Download (I) EML4-ALK

EML4-ALK
non-small cell
lung cancer
20120604
LM R2 Yang Jeongseok
 Non-small cell lung cancer (NSCLC)
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80-85% of all cases of lung cancer
Leading cause of cancer deaths
Only 15% patients survive for more than 5 yr
Cytotoxic chemotherapy – mainstay treatment
Molecular-targeted therapy
 For genetically defined subsets
of patients
 Identification of genetic alterations of key oncogene is
important
EML4-ALK Fusion Gene

echinoderm microtubule-associated protein-like 4 (EML4)
- anaplastic lymphoma kinase (ALK) fusion oncogene
 Chromosome inversion inv(2)(p21;23)
 Novel molecular target in NSCLC
 3-7% of NSCLCs (Sasaki T et al. Eur J Cancer 2010;46: 1773-80)
 ALK kinase inhibitors – shown to be highly effective
 EML4-ALK variants
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All involve the tyrosine kinase domain of ALK (exon 20)
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Variably truncation of EML4
(exon 2,6,13,14,15,18 and 20)

E13;A20 (33%)
E5a/b;A20 (29%)

Sasaki T, Rodig SJ, Chirieac LR, Janne PA. The biology and treatment of
EML4-ALK non-small cell lung cancer. Eur J Cancer 2010; 46: 1773-80.
 Prevalence
of different EML4-ALK fusion variants
in Korean NSCLCs?
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Not studied well
May differ from other countries
To answer this (In this study)
 EML4-ALK fusion variants
 Reverse-transcriptase-polymerase
 Korean NSCLCs
chain reaction
Specimens
 National Biobank of Korea
Kyungbook-National University Hospital, Daegu
(KNUHBIO_10_1016)
 167 Korean NSCLC patients (Jan 2001 ~ Dec 2009)
 Curative resection at KNUH
 Exclusion Criteria

Chemotherapy or radiation therapy prior to surgery
(to avoid effects on RNA)
Specimen Description
 167 NSCLC
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46 squamous cell carcinoma (SCC)
121 adenocarcinoma (AC)
 93 never
smokers
 85 males / 82 females
 Smoking history
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94 never smokers
73 smokers
Experiment (I)
RNA extration
1.
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RNeasy Mini Kit (Qiagen Valencia, CA, USA)
RNA incubation
2.
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RNase-free DNase I (Qiagen)
Reverse transcription of total RNA
3.
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cDNA Qiagen kit
Experiment (II)
 Two sense
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primers and single antisense primer
(5´-TCACTGTGCTAAAGGCGGCTTTGG-3´, on exon 2 of EML4)
(5´-CCACACCTGGGAAAGGACCTAAAG-3´, on exon 13 of EML4)
(5´-CAGGGCTTCCATGAGGAAATCCAG-3´, on exon 22 of ALK)
 PCR reaction
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cDNA, primers, Taq polymerase
GAPDH – internal control
 PCR product purification
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GENECLEAN Turbo kit (Q-Biogene, CA, USA)
 Sequencing
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ABI Prism 3100 Genetic Analyzer (PE Biosystems, CA, USA)
Experiment (III)
 Additional Mutation Analysis
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EGFR (exon 18-21)
ERBB2 (exon 19-20)
KRAS (exon 2)
Results (I)
 EML4-ALK fusion transcripts
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10 (6.0%) of 167 NSCLCs
More common in
 Younger patients
(P = 0.002)
 Females
 Never smokers
 Adenocarcinoma (AC)
Results (II)
 Of 10 EML4-ALK fusion transcripts
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No EGFR, ERBB2 and KRAS mutation
8 variant 1 (E13;A20)
2 variant 3b (E6;A20)
Results (III)
 Frequency of
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EML4-ALK fusion gene
6.0% in NSCLCs (7.4% in ACs)
Consistent with reported frequencies of
East Asian patients with NSCLCs (3-13%)
Similar to previous Korean study
(4.2% of NSCLCs and 6.8% in ACs)
Discussion
 Current methods of detection
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FISH (ALK rearrangement) – standard?
 Unlike PCR, cannot distinguish different
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IHC method
RT-PCR assay for this study
fusion variants
Limitations
 Only cases with available RNA
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Demographic and clinicopathologic characteristics
were somewhat different from
nationwide lung cancer survey
Lee C, Kang KH, Koh Y, Chang J, Chung HS, Park SK, Yoo K, Song JS. Characteristics
of lung cancer in Korea, 1997. Lung Cancer 2000; 30: 15-22.

RT-PCR results were not confirmed by FISH
 False
positive results are possible
Application of LD-/LDI-PCR
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LD-/LDI-PCR
fusion breakpoint analysis method

Uses stable genomic DNA
sensitivity as FISH
screening all types of fusion genes
specific as RT-PCR method
confirming specific subtypes of a fusion gene

Intron 19 of the ALK
size of about 1,932 bp (breakpoint cluster region, BCR)

expected to be a powerful method
to analyze all EML4-ALK variants
and also other ALK rearrangements
with both known and unknown fusion partner genes
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I agree with the comment that
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RT-PCR technology for identification of ALK fusion variants has several
limitations.
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Another limitation
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there are multiple EML4-ALK fusion variants
non-EML4 fusion partners, such as KIF5B, and KLC1
most specimens from lung cancer patients are stored as formalin-fixed
paraffin embedded tissue
RNA may have been substantially degraded
PCR-based detection of EML4-ALK can yield false positive results
in the absence of detectable ALK-rearrangement
I agree with the suggestion that
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LD-/LDI-PCR
could be used to identify all EML4-ALK fusion variants
as well as other ALK rearrangements having known or unknown fusion
partner genes