Method
Sensitivity (% mutant DNA)
Application
Low sensitivity
Sanger sequencing
20%
Tumor tissue
Medium sensitivity
Pyro-sequencing
5–10%
Tumor tissue
TaqMan PCR
5–10%
Tumor tissue
SNAPSHOT
5–10%
Tumor tissue
dHPLC
5%
Tumor tissue
HRMA (melt analysis)
5%
Tumor tissue
Fragment analysis
5%
Tumor tissue
MALDI-TOF MS
5%
Tumor tissue
High sensitivity
Scorpion ARMS
1%
Tumor tissue/CTC/CF-DNA
PNA/LNA clamp
0.1–1%
Tumor tissue/CTC/CF-DNA
Ultra-high sensitivity
BEAMing
0.01%
CTC/CF-DNA
Digital PCR
0.01%
CTC/CF-DNA
Digital Droplet PCR
Digital PCR offers an alternate methodology to that of traditional real-time PCR for lung cancer biomarker testing. Like RT-PCR, digital droplet PCR offers ultrahigh sensitivity and precision for lung cancer mutation screening (Table 7.1) [18, 19]. This method involves partitioning DNA into numerous individual or parallel PCR reactions (droplets). The result of this process is a decrease in the amount of competing DNA for amplification and the ability to quantify lung cancer mutations (such as EGFR or KRAS) at the single-molecule level. Individual droplets with more than one copy of a target molecule can be identified using a Poisson model for exclusion from analysis. Applications include detecting copy number variation and identification of low abundant mutations in EGFR (including T790 M mutation) in pretreatment biopsy samples [20]. Advantages include no need for standards or references, scalable assay precision (increase number of droplets), linear detection of small fold changes, and simple workflow. A major disadvantage is the requirement of dedicated instrumentation.
nCOUNTER
Methods discussed above focused primarily on mutation detection including substitution and small in/dels which are applicable for EGFR and KRAS testing in NSCLC . However, molecular methods available for detecting a single or multiple gene translocation events are limited. Florescent in situ hybridization (FISH), immunohistochemistry (IHC), and next-generation sequencing (NGS) can be applied to detect translocations; however, each assay has its own strengths and weakness. A molecular assay for simultaneous detection of multiple gene translocations (ALK, RET, and ROS1 in NSCLC) has been previously demonstrated using the nCOUNTER system (NanoString) [21]. The nCOUNTER multiplex assay utilizes detection of gene expression differences in known translocations based on non-amplified mapping of mRNA transcripts. The basic idea is in the event of a translocation, there will be an imbalance of probes in the 5′ and 3′ end of the translocated gene. The advantages include the ability to utilize formalin-fixed paraffin-embedded tissue, no amplification steps (no PCR artifacts), low hands on time, and a high level of multiplexing for simultaneous detection of all relevant NSCLC clinical translocations. Disadvantages include dependence on high-quality RNA extraction from FFPE and dedicated instrumentation.
Minimal Invasive Lung Biomarker Testing
Recently there has been a large emphasis and excitement surrounding the detection of actionable variants utilizing nucleic acid isolation following a blood draw (i.e., liquid biopsy). This has taken the form of detecting cell-free DNA (CF-DNA ) and/or isolated circulating tumor cells (CTC ) . Both methods represent an important evolution in testing but push the limitations of assay performance, as clinical lung cancer biomarker assays need to possess both an exceptionally high sensitivity and specificity in order to limit false positive/negative interpretations. CTC methods require a CTC capture step which is most commonly performed using an antibody-mediated capture procedure (such as anti-EMT capture). This form of capture has inherent bias due to the inefficiency of capture which influences downstream molecular biomarker testing. CF-DNA methods are hindered by collection of low-quantity tumor-specific DNA released in circulation and stability of free nucleic acids in circulation. Overcoming both CF-DNA and CTC limitations requires a downstream ultrahigh sensitive molecular methodology. Mutation screening methods for EGFR and KRAS have been successfully implemented in plasma using PNA-LNA clamping and digital PCR (Table 7.1) [22–25]. While these assays offer ultrahigh sensitivity in mutation detection, their utility in comparison to standard tumor biopsy-based methodologies is still an active area of investigation.
