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Lung cancer is the leading cause of cancer-associated mortality worldwide for both men and women. Non-small cell lung carcinoma (NSCLC) encompasses a heterogeneous group of lung carcinomas including adenocarcinoma, squamous cell carcinoma, and large cell carcinoma, the 3 most common types. Each type has many morphologic subtypes and some lung adenocarcinoma subtypes have been associated with different molecular abnormalities. NSCLC comprises 75% to 80% of all lung cancers and the currently available conventional chemotherapeutic regimens have done relatively little to improve outcomes in NSCLC patients over the past decades. However, several recently introduced agents show promising results, with improved survival of patients who have appropriate molecular targets in their tumors.
Targeted inhibitors have proven to be remarkably successful against epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) in patients with certain EGFR mutations and ALK rearrangements, and this has opened a new era of targeted therapy in advanced NSCLC. Other molecular changes have been found via recent genomic studies in NSCLC patients, including gene rearrangements of ROS1 and RET, amplification of MET, and activating mutations in the BRAF, HER2, and KRAS genes. NSCLCs harboring these genetic changes can potentially be treated with agents approved for other extrapulmonary cancers or with agents currently undergoing clinical trials. These targetable oncogenes are primarily identified in lung adenocarcinomas (Figure 1) and possibly in some large cell carcinomas, but are essentially absent in squamous cell carcinomas.1 Several targetable genes in squamous cell carcinomas of the lung also have been recognized but will not be covered in this article.
According to guidelines from the College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology (CAP/IASLC/AMP), molecular analysis for EGFR mutations and ALK gene rearrangements should be considered upon diagnosis of all advanced-stage lung adenocarcinomas.2 The guidelines also state that testing for EGFR mutations and ALK gene rearrangements is acceptable for some large cell carcinomas and adenosquamous carcinomas (having the features of both adenocarcinoma and squamous cell carcinoma), but not on pure squamous cell carcinomas.2 Patients’ gender, age, smoking status, and histologic grade or subtype of adenocarcinomas are not completely sensitive or specific for predicting any molecular changes and, thus, cannot be used as criteria for selecting patients for biomarker testing.
Both quantity and quality of tumor tissue are critical for successful genomic testing, and an emphasis must be placed on obtaining adequate tumor material at the time of diagnostic sampling. In patients with advanced-stage NSCLC, treatment decisions frequently rely on small biopsies or cytology specimens such as pleural fluid or fine-needle aspirates. Biopsies with small (18- to 20-gauge) core needles or cell blocks from any cytology specimen can yield sufficient and reliable samples for genomic testing. Some studies have demonstrated that cytology smear or cytospin specimens can be used for both mutational analysis and fluorescence in situ hybridization (FISH), though further validation studies are needed before recommending the routine use of the cells from smear or cytospin preparations. Generally, a sample with 300 to 500 tumor cells is sufficient for DNA sequencing. Though 50% tumor content is ideal, more sensitive tests detecting mutations in specimens with as little as 10% cancer cells are used in many laboratories. The bare minimum of 50 analyzable cells is needed for FISH though 100 or more tumor cells is ideal. Most molecular assays have been optimized and validated on tissues fixed in 10% neutral buffered formalin or alcohol (70% ethanol). Samples processed with heavy metal fixatives (such as Zenker’s and B5) or acids (such as Bouin’s solution) cannot be used for molecular testing. While decalcified specimens are not ideal for molecular testing, light decalcifications for less than 30 minutes may still be amenable for molecular evaluation and, thus, close scrutiny of the decalcification conditions among individual cases may allow for salvage of a usable specimen that otherwise may be rejected.
The role of the pathologist is crucial because he or she is in the central position to ensure the adequacy of the specimen for testing and to communicate with the clinicians and laboratory personnel. Turnaround time for genomic testing must be reasonably short to allow timely clinical decisions based on the prerequisite results of molecular analysis. CAP/IASLC/AMP guidelines recommend that EGFR and ALK testing be completed within 10 working days after receiving the specimen in the testing laboratory.2 Given the fast-paced changes in molecular testing fields, pathologists and laboratories must be ready to incorporate the emerging new techniques and platforms such as next-generation sequencing, which may offer much more rapid and comprehensive information on actionable oncogenic alterations and targeted treatment options.
