Fine Needle Aspiration Cytology: A Tool to Study ... - Springer Link

Mol Biotechnol (2015) 57:549–557 DOI 10.1007/s12033-015-9848-3

RESEARCH

Fine Needle Aspiration Cytology: A Tool to Study NHERF1 Expression as a Potential Marker of Aggressiveness in Lung Cancer Anita Mangia • Giulia Partipilo • Laura Schirosi • Concetta Saponaro • Domenico Galetta • Annamaria Catino Anna Scattone • Giovanni Simone

•

Published online: 6 March 2015 Ó Springer Science+Business Media New York 2015

Abstract Non-small cell lung cancer (NSCLC) is the leading cause of cancer death worldwide and cytology is often the only diagnostic approach. Na?/H? exchanger regulatory factor 1 (NHERF1) is a protein implicated in various cancers. However, the function of NHERF1 in lung cancer has not been described. The aim of this study was to evaluate the biological role of NHERF1 in lung tumors using fine needle aspirates cytology (FNAC) and to verify the utility and reliability of FNACs. We assessed NHERF1 expression by immunohistochemistry on 26 advanced NSCLC

A. Mangia (&) G. Partipilo L. Schirosi C. Saponaro Functional Biomorphology Laboratory, National Cancer Research Centre, Istituto Tumori ‘‘Giovanni Paolo II’’, Bari, Italy e-mail: [email protected] G. Partipilo e-mail: [email protected] L. Schirosi e-mail: [email protected] C. Saponaro e-mail: [email protected] D. Galetta A. Catino Medical Oncology Department, National Cancer Research Centre, Istituto Tumori ‘‘Giovanni Paolo II’’, Bari, Italy e-mail: [email protected] A. Catino e-mail: [email protected] A. Scattone G. Simone Pathology Department, National Cancer Research Centre, Istituto Tumori ‘‘Giovanni Paolo II’’, Bari, Italy e-mail: [email protected] G. Simone e-mail: [email protected]

FNAC and on 18 surgical NSCLC samples. The relationship of its expression with clinicopathological features including stage and histotype was analyzed. In FNACs, cytoplasmic and nuclear NHERF1 expression was significantly higher than membranous expression. In surgical samples, cytoplasmic NHERF1 was significantly higher than both membranous and nuclear NHERF1 expression. In these tumors, a higher cytoplasmic and lower nuclear NHERF1 expression was observed compared to advanced NSCLCs (p \ 0.0001, p \ 0.05, respectively). In all 44 NSCLCs, a significant correlation was found between cytoplasmic NHERF1 expression and stage (p = 0.001), and between nuclear NHERF1 expression and histotype (p = 0.015). To our knowledge, this is the first study analyzing the immunohistochemical expression of NHERF1 in lung cancer using FNAC samples. We conclude that FNACs provide useful material for detection of NHERF1 localization and expression, and that high nuclear NHERF1 expression may be a potential marker of aggressiveness in NSCLC. Keywords Fine needle aspiration cytology Non-small cell lung cancer NHERF1 EBP50 Immunohistochemistry Abbreviations NSCLC NHERF1 FNAC ADC SCC LCC NSCLC-NOS cNHERF1 nNHERF1 mNHERF1

Non-small cell lung cancer Na?/H? exchanger regulatory factor 1 Fine needle aspiration cytology Adenocarcinoma Squamous cell carcinoma Large cell carcinoma NSCLC-not otherwise specified Cytoplasmic NHERF1 Nuclear NHERF1 Membranous NHERF1

