estimated cross-sectional associations of immune cell subsets with liver fat parameters adjusted for risk factors. We hypothesized that higher proportions of non-classical monocytes, Th1, Th17, and memory CD4+ T cells, and lower proportions of classical monocytes and naive CD4+ T cells, were associated with liver fat. Exploratory analyses evaluated additional immune cell phenotypes (n = 19). None of the hypothesized cells were associated with presence of liver fat. Higher memory CD4+ T cells were associated with >30% liver fat content, but this was not significant after correction for multiple hypothesis testing (odds ratio (OR): 1.31, 95% confidence interval (CI): 1.03, 1.66). In exploratory analyses unadjusted for multiple testing, higher proportions of CD8+CD57+ T cells were associated with liver fat presence (OR: 1.21, 95% CI: 1.02, 1.44) and >30% liver fat content (OR: 1.34, 95% CI: 1.07, 1.69). Higher circulating memory CD4+ T cells may reflect liver fat severity. CD8+CD57+ cells were associated with liver fat presence and severity, but replication of findings is required.

Abstract 7363: Clinical, genomic, transcriptomic and immunologic profile of BRAF mutant colorectal tumors

Abstract Background: Colorectal cancer (CRC) is the second leading cause of cancer-related death worldwide. BRAF, a protein kinase of the Mitogen-Activated Protein Kinase (MAPK) pathway, is mutated in 12% of colorectal cancers and is a poor prognostic factor for patients. BRAF mutations at the V600 codon are most studied but non-V600 BRAF mutations also exist and behave differently. BRAF V600 CRC treated with BRAF+MEK inhibitors show 12% response rates while we observe 65% response rates in BRAF V600 Melanoma. Additionally, no approved targeted therapies exist for non-V600 BRAF mutant cancers and retrospective studies show poorer PFS with BRAF+MEK inhibitors vs BRAF V600 cancers. We interrogated publicly available data of BRAF mutant CRC to uncover differences in the tumor microenvironment and signalling pathways of BRAF V600 and non-V600 tumors in hopes of uncovering better treatments. Methods: We analyzed the clinical and genomic profile of BRAF mutant CRC tumors from the GENIE v12 data. Gene Set Enrichment Analysis was performed on RNAseq from the TCGA data (discovery cohort) and the combined data from CPTAC and Sidra-LUMC data (validation cohort). We investigated the composition of tumor infiltrating immune cells using CIBERSORTx (deconvolution algorithm for bulk RNAseq). Results: Non-V600 mutations (n=219/1061) were most represented in patients of Asian or Black race compared to White patients (p&amp;lt;0.0001) and importantly, were found in younger patients (p&amp;lt;0.0001). We identified n=67 and n=74 BRAF mutant RNAseq samples in the discovery and validation cohorts, respectively. In the discovery cohort: BRAF V600 CRC were enriched for Interferon Gamma Signaling, Complement pathway, and IL6 JAK STAT3 Signaling (p&amp;lt;0.05) while the non-V600 BRAF mutant CRC showed an enrichment for Wnt-beta Catenin, Notch, and Hedgehog Signaling pathways (p&amp;lt;0.05). In the validation cohorts these same pathways were enriched in V600 and non-V600 BRAF mutant tumors. Interestingly, enrichment of genes co-mutated (GENIE data) in non-V600 BRAF mutant CRC also revealed the Wnt pathway. Non-V600 CRC tumors have a higher proportion of M0 macrophages (p=0.0035) and CD4 memory resting T cells (p&amp;lt;0.0001) while patients with V600 CRC have higher proportions of CD8 T cells (p=0.006) and activated mast cells (p=0.0341). Conclusion: No optimal therapeutic strategies have been defined for these potentially actionable non-V600 BRAF mutant CRCs. By exploring the multi-omic landscape of these tumors we have identified unique characteristics of non-V600 BRAF mutant CRC that could be therapeutically exploited. Citation Format: Emmanuelle Rousselle, Suzanne Kazandjian, April Rose. Clinical, genomic, transcriptomic and immunologic profile of BRAF mutant colorectal tumors [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 7363.

