Non-small cell lung cancer (NSCLC) is a major cause of

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1 Original Article The BIM Deletion Polymorphism and its Clinical Implication in Patients with EGFR-Mutant Non Small-Cell Lung Cancer Treated with EGFR Tyrosine Kinase Inhibitors Ji Yun Lee, MD,* Bo Mi Ku, PhD, Sung Hee Lim, MD,* Min-Young Lee, MD,* Haesu Kim, MD,* Moonjin Kim, MD,* Sungmin Kim, MD,* Hyun Ae Jung, MD,* Jong-Mu Sun, MD, PhD,* Jin Seok Ahn, MD, PhD,* Keunchil Park, MD, PhD,* and Myung-Ju Ahn, MD, PhD* Introduction: A germline BIM deletion polymorphism has been proposed to predict poor treatment response to certain kinase inhibitors. The purpose of this study was to explore whether the BIM deletion polymorphism predicts treatment efficacy of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in Korean patients with EGFR-mutant non small-cell lung cancer (NSCLC). Methods: Peripheral blood samples from a total of 205 patients with EGFR-mutant NSCLC who were treated with EGFR TKIs between July 2008 and April 2013 were included. The incidence of BIM deletions in these samples was detected by polymerase chain reaction. We compared the clinical outcomes in patients with and without the polymorphism after treatment with EGFR TKIs (gefitinib or erlotinib). Results: The BIM deletion polymorphism was present in 15.6% (32 of 205) of patients. One patient was homozygous for the deletion, and the remaining 31 had heterozygous deletions. The majority of patients were younger than 65 years (74%), female (68%), never smokers (76%), and had stage IV NSCLC (67%). There were no associations between the BIM deletion polymorphism and clinicopathological features including gender, age, smoking status, histology, stage, and number of metastasis sites. Patients with and without the BIM deletion polymorphism had similar objective response rates (91 vs. 84%, p = 0.585). Progression-free survival and overall survival did not differ significantly between patients with and without the BIM deletion polymorphism (median progression-free survival 12 vs. 11 months, p = 0.160; median overall survival 31 vs. 30 months, p = 0.452). Multivariate analysis identified significantly predictive markers for clinical outcomes of EGFR TKIs including Eastern Cooperative Oncology Group performance status 0 1, adenocarcinoma histology, recurrent disease, and EGFR mutation type. *Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, South Korea; and Samsung Biomedical Research Institute, Samsung Cancer Research Institute, Samsung Medical Center, Seoul, South Korea. Ji Yun Lee and Bo Mi Ku equally contributed to this work. Disclosure: The authors declare that there are no conflicts of interest. Address for correspondence: Myung-Ju Ahn, MD, PhD, Division of Hematology Oncology, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, 81 Irwon-ro, Gangnam-gu, Seoul , South Korea. silkahn@skku.edu, silk.ahn@samsung.com DOI: /JTO Copyright 2015 by the International Association for the Study of Lung Cancer ISSN: /15/ The results were validated in an independent cohort of 69 NSCLC patients. Conclusions: It remains to be determined whether the BIM deletion polymorphism provides intrinsic resistance or decreased sensitivity to EGFR TKIs in EGFR-mutant NSCLC patients. Key Words: Non small-cell lung cancer, Epidermal growth factor receptor tyrosine kinase inhibitors, BIM deletion polymorphism. (J Thorac Oncol. 2015;10: ) Non-small cell lung cancer (NSCLC) is a major cause of cancer-related death. 1,2 Among patients with advanced NSCLC, cytotoxic chemotherapy has a response rate of 20% to 35% and a median survival time of 10 to 12 months. 3,4 Recent insights into the molecular mechanisms of NSCLC have led to the development of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), including gefinib, erlotinib, and afatinib. These EGFR TKIs are superior to chemotherapy as first-line treatments of EGFR-mutant NSCLC However, 20% to 30% of patients with EGFR-mutations do not respond to EGFR TKIs because of de novo or intrinsic resistance. 