Medical Policy Manual. Date of Origin: August Topic: Molecular Analysis for Targeted Therapy of Non- Small Cell Lung Cancer (NSCLC)

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1 Medical Policy Manual Topic: Molecular Analysis for Targeted Therapy of Non- Small Cell Lung Cancer (NSCLC) Section: Genetic Testing Policy No: 56 Date of Origin: August 2010 Last Reviewed Date: December 2014 Effective Date: April 10, 2015 IMPORTANT REMINDER Medical Policies are developed to provide guidance for members and providers regarding coverage in accordance with contract terms. Benefit determinations are based in all cases on the applicable contract language. To the extent there may be any conflict between the Medical Policy and contract language, the contract language takes precedence. PLEASE NOTE: Contracts exclude from coverage, among other things, services or procedures that are considered investigational or cosmetic. Providers may bill members for services or procedures that are considered investigational or cosmetic. Providers are encouraged to inform members before rendering such services that the members are likely to be financially responsible for the cost of these services. DESCRIPTION Targeted Therapy for Non-small Cell Lung Cancer (NSCLC) Treatment options for NSCLC depend on disease stage and include various combinations of surgery, radiation therapy, chemotherapy, and best supportive care. In up to 85% of cases, the cancer has spread locally beyond the lungs at diagnosis, precluding surgical eradication. In addition, up to 40% of patients with NSCLC present with metastatic disease. [1] Treatment of advanced NSCLC has generally been with platinum-based chemotherapy, with a median survival of 8 to 11 months and a 1-year survival of 30% to 45%. [2,3] More recently, the identification of specific, targetable oncogenic driver mutations in a subset of NSCLCs has resulted in a reclassification of lung tumors to include molecular subtypes, which are predominantly of adenocarcinoma histology. Epidermal Growth Factor Receptor (EGFR) Gene Mutations EGFR is a receptor tyrosine kinase (TK) frequently overexpressed and activated in NSCLC. Mutations in two regions of the EGFR gene (exons 18-24) small deletions in exon 19 and a point mutation in exon 21 (L858R) appear to predict tumor response to tyrosine kinase inhibitors (TKIs) such as erlotinib. [4,5] These can be detected by direct sequencing or polymerase chain reaction (PCR) technologies. 1 GT56

2 Testing is intended for use in patients with advanced NSCLC. Laboratory and animal experiments have shown that therapeutic interdiction of the EGFR pathway could be used to halt tumor growth in solid tumors that express EGFR. [6] These observations led to the development of two main classes of anti- EGFR agents for use in various types of cancer: small molecule TKIs and monoclonal antibodies (MAbs) that block EGFR-ligand interaction. [7] Patients with either small deletions in exon 19 or a point mutation in exon 21 (L858R) of the tyrosine kinase domain of the EGFR gene are considered good candidates for treatment with erlotinib. Patients found to be wild type are unlikely to respond to erlotinib, so other treatment options should be considered. KRAS Gene Mutations KRAS is a G-protein involved in the EGFR-related signal transmission. The KRAS gene, which encodes RAS proteins, can harbor oncogenic mutations that result in a constitutively activated protein, independent of signaling from the EGF receptor, possibly rendering a tumor resistant to therapies that target the EFG receptor. Mutations in the KRAS gene, mainly codons 12 and 13, have been reported in 20-30% of NSCLC, and occur most often in adenocarcinomas in heavy smokers. ROS1 ROS1 codes for a receptor tyrosine kinase of the insulin receptor family, and chromosomal rearrangements result in fusion genes. The prevalence of ROS1 fusions in NSCLC varies from %. [8] Patients with ROS1 fusions are typically never smokers with adenocarcinoma. RET RET (rearranged during transfection) is a proto-oncogene that encodes a receptor TK growth factor. Translocations that result in fusion genes with several partners have been reported. [8] RET fusions occur in 0.6-2% of NSCLCs and in 1.2-2% of adenocarcinomas. MET MET amplification is one of the critical events for acquired resistance in EGFR-mutated adenocarcinomas refractory to EGFR-TKIs. [8] BRAF RAF proteins are serine/threonine kinases that are downstream of RAS in the RAS-RAF-ERK-MAPK pathway. In this pathway, the BRAF gene is the most frequently mutated in NSCLC, in approximately 1-3% of adenocarcinomas. Unlike melanoma, about 50% of the mutations in NSCLC are non-v600e mutations. [8] Most BRAF mutations occur more frequently in smokers. HER2 Human epidermal growth factor receptor 2 (HER2) is a member of the HER (EGFR) family of TK receptors, and has no specific ligand. [8] When activated, it forms dimers with other EGFR family members. HER2 is expressed in approximately 25% of NSCLC. HER2 mutations are detected mainly in exon 20 in 1-2% of NSCLC, predominantly in adenocarcinomas in nonsmoking women. 2 GT56

3 Regulatory Status There are two U.S. Food and Drug Administration (FDA)-approved tests for EGFR mutation testing: The cobas EGFR Mutation Test is a companion diagnostic for the cancer drug Tarceva (erlotinib). [9] This diagnostic test detects EGFR gene mutations. The therascreen EGFR Rotor Gene Q polymerase chain reaction (PCR) Kit is an automated molecular assay designed to detect the presence of EGFR mutations for selecting NSCLC patients for treatment with GILOTRIF (afatinib). If the test results indicate that EGFR exon 19 deletion or exon 21 (L858R) substitution mutation is present in NSCLC cells, then the patient may be considered for treatment with GILOTRIF (afatinib). MEDICAL POLICY CRITERIA I. Analysis of two types of somatic mutation within the EGFR gene small deletions in exon 19 and a point mutation in exon 21 (L858R) may be considered medically necessary to predict treatment response to erlotinib (Tarceva ) or afatinib (GILOTRIF ) in patients with advanced or metastatic (stage III or IV) non-squamous cell-type non-small cell lung cancer (NSCLC). II. The following analyses/tests are considered investigational: A. Analysis of two types of somatic mutation within the EGFR gene small deletions in exon 19 and a point mutation in exon 21 (L858R) for patients with advanced NSCLC of squamous cell-type B. Analysis for other mutations within exons of the EGFR gene, or for other applications related to NSCLC C. Analysis of somatic mutations of the KRAS gene as a technique to predict treatment nonresponse to anti-egfr therapy with tyrosine kinase inhibitors and for the use of the anti-egfr monoclonal antibody cetuximab in NSCLC D. Testing for genetic alterations in the genes ROS, RET, MET, BRAF, and HER2 for targeted therapy in patients with NSCLC SCIENTIFIC EVIDENCE [10] The focus of the following review is on evidence related to the ability of test results to: Guide decisions in the clinical setting related to either treatment, management, or prevention, and Improve health outcomes as a result of those decisions. EGFR Two publications demonstrated that the underlying molecular mechanism underpinning dramatic responses in these favorably prognostic groups appeared to be the presence of activating somatic 3 GT56

