Oncogene amplifications in non small cell lung cancers with supernumerary
ring chromosomes: characterization by tiling resolution bacterial artificial
chromosome microarrays
Maria Soller, MD, PhD1, Anna Karlsson, MSc2, Anna Collin, PhD1, Göran Jönsson,
PhD2, Maria Planck, MD, PhD2*
Departments of Clinical Genetics 1 and Oncology 2 at Lund University Hospital,
Sweden.
* Correspondence to: Maria Planck, Lund University, Clinical Sciences, Lund,
Department of Oncology, Barngatan 2:1, SE-221 85 Lund, Sweden
Phone +46-46-177501, Fax +46-46-147327, E-mail maria.planck@med.lu.se
2
Abstract
A small fraction of lung cancers have a karyotype displaying supernumerary ring chromosomes.
We obtained gene copy number profiles by array based comparative genomic hybridization,
using whole-genome tiling resolution bacterial artificial chromosome microarrays, for five
primary non small cell lung cancers (NSCLC) with a supernumerary ring chromosome as their
sole clonal karyotypic aberration. Regions with about 2- to 3-fold amplifications were revealed in
4/5 tumors. Amplicons within the chromosomal regions 3q26.1-3q29, 17q11.1-17q21.2, and
19q12-19q13.2 were shared between the two squamous cell carcinomas whereas the two
adenocarcinomas demonstrated a single high-level amplification on 14q13.2-14q21.2 and
amplifications on 7p22.3-7p11.2 and 21q22.13-21q22.3, respectively. Several known oncogenes
were identified as presumed targets of amplification, e.g. PIK3CA, ERBB2, EGFR and CCNE1.
The rings and the candidate gene-containing amplifications may simply reflect independent,
coexisting, alterations detected by different techniques. Alternatively, supernumerary ring
chromosomes in NSCLC harbor amplified oncogenes and thus have a similar role in
tumorigenesis as described for some mesenchymal tumors.
Keywords: Lung cancer, ring chromosome, array-based CGH, FISH, chromosomal imbalances,
gene amplification
3
Introduction
Lung cancer, with 1.35 million cases diagnosed worldwide each year, is a common and
increasing type of cancer, with the majority of cases attributable to cigarette smoking. The
disease is the leading cause of cancer death in the world and, despite diagnostic and therapeutic
improvements during the past two decades, the overall 5-year survival rate is still below 15% (1).
Lung carcinomas are histopathologically divided into two major categories; small cell lungcancer
(SCLC) and non small cell lung cancer (NSCLC). All major histological types of invasive lung
carcinoma display multiple genetic alterations, generally believed to have accumulated during a
multistep carcinogenesis. The tumors frequently exhibit multiple karyotypic changes with both
numerical and structural aberrations that may involve any chromosome (2). In NSCLC,
frequently reported karyotypic changes include losses on chromosomal arms 3p, 6q, 8p, 9p, 9q,
13q, 17p, 18q, 19p, 21q, and 22q. The chromosomal arms most frequently involved in gains
revealed by karyotyping are 1q, 3q, 5p, 7p, 7q, 11q and 12q (3). Studies of recurrent alterations
by comparative genomic hybridization (CGH) have made it possible to identify cancer-related
genes through observations of lost or gained chromosome regions (4). CGH analysis has
confirmed the multiple genomic imbalances in NSCLC, including gains of chromosome arms 1q,
3q, 5p and 8q, and loss of 3p, 8p, 9p, 13q, and 17p (3). Some imbalances seem to occur at
different frequencies in the two major subtypes of NSCLC, squamous cell carcinoma (SCC) and
adenocarcinoma (AC). Indeed, gain of 3q24-3q26 is twice as common in SCCs as in ACs, with
PIK3CA, one of the most highly mutated oncogenes identified in human cancers, located at 3q26
as a presumed target of amplification (3, 5). On the other hand, gains in 1q22-32 are more
frequent in ACs (3).
