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Lung Cancer 69 (2010) 289–295

Contents lists available at ScienceDirect

Lung Cancer

journa l homepage: www.e lsev ier .com/ locate / lungcan

nail nuclear expression parallels higher malignancy potential in neuroendocrineung tumors

.A. Galvána, M.V. Gonzálezb, G. Crespoc, M.V. Folguerasd, A. Astudilloa,d,∗

Laboratorio del Banco de Tumores, Instituto Universitario de Oncología del Principado de Asturias, Obra Social CajAstur (IUOPA), c/Celestino Villamil s/n,3006 Oviedo, Asturias, SpainOncologia Quirúrgica, Instituto Universitario de Oncologia del Principado de Asturias, Obra Social CajAstur (IUOPA), c/Celestino Villamil s/n,3006 Oviedo, Asturias, SpainServicio de Oncología Médica, Hospital Universitario Central de Asturias, 33006 Oviedo, SpainServicio de Anatomia Patológica, Hospital Universitario Central de Asturias, 33006 Oviedo, Spain

r t i c l e i n f o

rticle history:eceived 24 September 2009eceived in revised form 9 December 2009ccepted 18 December 2009

eywords:euroendocrine lung tumors

mmunohistochemistry-cadherin-cateninnail

a b s t r a c t

Introduction: The aim of our study was to determine the integrity of the cell–cell adhesion E-cadherin–�-catenin complex in neuroendocrine lung tumors (NELTs) and the possible involvement of Snail in itsderegulation.Methods: The studied series consisted of formalin-fixed-paraffin-embedded tissue samples from 70patients diagnosed with NELT (2000–2006) including tumors of low malignacy potential (3 tumorlets, 33typical carcinoids), intermediate malignancy potential (3 atypical carcinoids) and tumors of high malig-nancy potential (10 large cell neuroendocrine carcinomas—LCNEC and 21 small cell carcinoma—SCLC).E-cadherin, �-catenin and Snail expression were immunohistochemically evaluated and mRNA levelswere assessed by Q-RT-PCR for E-cadherin and Snail.Results: Nuclear Snail signal was high in 46% tumors with the strongest level observed in high malignancytumors. Furthermore, Snail levels correlated with tumor size, lymph node involvement and tobacco con-sumption. E-cadherin expression was downregulated in 24% cases and it was absent from the membranein 31%, all of them cases of high malignancy potential. High E-cadherin levels and a membrane pat-tern were associated with tumor-free lymph node patients and inversely proportional to Snail protein

expression. �-catenin levels were weak in 43% and absent from the membrane in 59% cases. Interest-ingly, among high malignancy potential tumors, �-catenin levels were significantly higher in LCNECthan in SCLC. The integrity of the E-cadherin–�-catenin complex was retained in 37% cases, most ofthem carcinoid tumors, and correlated with low Snail levels, low malignancy potential and free lymphnodes.Conclusion: Snail nuclear expression and loss of integrity of cell adhesion complex E-cadherin/�-catenin

cy po

parallels higher malignan

. Introduction

Neuroendocrine lung tumors (NELTs) represent a wide spec-rum of entities which share morphologic, ultrastructural,mmunohistochemical and molecular characteristics, with differ-nces in progression and aggressiveness. According to the WHO

riteria, NELTs are histologically classified as: small cell carcinomaSCLC), large cell neuroendocrine carcinoma (LCNEC), typical car-inoid (TC), atypical carcinoid (AC). Tumorlets (TL) are additionallyonsidered as benign proliferative lesions [1].

∗ Corresponding author at: Servicio de Anatomía Patológica, Hospital Universi-ario Central de Asturias, 33006 Oviedo, Spain. Tel.: +34 985107891.

E-mail address: astudillo@uniovi.es (A. Astudillo).

169-5002/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.lungcan.2009.12.010

tential in NELTs.© 2009 Elsevier Ireland Ltd. All rights reserved.

NELTs are diagnosed by light microscopy according to theirgrowth pattern and neuroendocrine differentiation. Immunohis-tochemically, these tumors stain for neuroendocrine markers:neural cell adhesion molecule, chromogranin A, synaptophysinand enolase. Additional features, such as the mitotic index andthe presence of necrosis, are helpful for tumor subclassification[2,3].

