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Lung Cancer 79 (2013) 180– 186

Contents lists available at SciVerse ScienceDirect

Lung Cancer

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urvivin expression impacts prognostically on NSCLC but not SCLC

ntonio Rosatoa,b,∗,1, Chiara Meninb,1, Daniela Boldrinb, Silvia Dalla Santab, Laura Bonaldib,aria Chiara Scainia, Paola Del Biancob, Davide Zardoc, Matteo Fassanc, Rocco Cappellessoc,

mbrogio Fassinac

Department of Surgery, Oncology and Gastroenterology, University of Padua, Padua, ItalyIstituto Oncologico Veneto IRCCS, Padua, ItalyDepartment of Medicine, University of Padua, Padua, Italy

r t i c l e i n f o

rticle history:eceived 15 May 2012eceived in revised form 30 October 2012ccepted 7 November 2012

eywords:SCLCCLCurvivineal-time RT-PCRNA-sequencing

a b s t r a c t

Survivin is expressed in lung cancer and in most cancer tissues and has a significant impact on prognosis.This work aimed to comparatively assess survivin expression and significance in Non-Small (NSCLC) andSmall Cell Lung Cancers (SCLC). Sixty-five NSCLC and 35 SCLC samples were analyzed by semi-quantitativereal-time RT-PCR. Survivin mRNA levels were significantly higher in tumors than in normal tissue, andin SCLC than in NSCLC samples. Immunohistochemistry and FISH analyses were performed in 59 and26 tumor specimens, respectively. In SCLC survivin was only present in cytoplasm, while in some NSCLCcases it also showed nuclear or mixed patterns. FISH analysis did not disclose survivin gene amplification,except for one NSCLC case. Finally, 90 samples were genotyped for the −31G/C SNP of survivin promoterby direct sequencing; the −31G/C SNP genotype status showed a significant association only with nodalNSCLC metastasis, but not with survivin expression in any tumor group. A better prognosis was correlated

mmunohistochemistryISH

to higher levels of survivin mRNA and to the presence of at least one G allele at −31 SNP in NSCLC, whilethese parameters did not correlate with overall survival in SCLC. Moreover, this SNP would appear tohave no effect on the risk of lung cancer in our samples.

The different prognostic role played by survivin in NSCLC and SCLC highlights the biological differencesbetween these lung tumor histotypes and stresses the need to clarify the molecular pathways leading totheir neoplastic transformation.

. Introduction

Lung cancer remains a leading cause of tumor-related deathorldwide [1–3]. According to the World Health Organization, lung

ancer has been classified into Small Cell Lung Cancer (SCLC) andon-Small Cell Lung Cancer (NSCLC) [4,5]. Surgery is the treatmentf choice for early-stage NSCLC, but nonetheless the majority of

atients present recurrence of disease [6]. Chemotherapy alone issed in SCLC, even though this tumor is often resistant to a broadpectrum of anti-cancer agents [7].

∗ Corresponding author at: Department of Surgery, Oncology and Gastroenter-logy, University of Padua, Via Gattamelata 64, I-35128, Padua, Italy. Tel.: +39 049215858; fax: +39 049 8072854.

E-mail addresses: antonio.rosato@unipd.it (A. Rosato), chiara.menin@ioveneto.itC. Menin), daniela.boldrin@yahoo.it (D. Boldrin), silvia.dallasanta@unipd.itS.D. Santa), laura.bonaldi@unipd.it (L. Bonaldi), mariachiara.scaini@unipd.itM.C. Scaini), paola.delbianco@ioveneto.it (P. Del Bianco), zardo.davide@gmail.comD. Zardo), matteo.fassan@gmail.com (M. Fassan), rocco.cappellesso@gmail.comR. Cappellesso), ambrogio.fassina@unipd.it (A. Fassina).

1 These authors contributed equally to the work.

169-5002/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.lungcan.2012.11.004

© 2012 Elsevier Ireland Ltd. All rights reserved.

Notwithstanding important progress made in molecularlytargeted therapies [8], at present, clinical and pathological char-acteristics do not predict disease outcome, and new molecularmarkers for high-risk patients recognition need to be identified [9].

