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Store-operated Ca2+ entry (SOCE) provides a major Ca2+ entry route in cancer cells. SOCE is mediated by the assembly of Stim and Orai proteins at endoplasmic reticulum (ER)-plasma membrane junctions upon depletion of the ER Ca2+ store. Additionally, Stim and Orai proteins underpin constitutive Ca2+ entry in a growing number of cancer cell types due to the partial depletion of their ER Ca2+ reservoir. Herein, we investigated for the first time the structure and function of SOCE in primary cultures of colorectal carcinoma (CRC) established from primary tumor (pCRC) and metastatic lesions (mCRC) of human subjects. Stim1-2 and Orai1-3 transcripts were equally expressed in pCRC and mCRC cells, although Stim1 and Orai3 proteins were up-regulated in mCRC cells. The Mn2+-quenching technique revealed that constitutive Ca2+ entry was significantly enhanced in pCRC cells and was inhibited by the pharmacological and genetic blockade of Stim1, Stim2, Orai1 and Orai3. The larger resting Ca2+ influx in pCRC was associated to their lower ER Ca2+ content as compared to mCRC cells. Pharmacological and genetic blockade of Stim1, Stim2, Orai1 and Orai3 prevented ER-dependent Ca2+ release, thereby suggesting that constitutive SOCE maintains ER Ca2+ levels. Nevertheless, pharmacological and genetic blockade of Stim1, Stim2, Orai1 and Orai3 did not affect CRC cell proliferation and migration. These data provide the first evidence that Stim and Orai proteins mediate constitutive Ca2+ entry and replenish ER with Ca2+ in primary cultures of CRC cells. However, SOCE is not a promising target to design alternative therapies for CRC.

Two types of polyubiquitin-reactive cytoplasmic bodies, particulate cytoplasmic structures (PaCS) and dendritic cell (DC) aggresome-like induced structures (DALIS), were analyzed by electron microscopy, immunocytochemistry, immunoblotting, and flow cytometry in DC obtained from human blood monocytes incubated with GM-CSF plus IL-4 (IL4-DC), GM-CSF plus IFNα (IFN-DC), or GM-CSF alone (GM-DC), with or without LPS maturation. PaCS developed as monomorphic aggregates of proteasome-reactive barrel-like particles only in ribosomes-rich cytoplasmic areas of differentiating IL4-DC. In contrast, DALIS formed as vesicular bodies storing K63-linked ubiquitinated proteins by coalescence of increased endosomal structures, in IFN-DC or after LPS maturation of GM-DC. DALIS-forming cells showed incomplete morphological and functional DC-type differentiation when compared to PaCS-forming IL4-DC. PaCS and DALIS may have different function as well as different origin and cytochemistry. DALIS may be a transient accumulation site of potentially antigenic polyubiquitinated proteins during their processing and presentation. PaCS are found under physiologic or pathologic conditions associated with increased/deranged protein synthesis and increased ubiquitin–proteasome activity. Given its high heat-shock protein content PaCS may work as a quality control structure for newly synthesized, cytosolic proteins. This comparative analysis suggests that PaCS and DALIS have distinctive roles in DC.