Biomarkers Utilized for Lung Adenocarcinoma
KRAS (Mutation; Frequency ~25%)
Kirsten rat sarcoma ( KRAS ) viral oncogene homolog is a member of the guanosine triphosphate (GTP)-binding protein family and is responsible for pro-survival signaling. KRAS represents the most commonly mutated gene in NSCLC adenocarcinomas [26]. Mutations cluster at hotspots in codons 12, 13, and 61 [27]. Mutations include substitutions, insertions, and deletions. Mutations in KRAS are most frequently observed in smokers; however, incidence in nonsmokers approaches 15%, and therefore smoking history provides a poor estimation of KRAS mutation status [28–30]. KRAS mutations result in stimulus-free and constitutively active signaling, which results in cell proliferation and survival. Furthermore, KRAS mutations are nearly mutually exclusive from EGFR mutations or ALK translocations [31]. Currently, there is no direct targeted therapy for KRAS-mutated NSCLC. However, MEK inhibition (selumetinib and trametinib) is under investigation as a strategy to target the downstream aberrant signaling in KRAS-mutated NSCLC. In addition to MEK inhibition, CDK4/CDK6 inhibition also shows antitumor activity in KRAS-mutated NSCLC [32]. Likely improved drug targeting will include combination therapy such as MEK or CDK4/CDK6 in combination with AKT inhibitors (MK-2206) to circumvent resistance mechanisms [33].
EGFR (Mutation; Frequency ~15%)
Epidermal growth factor receptor ( EGFR ) , also known as HER1/ERBB1 , represents one of the most highly investigated and currently utilized biomarkers in NSCLC. Presence of an EGFR activating mutation serves as a predictor of response to tyrosine kinase inhibitors (TKI) including erlotinib, gefitinib, and afatinib [34–36]. EGFR mutations most commonly occur in nonsmokers and women of Asian descent. While TKIs show clinical benefit (improved response rate and progression-free survival) [12, 15, 37], the likelihood of response is relative to location of the mutation site. EGFR mutations in exons 18–21 (tyrosine kinase domain of EGFR) are associated with sensitivity to TKIs [34, 35]. Within these exons, short in frame deletion amino acids (747–750) and a point mutation (L858R) account for approximately 90% of activating EGFR mutations [34, 35, 38]. One key clinical mutation for screening includes the T790 M mutation in exon 20 which is estimated to confer resistance to first-generation TKIs in approximately 50% of cases [39].
BRAF (Mutation; Frequency ~4%)
The v-Raf murine sarcoma viral oncogene homolog B ( BRAF ) protein is a serine/threonine protein kinase. Mutations in BRAF, particularly the glutamate substitution mutation at codon 600 (V600E), are common in disease types such as papillary thyroid carcinoma and melanoma where they are observed in approximately 50% of cases [40–42]. However, in NSCLC BRAF mutations are relatively uncommon (approximately 3–4%) [43, 44]. In addition, only about half of the mutations in NSCLC are isolated to the single amino acid V600. While BRAF mutations can be targeted with inhibitors (vemurafenib and dabrafenib), the low frequency and nonuniform mutation profile in NSCLC dilutes the attractiveness of BRAF as a routine predictive biomarker.