Epidermal Growth Factor Receptor Gene (EGFR)
EGFR gene mutations are present in 10% to 15% of lung adenocarcinoma patients of European background and are more common in those who have never smoked, in women, and in patients of Asian descent. Activating somatic mutations in exons 18–21 of the tyrosine kinase (TK) domain of EGFR is recognized as the predictor of responsiveness to small molecule EGFR tyrosine kinase inhibitors (TKI). Therefore, the new guidelines from CAP/IASLC/AMP2 recommend all patients with lung adenocarcinoma in advanced stage be tested for those genetic abnormalities that indicate suitability for treatment with targeted agents, specifically EGFR inhibitors. The most common mutations include in-frame deletions in exon 19 and L858R point mutations in exon 21, which account for 90% of all EGFR mutations.3 Many less common mutations are associated with response and, therefore, EGFR mutation analysis should be designed to capture all mutations reported to be predictors of response to EGFR TKIs.2 Multiple test platforms for EGFR mutation testing are available and each laboratory should establish the test with acceptable performance during internal validation.
After the initial response to EGFR inhibitors in patients with EGFR-mutant NSCLC, the vast majority of these patients will develop resistance to the EGFR TKIs. Strategies for overcoming and preventing acquired resistance to EGFR TKIs have been the subject of many studies. The most common mechanism of resistance is emergence of the EGFR T790M mutation.4 The T790M mutation is rarely found in pretreatment specimens, but has been identified as a germ line mutation in cases of familial lung cancer.5 Clinical trials targeting this mutation are underway, and laboratories should employ assays of sufficient sensitivity to detect the T790M mutation in as few as 5% of tumor cells.2 Other important mechanisms of resistance include MET amplification or polysomy, HER2 amplification, transformation to small cell carcinoma, MAPK1 amplification, and PIK3CA mutation.6 In about 15% of patients, the mechanism for resistance to EGFR TKIs is unknown.
FISH/chromogenic in situ hybridization (CISH) is less predictive of the response to EGFR TKIs than mutation testing and, therefore, is not recommended in patient selection for treatment at this time.2 When the available specimen is insufficient for molecular analysis, EGFR immunohistochemical (IHC) testing might be an option and be potentially informative if positive for mutation-specific rabbit monoclonal antibodies detecting the 2 most common mutated EGFR proteins (EGFR E746-A750del and EGFR L858R). These antibodies, especially mutant-specific antibody against L858R mutation, offer clinically acceptable performance, with 95% sensitivity and 99% positive predictive value. However, the coverage by these 2 antibodies is incomplete and negative IHC results do not exclude the presence of sensitizing EGFR mutations.
Anaplastic Lymphoma Kinase Gene (ALK)
The ALK gene encodes a receptor tyrosine kinase that is normally expressed only in select neuronal cell types, but not in any lung cells. ALK can be constitutively activated by a translocation that results in a fusion with other genes such as echinoderm microtubule-associated protein-like 4 (EML4). The EML4-ALK fusion gene is a recently identified oncogenic driver that is found in approximately 3% to 5% of nonselected NSCLC patients.7 Several studies have reported the demographics, smoking status, and other clinical features associated with ALK gene rearrangement. Previous studies have reported that EML4-ALK mutant tumors are associated with younger age, never smoked status, advanced clinical stage, a solid and/or signet ring cell histology, and a higher risk of brain and liver metastases.8
Crizotinib, an ALK inhibitor, was approved by the United States Food and Drug Administration (FDA) in August 2011 for locally advanced or metastatic NSCLC having ALK gene rearrangement. The phase I and II clinical trials conducted with crizotinib used a dual-color break-apart FISH assay (Vysis, Abbott Molecular) to detect ALK gene rearrangement. Accordingly, the FDA approved crizotinib along with a companion diagnostic FISH test using the Vysis ALK break-apart FISH probe kit to detect ALK-positive cells.