11 Although several possible mechanisms have been investigated in preclinical and retrospective studies, the factors that predict treatment response to EGFR TKIs remain elusive The BIM (also known as BCL2L11) gene encodes a BH3-only protein that is a proapoptotic member of the BCL2 family of proteins and is required for promoting cell death. 15 Prior studies have demonstrated that BIM is essential for EGFR TKIs-induced killing of NSCLC cells. Similarly, suppressing BIM expression is sufficient to confer in vitro TKI resistance Given these findings, it is important to describe the impact of the BIM deletion polymorphism on EGFR TKIs treatment in EGFR-mutant NSCLC patients. A recent study demonstrated that patients with the BIM deletion polymorphism had a lower median progression-free survival (PFS) than did those without the polymorphism (6.6 vs months; p = 0.003). 20 However, the relationship between the BIM deletion polymorphism and the efficacy of EGFR TKIs against EGFR-mutant NSCLC has not been completely elucidated. Therefore, the aim of this study was to determine whether the BIM deletion polymorphism influences the clinical outcomes of NSCLC with EGFR TKI treatment. Journal of Thoracic Oncology Volume 10, Number 6, June

2 Lee et al. Journal of Thoracic Oncology Volume 10, Number 6, June 2015 MATERIALS AND METHODS Study Populations A total of 227 patients with EGFR-mutant NSCLC who received EGFR TKIs between July 2008 and April 2013 were retrospectively reviewed from the lung cancer database of Samsung Medical Center. Patients were excluded if they had insufficient archival tissue (n = 14) or EGFR exon 20 mutations (n = 8: 4 patients with insertion and/or deletion mutations, 2 with missense mutations, 2 with duplication mutations). Previous research has suggested that exon 20 mutations are associated with poor treatment responses to EGFR TKIs. 21,22 Therefore, the eight patients with EGFR exon 20 mutations were excluded from this study. Patients either received oral gefitinib (250 mg per day) or oral erlotinib (150 mg per day). Every two cycles, patients treatment responses were evaluated using a spiral computed tomography scan and the Response Evaluation Criteria in Solid Tumors version The primary objective of this study was to analyze the effect of the BIM deletion polymorphism on the PFS of patients with EGFR-mutant NSCLC who were treated with EGFR TKIs. The secondary objectives included comparison of the following clinical features between patients with and without the BIM deletion polymorphism: (1) clinicopathologic distribution according to BIM status; (2) objective response rate (ORR) and disease control rate (DCR) for EGFR TKIs; and (3) overall survival (OS). This study was approved by the Institutional Review Board of Samsung Medical Center. Informed consent was waived because the study was based on retrospective administrative and clinical data. Tissue Analysis and EGFR Mutation Testing A pathologist reviewed the tumor contents and histologic review of all of the samples. EGFR (exons 18 21) mutations were analyzed by directional sequencing of polymerase chain reaction (PCR) fragments amplified with genomic DNA from formalin-fixed paraffin embedded (FFPE) tissue. Either standard methodology or the peptide nucleic acid-locked nucleic acid PCR clamp method was used. 24,25 Genotyping of BIM Deletion Polymorphism Because previous studies showed concordance between matched germline (peripheral blood) and somatic (fresh/frozen tumor tissue) DNA, we used peripheral blood samples for genotyping of BIM deletion polymorphism. 26,27 Genomic DNA was extracted from peripheral blood using the DNeasy Blood & Tissue Kit (Qiagen, Tokyo, Japan) according to the manufacturer s protocol. PCR was carried out to detect the BIM deletion polymorphism. The primer sequence and cycling conditions have been previously described in detail. 