Elsevier

European Journal of Cancer

Volume 158, November 2021, Pages 123-132
European Journal of Cancer

Original Research
Immune infiltrates in patients with localised high-risk soft tissue sarcoma treated with neoadjuvant chemotherapy without or with regional hyperthermia: A translational research program of the EORTC 62961-ESHO 95 randomised clinical trial

https://doi.org/10.1016/j.ejca.2021.09.015Get rights and content

Highlights

  • Preoperative therapy turned the non-inflamed tumour into an immune infiltrated tumour.

  • Immune infiltrate associated with response and survival when hyperthermia was added.

  • Neoadjuvant chemotherapy with hyperthermia primed an anti-tumour microenvironment.

Abstract

Background

The EORTC 62961-ESHO 95 randomised trial showed improved long-term survival of patients with high-risk soft-tissue sarcoma by adding regional hyperthermia to neoadjuvant chemotherapy. We hypothesised that immune infiltrate of patients treated with neoadjuvant therapy associate with clinical outcome.

Methods

Tumour infiltrating lymphocytes (TILs) and CD8, FOXP3, PD-1, and PD-L1 were evaluated in sequential biopsies of patients after four cycles of therapy.

Results

From a subgroup of 109 patients who had been randomised between July 1997 and November 2006 to neoadjuvant chemotherapy (53 patients) or neoadjuvant chemotherapy with regional hyperthermia (56 patients), 137 biopsies were obtained. TILs increased in paired second biopsies independent of treatment allocation (p < 0.001). FOXP3 regulatory T cells decreased (p = 0.002), and PD-L1 expression of tumours became undetectable. In the multivariate analysis, post-treatment high TILs correlated to LPFS (HR: 0.34; 95% CI 0.15–0.75; p = 0.008) and DFS (HR: 0.38; 95% CI 0.17–0.82; p = 0.015). In comparing post-treatment immune infiltrate between treatment arms, tumour response was associated with neoadjuvant chemotherapy with regional hyperthermia (p = 0.013) and high TILs (p = 0.064). High CD8 cell infiltration was associated with improved LPFS (HR: 0.27; 95% CI 0.09–0.79; Log-rank p = 0.011) and DFS (HR: 0.25; 95% CI 0.09–0.73; Log-rank p = 0.006). Improved survival at 10 years was associated with immune infiltrate after neoadjuvant chemotherapy with regional hyperthermia.

Conclusion

Preoperative therapy re-programs a non-inflamed tumour at baseline into an inflamed tumour. The post-treatment immune infiltrate became predictive for clinical outcomes. The combination with regional hyperthermia primes the tumour microenvironment, enabling enhanced anti-tumour immune activity in high-risk soft tissue sarcomas.

Trial registration

ClinicalTrials.gov, NCT00003052.

Introduction

The growing field of immuno-oncology and the success of immune checkpoint inhibitors (ICI) in distinct types of malignancies has led to considerable interest in the evaluation of immune infiltrates of sarcomas [1,2]. The binding of the programmed death receptor ligand 1 (PD-L1) upregulated on tumour cells to its cognate programmed death receptor 1 (PD-1) on immune cells provides an immune escape mechanism that can block infiltrating cytotoxic T cells, subsequently allowing tumour progression [3]. Soft tissue sarcomas (STS) are considered ‘non-immunogenic’ and are typically characterised by low tumour mutational burden (TMB) [4]. However, high throughput profiling recently identified two main subgroups with distinct immune profiles, clinical outcome and sensitivity to targeted therapies [5]. One of the greatest challenges lies in the therapy of immunologically “cold” tumours that are devoid of tumour infiltrating immune cells at baseline, underscoring the need for strategies that increase the immune infiltrate into the tumour, i.e. convert a cold tumour into a hot tumour [6]. Retrospective studies mostly refer to the sarcoma-associated immune infiltrate at the time of diagnosis. Low TILs and expression of PD-1/PD-L1 were predictors of poor prognosis [[7], [8], [9]]. As recently presented, neoadjuvant checkpoint blockade therapy with concurrent radiotherapy showed remarkable pathological responses [10]. For the same subgroup of patients, correlative analyses in the SARC028 trial showed an association between radiographic response to pembrolizumab and sarcoma-associated immune infiltrate [11]. The results support the notion that specific immune characteristics of the TMA may impact the potential clinical benefit of ICIs [12].