A small fraction of lung cancers demonstrate a karyotype displaying supernumerary ring
chromosomes. These are unstable chromosomal structures that are generally rare in epithelial
4
tumors but frequently observed in mesenchymal tumors (2, 6). Ring chromosomes are typically
formed through breakage of both chromosomal arms and fusion of the proximal ends into a ringshaped structure that thus may contain amplified chromosome material. The size and number of
rings may vary greatly between cells within a tumor, perhaps due to frequent recombination
based on breakage-fusion-bridge cycles (7). The flexible/unstable structure of ring chromosomes
may constitute a useful mechanism for rapid change in oncogene copy number during tumor
evolution (6). In mesenchymal tumors, ring chromosomes do typically contain amplified material
from chromosome arm 12q that include known oncogenes such as MDM2, SAS and CDK4. The
pathogenic effects of ring chromosomes in lung cancer, as in any epithelial tumor type, are,
however, largely unknown. Cytogenetic analyses of a consecutive series of primary NSCLCs
collected at our hospital revealed ring chromosomes as sole abnormality in 4/114 ACs (8), 1/26
large cell carcinomas (9) and in 2/111 SCCs (10). The origin of the supernumerary ring
chromosomes could not be disclosed by conventional cytogenetic methods. In the present study,
five ring-containing primary NSCLC were selected for characterization by array based CGH
(aCGH), using whole-genome tiling resolution bacterial artificial chromosome (BAC)
microarrays. This recently developed method allows for characterization of DNA copy number
changes at a resolution only limited by the number of BAC clones used for the arrays (11, 12).
We used arrays encompassing 32 433 overlapping BAC clones covering the whole genome, i.e.
the tumor DNA could be analyzed with an average resolution of 70 kbp.
Results
We obtained gene copy number profiles by aCGH for five primary NSCLCs with a
supernumerary ring chromosome as their sole karyotypic aberration (Figure 1). The aCGH
demonstrated a high frequency of DNA copy number changes (range 7-90 regions per tumor),
5
most of which were at modest levels (about 1.5-fold) and with gains dominating over losses. A
summary of all imbalances (log2 ratio > ± 0.2) is available as supplementary material. On
14q13.2-14q21.2 in tumor no. 5 (AC), on 3q26.1-3q29, 22q12.2 and 22q13.33 in tumor no. 2
(SCC), and on 17q11.1-17q21.2 and 19q12-19q13.2 in tumor no. 1 (SCC), we found high level
amplifications (log2 ratio >1.5, i.e. at least 3-fold amplification), some of which contained known
cancer-related genes (Figure 2, Table 2). Furthermore, the amplifications of the regions 3q263q29, 17q11-17q21, and 19q12-19q13 were seen in both SCC, although at different levels (see
Table 2). Tumor no. 3 (AC) harbored distinct gains in the regions 7p22.3-7p11.2 and 21q22.1321q22.3, at amplification levels that were approximately 2-fold (Table 2). Few (a total of 7), and
modestly altered, regions of gains/losses were detected by aCGH in tumor no. 4 (AC).
Interphase FISH analysis was done using probes targeting the amplified genomic sequences
mentioned above, including known oncogenes when possible. The results of the FISH analysis
for the amplifications seen on the aCGH are shown in Table 2 and exemplified by Figure 3. The
analysis confirmed the presence of these alterations, although the number of gene copies in the
amplified region 14q13.2-14q21.2 (tumor no. 5) could not be examined, probably due to poor
quality of the BAC clone selected as FISH probe.
Discussion
Ring chromosomes have been observed in various types of cancer but the knowledge on the
tumor biology behind these changes is limited. Although common in a subgroup of mesenchymal
tumors, ring-shaped chromosomal structures are rarely observed in epithelial tumors (2, 6). Rings
were detected in only 7 of 251 primary NSCLCs that were cytogenetically analyzed at our
hospital. The origin of the supernumerary ring chromosomes and their role in initiation and
6
development of these tumors is unknown (8-10). All cases in which the ring constituted the sole
karyotypic alteration (2 SCCs and 3 ACs) were selected for aCGH analysis.
