Mass Cytometry Research Articles

Hallmarks of tumor-experienced T cells are absent in multiple myeloma patients from diagnosis through maintenance therapy.

Dysregulation of the bone marrow (BM) niche in multiple myeloma (MM) alters the composition and state of resident immune cells, potentially impeding anti-tumor immunity. One common mechanism of immune inhibition in solid tumors is the induction of exhaustion in tumor-specific T cells. However, the extent of T cell tumor recognition and exhaustion is not well-characterized in MM. As the specific mechanisms of immune evasion are critical for devising effective therapeutic strategies, we deeply profiled the CD8+ T cell compartment of newly-diagnosed MM (NDMM) patients for evidence of tumor reactivity and T cell exhaustion. We applied single-cell multi-omic sequencing and antigen-specific mass cytometry to longitudinal BM and peripheral blood (PB) samples taken from timepoints spanning from diagnosis through induction therapy, autologous stem cell transplant (ASCT), and maintenance therapy. We identified an exhausted-like population that lacked several canonical exhaustion markers, was not significantly enriched in NDMM patients, and consisted of small, nonpersistent clones. We also observed an activated population with increased frequency in the PB of NDMM patients exhibiting phenotypic and clonal features consistent with homeostatic, antigen-nonspecific activation. However, there was no evidence of "tumor-experienced" T cells displaying hallmarks of terminal exhaustion and/or tumor-specific activation/expansion in NDMM patients at any timepoint.

SpillR: spillover compensation in mass cytometry data.

Channel interference in mass cytometry can cause spillover and may result in miscounting of protein markers. Chevrier et al. introduce an experimental and computational procedure to estimate and compensate for spillover implemented in their R package CATALYST. They assume spillover can be described by a spillover matrix that encodes the ratio between the signal in the unstained spillover receiving and stained spillover emitting channel. They estimate the spillover matrix from experiments with beads. We propose to skip the matrix estimation step and work directly with the full bead distributions. We develop a nonparametric finite mixture model and use the mixture components to estimate the probability of spillover. Spillover correction is often a pre-processing step followed by downstream analyses, and choosing a flexible model reduces the chance of introducing biases that can propagate downstream. We implement our method in an R package spillR using expectation-maximization to fit the mixture model. We test our method on simulated, semi-simulated, and real data from CATALYST. We find that our method compensates low counts accurately, does not introduce negative counts, avoids overcompensating high counts, and preserves correlations between markers that may be biologically meaningful. Our new R package spillR is on bioconductor at bioconductor.org/packages/spillR. All experiments and plots can be reproduced by compiling the R markdown file spillR_paper.Rmd at github.com/ChristofSeiler/spillR_paper.

Integrated spatial and multimodal single-cell transcriptomics reveal patient-dependent cell heterogeneity in splenic marginal zone lymphoma.

Biological hallmarks of splenic marginal zone lymphoma (SMZL) remain poorly described. Herein, we performed in-depth SMZL characterization through multimodal single-cell analyses of paired blood/spleen samples. The 3'-single-cell RNA-sequencing, Cellular Indexing of Transcriptomes and Epitopes by sequencing, and 5'-V(D)J single-cell RNA-sequencing datasets were integrated to characterize SMZL transcriptome profiles, including B-cell receptor and T-cell receptor repertoires. Hyperexpanded B-cell clones in the spleen were at a memory-like stage, whereas recirculating tumor B-cells in blood encompassed multiple differentiation stages, indicating an unexpected desynchronization of the B-cell maturation program in SMZL cells. Spatial transcriptomics showed the enrichment of T-effector and T-follicular helper (TFH) signatures in the nodular subtype of SMZL. This latter also exhibited gene-based cell-cell interactions suggestive of dynamic crosstalk between TFH and cancer cells in transcriptomics, further substantiated by using imaging mass cytometry. Our findings provide a comprehensive high-resolution description of SMZL biological hallmarks and characterize, for the first time in situ, inter- and intra-patient heterogeneity at both transcriptomic and protein levels. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

