Companion Diagnostic Testing for Targeted Cancer Therapies: An Overview

Abstract

Genes and their products involved in the biological pathways of human cancers have been studied as either targets of new therapies, or predictive markers for the sensitivity of or resistance to the therapies. Companion diagnostic testing on biological markers for targeted cancer therapies has become a vital component of personalized cancer treatment. This article provides an overview on the biological pathways and biomarkers, including EGFR, KRAS, BRAF, ALK, ROS1, HER2, and KIT for targeted treatment of lung, gastrointestinal, colorectal, and breast cancers as well as malignant melanoma. The current testing approach appears to focus on single biomarkers. However, a comprehensive approach that includes testing multimarkers involved in the mitogen-activated protein kinase, phosphoinositide 3-kinase/protein kinase B, and mammalian target of rapamycin pathways may become more desirable for some cancers, because of therapy resistance that can be caused by mutations in different genes and the availability of new therapies that may aim at multiple targets in the pathways. Only a few companion diagnostic kits have been approved by FDA, and the use of an FDA approved kit for some biomarkers, such as BRAF and KRAS, can be controversial.

Introduction

The signaling pathways involved in the regulation of cell growth and apoptosis provide the basis for targeted therapies and companion diagnostic testing for common human cancers (Snozek et al., 2009; Smalley, 2010; Pao and Girard, 2011; Vakiani and Solit, 2011) (Fig. 1). The mitogen-activated protein kinase (MAPK) pathway can be activated by growth factors released from the local microenvironment, such as stem cell factors, stimulating hormones, or specific growth factors of the cell type. Under normal physical conditions, these growth factors only induce a weak stimulation of the pathway for normal cell growth through the receptor protein kinases located on the membrane of the cell. In cancer cells, the MAPK pathway can be activated by acquisition of an oncogenic mutation in the growth factor receptor genes such as EGFR, KIT, HER2, and PDGFR. The receptor protein kinases produced by mutant growth factor receptor genes activate RAS, and then the downstream effectors such as the RAF family. Once activated, RAF stimulates the MAPK cascade, resulting in sequential activation of MAPK or ERK kinase (MEK), and then the extracellular signaling-regulated kinase (ERK), which activates cytoplasmic targets or migrates into the nucleus to phosphorylate transcription factors. The MAPK cascade can also be activated by activating mutations in the RAS and RAF genes, so it has also been called as the RAS-RAF-MEK-ERK pathway. The MAPK pathway intersects with other pathways such as phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) and mammalian target of rapamycin (mTOR) pathways to regulate cell proliferation and apoptosis. AKT has a critical role in cancer development through its ability to regulate apoptosis, while phosphatase and tensin homolog (PTEN) prevents the activation of AKT. Dysregulation of these pathways caused by activating oncogenic mutations or loss of functions of tumor suppressor genes leads to accelerated cell cycle, angiogenesis, apoptosis inhibition, invasion, and metastasis of tumor cells. The mutations that drive tumor proliferation and maintenance are called driver mutations. With the recently developed new cancer therapies targeting the activating mutations of the genes in signaling pathways, companion diagnostic testing has become essential for effective treatment of cancer patients (Ho and Laskin, 2009; Keedy et al., 2011; Pao and Girard, 2011; Vakiani and Solit, 2011)

FIG. 1.

The biological pathways as the targets of cancer therapies.

Nonsmall Cell Lung Cancer

Lung cancer causes more than 1 million deaths per year worldwide. Nonsmall cell lung cancers (NSCLCs) account for about 80% of lung cancers, comprising three different subtypes: adenocarcinoma (40%), squamous cell carcinoma (30%), and large cell carcinoma (10%).

Testing EGFR mutations for EGFR TKIs

EGFR activating mutations occur in about 10%-20% of Caucasian patients and in 30%-60% of Asian patients with NSCLC. Patients with these mutations respond to the EGFR tyrosine kinase inhibitors (EGFR-TKIs) erlotinib (Tarceva^®) and gefitinib (Iressa^®). Current molecular testing on EGFR detects activating mutations, including deletions in exon 19, and point mutations in exon 18 (codon 719), exon 20 (codon 768 and 790), and exon 21 (codon 858 and 861). The primary activating mutations are associated with a favorable response to the EGFR TKIs (Mok et al., 2009; Maemondo et al., 2010; Mitsudomi et al., 2010), and the response rates generally range from 62% to 83% and the overall survival (OS) ranges from 23 to 39 months (Mok et al., 2009; Rosell et al., 2009; Mitsudomi et al., 2010). Compiling clinical evidence from large clinical trials has shown the necessity of testing EGFR mutations for NSCLC patients, as recently stated by the American Society of Clinical Oncology Provisional Clinical Opinion: Patients with NSCLC, who are being considered for first-line therapy with an EGFR TKI, should have their tumor tested for EGFR mutations to determine whether an EGFR TKI or chemotherapy is the appropriate first-line therapy (Keedy et al., 2011). Other biomarkers, including EGFR copy number measured by fluorescence in situ hybridization (FISH) and EGFR expression measured by immunohistochemistry (IHC), are not predictive for outcomes with erlotinib (Brugger et al., 2011; Fukuoka et al., 2011). The College of American Pathologists (CAP), the International Association for the Study of Lung Cancer (IASLC), and the Association for Molecular Pathology (AMP) have recently provided guideline recommendations for testing EGFR and anaplastic lymphoma kinase (ALK) in lung cancer (CAP IASLC and AMP, 2011).

Despite the dramatic efficacy of EGFR TKIs, ∼30% of patients with an EGFR activating mutation experience de novo resistance to the treatment and virtually all patients who initially respond will ultimately develop acquired resistance. Some uncommon primary EGFR mutations are known to be associated with a poor EGFR TKI response, including T790M, D761Y, L747S, T854A, mutations on G719 and L861, and small insertion or duplication in exon 20 (Balak et al., 2006; Bean et al., 2008; Costa et al., 2008; Maheswaran et al., 2008; Wu et al., 2008; Wu et al., 2011). The secondary T790M mutation frequently occurs and confers resistance to these therapies (Balak et al., 2006; Kosaka et al., 2006). Compound mutations (double or complex) comprise 14% of all mutations identified in exons 18-21 and some of them are associated with EGFR TKI resistance (Kobayashi et al., 2013). The reports on the response of the uncommon mutations to EGFR TKIs provide useful data for interpretation of the clinical significance of individual mutations.

