Lung Cancer Genotype-Based Therapy and Predictive Biomarkers: Present and Future

Epidermal growth factor receptor (EGFR) is a member of the HER/ErbB family of cell surface receptor tyrosine kinases that controls intracellular signaling pathways that regulate cell proliferation and apoptosis.^{3,35–40,45,46}  During the first several years of the 21st century, a first generation of oral, selective, reversible EGFR tyrosine kinase inhibitors (TKIs), gefitinib (Iressa; AstraZeneca, London, United Kingdom) and erlotinib (Tarceva; Genentech, South San Francisco, California, and OSI Pharmaceuticals, Long Island, New York), were investigated in clinical trials of patients with advanced NSCLC.^47–57  Beginning in 2004, somatic mutations in the EGFR gene were identified as driver mutations causing oncogene addiction of a percentage of NSCLCs, making them likely to respond to EGFR TKI therapy.^58–60 

Therefore, this first generation of EGFR TKIs were found to be of limited value in treating unselected NSCLC patients in early clinical trials but had a significant response rate (RR), improved median progression-free survival (PFS), and improved overall survival in NSCLC patients with activating EGFR mutations.^61–69  In 2009, Tony Mok and colleagues^70,71  reported results of the IRESSA Pan-Asia Study, which demonstrated that patients with EGFR mutation–positive NSCLC had a better RR and PFS with gefitinib therapy compared with conventional chemotherapy. In this clinical trial, patients whose NSCLC lacked EGFR mutations had better RR and PFS with conventional chemotherapy than with gefitinib therapy. In 2010, 2 other clinical trials, WJTOG3405⁷²  and NEJ002,⁷³  confirmed better RR and PFS with gefitinib compared with conventional chemotherapy in patients with EGFR mutation–positive NSCLC. Clinical trials of erlotinib versus conventional chemotherapy in patients with EGFR mutation–positive NSCLC such as the OPTIMAL study⁷⁴  and EURTAC study⁷⁵  have found similar results for erlotinib.

The first-generation EGFR TKIs have been found to be useful as first-line, second-line, or subsequent-line therapies in advanced NSCLC with activating EGFR mutations.^69,76–86  No overall survival advantage for EGFR TKIs over conventional chemotherapy was demonstrated in these clinical trials, but presumably this is because of patient crossover to EGFR TKI therapy during the clinical trials.⁶⁹  Whether or not patients with early-stage NSCLC, those that might be potentially amenable to surgical resection but often later die from their cancer, will benefit from EGFR TKI therapy remains to be elucidated.^87,88  A major issue with first-generation reversible EGFR TKI therapy is the eventual development of resistance to the drugs and relapse of the cancer in patients who initially respond to the drugs.^89–91  This is explored in greater detail below.

The observation that activating EGFR mutations are a predictive biomarker for response to EGFR TKI therapy introduced a new role for pathologists in precision medicine of lung cancer patients. Two mutations account for 90% of the activating EGFR mutations, short in-frame mutations in exon 19 and the L858R point mutation in exon 21, but there are a number of less frequent EGFR mutations that are also clinically relevant.^† Multiplex testing allows simultaneous detection of multiple EGFR mutations, not just the major 2.

The frequency of EGFR mutations in lung cancers ranges up to 32% in East Asians, ranges from 7% to 15% in Caucasians, and occurs in about 2% of African Americans.^94–96  It is estimated that there are 30 000 new cases of EGFR mutation–positive NSCLC in the United States each year.^1,69,97  Activating EGFR mutations are more common in NSCLCs from women than men, from never smokers than former or current smokers, and from Asian than other ethnic groups. The frequency of EGFR mutations in NSCLC in East Asian women who have never smoked is very high, as high as 50%.^‡ However, although the demographic associations may suggest the likelihood of EGFR mutations in tumors from those patients who fit the demographic profile, use of these criteria alone would exclude too many patients who do not meet these demographic criteria, but might benefit from EGFR TKI therapy.^34,98 

