Med Oncol (2014) 31:79 DOI 10.1007/s12032-014-0079-4

ORIGINAL PAPER

Nuclear survivin expression: a prognostic factor for the response to taxane–platinum chemotherapy in patients with advanced non-small cell lung cancer Yao-Kuang Wu • Chun-Yao Huang • Mei-Chen Yang • Chou-Chin Lan • Chih-Hsin Lee Err-Cheng Chan • Kuei-Tien Chen

•

Received: 17 April 2014 / Accepted: 13 June 2014 / Published online: 25 June 2014 Ó Springer Science+Business Media New York 2014

Abstract Survivin, a structurally unique protein expressed in most common human neoplasms, is thought to support cell cycle progression and suppress apoptosis. Survivin expression is highly correlated with advanced non-small cell lung cancer (NSCLC) and poor prognosis. In this retrospective study of banked pathology tissue of patients with advanced NSCLC, we tested for correlations of N-survivin expression in tumor tissues and responsiveness to treatment with platinum-based regimens containing paclitaxel or docetaxel. The 48 patients with NSCLC included 32 (66.7 %) males and 16 (33.3 %) females. Mean age at diagnosis was 59.4 years (range 36–83 years), and median follow-up time was 20.4 months (range 3.4–59.0 months). Patients with high tumor N-survivin expression had significantly better responses to taxane– platinum chemotherapy than those with low tumor N-survivin expression (P \ 0.001). Adjusted multivariate modeling found high tumor N-survivin expression to be an independent prognostic factor for a clinical response to chemotherapy (high vs. low, OR 6.14, 95 % CI 1.62–23.29; P = 0.008). Median overall survival differed significantly between those with high tumor N-survivin expression who did/did not respond to chemotherapy and Y.-K. Wu  C.-Y. Huang  M.-C. Yang  C.-C. Lan  C.-H. Lee Division of Pulmonary Medicine, Department of Internal Medicine, Taipei Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Taipei, Taiwan Y.-K. Wu  M.-C. Yang  C.-C. Lan  C.-H. Lee School of Medicine, Tzu-Chi University, Hualien, Taiwan E.-C. Chan  K.-T. Chen (&) Department of Medical Biotechnology and Laboratory Science, Chang Gung University, 259 Wen-Hua 1st Road, Kweishan, Taoyuan 333, Taiwan e-mail: [email protected]; [email protected]

between those with low tumor N-survivin expression who did/did not respond to chemotherapy (P \ 0.05). Tumor N-survivin expression shows promise as a predictive biomarker in the chemotherapy setting as a surrogate marker of high proliferation status. Keywords Non-small cell lung cancer  Survivin expression  Platinum–taxane chemotherapy  Paclitaxel  Docetaxel

Introduction Lung cancer, which accounted for 1.6 million new registered cases and 1.37 million deaths in 2008, remains a leading cause of cancer and cancer-related deaths worldwide [1]. Non-small cell lung carcinoma (NSCLC) represents approximately 85 % of all lung cancer cases, with most patients presenting with locally advanced or distant metastatic disease (stage III/IV) [2–4]. The current treatment of choice for patients with early-stage disease is surgical resection in combination with adjuvant chemotherapy. Cisplatin-based adjuvant chemotherapy, in particular, has been shown to improve 5-year survival and disease-free survival among patients with completely resected NSCLC. Nonetheless, a large number of patients diagnosed at an early stage will eventually experience disease relapse and require treatment for metastases. In advanced NSCLC, use of platinum-based doublet regimens is the accepted standard of care [5], but these regimens are associated with limited efficacy and toxicity concerns. Even with modern treatment regimens, the 5-year survival rate for all stages of NSCLC combined remains at 15 %. Thus, an urgent need exists for adjuvant therapies that can reduce the risk of relapse and improve survival.

