Aneuploidy of chromosome 7 can be detected in invasive lung cancer and associated premalignant lesions of the lung by fluorescence in situ hybridisation

Lung Cancer 28 (2000) 225 – 235 www.elsevier.nl/locate/lungcan Aneuploidy of chromosome 7 can be detected in invasive lung cancer and associated premalignant lesions of the lung by fluorescence in situ hybridisation Niklas Zojer a,c, Gerhard Dekan b, Jutta Ackermann a, Michael Fiegl a, Hannes Kaufmann a, Johannes Drach a,*, Heinz Huber a a First Department of Internal Medicine, Di6ision of Clinical Oncology, Wahringer Gurtel 18 -20, 1090, Vienna, Austria b Institute of Clinical Pathology, Uni6ersity of Vienna, Wilhelminenspital, Vienna, Austria c First Department of Medicine and Medical Oncology, Wilhelminenspital, Vienna, Austria Received 29 September 1999; received in revised form 16 December 1999; accepted 17 December 1999 Abstract In the present study the chromosomal status of seven invasive non small cell lung cancer specimens and associated premalignant lesions was investigated. By fluorescence in situ hybridisation (FISH) with centromere specific probes, an increase in the percentage of aneuploid cells from pre-invasive to invasive lesions could be demonstrated (mean 8.5 and 59%, respectively, for chromosome 7). Furthermore, mean chromosome copy numbers were higher in invasive carcinomas as compared to premalignant lesions, indicating polyploidization during tumor development. Increasing evidence suggests that aberrations of chromosome 7 occur early in the development of lung cancer. Whether these aberrations can be used as a biomarker for future neoplastic progression remains to be determined. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Lung cancer; Premalignant lesions; Atypical adenomatous hyperplasia; Cytogenetic; Chromosomal aberrations; FISH 1. Introduction Genetic alterations which characterize premalignant lesions of the lung are increasingly identified. Dysregulation of cell cycle regulatory genes and their respective gene products have been described [2,3,11,12,14,19,20,23,24,28,40,55,58] as * Corresponding author. Tel.: + 43-1-404004429 ;fax: + 431-404004451. E-mail address: johannes.drach@akh-wien.ac.at (J. Drach) well as mutations of the K-ras oncogene [5,34,47,54] and allelic losses at chromosomal arms 3p and 9p [10,13,16,17,46,51,52] and 8p [56]. Furthermore, sequential molecular alterations have been suggested to occur during development to lung cancer, with deletions of 3p preceeding p53-gene alterations [4]. Distinct chromosomal aberrations have however, been rarely reported for preinvasive lesions of the lung. This may be due to difficulties in obtaining material suitable for conventional cyto- 0169-5002/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 9 - 5 0 0 2 ( 0 0 ) 0 0 0 9 7 - 0 226 N. Zojer et al. / Lung Cancer 28 (2000) 225–235 genetic analysis as well as the low proliferative index of these lesions in vitro. In fact only three cases of successfully karyotyped dysplasias of the bronchial epithelium have been reported, with deletion of 17p13 or deletion of 3p as sole clonal abnormality [43,44]. Numerical chromosomal alterations are a common finding in invasive lung cancer [37,49,50]. Polysomies of chromosome 7 were shown to be particularly frequent, occuring alone or in combination with other aberrations in 41% of non small cell lung cancer specimens [50]. Furthermore, aberrations of chromosome 7 could be demonstrated even in non neoplastic tissue adjacent to lung tumors [27] and in premalignant lesions of the upper aerodigestive tract. Polysomies of chromosomes 7 and 17 were detected with increasing frequency in normal epithelium, hyperplastic lesions and dysplastic lesions of the oral cavity [26]. Similar data were obtained for premalignant lesions and invasive cancer of the head and neck [42,53]. Moreover, the presence of genetic aberrations in premalignant lesions appeared to be associated with progression to invasive cancer. In the studies on preinvasive lesions of the head and