*From the Institute of Immunology (Drs. Mecklenburg, Riethmüller, and Kufer), Ludwig-Maximilians-University, Munich; Department of Pneumology (Drs. Stratakis and Huber), Medizinische Klinik Innenstadt, Ludwig-Maximilians-University, Munich; and Departments of Pneumology (Dr. Häussinger) and Pathology (Dr. Morresi-Hauf), Asklepios Fachkliniken München-Gauting, Munich, Germany.
Correspondence to: Ingo Mecklenburg, Institute of Immunology, Ludwig-Maximilians-University, Goethestrasse 31, 80336 Munich, Germany; e-mail: firstname.lastname@example.org
Lung cancer is the leading cause of cancer death in men and women in industrialized nations. Several approaches have been evaluated for the early detection of lung cancer, but screening strategies have failed to decrease disease-specific mortality.1There has been intensive research on molecular abnormalities in lung cancer and a broad evaluation of methods to diagnose early malignant transformations.2Exfoliated cells shed from the lower respiratory tract can be detected in (induced) sputum or in BAL samples and can be helpful for the detection of central tumors of the larger bronchi. Several molecular biomarkers have been evaluated to detect neoplastic lesions in the lung. However, only a subset of tumors display specific mutations resulting in insufficient sensitivity. More importantly, stage IA tumors only infrequently show molecular abnormalities,3 making the search for new biomarker that detect premalignant lesions inevitable.
Individual members of the melanoma antigen (MAGE) family4are frequently expressed in lung cancers of different histologic origin.5–6 No expression has been observed in any normal adult tissues with the only exception of testicular germ cells. MAGE genes belong to the family of cancer/testis antigens, and are known to be the most tumor-specific marker so far.7Recently MAGE expression has been found in normal appearing lung tissue adjacent to lung cancer in striking contrast to lung tissue from healthy individuals.8Expression of MAGE genes seems to be an early event in lung carcinogenesis, and the finding of significant expression in the bronchial epithelium of former smokers demonstrates the potential use as marker for preneoplastic lesions in the lung. We recently developed a multimarker reverse transcription polymerase chain reaction (RT-PCR) for the detection of individual MAGE markers in blood and bone marrow of tumor patients,9 and assessed the potential use of a multimarker MAGE RT-PCR on sputum and BAL samples for the detection of (pre-) neoplastic cells.
After receiving informed consent, we obtained sputum samples from 15 patients with malignant lung tumors and 2 patients with benign pulmonary diseases. Induced sputum was collected after 30 min of inhalation with a 4.5% saline solution and diluted with two volumes of 1% dithiotreitol. After washing and dilution in phosphate-buffered saline solution, a 2-mL aliquot was dispensed in 8 mL of denaturating guanidine isothiocyanate nucleic acid extraction buffer and stored at 4°C until RNA isolation the next day. We further collected bronchial lavage fluid during bronchoscopy performed for suspected malignant disease in 30 patients. Two-milliliter samples of the lavage fluid were immediately mixed with the nucleic acid extraction buffer and stored at 4°C until transportation to the laboratory within a few days. A detailed protocol for RNA preparation, complementary DNA synthesis, and MAGE-polymerase chain reaction with all primer sequences is described elsewhere.9
Expression of at least one MAGE gene was detected in 5 of 15 sputum specimens from patients with lung cancer (33%), with negative findings in the control samples. In BAL fluid we found MAGE expression in 18 cases of 23 patients with confirmed lung cancer (78%). Conventional cytology demonstrated dysplastic cells in eight samples (35%). In two patients, MAGE expression was detected in lavage fluid with actinomycosis and severe bronchitis and histologically severe inflammation with concomitant tissue regeneration. The results are summarized in Tables 1, 2
Exfoliated lung cancer cells can be detected in induced sputum and BAL fluid by their expression of MAGE genes. Because of the early activation of MAGE gene expression in lung carcinogenesis,8 a multimarker MAGE RT-PCR may further have the attractive capability to reveal preneoplastic lesions. MAGE genes are possibly expressed when cells change differentiation and tissue is regenerated, eg, in invasive cancers or severe bronchitis with bronchiectasis. The specificity of this marker may therefore be restricted to patients without severe inflammatory processes, but a multimarker MAGE polymerase chain reaction in sputum may complement noninvasive imaging studies, and may provide a sensitive screening tool for secondary prevention of lung cancer.
Abbreviations: MAGE = melanoma antigen; RT-PCR = reverse transcription polymerase chain reaction
+ and ++ denote expression intensity; − denotes negative result; ND = not determined; SCLC = small cell lung cancer.
+ through ++++ denote expression intensity; − denotes negative result; NA = not appraisable; NSCLC = non-small cell lung cancer. See Table 1 for expansion of other abbreviations.
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