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An increase in the understanding of the molecular biology of NSCLC is anticipated to lead to the identification of specific genetic alterations in individual cases such that more effective treatments are achieved [6]. Identification of molecular biological prognostic factors is expected to aid in a more accurate prediction of clinical outcome and reveal novel predictive factors and therapeutic targets [7]. Of current interest for lung cancer are prognostic factors associated with cell cycle progression and suppression of apoptosis. Survivin, a structurally unique member of the inhibitors of apoptosis (IAP) family of proteins, is expressed in most common human neoplasms but is not usually detected in normal tissue [8, 9]. Survivin expression is regulated in a highly cell cycle-dependent manner, with a marked increase in the protein observed during the G2M phase. A wealth of evidence favors roles for the protein in the inhibition of apoptosis, promotion of cell cycle progression, and enhancement of DNA repair in cancer cells. Survivin has been localized to the nucleus, cytoplasm, mitochondria, and extracellular space, but its specific functions in these compartments are unclear [10]. Phosphorylation, acetylation, and ubiquitination are hypothesized to regulate the anti-apoptotic and mitotic actions of the protein and its localization to the nucleus. Expression of nuclear survivin (N-survivin) is reported to have both negative and positive prognostic values in certain forms of cancer, but the prognostic value of survivin expressed in other subcellular compartments of cancer cells is currently unclear [reviewed in [11] ]. Overexpression of survivin is highly correlated with advanced NSCLC and is generally considered predictive of a poor prognosis in terms of overall survival (OS) and responses to surgery, radiation treatments, or platinum-based chemotherapies [12–19]. Paclitaxel and docetaxel, members of the taxane class of anticancer agents, have central roles in the therapy of lung cancers. These agents bind to the b-tubulin subunit of the tubulin dimer to accelerate microtubule polymerization and stabilization such that the cell cycle is arrested principally at the G2/M phase and apoptosis is triggered through a cellsignaling cascade [20]. In platinum-based doublet regimens for treatment of metastatic NSCLC, the taxanes, like other third-generation chemotherapeutic agents, have been found to be more effective in terms of response rates and survival and better tolerated than older platinum-based combinations [2]. Although the anticancer mechanisms of taxanes have been proposed to involve survivin [21, 22], no studies have been reported that evaluate the relationship between survivin expression in tumor tissues and responsiveness to treatments with taxane-containing regimens. The present retrospective study of banked pathology tissue was performed to test for potential correlations between N-survivin

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expression in tumor tissues from patients with stage IIIA to IV NSCLC and the responsiveness of these patients to treatment with platinum-based regimens containing paclitaxel or docetaxel. It was hypothesized that platinum/ taxane regimens would prove especially beneficial for patients with advanced NSCLC and high tumor N-survivin expression.

Methods Eligibility All subjects whose banked tissue was used in this retrospective study received treatment at the Taipei Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation. Trial eligibility required pathologically confirmed NSCLC; stage III A, IIIB, or IV disease; age of 18 years of greater; and adequate bone marrow, liver, and kidney function. Clinical records and histopathological diagnoses were fully reviewed and included gender, age, smoking status, histology, initial performance status (Eastern Cooperative Oncology Group Performance Status; ECOG PS), and pathologic stage, which was classified according to the seventh edition of the American Joint Committee on Cancer revised tumor-node-metastasis (TNM) system [23]. Patients receiving platinum-based taxane-containing regimens as their first treatment were eligible; those with central nervous system metastases were eligible if no immediate intervention was required or if radiation therapy was completed more than 28 days before the planned chemotherapy. Patients with prior malignancies were also eligible providing no recurrence was observed during the period of July 2004–November 2009 in the Division of Pulmonary Medicine. Informed consent was obtained in all cases from patients or family members. Treatment regimens All eligible patients were treated with standard taxane– platinum chemotherapy involving paclitaxel (50 mg/m2) or docetaxel (31 mg/m2) on days 1, 8, and 15 and cisplatin (75 mg/m2 on day 15) for each 28-day cycle. Disease response was assessed sequentially after every third cycle by computed tomography (CT) of the chest, upper abdomen, and other areas as indicated. Other imaging modalities used for disease response assessment included magnetic resonance imaging of the brain and soft tissues. Patients without disease progression received at least three additional cycles of chemotherapy. Subsequent clinical management was dependent on the judgment of the treating physician.