MET (Mutation; Frequency ~3%)
The mesenchymal-to-epithelial transition (MET) gene was discovered in the late 1980s and encodes a RTK [45]. The MET-RTK regulates a multitude of cell processes including cell scattering, invasion, anti-apoptosis, and angiogenesis [46]. Aberrant MET signaling has been identified in multiple human malignancies [46–48]. Likewise, variations in MET signaling can occur due to increased ligand (hepatocyte growth factor, HGF) binding, mutation, amplification, or increased proteolytic degradation. MET overexpression is commonly seen in NSCLC (25–75% depending on antibody and cutoff criteria) and has been linked to poor clinical outcome [49, 50]. MET alterations in NSCLC also include mutations resulting in exon 14 skipping, which increase MET signaling and promote oncogenesis [51, 52]. These mutations are most common in smokers and older patients. Therapy options include multikinase TKIs [53–56]. However, alternative therapies under evaluation include targeted MET-TKI inhibitor (tivantinib) and monoclonal antibody (onartuzumab) [57–59].
ERBB2 (HER2) (Mutation; Frequency ~2–4%)
Human epidermal growth factor receptor 2 (HER2) belongs to a family of membrane receptors known as the erbB family. HER2, also known as ERBB2, functions by homo- and hetero-dimerization with erbB family members. Dimerization results in signaling via multiple pathways including PI3K, MAPK, and JAK/STAT [60]. ERBB2 represents a widely utilized biomarker in breast cancer due to amplification being present in 15–25% cases. However, in NSCLC, the frequency of ERBB2 mutation is low (2–4% exon 20 in frame insertion). ERBB2 mutations in NSCLC occur most commonly in nonsmokers, females, and Asian ethnicity [61]. ERBB2 amplification is observed more frequently than mutation in NSCLC, with an incidence of approximately 20% [62, 63]. Clinical response to anti-HER2-directed therapy (trastuzumab, neratinib, afatinib, and lapatinib) in NSCLC with HER2 overexpression has shown limited therapeutic efficacy [64–69].
ALK (Translocation; Frequency ~5%)
Anaplastic lymphoma kinase (ALK) gene encodes a receptor tyrosine kinase with high sequence similarity to the insulin receptor [70]. Translocations involving ALK and EML4 in NSCLC were first described in 2007 [71]. The most common gene rearrangement in NSCLC is EML4-ALK at a frequency of 4–5% and is most commonly observed in young women with a history of light smoking or never-smoking [26]. Subsequently, nearly 30 ALK translocation partners have been identified [70]. Therapy options include multikinase TKIs (crizotinib, ceritinib, and alectinib) [72–76]. Resistance occurs in the majority of cases treated with first-line crizotinib. New second-generation TKIs including ceritinib are being investigated in patients that progress or are intolerant of crizotinib [75].
RET (Translocation; Frequency 1–2%)
The RET gene encodes a RTK that is essential for normal cell development and maturation. RET translocations have clinical significance in a variety of human malignancies including papillary thyroid carcinoma (20–40%) and NSCLC (1–2%) [77–79]. Like ALK translocations in NSCLC, RET translocations occur most frequently in young females with a history of light smoking or nonsmoking. In NSCLC RET has six identified translocation partners (KIF5B, CCDC6, NCOA4, TRIM33, CUX1, and KIAA1468) [80]. The most frequent RET translocation partners include KIF5B and CCDC6. In in vitro models, several TKI’s (sunitinib, sorafenib, vandetanib, and cabozantinib) have shown efficacy in blocking RET translocation signaling [81]. However, clinical trials evaluating human therapeutic efficacy are ongoing (NCT01823068, NCT01639508, NCT01866410, and NCT01708954).
ROS1 (Translocation; Frequency 1–2%)
The ROS1 gene encodes a RTK that is involved in pro-survival and anti-apoptotic signaling through multiple pathways [82]. In NSCLC, ROS1 translocations occur with a variety of gene partners including but not limited to CD74, EZR, SDC4, and TPM3 [82]. ROS1 translocations result in neoplastic transformation both in vitro and in vivo. Translocations occur in multiple malignancies but are most prevalent in papillary thyroid carcinoma (approximately 40%) [83]. ROS1 translocations are also observed in NSCLC adenocarcinomas at a frequency of 1–2%, but do not represent a clinically actionable finding [84, 85]. The patient population that harbors ROS1 translocations is similar to ALK translocations and includes young nonsmokers or light smokers [84]. Early preclinical evaluation of ROS1 translocation-positive NSCLC showed strong sensitivity to crizotinib [83]. Recently NCCN guidelines added ROS1 to include testing in all NSCLCs similar to ALK [86].
