The clinical implications and biologic relevance of neurofilament

Oct 11, 2011 ... lated with tumor size, infiltration, and grade (P < .05). CONCLUSIONS: ... Group, Department of Gastroenterological Surgery, Yale Uni...
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Original Article

The Clinical Implications and Biologic Relevance of Neurofilament Expression in Gastroenteropancreatic Neuroendocrine Neoplasms Simon Schimmack, MD1,2; Ben Lawrence, MB, ChB, MSc1; Bernhard Svejda, MD1; Daniele Alaimo, BS1; ¨ chler, MD2; Mark Kidd, PhD1; Hubertus Schmitz-Winnenthal, MD2; Lars Fischer, MD2; Markus W. Bu and Irvin Modlin, MD, PhD1

BACKGROUND: Although gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs) exhibit widely divergent behavior, limited biologic information (apart from Ki-67) is available to characterize malignancy. Therefore, the identification of alternative biomarkers is a key unmet need. Given the role of internexin alpha (INA) in neuronal development, the authors assessed its function in neuroendocrine cell systems and the clinical implications of its expression as a GEPNEN biomarker. METHODS: Functional assays were undertaken to investigate the mechanistic role of INA in the pancreatic BON cell line. Expression levels of INA were investigated in 50 pancreatic NENs (43 primaries, 7 metastases), 43 small intestinal NENs (25 primaries, 18 metastases), normal pancreas (n ¼ 10), small intestinal mucosa (n ¼ 16), normal enterochromaffin (EC) cells (n ¼ 9), mouse xenografts (n ¼ 4) and NEN cell lines (n ¼ 6) using quantitative polymerase chain reaction, Western blot, and immunostaining analyses. RESULTS: In BON cells, decreased levels of INA messenger RNA and protein were associated with the inhibition of both proliferation and mitogen-activated protein kinase (MAPK) signaling. INA was not expressed in normal neuroendocrine cells but was overexpressed (from 2fold to 42-fold) in NEN cell lines and murine xenografts. In pancreatic NENs, INA was overexpressed compared with pancreatic adenocarcinomas and normal pancreas (27-fold [P ¼ .0001], and 9-fold [P ¼ .02], respectively). INA transcripts were correlated positively with Ki-67 (correlation coefficient [r] ¼ 0.5; P < .0001) and chromogranin A (r ¼ 0.59; P < .0001). INA distinguished between primary tumors and metastases (P ¼ .02), and its expression was correlated with tumor size, infiltration, and grade (P < .05). CONCLUSIONS: INA is a novel NEN biomarker, and its expression was associated with MAPK signaling and proliferation. In clinical samples, elevated INA was correlated with Ki-67 and identified malignancy. INA may provide additional biologic information relevant to delineation of both pancreatic C 2011 American Cancer Society. NEN tumor phenotypes and clinical behavior. Cancer 2012;118:2763-75. V KEYWORDS: biomarker, BON pancreatic cell line, carcinoids, internexin alpha, Ki-67, metastasis, neuroendocrine neoplasm, neurofilament, pancreatic neuroendocrine tumor.

INTRODUCTION Most gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs) are sporadic lesions, although some, especially pancreatic neuroendocrine neoplasms (pNENs), may occur as part of familial tumor syndromes, such as multiple endocrine neoplasia type 1 (MEN1 syndrome), von Hippel-Lindau disease, neurofibromatosis type 1 (NF-1), and tuberous sclerosis.1 Overall, the incidence of GEP-NENs has increased exponentially during the last 3 decades.2 Thus, they comprise 1% of all malignancies; and, in terms of prevalence, GEP-NENs represent the second most common gastrointestinal malignancy after colorectal cancer.3 The diagnosis of GEP-NEN is late, and metastases are evident in 60% to 80% of patients at presentation.4 Neuroendocrine carcinomas (NECs) often have the same poor prognosis as adenocarcinomas of the same organ, for example, Corresponding author: Mark Kidd, PhD, Gastrointestinal Pathobiology Research Group, Department of Gastroenterological Surgery, Yale University School of Medicine, PO Box 208602, New Haven, CT 06520-8062; Fax: (203) 737-4067; [email protected]; Irvin Modlin, MD, PhD, Gastrointestinal Pathobiology Research Group, Department of Gastroenterological Surgery, Yale University School of Medicine, P.O. Box 208602, New Haven, CT 06520-80762; Fax: (203) 737-4067; [email protected] 1 Gastrointestinal Pathobiology Research Group, Department of Gastroenterological Surgery, Yale University School of Medicine, New Haven, Connecticut; 2University Hospital of General, Visceral, and Transplantation Surgery of Heidelberg, Heidelberg, Germany