Background The Wilms tumor antigen 1 (WT1) is over-expressed in a vast majority of adult and childhood acute leukemia and myelodysplastic syndromes, being lowly or transiently expressed in normal tissues and hematopoietic stem cells (HSCs). A number of HLA-restricted WT1 epitopes are immunogenic, allowing the in vitro induction of WT1-specific cytotoxic T lymphocytes (CTLs) from patients and healthy donors. Aim The aim of the study was to investigate the feasibility of producing WT1-specific CTLs suitable for somatic cell therapy to prevent or treat relapse in children with acute myeloid or lymphoblastic leukemia given haploidentical HSC transplantation (haplo-HSCT). Methods For WT1-specific CTL production, donor-derived either peripheral blood mononuclear cells (PBMCs) or CD8+ lymphocytes were stimulated with WT1 peptide-loaded donor dendritic cells in the presence of interleukin (IL)-7 and IL-12. Effector cells were re-stimulated once with irradiated donor PBMCs pulsed with WT1-peptides, and then expanded in an antigen-independent way. Results WT1-specific CTLs, displaying high-level cytotoxicity against patients’ leukemia blasts and negligible activity against patients’ non-malignant cells, were obtained from both PBMCs and CD8+ lymphocytes. WT1-specific CTLs obtained from PBMCs showed a better expansion capacity and better anti-leukemia activity than those obtained from CD8+ lymphocytes, even though the difference was not statistically significant. In CTLs derived from PBMCs, both CD8+ and CD4+ subpopulations displayed strong anti-leukemia cytotoxic activity. Discussion Results of this pre-clinical study pave the way to a somatic cell therapy approach aimed at preventing or treating relapse in children given haplo-HSCT for WT1-positive leukemia.

An increase in intracellular Ca2+ concentration plays a key role in the establishment of many cancer hallmarks, including aberrant proliferation, migration, invasion, resistance to apoptosis and angiogenesis. The dysregulation of Ca2+ entry is one of the most subtle mechanisms by which cancer cells overwhelm their normal counterparts and gain the adaptive advantages that result in tumour growth, vascularisation and dissemination throughout the organism. Both constitutive and agonist-induced Ca2+ influx may be mediated by store-dependent as well as store-independent Ca2+ entry routes. A growing body of evidences have shown that different isoforms of Stromal Interaction Molecules (Stim1) and Orai proteins, i.e. Stim1, Stim2, Orai1 and Orai3, underlie both pathways in cancer cells. The alteration in either the expression or the activity of Stim and Orai proteins has been linked to the onset and maintenance of tumour phenotype in many solid malignancies, including prostate, breast, kidney, esophageal, skin, brain, colorectal, lung and liver cancers. Herein, we survey the existing data in support of Stim and Orai involvement in tumourigenesis and provide the rationale to target them in cancer patients. Besides, we summarize the most recent advances in the identification of novel pharmacological tools that could be successfully used in clinical therapy.

Treatment of advanced metastatic colorectal cancer (mCRC) patients is associated with a poor prognosis and significant morbidity. Moreover, targeted therapies such as anti-epidermal growth factor receptor (EGFR) have no effect in metastatic patients with tumors harboring a mutation in the RAS gene. The failure of conventional treatment to improve outcomes in mCRC patients has prompted the development of adoptive immunotherapy approaches including natural killer (NK)-based therapies. In this study, after confirmation that patients' NK cells were not impaired in their cytotoxic activity, evaluated against long-term tumor cell lines, we evaluated their interactions with autologous mCRC cells. Molecular and phenotypical evaluation of mCRC cells, expanded in vitro from liver metastasis, showed that they expressed high levels of polio virus receptor and Nectin-2, whereas UL16-binding proteins were less expressed in all tumor samples evaluated. Two different patterns of MICA/B and HLA class I expression on the membrane of mCRC were documented; approximately half of mCRC patients expressed high levels of these molecules on the membrane surface, whereas, in the remaining, very low levels were documented. Resting NK cells were unable to display sizeable levels of cytotoxic activity against mCRC cells, whereas their cytotoxic activity was enhanced after overnight or 5-day incubation with IL-2 or IL-15. The susceptibility of NK-mediated mCRC lysis was further significantly enhanced after coating with cetuximab, irrespective of their RAS mutation and HLA class I expression. These data open perspectives for combined NK-based immunotherapy with anti-epidermal growth factor receptor antibodies in a cohort of mCRC patients with a poor prognosis refractory to conventional therapies.

