Lung cancer ranks as the leading cause of cancer-related

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1 Review Article Reflex Testing for Epidermal Growth Factor Receptor Mutation and Anaplastic Lymphoma Kinase Fluorescence In Situ Hybridization in Non Small Cell Lung Cancer Mari Mino-Kenudson, MD; Eugene J. Mark, MD N Context. Non small cell lung cancer (NSCLC) is a poorprognosis malignancy for which more effective treatments are needed, with accumulating clinical experiences supporting benefits of receptor tyrosine kinase inhibitors for patients with tumors harboring an epidermal growth factor receptor (EGFR) mutation or anaplastic lymphoma kinase (ALK) rearrangement. Objective. To review completed and ongoing clinical trials of EGFR tyrosine kinase inhibitors for EGFR mutation positive NSCLC and an ALK inhibitor for those with ALK rearrangement, while also exploring practical issues surrounding the implementation of molecular testing as a routine component of the diagnostic workup of NSCLC in the United States. Data Sources. Published biomedical literature, abstracts presented at recent major oncology meetings, and ClinicalTrials.gov. Conclusions. Continually evolving evidence indicates the possible efficacy of molecularly targeted agents for the treatment of advanced NSCLC, especially adenocarcinoma. To identify patients who will most likely benefit from the targeted therapy, routine determination of the corresponding genetic alterations after histologic diagnosis of NSCLC (reflex molecular testing for EGFR mutations and ALK rearrangement) should be considered. (Arch Pathol Lab Med. 2011;135: ) Lung cancer ranks as the leading cause of cancer-related mortality in the United States, where the estimated deaths in 2010 were approximately equivalent to those for breast, prostate, colorectal, and pancreatic cancer combined. 1 Non small cell lung cancer (NSCLC), mainly consisting of squamous cell carcinoma, adenocarcinoma, and large cell carcinoma, is estimated to account for approximately 85% of lung cancer diagnoses 2,3 and most lung cancer related morbidity and mortality. Clinical practice decisions regarding the use of surgery, radiation, and systemic therapy for patients with NSCLC are largely guided by the stage of disease. 3 Although surgery is used with curative intent for stage I to IIIA NSCLC, an analysis of the Surveillance, Epidemiology, and End Results registry found that disease will ultimately recur and result in death within 5 years of resection in approximately 30% of patients with stage I disease, in 60% with stage II disease, and in 75% with stage IIIA disease. 4 Accepted for publication January 19, From the Department of Pathology, Massachusetts General Hospital, Boston. This work is supported by Boehringer Ingelheim Pharmaceuticals, Inc (BIPI; Ridgefield, Connecticut). Editorial assistance is provided by Alyssa Tippens, PhD, of MedErgy (Yardley, Pennsylvania), which is contracted by BIPI for these services. The authors report the payment of $483 by BIPI to the Nature Publishing Company for permission to publish Figure 1, A through F. The authors have received no compensation related to the development of the manuscript. The authors have no relevant financial interest in the products or companies described in this article. Reprints: Mari Mino-Kenudson, MD, Department of Pathology, Massachusetts General Hospital, 55 Fruit St, Warren 122, Boston, MA ( mminokenudson@partners.org). Adjuvant chemotherapy has an established role for improving survival among patients with completely resected NSCLC, 5 but with substantial variability in influencing survival by pathologic stage (ie, more pronounced benefit for stage II/III disease relative to stage IB disease, with potential for an adverse risk to benefit ratio for patients with stage IA disease). 6 More effective treatments are needed to reduce the high rate of disease recurrence after surgical resection of NSCLC, as well as to improve the historically poor long-term outcomes for patients presenting with unresectable locally advanced or metastatic disease. Non small cell lung cancer has become a major platform for studying molecularly targeted agents as an adjunct or alternative to traditional chemotherapy, which is widely used for the treatment of most patients with early-stage or advanced NSCLC. The most commonly studied novel targeted therapies include tyrosine kinase inhibitors (TKIs) of epidermal growth factor receptor (EGFR), such as erlotinib (Tarceva, Genentech, South San Francisco, California) and gefitinib (Iressa, AstraZeneca, Wilmington, Delaware), and anaplastic lymphoma kinase (ALK), crizotinib (PF , Pfizer, Groton, Connecticut). In this review, we will provide (1) a brief overview of clinical data supporting an important role for the TKIs in patients with NSCLC with specific genetic alterations and (2) a discussion of practical issues surrounding the implementation of molecular testing as a routine component of the diagnostic workup of NSCLC (reflex testing) in the United States. Given the limited information available for the ALK TKI, we will focus on the discussion associated with EGFR and briefly touch upon ALK. Arch Pathol Lab Med Vol 135, May 2011 Reflex Molecular Testing in NSCLC Mino-Kenudson & Mark 655