DOI: 10.1002/cncr.26592, Received: July 8, 2011; Revised: August 31, 2011; Accepted: September 12, 2011, Published online October 11, 2011 in Wiley Online Library (wileyonlinelibrary.com)

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Figure 1. The schematic structure of neurofilaments (NFs), chromosomal (chr) location, and coding size is illustrated. Each NF has an amino-terminal and flexible phosphorylation target site (orange) at the head. The tail exhibits a carboxyterminal flexible phosphorylation target site (brown). The long middle rod domain contains 4 alpha-helices separated by short amino acid sequences (black). INA indicates internexin alpha; NF-L, light neurofilament; NF-M, medium neurofilament; NF-H, heavy neurofilament.

colorectal neoplasms.5 The reasons for these poor outcomes reflect late diagnosis because of failure to identify nonspecific symptoms as well as topographic investigations that fail to identify tumors at an early stage and the lack of well characterized biomarkers. Currently, Ki-67 is the best characterized proliferation-associated marker and has been proposed as an important parameter in defining neuroendocrine neoplasia.6,7 This cell cycle-associated protein has some utility as a prognostic predictor of 5-year survival in pNENs8 but is less accurate in other NENs.9,10 This may reflect both the different cells implicated in the genesis of this heterogeneous group of neoplasms (GEP-NENs) and the reality that the origin of the cells from which GEP-NENs arise is not well understood (intestine and pancreas contain at least 17 different neuroendocrine cell types).3 The hybrid term ‘‘neuroendocrine’’ is a composite description of a cell type that exhibits mixed morphologic and physiologic attributes of both the neural and endocrine regulatory systems.3 These congruencies also are features of neoplasia; neuroblastomas can transdifferentiate into cells with neuroendocrine features, highlighting the plasticity of their interchangeable phenotypes.11 Given these phenotypic commonalities,12,13 we hypothesized that biomarkers relevant to a neural cell system also may be implicated in defining the phenotype of GEP-NENs. Because internexin alpha (INA) and its family members (Fig. 1) are components of a cytoskeletal protein class14 (key to the neural system), we investigated this group of class IV intermediate filaments or neurofilaments (NFs) as potential biomarkers. The basis for this analysis reflected our assessment using gene-interactive pathway analysis15 of published GEP-NEN microarray data from our group. These studies demonstrated that

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INA was overexpressed (from 3-fold to 10-fold) in both small intestinal NENs (SI-NENs) and pNENs.16 Both the level of expression and the observation that INA was coexpressed with several genes associated with neuronal development (synapsin 1 and chromogranin B) suggested that GEP-NENs and neuronal tumors express common biomarkers and that the INA protein family may play a role in neuroendocrine cell growth and development. Class IV neurofilaments have been identified previously in postmitotic neurons during the development of the central nervous system (CNS) and the peripheral nervous system (PNS)17,18 and are expressed in enteric nerves.19 In adults, INA is present predominantly in the CNS and at low levels in the PNS, where it is colocalized with light neurofilament (NF-L), medium neurofilament (NF-M), and heavy neurofilament (NF-H) in most axons.14 In neoplasia of the nervous system, INA protein expression has been demonstrated in neuroblastomas,20,21 medulloblastomas,22 and oligodendrogliomas23; elevated expression is considered to have prognostic significance.24 In the current study, we investigated whether neurofilaments were expressed in normal neuroendocrine cells, GEP-NEN cell lines, and a xenograft model. In particular, we assessed whether neurofilament expression was related to proliferation. To assess whether this was representative of all NENs or specific to pNENs, we also investigated whether transcript and protein levels were expressed differentially in normal pancreatic tissue, pNEN, and pancreatic adenocarcinoma (pAC). To evaluate the clinical relevance and specificity of our observations, we assessed the expression of INA in SI-NENs, Crohn’s disease, and in normal SI mucosa. Although we focused particularly on INA, we also investigated expression levels of other members of this family (NFL, NF-M, and NF-H) to assess their utility as potential biomarkers of pNENs. In addition, to evaluate whether differential expression provided information with respect to biologic behavior, we examined whether transcripts or protein levels of these neurofilaments differed between GEPNEN primaries and metastases. To evaluate the general utility of INA as a marker of NENs, we correlated expression with known neuroendocrine tumor biomarkers and progression parameters, including chromogranins A and B, Ki67, tumor size, ‘‘differentiation,’’ and metastatic spread.8,25

