Normal cells Normalcells
ALK DNA ALK DNA ALK DNA mRNA mRNA mRNA
Kinase Kinase domain 42 domain
ALK+ cells
EML4 Coiled EML4 Coiled coil domain coil domain ALK Kinase ALK Kinase domain domain
IASLC ATLAS OF ALK TESTING IN LUNG CANCER
mRNA mRNA mRNA
from FFPE samples enables the application of this method to clinical practice, as quantitative RT-PCR generally requires short-length products (approximately 150 bp), which are usually retained, even in FFPE samples (Figure 4).
ALK ALK ALK
Comparison between 5' and !"#$%&'(")*+,-.,,)*/0*%)1*20* 3' Comparison between 5' and 3' regions of ALK transcript regions of ALK transcript &,3'")(*"4*567*-&%)(8&'$-*
10 8 >9* 6=* 4 <* 2;* 0 :*
9*
Kinase Kinase domain domain 3' region 3’ region region
10 6 4 2 0
34.83
5' region 5’ region 5' region
800
10 5'-3' product comparative RT-PCR8 >9*
=* <* ;* :* 9*
3' products
Fluorescence
600 400 200 3' region region
50.0
5' region region
5' region
3' region
-200
0
5' region region 3' region region 5' products
5' region
20 30 40 50
3' region
60
A
B
10
Cycles
ALK exon 20
EML4 exon 13
C
D
Figure 4. An example of ALK-positive lung adenocarcinoma (A), using comparative real-time PCR on RNA in a FFPE specimen. RNA was extracted from the FFPE and the expression level of the 3' and 5' regions of ALK was analyzed (B). In this tumor, the difference between the two regions was substantial, suggesting ALK rearrangement. The fusion was confirmed with RT-PCR direct sequencing (C) and break-apart FISH (D).
Multiple Gene Assays
There is an increasing need in lung cancer to develop clinically applicable assays that can simultaneously determine the mutation, rearrangement, and expression status of many genes, including ALK. These multiplexed methodologies are important to maximize the use of small diagnostic lung tumor tissue and cytology specimens. Although several multiplexed genomic assays can be applied to determine ALK fusions at the DNA and RNA levels, published reports are limited (Li T 2013). The rapid development of technologies for large-scale sequencing (next-generation sequencing [NGS]) has facilitated high-throughput molecular analysis that offers various advantages over traditional sequencing, including the ability to fully sequence large numbers of genes in a single test and simultaneously detect deletions, insertions, copy number alterations, rearrangements, and exomewide base substitutions (including known hot-spot mutations) in all known cancer-related genes (Ross 2011). Currently, NGS platforms, including whole genome, whole exome, and targeted gene sequencing, represent emerging diagnostic methodologies for the detection of oncogene fusions