In addition to ERCC1, there are several other phar­macogenomic markers that could have prognostic impact, although they act through different mechanisms. Angio­genesis plays an important role in tumor growth and has attracted interest as a potential therapeutic target.9 Studies have shown that 14-3-3 sigma (14-3-3σ) protein levels are more highly expressed in human lung cancers compared to normal tissues.

The aim of this study was to correlate clinical factors and pharmacogenomic markers, such as ERCC1 and 14-3-3σ, with overall survival in patients with advanced stage NSCLC and also to determine the feasibility of utilizing pharmacogenomic markers as prognostic markers and to determine their value for incorporation into future treatment protocols.


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Materials and Methods

Fifty patients with advanced stage NSCLC were included in the study in the period between January 2007 and December 2008. We included patients with advanced stage NSCLC (recurrent NSCLC or stage III or IV NSCLC; squamous, adenocarcinoma, or large cell undifferentiated NSCLC) with adequate tissue to undergo further analysis.

Most of the patients with advanced stage were only diagnosed with fine needle aspiration or small biopsies. Three patients had surgical resection, followed by adjuvant systemic chemotherapy, however, were included in the study as they became progressive with recurrent advanced NSCLC.

Pharmacogenomic testing was carried out in the majority of the 50 patients using the biopsy samples of the advanced disease, but for patients who had prior surgical resection, the available surgically resected tumor was tested. Five patients received radiation for limited periods of time.

All patients had chest X-ray and a computed tomography (CT) scan of the chest and upper abdomen before entry into the study and underwent repeated evaluation at least every 6 weeks. All patients gave signed informed consent, and the study was approved by the institutional ethics review boards. Archival tumor specimens from each patient were retrieved from the pathology core facility after review of the H&E (Hematoxylin and eosin) stained slides.

This was a retrospective study, approved by the University of Cincinnati Institutional Review Board, in which 50 patients with stages III and IV NSCLC from the University Medi­cal Center and the VA medical center were included between 1998 until 2008.

The patients’ medical records were reviewed and their gender, race, PS, chemotherapy regimens, and phar­macogenomic marker levels were documented and evaluated for correlation with survival. Pharmacogenomic markers were measured by pathologists at the University of Cincinnati.

The evaluation was blinded and they used immunohistochemical (IHC) staining and included: ERCC1, vascular endothelial growth factors (VEGF) and VEGF receptor, nuclear fac­tor kappa beta (NF-kB [p65]), 14-3-3σ, and phosphorylated AKT (pAKT) and PTEN.

Immunohistochemical staining. IHC staining was performed using Vectastain Elite ABC universal staining kit (Vector Laboratories, CA, USA) according to the manufac­turer’s protocol.

Paraffin blocks of stage III and IV NSCLC were sectioned at 4 microseconds and de-waxed for 2 hours at 65 °C. Sections were boiled at 95 °C in antigen demasking buffer (10 mM sodium citrate at pH 6.0) for 15 minutes. These sections were further incubated with 3% H2O2 in 100% methanol to quench the endogenous peroxidase activity and blocked with 10% goat or horse serum for 2 hours.

The treated sections were incubated overnight at 4 °C with anti-VEGF (A-20) rabbit polyclonal at 1:100 dilution; anti-p65 (C-20) rabbit poly­clonal at 1:100 dilution; anti-14-3-3σ (N-14) goat polyclonal at 1:500 dilution (Santa Cruz Biotechnology, CA, USA); anti-VEGFR2 (KDR-55B11) rabbit monoclonal at 1:250 dilu­tion; anti-pAKT (ser 473) rabbit monoclonal at 1:100 dilution (Cell Signaling, MA, USA); anti-PTEN rabbit polyclonal at 1:100 dilution (Abcam, MA, USA); and anti-ERCC1 (8F1) rabbit polyclonal at 1:200 dilution (Thermo Fisher Scientific, CA, USA). Peroxidase staining was revealed using a DAB-enhanced liquid substrate system (3,3′-diaminobenzidine tetrahydrochloride) (Sigma, MO, USA).