MATERIALS AND METHODS Cell Lines The SI-NEN cell lines KRJ-1, P-STS (both primary tumors), H-STS (liver metastasis), and L-STS (lymph

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node metastasis) were cultured as described previously.26 The human metastasized pNEN-adherent cell line BON was cultivated in medium containing RPMI 1640, Hams F12 medium (1:1 ratio), 10% fetal calf serum, and penicillin and streptomycin (100 IU/mL).27 To assess the role of INA in cell proliferation, we cultured BON cells (2.5  105 cells per well) in 6-well plates and harvested them after 2 days, 3 days, 5 days, and 7 days to investigate messenger RNA (mRNA) and protein levels. In separate experiments, to evaluate the effect of growth signaling on INA expression, BON cells (1  105 cells/mL) were seeded into 6-well plates (Falcon; BD, Franklin Lakes, NJ) and tested with PD98059 (a mitogen-activated protein kinase [MAPK] signaling pathway inhibitor; 105 M) or Wortmannin (a phosphatidylinositol 3-kinase/protein kinase B [PI3K/PKB] inhibitor; 108 M) for 1 hour before mRNA collection. Cells were harvested by adding TRIZOL (Invitrogen, Carlsbad, Calif) or lysis buffer (see below). Murine Xenograft Model The proliferation of the BON cell line was determined in vivo by subcutaneous injection into the flanks of Nu/J mice (Jackson Laboratory, Bar Harbor, Me). Animals were handled and stored according to institutional review board standards in the animal facility at the Yale University School of Medicine. Approximately 1  107 BON cells suspended in 0.2 mL saline were injected subcutaneously into the flanks of 4 mice, and the mice were killed after 60 days. Three pieces of tumor were harvested from each xenograft. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and Western blot analyses were undertaken on each specimen to detect INA, and qRT-PCR was used to detect the proliferation marker Ki-67. We normalized in vivo data to INA levels obtained in BON cells from day 2 (logarithmic growth phase; in vitro). This allowed us to directly compare xenograft expression with the expression in rapidly growing cells. Human Sample Collection Pancreatic tissues from patients with pNENs (n ¼ 50), pACs (n ¼ 21), and normal pancreas (n ¼ 10) as well as SI tissues from patients with SI-NENs (n ¼ 43) and normal SI mucosa (n ¼ 16) were obtained according to the ethics committee requirements for Heidelberg and a standard institutional review board protocol for Yale University School of Medicine. Protocols included steps to minimize the time from resection to processing and freezing. Clinical sample details are provided in Table 1.

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Table 1. Sample Details

Tissue Type/Organ

No.

Total

43 7 25 18

50

Neuroendocrine neoplasms Primary pNEN Liver metastasis of pNEN Primary SI-NEN Liver metastasis of SI-NEN

43

Control cancer 21

Pancreatic adenocarcinoma

Non-neoplastic tissue Normal pancreas Normal small bowel mucosa Croh’n disease

10 16 9

35

NEN cell lines BON (2), STS (3), KRJ-1 (1)

6

Abbreviations: NEN, neuroendocrine neoplasm; pNEN, pancreatic neuroendocrine neoplasm; SI-NEN, small intestinal neuroendocrine neoplasm.