2 EGFR AND EGFR TKIs Epidermal growth factor receptor is 1 of 4 receptor tyrosine kinases of the EGFR family, which is composed of the following members: EGFR and the human epidermal growth factor receptors 2, 3, and 4 (HER2, HER3, and HER4). The binding of ligands, such as epidermal growth factor, induces the formation of receptor homodimers or heterodimers and activates EGFR tyrosine kinase that, in turn, phosphorylates its substrates rapidly, thus relaying signals to the phosphatidylinositol 3-kinase (PI3K) protein kinase B (AKT) mammalian target of rapamycin (mtor) pathway involved in cell survival, or to the retrovirus-associated DNA sequences (RAS) v-raf-1 murine leukemia viral oncogene homolog 1 (RAF) mitogenactivated protein kinase kinase (MEK) extracellular signal-regulated kinase (ERK) pathway involved in cell proliferation. A high response rate (60% to 90%) to treatment with EGFR TKIs was observed in patients with mutations in exons 18 to 21 of the tyrosine kinase domain of EGFR. 7 9 These mutations can be identified in about 10% of Western patients with NSCLC and 35% of East Asian patients. Of the reported mutations, exon 19 deletions and exon 21 L858R point mutations comprise approximately 90% of all mutations and are responsible for the high response rate. Regardless of ethnicity, EGFR mutations are predominantly found in tumors from never-smokers (defined as,100 cigarettes in a patient s lifetime) and are most frequently seen in adenocarcinomas. 10,11 Reversible EGFR TKIs were the first targeted agents to be approved for treating advanced NSCLC in the United States. Erlotinib has been indicated since 2004 for use in platinum-pretreated disease on the basis of the significant prolongation of overall survival (OS) in the BR.21 phase III trial to a median of 6.7 months versus 4.7 months with placebo (hazard ratio [HR], 0.70; 95% confidence interval [CI], ; P,.001). 12 Gefitinib was approved by the US Food and Drug Administration in May 2003 for use in chemotherapy-pretreated advanced NSCLC (current indication is for the continued treatment of patients after the failure of platinum and docetaxel therapy) because of early observations of objective responses in a small subset of patients and symptomatic benefits 13,14 ; however, erlotinib may be the preferred EGFR TKI owing to the lack of OS benefit with gefitinib in this setting. EGFR Mutations and Response to EGFR TKIs Mutations in the EGFR gene contributing to the variability in responses observed with erlotinib and gefitinib became evident during 2004, when a series of reports involving small numbers of patients collectively demonstrated the sensitivity of EGFR mutation positive tumors to EGFR TKI monotherapy It is now well appreciated that mutations of the EGFR tyrosine kinase domain tend to occur in patients with adenocarcinoma who are never-smokers or have a light-smoking history, most commonly manifesting as in-frame deletions in exon 19 or L858R point mutations in exon ,19 Since 2005, a number of biomarker analyses of the phase III trials of erlotinib or gefitinib as monotherapy for pretreated disease or as a component of first-line chemotherapy in unselected populations have been published (Table 1), overall supporting a heightened propensity for patients with EGFR mutation positive tumors to respond to these agents. However, the enhanced tumor sensitivity suggested by these exploratory biomarker analyses did not appear to translate into prolonged OS. As an accumulating number of patients with EGFR mutation positive advanced NSCLC have been treated within clinical trials of erlotinib or gefitinib, several combined analyses have been conducted to characterize the benefits of EGFR TKI monotherapy for patients with EGFR mutation positive advanced NSCLC Analyses focused on previously untreated patients have yielded particularly intriguing findings supporting the use of EGFR TKI monotherapy in previously untreated EGFR mutation positive patient populations. 26,27 For example, within an analysis of data for 148 patients with EGFR mutation positive advanced NSCLC who participated in 7 prospective Japanese phase II trials of gefitinib, first-line use of gefitinib conferred a significantly higher objective response rate of 79.3% versus 24.6% with first-line chemotherapy (P,.001) and significantly longer progression-free survival (PFS) (10.7 months versus 6.0 months; HR, 0.35; 95% CI, ; P,.001), but no OS benefit (27.7 months versus 25.7 months; HR, 1.1; 95% CI, ; P 5.78). 26 Similarly, in a combined analysis of 5 clinical trials in which patients with EGFR mutation positive NSCLC received erlotinib or gefitinib as first-line monotherapy (predominantly Western/white patients [95% of 223 patients]), the objective response rate was 67%, median time to progression was 11.8 months, and median OS was 23.9 months. 