Although testing for ALK gene rearrangement using break-apart ALK FISH probes is generally recognized as a standard diagnostic criterion for ALK TKIs therapy, the screening algorithm for detection of ALK-rearranged lung cancer is still under development. Groups in all parts of the world are conducting studies to develop national or regional guidelines. Most studies focus on the standardization of both IHC and FISH (Figure 2) tests as well as evaluation of the sensitivity and specificity of IHC as a screening tool. As results from these studies are published, global consensus on an ALK screening algorithm likely may evolve within the next few years. The 2013 CAP/IASLC/AMP guidelines include ALK IHC as a screening methodology to select specimens for ALK FISH if carefully validated.2 Real-time polymerase chain reaction (RT-PCR), however, was not recommended as an alternative to FISH for selecting patients for ALK inhibitor therapy.2
Similar to EGFR TKIs, acquired resistance ultimately limits the clinical benefit of crizotinib. Clinical trials of second-generation ALK inhibitors are ongoing and the strategies aimed at overcoming and preventing the emergence of resistance in ALK-positive NSCLC are being explored.4 Currently, testing for secondary mutations in ALK associated with acquired resistance to ALK inhibitors has not been required for clinical management.2 However, laboratories should be prepared to cover these secondary mutations with newer, comprehensive techniques such as next-generation sequencing.
c-ros Oncogene 1, Receptor Tyrosine Kinase Gene (ROS1)
Chromosomal rearrangements involving ROS1, which encodes a receptor tyrosine kinase with homology to the insulin receptor, were originally described in glioblastomas before their identification in NSCLC, in which several ROS1 fusion partners have since been reported. These ROS1 rearrangements lead to constitutive kinase activity. Approximately 1% to 2% of lung adenocarcinomas harbor ROS1 rearrangements according to a study using a break-apart FISH assay.9 (Figure 3) In one study, ROS1 rearrangements were found to be more common in never or light smokers and were associated with younger age at diagnosis—a clinical profile similar to that of patients with ALK-rearranged NSCLC.9 In vitro, crizotinib inhibits the growth of ROS1-positive lung cancers at achievable serum concentrations. Among 14 ROS1-positive patients treated with crizotinib, the response rate was 57%, remarkably similar to the rate seen in ALK-positive patients treated with crizotinib.9 ROS1-specific kinase inhibitors are also in clinical development. The identification of ROS1-rearranged NSCLC is expected to build on the ALK model for rapid validation of a biomarker that will likely impact the diagnosis and treatment of lung cancer. The current National Comprehensive Cancer Network (NCCN) guidelines suggest crizotinib therapy in patients with advanced NSCLC bearing a ROS1 rearrangement.10
v-Raf Murine Sarcoma Viral Oncogene Homolog B1 (BRAF)
BRAF is a serine/threonine kinase that lies downstream of RAS in the RAS/RAF/MEK/ERK signaling pathway. Mutations in BRAF are seen in approximately one-half of melanomas in which BRAF V600E is a driver mutation that can be effectively targeted with selective BRAF and/or MEK inhibitors. BRAF mutations are also detected in 2% to 4% of NSCLCs.11 The BRAF mutations found in lung cancers appear distinct from the melanoma setting in that lung adenocarcinomas harbor non-V600E mutations in 40% to 50% of cases whereas BRAF-mutated melanomas harbor a V600E mutation in more than 80% of cases.11 Many non-V600E mutations show only intermediate or low kinase activity, which casts some doubt as to their status as driver events. Two recent studies suggested that non-V600E BRAF mutations occur almost exclusively in smokers, while one group found that BRAF V600E was more common in those who have never smoked and in women.11 Because of the predominance of BRAF V600E mutations in melanoma, drugs targeting BRAF, including vemurafenib and dabrafenib, are designed to have specific activity against the V600E-mutant BRAF kinase. Preliminary results from an ongoing phase II trial of dabrafenib described the efficacy in 8 of 20 evaluable patients with BRAF V600E-mutant NSCLC. However, preclinical data suggest that non-V600E- mutant BRAF kinases are resistant to BRAF inhibitors.
Human Epidermal Growth Factor Receptor 2 Gene (HER2)
HER2 is a member of the HER family of receptors that is activated in 25% to 30% of breast cancers by focal genomic amplification. HER2-amplified breast cancer can be effectively treated with the anti-HER2 monoclonal antibody trastuzumab and the HER2 TKI lapatinib. HER2 gene amplification detected by FISH using criteria for amplification in breast cancer is present in approximately 2% of NSCLCs. Data are incomplete regarding the efficacy of trastuzumab or other HER2-targeted agents in a population with this more rigorous definition of HER2 amplification.12 HER2 is also activated by exon 20 in-frame insertion mutations in approximately 2% of lung adenocarcinomas; these mutations are not seen in breast cancer. Several TKIs are being tested in HER2-dependent lung adenocarcinomas and some in phase I or II clinical trials have shown promising results.
