EML4-ALK fusion transcript is not found in gastrointestinal and breast cancers.

Abstract

Fusion genes have been identified as chromosomal rearrangements in certain cancers, such as leukaemia, lymphoma, and sarcoma. The EML4-ALK (EML4: echinoderm microtubule-associated-protein-like 4; ALK: anaplastic lymphoma kinase) fusion gene has been identified as an oncogene in non-small-cell lung cancer (NSCLC). This study examined the presence of this fusion transcript in gastrointestinal and breast cancers. We evaluated the expression of the EML4-ALK transcript in 104 lung cancer cases and in 645 gastrointestinal and breast cancer samples. Only one of the lung cancer samples tested positive for the EML4-ALK fusion transcript, whereas none were detected in 555 gastrointestinal and 90 breast cancer cases. Our data suggest that the EML4-ALK fusion transcript is not present in gastrointestinal or breast cancers and is specific to NSCLC.

Full text

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EML4–ALK fusion transcript is not found in gastrointestinal and
breast cancers
Y Fukuyoshi1,4, H Inoue1,4, Y Kita2, T Utsunomiya3, T Ishida3and M Mori*,1
1Department of Surgery, Medical Institute of Bioregulation, Kyushu University, 4546 Tsurumihara, Beppu 874-0838, Japan;2Department of Surgery,
Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan;3Department of Surgery, Hiroshima Red Cross Hospital, 1-9-6 Senda,
Hiroshima 730-8619, Japan
Fusion genes have been identified as chromosomal rearrangements in certain cancers, such as leukaemia, lymphoma, and sarcoma.
The EML4–ALK (EML4: echinoderm microtubule-associated-protein-like 4; ALK: anaplastic lymphoma kinase) fusion gene has been
identified as an oncogene in non-small-cell lung cancer (NSCLC). This study examined the presence of this fusion transcript in
gastrointestinal and breast cancers. We evaluated the expression of the EML4–ALK transcript in 104 lung cancer cases and in 645
gastrointestinal and breast cancer samples. Only one of the lung cancer samples tested positive for the EML4–ALK fusion transcript,
whereas none were detected in 555 gastrointestinal and 90 breast cancer cases. Our data suggest that the EML4–ALK fusion
transcript is not present in gastrointestinal or breast cancers and is specific to NSCLC.
British Journal of Cancer (2008) 98, 1536–1539. doi:10.1038/sj.bjc.6604341
Published online 15 April 2008
& 2008 Cancer Research UK
www.bjcancer.com
Keywords: EML4–ALK; fusion transcript; non-small-cell lung cancer; gastrointestinal cancer; breast cancer
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Chromosomal aberrations, in particular, translocations and their
corresponding gene fusions, are common occurrences in certain
cancers, such as leukaemia, lymphoma, and sarcoma; however,
these abnormalities have not been as readily identified in solid
cancers (Mitelman et al, 2007; Rikova et al, 2007). Two
characteristics of chromosomal rearrangements, gene fusions,
and fusion proteins make them ideal targets for cancer diagnosis
and treatment. First, chromosomal rearrangements and gene
fusions are specific to tumour cells, are associated with tumour
development, and are not found in normal cells and tissues. Target
cancer cells are easily detected with high specificity using reverse
transcription-polymerase chain reaction (RT–PCR), with primers
designed for the corresponding gene fusion partners. Second,
treatments targeting fusion proteins are tumour-specific and show
fewer adverse effects. For example, imatinib mesylate (Gleevec),
which targets the Bcr–Abl fusion protein, is a highly effective
treatment for chronic myelogenous leukaemia (Druker et al, 1996,
2006). Recently, Soda et al (2007) reported the existence of a fusion
gene in non-small-cell lung cancer (NSCLC). They reported that a
small inversion within chromosome 2p results in the formation of
a fusion comprising portions of the echinoderm microtubule-
associated-protein-like 4 (EML4) gene and the anaplastic lymphoma
kinase (ALK) gene. This fusion was detected in 5 out of the 75
cases studied. The identification of this and other chromosomal
aberrations would be an important step in understanding the
mechanisms underlying the development of solid cancers. There-
fore, this study was undertaken to evaluate the presence of the
EML4–ALK transcript in 749 cases of lung cancer and 2 major
solid carcinomas (gastrointestinal and breast cancers). We
established that the EML4–ALK gene alteration is not found in
both these carcinomas and is specific to NSCLC.
MATERIALS AND METHODS
Clinical samples
Fresh surgical specimens were obtained from 749 patients, as
described in Table 1. The patients had undergone surgery at the
Kyushu University Hospital at Beppu, Hiroshima Red Cross
Hospital, or Kagoshima University Hospital. Written informed
consent was obtained from all patients according to the guidelines
approved by the Institutional Research Board, and this study was
conducted under the supervision of the ethical board of Kyushu
University.
RNA preparation and reverse transcription
Total RNA was isolated using a modified acid guanidinium-
phenol-chloroform procedure with DNase. Complementary DNA
was synthesized from 2.5mg of total RNA, as described previously
(Inoue et al, 1995).
RT–PCR of EML4–ALK fusion transcript
The following primers were designed to amplify 247bp of the
EML–ALK fusion transcript (Soda et al, 2007): 50-TGCAGTGTT
Received 11 December 2007; revised 27 February 2008; accepted 10
March 2008; published online 15 April 2008
*Correspondence: Dr M Mori, Department of Surgery and Molecular
Oncology, Medical Institute of Bioregulation, Kyushu University, 4546
Tsurumihara, Beppu 874-0838, Japan;
E-mail: mmori@beppu.kyushu-u.ac.jp
4These authors contributed equally to this study.
British Journal of Cancer (2008) 98, 1536–1539
& 2008 Cancer Research UK All rights reserved 0007– 0920/08$30.00
www.bjcancer.com
Molecular Diagnostics

