The Prognostic Role of the Class III β-Tubulin in Non-Small Cell Lung Cancer (NSCLC) Patients Receiving the Taxane/Vinorebine-Based Chemotherapy: A Meta-Analysis

Background A number of studies have examined the relationship between the expression of the class III β-tubulin (TUBB3) and the treatment responses to the taxane/vinorebine-based chemotherapy in patients with non-small cell lung cancer (NSCLC). However, the results of these studies were inconsistent and inconclusive. Therefore, we conducted an up-to-date meta-analysis to evaluate the prognostic role of TUBB3 in the taxane/vinorebine-based chemotherapy. Methods A literature search for relevant studies was conducted in PubMed, Embase, and CNKI. The inclusion criteria were the taxane/vinorebine-based chemotherapy in patients with NSCLC and the evaluation of the clinical outcomes in relation to the expression of TUBB3. The clinical outcomes analyzed in this study included the overall response rate (ORR), overall survival (OS), and event-free survival (EFS). Odds ratio (OR) or hazard ratio (HR) with 95% confidence interval (CI) were calculated to assess the risk associated with the TUBB3 expression in the taxane/vinorebine-based chemotherapy. Results A total of 28 studies with 2401 NSCLC patients were qualified for this meta-analysis. We found that the positive or high level of TUBB3 expression was associated with a poorer ORR (OR = 0.24, 95% CI = 0.16–0.36, p<0.001), an unfavorable OS (HR = 1.52, 95% CI = 1.27–1.82, p<0.001), and a worse EFS (HR = 1.47, 95% CI = 1.24–1.74, p<0.001) compared to the negative or low level of TUBB3 expression. The statistically significant associations between TUBB3 and chemotherapy responses were also observed in the stratified subgroup analysis, which included the analysis by ethnic subgroup (Asian and Caucasian), chemotherapy regimen (taxane-based and vinorebine-based), TUBB3 detection method (IHC and PCR), and treatment strategy (surgery plus adjuvant chemotherapy and palliative chemotherapy). Conclusions The expression level of TUBB3 may be a useful biomarker to predict the clinical outcomes of the taxane/vinorebine-based chemotherapy in patients with NSCLC.


Introduction
Lung cancer is the most common type of cancer and a leading cause of cancer-related death for both men and women worldwide [1]. The non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancer cases. Two-thirds of NSCLC cases are diagnosed at advanced stages because the patients are typically asymptomatic at early stages [2]. Due to the recent advancements in the surgical techniques and chemoradiation therapy, the one-year relative survival rate for lung cancer has increased from 35.7% to 44.5% [3]. However, the prognosis of lung cancer, especially for the advanced stage of NSCLC, is still poor. The high morbidity and mortality rates of lung cancer has continued to be a major public health concern worldwide [4].
Approximately half of NSCLC patients receive chemotherapy as part of their treatments, indicating that chemotherapy has become a common treatment method for NSCLC [5]. However, chemoresistance has emerged to be a major problem that has greatly limited the benefits of chemotherapy in patients with NSCLC [5,6]. The clinical outcomes of chemotherapy are usually very heterogeneous and unpredictable even in NSCLC patients with similar clinical and pathologic features [7]. In addition, chemoresistance results in a waste of public health budget and makes patients suffer from unnecessary adverse effects of chemotherapy [8]. Since genetic factors may play an important role in the development of chemoresistance, it would be of great value to identify useful biomarkers that can predict the clinical outcomes of chemotherapy in NSCLC [8].
Tubulin-binding agents (TBAs), such as taxanes (paclitaxel, docetaxel) and vinca alkaloids (vinorelbine, vincristine), have been widely used in the treatment of NSCLC [9][10][11]. These agents block cell division by inhibiting the mitotic spindles. Previous in vitro studies have shown that high level of class III b-tubulin (TUBB3) expression was associated with chemoresistance to paclitaxel, docetaxel, and vinblastine [12]. On the basis of these preclinical results, several studies have investigated the clinical role of TUBB3 in various human cancers, mostly in NSCLC [12][13][14][15]. To date, a large number of clinical studies have examined the relationship between the expression of TUBB3 and the clinical outcomes of taxane/vinorebine-based chemotherapy in patients with NSCLC. However, the results were still inconclusive . Some studies showed a significant association between TUBB3 and taxane/vinorebine-based chemotherapy, while the others found no correlation. Considering the relatively small sample size and the limited statistical power of an individual study, it is necessary to conduct a comprehensive up-to-date meta-analysis to evaluate the association between the expression of TUBB3 and the clinical outcomes of chemotherapy, such as the objective response rate (ORR), overall survival (OS), and event-free survival (EFS) in NSCLC patients receiving the taxane/vinorebine-based chemotherapy.

