Immunohistochemichal expression of biomarkers: a comparative study between diagnostic bronchial biopsies and surgical specimens of non-small-cell lung cancer

original article

Annals of Oncology 18: 1043–1050, 2007 doi:10.1093/annonc/mdm072 Published online 12 March 2007

Immunohistochemichal expression of biomarkers: a comparative study between diagnostic bronchial biopsies and surgical specimens of non-small-cell lung cancer

Department of Medicine, Institut Gustave Roussy (IGR), Villejuif; 2Institut National de la Sante´ et de la Recherche Me´dicale UMR 484 and Department of Pathology, Centre Jean Perrin, Clermont-Ferrand; 3Department of Biostatistics; 4Laboratory of Translational Research, BU Thoracic/Head and Neck Cancer, IGR, Villejuif; 5 Department of Pathology; 6Department of Pneumology, Institut Mutualiste Montsouris; 7Department of Thoracic Surgery, Hoˆpital Tenon, Paris, France

Received 11 October 2006; revised 30 January 2007; accepted 1 February 2007

Background: The increasing use of biomarkers as molecular determinants of responsiveness to conventional chemotherapy or molecular targeted therapy has raised the question of the reliability and reproducibility of their evaluation in bronchial biopsies as compared with corresponding resected surgical specimens. Patients and methods: Immunohistochemical expression of five markers related to signal transduction [epidermal growth factor receptor (EGFR), phospho-Akt], cell proliferation (Ki-67), DNA repair [excision repair crosscomplementing (ERCC)1] and cellular ÔimmortalityÕ [human telomerase catalytic component (hTERT)], was assessed in 41 patients with operable non-small-cell lung cancer in both bronchial biopsies and whole surgical specimens. Results: High correlation coefficients were observed between the expression of ERCC1, hTERT and Ki-67 in the biopsies and the surgical specimens [0.83 (P < 0.0001); 0.55 (P < 0.001) and 0.64 (P < 0.0001), respectively]. On the other hand, biomarker expression in biopsy was less correlated with the expression in the whole tissue sample for the markers of signal response and transduction [0.24 (P = 0.17) and 0.29 (P = 0.09) for EGFR and phospho-Akt, respectively]. Conclusions: Our results indicate a lack of association in the expression of important biomarkers between lung biopsies and corresponding resected tumors, with discordance rates ranging between 9% and 41%. Although these results need to be further validated in larger cohorts, they indicate that the evaluation of the expression of biomarkers in bronchial biopsies can be misleading. Key words: biological markers, biopsy, correlation, immunohistochemistry, non-small-cell lung cancer, surgical specimen

introduction Lung cancer is the leading cause of cancer death worldwide accounting for 31% and 26% of cancer deaths in men and women, respectively [1]. Non-small-cell lung cancer (NSCLC) accounts for 80% of all cases of lung cancer and it is estimated that 55% of patients present with inoperable disease at the time of diagnosis [stage tumor–node–metastasis (TNM) IIIB or IV]. The determination of the molecular pathways implicated in NSCLC etiology and pathogenesis, has already led to identification of potential new therapeutic targets. Moreover, elucidation of the mechanisms of cytotoxic *Correspondence to: Dr J.-C. Soria, Department of Cancer Medicine, Institut Gustave Roussy, 39 Rue Camille Desmoulins, 94805 Villejuif, France. Tel: +33-4211-4291; Fax: +33-4211-5230; E-mail: [email protected]

ª 2007 European Society for Medical Oncology

or molecular drug interactions resulted in identification of novel biological markers within the cancer cell which may be associated with individual patient chemosensitivity, allowing thus the application of chemotherapy or molecular targeted therapy to patients who are most likely to benefit from it [2]. The epidermal growth factor receptor family of genes (EGFR) encodes widely expressed transmembrane molecules that have been implicated in the development and progression of cancer [3]. After epidermal growth factor binding, the receptor autophosphorylates tyrosine residues in its cytoplasmic domain and triggers a cascade that leads to cellular proliferation, angiogenesis, metastasis and inhibition of apoptosis [3]. High EGFR expression has been proposed as a poor prognostic factor for survival in patients with NSCLC

