Evidence has shown that T-cell receptors (TCRs) that recognize the same epitopes may not be the exact TCR clonotypes but have slightly different TCR sequences. However, the changes in the genomic and transcriptomic signatures of these highly homologous T cells during immunotherapy remain unknown. Here, we examined the evolutionary features in circulating TCR clonotypes observed in tumors (tumor-infiltrating lymphocyte (TIL)-TCRs) by combining single-cell RNA/TCR sequencing of longitudinal blood samples and TCR sequencing of tumor tissue from a patient treated with anti-cytotoxic T-lymphocyte-associated protein 4/programmed cell death protein-1 therapy. We found frequent circulating CD8+TIL-TCRs with identical complementarity determining region 3 (CDR3)α amino acid sequences but quasi-identical CDR3β and TCR α/β (TRA/TRB) sequences. Despite their sequence similarities, these highly homologous TIL-TCRs responded differently to immunotherapy, and exhibited distinct transcriptional signatures that were uniquely distinguished by the expression ofGZMK. Overall, the expression ofIFNGin CD8+T-cell subsets including highly homologous TIL-TCRs increased when the patient achieved a response, but gradually decreased as the patient developed acquired resistance. Our findings provide insight into the cross-talk between T cells in the tumor microenvironment and those in the blood, and highlight that CD8+T cells with highly homologous TCR sequences might display divergent transcriptional states and kinetics in response to immunotherapy.

Corticosteroids are frequently prescribed to patients with non-small cell lung cancer (NSCLC) for palliation of cancer-related symptoms; however, the potential impact of baseline steroid use on immune checkpoint inhibitor (ICI) therapy and its underlying mechanisms remain unclear. In this study, we evaluated clinical outcomes of 277 patients with NSCLC treated with ICI therapy at two academic institutions. Twenty-one patients (8%) were taking steroids at the start of ICIs. Patients on baseline steroids had a lower overall response rate with markedly shorter progression-free survival and overall survival compared with those not receiving steroids. In multivariate analysis, steroid use was the only significant independent risk factor for disease progression and mortality in both independent cohorts, Roswell Park Comprehensive Cancer Center (n = 88) and University of Southern California (n = 189). A baseline peripheral blood neutrophil-to-lymphocyte ratio <5 was a strong prognostic indicator; however, the prognostic value of neutrophil-to-lymphocyte ratio was absent in patients receiving steroids. Additionally, the baseline frequency of circulating CX3CR1+CD8+ T cells was substantially lower in patients on steroids. Using a bedside-to-bench approach, we found that concurrent steroid use significantly decreased antitumor efficacy of anti-PD-1 therapy and attenuated the increase of CX3CR1+CD8+ T cells in mice bearing MC38 tumors whereas discontinuation of steroid at the start of treatment did not make a negative impact on survival. Collectively, baseline steroid use was associated with worse outcomes and decreased frequency of circulating differentiated effector T cells in patients with NSCLC. Caution should be taken when interpreting the results from circulating immune-related biomarkers in patients on steroids. SIGNIFICANCE:The impact of corticosteroids, widely prescribed for palliation of cancer-related symptoms, on ICI therapy remains unclear. This study shows that baseline steroid use is a negative independent prognostic factor in patients with NSCLC undergoing ICI therapy and provides insights into the decreased T-cell effector differentiation and utility of predictive blood-based markers by steroids.

