Chromosome Abnormalities in Human Non-Small Cell Lung Cancer1

[CANCER RESEARCH (SUPPL.) 52. 2702s-2706s, May I, 1992] Chromosome Abnormalities in Human Non-Small Cell Lung Cancer1

Joseph R. Testa2 and Jill M. Siegfried

Department of Medical Oncology, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111 [J. R. T.], and Department of Pharmacology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261 fJ. M. S.J

Abstract difficult to obtain adequate numbers of well banded metaphase cells for detailed cytogenetic analysis. Moreover, the karyotypes Clonal cytogenetic abnormalities found in 30 non-small cell lung can be extremely complex, with many additional chromosomes, carcinomas (NSCLC), including 28 newly diagnosed primary tumor spec complicating efforts to identity consistent changes. Recently, imens, are summarized. Multiple chromosome alterations were identified in every case, and 19 of 30 tumors had near-triploid or near-tetraploid several groups have reported detailed karyotypic findings in karyotypes. Polysomy 7 and partial gains of 7p, including 7pl l-pl3 (site primary NSCLC (2-4); these studies have revealed multiple of the EGFR gene), were particularly frequent, occurring alone or in numerical and structural alterations in this neoplasm. combination in 26 tumors. Recurrent losses involving Ip, 3p, 6q, 9p, lip, In this report, we summarize clonal cytogenetic changes 15p, and 17p (where the TP53 gene is located) were each seen in 16-25 found in 30 patients with NSCLC. We also validate the culture cases. Five tumors exhibited double minutes, which were associated with model used to produce actively dividing tumor cells, which amplified MYC1 (1 case) and EGFR (1 case), as determined by Southern provide better quality karyotypes. Although the karyotypic pat analysis. The cytogenetic data were compiled from either short term tern appears to be very complex in NSCLC, several recurrent cultures of tumor tissue harvested within 1-9 days (18 cases) or later abnormalities have been identified, which should help to target harvests performed on long term cultures or cell lines (6 cases); in the specific chromosome sites for future molecular investigations. other 6 cases results were obtained from both short term and long term cultures. Two studies were performed to validate the use of long term The data indicate that, as in colon cancer (5), tumorigenesis in culture for cytogenetic analysis of solid lung tumors. First, in order to NSCLC is characterized by the accumulation of multiple ge determine whether cytogenetic results from cultures are representative netic alterations. of the original tumor, the modal chromosome number of 13 specimens placed into culture was compared to the DNA index of the original tumor Materials and Methods tissue, as measured by flow cytometry. The DNA indices of the solid tumor biopsies agreed with the degree of aneuploidy observed by cyto Cytogenetic analysis was performed on 28 newly diagnosed, fresh, genetic analysis in every case. Second, in 6 cases we performed direct primary NSCLC tumors and 2 pleural effusions. Findings in 21 of comparisons of karyotypes obtained from cells cultured by both methods. these patients have been described in detail in an earlier report (4). Identical chromosome abnormalities were detected in short term cultures Solid specimens were disaggregated mechanically. Hard specimens and later harvests of the same specimen. Overall, our findings indicate were also dissociated further by shaking the minced tumor pieces for that tumorigenesis in NSCLC is characterized by the accumulation of 3-5 h in medium to which collagenase A was added, to a final concen multiple chromosome alterations. Furthermore, these data demonstrate tration of 0.5 mg/ml. Following disaggregation, the cells were washed that recurrent cytogenetic changes can be identified in NSCLC and that twice in Hanks1 balanced salt solution, and then single cells and cell detailed karyotypes from long term cultures are relevant to the original aggregates were suspended in complete medium and placed in tissue tumor. Chromosome abnormalities detected by these techniques may culture flasks, as described in detail earlier (4). Fresh specimens were have clinical and biological significance. However, the complex pattern usually harvested for Cytogenetics after 1, 2, and 3 days, in order to of karyotypic changes seen in newly diagnosed NSCLC emphasizes the minimize any selection in vitro. In some cases, later harvests were done need for future investigations of premalignant bronchial lesions in order because of low yields of metaphase cells. For 24 of these cases, the data to identify primary genetic changes important for early detection and presented were obtained from cells cultured for 9 days or less; of these intervention in this aggressive neoplasm. 24, results were also obtained on six long term cultures (up to 80 days). In the other six cases, cell lines that were established in culture were used for harvests at passage 3-20. Long term cultures were established Introduction using 3T3 feeder cell layers and conditioned medium from a lung Cytogenetic analysis of lung tumors may reveal specific chro carcinoma cell line, as described (6). Metaphase cells were arrested by mosome alterations that have clinical significance in prognosis exposing cells overnight to colcemid (0.01 Mg/ml). Cells were detached from the surface of flasks using trypsin and a cell scraper, treated with and therapy. These clonal abnormalities may be indicative of 0.075 M KC1 hypotonie solution for 30 min at 37Â°C,and then fixed in critical molecular events in the etiology of lung carcinomas. NSCLC3 represent 75-80% of all lung tumors. Despite the a 3:1 mixture of methanol:acetic acid. Chromosomes were analyzed using G-banding or, occasionally, Q- high incidence of NSCLC, the cytogenetic data available are banding techniques. For each specimen, we attempted to obtain chro extremely limited in this neoplasm, compared with those for mosome counts on at jeast 20 metaphase cells to determine the modal the less frequent hematological malignancies (1). Primary chromosome number. Whenever possible, at least five karyotypes were NSCLC specimens often have a low mitotic index, making it cut out for each case. 1Presented at the the NCI Workshop "Investigational Strategies for Detection and Intervention in Early Lung Cancer," April 21-24, 1991, Annapolis. MD. Results This work was supported by NIH Grants CA-45745. CA-06927, and CA-50694, by a grant from the United States Environmental Protection Agency (Co-operative All 30 tumors exhibited complex karyotypes with many struc Agreement CR-816188), and by an appropriation from the Commonwealth of Pennsylvania. Most of the data reported herein were obtained while J. R. T. was tural and numerical changes. While clonal abnormalities were a Scholar of the Leukemia Society of America. J. M. S. is the recipient of an present in all of these cases, every specimen showed consider American Cancer Society Junior Faculty Award. 2To whom requests for reprints should be addressed, at Section of Molecular able karyotypic heterogeneity. Aneuploidy was observed in all Cytogenetics, Fox Chase Cancer Center, 7701 Burholme Avenue. Philadelphia, samples, with modal chromosome numbers clustering in the PA 19111. near-triploid to near-tetraploid range (19 specimens). Four 3The abbreviations used are: NSCLC, non-small cell lung cancer(s); dmin, double-minute chromosomes; MYCI, avian myelocytomatosis viral (\-myc) on cases had hypodiploid modal numbers, and five others were cogene homologue; EGFR, epidermal growth factor receptor. hyperdiploid, with 47-57 chromosomes. Two tumors had near- 2702s