Testing ALK and ROS1 rearrangement for ALK inhibitors

The echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusion oncogene leads to constitutive ALK activation with a potent tyrosine kinase activity (Soda et al., 2007). A study has shown that expression of EML4-ALK induces marked activation of ERK (Takezawa et al., 2011), which is consistent with the earlier finding that NPM-ALK induces RAS activation and phosphorylation of the ERK (Turner et al., 2007). EML4-ALK fusion and its variants occur in 2%-7% of NSCLCs, and the patients with ALK rearrangement positive lung adenocarcinoma benefit from an ALK inhibitor, crizotinib (Koivunen et al., 2008; Kwak et al., 2010). Crizotinib (Xalkori^®) was approved by FDA for treatment of ALK rearrangement positive NSCLC, with ALK FISH as a companion diagnostic test. FISH detects the EML4-ALK fusion and its variants. Despite remarkable initial responses, cancers eventually develop resistance to crizotinib, usually within 1 year. A recent study on an EML4-ALK-positive NSCLC cell line has shown that amplification of the EML4-ALK fusion gene and a gatekeeper mutation, L1196M, within the kinase domain may render EML4-ALK insensitive to crizotinib and that secondary generation of ALK inhibitors can be highly active against the crizotinib-resistant cancers in vitro and in vivo (Katayama et al., 2011).

Rearrangement of the c-ros oncogene 1 (ROS1) with different partner genes (TPM3, SDC4, SLC34A2, CD74, and EZR) leads to ROS1 fusion proteins as receptor tyrosine kinase that drives NSCLC development (Rikova et al., 2007; Takeuchi et al., 2012). ROS1 fusion occurs in about 2% NSCLC, which can be detected by FISH, RT-PCR, or IHC. ROS1 rearrangement defines a molecular subset of NSCLC with distinct clinical characteristics that are similar to those observed in patients with ALK-rearranged NSCLC. Patient with ROS1 fusion are resistant to gefitinib and erlotinib, but sensitive to crizotinib (Bergethon et al., 2012; Davies et al., 2012; Rimkunas et al., 2012). It is likely that FISH for ROS1 fusion will quickly become a routine clinical test for targeted NSCLC treatment.

KRAS and other driver mutations

Mutations in KRAS, which encodes a GTP as downstream of EGFR, are present in about 25% of patients with lung adenocarcinoma (Riely et al., 2009; Paik et al., 2011). Somatic KRAS mutations commonly occur in codon 12, 13, and 61, and are associated with resistance to gefitinib and erlotinib. Emerging data suggest that KRAS mutations are negative predictors of benefit from both adjuvant chemotherapy and EGFR KTIs. Studies have also shown that patients with a KRAS mutant tumor have a shorter OS than those with EGFR mutant tumors. New driver mutations in BRAF, HER2, PIK3CA, AKT1, MAP2K1, and MET have been recently identified in NSCLC, each occurring in ≤5% of tumors (Paik et al., 2011; Pao and Girard, 2011). BRAF mutations are mutually exclusive to EGFR and KRAS, and so are HER2 mutations. In contrast to patients with an EGFR mutation or an ALK rearrangement who are mostly never smokers, all patients with BRAF mutations are current or former smokers (Paik et al., 2011). Different from malignant melanoma, about half of the BRAF mutations in lung adenocarcinomas are non-V600E type. While the BRAF inhibitor vemurafenib has been approved for treatment of melanoma, its usefulness in treatment of NSCLC has not been assessed. Other agents targeting BRAF, HER2, PI3K, and mTOR proteins are currently under development. Testing multiple markers for lung cancer can be desirable given the ongoing development of new therapies and clinical trials (Bunn and Doebele, 2011).

FGFR1 and other markers in lung squamous cell carcinomas

Lung squamous cell carcinoma (LSCC) is a smoking associated lung cancer, accounting for about 30% of the NSCLCs. Several recent studies have shown amplification of the fibroblast growth factor receptor 1 (FGFR1) in LSCCs and therapeutic potentials of FGFR1 inhibitors (Weiss et al., 2010; Dutt et al., 2011; Sasaki et al., 2012; Zhang et al., 2012). Currently, clinical phase 1 trials are underway to examine whether patients with FISH detected FGFR1 amplification-positive LSCCs benefit from a targeted therapy approach using small molecule inhibitors (Goeke et al., 2012). A standardized reading and evaluation strategy for FGFR1 FISH on LSCC has been established (Schildhaus et al., 2012). Along with FGFR1, recent discoveries of SOX2 amplification, NFE2L2 and KEAP1 mutations, PI3K pathway changes, and DDR2 mutations have ushered in a new era of targeted therapeutic agents for LSCCs (Drilon et al., 2012).

Colorectal Cancer

Colorectal cancer (CRC) is the third most common cancer worldwide, and about 50% of the patients have a metastatic disease at diagnosis. Patients with metastatic CRC have a median OS of 18-21 months. Development of CRC also involves the MAPK signaling pathway and anti-EGFR monoclonal antibodies (anti-EGFR moAbs), such as panitumumab (Vectibix™) and cetuximab (Erbitux^®), have been used for treatment of metastatic CRC. However, only 10% to 20% of patients with metastatic CRC clinically benefit from anti-EGFR moAbs (Saltz et al., 2004; Chung et al., 2005; Cunningham et al., 2008). EGFR somatic mutations occur extremely rarely in CRC and are not associated with response (Barber et al., 2004; Moroni et al., 2005)

Testing KRAS and BRAF for anti-EGFR therapy resistance

A number of studies and large clinical trials have shown that patients with mCRC harboring an activating KRAS mutation do not respond to cetuximab or panitumumab (Jonker et al., 2007; Amado et al., 2008; Karapetis et al., 2008; Lièvre et al., 2008; Bokemeyer et al., 2009; Hecht et al., 2009; Tol et al., 2009). KRAS mutations account for ∼35%-45% of nonresponsive CRC patients and have been used as a major negative predictor of efficacy in patients receiving cetuximab or panitumumab. Testing of KRAS mutations has become an essential determinant of therapy selection (Allegra et al., 2009). Most of clinical trials assessed patient's response to anti-EGFR moAbs relating to KRAS mutations in codon 12 and 13. Recent studies have shown that activating mutations also occur in codons 61 and 146, and the tumors with these mutations do not respond to anti-EGFR moAbs (Loupakis et al. 2009; Vaughn et al., 2011). It was reported that when chemotherapy refractory CRC was treated with cetuximab, the patients with a KRAS G13D mutant tumor had a longer overall and progression-free survival than those with other KRAS mutant tumors (De Roock et al., 2010b). Very recently, the FDA has approved cetuximab as the first-line treatment for KRAS mutation-negative and EGFR-expressing metastatic CRC in combination with irinotecan, 5-fluorouracil, and leucovorin. Concurrently, the FDA also approved the Therascreen^® KRAS RGQ PCR Kit (Qiagen) as a companion diagnostic test. Currently, there are no drugs available for specific and direct inhibition of KRAS for CRC treatment.