20 The PCR reactions were performed using the GoTaq Long PCR Master Mix (Promega, Madison, WI), and human genomic DNA included in the GoTaq Long PCR Master Mix kit was used as positive control. The PCR products (1323 base pair for deleted BIM and 4226 base pair for wild-type BIM) were analyzed on 1% agarose gels (Supplemental Fig. 1, Supplemental Digital Content 1, Statistical Analysis We calculated the PFS from the initiation of EGFR TKIs therapy until tumor progression or until death from any cause. OS was defined as the time from the initiation of EGFR TKIs therapy to death from any cause. Either the χ 2 test or Fisher exact test was used to analyze the differences between the clinicopathologic characteristics of the BIM-deleted and wild-type populations. Kaplan Meier methodology was used to estimate survival, which was expressed as a mean with a range and twosided 95% confidence interval (CI). Using Cox proportional hazard regression analyses, univariate and multivariate hazard ratios were generated for the following factors: age, gender, smoking history, performance status (Eastern Cooperative Oncology Group [ECOG]), pathology, stage, number of metastasis, type of EGFR mutation, type of EGFR TKIs, and line of EGFR TKIs. Two-sided p values less than 0.05 were considered statistically significant. All analyses were performed using SPSS (version 19.0, SPSS Inc., Chicago, IL). RESULTS Prevalence and Clinicopathological Characteristics of the BIM Deletion Polymorphism Table 1 summarizes the demographic and clinicopathologic characteristics of the 205 patients. The median patient age was 59 years, and 68% were female. The majority of patients were never smokers (76%) and had ECOG performance status (PS) of 0 1 (94%), tumor stage IV (67%) with adenocarcinoma (99%), and one or two metastatic sites (79%). Among 205 patients, 68 (33%) received EGFR TKIs as firstline treatment. More patients were treated with gefitinib than with erlotinib (67% vs. 33%, respectively). The exon 19 deletion was the most common mutation, accounting for 56% of mutations. The second most common was the L858R mutation (39%). Other EGFR mutations such as rare or complex mutations included exon 18 point mutations (Gly 719 to Ser, Ala or Cys [G719S/A/C], n = 6 patients) and complex mutations (deletion and missense mutations in exon 19 or 21, n = 5; Table 1). A total of 32 patients (15.6%) were found to have the BIM deletion polymorphism. The deletion was heterozygous in 31 patients, and homozygous in 1 patient. The BIM deletion polymorphism had no association with basic clinicopathologic variables. Response By the end of data collection in June 2014, 22 patients (11%) were still receiving EGFR TKIs. The most common reasons for treatment discontinuation were disease progression (n = 171), drug toxicity (n = 6), and loss to follow-up (n = 5). Patients had similar ORRs regardless of their BIM deletion polymorphism status (91% [with mutation] vs. 84% [without], p = 0.585). Similarly, there was no difference between the DCR values of the two groups (94% [with mutation] vs. 94% [without], p = 0.614; Table 1). Survival The median follow-up duration was 21.8 months (range ). In the total population, the median PFS and OS were 904

3 Journal of Thoracic Oncology Volume 10, Number 6, June 2015 Non Small Cell Lung Cancer and BIM Deletion Polymorphism TABLE 1. Patients Characteristics and Comparisons Between Patients With and Without the BIM Deletion Polymorphism (n = 205) Total (n = 205) BIM Deletion + (n = 32) BIM deletion (n = 173) P value Basic clinicopathologic variables Age, mean ± SD 59 ± ± ± Age, n (%) < (74) 23 (72) 129 (75) (26) 9 (28) 44 (25) Sex, n (%) Male 66 (32) 7 (22) 59 (34) Female 139 (68) 25 (78) 114 (66) Smoking history, n (%) Never 156 (76) 26 (81) 130 (75) Former/current 49 (24) 6 (19) 43 (25) ECOG PS, n (%) (94) 31 (97) 161 (93) (6) 1 (3) 12 (7) Pathology, n (%) Adenocarcinoma 203 (99) 32 (100) 171 (99) Squamous 2 (1) 0 2 (1) Stage, n (%) IIIB 8 (4) 0 8 (5) IV 138 (67) 19 (59) 119 (69) Postoperative relapse 59 (29) 13 (41) 46 (27) No. of metastasis, n (%) (79) 25 (78) 136 (79) 3 44 (21) 7 (22) 37 (21) EGFR mutation, n (%) Exon 19 deletion 114 (56) 20 (63) 94 (54) L858R mutation 80 (39) 11 (34) 69 (40) Others