neck, the technical limitations of conventional karyotypic analysis were overcome by interphase fluorescence in situ hybridisation (FISH) with chromosome specific probes, which provides the opportunity to evaluate cytogenetic aberrations on the single cell level. Moreover, since FISH can be performed on paraffin sections, chromosomal status can be related to distinct histological lesions. In the present study, we therefore, used FISH with chromosome specific probes to investigate the chromosomal status of both invasive lung cancer and adjacent premalignant lesions. Chromosome 7 was targeted in all experiments. We aimed at demonstrating a clonal relationship of invasive and preinvasive lesions and thus determining aberrations which can be preferentially used in lung cancer screening procedures. 2. Materials and methods Paraffin-sections from seven surgically resected non small cell lung cancer specimens (five adeno- carcinomas, two squamous cell carcinomas) were selected according to the concomitant presence of invasive carcinoma and premalignant lesions. The pathologic stage and grade of primary tumors is specified in Table 1. Premalignant and malignant lesions were identified on hematoxylin/eosin (H/E) stained deparaffinized sections and adjacent paraffin-sections were used in the FISH experiments. The typical histopathological features of premalignant lesions of the lung (squamous metaplasia and atypical adenomatous hyperplasia) have been reviewed elsewhere [18,25,41,48] (Figs. 1 and 2). Case numbers 1 and 3 represent foci of atypical adenomatous hyperplasia not contiguous with the invasive carcinoma, whereas case numbers 2 and 4 represent adenomatous hyperplasia — like lesions directly adjacent to the invasive tumor (bronchioloalveolar carcinoma). A focus of squamous metaplasia near a squamous cell cancer of the lung was investigated in case number 6. Pleural effusion specimens were obtained from six lung cancer patients during routine diagnostic or therapeutic procedures. All patients had advanced disease at initial diagnosis (stage IIIA-IV), and did not undergo surgical resection of the primary tumor. An aliquot of the effusion specimens was submitted to the department of pathology for cytological evaluation. Pleural effusion cells for FISH-studies were gained by centrifugation, washed twice in phosphate-buffered saline (PBS), fixed in methanol-acetic acid (3:1, v/v) and stored at −80°C. For FISH-experiments, paraffin-embedded tissue specimens were applied to silanized slides, dewaxed in xylene at 40° Celsius and fixed in methanol for 10 min each. Deparaffinization and fixation were repeated three times, followed by rehydration in graded ethanol. To enhance probe accessibility to the nucleus, cells were incubated at 80°C in 1 mol/l sodium-thiocyanat and at 37°C in 0.4% Pepsin (in 0.2 N HCl) for 2 and 4 min, respectively [35]. Interphase FISH for numerical chromosomal aberrations was performed using centromere-specific probes for chromosomes 7, 8, 9 and 18, directly labeled with fluorochromes (obtained from VYSIS, Downers Grove, IL). Prehybridisation and hybridisation was carried out as previ- N. Zojer et al. / Lung Cancer 28 (2000) 225–235 ously described [8,9]. Briefly, slides were immersed in 0.1 N HCl/0.05% TritonX-100, washed twice in saline sodium citrate (SSC) and PBS and treated with formaldehyde (1% in PBS). After washes with PBS and SSC, cells were dehydrated through 70, 85 and 100% ethanol. Denaturation temperature and time were adjusted for the use in paraffin-embedded lung sections [15], i.e. DNA was denaturated by incubation with formamide 70% at 85°C for 5 min (in case of effusion cells: formamide 70% at 70°C for 5 min). Cells were again dehydrated through ethanol. Hybridisation mixture (with 2 mg/ml of the specific probe) was then applied to each slide, which was coverslipped and sealed with rubber cement. Hybridisation was performed in a humidified chamber overnight at 37°C. After two washes with SSC nuclei were counterstained with DAPI, and cells were analyzed by fluorescence microscopy. Illustrations of hybridisation results were produced using a cooled charged coupled device (CCD) camera (Photometrics, Tucson, Ariz) mounted on an Axioplan-Zeiss immunofluorescence microscope (Zeiss, Austria). 