The best criterion for selecting NSCLCs that should be sent for EGFR mutation testing is cell type, a diagnosis that is dependent upon the pathologist. Throughout the literature, EGFR mutations are mostly detected in adenocarcinomas.^§ Based on recent studies, it seems likely that many EGFR mutation–positive lung cancers given a nonadenocarcinoma diagnosis in older studies may have been misdiagnosed adenocarcinomas. As already discussed, for poorly differentiated NSCLC, additional studies such as immunohistochemistry may be needed to make an accurate diagnosis of cell type.^8,17,21–24 Many tumors previously called large cell carcinomas are now known to be specific cell types, mostly poorly differentiated adenocarcinomas. Also, it may be difficult to differentiate a solid-pattern adenocarcinoma from a squamous cell carcinoma based on routine histology. Even with special studies, a few lung tumors cannot be classified as a specific cell type.^8,17,21–24 In addition, adenosquamous carcinomas make up about 1% of lung cancers and have at least a 10% adenocarcinoma component and at least a 10% squamous cell carcinoma component. These rare tumors may have EGFR mutations and respond to EGFR TKI therapy.¹⁰⁰ 

Although EGFR mutations have been reported in less than 1% of squamous cell carcinomas, it is possible that these are also misdiagnosed adenocarcinomas. Rekhtman et al¹⁰¹  looked at this issue in a large series of lung cancers. Upon further workup of 16 tumors initially diagnosed as squamous cell carcinomas that had EGFR/KRAS mutations, they found that 10 (63%) were adenosquamous carcinomas and 5 (31%) were poorly differentiated adenocarcinomas that morphologically mimicked squamous cell carcinomas (adenocarcinomas with squamoid morphology); 1 case (6%) had no follow-up. Although it is likely that some investigators will continue to report EGFR mutations in NSCLCs other than adenocarcinoma, there is no doubt that the association between adenocarcinoma cell type and EGFR mutations is very strong and that misdiagnosed adenocarcinomas should be ruled out before acknowledging cell type exceptions.

Patients who initially respond to first-generation EGFR TKIs eventually develop resistance to the drug and relapse while still under EGFR TKI therapy.^89–91  A clinical definition of acquired resistance to EGFR TKI therapy is used in many clinical trials. The criteria consist of previous treatment with a single-agent EGFR TKI; either or both of lung cancer with an EGFR mutation associated with TKI sensitivity or objective clinical benefit from treatment with an EGFR TKI; systemic progression of disease while on continuous treatment with gefitinib or erlotinib within the last 30 days; and no intervening systemic therapy between cessation of gefitinib or erlotinib and initiation of new therapy.⁹⁰  Resistance to EGFR TKIs can be acquired by several mechanisms, most notably secondary mutations in the EGFR gene. The lung cancer may develop an EGFR T790M mutation, which causes about 50% of cases of acquired resistance to first-generation reversible EGFR TKIs.^{89,91,102–105}  It should be noted that T790M mutations may develop in lung cancers that have not been treated with EGFR TKIs. Other EGFR mutations, such as T854A, D761Y, and L747S, have also been reported to cause acquired resistance to first-generation EGFR TKIs.^102,104,106  Secondary overexpression and/or amplification of the receptor tyrosine kinase c-MET or its ligand, hepatocyte growth factor, is associated with about 18% of cases of acquired resistance to EGFR TKIs by activating the HER3/ERBB3 pathway or causing secondary KRAS activation.^{103,107–110}  Less frequent causes of acquired resistance to EGFR TKIs include acquired mutations of phosphatidylinositol-3-kinase (PI3K), transformation to small cell lung cancer, epithelial to mesenchymal transition, and KRAS mutations.^69.89 