The immune landscape of uterine fibroids as determined by mass cytometry

ObjectiveTo study the differences in immune cell profile in uterine fibroids (Fib) and matched myometrium (Myo). DesignObservational Study SettingLaboratory Study PatientsThe study included tissue that was collected from ten pairs of fibroid and matched myometrium from women, not on hormonal medications, undergoing hysterectomy and myomectomy. InterventionsNone Main Outcome MeasuresDifferences in immune cell and cytokine composition between fibroid and matched myometrium Result(s)The mass cytometry analysis indicated that fibroids had a significantly higher number of NK cells, total macrophages, M2 macrophages, and conventional dendritic cells when compared to matched myometrium from the same patient. In contrast, fibroids had significantly fewer CD3 and CD4 T cells when compared to myometrium. The mass cytometry analysis did not show any significant difference in the number of resting mast cells. IFC and IHC imaging confirmed the CytoF results, showing a significantly higher (p<0.05) number of NK, tryptase-positive mast cells indicative of mast cell activation, total macrophage, and M2 cells in Fibroids and a significantly lower (p<0.05) number of CD3 and CD4 T cells. The cytokine assay revealed significantly increased (p<0.05) levels of IFNA2, Il-1α, and PDGF-AA and significantly lower levels of M-CSF and IL-1RA in Fib. ConclusionOur results show significant differences in immune cell populations and cytokine levels between Fib and Myo. There was a significant increase in total number of macrophages, M2 macrophages, NK cells, and dendritic cells and a significant decrease in CD3 and CD4 T cells in Fib. IHC confirmed no differences in total resting mast cell count, but a significant increase in tryptase-positive mast cells in Fib. Fibs also expressed significantly higher levels of IFNA2, IL-1α, and PDGF-AA and significantly lower levels of IL-1RA and M-CSF as compared with matched myometrium. These findings provide a foundation for further studies exploring the role of immune cells in Fib development.

A phase 2 study evaluating response and biomarkers in patients with microsatellite stable (MSS) advanced colorectal cancer (CRC) treated with nivolumab/relatlimab.

3554 Background: Lymphocyte-activation gene 3 (LAG-3) is an immunosuppressive signal, impairing T effector (Teff) activation. Pre-clinical studies suggest LAG-3 inhibition promotes anti-tumor immunity by enhancing Teff function. Correlative studies linked increased LAG-3 levels with reduced overall survival (OS) in CRC. These data prompted optimism that combined LAG-3/PD-1 inhibition might promote anti-tumor responses in MSS CRC. Methods: We conducted a phase 2 study of nivolumab (Nivo) (aPD1)/relatlimab (Rela) (aLAG3) in patients with previously treated advanced MSS CRC. This study enrolled patients onto 1 of 3 arms. Arms A and B used a Composite PD-L1 and acellular Mucin (CPM) score to define patients as CPM(+) in Arm A (CPM≥ 15%) and CPM(-) in Arm B (CPM&lt; 15%). Patients in Arm A and B received Nivo 480mg/Rela 160mg IV q4 weeks. Arm C enrolled patients without biomarker selection who received Nivo 480mg/Rela 960mg or Rela 480mg/Nivo 480mg. The primary endpoint was objective response, and each arm was powered to detect an objective response rate (ORR) of ≥20%. Results: Fifty nine evaluable patients were enrolled (Arm A: 12, Arm B: 15, Arm C: 32). Patient characteristics and responses are outlined. Treatment was well tolerated with no new safety signals over aPD1 alone. Three (5%) patients achieved a partial response (PR), 6 (10%) stable disease (SD), and 50 with progression of disease (PD) as best response. The median progression-free survival was (Arm A: 2.2 mo., Arm B: 2.8 mo., Arm C: 2.6 mo.) while median OS was (Arm A: 4.3 mo., Arm B: 14.2 mo., Arm C: 12.4 mo.). In N=6 patients with lung-only metastases, 2 (33%) experienced a PR, and 1 (17%) a SD, for a clinical benefit rate of 50%. The trial incorporated translational correlates. We will report mass cytometry on PBMCs, RNAseq on baseline biopsies, and imaging mass cytometry on pre- and on-treatment biopsies. Conclusions: This combination did not meet the primary endpoint of 20% ORR for any of the 3 cohorts, but there was intriguing possible activity in patients with lung only metastases, consistent with emerging data. Clinical trial information: NCT03642067 .

Identification and Clinical Correlation of Circulating MAIT, γδ T, ILC3, and Pre-Inflammatory Mesenchymal Cells in Patients with Rheumatoid Arthritis and Spondyloarthritis.

Inflammatory rheumatic diseases (IRDs), such as rheumatoid arthritis (RA) and spondyloarthropathy (SpA), comprise a heterogeneous group of immune-mediated disorders, characterised by the presence of localised and/or systemic inflammation. The limited knowledge of the pathogenesis and the complex mechanisms involved in the induction and maintenance of inflammation in IRDs have impeded the development of reliable biomarkers and the discovery of new therapeutic targets. Although the involvement of heterogeneous cell populations in the pathogenesis of IRDs has been recognised, the characterisation of these cellular subsets in the peripheral blood of patients has not been studied yet. Mass cytometry, allowing the simultaneous detection of more than 120 different parameters in single-cell resolution, will enable the identification of circulating cell subpopulations that might play a pivotal role in IRDs pathophysiology and their potential use as therapeutic targets.

Roginolisib, an oral, highly selective and allosteric modulator of phosphoinositide 3-kinase inhibitor delta (PI3Kδ) in patients with uveal melanoma and advanced cancers.