4 mutations in the TK domain of the EGFR gene, notably small deletions in exon 19 and a point mutation in exon 21 (L858R). [4,5] Three orally administered EGFR-selective small molecules (quinazolinamine derivatives) have been identified for use in treating NSCLC: erlotinib (Tarceva, Genentech BioOncology), afatinib (GILOTRIF, Boehringer Ingelheim Pharmaceuticals, Inc), and gefitinib (Iressa, AstraZeneca). Erlotinib and afatinib are available for use in patients in the U.S. The marketing status for gefitinib is classified as discontinued by the FDA. [11] Therefore, the following literature review focused on studies that did not include gefitinib. Erlotinib Systematic Reviews A 2010 update of a BlueCross BlueShield Association Technology Evaluation Center (TEC) Assessment on this topic concluded that the evidence was sufficient to determine that EGFR mutation testing had clinical utility in selecting or deselecting patients for treatment with erlotinib. [12] Petrelli et al. reported a meta-analysis of 13 randomized trials of 1,260 patients receiving TKIs for first line, second line, or maintenance therapy and compared outcomes to standard therapy. [13] Overall they noted that in patients with EGFR mutations, use of EGFR TKIs increased the chance of obtaining an objective response almost 2-fold when compared to chemotherapy. Response rates were 70% vs. 33% in first line trials and 47% versus 28.5% in second line trials. TKIs reduced the hazard of progression by 70% in all trials and by 65% in first-line trials; however, they did not improve overall survival (OS). A 2013, Lee and colleagues published a meta-analysis of 23 trials (n=14,570) of erlotinib, gefitinib, and afatinib in patients with advanced NSCLC reported improved progression free survival (PFS) in EGFR mutation-positive patients treated with EGFR TKIs in the first- and second-line settings and for maintenance therapy. [14] Comparisons were with chemotherapy, chemotherapy and placebo, and placebo in the first-line, second-line, and maintenance therapy settings, respectively. Among EGFR mutation-negative patients, progression-free survival (PFS) was improved with EGFR TKIs compared with placebo maintenance but not in the first- and second-line settings. OS did not differ between treatment groups in either mutation-positive or mutation-negative patients. Statistical heterogeneity was not reported for any outcome. The authors concluded that EGFR mutation testing is indicated to guide treatment selection in NSCLC patients. A 2013 meta-analysis of 3 trials [15-17] in patients with wild-type EGFR reported improved overall survival with erlotinib treatment in second and third line and maintenance settings. [18] However, 75% of patients in the control arms in this analysis received placebo. Data were not available to perform PFS and relative risk (RR) analysis. Randomized Controlled Trials (RCTs) In a phase 3 prospective clinical trial, Zhou et al. reported the results of first line treatment of patients with EGFR-mutation positive NSCLC randomized to treatment with erlotinib (n=83) versus standard chemotherapy (gemcitabine plus carboplatin) (n=82). [19] They observed a significant increase in progression-free survival compared to treatment with chemotherapy (13.1 vs. 4.5 months; hazard ratio 0.16 (p<0.0001). Patients treated with erlotinib experienced fewer grade 3 and 4 toxic effects than those on chemotherapy. These results were duplicated in the EURTAC trial (NCT ), a multicenter, open-label, randomized phase 3 trial. Adult patients with EGFR-mutations (exon 19 deletion or L858R mutation 4 GT56

5 in exon 21) with NSCLC were randomized. [20] Eighty-six received erlotinib and 87 received standard chemotherapy. A planned interim analysis showed that the primary endpoint had been met. At the time the study was halted (Jan 26, 2011), median PFS 9.7 ( ) months versus 5.2 ( ) in the erlotinib and standard chemotherapy groups respectively. Hazard ratio 0.37 ( ); p<0.0001). Six percent of patients on erlotinib had treatment-related severe adverse events compared to 20% of those receiving a standard chemotherapy regimen. In 2013, Garassino et al. compared the efficacy of erlotinib and docetaxel as second-line therapy in 222 EGFR wild-type patients with metastatic NSCLC who had received previous platinum-based therapy. [21] Most patients (69%) had adenocarcinoma; 25% had squamous cell carcinoma (SCC). With a median follow-up of 33 months, median PFS was 2.9 months with docetaxel and 2.4 months with erlotinib (adjusted HR=0.71 [95% CI, 0.53 to 0.95]; p=0.02). Median overall survival was 8.2 months with docetaxel and 5.4 months with erlotinib (adjusted HR=0.73 [95% CI, 0.53 to 1.00]; p=0.05). Grade 3 or higher skin adverse events occurred in 14% of the erlotinib group and did not occur in the docetaxel group. Grade 3 or higher neutropenia occurred only in the docetaxel group (20%). As stated in an accompanying editorial, [T]he efficacy of EGFR tyrosine kinase inhibitors is very limited for second-line treatment of wild-type EGFR NSCLC. [22] Nonrandomized Studies Thirteen publications provided data on EGFR mutations in tumor samples obtained from NSCLC patients in erlotinib treatment studies. Nine of these were nonconcurrent, prospective studies of patients treated with erlotinib and then studied for the presence or absence of mutations. [23-31] Data comparing erlotinib results in EGFR mutation-positive versus wild-type patients have been reported in 9 studies of 630 patients. [23-27,29-31] EGFR mutations appear to provide prognostic, as well as predictive information about the behavior of tumors. In the study by Eberhard et al., improved outcome parameters were observed in EGFR-positive patients compared with wild-type patients for the population as a whole (standard chemotherapy and standard chemotherapy with erlotinib) in all measurement categories with objective radiologic response of 38% versus 23% (p=0.01), time to progression of 8 months versus 5 months (p<0.001), and overall survival (OS) (not reached versus 10 months [p<0.001]). [28] Four studies were prospective one-arm enrichment studies of mutation-positive patients and all patients had stage IIIA/IV NSCLC: [32-35] Rosell et al. compared EGFR-positive patients with erlotinib in treatment failure and chemotherapy naïve patients [33] and concluded that 350 patients out of 2105 had mutations. The objective radiologic response rates were 70%, progression-free survival (PFS) times were 14 months, and overall survival (OS) times were 27 months. Additional results in this study reported mutations in 16.6% of the total patients studied but noted these were found more frequently in women (69.7%), in patients who had never smoked (66.6%), and in patients with adenocarcinomas (80.9%). Based on these findings, the authors recommended EGFR-mutation screening in women with lung cancer with nonsquamous cell tumors who have never smoked. A study by Jackman et al. combined patient data from five trials (N=223 patients) in predominantly Western populations to assess the impact of EGFR and KRAS mutations on first-line therapy with an EGFR-tyrosine kinase inhibitor (TKI) and compare clinical versus molecular predictors of sensitivity. [32] Sensitizing EGFR mutations were associated with a 67% response rate, time to 5 GT56

