National Medical Policy

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1 National Medical Policy Subject: Policy Number: Molecular Tumor Markers for Non-Small Cell Lung Cancer (NSCLC) NMP206 Effective Date*: November 2010 Update: June 2016 This National Medical Policy is subject to the terms in the IMPORTANT NOTICE at the end of this document For Medicaid Plans: Please refer to the appropriate State's Medicaid manual(s), publication(s), citations(s) and documented guidance for coverage criteria and benefit guidelines prior to applying Health Net Medical Policies The Centers for Medicare & Medicaid Services (CMS) For Medicare Advantage members please refer to the following for coverage guidelines first: Use Source Reference/Website Link National Coverage Determination (NCD) National Coverage Manual Citation Local Coverage Determination (LCD)* Article (Local)* Other X None Use Health Net Policy Instructions Medicare NCDs and National Coverage Manuals apply to ALL Medicare members in ALL regions. Medicare LCDs and Articles apply to members in specific regions. To access your specific region, select the link provided under Reference/Website and follow the search instructions. Enter the topic and your specific state to find the coverage determinations for your region. *Note: Health Net must follow local coverage determinations (LCDs) of Medicare Administration Contractors (MACs) located outside their service area when those MACs have exclusive coverage of an item or service. (CMS Manual Chapter 4 Section 90.2) If more than one source is checked, you need to access all sources as, on occasion, an LCD or article contains additional coverage information than contained in the NCD or National Coverage Manual. If there is no NCD, National Coverage Manual or region specific LCD/Article, follow the Health Net Hierarchy of Medical Resources for guidance. Molecular Tumor Markers for Non-Small Cell Lung Cancer Jun 16 1

2 Current Policy Statement (Please refer to the Health Net Medical Policy: K-RAS Mutation Analysis of Colon Cancer) Health Net, Inc. considers the following serum molecular tumor markers* or mutation analyses medically necessary for the prediction of sensitivity and/or resistance of chemotherapy that is receptive to non-small cell lung cancer (NSCLC). 1. Epidermal growth factor receptor (EGFR) mutation testing in individuals with nonsquamous NSCLC (i.e., adenocarcinoma, large cell) or in NSCLC NOS to predict treatment benefit from EFGR tyrosine kinase inhibitors (EGFR-TKI ) therapy. The presence of the EFGR mutation is predictive of treatment benefit from EFGR-TKI therapy. Erlotinib (Tarceva) is recommended for patients who are positive for the EGFR mutations. Osimertinib is recommended as subsequent therapy as a treatment option for patients with metastatic EGFR T790M mutation-positive tumors, as determined by FDA approved tests or other validated laboratory-developed tests test performed in a CLIA-approved laboratory (e.g., cobas EGFR Mutation Test v2) 2. ALK testing* in individuals with nonsquamous NSCLC (i.e., adenocarcinoma, large cell) or in NSCLC NOS for prediction of response to crizotinib therapy. Crizotinib, an oral ALK inhibitor, is approved for patients with locally advanced or metastatic NSCLC who have the ALK gene rearrangement (i.e. ALK positive).. Individuals with rearrangements of the EML4-ALK gene are resistant to EGFR TKI s. *NOTE: There is a FDA approved test for the anaplastic lymphoma kinase (ALK) fusion gene (i.e., Vysis ALK Break Apart FISH Probe Kit) for individuals who are considering the medication Crizotinib for the treatment of locally advanced or metastatic NSCLC. 3. KRAS gene sequencing for the selection of individuals who are candidates for tyrosine kinase inhibitor (TKI) therapy. K-RAS mutations are predictive of lack of benefit from platinum/vinorelbine chemotherapy or EGFR TKI therapy (erlotinib). *K-RAS mutations, the epidermal growth factor receptor (EGFR) mutations and the anaplastic lymphoma kinase fusion protein test (EML4-ALK fusion gene) ALK mutations, are almost always mutually exclusive, (i.e. mutations of only one of the three genes occur within any individual tumor), in NSCLC. Note: Per the NCCN, EGFR testing and ALK testing are NOT routinely recommended in patients with squamous cell carcinoma. Note: NCCN guidelines recommend that EGFR mutation testing be done as part of broad molecular profiling. Testing for ALK gene rearrangements can be done with FISH or NGS. 4. Testing for genetic alterations using multiplex/ngs for any of the following, to ensure that individuals with advanced NSCLC receive the most appropriate treatment: HER2 mutations to predict response to trastuzumab, afatinib BRAF mutations to predict response to vemurafenib, dabrafenib Molecular Tumor Markers for Non-Small Cell Lung Cancer Jun 16 2

3 MET amplification to predict response to crizotinib ROS1 rearrangements to predict response to crizotinib RET rearrangements to predict response to cabozantinib Medically Necessary Health Net, Inc. considers proteomic testing* (i.e., Veristrat testing) for advanced NSCLC and wild-type (i.e., no mutation detected) EGFR or with unknown EGFR status, medically necessary, to determine second-line treatment, for individuals who have failed first-line system chemotherapy. A patient with a poor * classification should not be offered erlotinib in the second-line setting. Test results will be used to decide whether to proceed with erlotinib (Tarceva) therapy or chemotherapy. *NOTE: NCCN 2015 recommendation. Investigational Health Net, Inc. considers either of the following tests investigational: 1. ERCC1 expression analysis for individuals with NSCLC for whom treatment with a platinum-based chemotherapy regimen is being considered. Although it has been proposed as a prognostic and predictive marker for NSCLC, studies have not demonstrated the level of evidence necessary to use ERCC1, as a biomarker. In addition, since NSCLC patients are typically treated with multiple chemotherapy agents that function in different manners, it is believed that the assessment of multiple biomarkers may be necessary in order to select the optimal treatment regimen for a given NSCLC patient; or 2. Biodesix GeneStrat, a genetic test that uses a targeted liquid biopsy method to identify variants that provide information that may inform the choice of treatments for NSCLC. The GeneStrat genomic test is a panel that includes testing of the following: EGFR sensitizing mutations (Exon 19 ΔE746-A750; Exon 21 L858R), EGFR Resistance Mutation (Exon 20 T790M), ALK Fusion Products (EML4-ALK fusion variants 1, 2, 3), KRAS Mutations (G12C; G12D; G12V) and BRAF Mutation (V600E). Molecular Tumor Markers for Non-Small Cell Lung Cancer Jun 16 3