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1992 American Association for Cancer Research. CHROMOSOMES IN NON-SMALL CELL LUNG CANCER pentaploid or near-hexaploid modal numbers. All chromosomes contributed to numerical changes. The most frequent numerical alteration was polysomy 7, which was observed in 14 of 30 specimens (Table 1). In another six specimens there was partial trisomy of 7, with the shortest region of overlap of additional bands consisting of 7p 11-pi3. i Six others had both polysomy 7 and +7p. Overall, gain of part or all of chromosome 7 was observed in 26 tumors; in 19 of these cases the number of 7/7p exceeded the number of copies n expected based on the ploidy of the tumor. Polysomies of other t chromosomes (e.g., 2, 11, and 20) were also prevalent, but their l 2 3 incidence did not appear to be remarkable when the ploidy of : individual tumors was taken into account. Ss::â„¢ Among the structural rearrangements identified, some chro mosomes tended to be involved repeatedly. Included in this group are chromosomes 1, 3, 6, 7, 8, 11, 13, 15, 17, and 19. Breakpoints in clonal rearrangements appeared to be distrib g 10 n l? 13 uted in a nonrandom manner (Fig. 1). The most frequently affected bands were Ipl3, 3pl3, 8pll-qll, 15pll-qll, and 17pll, each of which participated in rearrangements in 8-14 r different specimens. There were no obvious differences among histolÃ³gica! subtypes with regard to the particular kinds of rearrangements observed, except that isochromosomes for 8q Fig. 1. Idiogram depicting breakpoints (â€¢)of all clonal chromosome re (a clonal change in 6 cases) and 13q (two cases) and der(9ql5q) arrangements seen in 30 patients with non-small cell lung cancer. Whenever a (two cases) were seen only in adenocarcinomas. An isochro- single case contained two or more rearrangements with breaks at identical bands, mosome of 5p was identified in four tumors (two adenocarci only one breakpoint is depicted. nomas and two squamous cell tumors). Balanced translocations seemed to be relatively rare in cytometry, a comparable wide range of chromosome numbers NSCLC. In contrast, chromosomal losses due to missing chro was found in culture. mosomes or apparently unbalanced rearrangements (deletions Results from short and long term cultures were also com and derivative chromosomes) were often observed (Table 2). In pared in six cases, in order to determine how stable lung tumor this series, there were recurrent losses of Ip, 3p, 6q, 9p, lip, karyotypes are in culture. The number of analyzable mitoses 15p, and 17p. Each of these losses was identified in at least and the quality of chromosome banding typically were much 50% of all cases, with loss of 9p being the most frequent (25 of higher in long term cultures than in short term harvests (Table 30 cases). In some of these cases there was a 9p- or another 4). In addition, cytogenetic abnormalities were found to be unbalanced rearrangement affecting 9p, and in others the num virtually identical in cultures analyzed at different times. How ber of copies of chromosome 9 was less than that expected ever, occasionally there was duplication of all chromosomes in based on the ploidy of the specimen. some cells from long term cultures. This may mimic a "poly- Tumor cells from five patients contained variable numbers of ploidization" process which occurs in vivo to produce multiple, dmin. A sufficient amount of tumor specimen was available for highly aneuploid populations. molecular analysis in four cases, and evidence for gene ampli fication was found in two of these; one case had amplified Discussion MYC1, and the other had amplification of the EGFR gene. To validate the culture system, the modal chromosome num Our findings demonstrate that karyotypes in NSCLC gener ber of 13 cases was compared to the DNA index of the original ally are very complicated, even in newly diagnosed primary solid tumor, as measured by flow cytometry. These were cases tumors. We have focused our investigations on samples ob for which tissue was available for flow cytometry and a success tained prior to initiating cytotoxic therapy; thus, the cytogenetic ful chromosome harvest was also obtained. Agreement between complexity seen in these cases appears to be part of the natural the predicted ploidy of the aneuploid or near-diploid peaks course of NSCLC. Furthermore, it is unlikely that this overall observed in the solid tumors and the modal numbers of cultures complicated pattern can be attributed to in vitro karyotypic was found in all 13 cases (Table 3). Agreement was observed evolution, because most of the analyses presented here are from even in cases where the tumor population in the solid tissue relatively short term cultures of tumor cells. In most cases it was a minority of the cells in the flow cytometry analysis. In was possible to obtain at least a few highly abnormal karyotypes one case where two aneuploid peaks were detected by flow from cells cultured for 9 days or less, and in approximately one half of all cases these data were from cells cultured for only 1- 3 days. The agreement we found between modal chromosome Table 1 Gain of 7 or 7p in 30 NSCLC specimens number and DNA index further demonstrates that cells which Gains No. of cases proliferate in culture usually come from the major aneuploid Polysomy 7 14 or near-diploid component of the solid tumor. This suggests Gain of 7p 6 that our culture techniques allow us to draw valid conclusions +7 and +7p 6 35Â° about the chromosome abnormalities found in solid tumors. Â°In 19 cases the number of copies of 7 or 7p exceeded the number expected Some lung tumor specimens were cultured on 3T3 feeder based on the ploidy of the tumor. layers, from which we were able to obtain long term cultures. 2703s

