EGFR (ERBB1) gene, located on chromosome 7p12, encodes for a protein which is one of the four receptor tyrosine kinases belonging to ERBB family receptor tyrosine kinases. Receptor binding with ligands such as epidermal growth factor (EGF), transforming growth factor, alpha (TGFA), amphiregulin (AREG), epithelial mitogen/epigen (EPGN), betacellulin (BTC), epiregulin (EREG), and heparin-binding EGF-like growth factor (HBEGF), triggers homo- and/or heterodimerization of the receptors leading to autophosphorylation on key cytoplasmic residues. This in turn activates complex downstream signaling cascades in at least four major downstream signaling pathways including the RAS-RAF-MEK-ERK, PI3K-AKT, PLCG1-PRRT2 (PLCgamma-PKC), and STATs modules and sometimes, G protein-coupled receptor signaling and NFKB1 (NF-kappa-B) signaling cascade. These activated signaling pathways drive many cellular responses such as changes in gene expression, cytoskeletal rearrangement, and anti-apoptosis and increased cell proliferation. Due to activating mutations in cancer cells, there is a constitutive or sustained active state that leads to pro-survival signaling which increases potential for invasiveness, proliferation, metastasis, and angiogenesis [14].

The EGFR mutation testing is not used as a diagnostic test for lung adenocarcinoma. Tumors with EGFR mutations in exons 18–21 lead to constitutive activation of the downstream signaling and this is thought to yield more aggressive tumor phenotypes. EGFR mutation testing is recommended for patients being considered TKI therapy. To that effect CPG for EGFR testing were published in 2013.

Briefly, after a systematic review of extensive published data on EGFR-mutated lung cancer and ALK rearrangements in lung cancers, CAP/IASLC/AMP with interest in the diagnosis and management of lung cancer published evidence-based recommendations for the molecular testing of lung cancers for these two predictive biomarkers in a CPG in 2013 [13].

Since close to 90 % of the mutations in lung adenocarcinomas have deletions in exon 19 or point mutation in exon 21 (L858R) of EGFR gene, most laboratories offer molecular tests designed to detect these alterations. However, after the publication of EGFR mutations and ALK rearrangements testing guidelines for selecting patients for TKI therapy, the current recommendation for clinical EGFR mutation testing includes ability to detect all individual mutations that have been reported with a frequency of at least 1 % of EGFR-mutated lung adenocarcinomas (Table 8.3 from guidelines). This is not a concern for laboratories offering Sanger sequencing, which provides information on mutations sequenced throughout the exons 18–21. But it is an important consideration in the selection or design of mutation-specific assays.

EGFR molecular testing should be used to select patients for EGFR-targeted TKI therapy. Patients with lung adenocarcinoma should not be excluded from testing on the basis of clinical characteristics. All patients with advanced lung adenocarcinomas at the time of diagnosis irrespective of morphology and grade and/or at the time of recurrence or progression in patients who originally presented with lower stage disease but were not previously tested are eligible for molecular testing. Lower stage tumors may be tested for mutations but the decision to do so should be made locally by each laboratory, in collaboration with its oncology team. Current summary of guideline recommendation is listed in Table 8.4 [13].

Deletions in exon 19 of EGFR : Most commonly used assay to detect deletions in exon 19 of EGFR is length analysis of fluorescently labeled PCR products. If there is presence of mutation, the mutant allele is of smaller size than the wild type and therefore can be easily detected by capillary electrophoresis (Fig. 8.1). Different size deletions can be discriminated from wild-type allele by the difference in size of the length of PCR product.

EGFR exon 21 point mutation: The 2573 T>G (L858R) mutation creates a new Sau96I restriction site, GGNCC. Incubation and digestion of PCR product with restriction enzyme Sau96I generate an additional shorter fragment, the basis for a PCR restriction fragment length polymorphism (PCR-RFLP) assay design. The digested fluorescently labeled PCR products are then analyzed by capillary electrophoresis [32] (Fig. 8.2). The sensitivity or limit of detection for both the assays is estimated between 3 and 6 %.

ARMS-PCR uses the ability of Taq DNA polymerase effectively distinguishing between a match and a mismatch at the 3′ end of a PCR primer. Specific mutated sequences are selectively amplified with maximum efficiency only when the primer is fully matched, in a background of the sequences that do not carry the mutation including wild-type sequences. Only a low-level background amplification occurs when the 3′ base is mismatched. The fluorescently labeled probes are called scorpions which have fluorophore and quencher in close proximity leading to reduced fluorescence [fluorescence resonance energy transfer (FRET)]. Upon successful PCR, the fluorophore separates leading to increased fluorescence. A commercially available assay detecting 29 mutations in EGFR gene in exons 18–21 is available using this method (Fig. 8.3). The sensitivity for all the mutations except T790M is about 1 %. For T790M, the sensitivity is about 10 %.