4 Definitions K-RAS Kirsten rat sarcoma EGFR Epidermal Growth Factor Receptor TKI Tyrosine kinase inhibitors NSCLC Non-small cell lung cancer GTP Guanosine diphosphate GAP GTPase-activating protein GDP Guanosine diphosphate PCR Polymerase chain reaction HR Hazard ratio HER1 Human epidermal growth factor receptor type 1 ALK anaplastic lymphoma kinase fusion protein test (EML4-ALK fusion gene) ALK mutations QOL Quality of life ERCC1 Excision repair cross-complementation group 1 protein NER Nucleotide excision repair MALDI Matrix-assisted laser desorption ionization MS Mass spectrometry SELDI/TOF Surface-enhanced laser desorption ionization/time-of-flight Codes Related To This Policy NOTE: The codes listed in this policy are for reference purposes only. Listing of a code in this policy does not imply that the service described by this code is a covered or noncovered health service. Coverage is determined by the benefit documents and medical necessity criteria. This list of codes may not be all inclusive. On October 1, 2015, the ICD-9 code sets used to report medical diagnoses and inpatient procedures have been replaced by ICD-10 code sets. ICD-9 Codes Malignant neoplasm of bronchus and lung [covered for metastatic or recurrent epidermal growth factor receptor (EGFR) positive non-small cell lung cancer only] ICD-10 Codes C34.00-C34.92 Malignant neoplasm of bronchus and lung CPT Codes EFGR(epidermal growth factor receptor) e.g., non-small cell lung cancer) gene analysis, common variants (e.g. exon 19 LREA deletion,l858r, T790M, G719A, G179S, L861Q) KRAS (v-ki-ras2 Kirsten rat sarcoma viral oncogene) (eg, carcinoma) gene analysis, variants in condons 12 and Molecular diagnostics; isolation or extraction of highly purified nucleic acid, each nucleic acid type (i.e., DNA or RNA) (code deleted 12/2012) Molecular diagnostics; mutation identification by sequencing, single segment, each (code deleted 12/2012) Molecular diagnostics; interpretation and report (code deleted 12/2012) Unlisted chemistry procedure Molecular Tumor Markers for Non-Small Cell Lung Cancer Jun 16 4

5 HCPCS Codes N/A Scientific Rationale Update June 2016 According to Biodesix, GeneStrat is a genetic test that uses a targeted liquid biopsy method to identify variants that provide information that may inform the choice of treatments for NSCLC. According to the testing laboratory, cell-free DNA is extracted from plasma and emulsified with oil into surfactant-stabilized droplets. In each droplet, fluorescently labeled oligonucleotide probes hybridize to the region of interest in the PCR reaction. Once the target is amplified, the fluorescent probe is cleaved and quantification of the wild-type DNA and variant DNA is performed by comparing the intensity of each fluorescent signal. The fraction of variant and wildtype droplets is compared using unstated analytical software tools. According to Biodesix, deleterious variants in 4 genes (epidermal growth factor receptor [EGFR]; anaplastic lymphoma receptor tyrosine kinase [ALK]; Kirsten rat sarcoma viral oncogene homolog [KRAS]; B-raf proto-oncogene, serine/threonine kinase [BRAF]) are evaluated in the GeneStrat full panel: EGFR exon 19 ΔE746-A750, EGFR exon 21 L858R, EGFR exon 20 T790M, ALK fusion products (EML4-ALK fusion variants 1, 2, 3), KRAS G12C, KRAS G12D, KRAS G12V, and BRAF V600E. According to Biodesix the tests can also be ordered indivudally or as a GeneStrat/VeriStrat Reflex Panel (i.e., VeriStrat Proteomic Test will be run following the GeneStrat Genomic Test (all genes) only if patient does not have an EGFR sensitizing mutation). At this time, no studies evaluating the GeneStrat test were identified, thus the clinical utility and analytic and clinical validity have not been demonstrated. Per NCCN guidelines on NSCLC (4.2016), The NCCN panel strongly endorses broader molecular profiling (also known as precision medicine) to identify rare driver mutations to ensure that patients receive the most appropriate treatment; patients may be eligible for clinical trials for some of these targeted agents. The guidelines do not mention panels such as the GeneStrat test in the current guidelines. In the updated 2016 NCCN guidelines on NSCLC (version 2), the NCCN panel added a 2A recommendation for osimertinib as second-line and beyond (subsequent) therapy for patients with metastatic EGFR T790M mutation who have progressed on sensitizing EGFR TKI therapy (e.g., erlotinib, gefitinifb, afatanib) A footnote was added to the algorithmn noting that osimertinib is approved for patients with metastatic EGFR T790M mutation-positive tumors, as determined by approved tests or other validated laboratory-developed tests performed in a CLIA-approved laboratory. In November 2015, the FDA granted approval to the cobas EGFR Mutation Test v2. The cobas EGFR Mutation Test v2 is a real-time PCR test for the qualitative detection of defined mutations of the epidermal growth factor receptor (EGFR) gene in DNA derived from formalin-fixed paraffin-embedded tumor tissue (FFPET) from non-small cell lung cancer (NSCLC) patients. The first version of the test was initially approved in 2013 as an aid in selecting patients with NSCLC for whom Tarceva (erlotinib), an EGFR tyrosine kinase inhibitor (TKI), was indicated. Version 2 expands the use to aid in identifying patients with NSCLC whose tumors have defined EGFR mutations and for whom safety and efficacy of a drug have been established as follows: Tarceva (erlotinib) -Exon 19 deletions and L858R Tagrisso (osimertinib) -T790M Molecular Tumor Markers for Non-Small Cell Lung Cancer Jun 16 5

