OS and PFS medians with the corresponding confidence intervals (CI) were used to summarize the time-to-event distributions. mounting ADCC response had a higher likelihood of stable disease (= 0.01) and showed a trend toward increased PFS: 14 weeks versus 6.8 weeks, respectively (= 0.13). Enhanced ADCC and innate immunity responses were more predictive of clinical response than FcRIIIa and may offer a functional assay to select patients suitable for cetuximab therapy. Introduction Cetuximab has established benefit in squamous cell carcinoma of the head and 666-15 neck (SCCHN) and colorectal cancer and, by virtue of being an IgG1 antibody, can activate antibody-dependent cellular cytotoxicity (ADCC). In ADCC, the constant (Fc) region of IgG1-based mAbs that are bound to cell surface targets can engage and crosslink low-affinity canonical Fc receptors (FcRIIIa) expressed on natural killer (NK) cells, resulting in NK activation, degranulation, and lysis of the target cell (1, 2). There has been considerable effort to identify biomarkers that reflect the capacity to mount ADCC. Presently, the most studied biomarkers are single amino acid polymorphisms at the 158 position of FcRIIIa. Substitution of phenylalanine [polymorphism were more effective in killing K562 leukemia cells, even in the absence of antibody (innate immunity), compared with NK cells homozygous for the low-affinity polymorphism (10). These data suggest that FcRIIIa-158 polymorphisms may correlate not only with the FcRIIIa-binding affinity, but also with an NK phenotype that has a broader cytotoxicity profile. In this study, we sought 666-15 to more clearly define the link between FcRIIIa-158-and polymorphisms and ADCC response, both and polymorphism is not the sole determinant of the magnitude of cetuximab-mediated ADCC ADCC; when NK cells from patients were 666-15 able to effectively initiate ADCC best predicted improved clinical response, regardless of FcRIIIa polymorphisms. In concert, these data support that innate NK-cell cytotoxicity and the capacity to mount ADCC are more important than FcRIIIa polymorphisms in determining clinical response to cetuximab. Materials and Methods ADCC assays Whole blood was obtained from enrolled SCCHN patients for the isolation of NK cells to perform ADCC assays, which in this study is distinguished from ADCC assays involving healthy blood donors utilized for colorectal cancer studies. From each enrolled patient, 150 mL of whole blood was processed and centrifuged; the buffy coat layer was isolated to harvest peripheral blood mononuclear cells (PBMC) using the FicollCHypague centrifugation method; NK cells were negatively selected using a MACS human NK cell isolation kit (Miltenyi Biotec). ADCC assays were performed using SCCHN cells (TU167) as targets, and purified NK cells as effectors from enrolled SCCHN patients. ADCC assays were also performed using a leukemia cell line (K562) for positive controls, two colon cancer cell lines (HT29, SW480) as target cells, and purified NK cells from healthy donors. Target cells were incubated with 150 Ci of chromium-51 (51Cr; Amersham) at 37C for 1 hour, mixing thoroughly every 15 minutes, and washed twice with media. Cells were subsequently incubated with 10 g/mL of cetuximab, 10 g/mL of control human IgG1 isotype, or with media alone for another 30 minutes at 37C, and washed twice with media to remove unbound antibodies. The concentration of cetuximab was established based upon our prior work and also on physiologic serum concentration ranges: 4C8 g/mL to 16C23 g/mL for peak and trough, respectively, according to packet insert. Effector and target cells were plated at 50:1, 25:1, and 12.5:1 in 96-well plates and incubated for 14 Rabbit polyclonal to PECI to 16 hours. Each assay was performed in triplicate. Cell lysis supernatant was collected and mixed with the Optiphase Supermix scintillation fluid (Perkin Elmer) and counted in a MicroBeta 1450 scintillation counter (Wallac). Activity against NK-sensitive K562 tumor cell line was used as a positive control for all ADCC experiments and provided the measure for innate cytotoxicity. Results were expressed as the percentage of specific lysis according to the following formula: ADCC response (NK cells harvested from enrolled patients) with PFS, OS, and FcRIIIa polymorphisms. Patient eligibility Patients with recurrent or metastatic squamous cell or undifferentiated carcinoma of the head and neck not amenable to curative therapy were screened for eligibility. Patients were eligible if they were at least 18 years of age, able to understand and provide voluntary consent, had an Eastern Cooperative Oncology Group (ECOG) status of at least 2, serum chemistry reflecting normal kidney, liver, and hematologic function, and had not received.