227 Lesions were identified after FISH according to their histopathological features and diagnosis was confirmed by concomitantly evaluating adjacent H/E stained sections. At least 50 non overlapping nuclei were evaluated for each lesion (premalignant and invasive) and each targeted chromosome (at least 200 cells were scored in case of effusion samples). Mean chromosome copy numbers were calculated for each lesion/effusion sample and chromosome by dividing the sum of the centromeric signals by the number of nuclei scored [60]. After FISH study, tissue specimens were again stained with H/E to determine the degree of tissue preservation. Despite sodium-thiocyanat treatment and Pepsin digestion, only minor tissue desintegration was observed and the architecture of preinvasive lesions was easily discernible on the H/E stained FISH slides. To determine unspecific background aneuploidy, normal bronchial mucosa specimens (n = 4) were targeted by FISH and numerical probes (Fig. 3). The mean frequency of monosomy 7 in control specimens was 12.2%, mainly due to truncated nuclei in the 5 mm sections with consecutive Table 1 Chromosomal status of invasive lung carcinoma and adjacent premalignant lesiona Aneupoidy rate (mean chromosone copy number) 1. 2. 3. 4. 5. 6. 7. Chromosome Chromosome Chromosome Chromosome Chromosome Chromosome Chromosome Chromosome Chromosome Chromosome 7 9 7 9 7 18 7 18 8 7 Chromosome 7 Chromosome 9 Chromosome 7 Atypical ad. hyperplasia/AAH-like lesions Invasive carcinoma Stage, grading 0% 0% 6% (2.0) 0% 11% (1.6) 0% 5% (3.0) 10% (3.2) 0% – Squamous metaplasia 21% (3.3) 0% – 10% (3.2) 0% 31% (3.2) 17% (1.0) 13% (1.0) 4% (1.0) 94% (\6.0) 23% (3.4) 6% (3.0) 59% (3.8) Invasive carcinoma 79% (4.4) 36% (3.2) 73% (3.5) pT2, G2 pT4, G2 pT2pN0pM1, G1 (Multicentric) pT1, G2 pT1pN2, G2 pT3pN2, G2 pT2pN0, G2 a Lists of the percentages of aberrant nuclei for each lesion and chromosome (aneuploidy rate). Values are corrected for background aneuploidy, e.g. in sample Number 3 the invasive carcinoma had 35.4% monosomic nuclei, when targeting chromosome 7 with a centromere specific probe. Correction for background monosomy (22.4%), left an aneuploidy rate of 13%; The number in brackets indicates the mean chromosome copy number, which is calculated by dividing the sum of signals by the number of nuclei enumerated; The pathologic stage and grade of the primary tumor is specified in the last column. 228 N. Zojer et al. / Lung Cancer 28 (2000) 225–235 Fig. 1. The alveolar lining is replaced by slightly atypical cylindrical cells in this focus of adenomatous hyperplasia (case no. 1; H/E). Fig. 2. Cells with moderate to severe atypia, indicative of progression of atypical adenomatous hyperplasia to adenocarcinoma in situ (case number 4; H/E). loss of chromosomal signals. No cells with trisomy 7 were detected in control specimens. True monosomy 7 in premalignant lesions and/or lung cancer specimens thus required the presence of more than 22.4% (12.2% +3 S.D.) monosomic cells. For effusion samples the cutoff was set at N. Zojer et al. / Lung Cancer 28 (2000) 225–235 1.5% for trisomic and tetrasomic nuclei each, according to our data from non malignant effusions. 3. Results All seven invasive carcinoma specimens were successfully investigated by FISH with centromeric probes. However, due to tissue artifacts, enumeration of signals was not possible in two of the seven premalignant lesions (Table 1). Aneuploidy of chromosome 7 was detected in 100% (7/7) primary tumors. The percentage of aneuploid cells ranged from 10– 94% (mean 59%). Premalignant lesions exhibited abnormalities of chromosome 7 in 80% (4/5) (range of aneuploid cells 5 – 21%; mean 8.5%; Fig. 4). Other chromosomes (chromosomes 8, 9 and 18) were also frequently altered in invasive carcinoma, however, abnormalities