Isochromosome (8q) in Four Patients with Adenocarcinoma of the Lung Ikuo Miura, James Resau, Takafumi Tomiyasu, and Joseph R. Testa

ABSTRACT: We report an isochromasome, i(8q ), in combination with many othercytogenetic changes in tumor cells from four patients with lung cancer. In each case, the tumor subtype was adenocarcinoma. This isochromosame has not been identified in primary tumors from patients with other histological types of lung cancer. Among the few previously reported cytogenetic analyses of pulmonary adenocarcinomas, i(Sq) has been observed in four additional patients. Therefore, i(8q ) represents a recurring change in this specific type of lung cancer. In addition to i(8q), tumor cells from each of our four patients had different abnormalities of 17p, and two patients had alterations of 3p as well.

INTRODUCTION In a d d i t i o n to showing specific chromosome changes that are of diagnostic value, the karyotypic pattern can have important prognostic implications in leukemia. Considerably less cytogenetic information is available from solid tumors, however. Few cytogenetic analyses of primary non-small cell lung cancer (NSCLC) have been reported previously [1]. Recently, Jin et al. [2] reported two cases of primary a d e n o c a r c i n o m a of the lung associated with i(8q) and suggested that this abnormality may represent a primary change in this tumor type. We report i(8q) and numerous other chromosome alterations in four adenocarcinomas of the lung. MATERIALS A N D METHODS

Tumor specimens were primary lung carcinomas resected surgically from three patients and one pleural effusion from a primary lung carcinoma. Solid tissue specimens were disaggregated by mechanical or enzymatic methods described in detail previously [3]. Specimens were cut into small pieces with scissors, and both supernatant and s e d i m e n t were placed into several tissue culture flasks. For enzymatic disaggregation, specimens were first m i n c e d into small pieces and then dissociated by overnight incubation at 37°C in collagenase A (Boehringer Mannheim, Indianapolis, Ind.) at a concentration of 0.5 mg/ml in RPMI 1640 m e d i u m containing 10% fetal calf serum. In one patient (patient 2), we also used mouse Swiss 3T3 cell feeder layers to facilitate From the Cancer Center and Department of Pathology,University of Maryland at Baltimore,Baltimore, Maryland. Address reprint requests to: Joseph R. Testa, Ph.D., Section of Molecular Cytogenetics, Fox Chase Cancer Center, 7701 Burholme Ave., Philadelphia, PA 19111. Received August 31, 1989; accepted December 1, 1989. The present address of Drs. Ikuo Miura and Joseph R. Testa is Fox Chase Cancer Center, Philadelphia, PA 19111.

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culture of tumor cells according to the method of Siegfried [4]. The pleural effusion was first centrifuged, and then pelleted cells were resuspended in 10 ml RPMI 1640 m e d i u m , gently layered over Histopaque (Sigma, St. Louis, MO), amt centrifuged at 2,000 rpm for 10 m i n u t e s at room temperature. Cells at the interface were then washed twice before being processed by short-ternl culture methods 13I. Each specimen was cultured in RPMI 1640 m e d i u m supplemented with 17% fetal bovine serum plus antibiotics and Fungizone (Life Technologies, Gaithersburg, Md.). The effusion specimen was harvested for cytogenetic analysis after 1, 2, and 3 clays by exposing cells overnight to Colcemid (0.01 #g/ml). Solid tissue specimens were harvested after 3 14 days of culture except in patient 3 (see Results section). Cells were detached from the surface of the flasks using trypsin and a cell scraper and were then treated with 0.075 M KC1 hypotonic solution for 20 minutes, followed by fixation in a 3 : 1 mixture of methanol :acetic acid. Cells from patient 3 were exposed to ethidium b r o m i d e / 1 0 ~g/ ml) for 2 hours before harvesting to increase the yield of metaphase cells with elongated chromosomes [5, 6]. Metaphase preparations were made either by air- or flamedrying methods. Chromosomes were analyzed using Q- or G-banding techniques.

