Clinical Translational Research Received: July 24, 2013 Accepted: September 24, 2013 Published online: November 9, 2013

Oncology 2013;85:312–316 DOI: 10.1159/000356019

The Germline BIM Deletion Polymorphism Is Not Associated with the Treatment Efficacy of Sorafenib in Patients with Advanced Hepatocellular Carcinoma Yu-Yun Shao a, c Yung-Lin Chang a Chung-Yi Huang a Chih-Hung Hsu a, c Ann-Lii Cheng a–c Departments of a Oncology and b Internal Medicine, National Taiwan University Hospital, and c Graduate Institutes of Oncology, College of Medicine, National Taiwan University, Taipei, Taiwan, ROC

Abstract Objectives: A germline BIM deletion polymorphism has been proposed to predict a poor treatment efficacy of certain kinase inhibitors. The current study aimed to explore whether the BIM deletion polymorphism predicts the treatment efficacy of sorafenib for advanced hepatocellular carcinoma (HCC). Methods: All patients who were enrolled in clinical trials to receive sorafenib-containing regimens as first-line therapy for advanced HCC and consented to providing peripheral blood samples were included. Polymerase chain reaction followed by gel electrophoresis was used to detect the germline BIM deletion polymorphism. Results: A total of 89 patients were enrolled; 69 (77%) patients had chronic hepatitis B infection, and 18 (20%) had chronic hepatitis C infection. The heterozygous BIM deletion polymorphism was identified in 9 (10%) patients. Patients with and without the BIM deletion polymorphism had similar response rates (11 vs. 6%) and disease control rates (56 vs. 61%). The time to progression, progression-free survival, and overall survival were similar between patients with and with-

© 2013 S. Karger AG, Basel 0030–2414/13/0855–0312$38.00/0 E-Mail [email protected] www.karger.com/ocl

out the BIM deletion polymorphism. After adjusting for basic clinicopathologic variables and treatment regimens, the BIM polymorphism still could not predict treatment outcomes. Conclusions: The BIM deletion polymorphism was not associated with the treatment efficacy of sorafenib for advanced HCC. © 2013 S. Karger AG, Basel

Introduction

Apoptosis is one of the pivotal mechanisms for cancer cell death induced by targeted therapy. The B-cell lymphoma 2 (BCL2) interacting mediator of cell death (Bim) is a proapoptotic protein that has been demonstrated to mediate and convey the apoptotic signals [1]. Its proapoptotic BCL2 homology domain 3 (BH3) is crucial for its function. Recently, a common germline deletion polymorphism of BIM, the coding gene of Bim, was identified in approximately 12% of the East Asian population. The deletion resulted in the expression of a Bim isoform missing BH3, which led to the intrinsic resistance and inferior responses to two kinase inhibitors, i.e. imatinib and gefitinib for chronic myelogenous leukemia (CML) and epidermal growth factor receptor (EGFR)-mutated nonChih-Hung Hsu, MD, PhD Department of Oncology, National Taiwan University Hospital 7 Chung-Shan South Road Taipei 10002, Taiwan (ROC) E-Mail chihhunghsu @ ntu.edu.tw

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Key Words BIM · Germline polymorphism · Sorafenib · Hepatocellular carcinoma

small-cell lung cancer (NSCLC), respectively [2]. Imatinib and gefitinib target two distinct signaling pathways, but both of their efficacies can be impaired by BIM deletion. It is thus intriguing whether BIM deletion has an impact on other kinase inhibitors. Sorafenib is the current standard treatment for patients with advanced hepatocellular carcinoma (HCC) because randomized studies have demonstrated the survival benefit of sorafenib compared to placebo [3, 4]. Sorafenib is a kinase inhibitor targeting multiple kinases, including B-Raf and vascular endothelial growth factor receptor (VEGFR) [5]. Despite the survival benefits, the efficacy of sorafenib for advanced HCC is modest, with an objective tumor response around 2–3% [3, 4]. Therefore, a biomarker that can predict the efficacy of sorafenib is important for clinical practice. However, such predictive biomarkers are currently unavailable [6, 7]. In the current study, we explored the potential of the germline deletion polymorphism of BIM to be a predictive biomarker for sorafenib in HCC, by evaluating the associations between the BIM deletion polymorphism and the treatment outcomes of patients who received sorafenib for advanced HCC.