Survivin is a member of the human Inhibitors of Apoptosis Pro-tein (IAP) family that plays an important role in the developmentand progression of neoplastic processes [10–13]. Its higher andoften almost exclusive expression in many tumors compared tothe corresponding normal tissues has led to its being proposed as adiagnostic marker and potential therapeutic target; moreover, sur-vivin can be also considered a prognostic marker in different cancerhistotypes [10–13]. In lung cancer, its prognostic value is still con-troversial and has been investigated mainly only in NSCLC [14–26],while data on SCLC is almost completely lacking.

Survivin gene expression might be modified by a variety offactors, with recent work focusing on single-nucleotide polymor-phisms (SNP) on its promoter region. In particular, the −31G/C SNP

was shown to influence survivin expression, thereby modulatingoverall susceptibility to cancer [27]. The role of this polymorphismin lung cancer is not well defined [28,29], and data concerningSCLC is not yet available. Based on these premises, we aimed at

Cancer 79 (2013) 180– 186 181

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Fig. 1. Survivin mRNA expression in lung tissues. (A) Comparison of normal pul-monary tissue and lung cancer (Kruskal–Wallis test, P < 0.0001). (B) Differences inexpression in NSCLC and SCLC tumors (Kruskal–Wallis test, P = 0.0012). (C) The accu-racy of survivin expression in discriminating between normal and tumor tissue was

A. Rosato et al. / Lung

valuating and comparing the role of survivin in both NSCLC andCLC, and investigating potential biological mechanisms underly-ng its overexpression.

. Materials and methods

.1. Patients

A total of 111 cases of lung cancer from patients who hadndergone surgical tumor resection or trans-bronchial biopsyetween 2002 and 2009, were selected from the files of theadua University Surgical Pathology & Cytopathology Unit. Clini-opathological characteristics of the study population are reportedn Supplementary Table 1. NSCLC histological specimens derivedrom tumor resections, while SCLC samples were obtained byrans-bronchial biopsies; 10 biopsies from healthy lung tissueesected from 10 different patients who died for extra-pulmonaryauses were used as controls. Moreover, 240 healthy blood donorsatched to cases for sex and residential area, were included as

ontrols in the SNP analysis.

.2. cDNA microarray analysis

The Oncomine database and gene microarray analy-is tool, a repository for published cDNA microarray datahttp://www.oncomine.org), was explored for survivin mRNAxpression in non-neoplastic lung tissues, NSCLC and SCLC tumors.ncomine algorithms were used to perform a statistical analysis of

he differences in survivin expression within any single study. Onlytudies with analytical results with a P < 0.05 were considered.

.3. RNA extraction and real-time PCR assay

Total RNA was extracted from 2 sections (10 �m) oformalin-fixed and paraffin-embedded (FFPE) specimen usingureLinkTM FFPE Total RNA Isolation Kit (Invitrogen, San Giu-iano Milanese, Italy), following the manufacturer’s specifications.onversion of RNA into cDNA and gene expression was quan-ified as previously reported [30] using commercial on-demandssays (Hs00153353 m1, BIRC5; Hs99999905 m1, GAPDH; Appliediosystems, Foster City, CA). The expression of survivin in eachample was determined using the comparative C(T) method alsoeferred to as the 2−��Ct method [31]; fold change values of neo-lastic samples were determined using the pool of normal lungpecimens as calibrator, after normalization with GAPDH as inter-al control reference gene.

.4. Immunohistochemistry (IHC)

Immunohistochemistry was performed with an anti-survivinouse monoclonal antibody (clone 8E2, Novus Biologicals, Little-

on, CO, USA; 1:100 diluted), as previously reported [30].

.5. Fluorescence in situ hybridization (FISH) analysis

FISH was carried out with BAC clone RP11-219G17 (BACAC Resources, http://bacpac.chori.org), which includes BIRC5, theurvivin-encoding gene, and a centromeric probe for chromosome7 (CEP17) (Visys-Abbott, Downers Grove, IL, USA). The BAC probe

as prepared from bacterial cultures using Qiagen-Plasmid Midi kit

Qiagen GmnH, Düsseldorf, Germany) and labeled by nick transla-ion with SpectrumOrange-dUTP (Visys). FISH was performed on

�m FFPE sections as previously described [32].

verified by the area under the ROC curve (AUC, 95% CI = 0.95; 0.90–0.98). (D) Com-parison of survivin expression in male and female SCLC patients (Kruskal–Wallistest, P = 0.0150).