E-cadherin is a transmembrane glycoprotein involved in Ca2+-dependent epithelial cell–cell homophylic interaction at theadherens junctions [4]. The cytoplasmic domain of E-cadherin

interacts with a group of closely related proteins, termed �-,�- and �-catenin, which connect the cell membrane tightly tothe cytoskeleton. Moreover, �-catenin acts as a link betweenE-cadherin and Wnt signaling pathways, acting as a cellular trans-ducer to the nucleus, where it activates the transcription of several

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90 J.A. Galván et al. / Lung

enes [5,6]. Dysregulation of the E-cadherin/�-catenin-dependentdhesion complex has been associated with the developmentnd progression of many solid tumors, including several types ofndocrine tumors [5,7–11].

A decreased expression of E-cadherin represents therototypical epithelial cell marker of the epithelial–esenchymal transitions (EMTs). Moreover, loss of E-cadherin

unction promotes EMT [12–14]. EMTs have recently been classi-ed into three general subgroups: type 1 EMT involves transitionshat occur during embryonic development, type 2 refers tohose that take place during tissue fibrosis, and type 3 refers topithelial carcinoma cells in primary nodules transitioning toetastatic tumor cells in order to migrate through the blood

tream and, in some cases, form secondary nodules in distantetastatic sites by mesenchymal–epithelial transitions (METs)

15,16].The reduction of E-cadherin expression may result from

he transcriptional repression by the Snail family proteins, alsonvolved in the triggering of type 1–3 EMTs [17–20]. Snail is

zinc finger transcription factor that interacts with the con-ensous sequence at the CDH1 promoter through its C-terminalomain, evolutionary conserved in vertebrates and invertebrates21], where it plays a key role in mesoderm development, neuralifferentiation and the formation of the neural crest in vertebrates22].

Since Snail has been shown to be involved in E-cadherin repres-ion during EMT type 3, its expression has been analyzed inifferent types of tumors: breast, melanoma and hepatocellulararcinomas [23–27].

In embryonic development, neural crest cells undergo EMT typeto expand to the periphery. Thus, it is likely that tumoral neu-oendocrine cells newly express genes involved in EMT, such asnail. However, there are no reports on Snail expression in neu-

oendocrine lung tumors.

Our study analyzes Snail and E-cadherin/�-catenin complexxpression in the whole spectrum of neuroendocrine lung tumors,ombining these data with histological and clinicopathologic fea-ures.

able 1linicopathologic features of the studied patients and their tumors (N = 70).

Characteristics Histologic classifi

TL T

Total (%) 3 (4) 3

AgeMin 33 1Max 56 7Mean 47 5

GenderFemale 1 1Male 2 1

Toxic HabitsSmoker 0Non-smoker 2 1Unknown 1 1

Tumor Size<Median (2.7 cm) 1 2>Median (2.7 cm) 0 1Unknown 2

Lymph node statusFree 3 2Affected 0Unknown 0

Treatment

Surgery 3 3Surgery + adyuvant 0Chemotherapy 0Unknown 0

Disease StatusAlive 3 3Death of tumor 0Death other causes 0

er 69 (2010) 289–295

2. Materials and methods

2.1. Patients and samples

70 patients with neuroendocrine lung tumors were collected atthe Hospital Universitario Central de Asturias between February2000 and October 2006 with institutional review board approvalfor guidelines on ethical procedures. Tumor samples were obtainedfrom surgical resection, except for SCLC, obtained from trans-bronchial biopsy (21 cases). The diagnosis of neuroendocrinetumors was based on morphologic criteria according to the updatedWorld Health Organization classification. Criteria include mitoticactivity counted in 10 high-power fields (HPFs) of light microscope(40×), presence or absence of necrosis and immunophenotypicalfindings, including reactivity for neuroendocrine markers, synapto-physin and chromogranin A [28]. The characteristics of the studiedpatients (age, gender, toxic habits) and the clinicopathological fea-tures of their tumors (tumor size, lymph node status, treatment,and disease status, according to their histologic classification) areshown in Table 1.

Tissues obtained from biopsies were fixed in 10% formaldehydeand paraffin embedded, then cut, 4 �m thick, mounted on treatedslides and stained with H&E.

Frozen tissue from 28 out of those 70 cases (tumor and normaltissue counterpart) was available at Banco de Tumores at HUCA,being processed for RNA extraction and expression analysis.

Normal epithelial tissue, of every section, was considered as areference.

