Nobuaki Ochi

Twenty-Seven Years of Phase III Trials for Patients with Extensive Disease Small-Cell Lung Cancer: Disappointing Results

Background Few studies have formally assessed whether treatment outcomes have improved substantially over the years for patients with extensive disease small-cell lung cancer (ED-SCLC) enrolled in phase III trials. The objective of the current investigation was to determine the time trends in outcomes for the patients in those trials. Methods and Findings We searched for trials that were reported between January 1981 and August 2008. Phase III randomized controlled trials were eligible if they compared first-line, systemic chemotherapy for ED-SCLC. Data were evaluated by using a linear regression analysis. Results: In total, 52 trials were identified that had been initiated between 1980 and 2006; these studies involved 10,262 patients with 110 chemotherapy arms. The number of randomized patients and the proportion of patients with good performance status (PS) increased over time. Cisplatin-based regimens, especially cisplatin and etoposide (PE) regimen, have increasingly been studied, whereas cyclophosphamide, doxorubicin, and vincristine–based regimens have been less investigated. Multiple regression analysis showed no significant improvement in survival over the years. Additionally, the use of a PE regimen did not affect survival, whereas the proportion of patients with good PS and the trial design of assigning prophylactic cranial irradiation were significantly associated with favorable outcome. Conclusions and Significance The survival of patients with ED-SCLC enrolled in phase III trials did not improve significantly over the years, suggesting the need for further development of novel targets, newer agents, and comprehensive patient care.

Non–Small Cell Lung Cancer Cells Acquire Resistance to the ALK Inhibitor Alectinib by Activating Alternative Receptor Tyrosine Kinases

Crizotinib is the standard of care for advanced non-small cell lung cancer (NSCLC) patients harboring the anaplastic lymphoma kinase (ALK) fusion gene, but resistance invariably develops. Unlike crizotinib, alectinib is a selective ALK tyrosine kinase inhibitor (TKI) with more potent antitumor effects and a favorable toxicity profile, even in crizotinib-resistant cases. However, acquired resistance to alectinib, as for other TKIs, remains a limitation of its efficacy. Therefore, we investigated the mechanisms by which human NSCLC cells acquire resistance to alectinib. We established two alectinib-resistant cell lines that did not harbor the secondary ALK mutations frequently occurring in crizotinib-resistant cells. One cell line lost the EML4-ALK fusion gene, but exhibited increased activation of insulin-like growth factor-1 receptor (IGF1R) and human epidermal growth factor receptor 3 (HER3), and overexpressed the HER3 ligand neuregulin 1. Accordingly, pharmacologic inhibition of IGF1R and HER3 signaling overcame resistance to alectinib in this cell line. The second alectinib-resistant cell line displayed stimulated HGF autocrine signaling that promoted MET activation and remained sensitive to crizotinib treatment. Taken together, our findings reveal two novel mechanisms underlying alectinib resistance that are caused by the activation of alternative tyrosine kinase receptors rather than by secondary ALK mutations. These studies may guide the development of comprehensive treatment strategies that take into consideration the various approaches ALK-positive lung tumors use to withstand therapeutic insult.

Synergistic Effect of Olaparib with Combination of Cisplatin on PTEN-Deficient Lung Cancer Cells

