Annals of Oncology Advance Access originally published online on March 8, 2006
Annals of Oncology 2006 17(6):981-985; doi:10.1093/annonc/mdl038
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
© 2006 European Society for Medical Oncology
Comparison of the epidermal growth factor receptor gene and protein in primary non-small-cell-lung cancer and metastatic sites: implications for treatment with EGFR-inhibitors
1 Department of Medical Oncology, Centre Antoine-Lacassagne, Nice; 2 Laboratory of Solid Tumors Genetics, Centre Hospitalier Universitaire de Nice and CNRS UMR 6543, Faculté de Médecine, Canceropôle PACA, Nice; Departments of 3 Pathology, 4 Thoracic Surgery, 5 Department of Neurosurgery, Centre Hospitalier Universitaire de Nice, Nice, France
* Correspondence to: Dr A. Italiano, Laboratory of Solid Tumors Genetics, Faculty of Medicine, 28 avenue de Valombrose, 06107 Nice, France. Tel: +33 04 93 37 70 12, Fax: +33 04 92 03 10 46; E-mail: antoineitaliano{at}cario.fr
 |
Abstract |
|---|
 Top Abstract introductionpatients and methodsresultsdiscussionReferences |
|---|
Background: Very recent studies have suggested that EGFR gene copy number and expression obtained by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) should be used to predict which lung cancer patients are expected to respond to anti-EGFR treatments. However, it is still not known whether EGFR expression differs in metastases compared to primary non-small cell lung cancer (NSCLC).
Patients and methods: EGFR status was analysed by IHC and FISH on tumor samples of primary NSCLC and at least one distant metastatic lesion in 30 patients.
Results: Ten cases (33.3%) showed primary tumor/metastasis discordance by IHC analysis (n = 30): in seven cases, EGFR was expressed in the primary tumor but not in the metastasis, while three samples showed EGFR expression in the metastasis but not in the primary tumor (Pearson correlation coefficient = 0.331, P = 0.0074). By FISH (n = 26), seven (27%) cases were discordant: six cases showed a high-level of EGFR polysomy in the primary tumor but not in the metastasis and one case showed a high-level of EGFR polysomy in the metastasis but not in the primary sample (Pearson correlation coefficient = 0.52, P = 0.007).
Conclusion: EGFR expression is not stable during metastatic progression in a significant proportion of NSCLC. These findings have to be considered in future prospective studies.
Key words: epidermal growth factor receptor, non small cell lung cancer, EGFR inhibitor
|
introduction |
|---|
|
TopAbstractintroductionpatients and methodsresultsdiscussionReferences |
|---|
Lung cancer is the major cause of cancer death in both men and women throughout the world. The current standard of care for patients with metastatic non-small cell lung cancer (NSCLC) is systemic chemotherapy. However, this treatment does not result in cure and nearly all patients succumb to the disease. During the last few years, significant advances in the development of new molecularly targeted agents have been made. These agents aim to inhibit specific pathways and key molecules in tumor growth and progression. One example of such targets is the epidermal growth factor receptor (EGFR), a tyrosine kinase receptor that belongs to the ErbB family. EGFR is overexpressed in many human epithelial malignancies, including NSCLC [1
–2] where this expression appears to be associated with poor survival [3]. Following stimulation by its natural ligands, this receptor initiates signal transduction cascades that promote cell division, migration and angiogenesis, and inhibit apoptosis. Several molecules have been synthesized that inhibit the EGFR tyrosine kinase domain [4–5]. Gefitinib and erlotinib are small molecules that reversibly target EGFR. These inhibitors produce objective response rates of 12%–27% in previously treated or untreated advanced NSCLC [6–9].