a 11 (5) 1 (3) 10 (6) EGFR TKIs, n (%) Gefitinib 124 (61) 22 (69) 102 (59) Erlotinib 81 (39) 10 (31) 71 (41) Line of EGFR TKIs, n (%) First 68 (33) 12 (38) 56 (32) Second or more line 137 (67) 20 (62) 117 (68) Treatment response Objective tumor response 175 (85) 29 (91) 146 (84) Disease control 193 (94) 30 (94) 163 (94) a Others mutation: Exon 18 point mutation (Gly 719 change to Ser, Ala or Cys [G719S/A/C]), six patients; complex mutation (deletion and missense mutation in exon 19 or 21), seven patients. ECOG PS, Eastern Cooperative Oncology Group performance status; EGFR, epidermal growth factor receptor, TKIs, tyrosine kinase inhibitors (95% CI: ) and 30.3 months (95% CI: ), respectively. The possible predictive factors of EGFR TKIs treatment efficacy were analyzed as subgroups in terms of PFS (Table 2). Univariate analysis revealed several factors that are significantly associated with poor PFS. These include male sex, former or current smoking, ECOG PS 2 4, squamous cell carcinoma, stage IV, and number of metastasis sites. There were similar PFS values regardless of BIM deletion polymorphism status (median PFS 11.9 [with mutation] vs (without) months, p = [Fig. 1A]). Similarly, OS was comparable between the groups (median OS 31.2 [with mutation] vs months [without], p = [Fig. 1B]). Multivariate analysis revealed that ECOG PS 0 1, adenocarcinoma histology, recurrent disease, and EGFR mutation type were all significant predictive markers for clinical outcomes with EGFR TKI treatment (Table 2 and Supplemental Table 1, Supplemental Digital Content 2, Validation from Independent NSCLC Cohort Because the results in this study were inconsistent with previous studies, we studied BIM deletion polymorphism using peripheral blood from an independent cohort of 69 NSCLC patients (Supplemental Table 2, Supplemental Digital Content 2, The BIM deletion polymorphism was present in 14.5% (10 of 69) of patients. Two patients were homozygous for the deletion, and the remaining eight had heterozygous deletions. Patients with and without the BIM deletion polymorphism had similar ORRs (70.0% vs. 74.6%, p = 0.713). PFS did not differ significantly between patients with and without the BIM deletion polymorphism (median PFS 11.6 vs. 9.7 months, p = 0.599; Supplemental Fig. 2, Supplemental Digital Content 3, JTO/A823). These results validated that the presence of the BIM deletion polymorphism was not associated with treatment response to EGFR TKIs. DISCUSSION Recent data have demonstrated that the BIM deletion polymorphism, a germline mutation, is particularly influential in the targeted therapy-induced apoptosis of both blood and solid tumor cancers In particular, this polymorphism is thought to be associated with intrinsic resistance to targeted therapies such as EGFR TKIs. Despite a large sample size, this study fails to demonstrate a correlation between BIM deletion polymorphism status and clinical outcomes in EGFR mutant NSCLC treated with EGFR TKIs. It is noteworthy that the BIM deletion polymorphism is relatively common in East Asian populations but is absent in Africans and Europeans. 20 In the current study, the BIM deletion polymorphism was present in 32 of 205 patients (15.6%). One patient (0.5%) was homozygous for this deletion. These proportions are relatively consistent with prior reports. 20,31 33 We also found that the distribution of BIM deletion polymorphisms was not associated with any specific clinicopathologic features. In the previous study by Ng et al., 20 patients with BIM deletion polymorphism had significantly shorter PFS than did patients without the polymorphism after EGFR TKIs (6.6 vs months, respectively, p = ). Others have previously demonstrated the correlation between the BIM deletion polymorphism and poor treatment response rates (to EGFR TKIs) and short PFS in patients with EGFR-mutant NSCLC (Table 3) On the basis of these results, studies have highlighted the clinical benefits of BH3-mimetic drugs or histone 905

