Yen, Po-Min Chen, Division of Medical Oncology, Department of
Medicine, Taipei Veterans
General Hospital and National Yang-Ming University School of
Medicine, Taipei, Taiwan, China
Yann-Jang Chen, Faculty of Life Science, National Yang-Ming
University, Taipei, Taiwan, China
Chin-Chen Pan, Paul Chih-Hsueh Chen, Department of Pathology,
National Yang-Ming University and Taipei Veterans General Hospital,
Kai-Hsi Lu, Institute of Microbiology and Immunology,
National Yang-Ming University, Taipei, Taiwan, China
Jiun-Yi Hsia, Division of Thoracic Surgery, Department of
Surgery, Taichung Veterans General Hospital, Taichung, Taiwan, China
Jung-Ta Chen, Department of Pathology, Taichung Veterans
General Hospital, and Department of Pathology, Chung-Shan Medical
University, Taichung, Taiwan, China
Yu-Chung Wu, Wen-Hu Hsu, Liang-Shun Wang, Min-Hsiung Huang,
Biing-Shiung Huang, Division of Thoracic Surgery, Department of
Surgery, Taipei Veterans General Hospital and National Yang-Ming
University, Taipei, Taiwan, China
Cheng-Po Hu, Institute of Microbiology and Immunology,
National Yang-Ming University, and Department of Medical Research
and Education, Taipei Veterans General Hospital, Taipei, Taiwan,
Chi-Hung Lin, Institute of Microbiology and Immunology, and
Institute of Biophotonics, National Yang-Ming University, Taipei,
Supported by the National Microarray and Gene Expression
Analysis Core Facility of the National Research Program for Genomic
Medicine at National Yang-Ming University (http://www.ym.edu.tw/microarray),
and annual project Grant From National Science Council (Grant NO.
NSC 92-2314-B-075-055), Taiwan, China
Correspondence to: Dr. Chi-Hung Lin, Institute of
Microbiology and Immunology, National Yang-Ming University, NO. 155,
Li-Non St., Section 2, Taipei 112, Taiwan, China.
Aim: By using
comparative genomic hybridization, gain of 3q was found in 45-86%
cases of esophageal squamous cell carcinoma (EC-SCC). Chromosome
3q25.3-qter is the minimal common region with several oncogenes
found within this region. However, amplification patterns of these
genes in EC-SCC have never been reported. The possible association
of copy number changes of these genes with pathologic
characteristics is still not clear.
Real-time quantitative PCR (Q-PCR) was performed to analyze the copy
number changes of 13 candidate genes within this region in 60
primary tumors of EC-SCC, and possible association of copy number
changes with pathologic characteristics was analyzed by statistics.
Immunohistochemistry (IHC) study was also performed on another set
of 111 primary tumors of EC-SCC to verify the association between TP63
expression change and lymph node metastasis status.
average copy numbers (±SE) per haploid genome of individual genes
in 60 samples were (from centromere to telomere): SSR3: 4.19
(±0.69); CCNL1: 5.24 (±0.67); SMC4L1: 2.01 (±0.16);
EVI1: 2.02 (±0.12); hTERC: 5.28 (±0.54); SKIL:
2.71 (±0.14); EIF5A2: 1.95 (±0.12); ECT2: 9.18 (±1.68);
PIK3CA: 8.13 (±1.17); EIF4G1: 1.07 (±0.05); SST:
3.07 (±0.25); TP63: 2.51 (±0.22); TFRC: 2.42 (±0.19).
Four clusters of amplification were found: SSR3 and CCLN1
at 3q25.31; hTERC and SKIL at 3q26.2; ECT2 and PIK3CA
at 3q26.31-q26.32; and SST, TP63 and TFRC at
3q27.3-q29. Patients with lymph node metastasis had significantly
lower copy number of TP63 in the primary tumor than those
without lymph node metastasis. IHC study on tissue arrays also
showed that patients with lymph node metastasis have significantly
lower TP63 staining score in the primary tumor than those
without lymph node metastasis.
This study showed that different amplification patterns were seen
among different genes within 3q25.3-qter in EC-SCC, and several
novel candidate oncogenes (SSR3, SMC4L1, ECT2,
and SST) were identified. TP63 is amplified in early
stage of EC-SCC carcinogenesis but down-regulated in advanced stage
© 2005 The WJG Press and Elsevier Inc. All rights reserved.