9597 Background: The highly selective, oral allosteric modulator of PI3Kδ, roginolisib (IOA-244), blocks the activity of PI3Kδ-dependent signaling, in both tumor cells and Tregs. Methods: Continuous daily dosing of roginolisib at 10, 20, 40 and 80 mg in patients (pts) with pretreated solid tumours and Follicular Lymphoma (FL) were evaluated (Part A). Part B consists of a dose confirmation cohort in uveal melanoma (UM) pts of 2 parts, B.1 and B.2. Primary objective: safety of the anticipated biologically effective dose (BED). Secondary objectives: PK; PD (e.g., inhibition of CD63 expression on basophils, changes in immune cell subsets in peripheral blood); RECIST 1.1 or Lugano-based responses; PFS and OS. Exploratory studies: phenotypic changes in circulating immune cells by mass cytometry (i.e., CyTOF); response assessments by radiomics; analysis of circulating protein signatures via Olink proteomics. Results: Part A Solid Tumour (completed): Sixteen pts were treated in 4 cohorts of 4 pts each. Pts characteristics: uveal melanoma (9/16; 56%), cutaneous melanoma (5/16; 31%) and pleural mesothelioma (2/16; 13%). Only Grade 1 and 2 AEs by CTCAE v.5 were observed, including 2 cases of transient (lasting &lt; 24 hrs) diarrhoea and 2 of AST/ALT elevation. Part A FL Cohort (completed): 8 pts were treated at 20 mg (n=4) and 80 mg (n=4). Grade 3 toxicities were transient and observed in 2/8 (25%) pts. 2/4 pts (50%) at the 80 mg had PR as per Lugano criteria. Treatment-emergent adverse events (TEAEs) did not result in study drug discontinuation, immune-related toxicity, or dose-limiting toxicity in either the solid tumor or hematological patients. UM pts (Part A and Part B)&gt;: 29 pts (Part A: 9 pts; Part B.1: 7 pts, Part B.2: 13 pts), of which 23 were treated at 80 mg QD (RP2D). Mean time on treatment: 10.7 mo (range: 1.5-39.6 mo). ORR (RECIST 1.1): PR: 1/29 (3%); SD: 21/29 (72%). Median OS for Part A: 20.8 mo (5/9 with 4 pts alive); Part B.1 20.1 (4/7 with 3 pts alive); Part B.2 not determined; 1-year OS rate: Part A 66.7%; Part B.1 71%; Part B.2 immature. Exploratory Investigations: At 3.7 mo (Cycle 5), there were 2 groups: one with SD (13/29; 45%) and the other with PD (16/29; 55%). At 6 mo, this split continued with 38% (11/29) showing SD and 62% having PD (18/29). Volumetric changes matched durable SDs while having higher sensitivity in the 15 analyzed patients. Interestingly, tumor burden reduction in mediastinal lymph nodes was indicative of SD and 70% of pts had volumetric spleen size reduction. Volumetric and radiomic changes were matched with peripheral changes in plasma proteins and immune cell changes. Conclusions: Roginolisib is well tolerated at 80 mg, demonstrates efficacy, and induces phenotypic changes in circulating immune cells. At the RP2D, the most robust anti-tumour activity was observed in FL (50% PR) and UM pts (3% PR; 72% SD). Clinical trial information: NCT04328844 .

The immunological milieu of oropharyngeal squamous cell carcinoma (OPC) according to HPV and smoking status and its influence on prognosis.

3147 Background: OPC exhibits distinct epidemiological, genetic, and clinical characteristics, significantly influenced by the presence of HPV. Despite multidisciplinary therapy, approximately 40% of OPC patients (pts) experience recurrence, and those who achieve cure often endure late and morbid sequelae of treatment. In this study, we comprehensively explored the composition and functional status of the tumor immune microenvironment (TIME) in a cohort of OPC pts treated with curative intent and investigated TIME differences based on HPV infection, smoking status, and clinical outcomes. Methods: Two tissue microarrays with baseline OPC tissues were stained with a previously validated panel containing 33 metal-tagged markers. Imaging mass cytometry was performed with the Fluidigm Helios CyTOF instrument, and analysis performed with Visiopharm software. HPV status was determined by p16 IHC and/or DNA testing. Comparison of cell types and markers densities among subgroups (HPV positive vs negative) were analyzed with Wilcoxon signed-rank test and its association with clinical outcomes (recurrence vs no-recurrence) with log-rank and Cox Proportional Hazards. Multiple comparisons were corrected by Benjamini Hochberg procedure. Results: A total of 99 samples from 61 pts from a single institution were included in this study. The majority of pts were male (N=47, 77%) and heavy smokers (N=55, 90%); 72% were HPV-negative. The median age was 61 years (range, 39 – 87 years), and 52% of cases were diagnosed with stages III-IVB (AJCC 8th edition). Most pts (57%) was treated with surgery (±RT). Thirty-three pts (54%) experienced recurrent disease. Smoking, stage III-IVB, and HPV-negative tumors were associated with higher recurrence rates (p &lt;0.01). Regarding the OPC TIME, the most prevalent cell populations in the stroma were fibroblasts (22%), followed by T-cells (17%) and endothelial cells (10%). The stroma of HPV+ tumors exhibited higher densities of memory T cells (FDR &lt; 0.01), B cells (FDR = 0.02), and T cells (FDR = 0.02) compared to HPV negative counterparts, which displayed an increased density of neutrophils (FDR = 0.04). The density of cancer stem cells was directly associated with smoking history (FDR&lt;0.001), and an increased density of intra-tumoral B-cells was found in non-smokers (FDR=0.01). Higher intra-tumoral neutrophil density was linked to recurrent disease (FDR &lt; 0.001). Conversely, higher densities of memory CD8+ T cells and non-M2 macrophages in the TIME were associated with a lower chance of recurrence (FDR &lt; 0.1). Conclusions: The intricate analysis of the OPC TIME revealed significant differences according to HPV and smoking status and highlighted the prognostic role of neutrophils, memory CD8+ T cells, and non-M2 macrophages. These findings may guide the development of personalized therapeutic strategies for OPC.