27 Significantly prolonged OS was observed for patients with exon 19 deletions compared with L858R mutations (30.8 months versus 14.8 months; P,.001). 27 Phase III Trials of Reversible EGFR TKI Monotherapy Versus Chemotherapy as First-Line Treatment of EGFR Mutation Positive Advanced NSCLC In 2006, 3 phase III trials 7 9 were initiated in Asia to evaluate EGFR TKI monotherapy with gefitinib versus standard platinum-based doublet chemotherapy in the first-line treatment of patients with EGFR mutation positive advanced NSCLC. Two of these studies 7,8 were Japanese studies that required documentation of an EGFR mutation, whereas the cohort of the other study 9 consisted of a clinically selected East Asian population (ie, female sex, Asian ethnicity, history of never smoking, and diagnosis of pulmonary adenocarcinoma). The results, as summarized in Table 2, are consistent with respect to significantly greater efficacy for gefitinib versus chemotherapy in patients with EGFR mutation positive advanced NSCLC. The results of these trials have revolutionized first-line therapy for advanced NSCLC, and clinical practice guidelines have been updated to recommend the first-line use of EGFR TKIs as monotherapy for advanced disease, specifically for patients with tumors harboring EGFR mutations. 3,28 In contrast, molecular diagnostics of lung cancers have not been standardized yet. Second-Generation (Irreversible) EGFR TKIs in EGFR Mutation Positive Advanced NSCLC To date, the major barrier to long-term success with the reversible EGFR TKIs gefitinib and erlotinib has been the 656 Arch Pathol Lab Med Vol 135, May 2011 Reflex Molecular Testing in NSCLC Mino-Kenudson & Mark

3 Table 1. Biomarker Analyses of Phase III Trials of Erlotinib or Gefitinib in Unselected NSCLC Populations Trial INTEREST 21 ISEL 23 INTACT-1 and INTACT-2 20 TRIBUTE 22 BR Treatment and Setting Gefitinib versus docetaxel in pretreated advanced NSCLC (N ) Gefitinib versus placebo in pretreated advanced NSCLC (N ) CT 6 gefitinib in previously untreated advanced NSCLC (N ) CT 6 erlotinib in previously untreated advanced NSCLC (N ) Erlotinib versus placebo in pretreated advanced NSCLC (N 5 731) Patients With EGFR Mutation, No. (%) Active Treatment Arms 44/297 (15) Gefitinib Docetaxel Results in EGFR Mutation Positive Patients Objective Response Rates, No. (%) Median PFS or TTP, mo Median OS, mo 8/19 (42.1) 4/19 (21.1) P /215 (12) Gefitinib 6/16 (37.5) [versus 3/116 (2.6) for EGFR mutation negative disease] 32/312 (10) Gefitinib/CT CT/placebo 29/228 (13) Erlotinib/CT CT/placebo 13/18 (72) 2/5 (40) P /15 (53) 3/14 (21) P /177 (23) Erlotinib 3/19 (16) [versus 6/81 (7) for EGFR mutation negative disease] PFS: HR, 0.16; 95% CI, ; P NR PFS: Not reached 6.7 HR, 0.55; 95% CI, TTP: P 5.09 NR HR, 0.83; 95% CI, ; P 5.60 NR HR, 1.77; 95% CI, Not estimable Not estimable P 5.96 NR HR versus placebo, 0.77; 95% CI, ; P 5.45 Abbreviations: CI, confidence interval; CT, chemotherapy; EGFR, epidermal growth factor receptor; HR, hazard ratio; NR, not reported; NSCLC, non small cell lung cancer; OS, overall survival; PFS, progression-free survival; TTP, time to progression. emergence of drug resistance in nearly all patients (regardless of the degree of initial response to these agents), usually 8 to 12 months after the initiation of EGFR TKI therapy. Several resistance mechanisms with development of T790M mutations representing about 40% of resistant cases have been reported. 18 Second generation irreversible EGFR TKIs targeting multiple tyrosine kinases are currently in development, with several of these agents demonstrating potential activity against EGFR mutation positive NSCLC. EGFR Mutations and Other Predictors of Response to EGFR TKIs In addition to EGFR mutations, several other markers can predict response to and benefit from EGFR inhibitors, including clinicopathologic features, EGFR protein expression by immunohistochemistry, EGFR gene copy number changes, and other oncogenic mutations such as Kirsten rat sarcoma viral oncogene homolog (KRAS) gene mutations as a negative predictor. This section will discuss the efficacy of each marker in predicting the benefit of treatment with EGFR inhibitors and the potential for each marker to predict the presence of EGFR mutations. Clinicopathologic Features. Accumulating evidence suggests that mutations of the EGFR tyrosine kinase domain tend to occur more frequently than expected in female patients with adenocarcinoma who are of Asian ethnicity and have never smoked or have a light-smoking history. 