Early in vitro studies showed that granulocytes mediate efficient ADCC of NB. Cytotoxicity was dependent on FcγRIIA (CD32) and CD11b/CD18 integrins but was not dependent on oxidative intermediates.14–16
Subsequent studies using Lym-1 antibody in B-cell lymphoma showed similar findings.33
In the presence of GM-CSF, anti-GD2 MoAb was effective against NB.13,23
Combining GM-CSF with anti-CD20 antibody was also found to have benefit in patients with advanced non-Hodgkin's lymphoma and Hodgkin's disease.34
Despite compelling in vitro evidence, the in vivo role of neutrophils in these antitumor responses was uncertain. This report provides the first evidence, to our knowledge, that granulocyte activation by GM-CSF in patients, as measured by the CD11b activation epitope CBRM1/5, may be an independent prognostic factor on outcome. This phenomenon held true for patients who were in first and second CR/VGPR, as well as those who had primary refractory NB before immunotherapy. Moreover, PB from patients with GM-CSF administered by SC route elicited greater granulocyte activation when compared with the IV route. This finding may account, in part, for the superior survival outcome of patients receiving SC GM-CSF (NCT00072358) when compared with patients receiving IV GM-CSF (NCT00002560).35
These data, together with the prognostic importance of FCGR2A
(CD32) polymorphism among patients with primary refractory NB,26
gave credence that myeloid cells, specifically granulocytes, contributed to the antitumor response after combination therapy using anti-GD2 antibody and GM-CSF.
This study demonstrated that an increase in frequency or MFI of CBRM1/5-positive granulocytes during the first treatment cycle was a key favorable determinant of patient outcome. This correlation was somewhat unexpected, because multiple cycles were given to patients for up to 2 years on this protocol. These data suggest that the level of granulocyte excitability in patients receiving SC GM-CSF was similar throughout the repeated cycles and that the excitability determined as early as the first cycle was prognostic of treatment outcome. In addition, because the size of the circulating granulocyte population, relative to the marginating pool or to the marrow reserve, was variable between patients, the absolute granulocyte count or CBRM1/5-positive granulocyte count per microliter of blood might not be representative of the patient's total-body granulocyte pool.
The biologic effects of GM-CSF can be multifold.36
It stimulates production of myeloid cells, including granulocytes and monocytes/macrophages, accelerating hematopoietic recovery after dose-intensive chemotherapy.36
It also stimulates the maturation of monocytes into antigen-presenting cells. When locally combined with cancer vaccines,37
GM-CSF has shown clinical promise.36
However, GM-CSF is also a potent stimulator of myeloid-derived suppressor cells (MDSCs), which can interfere with both the innate and adaptive immune response.38,39
In mouse models, MDSCs are Gr-1+
myeloid precursor cells that can differentiate into dendritic cells, macrophages, and neutrophils. MDSCs accumulate in the blood, lymph nodes, and bone marrow and at tumor sites to inhibit both adaptive and innate immunity.40–42
Tumor-infiltrating MDSCs have also been associated with adverse patient outcome.43
Furthermore, the stimulation of MDSCs by high GM-CSF levels could reduce vaccine response and possibly decrease antitumor efficacy in patients.38,39
It was reassuring that CD11b activation in our patients had positive instead of negative impact on patient outcome.
In addition to inducing MDSCs,44
high doses of GM-CSF (> 250 μg/m2
/d) have also been implicated to have a negative effect on monocyte and lymphocyte ADCC.45
Hence, optimal doses of GM-CSF of 40 to 80 μg/d were suggested for vaccine development,46
and doses of 80 to 150 μg/d (< 150 μg/m2
/d) were suggested for antibody therapy.45
However, in the randomized studies of ch14.18, GM-CSF at 250 μg/m2
/d seemed beneficial for patient outcome.13
In our study, at an even higher dose (500 μg/m2
/d for 3 days; ), there was further granulocyte activation, which correlated with superior PFS.
Previous studies have also shown that after repeated daily injections of GM-CSF 250 μg/m2
/d, peak serum concentration of GM-CSF gradually decreased on days 5 and 10 when compared with day 1. Such a decline in serum level was less pronounced in cycle 4 compared with cycle 1.45
Thus, serum GM-CSF levels could not have accounted for the smaller increase in CBRM1/5-positive granulocytes in PB in cycle 4 when GM-CSF was administered IV (A). These results suggest that the SC route of GM-CSF administration was more effective than the IV route in granulocyte activation.
In addition to CBRM1/5 activation, two other independent prognostic factors were identified in the multivariable analysis, namely the favorable impact of missing KIR ligand and the negative impact when additional cyclophosphamide-based therapy was required to consolidate remission (). Inadequate response to induction therapy is well known to be an adverse prognostic factor in high-risk NB. In the randomized study carried out by the Children's Oncology Group, achieving CR/VGPR was a highly favorable predictor of outcome.47
In our study, patients with refractory disease or relapsed disease were treated with high-dose cyclophosphamide-based regimens containing vincristine with either topotecan or irinotecan.31
Once they achieved CR/VGPR or stable disease, they were enrolled onto the 3F8/GM-CSF protocol. Thus, it is not surprising that in the multivariable model, the necessity of a cyclophosphamide-based second-line regimen was a poor prognostic marker associated with refractory or high-risk disease.
Both in the context of autologous stem-cell transplantation27
and high-dose cyclophosphamide,48
missing KIR ligand seemed to be highly prognostic for patient outcome after treatment with antibody 3F8 plus GM-CSF. Although NK cells alone were suboptimal in killing NB,49
killing by both licensed and unlicensed NK cells was much more efficient in the presence of antibody 3F8.50
Previous studies in patients undergoing autotransplantation51
and more recently patients receiving hu14.18–interleukin-2 fusion protein52
have also implicated missing KIR ligand as a prognostic factor. In our analysis, lymphocytes had low CBRM1/5 expression, and as expected, it did not increase with GM-CSF. CBRM1/5 among the NK subset was not tested, but this may be an important marker to monitor in future studies. Although supporting the role played by NK cells in mediating antitumor response,27
this study strongly suggests that granulocyte and myeloid cell activation may be a crucial component in the overall strategy of antibody therapy in NB.53