The DNA copy number profiles generated by using the tiling resolution BAC arrays revealed
regions with about 2- to 3-fold amplifications in 4/5 tumors. The loci for these imbalances, as
well as known cancer related genes within the amplicons, are summarized in Table 2. Amplicons
within the chromosomal regions 3q26.1-3q29 (Figure 2), 17q11.1-17q21.2, and 19q12-19q13.2
were shared between the two SCCs, a finding consistent with observations from studies on
sarcomas harboring ring chromosomes, with each ring often containing material from multiple
chromosomes (14). Similarly, tumor 3 (AC) contained amplifications of the regions 7p22.37p11.2 and 21q22.13-21q22.3. In contrast, in tumor 5 (AC), we demonstrated a single high-level
amplification on 14q13.2-14q21.2 (Figure 2). A relatively low proportion of ring-containing cells
in the tumors was observed by previous karyotyping (8, 10) and the remaining AC, tumor no. 4,
may thus harbor too few amplicon-containing cells to show a sufficient copy number change in
the aCGH analysis. Recurrent 14q13-14q21 amplification, or any other copy number change that
could have explained the existence of the rings, can still exist undetected in this sample. The
great similarities between the aCGH-patterns regarding presumed oncogenes in the two SCC with
supernumerary rings as solitary cytogenetic abnormality may indicate that the amplifications
reflect the presence of the ring chromosomes.
Probes targeting the regions that were amplified according to the aCGH analysis (log2 ratio >1.5)
were used for subsequent FISH analysis that confirmed the amplifications at 3q11, 3q26, 3q29,
7p11, 17q12, 17q25, 19q12, 19p13, 21q22, and 22q12 (Table 2, Figure 3). The number and
configuration of the corresponding extra gene copies demonstrated by interphase FISH in this
study were well in line with the interphase structure of ring amplifications previously
demonstrated in sarcomas (15).
7
In addition to the amplifications described above, the five tumors also displayed frequent lowlevel copy number alterations involving all chromosomes, despite having almost normal
karyotypes according to previous cytogenetic analysis. All copy number changes are summarized
in table and in frequency plot, available as supplementary material. Although of unknown
importance, these imbalances illustrates the high resolution of the whole-genome tiling resolution
32k BAC arrays used, which, compared to conventional methods, enabled us to detect more and
smaller changes (11, 12, 14). As expected from the karyotypes, gains dominated over losses.
In order to ensure the specificity of the high-level amplifications, we also used FISH probes
targeting regions that were not gained as revealed by aCGH (log2 ratio < 0,2), i.e. presumed to be
representative of the general DNA content of the tumors. By using these probes, we observed a
polyploidy in all tumors, corresponding to approximately 4-5 copies per gene and thus at a level
not comparable to the high number of copies demonstrated with the FISH probes specific for the
amplified regions.
Ring chromosomes are frequently observed in bone and soft tissue tumors as well as in leukemias
and lymphomas (2, 6, 14). The role of these chromosomal structures in other tumor types is
largely unknown. There are, however, a few reported cases of acquired supernumerary ring
chromosomes in cancers of the esophagus, ovary, urinary bladder, lung, brain, and kidney (10,
16-18). A number of difficulties must be addressed when studying the formation, structure and
implications of somatically acquired ring chromosomes in tumors; There is an intratumoral
variation of number and size of the rings and the karyotypic banding pattern is typically diffuse
and the chromosomal origin hard to establish. Moreover, the highly unstable ring structures may
be completely changed during tumor development, probably allowing changes up and down in
copy numbers each cell division (6). The rings in our study may represent unimportant changes
8
that simply coexist with the more obviously cancer-related amplifications observed by aCGH.
Indeed, the rings may represent clones that simply had a growth advantage during cell culturing,
a process that do not always allow the relevant abnormal tumor cells to grow. Given the low
proportion of ring-containing clones in the tumor samples, the aCGH does not necessarily reflect
the ring-containing karyotype but rather DNA copy number changes that were not possible to
pin-point by cytogenetics or conventional CGH. Furthermore, the presumed importance of the
oncogene-containing amplifications observed by aCGH for the development of the tumors in our
study can also be argued against. Indeed, previous studies have demonstrated that far from all
gene amplifications are overexpressed according to their gene expression profiles (14, 19). Thus,
the exact role of ring chromosomes in the development of the lung cancers in our study remains
to be elucidated. However, we believe that the specific amplicons, as revealed by aCGH, may
constitute the origin of the rings. This hypothesis is strengthened by the interphase FISH
analyses, which confirm the presence of the amplifications and display a pattern very similar to
interphase FISH on ring-containing sarcomas (15), and the co-existence of similar amplifications
in ring-containing tumors of the same histological type. The rings in the present study may thus
harbor cancer-related genes in a mode consistent with the model suggested for MDM2
amplifications in ring chromosomes in malignant fibrous histiocytomas (6). Of the genes located
within the amplified regions, Table 2 aims to summarize those with previously reported
oncogenic function, involved in e.g. cell motility, transcription, or signal transduction. For
example, the well characterized oncogene PIK3CA has been implicated in SCC tumor
progression and the region 3q11.2-3q29 was accordingly amplified in both our SCC tumors (20).