18,19 In a study of 317 chemotherapy-naive patients treated with erlotinib or gefitinib in 1 of 5 clinical trials, 223 patients had undergone successful mutation testing for EGFR and/or KRAS. 27 When stratified by numbers of clinicopathologic predictors of EGFR mutations present in each case, 64% (38 of 59) of patients harboring 3 or more of the 4 predictors were found to have the sensitizing mutations, whereas only 29% (44 of 164) of patients with 2 or fewer predictors harbored the sensitizing mutations. Looking at response to EGFR TKIs, only 49% of the 59 patients with 3 or more predictors responded to TKI therapy, while 20% of the 164 patients with 2 or fewer predictors responded to TKI therapy. Conversely, twothirds (68%) of the patients with EGFR-sensitizing mutations responded to EGFR inhibitors as opposed to 4% of those without mutations. It appears that those clinicopathologic features are not as powerful in predicting response to the TKI therapy as EGFR mutations. Furthermore, they are not an optimal predictor for the presence of EGFR mutations, with a false-negative rate of 54%. Genotype-Phenotype Correlation. There have been many studies reporting subtypes of lung adenocarcinoma as histologic predictors of EGFR mutations. Some studies Arch Pathol Lab Med Vol 135, May 2011 Reflex Molecular Testing in NSCLC Mino-Kenudson & Mark 657

4 Trial IPASS 9,74 Table 2. Treatment and Setting Gefitinib versus paclitaxel/ carboplatin in nonsmokers or former light smokers with advanced NSCLC of adenocarcinoma histology (N ) WJTOG Gefitinib versus docetaxel/cisplatin in EGFR mutation positive advanced or postoperatively recurring NSCLC (N 5 177) North-East Japan Study Group Gefitinib 002 trial 7 Gefitinib versus paclitaxel/ carboplatin in EGFR mutation positive advanced or postoperatively recurring NSCLC (N 5 228) Published Results of Phase III Trials of Gefitinib in EGFR Mutation Positive NSCLC Patients With EGFR Mutation, No. (%) Active Treatment Arms 261/437 (59.7) Gefitinib Paclitaxel/ carboplatin 172/172 (100) Gefitinib Docetaxel/ cisplatin 228/228 (100) Gefitinib Paclitaxel/ carboplatin found the correlation of bronchioloalveolar carcinoma or a predominant bronchioloalveolar carcinoma pattern with EGFR mutations, 10,29 31 while others identified a predominant papillary pattern to be a predictor of EGFR mutations A micropapillary and hobnail cell type has also been reported to be associated with EGFR mutations. 31 The difference in the results may well be attributed to subjectivity in morphologic interpretation of the World Health Organization criteria for classifications of lung tumors. 35 Furthermore, different types of samples, including surgical resection and cytology specimens, were used for morphologic analysis in those studies. Given the fact that most lung adenocarcinomas represent a morphologically heterogenous group of tumors, 34 precise assessment of different growth patterns may be achieved only by examining multiple histologic sections of resected specimens. It indicates that the adequate histologic assessment, including prediction of EGFR mutations, is only possible for 15% or fewer of all patients with lung cancer, since 85% or more present with a tumor at advanced stage, the diagnosis of which is usually made on small biopsy or cytology specimens. 36 EGFR Gene Copy Changes. Several substudies of clinical trials using gefitinib or erlotinib have reported that the high EGFR gene copy number depicted by fluorescence in situ hybridization (FISH), as determined by the Colorado scoring criteria, 37 was associated with either increased response rates or an improved survival benefit. 23,24,38 However, high EGFR gene copy number seems less predictive of response to EGFR TKIs than EGFR mutations. Dahabreh and colleagues 25 recently conducted a literature search for EGFR status (somatic mutations and copy number aberrations) in patients with advanced Results in EGFR Mutation Positive Patients Objective Response Rates, No. (%) Median PFS, mo Median OS, mo 94/132 (71.2) 61/129 (47.3) P, /58 (62.1) 19/59 (32.2) P, /114 (73.7) 35/114 (30.7) P,.001 NR (73.5% event rate) NR (86.0% event rate) HR, 0.48; 95% CI, ; P, HR, 0.489; 95% CI, ; P, HR, 0.36; 95% CI, ; P, HR, 1.002; 95% CI, Not reached HR, 1.638; 95% CI, ; P P 5.31 Abbreviations: CI, confidence interval; EGFR, epidermal growth factor receptor; HR, hazard ratio; NR, not reported; NSCLC, non small cell lung cancer; OS, overall survival; PFS, progression-free survival. NSCLC who were treated with EGFR TKIs (monotherapy). Among 222 retrieved articles, 59 articles were considered eligible for the EGFR mutation meta-analysis (1020 mutations among 3101 patients) and 21 were considered eligible for the EGFR gene copy number meta-analysis (542 gene gains among 1539 patients). For EGFR mutations, the pooled sensitivity was 0.78 (95% CI, ) and pooled specificity was 0.86 (95% CI, ) for predicting a response to EGFR TKIs. For EGFR gene copy number, the pooled sensitivity was 0.61 (95% CI, ) and the pooled specificity was 0.71 (95% CI, ) for predicting a response to EGFR TKIs. The analysis provides empirical evidence that EGFR mutations are a sensitive and specific predictor of response to singleagent EGFR TKIs in advanced NSCLC. The performance of EGFR gene gain seems worse than that of mutations, and EGFR gene copy number analysis by FISH or chromogenic in situ hybridization cannot be considered clinically suitable for patient selection. 