6 Gao et al (2016) aimed to evaluate the feasibility of the NextDaySeq-Lung panel, which is an NGS-based assay for mutation analysis of key driver genes in lung cancer, in a clinical setting. A total of 138 FFPE samples of non-small cell lung cancer (NSCLC) were examined in parallel with assays developed on the nextgeneration sequencing (NGS), quantitative PCR (QPCR), and Sanger sequencing (Sanger) platforms for somatic mutations in EGFR, KRAS, PIK3CA, and BRAF. The assays with the three platforms were compared and analyzed. Compared with Sanger, NGS and QPCR assays have significant higher sensitivity, as Sanger failed to detect variants with mutation rates lower than 15%. Meanwhile, NGS and QPCR assays showed similar analytical sensitivity, specificity, and high concordance. In addition, the NGS assay exhibited advantages over QPCR in providing accurate information of allele sequence and mutation frequency, and detecting non-hotspot mutations. The authors concluded the NGS assay has significant technical advantages over Sanger and QPCR assays. It shows good potential as a solid molecular diagnostics assay in the clinical setting. Zhang et al (2016) reported KRAS mutation is one of the major driver genes of NSCLC. KRAS is a resistant predictor of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), which raises controversy because of its role in chemotherapy sensitivity and prognosis. The authors sought to accumulate clinical experience in treating NSCLC patients harboring KRAS mutation. A total of 107 NSCLC patients harboring KRAS mutation were analyzed retrospectively. The efficacy was analyzed in terms of first-line chemotherapy or EGFR-TKIs therapy. The objective response rate (ORR) to first-line chemotherapy of 52 patients with advanced disease harboring KRAS mutation was 9.6%. The disease control rate (DCR) was 53.8%, and the median progression-free survival (PFS) was 3 months. The ORR to EGFR-TKIs therapy in 21 patients harboring KRAS mutation and EGFR/KRAS co-mutation was 9.5%; the DCR was 23.8%, and the median PFS was 1 month. The ORR and DCR to EGFR-TKIs therapy of patients with EGFR/KRAS comutation were significantly higher than those of patients with KRAS mutation (50% vs 0, P=0.029; 75% vs 11.8%, P=0.043); the median PFS was also significantly longer (3 months vs 1 month, P=0.004). The authors concluded the efficacy to firstline chemotherapy and EGFR-TKIs therapy in NSCLC patients harboring KRAS mutation was poor; thus, new drugs should be developed. Furthermore, the existence of EGFR/KRAS co-mutation was confirmed. Hence, EGFR-TKIs therapy should be administered to patients with EGFR/KRAS co-mutation. Zheng et al (2016) evaluated the clinicopathologic characteristics, oncogenic drivers, and prognosis of former smokers with NSCLC, and to compare them with those of the current and never smokers. This investigation was a single-institution retrospective study of 2289 NSCLC patients, who were classified as former, current, or never smokers. A collection was made of the clinicopathological characteristics, spectra of well-identified driver genes and survival rates. The survival rates were compared using log-rank test, and independent prognostic factors, identified using Cox regression analysis. Of 2289 NSCLC patients, 257 (11.2%) were former smokers; 868 (37.9%), current smokers; and 1164 (50.9%), never smokers. Compared with the current, the former were characterized by older age at diagnosis (64.3y vs. 59.9y; P < 0.001), earlier TNM stage (stage I, 47.9% vs. 39.5%; P = 0.017), fewer solid predominance in adenocarcinomas (16.2% vs. 29.5%; P = 0.005), and more EGFR mutation (33.2% vs. 20.7%; P < 0.001) but less KRAS mutation (6.7% vs. 11.9%, P = 0.041). No statistically significant survival differences were observed between the former and current. However, the light former smokers presented favorable overall survival when compared with the light current and heavy former or current (the light former vs. the heavy former, P = 0.028; the light former vs. the light current, P = 0.048; and the light former vs. the heavy current, P = 0.048). Our findings suggest that the former smokers with Molecular Tumor Markers for Non-Small Cell Lung Cancer Jun 16 6

7 NSCLCs can have distinctive clinicopathologic characteristics, oncogenic drivers, and prognosis, and they, especially the light former, can benefit from smoking cessation. Matam et al (2015) reported EGFR is one of the targeted molecular markers in many cancers including lung malignancies. Gefitinib and erlotinib are two available therapeutics that act as specific inhibitors of tyrosine kinase (TK) domains. The authors performed a case-control study with formalin-fixed paraffin-embedded tissue blocks (FFPE) from tissue biopsies of 167 NSCLC patients and 167 healthy controls. The tissue biopsies were studied for mutations in exons of the EGFR gene. This study was performed using PCR followed by DNA sequencing. The authors identified 63 mutations in 33 men and 30 women. Mutations were detected in exon 19 (dele746-a750, dele746-t751, dell747-e749, dell747-p753, dell747-t751) in 32 patients, exon 20 (S786I, T790M) in 16, and exon 21 (L858R) in 15. No mutations were observed in exon 18. The 63 patients with EFGR mutations were considered for upfront therapy with oral tyrosine kinase inhibitor (TKI) drugs and have responded well to therapy over the last 15 months. The control patients had no mutations in any of the exons studied. The advent of EGFR TKI therapy has provided a powerful new treatment modality for patients diagnosed with NSCLC. The authors concluded the study emphasizes the frequency of EGFR mutations in NSCLC patients and its role as an important predictive marker for response to oral TKI in the south Indian population. Scientific Rationale Update May 2015 Proteomic testing has been proposed as a way to predict outcomes, response and selection of targeted therapy for patients with non-small-cell lung cancer (NSCLC). The VeriStrat assay, has been investigated as a predictive marker for response to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). The test relies on a predictive algorithm based on matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) analysis of pretreatment serum to generate a good or poor assessment for response to TKIs. VeriStrat has been proposed as a method to predict response to erlotinib in patients with NSCLC after failure of treatment with first-line therapy. Proposed uses have been in addition to EGFR testing, or in patients who do not have tumor samples available for EGFR testing. Although the VeriStrat MALDI-MS-based predictive algorithm has the most literature associated with it, other investigators have used alternative MS methods, such as surface-enhanced laser desorption ionization/time-of-flight (SELDI/TOF) mass spectrometry, and alternative predictive algorithms, in the assessment of proteomic predictors of lung cancer risk. Lazzari et al. (2012) The authors previous study showed that pretreatment serum or plasma Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry may predict clinical outcome of non-small cell lung cancer (NSCLC) patients treated with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). In this study, plasma proteomic profiles of NSCLC patients were evaluated in the course of EGFR TKI therapy. Plasma samples were collected at baseline, in the course of gefitinib therapy and at treatment withdrawal. Samples were analyzed by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry. Acquired spectra were classified by the VeriStrat test into "good" and "poor" profiles. The association between VeriStrat classification and progression-free survival (PFS) and overall survival (OS), and types of clinical progression, was analyzed. Plasma samples from 111 NSCLC patients treated with gefitinib were processed. VeriStrat "good" classification at baseline correlated with longer PFS (hazard ratio [HR], 0.54; 95% confidence interval, ; p = 0.005) and OS (HR, 0.40; 95% confidence interval, ; p < ), when compared with VeriStrat "poor." Multivariate analysis confirmed longer PFS (HR, 0.52; p = 0.025) and OS (HR, 0.44; p = 0.001) in patients classified as VeriStrat "good", when Molecular Tumor Markers for Non-Small Cell Lung Cancer Jun 16 7