Protein Extraction and Western Blot Analysis Small pieces of tissue (1  2 mm) or cell line lysates were manually homogenized (PYREX homogenizer; Corning Incorporated, Corning, NY) and were prepared by adding 500 lL of ice-cold cell lysis buffer (10 times radioimmunoprecipitation assay lysis buffer [RIPA; Millipore, Temecula,Calif], complete protease inhibitor [Roche, Indianapolis, Ind], phosphatase inhibitor sets 1 and 2 [Calbiochem, La Jolla, Calif], 100 mM phenylmethylsulfonyl fluoride [Roche], 200 mM Na3VO4 [Acros Organics, Geel, Belgium], and 12.5 mg/mL sodium dodecyl sulfate [SDS] [American Bioanalytical, Natick, Mass]).2 Tubes were centrifuged for 20 minutes at 12,000 g, and supernatant protein was quantified with the BCA protein assay kit (Thermo Fisher Scientific, Rockford, Ill). Total protein lysates (15 lg) were denaturated in SDS sample buffer, separated on an SDS-polyacrylamide electrophoresis gel (10%), and transferred to a polyvinylidene fluoride membrane (pore size, 0.45 mm; Bio-Rad, Hercules, Calif). After blocking in 5% bovine serum albumin for 60 minutes at room temperature, the membrane was incubated with primary antibodies (ABCAM, Cambridge, Mass) in 5% bovine serum albumin/phosphate-buffered saline/Tween-20 overnight at 4 C. The membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology, Beverly, Mass) for 60 minutes at room temperature, and immunodetection was performed using Western Lightning PlusECL (Perkin Elmer, Waltham, Mass). Blots were exposed on X-Omat AR film (Eastman Kodak, Rochester, NY).28 Cross-detection was avoided by stripping the membranes; in cell lines, protein expression was reported relative to

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that of b-actin (Sigma-Aldrich, St. Louis, Mo). The optical density of the appropriately sized bands was measured using ImageJ software (National Institutes of Health, Bethesda, Md). The ratio between neurofilament expression in neuroendocrine neoplasms or adenocarcinoma specimens and control protein was calculated.29 RNA Isolation and Reverse Transcription Messenger RNA was extracted from each tissue sample using TRIZOL (Invitrogen) and then was cleaned (RNeasy kit; Qiagen, Valencia, Calif). After conversion to complementary DNA (cDNA) (High Capacity cDNA Archive Kit, Applied Biosystems, Carlsbad, Calif),29 RTPCR analyses were performed using Assays-on-Demand and the ABI 7900 Sequence Detection System (Applied Biosystems).30 All primer sets were obtained from Applied Biosystems, and a PCR mix on gels was performed to confirm the presence of single bands for each primer set. PCR data were normalized using the DDCT method; the asparagine-linked glycosylation 9 gene ALG9 was used as a housekeeping gene.31 Immunostaining An established immunohistochemical approach was used to identify target protein.32 Briefly, deparaffinized sections were incubated with monoclonal antibodies (also used for Western blot analysis; INA [Abcam, Cambridge, United Kingdom]; 1:500 dilution) overnight at 4 C and then with fluorescein isothiocyanate (FITC) antirabbit immunoglobulin G (1:250 dilution) for 60 minutes at room temperature. Sections were rinsed with phosphate-buffered saline (American Bioanalytical), and nuclei were stained with 40 ,6-diamidino-2-phenylindole (1:100 dilution). Bound antibodies were observed using immunofluorescent microscopy. Statistical Evaluation All statistical analyses were performed using Microsoft Excel (Microsoft, Redmond, Wash), SPSS (version 14; SPSS Inc., Chicago, Ill), and Prism 5 (GraphPad Software, San Diego, Calif). Comparison between NENs, adenocarcinomas, and controls were performed using the Kruskal-Wallis test followed by the Dunn post hoc test when appropriate. Binary comparisons were made using a 2-tailed Mann-Whitney test. Correlations were determined using the Spearman correlation. All P values < .05 were considered significant. Data points >20 standard deviation (SD) above the mean were excluded as outliers; in total, 3 data points (pAC [data points >28 SD above the mean], grade 1 pNET [data points >103 SD above

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the mean], and SI-NEN metastases [data points >3 SD above the mean]) were excluded.