EGFR mutations predict response to EGFR TKI therapy and, therefore, EGFR mutation testing is the basis for selecting patients for EGFR therapy. There are multiple EGFR mutation assays available, but because tumor samples may be very small and some relevant mutations may be uncommon, sensitive tests that can detect mutations in specimens with as few as 10% malignant cells are preferred.^{46,93,111–124}  Multiplex platforms offer advantages of simultaneous investigation of multiple mutations at one time. Epidermal growth factor receptor polysomy and amplification are associated with the presence of EGFR mutations, but in clinical trials EGFR polysomy and amplification do not predict response to EGFR TKI therapy nearly as well as EGFR mutation.^‖ Therefore, EGFR fluorescence in situ hybridization (FISH) is not as reliable for selecting patients for EGFR TKI therapy. Traditional EGFR immunohistochemistry is not mutation specific and, therefore, not useful as a predictive biomarker test for EGFR TKI therapy.^¶ New EGFR mutation–specific antibodies and possible new roles for EGFR FISH and EGFR immunohistochemistry are discussed in a later section.

KRAS mutation testing has an established role in selecting EGFR antibody therapy for metastatic colon cancer, but the role of KRAS mutation testing in EGFR therapy is less clear for NSCLC. Although KRAS mutations and EGFR mutations are usually mutually exclusive in NSCLC, clinical trials have not yet confirmed a predictive value for KRAS testing for determining whether or not to give EGFR TKI or EGFR antibody therapy in NSCLC. However, some laboratories perform KRAS testing as an early step in an algorithm to exclude the need to test for other biomarkers, including EGFR, if KRAS mutation is positive.^{38,69,85,93,128–131} 

In 2007, a new fusion oncogene, the echinoderm microtubule-associated proteinlike 4 (EML4) anaplastic lymphoma kinase (ALK) fusion tyrosine kinase, was described in NSCLC.¹³²  EML4-ALK is an oncogenic driver and activates downstream signaling pathways. Non–small cell lung carcinoma cells become addicted to EML4-ALK, making it a potential target for ALK TKIs. In NSCLC, there are multiple variants of the EML4-ALK fusion, and ALK may sometimes have other fusion partners such as TFG and KIF5B.^133–136  Anaplastic lymphoma kinase rearrangements are associated with younger patient age, never or light smokers, and adenocarcinoma histology.^137–141  The general frequency of ALK fusion genes is about 4% of adenocarcinomas, but frequency of ALK fusion genes has been reported to be 13.7% or even higher in advanced stage adenocarcinomas in never smokers.^69,140–143  It is estimated that there are about 10 000 new cases of ALK fusion gene–positive NSCLC in the United States each year.^1,69,97  Cytogenetic methods such as FISH are best for identifying these chromosomal rearrangements. Reverse transcriptase polymerase chain reaction (RT-PCR) may miss fusion variants that are not specifically tested for.^{142,144–146}  New methods for ALK detection are discussed later.

Early clinical trials of the first-generation ALK TKI crizotinib produced improved RR and PFS in ALK-positive NSCLC.^{141,142,144–147}  This led to accelerated approval of crizotinib or Xalkori (Pfizer, New York, New York) by the Food and Drug Administration for treatment of advanced NSCLC with ALK rearrangements. The US Food and Drug Administration also approved a specific companion test (Vysis ALK Break-Apart FISH Probe Kit; Abbott Molecular, Des Plaines, Illinois) to select patients for therapy with Xalkori.^148–151 

As with EGFR TKIs, patients receiving crizotinib relapse within a year because of acquired resistance. Acquired resistance to crizotinib is due to secondary mutations in the ALK tyrosine kinase domain in about one-fourth of cases, ALK gene amplification, amplification of KIT, aberrant activation of other kinases, and increased autophosphorylation of EGFR. Multiple resistance mechanisms may develop simultaneously in one tumor.^152–155 

Detection of predictive biomarkers (EGFR mutations and ALK fusion genes) is the most reliable basis for selecting NSCLC patients who are likely to respond to selective first-generation TKIs.^# The pathologist has a crucial role in the preanalytic steps before the tests are performed in a molecular diagnostics laboratory.