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Introduction Non-small cell lung cancer (NSCLC) accounts for approximately 85 % of all lung cancer subtypes and is the leading cause of cancer death worldwide. NSCLC comprises several distinct histologic subtypes [1], the most common being adenocarcinoma (ADC) [2], squamous cell carcinoma (SCC), and large cell carcinoma (LCC). Since lung cancer does not usually become clinically evident until it reaches an advanced stage, most patients are diagnosed with metastatic and advanced disease, and only a small proportion is eligible for surgical resection [3]. For these patients, the only pathologic material evaluable for systemic therapy is small biopsy or cytology specimens [4, 5]. The performance characteristics of cytology in NSCLC predictive marker testing, however, are not well established. Fine needle aspiration cytology (FNAC) and core needle biopsies are clinically accepted, minimally invasive techniques which allow sampling of tumors for diagnosis [6]. Even though conventional cytology often provides a small amount of cellular samples, the availability of FNAC cell blocks allows researchers to perform multiple analyses with the aim of obtaining correct characterization of the tumor histotype or finding a large number of potential tumor-related markers [7–9]. Several studies have demonstrated that extremely small specimens, such as cytology cell blocks, can also be successfully utilized to perform gene analysis [4, 10–13]. It is important to understand whether small samples could be utilized to evaluate potential prognostic markers. In recent years, there has been a remarkable increase in the understanding of the biological mechanisms that underlie lung cancer development, which has led to the identification of novel markers and therapeutic targets. Indeed, many studies have focused on the identification of biomarkers for patient selection and the development of targeted agents [7–9]. In this preliminary study, we report our experience with Na?/H? exchanger regulatory factor 1 (NHERF1; also named SLC9A3R1 and ezrin-radixin-moesin (ERM)-binding phosphoprotein 50 EBP50), analyzing its expression and subcellular localization in cell blocks obtained by FNAC and in surgical samples. NHERF1 is a 50-kDa adaptor protein composed of two tandem PDZ domains [14] and a carboxyl C-terminal ERM-binding region [15, 16]. Several studies have identified a central role of NHERF1 in different types of cancer such as hepatocellular carcinoma [17], schwannoma [18], breast [19–21], colorectal cancers [22–24], and glioblastoma [25]. NHERF1 has been described as a new marker of progression that undergoes progressive alterations of expression, distribution, and subcellular localization during normal-to-carcinoma transition, ranging from loss of normal apical membrane

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Mol Biotechnol (2015) 57:549–557

distribution to cytoplasmic and/or nuclear localization [20, 23]. Up to now, there have been no data published on the significance of NHERF1 expression in lung cancer. The objective of the study was to assess the expression and the intracellular distribution of the NHERF1 protein in lung tumors using FNAC samples, firstly to evaluate its biological role, and secondly to verify the utility and reliability of FNACs.

Materials and Methods Case Selection Cell blocks from paraffin-embedded FNAC were retrieved from the archives of the Department of Pathology, National Cancer Research Centre, Bari, Italy. We examined the expression and the subcellular localization of NHERF1 on 26 advanced NSCLC FNACs with an adequate percentage of tumor cells, and on 18 primary NSCLC surgical samples and their contiguous non-tumor lung tissue. Finally, as control tissue we used ten samples of lung parenchyma obtained from patients undergoing surgery for non-neoplastic lung disease. Specifically, the median age of NSCLC patients was 67 years (ranging between 31 and 84 years old) and 24 (92 %) of the patients were male. These patients were of stage IIIB (58 %) and IV (42 %). Histopathologically, eight were diagnosed as squamous cell carcinomas (SCC), four as large NSCLC-not otherwise specified (NOS), ten as adenocarcinomas (ADC), and four as large cell carcinomas (LCC) (Table 1). Moreover, 18 tumor samples from patients with operable NSCLC who had undergone surgery were retrieved from the archives of the Department of Pathology at the San Paolo Hospital of Bari. Surgical samples were morphologically classified according to the 2004 World Health Organization classification criteria [1] and included 3 SCC, 13 ADC, 1 sarcomatoid carcinoma, and 1 adenosquamous carcinoma. The 18 operable patients (15 males and 3 females) ranged in age from 44 to 80 years (mean age 65 years). The post-surgical pathologic stage of each tumor was classified according to the international Tumor, Node, and Metastasis (TNM) classification [26]. Stages were IA in 3 (16.7 %), IB in 3 (16.7 %), IIA in 3 (16.7 %), and IIIA in 9 (50 %) patients (Table 1). None of the patients had received neoadjuvant chemotherapy or radiation therapy prior to surgery. Cell Block Procedure and Immunohistochemistry (IHC) FNAC sampling had been performed using 21- to 23-gauge fine needles. Cell blocks were prepared by spinning the


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Table 1 Clinicopathological characteristics of 44 NSCLC patients Characteristics

Advanced NSCLCs No. of pts (%)

Surgical NSCLCs No. of pts (%)

Total no. of patients

26

18

Median age (range)

67 (31–84)

65 (44–80)

Gender Male

24 (92)

15 (83)

Female

2 (8)

3 (17)