4 Lee et al. Journal of Thoracic Oncology Volume 10, Number 6, June 2015 TABLE 2. Univariate and multivariate analysis for progression-free survival Variable Univariate Analysis Multivariate Analysis Hazard Ratio P Value Hazard Ratio P Value Age, n (%) < ( ) Sex, n (%) Female Male 1.55 ( ) ( ) Smoking history, n (%) Never Former/current 1.47 ( ) ( ) ECOG PS, n (%) ( ) ( ) Pathology, n (%) Adenocarcinoma Squamous 5.06 ( ) ( ) Stage, n (%) Postoperative relapse IIIB 2.29 ( ) ( ) IV 1.99 ( ) ( ) No. of metastasis, n (%) ( ) ( ) EGFR mutation, n (%) Exon 19 deletion L858R 0.94 ( ) ( ) Others a 1.81 ( ) ( ) EGFR TKI, n (%) Gefitinib 1.00 Erlotinib 0.94 ( ) Line of EGFR TKI, n (%) First 1.00 Second or more line 1.02 ( ) BIM polymorphism, n (%) Absent 1.00 Present 0.74 ( ) a Others mutation: Exon 18 point mutation (Gly 719 change to Ser, Ala or Cys [G719S/A/C]), six patients; complex mutation (deletion and missense mutation in exon 19 or 21), five patients. ECOG PS, Eastern Cooperative Oncology Group performance status; EGFR, epidermal growth factor receptor; TKIs tyrosine kinase inhibitors. deacetylase inhibitors in patients with the BIM deletion polymorphism. 19,32 However, the BIM deletion polymorphism was not predictive of EGFR TKIs outcome in this study, which is consistent with previous findings in Korea from Lee et al. 31 Data from Lee et al. 31 demonstrated no difference in the PFS among patients with and without the BIM deletion polymorphism (11.9 months with BIM deletion vs months without, p = 0.791). Similarly, Isobe et al. 32 reported that patients with the BIM deletion polymorphism had similar ORR and DCR but shorter PFS than did patients without the mutation. Different BIM expression levels may explain the contradictory findings regarding the deletion polymorphism and EGFR TKI outcomes. BIM upregulation, especially of the proapoptotic isoform containing the BH3 domain, is required for TKIs to induce apoptosis. However, a deletion polymorphism in the BIM gene leads to alternatively spliced BIM isoforms that lack this crucial BH3 domain. 18,36 Therefore, one hypothesis is that low BIM expression of the BH3 domain is associated with an unfavorable response to EGFR TKIs. 34 The current study and a report by Lee et al. 31 analyzed the BIM deletion polymorphism using genomic DNA extracted from peripheral blood and FFPE tissues. To establish the role of BIM as a predictor of treatment efficacy, it is useful to measure the BIM RNA levels in patients before treatment with EGFR TKIs. 906

5 Journal of Thoracic Oncology Volume 10, Number 6, June 2015 Non Small Cell Lung Cancer and BIM Deletion Polymorphism FIGURE 1. Kaplan Meier curves for progression-free survival (A) and overall survival (B) according to BIM status. TABLE 3. Results from Recent Studies of BIM for EGFR-Mutant NSCLC Treated With EGFR TKIs Reference No. Method Specimen Detection of BIM Deletion Overall Response Rate Median PFS (Months) Median OS (months) Ng et al DNA polymorphism Tumor tissue or peripheral blood 26 (18.4%) Not reported 6.6 vs a Not reported Faber et al mrna expression Tumor tissue 9 (37.5%) b 29% vs. 57% a 4.7 vs a Not reported Costa et al mrna expression Tumor tissue 26 (52.0%) c 32% vs. 88% a 7.2 vs a 20.8 vs Zhao et al DNA polymorphism Tumor tissue 16 (9.6%) 25% vs. 66% a 4.7 vs a Not reported Lee et al DNA polymorphism Tumor tissue 21 (10.9%) Not reported 11.3 vs Not reported Isobe et al DNA polymorphism Tumor tissue or peripheral 13 (18.6%) 62% vs. 65% 7.6 vs a 39.2 vs Current study 205 DNA polymorphism Blood, peripheral blood 32 (15.6%) 91% vs. 84% 11.9 vs vs a p < b Low BIM levels, defined as relative mrna to β-actin less than 30. c Low/intermediate BIM levels, defined as low (<1.83) or intermediate ( ). PFS, progression-free survival; EGFR, epidermal growth factor receptor; TKIs, tyrosine kinase inhibitors; OS, overall survival. It is also worth noting that proapoptic BCL2 family members such as BAX, BAK, and other BH3-only proteins including BAD and PUMA might be key players in the apoptotic response in oncogene-addicted cancer. BAX and BAK are often referred to as effector proteins as they are required for activation of the apoptosis. 