Furthermore, the amplified region on 17q11.1-17q21.2 harbors the oncogene ERBB2. Somatic
mutation of ERBB2 or partial gain of chromosome 17 has been associated with a poor prognosis
of NSCLC (21). The amplicon encompassing 7p22.3-7p11.2 contains, among other presumed
9
oncogenes, the EGFR gene, with prognostic and therapeutic implications in NSCLC. In a
previous study that used whole genome tiling BAC microarray for characterization of NSCLC
cell lines, the regions 7p22.1-7p22.3 and 7p15.3-7p11.2 were amplified in more than 80% of the
cases (22). We detected a high level amplification at 14q13.2-14q21.2 and part of this region
(14q13.2-14q13.3) was highly amplified also in 6/28 NSCLC cell lines in the study by Garnis et
al. (22). This region harbors for example the gene PAX9 which is frequently expressed in cancer
and required for the growth and survival of cancer cells (23). In a study using single nucleotide
polymorphism (SNP) arrays for determination of copy number alterations, this same 14q13.3
amplification was described as the most common (6%) focal event in lung adenocarcinomas, and
the transcription factor NKX2-1 (essential for pneumocyte formation) was identified as the target
gene and thus suggested as an important protooncogene in lung adenocarcinoma (Ref Weir et al.
Nature 2007). Since the karyotypes have not been investigated in these studies, the occurrence of
ring chromosomes as the mechanism of 14q amplification in these tumors is unknown (22 +
Weir).
In summary, we identified several known oncogenes as targets of amplifications in primary
NSCLC with supernumerary ring chromosomes. We observed these same amplifications as an
interphase FISH pattern that is consistent with the behavior of interphase ring structures
previously demonstrated in sarcomas. It is not finally proved whether ring chromosomes in
NSCLC contribute to oncogenic change or constitute innocent cytogenetic alterations that exist in
parallel with more significant amplifications of known oncogenes. Future aCGH studies on
tumors available for metaphase FISH could hopefully give direct evidence for the presence of the
aCGH amplifications within the rings.
10
Materials and methods
Cytogenetics
Fresh tumor samples were received directly after surgery between years 1987-1993 and an
adjacent tissue block of the tumor was used for histological classification. The tumor samples
were then cell cultured and analyzed cytogenetically. The methods used for cell culturing,
harvesting, preparation, and G-banding have previously been described in detail (8, 10). In brief,
the tumors were cultured 3-8 days and thereafter harvested and G-banded with Wright’s stain.
The tumors selected for this study all contained an extra ring chromosome as the single clonal
aberration in all cases (Figure 1, Table 1). The chromosomes of origin could not be reliably
identified by the G-bands in any of the cases. The description of the karyotypes of the five ringcontaining tumors in this study have previously been published (8-10) and followed the
guidelines provided by ISCN (1991).