IA IB

– –

3 (16.7) 3 (16.7)

IIA

–

3 (16.7)

IIIA

–

9 (50)

IIIB

15 (58)

–

IV

11 (42)

–

SCC

8 (31)

3 (17)

NSCLC-NOS

4 (15)

–

ADC

10 (39)

13 (72)

LCC

4 (15)

–

Othersa

–

2 (11)

Well differentiated

–

2 (11)

Moderately differentiated

13 (50)

9 (50)

Poorly differentiated

13 (50)

7 (39)

Tumor stage

Histological type

Histological differentiation

pts patients, FNAC fine needle aspiration cytology, NSCLC-NOS nonsmall cell lung cancer-not otherwise specified, LCC large cell carcinoma, SCC squamous cell carcinoma, ADC adenocarcinoma a

Others include 1 sarcomatoid carcinoma and 1 adenosquamous carcinoma

FNAC cellular specimens, fixed in 10 % buffered formalin, at 1,000 revolutions per minute for 10 min as previously described [4]. The sediment was re-suspended in 95° ethyl alcohol for 10 min and then underwent a second centrifugation. The packed sediment was then removed and wrapped in lens paper. The wrapped sediment was embedded in paraffin according to conventional histological techniques after a short processing cycle with xylene. Cytology cell block specimens were reviewed by the pathologist (GS) to assess the quality and quantity of the tumor tissue. FNAC samples showed a percentage of neoplastic cells ranging from 30 to 70 %. Immunohistochemical detection of NHERF1 expression was performed on 26 FNAC and 18 surgical NSCLC samples using standard immunoperoxidase techniques as previously described [20]. The corresponding hematoxylin and eosin stains were assessed to confirm the diagnoses of these patients. The sections of 4 lm thickness were cut from formalinfixed and paraffin-embedded histological blocks of FNAC and surgical samples, deparaffinised in xylene and rehydrated in graded ethanol solution. The slides were then

immersed in 10 mM sodium citrate buffer (pH 6.0) to enhance antigen retrieval, boiled for 30 min on a hot plate, and then allowed to cool for 20 min. Sections were incubated for 10 min in 3 % hydrogen peroxide in distilled water, and then washed in PBS three times for 5 min. Subsequently, the rabbit polyclonal antihuman EBP50 antibody (PA1-090 Affinity Bioreagents, Golden, CO, USA; dilution 1:150) was applied overnight. Sections were then washed with PBS and incubated with biotinylated link for 30 min and peroxidase-labeled streptavidin for 30 min. The peroxidase reaction was developed using the 3-amino9-ethylcarbazole substrate-chromogen (LSAB2 SystemHRP; DakoCytomation) for 15 min in the dark followed by hematoxylin for 5 min and mounted. As a positive internal control, we used paraffin-embedded cell pellets from MCF7 cell line, expressing high levels of NHERF1. For negative control, the primary antibody was omitted and replaced by PBS pH 7.6. For each case, two consecutive sections were processed immunohistochemically. The sections were scored separately by two independent observers (AM, GS) blinded to the clinicopathological data, with a consensus decision made in all cases of disagreement. We considered concordant the cases with a difference of less than 5 %. In discordant cases (2/44), an agreement was reached after reading peer review. Membranous, cytoplasmic, and nuclear expressions were examined in morphologically confirmed neoplastic cells. NHERF1 expression was quantified by counting the positive cells in three representative areas of tumor for each section at 209 magnification and expressed as a percentage of positive cells/section as described in our previous publication [20].

Ethics Statement This retrospective study was approved by the Institutional Review Board of the National Cancer Research Centre, Bari, Italy. All patients had signed an informed consent form authorizing the Institute to utilize their removed biological tissue for research purposes according to ethical standards.

Statistical Analysis The two-tailed non parametric Kruskal–Wallis and Mann– Whitney tests were used to compare the different expression levels of NHERF1. Statistical significance was calculated for a 95 % confidence interval (p \ 0.05). Calculations were performed using the Prism version 5.00 software package (GraphPad Software, San Diego, CA,

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USA). Fisher’s exact tests were used for analysis of association between NHERF1 expression and clinicopathological variables and were performed using SPSS 15.0 statistical software (SPSS Inc., Chicago, IL, USA). The statistical correlations were considered significant at level of p value less than 0.05 (p \ 0.05).