Key words: Chromosomal aberration; Comparative genomic
hybridization; Esophageal neoplasm; Immunohistochemistry;
Quantitative real-time PCR; Tissue array; Tumor protein 63
Yen CC, Chen YJ, Pan CC, Lu KH, Chen PCH, Hsia JY, Chen JT, Wu YC,
Hsu WH, Wang LS, Huang MH, Huang BS, Hu CP, Chen PM, Lin CH. Copy
number changes of target genes in chromosome 3q25.3-qter of
esophageal squamous cell carcinoma: TP63 is amplified in
early carcinogenesis but down-regulated as disease progressed.
World J Gastroenterol 2005;
Esophageal cancer (EC) is one of the most lethal cancers in the
well as in Taiwan (http://www.doh.gov.tw/dohenglish/Upload/Statistics/S02/9110-eng.xls).
In most Asian countries, squamous cell carcinoma (EC-SCC) is the
most frequent histological subtype of EC. By using modern molecular
cytogenetic study such as comparative genomic hybridization (CGH)
and spectral karyotyping (SKY),
several recurrent chromosomal aberrations of EC-SCC were identified.
Among them, chromosome 3q is especially important, because 45-86%
cases of EC-SCC were found to have amplification in this region,
with minimal overlapping region over 3q25.3-qter[4-8].
Gain of chromosome 3q was also reported in many other tumors, such
as cancer of the lung,
and head and neck squamous cell carcinoma (HNSCC)[13,14].
The size of 3q25.3-qter is about 40 MB,
containing about 367 known genes within this region, not to mention
the unknown ones. Several candidate oncogenes were located in this
chromosomal region, such as cyclin L1 (CCNL1), at 3q25; human
telomerase RNA component (hTERC), at 3q26;
phosphoinositide-3-kinase, catalytic, alpha polypeptide (PIK3CA),
at 3q26; Ski-like protein (SKIL), at 3q26; ecotropic
viral integration site 1 (EVI1), at 3q26; eukaryotic
translation initiation factor 5A2 (EIF5A2), at 3q26;
eukaryotic translation initiation factor 4gamma1 (EIF4G1), at
3q27; tumor protein 63 (TP63), at 3q28; and transferrin
receptor (TFRC), at 3q29 (GeneCardsTM,
http://bioinformatics.weizmann.ac.il/cards/). However, amplification
patterns of these genes in EC-SCC have never been reported.
A recently introduced method, real-time
quantitative PCR (Q-PCR), could be used to accurately evaluate copy
numbers of genes with only minimal amounts of tumor materials with
In previous study, the accuracy of assessing copy number changes by
Q-PCR in comparison with fluorescence in situ hybridization
(FISH) had been demonstrated.
In this study, Q-PCR was used to study the copy number changes of
the afore mentioned nine candidate oncogenes, together with four
genes [signal sequence receptor gamma (SSR3) at 3q25.31,
structural maintenance of chromosomes 4-like 1 (SMC4L1) at
3q26.1, epithelial cell transforming sequence 2 oncogene (ECT2)
at 3q26.31, and somatostatin (SST) at 3q27.3] that
were not down-regulated in our preliminary expression profile study,
within chromosome 3q25.3-qter region in 60 primary tumors of EC-SCC,
and the possible associations of these changes with disease
progression were analyzed. Immunohistochemistry (IHC) was also used
to verify the association of expression change of TP63 with
lymph node metastasis status on another 111 cases of EC-SCC.
MATERIALS AND METHODS
Primary tumors and cell lines of EC-SCC
From 1995 to 1997, 60 ethic Chinese patients with EC-SCC
were enrolled for Q-PCR study. All patients underwent primary
surgical resection without neoadjuvant chemotherapy or radiotherapy.
Only patients with written informed consent were included.
Pathological evaluation of depth of tumor invasion, tumor
differentiation and lymph node metastasis were done by one of our
pathologists (Jung-Ta Chen), and staging and grading of tumor were
defined according to the Cancer Staging Manual (5th edition;
American Joint Committee on Cancer). Portions of tumor from the
paraffin-embedded primary tumor samples, of which at least 70% were
tumor cells, were identified under the microscope by one of our
pathologists (Jung-Ta Chen) and were cut out for study. The
procedure of DNA extraction was modified from a previously described
CGH analysis of part of these patients had been previously reported.