The development of immune checkpoint inhibitors (ICIs) has transformed cancer treatment, but patient responses remain inconsistent. Evidence has shown that T-cell receptors (TCRs) recognizing the same epitopes may not always be identical clonotypes but instead exhibit subtle sequence variations. However, the genomic and transcriptomic changes in these closely related T cells during immunotherapy remain unclear. In this study, we explored the evolutionary dynamics of circulating TCR clonotypes identified in tumors (TIL-TCRs) by integrating single-cell RNA/TCR sequencing from longitudinal blood samples with TCR sequencing from tumor tissue in a patient undergoing anti-CTLA-4/PD-1 therapy. We identified circulating CD8+ TIL-TCRs with identical CDR3α amino acid sequences but nearly identical CDR3β and TCR α/β (TRA/TRB) sequences. Despite their high sequence similarity, these quasi-identical TIL-TCRs displayed differential responses to immunotherapy and distinct transcriptional profiles, particularly marked by GZMK expression. Furthermore, IFNG expression within CD8A+ T-cell subsets, including these quasi-identical TIL-TCRs, increased during the patient’s response to treatment but gradually declined as acquired resistance developed. These findings highlight the interplay between tumor-infiltrating and circulating T cells, underscoring that CD8+ T cells with highly homologous TCR sequences can exhibit diverse transcriptional behaviors and dynamics in response to immunotherapy. Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

An elevated baseline neutrophil-to-lymphocyte ratio (NLR) can signal an imbalance between pro-tumor and anti-tumor inflammatory responses and has been associated with worse overall survival (OS) and lower response rates in patients with non-small cell lung cancer (NSCLC) treated with immune checkpoint inhibitors (ICIs). Corticosteroids, known to cause neutrophilia and lymphopenia, are widely used in NSCLC, but their impact on biomarker performance remains elusive. This study investigates how baseline steroid use affects biomarker performance in NSCLC patients on ICIs. We retrospectively evaluated 252 patients with NSCLC treated with ICIs at the Roswell Park Comprehensive Cancer Center (RPCCC) (n=81) (discovery cohort) and the University of Southern California (USC) (n=171) (validation cohort). We analyzed the association between baseline NLR at ICI initiation with overall response rate (ORR), progression free survival (PFS), and OS. NLR was defined as absolute neutrophil count divided by absolute lymphocyte count. Patients were stratified by NLR: NLR ≥ 5 (high) and NLR < 5 (low). ORR was estimated using Fisher’s exact test, and Kaplan-Meier survival distributions for PFS and OS were generated. In our cohort, 159 (63.1%) of 252 patients had an NLR < 5 and 93 (33.9%) had NLR ≥ 5. NLR < 5 at baseline was correlated with longer PFS (RPCCC: p<0.0005; USC: p<0.005) and OS (RPCCC: p<0.0005; USC: p<0.05) although there was no significant relationship between NLR and treatment response rates in RPCCC or USC cohorts. We identified 19 patients who were on systemic steroids at the time of initiation of ICI therapy. Indications for corticosteroids were brain metastases or comorbid lung conditions such as COPD, and all 19 patients remained on steroids for at least 12 weeks from the initiation of ICI therapy. When examining the predictive value of NLR by steroid and non-steroid subgroups in both cohorts, we found no significant association between NLR and treatment response rates for patients taking steroids and patients not taking steroids. Patients not taking steroids with low NLR showed markedly longer PFS (RPCCC: p<0.005; USC: p<0.05) and OS (RPCCC: p<0.001; USC: p<0.05). However, patients taking steroids showed no significant correlation of NLR with PFS or OS. Baseline NLR was associated with prognosis of NSCLC patients undergoing ICI therapy who do not receive steroids at the initiation of ICI therapy but was not correlated with response to ICI therapy. The results of NLR can be obtained via routine bloodwork and readily available for prompt feedback to oncologists and patients; however, clinicians must take care in the prognostic value of NLR in patients who were on steroids at the initiation of ICI therapy. Angelina Lim, Alexandra Meyer, Lauren Polyakov, Ryan Cooper, Aubree Mades, Shirley Ye, Joshua Ni, Hongbin Chen, Leslie Contreras, Joy Sarkar, Rajeev Sharma, Brahm H. Segal, Robert Hsu, Jorge Nieva, Grace K. Dy, Fumito Ito. Impact of corticosteroids on predictive and prognostic value of neutrophil-to-lymphocyte ratio in patients with non-small cell lung cancer treated with immune checkpoint inhibitor therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2021.