25 A possible role of EGFR FISH in predicting the presence of EGFR mutations was also studied. Dacic and colleagues 39 recently correlated the results of EGFR FISH with EGFR and KRAS mutation profiles in 345 surgically resected lung adenocarcinomas and showed that EGFR FISH positivity, defined by the Colorado criteria, was a good predictor of the presence of EGFR mutations (P 5.02). There were a significant number of cases exhibiting high polysomy of chromosome 7 alone particularly in the EGFR-mutated group, and logistic regression showed that high polysomy was the best predictor of EGFR mutations (P 5.001). However, EGFR FISH positivity was also observed in non- EGFR mutants (Figure 1, A through F), indicating that FISH analysis cannot replace mutational analysis Arch Pathol Lab Med Vol 135, May 2011 Reflex Molecular Testing in NSCLC Mino-Kenudson & Mark

5 Immunohistochemistry for EGFR Protein Expression. Studies using different antibody sources 40 have reported a high prevalence of EGFR immunostaining in NSCLCs; however, the rate is consistently higher in squamous cell carcinomas (55% 100%) than in adenocarcinomas (35% 60%) or large-cell carcinomas (20% 60%). There have been conflicting results as to whether EGFR protein expression has a value in predicting survival benefits with EGFR TKIs, 38 and the lack of standardization in staining procedures and scoring system is the major reason for the conflicting results across studies. Recently, mutation-specific rabbit monoclonal antibodies detecting the 2 most common mutations (exon 19 deletion and exon 21 L858R mutation) have been reported. 41 Subsequently, Brevet and colleagues 42 examined the performance of the mutationspecific antibodies on 218 paraffin-embedded lung adenocarcinomas with known mutation profiles determined by standard molecular mutational assays. The EGFR L858R mutant antibody showed a sensitivity of 95% and a positive predictive value of 99%, with a positivity cutoff of 1+, and showed a sensitivity of 76% and a positive predictive value of 100%, with a positivity cutoff of 2+. The EGFR exon 19 mutant specific antibody showed reduced sensitivity for exon 19 deletions other than 15 base pairs. These results indicate a potential use of these antibodies as a screen to identify most candidates for EGFR inhibitors; however, their performance is inferior when compared with standard mutational assays. 42 KRAS Mutation. RAS genes encode G-proteins downstream of receptor tyrosine kinases including EGFR. Upon ligand binding, activated EGFR leads to the activation of downstream RAS proteins by switching from an off state (RAS guanosine-59-diphosphate [GDP]) to an on state (RAS guanosine-59-triphosphate [GTP]). Activated GTP-bound RAS proteins regulate cell growth, proliferation, and apoptosis through various pathways, including the RAF-MEK-ERK cascade. The RAS family of proteins is encoded by 3 genes: KRAS, NRAS, and HRAS. All are commonly mutated in human cancers, with mutations leading to constitutively activated proteins locked in the GTP-bound on state. 11 Of those, most mutations found in NSCLC involve KRAS and predominantly occur in adenocarcinomas (approximately 30%) and less frequently in squamous cell carcinomas (approximately 5%). KRAS mutations are mutually exclusive with EGFR. Given that the EGFR-KRAS signaling cascade is considered to function as unidirectional linear outside-in signaling, it is hypothesized that tumors harboring mutant KRAS are independent of EGFR activation, because KRAS is already activated, and are resistant to EGFR-directed therapy. 43 However, there have been conflicting results in the recent phase III trials as to whether KRAS mutations identify patients who do not benefit from EGFR-directed therapy including gefitinib and erlotinib. 21,44,45 Thus, KRAS mutation analysis is not recommended for making EGFRdirected treatment decisions, although there may be a place in the near future for this molecular test in making other treatment decisions. 43 Because many KRAS mutant tumors display dependence on continued mutant KRAS signaling for survival, multiple efforts are being made to develop specific inhibitors, including combinations of PI3K and MEK inhibitors. 46,47 Figure 1. An example of acinar-type adenocarcinoma (A) with Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation; (B) another example of acinar-type adenocarcinoma (D) with epidermal growth factor receptor (EGFR) mutation. E, Both tumors were EGFR fluorescence in situ hybridization positive. C, Fluorescence in situ hybridization positivity defined by EGFR gene amplification (green, centromeric probe binding to the centromere of chromosome 7; red, EGFR probe). F, Fluorescence in situ hybridization positivity defined by high polysomy in the absence of EGFR gene amplification (hematoxylin-eosin, original magnifications 3200 [A] and 3100 [D]; original magnification [C] and 3600 [F]). Reprinted with permission from Macmillan Publishers Ltd. 39 Arch Pathol Lab Med Vol 135, May 2011 Reflex Molecular Testing in NSCLC Mino-Kenudson & Mark 659