DNA extracted from five volunteer donor lymphocytes was used as
control. EC-SCC cell lines CE 48T/VGH, CE 81T/VGH, TE6 and TE9,
which have been previously characterized by molecular cytogenetics,
were used to validate the accuracy of Q-PCR.
Fluorescence in situ hybridization (FISH)
FISH was performed using methods as previously suggested.
The search for FISH probes covering the 13 genes was done by
browsing Ensembl Genome Browser http://www.ensembl.org/ and UCSC
Genome Browser http://genome.ucsc.edu for candidate bacterial
artificial chromosome (BAC) clones. The resulting clones were then
obtained from RPCI-11 BAC library (Table 1).
Their identities were verified by FISH-mapping onto normal
lymphocyte metaphases. For each cell line, FISH signals were
counted in 10 metaphases, and FISH signals per haploid genome were
calculated by using average FISH signals per cell ×23/ average
number of chromosomes per cell.
Real-time quantitative PCR (Q-PCR)
All primers were designed with Primer Express 3.0 software (Applied
Biosystems Foster City, CA) using default parameters, with modified
minimum amplicon length requirements (85 bp). An additional
requirement consisted of a maximum GC content of 40% for the five
last 3’ end nucleotides. The sequences of the primers are listed
in Table 1. PCR reactions were performed as previously described.
DNA content was normalized to that of long interspersed elements (LINE1),
a repetitive element for which copy numbers per haploid genome are
similar both in normal or neoplastic cells.
Copy number changes per haploid genome were calculated by using the
where Nt is the threshold cycle number observed for an experimental
primer in the normal DNA sample, Nline is the threshold cycle number
observed for a LINE1 primer in the normal DNA sample, and Tt
and Tline are the threshold cycle numbers observed for the
experimental primer and LINE1 primer in test DNA sample,
For normal cell the copy number of a gene per haploid genome should
be one. PCR for each primer set were performed in triplicate, and
calculated copy number changes per haploid genome were averaged.
Construction of tissue arrays
Tissue arrays of another 120 EC-SCC tumor samples were
constructed using method as previously described.
Briefly, the H&E-stained slides of selected tumor samples were
examined under a light microscope. The areas of interest were
circled with a color pen, and a 16-gauge bone marrow biopsy trephine
apparatus was used to punch at the circled areas, extracting a
tissue cylinder with 2.0 mm diameter. At least three cylindrical
core biopsies were taken from different sites of each tumor. About
40 cylinders (8×5) were carefully transferred with forceps to a
recipient metal paraffin block box. The recipient box was then
covered with a plastic cassette and then liquid wax was gently
poured into the box until it was full. The box was then put on a hot
plate for 1 min to homogenize the wax, after which the box was
removed from the hot plate and cooled to room temperature slowly.
Four-micrometer sections were cut and mounted on silane-coated
Immunohistochemistry (IHC) for TP63
Immunostaining for TP63 was performed on tissue array slides
using a mouse anti-human TP63 monoclonal antibody clone: 7JUL
(1:30; Novocastra, New Castle Upon Tyne, UK), which recognized all TP63
isoforms, and high temperature antigen unmasking technique
(autoclave in pH 8.0 EDTA buffer) as previously described.
The bound antibodies were detected using the DAKO Envision system.
Positive control (non-neoplastic tonsil tissue) and negative control
(replacement of primary antibody by PBS) were included. The slides
were independently reviewed by two of the authors (Chin-Chen Pan,
Paul Chih-Hsueh Chen) who were blinded to the clinicopathologic
data. TP63 immunoreactivity was semi-quantified using a
combined intensity and percentage of positive scoring method.
Intense nuclear staining was scored as 2, weak as 1, and negative as
0 (Figure 1).
The percentage of cells with each intensity score was estimated. A TP63
staining score was defined as the sum of the percentage of positive
cells with each intensity level multiplied by the intensity score
[e.g., a case with 30% intense staining and 40% weak staining would
be scored as 100 (30×2+40×1)].
