Gefitinib Resistance In Non Small Cell Lung Cancer With Active EGFR Mutations And Potential Therapeutic Significance Of Cytokine Induced Killer Cells

Background and ObjectiveThe advent of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor gefitinib for treatment of non-small cell lung cancer (NSCLC) had added significantly to our armamentarium as it had led to remarkable improvements in response and survival. Recent studies had demonstrated such a link between somatic EGFR exon19 deletions and exon21 L858R mutations and the responses to gefitinib. All patients, however, ultimately developed resistance to these agents. Thus, one key aspect toward realizing the potential of gefitinib in future requires a better understanding of the intrinsic or acquired resistance mechanisms that limit their efficacy, the development of new generations of rationally designed targeted agents, and translating this information to the clinic to select patients for appropriate therapy. In the present study, we therefore tried to analyze the factors and the potential mechanisms associated with gefitinib resistance in patients with EGFR-mutated NSCLC and evaluated the drug-resistance reversal to gefitinb by the cytokine induced killer cells (CIK cells).Materials and Methods1. The efficacy and predictive markers of gefitinib in patients with EGFR-mutated NSCLC.Paraffin-embedded specimens from 50 NSCLC patients were performed for EGFR gene typing. In addition, Plasma Samples were taken from 33 patients with advanced NSCLC of either EGFR mutations or wild type prior to gefitinib therapy. The EGFR exon19 deletions and exon21 L858R mutations were assessed by the mutant-enriched PCR assay. Immunohistochemistry stainings with antibodies against EGFR, p-Erk, p-Akt, p-Stat3 and p53 were performed for investigation of the relationship between smoking and gefitinib response in NSCLC with EGFR mutation.2. Establishment of a PC-9/ gefitinib resistant (GR) cell line.A NSCLC PC-9 cell line harbors EGFR exon19 E746-A750 deletion and p53 mutation, which was sensitive to gefitinib. The PC-9/ GR cells developed an acquired resistance to gefitinib following a stepwise increasing dose of gefitinib combined with intermittent high-dose challenge. Cytolysis was measured with a colorimetric cell-counting kit (CCK-8; Dojindo Laboratories, Kumamoto, Japan) assay according to the manufacturer's instructions. A soft agar colony formation assay was performed for in vitro chemosensitivity testing. Cell cycle and apoptosis were detected by flow cytometry analysis with staining of PI and Annexin-v/7-AAD respectively. Both of PC-9 and PC-9/GR cell lines were subjected to sequencing of EGFR gene exon 19 and 20. EGFR pathway signaling targets were also evaluated by the western blot analysis in the both cell lines. Expression of p53 protein was detected using immunocytochemistry.3. CIK cell-mediated cytotoxicity in the PC-9/GR cells.Effects of CIK cells alone or combined with gefitinib on PC-9/GR cell line was determined while the nude mice bearing with PC-9/GR-tumor were used for evaluation of CIK cells immunotherapy in treating gefitinib resistant tumor.4. CIK cell-mediated immune modulation in patients with tumorsAutomated Peripheral blood mononuclear cells (PBMC) were obtained from patients by a blood cell separator and were subjected to a disposable wave bioreactor for perfusion CIK cell culture. The cultured PBMCs were added timely with interferon-γ, OKT3 and IL-2. CIK cells phenotyping was performed by flow cytometry analysis while the cytolytic activity was evaluated by the CCK-8 assay. Peripheral blood samples were collected before CIK cells infusion and one day, one week, two weeks, one month after CIK cells infusion separately, and then the immune cell subsets and expression of cotokines/chemokines were analyzed by flow cytometry and liquid chip assay respectively. Meanwhile, we made a comparison of proliferation efficiency of CIK cells cultured in vitro between wave bioreactor and static flask situations. Moreover, the effects of IL-2 withdrawal and add-back on the cytolytic activity in the CIK cells were evaluated. In addition, we compared the cytolytic activity in the CIK cells between fresh and frozen-thawed situations.Results1. The efficacy and predictive markers of gefitinib in patients with EGFR-mutated NSCLC.In all the 77 cases, 35 (45.5%) patients were detected with EGFR exon19 deletions and exon21 L858R mutations using mutant-enriched PCR. The mutations were most found in non-smokers compared to smokers (56.1% vs. 33.3%,P=0.045). The overall objective response (CR+PR) rate and median progression-free survival in the 33 patients treated with gefitinib were 42.4% and 3.000 (1.972~4.028) months. The objective response rate was significantly higher in the EGFR mutation-positive cases or non-smokers than that in the EGFR mutation-negative cases or smokers, P<0.05, which was also been found in the median progression-free survival of patients treated with geifitnib. Logistic multivariate analysis showed that only EGFR mutation was significantly associated with the objective response rate of gefitinib therapy, while EGFR gene mutation and smoking history were the major risk factors that were contributing to progression-free survival revealed by Cox regression model multivariate analysis, P<0.05. Hierarchical analysis showed that the progression-free survival of smokers were significantly shorter than that of non-smokers in patients with EGFR-mutated NSCLC.Positive immunostainings of p53, EGFR and p-Akt were detected in 24 out of 50 specimens of NSCLC (48.0%), and p-Erk in 18 of 50 (36.0% ), and