The literature indicates that many types of tumor samples can be used for predictive biomarker testing, including formalin-fixed, paraffin-embedded tissue, fresh tissue, and frozen tissue. Fortunately, because the only tissue ordinarily obtained for 70% of lung cancers is small biopsies and/or cytology specimens, these types of small specimens are amenable to biomarker testing, including transbronchial biopsies, needle biopsies, aspirates, cell blocks, direct smears, and touch preparations.^157–166  Whether or not to do reflex testing in which all NSCLCs or all adenocarcinomas are automatically sent for biomarker testing is currently a local decision, but is likely to increase in frequency.^87,167 

It is the responsibility of the pathologist to select the tissue sample that is to be submitted for biomarker testing. The pathologist must differentiate cancer from noncancer and viable tissue from nonviable tissue and, where applicable, select a representative block from several blocks for submission to the molecular diagnostics laboratory. Adenocarcinoma cell type is a clear indication to send a tumor specimen for predictive biomarker testing, and it is the pathologist who makes this diagnosis. Small biopsies and cytology specimens in which a diagnosis of adenocarcinoma can be neither confirmed nor excluded, including when other cell types such as squamous cell carcinoma are identified, are more problematic. A decision may be made to send these for biomarker testing even if adenocarcinoma can not be confirmed. Whether or not cell subtypes within a tumor should be selected for specific biomarker testing is controversial, but certainly not yet confirmed. However, subtypes may be selected for testing as part of a clinical trial or other study.^**

The clinical need for new predictive biomarker tests is driven by the limitations of the currently available molecular targeted therapies: (1) Current clinically validated targets, EGFR and ALK, are present in only a minority of lung cancers. As discussed previously, EGFR mutations and ALK rearrangements are largely restricted to adenocarcinomas, about 15% and 4%, respectively. Presence of one driver mutation in a lung cancer often excludes others, so that other target mutations may be present in lung cancers that are negative for EGFR mutations and ALK rearrangements.^{69,94–96,140–143}  Therefore, identification of additional validated targets is needed for SCLCs, squamous cell carcinomas, and the 80% or so of adenocarcinomas that are not positive for EGFR mutation or ALK rearrangement. (2) Acquired resistance to currently available targeted therapies eventually develops. Over time, alterations in the cancer genome, particularly the emergence of secondary mutations that bestow resistance to an EGFR TKI or crizotinib, confer resistance to a tumor that was previously susceptible to these agents.^†† Therefore, druggable targets are needed for lung cancers that develop resistance to current first-generation EGFR or ALK inhibition therapies. Strategies to address these needs include a mixture of (1) second-generation drugs directed at targets that have become resistant to first-generation drugs, (2) clinical validation of drugs that act on other potential molecular targets in lung cancers, and (3) inhibition of more than one target in a cancer by using multiple agents to impact multiple targets or by using single agents that act on more than one target.^172–179  All of these approaches have implications for the future role of the pathologist in personalized health care of lung cancer patients.

As discussed previously, pulmonary adenocarcinomas that are initially responsive to first-generation reversible EGFR TKIs eventually develop acquired resistance due to one or more of several possible mechanisms. Several second-generation drugs are under investigation and some are in advanced clinical trials. These second-generation drugs are higher-affinity, irreversible EGFR tyrosine kinase blockers that also inhibit HER2 and sometimes HER4 and may have modest activity against T790M or other mutations that cause acquired resistance to first-generation EGFR TKIs.^{172,173,180–184} 