Covering BAC clones and position of 13 genes and sequence of
Q-PCR primers of LINE1 and 13 genes over 3q25.3-qter
long interspersed elements; SSR3: signal sequence receptor
gamma; CCNL1: cyclin L1; SMC4L1:structural maintenance
of chromosomes 4-like 1; EVI1: ecotropic viral integration
site 1; hTERC: human telomerase RNA component; SKIL:
Ski-like protein; EIF5A2: eukaryotic translation initiation
factor 5A2; ECT2: epithelial cell transforming sequence 2
oncogene; PIK3CA: phosphoinositide-3-kinase, catalytic, alpha
polypeptide; EIF4G1: eukaryotic translation initiation factor
4gamma1; SST: somatostatin; TP63:tumor protein 63; TFRC:
transferrin receptor. BAC clones, clones of bacterial artificial
chromosomes covering the genes obtained from RPCI-11 library.
Immunoreactivity for TP63. A:
Intense nuclear TP63 staining; B:
Weak nuclear TP63 staining; C:
Negative staining. (Original magnification: ×400).
Statistical comparisons were performed using SPSS 12.0 software.
Pearson coefficient of correlation was used to determine the
correlation between the copy number changes assessed by FISH and Q-PCR.
Student’s t-test was used to test the association between
the results of Q-PCR or the TP63 staining score and lymph
node metastasis or differentiation status. Kruskal-Wallis test was
used to test the association between the results of Q-PCR or the TP63
staining score and primary tumor extent.
Evaluation of Q-PCR accuracy
The accuracy of the assay was tested on 4 EC-SCC cell lines.
In Figure 2, copy number changes of 13 genes on these 4 EC-SCC cell
lines, assessed by both FISH and Q-PCR, were quite compatible. The
Pearson coefficients of correlation for comparing FISH and Q-PCR of
13 genes in 4 cell lines were all >0.9.
Comparison of copy numbers assessed by FISH (stripped bar) and Q-PCR
bar) of 13 genes in 4 EC-SCC cell lines [CE 48T/VGH (pink), CE 81T/VGH
(yellow), TE6 (green), TE9 (blue)]. Pearson coefficients of
correlation for comparing FISH and Q-PCR of 13 genes in 4 cell lines
were all >0.9.
Assessment of copy number changes of candidate genes in
primary tumors by Q-PCR
Copy number changes of 13 candidate genes were assessed by
Q-PCR in 60 primary EC-SCC tumors. The average copy numbers (±SE)
per haploid genome of individual genes in 60 samples were (from
centromere to telomere): SSR3: 4.19 (±0.69); CCNL1:
5.24 (±0.67); SMC4L1: 2.01 (±0.16); EVI1: 2.02 (±0.12);
hTERC: 5.28 (±0.54); SKIL: 2.71 (±0.14); EIF5A2:
1.95 (±0.12); ECT2: 9.18 (±1.68); PIK3CA: 8.13 (±1.17);
EIF4G1: 1.07 (±0.05); SST: 3.07 (±0.25); TP63:
2.51 (±0.22); TFRC: 2.42 (±0.19). Four clusters of
amplification could be found: SSR3 and CCLN1 at
3q25.31; hTERC and SKIL at 3q26.2; ECT2 and PIK3CA
at 3q26.31-q26.32; and SST, TP63 and TFRC at
3q27.3-q29 (Figure 3).
Association of copy number changes of candidate genes in
primary tumors with disease staging or differentiation
The association between copy number changes of 13 genes and
primary tumor extent, lymph node metastasis or tumor differentiation
was analyzed. Patients with lymph node metastasis have significantly
lower copy number of TP63 in the primary tumor than those
without lymph node metastasis (2.05 vs 3.08; P =
0.017) (Figure 4). There were no significant association between
copy number changes of the other 12 genes and lymph node metastasis.
Also the copy number changes of all 13 genes had no statistical
association with either primary tumor extent or tumor
Association of TP63 staining score in primary tumors with
disease staging or differentiation
Immunostaining of TP63 could be evaluated in 111 cases. The
association between TP63 staining score and primary tumor
extent, lymph node metastasis or tumor differentiation was analyzed.