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L. Taillade1, F. Penault-Llorca2, T. Boulet3, P. Fouret4, S. Michiels3, E. Taranchon4, G. Mountzios1, P. Validire5, J. Domont1, P. Girard6, D. Grunenwald7, T. Le Chevalier1 & J.-C. Soria1*

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has led to the adoption of a therapeutic strategy mainly on the basis of the histological and molecular analysis of bronchial biopsy. Given the potential heterogeneity of these tumors and the small amount of tissue analysed in biopsy, we evaluated the correlation of immunohistochemical expression of markers with a prognostic or predictive potential (EGFR, pAkt, ERCC1, hTERT and Ki-67) between biopsy and corresponding surgical specimen.

materials and methods study population The study population consisted of patients with operable and resectable NSCLC who underwent both diagnostic lung biopsies and definitive surgical resection between July 2001 and April 2004 at the Department of Thoracic Surgery of the Institut Mutualiste Montsouris, Paris, France. To be eligible for the study, patients should have not received chemotherapy or radiotherapy before surgery. A meticulous search of the Patient Registry database identified retrospectively 41 eligible patients. The diagnostic procedures included bronchial biopsies during fiberoptic bronchoscopy for 23 patients and percutaneous computed tomographyguided bronchopulmonary biopsy with an 18-gauge needle for 18 patients. Biopsies were fixed in formalin–acetic acid–alcohol (FAA) for 24 h and then embedded in paraffin. The surgical procedure was a lobectomy with homolateral lymph node dissection for 29 patients and a pneumonectomy with homolateral lymph node dissection in 12 patients. Surgical specimens were fixed in 10% buffered formaldehyde during 48 h and then embedded in paraffin. According to the World Health Organization classification [21], the histological types were 23 squamous cell carcinomas, 16 adenocarcinomas and two large-cell carcinomas. According to the TNM staging system [22], there were two patients (5%) with stage IA disease, 15 (37%) with stage IB, seven (17%) with stage IIB, seven (17%) with stage IIIA and 10 (24%) with operable stage IIIB disease, including seven cases of tumors with two localizations within the same lobe and three cases of invasion of vital thoracic vessels. The median tumor size at the time of intervention was 4.8 cm (range: 0.5–10 cm).

immunohistochemical procedure The immunohistochemical procedure was similar for biopsies and surgical specimens. Paraffin-embedded sections of 4 lm were deparaffinized through xylen and a graded series of ethanol. To retrieve epitopes, slides were preheated at 98
C with 10-mmol/l citrate buffer at pH 6.0 for 30 min (pAkt, ERCC1, hTERT and Ki-67). For the EGFR antibody (Ab), the unmasking was carried out by incubation for 8 min with prediluted protease 1 (Ventana Medical Systems, Illkirch, France). Endogenous peroxidase activity was quenched by incubation in peroxidase blocking solution (Dako, Glostrup, Denmark) for 10 min and then washed out with phosphate-buffered saline (PBS) for 5 min. Nonspecific sites were blocked by incubation with 2.5% horse serum of the Vectastain kit (Vector Laboratories Inc., Burlingame, Canada). Slides were then incubated with primary Ab according to the manufacturersÕ instructions. For EGFR, we used an anti-EGFR (3C6) primary Ab (Ventana Medical Systems). A Ventana NexES Immunohistochemistry platform (Ventana Medical Systems) was used to carry out the staining. The Ab was incubated for 32 min. For pAkt, the primary Ab (Cell Signaling Technology, Beverly, MA) was produced by immunizing rabbits with a synthetic phospho-Ser473 peptide corresponding to residues around Ser473 of mouse Akt. The Ab was applied

