Galvan et al. - AJCP 2013

Am J Clin Pathol 2013;140:61-72 6161 DOI: 10.1309/AJCPIV40ISTBXRAX 61

© American Society for Clinical Pathology

Anatomic Pathology / EMT in GEP NET Differential Diagnosis

Epithelial-Mesenchymal Transition Markers in the Differential Diagnosis of Gastroenteropancreatic Neuroendocrine Tumors

Jose A. Galván, PhD,1 Aurora Astudillo, PhD,2 Aitana Vallina,1 Paula J. Fonseca, PhD,3 Lourdes Gómez-Izquierdo, PhD,4 Rocío García-Carbonero, PhD,5 and Maria Victoria González, PhD6

Key Words: Neuroendocrine tumors; EMT; Snail1; Snail2; Foxc2; E-cadherin; b-catenin

DOI: 10.1309/AJCPIV40ISTBXRAX

A b s t r a c t

Objectives: To elucidate the role of epithelial-mesenchymal transition markers in gastroenteropancreatic neuroendocrine tumors (GEP NETs) and the potential usefulness in their clinical management.

Methods: One hundred ten GEP NET paraffin-embedded samples were immunohistochemically analyzed for E-cadherin, N-cadherin, b-catenin, vimentin, Snail1, Snail2, Twist, and Foxc2 protein expression.

Results: The 5-year survival rate was reduced for those patients showing high Snail1 protein levels, a cytoplasmic E-cadherin pattern, reduced N-cadherin expression, and loss of E-cadherin/b-catenin adhesion complex integrity at the cell membrane. Interestingly, high b-catenin expression was useful in identifying a grade 1 NET subgroup with a favorable clinical course. Importantly, it also helped to discriminate small-cell vs large-cell grade 3 neuroendocrine carcinomas.

Conclusions: b-Catenin and N-cadherin immunohistochemical detection might be a useful tool in the differential diagnosis of small-cell vs large-cell G3 neuroendocrine carcinomas. High Snail1 and Foxc2 expression is associated with the invasion and metastatic spread of GEP NETs.

Gastroenteropancreatic neuroendocrine tumors (GEP NETs) are a poorly understood heterogeneous group of lesions derived from the neuroendocrine cells in the gastro-intestinal mucosa and the pancreas.1,2 The overall incidence of GEP NETs is about 2.5 to 5 cases per 100,000 and has increased significantly over the past 3 decades.3

These tumors are diagnosed by light microscopy based on their growth pattern and neuroendocrine differentiation. According to the recent World Health Organization (WHO) classification, they are classified as grade 1 neuroendocrine tumors (G1 NETs), grade 2 neuroendocrine tumors (G2 NETs), and grade 3 neuroendocrine carcinoma, large cell (G3 NEC-LC) or small cell (G3 NEC-SC) subtype. Immunohis-tochemically, these tumors stain for neuroendocrine markers chromogranin A and synaptophysin. Some features, such as the mitotic index, Ki-67 labeling index, tumor size, and the presence of necrosis or blood vessel invasion, are required for tumor classification.4

The GEP NETs show a variable biologic aggressiveness and a clinical course that is poorly predicted by the clinical prognostic factors currently available in the clinical setting. Some patients showing low-risk pathologic features develop metastasis unexpectedly, compromising their life expectancy. The molecular basis underlying GEP NET development is poorly understood, and few studies have been reported to date.

The epithelial-mesenchymal transition (EMT) is a biologic process whereby epithelial cells express proteins (eg, vimentin) and show morphologic features of a mesen-chymal phenotype.5,6 Epithelial cells are characterized by a cuboidal morphology and maintenance of cell polarity. Cells tightly interact with each other through homotypic adhesion complexes mediated by cell-cell junction proteins

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62 Am J Clin Pathol 2013;140:61-7262 DOI: 10.1309/AJCPIV40ISTBXRAX


Galván et al / EMT in GEP NET Differential Diagnosis

(composed mainly of the cadherin family). In EMT, the cells lose their polarity and cell-cell adhesion and gain motility and invasive capacities.7

Epithelial-mesenchymal transitions have recently been classified into 3 general subgroups: type 1 EMT involves tran-sitions during embryonic development (gastrulation, devel-opment of the neural crest, etc), type 2 EMT during tissue fibrosis, and type 3 EMT in the carcinogenic process.8,9

In the carcinogenic setting, EMT takes place in the first phase of the metastatic cascade, prior to the local invasion. Once a distant organ is colonized, tumor cells must return to the epithelial phenotype by the reverse process known as the mesenchymal-epithelial transition.