who had the heterozygous BIM deletion polymorphism that had been confirmed by direct sequencing served as a positive control in the genotyping experiments. Statistical Methods Statistical analyses were performed using SAS statistical software (version 9.1.3; SAS Institute Inc., Cary, N.C., USA). In statistical testing, a two-sided p value ≤0.05 was considered statistically significant. To study the associations between the germline BIM deletion polymorphism and basic clinicopathologic variables, Fisher’s exact test was used for nominal variables and an independent t test was used for continuous variables. We also used Fisher’s exact test to examine the associations between the germline BIM deletion polymorphism and objective tumor response or disease control. The Kaplan-Meier method was used to estimate the time to tumor progression (TTP), progression-free survival (PFS), and overall survival (OS). The log-rank test was used to compare these outcomes between patients with and without the BIM deletion polymorphism univariately. Cox’s proportional hazards model was used in multivariate analysis to examine whether BIM deletion was an independent predictor of TTP, PFS, or OS, while adjusting for treatment regimens, sex, age, hepatitis B virus (HBV) surface antigen (HBsAg), antibodies against hepatitis C virus (HCV) (antiHCV), α-fetoprotein level, macrovascular invasion, extrahepatic metastases, Eastern Cooperative Oncology Group (ECOG) performance status, and Cancer of the Liver Italian Program (CLIP) scores.

Methods Results

BIM Genotyping Mononuclear cells from peripheral blood were separated, and genomic DNA was extracted using a QIAamp® DNA Mini Kit (Qiagen, Valencia, Calif., USA). We performed PCR for BIM genotyping using FastStart Taq DNA Polymerase (Roche Applied Science, Indianapolis, Ind., USA) with the following conditions: 95 ° C for 5 min, (95 ° C for 50 s, 58 ° C for 50 s, and 72 ° C for 1 min) × 39 and 72 ° C for 10 min. The primers for detection of the deletion polymorphism were 5′-CCACCAATGGAAAAGGTTCA-3′ and 5′-GGCACAGCCTCTATGGAGAA-3′. The primers for the detection of no deletion polymorphism were 5′-CCACCAATGGAAAAGGTTCA-3′ and 5′-CTGTCATTTCTCCCCACCAC-3′. The products were then analyzed on a 2% agarose gel. The PCR product of DNA with BIM deletion should be 284 bp, and that of DNA without BIM deletion should be 362 bp. DNA from a patient

A total of 89 patients were enrolled; 64 (72%) patients received sorafenib combined with metronomic tegafur/ uracil [8], and 25 (28%) patients received sorafenib alone. HBsAg was positive in 68 (76%) patients, and anti-HCV was positive in 18 (20%) patients. Most (89%) patients had Barcelona Clinic Liver Cancer stage C di sease (table 1). The objective response rate (RR) was 7%, and the disease control rate (DCR) was 61%. The treatment regimen was not associated with the RR (p = 1.000) or the DCR (p = 0.377) (table 1). Nine patients (10%) were found to have the heterozygous BIM deletion polymorphism. No patients had homozygous BIM deletion. The BIM deletion polymorphism had no association with the basic clinicopathologic variables, including sex, age, hepatitis etiologies, tumor stages, and α-fetoprotein levels (table 1). Patients with the BIM deletion polymorphism, compared to patients without the BIM deletion polymorphism, had similar RR (11% vs. 6%, p = 0.483) and DCR (56% vs. 61%, p = 0.740) (table 1). Patients with and without the BIM deletion polymorphism had similar TTP (median 4.2 vs. 4.0 months, p = 0.981; fig. 1a), PFS (me-

Germline BIM Deletion and the Efficacy of Sorafenib

Oncology 2013;85:312–316 DOI: 10.1159/000356019

 

 

 

 

 

 

 

 

 

 

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Study Population We searched for patients who were enrolled in clinical trials and receiving first-line sorafenib-containing systemic therapy for advanced HCC from April 2007 to December 2011 at the National Taiwan University Hospital (NTUH), Taipei, Taiwan. All patients who consented to providing peripheral blood samples were included in the current study. Basic tumor characteristics, liver reserve, objective tumor response, treatment duration, date of disease progression, and survival data were retrieved from the original clinical trial records. The patients consented to participating in this study, which was approved by the Institute Research Ethical Committee of NTUH.