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[4]. Some studies indicate that EGFR expression may be correlated to higher responsiveness to inhibitors of the tyrosine kinase of EGFR, although specific mutations or amplification of the gene encoding for EGFR seem to be better molecular predictors of response to molecular therapy [5]. Akt, or protein kinase B, is a serine/threonine kinase that has been implicated in the control of major cellular functions such as transcription and protein synthesis and is a downstream effector of growth factor-mediated cell survival [6]. Activation of Akt, as measured by phosphorylation of the protein [phosphorylated Akt (phospho-Akt or pAkt)], is also increased in multiple tumor types including lung cancer [7]. pAkt overexpression is implicated in NSCLC carcinogenesis [7], radioresistance [8], chemoresistance [8] and has been shown to be associated with reduced patient survival [9]. Interestingly enough, its expression was indicated to be predictive for EGFR tyrosine kinase inhibition efficacy [10]. The excision repair cross-complementing (ERCC) gene family reduces damage to DNA by mismatched nucleotide excision and repair by substitution with the corresponding nucleotide. Modified as well as adjacent nucleotides are removed from the damaged strand during the first step (excision), which is followed by synthesis of an intact strand through DNA polymerase activity (repair synthesis) [11]. The ERCC1 gene encodes a protein of 297 amino acids that is required in both phases of DNA damage repair [12]. Increased ERCC1 expression has been proved to be a Ôdouble-edge swordÕ in NSCLC, being at the same time a significant and independent prognostic factor of survival in operated patients with NSCLC [13] and associated with increased resistance to platinum-based chemotherapy [14]. A specific RNA-dependent DNA polymerase, called telomerase, has been identified as responsible for the maintenance of length of the telomeric DNA parts. This ribonucleoprotein enzyme is a reverse transcriptase composed of two essential subunits, a human telomerase RNA component and a human telomerase catalytic component (hTERT) [15]. The expression of the catalytic subunit of telomerase, hTERT, is restricted only to cells that exhibit telomerase activity [16] and plays a critical role in sustaining cellular immortality and carcinogenesis [17]. Positive hTERT expression in NSCLC is significantly associated with worse overall and disease-specific survival [18]. Ki-67, a DNA-binding nuclear protein, is directly associated with cell proliferation. Being present during all active phases of the cell cycle (G1, S, G2 and mitosis) and absent from resting cells (G0) makes it an excellent marker for determining the so-called growth fraction of a given cell population [19]. Although the question of its possible prognostic value remains controversial, a recent meta-analysis has shown that high expression of Ki-67 may indicate poor survival in NSCLC [20]. When surgical resection of the primary tumor is feasible (only 25%–30% of NSCLC at the time of diagnosis), most of the above-mentioned biological parameters are usually immunohistochemically evaluated on paraffin-embedded tissue blocks, originating from the surgical specimen. Nevertheless, the high occurrence of inoperable disease at the time of diagnosis (locally advanced and metastatic setting), as well as the increasing use of chemotherapy in the neo-adjuvant setting,

Annals of Oncology

original article

Annals of Oncology

immunohistochemichal evaluation The presence of tumor cells, as well as the cellularity of the tumor, was evaluated by a pathologist (FP-L). A biopsy sample was considered eligible for the study if tumor morphology was well preserved and if a minimum of 20 cancer cells were present in the sample. Immunohistochemical staining in both biopsies and surgical specimens was independently assessed by two researchers (LT and FP-L) blinded to clinical data. Tumor cells were counted at a ·400 magnification. Only the percentage of positive cells was taken into account for the scoring. The stain was nuclear for pAkt, ERCC1, hTERT and Ki-67 and membranous for EGFR. Since nucleolar hTERT is associated with telomere function [23], only cells with nucleolar staining were considered as positively stained. The number of neoplastic cells with positive staining in representative high-power fields of the tumor was divided by the total number of neoplastic cells in those fields, and the result was expressed as a percentage. When discrepancy between the two researchers was superior to 20%, the slides were reviewed in order to obtain a consensus. In case of discrepancy <20%, the highest value was validated for analysis. In addition, expression values of the markers were dichotomized into negative and positive, using the following cut-off values: 10% for pAkt, ERCC1, hTERT and Ki-67 and 1% for EGFR.

statistical analysis Statistical analysis was carried out by using the SAS software system, version 8.2. For each biomarker, the relationship between the percentage of expression in the diagnostic biopsies and in the surgical specimens was studied using Spearman’s rank correlation coefficient and tested using Spearman’s rank test. Two-sided P values < 0.01 were considered statistically significant in order to account for the multiple testing. For the comparison of the dichotomized expression values, we calculated the proportion of discordances between the two techniques together with 95% confidence intervals (CIs).

