Clinical significance of detecting survivin-expressing circulating cancer cells in patients with non-small cell lung cancer

Abstract

We previously demonstrated that the detection of circulating cancer cells (CCC) expressing survivin mRNA could provide valuable information for predicting metastasis and recurrence in breast cancer. The objective of this study was to investigate the significance of detecting survivin-expressing CCC on the clinical outcomes of patients with non-small cell lung cancer (NSCLC). Peripheral blood samples collected from 143 NSCLC patients and 177 healthy volunteers were quantitatively evaluated using a technique developed in our laboratory that detected reverse transcription-polymerase chain reaction (RT-PCR) products based on a hybridisation-enzyme linked immunosorbant essay (ELISA), which we called RT-PCR ELISA. The presence of survivin-expressing CCC was detected in 63 cancer patients (44.1%) and was significantly associated with pathological T classification, nodal status, and disease stages (all P < 0.001). During a follow-up period of 36 months, patients who had positive survivin expressions at the time of the initial assay test had a higher relapse rate and shorter survival time when compared to those who had negative survivin expressions (all P < 0.001). Through multivariate analysis, the detection of survivin-expressing CCC was found to be an independent predictor for cancer recurrence (HR = 43.5; 95% CI = 2.67–70.9; P = 0.008) and survival (HR = 1.35; 95% CI = 1.02–4.31; P = 0.049). Thus, detection of survivin-expressing CCC could be used in the prediction of disease recurrence as well as in the prognosis of NSCLC.

Introduction

Lung cancer is one of the most common causes of cancer-related deaths around the world with non-small cell lung cancer representing approximately 80% of the cases [1], [2]. Despite advances made in cancer diagnosis and treatment over the last few decades, the prognosis of NSCLC still remains poor, with a 5-year overall survival rate of less than 15% in many countries [1], [3].

The current staging method for lung cancer employs the American Joint Committee on Cancer Tumour-Node-Metastasis (TNM) system, which classifies patients into groups based on the extent of cancer. This system relies heavily on the pathological evaluation of the primary tumour (T), regional nodes (N), and distant metastases (M). Using such an evaluation, approximately one third of NSCLC patients presented with early stages of the disease are considered amenable to potentially curative resection and multimodality therapy [4]. However, even in the early stages of the disease, 30% of patients suffer from relapse and die within 5 years of surgery [4]. These statistics indicate that the current staging method lacks the sensitivity necessary to separate patients into low-risk and high-risk groups in terms of recurrence and the need for adjuvant therapy. Therefore, there is a need to look for other novel prognostic factors.

Metastatic spreading through blood vessels is the most important factor affecting the prognosis of patients with primary carcinomas such as breast, colon or lung. The metastatic process is a complex cascade of events in which the tumour cells at the primary site must first erode the basement membrane, and then penetrate a blood vessel to finally spread to distant sites [5]. In this regard, detection of cancer cells in the blood could be important in identifying cancer patients at high risks of relapse.

Studies have reported the presence of epithelial cancer cells in the peripheral blood of patients with NSCLC [6]. RT-PCR and variations of this technique have been used to detect tumour markers that indicate the presence of circulating cancer cells in the blood [7]. The development of such diagnostic tests to detect circulating cancer cells in the blood has been shown to be feasible in a number of studies [8], [9], [10], [11], [12], [13], [14], [15], [16]. However, these studies have also revealed a major problem in that some tumour markers are expressed in the normal cells of the peripheral blood and frequently in normal epithelial cells as well. This inevitably leads to false-positive results that greatly diminish the correlation between tumour markers and some of the well-known clinical and pathological prognostic factors. In order to avoid false-positive results, careful selection must be made on a particular tumour marker that is uniquely expressed in lung cancer cells but not in normal cells.

Survivin, a novel inhibitor of the apoptosis protein family, has been found to be expressed in tissues during fetal development and selectively to cancer cells in many common human neoplasm [17]. Functionally, survivin has been shown to inhibit apoptosis, promote cell proliferation and enhance angiogenesis. Consistent with its role in these processes, survivin has been shown to play a key role in cancer progression [18]. Survivin was also demonstrated to be involved in tumor cell resistance to anticancer agents and ionizing radiation [19]. Recently, extensive investigation has been conducted to examine the role of survivin as a potential therapeutic target [20]. Moreover, survivin has been suggested as an ideal tumour marker in the diagnosis and prognosis of many common cancers [21].

In NSCLC, reports reveled that survivin gene transcript was either identified or shown to be massively elevated in a majority of NSCLC cases [22], [23]. Although the clinical prognostic value of survivin on the survival of NSCLC patients remains controversial [24], several studies have been carried out that suggest that survivin could serve as a potential tumour marker in evaluating recurrence and prognosis of patients with NSCLC [22], [23], [25], [26], [27], [28].

In our previous study, by using the RT-PCR ELISA technique developed in our laboratory, we demonstrated that: (1) survivin-expressing CCC were detected in 50% of the peripheral blood samples from breast cancer patients, but not in the healthy women that were used as controls; (2) the presence of survivin-expressing CCC was found to be significantly associated with various clinicopathological parameters; (3) the detection of circulating cancer cells expressing survivin mRNA could provide valuable information for predicting metastasis and recurrence of breast cancer [29].

On the basis of these findings, we hypothesised that the detection of survivin mRNA in the peripheral blood might also serve as an ideal tumor marker to in identifying circulating cancer cells in patients with NSCLC. Therefore, we focused our current study on whether or not the detection of survivin-expressing CCC in NSCLC patients could be used for predicting recurrence and survival. To do so, survivin-expressing CCC were analysed in 143 patients with NSCLC using the RT-PCR ELISA technique. Their correlation with various clinicopathological parameters and their value in predicting recurrence and survival were subsequently evaluated.