At the molecular level, EMT transcriptional reprogram-ming occurs through transcription factors, such as Snail1, Snail2, Foxc2, and Twist, and the subsequent repression of adhesion molecules expression, such as E-cadherin.10-14 Another event triggered in EMT is the so-called cadherin switch, defined as an increase in N-cadherin expression with or without a decrease in E-cadherin expression.15 In addition, Twist has recently been shown to be involved in the cadherin switch as an inducer of N-cadherin expression.16

Additional markers are urgently needed for the cor-rect handling of these patients and clinical decision making regarding treatment options and patient follow-up. Our study analyzes EMT regulator expression involved in E-cadherin/b-catenin complex alteration in the whole spectrum of GEP NETs and patients’ prognosis.

Materials and Methods

Patients and SamplesSamples from 110 patients with GEP NETs were col-

lected at the Hospital Universitario Central of Asturias and Hospital Virgen del Rocío of Seville, Spain, between Febru-ary 2000 and October 2009, with institutional review board approval for guidelines on ethical procedures. The diagnosis of NETs was based on morphologic criteria according to the updated WHO grading classification in 2010.4 The charac-teristics of the studied patients (age, sex, and risk behaviors) and the clinicopathologic features of their tumors (WHO clas-sification, tumor site and size, differentiation, stage, mitotic index, proliferation index, vascular invasion, lymph node status, necrosis, and the presence of metastasis) are shown in ❚Table 1❚. The anatomic locations of the 110 tumors were the stomach (18), small intestine (28), pancreas (22), colon/rectum (23), and appendix (19).

Tissues obtained from biopsy specimens were fixed in 10% formaldehyde and paraffin embedded, cut 4 mm thick, mounted on treated slides, and stained with H&E. The

❚Table 1❚Clinicopathologic Parameters of Gastroenteropancreatic Neuroendocrine Tumors

Characteristic No. (%) (n = 91)a

Age, y (mean, 60 y; range, 36-87 y) ≤60 49 (53.8) >60 42 (46.2)Sex Male 49 (53.8) Female 42 (46.2)Tobacco consumption Nonsmoker 26 (28.6) Smoker 29 (31.9) Unknown 36 (39.6)Alcohol consumption Nondrinker 31 (34.1) Drinker 31 (34.1) Unknown 29 (31.9)WHO diagnosis G1 NET 49 (53.8) G2 NET 17 (18.7) G3 NEC-LC 5 (5.5) G3 NEC-SC 20 (22.0)Tumor site Stomach 18 (19.8) Small intestine 28 (30.8) Pancreas 22 (24.2) Colon and rectum 23 (25.3)Mean tumor size, cm ≤2.7 49 (53.8) >2.7 36 (39.6) Unknown 6 (6.6)Histologic differentiation Well 68 (74.7) Poor 23 (25.3)Tumor stage I 23 (25.3) IIA 7 (7.7) IIB 7 (7.7) IIIA 1 (1.1) IIIB 24 (26.4) IV 21 (23.1) Unknown 8 (8.8)Mitotic index, 10× high-power fields ≤2 59 (64.8) 3-20 16 (17.6) >20 16 (17.6)Proliferation index (Ki-67) <2% 50 (54.9) 2%-20% 17 (18.7) >20% 24 (26.4)Vascular invasion Absent 48 (52.7) Present 43 (47.3)Lymph node metastasis Absent 59 (64.8) Present 32 (35.2)Necrosis Absent 71 (78.0) Present 20 (22.0)Distant metastasis Absent 70 (76.9) Present 21 (23.1)

G1, grade 1; G2, grade 2; G3, grade 3; NEC-LC, neuroendocrine carcinoma, large cell subtype; NEC-SC, neuroendocrine carcinoma, small cell subtype; NET, neuroendocrine tumor; WHO, World Health Organization.

a Appendix tumors excluded.

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Anatomic Pathology / Original Article

neuroendocrine phenotype was confirmed by chromogranin A and synaptophysin immunohistochemical staining. Nor-mal tissue present in each tissue section was considered a reference.