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Response to therapy and survival assessment To determine the response of the cancer to therapy, the size of cancer lesions (at least one and up to eight separate lesions) was measured at the site of greatest diameter using images obtained with intravenous contrast on a multichannel helical CT scanner. Measurements were performed at intervals of 12 weeks. The percentage change in the sum of tumor diameters for post- as compared to pretreatment measurements was calculated. A positive value was considered to indicate tumor shrinkage and a negative value to indicate tumor growth. The appearance of a new or previously unobserved tumor lesion in images or during the physical exam was coded as disease progression. Response categories were progressive disease (PD), stable disease (SD), partial response (PR), and complete remission (CR) according to the RECIST criteria [24]. OS was defined as the percentage of patients still alive at 5 years following the initial treatment. Specimen collection and processing Collection of tumor specimens was required prior to initiation of therapy and was performed by core needle biopsy (20-gauge needle) to obtain a specimen with a diameter of 0.8 mm. The biopsy was performed percutaneously or endoscopically under radiographic or sonographic guidance. Malignant cells were selectively procured from serially microdissected tissue sections of 10 lm and were stained with nuclear Fast Red using the Arcturus LCM system (Mountain View, CA; 60 mW, 1.5 ms, intensity 100, spot size of approximately 20 lm). Immunohistochemistry Specimens were fixed in 4 % paraformaldehyde and embedded in paraffin. Tissue sections (5 lm thick) were obtained from paraffin-embedded tissue blocks, mounted on silanized slides (Superfrost, Menzel, Braunschweig, Germany), de-paraffinized by two 10-min treatments with xylene, and rehydrated with an ethanol gradient as previously described [25]. To eliminate endogenous peroxidase activity, sections were incubated with 3 % H2O2 at room temperature for 30 min. The specimens were subsequently heated in a microwave oven for antigen retrieval (10 mmol/L citrate buffer, pH 6.0, 20 min, 700 W). To block non-specific binding, the slides were preincubated with 10 % non-immune goat serum at 37 °C for 30 min. The samples were then incubated with anti-survivin monoclonal antibody generated previously (1:1,000) for 30 min at room temperature [26]. Slides were then washed with phosphate-buffered saline (pH 7.4), incubated with horseradish peroxidase–labeled antimouse IgG secondary

Fig. 1 Immunohistochemical analysis of survivin expression in lung tumor tissues from patients with advanced NSCLC. Expression of survivin was validated by immunohistochemistry using tissue sections from formalin-fixed, paraffin-embedded lung tumor tissue samples. Representative immunohistochemical images of tumors from patients with advanced stage NSCLC are shown (magnification = 9400). a Tumor tissues with low survivin expression; b tumor tissues with high survivin expression (note the expression of survivin in both cytoplasm and nucleus)

antibody (rabbit, 1:2,000 dilution; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 30 min at room temperature, and treated with 3,30 -diaminobenzidine (Sigma, St. Louis, MO, USA) for color development. The sections were subsequently counterstained with hematoxylin, washed in running tap water, dehydrated, and mounted in Neo-Mount (Merck, Darmstadt, Germany), and observed under a microscope (E600, Nikon, Tokyo, Japan). Samples incubated without primary antibody served as controls. Determination of nuclear survivin expression score Expression of N-survivin in the original diagnostic tumor biopsy samples was scored by two independent pathologists blinded to outcomes using a simplified H scoring system [27]. This system is based on two distinct characteristics: the percentage of positivity and the intensity grade

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of N-survivin staining in the slide. Positivity was determined according to the percentage of positive tumor cells per slide as follows: 0–9 %, score = 0; 10–49 %, score = 1; 50–70 %, score = 2; 70–90 %, score = 3; [90 %, score = 4. The intensity grade of the marker was scored as follows: absence, score = 0; weak, score = 1; moderate, score = 2; strong, score = 3. The two scores were then multiplied, yielding intermediate scores ranging from 0 to 12. The final score considered for analysis was the mean score obtained by the pathologists. Representative images of nuclear and cytoplasmic survivin expression in tumor tissues from patients with advanced NSCLC are shown in Fig. 1. Statistical analysis Continuous variables were summarized by mean ± SD or by medians with inter-quartile ranges (IQR: the range between the 25th and 75th percentile) depending on normality of data. Categorical variables were expressed by frequencies and percentages. Significant differences between patients with high (scores of 7–12) and low (scores of 0–6) N-survivin expression were detected by the independent t test or Wilcoxon rank-sum test for continuous variables and by the chi-square test or Fisher’s exact test for categorical variables, as appropriate. The OS time was determined from the date of treatment to the date of death or last follow-up, and patients were censored in the OS analysis if they were alive at last visit. Survival curves were constructed by the Kaplan–Meier method with the log-rank test used to detect differences between specific groups. To identify prognostic factors for clinical responsiveness, logistic regression models were constructed to calculate crude and adjusted odds ratios (OR) with 95 % confidence intervals (CIs). In the multivariate logistic regression model, variables that were significant in the univariate analysis were candidates in the backward selection procedure wherein variables that did not improve model fit at P \ 0.1 were discarded; however, age and gender were always forced in the model for adjustment. All statistical analyses were performed using SAS software version 9.2 (SAS Institute Inc., Cary, NC, USA). A twotailed P \ 0.05 indicated statistical significance.

