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    Prediction of lymph node metastasis by tumor-infiltrating lymphocytes in T1 breast cancer. Takada Koji,Kashiwagi Shinichiro,Asano Yuka,Goto Wataru,Kouhashi Rika,Yabumoto Akimichi,Morisaki Tamami,Shibutani Masatsune,Takashima Tsutomu,Fujita Hisakazu,Hirakawa Kosei,Ohira Masaichi BMC cancer BACKGROUND:Lymph node metastasis is more likely in early-stage breast cancer with lower tumor-infiltrating lymphocyte (TIL) density. Therefore, we investigated the correlation between TILs and lymph node metastasis in cT1 breast cancer patients undergoing surgery and the usefulness of TILs in predicting sentinel lymph node metastasis (SLNM) in cT1N0M0 breast cancer. METHODS:We investigated 332 breast cancer patients who underwent surgery as the first-line treatment after preoperative diagnosis of cT1. A positive diagnosis of SLNM as an indication for axillary clearance was defined as macrometastasis in the sentinel lymph node (SLN) (macrometastasis: tumor diameter > 2 mm). Semi-quantitative evaluation of lymphocytes infiltrating the peritumoral stroma as TILs in primary tumor biopsy specimens prior to treatment was conducted. RESULTS:For SLN biopsy (SLNB), a median of 2 (range, 1-8) SLNs were pathologically evaluated. Sixty cases (19.4%) of SLNM (macrometastasis: 46, micrometastasis: 16) were observed. Metastasis was significantly greater in breast cancers with tumor diameter > 10 mm than in those with diameter ≤ 10 mm (p = 0.016). Metastasis was significantly associated with lymphatic invasion (p < 0.001). These two clinicopathological factors correlated with SLNM even in patients diagnosed with cN0 (tumor size; p = 0.017, lymphatic invasion; p = 0.002). Multivariate analysis for SLNM predictors revealed lymphatic invasion (p = 0.008, odds ratio [OR] = 2.522) and TILs (p < 0.001, OR = 0.137) as independent factors. CONCLUSIONS:Our results suggest a correlation between lymph node metastasis and tumor immune-microenvironment in cT1 breast cancer. TIL density may be a predictor of SLNM in breast cancer without lymph node metastasis on preoperative imaging. 10.1186/s12885-020-07101-y
    Characterization of the immune microenvironment of NSCLC by multispectral analysis of multiplex immunofluorescence images. Surace Michael,Rognoni Lorenz,Rodriguez-Canales Jaime,Steele Keith E Methods in enzymology Multiplex immunofluorescence (MIF) staining of tumor sections combined with computational pathology quantifies phenotypic variants of tumor and immune cells and assesses their spatial relationships. Here, we discuss a MIF panel composed of cytokeratin, PD-L1, PD1, CD8, CD68, and Ki67 applied to non-small cell lung cancer (NSCLC) to demonstrate key components of the immune response to this cancer. We also describe a method of whole-slide multiplex imaging and digital multispectral image analysis. Key aspects of marker labeling and digital tissue and cellular classification are highlighted. We then illustrate how digital analysis can measure the spatial relationships among important cell types. This approach is presented in the context of a multidisciplinary team of scientists who together can optimize the combined methods to increase the impact of the study findings. Recommendations are provided to assist others to apply similar methods to further understand the immune response to NSCLC. 10.1016/bs.mie.2019.07.039
    Close proximity of immune and tumor cells underlies response to anti-PD-1 based therapies in metastatic melanoma patients. Gide Tuba N,Silva Ines P,Quek Camelia,Ahmed Tasnia,Menzies Alexander M,Carlino Matteo S,Saw Robyn P M,Thompson John F,Batten Marcel,Long Georgina V,Scolyer Richard A,Wilmott James S Oncoimmunology Immune checkpoint blockade has greatly improved the clinical outcomes of many patients with metastatic melanoma, however, almost half do not respond. Whether the interspatial distribution of immune and tumor cells predicts response to anti-PD-1-based therapies and patient outcomes in any cancer, including melanoma, is currently unknown. Here, we examined the spatial distribution of immune and tumor cells via multiplex immunofluorescence. Pre-treatment melanoma specimens from 27 patients ( = 18 responders; = 9 non-responders) treated with anti-PD-1 monotherapy and 34 patients ( = 22 responders; = 12 non-responders) treated with combined ipilimumab and anti-PD-1 immunotherapy were studied. Responders displayed significantly higher densities of CD8 tumor-infiltrating lymphocytes within a 20 µM distance from a melanoma cell compared to non-responders in both anti-PD-1 alone ( = .0024) and combination-treated patients ( = .0096), that were associated with improved progression-free survival for both therapies (anti-PD-1 = .0158; combination therapy = .0088). In multivariate analysis, the best model for 12-month progression-free survival for anti-PD-1 monotherapy included PD-L1 cells within proximity to tumor cells and intratumoral CD8 density (AUC = 0.80), and for combination therapy included CD8 cells in proximity to tumor cells, intratumoral PD-L1 density and LDH (AUC = 0.85). Assessment of the spatial distribution of immune cells in relation to tumor cells provides insight into their role in modulating immune response and highlights their potential role as predictors of response to anti-PD-1 based therapies. 10.1080/2162402X.2019.1659093