Obtaining human tumor cell lines from fresh tumors is essential to advance our understanding of antitumor immune surveillance mechanisms and to develop new ex vivo strategies to generate an efficient anti-tumor response. The present study delineates a simple and rapid method for efficiently establishing primary cultures starting from tumor samples of different types, while maintaining the immuno-histochemical characteristics of the original tumor. We compared two different strategies to disaggregate tumor specimens. After short or long term in vitro expansion, cells analyzed for the presence of malignant cells demonstrated their neoplastic origin. Considering that tumor cells may be isolated in a closed system with high efficiency, we propose this methodology for the ex vivo expansion of tumor cells to be used to evaluate suitable new drugs or to generate tumor-specific cytotoxic T lymphocytes or vaccines.

Background aims. Adoptive T-cell therapy with tumor-specifi c T cells has emerged as a potentially useful approach for treating patients with advanced malignancies. We have demonstrated previously the feasibility of obtaining large numbers of autologous anti-tumor-specifi c cytotoxic T lymphocytes (CTL) generated by stimulation of patients ’ peripheral blood mononuclear cells with dendritic cells pulsed with apoptotic tumor cells. Methods . Six patients with progressing metastatic solid tumors (one renal cell carcinoma, two ovarian cancers, two extraosseous peripheral neuroectodermal tumors, one soft tissue sarcoma) not eligible for conventional therapies were treated with adoptive immunotherapy. Anti-tumor CTL, proven to be reactive in vitro against patient tumor cells, but not against normal cells, were infused following lymphodepleting chemotherapy administered to favor T-cell proliferation in vivo. Results . Patients received a median of nine CTL infusions (range 2 – 19). The median number of CTL administered per infusion was 11 10 8 (range 1 – 55 10 8 ). No patient experienced acute or late adverse events related to CTL infusion, even when large numbers of cells were given. Post-infusion laboratory investigations demonstrated an increase in the frequency of circulating anti-tumor T-cells and, in patients with a longer follow-up receiving two CTL infusions/year, a stabilization of these values. Conclusions. Our study demonstrates that autologous ex vivo -generated anti-tumor CTL can be administered safely in patients with advanced solid tumors and can improve the immunologic reactivity of recipients against tumor. These preliminary results provide a rationale for evaluating the clinical effi cacy of this immunotherapeutic approach in phase I/II studies.

The adoptive transfer of ex vivo-induced tumor-specific T-cell lines provides a promising approach for cancer immunotherapy. We have demonstrated previously the feasibility of inducing in vitro long-term anti-tumor cytotoxic T-cell (CTL) lines directed against different types of solid tumors derived from both autologous and allogeneic PBMC. We have now investigated the possibility of producing large amounts of autologous anti-tumor CTL, in compliance with good manufacturing practices, for in vivo use.Four patients with advanced solid tumors (two sarcoma, one renal cell cancer and one ovarian cancer), who had received several lines of anticancer therapy, were enrolled. For anti-tumor CTL induction, patient-derived CD8-enriched PBMC were stimulated with DC pulsed with apoptotic autologous tumor cells (TC) as the source of tumor Ag. CTL were then restimulated in the presence of TC and expanded in an Ag-independent way.Large amounts of anti-tumor CTL (range 14-20 x 10(9)), which displayed high levels of cytotoxic activity against autologous TC, were obtained in all patients by means of two-three rounds of tumor-specific stimulation and two rounds of Ag-independent expansion, even when a very low number of viable TC was available. More than 90% of effector cells were CD3(+) CD8(+) T cells, while CD4(+) T lymphocytes and/or NK cells were less than 10%.Our results demonstrate the feasibility of obtaining large quantities of anti-tumor specific CTL suitable for adoptive immunotherapy approaches.

Aims and background Adoptive immunotherapy can be a therapeutic option to treat cancer patients with advanced-stage disease refractory to conventional therapies. Methods and study design We used T-cell therapy with autologous antitumor cytotoxic T lymphocytes (CTLs) in a patient with metastatic renal cell carcinoma. Autologous antitumor CTLs obtained by stimulating CD8-enriched cells with dendritic cells pulsed with irradiated apoptotic tumor cells and expanded in vitro were transfused on days +14 and +28 following chemotherapy and every 2 to 4 months thereafter. Results and Conclusions Treatment with high doses of antitumor CTLs proved to be safe and induced immunological responses and long-lasting clinical benefit in our patient.