8555 Background: Pulmonary sarcomatoid carcinoma (PSC) is a rare subtype of non-small cell lung cancer (NSCLC), with historically poor outcome. Excellent response of metastatic PSC to immunotherapy was previously reported, but its outcome compared with other NSCLCs is unknown in the immunotherapy era. Methods: NCDB was queried for stage IV NSCLC diagnosed from 2016 to 2020. Patients who were treated with chemotherapy only or immunotherapy only in the first course of treatment were included. Log-rank was used for survival analysis and adjusted by cox regression for age, gender, race, academic/integrated center, insurance, and comorbidity. Results: A total of 37,617 patients were identified, including 201 PSCs and 37,416 other NSCLCs. Pleomorphic (54%) was the most common PSC subtype. There was significant interaction between the survival effect of treatment and histology (interaction p = 0.024). When treated with immunotherapy alone, no significant difference was found between PSC and other NSCLCs (median survival 11.4 vs 12.4 months, logrank p = 0.56, HR 1.02, 95% CI 0.81 – 1.30). However, when treated with chemotherapy alone, PSC remained significantly associated with worse survival (median survival 4.5 vs. 8.5 months, logrank p <0.001, HR 0.67, 95% CI 0.54 – 0.83). Among PSC subtypes, spindle/giant cell PSCs were more likely to benefit from immunotherapy alone than pleomorphic PSCs (interaction p = 0.005), when compared with chemotherapy (Spindle/giant cell subtype: HR 0.24, 95% CI 0.14 – 0.42; Pleomorphic subtype: HR 0.60, 95% CI 0.37 – 0.99). Conclusions: Compared with other NSCLCs, PSC may not be associated with worse survival when treated with immunotherapy alone as it was when treated with chemotherapy. Distinct PSC subtypes may respond differently to immunotherapy and warrants further research. [Table: see text]

This study investigates patient preferences for adjuvant Osimertinib in EGFR-mutated NSCLC without complete OS data. This prospective survey assessed thresholds for DFS benefits and OS justifications. Findings reveal diverse patient preferences and highlight the importance of shared decision-making. Factors like employment, education, and willingness to pay significantly influenced decisions. Background: The ADAURA trial confirmed adjuvant Osimertinib's efficacy in EGFR-mutated Non-small-cell lung cancer (NSCLC), yet the limited mature overall survival (OS) data at approval poses a challenge. This study explores patient preferences in the absence of complete OS information, hypothesizing that disease-free survival (DFS) benefit alone may influence adjuvant Osimertinib pursuit. Methods: At Roswell Park Comprehensive Cancer Center (Jan-Dec 2021), patients assessed for adjuvant therapy received a survey probing OS and DFS preferences. Scenarios were (a) minimum OS justifying Osimertinib, (b) minimum DFS improvement justifying 3-years of adjuvant Osimertinib, (c) minimum 5-year DFS percent change, and (d) minimum OS justifying copay changes. Results were analyzed. Results: Of 524 NSCLC patients, 51 participated. Scenario 1 saw 56% requiring a 12-month OS benefit for Osimertinib justification. In scenario 2, 72% deemed a 12-month DFS benefit sufficient. Scenario 3 revealed 31% opting out despite a 10% OS increase. Scenario 4 showed varied willingness to pay, with 33% unwilling to any shoulder copayment even with a 10-year OS benefit. Conclusion: This study explores patient preferences without complete OS data, revealing diverse thresholds. Factors include employment, education, and willingness to pay. Findings underscore shared decision-making importance. Limitations include sample size, potential biases, and regional focus; larger cohorts are needed for validation.