in preinvasive lesions were detected in only 1/5 cases. Regarding aneuploidy of chromosome 7, there was a clear progression from preinvasive lesion to invasive cancer. The frequency of aberrant cells increased from 8.5% in preinvasive lesions to 59% in invasive carcinoma, indicating clonal outgrowth. Also, there was an increase in chromosomal copy numbers, which propably is due to polyploidization in the context of genetic instability. As indicated by the mean chromosome copy numbers in Table 1, a gain of chromosomes was 229 evident in most cases, however monosomy 7 and monosomy 18 were detected in primary tumor number 3 and monosomy 9 in primary tumor number 2. As reported previously [37], numerical chromosomal aberrations are present invariably in samples of metastatic lung cancer cells. Similarily, we detected numerical chromosomal aberrations in 100% (6/6) of malignant pleural effusion specimens from lung cancer patients (Table 2). No case of chromosomal loss was observed. 4. Discussion Neoplasms of the upper aerodigestive tract and lung arise when genetic damage accumulates in a multistep process of malignant transformation, e.g. due to exposure to inhaled carcinogens. Chromosome 7 was shown to be an early marker of tumorigenesis in case of head and neck cancer, where polysomies of this chromosome have been demonstrated in hyperplastic and dysplastic epithelium by fluorescence in situ hybridisation [26,42,53]. Similar data for lung cancer are lacking, although Crowell et al. [6] could detect trisomy 7 in cytologic material obtained during fiber bronchoscopy from lung cancer patients. Tumor cells were not present in the samples unequivocally as judged by cytologic criteria. Instead cytology revealed squamous metaplasia or atypical glandular cells in 32% of cases, whereas Table 2 Chromosomal status of effusions from patients with lung cancera Aneuploidy rate (mean chromosome copy number) a b c d e f Chromosome 7 Chromosome 9 Chromosome 18 Cytology 3.1% 95.1% 34.4% 59.9% 0% 16.4% 2.1% 63.2% 7.1% 0.4% 5.4% 19.2% 1.2% 17.6% 3.8% 0.4% 0% 12.1% Squamous cell cancer Adenocarcinoma Adenocarcinoma Squamous cell cancer Adenocarcinoma Adenocarcinoma (4.0) (3.7) (4.1) (3.9) (3.0) (4.0) (5.5) (\6.0) (3.0) (3.7) (3.6) (3.8) (3.6) (4.0) (3.0) (3.0) a Lists of the percentages of aberrant nuclei for each effusion and chromosome (aneuploidy rate). Values are corrected for background aneuploidy. Cut-off was set at 1.5% for trisomic and tetrasomic nuclei each, according to our data from non-malignant effusions. The number in brackets indicates the mean chromosome copy number, which is calculated by dividing the sum of signals by the number of nuclei enumerated. 230 N. Zojer et al. / Lung Cancer 28 (2000) 225–235 Fig. 3. Normal bronchial mucosa from a lung cancer patient. As shown by FISH, a diploid signal pattern for chromosome 7 is present. Fig. 4. FISH-study of adenomatous hyperplasia-like lesion (case number 4). Some nuclei exhibit three signals for chromosome 7 in this preinvasive lesion, which demonstrates replacement of normal (diploid) cells by an aberrant cell population. N. Zojer et al. / Lung Cancer 28 (2000) 225–235 231 Table 3 Frequency of genetic alterations in lesions preceeding squamous cell cancer of the lung as reported in the literature e Alteration Hyperplasia/metaplasia Dysplasia Carc. in situ References p53a LOH 3p LOH 9p Loss of Fhit Cyclin D1 Cyclin E p16b LOH 8p21-23 LOH 5q Telomerasec Rbd 7% 76% 38% 0–47% 6% 0% 17% 15% 11% 71–80% 0% 37% 86% 80% ns 17–46% 0–33% 24% 50% 33% 82% 0% 75% 100% 100% 93% 38% ns 50% 92% 40% 100% 0% [3,12,23,28,55] [13,23,51] [16,23,51] [10,46,52] [3,28] [28] [2,3] [56] [51] [58] [3] a p53 alterations (p53-gene deletion as determined as loss of heterozygosity 17p13, overexpression of p53 as determined by immunohistochemistry, p53 gene mutations). b p16 alterations (aberrant methylation of p16, loss of p16 expression by immunohistochemistry). c Telomerase enzyme activity. d Loss of retinoblastoma protein expression as determined by