As in the study of Q-PCR, patients with lymph node metastasis have
significantly lower TP63 staining score in the primary tumor
than those without lymph node metastasis (51.03 vs 79.62; P
= 0.034) (Figure 5). There was no statistical association between TP63
staining score and either primary tumor extent or tumor
Average copy number changes (indicated by the circles with number
listed beside them; crossbar represented 2×standard
error) of 13 genes in association of their relative position on
chromosome 3q. Four clusters of amplifications could be found: SSR3
and CCLN1 at 3q25.31; hTERC and SKIL at 3q26.2;
ECT2 and PIK3CA at 3q26.31-q26.32; and SST, TP63
and TFRC at 3q27.3-q29.
In Q-PCR analysis of 60 EC-SCC tumors, patients with lymph node
metastasis [LN (+)] have significantly lower copy number of TP63
in the primary tumor than those without lymph node metastasis [LN
(-)] (2.05 vs 3.08; P = 0.017).
In IHC study of 111 EC-SCC tumors, patients with lymph node
metastasis [LN (+)] have significantly lower TP63 staining
score in the primary tumor than those without lymph node metastasis
[LN (-)] (51.03 vs 79.62; P = 0.034).
In this study, the pattern of copy number changes of 13
potential target genes located on 3q25.3-qter in EC-SCC and the
association of these changes with disease progression were analyzed.
In general, all genes except FIE4G1 had increased copy
number. However, four clusters of genes with higher amplification
were found: SSR3 and CCNL1 at 3q25.31; hTERC
and SKIL at 3q26.2; ECT2 and PIK3CA at
3q26.31-q26.32; and SST, TP63 and TFRC at
3q27.3-q29, with highest peaks over ECT2 and PIK3CA
(Figure 3). In previous study, it was demonstrated that copy number
of PIK3CA was significantly higher than that of TP63
Therefore, in EC-SCC, 3q26.31-q26.32 might be the mostly amplified
area within this region.
and TFRC 
were reported as potential oncogenes in different diseases .
However, SSR3, SMC4L1, ECT2 and SST have
never been previously identified as oncogenes. SSR3 is one of
the 4 members of translocon-associated protein (TRAP) over
endoplasmic reticulum (ER) membrane. TRAP is responsible for the
passage of peptide through ER membrane.
SMC4L1 is one of the members of structural maintenance of
chromosomes (SMC) proteins. The eukaryotic SMC proteins could form
heterodimers, which may be involved in chromosome condensation,
sister chromatid cohesion, and DNA recombination.
ECT2 was identified as an exchange factor for Rho GTPases,
phosphorylated in G2/M phases, and involved in cytokinesis.
Recent studies have showed that N-terminal truncation of ECT2
may render it as an oncogenic protein, which may cause malignant
transformation of cell.
SST (somatostatin) and its analogues have been used in the
treatment of endocrine tumor. Whether these genes play roles in
carcinogenesis of EC-SCC deserve further investigation.
It is very interesting to find that the copy
number changes as well as expression of TP63 decrease in more
advanced stage of disease in EC-SCC. Heselmeyer et al.
reported that gain of chromosome 3q could be found in the early
dysplasia lesion as well as in the invasive carcinoma of cervical
cancer, but at reduced frequency in advanced stage of disease[11,12].
However, this phenomenon has never been reported in EC-SCC.
TP63 gene, one of TP53 gene family,
is a well-known oncogene within this region. Overexpression of TP63
was found in most squamous cell carcinoma[33-37].
In a IHC study of EC-SCC, TP63 protein was found highly
expressed in carcinoma (50/51), dysplasia (10/11), and even in
histologically normal epithelia of esophagus adjacent to the
cancerous tissues (45/47).
On the contrary, in the study of TP63 expression in
urothelial carcinoma, Koga et al. reported that lower TP63
expression was significantly associated with higher Tumor-Node-
Metastasis (TNM) stage (P = 0.0004) and lymph-node metastasis
(P = 0.013). It was found that cancer cells with lower TP63
staining had higher chance of lower membranous b-catenin
expression, which plays a role in cell-cell adherent junctions, and
cancer invasion and metastasis could be promoted by reduced
membranous b-catenin expression.