Table 2 Recurrent losses in 30 NSCLC cases and increased EGFR levels in NSCLC is unclear at this time. Losses"lp ofcases16 While recent advances in cell culture and cytogenetics have improved the success rate and quality of karyologic investiga 3p 20 tions in NSCLC, currently it is not possible to examine kary 6q9P IS 25 otypes of the malignant cells in every specimen. In some cases, lip 17 the mitotic rate of the tumor cells is very low and analyzable 15p17pNo. 21 21 abnormal metaphase spreads are not found. However, it is now " Losses due to missing chromosomes or apparently unbalanced re possible to use nonisotopic DNA probes to examine interphase arrangements. cells for chromosomal alterations. Nonisotopic in situ hybridi zation techniques provide a rapid and precise method for label Table 3 Expected and experimentally determined modal chromosome numbers of ing specific targets on metaphase chromosomes and interphase primary lung tumors nuclei. For example, in situ hybridization with fluoresceinated modal number of number of repetitive a-satellite DNA probes to the centromeres of specific Caseno.12345678910111213Expectedtumor (approximate)">466973.6, tumorculture51-5364-6961-924562-7482-858467-7168-7648-5562-68136-14662-68chromosomes has been used successfully for the targeted karyotypic analysis of numerical abnormalities within interphase nuclei of gastric tumors (12). Using such a fluorescence-based 1424678.287.487.473.687.44678.2147.282.8Modal detection system, hybridization of an a-satellite probe to a specific interphase chromosome appears as a brightly fluores cent spot. The number of spots corresponds to the number of copies of the specific chromosome in the cell. In a preliminary experiment, we hybridized a chromosome 7-specific probe to cells from both normal tissue and a NSCLC cell line that exhibits polysomy 7. Two intensely fluorescent spots were " Based on DNA index of original solid tumor biopsy. detected in a high percentage (>90%) of normal diploid cells, whereas three or more spots were found in the NSCLC cells (Fig. 2). Besides enhancing karyological investigations of tumor In six of these cases we also harvested conventional short term specimens, such interphase cytogenetics techniques may repre sent a useful approach to assess "noninvolved" bronchial tissues cultures. In each of the latter cases the karyotypes obtained with the two methods were very similar, but the quality of and premalignant lesions for the presence of cells with early chromosome preparations was superior with the feeder layer (primary) chromosome changes. method. Thus, our preliminary studies suggest that the feeder Loss of all or part of the short arm of chromosome 3 was layer method can be a useful procedure for the cytogenetic identified in 20 (67%) cases in this series. Loss of heterozygosity analysis of NSCLC specimens. Cell lines established by this for alÃeleson 3p has been reported in 25-100% of NSCLC method will also be useful for molecular analysis. cases in different reports (13-18). The shortest region of overlap Polysomy 7 was a frequent numerical alteration, occurring of chromosome losses appears to be at 3p21. AIk-lie loss at in 20 of 30 (67%) specimens. Polysomy for all or part of 3p21 has been reported in all major types of lung cancer (14, chromosome 7 was identified in each of three lung adenocar- 19). Deletion of 3pl4-p23 was initially reported as a specific cinomas examined by Jin et al. (2) and in all four lung adeno- chromosome aberration in small cell lung cancer (20). More carcinoma cell lines reported by Fan and Li (7). Lee et al. (8) recently, deletions of 3p have been reported in a number of suggested that trisomy 7 is a very early change in NSCLC, other malignancies, including renal cell carcinoma, meso- which can be found in premalignant lung tissue in a subset of thelio ma, ovarian cancer, and breast cancer (21). Thus, loss of patients. Twelve patients in our series had gains of part of 7p 3p may represent an important generalized tumorigenic event (in six of these cases this change was accompanied by polysomy common to various solid tumors, including NSCLC. 7). The shortest region of overlap of partial gains of 7p was at As noted earlier, 9p was the most frequently lost chromosome 7pl l-pl3. Interestingly, the EGFR gene is located within this segment in this series (25 of 30 cases). In a previous report, same region on chromosome 7 (9). Increased levels of expres Lukeis et al. (3) identified deletions of 9p in 9 of 10 NSCLC sion of EGFR have been demonstrated in NSCLC (10, 11), but and suggested that this change may represent a critical event in the relationship between additional copies of chromosome 7 this neoplasm. While only a minority of our cases had a 9p-,