6 progression (TTP) of 11.8 months, and OS of 23.9 months. Exon 19 deletions were associated with longer median TTP and overall survival compared with L858R mutations. Wild-type EGFR was associated with poorer outcomes (response rate, 3%; TTP, 3.2 months) irrespective of KRAS status. No difference in outcome was seen between patients harboring KRAS transition versus transversion mutations. Authors concluded that EGFR genotype was more effective than clinical characteristics at selecting appropriate patients for consideration of first-line therapy with an EGFR-TKI. Sun and others studied a total of 77 patients with either an exon 19 deletion (n = 58) or L858R mutation (n = 19) who were treated with gefitinib or erlotinib. [34] The overall response rate was 69%. Patients with an exon 19 deletion had a significantly longer PFS compared with patients with L858R mutation (9.5 vs. 7.7 months; P = 0.029). The L858R mutation was independently associated with a shorter PFS compared with an exon 19 deletion, even after adjusting for other clinical factors (hazard ratio 2.72; 95% CI ). However, there were no significant differences in response rate (71 vs. 63%) and OS (21.4 vs months) between subjects with exon 19 deletions and L858R mutations, respectively. Authors concluded, in Korean NSCLC patients, EGFR exon 19 deletions are associated with longer PFS compared with EGFR L858R mutations. In the first prospective biomarker study by Yoshioka and others, 30 patients were enrolled, and results for erlotinib therapy for pretreated patients with EGFR- tumors were described. [35] Objective response was observed in one patient (3.3%), and the disease became stable in 18 patients (60.0%). Skin rash was the most common side effect. Grades 3-4 adverse events included pulmonary embolism, keratitis, and anemia. Two other patients developed interstitial lung disease (grades 1 and 2). Nevertheless, all these events were reversible, resulting in no treatment-related deaths. With a median follow-up time of 10.7 months, the median survival time and median progression-free survival times were 9.2 and 2.1 months, respectively. Specific Populations An increased incidence of mutations is clearly seen in these special populations (women, patients with adenocarcinoma, nonsmokers, and/or Asians); however, it does appear that a substantial number of patients without these selected demographics still exhibit EGFR mutations and would benefit from erlotinib treatment. In a comprehensive analysis of 14 studies involving 2,880 patients, Mitsudomi et al. noted mutations were observed in 10% of men, 7% of non-asian patients, and 7% of current or former smokers, but only 2% of patients with non-adenocarcinoma histologies. [36] Park et al., reported on EGFR status in a preselected set of Korean patients treated with gefitinib. EGFR mutations were present in 3 out of 20 (15%) male smokers with squamous cell carcinoma of lung (SCC), a patient subgroup that based on demographics should have a low yield of EGFR mutations. [37] Two of the 3 patients identified with the mutation exhibited a response to the drug versus a response in 1 of 17 wild-type patients. The PFS in patients with EGFR was 5.8 months, compared to 2.4 months in the wild-type group (not statistically significant, p=0.07, but suggesting a trend favoring a treatment response in patients with the mutation). In vivo studies by Dobashi et al. have been reported, showing that in tumors in Japanese patients with both adenocarcinomas and SCCs, EGFR mutations are associated with downstream phosphorylation of EGFR and constitutive activation of the EGFR pathway. [38] Conclusion In a pooled analysis of studies, EGFR mutations appear to demonstrate improved patient outcomes for patients treated with erlotinib, as compared to standard chemotherapy (median PFS of 13.2 versus GT56