In a similar study of upper urinary tract urothelial carcinoma,
Zigeuner et al. found that decreased TP63
immunoreactivity was significantly associated with advanced tumor
stages and poor prognosis. They also found that cases with decreased
TP63 immunoreactivity had higher chance of TP53
overexpression in comparison with cases with normal TP63
By combining results of this study with other
reports, it is very likely that amplification of genes located on
3q25.3-qter may occur in quite early stage in the carcinogenesis of
EC-SCC. But some of the genes, such as TP63, may be
down-regulated as disease progressed. One possible explanation is
that the alterations of genes are no longer necessary for
maintenance of cancer cells survival. The other possibility is that
down-regulation of these genes may accompany alterations of other
genes (such as b-catenin or TP53), which may render
cancer cell more invasive or malignant.
In conclusion, this study demonstrated different amplification
patterns of different genes within 3q25.3-qter in EC-SCC, with
highest amplification over 3q26.31-q26.32. SSR3, SMC4L1,
ECT2 and SST were identified as novel candidate
oncogenes within this region. TP63 is amplified in early
stage of EC-SCC carcinogenesis but down-regulated in advanced stage
The authors gratefully acknowledge the technical support from
National Yang-Ming University Genome Research Center (http://genome.ym.edu.tw/).
Wobst A, Audisio RA, Colleoni M, Geraghty JG.
Oesophageal cancer treatment: Studies, strategies and facts. Ann
Oncol 1998; 9: 951-962
Kallioniemi A, Kallioniemi OP, Sudar D, Rutovitz D,
Gray JW, Waldman F, Pinkel D. Comparative genomic
for molecular cytogenetic
analysis of solid tumors. Science 1992; 258: 818-821
Schrock E, Du MS, Veldman T, Schoell B, Wienberg J,
Ferguson-Smith MA, Ning Y, Ledbetter DH, Bar-Am I, Soenksen
D, Garini Y, Ried T.
Multicolor spectral karyotyping of human chromosomes. Science
1996; 273: 494-497
Du PL, Dietzsch E, Van GM, Van RN, Van HP, Parker MI,
Mugwanya DK, De GM, Marx MP, Kotze MJ, Speleman F.
Mapping of novel regions of
DNA gain and loss by comparative genomic hybridization in esophageal
in the black and colored populations of South Africa. Cancer Res
1999; 59: 1877-1883
Pack SD, Karkera JD, Zhuang Z, Pak ED, Balan KV, Hwu
P, Park WS, Pham T, Ault DO, Glaser M, Liotta
L, Detera-Wadleigh SD,
Wadleigh RG. Molecular cytogenetic fingerprinting of esophageal
squamous cell carcinoma
by comparative genomic
hybridization reveals a consistent pattern of chromosomal
Chromosomes Cancer 1999; 25:
Tada K, Oka M, Tangoku A, Hayashi H, Oga A, Sasaki K.
Gains of 8q23-qter and 20q and loss of 11q22-qter in
esophageal squamous cell
carcinoma associated with lymph node metastasis. Cancer 2000;
Ueno T, Tangoku A, Yoshino S, Abe T, Toshimitsu H,
Furuya T, Kawauchi S, Oga A, Oka M, Sasaki K. Gain of
5p15 detected by comparative
genomic hybridization as an independent marker of poor prognosis in
with esophageal squamous cell
carcinoma. Clin Cancer Res 2002; 8: 526-533
Yen CC, Chen YJ, Chen JT, Hsia JY, Chen PM, Liu JH,
Fan FS, Chiou TJ, Wang WS, Lin CH. Comparative
genomic hybridization of
esophageal squamous cell carcinoma: Correlations between chromosomal
progression/prognosis. Cancer 2001; 92: 2769-2777
Lu YJ, Dong XY, Shipley J, Zhang RG, Cheng SJ.