Results NHERF1 was analyzed at the protein level by IHC in 26 advanced NSCLC FNACs and 18 surgical NSCLC specimens. NHERF1 showed a different localization (membrane, cytoplasm, and nucleus) and pattern of expression both in the FNACs and the surgical NSCLC samples. Membranous NHERF1 staining was low in tumor cells both in FNACs and surgical NSCLC samples, and completely absent in a few cases. In lung parenchyma tissue, NHERF1 immunoreactivity showed mostly an apical membranous reactivity in the bronchial epithelial cells, whereas it was negative in the alveoli (Fig. 1). In the 26 FNACs, cytoplasmic NHERF1 (cNHERF1) expression was observed in 23/26 (88 %) of cases and nuclear NHERF1 (nNHERF1) expression in 24/26 (92 %) of cases, while both cNHERF1 and nNHERF1 expressions were observed in 21/26 (81 %) of cases. In tumor cells of the 26 FNACs, the median value of cNHERF1 (20 %, range 0–80 %) and nNHERF1 (30.5 %, range 0–80 %) expressions was significantly higher than membranous NHERF1 (mNHERF1) expression (5 %, range 0–70 %) (p = 0.01, p = 0.004, respectively) (Fig. 2). Representative images of membranous, cytoplasmic, and nuclear NHERF1 immunohistochemical staining in tumor specimens from advanced lung cancer are shown in Fig. 3a, b, c, respectively.

Fig. 1 NHERF1 immunoreactivity in lung parenchyma samples. a Immunoreactivity of NHERF1 in MCF7 cell line used as positive control in IHC. Representative images of apical membranous

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In the 18 surgical NSCLCs samples, cNHERF1 expression was present in all cases and 10/18 (55 %) of tumors also presented nNHERF1 expression. In contiguous non-tumor lung tissue, we mainly observed a strong membrane immunoreactivity along apical cells. Representative images of cNHERF1 and nNHERF1 expressions in tumor cells and membrane immunoreactivity in non-tumor cells are shown in Fig. 4a, b, c. The median expression of cNHERF1 (80 %, range 10–95 %) was significantly higher than both membranous (0 %, range 0–60 %) and nuclear expressions (8.5 %, range 0–70 %), (p \ 0.0001, for both) (Fig. 4d). In the 18 surgical NSCLC samples, the cNHERF1 expression was significantly higher than that of the 26 advanced NSCLCs (p \ 0.0001) (Fig. 5a), whereas a significantly lower nNHERF1 expression was found in surgical compared to advanced NSCLCs (p \ 0.05) (Fig. 5b). In addition, cytoplasmic and nuclear NHERF1 expression of the 44 NSCLCs (26 advanced and 18 surgical samples) was correlated to the clinicopathological characteristics. The median values of cNHERF1 and nNHERF1 expression were computed from the 44 NSCLCs (18 surgical and 26 FNAC) set and were considered as cut-off. According to the median value, the cases were classified positive when cNHERF1 immunoreactivity was present in [40 % of tumor cells (median value 40 %) or when nNHERF1 expression was detected in [18 % of tumor cells examined (median value 18 %). We observed a heterogeneous distribution of cytoplasmic and nuclear NHERF1 expression in different stages and histotypes of lung cancers, respectively (Table 2). A statistically significant correlation was found between cNHERF1 expression and the stage (p = 0.001). In particular, all tumors with IIA–IIIA stage showed cNHERF1 positive, while negative cNHERF1 expression was observed in almost all of the tumors with IV stage. Moreover, a statistically

immunoreactivity of NHERF1 in the bronchial epithelial cells (b) and of the negative control (c) (original magnification 9 20)


Fig. 2 Percentage of NHERF1 protein expression in membrane, cytoplasm, and nucleus of FNAC NSCLCs. Bars in the histogram represent percentage range and median value of NHERF1 protein expression in membrane, cytoplasm, and nucleus in FNAC NSCLCs. The level of significance has been reported for each statistical comparison performed

significant correlation was also found between nNHERF1 expression and histotypes (p = 0.015) (Table 2).