Store-operated Ca2+entry (SOCE) is activated following depletion of the inositol-1,4,5-trisphosphate (InsP3)-sensitive Ca2+pool to regulate proliferation in immortalized cell lines established from either primary or metastatic lesions. The molecular nature of SOCE may involve both Stim1, which senses Ca2+levels within the endoplasmic reticulum (ER) Ca2+reservoir, and a number of a Ca2+-permeable channels on the plasma membrane, including Orai1, Orai3, and members of the canonical transient receptor (TRPC1–7) family of ion channels. The present study was undertaken to assess whether SOCE is expressed and controls proliferation in primary cultures isolated from secondary lesions of heavily pretreated metastatic renal cell carcinoma (mRCC) patients. SOCE was induced following pharmacological depletion of the ER Ca2+store, but not by InsP3-dependent Ca2+release. Metastatic RCC cells express Stim1-2, Orai1–3, and TRPC1–7 transcripts and proteins. In these cells, SOCE was insensitive to BTP-2, 10 µM Gd3+and Pyr6, while it was inhibited by 100 µM Gd3+, 2-APB, and carboxyamidotriazole (CAI). Neither Gd3+nor 2-APB or CAI impaired mRCC cell proliferation. Consistently, no detectable Ca2+signal was elicited by growth factor stimulation. Therefore, a functional SOCE is expressed but does not control proliferation of mRCC cells isolated from patients resistant to multikinase inhibitors.

We demonstrated in previous studies that interleukin (IL) -2 supports in vitro cell proliferation of donor-derived cytotoxic T lymphocyte (CTL) lines directed against different types of leukemia blasts. The aim of this study was to compare the capacity of IL-15 with that of IL-2 in supporting the proliferation and cytotoxic activity of antileukemia CTL cultures, and their influence on T-cell memory compartment differentiation. Antileukemia CTL lines were generated using donor-derived dendritic cells pulsed with apoptotic leukemia blasts, in the presence of IL-12 and IL-7, during the primary culture, and expanded through 2 rounds of leukemia-specific stimulation and 1 round of antigen-independent expansion, each supplemented with either IL-2 or IL-15. Both IL-2-supplemented (IL-2-CTLs) and IL-15-supplemented (IL-15-CTLs) lines contained predominant numbers of CD45RA/CCR7 effector memory (TEM) and CD45RA/CCR7 (TEMRA+) T cells. Significantly higher numbers (P

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.

In previous studies, we showed the possibility of expanding in vitro polyclonal CTL lines directed against patient leukemia cells using effector cells derived from both HLA-matched and HLA-mismatched hematopoietic stem cell donors. Some CTL lines, especially those derived from an HLA-disparate donor, displayed residual alloreactivity against patient nonmalignant cells. In this study, we evaluated the possibility of separating in vitro CTLs with selective graft-versus-leukemia (GVL) activity from those potentially involved in the development of graft-versus-host disease (GVHD) through single T-cell cloning of antileukemia polyclonal CTL lines. We showed that CTLs that were expanded from a single T-cell clone (TCC), able to selectively kill leukemia blasts and devoid of alloreactivity towards nonmalignant cells, can be obtained from antileukemia alloreactive polyclonal CTL lines. TCCs expressed a wide repertoire of different T-cell receptor (TCR)-Vβ families, mainly produced IFNγ and interleukin 2, irrespective of CD8 or CD4 phenotype, and could be extensively expanded in vitro without losing their peculiar functional features. The feasibility of our approach for in vitro separation of GVL from GVH reaction opens perspectives for using TCCs, which are selectively reactive towards leukemia blasts, for antileukemia adoptive immune therapy approaches after hematopoietic stem cell transplantation, in particular from HLA-mismatched donors. (Cancer Res 2006; 66(14): 7310-6)