2.6. Survivin −31G/C SNP genotyping

DNA was extracted from FFPE samples using PureLink GenomicDNA mini kit (Invitrogen) and following the manufacturer’sspecifications. Survivin promoter-specific PCR was performed asdescribed [33]. PCR products were sequenced using the Big Dye ter-minator v1.1 Cycle Sequencing kit and analyzed on the ABI Prism3130 sequencer (Applied Biosystems, CA, USA).

2.7. Statistical analysis

Differences in survivin expression between normal and tumorsamples and between the two different histotypes were eval-uated by Mann–Whitney test. To estimate survivin associationwith clinicopathological characteristics the Mann–Whitney test,the Spearman’s rho coefficient, and the Kruskal–Wallis test wereperformed, as appropriate. The accuracy of survivin expression todiscriminate between normal and tumor lung tissue was verifiedby the area under the receiver operating characteristic (ROC) curve

[34]. Fischer’s Exact test was run to find difference in frequen-cies of polymorphism between the healthy subject population andpatients with NSCLC or SCLC. For survivin expression and eachclinicopathological characteristic, overall survival analysis was

182 A. Rosato et al. / Lung Cancer 79 (2013) 180– 186

Table 1Correlation between survivin expression (fold change) and clinicopathological and genetic characteristics of patients.

Characteristics NSCLC SCLC

No. Median (Q1–Q3) P No. Median (Q1–Q3) P

All patients 65 11.8 (4.8–27.9) 35 28.4 (12.1–63.6)Gender

Male 48 11.8 (4.1–29.1) 23 16.0 (6.7–46.8)Female 17 11.7 (7.6–20.6) 0.4737 12 48.9 (30.5–135.3) 0.0150

HistotypeAdenocarcinoma 48 11.6 (4.6–25.0)Squamous carcinoma 17 16.0 (5.0–30.3) 0.7426

Histological gradeG1 16 10.0 (4.0–20.3)G2 27 10.7 (7.2–25.0)G3 22 15.3 (4.0–44.9) 0.6727

TNM stage [51]IA–IB 23 14.3 (8.9–29.2)IIA–IIB 42 11.1 (4.0–27.9) 0.2606

IASLC stage [52]Limited 10 21.8 (11.8–63.6)Extended 24 30.5 (13.5–53.0) 0.7337

Node metastasisNegative 36 11.8 (5.7–27.1)Positive 29 11.6 (3.6–27.9) 0.4519

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erformed using the Kaplan–Meier method and compared with theog-rank test. A cut-off value for dividing patients into subgroupsased on survivin mRNA levels was identified using the method byontal and O’ Quigley [35].

Hazard ratios were estimated using univariate Cox proportionalazard models and reported with 95% confidence interval. Mul-ivariate survival analysis considered all significant variables innivariate survival analysis except for stage, being this a nodeetastasis-dependent variable. To analyze the Oncomine data, dif-

erences and correlations between groups were tested by applyinghe one way ANOVA with Bonferroni post-test and the modifiedruskal–Wallis non parametric test for trend, where appropriate.tatistical analyses were carried out with SAS statistical softwareersion 9.1 (SAS Institute, Cary, NC) and with MedCalc® version.3.1.0 software.

. Results

.1. Analysis of survivin transcript in normal and neoplastic lungissues

Sixty-five NSCLC, 35 SCLC, and 10 normal tissue samples werevailable for quantitative RT-PCR analysis. Survivin mRNA wasetected in all cancer specimens and only in 4 out of 10 normal sam-les. Overall, survivin mRNA was expressed at significantly higher

evels in either tumors than in normal tissue (P < 0.0001, Fig. 1a),nd more in SCLC than in NSCLC (P = 0.0021, Fig. 1b). The ROC curvenalysis identified an optimal cut-off value of 3.456, with sensitiv-ty of 91% (95% CI: 83–96) and specificity of 90% (95% CI: 56–100),iscriminating between normal and pathological samples (Fig. 1c).

No correlation was found between survivin mRNA expressionnd histopathological characteristics of either tumors (Table 1). Aignificantly higher survivin expression was found in female thann male SCLC subjects (P = 0.0150, Table 1 and Fig. 1d).