Direct Sanger sequencing has been the traditional method to detect mutations in different genes including EGFR exons 18–21. It is generally accepted that direct sequencing is likely to miss mutations when the tumor cell content is less than 25 % of the sample. Therefore, although a gold standard, SS may not be sensitive enough to detect mutations in a limited tissue sample such as needle biopsies or fine-needle aspiration cytology material. In a limited sample, enrichment strategies for mutant allele using LNA or PNA probe may be necessary for acceptable clinical sensitivity. An example of deletion in exon 19 of EGFR is shown in Fig. 8.4.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) MassARRAY system (Sequenom), originally designed to analyze single-nucleotide polymorphisms in amplified DNA fragments, is also used to detect known somatic point mutations [33]. Genotyping is done by single-allele base extension reaction products that discriminate mutant and wild-type alleles for a given point mutation based on mass differences that are specific to the sequence of the wild-type product and the mutant products as resolved by MALDI-TOF MS [34].

The sensitivity of detection of mutation is generally accepted between 1 and 10 % depending upon plex level of the assay. Example of positive exon 21 L858R mutation is shown in Fig. 8.5. The wild-type and mutant peaks are discriminated based on differences in the masses of the single-base extension with mass-modified dNTPs.

Immunohistochemistry (IHC) for total EGFR is not recommended for selection of EGFR TKI therapy due to poor correlation between total EGFR expression and mutation status. In addition, response to EGFR TKI does not correlate with total EGFR expression detected by using immunohistochemistry [19, 35, 36]. IHC for phosphorylated EGFR is limited as the stability of phosphorylation status in FFPE material is variable. EGFR mutation-specific monoclonal antibodies against mutant protein, viz. L858R point mutation in exon 21 and 15 bp deletion in exon 19 (most common deletion), have been shown to be sensitive and specific to detect these mutations in the published literature [37–39]. But for exon 19 deletions, the sensitivity is reduced to detect other sized deletions. These mutation-specific antibodies could be used to identify patients eligible for EGFR TKI therapy. However, mutation-specific IHC is very insensitive to be used as a stand-alone assay. Also, for negative results, currently there is no algorithm that exists to test for mutations and molecular testing is still needed. In addition, no prospective clinical experience is available for validation. IHC may be an option in small low-cellularity diagnostic specimens with marginal tumor cellularity for DNA-based analysis, but again negative results do not replace mutation testing [40]. Per published guidelines for EGFR testing for selecting patients eligible for EGFR TKI, the body of published data is insufficient to make an evidence-based recommendation regarding the use of EGFR mutation-specific IHC at this time [13].

EGFR amplification or polysomy is frequently encountered in patients with lung adenocarcinomas but the response rate with EGFR TKI for these patients is well below the response rate in patients with EGFR mutations (30 vs. 68 %) [35, 36]. Amplification of mutant allele is noted commonly [19] and that is strongly associated with EGFR polysomy/amplification. This association is thought to be related to EGFR TKI response. But in cases where there is discrepancy between EGFR mutation and copy number, the mutation status is better predictor response to EGFR TKI [36]. Therefore, EGFR copy number analysis (FISH or chromogenic in situ hybridization) is not recommended for selection of EGFR TKI therapy [13].

ALK gene, located on chromosome 2p23, encodes a receptor tyrosine kinase, which belongs to the insulin receptor superfamily [41]. ALK plays an important role in the development of the brain and exerts its effects on specific neurons in the nervous system. This gene is frequently rearranged with different genes with novel fusions, mutated, or amplified in many tumors including anaplastic large-cell lymphomas, neuroblastoma, and NSCLC [41].

In 2007, Soda and co-workers reported an interstitial deletion and inversion within the short arm of chromosome arm 2 that resulted in the formation of a novel fusion gene comprising portions of the echinoderm microtubule-associated protein-like 4 (EML4) gene and the ALK gene in a subset of lung adenocarcinoma [42]. The chimeric fusion of EML4-ALK results in constitutive dimerization leading to oncogenic activity of ALK gene [43]. The original report from Soda et al. showed 7 % positive rate in NSCLC in Japanese patients (5 of 75). Subsequent studies in the USA have shown the gene fusion positive rate in about 2–7 % of all NSCLCs. This gene fusion is more frequent in adenocarcinomas in never smokers or light smokers.