PARP enzyme plays a key role in the cellular machinery responsible for DNA damage repair. PTEN is a tumor-suppressor gene deactivating PI3K downstream of EGFR signaling. We hypothesize that PTEN-deficient lung cancer cells suppressed DNA damage signaling and that the absence of PTEN can sensitize these cells to a concurrent treatment of a DNA-damaging agent (cisplatin) and a PARP inhibitor (olaparib). To investigate the effect of olaparib and cisplatin on PTEN-deficient lung tumors, two EGFR-mutant (deletion in exon19) non–small cell lung cancer (NSCLC) cell lines, PC-9 (PTEN wild-type) and H1650 (PTEN loss), were used. We transfected intact PTEN gene into H1650 cells (H1650PTEN+) and knocked down PTEN expression in the PC-9 cells (PC-9PTEN−) using short hairpin RNA (shRNA). Combination of cisplatin with olaparib showed a synergistic effect in vitro according to the combination index in H1650 cells. Restoration of PTEN in the H1650 cells decreased sensitivity to the combination. Ablation of PTEN in PC-9 cells increased sensitivity to olaparib and cisplatin. We also examined the effectiveness of cisplatin and olaparib in a xenograft model using H1650 and PC-9PTEN− cells. The combination of cisplatin with olaparib was more effective than each agent individually. This effect was not observed in a xenograft model using H1650PTEN+ and PC-9 cells. Mechanistic investigations revealed that PTEN deficiency caused reductions in nuclear RAD51 and RPA focus formation and phosphorylated Chk1 and Mre11. Thus, genetic inactivation of PTEN led to the suppression of DNA repair. Mol Cancer Res; 11(2); 140–8. ©2012 AACR.

Subpopulation of small-cell lung cancer cells expressing CD133 and CD87 show resistance to chemotherapy.

Tumors are presumed to contain a small population of cancer stem cells (CSCs) that initiate tumor growth and promote tumor spreading. Multidrug resistance in CSCs is thought to allow the tumor to evade conventional therapy. This study focused on expression of CD133 and CD87 because CD133 is a putative marker of CSCs in some cancers including lung, and CD87 is associated with a stem‐cell‐like property in small‐cell lung cancer (SCLC). Six SCLC cell lines were used. The expression levels of CD133 and CD87 were analyzed by real‐time quantitative reverse transcription‐polymerase chain reaction and flow cytometry. CD133+/− and CD87+/− cells were isolated by flow cytometry. The drug sensitivities were determined using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay. Non‐obese diabetic/severe combined immunodeficiency mice were used for the tumor formation assay. SBC‐7 cells showed the highest expression levels of both CD133 and CD87 among the cell lines. CD133−/CD87−, CD133+/CD87−, and CD133−/CD87+ cells were isolated from SBC‐7 cells; however, CD133+/CD87+ cells could not be obtained. Both CD133+/CD87− and CD133−/CD87+ subpopulations showed a higher resistance to etoposide and paclitaxel and greater re‐populating ability than the CD133−/CD87− subpopulation. CD133+/CD87− cells contained more G0 quiescent cells than CD133−/CD87− cells. By contrast, CD133−/CD87− cells showed the highest tumorigenic potential. In conclusion, both CD133 and CD87 proved to be inadequate markers for CSCs; however, they might be beneficial for predicting resistance to chemotherapy. (Cancer Sci 2013; 104: 78–84)

JAK2-related pathway induces acquired erlotinib resistance in lung cancer cells harboring an epidermal growth factor receptor-activating mutation

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors, such as gefitinib and erlotinib, are effective for non‐small cell lung cancer with activating EGFR mutations. However, even in patients with an initial dramatic response to such a drug, acquired resistance develops after 6–12 months. A secondary mutation of T790M in EGFR and amplification of the MET gene account for this resistance; however, the mechanism(s) of approximately 30% of acquired resistance cases remain unknown. We established an erlotinib‐resistant lung cancer cell line named PC‐9/ER3 that harbors an EGFR mutation after continuously exposing PC‐9 cells to erlotinib. PC‐9/ER3 cells were 136‐fold more resistant to erlotinib than the parental cells. Although the PC‐9/ER3 cells did not carry the T790M mutation or MET amplification and had similar levels of phosphorylated (p) STAT3, pJAK2 increased in the resistant cells. It was found in the present study that 3–12 h of exposure to erlotinib in both cell lines did not affect pJAK2 expression, but did result in increased pSTAT3 expression. pAkt in PC‐9/ER3 cells was less suppressed than in PC‐9 cells, although pEGFR and pMAPK were markedly suppressed in both cell lines. The combined treatment of erlotinib plus a JAK2 inhibitor (JSI‐124) suppressed pAkt in PC‐9/ER3 cells. Similarly, the combination of erlotinib plus JSI‐124 or siRNA against JAK2 restored sensitivity to erlotinib in PC‐9/ER3 cells. The combination of erlotinib plus JSI‐124 was also effective for reducing PC‐9/ER3 tumors in a murine xenograft model. Our results suggest that the activation of JAK2 partially accounts for acquired erlotinib resistance.(Cancer Sci, doi: 10.1111/j.1349‐7006.2012.02363.x, 2012)