Stained sections were counterstained using Mayers hematoxylin. Results were interpreted independently by two pathologists and graded by the following criteria: Grade 0 (negative or non-reactive expression), Grade 1 (weak or focal expression), Grade 2 (moderate expression), and Grade 3 (strong or diffused expression).

Grades #5% were considered negative. All tumor samples were graded using normal samples as a control. Figures 1A and 1B demonstrate ERCC1-positive and -negative expressions, respectively.

Statistical Analysis

The main objective of this study was to identify molecular determinants of clinical outcomes related to cancer prognosis and survival. The primary outcomes were overall survival and progression-free survival.

The expression rates of the protein markers VEGF receptor and 14-3-3σ, as determined by IHC labeling, were treated as continuous variables, while the remaining markers (VEGF, P65, PTEN, pAKT, and ERCC1) were treated as ordinal categorical variables. Cells with protein expression rates above 0 (VEGF receptor and 14-3-3σ) or stained as 1+ or 2+ (VEGF, P65, PTEN, pAKT, and ERCC1) were considered positive. Cells with 0 expression or stained as 0+ were considered negative for the marker.

Overall survival was defined as the time from the date of diagnosis to the time of death from any cause or the time at the last follow-up. Progression-free survival was defined as the duration between the date of diagnosis and disease progression or the last follow-up.

The Kaplan–Meier method was used to estimate the distribution of survival time of the patients, overall and after stratifying by race and gender. Univariate analysis was conducted to examine the survival curves across the protein expression levels of the molecular markers (classified as positive and negative) using the log rank test.

In order to determine the joint effect of the predictors (age, gender, race, type of chemotherapy, and protein expression of each marker) on the overall survival and progression-free survival of the patients, a Cox regression model was used. Covariates were chosen using the backward selection technique at the 10% significance level.

The association between the molecular markers and demographic and clinical parameters was examined. Comparisons of categorical data were performed using the Chi-square test or Fisher’s exact test.

The Wilcoxon two-sample test with t approximation was used to compare continuous data that were not normally distributed. Continuous variables, including the overall survival and progression-free survival, were correlated with expression levels of the markers using Spearman correlation coefficients. A two-tailed P value ≤0.1 was considered significant in this preliminary analysis.

Results

Fifty patients with histologically proven NSCLC of advanced stage (stage III or IV) were included in our study. Forty patients received a platinum-based chemotherapy regimen, while 10 received non-platinum based regimens. Median age was 65 years (range 42–84); age did not correlate with OS (Overall Survival) (P = 0.24). ECOG PS was 0 in 8 patients (16%), 1 in 29 patients (58%), 2 in 7 patients (14%), and 3 in 6 patients (14%).

ECOG PS of 0 and 1 were significantly associated with higher overall survival (P = 0.004). Females had significantly longer survival compared to males (31.7 months versus 12.4 months, P = 0.04) as shown in Figure 2 and Table 1.

Caucasians had better overall survival in comparison to African Americans with median survival of 14.8 months versus 10.4 months, although this did not reach statistical significance (P = 0.1) as shown in Table 1.

Patients treated with gemcitabine therapy had a median survival of 19 months versus 13 months in the non-gemcitabine therapy group (P = 0.003); patients treated with platinum-based therapy had a median survival of 15 months versus 8 months in the non-platinum based therapy group, but this did not reach statistical significance. 

However, in patients treated with platinum, ERCC1 negativity was strongly associated with longer survival (P = 0.007) as shown in Figure 3.

Higher expression of the VEGF receptor was associated with improved overall survival of 14 months versus 9.7 months with lower expression, but the difference did not reach statistical significance. 14-3-3σ, pAKT, and PTEN tissue expression did not correlate with overall survival.

NF-kB (p65) expression was positive in 4 out of 46 patients’ lung cancer tissue. The positivity of NF-kB (p65) was associated with shorter survival of 9.9 months versus 13 months in patients with negative expression of the same receptor, with only a statistical trend of P = 0.1 (see Table 1).