Abstract 2593: Spatial multi-omics profiling of tumor cell phenotypes in primary cutaneous melanoma at single-cell resolution

Abstract Primary cutaneous melanomas develop in stages from melanoma precursor cells that may progress to melanoma in situ (MIS) and eventually to invasive disease. Molecular changes, such as tumor cell state switches from melanocytic to undifferentiated and neural-crest-like phenotypes, have been associated with the development of melanoma metastases and therapeutic resistance. However, it is unknown whether cell states or gene expression changes enriched in advanced tumors exist at the premalignant stage and if they can elicit tumor-infiltrating lymphocytes (TILs). To address this gap, we analyzed a cohort of clinical samples from 43 patients with primary cutaneous melanoma using whole-slide cyclic multiplex immunofluorescence imaging (CyCIF), morphology-guided micro-regional RNA sequencing, and high-resolution 3D imaging. Each specimen contained multiple histologically-distinct regions representing a progression axis from normal to precursor to melanoma in situ to invasion. Subsequently, we examined the molecular profiles of both atypical melanocytes and tumor cells, and the gene expression and tumor microenvironmental (TME) changes that occurred during these progression stages. We identified major intra- and inter-specimen heterogeneity in the tumor intrinsic and extrinsic features across progression stages. Multiple tumor samples contained regions where publicly available gene signatures linked to both melanocytic and undifferentiated cell phenotypes were enriched. CyCIF analysis confirmed that various tumor cell phenotypes can coexist in close proximity to each other in early-stage untreated melanomas, and that the majority of undifferentiated tumor cells were located within regions of dermal invasion. Moreover, the cellular neighborhoods around undifferentiated tumor cells had a higher proportion of CD8+ T cells and cells of myeloid lineage compared to the neighborhoods around melanocytic tumor cell phenotypes. When assessing the localization and abundance of various tumor cell phenotypes, the levels of spatial and regional heterogeneity increased proportionally along normal to premalignant to malignant progression. Furthermore, the regional entropy of tumor cell phenotypes decreased along tumor depth in the majority (63%) of the samples, and even the melanoma in situ regions showed varying levels of spatial heterogeneity. Our in-depth multimodal molecular analyses highlight the presence of spatial heterogeneity among tumor cell phenotypes as well as the organization of early-stage primary melanomas, and are suggestive of tumor cell adaptation to the local environment. Citation Format: Tuulia Vallius, Edward Novikov, Ajit Nirmal Johnson, Shi Yingxiao, Roxanne Pelletier, Shishir Pant, Zoltan Maliga, Alyce Chen, Clarence Yapp, Sabrina Chan, Christine Lian, George F. Murphy, Sandro Santagata, Peter Sorger. Spatial multi-omics profiling of tumor cell phenotypes in primary cutaneous melanoma at single-cell resolution [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 2593.

Abstract 1535: Differential immune microenvironments in triple-negative breast cancer and hormone receptor-positive HER2-negative breast cancer: A spatial analysis via multiplex immunofluorescence

Abstract Objective: To elucidate differences in immune cell (IC) composition and their spatial distribution between triple-negative breast cancer (TNBC) and hormone receptor-positive HER2-negative breast cancer (HR+HER2-BC), and to evaluate the effect of PD-L1 status on these parameters. Methods: We selected 32 patients with ≥60% tumor-infiltrating lymphocytes (TIL) (TNBC, n=18; HR+HER2- BC, n=14). Multiplex immunofluorescence was employed on resected tumor tissues. Using markers such as CK, CD20, CD8, CD4, and FOXP3, IC types and tumor cells (TC) were identified in both stromal and tumor areas. We analyzed and compared cellular density and proportional distribution of IC, and spatial interactions (both IC-TC and IC-IC) based on tumor subtype and PD-L1 status. Results: TNBC exhibited a unique IC composition with a higher proportion of CD8+IC (stroma: 27% vs 17%, p&amp;lt;0.001; tumor: 54% vs 31%, p&amp;lt;0.001) and CD4+FOXP3+IC (stroma: 3.9% vs 3.0%, p=0.036) than HR+HER2-BC. Among PD-L1 positive cases, TNBC had a denser stromal population of CD4+FOXP3+IC (146.4±67.1/mm2 vs 114.3±146.9/mm2, p=0.036). Both tumor subtypes displayed varied IC compositions based on PD-L1 status. Density plots revealed a pronounced clustering of IC near TC in TNBC, especially in PD-L1 positive cases. Analysis of cell-cell interactions showed diverse adjacency patterns between TC and various IC combinations with frequencies differing by tumor subtype and PD-L1 status. Conclusion: The immune landscape, typified by IC composition and spatial distribution, varies between TNBC and HR+HER2- BC, with PD-L1 status further modulating these patterns. Citation Format: Yoon Jin Cha, Inho Park, Su-Jin Shin. Differential immune microenvironments in triple-negative breast cancer and hormone receptor-positive HER2-negative breast cancer: A spatial analysis via multiplex immunofluorescence [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 1535.