Afatinib (BIBW2992; Boehringer Ingelheim, Ingelheim, Germany) binds irreversibly to EGFR, HER2 and HER4, including receptors with the T790M mutation. Clinical trials of afatinib have found modest results in patients with advanced pulmonary adenocarcinoma who progressed after receiving first-generation EGFR TKIs, but ongoing clinical trials may define a role for afatinib as first-line therapy for lung cancers with activating EGFR mutations.^{173,181,184–187}  Other second-generation ERBB family blockers are also under investigation,^172,173  including dacomitinib^183,188,189  and XL647.^190–192 

From the pathologist's perspective, the growing number of treatment options may create a need to rebiopsy and repeat EGFR testing during the course of a lung cancer patient's treatment. This permits monitoring for the development of mutations that may impact response to the patient's current therapy and warrant a change to a different drug. Not only might rebiopsy/repeat testing be needed after the patient relapses, but a surveillance protocol could conceivably screen for the early detection of acquired mutations such as T790M before significant clinical deterioration, when an alteration in drugs might be most effective.^193,194  A similar situation exists for acquired resistance to the first-generation ALK TKI crizotinib, and investigations are underway for second-generation ALK TKIs.¹⁵⁴ 

Cetuximab (Erbitux; Bristol-Myers Squibb, New York, New York, and Eli Lilly and Company, Indianapolis, Indiana) is an anti-EGFR immunoglobulin G1 monoclonal antibody that is currently undergoing clinical trials for lung cancer therapy.¹⁹⁵  The First Line Erbitux in Lung Cancer clinical trials produced modest results for advanced NSCLC patients treated with cetuximab and chemotherapy. However, subgroup analysis of the First Line Erbitux in Lung Cancer phase III trial found that high EGFR expression based on an immunohistochemistry score of 200 or more using the Dako pharmDx kit (Glostrup, Denmark) was associated with increased overall survival in patients receiving first-line chemotherapy plus cetuximab in patients with advanced NSCLC compared with chemotherapy alone.^196–198  The Southwest Oncology Group study SO342 of advanced NSCLC patients receiving cetuximab plus chemotherapy found a doubling of median PFS among EGFR FISH–positive patients compared with EGFR FISH–negative patients.¹⁹⁹  The Southwest Oncology Group phase III trial SO819 is prospectively evaluating both therapeutic response to cetuximab in advanced NSCLC patients and EGFR FISH as a predictive biomarker for cetuximab response in these patients.²⁰⁰  Therefore, although EGFR mutation testing is recommended as the best predictive biomarker test for EGFR TKIs, in the future, EGFR IHC and EGFR FISH may prove to be preferred predictive biomarker tests for cetuximab therapy for lung cancer. FISH and, especially, IHC are conventional techniques familiar to surgical pathologists and are more likely to be available in pathology laboratories that lack their own molecular diagnostics laboratory.

The search for driver mutations that can serve as targets for currently available or investigational new drugs has identified a number of candidate targets. Most kinase inhibitors can inhibit multiple kinase targets, and the use of agents that can simultaneously inhibit several targets is one approach to circumvent acquired resistance.

An example of a drug that inhibits multiple tyrosine kinases is crizotinib, which inhibits ALK, MET, RON, and ROS1.^201–203  Already Food and Drug Administration–approved for anti-ALK therapy, crizotinib is likely to find uses in inhibiting other targets. Similar to ALK, ROS1 rearrangements are found in a small subset of pulmonary adenocarcinomas and have a tendency to occur in patients who are younger and never smokers. ROS1 fusion genes are detected in general by FISH, and specific fusion partners are detected by RT-PCR, including CD74-ROS1, SLC34A22-ROS1, and FIG-ROS1; an antibody for immunohistochemistry has also been described. Preliminary studies show that crizotinib will be clinically useful in treating NSCLC with ROS1 fusion genes.^{202,204–207} 

The KIF5B-RET fusion gene has been reported in 1% to 2% of pulmonary adenocarcinomas and is a prospective target of the RET TKI vandetanib.^208–212 