RESULTS Neurofilament Expression in Normal Neuroendocrine Cells, Neuroendocrine Neoplasm Cell Lines, and Murine Xenografts Before we examined neurofilament expression in clinical samples, we screened a range of human normal and NEN cell lines for different neurofilament expression (INA, NF-L, NF-M, and NF-H). We used normal human EC cells (n ¼ 9 preparations), 6 different NEN cell lines, and 4 murine xenografts. By using PCR in normal EC cells, we observed that most neurofilaments either were not expressed or were marginally evident (NF-L) (Fig. 2A). However, INA was expressed significantly in all 6 NEN cell lines (P ¼ .0008), with the greatest expression in the pancreatic BON cell line (from 24-fold to 42-fold). This was confirmed by Western blot analysis (Fig. 2C), which demonstrated significant overexpression (P ¼ .03) of this 66-kDa protein in BON cells compared with the 2 SINEN cell lines KRJ-1 and H-STS. Both NF-L and NF-M were highly expressed in NEN cell lines (however, because of a high SD, the difference was not significant vs normal cells), whereas NF-H was not identified. In BON xenografts, INA mRNA (Fig. 2B) and protein (Fig. 2D) were expressed at levels higher than in cultured cells, demonstrating continued expression under in vivo conditions. To assess a potential biologic role for INA in GEPNENs, we evaluated expression as a function of cell proliferation. Levels of INA (mRNA [Fig. 3A] and protein [Fig. 3B]) decreased after 3 up to 7 days of culture, and that correlated with decreasing expression of Ki-67 (Spearman correlation coefficient [r] ¼ 1.0; P < .0001) (Fig. 3C), suggesting that INA expression may be a function of or related to proliferation. Because both MAPK and PI3K/AKT (PKB) are key proliferative regulatory pathways in BON cells,33,34 we investigated the role of these signaling pathways in INA transcription. The MAPK inhibitor PD98059 inhibited INA mRNA levels (P ¼ .03) using a 1 hour assay, whereas Wortmannin had no effect (Fig. 3D). This suggests that proliferation and signaling through MAPK is associated with the regulation of INA transcription. Internexin Alpha Expression in the Normal and Neoplastic Pancreas After we identified increased expression of INA in NEN cell lines and correlated those levels with the proliferation

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Figure 2. RNA expression of neurofilaments (NFs) in normal enterochromaffin (EC) cells (n ¼ 9 preparations), 6 individual neuroendocrine neoplasm (NEN) cell lines (2 BON [pancreatic] cell lines, 3 small intestinal NEN [STS] cell lines, and 1 small intestine EC cell-derived neuroendocrine tumor cell line [KRJ-1]) and murine xenograft models. (A) Internexin alpha (INA) messenger RNA (mRNA) was expressed at significantly higher levels in NEN cell lines compared with the expression in normal EC cells (a single asterisk indicates P ¼ .0008) NF-L indicates light neurofilament; NF-M, medium neurofilament; NF-H, heavy neurofilament. (C) INA protein was expressed in all 3 different cell lines (3 BON cell samples, 3 H-STS samples, and 4 KRJ-1 cell samples),but its expression was significantly higher in the adherent BON cells (a single asterisk indicates P ¼ .03). (B,D) BON cell xenografts that were grown subcutaneously in mice exhibited higher INA levels at (B) the mRNA level and (D) the protein level compared with BON cells (logarithmic growth phase; Day 2 [d2]) in vitro.

marker Ki-67, we examined whether mRNA and protein were expressed in human pNENs and were increased compared with their expression in normal pancreas. Figure 4A demonstrates the differences in mRNA expression in the normal pancreas, pNENs, and pACs. The levels differed between normal pancreas and pNENs (P ¼ .02) as well as between pACs and pNENs (P ¼ .0001). Protein levels (Fig. 4B,C) followed a similar pattern; INA levels were significantly higher in pNENs than in normal pancreas (P ¼ .0072) and were higher than in pACs (P value not significant). Immunohistochemical staining (Fig. 4D) demonstrated that INA was restricted cytoplasmically in

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NENs, suggesting that it played no membrane receptor or nuclear role. Examination of the Correlation Between Internexin Alpha and Other Pancreatic Neuroendocrine Neoplasms Biomarkers To further delineate a role for INA as a pNEN biomarker, we compared and correlated INA expression with common NEN markers, including chromogranin A, chromogranin B, and Ki-67. There was a strong, positive correlation (Spearman) between INA mRNA and each marker (chromogranin A: r ¼ 0.59; P < .0001; chromogranin B: r ¼

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Figure 3. Internexin alpha (INA) and BON (pancreatic) cell proliferation is illustrated. (A) INA and Ki-67 messenger RNA (mRNA) levels were reduced progressively in BON cells that were harvested on Day 2 (d2), d3, and d7 after subculture (ddct indicates DDCT). (B) INA protein levels were similarly decreased. (C) Although the decreasing INA and Ki-67 mRNA expression over 7 days was not significant, the expression of both genes was strongly correlated (Spearman correlation coefficient [r] ¼ 1.0; P < .0001). (D) The mitogen-activated protein kinase (MAPK) inhibitor PD98059 significantly deceased INA mRNA in subcultured BON cells (a single asterisk indicates P ¼ .03), indicating that INA transcription was regulated by MAPK signaling. Wortmannin is a phosphatidylinositol 3-kinase/protein kinase B inhibitor.

