Insights from prospective multi-omic profiling of lymphocytes in resected lung cancer

In this issue of Annals of Oncology, Federico and colleagues report prospectively collected multi-omic data from 150 patients with localized non-small-cell lung cancer (NSCLC) who underwent resection at the MD Anderson Cancer Center from 2016 to 2018 (ICON cohort).1Federico L. McGrail D.J. Bentebibel S.-E. et al.Distinct tumor-infiltrating lymphocyte landscapes are associated with clinical outcomes in localized non-small cell lung cancer.Ann Oncol. 2022; 33: 42-56Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar Tissue from 76 of these patients were collected freshly to allow for flow cytometric analysis to be assessed in tandem with other readouts that can be carried out on standard workflow formalin-fixed paraffin-embedded material, including multiplex immunofluorescence, T-cell receptor (TCR) sequencing, bulk RNA sequencing, and bulk whole-exome sequencing. The authors confirm prior data that total T cells, regulatory T cells, and T cells with activation/exhaustion features are enriched in the tumor relative to the adjacent normal tissue.2Lavin Y. Kobayashi S. Leader A. et al.Innate immune landscape in early lung adenocarcinoma by paired single-cell analyses.Cell. 2017; 169: 750-765.e17Abstract Full Text Full Text PDF PubMed Scopus (633) Google Scholar, 3Guo X. Zhang Y. Zheng L. et al.Global characterization of T cells in non-small-cell lung cancer by single-cell sequencing.Nat Med. 2018; 24: 978-985Crossref PubMed Scopus (515) Google Scholar, 4Lizotte P.H. Ivanova E.V. Awad M.M. et al.Multiparametric profiling of non-small-cell lung cancers reveals distinct immunophenotypes.JCI Insight. 2016; 1: e89014Crossref PubMed Google Scholar, 5Stankovic B. Bjørhovde H.A.K. Skarshaug R. et al.Immune cell composition in human non-small cell lung cancer.Front Immunol. 2018; 9: 3101Crossref PubMed Scopus (111) Google Scholar Using orthogonal methods, the authors show that neither total T cells, nor total CD8 T cells, were associated with recurrence-free survival (RFS) and the authors confirmed this finding in a larger dataset of The Cancer Genome Atlas (TCGA). This result contrasts with prior reports that found total CD8 T cells associated with survival in smaller patient cohorts.6Kim S.H. Go S.I. Song D.H. et al.Prognostic impact of CD8 and programmed death-ligand 1 expression in patients with resectable non-small cell lung cancer.Br J Cancer. 2019; 120: 547-554Crossref PubMed Scopus (19) Google Scholar,7Mazzaschi G. Madeddu D. Falco A. et al.Low PD-1 expression in cytotoxic CD8(+) tumor-infiltrating lymphocytes confers an immune-privileged tissue microenvironment in NSCLC with a prognostic and predictive value.Clin Cancer Res. 2018; 24: 407-419Crossref PubMed Scopus (140) Google Scholar Beyond total and CD8 T cells, there was also no association of 220 individual T-cell subpopulations assessed by flow cytometry and RFS in this ICON cohort. One caveat is that the flow cytometry dataset was ultimately based on 47 patient samples due to certain samples being excluded for quality control, which raises the possibility that the lack of association of certain T-cell subpopulations with RFS may be due to insufficient statistical power. Nonetheless, since single T-cell parameters were not individually prognostic, the authors generated two immunotypes (IM1 and IM2) utilizing t-distributed stochastic neighbor embedding (tSNE) on their flow cytometry data. IM1 was associated with improved RFS and was enriched in natural killer cells, BTLA+, granzyme B+, CD103−, CD103+ PD-1− Tim-3− CD8 T cells, and also CD103−, CD103+ PD-1− Tim-3−, and total CD4 T cells. These results indicate that features of T-cell exhaustion on both CD4 and CD8 T cells are associated with worse outcomes after resection. These results are very much in line with two prior studies7Mazzaschi G. Madeddu D. Falco A. et al.Low PD-1 expression in cytotoxic CD8(+) tumor-infiltrating lymphocytes confers an immune-privileged tissue microenvironment in NSCLC with a prognostic and predictive value.Clin Cancer Res. 2018; 24: 407-419Crossref PubMed Scopus (140) Google Scholar,8Koh J. Go H. Keam B. et al.Clinicopathologic analysis of programmed cell death-1 and programmed cell death-ligand 1 and 2 expressions in pulmonary adenocarcinoma: comparison with histology and driver oncogenic alteration status.Mod Pathol. 2015; 28: 1154-1166Crossref PubMed Scopus (131) Google Scholar and with poor survival seen in early-stage lung patients in the TRAcking Cancer Evolution through therapy (TRACERx) studies whose resected tumor tissues showed ‘differentiation skewing’, which is defined as loss of progenitor-like CD4 T cells and increased abundance of dysfunctional CD4 and CD8 T cells.9Ghorani E. Reading J.L. Henry J.Y. et al.The T cell differentiation landscape is shaped by tumour mutations in lung cancer.Nat Cancer. 2020; 1: 546-561Crossref PubMed Scopus (40) Google Scholar As the authors note in their discussion, although enhanced T-cell exhaustion is associated with worse prognosis in this cohort, it is also predictive of improved response to immune checkpoint blockade (ICB) in other studies.10Kumagai S. Togashi Y. Sakai C. et al.The PD-1 expression balance between effector and regulatory T cells predicts the clinical efficacy of PD-1 blockade therapies.Nat Immunol. 