Array CGH
DNA for array-based CGH analysis was extracted from freshly frozen tumor biopsies using
Proteinase K (20mg/µl) digestion followed by phenol-chloroform purification according to
published protocols. Microarrays used for the present aCGH investigations were produced at the
Swegene DNA Microarray Resource Center, Department of Oncology, Lund University, Sweden
(http://swegene.onk.lu.se) using the 32k BAC Re-Array set Ver. 1.0 (BACPAC Resource Center,
Children’s Hospital, Oakland Research Institute, US). This platform, including a total of 32 433
BAC clones, provides 99 % coverage of the BAC fingerprint map (November 2001) and
comprises clones sampled from the RPCI-11, RPCI-13 (94%) and Caltech-D (6%) libraries. For
all samples, 2µg of tumor DNA and 1.5µg reference DNA (Promega Corporation, Madison,
11
USA) was labeled with Cy3-dCTP and Cy5-dCTP (Amersham Biosciences, Uppsala, Sweden),
respectively, using BioPrime Array CGH Genomic Labeling System (Invitrogen Life
Technologies, Carlsbad, US). Purification of labeled DNA was done using the Cyscribe GFX
purification kit (Amershamn Biosciences) and the pooled tumor and reference DNA were mixed
with Human COT-1 DNA (1µg/µl, Invitrogen Life Technologies) and resuspended in a
hybridization buffer containing de-ionized formamide, Yeast-tRNA (Invitrogen Life
Technologies) and dextrane sulphate. Array slides were UV cross-linked (500 mJ/cm2) and pretreated using Pronto! Universal Microarray Hybridization Kit (Corning BV, Schiphol-Rijk, the
Netherlands). Denatured and re-annealed DNA was applied to the arrays and hybridization was
performed in hybridization chambers (Corning BV) for 48-72 hours in a 37°C water bath. Array
slides were washed in post hybridization buffers with different contents of 20xSSC, 10% SDS
and de-ionized formamide and scanned in an Agilent Microarray Scanner (Agilent Technologies,
Palo Alto, CA).
Spots were identified using GenePix Pro. 4.1 (Axon Instruments) and the data was uploaded in
BioArray Software Environment (BASE) (13). Background correction of the two channels was
calculated using the median-feature and median-local background intensities of the uploaded file.
A signal to noise ratio (SNR) was set to ≥ 5 for both channels and data was normalized using a
pin-based lowess algorithm excluding chromosome X. A moving average of 250 kbp was applied
to the CGH-plotter tool and plots were created excluding the X and Y chromosomes, a noise
constant was set to 25 and gain/loss limits were set to log2 +/-0.2.
FISH
Interphase FISH analysis was done on imprint preparations made from frozen tumor tissue
(tumors no. 1-3) or on nuclei isolated from formalin fixed and paraffin-embedded material (no. 4
12
and 5). A total of 10 BAC clones, selected on the basis of previous aCGH findings, were used as
probes for FISH analysis (Table 2). BAC DNA was extracted by alkaline lysis with SDS using
standard protocols and labeled with Cy3-dUTP (Amersham Biosciences, Uppsala, Sweden) or
Fluorescein-12-dUTP (FITC) (Roche, Mannheim, Germany) using the Megaprime DNA
Labelling System (Amersham Biosciences). Fresh imprint preparations were placed in Carnoy’s
fixative for 10 min, aged over-night at 60 ºC and incubated in 2x SSC/0.05% Tween-20 (SigmaAldrich, Stockholm, Sweden) for 1 h at 60 ºC. As regards the paraffin-embedded tumors, nuclei
were isolated from a 40 µm-section cut from the paraffin block. Paraffin was removed by
treatment with xylene (3 x 10 min) followed by rehydration in an ethanol (100%, 85%) to water
series, 2 x 5 min in each step. Nuclei were isolated during incubation with a 4 mg/ml pepsin
(Serva, Heidelberg, Germany) solution for 2 h at 37 °C in a shaker incubator. The sample was
thoroughly vortexed and the suspension was filtered through a 41 µm nylon filter (Millipore,
Solna, Sweden) followed by centrifugation for 10 min at 2000 rpm. Isolated nuclei were washed
twice in 1x PBS, resuspended in 50 l, spread on SuperFrost Plus slides (Menzel-Gläser,
Brunschweig, Germany) and air-dried. The preparations were then placed in Carnoy’s fixative for
10 min followed by incubation in 2x SSC/0.05% Tween-20 for 1 h at 60 ºC. All preparations
were pretreated with 20 mg/ml pepsin for 10 min at 37°C, postfixed in 1% formaldehyde for 10
min and finally dehydrated in an ethanol series (70%, 85%, 100%). For each hybridization, a total
of 100ng labeled DNA/probe in 50% formamide/2x SSC was applied to the preparation and
sealed with a cover slip. Imprint preparations and isolated nuclei were denaturated on a hot plate
for 3 min at 74°C and 3 min at 90°C, respectively, and subsequently incubated over-night in a
humid chamber at 37°C. Post-hybridization washes were done in 0.4x SSC/0.05% Tween-20 and
0.2x SSC/0.05% Tween-20 for imprint preparations and isolated nuclei, respectively, followed by
13
dehydration in an ethanol series. For detection of the nuclei and evaluation of the probes, the
preparations were counterstained with 0.5 mg/ml 4,6,diamidino-2-phenylindole (DAPI, Roche)
and mounted in a 2% 1,4-diazabicyclo-[2,2,2]-octane (DABCO, Sigma) solution. Conventional
epifluorescence microscopy was carried out with a Zeiss Axioplan 2 imaging microscope (Zeiss,
Jena, Germany) equipped with an 100 W HBO 103 mercury vapor lamp (Osram, München,
Germany) and a CoolSNAPES camera (Photometrics, Tucson, US) linked to a CytoVision Ultra
system (Applied Imaging, San José, US).