0.74; P < .0001; Ki-67: r ¼ 0.5; P < .0001) (Fig. 5A-C). These results indicate INA may be a neuroendocrine biomarker that positively correlates with proliferation. Internexin Alpha Expression and Tumor Size, Differentiation, and Pancreatic Neuroendocrine Neoplasm Metastasis Next, we examined whether INA expression was associated with clinical features, including stage and grade in pNENs. According to the tumor-lymph node-metastasis (TNM) classification (Union for International Cancer Control), larger tumors (T3) at resection were associated with higher

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INA levels (P ¼ .015) (Fig. 6A). In addition, INA levels were correlated with tumor size (measured as the greatest tumor dimension in cm; r ¼ 0.36; P ¼ .03; data not shown). According to tumor grade, INA was expressed differentially between grade 1 and grade 2 neoplasms. Grade 2 pNENs in particular had 7-fold higher INA expression than grade 1 pNENs (P < .05) and normal pancreas (P ¼ .002) (Fig. 6B). Comparisons of INA mRNA levels between pNEN primaries and metastasis demonstrated that levels were elevated significantly (6-fold) in metastases (P ¼ .007) (Fig. 6D). No differences were noted for angioinvasion, whereas tumors that were classified as

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INA in Neuroendocrine Neoplasms/Schimmack et al

Figure 4. (A) Transcript levels of internexin alpha (INA) are illustrated in normal pancreas (n ¼ 10), pancreatic adenocarcinomas (pACs) (n ¼ 21), and pancreatic neuroendocrine neoplasms (pNENs) (n ¼ 50). There was a significant difference in messenger RNA (mRNA) expression between normal pancreas and pNENs (a single asterisk indicates P ¼ .02) and between pNENs and pACs (a pound sign indicates P ¼ .0001; ddct indicates DDCT). (B,C) Significant differences also were observed for protein levels of INA in pancreatic adenocarcinomas (n ¼ 4) compared with pNENs (n ¼ 2) and normal pancreas (n ¼ 2; a single asterisk indicates P ¼ .0072). (B) A post hoc test demonstrated a significant difference in INA expression levels between pNENs and normal pancreas. (D) Immunohistochemical staining for INA expression in a pNEN is shown (Left) with 40 ,6-diamidino-2-phenylindole staining (nuclei; blue), (Middle) with fluorescein isothiocyanate INA staining (green), and (Right) in a composite view. INA expression was restricted to the cytoplasm in NENs, suggesting that it played no membrane receptor or nuclear role.

‘‘functioning’’ did not exhibit different INA expression compared with ‘‘nonfunctioning’’ tumors. With respect to outcome, only 6 of the 50 patients with pNENs had died at follow-up; an analysis of INA expression indicated that it was increased (but not significantly; data not shown) in the nonsurvivors. Western blot analysis confirmed elevated expression in metastases (P < .01) (Fig. 6C,E). Internexin Alpha in Normal Small Intestinal Mucosa and in Small Intestinal Neuroendocrine Neoplasms An analysis of primary SI-NENs and metastases demonstrated significantly increased INA mRNA levels (>10-fold; P ¼ .0002 and P ¼ .0001, respectively) compared with normal mucosa (Fig. 7A). These differences indicated a trend at

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the protein level, particularly for metastases (Fig. 7B,C). An analysis of clinical features indicated that INA was expressed differentially between grade 1 and grade 2 neoplasms. Grade 2 SI-NENs in particular had 2-fold higher INA expression compared with grade 1 pNENs (P < .05). An outcome analysis could not be performed, because only 2 of the 43 patients with SI-NENs had died at follow-up. Immunohistochemically, INA exhibited the same cytoplasmic expression (Fig. 7D) that was noted in pNENs. Gastroenteropancreatic Neuroendocrine Neoplasms With Low, Medium, and Heavy Neurofilament Expression Next we examined mRNA and protein expression of the other 3 class IV intermediate neurofilaments in pNENs

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Figure 5. The utility of internexin alpha (INA) as a biomarker in pancreatic neuroendocrine neoplasms (pNENs) is illustrated in a correlation analysis of INA transcripts with chromogranin (Cg) and Ki-67. Significant associations were identified for both (A) CgA and (B) CgB (Spearman correlation coefficient [r] ¼ 0.59) and CgB (r ¼ 0.74; P < .0001) and (C) for the proliferation marker Ki-67 (r ¼ 0.5; P < .0001) (ddct indicates DDCT).

and SI-NENs to evaluate whether their expression was similar to that of INA. Differences in transcript levels of NF-L and NF-M were noted between normal pancreas and pNENs (P < .04) (Fig. 8A). NF-H mRNA levels were not different. In contrast, these neurofilaments were not expressed differentially in SI-NENs (Fig. 8B). No significant differences between primaries, metastases, and normal tissues were noted at the protein level (Fig. 8C,D).