Objective Studies of memory T cells transferred with the graft are relevant to better understand the early immune reconstitution of patients given autologous bone marrow transplantation (A-BMT). A critical question is whether memory T cells resident in bone marrow (BM) of patients with hematological malignancies are resistant to either pretransplant chemotherapy or ex vivo pharmacological purging. Patients and methods To address these issues, we evaluated the frequency of tetanus-toxoid (TT)-specific proliferating T-cell precursors (TT-PTCp) in BM and peripheral blood (PB) of eight patients with acute myeloid leukemia (AML) given A-BMT after in vitro purging of BM with mafosfamide. Patients were studied at the time of BM harvesting and five of them also after A-BMT. Results The range of TT-PTCp frequencies found after A-BMT were comparable with those observed in PB and in BM at the time of harvesting and did not differ significantly from those of eight age-matched healthy subjects who donated BM for a human leukocyte antigen-identical sibling. TT-PTCp frequencies in BM, studied before and after ex vivo purging, appeared not to be affected by incubation with mafosfamide. We also compared the T-cell receptor (TCR)-Vβ-repertoire usage of TT-specific T-cell lines (TT-TCL) in BM of patients at the time of harvesting and in their PB 2 months after transplantation. The same TCR-clonotypes were detected in TT-TCL at time of harvesting and after A-BMT. Conclusion These data indicate that BM-resident memory T cells of patients with AML are resistant to both pretransplant chemotherapy and ex vivo pharmacological purging and may contribute to immune reconstitution after A-BMT.

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.

Cerebellar granule cells (CGCs) are a sensitive target for methylmercury (MeHg) neurotoxicity. In vitro exposure of primary cultures of rat CGCs to MeHg resulted in a time- and concentration-dependent cell death. Within 1 hr exposure, MeHg at 5-10 microM caused impairment of mitochondrial activity, de-energization of mitochondria and plasma membrane lysis, resulting in necrotic cell death. Lower MeHg concentrations (0.5-1 microM) did not compromise cell viability, mitochondrial membrane potential and function at early time points. Later, however, the cells progressively underwent apoptosis and 100% cell death was reached by 18 hr treatment. Neuronal network fragmentation and microtubule depolymerization were detected as early as within 1.5 hr of MeHg (1 microM) exposure, long before the occurrence of nuclear condensation (6-9 hr). Neurite damage worsened with longer exposure time and proceeded to the complete dissolution of microtubules and neuronal processes (18 hr). Microtubule stabilization by taxol did not prevent MeHg-induced delayed apoptosis. Similarly ineffective were the caspase inhibitors z-VAD-fluoromethylketone and z-DEVD-chloromethylketone, the L-type calcium channel inhibitor nifedipine, the calcium chelator EGTA and BAPTA, and the NMDA receptor antagonist MK-801. On the other hand, insulin-like growth factor-I partially rescued CGCs from MeHg-triggered apoptosis. Altogether these results provide evidence that the intensity of MeHg insult is decisive in the time of onset and the mode of neuronal death that follows, i.e., necrosis vs. apoptosis, and suggest that cytoskeletal breakdown and deprivation of neurotrophic support play a role in MeHg delayed toxicity.