Recently, the National Cancer Institute of Canada conducted a multicenter randomized placebo-controlled trial of erlotinib (150 mg/day) in patients with advanced NSCLC following failure of first-line or second-line chemotherapy (trial BR.21). Results of this study involving 731 patients showed, for the first time in a randomized trial, that single-agent erlotinib prolonged patient survival after a first or second-line chemotherapy [9]. Overall response to erlotinib was 9%, and the overall survival was 6.7 months for erlotinib versus 4.7 months for placebo (P = 0.001). On the basis of these studies, gefitinib has been approved for use in NSCLC in several countries and erlotinib received approval by the US Food and Drug Administration in November 2004. However, an emerging issue concerning EGFR targeted therapy in NSCLC is to identify the best method for selecting patients who will most likely benefit from EGFR inhibition. Very recent studies have suggested that EGFR gene copy number assessed by fluorescence in situ hybridization (FISH) or EGFR immunohistochemistry (IHC) should be used to predict which patients will respond to gefitinib therapy [10–12]. However, it is still unknown whether EGFR expression differs in metastases compared to primary NSCLC. The aim of our study was to identify the potential changes in EGFR biology between primary and metastatic disease, by correlating EGFR gene copy number and EGFR immunohistochemical expression in primary NSCLC and corresponding metastases.
|
patients and methods |
|---|
|
TopAbstractintroductionpatients and methodsresultsdiscussionReferences |
|---|
patients
Between January 1990 and December 2003, tumor specimens from 32 consecutive patients with NSCLC, who underwent surgery for excision of a primary tumor and corresponding metastases, were analyzed in the Pathology Department of the University Hospital of Nice, France. Two patients were excluded from EGFR status analysis because tumor specimens were not available.
EGFR protein expression evaluation
All slides from the lung resection specimens and metastasectomies were reviewed by the same pathologist. EGFR protein expression was evaluated by IHC using methods described elsewhere [13] with the immunohistochemical system kit EGFR pharmDx (DakoCytomation, Carpinteria, CA) on freshly cut, formalin-fixed, paraffin-embedded tissue from the most representative block with adequate viable tumor (avoiding areas of necrosis). The cell lines HT-29 and CAMA-1 supplied by the manufacturer as positive and negative controls were pelleted, formalin-fixed and paraffin-embedded, respectively.
Sections were analyzed by two observers using light microscopy. The observers were unaware of the clinicopathological details. Both the primary and metastatic neoplasms were considered positive for EGFR when > 10% of the tumor cells had membranous staining [12]. Cytoplasmic staining without associated membrane staining was reported as negative.
EGFR gene copy number evaluation
EGFR gene copy number per cell was investigated by FISH on paraffin embedded sections, from the lung resection specimens and metastasectomies. The procedure for deparaffinization and FISH was done according to Coindre et al. [14]. The 5-µm sections on polysinated slides were dewaxed for 3 x 10 min in xylene, washed in 100% ethanol, air-dried and incubated in 2 x SSC (sodium saline citrate) at 75°C for 20 min and a proteinase K solution (Roche, Meylan, France) (20 U/ml in 2 x SSC) at 45°C for 50 min. They were then washed in 2 x SSC for 5 min at room temperature and stored in 70% ethanol at 4°C before hybridization with the LSI EGFR SpectrumOrange/CEP 7 SpectrumGreen probe (Vysis, Abbott Laboratories Inc, Rungis, France) according to the manufacturer's instructions. At least 100 non-overlapping interphase nuclei per slide were analyzed, independently, by a single observer who was unaware of the patients' clinical characteristics. Patients were classified into two strata: (i) FISH-negative, with no or low genomic gain (<four copies of the gene in >40% of cells) or (ii) FISH-positive with either a high level of polysomy (
four copies of the gene in > 40% of cells) or with gene amplification. Gene amplification was defined by the presence of tight gene clusters, and a gene/chromosome per cell ratio two, or 15 copies of the gene per cell in 10% of analyzed cells [10, 12].
statistical analysis
The ‘R 1.7.1’ statistical software package was used for all calculations. Data management was performed using Windows ‘ACCESS-2000’ software. Pearson's correlation test confirmed by Spearman's correlation test was used to compare the EGFR status between primary tumors and related metastatic sites. A strong correlation was defined as a correlation coefficient r 0.8 (perfect correlation = 1.0). Differences were considered to be statistically significant when the p value was 0.05 or less. All statistical tests were two-sided.