Biomarkers Utilized for Lung Squamous Cell Carcinoma
FGFR1 (Amplification; Frequency ~17–20%)
The fibroblast growth factor receptor 1 (FGFR1) is a RTK that regulates proliferation via MAPK and PI3K signaling pathways. FGFR1 amplification is the most common alteration seen in squamous cell carcinoma (20%) [87, 88]. FGFR1 amplification has oncogenic potential as seen in NSCLC cell lines, which show sensitivity to RTK targeted therapy [87]. Clinical-based RTKs targeted therapies including dovitinib and nintedanib are currently ongoing (NCT01861197 and NCT01948141) [89, 90].
PIK3CA (Mutation; Frequency ~16%)
There are three classes of phosphatidylinositol-3 kinases, Class 1A PI3Ks (PIK3CA ) are the most relevant to human cancer [91]. PIK3CA is responsible for production of phosphatidylinositol-3,4,5-trisphosphate which activates the AKT/mTOR pathway [92]. This pathway is essential for cell growth, survival, and motility. Amplification of PIK3CA is more common in NSCLC squamous cell carcinoma than adenocarcinoma (33% vs. 6%, respectively) [93]. Mutations are also more prevalent in squamous cell carcinoma and occur at approximately 2–5% of cases [93–95]. Unlike most mutations in NSCLC, PIK3CA mutations can occur in conjunction with other genes and is therefore not always mutually exclusive [96]. Furthermore, PIK3CA mutations have been implicated as a resistance method to EGFR-based TKI therapy [97].
PTEN (Mutation; Frequency ~10–15%)
PTEN inhibits the PI3K/AKT/mTOR signaling cascade through dephosphorylating PI-(3,4,5)-triphosphate [98]. Inactivation of PTEN removes pathway inhibition and therefore leads to nonrestricted activation of AKT. Mutations are almost exclusively seen in squamous cell carcinoma (approximately 10–15%), but are rarely observed in adenocarcinoma (approximately 1–2%) [99]. Current targeted therapy under evaluation includes AKT and mTOR inhibitors (MK-2206 and ridaforolimus) [100, 101].
FGFR2/FGFR3 (Mutation and Translocation; Frequency ~6%)
Fibroblast growth factor receptors 2 and 3 (FGFR2 and FGFR3 ) are RTKs that play essential roles in cell proliferation, differentiation, angiogenesis, and development. Activation of FGFR2/FGFR3 results in downstream activation of Ras/MAPK and PI3K/AKT [102]. FGFR2 and FGFR3 mutations are most commonly observed in endometrial carcinoma (~12%) and urothelial carcinoma (~30%) [87, 103–105]. It is worth noting that the cancer genome atlas data first reported FGFR2/FGFR3 somatic mutations in NSCLC [26]. Targeted therapies for FGFR2/FGFR3 under investigation include multi-RTK inhibitors (sunitinib, sorafenib, pazopanib, and vandetanib). More recently, to address side effects and low efficacy of multi-TKIs, new selective and highly potent FGFR TKIs are under evaluation (AZD4547, BGJ-398, and JNJ42756493 [106].
DDR2 (Mutation; Frequency ~2–4%)
Discoidin domain receptor 2 ( DDR2 ) is a RTK involved with tissue repair and tumor progression [107]. DDR2 mutation rate in squamous cell carcinoma is approximately 2–4% [108, 109]. Early clinical evidence has demonstrated potential therapeutic effectiveness with dasatinib [110, 111] and clinical trials are in progress (NCT011514864).
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