7 months, respectively). [39] Patients with EGFR mutations appear to be ideal candidates for treatment with erlotinib, whereas wild-type patients appear to derive little detectable benefit from erlotinib. Identification of patients likely to respond or to fail to respond to erlotinib treatment leads to tailored choices of treatment likely to result in predictable and desirable outcomes. In studies of treatment with erlotinib, objective radiologic response rates in patients with EGFRmutation-positive tumors ranged from 0% to 83% (median 45%) compared with objective radiologic response rates in patients with wild-type tumors of between 0% and 18% (median 5.5%). In studies statistically evaluating results, patients with EGFR-mutation-positive tumors always demonstrated statistically significant increases in objective radiologic response. Afatinib Studies on afatinib demonstrate the preclinical efficacy in NSCLC with common EGFR-activating mutations and the T790M mutation typically associated with EGFR TKI resistance. Clinically, afatinib has been evaluated in the LUX Lung trial program, with significant activity seen in the first and laterline settings, and these studies are described below. Randomized Controlled Trials (RCTs) In the LUX-Lung 2 phase III study by Sequist et al., eligible patients with stage IIIB/IV lung adenocarcinoma were screened for EGFR mutations. [40] A total of 1,269 patients were screened, and 345 were randomly assigned to treatment. Seventy-two percent of patients were Asian, 26% were white, and 90% (308 patients) had common EGFR mutations (exon 19 deletion or L858R substitution mutation in exon 21). Patients received either afatinib or chemotherapy (cisplatin plus pemetrexed). Median PFS was 11.1 months for afatinib and 6.9 months for chemotherapy (hazard ratio [HR], 0.58; 95% CI, 0.43 to 0.78; P =.001). Median PFS among those with exon 19 deletions and L858R EGFR mutations (n = 308) was 13.6 months for afatinib and 6.9 months for chemotherapy (HR, 0.47; 95% CI, 0.34 to 0.65; P =.001). Incidence of objective response in the entire patient sample was 56% in the afatinib group and 23% in the chemotherapy group (p=0.001). With a median follow-up of 16.4 months, median OS was not reached in any group; preliminary analysis indicated no difference in OS between the 2 treatment groups in the entire patient sample (HR=1.12 [95% CI, 0.73 to 1.73]; p=0.60). The most common treatment-related adverse events were diarrhea, rash/acne, and stomatitis for afatinib and nausea, fatigue, and decreased appetite for chemotherapy. Authors concluded that afatinib is associated with prolongation of PFS when compared with standard doublet chemotherapy in patients with advanced lung adenocarcinoma and EGFR mutations. Additional RCTs demonstrated significant improvements in PFS with afatinib over standard chemotherapy regimens in patients with EGFR mutations. [41] In the LUX-Lung 1 phase 2b/3 trial, Miller et al. enrolled patients with stage IIIB or IV adenocarcinoma and an Eastern Cooperative Oncology Group performance (ECOG) performance score of 0-2 who had received one or two previous chemotherapy regimens and had disease progression after at least 12 weeks of treatment with erlotinib or gefitinib. [42] Authors identified 697 patients, 585 of whom were randomly allocated to treatment (390 to afatinib, 195 to placebo). Median overall survival was 10 8 months (95% CI ) in the afatinib group and 12 0 months ( ) in the placebo group (hazard ratio 1 08, 95% CI ; p=0 74). Median progression-free survival was longer in the afatinib group (3 3 months, 95% CI ) than it was in the placebo group (1 1 months, ; hazard ratio 0 38, 95% CI ; p<0 0001). No complete responses to treatment were noted; 29 (7%) patients had a partial response in the afatinib group, as did one patient in the placebo group. Subsequent cancer treatment was given to 257 (68%) patients in the afatinib group and 153 (79%) patients in the placebo group. The most common adverse events in the afatinib group were diarrhea (339 7 GT56

8 [87%] of 390 patients; 66 [17%] were grade 3) and rash or acne (305 [78%] patients; 56 [14%] were grade 3). These events occurred less often in the placebo group (18 [9%] of 195 patients had diarrhoea; 31 [16%] had rash or acne), all being grade 1 or 2. Drug-related serious adverse events occurred in 39 (10%) patients in the afatinib group and one (<1%) patient in the placebo group. Authors recorded two possibly treatment-related deaths in the afatinib group. Authors concluded that findings for progressionfree survival and response to treatment suggest that afatinib could be of some benefit to patients with advanced lung adenocarcinoma who have failed at least 12 weeks of previous EGFR tyrosine-kinase inhibitor treatment. Nonrandomized Studies In the LUX-Lung 2 phase 2 study by Yang and others, authors enrolled 129 patients from 30 centers with lung adenocarcinoma (stage IIIb with pleural effusion or stage IV) with EGFR mutations, who had no more than one previous chemotherapy regimen for advanced disease, an Eastern Cooperative Oncology Group performance status of 0-2, and no previous treatment with EGFR tyrosine-kinase inhibitors. [43] Patients were treated with afatinib, 99 with a starting dose of 50 mg and 30 with a starting dose of 40 mg. 79 (61%) of 129 patients had an objective response (two complete responses, 77 partial responses). 70 (66%) of the 106 patients with the two common activating EGFR mutations (deletion 19 or L858R) had an objective response, as did nine (39%) of 23 patients with less common mutations. Similar proportions of patients had an objective response when analyzed by starting dose (18 [60%] of 30 patients at 40 mg vs 61 [62%] of 99 patients at 50 mg). Of the two most common adverse events (diarrhea and rash or acne), grade 3 events were more common in patients receiving a 50 mg starting dose (22 [22%] of 99 patients for diarrhea and 28 [28%] of 99 patients for rash or acne) than they were in those receiving a 40 mg starting dose (two [7%] of 30 patients for both diarrhea and rash or acne); possibly treatment-related serious adverse events were also less common in patients receiving a 40 mg starting dose (two of 30 patients vs 14 of 99 patients). Authors recorded one possibly drug-related death (interstitial lung disease). Authors concluded that afatinib shows activity in the treatment of patients with advanced lung adenocarcinoma with EGFR mutations, especially in patients with deletion 19 or L858R mutations. In the LUX-Lung 4 single-arm phase II trial conducted in patients with stage IIIB to IV pulmonary adenocarcinoma who progressed after 12 weeks of prior erlotinib and/or gefitinib, patients received afatinib 50 mg per day. [44] Of 62 treated patients, 45 (72.6%) were EGFR mutation positive in their primary tumor according to local and/or central laboratory analyses. Fifty-one patients (82.3%) fulfilled the criteria of acquired resistance to erlotinib and/or gefitinib. Of 61 evaluable patients, five (8.2%; 95% CI, 2.7% to 18.1%) had a confirmed objective response rate (partial response). Median PFS was 4.4 months (95% CI, 2.8 to 4.6 months), and median OS was 19.0 months (95% CI, 14.9 months to not achieved). Two patients had acquired T790M mutations: L858R + T790M, and deletion in exon 19 + T790M; they had stable disease for 9 months and 1 month, respectively. The most common afatinib-related adverse events (AEs) were diarrhea (100%) and rash/acne (91.9%). Treatment-related AEs leading to afatinib discontinuation were experienced by 18 patients (29%), of whom four also had progressive disease. Authors concluded that afatinib demonstrated modest but noteworthy efficacy in patients with NSCLC who had received third- or fourth-line treatment and who progressed while receiving erlotinib and/or gefitinib, including those with acquired resistance to erlotinib, gefitinib, or both. In 2013, Fang et al., reported EGFR mutations (all L858R) in 2% (3 patients) of 146 consecutively treated Chinese patients with early stage SCC. [45] In a separate cohort of 63 Chinese patients with SCC who received erlotinib or gefitinib as second- or third-line treatment (63% never smokers, 21% women), EGFR mutation prevalence (all exon 19 deletion or L858R) was 23.8%. Objective response occurred in 26.7% of 15 EGFR mutation-positive and 2.1% of 48 mutation-negative patients 8 GT56