2.2. Immunohistochemistry

Sections were deparaffinized in xylene for 20 min and rehy-drated in decreasing graded ethanol solutions. Antigen retrieval

was done by heating in 10 mM citrate buffer solution (pH 6.5) ina pressure cooker for 7 min. Non-specific antibody binding wasblocked using 1% bovine serum albumin (BSA) in PBS for 20 min.Endogenous peroxidase activity was blocked with H2O2 solution(3%) for 10 min. Samples were incubated overnight with the fol-

cation

C AC LCNEC SCLC Total (%)

3 (47) 3 (4) 10 (15) 21 (30) 70 (100)

7 37 44 546 69 77 851 58 60 70

4 2 5 2 24 (34.29)9 1 5 19 46 (65.71)

8 3 7 3 21 (30)1 0 0 0 13 (18.57)4 0 3 18 36 (51.43)

0 0 1 1 23 (32.86)0 3 9 1 23 (32.86)3 0 0 19 24 (32.28)

9 2 8 9 51 (72.86)0 0 2 12 14 (20)4 1 0 0 5 (7.14)

1 2 2 2 40 (57.14)2 1 7 2 12 (17.14)0 0 0 17 17 (24.28)0 0 1 0 1 (0)

0 2 5 5 45 (64)2 1 5 13 21 (30)1 0 0 3 4 (6)

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J.A. Galván et al. / Lung

owing primary antibodies in a humid chamber at 4 ◦C: polyclonalnti-Snail (Abcam 17732, Cambridge, UK) (1:800 dilution in goaterum); monoclonal anti-E-cadherin (DakoCytomation, Glostrup,enmark) (1:100 dilution) and monoclonal anti-�-catenin (Sigma,O, USA) (1:800 dilution).The slides were incubated with the EnVision system (Dako-

ytomation, Denmark) for 30 min at room temperature. Thenhe samples were visualized with DAB (3-3′-Diaminobenzidine)DakoCytomation, Denmark). Finally, samples were counterstainedith Mayer’s hematoxyline, dehydrated and mounted in Entellan®

Merck, Germany). The sections were studied and photographedsing an Olympus light microscope.

.3. Immunohistochemistry assessment

According to the protein expression levels the specimens werelassified by the pathologist following these criteria.

�-catenin and E-cadherin immunostaining intensities werevaluated as weak vs intense, and their presence/absence in the cellembrane was recorded. A variable reflecting the integrity of the

-cadherin–�-catenin complex in the cellular membrane was cre-ted, including two categories: integrity retained (both moleculesn a membranous pattern) and integrity lost (expression of at leastne of them not observed in the membrane).

Snail immunostaining was evaluated attending to the nuclearignal as the product of two parameters: extent of inmunoreactivitycored on a 0–5 scale according to the proportion of positive tumorells: (0) 0%; (1) <1%; (2) 1-10%; (3) 11–33%; (4) 34–66% and (5)7–100%, and signal intensity on a 0–3 point scale. Score valuesanged from 0 to 15.

.4. RNA extraction

Total RNA was isolated following TRIZOL Reagent®

InvitrogenTM Ambion) phenolic extraction based in the method byhomczynski[29]. RNA quality was assessed by the Agilent 2100ioanalyzer RNA Nano Labchip® (Agilent Technologies). Twentyases out of 28 were considered to yield high quality RNA andere included in our study.

Samples were stored at −80 ◦C until use.

.5. Real time RT-PCR

cDNA was synthesized by RT-PCR using GeneAmp RNA-PCRApplied Biosystems) from 1 �g total RNA and random hexamers,n a final volume of 20 �l.

Snail and E-cadherin mRNA expression levels were assessed inaired normal and tumor tissues, with GAPDH as endogenous con-rol gene. TaqMan® Gene Expresión Assays with TaqMan UniversalCR Master Mix (AmpErasa® UNG) (Applied Biosystems) were usednd reaction duplicates were carried out in a final volume of 20 �ln 96 well-plates.

.6. Statistical analysis

The molecular data distributed among the different clinicalroups of tumors were tested for significance employing the �2 testwith Yates’ correction, when appropriate). Survival curves werealculated using the Kaplan–Meier product limit estimate. Deathsrom causes other than the index tumor or its metastases were notonsidered treatment failures, and these patients were censored in

ll analysis involving length of survival. Differences between sur-ival times were analyzed by the log-rank method. Multivariate Coxroportional hazards models (forward Wald method) were used toxamine the relative impact of those variables statistically signif-cant in univariate analysis. All statistical analysis was carried out

er 69 (2010) 289–295 291

with the help of the software package SPSS 12.0 (SPSS, Inc., Chicago,IL). All tests were two-sided. p < 0.05 values were considered statis-tically significant. The mean follow up period was 54 months (min,16 months; max 97 months).