Tissue Microarray ConstructionRepresentative tumor regions were identified on H&E-

stained paraffin sections in each sample and selected to make 4 tissue microarrays containing 3 tissue cores from each of the 110 GEP NETs. Core cylinders 1 mm in diameter were processed using Microarrayer (Beecher Instruments, Sun Prairie, WI). Three core cylinders, punched from the donor block, were then deposited in the recipient paraffin block. After 5 minutes at 60°C, the tissue microarray blocks were cut in 4-mm-thick sections, ready for immunohisto-chemical techniques.

ImmunohistochemistryThe automated system DISCOVERY (Ventana Medical

Systems, Tucson, AZ) was used to carry out the immuno-histochemical protein detection of interest. Sections were deparaffinized and rehydrated in EZ Prep (Ventana Medi-cal Systems) for 20 minutes. Antigen retrieval was done by heating citrate buffer solution (pH 6.5) and HCl-Tris buffer solution (pH 9.0). Nonspecific antibody binding was blocked using casein (Antibody Block, Ventana Medical Systems) for 20 minutes. Endogenous peroxidase activity was blocked with H2O2 solution (Inhibitor, Ventana Medical Systems) for 4 minutes. Samples were incubated at 37°C with primary anti-bodies described in ❚Table 2❚. The slides were incubated with the secondary antibody (OmniMap, Ventana Medical Sys-tems) for 30 minutes at room temperature. Then the samples were visualized with 3,3'-diaminobenzidine (DAB, Ventana Medical Systems). Finally, samples were counterstained with hematoxylin (Ventana Medical Systems) and dehydrated and mounted in Entellan (Merck, Darmstadt, Germany). The sec-tions were studied and photographed (20× objective) under a light microscope (Eclipse 80i, Nikon, Tokyo, Japan).

Double ImmunohistochemistryTo assess the expression of EMT markers in healthy

neuroendocrine cells, we performed double immunohisto-chemistry in normal intestinal mucosa, using synaptophysin antibody (MRQ-40, Ventana Medical Systems) with an ultra-View Universal Alkaline Phosphatase Red Detection Kit as chromogen (Ventana Medical Systems). The EMT markers were visualized with DAB as chromogen.

Immunohistochemistry AssessmentThe protein expression levels were evaluated by a semi-

quantitative method by 2 independent observers (A.A. and L.G.I., with a third observer [M.V.G.] in case of disagreement),

taking into account 2 parameters: immunohistochemical sig-nal intensity (graded on a 0- to 3-point scale) and the percent-age of positive cells (0-100). All tumor cells in a core were considered to assess the percentage of positive tumor cells in a 20× field (1.2 mm). The product of both parameters ren-dered a score for each specimen. For statistical purposes, the tumors were divided into 2 groups, taking the median score value for each marker as a cutoff point.

For cadherins and b-catenin, their presence or absence in the cell membrane was recorded. A variable reflecting the integrity of the E-cadherin/b-catenin complex in the cel-lular membrane was created, including 2 categories: integrity retained (both molecules showing a membranous pattern) and integrity lost (expression of at least 1 that was not observed in the membrane). For EMT markers (Snail1, Snail2, Twist, and Foxc2), only the nuclear immunostaining signal was considered.

Statistical AnalysisThe experimental results distributed among the different

clinical groups of tumors were tested for significance employ-ing the χ2 test (with Yates correction, when appropriate). Sur-vival curves were calculated using the Kaplan-Meier product limit estimate. Deaths from causes other than the index tumor or its metastases were not considered treatment failures, and these patients were censored in all analyses involving length of survival. Differences between survival times were analyzed by the log-rank method. Multivariate Cox proportional haz-ards models (forward Wald method) were used to examine the relative impact of statistically significant variables in a univariate analysis. All statistical analysis was performed with SPSS 19.0 (SPSS, Chicago, IL). All tests were 2-sided. P values less than .05 were considered statistically significant.

❚Table 2❚Antibodies Used in Immunohistochemical Analysis

Incubation Antibody (Species) Antigen Retrieval Dilution Time, min

Snail1 (P)a CB, pH 6.5 1:300 60Snail2 (P)a CB, pH 6.5 1:100 60Twist (P)a CB, pH 6.5 1:1000 30Foxc2 (P)b CB, pH 6.5 1:600 30E-cadherin (M)c TB, pH 9 1:100 20b-catenin (M)d TB, pH 9 1:1000 20N-cadherin (M)e TB, pH 9 1:200 30Vimentin (M)f TB, pH 9 1:100 20Synaptophysin (M)f TB, pH 9 1:100 30

CB, citrate buffer; M, monoclonal mouse; P, polyclonal rabbit; TB, Tris-HCl buffer.a Abcam, Cambridge, UK.b ABR Affinity Bioreagents, Golden, CO.c DAKO, Glostrup, Denmark.d Sigma, St Louis, MO.e Becton Dickinson, Franklin Lakes, NJ.f Ventana Medical Systems, Tucson, AZ.