8 VeriStrat was considered as a time-dependent variable. About one-third of baseline "good" classifications had changed to "poor" at the time of treatment withdrawal; progression in these patients was associated with the development of new lesions. The author s findings support the role of VeriStrat in the assistance in treatment selection of NSCLC patients for EGFR TKI therapy and its potential utility in treatment monitoring. Ciuleanu et al. (2012) Erlotinib, docetaxel, and pemetrexed are approved for the second-line treatment of non-small-cell lung cancer (NSCLC), but no data from large clinical trials are available. The authors completed the Tarceva In Treatment of Advanced NSCLC (TITAN) study to assess the efficacy and tolerability of second-line erlotinib versus chemotherapy in patients with refractory NSCLC. TITAN was an international, randomised multicentre, open-label, phase 3 study that was done at 77 sites in 24 countries. Chemotherapy-naive patients with locally advanced, recurrent, or metastatic NSCLC received up to four cycles of first-line platinum doublet chemotherapy, after which patients with disease progression during or immediately after chemotherapy were offered enrolment into TITAN. Enrolled patients were randomly assigned (1:1) by a minimisation method to ensure balanced stratification, to receive erlotinib 150 mg/day or chemotherapy (standard docetaxel or pemetrexed regimens, at the treating investigators' discretion), until unacceptable toxicity, disease progression, or death. Patients were stratified by disease stage, Eastern Cooperative Oncology Group performance status, smoking history, and region of residence. The primary endpoint was overall survival in the intention-totreat population. TITAN was halted prematurely because of slow recruitment. This study is registered with ClinicalTrials.gov, number NCT Between April 10, 2006, and Feb 24, 2010, 2590 chemotherapy-naive patients were treated with firstline platinum doublet chemotherapy, of whom 424 had disease progression and were enrolled into TITAN. 203 patients were randomly assigned to receive erlotinib and 221 were assigned to receive chemotherapy. Median follow-up was 27 9 months (IQR ) in the erlotinib group and 24 8 months ( ) in the chemotherapy group. Median overall survival was 5 3 months (95% CI ) with erlotinib and 5 5 months ( ) with chemotherapy (hazard ratio [HR] 0 96, 95% CI ; log-rank p=0 73). The adverse-event profile of each group was in line with previous studies. Rash (98/196 [50%] in the erlotinib group vs 10/213 [5%] in the chemotherapy group for all grades; nine [5%] vs none for grade 3 or 4) and diarrhea (36 [18%] vs four [2%] for all grades; five [3%] vs none for grade 3 or 4) were the most common treatment-related adverse events with erlotinib, whereas alopecia (none vs 23 [11%] for all grades; none vs one [<1%] for grade 3/4) was the most common treatment-related adverse event with chemotherapy. No significant differences in efficacy were noted between patients treated with erlotinib and those treated with docetaxel or pemetrexed. Since the toxicity profiles of erlotinib and chemotherapy differ, second-line treatment decisions should take into account patient preference and specific toxicity risk profiles. Karampeazis et al. (2013) Patients with stage IIIB/IV NSCLC who progressed after first-line or second-line treatment were randomized to receive either pemetrexed or erlotinib. In total, 21.7% of patients in the pemetrexed arm and 23.5% of patients in the erlotinib arm had squamous cell histology, and treatment was third line in 39.2% and 46.4% of patients, respectively. The primary study endpoint was time to tumor progression (TTP). Epidermal growth factor receptor/v-ki-ras2 Kirsten rat sarcoma viral oncogene homolog (EGFR/KRAS) mutation status also was investigated. There was no difference in terms of the TTP (P =.195), the objective response rate (P =.469), or overall survival (P =.986) between the 2 treatment arms. In patients who had squamous cell histology, erlotinib resulted in a superior TTP compared with pemetrexed (4.1 months vs 2.5 months, respectively; P =.006). The incidence of grade 3 and 4 neutropenia, thrombocytopenia, and asthenia was significantly higher Molecular Tumor Markers for Non-Small Cell Lung Cancer Jun 16 8