Patients with tumors containing EML4-ALK mutation respond effectively with ALK kinase inhibitors alone or in combination [44, 45]. In a large series ALK rearrangement-positive (about 5 % of 1,500 NSCLC patients screened) patients treated with EGFR inhibitor, crizotinib, showed an overall response rate of 57 %, with 72 % having a PFS of 6 months or greater [46]. The US Food and Drug Administration (FDA) approved crizotinib for advanced-stage, ALK-positive lung cancer in November 2013. This targeted therapy is also recommended by guidelines from professional organizations, including the American Society of Clinical Oncology (ASCO), European Society for Medical Oncology, and National Comprehensive Cancer Network (NCCN). To that effect as described above CAP/IASLC/AMP published evidence-based recommendations for the molecular testing of lung cancers for ALK as second predictive biomarkers in a CPG in 2013 [13]

To detect all fusion products, RT-PCR has limitations as there are many fusion combinations between different exons on EML4 and ALK genes and each needs a separate primer pair. Also, rare other fusion partners with ALK (KIF5B–ALK, TFG–ALK [47, 48]) will be missed by RT-PCR as the primers are designed for specific breakpoints in different exons [49]. Thus there are concerns for a higher failure rate of an RNA-based assay in routine FFPE pathology material and RT-PCR is currently not recommended as a first-line diagnostic method for determining ALK fusion status

IHC is an attractive and cheaper way to detect ALK overexpression as most anatomic pathology departments have IHC laboratory and pathologists use IHC extensively in their routine practise. However, due to low expression of ALK protein in ALK-rearranged lung adenocarcinomas, IHC is not reliable using the same antibody clone compared to anaplastic large-cell lymphoma (mouse monoclonal anti-human CD246, clone ALK1). Recently two rabbit monoclonal anti-human ALK antibodies (clones D5F3 and D9E4) have shown high sensitivity, specificity, and reproducibility compared to ALK FISH. These reports show promising results and IHC assays may potentially facilitate the routine identification of ALK-rearranged lung adenocarcinoma [50]. The published view by CAP/IASLC/AMP based on current literature is that the data is still limited and more studies are needed to recommend developing a specific recommendation on the use of ALK IHC as a sole determinant of ALK TKI therapy [13, 124].

The commercial availability of a dual-probe “break-apart” (FISH) assay for ALK fusions/rearrangements is the most efficient and consistent way to detect ALK gene rearrangements for clinical use. This is the same ALK FISH dual probe used to detect anaplastic large-cell lymphoma [51]. This commercially available ALK FISH assay is FDA approved to be used as a companion diagnostic assay for selecting patients for crizotinib therapy.

Negative results have a yellow fusion signal or narrowly split orange/red and green signals using break-apart dual-labeled FISH assay (Fig. 8.6). The most common positive result in lung cancer will result in one separate orange/red and one separate green signal (Fig. 8.7). The separation of orange/red and green signals should be a gap larger than two signal diameters, to be indicative of an ALK gene rearrangement. The germline unaltered ALK region servers as a built-in negative control which stays as a yellow fusion signal or appears as two narrowly split orange/red and green signals. The second positive result is loss of the green 5′ probe with a remaining unpaired 3′ orange/red probe, indicating an unbalanced rearrangement (Fig. 8.8). Minimum of 50 tumor nuclei in a tumor-rich area are recommended for adequate scoring. The tumors are considered positive if 15 % or more of 50 nuclei assessed show the classic split-signal pattern or loss of the green 5′ probe with remaining red 3′ probe [51, 52]. This cutoff is part of the labeling of the FDA-approved commercial FISH assay.

KRAS mutations are seen in 15–25 % of lung adenocarcinomas and are uncommon in squamous cell carcinomas (SCC) [54]. Most mutations are point mutations producing an amino acid substitution at codons 12, 13, or 61 leading to constitutive activation of downstream signaling pathways. KRAS, EGFR, and ALK mutations are mutually exclusive in NSCLC. KRAS mutations are more frequently encountered in adenocarcinoma with mucinous morphology compared to other morphologies and in patients with history of smoking, both former/current smokers and never smokers [55, 56]. Five percent of never smokers with lung adenocarcinoma have been shown to harbor KRAS mutations [57]. In contrast to colorectal adenocarcinomas, in advanced NSCLC, the role of KRAS as a biomarker either as a prognostic or predictive marker is uncertain at this time as very few prospective randomized trials have been completed [55, 58].