Results Demographic and clinicopathological characteristics A total of 48 patients with NSCLC were included in this study which was comprised of 32 (66.7 %) males and 16 (33.3 %) females. The mean age at diagnosis was 59.4 years (range 36–83 years), and the median follow-up

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time was 20.4 months (range 3.4–59.0 months). Forty-two (87.5 %) patients were deceased at the last follow-up. The baseline characteristics of the patients in this study are shown in Table 1. Most (87.5 %) patients were classified as having a tumor status of T4. The most frequently observed nodal stages were N2 (43.8 %) and N3 (39.6 %). Thirty-six (75 %) patients had distant metastases, with metastasis to the bone (54.2 %) observed most frequently. Regarding pathological stage, the majority (75 %) of patients had stage IV cancer. According to histology, 40 (83.3 %) tumors were classified as adenocarcinoma and 8 (16.7 %) were classified as squamous cell carcinoma. With regard to type of taxane administered, the percentages of subjects receiving paclitaxel (52.1 %) or docetaxel (47.9 %) were roughly comparable. More than one-third (35.4 %) of patients received six courses of chemotherapy. Forty-four (91.7 %) patients had good performance scores (0–1) according to ECOG analysis. Half (50 %) of the patients in this study were current smokers, whereas 19 (39.6 %) had never smoked. High tumor expression of N-survivin was observed for 26 (54.2 %) patients, whereas low tumor expression of the protein was observed for 22 (45.8 %) patients. No significant differences in any demographic or clinicopathological characteristic were observed between patients with high as compared to low tumor N-survivin expression (Table 1). Expression of N-survivin and response to chemotherapy Responses to taxane–platinum chemotherapy (CR, PR, SD, PD) differed significantly between patients with high as compared to low tumor N-survivin expression, with significantly better responses observed for the high expression group (P \ 0.001, Table 2). Patients with high tumor N-survivin expression also had significantly higher disease control rates when compared with patients with low tumor N-survivin expression (96.2 and 45.5 %, respectively; P \ 0.001). Furthermore, clinical response rates were significantly higher for patients with high as compared to low tumor N-survivin expression (57.7 vs. 18.2 %; P = 0.005). Prognostic factors associated with clinical response to chemotherapy Clinical response rates as a function of different characteristics of the patients in this study are presented in Table 3. Using univariate logistic regression, it was observed that patients without metastases (M0 vs. M1, OR 4.55, 95 % CI 1.13–18.32; P = 0.033), with pathological stage IIIB disease (IIIB vs. IV, OR 5.30, 95 % CI 1.15–24.42; P = 0.032), and with high tumor N-survivin expression (high vs. low, OR 6.14, 95 % CI 1.62–23.29; P = 0.008) had significantly higher clinical response rates.

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Table 1 Demographic and clinicopathological characteristics of NSCLC patients according to nuclear survivin expression Total (n = 48)

High N-survivin expressiona (n = 26)

Low N-survivin expression (n = 22)

P value

59.4 ± 11.7

61.8 ± 11.5

56.5 ± 11.5

0.113 

32 (66.7) 16 (33.3)

17 (65.4) 9 (34.6)

15 (68.2) 7 (31.8)

0.838à

T2

4 (8.3)

1 (3.9)

3 (13.6)

0.282§

T3

2 (4.2)

2 (7.7)

0 (0.0)

T4

42 (87.5)

23 (88.5)

19 (86.4)

N0

4 (8.3)

2 (7.7)

2 (9.1)

N1

4 (8.3)

3 (11.5)

1 (4.6)

N2

21 (43.8)

11 (42.3)

10 (45.5)

N3

19 (39.6)

10 (38.5)

9 (40.9)

M0

12 (25.0)

9 (34.6)

3 (13.6)

M1

36 (75.0)

17 (65.4)