Activating BRAF mutations have been reported in 5%-10% CRC patients treated with cetuximab or panitumumab (Di Nicolantonio et al., 2008; Bardelli and Siena, 2010; Tol et al., 2010). BRAF and KRAS mutations are known to be mutually exclusive in these patients. An initial study on 132 patients detected the BRAF V600E mutation in 14% (11 of 79) nonresponders, but not in any of the patients who responded to cetuximab or panitumumab, suggesting that BRAF testing can be used for patient selection (Di Nicolantonio et al., 2008). The study also showed that BRAF-mutated patients have a significantly shorter progression-free survival and OS than wild-type patients. Similar conclusion was made with a recent large retrospective analysis and a systemic review (De Roock et al., 2010a; Tol et al., 2010; Lin et al., 2011).

PIK3CA, PTEN, and NRAS mutations

PIK3CA mutations of which 68.5% are located in exon 9 and 20.4% in exon 20 occur in 5% of CRC patients, and NRAS mutations in 2.6% of patients (De Roock et al., 2010a). Either activating mutations in the PIK3CA gene or inactivation of the PTEN phosphatase can activate the cascade of PI3K, AKT, and mTOR, which then activates MEK at the downstream of the EGFR-RAS-RAF pathway. Although KRAS and BRAF mutations seem to be mutually exclusive, PIK3CA mutations or PTEN inactivation can coexist with either KRAS or BRAF mutations. NRAS and PIK3CA exon 20 mutations appear to show a worse outcome based on a small number of identified mutations. Multivariate analysis and conditional inference trees have confirmed that, if KRAS is not mutated, assessing BRAF, NRAS, and PIK3CA exon 20 mutations (in that order) gives additional information about outcome (Lin et al., 2011). A recent study (Liao et al., 2012) showed that use of aspirin among CRC patients with mutated PIK3CA was associated with a 46% reduction of overall mortality and an 82% reduction in CRC-specific mortality. In contrast, aspirin use in CRC patients with wild-type PIK3CA did not affect either the overall or CRC-specific mortality. Because aspirin downregulates PI3K signaling activity, testing the PIK3CA mutation status will guide targeted use of aspirin adjuvant therapy for CRC patients if this preliminary result is confirmed by further studies.

Malignant Melanoma

Melanoma arises from the malignant transformation of melanocytes of the skin. There are more than 60,000 new cases in USA each year and metastasis develops in 10%-15% of patients with cutaneous melanoma. Patients with metastatic melanoma have a median survival of <6 months.

Testing BRAF V600 mutation for RAF inhibitors

BRAF mutations occur in >60% of malignant melanomas with a single substitution V600E accounting for 80% of the mutations (Davies et al., 2002). The mutant BRAF protein has an elevated kinase activity, which provides the target of treatment. Initial clinical trials showed clear evidence that the class I RAF inhibitors vemurafenib and dabrafenib have a remarkable antitumor activity and that the patients with advanced melanoma and a BRAF V600E mutation have a high response rate (Flaherty et al., 2010; Ribas and Flaherty, 2011). A V600K mutation is present in about 12% of the BRAF mutation-positive melanomas, while several other V600 mutations, including V600R, V600E2, V600D, and V600M, collectively account for about 5% of the cases (Anderson et al., 2012). Clinical responses have been observed in some patients with V600K mutant melanomas in both the phase II and phase III trials of vemurafenib (Chapman et al., 2011; Sosman et al., 2012). The FDA has recently approved vemurafenib (Zelboraf^®, Roche) for treatment of late-stage melanoma and the Cobas^® 4800 BRAF V600 Mutation Test (Roche) as its companion diagnostic kit. BRAF mutation testing has become an essential step for RAF-targeted treatment of malignant melanoma.

Markers associated with resistance to RAF inhibition

While RAF inhibitors can lead to drastic and impressive early tumor responses in patients, it has become clear that resistance to therapy quickly occurs in most of the patients. Approximately 20% of patients with mutant BRAF melanoma show no response, and most of the patients relapse with a median progression-free survival of 8-9 months (Ribas and Flaherty, 2011). Direct sequencing of BRAF exons has not revealed secondary mutations as the cause of resistance (Nazarian et al., 2010). The same study showed evidence that resistance to the RAF inhibitor is acquired by mutually exclusive PDGFRB upregulation or NRAS mutation. An NRAS mutation may restore the MAPK pathway signaling via CRAF, even though BRAF signaling is still inhibited. Deletions in PTEN and increased PI3K signaling are thought to be one potential driver of resistance. Secondary skin lesions such as well-differentiated squamous cell carcinomas and keratoacanthomas may develop rapidly in ∼15% to 30% of patients treated with vemurafenib or dabrafenib (Nazarian et al., 2010; Ribas and Flaherty, 2011). A very recent study has shown that mutations in RAS, particularly HRAS, are frequent in cutaneous squamous cell carcinomas and keratoacanthomas in patients treated with vemurafenib (Su et al., 2012). The molecular mechanism is consistent with the paradoxical activation of MAPK signaling, which leads to accelerated growth of these lesions.

Gastrointestinal Stromal Tumors

Gastrointestinal stromal tumor (GIST) is the most common mensenchymal neoplasm arising in the digestive tract with an estimated prevalence of 15-20 per million. Colorectal GISTs represent about 5%-10% of the cases. While surgery is the first-line treatment for resectable nonmetastatic colorectal GISTs, half of the patients develop local recurrence or distant metastasis after R0 operation (complete removal of all tumors with microscopic examination of margins showing no tumor cells). GIST is also known to be notoriously refractory to conventional chemotherapy or radiation.

Testing primary KIT and PDGFRA mutations for TKI therapy

KIT is a member of the transmembrane receptor tyrosine kinase family. Oncogenic KIT mutations occur in 80%-85% of GISTs as a central mechanism of pathogenesis, while mutations in PDGFRA, which also encode a receptor tyrosine kinase, occur in about 10% of GISTs (Antonescu, 2011). Imatinib, as a selective tyrosine kinase inhibitor of KIT and PDGFR, achieves partial response or stable disease in nearly 80% of GIST patients, and remarkably, a 2-year survival in 75%-80% of patients with advanced disease (Demetri et al., 2002). The types of KIT mutations are heterogeneous, including in-frame deletions, insertions, and substitutions. The presence of deletions in exon 11 predicts a more aggressive disease, while a less common-type mutation, the internal tandem duplications at the 3′ end of exon 11, allows a more indolent clinical course (Antonescu et al., 2003; Wardelmann et al., 2003). The exon 9 mutations are characterized by a small bowel location and aggressive behavior (Antonescu et al., 2003). Primary KIT mutations associated with TKI sensitivity frequently occur in exon 11 (65%-70%), exon 9 (10%-20%), and exon 13 (12%). Primary PDGFRA mutations commonly occur in exon 12, 14, and 18. A particular notice is that patients with one of the mutations in codon 18, D842V, are not usually sensitive to imatinib (Maleddu et al., 2009). Mutations in PDGFRA and KIT are mutually exclusive, and most GISTs with a mutant PDGFRA have a distinct phenotype (Lasota et al., 2004).