Dual analysis of IL-8 in non-small cell lung cancer: A clinical perspective on protein and RNA evaluation via imaging mass cytometry.

e15068 Background: The cytokine Interleukin-8 (IL-8) plays a pivotal role in the tumorigenesis and progression of non-small cell lung cancer (NSCLC), influencing both the tumor microenvironment and immune escape mechanisms. Given its significance, accurately assessing IL-8 levels at the protein and RNA levels could provide valuable insights into its prognostic value and inform therapeutic targeting strategies. This study utilizes imaging mass cytometry (IMC) to conduct a comprehensive evaluation of IL-8 expression in NSCLC, integrating both protein presence and gene expression analysis within the spatial context of the tumor microenvironment. Methods: Our approach leverages the capabilities of IMC to simultaneously detect protein and RNA levels of IL-8 in NSCLC tissue samples. For protein analysis, antibodies tagged with heavy metal isotopes specifically target IL-8, allowing for precise quantification and spatial localization within the tumor. Concurrently, RNA detection is achieved through hybridization probes designed for IL-8 mRNA, enabling the direct observation of gene expression patterns. This method provides a unique opportunity to correlate IL-8's protein abundance with its mRNA expression, offering insights into the regulatory mechanisms at play and their impact on the tumor's behavior and patient outcomes. Results: Preliminary data demonstrate a marked heterogeneity in IL-8 distribution at both the protein and RNA levels, with distinct patterns emerging that relate to tumor aggressiveness and immune infiltration. High IL-8 expression, identified through our integrated IMC approach, correlates with poor prognosis and reduced response to standard therapies, underscoring its potential as a biomarker for NSCLC management. Furthermore, the spatial analysis afforded by IMC highlights the microenvironmental niches where IL-8-mediated interactions could drive tumor progression and immune evasion. Conclusions: By employing IMC for the dual analysis of IL-8 protein and RNA in NSCLC, our study provides a comprehensive view of this cytokine's role in lung cancer pathology. The ability to visualize and quantify IL-8 expression within the spatial context of the tumor microenvironment opens new avenues for understanding its biological impact and for developing targeted therapies. Our findings advocate for the inclusion of IL-8 in the biomarker panel for NSCLC, with potential implications for patient stratification and personalized treatment planning. Future research will focus on expanding these observations to a larger cohort and exploring the therapeutic modulation of IL-8 as a strategy for improving NSCLC outcomes.

Interrogating the tumor microenvironment (TME) in patients (pts) with head and neck mucosal squamous cell carcinoma (HNmSCC) treated with programmed death-1 (PD-1) inhibitors.