6 Practical Issues Regarding EGFR Mutation Reflex Testing in NSCLC Clinical Feasibility and Utility in Influencing Course of Treatment. Overall, there is sufficient evidence supporting the role of EGFR mutational analysis in predicting patients response to EGFR TKIs. One contemporary question surrounding EGFR mutation testing is whether routine screening is both feasible and worthwhile in terms of improving outcomes in clinical practice, without negative consequences from delaying treatment until results are available. With this question in mind, the Spanish Lung Cancer Group 48 conducted a large-scale, prospective, EGFR mutation screening study in which biopsy-derived tumor tissue samples from 2105 patients treated across 129 centers in Spain were submitted to a central laboratory for assessment of EGFR mutations, to guide decisions regarding the use of erlotinib. The lagtime from laboratory receipt of the sample to investigator availability of results ranged from 5 to 9 days, with a median of 7 days. EGFR mutations were detected in 350 patients (17%), of whom 296 were considered for treatment with erlotinib. For all 217 patients who ultimately received erlotinib as first-line, second-line, or third-line treatment, the objective response rate among response-evaluable patients was 71% (139 of 197) and median PFS and OS were 14.0 and 27.0 months, respectively, with the authors noting that these durations were longer than expected meaning that EGFR mutation testing improved patient outcomes. Subsequently, D Angelo and colleagues, 49 from Memorial Sloan-Kettering Cancer Center (MSKCC, New York, New York), published their 3-year experience with EGFR mutation and KRAS mutation reflex testing of resected pulmonary adenocarcinoma specimens. As previously mentioned, KRAS mutations are thought by some to be mutually exclusive with EGFR mutations and are associated with poor response to EGFR TKI therapy. 18 Among 1831 patients with stage I to IV adenocarcinoma who underwent reflex testing, 364 patients had an EGFR mutation and 448 had a KRAS mutation. Among patients with stage I to III disease who underwent curative-intent surgical resection at MSKCC, a high rate of patients (78%, 855 of 1097) had undergone reflex testing, with EGFR and KRAS mutations detected in 158 and 207 patients, respectively. For the 15% of patients with stage I to III disease who experienced recurrence to date, the prior reflex testing results were used in the decisionmaking for subsequent therapy. The authors concluded that EGFR and KRAS reflex testing is feasible, with the data derived from this effort applicable to clinical decisions for treatment as well as to future research directives (as the data collected in this 3-year period have been used in a number of retrospective analyses). Finally, recently presented results from the M.D. Anderson Cancer Center s BATTLE study 50 (Houston, Texas) a phase II trial of novel design involving the screening of biopsy samples for a total of 11 biomarkers across 4 molecular pathways of importance in NSCLC (EGFR, RAS/RAF, vascular endothelial growth factor, and cyclin D1/retinoid X receptor) before randomization to receive 1 of 4 targeted therapy regimens lend further support to the feasibility of individualizing therapy based on EGFR mutation status and a number of other molecular characteristics of individual tumors. Methods to Determine EGFR Mutation Status. Sanger DNA direct sequencing is still the most common mutational technique used by many laboratories, but it may not detect mutations if tumor cells represent less than 25% of the sample, which may be an issue with small biopsies or fine-needle aspirates. Furthermore, direct sequencing is a very laborious process, frequently resulting in prolonged turnaround time for reporting results. This problem may be solved by implementation of molecular testing as reflex testing with a turnaround time of 5 to 6 working days (written communication, January 2011). Many other mutation detection techniques have been reported that are more sensitive, do not require a large amount of tissue, and are associated with reduced time and cost. 10,51 55 Similar to DNA direct sequencing, these assays perform better when fresh tissue, rather than formalin-fixed and paraffin-embedded (FFPE) tissue, is used. However, there are several reasons why direct sequencing is still considered to be a gold standard. Some of these more sensitive assays (ie, EGFR mutation assays using the Scorpion Amplification Refractory Mutation System, single-strand confirmation polymorphism, or denaturing high-pressure liquid chromatography) can screen a large number of samples