Testing secondary KIT and PDGFRA mutations for TKI resistance

Although most patients have a good durable response, they develop resistance to imatinib after a median time of 14 months. Acquisition of new mutations in KIT or PDGFRA is considered the most important and the most frequent cause of TKI resistance (Maleddu et al., 2009). More than 20 different secondary KIT mutations have been identified in exon 13, 14, 17, and 18, with V654A in exon 13 as the most frequent. The same mutation can be primary and secondary, such as the exon 13 mutation N822K. Two secondary mutations, H687Y and D842V, have been identified in PDGFRA. When the primary mutations are tested for selection of TKI therapy, testing secondary mutations appears to be even more important for understanding the mechanisms of TKI resistance and selection of second-line therapy. Sunitinib (Pfizer) is the only FDA approved second-line therapy for patients with imatinib-resistant or imatinib-intolerant GISTs. Although targeting VEGFR in addition to KIT and PDGFRA, sunitinib resistance shares similar mechanisms seen in imatinib failure, that is, acquisition of secondary mutations in the activation loop conferring resistance to both drugs (Guo et al., 2009).

BRAF mutations in GIST

A study revealed a primary BRAF mutation in 7% of adult GIST patients lacking KIT/PDGFRA mutations (Agaram et al., 2008). The BRAF mutant GISTs show predilection for small bowel location and a high risk of malignancy. The mutations seen in GISTs are also located within the exon 15 V600E hot-spot. The BRAF V600E mutation was identified in a patient who developed acquired resistance to imatinib, suggesting that secondary BRAF mutations may represent an alternative mechanism of imatinib resistance. Clinical evidence is needed to show if patients with a BRAF mutation will benefit from BRAF inhibitors such as vemurafenib.

Breast Cancer

There are about 1 million new cases of breast cancer each year worldwide. Breast cancer is subclassified into 4 major categories: luminal-A, luminal-B, basal, and HER2 amplification-positive. On the molecular basis, a breast cancer can be classified as estrogen receptor (ER)-positive/progesterone receptor (PR)-positive, HER2-positive, or ER/PR/HER2-negative (triple negative). Luminal-type cancers are primarily ER+/PR+, accounting for 60%-70% of patients, treated by antiestrogen therapy such as tamoxifan. About 30% of invasive breast cancers are HER2+. Most triple-negative cancers are made up of the basal-like subtype, representing 10% of primary breast cancers. Triple-negative breast cancers are aggressive, showing early metastasis with poor prognosis. Several clinical trials using PARP inhibitors such as olaparib, valiparib, and BSI-210 in treatment of triple-negative breast cancers appear to show promising results (Anders et al., 2010).

IHC, FISH, CISH, and SISH for anti-HER2 therapy

Activated or amplified HER2 stimulates the downstream signaling of the MAPK and other pathways. A monoclonal anti-HER2 antibody, trastuzumab, which specifically binds the extracellular portion of the HER2 transmembrane receptor, was approved by the FDA for treatment of metastatic breast cancer in 1998. In 2007, the FDA approved lapatinib, an EGFR and HER2 protein kinase inhibitor, for patients with metastatic cancer that overexpresses the HER2 protein. In 2008, trastuzumab was further approved for treatment of lymph node-negative breast cancer in the adjuvant setting. Several FDA approved kits for IHC HER2 expression, FISH, and chromogenic in situ hybridization (CISH) for HER2 amplification have been in the market.

A precise test result on HER2 status is very important for the appropriate use of trastuzumab, which costs about $100,000/year/patient in the United States (Wolff et al., 2007). There have been numerous debates and discussions on the advantage and disadvantage of FISH versus IHC. Although many studies have concluded that FISH is more precise than IHC (Ross et al., 2009), IHC remains the most frequent initial test for HER2 status and performed on ∼80% of newly diagnosed breast cancers in the USA. Based on the American Society of Clinical Oncology/CAP guideline (Wolff et al., 2007), a positive HER2 result is IHC staining of 3+ (uniform, intense membrane staining of >30% of invasive tumor cells), or FISH showing a signal ratio (the HER2 gene signal to chromosome 17 centromere signal) of >2.2. A negative result is an IHC staining of 0 or 1+, or a FISH ratio of <1.8. A result of IHC 2+ or FISH ratio of 1.8-2.2 is considered equivocal. An equivocal IHC is referred for FISH and an equivocal FISH needs counting additional cells or a repeat study. Patients with an equivocal FISH, but a ratio of ≥2.0 are qualified for trastuzumab treatment in the USA. CISH is an enzymatic ISH with the HER2 copy number examined using a bright-field microscope. Similar to FISH, CISH has a high correlation with IHC 0, 1+, and 3+ results, but a low correlation with IHC 2+. Silver in situ hybridization, SISH, is a recently developed technology that shows similar results as FISH and CISH, pending on FDA approval.

Trastuzumab resistance and new targeted therapies

Many clinical trials have shown convincing evidence that all HER2+ breast cancers benefit from the addition of trastuzumab. However, de novo trastuzumab resistance is seen in roughly 50% of patients with HER2+ metastatic breast cancer and acquired resistance after an initial response develops in most patients (Callahan and Hurvitz, 2011). Multiple molecular mechanisms contributing to trastuzumab resistance have been proposed, including truncated HER2, PI3K mutation, PTEN loss, the activity of HER3, IGF1R, or c-MET, and increased expression of Muc4 (Galanina et al., 2011; Nahta, 2012). Studies evaluating trastuzumab resistance breast cancer have shown the benefit of a combination of trastuzumab with chemotherapy, or with a second HER2-targeted agent such as lapatinib, or with inhibitors of PI3K, mTOR, or VEGF (Callahan and Hurvitz, 2011; Gajria and Chandarlapaty, 2011). Phase II and III clinical trials have demonstrated an impressive antitumor activity of the newly approved pertuzumab (Perjeta^™; Genentech) in combination with trastuzumab (Sendur et al., 2012). Pertuzumab is a monoclonal antibody that blocks ligand-dependent heterodimerization of HER2 with EGFR, HER3, and HER4. Another new drug, trastuzumab-DM1 (T-DM1), represents an approach of targeted delivery of the biological activity of trastuzumab by a highly potent antimicrotubule agent, DM1, to HER2-overexpressing breast cancer cells. Completed phase I and phase II clinical trials have shown the clinical activity of T-DM1 as a single agent or in combination with chemotherapies or pertuzumab (Barginear et al., 2013).