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TAGCATTCTTGGGG-30(forward) and 50-TCTTGCCAGCAAAG
CAGTAGTTGG-30(reverse).
Either of the fused gene variants (1 or 2) could be amplified with
the primer sets described by Soda et al (2007).
The primers for nested RT–PCR were also employed: 50-GC
AGTGTTTAGCATTCTTGGGGA-30(forward) and 50-CTTGCCAGC
AAAGCAGTAGTTGGG-30(reverse).
The forward primer was located in exon 2 of the EML gene,
whereas the reverse primer was located in exon 3 of ALK.
Amplification was performed for 35 cycles of 30s at 951C, 30s at
661C, and 1min at 721C. A 10ml aliquot of each reaction mixture
was size-fractionated in 2% agarose gel and visualised by ethidium
bromide staining. Amplification of the GAPDH (glyceraldehyde-3-
phosphate dehydrogenase) gene transcript was used as a positive
control (of RNA integrity and assay conditions). GAPDH was
amplified using the following cycling parameters: 22 cycles of 30s
at 951C, 30s at 561C, and 30s at 721C. The following GAPDH
primer sequences were used: 50-TTGGTATCGTGGAAGGACTCA-
30(forward) and 50-TGTCATCATATTTGGCAGGTT-30(reverse)
(Inoue et al, 1995). The RT–PCR product was cloned into pCR
vector using TA Cloning Kit Dual Promoter pCR II (Invitrogen
Corp., Carlsbad, CA, USA) and sequenced with ABI PRISM 3100-
Avant Genetic Analyzer (Applied Biosystems, Foster, CA, USA).
DNA extraction
Frozen tissue was dissolved in 200mg of buffer ATL containing
10% proteinaseK, and genomic DNA was extracted and purified
using QIAamp DNA Micro Kit (Qiagen, Hilden, Germany), in
accordance with the manufacturer’s protocols.
Long PCR of EML4–ALK fusion gene
The following primers were designed to amplify the genomic
sequence flanking the breakpoint of the EML4–ALK fusion gene
(Soda et al, 2007): 50-CCACACCTGGGAAAGGACCTAAAG-30
(forward) and 50-AGCTTGCTCAGCTTGTACTCAGGG-30(reverse).
Amplification was performed for 35 cycles of 30s at 941C, 30s at
601C, and 5min at 721C. A 20ml aliquot of each reaction mixture
was size-fractionated in 2% agarose gel and visualised by ethidium
bromide staining.
RESULTS AND DISCUSSION
Expression of the EML4–ALK fusion gene was studied in 749 solid
tumours (Table 1). A 247bp transcript was amplified in only 1 out
of the 104 lung cancers studied (Figure 1). Sequencing of this
transcript confirmed that it was the product of the EML4–ALK
fusion (type 1 variant) (Figure 2). However, this transcript was
not detected in 555 gastrointestinal and 90 breast cancers. The
RT–PCR experiments were repeated three times, including one
using nested inner primer set; we thereby confirmed the above
results. Thus, the EML4–ALK fusion gene appears to be specific to
NSCLC. Very recently, Inamura et al (2008) investigated the
presence of EML4–ALK fusion in lung cancers: 149 adenocarci-
nomas, 48 squamous cell carcinomas, 3 large-cell neuroendocrine
carcinomas, and 21 small-cell carcinomas. They reported that 5 out
of 149 adenocarcinomas showed EML4–ALK fusion, but this was
not found in carcinomas of other types. The case expressing
EML4–ALK in our report was also adenocarcinoma. The EML4–
ALK fusion gene is thus considered to be specific to NSCLC,
especially adenocarcinomas.
We next surveyed the genomic alterations in nine cases of
NSCLC, including one that was positive for EML4–ALK fusion
transcript. A 2.5kb PCR product was detected only in the EML4–
ALK-positive case (Figure 3). The size of the long PCR product was
different from the one originally reported by Soda, indicating that
our case had a different breakpoint. The genomic DNA involved by
the alteration was sequenced to identify the breakpoints in the
EML4 and ALK genes. As a result, EML4 was disrupted at
a position 1485bp downstream of exon 13 and was ligated to a
position 1254bp upstream of exon 21 of ALK (Figure 4). In the
report by Soda, EML4 was disrupted at a position approximately
3.6bp downstream of exon 13 and was ligated to a position 297bp
upstream of exon 21 of ALK. Thus, we confirmed the difference of
genomic rearrangements between the present case and the original
report by Soda.
Earlier, fusion genes were identified based on the presence of
large-scale chromosomal rearrangements. However, genome pro-
ject completions and advances in microarray technology have
Lung OesophagusLiver
Marker
EML4–ALK
GAPDH
Figure 1
cases of lung, liver, and oesophageal carcinoma were subjected to reverse
transcription-polymerase chain reaction with primers for EML4–ALK fusion
transcript. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA is
also shown. Marker, 100bp DNA ladder.
Screening of specimens for EML4–ALK mRNA. Representative
Table 1 Specimens examined for EML4–ALK transcript
Number Positive case
Lung
Colorectum
Stomach
Oesophagus
Breast
Liver
Cholangiocellular
Pancreas
Total
104
96
96
112
90
232
11
1
0
0
0
0
0
0
0
1
8
749
ALK¼anaplastic lymphoma kinase; EML4¼echinoderm microtubule-associated-
protein-like 4.
ALK4
Exon 21
EML
Exon 13
12 Mb
…AAAG
TGTA…
Figure 2
revealing fusion of EML4 with ALK in a case with non-small-cell lung cancer
(adenocarcinoma). Line graph shows the position and automated DNA
sequencing of the fusion points (identical to the variant 1 transcript).
Schematic of reverse transcription-polymerase chain reaction
Absence of EML4–ALK transcript in GI tract and breast cancers
Y Fukuyoshi et al
1537
British Journal of Cancer (2008) 98(9), 1536–1539
& 2008 Cancer Research UK
Molecular Diagnostics