9 (p=0.002). Median PFS was 3.9 months and 1.9 months, respectively (p=0.19). Based on these findings, the authors concluded that routine EGFR mutation testing of all SCC specimens is not justified. Conclusion EGFR tyrosine kinase inhibitors (TKIs) are valuable treatments for EGFR-mutated non-small cell lung cancer (NSCLC). First-generation, reversible EGFR inhibitors erlotinib and gefitinib therapies may eventually fail due to primary or acquired resistance. Irreversible inhibitors targeting ErbB family receptor tyrosine kinases, such as afatinib, have been developed to confer sustained disease control in ErbB-dependent cancers. Clinically, afatinib has been evaluated in the broad-reaching LUX Lung trial program, with significant activity seen in the first and later-line settings. Clinical Practice Guidelines National Comprehensive Cancer Network (NCCN) [46] The NCCN guidelines (v1.2015) on NSCLC make the following recommendations: EGFR mutational analysis in patients with advanced NSCLC. NCCN recommends (category 1) both erlotinib and afatinib as first-line therapy for the treatment of NSCLC for patients with sensitizing EGFR mutations based on category 2A evidence: 1) Erlotinib is recommended as a first-line therapy in patients with sensitizing EGFR mutations and should not be given as first-line therapy to patients negative for these EGFR mutations or with unknown EGFR status and 2) Afatinib is indicated for select patients with sensitizing EGFR mutations. In patients with squamous cell carcinoma (SCC), the low incidence of EGFR mutations (2.7%) [47] does not justify routine testing of all tumor specimens. However, it is reasonable to test for EGFR mutations or ALK rearrangements in squamous cell histology if patients are never smokers, small biopsy specimens were used for testing, or mixed histology was reported. This recommendation was based on a case series of 13 patients with squamous or pseudosquamous histology. [48,49] However, 7 patients were subsequently determined to have adenocarcinoma histology. The six remaining patients were nonsmokers with an exon 19 deletion or L858R substitution mutation in EGFR. American Society of Clinical Oncology (ASCO) [50] In a 2011 publication the American Society of Clinical Oncology (ASCO) issued a provisional clinical opinion on EGFR mutation testing for patients with advanced non-small-cell lung cancer considering first- line EGFR tyrosine kinase inhibitor therapy. It concluded patients with NSCLC being considered for first-line therapy with an EGFR tyrosine kinase inhibitor should have their tumor tested for EGFR mutations to determine whether an EGFR tyrosine kinase inhibitor or chemotherapy is the appropriate first-line therapy. College of American Pathologists [51] In 2013, the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology published evidence-based guidelines for molecular testing to select patients with lung cancer for treatment with EGFR TKI therapy. Based on 9 GT56

10 excellent quality evidence (category A), the guidelines recommended EGFR mutation testing in patients with lung adenocarcinoma regardless of clinical characteristics, such as smoking history. The joint guideline reported an EGFR mutation incidence of 0% to 5% in patients with SCC was reported. Recommendations for EGFR mutation testing in patients with SCC depend on tumor sample availability: For fully excised lung cancer specimens, EGFR testing is not recommended when an adenocarcinoma component is lacking, e.g., tumors with pure squamous cell histology with no immunohistochemistry evidence of adenocarcinoma differentiation (e.g., thyroid transcription factor 1 [TTF-1] or mucin positive). When lung cancer specimens are limited (e.g., biopsy, cytology) and an adenocarcinoma component cannot be completely excluded, EGFR testing may be performed in cases showing squamous cell histology; clinical criteria (e.g., lack of smoking history) may be useful to select a subset of these samples for testing. Despite these recommendations, evidence demonstrating how EGFR mutation testing in all patients with lung adenocarcinoma results in improved health outcomes is lacking. [45,51] American College of Chest Physicians (ACCP) [52] ACCP updated its evidence-based clinical practice guidelines on the treatment of stage IV NSCLC in Based on their review of the literature, guideline authors reported improved response rates, progression-free survival (PFS), and toxicity profiles with first-line erlotinib or gefitinib compared with first-line platinum-based therapy in patients with EGFR mutations, especially exon 19 deletion and L858R. ACCP recommended, testing patients with NSCLC for EGFR mutations at the time of diagnosis whenever feasible, and treating with first-line EGFR TKIs if mutation-positive. KRAS KRAS and EGFR Tyrosine Kinase Inhibitors (TKIs) Studies suggest that NSCLC patients with KRAS mutations may be nonresponsive to treatment with EGFR TKIs; however, the number of patients in the currently published studies who had KRAS-mutated tumors is relatively small and studies are mostly retrospective in nature. Systematic Reviews In a 2008 systematic review and meta-analysis, Linardou et al. assessed whether KRAS mutations represent a candidate predictive biomarker for anti-egfr-targeted therapeutic strategies in NSCLC. [53] Authors stated substantial evidence was found in the literature that determined KRAS mutations were appropriate markers for the identification of a subgroup of patients (20% of patients with NSCLC) with a limited probability of responding to EGFR-targeted treatments. In the metaanalysis, the presence of KRAS mutations was significantly associated with an absence of response to TKIs; however the pooled sensitivity was low (0.21 [95% CI ]). In summary, the findings of this study suggested that somatic mutations leading to gain-of-function and constitutive signaling of the KRAS pathway(s) represent a strong candidate predictive biomarker for nonresponsiveness to TKI-based strategies. Authors advocated for a large cooperative prospective study that would address the prognostic and predictive value of KRAS in predicting the efficacy of EGFR- 10 GT56