9 in the pemetrexed arm, whereas the incidence of grade 3 and 4 skin rash was higher in the erlotinib arm. Both pemetrexed and erlotinib had comparable efficacy in pretreated patients with metastatic NSCLC, and the current results indicated that genotyping of tumor cells may have an important effect on treatment efficacy. Gregorc et al. (2014) An established multivariate serum protein test can be used to classify patients according to whether they are likely to have a good or poor outcome after treatment with EGFR tyrosine-kinase inhibitors. The authors assessed the predictive power of this test in the comparison of erlotinib and chemotherapy in patients with non-small-cell lung cancer, (PROSE STUDY). From Feb 26, 2008, to April 11, 2012, patients (aged 18 years) with histologically or cytologically confirmed, second-line, stage IIIB or IV non-small-cell lung cancer were enrolled in 14 centres in Italy. Patients were stratified according to a minimization algorithm by Eastern Cooperative Oncology Group performance status, smoking history, centre, and masked pretreatment serum protein test classification, and randomly assigned centrally in a 1:1 ratio to receive erlotinib (150 mg/day, orally) or chemotherapy (pemetrexed 500 mg/m (2), intravenously, every 21 days, or docetaxel 75 mg/m (2), intravenously, every 21 days). The proteomic test classification was masked for patients and investigators who gave treatments, and treatment allocation was masked for investigators who generated the proteomic classification. The primary endpoint was overall survival and the primary hypothesis was the existence of a significant interaction between the serum protein test classification and treatment. Analyses were done on the per-protocol population. This trial is registered with ClinicalTrials.gov, number NCT patients were randomly assigned to chemotherapy and 143 to erlotinib, and 129 (91%) and 134 (94%), respectively, were included in the per-protocol analysis. 88 (68%) patients in the chemotherapy group and 96 (72%) in the erlotinib group had a proteomic test classification of good. Median overall survival was 9 0 months (95% CI ) in the chemotherapy group and 7 7 months ( ) in the erlotinib group. We noted a significant interaction between treatment and proteomic classification (pinteraction=0 017 when adjusted for stratification factors; pinteraction=0 031 when unadjusted for stratification factors). Patients with a proteomic test classification of poor had worse survival on erlotinib than on chemotherapy (hazard ratio 1 72 [95% CI ], p=0 022). There was no significant difference in overall survival between treatments for patients with a proteomic test classification of good (adjusted HR 1 06 [ ], p=0 714). In the group of patients who received chemotherapy, the most common grade 3 or 4 toxic effect was neutropenia (19 [15%] vs one [<1%] in the erlotinib group), whereas skin toxicity (one [<1%] vs 22 [16%]) was the most frequent in the erlotinib group. The authors findings indicate that serum protein test status is predictive of differential benefit in overall survival for erlotinib versus chemotherapy in the second-line setting. Patients classified as likely to have a poor outcome have better outcomes on chemotherapy than on erlotinib. This proteomic signature assay (i.e., VeriStrat) was validated in this phase III PROSE trial. The above PROSE study by Gregorc et al. (2014) was based on the Clinical Trial Study of Blood and Tissue Samples in Predicting Response to Second-Line Therapy Using Erlotinib Hydrochloride or Chemotherapy in Patients With Advanced Non-Small Cell Lung Cancer with the ClinicalTrials.gov Identifier of NCT This Clinical Trial notes that the recruitment status of this study is unknown because the information has not been verified recently. It was last updated on August 9, This randomized phase III trial is studying blood and tissue samples in predicting response to second-line therapy using erlotinib hydrochloride or chemotherapy in patients with advanced non-small cell lung cancer. The objectives of this study were: To evaluate the predictive value of proteomic profiling on the effect of secondline therapy with erlotinib hydrochloride versus standard chemotherapy Molecular Tumor Markers for Non-Small Cell Lung Cancer Jun 16 9

10 (pemetrexed disodium or docetaxel) in patients with advanced non-small cell lung cancer. To assess the role of other known tissue-based predictive markers (e.g., EGFRgene copy number, EGFR-protein expression, pakt, pmapk, EGFR mutations, EMT markers, and k-ras mutation). Ruiz et al. (2015) completed a clinical prospective on PROSE, noted above by Gregorc et al. (2014). The authors note that PROSE was a well-designed clinical trial with a few limitations. Although the name, VeriStrat test, was not noted in PROSE, it does seem to refer to this test, which confirmed its prognostic ability reporting a different progression free survival (PFS) and overall survival (OS) independent of the treatment given. These results include a 1.4 month difference in median PFS and a 4.5 month difference in median OS between VG and VP patients. This magnitude of effect on prognosis indicates that VeriStrat is measuring factors in the blood stream that strongly correlate with the outcomes of patients. Since this is a black-box test, the details of what is being measured have not yet been made available or are not understood. Understanding what these factors are, could potentially lead to identification of new targets and better understanding of resistance to tyrosine kinase inhibitors. As for the predictive value of VeriStrat, PROSE reports a differential treatment response in VP patients. The VP group derived little benefit from erlotinib with worse OS when compared with to VP patients who received chemotherapy. A small number of EGFR activating mutations were included in this study which was performed predating EGFR mutation testing as standard of care. In addition, there were a small number of patients with squamous cell histology, and although tumors were evaluated for KRAS status, the role is in predicting response in KRAS positive patients was not reported. In summary, VeriStrat is a test with powerful prognostic utility. For patients with EGFR wild-type cancers who would prefer to take erlotinib rather than chemotherapy in the second-line setting, the results of PROSE support the use of VeriStrat testing to help those patients avoid using an ineffective medication if they fall into a highly resistant group. For patients who prefer intravenous treatment, the value of VeriStrat testing in the second-line is somewhat more limited. Given that patients may not know what their future preferences may be, it is likely that many clinicians will obtain VeriStrat testing to help their patients make informed decisions using the best technology currently available for weighing risks and benefits. Mazzoni et al. (2015) European Society of Medical Oncology (ESMO) guidelines say that patients clinically or radiologically progressing after first-line chemotherapy, irrespective of administration of maintenance chemotherapy, and with an Eastern Cooperative Oncology Group (ECOG) Performance Status (PS) 0-2, should be offered second-line chemotherapy. In this setting, single agent chemotherapy improve disease-related symptoms and overall survival (OS) to nearly months, with 30% of patients alive at 1 year. Comparable options in the second-line therapy consist of pemetrexed, for non-squamous histology only, or docetaxel. Erlotinib is an additional potential option in patients with PS 0-2 (1). Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are the standard treatment option for advanced NSCLC patients harboring EGFR-activating mutations. Large RCTs enriching for the patients harboring EGFR-activating mutations showed the superiority of TKI treatment over conventional cytotoxic drugs in terms of progression-free survival (PFS) and objective response rate (ORR). However, the majority of patients with advanced NSCLC worldwide do not have tumors harboring EGFR-activating mutations. Erlotinib was approved as a second or third-line standard treatment based on the results of BR.21 trial, which demonstrated the prolongation of OS compared with the best supportive care in all NSCLC histological subtype patients EGFR-unselected, not eligible for further chemotherapy, including patients with PS 3. Although the EGFR TKI treatment has been widely used in patients with Molecular Tumor Markers for Non-Small Cell Lung Cancer Jun 16 10