19 (86.4)

Liver

4 (8.3)

4 (15.4)

0 (0.0)

0.114§

Brain Lung

7 (14.6) 12 (25.0)

3 (11.5) 4 (15.4)

4 (18.2) 8 (36.4)

0.687§ 0.094à

Bone

26 (54.2)

13 (50.0)

13 (59.1)

0.529à

IIIa

2 (4.2)

2 (7.7)

0 (0.0)

0.257§

IIIb

10 (20.8)

7 (26.9)

3 (13.6)

IV

36 (75.0)

17 (65.4)

19 (86.4)

Adenocarcinoma

40 (83.3)

21 (80.8)

19 (86.4)

Squamous cell carcinoma

8 (16.7)

5 (19.2)

3 (13.6)

Paclitaxel

25 (52.1)

13 (50.0)

12 (54.6)

Docetaxel

23 (47.9)

13 (50.0)

10 (45.5)

3

11 (22.9)

4 (15.4)

7 (31.8)

4

11 (22.9)

7 (26.9)

4 (18.2)

5 6

9 (18.8) 17 (35.4)

4 (15.4) 11 (42.3)

5 (22.7) 6 (27.3)

0

7 (14.6)

4 (15.4)

3 (13.6)

1

37 (77.1)

18 (69.2)

19 (86.4)

2

4 (8.3)

4 (15.4)

0 (0.0)

Never smoked

19 (39.6)

11 (42.3)

8 (36.4)

Ex-smoker

5 (10.4)

4 (15.4)

1 (4.6)

Current smoker

24 (50.0)

11 (42.3)

13 (59.1)

Age (years), mean ± SD Gender, n (%) Male Female Tumor status, n (%)

Nodal stage, n (%) 0.929§

Metastasis, n (%) 0.094à

Pathological stage, n (%)

Histology, n (%) 0.710§

Taxane administered, n(%) 0.780§

Cycles of chemotherapy, n (%) 0.439§

ECOGb, n (%) 0.200§

Smoking status, n (%) 0.376§

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Table 1 continued

Follow-up time (month), median (IQR) a

Nuclear expression

b

ECOG, Eastern Cooperative Oncology Group

 

Independent t test

à

Chi-square test

§

Fisher’s exact test Wilcoxon rank-sum test

}

Total (n = 48)

High N-survivin expressiona (n = 26)

Low N-survivin expression (n = 22)

P value

20.4 (10.7, 35.7)

18.6 (9.6, 30.6)

24.4 (15.4, 38.4)

0.296}

After the backward selection procedure only, age, gender, metastasis, and tumor N-survivin expression were included in the final multivariate model (Table 4). After adjustment for other variables, high tumor N-survivin expression was found to be an independent prognostic factor for a clinical response to chemotherapy (high vs. low, OR 6.14, 95 % CI 1.62–23.29; P = 0.008). Overall survival as a function of tumor N-survivin expression and clinical response to chemotherapy For the 48 patients included in this study, the median OS time was 20.4 months (95 % CI 15.7–28.5 months). Four groups of patients were examined with respect to OS time: (1) those with high tumor N-survivin expression and a clinical response to the chemotherapy (n = 15); (2) those with high tumor N-survivin expression but no clinical response to the chemotherapy (n = 11); (3) those with low tumor N-survivin expression and a clinical response to the chemotherapy (n = 4); and (4) those with low tumor N-survivin expression but no clinical response to the chemotherapy (n = 18). The OS curves differed significantly among these four groups (Fig. 2, P \ 0.001). Median OS times for these groups were as follows: (1) 30 months, (2) 8.7 months, (3) 33.2 months, and (4) 20.6 months. The difference in median OS survival time between group (1) and group (2) was significant (P = 0.013) as well as between group (2) and group (3) (P = 0.006).