11 targeted agents in lung cancer due to the limitations of this study. These limitations included the unavailability of individual patient data, inadequate reporting of survival data, heterogeneity of response endpoints, intrinsic differences in the treatment regimens, patient selection criteria, and retrospective analysis of studies. In the most recent meta-analysis identified, authors suggested it was the first study to demonstrate a survival benefit of combining targeted therapy for advanced NSCLC; however, progression-free survival for patients with EGFR-mutation or wild type KRAS favored monotherapy erlotinib. [54] Though more studies are still needed to identify patients who will most likely benefit from the appropriate combining targeted therapy, 8 randomized controlled trials (N=2,417) with significant methodological limitations were included. Sub-group analysis based on phases of trials showed a tendency to improve PFS and OS in combining targeted therapy. Moreover, it should be noted that not all of the trials analyzed, including 2 phase III trials, demonstrated OS benefits from combining therapies. There were several limitations in this meta-analysis such as the lack of individual patient data; an individual patient data-based meta-analysis produces a more reliable estimation than one based on abstracted data. Possible survival benefits could not be determined in studies when patient clinical variables (staging, age, histologic types and general physical conditions) were unknown. In addition, different treatment duration and different combining of targeted therapies were both potential factors that increased heterogeneity amongst trials. Phase II and Phase III trials were combined in this study and thus presented an additional study limitation. Finally, publication bias was possible because papers with null results tend not to be published. Due to inconclusive results on studies that have evaluated the association between KRAS mutations and resistance to TKIs with NSCLC, authors in the second cited meta-analysis analyzed 22 studies that included 1,470 NSCLC patients, of whom 16% had KRAS mutations (N=231). [55] This study suggests that KRAS mutations may represent negative predictive biomarkers for tumor response in NSCLC patients treated with EGFR-TKIs. However, due to a mutually exclusive relationship between KRAS and EGFR mutation and no difference in survival between KRAS mutant/egfr wildtype and KRAS wild-type/egfr wild-type NSCLC, the clinical usefulness of KRAS mutation as a selection marker for EGFR -TKIs sensitivity in NSCLC is limited. Randomized Controlled Trials Data on the role of KRAS mutations in NSCLC and response to erlotinib are available from a small number of Phase II and Phase III trials and retrospective single-arm studies. [26-31,56-58] The majority of identified studies had significant methodological limitations including small sample size, variance in study populations (older individuals 70 and females), and inconsistent staging information. Representative studies are described below: Guan et al. (2013) reported on 1,935 consecutive patients with NSCLC who were treated at a single institution. [59] Patients with mutated KRAS were randomly matched on tumor, node, metastasis (TNM) stage, time of first visit within 1 year, and histology, to both EGFR mutation-positive and KRAS/EGFR wild-type patients. Seventy patients (4%) received EGFR TKI therapy. In this group, median progression free-survival (PFS) was 11.8and 2.0 months in patients with EGFR and KRAS mutations, respectively, and 1.9 months in wild-type patients; in comparison to wild-type patients, PFS was statistically longer in patients with EGFR mutations (p<0.001) but not different in patients with KRAS mutations (p=0.48). The authors observed that the presence of an EGFR mutation, but not a KRAS mutation, was predictive of responsiveness to EGFR TKI treatment. In 2013, Fiala et al. reported on a retrospective analysis of patients with squamous cell NSCLC who underwent EGFR, KRAS, and PIK3CA (phosphatidylinositide-3-kinase catalytic subunit-alpha) mutation testing. [60] Of 215 patients tested, 16 (7.4%) had mutated KRAS. Of 174 tested patients who 11 GT56

12 were treated with an EGFR TKI (erlotinib or gefitinib), median PFS in 14 KRAS-mutated patients was 1.3 months versus 2.0 months in KRAS wild-type patients (n=160 [92%]); the difference was not statistically significant (Kaplan-Meier [KM] log-rank test, p=0.120). Median OS in this treated group was 5.7 months in KRAS-mutated patients versus 8.2 months in KRAS wild-type patients, a statistically significant difference (KM log-rank test; p=0.039). The authors concluded that there was no role identified for EGFR, KRAS, PIK3CA mutations in the prediction of EGFR -TKIs efficacy in patients with advanced-stage squamous cell NSCLC. Pao and others provided analysis on 60 drug-sensitive adenocarcinomas; 9 out of 38 (24%) had KRAS mutations, while none of the drug-sensitive tumors had mutations. [56] These data suggest that tumors with the KRAS mutation are associated with a lack of response to these kinase inhibitors. These findings suggested that patients whose lung adenocarcinomas have KRAS mutations will not experience significant tumor regression with either gefitinib or erlotinib. Whether KRAS mutational status can be used to predict responses to erlotinib in patients is still under investigation. Data presented here suggested that clinical decisions regarding the use of these agents in patients with lung adenocarcinomas might be improved in the future by pre-treatment mutational profiling of KRAS. These findings warrant validation in large prospective trials using standardized mutation detection techniques. KRAS is frequently activated in NSCLC, and the relationship of KRAS mutations to outcome after EGFR inhibitor treatment has not been described. Eberhard and others detected KRAS mutations in 21% of tumors from their patient population and determined an association of the mutation with significantly decreased time to progression and survival in erlotinib plus chemotherapy-treated patients. [28] However, authors stated that further studies are needed to confirm the findings of their retrospective subset analysis. In an additional study, the effect of KRAS mutation on the response to erlotinib treatment was analyzed in 206 tumors; 15% of patients had KRAS mutations. [31] Erlotinib response rates were 10% for wild-type and 5% for mutant KRAS. Significant survival benefit from erlotinib therapy was observed for patients with wild-type KRAS but not for patients with mutant KRAS. In multivariate analysis, KRAS was not a prognostic for poorer survival or predictive of differential survival benefit from erlotinib. Authors sequenced tumor samples from patients with stage IIIB/IV NSCLC. [27] None of 17 patients with a KRAS mutation had a tumor response. Authors suggest prospective, placebo-controlled studies are needed to determine the predictive value of the putative biomarkers. A recent single prospective study with study limitations is described below: [58] In a study of 246 NSCLC patients, the presence of KRAS mutations in plasma was suggested to be a marker of poor prognosis and thought to hold predictive value. [61] Patients with a detectable plasma- KRAS mutation had a significantly shorter overall survival and progression-free survival compared to patients without the KRAS mutation. The response rate to chemotherapy was significantly lower in the group of patients with a mutation compared to patients without the mutation. Further validation of an independent cohort is needed. Conclusions It remains unclear whether assessment of KRAS mutation status will be clinically useful with regard to anti-egfr therapy in the treatment of NSCLC. Data on the role of KRAS mutations in NSCLC and response to erlotinib are available from 2 Phase III trials that conducted non-concurrent subgroup analyses of the efficacy of TKIs in patients with wild-type (non-mutated) versus mutated KRAS lung tumors, Phase II trials, retrospective single-arm studies, 2 meta-analyses, 1 systematic review, and 1 12 GT56

