Significant involvement of chromosome 13q deletions in progression of larynx cancer, detected by comparative genomic hybridization.
ABSTRACT Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous group of tumours with various clinical characteristics. These tumours generally exhibit complex karyotypes. Few studies of genomic imbalances have been performed exclusively in subgroups of larynx cancer samples at different stages of the disease. In the present study, chromosomal gains and losses were investigated in 52 larynx tumours, by using comparative genomic hybridization (CGH). The mean number of observed alterations was 37.7 per tumour. The most common sites of losses were 1p, 13q, Xp, and the most common gains were located in 1p, 9q, 16q. The overall number of gains was negatively associated with cancer grading. G1 tumours were also characterized by a higher frequency of deletions in 13q32 and amplifications in 1q23, than tumours in other grades (p < 0.05). The frequency of losses of 13q22 also positively associated with tumour size. There was no association between the frequency of losses in 13q and the presence of lymph node metastases at the time of diagnosis. Another statistically significant association was observed for gains at 1q22-23 and tumour size (p < 0.01). No statistically significant difference in the frequency of most common imbalances was detected between primary tumours with lymph node metastases and those without metastases. In conclusion, we discovered a significant involvement of 13q deletions in the progression of larynx cancer. All the other significant changes observed in the present study were reported previously as being important for HNSCC progression. It seems that multiple genes are disrupted in the process of neoplastic transformation in the larynx, and the networks of events remain to be elucidated.
- [show abstract] [hide abstract]
ABSTRACT: Comparative genomic hybridization was performed on 38 primary laryngeal carcinomas divided into two groups according to the metastatic phenotype. DNA copy number changes were detected in 22 of the 38 cases (57.9%). Gains were most frequently observed at 3q, 8q, and 9q, and losses were found in decreasing order at 18q, 3p, and 4. The mean value of losses was 2.5 times as high in metastasizing primary tumors (23/38) as in nonmetastasizing tumors. The most frequent losses in metastasizing tumors were at 18q, 3p, and 5q.Cancer Genetics and Cytogenetics 11/1999; 114(1):31-4. · 1.93 Impact Factor
Head and neck squamous cell carcinomas
(HNSCCs) represent 2–3% of all malignancies
diagnosed in the world and constitute about 5% of
annually diagnosed cancers in Europe, including
Poland. Larynx cancer (in 95% squamous cell
carcinoma of the larynx; SCCL) is the most
frequent tumour of the head and neck (Bergamo
et al. 2000, Jassem et al. http://www.puo.pl/
ksiazka.php). The complex aetiology of HNSCC
involves both genetic and environmental factors
viruses) (Papadimitrakopoulou 2000). Genetic
alterations in HNSCC are observed at both
the cytogenetic level (chromosomal imbalances
and structural aberrations) and the molecular level
(point mutations, microsatellite instability, loss of
Clonal chromosomal changes have been diag-
nosed with the use of classical banding techniques
and FISH in over 250 HNSCCs (Mitelman et al.
2000). Other recurrent structural aberrations in-
clude isochromosomes of 3q, 5p, 8q, and a homo-
geneously staining region (hsr) at 11q13 (Jin et al.
1993; van Dyke et al. 1994; Jin et al. 1995; Gollin
J Appl Genet 46(4), 2005, pp. 407-413
Significant involvement of chromosome 13q deletions
in progression of larynx cancer,
detected by comparative genomic hybridization
Kamilla Schlade-Bartusiak1,*, Agnieszka Stembalska1, David Ramsey2
1Department of Genetics, Wroc³aw Medical University, Poland
2Institute of Mathematics, Wroc³aw University of Technology, Poland
Abstract. Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous group of tumours with various
clinical characteristics. These tumours generally exhibit complex karyotypes. Few studies of genomic imbal-
ances have been performed exclusively in subgroups of larynx cancer samples at different stages of the disease.
genomic hybridization (CGH). The mean number of observed alterations was 37.7 per tumour. The most com-
of gains was negatively associated with cancer grading. G1 tumours were also characterized by a higher fre-
quency of deletions in 13q32 and amplifications in 1q23, than tumours in other grades (p < 0.05). The frequency
association was observed for gains at 1q22-23 and tumour size (p < 0.01). No statistically significant difference
in the frequency of most common imbalances was detected between primary tumours with lymph node
in the progression of larynx cancer. All the other significant changes observed in the present study were reported
previously as being important for HNSCC progression. It seems that multiple genes are disrupted in the process
of neoplastic transformation in the larynx, and the networks of events remain to be elucidated.
Key words: CGH, genomic imbalances, larynx cancer.