Discussion The present study is the first to evaluate the prognostic value of tumor survivin expression in the response of lung cancer patients to chemotherapy including a taxane. The study was confined to subjects with advanced NSCLC and who received a platinum/taxane regimen as the first treatment for their cancer. Most (95 %) subjects had tumors

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expressing N-survivin, with approximately half having tumors with high N-survivin expression. No significant differences in any demographic or clinicopathological characteristic were observed between patients with high as compared to low tumor N-survivin expression. Responses to platinum/taxane chemotherapy (CR, PR, SD, PD) were significantly better for patients with high as compared to low tumor N-survivin expression. Following treatment with platinum/taxane regimens, patients with high tumor N-survivin expression also had significantly higher disease control rates and clinical response rates when compared with patients with low tumor N-survivin expression. Using a multivariate model following a backward selection procedure for age, gender, metastasis, and tumor N-survivin expression and adjustments for other variables, high tumor N-survivin expression was found to be an independent prognostic factor for a clinical response to taxane-containing chemotherapy. Finally, the median OS time for patients with high tumor N-survivin expression and a clinical response to the chemotherapy was found to be greater than that for patients with high tumor N-survivin expression and no clinical response to the chemotherapy. The majority of studies published to date favor the hypothesis that survivin expression in NSCLC tumors serves as an unfavorable prognostic factor in terms of OS. For example, Wang et al. [13] examined tumors from patients with stage III NSCLC and concluded that survivin expression is a valuable marker of tumor size and poor survival in these patients. A meta-analysis conducted by Huang et al. [16] led these investigators to conclude that patients carrying NSCLC tumors in which survivin is overexpressed predicts a poor outcome. Xu et al. [28] reported that expression of survivin in NSCLC tumors from Chinese subjects undergoing surgery predicts a poor prognosis. A meta-analysis performed by Zhang et al. [15] led these investigators to conclude that tumor survivin expression is a prognostic marker of advanced stage, but not early stage, NSCLC and is a pejorative prognostic

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Table 2 Expression of N-survivin and responses to chemotherapy High N-survivin expression (n = 26)

Low N-survivin expression (n = 22)

P value

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Table 3 Univariate analysis of prognostic factors associated with a clinical response to chemotherapy Clinical response rate (%)

OR (95 % CI)

P value

Response to chemotherapy, n (%) Complete response

4 (15.4)

Partial response

11 (42.3)

Stable disease

10 (38.5)

Progressive disease

0 (0.0)

\0.001 

Age (years)

4 (18.2)

0.51 (0.16, 1.66)

0.265

13/32 (40.6)

1.14 (0.33, 3.92)

0.835

6/16 (37.5)

1.00 (reference)

T2

1/4 (25.0)

0.54 (0.05, 5.66)

0.609

T3

2/2 (100.0)

NA

0.980

Female

1 (3.9)

12 (54.6)

Tumor status

25 (96.2)

10 (45.5)

No

1 (3.9)

12 (54.6)

\0.001à

T4

15 (57.7) 11 (42.3)

4 (18.2)

à

0.005

18 (81.8)

a

Disease control = complete response ? partial response ? stable disease b

Clinical response = complete response ? partial response

 

Fisher’s exact test

16/42 (38.1)

1.00 (reference)

Nodal stage

Clinical responseb, n (%)

à

1.00 (reference)

8/25 (32.0)

Male

6 (27.3)

Yes

No

11/23 (47.8)

C60 Gender

Disease controla, n (%)

Yes

\60

Chi-square test

factor in terms of OS for surgically treated NSCLC. Similarly, high tumor survivin expression is associated with poorer responses of NSCLC patients to standard platinumbased chemotherapies. For example, Karczmarek-Borowska et al. [29] observed that patients with stage IIB and III NSCLC and high tumor survivin expression exhibited poorer responses to cisplatin plus etoposide as compared to those with low tumor survivin expression. Chen et al. [30] reported that patients with advanced NSCLC and low tumor survivin expression showed a better response to cisplatin plus vinorelbine or gemcitabine than did those with high tumor expression of the protein. Similar findings were made by Dai et al. [31] of patients treated with adjuvant chemotherapy following surgery. Survivin expression is now generally accepted as a biomarker of resistance to chemotherapeutic agents and radiation treatment, regardless of cancer type [12, 14, 18, 32]. In the present study, however, high tumor N-survivin expression was found to serve as an independent positive prognostic factor for a clinical response to chemotherapy. The discrepancies between this finding and those of others regarding NSCLC and clinical responses to platinum-based chemotherapies in NSCLC [29–31] may reflect the use of non-taxane-containing chemotherapies, the presence or absence of advanced disease, measurements of survivin in

N0

0/4 (0.0)

NA

N1

1/4 (25.0)

0.37 (0.03, 4.23)

0.972 0.424

N2

9/21 (42.9)