immunohistochemistry. e Percentages given in the table were taken from the first reference cited in the fifth column; LOH=loss of heterozygosity; , overexpression of cyclins as determined by immunohistochemistry; ns, not stated. in all other samples only normal cells were detected. The occurrence of trisomy 7 in premalignant bronchial cells was also suggested by the presence of this aberration in bronchial epithelium from cancer free smokers and uranium miners. However, the unequivocal identification of malignant cells by cytologic criteria alone may be difficult, and indeed our group repeatedly demonstrated the superiority of FISH with chromosome specific probes as compared to cytology in terms of demonstrating malignancy [37,59,60]. Although cytogenetic data on premalignant lesions of the lung are rare [43,44], there is increasing evidence for the presence of multiple genetic abnormalities in preinvasive lesions of the lung, as evidenced by immunohistochemistry, LOH- analysis, and analysis of p53-gene and K-ras mutations (Tables 3 and 4). E.g. Wistuba et al. [55] found loss of heterozygosity (LOH) at one or more chromosomal regions in 31 and 42% of normal bronchial mucosa and mildly abnormal specimens, respectively. Allelic losses on 3p and 9p were particularily frequent. They also demonstrated a progressive increase of LOH frequency with increasing severity of histopathological changes. In the present study, four cases of atypical adenomatous hyperplasia (or adenomatous hyperplasia-like lesions) of the lung and Table 4 Frequency of genetic alterations in lesions preceeding adenocarcinoma of the lung as reported in the literature Alteration AAH Referencesf p53a LOH 3p LOH 9p Cyclin D1 p16b K-rasc c-erbB-2d Rbe Telomerase 11% 18% 13% 47–89% 11–25% 39% 7% 0–18% 0% [14,19,20,40] [17] [17] [24] [24] [5,34,47,54] [14] [24] [58] a Overexpression of p53 as determined by immunohistochemistry. b Loss of p16 expression by immunohistochemistry. c K-ras mutations. d Overexpression of c-erbB-2 by immunohistochemistry. e Loss of retinoblastoma protein expression as determined by immunohistochemistry. f Percentages given in the table were taken from the first reference cited in the third column; AAH, atypical adenomatous hyperplasia; LOH, loss of heterozygosity; , overexpression of cyclins as determined by immunohistochemistry. 232 N. Zojer et al. / Lung Cancer 28 (2000) 225–235 associated invasive adenocarcinomas and one case of squamous metaplasia and associated invasive squamous cell cancer were successfully targeted by FISH and centromeric probes. Squamous cell lung cancer has long been thought to arise from premalignant lesions [1,36]. However, only re-cently an adenoma-carcinoma sequence has been proposed for the development of adenocarcinoma of the lung [22,30 – 32]. The frequency at which atypical adenomatous hyperplasia is detected in resection specimens of primary carcinoma of the lung is 5 – 15% [30,32]. These lesions may present as several diffuse foci or as a single circumscribed focus, in which the alveolar lining is replaced by cuboidal cells with signs of mild to moderate atypia. Foci of cell proliferation indistinguishable from solitary adenomatous hyperplasia can sometimes be observed in continuity with overt adenocarcinoma [18]. These lesions are thought to give rise to the invasive part of the tumor and were thus included in our study as precursor lesions. The potential for progression to invasive cancer is suggested by the presence of aneuploidy as determined by flow cytometry in 54% [33] and LOH of 3p or 9p in 10– 20% [23] of cases of atypical adenomatous hyperplasia. The genetic alterations which were detected in adenomatous hyperplasia to date are reviewed in Table 4. A clonal relationship between a premalignant lesion and associated lung carcinoma has rarely been demonstrated. Wistuba et al. [55] found identical genetic aberrations in invasive lung cancer and associated preinvasive lesion in only 6% of cases, although a clonal relationship was considered possible in 48% of