Background:The Epithelial-Mesenchymal Transition (EMT) is a very important process involved in cancer invasion and metastasis. Additionally, the Cathepsin K (CTSK) gene is closely related to the degradation of the extracellular matrix, which is a critical component of the EMT. The purpose of this study was to determine the relationships between EMT-related genes and immune cell infiltration and their prognostic value in Thyroid carcinoma (THCA). The effect of the CTSK gene on the aggressive biological features of THCA was assessed. Methods:Within the framework of the present study, the THCA cohort was analyzed in detail based on data obtained from The TCGA database in the context of the EMT. The TCGA-THCA cohort was then divided into two groups, namely, high- and low-risk groups, based on the calculated EMT scores. Finally, based on the findings from the Weighted Gene Co-Expression Network Analysis (WGCNA) algorithm, LASSO regression analysis, and Kaplan-Meier plotter, we selected five genes (CTSK, C3ORF80, FBLN2, PRELP and SRPX2) associated with patient prognosis. Furthermore, this study examined the presence of various immune cells within the THCA samples using three distinct algorithms, namely ssGSEA, xCell, and MCPcounter. Additional studies have been conducted to establish the roles of CTSK in THCA cell proliferation and migration using various assays, such as CCK8, colony formation, EdU proliferation, Transwell migration and wound healing assays. Additionally, the involvement of CTSK in the regulation of various EMT-related markers was confirmed using Western blot analysis. Results:Based on EMT scores, TCGA-THCA patients were further divided into two groups, and the study revealed that patients in the high-risk group had a worse prognosis than those in the low-risk group. Among the five genes linked to the prognostic value of EMT (CTSK, C3ORF80, FBLN2, PRELP, and SRPX2), CTSK exhibited notably elevated expression in the high-risk cohort. This group also exhibited pronounced immune cell infiltration, with a marked correlation observed between CTSK expression and the levels of macrophages, MDSCs, and various T-cell subtypes. Furthermore, in vitro studies demonstrated that reducing CTSK expression led to significant reductions in THCA cell viability; clonogenic, proliferative, motility and migratory capacities; and the expression of key EMT-related proteins, including N-cadherin, vimentin, slug, and snail. Conclusion:Our results suggest that the expression of CTSK, a gene associated with the EMT, may be associated with THCA onset and progression and thus may serve as a promising prognostic biomarker.

<p>Supplementary Figure 1: Single-cell RNA/TCR sequencing (scRNA/TCR-seq) quality assessment. A Gating strategy of identifying single live CD45+CD19–CD3+CD56– cells from cryopreserved peripheral mononuclear blood cells for scRNA/TCR-seq. B Representative flow cytometric plots showing the frequency of CD45+CD19–CD3+CD56– cells after flow-sort. C Scatterplots depicting total features, counts, doublet scores, mitochondrial content and ribosomal content across all cells prior to filtering. Filtering thresholds applied are shown. D Elbow plot of PCA (top 50 components). PC selection for downstream analysis was limited to those components accounting for at least 0.1 % of total variation (n = 44). E UMAP representations of all-post filtered, normalized cells showing cellular distributions of total unique counts, mitochondrial scores, ribosomal scores, G2M/S phase scores and doublet scores. F UMAP plot identifying doublets by hashtag oligos (HTO).</p>

<p>Supplementary Figure 3. An increase of the CX3CR1 score is associated with better survival in NSCLC patients undergoing chemo-immunotherapy. Related to Fig. 1E Progression free survival (PFS) and overall survival (OS) for high (≥ 20%) versus low (< 20%) CX3CR1 score. P values were calculated by a log-rank (Mantel-Cox) test.</p>

<p>Supplementary Figure 7. Related to Fig. 3 Bar graphs displaying the counts of tumor-infiltrating lymphocytes (TILs) expressing a given TCRβ clonotype on the y axis and individual TIL clonotypes ordered by increasing frequency on the x axis for a patient in supplementary figure 4.</p>

<p>Supplementary Figure 4. Related to Fig. 2, 3 and Supplementary Fig. 5-7. 68-year-old female with bilateral lung metastases and multiple mediastinal lymph node metastasis was treated with chemo-immunotherapy (carboplatin, pemetrexed, and pembrolizumab). PD-L1 expression in the pre-treatment tumor specimen was 2%. The patient had stable disease for 621 days with overall survival > 3 years. A Contrast-enhanced cross-sectional imaging obtained at prior to and during treatment. B Expression of CX3CR1 in peripheral blood CD8 T cells (left) and the CX3CR1 score (right) at different time points as indicated.</p>

