Activation of peripheral-blood granulocytes is strongly correlated with patient outcome after immunotherapy with anti-GD2 monoclonal antibody and granulocyte-macrophage colony-stimulating factor

Abstract

Purpose: Adjuvant therapy using anti-GD2 monoclonal antibody and granulocyte-macrophage colony-stimulating factor (GM-CSF) has shown treatment success for patients with high-risk neuroblastoma (NB). Although there is ample evidence on how the antibody targets NB, in vivo contribution by GM-CSF remains unclear. This report investigates granulocyte activation and its correlation with treatment outcome.

Patients and methods: Patients enrolled onto NCT00072358 received multiple treatment cycles, each consisting of anti-GD2 antibody 3F8 plus subcutaneous (SC) GM-CSF. Peripheral-blood (PB) samples from 151 patients were collected on day 0 and day 4 of cycle 1. PB from a subgroup of 35 patients had intravenous (IV) instead of SC GM-CSF during cycle 4. Samples were analyzed by flow cytometry for CD11a, CD63, CD87, and CD11b and its activation epitope CBRM1/5.

Results: Comparing cycle 1 day 4 PB samples with day 0 PB samples, five of five activation marker-positive granulocytes were significantly higher. The change in frequency and mean fluorescence intensity of CBRM1/5-positive granulocytes correlated with progression-free survival (PFS; P = .024 and P = .008, respectively). A multivariable analysis identified increasing CBRM1/5-positive granulocytes and missing killer immunoglobulin-like receptor ligand as positive independent prognostic factors for PFS, whereas second-line cyclophosphamide-based therapy before protocol entry negatively influenced outcome. Thirty-five patients who received SC GM-CSF at cycle 1 and IV GM-CSF at cycle 4 had significantly less CBRM1/5 activation after IV GM-CSF. In contrast, 63 patients who received SC GM-CSF at both cycles had comparable CBRM1/5 activation.

Conclusion: GM-CSF-induced granulocyte activation in vivo is associated with improved patient outcome. This activation was more apparent when GM-CSF was given by the SC route instead of IV route.

Conflict of interest statement

Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.

Figures

Fig 1.

Distribution of leukocyte population during cycle 1 of anti-GD2 antibody 3F8 plus subcutaneous granulocyte-macrophage colony-stimulating factor (GM-CSF). Day 0 (pre) samples were obtained after 5 days of GM-CSF at 250 μg/m2, and day 4 (post) samples were obtained after 4 more days of GM-CSF (2 days at 250 μg/m2/d, followed by 2 additional days at 500 μg/m2/d; mean + SEM; P < .001).

Fig 2.

Frequency of activation marker in leukocyte population in day 0 (pre) and day 4 (post) peripheral blood of patients during cycle 1 of anti-GD2 antibody 3F8 plus subcutaneous granulocyte-macrophage colony-stimulating factor: (A) granulocytes, (B) lymphocytes, and (C) monocytes. Frequency was defined as the percentage of cells positive for the specific activation marker in the respective leukocyte population. (*) P < .03. (†) P < .0001. (‡) P < .001.

Fig 3.

Correlation between mean fluorescence intensity (MFI) of CBRM1/5-positive granulocytes and progression-free survival among 147 patients treated with anti-GD2 antibody 3F8 plus subcutaneous granulocyte-macrophage colony-stimulating factor (GM-CSF). Median ratio of day 4 MFI over day 0 MFI was used as the cut point (solid line, > median, n = 73; dotted line, P = .008).

Fig 4.

Comparing frequency change of CBRM1/5-positive granulocytes from day 0 to day 4 peripheral blood when patients were treated with (A) subcutaneous (sc) granulocyte-macrophage colony-stimulating factor (GM-CSF; cycle 1) and intravenous (iv) GM-CSF (cycle 4; n = 35) or (B) SC GM-CSF (cycles 1 and 4; n = 63).