Discussion This study provides a first analysis of the expression of NHERF1 in human lung cancer using immunohistochemistry. We demonstrated that the material from FNACs contains an important source of cells suitable for examining the expression of the NHERF1 protein and its different subcellular localization similarly to surgical sample. It is

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noteworthy that NHERF1 immunoreactivity was well preserved in the FNAC cell block, and we believe this may be due to the immediate fixation of the cytology samples. In our study, the FNACs showed a suitability in subtyping NSCLCs non-different than that obtained using surgical NSCLC specimens containing a higher percentage of cells. In recent years, there has been increasing interest in the role of NHERF1 in cancer progression for a number of solid malignancies, including breast cancer [19, 20], colorectal cancer [22, 23], hepatocellular carcinoma [17], and prostatic adenocarcinoma [27]. NHERF1 is expressed in the epithelia of many tissues and is primarily localized at the plasma membrane of polarized epithelial cells, such as small intestine, pancreas, liver, kidney, and stomach [28– 30]. Overexpression of NHERF1 has been found in many human neoplasms [17–19]. Several studies have reported the shift of NHERF1 subcellular localization from membrane to cytoplasm and nucleus in different tumors [17, 20, 22]. A mechanistic model has been proposed in which NHERF1 has a double role as a tumor suppressor when it is present at membrane level and as an oncogenic protein when present in the cytoplasmic and nuclear compartments [23, 30]. This evidence led us to investigate the different expression and localization of NHERF1 in NSCLC samples still unexplored. In line with our previous findings [20, 23] and those of other authors [17, 22, 31], we observed a heterogeneous distribution of the protein expression in lung cancer samples. As expected, apical membranous NHERF1 immunoreactivity was high in the bronchial epithelial cells and in the contiguous non-tumor tissue of the surgical samples, and scarce or absent in FNAC and surgical tumor

Fig. 3 Representative images of NHERF1 immunohistochemical staining in FNACs NSCLCs. Representative images of negative control (a), membranous (b; marked by black arrows), cytoplasmic (c) and nuclear (d) NHERF1 expression (original magnification 9 40)

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Fig. 4 Immunoreactivity and localization of NHERF1 in surgical NSCLC tissues. Representative images of immunohistochemical staining: a cytoplasmic and b nuclear in ADC cells, c membranous NHERF1 expression in contiguous non-tumor epithelia cells (original magnification 9 20). d Bars in the histogram represent percentage


range and median value of NHERF1 expression in membrane, cytoplasm, and nucleus in surgical NSCLC samples. The level of significance has been reported for each statistical comparison performed

Fig. 5 Comparison of cytoplasmic and nuclear NHERF1 expression between 26 advanced and 18 surgical NSCLC samples. Percentage and median value of cytoplasmic (a) and nuclear (b) NHERF1 expression in advanced and surgical NSCLCs. The level of significance has been reported for each statistical comparison

cells. These findings confirmed the hypothesis that NHERF1 may have different functions in relation to subcellular localization. In fact, in tumor cells of operable and advanced NSCLCs, we observed a higher accumulation of NHERF1 protein in cytoplasmic and nuclear compartments compared to membranous localization. In this study, taking into consideration the cytoplasmic protein staining, we found a higher NHERF1 expression in operable compared to advanced NSCLC samples. Moreover,

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when we considered the 44 NSCLCs (18 surgical and 26 FNAC samples), we also obtained a statistically high significant correlation between cytoplasmic NHERF1 expression and stage. It is noted that positive cNHERF1 expression was associated at all IIA–IIIA stages. In contrast, almost all patients with IV stage had a negative cytoplasmic NHERF1 expression. Altogether, these results suggest that the presence of NHERF1 in the cytoplasmic compartment could characterize a less aggressive phenotype. In support of this


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Table 2 Correlation of cytoplasmic and nuclear NHERF1 expression with clinicopathological characteristics of 44 NSCLCs Characteristics

No. of pts

cNHERF1 expression Positive (%)

Negative (%)

p value*

nNHERF1 expression Positive (%)

Negative (%)

p value*

Histotypes ADC

23

12 (52.2)

11 (47.8)

11 (47.8)

12 (52.2)

SCC

11

5 (45.5)

6 (54.5)

0.493

2 (18.2)

9 (81.8)

LCC

4

1 (25.0)

3 (75.0)

3 (75.0)

1 (25.0)

NSCLC-NOS

4

1 (25.0)

3 (75.0)

4 (100)

0 (0)

Other

2

2 (100)

0 (0)

0 (0)

2 (100)

Grade G1

2

2 (100)

0 (0)

1 (50.0)