Background Immune checkpoint inhibitors (ICIs) have become a cornerstone of treatment for patients with advanced non-small cell lung cancer (NSCLC). Many patients take corticosteroids, which can potentially decrease the efficacy of treatment; however, the potential impact of baseline corticosteroids at the time of treatment initiation and the underlying mechanisms remain elusive. Methods We retrospectively evaluated 88 patients with NSCLC who received ICIs with or without peri-treatment steroids at medium or high doses as defined by a medium dose of >7.5 mg, but ≤30 mg prednisone equivalent a day and a high dose of >30 mg, prednisone equivalent daily. We assessed the progression-free survival (PFS), overall survival (OS), and overall response rate (ORR) Results Eleven (13%) of 88 patients were taking corticosteroid at the start of ICI therapy. Patients who received steroids at any dose had a significantly lower PFS (mPFS: 324 days vs. 86.5 days and 96 days for no steroids (n=77) vs. medium (n=7) and high (n=4) steroids; p = 0.0152) while significantly lower OS was identified in patients with high doses of steroids (mOS: 639 days and 613 days vs. 207 days for no and medium steroids vs. high steroids; p = 0.0249). Patients who received steroids tend to have lower ORR (9.1% vs. 36.3% for steroids vs. no steroids, p = 0.0635). We analyzed the frequency of peripheral blood (PB) CD8+ T cells expressing a differentiation marker, CX3CR1, and discovered that the baseline frequency of CX3CR1+CD8+ T cells was significantly lower in patients who were on steroids (p = 0.0293). Using a bedside-to-bench approach, we confirmed that concurrent steroid use significantly decreased anti-tumor efficacy of anti-PD-1 therapy and PB CX3CR1+CD8+ T cells in mice bearing MC38 tumors while discontinuation of steroids at the start of treatment did not negatively impact survival and T-cell differentiation. Conclusions Baseline corticosteroid use was associated with worse outcome in NSCLC patients treated with ICI therapy and decreased frequency of circulating CX3CR1+CD8+ differentiated T cells. Results from a pre-clinical model suggested that the discontinuation of steroid at the time of treatment initiation could maintain the efficacy of anti-PD-1 therapy and T-cell differentiation. Taken together, these findings support the recommendation to taper steroids to the lowest possible dose before ICI administration to alleviate symptoms without compromising the benefits of ICIs, if possible, and provide mechanistic insight into the impact of corticosteroid on T-cell differentiation. Acknowledgements We would like to thank all our patients and their families who generously participated in our study. This work was supported by National Cancer Institute grants P30CA016056 and by the National Center for Advancing Translational Sciences of the National Institute of Health UL1TR001412. This work was further supported by the Department of Defense Lung Cancer Research Program (LC180245) and the National Cancer Institute grant K08CA197966, R01CA255240–01A1. Ethics Approval All patients gave informed consent. All patient information including blood samples, archived tumor tissue, and medical records was reviewed under the Institutional Review Boards of Roswell Park Comprehensive Cancer Center protocol approval (I 188310) and the University of Southern California protocol approval (HS-22–00134).

<p>Supplementary Figure 5. Related to Figure 2 and Supplementary Table 4. Analysis of peripheral blood CD45+CD19–CD3+CD56– cells by scRNA/TCR-seq. Heatmap of all cells showing the expression levels of the 10 most discriminative genes per cell type (in rows) across all the identified cell populations (in columns). Gene expression in each clusters are also listed in Supplementary Table 4. Color-code layout: scale of purple to yellow; from lowest expression to highest expression.</p>

<p>Supplementary Figure 5. Related to Figure 2 and Supplementary Table 4. Analysis of peripheral blood CD45+CD19–CD3+CD56– cells by scRNA/TCR-seq. Heatmap of all cells showing the expression levels of the 10 most discriminative genes per cell type (in rows) across all the identified cell populations (in columns). Gene expression in each clusters are also listed in Supplementary Table 4. Color-code layout: scale of purple to yellow; from lowest expression to highest expression.</p>