in a short period of time, but require direct sequencing for confirmation of results. Furthermore, the clinical and predictive significance of low-abundance mutations detected in heterogeneous samples with these more sensitive assays is uncertain. Development of new targeted therapies against oncogenic targets other than EGFR has resulted in the expansion of clinical mutation testing for NSCLC. At Massachusetts General Hospital, the SNaPshot assay (Applied Biosystems, Inc, Carlsbad, California) is being used to examine 120 previously described mutations in 13 cancer genes, including responsive mutations and T790M of EGFR, and KRAS and BRAF mutations. It consists of a multiplexed polymerase chain reaction (PCR) step followed by a single-base extension reaction that generates allele-specific, fluorescently labeled probes and performs very well with FFPE tissue derived DNA with low background noise. The sensitivity of the SNaPshot assay is high enough to successfully detect mutations in scant tumor tissues, such as cell blocks made from fine-needle aspiration. 56 One of the main limitations of molecular testing in patients with advanced lung cancer is lack of adequate tissue sample. To overcome this problem, mutational analysis of collected circulating tumor cells, with a microfluidic-based device called the CTC-chip, 57 and/or plasma DNA 58 may represent an optimal alternative in the future. ALK TRANSLOCATION AND ALK INHIBITOR Anaplastic lymphoma kinase is a member of the insulin receptor superfamily of receptor tyrosine kinases, and its ligand is unknown. The enzyme was first found as a chimeric protein resulting from a chromosomal translocation, t(2;5)(p23;q35), in anaplastic large cell lymphoma. The translocation results in a fusion protein, nucleophosmin-alk. The truncated nucleophosmin constitutively activates ALK tyrosine kinase activity. In 2007, a different ALK translocation that is generated by inversion (2)(p21p23) was found in NSCLC. The resultant fusion protein contains the N-terminal part of echinoderm 660 Arch Pathol Lab Med Vol 135, May 2011 Reflex Molecular Testing in NSCLC Mino-Kenudson & Mark

7 microtubule-associated protein-like 4 (EML4) and the tyrosine kinase domain of ALK protein (EML4-ALK fusion protein) and is constitutively active and oncogenic. EML4-ALK is mutually exclusive with EGFR and KRAS mutations and is uncommon, occurring in 2% to 7% of all NSCLCs, but each year newly diagnosed patients with NSCLC are ALK positive worldwide. 59 At least 9 EML4-ALK fusion variants have been reported, and ALK variants can also have 5 other partners, such as KIF5B and TGF, with all variants containing the kinase domain of ALK Anaplastic lymphoma kinase fusion positive lung cancers are highly sensitive to treatment with an ALK TKI. A recent phase I/II clinical trial (ClinicalTrials.gov Identifier: NCT ) showed that 47 of 82 patients (57%) with ALK fusion positive lung cancer (confirmed by ALK FISH) displayed objective responses to an oral ALK inhibitor, crizotinib, and an additional 27 patients (33%) had stable disease. 59 By contrast, tumors harboring ALK fusions are resistant to EGFR TKIs. 64 A total of 63 of 82 patients (77%) continued to receive crizotinib at the time of data cutoff, and the estimated probability of 6-month PFS was 72%, with no median PFS reached in the study. Given that most patients had received chemotherapies (on average 2 therapies), the findings compare favorably with those in a meta-analysis involving patients with NSCLC who were treated with secondline multiagent chemotherapy that showed a median duration of PFS of 14 weeks and a 6-month rate of PFS of 27%. 9,59,68 Currently, a phase III trial of crizotinib versus standard of care (pemetrexed or docetaxel) for patients with NSCLC (NCT ) and the aforementioned phase II trial are recruiting participants but are restricted to those with ALK rearrangement confirmed by FISH analysis. Clinical characteristics associated with ALK translocation include adenocarcinoma histology, younger patients, and never-smoker or light-smoker status, similar to those associated with EGFR mutations. Pathologically, a solid pattern with abundant signet ring cells (greater than 10%) has been associated with ALK rearrangement in the Western population, especially in persons with advanced NSCLC (Figure 2, A and B) 63 ; however, resected NSCLCs of Asian patients appear to have a papillary, acinar, or mixed papillary, micropapillary, and/or cribriform growth pattern in ALK-rearranged tumors. 67,69 Among patients with a history of never or light smoking who have adenocarcinoma, driver mutations include EGFR in 50% Figure 2. Adenocarcinoma with anaplastic lymphoma kinase (ALK) rearrangement showing solid growth (A) and greater than 10% signet ring cells (B). ALK-rearranged non small cell lung cancer with expression of ALK protein (C), and split signals (arrows) of fluorescence in situ hybridization probes flanking the ALK gene (D). Arrowheads indicate nonsplit signals (hematoxylin-eosin, original magnifications 3100 [A] and 3600 [B]; D5F3 antibody, original magnification 3400 [C]; original magnification [D]). Arch Pathol Lab Med Vol 135, May 2011 Reflex Molecular Testing in NSCLC Mino-Kenudson & Mark 661