the remaining cases. A combined cytogenetic, immunocytochemical and molecular approach suggested that simultaneously occurring, multiple invasive and/ or premalignant lesions of the lung are of independent origin [44]. Other authors achieved similar results, with identical molecular alterations found in the premalignant and invasive lesions only infreqently [23,54]. We targeted chromosome 7 by FISH with a centromere specific probe in all lung sections. As shown in Table 1, alterations of chromosome 7 were detected in 7/7 (100%) of primary tumors and 4/5 (80%) of pre-invasive lesions. The increase in the frequency of aberrant cells during the progression from pre-invasive lesion to invasive lung cancer is indicative of a cumulative replacement of non-neoplasic cells by malignant cells. There also was an increase in chromosomal copy numbers occuring in the transformation from pre-invasive to invasive lesion. Furthermore, aneuploidy of additional chromosomes was demonstrated in invasive lesions and metastatic bronchial carcinoma cells derived from effusions (e.g. polysomy of chromosome 9). This can be related to genetic instability and cumulative gain of genetic material, i.e. by polyploidization, during tumor development. Genetic instability also leads to intratumor heterogeneity regarding chromosomal status, which was evident in primary tumors and malignant effusion cells (e.g. in effusion d. the percentage of aneuploid cells ranged from 0.4 to 59.9% for different chromosomes). Alterations of chromosome 7 have been described in tumor-infiltrating lymphocytes and non neoplastic tubular epithelial cells of the kidney [7,21]. In the present study the clonal nature of chromosome 7 aberrations in bronchial premalignant and malignant lesions is supported by the absence of this alteration in normal lung tissue as shown by FISH. Chromosome 7 harbours the proto-oncogene c-met, the platelet derived growth factor A-chain gene, the plasminogen activator inhibitor type-1 gene and the gene for the epidermal growth factor receptor (EGFR). Overexpression of EGFR was shown to be an early event in head and neck and lung tumorigenesis [39,45]. Thus, numerical alterations of chromosome 7 in conjunction with upregulation of the EGFR-gene may be of major importance in these tumor types. Several studies demonstrated the presence of genetic alterations (by LOH analysis and analysis of K-ras mutations) in bronchoscopically gained material from smokers and patients at risk for second primary lung cancer [29,38, 51,57]. However, it is unclear whether the presence of such alterations is associated with an increased risk for lung cancer. Since FISH was N. Zojer et al. / Lung Cancer 28 (2000) 225–235 shown to be feasible to detect aberrant cells in material obtained by fiber bronchoscopy [37], we propose to study cytogenetic markers by FISH in bronchoscopically gained material from lungs of smokers prospectively and to target chromosome 7 in these experiments. Indeed, Crowell et al. [6] detected trisomy 7 in bronchial epithelium from cancer-free smokers and uranium miners and two of the former uranium miners with aberration of chromosome 7 developed lung cancer within 2 years of bronchial cell collection. A study including a greater number of patients will be necessary to accurately define the role of chromosome 7 as a biomarker for lung cancer development. 5. Conclusion In the present study we demonstrated numerical aberrations of chromosome 7 to be present in invasive lung cancer and adjacent premalignant lesions of the lung. In contrast, we did not detect aneuploidy of chromosome 7 by FISH in normal bronchial mucosa /alveolar cells of lung cancer patients. These results suggest that alterations of chromosome 7 accompany the transition from normal lung tissue to hyperplastic lesions and may indicate a clonal relationship between primary tumor and associated premalignant lesion. Acknowledgements This study was supported by a grant from Kommission Onkologie. 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