Gastric Cancer and HER2

Gastric and gastroesophageal cancers are diagnosed in 1.4 million new cases annually, representing a major global health problem. HER2 amplification occurs in ∼20% of the gastric cancers. Preclinical in vitro and in vivo studies showed that both trastuzumab and lapatinib were effective in gastric cancer models (Fujimoto-Ouchi K et al., 2007; Wainberg et al., 2010). In the first phase III study, the ToGA trial demonstrated that patients with HER2+ advanced gastroesophageal and gastric adenocarcinoma had a significant gain in OS when treated with a combination of trastuzumab with chemotherapy (Bang et al., 2010). The study also showed that the survival benefit associated with trastuzumab was the greatest in patients with high HER2-expressing tumors (IHC 3+ or IHC 2+ and FISH-positive). Further studies have shown highly concordant results between IHC using different antibodies and between FISH and SISH (Park et al., 2012).

Companion Diagnostic Tests and FDA Approved Kits

In the USA, a diagnostic cancer test must be performed in a laboratory that is State licensed, and CLIA certified. The laboratory may or may not be accredited by CAP, but must participate in the CAP proficiency surveys as one of the quality management measures. EGFR, ALK, KRAS, and BRAF have been described as “cancer biomarkers” in the CAP and AMP guidelines. Only a small number of cancer biomarkers have been recommended by CAP and AMP to be used as routine clinical tests (Table 1). A clinical test on a biomarker can be performed with different methodologies, such as PCR, direct sequencing, or pyrosequencing, accordingly with use of different reagents or products. When a cancer biomarker is used as a clinical test, a CLIA certified laboratory must have performed technical validation on the marker following the CAP guidelines.

Table 1.

Current Clinical Tests and FDA Approved Companion Diagnostic Kits for Targeted Cancer Therapies

Disease	Therapies	Markers as clinical tests	FDA approved kits
Nonsmall cell lung cancer	Getifinin Erlotinib Crizotinib	EGFR ALK ROS1	ALK FISH Kit (Abbott)
Colon cancer	Cetuximab Panitumumab	KRAS BRAF	Therascreen^® KRAS (Qiagen)
Malignant melanoma	Vemurafenib Dabrafenib	BRAF	Cobas^® 4800 BRAF V600E (Roche)
Gastrointestinal stromal tumors	Imatinib Sunitinib	KIT PDGFRA	None
Breast cancer	Trastuzumab Lapatinib Pertuzumab Trastuzumab-DM1	HER2	Herceptest™ HER2 IHC(Dako); Pathway™ HER2 IHC (Ventana); PathVysion HER2 FISH (Abbott); Spot-Light™ HER2 CISH (Invitrogen)
Gastric cancer	Trastuzumab Lapatinib	HER2

On July 14, 2011, FDA released a Draft Guidance for Industry and Food and Drug Administration Staff, In Vitro Companion Diagnostic Devices (FDA, 2011). “IVD companion diagnostic device” was defined as “an in vitro diagnostic device that provides information that is essential for the safe and effective use of a corresponding therapeutic product.” So far, only a few IVD companion diagnostic devices (kits) for cancer treatment have been approved by the FDA (Table 1), including the two recently approved RT-PCR-based kits: the Cobas^® 4800 BRAF V600E (Roche) for zelboraf treatment of melanoma and Therascreen^® KRAS (Qiagen) as a companion diagnostic kit for cetuximab treatment of CRC.

Many AMP members expressed concerns about the FDA guidance and approval of the Cobas^® 4800 BRAF V600E because BRAF is commonly tested for diagnosis and treatment of different cancers, and each laboratory may have used different techniques or reagents from different manufacturers. Based on the discussion of the members, AMP made official comments as a response to the FDA guidance in October, 2011 (AMP, 2011). Here are some of the points stated in the comments, which are more relevant to the diagnostic laboratories: (1) AMP applauds FDA's discouraging the inclusion of test brand names or manufacturers in therapeutic product labeling; (2) FDA's primary focus should be on the companion biomarker rather than associated tests; (3) the FDA should support introduction and use of alternative test products in promoting laboratory efficiency and enhancing assay performance, and thereby improving the care of our patients; (4) In many instances, CLIA-certified laboratories may offer laboratory developed assays for a biomarker that utilize standard molecular diagnostic techniques with which there is significant clinical experience; (5) It is not feasible for most laboratories to maintain multiple assays for the identical biomarker, and it would be unreasonable to expect them to do so; (6) Inclusion in therapeutic product labeling of implicit or explicit requirements mandating the use of the FDA cleared or approved tests for a biomarker will in many instances be harmful to patients and injurious to their care. The laboratories testing for KRAS mutations may have similar concerns about the very recent FDA approval of RT-PCR-based Therascreen^® KRAS (Qiagen) as a companion diagnostic kit for colon cancer treatment: First, the FDA approved kit does not detect mutations in KRAS codon 61 and 164, which are well known to be associated with resistance to anti-EGFR therapies for CRCs; Second, other sensitive technologies such as pyrosequensing have been well established for KRAS testing.

Conclusion

The therapies targeting the biological pathways represent a major advance in cancer treatment. However, therapy resistance has been a great challenge. To improve a patent's response, new approaches, including those with multiple targets, the use of a combination of targeted therapy with chemotherapies, or a combination of two or more targeted therapies, are being investigated with clinical trials. Companion diagnostic testing is crucial to maximize the efficacy, to minimize toxicity, and to reduce the cost of the therapies. Current clinical and laboratory practice appears focusing on testing individual biomarkers. However, a comprehensive or multimarker approach may become desirable and cost-effective, because of the availability of multitarget therapies in the future and the complex mechanisms of therapy resistance that usually involves multiple genes in the cancer biological pathways. While a clinical test must be validated by a CLIA certified laboratory, use of a FDA approved kit or device may not always be practical for laboratory operations or for its clinical applications.

Footnotes

Author Disclosure Statement

No financial interests or conflicts exist.

References

Agaram

, Laquaglia

, Ustun

et al. 2008. Molecular characterization of pediatric gastrointestinal stromal tumors. Clin Cancer Res, 14:3204-3215.

Allegra

, Jessup

, Somerfield

et al. 2009. American Society of Clinical Oncology provisional clinical opinion: testing for KRAS gene mutations in patients with metastatic colorectal carcinoma to predict response to anti-epidermal growth factor receptor monoclonal antibody therapy. J Clin Oncol, 27:2091-2096.

