Expression of Chemokine Receptor CCR6 as a Molecular Determinant of Adrenal Metastatic Relapse in Patients With Primary Lung Cancer

Christophe M. Raynaud; Olaf Mercier; Philippe Dartevelle; Frédéric Commo; Ken André Olaussen; Vincent de Montpreville; Fabrice André; Laure Sabatier; Jean-Charles Soria

Disclosures

Clin Lung Cancer. 2010;11(3):187-191. 

In This Article

Patients and Methods

Patients

The recruitment period of patients for the present study occurred between April 1992 and September 2005 and involved 21 consecutive patients with NSCLC who had undergone surgery for the excision of a primary tumor and corresponding adrenal metastasis at the Centre Chirurgical Marie Lannelongue (CCML; Le Plessis Robinson, France). All surgical formalin-fixed, paraffin-embedded samples (primary tumors and metastases) were histologically analyzed by a pathologist in the Department of Pathology at CCML.

Antibodies

Antibodies recognizing the different proteins included CCR6 (catalogue number MAB 195; R & D Systems; Minneapolis, MN) at a final dilution of 1/500, CCR7 (ser139; catalogue number 550937; Pharmingen; San Diego, CA) at a final dilution of 1/200, CX3CR1 (catalogue number AB-1892; Chemicon International; Temecula, CA) at a final dilution of 1/1000, CXCR4 (catalogue number MAB 172; R & D Systems) at a final dilution of 1/2500, and CCL20 (catalogue number AF 360; R & D Systems) at a final dilution of 1/20.

Immunohistochemical Staining for Chemokine Receptors

Indirect immunoperoxidase staining was performed on formalinfixed tissue sections or cultured cells using a Vectastain Elite kit (catalogue number PK-6200; Vector Laboratories; Burlingame, CA) and a standard protocol. Briefly, using xylene baths, paraffin was removed from serial tumor sections, which were then rehydrated in a series of graded ethanol solutions. Antigens were retrieved by heating (98°C) using different prewarmed buffers, ie, citrate buffer at pH 6 for CCR6, citrate buffer at pH 7.3 for CX3CR1, and citrate buffer at pH 9.9 for CXCR4 for 20, 10, and 30 minutes, respectively. No antigen retrieving was used for CCR7 or CCL20, according to the manufacturer's instructions. Endogenous peroxidase activity was inhibited by immersing the tumor sections in 3% hydrogen peroxide for 5 minutes. Slides were then re-equilibrated with phosphate-buffered saline and incubated with 1% normal horse serum for 20 minutes. Slides were incubated with the primary antibody (diluted in 1% horse serum buffer) at room temperature for 2 hours or overnight at 4°C. Positive control tissues were used according to the manufacturer's instructions. Negative control consisted of omitting the primary antibody, and incubation with immunoglobulins of the same species. Slides were then incubated with a biotinylated secondary antibody, followed by avidin-biotin peroxidase complex (catalogue number PK-6200; Vector Laboratories) for 30 minutes each. A color reaction was developed, using diaminobenzidine as substrate. Slides were lightly counterstained with hematoxylin and mounted in permanent mounting medium.

Scoring of Stained Cells

Levels of protein expression were scored using a variant histologic score (H-score), involving the product of the percentage of positive tumor cells according to staining intensity (0, null; 1, faint; 2, moderate; 3, strong). Consequently, H-scores range from 0–0. For samples with heterogenous staining (rarely observed), a mean value of different scores from representative fields was calculated in each case. Only a cytoplasmic staining pattern was considered positive, as previously described.[17]

Statistical Analysis

Tumor chemokine expressions were compared using Wilcoxon tests. Differences were considered significant if a 2-sided P < .05 was obtained. All statistical analyses were performed using SAS, version 9.13 (SAS Institute, Inc.; Cary, NC).

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