BACKGROUND: Currently, clinical implications of immune system cells in chordoma remain to be elucidated. OBJECTIVE: To characterize in situ immune cell infiltrates, the Immunoscore, and investigate their correlation with clinicopathologic data of spinal chordoma patients and outcome. METHODS: Tumor-infiltrating lymphocytes (TILs) subtypes were assessed in 54 tumor specimens using immunohistochemistry for CD3, CD4, CD8, CD20, Foxp3, PD-1, and PD-L1. RESULTS: Overall, immune cell infiltrates were present in all samples and there was low or moderate correlation among several TILs subsets. PD-1⁺ TILs density, CD3⁺, and CD8⁺ TILs densities in the tumor interior (TI) subarea were associated with surrounding muscle invasion by tumor, whereas PD-L1⁺ TILs showed inverse association with tumor pathological grade and stage. The density of PD-1⁺ TILs, PD-L1⁺ TILs, CD4⁺ TILs, and CD3⁺ TILs both in the TI and combined tumor regions (TI and invasion margin) were significantly associated with local recurrence-free survival and overall survival (OS). However, Foxp3⁺ TILs (P = .024) and CD8⁺ TILs evaluated in the TI (P < .001) only correlated with OS. The Immunoscore predicted less aggressive clinical features and favorable outcomes. Patients with an Immunoscore of 4 had a median OS of 128 mo, while I0 (Immunoscore of 0) patients survived only 27 mo. Multivariate analysis demonstrated that the Immunoscore was an independent favorable prognostic factor of both local recurrence-free survival (P = .026) and OS (P = .046). CONCLUSION: Our data suggest a clinically relevant role of the immune microenvironment in spinal chordoma and identify the Immunoscore as promising prognostic marker.

BACKGROUND: Currently, little is known about the clinical relevance of tumor-stroma ratio (TSR) in chordoma and data discussing the relationship between TSR and immune status of chordoma are lacking. OBJECTIVE: To characterize TSR distribution in spinal chordoma, and investigated its correlation with clinicopathologic or immunological features of patients and outcome. METHODS: TSR was assessed visually on hematoxylin and eosin-stained sections from 54 tumor specimens by 2 independent pathologists. Multiplex immunofluorescence was used to quantify the expression levels of microvessel density, Ki-67, Brachyury, and tumor as well as stromal PD-L1. Tumor immunity status including the Immunoscore and densities of tumor-infiltrating lymphocytes (TILs) subtypes were obtained from our published data and reanalyzed. RESULTS: Bland-Altman plot showed no difference between mean TSR derived from the two observers. TSR was positively associated with stromal PD-L1 expression, the Immunoscore and CD3⁺ as well as CD4⁺ TILs density, but negatively correlated with tumor microvessel density, Ki-67 index, surrounding muscle invasion by tumor and number of Foxp3⁺ and PD-1⁺ TILs. Low TSR independently predicted poor local recurrence-free survival and overall survival. Moreover, patients with low TSR and low Immunoscore chordoma phenotype were associated with the worst survival. More importantly, combined TSR and Immunoscore accurately reflected prognosis and enhanced the ability of TSR or Immunoscore alone for outcome prediction. CONCLUSION: These data reveal the significant impact of TSR on tumor progression and immunological response of patients. Subsequent use of agents targeting the stroma compartment may be an effective strategy to treat chordoma especially in combination with immune-based drugs.

Background: Currently, the measurement of immune cells in previous studies is usually subjective, and no immune‐based prognostic model has been established for chordoma. In this study, we sought to simultaneously measure tumor‐infiltrating lymphocyte (TIL) subtypes in chordoma samples using an objective method and develop an immune risk score (IRS) model for survival prediction.Methods: Multiplexed quantitative immunofluorescence staining was used to determine the TIL levels in the tumoral and stromal subareas of 114 spinal chordoma specimens (54 in the training and 60 in the validation cohort) for programmed death‐1 (PD‐1), CD3, CD8, CD20 (where CD is cluster of differentiation), and FOXP3. Flow cytometry was performed to validate the immunofluorescence assay for lymphocyte measurement on an additional five fresh chordoma specimens. Subsequently, the IRS model was built using the least absolute shrinkage and selection operator (LASSO) Cox regression method.Results: Flow cytometry and quantitative immunofluorescence showed similar lymphocytic percentages and TIL subpopulation proportions in the fresh tumor specimens. With the training data, the LASSO model identified four immune features for IRS construction: _tumoralFOXP3, _tumoralPD‐1, _stromalFOXP3, and _stromalCD8. In both cohorts, a high IRS was significantly associated with tumoral programmed cell death‐1 ligand 1 expression, Enneking inappropriate tumor resection, and surrounding muscle invasion by tumor. Multivariate Cox regression and stratified analysis in the two cohorts revealed that the IRS was an independent predictor and could effectively separate patients with similar Enneking staging into different risk subgroups, with significantly different survival rates. Further receiver operating characteristic analysis found that the IRS classifier had a better prognostic value than the traditional clinicopathological factors and compensated for the deficiency of Enneking staging for outcome prediction. More importantly, a nomogram based on the IRS and clinical predictors showed adequate performance in estimating disease recurrence and survival of patients.Conclusions: These data support the use of the IRS signature as a reliable prognostic tool in spinal chordoma and may facilitate individualized therapy decision making for patients.