Afatinib Prolongs Survival Compared with Gefitinib in an Epidermal Growth Factor Receptor-Driven Lung Cancer Model

An irreversible ErbB family blocker is expected to inhibit tumors with activating epidermal growth factor receptor (EGFR) mutations more strongly than reversible EGFR tyrosine kinase inhibitors and to overcome acquired resistance to the T790M secondary mutation. Eleven-week-old transgenic mice with Egfr exon 19 deletion mutation were treated with afatinib, gefitinib, or vehicle for 4 weeks. All mice were sacrificed at 15 weeks of age, and the number of superficial left lung tumors with a long axis exceeding 1 mm was counted. The afatinib-treated group had significantly fewer tumors than the vehicle group (P < 0.01) and tended to have fewer tumors than the gefitinib-treated group (P = 0.06). Pathologically, gefitinib-treated mice had clearer, more nodular tumors than afatinib-treated mice. Immunoblotting showed that afatinib suppressed not only pEGFR but also pHER2, and induced apoptosis for longer periods than gefitinib. Subsequently, when each drug was administered 5 days per week until death, afatinib significantly enhanced mouse survival compared with gefitinib (median survival time: 456 days vs. 376.5 days; log-rank test, P < 0.01). Finally, the combination of afatinib with bevacizumab was found to be superior to either drug alone in exon 19 deletion/T790M and L858R/T790M xenograft tumors. Overall, afatinib was more potent than gefitinib in tumors harboring an exon 19 deletion mutation, and the combination of afatinib with bevacizumab efficiently suppressed tumors harboring the T790M secondary mutation. Mol Cancer Ther; 12(5); 589–97. ©2013 AACR.

Association between incremental gains in the objective response rate and survival improvement in phase III trials of first-line chemotherapy for extensive disease small-cell lung cancer

BACKGROUND The duration of, resources required for and cost of clinical trials could be reduced if a surrogate end point was to be used in place of survival. We assessed the extent to which the objective response rate (ORR) is predictive of mortality, how much difference in the ORR is needed to predict an obvious survival difference and what factors could affect the association between the two parameters during the first-line treatment of extensive disease (ED)-small-cell lung cancer (SCLC). METHODS We used the ORRs and median survival times (MSTs) from 48 phase III trials of first-line chemotherapy involving 8779 randomised patients with ED-SCLC in a linear regression analysis. The MST difference was calculated as the difference in MST between the investigational and reference arms; the ORR difference was similarly defined. RESULTS ORR difference between the treatment arms was modestly associated with the MST difference in the overall trials (R(2) = 0.3314). In contrast, the relationship was stronger among only trials in which prophylactic cranial irradiation was given to those having an objective response to the initial chemotherapy (R(2) = 0.6279). In this trial setting, large differences in ORR were needed to predict a survival advantage (1.2-day survival advantage per 2% increase in ORR). CONCLUSIONS In the first-line treatment of ED-SCLC, a favourable relationship was detected between the two parameters in the selected trial setting. Large ORR differences were needed to predict a survival benefit, clearly suggesting the need for new chemotherapeutic agents.

Src mediates ERK reactivation in gefitinib resistance in non-small cell lung cancer

To study epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) resistance mechanisms, we established a novel gefitinib-resistant lung cancer cell line derived from an EGFR-mutant non-small cell lung cancer cell line (PC-9) pretreated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (designated PC9-GR). We found that gefitinib substantially suppressed the EGFR signaling pathway, whereas ERK was reactivated after several hours in PC9-GR but not in PC-9. The combination of gefitinib with ERK inhibition (by U0126) restored gefitinib susceptibility in PC9-GR, but PI3K-Akt inhibition with LY294002 did not. Although the levels of phosphorylated Src were up-regulated simultaneously with ERK reactivation, neither ERK suppression using U0126 nor an ERK-specific siRNA induced Src phosphorylation. Furthermore, dual inhibition of EGFR and Src restored gefitinib sensitivity in PC9-GR in vitro and in vivo. In conclusion, our results indicate that Src-mediated ERK reactivation may play a role in a novel gefitinib resistance mechanism, and that the combined use of gefitinib with a Src inhibitor may be a potent strategy to overcome this resistance.