Acknowledgements
We thank Dr. Mike Dictor at the Department of Pathology in Lund, for kindly providing the
DNA extraction protocol used for FISH analysis of the paraffin-embedded tumors, and Drs Leif
Johansson and Jens Enoksson at the Department of Pathology in Lund, for histopathological
review. This work is supported by the Knut and Alice Wallenberg Foundation via the SWEGENE
program. The support from Pieter de Jong and Kazutoyo Osoegawa, BACPAC Resource Center
at CHORI, is acknowledged. Financial support was granted from the Royal Physiographic
Society of Lund, the Berta Kamprad Foundation, the Anna Lisa and Sven-Eric Lundgren
Foundation for Medical Research, the Region Skåne Research Foundation, and the Medical
Faculty ALF-committee of Lund University.
14
References
1. Parkin DM, Bray F, Ferlay J,Pisani P. Global cancer statistics, 2002. CA Cancer J Clin 2005;55:74-108.
2. Mitelman F, Johansson B,Mertens F. Mitelman database of chromosome aberrations in cancer.
http://cgap.nci.nih.gov/Chromosomes/Mitelman 2006;
3. Balsara BR,Testa JR. Chromosomal imbalances in human lung cancer. Oncogene 2002;21:6877-83.
4. Kallioniemi A, Kallioniemi OP, Sudar D, Rutovitz D, Gray JW, Waldman F, et al. Comparative genomic
hybridization for molecular cytogenetic analysis of solid tumors. Science 1992;258:818-21.
5. Samuels Y,Velculescu VE. Oncogenic mutations of PIK3CA in human cancers. Cell Cycle 2004;3:1221-4.
6. Gisselsson D, Höglund M, Mertens F, Johansson B, Dal Cin P, Van den Berghe H, et al. The structure and
dynamics of ring chromosomes in human neoplastic and non-neoplastic cells. Hum Genet 1999;104:315-25.
7. Gisselsson D, Höglund M, Mertens F, Mitelman F,Mandahl N. Chromosomal organization of amplified
chromosome 12 sequences in mesenchymal tumors detected by fluorescence in situ hybridization. Genes
Chromosomes Cancer 1998;23:203-12.
8. Johansson M, Berker Karaüzum S, Dietrich C, Mandahl N, Hambraeus G, Johansson L, et al. Karyotypic
abnormalities in adenocarcinomas of the lung. Int J Oncol 1994;17-26.
9. Johansson M, Dietrich C, Mandahl N, Hambraeus G, Johansson L, Clausen PP, et al. Karyotypic characterization
of bronchial large cell carcinomas. Int J Cancer 1994;57:463-7.
10 Johansson M, Jin Y, Mandahl N, Hambraeus G, Johansson L, Mitelman F, et al. Cytogenetic analysis of shortterm cultured squamous cell carcinomas of the lung. Cancer Genet Cytogenet 1995;81:46-55.
11. Ishkanian AS, Malloff CA, Watson SK, DeLeeuw RJ, Chi B, Coe BP, et al. A tiling resolution DNA microarray
with complete coverage of the human genome. Nat Genet 2004;36:299-303.
12. Jönsson G, Bendahl PO, Sandberg T, Kurbasic A, Staaf J, Sunde L, et al. Mapping of a novel ocular and
cutaneous malignant melanoma susceptibility locus to chromosome 9q21.32. J Natl Cancer Inst 2005;97:1377-82.
13. Saal LH, Troein C, Vallon-Christersson J, Gruvberger S, Borg A,Peterson C. BioArray Software Environment
(BASE): a platform for comprehensive management and analysis of microarray data. Genome Biol
2002;3(8):software0003.1-0003.6.