results Among the 41 eligible patients, there were 30 male and 11 female (median age, 63 years; range: 44–82 years). Both biopsies and surgical specimens were available and assessable for 34, 34, 27, 35 and 34 patients concerning EGFR, pAkt, ERCC1, hTERT and Ki-67, respectively. Missing biopsies were due to either insufficient tissue samples or containing extensive necrotic areas. For ERCC1, seven cases without valid internal control were identified and thus excluded from the study. The level of discordance (discrepancy in staining percentage

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evaluation >20%) between the two independent researchers was 7%, 3%, 6%, 3% and 6% for ERCC1, Ki-67, hTERT, EGFR and pAkt, respectively. For EGFR, the low correlation (r = 0.24) observed between the percentage of positive cells in biopsies and surgical specimens was not statistically significant (P = 0.17). Using the cut-off of 1%, 29 biopsies (85%) and 31 surgical specimens (91%) were considered positive for EGFR. The percentage of discordant cases was 18% (95% CI: 15–31) (Figures 1A, 1B and 2A and Table 1). A low correlation coefficient (r = 0.29) was also observed between the percentage of pAkt positive cells in biopsies and surgical specimens that was not statistically significant (P = 0.09). Using the cut-off of 10%, 17 biopsies (50%) and 10 resected tumors (30%) were considered positive for pAkt. The percentage of discordant cases was 41% (95% CI: 25–58) (Figures 1C and 2B and Table 2). On the other hand, a high correlation coefficient (r = 0.83) was observed when the percentage of ERCC1 positive cells of biopsies was compared with that of surgical specimens and this correlation was statistically significant (P < 0.0001). Using the cut-off of 10%, eight biopsies (30%) and 11 resected tumors (41%) were considered positive for ERCC1. The percentage of discordant cases was 9% (95% CI: 0–18.4) (Figures 1D, 2C and Table 3). As far as hTERT is concerned, a high correlation coefficient (r = 0.55) was observed between biopsies and surgical specimens in statistically significant manner (P < 0.001). Using the cut-off of 10%, eight biopsies (23%) and nine resected tumors (25%) were considered positive for hTERT. The percentage of discordant cases was 14% (95% CI: 3–26) (Figures 1E and 2D and Table 4). Finally, the Spearman’s rank test demonstrated a high correlation coefficient (r = 0.64) in percentage of Ki-67 positive cells between biopsies and surgical specimens in a notably significant level (P < 0.0001). Using the cut-off of 10%, 29 biopsies (85%) and 28 resected tumors (82%) were considered positive for Ki-67. The percentage of discordant cases was 21% (95% CI: 7.0–34) (Figures 1F, 1G and 2E and Table 5).

discussion The aim of our study was to compare immunohistochemichal expression of five biomarkers between preoperative biopsies and complete surgical specimens. The significant proportion of NSCLC patients with inoperable disease at the time of diagnosis, along with the increasing experience in neo-adjuvant chemotherapy protocols, has established the bronchial or the percutaneous lung biopsy as a very useful means of obtaining information about tumor biology that could define and orientate the therapeutic strategy in the neo-adjuvant or metastatic setting of NSCLC. This remark becomes even more important, taken under consideration that the expression of several biological markers can be modified by neo-adjuvant chemotherapy. For example, Horii et al. [24] reported a significant decrease in Ki-67 expression after neo-adjuvant chemotherapy for esophageal cancer. Moreover, better and more accurate definition of the biological characteristics of the tumor in the individual level is urgently needed, especially

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with a dilution of 1 : 50 and was incubated overnight at 4
C in humid chamber. To determine ERCC1 expression, the clone 8F1 of mouse monoclonal anti-ERCC1 was used as the primary Ab (Neomarkers, Fremont, CA) in dilution 1 : 300 and was incubated for 60 min at room temperature. Monoclonal antibody (mAb) Ki-67 (Dako) was applied in dilution 1 : 50 for 1 h at room temperature. Finally, primary monoclonal mouse hTERT 44F12 Ab (Novocastra, Newcastle on Tyne, UK) at a dilution of 1 : 500 was applied on slides for 60 min at room temperature. All slides were washed in PBS before addition and incubation of secondary Ab (Vectastain Elite ABC kit). Slides were then washed and the avidin–biotin–peroxydase complex was used to amplify the signal (Vectastain Elite ABC kit), except for EGFR, where a Ventana visualization kit was used. The chromogen used to localize the antigen was diaminobenzidine. All slides were finally counterstained with Mayer’s hematoxylin. As a negative control, the staining procedure was carried out with the primary Ab omitted.

