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Although the graft-versus-leukemia effect of allogeneic bone marrow transplantation (BMT) is of paramount importance in the maintenance of disease remission, the role played by the autologous T-cell response in antitumor immune surveillance is less defined. We evaluated the emergence of antileukemia cytotoxic T-lymphocyte precursors (CTLp's) and the correlation of this phenomenon with maintenance of hematologic remission in 16 children with acute myeloid leukemia (AML), treated with either chemotherapy alone (5 patients) or with autologous BMT (A-BMT, 11 patients). Antileukemia CTLp's were detectable in 8 patients in remission after induction chemotherapy; none of them subsequently had a relapse. Of the 8 patients who did not show detectable CTLp frequency while in remission after induction chemotherapy, 7 subsequently experienced leukemia relapse. In patients undergoing A-BMT, molecular fingerprinting of the TCR-Vβ repertoire, performed on antileukemia lines, demonstrated that selected antileukemia T-cell clonotypes, detectable in bone marrow before transplantation, survived ex vivo pharmacologic purging and were found in the recipient after A-BMT. These data provide evidence for an active role of autologous T cells in the maintenance of hematologic remission and also suggest that quantification of antileukemia CTLp frequency may be a useful tool to identify patients at high risk for relapse, thus potentially benefiting from an allogeneic antitumor effect.

Background The failure of conventional treatment and target therapies to significantly improve outcomes in metastatic colorectal cancer (mCRC) has prompted the development of immune-based therapies, including natural killer (NK) cells-based strategies. NK cells can kill target cells directly, as well as mediate antibody (Ab)-dependent cellular cytotoxicity (ADCC) via the membrane receptor FcgRIII, which binds to the Fc portion of IgG Ab. This feature makes them of particular importance in the immunotherapy of mCRC patients harboring the KRAS mutation that cannot benefit from administration of anti-epidermal growth factor receptor (EGFR) drugs. In this study we evaluated the capacity of patients derived NK cells, either resting or after cytokine activation, to lyse autologous mCRC cells. mCRC cells were analyzed for expression of ligands for adhesion and triggering NK receptors involved in their recognition and killing. We also evaluated whether KRAS mutated mCRC cells were susceptible to anti-EGFR-induced ADCC mediated by NK cells. Patients and methods. After obtaining informed signed consent, 25 mCRC patients have been enrolled to date. Tumor cells were disaggregated by GentleMACS Dissociator (Miltenyi Biotec,Germany), in vitro expanded and analyzed for the expression of ligands for NK triggering receptors. Ligands expression was evaluated by cytofluorimetric analysis and gene expression by qPCR on mCRC cultured cells and by immunohistochemistry in sections of samples embedded in paraffin. Resting and IL-2- or IL-15-activated NK cells, were analyzed for expression of triggering and inhibitory receptors and for their ability to kill autologous mCRC cells alone or after incubation with anti-EGFR monoclonal antibodies (mAbs) in a 51Cr release cytotoxicity assay. Results. Tumor cells were successfully expanded from 21 of 25 samples. Experiments performed in 10 patients showed the inability of patients resting NK cells to lyse mCRC cells (< 10% at effector:target ratio (E:T) of 20:1. Cytokine overnight (ON) activation resulted in an increased NK cytotoxic activity (IL-2: mean 28%; range:10-71; and IL-15: mean 40%; range 16-76 at E:T ratio of 20:1). Additional days of NK cell activation were able to further enhance their lytic capability. In resting NK cells the mean surface expression of activating receptors DNAM-1 and NKG2D was 79%, (range 75-91) and 39 % (range 29-58), respectively. The latter was up-regulated by ON cytokine activation (IL-2: mean 53%, range 48-70; IL-15: mean 70%, range 63-87). Among activating natural cytotoxicity receptors (NCRs), NKp46 is highly expressed both on resting and activated cells (>90%), while low surface expression of NKp30 and NKp44 was documented on resting NK cells ( Conclusions. The evidence that ex vivo activated autologous NK cells are able to lyse patients tumor cells can offer new therapeutic options to a cohort of mCRC patients with a poor prognosis. Disclosures No relevant conflicts of interest to declare.

Abstract 5119 Purpose Prognosis for metastatic colorectal-cancer (mCRC) patients is unfavorable, median survival from diagnosis varying between sixteen and twenty-four months. Numerous clinical trials have demonstrated a clinical advantage for patients treated with a combination of chemotherapy and cetuximab. However recent retrospective evidence from several randomized studies, has established that mCRC patients with tumors harboring a mutation in the KRAS gene do not derive benefit from the administration of cetuximab. We hypothesized that KRAS mutated CRC cells may be susceptible to cetuximab-induced ADCC mediated by healthy donor NK cells. Experimental Design 7 different CRC cell lines (DLD 1, HCT-116, HT-29, LOVO, SW620, SW48, SW480) were analyzed for EGFR membrane expression levels, tested for KRAS mutational status and MHC class I-II expression. CD56+CD3- NK cells were purified from volunteer healthy donors peripheral blood mononuclear cells, by a two-steps CD3+ cells negative selection followed by CD56+ cells positive selection by MACS cell separation columns (Miltenyi biotec). Purified NK cells were incubated overnight with medium alone or stimulated with IL-2 (100U/ml) or IL-15 (20U/ml). CRC cell lines, were labeled with 51Cr overnight, and used as target cells for NK assay or incubated at 4°C for 30 minutes with Cetuximab 100 ug/ml for ADCC assay. Cytotoxic activity was evaluated in a 4-hour cytotoxicity assay, at an effector/target (E/T) ratio ranging between 100:1 and 2:1. Results all but one (SW620) CRC cell lines express EGFR (range 40-90%) and MHC class I-II molecules; 4/7 harbor KRAS mutation (DLD 1, HCT-116, SW620, SW480). All CRC cell lines were susceptible to unstimulated allogeneic NK cells lysis (mean 20% ± 4,57, at ratio 25:1; mean 15% ± 3,65, at ratio 12:1) and lytic activity is significantly enhanced (p Conclusions allogeneic NK cells from different healthy donors are able to lyse all tested CRC lines (4 KRAS mutated). IL-2 and IL-15 activation significantly enhances NK cytotoxicity; moreover lytic activity of thawed and fresh NK cells is similar allowing the use of both populations in clinical trials. In ADCC assay NK cell lytic activity is significantly enhanced by cetuximab; surprisingly cetuximab mediated ADCC activity is independent from CRC cell lines KRAS oncogene mutational status and require EGFR membrane expression. Pre-coating with cetuximab also increase lytic activity of IL-2 and IL-15 activated NK cells. Further experiments are in progress to evaluate the susceptibility to the lysis of primary tumor cells derived from KRAS mutated and wild type CRC patients, in NK and ADCC assays in order to confirm data obtained against established CRC cell lines and to correlate cetuximab-mediated ADCC with EGFR membrane expression. These findings support a phase I study with allogeneic NK cells infusions in association with cetuximab and IL-2 in mCRC patients harboring a mutation in the KRAS gene. Disclosures No relevant conflicts of interest to declare.