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High Rate of Molecular Alteration in Histologically Tumour-Free Bronchial Epithelium of NSCLC Patients Detected by Multicolour Fluorescence in Situ Hybridisation

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High Rate of Molecular Alteration in Histologically Tumour-Free Bronchial Epithelium of NSCLC Patients Detected by Multicolour Fluorescence in Situ Hybridisation 1233-1240 28/3/06 14:14 Page 1233 ONCOLOGY REPORTS 15: 1233-1240, 2006 High rate of molecular alteration in histologically tumour-free bronchial epithelium of NSCLC patients detected by multicolour fluorescence in situ hybridisation WOLFGANG HILBE1, JUTTA AUBERGER1, STEPHAN DIRNHOFER5, THOMAS SCHMID3, MARTIN ERDEL2 and HANS-CHRISTOPH DUBA2,4 Departments of 1Internal Medicine, 2Medical Biology and Human Genetics, and 3Surgery, Medical University Innsbruck, Innsbruck; 4Department of Human Genetics, General Women and Children Hospital, Linz, Austria; 5Department of Pathology, University of Basel, Switzerland Received January 18, 2006; Accepted February 7, 2006 Abstract. Detection of molecular abnormalities could Introduction provide an essential tool for the diagnosis of non-small cell lung cancer (NSCLC) and defining patients at risk for early Non-small cell lung cancer (NSCLC) is the leading cause of relapse. Fluorescence in situ hybridisation (FISH) targeting cancer-related deaths in the Western world. The failure of 17 gene loci was applied to determine the frequency of antismoking campaigns, limitations in cytostatic treatment, molecular alteration in NSCLC probes and adjacent tumour- as well as the lack of efficient screening tests resulting in free bronchial epithelium. FISH was performed on fresh frozen delayed diagnoses are the reasons that patient outcome has specimens from 76 patients with histologically confirmed not improved substantially in recent years. New developments NSCLC and 54 specimens of adjacent tumour-free tissue. in the field of molecular biology have raised hopes for Routine autopsy lung tissue probes from 7 cancer-free patients improving the patient's clinical course. Molecular typing of served as a control group. Locus-specific (3p14.2, 3p21.2, bronchial epithelium could provide an essential tool for 3p21.3, 3p25.3, 5p15.2, 7p12, 8q24.12, 9p21, 13q14, and screening programs and, thus, early diagnosis (1-3). After 17p13.1) as well as centromere probes (4, 6, 7, 9, 11 and 16) curative surgery, a prognostic assessment identifying patients were used. Molecular alterations using FISH on interphase at risk for early relapse would be of major importance for nuclei were detected in 100% of NSCLC tumour specimens planning adjuvant treatment modalities. Furthermore, the and 89% of microscopically tumour-free tissues of NSCLC knowledge of molecular alterations has led to the development patients. In histologically ‘normal’ epithelium, the most of targeted therapies, proving their therapeutic efficacy (4,5). frequent alterations were seen with locus-specific probes for NSCLC emanates from a multistep carcinogenic process 3p14.2, 3p.21, 3p21.3, 3p25.3 and 7p12 and centromere- leading to an accumulation of genetic mutations in the specific probes 11 and 16 (12-93%). As expected, the bronchial epithelium (2,3,6-8), first generating histologically majority of genetic alterations seen in ‘premalignant’ identifiable ‘premalignant’ lesions and finally resulting in specimens were found in the correlating tumour probes. invasive carcinoma. These genetic alterations simultaneously None of the tested parameters revealed prognostic significance occur in multiple sites of the respiratory epithelium, a in univariate Cox analysis. FISH analysis, performing phenomenon called ‘field cancerisation’ (9-12). Allelic multicolour strategies, demonstrated its power in detecting deletions detected as a loss of heterozygosity (LOH) were genetic abnormalities in NSCLC specimens and even in reported for multiple chromosomal loci, indicating inactivation tumour-free sections of tumour patients. of several tumour suppressor genes and finally leading to oncogenesis and progression to lung cancer. Recurrent _________________________________________ alterations in tumours at numerous chromosomal loci of any chromosome have been seen in NSCLC at a rate between 8% and 100% (3,7,13,14). Correspondence to: Professor Wolfgang Hilbe, Department of Several methods (PCR, FISH, immunochemistry, etc.) Internal Medicine, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck, Austria have been applied to identify LOH and amplifications of E-mail: [email protected] chromosomal regions (6,14-19). Among those, fluorescence in situ hybridisation (FISH) is a simple and sensitive method Key words: lung cancer, non-small cell lung cancer, fluorescence to detect genetic deletions and amplifications (20-23). in situ hybridisation, multicolour-FISH, screening, field cancerisation Chromosomal abnormalities within interphase nuclei were targeted by using chromosome-specific DNA probes (21). Moreover, FISH offers the possibility of analysing cells directly from tissue sections or cell suspensions without prior 1233-1240 28/3/06 14:14 Page 1234 1234 HILBE et al: FISH AND NSCLC Table I. Comparison between tumour and premalignant specimens from results of FISH analysis. ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Tumour specimens (n=76) Premalignant specimens (n=54) ––––––––––––––––––––––––––– ––––––––––––––––––––––––––– Cut-offa No. of positive No. of positive Parameters (%) cases (%) Median (range) cases (%) Median (range) p-value ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– LOH13q14 (RB) 18.4 28 (37%) 15.8 (6.1-40.9) 5 (9.3%) 13.1 (3.5-22.0) 0.000 LOH17p13.1 (p53) 19.7 26 (34%) 16.9 (6.0-48.6) 4 (7.5%) 10.7 (4.2-26.2) 0.000 LOH9p21 (p16) 17.3 13 (18%) 13.9 (6.5-22.0) 3 (5.9%) 13.0 (20.0-6.0) 0.045 LOH 3p14.2 (FHIT) 5.2 63 (90%) 10.0 (2.4-35.3) 35 (81%) 7.5 (1.5-16.7) NS LOH 3p21 7.0 54 (78%) 10.5 (3.0-46.0) 33 (70%) 8.0 (3.4-13.0) NS LOH3p21.3 4.8 66 (94%) 9.5 (4.2-56.0) 39 (93%) 7.4 (2.4-15.0) NS LOH 3p25.3 5.6 62 (89%) 9.0 (1.1-26.8) 39 (83%) 8.2 (3.4-12.5) NS Mon.Chr.4 19.7 5 (7.5%) 11.7 (1.6-24.9) 1 (2.0%) 11.4 (3.8-21.3) NS Mon.Chr.7 25.7 2 (2.7%) 11.6 (0.8-25.7) 1 (1.9%) 11.0 (3.0-31.7) NS Mon.Chr.9 20.6 4 (5.5%) 13.5 (5.0-35.0) 0 (0%) 13.0 (6.5-20.1) 0.091 Mon.Chr.11 15.5 14 (19%) 11.5 (3.0-25.2) 6 (12%) 10.6 (4.5-29.9) NS Mon.Chr.16 16.5 9 (13%) 12.1 (1.5-22.5) 13 (28%) 11.8 (4.4-23.5) 0.041 Mon.Chr.17 17.5 5 (6.9%) 14.0 (7.3-24.5) 3 (5.7%) 13.5 (4.8-19.0) NS Ampl.8q24 (c-myc) 3.0 64 (93%) 9.1 (0.0-51.8) 2 (3.7%) 3.7 (0.0-12.4) 0.000 Ampl.7p12 (EGFR) 1.7 66 (96%) 8.1 (0.0-44.0) 9 (17%) 2.9 (0.0-9.3) 0.000 Ampl.5p15.2 8.9 29 (45%) 8.1 (0.0-57.4) 0 (0%) 3.5 (0.0-13.4) 0.000 Ampl.CEP Chr.6 4.6 52 (75%) 6.4 (0.6-40.5) 2 (3.7%) 4.2 (0.9-10.8) 0.000 ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– aCut-off level was defined by mean + 2SD of the controls. NS, not signficant. ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– cell culture. Due to the standardisation of preparation and carcinoma (SCC), 35 cases; large cell carcinoma, 3 cases; and ease of analysis, this method has gained broad acceptance. carcinoids, 2 cases. According to UICC recommendations The current study is aimed at defining the frequency of (25), 49 patients were classified as stage I, 16 patients as molecular alteration in NSCLC patients. Therefore, 76 tumour stage II, 9 patients as stage III and 2 patients as stage IV. All specimens were analysed using a panel of centromeric DNA patients were surgically treated between 1994 and 2001 probes for the chromosomes 4, 6, 9, 11, 16 and 17. Probes for (pneumectomy in 26 cases, lobectomy in 47 cases and wedge specific chromosomal regions, such as 3p14.2, 3p21.2, 3p21.3, resection in 3 cases). Patients in advanced clinical stages 3p25.3, 5p15.2, 7p12, 8q24.12, 9p21, 13q14 and 17p13.1, underwent chemotherapy and/or radiotherapy. The median were applied to test either LOH or amplification. Furthermore, observation time was 31 months (range, 1-127 months), the the same markers were examined in 54 microscopically 50% survival probability was reached at 65 months, and 30 tumour-free specimens of lung cancer patients to define the patients died. At the time of surgery, the median age of patients premalignant molecular profile in NSCLC patients. was 61 years (range, 37-79 years). The male:female ratio was 3.2:1.0 (58/18). Patients and methods FISH analysis. Frozen lung tissue specimens were cut at 5 μm Patients. In total, 76 tumour and 54 tumour-free specimens in a cryostat. Before nucleus extraction, cryostat sections from surgically-treated NSCLC patients were analysed. were immunohistochemically evaluated to quantify tumour Freshly resected tissue was collected for routine diagnosis. cell infiltration (median, 60% tumour cells). Malignant cells One portion of the sample was fixed in 10% formalin and were mechanically disaggregated and suspended in phosphate- routinely processed for paraffin embedding. All surgical buffered saline solution (PBS), pelleted at 1000 x g, fixed in tumour specimens were classified histopathologically methanol/acetic acid and stored at -20˚C. After nucleus according to the WHO classification (24). Another portion of extraction (26), fluorescence in situ hybridisation (FISH) was surgical samples was used for molecular analysis. The probes performed as described (22,27). The following probes were were immediately snap-frozen in liquid nitrogen and stored used: a) locus-specific probes: LSI® p16 (9p21)/CEP® 9 Dual at -80˚C until sectioning. Color Probe; LSI 13 (RB1) 13q14 Probe; LSI p53 (17p13.1) All 76 patients showed histologically quantified tumour (Vysis Inc.); and probes for 3p14.2, 3p21.2, 3p21.3 and cell infiltration. Routine autopsy lung tissue probes from 7 3p25.3.
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