1 (50.0)

G2

22

12 (54.5)

10 (45.5)

10 (45.5)

12 (54.5)

G3

20

7 (35.0)

13 (65.0)

9 (45.0)

11 (55.0)

3 (50.0)

3 (50.0)

0.116

0.015

1.000

Stage IA–IB

6

IIA–IIIA

12

12 (100)

IIIB

15

5 (33.3)

IV

11

1 (9.1)

2 (33.3)

4 (66.7)

4 (33.3)

8 (66.7)

10 (66.7)

8 (53.3)

7 (46.7)

10 (90.9)

6 (54.5)

5 (45.5)

0 (0)

0.001

0.653

pts patients, cNHERF1 cytoplasmic NHERF1, nNHERF1 nuclear NHERF1, ADC adenocarcinoma, SCC squamous cell carcinoma, LCC large cell carcinoma, NSCLC-NOS non-small cell lung cancer-not otherwise specified * p values were calculated with the Fisher test

hypothesis, in surgical tumors we also found a significantly higher cytoplasmic NHERF1 expression compared to the nuclear one. However, these data are contradictory to previous studies, which indicated that cytoplasmic NHERF1 overexpression in breast cancer was associated with aggressive tumor behavior and poorer prognosis, and it increases during breast tumor progression, suggesting a role in this process [19, 20, 31, 32]. Based on our findings, it seems it may be difficult to establish a clear biological significance and suggest an oncogenic function of cytoplasmic NHERF1 expression in lung cancer. We hypothesize that a different tumor microenvironment in lung compared to breast cancer could influence the different behavior of cytoplasmic NHERF1 expression. Further investigation will be required before definitive conclusions can be drawn in order to quantify the NHERF1 gene transcript and to valuate the colocalization with other proteins involved in NHERF1 gene pathway activation. Little information is available on the role of nuclear NHERF1 expression in human cancers. Shibata T et al. found that nuclear accumulation of the NHERF1 protein was present in cases of hepatocellular carcinoma compared with the surrounding non-cancerous liver tissue [17]. In a previous study, we observed significant changes in the expression of nuclear NHERF1 in colorectal adenoma– carcinoma sequence in comparison to non-neoplastic tissue [23]. In the present study, when nuclear NHERF1 staining was taken into account, we found a lower NHERF1

expression in the operable compared to the advanced NSCLC samples. The diffuse and strong nuclear NHERF1 staining in advanced NSCLC could imply that it has an important role in the development and progression of the disease. The nuclear NHERF1 localization in our study is in agreement with previous reports on colorectal cancer [22–24] and suggests a possible correlation between high nuclear NHERF1 expression and tumor aggressiveness. The results are in line with the hypothesis [30] associating strong nuclear expression with oncogenic function of NHERF1. When nuclear NHERF1 expression of all the NSCLCs was correlated to the clinicopathological characteristics, we found a statistically significant correlation between this expression and histotypes. The heterogeneous distribution of nuclear NHERF1 expression in different histotypes of lung cancers might indicate NHERF1 expression as a diagnostic tool to distinguish the different phenotypes. However, an increase of the series of lung samples and a balanced representation of the different histotypes would be necessary to confirm this hypothesis. In conclusion, our experimental results show that FNAC samples contain sufficient material for the analysis of NHERF1 expression and its subcellular localization. The results obtained related to cytoplasmic and nuclear NHERFl expression could suggest a different significance of protein localization in lung cancer. Cytoplasmic NHERF1 expression could characterize a less aggressive phenotype of lung cancer. Elevated nuclear NHERF1

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expression, found in advanced NSCLC compared to surgical NSCLC samples, could be specific for aggressive tumors. Finally, nuclear NHERF1 expression might be able to distinguish between different phenotypes. Taken together these findings suggest that nuclear NHERF1 expression may be a potential marker of aggressiveness in lung cancer. Future studies on a larger series of NSCLC patients are necessary to draw more reliable conclusions and to correlate NHERF1 expression directly with patient outcomes. Acknowledgments The authors thank Rossana Daprile for technical assistance. The authors would also like to thank Caroline Oakley for manuscript revision and Giusi Graziano for statistical analyses. The authors would also like to thank Dr. Stefania Tommasi for her collaboration in the critical revision of the paper. Conflict of interest peting interests.

The authors declare that they have no com-

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