37 Altered BAX and BAK expression is frequently reported in NSCLC None of these studies conclude that altered BAX or BAK expression has significant value as a prognostic marker. Sun et al. 41 demonstrated that PUMA induction is correlated with EGFR- TKI sensitivity and inhibition of the PI3K/AKT pathway. The crucial role the proapoptotic proteins BAX and BAK play in response to EGFR TKIs warrants further study. Other concomitant genetic alterations beyond EGFR mutations could conceivably accelerate or delay cancer progression. For example, the T790M mutation, found in 50% to 63% of patients, is well known to induce acquired resistance to EGFR TKIs. 42,43 However, the impact of pretreatment T790M mutations on the sensitivity of EGFR TKIs remains controversial The incidence of pretreatment T790M mutations is highly dependent on the sensitivity of the detection method. Therefore, the true frequency associated with drug resistance remains unknown. We did not identify any patients harboring germline T790M mutations in the current study. However, coexistence of the T790M mutation before treatment may have been underappreciated in this report, because the peptide 907

6 Lee et al. Journal of Thoracic Oncology Volume 10, Number 6, June 2015 nucleic acid clamp method (which is a more sensitive detector of EGFR mutations than of T790M mutations) was used. In the future, patients lacking sensitivity to EGFR TKIs will need to be worked-up further using additional genetic analyses such as TP53, 35,47 PTEN loss, 48 and PIK3CA mutations. 49 There may be additional ethnic differences in BIM polymorphisms between Far East Asian and East Asian. We validated the clinicopathologic features that were predictive of outcomes to EGFR TKIs. Patients with postoperative relapse had significantly better prognoses than did those with stage IIIB/IV disease, which is consistent with previously reported findings. 6 The more favorable prognosis in patients with relapse can be explained by the lower tumor burden in these patients compared with that in advanced metastatic disease (ρ correlation efficient 0.149, p = 0.033). In addition, the number of metastatic sites at baseline was significantly associated with poor survival. The present study had several limitations. Because the study was retrospective in nature, there may have been undefined bias regarding clinical outcomes. We analyzed the BIM deletion polymorphisms using genomic DNA from peripheral blood samples only. A report by Isobe et al. 32 showed that there is no discordance between the BIM status of peripheral blood and FFPE samples; still, validating the BIM status in the tumor tissue would have been ideal. We did not analyze other coexisting genetic abnormalities beyond that of the BIM deletion polymorphism. Therefore, the underlying complex biology of EGFR-mutant NSCLC and tumor heterogeneity were not fully evaluated. In this study, the presence of the BIM deletion polymorphism was not associated with treatment response to EGFR TKIs. Well-designed prospective studies are required to determine whether this polymorphism contributes to the heterogeneity of EGFR TKI responses among patients with EGFR-mutant NSCLC. ACKNOWLEDGMENTS This study was supported by the Samsung Biomedical Research Institute Grant (GE1-B ). REFERENCES 1. Jemal A, Siegel R, Xu J, Ward E. Cancer statistics, CA Cancer J Clin 2010;60: Jung KW, Park S, Kong HJ, et al. Cancer statistics in Korea: incidence, mortality, survival, and prevalence in Cancer Res Treat 2011;43: Schiller JH, Harrington D, Belani CP, et al.; Eastern Cooperative Oncology Group. Comparison of four chemotherapy regimens for advanced nonsmall-cell lung cancer. N Engl J Med 2002;346: Ohe Y, Ohashi Y, Kubota K, et al. Randomized phase III study of cisplatin plus irinotecan versus carboplatin plus paclitaxel, cisplatin plus gemcitabine, and cisplatin plus vinorelbine for advanced non-smallcell lung cancer: Four-Arm Cooperative Study in Japan. 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