14. Heidenblad M, Hallor KH, Staaf J, Jönsson G, Borg Å, Höglund M, et al. Genomic profiling of bone and soft
tissue tumors with supernumerary ring chromosomes using tiling resolution bacterial artificial chromosome
microarrays. Oncogene 2006;29:
15. Gisselsson D, Pålsson E, Höglund M, Domanski H, Mertens F, Pandis N, et al. Differentially amplified
chromosome 12 sequences in low- and high-grade osteosarcoma. Genes Chromosomes Cancer 2002;33:133-40.
16. Alimov A, Sundelin B, Bergerheim U, Pavlenko M, Pisa P, Zetterberg A, et al. Molecular cytogenetic
characterization shows higher genetic homogeneity in conventional renal cell carcinoma compared to other kidney
cancers. Int J Oncol 2004;25:955-60.
17. Fadl-Elmula I, Kytola S, Pan Y, Lui WO, Derienzo G, Forsberg L, et al. Characterization of chromosomal
abnormalities in uroepithelial carcinomas by G-banding, spectral karyotyping and FISH analysis. Int J Cancer
2001;92:824-31.
15
18. Lopez-Gines C, Cerda-Nicolas M, Gil-Benso R, Pellin A, Lopez-Guerrero JA, Benito R, et al. Primary
glioblastoma with EGFR amplification and a ring chromosome 7 in a young patient. Clin Neuropathol 2006;25:1939.
19. Platzer P, Upender MB, Wilson K, Willis J, Lutterbaugh J, Nosrati A, et al. Silence of chromosomal
amplifications in colon cancer. Cancer Res 2002;62:1134-8.
20. Massion PP, Kuo WL, Stokoe D, Olshen AB, Treseler PA, Chin K, et al. Genomic copy number analysis of nonsmall cell lung cancer using array comparative genomic hybridization: implications of the phosphatidylinositol 3kinase pathway. Cancer Res 2002;62:3636-40.
21. Nakamura H, Kawasaki N, Taguchi M,Kabasawa K. Association of HER-2 overexpression with prognosis in
nonsmall cell lung carcinoma: a metaanalysis. Cancer 2005;103:1865-73.
22. Garnis C, Lockwood WW, Vucic E, Ge Y, Girard L, Minna JD, et al. High resolution analysis of non-small cell
lung cancer cell lines by whole genome tiling path array CGH. Int J Cancer 2006;118:1556-64.
23. Muratovska A, Zhou C, He S, Goodyer P,Eccles MR. Paired-Box genes are frequently expressed in cancer and
often required for cancer cell survival. Oncogene 2003;22:7989-97.
16
Figure 1.
A representative karyogram of case 2. (Loss of chromosome 11 is a nonclonal aberration.)
17
Figure 2.
DNA copy number profiles based on array CGH results from cases 1-5. A genome-wide
overview, with each row representing a separate sample, is shown. Each column represents one
of the 32 433 BAC clones used on the array CGH platforms ordered according to their sequence
positions, from terminal part of chromosomal arm 1p to Yq. Tumor over reference-log2 ratios,
where red represents amplification and green represents deletion, are shown for each clone.
Enlarged views of chromosomes 3 and 14 are shown in detail for tumors no. 2 and 5,
respectively, illustrating high level amplifications at 3q26.1-3q29 and 14q13.2-14q21.2 in these
samples.
18
Figure 3.
Representative interphase FISH analyses of selected DNA copy number changes revealed by
aCGH. a) FISH confirmation of 19q12 (red) and 17q12 (green) amplifications in a tumor cell
from case 1. Normal copy number is seen in a non-malignant cell within the same sample. b)
FISH confirmation of amplifications at 3q26.1 (red) and 3q29 (green) in tumor cells (left) but not
in non-malignant cells (right) from case 2. c) FISH result from case 3, confirming amplifications
at 7p11.2 (red) and 21q22.3 (green) in tumor cell but not in surrounding normal cells.
19
20
TABLE 1. Clinicopathological and cytogenetic data on the five ring-containing tumors
Tumor Histopathology Age/sex Smoking Karyotype
TNM
T-size, lobe
1
SCC, pd
51/M
Yes
T2N1M0 4x4x2, LL
47,XY,+r[7]
2
SCC, md
71/M
No
47,XY,+r[2]
T2N0M0 7x5x4, RM
3
AC, p, ba*
76/F
No
47,XX,+r[?]