Table 4 Comparison of cytogenetic findings using two different culture methods in culture or passage number karyotypic of analyzable quality and Caseno.1 atharvest9/9, analysisYes/yes karyotype"Yes/yes cells(%)100/43 mitoses/slide*4.2/2.5 bandingquality++/++ 27 2 12/7 Yes/yes Yes/yesc 100/100 4.8/=l ++/+ 3 Passage 3/3 Yes/no Yes/- 100/- 6.2/- ++++/- 4 Passage 4/5 Yes/yes Yes/yes" 100/100 19/=1 +++/+ 5 80/1,2 Yes/yes Yes/yes 100/100 23/=l +++/++ 6Days 20/1, 2Successful Yes/yesAbnormal Yes/yes'Abnormal 100/100Number 4.2/=lChromosome +++/++ " Karyotypic findings were similar in both types of cultures, unless indicated. â€”¿,culturenot successful. * Six to 10 slides were examined per case. We attempted to keep the density of nuclei equal for each case. ' Modal number and markers were similar in both cultures, but no completely analyzable metaphases were seen in short term culture. A few new changes were seen only in long term culture [i.e., +1, +der(3), +some cells with dmin]; also, unlike short term culture, the cell line exhibited a few polyploid cells (2 of 19). Breakpoint localization and interpretation of rearranged chromosomes were more precise using the cell line. ' A few minor differences were seen between cultures (i.e., -15 seen only in short term culture; also -X and derivative marker were seen in several cells in short term culture but not long term culture). Polyploid cells were found in 3 of 9 cells in long term culture but in 0 of 6 cells from short term culture. 2704s