6048 Background: Survival in recurrent/metastatic HNmSCC remain poor. PD-1 inhibitors have become standard of care, demonstrating improved overall survival and toxicity when compared to chemotherapy and targeted therapy. Biomarkers such as PD-Ligand(L)1 combined proportion score (CPS) remain rudimentary, with CPS &gt;20 showing a response rate of only 23% to pembrolizumab (KEYNOTE-048). We used high-dimensional imaging mass cytometry (IMC) to explore predictive biomarkers in HNmSCC pts receiving PD-1 inhibitor-based therapy. Methods: We retrospectively analysed 27 formalin-fixed paraffin embedded tissue samples from 24 pts prior to receiving PD-1 inhibitor-based therapy between May 2016 – April 2021. Clinicopathological characteristics including PD-L1, p16 status, prior treatment and survival data were collected. Pts were classified into responders (RES, Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 complete response (CR) or partial response (PR), stable disease (SD) &gt;6 months (mths)) and non-responders (non-RES, RECIST SD &lt;6 mths or disease progression (PD)). An antibody panel (n = 40) was created to interrogate specific components of interest within the TME and was analysed by IMC using the HyperionTM Imaging System. Results: Of the 24 patients, 16/24 were male and median age 57.6 year. 8 pts were RES (RECIST CR, n = 1; PR, n = 3; SD &gt; 6 mths, n = 4) and 14 pts were non-RES (RECIST SD &lt; 6 mths, n = 1; PD, n = 11; clinical PD, n = 4). Four patients underwent rapid clinical disease progression prior to progress imaging and were categorised as non-RES. At time of data cut off on January 2024, and 23/24 pts had progressed on treatment. The cellular landscape within the TME was similar, irrespective of the location of the primary and p16 status. However, distinct immune profiles were observed between RES vs non-RES: RES showed higher infiltrates of CD4+ T cells, B cells, PD-1+ CD8+ T cells (P &lt; 0.05) and both central memory and effector memory T cell subsets (p &lt; 0.01). In contrast, non-RES showed high frequencies of CD44+ NK cells. Key cell interactions within the TME identified proliferating malignant squamous cells closely interacting with CD8+ T cells, CD4+ Tregs and endothelial cell in RES but not interacting in non-RES. Further spatial regional analysis identified a distinct tissue architecture with hallmarks of Tertiary Lymphoid-like Structures (TLS), present in higher proportions in RES. RES pts with TLS proportions &gt;20% (n = 3) had a progression free survival of 80.3 mths, 26.8 mths and NE (unrelated death at 15.6 mths). Conclusions: The findings of this study identify mechanisms of PD-1 inhibitor response and resistance in HNmSCC pts, providing a unique opportunity to guide combination strategies and improve outcome.

Axitinib plus avelumab for recurrent/metastatic adenoid cystic carcinoma (R/M ACC): Biomarker analysis of the phase II trial.

6104 Background: In the phase II axitinib (VEGFR inhibitor) plus avelumab (PD-L1 inhibitor) trial in R/M ACC, response rate was 18% with a median progression-free survival (PFS) of 7.3 months, with subsequent inclusion of the combination in NCCN guidelines as a possible treatment for ACC (category 2B). We sought to identify potential biomarkers predictive of axitinib plus avelumab benefit in R/M ACC. Methods: The phase II trial included 28 R/M ACC patients (pts). Pre-treatment tumors were analyzed by whole exome sequencing (n=24), using Twist Human Core Exome V2 kit, and transcriptome profiling (n=26), using HTG Transcriptome Panel (19,616 probes). For 17 pts, imaging mass cytometry (IMC) was performed with 35 metal-tagged markers and analyzed with Visiopharm software. Microbiome composition was assessed in tumor (n=20), oral rinse (n=20) and fecal (n=19) samples via 16s rRNA gene sequencing. All results were assessed for association with PFS on therapy with a Cox proportional hazards model, maintaining ACC subtype (ACC-I vs. ACC-II) as a covariate. Results: Only 1 (6%) of 17 assessed tumors were PD-L1 positive. By WES, tumor mutational burden (TMB) was overall low (median 1.4 mut/Mb, range 0.7 – 18.7), but higher TMB was associated with worse PFS. Four mutational signatures significantly correlated with worse PFS, including SBS86, associated with previous chemotherapy exposure. Immune deconvolution of RNA-seq data revealed that a higher neutrophil-to-lymphocyte ratio and more T follicular helper cells associated with worse PFS, while presence of cytotoxic cells, T effector memory cells, and mast cells associated with longer PFS. Notably, a 167-gene predictive signature was developed and validated in the phase II ipilimumab plus nivolumab salivary gland cancer trial. This 167-gene signature was not prognostic in ACC pts who did not receive immunotherapy. IMC single-cell immune mapping revealed that pts with longer PFS had increased presence of CD8 T cells, CD73+ macrophages in tumor, a higher stromal CD8 T cell:Regulatory T cell ratio, increased stromal SIGLEC15 positive cells, and a higher tumor-to-stroma ratio of fibroblasts. Conversely, Ki67-positive T cells, cancer stem cells, and M2 macrophages were associated with worse PFS. Through microbiome analysis, 13 bacterial genera in tumor samples, 25 in stool, and 8 in oral rinse were significantly associated with therapy outcomes. Of note, in stool microbiome, presence of Akkermansia spp. and Bifidobacterium spp. were significantly correlated with improved PFS, with the latter association also seen in oral rinse. Conclusions: Correlative analysis of the phase II axitinib plus avelumab trial revealed potential biomarkers predictive of combination clinical benefit in ACC, including a gene-expression signature. These findings may guide patient stratification for combinatorial therapy.

ICARUS-LUNG01: A phase 2 study of datopotomab deruxtecan (Dato-DXd) in patients with previously treated advanced non-small cell lung cancer (NSCLC), with sequential tissue biopsies and biomarkers analysis to predict treatment outcome.