Literature search strategy
Relevant studies published before August 2013 were retrieved from online databases, including the PubMed, EMBASE, and China National Knowledge Infrastructure (CNKI) using the following terms and combinations: ''class III b-tubulin'' or ''tubulin'' or ''TUBB3'' or ''lung cancer'' or ''neoplasm, lung''. The search was limited to full-text papers written in English or Chinese. Furthermore, the references of the retrieved studies were manually screened for additional relevant studies. If necessary, the authors of the original articles were contacted for additional data, such as the relationship between the expression level of TUBB3 and the ORR, OS, and EFS.

Inclusion and exclusion criteria
Eligible studies were identified according to the following criteria: (1) human-based investigations; (2) pathologically confirmed non-small cell lung cancer; (3) articles published in English or Chinese; (4) taxane/vinorebine-based chemotherapy treatment; (5) investigation of the association between the expression of class III b-tubulin and the clinical outcomes of chemotherapy, including the ORR, OS, and EFS (including progression-free survival (PFS), disease-free survival (DFS), and recurrence-free survival (RFS)); (6) the full-text of the published articles were available. The exclusion criteria were as the follows: (1) patients younger than 18 years old; (2) studies in which necessary data were not provided; (3) for overlapped studies, the studies with a smaller dataset were excluded.

Data extraction
Two investigators (YL Yang and XP Luo) independently extracted the following information from the qualified publications: surname of the first author, publication year, country, ethnicity, sample size, disease stage, ECOG performance status, chemotherapy regimen, detection method of TUBB3, and clinical outcome (ORR, OS, or EFS). All data were then examined by two investigators independently (YL Yang and XP Luo). Disagree-ments between the investigators were resolved by discussion. When necessary, a third investigator (L Xian) helped to reach a consensus with all investigators.

Quality Assessment
The quality of the methodology of the included studies was assessed by the Newcastle-Ottawa scale (NOS) recommended by the Cochrane Non-Randomized Studies Methods Working Group [44]. Studies with five or more stars were defined as high quality studies. Quality assessment was performed by two investigators (YL Yang and XP Luo) independently. Disagreements were resolved by discussion.

Statistical analysis
The ORR was assessed by the Response Evaluation Criteria In Solid Tumors (RECIST) criteria [45] or the World Health Organization (WHO) criteria (Good response = complete response + partial response; poor response = stable disease + progressive disease) [46]. The HRs and their 95% CIs for the OS and EFS were directly extracted from reports, or indirectly estimated from the Kaplan-Meier curves, or calculated by methods described by Tierney [47]. The association between the expression of TUBB3 and the clinical responses to the taxane/ vinorebine-based chemotherapy was evaluated by the odds ratio (OR) with 95% CIs in the following comparison: high or positive expression of TUBB3 vs. low or negative expression of TUBB3. For the OS and EFS, the pooled HRs and 95% CIs were calculated from the HRs and 95% CIs extracted from each eligible study. Heterogeneity between studies was detected by the Q test and the I 2 metric (no heterogeneity: I 2 = 0%-25%; moderate heterogeneity: 25%-50%; large heterogeneity: 50%-75%; and extreme heterogeneity: 75%-100%) [48]. A fixed effect model analysis was performed when p$0.10 in the Q test or when I 2 ,50% [49], otherwise a random effect model analysis was conducted [50]. Subgroup analyses by ethnicity (Asian or Caucasian), chemotherapy regimen (taxane alone, vinorebine alone, or taxane and vinorebine combined), TUBB3 detection method (IHC, PCR, or Western blot), and treatment strategy (surgery plus adjuvant chemotherapy or palliative chemotherapy) were also performed. Publication bias were tested by the Begg's funnel plot [51] and the Egger's test [52]. All p values were twotailed and a p value less than 0.05 was considered statistically significant. Most of the statistical analyses in this study were conducted by the STATA software (version 11.2; StataCorp, College Station, Texas USA).

Publication bias
Publication bias was evaluated by the funnel plots qualitatively and tested by the Begg's and the Egger's tests quantitatively. Visual inspection of the Begg's funnel plots did not show significant evidences of asymmetry in the ORR, OS, and EFS (figure not shown), suggesting no publication bias in this meta-analysis. In addition, no significant bias was found in the Begg's test and the Egger's test. For the Begg's test, P was 0.17, 0.48, and 0.16 for the ORR, OS, and EFS respectively; for the Egger's test, P was 0.20, 0.07, and 0.06 for the ORR, OS, and EFS respectively (figure not shown).