Fig. 2. Fluorescence in situ hybridization of a chromosome 7-specific, Â«-satelliteprobe to interphase nuclei and metaphase chromosomes (arrows) from normal diploid lymphocytes (A and B) and an aneuploid NSCLC cell line known from a previous karyotypic analysis to exhibit polysomy 7 (Cand D). Note thai lymphocytes have two fluorescent spots in each interphase nucleus and, correspondingly, two labeled chromosomes in the metaphase spread shown in B. The NSCLC cell line had three or four fluorescent spots in interphase nuclei (four in each nucleus shown here): three labeled metaphase chromosomes can be seen in the mclaphase spread in D. The biotin-labeled probe is fluoresceinated and appears green under the microscope; the chromosomes are counterstained with the red-fluorescing DNA-specific dye propidium iodide. others had either an apparently unbalanced rearrangement (var DNA analyses have demonstrated that loss of alÃeleson 17p ious derivatives) affecting 9p or less than the expected number often occurs in NSCLC (16). The TP53 tumor suppressor gene of copies of chromosome 9 (e.g., only two copies of chromo is located at band 17pl3.1, and 77*53 has been shown to be a some 9 in a triploid tumor). frequent target for molecular alteration in lung cancer (23-25). The pericentromeric regions of chromosomes 13-15 fre Our data suggest that visible cytogenetic changes may be the quently participate in structural rearrangements in NSCLC. cause of the allelic loss detected by molecular methods, at least The 15pl 1-ql 1 region seems to be especially prone to cytoge- in some cases. Taken together, the cytogenetic and molecular netic change. All rearrangements affecting this site appeared to evidence suggests that loss of 17p containing a normal TP53 involve unbalanced derivative chromosomes which would result alÃeleunmasks a remaining mutant gene on the other (kary- in loss of part of 15p. Thus, 15ql 1-pter could represent a site otypically normal) homologue. of a putative tumor suppressor genes or antioncogene (22). While no balanced reciprocal rearrangements have been iden Among the abnormalities of chromosome 15 seen in this series tified that are specific for a histological subtype of NSCLC, was a der(9ql5q), which was found in two adenocarcinomas i(8q) was found only in patients with adenocarcinomas (a clonal and may represent a recurrent rearrangement in NSCLC. An change in six cases). Even though this isochromosome appears apparently identical 9; 15 rearrangement was identified in a lung to be a recurrent finding in adenocarcinoma of the lung, i(8q) adenocarcinoma cell line reported by Fan and Li (7), and a is not specific for this tumor. This abnormality has been re der(15)t(15;?)(p?;?) was identified in a lung adenocarcinoma ported in various types of leukemia and in several different examined by Jin et al. (2). solid tumors (1). The most consistent breakpoint in this series was at band Variable numbers of dmin were seen in 5 of 30 (17%) speci 17pl 1 (Fig. 1). Overall, rearrangements of 17p were seen in 21 mens. It is now well known that these chromatin bodies are (70%) cases. These structural changes include partial deletions associated with amplification of oncogenes and genes involved and different derivative chromosomes involving 17p. Previous in drug resistance (26, 27). We identified an amplified DNA 2705s