Abstract 1160: Investigating spatial gene expression profiling in colorectal cancer: Tumor and stroma comparison between primary lesions and matched liver metastases

Abstract The nanoString GeoMx® Digital Spatial Profiling (DSP) enables the investigation of spatial assessment of tumors through high-plex profiling at the RNA and protein levels. Recent genomic analyses have revealed the intertumor heterogeneity between primary and metastatic lesions in colorectal cancer (CRC) patients. In CRC with liver metastases, current treatment strategies are mainly based on the parameters of primary tumors, and metastatic heterogeneity is a challenge since molecular heterogeneity contributes to therapeutic resistance. Furthermore, spatial intratumor heterogeneity exists within a single tumor between cancer cells (tumor) and their microenvironment (stroma) in human cancers. However, whether there are distinct spatial gene expression patterns of tumor and stroma between primary lesions and liver metastases remains unclear in CRCs. We examined 24 formalin-fixed paraffin-embedded tissue samples, including primary lesions and matched liver metastases from 12 patients (6 with synchronous and 6 with metachronous metastases) using NanoString GeoMx® DSP. The best regions of interest (ROI) with the invasive front boundary of the tumor were selected by pathologists. The ROI was segmented into PanCK-positive (tumor) and PanCK-negative (stroma), followed by a collection of indexed oligonucleotides and sequencing on Illumina instrument. Differential expression and pathways enrichment analyses were performed using R BioConductor package standR, limma, and GSEABase. Statistical significances were based on |Log2 fold change| &amp;gt; 1 and Benjamini-Hochberg-corrected P &amp;lt; 0.05. Immune cell abundance was estimated using the SpatialDecon package. In 12 metastatic patients (mean age, 61 years ± 7 [standard deviation]), liver metastatic stroma was associated with 68 upregulated and 93 downregulated genes compared to the primary stroma, with enrichment of 'Immune response' terms in gene set enrichment analysis (GSEA). A higher proportion of CD4 T memory cells and increased expression of cytotoxic genes (IL7R [2.02-fold], CD3E [1.75-fold], KLRB1 [1.68-fold], and GZMK [1.54-fold]) were observed in liver stroma compared to primary stroma. Conversely, liver metastatic tumors were associated only with 23 upregulated and 6 downregulated genes compared to primary tumors, with 'humoral response' terms enriched in GSEA. Similar patterns were observed in stratified analyses with synchronous and metachronous metastases, although a more prominent ‘immune response’ was observed in synchronous metastatic stroma compared to the primary stroma. We revealed that higher immune cell proportion and cytotoxic activity were observed in liver metastatic stroma compared to CRC stroma, providing novel insights into a unique etiology and may yield clinical implications for developing targeted treatment modalities for liver metastatic CRC patients. Citation Format: Jongwon Lee, Yeseul Kim, Hyo Seon Ryu, Jongmin Sim, Chungyeul Kim, Jong Min Park, Ah-Reum Lim, Jung Sun Kim, Hwa Jung Sung, Xingyi Guo, Jungmin Choi, Jungyoon Choi. Investigating spatial gene expression profiling in colorectal cancer: Tumor and stroma comparison between primary lesions and matched liver metastases [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 1160.

Association of lymphocyte subsets with the efficacy and prognosis of PD‑1 inhibitor therapy in advanced gastric cancer: results from a monocentric retrospective study