    Relevance of Spatial Heterogeneity of Immune Infiltration for Predicting Risk of Recurrence After Endocrine Therapy of ER+ Breast Cancer. Heindl Andreas,Sestak Ivana,Naidoo Kalnisha,Cuzick Jack,Dowsett Mitchell,Yuan Yinyin Journal of the National Cancer Institute Background:Despite increasing evidence supporting the clinical utility of immune infiltration in the estrogen receptor-negative (ER-) subtype, the prognostic value of immune infiltration for ER+ disease is not well defined. Methods:Quantitative immune scores of cell abundance and spatial heterogeneity were computed using a fully automated hematoxylin and eosin-stained image analysis algorithm and spatial statistics for 1178 postmenopausal patients with ER+ breast cancer treated with five years' tamoxifen or anastrozole. The prognostic significance of immune scores was compared with Oncotype DX 21-gene recurrence score (RS), PAM50 risk of recurrence (ROR) score, IHC4, and clinical treatment score, available for 963 patients. Statistical tests were two-sided. Results:Scores of immune cell abundance were not associated with recurrence-free survival. In contrast, high immune spatial scores indicating increased cell spatial clustering were associated with poor 10-year, early (0-5 years), and late (5-10 years) recurrence-free survival (Immune Hotspot: LR-χ2 = 14.06, P < .001, for 0-10 years; LR-χ2 = 6.24, P = .01, for 0-5 years; LR-χ2 = 7.89, P = .005, for 5-10 years). The prognostic value of spatial scores for late recurrence was similar to that of IHC4 and RS in both populations, but was not as strong as other tests in comparison for recurrence across 10 years. Conclusions:These results provide a missing link between tumor immunity and disease outcome in ER+ disease by examining tumor spatial architecture. The association between spatial scores and late recurrence suggests a lasting memory of protumor immunity that may impact disease progression and evolution of endocrine treatment resistance, which may be exploited for therapeutic advances. 10.1093/jnci/djx137
    Tumor-Infiltrating Lymphocytes-Location for Prognostic Evaluation. Peled Michael,Onn Amir,Herbst Roy S Clinical cancer research : an official journal of the American Association for Cancer Research Tumor-infiltrating lymphocytes (TIL) are crucial for the success of immunotherapy, and their density can predict prognosis. It has now been shown that computer-based analysis of the spatial organization of TILs adds prognostic value in early-stage non-small cell lung cancer, possibly improving the treatment algorithm to prevent recurrence.. 10.1158/1078-0432.CCR-18-3803
    The non-small cell lung cancer immune landscape: emerging complexity, prognostic relevance and prospective significance in the context of immunotherapy. Anichini Andrea,Tassi Elena,Grazia Giulia,Mortarini Roberta Cancer immunology, immunotherapy : CII Immunotherapy of non-small cell lung cancer (NSCLC), by immune checkpoint inhibitors, has profoundly improved the clinical management of advanced disease. However, only a fraction of patients respond and no effective predictive factors have been defined. Here, we discuss the prospects for identification of such predictors of response to immunotherapy, by fostering an in-depth analysis of the immune landscape of NSCLC. The emerging picture, from several recent studies, is that the immune contexture of NSCLC lesions is a complex and heterogeneous feature, as documented by analysis for frequency, phenotype and spatial distribution of innate and adaptive immune cells, and by characterization of functional status of inhibitory receptor T cells. The complexity of the immune landscape of NSCLC stems from the interaction of several factors, including tumor histology, molecular subtype, main oncogenic drivers, nonsynonymous mutational load, tumor aneuploidy, clonal heterogeneity and tumor evolution, as well as the process of epithelial-mesenchymal transition. All these factors contribute to shape NSCLC immune profiles that have clear prognostic significance. An integrated analysis of the immune and molecular profile of the neoplastic lesions may allow to define the potential predictive role of the immune landscape for response to immunotherapy. 10.1007/s00262-018-2147-7
