PDF file - 45K, In-vitro expansion of EGFR-specific CTL in the presence of NK cells.

PDF file - 441K, Importance of IFN-γ released by cetuximab activated NK cells in the enhancement of DC maturation.

PDF file - 160K, The absence of cetuximab in the NK:DC co-culture (without HNC cells) or DC:PCI-15B co-culture (without NK cells) abrogated the IFN-γ secretion.

Abstract Purpose: Tumor antigen–specific monoclonal antibodies (mAb) block oncogenic signaling and induce Fcγ receptor (FcγR)–mediated cytotoxicity. However, the role of CD8+ CTL and FcγR in initiating innate and adaptive immune responses in mAb-treated human patients with cancer is still emerging. Experimental Design: FcγRIIIa codon 158 polymorphism was correlated with survival in 107 cetuximab-treated patients with head and neck cancer (HNC). Flow cytometry was carried out to quantify EGF receptor (EGFR)–specific T cells in cetuximab-treated patients with HNC. The effect of cetuximab on natural killer (NK) cell, dendritic cell (DC), and T-cell activation was measured using IFN-γ release assays and flow cytometry. Results: FcγRIIIa polymorphism did not predict clinical outcome in cetuximab-treated patients with HNC; however, elevated circulating EGFR853–861–specific CD8+ T cells were found in cetuximab-treated patients with HNC (P < 0.005). Cetuximab promoted EGFR-specific cellular immunity through the interaction of EGFR+ tumor cells and FcγRIIIa on NK cells but not on the polymorphism per se. Cetuximab-activated NK cells induced IFN-γ–dependent expression of DC maturation markers, antigen processing machinery components such as TAP-1/2 and T-helper cell (TH1) chemokines through NKG2D/MICA binding. Cetuximab initiated adaptive immune responses via NK cell–induced DC maturation, which enhanced cross-presentation to CTL specific for EGFR as well as another tumor antigen, MAGE-3. Conclusion: Cetuximab-activated NK cells promote DC maturation and CD8+ T-cell priming, leading to tumor antigen spreading and TH1 cytokine release through “NK–DC cross-talk.” FcγRIIIa polymorphism did not predict clinical response to cetuximab but was necessary for NK–DC interaction and mAb-induced cross-presentation. EGFR-specific T cells in cetuximab-treated patients with HNC may contribute to clinical response. Clin Cancer Res; 19(7); 1858–72. ©2013 AACR.

Abstract Despite over-expression of epidermal growth factor receptor (EGFR) in 80-90% of head and neck cancers (HNC), only ~20% of HNC patients are clinically responsive the EGFR-specific therapeutic monoclonal antibody (mAb) cetuximab. In addition to blocking EGFR signaling that leads to tumor cell survival and proliferation, increasing evidence suggests that innate and adaptive immunity likely plays a significant role in patient response to cetuximab. Natural killer (NK) cells recognize the Fc-region of EGFR-bound cetuximab via their activating Fcγ receptor IIIa (FcγRIIIa, also known as CD16) and mediate tumor cell lysis by antibody dependent cellular cytotoxicity (ADCC). NK cells are increasingly recognized as key mediators of crosstalk bridging the innate and adaptive arms of the immune response to human malignancies. We have previously shown that in addition to being direct effectors of ADCC against cetuximab-coated HNC cells, NK cells are instrumental in indirectly promoting an anti-tumor adaptive immune response via secretion of immunomodulatory cytokines such as IFNγ, which is known to enhance dendritic cell (DC) antigen processing and presentation to CD8+ T cells. Multiple studies have consistently demonstrated a relative decrease in the CD56dimCD16bright NK cell subset, traditionally considered to be primarily cytotoxic effectors, and an increase in the immunoregulatory CD56brightCD16dim NK cell subset in the peripheral blood of patients with various malignancies. However, given that the immunoregulatory CD56brightCD16dim NK cells are generally considered to be the more prolific cytokine producing subset, it was unclear how this redistribution of NK cell subsets might affect the dynamics of cytokine-mediated NK:DC crosstalk in the presence of mAb-coated tumor cells. Using primary NK cells and monocyte-derived DC isolated from PBMC of healthy donors, we therefore sought to better elucidate the patterns and kinetics of IFNγ secretion by the two NK cell subsets in response to cetuximab-coated HNC cells in the presence of DC. In this context, we found that while CD56brightCD16dim NK cells express more of the activating NKG2D receptor, rapid (by 4hrs) IFNγ secretion by CD56dimCD16bright NK cells is a CD16-dependent process. CD56brightCD16dim NK cells only produced IFNγ following stimulation for at least 12 hours with a combination of IL-12 and IL-15. IL-12/IL-15 stimulation also induced IFNγ secretion by CD56dimCD16bright NK cells, though to a lesser extent as compared to the CD56brightCD16dim subset. Taken together, these data contribute significantly to a mechanistic understanding of how NK cells function to bridge innate and adaptive anti-tumor responses in the context of mAb cancer therapies. Understanding this link is of broad relevance to the optimization of current therapeutic mAbs and will help devise novel ways to improve clinical responses. Citation Format: Christopher A. Lord, Raghvendra M. Srivastava, Robert L. Ferris. Patterns and kinetics of interferon-γ secretion by natural killer cell subsets co-cultured with cetuximab-coated head and neck cancer cells and dendritic cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A41.