11 unknown (UK) or wild-type (WT) EGFR status NSCLC, its benefit is less pronounced and more controversial than in those with EGFR-activating mutations. Three randomized trials (INTEREST, TITAN and HORG study), comparing EGFR TKI with second-line chemotherapy agents (docetaxel or pemetrexed), failed to show greater efficacy of chemotherapy in patients with UK or WT EGFR tumors, with a better toxicity profile and quality of life (QoL) for TKI. However, the recently reported TAILOR and DELTA trials demonstrated a significant improvement in PFS with second-line chemotherapy compared with erlotinib in patients with WT EGFR NSCLC. A meta-analysis of these trials demonstrated that for patients with advanced NSCLC harboring WT EGFR tumors conventional chemotherapy was associated with improvement in PFS and with a higher ORR, compared with first-generation EGFR TKIs. However, there was no statistically significant difference in terms of OS between the two treatment groups. In this particular and complicated background, it is becoming increasingly important to identify other markers in order to select patients to treat with an EGFR TKI or with a cytotoxic agent. The PROSE trial aim was trying to indentify one of these markers; this trial was a biomarker-stratified, randomized phase 3 trial, written by Gregorc et al. The biomarker status was used to guide analysis but not to assign treatment. The primary aim of this trial was to assess the predictive power of the proteomic test in the comparison of two approved treatments in second line, erlotinib and chemotherapy, in patients with NSCLC. The proteomic test was developed by Taguchi et al. (2007) It is commercially available as VeriStrat and was used to assign one of two classifications (i.e., good or poor) by comparison of the intensity of eight regions in the mass spectra obtained from patients pretreatment serum samples with the intensity of those of a reference set. The test was used for the analysis of serum to identify patients likely to have good or poor survival when treated with EGFR TKIs. The patients enrolled in PROSE study were submitted to the serum test and randomized to receive erlotinib or chemotherapy. The trial enrolled 263 patients: 184 (70%) were classified good and 79 (30%) poor. Patients with a poor proteomic test classification had significantly shorter OS when treated with erlotinib than did those given chemotherapy [median 3.0 months (95% CI: ) vs. 6.4 months (95% CI: ); HR 1.72 (95% CI: ), P=0.022]; instead, in the good classification group, there was no significant difference in OS between the treatment groups and median OS was 10.9 months (95% CI: ) in the chemotherapy group and 11.0 months (95% CI: ) in the erlotinib group [HR 1.06 (95% CI: ), P=0.714]. In clinical practice, patients considered to have a poor prognosis usually are delivered to TKI therapy, due to its better toxicity profile in comparison with chemotherapy. This trial highlight the fact that this practice is no longer effective, in fact patients with a proteomic test classification of poor (30%) should not receive erlotinib. Conversely, patients classified as good seems to have similar results both with erlotinib therapy and with chemotherapy; but, this study was not originally designed and powered to detect survival benefit of erlotinib versus chemotherapy within the good proteomic classification group, indeed the study was designed to evaluate the interaction test between VeriStrat status and treatment effect. In hindsight, this is a limitation of the trial and the study design could have been improved by enlarging the study to address this additional analysis. In the study no statistical significant differences were detected in terms of PFS and ORR between the two treatment groups, irrespective of the proteomic test. Therefore the trial demonstrate that VeriStrat can be utilized as a predictive factor in order to select which patients are not to be treat with EGFR TKI; in fact poor classified patients exhibit a statistical significant survival advantage when treated with chemotherapy compared to erlotinib; while no significant informations were showed for good classified patients, for whom both treatment options remain effectives. Similar results were showed also for EGFR WT population. Furthermore, this was the first prospective study to confirm the prognostic role of VeriStrat test, in fact patients with a classification of good had better OS and PFS than did those with a classification of poor, even when the data is Molecular Tumor Markers for Non-Small Cell Lung Cancer Jun 16 11

12 correct considering the other prognostic factors. Median overall survival was 11.0 months (95% CI: ) and 3.7 months (95% CI: ) for good and poor classifications, respectively [HR 2.50 (95% CI: ), P<0.0001]. Median PFS was 3.4 months (95% CI: ) and 2.0 (95% CI: ) for good and poor classification groups, respectively [HR 1.75 (95% CI: ), P<0.0001]. The PROSE study is a well conducted and designed trial, however, open issues that remain include the accessibility of the test on a large scale, and it still remains unclear the biological rational of VeriStrat test and its correlation with EGFR. There is a Randomized Phase III Clinical Trial on Testing of Drugs Erlotinib and Docetaxel in Lung Cancer Patients Classified Regarding Their Outlook Using VeriStrat, which is ongoing but not currently recruiting participants. The ClinicalTrials.gov Identifier is NCT , and it was last updated on February 9, Using a laboratory test (VeriStrat), patients with relapsed squamous cell lung cancer are assigned to two strata, VSG (VeriStrat Good) and VSP (VeriStrat Poor). They are then randomized between an EGFR-TK inhibitor (erlotinib) and chemotherapy (Docetaxel). It is hypothesized that the VeriStrat test results are able to predict the benefit of treatment with erlotinib vs docetaxel. This would suggest a significant improvement in progression-free survival for VSG patients when treated with Erlotinib, and no significant improvement in VSP patients who receive the same treatment. The estimated study completion date is August There is another Clinical Trial on Veristrat as Predictor of Benefit of First Line Non- Small Cell Lung Cancer (NSCLC) Patients From Standard Chemotherapy, which is currently recruiting participants. The ClinicalTrials.gov Identifier is NCT , and it was last updated on November 23, VeriStrat is a pretreatment bloodbased test correlated with clinical outcome after EGFR-TKI therapy in non-small cell lung cancer (NSCLC) patients. The investigators hypothesis is that VeriStrat could be also employed as a biomarker of benefit from treatment with standard chemotherapy regimens in first line NSCLC patients. The primary objective of this study is to evaluate the potential role of VeriStrat test as a biomarker of benefit from treatment with standard chemotherapy regimens in first line NSCLC patients in terms of progression-free survival (PFS) (primary endpoint) in two populations: patients with non-squamous histology treated with cisplatin and pemetrexed, and patients with squamous histology treated with cisplatin and gemcitabine. The estimated study completion date is May 2015, however, it is now June 2015, and this study is still recruiting participants. An additional Clinical Trial is on Effect of Symptom Management on Inflammation and Survival in Metastatic Lung Cancer which is currently recruiting participants. The ClinicalTrials.gov Identifier is NCT and it was last updated on October 20, There is a growing body of evidence that implicates inflammation as a mechanism of disease progression and reduced survival in patients with advanced cancer. Elevated c-reactive protein levels have been shown to be associated with reduced performance status, specific cancer related symptoms and reduced overall survival. C-reactive protein levels are a surrogate for IL-6 expression. IL-6 is part of an inflammatory signature predicting cancer recurrence. VeriStrat is a multivariate test which measures protein expression related to a host/tumor interaction mediated by inflammation. The investigators hope to examine the relationship between quality of life outcomes per FACT-L survey and correlate them with changes in c-reactive protein levels and the VeriStrat status. The hypothesis of this study is that the remarkable survival benefit in the Temel study is mediated by reduced inflammation with improvement of symptom control. The estimated study completion date is July Molecular Tumor Markers for Non-Small Cell Lung Cancer Jun 16 12