BIM deletion (–) BIM deletion (+)

80 TTP probability (%)

Color version available online

100

60 40

p = 0.981

0 0

6

12

18

a

24

30

36

42

48

54

60

Time (months)

100

BIM deletion (–) BIM deletion (+)

80 PFS probability (%)

between patients with and without the BIM deletion polymorphism All

Total

20

60 40 20

p = 0.857

0

6

12

18

b

24

30

36

42

48

54

60

Time (months)

100

BIM deletion polymorphism

89

Basic clinicopathologic variables Gender Female Male Mean age, years 55.8 HBsAg positive 68 (76) Anti-HCV positive 18 (20) Macrovascular invasion 51 (57) Extrahepatic metastasis 57 (64) AFP >400 ng/ml 43 (48) BCLC stage B C AJCC stage II III IV Treatment response Objective tumor responsec 6 (7) 54 (61) Disease controld

0

yes

no

9

80

p valuea

0.225 2 (22) 7 (8) 52.7 7 (78) 1 (11) 6 (67) 6 (67) 6 (67)

7 (9) 73 (91) 56.2 61 (76) 17 (21) 45 (56) 51 (64) 37 (46)

0.438b 1.000 0.680 0.727 1.000 0.305 0.590

0 (0) 10 (13) 9 (100) 70 (88) 0.662 0 (0) 6 (8) 3 (33) 21 (26) 6 (67) 53 (66) 1 (11) 5 (6) 5 (56) 49 (61)

0.483 0.740

Values are presented as numbers (%) unless otherwise stated. AFP = α-Fetoprotein; BCLC = Barcelona Clinic Liver Cancer; AJCC = American Joint Committee on Cancer. a Fisher’s exact test. b Independent t test. c Complete response + partial response. d Complete response + partial response + stable disease.

BIM deletion (–) BIM deletion (+)

80 OS probability (%)

Table 1. Basic demographic data of the patients and comparisons

dian 4.2 vs. 3.7 months, p = 0.857; fig. 1b), and OS (median 8.1 vs. 7.4 months, p = 0.762; fig. 1c). After adjusting for treatment regimens and basic clinicopathologic variables in multivariate analyses, the BIM deletion polymorphism was still not a predictor for TTP, PFS, or OS (table 2).

60 40 20

p = 0.762

0 6

12

18

24

30

36

42

48

54

60

Fig. 1. Kaplan-Meier analysis of the TTP (a), PFS (b), and OS (c)

of patients receiving first-line sorafenib-containing chemotherapy for advanced HCC with or without the BIM deletion polymorphism. p values were determined using the log-rank test.

314

Discussion

Time (months)

Oncology 2013;85:312–316 DOI: 10.1159/000356019

Ng et al. [2] demonstrated that the BIM deletion polymorphism was sufficient to confer resistance to imatnib and gefitinib in CML and EGFR-mutated NSCLC cell lines, respectively. Moreover, Ng et al. [2] showed that patients with CML and EGFR-mutated NSCLC harboring the germline BIM deletion polymorphism experiShao /Chang /Huang /Hsu /Cheng  

 

 

 

 

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0

c

Table 2. Cox’s proportional hazards model for potential predictors of TTP, PFS, and OS

Covariates

BIM deletion Treatment regimena Male Age HBsAg positive Anti-HCV positive Macrovascular invasion α-Fetoprotein >400 ng/ml Extrahepatic metastasis ECOG PS ≥1 CLIP >3

TTP

PFS

OS

p

HR

95% CI

p

HR

95% CI

p

HR

95% CI

0.963 0.017 0.047 0.016 0.786 0.406 0.487 0.685 0.019 0.972 0.123

1.019 2.136 2.633 0.971 1.088 0.721 1.205 1.111 1.972 1.011 1.895

0.5 – 2.3 1.1 – 4.0 1.0 – 6.8 0.9 – 1.0 0.6 – 2.0 0.3 – 1.6 0.7 – 2.0 0.7 – 1.8 1.1 – 3.5 0.5 – 1.9 0.8 – 4.3