p-Stat3 in 12 of 50(24.0% ). It was found that mutated p53 protein was more frequently observed in smokers (61.5%,16/26) and patients with EGFR wild type (60.7%,17/28) than that in non-smokers (33.3%,8/24) and patients with mutated EGFR (31.8%,7/22), P <0.05. However, there was no significant correlation between the expression of p-Akt, p-Erk, p-Stat3 and smoking history or EGFR mutation. Further analysis revealed that p53 protein was highly expressed in smokers compared to non-smokers in patients with mutated NSCLC, 75.0% vs. 7.1%, P =0.02.2. Establishment of a PC-9/GR gefitinib resistant cell line.The gefitinib resistant human NSCLC cell line PC-9/GR was developed after gefitinib selection. The doubling time of growth was shorter in PC-9/GR cell line, as compared with that in the parental cell line PC-9 (24.79h vs. 33.47h). Resistance index to gefitinib in PC-9/GR cell line was 100.16, whereas the resistance index to cisplatin in PC-9/GR cell line was only 1.19. Induction of G0/G1 Arrest and Apoptosis by gefitinib was attenuated in PC-9/GR cell line. Sequencing of EGFR gene exon 19 and 20 showed an additional EGFR exon 20 T790M mutation presented in PC-9/GR cell line, but not in the parental cell line PC-9. Western blot analysis showed that gefitinib significantly inhibited activation of p-EGFR, p-Erk and p-Akt dose dependently in PC-9 cell line, whereat p-EGFR, p-Erk and p-Akt were sustained activation in PC-9/GR cell line in spite of geifitinib treatment. Immunocytochemistry revealed that both of PC-9 and PC-9/GR cell line express p53 protein.3. CIK cell-mediated cytotoxicity in the PC-9/GR cellsThere was no significant difference in the cytolytic activity of CIK cells against PC-9 and PC-9/GR targets. The growth of the tumor was significantly suppressed in the PC-9 and PC-9/GR cell-bearing nude mice after CIK cells infusion when compared with the control groups, in terms as an evident inhibition of tumor volume and weight, P<0.05. However, there was no significant difference of tumor growth inhibition between PC-9 and PC-9/GR cell-bearing nude mice after CIK cells therapy. In addition, a synergistic growth inhibitory effect was obtained by the combination CIK cells and geifitinb in PC-9/GR cell line.4. CIK cell-mediated immune modulation in patients with tumorsThe starting population contained a total PBMCs ranging from 1.2×109 to 1.6×109 with a very small amount of CD3+CD56+ cells varied from 2.1% to 4.4%. The target CIK cell dose was achieved in those cultures. A total cells population was about 1.0×1010 to 1.5×1010. The median percentage of CD3+CD56+ cells was 21.08% (range, 14.7%~31.0%). The absolute number of CD3+CD56+ cells obtained from CIK cultures varied from 1.26×109 to 4.65×109. There were no severe side effects in all the 4 patients after CIK cells infusion. We showed that the number of NK cells in peripheral blood was increased in the patients after CIK cells therapy, while the frequency of Treg cells was decreased; the differences were significant (P<0.05) at day 14 or day 30 after CIK cells infusion compared to before the therapy. Moreover, we found that the frequency of CD3-CD16dimCD56bright NK cell was decreased in peripheral blood by the treatment of CIK cells infusion, whereas the frequency of CD3-CD16brightCD56dim NK cell was increased. In accordance, there was a significant elevation in plasma levels of IFN-γ,IP-10,RATENS at day 1 or day 14 after CIK cells infusion and TNF-α,IL-15,MCP-3 at day 14 after the addition, P<0.05. There was also an increase in MCP-1 level in the plasma of the treated patients with a peak at day 14 , but a decrease in MIP-1αlevel with the lowest level at day 7, P<0.05. Importantly, a significant decrease in the level of TGF-βwas observed after the CIK cell therapy in the plasma of those patients who were detected with a high level of TGF-βprior to the therapy. TGF-βlevel was decreased approximately 54.85% at day 30 after the infusion. In addition, we showed a decreased cytotoxicity against a human chronic myelogenous leukemia cell line K562 in the CIK cells with IL-2 withdraw or frozen-thawed. Addition of IL-2 could restore cytolytic activity of the CIK cells.Conclusion1. EGFR exon19 deletions and exon21 L858R mutations are powerful predictive marker for the therapy response to gefitinib in patients with NSCLC, while smoking history plays a crucial role in affecting the therapeutic response to geifitinib in the patients with EGFR-mutated NSCLC. p53 mutation induced by smoking maybe an important factor related to the decreased therapeutic response to geifitinib.2. Gefitinib resistant cell line PC-9/GR was successfully derived from the parental NSCLC cell PC-9 with EGFR exon19 E746-A750 deletion and p53 mutation concomitantly. The sustained activation of PI3K/Akt, MAPK/Erk1/2 signaling in spite of geifitinib treatment was found in PC-9/GR cell line, with an acquired EGFR exon20 T790M mutation.3. CIK cells represent a strong cytolytic activity to gefitinib resistant cell line PC-9/GR, as well as a synergistic effect in PC-9/GR with the treatment of gefitinib.4. Adoptive immunotherapy with CIK cells could significantly enhances immune functions increasing the frequency of NK cell and decreasing the frequency of Treg cell and level of core immunosuppressive cytokine TGF-βwithout side effects, which represents a clinical immunoregulation therapy and may have a favorable impact on conventional treatment strategy of NSCLC patients with EGFR mutation.5. We have developed a disposable wave bioreactor for perfusion CIK cell culture.with advanced biosafety, high-efficiency and ease-of-operation for clinical application.