0.83 (0.24, 2.90)

0.775

N3

9/19 (47.4)

1.00 (reference)

M0

8/12 (66.7)

4.55 (1.13, 18.32)

M1

11/36 (30.6)

Metastasis 0.033

1.00 (reference)

Pathological stage IIIa

1/2 (50.0)

2.27 (0.13, 39.73)

0.574

IIIb

7/10 (70.0)

5.30 (1.15, 24.42)

0.032

IV

11/36 (30.6)

1.00 (reference)

15/40 (37.5)

1.00 (reference)

Histology Adenocarcinoma Squamous cell carcinoma

4/8 (50.0)

1.67 (0.36, 7.67)

0.512

Taxane administered Paclitaxel

10/25 (40.0)

1.00 (reference)

Docetaxel

9/23 (39.1)

0.96 (0.30, 3.07)

0.951

Cycles of chemotherapy \5

6/22 (27.3)

1.00 (reference)

C5

13/26 (50.0)

2.67 (0.79, 8.97)

0.113

High

15/26 (57.7)

6.14 (1.62, 23.29)

0.008

Low

4/22 (18.2)

1.00 (reference)

0

4/7 (57.1)

1.00 (reference)

1

14/37 (37.8)

0.46 (0.09, 2.35)

0.348

2

1/4 (25.0)

0.25 (0.02, 3.77)

0.317

9/19 (47.4)

1.00 (reference)

N-Survivin expression

ECOGa

Smoking status Never smoked Ex-smoker Current smoker

4/5 (80.0)

4.44 (0.42, 47.50)

0.217

6/24 (25.0)

0.37 (0.10, 1.35)

0.131

OR odds ratio, CI confidence interval a

ECOG, Eastern Cooperative Oncology Group

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Table 4 Multivariate analysis of prognostic factors associated with a clinical response to chemotherapy Adjusted OR (95 % CI)

P value

Age (years) \60

1.00 (reference)

C60

0.36 (0.09, 1.47)

0.152

Male

1.50 (0.36, 6.31)

0.577

Female

1.00 (reference)

Gender

Metastasis M0 M1

3.55 (0.79, 15.84)

0.097

1.00 (reference)

N-Survivin expression High

6.16 (1.44, 26.32)

Low

1.00 (reference)

0.014

OR odds ratio, CI confidence interval

different subcellular compartments, or methodological differences. For example, the RT-PCR method used by Chen et al. [30] is likely to have detected both nuclear and cytoplasmic forms of survivin, whereas immunohistochemistry distinguishes between these two compartments. Survivin gene splice variants and polymorphisms have been identified [33–35], whether these variations influence the accuracy of current methodologies for determining survivin expression is unclear. The recent observation [19] that tumor survivin expression is an independent negative prognostic marker in NSCLC patients with surgically resected adenocarcinoma, but not squamous cell carcinoma may also explain these discrepancies. A possible functional relation between survivin and taxanes during mitosis may be relevant to the findings of this report. Survivin is a member of the chromosomal passenger complex [22] which controls many aspects of mitosis, including regulation of the mitotic spindle checkpoint and mitotic progression. The protein is required for the maintenance of the spindle assembly checkpoint arrest in the presence of taxol, and this event has been shown to be essential for cellular sensitivity to taxol [36, 37]. Accordingly, survivin has been found to be down-regulated in beta-tubulin mutant ovarian cancer cells with a compromised mitotic response to taxol [21]. Additionally, class III b-tubulin expression was recently found to be associated with the response of NSCLC patients to paclitaxel/vinorelbine-based chemotherapy [38]. Responses to chemotherapy in advanced lung cancer range from 16 to 21 % [5]. In the present study, 39.58 % of patients exhibited a clinical response to taxane-containing chemotherapy. More importantly, high tumor N-survivin expression was significantly associated with response rate. This association, which was demonstrated for a small cohort