MNNG-HOS Transforming Gene (MET)
The MET protooncogene encodes a transmembrane tyrosine kinase receptor that activates downstream signaling molecules involved in cell proliferation, survival, motility, and invasion. A previous study reported rapid and durable clinical response in a patient with NSCLC with de novo MET amplification and no rearrangement of the ALK gene who was enrolled in the MET-enriched cohort of the ongoing phase I study of crizotinib.This suggests that primary MET amplification may be an oncogenic driver in a subset of NSCLCs and a potential therapeutic target.13 Amplification of the MET gene has been associated with the development of resistance to gefitinib or erlotinib in approximately 5% of patients with sensitizing EGFR mutations who were initially responsive to the drug. Many ongoing preclinical studies and clinical trials are assessing the potential effects of small molecule inhibitors that target the MET pathway. FISH for MET amplification and IHC for MET overexpression are important for selecting patients and are under active development.
Rearranged During Transfection Gene (RET)
The RET protooncogene encodes a tyrosine-kinase receptor that is involved in cell proliferation, migration, differentiation, and neuronal navigation. Germ line and somatic gain-of-function RET mutations are known to predispose to multiple endocrine neoplasia type 2 and sporadic medullary thyroid carcinoma, respectively, whereas somatic RET gene fusions account for the majority of radiation-induced and sporadic papillary thyroid cancers. In 2011, a fusion gene involving RET partnered with KIF5B was discovered in a young patient with lung adenocarcinoma who had never smoked.14 KIF5B-RET is the predominant RET fusion in NSCLC and found in approximately 1% to 2% of lung adenocarcinoma or adenosquamous carcinoma patients. (Figure 4) Patients with RET-positive NSCLC tended to be younger and to have never smoked, sharing similar features with ALK- and ROS1-positive patients. KIF5B-RET and other RET fusions induce constitutive activation of the oncoprotein and the cells expressing KIF5B-RET are sensitive to RET inhibitors vandetanib, sorafenib, and sunitinib. Cabozantinib, a multi-TKI and more potent inhibitor of RET, is approved by the FDA to treat advanced medullary thyroid cancer and is being tested in a phase II trial for RET fusion-positive NSCLC with promising results.
Kirsten Rat Sarcoma Viral Oncogene Homolog Gene (KRAS)
KRAS, a RAS family gene, is the most frequently activated gene known in lung adenocarcinomas (about 20%). Most KRAS mutations are single amino acid substitutions in codons 12, 13, or 61. These mutations render KRAS constitutively active, leading to activation of downstream effectors, including the RAF/MEK/ERK and PI3K/AKT/mTOR signaling cascades. KRAS mutations are more common in smokers. A randomized phase II study comparing selumetinib, an oral MEK1/MEK2 inhibitor, in combination with docetaxel and with placebo has shown promising results in some patients with advanced NSCLCs bearing KRAS mutations. Several other agents in ongoing clinical trials are targeting KRAS or downstream pathways.
Mayo Medical Laboratories offers EGFR analysis (EGFRX / Lung Cancer, EGFR with ALK Reflex, Tumor) to identify non-small cell lung cancers that may benefit from treatment with EGFR- or ALK-TKIs. Drug-resistant EGFR mutations are also identified by this test. If no mutations are identified in the 29 mutation panel, ALK (2p23) FISH testing (FLCA / Lung Cancer, ALK (2p23) Rearrangement, FISH, Tissue) will be performed. EGFR IHC for 2 classes of mutants (exon 19 deletions and exon 21 L858R mutation) is available for cases with insufficient tissue for molecular testing. Analysis for ALK IHC, KRAS and BRAF mutations, and FISH testing for ROS1 and RET rearrangements are also available. See the “Utilization Spotlight” section within this issue of the Communique for complete test information and appropriate test utilization.
Biomarker testing is a crucial step in selecting patients for targeted therapy in NSCLC. Currently, there are FDA-approved TKIs effective in lung cancer patients with certain EGFR mutations or ALK translocations. There are many more promising agents targeting other driver oncogenes of NSCLCs at various phases of clinical trials. Sensitive and specific tests are available to detect the biomarkers associated with responsiveness to these new drugs. The recent advances in molecular techniques might revolutionize the current practice and offer more comprehensive information at a significantly shorter turnaround time and potentially at a lower cost.
Authored by: Joanne E Yi, MD