|
results |
|---|
|
TopAbstractintroductionpatients and methodsresultsdiscussionReferences |
|---|
clinical and pathological features
The characteristics of the 30 NSCLC patients are described in Table 1. The median age of the patients at initial surgery was 57 years old (with a range of 39 to 77 years). Thirty metastatic samples, including 20 brain metastases, were analyzed. Metastases were synchronous in 10 cases (33.3%) and metachronous in 20 cases (66.7%). The median time between resection of the primary and the corresponding metastatic site was 8 months. None of the patients received prior EGFR-targeted therapy.
|
EGFR status in primary and metastatic NSCLC tumors (Table 2)
Using IHC, we found EGFR overexpression in 20 of total 30 primary tumors (66.7%) and in 16 of total 30 metastatic lesions (53.3%). Analysis of metastatic sites showed EGFR expression in 12 of total 20 brain metastases (60%). By using FISH analysis, high level of polysomy (
four copies of the gene in > 40% of cells) was detected in 16 of total 28 primary tumors (57.1%) and 12 of total 28 metastatic lesions (42.9%). EGFR amplification (> 10 copies/cell) was detected in none of the cases. Two primary tumor and two metastatic lesions were not evaluable by FISH analysis (uninterpretable results).
|
comparison of the EGFR status evaluated by IHC and FISH analysis
FISH and IHC methods were concomitantly successful for detection of EGFR status in 28 of total 30 primary tumors (93.4%) and in 28 of total 30 metastatic samples (93.4%).
Regarding the primary tumors, the two techniques showed discordant results in nine cases (32%): three of these cases were IHC-negative and FISH-positive, while the other six cases were HIC-positive and FISH-negative. Among the metastatic samples, there were seven cases (25%) of discordance between IHC and FISH results: two of seven cases were IHC-negative and FISH-positive, while five of seven cases were IHC-positive and FISH-negative. Therefore, the EGFR status evaluated by IHC was not perfectly correlated to the FISH results (r < 0.8) (primary tumors: Pearson correlation coefficient r = 0.331, P = 0.0085; metastatic tumors: Pearson correlation coefficient r = 0.512, P = 0.005).
comparison of the EGFR status between primary tumors and metastases (Table 3)
Thirty paired primary and metastatic tumors were available for IHC analysis. A discordance was observed in 10 cases (33.3%): in 7 cases, EGFR was expressed in the primary tumor but not in the metastasis, while three samples showed EGFR expression in the metastasis but not in the primary tumor (Pearson correlation coefficient = 0.331, P = 0.0074).
|
Regarding FISH results, seven out the 26 paired primary and metastatic tumors (27%) were discordant for EGFR status between the primary site and the metastases: six cases showed high-level of EGFR polysomy in the primary tumor but not in the metastasis while one case showed high-level of EGFR polysomy in the metastasis but not in the primary sample (Pearson correlation coefficient = 0.52, P = 0.007).
Because EGFR status determined by IHC is not always concordant with the status determined by FISH, we selected 12 paired primary and metastatic tumors that had shown perfectly consistent EGFR status results by IHC and FISH. In three of these 12 cases (25%) both positive immunohistochemical expression and polysomy were detected in the primary tumor but were not identified in the metastasis (Pearson correlation coefficient = 0.57, P = 0.049), showing that EGFR status was not always identical in primary tumors and metastases (Figure 1).
|
|
discussion |
|---|
|
TopAbstractintroductionpatients and methodsresultsdiscussionReferences |
|---|
To the best of our knowledge, the present study is the first to compare EGFR status in primary NSCLC with their distant metastases using both IHC and FISH techniques, in a significant number of patients.