8501 Background: Few treatments with limited benefit are currently available after failure of targeted therapies, immunotherapy and platinum-based chemotherapy in patients (pts) with advanced NSCLC. Dato-DXd is an antibody-drug conjugate (ADC) composed of a TROP-2-directed monoclonal antibody linked to a topoisomerase I inhibitor via a peptide cleavable linker. In the phase 3 TROPION-Lung01 study, Dato-DXd demonstrated a statistically significant improvement of PFS over docetaxel in previously treated pts with advanced NSCLC. Here we report the results of ICARUS-Lung01 (NCT04940325), a multi-center, single-arm, phase 2 study evaluating activity, safety, and biomarkers of response/resistance to Dato-DXd in pretreated pts with advanced NSCLC. Methods: Intravenous Dato-DXd 6 mg/kg was given every 21 days to pts with advanced NSCLC, ECOG 0/1, who progressed on 1-3 lines of therapy (including actionable genomic alteration (AGA)-specific therapy if indicated). All pts underwent fresh tumor tissue biopsies at baseline, on-treatment (week 3 or 6) and end of treatment. Primary endpoint: investigator-assessed confirmed objective response rate (ORR). A set of translational analyses was performed to determine biomarkers associated with response/resistance, including TROP2 tumor membrane expression, TROP2-dynamics and spatial distribution (by AI-digital pathology), genomics, transcriptomic, spatial proteomics (by imaging mass cytometry) and CTCs. Results: A total of 100 pts received ≥1 dose: median age 60 years (26-83); 62% males, 89% smokers, 82% non-squamous (NSQ) histology, 23% AGA, median number of prior therapy lines 2 (1-5). At data cut-off (Dec 04, 2023), 92% discontinued therapy, 92 % had disease event, 67% died. Median treatment duration was 2.8 months (mos) (95%CI, 2.1-4.8), median follow-up 19.4 mos (95%CI, 18.2-20.4). Efficacy and safety results are shown in the table. Conclusions: In this heavily pretreated population, Dato-DXd showed similar efficacy and safety results to those reported in TROPION-Lung01. Patients with NSQ appeared to derive the greatest benefit. Translational analyses to determine biomarkers associated with response and/or resistance will be presented. Clinical trial information: NCT04940325 . [Table: see text]

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 .

Coarsened Mixtures of Hierarchical Skew Normal Kernels for Flow and Mass Cytometry Analyses

Cytometry is the standard multi-parameter assay for measuring single cell phenotype and functionality. It is commonly used for quantifying the relative frequencies of cell subsets in blood and disaggregated tissues. A typical analysis of cytometry data involves cell classification—that is, the identification of cell subgroups in the sample—and comparisons of the cell subgroups across samples or conditions. While modern experiments often necessitate the collection and processing of samples in multiple batches, analysis of cytometry data across batches is challenging because differences across samples may occur due to either true biological variation or technical reasons such as antibody lot effects or instrument optics across batches. Thus a critical step in comparative analyses of multi-sample cytometry data—yet missing in existing automated methods for analyzing such data—is cross-sample calibration, whose goal is to align corresponding cell subsets across multiple samples in the presence of technical variations, so that biological variations can be meaningfully compared. We introduce a Bayesian nonparametric hierarchical modeling approach for accomplishing both calibration and cell classification simultaneously in a unified probabilistic manner. Three important features of our method make it particularly effective for analyzing multi-sample cytometry data: a nonparametric mixture avoids prespecifying the number of cell clusters; a hierarchical skew normal kernel that allows flexibility in the shapes of the cell subsets and cross-sample variation in their locations; and finally the “coarsening” strategy makes inference robust to departures from the model not captured by the skew normal kernels. We demonstrate the merits of our approach in simulated examples and carry out a case study in the analysis of a multi-sample cytometry data set. We provide an R package for our method.

Spatially mapping the single-cell immune landscapes of melanoma brain metastases using imaging mass cytometry.