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ResultsResults are reported following the REMARK guidelines

(REporting recommendations for tumor MARKer prognostic studies).17

Previous ConsiderationsAn initial survival analysis of the entire series revealed

a striking prognostic difference when the primary tumor anatomic site was considered. Neuroendocrine tumors aris-ing in the appendix (n = 19) showed a cumulative 5-year survival rate of 94.4% ❚Figure 1❚. They belonged to favor-able categories: G1 grade; good differentiation; early stage; no vascular invasion, lymph node involvement, or necrosis; small size (<1 cm); and low proliferation rate (<2 mitosis 10× high-power field and <2% Ki-67 expression). These observations suggest that the biology underlying the devel-opment of NETs of the appendix may differ from NETs of other gastrointestinal anatomic origin. For this reason, we decided to exclude these cases from the series and study them separately.

No significant survival differences were observed among the other anatomic sites (Figure 1). A subset analysis based on site was performed, but strong statistical conclu-sions could not be drawn due to sample size. Thus, the results reported here refer to GEP NETs as a whole, with the appendix excluded.

Protein Expression in Healthy vs Tumor TissueIn the healthy intestinal neuroendocrine cells, E-cad-

herin and b-catenin immunostaining was detected in the membrane with a linear pattern ❚Image 1A❚ and ❚Image 1B❚. Both proteins form the E-cadherin/b-catenin complex, which is essential in maintaining tissue homeostasis. N-cad-herin and vimentin expression was negative in healthy intestinal neuroendocrine cells. None of the transcrip-tional repressors showed a nuclear signal in healthy intes-tinal neuroendocrine cells ❚Image 1C❚ and ❚Image 1D❚. In tumors, E-cadherin, b-catenin, and N-cadherin were highly expressed in 43 (47.3%), 46 (50.5%), and 61 (67.0%) of 91 cases, respectively. Vimentin expression was detected in 10 (11.0%) of 91 cases, 7 of which were G1 NETs. According to the E-cadherin/b-catenin complex integrity status, 37 (41.6%) of 89 tumors were classified as having lost com-plex integrity. In tumors, Snail1, Snail2, Twist, and Foxc2 were positive in 45 (50.0%) of 90 cases and in 40 (44.0%), 43 (47.3%), and 52 (57.1%) of 91 cases, respectively. Representative immunostainings of E-cadherin, b-catenin, Snail, and Foxc2 are shown in ❚Image 2❚ (G1 NETs), ❚Image 3❚ (G2 NETs), ❚Image 4❚ (G3 NEC-LC), and ❚Image 5❚ (G3 NEC-SC).

Most tumors arising in the appendix showed no Snail1, Snail2, Twist, Foxc2, or vimentin expression and high E-cadherin expression (P < .05 for all).

Associations Between Clinicopathologic and Protein Expression Variables in GEP NETs

Grade 3 NEC-LC and NEC-SC showed the following changes in EMT marker expression more often than G1 and G2 NETs: E-cadherin cytoplasmic pattern (P = .001), weak b-catenin expression (P = .04), high Foxc2 expression (P = .02), and a trend toward high Snail1 expression (P = .08). Tumor grade and the presence of necrosis were associated with a cytoplasmic pattern of E-cadherin (P = .001 and P = .047, respectively), weak N-cadherin expression (P = .003 and P = .004, respectively), high Snail1 expression (P = .04 and P = .04, respectively), and high Foxc2 expression (P = .005 and P = .004, respectively).

High N-cadherin expression was associated with the favorable findings of absence of necrosis (P = .004) and G1 status (P = .0001). Moreover, a cytoplasmic N-cadherin pat-tern was associated with an absence of nodal involvement (P = .04) and early stage tumors (I-II; P = .049). Tumors of patients with excessive alcohol consumption showed an altered E-cadherin/b-catenin complex (P = .01).