Chromosome 3 imbalances are the most frequent aberration found
in non-small cell lung
carcinoma. Lung Cancer 1999; 23: 61-66
Arnold N, Hagele L, Walz L, Schempp W, Pfisterer J,
Bauknecht T, Kiechle M. Overrepresentation of 3q and 8q
material and loss of 18q
material are recurrent findings in advanced human ovarian cancer. Genes
Cancer 1996; 16:
Heselmeyer K, Macville M, Schrock E, Blegen H,
Hellstrom AC, Shah K, Auer G, Ried T. Advanced-stage
cervical carcinomas are
defined by a recurrent pattern of chromosomal aberrations revealing
high genetic instability
and a consistent gain of
chromosome arm 3q. Genes Chromosomes Cancer 1997; 19:
Heselmeyer K, Schrock E, du Manoir S, Blegen H, Shah
K, Steinbeck R, Auer G, Ried T. Gain of chromosome 3q
defines the transition
from severe dysplasia to invasive carcinoma of the uterine cervix. Proc
Natl Acad Sci USA
1996; 93: 479-484
Fang Y, Guan X, Guo Y, Sham J, Deng M, Liang Q, Li H,
Zhang H, Zhou H, Trent J. Analysis of genetic alterations
nasopharyngeal carcinoma by comparative genomic hybridization. Genes
Chromosomes Cancer 2001;
Liehr T, Ries J, Wolff E, Fiedler W, Dahse R, Ernst G,
Steininger H, Koscielny S, Girod S, Gebhart E. Gain of DNA
copy number on
chromosomes 3q26-qter and 5p14-pter is a frequent finding in head
and neck squamous
cell carcinomas. Int
J Mol Med 1998; 2: 173-179
Bieche I, Olivi M, Champeme MH, Vidaud D, Lidereau R,
Vidaud M. Novel approach to quantitative polymerase
chain reaction using
real-time detection: Application to the detection of gene
amplification in breast cancer. Int J
Cancer 1998; 78:
De PK, Speleman F, Combaret V, Lunec J, Laureys G,
Eussen BH, Francotte N, Board J, Pearson AD, De PA, Van
RN, Vandesompele J.
Quantification of MYCN, DDX1, and NAG gene copy number in
neuroblastoma using a
PCR assay. Mod Pathol 2002; 15: 159-166
Wang TL, Maierhofer C, Speicher MR, Lengauer C,
Vogelstein B, Kinzler KW, Velculescu VE. Digital karyotyping.
Proc Natl Acad Sci USA
2002; 99: 16156-16161
Yen CC, Chen YJ, Lu KH, Hsia JY, Chen JT, Hu CP, Chen
PM, Liu JH, Chiou TJ, Wang WS, Yang MH, Chao TC, Lin
CH. Genotypic analysis
of esophageal squamous cell carcinoma by molecular cytogenetics and
chain reaction. Int J Oncol 2003; 23: 871-881
Isola J, DeVries S, Chu L, Ghazvini S, Waldman F.
Analysis of changes in DNA sequence copy number by
hybridization in archival paraffin-embedded tumor samples. Am J
Pathol 1994; 145: 1301-1308
Pinkel D, Landegent J, Collins C, Fuscoe J, Segraves
R, Lucas J, Gray J. Fluorescence in situ hybridization with
chromosome-specific libraries: Detection of trisomy 21 and
translocations of chromosome 4. Proc Natl Acad
Sci USA 1988; 85:
Osoegawa K, Mammoser AG, Wu C, Frengen E, Zeng C,
Catanese JJ, de Jong PJ. A bacterial artificial
chromosome library for
sequencing the complete human genome. Genome Res 2001; 11:
Pan CC, Chen PC, Chiang H. KIT (CD117) is frequently
overexpressed in thymic carcinomas but is absent in thymomas.