RESULTS

Case 1

Six abnormal metaphase cells from a 50-year-old w o m a n were karyotyped. The chromosome counts were near-triploid (modal chromosome number, 71). All cells examined had an i(8q), and most had two copies of this isochromosome. Double m i n u t e chromosomes (dmin) were found in four of six cells (range, 1 - 4 per cell). Clonal abnormalities noted in at least one half of all cells analyzed included the following: + 1,+ 5,+ 8, + 8,+ 10,+ 10, + 11,+ 12,+ 14, + 14,+ 15,+ 17, + 18, + 18,+ 20,+ 21, + 21 ,del(6)(q15q23),2i(8q),2del(lO)(p12), + der(2)t(2;?)(q35;?), + der(3)t(3;?)(p21 ;?), + der(3)t(3;?)(pl?3;?), + der(6)t(6;?)(p21.3;?), + 2der(7)t(7;?)(p15;?), + der(7)t(7:?) (7p13--+7q36::?), + der(7)t(?;7;?)(?::7p15-+7q36::?), + der(10)t(10;?)(q22;?), + 2der(13)t(7;13)(p12;p1?3), + derI17)t(17;?)(p11.2;?), + 3-6Inar, + O-4dmin. A representative karyotype is shown in Figure 1.

Case 2

Six abnormal metaphase cells from a 63-year-old man were karyotyped. All cells were hypotetraploid with a mode of 82-85 chromosomes per cell. There were multiple copies of some of the rearranged chromosomes, i n c l u d i n g the isochromosome (8q) (Fig. la). Clonal abnormalities included the following: + X, + Y, - 1, - 1, + 2, + 2, + 3, + 3,+ 5,+ 6,+ 6,+ 7,+ 7,+ 8 , + 8 , + 9,+ 9,+ 10,+ 11,+ 11, + 13,+ 13,+ 13,+ 1 3 , - 14, - 14, + 16, + 16, + 19, + 20, + 20, + 20, + 21, + 22,2i(8q),i(13q),del(17)(pl?2), + 2der(1) t(1;?}(1p32-->1q32::?), + 3der(1)t(?;1;?)(?::1p3?1--+1q44::?),+ 2der(8)t(8;?)(p12~?), + der(14)t(14;?)(q24;?), + 4 - 7 m a r .

Case 3

Initial active growth of tumor cells from a 54-year-old man was not evident in shortterm cultures, and successful cytogenetic analysis was possible only in long-term cultures and after establishment of a cell line. Twenty-six metaphase cells were analyzed in detail at passage 12; all of these cells were hyperdiploid, with a modal chromosome n u m b e r of 52. Some of the consistent abnormalities identified in these tumor cells i n c l u d e d the following: - Y, - 7, + 8, - 9, - 9, - 12, - 12, - 13, - 15, - 15,

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liz,J mar Figure 1 Karyotype of a G-banded metaphase cell from disaggregated solid tumor tissue cultured for 7 days (case 1). Arrows indicate i(8q) and rearrangements of 3p and 17p. Inset: i(8q) (arrows) from cases 2 (a), 3 (b), and 4 (c). In patient 2, the second chromosome 8 is also rearranged.

- 1 7 , - 1 9 , - 19,del(2)(q13),del(6)(q11.2),i(8q), + der(3)t(3;?)(p11;?), + der(5)t(5;?) (p15;?), + 2der(9)t(9;15)(p11;q1?1), + 3der(12)t(12;?)(q24.3;?), + der(17)t(17;?) (p11.2;?),+2der(19)t(19;?)(q13.3;?),+ 2mar. Chromosome pair 8 is shown in Figure lb. Detailed investigations on the established cell line derived from this tumor will be described elsewhere. Case 4

Seven abnormal metaphase cells from a 63-year-old w o m a n were karyotyped. There was no distinct mode, with chromosome numbers ranging from 67 to 84. Six other analyzed cells were cytogenetically normal. All abnormal cells had highly complex karyotypes. Most cells had two copies of i(8q) (Fig. lc) and each of several other markers. Clonal abnormalities included the following: + X, + 2, + 2, + 3, + 3, + 4, + 5, + 7,+ 7, + 8, + 8, + 9,+ 1 0 , - 1 1 , - 11, + 1 2 , - 13, + 1 4 , - 15, + 16,+ 19, + 19,+ 20,del(1)