13 prospective study. Although studies have shown that a KRAS mutation in patients with NSCLC confers a high level of resistance to TKIs, data are insufficient to make a determination about an association between KRAS mutation status and survival in these patients. KRAS and Anti-EGFR Monoclonal Antibodies Two Phase III trials, BMS-099 and FLEX, investigated platinum-based chemotherapy with and without cetuximab in the first-line setting for advanced NSCLC. [62,63] Subsequently, an investigation of KRAS mutational status and cetuximab treatment was performed from both trials. [64,65] Outcomes observed (overall survival and/or progression free survival) in the cetuximab-containing and chemotherapy alone arms were similar between patients with mutant and wild-type KRAS. However, these findings should be interpreted with caution given the small subgroup sample size and retrospective nature of the analysis. In addition, findings from the FLEX trial have not yet been published in full and are currently available only in abstract. Conclusions While a lack of response to the EGFR monoclonal antibodies has been established in metastatic colorectal cancer, the expectation that KRAS mutation status would also be an important predictive marker for cetuximab use in NSCLC has not been shown. In 2 randomized trials with non-concurrent subgroup analyses of KRAS mutation status and the use of cetuximab with chemotherapy, KRAS mutations did not appear to identify patients who would not benefit from anti-egfr antibodies, as the outcomes observed with cetuximab were regardless of KRAS mutational status. Clinical Practice Guidelines National Comprehensive Cancer Network (NCCN) [46] The NCCN guidelines (v1.2015) for treatment of NSCLC state that KRAS mutations are associated with intrinsic TKI resistance, and KRAS gene sequencing could be useful for the selection of patients as candidates for TKI therapy. KRAS testing may identify patients who may not benefit from further molecular diagnostic testing. However, the guidelines make no specific recommendations. The guidelines state the presence of KRAS mutations is prognostic of poor survival for patients with NSCLC when compared to absence of KRAS mutations, independent of therapy. KRAS mutations are also predictive of lack of benefit from platinum/vinorelbine chemotherapy or EGFR TKI therapy. No specific recommendation for KRAS testing is made in the NCCN guidelines. College of American Pathologists (CAP) Joint Guidelines [51] In 2013 CAP, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology published evidence-based guidelines for molecular testing to select patients with lung cancer for treatment with EGFR and alkaline phosphatase TKI therapy. Based on good quality evidence (category B), KRAS mutation testing is not recommended as a sole determinant of EGFR TKI therapy. The guideline authors stated that, The significance of KRAS mutational analysis may become increasingly important with the further development of new therapies targeting downstream RAS pathways, such as PI3K/AKT/mTOR and RAS/RAF/MEK, but at this time, the absence of a KRAS mutation does not add clinically useful information to the EGFR mutation result and should not be used as a determinant of EGFR TKI therapy. 13 GT56

14 Other Oncogenic Mutations Other potentially targetable oncogenic mutations have been characterized in lung adenocarcinomas including in the genes ROS, RET, MET, BRAF, and HER2. The data on the use of targeted therapies in NSCLC with a mutation in 1 of these genes is preliminary in that much of the demonstrated sensitivity of tumor to the various drugs has been in vitro or in animal studies, and published data on patient tumor response and survival outcomes are extremely limited, consisting of case reports and small case series. The following studies are representative of the available published evidence for these genes. ROS Bergethon et al. conducted a retrospective analysis of the clinical characteristics and treatment outcomes of patients with NSCLC with a ROS1 rearrangement. [66] The authors screened 1,073 patients from multiple institutions for ROS1 rearrangements using a FISH assay and correlated ROS1 status with clinical characteristics, OS, and when available, ALK rearrangement status. Clinical data were extracted from medical record review. In vitro studies with human NSCLC cell lines were also conducted to assess the responsiveness of cells with ROS1 rearrangements to crizotinib. Of the tumors that were screened, 18 (1.7%) had ROS1 rearrangements, and 31 (2.9%) had ALK rearrangements. All of the ROS1-positive tumors were adenocarcinomas. The patients with ROS1 rearrangements were significantly younger (median age 49.8 years) and more likely to be neversmokers, when compared with the ROS1-negative group (each p<.001). There was no survival difference observed between the ROS1-positive and negative groups. The in vitro studies showed evidence of sensitivity to crizotinib. Finally, the authors reported the clinical response of 1 patient in their study with a ROS1 rearrangement. The patient was enrolled as part of an expanded phase 1 cohort, in an open-label, multicenter trial of crizotinib. The patient had no EGFR mutation or ALK rearrangement. After treatment failure with erlotinib, the patient was treated with crizotinib and had near complete resolution of tumor without evidence of recurrence at 6 months. Kim et al. reported clinical outcomes in 208 never-smokers with NSCLC adenocarcinoma, according to ROS1-rearrangement status. [67] ALK rearrangements and EGFR mutations were concurrently analyzed. The patients had clinical stages ranging from I-IV, but most were stage IV (41.3%). Of the 208 tumors, 3.4% (n=7) were ROS1 rearranged. ROS1 rearrangement was mutually exclusive from ALK rearrangement, but 1 of 7 ROS1-positive patients had a concurrent EGFR mutation. Patients with ROS1 rearrangement had a higher objective response rate and longer median PFS on pemetrexed than those without a rearrangement. In patients with ROS1 rearrangement, PFS with EGFR-TKIs was shorter than those patients without the rearrangement. None of the ROS1 positive patients received ALK inhibitors (e.g., crizotinib), which is the proposed targeted therapy for patients with NSCLC and this genetic alteration. Jin et al. found ROS1 gene copy number gain (CNG) to be significantly associated with shorter disease-free survival (DFS) and OS than patients without CNG. Of 357 samples, ROS1 protein overexpression was observed in 18 (5%). [68] There was no statistically significant correlation between ROS1 gene CNG and protein overexpression. ROS1 gene rearrangement was detected in 0.8% of surgically resected NSCLC and ROS1 gene CNG was an independent factor for poor prognosis. Clinical utility was not addressed in this study. RET 14 GT56