3. Results

For statistical purposes, clinicopathologic variables were cat-egorized as follows: tumor type according to their malignancypotential: low (LMP: tumorlets and TC, 52%), intermediate (IMP:AC, 4%) and high (HMP: LCNEC and SCLC, 44%); tumor size: accord-ing to the median value (2.7 cm); lymph node involvement: free vsaffected nodes; age: according to the median value (58 years); toxichabits: smoker vs non-smoker.

3.1. E-cadherin protein expression

Healthy epithelium showed an intense and membranous E-cadherin expression pattern (Fig. 1B).

In tumors, E-cadherin was highly expressed in 53 cases (76%)and weakly in 17 cases (24%). 69% (46/67 cases) retained E-cadherinin the plasma membrane and showed a lineal pattern. When E-cadherin was detected in the cytoplasm, a granulated pattern couldbe observed (Fig. 1H and J).

3.2. ˇ-catenin protein expression

�-catenin showed an intense signal and a membranous patternin the normal epithelia (Fig. 2A). 39/68 tumors (57%) displayed high�-catenin levels. The membranous pattern was observed in 26/63cases (41%), while cytoplasmic and/or nuclear in the rest. A nuclearsignal was detected in 28 cases.

According to the E-cadherin–�-catenin complex integrity sta-tus, 22/60 (37%) tumors were classified as complex integrityretained and 38/60 (63%) as complex integrity lost.

3.3. Snail protein expression

A weak nuclear and cytoplasmic Snail signal was detected inhealthy epithelia (Fig. 1A). Taking into account the proposed roleof Snail as a transcription factor, we only considered the nuclear sig-nal, as suggested by other authors [30]. A score (0–15) was assignedto each case as described in the methods section. For statisticalpurposes tumors were divided into two groups taking the medianscore value (8) as a cutoff point. Thus, 32 out of the 70 cases (46%)displayed high Snail levels, while they were low in the rest.

3.4. E-cadherin and Snail mRNA Q-RT-PCR assessment

E-cadherin and Snail mRNA levels could be assessed for ninecases (5 CT, 1 CA, 2 LCNEC and 1 SCLC), for which pairedtumor and normal tissue were available. E-cadherin mRNA lev-els were not altered in 6/9 tumors. Two tumors showed lowerlevels and one higher levels than their corresponding normaltissues.

Snail mRNA levels were lower in 7/9 tumors and similar to thecorresponding normal tissue in the rest.

Neither E-cadherin or Snail mRNA levels correlated with levelsof the corresponding proteins.

3.5. Molecular findings correlations

An inverse correlation was observed for Snail expression andE-cadherin protein levels (p < 0.001) (Fig. 1). This pattern was notobserved at the mRNA level. The integrity of the adhesion complexwas more frequently retained among tumors with reduced Snaillevels (20/37, 54%) vs those with high levels (2/23, 9%) (p < 0.001).

292 J.A. Galván et al. / Lung Cancer 69 (2010) 289–295

F n nort especp l nuclef

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ig. 1. Illustration of the inverse expression levels of Snail and E-cadherin protein iumorlet (C and D, respectively), carcinoid (E and F, respectively), LCNECs (G and H, rattern (observed in normal tissue) is lost in high malignancy potential tumors. Snaior each case. Scale bar, 50 �m.

e also observed correlation between the presence of both E-adherin and �-catenin in the membrane (p = 0.034): �-catenin wasbsent from the membrane more frequently in tumors with low-cadherin levels (p = 0.02).

mal tissue (A and B, respectively) and in NELTs of increasing malignancy potential:tively) and SCLC (I and J, respectively). The linear membrane and intense E-cadherinar expression increases with malignancy potential. Consecutive sections are shown

3.6. Correlations with clinicopathologic features

HMP tumors showed the following alterations more frequentlythan LMP ones: high Snail levels (29/31, 94% HMP tumors vs 2/36,

J.A. Galván et al. / Lung Cancer 69 (2010) 289–295 293

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ig. 2. Representative �-catenin immunostaining patterns in normal tissue (A) andmembrane disrupted pattern is observed in LCNECs (C) and SCLCs (D). Nuclear acc

cale bar, 50 �m.