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(p13),2i(8q),2i(14q), + 3der{?)t(?;1)(?;ql ?2), + 2der(1 )t(1 ;?)(p36;?), + der(6)t(6;17) (q15;q12), + 2der(11)t(11;11)(p15;q13), + der(?)t(?;13)(?;q12), + 2der(17)t(15:17) (q11:p11),+ 3 11mar.

DISCUSSION

The karyotypes in these four adenocarcinomas were extremely complicated, but in each case one or more copies of i(8q) was found. Recently, Jin et al. [2] reported i s o c h r o m o s o m e s i(8q) or i(9q) in three primary adenocarcinomas of the lung. An i(8q) was also reported in metastatic specimens from two other patients with lung a d e n o c a r c i n o m a [7, 8]. Jin et al. [2] suggested that these isochromosomes might constitute p r i m a r y aberrations in this type of tumor. We did not identify an isochromosome (9q) in any of our patients with NSCLC, but we did find i(8q) in four of our 13 successfully analyzed, cytogenetically abnormal cases of NSCLC. We did not find an i(8q) in any of the six cases of small cell lung cancer {SCLC) e x a m i n e d in our laboratory. Even though this i s o c h r o m o s o m e appears to be a frequent finding in adenocarcinoma of the lung, i(8q) is not specific for this tumor. This abnormality has been reported in various types of leukemias and in several types of solid tumors [1 ]. Muleris et al. [9] reported an i(8q) in three of 11 cases of h u m a n large bowel cancer. Ferti et al. [10] reported an identical isochromosome (Sq) in five of 10 cases of rectosigmoidal adenocarcinoma, and Gibas et al. [11, 12] reported i(8q) in three of 16 cases of transitional cell carcinoma of the bladder. Thus, these data suggest that i(8q) is a relatively c o m m o n abnormality in many types of malignancies. Among lung tumors studied thus far, however, i(8q) appears to be a frequent occurrence in lung adenocarcinoma but not in any other type of NSCLC or in SCLC. Besides this abnormality of chromosome 8, we also noted that two of our four patients with a d e n o c a r c i n o m a of the lung had alterations involving the short arm of c h r o m o s o m e 3. Deletion of 3p was originally reported as a specific change associated with SCLC [13], but more recently it has been reported in NSCLC and in various other solid tumors [1]. Our patient 1 had two different rearrangements involving 3p, as well as two a p p a r e n t l y normal copies of #3. Patient 3 had a derivative c h r o m o s o m e 3 with apparent loss of p11-+pter. In addition to abnormalities of #8 and #3, chromosome 17 was altered in each of the four cases reported. Three cases had different derivative c h r o m o s o m e s involving the short arm of chromosome 17. The fourth patient (case 2) had a 17p - . In each of these cases there is a rearrangement affecting 17p which could have resulted in loss of alleles w i t h i n this region. Various investigators have suggested that loss of alleles on 17p is a c o m m o n finding in lung cancer and other types of solid tumors [9, 14]. At present, it is impossible to determine if i(8q) or abnormalities of 3p and 17p represent initial events in p u l m o n a r y adenocarcinoma. Gibas et al. [11] provided evidence suggesting that i(8q) may be a secondary, noninitiating event in the karyotypic evolution of transitional cell bladder carcinoma. Clones with an i(8q) may have a proliferative growth advantage as c o m p a r e d to that of other cell populations. Because karyotypes are often extremely c o m p l e x in NSCLC, it will be necessary to study m a n y more cases, using improved culture methods, to identify recurring changes that are critical in the pathogenesis of lung cancer.

This study was supported in part by NCI Grant No. CA45745 and contract NO1-CP-51000. I. R. T. is a Scholar of the Leukemia Society of America.

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