Overexpression and/or amplification of the receptor tyrosine kinase c-MET or its ligand, hepatocyte growth factor, is associated with about 18% of cases of acquired resistance to EGFR TKIs. Several c-MET inhibitors and c-MET or hepatocyte growth factor antibodies are under investigation for several types of cancer.²¹³  ARQ 197 or tivantinib is a TKI that targets c-MET. Tivantinib plus erlotinib is currently undergoing phase III trials in previously treated patients with locally advanced or metastatic nonsquamous NSCLC, referred to as the MARQUEE (Met Inhibitor ARQ 197 plus Erlotinib versus Erlotinib plus placebo in NSCLC) trial.^214,215 

Signaling in the PI3K/AKT/mTOR pathway is initiated by activation of transmembrane receptor tyrosine kinases such as EGFR or HER2, and mutations in the PI3K/AKT/mTOR pathway have been implicated in NSCLC and SCLC. A number of inhibitors of the components of the PI3K/AKT/mTOR pathway are undergoing clinical trial for NSCLC, including RAD001 or everolimus (mTOR inhibitor), BEZ235 (PI3K/mTOR inhibitor), GDC-0941 (PI3K inhibitor), XL147 (PI3K inhibitor) and MK-2206 (AKT inhibitor).^216–227  In addition to clinical trials for NSCLC, mTOR inhibitors, including everolimus and temsirolimus, are under investigation as potential therapies for SCLC.^228–232  Other targets and their selective inhibitors may prove useful in the future, but studies are early or have not yet yielded results. Members of the IL-6/JAK/STAT pathway are potential targets for lung cancer therapy, with several agents proposed for further investigation, including enzastaurin (JAK1 inhibitor), AZD1480 (JAK 1/2 inhibitor) and NSC-743380 (STAT inhibitor).^233–236  Src TKIs such as dasatinib have also undergone phase II clinical trials with modest or disappointing results.^237–239  Interestingly, although KRAS is the most frequently mutated oncogene in pulmonary adenocarcinomas, found in approximately 30% of tumors, no selective KRAS inhibitors that are effective have yet been identified.²⁴⁰  MEK is a potential target downstream of KRAS, but phase II clinical trials of selective MEK inhibitors have had mediocre results.^241–243 

Next-generation technologies allow for multiplexed genotyping of lung cancers to simultaneously identify the mutational status of many genes in a tumor specimen.^{116–124,244–249}  Many of these platforms are becoming commercially available, and, although they are expensive, this approach eliminates the need for an algorithm of sequential tests, which takes a much longer time period to complete.

Recently, antibodies specific to ALK^250–252  and EGFR^253,254  have been under investigation, and an ALK antibody is now commercially available in the United States (ALK [D5F3] XP Rabbit mAb [Biotinylated] No. 8936; Cell Signaling Technology, Danvers, Massachusetts). Immunohistochemistry allows for direct visualization of viable cancer cells within a small biopsy or cell block section, providing a rapid, cost-effective identification of immunopositivity without attempting to extract RNA or DNA from a potentially inadequate tissue sample. Immunohistochemistry also uses routine equipment and routine laboratory procedures familiar to pathologists who are not specialized in molecular diagnostics.^3,4,43 

Lung cancer remains the most significant cause of cancer death in the United States and in the world. The advent of genotype-based therapy has created great promise for lung cancer patients and the identification of predictive biomarkers to select patients for therapy has assured a vital role for pathologists in precision medicine of lung cancer. The need for druggable targets for the majority of lung cancers that do not harbor the 2 targets that are currently clinically validated and the need for additional therapies for patients whose lung cancers develop acquired resistance to first-generation TKIs are being addressed by investigation of second-generation TKIs and new druggable targets. These endeavors and the development of multiplex platforms for simultaneous detection of multiple mutations and antibodies for sensitive and specific detection of predictive biomarkers promise to enhance the role of pathologists in precision medicine of lung cancer for years to come.