% LMP tumors, p < 0.001) (Fig. 1G and I vs C); absence of E-cadherint the membrane (21/28, 75% HMP tumors vs a 0% LMP, p < 0.001)Fig. 2H and J vs D) and absence of �-catenin at the membrane21/25, 84%) HMP vs 14/35, 40% LMP, p = 0.003) (Fig. 2C and D vs B).

Tumors with high Snail protein levels had a greater sizep = 0.001), corresponded to smoker patients (p = 0.056) and cor-elated with affected nodes (p = 0.001). High E-cadherin levels andembrane pattern were more frequently observed among tumor-

ree lymph node patients (p = 0.003 and p < 0.001, respectively).Interestingly, �-catenin levels were significantly higher in

CNEC than in SCLC (p < 0.001) (Fig. 2C and D).The adhesion complex integrity conservation correlated with

he malignancy potential (21/33, 64% LMP vs 0% HMP, p < 0.001),ymph node status (21/45, 47% lymph node-free cases retained theomplex integrity vs 0/11 with affected nodes, p = 0.004) and toxicabits (9/12, 75% non-smokers conserved the integrity vs 5/20, 25%mokers, p = 0.006).

.7. Survival analysis

The 5-year survival rate of the studied series was of 60%S.E.: 6). Survival rates were significantly better for those patientsarbouring LMP tumor (p < 0.0001) and tumor-free lymph nodesp = 0.0001). Regarding molecular variables, survival times were

horter for those patients showing high Snail protein levelsp < 0.0001) (Fig. 3), weaker E-cadherin and �-catenin levelsp = 0.0001 and p = 0.0445, respectively), and for those cases inhich the adhesion complex integrity at the cell membrane was

ost (p < 0.0001).

ig. 3. Cumulative Kaplan–Meier survival curves stratified according to Snail pro-ein expression levels.

s. A membrane-linear pattern is typically seen in well-differentiated carcinoids (B).tion of �-catenin is present exclusively in high-grade neuroendocrine lung tumors.

Multivariate survival analysis revealed that diagnosis wasthe only variable with independent prognostic value (p < 0.001)(Table 2).

4. Discussion

NELTs include a broad range of neoplasias of different malig-nancy potential. Although they share neuroendocrine features,biological differences must account for their different clinicalcourse, being favourable for the well-differentiated lesions andadverse for the poorly differentiated ones.

Several studies have shown that the E-cadherin–�-catenin cel-lular adhesion complex is disrupted during the early stages oftumor invasion [31–33]. The aim of our study was to check theintegrity of such complex and to evaluate the possible role of Snailin epithelial adhesion maintenance in NELTs. Linear membrane dis-tribution and intense signal could be observed for both E-cadherinand �-catenin proteins, compatible with their described role inmediating adherens intercellular junctions in intact epithelia.

Tumors were grouped according to the preserved/lost E-cadherin–�-catenin complex integrity (when both were retainedat the membrane or when at least one of them was absent). Lowmalignancy potential tumors, cases with tumor-free lymph nodesand those from non-smoker patients showed an intact complexmore frequently than the rest. This observation reflects the impor-tance of the loss of E-cadherin–�-catenin interaction at the cellularmembrane in NELTs development. Clavel et al. [7] did not find anycorrelation between the complex expression and clinicopathologicfeatures probably due to the limited number of the cases of theirseries.

The observation of different subcellular localizations of �-catenin (membrane and nucleus) is particularly interesting. Thedynamic regulation of �-catenin protein levels by exchange ofmolecules between the spatially separated pools is poorly under-stood [34]. Disruption of adherens junctions releases �-catenin into

the cytoplasm. Moreover, the inhibition of �-catenin proteasomaldegradation resulting from the stimulation of the Wnt signallingpathway by Wnt extracellular factors or from stabilizing mutationsin the CTNNB1 gene (or genes that control �-catenin stability, suchas APC or axins), contributes to �-catenin cytoplasmic accumula-

Table 2Statistical significance (p value) of the influence of clinicopathological and molecularvariables on survival in a univariate (Kaplan–Meier) or multivariate (Cox Regression)analysis basis.

Variable p value univariateanalysis

p value multivariateanalysis

Malignancy potential <0.0001 0.036Lymph node status 0.0001 0.407Snail protein level <0.0001 0.477E-cadherin protein level 0.0001 0.125�-catenin protein levels 0.0445 0.254Complex integrity <0.0001 0.93

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ion. This accumulation leads to its nuclear translocation, where itnduces expression of target genes.