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Figure 1. Panels A and B: adenocarcinoma with positive membranous staining for epidermal growth factor receptor (surgical specimens). Panel C: adenocarcinoma with positive nuclear staining for phosphorylated Akt (surgical specimen). Panel D: squamous cell carcinoma with positive nuclear staining for excision repair cross-complementing 1 (surgical specimen). Panel E: adenocarcinoma with positive nuclear staining for human telomerase catalytic component (surgical specimen). Panels F and G: corresponding slides of positive nuclear staining for Ki-67 (F: biopsy, G: surgical specimen).

in the new era of molecular agents that target a specific biological pathway that is activated in a certain tumor. Our study showed that the expression of important biological markers of cell proliferation (Ki-67), ÔimmortalityÕ (hTERT) and DNA repair (ERCC1) assessed in biopsy specimens was significantly correlated with that of the corresponding resected tumor. Consequently, one could presume that immunohistochemical evaluation of these markers may provide reproducible and thus reliable information that could guide the therapeutic strategy.

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For example, it has been demonstrated that high ERCC1 expression can serve as a predictor of clinical resistance to platinum-based chemotherapy [14]. Subsequently, we dichotomized the expression values in order to investigate the hypothesis that treatment decisions could be on the basis of the positivity or negativity of these markers in bronchial biopsies. We found discordance rates between the positivity/negativity in the biopsy as compared with the surgical specimen for 9%, 14 and 21% of the patients for the expression of ERCC1, hTERT and Ki-67, respectively.

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Surgery Ki-67 (% positive cells) Figure 2. (A) Epidermal growth factor receptor expression in biopsies versus the expression in paired resected tumors. (B) phosphorylated Akt expression in biopsies versus the expression in paired resected tumours. (C) Excision repair cross-complementing 1 expression in biopsies versus the expression in paired resected tumours. (D) Human telomerase catalytic component expression in biopsies versus the expression in paired resected tumours. (E) Ki-67 expression in biopsies versus the expression in paired resected tumours.

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Surgery EGF-R (% positive cells)

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Table 1. Expression of EGFR using the cut-off ‡1% EGFR Biopsy Negative Positive Total

Surgery Negative

Positive

Total

1 2 3

4 27 31

5 29 34

EGFR, epidermal growth factor receptor. Table 2. Expression of pAkt using the cut-off ‡10% pAkt

Positive

Total

13 11 24

3 7 10

16 18 34

pAkt, phosphorylated Akt. Table 3. Expression of ERCC1 using the cut-off ‡10% ERCC1 Biopsy Negative Positive Total

Surgery Negative

Positive

Total

16 0 16

3 8 11

19 8 27

ERCC1, excision repair cross-complementing 1. Table 4. Expression of hTERT using the cut-off ‡10% hTERT Biopsy Negative Positive Total

Surgery Negative

Positive

Total

24 2 26

3 6 9

27 8 35

hTERT, human telomerase catalytic component. Table 5. Expression of Ki-67 using the cut-off ‡10% Ki-67 Biopsy Negative Positive Total

Surgery Negative

Positive

Total

2 4 6

3 25 28

5 29 34

This clearly illustrates that a high correlation of biomarker expression between the two techniques does not necessarily mean that the substitution of the whole tumor sample by the bronchial biopsy is always applicable. According to our results,

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Biopsy Negative Positive Total