Amado

, Wolf

, Peeters

et al. 2008. Wild-type KRAS is required for panitumumab efficacy in patients with metastatic colorectal cancer. J Clin Oncol, 26:1626-1634.

AMP. 2011. Comments Regarding FDA Draft Guidance for “In Vitro Companion Diagnostic Devices” October 2011. www.amp.org/publications_resources/position_statements_letters/documents/AMPResponseCompanionDx_Final.pdf

Anders

, Winer

, Ford

et al. 2010. Poly(ADP-Ribose) polymerase inhibition: “targeted” therapy for triple-negative breast cancer. Clin Cancer Res, 16:4702-4710

Anderson

, Bloom

, Vallera

et al. 2012. Multisite analytic performance studies of a real-time polymerase chain reaction assay for the detection of BRAF V600E mutations in formalin-fixed paraffin-embedded tissue specimens of malignant melanoma. Arch Pathol Lab Med, 136:1385-1391.

Antonescu

. 2011. The GIST paradigm: lessons for other kinase-driven cancers. J Pathol, 223:251-261.

Antonescu

, Sommer

, Sarran

et al. 2003. Association of KIT exon 9 mutations with nongastric primary site and aggressive behavior: KIT mutation analysis and clinical correlates of 120 gastrointestinal stromal tumors. Clin Cancer Res, 9:3329-3337.

Balak

, Gong

, Riely

et al. 2006. Novel D761Y and common secondary T790M mutations in epidermal growth factor receptor-mutant lung adenocarcinomas with acquired resistance to kinase inhibitors. Clin Cancer Res, 12:6494-6501.

10.

Bang

, Van Cutsem

, Feyereislova

et al. 2010. Trastuzumab in combination with chemotherapy versus chemotherapy alone for treatment of HER2-positive advanced gastric or gastro-oesophageal junction cancer (ToGA): a phase 3, open-label, randomised controlled trial. Lancet, 376:687-697.

11.

Barber

, Vogelstein

, Kinzler

et al. 2004. Somatic mutations of EGFR in colorectal cancers and glioblastomas. N Engl J Med, 351:2883.

12.

Bardelli

, Siena

. 2010. Molecular mechanisms of resistance to cetuximab and panitumumab in colorectal cancer. J Clin Oncol, 28:1254-1261.

13.

Barginear

, John

, Budman

. 2013. Trastuzumab-DM1: a clinical update of the novel antibody-drug conjugate for HER2-overexpressing breast cancer. Mol Med, 18:1473-1479.

14.

Bean

, Riely

, Balak

et al. 2008. Acquired resistance to epidermal growth factor receptor kinase inhibitors associated with a novel T854A mutation in a patient with EGFR-mutant lung adenocarcinoma. Clin Cancer Res, 14:7519-7525.

15.

Bergethon

, Shaw

, Ou

et al. 2012. ROS1 rearrangements define a unique molecular class of lung cancers. J Clin Oncol, 30:863-870.

16.

Bokemeyer

, Bondarenko

, Makhson

et al. 2009. Fluorouracil, leucovorin, and oxaliplatin with and without cetuximab in the first-line treatment of metastatic colorectal cancer. J Clin Oncol, 27:663-671.

17.

Brugger

, Triller

, Blasinska-Morawiec

et al. 2011. Prospective molecular marker analyses of EGFR and KRAS from a randomized, placebo-controlled study of erlotinib maintenance therapy in advanced non-small-cell lung cancer. J Clin Oncol, 29:4113-4120.

18.

Bunn

Jr. , Doebele

. 2011. Genetic testing for lung cancer: reflex versus clinical selection. J Clin Oncol, 29:1943-1945.

19.

Callahan

, Hurvitz

. 2011. Human epidermal growth factor receptor-2-positive breast cancer: Current management of early, advanced, and recurrent disease. Curr Opin Obstet Gynecol, 23:37-43.

20.

CAP IASLC and AMP. 2011. Lung cancer biomarkers guideline draft recommendations. http://www.cap.org/apps/docs/membership/transformation/new/lung_public_comment_supporting_materials.pdf

21.

Chapman

, Hauschild

, Robert

et al. 2011. Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N Engl J Med, 364:2507-2516.

22.

Chung

, Shia

, Kemeny

et al. 2005. Cetuximab shows activity in colorectal cancer patients with tumors that do not express the epidermal growth factor receptor by immunohistochemistry. J Clin Oncol, 23:1803-1810.

23.

Costa

, Schumer

, Tenen

et al. 2008. Differential responses to Erlotinib in Epidermal Growth Factor Receptor (EGFR)-mutated lung cancers with acquired resistance to gefitinib carrying the L747S or T790M secondary mutations. J Clin Oncol, 26:1182-1184.

24.

Cunningham

, Starling

, Rao

et al. 2008. Upper Gastrointestinal Clinical Studies Group of the National Cancer Research Institute of the United Kingdom: Capecitabine and oxaliplatin for advanced esophagogastric cancer. N Engl J Med, 358:36-46.

25.

Davies

, Bignell

, Cox

et al. 2002. Mutations of the BRAF gene in human cancer. Nature, 417:949-954.

26.

Davies

, Le

, Theodoro

et al. 2012. Identifying and targeting ROS1 gene fusions in non-small cell lung cancer. Clin Cancer Res, 18:4570-4579.

27.

Demetri

, von Mehren

, Blanke

et al. 2002. Efficacy and safety of imatinib mesylate in advanced gastrointestinal stromal tumors. N Engl J Med, 347:472-480.

28.

De Roock

, Claes

, Bernasconi

et al. 2010a. Effects of KRAS, BRAF, NRAS, and PIK3CA mutations on the efficacy of cetuximab plus chemotherapy in chemotherapy-refractory metastatic colorectal cancer: a retrospective consortium analysis. Lancet Oncol, 11:753-762.

29.

De Roock

, Jonker

, Di Nicolantonio

et al. 2010b. Association of KRAS p.G13D mutation with outcome in patients with chemotherapy-refractory metastatic colorectal cancer treated with cetuximab. JAMA, 304:1812-1820.

30.

Di Nicolantonio

, Martini

, Molinari

et al. 2008. Wild-type BRAF is required for response to panitumumab or cetuximab in metastatic colorectal cancer. J Clin Oncol, 26:5705-5712.

31.

Drilon

, Rekhtman

, Ladanyi

et al. 2012. Squamous-cell carcinomas of the lung: emerging biology, controversies, and the promise of targeted therapy. Lancet Oncol, 13:e418-e426.

32.

Dutt

, Ramos

, Hammerman

et al. 2011. Inhibitor-sensitive FGFR1 amplification in human non-small cell lung cancer. PLoS One, 6:e20351.

