Granzyme B as a predictive biomarker for therapeutic outcomes in non-small cell lung cancer patients undergoing anti-PD-1 therapy
Abstract
BackgroundImmune checkpoint inhibitors (ICIs) targeting the PD-1/PD-L1 axis have transformed the management of advanced non-small cell lung cancer (NSCLC). However, durable clinical benefit is restricted to a subset of patients, necessitating reliable, minimally invasive pharmacodynamic biomarkers to monitor treatment engagement and response.MethodsIn this prospective observational study evaluating an expanded patient cohort overlapping with our previous reporting, 64 patients with advanced NSCLC receiving second-line nivolumab monotherapy and 30 healthy controls were analyzed. Peripheral blood samples were collected longitudinally at baseline (T0), 6–8 weeks (T1), and 14–16 weeks (T2) post-treatment initiation. Plasma Granzyme B (GrmB) concentrations (pg/mL) were quantified via ELISA, and cell subset-specific intracellular expression was characterized using multiparametric flow cytometry. Archival tumor tissue was evaluated for PD-L1 expression, tumor mutational burden, microsatellite instability, and TP53 status. Exploratory in-vitro experiments evaluated A549 and NCI-H23 cell lines exposed to direct nivolumab treatment (0-200 µg/mL). Longitudinal dynamics were evaluated using repeated-measures linear mixed-effects models with multiplicity adjustments, and survival outcomes (OS and PFS) were assessed using Kaplan–Meier.ResultsResponders (R) demonstrated significantly higher baseline tumor PD-L1 expression compared to non-responders (NR). Flow cytometric immunophenotyping revealed dynamic expansions in responders, including a three-fold increase in CD3+CD56+ NKT-like cells, a two-fold increase in NK cells, a ten-fold elevation in CD8+Granzyme B+ T cells, and a 2.5-fold increase in proliferating Ki67+CD8+PD-1+ T cells. Circulating plasma Granzyme B concentrations were markedly elevated in responders compared to non-responders and healthy controls (P < 0.0001). In exploratory cell-line assays, direct nivolumab exposure increased Granzyme B secretion and expression in A549 and NCI-H23 cells without impacting short-term cell viability. While higher circulating Granzyme B trajectory correlated with superior progression-free and overall survival, multivariable Cox regression indicated that Granzyme B kinetics serve as a dynamic marker of active immune engagement rather than an independent pre-treatment predictive biomarker when adjusting for overall clinical response.ConclusionLongitudinal monitoring of plasma Granzyme B kinetics and cytotoxic lymphocyte activation particularly CD3+CD56+ NKT-like and effector CD+ T-cell subsets provides a valuable non-invasive pharmacodynamic indicator of systemic anti-tumor immune activation during nivolumab therapy in advanced NSCLC.
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BibTeX
@article{jain2026granzyme,
title = {{Granzyme B as a predictive biomarker for therapeutic outcomes in non-small cell lung cancer patients undergoing anti-PD-1 therapy}},
author = {Kriti Jain and Asees Singh and Shyam Aggarwal and Amit Awasthi and Nirmal Kumar Ganguly and Evanka Madan},
journal = {Frontiers in Oncology},
year = {2026},
month = nov,
doi = {10.3389/fonc.2026.1964690},
url = {https://www.frontiersin.org/articles/10.3389/fonc.2026.1964690},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Granzyme B as a predictive biomarker for therapeutic outcomes in non-small cell lung cancer patients undergoing anti-PD-1 therapy
AU - Kriti Jain
AU - Asees Singh
AU - Shyam Aggarwal
AU - Amit Awasthi
AU - Nirmal Kumar Ganguly
AU - Evanka Madan
PY - 2026
DA - 2026/11/09
JO - Frontiers in Oncology
DO - 10.3389/fonc.2026.1964690
UR - https://www.frontiersin.org/articles/10.3389/fonc.2026.1964690
AB - BackgroundImmune checkpoint inhibitors (ICIs) targeting the PD-1/PD-L1 axis have transformed the management of advanced non-small cell lung cancer (NSCLC). However, durable clinical benefit is restricted to a subset of patients, necessitating reliable, minimally invasive pharmacodynamic biomarkers to monitor treatment engagement and response.MethodsIn this prospective observational study evaluating an expanded patient cohort overlapping with our previous reporting, 64 patients with advanced NSCLC receiving second-line nivolumab monotherapy and 30 healthy controls were analyzed. Peripheral blood samples were collected longitudinally at baseline (T0), 6–8 weeks (T1), and 14–16 weeks (T2) post-treatment initiation. Plasma Granzyme B (GrmB) concentrations (pg/mL) were quantified via ELISA, and cell subset-specific intracellular expression was characterized using multiparametric flow cytometry. Archival tumor tissue was evaluated for PD-L1 expression, tumor mutational burden, microsatellite instability, and TP53 status. Exploratory in-vitro experiments evaluated A549 and NCI-H23 cell lines exposed to direct nivolumab treatment (0-200 µg/mL). Longitudinal dynamics were evaluated using repeated-measures linear mixed-effects models with multiplicity adjustments, and survival outcomes (OS and PFS) were assessed using Kaplan–Meier.ResultsResponders (R) demonstrated significantly higher baseline tumor PD-L1 expression compared to non-responders (NR). Flow cytometric immunophenotyping revealed dynamic expansions in responders, including a three-fold increase in CD3+CD56+ NKT-like cells, a two-fold increase in NK cells, a ten-fold elevation in CD8+Granzyme B+ T cells, and a 2.5-fold increase in proliferating Ki67+CD8+PD-1+ T cells. Circulating plasma Granzyme B concentrations were markedly elevated in responders compared to non-responders and healthy controls (P < 0.0001). In exploratory cell-line assays, direct nivolumab exposure increased Granzyme B secretion and expression in A549 and NCI-H23 cells without impacting short-term cell viability. While higher circulating Granzyme B trajectory correlated with superior progression-free and overall survival, multivariable Cox regression indicated that Granzyme B kinetics serve as a dynamic marker of active immune engagement rather than an independent pre-treatment predictive biomarker when adjusting for overall clinical response.ConclusionLongitudinal monitoring of plasma Granzyme B kinetics and cytotoxic lymphocyte activation particularly CD3+CD56+ NKT-like and effector CD+ T-cell subsets provides a valuable non-invasive pharmacodynamic indicator of systemic anti-tumor immune activation during nivolumab therapy in advanced NSCLC.
SN - 2234-943X
ER -