Surgery Negative

for a nonnegligible percentage of patients, the assessment of positivity/negativity of expression would have lead to a misleading interpretation. To go even further, when using these results in the hypothetical setting of determining the therapeutic strategy (e.g. with a specific molecular targeted agent), a significant proportion of patients might have received a different therapeutic decision in the clinical setting. A few other studies have been published concerning the evaluation of biological markers in preoperative biopsies of NSCLC. Viberti et al. [25] have compared 26 bronchial biopsies and 40 fine needle aspiration biopsies of NSCLC with the corresponding 66 surgical specimens in terms of Ki-67 expression. Bozzetti et al. [26] compared, in 29 cases of NSCLC, p53, Ki-67 and Bcl-2 expression in fine needle aspirates from surgical specimens with immunohistochemical determination in whole tissue sections. Meert et al. [27, 28] compared EGFR expression in bronchial biopsies and resected tumors in 27 patients and in a second study the expression of c-erbB-2, EGFR, Ki-67 and p53 between biopsies and the paired resected tumors in 28 patients. The results of all these studies showed that p53, bcl-2, EGFR, c-erbB-2 and Ki-67 expressions in biopsy specimens are highly correlated with those of the corresponding resected tumors. Our study did not confirm the high correlation between the percentage of EGFR positive cells in biopsies and surgical specimens, observed in the study of Meert et al. [27, 28]. These authors have used the EGFR clone EGFR 113 from Novocastra whereas in our study, we have used the mAb clone 3C6 from Ventana because in our experience, this Ab showed higher sensitivity when compared with the Food and Drug Administration-approved Dako pharmaDx kit [29]. In four cases of our study, EGFR expression was positive in the surgical specimen (with 100% of stained cells) and negative in the biopsy. Ferrigan et al. [30] have demonstrated considerable intratumor heterogeneity in terms of EGFR expression in surgical specimens, and this was associated with a low level of concordance with the result obtained by the diagnostic biopsy of NSCLC. The panel A of Figure 1 illustrates the heterogeneity of EGFR expression on a surgical specimen of our study. In addition to this heterogeneity in EGFR expression, one could argue that the relatively large size of the resected tumor samples in our study (median size of tumors: 4.8 cm) may increase the possibility of negative expression of EGFR in a small tissue sample obtained with biopsy. In order to avoid misleading interpretations, we suggest the immunohistochemical analysis of several biopsies (at least two or three), should a therapeutic decision depend on EGFR expression on a small amount of tissue. Furthermore, in two cases, EGFR expression was positive on the biopsy and negative on the resected tumor. In these cases, one could presume that differences in the fixative procedure and the storage time between biopsies and surgical specimens may result in unexpected discrepancies. Indeed, whereas FAA fixative used for biopsy samples reacts quickly, reactions of formaldehyde with proteins, especially crosslinking, occur gradually and slowly and may partially explain the negative expression of EGFR in several resected tumors. Moreover, Atkins et al. [31] demonstrated that long-term storage of cut slides at room temperature directly affects the EGFR epitope preservation and Van den Broek et al. [32]

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bronchial biopsies can be misleading, raising thus an important issue of reliability when integrating these data in the clinical setting. In this context, the necessity of obtaining multiple biopsies from different areas of the tumor in an effort to enhance the validity of the results of immunohistochemical evaluation should be emphasized. Moreover, in order to reduce antigenicity loss (especially for pAkt), the time between surgical intervention and fixation of the resected tissue should be minimized (ideally 20 min). If the pathology department is not near to the operating room, the fixation procedure could alternatively be initiated by a technician within the operating room. Further control studies in larger patient populations studying a wide variety of biological markers are also urgently needed in order to validate the reliability of immunohistochemical expression in biopsy specimens and to optimize the therapeutic strategy of NSCLC in the neoadjuvant and metastatic setting.