Background:Immune checkpoint inhibitors (ICIs) such as programmed cell death protein-1 (PD-1) inhibitors or PD-1 ligand-1 (PD-L1) inhibitors have led to remarkable improvement in outcomes of non-small cell lung cancer (NSCLC). Unfortunately, the significant benefits of ICI therapy are frequently limited by resistance to treatment and adverse effects, and the predictive value of pre-treatment tumor tissue PD-L1 expression is limited. Development of less invasive biomarkers that could identify responders and non-responders in early on-treatment could markedly improve the treatment regimen. Accumulating evidence suggests that baseline gut microbiota profile is associated with response to PD-1/PD-L1 blockade therapy. However, change in the gut microbiome composition during PD-1/PD-L1 blockade therapy and its relation to response remain unclear.Methods:Here, we analyzed pre- and on-treatment fecal samples from five NSCLC patients receiving anti-PD-1 immunotherapy, alone or in tandem with chemotherapy, and performed 16S rRNA sequencing.Results:The overall alpha diversity of the baseline gut microbiome was similar between three responders and two non-responders. While the gut microbiome composition remained stable overall during treatment (R2 = 0.145), responders showed significant changes in microbiome diversity between pre- and on-treatment samples during anti-PD-1 therapy compared to non-responders (P = 0.0274). Within the diverse microbiota, responders showed decreases in the abundance of genera Odoribacter, Gordonibacter, Candidatus Stoquefichus, Escherichia-Shigella, and Collinsella, and increase in abundance of Clostridium sensu stricto 1. In contrast, non-responders demonstrated on-treatment increases in genera Prevotella, Porphyromonas, Streptococcus, and Escherichia-Shigella, and decrease in abundance of Akkermansia.Conclusions:This pilot study identified a substantial change in gut microbiome diversity between pre- and on-treatment samples in NSCLC patients responding to anti-PD-1 therapy compared to non-responders. Our findings highlight the potential utility of gut microbiota dynamics as a noninvasive biomarker to predict response to PD-1/PD-L1 blockade therapy for a wide variety of malignancies, which sets a path for future investigation in larger prospective studies.

<p>Supplementary Figure 1: Single-cell RNA/TCR sequencing (scRNA/TCR-seq) quality assessment. A Gating strategy of identifying single live CD45+CD19–CD3+CD56– cells from cryopreserved peripheral mononuclear blood cells for scRNA/TCR-seq. B Representative flow cytometric plots showing the frequency of CD45+CD19–CD3+CD56– cells after flow-sort. C Scatterplots depicting total features, counts, doublet scores, mitochondrial content and ribosomal content across all cells prior to filtering. Filtering thresholds applied are shown. D Elbow plot of PCA (top 50 components). PC selection for downstream analysis was limited to those components accounting for at least 0.1 % of total variation (n = 44). E UMAP representations of all-post filtered, normalized cells showing cellular distributions of total unique counts, mitochondrial scores, ribosomal scores, G2M/S phase scores and doublet scores. F UMAP plot identifying doublets by hashtag oligos (HTO).</p>

<p>Supplementary Figure 4. Related to Fig. 2, 3 and Supplementary Fig. 5-7. 68-year-old female with bilateral lung metastases and multiple mediastinal lymph node metastasis was treated with chemo-immunotherapy (carboplatin, pemetrexed, and pembrolizumab). PD-L1 expression in the pre-treatment tumor specimen was 2%. The patient had stable disease for 621 days with overall survival > 3 years. A Contrast-enhanced cross-sectional imaging obtained at prior to and during treatment. B Expression of CX3CR1 in peripheral blood CD8 T cells (left) and the CX3CR1 score (right) at different time points as indicated.</p>

<p>Supplementary Figure 6. Related to Fig. 2 and Supplementary Table 5A, B. Heat map showing the top 10 top significantly enriched pathways found in each T-cell cluster. Gene sets from Reactome (A) and Gene Ontology-Biological Processes (GO-BP) (B) are shown separately. Only gene sets with Benjamini-Hochberg-adjusted p < 0.05 were considered as significantly enriched.</p>