Accordingly, low E-cadherin levels were associated with thebsence of �-catenin at the cell membrane in our NELTs series. Thebserved �-catenin nuclear signal might result from mutations inTNNB1 or APC genes. However, previous studies reported nuclearccumulation of �-catenin in a subset of pulmonary high-gradeeuroendocrine carcinomas in the absence of loss-of-functionutations or polymorphisms of either CTNNB1 or APC [7,8,35], so

nother mechanism should be considered. Stimulation of the Wntignalling pathway or alterations in other members of the complex,uch as axin and the protein kinases GSK-3� and CKIa could alsoontribute to �-catenin stabilization [36].

The CDH1 gene (coding for E-cadherin) has been proposed as annvasion suppressor gene, since its downregulation correlates withhe invasion potential of human tumors [37,38]. In our NELTs series,educed E-cadherin levels were associated with lymph node tumornvasion. Moreover, patients whose tumors lacked E-cadherin athe membrane displayed affected lymph nodes more frequently77%) than those that retained the membranous pattern. Thus, ouresults, along with those by Salon et al. [35] and Pelosi et al. [8],upport the role of CDH1 as an invasion suppressor.

To gain insight into the molecular events involved in the main-enance of the E-cadherin–�-catenin complex integrity, we set totudy Snail expression in our tumor series. This is the first reporthowing that human NELTs express Snail. In agreement with theroposed role of Snail as a transcriptional E-cadherin repressor,heir expression were inversely correlated. This result supportshose described in hepatocarcinomas, breast, ovarian and gastricarcinomas [26,39,40]. However, most of the studies rely on cellularodels while human tumor samples are only assessed in a reduced

umber of reports on breast cancer, melanoma, colon carcinoma,brosarcomas and gastrointestinal carcinomas [24,25,41–43].

Moreover, high Snail levels were associated to the loss of the E-adherin–�-catenin complex at the membrane and to lymph nodepread, thus contributing to the malignancy potential of the NELTs.

Some subtypes of NELTs might present diagnostic difficulties,ince occasionally they share pathological features. To shed lightnto this question, molecular findings were tested for significance toiscriminate between different histologic subtypes. Significant dif-erences were detected for �-catenin expression levels in SCLC andCNEC, as suggested by other authors [44]. Thus, it might constitutenew helpful diagnostic tool.

Tumors of smoker patients harboured elevated Snail expression.ntracellular pathways activated during EMTs (TGF-�, Akt, Wnt andas/MAPK) also respond to the action of tobacco components [45].aken together, these observations point to a role of Snail in NELTsf smoker patients.

Tumor Snail and E-cadherin mRNA expression results comparedo their normal tissue counterparts must be considered cautiously.irst, taking into account the high proportion of inflammatoryomponent in healthy tissue (not so abundant in tumor samples),e cannot consider these samples to be comparable, since the

ignal registered from the former would not actually reflect theRNA level of healthy epithelial cells. Secondly, when tumors were

lassified according to their Snail or E-cadherin mRNA levels, no dif-erences could be found between tumors of different malignancyotential but differences were observed at the protein level, whenuclear the membrane signals were considered. The lack of correla-ion between Snail mRNA and protein levels observed in our seriesould reflect its low stability, with a short half-life controlled by

osttranslational modifications.

Molecular findings such as high Snail levels, reduced E-cadherinnd �-catenin protein expression, as well as loss of the integrity ofhe E-cadherin–�-catenin complex were predictors of poor prog-osis in univariate analysis. However, the malignancy potential

[

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er 69 (2010) 289–295

was the only variable with an independent prognostic value (ona multivariate analysis basis).

5. Conclusions

In summary, our results point to a central role of the E-cadherin–�-catenin adhesion complex integrity, influenced by theSnail protein expression levels, in NELTs spectrum, and suggest thepotential diagnostic value of an immunohistochemical detection of�-catenin protein levels in LCNEC and SCLC discrimination.

Conflict of interest

The authors have nothing to disclose.

Acknowledgements

We thank Aitana Vallina, Olivia García Suárez and Marta SánchezPitiot for an excellent technical help and Inigo Santamaría for tech-nical advice and critical review of the manuscript. This work wassupported by Obra Social CajAstur, FICYT (to JAG) and ProgramaRamon y Cajal (Ministerio de Educacion y Ciencia-Spain, to MVG).The HUCA Tumor Bank, is supported by RTICC.

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