    Spatial heterogeneity of the T cell receptor repertoire reflects the mutational landscape in lung cancer. Joshi Kroopa,de Massy Marc Robert,Ismail Mazlina,Reading James L,Uddin Imran,Woolston Annemarie,Hatipoglu Emine,Oakes Theres,Rosenthal Rachel,Peacock Thomas,Ronel Tahel,Noursadeghi Mahdad,Turati Virginia,Furness Andrew J S,Georgiou Andrew,Wong Yien Ning Sophia,Ben Aissa Assma,Sunderland Mariana Werner,Jamal-Hanjani Mariam,Veeriah Selvaraju,Birkbak Nicolai J,Wilson Gareth A,Hiley Crispin T,Ghorani Ehsan,Guerra-Assunção José Afonso,Herrero Javier,Enver Tariq,Hadrup Sine R,Hackshaw Allan,Peggs Karl S,McGranahan Nicholas,Swanton Charles, ,Quezada Sergio A,Chain Benny Nature medicine Somatic mutations together with immunoediting drive extensive heterogeneity within non-small-cell lung cancer (NSCLC). Herein we examine heterogeneity of the T cell antigen receptor (TCR) repertoire. The number of TCR sequences selectively expanded in tumors varies within and between tumors and correlates with the number of nonsynonymous mutations. Expanded TCRs can be subdivided into TCRs found in all tumor regions (ubiquitous) and those present in a subset of regions (regional). The number of ubiquitous and regional TCRs correlates with the number of ubiquitous and regional nonsynonymous mutations, respectively. Expanded TCRs form part of clusters of TCRs of similar sequence, suggestive of a spatially constrained antigen-driven process. CD8 tumor-infiltrating lymphocytes harboring ubiquitous TCRs display a dysfunctional tissue-resident phenotype. Ubiquitous TCRs are preferentially detected in the blood at the time of tumor resection as compared to routine follow-up. These findings highlight a noninvasive method to identify and track relevant tumor-reactive TCRs for use in adoptive T cell immunotherapy. 10.1038/s41591-019-0592-2
    Spatial Architecture and Arrangement of Tumor-Infiltrating Lymphocytes for Predicting Likelihood of Recurrence in Early-Stage Non-Small Cell Lung Cancer. Corredor Germán,Wang Xiangxue,Zhou Yu,Lu Cheng,Fu Pingfu,Syrigos Konstantinos,Rimm David L,Yang Michael,Romero Eduardo,Schalper Kurt A,Velcheti Vamsidhar,Madabhushi Anant Clinical cancer research : an official journal of the American Association for Cancer Research PURPOSE:The presence of a high degree of tumor-infiltrating lymphocytes (TIL) has been proven to be associated with outcome in patients with non-small cell lung cancer (NSCLC). However, recent evidence indicates that tissue architecture is also prognostic of disease-specific survival and recurrence. We show a set of descriptors (spatial TIL, SpaTIL) that capture density, and spatial colocalization of TILs and tumor cells across digital images that can predict likelihood of recurrence in early-stage NSCLC. EXPERIMENTAL DESIGN:The association between recurrence in early-stage NSCLC and SpaTIL features was explored on 301 patients across four different cohorts. Cohort D ( = 70) was used to identify the most prognostic SpaTIL features and to train a classifier to predict the likelihood of recurrence. The classifier performance was evaluated in cohorts D ( = 119), D ( = 112), and D ( = 112). Two pathologists graded each sample of D and D; intraobserver agreement and association between manual grading and likelihood of recurrence were analyzed. RESULTS:SpaTIL was associated with likelihood of recurrence in all test sets (log-rank < 0.02). A multivariate Cox proportional hazards analysis revealed an HR of 3.08 (95% confidence interval, 2.1-4.5, = 7.3 × 10). In contrast, agreement among expert pathologists using tumor grade was moderate (Kappa = 0.5), and the manual TIL grading was only prognostic for one reader in D ( = 8.0 × 10). CONCLUSIONS:A set of features related to density and spatial architecture of TILs was found to be associated with a likelihood of recurrence of early-stage NSCLC. This information could potentially be used for helping in treatment planning and management of early-stage NSCLC.. 10.1158/1078-0432.CCR-18-2013