Page 3

revealed chromosomal rearrangements in several kinds of cancers
(Mitelman et al, 2004; Hirasaki et al, 2007). The report of the
TMPRSS2–ERG fusion in prostate cancer by Tomlins et al (2005)
is probably the first description of such a gene fusion in solid
cancers. This altered gene was recognized in 22 out of 24 examined
cases, and activity of the TMPRSS2–ERG fusion protein might be
correlated with prostate cancer development. Of course, as this
gene does not exist in normal cells, it could prove useful for cancer
diagnosis and treatment. The TMPRSS2–ERG fusion does not
show a chromosomal translocation, but originates from a 2.7Mb
deletion in chromosome 19 (Tomlins et al, 2005, 2007). The
EML4–ALK fusion gene reported by Soda et al (2007) is also
derived from a genomic inversion of less than 12Mb in
chromosome 2. These alterations (deletions or inversion) are too
small to be detected by traditional cytogenetic techniques.
Microarrays allow a more detailed analysis and the possibility of
detecting novel fusion genes in the absence of chromosomal
translocations.
The TMPRSS2–ERG fusion gene is specific to prostate cancer,
which may be due to the strong induction of TMPRSS2 by
androgen (Perner et al, 2006). In contrast, the reason behind the
NSCLC specificity of EML4–ALK is unclear. This alteration was
not detected in two of the most prevalent carcinomas, gastro-
intestinal and breast cancers. However, as mentioned above, there
is a possibility that novel fusion genes will be identified, which can
open a new era in the diagnosis and treatment of these common
solid cancers.
ACKNOWLEDGEMENTS
We thank T Shimooka, K Ogata, N Kasagi, and Y Nakagawa for
their excellent technical assistance. This study was supported by
Grant-in-Aid for Scientific Research (S) (17109013), Grant-in-Aid
for Scientific Research (B) (18390367), and Grant-in-Aid for
Exploratory Research (18659384) from the Japan Society for the
Promotion of Science and Core Research for Evolutional Science
and Technology of Japan Science and Technology.
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The breakpoints in EML4 and ALK genes. The upper figure
Absence of EML4–ALK transcript in GI tract and breast cancers
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British Journal of Cancer (2008) 98(9), 1536–1539
& 2008 Cancer Research UK
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Absence of EML4–ALK transcript in GI tract and breast cancers
Y Fukuyoshi et al
1539
British Journal of Cancer (2008) 98(9), 1536–1539
& 2008 Cancer Research UK
Molecular Diagnostics