Retrospective analyses of gefitinib [6–7] or erlotinib [8] studies revealed several clinical predictors of response. Responses were more frequent among never-smokers, women, patients with adenocarcinomas, and patients of East Asian ethnicity. In addition, dramatic advances in EGFR mutations research have been presented in recent months. These studies suggested that acquired mutations of EGFR in exons 18–21 account for the increased sensitivity of NSCLC patients to gefitinib. Moreover, patients who did not express a mutation had a low probability of responding [15–16]. The group from Memorial Sloan-Kettering Cancer Center extended these data and showed that similar EGFR mutations also were associated with responses to erlotinib [8]. These findings were supported by a report that showed correlation between clinical predictors of response mentioned above and EGFR mutation status [17]. Compiling these data revealed that EGFR mutations were identified in 80% of responders to EGFR small molecule tyrosine kinase inhibitors, whereas mutations in K-RAS (exon 2) were associated with lack of sensitivity to either erlotinib or gefitinib [18]. However, the most recent and, perhaps, the most clinically significant advance in the optimal selection of patients for EGFR-targeted therapies comes from two studies that demonstrated a significant survival benefit in EGFR FISH and/or IHC-positive patients, suggesting that increase in EGFR gene copy numbers or in EGFR protein expression can be used as markers to assess survival potential in NSCLC patients to be treated with EGFR tyrosine kinase inhibitors [10–11]. These findings have considerable clinical implications because IHC and FISH techniques, which are performed routinely on paraffin-embedded material, are more convenient tools than DNA sequencing for extensive clinical use. However, contradictory results have been reported by Tsao et al., in a recent study [12]. In the univariate analysis, the authors showed that a survival advantage was associated with increased numbers of gene copies or EGFR expression in patients treated with erlotinib; however, this association was not found in the multivariate analyses [12]. So, before any clinical recommendation can be made, the predictive value of EGFR gene copy number and/or EGFR expression should be validated by prospective studies. To this end, we raised the question of the stability of EGFR expression during the metastatic process in NSCLC. Indeed, the main targets of any therapy in metastatic NSCLC are the metastases. However, in the great majority of studies investigating the EGFR biology in NSCLC, EGFR status was determined on the primary tumor, and data referring to distant metastases are almost inexistent.
Our study revealed that correlation between EGFR status in primary NSCLC cancer and that in corresponding metastatic sites is relatively poor (r < 0.8) since 33.3% and 27% of cases showed discordance by IHC and FISH, respectively. This discordance also has been observed by Petersen et al. [19] who identified chromosomal imbalances of the 7p11 region, where the EGFR gene is mapped, by comparative genomic hybridization (CGH) in a series of paired primary tumor and metastases of NSCLC. The proportion of patients with primary tumors that were EGFR-positive by IHC (66.7%) or FISH (57.1%) in our study is consistent with the report of Tsao et al. using the same scoring system [12]. Moreover, as has been previously shown [2], we found that the correlation between increased EGFR gene copy number per cell and protein expression was not absolute (r < 0.8). IHC is not a strictly quantitative method, as there is no uniform scoring system and the interpretation of staining intensity is highly subjective. In addition, variations in protocols, such as in fixation procedures, antibodies and storage time of the tissue samples, are likely to affect the sensitivity of these assays, making comparison of results from different laboratories difficult [20]. So, it is not known yet whether the discrepancy between FISH and IHC is due to false-positive or false-negative IHC results or to mechanisms controlling EGFR expression unrelated to the number of genes per cell, such as post-transcriptional regulation.
Our finding that EGFR status may change between primary NSCLC and corresponding distant metastatic sites is novel and contributes to the debate about the best method for predicting response of NSCLC tumors to EGFR targeted therapies. Should any available neoplastic tissue (primary tumor or metastasis) be considered adequate to study EGFR biology in NSCLC? Are the patients carrying EGFR overexpression or increased gene copy number in a stable way during tumor progression the best candidates for EGFR targeted therapies? Both these questions should be considered in future prospective studies.
|
Acknowledgements |
|---|
We thank R. Auvergne for skilful technical assistance and K. L. Huyck for help in the preparation of the manuscript.
Received for publication December 8, 2005. Revision received February 1, 2006. Accepted for publication February 3, 2006.
|
References |
|---|
|
TopAbstractintroductionpatients and methodsresultsdiscussionReferences |
|---|
1. Salomon DS, Brandt R, Ciardiello F et al. Epidermal growth factor-related peptides and their receptors in human malignancies. Crit Rev Onco Hematol 1995; 19: 183–232.