Received: July 25, 2005. Accepted: September 30, 2005.
Correspondence: A. Stembalska, Department of Genetics, Wroc³aw Medical University, Marcinkowskiego 1, 50-368
Wroc³aw, Poland, e-mail: email@example.com; *present address: Department of Medical Genetics, University of Alberta,
2001). The loss of genetic material was observed
and 18q, whereas chromosomal gains were de-
tected mostly in 1q, 3q, 8q, 15q and 11q13 (Jin
et al. 1993; Jin et al. 1995; Mertens et al. 1997).
Another technique widely used in recent years to
study chromosomal imbalances in cancer is com-
parative genomic hybridization (CGH), which en-
ables the detection of changes at a resolution of
3–10 Mb. The results of studies on head, neck
and buccal cavity tumours (premalignant lesions,
primary, metastatic and cell lines) by using this
technique were reviewed by Struski et al. (2002).
In 494 cases summarised from 21 publications,
chromosomal changes were observed most often
(> 40% of cases) in 3q, 5p, 7p, 8q, 11q, 17q
and 20q. A variety of oncogenes localised in those
regions were hypothesised to play a role in the ae-
tiology of HNSCC.
An earlier study on 20 pairs of larynx cancer
samples (tumour specimens and metastases to ad-
jacent lymph nodes) revealed a significant in-
crease in frequency of 13q deletions in metastases
(74% of samples) in comparison with primary tu-
mours (42% of samples) (Kujawski et al. 2002).
The relationship between the frequency of 13q de-
letions and the histological grade of primary tu-
mours remains unclear. The aim of the present
study was to analyse chromosomal imbalances in
a set of 52 primary squamous cell carcinoma of
the larynx (SCCL) and to compare the frequency
of observed aberrations with regard to tumour stage
(according to TNM classification) and grade.
Material and methods
The study was performed on 52 SCCL patients
(46 males and 6 females). Unlike most other stud-
ies of genomic imbalances in HNSCC, the present
cases. None of the patients had a family history of
cancer. The fresh tumour material and blood sam-
ples were obtained before any treatment at the De-
partment of Otolaryngology, Wroc³aw Medical
histopathologically according to the guidelines
of the World Health Organisation. Three tumours
were stage I, one was stage II, twenty four were
stage III and twenty four were stage IV. Sixteen
patients had lymph node metastases. No patient
had distant metastases at the time of diagnosis.
Histological grade was well differentiated (G1)
in 9 cases, moderately differentiated (G2) in
30 cases, and poorly differentiated (G3) in
13 cases. Due to the small number of patients with
tumours in stages I and II, the group was divided
into only two subgroups (I-III stage versus IV
stage) for the purpose of statistical analysis of the
The experimental protocol was approved by
the Ethics Committee of the Wroc³aw Medical
Comparative genomic hybridization (CGH)
CGH was performed according to standard proce-
dures (Kallioniemi et al. 1994). Normal male
(46,XY) metaphase slides (Metaphase CGH
RNAse (Sigma) for 20 min and 0.001% pepsin A
(Sigma) for 5 min at 37oC, denatured in 70%
series (–20oC; 70, 90, 100%) for 2 min,
and air-dried. Human reference DNA was pre-
rhodamine-5-dUTP (R-5-dUTP, Roche) after
(Telenius et al. 1992). Tumour DNA was isolated
from larynx cancer specimens and labelled with
through DOP-PCR. DOP-PCR was performed
in 25 ?L of a mixture containing 100 ng of
genomic DNA, 0.2 mM of each dATP, dGTP,
dCTP, dTTP (Finnzymes), 0.1 mM F-12-dUTP
or R-5-dUTP, 3 mM MgCl2, 20 ?M of a universal
degenerate primer (5’-CCG ACT CGA NNN
ase (Gibco BRL). PCR amplification was done
with an initial denaturation at 94oC for 1 min, fol-
and 4 min at 72oC. This was followed by 20 cycles
for 1 min at 94oC, 1 min at 56oC, 2 min at 72oC
and one additional cycle for 1 min at 94oC, 1 min
at 56oC, and 5 min at 72oC. DOP-PCR products
were treated with 0.01 U ?L–1DNAse for 3 min at
95oC, resulting in probe size between 200
and 500 bp. Fluorescein- and rhodamine-labelled
DNA were precipitated together in the presence of
10 ?g human Cot I DNA and 25 ?g herring-sperm
DNA (Gibco BRL). The pellet was dissolved in
12 ?L of hybridization solution (20% dextran sul-
fate, 4 × SSC, 50% formamide), denatured for
10 min at 96oC, and preannealed for 30 min
408 K. Schlade-Bartusiak et al.
at 37oC. The probe mixture was then applied onto
was carried out for 48 h at 37oC in a moisture
with rubber cement. The slides were washed at
2 × SSC, and 3 times in 0.1 × SSC, 5 min each time.