e21517 Background: Up to 60% of metastatic melanoma patients develop brain metastases, and 5% develop leptomeningeal disease (LD), which presents with the worst prognosis of any melanoma brain metastasis (MBM) infiltration pattern. Median survival duration for MBM is ~12 months, however for LD patients it can be &lt; 10 weeks. The spatial immune landscape of MBM with and without LD remains poorly understood. Methods: To spatially characterize the tumor microenvironment (TME) of MBM, we performed CyTOF Imaging Mass Cytometry (CyTOF-IMC) on a highly multiplexed panel of 35 antibodies for 21 MBMs (13 with LD, 8 without LD). We performed a novel cell segmentation, cell type assignment and identification approach to identify cell lineages to spatially characterize the TME of MBMs, segmenting and classifying 130,000 cells into 19 cell types. Results: We found enrichment of dendritic cells in the direct neighbourhood of melanoma cells of patients with LD (p = 0.019) and trends towards elevation of anti-tumoral monocyte populations in the TME of patients without LD. To investigate survival outcomes, we divided our cohort into long (≥365days) and short-survivors ( &lt; 365 days), and found significant elevation of anti-tumoral T-cell populations (Total T-cells, CD8+ T-cells, CD4+ T-cells and CD3+CD4-CD8- T-cells, p &lt; 0.05), and closer proximity of T-cells (CD8+ &amp; CD4+ T-cells) to melanoma cells (p &lt; 0.05). Survival analysis using median cell-proportion for each T-cell subset showed statistically significant (p &lt; 0.05) survival differences between patients with high and low cell-proportions of CD8+ T-cells and CD4+ T-cells, with similar trends observed for a subset of patients (n = 13) with LD and a subset of patients treated with ICIs (n = 10), and results were confirmed by a Cox Regression Model. Cellular neighborhood (CN) analysis was performed generating N = 12 stable CNs. We found patients with higher proportions of CN9 (M1-like-microglia/M2-like-microglia/melanoma), CN10 (M1-like-macrophages/M2-like-macrophages/melanoma) and CN12 (Cytotoxic-T-cells/Helper-T-cells/melanoma) was associated with longer overall survival after diagnosis with MBM (p &lt; 0.01). Conclusions: Proportion and proximity of anti-tumoral monocyte and T-cell populations may play a key role in modulating tumor response and overall survival for patients with MBM, including for patients with LD or for patients treated with ICIs.

Metabolomics reveals ascorbic acid inhibits ferroptosis in hepatocytes and boosts the effectiveness of anti-PD1 immunotherapy in hepatocellular carcinoma

BackgroundImmunotherapy combined with molecular targeted therapy is increasingly popular in patients with advanced hepatocellular carcinoma (HCC). However, immune-related adverse events(irAEs) brought on by immunotherapy increase the likelihood of side effects, thus it is important to look into ways to address this issue.MethodsDifferent metabolite patterns were established by analyzing metabolomics data in liver tissue samples from 10 patients(divided into severe and mild liver injury) before and after immuno-targeted therapy. After establishing a subcutaneous tumor model of HCC, the mice were divided into PBS group, ascorbic acid(AA) group, and anti-PD1 + tyrosine kinase inhibitor (TKI) group, anti-PD1 + TKI + AA group. Liver tissue were stained with hematoxylin-eosin staining(HE) and the content of aspartate transaminase (AST) and alanine transaminase(ALT) in blood were determined. The mechanism was confirmed by western blotting, mass cytometry, and other techniques.ResultsThrough metabolomics analysis, AA was significantly reduced in the sample of patients with severe liver injury caused by immuno-targeted therapy compared to patients with mild liver injury. The addition of AA in vivo experiments demonstrated a reduction in liver injury in mice. In the liver tissues of the anti-PD1 + TKI + AA group, the protein expressions of SLC7A11,GPX4 and the level of glutathione(GSH) were found to be higher compared to the anti-PD1 + TKI group. Mass cytometry analysis revealed a significant increase in the CD11b+CD44+ PD-L1+ cell population in the AA group when compared to the PBS group.ConclusionsAA could reduce liver injury by preventing hepatocyte SLC7A11/GPX4 ferroptosis and improve the immunotherapy effect of anti-PD1 by boosting CD11b+CD44+PD-L1+cell population in HCC.

Direct comparison of mass cytometry and single-cell RNA sequencing of human peripheral blood mononuclear cells

Single-cell methods offer a high-resolution approach for characterizing cell populations. Many studies rely on single-cell transcriptomics to draw conclusions regarding cell state and behavior, with the underlying assumption that transcriptomic readouts largely parallel their protein counterparts and subsequent activity. However, the relationship between transcriptomic and proteomic measurements is imprecise, and thus datasets that probe the extent of their concordance will be useful to refine such conclusions. Additionally, novel single-cell analysis tools often lack appropriate gold standard datasets for the purposes of assessment. Integrative (combining the two data modalities) and predictive (using one modality to improve results from the other) approaches in particular, would benefit from transcriptomic and proteomic data from the same sample of cells. For these reasons, we performed single-cell RNA sequencing, mass cytometry, and flow cytometry on a split-sample of human peripheral blood mononuclear cells. We directly compare the proportions of specific cell types resolved by each technique, and further describe the extent to which protein and mRNA measurements correlate within distinct cell types.

