Chapter 5 Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) and Multiple Gene Assays By Yasushi Yatabe, Kengo Takeuchi, and Ignacio Wistuba
PCR is clinically feasible, as this technique is used in most assays to detect EGFR mutations in NSCLC. However, assays with reverse transcriptase (RT)-PCR are used primarily for hematologic malignancies, as high-quality RNA is required, which is difficult to obtain in clinical practice. Indeed, in the CAP/ IASLC/AMP guideline, RT-PCR is not recommended for testing to select patients for ALK inhibitor therapy for lung cancer because of the risk of false-negative results and a high failure rate for RNAbased assays on FFPE samples. However, RT-PCR provides the most robust and detailed information about ALK fusion patterns. Furthermore, some recently developed techniques can be applied to clinical samples with a high success rate.
RT-PCR EML4-ALK shows many fusion variants. The breakpoint of ALK is constantly located before the 5'-end of exon 20 (ENST00000389048) where the kinase domain starts, and this constant retention is seen in other cancers with ALK translocation, such as lymphoma and sarcoma. In contrast, the breakpoint of EML4 may distribute various exons. Following the discovery of the first two fusion variants (Soda 2007), more than 13 variants and three fusion partners have been found (Figure 1). In the six most common EML4-ALK variants, the exons of EML4 and ALK are directly fused in an in-frame fashion without any insertion or deletion. The three major variants (v1: E13;A20, v2: E20;A20, and v3: E6;A20) account for more than 90% of lung cancers associated with EML4-ALK. Among the minor variants, E2;A20 (v5) and E18;A20 (v5') are found in 1% to 2% of lung cancers with EML4-ALK (Takeuchi 2008, Wong 2009). E21;A20 has been identified in colorectal cancer (Lin 2009), but not yet in lung cancer. RT-PCR on fresh or snap-frozen tumor samples may be more sensitive than other methods in terms of the number of cancer cells required. However, high sensitivity is achieved only when the fusion pattern is within a detectable range of primer pairs. To detect all the possible EML4-ALK variants, primer sets should be designed in a comprehensive way (Table 1), and even a comprehensive design may not detect irregular variants with deletions in the annealing site of the primers. Furthermore, RT-PCR systems designed for detecting EML4-ALK cannot detect ALK fusions with other partner sets such as kinesin family member 5B (KIF5B) and kinesin light chain 1 (KLC1) (Takeuchi 2009, Togashi 2012). To overcome this limitation, primers for other fusion partners have been designed, enabling simultaneous one-tube RT-PCR detection of EML4-ALK, KIF5B-ALK (Figure 2), and ret proto-oncogene (RET) fusions (Takeuchi 2012). Despite these efforts, fusions with unknown partners have remained undetectable. Therefore, RT-PCR is not recommended for screening of patients for treatment with