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104 | Chapter 4 NK cells efficiently target and kill primary colon tumor cells As an indication that our observations regarding combined cetuximab/NK cell antitumor efficacy could be extrapolated to the clinical situation, primary tumor material from five patients with CRC was subjected to NK cell killing in the presence or absence of cetuximab in an experimental set-up that was essentially the same as used for the cytotoxicity experiments with colon cancer cell lines described above. In all 5 patients, tumor cells were effectively lysed by NK cells, but only 3 out of 5 tumors responded to cetuximab monotherapy. Importantly, and as shown in Figure 5A and B, cetuximab increased NK cell tumor cell lysis regardless of whether tumors were susceptible to cetuximab monotherapy. The differences in response to cetuximab monotherapy were related to the RAS mutation status of the tumors as the 2 tumors not responding to cetuximab monotherapy had a mutation (KRAS exon 2, c.35>T; p.G12V) in the RAS gene. Figure 5C and D show tumor cell EGFR and HLA-ABC expression levels respectively; all tumors tested had low levels of EGFR and HLA-ABC. Together, these experiments have demonstrated that NK cells have the ability to kill primary tumor cells, and that this can be further increased via cetuximab- mediated ADCC in both RAS wt and RAS mut tumors. Discussion Anti-EGFR therapies currently in practice are not adequate to prevent mCRC. The main aim of this study was to determine whether NK cells and cetuximab could be combined to improve their anti-tumor efficacy and widen their applicability in mCRC independent of EGFR and RAS status. NK cells are the immune effectors of choice for ADCC induced by cetuximab 17,18 . Higher frequencies of NK cells in the peripheral blood and increased NK tumor infiltration have both been associated with a favorable prognosis in CRC 19 . The ability of NK cells to target cancer stem cells has also been reported 20 . Thus, the presence of an adequate number of cytolytic NK cells has the potential to drive potent ADCC in combination with cetuximab. EGFR is overexpressed in various solid tumors, making it an attractive biomarker for anti-EGFR therapy 21 . Cetuximab, when administered following chemotherapy or in chemo- refractory RAS wt mCRC patients improves their progression-free survival 22 . However, although the ensuing anti-tumor response is reliant on the presence of a non-mutated RAS gene, the overall response rate of cetuximab is still only around 23%, even among patients with RAS wt tumors 23-25 . We hypothesized that NK cells in combination with cetuximab could enhance the cytotoxic effects of cetuximab in EGFR + RAS wt/mut BRAF mut CRCs, besides NK cell natural cytotoxicity can mediate anti-tumor activity on EGFR - CRCs that do not respond to anti-EGFR therapy. In our experiments, a group of solid tumor cell lines was selected based on EGFR expression, RAS and BRAF status. Titrating cetuximab in cultures of A431 cells (EGFR +++ ,