8 Figure 3. Reflex testing flowchart. Reflex testing is performed on nonsquamous cell carcinomas. These tests include the identification of anaplastic lymphoma kinase (ALK) rearrangements by fluorescence in situ hybridization (FISH) and the identification of responsible mutations in epidermal growth factor receptor (EGFR). Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation analysis may also be performed. and ALK in an additional 20%. In addition, up to 15% of patients with EGFR-positive NSCLC and about 6% of those with an ALK-rearranged tumor had a heavysmoking history but responded to each targeted therapy, indicating that definitive molecular characterization is critical to guide the selection of the appropriate targeted therapy. 59 Detection of ALK Rearrangement There are 3 methods that can detect ALK rearrangement: FISH, reverse transcriptase PCR (RT-PCR; multiplex RT- PCR), and immunohistochemistry for aberrant expression of ALK protein. Multiplex RT-PCR can identify all possible EML4-ALK fusion complementary DNAs in a single-tube experiment 66 ; however, this technique is not done in many routine pathology laboratories. In addition, the technique may not be able to detect all the translocations involving the ALK gene, especially when the translocation partner is not known; and most importantly, it may not be entirely specific. 70,71 Immunohistochemistry for ALK protein could prove to be a fast and cost-effective method that facilitates the routine identification of ALK-rearranged NSCLC because immunohistochemistry remains a preferred technique for screening and diagnosis using FFPE tissues in routine surgical pathology practice. ALK-rearranged NSCLCs, irrespective of the fusion partner, invariably express ALK protein but at much lower levels than anaplastic large cell lymphomas. As a result, greater than 30% of ALK-rearranged NSCLCs are not identified by standard immunohistochemistry assays using the ALK antibody that has been used most prevalently worldwide (ALK1, Dako USA, Carpinteria, California). 70 To improve sensitivity, a few techniques to enhance the protein expression have been introduced 72 ; however, they are not being applied in routine pathology laboratories. A novel antibody (D5F3, Cell Signaling Technology, Danvers, Massachusetts) that has high reproducibility, sensitivity (100%), and specificity (99%) in identifying ALK-rearranged NSCLCs (compared with the results of FISH analysis) 70 is not yet commercially available (Figure 2, C). ALK FISH assays usually consist of 2 multicolor breakapart probes that span the ALK gene (Figure 2, D). If the gene is intact the 2 colors merge together, but if the gene is rearranged the 2 colors are separated. Thus, the use of break-apart probes can inform whether a rearrangement is present. However, there are a few limitations: (1) the actual gene rearrangement will not be known, although further studies can then be performed as needed on tumor-derived complementary DNAs with PCR primers spanning multiple potential fusions 73 ; (2) the break-apart signal pattern resulting from the intrachromosomal deletion and inversion event in the setting of polysomy typical of lung cancer is subtle and may be easily missed; and (3) morphologic indicators of tumor versus nonneoplastic stromal or normal epithelial tissue are lost under fluorescence. 70 Despite the shortcomings, FISH analysis is currently considered as the gold standard, and FISH positivity for ALK rearrangement is required for entry into the clinical trials. In the near future, however, novel ALK antibodies with high sensitivity and high specificity may become commercially available and replace FISH analysis for detecting NSCLCs with ALK rearrangement. CONCLUSIONS Continually evolving evidence, mainly through clinical trials, indicates the possible efficacy of molecularly targeted agents EGFR and ALK TKIs for the treatment of advanced NSCLC, especially adenocarcinoma. To identify patients who will most likely benefit from the targeted therapy, routine determination of the corresponding genetic alterations after histologic diagnosis of NSCLC, with implementation of EGFR mutation assay and ALK FISH as part of the initial diagnostic workup, should be strongly considered (Figure 3). References 1. Jemal A, Siegel R, Xu J, Ward E. Cancer Statistics, CA Cancer J Clin. 2010;60(5): American Cancer Society. Cancer Facts & Figures, Atlanta, GA: American Cancer Society; National Comprehensive Cancer Network. NCCN Clinical Practice Guidelines in Oncology TM : Non-Small Cell Lung Cancer. Version Arch Pathol Lab Med Vol 135, May 2011 Reflex Molecular Testing in NSCLC Mino-Kenudson & Mark

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