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Fig. 2 Kaplan–Meier curves of overall survival as a function of tumor N-survivin expression and clinical response to chemotherapy. Curves are presented for four groups of patients with NSCLC: (1) those with high tumor N-survivin expression and a clinical response to chemotherapy; (2) those with high tumor N-survivin expression but no clinical response to chemotherapy; (3) those with low tumor N-survivin expression and a clinical response to chemotherapy; and (4) those with low tumor N-survivin expression but no clinical response to chemotherapy

of lung cancer patients, reinforces the value of survivin as a predictive biomarker for a clinical response of patients with NSCLC to chemotherapy containing a taxane and warrants studies in a larger study population. In the present study, high tumor N-survivin expression was particularly associated with clinicopathological characteristics of patients with a poor prognosis, i.e., those with high-grade tumors and younger patients. Patients with high-grade tumors generally have a poorer prognosis irrespective of therapy. The finding that patients with NSCLC tumors showing high N-survivin expression responded better to the chemotherapy may reflect the commonly observed sensitivity of highly proliferating cells to various chemotherapies. In the present study, expression of survival in tumor nuclei was found to be a positive prognostic factor for a clinical response to taxane/cisplatin chemotherapy in advanced NSCLC. Nonetheless, the prognostic significance of tumor N-survivin expression, as compared to expression in other subcellular compartments, for OS or responsiveness to treatments in NSCLC remains controversial. When expression and localization of tumor N-survivin in advanced NSCLC were examined in relation to clinicopathological parameters, to OS and to response to chemotherapy, localization of survivin to the nucleus was found to be a positive prognostic factor for survival [39]. By contrast, NSCLC tumor N-survivin was identified as an independent biomarker for disease recurrence and decreased OS in patients with resected stage I and II

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NSCLC [40] and was more predictive of poor survival than was cytoplasmic survivin expression in this disease [41]. Meta-analysis [42] also revealed no positivity of tumor N-survivin expression in terms of survival in NSCLC. Additional studies are needed to clarify the prognostic value of expression of survivin in tumor cell nuclei as compared to other subcellular compartments in NSCLC. No significant differences in any demographic or clinicopathological characteristics were observed between patients with high as compared to low tumor N-survivin expression in the present report. However, tumor N-survivin expression in NSCLC was found to correlate with certain clinicopathological variables in other studies. For example, positive correlations between tumor N-survivin staining and age [39, 40] and between tumor survivin expression and TNM stage and lymph node metastasis [28] have been reported. These discrepancies may reflect the presence or absence of advanced disease, different treatment modalities, measurements of survivin in different subcellular compartments, methodological differences, the absence or presence of survivin splice variants/polymorphisms, and the relative proportions of adenocarcinoma and squamous cell carcinoma in these studies. Limitations of the present study include its retrospective nature, the small cohort of subjects examined, and the lack of clarity of the mechanism(s) through which high tumor N-survivin expression renders patients with advanced NSCLC more susceptible to the effects of platinum/taxane chemotherapy. It should also be noted that the OS of patients with high tumor N-survivin expression and a clinical response to the chemotherapy did not differ significantly from that of patients with low tumor N-survivin expression regardless of response to chemotherapy. The availability of new treatments that enhance the efficacy of chemotherapies for patients with advanced NSCLC is greatly needed. In isolated lung cancer systems, down-regulation of survivin is reported to enhance the cytotoxic effects of cisplatin, paclitaxel, and docetaxel [43, 44]. Regarding the down-regulation of survivin in cancer patients, current efforts are underway to evaluate the therapeutic benefits of targeting survivin through small molecule inhibitors, molecular antagonists, vaccination-based therapies, antisense oligonucleotides, and small hairpin RNA [9, 12, 45]. Nanotherapy utilizing survivin targeting is anticipated to play a key role in the development of personalized medicine for cancer [46].

Conclusions Based on the findings of the present study, expression of N-survivin in tumors from patients with advanced NSCLC is proposed as a prognostic indicator of the response of these patients to taxane-containing chemotherapies.

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Measurement of N-survivin expression in tumors from patients with advanced NSCLC should therefore prove valuable in the selection of patients for taxane-based therapies as their initial treatment. However, larger clinical trials with widely accepted assessment methods are needed to establish the prognostic significance of tumor N-survivin expression in the response of NSCLC patients to such therapies. It is recommended that tumor N-survivin expression be incorporated as a predictive biomarker in the chemotherapy setting and be considered a surrogate marker of high proliferation status. Acknowledgments The authors are grateful to Chuen Hsueh (Pathology Core Laboratory, Molecular Medicine Research Center, Chang Gung University, Taiwan) and Yue Chung Tai (Department of Pathology, Taipei Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation.) for help with the immunohistochemical preparation and evaluation. Conflicts of interest

None.

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