4575 Background: Immunotherapy still remains the only treatment which proved able to induce long-lasting complete responses in aRCC. The aim of the present study was to evaluate the feasibility and safety of adoptive immunotherapy with ex vivo-generated autologous CTLs pulsed with patient-derived tumor cells in heavily pre-treated subjects with aRCC. Methods: This study was performed in accordance with local regulations on the production of cell derivatives; local Ethical Committee approval was obtained on a named patient basis, after obtaining each patient’s informed consent. CTLs were generated stimulating CD8-enriched lymphocytes with dendritic cells (DCs) pulsed with fresh autologous tumor cells in the presence of IL-12 and IL-7; these specific anti-tumor CTLs were expanded and then reinfused after a lymphodepleting chemotherapy with CTX and fludarabine, followed by s.c. administration of low-dose IL-2. Disease status was assessed after every two re-infusions. Eight patients were enrolled, 6 with a clear cell RCC, 1 with a papillary RCC, and 1 with a mixed, papillary and collecting ducts, RCC. All patients have been previously treated with a median of 5 prior treatment lines; the median time between diagnosis of aRCC and tumor biopsy was 55 months (15-185). Results: Six of the 8 enrolled patients have produced suitable tumor cell lines, and 5 of them had been already re-infused with tumor-specific CTLs. All patients received at least 2 CTL infusions (range: 2-5+); mean of totally infused CTLs was 10.7 x 109(range: 4-26.6). All CTLs infusions were well tolerated, with no evidence of autoimmune reactions. In terms of antitumor activity, one patient achieved a partial response, 3 had a slowly progressing disease, and one patient is still not evaluable; furthermore, in 3 patients disease-related symptoms improved. Conclusions: Our preliminary results suggest that immunotherapy with autologous CTLs pulsed ex-vivo with patient-derived tumor cells is feasible and safe in heavily pre-treated aRCC patients. Signs of activity were recorded, suggesting the need to further expand this cohort of patients.

e14057 Background: Prognosis for metastatic colorectal-cancer (mCRC) patients is unfavorable. Futhermore 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. Methods: Purified allogeneic NK cells or preincubated with IL-2 or IL-15 were used as effectors. Seven different CRC cell lines (DLD1, HCT-116, HT-29, LOVO, SW620, SW48, SW480) labeled with 51Cr were used as target cells alone or preincubated with cetuximab for ADCC assay. Results: All but one (SW620) cell lines express EGFR and MHC molecules; 4/7 harbor KRAS mutation (DLD 1, HCT-116, SW620, SW480). All cell lines were susceptible to unstimulated NK cells lysis (mean 20% ± 4.57, at ratio 25:1; mean 15% ± 3.65, at ratio 12:1) and lytic activity is strongly enhanced by IL-2 or IL-15 (mean 48% ± 5.8, at ratio 25:1; mean 38% ± 4.3, at ratio 12:1) (p < 0.05). No ...

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.

e13103 Background: Several immune-based approaches for treatment of metastatic colorectal cancer (mCRC) are currently under investigation. Aims of our study were to grow and characterize tumor cells (TC) obtained from mCRC patients, and to investigate their susceptibility to autologous natural killer (NK) cell recognition and killing in vitro. Methods: Tumor samples and peripheral blood mononuclear cells from 10 mCRC patients undergoing surgery were collected. Establishment of TC lines that are representative of the original tumor and could be expanded in vitro for a significant number of passage was performed according with our previously described method (Turin I et al, Cytotherapy 2007). CD56+CD3- NK cells were incubated overnight with medium alone or activated with IL-2 (100U/ml) or IL-15 (20ng/ml) and tested against 51Cr-labeled primary TC in NK assay or in ADCC assay after pre-coating of TC with anti-EGFR monoclonal antibody cetuximab. Results: Eight stable mCRC lines have been obtained and characterized to confirm their neoplastic origin. Patient NK cells display low capacity to lyse autologous TC (median 12%, range 5%-17%). IL-2 and IL-15 incubation significantly potentiate NK cytotoxic capacity (median18%, range 6%-62% and median 35%, range 28%-65%, respectively). These results refer to an effector:target ratio of 25:1. ADCC assay has shown that cetuximab pre-coating of TC results on an increasing of their susceptibility to NK-mediated lysis. Phenotypical and molecular analysis of activating NK receptors and their ligands present on surface of TC are currently under evaluation. Conclusions: NK cells derived from mCRC patients display measurable cytotoxic activity against autologous primary TC. Susceptibility of NK-mediated lysis is strongly enhanced by pre-incubation of NK cell with cytokines and by pre-coating of TC with cetuximab. These findings, presented upon approval by the study’s data monitoring committee and undergoing confirmation in a larger series, may support a pilot study of NK cells pre-activated ex vivo for treatment of mCRC patients.