?
?, LL
4
AC,a/t
65/F
Yes
47,XX,+r[6]
T1N0M0 3x3x2, LL
5
AC,a/t
43/F
Yes
47,XX,+r[7]
T2N1M1 8x8x6, LU
Reference
Johansson et al, 1995, case 40
Johansson et al, 1995, case 19
Johansson et al, 1994a, case 48
Johansson et al, 1994a, case 17
Johansson et al, 1994a, case 18
SCC=squamous cell carcinoma
AC=adenocarcinoma
pd=poorly differentiated
md=moderately differentiated
p=papillary
ba=bronchioalveolar
a=acinar
t=tubular
*minor component
? = unknown
LL=left lower
RM=right middle
LU=left upper
TABLE 2. Summary of high level-amplifications as revealed by aCGH and FISH.
Tumor Locus
Start position Size
Log2 ratio
Examples of genes within the region
(bp)
(bp)
(range)
name
RPL29
CASR, MLCK
MCM2, RHO
TOPBP1, TF, RYK, AMOTL2, EPHB1
SAG
3q11.2-3q29
96425332
102818857 0.77-1.37
SSR3
SMC4
1
ECT2
WIG, PIK3CA
SST, BCL6
KSR
17q11.1-17q21.2 22594930
14765889 0.58-1.54 RPL23, RPL19, ERBB2
TOP2A
CCNE1
19q12-19q13.2
34914348
10211575 1.06-1.89
RASGRP4, MAP4K1, PAK4
ECT2
3q26.1-3q29
165423841
33820348 1.53-2.12 WIG, PIK3CA
SST, BCL6
KSR
RPL23, RPL19, ERBB2
TOP2A, CCR10
ETV4, NMT1
WNT3, WNT9B, SP2, SP6, HOXB1-9, HOXB13, PRAC, PRAC2, IMP-1
17q11.1-17q25.3 22594930
53294804 0.61-1.33 ITGA3, COL1A1
PPM1D, BCAS3, TBX2
2
MAP3K3, SMURF2
PRKCA
FOXJ1
SEPT9,
LYL1
19p13.13-19p13.11 12968669
6934564
0.61-1.02 NOTCH3, BRD4
EDG4, MECT1
CCNE1
19q12--19q13.2
34914348
12612519 0.43-0.86
RASGRP4, MAP4K1, PAK4
22q12.2
28095075
2000172
0.53-1.96
22q13.33
48456823
162950
1.83-2.08
MAFK
7p22.3-7p11.2
135318
55161172 0.48-1.21 ETV1
EGFR
3
ERG
21q22.13-21q22.3 37302110
9624382
0.40-0.99
PTTG1IP, TMPRSS2, TMPRSS3
locus
3q13.31
3q21.1
3q21.3
3q22.1
3q23
3q25.31
3q25.33
3q26.31
3q26.32
3q27.3
17q11.2
17q12
17q21.2
19q12
19q13.2
3q26.31
3q26.32
3q27.3
17q11.2
17q12
17q21.2
17q21.31
17q21.32
17q21.33
17q23.2
17q23.3
17q24.1
17q25.1
17q25.2
19p13.13
19p13.12
19p13.11
19q12
19q13.2
22q12.2
22q13.33
7p22.3
7p21.2
7p11.2
21q22.2
21q22.3
4
5
14q13.2-14q21.2
34982125
9565294
0.52-1.53
PAX9
FOX1A, SIP1
14q13.3
14q21.1
clone
FISH probe
gene
RP11-2F4
LMLN
RP11-192L23
RP11-62N23
ERBB2
RP11-104J24
CCNE1
RP13-616I7
RP11-192L23
RP11-62N23
ERBB2
RP11-781F24
RP11-794L5
STX10
RP11-104J24
CCNE1
RP11-598M6
RP11-708P5
EGFR
RP11-734C17
PTTG1IP
RP11-505P11
RP11-505P11
RP11-722P2
SLC25A21, MIPOL1
SLC25A21, MIPOL1
RP11-505P11
SLC25A21, MIPOL1
RP11-722P2