    Hyperspectral cell sociology reveals spatial tumor-immune cell interactions associated with lung cancer recurrence. Enfield Katey S S,Martin Spencer D,Marshall Erin A,Kung Sonia H Y,Gallagher Paul,Milne Katy,Chen Zhaoyang,Nelson Brad H,Lam Stephen,English John C,MacAulay Calum E,Lam Wan L,Guillaud Martial Journal for immunotherapy of cancer BACKGROUND:The tumor microenvironment (TME) is a complex mixture of tumor epithelium, stroma and immune cells, and the immune component of the TME is highly prognostic for tumor progression and patient outcome. In lung cancer, anti-PD-1 therapy significantly improves patient survival through activation of T cell cytotoxicity against tumor cells. Direct contact between CD8+ T cells and target cells is necessary for CD8+ T cell activity, indicating that spatial organization of immune cells within the TME reflects a critical process in anti-tumor immunity. Current immunohistochemistry (IHC) imaging techniques identify immune cell numbers and densities, but lack assessment of cell-cell spatial relationships (or "cell sociology"). Immune functionality, however, is often dictated by cell-to-cell contact and cannot be resolved by simple metrics of cell density (for example, number of cells per mm). To address this issue, we developed a Hyperspectral Cell Sociology technology platform for the analysis of cell-cell interactions in multi-channel IHC-stained tissue. METHODS:Tissue sections of primary tumors from lung adenocarcinoma patients with known clinical outcome were stained using multiplex IHC for CD3, CD8, and CD79a, and hyperspectral image analysis determined the phenotype of all cells. A Voronoi diagram for each cell was used to approximate cell boundaries, and the cell type of all neighboring cells was identified and quantified. Monte Carlo analysis was used to assess whether cell sociology patterns were likely due to random distributions of the cells. RESULTS:High density of intra-tumoral CD8+ T cells was significantly associated with non-recurrence of tumors. A cell sociology pattern of CD8+ T cells surrounded by tumor cells was more significantly associated with non-recurrence compared to CD8+ T cell density alone. CD3+ CD8- T cells surrounded by tumor cells was also associated with non-recurrence, but at a similar significance as cell density alone. Cell sociology metrics improved recurrence classifications of 12 patients. Monte Carlo re-sampling analysis determined that these cell sociology patterns were non-random. CONCLUSION:Hyperspectral Cell Sociology expands our understanding of the complex interplay between tumor cells and immune infiltrate. This technology could improve predictions of responses to immunotherapy and lead to a deeper understanding of anti-tumor immunity. 10.1186/s40425-018-0488-6
    Spatial interaction of tumor cells and regulatory T cells correlates with survival in non-small cell lung cancer. Barua Souptik,Fang Penny,Sharma Amrish,Fujimoto Junya,Wistuba Ignacio,Rao Arvind U K,Lin Steven H Lung cancer (Amsterdam, Netherlands) OBJECTIVES:To determine the prognostic significance of spatial proximity of lung cancer cells and specific immune cells in the tumor microenvironment. MATERIALS AND METHODS:We probed formalin-fixed, paraffin-embedded (FFPE) tissue microarrays using a novel tyramide signal amplification multiplexing technique labelling CD8, CD4, Foxp3, and CD68+ cells. Each multiplex stained immunohistochemistry slide was digitally processed by Vectra INFORMS software, and an X- and Y-coordinate assigned to each labeled cell type. The abundance and spatial location of each cell type and their proximity to one another was analyzed using a novel application of the G-cross spatial distance distribution method which computes the probability of finding at least one immune cell of any given type within a rμm radius of a tumor cell. Cox proportional hazards multiple regression was used for multivariate analysis of the influence of proximity of lymphocyte types. RESULTS:Pathologic tumor specimens from 120 NSCLC patients with pathologic tumor stage I-III disease were analyzed. On univariate analysis, age (P = .0007) and number of positive nodes (P = .0014) were associated with overall survival. Greater area under the curve (AUC) of the G-cross function for tumor cell-Treg interactions was significantly associated with worse survival adjusting for age and number of positive nodes (HR 1.52 (1.11-2.07), P = .009). Greater G-cross AUC for T-reg-CD8 was significantly associated with better survival adjusting for age and number of positive lymph nodes (HR 0.96 (0.92-0.99), P = .042). CONCLUSION:Increased infiltration of regulatory T cells into core tumor regions is an independent predictor of worse overall survival in NSCLC. However, increased infiltration of CD8+ cytotoxic T cells among regulatory T cells seems to mitigate this effect and was significantly associated with better survival. Validation of the G-cross method of measuring spatial proximity between tumor and immune cell types and exploration of its use as a prognostic factor in lung cancer treatment is warranted. 10.1016/j.lungcan.2018.01.022