1. EML4-ALK non-small cell lung cancer
LM R2 Yang Jeongseok

3. Non-small cell lung cancer (NSCLC)
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

4. 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: )
ALK kinase inhibitors – shown to be highly effective

5. EML4-ALK variants
All involve the tyrosine kinase domain of ALK (exon 20)
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:

6. Prevalence of different EML4-ALK fusion variants in Korean NSCLCs?
Not studied well
May differ from other countries
To answer this (In this study)
EML4-ALK fusion variants
Reverse-transcriptase-polymerase chain reaction
Korean NSCLCs

7. 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)

8. Specimen Description 167 NSCLC 85 males / 82 females Smoking history
46 squamous cell carcinoma (SCC)
121 adenocarcinoma (AC)
93 never smokers
85 males / 82 females
Smoking history
94 never smokers
73 smokers

9. Experiment (I) RNA extration RNA incubation
RNeasy Mini Kit (Qiagen Valencia, CA, USA)
RNA incubation
RNase-free DNase I (Qiagen)
Reverse transcription of total RNA
cDNA Qiagen kit

10. Experiment (II) Two sense 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
cDNA, primers, Taq polymerase
GAPDH – internal control
PCR product purification
GENECLEAN Turbo kit (Q-Biogene, CA, USA)
Sequencing
ABI Prism 3100 Genetic Analyzer (PE Biosystems, CA, USA)

11. Experiment (III) Additional Mutation Analysis EGFR (exon 18-21)
ERBB2 (exon 19-20)
KRAS (exon 2)

12. Results (I) EML4-ALK fusion transcripts 10 (6.0%) of 167 NSCLCs
More common in
Younger patients (P = 0.002)
Females
Never smokers
Adenocarcinoma (AC)

13. Results (II) Of 10 EML4-ALK fusion transcripts
No EGFR, ERBB2 and KRAS mutation
8 variant 1 (E13;A20)
2 variant 3b (E6;A20)

14. Results (III) Frequency of 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)

15. Discussion Current methods of detection
FISH (ALK rearrangement) – standard?
Unlike PCR, cannot distinguish different fusion variants
IHC method
RT-PCR assay for this study

16. Limitations Only cases with available RNA
Demographic and clinicopathologic characteristics were somewhat different from nationwide lung cancer survey
RT-PCR results were not confirmed by FISH
False positive results are possible
Lee C, Kang KH, Koh Y, Chang J, Chung HS, Park SK, Yoo K, Song JS. Characteristics
of lung cancer in Korea, Lung Cancer 2000; 30:

18. Application of LD-/LDI-PCR
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

19. 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

21. I agree with the comment that
RT-PCR technology for identification of ALK fusion variants has several limitations.
there are multiple EML4-ALK fusion variants
non-EML4 fusion partners, such as KIF5B, and KLC1
Another limitation
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
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