2020; 21: 1346-1358Crossref PubMed Scopus (163) Google Scholar,11Thommen D.S. Koelzer V.H. Herzig P. et al.A transcriptionally and functionally distinct PD-1(+) CD8(+) T cell pool with predictive potential in non-small-cell lung cancer treated with PD-1 blockade.Nat Med. 2018; 24: 994-1004Crossref PubMed Scopus (453) Google Scholar Hence, there may be an opportunity to turn an unfavorable prognosis into a therapeutically actionable one with ICB therapies in the neoadjuvant and/or adjuvant settings. Notably, despite prior reports that CD103 on CD8 T cells (a marker of tissue residence) were associated with improved survival in NSCLC,12Koh J. Kim S. Kim M.Y. Go H. Jeon Y.K. Chung D.H. Prognostic implications of intratumoral CD103+ tumor-infiltrating lymphocytes in pulmonary squamous cell carcinoma.Oncotarget. 2017; 8: 13762-13769Crossref PubMed Scopus (55) Google Scholar the authors report that CD103+ CD8 T cells were in fact lower in the IM1 favorable subtype and the prognostic significance of CD103 was modulated by the concomitant expression of T-cell exhaustion markers. Using patient samples with the IM1 or IM2 immunotypes, the authors analyzed linked bulk RNA sequencing to generate a 70-gene expression signature derived from the IM1 immunotype. When mapped on to TCGA data from lung cancer patients, this derived signature was associated with a modest increase in overall survival. The gene signature extracted from the IM1 immunotype also featured enrichment of B cells. The authors used orthogonal modalities in the ICON cohort and TCGA data to show that high levels of the B-cell marker CD20 was associated with enhanced survival. When re-evaluating the data as a combined biomarker with total T-cell infiltration, they show that high levels of both B cells and T cells were associated with the greatest survival in both the ICON and TCGA cohorts. Tertiary lymphoid structures (TLS) are ectopic lymphoid aggregates that include B and T cells and are reported to propagate local immune responses in non-lymphoid tissues. Concordant with the combined prognostic importance of B and T cells, the authors find more TLS in the IM1 immunotype biospecimens. These findings are in line with recent studies highlighting B cells and TLS as integral to the efficacy of ICB therapy.13Helmink B.A. Reddy S.M. Gao J. et al.B cells and tertiary lymphoid structures promote immunotherapy response.Nature. 2020; 577: 549-555Crossref PubMed Scopus (691) Google Scholar, 14Cabrita R. Mitra S. Sanna A. et al.Tertiary lymphoid structures improve immunotherapy and survival in melanoma.Nature. 2020; 577: 561-565Crossref PubMed Scopus (569) Google Scholar, 15Petitprez F. de Reyniès A. Keung E.Z. et al.B cells are associated with survival and immunotherapy response in sarcoma.Nature. 2020; 577: 556-560Crossref PubMed Scopus (600) Google Scholar The authors carried out rigorous secondary analyses to ensure that captured covariates were not differentially represented in IM1 and IM2. One limitation of the prospective collection that occurred from 2016 to 2018 is that driver mutation status was not annotated in this dataset. Although driver mutation status has generally had only modest, if any, effects on disease-free status in early-stage resected lung cancer,16Suda K. Mitsudomi T. Shintani Y. et al.Clinical Impacts of EGFR mutation status: analysis of 5780 surgically resected lung cancer cases.Ann Thorac Surg. 2021; 111: 269-276Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 17Graziano S.L. Gamble G.P. Newman N.B. et al.Prognostic significance of K-ras codon 12 mutations in patients with resected stage I and II non-small-cell lung cancer.J Clin Oncol. 1999; 17: 668-675Crossref PubMed Google Scholar, 18Shepherd F.A. Domerg C. Hainaut P. et al.Pooled analysis of the prognostic and predictive effects of KRAS mutation status and KRAS mutation subtype in early-stage resected non-small-cell lung cancer in four trials of adjuvant chemotherapy.J Clin Oncol. 2013; 31: 2173-2181Crossref PubMed Scopus (217) Google Scholar NSCLC subtypes do generally display differential immune profiles.19Toki M.I. Mani N. Smithy J.W. et al.Immune marker profiling and programmed death ligand 1 expression across NSCLC mutations.J Thorac Oncol. 2018; 13: 1884-1896Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar Thus, it would be useful to re-analyze this data after driver mutation status can be annotated, which should theoretically be possible from the paired whole-exome data. The authors should be commended for planning, undertaking, and completing this comprehensive study. This study has produced an invaluable dataset for the lung cancer community, which the authors have thankfully made available (raw tissue flow cytometry, pathology, and clinical annotation https://data.mendeley.com/datasets/2j3dpx83w6/1; raw TCRseq through ImmunoSeq website, https://doi.org/10.21417/LF2021AO). Beyond the robust specific findings detailed earlier, the study has shown that comprehensive multi-omic profiling that includes fresh tissue processing on this scale is feasible. Fresh tissue processing enables single-cell approaches that include not only the high-dimensional flow cytometry featured in this study but also simultaneous single-cell protein, transcriptomic, and TCR profiling. Moreover, once in a fresh single-cell suspension, this material can be cryopreserved for future functional immunological correlates, such as suppression and cytotoxicity assays. This ICON study can serve as a model on which to build future prospective collections to maximize what we can learn from valuable human biospecimens. This work was supported by an NIH Clinical Investigator Award [grant number K08 CA-248723 ] to [AC]; the Damon Runyon Cancer Research Foundation [grant number CI-98-18 ] to [MDH]; the Memorial Sloan Kettering Cancer Center support grant/core grant [grant number P30 CA008748 ] to [AC and MDH].