Discussion
In this meta-analysis, we explored the predictive role of TUBB3 in patients receiving the taxane/vinorebine-based chemotherapy. We found that the high level of TUBB3 expression was associated with a lower objective response rate (ORR), a shorter overall survival (OS), and a worse event-free survival (EFS) compared to the low level of TUBB3 expression. Consistently, a previous metaanalysis by Zhang et al. [54] reported that TUBB3 was a biomarker for the sensitivity of paclitaxel/vinorebine-based chemotherapy in patients with NSCLC.
We think it is necessary to conduct an updated meta-analysis to re-evaluate the association between the expression of TUBB3 and the efficacy of taxane/vinorebine-based chemotherapy for the following reasons. First of all, the latest studies included in the previous meta-analysis by Zhang et al. [54] were published before 2009. There have been a number of studies with large patient sample size being published since 2009. In fact, our study has a much larger sample size (2401 patients from 28 studies) compared to the study by Zhang et al. (552 patients from 10 studies), which gives more reliable results in our analysis. In addition, Zhang and colleagues only examined the objective response rate and the median survival time in their analysis. In our study, we used the objective response rate, overall survival, and event free survival as the primary parameters to assess the association between the expression of TUBB3 and the clinical outcomes of taxane/ vinorebine-based chemotherapy. Because a low ORR suggests tumor resistance to the chemotherapy regimen and a short OS/ EFS indicates a poor prognosis, it is necessary to include all three parameters in order to make a comprehensive assessment about the treatment response to a chemotherapy regimen [55]. In our analysis, we demonstrated that the high level of TUBB3 expression was associated with a lower ORR, a shorter OS, and a worse EFS, which strongly supported that TUBB3 had a prognostic value in predicting the treatment response to the taxane/vinorebine-based chemotherapy. Furthermore, we performed a subgroup analysis by the TUBB3 detection method and treatment strategy, which were not done in the study by Zhang and colleagues.
Microtubule is composed of polymers of tubulin dimers and plays an important role in the development and maintenance of cell polarity, vesicle and organelle transportation, cellular signaling, and cell division. Tubulin and microtubules are the main targets of the vinca alkaloids. Vinorelbine binds to the b-subunit of tubulin dimers at a distinct region called the vinca-binding domain. In contrast, paclitaxel binds to b-tubulin within the lumen of microtubule. This binding event affects a protein loop, called the M-loop, which is thought to stabilize the lateral interactions between the adjacent protofilaments of microtubule [12,57]. Binding of taxane/vinorebine inhibits the association/dissociation of tubulin dimers in microtubule and thus disrupts the spindle dynamics, resulting in cell cycle arrest in the transition from metaphase to anaphase and eventually causes apoptotic cell death [56]. However, the molecular mechanism underlying the association between the expression level of TUBB3 and the sensitivity of taxane/vinorebine chemotherapy remains an unanswered question. Further studies are needed to address this question.
Our meta-analysis showed that the overexpression of TUBB3 may be an important factor for the development of chemoresistance to taxane/vinorebine. Hence, the expression level of TUBB3 may serve as a useful biomarker in the individually-tailored personalized chemotherapy in the future. The patient with low level of TUBB3 may benefit from the taxane/vinorebine-based regimens, whereas those with high level of TUBB3 should avoid the taxane or vinorebine-based chemotherapy and replace them with chemotherapy drugs that can overcome chemoresistance, such as ixabepilone [57].
There are some limitations in our meta-analysis. Firstly, the studies included in this meta-analysis had different study designs, such as the patient selection criteria and chemotherapy protocols. Some studies included radiotherapy in addition to the chemotherapy while the others did not. These differences could contribute to the heterogeneity among those studies.
Secondly, some factors such as age, sex, smoking history, histology type, tumor stage, and treatment method may affect the prognosis in patients receiving chemotherapy treatment. However, we could not conduct a stratified analysis to assess the effects of confounding factors on the predictive role of TUBB3 in NSCLC patients because of the limited information provided in the original publications.
Moreover, the HRs and their 95% CI we extracted from the time-to-event data were not consistent. We have to estimate the HRs by reading the Kaplan-Meier curves because some studies did not report the HRs. Some studies reported the unadjusted HRs while the others provided the adjusted HRs. Moreover, the cofounders they adjusted were not the same for the adjusted HRs. All of these factors more or less contributed to the heterogeneity in this study.
At last, potential publication biases may exist. Articles were not written in English or Chinese and studies failed to get published because of negative or null results cannot be identified in our literature search and thus were not included in this analysis. In addition, some reports did not provide sufficient data were also excluded from our analysis.
In conclusion, our meta-analysis indicated that the expression of TUBB3 may be a useful biomarker to predict the clinical outcomes of taxane/vinorebine-based chemotherapy in patients with NSCLC. With the limitations, heterogeneities, and bias of meta-analysis, our conclusions in this study need to be interpreted with caution. Future large prospective studies with rigorously designed methodology are warranted to confirm our results.