Abstract Objectives To determine the difference in operative times and associated complications for cases performed solely by attending-level surgeons versus cases assisted by surgeons-in-training for endoscopic endonasal pituitary surgeries. Design Retrospective chart review. Setting Tertiary-care academic medical center. Participants A total of 228 patients having undergone endoscopic endonasal pituitary surgery from 2005 to 2011. Main Outcome Measure Duration of surgery comparing attending only (AO) and trainee-assisted (TA) surgeries. Results Thirty-seven (19%) of 198 cases were identified as AO surgeries, the remaining 161 (81%) were TA. Operative times (minutes) for the AO group were significantly shorter than the TA group (149.1 ± 54.8 vs 219.5 ± 83.7, p < 0.001). The AO group had fewer intraoperative cerebrospinal fluid leaks (30% vs 39%, p = 0.318), decreased estimated blood loss (408 mL vs 523 mL, p = 0.176), fewer postoperative complications (27% vs 37%, p = 0.268), and shorter length of stay (3.5 vs 4.3 days, p = 0.294). Conclusions This is the first study in otolaryngology or neurosurgery to compare operative times and outcomes for AO versus TA cases at a single academic institution. Operative times were significantly decreased and a trend toward a decrease in patient morbidity was noted for cases performed solely by attendings. The valuation of teaching activities in the operating room is a necessary first step toward optimizing the allocation of resources and funding of surgical education.

Despite over-expression of epidermal growth factor receptor (EGFR) in 80-90% of head and neck cancers (HNC), only ∼20% of HNC patients are clinically responsive to treatment with the EGFR-specific monoclonal antibody (mAb) cetuximab. To date, no reliable biomarker of clinical efficacy has been identified to predict clinical response to cetuximab therapy in HNC. Modest, yet statistically significant correlations of Fcγ receptor (FcγR) IIIa polymorphisms with clinical outcome have been noted in B cell lymphoma, breast, and colorectal cancer patients treated with rituximab, trastuzumab, and cetuximab, respectively. Therefore, we have investigated whether a known FcγRIIIa polymorphism in natural killer (NK) cells at amino acid position 158 (valine [V] vs. phenylalanine [F]) correlates with the anti-tumor activity of cetuximab in immunodeficient mice engrafted with human HNC tumors as well as with the induction of EGFR-specific cytotoxic T lymphocytes (CTL) and clinical course in HNC patients treated with cetuximab. When injected into immunodeficient mice, human NK cells expressing the V allele at this position demonstrated significantly greater ability to control the growth of xenografted HNC tumors. However, we did not find a significant correlation between FcγRIIIa genotype and disease free survival (p=0.683) in a cohort of 107 consecutive HNC patients treated with regimens incorporating cetuximab. Furthermore, we demonstrate for the first time that in the presence of cetuximab-opsonized HNC cells, NK cells trigger maturation of dendritic cells (DC), leading to augmented TA-specific cross-priming of EGFR and MAGE-specific CTL in vitro, and in vivo in cetuximab-treated HNC patients. The significantly higher frequencies of EGFR-specific CTL we found in HLA-A2+ PBMC from cetuximab-treated HNC patients relative to cetuximab-naive HNC patients (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-431. doi:1538-7445.AM2012-LB-431