0.706 0.038 0.054 0.030 0.385 0.815 0.213 0.832 0.055 0.650 0.034

0.866 1.867 2.299 0.975 1.298 0.921 1.372 1.052 1.649 0.873 2.185

0.4 – 1.8 1.0 – 3.4 1.0 – 5.4 1.0 – 1.0 0.7 – 2.3 0.5 – 1.8 0.8 – 2.3 0.7 – 1.7 1.0 – 2.7 0.5 – 1.6 1.1 – 4.5

0.309 0.942 0.281 0.907 0.210 0.982 0.032 0.230 0.495 0.980 0.001

0.668 1.021 1.642 0.999 1.530 0.992 1.798 1.368 1.192 1.007 3.441

0.3 – 1.5 0.6 – 1.8 0.7 – 4.0 1.0 – 1.0 0.8 – 3.0 0.5 – 2.0 1.1 – 3.1 0.8 – 2.3 0.7 – 2.0 0.6 – 1.8 1.7 – 7.2

Hazard ratios represent the comparative risks of developing the indicated events. HR = Hazard ratio; CI = confidence interval; BCLC = Barcelona Clinic Liver Cancer; PS = performance status. a Sorafenib vs. sorafenib + tegafur/uracil.

enced significantly inferior responses to kinase inhibitors compared to those without the polymorphism [2]. In the current study, we could not find such an association in patients who were treated with sorafenib for advanced HCC. The BIM deletion polymorphism was not correlated with other clinicopathologic variables of HCC. The polymorphism rate we found (10%) was similar to that of the Singaporean and Malaysian cohort reported by Ng et al. [2]. Based on the findings of Ng et al. [2] and us, the prediction of efficacy by BIM deletion appears restricted to cancers with oncogene addiction that have been treated with the corresponding targeted agents. The chimeric Bcr-Abl protein and the mutated EGFR are well-established addicted oncogenes that drive the growth of CML and NSCLC cells, respectively [9–11]. In another study of cancer cell lines with oncogene addiction, Faber et al. [12] demonstrated that higher basal expression levels of the proapoptotic Bim isoform were associated with more robust apoptosis induced by inhibitors of EGFR, human EGFR 2 (HER2), and phosphatidylinositol 3-kinases (coded by PIK3CA) in EGFR-mutant, HER2-amplified, and PIK3CA-mutant cell lines, respectively [12]. The data presented here may suggest that the two pharmacologic targets of sorafenib, i.e. VEGFR and B-Raf, are not the only drivers of the carcinogenicity of HCC. Clinically, the efficacy of sorafenib for advanced HCC, including an objective RR of 2–3% and a survival prolongation of 2–3 months, is not as overwhelming as the efficacy of other

kinase inhibitors for cancers with oncogene addiction [3, 4, 8, 13]. One limitation of our study is that some patients received metronomic chemotherapy along with sorafenib. However, we did not find any direct association or interaction between the different treatment regimens and the predictive values of BIM deletion. When we adjusted all clinicopathologic variables including the treatment regimen, the BIM deletion polymorphism still failed to predict the efficacy of sorafenib for advanced HCC. Although this was a retrospective study, data about tumor response and TTP were prospectively collected because all patients were enrolled in clinical trials. Collectively, although the germline BIM deletion polymorphism may serve as a biomarker for the response of imatinib to CML and gefitinib to EGFR-mutated NSCLC, respectively, the current report indicates that the germline BIM deletion polymorphism does not predict the efficacy of sorafenib for advanced HCC.

Germline BIM Deletion and the Efficacy of Sorafenib

Oncology 2013;85:312–316 DOI: 10.1159/000356019

Acknowledgement

315

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This study was supported by grants from the NTUH (NTUH.101-N1965) and the National Science Council, Taiwan (NSC-101-2314-B-002-141, 102-2314-B-002-120, and 100CAP 1020-2).

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The germline BIM deletion polymorphism is not associated with the treatment efficacy of sorafenib in patients with advanced hepatocellular carcinoma.

A germline BIM deletion polymorphism has been proposed to predict a poor treatment efficacy of certain kinase inhibitors. The current study aimed to e...
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