Slides were then dehydrated in an ethanol series
(70, 90, 100%) for 2 min at room temperature
and air-dried. Chromosomes were counterstained
Image acquisition and evaluation were done
using a Leica DM-RB epifluorescence micro-
scope equipped with a Kappa CF 8/1 DX camera
controlled by the ISIS software (MetaSystems
GmbH). Three colour images, green for tumour
DNA, red for reference DNA, and blue for
the DAPI counterstain were acquired from 10–20
metaphases per sample. The threshold values
for detection of genomic imbalances were: 0.8 for
losses and 1.25 for gains. All centromeres, as well
as the heterochromatic regions of chromosomes
sis, because these regions can yield unreliable
CGH data, due to incompletely suppressed repeti-
tive DNA sequences.
The test for difference in proportions was used to
analyse the frequency of gains and losses. Stu-
dent’s t-test, Mann-Whitney test, and Fisher exact
test for independence were used to analyse associ-
ations between the group of tumour samples with
deletions in the 1q22-23 and 13q regions detected
by CGH and the group of tumour samples without
deletion in these regions. All the calculations were
carried out with the STATISTICA package.
In the present study, the CGH analysis of chromo-
somal imbalances was performed on 52 SCCL
samples. An overview of the CGH results is pro-
vided in Figure 1. Altogether, 1961 unbalanced
chromosomal changes were detected: 1042 losses
37.7 per tumour. Statistically significant changes
(p < 0.05) in DNA copy number are presented in
Table 1. The most common sites of losses were
1p34.1-p36.3 (33% of cases), 13q21-q32 (42%),
Xp21-p22.1 (37%), and the most common gains
were located in 1p13-q23 (62%), 9q13-q21 (54%)
and 16q13 (37%).
Deletion of 13q in larynx cancer409
Figure 1. Summary of DNA copy number changes in 52 patients with larynx cancer. Bars on the left side of each
chromosome ideogram denote losses of sequences in the tumour genome, and bars on the right side denote gains.
We analysed associations between clinico-
and the overall number of gains and losses (Ta-
ble 2), as well as statistically significant gains and
losses (Table 3). The overall number of gains was
negatively correlated with cancer grading. G1 tu-
mours were characterized by a higher overall num-
ber of gains and a higher frequency of deletions in
13q32 and amplifications in 1q23, than tumours in
other grades (p < 0.05 and p < 0.01, respectively).
Statistically significant associatons (p < 0.01) were
also observed between tumour size and gains at
1q22-23 and loss of 13q22 (Table 3). No statisti-
tumours with lymph node metastases and those
Tumour samples from HNSCC patients usually
present a high heterogeneity of genetic imbal-
ances. Although a few common imbalances de-
tected by CGH and karyotype analysis were
repeatedly reported, it is expected that tumours di-
agnosed in different sites of the head and neck
could be characterized by specific patterns of
chromosomal aberrations. In the present study,
multiple imbalances were found. Chromosomal
losses were observed more often than gains (1042
410K. Schlade-Bartusiak et al.
observed in 52 SCCL samples
1. Regionswithstatistically significant
% of cases
Table 2. Associations between number of imbalances
and histopathological features of cancer cells observed
in 52 larynx cancer samples
Mean no. of
Mean no. of
*statistically significant difference (Student’s t-test, p < 0.05)
or losses and histopathological features of cancer cells
observed in 52 larynx cancer samples
4 (33.3%) 7 (58.3%)1 (8.3%)
5 (12.8%)22 (56.4%)12 (30.8%)
Association between tumour grading (G) and deletions in 13q32
(Mann-Whitney test, p < 0.05)
Tumours with deletion4 (28.6%) 10 (71.4%)
Tumours without deletion 23 (63.9%) 13 (36.1%)
exact test, p < 0.05)
Tumours with amplification 7 (31.8%)15 (68.2%)
Tumours without amplification18 (69.2%)8 (30.8%)
Tumours with amplification
2 (16.7%) 10 (83.3%)
Tumours without amplification
26 (65.0%)14 (35.0%)
Association between tumour size (T) and amplifications in 1q22
and 1q23 (Fisher’s exact test, p < 0.05 and p < 0.01, respectively)
1q23 G1G2 G3
5 (41.7%) 7 (58.3%) 0 (0.0%)
4 (10.0%)23 (57.5%)13 (32.5%)
Association between tumour grading (G) and amplification in 1q23
(Mann-Whitney test, p < 0.01)
and 919, respectively). A similar result was ob-
tained by Kujawski et al. (1999) in a different
group of larynx cancer cases.