Differential Diagnosis of GEP NETsOne of the aims of our study was to find molecular

variables that might help in the differential diagnosis of NET subgroups. Thus, we established comparisons of each

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❚Figure 1❚ Primary tumor location and survival rates. Kaplan-Meier survival analysis of the 110 patient series is stratified according to the primary tumor anatomic site of origin.

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protein expression variable between the following sub-groups: G1 NET vs G2 NET and G3 NEC-LC vs G3 NEC-SC. We found that weak b-catenin and N-cadherin expres-sion discriminated between G3 NECs, being more common in the small cell subtype than in the large cell subtype (P = .009 and P = .002, respectively).

Associations Between Protein Expression FindingsE-cadherin protein expression levels were independent

of the Snail1 levels. However, E-cadherin cytoplasmic localization was correlated with high Snail1 expression (P = .03) and, as expected, with the loss of the integrity of the E-cadherin/b-catenin complex (P = .001). Also, elevated N-cadherin expression was associated with the conserved

E-cadherin/b-catenin complex (P = .04). An association between high Twist expression and high N-cadherin level at the membrane (P = .007) was observed. The elevated Foxc2 expression levels correlated with high Snail1 (P = .0001), Snail2 (P = .002), and Twist expression (P = .001). No evi-dence of the described “cadherin switch” was found.

Survival AnalysisThe mean follow-up was 58 months (range, 1-130

months), and the 5-year cumulative survival rate was 56.8%. A negative impact on patient survival was found for the follow-ing clinicopathologic variables: age, diagnosis, tumor grade, mitotic index, proliferation index (Ki-67), histologic differen-tiation, and necrosis. The molecular variables with a negative

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❚Image 1❚ Representative double immunostaining of healthy intestinal mucosa: synaptophysin expression identifies the neuroendocrine cell (red) with normal E-cadherin (brown) (A) and b-catenin (brown) (B) expression (arrows). Nuclear expression of Snail1 (C) and Foxc2 (D) is negative (arrows) in neuroendocrine cells (×400).

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impact on survival were loss of integrity complex ❚Figure 2A❚, reduced N-cadherin expression ❚Figure 2B❚, high Snail1 level ❚Figure 2C❚, and the cytoplasmic E-cadherin pattern ❚Figure 2D❚. Mean survival times for each category are shown in ❚Figure 3❚. In the multivariate survival analysis (Cox) in which significant variables were included according to Kaplan-Meier analysis, the variables with an independent prognostic value were the mitotic index (2.6-fold increased risk of death by disease, P = .0001) and Snail1 expression (2.17-fold increased risk of death by disease, P = .05).

Differences in Survival Between G1 NETsGrade 1 NETs show the peculiarity that their histologic

differentiation does not correlate with tumor invasion and metastasis. This type of tumor was the most prevalent in our

series (n = 49), which allowed for a statistical analysis. In a search of variables with prognostic significance, we performed a survival analysis and found that age younger than 60 years and high b-catenin expression identified subgroups of patients with better survival rates (P = .04 and P = .04, respectively).

Discussion

Gastroenteropancreatic NETs are a heterogeneous group of rare neoplasms with an incidence that has increased over the past 30 years. Frequently, their histologic differentiation does not correlate with their clinical aggressiveness. Thus, additional features might determine their biologic behavior. With the aim of gaining insight into the molecular alterations present in GEP NETs, we focused our study on the EMT in

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❚Image 2❚ Representative immunostaining of grade 1 neuroendocrine tumors: E-cadherin (A), b-catenin (B), Snail1 (C), and Foxc2 (D) (×200).

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tissues, noting that the E-cadherin/b-catenin complex showed a membrane distribution in the intestinal neuroen-docrine cells, with a linear pattern and a high expression level of both molecules. This observation is consistent with the function of this complex in mediating intercellular adherens junctions.

To determine the impact of the E-cadherin/b-catenin complex integrity loss in the tumor cells, we divided the samples into 2 groups: those that retained the expression of both components in the membrane and those in which at least 1 was absent. Integrity was lost in 43% of tumors. There was no association with any clinicopathologic vari-able except for excessive alcohol consumption, an observa-tion also described by Wang et al.22 The cytoplasmic E-cad-herin pattern was significantly associated with high-grade

these neoplasms. The present work, based on a represen-tative series of human primary tumors with a significant number of samples, provides a way to identify pathologic or molecular parameters possessing potential application in the clinical setting with a diagnostic and/or prognostic value.