15 In a phase 2 prospective trial for patients with RET fusion-positive tumors, preliminary data on 3 patients treated with cabozantinib showed a partial response in 2 patients, and 1 with stable disease approaching 8 months. [69] MET A phase 2 trial of MET-positive NSCLC, in which patients were treated with an anti-met antibody plus erlotinib, showed improved PFS and OS. [70] BRAF Rare case reports have documented a response to vemurafenib in patients with NSCLC and a BRAF mutation. [71-73] Human Epidermal Growth Factor Receptor 2 Mazières et al. reported on a retrospective review of a consecutive series of patients with NSCLC who were tested for a HER2 mutation, and the authors assessed clinicopathologic characteristics and patient outcomes according to mutation status. [74] A HER2 mutation was identified in 65 of 3800 (1.7%) patients, and was mutually exclusive of other driver mutations (EGFR, ALK, BRAF), with the exception of 1 case in which both a HER2 and KRAS mutation were identified. The patient population in which a HER2 mutation was found had a median age of 60 years (range, 31-86), 69% were women, and 52% were never-smokers. All of the tumors were adenocarcinomas, and 50% were stage IV (n=33). The patients with stage IV disease received conventional chemotherapy, and of these, 16 patients also received HER2-targeted therapy as additional lines of therapy (for a total of 22 individual anti-her2 treatments that were evaluable). Four patients had progressive disease, 7 had disease stabilization, and 11 with partial response. PFS for patients with HER2 therapies was 5.1 months. Conclusion The data on the use of targeted therapies in NSCLC with a mutation in ROS, RET, MET, BRAF, or HER2 is preliminary and limited to a few case reports, retrospective analyses, and case series. Further studies are needed to determine whether testing for genetic alternation in these genes may be useful for targeted therapy in patients with NSCLC. Clinical Practice Guidelines National Comprehensive Cancer Network (NCCN) [46] The NCCN guidelines for NSCLC (v1.2015) note that driver mutations and gene rearrangements (i.e., driver events) are being identified such as HER2 (also known as ERBB2) and BRAF mutations, ROS1 RET gene arrangements, and MET amplification. Targeted agents are available for patients with NSCLC who have these genetic alterations, although they are FDA approved for other indications. Thus, the NCCN Guidelines recommend testing for genetic alterations using multiplex/ngs to ensure that patients receive the most appropriate treatment; patients may be eligible for clinical trials for some of these targeted agents. Summary 15 GT56

16 EGFR The evidence demonstrates that the detection of epidermal growth factor receptor (EGFR) gene mutations identifies patients with advanced non-squamous cell-type non-small cell lung cancer (NSCLC) who are likely to benefit from use of erlotinib and afatinib, and therefore represent ideal candidates for treatment with these drugs. These observations have been made in a population composed primarily of tumors with adenocarcinoma histology. Patients who are found to have wild-type tumors are unlikely to respond to erlotinib or afatinib, and they should be considered candidates for alternative therapies. Therefore, EGFR mutational analysis may be considered medically necessary to predict treatment response to erlotinib and afatinib in patients with advanced non-squamous cell-type NSCLC. There is limited evidence to indicate whether mutations within exons of the epidermal growth factor receptor (EGFR) gene are seen in patients with squamous cell-type non-small cell lung cancer (NSCLC); therefore, EGFR mutational analysis is considered investigational in these patients. KRAS KRAS mutation may be an indicator of poor prognosis in non-small cell lung cancer (NSCLC) and may predict a lack of response to tyrosine kinase inhibitors (TKIs). However, impact of testing for KRAS mutations on clinical management or to predict treatment benefit is unknown. Studies have not shown that KRAS mutations identify a population that may benefit from the use of anti-egfr monoclonal antibodies. Therefore, analysis of mutations of the KRAS gene is considered investigational as a technique to predict treatment non-response to anti-egfr monoclonal antibody therapy (e.g., cetuximab) in non-small cell lung cancer. Other Oncogenic Mutations Mutations in the genes ROS, RET, MET, BRAF, and HER2 have been characterized as potentially targetable oncogenic mutations in lung adenocarcinomas. Current evidence is limited to preliminary data from a few case reports, retrospective analyses, and case series. Further studies are needed, particularly for tumor sensitivity to various drugs and for survival outcomes, to determine whether mutations in these genes can be used in targeted therapy. Therefore testing for genetic alternations in the genes ROS, RET, MET, BRAF, and HER2 for targeted therapy in patients with non-small cell lung cancer is considered investigational. REFERENCES 1. Fathi, AT, Brahmer, JR. Chemotherapy for advanced stage non-small cell lung cancer. Semin Thorac Cardiovasc Surg Fall;20(3): PMID: Martoni, A, Marino, A, Sperandi, F, et al. Multicentre randomised phase III study comparing the same dose and schedule of cisplatin plus the same schedule of vinorelbine or gemcitabine in advanced non-small cell lung cancer. Eur J Cancer Jan;41(1): PMID: Rudd, RM, Gower, NH, Spiro, SG, et al. Gemcitabine plus carboplatin versus mitomycin, ifosfamide, and cisplatin in patients with stage IIIB or IV non-small-cell lung cancer: a phase III randomized study of the London Lung Cancer Group. J Clin Oncol Jan 1;23(1): PMID: Lynch, TJ, Bell, DW, Sordella, R, et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med May 20;350(21): PMID: GT56

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