The mean copy number of chromosomal im-
balances per case was 15.65 for deletions
and 17.42 for gains. Singh et al. (2001) in their
study on 11 HNSCC cell lines observed a median
of 16 copy number changes. Thus, HNSCC seems
to be more cytogenetically unstable than many
other solid tumours, such as lung cancer (median
of 13 copy number changes) and colorectal carci-
noma (median of 5.6 copy number changes) (Ried
et al. 1996; Petersen et al. 1997). However, cell
lines derived from breast cancer are characterised
by greater instability, with about 19.6 copy num-
ber changes per case (Kytola et al. 2000).
somal losses at 1p34.1-p36.3 and 13q21-q32 were
types of cancers, e.g. colon, breast, ovary, oral cav-
(Kaghad et al. 1997; di Vinci et al. 1998; Alvarez
et al. 2001). Allelic loss in 1p36 was correlated
with advanced histological grade in ovarian carci-
noma (Alvarez et al. 2001). Deletion in 1p32-pter
cer development, important for the initiation of
carcinogenesis (Bardi et al. 1993; di Vinci et al.
1998). Ogunbiyi et al. (1997) showed that allelic
an independent marker of unfavourable clinical
outcome in this type of cancer. Putative apoptosis
genes, p73, DR3 and Cdc2L1/2, are located in this
chromosomal region (Lathi et al. 1995; Kaghad
et al. 1997; Grenet et al. 1998; Sunahara et al.
Deletions in 13q were described in lung
and head and neck cancers by several authors
(Field et al. 1996; Larramendy et al. 2000;
Luk et al. 2001; Gollin et al. 2001). Califano et al.
(1996, 1999) suggested that deletions in chromo-
some 13 play an important role in genetic progres-
tumours can be the source of clonal premalignant
cells and evolveto
(Califano et al. 1999). Kujawski et al. (1999,
2002) suggested that losses in 13q play a role in
metastasis formation in HNSCC. In the present
study we replicated the finding of significant in-
volvement of 13q deletions in larynx cancer.
The deletions were significantly more frequent in
well-differentiated tumours, as compared with G3
tumours. We did not find any correlation between
the frequency of losses in 13q and the presence of
lymph node metastases at the time of diagnosis,
but the frequency of 13q22 deletions did correlate
with tumour size. It seems that accumulation
of 13q changes is associated with less aggressive
types of tumours, independently of metastase for-
mation. However, the lack of tissue material from
metastases did not allow us to compare 13q
changes in primary versus metastatic tumours,
and such an association cannot be excluded.
Several genes located in 13q were implicated
in cancer formation and progression. Retinoblas-
toma gene RB1 is located in 13q14, proximal to
13q21-q32. Pack et al. (1999) reported a high fre-
quency of LOH at the RB1 locus and deletion at
13q21-q31 in oesophageal cancers. The authors
suggested that the candidate tumour-suppressor
gene on 13q implicated in oesophageal squamous
cell carcinoma is a novel gene, distinct from
the RB1 locus. The BRCA2 gene is located in
ported to predispose to various types of cancer, in-
cluding larynx cancer (Easton et al. 1997).
The loss of heterozygosity in the region proximal
to 13q12-q13 suggested that this region contains
an unknown tumour suppressor gene (Krikpatrick
et al. 1997).
The overall number of gains observed in
the present study was negatively correlated with
cancer grading. Among regions in which the gains
were observed most frequently, 1q22 and 1q23
seem to be particularly interesting, because gains
in these regions are associated with tumour size
(and IV clinical stage). Gains at 1q in HNSCC
were previously described and can be important
for cancer progression (Speicher et al. 1995;
Kujawski et al. 1999). A significant number of
losses in chromosome X, detected in the present
study, were also previously observed. Van Dyke
et al. (1994) found a loss of the short arm of chro-
mosome X in 70% of patients with HNSCC.
ances in different types of squamous cell carci-
noma suggests that shared genes are involved in
tumour progression. As a variety of chromosomal
alterations are associated with this tumour type, it
appears that multiple genes are disrupted in
the process of HNSCC neoplastic transformation,
and the networks of events remain to be eluci-
Prof. Tomasz Krêcicki from the Department
and Clinic of Otolaryngology, Medical University
of Wroc³aw for tissue samples.
Wewish to thank
Deletion of 13q in larynx cancer 411
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Deletion of 13q in larynx cancer 413