PurposeThis retrospective study aimed to investigate the changes in peripheral blood lymphocyte subsets before and after immunotherapy in patients with advanced gastric cancer and their relationship n with the therapeutic efficacy and clinical prognosis.MethodsPeripheral blood lymphocyte subsets, including CD4 + T cells, CD8 + T cells, CD4+/CD8 + ratio, NK cells, Treg cells, and B cells, were collected from 195 patients with advanced gastric cancer who were admitted to the First Hospital of Shanxi Medical University with immunotherapy from January 2020 to October 2021, at the time of diagnosis of advanced gastric cancer, before immunotherapy and after 3 cycles of immunotherapy. T-tests were used to examine the factors influencing the patients’ peripheral blood lymphocyte subsets and the changes after immunotherapy. To examine the relationship between lymphocyte subsets and treatment outcomes, ROC curves were plotted using a logistic regression. Kaplan–Meier curve was drawn, and the Log Rank test was carried out to compare the differences in PFS between the different groups. Cox proportional hazards regression model was used to analyze the factors affecting PFS after calibration of other variables.ResultsThe proportion of peripheral blood lymphocyte subsets in patients with advanced gastric cancer was affected by age and PD-L1 level. Compared to the baseline, the treatment effective group had higher proportions of CD4 + T cells, a higher CD4+/CD8 + ratio, NK cells and Treg cells, and lower proportions of CD8 + T cells and B cells in the peripheral blood after three cycles of immunotherapy. In the treatment-naive group, there were no significant differences in the lymphocyte subsets. With cut-off values of 30.60% and 18.00%, baseline CD4 + T cell and NK cell ratios were independent predictors of immunotherapy efficacy and PFS. Treg cell ratio, gender, PD-L1 levels, and MMR status all predicted PFS independently.ConclusionThe proportion of peripheral blood lymphocyte subsets was modified in patients who responded to PD-1 inhibitors. Different lymphocyte subpopulation levels can be used as biomarkers to predict immunotherapy efficacy and clinical prognosis in patients with advanced gastric cancer.

In-depth human immune cellular profiling from newborn to frail.

Immune functional decline and remodeling accompany aging and frailty. It is still largely unknown how changes in the immune cellular composition differentiate healthy individuals from those become frail at a relatively early age. Our aim in this exploratory study was to investigate immunological changes from newborn to frailty, and the association between health statute and various immune cell subtypes. The participants analyzed in this study covered human cord blood cells and peripheral blood cells collected from young adults, healthy and frail old individuals. A total of 30 immune cell subsets was performed by flow cytometry based on the surface markers of immune cells. Furthermore, frailty was investigated for its relations with various leukocyte subpopulations. Frail individuals exhibited a higher CD4/CD8 ratio, a higher proportion of CD4+ central memory T (TCM) cells, CD8+ effector memory T cells, CD27- switched memory B (CD27-BSM) cells, CD27+ switched memory B cells, age-associated B cells (ABCs) and CD38-CD24- B cells, and a lower proportion of naïve CD8 + T cells and progenitor B cells. The Frailty index score was found to be associated with naïve T cells, CD4/CD8 ratio, ABCs, CD27-BSM cells, and CD4+ TCM cells. Our findings conducted a relatively comprehensive and extensive atlas of age- and frailty-related changes in peripheral leukocyte subpopulations from newborn to frailty. The immune phenotypes identified in this study can contribute to a deeper understanding of immunosenescence in frailty and may provide a rationale for future interventions and diagnosis.

Targeting oxidative phosphorylation to increase the efficacy of immune-combination therapy in renal cell carcinoma

BackgroundImmune checkpoint inhibitors (ICIs) are the standard of care for metastatic renal cell carcinoma (RCC); however, most patients develop de novo or acquired resistance to ICIs. Oxidative phosphorylation (OXPHOS) has...

Identification and validation of platelet-related diagnostic markers and potential drug screening in ischemic stroke by integrating comprehensive bioinformatics analysis and machine learning.

Ischemic stroke (IS), caused by blood and oxygen deprivation due to cerebral thrombosis, has links to activated and aggregated platelets. Discovering platelet-related biomarkers, developing diagnostic models, and screening antiplatelet drugs are crucial for IS diagnosis and treatment. Combining and normalizing GSE16561 and GSE22255 datasets identified 1,753 upregulated and 1,187 downregulated genes. Fifty-one genes in the platelet-related module were isolated using weighted gene co-expression network analysis (WGCNA) and other analyses, including 50 upregulated and one downregulated gene. Subsequent enrichment and network analyses resulted in 25 platelet-associated genes and six diagnostic markers for a risk assessment model. This model's area under the ROC curve outperformed single genes, and in the peripheral blood of the high-risk group, immune infiltration indicated a higher proportion of CD4, resting CD4 memory, and activated CD4 memory T cells, along with a lower proportion of CD8 T cells in comparison to the low-risk group. Utilizing the gene expression matrix and the CMap database, we identified two potential drugs for IS. Finally, a rat MACO/R model was used to validate the diagnostic markers' expression and the drugs' predicted anticoagulant effects. We identified six IS platelet-related biomarkers (APP, THBS1, F13A1, SRC, PPBP, and VCL) for a robust diagnostic model. The drugs alpha-linolenic acid and ciprofibrate have potential antiplatelet effects in IS. This study advances early IS diagnosis and treatment.