The EMT is physiologically present as an essential process in embryonic development and wound healing. Its pathologic activation during carcinogenesis triggers tumor invasion, promoting the metastatic cascade.10,13,14,18 In recent years some studies have shown that during the early stages of tumor invasion there is a loss of intercellular adhesion that is normally preserved by the E-cadherin/b-catenin complex at the cell membranes.19-21 This complex is essential in maintaining the integrity of epithelia. We first evaluated the status of those molecules in normal

❚Image 3❚ Representative immunostaining of grade 2 neuroendocrine tumors: E-cadherin (A), b-catenin (B), Snail1 (C), and Foxc2 (D) (×200).

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Next, we evaluated the expression of the transcription fac-tors responsible for E-cadherin gene repression and thus EMT triggering. The Snail1, Snail2, Twist, and Foxc2 transcription factors have been extensively studied in recent years and have been involved in the progression of many tumor types. Nuclear Snail1 has been evaluated using specific antibodies in studies on melanoma,27 breast cancer,28 colon cancer,29 gastrointesti-nal cancer,30 and fibrosarcomas.31 For the first time, our group reported the contribution of Snail to the aggressiveness of pulmonary NETs.32 Few studies have revealed the involve-ment of these factors on GEP NETs, mostly related to the E-cadherin/b-catenin complex.33-37

In the present study, we found that immunostaining for these factors was negative in normal neuroendocrine cells. In the tumor series, high Snail1 expression was associated with

tumors (G3 NEC-LC and G3 NEC-SC), results in line with those reported by Kawahara et al.23

An interesting finding was the absence of the cadherin switch (defined as the increased expression of N-cadherin, with or without reduction of E-cadherin), a process also described in embryonic development and in the carcinogen-esis of other tumor types such as breast, prostate, bladder, and melanoma.15,24 Our results show that N-cadherin expression does not increase with tumor malignancy but tends to decrease, in parallel with E-cadherin expression. Similar results have been described for osteosarcomas,25 in which high levels of N-cadherin are associated with inhibition of cell migration and metastasis. Another team found that N-cadherin levels were independent of tumor malignancy in astrocytomas, although they seemed to facilitate the spread of local recurrences.26

❚Image 4❚ Representative immunostaining of grade 3 large-cell neuroendocrine carcinomas: E-cadherin (A), b-catenin (B), Snail1 (C), and Foxc2 (D) (×200).

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chemotherapy regimen. However, given the current uncertainty as to whether they show different responses to treatment, it is now of paramount importance to find a clear molecular distinction between them, especially when new molecularly targeted therapies are considered. Some recent revisions reveal differences between the 2 types of tumors. Shia et al40 suggest that most of the HGNECs arising in the squamous lined parts (esophagus and anal canal) are of the small cell type, whereas most involving the glandular mucosa are of the large cell or mixed type. No significant differences in survival have been found until now. In the present study, we observed that weak N-cadherin and b-catenin expression discriminated between G3 NEC-LC and G3 NEC-SC. This is a novel result that pro-vides protein expression information with potential usefulness in the clinical management of these tumors (diagnosis).

a cytoplasmic E-cadherin pattern, as previously described.33 There was also a correlation between Twist and N-cadherin levels, consistent with the role described for Twist as a direct inducer of N-cadherin.16 For the first time, we found Foxc2 as the only transcription factor whose expression was associ-ated with high malignancy potential, being more frequent in G3 NECs vs G1 and G2 NETs. Another objective was to find molecular variables that could discriminate between dif-ferent tumor subtypes, facilitating the pathologic differential diagnosis. The NEC-LCs and NEC-SCs are highly aggressive types of cancers (grouped as high-grade malignancy tumors, HGNECs), considered distinct morphologic entities, with specific clinical manifestations, in the WHO 2010 lung tumor classification.38 The actual suggested treatment algorithm39 includes both morphologic entities to be treated with the same

❚Image 5❚ Representative immunostaining of grade 3 small-cell neuroendocrine carcinomas: E-cadherin (A), b-catenin (B), Snail1 (C), and Foxc2 (D) (×200).

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expression, and cytoplasmic E-cadherin pattern reduced sur-vival rates of patients with GEP NET.

Importantly, among the G1 tumor group, we identified 2 variables—age older than 60 years and weak b-catenin expression—that identified those cases with worse progno-sis. This result might be of importance considering that the prognosis of these cases is difficult to establish.

