Neutrophil Extracellular Trap is Surrogate Biomarker for Prognosis and Response to Neoadjuvant Therapy in Locally Advanced Rectal Cancer

Abstract

Purpose

To demonstrate the intrinsic association of Neutrophil extracellular traps (NETs) with outcome and neoadjuvant therapy response of locally advanced rectal cancer (LARC), and the mechanisms.

Patients and Methods

We enrolled 240 patients with LARC who underwent surgery without neoadjuvant therapy in two independent sets (training and validation), and 153 patients who received neoadjuvant therapy with biopsy followed by surgery. Immunohistochemistry, immunofluorescence staining and bioinformatics analysis were performed in formalin-fixed paraffin-embedded sections. NETs were identified by costaining for myeloperoxidase and citrullinated histone H3.

Results

NETs were associated with recurrence-free survival in the surgical training and validation sets. High-NET density predicted poor postoperative survival of patients with LARC. Multivariate analysis identified NETs, TNM stage, and neutrophil-to-lymphocyte ratio as independent prognostic factors for recurrence-free survival. Low-NETs LARC demonstrated increased CD8⁺ T cell and lower T regulatory cell infiltration, which indicated a tumor immune microenvironment with strong antitumor capacity. High-NET density was associated with epithelial–mesenchymal transition, which is considered to contribute to tumor progression. In the neoadjuvant therapy cohort, high-NET density on biopsy was significantly associated with reduced likelihood of complete/near complete response.

Conclusion

NET was an independent prognostic factor in LARC that were associated with patients’ survival, and NET density in pretreatment biopsies was an independent predictive biomarker of response to neoadjuvant therapy. This biomarker may be helpful in predicting survival in LARC with improved accuracy and selecting patients who will respond to neoadjuvant therapy.

Keywords:

• NETs
• tumor microenvironment
• locally advanced rectal cancer
• outcome

Abbreviations

NETs, Neutrophil extracellular traps; LARC, Locally advanced rectal cancer; nCRT, neoadjuvant chemoradiotherapy; MPO, Myeloperoxidase; NE, Neutrophil elastase; AJCC, American Joint Committee of Cancer; RFS, recurrence-free survival; TRS, Tumor regression score; pCR, pathological complete response; i-pCR, incomplete pathological remission; FFPE, formalin fixed and paraffin embedded; CitH3, citrullinated histone H3; Treg, T regulatory; BSA, bovine serum albumin; DAB, diaminobenzidine; GSEA, Gene Set Enrichment Analysis; ssGSEA, Single sample gene set enrichment analysis; NLR, neutrophil-to-lymphocyte ratio; EMT, epithelial–mesenchymal transition; TME, tumor microenvironment; DEGs, differentially expressed genes.

Data Sharing Statement

Most of the data generated and analyzed during the current study are available from the Supplementary Data and the corresponding author upon reasonable request.

Ethics Approval and Consent to Publication

The studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards and were reviewed and approved by the Institute Research Ethics Committees of the 7th Medical Center of Chinese PLA General Hospital and Affiliated Drum Tower Hospital of Nanjing University Medical School (2023-38). The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.

Acknowledgments

We thank Majorbio Company for offering the gene expression sequencing.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Additional information

Funding

This work was supported by the National Natural Science Foundation of China [No. 81870393, JFD, and 81970500, XFS].