2. Hirsch FR, Varella-Garcia M, Bunn PA Jr et al. Epidermal growth factor receptor in non-small-cell lung carcinomas: correlation between gene copy number and protein expression and impact on prognosis. J Clin Oncol 2003; 21: 3798–807.
3. Selvaggi G, Novello S, Torri V et al. Epidermal growth factor receptor overexpression correlates with a poor prognosis in completely resected non-small-cell lung cancer. Ann Oncol 2004; 15: 28–32.
4. Levitzki A, Gazit A. Tyrosine kinase inhibition: an approach to drug development. Science 1995; 267: 1782–8.
5. Levitt ML, Koty PP. Tyrosine kinase inhibitors in preclinical development. Invest New Drugs 1999; 17: 213–26.[CrossRef][Web of Science][Medline]
6. Fukuoka M, Yano S, Giaccone G et al. Multi-institutional randomized phase II trial of gefitinib for previously treated patients with advanced non-small-cell lung cancer. J Clin Oncol 2003; 21: 2237–46.
7. Kris MG, Natale RB, Herbst RS et al. Efficacy of gefitinib, an inhibitor of the epidermal growth factor receptor tyrosine kinase, in symptomatic patients with non-small-cell lung cancer: a randomized trial. JAMA 2003; 290: 2149–58.
8. Perez-Soler R, Chachoua A, Hammond L et al. Determinants of tumor response and survival with erlotinib in patients with non-small-cell lung cancer (NSCLC). J Clin Oncol 2004; 22: 3238–47.
9. Shepherd FA, Rodrigues Pereira J, Ciuleanu T et al. National Cancer Institute of Canada Clinical Trials Group. Erlotinib in previously treated non-small-cell lung cancer. N Engl J Med 2005; 353: 123–32.
10. Cappuzzo F, Hirsch FR, Rossi E et al. Epidermal growth factor receptor gene and protein and gefitinib sensitivity in non-small-cell lung cancer. J Natl Cancer Inst 2005; 97: 643–55.
11. Hirsch FR, Varella-Garcia M, McCoy J et al. Increased epidermal growth factor receptor gene copy number detected by fluorescence in situ hybridization associates with increased sensitivity to gefitinib in patients with bronchioloalveolar carcinoma subtypes: a southwest oncology group study. J Clin Oncol 2005; 23: 6838–45.
12. Tsao MS, Sakurada A, Cutz JC et al. Erlotinib in lung cancer – molecular and clinical predictors of outcome. N Engl J Med 2005; 353: 133–44.
13. Italiano A, Saint-Paul MC, Caroli-Bosc FX et al. Epidermal growth factor receptor (EGFR) status in primary colorectal tumors correlates with EGFR expression in related metastatic sites: biological and clinical implications. Ann Oncol 2005; 16: 1503–7.
14. Coindre JM, Hostein I, Maire G et al. Inflammatory malignant fibrous histiocytomas and dedifferentiated liposarcomas: histological review, genomic profile, and MDM2 and CDK4 status favour a single entity. J Pathol 2004; 203: 822–30.[CrossRef][Web of Science][Medline]
15. Lynch TJ, Bell DW, Sordella R et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness fo non-small-cell lung cancer to gefitinib. N Engl J Med 2004; 350: 2129–2139.
16. Paez JG, Janne PA, Lee JC et al. EGFR mutations in lung cancer: Correlation with clinical response to gefitinib therapy. Science 2004; 304: 1497–1500.
17. Shigematsu H. Clinical and biological features associated with epidermal growth factor receptor gene mutations in lung cancers. J Natl Cancer Inst 2005; 97: 339–346.
18. Pao W, Miller VA. Epidermal growth factor receptor mutations, small-molecule kinase inhibitors, and non-small-cell lung cancer: Current knowledge and future directions. J Clin Oncol 2005; 23: 2268–2556.