.2. Searching the Oncomine database

Survivin gene expression was also analyzed in silico by check-ng different publicly available lung microarray studies, using thencomine database and gene microarray data analysis tools. In

23 25.2 (12.1–63.6)0.3965 6 21.4 (12.6–51.2) 0.6667

seven independent studies [36–42], a statistically significant up-regulation of survivin mRNA expression levels was identified inlung tumors (both NSCLC and SCLC) by comparison with normallung parenchyma. Interestingly, two independent data sets [37,41]showed a significant up-regulation trend of survivin mRNA expres-sion levels when normal, NSCLC and SCLC samples were considered(Supplementary Fig. 1).

3.3. Assessment of survivin protein expression

IHC analysis was carried out in 31 NSCLC, 28 SCLC and in all 10normal samples (Supplementary Fig. 2a–c); positive staining wasdemonstrated in 65% and 46% of NSCLC and SCLC, respectively,while all 10 normal control samples were negative. Immunore-activity pattern was variable, but mainly with a cytoplasmiclocalization in either histological lung cancer types; indeed, 60%(n = 12) of positive NSCLC samples showed a cytoplasmic patternonly, 35% (n = 7) stained in both cytoplasmic and nuclear compart-ments, and only one sample (5%) exhibited nuclear staining alone;on the other hand, all positive SCLC specimens disclosed exclu-sively a cytoplasmic localization of survivin protein. Consideringthe samples analyzed by both IHC and real-time RT-PCR (48 tumortissues and 10 normal samples), a direct correlation between sur-vivin mRNA and protein levels was found (P = 0.003).

3.4. FISH analysis of genomic amplification

FISH was performed to determine whether the increasingsurvivin expression would correlate to the BIRC5 copy number(Supplementary Fig. 2d). The analysis was conducted in 18 NSCLCsamples and in 8 SCLC specimens. Among the NSCLC, 12 out of 18samples (67%) showed a BIRC5 gain, while only one (5%) was clearlyamplified; the remaining 5 cases displayed a normal disomic pat-tern. On the other hand, all 8 SCLC specimens tested presented anormal pattern with two copies of the gene. Therefore, althoughFISH analysis apparently revealed some differences in genetic pro-

files between NSCLC and SCLC, BIRC5 gene amplification did notappear a specific feature of lung tumors. Moreover, no correla-tion was found between FISH data and survivin mRNA or proteinexpression by IHC (data not shown). Interestingly, such results were

A. Rosato et al. / Lung Cancer 79 (2013) 180– 186 183

Table 2Correlation between survivin −31G/C SNP and clinicopathological characteristics of patients.

Characteristics NSCLC SCLC

G/G + G/C C/C P G/G + G/C C/C P

All patients 48 (88.9%) 6 (11.1%) 29 (80.6%) 7 (19.4%)Histotype

Adenocarcinoma 34 (87.2%) 5 (12.8%)Squamous carcinoma 14 (93.3%) 1 (6.7%) 1.0000

Histological gradeG1 13 (100%) –G2–G3 35 (85.4%) 6 (14.6%) 0.3170

TNM stageIA–IB 20 (95.2%) 1 (4.8%)IIA–IIB 28 (84.8%) 5 (15.1%) 0.3863

IASLC stageLimited 11 (100%) –Extended 17 (70.8%) 7 (29.2%) 0.0721

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upported by data from a single Oncomine copy number study thatailed to evidence BIRC5 gene amplification [42] (Supplementaryig. 3).

.5. −31G/C SNP analysis of survivin promoter

To verify the role of the −31G/C SNP in survivin expression,enotype was determined in 54 NSCLC and 36 SCLC (Table 1). Theistribution of genotypes did not disclose significant differencesetween the two tumor histotypes. Moreover, this SNP appearedo have no effect on the risk of lung cancer (genotype frequencies inonors: G/G: 39.2%; G/C: 45.8%; C/C: 15.0%). The observed genotyperequencies in all groups considered were in Hardy–Weinberg equi-ibrium (controls: �2 = 0.168, P = 0.682; NSCLC: �2 = 0.04, P = 0.838;CLC: �2 = 0.646, P = 0.422).