Establishment of pemetrexed-resistant non-small cell lung cancer cell lines

Pemetrexed (PEM), a multitargeted antifolate with manageable toxicity, is active against non-squamous non-small cell lung cancer; however, most patients eventually acquire resistance to PEM. To elucidate the resistant mechanism, we established PEM-resistant lung adenocarcinoma cell lines. Two parental cell lines, PC-9 and A549, were treated with step-wise increasing concentrations of PEM. Growth inhibition was determined by the 3-[4,5-dimethyl-thizol-2-yl]-2,5-diphenyltetrazolium bromide assay. Expression of the genes encoding thymidylate synthase (TS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyltransferase (GARFT) was analyzed by quantitative real-time reverse transcriptase polymerase chain reaction. The four PC-9 sublines were more resistant than the PC-9 cell line to PEM (2.2-, 2.9-, 8.4-, and 14.3-fold, respectively). The four A549 sublines also showed more resistance to PEM (7.8-, 9.6-, 42.3-, and 42.4-fold, respectively) than the parent cell line. All resistant sublines showed cross-resistance to cisplatin, but not to docetaxel, vinorelbine, 5-fluorouracil, or the active metabolite of irinotecan, SN-38. All PEM-resistant sublines expressed more TS than the parental cells, by polymerase chain reaction and Western blotting. DHFR was significantly increased in the four PEM-resistant A549 sublines. GARFT did not correlate with resistance to PEM. In summary, PEM-resistant cells remained sensitive to docetaxel, vinorelbine, 5-fluorouracil, and irinotecan. TS expression appeared to be associated with resistance to PEM.

Effect of AZD1480 in an epidermal growth factor receptor-driven lung cancer model

OBJECTIVE STAT3 plays a vital role in inducing and maintaining a pro-carcinogenic inflammatory microenvironment and is reported to be a critical mediator of the oncogenic effects of EGFR mutations. STAT3 activation is mediated through JAK family kinases. We investigated the effect of the JAK1/2 inhibitor AZD1480 on lung tumors induced by an activating EGFR mutation. MATERIALS AND METHODS Three EGFR tyrosine kinase inhibitor-resistant cell lines (RPC-9, PC-9/Van-R and PC-9/ER3) established from PC-9 harboring an EGFR exon19 deletion mutation were used. Growth inhibition was measured using an MTT assay. Effects of AZD1480 were also evaluated in the xenograft model and in the EGFR transgenic mice model. Protein expressions were assessed by immunoblotting and immunohistochemistry. Group differences were compared using Students t-test. To evaluate the efficacy of AZD1480 on survival, AZD1480 or vehicle was administered orally from 7 weeks of age of the transgenic mice. Overall survival curves were calculated using the Kaplan-Meier method. RESULTS The sensitivities of resistant and parent cells to AZD1480 were similar in vitro. AZD1480 (30 or 50 mg/kg/day, per os) reduced angiogenesis and revealed significant tumor regression in a mouse xenograft model. Subsequently, the transgenic mice were treated with AZD1480 (30 mg/kg/day) or vehicle alone. The numbers of lung tumors (long axis exceeding 1mm) in the AZD1480-treated group and control group were 0.37±0.18 and 2.25±0.53 (p<0.001), respectively. AZD1480 treatment suppressed pSTAT3, pJAK1, pJAK2 and angiogenesis. The median survival time in the AZD1480-treated group (217 days) was significantly greater than that in the control group (106 days) (log-rank test, p<0.0001). CONCLUSION AZD1480 may be effective against lung tumors driven by an activating EGFR mutation.