EML4-ALK non-small cell lung cancer

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Presentation transcript:

EML4-ALK non-small cell lung cancer 20120604 LM R2 Yang Jeongseok

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

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 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: 1773-80.

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

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 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

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

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)

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

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)

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

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)

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

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, 1997. Lung Cancer 2000; 30: 15-22.

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

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

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