19. Petersen S, Aninat-Meyer M, Schluns K et al. Chromosomal alterations in the clonal evolution to the metastatic stage of squamous cell carcinomas of the lung. Br J Cancer 2000; 82: 65–73.[CrossRef][Web of Science][Medline]
20. 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: 893–901.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  H. West, D. Harpole, and W. Travis Histologic Considerations for Individualized Systemic Therapy Approaches for the Management of Non-small Cell Lung Cancer Chest, October 1, 2009; 136(4): 1112 - 1118. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-C. Soria, F. A. Shepherd, J.-Y. Douillard, J. Wolf, G. Giaccone, L. Crino, F. Cappuzzo, S. Sharma, S. H. Gross, S. Dimitrijevic, et al. Efficacy of everolimus (RAD001) in patients with advanced NSCLC previously treated with chemotherapy alone or with chemotherapy and EGFR inhibitors Ann. Onc., October 1, 2009; 20(10): 1674 - 1681. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Holm, A. Mellemgaard, T. Skov, and B. G. Skov Different Impact of Excision Repair Cross-Complementation Group 1 on Survival in Male and Female Patients With Inoperable Non-Small-Cell Lung Cancer Treated With Carboplatin and Gemcitabine J. Clin. Oncol., September 10, 2009; 27(26): 4254 - 4259. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C.-H. Gow, Y.-L. Chang, Y.-C. Hsu, M.-F. Tsai, C.-T. Wu, C.-J. Yu, C.-H. Yang, Y.-C. Lee, P.-C. Yang, and J.-Y. Shih Comparison of epidermal growth factor receptor mutations between primary and corresponding metastatic tumors in tyrosine kinase inhibitor-naive non-small-cell lung cancer Ann. Onc., April 1, 2009; 20(4): 696 - 702. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V Martin, L Mazzucchelli, and M Frattini An overview of the epidermal growth factor receptor fluorescence in situ hybridisation challenge in tumour pathology J. Clin. Pathol., April 1, 2009; 62(4): 314 - 324. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Personeni, S. Fieuws, H. Piessevaux, G. De Hertogh, J. De Schutter, B. Biesmans, W. De Roock, A. Capoen, M. Debiec-Rychter, J.-L. Van Laethem, et al. Clinical Usefulness of EGFR Gene Copy Number as a Predictive Marker in Colorectal Cancer Patients Treated with Cetuximab: A Fluorescent In situ Hybridization Study Clin. Cancer Res., September 15, 2008; 14(18): 5869 - 5876. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Cappuzzo, G. Finocchiaro, E. Rossi, P.A. Janne, C. Carnaghi, C. Calandri, K. Bencardino, C. Ligorio, F. Ciardiello, T. Pressiani, et al. EGFR FISH assay predicts for response to cetuximab in chemotherapy refractory colorectal cancer patients Ann. Onc., April 1, 2008; 19(4): 717 - 723. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. A. Eberhard, G. Giaccone, and B. E. Johnson Biomarkers of Response to Epidermal Growth Factor Receptor Inhibitors in Non-Small-Cell Lung Cancer Working Group: Standardization for Use in the Clinical Trial Setting J. Clin. Oncol., February 20, 2008; 26(6): 983 - 994. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C.-H. Gow, C.-R. Chien, Y.-L. Chang, Y.-H. Chiu, S.-H. Kuo, J.-Y. Shih, Y.-C. Chang, C.-J. Yu, C.-H. Yang, and P.-C. Yang Radiotherapy in Lung Adenocarcinoma with Brain Metastases: Effects of Activating Epidermal Growth Factor Receptor Mutations on Clinical Response Clin. Cancer Res., January 1, 2008; 14(1): 162 - 168. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Zlobec, R. Steele, L. Terracciano, J. R Jass, and A. Lugli Selecting immunohistochemical cut-off scores for novel biomarkers of progression and survival in colorectal cancer J. Clin. Pathol., October 1, 2007; 60(10): 1112 - 1116. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-C. Soria ERCC1-Tailored Chemotherapy in Lung Cancer: The First Prospective Randomized Trial J. Clin. Oncol., July 1, 2007; 25(19): 2648 - 2649. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||