13 Per the NCCN Guidelines, Version , updates on Non-Small Cell Lung Cancer, the following 2A recommendations are noted: Proteomic testing for patients with NSCLC and wild-type EGFR or with unknown EGFR status. A patient with a poor classification should not be offered erlotinib in the second-line setting. Scientific Rationale Update September 2014 Per the NCCN guidelines on systemic therapy for advanced or metastatic NSCLC (4.2014): The drug regimen with the highest likelihood of benefit with toxicity deemed acceptable to both the physician and the patient should be given as initial therapy for advanced lung cancer. Stage, weight loss, performance status, and gender predict survival Platinum-based chemotherapy prolongs survival, improves symptom control, and yields superior quality of life compared to best supportive care Histology of NSCLC is important in the selection of systemic therapy New agent/platinum combinations have generated a plateau in overall response rates (approx 25-35%), time to progression (4-6 mo), median survival (8-10 mo), 1 year survival rate (30-40%), and 2 year survival rate (10-15%) in fit patients Unfit at any age (performance status 3-4) do not benefit from cytotoxic treatment, except erlotinib for EGFR mutation-positive patients. NCCN recommends testing for genetic alterations (i.e., driver events) in select patients with NSCLC, because targeted therapy has been shown to decrease tumor burden, decrease symptoms, and dramatically improve the quality of life for patients with specific genetic alterations. NCCN notes that the number of available targeted agents is increasing. They note the following targeted agents are now recommended for patients with specific genetic alterations: afatinib, cabozantinib, certitinib, crizotinib, dabreafenib, vemurafenib. Crizotinib, an inhibitor of ALK, ROS1, and MET tyrosine kinases, is approved by the FDA for patients with locally advanced or metastatic NSCLC who have ALK gene rearrangements. The guidelines note that Crizotinib has been shown to yield very high response rates (>60%) when used in patients with advanced NSCLC who have ALK rearrangements. ROS1 rearrangement ROS1 is a receptor tyrosine kinase of the insulin receptor family that acts as a driver oncogene in 1 to 2 percent of NSCLC via a genetic translocation between ROS1 and the partners CD74, SLC24A2, or FIG. Histologic and clinical features that are associated with ROS1 translocations include adenocarcinoma histology, younger patients, and never-smokers. ROS1 translocations are identified by a FISH breakapart assay similar to that used for ALK translocations. The ROS1 tyrosine kinase is highly sensitive to crizotinib in preclinical models, due to a high degree of homology between the ALK and ROS tyrosine kinase domains. An open label, international study of crizotinib is in progress for patients whose tumor contains the ROS1 translocation (NCT ). MET Expression MET is a tyrosine kinase receptor for hepatocyte growth factor (HGF). While MET mutations are quite infrequent and of unknown clinical significance in NSCLC, increased MET expression may predict response to MET targeted drugs. Standard Molecular Tumor Markers for Non-Small Cell Lung Cancer Jun 16 13