Next, the role of −31G/C SNP on survivin expression was eval-ated by comparing the mRNA levels of patients with distinctenotypes: no significant difference was found in mRNA survivinevels of the 51 NSCLC or 29 SCLC patients analyzed having dis-inct genotypes (Supplementary Fig. 4). No significant associationmerged between the −31G/C SNP genotype and other clinico-athological characteristics of patients, except between the −31C/CNP status and the presence of lymph node metastases in NSCLCP = 0.0355). In SCLC an association (significant at 10%, P = 0.0721)etween the −31C/C SNP status and extended stage was foundTable 2).

.6. Survival analysis.

Univariate analysis (Table 3) showed no correlation in NSCLCetween survivin expression and overall survival when the sur-ivin levels were considered as a continuous variable (P = 0.1857).onversely, the selection of a cut-off value (3.96-fold change)

or survivin mRNA corresponding to the most significant differ-nce in survival [35], evidenced that NSCLC patients with tumorsxpressing very low survivin (≤3.96-fold change) had a partic-larly bad prognosis (HR = 2.3, 95% CI = 1.1–4.7, P = 0.0216). Thisata was confirmed by Kaplan–Meier survival analysis (Log Rank

= 0.0143, Fig. 2a). Interestingly, 9 out of 12 of these patients weret Stage II and 8 of them (67%) presented lymph node involvement;onversely, among patients with high survivin levels (>3.96-fold

hange), only 21 out of 53 (40%) had lymph node metastasis. More-ver, even though not statistically significant, a trend for a betterrognosis was also seen for individuals having a IHC pattern posi-ive for survivin in cytoplasm, as these patients presented a median

Fig. 2. Kaplan–Meier survival curves of NSCLC patients. (A) Stratified for survivinexpression (cut off = 3.96-fold change, Log Rank P = 0.0143) or (B) According to their−31G/C SNP genotype status (Log Rank P = 0.0332).

184 A. Rosato et al. / Lung Cancer 79 (2013) 180– 186

Table 3Univariate survival analysis.

Variable NSCLC

Events/n Median survival (months) (95% CI) HR 95% CI (HR) P

Survivin (real time) 45/65 0.989 0.975–1.005 0.1857Survivin (real time)

≤3.96 fc 10/12 3.0 (1.0;5.0) 2.3 1.1–4.7 0.0216>3.96 fc 35/53 10.0 (7.0;33.0) 1

Survivin IHC (C)Negative 8/12 – 1Positive 10/19 – 0.6 0.2–1.5 0.2960

Survivin IHC (N)Negative 15/23 11.0 (9.0;) 1Positive 3/8 –(8.0;) 0.5 0.1–1.6 0.2185

SNP −31G/CG/G + G/C 31/48 10.0 (8.0;34.0) 1C/C 5/6 3.0 (1.0;5.0) 2.6 1.0–6.9 0.0467

GenderM 38/50 7.5 (5.0;10.0) 2.4 1.1–5.2 0.0238F 8/19 –(10.0;) 1

HistotypeAdenocarcinoma 32/51 10.0 (5;) 1Squamous carcinoma 14/18 8.5 (4.0;11.0) 1.3 0.7–2.5 0.3861

Histological gradeG1 8/16 –(5.0;) 1G2 18/29 10.0 (5.0;) 1.4 0.6–3.1 0.4729G3 20/24 5.5 (4.0;11.0) 2.2 1.0–5.0 0.0610

TNM StageIA–IB 9/25 –(10.0;) 1IIA–IIB 37/44 5.0 (4.0;9.0) 3.8 1.8–8.0 0.0003

Node metastasisNegative 17/38 –(10.0;) 1Positive 29/31 4.0 (3.0–5.0) 4.4 2.4–8.2 <0.0001

Variable SCLC

Events/n Median survival (months) (95% CI) HR 95% CI (HR) P

Survivin (real time) 32/35 1.0 0.995–1.003 0.2736Survivin (real time)

≤36.79 fc 19/20 10.0 (7.0;13.0) 1>36.79 fc 13/15 16.0 (4.0;17.0) 0.7 0.3–1.4 0.2683

Survivin IHC (C)Negative 13/15 10.0 (7.0;19.0) 1Positive 13/13 6.0 (2.0;16.0) 1.8 0.8–4.0 0.1572