The translational research program of the EORTC-ESHO trial included the objective of whether immune mechanisms in addition to the well-known drug synergy with hyperthermia might be involved in the therapy associated efficacy of the combined treatment. Therefore, we analysed the immune infiltrate in biopsies of patients at baseline and/or after four cycles of neoadjuvant therapy to decipher the characteristics that may associate with clinical tumour response and survival.

Section snippets

Methods

The study design and the results of the European Organisation for Research and Treatment of Cancer (EORTC) 62961- European Society of Hyperthermia Oncology (ESHO) 95 randomised trial (NCT 00003052) have been previously published and discussed in the context of other neoadjuvant chemotherapy trials [[13], [14], [15]] The study protocol included an optional accompanying translational program, to determine immune cells of tumour tissue. Adult patients with histologically proven STS with the

Results

From 329 patients of the EORTC -ESHO randomised trial, 109 (33%) patients with available tumour biopsies were included in the analysis of immune infiltrate. Between July 1997 and November 2006, patients had received either four cycles of preoperative chemotherapy combined with regional hyperthermia (N = 56patients; NAC + RHT) or preoperative chemotherapy alone (N = 53 patients; NAC) as the allocated treatment followed by evaluation of tumour response and surgery (Fig. 1A). From these 109

Discussion

One of our main results was that preoperative chemotherapy without or with concomitantly applied RHT turned the state of a cold, non-immunogenic sarcoma into a more immunogenic tumour with high TILs, a decrease of immune-suppressive FOXP3 regulatory T-cells, and absence of PD-L1 expression. Chemotherapy alone has been shown to induce cancer immunogenic cell death (ICD) with the release of danger signals that can promote tumour immunogenicity via activation of cytotoxic T-cells and depletion of

Conclusion

Several consequences for clinical practice can be deduced from our results. First, preoperative therapy re-programs a non-inflamed tumour at baseline into an inflamed tumour. Second, a survey of immune infiltration prior to initiation of neoadjuvant therapy does not provide predictive evidence for the outcome. Third, the combination with regional hyperthermia primes the tumour microenvironment, enabling enhanced anti-tumour immune activity in high-risk STS.

Author contributions

Dr. Issels has full access to all of the data and takes responsibility for the accuracy of the data analysis.

Translational study program and design: Issels, Buecklein.

Acquisition, analysis, or interpretation of data: Knoesel, Schmidt, Issels, Noessner, Lindner, Altendorf-Hofmann, Xu, Mansmann, Albertsmeier, Stutz, Blay, Abdel-Rahman.

Drafting of the manuscript: Issels, Noessner, Lindner.

Critical revision of the manuscript for important intellectual content: Issels, Noessner, Lindner, Stutz,

Funding

The study was funded by the European Organisation for Research and Treatment of Cancer Soft Tissue and Bone Sarcoma Group (EORTC -STBSG 62961).

Conflict of interest statement

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: R.D.I. received travel support and honoraria from Pyrexar, PharmaMar, MedTherm, and Bayer. E.N. received travel support and honoraria from Roche Pharma, AstraZeneca, Novartis, IPSEN, Definiens AG, Visiopharm, Hexal, Solutions Akademie GmbH, Phio Pharmaceuticals Corp, and Medigene Immunotherapeutics GmbH. M.A. received travel support and honoraria from PharmaMar

Acknowledgments

The statistical analysis was performed independently from the sponsor at the Institute of Medical Information Sciences, Biometrics, and Epidemiology (LMU), headed by Ulrich Mansmann. The senior investigator, Thomas Knoesel, organised the acquisition of tumour samples in close collaboration with the Department of Pathology at the LMU. Manuela Gruber provided editorial assistance with the final draft of the manuscript and coordinated the submission.

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