14 testing methods for MET expression testing include immunohistochemistry (IHC), which is positive in 25 to 50 percent of NSCLC specimens, and FISH for MET gene amplification, which occurs at an intermediate or high level in approximately 6 percent of patients with NSCLC and appears to be smoking related. MET expression also appears to be associated with a worse prognosis. HER2 mutation HER2 (ERBB2) is an EGFR family receptor tyrosine kinase. Mutations in HER2 have been detected in approximately 1 to 2 percent of NSCLC tumors. They usually involve small in-frame insertions in exon 20, but point mutations in exon 20 have also been observed. These tumors are predominantly adenocarcinomas, are more prevalent among never-smokers, and a majority of these patients are women. There is no obvious association between HER2 amplification and HER2 mutations, and previous trials demonstrated no benefit for trastuzumab in HER2 amplified NSCLC. newer case series suggest that patients with tumors harboring HER2 insertions often respond to trastuzumab and chemotherapy or to afatinib, an EGFR/HER2 tyrosine kinase inhibitor, with time to progression generally around four months. Larger clinical trials are ongoing to further define efficacy of these classes of agents in this lung cancer subtype. BRAF mutation BRAF is a downstream signaling mediator of KRAS which activates the MAP kinase pathway. BRAF mutations have been observed in 1 to 3 percent of NSCLC and are usually associated with a history of smoking. BRAF mutations have been identified predominantly in patients with adenocarcinoma. Activating BRAF mutations can occur either at the V600 position of exon 15, like in melanoma, or outside this domain. BRAF mutations have also been described as a resistance mechanism in EGFR mutation positive NSCLC. The BRAF inhibitor dabrafenib is being studied in a phase I/II study in patients with previously treated advanced NSCLC whose tumors contain the V600E mutation. RET rearrangements The RET gene encodes a cell surface tyrosine kinase receptor that is frequently altered in medullary thyroid cancer. Recurrent translocations between RET and the various fusion partners (CCDC6, KIF5B, NCOA4) have been identified in approximately 1 percent of patients with adenocarcinoma or adenosquamous carcinoma of the lung. Case reports have described responses to both vandetanib and cabozantinib in patients whose tumors contained a RET translocation. Clinical trials with RET inhibitors are ongoing in patients with tumors known to contain a RET translocation. Bergethon et al (2012) reported chromosomal rearrangements involving the ROS1 receptor tyrosine kinase gene have recently been described in a subset of NSCLCs. The authors examined the clinical characteristics and treatment outcomes of patients with NSCLC with ROS1 rearrangement. Using a ROS1 fluorescent in situ hybridization (FISH) assay, the authors screened 1,073 patients with NSCLC and correlated ROS1 rearrangement status with clinical characteristics, overall survival, and when available, ALK rearrangement status. In vitro studies assessed the responsiveness of cells with ROS1 rearrangement to the tyrosine kinase inhibitor crizotinib. The clinical response of one patient with ROS1-rearranged NSCLC to crizotinib was investigated as part of an expanded phase I cohort. Of 1,073 tumors screened, 18 (1.7%) were ROS1 rearranged by FISH, and 31 (2.9%) were ALK rearranged. Compared with the ROS1-negative group, patients with ROS1 rearrangements were significantly younger and more likely to be never-smokers.. All of the ROS1-positive tumors were adenocarcinomas, with a tendency toward higher grade. ROS1-positive and -negative groups showed no difference in overall survival. Molecular Tumor Markers for Non-Small Cell Lung Cancer Jun 16 14

15 The HCC78 ROS1-rearranged NSCLC cell line and 293 cells transfected with CD74- ROS1 showed evidence of sensitivity to crizotinib. The patient treated with crizotinib showed tumor shrinkage, with a near complete response. The authors concluded ROS1 rearrangement defines a molecular subset of NSCLC with distinct clinical characteristics that are similar to those observed in patients with ALK-rearranged NSCLC. Crizotinib shows in vitro activity and early evidence of clinical activity in ROS1-rearranged NSCLC. Cha et al (2014) investigated the usefulness of ROS1 immunohistochemistry (IHC) for the detection of patients who harbor ROS1 rearrangements in two separate cohorts. They also compared ROS1 IHC with ALK IHC in terms of diagnostic performance to predict each gene rearrangement. In a retrospective cohort, IHC was performed in 219 cases of lung adenocarcinoma with already known genetic alterations. In a prospective cohort, they performed IHC for 111 consecutive cases of lung adenocarcinoma and confirmed the results by subsequent FISH. In the retrospective cohort, all 8 ROS1-rearranged tumors were immunoreactive, and 14 of 211 ROS1-wild cases were immunoreactive (sensitivity 100% and specificity 93.4%). In the prospective cohort, all IHC-negative cases were FISH-negative, and 5 of 34 ROS1 immunoreactive cases were ROS1-rearranged (sensitivity 100% and specificity 72.6%). In ROS1-wild tumors, ROS1 protein was more expressed in the tumors of ever-smokers than in those of never-smokers. ALK IHC showed 100% sensitivity and 98.1 to 100% specificity in both patient cohorts. The authors concluded, ROS1 IHC is highly sensitive, but less specific compared with ALK IHC for detection of the corresponding rearrangement. ROS1 IHC-reactive tumors, especially when the tumor is stained with moderate to strong intensity or a diffuse pattern, are recommended to undergo FISH to confirm the gene rearrangement. Han et al (2014) investigated the clinical utility of targeted next-generation sequencing (NGS) for predicting the responsiveness to epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) therapy. The authors compared the efficacy with conventional sequencing in never-smokers with lung adenocarcinoma (NSLAs). Investigators obtained DNA from 48 NSLAs who received gefitinib or erlotinib for their recurrent disease after surgery. Sanger sequencing and peptide nucleic acid clamp polymerase chain reaction (PCR) were used to analyze EGFR, KRAS, BRAF, and PIK3CA mutations. Investigatrors analyzed ALK, RET, and ROS1 rearrangements by fluorescent in situ hybridization or reverse transcriptase-pcr and quantitative real-time PCR. After molecular screening, Ion Torrent NGS was performed in 31 cases harboring only EGFR exon 19 deletions (19DEL), an L858R mutation, or none of the above mutations. The 31 samples were divided into four groups: responders to EGFR-TKIs with only 19DEL or L858R (n=15); primary resistance to EGFR-TKI with only 19DEL or L858R (n=4); primary resistance to EGFR-TKI without any mutations (n=8); responders to EGFR-TKI without any mutations (n=4). With NGS, all conventionally detected mutations were confirmed except for one L858R in group 2. Additional uncovered predictive mutations with NGS included one PIK3CA E542K in group 2, two KRAS (G12V and G12D), one PIK3CA E542K, one concomitant PIK3CA and EGFR L858R in group 3, and one EGFR 19DEL in group 4. Investigators concluded targeted NGS provided a more accurate and clinically useful molecular classification of NSLAs. It may improve the efficacy of EGFR-TKI therapy in lung cancer. Pan et al (2014) performed a comprehensive investigation of the clinicopathologic, histologic and cytologic features of fusion-positive lung adenocarcinomas. Quantitative real-time reverse transcriptase PCR (qrt-pcr) and reverse transcriptase PCR (RT-PCR) were simultaneously performed to screen ALK, ROS1 and RET fusions in resected tumor samples from 1139 Chinese lung adenocarcinoma patients, with validation of positive results using fluorescent in situ hybridization. Molecular Tumor Markers for Non-Small Cell Lung Cancer Jun 16 15