SNP −31G/CG/G + G/C 27/29 10.0 (6.0–14.0) 1C/C 6/7 16.0 (4.0–24.0) 0.6 0.3–1.5 0.3133

GenderM 26/27 10.0(6.0–13.0) 1.5 0.8–3.0 0.2099F 13/15 16.0 (4.0–17.0) 1

IASLC stageLimited 12/13 14.0 (7.0–17.0) 1Extended 25/27 8.0 (4.0–13.0) 1.4 0.7–2.8 0.3422

fc: fold change; C: cytoplasmatic; N: nuclear.

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urvival of 33 months vs 10 months in patients negative for cyto-lasmic survivin (data not shown).

In NSCLC patients the univariate survival analysis confirmedhe prognostic role of tumor stage with patients at stage II hav-ng a median survival of only 5 months (HR = 3.8; 95% CI = 1.8–8.0;

= 0.0003). This survival is worse than previously reported [43]nd is likely due to the advanced age at diagnosis (median, 69.5ears) and the high percentage of patients with node metastasis31/44, 70%), a clinicopathological feature endowed with a strongrognostic relevance (HR = 4.4; 95% CI = 2.4–8.2; P < 0.0001). As alsoeported by others [44], gender was another prognostic factor, withales having a worse prognosis than female subjects (HR = 2.4; 95%

I = 1.1–5.2; P = 0.0238). Finally, considering the −31G/C SNP status,/C carriers showed a shorter survival than patients with differentenotypes (HR = 2.6; 95% CI = 1.0–6.9; P = 0.0467), as confirmed byaplan–Meier curves (Log Rank P = 0.0332, Fig. 2b).

In SCLC, survivin expression, clinicopathological characteristicsand presence of −31G/C survivin SNP did not correlate with patientprognosis (Table 3).

Multivariate analysis (Table 4) confirmed the prognostic roleof survivin expression in NSCLC as dichotomic variable, disclosingthat survivin mRNA levels ≤ 3.96-fold change were associated witha shorter survival rate (HR = 5.5, 95% CI = 1.8–17.1, P = 0.0033), aswell as −31C/C status (HR = 2.8, 95% CI = 1.005–8.026, P = 0.0488)and the presence of node metastasis (HR = 3.0, 95% CI = 1.412–6.211,P = 0.0041).

4. Discussion

Survivin has been widely studied as prognostic marker in NSCLC[14–18,20–26,45], but only very limited information is available forSCLC [46,47].

A. Rosato et al. / Lung Cance

Table 4Multivariate survival analysis.

Variable NSCLC

HR 95% CI (HR) P

Survivin (real time)≤3.96 fc 5.5 1.8–17.1 0.0033>3.96 fc 1

SNP −31G/CG/G + G/C 1C/C 2.8 1.0–8.0 0.0488

GenderM 1.7 0.7–4.1 0.2298F 1

Node metastasisNegative 1Positive 3.0 1.4–6.2 0.0041

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c: fold change.

We found that survivin transcripts were more expressed inung tumors than in healthy counterpart tissues, an aspect alsovidenced by IHC; moreover, survivin mRNA was more representedn SCLC than in NSCLC. Interestingly, higher survivin expression inCLC than in NSCLC was also observed by querying the Oncomineatabase. Overall, these findings suggest that survivin may play aole in lung cancer progression and contribute to cancer aggres-iveness, improving cell proliferation and resistance to apoptosis,specially in SCLC.

A recent meta-analysis on the role of survivin in NSCLC con-luded that high survivin expression may be a negative prognosticactor for overall survival [45]. In our study, survivin expressionas related neither to the clinicopathological characteristics or the31SNP G/C status of patients, nor to the prognosis, when consid-red as a continuous variable. Conversely, by evaluating survivinxpression as a dichotomic variable only, we identified a group ofatients with a very poor prognosis and very low survivin expres-ion levels in tumors. Although such cohort is composed only of2 patients and therefore this may represent a limit of the study,onetheless the high percentage of patients with node metastasis67%) in such group compared to that present in patients with highurvivin levels (40%), and the fact that both the presence of nodeetastasis and survivin levels resulted independent prognostic fac-