33.

FDA. 2011. Draft Guidance for Industry and Food and Drug Administration Staff, In Vitro Companion Diagnostic Devices. July 14, 2011. www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/GuidanceDocuments/ucm262292.htm

34.

Flaherty

, Puzanov

, Kim

et al. 2010. Inhibition of mutated, activated BRAF in metastatic melanoma. N Engl J Med, 363:809-819.

35.

Fukuoka

, Wu

, Thongprasert

et al. 2011. Biomarker analyses and final overall survival results from a phase III, randomized, open-label, first-line study of gefitinib versus carboplatin/paclitaxel in clinically selected patients with advanced non-small-cell lung cancer in Asia (IPASS) J Clin Oncol, 29:2866-2874.

36.

Fujimoto-Ouchi

, Sekiguchi

, Yasuno

et al. 2007. Antitumor activity of trastuzumab in combination with chemotherapy in human gastric cancer xenograft models. Cancer Chemother Pharmacol, 59:795-805.

37.

Gajria

, Chandarlapaty

. 2011. HER2-amplified breast cancer: mechanisms of trastuzumab resistance and novel targeted therapies. Expert Rev Anticancer Ther, 11:263-275.

38.

Galanina

, Bossuyt

, Harris

. 2011. Molecular predictors of response to therapy for breast cancer. Cancer J, 17:96-103.

39.

Goeke

, Franzen

, Menon

et al. 2012. Rationale for treatment of metastatic squamous cell carcinoma of the lung using FGFR Inhibitors. Chest, 142:1020-1026.

40.

Guo

, Hajdu

, Agaram

et al. 2009. Mechanisms of sunitinib resistance in gastrointestinal stromal tumors harboring KITAY502-3ins mutation: an in vitro mutagenesis screen for drug resistance. Clin Cancer Res, 15:6862-6870.

41.

Hecht

, Mitchell

, Chidiac

et al. 2009. A randomized phase IIIB trial of chemotherapy, bevacizumab, and panitumumab compared with chemotherapy and bevacizumab alone for metastatic colorectal cancer. J Clin Oncol, 27:672-680.

42.

, Laskin

. 2009. EGFR-derived therapies to treat non-small-cell lung cancer. Expert Opin Investig Drugs, 18:1133-1145.

43.

Jonker

, O'Callaghan

, Karapetis

et al. 2007. Cetuximab for the treatment of colorectal cancer. N Engl J Med, 357:2040-2048.

44.

Karapetis

, Khambata-Ford

, Jonker

et al. 2008. K-ras mutations and benefit from cetuximab in advanced colorectal cancer. N Engl J Med, 359:1757-1765.

45.

Katayama

, Khan

, Benes

et al. 2011. Therapeutic strategies to overcome crizotinib resistance in non-small cell lung cancers harboring the fusion oncogene EML4-ALK. Proc Natl Acad Sci U S A, 108:7535-7540.

46.

Keedy

, Temin

, Somerfield

et al. 2011. American Society of Clinical Oncology provisional clinical opinion: epidermal growth factor receptor (EGFR) mutation testing for patients with advanced non-small-cell lung cancer considering first-line EGFR tyrosine kinase inhibitor therapy. J Clin Oncol, 29:2121-2127.

47.

Kobayashi

, Canepa

, Bailey

et al. 2013. Compound EGFR Mutations and Response to EGFR Tyrosine Kinase Inhibitors. J Thorac Oncol, 8:45-51.

48.

Koivunen

, Mermel

, Zejnullahu

et al. 2008. EML4-ALK fusion gene and efficacy of an ALK kinase inhibitor in lung cancer. Clin Cancer Res, 14:4275-4283.

49.

Kosaka

, Yatabe

, Endoh

et al. 2006. Analysis of epidermal growth factor receptor gene mutation in patients with non-small cell lung cancer and acquired resistance to gefitinib. Clin Cancer Res, 12:5764-5769.

50.

Kwak

, Bang

, Camidge

et al. 2010. Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer. N Engl J Med, 363:1693-1703.

51.

Lasota

, Dansonka-Mieszkowska

, Sobin

et al. 2004. A great majority of GISTs with PDGFRA mutations represent gastric tumors of low or no malignant potential. Lab Invest, 84:874-883.

52.

Liao

, Lochhead

, Nishihara

et al. 2012. Aspirin use, tumor PIK3CA mutation, and colorectal-cancer survival. N Engl J Med, 367:1596-1606.

53.

Lièvre

, Bachet

, Boige

et al. 2008. KRAS mutations as an independent prognostic factor in patients with advanced colorectal cancer treated with cetuximab. J Clin Oncol, 26:374-379.

54.

Lin

, Webber

, Senger

et al. 2011. Systematic review of pharmacogenetic testing for predicting clinical benefit to anti-EGFR therapy in metastatic colorectal cancer. Am J Cancer Res, 1:650-662.

55.

Loupakis

, Ruzzo

, Cremolini

et al. 2009. KRAS codon 61, 146 and BRAF mutations predict resistance to cetuximab plus irinotecan in KRAS codon 12 and 13 wild-type metastatic colorectal cancer. Br J Cancer, 101:715-721.

56.

Maemondo

, Inoue

, Kobayashi

et al. 2010. Gefitinib or chemotherapy for non-small-cell lung cancer with mutated EGFR. N Engl J Med, 362:2380-2388.

57.

Maheswaran

, Sequist

, Nagrath

et al. 2008. Detection of mutations in EGFR in circulating lung-cancer cells. N Engl J Med, 359:366-377.

58.

Maleddu

, Pantaleo

, Nannini

et al. 2009. Mechanisms of secondary resistance to tyrosine kinase inhibitors in gastrointestinal stromal tumours (Review) Oncol Rep, 21:1359-1366.

59.

Mitsudomi

, Morita

, Yatabe

et al. 2010. Gefitinib versus cisplatin plus docetaxel in patients with non-small-cell lung cancer harbouring mutations of the epidermal growth factor receptor (WJTOG3405): an open label, randomised phase 3 trial. Lancet Oncol, 11:121-128.

60.

Mok

, Wu

, Thongprasert

et al. 2009. Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma. N Engl J Med, 361:947-957.

61.

Moroni

, Veronese

, Benvenuti

et al. 2005. Gene copy number for epidermal growth factor receptor (EGFR) and clinical response to antiEGFR treatment in colorectal cancer: a cohort study. Lancet Oncol, 6:279-286.

62.

Nahta

. 2012. Molecular mechanisms of trastuzumab-based treatment in HER2-overexpressing breast cancer. ISRN Oncol, 2012:428062.

63.