J Pathol 2004; 202:
Pan CC, Chen PC, Chou TY, Chiang H. Expression of
calretinin and other mesothelioma-related markers in
thymic carcinoma and
thymoma. Hum Pathol 2003; 34: 1155-1162
Koga F, Kawakami S, Fujii Y, Saito K, Ohtsuka Y, Iwai
A, Ando N, Takizawa T, Kageyama Y, Kihara K. Impaired
associates with poor prognosis and uroplakin III expression in
invasive urothelial carcinoma of
the bladder. Clin
Cancer Res 2003; 9: 5501-5507
Redon R, Hussenet T, Bour G, Caulee K, Jost B, Muller
D, Abecassis J, Du MS. Amplicon mapping and
pinpoint cyclin L as a candidate oncogene in head and neck cancer. Cancer
Soder AI, Hoare SF, Muir S, Going JJ, Parkinson EK,
Keith WN. Amplification, increased dosage and in situ
of the telomerase RNA
gene in human cancer. Oncogene 1997; 14: 1013-1021
Hiyama T, Yokozaki H, Kitadai Y, Haruma K, Yasui W,
Kajiyama G, Tahara E. Overexpression of human telomerase
RNA is an early event in
oesophageal carcinogenesis. Virchows Arch 1999; 434:
Yokoi S, Yasui K, Iizasa T, Imoto I, Fujisawa T,
Inazawa J. TERC identified as a probable target within the
3q26 amplicon that is
detected frequently in non-small cell lung cancers. Clin Cancer
Res 2003; 9: 4705-4713
Imoto I, Pimkhaokham A, Fukuda Y, Yang ZQ, Shimada Y,
Nomura N, Hirai H, Imamura M, Inazawa J. SNO is a
probable target for gene
amplification at 3q26 in squamous-cell carcinomas of the esophagus. Biochem
Res Commun 2001; 286:
Redon R, Muller D, Caulee K, Wanherdrick K, Abecassis
J, du Manoir S. A simple specific pattern of
at early stages of head and neck squamous cell carcinomas: PIK3CA
but not p63 gene as
a likely target of
3q26-qter gains. Cancer Res 2001; 61: 4122-4129
Ma YY, Wei SJ, Lin YC, Lung JC, Chang TC, Whang-Peng
J, Liu JM, Yang DM, Yang WK, Shen CY. PIK3CA as
an oncogene in cervical
cancer. Oncogene 2000; 19: 2739-2744
Woenckhaus J, Steger K, Werner E, Fenic I, Gamerdinger
U, Dreyer T, Stahl U. Genomic gain of PIK3CA and
increased expression of
p110alpha are associated with progression of dysplasia into invasive
squamous cell carcinoma.
J Pathol 2002; 198:
Hibi K, Trink B, Patturajan M, Westra WH, Caballero OL,
Hill DE, Ratovitski EA, Jen J, Sidransky D. AIS is an
oncogene amplified in
squamous cell carcinoma. Proc Natl Acad Sci USA 2000; 97:
Hibi K, Nakayama H, Taguchi M, Kasai Y, Ito K, Akiyama
S, Nakao A. AIS overexpression in advanced esophageal
cancer. Clin Cancer
Res 2001; 7: 469-472
Hu H, Xia SH, Li AD, Xu X, Cai Y, Han YL, Wei F, Chen
BS, Huang XP, Han YS, Zhang JW, Zhang X, Wu M, Wang
MR. Elevated expression
of p63 protein in human esophageal squamous cell carcinomas. Int
J Cancer 2002;
Glickman JN, Yang A, Shahsafaei A, McKeon F, Odze RD.
Expression of p53-related protein p63 in the
and in esophageal metaplastic and neoplastic disorders. Hum
Pathol 2001; 32: 1157-1165
Wang TY, Chen BF, Yang YC, Chen H, Wang Y, Cviko A,
Quade BJ, Sun D, Yang A, McKeon FD, Crum CP. Histologic
classification of cervical carcinomas by expression of the p53
homologue p63: A study of
250 cases. Hum Pathol
2001; 32: 479-486
Tanaka N, Sugihara K, Odajima T, Mimura M, Kimijima Y,
Ichinose S. Oral squamous cell carcinoma:
Electron microscopic and
immunohistochemical characteristics. Med Electron Microsc
2002; 35: 127-138
Hartmann E, Gorlich D, Kostka S, Otto A, Kraft R,
Knespel S, Burger E, Rapoport TA, Prehn S. A tetrameric
of membrane proteins in
the endoplasmic reticulum. Eur J Biochem 1993; 214:
Strunnikov AV, Jessberger R. Structural maintenance of
chromosomes (SMC) proteins: Conserved
molecular properties for
multiple biological functions. Eur J Biochem 1999; 263:
Tatsumoto T, Xie X, Blumenthal R, Okamoto I, Miki T.
Human ECT2 is an exchange factor for Rho
in G2/M phases, and involved in cytokinesis. J Cell Biol
1999; 147: 921-928
Saito S, Liu XF, Kamijo K, Raziuddin R, Tatsumoto T,
Okamoto I, Chen X, Lee CC, Lorenzi MV, Ohara N, Miki
T. Deregulation and
mislocalization of the cytokinesis regulator ECT2 activate the Rho
signaling pathways leading
transformation. J Biol Chem 2004; 279: 7169-7179
Assistant Editor Guo SY Edited by Gabbe M