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1992 American Association for Cancer Research. CHROMOSOMES IN NON-SMALL CELL LUNG CANCER sequence in two of our cases with dmin; one case had amplified 11. Haeder, M., Rotsch, M., Bepler, G., Hennig, C., Havemann, K., Heimann, B., and Moelling, K. Epidermal growth factor receptor expression in human MYCl and the other had amplified EGFR. In two series of lung cancer cell lines. Cancer Res., 48: 1132-1136, 1988. NSCLC specimens, amplification of the MYCl oncogene was 12. van Dekken. H.. Pizzolo. J. G., Kelsen. D. P., and Melamed, M. R. Targeted cytogenetic analysis of gastric tumors by in situ hybridization with a set of documented in 3 of 36 (8%) and 4 of 25 (16%) cases (28, 29). chromosome-specific DNA probes. Cancer (Phila.), 66: 491-497, 1990. Amplification of KRAS2 has also been reported in a case of 13. Brauch, H., Johnson, B., Hovis, J., Yano, T., Gazdar, A.. Pettengill, O. S., NSCLC which contained dmin (30). Graziano, S.. Sorenson, G. D., Poiesz, B. J., Minna, J., Linehan, M., and Zbar, B. Molecular analysis of the short arm of chromosome 3 in small-cell Overall, our cytogenetic findings indicate that chromosome and non-small-cell carcinoma of the lung. N. Engl. J. Med., 317:1109-1113, alterations in NSCLC are often extremely complex, even in 1987. newly diagnosed primary tumors, but recurrent karyotypic 14. Kok, K., Osinga, J., CarriÂ«. B., Davis, M. B., van der Hout, A. H., van der Veen, A. Y., Landsvater, R. M., de Leij, L. F. M. H., Berendsen, H. H., changes can be found and these changes may have clinical and Postmus, P. E., Poppema, S.. and Buys, C. H. C. M. Deletion of a DNA biological significance. The abundance of cytogenetic altera sequence at the chromosomal region 3p21 in all types of lung cancer. Nature (Lond.), 550:578-581, 1987. tions identified in these tumors indicates that numerous genetic 15. Yokota, J., Wada, M., Shimosato, Y., Terada, M., and Sugimura, T. Loss of events can occur in NSCLC tumorigenesis. The recurrent loss heterozygosity on chromosomes 3, 13, and 17 in small-cell carcinoma and of several specific chromosomal segments, including 3p, 9p, on chromosome 3 in adenocarcinoma of the lung. Proc. Nati. Acad. Sci. USA, 84: 9252-9256, 1987. and 17p, provides additional evidence for a critical role of 16. W'eston, A., Willey, J. C., Modali, R., Sugimura, H., McDowell, E. M., tumor suppressor genes at these sites in NSCLC. Other chro Resau, J., Light, B., Haugen, A., Mann, D. L., Trump, B. F., and Harris, C. C. Differential DNA sequence deletions from chromosomes 3, 11, 13, and mosomal imbalances, especially of chromosome 7, appear to 17 in squamous-cell carcinoma, large-cell carcinoma, and adenocarcinoma have an important but as yet undetermined role in this neo of the human lung. Proc. Nati. Acad. Sci. USA, 86: 5099-5103, 1989. plasm. The complex pattern of cytogenetic changes seen in 17. 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2706s

Joseph R. Testa and Jill M. Siegfried

Cancer Res 1992;52:2702s-2706s.

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