Nazarian

, Shi

, Wang

et al. 2010. Melanomas acquire resistance to B-RAF (V600E) inhibition by RTK or N-RAS upregulation. Nature, 468:973-977.

64.

Paik

, Arcila

, Fara

et al. 2011. Clinical characteristics of patients with lung adenocarcinomas harboring BRAF mutations. J Clin Oncol, 29:2046-2051.

65.

Pao

, Girard

. 2011. New driver mutations in non-small-cell lung cancer. Lancet Oncol, 12:175-180.

66.

Park

, Hwang

, Park

et al. 2012. Comprehensive analysis of HER2 expression and gene amplification in gastric cancers using immunohistochemistry and in situ hybridization: which scoring system should we use? Hum Pathol, 43:413-422.

67.

Ribas

, Flaherty

. 2011. BRAF targeted therapy changes the treatment paradigm in melanoma. Nat Rev Clin Oncol, 8:426-433.

68.

Riely

, Marks

, Pao

. 2009. KRAS mutations in non-small cell lung cancer. Proc Am Thorac Soc, 6:201-205.

69.

Rikova

, Guo

, Zeng

et al. 2007. Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung cancer. Cell, 131:1190-1203.

70.

Rimkunas

, Crosby

, Li

et al. 2012. Analysis of receptor tyrosine kinase ROS1-positive tumors in non-small cell lung cancer: identification of a FIG-ROS1 fusion. Clin Cancer Res, 18:4449-4457.

71.

Rosell

, Moran

, Queralt

et al. 2009. Screening for epidermal growth factor receptor mutations in lung cancer. N Engl J Med, 361:958-967.

72.

Ross

, Slodkowska

, Symmans

et al. 2009. The HER-2 receptor and breast cancer: ten years of targeted anti-HER-2 therapy and personalized medicine. Oncologist, 14:320-368.

73.

Saltz

, Meropol

, Loehrer

Sr.

et al. 2004. Phase II trial of cetuximab in patients with refractory colorectal cancer that expresses the epidermal growth factor receptor. J Clin Oncol, 22:1201-1208.

74.

Sasaki

, Shitara

, Yokota

et al. 2012. Increased FGFR1 copy number in lung squamous cell carcinomas. Mol Med Report, 5:725-728.

75.

Schildhaus

, Heukamp

, Merkelbach-Bruse

et al. 2012. Definition of a fluorescence in-situ hybridization score identifies high- and low-level FGFR1 amplification types in squamous cell lung cancer. Mod Pathol, 25:1473-1480.

76.

Sendur

, Aksoy

, Altundag

. 2012. Pertuzumab in HER2-positive breast cancer. Curr Med Res Opin, 28:1709-1716.

77.

Smalley

KSM

. 2010. Understanding melanoma signaling networks as the basis for molecular targeted therapy. J Invest Dermatol, 130:28-37.

78.

Snozek

, O'Kane

, Algeciras-Schimnich

. 2009. Pharmacogenetics of solid tumors: directed therapy in breast, lung, and colorectal cancer: a paper from the 2008 william beaumont hospital symposium on molecular pathology. J Mol Diagn, 11:381-389.

79.

Soda

, Choi

, Enomoto

et al. 2007. Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer. Nature, 448:561-566.

80.

Sosman

, Kim

, Schuchter

et al. 2012. Survival in BRAF V600-mutant advanced melanoma treated with vemurafenib. N Engl J Med, 366:707-714.

81.

, Viros

, Milagre

et al. 2012. RAS mutations in cutaneous squamous-cell carcinomas in patients treated with BRAF inhibitors. N Engl J Med, 366:207-215.

82.

Takeuchi

, Soda

, Togashi

et al. 2012. RET, ROS1 and ALK fusions in lung cancer. Nat Med, 18:378-381.

83.

Takezawa

, Okamoto

, Nishio

et al. 2011. Role of ERK-BIM and STAT3-survivin signaling pathways in ALK inhibitor-induced apoptosis in EML4-ALK-positive lung cancer. Clin Cancer Res, 17:2140-2148.

84.

Tol

, Dijkstra

, Klomp

et al. 2010. Markers for EGFR pathway activation as predictor of outcome in metastatic colorectal cancer patients treated with or without cetuximab. Eur J Cancer, 46:1997-2009.

85.

Tol

, Koopman

, Cats

et al. 2009. Chemotherapy, bevacizumab, and cetuximab in metastatic colorectal cancer. N Engl J Med, 360:563-572.

86.

Turner

, Yeung

, Hadfield

et al. 2007 Cook

, Alexander

. The NPM-ALK tyrosine kinase mimics TCR signalling pathways, inducing NFAT and AP-1 by RAS-dependent mechanisms. Cell Signal, 19:740-747.

87.

Vakiani

, Solit

. 2011. KRAS and BRAF: drug targets and predictive biomarkers. J Pathol, 223:219-229.

88.

Vaughn

, Zobell

, Furtado

et al. 2011. Frequency of KRAS, BRAF, and NRAS mutations in colorectal cancer. Genes Chromosomes Cancer, 50:307-312.

89.

Wainberg

, Anghel

, Desai

et al. 2010. Lapatinib, a dual EGFR and HER2 kinase inhibitor, selectively inhibits HER2-amplified human gastric cancer cells and is synergistic with trastuzumab in vitro and in vivo. Clin Cancer Res, 16:1509-1519.

90.

Wardelmann

, Losen

, Hans

et al. 2003. Deletion of Trp-557 and Lys-558 in the juxtamembrane domain of the c-kit protooncogene is associated with metastatic behavior of gastrointestinal stromal tumors. Int J Cancer, 106:887-895.

91.

Weiss

, Sos

, Seidel

et al. 2010. Frequent and focal FGFR1 amplification associates with therapeutically tractable FGFR1 dependency in squamous cell lung cancer. Sci Transl Med, 2:62ra93.

92.

Wolff

, Hammond

, Schwartz

et al. 2007. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. Arch Pathol Lab Med, 131:18-43.

93.

, Wu

, Yang

et al. 2008. Lung cancer with epidermal growth factor receptor exon 20 mutations is associated with poor gefitinib treatment response. Clin Cancer Res, 14:4877-4882.

94.

, Yu

, Chang

et al. 2011. Effectiveness of tyrosine kinase inhibitors on “uncommon” epidermal growth factor receptor mutations of unknown clinical significance in non-small cell lung cancer. Clin Cancer Res, 17:3812-3821.

95.

Zhang

, Zhang

, Su

et al. 2012. Translating the therapeutic potential of AZD4547 in FGFR1-amplified non-small cell lung cancer through the use of patient-derived tumor xenograft models. Clin Cancer Res, 18:6658-6667.