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indicated that nuclear and cytoplasmic stains are less affected by storage time and temperature than membranous stains. Whereas EGFR expression was more likely to be negative in biopsies than in surgical specimens, this was not the case for pAkt: in 11 cases, a negative expression of the marker in the resected tumor and a positive one in the paired biopsy was observed. Baker et al. [33] studied the stability of protein phosphorylation after interruption of blood supply to the tumor and before the tumor is fixed, to prevent dephosphorylation of the marker. The authors encountered a striking difference in the phospho-Ser473-Akt stains between biopsies where staining was strong and postoperative surgical samples where staining was absent and found that the half-life of phospho-Ser473-Akt at room temperature was 20 min, meaning that almost total Akt was lost within 180 min. The time between the surgical excision of a lung tumor and the fixation of the specimen can exceed 20 min because of the different steps before fixation (transport of the surgical specimen to the laboratory, insufflation of the lung with fixative) and may lead to the negativity of pAkt immunostaining expression. Owing to this protein instability, some authors propose different methods to preserve antigenicity [34]. In our study, we preferred to assess expression of biological markers with standard fixative and immunohistochemical methods because these techniques are not expensive and routinely carried out in most pathology laboratories worldwide. An important concern of our study has been the determination of cut-off values, since for many markers, the etiological and clinical relevance of minimal staining has not been elucidated and the cut points for scoring stains have not yet been standardized [35]. In our study, we used the values most consistently reported in the literature. As far as Ki-67 is concerned, although a relatively high correlation was observed between biopsies and surgical specimens, the application of Fisher’s exact test with a cut-off of 10% did not allow to reject the hypothesis of independence. It is of note that in three cases, the percentage of positive cells in biopsy was 5% (considered negative) whereas the paired surgical sample was found to be positive. Taken altogether, our results indicate that the analysis of the immunohistochemical expression of bronchial or percutaneous lung biopsies in NSCLC may provide valid information concerning ERCC1, hTERT and, to a lesser extent, Ki-67 expression in resected tumors and could potentially help to elaborate a therapeutic strategy in the neo-adjuvant or metastatic setting of the disease. On the other hand, our results indicate a lack of association regarding EGFR expression between lung biopsies and corresponding resected tumors, probably due to the heterogeneity of the marker’s distribution in tumors, the different fixative procedure and the difference in storage time of the samples. The instability of the phosphorylated protein pAkt may also explain the low correlation of immunohistochemical expression between biopsies and surgical specimens. Since the number of discordant cases regarding EGFR and pAkt expression in the current study is rather limited, we emphasize the need to further validate these data in larger cohorts. Nevertheless, from the current data we can presume that in a significant number of cases the evaluation of the expression of biomarkers in

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28. Meert AP, Martin B, Verdebout JM et al. Correlation of different markers (p53, EGF-R, c-erbB-2, Ki-67) expression in the diagnostic biopsies and the corresponding resected tumors in non-small cell lung cancer. Lung Cancer 2004; 44(3): 295–301. 29. Penault-Llorca F, Cayre A, Arnould A et al. Is there an immunohistochemical technique definitively valid in EGFR assessment? Oncol Rep 2006; 16(6): 1173–1179. 30. Ferrigan L, Wallace WA. Predicting non-small cell lung cancer expression of epidermal growth factor receptor and matrix metalloproteinase 9 from immunohistochemical staining of diagnostic biopsy samples. Eur J Cancer 2004; 40(10): 1589–1592. 31. Atkins D, Reiffen KA, Tegtmeier CL et al. Immunohistochemical detection of EGFR in paraffin-embedded tumor tissues: variation in staining intensity due to choice of fixative and storage time of tissue sections. J Histochem Cytochem 2004; 52(7): 893–901. 32. Van den Broek LJ, Van de Vijver MJ. Assessment of problems in diagnostic and research immunohistochemistry associated with epitope instability in stored paraffin sections. Appl Immunohistochem Mol Morphol 2000; 8: 316–321. 33. Baker AF, Dragovich T, Ihle NT et al. Stability of phosphoprotein as a biological marker of tumor signaling. Clin Cancer Res 2005; 11(12): 4338–4340. 34. DiVito KA, Charette LA, Rimm DL, Camp RL. Long-term preservation of antigenicity on tissue microarrays. Lab Invest 2004; 84(8): 1071–1078. 35. Fergenbaum JH, Garcia-Closas M, Hewitt SM et al. Loss of antigenicity in stored sections of breast cancer tissue microarrays. Cancer Epidemiol Biomarkers Prev 2004; 13(4): 667–672.

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