Maus MK et al. (2012), Identification of Novel Variant of EML4-ALK Fus...

ECB-ART-50131

Int J Biomed Sci 2012 Mar 01;81:1-6.

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Identification of Novel Variant of EML4-ALK Fusion Gene in NSCLC: Potential Benefits of the RT-PCR Method.

Maus MK , Stephens C , Zeger G , Grimminger PP , Huang E .

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BACKGROUND: The discovery of the transforming fusion gene of the anaplastic lymphoma kinase (ALK) with the echinoderm microtubule-associated protein like 4 (EML4) as an oncogene in 2007 has led to its validation as a clinical target in NSCLC patients in a short period of time. The inhibition of the anaplastic lymphoma receptor tyrosine kinase has demonstrated to prolong progression-free survival compared to the standard of care chemotherapy in patients with advanced NSCLC that are ALK positive. However, the clinical implications of the 15 different variants of the EML4-ALK transforming gene described so far are currently not defined. Here we present a novel variant of the EML4-ALK fusion gene which we named variant 3c. METHODS: RNA extracted from formalin fixed paraffin embedded (FFPE) specimens from patients with advanced and metastatic NSCLC was amplified, using primers and probes designed to detect specific EML4-ALK fusion gene fragments. Gel electrophoresis showed a different band for the new variant 3c compared to the known bands of positive cell lines for variant 3a and 3b. These findings were further investigated by dye-terminator Sequencing and FISH. RESULTS: The novel variant, detected in two NSCLC specimens, is longer than v3a and shorter than v3b, representing an 18 base pair insertion of intron 19 of ALK between exon 6 of EML4 and exon 20 of ALK. All of the two samples showed exactly the same sequencing result. One of the samples was negative for FISH break apart testing and the other one showed a positive result, defined by ≥15% split nuclei as indicative of an ALK rearrangement. CONCLUSIONS: Compared to FISH technology, RT-PCR enables the detection of different isoforms of the EML4-ALK transforming gene, which can be validated by sequencing. Only one out of two samples that were positive for the new variant by RT-PCR could be confirmed by FISH. The clinical significance of the different variants, notably to resistance and response to ALK-Inhibitors and the concordance and sensitivity of FISH and RT-PCR should be subject to further investigations.

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References [+] :

Choi, Identification of novel isoforms of the EML4-ALK transforming gene in non-small cell lung cancer. 2008, Pubmed

	Figure 1. H&E slides from the two patients, that were tested positive for EML4-ALK Variant 3c. Image A shows poorly differenciated squamous cell carcinoma including bone (blue arrow) from a patient with history of squamous cell lung cancer. Image B shows moderately differenciated adenocarcinoma of a primary lung cancer. The green arrows indicate scattered signet ring cells.
	Figure 2. Frequency of EML4-ALK variants from the Response Genetics Inc. dataset 2010-2011.
	Figure 3. Image of Gel-Electrophoresis showing the bands of variant 3a/b and the band of variant 3c (sample & sample double) between them.
	Figure 4. Chromogram of the sequence of variant 3c containing the 18 base pair insertion of Intron 19 of ALK (red box) and parts of Exon 6 of EML4 (black arrow) and Exon 20 of ALK (green arrow).
	Figure 5. Sequence of the new variant 3c, showing the forward primer (blue), the intron 19 insertion (green), the probe (yellow) and the reverse primer (red). The EML4 gene is represented by black letters, the ALK gene by red letters. This seyuence is complimentary to the sequnce shown in the chromogram above (Fig. 3).
	Figure 6. FISH Image showing two negative nuclei and one positive split nucleus (upper right).