Deep proteome profiling of reticulated platelets in patients with chronic coronary syndrome using mass cytometry

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Else Körner Fresenius Stiftung (EKFS) Background Immature or reticulated platelets (RPs), a subgroup of larger and RNA-rich platelets, which are newly released by megakaryocytes from the bone marrow into blood, have gained rising interest because of the further decryption of RPs’ prothrombotic biology and the potential as biomarkers for predicting adverse events in cardiovascular disease. Purpose The biological features of RPs in patients with chronic coronary syndrome (CCS) are largely unknown. With this project, we aimed to perform the first high resolution proteomic characterisation of reticulated platelets using mass cytometry(CyTOF). Method Two samples of thrombocytes from peripheral blood (non-stimulated, TRAP-stimulated) were prepared from each patient with a custom-made CyTOF-antibody panel of 20 antibodies targeting essential transmembrane proteins(anti-CD9, anti-CD29, anti-CD31, anti-CD36-, anti-CD40, anti-CD41, anti-CD42a, anti-CD42b-, anti-CD47, anti-CD61, anti-CD62P-, anti-CD63, anti-CD69, anti-CD107a, anti-CD154, anti-GPVI, anti-GPIIbIIa complex, anti-Par1, anti-PEAR-1 and the negative control anti-CD3 coupled with different metal isotopes). RPs and mature platelets (MPs) were detected based on their RNA content. Earth mover’s distance (EMD) was assessed as a measure of differential expression. Results CyTOF detected an upregulation of important transmembrane receptors in RPs compared to MPs in quiescent platelets (Fig.1A and B). The collagen receptor GPVI (EMD 33.95), the collagen integrin receptor unit ITGB1 (EMD 32.37), the adhesion protein CD9 (EMD 31.78) and the von Willebrand receptor unit CD42b (EMD 30.08) were upregulated in RPs which all serve as crucial regulators of platelet activation and aggregation. RPs show higher median signal intensities of typical activation markers compared to MPs upon TRAP stimulation (Fig.1C). Conclusion We provide the first proteomic characterization of RPs’ pro-thrombotic profile offering the first biomolecular explanation of their hyperactivity and detrimental role in CCS patients.

Multimodal Mass Spectrometry Imaging of an Osteosarcoma Multicellular Tumour Spheroid Model to Investigate Drug-Induced Response.

A multimodal mass spectrometry imaging (MSI) approach was used to investigate the chemotherapy drug-induced response of a Multicellular Tumour Spheroid (MCTS) 3D cell culture model of osteosarcoma (OS). The work addresses the critical demand for enhanced translatable early drug discovery approaches by demonstrating a robust spatially resolved molecular distribution analysis in tumour models following chemotherapeutic intervention. Advanced high-resolution techniques were employed, including desorption electrospray ionisation (DESI) mass spectrometry imaging (MSI), to assess the interplay between metabolic and cellular pathways in response to chemotherapeutic intervention. Endogenous metabolite distributions of the human OS tumour models were complemented with subcellularly resolved protein localisation by the detection of metal-tagged antibodies using Imaging Mass Cytometry (IMC). The first application of matrix-assisted laser desorption ionization-immunohistochemistry (MALDI-IHC) of 3D cell culture models is reported here. Protein localisation and expression following an acute dosage of the chemotherapy drug doxorubicin demonstrated novel indications for mechanisms of region-specific tumour survival and cell-cycle-specific drug-induced responses. Previously unknown doxorubicin-induced metabolite upregulation was revealed by DESI-MSI of MCTSs, which may be used to inform mechanisms of chemotherapeutic resistance. The demonstration of specific tumour survival mechanisms that are characteristic of those reported for in vivo tumours has underscored the increasing value of this approach as a tool to investigate drug resistance.

Natural killer cells associate with epithelial cells in the pancreatic ductal adenocarcinoma tumor microenvironment.

Pancreatic ductal adenocarcinoma (PDAC) is the most common form of pancreatic cancer. PDAC's poor prognosis and resistance to immunotherapy are attributed in part to its dense, fibrotic tumor microenvironment (TME), which is known to inhibit immune cell infiltration. We recently demonstrated that PDAC patients with higher natural killer (NK) cell content and activation have better survival rates. However, NK cell interactions in the PDAC TME have yet to be deeply studied. We show here that NK cells are present and active in the human PDAC TME. We used imaging mass cytometry (IMC) to assess NK cell content, function, and spatial localization in human PDAC samples. Then, we used CellChat, a tool to infer ligand-receptor interactions, on a human PDAC scRNAseq dataset to further define NK cell interactions in PDAC. Spatial analyses showed for the first time that active NK cells are present in the PDAC TME, and both associate and interact with malignant epithelial cell ducts. We also found that fibroblast-rich, desmoplastic regions limit NK cell infiltration in the PDAC TME. CellChat analysis identified that the CD44 receptor on NK cells interacts with PDAC extracellular matrix (ECM) components such as collagen, fibronectin and laminin expressed by fibroblasts and malignant epithelial cells. This led us to hypothesize that these interactions play roles in regulating NK cell motility in desmoplastic PDAC TMEs. Using 2D and 3D in vitro assays, we found that CD44 neutralization significantly increased NK cell invasion through matrix. Targeting ECM-immune cell interactions may increase NK cell invasion into the PDAC TME.