DISCUSSION Overall, GEP-NENs represent a conundrum, because these tumors comprise a diverse group of lesions of different cell types that exhibit dissimilar biologic and clinical behaviors. Despite this, they are largely treated as a uniform neoplastic entity. A key unmet need in the management of GEP-NENs is the identification of specific biomarkers that typify these lesions and, hence, provide appropriate clinical guidance for the optimal management strategy of individual tumors.35 To date, biomarker discovery in GEP-NENs has been based on analysis of proliferation, eg, the contentious Ki-67.36 In the current study, we sought to identify and examine the utility of neural tumor biomarkers as an alternative strategy, because these lesions demonstrate overlapping phenotypes with NENs. INA, a class IV intermediate filament, is an example of a neural biomarker that has prognostic significance in oligodendroglial grade 3 tumors.24 In these tumors, the overexpression of INA is associated both with longer progression-free survival and with longer overall survival (50.4 months vs 6.6. months and

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16 months vs median not reached, respectively) regardless of the treatment received.24 Therefore, the identification of INA expression in oligodendroglial tumors may be reflective of a ‘‘proneural’’ gene profile, as described by Ducray et al.37 This suggests that INA plays a role both in neurogenesis and in neurosurvival in these lesions through protection or regeneration of the neuronal cells as its original tissue. In the current study, in GEP-NENs, INA was overexpressed in the neoplastic phenotype (particularly in tumors of pancreatic origin) as well as in proliferating NEN cell lines and murine xenograft tumors compared with normal tissues or nontransformed neuroendocrine cells. This expression was more evident in metastases and was associated both with increasing lesion size and with higher tumor grades. Transcript levels of INA correlated well with aggressive and advancing disease, with decreasing differentiation, and with high levels of Ki-67, markers commonly associated with a poor outcome.8,25 It is important to acknowledge that normal tissue may be an imperfect comparator for tumor expression of a biomarker. However, given the absence of any other appropriate normal neuroendocrine controls as well as the current concept that neuroendocrine cells (as well as adenocarcinomas) are derived from the same multipotential stem cells,38-43 the use of normal pancreatic tissue or normal mucosal tissue provides the most appropriate baseline against which to assess INA values. Our findings support the hypothesis that the expression of INA may be reflective of a malignant phenotype and tumor aggressiveness in GEP-NENs. This raises the

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Figure 6. Internexin alpha (INA) expression in pancreatic neuroendocrine neoplasms (pNENs) was analyzed as a function of tumor size, grade, and metastasis. INA transcripts were significantly over-expressed (A) in T3 pNENs compared with T1 pNENs (P ¼ .015) and (B) in grade 2 pNENs compared with normal pancreas (a single asterisk indicates P ¼ .002) and compared with grade 1 pNENs (a pound sign indicates P < .05; ddct indicates DDCT). (D) INA messenger RNA (mRNA) levels were elevated significantly in pNEN metastases compared with the levels in normal pancreas (an asterisk indicates P ¼ .003) and in primary tumors (a pound sign indicates P ¼ .02). (C,E) At the protein level, INA expression was higher in metastases (P < .01).

key biologic question of the role played by INA in the regulation of GEP-NEN proliferation or metastasis. Evaluation of this hypothesis by functional assessment of INA in the BON cell line indicated that expression levels (mRNA and protein) were correlated with logarithmic growth (day 2) and Ki-67 transcription and that mRNA levels were regulated by MAPK—a key component of the proliferation-signaling pathway in this cell line.33 These results suggest that INA is expressed preferentially in rapidly growing cells and that its expression may be a downstream response to proliferation signaling (MAPK) pathway activation. The elevated expression of this neuro-