In our series, as expected, clinicopathologic variables such as the mitotic index, Ki-67, tumor grade, tumor dif-ferentiation, and necrosis had a significant influence on sur-vival times. When the tumors arising in the appendix were excluded, no significant survival difference was observed for anatomic site. Among the molecular variables, the alteration of the E-cadherin/b-catenin complex, high Snail1

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❚Figure 2❚ Molecular findings affecting survival rates. Cumulative Kaplan-Meier survival curves are stratified according to the integrity of the E-cadherin/b-catenin complex (P = .04) (A), N-cadherin expression level (P = .055) (B), Snail1 protein expression levels (P = .02) (C), and E-cadherin pattern (P = .005) (D).

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Address reprint requests to Dr Galván: Laboratorio del Banco de Tumores–Anatomia Patológica, c/ Celestino Villamil, s/n, 33006 Oviedo, Spain; [email protected].

Acknowledgments: We thank the Pathology Department and Tumor Registry of Hospital Universitario Central de Asturias (HUCA) for technical help and clinical information and Jorge Martinez Aranda for graphic design support.

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gastroenteropancreatic endocrine system and related tumors. Gastroenterol Clin North Am. 1989;18:671-693.

2. Kloppel G, Rindi G, Anlauf M, et al. Site-specific biology and pathology of gastroenteropancreatic neuroendocrine tumors. Virchows Arch. 2007;451(suppl 1):S9-S27.

3. Modlin IM, Lye KD, Kidd M. A 5-decade analysis of 13,715 carcinoid tumors. Cancer. 2003;97:934-959.

4. Bosman FT, Carneiro F, Hruban RH, et al. WHO Classification of Tumours of the Digestive System. Sterling, VA: Stylus; 2010.

5. Thiery JP. Epithelial-mesenchymal transitions in tumour progression. Nat Rev. 2002;2:442-454.

6. Thiery JP, Acloque H, Huang RY, et al. Epithelial-mesenchymal transitions in development and disease. Cell. 2009;139:871-890.

7. Thiery JP. Transitions epithelium mesenchyme dans le developpement et la progression des carcinomes [Epithelial-mesenchymal transitions in cancer onset and progression]. Bull Acad Natl Med. 2009;193:1969-1979.

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Regarding the NETs arising in the appendix, our results (no Snail1, Snail2, Foxc2, or vimentin expression and high E-cadherin expression) are compatible with an absence of EMT. This could help to explain the low malignant pheno-type of these tumors.

In summary, our results show the importance of the loss of integrity of the E-cadherin/b-catenin complex in the development of GEP NETs. Furthermore, this study provides molecular tools with potential utility in GEP NET management, helping in the differential diagnosis and pro-viding information about prognosis.

From the 1Laboratorio del Banco de Tumores, Instituto Universitario de Oncología del Principado de Asturias, Obra Social CajAstur, Oviedo, Spain; 2Servicio de Anatomía Patológica, Hospital Universitario Central de Asturias, Oviedo, Spain; 3Servicio de Oncología Médica, Hospital Universitario Central de Asturias, Oviedo, Spain; 4Servicio de Anatomía Patológica and 5Servicio de Oncología Médica, Hospital Universitario Virgen del Rocío–Instituto de Biomedicina de Sevilla, Sevilla, Spain; and 6Unidad de Oncología de Cabeza y Cuello, Instituto Universitario de Oncología del Principado de Asturias, Obra Social CajAstur, Oviedo, Spain.

The Instituto Universitario de Oncologiá del Principado de Asturias (IUOPA) is supported by Obra Social CajAstur. The HUCA Tumor Bank is supported by Red Temática de Investigación Cooperativa en Cancer (RTICC). J.A.G. was supported by FICYT (Fundación para el fomento en Asturias de la Investigación Científica Aplicada y la Tecnología) and M.V.G. by Programa Ramon y Cajal (former Ministerio de Ciencia e Innovación–Spain).

❚Figure 3❚ Mean survival times for each category of the molecular variables with an impact on survival.

Integrity ofE-Cadherin/

β-Catenin Complex

Preserved0

10

20

30

40

50

60

70

80

90

100

Lost

N-Cadherin/Expression

Mea

n S

urvi

val T

ime

(mo

)

Weak High

E-Cadherin/Pattern

Membrane Cytoplasm

Snail1Expression

Weak High

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