ors in multivariate survival analysis, support the concept that sucharameters play a real prognostic role. The relationship betweenhe low survivin levels and bad prognosis, although apparentlyiscordant from some of previous reports [15,17,18,23,25,26,45],ight find alternative explanations. Indeed, this association could

e due to a lower or absent expression of the survivin splicingariant endowed with pro-apoptotic activity compared to that hav-ng anti-apoptotic effects. Interestingly, it has been reported thatelatively high levels of pro-apoptotic isoform are significantlyssociated with better prognosis [16]. Alternatively, the differentialntracellular protein distribution might play a role: in fact, a pre-ious report showed a poor prognostic value of nuclear survivin inSCLC [48]; therefore, the prevalent cytoplasmic localization foundven in samples with high survivin levels might act as a contribut-ng factor for the better outcome.

Otherwise, the high survivin levels in SCLC would appear to ben intrinsic characteristic of this histotype, independent of prog-osis: in this case, survivin can be considered a marker of highroliferation rather than a real cancer-specific gene [49].

To discover biological factors potentially influencing survivinxpression, we first focused on the genetic alteration of sur-ivin locus, since the amplification of the 17q25 region in tumors

as been reported as a cause of higher survivin expression [50].lthough only a limited number of samples could be studied, FISHnalysis did not disclose BIRC5 gene amplification in either NSCLC

r 79 (2013) 180– 186 185

or SCLC, and no correlation was found with gene expression or IHC,findings that probably argue against the involvement of gene copynumber in determining the increase of survivin expression in lungcancer.

Subsequently, careful attention was paid to the polymorphismson the promoter region of survivin gene, since the survivin pro-moter is largely inactive in normal tissues, but is functional in tumorcells. Because of its localization, the −31G/C SNP may affect thebinding of elements that regulate cell cycle-dependent transcrip-tion of the survivin gene, and was previously described as havingmodified transcriptional activities in cancer cell lines [51]. Recently,higher mRNA survivin levels have been described in colorectalpatients carrying the C allele at −31 SNP compared to non-carriers[52]. On the other hand, other epidemiological studies have shownthis SNP to be associated with an increased risk of cancer in differenttumor histotypes [27].

In lung cancer, the few available data are controversial eitherregarding the role of −31G/C SNP in promoting the susceptibil-ity to tumor and also concerning its prognostic significance [28].In this study, the −31 SNP of the survivin promoter was not cor-related with lung cancer risk. Moreover, we found that survivinexpression in patients with distinct genotypes was not different,thus arguing against a role of −31G/C SNP in inducing the overex-pression of survivin mRNA. On the other hand, in NSCLC we foundthat patients homozygous for −31C/C genotype exhibited a sig-nificantly higher presence of lymph node metastases and worseprognosis with respect to patients with different genotypes (G/Cand G/G). Although the −31C/C patients were only 6, the effect ofSNP was also maintained in multivariate analysis, thus supportingits potential prognostic role. In SCLC, a weak association betweenthe −31C/C SNP status and extended stage was found. The analysisof a more cases may strength the significance of the association andhence link the polymorphism to survival.

5. Conclusion

This work assessed survivin expression and significance inNSCLC and SCLC. Survivin mRNA levels were significantly higherin tumors than in normal tissue, and in SCLC than in NSCLCsamples. A better prognosis was correlated to higher levels ofsurvivin mRNA and to the presence of at least one G allele at−31 G/C SNP on survivin promoter region only in NSCLC, whilethese parameters did not correlate with overall survival in SCLC.The different prognostic role played by survivin expression in thecontext of NSCLC and SCLC highlights the biological differencesexisting between these lung tumor histotypes, and stresses theneed to clarify the molecular pathways leading to their neoplastictransformation.

Conflict of interest statement

None declared.

Acknowledgements

The authors would like to thank Vincenza Guzzardo and Elisa-betta Tebaldi for their excellent technical assistance. This study was

Italian Association for Cancer Research (AIRC), the Veneto Region(Ricerca Finalizzata 2006) and the Cariparo Foundation Excellence-grant.

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86 A. Rosato et al. / Lung

ppendix A. Supplementary data

Supplementary data associated with this article can beound, in the online version, at http://dx.doi.org/10.1016/j.ungcan.2012.11.004

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