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filament in invasive GEP-NENs (and xenograft models) also suggests that INA may play a role in NEN metastasis. Because the protein is a cytoplasmically restricted cytoskeletal filament, we postulate that INA may play a role in regulating cell-cell interactions, a fundamental component of tumor growth and invasion. The heterogeneity of GEP-NENs, particularly their individual phenotypes, may provide further insight into the role of INA. Thus, in pNENs, which are relatively faster growing and more aggressive, INA expression correlated well with disease extent and aggression; whereas, in SI-NENs, which have a lower proliferation and low Ki-67

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Figure 7. Transcript expression of internexin alpha (INA) is illustrated in normal small intestinal mucosa (n ¼ 16) and in small intestinal neuroendocrine neoplasms (SI-NENs) (n ¼ 43). (A) The expression of messenger RNA (mRNA) was significantly higher in SI-NEN primaries and metastasis compared with the expression in normal mucosa (single asterisks indicate P ¼ .0002 and P ¼ .0001, respectively; ddct indicates DDCT). (B) Western blot analysis identified a trend toward increased INA expression in metastases (P ¼ .057). (C) Protein levels of INA are indicated in SI-NEN primaries (n ¼ 4), SI-NEN metastasis (n ¼ 4), and normal mucosa (n ¼ 3). (D) Immunostaining of (Left) normal mucosa did not identify significant INA expression in the mucosa; expression was evident in the mesenteric plexus. Both (Center) SI-NEN primaries and (Right) liver metastases exhibit strong cytoplasmic staining, suggesting that INA has no membrane receptor or nuclear role. Blue staining indicates nuclei (40 ,6-diamidino-2-phenylindole); green staining, INA (fluorescein isothiocyanate).

expression, INA was less well correlated. It is well known that pNENs represent a far more aggressive group of NENs with greater malignancy and a substantially worse prognosis.44 The finding that INA was expressed differentially in pNEN primaries and metastasis compared with its expression in SI-NENs may reflect the functional role INA plays in tumor growth and invasion in this group of more aggressive tumors. An assessment of outcomes indicated that INA expression was elevated in the patients who died, but the small number (n ¼ 6) meant that the difference did not reach statistical significance. Longer

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term follow-up will identify whether INA is a prognostic marker for pNENs. In conclusion, INA and the other 3 class IV intermediate filaments are highly expressed at the mRNA and protein levels in GEP-NENs. On the basis of these observations and the confirmation that these proteins also are expressed in neuronal tissue,45 we propose that it may be better to consider this class of proteins not only as neural filament proteins but as neuroendocrine filaments. Although the precise functional role of these neurofilaments in the evolution of NEN genesis remains to be

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Figure 8. Expression levels of light neurofilament (NF-L), medium neurofilament (NF-M), and heavy neurofilament (NF-H) are illustrated in (A) pancreatic neuroendocrine neoplasms (pNENs) and (B) small intestinal neuroendocrine neoplasms (SI-NENs). NF-L and NF-M messenger RNA (mRNA) expression was elevated significantly in primary pNENs compared with normal pancreas (single asterisks indicate P ¼ .0005 and P ¼ .0076, respectively). However, there was no significant difference in expression between SI-NEN primaries or metastasis (ddct indicates DDCT). (C) At the protein level, NF-L expression was increased in pancreatic metastases (PM) compared with NF-L expression in normal pancreas (PN); however, because of the small number of samples in each group, the difference was not significant. (D) NF-M expression levels were similar in all tissue types. Note that the extremely high neurofilament levels in SI-NENs (log scale) (8B) were detected only in the more aggressive neuroendocrine carcinomas, particularly in metastasized neoplasms. PP indicates pNEN primary; PM, pNEN metastasis; PC, pancreatic adenocarcinoma; PN, normal pancreas; SP, SI-NEN primary; SM, SI-NEN metastasis; SN, normal small intestinal mucosa.

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Original Article

elucidated, we postulate that they may play a role in tumor growth and invasion, particularly in pNENs. If this is the case, then the demonstration of their expression in an individual neoplastic lesion may provide the opportunity to consider the class as representative of a target group for cytostatic therapies.

FUNDING SOURCES S.S. is supported by the Deutsche Forschungsgemeinschaft (SCHI 1177/1-1). M.K. is supported by the National Institutes of Health (DK080871). B.L. is supported in part by a Murray Jackson Clinical Fellowship from the Genesis Oncology Trust Auckland, New Zealand.

CONFLICT OF INTEREST DISCLOSURES The authors made no disclosures.

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